Anti-CD25 antibody

Abstract

The present invention relates to novel anti-human CD25 antibodies and their use to treat cancer and infectious diseases.

Description

[Technical Field]

[0001] This invention relates to the field of cancer and infectious disease treatment, and more particularly discloses novel anti-human CD25 antibodies that can be used to treat cancer and infectious diseases. [Background technology]

[0002] Regulatory T cells (Tregs) are important mediators of immune tolerance, which is involved in the body's protection against autoimmunity. However, in cancer, Tregs appear to play a controversial role. Indeed, the tumor microenvironment can favor the differentiation and recruitment of Tregs, and thus suppress the function of antitumor effector T cells. Therefore, Tregs can be a major obstacle to immunotherapy. This phenomenon has been described in many human cancers and most mouse models of tumor growth, with increased Treg frequency and their suppressor function compared to those reported in healthy subjects. In particular, Tregs have been shown to accumulate in tumors in the presence of tumor-derived chemokines, and once in place, progress to prevent or blunt the antitumor response mediated by immune cells infiltrating the tumor microenvironment. Therefore, Treg accumulation may contribute to the tumor's escape from the host immune system by silencing antitumor immune effector cells.

[0003] As a result, there is a need to develop methods to treat cancer by silencing or eliminating Treg cells from tumors, thereby restoring the antitumor function of T effector cells.

[0004] Tregs are mice that express constitutively high levels of CD25, which is the interleukin-2 receptor α chain that binds to interleukin-2 (IL-2) and regulates Treg development and homeostasis. + It was first described by Sakaguchi et al. as a circulating subset of T cells.

[0005] The use of anti-CD25 antibodies and their modulation of Treg function or activity has been described in the Art. For example, basiliximab is a chimeric mouse-human CD25 antibody that can be used to prevent graft-versus-host disease. Furthermore, daclizumab is a chimeric mouse-human CD25 antibody approved for the treatment of relapsing forms of multiple sclerosis. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Sakaguchi et al. [Overview of the project] [Problems that the invention aims to solve]

[0007] However, to the best of the applicant's knowledge, anti-CD25 antibodies have not been clinically validated for the treatment of cancer in humans. Therefore, there is still a need for effective antitumor immunotherapy based on the modulation of the IL-2 / CD25 pathway. [Means for solving the problem]

[0008] The present invention relates to an isolated anti-human CD25 antibody or its antigen-binding fragment, wherein the variable region (VH) of the heavy chain comprises the following three complementarity-determining regions (CDR): -CDR1:NHAMA(Sequence ID 1); -CDR2:VISYDGX1NX2YYX3DSVKG(Sequence ID 2)(X1 is S or D, X2 is K or T, X3 is A or R) -CDR3:GX4NSGYD(Sequence ID 3)(X4 is either W or L) Or, any CDR having an amino acid sequence that shares at least approximately 95% identity with SEQ ID NOs: 1-3, The variable region (VL) of the light chain consists of the following three CDRs: -CDR1:RASQX5X6X7X8X9LN(Sequence ID 4), (X5 is S or N, X6 is V or I, X7 is N or S, X8 is S or K, X9 is F or Y) and -CDR2:GTX 10 SLQS(Sequence ID 5), (X 10 is S or N); and -CDR3:QQYX 11 SWPWT(Sequence No. 6)(X 11 (is T or N) Or it includes any CDR having an amino acid sequence that shares at least approximately 95% identity with SEQ ID NOs: 4-6.

[0009] In one embodiment, the VH of the antibody or its antigen-binding fragment is the following CDR: -CDR1:NHAMA(Sequence ID 1); -CDR2:VISYDGSNKYYADSVKG(Sequence ID 7); and -CDR3:GWNSGYD(Sequence ID 8) Includes, The VL of the antibody or its antigen-binding fragment is as follows: -CDR1:RASQSVNSFLN(Sequence ID 9); -CDR2:GTSSLQS(sequence number 10); and -CDR3:QQYTSWPWT(Sequence ID 11) Includes.

[0010] In one embodiment, the VH of the antibody or its antigen-binding fragment is the following CDR: -CDR1:NHAMA(Sequence ID 1); -CDR2:VISYDGSNKYYADSVKG(Sequence ID 7); and -CDR3:GWNSGYD(Sequence ID 8) Includes, The VL of the antibody or its antigen-binding fragment is as follows: -CDR1:RASQSVNSYLN(sequence number 12); -CDR2:GTNSLQS(sequence number 13); and -Includes CDR3:QQYNSWPWT (Sequence ID 14).

[0011] In one embodiment, the VH of the antibody or its antigen-binding fragment is the following CDR: -CDR1:NHAMA(Sequence ID 1); -CDR2:VISYDGSNKYYADSVKG(Sequence ID 7); and -CDR3:GWNSGYD(Sequence ID 8) Includes, The VL of the antibody or its antigen-binding fragment is as follows: -CDR1:RASQSISSYLN(sequence number 15); -CDR2:GTNSLQS(sequence number 13); and -CDR3:QQYNSWPWT(Sequence ID 14) Includes.

[0012] In one embodiment, the VH of the antibody or its antigen-binding fragment is the following CDR: -CDR1:NHAMA(Sequence ID 1); -CDR2:VISYDGSNKYYADSVKG(Sequence ID 7); and -CDR3:GWNSGYD(Sequence ID 8) Includes, The VL of the antibody or its antigen-binding fragment is as follows: -CDR1:RASQSVSSFLN(Sequence ID 16); -CDR2:GTNSLQS(sequence number 13); and -CDR3:QQYTSWPWT(Sequence ID 11) Includes.

[0013] In one embodiment, the VH of the antibody or its antigen-binding fragment is the following CDR: -CDR1:NHAMA(Sequence ID 1); -CDR2:VISYDGSNKYYADSVKG(Sequence ID 7); and -CDR3:GLNSGYD(sequence number 17); Includes, The VL of the antibody or its antigen-binding fragment is as follows: -CDR1:RASQSVSKFLN(Sequence ID 18); -CDR2:GTNSLQS(sequence number 13); and -CDR3:QQYNSWPWT(Sequence ID 14) Includes.

[0014] In one embodiment, the VH of the antibody or its antigen-binding fragment is the following CDR: -CDR1:NHAMA(Sequence ID 1); -CDR2:VISYDGSNKYYADSVKG(Sequence ID 7); and -CDR3:GLNSGYD(sequence number 17); Includes, The VL of the antibody or its antigen-binding fragment is as follows: -CDR1:RASQNINSFLN(Sequence ID 19); -CDR2:GTSSLQS(sequence number 10); and -CDR3:QQYNSWPWT(Sequence ID 14) Includes.

[0015] In one embodiment, the VH of the antibody or its antigen-binding fragment is the following CDR: -CDR1:NHAMA(Sequence ID 1); -CDR2:VISYDGSNKYYADSVKG(Sequence ID 7); and -CDR3:GLNSGYD(sequence number 17); Includes, The VL of the antibody or its antigen-binding fragment is as follows: -CDR1:RASQNISSFLN(Sequence ID 20); -CDR2:GTNSLQS(sequence number 13); and -CDR3:QQYNSWPWT(Sequence ID 14) Includes.

[0016] In one embodiment, the VH of the antibody or its antigen-binding fragment is the following CDR: -CDR1:NHAMA(Sequence ID 1); -CDR2:VISYDGSNKYYADSVKG(Sequence ID 7); and -CDR3:GLNSGYD(sequence number 17); Includes, The VL of the antibody or its antigen-binding fragment is as follows: -CDR1:RASQSISSFLN(Sequence ID 21); -CDR2:GTNSLQS(sequence number 13); and -CDR3:QQYTSWPWT(Sequence ID 11) Includes.

[0017] In one embodiment, the VH of the antibody or its antigen-binding fragment is the following CDR: -CDR1:NHAMA(Sequence ID 1); -CDR2:VISYDGSNKYYADSVKG(Sequence ID 7); and -CDR3:GLNSGYD(sequence number 17); Includes, The VL of the antibody or its antigen-binding fragment is as follows: -CDR1:RASQSVSSFLN(Sequence ID 16); -CDR2:GTNSLQS(sequence number 13); and -CDR3:QQYNSWPWT(Sequence ID 14) Includes.

[0018] In one embodiment, the VH of the antibody or its antigen-binding fragment is the following CDR: -CDR1:NHAMA(Sequence ID 1); -CDR2:VISYDGSNKYYADSVKG(Sequence ID 7); and -CDR3:GLNSGYD(sequence number 17); Includes, The VL of the antibody or its antigen-binding fragment is as follows: -CDR1:RASQNVSSFLN(Sequence ID 22); -CDR2:GTSSLQS(sequence number 10); and -CDR3:QQYTSWPWT(Sequence ID 11) Includes.

[0019] In one embodiment, the VH of the antibody or its antigen-binding fragment is the following CDR: -CDR1:NHAMA(Sequence ID 1); -CDR2:VISYDGSNKYYADSVKG(Sequence ID 7); and -CDR3:GLNSGYD(sequence number 17); Includes, The VL of the antibody or its antigen-binding fragment is as follows: -CDR1:RASQSINSFLN(Sequence ID 23); -CDR2:GTNSLQS(sequence number 13); and -CDR3:QQYNSWPWT(Sequence ID 14) Includes.

[0020] In one embodiment, the VH of the antibody or its antigen-binding fragment is the following CDR: -CDR1:NHAMA(Sequence ID 1); -CDR2:VISYDGSNKYYADSVKG(Sequence ID 7); and -CDR3:GLNSGYD(sequence number 17); Includes, The VL of the antibody or its antigen-binding fragment is as follows: -CDR1:RASQNINSFLN(Sequence ID 19); -CDR2:GTNSLQS(sequence number 13); and -CDR3:QQYTSWPWT(Sequence ID 11) Includes.

[0021] In one embodiment, the VH of the antibody or its antigen-binding fragment is the following CDR: -CDR1:NHAMA(Sequence ID 1); -CDR2:VISYDGSNKYYADSVKG(Sequence ID 7); and -CDR3:GLNSGYD(sequence number 17); Includes, The VL of the antibody or its antigen-binding fragment is as follows: -CDR1:RASQSVSSYLN(sequence code 24); -CDR2:GTNSLQS(sequence number 13); and -CDR3:QQYTSWPWT(Sequence ID 11) Includes.

[0022] In one embodiment, the VH of the antibody or its antigen-binding fragment is the following CDR: -CDR1:NHAMA(Sequence ID 1); -CDR2:VISYDGSNKYYRDSVKG(Sequence ID 25); and -CDR3:GLNSGYD(sequence number 17); Includes, The VL of the antibody or its antigen-binding fragment is as follows: -CDR1:RASQSISSFLN(Sequence ID 21); -CDR2:GTNSLQS(sequence number 13); and -CDR3:QQYNSWPWT(Sequence ID 14) Includes.

[0023] In one embodiment, the VH of the antibody or its antigen-binding fragment is the following CDR: -CDR1:NHAMA(Sequence ID 1); -CDR2:VISYDGSNKYYRDSVKG(Sequence ID 25); and -CDR3:GLNSGYD(sequence number 17); Includes, The VL of the antibody or its antigen-binding fragment is as follows: -CDR1:RASQNINSFLN(Sequence ID 19); -CDR2:GTSSLQS(sequence number 10); and -CDR3:QQYNSWPWT(Sequence ID 14) Includes.

[0024] In one embodiment, the VH of the antibody or its antigen-binding fragment is the following CDR: -CDR1:NHAMA(Sequence ID 1); -CDR2:VISYDGSNKYYRDSVKG(Sequence ID 25); and -CDR3:GLNSGYD(sequence number 17); Includes, The VL of the antibody or its antigen-binding fragment is as follows: -CDR1:RASQNVSSFLN(Sequence ID 22); -CDR2:GTNSLQS(sequence number 13); and -CDR3:QQYNSWPWT(Sequence ID 14) Includes.

[0025] In one embodiment, the VH of the antibody or its antigen-binding fragment is the following CDR: -CDR1:NHAMA(Sequence ID 1); -CDR2:VISYDGSNTYYADSVKG(Sequence ID 26); and -CDR3:GLNSGYD(sequence number 17); Includes, The VL of the antibody or its antigen-binding fragment is as follows: -CDR1:RASQNVSSFLN(Sequence ID 22); -CDR2:GTNSLQS(sequence number 13); and -CDR3:QQYNSWPWT(Sequence ID 14) Includes.

[0026] In one embodiment, the VH of the antibody or its antigen-binding fragment is the following CDR: -CDR1:NHAMA(Sequence ID 1); -CDR2:VISYDGDNKYYADSVKG(Sequence ID 27); and -CDR3:GLNSGYD(sequence number 17); Includes, The VL of the antibody or its antigen-binding fragment is as follows: -CDR1:RASQSINSFLN(Sequence ID 23); -CDR2:GTNSLQS(sequence number 13); and -CDR3:QQYTSWPWT(Sequence ID 11) Includes.

[0027] In one embodiment, the isolated anti-human CD25 antibody or its antigen-binding fragment is chimeric, humanized, or human. In one embodiment, the antibody or antigen-binding fragment is monoclonal.

[0028] In one embodiment, the isolated anti-human CD25 antibody or its antigen-binding fragment mediates antibody-dependent cytotoxicity, complement-dependent cytotoxicity, and / or antibody-dependent phagocytosis.

[0029] In one embodiment, the isolated anti-human CD25 antibody or its antigen-binding fragment described above is a bispecific antibody.

[0030] The present invention further relates to a fusion protein comprising an isolated anti-human CD25 antibody or its antigen-binding fragment as described above herein.

[0031] The present invention further relates to nucleic acids encoding isolated anti-human CD25 antibodies or their antigen-binding fragments, or fusion proteins, as described herein.

[0032] The present invention further relates to a pharmaceutical composition comprising an isolated anti-human CD25 antibody or its antigen-binding fragment or fusion protein as described above herein, and at least one pharmaceutically acceptable excipient.

[0033] The present invention further relates to isolated anti-human CD25 antibodies, antigen-binding fragments thereof, fusion proteins, or pharmaceutical compositions, as described above herein, for use as pharmaceuticals.

[0034] The present invention further relates to isolated anti-human CD25 antibodies, or their antigen-binding fragments, fusion proteins, or pharmaceutical compositions, as described above herein, for use in subjects requiring such treatment for cancer or infectious diseases.

[0035] The present invention further relates to a combination of an immunotherapy, as described above herein, and an isolated anti-human CD25 antibody or its antigen-binding fragment, fusion protein, or pharmaceutical composition, for use in subjects requiring such treatment for cancer or infectious diseases. [Brief explanation of the drawing]

[0036] [Figure 1] This is a combination of two graphs showing that the anti-CD25 antibody of the present invention specifically binds to human CD25 (huCD25) expressed on the surface of transfected HEK293 cells. Figure 1A shows the geometric mean fluorescence intensity (GeoMFI) of isotype controls, the anti-CD25 antibody of the present invention (H07, H09, G02, E04, D01, E04-2, B05, G09, B01, C01, G01, H01, G02-2, H02, F03, D05, B07, H08, B12), or basiliximab at different concentrations (0.01 nM to 500 nM) in HEK293 cells transfected with huCD25. Figure 1B shows the geometric mean fluorescence intensity (GeoMFI) of isotype controls, the anti-CD25 antibodies of the present invention (H07, H09, G02, E04, D01, E04-2, B05, G09, B01, C01, G01, H01, G02-2, H02, F03, D05, B07, H08, B12), or basiliximab at different concentrations (0.01 nM to 500 nM) in HEK293 WT cells. [Figure 2]Figure 2A is a combination of two histograms showing the effect of the anti-CD25 antibodies (H09, D01, E04-2, B05, C01, G01, G02-2, F03, D05, B07, B12) of the present invention on IL-2-induced effector T cell proliferation. Figure 2A is a histogram showing the effect of the anti-CD25 antibodies (H09, D01, E04-2, B05, C01, G01, G02-2, F03, D05, B07, B12) of the present invention on IL-2-induced effector T cell proliferation compared to human IgG1 control antibody, basiliximab, and 7G7B6. **p<0,01 vs H09, D01, E04-2, B05, C01, G01, G02-2, F03, D05, B07, B12; one-way ANOVA. Figure 2B is a histogram showing the effect of the anti-CD25 antibodies of the present invention (H09, D01, E04-2, B05, C01, G01, G02-2, F03, D05, B07, B12) on IL-2-induced effector T cell proliferation compared to human IgG1 control antibody, basiliximab, and MA-251. **p<0,01 vs H09, D01, E04-2, B05, C01, G01, G02-2, F03, D05, B07, B12; one-way ANOVA. [Figure 3] Figure 3A shows a combination of two histograms illustrating the effect of the anti-CD25 antibody of the present invention on Treg cell depletion within the CD45+ lymphocyte population. Figure 3A shows the percentage of Treg cells within the CD45+ lymphocyte population after incubation with 1 μg / ml human IgG1 control antibody, the anti-CD25 antibody of the present invention (H09, D01, E04-2, B05, C01, G01, G02-2, F03, D05, B07, B12), or basiliximab. Figure 3B shows the percentage of Treg cell depletion within the CD45+ lymphocyte population after incubation with 1 μg / ml human IgG1 control antibody, the anti-CD25 antibody of the present invention (H09, D01, E04-2, B05, C01, G01, G02-2, F03, D05, B07, B12), or basiliximab. [Figure 4]Figure 4B shows two histograms (A, B) illustrating the effect of the anti-CD25 antibodies of the present invention on the depletion of CD4+ effector T cells and CD8+ effector T cells within a CD45+ lymphocyte population. Figure 4A shows the percentage of CD4+ effector T cells within a CD45+ lymphocyte population after incubation with a 1 μg / ml human IgG1 control antibody and the anti-CD25 antibodies of the present invention (E04-2, B05, C01, G01, G02-2). Figure 4B shows the percentage of CD8+ effector T cells within a CD45+ lymphocyte population after incubation with a 1 μg / ml human IgG1 control antibody and the anti-CD25 antibodies of the present invention (E04-2, B05, C01, G01, G02-2). [Figure 5] This histogram shows the percentage of antibody-dependent phagocytosis (ADCP) induced by the 10 μg / mL anti-CD25 antibodies (E04-2, B05, C01, G01, G02-2, B07) of the present invention, compared to human IgG1 control antibodies. The data are expressed as mean ± SEM. [Modes for carrying out the invention]

[0037] definition In this invention, the following terms have the following meanings.

[0038] The word "approximately" preceding a number includes a range of ±10% or less of the given number. It should be understood that the value referred to by the term "approximately" is also preferably disclosed specifically.

[0039] "Adnectin" is a protein that is known both as a monobody and in the art, and is designed to bind to antigens with high affinity and specificity. Collectively, they belong to a class of molecules called "antibody mimes."

[0040] "Alpha bodies," also known as cell-permeable alpha bodies, refer to a type of antibody mimetic consisting of small 10kDa proteins engineered to bind to various antigens. Alpha bodies can reach and bind to intracellular protein targets.

[0041] "Affibody" refers to affinity proteins based on a 58-amino acid protein domain derived from one of the IgG-binding domains of Staphylococcus protein A (Frejd & Kim, 2017. Exp Mol Med. 49(3):e306; U.S. Patent No. 5,831,012).

[0042] "Affilins" refer to artificial proteins designed to selectively bind to antigens. They are similar to antibodies in their affinity and specificity for antigens, but not in their structure, which makes them antibody mimetic types.

[0043] "Affinity" and "binding activity" are used to define the strength of an antibody-antigen complex. Affinity measures the strength of the interaction between the epitope and the antigen-binding site on the antibody. It is the affinity constant K. A or the dissociation constant K D It can be expressed by: (i) binding activity (or functional affinity), which gives a measure of the overall strength of the antibody-antigen complex. It can depend on a variety of parameters, including (ii) the affinity of the antibody or its antigen-binding fragment to the epitope, (ii) the binding titer of both the antibody and the antigen, and (iii) the structural arrangement of the interacting parts.

[0044] Where used herein, “antibody” and “immunoglobulin” may be used interchangeably and refer to a protein having a combination of two heavy chains and two light chains, with or without any relevant specific immunoreactivity. “Antibody” refers to such an assembly having significant known specific immunoreactive activity against an antigen of interest (e.g., human CD25). The term “anti-hCD25 antibody” is used herein to refer to an antibody that exhibits immunological specificity to the human CD25 protein. As described elsewhere herein, “specificity” to human CD25 (hCD25) does not exclude cross-reactivity with species homologs of hCD25, e.g., monkey CD25. Antibodies and immunoglobulins include light and heavy chains, with or without interchain covalent bonds between them. The basic immunoglobulin structure in vertebrate systems is relatively well understood. The general term “immunoglobulin” includes five distinct classes of antibodies that can be biochemically distinguished. The following discussion generally concerns the IgG class of immunoglobulin molecules, but all five classes of antibodies are within the scope of this invention. Regarding IgG, immunoglobulins consist of two identical polypeptide light chains with a molecular weight of approximately 23 kDa and two identical heavy chains with a molecular weight of approximately 53–70 kDa. The four chains are linked by disulfide bonds in a "Y" configuration, where the light chains support the heavy chains that begin at the "Y" mouth and continue through the variable region. The light chains of antibodies are classified as either kappa (κ) or lambda (λ). Each heavy chain class may be linked to either a κ or λ light chain. Generally, when immunoglobulins are produced by hybridomas, B cells, or genetically engineered host cells, the light and heavy chains are covalently linked to each other, and the "tail" regions of the two heavy chains are linked to each other by covalent disulfide bonds or non-covalent bonds. In the heavy chains, the amino acid sequence extends from the N-terminus of the branched ends of the Y configuration to the C-terminus at the bottom of each chain. Those skilled in the art will understand that heavy chains are classified as gamma (γ), mu (μ), alpha (α), delta (δ), or epsilon (ε), and that there are several subclasses within them (e.g., γ1-γ4). It is the properties of these chains that determine the "class" of an antibody as IgG, IgM, IgA, IgD, or IgE, respectively.Immunoglobulin subclasses or "isotypes" (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, etc.) are well-characterized and known to confer functional specialization. Modified versions of each of these classes and isotypes are readily identifiable to those skilled in the art in light of this disclosure and are therefore within the scope of the present invention. As described above, the variable region of an antibody enables the antibody to selectively recognize and specifically bind to an epitope on an antigen. That is, the light chain variable domain (VL domain) and heavy chain variable domain (VH domain) of the antibody combine to form a variable region that defines a three-dimensional antigen-binding site. This quaternary antibody structure forms antigen-binding sites located at the ends of each arm of the "Y". More specifically, the antigen-binding site is defined by three complementarity-determining regions (CDRs) of the VH and VL chains, respectively.

[0045] "Afitin" refers to highly stable, engineered affinity proteins originally derived from two 7kDa DNA-binding polypeptides, Sac7d and Sso7d, belonging to the genus Sulfolobus.

[0046] "Anticalin" refers to an antibody-mimicking technique whose binding specificity is derived from lipocalin. Anticalin can also be formatted as a dual-targeting protein called duocalin.

[0047] As used herein, “antigen-binding fragment” refers to a portion or region of an antibody containing fewer amino acid residues than the entire antibody. The “antigen-binding fragment” binds to an antigen and / or competes for antigen binding with the entire antibody from which it originates (e.g., specific binding to human CD25). Antibody-antigen-binding fragments include, but are not limited to, single-chain antibodies, Fv, Fab, Fab', Fab'-SH, F(ab)'2, Fd, defucosylated antibodies, diabodies, triabodies, and tetrabodies.

[0048] As used herein, "armadillo repeat protein-based scaffold" refers to a type of antibody mimetic corresponding to an artificial peptide-binding scaffold based on an armadillo repeat protein. An armadillo repeat protein is characterized by an armadillo domain composed of tandem armadillo repeats of approximately 42 amino acids that mediate interaction with a peptide or protein.

[0049] "Atomer" refers to a binding molecule for a target protein that trimerizes into a refined form for biological activity. They are relatively large compared to other antibody mimetic scaffolds.

[0050] "Avimer" refers to antibody mimetic technology.

[0051] "CD25", unless otherwise indicated, refers to any native CD25 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). The interleukin-2 receptor alpha chain (also called CD25) protein is encoded by the IL2RA gene. Two forms of the IL-2 receptor have been described: the first form includes the α subunit (CD25), the β subunit (CD122), and the γ subunit (CD132), and the second form includes only the β and γ subunits (i.e., CD122 and CD132). This term encompasses "full-length" or unprocessed CD25, as well as any form of CD25 resulting from intracellular processing. This term also encompasses naturally occurring variants of CD25 (e.g., splice variants or allelic variants). In certain embodiments, CD25 is human CD25. For example, CD25 is expressed by activated T lymphocytes and activated B lymphocytes in response to antigen or mitogen stimulation. CD25 is also expressed by regulatory T cells (CD25 high FoxP3 + regulatory T cells). In one embodiment, CD25 refers to human CD25 (Uniprot accession number P01589, SEQ ID NO: 92). SEQ ID NO: 92 MDSYLLMWGLLTFIMVPGCQAELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPGEEKPQASPEGRPESETSCLVTTTDFQIQTEMAATMETSIFTTEYQVAVAGCVFLLISVLLLSGLTWQRRQRKSRRTI

[0052] A “CDR” or “complementarity-determining region” refers to a discontinuous antigen-binding site found within the variable regions of both heavy-chain and light-chain polypeptides. The precise amino acid sequence boundary of a given CDR can be determined using one of numerous well-known schemes, including those described in Kabat et al. (1991), “Sequences of Proteins of Immunological Interest” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (“Kabat” numbering scheme), Al-Lazikani et al., (1997) JMB 273, 927-948 (“Chothia” numbering scheme), or combinations thereof. More recently, a universal numbering system has been developed and widely adopted: ImMunoGeneTics (IMGT) Information System (registered trademark) (Lefranc et al., Nucleic Acids Res. 27:209-212 1999). IMGT is an integrated information system specifically for human and other vertebrate immunoglobulins (IG), T cell receptors (TR), and major histocompatibility complexes (MHC). In this specification, CDRs are referred to in terms of both amino acid sequence and location within the light or heavy chain. Since the “location” of CDRs within the structure of immunoglobulin variable domains is conserved across species and resides within structures called loops, CDRs and framework residues can be easily identified by using a numbering system that aligns variable domain sequences according to their structural features. This information can be used when grafting and substituting CDR residues from immunoglobulins of one species into receptor frameworks, typically from human antibodies. The correspondence between Kabat numbering and IMGT-specific numbering systems is also well known to those skilled in the art (e.g., Lefranc et al. above).Therefore, in one embodiment, the CDR region or CDR is intended to indicate the hypervariable regions of the heavy and light chains of immunoglobulins as defined by the IMGT® numbering system (e.g., Lefranc et al. above).

[0053] "DARPins" (Designed Ankyrin Repeat Proteins) refers to antibody-mimicking DRP (Designed Repeat Protein) technology developed to utilize the binding ability of non-antibody polypeptides.

[0054] As used herein, “diabody” refers to a small antibody fragment prepared by constructing an scFv fragment having a short linker (about 5-10 residues) between the VH and VL such that interchain pairing, rather than intrachain pairing, is achieved in the variable domain, resulting in a bivalent fragment, i.e., a fragment having two antigen-binding sites. A bispecific diabody is a heterodimer of two “crossover” scFv fragments in which the VH and VL of two antibodies are located on different polypeptide chains. Diabodies are described, for example, in European Patent No. 0404097 or International Publication No. 1993011161.

[0055] A "domain antibody" refers to the smallest functional binding unit of an antibody, corresponding to the variable region of either the heavy chain or the light chain of the antibody.

[0056] "Domain Knitz peptide" refers to a type of antibody mimetic, based on the active domain of a protein that inhibits the function of a protease.

[0057] "Effector T cells" are several types of T cells (e.g., CD4) + and CD8 + This refers to a group of cells including T cells. It includes helper T cells (Th cells) that assist other leukocytes in immunological processes, including the maturation of B cells into plasma cells and memory B cells, as well as cytotoxic T cells (Tc cells, CTLs, T killer cells, killer T cells) that destroy virus-infected cells and tumor cells, and are also involved in transplant rejection.

[0058] An "epitope" refers to a specific sequence of amino acids located on one or more proteins to which an antibody, its antigen-binding fragment, or antibody mimetic binds. Epitopes often consist of chemically active surface groups of molecules such as amino acids or sugar side chains, and possess specific three-dimensional structural and charge properties. Epitopes can be linear (or continuous) or conformated, meaning they contain two or more sequences of amino acids from various regions of an antigen, which do not necessarily have to be continuous.

[0059] "Evasin" refers to a class of chemokine-binding proteins.

[0060] "Fab" refers to a monovalent fragment containing the following regions linked by intramolecular disulfide bonds: VH, VL, CH1, and CL. As used herein, F(ab')2 refers to a fragment containing two antigen-binding regions linked by disulfide bonds. As used herein, Fab' refers to a fragment obtained by reduction of the F(ab')2 fragment.

[0061] The "framework region" or "FR region" contains amino acid residues that are part of the variable region but not part of the CDR (e.g., using the IMGT® numbering definition for CDRs). The framework region of the light chain is similarly separated by each of the CDRs of the VL. In naturally occurring antibodies, the six CDRs present on each monomeric antibody are short discontinuous sequences of amino acids that are specifically positioned to form antigen-binding sites when the antibody takes its three-dimensional configuration in an aqueous environment. The rest of the heavy chain variable domain and light chain variable domain exhibit less intermolecular variability in the amino acid sequence and are called the framework region. The framework region primarily takes a β-sheet conformation, and the CDRs form loops that connect, and sometimes form part of, the β-sheet structure. Thus, these framework regions act to form a scaffold that positions the six CDRs in the correct orientation through non-covalent interactions between the chains. The antigen-binding site formed by the positioned CDRs defines a surface complementary to the epitope on the immunoreactive antigen. This complementary surface promotes non-covalent binding of antibodies to immunoreactive antigen epitopes. The location of the CDR can be easily identified by those skilled in the art.

[0062] "Fc domain," "Fc portion," and "Fc region" refer to the C-terminal fragment of an antibody heavy chain, for example, approximately amino acids (aa)230 to approximately aa450 of a human gamma heavy chain, or its corresponding sequence in other types of antibody heavy chains (e.g., α, δ, ε, and μ of human antibodies), or its naturally occurring allotype.

[0063] The term "Fd fragment" refers to the heavy chain of the Fab fragment, which includes the VH region and the CH1 region.

[0064] "Finomers" refer to proteins belonging to the class of antibody mimetic molecules. They are attractive binding molecules due to their high thermal stability and low immunogenicity.

[0065] As used herein, "Fv" refers to the smallest antibody fragment containing the complete antigen recognition and binding site. This fragment consists of a dimer in which one VH and one VL are closely non-covalently bonded. The folding of these two domains gives rise to six hypervariable loops (three from the heavy chain and three from the light chain, respectively) that contribute to antigen binding and confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of the Fv containing only the three antigen-specific CDRs) has the ability to recognize and bind to the antigen, albeit with lower affinity than the entire binding site.

[0066] The "heavy chain region" contains an amino acid sequence derived from the constant domain of the immunoglobulin heavy chain. Proteins containing a heavy chain region are C H 1 domain, hinge (e.g., upper hinge region, middle hinge region, and / or lower hinge region) domain, C H 2 domains, C H The antibody or antigen-binding fragment thereof comprises at least one of the three domains, or their variants or fragments. In one embodiment, the antibody or antigen-binding fragment according to the present invention comprises an immunoglobulin heavy chain (e.g., hinge portion, C H 2 domains and C H It may include an Fc region of 3 domains. In another embodiment, the antibody or antigen-binding fragment thereof according to the present invention may include at least a constant domain (e.g., C H It lacks a region (all or part of 2 domains). In certain embodiments, at least one, preferably all, of the constant domains is derived from a human immunoglobulin heavy chain. For example, in one embodiment, the heavy chain region includes a fully human hinge domain. In other embodiments, the heavy chain region is a fully human Fc region (e.g., a hinge, C domain derived from human immunoglobulin). H 2 and C H It includes a 3-domain sequence. In certain embodiments, the constituent constant domains of the heavy chain region are derived from different immunoglobulin molecules. For example, the heavy chain region of a protein is derived from the IgG1 molecule. HIt may include two domains and a hinge region derived from an IgG3 or IgG4 molecule. In other embodiments, the constant domain is a chimeric domain containing regions from different immunoglobulin molecules. For example, the hinge may include a first region derived from an IgG1 molecule and a second region derived from an IgG3 or IgG4 molecule. In some embodiments, the constant domain of the heavy chain region may be modified so that its amino acid sequence differs from that of a naturally occurring (wild-type) immunoglobulin molecule. That is, the antibody or antigen-binding fragment thereof according to the present invention may have one or more heavy chain constant domains (C H 1. Hinge, C H 2 or C H 3) and / or light chain constant domain (C L This may include changes or modifications to the ) domain. Exemplary modifications include the addition, deletion, or substitution of one or more amino acids in one or more domains.

[0067] The "hinge region" is C H 1 domain C H It contains a region of heavy chain molecules that bind to two domains. This hinge region contains approximately 25 residues, is flexible, and therefore allows the two N-terminal antigen-binding regions to move independently. The hinge region can be subdivided into three distinct domains, namely the upper, middle, and lower hinge domains (Roux et al., 1998. J Immunol. 161(8):4083-90).

[0068] While "hypervariable loops" are defined based on structure, complementarity-determining regions (CDRs) are defined based on sequence variability (Kabat et al., 1991. Sequences of proteins of immunological interest (5th ed.). Bethesda, MD: USD ep. of Health and Human Services), and the limits of HV and CDRs are several V H and V L Because it can vary by domain, hypervariable loop (HV) is not strictly synonymous with CDR.

[0069] When used herein in relation to the relationship between two or more amino acid sequences or two or more nucleic acid sequences, “identity” refers to the degree of sequence relevance between amino acid sequences or nucleic acid sequences, determined by the number of matches between strings of two or more amino acid residues or nucleic acid residues. “Identity” measures the percentage of identical matches between the smaller of two or more sequences that have gap alignments (if any) addressed by a specific mathematical model or computer program (i.e., “algorithm”). The identity of related amino acid sequences or nucleic acid sequences can be readily calculated by known methods. Such methods include, but are not limited to, those described in Lesk AM (1988). Computational molecular biology: Sources and methods for sequence analysis. New York, NY: Oxford University Press; Smith DW (1993). Biocomputing: Informatics and genome projects. San Diego, CA: Academic Press; Griffin AM & Griffin HG (1994). Computer analysis of sequence data, Part 1. Totowa, NJ: Humana Press; von Heijne G (1987). Sequence analysis in molecular biology: treasure trove or trivial pursuit. San Diego, CA: Academic Press; Gribskov MR & Develeux J (1991). Sequence analysis primer. New York, NY: Stockton Press; Carillo et al., 1988. SIAM J Appl Math. 48(5):1073-82. The preferred method for determining identity is designed to give the greatest match between the sequences being tested. Methods for determining identity are described in publicly available computer programs.Preferred computer programming methods for determining the identity between two sequences include the GCG program package containing GAP (Genetics Computer Group, University of Wisconsin, Madison, WI; Devereux et al., 1984. Nucleic Acids Res. 12(1 Pt 1):387-95), BLASTP, BLASTN, and FASTA (Altschul et al., 1990. J Mol Biol. 215(3):403-10). The BLASTX program is publicly available from the National Center for Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul et al. NCB / NLM / NIH Bethesda, Md. 20894). Identity can also be determined using the well-known Smith-Waterman algorithm.

[0070] As used herein, “interleukin-2” or “IL-2” refers to any natural IL-2 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise specified. The term encompasses untreated IL-2 and any form of IL-2 resulting from intracellular processing. The term also encompasses naturally occurring variants of IL-2 (e.g., splice variants or allele variants). In one embodiment, IL-2 is human IL-2 having the sequence of SEQ ID NO: 93. Sequence ID 93 MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT

[0071] "Nottin" (also known as the inhibitor cystine-not) refers to an antibody mime that contains a protein structural motif with three disulfide crosslinks.

[0072] "Mammals" refers to any mammal, including humans, non-human primates, livestock and agricultural animals, as well as animals in zoos, sports, or as pets, such as dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, monkeys, etc. Preferably, the mammal is a human.

[0073] A "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies; that is, the individual antibodies in the population are identical except for any naturally occurring variations that may be present in small amounts. Monoclonal antibodies are highly specific and directed to a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations, which contain different antibodies against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies have the advantage of being able to be synthesized without contamination by other antibodies. The modifier "monoclonal" should not be interpreted as requiring the production of the antibody by any particular method. For example, the monoclonal antibody or its antigen-binding fragment according to the present invention can be prepared by the hybridoma methodology first described by Kohler et al., 1975. Nature. 256(5517):495-7, or can be produced using recombinant DNA methods in bacterial, eukaryotic, or plant cells (U.S. Patent No. 4,816,567). Monoclonal antibodies can also be isolated from phage antibody libraries using techniques described, for example, Clackson et al., 1991. Nature. 352(6336):624-8 and Marks et al., 1991. J Mol Biol. 222(3):581-97.

[0074] "Nanobodies" refer to antibody-derived therapeutic proteins that possess the unique structural and functional characteristics of naturally occurring heavy chain antibodies (Muyldermans, 2013. Annu Rev Biochem. 82:775-97). These heavy chain antibodies have a single variable domain (VHH) and two constant domains (C H 2 and C H 3) may be included.

[0075] "Preventing," "preventing," and "prevention" refer to preventive and protective measures aimed at reducing the likelihood that a subject will develop a pathological condition or disability over a given period of time. Such reduction may be reflected, for example, in the delay of the onset of at least one symptom of a pathological condition or disability in the subject.

[0076] "Regulatory T cells" or "Treg cells" are a special type of T cell that can suppress the response of other T cells, particularly CD4 cells. + This refers to T cells. Treg cells are generally characterized by the expression of CD4, the α subunit of the IL-2 receptor (CD25), and the transcription factor forkheadbox P3 (Foxp3), and play a crucial role in inducing and maintaining peripheral self-tolerance to antigens, including those expressed by tumors. More recently, CD8 Tregs have also been described.

[0077] As used herein, "single-chain antibody" means any antibody or fragment thereof that is a protein having a primary structure consisting of or containing a single uninterrupted sequence of consecutive amino acid residues, but is not limited to, (1) a single-chain Fv molecule (scFv); (2) a single-chain protein containing only one light chain variable domain, or a fragment thereof containing three CDRs of a light chain variable domain and not having an associated heavy chain portion; and (3) a single-chain protein containing only one heavy chain variable region, or a fragment thereof containing three CDRs of a heavy chain variable region without an associated light chain portion.

[0078] "Single-chain Fv" is also abbreviated as "sFv" or "scFv," and refers to a single amino acid chain linked to V. H and V L Refers to an antibody fragment containing an antibody domain. Preferably, the scFv amino acid sequence contains a peptide linker that allows the scFv to form a desired structure for antigen binding. H Domain and V L Further include between the domain and the domain.

[0079] As used herein, “subject” refers to a mammal, preferably a human. In one embodiment, the subject may be a “patient,” i.e., a warm-blooded animal, more preferably a human, who is awaiting acceptance of medical treatment, receiving medical treatment, or has been / is / will be subject to medical treatment, or is being monitored for the onset of a disease.

[0080] "Therapeutic dose" means the level or amount of antibodies described herein that, without causing significant negative or adverse side effects against the target, is intended to (1) delay or prevent the onset of a disease, disorder, or condition; (2) slow or halt the progression, worsening, or aggravation of one or more symptoms of a disease, disorder, or condition; (3) improve the symptoms of a disease, disorder, or condition; (4) reduce the severity or incidence of a disease, disorder, or condition; or (5) cure a disease, disorder, or condition. Therapeutic dose may be administered prophylactically or for a prophylactic effect before the onset of a disease, disorder, or condition. Alternatively or additionally, the therapeutic dose may be administered after the onset of a disease, disorder, or condition for a therapeutic effect.

[0081] "To treat," "to cure," or "to alleviate" refers to both therapeutic treatment and preventive or prophylactic measures, the purpose of which is to prevent or slow (reduce) the targeted pathological condition or disability. Those who require treatment include those who already have a disability, as well as those who are prone to developing a disability or who should be prevented from developing a disability. In one embodiment, a subject is considered successfully "treated" for cancer or infectious disease if, after receiving a therapeutic dose of antibodies according to the present invention, he or she exhibits at least one of the following: a reduction in the number of cancer cells (or tumor size) or pathogenic cells; a reduction in the percentage of all cells that are cancerous or pathogenic; some degree of relief of one or more symptoms associated with the cancer or infection being treated; a reduction in morbidity and mortality; or an improvement in quality of life. The above parameters for evaluating successful treatment and improvement of a disease are readily measurable by routine procedures well known to physicians.

[0082] "Tumor-infiltrating Treg" is a conventional CD4+ or CD8 + CD25 accumulates within neoplastic lesions as a result of several different mechanisms, including infiltration from cells, localized spread, survival advantage, and increased in situ occurrence. + / hi Foxp3 + Regarding regulatory T cells.

[0083] A "unibody" refers to an antibody fragment that lacks the hinge region of an IgG4 antibody. The absence of the hinge region results in a molecule that is essentially half the size of a traditional IgG4 antibody and has a monovalent binding region instead of the bivalent binding region of the IgG4 antibody.

[0084] "Variable" refers to the variable domain V. H and V L This refers to the fact that specific regions of the antibody have significantly different sequences across antibodies, and these are used for the binding and specificity of each particular antibody to its target antigen. However, variability is not uniformly distributed throughout the variable domain of the antibody. This is because the V region, which forms part of the antigen-binding site, is not uniformly distributed across the entire variable domain. L Domain and V H Each domain is concentrated in three segments called "hypervariable loops." The first, second, and third hypervariable loops of the Vλ light chain domain are referred to herein as L1(λ), ​​L2(λ), and L3(λ), respectively. L The domain can be defined as containing residues 24-33 (L1(λ), ​​consisting of 9, 10, or 11 amino acid residues), 49-53 (L2(λ), consisting of 3 residues), and 90-96 (L3(λ), consisting of 6 residues) (Morea et al., 2000. Methods. 20(3):267-79). The first, second, and third hypervariable loops of the Vκ light chain domain are referred to herein as L1(κ), L2(κ), and L3(κ), and V L It can be defined as containing residues 25-33 (L1(κ), consisting of 6, 7, 8, 11, 12, or 13 residues), 49-53 (L2(κ), consisting of 3 residues), and 90-97 (L3(κ), consisting of 6 residues) within the domain (Morea et al., cited above). V HThe first, second, and third hypervariable loops of the domain are referred to herein as H1, H2, and H3, and may be defined as containing residues 25-33 (H1, consisting of 7, 8, or 9 residues), 52-56 (H2, consisting of 3, or 4 residues), and 91-105 (H3, with a highly variable length) within the VH domain (Morea et al., cited above). Unless otherwise indicated, the terms L1, L2, and L3 refer to the first, second, and third hypervariable loops of the VL domain, respectively, and encompass the hypervariable loops obtained from both the Vκ and Vλ isotypes. The terms H1, H2, and H3 refer to the V H This refers to the first, second, and third hypervariable loops of the domain, and includes hypervariable loops obtained from any known heavy-chain isotype, including gamma (γ), mu (μ), alpha (α), delta (δ), or epsilon (ε). Each of the hypervariable loops L1, L2, L3, H1, H2, and H3 may include a portion of the “complementarity-determining region” or “CDR” as defined herein above.

[0085] "Versabodies" refers to antibody mimicry technology. These are small 3-5 kDa proteins with >15% cysteine ​​that form a scaffold with a high disulfide density, replacing the hydrophobic core of typical proteins. Replacing the numerous hydrophobic amino acids, including the hydrophobic core, with a small number of disulfides results in a smaller, more hydrophilic protein (less aggregation and nonspecific binding), more resistant to proteases and heat, and with a lower density of T cell epitopes, because the residues that contribute most to MHC presentation are hydrophobic. All four of these properties are well known to affect immunogenicity, and together they are expected to cause a significant decrease in immunogenicity.

[0086] Detailed explanation Numerous studies have analyzed the interaction between IL-2 and CD25 in mouse models. Blocking IL-2 binding to CD25 with anti-CD25 antibodies (e.g., PC61) in tumor-bearing mice has been shown to result in loss of both FoxP3 expression and Treg suppression function. These results suggest that blocking IL-2 binding to CD25 is a promising method for preventing cancer development. Recently, Vargas et al. (2017, Immunity 48(6), 577-586) developed an Fc-optimized form of the PC61 antibody that enables intratumoral Treg depletion via antibody-dependent cytotoxicity (ADCC) or complement-mediated cytotoxicity (CDC) mechanisms, providing significant therapeutic benefits in mouse tumor models. Given the physiological importance of the CD25 and IL-2 pathways in Tregs, blocking these pathways was therefore considered a potent and promising antitumor immunotherapy.

[0087] Despite the clear potential of IL-2 pathway blockade as an antitumor immunotherapy, manipulation of the IL-2 pathway should be carefully considered because it modulates both immunostimulatory and immunomodulatory functions. Indeed, the IL-2 pathway plays a crucial role in regulating immune responses and maintaining peripheral self-tolerance, but it also acts as a T cell growth factor essential for T cell proliferation and survival, as well as the generation of effector and memory T cells. Furthermore, the IL-2 receptor is also transiently expressed in effector T cells and bone marrow dendritic cells; therefore, IL-2 pathway manipulation may lead to unpredictable consequences, such as alterations in the function of antitumor effector T cells, particularly CD8+ effector T cells, potentially resulting in cancer progression.

[0088] As a component of the immune system, effector CD8+ T cells play a crucial role in tumor suppression. For example, effector CD8+ T cells can kill tumor cells with cytotoxic molecules such as granzymes and perforins. IFN-γ produced by CD8+ T cells increases the expression of MHC class I antigens by tumor cells, thereby making them better targets for CD8+ T cells. Therefore, effector CD8+ T cells are important for eliminating neoplastic cells during cancer.

[0089] Herein, the applicants aimed to eliminate or silence Tregs while maintaining an efficient effector T cell response in cancer. Accordingly, the present invention relates to a novel anti-CD25 antibody (particularly an anti-human CD25 antibody) that exhibits a potent anti-cancer effect by depleting Tregs in particular without blocking the IL-2 signaling pathway, thereby enabling IL-2 to stimulate effector T cells.

[0090] Therefore, the present invention first relates to an isolated protein that binds to human CD25 (hCD25).

[0091] In one embodiment, the isolated protein according to the present invention is an isolated antibody or its antigen-binding fragment, which binds to human CD25 (hCD25).

[0092] As used herein, “isolated protein,” and in particular “isolated antibody,” is intended to mean a protein, and especially an antibody that is substantially free of other proteins or antibodies having different antigen specificity (for example, an isolated protein or antibody that specifically binds to hCD25 substantially does not contain proteins or antibodies that specifically bind to antigens other than hCD25). However, an isolated protein, and especially an isolated antibody that specifically binds to hCD25, may have cross-reactivity to other antigens, such as hCD25 molecules from other species. Furthermore, an isolated protein or antibody may not substantially contain other cellular material and / or chemicals, in particular, but not limited to enzymes, hormones, and other proteinaceous or nonproteinaceous components that would interfere with the therapeutic use of the protein or antibody.

[0093] In one embodiment, isolated proteins, particularly isolated antibodies or their antigen-binding fragments, are purified.

[0094] In one embodiment, the isolated protein or antibody (or its antigen-binding fragment) (1) As determined by the Lowry method, 80% by weight, 85% by weight, 90% by weight, 91% by weight, 92% by weight, 93% by weight, 94% by weight, 95% by weight, or more, most preferably 96% by weight, 97% by weight, 98% by weight, or 99% by weight of protein or antibody (or antigen-binding fragment thereof); (2) To a sufficient extent to obtain at least 15 residues of the N-terminal or internal amino acid sequence using a spinning cup sequencer; or (3) Uniformity shown by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or preferably silver staining. It is refined to this extent.

[0095] According to the present invention, isolated proteins, particularly isolated antibodies or their antigen-binding fragments, do not inhibit interleukin-2 (IL-2) signaling via CD25. In one embodiment, the isolated protein does not inhibit the binding of IL-2 to human CD25. In one embodiment, the isolated antibody or its antigen-binding fragment does not inhibit the binding of IL-2 to human CD25 and may therefore be referred to herein as a "non-blocking antibody."

[0096] In one embodiment, the antibody protein, antibody, or antigen-binding fragment according to the present invention inhibits IL-2 signaling by less than 50% compared to IL-2 signaling in the absence of the antibody protein, antibody, or antigen-binding fragment. In another embodiment, the antibody protein, antibody, or antigen-binding fragment according to the present invention inhibits IL-2 signaling by 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, and less than 5% compared to IL-2 signaling in the absence of the protein, antibody, or antigen-binding fragment.

[0097] Methods for measuring IL-2 signaling are well known in the art and include, for example, measuring the induction of IL-2 receptor signaling (e.g., by detection of phosphorylated STAT5a), measuring the induction of T cell proliferation (e.g., by detection of Ki-67, particularly using the CellTrace® cell proliferation kit, by direct evaluation of T cell proliferation in the presence of IL-2, in MLR experiments (e.g., including cell activation by CD3 and CD28 in the presence of IL-2), or using IL-2-dependent cell lines, e.g., the CTLL2 cell line), and / or measuring the upregulation of the expression of activation markers (e.g., cytotoxic molecules such as CD25 and CD69, e.g., granzyme B).

[0098] In one embodiment, the protein, antibody, or antigen-binding fragment of the present invention is CD4 + and CD8 + It does not inhibit the proliferation and / or activation of T cells. In one embodiment, the protein, antibody, or antigen-binding fragment of the antibody of the present invention is CD4 + and CD8+ It does not inhibit IL-2-induced proliferation of T cells. An example of a method that may be used to measure IL-2-induced proliferation is flow cytometry measurement and monitoring of cell division in T cells cultured in the presence of IL-2. An example of such a method is provided in the Examples section.

[0099] In one embodiment, the protein, antibody, or antigen-binding fragment of the present invention is CD4 using an isotype control antibody. + and CD8 + Compared to IL-2-induced proliferation of T cells, CD4 + and CD8 + It inhibits IL-2-induced proliferation of T cells by less than 30%, preferably by less than 25%, 20%, 15%, 10%, or less.

[0100] In one embodiment, the protein, antibody, or antigen-binding fragment of the antibody according to the present invention is CD4 + and CD8 + It does not inhibit the phosphorylation of STAT5a in T cells.

[0101] In one embodiment, the protein, antibody, or antigen-binding fragment of the antibody according to the present invention inhibits IL-2 binding to CD25 by less than 50% compared to IL-2 binding to CD25 in the absence of the protein, antibody, or antigen-binding fragment, respectively. In another embodiment, the antibody protein, antibody, or antigen-binding fragment of the present invention inhibits IL-2 binding to CD25 by 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, and less than 5% compared to IL-2 binding to CD25 in the absence of the protein, antibody, or antigen-binding fragment, respectively.

[0102] Examples of methods for measuring IL-2 binding to CD25 are well known to those skilled in the art, but include the detection of labeled IL-2 on CD25, for example, biotinylation or radiolabeling of IL-2 on CD25.

[0103] In one embodiment, the protein, antibody, or antigen-binding fragment of an antibody according to the present invention is specific to human CD25 (hCD25).

[0104] The protein, antibody, or its antigen-binding fragment is preferably about 10 times the antigen (e.g., CD25) at a detectable level. 6 M -1 Preferably about 10 7 M -1 , 10 8 M -1 , 5×10 8 M -1 , 10 9 M -1 , 5×10 9 M -1 The affinity constant (K A When a reaction occurs with ), it is said to be "specific," "immunospecific," or "specifically binding" to the antigen. The affinity of a protein, antibody, or its antigen-binding fragment to its homologous antigen is also generally expressed by the equilibrium dissociation constant (K). D ) is expressed as. The antibody or its antigen-binding fragment is detected at a detectable level with the antigen (e.g., CD25) and 10 -6 M or less, preferably 10 -7 M, 5.10 -8 M, 10 -8 M, 5.10 -9 M, 10 -9 K below M D When a reaction occurs, it is said that the antigen is "immunely specific," "specific," or "specifically bound."

[0105] The affinity of an antibody or its antigen-binding fragment can be readily determined using conventional techniques, such as those described in Scatchard, 1949. Ann NY Acad Sci. 51:660-672. The binding properties of an antibody or its antigen-binding fragment to an antigen, cell, or tissue can generally be determined and evaluated using immunofluorescence-based assays such as immunodetection methods including ELISA, immunohistochemistry (IHC), and / or fluorescence-activated cell sorting (FACS), or by surface plasmon resonance (SPR, e.g., using BIAcore®).

[0106] In one embodiment, the protein according to the present invention (particularly an antibody or its antigen-binding fragment) is approximately 30.10-9 M or less, preferably about 20.10 -9 M or less, preferably about 10.10 -9 M or less, preferably about 5.10 -9 M or less, preferably about 1.10 -9 K for binding to human CD25 of M or less D This shows the K of the present invention for binding to human CD25. D It is approximately 1.10 -10 M~20.10 -9 M, preferably about 6.10 -10 M ~ approx. 10.10 -9 It is within the range of M.

[0107] In one embodiment, the protein, antibody, or antigen-binding fragment thereof according to the present invention is polyclonal.

[0108] In another embodiment, the protein, antibody, or antigen-binding fragment thereof according to the present invention is monoclonal.

[0109] In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention is a molecule selected from the group comprising or consisting of whole antibodies, humanized antibodies, single-chain antibodies, dimeric single-chain antibodies, Fv, Fab, Fab', Fab'-SH, F(ab)'2, Fd, defucosylated antibodies, bispecific antibodies, diabodies, triabodies, and tetrabodies.

[0110] In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention is a molecule selected from the group comprising or consisting of whole antibodies, single-chain variable fragments (scFv), Fv, Fab, Fab', Fab'-SH, F(ab)'2, defucosylated antibodies, bispecific antibodies, diabodies, triabodies, and tetrabodies.

[0111] Antigen-binding fragments of antibodies can be obtained using standard methods. For example, Fab or F(ab')2 fragments can be produced by protease digestion of isolated antibodies according to conventional techniques.

[0112] It will also be understood that the proteins, antibodies, or antigen-binding fragments according to the present invention can be modified using known methods. For example, proteins, antibodies, or antigen-binding fragments can be modified with polyethylene glycol (PEG) to delay in vivo clearance and obtain a more desirable pharmacokinetic profile. Methods for coupling PEG to antibodies or antigen-binding fragments and conjugating them site-specifically are, for example, Leong et al., 2001. Cytokine. 16(3):106-19; Delgado et al., 1996. Br J Cancer. 73(2):175-82.

[0113] In one embodiment, the antibody or antigen-binding fragment according to the present invention is a molecule comprising a single antibody, a domain antibody, and a nanobody, or selected from the group consisting of these. In one embodiment, the antibody or antigen-binding fragment according to the present invention is a single antibody.

[0114] In one embodiment, the isolated protein according to the present invention is an antibody mimetic selected from the group comprising or consisting of afibody, alphabody, armadillo repeat protein-based backbone, Nottin, domain Kunitz peptide, affilin, afitin, adnectin, atrimer, evasin, DARPin, antikalin, avimer, finomer, versabody, or duokalin.

[0115] In one embodiment, an antibody, its antigen-binding fragment, or antibody mimetic binds to an epitope of CD25, preferably human CD25. In one embodiment, the epitope is a conformational epitope, for example, an epitope containing two or three amino acid sequences in CD25 (preferably human CD25). In one embodiment, the epitope does not contain amino acids involved in the binding of IL-2 by CD25.

[0116] In the following, unless otherwise specified, the numbering and definitions of CDRs will follow the IMGT® numbering system.

[0117] In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention has at least one, preferably at least two, more preferably three, complementarity-determining regions (CDRs): CDR1:NHAMA(Sequence ID 1); CDR2:VISYDGX1NX2YYX3DSVKG(Sequence ID 2)(X1 is S or D, X2 is K or T, X3 is A or R) and / or CDR3:GX4NSGYD (Sequence ID 3) (X4 is either W or L) It includes a heavy chain variable region (abbreviated as VH in this specification).

[0118] In one embodiment, the antibody or antigen-binding fragment according to the present invention comprises the following three CDRs: CDR1:NHAMA(Sequence ID 1); CDR2:VISYDGX1NX2YYX3DSVKG(Sequence ID 2)(X1 is S or D, X2 is K or T, X3 is A or R); and CDR3:GX4NSGYD (Sequence ID 3) (X4 is either W or L) Includes VH.

[0119] In one embodiment, the antibody or antigen-binding fragment according to the present invention is the following CDR, preferably the following three CDRs: CDR1:NHAMA(Sequence ID 1); CDR2:VISYDGSNKYYADSVKG(Sequence ID 7); and CDR3:GWNSGYD (Sequence ID 8) Includes a VH that includes at least one of the following (e.g., 1, 2, or 3).

[0120] In one embodiment, the antibody or antigen-binding fragment according to the present invention is the following CDR, preferably the following three CDRs: CDR1:NHAMA(Sequence ID 1); CDR2:VISYDGSNKYYADSVKG(Sequence ID 7); and CDR3:GLNSGYD (Sequence ID 17) Includes a VH that includes at least one of the following (e.g., 1, 2, or 3).

[0121] In one embodiment, the antibody or antigen-binding fragment according to the present invention is the following CDR, preferably the following three CDRs: CDR1:NHAMA(Sequence ID 1); CDR2:VISYDGSNKYYRDSVKG(Sequence ID 25); and CDR3:GLNSGYD (Sequence ID 17) Includes a VH that includes at least one of the following (e.g., 1, 2, or 3).

[0122] In one embodiment, the antibody or antigen-binding fragment according to the present invention is the following CDR, preferably the following three CDRs: CDR1:NHAMA(Sequence ID 1); CDR2:VISYDGSNTYYADSVKG(Sequence ID 26); and CDR3:GLNSGYD (Sequence ID 17) Includes a VH that includes at least one of the following (e.g., 1, 2, or 3).

[0123] In one embodiment, the antibody or antigen-binding fragment according to the present invention is the following CDR, preferably the following three CDRs: CDR1:NHAMA(Sequence ID 1); CDR2:VISYDGDNKYYADSVKG(Sequence ID 27); and CDR3:GLNSGYD (Sequence ID 17) Includes a VH that includes at least one of the following (e.g., 1, 2, or 3).

[0124] In one embodiment, any of the CDR1, CDR2, and / or CDR3 of the VH of SEQ ID NOs. 1-3, 7, 8, 17, 25-27 can be characterized as having 1, 2, 3 or more amino acids that are substituted with different amino acids.

[0125] In one embodiment, any one of the CDR1, CDR2, and / or CDR3 of the VH of SEQ ID NOs: 1 to 3, 7, 8, 17, 25 to 27 can be characterized as having an amino acid sequence that shares at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with a specific CDR or set of CDRs listed in the corresponding SEQ ID NO.

[0126] In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention comprises at least one, preferably at least two, more preferably three of the following complementarity-determining regions (CDRs): CDR1: RASQX5X6X7X8X9LN (SEQ ID NO: 4) (X5 is S or N, X6 is V or I, X7 is N or S, X8 is S or K, X9 is F or Y); CDR2: GTX 10 SLQS (SEQ ID NO: 5) (X 10 is S or N); and / or CDR3: QQYX 11 SWPWT (SEQ ID NO: 6) (X 11 is T or N) and includes a variable light chain region (abbreviated herein as VL).

[0127] In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention comprises the following three CDRs: CDR1: RASQX5X6X7X8X9LN (SEQ ID NO: 4) (X5 is S or N, X6 is V or I, X7 is N or S, X8 is S or K, X9 is F or Y); CDR2: GTX 10 SLQS (SEQ ID NO: 5) (X 10 is S or N); and CDR3: QQYX 11 SWPWT (SEQ ID NO: 6) (X 11 is T or N) and includes VL.

[0128] In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention comprises the following CDRs, preferably the following three CDRs: CDR1:RASQSVNSFLN(Sequence ID 9); CDR2:GTSSLQS(sequence number 10); and CDR3:QQYTSWPWT(Sequence ID 11) Includes a VL that includes at least one of the following (e.g., 1, 2, or 3).

[0129] In one embodiment, the antibody or antigen-binding fragment according to the present invention is the following CDR, preferably the following three CDRs: CDR1:RASQSVNSYLN(Sequence ID 12); CDR2:GTNSLQS(sequence number 13); and CDR3:QQYNSWPWT(Sequence ID 14) Includes a VL that includes at least one of the following (e.g., 1, 2, or 3).

[0130] In one embodiment, the antibody or antigen-binding fragment according to the present invention is the following CDR, preferably the following three CDRs: CDR1:RASQSISSYLN(Sequence ID 15); CDR2:GTNSLQS(sequence number 13); and CDR3:QQYNSWPWT(Sequence ID 14) Includes a VL that includes at least one of the following (e.g., 1, 2, or 3).

[0131] In one embodiment, the antibody or antigen-binding fragment according to the present invention is the following CDR, preferably the following three CDRs: CDR1:RASQSVSSFLN(Sequence ID 16); CDR2:GTNSLQS(sequence number 13); and CDR3:QQYTSWPWT(Sequence ID 11) Includes a VL that includes at least one of the following (e.g., 1, 2, or 3).

[0132] In one embodiment, the antibody or antigen-binding fragment according to the present invention is the following CDR, preferably the following three CDRs: CDR1:RASQSVSKFLN(Sequence ID 18); CDR2:GTNSLQS(sequence number 13); and CDR3:QQYNSWPWT(Sequence ID 14) Includes a VL that includes at least one of the following (e.g., 1, 2, or 3).

[0133] In one embodiment, the antibody or antigen-binding fragment according to the present invention is the following CDR, preferably the following three CDRs: CDR1:RASQNINSFLN(Sequence ID 19); CDR2:GTSSLQS(sequence number 10); and CDR3:QQYNSWPWT(Sequence ID 14) Includes a VL that includes at least one of the following (e.g., 1, 2, or 3).

[0134] In one embodiment, the antibody or antigen-binding fragment according to the present invention is the following CDR, preferably the following three CDRs: CDR1:RASQNISSFLN(Sequence ID 20); CDR2:GTNSLQS(sequence number 13); and CDR3:QQYNSWPWT(Sequence ID 14) Includes a VL that includes at least one of the following (e.g., 1, 2, or 3).

[0135] In one embodiment, the antibody or antigen-binding fragment according to the present invention is the following CDR, preferably the following three CDRs: CDR1:RASQSISSFLN(Sequence ID 21); CDR2:GTNSLQS(sequence number 13); and CDR3:QQYTSWPWT(Sequence ID 11) Includes a VL that includes at least one of the following (e.g., 1, 2, or 3).

[0136] In one embodiment, the antibody or antigen-binding fragment according to the present invention is the following CDR, preferably the following three CDRs: CDR1:RASQSVSSFLN(Sequence ID 16); CDR2:GTNSLQS(sequence number 13); and CDR3:QQYNSWPWT(Sequence ID 14) Includes a VL that includes at least one of the following (e.g., 1, 2, or 3).

[0137] In one embodiment, the antibody or antigen-binding fragment according to the present invention is the following CDR, preferably the following three CDRs: CDR1:RASQNVSSFLN(Sequence ID 22); CDR2:GTSSLQS(sequence number 10); and CDR3:QQYTSWPWT(Sequence ID 11) Includes a VL that includes at least one of the following (e.g., 1, 2, or 3).

[0138] In one embodiment, the antibody or antigen-binding fragment according to the present invention is the following CDR, preferably the following three CDRs: CDR1:RASQSINSFLN(Sequence ID 23); CDR2:GTNSLQS(sequence number 13); and CDR3:QQYNSWPWT(Sequence ID 14) Includes a VL that includes at least one of the following (e.g., 1, 2, or 3).

[0139] In one embodiment, the antibody or antigen-binding fragment according to the present invention is the following CDR, preferably the following three CDRs: CDR1:RASQNINSFLN(Sequence ID 19); CDR2:GTNSLQS(sequence number 13); and CDR3:QQYTSWPWT(Sequence ID 11) Includes a VL that includes at least one of the following (e.g., 1, 2, or 3).

[0140] In one embodiment, the antibody or antigen-binding fragment according to the present invention is the following CDR, preferably the following three CDRs: CDR1:RASQSVSSYLN(Sequence ID 24); CDR2: GTNSLQS (SEQ ID NO: 13); and CDR3: QQYTSWPWT (SEQ ID NO: 11) comprises a VL comprising at least one (e.g., 1, 2, or 3) of them.

[0141] In one embodiment, the antibody or antigen-binding fragment thereof according to the invention comprises a VL comprising at least one of the following CDRs, preferably the following three CDRs: CDR1: RASQSISSFLN (SEQ ID NO: 21); CDR2: GTNSLQS (SEQ ID NO: 13); and CDR3: QQYNSWPWT (SEQ ID NO: 14) comprises a VL comprising at least one (e.g., 1, 2, or 3) of them.

[0142] In one embodiment, the antibody or antigen-binding fragment thereof according to the invention comprises a VL comprising at least one of the following CDRs, preferably the following three CDRs: CDR1: RASQNVSSFLN (SEQ ID NO: 22); CDR2: GTNSLQS (SEQ ID NO: 13); and CDR3: QQYNSWPWT (SEQ ID NO: 14) comprises a VL comprising at least one (e.g., 1, 2, or 3) of them.

[0143] In one embodiment, the antibody or antigen-binding fragment thereof according to the invention comprises a VL comprising at least one of the following CDRs, preferably the following three CDRs: CDR1: RASQSINSFLN (SEQ ID NO: 23); CDR2: GTNSLQS (SEQ ID NO: 13); and CDR3: QQYTSWPWT (SEQ ID NO: 11) comprises a VL comprising at least one (e.g., 1, 2, or 3) of them.

[0144] In one embodiment, any one of CDR1, CDR2, and / or CDR3 of the VL having SEQ ID NOs: 4-6, 9-16, 18-24 may be characterized in that 1, 2, 3, 4, 5 or more amino acids are substituted with different amino acids.

[0145] In one embodiment, any of the VLs CDR1, CDR2, and / or CDR3 of SEQ ID NOs. 4-6, 9-16, and 18-24 can be characterized as having an amino acid sequence that shares at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more identity with a specific CDR or set of CDRs listed in the corresponding SEQ ID NO.

[0146] In one embodiment, the antibody or antigen-binding fragment of the present invention is - At least one, preferably at least two, more preferably three of the following CDRs: CDR1:NHAMA(Sequence ID 1); CDR2:VISYDGX1NX2YYX3DSVKG(Sequence ID 2)(X1 is S or D, X2 is K or T, X3 is A or R); and / or CDR3:GX4NSGYD(Sequence ID 3)(X4 is either W or L); VH including, - At least one, preferably at least two, more preferably three of the following CDRs: CDR1:RASQX5X6X7X8X9LN(Sequence ID 4)(X5 is S or N, X6 is V or I, X7 is N or S, X8 is S or K, and X9 is F or Y); CDR2:GTX 10 SLQS(Sequence ID 5)(X 10 (is S or N) and / or CDR3:QQYX 11 SWPWT(Sequence No. 6)(X 11 (is T or N) VL including, Includes.

[0147] In one embodiment, the antibody or antigen-binding fragment of the present invention is The following three CD-Rs: CDR1:NHAMA(Sequence ID 1); CDR2: VISYDGX1NX2YYX3DSVKG (SEQ ID NO: 2) (where X1 is S or D, X2 is K or T, and X3 is A or R); and CDR3: GX4NSGYD (SEQ ID NO: 3) (where X4 is W or L); comprising a VH and the following three CDRs: CDR1: RASQX5X6X7X8X9LN (SEQ ID NO: 4) (where X5 is S or N, X6 is V or I, X7 is N or S, X8 is S or K, and X9 is F or Y); CDR2: GTX 10 SLQS (SEQ ID NO: 5) (where X 10 is S or N); and CDR3: QQYX 11 SWPWT (SEQ ID NO: 6) (where X 11 is T or N) comprising a VL and is included.

[0148] In one embodiment, any of CDR1, CDR2, and / or CDR3 of VH of SEQ ID NOs: 1 - 3 and / or VL of SEQ ID NOs: 4 - 6 can be characterized as having an amino acid sequence that shares at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with the specific CDR or set of CDRs listed in the corresponding SEQ ID NO.

[0149] In one embodiment, the antibody or antigen - binding fragment thereof of the present invention has the following three CDRs: CDR1: NHAMA (SEQ ID NO: 1); CDR2: VISYDGSNKYYADSVKG (SEQ ID NO: 7); and CDR3: GWNSGYD (SEQ ID NO: 8) comprising a VH and the following three CDRs: CDR1: RASQSVNSFLN (SEQ ID NO: 9); CDR2: GTSSLQS (SEQ ID NO: 10); and CDR3: QQYTSWPWT (SEQ ID NO: 11) VL including, Includes.

[0150] In one embodiment, any of the VH sequences of SEQ ID NOs. 1, 7-8 and / or the VL sequences of SEQ ID NOs. 9-11 can be characterized as having an amino acid sequence that shares at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with a specific CDR or set of CDRs listed in the corresponding SEQ ID NO.

[0151] An example of an antibody containing a heavy chain with CDR1, CDR2, and CDR3 from SEQ ID NOs. 1, 7-8, and a light chain with CDR1, CDR2, and CDR3 from SEQ ID NOs. 9-11 is H07.

[0152] In one embodiment, the antibody or antigen-binding fragment of the present invention is The following three CD-Rs: CDR1:NHAMA(Sequence ID 1); CDR2:VISYDGSNKYYADSVKG(Sequence ID 7); and CDR3:GWNSGYD (Sequence ID 8) VH including, The following three CD-Rs: CDR1:RASQSVNSYLN(Sequence ID 12); CDR2:GTNSLQS(sequence number 13); and CDR3:QQYNSWPWT(Sequence ID 14) VL including, Includes.

[0153] In one embodiment, any of the VH sequences of SEQ ID NOs. 1, 7-8 and / or the VL sequences of SEQ ID NOs. 12-14 can be characterized as having an amino acid sequence that shares at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with a specific CDR or set of CDRs listed in the corresponding SEQ ID NO.

[0154] An example of an antibody containing a heavy chain with CDR1, CDR2, and CDR3 having SEQ ID NOs. 1, 7-8, and a light chain with CDR1, CDR2, and CDR3 having SEQ ID NOs. 12-14 is G02.

[0155] In one embodiment, the antibody or antigen-binding fragment of the present invention is The following three CD-Rs: CDR1:NHAMA(Sequence ID 1); CDR2:VISYDGSNKYYADSVKG(Sequence ID 7); and CDR3:GWNSGYD (Sequence ID 8) VH including, The following three CD-Rs: CDR1:RASQSISSYLN(Sequence ID 15); CDR2:GTNSLQS(sequence number 13); and CDR3:QQYNSWPWT(Sequence ID 14) VL including, Includes.

[0156] In one embodiment, any of the VH sequences of SEQ ID NOs. 1, 7-8 and / or the VL sequences of SEQ ID NOs. 13-15 can be characterized as having an amino acid sequence that shares at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more identity with a specific CDR or set of CDRs listed in the corresponding SEQ ID NO.

[0157] An example of an antibody containing a heavy chain with CDR1, CDR2, and CDR3 having SEQ ID NOs. 1, 7-8, and a light chain with CDR1, CDR2, and CDR3 having SEQ ID NOs. 13-15 is E04.

[0158] In one embodiment, the antibody or antigen-binding fragment of the present invention is The following three CD-Rs: CDR1:NHAMA(Sequence ID 1); CDR2:VISYDGSNKYYADSVKG(Sequence ID 7); and CDR3:GWNSGYD (Sequence ID 8) VH including, The following three CD-Rs: CDR1:RASQSVSSFLN(Sequence ID 16); CDR2:GTNSLQS(sequence number 13); and CDR3:QQYTSWPWT(Sequence ID 11) VL including, Includes.

[0159] In one embodiment, any of the VH sequences of SEQ ID NOs. 1, 7-8 and / or the VL sequences of SEQ ID NOs. 11, 13 and 16 can be characterized as having an amino acid sequence that shares at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with a specific CDR or set of CDRs listed in the corresponding SEQ ID NO.

[0160] An example of an antibody containing a heavy chain with CDR1, CDR2, and CDR3 having SEQ ID NOs. 1, 7-8, and a light chain with CDR1, CDR2, and CDR3 having SEQ ID NOs. 11, 13, and 16 is D05.

[0161] In one embodiment, the antibody or antigen-binding fragment of the present invention is The following three CD-Rs: CDR1:NHAMA(Sequence ID 1); CDR2:VISYDGSNKYYADSVKG(Sequence ID 7); and CDR3:GLNSGYD (Sequence ID 17) VH including, The following three CD-Rs: CDR1:RASQSVSKFLN(Sequence ID 18); CDR2:GTNSLQS(sequence number 13); and CDR3:QQYNSWPWT(Sequence ID 14) VL including, Includes.

[0162] In one embodiment, any of the VH sequences of SEQ ID NOs. 1, 7, and 17 and / or the VL sequences of SEQ ID NOs. 13, 14, and 18 can be characterized as having an amino acid sequence that shares at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more identity with a specific CDR or set of CDRs listed in the corresponding SEQ ID NO.

[0163] An example of an antibody containing a heavy chain with CDR1, CDR2, and CDR3 of SEQ ID NOs. 1, 7, and 17, and a light chain with CDR1, CDR2, and CDR3 of SEQ ID NOs. 13, 14, and 18 is H09.

[0164] In one embodiment, the antibody or antigen-binding fragment of the present invention is The following three CD-Rs: CDR1:NHAMA(Sequence ID 1); CDR2:VISYDGSNKYYADSVKG(Sequence ID 7); and CDR3:GLNSGYD (Sequence ID 17) VH including, The following three CD-Rs: CDR1:RASQNINSFLN(Sequence ID 19); CDR2:GTSSLQS(sequence number 10); and CDR3:QQYNSWPWT(Sequence ID 14) VL including, Includes.

[0165] In one embodiment, any of the VH sequences of SEQ ID NOs. 1, 7, and 17 and / or the VL sequences of SEQ ID NOs. 10, 14, and 19, CDR1, CDR2, and / or CDR3, can be characterized as having an amino acid sequence that shares at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more identity with a specific CDR or set of CDRs listed in the corresponding SEQ ID NO.

[0166] An example of an antibody containing a heavy chain with CDR1, CDR2, and CDR3 having SEQ ID NOs: 1, 7, and 17, and a light chain with CDR1, CDR2, and CDR3 having SEQ ID NOs: 10, 14, and 19, is E04-2.

[0167] In one embodiment, the antibody or antigen-binding fragment of the present invention is The following three CD-Rs: CDR1:NHAMA(Sequence ID 1); CDR2:VISYDGSNKYYADSVKG(Sequence ID 7); and CDR3:GLNSGYD (Sequence ID 17) VH including, The following three CD-Rs: CDR1:RASQNISSFLN(Sequence ID 20); CDR2:GTNSLQS(sequence number 13); and CDR3:QQYNSWPWT(Sequence ID 14) VL including, Includes.

[0168] In one embodiment, any of the VH sequences of SEQ ID NOs. 1, 7, and 17 and / or the VL sequences of SEQ ID NOs. 13, 14, and 20 can be characterized as having an amino acid sequence that shares at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more identity with a specific CDR or set of CDRs listed in the corresponding SEQ ID NO.

[0169] An example of an antibody containing a heavy chain with CDR1, CDR2, and CDR3 of SEQ ID NOs. 1, 7, and 17, and a light chain with CDR1, CDR2, and CDR3 of SEQ ID NOs. 13, 14, and 20 is B01.

[0170] In one embodiment, the antibody or antigen-binding fragment of the present invention is The following three CD-Rs: CDR1:NHAMA(Sequence ID 1); CDR2:VISYDGSNKYYADSVKG(Sequence ID 7); and CDR3:GLNSGYD (Sequence ID 17) VH including, The following three CD-Rs: CDR1:RASQSISSFLN(Sequence ID 21); CDR2:GTNSLQS(sequence number 13); and CDR3:QQYTSWPWT(Sequence ID 11) VL including, Includes.

[0171] In one embodiment, any of the VH sequences of SEQ ID NOs. 1, 7, and 17 and / or the VL sequences of SEQ ID NOs. 11, 13, and 21 can be characterized as having an amino acid sequence that shares at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more identity with a specific CDR or set of CDRs listed in the corresponding SEQ ID NO.

[0172] An example of an antibody containing a heavy chain with CDR1, CDR2, and CDR3 having SEQ ID NOs. 1, 7, and 17, and a light chain with CDR1, CDR2, and CDR3 having SEQ ID NOs. 11, 13, and 21 is C01.

[0173] In one embodiment, the antibody or antigen-binding fragment of the present invention is The following three CD-Rs: CDR1:NHAMA(Sequence ID 1); CDR2:VISYDGSNKYYADSVKG(Sequence ID 7); and CDR3:GLNSGYD (Sequence ID 17) VH including, The following three CD-Rs: CDR1:RASQSVSSFLN(Sequence ID 16); CDR2:GTNSLQS(sequence number 13); and CDR3:QQYNSWPWT(Sequence ID 14) VL including, Includes.

[0174] In one embodiment, any of the VH sequences of SEQ ID NOs. 1, 7, and 17 and / or the VL sequences of SEQ ID NOs. 13, 14, and 16 can be characterized as having an amino acid sequence that shares at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more identity with a specific CDR or set of CDRs listed in the corresponding SEQ ID NO.

[0175] An example of an antibody containing a heavy chain with CDR1, CDR2, and CDR3 of SEQ ID NOs. 1, 7, and 17, and a light chain with CDR1, CDR2, and CDR3 of SEQ ID NOs. 13, 14, and 16 is G01.

[0176] In one embodiment, the antibody or antigen-binding fragment of the present invention is The following three CD-Rs: CDR1:NHAMA(Sequence ID 1); CDR2:VISYDGSNKYYADSVKG(Sequence ID 7); and CDR3:GLNSGYD (Sequence ID 17) VH including, The following three CD-Rs: CDR1:RASQNVSSFLN(Sequence ID 22); CDR2:GTSSLQS(sequence number 10); and CDR3:QQYTSWPWT(Sequence ID 11) VL including, Includes.

[0177] In one embodiment, any of the VH sequences of SEQ ID NOs. 1, 7, and 17 and / or the VL sequences of SEQ ID NOs. 10, 11, and 22 can be characterized as having an amino acid sequence that shares at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more identity with a specific CDR or set of CDRs listed in the corresponding SEQ ID NO.

[0178] An example of an antibody containing a heavy chain with CDR1, CDR2, and CDR3 of SEQ ID NOs. 1, 7, and 17, and a light chain with CDR1, CDR2, and CDR3 of SEQ ID NOs. 10, 11, and 22 is H01.

[0179] In one embodiment, the antibody or antigen-binding fragment of the present invention is The following three CD-Rs: CDR1:NHAMA(Sequence ID 1); CDR2:VISYDGSNKYYADSVKG(Sequence ID 7); and CDR3:GLNSGYD (Sequence ID 17) VH including, The following three CD-Rs: CDR1:RASQSINSFLN(Sequence ID 23); CDR2:GTNSLQS(sequence number 13); and CDR3:QQYNSWPWT(Sequence ID 14) VL including, Includes.

[0180] In one embodiment, any of the VH sequences of SEQ ID NOs. 1, 7, and 17 and / or the VL sequences of SEQ ID NOs. 13, 14, and 23 can be characterized as having an amino acid sequence that shares at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more identity with a specific CDR or set of CDRs listed in the corresponding SEQ ID NO.

[0181] An example of an antibody containing a heavy chain with CDR1, CDR2, and CDR3 of SEQ ID NOs. 1, 7, and 17, and a light chain with CDR1, CDR2, and CDR3 of SEQ ID NOs. 13, 14, and 23 is G02-2.

[0182] In one embodiment, the antibody or antigen-binding fragment of the present invention is The following three CD-Rs: CDR1:NHAMA(Sequence ID 1); CDR2:VISYDGSNKYYADSVKG(Sequence ID 7); and CDR3:GLNSGYD (Sequence ID 17) VH including, The following three CD-Rs: CDR1:RASQNINSFLN(Sequence ID 19); CDR2:GTNSLQS(sequence number 13); and CDR3:QQYTSWPWT(Sequence ID 11) VL including, Includes.

[0183] In one embodiment, any of the VH sequences of SEQ ID NOs. 1, 7, and 17 and / or the VL sequences of SEQ ID NOs. 19, 13, and 11 can be characterized as having an amino acid sequence that shares at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more identity with a specific CDR or set of CDRs listed in the corresponding SEQ ID NO.

[0184] An example of an antibody containing a heavy chain with CDR1, CDR2, and CDR3 of SEQ ID NOs. 1, 7, and 17, and a light chain with CDR1, CDR2, and CDR3 of SEQ ID NOs. 19, 13, and 11 is B07.

[0185] In one embodiment, the antibody or antigen-binding fragment of the present invention is The following three CD-Rs: CDR1:NHAMA(Sequence ID 1); CDR2:VISYDGSNKYYADSVKG(Sequence ID 7); and CDR3:GLNSGYD (Sequence ID 17) VH including, The following three CD-Rs: CDR1:RASQSVSSYLN(Sequence ID 24); CDR2:GTNSLQS(sequence number 13); and CDR3:QQYTSWPWT(Sequence ID 11) VL including, Includes.

[0186] In one embodiment, any of the VH sequences of SEQ ID NOs. 1, 7, and 17 and / or the VL sequences of SEQ ID NOs. 11, 13, and 24 can be characterized as having an amino acid sequence that shares at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more identity with a specific CDR or set of CDRs listed in the corresponding SEQ ID NO.

[0187] An example of an antibody containing a heavy chain with CDR1, CDR2, and CDR3 of SEQ ID NOs. 1, 7, and 17, and a light chain with CDR1, CDR2, and CDR3 of SEQ ID NOs. 11, 13, and 24 is H08.

[0188] In one embodiment, the antibody or antigen-binding fragment of the present invention is The following three CD-Rs: CDR1:NHAMA(Sequence ID 1); CDR2:VISYDGSNKYYRDSVKG(Sequence ID 25); and CDR3:GLNSGYD (Sequence ID 17) VH including, The following three CD-Rs: CDR1:RASQSISSFLN(Sequence ID 21); CDR2:GTNSLQS(sequence number 13); and CDR3:QQYNSWPWT(Sequence ID 14) VL including, Includes.

[0189] In one embodiment, any of the VH sequences of SEQ ID NOs. 1, 17, and 25 and / or the VL sequences of SEQ ID NOs. 13, 14, and 21 can be characterized as having an amino acid sequence that shares at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more identity with a specific CDR or set of CDRs listed in the corresponding SEQ ID NO.

[0190] An example of an antibody containing a heavy chain with CDR1, CDR2, and CDR3 of SEQ ID NOs. 1, 17, and 25, and a light chain with CDR1, CDR2, and CDR3 of SEQ ID NOs. 13, 14, and 21 is D01.

[0191] In one embodiment, the antibody or antigen-binding fragment of the present invention is The following three CD-Rs: CDR1:NHAMA(Sequence ID 1); CDR2:VISYDGSNKYYRDSVKG(Sequence ID 25); and CDR3:GLNSGYD (Sequence ID 17) VH including, The following three CD-Rs: CDR1:RASQNINSFLN(Sequence ID 19); CDR2:GTSSLQS(sequence number 10); and CDR3:QQYNSWPWT(Sequence ID 14) VL including, Includes.

[0192] In one embodiment, any of the VH sequences of SEQ ID NOs. 1, 17, and 25 and / or the VL sequences of SEQ ID NOs. 10, 14, and 19 can be characterized as having an amino acid sequence that shares at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more identity with a specific CDR or set of CDRs listed in the corresponding SEQ ID NO.

[0193] An example of an antibody containing a heavy chain with CDR1, CDR2, and CDR3 of SEQ ID NOs. 1, 17, and 25, and a light chain with CDR1, CDR2, and CDR3 of SEQ ID NOs. 10, 14, and 19 is B05.

[0194] In one embodiment, the antibody or antigen-binding fragment of the present invention is The following three CD-Rs: CDR1:NHAMA(Sequence ID 1); CDR2:VISYDGSNKYYRDSVKG(Sequence ID 25); and CDR3:GLNSGYD (Sequence ID 17) VH including, The following three CD-Rs: CDR1:RASQNVSSFLN(Sequence ID 22); CDR2:GTNSLQS(sequence number 13); and CDR3:QQYNSWPWT(Sequence ID 14) VL including, Includes.

[0195] In one embodiment, any of the VH sequences of SEQ ID NOs. 1, 17, and 25 and / or the VL sequences of SEQ ID NOs. 13, 14, and 22 can be characterized as having an amino acid sequence that shares at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more identity with a specific CDR or set of CDRs listed in the corresponding SEQ ID NO.

[0196] Examples of antibodies containing heavy chains with CDR1, CDR2, and CDR3 having SEQ ID NOs. 1, 17, and 25, and light chains with CDR1, CDR2, and CDR3 having SEQ ID NOs. 13, 14, and 22 are G09 and H02.

[0197] In one embodiment, the antibody or antigen-binding fragment of the present invention is The following three CD-Rs: CDR1:NHAMA(Sequence ID 1); CDR2:VISYDGSNTYYADSVKG(Sequence ID 26); and CDR3:GLNSGYD (Sequence ID 17) VH including, The following three CD-Rs: CDR1:RASQNVSSFLN(Sequence ID 22); CDR2:GTNSLQS(sequence number 13); and CDR3:QQYNSWPWT(Sequence ID 14) VL including, Includes.

[0198] In one embodiment, any of the VH sequences of SEQ ID NOs. 1, 17, and 26 and / or the VL sequences of SEQ ID NOs. 13, 14, and 22 can be characterized as having an amino acid sequence that shares at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more identity with a specific CDR or set of CDRs listed in the corresponding SEQ ID NO.

[0199] An example of an antibody containing a heavy chain with CDR1, CDR2, and CDR3 of SEQ ID NOs. 1, 17, and 26, and a light chain with CDR1, CDR2, and CDR3 of SEQ ID NOs. 13, 14, and 22 is F03.

[0200] In one embodiment, the antibody or antigen-binding fragment of the present invention is The following three CD-Rs: CDR1:NHAMA(Sequence ID 1); CDR2:VISYDGDNKYYADSVKG(Sequence ID 27); and CDR3:GLNSGYD (Sequence ID 17) VH including, The following three CD-Rs: CDR1:RASQSINSFLN(Sequence ID 23); CDR2:GTNSLQS(sequence number 13); and CDR3:QQYTSWPWT(Sequence ID 11) VL including, Includes.

[0201] In one embodiment, any of the VH sequences of SEQ ID NOs. 1, 17, and 27 and / or the VL sequences of SEQ ID NOs. 11, 13, and 23 can be characterized as having an amino acid sequence that shares at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more identity with a specific CDR or set of CDRs listed in the corresponding SEQ ID NO.

[0202] An example of an antibody containing a heavy chain with CDR1, CDR2, and CDR3 of SEQ ID NOs. 1, 17, and 27, and a light chain with CDR1, CDR2, and CDR3 of SEQ ID NOs. 11, 13, and 23 is B12.

[0203] In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention comprises or contains VH derived from SEQ ID NO 94, where X1 is S or D, X2 is K or T, X3 is A or R, X4 is A or S, X5 is K or Q, X6 is N or S, and X7 is W or L. Sequence ID 94 QVQLVESGGGVVQPGRSLRLSCAASGFTFSNHAMAWVRQAPGKGLEWVAVISYDGX1NX2YYX3DSVKGRFTISRDNX4X5X6TLYLQMNSLRAEDTAVYYCTTGX7NSGYDWGQGTLVTVSS

[0204] In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention comprises or contains VH derived from SEQ ID NO: 96, where X1 is A or S and X2 is N or S. Sequence ID 96 QVQLVESGGGVVQPGRSLRLSCAASGFTFSNHAMAWVRQAPGKGLEWVAVISYDGSNKYYRDSVKGRFTISRDNX1QX2TLYLQMNSLRAEDTAVYYCTTGLNSGYDWGQGTLVTVSS

[0205] In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention comprises a sequence selected from the group including SEQ ID NOs: 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, and 39, or comprises a VH consisting of such a sequence. Sequence ID 28 QVQLVESGGGVVQPGRSLRLSCAASGFTFSNHAMAWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCTTGWNSGYDWGQGTLVTVSS Sequence ID 29 QVQLVESGGGVVQPGRSLRLSCAASGFTFSNHAMAWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSQNTLYLQMNSLRAEDTAVYYCTTGGLNSGYDWGQGTLVTVSS Sequence ID 30 QVQLVESGGGVVQPGRSLRLSCAASGFTFSNHAMAWVRQAPGKGLEWVAVISYDGSNKYYRDSVKGRFTISRDNAQSTLYLQMNSLRAEDTAVYYCTTGNLNSGYDWGQGTLVTVSS Sequence ID 31 QVQLVESGGGVVQPGRSLRLSCAASGFTFSNHAMAWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCTTGNLNSGYDWGQGTLVTVSS Sequence ID 32 QVQLVESGGGVVQPGRSLRLSCAASGFTFSNHAMAWVRQAPGKGLEWVAVISYDGSNKYYRDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTTGNLNSGYDWGQGTLVTVSS Sequence ID 33 QVQLVESGGGVVQPGRSLRLSCAASGFTFSNHAMAWVRQAPGKGLEWVAVISYDGSNKYYRDSVKGRFTISRDNAQNTLYLQMNSLRAEDTAVYYCTTGNLNSGYDWGQGTLVTVSS Sequence ID 34 QVQLVESGGGVVQPGRSLRLSCAASGFTFSNHAMAWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNAKSTLYLQMNSLRAEDTAVYYCTTGNLNSGYDWGQGTLVTVSS Sequence ID 35 QVQLVESGGGVVQPGRSLRLSCAASGFTFSNHAMAWVRQAPGKGLEWVAVISYDGSNKYYRDSVKGRFTISRDNSQSTLYLQMNSLRAEDTAVYYCTTGNLNSGYDWGQGTLVTVSS Sequence ID 36 QVQLVESGGGVVQPGRSLRLSCAASGFTFSNHAMAWVRQAPGKGLEWVAVISYDGSNTYYADSVKGRFTISRDNAKSTLYLQMNSLRAEDTAVYYCTTGNLNSGYDWGQGTLVTVSS Sequence ID 37 QVQLVESGGGVVQPGRSLRLSCAASGFTFSNHAMAWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTTGWNSGYDWGQGTLVTVSS Sequence ID 38 QVQLVESGGGVVQPGRSLRLSCAASGFTFSNHAMAWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTTGNLNSGYDWGQGTLVTVSS Sequence ID 39 QVQLVESGGGVVQPGRSLRLSCAASGFTFSNHAMAWVRQAPGKGLEWVAVISYDGDNKYYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCTTGNLNSGYDWGQGTLVTVSS

[0206] In one embodiment, VH includes or consists of the sequences of SEQ ID NOs: 28, SEQ ID NOs: 29, SEQ ID NOs: 30, SEQ ID NOs: 31, SEQ ID NOs: 32, SEQ ID NOs: 33, SEQ ID NOs: 34, SEQ ID NOs: 35, SEQ ID NOs: 36, SEQ ID NOs: 37, SEQ ID NOs: 38, or SEQ ID NOs: 39, which have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or more amino acids substituted with different amino acids. In one embodiment, VH includes or consists of the sequence of SEQ ID NO: 94 or SEQ ID NO: 96 having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or more amino acids substituted with different amino acids.

[0207] In one embodiment, VH has an amino acid sequence that shares at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more identity with SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, or SEQ ID NO: 39. In one embodiment, VH has an amino acid sequence that shares at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more identity with SEQ ID NO: 94 or SEQ ID NO: 96.

[0208] In one embodiment, the antibody or antigen-binding fragment according to the present invention comprises the sequence of SEQ ID NO: 95 or comprises VL consisting thereof, where X1 is S or N, X2 is V or I, X3 is N or S, X4 is S or K, X5 is F or Y, X6 is K or E, X7 is R or K, X8 is S or N, X9 is Y or F, X 10 It is either T or N. Sequence ID 95 DIQMTQSPSSLSASVGDRVTITCRASQX1X2X3X4X5LNWYQQKPGX6APX7RLIYGTX8SLQSGVPSRFSGSGSGTDX9TLTISSLQPEDFATYYCQQYX 10 SWPWTFGQGTKLEIK

[0209] In one embodiment, the antibody or antigen-binding fragment according to the present invention includes or comprises a VL consisting of a sequence selected from the group including SEQ ID NOs: 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, and 57. Sequence ID 40 DIQMTQSPSSLSASVGDRVTITCRASQSVNSFLNWYQQKPGKAPRRLIYGTSSLQSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQYTSWPWTFGQGTKLEIK Sequence ID 41 DIQMTQSPSSLSASVGDRVTITCRASQSVSKFLNWYQQKPGKAPRRLIYGTNSLQSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQYNSWPWTFGQGTKLEIK Sequence ID 42 DIQMTQSPSSLSASVGDRVTITCRASQSVNSYLNWYQQKPGKAPRRLIYGTNSLQSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQYNSWPWTFGQGTKLEIK Sequence ID 43 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPRRLIYGTNSLQSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQYNSWPWTFGQGTKLEIK Sequence ID 44 DIQMTQSPSSLSASVGDRVTITCRASQSISSFLNWYQQKPGKAPRRLIYGTNSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSWPWTFGQGTKLEIK Sequence ID 45 DIQMTQSPSSLSASVGDRVTITCRASQNINSFLNWYQQKPGKAPRRLIYGTSSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSWPWTFGQGTKLEIK Sequence ID 46 DIQMTQSPSSLSASVGDRVTITCRASQNINSFLNWYQQKPGKAPRRLIYGTSSLQSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQYNSWPWTFGQGTKLEIK Sequence ID 47 DIQMTQSPSSLSASVGDRVTITCRASQNVSSFLNWYQQKPGEAPRRLIYGTNSLQSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQYNSWPWTFGQGTKLEIK Sequence ID 48 DIQMTQSPSSLSASVGDRVTITCRASQNISSFLNWYQQKPGEAPRRLIYGTNSLQSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQYNSWPWTFGQGTKLEIK Sequence ID 49 DIQMTQSPSSLSASVGDRVTITCRASQSISSFLNWYQQKPGEAPRRLIYGTNSLQSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQYTSWPWTFGQGTKLEIK Sequence ID 50 DIQMTQSPSSLSASVGDRVTITCRASQSVSSFLNWYQQKPGKAPKRLIYGTNSLQSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQYNSWPWTFGQGTKLEIK Sequence ID 51 DIQMTQSPSSLSASVGDRVTITCRASQNVSSFLNWYQQKPGKAPRRLIYGTSSLQSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQYTSWPWTFGQGTKLEIK Sequence ID 52 DIQMTQSPSSLSASVGDRVTITCRASQSINSFLNWYQQKPGKAPRRLIYGTNSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSWPWTFGQGTKLEIK Sequence ID 53 DIQMTQSPSSLSASVGDRVTITCRASQNVSSFLNWYQQKPGKAPRRLIYGTNSLQSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQYNSWPWTFGQGTKLEIK Sequence ID 54 DIQMTQSPSSLSASVGDRVTITCRASQSVSSFLNWYQQKPGEAPRRLIYGTNSLQSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQYTSWPWTFGQGTKLEIK Sequence ID 55 DIQMTQSPSSLSASVGDRVTITCRASQNINSFLNWYQQKPGEAPRRLIYGTNSLQSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQYTSWPWTFGQGTKLEIK Sequence ID 56 DIQMTQSPSSLSASVGDRVTITCRASQSVSSYLNWYQQKPGKAPRRLIYGTNSLQSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQYTSWPWTFGQGTKLEIK Sequence ID 57 DIQMTQSPSSLSASVGDRVTITCRASQSINSFLNWYQQKPGEAPRRLIYGTNSLQSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQYTSWPWTFGQGTKLEIK

[0210] In one embodiment, VL includes or consists of the sequences of SEQ ID NOs: 40, SEQ ID NOs: 41, SEQ ID NOs: 42, SEQ ID NOs: 43, SEQ ID NOs: 44, SEQ ID NOs: 45, SEQ ID NOs: 46, SEQ ID NOs: 47, SEQ ID NOs: 48, SEQ ID NOs: 49, SEQ ID NOs: 50, SEQ ID NOs: 51, SEQ ID NOs: 52, SEQ ID NOs: 53, SEQ ID NOs: 54, SEQ ID NOs: 55, SEQ ID NOs: 56, or SEQ ID NOs: 57, which have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or more amino acids substituted with different amino acids. In one embodiment, VL includes or consists of sequences of Sequence ID No. 95 having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or more amino acids substituted with different amino acids.

[0211] In one embodiment, VL has an amino acid sequence that shares at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more identity with SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, or SEQ ID NO: 57. In one embodiment, VL has an amino acid sequence that shares at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more identity with SEQ ID NO: 95.

[0212] As used herein, the phrase "[...] characterized by the substitution of different amino acids with respect to a given sequence" refers to the occurrence of a conservative amino acid modification within that sequence.

[0213] As used herein, the term "conservative amino acid modification" refers to modifications that do not significantly affect or alter the binding properties of an antibody or its antigen-binding fragment, which include an amino acid sequence. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into an antibody or its antigen-binding fragment by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis.

[0214] Conservative amino acid substitutions are typically those in which an amino acid residue is replaced by an amino acid residue having a side chain with similar physicochemical properties. The specified variable region and CDR sequence may include insertions, deletions, and / or substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or more amino acids. Where substitutions are made, preferred substitutions are conservative modifications. Families of amino acid residues with similar side chains are defined in the art. These families include amino acids having basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), non-charged side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, one or more amino acid residues in the CDR and / or variable region of the antibody or its antigen-binding fragment according to the present invention can be replaced with other amino acid residues from the same side-chain family, and the modified antibody can be tested for retained function (i.e., the properties described herein, e.g., binding to hCD25) using the assay described herein. In another embodiment, the amino acid string in the CDR and / or variable region of the antibody or antigen-binding fragment according to the present invention can be replaced with a structurally similar string that differs in the order and / or composition of its side-chain family members.

[0215] In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention is - A sequence of sequence number 94 (where X1 is S or D, X2 is K or T, X3 is A or R, X4 is A or S, X5 is K or Q, X6 is N or S, and X7 is W or L); or sequence number 96 (where X1 is A or S, and X2 is N or S), and a VH comprising or consisting of a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with sequence number 94 or 96, - Sequence ID 95 (X1 is S or N, X2 is V or I, X3 is N or S, X4 is S or K, X5 is F or Y, X6 is K or E, X7 is R or K, X8 is S or N, X9 is Y or F, X 10 A VL comprising a sequence (where is T or N) and a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with sequence number 95, Includes.

[0216] In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention is - A VH comprising a sequence selected from the group comprising sequences having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with sequence numbers 28-39, or a VH consisting of such a sequence, - A VL comprising or consisting of sequences selected from the group comprising sequences having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with sequences 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57 and sequences 40-57, Includes.

[0217] In one embodiment, the antibody or its antigen-binding fragment is VH containing or consisting of the sequence of sequence number 28, A VL containing or consisting of the sequence of sequence number 40, Includes.

[0218] An example of such an antibody is H07.

[0219] In one embodiment, the antibody or its antigen-binding fragment is VH containing or consisting of the sequence of sequence number 29, A VL containing or consisting of the sequence of sequence number 41, Includes.

[0220] An example of such an antibody is H09.

[0221] In one embodiment, the antibody or its antigen-binding fragment is VH containing or consisting of the sequence of sequence number 28, A VL containing or consisting of the sequence of sequence number 42, Includes.

[0222] An example of such an antibody is G02.

[0223] In one embodiment, the antibody or its antigen-binding fragment is VH containing or consisting of the sequence of sequence number 28, A VL containing or consisting of the sequence of sequence number 43, Includes.

[0224] An example of such an antibody is E04.

[0225] In one embodiment, the antibody or its antigen-binding fragment is VH containing or consisting of the sequence of sequence number 30, A VL containing or consisting of the sequence of sequence number 44, Includes.

[0226] An example of such an antibody is D01.

[0227] In one embodiment, the antibody or its antigen-binding fragment is VH containing or consisting of the sequence of sequence number 31, A VL containing or consisting of the sequence of sequence number 45, Includes.

[0228] An example of such an antibody is E04-2.

[0229] In one embodiment, the antibody or its antigen-binding fragment is VH containing or consisting of the sequence of sequence number 32, A VL containing or consisting of the sequence of sequence number 46, Includes.

[0230] An example of such an antibody is B05.

[0231] In one embodiment, the antibody or its antigen-binding fragment is VH containing or consisting of the sequence of sequence number 33, A VL containing or consisting of the sequence of sequence number 47.

[0232] An example of such an antibody is G09.

[0233] In one embodiment, the antibody or its antigen-binding fragment is VH containing or consisting of the sequence of sequence number 34, A VL containing or consisting of the sequence of sequence number 48, Includes.

[0234] An example of such an antibody is B01.

[0235] In one embodiment, the antibody or its antigen-binding fragment is VH containing or consisting of the sequence of sequence number 29, A VL containing or consisting of the sequence of sequence number 49, Includes.

[0236] An example of such an antibody is C01.

[0237] In one embodiment, the antibody or its antigen-binding fragment is VH containing or consisting of the sequence of sequence number 29, A VL containing or consisting of the sequence of sequence number 50, Includes.

[0238] An example of such an antibody is G01.

[0239] In one embodiment, the antibody or its antigen-binding fragment is VH containing or consisting of the sequence of sequence number 29, A VL containing or consisting of the sequence of sequence number 51, Includes.

[0240] An example of such an antibody is H01.

[0241] In one embodiment, the antibody or its antigen-binding fragment is VH containing or consisting of the sequence of sequence number 31, A VL containing or consisting of the sequence of sequence number 52, Includes.

[0242] An example of such an antibody is G02-2.

[0243] In one embodiment, the antibody or its antigen-binding fragment is VH containing or consisting of the sequence of sequence number 35, A VL containing or consisting of the sequence of sequence number 47, Includes.

[0244] An example of such an antibody is H02.

[0245] In one embodiment, the antibody or its antigen-binding fragment is VH containing or consisting of the sequence of sequence number 36, A VL containing or consisting of the sequence of sequence number 53, Includes.

[0246] An example of such an antibody is F03.

[0247] In one embodiment, the antibody or its antigen-binding fragment is VH containing or consisting of the sequence of sequence number 37, A VL containing or consisting of the sequence of sequence number 54, Includes.

[0248] An example of such an antibody is D05.

[0249] In one embodiment, the antibody or its antigen-binding fragment is VH containing or consisting of the sequence of sequence number 29, A VL containing or consisting of the sequence of sequence number 55, Includes.

[0250] An example of such an antibody is B07.

[0251] In one embodiment, the antibody or its antigen-binding fragment is VH containing or consisting of the sequence of sequence number 38, A VL containing or consisting of the sequence of sequence number 56, Includes.

[0252] An example of such an antibody is H08.

[0253] In one embodiment, the antibody or its antigen-binding fragment is VH containing or consisting of the sequence of sequence number 39, A VL containing or consisting of the sequence of sequence number 57, Includes.

[0254] An example of such an antibody is B12.

[0255] In one embodiment, the antibody or its antigen-binding fragment is VH containing or consisting of the sequence of sequence number 96 (wherein X1 is A or S and X2 is N or S), A VL containing or consisting of the sequence of sequence number 47, Includes.

[0256] Examples of such antibodies are G09 and H02.

[0257] In one embodiment, VL and / or VH preferably further include a leader sequence located at the N-terminus of the VL amino acid sequence or at the N-terminus of the VH amino acid sequence. Examples of leader sequences include, but are not limited to, SEQ ID NOs. 58 and 59. Sequence ID 58 MDIRLSLAFLVLFIKGVQC Sequence ID 59 MAAVQLLGLLLLWLPAMRC

[0258] In one embodiment, VH includes an amino acid sequence reader sequence SEQ ID NO: 58 located at the N-terminus of a VH amino acid sequence (e.g., SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, or SEQ ID NO: 39). Other examples of VH amino acid sequences that may include the amino acid reader sequence SEQ ID NO: 58 include SEQ ID NO: 94 or SEQ ID NO: 96.

[0259] In one embodiment, the VL includes an amino acid leader sequence, SEQ ID NO: 59, located at the N-terminus of a VL amino acid sequence (e.g., SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, or SEQ ID NO: 57, etc.). Another example of a VL amino acid sequence that may include the amino acid leader sequence SEQ ID NO: 59 is SEQ ID NO: 95.

[0260] The present invention further relates to H07-like antibodies, i.e., antibodies that bind to the same epitope as H07 or substantially the same epitope as H07. Therefore, the present invention further relates to antibodies that compete with H07 for binding to CD25.

[0261] The present invention further relates to H09-like antibodies, i.e., antibodies that bind to the same epitope as H09 or substantially the same epitope as H09. Therefore, the present invention further relates to antibodies that compete with H09 for binding to CD25.

[0262] The present invention further relates to G02-like antibodies, i.e., antibodies that bind to the same epitope as G02 or substantially the same epitope as G02. Therefore, the present invention further relates to antibodies that compete with G02 for binding to CD25.

[0263] The present invention further relates to E04-like antibodies, i.e., antibodies that bind to the same epitope as E04 or substantially the same epitope as E04. Therefore, the present invention further relates to antibodies that compete with E04 for binding to CD25.

[0264] The present invention further relates to D01-like antibodies, i.e., antibodies that bind to the same epitope as D01 or substantially the same epitope as D01. Therefore, the present invention further relates to antibodies that compete with D01 for binding to CD25.

[0265] The present invention further relates to E04-2-like antibodies, i.e., antibodies that bind to the same epitope as E04-2 or substantially the same epitope as E04-2. Therefore, the present invention further relates to antibodies that compete with E04-2 for binding to CD25.

[0266] The present invention further relates to B05-like antibodies, i.e., antibodies that bind to the same epitope as B05 or substantially the same epitope as B05. Therefore, the present invention further relates to antibodies that compete with B05 for binding to CD25.

[0267] The present invention further relates to G09-like antibodies, i.e., antibodies that bind to the same epitope as G09 or substantially the same epitope as G09. Therefore, the present invention further relates to antibodies that compete with G09 for binding to CD25.

[0268] The present invention further relates to B01-like antibodies, i.e., antibodies that bind to the same epitope as B01 or substantially the same epitope as B01. Therefore, the present invention further relates to antibodies that compete with B01 for binding to CD25.

[0269] The present invention further relates to C01-like antibodies, i.e., antibodies that bind to the same epitope as C01 or substantially the same epitope as C01. Therefore, the present invention further relates to antibodies that compete with C01 for binding to CD25.

[0270] The present invention further relates to G01-like antibodies, i.e., antibodies that bind to the same epitope as G01 or substantially the same epitope as G01. Therefore, the present invention further relates to antibodies that compete with G01 for binding to CD25.

[0271] The present invention further relates to H01-like antibodies, i.e., antibodies that bind to the same epitope as H01 or substantially the same epitope as H01. Therefore, the present invention further relates to antibodies that compete with H01 for binding to CD25.

[0272] The present invention further relates to G02-2-like antibodies, i.e., antibodies that bind to the same epitope as G02-2 or substantially the same epitope as G02-2. Therefore, the present invention further relates to antibodies that compete with G02-2 for binding to CD25.

[0273] The present invention further relates to H02-like antibodies, i.e., antibodies that bind to the same epitope as H02 or substantially the same epitope as H02. Therefore, the present invention further relates to antibodies that compete with H02 for binding to CD25.

[0274] The present invention further relates to F03-like antibodies, i.e., antibodies that bind to the same epitope as F03 or substantially the same epitope as F03. Therefore, the present invention further relates to antibodies that compete with F03 for binding to CD25.

[0275] The present invention further relates to D05-like antibodies, i.e., antibodies that bind to the same epitope as D05 or substantially the same epitope as D05. Therefore, the present invention further relates to antibodies that compete with D05 for binding to CD25.

[0276] The present invention further relates to B07-like antibodies, i.e., antibodies that bind to the same epitope as B07 or substantially the same epitope as B07. Therefore, the present invention further relates to antibodies that compete with B07 for binding to CD25.

[0277] The present invention further relates to H08-like antibodies, i.e., antibodies that bind to the same epitope as H08 or substantially the same epitope as H08. Therefore, the present invention further relates to antibodies that compete with H08 for binding to CD25.

[0278] The present invention further relates to B12-like antibodies, i.e., antibodies that bind to the same epitope as B12 or substantially the same epitope as B12. Therefore, the present invention further relates to antibodies that compete with B12 for binding to CD25.

[0279] In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention comprises a complete or substantially complete human heavy chain constant region (C in this specification). H (abbreviated as C) and / or light chain steady region (C in this specification) L (abbreviated as ). In one embodiment, the steady-state region is of human origin.

[0280] In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention is a complete or substantially complete mouse C H and / or C L This includes. In one embodiment, the steady region is of mouse origin.

[0281] In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention is a mouse antibody or a fragment thereof.

[0282] In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention is a chimeric antibody or a fragment thereof.

[0283] As used herein, “chimeric antibody” refers to an antibody or its antigen-binding fragment containing a first amino acid sequence linked to a second amino acid sequence that is not naturally linked in its natural state. The amino acid sequences may typically be present in separate proteins joined together in a fusion protein, or they may typically be present in the same protein but in a new configuration within the fusion protein. Chimeric proteins can be produced, for example, by chemical synthesis, or by constructing and translating polynucleotides in which peptide regions are encoded in a desired relationship. The term “chimeric antibody” as used herein encompasses antibodies and their antigen-binding fragments, and here, (a) The constant region or a portion thereof is altered, substituted or exchanged so that the variable region is linked to a constant region of a different or altered class, effector function and / or species, or to an entirely different molecule, such as an enzyme, toxin, hormone, growth factor, drug, etc., which confers new characteristics to the chimeric antibody, or (b) The variable region or any part thereof is modified, substituted, or replaced by a variable region or any part thereof having different antigen specificity or modified antigen specificity, or by a corresponding sequence from another species or another antibody class or subclass.

[0284] In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention is a humanized antibody or a fragment thereof.

[0285] As used herein, “humanized antibody” refers to a chimeric antibody or its antigen-binding fragment containing a minimal sequence derived from a non-human immunoglobulin. This includes, for example, antibodies produced by non-human cells having variable and constant regions that have been modified to more closely resemble antibodies produced by human cells by altering the non-human antibody amino acid sequence to incorporate amino acids found in human germline immunoglobulin sequences. The humanized antibody or its antigen-binding fragment according to the present invention may, for example, include amino acid residues in the CDR that are not encoded by a human germline immunoglobulin sequence (e.g., mutations introduced by random or site-directed mutagenesis in vitro or somatic mutation in vivo). The term “humanized antibody” also includes antibodies and their antigen-binding fragments in which a CDR sequence derived from the germline of another mammalian species, such as mouse, is grafted onto a human framework sequence. In other words, the term “humanized antibody” refers to an antibody or its antigen-binding fragment in which the CDR of a recipient human antibody is replaced by a CDR derived from a donor non-human antibody. The humanized antibody or its antigen-binding fragment may also contain donor-derived residues in the framework sequence. Humanized antibodies or their antigen-binding fragments may also contain at least a portion of the constant region of human immunoglobulin. Humanized antibodies or their antigen-binding fragments may also contain residues not found in the recipient antibody or in the imported CDR or framework sequence.Humanization includes techniques such as "superhumanized" antibodies (e.g., Tan et al., 2002. J Immunol. 169(2):1119-25) and "resurfacing" (e.g., Staelens et al., 2006. Mol Immunol. 43(8):1243-57; Roguska et al., 1994. Proc Natl Acad Sci USA. 91(3):969-73), as well as methods known in the art (e.g., Jones et al., 1986. Nature. 321(6069):522-5; Riechmann et al., 1988. Nature. 332(6162):323-7; Verhoeyen et al., 1988. Science. 239(4847):1534-6; Presta, 1992. Curr Opin This can be done using Biotechnol.3(4):394-8; U.S. Patent No. 4,816,567.

[0286] Humanized antibodies may retain similar antigen specificity to the original antibody. However, specific humanization methods can be used to increase the affinity and / or specificity of antibody binding.

[0287] Methods for humanizing antibodies or antigen-binding fragments thereof according to the present invention are well known in the art. The selection of human variable domains (both light and heavy chains) used in the preparation of humanized antibodies or antigen-binding fragments is crucial for reducing antigenicity. According to the so-called "best-fit" method, the sequence of the variable domain of the antibody or antigen-binding fragment according to the present invention is screened against an entire library of known human variable domain sequences. The human sequence that most closely resembles the mouse sequence is then accepted as the human framework (FR) for the humanized antibody (Sims et al., 1993. J Immunol. 151(4):2296-308; Chothia & Lesk, 1987. J Mol Biol. 196(4):901-17).

[0288] Another method for humanizing antibodies or their antigen-binding fragments according to the present invention uses a specific framework from the consensus sequences of all human antibodies of a particular subgroup of light or heavy chain. The same framework can be used for several different humanized antibodies (Carter et al., 1992. Proc Natl Acad Sci USA. 89(10):4285-9; Presta et al., 1993. J Immunol. 151(5):2623-32). It is even more important to humanize the antibodies while retaining high affinity for hCD25 and other desirable biological properties. To achieve this objective, according to a preferred method, humanized antibodies and their antigen-binding fragments are prepared by a process of analyzing the parent sequence and various conceptual humanized products using a three-dimensional model of the parent sequence and the humanized sequence. Three-dimensional immunoglobulin models are generally available and well known to those skilled in the art. Computer programs are available that illustrate and display the possible three-dimensional structures of selected candidate immunoglobulin sequences. Examination of these displays allows for the analysis of the likely roles of residues in the function of candidate immunoglobulin sequences, i.e., the analysis of residues that affect the candidate immunoglobulin's ability to bind to its epitope. In this way, CDR residues can be selected and combined from consensus and import sequences so that desired antibody characteristics, such as increased affinity for hCD25, are achieved. Generally, CDR residues are directly and most substantially involved in the effect on antigen binding.

[0289] Another method for humanizing antibodies or their antigen-binding fragments according to the present invention is to use transgenic or transchromosomal animals that carry a portion of the human immune system for immunization. As hosts, these animals have replaced their immunoglobulin genes with functional human immunoglobulin genes. Thus, antibodies produced by these animals or in hybridomas prepared from the B cells of these animals are already humanized. Examples of such transgenic or transchromosomal animals, but are not limited to, include: - XenoMouse (Abgenix, Fremont, CA) as described in U.S. Patents No. 5,939,598, No. 6,075,181, No. 6,114,598, No. 6,150,584 and No. 6,162,963; -Lonberg et al.,1994.Nature.368(6474):856-859;Lonberg&Huszar,1995.Int Rev Immunol.13(1):65-93;Harding&Lonberg,1995.Ann NY Acad Sci.764:536-46;Taylor et al.,1992.Nucleic Acids Res.20(23):6287-95;Chen et al.,1993.Int Immunol.5(6):647-56;Tuaillon et al.,1993.Proc Natl Acad Sci USA.90(8):3720-4;Choi et al.,1993.Nat Genet.4(2):117-23;Chen et al. al.,1993.EMBO HuMAbMouse (registered trademark) (Medarex, Inc.) is described in J.12(3):821-30; Tuaillon et al., 1994. J Immunol.152(6):2912-20; Taylor et al., 1994. Int Immunol.6(4):579-91; Fishwild et al., 1996. Nat Biotechnol.14(7):845-51. -KMMouse (registered trademark) as described in International Patent Application Publication No. 2002043478; -TC mice described in Tomizuka et al., 2000. Proc Natl Acad Sci USA. 97(2):722-7; and OmniRat (trademark) (OMT, Inc.) is described in International Patent Application Publication No. 2008151081; Geurts et al., 2009. Science. 325(5939):433; Menoret et al., 2010 Eur J Immunol. 40(10):2932-41.

[0290] Humanized antibodies and their antigen-binding fragments can also be produced according to various other techniques, for example, by using other transgenic animals engineered to express a human antibody repertoire for immunization (Jakobovitz et al., 1993. Nature. 362(6417):255-8), or by selection of the antibody repertoire using phage display. Such techniques are known to those skilled in the art and can be implemented starting from the monoclonal antibodies or their antigen-binding fragments disclosed in this application.

[0291] In some embodiments, an antibody or antigen-binding fragment comprising VH and VL or its CDR has a first constant domain (C H 1 and / or C L ) may contain such amino acids that their amino acid sequence is entirely or substantially human.

[0292] In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention is a complete or substantially human antibody or fragment thereof.

[0293] In some embodiments, particularly when the antibody or antigen-binding fragment according to the present invention is intended for human therapeutic use, it is typical that the entire constant region or at least a portion thereof has a complete or substantially human amino acid sequence. Therefore, C H 1 domain, hinge region, C H 2 domains, C H 3 domains and C L Domain (and C if it exists) H One or any combination of the four domains may be entirely or substantially human with respect to their amino acid sequence. Advantageously, C H 1 domain, hinge region, C H 2 domains, C H 3 domains and C L Domain (and C if it exists) H All four domains may have a complete or substantially human amino acid sequence.

[0294] The term "substantially human" refers to at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more amino acid sequence identity with the human constant region in the context of the constant region of a humanized antibody or chimeric antibody or its antigen-binding fragment.

[0295] The term “human amino acid sequence” in this context refers to the amino acid sequence encoded by human immunoglobulin genes, including germline genes, rearranged genes, and somatic mutation genes. The present invention also intends to include a protein comprising a constant domain of a “human” sequence that has been altered by the addition, deletion, or substitution of one or more amino acids with respect to the human sequence, except in embodiments where the presence of a “complete human hinge region” is explicitly required.

[0296] The presence of a “complete human hinge region” in the antibody or its antigen-binding fragment according to the present invention may be beneficial for both minimizing immunogenicity and optimizing antibody stability. Substitutions, insertions, or deletions of one or more amino acids are considered possible within the constant regions of the heavy and / or light chains, particularly within the Fc region. Amino acid substitutions may result in the substitution of the substituted amino acid with a different native amino acid, or with a non-natural or modified amino acid. Other structural modifications are also possible, such as changes in the glycosylation pattern (e.g., by the addition or deletion of N or O-linked glycosylation sites). Depending on the intended use of the antibody or its antigen-binding fragment, it may be desirable to modify the antibody or its antigen-binding fragment according to the present invention with respect to its binding properties to the Fc receptor, for example, to modulate effector function. For example, cysteine ​​residues may be introduced into the Fc region to form interchain disulfide bonds in this region. Homodimeric antibodies produced in this manner may possess improved effector function (Caron et al., 1992. J Exp Med. 176(4): 1191-5; Shopes, 1992. J Immunol. 148(9): 2918-22).

[0297] In one embodiment, the antibody or its antigen-binding fragment is derived from the IgG class.

[0298] In one embodiment, the antibody or its antigen-binding fragment is derived from the human IgG1 subclass. Therefore, in one embodiment, the antibody or its antigen-binding fragment according to the present invention is an IgG1 antibody, preferably a human IgG1 antibody.

[0299] In another embodiment, the antibody or its antigen-binding fragment is derived from the human IgG2 subclass.

[0300] The Fc region of IgG antibodies interacts with cellular Fcγ receptors (FcγRs) to stimulate and modulate downstream effector mechanisms. There are five activating receptors, namely FcγRI(CD64), FcγRIIa(CD32a), FcγRIIc(CD32c), FcγRIIIa(CD16a), and FcγRIIIb(CD16b), as well as one inhibitory receptor, FcγRIIb(CD32b). Communication between IgG antibodies and the immune system is regulated and mediated by FcγRs, which relay information sensed and collected by the antibody to the immune system, providing connections between the innate and adaptive immune systems, particularly in biotherapy (Hayes J et al., 2016. J Inflamm Res 9:209-219).

[0301] IgG subclasses differ in their ability to bind to FcγR, and this differential binding determines their ability to induce a range of functional responses. For example, in humans, FcγRIIIa is the primary receptor involved in the activation of antibody-dependent cell-mediated cytotoxicity (ADCC), and IgG3 (closely followed by IgG1) shows the highest affinity for this receptor, reflecting its ability to potently induce ADCC.

[0302] IgG2 has been shown to have weak binding to this receptor.

[0303] In one embodiment, the antibody or antigen-binding fragment of the present invention binds to FcγR with high affinity, and preferably to the activating receptor with high affinity.

[0304] In one embodiment, the antibody of the present invention or its antigen-binding fragment binds with high affinity to FcγRI and / or FcγRIIa and / or FcγRIIc and / or FcγRIIIa and / or FcγRIIIb.

[0305] In one embodiment, the antibody or antigen-binding fragment of the present invention is an IgG1 antibody (preferably a human IgG1 antibody) or a fragment thereof, and binds to at least one Fc-activated receptor. For example, the antibody or antigen-binding fragment may bind to one or more receptors selected from FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa, and FcγRIIIb. In one embodiment, the antibody or antigen-binding fragment can bind to FcγRIIIa. In one embodiment, the antibody or antigen-binding fragment can bind to FcγRIIa. In one embodiment, the antibody or antigen-binding fragment can bind to FcγRIIIa, FcγRIIc, and optionally FcγRI. In one embodiment, the antibody or antigen-binding fragment can bind to FcγRIIIa, FcγRIIa, and optionally FcγRI.

[0306] In one embodiment, the antibody or antigen-binding fragment of the present invention is approximately 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 It binds to at least one activated Fcγ receptor with a dissociation constant of less than M.

[0307] In one embodiment, the antibody or antigen-binding fragment of the present invention is an IgG1 antibody (preferably a human IgG1 antibody) or a fragment thereof, which binds to FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa, and / or FcγRIIIb with a higher affinity and lower affinity than it binds to FcyRIIb.

[0308] In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention may contain a human heavy chain constant region sequence, which can target, block, and / or deplete CD25-expressing cells to which it is bound.

[0309] In one embodiment, the protein, antibody, or antigen-binding fragment of the present invention depletes the CD25-expressing cells to which they are bound. In one embodiment, the protein, antibody, or antigen-binding fragment of the present invention depletes the Treg cells to which they are bound. In one embodiment, the protein, antibody, or antigen-binding fragment of the present invention also depletes or reduces the tumor-infiltrating regulatory T cells to which they are bound.

[0310] With respect to CD25-expressing cells or Tregs, the terms “deplete” or “to deplete” refer to killing, elimination, lysis, or induction of such killing, elimination, or lysis that adversely affects the number or proportion of CD25-expressing cells present in a sample or subject. In one embodiment, the protein, antibody, or antigen-binding fragment thereof according to the present invention enables the targeting, blockade of proliferation, and / or depletion of CD25-expressing cells or Treg cells. In one embodiment, depletion is mediated by ADCC. In one embodiment, depletion is mediated by ADCP. In one embodiment, depletion is mediated by CDC.

[0311] Therefore, in one embodiment, the protein, antibody, or antigen-binding fragment of the antibody of the present invention directly or indirectly results in the depletion of CD25-expressing cells (for example, resulting in the elimination or reduction of 10%, 20%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 80%, 85%, or more of the number of CD25-expressing cells).

[0312] In one embodiment, a protein, antibody, or antigen-binding fragment of an antibody does not inhibit the binding of interleukin-2 (IL-2) to CD25, but rather depletes the Treg to which they are bound.

[0313] In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention induces antibody-dependent cell-mediated cytotoxicity (ADCC).

[0314] The term "antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to cell-mediated cytotoxicity induced in an antibody-dependent manner when the Fc region of an antibody bound to an antigen binds to an Fc receptor on effector cells such as natural killer cells, macrophages, neutrophils, eosinophils, and mononuclear cells (e.g., peripheral blood mononuclear cells), thereby causing lysis of target cells. ADCC can be measured using assays known and available in the art (e.g., Clynes et al. (1998) Proc Natl Acad Sci USA 95, 652-6).

[0315] In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention is derived from the IgG1 (preferably human IgG1) subclass and has ADCC activity.

[0316] In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention induces antibody-dependent cell-mediated phagocytosis (ADCP).

[0317] The terms "antibody-dependent cell-mediated phagocytosis" (ADCP) or "opsonization" refer to a cell-mediated response in which nonspecific cytotoxic cells expressing Fc receptors (FcRs) (e.g., phagocytes, macrophages) recognize antibodies bound to target cells and induce phagocytosis of those target cells. ADCP can be measured using assays known and available in the art (e.g., Clynes et al. (1998) Proc Natl Acad Sci USA 95, 652-6).

[0318] In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention is derived from the IgG1 (preferably human IgG1) subclass and has ADCP activity.

[0319] In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention induces complement-dependent cell-mediated cytotoxicity (CDC).

[0320] The term "complement-dependent cell injury" (CDC) refers to the induction of lysis of antigen-expressing cells recognized by the antibody or its antigen-binding fragment of the present invention in the presence of complement. The complement activation pathway is initiated by the binding of the first component of the complement system (C1q) to a molecule (e.g., an antibody) complexed with a congener antigen. CDC can be measured using assays known and available in the art (e.g., Clynes et al. (1998) Proc Natl Acad Sci USA 95,652-6; Gazzano-Santaro et al., J.Immunol.Methods, 202:163 (1996)).

[0321] In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention is derived from the IgG1 (preferably human IgG1) subclass and has CDC activity.

[0322] The constant region of an antibody is crucial for its ability to fix complement and mediate cell-dependent cytotoxicity and phagocytosis. Therefore, as discussed herein, antibody isotypes may be selected based on whether it is desirable for the antibody to mediate cytotoxicity / phagocytosis.

[0323] In one embodiment, the antibody or antigen-binding fragment according to the present invention is conjugated to, for example, a toxic moiety. In another embodiment, the antibody or antigen-binding fragment according to the present invention is linked to a toxic moiety.

[0324] In one embodiment, the antibody or antigen-binding fragment according to the present invention is not conjugated to, for example, a toxic moiety. In another embodiment, the antibody or antigen-binding fragment according to the present invention is not linked to a toxic moiety.

[0325] In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention lacks an Fc domain (for example, C H 2 and / or C H It lacks three domains, or contains an Fc domain of an IgG2 or IgG4 isotype (preferably human IgG2 or IgG4).

[0326] In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention does not contain an Fc region that mediates ADCC, ADCP, and / or CDC.

[0327] In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention does not induce ADCC, ADCP, and / or CDC.

[0328] Therefore, in one embodiment, the antibody or its antigen-binding fragment according to the present invention does not directly or indirectly cause depletion of CD25-expressing cells (for example, it does not cause the elimination or reduction in number of 10%, 20%, 50%, 60% or more of CD25 cells). For example, the antibody according to the present invention does not contain an Fc domain that can substantially bind to the FcγRIIIA(CD16) polypeptide.

[0329] In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention is a manipulated antibody or fragment thereof.

[0330] The manipulated antibodies of the present invention include those in which framework residues within the VH and / or VL have been modified, for example, to improve the properties of the antibody. Typically, such framework modifications are made to reduce the immunogenicity of the antibody. For example, one approach is to “reverse-mutate” one or more framework residues to the corresponding germline sequence. More specifically, an antibody that has undergone somatic mutation may contain framework residues different from the germline sequence from which the antibody originates. Such residues can be identified by comparing the antibody framework sequence with the germline sequence from which the antibody originates. To return the framework region sequence to its germline arrangement, the somatic mutation can be “reverse-mutated” to the germline sequence, for example, by site-directed mutagenesis or PCR-mediated mutagenesis. Such “reverse-mutated” antibodies are also intended to be included in the present invention. Another type of framework modification involves mutating one or more residues within the framework region, or even within one or more CDR regions, to remove a T cell epitope and thereby reduce the potential immunogenicity of the antibody. This approach, also known as "deimmunization," is described in more detail in U.S. Patent Application Publication No. 20030153043 by Carr et al.

[0331] In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention is engineered to induce an enhanced, increased, or improved ADCC, ADCP, and / or CDC response.

[0332] As used herein, the term “enhancement, increase, or improvement of ADCC, ADCP, and / or CDC response” refers to the ADCC, ADCP, and / or CDC response induced by the antibody or fragment thereof according to the present invention, compared to the ADCC, ADCP, and / or CDC response induced by other anti-CD25 antibodies, including those that do not inhibit the binding of IL-2 to CD25, and by, for example, an unmodified anti-CD25 monoclonal antibody.

[0333] Methods for increasing ADCC, ADCP, and / or CDC are well known in the art. For example, ADCC can be increased by removing the fucose moiety from antibody glycans, for example by antibody production in the YB2 / 0 cell line, or by introducing specific mutations into the Fc portion of human IgG1.

[0334] (For example, S298A / E333A / K334A, S239D / I332E / A330L, G236A / S239D / A330L / I332E) (Lazar et al. (2006) Proc Natl Acad Sci USA 103, 2005-2010; Smith et al. (2012) Proc Natl 25 Acad Sci USA 109, 6181-6). ADCP can also be increased by introducing specific mutations into the Fc portion of human IgG1 (Richards et al. (2008) Mol Cancer Ther 7, 2517-27). CDC response can be increased by mutations in antibodies that increase the affinity for C1q binding (Idusogie et al. (2001) J Immunol 166, 2571-5).

[0335] Notably, methods for reducing or eliminating ADCC, ADCP, and / or CDC are also well known in the art. For example, ADCC can be reduced or eliminated by modifying the glycosylation profile of the Fc domain of an immunoglobulin. CDC can be reduced or eliminated by substituting one or more amino acids with other amino acids so that the antibody alters the C2q binding (U.S. Patent No. 6,194,551 by Idusogie et al.).

[0336] In one embodiment, an antibody or its antigen-binding fragment according to the present invention is manipulated to modify its glycosylation. For example, the antibody according to the present invention is aglycosylated (i.e., the antibody lacks glycosylation). Glycosylation can be modified, for example, to increase the affinity of the antibody to an antigen or to alter the ADCC activity of the antibody. Such carbohydrate modification can be achieved, for example, by altering one or more sites of glycosylation in the antibody sequence. For example, glycosylation can be eliminated by making one or more amino acid substitutions that result in the exclusion of one or more variable region framework glycosylation sites. Such aglycosylation may increase the affinity of the antibody to an antigen. Such approaches are described in further detail in U.S. Patents 5,714,350 and 6,350,861 by Co et al. (incorporated herein by reference). In addition to or instead of this, antibodies with altered glycosylation types can be produced, for example, low-fucosylated or non-fucosylated antibodies with a small amount of fucosyl residues or no fucosyl residues, or antibodies with increased bisected GlcNac structures. Such altered fucosylation patterns have been demonstrated to increase the ADCC capacity of antibodies. Such carbohydrate modification can be achieved, for example, by expressing antibodies in host cells having an altered glycosylation mechanism. Cells having an altered glycosylation mechanism have been described in the Art and can be used as host cells to express the recombinant antibodies of the present invention and thereby produce antibodies having altered glycosylation. For example, European Patent No. 1176195 (incorporated herein by reference) describes a cell line having a functionally disrupted FUT8 gene encoding a fucosyltransferase, in which antibodies expressed in such cell lines exhibit fucosylation failure or lack fucosyl residues. Therefore, in some embodiments, the antibodies or antigen-binding fragments of the present invention may be produced by recombinant expression in cell lines exhibiting low-fucosylation or non-fucosylation patterns, such as mammalian cell lines lacking expression of the FUT8 gene encoding fucosyltransferase.PCT Public International Publication No. 03 / 035835 (incorporated herein by reference) describes a mutant CHO cell line, Lecl3 cells, which have a reduced ability to bind fucose to Asn(297)-binding carbohydrates and also result in hypofucosylation of antibodies expressed in its host cells (see also Shields, R. Let al, 2002 J. Biol. Chem. 277:26733-26740). PCT Public International Publication No. 99 / 54342 (incorporated herein by reference) describes cell lines that express glycoprotein-modified glycosyltransferases (e.g., β(l,4)-N-acetylglucosaminyltransferase III (GnTIII)) and are engineered so that antibodies expressed in the engineered cell lines exhibit an increase in bisected GlcNac structures, resulting in increased ADCC activity of the antibodies (see also Umana et al, 1999 Nat. Biotech. 17:176-180). Eureka Therapeutics further describes genetically engineered CHO mammalian cells capable of producing antibodies with altered mammalian glycosylation patterns lacking fucosyl residues (http: / / www.eurekainc.com / a&boutus / companyoverview.html). Alternatively, the antibody of the present invention (preferably a monoclonal antibody) can be produced in yeast or filamentous fungi that can be manipulated for a mammalian-like glycosylation pattern and produce an antibody lacking fucose as a glycosylation pattern (see, for example, European Patent No. 1,297,172).

[0337] In one embodiment, the antibody or antigen-binding fragment thereof according to the present invention is a pegylated antibody or a fragment thereof.

[0338] Antibodies can be pegylated to increase their biological (e.g., serum) half-life, for example. To pegylate an antibody or a fragment thereof, the antibody or antibody fragment is typically reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions that one or more PEG groups bind to the antibody or antibody fragment. Pegylation can be carried out by an acylation or alkylation reaction with a reactive PEG molecule (or a similar reactive water-soluble polymer). As used herein, the term "polyethylene glycol" is intended to encompass any form of PEG used to derivatize other proteins, such as mono(DY12-DY120)alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. In certain embodiments, the antibody to be pegylated is a non-glycosylated antibody. Methods for pegyrating proteins are known in the art and can be applied to the antibodies of the present invention, for example, as described in European Patent No. 0154316 and European Patent No. 0401384 (incorporated herein by reference).

[0339] The present invention further relates to proteins as described herein, and more particularly to fusion proteins comprising antibodies or antigen-binding fragments thereof as described herein.

[0340] In one embodiment, the fusion protein includes a second antigen-binding moiety.

[0341] In one embodiment, the fusion protein is a multispecific antibody, such as a bispecific antibody.

[0342] In one embodiment, the antibody or its antigen-binding fragment is bispecific and can further bind to another molecule.

[0343] In one embodiment, the other molecule is an immune receptor. Examples of immune receptors that can be bound by the bispecific antibody of the present invention include, but are not limited to, CTLA4, PD-1, PD-L1, TIM-3, LAG-3, B7H3, B7H4, B7H6, 4-1BB, OX40, ICOS, GITR, TIGIT, CD27-CD70, CD40, BTLA, HVEM, CD160, CCR8, and CEACAM-1.

[0344] In one embodiment, the antibody or its antigen-binding fragment is bispecific and can further bind to a costimulatory molecule. Examples of costimulatory molecules include, but are not limited to, 4-1BB, ICOS, GITR, CD27-CD70, CD40, and OX40.

[0345] In one embodiment, the antibody or its antigen-binding fragment is bispecific and can bind to OX40. In one embodiment, the antibody or its antigen-binding fragment is bispecific and can bind to GITR. In one embodiment, the antibody or its antigen-binding fragment is bispecific and can bind to ICOS.

[0346] In another embodiment, the antibody or its antigen-binding fragment is bispecific and can further bind to a co-inhibitory molecule. Examples of co-inhibitory molecules include, but are not limited to, CTLA4, PD-1, PD-L1, TIM-3, LAG-3, TIGIT, BTLA, HVEM, CD160, and CEACAM-1.

[0347] In one embodiment, the antibody or its antigen-binding fragment is bispecific and can bind to CTLA4. In one embodiment, the antibody or its antigen-binding fragment is bispecific and can bind to PD-1. In one embodiment, the antibody or its antigen-binding fragment is bispecific and can bind to TIGIT.

[0348] In one embodiment, the fusion protein includes a second antigen-binding moiety that binds to an immune checkpoint protein. Immune checkpoint proteins include checkpoint inhibitors and checkpoint agonists.

[0349] Checkpoint inhibitors (also known as immune checkpoint inhibitors or CPIs), often antibodies, block the interaction between inhibitory receptors (IRs) expressed on T cells and their ligands.

[0350] Examples of checkpoint inhibitors include, but are not limited to, inhibitors of the cell surface receptor PD-1 (programmed cell death protein 1), also known as CD279 (cluster differentiation 279); inhibitors of ligand PD-L1 (programmed cell death ligand 1), also known as CD274 (differentiation cluster 274) or B7-H1 (B7 homolog 1); inhibitors of the cell surface receptor CTLA4 or CTLA-4 (cytotoxic T lymphocyte-associated protein 4), also known as CD152 (differentiation cluster 152); inhibitors of LAG-3 (lymphocyte activation gene 3), also known as CD223 (cluster differentiation 223); and HAVCR2 (hepatitis A virus). Examples include inhibitors of TIM-3 (containing T cell immunoglobulin and mucin domains), also known as cell receptor2 or CD366 (cluster differentiation366); inhibitors of TIGIT (T cell immune receptor with Ig and ITIM domains), also known as VSIG9 (protein containing V-Set and immunoglobulin domains9) or VSTM3 (protein containing V-Set and transmembrane domain3); inhibitors of BTLA (B and T lymphocyte depressant), also known as CD272 (cluster differentiation272); and inhibitors of CEACAM-1 (carcinoembryonic antigen-associated cell adhesion molecule1), also known as CD66a (cluster differentiation66a).

[0351] Checkpoint agonists act by activating stimulus receptors (costimulatory receptors) expressed on immune cells such as T cells. As used herein, the term "stimulatory receptor" refers to a receptor that, upon activation, induces a stimulus signal and thus enhances the immune response.

[0352] Examples of checkpoint agonists include, but are not limited to, agonists of CD137 (cluster differentiation 137), also known as 4-1BB or TNFRS9 (tumor necrosis factor receptor superfamily, member 9); agonists of the OX40 receptor, also known as CD134 (cluster differentiation 134) or TNFRSF4 (tumor necrosis factor receptor superfamily, member 4); agonists of GITR (glucocorticoid-induced TNF receptor family-related protein); agonists of ICOS (inducing costimulator); agonists of CD27-CD70 (cluster differentiation 27-cluster differentiation 70); and agonists of CD40 (cluster differentiation 40).

[0353] In one embodiment, the fusion protein includes a second antigen-binding moiety that binds to a T cell marker, such as CD2, CD3, or CD28.

[0354] In one embodiment, the fusion protein includes a second antigen-binding moiety that binds to an NK cell marker, such as an activated NK receptor. Examples of activating NK receptors include, but are not limited to, activated forms of KIR proteins (e.g., KIR2DS protein), CD160-TM, NKG2D, IL-2R, IL-12R, IL-15R, IL-18R, and IL-21R.

[0355] In one embodiment, an antibody or its antigen-binding fragment is conjugated with a therapeutic moiety, i.e., a drug. The therapeutic moiety may be, for example, a chemotherapeutic agent, an immunosuppressant, a soluble peptide, a radionuclide, or a toxin. Thus, in one embodiment, the fusion protein comprises the therapeutic moiety and protein, antibody or its antigen-binding fragment as described herein.

[0356] In one embodiment, the antibody or its antigen-binding fragment is not conjugated with a radionuclide (i.e., the antibody or its antigen-binding fragment is not radiolabeled) and / or conjugated with a toxin.

[0357] Examples of radionuclides include: 90 Y, 131 I, or 67 This includes, but is not limited to, cu.

[0358] Examples of toxins include, but are not limited to, doxorubicin and calicheamycin.

[0359] In one embodiment, an antibody or its antigen-binding fragment is conjugated with a cytotoxic moiety. The cytotoxic portion includes, for example, taxol; cytochalasin B; gramicidin D; ethidium bromide; emetine; mitomycin; etoposide; tenoposide; vincristine; vinblastine; colchicine; doxorubicin; daunorubicin; dihydroxyanthracine dione; meitansine or its analogs or derivatives, which are tubulin inhibitors; monomethyl auristatin E or F or its analogs or derivatives, which are mitotic inhibitors; drastatin 10 or 15 or its analogs; irinotecan or its analogs; mitoxantrone; mitramycin; actinomycin D; 1-dehydrotestosterone; glucocorticoids; procaine; tetracaine; lidocaine; propranolol; puromycin; calicheamicin or its analogs or derivatives; methotrexate, 6-mercaptopurine, 6-thioguanine, and cytarabi. Antimetabolites such as fludarabine, 5-fluorouracil, decarbazine, hydroxyurea, asparaginase, gemcitabine, and cladribine; alkylating agents such as mechloretamine, thioepa, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, dacarbazine (DTIC), procarbazine, and mitomycin C; platinum derivatives such as cisplatin and carboplatin; duocalmycin A, duocalmycin SA, and lacemycin (CC-1065); antibiotics such as dactinomycin, bleomycin, daunorubicin, doxorubicin, idarubicin, mitramycin, mitomycin, mitoxantrone, plicamycin, and anthramycin (AMC); and pyrrolo[2,lc][l,4]-benzodiazepine (PDB).Diphtheria toxin and related molecules, e.g., diphtheria A chain and its active fragments and hybrid molecules, lysine toxin, e.g., lysine A or deglycosylated lysine A chain toxin, cholera toxin, Shiga-like toxin, e.g., SLT I, SLT II, ​​SLT IIV, LT toxin, C3 toxin, Shiga toxin, pertussis toxin, tetanus toxin, soybean Bowman-Birk protease inhibitor, Pseudomonas exotoxin, allorin, saporin, modesin, geranine, abrin A chain, modesin A chain, α-sarcin, Aleurites fordii protein, dianthin protein, Phytolacca americana protein, e.g., PAPI, PAPII and PAP-S, momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor The following may be selected from the group consisting of officinalis inhibitors, geronin, mitojerin, restrictosin, phenomycin, and enomycin toxin; ribonuclease (RNase); DNase I, Staphylococcus enterotoxin A; chickweed antiviral protein; diphtherin toxin; and Pseudomonas endotoxin.

[0360] In one embodiment, an antibody or its antigen-binding fragment is conjugated with a cytokine. Suitable cytokines include, but are not limited to, interferons, interleukins, and colony-stimulating factors. Thus, in one embodiment, the fusion protein comprises the cytokines and proteins, antibodies or their antigen-binding fragments described herein.

[0361] In one embodiment, an antibody or its antigen-binding fragment is conjugated with a cytokine mimetic. Thus, in one embodiment, the fusion protein comprises the cytokine mimetic and protein described herein, and an antibody or its antigen-binding fragment.

[0362] Techniques for conjugating molecules to antibodies or their antigen-binding fragments are well known in the art. Typically, nucleic acid molecules are covalently bound to lysine or cysteine ​​on the antibody or its fragment via N-hydroxysuccinimide ester or maleimide functional groups, respectively. Methods of conjugation using engineered cysteine ​​or incorporation of non-natural amino acids have been reported to improve the uniformity of the conjugate.

[0363] Another object of the present invention is an isolated protein that binds to human CD25 according to the present invention, in particular an isolated nucleic acid encoding an antibody or an antigen-binding fragment thereof.

[0364] Another object of the present invention is an isolated nucleic acid encoding a fusion protein according to the present invention.

[0365] As used herein, “isolated nucleic acids” is intended to refer to nucleic acids substantially isolated from other genomic DNA sequences naturally associated with a native sequence, as well as from proteins or complexes such as ribosomes and polymerases. The term encompasses nucleic acid sequences removed from their naturally occurring environment and includes recombinant or cloned DNA isolates and chemically synthesized analogs or analogs biologically synthesized by heterologous systems. Substantially pure nucleic acids include nucleic acids in isolated forms.

[0366] Of course, this refers to the initially isolated nucleic acid and does not exclude genes or sequences that may be later added to nucleic acids isolated by humans.

[0367] In one embodiment, the isolated nucleic acid is purified.

[0368] In one embodiment, the isolated nucleic acid is purified to the following extent. (1) By absorbance method or fluorescence method (for example, the absorbance ratio at 260 nm and 280 nm (A 260 / 28080% by weight, 85% by weight, 90% by weight, 91% by weight, 92% by weight, 93% by weight, 94% by weight, 95% by weight or more, most preferably 96% by weight, 97% by weight, 98% by weight or 99% by weight of nucleic acids, determined by measuring ); or (2) Homogeneity demonstrated by agarose gel electrophoresis and the use of inserts, such as ethidium bromide, SYBR Green, GelGreen, etc.

[0369] In one embodiment, the nucleic acid encodes at least the heavy chain variable region or the light chain variable region of the antibody or antigen-binding fragment according to the present invention. In one embodiment, the nucleic acid may encode the variable region and the constant region of the antibody or antigen-binding fragment according to the present invention. In one embodiment, the nucleic acid may encode the heavy chain and light chain of the antibody or antigen-binding fragment on separate nucleic acids or on the same nucleic acid molecule.

[0370] In one embodiment, the nucleic acid according to the present invention comprises or consists of a sequence encoding the VH of the antibody or its antigen-binding fragment according to the present invention.

[0371] In one embodiment, the nucleic acid according to the present invention comprises or comprises a sequence encoding the VH of the antibody or antigen-binding fragment thereof according to the present invention, the sequence comprising or comprising any sequence that shares at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more identity with SEQ ID NOs. 60, SEQ ID NOs. 61, SEQ ID NOs. 62, SEQ ID NOs. 63, SEQ ID NOs. 64, SEQ ID NOs. 65, SEQ ID NOs. 66, SEQ ID NOs. 67, SEQ ID NOs. 68, SEQ ID NOs. 69, SEQ ID NOs. 70, SEQ ID NOs. 71, and SEQ ID NOs. 60-71, and is selected from the group comprising such sequences. Sequence ID 60 TAATACGACTCACTATAGGGCGTCTCACTCTCAAGTGCAGCTGGTGGAATCCGGAGGAGGCGTGGTGCAGCCCGGAAGGTCTCTGAGACTGAGCTGTCGCCAGCGGCTTCACATTCAGCAATCACGCCATGGCTTTGGGTGAGACAAGCCCCCGGCAAGGGACTGGAATGGGTCGCCGTGATCTCCTACGACGGCAGCAACAA GTACTACGCCGATAGCGTGAAGGGAAGGTTCACTATCTCTAGGGACAACGCCAAGAACACACTGTATCTGCAGATGAAACAGCCTCAGAGCCGAGGACACAGCTGTCTACTACTGCACAACTGGCTGGAACAGCGGCTATGATTGGGGCCAAGGCACTCTGGTGACAGTGAGCAGCGCCAGTGAGAGGCCTCGACTGTGCCTTCTA sequence no.61 TAATACGACTCACTATAGGGCGTCTCACTCTCAAGTGCAGCTGGTGGAAAGCGGAGGCGAGTGGTGCAGCCCGGCAGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACATTCAGCACACGCCATGGCTTTGGGTGAGACAAGCCCCCGGCAAGGGACTGGAATGGGTCGCCGTGATCAGCTACGATGGCAGCAACAA GTACTACGCCGACTCCGTGAAGGGAAGGTTCACAATCTCTAGGGACAACAGCCAGAACACTCTGTATCTGCAGATGAACTCTCTGAGGGCTGAGGATACAGCCGTCTACTACTGCACAACTGGACTGAATAGCGGATACGATTGGGGCCAAGGCACACTGGTGACAGTGAGCAGCGCCAGTGAGAGGCCTCGACTGTGCCTTCTA sequence no.62 TAATACGACTCACTATAGGGCGTCTCACTCTCAAGTGCAGCTGGTGGAAAGCGGAGGAGGATGGTGCAGCCCGGAAGGTCTCTGAGGCTGAGCTGTGCTGCTAGCGGCTTCACTTTCAGCAATCACGCCATGGCTTTGGGTGAGACAAGCCCCCGGCAAAGGACTGGAGTGGGTGGCGTGATCAGCTACGGCAGCAACAA GTACTATAGGGACAGCGTCAAGGGAAGGTTCACTATCTCTAGGGATAACGCCCAGAGCACTCTGTATCTGCAGATGAATTCTCTGAGGGCTGAGGATACAGCCGTCTACTACTGCACTACTGGACTGAACAGCGGCTACGATTGGGGACAAGGCACACTGGTGACTGTGAGCAGCGCCAGTGAGAGGCCTCGACTGTGCCTTCTA sequence no. 63 TAATACGACTCACTATAGGGCGTCTCACTCTCAAGTGCAGCTGGTCGAGAGCGGAGGAGGAGTGGTGCAGCCCGGAAGGTCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACATTCAGCAATCACGCCATGGCTTTGGGTGAGACAAGCCCCCGGCAAGGGACTGGAATGGGTGCCGTGATCTCCTACGACGGCAGCAACAA GTACTACGCCGACTCCGTGAAGGGAAGGTTCACTATCTCTAGGGACAATGCCAAGAACACTCTCTATCTGCAGATGAACTCTCTGAGGGCTGAGGATACAGCCGTCACTACTGCACAACTGGACTGAACAGCGGCTACGATTGGGGCCAAGGCACTCTGGTGACAGTGAGCAGCGCCAGTGAGAGGCCTCGACTGTGCCTTCTA sequence no. 64 TAATACGACTCACTATAGGGCGTCTCACTCTCAAGTGCAGCTGGTGGAATCCGGAGGCGGCGTGGGTGCAACCCGGAAGGTCTCTGAGACTGAGCTGTCGCCAGCGGCTTCACATTCAACCACGCCATGGCTTTGGGTGAGACAAGCCCCCGGCAAGGGACTGGAATGGGTCGCCGTGATCTCCTAACGACGGCAGCAACAA GTACTATAGGGACAGCGTGAAGGGAAGGTTCACAATCTCTAGGGACAACAGCAAGAACACTCTCTATCTGCAGATGAACTCTCTGAGGGCTGAGGATACTGCCGTCTACTACTGCACTACTGGACTGAATAGCGGCTACGATTGGGGCCAAGGCACTCTGGTGACAGTCAGCAGCGCCAGTGAGAGGCCTCGACTGTGCCTTCTA sequence number 65 TAATACGACTCACTATAGGGCGTCTCACTCTCAAGTGCAGCTGGTGGAATCCGGAGGCGGCGTGGGTGCAACCCGGCAGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACATTCAGCAACCACGCCATGGCTTGGGTGAGACAAGCCCCCGGCAAGGGACTGGAATGGGTCGCCGTGATCTCCTAACGACGGCAGCAACAA GTACTATAGGGACAGCGTCAAGGGAAGGTTCACAATCTCTAGGGACAATGCCCAGAACACTCTGTATCTGCAGATGAACTCTCTGAGGGCTGAGGATACAGCCGTCACTACTGCACAACTGGACTGAATTCCGGCTACGATTGGGGCCAAGGCACTCTGGTGACAGTCAGCTCCGCCAGTGAGACGCCTCGACTGTGCCTTCTA sequence no.66 TAATACGACTCACTATAGGGCGTCTCACTCTCAAGTGCAGCTGGTGGAAAGCGGAGGCGGCGTGGGTGCAACCCGGCAGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTTACTTTCAGCAACCACGCCATGGCTTGGGTGAGACAAGCCCCCGGCAAGGGACTGGAATGGGTCGCCGTGATCTCCTACGATGGCAGCAACAA GTACTACGCCGACAGCGTGAAGGGAAGGTTCACAATCTCTAGGGACAACGCCAAGAGCACTCTGTATCTGCAGATGAACTCTCTGAGAGCCGAGGATACAGCCGTGTACTACTGCACAACTGGACTGAACAGCGGCTACGATTGGGGCCAAGGCACTCTGGTCACTGTGAGCAGCGCCAGTGAGACGCCTCGACTGTGCCTTCTA sequence number 67 TAATACGACTCACTATAGGGCGTCTCACTCTCAAGTGCAGCTGGTGGAAAGCGGCGGAGGAGTGGTGCAGCCCGGCAGATCTCTGAGGCTGAGCTGTGCCGCTAGCGGCTTCACTTCAGCAACCACGCCATGGCTTGGGTGAGACAAGCCCCCGGCAAAGGACTGGAGTGGGTGGCCGTGATCTCCTAACGACGGCAGCAACAC ATACTACGCTGACAGCGTCAAGGGAAGGTTCCAATCTCTAGGGGACAATGCCAAGTCCACTCTCTATCTGCAGATGAACTCTCTGAGGGCTGAGGATACAGCCGTCTACTACTGCACTACTGGACTGAACAGCGGCTACGATTGGGGCCAAGGCACTCTGGTGACAGTGAGCAGCGCCAGTGAGACGCTCGACTGTGCCTTCTA sequence number 68 TAATACGACTCACTATAGGGCGTCTCACTCTCAAGTGCAGCTGGTGGAAAGCGGAGGCGGAGTGGTCCAGCCCGGAAGGTCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTTACTTTCAGCAACCATGCCATGGCTTTGGGTGAGACAAGCCCCCGGCAAGGGACTGGAATGGGTCGCCGTGATCTCCTACGACGGCAGCAACAA GTACTACGCCGACAGCGTGAAGGGAAGGTTCACAATCTCTAGGGACAACAGCAAGAACACTCTCTATCTGCAGATGAACTCTCTGAGGGCCGAGGATACAGCCGTCACTACTGCACTACTGGCTGGAACAGCGGCTATGATTGGGGCCAAGGCACACTGGTGACAGTGAGCAGCGCCAGTGAGAGGCCTCGACTGTGCCTTCTA sequence no.69 TAATACGACTCACTATAGGGCGTCTCACTCTCAAGTGCAGCTGGTGGAAAGCGGAGGAGCGTGGTGCAGCCCGGAAGGTCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACATTCACGACACGCCATGGCTTGGGTGAGACAAGCCCCCGGCAAGGGACTGGAATGGGTCGCCGTGATCTCCTAACGATGGCAGCAACAA GTACTACGCCGACTCCGTGAAGGGAAGGTTCACTATCTCTAGGGACAACAGCAAGAACACTCTGTATCTGCAGATGAACTCTCTGAGGGCTGAGGATACAGCCGTGTACTACTGCACAACTGGACTGAATAGCGGCTACGATTGGGGCCAAGGCACTCTGGTGACTGTGAGCAGCGCCAGTGAGAGGCCTCGACTGTGCCTTCTA sequence no. 70 TAATACGACTCACTATAGGGCGTCTCACTCTCAAGTGCAGCTGGTGGAAAGCGGAGGCGGCGTGGTGCAACCCGGCAGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACATTCAGCAACCACGCCATGGCTTGGGTGAGACAAGCCCCCGGCAAGGGACTGGAATGGGTGGCCGTGATCTCCTACGACGGCGACAACAA GTACTACGCCGACAGCGTGAAGGGAAGGTTCACAATCTCTAGGGACAACGCCAAGAACACTCTCTATCTGCAGATGAACTCTCTGAGGGCTGAGGATACAGCCGTCTACTACTGCACAACTGGACTGAATAGCGGCTACGATTGGGGCCAAGGCACTCTGGTCACTGTGAGCAGCGCCAGTGAGACGCCTCGACTGTGCCTTCTA Sequence ID 71 TAATACGACTCACTATAGGGCGTCTCACTCTCAAGTGCAGCTGGTGGAATCCGGAGGAGGCGTGGTGCAGCCCGGCAGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACATTCAGCAATCACGCTATGGCTTGGGTGAGACAAGCCCCCGGCAAGGGACTGGAATGGGTCGCCGTGATCAGCTACGATGGCAGCAACAA GTACTATAGGGACAGCGTGAAGGGCAGATTCACTATCTCTAGGGACAACTCCCAGAGCACTCTGTATCTGCAGATGAATTCTCTGAGGGCCGAGGATACAGCCGTCTACTACTGCACAACTGGACTGAATAGCGGCTACGATTGGGGCCAAGGCACTCTGGTCACAGTGAGCAGCGCCAGTGAGACGCCTCGACTGTGCCTTCTA

[0372] In one embodiment, the nucleic acid according to the present invention comprises or consists of a sequence encoding the VL of an antibody or its antigen-binding fragment according to the present invention.

[0373] In one embodiment, the nucleic acid according to the present invention comprises or comprises a sequence encoding the VL of the antibody or its antigen-binding fragment according to the present invention, the sequence comprising or comprising any sequence that shares at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more identity with SEQ ID NOs. 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, and SEQ ID NOs. 72-89. Sequence ID 72 TAATACGACTCACTATAGGGCGTCTCACTCTGATATCCAGATGACTCAGAGCCCTAGCTCTCTGAGCGCTAGCGTCGGAGATAGGGTGACTATCACATGTAGGGCCAGCCAGAGCGTGAACAGCTTTCTGAACTGGTATCAGCAGAAGCCCGGCAAGGCCCCTAGGAGGCTGATCTATGGCACATCCTCTC TGCAGAGCGGCGTCCCAAGCAGATTCAGCGGCTCCGGCAGCGGCACTGACTACACACTGACTATCAGCAGCCTCCAGCCAGAGGACTTCGCCACATACTACTGCCAGCAGTACACTAGCTGGCCTTGGACATTCGGCCAAGGCACAAAGCTGGAGATCAAGAGAACTGAGACGCCTCGACTGTGCCTTCTA Sequence ID 73 TAATACGACTCACTATAGGGCGTCTCACTCTGATATCCAGATGACTCAGAGCCCAAGCTCTCTGAGCGCTAGCGTGGGGATAGGGTGACTATCACTTGTAGGGCCAGCCAGAGCGTGAGCAAGTTTCTGAACTGGTATCAGCAGAAGCCCGGCAAGGCCCCTAGGAGGCTGATCTACGGCACTAACTCTC TGCAGTCCGGCGTGCCTAGCAGATTCAGCGGAAGCGGCAGCGGCACTGACTACACTCTGACTATCAGCTCTCTGCAGCCAGAGGACTTCGCCACTTACTACTGCCAGCAGTACAACAGCTGGCCTTGGACTTTCGGCCAAGGCACTAAGCTGGATCAAGAGAACTGAGACGCCTCGACTGTGCCTTCTA sequence no. 74 TAATACGACTCACTATAGGGCGTCTCACTCTGATATCCAGATGACTCAGAGCCCTAGCTCTCTGAGCGCTAGCGTGGGAGATAGGGTGACTATCACTTGCAGAGCCAGCCAGAGCGTGAACAGCTATCTGAACTGGTATCAGCAGAAGCCCGGCAAGGCCCCAAGGAGGCTGATCTACGGCACAAATTCTC TGCAGAGCGGCGTGCCAAGCAGATTTAGCGGCAGCGGAAGCGGCACAGACTACACACTGACTATCAGCTCTCTGCAGCCAGAGGACTTCGCCACTTACTACTGCCAGCAGTACAACAGCTGGCCTTGGACATTCGGCCAAGGCACTAAGCTGGAGATCAAGAGAACTGAGACGCCTCGACTGTGCCTTCTA sequence no. 75 TAATACGACTCACTATAGGGCGTCTCACTCTGACATCCAGATGACACAGAGCCCTAGCTCTCTGAGCGCTAGCGTGGGAGATAGGGTGACAATCACATGTAGGGCCAGCCAGAGCATCAGCTCCTATCTGAACTGGTATCAGCAGAAACCCGGCAAGGCCCCTAGGAGGCTGATCTACGGCACAAATTCTC TGCAGAGCGGCGTGCCTAGCAGATTTAGCGGCAGCGGAAGCGGCACAGACTACACTCTGACTATCAGCTCTCTGCAGCCAGAGGACTTCGCCACATACTACTGCCAGCAGTACAACAGCTGGCCTTGGACTTTCGGCCAAGGCACTAAGCTGGATCAAGAGAACTGAGACGCCTCGACTGTGCCTTCTA sequence no. 76 TAATACGACTCACTATAGGGCGTCTCACTCTGATATCCAGATGACACAGAGCCCTAGCTCTCTGAGCGCTAGCGTGGGAGATAGGTGACTATCACTTGTAGGGCCAGCCAGAGCATCAGCAGCTTTCTGAACTGGTATCAGCAGAAGCCCGGCAAGGCCCCAAGGAGGCTGATCTACGGCACAAATTCTC TGCAGTCCGGCGTGCCAAGCAGATTCAGCGGCAGCGAAGCGGCACTGACTTCACACTGACTATCAGCTCTCTGCAGCCAGAGGACTTCGCCACTTACTACTGCCAGCAGTACAACAGCTGGCCTTGGACATTCGGCCAAGGCACAAAGCTGGAGATCAAGAGAACTGAGACGCCTCGACTGTGCCTTCTA sequence no. 77 TAATACGACTCACTATAGGGCGTCTCACTCTGATATCCAGATGACTCAGTCCCCTAGCTCTCTGAGCGCTAGCGTGGGGATAGGGTGACTATCACATGTAGGGCCTCCCAGAACATCAACAGCTTTCTGAACTGGTATCAGCAGAAGCCCGGCAAGGCCCCTAGGAGGCTGATCTATGGCACTTCCTTC TGCAGAGCGGAGTGCCATCCAGATTTAGCGGCAGCGGAAGCGGCACAGACTTCACTCTGACTATCAGCTCTCTGCAGCCAGAGGACTTCGCCACTTACTACTGCCAGCAGTACAACAGCTGGCCTTGGACATTCGGCCAAGGCACAAAGCTGGAGATCAAGAGAACTGAGACGCCTCGACTGTGCCTTCTA sequence no. 78 TAATACGACTCACTATAGGGCGTCTCACTCTGATATCCAGATGACTCAGAGCCCTAGCTCTCTGAGCGCTAGCGTGGGGATAGGGTGACAATCACTTGTAGGGCCAGCCAGAACATCAACAGCTTTCTGAACTGGTATCAGCAGAAGCCCGGCAAGGCCCCTAGGAGACTGATCTATGGCACTTCCTTCTC TGCAGAGCGGCGTGCCTAGCAGATTTAGCGGCAGCGGAAGCGGCACAGACTACACTCTGACTATCAGCAGCCTCCAGCCAGAGGACTTCGCCACATACTACTGCCAGCAGTACAACAGCTGGCCTTGGACTTTCGGCCAAGGCACAAAGCTGGAGATCAAGAGAACTGAGACGCCTCGACTGTGCCTTCTA sequence no. 79 TAATACGACTCACTATAGGGCGTCTCACTCTGATATCCAGATGACACAGGCCCTAGCTCTCTGAGCGCTAGCGTGGGAGACAGAGTGACTATCACTTGCAGAGCCAGCCAGAATGTGAGCAGCTTTCTGAACTGGTATCAGCAGAAGCCCGGCGAGGCCCCTAGGAGGCTGATCTACGGCACTAACTCTC TGCAGAGCGGCGTGCCTAGCAGATTTAGCGGCAGCGGAAGCGGCACAGACTACACTCTGACTATCAGCTCTCTGCAGCCAGAGGACTTCGCCACATACTACTGCCAGCAGTACAACAGCTGGCCTTGGACTTTCGGCCAAGGCACAAAGCTGGAGATCAAGAGAACTGAGACGCCTCGACTGTGCCTTCTA sequence no.80 TAATACGACTCACTATAGGGCGTCTCACTCTGATATCCAGATGACTCAGAGCCCTAGCTCTCTGAGCGCTAGCGTGGGAGATAGGTGACTATCACATGTAGGGCCAGCCAGAACATCAGCAGCTTTCTGAACTGGTATCAGCAGAAGCCCGGCGAGGCCCCTAGGAGGCTGATCTACGGCACAAATTCTC TGCAGAGCGGCGTGCCTTCTAGGTTTTCGGCAGCGGCTCCGGCACAGACTACACTCTGACTATCAGCTCTCTGCAGCCAGAGGACTTCGCCACTTACTACTGCCAGCAGTACAACAGCTGGCCTTGGACATTCGGCCAAGGCACAAAGCTGGAGATCAAGAGAACTGAGACGCCTCGACTGTGCCTTCTA sequence no. 81 TAATACGACTCACTATAGGGCGTCTCACTCTGATATCCAGATGACACAGAGCCCTAGCTCTCTGAGCGCTAGCGTGGGAGATAGGTGACTATCACTTGCAGAGCCAGCCAGAGCATCAGCAGCTTTCTGAACTGGTATCAGCAGAAGCCCGGCGAGGCCCCTAGGAGGCTGATCTACGGCACTAACTCTC TGCAGAGCGGCGTGCCTAGCAGATTTAGCGGCAGCGGAAGCGGCACAGACTACACTCTGACTATCAGCTCTCTGCAGCCAGAGGACTTCGCCACTTACTACTGCCAGCAGTACACAAGCTGGCCTGGACTTTCGGCCAAGGCACTAAGCTGGAGATCAAGAGAACTGAGACGCCTCGACTGTGCCTTCTA sequence number82 TAATACGACTCACTATAGGGCGTCTCACTCTGATATCCAGATGACTCAGTCCCCTAGCTCTCTGAGCGCTTCCGTGGGAGATAGGGTGACTATCACTTGCAGAGCCAGCCAGAGCGTGAGCAGCTTTCTGAATTGGTATCAGCAGAAGCCCGGCAAAGCCCCTAAGAGGCTGATCTACGGCACAAACTCTC TGCAGTCCGGCGTGCCTTCTAGGTTTTCCGGCAGCGGCTCCGGCACAGACTACACTCTGACTATCAGCTCTCTGCAGCCAGAGGACTTCGCCACTTACTACTGCCAGCAGTACAACAGCTGGCCTTGGACATTCGGCCAAGGCACAAAGCTGGAGATCAAGAGAACTGAGACGCCTCGACTGTGCCTTCTA sequence number83 TAATACGACTCACTATAGGGCGTCTCACTCTGATATCCAGATGACTCAGAGCCCTAGCTCTCTGAGCGCTAGCGTGGGAGATAGGGTGACAATCACTTGTAGGGCCAGCCAGAATGTGAGCTCCTTCTGAACTGGTATCAGCAGAAGCCCGGCAAGGCTCCAAGAAGGCTGATCTATGGCACATCCTCTC TGCAGAGCGGCGTGCCAAGCAGATTCAGCGGCTCCGGCAGCGGCACAGACTACACTCTGACTATCAGCAGCCTCCAGCCAGAGGACTTCGCCACATACTACTGCCAGCAGTACACTAGCTGGCCTTGGACATTCGGCCAAGGCACAAAGCTGGAGATCAAGAGAACTGAGACGCCTCGACTGTGCCTTCTA sequence number 84 TAATACGACTCACTATAGGGCGTCTCACTCTGATATCCAGATGACTCAGAGCCCTAGCTCTCTGAGCGCTAGCGTGGGAGACAGAGTGACTATCACTTGCAGAGCCAGCCAGAGCATCAACAGCTTTCTGAACTGGTATCAGCAGAAGCCCGGCAAGGCCCCTAGGAGGCTGATCTACGGCACTAATTCTC TGCAGAGCGGCGTCCCTAGCAGATTTAGCGGCAGCGGAAGCGGCACAGACTTCACTCTGACTATCAGCTCTCTGCAGCCAGAGGACTTCGCCACATACTACTGCCAGCAGTACAACAGCTGGCCTTGGACATTCGGCCAAGGCACAAAGCTGGAGATCAAGAGAACTGAGACGCCTCGACTGTGCCTTCTA sequence number 85 TAATACGACTCACTATAGGGCGTCTCACTCTGACATCCAGATGACTCAGAGCCCTAGCTCTCTGAGCGCTAGCGTGGGAGATAGGTGACTATCACATGTAGGGCCAGCCAGAATGTGAGCAGCTTTCTGGAACTGGTATCAGCAGAAGCCCGGCAAGGCTCCTAGGAGGCTGATCTACGGCACTAACTCTC TGCAGAGCGGCGTCCCTAGCAGATTTAGCGGCAGCGGAAGCGGCACAGACTACACACTGACTATCAGCTCTCTGCAGCCAGAGGACTTCGCCACATACTACTGCCAGCAGTACAACAGCTGGCCTTGGACTTTCGGCCAAGGCACAAAGCTGGAGATCAAGAGAACTGAGACGCCTCGACTGTGCCTTCTA sequence number 86 TAATACGACTCACTATAGGGCGTCTCACTCTGATATTCAGATGACACAGAGCCCTAGCTCTCTGAGCGCTAGCGTGGGAGATAGGGTCACAATCACATGCAGAGCCAGCCAGAGCGTGAGCAGCTTTCTGGAACTGGTATCAGCAGAAGCCCGGCGAGGCCCCAAGGAGGCTGATCTACGGCACTAACTCTC TGCAGAGCGGCGTGCCAAGCAGATTTAGCGGCAGCGGAAGCGGCACAGACTACACTCTGACTATCAGCTCTCTGCAGCCAGAGGACTTCGCCACATACTACTGCCAGCAGTACACTAGCTGGCCTTGGACATTCGGCCAAGGCACTAAGCTGGAGATCAAGAGAACTGAGACGCCTCGACTGTGCCTTCTA sequence no. 87 TAATACGACTCACTATAGGGCGTCTCACTCTGACATCCAGATGACACAGAGCCCTTCCTCTCTGAGCGCTAGCGTCGGAGATAGGGTGACAATCACATGTAGGGCCAGCCAGAACATCAACAGCTTTCTGAACTGGTATCAGCAGAAGCCCGGCGAGGCTCCTAGGAGGCTGATCTACGGCACAAATTCTC TGCAGAGCGGCGTCCCTTTCTAGGTTCAGCGGATCGGCAGCGGCACTGACTACACACTGACAATCAGCTCTCTGCAGCCAGAGGACTTCGCCACTTACTACTGCCAGCAGTACACTAGCTGGCCTTGGACATTCGGCCAAGGCACAAAGCTGGAGATCAAGAGAACTGAGACGCCTCGACTGTGCCTTCTA sequence number88 TAATACGACTCACTATAGGGCGTCTCACTCTGATATCCAGATGACTCAGAGCCCTTCCTCTCTGAGCGCTAGCGTGGGGACAGAGTGACAATCACATGTAGGGCCAGCCAGAGCGTGAGCAGCTATCTGAACTGGTATCAGCAGAAGCCCGGCAAGGCTCCTAGGAGGCTGATCTACGGCACTAATTCTC TGCAGAGCGGCGTGCCAAGCAGATTTAGCGGCAGCGGAAGCGGCACAGACTACACTCTGACTATCAGCTCTCTGCAGCCAGAGGACTTCGCCACATACTACTGCCAGCAGTACACTAGCTGGCCTTGGACATTCGGCCAAGGCACAAAGCTGGAGATCAAGAGAACTGAGACGCCTCGACTGTGCCTTCTA sequence number 89 TAATACGACTCACTATAGGGCGTCTCACTCTGATATCCAGATGACTCAGAGCCCTAGCTCTCTGAGCGCTAGCGTGGGAGATAGGGTGACTATCACATGCAGAGCCAGCCAGAGCATCAACAGCTTTCTGAACTGGTATCAGCAGAAGCCCGGCGAGGCTCCTAGGAGGCTGATCTACGGCACTAATTCTC TGCAGAGCGGCGTGCCTAGCAGATTTAGCGGCAGCGGAAGCGGCACAGACTACACTCTGACTATCAGCTCTCTGCAGCCAGAGGACTTCGCCACATACTACTGCCAGCAGTACACTAGCTGGCCATGGACATTCGGCCAAGGCACTAAGCTGGAGATCAAGAGAACTGAGACGCCTCGACTGTGCCTTCTA

[0374] In one embodiment, the nucleic acid according to the present invention is - A sequence encoding the VH of the antibody or its antigen-binding fragment according to the present invention, - A sequence encoding the VL of the antibody or its antigen-binding fragment according to the present invention, It includes or consists of.

[0375] In one embodiment, the nucleic acid according to the present invention is -A sequence that includes or is selected from a group of sequences that share at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with sequence numbers 60-71, -A sequence that includes or is selected from a group of sequences that share at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with any sequence 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89 and sequence 72-89, It includes or consists of.

[0376] In one embodiment, the nucleic acid according to the present invention is A sequence encoding VH that includes or consists of the sequence of sequence number 60, A sequence that encodes a VL containing or consisting of the sequence of sequence number 72, It includes or consists of.

[0377] In one embodiment, the nucleic acid encodes VH and VL of the H07 antibody.

[0378] In one embodiment, the nucleic acid according to the present invention is A sequence encoding VH that includes or consists of the sequence of sequence number 61, A sequence that encodes a VL containing or consisting of the sequence of sequence number 73, It includes or consists of.

[0379] In one embodiment, the nucleic acid encodes VH and VL of the H09 antibody.

[0380] In one embodiment, the nucleic acid according to the present invention is A sequence encoding VH that includes or consists of the sequence of sequence number 60, A sequence that encodes a VL containing or consisting of the sequence of sequence number 74, It includes or consists of.

[0381] In one embodiment, the nucleic acid encodes VH and VL of the G02 antibody.

[0382] In one embodiment, the nucleic acid according to the present invention is A sequence encoding VH that includes or consists of the sequence of sequence number 60, A sequence that encodes a VL containing or consisting of the sequence of sequence number 75, It includes or consists of.

[0383] In one embodiment, the nucleic acid encodes the VH and VL of the E04 antibody.

[0384] In one embodiment, the nucleic acid according to the present invention is A sequence encoding VH that includes or consists of the sequence of sequence number 62, A sequence that codes for a VL containing or consisting of the sequence of sequence number 76, It includes or consists of.

[0385] In one embodiment, the nucleic acid encodes VH and VL of the D01 antibody.

[0386] In one embodiment, the nucleic acid according to the present invention is A sequence encoding VH that includes or consists of the sequence of sequence number 63, A sequence that encodes a VL containing or consisting of the sequence of sequence number 77, It includes or consists of.

[0387] In one embodiment, the nucleic acid encodes VH and VL of the E04-2 antibody.

[0388] In one embodiment, the nucleic acid according to the present invention is A sequence encoding VH that includes or consists of the sequence of sequence number 64, A sequence that codes for a VL containing or consisting of the sequence of sequence number 78, It includes or consists of.

[0389] In one embodiment, the nucleic acid encodes VH and VL of the B05 antibody.

[0390] In one embodiment, the nucleic acid according to the present invention is A sequence encoding VH that includes or consists of the sequence of sequence number 65, A sequence that encodes a VL containing or consisting of the sequence of sequence number 79, It includes or consists of.

[0391] In one embodiment, the nucleic acid encodes the VH and VL of the G09 antibody.

[0392] In one embodiment, the nucleic acid according to the present invention is A sequence encoding VH that includes or consists of the sequence of sequence number 66, A sequence that codes for a VL containing or consisting of the sequence of sequence number 80, It includes or consists of.

[0393] In one embodiment, the nucleic acid encodes VH and VL of the B01 antibody.

[0394] In one embodiment, the nucleic acid according to the present invention is A sequence encoding VH that includes or consists of the sequence of sequence number 61, A sequence that codes for a VL containing or consisting of the sequence of sequence number 81, It includes or consists of.

[0395] In one embodiment, the nucleic acid encodes the VH and VL of the CO1 antibody.

[0396] In one embodiment, the nucleic acid according to the present invention is A sequence encoding VH that includes or consists of the sequence of sequence number 61, A sequence that codes for a VL containing or consisting of the sequence of sequence number 82, It includes or consists of.

[0397] In one embodiment, the nucleic acid encodes VH and VL of the G01 antibody.

[0398] In one embodiment, the nucleic acid according to the present invention is A sequence encoding VH that includes or consists of the sequence of sequence number 61, A sequence that codes for a VL containing or consisting of the sequence of sequence number 83, It includes or consists of.

[0399] In one embodiment, the nucleic acid encodes VH and VL of the H01 antibody.

[0400] In one embodiment, the nucleic acid according to the present invention is A sequence encoding VH that includes or consists of the sequence of sequence number 63, A sequence that codes for a VL containing or consisting of the sequence of sequence number 84, It includes or consists of.

[0401] In one embodiment, the nucleic acid encodes VH and VL of the G02-2 antibody.

[0402] In one embodiment, the nucleic acid according to the present invention is A sequence encoding VH that includes or consists of the sequence of sequence number 71, A sequence that encodes a VL containing or consisting of the sequence of sequence number 79, It includes or consists of.

[0403] In one embodiment, the nucleic acid encodes VH and VL of the H02 antibody.

[0404] In one embodiment, the nucleic acid according to the present invention is A sequence encoding VH that includes or consists of the sequence of sequence number 67, A sequence that encodes a VL containing or consisting of the sequence of sequence number 85, It includes or consists of.

[0405] In one embodiment, the nucleic acid encodes the VH and VL of the F03 antibody.

[0406] In one embodiment, the nucleic acid according to the present invention is A sequence encoding VH that includes or consists of the sequence of sequence number 68, A sequence that codes for a VL containing or consisting of the sequence of sequence number 86, It includes or consists of.

[0407] In one embodiment, the nucleic acid encodes VH and VL of the D05 antibody.

[0408] In one embodiment, the nucleic acid according to the present invention is A sequence encoding VH that includes or consists of the sequence of sequence number 61, A sequence that codes for a VL containing or consisting of the sequence of sequence number 87, It includes or consists of.

[0409] In one embodiment, the nucleic acid encodes VH and VL of the B07 antibody.

[0410] In one embodiment, the nucleic acid according to the present invention is A sequence encoding VH that includes or consists of the sequence of sequence number 69, A sequence that codes for a VL containing or consisting of the sequence of sequence number 88, It includes or consists of.

[0411] In one embodiment, the nucleic acid encodes VH and VL of the H08 antibody.

[0412] In one embodiment, the nucleic acid according to the present invention is A sequence encoding VH that includes or consists of the sequence of sequence number 70, A sequence that codes for a VL containing or consisting of the sequence of sequence number 89, It includes or consists of.

[0413] In one embodiment, the nucleic acid encodes VH and VL of the B12 antibody.

[0414] In one embodiment, VH and / or VL preferably further include a leader sequence located 5' from the VH nucleic acid sequence or 5' from the VL nucleic acid sequence, respectively. Examples of leader sequences include, but are not limited to, sequence numbers 58 and 59, which are coded by sequence numbers 90 and 91, respectively. Sequence ID 90 ATGGACATCAGGCTCAGCTTGGCTTTCCTTGTCCTTTTCATAAAAGGTGTCCAGTGT Sequence ID 91 ATGGCTGCAGTTCAACTCTTAGGGCTGCTGCTGCTTTGGCTCCCAGCCATGAGATGT

[0415] In one embodiment, VH includes a nucleic acid reader sequence, SEQ ID NO: 90, located at 5' from the VH coding nucleic acid sequence (e.g., SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, or SEQ ID NO: 71).

[0416] In one embodiment, the VL includes a nucleic acid sequence reader sequence, SEQ ID NO: 91, located at 5' from the nucleic acid sequence encoding the VL (e.g., SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, or SEQ ID NO: 89).

[0417] In one embodiment, the nucleic acid according to the present invention is a complete or substantially complete human C2 antibody or antigen-binding fragment thereof according to the present invention. H and / or C L It includes a sequence encoding a constant region. In such embodiments, the constant region may be derived from any human antibody constant region.

[0418] In one embodiment, the nucleic acid according to the present invention is a complete or substantially complete mouse C antibody or antigen-binding fragment thereof according to the present invention. H and / or C L It includes a sequence encoding a constant region. In such embodiments, the constant region may originate from any mouse antibody constant region.

[0419] In one embodiment, the nucleic acid according to the present invention comprises or consists of a sequence encoding the heavy chain of the chimeric antibody or its antigen-binding fragment according to the present invention. In one embodiment, the nucleic acid according to the present invention comprises or consists of a sequence encoding the light chain of the chimeric antibody or its antigen-binding fragment according to the present invention.

[0420] In one embodiment, the nucleic acid according to the present invention comprises or consists of a sequence encoding the heavy chain of the humanized antibody or its antigen-binding fragment according to the present invention. In one embodiment, the nucleic acid according to the present invention comprises or consists of a sequence encoding the light chain of the humanized antibody or its antigen-binding fragment according to the present invention.

[0421] Typically, the nucleic acid is a DNA or RNA molecule that can be contained in any suitable vector, such as a plasmid, cosmid, episome, artificial chromosome, phage, or viral vector.

[0422] Therefore, another object of the present invention is an expression vector comprising a nucleic acid encoding a protein, antibody, or antigen-binding fragment thereof according to the present invention. Another object of the present invention is an expression vector comprising a nucleic acid encoding a fusion protein according to the present invention.

[0423] The terms “vector,” “cloning vector,” and “expression vector” refer to a vehicle capable of introducing a DNA or RNA sequence (e.g., a foreign gene) into host cells in order to transform the host and promote the expression (e.g., transcription and translation) of the introduced sequence. Such vectors may include regulatory elements, such as promoters, enhancers, and terminators, to induce or direct the expression of the protein, antibody, or its antigen-binding fragment or fusion protein upon administration to the host. Examples of promoters and enhancers used in animal cell expression vectors include the SV40 initial promoter and enhancer, the Moloney mouse leukemia virus LTR promoter and enhancer, and the immunoglobulin H chain promoter and enhancer. Any expression vector for animal cells can be used as long as it can insert and express a gene encoding a protein, antibody, or its fragment or fusion protein. Examples of suitable vectors include pAGE107, pAGE103, pHSG274, pKCR, and pSG1βd2-4. Other examples of plasmids include replication plasmids containing origins of replication, or integration plasmids such as pUC, pcDNA, and pBR. Other examples of viral vectors include adenovirus, retrovirus, herpesvirus, and AAV vectors. Such recombinant viruses can be produced by techniques known in the art, for example, by transfecting packaging cells or by transient transfection with helper plasmids or viruses. Typical examples of viral packaging cells include PA317 cells, PsiCRIP cells, GPenv+ cells, and 293 cells. Detailed protocols for producing such replication-deficient recombinant viruses can be found in the art.

[0424] In one embodiment, the expression vector according to the present invention includes a sequence encoding the VH of the antibody or its antigen-binding fragment according to the present invention, which is operably linked to a regulatory element.

[0425] In one embodiment, the expression vector according to the present invention includes the sequences of SEQ ID NOs. 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, and 71, operably linked to a regulatory element, or any sequence that shares at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more identity with SEQ ID NOs. 60-71.

[0426] In one embodiment, the expression vector according to the present invention includes a sequence encoding a VL of the antibody or its antigen-binding fragment according to the present invention, which is operably linked to a regulatory element.

[0427] In one embodiment, the expression vector according to the present invention includes the sequences of SEQ ID NOs. 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, and 89, which are operably linked to a regulatory element, or any sequence that shares at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NOs. 72-89.

[0428] In one embodiment, the expression vector according to the present invention is - A sequence encoding the VH of the antibody or antigen-binding fragment thereof according to the present invention, operably linked to a regulatory element, - A sequence encoding the VL of the antibody or its antigen-binding fragment according to the present invention, operably linked to a regulatory element, Includes.

[0429] In one embodiment, the expression vector according to the present invention is - An array encoding VH which includes or consists of any arrays that share at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with arrays 60-71, operably linked to the adjustment element, - An array encoding a VL containing or consisting of any arrays that share at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with arrays 72-89, operably linked to the adjustment element, Includes.

[0430] In one embodiment, the expression vector according to the present invention is - An array encoding VH, which includes or consists of the array of sequence number 60, operably linked to the adjustment element, A sequence encoding a VL containing or consisting of the sequence of sequence number 72, which is operably linked to the adjustment element, Includes.

[0431] In one embodiment, the expression vector according to the present invention is - An array encoding VH, which includes or consists of the array of sequence number 61, operably coupled to the adjustment element, An array encoding a VL containing or consisting of the array of sequence number 73, which is operably linked to the adjustment element, Includes.

[0432] In one embodiment, the expression vector according to the present invention is - An array encoding VH, which includes or consists of the array of sequence number 60, operably linked to the adjustment element, An array encoding a VL containing or consisting of the array of sequence number 74, which is operably linked to the adjustment element, Includes.

[0433] In one embodiment, the expression vector according to the present invention is - An array encoding VH, which includes or consists of the array of sequence number 60, operably linked to the adjustment element, A sequence encoding a VL containing or consisting of the sequence of sequence number 75, which is operably linked to the adjustment element, Includes.

[0434] In one embodiment, the expression vector according to the present invention is - An array encoding VH, which includes or consists of the array of sequence number 62, operably coupled to the adjustment element, An array encoding a VL containing or consisting of the array of sequence number 76, which is operably linked to the adjustment element, Includes.

[0435] In one embodiment, the expression vector according to the present invention is - An array encoding VH, which includes or consists of the array of sequence number 63, operably coupled to the adjustment element, An array encoding a VL containing or consisting of the array of sequence number 77, which is operably linked to the adjustment element, Includes.

[0436] In one embodiment, the expression vector according to the present invention is - An array encoding VH, which includes or consists of the array of sequence number 64, operably linked to the adjustment element, An array encoding a VL containing or consisting of the array of sequence number 78, which is operably coupled to the adjustment element, Includes.

[0437] In one embodiment, the expression vector according to the present invention is - An array encoding VH, which includes or consists of the array of sequence number 65, operably coupled to the adjustment element, A sequence encoding a VL containing or consisting of the sequence of sequence number 79, which is operably linked to the adjustment element, Includes.

[0438] In one embodiment, the expression vector according to the present invention is - An array encoding VH, which includes or consists of the array of sequence number 66, operably coupled to the adjustment element, A sequence encoding a VL containing or consisting of the sequence of sequence number 80, which is operably linked to the adjustment element, Includes.

[0439] In one embodiment, the expression vector according to the present invention is - An array encoding VH, which includes or consists of the array of sequence number 61, operably coupled to the adjustment element, A sequence encoding a VL containing or consisting of the sequence of sequence number 81, which is operably coupled to the adjustment element, Includes.

[0440] In one embodiment, the expression vector according to the present invention is - An array encoding VH, which includes or consists of the array of sequence number 61, operably coupled to the adjustment element, A sequence encoding a VL containing or consisting of the sequence of sequence number 82, which is operably linked to the adjustment element, Includes.

[0441] In one embodiment, the expression vector according to the present invention is - An array encoding VH, which includes or consists of the array of sequence number 61, operably coupled to the adjustment element, A sequence encoding a VL containing or consisting of the sequence of sequence number 83, which is operably coupled to the adjustment element, Includes.

[0442] In one embodiment, the expression vector according to the present invention is - An array encoding VH, which includes or consists of the array of sequence number 63, operably coupled to the adjustment element, An array encoding a VL containing or consisting of the array of sequence number 84, which is operably linked to the adjustment element, Includes.

[0443] In one embodiment, the expression vector according to the present invention is - An array encoding VH, which includes or consists of the array of sequence number 71, operably coupled to the adjustment element, A sequence encoding a VL containing or consisting of the sequence of sequence number 79, which is operably linked to the adjustment element, Includes.

[0444] In one embodiment, the expression vector according to the present invention is - An array encoding VH, which includes or consists of the array of sequence number 67, operably coupled to the adjustment element, A sequence encoding a VL containing or consisting of the sequence of sequence number 85, which is operably linked to the adjustment element, Includes.

[0445] In one embodiment, the expression vector according to the present invention is - An array encoding VH, which includes or consists of the array of sequence number 68, operably coupled to the adjustment element, A sequence encoding a VL containing or consisting of the sequence of sequence number 86, which is operably coupled to the adjustment element, Includes.

[0446] In one embodiment, the expression vector according to the present invention is - An array encoding VH, which includes or consists of the array of sequence number 61, operably coupled to the adjustment element, An array encoding a VL containing or consisting of the array of sequence number 87, which is operably coupled to the adjustment element, Includes.

[0447] In one embodiment, the expression vector according to the present invention is - An array encoding VH, which includes or consists of the array of sequence number 69, operably coupled to the adjustment element, A sequence encoding a VL containing or consisting of the sequence of sequence number 88, which is operably coupled to the adjustment element, Includes.

[0448] In one embodiment, the expression vector according to the present invention is - An array encoding VH, which includes or consists of the array of sequence number 70, operably connected to the adjustment element, A sequence encoding a VL containing or consisting of the sequence of sequence number 89, which is operably coupled to the adjustment element, Includes.

[0449] In one embodiment, the expression vector according to the present invention comprises a C of the antibody or its antigen-binding fragment according to the present invention operably linked to a regulatory element. H Includes an array that codes for the C H This is any human antibody C H It may originate from this.

[0450] In one embodiment, the expression vector according to the present invention comprises a C of the antibody or its antigen-binding fragment according to the present invention operably linked to a regulatory element. L Includes an array that codes for the C L This is any human antibody C L It may originate from this.

[0451] In one embodiment, the expression vector according to the present invention comprises a C of the antibody or its antigen-binding fragment according to the present invention operably linked to a regulatory element. H Includes an array that codes for the C H This is any mouse antibody C H It may originate from this.

[0452] In one embodiment, the expression vector according to the present invention comprises a C of the antibody or its antigen-binding fragment according to the present invention operably linked to a regulatory element. L Includes an array that codes for the C L This is any mouse antibody C L It may originate from this.

[0453] In one embodiment, the expression vector according to the present invention includes a sequence encoding the heavy chain of a chimeric antibody or its antigen-binding fragment according to the present invention, which is operably linked to a regulatory element.

[0454] In one embodiment, the expression vector according to the present invention includes a sequence encoding a light chain of a chimeric antibody or its antigen-binding fragment according to the present invention, which is operably linked to a regulatory element.

[0455] In one embodiment, the expression vector according to the present invention includes a sequence encoding the heavy chain of a humanized antibody or its antigen-binding fragment according to the present invention, which is operably linked to a regulatory element.

[0456] In one embodiment, the expression vector according to the present invention includes a sequence encoding a light chain of a humanized antibody or its antigen-binding fragment according to the present invention, which is operably linked to a regulatory element.

[0457] In one embodiment, the expression vector according to the present invention is monocistronic.

[0458] "Monocistronic" means that a single nucleic acid is expressed using a single expression vector.

[0459] In one embodiment, the expression vector according to the present invention is polycistronic.

[0460] "Polycistronic" means that at least two or more nucleic acids are expressed in a single expression vector.

[0461] Another object of the present invention is an isolated host cell containing the vector. The host cell can be used for recombinant production of the protein, antibody or its antigen-binding fragment, or fusion protein of the present invention.

[0462] In one embodiment, the host cells are prokaryotes, yeasts, or eukaryotic cells, preferably mammalian cells, such as the monkey kidney CV1 strain transformed with SV40 (COS-7, ATCC CRL 1651); human fetal kidney lineage (293 or 293 cells subcloned for growth in suspension culture; Graham et al., J. Gen. Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); mouse myeloma cells SP2 / 0-AG14 (ATCC CRL 1581; ATCC CRL This could include 8287) or NSO (HPA culture collection number 85110503); monkey kidney cells (CV1 ATCC CCL70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical cancer cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat hepatocytes (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocytes (Hep G2, HB 8065); mouse mammary tumor cells (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals NYAcad.Sci.383:44-68 (1982)); MRC5 cells; FS4 cells; and human hepatocarcinoma cell line (Hep G2), as well as the PERC-6 cell line of DSM. Expression vectors suitable for use in each of these host cells are also generally known in the art. It should be noted that the term “host cell” generally refers to a cultured cell line. In one embodiment, the entire human being into which an expression vector encoding a protein, antibody or its antigen-binding fragment, or fusion protein according to the present invention has been introduced is excluded from the definition of “host cell.”

[0463] Another object of the present invention is a method for producing and purifying isolated proteins of the present invention, in particular antibodies or antigen-binding fragments thereof as described herein.

[0464] In one embodiment, the method is -Introducing recombinant nucleic acids or vectors as described above into competent host cells in vitro or ex vivo, -Culturing host cells transformed with nucleic acids or expression vectors in vitro or ex vivo under conditions suitable for the expression of the protein of the present invention, particularly antibodies or their antigen-binding fragments, - If necessary, select cells that express and / or secrete the protein, - To recover the expressed protein (especially the expressed antibody or its antigen-binding fragment), Includes.

[0465] This recombination process can be used, for example, for the large-scale production of proteins such as antibodies or their antigen-binding fragments, including monoclonal antibodies intended for in vitro, ex vivo, and / or in vivo therapeutic applications.

[0466] In one embodiment, the expressed protein, particularly the expressed antibody or its antigen-binding fragment, is further purified.

[0467] Methods for purifying proteins, particularly antibodies or their antigen-binding fragments, are well known in the art and include, but are not limited to, affinity chromatography on protein A-Sepharose, gel electrophoresis, chromatography, preferably affinity chromatography, and more preferably affinity chromatography on protein L-agarose.

[0468] Another object of the present invention is a composition comprising, essentially, the same as, at least one protein that binds to human CD25 (hCD25) according to the present invention, in particular at least one antibody that binds to human CD25 (hCD25) as described herein, or at least one antigen-binding fragment of said antibody.

[0469] Another object of the present invention is a composition comprising, essentially comprising, or consisting of at least one fusion protein according to the present invention.

[0470] Another object of the present invention is a composition comprising, essentially comprising, or consisting of, at least one nucleic acid encoding a protein, antibody, or antigen-binding fragment of said antibody, or a fusion protein according to the present invention.

[0471] Another object of the present invention is a composition comprising, essentially, or consisting of at least one expression vector comprising at least one nucleic acid encoding a protein, antibody, or antigen-binding fragment or fusion protein of said antibody according to the present invention.

[0472] Another object of the present invention is a pharmaceutical composition comprising, essentially comprising, or comprising at least one protein that binds to hCD25 according to the present invention, in particular at least one antibody that binds to human CD25 (hCD25) as described herein or at least one antigen-binding fragment of said antibody, and at least one pharmaceutically acceptable excipient.

[0473] Another object of the present invention is a pharmaceutical composition comprising, essentially comprising, or consisting of at least one fusion protein according to the present invention and at least one pharmaceutically acceptable excipient.

[0474] Another object of the present invention is a pharmaceutical composition comprising, essentially comprising, or comprising at least one nucleic acid encoding a protein of the present invention, in particular an antibody that binds to human CD25 (hCD25) as described herein, or an antigen-binding fragment of said antibody, or a fusion protein according to the present invention, and at least one pharmaceutically acceptable excipient.

[0475] Another object of the present invention is a pharmaceutical composition comprising, essentially comprising, or comprising at least one expression vector comprising at least one nucleic acid encoding a protein of the present invention, in particular an antibody bound to human CD25 (hCD25) as described herein, or an antigen-binding fragment of said antibody, or a fusion protein according to the present invention, and at least one pharmaceutically acceptable excipient.

[0476] As used herein, "essentially consisting of" a composition means that at least one protein, antibody or its antigen-binding fragment, fusion protein, nucleic acid, or expression vector is the only therapeutic agent or drug having biological activity within the composition.

[0477] The term "pharmaceutically acceptable excipients" includes all solvents, dispersions, coatings, antimicrobial and antifungal agents, isotonic agents and absorption retarders, etc. Such excipients do not cause adverse allergic reactions or other undesirable reactions when administered to animals, preferably mammals, and more preferably humans. For administration to humans, preparations should meet the sterility, pyrogenicity, and general safety and purity standards required by regulatory authorities, such as the FDA Office or EMA.

[0478] Examples of pharmaceutically acceptable excipients that may be used in the compositions of the present invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, e.g., human serum albumin, buffers, e.g., phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, e.g., protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulosic substances (e.g., sodium carboxymethylcellulose), polyethylene glycol, polyacrylate, wax, polyethylene-polyoxypropylene-block polymer, polyethylene glycol, and lanolin.

[0479] In one embodiment, the pharmaceutical composition according to the present invention comprises a pharmaceutically acceptable vehicle for a formulation that can be injected into a subject. These may be dry compositions, particularly lyophilized compositions, that enable the formation of an injectable solution by adding isotonic sterile saline (monosodium or disodium phosphate, sodium chloride, potassium, calcium or magnesium, etc., or a mixture of such salts), or optionally sterile water or saline.

[0480] Another object of the present invention is a pharmaceutical product comprising, or comprising, at least one protein that binds to hCD25 according to the present invention, in particular at least one antibody that binds to human CD25 (hCD25) as described herein, or at least one antigen-binding fragment of said antibody.

[0481] Another object of the present invention is a pharmaceutical product comprising, essentially comprising, or comprising at least one fusion protein according to the present invention.

[0482] Another object of the present invention is a pharmaceutical product comprising, essentially comprising, a protein of the present invention, an antibody that binds to human CD25 (hCD25) as described herein, an antigen-binding fragment of said antibody, or at least one nucleic acid encoding a fusion protein according to the present invention.

[0483] Another object of the present invention is a pharmaceutical product comprising, essentially comprising, a pharmaceutical product, a pharmaceutical product comprising

[0484] For use in administration to a subject, a composition, pharmaceutical composition, or drug is formulated for administration to the subject.

[0485] In one embodiment, the composition, pharmaceutical composition, or pharmaceutical according to the present invention is administered (or will be administered) parenterally, by inhalation spray, rectally, nasally, or via an implanted reservoir.

[0486] In one embodiment, the composition, pharmaceutical composition, or pharmaceutical is administered (or will be administered) by injection, including but not limited to subcutaneous, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrathecal, intrahepatic, intralesional, and intracranial injection or infusion techniques.

[0487] Examples of forms suitable for injection include, but are not limited to, solutions such as sterile aqueous solutions, gels, dispersions, emulsions, and suspensions, and solid forms suitable for use in preparing solutions or suspensions by adding liquid before use, such as powders and liposomes.

[0488] The compositions, pharmaceutical compositions, or sterile injection forms of the present invention may be aqueous or oily suspensions. These suspensions can be formulated according to techniques known in the art using appropriate dispersants or wetting agents and suspending agents. The sterile injection preparations may also be sterile injection solutions or suspensions in non-toxic, parenterally acceptable diluents or solvents. Acceptable vehicles and solvents that may be used include water, Ringer's solution, and isotonic sodium chloride solution. Furthermore, sterile fixative oils have been conventionally used as solvents or suspension media. For this purpose, any sterile fixative oil containing synthetic monoglycerides or diglycerides can be used. Fatty acids such as oleic acid and its glyceride derivatives are useful in the preparation of injections, as are naturally pharmaceutically acceptable oils such as olive oil or castor oil, particularly their polyoxyethylated versions. These oil solutions or suspensions may also contain long-chain alcohol diluents or dispersants, such as similar dispersants commonly used in formulations of pharmaceutically acceptable dosage forms including carboxymethylcellulose or emulsions and suspensions. Other commonly used surfactants, such as Tween, Spans, and other emulsifiers or bioavailability enhancers, which are commonly used in the manufacture of pharmaceutically acceptable solids, liquids, or other dosage forms, may also be used for formulation purposes.

[0489] In one embodiment, an isolated protein, isolated antibody or its antigen-binding fragment, fusion protein, nucleic acid, expression vector, composition, pharmaceutical composition, or pharmaceutical according to the present invention is to be administered in a therapeutically effective amount to a subject in need.

[0490] However, it will be understood that the total daily dose of isolated proteins, isolated antibodies or their antigen-binding fragments, fusion proteins, nucleic acids, expression vectors, compositions, pharmaceutical compositions, or medicinal products according to the present invention is to be determined by the attending physician within the bounds of sound medical judgment. The specific therapeutically effective dose level for any particular patient will depend on a variety of factors, including: the disease being treated and its severity; the activity of the isolated protein, isolated antibody or its antigen-binding fragment, fusion protein, nucleic acid, or expression vector used; the subject's age, weight, overall health, sex, and diet; the timing, route of administration, and excretion rate of the specific isolated protein, isolated antibody or its antigen-binding fragment, fusion protein, nucleic acid, or expression vector used; the duration of treatment; drugs used in combination with or simultaneously with the specific isolated protein, isolated antibody or its antigen-binding fragment, fusion protein, nucleic acid, or expression vector used; and similar factors well known in the medical field. For example, it is well within the scope of the art to start with a dose of the compound at a level lower than necessary to achieve the desired therapeutic effect and gradually increase the dose until the desired effect is achieved. The total dose required for each treatment may be administered in multiple doses or single doses.

[0491] The daily dose of proteins, antibodies or their antigen-binding fragments, fusion proteins, nucleic acids, or expression vectors can vary widely from about 0.01 to about 1000 mg / day per adult. Compositions may contain about 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250, and about 500 mg of the active ingredient for symptomatic dose adjustments to the target being treated. Pharmaceutical compositions or drugs typically contain about 0.01 mg to about 500 mg of the active ingredient. A therapeutically effective dose of a drug is usually supplied at a dose level of about 0.0002 mg / kg to about 20 mg / kg body weight / day. For example, the proteins, antibodies or their antigen-binding fragments, fusion proteins, nucleic acids, or expression vectors present in the compositions, pharmaceutical compositions, or pharmaceuticals of the present invention can be supplied at concentrations ranging from about 1 mg / mL to about 100 mg / mL, for example, at concentrations of about 1 mg / mL, 5 mg / mL, 10 mg / mL, 50 mg / mL, or about 100 mg / mL. In one embodiment, the protein, antibody or its antigen-binding fragment, fusion protein, nucleic acid, or expression vector is supplied at a concentration of about 10 mg / mL in either a 100 mg (10 mL) or 500 mg (50 mL) single-use vial. These dosages are illustrative, and it should be understood that the optimal dosage may be adapted considering the affinity and tolerability of the specific protein, antibody or antigen-binding fragment, fusion protein, nucleic acid, or expression vector, which must be determined in clinical trials.

[0492] In one embodiment, the protein, antibody or its antigen-binding fragment, fusion protein, nucleic acid, or expression vector of the present invention should be administered to an adult at a dose level of approximately 0.01 mg, 0.05 mg, 0.1 mg, 0.5 mg, 1 mg, 5 mg, 10 mg, 50 mg, 100 mg, 500 mg, or approximately 1000 mg / day.

[0493] The present invention relates to at least one isolated protein as described herein, and more particularly to at least one antibody conjugated to human CD25 (hCD25) as described herein, or at least one antigen-binding fragment of such antibody, for use as a pharmaceutical, i.e., for treating (or for use in treatment of) a disease, disorder or symptom in a subject that requires it.

[0494] The present invention relates to at least one fusion protein described herein for use as a pharmaceutical, that is, for treating (or for use in treatment of) a target disease, disorder, or symptom requiring such treatment.

[0495] The present invention relates to at least one nucleic acid described herein for use as a pharmaceutical, that is, for treating (or for use in the treatment of) a target disease, disorder or symptom requiring such treatment.

[0496] The present invention relates to at least one expression vector described herein for use as a pharmaceutical, that is, for treating (or for use in treatment of) a target disease, disorder, or symptom requiring such treatment.

[0497] The present invention relates to compositions, pharmaceutical compositions, or pharmaceuticals, as described herein, for use in the treatment of diseases, disorders, or symptoms in subjects requiring treatment of diseases, disorders, or symptoms.

[0498] Accordingly, the present invention further relates to a method for treating a disease, disorder, or symptom in a subject requiring treatment of the disease, disorder, or symptom, and includes administering an isolated protein (particularly an antibody or its antigen-binding fragment), fusion protein, nucleic acid or vector, or composition, pharmaceutical composition or drug as described herein to the subject.

[0499] Examples of diseases that can be treated in this invention include, but are not limited to, cancer and infectious diseases.

[0500] In one embodiment, an isolated protein, antibody or its antigen-binding fragment, fusion protein, nucleic acid, or vector according to the present invention may be used to treat cancer in a subject requiring cancer treatment.

[0501] In one embodiment, a therapeutically effective amount of the protein, antibody, or its antigen-binding fragment, fusion protein, nucleic acid, or expression vector is administered to the subject, or will be administered to the subject.

[0502] As used herein, the term "cancer" has its general meaning in the art and includes, but is not limited to, solid tumors and hematological tumors. The term cancer includes, but is not limited to, diseases of the skin, tissues, organs, bones, cartilage, blood, and blood vessels. The term "cancer" further encompasses both primary and metastatic cancers.

[0503] Examples of cancers that can be treated by the methods and compositions of the present invention include, but are not limited to, cancer cells originating from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestinal tract, gums, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, endometrium, pancreas, or uterus.

[0504] Furthermore, cancer may be selected from the following non-restrictive list: malignant neoplasms; undifferentiated carcinoma; giant cell carcinoma and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; trichomalomatous carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; malignant gastrinoma; cholangiocarcinoma; hepatocellular carcinoma; mixed hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyps; adenocarcinoma associated with familial adenomatous polyposis; solid tumors; malignant carcinoid tumors; branch alveolar adenocarcinoma; papillary adenocarcinoma; pigmentaphobe carcinoma; eosinophilic carcinoma; eosinophilic adenocarcinoma; basophilic carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma Papillary and follicular adenocarcinoma; non-encapsulated sclerosing carcinoma; adrenal cortical carcinoma; endometrial carcinoma; cutaneous adnexal carcinoma; apocrine gland carcinoma; sebaceous gland carcinoma; otogonial gland carcinoma; mucosal epidermal carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; invasive ductal carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Paget's disease of the breast; acinar cell carcinoma; adenosquamous cell carcinoma; adenocarcinoma with squamous metaplasia; malignant thymoma; malignant ovarian stromal tumor; malignant medullary tumor; malignant granulosa cell tumor; malignant blastoma; Sertoli cell carcinoma; malignant Leydig cell tumor; malignant lipid cell tumor; malignant paraganglioma; malignant extramammary paraganglioma Pheochromocytoma; mammary angiosarcoma; malignant melanoma; chromosomal melanoma; superficial spreading melanoma; malignant melanoma in giant pigmented nevi; epithelial-like cell melanoma; malignant blue nevus; sarcoma; fibrosarcoma; malignant fibrous histiocytoma; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; fetal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; malignant mixed tumor; Müller mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; malignant mesenchymal tumor; malignant Brenner tumor; malignant phyllodes tumor; synovial sarcoma; malignant mesothelioma; undifferentiated germ cell tumor; embryonic carcinoma; malignant teratoma; malignant ovarian stroma; choriocarcinoma; malignant mesonephroma; angiosarcoma; malignant hemangioendothelioma; Posi sarcoma; malignant periangiocarcinoma; lymphangiosarcoma; osteosarcoma; paracortical osteosarcoma; chondrosarcoma; malignant chondroblastoma; mesenchymal chondrosarcoma; giant cell tumor of bone; Wing sarcoma; malignant odontogenic tumor; ameloblastic gingivoma; malignant ameloblastoma; ameloblastofibrosarcoma; malignant pineal gland; chordae tendineae; malignant glioma; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrous astrocytoma; astrocytoma; glioblastoma; oligodendroglioma; oligodendroblastoma; protoneurectoderm; cerebellar sarcoma; ganglioblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; malignant meningioma; neurofibrosarcoma; malignant schwannoma;Malignant granular cell tumor; malignant lymphoma; Hodgkin's disease; Hodgkin's lymphoma; paragranuloma; malignant lymphoma - small lymphocytic; malignant diffuse large cell lymphoma; malignant follicular lymphoma; mycosis fungoides; other certain non-Hodgkin lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative bowel disease; leukemia; lymphocytic leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia.

[0505] In another embodiment, isolated proteins, antibodies or antigen-binding fragments thereof, fusion proteins, nucleic acids or expression vectors according to the present invention may be used to treat infectious diseases, disorders or symptoms thereof in subjects requiring treatment of infectious diseases, disorders or symptoms thereof.

[0506] In one embodiment, a therapeutically effective amount of the protein, antibody or its antigen-binding fragment, fusion protein, nucleic acid, or expression vector of the present invention is administered to or will be administered to a subject.

[0507] As used herein, the term “infection” includes any infection caused by a virus, bacteria, protozoa, mold, or fungus.

[0508] In some embodiments, the viral infection involves infection with one or more viruses selected from the group including, but not limited to, viruses of the families Arenaviridae, Astroviridae, Birnaviridae, Bromoviridae, Bunyaviridae, Caliciviridae, Closteroviridae, Comoviridae, Cystoviridae, Flaviviridae, Flexiviridae, Hepevirus, Leviviridae, Luteoviridae, Mononegaviridae, Mosaicviridae, Nidoviridae, Nodaviridae, Orthomyxoviridae, Picovirnavirus, Picornaviridae, Pochiviridae, Reoviridae, Retroviridae, Sekiviridae, Tenuviridae, Togaviridae, Tombusviridae, Tochiviridae, Timoviridae, Hepadnaviridae, Herpesviridae, Paramyxoviridae, or Papillomaviridae. Related taxonomic families of RNA viruses include, but are not limited to, viruses of the Astroviridae, Birnaviridae, Bromoviridae, Caliciviridae, Closteroviridae, Comoviridae, Cystoviridae, Flaviviridae, Flexiviridae, Hepevirus, Leviviridae, Luteoviridae, Mononegaviridae, Mosaicviridae, Nidoviridae, Nodaviridae, Orthomyxoviridae, Picovirnavirus, Picornaviridae, Pochiviridae, Reoviridae, Retroviridae, Sekiviridae, Tenuviridae, Togaviridae, Tombusviridae, Tochiviridae, and Timoviridae families.

[0509] In some embodiments, viral infections include adenovirus, Alfivirus, Vanzivirus, Bovine diarrhea virus, coronavirus, Coxsackievirus, Crimean-Congo virus, Dengue virus, Ebola virus, Encephalitis virus (including Japanese encephalitis virus, California encephalitis virus, Venezuelan horse encephalitis virus, Western horse encephalitis virus, Eastern horse encephalitis virus, St. Louis encephalitis virus, and tick-borne encephalitis virus), Guanalitovirus, Hantavirus, Hepatitis virus, Ilhaeus virus, Immunodeficiency virus, Influenza viruses including influenza A and B viruses (including human, avian, and swine) and parainfluenza viruses, Junin virus, Cocobella virus, Kunzin virus, Kyasanur Forest disease virus, Lacrosse virus, Lassa virus, Looping disease virus, Lymphocytic choriomeningitis virus, Measles virus, Machupo virus, Marburg virus, Malay Valley virus, Pakinda virus, Pikinde virus, Poliovirus, Poissant virus, Punta This includes infection by one or more viruses selected from the group including, but not limited to, Toro virus, respiratory syncytial virus, rhinovirus, Rift Valley fever virus, rosiovirus, severe acute respiratory syndrome (SARS), smallpox virus, Takaribe virus, West Nile virus, and yellow fever virus.

[0510] Examples of bacterial infections that can be treated by the present invention include, but are not limited to, infections caused by: Staphylococcus; Streptococcus, including S. pyogenes; Enterococci; Bacillus, including, for example, Bacillus anthracis and Lactobacillus; Listeria; Corynebacterium diphtheriae; Gardnerella, such as G. vaginalis; Nocardia; Streptomyces; Thermoactinomyces vulgaris vulgaris); Treponema; Campylobacter (e.g., P. aeruginosa), Pseudomonas; Legionella; Neisseria (e.g., N. gonorrhoeae and N. meningitides), Flavobacterium (e.g., F. meningosepticum and F. odoraturn), Brucella la); Bordetella such as B. pertussis and B. bronchiseptica; Escherichia such as E. coli and Klebsiella; Enterobacter such as S. marcescens and S. liquefaciens, Serratia; Edwardsiella; P. mirabilis and P. vulgaris (P.Proteus (e.g., *Proteus vulgaris*); Streptobacillus; Rickettsiaceae (e.g., *R. fickettsfi*), Chlamydia (e.g., *Chlamydia psittaci* and *Chlamydia trachornatis*); M. tuberculosis, M. intracellulare Mycobacterium species such as M. intracellulare, M. folluiturn, M. laprae, M. avium, M. bovis, M. africanum, M. kansasii, and M. lepraernurium; as well as Nocardia.

[0511] Examples of protozoan infections that can be treated in this invention include, but are not limited to, infections caused by leishmania, coczidiosa, and trypanosoma.

[0512] A complete list of infectious diseases can be found on the National Center for Infectious Diseases (NCID) website of the Center for Disease Control (CDC) (www.cdc.gov / ncidod / diseases / ), which is incorporated herein by reference. All of these diseases are candidates for treatment using the isolated proteins, antibodies or their antigen-binding fragments, fusion proteins, nucleic acids, expression vectors, compositions, pharmaceutical compositions, or medicinal products according to the present invention.

[0513] In one embodiment, the isolated protein (particularly an antibody or its antigen-binding fragment), fusion protein, nucleic acid, or expression vector according to the present invention is used alone.

[0514] In another embodiment, the isolated protein (particularly an antibody or its antigen-binding fragment), fusion protein, nucleic acid, or expression vector according to the present invention is used in combination with at least one further therapeutic agent.

[0515] In one embodiment, the administration of at least one further therapeutic agent and an isolated protein (particularly an antibody or its antigen-binding fragment), fusion protein, nucleic acid, or expression vector according to the present invention may be simultaneous, separate, or sequential.

[0516] In one embodiment, for co-administration, at least one further therapeutic agent and the isolated proteins (particularly antibodies or their antigen-binding fragments), fusion proteins, nucleic acids, or expression vectors according to the present invention are administered, as a single composition or as separate compositions, as necessary.

[0517] Further examples of therapeutic agents include, but are not limited to, chemotherapeutic agents, targeted cancer therapies, radiotherapy, immunotherapeutic agents or anti-cancer immunogens, anti-cancer antibodies, cytotoxic agents, anti-angiogenic agents, cell cycle regulators / apoptotic regulators, hormone regulators, and other immunosuppressants and / or anti-inflammatory agents selected from corticoids, such as glucocorticoids.

[0518] In one embodiment, at least one further therapeutic agent is a therapeutic agent useful for treating a specific disease, disorder, or condition treated in the present invention. For example, to treat cancer, at least one further therapeutic agent may be selected from the group including, but not limited to, chemotherapeutic agents, targeted cancer therapy agents, radiotherapy agents, immunotherapy agents or anti-cancer immunogens, anti-cancer antibodies, cytotoxic agents, anti-angiogenic agents, cell cycle regulators / apoptotic regulators, hormone regulators, and other immunosuppressants and / or anti-inflammatory agents selected from corticoids such as glucocorticoids.

[0519] In one embodiment, an isolated protein (particularly an antibody or antigen-binding fragment thereof as described herein), a fusion protein, a nucleic acid, or an expression vector according to the present invention is used in combination with a chemotherapeutic agent.

[0520] The term "chemotherapeutic agent" refers to chemical compounds that are effective in inhibiting tumor growth.

[0521] Examples of chemotepas include thiotepa and cyclophosphamide; alkyl sulfonates, e.g., busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carbocon, metredopa, and uredopa; ethyleneimines and methylamelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphaarnide, and trimethylomellamine; acetogenins (especially bratacin and bratacinone); carumptothecin (including its synthetic analog topotecan); bryostatin; calistatin; CC-1065 (including its synthetic analogs adzeresin, carzeresin, and bizeresin); cryptophycin (especially cryptophycin 1 and cryptophycin 8); dorastatin; duocalmycin (including synthetic analogues KW-2189 and CBI-TMI); eleuterobin; pancratistatin; sarcodicin; spongstatin; chlorambucil, chlornafadin, chlorophosphamide, estralnustine, ifosfamide, mechloretamine, mechloretamine hydrochloride, melphalan, nobenbitin, fenestrine, prednimustine, trophosphamide, uracil mustard, and other nitrogen mustards; nitrosourea such as carmustine, chlorozotosine, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as engine antibiotics (e.g., calicheamicin, especially calicheamicin 11 and calicheamicin 211); dynemycin (including dynemycin A); esperamycin;Neocardinostatin chromophore and related pigment protein enediin antibiotic chromophore, acrasinomycin, actinomycin, ausuramycin, azaserin, bleomycin, kactinomycin, carabicin, canninomycin, cardinophilin, chromomycin, dactinomycin, daunorubicin, detrevicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), e Pyrubicin, esorubicin, idanrubicin, marcelomycin, mitomycin, mycophenolic acid, nogalarnicin, olibomycin, peplomycin, potiphyllomycin, puromycin, queramycin, rhodorubicin, streptoglin, streptozocin, tubercidine, ubenimex, dinostatin, dinorevicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, and trimethrexate; fludarabine, 6-mercaptopurine, thiamip Phosphorus, thioguanine and other purine analogs; ancitabine, azacitidine, 6-azauridine, carmoflu, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU and other pyrimidine analogs; androgens, e.g., carsterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone; anti-adrenaline, e.g., aminoglutethimide, mitotane, trilostane; folic acid supplements such as floric acid; acegraton; aldofsfarnide glycoside; aminolevulinic acid; amsacrin; vestra Bucyl; Bisanthren; Edatraxate; Defofamine; Demecolsin; Diadicone; Elhornitine; Erliptinium acetate; Epotilon; Etoglucide; Gallium nitrate; Hydroxyurea; Lentinan; Ronidamin; Maytansinoids such as Maytansine and Ansamitosine; Mitoguazone; Mitoxantrone; Mopidamol; Nitracrine; Pentostatin; Fenamet; Pirarubicin; Podophyllic acid; 2-Ethylhydrazide; Procarbazine; PSK(registered trademark); Lazoxane; Rhizoxin; Schizophyllan; Spirogennanium; Tenuazonic acid;Triadicone; 2,2',2”-Trichlorotriethylalunine; Trichothecenes (especially T-2 toxin, beracrine A, loridine A and angidin); Urethanes; Vindesine; Dacarbazine; Mannomustine; Mitobromitol; Mitractol; Pipobroman; Gacitosine; Arabinoside ("Ara-C"); Cyclophosphamide; Thiotepa; Taxoids, e.g., paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.) and doxetaxel (TAXOTERE®, Rhone-Poulenc This includes, but is not limited to, alkylating agents such as Rorer, Antony, France; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeroda; ibandronate; CPT-1; topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoic acid; capecitabine; and any pharmaceutically acceptable salts, acids, or derivatives of the above.

[0522] Antihormone agents that act to regulate or inhibit the hormonal effects on tumors, such as antiestrogens including tamoxifen, raloxifene, the aromatase inhibitor 4(5)-imidazole, 4-hydroxytamoxifen, trioxyfen, keoxyfen, LY117018, onapristone, and toremifene (Fareston); and antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; as well as any pharmaceutically acceptable salts, acids, or derivatives of the above, are also included in the definition of chemotherapeutic agents.

[0523] In one embodiment, the isolated protein, antibody or its antigen-binding fragment, fusion protein, nucleic acid, or vector according to the present invention is used in combination with targeted cancer therapy.

[0524] As used herein, the term “targeted cancer therapy” refers to a drug or other substance that blocks the growth and spread of cancer by interfering with specific molecules (“molecular targets”) involved in the growth, progression, and expansion of cancer. Targeted cancer therapy may also be called “molecular targeted drugs,” “molecular targeted therapies,” or “precision drugs.” In some embodiments, targeted therapy consists of administering a tyrosine kinase inhibitor to a subject.

[0525] The term "tyrosine kinase inhibitor" refers to any of the various therapeutic agents or drugs that act as selective or non-selective inhibitors of receptor and / or non-receptor tyrosine kinases. Compounds related to tyrosine kinase inhibitors will replicate the effects of tyrosine kinase inhibitors, and it will be understood by those skilled in the art that related compounds act on different members of the tyrosine kinase signaling pathway to produce the same effect as a tyrosine kinase inhibitor of that tyrosine kinase. Examples of tyrosine kinase inhibitors and related compounds suitable for use in the methods of the embodiments of the present invention include ABT-869, AEE-788, AEW-541, axitinib, AZM-475271, BEZ235, BMS-599626 (AC-480), bosutinib, brivanib (BMS-582664), canertinib (CI1033), cediranib, CEP-11981, CP-547632, and CP-7247. 14. Dasatinib (BMS-354825), Dovitinib, Enzastaurin, Erlotinib (Tarceva; OSI-1774), Gefitinib (Iressa), Imatinib (Gleevec; STI571), KRN-633, KRN-951, Lapatinib (GW572016; GW2016), Leflunomide (SU101), Restautinib, L-21649, Motasenib, Midostaurin, MKC-I (Ro- 317453;R-440), MK-2206 (8-[4-aminocyclobutyl]phenyl]-9-phenyl-1,2,4-triazolo[3,4-f][1,6]naphthirizine-3(2H)-one hydrochloride), MLN-8054, neratinib, nilotinib, OSI-930, pazopanib, PD-0325901, PD-0332991, PP2, salakatinib, semaxinib (SU5416), celecyclib, SN Examples include, but are not limited to, S-032, sorafenib (BAY43-9006), sunitinib (Sutent; SU11248), SU-14813, SU-6668 (TSU-68), TAK-165, tandutinib, teratinib, batalanib (PTK787 / ZK222584), vandetanib (Zactima; ZD6474), their derivatives, their analogues, and combinations thereof.

[0526] In one embodiment, the isolated protein, antibody or its antigen-binding fragment, fusion protein, nucleic acid, or vector according to the present invention is used in combination with radiotherapy.

[0527] The term “radiotherapy” may include the associated administration of radiation or radiopharmaceuticals to a patient. The radiation source may be either external or internal to the patient being treated (radiotherapy may take the form of, for example, external beam radiation therapy (EBRT) or close-range radiation therapy (BT)). Radioactive elements that may be used in carrying out such methods include, for example, radium, cesium-137, iridium-192, americium-241, gold-198, cobalt-57, copper-67, technetium-99, iodide-123, iodide-131, and indium-111.

[0528] In one embodiment, the isolated protein, antibody or its antigen-binding fragment, fusion protein, nucleic acid, or expression vector according to the present invention is used in combination with an immunotherapy agent or immunotherapy.

[0529] As used herein, the terms “immunotherapy agent” or “immunotherapy” refer to compounds, compositions or treatments that indirectly or directly enhance, stimulate or increase the body’s immune response to cancer cells and / or reduce the side effects of other anticancer therapies. Thus, immunotherapy is a treatment that directly or indirectly stimulates or enhances the immune system’s response to cancer cells and / or reduces the side effects that may be caused by other anticancer agents. Immunotherapy is also referred to in the art as immunotherapy, biological therapy, biological response modifier therapy, and biotherapy. Examples of common immunotherapy agents or immunotherapies known in the art include, but are not limited to, cytokines, checkpoint inhibitors, checkpoint agonists (also called T-cell agonists), antibodies including monoclonal antibodies, antibody domains, antibody fragments, bispecific antibodies, prophylactic and therapeutic vaccines, oncolytic viruses, and adoptive transfer of immune cells (T cells, NK cells, dendritic cells, B cells, etc.).

[0530] One of the central premises underlying cancer immunotherapy is the presence of antigens that are selectively or abundantly expressed or mutated in cancer cells, thus enabling the specific recognition and subsequent destruction of cancer cells. Such antigens are generally called tumor-specific antigens. Another central premises underlying cancer immunotherapy is the presence of lymphocytes in the tumor, namely tumor-infiltrating lymphocytes (TILs), particularly effector TILs that can target and kill tumor cells through the recognition of the aforementioned tumor-specific antigens.

[0531] Immunotherapeutic agents or therapies can be passive. Passive immunotherapeutic agents produce an immediate effect upon administration of immune cell factors, such as monoclonal antibodies. Since the results of passive immunotherapy are temporally related to the administration of the active agent, continuous administration may be necessary for a long-term response. In another embodiment, the immunotherapeutic agent or therapy is active. Active immunotherapeutic agents produce a sustained and persistent response by inducing immunological memory. This most closely resembles a normal immune response. However, just as immune system function changes in healthy populations, the level of response to active immunotherapeutic agents depends on individual factors.

[0532] Active immunotherapeutic agents include both nonspecific activators (i.e., agents that enhance the immune system to make the human body more effective in fighting the growth and / or spread of cancer cells) and specific activators (i.e., agents that induce the production of cell-mediated and antibody immune responses focused on specific antigens expressed by cancer cells). Nonspecific immunotherapeutic agents are used in addition to primary therapies, as well as alone as primary therapies for the treatment of cancer, in which case they function as adjuvants to enhance the effectiveness of other therapies (e.g., cancer vaccines).

[0533] Nonspecific immunotherapeutic agents can also function in the latter situation to reduce the side effects of other therapies, such as myelosuppression induced by certain chemotherapy agents. Nonspecific immunotherapeutic agents can act on important immune system cells and induce secondary responses such as increased production of cytokines and immunoglobulins. Alternatively, the agent itself may contain cytokines. Nonspecific immunotherapeutic agents are generally classified as cytokines or non-cytokine adjuvants.

[0534] In one embodiment, the isolated protein, antibody or its antigen-binding fragment, fusion protein, nucleic acid, or vector according to the present invention is used in combination with cytokine therapy.

[0535] As used herein, “cytokine therapy” is defined as the administration of at least one cytokine to a subject.

[0536] Numerous cytokines have been found to be applicable to the treatment of cancer, either as general nonspecific immunotherapies designed to enhance the immune system, or as adjuvants provided by other therapies. Appropriate cytokines include, but are not limited to, interferons, interleukins, and colony-stimulating factors. Interferons (IFNs) intended by this invention include general types of IFN, IFN-alpha (IFN-α), IFN-beta (IFN-β), and IFN-gamma (IFN-γ). IFNs can act directly on cancer cells, for example, by slowing their growth, promoting their development into cells with more normal behavior, and / or increasing antigen production, thus making it easier for the immune system to recognize and destroy cancer cells. IFNs can also act indirectly on cancer cells, for example, by slowing angiogenesis, enhancing the immune system, and / or stimulating natural killer (NK) cells, T cells, and macrophages. Recombinant IFN-α is commercially available as Roferon (Roche Pharmaceuticals) and IntronA (Schering Corporation). Interleukins intended by this invention include IL-2, IL-4, IL-11, IL-12, and IL-21. Examples of commercially available recombinant interleukins include Proleukin® (IL-2; Chiron Corporation) and Neumega® (IL-12; Wyeth Pharmaceuticals). Colony-stimulating factors (CSFs) intended by this invention include granulocyte colony-stimulating factor (G-CSF or filgrastim), granulocyte-macrophage colony-stimulating factor (GM-CSF or salglamostim), and erythropoietin (epoetin alfa, darbepoetin). Treatment with one or more growth factors may help stimulate the generation of new blood cells in subjects undergoing conventional chemotherapy. Therefore, treatment with CSFs may help reduce chemotherapy-related side effects and may allow the use of higher doses of chemotherapy agents.Various recombinant colony-stimulating factors, such as Neupogen® (G-CSF; Amgen), Neulasta (pelfilgrastim; Amgen), Leukine (GM-CSF; Berlex), Procrit (erythropoietin; Ortho Biotech), Epogen (erythropoietin; Amgen), and Arnesp (erytropoietin), are commercially available.

[0537] In one embodiment, the isolated protein, antibody or its antigen-binding fragment, fusion protein, nucleic acid or vector according to the present invention is used in combination with a cytokine mimetic, such as an IL-2 mimetic. In one embodiment, the IL-2 mimetic cannot bind to CD25. In one embodiment, the IL-2 mimetic preferentially binds to IL-2R containing β and γ subunits compared to IL-2R containing α, β and γ subunits. A non-limiting example of an IL-2 mimetic that may be used is NKTR-214 (Nektar Therapeutics).

[0538] In one embodiment, the isolated protein, antibody or its antigen-binding fragment, fusion protein, nucleic acid, or vector according to the present invention is used in combination with checkpoint inhibitor therapy.

[0539] As used herein, “checkpoint inhibitor therapy” is defined as the administration of at least one checkpoint inhibitor to a subject.

[0540] As a cancer treatment, checkpoint inhibitor therapy aims to prevent the activation of inhibitory receptors expressed on T cells by ligands expressed by tumor cells. Therefore, checkpoint inhibitor therapy aims to prevent the inhibition of T cells present in the tumor, i.e., tumor-infiltrating T cells, and thus enhance the target immune response against tumor cells.

[0541] Examples of checkpoint inhibitors are listed above.

[0542] In one embodiment, at least one checkpoint inhibitor is selected from the group comprising or consisting of PD-1 inhibitors, PD-L1 inhibitors, CTLA-4 inhibitors, and any mixture thereof.

[0543] In one embodiment, the isolated protein, antibody or its antigen-binding fragment, fusion protein, nucleic acid, or vector according to the present invention is used in combination with checkpoint agonist therapy.

[0544] As used herein, “checkpoint agonist therapy” is defined as the administration of at least one checkpoint agonist to a subject.

[0545] As a cancer treatment, checkpoint agonist therapy aims to activate stimulatory receptors expressed on immune cells present within tumors. In particular, T-cell agonist therapy aims to enhance the activation of T cells present in tumors, i.e., tumor-infiltrating T cells, and thus enhance the target immune response against tumor cells. Currently, numerous potential targets for checkpoint agonist therapy have been identified.

[0546] Examples of checkpoint agonists are listed above.

[0547] In one embodiment, at least one checkpoint agonist is selected from the group comprising or consisting of a CD137 agonist, an OX40 agonist, a GITR agonist, an ICOS agonist, a CD27-CD70 agonist, a CD40 agonist, and any mixture thereof.

[0548] In one embodiment, an isolated protein, antibody or its antigen-binding fragment, fusion protein, nucleic acid, or vector according to the present invention is used in combination with a second antibody specific to an immune receptor or costimulatory molecule.

[0549] Examples of antibodies specific to immune receptors include, but are not limited to, anti-CTLA4 antibodies (e.g., ipilimumab), anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-TIM-3 antibodies, anti-LAG-3 antibodies, anti-B7H3 antibodies, anti-B7H4 antibodies, anti-B7H6 antibodies, anti-4-1BB antibodies, anti-TIGIT antibodies, anti-ICOS antibodies, anti-GITR antibodies, anti-CD27-CD70 antibodies, anti-CD40 antibodies, anti-BTLA antibodies, anti-HVEM antibodies, anti-CD160 antibodies, anti-CCR8 antibodies, anti-CEACAM-1 antibodies, and anti-OX40 antibodies.

[0550] In some embodiments, the second antibody is specific to CD137. As used herein, the term “CD137” has its general meaning in the art and may also be called Ly63, ILA, or 4-1BB. CD137 is a member of the tumor necrosis factor (TNF) receptor family. Members of this receptor family and their structurally related ligands are important regulators of a wide variety of physiological processes and play a crucial role in regulating immune responses. CD137 is expressed by activated NK cells, T lymphocytes and B lymphocytes, as well as monocytes / macrophages. The gene encodes a 255-amino acid protein with three cysteine-rich motifs in a short N-terminal cytoplasmic portion containing an extracellular domain (characteristic of this receptor family), a transmembrane region, and potential phosphorylation sites. Expression in primary cells is strictly activation-dependent. The receptor ligand is TNFSF9. Human CD137 has been reported to bind only to its ligand. Agonists include natural ligands (TNFSF9), aptamers (see McNamara et al. (2008) J. Clin. Invest. 1 18:376-386), and antibodies.

[0551] In another embodiment, the antibody or its antigen-binding fragment is bispecific and can further bind to an immunoreceptor or a costimulatory molecule.

[0552] Examples of immune receptors include, but are not limited to, CTLA4, PD-1, PD-L1, TIM-3, LAG-3, B7H3, B7H4, B7H6, 4-1BB, TIGIT, ICOS, GITR, CD27-CD70, CD40, BTLA, HVEM, CD160, CCR8, CEACAM-1, and OX40.

[0553] Examples of co-stimulatory molecules include, but are not limited to, B7H3, B7H4, B7H6, 4-1BB, OX40, and GITR.

[0554] In one embodiment, an isolated protein (particularly an antibody or its antigen-binding fragment as described herein), fusion protein, nucleic acid, or vector according to the present invention is used in combination with a second antibody that induces cell death of an antigen to which the second antibody binds, via ADCC. In one embodiment, the second antibody (e.g., IgG1 or IgG3 isotype, particularly human IgG1 or IgG3 isotype) induces ADCC toward the cells to which the antibody binds. NK cells play a significant role in ADCC induction, and increased NK cell reactivity can be directed toward target cells through the use of such a second antibody. In one embodiment, the second antibody is specific to a cell surface antigen, e.g., a membrane antigen. In some embodiments, the second antibody is specific to tumor antigens (e.g., molecules specifically expressed by tumor cells), particularly lymphoma antigens (e.g., CD20), such as CD20, CD52, ErbB2 (or HER2 / Neu), CD33, CD22, CD25, MUC-1, CEA, KDR, αVβ3, etc.

[0555] Therefore, the present invention also provides a method for enhancing the antitumor effect of a monoclonal antibody against a tumor antigen(s).

[0556] In one embodiment, ADCC function is specifically enhanced by sequential administration of antibodies against one or more tumor antigens and the antibody of the present invention or its antigen-binding fragment, thereby enhancing the killing of target cells.

[0557] In one embodiment, an isolated protein (particularly an antibody or antigen-binding fragment thereof as described herein), fusion protein, nucleic acid, or vector according to the present invention is used in combination with a native ligand for an NK cell activating receptor or an antibody that binds to and activates an NK cell activating receptor.

[0558] In one embodiment, at least one further therapeutic agent is a drug that increases the presence of the native ligand for the NK cell activating receptor on the surface of target cells (e.g., infected cells or tumor cells).

[0559] As used herein, the term “activated NK receptor” refers to any molecule on the surface of an NK cell that, when stimulated, causes a measurable increase in any property or activity known in the art as relating to NK activity such as cytokine (e.g., IFN-γ and TNF-α) production, increased intracellular free calcium levels, the ability to target cells in retargeting killing assays, or the ability to stimulate NK cell proliferation.

[0560] Examples of "activating NK receptors" include, but are not limited to, the activation forms of KIR proteins (e.g., KIR2DS protein), CD160-TM, NKG2D, IL-2R, IL-12R, IL-15R, IL-18R, and IL-21R.

[0561] Examples of ligands that act as agonists in activating receptors include, for example, IL-2, IL-15, and IL-21 polypeptides.

[0562] In one embodiment, an isolated protein (particularly an antibody or antigen-binding fragment thereof as described herein), fusion protein, nucleic acid, or vector according to the present invention is used as a therapeutic vaccine or in combination with a therapeutic vaccine.

[0563] As used herein, a therapeutic vaccine is defined as the administration of at least one tumor-specific antigen (e.g., a synthetic long peptide or SLP) or a nucleic acid encoding such tumor-specific antigen; the administration of a recombinant viral vector that selectively enters and / or replicates in tumor cells; the administration of tumor cells; and / or the administration of immune cells (e.g., dendritic cells) that have been engineered to present tumor-specific antigens and induce an immune response to these antigens.

[0564] As a cancer treatment, therapeutic vaccines aim to enhance the target immune response against tumor cells.

[0565] Examples of therapeutic vaccines aimed at enhancing the target immune response against tumor cells include, but are not limited to, viral vector-based therapeutic vaccines such as adenoviruses (e.g., oncolytic adenoviruses), vaccinia viruses (e.g., modified vaccinia ankara (MVA)), alphaviruses (e.g., semliki forest virus (SFV)), measles virus, herpes simplex virus (HSV), and coxsackievirus; synthetic long peptide (SLP) vaccines; RNA-based vaccines; and dendritic cell vaccines.

[0566] In one embodiment, the isolated protein (particularly the antibody or antigen-binding fragment thereof described herein), fusion protein, nucleic acid, or vector according to the present invention is used in combination with oncolytic virus therapy.

[0567] As used herein, “oncolytic virus therapy” is defined as the administration of at least one oncolytic virus to a subject.

[0568] Oncolytic viruses are defined as viruses that preferentially infect and kill cancer cells over normal, non-cancerous cells. As a cancer treatment, oncolytic virus therapy aims to kill cancer cells and / or induce or enhance an immune response against cancer cells.

[0569] Examples of oncolytic viruses include, but are not limited to, modified herpes simplex virus type 1 such as Tarimogen laharpalepec (or T-VEC) or HSV-1716; modified adenovirus such as Ad5-DNX-2401; modified measles virus such as MV-NIS; modified vaccinia virus (VV) such as vaccinia virus TG6002; and modified poliovirus such as PVS-RIPO.

[0570] In one embodiment, the isolated proteins (particularly antibodies or antigen-binding fragments thereof), fusion proteins, nucleic acids, or vectors according to the present invention are used in combination with adoptive cell transfer, also known as adoptive cell therapy (both also called ACT), for example, adoptive transfer of T cells or NK cells, also known as adoptive T cell therapy or adoptive NK cell therapy, respectively.

[0571] As used herein, “adoptive cell transfer” or “adoptive cell therapy” is defined, for example, as transfer in the form of injection or reinjection of immune cells into a subject. As a cancer treatment, adoptive cell transfer into a subject aims to enhance the subject’s immune response to cancer cells.

[0572] Examples of immune cells that can be used in cell therapy include cytotoxic cells (e.g., natural killer (NK) cells, CD8 cells). + T cells, and natural killer (NK) T cells, effector T cells (e.g., CD4 + T cells and CD8 + Examples include, but are not limited to, T cells, alpha-beta (αβ) T cells and gamma-delta (γδ) T cells, antibody-expressing B cells or other antibody-producing or presenting cells and dendritic cells.

[0573] In one embodiment, the transferred immune cells are antigen-specific cells. In one embodiment, the transferred immune cells are antigen-specific immune cells, and the antigen is specifically and / or abundantly expressed by cancer cells. In one embodiment, the transferred immune cells described herein are tumor-specific immune cells, in other words, the transferred immune cells described herein specifically recognize cancer cells or tumor cells via an antigen that is specifically and / or abundantly expressed by the cancer cells or tumor cells. In one embodiment, the transferred immune cells are tumor-specific effector T cells. In one embodiment, the transferred immune cells are tumor-specific CD8 + Effector T cells, especially tumor-specific cytotoxic CD8 + These are T cells. In one embodiment, the transferred cells are tumor-infiltrating cells (TILs). In one embodiment, the transferred immune cells are tumor-specific cytotoxic cells. In one embodiment, the transferred immune cells are tumor-specific NK cells.

[0574] Examples of tumor-specific antigens, i.e., antigens specifically and / or abundantly expressed by cancer cells, include, but are not limited to, neoantigens (also called novel antigens or mutant antigens), 9D7, ART4, β-catenin, BING-4, Bcr-abl, BRCA1 / 2, calcium-activated chloride channel 2, CDK4, CEA (carcinoembryonic antigen), CML66, cyclin B1, CypB, EBV (Epstein-Barr virus) related antigens (e.g., LMP-1, LMP-2, EBNA1 and BARF1), Ep-CAM, EphA3, fibronectin, Gp100 / pmel17, Her2 / neu, HPV (human papillomavirus) E6, and HPV. Examples include E7, hTERT, IDH1, IDH2, immature laminin receptor, MC1R, Melan-A / MART-1, MART-2, mesothelin, MUC1, MUC2, MUM-1, MUM-2, MUM-3, NY-ESO-1 / LAGE-2, p53, PRAME, prostate-specific antigen (PSA), PSMA (prostate-specific membrane antigen), Ras, SAP-1, SART-I, SART-2, SART-3, SSX-2, Survivin, TAG-72, telomerase, TGF-βRII, TRP-1 / -2, tyrosinase, WT1, antigens of the BAGE family, antigens of the CAGE family, antigens of the GAGE ​​family, antigens of the MAGE family, antigens of the SAGE family, and antigens of the XAGE family.

[0575] As used herein, neoantigens (also called novel antigens or mutant antigens) correspond to antigens derived from proteins affected by somatic mutations or gene rearrangements acquired by tumors. Neoantigens can be specific to each individual target and therefore can provide targets for developing personalized immunotherapy. Examples of neoantigens include, but are not limited to, the R24C variant of CDK4, the R24L variant of CDK4, KRAS mutated at codon 12, mutated p53, the V599E variant of BRAF, and the R132H variant of IDH1.

[0576] In one embodiment, the transferred immune cells described above are specific to tumor antigens selected from the group including or comprising the classes of CTAs (cancer / testicular antigens also known as MAGE-type antigens), neoantigens, and viral antigens.

[0577] As used herein, the class of CTA corresponds to an antigen encoded by a gene that is expressed in tumor cells but not in normal tissues except male germ cells.

[0578] Examples of CTAs include, but are not limited to, MAGE-A1, MAGE-A3, MAGE-A4, MAGE-C2, NY-ESO-1, PRAME, and SSX-2.

[0579] As used herein, the class of viral antigens corresponds to antigens derived from viral oncogenic proteins. Examples of viral antigens include, but are not limited to, HPV (human papillomavirus)-related antigens such as E6 and E7, and EBV (Epstein-Barr virus)-related antigens such as LMP-1, LMP-2, EBNA1, and BARF1.

[0580] In one embodiment, the transferred immune cells are autoimmune cells, particularly autologous T cells. In another embodiment, the transferred immune cells are allogeneic (or homogeneous) immune cells, particularly allogeneic NK cells.

[0581] For example, autologous T cells can be generated ex vivo by either proliferating antigen-specific T cells isolated from a target or by reorienting the target's T cells through genetic manipulation.

[0582] In one embodiment, the immune cells to be injected are modified ex vivo before being injected into the target.

[0583] Methods for isolating T cells, particularly antigen-specific T cells, such as tumor-specific T cells, from subjects are well known in the art (see, for example, Rosenberg & Restifo, 2015, Science 348, 62-68; Prickett et al., 2016, Cancer Immunol Res 4, 669-678; or Hinrichs & Rosenberg, 2014, Immunol Rev 257, 56-71). Methods for proliferating T cells ex vivo are well known in the art (see, for example, Rosenberg & Restifo, 2015, Science 348, 62-68; Prickett et al., 2016, Cancer Immunol Res 4, 669-678; or Hinrichs & Rosenberg, 2014, Immunol Rev 257, 56-71). Protocols for T cell infusion in the target population, including pre-infusion therapy regimens, are well-known in the field (see, for example, Rosenberg & Restifo, 2015, Science 348, 62-68; Prickett et al., 2016, Cancer Immunol Res 4, 669-678; or Hinrichs & Rosenberg, 2014, Immunol Rev 257, 56-71).

[0584] In one embodiment, the isolated protein (particularly the antibody or antigen-binding fragment thereof described herein), fusion protein, nucleic acid, or vector according to the present invention is used in combination with CAR immunotherapy, particularly CAR T cell therapy or CAR NK cell therapy.

[0585] As used herein, CAR immunotherapy is adoptive cell therapy in which the transferred cells are genetically engineered to express chimeric antigen receptors (CARs), such as T cells or NK cells, which are among the aforementioned immune cells. As a cancer treatment, adoptive transfer of CAR immune cells to a target aims to enhance the target's immune response to cancer cells.

[0586] CARs are synthetic receptors consisting of a targeting moiety that associates with one or more signaling domains within a single fusion molecule or several molecules. Generally, the binding moiety of a CAR consists of the antigen-binding domain of a monoclonal antibody (scFv) containing the light chain and variable fragment of a monoclonal antibody linked by a flexible linker. Binding moieties based on receptor or ligand domains have also been successfully utilized. The signaling domain of first-generation CARs is typically derived from the cytoplasmic region of the CD3ζ or Fc receptor gamma chain. While first-generation CARs have been shown to successfully redirect the cytotoxicity of T cells, they have not been able to provide long-term expansion and antitumor activity in vivo. Therefore, signaling domains from costimulatory molecules, including CD28, OX-40 (CD134), and 4-1BB (CD137), have been added alone (second generation) or in combination (third generation) to enhance the survival and proliferation of CAR-modified T cells.

[0587] Therefore, in one embodiment, the transferred T cells as described above herein are CAR T cells. CAR expression allows T cells to be redirected to selected antigens, such as antigens expressed on the surface of cancer cells. In one embodiment, the transferred CAR T cells recognize tumor-specific antigens. In another embodiment, the transferred NK cells are CAR NK cells. CAR expression allows NK cells to be redirected to selected antigens, such as antigens expressed on the surface of cancer cells. In one embodiment, the transferred CAR NK cells recognize tumor-specific antigens.

[0588] Examples of tumor-specific antigens are mentioned above in this specification.

[0589] In one embodiment, the transferred CAR T cells or CAR NK cells recognize tumor-specific antigens selected from the group including or consisting of EGFR, particularly EGFRvIII, mesothelin, PSMA, PSA, CD47, CD70, CD133, CD171, CEA, FAP, GD2, HER2, IL-13Rα, αvβ6 integrin, ROR1, MUC1, GPC3, EphA2, CD19, CD21, and CD20.

[0590] In one embodiment, the CAR immune cells described above are autologous CAR immune cells, particularly autologous CAR T cells. In another embodiment, the CAR immune cells described above are allogeneic (or allogeneic) CAR immune cells, particularly allogeneic CAR NK cells.

[0591] In one embodiment, the isolated protein (particularly the antibody or antigen-binding fragment thereof described herein), fusion protein, nucleic acid, or vector according to the present invention is used in combination with an antibiotic. Examples of antibiotics include penicillin (e.g., penicillin, amoxicillin), tetracycline (e.g., doxicolate, tetracycline, minocycline), cephalosporin (e.g., cefuroxime, ceftriaxone, cefdinir), quinolone (e.g., ciprofloxacin, levofloxacin, moxifloxacin), lincomycin (e.g., clindamycin, lincomycin), macrolide (e.g., azithromycin, clinamycin), and Examples include, but are not limited to, rithromycin (rithromycin, erythromycin), sulfonamides (e.g., sulfamethoxazole-trimethoprim, sulfasalazine, sulfisoxazole), glycopeptides (e.g., dalbavancin, oritavancin, teravancin, vancomycin), aminoglycosides (e.g., gentamicin, tobramycin, amikacin), and carbapenems (e.g., imipenem, meropenem, doripenem, ertapenem).

[0592] In one embodiment, the isolated protein (particularly the antibody or antigen-binding fragment thereof described herein), fusion protein, nucleic acid, or vector according to the present invention is used in combination with an antiviral agent. Examples of antiviral agents include abacavir, acyclovir, adefovir, amantadine, amprenavir, amprigen, arbidol, atazanavir, atripra, paravir, cidofovir, combivir, dolutegravir, darunavir, delavirdin, didanosine, docosanol, edoxudine, efavirenz, emtricitabine, enfuvirtide, entecavir, ecolieva-famciclovir, homivirsen, fossamaprenavir, foscanet, phosfonet, ivacitabine, iminovir, doxuridine, imiquimod, indinavir, inosine, integrase inhibitors, interferon type III, interferon type II, interferon type I, interferon, lamivudine, lopinavir, and roviride. Examples of such inhibitors include, but are not limited to, maraviroc, moloxidine, methisazone, nelfinavir, nevirapine, nexavir, nitazoxanide, nucleoside analogs, norvir, oseltamivir, pegylated interferon alpha-2a, penciclovir, peramivir, preconalil, podophyllotoxin, protease inhibitors, raltegravir, reverse transcriptase inhibitors, ribavirin, rimantadine, ritonavir, pyramidine, saquinavir, sofosbuvir, stabudine, telaprevir, tenofovir, tenofovir disoproxil, tipranavir, trifluridine, trizivir, tromantadine, tolvada, valacyclovir, valganciclovir, bicriviroc, vidarabine, viramidine, zalcitabine, zanamivir, and zidovudine.

[0593] In one embodiment, the isolated protein (particularly the antibody or antigen-binding fragment thereof as described herein), fusion protein, nucleic acid, or vector according to the present invention is used in combination with an antifungal agent. Examples of antifungal agents include, but are not limited to, polyene antifungal agents (e.g., amphotericin B, candicidine, philipin, hamycin, natamycin, nystatin, rimocidine), imidazole antifungal agents (e.g., bifonazole, butoconazole, clotrimazole, econazole, fenticonazole, isoconazole, ketoconazole, luliconazole, miconazole, omoconazole, oxyconazole, sertaconazole, sulconazole) Examples include zole (thioconazole), triazole antifungal agents (e.g., albaconazole, efinaconazole, epoxyconazole, fluconazole, isabconazole, itraconazole, posaconazole, propiconazole, ravconazole, terconazole, voriconazole), thiazole antifungal agents (e.g., abafungin), allylamines, and echinocandins (e.g., anidurafungin, caspofungin, micafungin).

[0594] In one embodiment, the isolated proteins (particularly antibodies or antigen-binding fragments thereof), fusion proteins, nucleic acids, or vectors according to the present invention are used in combination with antiparasitic agents. Examples of antiparasitic agents include, but are not limited to, broad-spectrum antiparasitic agents (e.g., nitazoxanide), antiprotozoal agents (e.g., melarsoprol, eflornithine, metronidazole, tinidazole, miltefosine), anthelmintic agents (including, but not limited to, antinematodes (e.g., hookworms, mebendazole, pyrantel pamoate, thiabendazole, diethylcarbamazine, ivermectin), antitaeniaes (e.g., niclosamide, praziquantel, albendazole), antidendritic agents (e.g., praziquantel)), and anti-amebic agents (e.g., rifampin, amphotericin B).

[0595] Another object of the present invention relates to the use of isolated proteins (in particular antibodies or antigen-binding fragments thereof as described herein) or fusion proteins as described herein above, and other therapeutic agents as described herein above, in the treatment of diseases in subjects requiring such treatment, wherein the protein, antibody or antigen-binding fragment or fusion protein is used as an adjuvant for the therapeutic agent.

[0596] Accordingly, the present invention relates to isolated proteins (particularly antibodies or antigen-binding fragments thereof as described herein) or fusion proteins thereof (preferably in compositions, pharmaceutical compositions or pharmaceuticals) for use as adjuvants in cancer treatment. Accordingly, the present invention relates to isolated proteins (particularly antibodies or antigen-binding fragments thereof as described herein) or fusion proteins thereof (preferably in compositions, pharmaceutical compositions or pharmaceuticals) for use as adjuvants in therapies for infectious diseases.

[0597] In one embodiment, the present invention relates to the use of isolated proteins (in particular antibodies or antigen-binding fragments thereof as described herein) or fusion proteins as described herein for enhancing the immune response induced by cancer treatment in patients who require it.

[0598] In one embodiment, isolated proteins (particularly antibodies or antigen-binding fragments thereof) or fusion proteins according to the present invention may be used as immunotherapeutic agents for treating a wide variety of cancers, particularly cancers associated with immunosuppression and / or immune exhaustion.

[0599] In one embodiment, an isolated protein (in particular an antibody or its antigen-binding fragment) or fusion protein according to the present invention may enhance the immune response induced by cancer therapy in a patient, comprising administering the protein (e.g., an antibody or its fragment) or fusion protein to the patient in an amount effective in enhancing the immune response induced by cancer therapy in the patient.

[0600] As used herein, the term “adjuvant” refers to a compound or combination of compounds that enhances a treatment, such as cancer treatment. Adjuvants can increase an effective immune response against low-immunogenic or non-immunogenic tumor cells. In one embodiment, an adjuvant is used in conjunction with a well-known cancer treatment agent in the treatment of cancer, and thus enhances the immune response against cancer cells. For example, an adjuvant can enhance the immune response during cancer treatment, reduce T cell exhaustion (without reducing T cell activation), increase T cell survival, enhance NK cell cytotoxicity, reduce tumor growth and / or tumor size, and / or increase survival, and treat or prevent cancer metastasis. In one embodiment, the enhancement of cancer therapy in the presence of an adjuvant is defined by comparison with cancer therapy administered alone.

[0601] In another embodiment, the isolated proteins described herein (in particular, antibodies or antigen-binding fragments thereof) or fusion proteins can increase or improve the immune response of the subject.

[0602] As used herein, “immune response” refers to the response of cells of the immune system to a stimulus, such as B cells, T cells (CD4 or CD8), regulatory T cells, antigen-presenting cells, dendritic cells, monocytes, macrophages, NKT cells, NK cells, basophils, eosinophils, or neutrophils. In some embodiments of any aspect, the response is specific to a particular antigen (“antigen-specific response”) and refers to the response of CD4 T cells, CD8 T cells, or B cells via an antigen-specific receptor. In some embodiments of any aspect, the immune response is CD4 + Response or CD8 + These are T cell responses, such as cytotoxicity, proliferation, cytokine or chemokine production, transport, or phagocytosis, and may depend on the nature of the immune cells receiving the response.

[0603] As with other known immunotherapies, the ability of isolated proteins (particularly antibodies or their antigen-binding fragments, as described herein) or fusion proteins, as described above herein, to enhance the immune response in patients may have broader therapeutic significance beyond the field of cancer. For example, it has been suggested that immunostimulants may be useful in treating a wide variety of infectious diseases, particularly those caused by pathogens that promote immunosuppression and / or immune exhaustion. Furthermore, such immunostimulants may be useful in enhancing the immune efficacy of vaccines (e.g., infectious disease and cancer vaccines).

[0604] Another object of the present invention relates to the use of the isolated protein (particularly an antibody or antigen-binding fragment thereof as described herein), fusion protein, nucleic acid, or vector (preferably in a composition, pharmaceutical composition, or drug as described herein above) as described above, to deplete CD25-expressing Treg cells in a target requiring such use, in which a therapeutically effective amount of the isolated protein (particularly an antibody or antigen-binding fragment thereof as described herein), fusion protein, nucleic acid, or vector of the present invention is administered to the target.

[0605] Accordingly, the present invention further relates to a method for depleting CD25-expressing Treg cells in a target requiring such depletion, comprising administering an isolated protein (in particular an antibody or antigen-binding fragment thereof, as described herein), a fusion protein, a nucleic acid, a vector, a composition, a pharmaceutical composition, or a drug to the target.

[0606] In one embodiment, the isolated protein (particularly the antibody or antigen-binding fragment thereof as described herein), fusion protein, nucleic acid, or vector (preferably in a composition, pharmaceutical composition, or drug as described herein above) is intended for use in depleting CD25-expressing Treg cells.

[0607] In one embodiment, CD25-expressing Treg cells are tumor-infiltrating Treg cells.

[0608] In one embodiment, the antibody or antigen-binding fragment thereof, as described above herein, for use in depleting CD25-expressing Treg cells is IgG, preferably IgG1.

[0609] In one embodiment, the antibody or antigen-binding fragment thereof, used for depleting CD25-expressing Treg cells, preferably binds with high affinity to at least one activated Fcγ receptor selected from FcγRI, FcγRIIa, and FcγRIII.

[0610] In one embodiment, the antibody or antigen-binding fragment described herein for use in depleting CD25-expressing Treg cells induces an enhanced ADCC, ADCP, and / or CDC response, preferably an increased ADCC and / or ADCP response, more preferably an increased ADCC response.

[0611] In one embodiment, an antibody or antigen-binding fragment thereof, as described above herein, for use in depleting CD25-expressing Treg cells, does not inhibit CD25-mediated IL-2 signaling. Therefore, in one embodiment, an antibody or antigen-binding fragment thereof, as described above herein, for use in depleting CD25-expressing Treg cells, does not inhibit CD4 + and CD8 + It does not inhibit the proliferation and / or activation of T cells (or effector T cells). In another embodiment, an antibody or its antigen-binding fragment as described above herein for use in depleting CD25-expressing Treg cells is CD4 + and CD8 + It does not inhibit the phosphorylation of STAT5a in T cells (or effector T cells).

[0612] The present invention further relates to a method for inducing specific lysis of CD25-positive cells in T cells without inhibiting IL-2 signaling, and comprises administering a therapeutically effective amount of an isolated protein disclosed herein (in particular an antibody or its antigen-binding fragment as described herein), a fusion protein, a nucleic acid, a vector, a composition, a pharmaceutical composition, or a drug to a target.

[0613] The present invention further relates to a method for inducing specific lysis of CD25-positive cells by ADCC and / or ADCP without inhibiting IL-2 signaling in T cells, and comprises administering a therapeutically effective amount of an isolated protein disclosed herein (in particular an antibody or its antigen-binding fragment as described herein), a fusion protein, a nucleic acid, a vector, a composition, a pharmaceutical composition, or a drug to a target.

[0614] In some embodiments, the subjects are currently receiving or have previously received immunotherapy.

[0615] The present invention further relates to a method comprising the step of administering an immunotherapy to a subject, wherein the subject has received or is receiving a therapeutically effective amount of an isolated protein (in particular an antibody or its antigen-binding fragment as described herein), a fusion protein, a nucleic acid, a vector, a composition, a pharmaceutical composition, or a drug as disclosed herein.

[0616] In some embodiments, the therapeutically effective dose is an amount effective in inducing specific lysis of CD25-positive cells without inhibiting IL-2 signaling in T cells.

[0617] In some embodiments, the therapeutically effective dose is an amount effective in inducing specific lysis of CD25-positive cells by ADCC and / or ADCP without inhibiting IL-2 signaling in T cells.

[0618] The present invention further relates to the use of isolated proteins (in particular antibodies or antigen-binding fragments thereof, as described herein), fusion proteins, nucleic acids, and vectors disclosed herein in the manufacture of pharmaceuticals for subjects requiring treatment of diseases, disorders, or symptoms (e.g., cancer or infectious diseases) as described herein.

[0619] Compared to prior art anti-CD25 antibodies, the antibody or antigen-binding fragment of the present invention may offer at least one of the following advantages. -In some embodiments, the antibody or antigen-binding fragment thereof of the present invention exhibits increased affinity for CD25 compared to prior art CD25 antibodies. -In some embodiments, the antibody or antigen-binding fragment of the present invention exhibits increased binding activity to CD25 compared to prior art CD25 antibodies. -In some embodiments, the antibody or antigen-binding fragment of the present invention induces increased IL-2-dependent activation of T cells in culture, preferably increased proliferation of T cells in culture, compared to prior art CD25 antibodies. -In some embodiments, the antibody or antigen-binding fragment of the present invention induces lower inhibition of IL-2-induced T cell proliferation in culture compared to prior art CD25 antibodies. -In some embodiments, the antibody or antigen-binding fragment of the present invention is compared to the prior art CD25 antibody with respect to CD25 + Expression cells, preferably CD25 + T-cell expression, more preferably CD25 + It shows increased ADCC activity in Treg cells that express the gene. -In some embodiments, the antibody or antigen-binding fragment of the present invention is compared to the prior art CD25 antibody with respect to CD25 + Expression cells, preferably CD25 + T-cell expression, more preferably CD25 + It shows increased ADCP activity in Treg cells that express ADCP. -In some embodiments, the antibody or antigen-binding fragment of the present invention exhibits higher efficiency compared to prior art CD25 antibodies, thereby reducing the risk of CD25 contamination. + Expression cells, preferably CD25 + T-cell expression, more preferably CD25 + This induces depletion of expressing Treg cells (preferably in vivo depletion). -In some embodiments, the antibody of the present invention or its antigen-binding fragment exhibits lower immunogenicity compared to prior art CD25 antibodies. -In some embodiments, the antibody of the present invention or its antigen-binding fragment exhibits higher stability compared to prior art CD25 antibodies. -In some embodiments, the antibody or antigen-binding fragment of the present invention exhibits better cross-reactivity with cynomolgus monkey CD25 compared to prior art CD25 antibodies. [Examples]

[0620] The present invention will be further explained by the following examples.

[0621] Materials and methods CD25-specific mAb binding

[0622] HEK293WT (wild-type) and HEK293 cells transiently transfected with a human CD25 (huCD25) construct were incubated with either an Ultra-LEAF purified human IgG1 isotype (isotype control), the anti-CD25 antibody of the present invention (H07, H09, G02, E04, D01, E04-2, B05, G09, B01, C01, G01, H01, G02-2, H02, F03, D05, B07, H08, B12), or basiliximab at 11x serial 3-fold dilutions starting at 500 nM. Cells were then labeled with anti-human IgG (Fc-specific) FITC diluted 1:500 in FACS buffer, or with anti-mouse IgG-APC at a concentration of 2 μg / ml, for basiliximab detection. Cells were analyzed by FACS.

[0623] Antibody-dependent phagocytosis (ADCP) mediated specific lysis of CD25-positive cells Anti-CD25-induced ADCP was obtained by co-culturing THP-1 cells (as effector cells) and CFSE-stained SUDHL-1 cells (target cells) with either human IgG1 control or anti-CD25 antibodies (E04-2, B05, C01, G01, G02-2, and B07) at 37°C for 2 hours at 10 μg / ml. At this point, the cells were harvested, washed, and anti-CD33 APC antibody was added to the co-culture. The cells were washed before flow cytometry analysis. The percentage of CD33+CFSE+ in CD33+THP1 cells corresponds to the level of induced phagocytosis.

[0624] Effects on IL-2-induced T cell proliferation Newly isolated peripheral mononuclear cells (PBMCs) were cultured for 72 hours in RPMI medium (10% FCS, 2% glutamine, 1% antibiotic) supplemented with 5 μg / ml PHA. PBMCs were stained with CFSE and starved for 24 hours, after which activated T cells were isolated by magnetic cell sorting. The T cells were cultured for 72 hours with 50 U / ml IL-2 and 1 μg / ml anti-CD25 antibody (H09, D01, E04-2, B05, C01, G01, G02-2, F03, D05, B07, B12), basiliximab, 7G7B6, or MA-251. After cell division, flow cytometry was performed.

[0625] Treg cell depletion PBMC (2 x 10 6 Cells (1 / ml) were incubated with human IgG1 control isotype, the anti-CD25 antibody of the present invention (H09, D01, E04-2, B05, C01, G01, G02-2, F03, D05, B07, B12), or 1 μg / ml basiliximab + anti-CD3 / anti-CD28 conjugated beads (Dako). After 6 days of incubation, cells were labeled with a mixture of anti-CD8-FITC, -CD4-PE, CD39-PerCP-Cy5.5, -CD127-PE-Cy7, -CD3-APC, and CD45-Pacific Blue antibodies. Cells were analyzed by flow cytometry using Cytoflex (Beckman Coulter), and the data were analyzed with FlowJo software. Treg cells were identified as CD3+ CD4 + CD39 + CD127 low / - They were identified as a cell population. CD4+ T effector cells are CD3 + CD4 + (-)CD3 + CD4 + CD39 + CD127 low / - They were identified as a cell population. CD8+ T effector cells were identified as CD3 + CD8 + The cells were identified as a population. The results are expressed as the mean % ± SEM of Treg, CD4+ T effector cells, or CD8+ T effector cells within the CD45+ lymphocyte population.

[0626] result As shown in Figure 1, the anti-CD25 antibodies of the present invention (H07, H09, G02, E04, D01, E04-2, B05, G09, B01, C01, G01, H01, G02-2, H02, F03, D05, B07, H08, B12) can specifically bind to huCD25 expressed on the surface of transiently transfected HEK293 cells.

[0627] The ability of the present invention's anti-CD25 antibody to affect IL-2-induced effector T cell proliferation was measured. Figures 2A-B2 demonstrate that the present invention's anti-CD25 antibody did not significantly affect IL-2-induced effector T cell proliferation, while basiliximab, 7G7B6, and MA-251 all reduced effector T cell proliferation by at least approximately 50%.

[0628] Furthermore, the ability of the anti-CD25 antibody of the present invention to induce ADCP-induced cell lysis was tested. As shown in Figure 5, the anti-CD25 antibody of the present invention can induce ADCP-induced cell lysis.

[0629] Next, the anti-CD25 antibody of the present invention was tested in vitro for its ability to deplete Treg cells from anti-CD3+ anti-CD28 activated human peripheral mononuclear cells, and compared with a control antibody (huIgG1) or basiliximab. Figures 3A-3B show that the anti-CD25 antibody of the present invention depleted at least 70% of Treg cells, which is more efficient than basiliximab, which depleted approximately 50% of Treg cells. Importantly, no cell depletion was observed in either CD4+ effector T cells or CD8+ effector T cells, indicating that this effect is specific to Treg cells (Figures 4A, 4B).

[0630] In summary, these results demonstrate that the anti-CD25 antibody of the present invention has a significantly smaller impact on IL-2 activity than 7G7B6 and MA-251, and is a non-blocking antibody capable of efficiently and specifically depleting Treg cells.

[0631] Therefore, these results reveal the therapeutic potential of the antibodies of the present invention for treating cancer.

Claims

1. Isolated anti-human CD25 antibody or its antigen-binding fragment, The variable region (VH) of the heavy chain and the variable region (VL) of the light chain are one of the following combinations of complementarity-determining regions (CDR): (a) The following CD-Rs: VH-CDR1:NHAMA (Sequence ID 1); VH-CDR2:VISYDGSNKYYADSVKG (Sequence ID 7); VH-CDR3:GWNSGYD (Sequence ID 8); VL-CDR1:RASQSVNSFLN (Sequence ID 9); VL-CDR2:GTSSLQS (Sequence ID 10); and VL-CDR3:QQYTSWPWT (Sequence ID 11); or (b) The following CD-Rs: VH-CDR1:NHAMA (Sequence ID 1); VH-CDR2:VISYDGSNKYYADSVKG (Sequence ID 7); VH-CDR3:GWNSGYD (Sequence ID 8); VL-CDR1:RASQSVNSYLN (Sequence ID 12); VL-CDR2:GTNSLQS (Sequence ID 13); and VL-CDR3:QQYNSWPWT (Sequence ID 14); or (c) The following CD-Rs: VH-CDR1:NHAMA (Sequence ID 1); VH-CDR2:VISYDGSNKYYADSVKG (Sequence ID 7); VH-CDR3:GWNSGYD (Sequence ID 8); VL-CDR1:RASQSISSYLN (Sequence ID 15); VL-CDR2:GTNSLQS (Sequence ID 13); and VL-CDR3:QQYNSWPWT (Sequence ID 14); or (d) The following CD-Rs: VH-CDR1:NHAMA (Sequence ID 1); VH-CDR2:VISYDGSNKYYADSVKG (Sequence ID 7); VH-CDR3:GWNSGYD (Sequence ID 8); VL-CDR1:RASQSVSSFLN (Sequence ID 16); VL-CDR2:GTNSLQS (Sequence ID 13); and VL-CDR3:QQYTSWPWT (Sequence ID 11); or (e) The following CD-Rs: VH-CDR1:NHAMA (Sequence ID 1); VH-CDR2:VISYDGSNKYYADSVKG (Sequence ID 7); VH-CDR3:GLNSGYD (Sequence ID 17); VL-CDR1:RASQSVSKFLN (Sequence ID 18); VL-CDR2:GTNSLQS (Sequence ID 13); and VL-CDR3:QQYNSWPWT (Sequence ID 14); or (f) The following CD-Rs: VH-CDR1:NHAMA (Sequence ID 1); VH-CDR2:VISYDGSNKYYADSVKG (Sequence ID 7); VH-CDR3:GLNSGYD (Sequence ID 17); VL-CDR1:RASQNINSFLN (Sequence ID 19); VL-CDR2:GTSSLQS (Sequence ID 10); and VL-CDR3:QQYNSWPWT (Sequence ID 14); or (g) The following CDRs: VH-CDR1:NHAMA (Sequence ID 1); VH-CDR2:VISYDGSNKYYADSVKG (Sequence ID 7); VH-CDR3:GLNSGYD (Sequence ID 17); VL-CDR1:RASQNISSFLN (Sequence ID 20); VL-CDR2:GTNSLQS (Sequence ID 13); and VL-CDR3:QQYNSWPWT (Sequence ID 14); or (h) The following CD-Rs: VH-CDR1:NHAMA (Sequence ID 1); VH-CDR2:VISYDGSNKYYADSVKG (Sequence ID 7); VH-CDR3:GLNSGYD (Sequence ID 17); VL-CDR1:RASQSISFLN (Sequence ID 21); VL-CDR2:GTNSLQS (Sequence ID 13); and VL-CDR3:QQYTSWPWT (Sequence ID 11); or (i) The following CD-Rs: VH-CDR1:NHAMA (Sequence ID 1); VH-CDR2:VISYDGSNKYYADSVKG (Sequence ID 7); VH-CDR3:GLNSGYD (Sequence ID 17); VL-CDR1:RASQSVSSFLN (Sequence ID 16); VL-CDR2:GTNSLQS (Sequence ID 13); and VL-CDR3:QQYNSWPWT (Sequence ID 14); or (j) The following CD-Rs: VH-CDR1:NHAMA (Sequence ID 1); VH-CDR2:VISYDGSNKYYADSVKG (Sequence ID 7); VH-CDR3:GLNSGYD (Sequence ID 17); VL-CDR1:RASQNVSSFLN (Sequence ID 22); VL-CDR2:GTSSLQS (Sequence ID 10); and VL-CDR3:QQYTSWPWT (Sequence ID 11); or (k) The following CD-Rs: VH-CDR1:NHAMA (Sequence ID 1); VH-CDR2:VISYDGSNKYYADSVKG (Sequence ID 7); VH-CDR3:GLNSGYD (Sequence ID 17); VL-CDR1:RASQSINSFLN (Sequence ID 23); VL-CDR2:GTNSLQS (Sequence ID 13); and VL-CDR3:QQYNSWPWT (Sequence ID 14); or (l) The following CD-Rs: VH-CDR1:NHAMA (Sequence ID 1); VH-CDR2:VISYDGSNKYYADSVKG (Sequence ID 7); VH-CDR3:GLNSGYD (Sequence ID 17); VL-CDR1:RASQNINSFLN (Sequence ID 19); VL-CDR2:GTNSLQS (Sequence ID 13); and VL-CDR3:QQYTSWPWT (Sequence ID 11); or (m) The following CDRs: VH-CDR1:NHAMA (Sequence ID 1); VH-CDR2:VISYDGSNKYYADSVKG (Sequence ID 7); VH-CDR3:GLNSGYD (Sequence ID 17); VL-CDR1:RASQSVSSYLN (Sequence ID 24); VL-CDR2:GTNSLQS (Sequence ID 13); and VL-CDR3: QQYTSWPWT (Sequence ID 11) or (n) The following CD-Rs: VH-CDR1:NHAMA (Sequence ID 1); VH-CDR2:VISYDGSNKYYRDSVKG (Sequence ID 25); VH-CDR3:GLNSGYD (Sequence ID 17); VL-CDR1:RASQSISFLN (Sequence ID 21); VL-CDR2:GTNSLQS (Sequence ID 13); and VL-CDR3:QQYNSWPWT (Sequence ID 14) or (o) The following CD-Rs: VH-CDR1:NHAMA (Sequence ID 1); VH-CDR2:VISYDGSNKYYRDSVKG (Sequence ID 25); VH-CDR3:GLNSGYD (Sequence ID 17); VL-CDR1:RASQNINSFLN (Sequence ID 19); VL-CDR2:GTSSLQS (Sequence ID 10); and VL-CDR3: QQYNSWPWT (Sequence ID 14) or (p) The following CD-Rs: VH-CDR1:NHAMA (Sequence ID 1); VH-CDR2:VISYDGSNKYYRDSVKG (Sequence ID 25); VH-CDR3:GLNSGYD (Sequence ID 17); VL-CDR1:RASQNVSSFLN (Sequence ID 22); VL-CDR2:GTNSLQS (Sequence ID 13); and VL-CDR3:QQYNSWPWT (Sequence ID 14); or (q) The following CD-Rs: VH-CDR1:NHAMA (Sequence ID 1); VH-CDR2:VISYDGSNTYYADSVKG (Sequence ID 26); VH-CDR3:GLNSGYD (Sequence ID 17); VL-CDR1:RASQNVSSFLN (Sequence ID 22); VL-CDR2:GTNSLQS (Sequence ID 13); and VL-CDR3:QQYNSWPWT (Sequence ID 14); or (r) The following CDRs: VH-CDR1:NHAMA (Sequence ID 1); VH-CDR2:VISYDGDNKYYADSVKG (Sequence ID 27); VH-CDR3:GLNSGYD (Sequence ID 17); VL-CDR1:RASQSINSFLN (Sequence ID 23); VL-CDR2:GTNSLQS (Sequence ID 13); and VL-CDR3: QQYTSWPWT (Sequence ID 11) including, Isolated anti-human CD25 antibody or its antigen-binding fragment.

2. An isolated anti-human CD25 antibody or antigen-binding fragment thereof according to claim 1, which is monoclonal.

3. The isolated anti-human CD25 antibody or antigen-binding fragment according to any one of claims 1 to 2, wherein the antibody or antigen-binding fragment mediates antibody-dependent cytotoxicity, complement-dependent cytotoxicity, and / or antibody-dependent phagocytosis.

4. An isolated anti-human CD25 antibody or its antigen-binding fragment according to any one of claims 1 to 3, which is bispecific.

5. An isolated anti-human CD25 antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, which is conjugated with a drug.

6. A fusion protein comprising an isolated anti-human CD25 antibody or its antigen-binding fragment according to any one of claims 1 to 5.

7. A nucleic acid encoding an isolated anti-human CD25 antibody or its antigen-binding fragment according to any one of claims 1 to 5, or a fusion protein according to claim 6.

8. A pharmaceutical composition comprising an isolated anti-human CD25 antibody or its antigen-binding fragment according to any one of claims 1 to 5, or a fusion protein according to claim 6, and at least one pharmaceutically acceptable excipient.

9. A pharmaceutical composition according to claim 8, for use as a pharmaceutical product.

10. The pharmaceutical composition according to claim 8, for use in the treatment of cancer or infectious disease in a subject requiring treatment for cancer or infectious disease.

11. A pharmaceutical composition according to claim 9 or 10, to be used in combination with immunotherapy.

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