Anti-common gamma chain protein and compositions of matter

Abstract

The present invention details the design and production of antibodies and antigen-binding fragments (e.g., chimeric and humanized antibodies) that specifically bind to and inhibit the activation of the human common gamma chain (γc), a shared signaling receptor subunit utilized by six γc cytokines. Methods for production and functional assays to evaluate the efficacy of the generated antibodies are also described, as well as results showing the use of such anti-γc antibodies to treat or prevent γc- or γc cytokine-mediated autoimmune diseases (e.g., GVHD).

Description

[Technical Field]

[0001] Priority This application claims the right and benefit of U.S. Provisional Application No. 63 / 485,980, filed February 20, 2023, the entire contents of which are incorporated herein by reference.

[0002] Array List The above application contains a Sequence Listing in XML format, created on February 12, 2023, titled "SBL-011PCT_SL.xml," 227,306 bytes in size, which is incorporated herein by reference in its entirety.

[0003] The present invention relates to the development of antigen binding proteins that bind to γc proteins and assays to evaluate the effectiveness of those proteins in suppressing γc cytokine-induced functions in immune cells, suggesting their therapeutic potential in the treatment of autoimmune diseases. [Background technology]

[0004] The common γ chain (γc) was first discovered in 1992 as a component of the receptor for interleukin (IL)-2. γc was subsequently characterized as a shared signaling 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 and dimerize with γc to induce downstream pathways associated with a wide range of pleiotropic effects on the innate and adaptive immune systems. Dimerization of the two receptor subunits leads to activation of the Janus kinase signal transduction and activator of transcription (JAK-STAT) pathway. Autophosphorylation of JAK1 and JAK3 kinases after receptor dimerization phosphorylates STATs, which then dimerize and translocate to the nucleus. Currently, STATs function as transcriptional and epigenetic regulators of genes involved in survival, differentiation, proliferation, and immune responses.

[0005] The gene expressing γc is located on chromosome Xq13.1. γc is mutated in patients with X-linked severe combined immunodeficiency (XSCID) [Noguchi et al., Cell., 1993; 73(1):147-57.], which causes significant impairment of both cellular and humoral immunity due to underdevelopment of T cells and NK cells, as well as defective immunoglobulin synthesis due to the dysfunction and absence of fully mature B cells.

[0006] The role of γc cytokines in antitumor effects has been widely documented. IL-2, IL-7, IL-15, and IL-21 have shown the greatest potential in immunotherapy due to their ability to stimulate the proliferation and activation of NK cells, CD4+ helper T cells, and CD8+ cytotoxic T cells [Meazza et al., J Biomed Biotechnol., 2011;2011:861-920; Raeber et al., Immunol Rev., 2018;283(1):176-193.]. The administration of these γc cytokines has been tested as combination therapy in clinical trials [reviewed in Pulliam et al., Immunol Lett., 2016;169:61-72.]. However, these studies have shown that γc cytokines exhibit conflicting effects in hematopoietic cancers, making their use in anticancer therapy still controversial.

[0007] In contrast, elevated levels of γc cytokines have been consistently observed in human autoimmune disease pathologies and are associated with the progression of autoimmune diseases. IL-2 has been implicated in 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 is associated with atopic dermatitis (AD) [Chiricozzi et al., Immunotargets Ther., 2020; 9: 151-156], asthma [Pelaia et al., Front Pharmacol., 2022; 13: 851940.], and MS [Tahani et al., J Immunoassay Immunochem., 2019; 40(5):555-563.], alopecia areata (AA) [El-Latif et 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., 2015;72(2):146-53.] is associated with progression.IL-7 has been implicated in 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.], Sjögren'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 has been implicated in 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 has been shown to be involved in RA [Yang et al., Hum Immunol., 2015; 76(11):812-8.], psoriasis [Jesus-Gil et al., Exp Dermatol, 2020; 29(7):630-638.], vitiligo [Atwa et al., J Cosmet Dermatol., 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): 221-234.] is associated with progression.IL-21 is involved in 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. 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; Pages 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]. Summary of the Invention

[0008] The present invention provides antibodies or antigen-binding fragments (monospecific or multispecific) that bind to γc and are characterized by one or more of the following: i) binding to purified human γc with a KD of less than 10 M; ii) inhibiting IL-2-, IL-4-, IL-7-, IL-9-, IL-15-, and IL-21-induced STAT phosphorylation; and iii) inhibiting 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 resulting from the present invention may specifically bind to the same epitope on γc as commercially available antibodies or antigen-binding fragments, or may bind to a different epitope on γc than commercially available antibodies or antigen-binding fragments thereof, and it is variants of any antibody or fragment characterized by one or more of the above properties that constitute part of the present invention.

[0009] The present invention further provides antibodies or antigen-binding fragments that modulate at least one γc-cytokine family member-induced signaling pathway, leading to the suppression, amelioration, arrest, treatment, and / or prevention of at least one autoimmune disease.

[0010] The present invention further provides DNA sequences encoding antibodies or antigen-binding fragments, wherein the DNA sequence encoding CDR-H1, CDR-H2, and CDR-H3 of a heavy chain immunoglobulin or its variable region is set forth in SEQ ID NOs: 1, 17, 33, 49, 65, 81, 97, 113, 129, 145, 161, and / or 177, or a variant thereof, and / or (b) the DNA sequence encoding a light chain immunoglobulin or its variable region, including CDR-L1, CDR-L2, and CDR-L3 of a light chain immunoglobulin or its variable region, is set forth in SEQ ID NOs: 9, 25, 41, 57, 73, 89, 105, 121, 137, 153, 169, and / or 185, or a variant thereof.

[0011] The present invention further provides antibodies or antigen-binding fragments thereof, which comprise: (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 comprising the amino acid sequence set forth in SEQ ID NOs: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162, and / or 178, or a variant 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 comprising the amino acid sequence set forth in SEQ ID NOs: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170, and / or 186, or a variant thereof. In one embodiment of the present invention, the antibody or antigen-binding fragment can exhibit any of the following characteristics: (a) the ability to bind to the same γc epitope as a commercially available γc antibody, (b) the ability to bind to the same γc epitope as a competing γc antibody, or (c) the ability to bind to a different γc epitope as a commercially available γc antibody or a competing γc antibody.

[0012] In one embodiment of the invention, the antigen binding protein comprises (a) a heavy chain immunoglobulin or variable region thereof comprising an amino acid sequence having at least 90% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162 and / or 178, and / or (b) a light chain immunoglobulin or variable region thereof comprising an amino acid sequence having at least 90% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NOs: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170 and / or 186. For example, in one embodiment of the invention, the antigen binding protein comprises: (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 comprising the amino acid sequence set forth in SEQ ID NOs: 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 sequence set forth in SEQ ID NOs: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162 and / or 178; 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 comprising the amino acid sequence set forth 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 sequence set forth in SEQ ID NO: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170 and / or 186;

[0013] In one embodiment of the invention, the antibody or antigen binding protein comprises: (i) a heavy chain set of CDRs and / or (ii) a light chain set of CDRs; (i) Heavy chain set of CDRs: CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO:4, CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO:6, and CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO:8, and / or CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO:20, CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO:22, and CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO:24, and / or CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO:36, CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO:38, and CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO:40, and / or CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO:52, CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO:54, and CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO:56, and / or CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO:68, CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO:70, and CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO:72, and / or CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO:84, CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO:86, and CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO:88, and / or CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO:100, CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO:102, and CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO:104, and / or CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO:116, CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO:118, and CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO:120, and / or CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 132, CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 134, and CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 135.CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 136, and / or CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 148, CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 150, and CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 152, and / or CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 164, CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 166, and CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 168, and / or CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 180, CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 182, and CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 184; (ii) Light chain set of CDRs: CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 12, CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 14, and CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 16, and / or CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 28, CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 30, and CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 32, and / or CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 44, CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 46, and CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 48, and / or CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 60, CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 62, and CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 64, and / or CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 76, CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 78, and CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 80, and / or CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 92, CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 94, and CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 96, and / or CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 108, CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 110, and CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 112, and / or CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 124, CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 126, and CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 128, and / or 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: 143.CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 144, and / or CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 156, CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 158, and CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 160, and / or CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 172, CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 174, and CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 176, and / or CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 188, CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 190, and CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 192.

[0014] In one embodiment of the invention, the antigen binding protein according to the invention comprises the following set of heavy chain CDRs and light chain CDRs: (i) a heavy chain variable region comprising CDR-H1 having the amino acid sequence set forth in SEQ ID NO:4, CDR-H2 having the amino acid sequence set forth in SEQ ID NO:6, and CDR-H3 having the amino acid sequence set forth in SEQ ID NO:8, and a light chain variable region comprising CDR-L1 having the amino acid sequence set forth in SEQ ID NO:12, CDR-L2 having the amino acid sequence set forth in SEQ ID NO:14, and CDR-L3 having the amino acid sequence set forth in SEQ ID NO:16; (ii) a heavy chain variable region comprising CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 20, CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 22, and CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 24, and a light chain variable region comprising CDR-L1 having the amino acid sequence set forth in SEQ ID NO: 28, CDR-L2 having the amino acid sequence set forth in SEQ ID NO: 30, and CDR-L3 having the amino acid sequence set forth in SEQ ID NO: 32; (iii) a heavy chain variable region comprising CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 36, CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 38, and CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 40, and a light chain variable region comprising CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 44, CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 46, and CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 48; (iv) a heavy chain variable region comprising CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 52, CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 54, and CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 56, and a light chain variable region comprising CDR-L1 having the amino acid sequence set forth in SEQ ID NO: 60, CDR-L2 having the amino acid sequence set forth in SEQ ID NO: 62, and CDR-L3 having the amino acid sequence set forth in SEQ ID NO: 64; (v) a heavy chain variable region comprising CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 68, CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 70, and CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 72, and a light chain variable region comprising CDR-L1 having the amino acid sequence set forth in SEQ ID NO: 76, CDR-L2 having the amino acid sequence set forth in SEQ ID NO: 78, and CDR-L3 having the amino acid sequence set forth in SEQ ID NO: 80; (vi) a heavy chain variable region comprising CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 84, CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 86, and CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 88, and a light chain variable region comprising CDR-L1 having the amino acid sequence set forth in SEQ ID NO: 92, CDR-L2 having the amino acid sequence set forth in SEQ ID NO: 94, and CDR-L3 having the amino acid sequence set forth in SEQ ID NO: 96; (vii) a heavy chain variable region comprising CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 100, CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 102, and CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 104, and a light chain variable region comprising CDR-L1 having the amino acid sequence set forth in SEQ ID NO: 108, CDR-L2 having the amino acid sequence set forth in SEQ ID NO: 110, and CDR-L3 having the amino acid sequence set forth in SEQ ID NO: 112; (viii) a heavy chain variable region comprising CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 116, CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 118, and CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 120, and a light chain variable region comprising CDR-L1 having the amino acid sequence set forth in SEQ ID NO: 124, CDR-L2 having the amino acid sequence set forth in SEQ ID NO: 126, and CDR-L3 having the amino acid sequence set forth in SEQ ID NO: 128; (ix) a heavy chain variable region comprising CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 132, CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 134, and CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 136, and a light chain variable region comprising CDR-L1 having the amino acid sequence set forth in SEQ ID NO: 140, CDR-L2 having the amino acid sequence set forth in SEQ ID NO: 142, and CDR-L3 having the amino acid sequence set forth in SEQ ID NO: 144; (x) a heavy chain variable region comprising CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 148, CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 150, and CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 152, and a light chain variable region comprising CDR-L1 having the amino acid sequence set forth in SEQ ID NO: 156, CDR-L2 having the amino acid sequence set forth in SEQ ID NO: 158, and CDR-L3 having the amino acid sequence set forth in SEQ ID NO: 160; (xi) a heavy chain variable region comprising CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 164, CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 166, and CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 168, and a light chain variable region comprising CDR-L1 having the amino acid sequence set forth in SEQ ID NO: 172, CDR-L2 having the amino acid sequence set forth in SEQ ID NO: 174, and CDR-L3 having the amino acid sequence set forth in SEQ ID NO: 176; (xii) a heavy chain variable region comprising CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 180, CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 182, and CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 184, and a light chain variable region comprising CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 188, CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 190, and CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 192.

[0015] Also part of the invention are conjugates comprising an antigen-binding protein according to the invention, said antigen-binding protein binding to a γc polypeptide or an antigen fragment thereof conjugated to a carrier protein (e.g., mouse Fc, 6x His tag (SEQ ID NO:234)).

[0016] The present invention further provides methods for producing and purifying γc polypeptides (γc proteins) conjugated to carrier proteins (e.g., mouse Fc, 6x His tag (SEQ ID NO:234)) using mammalian cell expression systems (e.g., Expi-CHO cells).

[0017] The present invention further provides methods for producing and screening antigen-binding units, including (a) immunizing an animal (e.g., a mouse) by injection (e.g., subcutaneously, intravenously, or intraperitoneally) of purified human γc protein; (b) generating a phage library by randomly ligating a library of antigen-binding single-chain variable fragments (ScFvs) into a phage vector (e.g., pCANTAB5); (c) amplifying and screening the phage library using purified human γc protein or human cells expressing γc (e.g., Ramos B cells); and (d) producing and purifying soluble ScFvs for γc-binding and STAT phosphorylation blocking assays.

[0018] The present invention further provides methods for producing an antigen binding protein (e.g., an antibody or antigen-binding fragment thereof) or an immunoglobulin chain (e.g., VH, VL, HC or LC) thereof, comprising: (a) introducing one or more polynucleotides encoding one or more immunoglobulin chains of said antigen binding protein (or vectors comprising such polynucleotides) into a host cell expression system (e.g., Expi-CHO); (b) culturing the host cells under conditions favoring expression of the polynucleotides; and (c) isolating the antigen binding protein or immunoglobulin chain from the host cells and / or the host cell growth medium. The antigen binding proteins or immunoglobulin chains that are the products of such methods also form part of the present 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.

[0019] The present invention further provides a polypeptide, the polypeptide 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 comprising the amino acid sequence set forth in SEQ ID NOs: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162, and / or 178, or a variant 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 comprising the amino acid sequence set forth in SEQ ID NOs: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170, and / or 186, or a variant thereof; or (c) an amino acid sequence set forth in a member selected from the group consisting of SEQ ID NOs: 1 to 192, or a variant thereof. The present invention further provides polynucleotides encoding one or more polypeptides set forth in SEQ ID NOs: 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) comprising such polynucleotides. In some embodiments, the polypeptides further comprise a signal peptide (e.g., an IL-6 signal peptide) for active secretion from a host cell expression system.

[0020] The present invention further provides host cell expression systems (e.g., Expi-CHO) comprising the antigen binding proteins (e.g., antibodies or antigen-binding fragments thereof), immunoglobulin chains (e.g., VH, VL, HC or LC), polypeptides, polynucleotides or vectors described herein.

[0021] The present invention describes the construction of humanized anti-γc antibodies using the framework patching method (see, e.g., U.S. Patent No. 7,321,026 B2). In some embodiments, framework regions (FRs) derived from a mouse anti-γc antibody or / and antigen-binding protein were replaced with the corresponding FRs of the parent immunoglobulin. In some embodiments, amino acids within the humanized FRs were backmutated to the corresponding amino acids present in the mouse FRs. In some embodiments, framework substitutions were applied to the humanized antibody to increase binding affinity. In some embodiments, CDR sequences within the humanized antibody were altered to improve binding affinity and effector function (e.g., blocking downstream signaling pathways). When administered to human subjects, the resulting humanized antibody should exhibit reduced immunogenicity, enhanced effector function, and prolonged serum half-life. The amino acid sequences of the humanized variable heavy and variable light chains are set forth in SEQ ID NOs: 193-200. The humanized heavy chain FRs are shown in SEQ ID NOs: 221 (FR-H1), 222-223 (FR-H2), 224-225 (FR-H3), and 226 (FR-H4), respectively, and the humanized light chain FRs are shown in SEQ ID NOs: 227-228 (FR-L1), 229 (FR-L2), 230-231 (FR-L3), and 232-233 (FR-L4), respectively.

[0022] The invention further provides a container or injection device (e.g., a vial, syringe, prefilled syringe, or auto-injector) for administration to a subject (e.g., a human or mouse) comprising an antigen binding protein or composition (e.g., a pharmaceutical formulation) described herein.

[0023] The present invention further provides methods of administering an antigen binding protein or composition described herein to a subject (e.g., a human or mouse), the methods comprising, for example, introducing said antigen binding protein or composition into the subject's body (e.g., by subcutaneous, intravenous or intraperitoneal injection). The present invention suggests the possibility of treating a γc or γc cytokine mediated disease or condition (e.g., graft versus host disease (GVHD)) in a subject, comprising administering (e.g., injecting) an effective amount of an antigen binding protein or composition described herein to a subject in need thereof.

[0024] The present invention further provides methods for blocking STAT phosphorylation in γc-cytokine (IL-2, IL-4, IL-7, IL-9, IL-15, and / or IL-21)-induced PBMCs (e.g., T cells, B cells, and / or NK cells) or immune cell lines (e.g., mast cells), methods for blocking γc-cytokine (IL-2, IL-4, IL-7, IL-9, IL-15, and / or IL-21)-induced JAK-STAT (e.g., STAT3, STAT5, STAT6)-mediated intracellular signaling pathways and biological functions (e.g., cytokine and chemokine secretion, cell-mediated cytotoxicity, proliferation, cell survival, cell-cell interactions, differentiation, autoimmunity), and / or methods for reducing the population of CD45+ immune cells, NK cells, T cells, and / or B cells (e.g., excluding neutrophils, eosinophils, granulocytes) in PBMC cultures or in a subject (e.g., human or mouse), comprising administering to a subject an effective amount of a γc-binding protein, or a composition thereof, or a formulation thereof, as described herein. In one embodiment of the present invention, the subject is suffering from a γc or γc cytokine mediated disease or condition, such as GVHD, organ transplant rejection, birdshot chorioretinopathy, MS, uveitis, T1D, AD, RA, SLE, asthma, psoriasis, SS, vitiligo, celiac disease, IBD, AA, mast cell mediated disease, T cell lymphoma, NK cell lymphoma, and / or B cell lymphoma. [Brief explanation of the drawings]

[0025] [Figure 1] Figures 1A and 1B show the binding of various purified anti-γc ScFv clones to purified human γc protein in an ELISA assay. Data are expressed in arbitrary units, measuring the optical density at 450 nm. Figures 1A and 1B show the optical density of various anti-γc ScFv clones to purified human γc protein in an ELISA assay at 30°C (Figure 1A) and 37°C (Figure 1B), respectively. Figure 1C shows representative flow cytometry graphs of p-STAT5 blockade by anti-γc ScFv in HPB-ALL.

[0026] [Figure 2] Figure 2 shows an example of SDS-PAGE to assess the purity and stability of two purified γc-binding proteins. More specifically, Figure 2 shows the results of SDS-PAGE analysis of purified clones of anti-γc antibodies, in which signal peptides 2 and 3 were selected for full-length antibody purification, where HC = immunoglobulin heavy chain, LC = immunoglobulin light chain.

[0027] [Figure 3] Figure 3 shows the ELISA binding of A) SM-05A and B) SM-05E to purified γc protein from humans (Homo sapiens), cynomolgus monkeys (Macaca fascicularis), and chimpanzees (Pan troglodytes) in an ELISA assay. More specifically, Figure 3A shows the binding curve for SM-05A, and Figure 3B shows the binding curve for SM-05E. In each case, ELISA strips were coated with 1 μg / mL of γc protein from each species, and the antibodies were diluted 5-fold from 10 μg / mL to 0.00064 μg / mL, respectively.

[0028] [Figure 4]Figure 1 shows that anti-γc antibody clones selected using flow cytometry analysis and the competitor COMP2022 inhibit STAT phosphorylation induced by (A) IL-2, (B) IL-4, (C) IL-7, (D) IL-9, (E) IL-15, or (F) IL-21 in various cell lines. Cytokine concentrations in all groups were 10 ng / mL. The dose of all antibodies and inhibitors was 33 nM (5 μg / mL).

[0029] [Figure 5] This figure shows the survival of Ramos B cells after two consecutive days of pretreatment with IL-4 or IL-21 and various clones of anti-γc antibodies, the competitor COMP2022, or the JAK3-specific inhibitor ritrecitinib in a proliferation assay in the presence of anti-IgM. More specifically, the data shown in Figure 5 confirm that IL-4 and IL-21 rescue the anti-IgM-induced proliferation defect, while 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. The dose of all antibodies and inhibitors was 33 nM (5 μg / mL). In Figures 5A and 5C, * indicates p<0.05 compared to the indicated IgG control group by Student's t-test (N=5). In Figures 5B and 5D, one-way ANOVA (N=5) shows ***p<0.001, **p<0.01, *p<0.05 compared to the anti-IgM+IgG control group.

[0030] [Figure 6]Figure 6 shows cellular damage of Ramos B cells measured by apoptosis assay (AB) and Western blot (CD) in the presence of anti-IgM. Cells were pretreated for two consecutive days with IL-4 or IL-21 and various clones of anti-γc antibody, the competitor COMP2022, or the JAK3-specific inhibitor ritrecitinib. More specifically, the data shown in Figure 6 confirm that anti-γc antibody inhibits the restorative effects of IL-4 and IL-21 on anti-IgM-induced cell death (Figures 5A and 5B) and the expression of apoptosis / DNA damage markers (Figures 5C and 5D). The concentrations of IL-4 and IL-21 in all groups were 10 ng / mL. The dose of all antibodies and inhibitors was 33 nM (5 μg / mL). N=5.

[0031] [Figure 7] Figure 7 shows the suppression of (A) CD23 and (B) PRDM1 expression by anti-γc antibody clones selected in Ramos B cells after 24 hours of treatment. More specifically, the data shown in Figure 7 confirm that anti-γc antibodies suppressed CD23 (Figure 7A) and PRDM1 (Figure 7B) expression in Ramos B cells after IL-4 or IL-21 stimulation. The concentrations of IL-4 and IL-21 in all groups were 10 ng / mL. The dose of all antibodies and inhibitors was 33 nM (5 μg / mL).

[0032] [Figure 8] Figure 8 shows the survival of Jurkat T cells cultured for 24 to 72 hours in the presence of IL-9 and various anti-γc antibody clones, as measured by (A) proliferation assay and (B) p-ERK expression assay. More specifically, the data shown in Figure 8 confirm that anti-γc antibodies inhibited IL-9-induced ERK phosphorylation. The IL-9 concentration in all groups was 50 ng / mL. All antibodies were administered at 0 μg / mL to 10 μg / mL (5-fold intermediate dilutions).

[0033] [Figure 9]This figure shows HPB-ALL T cell homeostasis measured by measuring (A) BCL-2 expression, (B) CD127 internalization, and (C) CD127 degradation in the presence of IL-7 and various anti-γc antibody clones, the competitor COMP2022, or ritrecitinib (a JAK3-specific inhibitor). More specifically, the data shown in Figure 9 confirm that anti-γc antibodies suppressed IL-7-induced BCL-2 expression (Figure 9A), CD127 internalization (Figures 9B-9C), and CD127 degradation (Figures 9D-9E) in HPB-ALL. The IL-7 concentration in all groups was 50 ng / mL. The dose of all antibodies and inhibitors was 33 nM (5 μg / mL). **p<0.01, *p<0.05 compared to the IgG control. ##p<0.01, #p<0.05 compared to the IL-7 + IgG treatment group (treatment group). Statistical analysis was performed by Student's t-test, N=3-4.

[0034] [Figure 10] Figure 10 shows the establishment of molecular assays to study KHYG-1 homeostasis. Specifically, proliferation (Figure 10A), production of granzyme B (Figure 10B), and perforin (Figure 10C), and active secretion of granzyme A (Figure 10D), granzyme B (Figure 10E), and IFNγ (Figure 10F) were examined in KHYG-1 cultures after 3 days of cytokine stimulation. 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. The dose of all antibodies and inhibitors was 33 nM (5 μg / mL). ***p<0.001, **p<0.01, and *p<0.05 compared to the control. Statistical analysis was performed by Student's t-test. N=3.

[0035] [Figure 11]Figure 11 compares the inhibition of IL-2-, IL-15-, or IL-21-induced (AC) proliferation and (DF) granzyme A secretion in KHYG-1 NK cells after 3 days of treatment with selected anti-γc antibody clones, a commercially available anti-γc antibody, competitor COMP2022, or ritrecitinib (a JAK3-specific inhibitor). More specifically, the data shown in Figure 11 confirm that anti-γc antibodies inhibit KHYG-1 proliferation and granzyme A secretion (Figures 11D-11F) induced by IL-2 (Figure 11A), IL-15 (Figure 11B), and IL-21 (Figure 11C) after 3 days of incubation. IL-2 and IL-15 were administered at 20 ng / mL, and IL-21 at 25 ng / mL. The dose of all antibodies and inhibitors was 33 nM (5 μg / mL). Rit: ritrecitinib.

[0036] [Figure 12] Figure 12 shows the induction of STAT phosphorylation and proliferation by six γc cytokines in PBMC cultures. More specifically, the data shown in Figure 12 include a representative blot of STAT phosphorylation (Figure 12A), the ratio of STAT phosphorylation to the control group (Figure 12B), and the proliferation index in primary PBMC cultures induced with six γc cytokines (Figure 12C). The cytokine concentration in all groups was 10 ng / mL. **p<0.01, *p<0.05 compared to the control. Statistical analysis was performed by Student's t-test. N=3 for PBMCs collected from three independent donors.

[0037] [Figure 13] This figure shows the inhibition of STAT phosphorylation by selected anti-γc antibody clones and the competitor COMP2022 in PBMC cultures using flow cytometry analysis. The concentration of six γc cytokines (IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21) in all groups was 10 ng / mL. The dose of all antibodies and inhibitors was 66 nM (10 μg / mL).

[0038] [Figure 14] Figure 14 shows the proliferative effects of γc cytokines (excluding IL-9) in human PBMC cultures after 3 days of treatment with selected anti-γc antibody clones and the competing antibody COMP2022. B-D) The inhibitory effects of IL-2-, IL-7-, and IL-15-induced proliferation in PBMCs. More specifically, the results shown in Figure 14 show the proliferative effects of γc cytokines (excluding IL-9) in human PBMC cultures after 3 days of treatment with selected anti-γc antibody clones and the competing antibody COMP2022 (Figure 14A) and the inhibitory effects of IL-2- (Figure 14B), IL-7- (Figure 14C), and IL-15- (Figure 14D)-induced proliferation in PBMCs. The cytokine concentration in all groups was 50 ng / mL. The dose of all antibodies and inhibitors was 33 nM (5 μg / mL). **p<0.01, *p<0.05 compared to the IgG control group. Statistical analysis was performed by one-way ANOVA. N = 3–4 for PBMCs from independent donors.

[0039] [Figure 15]Figures 15A-15F show that IL-2, IL-15, and IL-21 induced the secretion of granzyme A and IFNγ from PBMC cultures as activation markers for T cells and NK cells, and that C-F show that IL-2 or IL-15 treatment for 3 days suppressed the secretion of granzyme A and granzyme B by various anti-γc antibody clones. More specifically, the results shown in Figure 15 show that IL-2, IL-15, and IL-21 induced the secretion of granzyme A (Figure 15A) and IFNγ (Figure 15B) as activation markers for T cells and NK cells from PBMC cultures. Figures 15C-15F show that IL-2 or IL-15 treatment for 3 days suppressed the secretion of granzyme A and IFNγ by various anti-γc antibody clones and ritrecitinib. IL-2 and IL-15 were administered at 20 ng / mL, and IL-21 at 25 ng / mL. The dose of all antibodies and inhibitors was 33 nM (5 μg / mL). ***p<0.001, **p<0.01, *p<0.05 compared with the indicated group. Statistical analysis was performed by Student's t-test. A-B) N=3-4 for PBMCs from independent donors.

[0040] [Figure 16] Figure 16 shows the comparison of suppression of FasL expression in IL-2- and IL-15-induced cytotoxic T cells after 3 days of treatment with SM-05A or ritrecitinib. Cytotoxic T cells were first gated via CD3+ and CD8+ channels (Figure 16A), and then gated using the FasL-positive population (Figure 16B). The dose of IL-2 and IL-15 was 20 ng / mL. The dose of all antibodies and inhibitors was 33 nM (5 μg / mL).

[0041] [Figure 17]Figure 17A shows the ELISA binding of humanized (h)SM-05A (Figure 17A) and hSM-05E (Figure 17B) to purified γc proteins from human (Homo sapiens), cynomolgus monkey (Macaca fascicularis), chimpanzee (Pan troglodytes), common marmoset (Callithrix jacchus), rhesus monkey (Macaca mulatta), mouse (Mus musculus), and rabbit (Oryctolagus cuniculus) in an ELISA assay. 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, respectively.

[0042] [Figure 18] Figures 18A and 18B show analytical plots for size-exclusion high-performance liquid chromatography (SEC-HPLC) for A-B) hSM-05A and C-D) hSM-05E at RT and 37°C after 12 days of PBS exchange. More specifically, the data in Figures 18A and 18B confirm that hSM-05A maintained stability and purity (aggregation-free) at RT (Figure 18A) and 37°C (Figure 18B). However, aggregation was observed for hSM-05E after incubation at RT (Figure 18C) and 37°C (Figure 18D). The blue arrows indicate the sharp peaks representing the monoclonal IgG antibody. The purity of hSM-05A was approximately 99.9%, while that of hSM-05E was approximately 80%.

[0043] [Figure 19] FIG. 1 shows representative plots of the binding affinity of humanized antibodies to AB) human γc protein and CD) common marmoset, as measured by Bio-Layer Interferometry (BLI).

[0044] [Figure 20]Figure 1 shows an in vivo binding assay of hSM-05A to γc protein expressed on the surface of human HEK-293 cells. Briefly, hSM-05A was fluorescently labeled with FITC. HEK-293 cells were transfected with plasmids expressing full-length γc proteins from different species for 2 days and then incubated with fluorescent antibodies for flow cytometry analysis. The results showed that hSM-05A could bind to native γc proteins from human and common marmoset. The dose of all antibodies was 33 nM (5 μg / mL). C = untransfected control; Hu: human; Rh: rhesus monkey; Cy: cynomolgus monkey; Ma: common marmoset. N = 3 for all groups.

[0045] [Figure 21] Figure 21 shows the workflow for the competition assay between hSM-05A and hSM-05E for human γc protein expressed on the surface of Ramos B cells. Briefly, hSM-05E was fluorescently labeled with FITC and then incubated with unconjugated hSM-05A in Ramos B cell cultures. Figures 21A and 21B show representative plots and the workflow for the competition assay. The statistical results in Figure 21C indicate that the two antibodies can bind to similar epitopes on human γc protein. Statistical analysis was performed by Student's t-test. N = 3 for all groups. The dose of all antibodies was 33 nM (5 μg / mL).

[0046] [Figure 22]Figure 22 shows the workflow for antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) of hSM-05A and hSM-05E in the human keratinocyte cell line, HaCaT. More specifically, Figure 22A shows the workflow for examining ADCC and CDC activity in HaCaT cells. ADCC was measured using PI to assess cell death under flow cytometry analysis, and CDC was examined in 96-well plates by WST-8 proliferation assay. Quantitative results showed that neither hSM-05A nor hSM-05E induced ADCC (Figure 22B) or CDC (Figure 22C) activity against HaCaT cells after incubation with human PBMCs or a complement mixture. Statistical analysis was performed by Student's t-test. N = 3 for all groups. The dose of all antibodies was 33 nM (5 μg / mL).

[0047] [Figure 23] Figure 23 shows the inhibition of STAT phosphorylation by hSM-05A and hSM-05E in PBMC cultures using flow cytometry analysis. More specifically, the results shown in Figure 23 confirm 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 STAT phosphorylation-induced expression of all six γc cytokines: IL-2 (Figure 23A), IL-7 (Figure 23B), IL-15 (Figure 23C), IL-4 (Figure 23D), and IL-21 (Figure 23E). The cytokine concentration in all groups was 50 ng / mL. Antibodies and inhibitors were diluted 5-fold from 33 nM to 0.0528 nM, respectively.

[0048] [Figure 24]Figure 24 shows the efficacy of hSM-05A and hSM-05E in restoring the B cell tolerance checkpoint in Ramos B cells stimulated with anti-IgM in the presence of IL-4 or IL-21. More specifically, the schematic diagram in Figure 24A shows that hSM-05A and hSM-05E can block IL-4-induced STAT6 phosphorylation and IL-21-induced STAT3 phosphorylation, pushing B cells back down the cell death pathway and suppressing the aberrant survival of autoreactive B cells. Quantitative data shown in Figures 24B and 24C confirm that both hSM-05A and hSM-05E can restimulate anti-IgM-induced cell death at levels comparable to ritrecitinib, as demonstrated by the WST-8 proliferation assay. The percentage of restimulation (restimulation rate) was calculated as (OD anti-IgM / cytokine-OD anti-IgM / cytokine / drug) * 100 / (OD anti-IgM / cytokine-OD anti-IgM). OD refers to the optical density observed in a microplate reader after adding WST-8 reagent. N = 3 for the ritrecitinib-treated group and N = 6 for the other groups. The dose of all antibodies and inhibitors was 33 nM (5 μg / mL).

[0049] [Figure 25]Figure 25 shows the efficacy of hSM-05A and hSM-05E in suppressing IL-7-induced BCL-2 upregulation and CD127 degradation in HPB-ALL T cells. More specifically, the schematic diagram in Figure 25A suggests that hSM-05A and hSM-05E can block IL-7-induced CD127 degradation and BCL2 upregulation by suppressing JAK3 activation. Quantitative data shown in Figures 25B and 25C confirm that both antibodies can suppress two IL-7-induced processes, but the effect was more potent in the hSM-05A group than in the ritrecitinib group. The percentage of inhibition (inhibition rate) was calculated as (PIL-7-PIL-7 / drug) * 100 / (PIL-7-P control). P refers to the percentage of cells showing positive intracellular expression of either BCL-2 or CD127 as measured by flow cytometry. All groups had an N = 3. The dose of all antibodies and inhibitors was 33 nM (5 μg / mL).

[0050] [Figure 26] Figure 26 shows the efficacy of hSM-05A and hSM-05E in inhibiting IL-9-induced ERK phosphorylation in Jurkat T cells. More specifically, the schematic diagram in Figure 26A suggests that hSM-05A and hSM-05E can block IL-9-induced ERK phosphorylation and survival by inhibiting STAT5 phosphorylation. Quantitative data shown in Figure 26B confirm that hSM-05A can significantly inhibit IL-9-induced protection against ceramide-induced cell death. Western blot results shown in Figure 26C demonstrate that hSM-05A can inhibit IL-9-induced ERK phosphorylation, and quantification confirms this observation. N = 2–3 for all samples. The dose of all antibodies was 33 nM (5 μg / mL). Analysis by Student's t-test showed ***p<0.001 and *p<0.05.

[0051] [Figure 27]Figure 27 shows the efficacy of hSM-05A and hSM-05E in suppressing IL-2- and IL-15-induced proliferation and cytotoxic factor secretion in human PBMCs. More specifically, the schematic diagram in Figure 27A suggests that hSM-05A and hSM-05E can block these IL-2- and IL-15-induced phenotypes by inhibiting STAT phosphorylation. Quantitative data shown in Figures 27B and 27C confirm that both antibodies were able to inhibit IL-2- and IL-15-induced proliferation at levels comparable to those of ritrecitinib. Similarly, data shown in Figures 27D-27G demonstrate that both antibodies were able to inhibit IL-2- and IL-15-induced IFNγ and granzyme B secretion, while ritrecitinib completely attenuated their secretion. Percentage of inhibition was calculated as (V cytokine - V cytokine / drug) * 100 / (V cytokine - V control). V refers to the OD value recorded by the microplate reader in either the proliferation experiment (Figures 27B-27C) or the ELISA experiment (Figures 27D-27G). N = 3-4 for all groups. The dose of all antibodies and inhibitors was 33 nM (5 μg / mL).

[0052] [Figure 28]These figures show that hSM-05A and hSM-05E can suppress the surface expression of activated NKp46 driven by IL-2 and IL-15 in human primary NK cells and cytotoxic T cells. More specifically, Figure 28A shows representative plots showing the number of NKp46+ NK cells after cytokine and drug treatment. Quantitative data shown in Figures 28B and 28C confirm the tendency of hSM-05A and hSM-05E to reduce IL-2- or IL-15-driven upregulation of NKp46 in primary NK cells. Similarly, quantitative data shown in Figures 28D and 28E confirm the tendency of hSM-05A and hSM-05E to reduce IL-2- or IL-15-driven upregulation of NKp46 in primary cytotoxic T cells. N=3 for Figures 28D-28E. The dose of all antibodies and inhibitors was 33 nM (5 μg / mL).

[0053] [Figure 29] These figures show that hSM-05A and hSM-05E can suppress the mixed lymphocyte reaction (MLR) in human PBMC cultures. More specifically, the schematic diagram in Figure 29A shows that T cells were isolated from donor 1 and T cells were depleted from donor 2. The isolated T cells were labeled with CFSE and then co-cultured with T cells depleted from donor 2. The number of proliferating (CFSE+) T cells can reflect the GvHD status in the cultures through flow cytometry analysis. Quantitative data shown in Figures 29B-C demonstrate that both hSM-05A and hSM-05E suppress the proliferation of cytotoxic and helper T cells driven by GvHD in the cultures. N=2 for each group.

[0054] [Figure 30] FIG. 1 is a schematic diagram showing how anti-γc antibodies suppress JAK / STAT activity on autoreactive T cells, B cells, and NK cells.

[0055] [Table overview] [Table 1] Heavy chain CDRs in the immunoglobulins of the present invention are shown. [Table 2] Light chain CDRs in the immunoglobulins of the present invention are shown. [Table 3] The heavy and light chain CDRs of each immunoglobulin according to the present invention are shown. [Table 4] shows the DNA sequences encoding the heavy and light chain CDRs of each immunoglobulin according to the present invention. [Table 5] The procedure for immunizing mice with γc protein for antibody production. [Table 6] shows the results of monoclonal phage ELISA for 406 positive clones after 2-3 rounds of panning process. Table 7 shows the inhibition of IL-15-induced STAT phosphorylation by various anti-γc ScFv clones in the KHYG-1 NK cell line using flow cytometry analysis. The dose of all ScFvs was 10 μg / mL. 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 using flow cytometry analysis. All ScFvs were administered at a dose of 10 μg / mL. Table 9 shows the inhibition of IL-7-induced STAT5 phosphorylation by various anti-γc ScFv clones in HPB-ALL T cell lines using flow cytometry analysis. The dose of all ScFvs was 10 μg / mL. Table 10 shows the inhibition of IL-9-induced A) STAT3 phosphorylation by various anti-γc ScFv clones in a Jurkat T cell line using flow cytometry analysis. The dose of all ScFvs was 10 μg / mL. [Table 11] Binding kinetics of humanized anti-γc antibodies to human γc protein. [Table 12] Binding kinetics of humanized anti-γc antibodies to γc proteins from other species. Table 13: Inhibition of STAT phosphorylation by hSM-05A and hSM-05E in PBMC cultures. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION

[0056] The present invention provides antibodies and antigen-binding fragments thereof that specifically bind to human and cynomolgus monkey γc and exhibit modulation of intracellular protein pathways and biological functions, including blocking γc cytokine-induced STAT phosphorylation, suppressing cytokine and enzyme secretion, inhibiting hyperactivation and autoreactivity, and apoptosis of autoreactive cells in T cell, B cell, NK cell, or / and mast cell cultures. The present invention has provided clues for the treatment of GVHD, as suggested by an international poster presentation (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 (identifiers: NCT03532958, NCT05589610).

[0057] The present invention further provides methods for designing, screening, and producing antibodies and antigen-binding fragments through conventional molecular biology, microbiology, and recombinant DNA techniques, phage display technology, and protein purification techniques. These techniques are described in the following publications: Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; DNA Cloning: A Practical Approach, Volumes I and II (DN Glover ed. 1985); Oligonucleotide Synthesis (MJ Gait ed. 1984); Nucleic Acid Hybridization (BD Hames & SJ Transcription And Translation (BD Hames & SJ Higgins, eds. (1984)); Animal Cell Culture (RI Freshney, ed. (1986)); Immobilized Cells And Enzymes (IRL Press, (1986)); B. Perbal, A Practical Guide To Molecular Cloning (1984); FM Ausubel, et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (1994); M. Hust and TS. Lim, Phage Display Methods and Protocols (2017), Humana Press; P. Meleady, Heterologous Protein Production in CHO Cells Methods and Protocols (2017), Humana Press; RKScope, Protein Purification: Principles and Practice, Second Edition (2013), Springer Science & Business Media.

[0058] The common gamma chain (γc), also known as p64, CIDX, IMD4, CD132, SCIDX, IL-2RG, and SCIDX1, is a signaling receptor subunit shared with several interleukin receptor subunits activated by six γc-cytokines, including IL-2R, IL-4R, IL-7R, IL-9R, IL-15R, and IL-21R.

[0059] Receptor dimerization occurs between receptor subunits and γc during induction of the corresponding γc cytokine, triggering the downstream JAK-STAT pathway for biological function. The heterotrimerization of IL-2Rα / IL-2Rβ / γc upon IL-2 stimulation and IL-15Rα / IL-2Rβ / γc upon IL-15 stimulation results in higher binding affinity and stability [Stauber et al., Proc Natl Acad Sci U S A., 2006;103(8):2788-93; Kobayashi et al., Blood., 2005;105(2):721-7.]

[0060] In one embodiment of the invention, human γc is encoded by the nucleotide sequence set forth in Genbank Accession No. NM_000206. In one embodiment of the invention, human γc comprises the amino acid sequence set forth in Genbank Accession No. NP_000197.

[0061] The complete γc protein consists of an extracellular domain, a transmembrane domain, and an intracellular domain [Waickman et al., Cell Mol Life Sci., 2016; 73(2): 253-269.] The extracellular domain is divided into two type III fibronectin domains (D1 and D2), which may form a disulfide-mediated tertiary structure required for interaction with the γc cytokine receptor subunit.

[0062] γc also contains the characteristic juxtamembrane 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 (Box1 and Box2 motifs) for JAK1 / JAK3 activation and downstream signaling events following receptor dimerization. A degradation motif is located adjacent to the Box2 motif for proper membrane localization, ligand-mediated internalization, and lysosomal degradation.

[0063] 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 T cells and CD8+ cytotoxic T cells, activated B cells, and dendritic cells (DCs). IL-4 is secreted by NK-like T cells (NKTs), 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 T helper cell type 9 (TH9), type 2 innate lymphoid cells (ILC2), mast cells, NKT cells, IL-9-producing cytotoxic T cells (TC9), T helper cell type 17 (TH17), and regulatory T cells (Tregs). IL-15 (or membrane-bound IL-15) is secreted or provided by DCs, monocytes, and macrophages. IL-21 is secreted by CD4+ helper T cells, NKT cells, T follicular helper cells (TFH), T17 cells, and gamma delta (γδ) T cells.

[0064] [Functions and pathologies related to γc cytokines] Because γc is widely expressed in the majority of immune cells, these γc cytokines contribute to various biological functions, such as proliferation, differentiation, maturation, activation, and immune function, in different immune cells, as shown in [reviewed in Leonard et al., Immunity., 2019; 50(4):832-850; Overwijk and Schluns, Clin Immunol., 2009; 132(2): 153-165].

[0065] IL-2 is characterized as a typical growth factor for T cells and NK cells. IL-2 is involved in T cell and NK cell proliferation, Treg development, B cell function enhancement, T cell activation-induced cell death (AICD), T cell differentiation (TH1, TH2, and TH9), and suppression of T17 and TfH differentiation. Impaired IL-2 production has been reported in SLE patients, leading to a reduced Treg population, impaired AICD, hyperinflammation, and renal impairment [Lieberman and Tsokos, J Biomed Biotechnol., 2010;2010:740619; Shao et al., J Interferon Cytokine Res., 2019;39(2):117-124.] However, overexpression / increased levels of IL-2 in serum induces preferential proliferation of Tregs [Antony et al., J. Immunol., 2006;176:5255-66.], leading to peripheral tolerance of immune function. Thus, IL-2 is considered to be both a negative and a positive regulator of autoimmunity.

[0066] 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 the differentiation / development of Th2, Th9, and Treg cells, activation of alternative macrophages (M2), and monocyte-mediated DC differentiation [Hiasa, et al., Blood., 2009;114(20):4517-26.] Although the effects of IL-4 deficiency have not been extensively studied in human subjects, animal models of IL-4 deficiency have reported increased Treg cell death, decreased granzyme secretion from Tregs [Yang, et al., Front Immunol., 2017;8:1508], neuronal hyperexcitability [Chen et al., Acta Pharm Sin B., 2020;10(9):1634-1645.], resistance to secondary pulmonary P. aeruginosa infection [Song, et al., J Infect Dis., 2015;211(10):1616-27.], and mechanical hypersensitivity [Uceyler, et al., PLoS One., 2011;6(12):e28205.]. Elevated serum levels of IL-4 have been observed in several autoimmune diseases and are thought to be involved in the progression of AD and allergic diseases, as demonstrated in animal studies [Elbe-Burger et al., J Invest Dermatol., 2002;118(5):767-78; Conde et al., Nat Commun., 2021;12(1):2574.].

[0067] IL-7 was discovered as a stromal factor mediating the development and homeostasis of naive CD4+ memory T cells and Tregs. IL-7 also acts synergistically with IL-15 to mediate CD8+ memory T cell homeostasis in humans. Loss of IL-7 expression in transgenic mice leads to significant T cell deficiency [Freeden-Jeffry et al., J Exp Med., 1995;181:1519-1526; Peschon et al., J Exp Med., 1994;180:1955-1960.]. Abnormal expression of IL-7 also causes T cell-related leukemia in mice [Fisher et al., Leukemia., 1993;2:S66-68.]. Elevated IL-7 levels have been shown in humans and mice to lead to preferential proliferation of CD4+ and CD8+ T cells, increasing the risk of allograft rejection [Schreiber et al., Front Immunol., 2019;10:742.], the development of anti-drug resistant gliomas [Cui et al., Cancer Biol Ther., 2012;13(7):496-503.], increased T cell reactivity to myelin basic protein (MBP) [Traggiai et al., J Neuroimmunol., 2001;121(1-2):111-9.], and the amplification of TH1-driven forms of MS [Lee et al., Sci Transl Med., 2011;3(93):93ra68.].

[0068] The function of IL-9 is relatively unknown compared to other γc cytokines. IL-9 is thought to be involved in T cell differentiation, antitumor effects, mucus production, and mast cell homeostasis. Although IL-9 is not essential for T cell development and immunoglobulin production, its deficiency leads to enhanced T cell 17 responses early in pulmonary infection [Li et al., Front Immunol., 2018;9:1118.], impaired goblet cell hyperplasia and mastocytosis upon pulmonary allergen challenge [Townsend et al., Immunity., 2000;13(4):573-83.], and elevated frequencies of Tregs, activated CD4+, and CD8+ T cells [Vieyra-Garcia et al., Clin Cancer Res., 2016;22(13):3328-39.]. Increased IL-9 expression may promote ulcerative colitis (UC) by impairing intestinal barrier function and inducing the production of proinflammatory cytokines by mucosal mononuclear cells [Bird, Nat Rev Immunol., 2014;14(7):432.], and may also directly induce immune pathology in the lung, as demonstrated in IL-9-overexpressing mice [Temann et al., J Clin Invest., 2002 Jan;109(1):29-39.].

[0069] IL-15 is primarily involved in the homeostasis of CD8+ cytotoxic T cells and NK cells. The interaction between transmembrane IL-15 and IL-15R on monocytes / DCs triggers the development, proliferation, and activation of CD8+ cytotoxic T cells and NK cells, and inhibits T cell differentiation. IL-15-mediated autocrine mechanisms may also 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 cells and CD8+ cytotoxic T cells [Suwanai et al., Proc Natl Acad Sci U S A., 2010;107(20):9305-10; Kennedy et al., J Exp Med., 2000;191(5):771-80.] and increases the susceptibility of mice to breast cancer metastasis by 10-fold [Gillgrass et al., J Immunol., 2014;193(12):6184-91.]. Overexpression of IL-15 can increase antigen-driven memory CD8+ T cells after microbial exposure [Yajima et al., J Immunol., 2002;168(3):1198-203.], promote epithelial damage in patients diagnosed with active celiac disease [Di Sabatino et al., Gut., 2006;55(4):469-477.], and cause large granular lymphocytic leukemia via chromosomal instability and DNA hypermethylation [Mishra et al., Cancer Cell., 2012;22(5):645-55.].

[0070] IL-21 is considered a multifunctional regulator of immunity. IL-21 is involved in enhancing antitumor activity mediated by CD8+ cytotoxic T cells and NK cells, regulating B cell apoptosis, inducing plasma cell differentiation and immunoglobulin production, stimulating T cell 17 and T cell differentiation, suppressing T cell 9 and Treg differentiation, and inhibiting DC maturation and function. IL-21 and / or IL-21R deficiency has been observed in patients with severe primary immunodeficiency syndrome (PID), mimicking common variable immunodeficiency (CVID), resulting in impaired B cell proliferation, immunoglobulin class switching, T cell effector function, and NK cell function [Kotlarz et al., Curr Opin Pediatr., 2014;26(6):704-12.] Overexpression of IL-21 can trigger the proliferation of hematopoietic progenitor cells in the spleen [Ozaki et al., Int J Hematol., 2006;84(3):224-30.], which aberrantly promotes the accumulation of CD8+ memory T cells and simultaneously reduces the number of naive T cells [Allard et al., Eur J Immunol., 2007;37(11):3069-77.], triggers alloimmunization by impairing Treg function [Petrelli et al., Diabetes., 2011;60(12):3223-3234.], and stimulates the proliferation and differentiation of autoreactive B cells in human SLE [Wang et al., Nat Commun., 2018;9(1):1758.].

[0071] [Pathologies associated with genetic mutations and polymorphisms in γc, γc cytokine, and γc cytokine receptor subunits] Hypomorphic mutations in the γc gene are recognized as a cause of XSCID [Lim et al., Allergy Asthma Clin Immunol., 2019;15:2.]. More than 300 unique mutations in the γc gene have been identified in patients with this disease. In typical XSCID, patients exhibit a complete absence of T cells and NK cells, with near-normal or abundant dysfunctional B cells. Infants with typical XSCID are highly susceptible to bacterial infections and usually die within the first year of life. Milder symptoms have been observed in atypical forms of XSCID with atypical phenotypic mutations (e.g., Tlow / -B+NK+ and dTlow / -BlowNK+ / low / -). The γc mutations associated with atypical XSCID disrupt the structure of γc and JAK3 binding to the cytoplasmic domain of γc, resulting in loss of JAK3 phosphorylation and immunodeficiency in patients.

[0072] 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 demonstrated that γc expression is directly associated with pancreatic cancer cell proliferation and poor prognosis in human gastric cancer.

[0073] Genetic polymorphisms in γc cytokines are highly correlated with the progression of human autoimmune diseases, as outlined in a review [Leonard et al., Immunity., 2019; 50(4):832-850.]. IL-2 is correlated with MS, T1D, and IBD. IL-4 is correlated with asthma and AR. IL-7 is correlated with MS. IL-9 is correlated with AR. IL-15 is correlated with celiac disease [Escudero-Hernandez et al., Cytokine., 2017; 99:73-79.]. IL-21 is correlated with SLE, T1D, and IBD.

[0074] Genetic polymorphisms in the γc cytokine receptor subunit are also associated with an increased risk of autoimmune disease [Leonard et al., Immunity., 2019; 50(4):832-850.] IL-2RA gene polymorphisms are associated with T1D, IBD, and MS; IL-4RA gene polymorphisms are associated with asthma and allergies; IL-7R gene polymorphisms are associated with MS and T1D; IL-9R gene polymorphisms are associated with AR; IL-15RA gene polymorphisms are associated with IBD; and IL-21RA gene polymorphisms are associated with SLE. Furthermore, IL2RA gene mutations are associated with inflammation and autoimmunity in patients with loss-of-function mutations; IL7R gene polymorphisms are associated with T, B, NK, SCID in patients with loss-of-function mutations; and IL21RA gene polymorphisms are associated with T and B function defects and various NK cell dysfunctions in patients with loss-of-function mutations.

[0075] Current strategies for the modulation of .GAMMA.c cytokine-mediated diseases Because γc cytokines are highly correlated with the progression of human autoimmune diseases, therapeutic strategies have been developed to regulate or treat γc cytokine-mediated diseases by inhibiting the activity of the γc cytokine family. These strategies include using ligands that share a similar structure to γc cytokines to compete for receptor binding sites, using antagonistic peptides that recognize and block binding sites on γc cytokines, using chemical inhibitors that target the JAK-STAT pathway to block downstream signaling pathways triggered by γc cytokines, using specific monoclonal antibodies against γc cytokines to neutralize the activity of target cytokines in vivo, and using monoclonal antibodies that target individual γc cytokine receptor subunits to selectively inhibit cytokine activity.

[0076] IL-2 muteins (mutated proteins) are engineered IL-2 variants with higher binding affinity to IL-2R and lower binding affinity to γc compared to wild-type human IL-2 (hIL-2). One example is H9-ERTR, which functions as a competitive inhibitor of IL-2 and IL-15. H9-ERTR has been engineered to have higher binding affinity to IL-2Rβ but lower affinity to γc, and can block IL-2- and / or IL-15-induced CD8+ cytotoxic T cell proliferation and NK cytotoxicity in vitro [Mitra et al., Immunity., 2015; 42(5): 826-838.]. In vivo studies have shown that it can reduce graft-versus-host disease and block the proliferation of chronic smoldering adult T-cell leukemia T cells. Despite the high efficacy of IL-2 muteins in animals, there is only one Phase I / II clinical trial (identifier: RPCEC00000234) for advanced solid tumors. Furthermore, no muteins have been developed for the remaining γc cytokines.

[0077] BNZ-1 is a pegylated peptide with enhanced binding affinity for γc receptor subunits but reduced affinity for other γc receptor subunits. It is a selective and simultaneous inhibitor of IL-2 and IL-15-mediated leukemia progression [Wang et al., Leukemia., 2019;33(5):1243-1255.]. Its promising effect in blocking IL-2 and IL-15 function has been tested in a Phase II clinical trial for AA (identifier: NCT03532958) and is currently in Phase II / III clinical trials for cutaneous T-cell lymphoma. These variants, BNZ-2 and BNZ-3, have subsequently been developed to target different combinations of γc cytokines.

[0078] The use of monoclonal antibodies is considered a specific and safer approach to addressing autoimmune diseases. Two monoclonal antibodies have been approved by the FDA for the treatment of autoimmune diseases. Daclizumab can be used to treat renal allograft rejection and MS by inhibiting the binding of IL-2Ra and IL-2R complexes. However, the drug was voluntarily withdrawn from the market as a result of its "complex and evolving benefit / risk profile." Daclizumab also improved symptoms of asthma (identifier: NCT03532958) and partially improved symptoms of uveitis (identifier: NCT00130637).

[0079] Dupilumab is a monoclonal antibody targeting IL-4Ra approved for the treatment of moderate to severe asthma and AD. It can inhibit both IL-4 signaling via the type 1 receptor (γc) and IL-4 and IL-13 signaling via the type 2 receptor. Dupilumab is also being tested in multiple clinical trials for the treatment of AA, AR, and conjunctivitis. Monoclonal antibodies targeting the remaining γc cytokine / receptor subunits are also being investigated in clinical trials. For example, OSE-127, which targets IL-7Ra, is currently being evaluated for symptomatic relief in UC and SS. The anti-IL-9 antibody enokizumab showed beneficial effects in asthma but failed to achieve its endpoint (identifier: NCT00968669). Ordesekimab can directly block IL-15 and is being investigated for the treatment of celiac disease, vitiligo, 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.

[0080] Although cytokine-specific antibodies and blocking peptides have shown beneficial effects in animals, multiple cytokines may cooperate to drive disease progression. Therefore, antibody approaches that neutralize a single γc cytokine or a single γc receptor subunit may be insufficient for the treatment of autoimmune diseases. Despite the development of IL-2 muteins and blocking peptides, their efficacy is limited to blocking the function of IL-2 and IL-15 without evidence that they modulate the activity of other γc cytokines. Alternative therapeutic strategies include the use of small molecules to inhibit key downstream proteins activated by γc cytokines and / or antibodies targeting specific protein receptors involved in disease pathogenesis whose activity and / or abundance are directly regulated by the γc cytokine signaling pathway.

[0081] JAK inhibitors are novel and widely used drugs that suppress downstream JAK1 and JAK3 phosphorylation after γc cytokine stimulation. The use of JAK inhibitors can effectively treat several autoimmune diseases by simultaneously blocking multiple cytokine-triggered signaling pathways. They can be classified into five categories: non-selective, JAK1-selective, JAK2-selective, TYK2-selective, and JAK3-selective (reviewed 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 hematological conditions. However, they are associated with a wide range of adverse effects (AEs), including severe and opportunistic infections.

[0082] Second-generation JAK inhibitors with selectivity for JAK1 (e.g., filgotinib and upadacitinib) and JAK2 (e.g., fedratinib) have been developed and approved over the past decade. These selective inhibitors exhibit a narrow spectrum of activity and offer improved safety profiles. However, patients treated with fedratinib may experience neutropenia and anemia, likely due to the involvement 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, elevated hemoglobin levels have been reported due to the anti-inflammatory effects of selective JAK1 inhibitors combined with the lack of erythropoietin blockade via JAK2 inhibition. JAK1-selective inhibitors also failed to reduce absolute lymphocyte or NK cell counts, likely due to a minor effect on IL-15 signaling [reviewed in Biggioggero et al., Drugs Context., 2019;8:212595.]

[0083] Because γc family cytokines require the common phosphorylation of JAK3 to trigger downstream signaling pathways, a novel discovery of selective JAK3 inhibitors has been proposed. Ritrecitinib is a covalent inhibitor that binds to the catalytic domain of JAK3, rather than to other JAK family members in which the compound's targeted cysteine ​​residue is replaced with a serine residue. Ritrecitinib is believed to provide a more specific method for blocking γc cytokine signaling because no other receptor molecules recruit JAK3 for signaling [Spinell et al., Eur J Immunol., 2021;51(7):1615-1627.] Ritrecitinib has demonstrated superior efficacy and a more favorable side effect profile than other JAK inhibitors in the treatment of autoimmune diseases, although long-term studies are needed to reach a definitive conclusion [Ramirez-Marin and Tosti, Drug Des Devel Ther., 2022;16:363-374.] Ritrecitinib is currently being studied in RA (identifiers: NCT04413617, NCT02969044), IBD (identifiers: NCT05636293, NCT02958865, NCT03395184), AA (identifiers: NCT05549934, NCT04006457, NCT03732807, NCT04517864), and vitiligo (identifier: NCT02974868).

[0084] JAK3-selective approaches are promising and provide clues for future drug development for autoimmune diseases. The present invention provides antibodies and / or antigen-binding fragments that target γc (a receptor that conjugates to JAK3) as a method for treating GVHD and autoimmune diseases in subjects (e.g., mice or humans) affected by either disease. The antibodies and / or antigen-binding fragments are believed to offer numerous advantages over JAK3-selective inhibitors (e.g., complete and specific attenuation of γc-related downstream effects, reduced toxicity, or extended half-life).

[0085] Recently, the anti-γc antibody REGN7257 has been enrolled in a phase I / II clinical trial for aplastic anemia (identifier: NCT04409080), and preclinical data suggest potential for disease indications such as 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 an anti-γc strategy to treat multiple autoimmune diseases.

[0086] γc-binding protein The present invention provides antigen-binding proteins, e.g., antibodies (e.g., humanized antibodies, monoclonal antibodies, and antibodies conjugated with additional therapeutic agents), and antigen-binding fragments thereof, that specifically bind to purified γc protein or antigenic 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 for binding to γc with any of the antigen-binding proteins described herein are also part of the present invention.

[0087] The present invention relates to SEQ ID NOs: 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, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 18 Further provided are any polypeptides and / or antigen binding proteins comprising the amino acid sequence set forth in Tables 26, 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 variants thereof. In some embodiments, the polypeptide is fused to one or more other polypeptides, for example, a human Fc (IgG1, IgG2, IgG3 or IgG4). In some embodiments, the mouse FRs in an anti-γc antibody (e.g., SM-05A, SM-05E) are replaced with the corresponding FRs present in a human antibody for humanization, comprising the amino acid sequence of the variable heavy chain (underlined amino acids: CDRs) shown in SEQ ID NOs: 193-194 and the amino acid sequence of the variable light chain (underlined amino acids: CDRs) shown in SEQ ID NOs: 195-196. In some embodiments, the mouse FRs in an anti-γc antibody (e.g., SM-05A, SM-05E) are replaced with the corresponding FRs present in a human antibody, but some amino acids are backmutated to amino acids present in the mouse variable heavy chain amino acid sequence (underlined amino acids: CDRs) shown in SEQ ID NOs: 197-198 and the variable light chain amino acid sequence (underlined amino acids: CDRs) shown in SEQ ID NOs: 199-200. Variable heavy and light chains from different antibody clones can be swapped to identify optimal humanized antibodies with sustained potency and binding affinity to the γc protein.

[0088] The present invention relates to SEQ ID NOs: 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, 113, 115, 117, 119, 121, 123, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 200, 201, 202, 203, 27, 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.

[0089] As used herein, the term "antibody" and its grammatical equivalents refer to an immunoglobulin molecule that recognizes and specifically binds to a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or any combination thereof, via at least one antigen-binding site, typically located within the variable region of the immunoglobulin molecule. Antibodies consist of four polypeptide chains, i.e., two heavy chains (HC) and two light chains (LC), interconnected 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 include, but are not limited to, mouse, rabbit, camel, primate, chimeric, humanized, and human antibodies. In one embodiment of the present invention, each antibody HC comprises a heavy chain variable region ("HCVR" or "VH") (e.g., SEQ ID NOs: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162, and / or 178, or variants thereof) and a heavy chain constant region (comprising domains CH1, CH2, and CH3). Each antibody light chain (LC) then comprises a light chain variable region ("LCVR" or "VL") (e.g., SEQ ID NOs: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170, and / or 186, or variants thereof) and a light chain constant region (CL). The VH and VL regions are further subdivided into regions of hypervariability called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In certain embodiments of the present invention, the FRs of an antibody (or antigen-binding fragment thereof) are identical to mouse germline sequences or are naturally or artificially modified.In certain embodiments of the present invention, the FRs of the antibody (or antigen-binding fragment thereof) are identical to human germline sequences or are naturally or artificially modified.

[0090] As used herein, the term "antigen-binding portion" or "antigen-binding fragment" of an antibody or antigen-binding protein includes naturally occurring, enzymatically derived, synthetic, or genetically engineered polypeptides or glycoproteins that specifically bind 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 the Fab fragment cleaved with papain), (iv) Fv fragments (VH or VL), and (v) ScFv molecules consisting of a VH fragment and a VL fragment linked by a linker region (e.g., a G4S linker (SEQ ID NO:236)). Other engineered molecules, such as domain-specific antibodies, single-domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies, and small modular immunopharmaceuticals (SMIPs), are also encompassed by the term "antigen-binding fragment" as used herein. In one embodiment of the present 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, SM-05J, SM-05K, or SM-05L.

[0091] The term "heavy chain" when used with respect to antibodies refers to a polypeptide chain of approximately 50-70 kDa, comprising a VH of approximately 120-130 amino acids at its amino-terminal end and a constant region at its carboxyl-terminal end. In some embodiments, the heavy chain constant region is composed of three domains, CH1, CH2, and CH3, with a short, flexible hinge region connecting the CH1 and CH2 domains. The constant region can be one of five different types, termed alpha (α), delta (δ), epsilon (ε), gamma (γ), or mu (μ), based on the amino acid sequence of the heavy chain constant region. These different heavy chains vary in size, with α, δ, and γ containing approximately 450 amino acids, and μ and ε containing approximately 550 amino acids. In one embodiment of the present invention, the γc-binding protein (e.g., antibody or antigen-binding fragment) comprises a heavy chain constant region, which gives rise to the five well-known classes of antibodies: IgA (IgA and IgA2), IgD, IgE, IgG (IgG1, IgG2, IgG3, and IgG4), and IgM, respectively.

[0092] The term "light chain," when used with respect to antibodies, refers to a polypeptide chain of approximately 25 kDa, comprising a VL of about 100 to about 110 or more amino acids at its amino terminal end and a constant region at its carboxyl terminal end. The light chain constant region is composed of one domain, CL. The approximate length of a light chain is 211 to 217 amino acids. Two distinct types exist, called kappa (κ) and lambda (λ), based on the amino acid sequence of the constant domain. The present 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, SM-05J, SM-05K, SM-05L), which are linked to heavy and / or light chain constant domains, e.g., as described above.

[0093] The term "variable domain" or "variable region" refers to a portion of an antibody's light or heavy chain, typically located at the amino terminus of the light or heavy chain, approximately 120-130 amino acids in length in the heavy chain and approximately 100-110 amino acids in the light chain, that is used for binding and specificity for a particular antibody's specific antigen. The sequences of variable domains vary significantly among different antibodies. While sequence variability resides in the CDRs, the FRs of different antibodies are highly similar. The CDRs of the light and heavy chains are primarily responsible for antibody-antigen interactions. In some embodiments, each VH and VL consists of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The numbering of amino acid positions used herein follows 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) 5thed.

[0094] CDR refers to any of the three hypervariable regions (H1, H2, or H3) within the non-framework region of the immunoglobulin (Ig or antibody) VH β-sheet framework, or any of the three hypervariable regions (L1, L2, or L3) within the non-framework region of the antibody VL β-sheet framework. Thus, CDRs are variable region sequences interspersed within the framework region sequences. CDR regions are well known to those skilled in the art and have been defined by various methods / systems. These systems and / or definitions include Kabat, Chothia, IMGT, AbM, and Contact, and have been developed and refined over the years. For example, Kabat defines the most hypervariable regions within antibody variable domains (V regions) as those with the highest hypervariability [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 structural loop regions and defines CDR region sequences as residues that are not part of the conserved β-sheet framework and can therefore adopt different conformations [Chothia and Lesk, Journal of Molecular Biology, Vol. 196, 4, 1987; 901-17]. Both terms are widely recognized in the art. Furthermore, 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 an analysis of available antibody crystal structures. Software programs (eg, abYsis) for analyzing antibody sequences and determining CDRs are available and known to those of skill in the art.The positions of CDRs within standard antibody variable domains have been determined by comparison of 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 hypervariable regions varies among different antibodies, it is customary to number additional residues relative to the standard position as a, b, c, etc., following the residue number in the standard variable domain numbering scheme. Such nomenclature is familiar to those skilled in the art.

[0095] In one embodiment of the present invention, the antigen-binding protein (e.g., an antibody or antigen-binding fragment thereof) according to the present invention comprises a heavy chain immunoglobulin comprising a VH comprising heavy chain CDRs (CDR-H1, CDR-H2, and CDR-H3). CDRs are defined in the present invention according to the Kabat (hypervariable) nomenclature. The composition of the antigen-binding protein is shown in Table 1 below.

[0096] In one embodiment of the present invention, the antigen-binding protein (e.g., an antibody or antigen-binding fragment thereof) according to the present invention comprises a light chain immunoglobulin comprising a VL comprising light chain CDRs (CDR-L1, CDR-L2, and CDR-L3). The CDRs are defined according to the Kabat (hypervariable) nomenclature in the present invention. The composition of the antigen-binding protein is shown in Table 2 below.

[0097] [Table 1] CDRs are defined according to the nomenclature of Kabat et al. (supra). *Numbers correspond to the amino acid sequence shown in that SEQ ID NO.

[0098] [Table 2] CDRs are defined according to the nomenclature of Kabat et al. (supra). *Numbers correspond to the amino acid sequence shown in that SEQ ID NO.

[0099] In one embodiment of the invention, an antigen-binding protein (e.g., an antibody or antigen-binding fragment thereof) according to the invention comprises heavy and light chain immunoglobulins each comprising a VH (e.g., HC) and a VL (e.g., LC), and comprises a combination of heavy and light chain CDRs (CDR-H1, CDR-H2 and CDR-H3, and CDR-L1, CDR-L2 and CDR-L3) as shown in Table 3 below.

[0100] [Table 3] CDRs are defined according to the nomenclature of Kabat et al. (supra). *Numbers correspond to the amino acid sequence shown in that SEQ ID NO.

[0101] The present invention includes antigen binding proteins (e.g., antibodies or antigen-binding fragments thereof) comprising a polypeptide pair comprising the following VH and VL amino acid sequences: (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.

[0102] The present invention includes polynucleotides comprising the following set of polynucleotides encoding CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, the DNA sequence organization of which is shown in Table 4 below.

[0103] [Table 4] *Numbers correspond to the amino acid sequence shown in that SEQ ID NO.

[0104] The present invention includes monoclonal anti-γc antigen-binding proteins (e.g., antibodies and antigen-binding fragments thereof), as well as 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 antibody population generated from a single clone (monoclon). That is, the antibody molecules comprising the population are identical in amino acid sequence, except for possible minor naturally occurring mutations. A "plurality" of such monoclonal antibodies and fragments in a composition refers to a concentration of identical (i.e., identical in amino acid sequence, except for possible minor naturally occurring mutations, as explained above) antibodies and fragments that exceeds the concentration of antibodies and fragments normally present in nature (e.g., in the blood of a host organism such as a mouse or human).

[0105] In some embodiments, the anti-γc antigen binding proteins provided herein are chimeric antibodies (e.g., antibodies comprising murine variable regions (VH and VL) and human constant regions (CH and CL)). In some embodiments, the anti-γc antigen binding proteins provided herein are humanized antibodies (e.g., antibodies comprising murine 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, FR-L4), and human constant regions (CH and CL). In some embodiments, amino acids in the humanized FRs are backmutated to amino acids from the original murine FRs. In some embodiments, amino acids within the CDRs are randomly mutated (e.g., from arginine to glutamic acid) to increase binding affinity. As used herein, the term "chimeric antibody" refers to an antibody having a variable region derived from a first antibody and a constant region derived from a second antibody derived from a different species than the first antibody (see, e.g., U.S. Pat. No. 4,816,567). As used herein, the term "humanized antibody" refers to an antibody in which variable region segments of human-derived amino acid residues are interspersed with foreign-derived amino acid segments (e.g., murine CDRs), and the humanized variable heavy and variable light chain domains are linked to human-derived heavy and light chain constant regions set forth in SEQ ID NOs:201-202 or SEQ ID NOs:203-204 [Meyler's Side Effects of Drugs (Sixteenth Edition), 2016]. Methods for producing humanized antibodies are well known in the art. The present invention uses framework repair to produce humanized versions (humanized forms) of anti-γc antigen-binding proteins (see, e.g., U.S. Pat. No. 7,321,026 B2). The resulting full-length humanized antibodies are designated hSM-05A and hSM-05E.

[0106] In some embodiments, the anti-γc antigen-binding protein provided herein is a murine ScFv fragment. As used herein, an "ScFv fragment" is a fusion protein of the variable regions of immunoglobulin VH and VL linked by a short linker peptide of 10 to approximately 25 amino acids (e.g., a G4S linker (SEQ ID NO: 236)). ScFv provides a convenient method for prescreening potential candidate substances with high binding affinity to γc protein and blocking activity against STAT phosphorylation.

[0107] The term "epitope" (also referred to as "antigenic determinant") is used interchangeably herein and refers to a site on the surface of a target molecule to which an antibody or antigen-binding fragment binds, e.g., a localized region on the surface of an antigen. Target molecules can 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 a target molecule with antigenic activity is a 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 is not necessarily immunogenic. Epitopes are often composed of chemically active surface groupings of molecules, such as amino acids or sugar side chains, and have specific three-dimensional structural characteristics and specific charge characteristics. The term "epitope" includes linear epitopes and conformational epitopes. The region of a target molecule (e.g., a polypeptide) that constitutes an epitope can be contiguous amino acids of the polypeptide, or the epitope can be linked from two or more noncontiguous regions of the target molecule. Epitopes formed by contiguous amino acids (also called linear epitopes) are typically retained upon protein denaturation, whereas epitopes formed by tertiary folding (also called conformational epitopes) are typically lost upon protein denaturation. Epitopes typically contain at least three amino acids, more commonly at least five, six, seven, or eight to ten amino acids, in a unique spatial conformation.

[0108] Methods for determining epitopes of antigen-binding proteins (such as antibodies, fragments, or polypeptides) include alanine scanning mutation analysis, peptide blot analysis [Reineke, Methods Mol. Biol., 2004;248:443-63], peptide truncation analysis, crystallography, and NMR analysis. In addition, methods such as epitope excision, epitope extraction, and chemical modification of antigens can also be used [Tomer, Prot. Sci., 2000;9:487-496]. Other methods include hydrogen / deuterium exchange with mass spectrometry detection [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 epitope analysis based on mass spectrometry and ultraviolet photodissociation [Mehaffey et al., Anal Chem., 2020;92(17):11869-11878.].

[0109] As used herein, the term "specific binding" refers to a situation in which a polypeptide or molecule interacts with an epitope, protein, or target molecule more frequently, more rapidly, for longer, with higher affinity, or a combination of these effects, than with other substances, including related and unrelated proteins. Binding moieties (e.g., antibodies) that specifically bind target molecules (e.g., antigens) can be identified, for example, by immunoassays, ELISA, Bio-Layer Interferometry (BLI), SPR (e.g., Biacore), or other techniques known to those skilled in the art. Typically, a specific response will be at least twice the background signal or noise and may be 10-fold higher than background. For a discussion of antibody specificity, see, for example, Paul, ed., 1989, Fundamental Immunology Second Edition, Raven Press, New York at pages 332-336. In some embodiments, a binding moiety that specifically binds to a target molecule can bind to the target molecule with an affinity that is at least 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold higher than the affinity for a different molecule. In some embodiments, a binding moiety that specifically binds to a particular target molecule binds to a different molecule with such low affinity that binding is undetectable using the assays described herein or other assays known in the art. In some embodiments, "specific binding" means, for example, that the binding moiety binds to a molecular target with a KD of about 0.1 mM or less. In some embodiments, "specific binding" means that a polypeptide or molecule binds to a target with a KD of about 10 μM or less or about 1 μM or less. In some embodiments, "specific binding" means that a polypeptide or molecule binds to a target with a KD of about 0.1 μM or less, about 0.01 μM or less, or about 1 nM or less. Due to sequence identity between homologous proteins in different species, specific binding can include polypeptides or molecules that recognize proteins or targets from more than one species.Similarly, specific binding can include polypeptides or molecules that recognize more than one protein or target due to homology in certain regions of the polypeptide sequences of different proteins. It should be understood that in some embodiments, a binding moiety (e.g., an antibody) that specifically binds to a first target may or may not specifically bind to a second target. Thus, "specific binding" does not necessarily require (although may include) exclusive binding, i.e., binding to a single target. Thus, in some embodiments, a binding moiety (e.g., an antibody) can specifically bind to more than one target.

[0110] As used herein, the term "binding affinity" generally refers to the strength of the sum of non-covalent interactions between a binding moiety (e.g., an antibody) and a target molecule (e.g., an antigen). Binding between a binding moiety and a target molecule is a reversible process, and the affinity of binding is usually reported as the equilibrium dissociation constant (KD). KD is the ratio of the dissociation rate (koff or kd) to the association rate (kon or ka). The lower the KD of a binding pair, the higher the affinity. KA is the equilibrium association constant, which is the reciprocal of the equilibrium dissociation constant, i.e., = 1 / KD. For antibody-antigen interactions, KD 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] × [antibody] / [antigen-antibody].

[0111] Various methods for measuring binding affinity are known in the art, any of which can be used for purposes of the present disclosure. Specific exemplary embodiments include the following: In some embodiments, the "KD" or "KD value" can be measured by an assay known in the art, such as a binding assay. The KD can be measured by a radiolabeled antigen binding assay (RIA) (Chen, Y et al. Journal of molecular biology vol. 293,4 (1999): 865-81). The KD or KD value can also be measured using biolayer interferometry (BLI), for example, using the Gator system (Probe Life) or the Octet-96 system (Sartorius, Gottingen, Germany). The KD or KD value can also be measured using a surface plasmon resonance assay using a BIAcore system (e.g., Pharmacia Biosensor AB, Uppsala, Sweden, Piscataway, NJ).

[0112] As used herein, the term "variant" in reference to a protein or polypeptide having specific sequence characteristics ("reference protein" or "reference polypeptide") refers to a different protein or polypeptide having one or more amino acid substitutions, deletions, and / or additions compared to the reference protein or polypeptide. The amino acid sequence changes may be amino acid substitutions. The amino acid sequence changes may be conservative amino acid substitutions. A functional fragment or functional variant of a protein or polypeptide maintains the basic structure and functional properties of the reference protein or polypeptide.

[0113] The terms "polypeptide," "peptide," and "protein," and their grammatical equivalents, used interchangeably herein, refer to polymers of amino acids of any length, which may be linear or branched, which may contain unnatural amino acids, modified amino acids, or may be interrupted by non-amino acids. A polypeptide, peptide, or protein may also be modified, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or other manipulations or modifications.

[0114] The terms "polynucleotide," "nucleic acid," and their grammatical equivalents, used interchangeably herein, refer to polymers of nucleotides of any length, including DNA and RNA. The nucleotides may be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase. Nucleic acid molecules can be single- or double-stranded.

[0115] As used herein, the term "encode" and its grammatical equivalents refer to the inherent property of a particular nucleotide sequence in a polynucleotide or nucleic acid (such as a gene, cDNA, or mRNA) to serve as a template for the synthesis of a particular nucleotide sequence (i.e., rRNA, tRNA, and mRNA) or other polymer or macromolecule having a particular amino acid sequence in a biological process, and the resulting biological properties. Thus, a gene encodes a protein if a protein is produced by transcription and translation of the mRNA corresponding to the gene. Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all mutually degenerate versions of nucleotide sequences that encode the same amino acid sequence. Protein- and RNA-encoding nucleotide sequences can include introns.

[0116] An "isolated" polypeptide, peptide, protein, antibody, polynucleotide, vector, cell, or composition is a polypeptide, peptide, protein, antibody, polynucleotide, vector, cell, or composition in a form not found in nature. Isolated polypeptides, peptides, proteins, antibodies, polynucleotides, vectors, cells, or compositions also include those that have been purified to the point that they are no longer in a form found in nature. In some embodiments, an isolated polypeptide, peptide, protein, antibody, polynucleotide, vector, cell, or composition is in a substantially pure state. In some embodiments, an isolated polypeptide, peptide, protein, antibody, polynucleotide, vector, cell, or composition is substantially free of other cellular material and / or chemicals.

[0117] The terms "identical," "identity," "percent identity," and their grammatical equivalents, as used herein in the context of two or more polynucleotides or polypeptides, refer to two or more sequences or subsequences that are identical, or that have a specified percentage of identical nucleotides or amino acid residues, when compared and aligned (with gaps inserted, if necessary) for maximum correspondence, where any conservative amino acid substitutions are not considered part of the sequence identity. Percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software that can be used to align amino acid or nucleotide sequences 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, two polynucleotides or polypeptides provided herein are substantially identical, meaning they share 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 for maximum correspondence, as determined using a sequence comparison algorithm or by visual inspection. In some embodiments, identity exists over a region of amino acid sequence that is at least about 10 residues, at least about 20 residues, at least about 40-60 residues, at least about 60-80 residues in length, or any integer value therebetween. In some embodiments, identity exists over a region longer than 60-80 residues (e.g., at least about 80-100 residues), and in some embodiments, the sequences are substantially identical over the entire length of the sequences being compared (e.g., the coding regions of the target proteins or antibodies).In some embodiments, identity exists over a region of nucleotide sequence that is at least about 10 bases, at least about 20 bases, at least about 40-60 bases, at least about 60-80 bases in length, or any integer value therebetween. In some embodiments, identity exists over a region longer than 60-80 bases (e.g., at least about 80-1000 or more bases), and in some embodiments, the sequences are substantially identical over the entire length of the sequences being compared (e.g., nucleotide sequences encoding proteins of interest).

[0118] As used herein, the term "conservative amino acid substitution" refers to an amino acid substitution in which one amino acid residue is replaced with another amino acid residue having a similar side chain. As used herein, "conservatively similar" amino acids or residues refer to non-identical amino acid residues with similar side chains. Families of amino acid residues with similar side chains have been 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).

[0119] The present invention includes antigen binding proteins that compete for binding between an antigen binding protein of the invention (e.g., SM-05A, SM-05B, SM-05C, SM-05D, SM-05E, SM-05F, SM-05G, SM-05H, SM-05I, SM-05J, SM-05K, SM-05L) and γc (e.g., a mutant γc epitope as described herein). As used herein, the term "compete" and grammatical equivalents refer to an antigen binding protein (e.g., an antibody or antigen-binding fragment thereof) that binds to an antigen (e.g., γc) and inhibits or blocks the binding of another antigen binding protein (e.g., an antibody or antigen-binding fragment thereof) to the antigen. Unless otherwise specified, the term also includes competition between two antigen binding proteins (e.g., antibodies) in two directions, i.e., a first antibody binds to an antigen and blocks the binding of a second antibody, and vice versa. Thus, in one embodiment of the present invention, competition occurs in one direction as described above. In certain embodiments, the first and second antigen-binding proteins (e.g., antibodies) can bind to the same epitope. Alternatively, the first and second antigen-binding proteins (e.g., antibodies) can bind to different, but e.g., overlapping or non-overlapping, epitopes, in which case binding of one of the antigen-binding proteins inhibits or blocks binding of the second antibody, e.g., through steric hindrance or conformational changes. Competition between antigen-binding proteins (e.g., antibodies) can be determined by methods known in the art, e.g., competitive standard enzyme-linked immunosorbent assay (ELISA) assays. Competition can also be measured by real-time, label-free biolayer interferometry assays using an Octet RED384 biosensor.

[0120] Typically, antibodies or antigen-binding fragments of the present invention, modified in some way, retain their specific binding affinity for γc, e.g., retain at least 10% of the γc-binding activity (compared to the parent antibody) when activity is expressed on a molar basis. Preferably, antibodies or antigen-binding fragments of the present invention retain at least 20%, 50%, 70%, 80%, 90%, 95%, or 100% or more of the γc-binding affinity of the parent antibody. It is also contemplated that antibodies or antigen-binding fragments of the present invention may include conservative or non-conservative amino acid substitutions (referred to as "conservative variants" or "function-conservative variants" of antibodies) that do not substantially alter their normal biological function (e.g., blockade of the γc cytokine-induced signaling pathway).

[0121] A "variant" of a polypeptide, e.g., an immunoglobulin chain (e.g., SM-05A, SM-05B, SM-05C, SM-05D, SM-05E, SM-05F, SM-05G, SM-05H, SM-05I, SM-05J, SM-05K, SM-05L, or a VH, VL, HC or LC comprising an amino acid sequence specifically described herein, or a CDR thereof), may be compared to a reference amino acid sequence described herein (e.g., SEQ ID NO: 1) when the comparison is performed by the BLAST algorithm. 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, 8 8, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 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) refers to a polypeptide comprising an amino acid sequence that is at least 70 to 99.9% (e.g., 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 to any of

[0122] Further, variants of a polypeptide can include, for example, a polypeptide that is an immunoglobulin chain (e.g., SM-05A, SM-05B, SM-05C, SM-05D, SM-05E, SM-05F, SM-05G, SM-05H, SM-05I, SM-05J, SM-05K, SM-05L, or a VH, VL, HC, or LC or CDR thereof), and can include the amino acid sequence of a reference polypeptide, which amino acid sequence is specifically described herein, but which has been modified by one or more (e.g., one, two, three, four, five, six, seven, eight, nine, or ten) modifications, such as one or more missense mutations (e.g., conservative substitutions), nonsense mutations, deletions, or insertions. In one embodiment of the present invention, the γc-binding protein includes an immunoglobulin heavy chain variant comprising CDR-H1, CDR-H2 and CDR-H3, wherein one or more (e.g., one or two or three) such CDRs have one or more such modifications (e.g., conservative substitutions), and / or an immunoglobulin light chain variant comprising CDR-L1, CDR-L2 and CDR-L3, wherein one or more (e.g., one or two or three) such CDRs have one or more such mutations (e.g., conservative substitutions).

[0123] Generation of a phage display library and production of human γc-binding proteins Phage display libraries are an advanced technology for generating and screening antigen-specific monoclonal antibodies beyond traditional hybridoma technology. After inserting a foreign polynucleotide (e.g., an anti-γc ScFv sequence) into a vector encoding a filamentous phage coat protein gene (e.g., pCANTAB5), the protein fragment of interest is "displayed" on the surface of the filamentous phage (e.g., M13 bacteriophage). The resulting engineered phage can be used to select γc-binding protein fragments with high affinity for γc protein.

[0124] As used herein, the term "vector" and its grammatical equivalents refer to a vehicle used to carry genetic material (e.g., a polynucleotide sequence) that can be introduced into a host cell and replicated and / or expressed therein. Applicable vectors include, for example, expression vectors, plasmids, phagemids, viral vectors, episomes, and artificial chromosomes, and can contain selection sequences or markers that enable stable integration into a host cell chromosome. Furthermore, a vector can contain one or more selection marker genes and appropriate expression control sequences. Selection marker genes can, for example, confer resistance to antibiotics or toxins, complement auxotrophic deficiencies, or supply important nutrients not contained in the culture medium. Expression control sequences include constitutive and inducible promoters, transcription enhancers, transcription terminators, and the like, all of which are well known in the art. When two or more polynucleotides are to be expressed simultaneously, the two polynucleotides can be inserted, for example, into a single expression vector or into separate expression vectors. For single vector expression, the encoding polynucleotides can be operatively linked to a common expression control sequence or to different expression control sequences (e.g., one inducible promoter and one constitutive promoter). Introduction of the polynucleotide into the host cell can be confirmed using methods well known in the art. One skilled in the art will understand that the polynucleotide will be expressed in an amount sufficient to produce the desired product (e.g., IgG containing γc-binding protein), and will also understand that expression levels can be optimized to obtain sufficient expression using methods well known in the art.

[0125] As used herein, the term "host cell" refers to a cell into which genetic material, such as a recombinant expression vector, can be introduced or has been introduced. Host cells include not only the subject cell into which exogenous genetic material has been introduced, but also the progeny of such a cell. Such progeny may not be identical to the parent cell because certain modifications may occur in successive generations due to mutations or environmental influences.

[0126] Eukaryotic and prokaryotic host cells, including mammalian cells, can be used as hosts for expressing γc-binding proteins (e.g., antibodies or 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, among others, Chinese hamster ovary (CHO) cells, mouse myeloma cells (NS0), SP2 cells, HeLa cells, baby 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 cell lines derived from various mammalian species. Other cell lines that can be used include insect cell lines (e.g., Spodoptera frugiperda or Trichoplusia ni), amphibian cells, bacterial cells (e.g., TOP10 E. coli, TG1 E. coli, HB2151 E. coli), plant cells, and fungal cells.Fungal cells include yeast and filamentous fungal cells, such as Pichia, Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia minuta (Ogataea minuta, Pichia lindnen), Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, and the like. methanolica, Pichia sp., Saccharomyces cerevisiae, Saccharomyces sp., Hansenula polymorpha, Kluyveromyces sp., Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fusarium sp. sp.), Fusarium gramineum, Fusarium venenatum, Physcomitrella patens, and Neurospora crassa.The present invention includes isolated host cells (e.g., Expi-CHO cells or any type of host cell described above) comprising polynucleotides (e.g., as described above) encoding antigen binding proteins, their VH, VL, HC, LC or CDRs (or variants thereof), e.g., SM-05A, SM-05B, SM-05C, SM-05D, SM-05E, SM-05F, SM-05G, SM-05H, SM-05I, SM-05J, SM-05K, SM-05L, and / or one or more immunoglobulin chains thereof.

[0127] Human γc protein (extracellular domain) with a mouse Fc tag, as 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 a physiological pH, the purified γc protein was buffer-exchanged into PBS using an Amicon Ultra-15 10k centrifugal filter. The resulting protein was diluted to 1 mg / mL in PBS for storage.

[0128] Mice were immunized with purified human γc protein as shown in Table 5. ELISA titrations of test bleeds were performed during the immunization steps to assess successful antibody generation in the animals.

[0129] [Table 5]

[0130] Total RNA was isolated from the spleens of mice at the end of immunization. All VH and VL fragments were identified and cloned using degenerate primers. All VH and VL fragments and linker regions (e.g., G4S linker (SEQ ID NO: 236)) were randomly combined to generate an ScFv library. To prepare ScFv genes, DNA fragments encoding the VH and VL domains are operatively linked to another fragment encoding a flexible linker (e.g., encoding the amino acid sequence (Gly4-Ser)3 (SEQ ID NO:237)), such that the VH and VL sequences are expressed as a contiguous single-chain protein with the VL and VH domains connected by the flexible linker [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].

[0131] The ScFv library was ligated into a phage vector (e.g., pCANTAB5), which was then transfected into bacterial host cells (e.g., TOP10-competent E. coli) to amplify a phage library containing all combinations of ScFv fragments. To select phages exhibiting strong binding affinity to human γc protein, the phage library was screened in 96-well plates with gradually decreasing concentrations of human γc protein. After two to three rounds of selection, each phage was cultured in a single clone format, and the binding affinity of each clone was evaluated by monoclonal phage ELISA, as shown in Table 6. Clones exhibiting OD values ​​five times higher than background (i.e., 0.3) were selected and subjected to DNA sequencing. The final DNA and amino acid sequences are shown in SEQ ID NOs: 1-192.

[0132] [Table 6] JPEG2026507610000007.jpg217170JPEG2026507610000008.jpg217170JPEG2026507610000009.jpg217170JPEG2026507610000010.jpg242166

[0133] Soluble ScFvs were produced using E. coli HB2151 strain after transfection of each host with phagemids. Bacterial periplasmic extracts containing soluble ScFvs were harvested by sonication and purified using Capto™. After measuring the concentration of the crude ScFv extract, the binding affinity of the ScFvs to human γc protein was assessed by ELISA using an antibody targeting the E-Tag on the ScFv. The results are summarized in Figure 1A-B.

[0134] To screen the potency of anti-γc ScFvs, several cell lines were preincubated with different anti-γc ScFvs (20 μg / mL) for 1 hour and then stimulated with individual cytokines (50 ng / mL) for 15 minutes. Cells were harvested, stained with FITC-labeled (conjugated) anti-pSTAT6 antibody, PE-labeled anti-pSTAT3 antibody, or APC-labeled anti-pSTAT5 antibody, and subjected to flow cytometry analysis. In some embodiments, purified ScFvs can block IL-4-induced STAT6 phosphorylation in cell cultures (e.g., Ramos). In some embodiments, purified ScFvs can block IL-7-induced STAT5 phosphorylation in cell cultures (e.g., HPB-ALL). In some embodiments, purified ScFvs 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 embodiments, purified ScFv can block IL-21-induced STAT3 phosphorylation in cell cultures (e.g., Ramos). Results are summarized in Tables 7-10. Representative flow cytometry graphs of p-STAT5 blockade by anti-γc ScFv in HPB-ALL are shown in Figure 1C.

[0135] [Table 7]

[0136] [Table 8a]

[0137] [Table 8b]

[0138] [Table 9]

[0139] [Table 10]

[0140] Methods for producing antibodies are well known in the art, see, for example, Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988) and 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.

[0141] In some embodiments, γc-binding proteins that can be used in the methods provided herein are recombinant, i.e., engineered or isolated. In some embodiments, γc-binding proteins disclosed herein can be prepared, for example, by introducing a recombinant expression vector into a host cell, by using a recombinant combinatorial human antibody library, by using antibodies isolated from animals (e.g., mice) transgenic for human immunoglobulin genes [Taylor, LD et al. Nucleic acids research vol. 20,23 (1992): 6287-95], or by using antibodies prepared, expressed, produced, or isolated by splicing human immunoglobulin gene sequences into other DNA sequences.

[0142] 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 a γc-binding protein, one or more recombinant expression vectors carrying immunoglobulin light and heavy chain DNA fragments encoding the γc-binding protein are introduced into the host cells, such that the light and heavy chains are expressed in the host cells, preferably secreted into the culture medium of the host cells, from which the γc-binding protein can be recovered. Standard recombinant DNA techniques are used to obtain the heavy and light chain genes of γc-binding proteins, incorporate these genes into recombinant expression vectors, and introduce the vectors into host cells, such as those described in Sambrook, Fritsch, and Maniais (eds.), Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, NY, (1989), Ausubel et al. (eds.) Current Protocols in Molecular Biology, Greene Publishing Associates, (1989), and U.S. Pat. No. 4,816,397.

[0143] To express a recombinant γc-binding protein, DNA fragments encoding the light and heavy chain variable regions are first obtained. These DNAs can be obtained by using the polymerase chain reaction (PCR) to amplify and modify hybridomas of the light and heavy chain variable sequences of a mouse antibody, or by oligo synthesis based on amino acid sequences encoding the light and heavy chain variable sequences designed using standard methods known to those skilled in the art. The coding DNA sequences can be further optimized to facilitate mammalian expression of the resulting antibody.

[0144] The VH and VL fragments of murine antibodies can be further mutated to encode humanized antibodies using framework repair methods (see, eg, US Pat. No. 7,321,026 B2).

[0145] After DNA fragments encoding γc-binding protein VH and VL fragments are obtained (e.g., by amplification and mutagenesis of the VH and VL genes from the original mouse as described above), these DNA fragments can be further manipulated by standard recombinant DNA techniques, e.g., to convert the variable region genes into full-length antibody chain genes or Fab fragment genes. In these manipulations, the VL- and VH-encoding DNA fragments are operatively linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker. As used herein, the term "operatively linked" means that the two DNA fragments are joined such that the amino acid sequences encoded by the two DNA fragments remain in frame.

[0146] 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 heavy chain constant regions (CH1, CH2, and CH3). Human heavy chain constant region gene sequences are known in the art (see, e.g., 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 region may be an IgG1, IgG2, IgG3, Ig4, IgA, IgE, IgM, or IgD constant region, with an IgG1 or IgG4 constant region being most preferred. In the case of a Fab fragment heavy chain gene, the VH-encoding DNA can be operatively linked to another DNA molecule encoding only the heavy chain CH1 constant region.

[0147] 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, e.g., 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 light chain constant region can be a kappa constant region or a lambda constant region, with a kappa constant region being most preferred.

[0148] To express a 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) so that the genes are operably linked to transcriptional and translational control sequences. As used herein, the term "operably linked" means that an antibody gene is ligated into a vector so that transcriptional and translational control sequences within the vector perform their intended function of controlling the transcription and translation of the antibody gene. The expression vector and expression control sequences are selected to be compatible with the expression host cells (e.g., Expi-CHO cells) used. The antibody light and heavy chain genes can be inserted into separate vectors (e.g., pEGFP-N1), or more typically, the two genes can be inserted into the same expression vector (e.g., pKS1). The antibody gene is inserted into the expression vector by standard methods (e.g., by ligating complementary restriction enzyme cleavage sites on the antibody gene fragment and the vector, or by blunt-end ligation if complementary restriction enzyme cleavage sites are not present). In some embodiments, prior to insertion of the protein light or heavy chain sequences, the expression vector already contains sequences encoding the heavy or light chain constant region, respectively, with the VH segment operably linked to the CH segment within the vector and the VL segment operably linked to the CL segment within the vector. Additionally, or alternatively, the recombinant expression vector can encode a signal peptide that facilitates secretion of the antibody chain from a host cell. The antibody chain gene can be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the antibody chain gene. The signal peptide can be either an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).

[0149] After transfection of 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 purified using MabSelect™ PrismA. To achieve greater stability and longer shelf life, the antibody is further exchanged into PBS as a storage buffer using an Amicon Ultra-15 10k centrifugal filter. If necessary, the mouse constant region is replaced with the desired human constant region (e.g., wild-type or modified IgG1 or IgG4) to generate a fully human anti-γc antibody. While the constant region selected will vary depending on the specific application, the high-affinity antigen-binding and target-specific characteristics reside in the variable region. In some specific examples, fully human anti-γc antibodies are isolated directly from antigen-positive B cells of the subject (e.g., mouse or human).

[0150] Pharmaceutical Composition Also provided herein are pharmaceutical compositions comprising γc-binding proteins that can be used in the methods of the present invention. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of a γc-binding protein of the present invention and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition can be used to treat immune cell lymphoma 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 mouse).

[0151] The amount of therapeutic γc-binding protein that can be combined with a carrier material in a pharmaceutical composition of the present invention can vary. In some embodiments, the amount of γc-binding protein present in the pharmaceutical composition is an amount that produces a therapeutic effect. Generally, this amount will range from about 0.01% to about 99%, about 0.1% to about 70%, or about 1% to about 30% of the active ingredient combined with a pharmaceutically acceptable carrier, based on a total amount of 100%.

[0152] Pharmaceutical compositions of the present invention comprise a γc-binding protein of the present invention. The γc-binding protein can be present at various concentrations. In some embodiments, pharmaceutical compositions of the present invention comprise a soluble γc-binding protein of the present invention at 1 to 1000 mg / mL. In some embodiments, pharmaceutical compositions of the present invention comprise a soluble γc-binding protein of the present invention at 10 to 500 mg / mL, 10 to 400 mg / mL, 10 to 300 mg / mL, 10 to 200 mg / mL, 10 to 100 mg / mL, 20 to 100 mg / mL, or 50 to 100 mg / mL. In some embodiments, pharmaceutical compositions of the present invention comprise a γc-binding protein of the present invention at 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. Dosages can be readily adjusted by one of skill in the art, e.g., as reduced purity necessitates increased dosages.

[0153] The pharmaceutical compositions of the present invention may be in a variety of forms. These forms include, for example, liquid, semi-solid, and solid dosage forms, such as liquid solutions (e.g., injectable or infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes, and suppositories. The preferred form depends on the intended mode 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).

[0154] Proper fluidity can be maintained, for example, by the use of coating materials (e.g., lecithin), by maintaining the desired particle size in the case of dispersions, and by the use of surfactants. In some embodiments, the pharmaceutical compositions of the present invention are in the form of an injectable or infusible solution. In some embodiments, the pharmaceutical composition is an aqueous formulation. Such formulations are typically solutions or suspensions, but can also include colloids, dispersions, emulsions, and multiphase materials. The term "aqueous formulation" is defined as a formulation containing at least 50% water by weight. Similarly, the term "aqueous solution" is defined as a solution containing at least 50% water by weight, and the term "aqueous suspension" is defined as a suspension containing at least 50% water by weight. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable for high drug concentration.

[0155] In some embodiments, the pharmaceutical compositions described herein are lyophilized, and the physician or patient adds solvents and / or diluents prior to use.

[0156] The pharmaceutical compositions provided herein can contain a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers include any and all physiologically acceptable solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonicity agents, and absorption delaying agents. Examples include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, and combinations thereof. In some embodiments, the pharmaceutically acceptable carrier includes an isotonicity agent, such as a sugar, a polyhydric alcohol (e.g., mannitol, sorbitol), or sodium chloride, in the composition.

[0157] In some embodiments, a pharmaceutically acceptable carrier further comprises minor amounts of auxiliary substances, such as wetting or emulsifying agents, preservatives, or buffers, which enhance the shelf life or effectiveness 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., administration by injection or infusion). Depending on the route of administration, the active ingredient (i.e., γc-binding protein) may be coated with a material to protect it from acids and other natural conditions that may inactivate the active ingredient.

[0158] Further provided herein is a kit for preparing a pharmaceutical composition comprising the γc-binding protein. In some embodiments, the kit comprises a γc-binding protein described herein and a pharmaceutically acceptable carrier in one or more containers. In another embodiment, the kit can comprise a γc-binding protein described herein for administration to a subject. In a specific embodiment, the kit includes instructions for preparing and / or administering the γc-binding protein.

[0159] In some embodiments, a pharmaceutical composition or formulation of the present invention comprises (a) a γc-binding protein disclosed herein, (b) a buffering agent, (c) a stabilizer, (d) a salt, (e) a bulking agent, and / or (f) a surfactant. In some embodiments, the pharmaceutical composition or formulation is 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 composition or formulation is stable when stored at 4°C, 25°C, or 40°C. In some embodiments, pharmaceutical compositions or formulations that improve the stability of a γc-binding protein and allow for long-term storage are further provided. The pharmaceutical compositions disclosed herein can further comprise one or more preservatives, tonicity agents, chelating agents, stabilizers, and / or surfactants, as well as various combinations thereof. The use of preservatives, tonicity agents, chelating agents, stabilizers, and surfactants in pharmaceutical compositions is well known to those of skill in the art. See Remington: The Science and Practice of Pharmacy (19th ed., 1995).

[0160] Buffers useful in the pharmaceutical compositions or formulations disclosed herein may be weak acids or weak bases used to maintain the acidity (pH) of a solution near a predetermined value after the addition of another acid or base. A suitable buffer can maximize the stability of a pharmaceutical formulation by maintaining pH control of the formulation. A suitable buffer can also ensure physiological compatibility or optimize solubility. Rheology, viscosity, and other properties may also depend on the pH of the formulation. Common buffers include, but are not limited to, histidine, citrate, succinate, acetate, and phosphate. In some embodiments, the buffer comprises histidine (e.g., L-histidine) and a tonicity agent, optionally with an acid or base known in the art for pH adjustment. In some embodiments, the buffer is L-histidine. In some embodiments, the pH of the formulation is maintained between about 2 and about 10, or between about 4 and about 8.

[0161] To stabilize pharmaceutical products, stabilizers are added to stabilize the product. Such pharmaceuticals can stabilize proteins in a variety of ways. Common 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), cyclodextrins and dextrans of any type and molecular weight, or PEG. In some embodiments, the stabilizer is selected to maximize the stability of the FIX polypeptide in the lyophilized formulation. In certain embodiments, the stabilizer is sucrose and / or arginine.

[0162] Fillers can be added to pharmaceutical compositions or dosage forms to increase the volume and mass of the product and facilitate accurate measuring and handling. Common fillers include, but are not limited to, lactose, sucrose, glucose, mannitol, sorbitol, calcium carbonate, or magnesium stearate.

[0163] Surfactants are amphiphilic substances with lyophilic and lyophobic groups. Surfactants may 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), cocamide MEA, cocamide DEA, polysorbates, or dodecyldimethylamine oxide. In some embodiments, the surfactant is polysorbate 20 or polysorbate 80.

[0164] The pharmaceutical compositions disclosed herein may further comprise a pharmaceutically acceptable antioxidant. 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 ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, etc., and (3) metal chelators, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[0165] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the presence of microorganisms can be ensured by the addition of various antibacterial and antifungal agents, such as parabens, chlorobutanol, and phenolsorbic acid, in addition to the sterilization procedures described above. It may also be desirable to include isotonic agents, such as sugars and sodium chloride, in the compositions. Furthermore, delayed absorption of the injectable pharmaceutical form can be achieved by incorporating agents that delay absorption, such as aluminum monostearate and gelatin.

[0166] Pharmaceutical compositions or formulations must usually be sterile and stable under the conditions of manufacture and storage. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Sterile injectable solutions can be prepared by mixing the required amount of a therapeutic antibody or antigen-binding fragment with an appropriate solvent containing one or a combination of the ingredients listed above, as needed, followed by filtered sterilization. The use of such vehicles and agents for pharmaceutically active substances is known in the art. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and any other desired ingredients listed above. For sterile powders used to prepare sterile injectable solutions, some methods of preparation are vacuum drying and freeze-drying (lyophilization), which produce a powder of the active ingredient and any additional desired ingredients from a previously sterile-filtered solution.

[0167] The pharmaceutical compositions disclosed herein can be prepared using carriers that will protect the active ingredient against rapid release, such as controlled-release formulations, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable and biocompatible polymers that can be used include ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for preparing such formulations are patented or generally known to those skilled in the art. See, for example, "Sustained and Controlled Release Drug Delivery Systems," edited by J.R. Robinson, Marcel Dekker, Inc., New York, 1978.

[0168] Anti-γc Protein Treatment and Administration In one embodiment of the present invention, the anti-γc antibodies or / and γc binding protein fragments can be used to treat a subject suffering from a γc or γc cytokine mediated disease or condition, such as GVHD, organ transplant rejection, Birdshott chorioretinopathy, MS, uveitis, T1D, AD, RA, SLE, asthma, psoriasis, SS, vitiligo, celiac disease, IBD, AA, mast cell mediated disease, T cell lymphoma, NK cell lymphoma, and / or B cell lymphoma.

[0169] Graft-versus-host disease (GVHD) refers to an autoimmune disease that can occur after allogeneic transplantation. For example, in GVHD, donated bone marrow or peripheral blood stem cells perceive the recipient as a foreign body, and the donated cells or bone marrow can attack the recipient. GVHD can occur, for example, in patients with hematopoietic cell transplantation (HCT, e.g., acute myeloid leukemia (AML) or acute lymphoblastic leukemia (ALL), and / or myelodysplastic syndrome or myeloproliferative neoplasm), blood transfusion, thymus transplant, or thymoma.

[0170] Graft-versus-host disease (GVHD) is less likely to occur when the donor and recipient are closely matched. For example, if the donor and recipient are related, the chance of GVHD occurring is approximately 35%-45%, but if the donor and recipient are unrelated, the chance is approximately 60%-80%.

[0171] Types of GVHD include steroid-resistant GVHD, acute GVHD, and chronic GVHD. Acute GVHD usually develops within a few days or at the latest within six months after transplantation, whereas chronic GVHD usually develops more than three months after transplantation and can persist lifelong. The overall mortality and non-relapse mortality rates within one year from the date of acute GVHD diagnosis are 35.2% and 25.5%, respectively, while patients with chronic GVHD tend to have oral and skin changes at the time of relapse [Holtan et al. Bone Marrow Transplant., 2022;57,1581-1585].

[0172] Symptoms of acute GVHD include a rash with burning and reddening of the skin, nausea, vomiting, abdominal cramps, loss of appetite, and diarrhea, jaundice (yellowing of the skin and / or eyes), and / or increased eye dryness / irritation.

[0173] Symptoms of chronic GVHD include: very dry mouth; sensitivity to hot, cold, spicy, and acidic foods, as well as to mints and carbonated drinks; painful mouth ulcers that may spread down the throat; difficulty eating; gum disease and tooth decay; rash; dry, tight, itchy skin; changes in skin color such as thickened or tight skin; intolerance to temperature changes due to sweat gland damage; changes in nail texture; hardened and brittle nails; nail loss or loss; hair loss on the head; premature graying of hair; thinning or loss of body hair; loss of appetite; unexplained weight loss; nausea, vomiting, diarrhea, stomach pain, shortness of breath and difficulty breathing; a persistent and chronic cough that does not subside; wheezing; abdominal distension; jaundice; abnormal liver function test results; muscle weakness and cramps; joint stiffness in the fingers, wrists, elbows, knees, and / or ankles that makes it difficult to fully extend them; and / or irritated genitals in both men and women.

[0174] Common treatments for acute GVHD include intensified immunosuppression in the form of corticosteroids and the application of JAK inhibitors (e.g., ruxolitinib). Patients with mild chronic GVHD are usually monitored closely or treated with topical steroid ointments. Furthermore, recent studies [Le Floc'h et al., Sci Transl Med., 2023;15(678):eabo0205; Le Floc'h et al., Hemasphere. 2022;6(Suppl ):694-695] have also recommended the use of anti-γc binding proteins as a treatment option.

[0175] AA is a disease that occurs when the immune system attacks hair follicles, causing hair loss. The cause of AA is still unknown, and both genetic and environmental factors (e.g., stress) are thought to play a role. People with certain autoimmune diseases, such as psoriasis, thyroid disease, and vitiligo, are also more likely to develop AA.

[0176] There are three main types of AA: patchy alopecia areata (hair loss occurring in one or more coin-sized patches on the scalp or other parts of the body), alopecia totalis (complete or near-complete loss of hair on the scalp), and alopecia universalis (complete or near-complete loss of hair on the scalp, face, and rest of the body).

[0177] Symptoms of AA generally include the sudden appearance of round or oval patches of hair loss on the scalp or other parts of the body (e.g., whisker area, eyebrows, or eyelashes in men), a tingling, burning, or itching sensation in the skin patch just before hair loss, and / or changes in the nails (e.g., bumps or pits). While AA is not a life-threatening condition, the psychological burden of AA may lead to self-harm, psychiatric disorders, and increased mortality associated with smoking-related malignancies [Lee et al., JAMA Dermatol. 2019;155(8):922-928].

[0178] Common treatments for AA include steroid injections, topical steroids (creams and ointments) and tablets, immunotherapy, dithranol cream (dichlorohydroxyanthracene cream), UV light therapy, and the use of minoxidil. Furthermore, recent clinical trials (identifiers: NCT03532958, NCT05589610) have also recommended the use of anti-γc binding protein as a treatment.

[0179] The present invention includes approaches for treating or preventing such autoimmune diseases (of any type) in cell models or in subjects (e.g., mice) comprising administering to the subject a therapeutically effective amount of anti-γc protein.

[0180] 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 to receive a particular treatment. The subject may be a human. The subject may be a patient suffering from a particular disease.

[0181] As used herein, the term "treatment" and its grammatical equivalents in relation to a disease or condition (disorder), or a subject suffering from a disease or condition, refer to the act of suppressing, eliminating, reducing, and / or ameliorating the symptoms, symptom severity, and / or symptom frequency associated with the disease or condition being treated. For example, when used in relation to GVHD, the term "treatment" and its grammatical equivalents refer to the act of reducing the severity of the disease or delaying or slowing the progression of the disease, including, but not limited to, (a) the absence of secondary systemic therapy, or (b) the absence of non-relapse mortality, or (c) the absence of recurrent or progressive malignancies [Martin et al., Blood., 2017; 130(3): 360-367].

[0182] The term "blocking" and its grammatical equivalents refer to the action of reducing the biological function of an alarmin, including, but not limited to, (a) directly competing for the binding site of an alarmin to its corresponding receptor, or (b) preventing heterodimerization of the corresponding receptor, thereby reducing the biological effect induced by alarmin occupancy.

[0183] As used herein, the term "administering" and its grammatical equivalents refer to the act of delivering or causing to be delivered a therapeutic agent or pharmaceutical composition into a subject, by methods described herein or other methods known in the art. The therapeutic agent may be a compound, polypeptide, or cell. Administering a therapeutic agent or pharmaceutical composition includes formulating the therapeutic agent or pharmaceutical composition for delivery into the subject. Exemplary administration forms include oral dosage forms (e.g., tablets, capsules, syrups, suspensions), injectable dosage forms (e.g., intravenous (IV) injections, intramuscular (IM) injections, intraperitoneal (IP) injections), subcutaneous (SC), transdermal dosage forms (e.g., creams, jellies, powders, or patches), buccal dosage forms, inhalation powders, sprays, suspensions, and rectal suppositories.

[0184] As used herein, the terms "effective amount," "therapeutically effective amount," and their grammatical equivalents refer to the administration of an agent, alone or as part of a pharmaceutical composition, in an amount capable of producing a detectable positive effect on the symptoms, aspects, or characteristics of a disease, disorder, or condition, either in a single dose or as part of a series of doses, to a subject when administered to the subject. A therapeutically effective amount can be determined by measuring the relevant physiological effect. The precise amount required will vary from subject to subject, depending on the subject's age, weight, and general condition, the severity of the condition being treated, the clinician's judgment, and the like. A therapeutically effective amount is also an amount in which any toxic or adverse effects of the therapeutic agent outweigh any therapeutically beneficial effects. In any individual case, an appropriate "effective amount" may vary depending on factors such as the individual's condition, age, sex, and weight, and can be determined by one of ordinary skill in the art using routine experimentation. A "prophylactically effective amount" refers to an amount effective, at the dosages and for the duration required, to achieve a desired prophylactic effect, such as delaying or preventing the onset of a disease or disorder. Typically, since a prophylactically effective amount is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will usually be less than the therapeutically effective amount.

[0185] As used herein and understood in the art, the term "EC" refers to the effective concentration of a drug (e.g., an antibody) and is commonly used in dose-response curves. The "effect" of a drug can be either a positive (activating) effect (positive effect) or a negative effect (negative effect). The term "EC50" refers to the concentration of an active substance (drug) (e.g., an antibody) that produces half of the maximum response. Also, as used herein and understood in the art, the term "IC" refers to the concentration of a drug that has an inhibitory effect and is commonly used in dose-response curves. The term "IC50" refers to the concentration that reduces the activity inhibited by a drug (e.g., an antibody) by half.

[0186] An effective or therapeutically effective amount of an anti-γc protein (e.g., an antibody or antigen-binding fragment) for treating or preventing a γc-mediated disease or condition refers to an amount of antigen-binding protein sufficient to alleviate one or more signs and / or symptoms of the disease or condition in a treated subject, by inducing regression or elimination of such signs and / or symptoms, or by inhibiting the progression of such signs and / or symptoms. In one embodiment of the present invention, the effective or therapeutically effective amount of anti-γc protein is approximately 0.05 to 50 mg / kg body weight. The dosage may vary depending on the age and size of the subject, the target disease, condition, route of administration, etc. In certain embodiments, the initial dose may be followed by a second or multiple subsequent doses of the antigen binding protein, which may be about the same as, less than, or greater than the initial dose, and the subsequent doses are spaced apart by at least 1 to 3 days, at least 1 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.

[0187] Prevention of a γc-mediated disease or condition, as it relates to the use of an anti-γc protein according to the present invention, refers to administration to a subject prior to the onset of the disease or condition in the subject's body, thereby preventing such onset from occurring.

[0188] [Immune complex] The present invention encompasses anti-γc proteins (e.g., antibodies or antigen-binding fragments) conjugated to another moiety, such as a therapeutic moiety ("immunoconjugate"). In one embodiment of the present invention, the anti-γc protein (e.g., antibodies or antigen-binding fragments) is conjugated to any of the other therapeutic agents described herein. As used herein, the term "immunoconjugate" refers to an antigen-binding protein (e.g., antibodies or antigen-binding fragments) chemically or biologically linked to another antigen-binding protein, a drug, a radioactive substance, a reporter molecule, an enzyme, a peptide, a protein, or a therapeutic agent.

[0189] [Effects and properties of anti-γc protein] The anti-γ c proteins described herein (chimeric or humanized forms), for example, contain variant immunoglobulin chains and have the following properties: binds to human γc (e.g., a fusion such as a 6x His tag (SEQ ID NO:234), or mouse Fc) at 25°C with KD values ​​shown in Table 11; Binding to common marmoset γc (e.g., fusion with a 6x His tag (SEQ ID NO: 234)) at 25°C with the KD values ​​shown in Table 12; partially binds to mouse γc (e.g., fusion with a 6x His tag (SEQ ID NO:234) or the like) at 25°C with the KD values ​​shown in Table 12; partially binds to rat γc (e.g., fusion with a 6x His tag (SEQ ID NO:234) or the like) at 25°C with the KD values ​​shown in Table 12; does not bind to cynomolgus γc (e.g., fusion with a 6x His tag (SEQ ID NO:234) or the like) at 25°C with the KD values ​​shown in Table 12; Maintain high structural stability at 37°C for up to two weeks. lack of ADCC and CDC activity against γc-expressing cell lines; suppressing six γc cytokine-induced STAT phosphorylation in cell lines, including NK cell cultures (e.g., the human NK cell line KHYG-1), B cell cultures (e.g., the human B cell line Ramos), and T cell cultures (e.g., the human T cell lines HPB-ALL and Jurkat), as measured, for example, by luciferase expression in cells containing a luciferase gene operatively linked to a STAT3 / STAT5 / STAT6 response element (response element) or by endogenous expression of phosphorylated STAT3 / STAT5 / STAT6 protein; Blocking STAT phosphorylation in six γc cytokine (except IL-9)-induced primary PBMC cultures; Blocking IL-4 and IL-21 rescue against anti-IgM-induced cell death and injury in B cell cultures (e.g., the human B cell line Ramos); Blocking IL-4 and IL-21-induced CD23 and PRDM1 expression in B cell cultures (e.g., the human B cell line Ramos); inhibiting cell proliferation in IL-2, IL-7, IL-15, or IL-21-induced human immune cells (e.g., PBMC cultures, primary T cells, primary NK cells, KHYG-1 cells); Suppression of the expression of BCL-2, a survival marker in IL-7-induced HPB-ALL T cells. inhibiting the internalization and degradation of CD127 in IL-7-induced HPB-ALL T cells; Attenuating IL-9-induced Jurkat T cell protection and proliferation in the presence of C2-ceramide Inhibiting ERK phosphorylation in IL-9-induced Jurkat T cells suppressing the secretion of soluble factors (e.g., proinflammatory cytokines and cytotoxic enzymes) from IL-2, IL-15, or IL-21-induced NK cell or PBMC cultures; suppressing the expression of surface activation receptors of cytotoxic T cells and NK cells in PBMC cultures; Attenuating primary T cell-induced MLR in allogeneic grafts; binding to the same epitope of human γc (e.g., on a γc-binding protein coated on an ELISA strip) as any one or more of the γc-binding proteins described herein; Competing for binding to human γc (e.g., on a γc-binding protein coated on an ELISA strip) as any one or more of the γc-binding proteins described herein. One or more of the following characteristics may be exhibited:

[0190] [Example] Experimental Example The following examples are put forward so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the methods and compositions of the present invention, and are not intended to limit the scope of what the inventors regard as their invention.

[0191] Example 1: Development and production of anti-γc antibodies Anti-γc antibodies were obtained from a phage display library after immunization of animals with the human γc protein (Sequence ID: NP_000197.1) shown in SEQ ID NO: 205, which consists of the extracellular domain of human γc protein (amino acids 1-262) linked by three repeats of a G4S linker (SEQ ID NO: 236) (amino acids 263-277) and a mouse Fc tag (underlined amino acids 278-509). Alternatively, anti-γc antibodies were obtained from a phage display library after immunization of animals with the human γc protein (SEQ ID NO: 206), which consists 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).

[0192] First, we constructed anti-γc ScFv using the HB2151 bacterial host cell system and evaluated the specificity and inhibitory properties of the anti-γc antibody. We used an ELISA method to evaluate the binding activity of the constructed anti-γc ScFv with human γc protein. Briefly, human γc protein produced in-house was diluted to 1 μg / mL in PBS, and 100 μL of γc protein was added to each well of an ELISA strip. The strip was sealed with parafilm and incubated overnight at 4°C for coating. The next day, the strip was washed three times with washing buffer (0.05% Tween 20 in PBS) and blocked with blocking buffer (3% BSA in PBS) at room temperature for 2 hours at 100 μL / well, followed by the addition of 10 μg / mL of anti-γc ScFv. After 2 hours of incubation at room temperature, the amount of bound anti-γc ScFv was determined at OD 450 nM by adding peroxidase-conjugated goat anti-human F(ab')2-specific antibody (Jackson ImmunoResearch, West Grove, USA) and TMB substrate (Sigma-Aldrich, St. Louis, USA) according to standard ELISA protocols known to those skilled in the art. The results show that all of the purified anti-γc ScFv bound to human-derived γc protein at 25°C and 37°C when compared with PBS and non-transfected TG1 controls (Figure 1).

[0193] Full-length chimeric anti-γc antibodies were produced using Expi-CHO host cells and purified using standardized methods. Each antibody consists of four peptides: two immunoglobulin heavy chains and two immunoglobulin light chains linked to form a "Y"-shaped molecule. Each heavy chain consists of a VH chain connected to a heavy chain constant region, and each light chain consists of a VL chain connected to a light chain constant region. 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 peptide 1 provided superior production yields. The stability of the purified antibodies was measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using a 10% non-reducing gel, demonstrating that all purified antibodies, except for SM-05E, remained intact (Figure 2).

[0194] Example 2: Flow cytometry analysis of p-STAT using anti-γc ScFv in multiple cell lines After cytokine induction and anti-γc ScFv treatment, the percentage of p-STAT-expressing cells was measured by flow cytometry. KHYG-1 (Creation BioArray, Shirley, NY11967, USA) cells were starved for 2 days in RPMI 1640 (ATCC modification, ThermoFisher Science, Waltham, MA, USA) containing 10% FBS (ThermoFisher Science) but without IL-2. Ramos, HPB-ALL, and Jurkat cell lines were used directly without starvation. Cells were replenished with fresh RPMI 1640 medium and pretreated with anti-γc ScFv (10 μg / mL) for 1 hour. After treatment 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 min, cells were fixed with 4% paraformaldehyde (PFA, Sigma, St. Louis, Missouri, USA) for 10 min, permeabilized with 0.1% Triton X-100 (Sigma) for 10 min, and then stained with FITC-conjugated anti-pSTAT6, PE-conjugated anti-pSTAT3, or APC-conjugated anti-pSTAT5 (all diluted 1:100, Biolegend, San Diego, California, USA) for 30 min at 37°C. The percentage of p-STAT-expressing cells was determined using a BD FACSLyric™ clinical flow cytometry system (BD Biosciences, New Jersey, USA). Data were analyzed using Flowjo (version 10, BD).

[0195] Different cell lines were tested. KHYG-1 is an NK cell line derived 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™) that 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 lymphoblastoid cell line (clone E6-1, TIB-152™) that has been widely used in T cell studies of IL-9 function. HPB-ALL is a T-cell leukemia model established in peripheral blood from a 14-year-old Japanese boy diagnosed with ALLL and thymoma in 1973, and can be used to study IL-7 function.

[0196] The results showed that four clones (SM-05A, SM-05E, and SM-05F) potentially suppressed the highest combination of γc cytokines that mediate p-STAT expression, thereby promoting the production of full-length antibodies (Tables 7-10).

[0197] [Example 3] EC50 of purified full-length anti-γc antibody Selected full-length antibody clones were evaluated by ELISA to confirm their binding specificity. The binding activity of the full-length anti-γc antibodies was investigated using γc proteins (extracellular domains) from human (SEQ ID NOs: 205-206), cynomolgus monkey (SEQ ID NOs: XP_005593949.1, SEQ ID NOs: 207-208), and chimpanzee (SEQ ID NOs: XP_008971810.1, SEQ ID NOs: 209-210). The γc proteins were purchased from Sino Biological Company or prepared in-house. The results showed that the anti-γc antibodies bound to human and chimpanzee γc proteins in a dose-responsive manner with similar affinities, but showed lower or no binding affinity to cynomolgus monkey γc protein (Figure 3).

[0198] [Example 4] Flow cytometry analysis of p-STAT using whole anti-γc antibody in multiple cell lines After cytokine induction and anti-γc antibody treatment, the percentage of p-STAT-expressing cells was measured by flow cytometry as described previously. IL-2 and IL-15 were tested in the KHYG-1 NK cell line, IL-4 and IL-21 in the Ramos B cell line, IL-7 in the HPB-ALL T cell line, and IL-9 in the Jurkat T cell line.

[0199] Both SM-05A and SM-05E were comparable in their potency in blocking STAT phosphorylation induced by IL-2 in KHYG-1 NK cells (Fig. 4A), IL-4 in Ramos B cells (Fig. 4B), IL-7 in HPB-ALL T cells (Fig. 4C), IL-9 in Jurkat T cells (Fig. 4D), IL-15 in KHYG-1 NK cells (Fig. 4E), and IL-21 in Ramos cells (Fig. 4F), and were comparable in potency to the competing antibody COMP2022 (sequence from WO2020160242A1). SM-05F did not show a strong inhibitory effect on STAT phosphorylation induced by the six γc cytokines.

[0200] Example 5: Bioassay for B cell research Ramos can serve as a model for B cell antigen receptor (BCR) activation, where cross-linking of the BCR with anti-IgM antibodies leads to cell cycle arrest and induction of apoptosis.

[0201] The effect of anti-γc antibodies in blocking IL-4 and IL-21 rescue of Ramos cell death following anti-IgM-induced hyperactivation was studied using a WST-8 proliferation assay. Briefly, Ramos cells (50,000 cells in 100 μL) were seeded into 96-well plates and stabilized at 37°C for 1 hour. Cells were pretreated with anti-γc antibodies or ritrecitinib for 1 hour and then incubated 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.

[0202] WST-8 can be reduced by cellular dehydrogenases to produce an orange formazan product, and the amount of formazan produced is directly proportional to the number of living Ramos cells. The optical density (OD) of each well was measured by absorbance at 450 nm using a Varioskan LUX Multimode Microplate Reader (ThermoFisher Scientific).

[0203] WST-8 assays confirmed that IL-4 and IL-21 can rescue anti-IgM-induced cell death in Ramos B cells, while application of anti-γc antibodies (SM-05A, SM-05E, SM-05F) can block the effects of IL-4 and IL-21. The overall effect of the anti-γc antibodies of the present invention is superior to that of COMP2022 and ritrecitinib when administered at the same molar concentration (33 nM) (Figures 5A-D).

[0204] The above effects were demonstrated by apoptosis assay using a Dead Cell Apoptosis Kit (#V13242, ThermoFisher Scientific) containing Annexin V (AV) for flow cytometry. Briefly, cells were 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) was analyzed by Flowjo.

[0205] All anti-γc antibodies tested partially reversed the IL-4 and IL-21 rescue of anti-IgM-induced apoptosis in Ramos B cells, with the strongest effect observed in the SM-05A-treated group (Figure 6A-B).

[0206] The levels of cleaved caspase 3 (an apoptotic protein) and μ-H2AX (a DNA damage marker) were studied by Western blot in Ramos B cells treated with the same paradigm. Briefly, total proteins were extracted from Ramos cells with RIPA lysis buffer supplemented with a cocktail of Halt™ protease and phosphatase inhibitors. Proteins were denatured and separated by electrophoresis and blotted onto a nitrocellulose membrane. The membrane was blocked with 5% nonfat milk diluted in TBST and incubated overnight at 4°C on a rotating wheel with primary antibodies diluted in 5% BSA / TBST. The next day, the membrane was washed with PBST and incubated with secondary antibodies diluted in 5% milk / TBST for 1 hour at room temperature. Protein band intensity was measured using an ECL substrate kit on a ChemiDoc imaging system.

[0207] Consistent with the results of the WST-8 and AV / PI assays, IL-4 and IL-21 were able to rescue anti-IgM-induced apoptosis and DNA damage markers. Application of anti-γc antibody and ritrecitinib blocked the protective effect of IL-4 and reinduce μ-H2AX levels, whereas SM-05A showed the strongest effect on cleaved caspases and μ-H2AX reinduction in the presence of IL-21 (Fig. 6C-D).

[0208] Genes related to B cell function were investigated by quantitative real-time PCR (qRT-PCR). Briefly, Ramos cells were starved in serum-free RPMI 1640 medium for 24 hours and then treated 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 gene expression was analyzed by quantitative real-time PCR using TB Green Premix Ex Taq on a LightCycler® 480 Real-Time PCR System. The housekeeping gene GAPDH was used for normalization.

[0209] The expression of CD23 and PRDM1 in Ramos was investigated after administration of 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, suppresses the production of membrane IgE, and regulates B cell proliferation and apoptosis [Veneri et al., Blood Transfus., 2009; 7(1): 29-34].

[0210] PRDM1 is a key transcription factor in B cells that regulates the terminal differentiation of antibody-secreting cells (ASCs) and IL-10-producing regulatory B cells (Bregs) [Nutt et al., Nat Rev Immunol., 2007; 7(12):923-7; Wang et al., Front Immunol., 2019; 10:1909].

[0211] The results show that SM-05A, SM-05E, and ritrecitinib significantly suppressed IL-4-induced CD23 expression, whereas only SM-05E inhibited IL-21-induced PRDM1 expression (Fig. 7 ).

[0212] Example 6: Bioassay for T cell research 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 disease / tumor biology [Lv et al., J Exp Clin Cancer Res., 2016;35(1):106].

[0213] Western blotting revealed that IL-9 can phosphorylate the pro-survival ERK signaling pathway, which can be blocked by application of SM-05A (Figure 8).

[0214] IL-7 has been shown to induce STAT5 phosphorylation and is involved in cell growth, proliferation, and survival of HPB-ALL cells [Ribeiro et al., Blood Adv., 2018; 2(17): 2199-2213], suggesting that HPB-ALL is a reliable cell line for studying the function of IL-7.

[0215] Several assays (e.g., an assay proposed to study 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 percentage of cells expressing BCL2 (an anti-apoptotic marker). Briefly, HPB-ALL cells were pretreated with antibodies or inhibitors for 1 hour and then stimulated with 50 ng / mL IL-7 for 3 days. Cells were harvested and stained with FITC-conjugated BCL-2 antibody (Biolegend) for flow cytometry analysis.

[0216] The results show that both SM-05A and SM-05E significantly attenuated IL-7-induced BCL-2 expression comparable to that of ritrecitinib (Figure 9A). The competing antibody COMP2022 showed variability in this assay.

[0217] A specific method for IL-7 function has been developed in HPB-ALL [Henriques et al., Blood., 2010; 115(16):3269-77]. IL-7 can internalize its receptor, CD127 (or IL-7Rα), into the cell compartment for degradation. Because CD127 degradation depends on the activation state of JAK3 after IL-7 stimulation, blocking γc binding to CD127 and the downstream signaling pathway should prevent CD127 internalization and degradation in HPB-ALL.

[0218] After pretreatment with antibodies or inhibitors, HPB-ALL cells were challenged with 50 ng / mL IL-7 for 3 hours. Surface CD127 expression was measured on live cells, whereas total CD127 expression was examined on PFA-fixed and permeabilized cells. Cells were stained with APC-conjugated CD127 antibody for flow cytometry analysis.

[0219] The results show that all anti-γc antibodies tested were able to block IL-7-induced CD127 internalization and degradation in HPB-ALL cells, with the highest efficacy observed in SM-05A-treated cells (Figure 9B-E).

[0220] Example 7: Bioassay for NK cell research The effect of anti-γc antibodies on NK cell homeostasis was studied 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 responded to IL-2 and IL-15 stimulation, but no information on other γc cytokines was obtained. Prior to antibody testing, the responsiveness of KHYG-1 to γc cytokines was tested.

[0221] Proliferation assays were performed on KHYG-1 cells starved overnight and then incubated with different combinations of γc cytokines for 3 days. The results showed that KHYG-1 cells could proliferate in response to IL-2 and IL-15, but no synergistic effects were observed among all combinations (Fig. 10A).

[0222] After 3 days of cytokine incubation, the production of cytotoxic factors was monitored by flow cytometry in KHYG-1 cells. Cells were fixed with 4% PFA and then permeabilized for intracellular staining using Pacific Blue-labeled granzyme B antibody and APC-labeled perforin antibody (Biolegend). The results showed that IL-2, IL-15, and IL-21 were able to increase the percentage of granzyme B / perforin-expressing cells after 3 days, but the strongest effect was observed in the IL-21-treated group (Figure 10B-C).

[0223] The results obtained by flow cytometry are consistent with the ELISA results obtained with the supernatants. IL-21 functions as the major γc cytokine that induces the production and secretion of granzymes and perforin from KHYG-1 cells (Figure 10D-E). The IFNγ ELISA showed a different result, in which IL-15 is the major 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).

[0224] To test the efficacy of anti-γc antibodies, proliferation and granzyme A secretion were selected as experimental models. Briefly, KHYG-1 cells were seeded in 48- or 96-well plates and starved overnight in medium without IL-2. After replenishing with fresh medium, KHYG-1 cells were preincubated with anti-γc antibodies or ritrecitinib for 1 hour, followed by treatment with γc cytokines for 3 days. The proliferation rate was measured by adding WST-8 reagent, and granzyme A secretion was estimated by ELISA.

[0225] The results showed that both SM-05A and SM-05E could suppress NK cell proliferation with IL-2, IL-15, and IL-21 compared with the JAK3 inhibitor ritrecitinib-treated group (Figures 11A-C).

[0226] Different results were observed regarding granzyme A secretion, where SM-05E and ritrecitinib significantly suppressed IL-2-, IL-15-, and IL-21-induced granzyme A secretion in NK cell cultures, while SM-05A provided less effect in suppression (Figure 11D-F).

[0227] Example 8: Bioassay of ex-vivo PBMC cultures PBMCs (iXCells Biotechnologies, San Diego, USA) isolated from healthy individuals were selected as primary immune cell cultures to estimate the efficacy of anti-γc antibodies in primary cells. STAT phosphorylation could be triggered by treatment with all six γc cytokines (except IL-9) in PBMC cultures (Figure 12A-B). Therefore, inhibition of p-STAT can be used as an indicator for confirming the efficacy of anti-γc antibodies.

[0228] SM-05A remained the most potent candidate for suppressing all six γc cytokine-induced STAT phosphorylation in PBMC cultures, whereas SM-05E and COMP2022 failed to suppress IL-4-induced p-STAT6 in PBMC cultures (Figure 13).

[0229] Proliferation was also studied in PBMC cultures following a similar protocol established for the KHYG-1 NK cell line, demonstrating that PBMCs respond primarily to IL-2, IL-7, and IL-15 for proliferation (Fig. 12C). After 3 days of antibody and cytokine incubation, WST-8 reagent was added to the wells, and proliferation was measured from the signal intensity measured at 450 nm using an enzyme-linked immunosorbent assay (Microplate Reader).

[0230] The SM-05A-treated group showed a significant antiproliferative effect (Figure 14A). Further quantification confirmed that both SM-05A and SM-05E could suppress IL-2-, IL-7-, and IL-15-induced proliferation, although SM-05A provided a slightly stronger effect. The competing antibody COMP2022 showed little antiproliferative effect in PBMC cultures (Figure 14B-D).

[0231] Activation of T cells and NK cells in PBMC cultures can be studied by measuring granzyme and IFNγ secretion [Carlin 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]. We have also obtained similar results, showing that IL-2 and IL-15, but not IL-21, can trigger granzyme A and IFNγ secretion in PBMC cultures (Figures 15A-B).

[0232] To investigate the inhibitory effects under IL-2 or IL-15 treatment, we added either the antibody or ritrecitinib to PBMC cultures. Similar to previous results, SM-05A remained the most potent anti-γc antibody at suppressing IL-2- and IL-15-induced granzyme A and IFNγ secretion. However, the levels of these two soluble factors were significantly reduced in ritrecitinib compared to the control group, suggesting potential toxicity of the JAK3 inhibitor (Figure 15C-F).

[0233] The suppression of T cell activation was further assessed by the expression of cell death-related receptors, such as Fas ligand (FasL), which is primarily expressed on activated T cells and may trigger apoptosis in Fas-expressing cells after binding [Garcia-Gonzalez and Selvi, Encyclopedia of Medical Immunology, 2014;pp 413-416].

[0234] Although treatment with IL-2 or IL-15 only slightly increased the cytotoxic T cell population (CD3+CD8+), both cytokines were able to significantly induce FasL expression in the gated cytotoxic T cell population. Compared with the ritrecitinib-treated group, SM-05A significantly reduced cytokine-induced FasL expression in cytotoxic T cells (Fig. 16).

[0235] [Example 9] Humanization of anti-γc antibody Preliminary results from cell lines and primary cultures suggested that both SM-05A and SM-05E were excellent candidates for suppressing γc cytokine-induced STAT phosphorylation and cell function. Therefore, for future clinical applications, the two anti-γc proteins were humanized using a framework repair approach (see, e.g., U.S. Patent No. 7,321,026 B2).

[0236] The humanized sequences of the heavy and light variable chains are set forth in SEQ ID NOs: 193-200. In some embodiments, the humanized antibody is maintained in a fully humanized form. In some embodiments, back mutations at specific amino acids in the framework region are introduced into the humanized antibody to improve biological activity. The humanized variable heavy and variable light domains are linked to human-derived heavy and light chain constant regions set forth in SEQ ID NOs: 201-202 or SEQ ID NOs: 203-204.

[0237] The humanized antibodies with back mutations (hSM-05A and hSM-05E) 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 for human and chimpanzee γc proteins (Figure 17).

[0238] Additional extracellular domains of γc proteins from common marmoset (SEQ ID NO: NP_001288775.1, SEQ ID NO: 211-212), rhesus macaque (SEQ ID NO: NP_001030606.1, SEQ ID NO: 213-214), rabbit (SEQ ID NO: XP_008270982.1, SEQ ID NO: 215-216), mouse (SEQ ID NO: NP_038591.1, SEQ ID NO: 217-218), and rat (SEQ ID NO: NP_543165.1, SEQ ID NO: 219-220) were purchased from Sino Biological Co., Ltd. or generated in-house. Results indicated that common marmoset is another biologically relevant species for the two γc antibodies, but no binding affinity was observed with the other studied species (Figure 17).

[0239] [Example 10] Stability and binding kinetics of anti-γc antibodies Next, the structural stability of hSM-05A and hSM-05E was studied by incubation at room temperature or 37°C for approximately two 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 two weeks.

[0240] Full-length humanized antibodies were evaluated by BLI to obtain their binding kinetics. The binding activity of full-length anti-γc antibodies was investigated using γc protein (extracellular domain) from human and common marmoset marmosets (purchased or produced in-house). Briefly, anti-γc antibodies (20 μg / mL) were immobilized on a biosensor via interaction with anti-human IgG. Following the standard operating protocol of the Octet ReD96 system, serially diluted γc proteins (158.7 nM, 79.4 nM, 39.7 nM, and 0 nM, respectively) were added to plot association-dissociation curves. An irrelevant antibody (SM03, anti-human CD22 chimeric IgG1 antibody, SinoMab BioScience Limited) was used as a control. The R-squared, Ka, Kdis, and KD values ​​for each antibody are summarized in Table 11. The KD is in the picomolar-single digit nanomolar range. The results show that the anti-γc antibodies bind to γc proteins from human and common marmoset with comparable binding kinetics (FIG. 19 and Tables 11-12).

[0241] [Table 11]

[0242] [Table 12]

[0243] The binding specificity of the humanized anti-γc antibody to native γc proteins was further evaluated by in vivo binding assays. Briefly, human HEK-293 cells were transfected with plasmids expressing full-length γc proteins from human, rhesus monkey, cynomolgus monkey, or common marmoset. After 2 days, transfected or untransfected control cells were incubated with FITC-labeled hSM-05A on ice for 1 hour and then analyzed by flow cytometry. The results showed that hSM-05A could specifically target native γc proteins from human and common marmoset expressed on HEK-293 cells (Figure 20).

[0244] [Example 11] Competition assay between anti-γc antibodies Purified anti-γc antibodies were conjugated with FITC fluorescent dye and used in a competition assay. Briefly, human Ramos B cells expressing human γc were first labeled with unlabeled (unconjugated) hSM-05A for 1 hour, and then further incubated with FITC-labeled hSM-05E. Flow cytometry analysis showed that the two anti-γc antibodies could compete for similar epitopes on the human γc protein (Figure 21).

[0245] [Example 12] Functional assay for measuring ADCC / CDC activity Immunomodulatory antibodies block activation of immune cells rather than inducing cell death, and therefore must circumvent ADCC and CDC in γc-binding proteins to prevent excessive damage to immune cells or the release of cytokines from multiple effector cells.

[0246] The human keratinocyte cell line, HaCaT, which expresses γc, was used as the target cell for ADCC and CDC assays. To determine ADCC activity, HaCaT cells were preconjugated with anti-γc antibody before coculture with human PBMCs. To determine CDC activity, HaCaT cells were preconjugated with anti-γc antibody and incubated with endotoxin-low complement proteins extracted from guinea pigs. ADCC was determined by the percentage of PI+ HaCaT cells in flow cytometry analysis, and CDC was measured by WST-8 proliferation assay of HaCaT cells in 96-well plates (Figure 22A).

[0247] Quantification revealed that hSM-05A and hSM-05E were unable to induce ADCC or CDC activity in HaCaT cells compared with the isotype antibody group. These assays provided evidence for the safe application of anti-γc antibodies in future therapeutic studies.

[0248] [Example 13] Functional assay for immunomodulatory effects of humanized antibodies STAT phosphorylation analysis was again performed to study the potency of the humanized anti-γc protein, as shown in Table 13. The results demonstrate that the two forms of the humanized antibody exhibited comparable p-STAT inhibitory activity compared to the chimeric form (FIG. 23).

[0249] [Table 13]

[0250] The values ​​shown represent the percentage of inhibition compared to the isotype control stimulated with the respective γc cytokine. The antibody concentration was 10 μg / mL.

[0251] The immunomodulatory effects of the humanized antibodies were again validated in Ramos B cells using a WST-8 proliferation assay. The results showed that both antibodies exhibited superior inhibitory effects against IL-4 compared to ritrecitinib, but not against IL-21 (Figure 24).

[0252] Compared with ritrecitinib, hSM-05A can provide better inhibitory effects on IL-7-induced BCL-2 upregulation and CD127 degradation in HPB-ALL T cells (Figure 25).

[0253] Previous studies have shown that IL-9 can rescue apoptosis driven by antitumor drugs in a B cell line model of diffuse large B cell lymphoma (DLBCL) [Lv et al., J Exp Clin Cancer Res., 2016;35(1):106]. Similar experiments were performed in Jurkat T cells to evaluate the efficacy of anti-γc antibodies.

[0254] Jurkat T cells were pretreated with anti-γc antibody or ritrecitinib, followed by administration of antitumor ceramide (50 μM) and IL-9 (50 ng / mL) for 3 days. To assess cell viability, 10 μL of WST-8 reagent was added to each well and incubated for 4 hours before measurement by spectrophotometry.

[0255] The results showed that IL-9 could rescue Jurkat cell death in the presence of ceramide, whereas application of hSM-05A could suppress the effect of IL-9 (Figure 26B). Furthermore, hSM-05A could more effectively suppress IL-9-induced ERK phosphorylation compared with hSM-05E (Figure 26C), indicating that hSM-05A could suppress T cell survival and proliferation in the presence of IL-9.

[0256] The roles of IL-2 and IL-15 in cell proliferation were determined in human PBMC cultures using a WST-8 proliferation assay. The results showed that both antibodies exhibited superior inhibitory effects on IL-2 compared with ritrecitinib, but not on IL-15 (Figures 27B-C).

[0257] The release of cytolytic agents from PBMC cultures was further evaluated by ELISA assay. hSM-05A was able to inhibit IFNγ secretion as effectively as ritrecitinib, whereas ritrecitinib was able to inhibit granzyme B secretion in culture (Figures 27D-G). hSM-05E only exhibited weaker inhibitory effects compared to the two drugs.

[0258] NK cell and cytotoxic T cell activation was studied by activating the receptor NKp46 on the surface of NK cells and cytotoxic T cells. PBMCs were pretreated with humanized antibodies or ritrecitinib for 1 hour and then incubated with IL-2 or IL-15 for 3 days. NK cells were gated as the CD3-CD56+ population, and cytotoxic T cells were identified as the CD3+CD8+ population by flow cytometry analysis. Quantification showed that the two humanized antibodies were effective in suppressing IL-2- and IL-15-induced cytotoxic T cell activation but only slightly inhibited NK cell activation compared to the ritrecitinib-treated group (Figure 28).

[0259] To provide insight into the efficacy of humanized antibodies in targeting GvHD, we developed an MLR assay using our PBMC system (Figure 29A). Briefly, total T cells were isolated from PBMCs of donor 1, and T cells were depleted from PBMCs of donor 2. T cells from donor 1 were activated with a cocktail of anti-CD3 and anti-CD28 antibodies in the presence of IL-2. Simultaneously, T cells were incubated with humanized antibodies or ritrecitinib and inducers. After 3 days, T cells were labeled with CFSE and then cocultured with T-cell-depleted cultures from donor 2 for 6 days. Proliferating T cells were identified by flow cytometry as CFSE-low or CSFE-negative populations.

[0260] The results showed that both hSM-05A and hSM-05E could significantly inhibit MLR-induced cytotoxic T cell proliferation in a dose-dependent manner, but the suppression of T helper cells was milder (Figure 29B-C).

[0261] [Summary of results and conclusions] The present invention describes the development procedures for anti-γc binding proteins and provides the DNA / amino acid sequences of the developed anti-γc binding proteins. These proteins suppressed γc-related downstream pathways (JAK / STAT) and biological functions (survival, proliferation, activation, cytokine secretion, cytotoxic enzyme secretion, differentiation, receptor internalization, and cytotoxicity) in multiple immune cell types (including, but not limited to, B cells, CD4+ T helper cells, CD8+ cytotoxic T cells, NK cells, mast cells, etc.) in single cell cultures or PBMC cultures (Figure 30). The present invention is suggested to show therapeutic potential in the treatment of γc or γc cytokine mediated diseases or conditions (e.g., GVHD, organ transplant rejection, Birdshott 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 a subject in need of treatment comprising, for example, administering (e.g., injecting) an effective amount of an antigen binding protein or composition described herein.

Claims

1. 1. An isolated antigen-binding protein that specifically binds to γc or an antigenic fragment thereof, comprising: The antigen-binding protein has the following characteristics: (a) binding to human γc or a fusion thereof with an acceptable K range of less than 10 M; (b) binding to chimpanzee and common marmoset γc or a fusion thereof with an acceptable KD range of less than 10-9 M; (c) partial binding to mouse and rat γc, or fusions thereof, with a KD range of 10-8 M; (d) exhibits no detectable binding to rhesus monkey, cynomolgus monkey, rabbit γc or fusions thereof; (e) inhibiting IL-4 and IL-21-induced STAT phosphorylation in Ramos B cells; (f) inhibiting IL-7-induced STAT phosphorylation in HPB-ALL T cells; (g) inhibiting IL-9-induced STAT phosphorylation in Jurkat T cells; (h) inhibiting IL-2 and IL-15-induced STAT phosphorylation in KHYG NK cells; (i) inhibiting IL-2, IL-4, IL-7, IL-9, IL-15, or IL-21-induced STAT phosphorylation in PBMC cultures; (j) blocking IL-4 and IL-21 rescue of anti-IgM-induced cell death and injury in B cell cultures; (k) blocking IL-4 and IL-21 inducible genes in B cell cultures; (l) inhibiting cell proliferation in human immune cells, including NK cells, T cells, and PBMC cultures; (m) attenuating IL-9-induced Jurkat T cell protection and proliferation in the presence of anti-tumor ceramide; (n) inhibiting the secretion of inflammatory cytokines and cytotoxic enzymes from NK cell or / and PBMC cultures; (o) inhibiting the activation of NK cells and T cells in primary immune cell cultures; (p) specifically binds to the same epitope on γc as a reference antibody or antigen-binding fragment thereof that is chimeric or humanized SM-05A, SM-05B, SM-05C, SM-05D, SM-05E, SM-05F, SM-05G, SM-05H, SM-05I, SM-05J, SM-05K, or SM-05L; (q) competing for binding to a γc polypeptide or an antigen-binding fragment thereof with a reference antibody or antigen-binding fragment thereof that is chimeric or humanized SM-05A, SM-05B, SM-05C, SM-05D, SM-05E, SM-05F, SM-05G, SM-05H, SM-05I, SM-05J, SM-05K, or SM-05L; (r) blocking binding to a heterodimer consisting of γc and the corresponding γc cytokine receptor subunit in the presence of IL-2, IL-4, IL-7, IL-9, IL-15, or IL-21; Antigen-binding proteins.

2. 2. The antigen binding protein of claim 1, wherein the protein is an isolated antibody.

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

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

5. (a) a heavy chain immunoglobulin or variable region thereof comprising an amino acid sequence having at least 90% amino acid sequence identity to the amino acid sequence set forth 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 a variable region thereof comprising an amino acid sequence having at least 90% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170, or 186; 3. The isolated antibody of claim 2, or the antigen-binding fragment of claim 3.

6. (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 comprising the amino acid sequence set forth 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 set forth 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 a variable region thereof comprising CDR-L1, CDR-L2 and CDR-L3 of a light chain immunoglobulin or a variable region thereof comprising the amino acid sequence set forth 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 set forth in SEQ ID NO: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170 or 186; 5. The isolated antibody or antigen-binding fragment of claim 4.

7. (s) a heavy chain immunoglobulin or variable region thereof having an amino acid sequence selected from the amino acid sequences set forth in Table 1; and / or (t) comprising a light chain immunoglobulin or a variable region thereof having an amino acid sequence selected from the amino acid sequences set forth in Table 2; 5. The isolated antibody or antigen-binding fragment of claim 4.

8. 8. The isolated antibody or antigen-binding fragment of claim 7, comprising one or more members selected from the group consisting of a heavy chain immunoglobulin and its light chain immunoglobulin selected from the amino acid sequences set forth in Table 3.

9. 1. An isolated antibody or antigen-binding protein that specifically binds to γc or an antigenic fragment thereof, comprising: The antibody or antigen-binding protein (a) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 2 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 10; (b) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 18 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 26; (c) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 34 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 42; (d) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 50 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 58; (e) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 66 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 74; (f) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 82 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 90; (g) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 98 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 106; (h) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 114 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 122; (i) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 130 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 138; (j) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 146 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 154; (k) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 162 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 170; (l) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 178 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 184; An isolated antibody or antigen-binding protein.

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

11. 1. An isolated humanized antibody or antigen-binding protein that specifically binds to γc or an antigenic fragment thereof, comprising: The antibody or antigen-binding protein (a) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 193 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 195; (b) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 197 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 199; (c) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 194 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 196; (d) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 198 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 200; An isolated humanized antibody or antigen-binding protein.

12. A heavy chain immunoglobulin G1 or G4 comprising the heavy chain variable region of claims 9 and 11 conjugated to either a heavy chain constant region, wherein the amino acid sequence of the heavy chain constant region is set forth in SEQ ID NO: 201 and 203. Heavy chain immunoglobulin G1 or G4.

13. A light chain immunoglobulin G1 or G4 comprising the light chain variable region of claims 9 and 11 conjugated to either a light chain constant region, wherein the amino acid sequence of the light chain constant region is set forth in SEQ ID NO: 202 and 204. Light chain immunoglobulin G1 or G4.

14. The antigen-binding protein of claim 1 , which has multiple specificities.

15. 10. A method for producing the antigen-binding protein or immunoglobulin chain thereof of claim 1, comprising: (a) immunizing an animal by injecting purified γc protein; (b) generating a phage library by randomly ligating the library of antigen-binding ScFvs into a phage vector; (c) amplifying and screening the phage library using purified γc protein or cells expressing γc; (d) producing and purifying soluble ScFv and antibodies from the host cells and / or the medium in which the host cells are grown; method.

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

17. 16. An antigen binding protein or immunoglobulin chain produced by the method of claim 15.

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

19. A vector comprising the polynucleotide of claim 18.

20. 1. A method of treating a γc or γc cytokine mediated autoimmune disease or condition in a subject in need thereof, comprising:

10. A method of administering by injection an effective amount of the antigen binding protein of claim 1, wherein the disease or condition is selected from GVHD, organ transplant rejection, Birdshott chorioretinopathy, MS, uveitis, T1D, AD, RA, SLE, asthma, psoriasis, SS, vitiligo, celiac disease, IBD, AA, mast cell mediated disease, T cell lymphoma, NK cell lymphoma, and B cell lymphoma.

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