Compositions against common gamma-chain proteins and substances

Abstract

Described herein are the design and production of antibodies and antigen-binding fragments (e.g., chimeric and humanized antibodies) that specifically bind and inhibit activation of human common gamma chain (gamma c) (shared signaling receptor subunits used by six gamma c cytokines). Methods of production and functional assays that assess the efficacy of the produced antibodies are also described, along with the results that such anti-gammac antibodies are indicated for use in the treatment or prevention of autoimmune cases mediated by gammac or gammac cytokines, such as GVHD.

Description

[0001] priority

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 485,980, filed February 20, 2023, the entire contents of which are incorporated herein by reference.

[0003] sequence list

[0004] This application contains a sequence list in XML format named "SBL-011PCT_SL.xml" created on February 12, 2023, which is 227,306 bytes in size, and the entire contents of which are incorporated herein by reference. Technical Field

[0005] This invention relates to the development of antigen-binding proteins that bind to γc proteins, and the assessment of the efficacy of those proteins in inhibiting γc cytokine-induced function in immune cells and demonstrating their therapeutic potential in treating autoimmune diseases. Background Technology

[0006] The common γ chain (γc) was first discovered in 1992 as a component of the receptor for interleukin (IL)-2. Subsequently, γc was characterized as a shared signal transduction receptor subunit used by six γc-cytokines: IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. γc is widely expressed on most immune cells, including B cells, T cells, natural killer (NK) cells, and innate lymphoid cells (ILCs). These γc-cytokines bind to their respective receptor subunits, which dimerize with γc to induce downstream pathways associated with broad pleiotropic effects on the innate and adaptive immune systems. Dimerization of the two receptor subunits triggers activation of the Janus kinase-signal transduction and transcription activator protein (JAK-STAT) pathway. Following receptor dimerization, autophosphorylation of JAK1 and JAK3 kinases can further phosphorylate STAT, which then dimerizes and translocates to the nucleus. Currently, STAT is recognized as a transcriptional and epigenetic regulator of genes involved in survival, differentiation, proliferation, and immune responses.

[0007] The gene for γc expression is located on chromosome Xq13.1. Mutations in γc in patients with X-linked severe combined immunodeficiency (XSCID) [Noguchi et al., Cell., 1993; 73(1):147-57.] lead to significant impairment of both cellular and humoral immunity due to failure of T and NK cell development, as well as abnormal immunoglobulin production due to dysfunction and absence of fully mature B cells.

[0008] The role of γc cytokines in antitumor effects has been described in various literatures. IL-2, IL-7, IL-15, and IL-21 are mentioned because they stimulate NK cells and CD4+. + Helper cells and CD8 + The role of cytotoxic T cells in proliferation and activation has shown great potential for immunotherapy [Meazza et al., J Biomed Biotechnol., 2011;2011:861920; Raeber et al., Immunol Rev., 2018;283(1):176-193.]. The administration of these γc cytokines has been tested in clinical trials as combination therapy [summarized in Pulliam et al., Immunol Lett.,2016;169:61-72.]. However, studies have shown that γc cytokines have ambivalent roles in hematopoietic cancers, and therefore the use of γc cytokines in anticancer therapy remains controversial.

[0009] In contrast, elevated levels of γc cytokines have been consistently observed in human autoimmune conditions, and these elevated levels are associated with the progression of autoimmune diseases. IL-2 is associated with the progression of asthma [clinical trial NCT01246414], vitiligo [Ranjkesh et al., Indian J Dermatol., 2021; 66(4): 366–370.], multiple sclerosis (MS) [Sharief and Thompson J Neurol Neurosurg Psychiatry., 1993; 56(2): 169–174.], and celiac disease [Tye-Din et al., Aliment Pharmacol Ther., 2019; 50(8):901-910.]. IL-4 and atopic dermatitis (AD) [Chiricozzi et al., Immunotargets Ther., 2020; 9:151–156], asthma [Pelaia et al., Front Pharmacol., 2022; 13: 851940.], MS [Tahaniet al., J Immunoassay Immunochem., 2019; 40(5):555-563.], alopecia areata (AA) [El-Latifet al., J Turk Acad Dermatol., 2021; 15(1):14-18.], allergic rhinitis (AR) [Liang et al., Front Pharmacol., 2020;11:291.], and rheumatoid arthritis (RA) [Talaat et al., Cytokine., The progress is related to [2015;72(2):146-53.].IL-7 is associated with the progression of asthma [Kelly et al., J Immunol., 2009; 182(3): 1404–1410.], psoriasis [Bonifati et al., Clin Immunol Immunopathol., 1997; 83(1): 41-4.], AA [Dai et al., Sci Adv., 2021; 7(14): eabd1866.], Sjogren's syndrome (SS) [Liang et al., Int Immunopharmacol., 2022; 108: 108758.], and type 1 diabetes (T1D) [Monti and Bonifacio Curr Diab Rep., 2014; 14(9): 518.]. IL-9 is associated with the progression of AD [Ma et al., Clin Exp Immunol., 2014; 175(1):25-31.], asthma [Mahdaviani et al., Acta Biomed., 2021; 92(3): e2021206.], systemic lupus erythematosus (SLE), and RA [Dantas et al., Dis Markers., 2015; 2015:519638.]. IL-15 and RA [Yang et al., Hum Immunol., 2015; 76(11):812-8.], psoriasis [Jesús-Gil et al., Exp Dermatol, 2020; 29(7):630-638.], vitiligo [Atwa et al., J CosmetDermatol., 2021; 20(8):2640-2644.], MS [Losy et al., Folia Neuropatho.l, 2002;40(3):151-3.], AA [Ebrahim et al., Int J Trichology., 2019; 11(1): 26–30.], celiac disease, SLE, and T1D [Abadie and Jabri Immunol Rev., 2014; 260(1): 812-8.] Related to the progress of 221–234.IL-21 and AD [Mizutani et al., Allergol Int., 2017; 66(3):440-444.], RA [Hao et al., BMC Musculoskelet Disord., 2021 Mar 5;22(1):246., 2021; 66(3):440-444.], Psoriasis [Wang et al., Am J Transl Res., 2016; 8(7): 3188–3196.], Vitiligo [Custurone et al., Int J Mol Sci., 2021; 22(21): 11429.], MS [Tzartos et al., Am J Pathol., 2011; 178(2): 794–802.], AA [Ahmed et al., The Egyptian Journal of Hospital Medicine, 2022; Page 831-836.], celiac disease [Iervasi et al., Autoimmunity., 2020;53(4):225-230.], SS [Kang et al., Arthritis Res Ther.,2011; 13(5): R179.], T1D [Ferreira et al., Diabetologia., 2015;58(4):781-90.], and SLE [Dolff et al., Arthritis Res Ther., 2011;13(5):R157.] are associated with the progression of these diseases. Summary of the Invention

[0010] This invention provides antibodies or antigen-binding fragments (monospecific or multispecific) that bind to γc, said antibody or antigen-binding fragment being characterized by one or more of the following: i) at a concentration of less than 10 -8 M of K DThe antibodies can bind to purified human γc; ii) inhibit STAT phosphorylation induced by IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21; and iii) inhibit γc cytokine-driven functions (e.g., proliferation and cytokine release) in immune cell lines, peripheral blood mononuclear cells (PBMCs), and isolated primary immune cell cultures. Antibodies generated by this invention can specifically bind to the same γc epitope with commercially available antibodies or antigen-binding fragments, or bind to different γc epitopes with commercially available antibodies or their antigen-binding fragments, which are variants of any antibody or fragment characterized by one or more of the properties described above, and constitute a part of this invention.

[0011] The present invention also provides antibody or antigen-binding fragments that can modulate signal transduction pathways induced by at least one member of the γc-cytokine family, resulting in the suppression, improvement, inhibition, treatment, and / or prevention of at least one autoimmune disease.

[0012] The present invention also provides DNA sequences for encoding antibody or antigen-binding fragments. DNA sequences for encoding CDR-H1, CDR-H2, and CDR-H3 of heavy chain immunoglobulins or their variable regions are shown in SEQ ID NO: 1, 17, 33, 49, 65, 81, 97, 113, 129, 145, 161, and / or 177; or variants thereof; and / or (b) DNA sequences for encoding light chain immunoglobulins containing CDR-L1, CDR-L2, and CDR-L3 of light chain immunoglobulins or their variable regions are shown in SEQ ID NO: 9, 25, 41, 57, 73, 89, 105, 121, 137, 153, 169, and / or 185; or variants thereof.

[0013] The present invention also provides an antibody or antigen-binding fragment comprising: (a) a heavy chain immunoglobulin or a variable region thereof, comprising CDR-H1, CDR-H2 and CDR-H3 of a heavy chain immunoglobulin or a variable region thereof containing the amino acid sequences shown in SEQ ID NO: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162 and / or 178, or variants thereof; and / or (b) a light chain immunoglobulin or a variable region thereof, comprising CDR-L1, CDR-L2 and CDR-L3 of a light chain immunoglobulin or a variable region thereof containing SEQ ID NO: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170 and / or 186, or variants thereof. In embodiments of the present invention, the antibody or antigen-binding fragment may exhibit one of the following characteristics: (a) it can bind to the same γc epitope as a commercially available γc antibody; (b) it can bind to the same γc epitope as a competing γc antibody; (c) it can bind to a different γc epitope as a commercially available or competing γc antibody.

[0014] In embodiments of the invention, the present invention comprises (a) a heavy chain immunoglobulin or its variable region comprising an amino acid sequence having at least 90% amino acid sequence identity with the amino acid sequences shown in SEQ ID NO: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162 and / or 178, and / or (b) a light chain immunoglobulin or its variable region comprising an amino acid sequence having at least 90% amino acid sequence identity with the amino acid sequences shown in SEQ ID NO: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170 and / or 186. For example, in embodiments of the present invention, the antigen-binding protein comprises (a) a heavy chain immunoglobulin or its variable region, comprising CDR-H1, CDR-H2, and CDR-H3 of the heavy chain immunoglobulin or its variable region containing the amino acid sequences shown in SEQ ID NO: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162, and / or 178, and having at least 90% amino acid sequence identity with the amino acid sequences shown in SEQ ID NO: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162, and / or 178, and / or (b) a light chain immunoglobulin or its variable region, comprising CDR-H1, CDR-H2, and CDR-H3 of the heavy chain immunoglobulin or its variable region containing ... The light chain immunoglobulin or its variable regions CDR-L1, CDR-L2 and CDR-L3 of the amino acid sequences shown in SEQ ID NO: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170 and / or 186, and having at least 90% amino acid sequence identity with the amino acid sequences shown in SEQ ID NO: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170 and / or 186.

[0015] In embodiments of the present invention, the antibody or antigen-binding protein comprises: (i) Heavy chain CDR group: CDR-H1, comprising the amino acid sequence shown in SEQ ID NO: 4; CDR-H2, comprising the amino acid sequence shown in SEQ ID NO: 6; and CDR-H3, comprising the amino acid sequence shown in SEQ ID NO: 8; and / or CDR-H1, comprising the amino acid sequence shown in SEQ ID NO: 20; CDR-H2, comprising the amino acid sequence shown in SEQ ID NO: 22; and CDR-H3, comprising the amino acid sequence shown in SEQ ID NO: 24; and / or CDR-H1, comprising the amino acid sequence shown in SEQ ID NO: 36; CDR-H2, comprising the amino acid sequence shown in SEQ ID NO: 38; and CDR-H3, comprising the amino acid sequence shown in SEQ ID NO: 40; and / or CDR-H1, comprising the amino acid sequence shown in SEQ ID NO: 52; CDR-H2, comprising the amino acid sequence shown in SEQ ID NO: 54 ... The amino acid sequence shown in SEQ ID NO: 56; and / or CDR-H1, which contains the amino acid sequence shown in SEQ ID NO: 68; CDR-H2, which contains the amino acid sequence shown in SEQ ID NO: 70; and CDR-H3, which contains the amino acid sequence shown in SEQ ID NO: 72; and / or CDR-H1, which contains the amino acid sequence shown in SEQ ID NO: 84; CDR-H2, which contains the amino acid sequence shown in SEQ ID NO: 86; and CDR-H3, which contains the amino acid sequence shown in SEQ ID NO: 88; and / or CDR-H1, which contains the amino acid sequence shown in SEQ ID NO: 100; CDR-H2, which contains the amino acid sequence shown in SEQ ID NO: 102; and CDR-H3, which contains the amino acid sequence shown in SEQ ID NO: 104; and / or CDR-H1, which contains the amino acid sequence shown in SEQ ID NO: 116; CDR-H2, which contains the amino acid sequence shown in SEQ ID NO: 118; and CDR-H3, which contains the amino acid sequence shown in SEQ ID NO: 68. The amino acid sequence shown in SEQ ID NO: 120; and / or CDR-H1, which contains the amino acid sequence shown in SEQ ID NO: 132; CDR-H2, which contains the amino acid sequence shown in SEQ ID NO: 134; and CDR-H3, which contains the amino acid sequence shown in SEQ ID NO: 136; and / or CDR-H1, which contains the amino acid sequence shown in SEQ ID NO: 148; and CDR-H2, which contains the amino acid sequence shown in SEQ ID NO: 150;And CDR-H3, which contains the amino acid sequence shown in SEQ ID NO: 152; and / or CDR-H1, which contains the amino acid sequence shown in SEQ ID NO: 164; CDR-H2, which contains the amino acid sequence shown in SEQ ID NO: 166; and CDR-H3, which contains the amino acid sequence shown in SEQ ID NO: 168; and / or CDR-H1, which contains the amino acid sequence shown in SEQ ID NO: 180; CDR-H2, which contains the amino acid sequence shown in SEQ ID NO: 182; and CDR-H3, which contains the amino acid sequence shown in SEQ ID NO: 184, and / or; (ii) Light chain CDR group: CDR-L1, comprising the amino acid sequence shown in SEQ ID NO: 12; CDR-L2, comprising the amino acid sequence shown in SEQ ID NO: 14; and CDR-L3, comprising the amino acid sequence shown in SEQ ID NO: 16; and / or CDR-L1, comprising the amino acid sequence shown in SEQ ID NO: 28; CDR-L2, comprising the amino acid sequence shown in SEQ ID NO: 30; and CDR-L3, comprising the amino acid sequence shown in SEQ ID NO: 32; and / or CDR-L1, comprising the amino acid sequence shown in SEQ ID NO: 44; CDR-L2, comprising the amino acid sequence shown in SEQ ID NO: 46; and CDR-L3, comprising the amino acid sequence shown in SEQ ID NO: 48; and / or CDR-L1, comprising the amino acid sequence shown in SEQ ID NO: 60; CDR-L2, comprising the amino acid sequence shown in SEQ ID NO: 62; and CDR-L3, comprising the amino acid sequence shown in SEQ ID NO: 12; The amino acid sequence shown in SEQ ID NO: 64; and / or CDR-L1, which contains the amino acid sequence shown in SEQ ID NO: 76; CDR-L2, which contains the amino acid sequence shown in SEQ ID NO: 78; and CDR-L3, which contains the amino acid sequence shown in SEQ ID NO: 80; and / or CDR-L1, which contains the amino acid sequence shown in SEQ ID NO: 92; CDR-L2, which contains the amino acid sequence shown in SEQ ID NO: 94; and CDR-L3, which contains the amino acid sequence shown in SEQ ID NO: 96; and / or CDR-L1, which contains the amino acid sequence shown in SEQ ID NO: 108; CDR-L2, which contains the amino acid sequence shown in SEQ ID NO: 110; and CDR-L3, which contains the amino acid sequence shown in SEQ ID NO: 112; and / or CDR-L1, which contains the amino acid sequence shown in SEQ ID NO: 124; CDR-L2, which contains the amino acid sequence shown in SEQ ID NO: 126; and CDR-L3, which contains the amino acid sequence shown in SEQ ID NO: 64; The amino acid sequence shown in SEQ ID NO: 128; and / or CDR-L1, which contains the amino acid sequence shown in SEQ ID NO: 140; CDR-L2, which contains the amino acid sequence shown in SEQ ID NO: 142; and CDR-L3, which contains the amino acid sequence shown in SEQ ID NO: 144; and / or CDR-L1, which contains the amino acid sequence shown in SEQ ID NO: 156; and CDR-L2, which contains the amino acid sequence shown in SEQ ID NO: 158;And CDR-L3, which contains the amino acid sequence shown in SEQ ID NO: 160; and / or CDR-L1, which contains the amino acid sequence shown in SEQ ID NO: 172; CDR-L2, which contains the amino acid sequence shown in SEQ ID NO: 174; and CDR-L3, which contains the amino acid sequence shown in SEQ ID NO: 176; and / or CDR-L1, which contains the amino acid sequence shown in SEQ ID NO: 188; CDR-L2, which contains the amino acid sequence shown in SEQ ID NO: 190; and CDR-L3, which contains the amino acid sequence shown in SEQ ID NO: 192.

[0016] In embodiments of the present invention, the antigen-binding protein of the present invention comprises the following heavy chain CDR group and light chain CDR group: (i) The heavy chain variable region comprising: CDR-H1, which comprises the amino acid sequence shown in SEQ ID NO: 4; CDR-H2, which comprises the amino acid sequence shown in SEQ ID NO: 6; and CDR-H3, which comprises the amino acid sequence shown in SEQ ID NO: 8; and the light chain variable region comprising: CDR-L1, which comprises the amino acid sequence shown in SEQ ID NO: 12; CDR-L2, which comprises the amino acid sequence shown in SEQ ID NO: 14; and CDR-L3, which comprises the amino acid sequence shown in SEQ ID NO: 16; (ii) The heavy chain variable region comprising: CDR-H1, which comprises the amino acid sequence shown in SEQ ID NO: 20; CDR-H2, which comprises the amino acid sequence shown in SEQ ID NO: 22; and CDR-H3, which comprises the amino acid sequence shown in SEQ ID NO: 24; and the light chain variable region comprising: CDR-L1, which comprises the amino acid sequence shown in SEQ ID NO: 28; CDR-L2, which comprises the amino acid sequence shown in SEQ ID NO: 30; and CDR-L3, which comprises the amino acid sequence shown in SEQ ID NO: 32; (iii) The heavy chain variable region comprising: CDR-H1, which comprises the amino acid sequence shown in SEQ ID NO: 36; CDR-H2, which comprises the amino acid sequence shown in SEQ ID NO: 38; and CDR-H3, which comprises the amino acid sequence shown in SEQ ID NO: 40; and the light chain variable region comprising: CDR-L1, which comprises the amino acid sequence shown in SEQ ID NO: 44; CDR-L2, which comprises the amino acid sequence shown in SEQ ID NO: 46; and CDR-L3, which comprises the amino acid sequence shown in SEQ ID NO: 48; (iv) The heavy chain variable region comprising: CDR-H1, which comprises the amino acid sequence shown in SEQ ID NO: 52; CDR-H2, which comprises the amino acid sequence shown in SEQ ID NO: 54; and CDR-H3, which comprises the amino acid sequence shown in SEQ ID NO: 56; and the light chain variable region comprising: CDR-L1, which comprises the amino acid sequence shown in SEQ ID NO: 60; CDR-L2, which comprises the amino acid sequence shown in SEQ ID NO: 62; and CDR-L3, which comprises the amino acid sequence shown in SEQ ID NO: 64; (v) Heavy chain variable region comprising: CDR-H1, comprising the amino acid sequence shown in SEQ ID NO: 68; CDR-H2, comprising the amino acid sequence shown in SEQ ID NO: 70; and CDR-H3, comprising the amino acid sequence shown in SEQ ID NO: 72; and light chain variable region comprising: CDR-L1, comprising the amino acid sequence shown in SEQ ID NO: 76; CDR-L2, comprising the amino acid sequence shown in SEQ ID NO: 78; and CDR-L3, comprising the amino acid sequence shown in SEQ ID NO: 80; (vi) The heavy chain variable region comprises: CDR-H1, which comprises the amino acid sequence shown in SEQ ID NO: 84; CDR-H2, which comprises the amino acid sequence shown in SEQ ID NO: 86; and CDR-H3, which comprises the amino acid sequence shown in SEQ ID NO: 88; and the light chain variable region comprises: CDR-L1, which comprises the amino acid sequence shown in SEQ ID NO: 92; CDR-L2, which comprises the amino acid sequence shown in SEQ ID NO: 94; and CDR-L3, which comprises the amino acid sequence shown in SEQ ID NO: 96; (vii) Heavy chain variable region comprising: CDR-H1, comprising the amino acid sequence shown in SEQ ID NO: 100; CDR-H2, comprising the amino acid sequence shown in SEQ ID NO: 102; and CDR-H3, comprising the amino acid sequence shown in SEQ ID NO: 104; and light chain variable region comprising: CDR-L1, comprising the amino acid sequence shown in SEQ ID NO: 108; CDR-L2, comprising the amino acid sequence shown in SEQ ID NO: 110; and CDR-L3, comprising the amino acid sequence shown in SEQ ID NO: 112; (viii) The heavy chain variable region comprising: CDR-H1, which comprises the amino acid sequence shown in SEQ ID NO: 116; CDR-H2, which comprises the amino acid sequence shown in SEQ ID NO: 118; and CDR-H3, which comprises the amino acid sequence shown in SEQ ID NO: 120; and the light chain variable region comprising: CDR-L1, which comprises the amino acid sequence shown in SEQ ID NO: 124; CDR-L2, which comprises the amino acid sequence shown in SEQ ID NO: 126; and CDR-L3, which comprises the amino acid sequence shown in SEQ ID NO: 128; (ix) Heavy chain variable region comprising: CDR-H1, comprising the amino acid sequence shown in SEQ ID NO: 132; CDR-H2, comprising the amino acid sequence shown in SEQ ID NO: 134; and CDR-H3, comprising the amino acid sequence shown in SEQ ID NO: 136; and light chain variable region comprising: CDR-L1, comprising the amino acid sequence shown in SEQ ID NO: 140; CDR-L2, comprising the amino acid sequence shown in SEQ ID NO: 142; and CDR-L3, comprising the amino acid sequence shown in SEQ ID NO: 144; (x) Heavy chain variable region comprising: CDR-H1, comprising the amino acid sequence shown in SEQ ID NO: 148; CDR-H2, comprising the amino acid sequence shown in SEQ ID NO: 150; and CDR-H3, comprising the amino acid sequence shown in SEQ ID NO: 152; and light chain variable region comprising: CDR-L1, comprising the amino acid sequence shown in SEQ ID NO: 156; CDR-L2, comprising the amino acid sequence shown in SEQ ID NO: 158; and CDR-L3, comprising the amino acid sequence shown in SEQ ID NO: 160; (xi) Heavy chain variable region comprising: CDR-H1, comprising the amino acid sequence shown in SEQ ID NO: 164; CDR-H2, comprising the amino acid sequence shown in SEQ ID NO: 166; and CDR-H3, comprising the amino acid sequence shown in SEQ ID NO: 168; and light chain variable region comprising: CDR-L1, comprising the amino acid sequence shown in SEQ ID NO: 172; CDR-L2, comprising the amino acid sequence shown in SEQ ID NO: 174; and CDR-L3, comprising the amino acid sequence shown in SEQ ID NO: 176; (xii) Heavy chain variable region comprising: CDR-H1, comprising the amino acid sequence shown in SEQ ID NO: 180; CDR-H2, comprising the amino acid sequence shown in SEQ ID NO: 182; and CDR-H3, comprising the amino acid sequence shown in SEQ ID NO: 184; and light chain variable region comprising: CDR-L1, comprising the amino acid sequence shown in SEQ ID NO: 188; CDR-L2, comprising the amino acid sequence shown in SEQ ID NO: 190; and CDR-L3, comprising the amino acid sequence shown in SEQ ID NO: 192.

[0017] Complexes containing the antigen-binding protein of the present invention are also part of the present invention, wherein the antigen-binding protein binds to a γc polypeptide or an antigen fragment conjugated to a carrier protein (e.g., mouse Fc, 6x His tag (SEQ ID NO: 234)).

[0018] The present invention also provides a method for producing and purifying γc polypeptides conjugated to carrier proteins (e.g., mouse Fc, 6x His tag (SEQ ID NO: 234)) using a mammalian cell expression system (e.g., Expi-CHO cells).

[0019] The present invention also provides a method for generating and screening antigen-binding units, comprising: (a) immunizing an animal (e.g., a mouse) with purified human γc protein by injection (e.g., subcutaneously, intravenously, or intraperitoneally); (b) generating a phage library by randomly ligating a library of antigen-binding single-stranded variable fragments (scFv) to a phage vector (e.g., pCANTAB5); (c) amplifying and screening the phage library using purified human γc protein or γc-expressing human cells (e.g., Ramos B cells); and (d) generating and purifying soluble scFv for γc binding and STAT phosphorylation blocking assays.

[0020] The present invention also provides for the preparation of antigen-binding proteins (e.g., antibodies or antigen-binding fragments thereof) or immunoglobulin chains thereof (e.g., V...). H V LA method (HC or LC) comprising: (a) introducing one or more polynucleotides (or a vector containing such polynucleotides) encoding one or more immunoglobulin chains of the antigen-binding protein into a host cell expression system (e.g., Expi-CHO); (b) culturing host cells under conditions favorable to the expression of the polynucleotide; and (c) isolating the antigen-binding protein or immunoglobulin chains from the host cells and / or the culture medium in which the host cells are grown. The antigen-binding protein or immunoglobulin chains that are products of such methods also constitute a part of this invention. In some embodiments, the antigen-binding protein further comprises a signal peptide (e.g., IL-6 signal peptide) for active secretion from the host cell expression system.

[0021] The present invention also provides polypeptides comprising: (a) a heavy chain immunoglobulin or a variable region thereof, comprising CDR-H1, CDR-H2 and CDR-H3 of a heavy chain immunoglobulin or a variable region thereof containing the amino acid sequences shown in SEQ ID NO: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162 and / or 178, or variants thereof; and / or (b) a light chain immunoglobulin or a variable region thereof, comprising CDR-L1, CDR-L2 and CDR-L3 of a light chain immunoglobulin or a variable region thereof containing the amino acid sequences shown in SEQ ID NO: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170 and / or 186, or variants thereof; or (c) an amino acid sequence shown in a member selected from SEQ ID NO: 1-192, or a variant thereof. The present invention also provides polynucleotides encoding one or more such polypeptides, said polypeptides being shown in SEQ ID NO: 1, 9, 17, 25, 33, 41, 49, 57, 65, 73, 81, 89, 97, 105, 113, 121, 129, 137, 145, 153, 161, 169, 177 and / or 185; or vectors (e.g., plasmids) containing such polynucleotides. In some embodiments, the polypeptides further comprise a signal peptide (e.g., IL-6 signal peptide) for active secretion from a host cell expression system.

[0022] The present invention also provides a host cell expression system (e.g., Expi-CHO) comprising the antigen-binding protein (e.g., antibody or antigen-binding fragment thereof) described herein, and immunoglobulin chains (e.g., V...). H V L (HC or LC), polypeptides, polynucleotides, or carriers.

[0023] This invention describes the construction of humanized anti-γc antibodies using a framework patching method (see, for example, U.S. Patent No. 7,321,026B2). In some embodiments, the framework region (FR) of a mouse anti-γc antibody and / or an antigen-binding protein is replaced by the corresponding FR of the parental immunoglobulin. In some embodiments, the amino acid reversion mutation within the humanized FR is performed to the corresponding amino acid present in the mouse FR. In some embodiments, framework substitution is applied to the humanized antibody to increase binding affinity. In some embodiments, the CDR sequence within the humanized antibody is altered to increase binding affinity and effector function (e.g., blocking downstream signaling pathways). When administered to a human subject, the resulting humanized antibody should exhibit reduced immunogenicity, increased effector function, and increased serum half-life. The amino acid sequences of the humanized variable heavy chain and variable light chain are shown in SEQ ID NO: 193-200. The corresponding humanized heavy chain FR is shown in SEQ ID NO: 221 (FR-H1), 222-223 (FR-H2), 224-225 (FR-H3) and 226 (FR-H4); the humanized light chain FR is shown in SEQ ID NO: 227-228 (FR-L1), 229 (FR-L2), 230-231 (FR-L3) and 232-233 (FR-L4).

[0024] The present invention also provides containers or injection devices (e.g., vials, syringes, pre-filled syringes or auto-injectors) for administration to a subject (e.g., human or mouse) containing an antigen-binding protein or composition (e.g., a pharmaceutical formulation) described herein.

[0025] The present invention also provides a method of administering the antigen-binding protein or composition described herein to a subject (e.g., a human or a mouse), comprising introducing the antigen-binding protein or composition into the body of the subject (e.g., by injection (e.g., subcutaneously, intravenously, or intraperitoneally)). The present invention also demonstrates the therapeutic potential for treating γc or γc cytokine-mediated diseases or conditions (e.g., graft-versus-host disease (GVHD)) in subjects with corresponding needs, including administering (e.g., by injection) an effective amount of the antigen-binding protein or composition described herein.

[0026] This invention also provides a method for blocking PBMCs induced by γc cytokines (IL-2, IL-4, IL-7, IL-9, IL-15 and / or IL-21). Methods for: STAT phosphorylation in (e.g., T cells, B cells, and / or NK cells) or immune cell lines (e.g., mast cells); methods for blocking JAK-STAT-mediated (e.g., STAT3, STAT5, STAT6) intracellular signaling pathways and biological functions (e.g., cytokine and chemokine secretion, cell-mediated cytotoxicity, proliferation, cell survival, cell-cell interactions, differentiation, autoimmunity) induced by γc cytokines (IL-2, IL-4, IL-7, IL-9, IL-15, and / or IL-21); and / or methods for reducing populations of CD45+ immune cells, NK cells, T cells, and / or B cells (e.g., excluding neutrophils, eosinophils, and granulocytes) in PBMC cultures or subjects (e.g., human or mouse), said methods comprising administering to the subject an effective amount of the γc-binding protein described herein or a combination thereof or a formulation thereof. In embodiments of the present invention, the subject suffers from a disease or condition mediated by γc or γc cytokines, such as GVHD, organ transplant rejection, shotgun chorioretinopathy, MS, uveitis, T1D, AD, RA, SLE, asthma, psoriasis, SS, vitiligo, celiac disease, IBD, AA, mast cell-mediated diseases, T-cell lymphoma, NK-cell lymphoma, and / or B-cell lymphoma. Attached Figure Description

[0027] Figure 1 The binding of various purified anti-γc ScFv clones to purified human γc protein in an ELISA assay is shown. Data are expressed in arbitrary units of optical density measured at 450 nm. Figure 1 A and Figure 1 B demonstrates at 30℃ ( Figure 1 A) and 37℃ Figure 1 B) Optical density of purified human γc protein against various anti-γc scFv clones in ELISA assays. Figure 1 C shows a representative flow cytometry plot of anti-γcscFv blocking p-STAT5 in HPB-ALL.

[0028] Figure 2 Examples of SDS-PAGE used to evaluate the purity and stability of two purified γc-binding proteins are shown. More specifically, Figure 2 The results of SDS-PAGE analysis of the purified anti-γc antibody clone are presented, in which signal peptides 2 and 3 were selected for purification of the full-length antibody, where HC = immunoglobulin heavy chain; LC = immunoglobulin light chain.

[0029] Figure 3 The ELISA assays show the binding of A) SM-05A and B) SM-05E to purified γc proteins from humans (Homo sapiens), cynomolgus monkeys (Macaca fascicularis), and chimpanzees (Pan troglodytes). More specifically, Figure 3 A shows the bonding curve of SM-05A, and Figure 3 B shows the binding curves of SM-05E. In each instance, ELISA strips were coated with 1 μg / ml γc protein from each species, and the antibodies were diluted 5-fold from 10 μg / ml to 0.00064 μg / ml.

[0030] Figure 4 The inhibition of STAT phosphorylation induced by selected anti-γc antibody clones and the competitor COMP2022 in various cell lines by (A) IL-2, (B) IL-4, (C) IL-7, (D) IL-9, (E) IL-15, or (F) IL-21 was shown using flow cytometry analysis. Cytokine concentrations in all groups were 10 ng / ml. All antibodies and inhibitors were administered at 33 nM (5 μg / ml).

[0031] Figure 5 The survival of Ramos B cells, as determined by proliferation assay, in the presence of anti-IgM was shown, with pretreatment for two days with IL-4 or IL-21 and various anti-γc antibody clones, the competitor COMP2022, or the JAK3-specific inhibitor ritlecitinib. More specifically, Figure 5 The data shown confirm that IL-4 and IL-21 rescue anti-IgM-induced proliferation defects, while the application of anti-γc antibodies (SM-05A, SM-05E, SM-05F) blocks the effects of IL-4 and IL-21. The concentrations of IL-4 and IL-21 in all groups were 10 ng / ml. All antibodies and inhibitors were administered at 33 nM (5 μg / ml). Figure 5 A and Figure 5 C, compared with the indicated IgG control group by Student's t-test. p < 0.05, N = 5. For Figure 5 B and Figure 5 D, compared with the anti-IgM+IgG control group by one-way ANOVA, p < 0.001, p < 0.01, p < 0.05, N = 5.

[0032] Figure 6Cell damage in Ramos B cells was shown by (AB) apoptosis assay and (CD) Western blot analysis in the presence of anti-IgM, with pretreatment for two days with IL-4 or IL-21 and various anti-γc antibody clones, the competitor COMP2022, or the JAK3-specific inhibitor litexitinib. More specifically, Figure 6 The data shown confirm that anti-γc antibodies inhibit IL-4 and IL-21, rescuing anti-IgM-induced cell death. Figure 5 Expression of A and 5B) and apoptosis / DNA damage markers (A and 5B) Figure 5 (C and 5D). The concentrations of IL-4 and IL-21 in all groups were 10 ng / ml. All antibodies and inhibitors were administered at 33 nM (5 μg / ml). N = 5.

[0033] Figure 7 The inhibition of (A)CD23 and (B)PRDM1 expression by selected anti-γc antibody clones in Ramos B cells after 24 hours of treatment is shown. More specifically, Figure 7 The data shown confirm that, following stimulation with IL-4 or IL-21, anti-γc antibodies inhibit CD23 (γc) in Ramos B cells. Figure 7 A) and PRDM1 Figure 7 B) Expression. The concentrations of IL-4 and IL-21 in all groups were 10 ng / ml. All antibodies and inhibitors were administered at 33 nM (5 μg / ml).

[0034] Figure 8 Jurkat T cell survival for 24 to 72 hours was demonstrated by (A) proliferation assay and (B) p-ERK expression assay in the presence of IL-9 and various anti-γc antibody clones. More specifically, Figure 8 The data shown confirm that the anti-γc antibody inhibits IL-9-induced ERK phosphorylation. The IL-9 concentration in all groups was 50 ng / ml. All antibodies were administered at concentrations ranging from 0 μg / ml to 10 μg / ml (with 5-fold dilutions in between).

[0035] Figure 9 The homeostasis of HPB-ALL T cells, as determined by measuring (A) BCL-2 expression, (B) CD127 internalization, and (C) CD127 degradation, is shown in the presence of IL-7 and various anti-γc antibody clones, the competitor COMP2022, or litexitinib (a JAK3-specific inhibitor). More specifically, Figure 9 The data shown confirm that anti-γc antibody inhibits IL-7-induced BCL-2 expression in HPB-ALL. Figure 9 A) CD127 internalization ( Figure 9 B- Figure 9C) and CD127 degradation ( Figure 9 D- Figure 9 E). IL-7 concentration in all groups was 50 ng / ml. All antibodies and inhibitors were administered at 33 nM (5 μg / ml). Compared with the IgG control, p < 0.01, p < 0.05; compared with the IL-7 + IgG treatment group, ##p < 0.01, #p < 0.05. Statistical analysis was performed using Student's t-test. N = 3–4.

[0036] Figure 10 The establishment of molecular assays for studying KHYG-1 homeostasis is illustrated. Specifically, proliferation was studied in KHYG-1 cultures 3 days after cytokine stimulation. Figure 10 A) Granzyme B ( Figure 10 B) and perforin ( Figure 10 The production of C), and granzyme A ( Figure 10 D) Granzyme B ( Figure 10 E) and IFNγ ( Figure 10 F) activity secretion. IL-2 was administered at 20 ng / ml or 100 ng / ml, IL-15 at 20 ng / ml, and IL-21 at 25 ng / ml. All antibodies and inhibitors were administered at 33 nM (5 μg / ml). Compared with the control, p < 0.001, p < 0.01, p < 0.05. Statistical analysis was performed using a Student's t-test. N = 3.

[0037] Figure 11 The inhibition of IL-2, IL-15, or IL-21-induced (AC) proliferation and (DF) granzyme A secretion in KHYG-1 NK cells was compared after 3 days of treatment with a selected anti-γc antibody clone, a commercially available anti-γc antibody, the competitor COMP2022, or litexitinib (a JAK3-specific inhibitor). More specifically, Figure 11 The data shown confirms that the anti-γc antibody inhibits IL-2 after 3 days of incubation. Figure 11 A) IL-15 Figure 11 B) and IL-21 Figure 11 C) Induces KHYG-1 proliferation and granzyme A secretion ( Figure 11 D- Figure 11 F). IL-2 and IL-15 were administered at 20 ng / ml, and IL-21 at 25 ng / ml. All antibodies and inhibitors were administered at 33 nM (5 μg / ml). Rit: litexitinib.

[0038] Figure 12 The induction of STAT phosphorylation and proliferation by six γc cytokines in PBMC cultures was demonstrated. More specifically, Figure 12 The data shown includes a representative imprint of STAT phosphorylation ( Figure 12 A) and the ratio of STAT phosphorylation relative to the control ( Figure 12 B) and the proliferation index in primary PBMC cultures induced by six γc cytokines ( Figure 12 C). The cytokine concentration in all groups was 10 ng / ml. Compared with the control, p < 0.01, p < 0.05. Statistical analysis was performed using Student's t-test. N = 3 PBMCs were harvested from 3 independent donors.

[0039] Figure 13 The inhibition of STAT phosphorylation by selected anti-γc antibody clones and the competitor COMP2022 in PBMC cultures, as determined by flow cytometry, is shown. The concentrations of six γc cytokines (IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21) in all groups were 10 ng / ml. All antibodies and inhibitors were administered at 66 nM (10 μg / ml).

[0040] Figure 14 This study demonstrates, after 3 days of treatment with the selected anti-γc antibody clone and the competitive antibody COMP2022, the proliferative effects of γc cytokines (except IL-9) in human PBMC cultures, and, conversely, the inhibition of IL-2, IL-7, and IL-15-induced proliferation in PBMCs. More specifically, Figure 14 The results shown demonstrate the proliferative effect of γc cytokines (except IL-9) in human PBMC cultures after 3 days of treatment with the selected anti-γc antibody clone and the competitive antibody COMP2022. Figure 14 A) and IL-2 in PBMCs Figure 14 B), IL-7 Figure 14 C) and IL-15 Figure 14 D) Inhibition of induced proliferation. Cytokine concentrations in all groups were 50 ng / ml. All antibodies and inhibitors were administered at 33 nM (5 μg / ml). Compared to the IgG control, p < 0.01, p < 0.05. Statistical analysis was performed using one-way ANOVA. N = 3–4 for PBMCs from independent donors.

[0041] Figure 15This study demonstrates (A) IL-2, IL-15, and IL-21-induced secretion of granzyme A and IFNγ from PBMC cultures as markers of T and NK cell activation. (C) Various anti-γc antibody clones inhibited granzyme A and granzyme B secretion after three days of treatment with IL-2 or IL-15. More specifically, Figure 15 The results shown demonstrate that IL-2, IL-15, and IL-21 induce granzyme A ( Figure 15 A) and IFNγ Figure 15 B) Secreted from PBMC cultures as a marker of T and NK cell activation. Figure 15 C- Figure 15 F depicts the inhibition of granzyme A and IFNγ secretion by various anti-γc antibody clones and litexitinib after three days of treatment with IL-2 or IL-15. IL-2 and IL-15 were administered at 20 ng / ml, and IL-21 at 25 ng / ml. All antibodies and inhibitors were administered at 33 nM (5 μg / ml). Compared with the indicated group, p < 0.001, p < 0.01, p < 0.05. Statistical analysis was performed using Student's t-test. (A) N = 3–4 for PBMCs from independent donors.

[0042] Figure 16 The inhibition of FasL expression on IL-2 and IL-15-induced cytotoxic T cells was compared after 3 days of treatment with SM-05A or litexitinib. First, cytotoxic T cells were gated via CD3+ and CD8+ channels. Figure 16 A), then using the FasL positive population to establish a phylogenetic group ( Figure 16 B). IL-2 and IL-15 were administered at 20 ng / ml. All antibodies and inhibitors were administered at 33 nM (5 μg / ml).

[0043] Figure 17 The humanized (h)SM-05A in the ELISA assay is shown. Figure 17 A) and hSM-05E ( Figure 17 B) ELISA with purified γc protein from humans (Homo sapiens), cynomolgus monkeys, chimpanzees, common marmosets (Callithrix jacchus), rhesus monkeys (Macaca mulatta), mice (Mus musculus), and rabbits (Oryctolagus cuniculus). ELISA strips were coated with 1 μg / ml of γc protein from each species. Antibodies were diluted 5-fold from 10 μg / ml to 0.00064 μg / ml.

[0044] Figure 18 The analytical chromatograms of AB) hSM-05A and CD) hSM-05E, performed by size exclusion-high performance liquid chromatography (SEC-HPLC) at RT and 37°C after 12 days of PBS exchange, are shown. More specifically, Figure 18 A and Figure 18 Data in B confirms that hSM-05A is at RT ( Figure 18 A) and 37℃ Figure 18 B) Maintain its stability and purity (no aggregation). However, in RT ( Figure 18 C) and 37℃ Figure 18 D) After incubation, aggregation was observed in hSM-05E. The blue arrows indicate peaks representing monoclonal IgG antibodies. The purity of hSM-05A was approximately 99.9%, while the purity of hSM-05E was approximately 80%.

[0045] Figure 19 Representative plots show the binding affinity of humanized antibodies to AB) human γc protein and CD) common marmosets, as measured by Bio-Layer Interferometry (BLI).

[0046] Figure 20 This study demonstrates the in vivo binding assay of hSM-05A to γc protein expressed on the surface of human HEK-293 cells. Briefly, hSM-05A was labeled with FITC fluorescence. HEK-293 cells were transfected with plasmids expressing full-length γc proteins from different species for 2 days, followed by incubation with fluorescent antibodies for flow cytometry analysis. Results showed that hSM-05A was able to bind to native γc proteins from both humans and common marmosets. All antibodies were administered at 33 nM (5 μg / ml). C = untransfected control; Hu: human; Rh: rhesus macaque; Cy: cynomolgus monkey; Ma: common marmoset. N = 3 for all groups.

[0047] Figure 21 The workflow for competitive assay of human γc protein expressed on the surface of Ramos B cells between hSM-05A and hSM-05E is shown. In short, hSM-05E is labeled with FITC fluorescence and then incubated with unconjugated hSM-05A in Ramos B cell culture. Figure 21 A and Figure 21 B shows a representative graph and workflow of the competitive determination. Figure 21 The statistical results in C showed that both antibodies could bind to similar epitopes on human γc protein. Statistical analysis was performed using a Student's t-test. N = 3 for all groups. All antibodies were administered at 33 nM (5 μg / ml).

[0048] Figure 22 The workflow for antibody-dependent cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) of hSM-05A and hSM-05E against the human keratinocyte HaCaT cell line is illustrated. More specifically, Figure 22 A workflow for studying ADCC and CDC activity on HaCaT cells is described. ADCC was determined using PI under flow cytometry analysis to assess cell death, while CDC was studied in 96-well plates via WST-8 proliferation assay. Quantitative analysis showed that neither hSM-05A nor hSM-05E induced ADCC in HaCaT cells after incubation with human PBMCs or a complement mixture. Figure 22 B) and CDC Figure 22 C) Activity. Statistical analysis was performed using Student's t-test. N = 3 for all groups. All antibodies were administered at 33 nM (5 μg / ml).

[0049] Figure 23 The inhibition of STAT phosphorylation by hSM-05A and hSM-05E in PBMC cultures, as determined by flow cytometry, is shown. More specifically, Figure 23 The results showed that administration of hSM-05A and hSM-05E to primary T cell cultures (IL-2 / IL-7 / IL-15) and Ramos B cells (IL-4 / IL-21) inhibited all six γc cytokines (IL-2, IL-7 / IL-15) induced by STAT phosphorylation. Figure 23 A) IL-7 Figure 23 B), IL-15 Figure 23 C), IL-4 Figure 23 D) and IL-21 Figure 23 E). The cytokine concentration in all groups was 50 ng / ml. The antibody and inhibitor were each diluted 5-fold from 33 nM to 0.0528 nM.

[0050] Figure 24 The efficacy of hSM-05A and hSM-05E in restoring B-cell tolerance checkpoints in anti-IgM-stimulated Ramos B cells was demonstrated in the presence of IL-4 or IL-21. More specifically, Figure 24 The schematic diagram in Figure A suggests that hSM-05A and hSM-05E can block IL-4-induced STAT6 phosphorylation and IL-21-induced STAT3 phosphorylation, thereby pushing B cells back to the cell death pathway and inhibiting the abnormal survival of autoreactive B cells. Figure 24 B and Figure 24The quantitative data shown in C confirm that both hSM-05A and hSM-05E can restimulate anti-IgM-induced cell death at levels comparable to litexitinib, as demonstrated in the WST-8 proliferation assay. The percentage of restimulation is calculated as (OD). 抗IgM / 细胞因子 – OD 抗IgM / 细胞因子 / 药物 ) 100 / (OD 抗IgM / 细胞因子 – OD 抗IgM OD refers to the optical density observed by a microplate reader after the addition of WST-8 reagent. The N=3 group was treated with litexitinib, and the N=6 group was treated with the other groups. All antibodies and inhibitors were administered at 33 nM (5 μg / ml).

[0051] Figure 25 This demonstrates the efficacy of hSM-05A and hSM-05E in inhibiting IL-7-induced upregulation of BCL-2 and CD127 degradation in HPB-ALL T cells. More specifically, Figure 25 The schematic diagram in Figure A suggests that hSM-05A and hSM-05E can block IL-7-induced CD127 degradation and BCL2 upregulation by inhibiting JAK3 activation. Figure 25 B and Figure 25 The quantitative data shown in C confirm that both antibodies can inhibit both IL-7-induced processes, and the effect in hSM-05A is stronger than that in the litexitinib treatment group. The percentage of inhibition is calculated as (P<0.05). IL-7 – P IL-7 / 药物 ) 100 / (P IL-7 – P 对照 P refers to the percentage of cells showing positive expression of BCL-2 or CD127, as determined by flow cytometry. N = 3 for all groups. All antibodies and inhibitors were administered at 33 nM (5 μg / ml).

[0052] Figure 26 The efficacy of hSM-05A and hSM-05E in inhibiting IL-9-induced ERK phosphorylation was demonstrated in Jurkat T cells. More specifically, Figure 26 The schematic diagram in Figure A suggests that hSM-05A and hSM-05E can block IL-9-induced ERK phosphorylation and survival by inhibiting STAT5 phosphorylation. For example... Figure 26 The quantitative data shown in B confirm that hSM-05A can significantly inhibit IL-9-induced protection against ceramide-induced cell death. Figure 26Western blot results shown in C indicate that hSM-05A inhibits IL-9-induced ERK phosphorylation, and this finding was quantitatively confirmed. N = 2–3 for all samples. All antibodies were administered at 33 nM (5 μg / ml). p < 0.001 p < 0.05, as analyzed by Student's t-test.

[0053] Figure 27 The efficacy of hSM-05A and hSM-05E in inhibiting IL-2 and IL-15-induced proliferation and cytotoxic factor secretion in human PBMCs was demonstrated. More specifically, Figure 27 The schematic diagram in Figure A suggests that hSM-05A and hSM-05E can block the two phenotypes induced by IL-2 and IL-15 by inhibiting STAT phosphorylation. Figure 27 B and Figure 27 The quantitative data shown in C confirm that both antibodies can inhibit IL-2 and IL-15-induced proliferation at levels comparable to litexitinib. Similarly, Figure 27 The data shown in D-27G confirm that both antibodies can inhibit IL-2 and IL-15-induced IFNγ and granzyme B secretion, while litexitinib can completely attenuate those secretions. The percentage of inhibition is calculated as (V 细胞因子 – V 细胞因子 / 药物 ) 100 / (V 细胞因子 – V 对照 V refers to the data obtained from the proliferation process via a microplate reader. Figure 27 B- Figure 27 C) or ELISA Figure 27 D- Figure 27 G) OD values ​​recorded in the experiment. N = 3-4 for all groups. All antibodies and inhibitors were administered at 33 nM (5 μg / ml).

[0054] Figure 28 The results showed that hSM-05A and hSM-05E can inhibit IL-2 and IL-15-driven activation of NKp46 surface expression on human primary NK and cytotoxic T cells. More specifically, Figure 28 A shows a representative graph to illustrate the number of NKp46+ NK cells after cytokine and drug treatment. Figure 28 B and Figure 28 The quantitative data shown in C confirm the trend in primary NK cells where hSM-05A and hSM-05E reduced IL-2 or IL-15-driven upregulation of NKp46. Similarly, Figure 28 D and Figure 28The quantitative data shown in E confirm the trend that hSM-05A and hSM-05E reduced the upregulation of IL-2 or IL-15 driven NKp46 in primary cytotoxic T cells. Figure 28 D- Figure 28 E's N = 3. All antibodies and inhibitors were administered at 33 nM (5 μg / ml).

[0055] Figure 29 This study demonstrated that hSM-05A and hSM-05E can inhibit the mixed lymphocyte response (MLR) in human PBMC cultures. More specifically, Figure 29 The schematic diagram in Figure A shows T cells isolated from donor 1 and consumed in donor 2. The isolated T cells were labeled with CFSE and then co-cultured with the consumed T cells from donor 2. Analysis of the number of proliferating (CFSE+) T cells by flow cytometry reflects the GvHD status in the culture. Figure 29 B- Figure 29 The quantitative data shown in C demonstrate that both hSM-05A and hSM-05E inhibited the proliferation of GvHD-driven cytotoxic T cells and helper T cells in the culture. N = 2 for each group.

[0056] Figure 30 This is an overview diagram showing how anti-γc antibodies inhibit JAK / STAT activity to block autoreactive T, B, and NK cells.

[0057] Table Overview

[0058] Table 1 shows the heavy chain CDRs in the immunoglobulins of the present invention.

[0059] Table 2 shows the light chain CDRs in the immunoglobulins of the present invention.

[0060] Table 3 shows the heavy and light chain CDRs in each immunoglobulin of the present invention.

[0061] Table 4 shows the DNA sequences encoding the heavy and light chain CDRs in each immunoglobulin of the present invention.

[0062] Table 5 shows the immunization program for γc protein in mice used to produce antibodies.

[0063] Table 6 shows the results of monoclonal phage ELISA on 406 positive clones after two to three rounds of screening.

[0064] Table 7 shows the inhibition of IL-15-induced STAT phosphorylation by various anti-γc ScFv clones in the KHYG-1 NK cell line, as determined by flow cytometry. All ScFv were administered at 10 μg / ml.

[0065] Table 8 shows the inhibition of (A)IL-4 and (B)IL-21-induced STAT phosphorylation by various anti-γc ScFv clones in the Ramos B cell line, as determined by flow cytometry. All ScFv were administered at 10 μg / ml.

[0066] Table 9 shows the inhibition of IL-7-induced STAT5 phosphorylation by various anti-γc ScFv clones in the HPB-ALL T cell line, as determined by flow cytometry. All ScFv were administered at 10 μg / ml.

[0067] Table 10 shows the inhibition of IL-9-induced α)STAT3 phosphorylation by various anti-γc ScFv clones in the Jurkat T cell line, as determined by flow cytometry. All ScFv were administered at 10 μg / ml.

[0068] Table 11 shows the binding kinetics between humanized anti-γc antibodies and human γc protein.

[0069] Table 12 shows the binding kinetics of humanized anti-γc antibodies to γc proteins from other species.

[0070] Table 13 shows the inhibition of STAT phosphorylation by hSM-05A and hSM-05E in PBMC cultures. Detailed Implementation

[0071] This invention provides antibodies and their antigen-binding fragments that specifically bind to human and cynomolgus monkey γc and demonstrate the regulation of intracellular protein pathways and biological functions, including blocking γc cytokine-induced STAT phosphorylation, inhibiting cytokine and enzyme secretion, suppressing overactivation and autoreactivity, and inhibiting apoptosis of autoreactive cells in T, B, NK and / or mast cell cultures. This invention also provides clues for the treatment of GVHD, as presented in international poster presentations [Azimi et al., American Association for Respiratory Care, 2021], recently published manuscripts [Le Floc'h et al., Sci Transl Med., 2023;15(678):eabo0205; Le Floc'h et al., Hemasphere.2022; 6(Suppl ): 694-695] and clinical trials (identified as NCT03532958, NCT05589610).

[0072] This invention also provides methods for designing, screening, and generating antibody and antigen-binding fragments using conventional molecular biology, microbiology and recombinant DNA technologies, phage display technologies, and protein purification technologies.These techniques are well described in the literature: Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; DNA Cloning: A Practical Approach, Volumes I and II (D.N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gait ed. 1984); Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. (1985)); Transcription And Translation (B. D. Hames & S. J. Higgins, eds. (1984)); Animal Cell Culture (R.I. Freshney, ed. (1986)); Immobilized Cells And Enzymes (IRL Press, (1986)); B. Perbal, A Practical Guide To Molecular Cloning (1984); F. M. Ausubel, et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (1994); M. Hust and T.S. Lim, Phage Display Methods and Protocols (2017), Humana Press; P. Meleady, Heterologous Protein Production in CHO Cells Methods and Protocols (2017), Humana Press; R.K. Scope, Protein Purification: Principles and Practice, Second Edition (2013), Springer Science & Business Media.

[0073] Overview

[0074] The common γ chain (γc), also known as P64, CIDX, IMD4, CD132, SCIDX, IL-2RG, and SCIDX1, is a shared signal transduction receptor subunit of several interleukin receptor subunits activated by six γc cytokines. The receptor subunits include IL-2R, IL-4R, IL-7R, IL-9R, IL-15R, and IL-21R.

[0075] Receptor dimerization occurs between the receptor subunit and γc during the induction of the corresponding γc cytokine to trigger the downstream JAK-STAT pathway for biological function. Higher binding affinity and greater stability are triggered during the heterotrimerization of IL-2Rα / IL-2Rβ / γc under IL-2 stimulation and IL-15Rα / IL-2Rβ / γc under IL-15 stimulation [Stauberet al., Proc Natl Acad Sci US A., 2006;103(8):2788-93;Kobayashi et al.,Blood., 2005;105(2):721-7.].

[0076] In an embodiment of the invention, human γc is encoded by the nucleotide sequence shown under Genbank accession number NM_000206. In an embodiment of the invention, human γc comprises the amino acid sequence shown under Genbank accession number NP_000197.

[0077] The complete γc protein consists of extracellular, transmembrane, and intracellular domains [Waickman et al., CellMol Life Sci., 2016; 73(2): 253–269.]. The extracellular domain is divided into two type III fibronectin regions (D1 and D2), which can form a disulfide bond-mediated tertiary structure necessary for interaction with the γc cytokine receptor subunit.

[0078] γc also possesses a characteristic proximal membrane WSXWS motif (SEQ ID NO: 235), a conserved amino acid sequence present in type I cytokine receptors and involved in receptor activation. The intracellular domain of γc contains two conserved JAK3-binding domains (Box 1 and Box 2 motifs) for JAK1 / JAK3 activation and, upon receptor dimerization, activation of downstream signaling events. A degradation motif, located near the Box 2 motif, is used for appropriate membrane localization, ligand-mediated internalization, and lysosomal degradation.

[0079] Six γc cytokines are secreted by various cell types for immune function [Leonard et al., Immunity., 2019; 50(4):832-850.]. IL-2 is secreted by both CD4+ helper cells and CD8+ cytotoxic T cells, activated B cells, and dendritic cells (DCs). IL-4 is secreted by NK-like T cells (NKT), basophils, CD4+ helper T cells, eosinophils, and mast cells. IL-7 is secreted by stromal cells, intestinal epithelial cells, and keratinocytes. IL-9 is secreted by helper T cells 9 (T 9). H 9) Type 2 innate lymphoid cells (ILC2), mast cells, NKT cells, and IL-9-producing cytotoxic T cells (T cells). C 9) Helper T cells 17 (T H 17) and regulatory T cells (Tregs) secrete IL-15. IL-15 (or membrane-bound IL-15) is secreted or provided by dendritic cells (DCs), monocytes, and macrophages. IL-21 is secreted by CD4+ helper T cells, NKT cells, and T follicular helper cells (T cells). FH ), T H 17 and Gamma delta (γδ) T cells secrete it.

[0080] Functions and pathology associated with γc cytokines

[0081] Since γc is widely expressed in most immune cells, those γc cytokines contribute to a variety of biological functions in different immune cells, including proliferation, differentiation, maturation, activation and immune function, as described below [in Leonard et al., Immunity., 2019; 50(4):832-850; Overwijk and Schluns, Clin Immunol., 2009; 132(2): 153–165].

[0082] IL-2 is characterized as a typical growth factor for T cells and NK cells. IL-2 affects T cell and NK cell proliferation, Treg development, B cell function enhancement, activation-induced cell death (AICD) of T cells, and T cell differentiation (T cells). H 1. T H 2 and T H 9) and T H 17 and T FHDifferentiation inhibition is responsible. Impaired IL-2 production has been reported in SLE patients, leading to a reduced Treg population, decreased AICD, excessive inflammation, and kidney damage [Lieberman and Tsokos, J Biomed Biotechnol., 2010;2010:740619; Shao et al., J Interferon Cytokine Res., 2019;39(2):117-124.]. However, overexpression / increased serum IL-2 induces preferential expansion of Tregs [Antony et al., J. Immunol., 2006;176:5255-66.], resulting in peripheral tolerance of immune function. Therefore, IL-2 is considered to be both a negative and positive regulator of autoimmunity.

[0083] IL-4 is one of the key cytokines required for B cell differentiation and immunoglobulin class switching in mature B cells. IL-4 is also involved in T cell differentiation. H 2. T H 9 and Treg differentiation / development, alternative macrophage (M2) activation and monocyte-mediated DC differentiation [Hiasa, et al., Blood., 2009;114(20):4517-26.]. The consequences of IL-4 deficiency have been poorly studied in human subjects, while animal models of IL-4 deficiency have shown increased Treg mortality, reduced granzyme secretion from Tregs [Yang, et al., Front Immunol., 2017;8:1508], excessive neurological excitation [Chen et al., Acta Pharm SinB., 2020;10(9):1634-1645.], resistance to secondary Pseudomonas aeruginosa infection [Song, et al., J Infect Dis., 2015;211(10):1616-27.], and mechanical hypersensitivity [Üçeyler, et al., PLoS One., 2011;6(12):e28205.]. Increased serum levels of IL-4 have been observed in a variety of autoimmune diseases and are believed to play a role in the progression of AD and allergic diseases, as demonstrated in animal studies [Elbe-Bürger et al., J Invest Dermatol., 2002;118(5):767-78; Conde et al., NatCommun., 2021;12(1):2574.].

[0084] IL-7 has been identified as a matrix factor mediating the development and homeostasis of naive T cells, CD4+ memory T cells, and Tregs. IL-7 also synergistically mediates CD8+ memory T cell homeostasis in humans with IL-15. Loss of IL-7 expression in transgenic mice leads to significant T cell deficiencies [Freeden-Jeffry et al., J Exp Med., 1995;181:1519–1526; Peschon et al., J Exp Med., 1994;180:1955–1960.]. Aberrant expression of IL-7 has also induced T cell-related leukemia in mice [Fisher et al., Leukemia., 1993;2:S66-68.]. Elevated IL-7 levels lead to preferential proliferation of CD4+ and CD8+ T cells, resulting in an increased risk of allogeneic graft rejection [Schreiber et al., Front Immunol., 2019;10:742.], the development of drug-resistant gliomas [Cui et al., Cancer BiolTher., 2012;13(7):496-503.], increased T cell responsiveness to myelin basic protein (MBP) [Traggiai et al., J Neuroimmunol., 2001;121(1-2):111-9.], and T... H 1-driven MS-based amplification [Lee et al., Sci Transl Med., 2011; 3(93): 93ra68.], as demonstrated in humans and mice.

[0085] The function of IL-9 is relatively unclear compared to other γc cytokines. IL-9 is believed to be involved in T... H IL-9 plays a role in T cell differentiation, antitumor activity, mucus production, and mast cell homeostasis. Although IL-9 is not essential for T cell development and immunoglobulin production, its deficiency leads to reduced T cell activity during early lung infections. HEnhanced 17 response [Li et al., Front Immunol., 2018;9:1118.], impaired goblet cell proliferation and mastocytosis during pulmonary allergen challenge [Townsend et al., Immunity., 2000;13(4):573-83.], and higher frequencies of Treg, activated CD4+ and CD8+ T cells [Vieyra-Garcia et al., Clin Cancer Res., 2016;22(13):3328-39.]. Increased IL-9 expression can promote ulcerative colitis (UC) by impairing intestinal barrier function and inducing mucosal mononuclear cells to produce pro-inflammatory cytokines [Bird, Nat Rev Immunol., 2014;14(7):432.] and directly induce immunopathology in the lungs [Temann et al., J Clin Invest., 2002 Jan;109(1):29-39.], as demonstrated in IL-9 overexpressing mice.

[0086] IL-15 is primarily involved in the homeostasis of CD8+ cytotoxic T cells and NK cells. The connection between transmembrane IL-15 and IL-15R on monocytes / DCs triggers the development, proliferation, and activation of CD8+ cytotoxic T cells and NK cells, as well as inhibits T cell homeostasis. H17. Differentiation. It is also suspected that IL-15-mediated autocrine mechanisms may be involved in the leukemic transformation of CD4+ T cells. [Azimi et al., Proc Natl Acad Sci US A., 1998; 95:2452-7;Azimi et al., J. Immunol.,1999;163:4064-72;Azimi et al., AIDS Res. Hum. Retroviruses, 2000;16:1717-22;Azimi et al., Proc Natl Acad Sci US A., 2001; 98:14559-64]. IL-15 deficiency directly attenuates the function of NK and CD8+ cytotoxic T cells [Suwanai et al., Proc Natl Acad Sci US A., 2010;107(20):9305-10; Kennedy et al., J Exp Med., 2000;191(5):771-80.], resulting in a 10-fold increase in sensitivity to breast cancer metastasis in mice [Gillgrass et al., J Immunol., 2014;193(12):6184-91.]. Although IL-15 overexpression can increase antigen-driven memory CD8+ T cells after microbial exposure [Yajima et al., J Immunol., 2002;168(3):1198-203.], it can also promote epithelial damage in patients diagnosed with active celiac disease [Di Sabatino et al., Gut., 2006; 55(4): 469–477.] and induce large granular lymphocytic leukemia through chromosomal instability and DNA hypermethylation [Mishra et al., Cancer Cell.,2012;22(5):645-55.].

[0087] IL-21 is considered a multifunctional regulator of immunity. IL-21 participates in enhancing the antitumor activity of CD8+ cytotoxic T cells and NK cells, regulating B cell apoptosis, inducing plasma cell differentiation and immunoglobulin production, and stimulating T cells. H 17 and T FHDifferentiation, inhibition of TH9 and Treg differentiation, and suppression of DC maturation and function. IL-21 and / or IL-21R deficiency was observed in patients with severe primary immunodeficiency exhibiting common variant immunodeficiency disease (CVID), leading to impairment of B-cell proliferation, immunoglobulin class switching, T-cell effector function, and NK cell function [Kotlarz et al., CurrOpin Pediatr., 2014;26(6):704-12.]. Overexpression of IL-21 can trigger the expansion of hematopoietic progenitor cells in the spleen [Ozaki et al., Int J Hematol., 2006;84(3):224-30.], which abnormally drives the accumulation of CD8+ memory T cells and is accompanied by a decrease in the number of naïve T cells [Allard et al., Eur J Immunol.,2007;37(11):3069-77.], triggering alloimmunity due to decreased Treg function [Petrelli et al., Diabetes., 2011; 60(12): 3223–3234.], and stimulate the expansion and differentiation of autoreactive B cells in human SLE [Wang et al., Nat Commun., 2018;9(1):1758.].

[0088] Pathology associated with gene mutations and polymorphisms in γc, γc cytokines, and γc cytokine receptor subunits

[0089] Sub-effect mutations in the γc gene are considered a cause of XSCID [Lim et al., Allergy AsthmaClin Immunol., 2019;15:2.]. More than 300 unique mutations in the γc gene have been identified in patients. In typical XSCID, T cells and NK cells are completely absent, but there are almost normal or a large number of dysfunctional B cells. Infants with typical XSCID are highly susceptible to bacterial infections and usually die within the first year of life. In patients with atypical phenotypic variants (e.g., T cells...) 低 / B + NK + and T 低 / B 低 NK + / 低 / Milder symptoms have been observed in atypical forms of XSCID. γc mutations associated with atypical XSCID disrupt the γc conformation and the binding of JAK3 to the cytoplasmic domain of γc, leading to the elimination of JAK3 phosphorylation and immunodeficiency in patients.

[0090] In contrast, overexpression of γc has been observed in patients with pancreatic ductal adenocarcinoma and gastric cancer [Ayars et al., Oncotarget., 2017;8(48):83370-83383; Wang et al., J Oncol. 2021;2021:6670834.]. In vitro and in vivo studies have also shown that γc expression is directly associated with pancreatic cancer cell growth and poor prognosis in human gastric cancer.

[0091] Genetic polymorphisms of γc cytokines are highly associated with the progression of human autoimmune status, as outlined in this review [Leonard et al., Immunity., 2019; 50(4):832-850.]. IL-2 is associated with MS, T1D, and IBD. IL-4 is associated with asthma and AR. IL-7 is associated with MS. IL-9 is associated with AR. IL-15 is associated with celiac disease [Escudero-Hernández et al., Cytokine., 2017; 99:73-79.]. IL-21 is associated with SLE, T1D, and IBD.

[0092] Genetic polymorphisms of the γc cytokine receptor subunit are also associated with an increased risk of autoimmune diseases [Leonard et al., Immunity., 2019; 50(4):832-850.]. IL-2RA gene polymorphisms are associated with T1D, IBD, and MS; IL-4RA with asthma and allergic reactions; IL-7R with MS and T1D; IL-9R with AR; IL-15RA with IBD; and IL-21RA with SLE. Furthermore, gene mutations in IL-2RA are associated with inflammation and autoimmunity in patients with loss-of-function mutations; IL-7R is associated with T1D in patients with loss-of-function mutations. - B + NK + SCID is associated with IL21RA; it is associated with T and B cell dysfunction and variable NK cell dysfunction in patients with loss-of-function mutations.

[0093] Current strategies for modulating γc cytokine-mediated diseases

[0094] Because γc cytokines are highly correlated with the progression of human autoimmune diseases, therapeutic strategies have been developed to modulate and / or treat γc cytokine-mediated diseases by inhibiting the activity of the γc cytokine family. These approaches include using ligands that share similar structures with γc cytokines to compete for receptor binding sites; using antagonistic peptides that recognize and block binding sites on γc; using chemical inhibitors that target the JAK-STAT pathway, blocking downstream signaling pathways triggered by γc cytokines; using specific monoclonal antibodies against γc cytokines to neutralize the activity of targeted cytokines in vivo; and using monoclonal antibodies that target individual γc cytokine receptor subunits to selectively inhibit cytokine activity.

[0095] IL-2 mutant proteins are genetically engineered IL-2 proteins that have a greater binding affinity for IL-2R and a reduced binding affinity for γc compared to wild-type human IL-2 (hIL-2). One example is H9-RETR, which acts as a competitive inhibitor of IL-2 and IL-15. H9-RETR is engineered to have a higher binding affinity for IL-2R β but a reduced affinity for γc, and it can block IL-2 and / or IL-15-induced CD8+ cytotoxic T cell proliferation and NK cell cytotoxicity in vitro [Mitra et al., Immunity., 2015; 42(5): 826–838.]. In in vivo studies, it has alleviated graft-versus-host disease and blocked the proliferation of T cells in chronic smoldering adult T-cell leukemia. Although the IL-2 mutant protein is highly effective in animals, there is only one phase I / II clinical trial for advanced solid tumors (ID: RPCEC00000234). Furthermore, no mutant proteins have been developed targeting the remaining γc cytokines.

[0096] BNZ-1 is a PEGylated peptide with a higher binding affinity for γc but a decreased affinity for other γc receptor subunits. BNZ-1 is a selective and simultaneous inhibitor with efficacy in blocking IL-2 and IL-15-mediated leukemia progression [Wang et al., Leukemia., 2019;33(5):1243-1255.]. Its promising role in blocking IL-2 and IL-15 function was tested in a phase II clinical trial at AA (ID: NCT03532958) and subsequently entered a phase II / III clinical trial in cutaneous T-cell lymphoma. Variants, BNZ-2 and BNZ-3, were subsequently developed to target different combinations of γc cytokines.

[0097] The use of monoclonal antibodies is considered a specific and safe approach to blocking autoimmune diseases. Two monoclonal antibodies have been approved by the FDA for the treatment of autoimmune diseases. Daclizumab, which inhibits the binding of the IL-2Ra to IL-2R complex, was used to treat renal allogeneic graft rejection and MS. However, it was automatically withdrawn from the market due to the drug's "complex and evolving benefit / risk profile." Daclizumab also improves symptoms in asthma (ID: NCT03532958) and partially improves the condition in uveitis (ID: NCT00130637).

[0098] Dupilumab is a monoclonal antibody targeting IL-4Ra approved for the treatment of moderate to severe asthma and Alzheimer's disease (AD). It inhibits both IL-4 signaling via receptor type 1 (γc) and IL-4 and IL-13 signaling via receptor type 2. Dupilumab is also being tested in multiple clinical trials for the treatment of atopic dermatitis (AA), acute rheumatoid arthritis (AR), and conjunctivitis. Monoclonal antibodies targeting the remaining γc cytokine / receptor subunits have also been investigated in clinical trials. For example, OSE-127, targeting IL-7Ra, is currently being tested to modulate symptoms in acute ulcerative colitis (UC) and symptomatic spondylitis (SS). Enokizumab, an anti-IL-9 antibody, showed beneficial effects in asthma but failed to meet its endpoint (ID: NCT00968669). Ordesekimab directly blocks IL-15 and is being investigated as a treatment for celiac disease, vitiligo, atopic dermatitis (RA), and psoriasis. NNC-0114-0006 can target IL-21 and is currently being tested in clinical trials for SLE, T1D, and Crohn's disease.

[0099] While cytokine-specific antibodies and blocking peptides have shown beneficial effects in animals, multiple cytokines can work together to induce disease progression. Therefore, antibody approaches involving the neutralization of a single γc cytokine or a single γc receptor subunit may be insufficient for treating autoimmune diseases. Although IL-2 mutant proteins and blocking peptides have been developed, their efficacy is limited to blocking IL-2 and IL-15 function, with no evidence of modulation of other γc cytokine activities. Alternative therapeutic strategies may include using small molecules to inhibit important downstream proteins activated by γc cytokines, and / or antibodies targeting specific protein receptors involved in disease pathogenesis, the activity and / or abundance of which are directly regulated by γc cytokine signaling pathways.

[0100] JAK inhibitors are a new class of widely used drugs that inhibit the phosphorylation of downstream JAK1 and JAK3 after stimulation by γc cytokines. JAK inhibitors are effective in treating several autoimmune diseases by simultaneously blocking signaling pathways triggered by multiple cytokines. They can be classified into five categories: non-selective, JAK1-selective, JAK2-selective, TYK2-selective, and JAK3-selective [summarized in Spinell et al., Eur J Immunol., 2021;51(7):1615-1627]. First-generation JAK inhibitors (e.g., tofacitinib) are non-selective and approved for the treatment of autoimmune, inflammatory, and hematologic conditions. However, they are associated with a wide range of adverse events (AEs), including serious and opportunistic infections.

[0101] Second-generation JAK inhibitors selective for JAK1 (e.g., filgotinib and upadacitinib) and JAK2 (e.g., fedratinib) have been developed and approved over the past decade. These selective inhibitors have shown a narrow spectrum of action and offer improved safety profiles. However, neutropenia and anemia may occur in patients treated with fedratinib due to the role of JAK2 in hematopoiesis [Spinell et al., Eur J Immunol., 2021;51(7):1615-1627.]. Furthermore, different classes of adverse events have been observed in patients treated with filgotinib. For example, an increase in hemoglobin levels has been reported due to the anti-inflammatory efficacy of selective JAK1 inhibitors and the lack of erythropoietin blockade mediated by JAK2 inhibition. Selective inhibitors of JAK1 also failed to reduce the absolute number of lymphocytes or NK cells, likely due to their minimal effect on IL-15 signaling [as outlined in Biggioggero et al., DrugsContext., 2019; 8: 212595.].

[0102] Since γc family cytokines require co-phosphorylation of JAK3 to trigger downstream signaling pathways, a recent discovery of selective JAK3 inhibitors has been proposed. Litexitinib is a covalent inhibitor that binds to the catalytic domain of JAK3 rather than other JAK family members in which the cysteine ​​residue targeted by the compound is replaced by a serine residue. Litexitinib is believed to provide a more specific way to block γc cytokine signaling because no other receptor molecule recruits JAK3 for signal transduction [Spinell et al., Eur J Immunol., 2021;51(7):1615-1627.]. Litexitinib has shown promising efficacy in treating autoimmune diseases and a more favorable adverse event profile than other JAK inhibitors; however, long-term studies are needed to draw conclusions [Ramírez-Marínand Tosti, Drug Des Devel Ther., 2022;16:363-374.]. Literxitinib is currently being investigated in RA (identifiers: NCT04413617, NCT02969044), IBD (identifiers: NCT05636293, NCT02958865, NCT03395184), AA (identifiers: NCT05549934, NCT04006457, NCT03732807, NCT04517864) and vitiligo (identifier: NCT02974868).

[0103] JAK3-selective approaches are promising and offer insights for future drug development targeting autoimmune diseases. This invention provides antibodies and / or antigen-binding fragments targeting γc (a receptor conjugated to JAK3) as a method for treating subjects (e.g., mice or humans) with GVHD and one of the autoimmune diseases. The antibodies and / or antigen-binding fragments are believed to offer more advantages than JAK3-selective inhibitors (e.g., complete and specific attenuation of γc-related downstream effects, lower toxicity, or longer half-life).

[0104] Recently, the anti-γc antibody REGN7257 was included in a phase I / II clinical trial for aplastic anemia (ID: NCT04409080), with preclinical data suggesting disease indications, including GVHD and MS [Le Floc'h et al., Sci Transl Med., 2023;15(678):eabo0205;Le Floc'h et al., Hemasphere. 2022; 6(Suppl ): 694-695]. These data suggest the use of anti-γc strategies to treat a variety of autoimmune diseases.

[0105] γc-binding protein

[0106] This invention provides antigen-binding proteins, such as antibodies (e.g., humanized antibodies, monoclonal antibodies, and antibodies conjugated to additional therapeutic agents) and their antigen-binding fragments, which specifically bind to purified γc protein or antigen fragments thereof (e.g., the extracellular domain of γc). Antigen-binding proteins that bind to the same epitope as a reference antibody, or that compete with any antigen-binding protein described herein for binding to γc, are also part of this invention.

[0107] The present invention also provides any polypeptide and / or antigen-binding protein, including SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 1 The amino acid sequences shown in 14, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, and / or 192, or variations thereof. In some embodiments, the polypeptide is fused with one or more other polypeptides (e.g., human Fc (IgG1, IgG2, IgG3, or IgG4)). In some embodiments, the mouse FR in the anti-γc antibody (e.g., SM-05A, SM-05E) is replaced by a corresponding FR for humanization present in the human antibody, said corresponding FR for humanization comprising the amino acid sequence of the variable heavy chain shown in SEQ ID NO: 193-194 (underlined amino acid: CDR) and the amino acid sequence of the variable light chain shown in SEQ ID NO: 195-196 (underlined amino acid: CDR). In some embodiments, the mouse FR in the anti-γc antibody (e.g., SM-05A, SM-05E) is replaced by a corresponding FR present in the human antibody, while some amino acids are reverted to amino acids present in mice having the amino acid sequence of the variable heavy chain shown in SEQ ID NO: 197-198 (underlined amino acid: CDR) and the variable amino acid sequence of the light chain shown in SEQ ID NO: 199-200 (underlined amino acid: CDR). The variable heavy and light chains from different antibody clones are interchangeable to identify the best humanized antibody with sustained potency and binding affinity to the γc protein.

[0108] The present invention also provides any polynucleotide for encoding polypeptides and / or antigen-binding proteins, including SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111. DNA sequences shown in 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189 and / or 191.

[0109] As used herein, the term "antibody" and its grammatical equivalents refer to an immunoglobulin molecule that recognizes and specifically binds to a target (e.g., a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or any combination thereof) through at least one antigen-binding site, wherein the antigen-binding site is typically located within a variable region of the immunoglobulin molecule. An antibody comprises four polypeptide chains: two heavy chains (HC) and two light chains (LC) linked together by disulfide bonds (i.e., a "complete antibody molecule") (e.g., IgG1), such as: SM-05A, SM-05B, SM-05C, SM-05D, SM-05E, SM-05F, SM-05G, SM-05H, SM-05I, SM-05J, SM-05K, and SM-05L. Antibodies also include, but are not limited to, mouse antibodies, rabbit antibodies, camel antibodies, primate antibodies, chimeric antibodies, humanized antibodies, and human antibodies. In embodiments of the invention, each antibody HC comprises a heavy chain variable region ("HCVR" or "V"). H (e.g., SEQ ID NO: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162 and / or 178, or variants thereof) and heavy chain constant regions (including domain C) H 1. C H 2 and C H 3); and each antibody light chain (LC) contains a light chain variable region (“LCVR” or “V”). L (e.g., SEQ ID NO: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170 and / or 186, or variants thereof) and light chain constant region (CL V H and V L The region can be further subdivided into hypervariable regions, called complementary determinant regions (CDRs), which are interspersed with more conservative regions, called frame regions (FRs). Each V H and V L It comprises three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In some embodiments of the invention, the FRs of the antibody (or its antigen-binding fragment) are identical to the mouse germline sequence or are naturally or artificially modified. In some embodiments of the invention, the FRs of the antibody (or its antigen-binding fragment) are identical to the human germline sequence or are naturally or artificially modified.

[0110] As used herein, the term "antigen-binding moiety" or "antigen-binding fragment" for antibodies or antigen-binding proteins includes any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments (the heavy chain portion of a Fab fragment cleaved with papain); and (iv) Fv fragments (V... H or V L (v) is derived from V H and V L The scFv molecule composed of fragments, the V H and V L The fragments are linked by linker regions (e.g., G4S linkers (SEQ ID NO: 236)). Other engineered molecules (e.g., domain-specific antibodies, single-domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-transplanted antibodies, bispecific antibodies, trispecific antibodies, tetraspecific antibodies, minibody, nanobody, and small modular immunopharmaceutical (SMIP)) are also included in the expression “antigen-binding fragment” as used herein. In embodiments of the invention, the antigen-binding fragment comprises three or more CDRs (e.g., CDR-H1, CDR-H2, and CDR-H3; or CDR-L1, CDR-L2, and CDR-L3) of SM-05A, SM-05B, SM-05C, SM-05D, SM-05E, SM-05F, SM-05G, SM-05H, SM-05I, SM05J, SM-05K, and SM-05L.

[0111] When used in the context of antibodies, the term "heavy chain" refers to a polypeptide chain of approximately 50-70 kDa, wherein the amino-terminal portion comprises approximately 120 to 130 or more amino acids. H The heavy chain constant region comprises a carboxyl-terminal portion. In some embodiments, the heavy chain constant region consists of three domains, CH1, CH2, and CH3, and a short, flexible hinge region connecting the CH1 and CH2 domains is present. Based on the amino acid sequence of the heavy chain constant region, the constant region can be one of five different types referred to as alpha (a), delta (δ), epsilon (ε), gamma (γ), and mu (μ). The different heavy chains differ in size: α, δ, and γ contain approximately 450 amino acids, while μ and ε contain approximately 550 amino acids. In embodiments of the invention, γc-binding proteins (e.g., antibodies or antigen-binding fragments) include the heavy chain constant region to generate five well-known classes of antibodies, IgA (IgA and IgA2), IgD, IgE, IgG (IgG1, IgG2, IgG3, and IgG4), and IgM, respectively.

[0112] When referring to antibody use, the term "light chain" refers to a polypeptide chain of approximately 25 kDa, in which the amino-terminal portion comprises approximately 100 to approximately 110 or more amino acids. L Furthermore, the carboxyl-terminal portion includes a constant region. The light chain constant region consists of a C domain. L Composition. The light chain has an approximate length of 211 to 217 amino acids. Based on the amino acid sequence of the constant domain, there are two distinct types, referred to as kappa (κ) and lambda (λ). This invention includes antigen-binding proteins comprising the variable domains described herein (e.g., SM-05A, SM-05B, SM-05C, SM-05D, SM-05E, SM-05F, SM-05G, SM-05H, SM-05I, SM05J, SM-05K, SM-05L), wherein the variable domains are linked to constant domains of the heavy chain and / or light chain, for example, as described above.

[0113] The term "variable domain" or "variable region" refers to a portion of the light or heavy chain of an antibody, typically located at the amino terminus of the light or heavy chain, and having a length of approximately 120 to 130 amino acids in the heavy chain and approximately 100 to 110 amino acids in the light chain. It is used for the binding and specificity of each specific antibody to its specific antigen. Variable domains differ significantly in sequence between different antibodies. Sequence variability resides in the CDR, while FRs (variable regions) are highly similar across different antibodies. The CDRs of both the light and heavy chains are primarily responsible for the antibody-antigen interaction. In some embodiments, each V H and V LIt consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The amino acid positions used in this paper are numbered according to the EU index, as described in Kabat et al. (1991) Sequences of proteins of immunological interest. (US Department of Health and Human Services, Washington, DC) 5th ed.

[0114] CDR refers to one of the three hypervariable regions (H1, H2, or H3) within the non-framework region of the VH β-sheet framework of an immunoglobulin (Ig or antibody), or one of the three hypervariable regions (L1, L2, or L3) within the non-framework region of the VL β-sheet framework of an antibody. Therefore, a CDR is a variable region sequence interspersed within a frame region sequence. CDR regions are well known to those skilled in the art and have been defined by various methods / systems. These systems and / or definitions have been developed and refined over many years, including Kabat, Chothia, IMGT, AbM, and Contact. For example, Kabat defined the most hypervariable region within the antibody variable (V) domain [Kabat et al., The Journal of Biological Chemistry vol. 252,19, 1977;6609-16.; Kabat, Advances in Protein Chemistry vol. 32, 1978;1-75.]. Chothia's definition is based on the location of the structural loop region, defining the CDR region sequence as those residues that are not part of the conserved β-sheet framework and are therefore capable of accommodating different conformations [Chothia, and Lesk, Journal of Molecular Biology vol. 196, 4, 1987; 901-17]. Both terms are widely accepted in the art. Additionally, the IMGT system is based on sequence variability and location within the variable region structure. The AbM definition is a compromise between Kabat and Chothia. The Contact definition is based on the analysis of available antibody crystal structures. Software programs (e.g., abYsis) are available and known to those skilled in the art for analyzing antibody sequences and determining CDRs. The location of CDRs within the variable domains of canonical antibodies has been determined by comparing numerous structures [Al-Lazikani, et al., Journal of Molecular Biology vol. 273, 4, 1997; 927-48; Morea et al., Methods (San Diego, Calif.) vol. 20, 3, 2000; 267-79]. Because the number of residues within the hypervariable region varies among different antibodies, in the canonical variable domain numbering scheme, the additional residues related to the canonical position are usually numbered a, b, c, etc., followed by the residue number. This nomenclature is well known to those skilled in the art.

[0115] In embodiments of the present invention, the antigen-binding protein (e.g., an antibody or its antigen-binding fragment) comprises V HHeavy chain immunoglobulins, the V H This includes heavy chain CDRs (CDR-H1, CDR-H2, and CDR-H3). The CDR is defined using the Kabat (hypervariant) nomenclature according to this invention. The composition of the antigen-binding protein is shown in Table 1 below.

[0116] In embodiments of the present invention, the antigen-binding protein (e.g., an antibody or its antigen-binding fragment) comprises V L The light chain immunoglobulin, the V L This includes light chain CDRs (CDR-L1, CDR-L2, and CDR-L3). The CDR is defined using the Kabat (hypervariant) nomenclature according to this invention. The composition of the antigen-binding protein is shown in Table 2 below.

[0117] Table 1: Heavy chain CDRs in immunoglobulins of the present invention

[0118] CDR by Kabat et al. (Same as above) Nomenclature definition.

[0119] The number corresponds to the amino acid sequence shown in SEQ ID NO.

[0120] Table 2: Light chain CDRs in the immunoglobulins of the present invention

[0121] CDR by Kabat et al. (Same as above) Nomenclature definition.

[0122] The number corresponds to the amino acid sequence shown in SEQ ID NO.

[0123] In embodiments of the present invention, the antigen-binding protein (e.g., an antibody or its antigen-binding fragment) comprises V H (e.g., HC) and V L (e.g., LC) heavy and light chain immunoglobulins, the V H and V L This includes the heavy and light chain CDRs shown in Table 3 below (CDR-H1, CDR-H2 and CDR-H3; and CDR-L1, CDR-L2 and CDR-L3).

[0124] Table 3: Heavy and light chain CDRs in each immunoglobulin of the present invention

[0125] CDR by Kabat et al. (Same as above) Nomenclature definition.

[0126] The number corresponds to the amino acid sequence shown in SEQ ID NO.

[0127] This invention includes antigen-binding proteins (e.g., antibodies or antigen-binding fragments thereof) comprising peptide pairs, said peptide pairs comprising the following V H and V L amino acid sequence: (i) SEQ ID NO: 2 and SEQ ID NO: 10; (ii) SEQ ID NO: 18 and SEQ ID NO: 26; (iii) SEQ ID NO: 34 and SEQ ID NO: 42; (iv) SEQ ID NO: 50 and SEQ ID NO: 58; (v) SEQ ID NO: 66 and SEQ ID NO: 74; (vi) SEQ ID NO: 82 and SEQ ID NO: 90; (vii) SEQ ID NO: 98 and SEQ ID NO: 106; (viii) SEQ ID NO: 114 and SEQ ID NO: 122; (ix) SEQ ID NO: 130 and SEQ ID NO: 138; (x) SEQ ID NO: 146 and SEQ ID NO: 154; (xi) SEQ ID NO: 162 and SEQ ID NO: 170; (xii) SEQ ID NO: 178 and SEQ ID NO: 186; This invention includes polynucleotides comprising the following polynucleotide groups encoding CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3. The composition of the DNA sequence of those polynucleotides is shown in Table 4 below.

[0128] Table 4: DNA sequences encoding heavy and light chain CDRs in each immunoglobulin of the present invention The number corresponds to the amino acid sequence shown in SEQ ID NO. This invention includes monoclonal anti-γc antigen-binding proteins (e.g., antibodies and their antigen-binding fragments), and monoclonal compositions comprising a plurality of isolated monoclonal antigen-binding proteins. As used herein, the term "monoclonal antibody" or "mAb" refers to a member of a substantially homogeneous group of antibodies produced by a monoclonal organism, i.e., the antibody molecules constituting the group are identical in amino acid sequence except for possible naturally occurring mutations present in small amounts. "A plurality of" such monoclonal antibodies and fragments in a composition refers to the concentration of identical (i.e., as discussed above, in amino acid sequence except for possible naturally occurring mutations present in small amounts) antibodies and fragments at concentrations higher than those typically found in nature, for example, in the blood of a host organism (e.g., a mouse or a human).

[0129] In some embodiments, the anti-γc antigen-binding protein provided herein is a chimeric antibody (e.g., containing a mouse variable region (V... H and V L ) and human constant region (C H and C L In some embodiments, the anti-γc antigen-binding protein provided herein is a humanized antibody (e.g., containing mouse CDRs (CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3), human FRs (FR-H1, FR-H2, FR-H3, FR-H4, FR-L1, FR-L2, FR-L3, and FR-L4) and human constant regions (C...). H and C L(Antibodies of the same type). In some embodiments, the amino acids in the humanized FR are reverted to amino acids from the original mouse FR. In some embodiments, the amino acids within the CDR are randomly mutated (e.g., arginine to glutamic acid) to increase binding affinity. As used herein, a “chimeric antibody” is an antibody having a variable domain from a first antibody and a constant domain from a second antibody, wherein the first and second antibodies are from different species (see, for example, U.S. Patent No. 4,816,567). As used herein, a “humanized antibody” is an antibody having exogenous amino acid segments (e.g., from a mouse-derived CDR) sandwiched between variable domain segments of human-derived amino acid residues, and humanized variable heavy and light chain domains linked to human-derived heavy and light chain constant regions as shown in SEQ ID NO: 201-202 or SEQ ID NO: 203-204 [Meyler's Side Effects of Drugs (Sixteenth Edition), 2016]. Methods for generating humanized antibodies are known in the art. This invention uses a framework repair method to generate humanized forms of anti-γc antigen-binding proteins (see, for example, U.S. Patent No. 7,321,026B2). The resulting full-length humanized antibodies refer to hSM-05A and hSM-05E.

[0130] In some embodiments, the anti-γc antigen-binding protein provided herein is a mouse scFv fragment. As used herein, "scFv fragment" is the V of immunoglobulin. H and V L The variable region of the fusion protein is linked by a short linker peptide of 10 to approximately 25 amino acids (e.g., the G4S linker (SEQ ID NO: 236)). ScFv provides a convenient method for pre-screening potential candidates with high binding affinity for γc protein and blocking activity against STAT phosphorylation.

[0131] The term “epitope” (also referred to as “antigenic determinant”) is used interchangeably herein to refer to a site on the surface of a target molecule to which an antibody or antigen-binding fragment binds, such as a localized region on the surface of an antigen. Target molecules may include proteins, peptides, nucleic acids, carbohydrates, or lipids. An epitope with immunogenic activity is a portion of a target molecule that is recognized by the immune system (e.g., antibodies, B cells, or T cells) to elicit an immune response in an animal. An epitope of an antigenically active target molecule is the portion of the target molecule to which an antibody binds, as determined by any method well known in the art (e.g., immunoassay). An antigenic epitope need not be immunogenic. Epitopes typically consist of chemically active surface groups of molecules, such as amino acids or sugar side chains, and have specific three-dimensional structural features and specific charge features. The term “epitope” includes linear epitopes and conformational epitopes. The region of a target molecule (e.g., a polypeptide) constituting an epitope may be a continuous amino acid of the polypeptide, or the epitope may be bound from two or more discontinuous regions of the target molecule. Epitopes formed from consecutive amino acids (also known as linear epitopes) are typically retained during protein denaturation, while epitopes formed from ternary folds (also known as conformational epitopes) are typically lost during protein denaturation. Epitopes usually contain at least 3 amino acids in a unique spatial conformation, and more often at least 5, 6, 7, or 8-10 amino acids.

[0132] Methods for identifying epitopes of antigen-binding proteins (e.g., antibodies, fragments, or peptides) include alanine scanning mutation analysis, peptide blotting [Reineke, Methods Mol. Biol., 2004;248:443-63], peptide cleavage analysis, crystallography, and NMR analysis. Additionally, methods such as epitope excision, epitope extraction, and chemical modification of antigens can be used [Tomer, Prot. Sci., 2000;9:487-496]. Other methods include hydrogen / deuterium exchange detected by mass spectrometry [Ehring, Analytical Biochemistry, 1999;267: 52-25; Engen and Smith, Anal.Chem., 2001;73: 256A-265A], shotgun mutagenesis [Davidson and Doranz, Immunology., 2014;143(1):13-20], and conformational epitopes based on mass spectrometry and ultraviolet photolysis [Mehaffey et al., Anal Chem.,2020;92(17):11869-11878.].

[0133] As used herein, the term "specific binding" refers to a combination of interactions between a polypeptide or molecule and an epitope, protein, or target molecule that are more frequent, faster, more persistent, have greater affinity, or have the aforementioned effects than interactions with other substances, including related and unrelated proteins. The binding moiety (e.g., antibody) that specifically binds to a target molecule (e.g., an antigen) can be identified by, for example, immunoassays, ELISA, biolayer interferometry (“BLI”), SPR (e.g., Biacore), or other techniques known to those skilled in the art. Typically, the specific response will be at least twice the background signal or noise, and may be up to 10 times higher than the background. See, for example, Paul, ed., 1989, Fundamental Immunology Second Edition, Raven Press, New York at pages 332-336, for a discussion of antibody specificity. In some embodiments, the binding portion of a specific target molecule can bind to the target molecule with an affinity at least 20, 30, 40, 50, 60, 70, 80, 90, or 100 times greater than its affinity for different molecules. In some embodiments, the binding portion of a specific target molecule binds to different molecules with such low affinity that the binding cannot be detected using the assays described herein or other assays known in the art. In some embodiments, "specific binding" refers, for example, to a binding portion with a K+ of about 0.1 mM or less. D Binding to molecular targets. In some implementations, "specific binding" refers to the peptide or molecule binding at a concentration of approximately 10 μM or less, or approximately 1 μM or less. D Binding to the target. In some implementations, "specific binding" refers to the peptide or molecule binding at a concentration of about 0.1 μM or less, about 0.01 μM or less, or about 1 nM or less. D Target binding. Due to sequence identity between homologous proteins in different species, specific binding can include peptides or molecules that recognize proteins or targets in more than one species. Similarly, due to homology in certain regions of the polypeptide sequences of different proteins, specific binding can include peptides or molecules that recognize more than one protein or target. It should be understood that in some embodiments, the binding portion (e.g., an antibody) that specifically binds to a first target may or may not specifically bind to a second target. Therefore, "specific binding" does not necessarily require (although it may include) exclusive binding, i.e., binding to a single target. Thus, in some embodiments, the binding portion (e.g., an antibody) may specifically bind to more than one target.

[0134] As used herein, the term "binding affinity" generally refers to the strength of the sum of non-covalent interactions between the binding moiety (e.g., antibody) and the target molecule (e.g., antigen). Binding between the binding moiety and the target molecule is a reversible process, and the affinity of binding is typically reported as the equilibrium dissociation constant (K0). D K D It is the dissociation rate (k off or k d ) and association rate (k on or k a The ratio of ) to K. D The lower the value, the higher the affinity. K a It is the equilibrium association constant, and it is also the reciprocal of the equilibrium dissociation constant, i.e., = 1 / K D Regarding antibody-antigen interactions, K D It can be calculated as the ratio of the product of the concentrations of free antibody and free antigen to the concentration of the antibody-antigen complex (i.e., [antigen] x [antibody] / [antigen-antibody]).

[0135] Various methods for measuring binding affinity are known in the art, and any of them can be used for the purposes of this disclosure. Specific illustrative embodiments include the following. In some embodiments, “K D "or "K D The value can be measured by methods known in the art, such as by combination determination. K D It can be measured in radiolabeled antigen binding assays (RIA) (Chen, Y et al. Journal of molecular biology vol. 293,4 (1999): 865-81). K D or K D The value can also be measured using biological layer interferometry (BLI) with systems such as the Gator system (Probe Life) or the Octet-96 system (Sartorius, Gottingen, Germany). D or K D The value can also be measured using surface plasmon resonance determination with the help of BIAcore systems (e.g., Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ).

[0136] As used herein in connection with a protein or polypeptide having specific sequence characteristics (“reference protein” or “reference polypeptide”), the term “variant” refers to a different protein or polypeptide compared to the reference protein or reference polypeptide by having one or more amino acid substitutions, deletions, and / or additions. Changes in amino acid sequence can be amino acid substitutions. Changes in amino acid sequence can be conserved amino acid substitutions. Functional fragments or functional variants of the protein or polypeptide maintain the basic structural and functional properties of the reference protein or polypeptide.

[0137] The terms “polypeptide,” “peptide,” “protein,” and their grammatical equivalents, used interchangeably herein, refer to polymers of amino acids of any length, which may be linear or branched. It may include non-natural amino acids, modified amino acids, or amino acid-interrupted molecules. Polypeptides, peptides, or proteins may also be modified by, for example, disulfide bond formation, glycosylation, esterification, acetylation, phosphorylation, or any other manipulation or modification.

[0138] The terms “polynucleotide,” “nucleic acid,” and their grammatical equivalents, used interchangeably in this document, refer to polymers of nucleotides of any length, including DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into the polymer by DNA or RNA polymerases. Nucleic acid molecules can be single-stranded or double-stranded.

[0139] As used herein, the term “encoding” and its syntactic equivalents refer to the inherent characteristics of a specific nucleotide sequence in a polynucleotide or nucleic acid (e.g., a gene, cDNA, or mRNA) that serves as a template for the synthesis of other polymers and macromolecules in biological processes, which have defined nucleotide sequences (i.e., rRNA, tRNA, and mRNA) or defined amino acid sequences and the resulting biological characteristics. Thus, if the transcription and translation of the mRNA corresponding to a gene produces a protein, then the gene encodes a protein. Unless otherwise stated, “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate to each other and encode the same amino acid sequence. Nucleotide sequences encoding proteins and RNA may include introns.

[0140] "Isolated" polypeptides, peptides, proteins, antibodies, polynucleotides, carriers, cells, or compositions are polypeptides, peptides, proteins, antibodies, polynucleotides, carriers, cells, or compositions in forms not found in nature. Isolated polypeptides, peptides, proteins, antibodies, polynucleotides, carriers, cells, or compositions include those that have been purified to the point that they are no longer in forms found in nature. In some embodiments, the isolated polypeptides, peptides, proteins, antibodies, polynucleotides, carriers, cells, or compositions are substantially pure. In some embodiments, the isolated polypeptides, peptides, proteins, antibodies, polynucleotides, carriers, cells, or compositions are substantially free of other cellular material and / or chemicals.

[0141] The terms “identical,” “identity,” “percentage of identity,” and their grammatical equivalents, used in the context of two or more polynucleotides or peptides herein, refer to two or more sequences or subsequences that are identical or have a specific percentage of nucleotide or amino acid residues that are identical when compared and aligned (with gaps introduced as needed) to obtain maximum correspondence, without considering any conserved amino acid substitutions as part of sequence identity. The percentage of identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software that can be used to obtain amino acid or nucleotide sequence alignments are well known in the art. These include, but are not limited to, BLAST, ALIGN, Megalign, BestFit, GCG Wisconsin Package, and variants thereof. In some embodiments, the two polynucleotides or peptides provided herein are substantially identical, meaning that they have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and in some embodiments at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% nucleotide or amino acid residue identity when compared and aligned using sequence comparison algorithms or by visual inspection to obtain maximum correspondence. In some embodiments, regions of at least about 10 residues, at least about 20 residues, at least about 40-60 residues, at least about 60-80 residues, or any integer value therebetween exist in the amino acid sequence. In some embodiments, regions longer than 60-80 residues (e.g., at least about 80-100 residues) exhibit identity, and in some embodiments, the sequences are substantially identical across the full length of the compared sequences (e.g., the coding region of a target protein or antibody). In some embodiments, regions of at least about 10 bases, at least about 20 bases, at least about 40-60 bases, at least about 60-80 bases, or any integer value therebetween exist in the nucleotide sequence. In some embodiments, regions longer than 60-80 bases (e.g., at least about 80-1000 bases or more) exhibit identity, and in some embodiments, the sequences are substantially identical across the full length of the compared sequences, such as the nucleotide sequence encoding a target protein.

[0142] As used herein, "conserved amino acid substitution" refers to an amino acid in which one amino acid residue is replaced by another amino acid residue having a similar side chain. As used herein, "conservatively similar" amino acids or residues refer to different amino acid residues having similar side chains. Families of amino acid residues with similar side chains are defined in the art and include basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0143] This invention includes antigen-binding proteins that compete with the antigen-binding proteins of this invention (e.g., SM-05A, SM-05B, SM-05C, SM-05D, SM-05E, SM-05F, SM-05G, SM-05H, SM-05I, SM05J, SM-05K, SM-05L) for binding to γc (e.g., the variant γc epitopes discussed herein). The term “competitive” and its grammatical equivalents as used herein refer to an antigen-binding protein (e.g., an antibody or its antigen-binding fragment) that binds to an antigen (e.g., γc) and inhibits or blocks the binding of another antigen-binding protein (e.g., an antibody or its antigen-binding fragment) to the antigen. Unless otherwise stated, the term also includes competition between two antigen-binding proteins (e.g., antibodies) in two directions, i.e., a first antibody binding to an antigen and blocking the binding of a second antibody, and vice versa. Thus, in embodiments of this invention, competition occurs in one such direction. In some embodiments, the first and second antigen-binding proteins (e.g., antibodies) may bind to the same epitope. Alternatively, the first and second antigen-binding proteins (e.g., antibodies) may bind to different, but e.g., overlapping or non-overlapping, epitopes, whereby the binding of one antigen-binding protein inhibits or blocks the binding of the second antibody, for example, by steric hindrance or conformational change. Competition between antigen-binding proteins (e.g., antibodies) can be determined by methods known in the art, e.g., by a competitive standard enzyme-linked immunosorbent assay (ELISA). Furthermore, competition can be measured by real-time, label-free biolayer interferometry using the Octet RED384 biosensor.

[0144] Typically, the antibody or antigen-binding fragments of the present invention, modified in some way, maintain a specific binding affinity for γc, for example, retaining at least 10% of their γc-binding activity (compared to the parent antibody) when the activity is expressed on a molar basis. Preferably, the antibody or antigen-binding fragments of the present invention retain at least 20%, 50%, 70%, 80%, 90%, 95%, or 100% or more of γc-binding affinity as a parent antibody. The antibody or antigen-binding fragments of the present invention are also intended to include conserved or non-conserved amino acid substitutions (referred to as “conserved variants” or “functionally conserved variants” of the antibody) that do not substantially alter their normal biological function (e.g., blocking γc cytokine-induced signaling pathways).

[0145] "Variants" of polypeptides (e.g., immunoglobulin chains such as SM-05A, SM-05B, SM-05C, SM-05D, SM-05E, SM-05F, SM-05G, SM-05H, SM-05I, SM05J, SM-05K, SM-05L; which contain the amino acid sequences specifically listed herein) H V L , HC or LC or CDR) refers to the sequence containing the amino acid sequence mentioned herein (e.g., SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114). At least 70% to 99.9% of the following values: 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, and / or 192. (For example, peptides with at least 70%, 72%, 74%, 75%, 76%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%) identical or similar amino acid sequences when compared using the BLAST algorithm.

[0146] In addition, variants of the peptide may include peptides such as immunoglobulin chains (e.g., SM-05A, SM-05B, SM-05C, SM-05D, SM-05E, SM-05F, SM-05G, SM-05H, SM-05I, SM05J, SM-05K, SM-05L; their V... H V L The γc-binding protein may include an amino acid sequence of a reference polypeptide, specifically listed herein but used for one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) regulatory mechanisms, such as one or more missense mutations (e.g., conserved substitutions), nonsense mutations, deletions, or insertions. In embodiments of the invention, the γc-binding protein includes immunoglobulin heavy chain variants comprising CDR-H1, CDR-H2, and CDR-H3 (where one or more (e.g., 1, 2, or 3) of such CDRs have one or more such regulatory mechanisms (e.g., conserved substitutions)) and / or immunoglobulin light chain variants comprising CDR-L1, CDR-L2, and CDR-L3 (where one or more (e.g., 1, 2, or 3) of such CDRs have one or more such mutations (e.g., conserved substitutions)).

[0147] The generation of phage display libraries and the generation of human γc-binding protein.

[0148] Phage display libraries are a more advanced technique than traditional hybridoma technology for generating and screening antigen-specific monoclonal antibodies. After inserting a foreign polynucleotide (e.g., an anti-γc ScFv sequence) into a vector encoding a filamentous phage coating protein gene (e.g., pCANTAB5), a fragment of the target protein is "displayed" on the surface of a filamentous phage (e.g., M13 phage). The resulting engineered phage can then be used to select γc-binding protein fragments with high affinity for γc proteins.

[0149] As used herein, the term "vector" and its grammatical equivalents refer to a medium for carrying genetic material (e.g., a polynucleotide sequence) that can be introduced into a host cell and replicated and / or expressed therein. Suitable vectors include, for example, expression vectors, plasmids, phage particles, viral vectors, episomes, and artificial chromosomes, which may include selective sequences or markers that can be used for stable integration into the host cell chromosome. Additionally, a vector may include one or more selective marker genes and appropriate expression control sequences. Selective marker genes may be included, for example, providing resistance to antibiotics or toxins, supplementing nutritional deficiencies, or providing key nutrients not present in the culture medium. Expression control sequences may include constitutive and inducible promoters, transcription enhancers, transcription terminators, etc., well-known in the art. When two or more polynucleotides are co-expressed, the two polynucleotides may be inserted into, for example, a single expression vector or a separate expression vector. For single-vector expression, the encoding polynucleotide may be operatively linked to a common expression control sequence or to different expression control sequences, such as an inducible promoter and a constitutive promoter. Methods well-known in the art can be used to confirm the introduction of the polynucleotide into the host cell. Those skilled in the art will understand that polynucleotides are expressed in sufficient quantities to produce the desired product (e.g., IgG composed of γc-binding proteins), and will also understand that expression levels can be optimized to achieve sufficient expression using methods well known in the art.

[0150] As used herein, the term "host cell" refers to a cell into which genetic material (e.g., a recombinant expression vector) can be introduced or has been introduced. Host cells include not only the test cells into which exogenous genetic material has been introduced, but also the progeny of such cells. Due to mutations or environmental influences, certain modifications may occur in subsequent generations, and such progeny may differ from the parent cells.

[0151] Eukaryotic and prokaryotic host cells (including mammalian cells) can be used as hosts for expressing γc-binding proteins (e.g., antibodies or their antigen-binding fragments). Such host cells are well known in the art, and many are available from the American Type Culture Collection (ATCC). These host cells include, in particular, Chinese hamster ovary (CHO) cells, mouse myeloma cells (NS0), SP2 cells, HeLa cells, young hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells, HEK-293 cells, and many other cell lines from various mammalian species. Other cell lines that can be used are insect cell lines (e.g., fall armyworm (Spodoptera frugiperda) or white armyworm (Trichoplusiani)), amphibian cells, bacterial cells (e.g., TOP10 Escherichia coli, TG1 Escherichia coli, HB2151 Escherichia coli), plant cells, and fungal cells.Fungal cells include yeast and filamentous fungal cells, including, for example, *Pichia*, *Pichia pastoris*, *Pichia finlandica*, *Pichia trehalophila*, *Pichia koclamae*, *Pichia membranaefaciens*, *Pichia minuta* (Ogataea minuta, *Pichia lindnen*), *Pichia opuntiae*, *Pichia athermotolerans*, *Pichia salictaria*, *Pichia guercuum*, *Pichia pijperi*, *Pichia stiptis*, *Pichia methanolica*, *Pichia sp.*, *Saccharomyces cerevisiae*, and *Saccharomyces*. *Hansenula polymorpha*, *Kluyveromyces* sp., *Kluyveromyces lactis*, *Candida albicans*, *Aspergillus nidulans*, *Aspergillus niger*, *Aspergillus oryzae*, *Trichoderma reesei*, *Chrysosporium lucknowense*, *Fusarium* sp., *Fusarium gramineum*, *Fusarium venenatum*, *Physcomi trella patens*, and *Neurospora crassa*. This invention includes isolated host cells (e.g., Expi-CHO cells or any of the above-mentioned types of host cells) containing antigen-binding proteins, their V... H V L, HC, LC or CDR (or their variants), such as SM-05A, SM-05B, SM-05C, SM-05D, SM-05E, SM-05F, SM-05G, SM-05H, SM-05I, SM05J, SM-05K, SM-05L; and / or polynucleotides encoding one or more of their immunoglobulin chains (e.g., as described above).

[0152] Human γc protein with the mouse Fc tag shown in SEQ ID NO: 201 was produced using a host cell system (e.g., Expi-CHO cells) and purified using ProSep Ultra Plus. To remove contaminants and maintain physiological pH, the purified γc protein was buffer-exchanged into PBS solution using an Amicon ultra-1510k centrifuge filter. The resulting protein was diluted to 1 mg / ml in PBS for storage.

[0153] Mice were immunized with purified human γc protein, as shown in Table 5. ELISA titrations with experimental bleeding were performed between immunization steps to assess successful antibody production in the animals.

[0154] Table 5: Immunotherapy methods for human γc protein

[0155] Total RNA was isolated from mouse spleens at the end of immunization. All V RNAs were identified using degenerate primers. H and V L Fragments and clones. All V H and V L Fragments and linker regions (e.g., the G4S linker (SEQ ID NO: 236)) are randomly combined to generate scFv libraries. To prepare the scFv gene, the region encoding V... H and V L The DNA fragment can be operatively linked to another fragment encoding a flexible linker, such as the amino acid sequence (Gly4-Ser)3 (SEQ ID NO: 237), such that V H and V L The sequence can be expressed as a continuous single-chain protein, where V L and V HThe regions are connected by flexible connectors [Bird, RE et al. Science (New York, NY) vol. 242,4877 (1988): 423-6; Huston, JS et al. Proceedings of the National Academy of Sciences of the United States of America vol. 85,16 (1988): 5879-83; McCafferty, J et al. Nature vol. 348,6301 (1990): 552-4].

[0156] After ligating the ScFv library to a phage vector (e.g., pCANTAB5), the vector was transfected into bacterial host cells (e.g., TOP10 competent *E. coli*) to amplify a phage library containing all ScFv fragment combinations. To select phages exhibiting strong binding affinity for human γc protein, phage libraries were screened against decreasing concentrations of human γc protein in 96-well plates. After two to three rounds of selection, each phage was cultured as a single clone, and the binding affinity of each clone was assessed by monoclonal phage ELISA, as shown in Table 6. Clones with an OD value 5-fold higher than the background (i.e., 0.3) were selected for DNA sequencing. The final DNA and amino acid sequences are shown in SEQ ID NO: 1-192.

[0157] Table 6: ELISA results of human γc protein monoclonal phage

[0158] Soluble scFv was generated using *E. coli* strain HB2151 after phage transfection into the appropriate host. Periplasmic extracts containing soluble scFv were harvested by sonication and purified using Capto™ L. After measuring the concentration of the crude scFv extract, the binding affinity of scFv to human γc protein was assessed by ELISA using an antibody targeting the E-Tag on scFv. Results are summarized in... Figure 1 In AB.

[0159] To screen the potency of anti-γcScFv, several cell lines were pre-incubated with different anti-γcScFv (20 μg / ml) for 1 hour, followed by stimulation with individual cytokines (50 ng / ml) for 15 minutes. Cells were harvested and stained with FITC-conjugated anti-pSTAT6, PE-conjugated anti-pSTAT3, or APC-conjugated anti-pSTAT5 antibodies for flow cytometry analysis. In some embodiments, purified ScFv can block IL-4-induced STAT6 phosphorylation in cell cultures (e.g., Ramos). In some embodiments, purified ScFv can block IL-7-induced STAT5 phosphorylation in cell cultures (e.g., HPB-ALL). In some embodiments, purified ScFv can block IL-9-induced STAT3 phosphorylation in cell cultures (e.g., Jurkat). In some embodiments, purified ScFv can block IL-15-induced STAT5 phosphorylation in cell cultures (e.g., KHYG-1). In some implementations, purified ScFv can block IL-21-induced STAT3 phosphorylation in cell cultures (e.g., Ramos). The results are summarized in Tables 7–10. Figure 1 C shows a representative flow cytometry plot of p-STAT5 blockade against γcScFv in HPB-ALL.

[0160] Table 7: Inhibition of IL-15-induced STAT phosphorylation

[0161] Table 8a: Inhibition of IL-4-induced STAT phosphorylation

[0162] Table 8b: Inhibition of IL-21-induced STAT phosphorylation

[0163] Table 9: Inhibition of IL-7-induced STAT phosphorylation

[0164] Table 10: Inhibition of IL-9-induced STAT phosphorylation

[0165] Antibody production methods are well known in the art. See, for example, Harlow et al., Antibodies: ALaboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling et al. , in: Monoclonal Antibodies and T-Cell Hybridomas 563, 681 (Elsevier, NY, 1981), each of which is incorporated herein by reference in its entirety.

[0166] In some embodiments, the γc-binding protein that can be used in the methods provided herein is recombinant, i.e., prepared, expressed, generated, or isolated by recombinant means. In some embodiments, the γc-binding protein disclosed herein can be prepared, for example, by introducing a recombinant expression vector into a host cell, a recombinant combined human antibody library, antibodies isolated from an animal (e.g., a mouse) that is transgenic for the human immunoglobulin gene [Taylor, LD et al. Nucleicacids research vol. 20,23 (1992): 6287-95], or by any other means involving splicing a human immunoglobulin gene sequence into another DNA sequence, to prepare, express, generate, or isolate antibodies.

[0167] In some embodiments, γc-binding proteins can be prepared by recombinant expression of immunoglobulin light and heavy chain genes in host cells (e.g., Expi-CHO cells). To express the γc-binding protein, one or more recombinant expression vectors carrying DNA fragments encoding the immunoglobulin light and heavy chains of the γc-binding protein are introduced into host cells, causing the light and heavy chains to be expressed in the host cells and preferably secreted into the culture medium from which the γc-binding protein can be recovered. The γc-binding protein heavy and light chain genes are obtained using standard recombinant DNA methods, these genes are incorporated into a recombinant expression vector, and the vector is introduced into host cells, for example, Sambrook, Fritsch and Maniais (eds), MOLECULAR CLONING: A LABORATORY MANUAL, Second Edition, Cold SpringHarbor, NY, (1989), Ausubel. et al. Those described in (eds.) Current Protocoles in Molecular Biology, Greene Publishing Associates, (1989) and U.S. Patent No. 4,816,397.

[0168] To express the recombinant γc-binding protein, DNA fragments encoding the light and heavy chain variable regions are first obtained. These DNA fragments can be obtained by amplifying and modifying hybridomas of mouse antibody light and heavy chain variable sequences using polymerase chain reaction (PCR), or by oligomerization based on the designed amino acid sequences encoding the light and heavy chain variable sequences using standard methods known to those skilled in the art. The encoding DNA sequences can be further optimized to facilitate mammalian expression of the resulting antibody.

[0169] The VH and VL fragments of mouse antibodies can be further mutated using a framework repair approach to encode humanized antibodies (see, for example, U.S. Patent No. 7,321,026B2).

[0170] Once the DNA fragments encoding the γc-binding proteins VH and VL are obtained (e.g., by amplification and mutagenesis of the original mouse VH and VL genes, as described above), these DNA fragments can be further manipulated using standard recombinant DNA techniques, such as converting variable region genes into full-length antibody chain genes or Fab fragment genes. In these operations, the DNA fragment encoding VL or VH is operatively linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker. The term "operatively linked" as used herein refers to the linking of two DNA fragments such that the amino acid sequences encoded by the two DNA fragments remain within the same reading frame.

[0171] Isolated DNA encoding the VH region can be converted into a full-length heavy chain gene by operatively linking the VH-encoding DNA to another DNA molecule encoding the heavy chain constant regions (CH1, CH2, and CH3). The sequences of human heavy chain constant region genes are known in the art (see, for example, Kabat, EA, et al (1991) SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242) and DNA fragments containing these regions can be obtained by standard PCR amplification. The heavy chain constant regions can be IgG1, IgG2, IgG3, Ig4, IgA, IgE, IgM, or IgD constant regions, but are most preferably IgG1 or IgG4 constant regions. For Fab fragment heavy chain genes, the VH-encoding DNA can be efficiently linked to another DNA molecule encoding only the heavy chain CH1 constant region.

[0172] Isolated DNA encoding the VL region can be converted into a full-length light chain gene (and a Fab light chain gene) by operatively linking the VL-encoding DNA to another DNA molecule encoding the light chain constant region CL. The sequences of human light chain constant region genes are known in the art (see, Kabat, EA, et al (1991) SEQUENCES OF PROTEINS OFIMMUNOLOGICAL INTEREST, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242) and DNA fragments containing these regions can be obtained by standard PCR amplification. The light chain constant region can be either a κ or λ constant region, but a κ constant region is preferred.

[0173] To express the γc-binding protein that can be used in the methods disclosed herein, DNA encoding the partial or full-length light and heavy chains, obtained as described above, is inserted into an expression vector (e.g., pEGFP-N1) such that the gene is operatively linked to transcriptional and translational control sequences. Herein, the term "operatively linked" means linking the antibody gene to a vector such that the transcriptional and translational control sequences within the vector perform their intended function of regulating the transcription and translation of the antibody gene. The expression vector and expression control sequences are chosen to be compatible with the expression host cells used (e.g., Expi-CHO cells). The antibody light chain gene and heavy chain gene can be inserted into separate vectors (e.g., pEGFP-N1), or more typically, both genes can be inserted into the same expression vector (e.g., pKS1). The antibody gene is inserted into the expression vector using standard methods (e.g., linking the antibody gene fragment to a complementary restriction site on the vector, or, if no restriction site is present, linking the blunt end). In some implementations, the expression vector already carries sequences encoding the heavy chain constant region or the light chain constant region, respectively, before inserting the protein light chain or heavy chain sequence, such that the VH fragment is operatively linked to the CH fragment within the vector, and the VL fragment is operatively linked to the CL fragment within the vector. Additionally or alternatively, the recombinant expression vector may encode a signal peptide that promotes the secretion of the antibody chain from the host cell. The antibody chain gene can be cloned into the vector such that the signal peptide is linked to the N-terminus of the antibody chain gene within the same reading frame. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide derived from a non-immunoglobulin protein).

[0174] After transfecting a vector containing the full-length antibody sequence into host cells (e.g., Expi-CHO cells), the antibody is actively secreted into the culture medium and MabSelect is used. TMPrismA purification. The antibody was further buffered in PBS as a storage buffer using an Amicon ultra-1510k centrifugation filter for greater stability and longer storage time. If desired, the mouse constant region was replaced with the desired human constant region (e.g., wild-type or modified IgG1 or IgG4) to produce a fully human anti-γc antibody. Although the selected constant region can vary depending on the specific application, high-affinity antigen binding and target-specific characteristics are present in the variable region. In some instances, the fully human anti-γc antibody was isolated directly from antigen-positive B cells from the subject (e.g., mouse or human).

[0175] Pharmaceutical Composition

[0176] This document also provides pharmaceutical compositions comprising a γc-binding protein that can be used in the methods disclosed herein. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of the γc-binding protein disclosed herein and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition can be used to treat immune cell lymphomas and autoimmune diseases. In some embodiments, the pharmaceutical composition can be used to treat GVHD. In some embodiments, the pharmaceutical composition can be used to inhibit the progression of GVHD in a subject (e.g., a human or a mouse).

[0177] The amount of therapeutic γc-binding protein that can be combined with the carrier material in the pharmaceutical compositions disclosed herein can vary. In some embodiments, the amount of γc-binding protein present in the pharmaceutical composition is the amount that produces a therapeutic effect. Typically, when the total amount is 100%, this amount will be about 0.01% to about 99% of the active ingredient, about 0.1% to about 70%, or about 1% to about 30% of the active ingredient in combination with a pharmaceutically acceptable carrier.

[0178] The pharmaceutical compositions provided herein contain the γc-binding protein provided herein. The γc-binding protein may be present in various concentrations. In some embodiments, the pharmaceutical compositions provided herein contain 1-1000 mg / ml of the soluble γc-binding protein provided herein. In some embodiments, the pharmaceutical compositions contain 10-500 mg / ml, 10-400 mg / ml, 10-300 mg / ml, 10-200 mg / ml, 10-100 mg / ml, 20-100 mg / ml, or 50-100 mg / ml of the soluble γc-binding protein provided herein. In some embodiments, the pharmaceutical compositions provided herein comprise about 10 mg / ml, about 20 mg / ml, about 30 mg / ml, about 40 mg / ml, about 50 mg / ml, about 60 mg / ml, about 70 mg / ml, about 80 mg / ml, about 90 mg / ml, about 100 mg / ml, about 120 mg / ml, about 150 mg / ml, about 180 mg / ml, about 200 mg / ml, about 300 mg / ml, about 500 mg / ml, about 800 mg / ml, or about 1000 mg / ml of the γc-binding protein provided herein. The dosage can be readily adjusted by those skilled in the art; for example, reducing purity requires increasing the dosage.

[0179] The pharmaceutical compositions described herein can be in various forms. These dosage forms include, for example, liquid, semi-solid, and solid dosage forms, such as liquid solutions (e.g., injectable and infusionable solutions), dispersions or suspensions, tablets, pills, powders, liposomes, and suppositories. Preferred forms depend on the intended method of administration and therapeutic application. Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions or formulations described herein include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils (e.g., olive oil), and injectable organic esters (e.g., ethyl oleate).

[0180] For example, by using a coating material (e.g., lecithin), the desired particle size can be maintained in the case of a dispersion, and by using a surfactant, appropriate flowability can be maintained. In some embodiments, the pharmaceutical compositions provided herein are in the form of injectable or infusionable solutions. In some embodiments, the pharmaceutical compositions are aqueous formulations. Such formulations are typically solutions or suspensions, but may also include colloids, dispersions, emulsions, and multiphase materials. The term "aqueous formulation" is defined as a formulation containing at least 50% w / w water. Similarly, the term "aqueous solution" is defined as a solution containing at least 50% w / w water, and the term "aqueous suspension" is defined as a suspension containing at least 50% w / w water. The compositions can be formulated as solutions, microemulsions, liposomes, or other ordered structures suitable for high drug concentrations.

[0181] In some embodiments, the pharmaceutical compositions disclosed herein are freeze-dried, and a physician or patient adds solvents and / or diluents to them prior to use.

[0182] The pharmaceutical compositions described herein may contain pharmaceutically acceptable carriers. Pharmaceutically acceptable carriers include any and all physiologically compatible solvents, dispersion media, coatings, antimicrobial and antifungal agents, isotonic agents, and absorption delay agents. Examples include one or more of water, saline, phosphate-buffered saline, dextran, glycerol, ethanol, and combinations thereof. In some embodiments, the pharmaceutically acceptable carrier includes an isotonic agent in the composition, such as a sugar, a polyol like mannitol, sorbitol, or sodium chloride.

[0183] In some embodiments, the pharmaceutically acceptable carrier also contains small amounts of excipients, such as wetting agents or emulsifiers, preservatives, or buffers, which improve the shelf life or efficacy of the antibody or antigen-binding fragment. In some embodiments, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active ingredient (i.e., γc-binding protein) may be coated in the material to protect it from acids and other natural conditions that can inactivate it.

[0184] This document also provides kits for preparing pharmaceutical compositions having the γc-binding protein disclosed herein. In some embodiments, the kit contains the γc-binding protein disclosed herein and a pharmaceutically acceptable carrier in one or more containers. In another embodiment, the kit may contain the γc-binding protein disclosed herein for administration to a subject. In a specific embodiment, the kit includes instructions for the preparation and / or administration of the γc-binding protein.

[0185] In some embodiments, the pharmaceutical compositions or formulations disclosed herein comprise: (a) the γc-binding protein disclosed herein; (b) a buffer; (c) a stabilizer; (d) a salt; (e) a filler; and / or (f) a surfactant. In some embodiments, the pharmaceutical compositions or formulations are stable for at least 1 month, at least 2 months, at least 3 months, at least 6 months, at least 1 year, at least 2 years, at least 3 years, at least 5 years, or longer. In some embodiments, the pharmaceutical compositions or formulations are stable when stored at 4°C, 25°C, or 40°C. In some embodiments, this document also provides pharmaceutical compositions or formulations that improve the stability of the γc-binding protein to allow for its long-term storage. The pharmaceutical compositions disclosed herein may further comprise one or more of preservatives, isotonic agents, chelating agents, stabilizers, and / or surfactants, as well as various combinations thereof. The use of preservatives, isotonic agents, chelating agents, stabilizers, and surfactants in pharmaceutical compositions is well known to those skilled in the art. See Remington: The Science and Practice of Phamacy, 19th edition, 1995.

[0186] Buffers that can be used in the pharmaceutical compositions or formulations disclosed herein can be weak acids or weak bases, used to maintain the acidity (pH) of the solution near a selected value after the addition of another acid or base. Suitable buffers can maximize the stability of the pharmaceutical formulation by maintaining pH control of the formulation. Suitable buffers can also ensure physiological compatibility or optimize solubility. Rheological, viscous, and other properties may also depend on the pH of the formulation. Commonly used buffers include, but are not limited to, histidine, citric acid, succinic acid, acetic acid, and phosphoric acid. In some embodiments, the buffer comprises histidine (e.g., L-histidine) with an isotonic agent, and potentially adjusts the pH with an acid or base known in the art. In some embodiments, the buffer is L-histidine. In some embodiments, the pH of the formulation is maintained at about 2 to about 10, or about 4 to about 8.

[0187] Stabilizers are added to pharmaceutical products to stabilize them. Such agents can stabilize proteins in various ways. Commonly used stabilizers include, but are not limited to, amino acids (e.g., glycine, alanine, lysine, arginine, or threonine), carbohydrates (e.g., glucose, sucrose, trehalose, raffinose, or maltose), polyols (e.g., glycerol, mannitol, sorbitol, cyclodextrin, or dextran of any type and molecular weight), or PEG. In some embodiments, stabilizers are selected to maximize the stability of the FIX peptide in the lyophilized formulation. In some embodiments, the stabilizer is sucrose and / or arginine.

[0188] Fillers can be added to pharmaceutical compositions or formulations to increase the volume and mass of the product, thereby facilitating precise measurement and handling. Commonly used fillers include, but are not limited to, lactose, sucrose, glucose, mannitol, sorbitol, calcium carbonate, or magnesium stearate.

[0189] Surfactants are amphiphilic substances having both hydrophilic and hydrophobic groups. Surfactants can be anionic, cationic, zwitterionic, or nonionic. Examples of nonionic surfactants include, but are not limited to, alkyl ethoxylates, nonylphenol ethoxylates, amine ethoxylates, polyethylene oxide, polypropylene oxide, fatty alcohols (e.g., cetyl alcohol or oleyl alcohol), cocoamide MEA, cocoamide DEA, polysorbates, or dodecyl dimethylamine oxide. In some embodiments, the surfactant is polysorbate 20 or polysorbate 80.

[0190] The pharmaceutical compositions disclosed herein may also include pharmaceutically acceptable antioxidants. Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.; (2) oil-soluble antioxidants, such as ascorbate palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, etc.; and (3) metal chelating agents, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[0191] These compositions may also contain adjuvants, such as preservatives, wetting agents, emulsifiers, and dispersants. Prevention of microbial presence can be ensured both through the aforementioned sterilization procedures and by including various antimicrobial and antifungal agents, such as parabens, chlorobutanol, and phenolic sorbic acid. It may also be desirable to include isotonic agents in the composition, such as sugars and sodium chloride. Furthermore, prolonged absorption of injectable drug forms can be achieved by including agents that delay absorption, such as aluminum monostearate and gelatin.

[0192] Pharmaceutical compositions or formulations must generally be sterile and stable under manufacturing and storage conditions. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the provisional preparation of sterile injectable solutions or dispersions. Sterile injectable solutions can be prepared by incorporating a therapeutic antibody or antigen-binding fragment in a desired amount into a suitable solvent, which, if desired, contains one or a combination of the components listed above, followed by filtration and sterilization. The use of such media and agents for pharmaceutical active substances is known in the art. Dispersions are typically prepared by incorporating the active compound into a sterile medium containing a basic dispersion medium and any other desired components from those listed above. In the case of sterile powders used to prepare sterile injectable solutions, some preparation methods include vacuum drying and lyophilization (freeze-drying), which produce a powder of the active ingredient plus any additional desired components from its previously sterile filtered solution.

[0193] The pharmaceutical compositions disclosed herein can be prepared using a carrier that protects the active ingredient from rapid release, such as controlled-release formulations, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Many methods for preparing such formulations are patented or commonly known to those skilled in the art. See, for example, *SUSTAINED AND CONTROLLED RELEASE DRUG DELIVERY SYSTEMS*, JR Robinson, ed., MarcelDekker, Inc., New York, 1978.

[0194] Treatment and administration of anti-γc protein

[0195] In embodiments of the present invention, anti-γc antibodies and / or γc-binding protein fragments can be used to treat patients with γc or γc cytokine-mediated diseases or conditions, such as GVHD, organ transplant rejection, shotgun chorioretinopathy, MS, uveitis, T1D, AD, RA, SLE, asthma, psoriasis, SS, vitiligo, celiac disease, IBD, AA, mast cell-mediated diseases, T-cell lymphoma, NK-cell lymphoma, and / or B-cell lymphoma.

[0196] GVHD refers to an autoimmune condition that can occur after allogeneic transplantation. For example, in GVHD, the donated bone marrow or peripheral blood stem cells may perceive the recipient's body as foreign, and the donated cells / bone marrow may attack the recipient's body. GVHD can occur in patients with hematopoietic stem cell transplantation (HCT; for example, in those with acute myeloid leukemia (AML) or acute lymphoblastic leukemia (ALL)) and / or myelodysplastic syndromes or myeloproliferative neoplasms), blood transfusions, thymus transplantation, or in patients with thymoma.

[0197] GVHD is less likely to occur when the match between the donor and recipient is close. For example, the chance of GVHD occurring is about 35% to 45% when the donor and recipient are related, and about 60% to 80% when the donor and recipient are not related.

[0198] Types of GVHD include steroid-refractory GVHD, acute GVHD, and chronic GVHD. Acute GVHD typically occurs within days or up to 6 months post-transplantation, while chronic GVHD usually begins more than 3 months post-transplantation and can persist for life. The one-year overall mortality rate from the date of diagnosis of acute GVHD and from the date of non-relapse is 35.2% and 25.5%, respectively, while patients with chronic GVHD tend to exhibit changes in the oral cavity and skin during relapse [Holtan et al. Bone Marrow Transplant., 2022;57, 1581–1585].

[0199] Symptoms of acute GVHD include a rash with burning and redness of the skin; nausea, vomiting, abdominal cramps, loss of appetite and diarrhea; jaundice (yellowing of the skin and / or eyes); and / or increased dryness / irritation of the eyes.

[0200] Symptoms of chronic GVHD include extreme dry mouth; sensitivity to hot, cold, spicy, and acidic foods, mint, and carbonated drinks; painful mouth ulcers that may extend down the throat; difficulty eating; gum disease and tooth decay; skin rash; dry, tight, and itchy skin; thickened and tightened skin; changes in skin color; intolerance to temperature changes due to damaged sweat glands; changes in nail texture; hard and brittle nails; missing nails; hair loss on the head; premature graying of hair; loss of body hair; loss of appetite, unexplained weight loss, nausea, vomiting, diarrhea, and stomach pain; shortness of breath and difficulty breathing; persistent and chronic cough that does not subside; wheezing; abdominal distension; jaundice; abnormal liver function test results; muscle weakness and cramps; joint stiffness that makes it difficult to fully extend the fingers, wrists, elbows, knees, and / or ankles; and / or genital irritation in women and men.

[0201] Common treatments for acute GVHD include enhanced immunosuppression in the form of corticosteroids and the use of JAK inhibitors (e.g., ruxolitinib). For milder cases of chronic GVHD, close observation or topical / surface treatment with topical steroid ointments is usually employed. Furthermore, a recently published manuscript [Le Floc'h et al., Sci Transl Med., 2023;15(678):eabo0205;Le Floc'h et al., Hemasphere. 2022; 6(Suppl ):694-695] also suggests the use of anti-γc-binding proteins as a treatment approach.

[0202] AA is a condition that occurs when the immune system attacks hair follicles and causes hair loss. The causes of AA are not fully understood, but both genetic and environmental factors (such as stress) are believed to play a role. People with certain autoimmune diseases (such as psoriasis, thyroid disease, or vitiligo) are also more likely to develop AA.

[0203] There are three main types of alopecia areata, including patchy alopecia (hair loss occurs in one or more coin-sized patches on the scalp or other parts of the body), total alopecia (all or almost all hair loss occurs on the scalp), and universal alopecia (all or almost all hair loss occurs on the scalp, face, and the rest of the body).

[0204] Typically, symptoms of AA include the sudden loss of round or oval patches of hair on the scalp or other parts of the body (e.g., the beard area in men, or eyebrows or eyelashes); stinging, burning, or itching sensations on the skin patches before the hair falls out; and / or nail changes such as ridges and pits. Although AA is not a life-threatening disease, the psychological burden of AA may lead to an increased mortality rate associated with self-harm, mental illness, and smoking-related malignancies [Lee et al., JAMADermatol. 2019;155(8):922-928].

[0205] Common treatments for AA include steroid injections, topical steroids (creams and ointments) and steroid tablets, immunotherapy, dithranol cream, UV phototherapy, and the use of minoxidil. In addition, recent clinical trials (IDs NCT03532958 and NCT05589610) have also recommended the use of anti-γc binding protein as a treatment option.

[0206] The present invention includes clues for treating or preventing (any type) of those autoimmune diseases in cell models or subjects (e.g., mice), which include administering a therapeutically effective dose of anti-γc protein to the subject.

[0207] As used herein, the term "subject" refers to any animal (e.g., mammal), including but not limited to humans, non-human primates, canines, felines, rodents, etc., that is a recipient of a specific treatment. A subject can be a human being. A subject can be a patient suffering from a specific disease.

[0208] As used herein, the term “treatment” and its grammatical equivalents in relation to a disease or condition or an object suffering from a disease or condition refer to actions that suppress, eliminate, alleviate and / or improve symptoms, severity and / or frequency of symptoms associated with the disease or condition being treated. For example, when referring to the use of GVHD, the term “treatment” and its grammatical equivalents refer to actions that reduce the severity of the disease or delay or slow the progression of the disease, including but not limited to (a) the absence of secondary systemic treatment, or (b) the absence of non-recurring mortality, or (c) the absence of recurrent or progressive malignancy [Martin et al., Blood., 2017; 130(3): 360–367].

[0209] The term “blocking” and its grammatical equivalents refer to the action of reducing the biological function of an alarm factor by means of, but not limited to, (a) directly competing for the binding site of the alarm factor on its corresponding receptor, or (b) preventing the heterodimerization of the corresponding receptor, thereby reducing the biological effects induced by alarm factor occupancy.

[0210] As used herein, the term "application" and its grammatical equivalents refer to the act of delivering or causing to deliver a therapeutic agent or pharmaceutical composition to the body of a subject by the methods described herein or other methods known in the art. Therapeutic agents can be compounds, peptides, or cells. Administering a therapeutic agent or pharmaceutical composition includes prescribing a therapeutic agent or pharmaceutical composition to be delivered to the body of a subject. Exemplary forms of administration include oral dosage forms such as tablets, capsules, syrups, and suspensions; injectable dosage forms such as intravenous (IV), intramuscular (IM), or intraperitoneal (IP); subcutaneous (SC), transdermal dosage forms including creams, gels, powders, or patches; sublingual dosage forms; and inhaled powders, sprays, suspensions, and rectal suppositories.

[0211] As used herein, the terms "effective amount," "therapeutic effective amount," and their grammatical equivalents refer to an amount of a pharmaceutical agent administered to a subject, alone or as part of a pharmaceutical composition and in a single dose or as part of a series of doses, that, when administered to the subject, would have any detectable positive effect on any symptom, aspect, or characteristic of a disease, symptom, or condition. Therapeutic effective amounts can be determined by measuring the associated physiological effects. The exact amount required varies depending on the subject's age, weight and general condition, the severity of the condition being treated, the clinician's judgment, etc. Therapeutic effective amounts are also the amount in which any toxic or harmful effects of the therapeutic agent exceed the beneficial therapeutic effects. In any individual case, the appropriate "effective amount" can vary depending on factors such as the individual's disease state, age, sex, and weight, and can be determined by a person skilled in the art using routine experiments. "Prophylactic effective amount" refers to the amount at the required dose and time period that effectively achieves the desired preventive outcome, such as delaying or preventing the onset of a disease or symptom. Typically, because preventive doses are used in subjects before or in the early stages of disease, prophylactic effective amounts are usually less than therapeutic effective amounts.

[0212] As used herein and as understood in the art, “EC” refers to the effective concentration of an agent (e.g., an antibody) and is commonly used in dose-response curves. The “effect” of an agent can be an active (activating) effect or a passive effect. The term “EC50” refers to the concentration of an active agent (e.g., an antibody) that produces a half-maximal response. Similarly, as used herein and as understood in the art, “IC” refers to the concentration of an agent that has an inhibitory effect and is also commonly used in dose-response curves. The term “IC50” refers to the concentration at which the inhibitory activity of an agent (e.g., an antibody) is reduced by half.

[0213] An effective or therapeutically effective dose of anti-γc protein (e.g., antibody or antigen-binding fragment) for the treatment or prevention of γc-mediated diseases or conditions refers to an amount of antigen-binding protein sufficient to alleviate one or more signs and / or symptoms of the disease or condition in the treated subject, whether by inducing the regression or elimination of these signs and / or symptoms or by inhibiting the progression of these signs and / or symptoms. In embodiments of the invention, an effective or therapeutically effective dose of anti-γc protein is about 0.05-50 mg / kg body weight. The dose may vary depending on the age and size of the subject to be administered, the target disease, the condition, the route of administration, etc. In some embodiments, a second or more subsequent doses of antigen-binding protein may be administered after the initial dose, the amount of which may be substantially the same as, less than or more than the initial dose, wherein the interval between subsequent doses is at least 1 to 3 days; at least one week; at least 2 weeks; at least 3 weeks; at least 4 weeks; at least 5 weeks; at least 6 weeks; at least 7 weeks; at least 8 weeks; at least 9 weeks; at least 10 weeks; at least 12 weeks; or at least 14 weeks.

[0214] Prevention of γc-mediated diseases or conditions refers to the administration of the anti-γc protein of the present invention to a subject prior to the manifestation of the disease or condition in the subject's body, in order to prevent such manifestation from occurring, since it involves the use of the anti-γc protein of the present invention.

[0215] Immunoconjugates

[0216] This invention includes an anti-γc protein, such as an antibody or antigen-binding fragment, conjugated to another portion, such as a therapeutic portion (“immunoconjugate”). In embodiments of this invention, the anti-γc protein (e.g., antibody or antigen-binding fragment) is conjugated to any other therapeutic agent described herein. As used herein, the term “immunoconjugate” refers to an antigen-binding protein (e.g., antibody or antigen-binding fragment) chemically or biologically linked to another antigen-binding protein, drug, radioactive agent, reporter portion, enzyme, peptide, protein, or therapeutic agent.

[0217] Functions and properties of anti-γc protein

[0218] The anti-γc proteins described herein (in chimeric or humanized forms), such as those comprising variant immunoglobulin chains, may exhibit one or more of the following properties: At 25°C, as indicated by K as listed in Table 11. D With human γc (e.g., its fusion, such as having a 6x His tag (SEQ ID NO: 234) or mouse Fc); At 25°C, as indicated by K as listed in Table 12. D It binds to common marmoset γc (e.g., its fusion forms, such as those with a 6xHis tag (SEQ ID NO: 234)); At 25°C, as indicated by K as listed in Table 12. D It binds to the mouse γc moiety (e.g., its fusion variant, such as one with a 6x His tag (SEQ ID NO: 234)); At 25°C, as indicated by K as listed in Table 12. D It binds to the rat γc moiety (e.g., its fusion variant, such as one with a 6x His tag (SEQ ID NO: 234)); At 25°C, as indicated by K as listed in Table 12. D It does not bind to cynomolgus monkey γc (e.g., its fusion, for example, has a 6x His tag (SEQ ID NO: 234)); It maintains high structural stability at 37°C for 2 weeks; Cell lines expressing γc did not exhibit ADCC and CDC activity; STAT phosphorylation induced by six γc cytokines in repressive cell lines (including NK cell cultures (e.g., human NK cell line KHYG-1), B cell cultures (e.g., human B cell line Ramos), and T cell cultures (e.g., human T cell lines HPB-ALL and Jurkat)), for example, as measured by luciferase expression in cells containing a luciferase gene operatively linked to a STAT3 / STAT5 / STAT6 response element or endogenous expression of phosphorylated STAT3 / STAT5 / STAT6 proteins; Blocking STAT phosphorylation in primary PBMC cultures induced by six γc cytokines (except IL-9); Blocking IL-4 and IL-21 rescues anti-IgM-induced cell death and damage in B cell cultures (e.g., human B cell line Ramos); Blocking IL-4 and IL-21 induces CD23 and PRDM1 expression in B cell cultures (e.g., human B cell line Ramos); Inhibits cell proliferation in human immune cells (e.g., PBMC cultures, primary T cells, primary NK cells, KHYG-1 cells) induced by IL-2, IL-7, IL-15, or IL-21; Inhibit the expression of BCL-2, a survival marker of HPB-ALL T cells induced by IL-7; Inhibits IL-7-induced CD127 internalization and degradation in HPB-ALL T cells; It weakens the protective and proliferative effects of IL-9 on Jurkat T cells in the presence of C2-ceramide; Inhibits IL-9-induced ERK phosphorylation in Jurkat T cells; Inhibit the secretion of soluble factors (e.g., pro-inflammatory cytokines and cytotoxic enzymes) from NK cells or PBMC cultures induced by IL-2, IL-15, or IL-21; Inhibits the expression of activation receptors on the surface of cytotoxic T cells and NK cells in PBMC cultures; Attenuate MLR induced by primary T cells from allogeneic grafts; The same epitope of human γc as any one or more γc-binding proteins described herein (e.g., on γc proteins coated in ELISA strips); It competes with any one or more γc-binding proteins described herein for binding to human γc (e.g., on γc proteins coated in ELISA strips).

[0219] Example

[0220] Experimental Examples

[0221] The following embodiments are provided to provide a complete disclosure and description of how to prepare and use the methods and compositions of the present invention to those skilled in the art, and are not intended to limit the scope of what the inventors consider to be their invention.

[0222] Example 1: Development and production of anti-γc antibodies

[0223] After immunizing animals with the human γc protein (sequence ID: NP_000197.1) shown in SEQ ID NO: 205, anti-γc antibodies were obtained from a phage display library, the human γc protein consisting of the extracellular domain of human γc protein (amino acids: 1-262) and a mouse Fc tag (underlined amino acids: 278-509) with three repeating G4S linkers (SEQ ID NO: 236) (amino acids: 263-277), or the human γc protein shown in SEQ ID NO: 206 consisting of the extracellular domain of human γc protein (amino acids: 1-262) and a 6x His tag (SEQ ID NO: 234) (underlined amino acids: 263-268).

[0224] First, an anti-γc ScFv was constructed using the HB2151 bacterial host cell system to evaluate the specificity and inhibitory properties of the anti-γc antibody. The binding activity of the constructed anti-γc ScFv to human γc protein was assessed using ELISA. Briefly, the internally generated human γc protein was diluted to 1 μg / ml in PBS, and 100 μl of γc protein was added to each well of an ELISA strip. The strips were sealed with sealing film and incubated overnight at 4°C for coating. On the second day, the strips were washed three times with washing buffer (0.05% Tween 20 in PBS) and blocked at RT with blocking buffer (3% BSA in PBS) at 100 μl / well for 2 hours, followed by the addition of 10 μg / ml anti-γc ScFv. After 2 hours of RT incubation, the amount of bound anti-γcScFv was determined by adding peroxidase-conjugated goat anti-human F(ab')2 specific antibody (Jackson Immuno Research, West Grove, USA) and TMB substrate (Sigma-Aldrich, St. Louis, USA). The assay was performed at OD 450 nm according to standard ELISA protocols known to those skilled in the art. Results showed that all purified anti-γcScFv bound to human γc protein at both 25°C and 37°C compared to PBS and the untransfected TG1 control. Figure 1 ).

[0225] Full-length chimeric anti-γc antibodies were generated using Expi-CHO host cells and purified using standardized methods. Each antibody consists of four polypeptides: two immunoglobulin heavy chains and two immunoglobulin light chains linked together to form a "Y"-shaped molecule. Each heavy chain is connected to a V-shaped linker in its constant region. H The chain consists of chains, and each light chain is connected to a constant region of the light chain by a V. L Chain composition. Each antibody belongs to one of five well-known antibody classes: IgA (IgA and IgA2), IgD, IgE, IgG (IgG1, IgG2, IgG3, and IgG4), and IgM. Three signal peptides were tested in the production strategy. Signal peptides 2 and 3 provided better yields than signal peptide 1. The stability of the purified antibodies was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using a 10% non-reducing gel, showing that all purified antibodies except SM-05E were intact. Figure 2 ).

[0226] Example 2: Flow cytometry analysis of p-STAT against γcScFv in multi-cell lines

[0227] Following cytokine induction and anti-γcScFv treatment, the percentage of cells expressing p-STAT was determined by flow cytometry. KHYG-1 cells (Creation BioArray, Shirley, NY 11967, USA) were starved for 2 days in IL-2-free RPMI1640 (ATCC modification, ThermoFisher Science, Waltham, MA, USA) containing 10% FBS (ThermoFisher Science). Ramos, HPB-ALL, and Jurkat cell lines were used directly without starvation. Cells were replenished with fresh RPMI1640 medium and pretreated with anti-γcScFv (10 μg / mL) for 1 hour. Cells were treated with IL-4 (10 ng / ml, Sino Biological), IL-7 (50 ng / ml, Sino Biological), IL-9 (50 ng / ml, Sino Biological), IL-15 (10 ng / ml, Sino Biological), or IL-21 (10 ng / ml, Sino Biological) for 15 minutes, then fixed in 4% paraformaldehyde (PFA, Sigma, St. Louis, Missouri, USA) for 10 minutes, permeabilized in 0.1% Triton X-100 (Sigma) for 10 minutes, and then stained at 37°C for 30 minutes with FITC-conjugated anti-pSTAT6, PE-conjugated anti-pSTAT3, or APC-conjugated anti-pSTAT5 (all diluted 1:100, Biolegend, San Diego, California, USA). The percentage of p-STAT-expressing cells was measured using the BD FACSLyric™ clinical flow cytometry system (BD Biosciences, New Jersey, USA). Analyze the data using Flowjo (version 10, BD).

[0228] Different cell lines were used for testing. KHYG-1 is an NK cell line harvested from the peripheral blood of a 45-year-old woman with aggressive NK cell leukemia. This cell line can respond to IL-2 and IL-15 stimulation in vitro [Yamasaki et al., Leuk Res., 2014;28(10):1023-31]. Ramos is a human Burkitt lymphoma cell line (CRL-1596™) and can be used as a model for IL-4 and IL-21 studies [Hui et al., Front Immunol., 2022;13:919854]. Jurkat is a human T lymphoblastic cell line (clone E6-1, TIB-152™) and is widely used for T cell studies of IL-9 function. HPB-ALL is a T cell leukemia model established from the peripheral blood of a 14-year-old Japanese boy diagnosed with ALL and thymoma in 1973 and can be used for IL-7 function studies.

[0229] The results showed that the four clones (SM-05A, SM-05E, and SM-05F) potentially inhibited the highest combination of γc cytokines mediating p-STAT expression, thereby promoting the production of full-length antibodies (Tables 7-10).

[0230] Example 3: EC50 of purified full-length anti-γc antibody

[0231] The binding specificity of selected full-length antibody clones was assessed by ELISA. The binding activity of full-length anti-γc antibodies was investigated using γc proteins (extracellular domains) from humans (SEQ ID NO: 205-206), cynomolgus monkeys (Sequence ID: XP_005593949.1, SEQ ID NO: 207-208), and chimpanzees (Sequence ID: XP_008971810.1, SEQ ID NO: 209-210), which were purchased from Sino Biological or internally generated. The results showed that the anti-γc antibodies bound to γc proteins from humans and chimpanzees in a dose-responsive manner with comparable affinity, but exhibited low / no binding affinity to cynomolgus monkey γc proteins. Figure 3 ).

[0232] Example 4: Flow cytometry analysis of p-STAT using full-length anti-γc antibody in multi-cell lines

[0233] As previously described, the percentage of cells expressing p-STAT was measured by flow cytometry after cytokine induction and anti-γc antibody treatment. IL-2 and IL-15 were tested in the KHYG-1 NK cell line; IL-4 and IL-21 were tested in the Ramos B cell line; IL-7 was tested in the HPB-ALL T cell line; and IL-9 was tested in the Jurkat T cell line.

[0234] Both SM-05A and SM-05E showed signs of blocking IL-2 in KHYG-1 NK cells ( Figure 4 A) IL-4 in Ramos B cells ( Figure 4 B) IL-7 in HPB-ALL T cells ( Figure 4 C) IL-9 in Jurkat T cells ( Figure 4 D) IL-15 in KHYG-1 NK cells ( Figure 4 E) and IL-21 in Ramos cells ( Figure 4 SM-05F showed comparable efficacy to F-induced STAT phosphorylation and was comparable to that of the competitive antibody COMP2022 (from the sequence of WO2020160242A1). SM-05F showed little to no strong inhibitory effect on STAT phosphorylation induced by the six γc cytokines.

[0235] Example 5: Bioassays for B cell research

[0236] Ramos can serve as a model for B-cell antigen receptor (BCR) activation, leading to cell cycle arrest and apoptosis by crosslinking the BCR with anti-IgM antibodies.

[0237] The WST-8 proliferation assay was used to investigate the rescue of Ramos cell death induced by anti-IgM-induced overactivation after blocking IL-4 and IL-21 with anti-γc antibody. In short, Ramos cells (50,000 cells per 100 μl) were seeded into 96-well plates and stabilized at 37°C for 1 hour. Cells were pretreated with anti-γc antibody or litexitinib for 1 hour, followed by incubation with IL-4 (10 ng / ml) or IL-21 (10 ng / ml) and anti-IgM (2 μg / ml, #109-006-129, Jackson ImmunoResearch). After 48 hours, 10 μl of WST-8 reagent (ab228554, Abcam) was added to each well and incubated for 4 hours.

[0238] WST-8 can be reduced to an orange formazan product by cellular dehydrogenases, and the amount of formazan produced is proportional to the number of live Ramos cells. The optical density (OD) of each well was measured at 450 nm using a Varioskan LUX Multimode Microplate Reader (ThermoFisherScientific).

[0239] WST-8 assays confirmed that IL-4 and IL-21 could rescue anti-IgM-induced cell death in Ramos B cells, while the application of anti-γc antibodies (SM-05A, SM-05E, SM-05F) blocked the effects of IL-4 and IL-21. When administered at the same molar concentration (33 nM), the overall efficacy of the anti-γc antibodies in this invention was superior to COMP2022 and litexitinib. Figure 5 AD).

[0240] The above effects can be explained by an apoptosis assay using a death cell apoptosis kit (# V13242, ThermoFisher Scientific) with annexin V (AV) for flow cytometry. In short, cells are treated with the same paradigm and then stained with AV / propidium iodide (PI) according to the manufacturer's protocol. The number of apoptotic cells (AV+PI- and AV+PI+ cells) is analyzed using Flowjo.

[0241] All tested anti-γc antibodies partially reversed the rescue effects of anti-IgM-induced apoptosis by IL-4 and IL-21 in Ramos B cells, with the strongest effect observed in the group treated with SM-05A. Figure 6 AB).

[0242] The levels of lysed caspase 3 (an apoptosis protein) and µ-H2AX (a DNA damage marker) were investigated in Ramos B cells treated with the same paradigm using Western blotting. Briefly, total protein was extracted from Ramos cells in RIPA lysis buffer supplemented with a mixture of Halt™ protease and phosphatase inhibitors. Proteins were denatured and separated by electrophoresis and blotted onto nitrocellulose membranes. The membranes were blocked with 5% skim milk diluted in TBST and then incubated overnight at 4°C with a primary antibody diluted in 5% BSA / TBST in a swirl wheel. The next day, the membranes were washed with PBST and incubated at RT for 1 hour with a secondary antibody diluted in 5% milk / TBST. The intensity of protein bands was determined using an ECL substrate kit on a ChemiDoc imaging system.

[0243] Consistent with WST-8 and AV / PI assays, IL-4 and IL-21 rescued anti-IgM-induced apoptosis and DNA damage markers. The application of anti-γc antibody and litexitinib blocked the protective effect of IL-4 and re-induced µ-H2AX levels, while SM-05A showed the strongest efficacy in re-inducing cleaved caspase 3 and µ-H2AX in the presence of IL-21. Figure 6 CD).

[0244] Genes related to B cell function were investigated using quantitative real-time PCR (qRT-PCR). In short, Ramos cells were starved in serum-free RPMI 1640 medium for 24 hours, followed by treatment with 10 ng / ml IL-4 or IL-21 for 24 hours. Total RNA was extracted using RNAzol according to the manufacturer's protocol. Reverse transcription was performed on 500 ng of total RNA using the PrimeScript RT reagent kit, and the results were analyzed using LightCycler. ® Gene expression was analyzed using quantitative real-time PCR with TB Green PremixEx Taq in a 480 real-time PCR system. The housekeeping gene GAPDH was used for normalization.

[0245] CD23 and PRDM1 expression were investigated in Ramos cells after treatment with IL-4 or IL-21. CD23 is a low-affinity receptor for IgE expressed on the surface of activated B cells and classical memory B cells. It enhances antibody responses, promotes the production of IgE in its soluble form, inhibits the production of membrane IgE, and controls B cell proliferation and apoptosis [Veneri et al., BloodTransfus., 2009; 7(1): 29–34].

[0246] PRDM1 is an important transcription factor in B cells. It controls the terminal differentiation of antibody-secreting cells (ASCs) and regulatory B cells (Bregs) that produce IL-10 [Nutt et al., Nat Rev Immunol., 2007; 7(12):923-7; Wang et al., Front Immunol., 2019; 10:1909].

[0247] The results showed that SM-05A, SM-05E, and litexitinib significantly inhibited IL-4-induced CD23 expression, while only SM-05E inhibited IL-21-induced PRDM1 expression. Figure 7 ).

[0248] Example 6: Bioassays for T cell research

[0249] The IL-9 receptor is expressed on the surface of Jurkat cells and can be used to study the relationship between IL-9 and autoimmune diseases / tumor biology [Lv et al., J Exp Clin Cancer Res., 2016;35(1):106].

[0250] Western blot analysis showed that IL-9 phosphorylates the pro-survival ERK signaling pathway, and that the application of SM-05A can block this effect. Figure 8 ).

[0251] It has been shown that IL-7 induces STAT5 phosphorylation and is responsible for the cell growth, proliferation and survival of HPB-ALL cells [Ribeiro et al., Blood Adv., 2018; 2(17): 2199–2213], indicating that HPB-ALL is a reliable cell line for studying IL-7 function.

[0252] Several assays (e.g., proposed for studying cell survival in HPB-ALL from the literature [Ribeiro et al., Blood Adv., 2018; 2(17): 2199–2213]) were measured by flow cytometry based on the proportion of cells expressing BCL2 (an anti-apoptotic marker). In short, HPB-ALL cells were pretreated with an antibody or inhibitor for 1 hour, followed by stimulation with 50 ng / ml IL-7 for 3 days. Cells were harvested and stained with a FITC-conjugated BCL-2 antibody (Biolegend) for flow cytometry analysis.

[0253] The results showed that both SM-05A and SM-05E significantly attenuated IL-7-induced BCL-2 expression, comparable to the litexitinib treatment group. Figure 9 A). The competitive antibody COMP2022 showed variability in this assay.

[0254] Specific methods for IL-7 function have been developed in HPB-ALL [Henriques et al., Blood., 2010; 115(16):3269-77]. IL-7 can trigger the internalization of its own receptor CD127 (or IL-7Rα) into cellular compartments for degradation. Since CD127 degradation depends on the JAK3 activation state after IL-7 stimulation, blocking the binding of γc to CD127 and downstream signaling pathways should prevent CD127 internalization and degradation in HPB-ALL.

[0255] After antibody or inhibitor pretreatment, HPB-ALL was challenged with 50 ng / ml IL-7 for 3 hours. Surface CD127 expression was investigated in live cells, while total CD127 expression was studied in PFA-fixed and permeabilized cells. Cells were stained with APC-conjugated CD127 antibody for flow cytometry analysis.

[0256] The results showed that all tested anti-γc antibodies could block IL-7-induced CD127 internalization and degradation in HPB-ALL cells, with the highest potency observed in cells treated with SM-05A. Figure 9 BE).

[0257] Example 7: Bioassays for NK cell research

[0258] The role of anti-γc antibodies in NK cell homeostasis was investigated in the KHYG-1 cell line (CSC-C0784, Creative BioArray), established from the peripheral blood of a 45-year-old woman with aggressive NK cell leukemia. As reported in the literature, KHYG-1 responds to IL-2 and IL-15 stimulation, but no information was found for other γc cytokines. We tested the responsiveness of KHYG-1 to γc cytokines prior to antibody assays.

[0259] Proliferation assays were performed on KHYG-1 cells that had been starved overnight, while simultaneously incubating them with different combinations of γc cytokines for 3 days. Results showed that KHYG-1 cells responded to IL-2 and IL-15 to proliferate, but no synergistic effect was observed in any of the combination groups. Figure 10 A).

[0260] The production of cytotoxic factors was monitored in KHYG-1 cells by flow cytometry after 3 days of cytokine incubation. Cells were fixed in 4% PFA and then permeabilized with Pacific Blue-conjugated granzyme B and APC-conjugated perforin antibodies (Biolegend) for intracellular staining. Results showed that IL-2, IL-15, and IL-21 increased the percentage of granzyme B / perforin-expressing cells after 3 days, with the strongest effect observed in the IL-21 treatment group. Figure 10 BC).

[0261] The results obtained from flow cytometry were consistent with those obtained from the supernatant via ELISA. IL-21 is used as a major γc cytokine to induce the production and secretion of granzyme and perforin from KHYG-1 cells. Figure 10 DE). Different results were observed for IFNγ ELISA, namely that IL-15 is the main γc cytokine that induces IFNγ secretion in KHYG-1 cells ( Figure 10F). All ELISA assays were performed using commercially available kits (R & D Systems, Minneapolis, USA).

[0262] To test the efficacy of the anti-γc antibody, proliferation and granzyme A secretion were selected as experimental models. Briefly, KHYG-1 was seeded into 48- or 96-well plates and starved overnight in medium without IL-2 supplementation. After supplementation with fresh medium, KHYG-1 was pre-incubated with anti-γc antibody or litexitinib for 1 hour, followed by administration of γc cytokines for 3 days. The proliferation rate was determined by the addition of WST-8 reagent, while granzyme A secretion was estimated by ELISA.

[0263] The results showed that, compared with the group treated with the JAK3 inhibitor litexitinib, both SM-05A and SM-05E could inhibit IL-2, IL-15, and IL-21-induced NK cell proliferation. Figure 11 AC).

[0264] Different results were observed in granzyme A secretion. SM-05E and litexitinib significantly inhibited IL-2, IL-15, and IL-21-induced granzyme A secretion in NK cell culture, while SM-05A contributed less to the inhibition. Figure 11 DF).

[0265] Example 8: Bioassays for in vitro PBMC cultures

[0266] PBMCs (iXCells Biotechnologies, San Diego, USA) isolated from healthy individuals were selected as primary immune cell cultures to infer the efficacy of anti-γc antibodies in primary cells. Since STAT phosphorylation can be triggered by treatment of all six γc cytokines (except IL-9) in PBMC cultures... Figure 12 Therefore, the inhibition of p-STAT can be used as an indicator to confirm the efficacy of anti-γc antibodies.

[0267] SM-05A remains the strongest candidate to inhibit STAT phosphorylation induced by all six γc cytokines in PBMC cultures, while SM-05E and COMP2022 failed to inhibit IL-4-induced p-STAT6 (…) in PBMC cultures. Figure 13 ).

[0268] Proliferation was also studied in PBMC cultures using a similar protocol established for the KHYG-1 NK cell line, and the results showed that PBMCs primarily responded to IL-2, IL-7, and IL-15 in terms of proliferation. Figure 12C). After 3 days of incubation with antibodies and cytokines, WST-8 reagent was added to the wells, and proliferation was measured by measuring the signal intensity at 450 nm using a microplate reader.

[0269] The SM-05A treatment group showed a significant inhibitory effect on proliferation. Figure 14 A). Further quantitative analysis confirmed that both SM-05A and SM-05E could inhibit IL-2, IL-7, and IL-15-induced proliferation, with SM-05A providing a slightly stronger effect. The competitive antibody COMP2022 showed almost no antiproliferative activity in PBMC cultures. Figure 14 BD).

[0270] Activation of T cells and NK cells in PBMC cultures can be investigated by the secretion of granzyme A and IFNγ [Carline et al., Blood., 2005;106(12):3874-9; Grossman et al., Blood., 2004;104(9):2840-8; Keppel et al., J Immunol., 2015; 194(4): 1954–1962; Tamang et al., Cytokine., 2006;36(3-4):148-59]. Similar results were obtained internally, with IL-2 and IL-15, but not IL-21, triggering the secretion of granzyme A and IFNγ in PBMC cultures. Figure 15 AB).

[0271] Antibodies or litexitinib were added to PBMC cultures to investigate their inhibitory effects in the presence of IL-2 or IL-15 treatment. Similar to previous results, SM-05A remained the most potent anti-γc antibody in inhibiting IL-2 and IL-15-induced granzyme A and IFNγ secretion, while the levels of both soluble factors in litexitinib were significantly reduced compared to the control group, indicating potential toxicity of JAK3 inhibitors. Figure 15 CF).

[0272] Further assessment of the inhibition of T cell activation was conducted by examining the expression of receptors associated with cell death. One example is the Fas ligand (FasL), which is primarily expressed on activated T cells and, upon binding, can trigger apoptosis in Fas-expressing cells [García-González and Selvi, Encyclopedia of Medical Immunology, 2014; pp 413–416].

[0273] Although treatment with IL-2 or IL-15 only slightly increased the cytotoxic T cell population (CD3+CD8+), both cytokines significantly induced FasL expression on the gated cytotoxic T cell population. Compared with the litexitinib treatment group, SM-05A significantly reduced cytokine-induced FasL expression on cytotoxic T cells. Figure 16 ).

[0274] Example 9: Humanization of anti-γc antibody

[0275] Preliminary results from cell lines and primary cultures indicate that both SM-05A and SM-05E are promising candidates for inhibiting γc cytokine-induced STAT phosphorylation and cellular function. Therefore, the humanization of both anti-γc proteins using a framework repair approach (see, for example, U.S. Patent No. 7,321,026B2) is proposed for future clinical applications.

[0276] Humanized sequences of the variable heavy and light chains are shown in SEQ ID NO: 193-200. In some embodiments, the humanized antibody is maintained in a fully humanized form. In some embodiments, reversion mutations in certain amino acids within the framework region are introduced into the humanized antibody to improve biological activity. The humanized variable heavy and light chain domains are linked to the constant heavy and light chain domains shown in SEQ ID NO: 201-202 or SEQ ID NO: 203-204.

[0277] Humanized antibodies (hSM-05A and hSM-05E) with reversion mutations were tested in the following assays. Binding affinity was investigated in an ELISA assay. The results showed that both hSM-05A and hSM-05E retained binding affinity and specificity to human and chimpanzee γc protein. Figure 17 ).

[0278] Other extracellular domains of the γc protein from common marmosets (Sequence ID: NP_001288775.1, SEQ ID NO: 211-212), rhesus monkeys (Sequence ID: NP_001030606.1, SEQ ID NO: 213-214), rabbits (Sequence ID: XP_008270982.1, SEQ ID NO: 215-216), mice (Sequence ID: NP_038591.1, SEQ ID NO: 217-218), and rats (Sequence ID: NP_543165.1, SEQ ID NO: 219-220) were purchased from Sino Biological or internally generated. The results indicate that the common marmoset is another species biologically related to both γc antibodies, while no binding affinity was observed in other studied species. Figure 17 ).

[0279] Example 10: Stability and Binding Kinetics of Anti-γc Antibody

[0280] Next, the structural stability of hSM-05A and hSM-05E was investigated by incubation at room temperature or 37°C for approximately 2 weeks. Antibody aggregation and degradation were measured using SEC-HPLC. The study showed that hSM-05A remained as a single molecule, while approximately 20% of hSM-05E formed aggregates after incubation. Figure 18 No antibody degradation was observed after 2 weeks.

[0281] Full-length humanized antibodies were evaluated using BLI to obtain their binding kinetics. The binding activity of full-length anti-γc antibodies was investigated using γc protein (extracellular domain) from humans and common marmosets (purchased or internally generated). Briefly, anti-γc antibodies (20 μg / mL) were immobilized on a biosensor by interaction with anti-human IgG; subsequently, serially diluted γc protein (concentrations of 158.7 nM, 79.4 nM, 39.7 nM, and 0 nM) were added according to the standard operating protocol of the OctetReD96 system to plot association and dissociation curves. An irrelevant antibody (SM03, anti-human CD22 chimeric IgG1 antibody, SinoMabBioScience Limited) was used as a control reference. R-squared, KA... a K dis and K D The values ​​are summarized in Table 11. K D Within the single picomolar to nanomolar range. Results indicate that the anti-γc antibody binds to γc protein from humans and common marmosets with comparable binding kinetics. Figure 19 (and Tables 11-12).

[0282] Table 11: Binding kinetics of hSM-05A and hSM-05E with human γc proteins from different species

[0283] Table 12: Binding kinetics of hSM-05A and hSM-05E with γc proteins from other species

[0284] The binding specificity of the humanized anti-γc antibody to native γc protein was further evaluated using in vivo binding assays. In short, human HEK-293 cells were transfected with plasmids expressing full-length γc protein from humans, rhesus macaques, cynomolgus monkeys, or common marmosets. Two days later, transfected cells or untransfected controls were incubated with FITC-conjugated hSM-05A on ice for 1 hour, followed by flow cytometry analysis. The results showed that hSM-05A specifically targets native γc protein expressed on HEK-293 cells from both humans and common marmosets. Figure 20 ).

[0285] Example 11: Competitive assay between anti-γc antibodies

[0286] Purified anti-γc antibodies were conjugated with FITC-conjugated fluorescent dyes for competitive assays. In short, Ramos B cells expressing human γc were first labeled with unconjugated hSM-05A for 1 hour, then further incubated with FITC-conjugated hSM-05E. Flow cytometry analysis showed that the two anti-γc antibodies could compete for similar epitopes on the human γc protein. Figure 21 ).

[0287] Example 12: Functional assay for ADCC / CDC activity determination

[0288] Since immunomodulatory antibodies block activation in immune cells rather than inducing cell death, ADCC and CDC need to be avoided in γc-binding proteins to prevent excessive killing of immune cells or release of cytokines from several effector cells.

[0289] The γc-expressing human keratinocyte HaCaT cell line was used as the target cells in ADCC and CDC assays. To determine ADCC activity, HaCaT cells were pre-bound with an anti-γc antibody before co-culturing with human PBMCs. To determine CDC activity, HaCaT cells were pre-bound with an anti-γc antibody and then incubated with a low-endotoxin complement protein extracted from guinea pigs. ADCC was determined by the percentage of PI+ HaCaT cells in flow cytometry analysis, while CDC was measured in 96-well plates by a WST-8 proliferation assay of HaCaT cells. Figure 22 A).

[0290] Quantitative studies clearly demonstrated that neither hSM-05A nor hSM-05E induced any ADCC or CDC activity in HaCaT cells compared to the isotype antibody treatment group. These assays provide evidence for the safe application of anti-γc antibodies in future therapeutic studies.

[0291] Example 13: Functional determination of the immunomodulatory effect of humanized antibodies

[0292] STAT phosphorylation analysis was performed again to investigate the efficacy of the humanized anti-γc protein, as shown in Table 13. The results show that both forms of the humanized antibody exhibited comparable p-STAT inhibitory activity compared to the chimeric form. Figure 23 ).

[0293] Table 13: Inhibition of p-STAT in PBMCs by humanized anti-γc antibodies

[0294] The given figures represent the percentage of inhibition compared to isotype controls stimulated with the corresponding γc cytokine. The antibody concentration is 10 μg / ml.

[0295] The immunomodulatory effects of the humanized antibodies were retested in Ramos B cells using the WST-8 proliferation assay. The results showed that both antibodies provided better inhibition of IL-4 than litexitinib, but their inhibition of IL-21 was less effective than that of litexitinib. Figure 24 ).

[0296] In HPB-ALL T cells, hSM-05A provided better inhibition of IL-7-induced BCL-2 upregulation and CD127 degradation compared to literxitinib. Figure 25 ).

[0297] Previous studies have shown that IL-9 can rescue antitumor drug-driven apoptosis in a B-cell model of diffuse large B-cell lymphoma (DLBCL) [Lv et al., J Exp Clin Cancer Res., 2016;35(1):106]. Similar experiments were performed on Jurkat T cells to evaluate the efficacy of the anti-γc antibody.

[0298] Jurkat T cells were pretreated with anti-γc antibody or litexitinib, followed by three days of treatment with anti-tumor ceramide (50 μM) and IL-9 (50 ng / ml). To assess cell viability, 10 μl of WST-8 reagent was added to each well, incubated for 4 hours, and then measured spectrophotometrically.

[0299] The results showed that IL-9 could rescue Jurkat cell death in the presence of ceramides, while the application of hSM-05A could inhibit the effect of IL-9. Figure 26 B). Furthermore, compared to hSM-05E, hSM-05A provides better inhibition of IL-9-induced ERK phosphorylation (B). Figure 26 C) indicates that hSM-05A can inhibit T cell survival and proliferation in the presence of IL-9.

[0300] In human PBMC cultures, the roles of IL-2 and IL-15 in cell proliferation were determined using the WST-8 proliferation assay. Results showed that both antibodies provided better inhibition of IL-2 than litexitinib, but their inhibitory effect on IL-15 was less effective than that of litexitinib. Figure 27 BC).

[0301] The release of cell lysing agents from PBMC cultures was further assessed by ELISA. Although hSM-05A provided comparable inhibition of IFNγ secretion to litexitinib, litexitinib provided superior inhibition of granzyme B secretion in cultures. Figure 27 DG). hSM-05E provides only weak inhibition against both agents.

[0302] Activation of NK cells and cytotoxic T cells was investigated by activating the expression of receptor NKp46 on the surface of NK cells and cytotoxic T cells. PBMCs were pretreated with humanized antibodies or litexitinib for 1 hour, followed by incubation with IL-2 or IL-15 for 3 days. NK cells were gated into a CD3-CD56+ population, while cytotoxic T cells were identified as a CD3+CD8+ population by flow cytometry. Quantitative analysis showed that both humanized antibodies were effective in inhibiting IL-2 and IL-15-induced cytotoxic T cell activation, while providing only a slight inhibition of NK cell activation compared to the litexitinib treatment group. Figure 28 ).

[0303] To provide clues about the role of humanized antibodies in targeting GvHD, an MLR assay was developed in our PBMC system. Figure 29 A). In short, total T cells were isolated from PBMCs of donor 1, while T cells were consumed from PBMCs of donor 2. T cells from donor 1 were activated with a mixture of anti-CD3 and anti-CD28 antibodies in the presence of IL-2. Simultaneously, the T cells were incubated with either a humanized antibody or litexitinib and an inducing agent. After 3 days, the T cells were labeled with CFSE and then co-cultured with the T cell consumption culture from donor 2 for 6 days. Flow cytometry identified proliferating T cells as either a CFSE-low or CSFE-negative population.

[0304] The results showed that both hSM-05A and hSM-05E significantly inhibited MLR-induced cytotoxic T cell proliferation in a dose-dependent manner, while exhibiting a milder inhibition of T helper cells. Figure 29 BC).

[0305] Results Summary and Conclusions

[0306] This invention describes a procedure for developing anti-γc-binding proteins and provides the DNA / amino acid sequences of the developed anti-γc-binding proteins. These proteins inhibit γc-related downstream pathways (JAK / STAT) and biological functions (survival, proliferation, activation, cytokine secretion, cytotoxic enzyme secretion, differentiation, receptor internalization, cytotoxicity) in various immune cell types (including but not limited to B cells, CD4+ T helper cells, CD8+ cytotoxic T cells, NK cells, and mast cells) in single-cell or PBMC cultures. Figure 30 This demonstrates the therapeutic potential of the present invention in treating diseases or conditions mediated by γc or γc cytokines (e.g., GVHD, organ transplant rejection, shotgun chorioretinopathy, MS, uveitis, T1D, AD, RA, SLE, asthma, psoriasis, SS, vitiligo, celiac disease, IBD, AA, mast cell-mediated diseases, T-cell lymphoma, NK-cell lymphoma, and / or B-cell lymphoma) in subjects of need, said treatment comprising, for example, administration or injection of an effective amount of the antigen-binding protein or composition described herein.

Claims

1. An isolated antigen-binding protein that specifically binds to γc or an antigen fragment thereof, wherein the antigen-binding protein exhibits one or more of the following characteristics: (a) at less than 10 -9 M's acceptable K D The range is associated with human γc or its fusion. (b) at less than 10 -9 M's acceptable K D The range is associated with chimpanzee and common marmoset γc or the aforementioned fusions; (c) with 10 -8 M of K D It partially binds to mouse and rat γc or the aforementioned fusions; (d) No binding of the earth to rhesus monkey, cynomolgus monkey and rabbit γc or the aforementioned fusions could be detected; (e) Inhibits IL-4 and IL-21-induced STAT phosphorylation in Ramos B cells; (f) Inhibits IL-7-induced STAT phosphorylation in HPB-ALL T cells; (g) Inhibits IL-9-induced STAT phosphorylation in Jurkat T cells; (h) Inhibits STAT phosphorylation induced by IL-2 and IL-15 in KHYG NK cells; (i) Inhibit STAT phosphorylation induced by IL-2, IL-4, IL-7, IL-9, IL-15 or IL-21 in PBMC cultures; (j) Blocking IL-4 and IL-21 in B cell cultures rescues anti-IgM-induced cell death and damage; (k) Blocking IL-4 and IL-21-induced genes in B cell cultures; (l) Inhibits the proliferation of human immune cells, including NK cells, T cells, and cells in PBMC cultures; (m) Attenuates the protection and proliferation of IL-9-induced Jurkat T cells in the presence of antitumor ceramides; (n) Inhibits the secretion of pro-inflammatory cytokines and cytotoxic enzymes from NK cells and / or PBMC cultures; (o) Inhibits the activation of NK and T cells in primary immune cell cultures; (p) Specifically binds to an epitope on γc that is identical to that of a reference antibody or its antigen-binding fragment, wherein the reference antibody or its antigen-binding fragment is a chimeric or humanized SM-05A, SM-05B, SM-05C, SM-05D, SM-05E, SM-05F, SM-05G, SM-05H, SM-05I, SM05J, SM-05K, or SM-05L; (q) Competing with a reference antibody or its antigen-binding fragment for binding to a γc peptide or its antigen fragment, wherein the reference antibody or its antigen-binding fragment is a chimeric or humanized SM-05A, SM-05B, SM-05C, SM-05D, SM-05E, SM-05F, SM-05G, SM-05H, SM-05I, SM05J, SM-05K, or SM-05L; and (r) Blocks the binding of heterodimers composed of γc and corresponding γc cytokine receptor subunits in the presence of IL-2, IL-4, IL-7, IL-9, IL-15 or IL-21.

2. The antigen-binding protein according to claim 1, wherein the protein is an isolated antibody.

3. The antigen-binding protein according to claim 1, wherein the protein is an antigen-binding fragment of an antibody.

4. The isolated antibody according to claim 2 or the antigen-binding fragment according to claim 3, which specifically binds to γc or an antigen fragment of γc, wherein the antibody or antigen-binding fragment comprises: (a) A heavy chain immunoglobulin or variable region comprising CDR-H1, CDR-H2, and CDR-H3 of a heavy chain immunoglobulin or a variable region thereof containing the amino acid sequences shown in SEQ ID NO: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162, 178 or variants thereof; and / or, (b) Light chain immunoglobulin or variable region comprising CDR-L1, CDR-L2 and CDR-L3 of a light chain immunoglobulin or a variable region thereof containing the amino acid sequence shown in SEQ ID NO: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170, 186 or a variant thereof.

5. The isolated antibody according to claim 2 or the antigen-binding fragment according to claim 3, comprising: (a) a heavy chain immunoglobulin or its variable region, comprising an amino acid sequence having at least 90% amino acid sequence identity with the amino acid sequence shown in SEQ ID NO: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162 or 178; and / or, (b) A light chain immunoglobulin or its variable region comprising an amino acid sequence having at least 90% amino acid sequence identity with the amino acid sequence shown in SEQ ID NO: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170 or 186.

6. The isolated antibody or antigen-binding fragment according to claim 4, comprising: (a) A heavy chain immunoglobulin or its variable region comprising CDR-H1, CDR-H2, and CDR-H3 of the heavy chain immunoglobulin or its variable region containing the amino acid sequence shown in SEQ ID NO: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162, or 178, and having at least 90% amino acid sequence identity with the amino acid sequence shown in SEQ ID NO: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162, or 178; and / or, (b) A light chain immunoglobulin or its variable region comprising CDR-L1, CDR-L2 and CDR-L3 of the light chain immunoglobulin or its variable region containing the amino acid sequence shown in SEQ ID NO: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170 or 186, and having at least 90% amino acid sequence identity with the amino acid sequence shown in SEQ ID NO: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170 or 186.

7. The isolated antibody or antigen-binding fragment according to claim 4, comprising: (s) heavy chain immunoglobulin or its variable region having an amino acid sequence selected from those shown in Table 1; and / or, (t) Light chain immunoglobulin or its variable region having an amino acid sequence selected from those shown in Table 2.

8. The isolated antibody or antigen-binding fragment according to claim 7, comprising one or more members selected from heavy chain immunoglobulins and their light chain immunoglobulins, wherein the heavy chain immunoglobulins and their light chain immunoglobulins are selected from the amino acid sequences shown in Table 3.

9. An isolated antibody or antigen-binding protein that specifically binds to γc or an antigen fragment thereof, wherein said antibody or antigen-binding protein comprises: (a) A heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 2 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 10; (b) A heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 18 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 26; (c) The heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 34 and the light chain variable region containing the amino acid sequence shown in SEQ ID NO: 42; (d) A heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 50 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 58; (e) A heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 66 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 74; (f) The heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 82 and the light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 90; (g) A heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 98 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 106; (h) A heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 114 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 122; (i) A heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 130 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 138; (j) The heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 146 and the light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 154; (k) A heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 162 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 170; (l) A heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 178 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:

184.

10. The antigen-binding protein according to claims 1 and 9, wherein the protein is a chimeric or humanized antibody constructed by a framework repair method.

11. An isolated humanized antibody or antigen-binding protein that specifically binds to γc or an antigen fragment thereof, wherein said antibody or antigen-binding protein comprises: (a) A heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 193 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 195; (b) A heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 197 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 199; (c) A heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 194 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 196; (d) A heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 198 and a light chain variable region containing the amino acid sequence shown in SEQ ID NO:

200.

12. Heavy chain immunoglobulin G1 or G4, comprising a heavy chain variable region according to claims 9 and 11 conjugated to any one of the heavy chain constant regions, wherein the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO: 201 and 203.

13. Light chain immunoglobulin G1 or G4, comprising a light chain variable region according to claims 9 and 11 conjugated to any one of the light chain constant regions, wherein the amino acid sequence of the light chain constant region is shown in SEQ ID NO: 202 and 204.

14. The antigen-binding protein according to claim 1, which is multispecific.

15. A method for preparing the antigen-binding protein or its immunoglobulin chain according to claim 1, the method comprising the following steps: (a) Immunizing animals by injecting purified γc protein; (b) Phage libraries were generated by randomly linking antigen-binding scFv libraries to phage vectors; (c) Use purified γc protein or cells expressing γc to expand and screen phage libraries; (d) Producing and purifying soluble ScFv and antibodies from host cells and / or the culture medium in which the host cells are grown.

16. The method according to claim 15, wherein the host cell is Escherichia coli or Chinese hamster ovary cells.

17. An antigen-binding protein or immunoglobulin chain, which is produced by the method according to claim 15.

18. A polynucleotide encoding one or more polypeptides of the antigen-binding protein according to claim 1.

19. A vector comprising the polynucleotide according to claim 18.

20. A method for treating individuals with γc or γc cytokine-mediated autoimmune diseases or conditions, said diseases or conditions selected from GVHD, organ transplant rejection, shotgun chorioretinopathy, MS, uveitis, T1D, AD, RA, SLE, asthma, psoriasis, SS, vitiligo, celiac disease, IBD, AA, mast cell-mediated diseases, T-cell lymphoma, NK-cell lymphoma, and B-cell lymphoma, said method comprising the step of administration by injecting an effective amount of the antigen-binding protein according to claim 1.

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