Anti-CD122 Antibodies, Anti-CD132 Antibodies, and Related Bispecific Binding Proteins

Abstract

Provided are bispecific CD122 / CD132 binding proteins in FIT-Ig and duobody formats prepared with antibodies capable of binding to CD122, antibodies capable of binding to CD132, and antigen-binding fragments thereof. The antibodies and bispecific binding proteins are useful for treating or preventing diseases, such as T cell dysfunction disorders or cancer.

Description

[Technical field]

[0001] The present disclosure relates to antibodies capable of binding to CD122, antibodies capable of binding to CD132, and bispecific binding proteins, such as bispecific CD122 / CD132 binding proteins (e.g., FIT-Ig and duobody formats). The antibodies and bispecific binding proteins disclosed herein may be useful for treating or preventing diseases, such as T cell dysfunction disorders or cancer. [Background technology]

[0002] IL-2 is a pleiotropic cytokine that regulates different immune cells, including T cells and natural killer (NK) cells. Two distinct receptor complexes for IL-2 have been identified on different cell types: the high affinity (K) receptor complex, which is composed of an alpha chain (IL-2Rα, also known as CD25), a beta chain (IL-2Rβ, also known as CD122) and a gamma chain (IL-2Rγ, also known as CD132). D Approximately 10 pM) IL-2 receptor complex; intermediate affinity (K D 1 nM) receptor. The high affinity complex is constitutively expressed on immunosuppressive regulatory T cells (Tregs) and transiently expressed on activated T cells, whereas the intermediate affinity receptor is typically expressed on memory phenotype (MP) CD8+ T cells and NK cells.

[0003] Heterodimerization of CD122 and CD132 is thought to be required for effective signal transduction upon binding to IL-2. Signaling occurs through several intracellular pathways, including the Janus kinase (JAK)-STAT pathway, the phosphoinositide 3-kinase (PI3K)-AKT pathway, and the mitogen-activated protein kinase (MAPK) pathway (Boyman, O. & Sprent, J. Nat. Rev. Immunol. 12, 180-190, 2012). CD25 does not directly participate in the IL-2 signaling pathway due to the lack of a cytoplasmic kinase activation domain.

[0004] Recombinant human IL-2 (rhIL-2) has been developed and approved for the treatment of metastatic melanoma and renal cell carcinoma. However, its clinical use is limited by its short half-life and severe adverse effects, including vascular leak syndrome (VLS), hypertension, and liver toxicity. According to Krieg et al. (Proc. Natl. Acad. Sci. 107, 11906-11911, 2010), vascular leak toxicity is related to the expression of high affinity IL-2 receptor complexes on vascular and pulmonary endothelial cells, leading to pulmonary edema. IL-2-induced pulmonary edema could be significantly reduced in vivo by depletion of CD25. On the other hand, the usefulness of IL-2 in cancer therapy may be further compromised by the preferential binding of IL-2 to high-affinity receptors on Treg cells, which may inhibit the expansion and activation of tumor-specific effector T cells, thus blunting antitumor efficacy (Sun, Z. et al. Nat. Commun. 10:3874, 2019). Summary of the Invention [Problem to be solved by the invention]

[0005] Various efforts have been made to design rhIL-2 variants that avoid binding to CD25 and extend half-life by PEGylation or other equivalent techniques. Although some of these designed proteins have the desired functional activity, many of them suffer from high levels of immunogenicity in vivo (Verhoef, JJF et al. Drug Discov. Today 19, 1945-1952 (2014)). Thus, there remains a need in the art to generate anti-tumor agonists of the IL-2 pathway with the desired biological activity and safety profile.

[0006] A bispecific antibody that simultaneously binds to IL-2Rβ (CD122) and IL-2Rγ (CD132) but does not bind to IL-2Rα (CD25), and thus has the activity of IL-2 while avoiding the problems associated with IL-2Rα, would be highly desirable, and would combine the biological activity of promoting the association of IL-2Rβ and IL-2Rγ and downstream signaling with the safety of an antibody molecule. [Means for solving the problem]

[0007] The present disclosure addresses the above needs by providing novel anti-CD122 antibodies, anti-CD132 antibodies, and engineered bispecific proteins that simultaneously bind IL-2Rβ (CD122) and IL-2Rγ (CD132) to induce activation of IL-2R signaling in human immune effector cells without preferentially activating T-regs, shifting the balance toward activation of effector T cells and NK cells.

[0008] In particular, in some embodiments, the disclosure provides anti-CD122 monospecific antibodies, e.g., anti-CD122 monospecific antibodies that have high binding capacity to CD122. In some embodiments, the disclosure also provides monospecific antibodies that bind to CD132, e.g., monospecific antibodies that bind to CD132 with high affinity. In some embodiments, the disclosure also provides CD122 / CD132 bispecific binding proteins that have the ability to bind to the medium affinity IL-2Rβγ receptor, which consists preferentially of CD122 and CD132. In some embodiments, the CD122 / CD132 bispecific binding proteins are in the format of Fabs-in-Tandem immunoglobulins (FIT-Ig) as described in PCT International Publication WO2015 / 103072, or in the format of a duobody as described in Labrijn et al., Proc Natl Acad Sci US A. (2013) 110(13):5145-50. In some embodiments, the bispecific multivalent binding proteins described herein are useful for stimulating signaling upon binding to a complex comprising CD122 and CD132, for preferentially stimulating the proliferation of effector T cells and / or NK cells over regulatory T cells, and for improving anti-tumor immunity of effector T cells and / or NK cells in vivo and / or in vitro. In some embodiments, the bispecific multivalent binding proteins described herein are useful for reducing tumor burden / growth / cell expansion.

[0009] In some embodiments, the disclosure also provides methods of making and using the anti-CD122 and anti-CD132 antibodies and CD122 / CD132 bispecific binding proteins described herein. Also disclosed are various compositions that may be used in methods of treating or preventing disorders in individuals associated with impaired effector T cell and / or NK cell function and / or down-regulation of immune responses. [Brief description of the drawings]

[0010] [Figure 1] FIG. 1 is a schematic diagram of the FIT-Ig structure. [Diagram 2] FIG. 2 is a schematic diagram of the Duobody structure. [Diagram 3] FIG. 3 shows that the CD122 / CD132 bispecific antibody FIT2019-86b binds to recombinant CD122 (triangles) and CD132 (diamonds) proteins but not recombinant CD25 (filled circles) protein. [Figure 4A] Figures 4A and 4B show that FIT2019-86b (triangles) and Duo2019-86 (inverted triangles) bind to cell surface CD122 (Figure 4A) and CD132 (Figure 4B). [Figure 4B] Figures 4A and 4B show that FIT2019-86b (triangles) and Duo2019-86 (inverted triangles) bind to cell surface CD122 (Figure 4A) and CD132 (Figure 4B). [Figure 4C] Figures 4C and 4D show that huFIT2019-86b-51 binds to cell surface CD122 (Figure 4C) and CD132 (Figure 4D). An irrelevant hIgG was used as a negative control. [Figure 4D] Figures 4C and 4D show that huFIT2019-86b-51 binds to cell surface CD122 (Figure 4C) and CD132 (Figure 4D). An irrelevant hIgG was used as a negative control. [Diagram 5] FIG. 5 shows that the CD122 / CD132 complex is activated by FIT2019-86b (triangles) but not by Duo2019-86 (inverted triangles). [Figure 6A] FIG. 6A shows activation of pSTAT5 on CD8+ T cells by FIT2019-86b (squares) and the reference molecules, neo2 / 15 (diamonds), H9 (open circles) and hIL-2 (triangles). [Figure 6B] FIG. 6B shows activation of pSTAT5 on CD8+ T cells by huFIT2019-86b-51 (filled circles) and reference molecules, neo2 / 15 (inverted triangles), H9 (filled squares) and hIL-2 (triangles). [Figure 7A] FIG. 7A shows activation of pSTAT5 on Treg cells by FIT2019-86b (squares) and reference molecules, neo2 / 15 (diamonds), H9 (open circles) and hIL-2 (triangles). [Figure 7B] Figure 7B shows activation of pSTAT5 on Treg cells by huFIT2019-86b-51 (filled circles) and reference molecules, neo2 / 15 (inverted triangles), H9 (filled squares) and hIL-2 (triangles). [Figure 8A-C] Figures 8A-8C show the proliferation profiles of CD8+ T cells (Figure 8A), CD4+ T cells (Figure 8B), and Treg cells (Figure 8C) upon exposure to FIT2019-86b, huFIT2019-86b-32 and reference molecules, neo2 / 15, H9, and IL-2 as indicated under each grouped bar. [Fig. 8D-F] Figures 8D-8F show the proliferation profiles of CD8+ T cells (Figure 8D), CD4+ T cells (Figure 8E), and Treg cells (Figure 8F) upon exposure to huFIT2019-86b-51 and reference molecules, neo2 / 15, H9, and IL-2 as indicated under each grouped bar. Media and CD3 / CD28 beads were used as controls. [Figure 9] FIG. 9 shows tumor growth curves of melanoma / PBMC combination implantation model in immunodeficient M-NSG mice treated with FIT2019-86b (inverted triangles) and vehicle control (filled circles). [Figure 10] FIG. 10 shows weight change in a melanoma / PBMC combination transplant model in immunodeficient M-NSG mice treated with FIT2019-86b (inverted triangles) and vehicle control (filled circles). [Figure 11] FIG. 11 shows tumor growth curves of a melanoma / PBMC combination transplant model in immunodeficient NCG mice treated with huFIT2019-86b-51 at four dosages of 1 mg / kg, 0.3 mg / kg, 0.1 mg / kg, and 0.03 mg / kg, as well as vehicle control. [Figure 12]FIG. 12 shows the body weight change of a melanoma / PBMC combination transplant model in immunodeficient NCG mice treated with huFIT2019-86b-51 at four dosages of 1 mg / kg, 0.3 mg / kg, 0.1 mg / kg, and 0.03 mg / kg, as well as vehicle control. [Figure 13] FIG. 13 shows tumor growth curves of the NSCLC cell / PBMC combination transplant model in immunodeficient NCG mice treated with huFIT2019-86b-51 at two dosages, 1 mg / kg and 0.3 mg / kg, and vehicle control. [Figure 14] FIG. 14 shows the body weight change of an NSCLC cell / PBMC combination transplant model in immunodeficient NCG mice treated with huFIT2019-86b-51 at two dosages, 1 mg / kg and 0.3 mg / kg, and vehicle control. [Figure 15] FIG. 15 shows the weight change in mice treated with FIT2019-86b (inverted triangles), IL-2 (squares) and vehicle control (filled circles). [Figure 16] Figure 16 shows the ratio of CD8+ to CD4+ T cells among PBMCs on days 1 (black), 4 (white) and 7 (horizontal stripes) after injection of FIT2019-86b, IL-2 or PBS control. Each group of bars represents data from an individual mouse and all data were normalized to the respective ratio on day 1 of the same mouse. [Figure 17A-B] Figure 17 shows the ability of huFIT2019-86b-51 and reference molecules to compete with the binding of IL2 to IL2Rβ. Figure 17A: Injection of IL2Rβ (C4.I - C4.R), IL2Rβ + huFIT2019-86b-51 (C5.I - C5.R) and running buffer (C6.I - C6.R) over a chip with immobilized IL2; Figure 17B: Injection of IL2Rβ (C4.I - C4.R), IL2Rβ + IL2 (C5.I - C5.R) and running buffer (C6.I - C6.R) over a chip with immobilized IL2. [Fig. 17C-D]Figure 17C: Injection of IL2Rβ (C4.I - C4.R), IL2Rβ + H9 (C5.I - C5.R) and running buffer (C6.I - C6.R) over chips with immobilized IL2; and Figure 17D: Injection of IL2Rβ (C4.I - C4.R), IL2Rβ + neo2 / 15 (C5.I - C5.R) and running buffer (C6.I - C6.R) over chips with immobilized IL2. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] The present disclosure relates to anti-CD122 antibodies, anti-CD132 antibodies, antigen-binding portions thereof, and bispecific binding proteins, such as FIT-Ig or duobody that binds both CD122 and CD132. Various aspects of the present disclosure relate to anti-CD122 and anti-CD132 antibodies and antibody fragments thereof, FIT-Ig and duobody binding proteins that bind human CD122 and human CD132, and pharmaceutical compositions thereof, as well as nucleic acids, recombinant expression vectors, and host cells for making such antibodies, functional antibody fragments, and binding proteins. Methods for improving effector T cell and / or NK cell function and / or upregulating immune responses, and treating diseases, particularly T cell dysfunction disorders or cancer, in vitro or in vivo using the antibodies, functional antibody fragments, and bispecific binding proteins of the present disclosure are also encompassed by the present disclosure.

[0012] definition Unless otherwise specified herein, scientific and technical terms used in connection with this disclosure shall have the meanings commonly understood by those skilled in the art. In the event of any potential ambiguity between this specification and dictionary or external definitions, the definitions provided herein shall prevail. Furthermore, unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular. In this application, the use of "or" means "and / or" unless otherwise indicated. Furthermore, the use of the term "including" as well as other forms such as "includes" and "included" is not limiting. Similarly, terms such as "element" or "component" encompass elements and components comprising one unit as well as elements and components comprising multiple subunits, unless otherwise specified.

[0013] As used herein, the amino acid positions of all heavy and light chain constant regions and domains are numbered according to the Kabat numbering system as described in Kabat, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991), and are referred to herein as "numbering according to Kabat." Specifically, the Kabat numbering system (see pages 647-660 of Kabat, et al., 1991) is used for the light chain constant domains CL of the kappa and lambda isotypes, and the Kabat EU index numbering system (see pages 661-723 of Kabat, et al., 1991) is used for the heavy chain constant domains (CH1, hinge, CH2 and CH3, which is further clarified herein by referring to the numbering according to the Kabat EU index in this case).

[0014] General information regarding human immunoglobulin light and heavy chain sequences is also provided in Kabat et al., 1991.

[0015] The term "interleukin-2" or "IL-2," as used herein, unless otherwise indicated, refers to any native IL-2 derived from any vertebrate source, such as mammals, for example, primates (e.g., humans) and rodents (e.g., mice and rats). The term encompasses unprocessed IL-2, as well as any form of IL-2 that results from processing within the cell. Unprocessed human IL-2 further includes an N-terminal 20 amino acid signal peptide that is not present in the mature IL-2 molecule.

[0016] The term "CD25" or "IL-2 receptor alpha" as used herein refers to any native CD25 from any vertebrate source, such as mammals, such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses "full-length," unprocessed CD25, and any form of CD25 that results from processing within the cell. The term also encompasses naturally occurring variants of CD25, such as splice variants or allelic variants. In a specific embodiment, the CD25 is human CD25.

[0017] As used herein, the term "high affinity IL-2 receptor" refers to the receptor γ-subunit (common cytokine receptor γ-subunit, γ c (also known as CD132), the receptor β-subunit (also known as CD122) and the receptor α-subunit (also known as CD25).

[0018] The term "intermediate affinity IL-2 receptor" or "IL-2 receptor βγ" refers to an IL-2 receptor that does not contain the α-subunit, but contains only the γ- and β-subunits.

[0019] "Conventional CD4 +The term "T cells" refers to CD4 T cells other than regulatory T cells. + Refers to T cells. Conventional CD4 + Memory T cells are characterized by expressing CD4 and CD3 but not FOXP3. + "Memory T cells" are the same as conventional CD4 + A subset of T cells expressing CD45RA, known as "conventional CD4 + In contrast to "naive T cells," they are further characterized by not expressing CD45RA.

[0020] The term "regulatory T cells" or "Treg cells" refers to CD4 T cells that can suppress the responses of other T cells (effector T cells). + Refers to a special type of T cell. Treg cells are characterized by expression of CD4, the α subunit of the IL-2 receptor (CD25), and the transcription factor forkhead box P3 (FOXP3) (Sakaguchi, Annu Rev Immunol 22, 531-62 (2004)). Treg cells play an important role in inducing and maintaining peripheral self-tolerance to antigens, including those expressed by tumors.

[0021] The term "isolated protein" or "isolated polypeptide" refers to a protein or polypeptide that, by reason of the origin or source of its derivation, is not associated with naturally associated components which accompany it in its natural state, is substantially free of other proteins from the same species, is expressed by cells from a different species, or is non-naturally occurring. A polypeptide that is chemically synthesized or synthesized in a cellular system different from the cell from which it is naturally derived is "isolated" from its naturally associated components. A protein can also be rendered substantially free of naturally associated components by isolation, using protein purification techniques well known in the art.

[0022] The terms "specific binding" or "specifically binds" with respect to the interaction of an antibody, binding protein, or peptide with a second chemical species means that the interaction is dependent on the presence of a particular structure (e.g., an antigenic determinant or epitope) on the second chemical species. For example, an antibody recognizes and binds to a specific protein structure rather than proteins in general. Generally, if an antibody is specific for epitope "A", in a reaction containing labeled "A" and an antibody, the presence of a molecule containing epitope A (or free, unlabeled A) reduces the amount of labeled A that binds to the antibody.

[0023] The term "antibody" refers broadly to any immunoglobulin (Ig) molecule composed of four polypeptide chains, two heavy (H) chains and two light (L) chains, or any functional fragment, mutant, variant, or derivative thereof that retains the essential epitope binding properties of an Ig molecule. Such mutant, variant, or derivative antibody formats are known in the art, and non-limiting embodiments are discussed below.

[0024] In a full-length antibody, each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains, CH1, CH2 and CH3. Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is composed of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged in the following order from amino terminus to carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The first, second and third CDRs of the VH domain are commonly enumerated as CDR-H1, CDR-H2 and CDR-H3. Similarly, the first, second and third CDRs of a VL domain are commonly enumerated as CDR-L1, CDR-L2 and CDR-L3. Immunoglobulin molecules may be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass.

[0025] The term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain, which can be generated by papain digestion of an intact antibody. The Fc region may be a native sequence Fc region or a variant Fc region. The Fc region of an immunoglobulin generally comprises two constant domains, namely a CH2 domain and a CH3 domain, and optionally, in the case of the Fc region of an IgM and IgE antibody, for example, a CH4 domain. The Fc regions of IgG, IgA and IgD antibodies comprise a hinge region, a CH2 domain and a CH3 domain. In contrast, the Fc regions of IgM and IgE antibodies lack a hinge region, but comprise a CH2 domain, a CH3 domain and a CH4 domain. Variant Fc regions having substitutions of amino acid residues in the Fc portion to alter antibody effector functions are known in the art (see, for example, Winter et al., U.S. Pat. Nos. 5,648,260 and 5,624,821). The Fc portion of an antibody mediates one or more effector functions, such as cytokine induction, ADCC, phagocytosis, complement-dependent cytotoxicity (CDC), and / or half-life / clearance rate of the antibody and antigen-antibody complex. In some cases, these effector functions are desirable for therapeutic antibodies, while in other cases, they may be unnecessary or even harmful depending on the therapeutic purpose. Certain human IgG isotypes, particularly IgG1 and IgG3, mediate ADCC and CDC through binding to FcγR and complement C1q, respectively. In yet another embodiment, at least one amino acid residue is replaced in the constant region of the antibody, e.g., the Fc region of the antibody, such that the effector function of the antibody is altered. Dimerization of two identical heavy chains of an immunoglobulin is mediated by dimerization of the CH3 domains and stabilized by disulfide bonds in the hinge region that connects the CH1 constant domain to the Fc constant domains (e.g., CH2 and CH3). The anti-inflammatory activity of IgG depends on sialylation of the N-linked glycans of the IgG Fc fragment. The precise glycan requirements for anti-inflammatory activity were determined such that a suitable IgG1 Fc fragment could be engineered, thereby generating a fully recombinant sialylated IgG1 Fc with greatly enhanced potency (see Anthony et al., Science, 320:373-376 (2008)).

[0026] The terms "antigen-binding portion," "antigen-binding fragment," and "functional fragment" in the context of antibodies are used interchangeably and refer to one or more fragments of an antibody that retain the ability to specifically bind to an antigen, i.e., the same antigen (e.g., CD122, or CD132) as the full-length antibody from which the portion or fragment is derived. It has been shown that the antigen-binding function of an antibody can be exerted by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include: (i) a Fab fragment (a monovalent fragment consisting of the VL, VH, CL, and CH1 domains); (ii) an F(ab')2 fragment (a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region); (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment comprising a single variable domain (Ward et al., Nature, 341:544-546 (1989); PCT Publication No. WO 90 / 05144); and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be linked by a synthetic linker, which allows them to be produced using recombinant methods as a single protein chain in which the VL and VH regions pair to form a monovalent molecule (known as single-chain Fv (scFv); see, e.g., Bird et al., Science, 242: 423-426 (1988); and Huston et al., Proc. Natl. Acad. Sci. USA, 85: 5879-5883 (1988)). Such single-chain antibodies are also encompassed within the term "antigen-binding portion" of an antibody and equivalent terms given above. Other forms of single-chain antibodies, such as diabodies, are also encompassed. Diabodies are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but use a linker that is too short to allow pairing between the two domains on the same chain, thereby allowing the domains to pair with the complementary domains of another chain and form two antigen-binding sites (see, e.g., Holliger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993)).Such antibody binding moieties are known in the art (Kontermann and Dubel, eds., Antibody Engineering (Springer-Verlag, New York, 2001), p. 790 (ISBN 3-540-41354-5)). In addition, single chain antibodies also include "linear antibodies" comprising a pair of tandem Fv segments (VH-CH1-VH-CH1) which form a pair of antigen-binding regions together with complementary light chain polypeptides. (Zapata et al., Protein Eng., 8(10): 1057-1062 (1995); and U.S. Patent No. 5,641,870).

[0027] Immunoglobulin constant (C) domain refers to either the heavy (CH) or light (CL) chain constant domain. Murine and human IgG heavy and light chain constant domain amino acid sequences are known in the art.

[0028] The term "monoclonal antibody" or "mAb" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic determinant (epitope). Furthermore, in contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each mAb is directed against a single determinant on the antigen. The modifier "monoclonal" should not be construed as requiring production of the antibody by any particular method.

[0029] The term "human sequence" refers to a sequence that is or is derived from a human immunoglobulin sequence, with respect to the light chain constant domain CL, the heavy chain constant domain CH, and the Fc region of an antibody or binding protein according to the present application. The human sequence of the present disclosure may be a native human sequence or a variant thereof that contains one or more (e.g., up to 20, 15, 10) amino acid residue changes.

[0030] The term "chimeric antibody" refers to an antibody that contains heavy and light chain variable region sequences from one species and constant region sequences from another species, e.g., an antibody having murine heavy and light chain variable regions linked to human constant regions.

[0031] The term "CDR-grafted antibody" refers to an antibody that comprises heavy and light chain variable region sequences from one species, but in which the sequences of one or more of the VH and / or VL CDR regions have been replaced with CDR sequences from another species, e.g., an antibody having human heavy and light chain variable regions in which one or more of the human CDRs have been replaced with murine CDR sequences.

[0032] The term "humanized antibody" refers to an antibody that comprises heavy and light chain variable region sequences from a non-human species (e.g., mouse), but in which at least a portion of the VH and / or VL sequences have been altered to be more "human-like", i.e., more similar to human germline variable sequences. One type of humanized antibody is a CDR-grafted antibody, in which CDR sequences from a non-human species (e.g., mouse) have been introduced into human VH and VL framework sequences. A humanized antibody is an antibody or variant, derivative, analog, or fragment thereof that immunospecifically binds to an antigen of interest and comprises framework and constant regions that have substantially the amino acid sequence of a human antibody, but a complementarity determining region (CDR) that has substantially the amino acid sequence of a non-human antibody. As used herein, the term "substantially" in the context of a CDR refers to a CDR that has an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of the non-human antibody CDR. A humanized antibody comprises substantially all of at least one, and typically two, variable domains (Fab, Fab', F(ab')2, Fv), in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin (i.e., donor antibody) and all or substantially all of the framework regions are of a human immunoglobulin consensus sequence. In one embodiment, the humanized antibody also comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. In some embodiments, the humanized antibody comprises both a light chain and at least the variable domains of a heavy chain. The antibody may also comprise the CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. In some embodiments, the humanized antibody comprises only a humanized light chain. In some embodiments, the humanized antibody comprises only a humanized heavy chain. In a specific embodiment, the humanized antibody comprises only a humanized variable domain of the light chain and / or a humanized heavy chain.

[0033] The humanized antibody can be selected from any immunoglobulin class, including IgM, IgG, IgD, IgA, and IgE, and any isotype, including but not limited to IgG1, IgG2, IgG3, and IgG4. The humanized antibody can comprise sequences from more than one class or isotype, and particular constant domains can be selected to optimize desired effector functions using techniques well known in the art.

[0034] The framework and CDR regions of a humanized antibody need not correspond exactly to the parental sequences (e.g., donor antibody CDRs), or the acceptor framework can be mutagenized by substitution, insertion and / or deletion of at least one amino acid residue such that the CDR or framework residue at that site does not correspond to either the donor antibody or the consensus framework. However, in exemplary embodiments, such mutations will not be extensive. Usually, at least 80%, at least 85%, at least 90%, or at least 95% of the humanized antibody residues will correspond to those of the parental FR and CDR sequences. Back mutations at specific framework positions that restore the same amino acid that appears at that position in the donor antibody are often used to preserve a particular loop structure or to orient the CDR sequence correctly for contact with the target antigen.

[0035] The term "CDR" refers to the complementarity determining region in an antibody variable domain sequence. There are three CDRs in each of the heavy and light chain variable regions, which are referred to as CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3. The term "CDR set" as used herein refers to a group of three CDRs present in a single variable region capable of binding to an antigen. The exact boundaries of these CDRs have been defined differently according to various systems. The system described by Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Maryland (1987) and (1991)) not only provides an unambiguous residue numbering system that is applicable to any variable region of an antibody, but also provides the exact residue boundaries that define the three CDRs.

[0036] The art-recognized term "Kabat numbering" refers to a system of numbering amino acid residues that are more variable (i.e., hypervariable) than other amino acid residues in the heavy and light chain variable regions of an antibody or antigen-binding portion thereof. See Kabat et al., Ann. NY Acad. Sci., 190: 382-391 (1971); and Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242 (1991).

[0037] The growth and analysis of extensive public databases of amino acid sequences of variable heavy and light chain regions over the past 20 years has allowed the typical boundaries between framework regions (FR) and CDR sequences within variable region sequences to be understood and allows one of skill in the art to precisely determine CDRs according to Kabat numbering, Chothia numbering, or other systems. See, e.g., Martin, "Protein Sequence and Structure Analysis of Antibody Variable Domains," Kontermann and Dubel, eds., Antibody Engineering (Springer-Verlag, Berlin, 2001), chapter 31, pages 432-433.

[0038] The term "multivalent binding protein" refers to a binding protein that contains two or more antigen binding sites. Multivalent binding proteins, in certain cases, are engineered to have three or more antigen binding sites and are generally not naturally occurring antibodies.

[0039] The term "bispecific binding protein" (which may be used interchangeably with the term "bispecific antibody" unless otherwise specified) refers to a binding protein capable of binding to two targets of different specificity. FIT-Ig of the present disclosure contains four antigen binding sites and is typically a tetravalent binding protein. Duobodies of the present disclosure have two antigen binding sites and are typically bivalent binding proteins. FIT-Ig or duobodies according to the present disclosure bind to both CD122 and CD132 and are bispecific.

[0040] FIT-Ig, which contains two long (heavy) VCVC-Fc chain polypeptides and four short (light) VC chain polypeptides, forms a hexamer exhibiting four Fab antigen binding sites (VH-CH1 and VL-CL pairs, sometimes denoted as VH-CH1::VL-CL). Each half of FIT-Ig contains a heavy chain polypeptide and two light chain polypeptides, and the complementary immunoglobulin pairing of the VH-CH1 and VL-CL elements of the three chains results in two Fab structure antigen binding sites arranged in tandem. In the present disclosure, the immunoglobulin domains containing the Fab elements are preferably fused directly in the heavy chain polypeptide without the use of an interdomain linker. That is, the N-terminal VC element of the long (heavy chain) polypeptide is fused at its C-terminus directly to the N-terminus of another VC element, which in turn is linked to the C-terminal Fc region. In a bispecific FIT-Ig molecule, the tandem Fab elements may react with different antigens. Each Fab antigen-binding site contains a heavy chain variable domain and a light chain variable domain, with a total of six CDRs per antigen-binding site.

[0041] A description of the design, expression and characterization of FIT-Ig molecules is provided in PCT Publication WO 2015 / 103072. One example of such a FIT-Ig molecule comprises a heavy chain and two distinct light chains. The heavy chain comprises a VH B Structural formula VL A -CL-VH B -CH1-Fc (i.e., "format LH") or CH1 is VL B The structural formula VH is directly fused to A -CH1-VL B -CL-Fc (i.e., "format HL"), and the two light chain polypeptides of FIT-Ig correspond to the formula VH A -CH1 and VL B -CL (for "Format LH"), or VL A -CL and VH B -CH1 (for "format HL"); where VL A is the variable light domain from the parent antibody that binds antigen A, and VLB is the variable light domain from the parent antibody that binds antigen B, and VH A is the variable heavy domain from the parent antibody that binds antigen A, and VH B is a variable heavy domain from a parent antibody that binds antigen B, CL is a light chain constant domain, CH1 is a heavy chain constant domain, and Fc is an immunoglobulin Fc region (e.g., the C-terminal hinge-CH2-CH3 portion of the heavy chain of an IgG1 antibody). In a bispecific FIT-Ig embodiment, antigen A and antigen B are different antigens or different epitopes of the same antigen. In the present disclosure, one of A and B is CD122 and the other is CD132, e.g., A is CD122 and B is CD132.

[0042] The duobody binding proteins of the present disclosure comprise Fc regions modified as described in Labrijn et al., Proc Natl Acad Sci US A. (2013) 110(13):5145-50, referred to as the "Duobody" format. In some embodiments, one of the CH3 regions comprises the substitution K409R and the other CH3 region of the Fc region comprises the substitution F405L.

[0043] As used herein, the term "k on (also "K on ", "k on "), as known in the art, refers to the on rate constant for association of a binding protein (e.g., an antibody) to an antigen to form an association complex, e.g., an antibody / antigen complex. As used interchangeably herein, "k on " is also known by the term "association rate constant" or "ka". This value indicates the rate of binding of an antibody to its target antigen or the rate of complex formation between an antibody and an antigen, as shown by the formula below: Antibody (“Ab”) + Antigen (“Ag”) → Ab-Ag

[0044] As used herein, the term "k off(also "Koff", "koff"), as known in the art, refers to the off rate constant or "dissociation rate constant" for dissociation of a binding protein (e.g., an antibody) from an association complex (e.g., an antibody / antigen complex). This value indicates the rate of dissociation of an antibody from its target antigen or the separation of the Ab-Ag complex into free antibody and antigen over time, as shown by the formula below: Ab+Ag←Ab-Ag

[0045] As used herein, the term "K D " (also "Kd") refers to the "equilibrium dissociation constant", measured by titration at equilibrium or by the association rate constant (k on ) to determine the dissociation rate constant (k off The association rate constant (k on ), dissociation rate constant (k off ) and the equilibrium dissociation constant (K D ) is used to express the binding affinity of an antibody to an antigen. Methods for determining association and dissociation rate constants are well known in the art. The use of fluorescence-based techniques offers high sensitivity and the ability to test samples in physiological buffers at equilibrium. Other experimental techniques and instruments can be used, such as the WAVEsystem (grating-coupled interferometry (GCI) assay (Creoptix AG, Switzerland), the BIAcore® (Biomolecular Interaction Analysis) assay (BIAcore International AB, Uppsala, Sweden), etc. Biolayer Interferometry (BLI), using, for example, the Octet® RED96 system (Pall ForteBio LLC), is another affinity assay technique. Additionally, the KinExA® (Kinetic Exclusion Assay) assay (available from Sapidyne Instruments, Boise, Idaho) can also be used.

[0046] The term "isolated nucleic acid" means a polynucleotide that is not associated, by human intervention, with all or a portion of the polynucleotides with which it is found in nature, operably linked to polynucleotides with which it is not naturally linked, or that does not occur in nature as part of a larger sequence (e.g., of genomic, cDNA, or synthetic origin, or a combination of portions thereof).

[0047] As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, in which additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell after introduction into the host cell, and thereby are replicated along with the host genome. In addition, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). In general, expression vectors that are useful in recombinant DNA techniques are often in the form of plasmids. As plasmids are the most commonly used form of vector, "plasmid" and "vector" can be used interchangeably herein. However, the disclosure is intended to include other forms of expression vectors, such as viral vectors (eg, replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

[0048] The term "operably linked" refers to a juxtaposition in which the described elements are in a relationship that allows them to function in their intended manner. A control sequence "operably linked" to a coding sequence is linked in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences. "Operatively linked" sequences include both expression control sequences that are contiguous with a gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest. As used herein, the term "expression control sequences" refers to polynucleotide sequences that are necessary to effect the expression and processing of the coding sequences to which they are linked. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals, such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequences); sequences that enhance protein stability; and, where appropriate, sequences that enhance protein secretion. The nature of such control sequences varies depending on the host organism. In prokaryotes, such control sequences generally include promoters, ribosomal binding sites, and transcription termination sequences. In eukaryotes, generally, such control sequences include promoters and transcription termination sequences. The term "control sequence" is intended to include elements whose presence is essential for expression and processing, and can also include additional elements whose presence is advantageous, for example, leader sequences and fusion partner sequences.

[0049] "Transformation", as defined herein, refers to any process by which exogenous DNA enters a host cell. Transformation can occur under natural or artificial conditions using a variety of methods well known in the art. Transformation can rely on any known method for inserting exogenous nucleic acid sequences into prokaryotic or eukaryotic host cells. The method is selected based on the host cell to be transformed and includes, but is not limited to, transfection, viral infection, electroporation, lipofection, and particle bombardment. Cells thus "transformed" include stably transformed cells in which the inserted DNA is capable of replicating either as an autonomously replicating plasmid or as part of the host chromosome. Such cells also include cells that transiently express the inserted DNA or RNA for a limited period of time.

[0050] The term "recombinant host cell" (or simply "host cell") refers to a cell into which exogenous DNA has been introduced. In one embodiment, a host cell (such as a host cell described in U.S. Pat. No. 7,262,028) contains two or more (e.g., a plurality) nucleic acids encoding an antibody. Such terms refer not only to the particular subject cell, but also to the progeny of such a cell. Since certain modifications may occur in subsequent generations due to mutations or environmental influences, in fact, such progeny may not be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein. In one embodiment, host cells include prokaryotic and eukaryotic cells selected from any of the kingdoms of life. In another embodiment, eukaryotic cells include protist, fungal, plant and animal cells. In another embodiment, host cells include, but are not limited to, the prokaryotic cell line Escherichia coli; the mammalian cell lines CHO, HEK293, Jurkat, COS, NS0, SP2, and PER.C6; the insect cell line Sf9; and the fungal cell yeast (Saccharomyces cerevisiae).

[0051] As used herein, the term "effective amount" refers to an amount of a therapy that is sufficient to reduce or ameliorate the severity and / or duration of a disorder or one or more symptoms thereof; prevent the progression of a disorder; cause regression of a disorder; prevent the recurrence, occurrence or progression of one or more symptoms associated with a disorder; detect a disorder; or enhance or improve the prophylactic or therapeutic effects of another therapy (e.g., a prophylactic or therapeutic agent).

[0052] The antibodies, functional fragments thereof, and binding proteins of the present disclosure may be purified (for the intended use) by using one or more of the various methods and materials available in the art for purifying antibodies and binding proteins, including, but not limited to, affinity chromatography (e.g., using resins, particles, or membranes bound to Protein A, Protein G, Protein L, or specific ligands of the antibody, functional fragment thereof, or binding protein), ion exchange chromatography (e.g., using ion exchange particles or membranes), hydrophobic interaction chromatography ("HIC"; e.g., using hydrophobic particles or membranes), ultrafiltration, nanofiltration, diafiltration, size exclusion chromatography ("SEC"), low pH treatment (to inactivate contaminating viruses), and combinations thereof to obtain a purity acceptable for the intended use. A non-limiting example of a low pH treatment to inactivate contaminating viruses includes lowering the pH of a solution or suspension containing an antibody, functional fragment thereof, or binding protein of the disclosure to pH 3.5 using 0.5 M phosphoric acid at 18° C. to 25° C. for 60 to 70 minutes.

[0053] Standard techniques can be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques can be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The techniques and procedures described above can generally be performed by conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout the specification. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).

[0054] Anti-CD122 and anti-CD132 monospecific antibodies The anti-CD122 and anti-CD132 antibodies of the present disclosure can be produced by any of a number of techniques known in the art, for example, by expression from a host cell in which expression vectors encoding the heavy and light chains have been transfected into the host cell by standard techniques. The various forms of the term "transfection" are intended to encompass a wide range of techniques commonly used for the introduction of exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, etc. Although the antibodies of the present disclosure can be expressed in either prokaryotic or eukaryotic host cells, expression of the antibodies in eukaryotic cells, for example mammalian host cells, is specifically contemplated, as they are widely used to assemble and secrete correctly folded and immunologically active antibodies.

[0055] In some embodiments, mammalian host cells for expressing recombinant antibodies of the disclosure include Chinese hamster ovary cells (CHO cells) (including dhfr- CHO cells, used with a DHFR selection marker (described in Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77: 4216-4220 (1980)), e.g., as described in Kaufman and Sharp, J. Mol. Biol., 159: 601-621 (1982)), NS0 myeloma cells, COS cells, and SP2 cells. When a recombinant expression vector encoding an antibody gene is introduced into a mammalian host cell, the antibody is produced by culturing the host cell for a period of time sufficient to allow for expression of the antibody in the host cell, or even secretion of the antibody into the culture medium in which the host cell is grown. The antibody can be recovered from the culture medium using standard protein purification methods.

[0056] Host cells can also be used to produce functional antibody fragments, such as Fab fragments or scFv molecules. It will be understood that variations on the above procedures fall within the scope of the present disclosure. For example, it may be desirable to transfect host cells with DNA encoding functional fragments of either the light and / or heavy chains of the antibodies of the present disclosure. Recombinant DNA technology can also be used to remove some or all of the DNA encoding either or both of the light and heavy chains that is not necessary for binding to the antigen of interest. Molecules expressed from such truncated DNA molecules are also encompassed by the antibodies of the present disclosure.

[0057] In an exemplary system for recombinant expression of the antibody, or antigen-binding portion thereof, of the present disclosure, a recombinant expression vector encoding both the antibody heavy chain and the antibody light chain is introduced into dhfr- CHO cells by calcium phosphate-mediated transfection. Within the recombinant expression vector, the antibody heavy and light chain genes are each operably linked to a CMV enhancer / AdMLP promoter regulatory element to drive high level transcription of the genes. The recombinant expression vector also carries a DHFR gene, allowing for the selection of CHO cells that have been transfected with the vector using methotrexate selection / amplification. The selected transfected host cells are cultured to allow expression of the antibody heavy and light chains, and intact antibody is recovered from the culture medium. Standard molecular biology techniques are used to prepare the recombinant expression vector, transfect the host cells, select for transfectants, culture the host cells, and recover the antibody from the culture medium. The present disclosure further provides a method of producing a recombinant anti-CD122 or anti-CD132 antibody of the present disclosure by culturing a transfected host cell of the present disclosure in a suitable culture medium until a recombinant antibody of the present disclosure is produced. The method can further include isolating the recombinant antibody from the culture medium.

[0058] Anti-CD122 antibody In some embodiments, the disclosure provides proteins that bind to CD122 at the extracellular domain of CD122. In some embodiments, the present disclosure discloses an isolated anti-CD122 antibody or antigen-binding fragment thereof that specifically binds to CD 122. In further embodiments, the anti-CD122 antibody or antigen-binding fragment thereof comprises a set of six CDRs: CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, where: CDR-H1 comprises the sequence DYVIS (SEQ ID NO: 44), CDR-H2 comprises the sequence EIYPGDGNTYYNEMFKG (SEQ ID NO: 45), CDR-H3 comprises the sequence GSYTYDNYAMDF (SEQ ID NO: 46), CDR-L1 comprises the sequence RSSQNIVHSNGNTYLE (SEQ ID NO: 50), CDR-L2 comprises the sequence KVSNRFS (SEQ ID NO: 51), CDR-L3 comprises the sequence FQGSHIPWT (SEQ ID NO: 52), The CDRs are defined according to the Kabat numbering system.

[0059] In some embodiments, the anti-CD122 antibody, or antigen-binding fragment thereof, comprises at least one, two, three, four, but not more than five residue modifications within the CDR sequences of SEQ ID NOs: 44-46 and 50-52. The amino acid modifications can be amino acid substitutions, deletions, and / or additions, e.g., conservative substitutions.

[0060] In one embodiment, an anti-CD122 antibody or antigen-binding fragment thereof according to the present disclosure comprises CDR-H1, CDR-H2, and CDR-H3 of the heavy chain variable domain VH of SEQ ID NO: 3, and CDR-L1, CDR-L2, and CDR-L3 of the light chain variable domain VL of SEQ ID NO: 4. The CDRs can be determined by one skilled in the art using the most widely used CDR definition schemes, e.g., the definitions according to Kabat, Chothia, or IMGT.

[0061] In one embodiment, an anti-CD122 antibody, or antigen-binding fragment thereof, according to the present disclosure comprises a heavy chain variable domain, VH, and a light chain variable domain, VL, where the VH domain comprises a sequence of SEQ ID NO:3 or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto; and / or The VL domain comprises the sequence of SEQ ID NO:4 or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto.

[0062] In some embodiments, an anti-CD122 antibody comprising a VH sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to a reference sequence while retaining the ability to bind to CD122 with the same or improved binding characteristics, e.g., Kd. In certain embodiments, the substitutions, insertions, or deletions occur in regions other than the CDRs (i.e., in the FRs). Optionally, the anti-CD122 antibody comprises a Q1E mutation compared to the VH sequence of SEQ ID NO: 3 to eliminate N-terminal pyroglutamic acid formation. In some embodiments, the anti-CD122 antibodies contain "DG" (Asp-Gly), "NT" (Asn-Thr), and "NG" (Asn-Gly) in CDR-H2 and / or CDR-L1 to mitigate post-translational modifications (PTMs) that may lead to heterogeneity during recombinant antibody production, such as "DG" in CDR-H2 set forth as SEQ ID NO: 45, "NT" in CDR-H2 set forth as SEQ ID NOs: 45, 48, and 49, "NG" in CDR-L1 set forth as SEQ ID NOs: 50 and 55, and "NT" in CDR-L1 set forth as SEQ ID NOs: 50 and 54.

[0063] In one embodiment, the isolated anti-CD122 antibody or antigen-binding fragment according to the present disclosure is a chimeric or humanized antibody. In some embodiments, the anti-CD122 antibody or antigen-binding fragment is a humanized antibody.

[0064] In some embodiments, a humanized isolated anti-CD122 antibody or antigen-binding fragment according to the present disclosure comprises one or more back mutations at positions within the framework regions to improve binding characteristics. In some embodiments, the VH domain of a humanized anti-CD122 antibody or antigen-binding fragment according to the present disclosure comprises back mutations from human residues to the following residues according to Kabat numbering: Thr at position 28 (28T), Thr at position 30 (30T), Arg at position 38 (38R), Met at position 48 (48M), Tyr at position 67 (67Y), Met at position 69 (68M), Asn at position 72 (70N), Thr at position 73 (70T), and / or Val at position 91 (70V). In one embodiment, the VL domain of a humanized anti-CD122 antibody or antigen-binding fragment according to the present disclosure comprises back mutations from human residues to the following residues according to Kabat numbering: Ser at position 7 (7S), Phe at position 36 (36F), Gln at position 37 (37Q), and / or Arg at position 46 (46R).

[0065] In some embodiments, an isolated anti-CD122 antibody or antigen-binding fragment according to the present disclosure is a humanized antibody comprising a set of six CDRs: CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, where: CDR-H1 comprises the sequence DYVIS (SEQ ID NO: 44), CDR-H2 comprises the sequence EIYPGDGNTYYNEMFKG (SEQ ID NO: 45), EIYPGDAQTYYNEMFKG (SEQ ID NO: 47), EIYPGDANTYYNEMFKG (SEQ ID NO: 48) or EIYPGEGNTYYNEMFKG (SEQ ID NO: 49), CDR-H3 comprises the sequence GSYTYDNYAMDF (SEQ ID NO: 46), CDR-L1 comprises the sequence RSSQNIVHSNGNTYLE (SEQ ID NO: 50), RSSQNIVHSEGQTYLE (SEQ ID NO: 53), RSSQNIVHSNANTYLE (SEQ ID NO: 54), RSSQNIVHSNGQTYLE (SEQ ID NO: 55), RSSQNIVHSNAQTYLE (SEQ ID NO: 56), CDR-L2 comprises the sequence KVSNRFS (SEQ ID NO: 51), CDR-L3 comprises the sequence FQGSHIPWT (SEQ ID NO: 52), The CDRs are defined according to the Kabat numbering system.

[0066] In some embodiments, the isolated anti-CD122 antibody or antigen-binding fragment of the present disclosure is a humanized antibody comprising the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 selected from the group consisting of: (i) SEQ ID NOs: 44, 45, 46; (ii) SEQ ID NOs: 44, 47, 46; (iii) SEQ ID NOs: 44, 48, 46; or (iv) SEQ ID NOs: 44, 49, 46, according to Kabat numbering.

[0067] In some embodiments, the isolated anti-CD122 antibody or antigen-binding fragment of the present disclosure is a humanized antibody comprising the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 selected from the group consisting of: (i) SEQ ID NOs: 50, 51, 52; (ii) SEQ ID NOs: 53, 51, 52; (iii) SEQ ID NOs: 54, 51, 52; (iv) SEQ ID NOs: 55, 51, 52; or (i) SEQ ID NOs: 56, 51, 52, according to Kabat numbering.

[0068] In one embodiment, an isolated anti-CD122 antibody or antigen-binding fragment according to the present disclosure comprises the following VH / VL sequence pairs: SEQ ID NOs: 21 / 30, 22 / 30, 23 / 30, 24 / 30, 25 / 30, 26 / 30, 21 / 31, 22 / 31, 23 / 31, 24 / 31, 25 / 31, 26 / 31, 21 / 32, 22 / 32, 23 / 32, 24 / 32, 25 / 32, 26 / 32, 21 / 33, 21 / 34, 21 / 35, 21 / 36, 21 / 37, 21 / 38, 21 / 39 ...1 / 39, 21 / 39, 21 / 39, 21 / 39, 21 / 39, 21 / 39, 21 / 39, 21 / 39, 21 / 39, 21 / 39, 21 / 39, 21 / 39, 21 / 39, 21 / 39, 21 / 39, 21 / 39, 21 / 39, 21 / 39, 2 and 21 / 41. The CDRs may be determined by one skilled in the art using the most widely used CDR definition schemes, such as those according to Kabat, Chothia or IMGT.

[0069] In some embodiments, an isolated anti-CD122 antibody or antigen-binding fragment according to the present disclosure comprises a combination of VH and VL sequences selected from the group consisting of:

[0070] TIFF2024541048000001.tif198125

[0071] In some embodiments, the antibody comprises a VH domain comprising or consisting of the sequence of SEQ ID NO:21, and a VL domain comprising or consisting of the sequence of SEQ ID NO:41.

[0072] In some embodiments of an anti-CD122 antibody or antigen-binding fragment according to the present disclosure, the antibody or antigen-binding fragment comprises an Fc region, which may be a native Fc region or a variant Fc region. In certain embodiments, the Fc region is a human Fc region derived from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, or IgD. Depending on the utility of the antibody, it may be desirable to use a variant Fc region to alter (e.g., reduce or eliminate) at least one effector function, such as ADCC and / or CDC. In some embodiments, the present disclosure provides an anti-CD122 antibody or antigen-binding fragment comprising an Fc region with one or more mutations, e.g., L234A and L235A, that alter at least one effector function.

[0073] In some embodiments, an antigen-binding fragment of an anti-CD122 antibody according to the present disclosure can be, for example, an Fv, Fab, Fab', Fab'-SH, F(ab')2; a diabody; a linear antibody; or a single-chain antibody molecule (e.g., scFv).

[0074] In one embodiment, the anti-CD122 antibodies or antigen-binding fragments thereof described herein bind to the CD122 extracellular domain or a portion thereof. In some embodiments, the CD122 is human CD122, e.g., human CD122 having Swiss Prot accession number PI4784, the complete sequence of which has 551 amino acids consisting of a signal peptide (residues 1-26), an extracellular domain (residues 27-240), a transmembrane domain (residues 241-265), and a cytoplasmic domain (residues 266-551). The anti-CD122 antibodies or antigen-binding fragments thereof described herein bind to an epitope within the extracellular domain of CD122. In one embodiment, the antibody binds to CD122 at the same epitope as an antibody having the VH / VL sequence pair of SEQ ID NOs: 3 and 4 (e.g., CD122-mAb77).

[0075] In one embodiment, the anti-CD122 antibodies or antigen-binding fragments thereof described herein have nanomolar (10 -7 ~10-9 ) range, e.g., 8×10 -7 Less than M, 5×10 -7 Less than M, 3 x 10 -7 Less than M, 1×10 -7 Less than M, 8 x 10 -8 Less than M, 5×10 -8 Less than M, 3 x 10 -8 Less than M, 2 x 10 -8 Less than M, 1×10 -8 Less than M, 8×10 -9 Less than M, 6×10 -9 Less than M, 4×10 -9 Less than M, 2 x 10 -9 Less than M or 1×10 -9 Dissociation constant (K D ).

[0076] Some cancers exhibit elevated CD122 levels compared to non-cancerous tissues of the same type, preferably from the same patient, and therefore the anti-CD122 antibodies or antigen-binding fragments thereof described herein can be applied to measure CD122 at the protein level (e.g., by immunoassay).

[0077] Anti-CD132 antibody In some embodiments, the disclosure provides proteins that bind to CD132 at the extracellular domain of CD132. In some embodiments, the disclosure discloses isolated anti-CD132 antibodies or antigen-binding fragments thereof that specifically bind to CD132. In further embodiments, the anti-CD132 antibodies or antigen-binding fragments thereof comprise a set of six CDRs: CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, where: CDR-H1 comprises the sequence SYWMH (SEQ ID NO:57), CDR-H2 comprises the sequence HIYLGGGATNYAEKFRS (SEQ ID NO:58), CDR-H3 comprises the sequence SQPYYYGMDS (SEQ ID NO:59), CDR-L1 comprises the sequence RASQDISNYLN (SEQ ID NO: 60), CDR-L2 comprises the sequence YKSRLHS (SEQ ID NO: 61), CDR-L3 comprises the sequence HQGHTIPFT (SEQ ID NO: 62), The CDRs are defined according to the Kabat numbering system.

[0078] In some embodiments, the anti-CD132 antibody, or antigen-binding fragment thereof, comprises at least one, two, three, four, but not more than five residue modifications within the CDR sequences of SEQ ID NOs: 57-59 and 60-62. The amino acid modifications can be amino acid substitutions, deletions, and / or additions, e.g., conservative substitutions.

[0079] In one embodiment, an anti-CD132 antibody or antigen-binding fragment thereof according to the present disclosure comprises CDR-H1, CDR-H2, and CDR-H3 of the heavy chain variable domain VH of SEQ ID NO: 5, and CDR-L1, CDR-L2, and CDR-L3 of the light chain variable domain VL of SEQ ID NO: 6. The CDRs can be determined by the skilled artisan using the most widely used CDR definition schemes, e.g., the definitions according to Kabat, Chothia, or IMGT.

[0080] In one embodiment, an anti-CD132 antibody, or antigen-binding fragment thereof, according to the present disclosure comprises a heavy chain variable domain, VH, and a light chain variable domain, VL, wherein: the VH domain comprises a sequence of SEQ ID NO:5 or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto; and / or The VL domain comprises the sequence of SEQ ID NO:6 or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto.

[0081] In some embodiments, an anti-CD132 antibody comprising a VH sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence while retaining the ability to bind to CD132 with the same or improved binding characteristics, e.g., Kd. In certain embodiments, the substitutions, insertions, or deletions occur in regions other than the CDRs (i.e., in the FRs). Optionally, the anti-CD132 antibody comprises a Q1E mutation compared to the VH sequence of SEQ ID NO:5 to eliminate N-terminal pyroglutamic acid formation.

[0082] In one embodiment, the isolated anti-CD132 antibody or antigen-binding fragment according to the present disclosure is a chimeric or humanized antibody. In some embodiments, the anti-CD132 antibody or antigen-binding fragment is a humanized antibody.

[0083] In some embodiments, a humanized isolated anti-CD132 antibody or antigen-binding fragment according to the present disclosure comprises one or more back mutations at positions within the framework regions to improve binding characteristics. In some embodiments, the VH domain of a humanized anti-CD132 antibody or antigen-binding fragment according to the present disclosure comprises back mutations from human residues to the following residues according to Kabat numbering: Thr at position 28 (28T), Thr at position 30 (30T), Arg at position 38 (38R), Met at position 48 (48M), Tyr at position 67 (67Y), Met at position 69 (68M), Asn at position 72 (70N), Thr at position 73 (70T), and / or Val at position 91 (70V). In one embodiment, the VL domain of a humanized anti-CD132 antibody or antigen-binding fragment according to the present disclosure comprises back mutations from the human residues to the following residues according to Kabat numbering: Glu at position 70 (70E) and / or Tyr at position 71 (71Y).

[0084] In some embodiments, the isolated anti-CD132 antibody or antigen-binding fragment according to the present disclosure is a humanized antibody comprising the six CDR sets set forth above in SEQ ID NOs: 57-59 and 60-62.

[0085] In one embodiment, the isolated anti-CD132 antibody or antigen-binding fragment according to the present disclosure is a humanized antibody comprising CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 of the heavy chain variable domain VH and the light chain variable domain VL selected from the group consisting of the following VH / VL sequence pairs: SEQ ID NOs: 14 / 19, 15 / 19, 16 / 19, 17 / 19, 18 / 19, 14 / 20, 15 / 20, 16 / 20, 17 / 20, and 18 / 20. The CDRs can be determined by the skilled artisan using the most widely used CDR definition schemes, e.g., the definitions according to Kabat, Chothia, or IMGT.

[0086] In some embodiments, an isolated anti-CD132 antibody or antigen-binding fragment according to the present disclosure comprises a combination of VH and VL sequences selected from the group consisting of:

[0087] TIFF2024541048000002.tif68144

[0088] In some embodiments, the antibody comprises a VH domain comprising or consisting of the sequence of SEQ ID NO:14, and a VL domain comprising or consisting of the sequence of SEQ ID NO:19.

[0089] In some embodiments of an anti-CD132 antibody or antigen-binding fragment according to the present disclosure, the antibody or antigen-binding fragment comprises an Fc region, which may be a native Fc region or a variant Fc region. In certain embodiments, the Fc region is a human Fc region derived from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, or IgD. Depending on the utility of the antibody, it may be desirable to use a variant Fc region to alter (e.g., reduce or eliminate) at least one effector function, such as ADCC and / or CDC. In some embodiments, the present disclosure provides an anti-CD132 antibody or antigen-binding fragment comprising an Fc region with one or more mutations, e.g., L234A and L235A, that alter at least one effector function.

[0090] In some embodiments, an antigen-binding fragment of an anti-CD132 antibody according to the present disclosure can be, for example, an Fv, Fab, Fab', Fab'-SH, F(ab')2; a diabody; a linear antibody; or a single-chain antibody molecule (e.g., scFv).

[0091] In one embodiment, the anti-CD132 antibodies or antigen-binding fragments thereof described herein bind to the CD132 extracellular domain or a portion thereof. In some embodiments, the CD132 is human CD132, e.g., human CD132 having Swiss Prot accession number P31785, the full sequence of which has 369 amino acids consisting of a signal peptide (residues 1-22), an extracellular domain (residues 23-262), a transmembrane domain (residues 263-283), and a cytoplasmic domain (residues 284-369). The anti-CD132 antibodies or antigen-binding fragments thereof described herein bind to an epitope within the extracellular domain of CD132. In one embodiment, the antibody binds to CD132 at the same epitope as an antibody having the VH / VL sequence pair of SEQ ID NOs: 5 and 6 (e.g., CD132-mAb17).

[0092] In one embodiment, the anti-CD132 antibodies or antigen-binding fragments thereof described herein have nanomolar (10 -7 ~10 -9 ) range, e.g., 8×10 -7 Less than M, 5×10 -7 Less than M, 3 x 10 -7 Less than M, 1×10 -7 Less than M, 8×10 -8 Less than M, 5×10 -8 Less than M, 3 x 10 -8 Less than M, 2 x 10 -8 Less than M, 1×10 -8 Less than M, 8×10 -9 Less than M, 6×10 -9 Less than M, 4×10 -9 Less than M, 2 x 10 -9 Less than M or 1×10 -9 Dissociation constant (K D ).

[0093] CD122×CD132 bispecific binding protein In another aspect, the present disclosure provides a CD122 / CD132 bispecific binding protein, such as a FIT-Ig (Fab-in-tandem immunoglobulin) or duobody, capable of binding to both CD122 and CD132. Each variable domain (VH or VL) in the FIT-Ig or duobody can be derived from one or more "parent" monoclonal antibodies that bind to one of the target antigens, i.e., CD122 or CD132. The FIT-Ig or duobody binding protein can be made using the variable domain sequences of the anti-CD122 and anti-CD132 monoclonal antibodies disclosed herein. For example, the parent antibody is a humanized antibody.

[0094] One aspect of the present disclosure relates to the selection of parent antibodies that have at least one or more properties desired in a FIT-Ig or duobody molecule. In one embodiment, the antibody properties are selected from the group consisting of antigen specificity, affinity for antigen, cell binding capacity, biological function, epitope recognition, stability, solubility, production efficiency, immunogenicity, pharmacokinetics, bioavailability, tissue cross-reactivity, and binding to orthologous antigens.

[0095] In some embodiments, the bispecific FIT-Ig proteins according to the present disclosure are constructed without an inter-domain peptide linker. It is generally understood in the art that in multivalent engineered immunoglobulin formats with tandem binding sites, adjacent binding sites will interfere with each other unless flexible linkers are used to spatially separate the binding sites. However, for the CD122 / CD132 FIT-Ig of the present disclosure, it has been discovered that the arrangement of immunoglobulin domains according to the chain formulas disclosed herein results in polypeptide chains that are sufficient for expression in transfected mammalian cells and are properly assembled and secreted as bispecific, multivalent immunoglobulin-like binding proteins that bind to the target antigens CD122 and CD132. See the Examples below. Furthermore, omission of the synthetic linker sequence from the binding protein can avoid the creation of antigenic sites recognizable by the mammalian immune system; thus, elimination of the linker reduces the immunogenic potential of FIT-Ig and results in a circulating half-life similar to that of natural antibodies, i.e., FIT-Ig is not rapidly cleared by immune opsonization and hepatic sequestration.

[0096] In some embodiments, the CD122×CD132 bispecific binding protein according to the present application comprises: a) a first antigen-binding site that specifically binds to CD122, and b) a second antigen-binding site that specifically binds to CD132 Includes.

[0097] In one embodiment, the bispecific binding proteins described herein comprise a set of six CDRs, CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, derived from any anti-CD122 antibody or antigen-binding fragment thereof described herein in accordance with the present application, to form the CD122 binding site of the bispecific binding protein. In some further embodiments, the bispecific binding proteins described herein comprise a VH / VL pair derived from any anti-CD122 antibody or antigen-binding fragment thereof described herein in accordance with the present application, to form the CD122 binding site of the bispecific binding protein.

[0098] In one embodiment, the bispecific binding proteins described herein further comprise a set of six CDRs, CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, derived from any anti-CD132 antibody or antigen-binding fragment thereof described herein in accordance with the present application, to form the CD132 binding site of the bispecific binding protein. In some further embodiments, the bispecific binding proteins described herein comprise a VH / VL pair derived from any anti-CD132 antibody or antigen-binding fragment thereof described herein in accordance with the present application, to form the CD132 binding site of the bispecific binding protein.

[0099] In one embodiment, the CD122 binding site and the CD132 binding site in a bispecific CD122 / CD132 binding protein according to the present application are humanized and comprise humanized VH / VL sequences, respectively.

[0100] Bispecific FIT-Ig binding protein In one embodiment, the CD122×CD132 bispecific binding protein according to the present application is a bispecific FIT-Ig binding protein capable of binding to CD122 and CD132. The FIT-Ig (Fab-in-tandem immunoglobulin) binding protein is a monomeric bispecific tetravalent binding protein comprising six polypeptide chains and having four functional Fab binding regions with two external Fab binding regions and two internal Fab binding regions. As shown in FIG. 1, the binding protein adopts a (external Fab-internal Fab-Fc)×2 format and binds both antigen A and antigen B. In one aspect, the CD122×CD132 bispecific binding protein according to the present application is a bispecific FIT-Ig binding protein, where the two Fab domains of the FIT-Ig protein form a first antigen binding site that specifically binds to CD122 and the other two Fab domains of the FIT-Ig protein form a second antigen binding site that specifically binds to CD132. In some embodiments, the FIT-Ig binding proteins according to the present disclosure do not utilize a linker between the immunoglobulin domains.

[0101] In a further embodiment, the present disclosure provides a bispecific FIT-Ig (Fab-in-tandem immunoglobulin) binding protein, comprising a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain, (i) In format LH, the first polypeptide chain is arranged from the amino terminus to the carboxyl terminus as follows: CL: VH B VL directly fused to A -CL-VH B -CH1-Fc; the second polypeptide chain comprises, from the amino terminus to the carboxyl terminus, A the third polypeptide chain comprises, from the amino terminus to the carboxyl terminus, B -Contains CL; or (ii) In format HL, the first polypeptide chain is arranged, from amino terminus to carboxyl terminus, in which CH1 is VL B VH directly fused toA -CH1-VL B the second polypeptide chain comprises, from the amino terminus to the carboxyl terminus, VL A the third polypeptide chain comprises, from the amino terminus to the carboxyl terminus, VH B - containing CH1; VL is a light chain variable domain, CL is a light chain constant domain, VH is a heavy chain variable domain, CH1 is a heavy chain constant domain, and Fc is an immunoglobulin Fc region, e.g., IgG1 Fc (optionally including, from amino terminus to carboxyl terminus, hinge-CH2-CH3); VL A -CL, VH A -CH1 to form a first Fab that specifically binds to a first antigen A, and VL B -CL, VH B - pairs with CH1 to form a second Fab that specifically binds to a second antigen B, The first antigen A is CD122 and the second antigen B is CD132, or the first antigen A is CD132 and the second antigen B is CD122; Two of the first polypeptide chains, two of the second polypeptide chains, and two of the third polypeptide chains associate to form the FIT-Ig protein.

[0102] In some embodiments of the bispecific FIT-Ig binding proteins of the present application, the first polypeptide chain comprises, from the amino terminus to the carboxyl terminus, a VL A -CL-VH B -CH1-Fc, where antigen A is CD122 and antigen B is CD132, or where antigen A is CD132 and antigen B is CD122.

[0103] In some embodiments of the FIT-Ig binding protein, the CD122 binding site is a Fab formed by pairing of VL-CL with VH-CH1 (e.g., when A is CD122, VL A -CL and VH A- Fab formed by CH1; or when B is CD122, VL B -CL and VH B -CH1) and comprises a set of six CDRs, i.e., CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, from any anti-CD122 antibody or antigen-binding fragment thereof according to the present application. In some further embodiments, CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 comprise, respectively, the sequence of SEQ ID NO: 44 as CDR-H1; the sequence of SEQ ID NO: 45, 47-49 as CDR-H2; the sequence of SEQ ID NO: 46 as CDR-H3; the sequence of SEQ ID NO: 50, 53-56 as CDR-L1; the sequence of SEQ ID NO: 51 as CDR-L2; and the sequence of SEQ ID NO: 52 as CDR-L3.

[0104] In some embodiments, the Fab in the FIT-Ig binding protein that binds to CD122 comprises a VH / VL pair derived from any anti-CD122 antibody or antigen-binding fragment thereof described herein in accordance with the present application. In some further embodiments, the VH / VL pair is selected from the following VH / VL sequence pairs: SEQ ID NOs: 3 / 4, 21 / 30, 22 / 30, 23 / 30, 24 / 30, 25 / 30, 26 / 30, 21 / 31, 22 / 31, 23 / 31, 24 / 31, 25 / 31, 26 / 31, 21 / 32, 22 / 32, 23 / 32, 24 / 32, 25 / 32, 26 / 32, 27 / 32, 28 / 32, 29 / 32, 30 / 32, 31 / 32, 32 / 32, 33 / 32, 34 / 32, 35 / 32, 36 / 32, 37 / 32, 38 / 32, 39 / 32, 40 / 32, 41 / 32, 42 / 32, 43 / 32, 44 / 32, 45 / 32, 46 / 32, 47 / 32, 48 / 32, 49 / 32, 50 / 32, 51 / 32, 52 / 32, 53 / 32, 54 / 32, 55 / 32, 56 / 32, 57 / 32, 58 / 32, 59 / 32, 60 / 32, 61 / 32, 62 / 32, 63 / 32, 64 / 32, 65 / 32, 66 / 32, 67 / 32, 68 / 3 1 / 33, 21 / 34, 21 / 35, 27 / 36, 27 / 37, 27 / 38, 27 / 39, 27 / 40, 21 / 36, 28 / 36, 29 / 36, 27 / 41, 27 / 42, 27 / 43, and 21 / 41, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity thereto. In some embodiments, the Fab that binds to CD122 in the FIT-Ig binding protein comprises the VH sequence of SEQ ID NO:21 and the VL sequence of SEQ ID NO:41.

[0105] In some embodiments of the FIT-Ig binding protein, the CD132 binding site is a Fab formed by pairing of VL-CL with VH-CH1 (e.g., when A is CD132, VL A -CL and VH A - Fab formed by CH1; or when B is CD132, VL B -CL and VH B and a Fab formed by pairing VL-CL with VH-CH1, which comprises a set of six CDRs, i.e., CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, from any anti-CD132 antibody or antigen-binding fragment thereof according to the present application. In some embodiments, the CD132 binding Fab formed by pairing VL-CL with VH-CH1 in the FIT-Ig binding protein comprises a set of six CDRs, where CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 comprise the sequences of SEQ ID NOs: 57, 58, 59, and 60, 61, 62, respectively. In some further embodiments, the Fab that binds to CD132 comprises a VH / VL pair comprising the sequences of SEQ ID NOs: 22 and 24, or a sequence with at least 80%, 85%, 90%, 95%, or 99% identity thereto, or the sequences of SEQ ID NOs: 5 / 6, 14 / 19, 15 / 19, 16 / 19, 17 / 19, 18 / 19, 14 / 20, 15 / 20, 16 / 20, 17 / 20, and 18 / 20, or a sequence with at least 80%, 85%, 90%, 95%, or 99% identity thereto.

[0106] In some embodiments, the Fab fragment of such a FIT-Ig binding protein comprises a VL fragment from a parent antibody that binds to one of the antigens CD122 and CD132. A -CL and VH A - VL from another parent antibody incorporating the CH1 domain and binding to the other of the antigens CD122 and CD132 B -CL and VH B -CH1 domain. In some embodiments, the tandem Fab portions recognizing CD122 and CD132, respectively, are formed by the VH-CH1::VL-CL pairing.

[0107] In the present disclosure, a CD122 / CD132 FIT-Ig binding protein comprises a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain, wherein the first polypeptide chain is arranged from the amino terminus to the carboxyl terminus to have a CL of VH CD132 VL directly fused to CD122 -CL-VH CD132 the second polypeptide chain comprises, from the amino terminus to the carboxyl terminus, a VH CD122 the third polypeptide chain comprises, from the amino terminus to the carboxyl terminus, VL CD132 In an alternative embodiment, the CD122 / CD132 FIT-Ig binding protein comprises a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain, wherein the first polypeptide chain comprises, from amino terminus to carboxyl terminus, CH1 comprises VL CD132 VH directly fused to CD122 -CH1-VL CD132 the second polypeptide chain comprises, from the amino terminus to the carboxyl terminus, VL CD122 the third polypeptide chain comprises, from the amino terminus to the carboxyl terminus, VH CD132 In some embodiments, the VL CD122 is the light chain variable domain of an anti-CD122 antibody, CL is the light chain constant domain, and VH CD122 is the heavy chain variable domain of an anti-CD122 antibody, CH1 is the heavy chain constant domain, and VL CD132 is the light chain variable domain of the anti-CD132 antibody, and VH CD132 is a heavy chain variable domain of an anti-CD132 antibody; optionally, the domain V CD132 -CL is the same as the light chain of the anti-CD132 parent antibody and has the domain VH CD132 -CH1 is the same as the heavy chain variable domain and the heavy chain constant domain of the anti-CD132 parent antibody, and the domain VL CD122 -CL is the same as the light chain of the anti-CD122 parent antibody and has the domain VH CD122-CH1 is identical to the heavy chain variable and heavy chain constant domains of the anti-CD122 parent antibody.

[0108] In the above formula for FIT-Ig binding protein, the Fc region can be a native or variant Fc region. In certain embodiments, the Fc region is a human Fc region from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, or IgD. In certain embodiments, the Fc is a human Fc from IgG1, or a modified human Fc that includes one or more mutations to reduce or eliminate at least one Fc effector function, such as Fc binding to FcγR, ADCC, and / or CDC. The mutations can be, for example, L234A / L235A (numbering according to Kabat EU index). In one embodiment, the Fc region corresponding to the CH1-hinge-CH2-CH3 from human constant IgG1 with the L234A / L235A mutations has the following amino acid sequence: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE AA GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 70)

[0109] In some embodiments of FIT-Ig binding proteins according to the present disclosure, the CH1, CL and Fc domains are of human sequences or are derived from human sequences. In some embodiments of FIT-Ig binding proteins according to the present disclosure, CH1 is a human IgG1 constant CH1 domain or a sequence having at least 90%, 95%, 97%, 98%, 99% or more identity thereto. In the above formula for FIT-Ig binding proteins, CL is a human constant kappa CL domain or a sequence having at least 90%, 95%, 97%, 98%, 99% or more identity thereto.

[0110] In one embodiment, the FIT-Ig binding proteins of the present disclosure retain one or more properties of the parent antibody. In some embodiments, FIT-Ig retains a binding affinity for a target antigen (i.e., CD132 and CD122) that is comparable to the binding affinity of the parent antibody, meaning that the binding affinity of the FIT-Ig binding protein for its antigen targets CD122 and CD132 does not vary by more than 10-fold compared to the binding affinity of the parent antibody for its respective target antigens, as measured by surface plasmon resonance or biolayer interferometry.

[0111] In one embodiment, a FIT-Ig binding protein of the present disclosure binds to CD122 and CD132 and is comprised of a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain; the first polypeptide chain comprises an amino acid sequence of SEQ ID NO:7, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto; the second polypeptide chain comprises an amino acid sequence of SEQ ID NO:8 or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto; the third polypeptide chain comprises an amino acid sequence of SEQ ID NO:9, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto.

[0112] In one embodiment, a FIT-Ig binding protein of the disclosure binds to CD122 and CD132 and is composed of a first polypeptide chain comprising, consisting essentially of, or consisting of the sequence of SEQ ID NO:7; a second polypeptide chain comprising, consisting essentially of, or consisting of the sequence of SEQ ID NO:8; and a third polypeptide chain comprising, consisting essentially of, or consisting of the sequence of SEQ ID NO:9.

[0113] Bispecific Binding Protein Duobodies In one embodiment, the CD122xCD132 bispecific binding protein according to the present application is a duobody capable of binding to CD122 and CD132. The duobody shown in Figure 2 is a monomeric bispecific bivalent binding protein comprising four polypeptide chains and having two functional Fab binding regions.

[0114] Duobodies can be generated by mixing two parent antibodies, each containing a single matching point mutation in the CH3 domain, and subjecting the two parent antibodies to controlled reducing conditions in vitro, where the antibodies are separated into HL half molecules, allowed to reassemble and reoxidize by a physiological process called Fab arm exchange (FAE), and the half molecules (HL pairs) recombine with half molecules from the other parent antibody molecule to form a highly pure bsAb.

[0115] In some embodiments, a duobody of the disclosure comprises an Fc region where one of the CH3 regions comprises the substitution K409R and the other CH3 region of the Fc region comprises the substitution F405L.

[0116] In some embodiments, monospecific anti-CD122 lgG1-K409R and anti-CD132 lgG1-F405L antibodies are mixed and then reduced with 2-MEA for several hours. The 2-MEA is removed by dialysis and the antibodies are reoxidized at 4° C. The fully formed bispecific duobody is purified by anion exchange chromatography.

[0117] In some embodiments, monospecific anti-CD132 lgG1-K409R and anti-CD122 lgG1-F405L antibodies are mixed and then reduced with 2-MEA for several hours. The 2-MEA is removed by dialysis and the antibodies are reoxidized at 4° C. The fully formed bispecific duobody is purified by anion exchange chromatography.

[0118] In some embodiments, the duobody of the disclosure comprises such an HL half molecule, wherein: (i) One HL half molecule from an anti-CD122 antibody of the present disclosure, comprising SEQ ID NO: 44 as CDR-H1, any one of SEQ ID NOs: 45, 47-49 as CDR-H2, SEQ ID NO: 46 as CDR-H3, and any one of SEQ ID NOs: 50, 53-56 as CDR-L1, SEQ ID NO: 51 as CDR-L2, and SEQ ID NO: 52 as CDR-L3. In some embodiments, the HL half molecule from an anti-CD122 antibody of the present disclosure comprises a VH / VL pair from any anti-CD122 antibody or antigen-binding fragment thereof described herein in accordance with the present application. In some further embodiments, the VH / VL pair comprises a sequence selected from the group consisting of the following VH / VL sequence pairs, or a sequence having at least 80%, 85%, 90%, 95% or 99% identity thereto: SEQ ID NOs: 3 / 4, 21 / 30, 22 / 30, 23 / 30, 24 / 30, 25 / 30, 26 / 30, 21 / 31, 22 / 3 1, 23 / 31, 24 / 31, 25 / 31, 26 / 31, 21 / 32, 22 / 32, 23 / 32, 24 / 32, 25 / 32, 26 / 32, 21 / 33, 21 / 34, 21 / 35, 27 / 36, 27 / 37, 27 / 38, 27 / 39, 27 / 40, 21 / 36, 28 / 36, 29 / 36, 27 / 41, 27 / 42, 27 / 43, and 21 / 41. In some embodiments, the HL half molecule from an anti-CD122 antibody comprises the VH sequence of SEQ ID NO:21 and the VL sequence of SEQ ID NO:41; (ii) one HL half molecule from an anti-CD132 antibody of the disclosure comprising SEQ ID NO:57 as CDR-H1, SEQ ID NO:58 as CDR-H2, SEQ ID NO:59 as CDR-H3, and SEQ ID NO:60 as CDR-L1, SEQ ID NO:61 as CDR-L2, and SEQ ID NO:62 as CDR-L3. In some embodiments, the HL half molecule from an anti-CD132 antibody of the disclosure comprises a VH / VL pair from any anti-CD132 antibody or antigen-binding fragment thereof described herein in accordance with the present application. In some further embodiments, the VH / VL pair comprises a sequence selected from the group consisting of the following VH / VL sequence pairs, or a sequence having at least 80%, 85%, 90%, 95% or 99% identity thereto: SEQ ID NOs: 5 / 6, 14 / 19, 15 / 19, 16 / 19, 17 / 19, 18 / 19, 14 / 20, 15 / 20, 16 / 20, 17 / 20, and 18 / 20. In some embodiments, the HL half molecule from an anti-CD132 antibody comprises the VH sequence of SEQ ID NO: 14 and the VL sequence of SEQ ID NO: 19; (iii) an Fc region, one of the CH3 regions of which contains the substitution K409R and the other CH3 region of which contains the substitution F405L.

[0119] In one embodiment, the duobody of the disclosure is composed of an HL half molecule derived from an anti-CD122 antibody that binds to CD122 and CD132 and comprises, consists essentially of, or consists of the sequence of SEQ ID NO: 12 / 13; and an HL half molecule derived from an anti-CD132 antibody that comprises, consists essentially of, or consists of the sequence of SEQ ID NO: 10 / 11.

[0120] Characteristics of bispecific binding proteins In some embodiments, a bispecific binding protein of the disclosure is capable of binding to cells expressing CD122, where the cell binding ability is reflected by an EC50 of about 5 nM or less, 4 nM or less, 3 nM or less, 2 nM or less, or 1 nM or less as measured by flow cytometry in a cell-based assay.

[0121] In some embodiments, the bispecific binding proteins of the present disclosure are capable of binding to cells expressing CD132, where the cell binding ability is reflected by an EC50 of about 80 nM or less, 60 nM or less, 40 nM or less, 20 nM or less, or 10 nM or less as measured by flow cytometry in a cell-based assay.

[0122] In some embodiments, the bispecific binding proteins of the disclosure have a K of less than about 30 nM, 25 nM, 20 nM, 15 nM, 10 nM, or 5 nM, as measured, for example, by a WAVEsystem or Biacore assay. D and binds to human CD122 and human CD132.

[0123] In other embodiments, the bispecific binding proteins of the disclosure have a K of less than about 100 nM, 80 nM, 60 nM, 40 nM, 20 nM, or 10 nM, as measured, for example, by a WAVE system or Biacore assay. D and binds to cynomolgus monkey CD122 and cynomolgus monkey CD132.

[0124] In some embodiments, the bispecific binding proteins of the present disclosure activate intracellular signaling upon contact with a cell expressing a complex comprising CD122 and CD132 at the cell surface. In some embodiments, the bispecific binding proteins of the present disclosure activate intracellular signaling upon contact with a cell expressing a medium affinity IL-2 receptor at the cell surface. In some embodiments, the bispecific binding proteins of the present disclosure activate intracellular signaling upon contact with a cell expressing a high affinity IL-2 receptor at the cell surface. Activation of intracellular signaling can be determined by detecting an increase in the level of phosphorylated STAT5 (i.e., pSTAT5). pSTAT5 can be detected, for example, using a reporter-based method as described in Example 6 and a flow cytometry-based STAT5 phosphorylation (pSTAT5) assay as described in Example 7.1.

[0125] In some embodiments, a bispecific binding protein of the disclosure can increase the amount of pSTAT5 by more than 1-fold, e.g., >1.1-fold, >1.2-fold, >1.3-fold, >1.4-fold, >1.5-fold, >1.6-fold, >1.7-fold, >1.8-fold, >1.9-fold, >2-fold, >3-fold, >4-fold, >5-fold, >6-fold, >7-fold, >8-fold, >9-fold, >10-fold, >20-fold, >30-fold, >40-fold, >50-fold, >60-fold, >70-fold, >80-fold, >90-fold, or >100-fold, the level of pSTAT5 detected following culture of cells expressing a complex comprising CD122 and CD132 on the cell surface in the absence of a bispecific binding protein of the disclosure or in the presence of a control antigen binding molecule (e.g., an isotype-matched control antigen binding molecule) in a comparable assay.

[0126] In some embodiments, the bispecific binding proteins of the present disclosure can stimulate the proliferation of cells expressing CD122 and CD132. In particular, the bispecific binding proteins of the present disclosure can preferentially stimulate the proliferation of effector T cells and / or NK cells over regulatory T cells. In some embodiments, the bispecific binding proteins of the present disclosure preferentially stimulate the proliferation of cells expressing intermediate affinity IL-2 receptors on the cell surface over cells expressing high affinity IL-2 receptors on the cell surface. Cell proliferation can be determined by analyzing cell division over a period of time. Cell division can be analyzed by CFSE dilution assay, for example, as described in Example 7.2.

[0127] In some embodiments, a bispecific binding protein of the disclosure can increase the number of proliferating cells by more than 1-fold, e.g., >1.1-fold, >1.2-fold, >1.3-fold, >1.4-fold, >1.5-fold, >1.6-fold, >1.7-fold, >1.8-fold, >1.9-fold, >2-fold, >3-fold, >4-fold, >5-fold, >6-fold, >7-fold, >8-fold, >9-fold, >10-fold, >20-fold, >30-fold, >40-fold, >50-fold, >60-fold, >70-fold, >80-fold, >90-fold, or >100-fold, in a comparable assay, the number of proliferating cells detected following culture of cells expressing a complex comprising CD122 and CD132 on the cell surface in the absence of a bispecific binding protein of the disclosure or in the presence of a control antigen binding molecule (e.g., an isotype-matched control antigen binding molecule).

[0128] In some embodiments, the bispecific binding proteins of the disclosure preferentially stimulate the proliferation / expansion of one or more of the following cell types over regulatory T cells (i.e., in preference to regulatory T cells): antigen-specific T cells (e.g., virus-specific T cells), antigen-specific CD4 T cells, antigen-specific CD8 T cells, effector memory CD4 T cells, effector memory CD8 T cells, central memory CD4 T cells, central memory CD8 T cells, cytotoxic CD8+ T cells (i.e., CTLs), NK cells, antigen-specific NK cells, or cells expressing a chimeric antigen receptor (CAR).

[0129] In some embodiments, the bispecific binding proteins of the disclosure increase the number of cells expressing CD122 and CD132 (i.e., expansion of a population of cells), e.g., inducing expansion of CD8+ and / or CD4+ T cells, or preferential expansion of CD8+ and / or CD4+ T cells over Treg cells.

[0130] Pharmaceutical Compositions The present disclosure also provides a pharmaceutical composition comprising an antibody, or antigen-binding portion thereof, or a bispecific multivalent binding protein (i.e., the main active ingredient) of the present disclosure, and a pharma- ceutically acceptable carrier. In a specific embodiment, the composition comprises one or more antibodies or binding proteins of the present disclosure. The present disclosure also provides a pharmaceutical composition comprising a combination of anti-CD122 and anti-CD132 antibodies, or antigen-binding fragment(s) thereof, described herein, and a pharma- ceutically acceptable carrier. In particular, the present disclosure provides a pharmaceutical composition comprising at least one FIT-Ig binding protein capable of binding to CD122 and CD132, and a pharma- ceutically acceptable carrier. In particular, the present disclosure provides a pharmaceutical composition comprising at least one duobody binding protein capable of binding to CD122 and CD132, and a pharma- ceutically acceptable carrier.

[0131] The pharmaceutical composition of the present disclosure may further comprise at least one additional active ingredient. In some embodiments, such additional ingredients include, but are not limited to, prophylactic and / or therapeutic agents, detection agents, such as antitumor drugs, cytotoxic agents, antibodies of different specificity or functional fragments thereof, detection labels or reporters. In one embodiment, the pharmaceutical composition comprises one or more additional prophylactic or therapeutic agents for treating or alleviating a disorder, i.e., agents other than the antibody or binding protein of the present disclosure. In one embodiment, the additional prophylactic or therapeutic agent is known, used, or currently being used to be useful in the prevention, treatment, management, or amelioration of a disorder or one or more symptoms thereof.

[0132] The pharmaceutical composition comprising the protein of the present disclosure is a pharmaceutical composition for, but not limited to, diagnosis, detection, or monitoring of a disorder; treatment, management, or amelioration of a disorder or one or more symptoms thereof; and / or use in research studies. In some embodiments, the composition may further comprise a carrier, diluent, or excipient. An excipient is generally any compound or combination of compounds other than the main active ingredient (i.e., other than the antibody, antigen-binding portion thereof, or binding protein of the present disclosure) that also provides a desired characteristic to the composition.

[0133] Nucleic Acids, Vectors, and Host Cells In further aspects, the present disclosure provides isolated nucleic acids encoding one or more amino acid sequences of the anti-CD122 antibodies or antigen-binding fragments thereof of the present disclosure; isolated nucleic acids encoding one or more amino acid sequences of the anti-CD132 antibodies or antigen-binding fragments thereof of the present disclosure; and isolated nucleic acids encoding one or more amino acid sequences of a bispecific binding protein, such as FIT-Ig (Fab-in-tandem immunoglobulin), capable of binding to both CD122 and CD132. Such nucleic acids can be inserted into vectors to perform various genetic analyses or to express, characterize, or improve one or more properties of the antibodies or binding proteins described herein. The vectors can include one or more nucleic acid molecules encoding one or more amino acid sequences of the antibodies or binding proteins described herein, operably linked to appropriate transcription and / or translation sequences that allow expression of the antibodies or binding proteins in a particular host cell harboring the vector. Examples of vectors for cloning or expressing nucleic acids encoding the amino acid sequences of the binding proteins described herein include, but are not limited to, pcDNA, pTT, pTT3, pEFBOS, pBV, pJV, and pBJ, and derivatives thereof.

[0134] The present disclosure also provides host cells that express or are capable of expressing vectors that include a nucleic acid encoding one or more amino acid sequences of the antibodies or binding proteins described herein. Host cells useful in the present disclosure can be prokaryotic or eukaryotic. An exemplary prokaryotic host cell is Escherichia coli. Eukaryotic cells useful as host cells in the present disclosure include protist cells, animal cells, plant cells, and fungal cells. An exemplary fungal cell is a yeast cell, including Saccharomyces cerevisiae. Exemplary animal cells useful as host cells according to the present disclosure include, but are not limited to, mammalian cells, avian cells, and insect cells. Exemplary mammalian cells include, but are not limited to, CHO cells, HEK cells, and COS cells.

[0135] Method for preparation In another aspect, the disclosure provides a method of making an anti-CD122 antibody or functional fragment thereof, comprising culturing a host cell comprising an expression vector encoding the antibody or functional fragment in a culture medium under conditions sufficient to cause the host cell to express the antibody or fragment capable of binding to CD122.

[0136] In another aspect, the disclosure provides a method of making an anti-CD132 antibody or functional fragment thereof, comprising culturing a host cell comprising an expression vector encoding the antibody or functional fragment in a culture medium under conditions sufficient to cause the host cell to express the antibody or fragment capable of binding to CD132.

[0137] In another aspect, the disclosure provides a method of making a bispecific, multivalent binding protein capable of binding to CD122 and CD132, in particular a FIT-Ig binding protein that binds to CD122 and CD132, comprising culturing a host cell comprising an expression vector encoding a FIT-Ig binding protein in a culture medium under conditions sufficient to cause the host cell to express a binding protein capable of binding to CD122 and CD 132. The proteins made by the methods disclosed herein can be isolated and used in the various compositions and methods described herein.

[0138] Uses of antibodies and binding proteins Given their ability to bind human CD122 and / or CD132, the antibodies, functional fragments thereof, and bispecific multivalent binding proteins described herein can be used to detect CD122 or CD132, or both, in biological samples containing cells expressing, for example, one or both of these target antigens. The antibodies, functional fragments, and binding proteins of the present disclosure can be used in conventional immunoassays, such as enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), or immunohistochemistry. The present disclosure provides a method of detecting CD122 or CD132 in a biological sample, comprising contacting the biological sample with an antibody, antigen-binding portion thereof, or binding protein of the present disclosure, and detecting whether binding to the target antigen occurs, thereby detecting the presence or absence of the target in the biological sample. The antibody, functional fragment, or binding protein may be directly or indirectly labeled with a detectable substance to facilitate detection of the bound or unbound antibody / fragment / binding protein. Suitable detection substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase. Examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin; examples of luminescent materials include luminol; examples of suitable radioactive materials include fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin; examples of suitable fluorescent materials include luminol, fluorescein isothiocyanate ... 3 H, 14 C. 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131 I, 177 Lu, 166 Ho, or 153 Including Sm.

[0139] In some embodiments, the disclosure provides an antibody or bispecific binding protein of the disclosure for use in generating / expanding a population of immune cells. In some embodiments, the disclosure provides an antibody or bispecific binding protein of the disclosure for use in treating any subject who would benefit from an increase in the number of cells expressing CD122 and CD132 (i.e., an expansion of a population of cells), e.g., an expansion of CD8+ T cells and / or CD4+ T cells, or a preferential expansion of CD8+ T cells and / or CD4+ T cells over Treg cells.

[0140] The antibodies (including functional fragments thereof) and binding proteins of the present disclosure can be incorporated into pharmaceutical compositions suitable for administration to a subject. Typically, the pharmaceutical composition comprises the antibodies or binding proteins of the present disclosure and a pharma- ceutically acceptable carrier. As used herein, a "pharmaceutically acceptable carrier" includes any / all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, that are physiologically compatible. Examples of pharma- ceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, and combinations thereof. In many cases, it will be preferable to include an isotonic agent, such as a sugar, a polyalcohol (e.g., mannitol or sorbitol), or sodium chloride in the composition. The pharma- ceutically acceptable carrier may further comprise minor amounts of auxiliary substances, such as humectants or emulsifiers, preservatives, or buffers, that enhance the shelf life or effectiveness of the antibodies or binding proteins present in the composition. A pharmaceutical composition of the disclosure is formulated to be compatible with its intended route of administration.

[0141] The disclosed method may include administering a composition formulated for parenteral administration by injection (e.g., by bolus injection or continuous infusion). Formulations for injection may be presented in unit dosage form (e.g., in ampoules or in multi-dose containers) with added preservatives. The compositions may take such forms, for example, as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents, such as suspending, stabilizing, and / or dispersing agents. Alternatively, the main active ingredient may be in powder form for constitution with a suitable vehicle (e.g., sterile pyrogen-free water) before use.

[0142] The use of the present disclosure may include administration of the composition formulated as a depot preparation. Such long-acting formulations may be administered by implantation (e.g., subcutaneous or intramuscular implantation) or by intramuscular injection. For example, the composition may be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil), may be formulated with ion exchange resins, or may be formulated as a poorly soluble derivative (e.g., as a poorly soluble salt).

[0143] The antibody, its functional fragment, or binding protein of the present disclosure may also be administered with one or more additional therapeutic agents that are useful in treating various diseases. The antibody, its functional fragment, and binding protein described herein may be used alone or in combination with an additional agent, e.g., an additional therapeutic agent, an additional agent selected by a person skilled in the art for its intended purpose. For example, the additional agent may be a therapeutic agent recognized in the art as being useful for treating the disease or condition treated by the antibody or binding protein of the present disclosure. The additional agent may also be an agent that imparts a beneficial attribute to the therapeutic composition, e.g., an agent that affects the viscosity of the composition.

[0144] Having now described the present disclosure in detail, the same will be more clearly understood with reference to the following examples, which are incorporated for purposes of illustration only and are not intended to be limitations of the disclosure.

[0145] Treatment methods and medical uses In some embodiments, the present disclosure provides a method for treating a T cell dysfunction disorder in a subject in need thereof, the method comprising administering to the subject an anti-CD122 antibody and / or an anti-CD132 antibody, or a bispecific multivalent binding protein capable of binding to CD122 and CD132, specifically a FIT-Ig binding protein that binds to CD122 and CD132, as disclosed herein. A T cell dysfunction disorder can be a disease or condition in which the function of normal T cells is impaired, causing downregulation of the subject's immune response to pathogenic antigens, such as infectious microorganisms, bacteria, viruses, etc.

[0146] In some embodiments, the present disclosure provides a method for treating cancer in a subject in need thereof, comprising administering to the subject an anti-CD122 antibody and / or an anti-CD132 antibody, or a bispecific multivalent binding protein capable of binding to CD122 and CD132, specifically a FIT-Ig binding protein that binds to CD122 and CD132, as disclosed herein. The cancer to be treated according to the invention described herein may be any undesired cell proliferation, neoplasm or tumor. The cancer may be benign or malignant, primary or secondary. The cancer may be metastatic.

[0147] In some embodiments, the cancer being treated may be a cancer of a tissue selected from the group consisting of colon, rectum, cervix, oropharynx, nasopharynx, liver, stomach, head and neck, oral cavity, esophagus, lips, mouth, tongue, tonsils, nose, pharynx, salivary glands, paranasal sinuses, pharynx, larynx, prostate, lung, bladder, skin, kidney, ovary, or mesothelium.

[0148] In some embodiments, the cancer to be treated is, for example, melanoma, metastatic melanoma, renal cell carcinoma, ovarian cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, brain cancer, head and neck cancer, breast cancer, colon cancer, colorectal cancer, cervical cancer, hepatocellular carcinoma, prostate cancer, or bladder cancer.

[0149] The treatment methods described herein may further comprise the step of administering to a subject in need thereof an additional active ingredient, which is suitably present in combination with the antibody or binding protein of the present disclosure for the intended treatment purpose, e.g., another drug having anti-tumor activity. In the treatment methods of the present disclosure, the additional active ingredient may be incorporated into a composition comprising the antibody or binding protein of the present disclosure and administered to a subject in need of treatment. In another embodiment, the treatment methods of the present disclosure may comprise the step of administering to a subject in need of treatment an antibody or binding protein described herein, and a separate step of administering to the subject an additional active ingredient prior to, contemporaneously with, or after the step of administering to the subject an antibody or binding protein of the present disclosure.

[0150] [Example] [Example 1] Generation and characterization of anti-CD122 and anti-CD132 antibodies SEQ ID NO:1; SEQ ID NO:1, HUMAN_CD122_ECD The recombinant extracellular domain of human CD122, shown as TIFF2024541048000003.tif28147, or SEQ ID NO: 2 below: SEQ ID NO:2, HUMAN_CD132_ECD Anti-CD122 and anti-CD132 antibodies, respectively, were obtained by immunizing Balb / c mice with the recombinant extracellular domain of human CD132, shown as TIFF2024541048000004.tif28147.

[0151] Mice were immunized at 2-week intervals and monitored for serum titers weekly after the second injection. After four immunizations, spleen cells were harvested and fused with mouse myeloma cells to form hybridoma cell lines. The fusion products were cultured at 1 × 10 in selective medium containing hypoxanthine-aminopterin-thymidine (HAT) in 96-well plates. 5 Spleen cells were plated per well. Hybridoma colonies were observed with the naked eye 7-10 days after fusion. The supernatants of hybridoma cells were then screened and selected to identify cell lines producing CD122-specific or CD132-specific mouse antibodies.

[0152] For preliminary characterization of antigen-specific binding to CD122 or CD132, an anti-CD122 hybridoma cell line designated clone #CD122-mAb77 and an anti-CD132 hybridoma cell line designated clone #CD132-mAb17 were selected. The variable domain sequences of the two murine antibodies are shown in Table 1. Complementarity determining regions (CDRs) are underlined according to Kabat numbering.

[0153] [Table 1]

[0154] The binding affinity and kinetic constants of anti-CD122 and anti-CD132 monospecific antibodies can be determined using grating-coupled interferometry (GCI) on the WAVEsystem (Creoptix AG, Switzerland) using label-free biosensors. Briefly, goat anti-human IgG Fc antibodies were loaded at approximately 1500 pg / mm on a PCP WAVEchip (Creoptix AG). 2 The antibody was pre-immobilized by amine coupling to a density of approximately 30 pg / mm 2The WAVEchip surface is captured to a density of 1000 ng / cm2 and then exposed to the respective antigen, recombinant human CD122 or CD132, serially diluted in 1× HBS-EP+ buffer (Cytiva, Cat No. BR100669) by injection at 60 μL / min for 250 s to assess association, followed by 1200 s of dissociation. After each round of association and dissociation, the WAVEchip surface can be regenerated by subjecting it to a pH 1.5 glycine-HCl buffer injection at 60 μL / min for 120 s. Sensorgrams are recorded at 25° C. and data can be analyzed on a WAVEcontrol (Creoptix AG) and double referenced by subtracting the signal from the blank injection and the reference channel. A Langmuir 1:1 model is used for data fitting.

[0155] [Example 2] Construction of bispecific antibodies targeting the CD122 / CD132 complex 2.1 Generation of bispecific anti-CD122 / CD132 FIT-Ig FIT-Ig molecules as depicted in FIG. 1 were constructed according to the general procedures described in PCT International Publication WO 2015 / 103072. Each FIT-Ig antibody having the ability to bind to CD122 and CD132 has the following structure: Chain #1 (long chain): VL A -CL-VH B - CH1-hinge-CH2-CH3; Chain #2 (first short chain): VH A - CH1; Chain #3 (second short chain): VL B -CL wherein each chain has, from N-terminus to C-terminus, "A" for antibody A directed against one antigen selected from CD122 and CD132, and "B" for antibody B directed against another antigen selected from CD122 and CD132.

[0156] The amino acid sequence of the bispecific anti-CD122 / CD132 FIT-Ig is exemplified in Table 2.

[0157] [Table 2]

[0158] To produce the FIT-Ig antibody, the plasmid constructs encoding the three polypeptide chains were mixed and co-transfected into HEK293 cells. The cells were cultured for 7 days, and the supernatant was harvested and subjected to Protein A purification. The purified FIT-Ig protein in the eluate was measured for concentration by A280 and for homogeneity by size exclusion chromatography (SEC). The exemplified CD122 / CD132 bispecific antibody is designated "FIT2019-86b".

[0159] 2.2 Generation of bispecific anti-CD122 / CD132 duobodies Fab-arm exchange based bispecific "duobodies" were generated according to the literature (Labrijn, AF et al. PNAS, 110, 5145-5150, 2013).

[0160] For example, the VH and VL of CD132-mAb17 were assembled with a human IgG1 constant domain containing a F405L mutation in CH3 and a human kappa constant domain. The VH and VL of CD122-mAb77 were assembled with a human IgG1 constant domain containing a K409R mutation in CH3 and a human kappa constant domain. The amino acid sequences of the four polypeptide chains are shown in Table 3.

[0161] [Table 3]

[0162] CD132-mAb17 (e.g., CD132-mAb17-IgG1-F405L antibody) and CD122-mAb77 (e.g., CD122-mAb77-IgG1-K409R antibody) were produced, respectively. Specifically, Expi293F cells were co-transfected with the corresponding heavy and light chain vectors and cultured for 7 days, then the supernatants were collected and subjected to Protein A chromatography (MabSelect SuRe™; GE Healthcare), and the eluates were dialyzed against PBS overnight and filter-sterilized on a 0.2 μM dead-end filter.

[0163] The purified IgG so obtained was measured for concentration by A280nm with the respective calculated specific extinction coefficient. High performance size exclusion chromatography (HP-SEC) evaluation of the IgG batches gave a monomeric purity of at least 94%. All IgG used in vivo had endotoxin levels below 0.1 endotoxin units per mg of IgG.

[0164] CD132-mAb17-IgG1-F405L and CD122-mAb77-IgG1-K409R antibodies were mixed and incubated with 25 mM 2-mercaptoethylamine (2-MEA, Sigma) at a final concentration of 1 mg / mL per antibody. The mixture was incubated at 37° C. for 90 minutes and then dialyzed overnight against PBS to remove 2-MEA through buffer exchange. Samples were stored overnight at 4° C. to allow for reoxidation for disulfide bonds and the generation of stable 1+1 asymmetric bispecific IgG1 molecules as shown in FIG. 2. The exemplified CD122 / CD132 bispecific antibody is designated “Duo2019-86b”.

[0165] [Example 3] Generation and evaluation of humanized anti-CD122 / CD132 FIT-Ig 3.1 Humanization of anti-CD122 and anti-CD132 antibodies 3.1.1 Humanization of anti-CD132 antibodies Utilizing available human sequences from the V BASE database (https: / / www2.mrc-lmb.cam.ac.uk / vbase / alignments2.php) for best matching of the most homologous human germline IgV gene sequences and framework sequences, humanized antibodies were generated using the sequences of the mAb CD132-mAb17 variable regions in Table 1. For the light chain, CDR-L1, CDR-L2, and CDR-L3 of CD132-mAb17 VL (SEQ ID NO: 6, underlined in Table 1) were grafted onto the framework sequence of the 012 gene (the closest human V gene match for the VL sequence in SEQ ID NO: 6) with the JK2 framework 2 sequence following CDR-L3. For the heavy chain, CDR-H1, CDR-H2, and CDR-H3 of CD132-mAb17 VH (SEQ ID NO:5, underlined in Table 1) were grafted onto the framework sequence of VH1-46 (the closest human match for the VH sequence of SEQ ID NO:5), with the JH6 framework 2 sequence following CDR-H3. The 3DFv model of CD132-mAb17 was generated by homology modeling, which can predict the three-dimensional (3D) structure of a query protein through sequence alignment of a template protein. Certain amino acids at framework positions that were deemed essential to support loop structures or VH / VL interfaces according to such models were backmutated to the corresponding murine residues to minimize problems with affinity / activity (indicated by double underlining in Table 4). Potential mutations were identified: V37M, R38K, M48I, R66K, V67A, M69L, R71A and V78A for the heavy chain, and D70E and F71Y for the light chain (all according to Kabat numbering). According to the ranking of the importance of each back mutation determined by the interaction with the CDRs, the most important back mutations were preferentially introduced into the humanized VH sequence, and other back mutations were subsequently introduced stepwise. Furthermore, the Q1E mutation (italicized and bold in Table 4) was always included to eliminate N-terminal pyroglutamic acid formation when applicable. The sequences so designed are shown in Table 4.

[0166] [Table 4]

[0167] 3.1.2 Humanized design of anti-CD122 antibody Using available human sequences from the V BASE database (https: / / www2.mrc-lmb.cam.ac.uk / vbase / alignments2.php) for best matching of the most homologous human germline IgV gene sequences and framework sequences, humanized antibodies were generated using the sequences of the CD122-mAb77 variable regions in Table 1. For the light chain, CDR-L1, CDR-L2, and CDR-L3 of CD122-mAb77 VL (SEQ ID NO: 4, underlined in Table 1) were grafted onto framework 2 of the A17 gene (the closest human V gene match for the VL sequence in SEQ ID NO: 4) with the JK4 framework 2 sequence following CDR-L3. For the heavy chain, CDR-H1, CDR-H2, and CDR-H3 (SEQ ID NO: 3, underlined in Table 1) of CD122-mAb77 VH were grafted onto the framework sequence of VH1-8 (the closest human match for the VH sequence of SEQ ID NO: 3) with the JH6 framework 2 sequence following CDR-H3. Certain amino acids at framework positions deemed essential to support loop structures or VH / VL interfaces according to 3D Fv model analysis by homology modeling were backmutated to the corresponding mouse residues to minimize problems with affinity / activity (indicated by double underlining in Table 5). Potential mutations (all according to Kabat numbering) of R28T, S30T, K38R, I48M, A67V, L69M, D72N, K73T, and F91Y for the heavy chain and T7S, Y36F, L37Q, and L46R for the light chain were identified. According to the ranking of the importance of each back mutation determined by the interaction with CDR, the most important back mutations were preferentially introduced into the humanized VH sequence, and other back mutations were subsequently introduced stepwise. Furthermore, the Q1E mutation (italicized and bold in Table 5) was always included to eliminate N-terminal pyroglutamate formation when applicable. "DG" (Asp-Gly), "NT" (Asn-Thr) and "NG" (Asn-Gly) in CDR-H2 or CDR-L1 are prone to post-translational modifications (PTM), which may result in heterogeneity during recombinant antibody production.Therefore, VH and VL CDRs containing the following point mutations (bold and highlighted): NG (Asn-Gly) to NA (Asn-Ala), DG (Asp-Gly) to DA (Asp-Ala), DG (Asp-Gly) to EG (Glu-Gly), NT (Asn-Thr) to QT (Gln-Thr) were also designed and evaluated. The sequences so designed are shown in Table 5.

[0168] [Table 5] TIFF2024541048000010.tif235165TIFF2024541048000011.tif217164

[0169] 3.2 Generation and binding evaluation of humanized anti-CD122 / 132 FIT-Ig 3.2.1 Generation of humanized anti-CD122 / 132 FIT-Ig Following the design and production process of FIT-Ig molecules as described in Example 2.1, VHs therein were prepared based on the peptide sequences listed in Table 2. A / VL A with the humanized anti-CD122 VH / VL sequences designed in Table 5, and B / VL B was replaced with the humanized anti-CD132 VH / VL sequences designed in Table 4 to construct a humanized FIT-Ig that recognizes both CD122 and CD123. Such replacements created the humanized anti-CD122 / CD132 FIT-Ig binding proteins listed in Table 6 below.

[0170] [Table 6]

[0171] 3.2.2 Target Binding Assessment of Humanized CD122 / CD132 FIT-Ig The binding affinity and kinetic constants of humanized CD122 / CD132 FIT-Ig were determined by grating coupling interferometry (GCI) using a label-free biosensor on the WAVEsystem (Creoptix AG, Switzerland). Briefly, a goat anti-human IgG Fc antibody was loaded at 3830 pg / mm on a PCP WAVEchip (Creoptix AG). 2 and used to immobilize humanized CD122 / CD132 FIT-Ig generated from Example 3.2.1 to a density of approximately 90 pg / mm 2 The WAVEchip surfaces were captured to a density of 1000 nm and then exposed to recombinant human / cynomolgus CD122 or CD132 serially diluted (200 nM to 823 pM for human CD122, human CD132 and cynomolgus CD132, respectively; 3-fold dilutions of 1000 nM to 1.37 nM for cynomolgus CD122) in 1× HBS-EP+ buffer (Cytiva, Cat No. BR100669) by injection at 60 μL / min for 250 s to assess association, followed by 1200 s of dissociation. After each round of association and dissociation, the WAVEchip surface was regenerated by subjecting it to a pH 1.5 glycine-HCl buffer injection at 60 μL / min for 120 s. Sensorgrams were recorded at 25° C. and data were analyzed on a WAVEcontrol (Creoptix AG) and double-referenced by subtracting the signal from the blank injection and reference channel. A Langmuir 1:1 model was used for data fitting. The kinetic constants obtained for the humanized CD122 / CD132 FIT-Igs generated from Example 3.2.1 showed that they all bind equally to human and cynomolgus CD122 and CD132 with high affinity. Table 7 illustrates the binding kinetic constants of huFIT2019-86b-51.

[0172] [Table 7]

[0173] [Example 4] CD25 binding evaluation of CD122 / CD132 bispecific antibodies CD122 / CD132 bispecific antibody, FIT2019-86b, was tested for CD25 binding activity by ELISA. Specifically, 96-well plates were coated with 1 μg / mL recombinant human CD25, CD122 or CD132, respectively, overnight at 4°C, washed once with washing buffer (PBST, PBS containing 0.05% Tween 20), blocked with blocking buffer (PBST containing 1% BSA) for 0.5 h at room temperature, and assigned concentrations of FIT2019-86b were added and incubated at RT for 0.5 h. Plates were washed three times with PBST, HRP-labeled anti-human IgG secondary antibody was added, incubated at RT for 15 min, and then washed five times with PBST. For color development, 100 μl of tetramethylbenzidine (TMB) chromogenic solution was added to each well, the reaction was quenched with 1N HCl, and the absorbance at 450 nm was measured on a microplate reader. Plot the binding signal against the antibody concentration using GraphPad Prism 6.0 software and calculate the EC 50 The value was calculated.

[0174] As shown in Figure 3, the FIT2019-86b antibody had no binding to CD25, but specific binding activity was observed for the CD122 and CD132 targets, respectively, with an EC of 0.3237 nM for the binding of the FIT2019-86b antibody to human CD122. 50 , EC of 0.6618 nM for binding of FIT2019-86b antibody to human CD132 50 It was.

[0175] [Example 5] Characterization of cell surface binding of CD122 / CD132 bispecific antibodies The CD122 / CD132 bispecific antibodies designated FIT2019-86b and Duo2019-86b, and the humanized anti-CD122 / CD132 antibody huFIT2019-86b-51 were assayed for cell binding activity using HEK293 cell lines overexpressing human CD122 and human CD132, respectively.

[0176] Briefly, 3 × 10 cells were placed in each well of a 96-well plate. 5 Cells were seeded and centrifuged at 400g for 5 min, the supernatant was discarded, then 100 μl of antibody (5-fold serial dilutions, 0.0064-100 nM) was added to each well and mixed gently. After 60 min incubation at 4°C, the plate was washed several times to remove excess antibody. A secondary fluorochrome-conjugated goat anti-human IgG antibody (Jackson Immunoresearch, Cat No. 109-606-098) was then added and incubated with the cells for 20 min at 4°C. After another round of centrifugation and washing, the cells were resuspended in assay buffer (1X PBS containing 2% FBS) for reading on the flow cytometer. Median fluorescence intensity (MFI) readouts were plotted against antibody concentration and analyzed with GraphPad Prism 6.0 software.

[0177] Figure 4 demonstrates that the CD122 / CD132 bispecific antibodies FIT2019-86b, Duo2019-86b and huFIT2019-86b-51 exhibited binding activity to both CD122 and CD132 targets expressed on the cell surface.

[0178] [Example 6] Activation of signal transduction pathways of the CD122 / CD132 complex by CD122 / CD132 bispecific antibodies Activation of the CD122 / CD132 complex signaling pathway was assessed using a STAT5-inducible secreted alkaline phosphatase (SEAP) reporter gene assay.

[0179] Briefly, 100ul of antibody (4-fold serial dilutions in DMEM with 10% FBS, 0.000031nM to 2nM) and 50,000 cells of HEK-Blue™ IL-2 cell (InvivoGen, Cat#hkb-il2) suspension were added to each well of a 96-well assay plate and incubated overnight in a tissue culture incubator (37°C, 5% CO2), 20uL of supernatant was then transferred from each well to another 96-well assay plate and mixed with 180uL of QUANTI-Blue solution (InvivoGen, Cat code rep-qbs). The latter assay plate was incubated at 37°C for 60 minutes for color development and the optical density at 630nm (OD630) was measured on a microplate reader. OD630 represents the level of STAT5 activation.

[0180] FIG. 5 shows that only FIT-2019-86b induced STAT5 activation (suggesting activation of the CD122 / CD132 complex), whereas Duo2019-86 did not. Given that the same variable regions of anti-CD122 and anti-CD132 antibodies were used in both formats, the difference in activation capacity may be format-dependent. Humanized anti-CD122 / CD132 FIT-Igs generated from Example 3.2.1 were also tested in a STAT5-inducible SEAP reporter gene assay (RGA) using bispecific FIT2019-86b as a positive control. EC50 values ​​were calculated to be in the range of about 0.1-0.3 nM. Lower EC50 values ​​indicate better STAT5 activation activity of the antibody. Table 8 shows the EC50 values ​​of some exemplary FIT-Igs.

[0181] [Table 8]

[0182] [Example 7] In vitro agonistic effect of CD122 / CD132 FIT-Ig 7.1 Induction of STAT5 phosphorylation in human lymphocytes The ability of FIT2019-86b and huFIT2019-86b-51 to stimulate IL-2R signaling in human CD4+ T cells, CD8+ T cells, Treg cells, and NK cells was assessed by a flow cytometry-based STAT5 phosphorylation (pSTAT5) assay.

[0183] Briefly, human PBMCs were prepared from frozen LeukoPak cells. PBMC cells were thawed (when necessary), washed twice with RPMI 1640 medium, and diluted to 5.5 × 10 6 Cells were resuspended at 100 μL / mL and then transferred to a 96-well plate at 90 μL / well and 10 μL of antibody or reference IL-2 / IL-2 variant as indicated (5-fold serial dilutions, 0.00128 nM to 100 nM) was added and incubated for 15 minutes at 37°C. The plate was then sealed and returned to the incubator for an additional hour. Immediately after the second incubation, cells were fixed with 100 μL pre-warmed BD Cytofix™ Buffer (Cat. No. 554655) for 10 minutes at 37°C and then centrifuged at 350g for 7 minutes. The supernatant was discarded and 200 uL pre-chilled BD Phosflow Perm Buffer III (BD, Cat#554655) was added and incubated with the cells on ice for 30 minutes in the dark. The cells were then washed twice by centrifugation at 450g for 7 min at 4°C and resuspended with 200uL of cell staining buffer (Biolegend, Cat#420201). The cell pellets were then stained with an antibody mix (anti-CD3, Biolegend, Cat. No. 344818; anti-CD4, Biolegend, Cat. No. 317408; anti-CD8, Biolegend, Cat. No. 565310; anti-CD25, BD, Cat. No. 562442; anti-Foxp3, Biolegend, Cat. No. 560852; anti-pSTAT5, BD, Cat. No. 562076; 2.5uL of each antibody per sample) and incubated in the dark at RT for 60 min. After staining, the cells were centrifuged again and washed twice. Subsequently, the cell pellet was resuspended in 200 uL of cell staining buffer and analyzed by flow cytometer.

[0184] As shown in Figures 6 and 7, treatment with IL-2, FIT2019-86b, and huFIT2019-86b-51 exhibited comparable pSTAT5 activation in human CD8+ T cells, but significantly weaker pSTAT5 activation in human Treg cells was observed from FIT2019-86b and huFIT2019-86b-51 treatment. Meanwhile, the "non-CD25 binding" reference molecules (neo2 / 15 described in PCT International Publication WO2020 / 005819A1; H9 described in PCT International Publication WO2018 / 234862A1) showed greater pSTAT5 activation in human CD8+ T cells than IL-2, but had stronger pSTAT5 activation in human Treg cells than FIT2019-86b and huFIT2019-86b-51.

[0185] 7.2 Induction of human PBMC proliferation Comparison of functional activity between the FIT-Ig of the present disclosure and the reference molecule was further performed in a T cell proliferation assay.

[0186] On day 1, human CD3+ T cells were resuspended at a density of 2E6 cells / mL with complete medium (RPMI1640 supplemented with 10% HI-FBS, 1% PenStrep and 55 μM 2-mercaptoethanol). T cells were washed twice with 1X PBS and transferred to 6-well plates pre-coated with 2 μg / mL anti-CD3 antibody (Biolegend, Cat No. 300332). Soluble anti-CD28 antibody (Biolegend, Cat No. 302934) was also added to the plates at a final concentration of 1 μg / mL. Plates were incubated at 37° C. until day 4, then T cells were washed once with 1X PBS and resuspended with complete medium to a density of 1E6 cells / mL and left overnight.

[0187] On day 5, T cells were harvested, stained with 1 μM CellTrace CSFE (Thermo, Cat No. C34554), resuspended in complete medium at a density of 2E6 cells / mL, dispensed at 100 μL / well into 96-well round-bottom plates, and then incubated with test articles or controls at 37°C for 4 days.

[0188] On day 9, cells were transferred to 96-well v-bottom plates, washed once with 1X DPBS by spinning at 400g for 6 min at 7°C, and then stained with viability dye (eBioscience, Cat No. 65-0865-14) diluted 1:1000 and Fc block (BD Bioscience, Cat No. 564219) diluted 1:200 by incubation at room temperature in the dark for 10 min. After incubation, 50 μL / well of surface staining mixture containing APC mouse anti-human CD4 (BD, Cat No. 555349), PE-Cy7 mouse anti-human CD8 (Biolegend, Cat No. 301012), BV421 mouse anti-human CD25 (Biolegend, 356114) was added and incubated with the cells for 20 min at 4°C. After another wash with 1X DPBS, cells were fixed with 100uL of freshly prepared Fix / Perm solution (Fix / Perm concentrate diluted 1:3, Invitrogen, Cat. No. 00-5123) for 45 minutes at RT in the dark. After incubation, cells were washed with Perm buffer (Perm concentrate diluted 1:10, Invitrogen, Cat. No. 00-8333) and incubated with PE Mouse Anti-Human FoxP3 antibody (Biolegend, Cat. No. 320108) for 1 hour at 4°C. After incubation, cells were centrifuged again and washed twice with Perm buffer. Subsequently, cell pellets were resuspended in 200uL of cell staining buffer (Biolegend, Cat#420201) and analyzed by flow cytometry.

[0189] Comparing FIT-Ig (FIT2019-86b or humanized CD122 / CD132 FIT-Ig generated from Example 3.2.1) with reference molecules (H9, neo2 / 15, or IL-2) based on the results of the PBMC proliferation assays as described above, the FIT-Igs of the present disclosure are comparable to the reference molecules in terms of inducing proliferation of CD8+ and CD4+ T cells, but are much less agonistic in expanding Treg cells. Figure 8 illustrates the results from FIT2019-86b, huFIT9-86b-32, and huFIT2019-8b-51.

[0190] [Example 8] In vivo tumor growth inhibition 8.1 In vivo tumor growth inhibition by FIT2019-86b in immunodeficient M-NSG mice In vivo tumor growth inhibition by FIT2019-86b was evaluated in a melanoma / PBMC combination transplant model in immunodeficient M-NSG mice. Briefly, A375 human melanoma cells (ATCC# CRL-1619) were routinely cultured for at least two passages prior to transplantation. Frozen human PBMCs were thawed and harvested according to standard procedures. On the day of transplantation, 5 × 10 cells were cultured for each mouse. 6 10 A375 cells and 2 x 10 5 PBMCs were mixed in 0.1 mL DPBS + 0.1 mL Matrigel. 0.2 mL of the resulting cell suspension was injected subcutaneously (sc) into the right flank of M-NSG mice (female, 6-7 weeks old) using a 26-gauge needle. 3 Mice with tumor volumes of 0.001 mg / kg were selected (on day 0, D0) and randomized into two groups for treatment with 3 mg / kg FIT2019-86b or vehicle control twice weekly (D3, D7, D10, and D14). Tumor growth and body weight were measured twice weekly. Tumor volume was calculated using the following formula: tumor volume = (length x width). 2 Tumor volumes were calculated based on tumor dimensions using the 1 / 2 ratio. Graft-versus-host disease (GVHD) was assessed in all animals by measuring weight loss over time. Animals that exhibited a 20% or greater weight loss were euthanized.

[0191] Figure 9 demonstrates that FIT2019-86b treatment resulted in significant tumor growth inhibition compared to vehicle controls. Animals treated with FIT2019-86b exhibited weight loss by day 14 (shown in Figure 10), indicating accelerated GVHD over vehicle controls.

[0192] 8.2 In vivo tumor growth inhibition by huFIT2019-86b-51 in immunodeficient NCG mice 8.2.1 In melanoma / PBMC combination transplantation model In vivo tumor growth inhibition by huFIT2019-86b-51 was evaluated in a melanoma / PBMC combination transplant model in immunodeficient NCG mice. Briefly, A375 human melanoma cells (ATCC# CRL-1619) were routinely cultured for at least two passages prior to transplantation. Frozen human PBMCs were thawed and harvested according to standard procedures. On the day of transplantation, 5 × 10 6 10 A375 cells and 2 x 10 5 PBMCs were mixed in 0.1 mL DPBS + 0.1 mL Matrigel. 0.2 mL of the resulting cell suspension was injected subcutaneously (sc) into the right flank of NCG mice (female, 6-7 weeks old) using a 26-gauge needle. 3 Mice with tumor volumes of 1000-1500 mg / kg were selected and randomized into five groups for 3 weeks of treatment twice weekly (day 0, D0) with huFIT2019-86b-51 in four dosages (1 mg / kg, 0.3 mg / kg, 0.1 mg / kg, 0.03 mg / kg) or vehicle control. Tumor growth and body weight were measured twice weekly. Tumor volume was calculated using the following formula: tumor volume = (length x width) x 1000 mg / kg. 2 Tumor volumes were calculated based on tumor dimensions using the 1 / 2 ratio. Graft-versus-host disease (GVHD) was assessed in all animals by measuring weight loss over time. Animals that exhibited a 20% or greater weight loss were euthanized.

[0193] Figure 11 demonstrates that huFIT2019-86b-51 treatment resulted in significant tumor growth inhibition compared to vehicle controls. The group treated with HuFIT2019-86b-51 (1 mg / kg) exhibited slight weight loss by day 14 (shown in Figure 12), indicating accelerated GVHD over vehicle controls.

[0194] 8.2.2 In NSCLC cell / PBMC combination transplantation model In vivo tumor growth inhibition by huFIT2019-86b-51 was also evaluated in an NSCLC cell / PBMC combination transplantation model in immunodeficient NCG mice. Briefly, H292 tumor cells (ATCC# CRL-1848) were routinely cultured for at least two passages prior to transplantation. Frozen human PBMCs were thawed and harvested according to standard procedures. On the day of transplantation, 2 × 10 6 10 H292 cells and 4 x 10 5 PBMCs were mixed in 0.1 mL DPBS. The cell suspension was injected subcutaneously (sc) into the right flank of NCG mice (female, 6-7 weeks old) using a 26-gauge needle. Seven days after inoculation (day 7, D7), mice were selected and randomized into three groups for treatment twice weekly (D7, D10, D14, D17 and D21) with huFIT2019-86b-51 in two dosages (1 mg / kg, 0.3 mg / kg), or vehicle control. Tumor growth and body weight were measured twice weekly. Tumor volume = (length × width) was calculated using the following formula: tumor volume = (length × width) × 100 × 100 = 0.01 mg / kg. 2 Tumor volumes were calculated based on tumor dimensions using the 1 / 2 ratio. Graft-versus-host disease (GVHD) was assessed in all animals by measuring weight loss over time. Animals that exhibited a 20% or greater weight loss were euthanized.

[0195] Figure 13 demonstrates that huFIT2019-86b-51 treatment resulted in significant tumor growth inhibition compared to vehicle control. Figure 14 shows the change in body weight during treatment with huFIT2019-86b-51 and vehicle control.

[0196] [Example 9] In vivo GVHD and CD8 expansion The functional activities of FIT2019-86b and recombinant human IL-2 (rhIL-2) were also compared in an accelerated GVHD model and in vivo effector T cell stimulation assay. Briefly, M-NSG mice (female, 6-7 weeks old) were injected with 5 × 10 6 Human PBMCs were adoptively transferred ip. After 18 days, mice were bled and human PBMC engraftment was monitored by measuring the number of human CD45+ cells in the peripheral blood. Mice with a human CD45+ percentage higher than 2% of total PBMCs were selected and randomized into three groups on day 0 (D0): mice in group 1 (G1) treated once with vehicle control (1X PBS) on D0; mice in group 2 (G2) treated with 50,000IU rhIL-2 every day for 5 consecutive days (D0 to D4); mice in group 3 (G3) treated with 5mg / kg FIT2019-86b by a single injection on D0. Body weight was measured every day from D0 onwards. Fresh whole blood samples were collected from each mouse on D1, D4 and D7 of the study. The percentages of human CD45+, CD4+ and CD8+ cells in peripheral blood were then measured by flow cytometry.

[0197] FIG. 15 shows that mice treated with FIT2019-86b exhibited a very rapid onset of GVHD, manifested by weight loss around D3. In contrast, vehicle control and rhIL-2 treated mice did not exhibit signs of GVHD by D4. This indicates an acceleration of GVHD compared to the vehicle control and rhIL-2 groups, consistent with enhanced activation of immune effector cells in treated mice. Specifically, the response in T cell proliferation profile is shown in FIG. 16 by the time course of CD8+ to CD4+ ratio normalized to the ratio of each individual mouse at D1. In comparison, mice treated with FIT2019-86b showed significantly increased percentages of cytotoxic human CD8+ T cells and relatively reduced human CD4+ T cells from D4 onwards, while the profile in the vehicle control group was relatively stable, but showed only a modest increase in human CD8+ T cells in mice treated with rhIL-2.

[0198] [Example 10] IL-2 / IL2Rβ binding competition assay The ability of huFIT2019-86b-51 and reference molecules to compete with IL2 binding to IL2Rβ was assessed by grating coupling interferometry (GCI) using the Creoptix WAVEsystem (Malvern Panalytical). 1X HBS-EP+ (Cytiva, Cat No. BR100669) containing an additional 350 mM NaCl was used as dilution and running buffer. Briefly, recombinant human IL2 was injected at approximately 2000 pg / mm on a PCP WAVEchip (Creoptix AG) according to the manufacturer's instructions. 2 For each round of testing, 500 nM IL2Rβ was first injected for 250 seconds, resulting in a density of approximately 35 pg / mm 2 immobilized IL2 on the chip. 2The WAVEchip was captured to a density of 1000 nm. 500 nM of test article (human IL2, H9, neo2 / 15 or huFIT2019-86b-51) was premixed with IL2Rβ (1:1 concentration ratio) and injected for 250 seconds to evaluate the binding of the mixture to the IL2 / IL2Rβ complex on the chip. At the end of each round, the WAVEchip was regenerated with pH 1.5 glycine-HCl buffer for 60 seconds at 60 μL / min. Sensorgrams were recorded at 25°C and data were analyzed with WAVEcontrol (Creoptix AG). Theoretically, if the test article / IL2Rβ mixture showed lower binding to the IL2 / IL2Rβ complex than IL2Rβ alone, the test article would be competing with IL2. If not, the test article has a different IL2Rβ binding epitope than IL2. IL2 was used as a competitive positive control in this assay.

[0199] As shown in FIG. 17, unlike IL2 and its derivatives, huFIT2019-86b-51 does not compete with IL2 / IL2Rβ binding.

[0200] Equivalent While specific embodiments of the subject invention have been discussed, the above specification is illustrative and not restrictive. Many variations of the invention will become apparent to those of skill in the art upon review of this specification and the claims that follow. The full scope of the invention should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations.

Claims

1. 1. A bispecific binding protein that specifically binds to CD122 and CD132, comprising a first antigen-binding site that specifically binds to CD122 and a second antigen-binding site that specifically binds to CD132, (a) the first antigen-binding site comprises a set of six CDRs, CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3; CDR-H1 comprises the sequence of DYVIS (SEQ ID NO: 44); CDR-H2 comprises the sequence EIYPGDAQTYYNEMFKG (SEQ ID NO: 47); CDR-H3 comprises the sequence GSYTYDNYAMDF (SEQ ID NO: 46); CDR-L1 comprises the sequence RSSQNIVHSNANTYLE (SEQ ID NO: 54); CDR-L2 comprises the sequence KVSNRFS (SEQ ID NO: 51); and CDR-L3 comprises the sequence FQGSHIPWT (SEQ ID NO: 52), and the second antigen-binding site comprises a set of six CDRs, CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3; CDR-H1 comprises the sequence SYWMH (SEQ ID NO: 57); CDR-H2 comprises the sequence HIYLGGGATNYAEKFRS (SEQ ID NO: 58); CDR-H3 comprises the sequence SQPYYYGMDS (SEQ ID NO: 59); CDR-L1 comprises the sequence RASQDISNYLN (SEQ ID NO: 60); CDR-L2 comprises the sequence YKSRLHS (SEQ ID NO: 61); and CDR-L3 comprises the sequence HQGHTIPFT (SEQ ID NO: 62), The CDRs are defined according to Kabat numbering. or (b) the first antigen-binding site comprises a set of six CDRs, CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3; CDR-H1 comprises the sequence of DYVIS (SEQ ID NO: 44); CDR-H2 comprises the sequence EIYPGDGNTYYNEMFKG (SEQ ID NO: 45); CDR-H3 comprises the sequence GSYTYDNYAMDF (SEQ ID NO: 46); CDR-L1 comprises the sequence RSSQNIVHSNANTYLE (SEQ ID NO: 54); CDR-L2 comprises the sequence KVSNRFS (SEQ ID NO: 51); and CDR-L3 comprises the sequence FQGSHIPWT (SEQ ID NO: 52), and the second antigen-binding site comprises a set of six CDRs, CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3; CDR-H1 comprises the sequence SYWMH (SEQ ID NO: 57); CDR-H2 comprises the sequence HIYLGGGATNYAEKFRS (SEQ ID NO: 58); CDR-H3 comprises the sequence SQPYYYGMDS (SEQ ID NO: 59); CDR-L1 comprises the sequence RASQDISNYLN (SEQ ID NO: 60); CDR-L2 comprises the sequence YKSRLHS (SEQ ID NO: 61); and CDR-L3 comprises the sequence HQGHTIPFT (SEQ ID NO: 62), The CDRs are defined according to Kabat numbering. or (c) the first antigen-binding site comprises a set of six CDRs, CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3; CDR-H1 comprises the sequence of DYVIS (SEQ ID NO: 44); CDR-H2 comprises the sequence EIYPGDANTYYNEMFKG (SEQ ID NO: 48); CDR-H3 comprises the sequence GSYTYDNYAMDF (SEQ ID NO: 46); CDR-L1 comprises the sequence RSSQNIVHSNANTYLE (SEQ ID NO: 54); CDR-L2 comprises the sequence KVSNRFS (SEQ ID NO: 51); and CDR-L3 comprises the sequence FQGSHIPWT (SEQ ID NO: 52), and the second antigen-binding site comprises a set of six CDRs, CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3; CDR-H1 comprises the sequence SYWMH (SEQ ID NO: 57); CDR-H2 comprises the sequence HIYLGGGATNYAEKFRS (SEQ ID NO: 58); CDR-H3 comprises the sequence SQPYYYGMDS (SEQ ID NO: 59); CDR-L1 comprises the sequence RASQDISNYLN (SEQ ID NO: 60); CDR-L2 comprises the sequence YKSRLHS (SEQ ID NO: 61); and CDR-L3 comprises the sequence HQGHTIPFT (SEQ ID NO: 62), The CDRs are defined according to Kabat numbering. or (d) the first antigen-binding site comprises a set of six CDRs, CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3; CDR-H1 comprises the sequence of DYVIS (SEQ ID NO: 44); CDR-H2 comprises the sequence EIYPGEGNTYYNEMFKG (SEQ ID NO: 49); CDR-H3 comprises the sequence GSYTYDNYAMDF (SEQ ID NO: 46); CDR-L1 comprises the sequence RSSQNIVHSNANTYLE (SEQ ID NO: 54); CDR-L2 comprises the sequence KVSNRFS (SEQ ID NO: 51); and CDR-L3 comprises the sequence FQGSHIPWT (SEQ ID NO: 52), and the second antigen-binding site comprises a set of six CDRs, CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3; CDR-H1 comprises the sequence SYWMH (SEQ ID NO: 57); CDR-H2 comprises the sequence HIYLGGGATNYAEKFRS (SEQ ID NO: 58); CDR-H3 comprises the sequence SQPYYYGMDS (SEQ ID NO: 59); CDR-L1 comprises the sequence RASQDISNYLN (SEQ ID NO: 60); CDR-L2 comprises the sequence YKSRLHS (SEQ ID NO: 61); and CDR-L3 comprises the sequence HQGHTIPFT (SEQ ID NO: 62), The CDRs are defined according to Kabat numbering. or (e) the first antigen-binding site comprises a set of six CDRs, CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3; CDR-H1 comprises the sequence of DYVIS (SEQ ID NO: 44); CDR-H2 comprises the sequence EIYPGDAQTYYNEMFKG (SEQ ID NO: 47); CDR-H3 comprises the sequence GSYTYDNYAMDF (SEQ ID NO: 46); CDR-L1 comprises the sequence RSSQNIVHSNGNTYLE (SEQ ID NO: 50); CDR-L2 comprises the sequence KVSNRFS (SEQ ID NO: 51); and CDR-L3 comprises the sequence FQGSHIPWT (SEQ ID NO: 52), and the second antigen-binding site comprises a set of six CDRs, CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3; CDR-H1 comprises the sequence SYWMH (SEQ ID NO: 57); CDR-H2 comprises the sequence HIYLGGGATNYAEKFRS (SEQ ID NO: 58); CDR-H3 comprises the sequence SQPYYYGMDS (SEQ ID NO: 59); CDR-L1 comprises the sequence RASQDISNYLN (SEQ ID NO: 60); CDR-L2 comprises the sequence YKSRLHS (SEQ ID NO: 61); and CDR-L3 comprises the sequence HQGHTIPFT (SEQ ID NO: 62), The CDRs are defined according to Kabat numbering. or (f) the first antigen-binding site comprises a set of six CDRs, CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3; CDR-H1 comprises the sequence of DYVIS (SEQ ID NO: 44); CDR-H2 comprises the sequence EIYPGDGNTYYNEMFKG (SEQ ID NO: 45); CDR-H3 comprises the sequence GSYTYDNYAMDF (SEQ ID NO: 46); CDR-L1 comprises the sequence RSSQNIVHSNGNTYLE (SEQ ID NO: 50); CDR-L2 comprises the sequence KVSNRFS (SEQ ID NO: 51); and CDR-L3 comprises the sequence FQGSHIPWT (SEQ ID NO: 52), and the second antigen-binding site comprises a set of six CDRs, CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3; CDR-H1 comprises the sequence SYWMH (SEQ ID NO: 57); CDR-H2 comprises the sequence HIYLGGGATNYAEKFRS (SEQ ID NO: 58); CDR-H3 comprises the sequence SQPYYYGMDS (SEQ ID NO: 59); CDR-L1 comprises the sequence RASQDISNYLN (SEQ ID NO: 60); CDR-L2 comprises the sequence YKSRLHS (SEQ ID NO: 61); and CDR-L3 comprises the sequence HQGHTIPFT (SEQ ID NO: 62), The CDRs are defined according to Kabat numbering. or (g) the first antigen-binding site comprises a set of six CDRs, CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3; CDR-H1 comprises the sequence of DYVIS (SEQ ID NO: 44); CDR-H2 comprises the sequence EIYPGDANTYYNEMFKG (SEQ ID NO: 48); CDR-H3 comprises the sequence GSYTYDNYAMDF (SEQ ID NO: 46); CDR-L1 comprises the sequence RSSQNIVHSNGNTYLE (SEQ ID NO: 50); CDR-L2 comprises the sequence KVSNRFS (SEQ ID NO: 51); and CDR-L3 comprises the sequence FQGSHIPWT (SEQ ID NO: 52), and the second antigen-binding site comprises a set of six CDRs, CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3; CDR-H1 comprises the sequence SYWMH (SEQ ID NO: 57); CDR-H2 comprises the sequence HIYLGGGATNYAEKFRS (SEQ ID NO: 58); CDR-H3 comprises the sequence SQPYYYGMDS (SEQ ID NO: 59); CDR-L1 comprises the sequence RASQDISNYLN (SEQ ID NO: 60); CDR-L2 comprises the sequence YKSRLHS (SEQ ID NO: 61); and CDR-L3 comprises the sequence HQGHTIPFT (SEQ ID NO: 62), The CDRs are defined according to Kabat numbering. or (h) the first antigen-binding site comprises a set of six CDRs, CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3; CDR-H1 comprises the sequence of DYVIS (SEQ ID NO: 44); CDR-H2 comprises the sequence EIYPGEGNTYYNEMFKG (SEQ ID NO: 49); CDR-H3 comprises the sequence GSYTYDNYAMDF (SEQ ID NO: 46); CDR-L1 comprises the sequence RSSQNIVHSNGNTYLE (SEQ ID NO: 50); CDR-L2 comprises the sequence KVSNRFS (SEQ ID NO: 51); and CDR-L3 comprises the sequence FQGSHIPWT (SEQ ID NO: 52), and the second antigen-binding site comprises a set of six CDRs, CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3; CDR-H1 comprises the sequence SYWMH (SEQ ID NO: 57); CDR-H2 comprises the sequence HIYLGGGATNYAEKFRS (SEQ ID NO: 58); CDR-H3 comprises the sequence SQPYYYGMDS (SEQ ID NO: 59); CDR-L1 comprises the sequence RASQDISNYLN (SEQ ID NO: 60); CDR-L2 comprises the sequence YKSRLHS (SEQ ID NO: 61); and CDR-L3 comprises the sequence HQGHTIPFT (SEQ ID NO: 62), The CDRs are defined according to Kabat numbering. or (i) the first antigen-binding site comprises a set of six CDRs, CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3; CDR-H1 comprises the sequence of DYVIS (SEQ ID NO: 44); CDR-H2 comprises the sequence EIYPGDAQTYYNEMFKG (SEQ ID NO: 47); CDR-H3 comprises the sequence GSYTYDNYAMDF (SEQ ID NO: 46); CDR-L1 comprises the sequence RSSQNIVHSEGQTYLE (SEQ ID NO: 53); CDR-L2 comprises the sequence KVSNRFS (SEQ ID NO: 51); and CDR-L3 comprises the sequence FQGSHIPWT (SEQ ID NO: 52), and the second antigen-binding site comprises a set of six CDRs, CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3; CDR-H1 comprises the sequence SYWMH (SEQ ID NO: 57); CDR-H2 comprises the sequence HIYLGGGATNYAEKFRS (SEQ ID NO: 58); CDR-H3 comprises the sequence SQPYYYGMDS (SEQ ID NO: 59); CDR-L1 comprises the sequence RASQDISNYLN (SEQ ID NO: 60); CDR-L2 comprises the sequence YKSRLHS (SEQ ID NO: 61); and CDR-L3 comprises the sequence HQGHTIPFT (SEQ ID NO: 62), The CDRs are defined according to Kabat numbering. or (j) the first antigen-binding site comprises a set of six CDRs, CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3; CDR-H1 comprises the sequence of DYVIS (SEQ ID NO: 44); CDR-H2 comprises the sequence EIYPGDGNTYYNEMFKG (SEQ ID NO: 45); CDR-H3 comprises the sequence GSYTYDNYAMDF (SEQ ID NO: 46); CDR-L1 comprises the sequence RSSQNIVHSEGQTYLE (SEQ ID NO: 53); CDR-L2 comprises the sequence KVSNRFS (SEQ ID NO: 51); and CDR-L3 comprises the sequence FQGSHIPWT (SEQ ID NO: 52), and the second antigen-binding site comprises a set of six CDRs, CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3; CDR-H1 comprises the sequence SYWMH (SEQ ID NO: 57); CDR-H2 comprises the sequence HIYLGGGATNYAEKFRS (SEQ ID NO: 58); CDR-H3 comprises the sequence SQPYYYGMDS (SEQ ID NO: 59); CDR-L1 comprises the sequence RASQDISNYLN (SEQ ID NO: 60); CDR-L2 comprises the sequence YKSRLHS (SEQ ID NO: 61); and CDR-L3 comprises the sequence HQGHTIPFT (SEQ ID NO: 62), The CDRs are defined according to Kabat numbering. or (k) the first antigen-binding site comprises a set of six CDRs, CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3; CDR-H1 comprises the sequence of DYVIS (SEQ ID NO: 44); CDR-H2 comprises the sequence EIYPGDANTYYNEMFKG (SEQ ID NO: 48); CDR-H3 comprises the sequence GSYTYDNYAMDF (SEQ ID NO: 46); CDR-L1 comprises the sequence RSSQNIVHSEGQTYLE (SEQ ID NO: 53); CDR-L2 comprises the sequence KVSNRFS (SEQ ID NO: 51); and CDR-L3 comprises the sequence FQGSHIPWT (SEQ ID NO: 52), and the second antigen-binding site comprises a set of six CDRs, CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3; CDR-H1 comprises the sequence SYWMH (SEQ ID NO: 57); CDR-H2 comprises the sequence HIYLGGGATNYAEKFRS (SEQ ID NO: 58); CDR-H3 comprises the sequence SQPYYYGMDS (SEQ ID NO: 59); CDR-L1 comprises the sequence RASQDISNYLN (SEQ ID NO: 60); CDR-L2 comprises the sequence YKSRLHS (SEQ ID NO: 61); and CDR-L3 comprises the sequence HQGHTIPFT (SEQ ID NO: 62), The CDRs are defined according to Kabat numbering. or (l) the first antigen-binding site comprises a set of six CDRs, CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3; CDR-H1 comprises the sequence of DYVIS (SEQ ID NO: 44); CDR-H2 comprises the sequence EIYPGEGNTYYNEMFKG (SEQ ID NO: 49); CDR-H3 comprises the sequence GSYTYDNYAMDF (SEQ ID NO: 46); CDR-L1 comprises the sequence RSSQNIVHSEGQTYLE (SEQ ID NO: 53); CDR-L2 comprises the sequence KVSNRFS (SEQ ID NO: 51); and CDR-L3 comprises the sequence FQGSHIPWT (SEQ ID NO: 52), and the second antigen-binding site comprises a set of six CDRs, CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3; CDR-H1 comprises the sequence SYWMH (SEQ ID NO: 57); CDR-H2 comprises the sequence HIYLGGGATNYAEKFRS (SEQ ID NO: 58); CDR-H3 comprises the sequence SQPYYYGMDS (SEQ ID NO: 59); CDR-L1 comprises the sequence RASQDISNYLN (SEQ ID NO: 60); CDR-L2 comprises the sequence YKSRLHS (SEQ ID NO: 61); and CDR-L3 comprises the sequence HQGHTIPFT (SEQ ID NO: 62), The CDRs are defined according to Kabat numbering. or (m) the first antigen-binding site comprises a set of six CDRs, CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3; CDR-H1 comprises the sequence of DYVIS (SEQ ID NO: 44); CDR-H2 comprises the sequence EIYPGDAQTYYNEMFKG (SEQ ID NO: 47); CDR-H3 comprises the sequence GSYTYDNYAMDF (SEQ ID NO: 46); CDR-L1 comprises the sequence RSSQNIVHSNGQTYLE (SEQ ID NO: 55); CDR-L2 comprises the sequence KVSNRFS (SEQ ID NO: 51); and CDR-L3 comprises the sequence FQGSHIPWT (SEQ ID NO: 52), and the second antigen-binding site comprises a set of six CDRs, CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3; CDR-H1 comprises the sequence SYWMH (SEQ ID NO: 57); CDR-H2 comprises the sequence HIYLGGGATNYAEKFRS (SEQ ID NO: 58); CDR-H3 comprises the sequence SQPYYYGMDS (SEQ ID NO: 59); CDR-L1 comprises the sequence RASQDISNYLN (SEQ ID NO: 60); CDR-L2 comprises the sequence YKSRLHS (SEQ ID NO: 61); and CDR-L3 comprises the sequence HQGHTIPFT (SEQ ID NO: 62), The CDRs are defined according to Kabat numbering. or (n) the first antigen-binding site comprises a set of six CDRs, CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3; CDR-H1 comprises the sequence of DYVIS (SEQ ID NO: 44); CDR-H2 comprises the sequence EIYPGDGNTYYNEMFKG (SEQ ID NO: 45); CDR-H3 comprises the sequence GSYTYDNYAMDF (SEQ ID NO: 46); CDR-L1 comprises the sequence RSSQNIVHSNGQTYLE (SEQ ID NO: 55); CDR-L2 comprises the sequence KVSNRFS (SEQ ID NO: 51); and CDR-L3 comprises the sequence FQGSHIPWT (SEQ ID NO: 52), and the second antigen-binding site comprises a set of six CDRs, CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3; CDR-H1 comprises the sequence SYWMH (SEQ ID NO: 57); CDR-H2 comprises the sequence HIYLGGGATNYAEKFRS (SEQ ID NO: 58); CDR-H3 comprises the sequence SQPYYYGMDS (SEQ ID NO: 59); CDR-L1 comprises the sequence RASQDISNYLN (SEQ ID NO: 60); CDR-L2 comprises the sequence YKSRLHS (SEQ ID NO: 61); and CDR-L3 comprises the sequence HQGHTIPFT (SEQ ID NO: 62), The CDRs are defined according to Kabat numbering. or (o) the first antigen-binding site comprises a set of six CDRs, CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3; CDR-H1 comprises the sequence of DYVIS (SEQ ID NO: 44); CDR-H2 comprises the sequence EIYPGDANTYYNEMFKG (SEQ ID NO: 48); CDR-H3 comprises the sequence GSYTYDNYAMDF (SEQ ID NO: 46); CDR-L1 comprises the sequence RSSQNIVHSNGQTYLE (SEQ ID NO: 55); CDR-L2 comprises the sequence KVSNRFS (SEQ ID NO: 51); and CDR-L3 comprises the sequence FQGSHIPWT (SEQ ID NO: 52), and the second antigen-binding site comprises a set of six CDRs, CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3; CDR-H1 comprises the sequence SYWMH (SEQ ID NO: 57); CDR-H2 comprises the sequence HIYLGGGATNYAEKFRS (SEQ ID NO: 58); CDR-H3 comprises the sequence SQPYYYGMDS (SEQ ID NO: 59); CDR-L1 comprises the sequence RASQDISNYLN (SEQ ID NO: 60); CDR-L2 comprises the sequence YKSRLHS (SEQ ID NO: 61); and CDR-L3 comprises the sequence HQGHTIPFT (SEQ ID NO: 62), The CDRs are defined according to Kabat numbering. or (p) the first antigen-binding site comprises a set of six CDRs, CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3; CDR-H1 comprises the sequence of DYVIS (SEQ ID NO: 44); CDR-H2 comprises the sequence EIYPGEGNTYYNEMFKG (SEQ ID NO: 49); CDR-H3 comprises the sequence GSYTYDNYAMDF (SEQ ID NO: 46); CDR-L1 comprises the sequence RSSQNIVHSNGQTYLE (SEQ ID NO: 55); CDR-L2 comprises the sequence KVSNRFS (SEQ ID NO: 51); and CDR-L3 comprises the sequence FQGSHIPWT (SEQ ID NO: 52), and the second antigen-binding site comprises a set of six CDRs, CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3; CDR-H1 comprises the sequence SYWMH (SEQ ID NO: 57); CDR-H2 comprises the sequence HIYLGGGATNYAEKFRS (SEQ ID NO: 58); CDR-H3 comprises the sequence SQPYYYGMDS (SEQ ID NO: 59); CDR-L1 comprises the sequence RASQDISNYLN (SEQ ID NO: 60); CDR-L2 comprises the sequence YKSRLHS (SEQ ID NO: 61); and CDR-L3 comprises the sequence HQGHTIPFT (SEQ ID NO: 62), The CDRs are defined according to Kabat numbering. or (q) the first antigen-binding site comprises a set of six CDRs, CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3; CDR-H1 comprises the sequence of DYVIS (SEQ ID NO: 44); CDR-H2 comprises the sequence EIYPGDAQTYYNEMFKG (SEQ ID NO: 47); CDR-H3 comprises the sequence GSYTYDNYAMDF (SEQ ID NO: 46); CDR-L1 comprises the sequence RSSQNIVHSNAQTYLE (SEQ ID NO: 56); CDR-L2 comprises the sequence KVSNRFS (SEQ ID NO: 51); and CDR-L3 comprises the sequence FQGSHIPWT (SEQ ID NO: 52), and the second antigen-binding site comprises a set of six CDRs, CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3; CDR-H1 comprises the sequence SYWMH (SEQ ID NO: 57); CDR-H2 comprises the sequence HIYLGGGATNYAEKFRS (SEQ ID NO: 58); CDR-H3 comprises the sequence SQPYYYGMDS (SEQ ID NO: 59); CDR-L1 comprises the sequence RASQDISNYLN (SEQ ID NO: 60); CDR-L2 comprises the sequence YKSRLHS (SEQ ID NO: 61); and CDR-L3 comprises the sequence HQGHTIPFT (SEQ ID NO: 62), The CDRs are defined according to Kabat numbering. or (r) the first antigen-binding site comprises a set of six CDRs, CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3; CDR-H1 comprises the sequence of DYVIS (SEQ ID NO: 44); CDR-H2 comprises the sequence EIYPGDGNTYYNEMFKG (SEQ ID NO: 45); CDR-H3 comprises the sequence GSYTYDNYAMDF (SEQ ID NO: 46); CDR-L1 comprises the sequence RSSQNIVHSNAQTYLE (SEQ ID NO: 56); CDR-L2 comprises the sequence KVSNRFS (SEQ ID NO: 51); and CDR-L3 comprises the sequence FQGSHIPWT (SEQ ID NO: 52), and the second antigen-binding site comprises a set of six CDRs, CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3; CDR-H1 comprises the sequence SYWMH (SEQ ID NO: 57); CDR-H2 comprises the sequence HIYLGGGATNYAEKFRS (SEQ ID NO: 58); CDR-H3 comprises the sequence SQPYYYGMDS (SEQ ID NO: 59); CDR-L1 comprises the sequence RASQDISNYLN (SEQ ID NO: 60); CDR-L2 comprises the sequence YKSRLHS (SEQ ID NO: 61); and CDR-L3 comprises the sequence HQGHTIPFT (SEQ ID NO: 62), The CDRs are defined according to Kabat numbering. or (s) the first antigen-binding site comprises a set of six CDRs, CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3; CDR-H1 comprises the sequence of DYVIS (SEQ ID NO: 44); CDR-H2 comprises the sequence EIYPGDANTYYNEMFKG (SEQ ID NO: 48); CDR-H3 comprises the sequence GSYTYDNYAMDF (SEQ ID NO: 46); CDR-L1 comprises the sequence RSSQNIVHSNAQTYLE (SEQ ID NO: 56); CDR-L2 comprises the sequence KVSNRFS (SEQ ID NO: 51); and CDR-L3 comprises the sequence FQGSHIPWT (SEQ ID NO: 52), and the second antigen-binding site comprises a set of six CDRs, CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3; CDR-H1 comprises the sequence SYWMH (SEQ ID NO: 57); CDR-H2 comprises the sequence HIYLGGGATNYAEKFRS (SEQ ID NO: 58); CDR-H3 comprises the sequence SQPYYYGMDS (SEQ ID NO: 59); CDR-L1 comprises the sequence RASQDISNYLN (SEQ ID NO: 60); CDR-L2 comprises the sequence YKSRLHS (SEQ ID NO: 61); and CDR-L3 comprises the sequence HQGHTIPFT (SEQ ID NO: 62), The CDRs are defined according to Kabat numbering. or (t) the first antigen-binding site comprises a set of six CDRs, CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3; CDR-H1 comprises the sequence of DYVIS (SEQ ID NO: 44); CDR-H2 comprises the sequence EIYPGEGNTYYNEMFKG (SEQ ID NO: 49); CDR-H3 comprises the sequence GSYTYDNYAMDF (SEQ ID NO: 46); CDR-L1 comprises the sequence RSSQNIVHSNAQTYLE (SEQ ID NO: 56); CDR-L2 comprises the sequence KVSNRFS (SEQ ID NO: 51); and CDR-L3 comprises the sequence FQGSHIPWT (SEQ ID NO: 52), and the second antigen-binding site comprises a set of six CDRs, CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3; CDR-H1 comprises the sequence SYWMH (SEQ ID NO: 57); CDR-H2 comprises the sequence HIYLGGGATNYAEKFRS (SEQ ID NO: 58); CDR-H3 comprises the sequence SQPYYYGMDS (SEQ ID NO: 59); CDR-L1 comprises the sequence RASQDISNYLN (SEQ ID NO: 60); CDR-L2 comprises the sequence YKSRLHS (SEQ ID NO: 61); and CDR-L3 comprises the sequence HQGHTIPFT (SEQ ID NO: 62), Depending on the situation, the first antigen-binding site comprises a VH domain and a VL domain; the VH domain comprises a sequence selected from any one of SEQ ID NOs: 21-29, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto, and / or the VL domain comprises a sequence selected from any one of SEQ ID NOs: 41, 30-40, 42-43, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto; or the VH domain comprises the sequence of SEQ ID NO: 3, 63, 64, or 65, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto, and / or the VL domain comprises the sequence of SEQ ID NO: 4, 66, 67, 68, or 69, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto; Furthermore, in some cases, the VH domain comprises amino acid residue 1E and 1 to 9 residues selected from 28T, 30T, 38R, 48M, 67V, 69M, 72N, 73T, 91Y, according to Kabat numbering; and the VL domain comprises 1 to 4 amino acid residues selected from 7S, 36F, 37Q, and 46R, according to Kabat numbering. Optionally, the antibody is and a combination of VH and VL sequences selected from the group consisting of: and the second antigen-binding site comprises a VH domain and a VL domain; the VH domain comprises a sequence selected from any one of SEQ ID NOs: 14 to 18, or a sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto, and / or the VL domain comprises a sequence selected from any one of SEQ ID NOs: 19 or 20, or a sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto; or the VH domain comprises the sequence of SEQ ID NO: 5, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto, and / or the VL domain comprises the sequence of SEQ ID NO: 6, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto; optionally, the VH domain comprises amino acid residue 1E and 1 to 8 amino acid residues selected from 37M, 38K, 48I, 66K, 67A, 69L, 71A and 78A, according to Kabat numbering; and the VL domain comprises 1 to 2 amino acid residues selected from 70E and 71Y, according to Kabat numbering; Optionally, the antibody is comprising a combination of VH and VL sequences selected from the group consisting of: Bispecific binding proteins.

2. comprising a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain; The first polypeptide chain is composed of, from the amino terminus to the carboxyl terminus, VL A -CL-VH B -CH1-hinge-CH2-CH3; the second polypeptide chain comprises, from the amino terminus to the carboxyl terminus, VH A the third polypeptide chain comprises, from the amino terminus to the carboxyl terminus, VL B - Contains CL; VL A -CL is VH A -CH1 to form a first Fab that specifically binds to a first antigen A, and VL B -CL is VH B - paired with CH1 to form a second Fab that specifically binds to a second antigen B; and the first antigen A and the second antigen B are CD122 and CD132, respectively, optionally wherein the first antigen A is CD122 and the second antigen B is CD132; the three polypeptide chains as half molecules associate with the three other polypeptide chains as other half molecules to form the FIT-Ig protein; 2. The bispecific binding protein of claim 1. or A duobody format based on Fab arm exchange of an antibody that specifically binds CD122 and an antibody that specifically binds CD132, optionally comprising (i) a heavy chain comprising the amino acid sequence of SEQ ID NO: 10, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto, and the amino acid sequence of SEQ ID NO: 11, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto, and (ii) a heavy chain comprising the amino acid sequence of SEQ ID NO: 12, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto, and a paired light chain comprising the amino acid sequence of SEQ ID NO: 13, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto.

2. The bispecific binding protein of claim 1, Optionally, in the bispecific binding protein: the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:7, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:8, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto; and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:9, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto; Depending on the situation, The bispecific binding protein has the following characteristics: (i) binds to CD122-expressing cells, wherein the cell-binding ability is reflected by an EC50 of about 5 nM or less, 4 nM or less, 3 nM or less, 2 nM or less, or 1 nM or less, as measured by flow cytometry in a cell-based assay; (ii) binds to CD132-expressing cells, wherein the cell-binding ability is reflected by an EC50 of about 80 nM or less, 60 nM or less, 40 nM or less, 20 nM or less, or 10 nM or less, as measured by flow cytometry in a cell-based assay; (iii) stimulating signal transduction upon binding to a complex containing CD122 and CD132; (iv) binds to human CD122 and human CD132 with a KD of less than about 30 nM, 25 nM, 20 nM, 15 nM, 10 nM, or 5 nM, as measured by a WAVE system or Biacore assay; and is cross-reactive with cynomolgus CD122 and cynomolgus CD132 with a KD of less than about 100 nM, 80 nM, 60 nM, 40 nM, 20 nM, or 10 nM; (v) stimulating the proliferation of cells expressing CD122 and CD132; (vi) preferentially stimulating the proliferation of CD8+ and / or CD4+ T cells over regulatory T cells; (vii) improving effector T cell and / or NK cell anti-tumor immunity in vivo and / or in vitro, e.g., reducing tumor burden / growth / cellular expansion, optionally including anti-tumor cytotoxicity. having one or more of: Bispecific binding proteins.

3. An isolated antibody or antigen-binding fragment thereof that specifically binds to CD122, comprising a set of six CDRs, CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, (a) CDR-H1 comprises the sequence of DYVIS (SEQ ID NO: 44); CDR-H2 comprises the sequence EIYPGDAQTYYNEMFKG (SEQ ID NO: 47); CDR-H3 comprises the sequence GSYTYDNYAMDF (SEQ ID NO: 46); CDR-L1 comprises the sequence RSSQNIVHSNANTYLE (SEQ ID NO: 54); CDR-L2 comprises the sequence KVSNRFS (SEQ ID NO: 51); and CDR-L3 comprises the sequence FQGSHIPWT (SEQ ID NO: 52), The CDRs are defined according to Kabat numbering. or (b) CDR-H1 comprises the sequence of DYVIS (SEQ ID NO: 44); CDR-H2 comprises the sequence EIYPGDGNTYYNEMFKG (SEQ ID NO: 45); CDR-H3 comprises the sequence GSYTYDNYAMDF (SEQ ID NO: 46); CDR-L1 comprises the sequence RSSQNIVHSNANTYLE (SEQ ID NO: 54); CDR-L2 comprises the sequence KVSNRFS (SEQ ID NO: 51); and CDR-L3 comprises the sequence FQGSHIPWT (SEQ ID NO: 52), The CDRs are defined according to Kabat numbering. or (c) CDR-H1 comprises the sequence of DYVIS (SEQ ID NO: 44); CDR-H2 comprises the sequence EIYPGDANTYYNEMFKG (SEQ ID NO: 48); CDR-H3 comprises the sequence GSYTYDNYAMDF (SEQ ID NO: 46); CDR-L1 comprises the sequence RSSQNIVHSNANTYLE (SEQ ID NO: 54); CDR-L2 comprises the sequence KVSNRFS (SEQ ID NO: 51); and CDR-L3 comprises the sequence FQGSHIPWT (SEQ ID NO: 52), The CDRs are defined according to Kabat numbering. or (d) CDR-H1 comprises the sequence of DYVIS (SEQ ID NO: 44); CDR-H2 comprises the sequence EIYPGEGNTYYNEMFKG (SEQ ID NO: 49); CDR-H3 comprises the sequence GSYTYDNYAMDF (SEQ ID NO: 46); CDR-L1 comprises the sequence RSSQNIVHSNANTYLE (SEQ ID NO: 54); CDR-L2 comprises the sequence KVSNRFS (SEQ ID NO: 51); and CDR-L3 comprises the sequence FQGSHIPWT (SEQ ID NO: 52), The CDRs are defined according to Kabat numbering. or (e) CDR-H1 comprises the sequence of DYVIS (SEQ ID NO: 44); CDR-H2 comprises the sequence EIYPGDAQTYYNEMFKG (SEQ ID NO: 47); CDR-H3 comprises the sequence GSYTYDNYAMDF (SEQ ID NO: 46); CDR-L1 comprises the sequence RSSQNIVHSNGNTYLE (SEQ ID NO: 50); CDR-L2 comprises the sequence KVSNRFS (SEQ ID NO: 51); and CDR-L3 comprises the sequence FQGSHIPWT (SEQ ID NO: 52), The CDRs are defined according to Kabat numbering. or (f) CDR-H1 comprises the sequence of DYVIS (SEQ ID NO: 44); CDR-H2 comprises the sequence EIYPGDGNTYYNEMFKG (SEQ ID NO: 45); CDR-H3 comprises the sequence GSYTYDNYAMDF (SEQ ID NO: 46); CDR-L1 comprises the sequence RSSQNIVHSNGNTYLE (SEQ ID NO: 50); CDR-L2 comprises the sequence KVSNRFS (SEQ ID NO: 51); and CDR-L3 comprises the sequence FQGSHIPWT (SEQ ID NO: 52), The CDRs are defined according to Kabat numbering. or (g) CDR-H1 comprises the sequence of DYVIS (SEQ ID NO: 44); CDR-H2 comprises the sequence EIYPGDANTYYNEMFKG (SEQ ID NO: 48); CDR-H3 comprises the sequence GSYTYDNYAMDF (SEQ ID NO: 46); CDR-L1 comprises the sequence RSSQNIVHSNGNTYLE (SEQ ID NO: 50); CDR-L2 comprises the sequence KVSNRFS (SEQ ID NO: 51); and CDR-L3 comprises the sequence FQGSHIPWT (SEQ ID NO: 52), The CDRs are defined according to Kabat numbering. or (h) CDR-H1 comprises the sequence of DYVIS (SEQ ID NO: 44); CDR-H2 comprises the sequence EIYPGEGNTYYNEMFKG (SEQ ID NO: 49); CDR-H3 comprises the sequence GSYTYDNYAMDF (SEQ ID NO: 46); CDR-L1 comprises the sequence RSSQNIVHSNGNTYLE (SEQ ID NO: 50); CDR-L2 comprises the sequence KVSNRFS (SEQ ID NO: 51); and CDR-L3 comprises the sequence FQGSHIPWT (SEQ ID NO: 52), The CDRs are defined according to Kabat numbering. or (i) CDR-H1 comprises the sequence of DYVIS (SEQ ID NO: 44); CDR-H2 comprises the sequence EIYPGDAQTYYNEMFKG (SEQ ID NO: 47); CDR-H3 comprises the sequence GSYTYDNYAMDF (SEQ ID NO: 46); CDR-L1 comprises the sequence RSSQNIVHSEGQTYLE (SEQ ID NO: 53); CDR-L2 comprises the sequence KVSNRFS (SEQ ID NO: 51); and CDR-L3 comprises the sequence FQGSHIPWT (SEQ ID NO: 52), The CDRs are defined according to Kabat numbering. or (j) CDR-H1 comprises the sequence of DYVIS (SEQ ID NO: 44); CDR-H2 comprises the sequence EIYPGDGNTYYNEMFKG (SEQ ID NO: 45); CDR-H3 comprises the sequence GSYTYDNYAMDF (SEQ ID NO: 46); CDR-L1 comprises the sequence RSSQNIVHSEGQTYLE (SEQ ID NO: 53); CDR-L2 comprises the sequence KVSNRFS (SEQ ID NO: 51); and CDR-L3 comprises the sequence FQGSHIPWT (SEQ ID NO: 52), The CDRs are defined according to Kabat numbering. or (k) CDR-H1 comprises the sequence of DYVIS (SEQ ID NO: 44); CDR-H2 comprises the sequence EIYPGDANTYYNEMFKG (SEQ ID NO: 48); CDR-H3 comprises the sequence GSYTYDNYAMDF (SEQ ID NO: 46); CDR-L1 comprises the sequence RSSQNIVHSEGQTYLE (SEQ ID NO: 53); CDR-L2 comprises the sequence KVSNRFS (SEQ ID NO: 51); and CDR-L3 comprises the sequence FQGSHIPWT (SEQ ID NO: 52), The CDRs are defined according to Kabat numbering. or (l) CDR-H1 comprises the sequence of DYVIS (SEQ ID NO: 44); CDR-H2 comprises the sequence EIYPGEGNTYYNEMFKG (SEQ ID NO: 49); CDR-H3 comprises the sequence GSYTYDNYAMDF (SEQ ID NO: 46); CDR-L1 comprises the sequence RSSQNIVHSEGQTYLE (SEQ ID NO: 53); CDR-L2 comprises the sequence KVSNRFS (SEQ ID NO: 51); and CDR-L3 comprises the sequence FQGSHIPWT (SEQ ID NO: 52), The CDRs are defined according to Kabat numbering. or (m) CDR-H1 comprises the sequence of DYVIS (SEQ ID NO: 44); CDR-H2 comprises the sequence EIYPGDAQTYYNEMFKG (SEQ ID NO: 47); CDR-H3 comprises the sequence GSYTYDNYAMDF (SEQ ID NO: 46); CDR-L1 comprises the sequence RSSQNIVHSNGQTYLE (SEQ ID NO: 55); CDR-L2 comprises the sequence KVSNRFS (SEQ ID NO: 51); and CDR-L3 comprises the sequence FQGSHIPWT (SEQ ID NO: 52), The CDRs are defined according to Kabat numbering. or (n) CDR-H1 comprises the sequence of DYVIS (SEQ ID NO: 44); CDR-H2 comprises the sequence EIYPGDGNTYYNEMFKG (SEQ ID NO: 45); CDR-H3 comprises the sequence GSYTYDNYAMDF (SEQ ID NO: 46); CDR-L1 comprises the sequence RSSQNIVHSNGQTYLE (SEQ ID NO: 55); CDR-L2 comprises the sequence KVSNRFS (SEQ ID NO: 51); and CDR-L3 comprises the sequence FQGSHIPWT (SEQ ID NO: 52), The CDRs are defined according to Kabat numbering. or (o) CDR-H1 comprises the sequence of DYVIS (SEQ ID NO: 44); CDR-H2 comprises the sequence EIYPGDANTYYNEMFKG (SEQ ID NO: 48); CDR-H3 comprises the sequence GSYTYDNYAMDF (SEQ ID NO: 46); CDR-L1 comprises the sequence RSSQNIVHSNGQTYLE (SEQ ID NO: 55); CDR-L2 comprises the sequence KVSNRFS (SEQ ID NO: 51); and CDR-L3 comprises the sequence FQGSHIPWT (SEQ ID NO: 52), The CDRs are defined according to Kabat numbering. or (p) CDR-H1 comprises the sequence of DYVIS (SEQ ID NO: 44); CDR-H2 comprises the sequence EIYPGEGNTYYNEMFKG (SEQ ID NO: 49); CDR-H3 comprises the sequence GSYTYDNYAMDF (SEQ ID NO: 46); CDR-L1 comprises the sequence RSSQNIVHSNGQTYLE (SEQ ID NO: 55); CDR-L2 comprises the sequence KVSNRFS (SEQ ID NO: 51); and CDR-L3 comprises the sequence FQGSHIPWT (SEQ ID NO: 52), The CDRs are defined according to Kabat numbering. or (q) CDR-H1 comprises the sequence of DYVIS (SEQ ID NO: 44); CDR-H2 comprises the sequence EIYPGDAQTYYNEMFKG (SEQ ID NO: 47); CDR-H3 comprises the sequence GSYTYDNYAMDF (SEQ ID NO: 46); CDR-L1 comprises the sequence RSSQNIVHSNAQTYLE (SEQ ID NO: 56); CDR-L2 comprises the sequence KVSNRFS (SEQ ID NO: 51); and CDR-L3 comprises the sequence FQGSHIPWT (SEQ ID NO: 52), The CDRs are defined according to Kabat numbering. or (r) CDR-H1 comprises the sequence of DYVIS (SEQ ID NO: 44); CDR-H2 comprises the sequence EIYPGDGNTYYNEMFKG (SEQ ID NO: 45); CDR-H3 comprises the sequence GSYTYDNYAMDF (SEQ ID NO: 46); CDR-L1 comprises the sequence RSSQNIVHSNAQTYLE (SEQ ID NO: 56); CDR-L2 comprises the sequence KVSNRFS (SEQ ID NO: 51); and CDR-L3 comprises the sequence FQGSHIPWT (SEQ ID NO: 52), The CDRs are defined according to Kabat numbering. or (s) CDR-H1 comprises the sequence of DYVIS (SEQ ID NO: 44); CDR-H2 comprises the sequence EIYPGDANTYYNEMFKG (SEQ ID NO: 48); CDR-H3 comprises the sequence GSYTYDNYAMDF (SEQ ID NO: 46); CDR-L1 comprises the sequence RSSQNIVHSNAQTYLE (SEQ ID NO: 56); CDR-L2 comprises the sequence KVSNRFS (SEQ ID NO: 51); and CDR-L3 comprises the sequence FQGSHIPWT (SEQ ID NO: 52), The CDRs are defined according to Kabat numbering. or (t) CDR-H1 comprises the sequence of DYVIS (SEQ ID NO: 44); CDR-H2 comprises the sequence EIYPGEGNTYYNEMFKG (SEQ ID NO: 49); CDR-H3 comprises the sequence GSYTYDNYAMDF (SEQ ID NO: 46); CDR-L1 comprises the sequence RSSQNIVHSNAQTYLE (SEQ ID NO: 56); CDR-L2 comprises the sequence KVSNRFS (SEQ ID NO: 51); and CDR-L3 comprises the sequence FQGSHIPWT (SEQ ID NO: 52), The CDRs are defined according to Kabat numbering. Depending on the situation, the antibody comprises a variable heavy domain VH and a variable light domain VL; the VH domain comprises the sequence of SEQ ID NO: 3, 63, 64, or 65, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto, and / or the VL domain comprises the sequence of SEQ ID NO: 4, 66, 67, 68, or 69, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto; or the VH domain comprises a sequence selected from any one of SEQ ID NOs: 21-29, or a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical thereto, and / or the VL domain comprises a sequence selected from any one of SEQ ID NOs: 30-43, or a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical thereto; Depending on the situation, the antibody is a chimeric or humanized antibody, optionally the antibody is a humanized antibody; and further optionally, the VH domain of the antibody comprises amino acid residues 1E and 1 to 9 residues selected from 28T, 30T, 38R, 48M, 67V, 69M, 72N, 73T, 91Y, according to Kabat numbering; and the VL domain comprises 1 to 4 amino acid residues selected from 7S, 36F, 37Q, and 46R, according to Kabat numbering. Depending on the situation, The antibody, and a combination of VH and VL sequences selected from the group consisting of: optionally, the antibody comprises a VH domain comprising the sequence of SEQ ID NO: 21 and a VL domain comprising the sequence of SEQ ID NO: 41; Depending on the situation, the antibody comprises an Fc region or an Fc region having the amino acid sequence of SEQ ID NO: 70; An isolated antibody or antigen-binding fragment.

4. A fusion or conjugate comprising the isolated antibody or antigen-binding fragment of claim 3.

5. A method for detecting CD122 in a biological sample, comprising contacting the biological sample with an isolated antibody or antigen-binding fragment described in claim 3 or a fusion or conjugate described in claim 4.

6. An isolated antibody or antigen-binding fragment thereof that specifically binds to CD132, comprising a set of six CDRs, CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, CDR-H1 comprises the sequence SYWMH (SEQ ID NO: 57); CDR-H2 comprises the sequence HIYLGGGATNYAEKFRS (SEQ ID NO: 58); CDR-H3 comprises the sequence SQPYYYGMDS (SEQ ID NO: 59); CDR-L1 comprises the sequence RASQDISNYLN (SEQ ID NO: 60); CDR-L2 comprises the sequence YKSRLHS (SEQ ID NO: 61); and CDR-L3 comprises the sequence HQGHTIPFT (SEQ ID NO: 62), In some cases, the CDRs are defined according to Kabat numbering. Depending on the situation, the antibody comprises a variable heavy domain VH and a variable light domain VL; the VH domain comprises the sequence of SEQ ID NO: 5, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto, and / or the VL domain comprises the sequence of SEQ ID NO: 6, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto; or the VH domain comprises a sequence selected from any one of SEQ ID NOs: 14 to 18, or a sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto, and / or the VL domain comprises a sequence selected from any one of SEQ ID NOs: 19 or 20, or a sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto; optionally, the VH domain of the antibody comprises amino acid residue 1E and 1 to 8 amino acid residues selected from 37M, 38K, 48I, 66K, 67A, 69L, 71A and 78A, according to Kabat numbering; and the VL domain comprises 1 to 2 amino acid residues selected from 70E and 71Y, according to Kabat numbering; Optionally, the antibody is and a combination of VH and VL sequences selected from the group consisting of: optionally, the antibody comprises a VH domain comprising the sequence of SEQ ID NO: 14 and a VL domain comprising the sequence of SEQ ID NO: 19; Depending on the situation, the antibody is a chimeric or humanized antibody, optionally the antibody is a humanized antibody; Depending on the situation, the antibody comprises an Fc region or an Fc region having the amino acid sequence of SEQ ID NO: 70; An isolated antibody or antigen-binding fragment.

7. A fusion or conjugate comprising the isolated antibody or antigen-binding fragment of claim 6.

8. A method for detecting CD132 in a biological sample, comprising contacting the biological sample with an isolated antibody or antigen-binding fragment described in claim 6 or a fusion or conjugate described in claim 7.

9. 10. A nucleic acid molecule encoding the bispecific binding protein of claim 2, or encoding the isolated antibody or antigen-binding fragment of claim 3 or 6.

10. A vector comprising the nucleic acid molecule of claim 9.

11. A host cell comprising the nucleic acid molecule of claim 9 or the vector of claim 10.

12. 10. A method for preparing the isolated antibody or antigen-binding fragment of claim 3 or 6, or the bispecific binding protein of claim 2, comprising: Culturing the host cell of claim 11 under conditions that allow for the production of the antibody, antigen-binding fragment, or bispecific binding protein; and Recovering the antibody, antigen-binding fragment, or bispecific binding protein from the culture. A method comprising:

13. 12. A pharmaceutical composition comprising the bispecific binding protein of claim 2, or the isolated antibody or antigen-binding fragment of claim 3 or 6, or the fusion or conjugate of claim 4 or 7, the nucleic acid of claim 9, the vector of claim 10, or the host cell of claim 11.

14. 14. The pharmaceutical composition according to claim 13 for treating or preventing diseases in which the function of effector T cells and / or NK cells is impaired and the immune response is downregulated.

15. 15. The pharmaceutical composition of claim 14, wherein the disease is a T-cell dysfunction disorder or cancer, such as melanoma, metastatic melanoma, renal cell carcinoma, ovarian cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, brain cancer, head and neck cancer, breast cancer, colon cancer, colorectal cancer, cervical cancer, hepatocellular carcinoma, prostate cancer, or bladder cancer.

16. Use of the pharmaceutical composition described in claim 13 for the manufacture of a medicament for treating or preventing a disease in which the function of effector T cells and / or NK cells is impaired and the immune response is downregulated.

17. The use of claim 16, wherein the disease is a T cell dysfunction disorder or cancer, such as melanoma, metastatic melanoma, renal cell carcinoma, ovarian cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, brain cancer, head and neck cancer, breast cancer, colon cancer, colorectal cancer, cervical cancer, hepatocellular carcinoma, prostate cancer, or bladder cancer.

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