Binding molecules to arginase ii (ARG2)

Abstract

To provide antibodies inhibiting the activity of ARG2.SOLUTION: An isolated antigen-binding protein is capable of binding specifically to human Arginase II (ARG2) and inhibiting the enzyme activity of human ARG2.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Technical Field The present invention relates to antigen-binding proteins, e.g., antibodies, that specifically bind to and inhibit human arginase II (ARG2). The present invention also relates to such anti-ARG2 antibodies for use in treating conditions associated with arginase activation and upregulation, such as cancer, immune cell dysfunction, infectious diseases, vascular disease, cardiovascular disease, endothelial dysfunction, aging and cellular senescence, CNS disease and injury, diabetes-related disease, or cystic fibrosis. [Background technology]

[0002] Background technology Activation and upregulation of arginase (ARG) have been implicated in numerous pathologies, including cancer, immune cell dysfunction, infectious diseases, vascular and cardiovascular diseases, endothelial dysfunction, aging and cellular senescence, CNS disease and injury, diabetes-related diseases, and cystic fibrosis or cystic fibrosis-associated infections (Munder, 2009; Caldwell et al., 2015; Caldwell et al., 2018). Arginase is a binuclear manganese metalloenzyme that catalyzes the hydrolysis of L-arginine to L-ornithine and urea. Two ARG isozymes (arginase I and II) have been identified in mammals. These catalyze the same biochemical reaction but differ in cellular expression, regulation, and subcellular localization (Jenkinson et al., 1996).

[0003] Arginase II (ARG2) is expressed as a mitochondrial protein in various peripheral mammalian tissues, most notably the kidney, prostate, small intestine, and mammary gland. ARG2 can be induced in many other cells, including endothelial cells and macrophages (Ming et al., 2012; Yepuri et al., 2012). The primary role of ARG2 is thought to be in L-arginine homeostasis (Morris, 2007; Durante et al., 2007), where ARG2 plays a crucial role in regulating the availability of L-arginine (or L-ornithine) for subsequent biosynthetic transformation. L-ornithine can be further metabolized into various metabolically important products, including polyamines such as putrescine, spermidine, and spermine, which participate in diverse cellular functions, such as proliferation and cell membrane transport. L-arginine also serves as a substrate for nitric oxide synthase (NOS), which produces nitric oxide (NO) and other reactive nitrogen intermediates (e.g., peroxynitrite). Thus, ARG2 activity, along with the recycling of L-arginine from L-citrulline via argininosuccinate synthase (ASS) and argininosuccinate lyase (ASL), plays a crucial role in both L-arginine and NO availability, with important therapeutic implications, including the following: extracellular L-arginine depletion by arginase can cause impaired lymphocyte responses to antigens during immune responses (Bronte and Zanavello, 2005); NO depletion can lead to increased risk of infection and endothelial dysfunction (Lewis et al., 2011; Sankaralingham et al., 2010; Xu et al., 2004; Bivalacqua et al., 2001).

[0004] The isoform arginase I (ARG1) is expressed in the liver as one of the enzymes of the urea cycle, which forms the main pathway for ammonia detoxification in mammals. This cycle is distributed across two cellular compartments (mitochondria / cytosol), with arginase acting as a cytosolic protein (Jenkinson et al., 1996). Non-malignant immature myeloid cells (called myeloid-derived suppressor cells (MDSCs)) have been described in cancer, infection, and inflammation and express arginase I (Monu et al., 2012) and inducible nitric oxide synthase (INS). Immunoglobulins such as iNOS (also known as NOS2) (Jayaraman et al., 2012) It has been shown that upregulation of the expression and production of immune suppressor factors can suppress T cell proliferation.

[0005] From the available literature, it is understood that ARG1 is primarily expressed in tumor-infiltrating myeloid cells, whereas ARG2 is primarily detected in cancer cells. Although immunohistochemical evidence is lacking in many studies, the frequent finding of ARGs in cancers suggests an important role for this enzyme in tumor biology and development. ARG-dependent tumor-promoting effects can range from proangiogenic activity, lymphocyte suppression, and support for tumor cell growth to stromal remodeling, all properties assigned to tumor-associated macrophages with distinct activation profiles (Mantovani et al., 2002; Balkwill et al., 2005). Several examples highlight this diverse ARG activity in both preclinical tumor models and clinical trials (Bronte and Zanovello, 2005).

[0006] In acute myeloid leukemia (AML), patient-derived AML blasts have been shown to suppress T cell proliferation, an effect mediated by the secretion of ARG2 (Mussai et al., 2015). AML blasts also polarize surrounding monocytes toward a suppressive M2-like phenotype, inhibiting hematopoiesis. They also demonstrate an arginase-dependent ability to suppress progenitor cell proliferation and differentiation. This study also demonstrates that the immunosuppressive activity of the described AML blasts can be modulated by small molecule inhibitors of ARG2 and iNOS. Together, these results support the hypothesis that ARG2 secretion by AML blasts plays an important role in generating an immunosuppressive microenvironment in both the bone marrow and blood, as well as in the development of the pancytopenia commonly observed in patients with AML. The discovery of the ability of AML blasts to induce an immunosuppressive phenotype provides a mechanistic basis for AML blasts whose role is reminiscent of MDSCs, which are involved in several human diseases, including cancer.

[0007] The discovery by Mussai et al. (ibid.) that AML blasts express ARG2, a poorly characterized arginase isoform of arginase with respect to immunosuppression. These findings represent a novel mechanism by which malignant disease can deplete arginine from the microenvironment. They showed that AML2, derived from AML blasts, is enzymatically active, converting arginine to urea and suppressing T-cell proliferation. Notably, ARG2 (but not ARG1) is released from AML blasts, resulting in significant concentrations of ARG2 in the plasma of AML patients. As a result, both plasma from AML patients and supernatants of cultured AML blasts showed enhanced ARG activity, which led to: i) suppression of T cell proliferation; ii) the polarization of peripheral monocytes towards an inhibitory M2-like phenotype in vitro and in transplanted NOD-SCID mice; and iii) Suppression of hematopoietic progenitor cell proliferation (leading to quiescence) in vitro (human CD34+ progenitors and murine GMP progenitors) (pancytopenia).

[0008] This study provides strong preclinical evidence of how AML blasts evade immune detection and provides clues for further research into understanding how they might overcome the immunosuppressive environment in cancer patients to improve overall survival.

[0009] The clinical development of ARG inhibitors is relevant for many indications. As mentioned above, there is accumulating data on the role of ARGs in tumor-associated MDSCs and their pathogenic role in pro-inflammatory immune suppression. ARG2 specifically inhibits the expression of ARGs in osteosarcoma (Setty et al., 2016), HCMV-driven It has been implicated in several cancers, including advanced GBM ( Costa et al., 2016 ), pancreatic cancer ( Ino et al., 2013 ), head and neck squamous cell carcinoma ( Bron et al., 2013 ), thyroid ( Sousa et al., 2010 ), prostate ( Mumenthaler et al., 2008 ), neuroblastoma ( Mussai et al., 2015 ), and breast cancer ( Polat et al., 2002 ).

[0010] Several specific ARG small molecule inhibitors have already been developed and validated in vitro. An intermediate in NO synthesis, NG-hydroxy-L-arginine (NOHA), is a known arginase inhibitor (Hecker et al., 1995). It inhibits NO in human prostate cancer organ cultures. The L-arginine derivative Nw-hydrogenase was successfully used to inhibit ARG activity and restore the responsiveness of tumor-infiltrating lymphocytes in synergy with NOS inhibitors (Bronte et al., 2005). Nor-L-arginine (nor-NOHA), for example, can completely reverse PMN-mediated T cell suppression in suppurative inflammation (Munder et al., 2006), and arginine It was able to restore airway responsiveness in an animal model of enzyme-mediated asthma (Maarsingh et al. al., 2006). The boronic acid 2(S)-amino-6-boronohexanoic acid (ABH) and S-(2-boronoethyl)-L-cysteine ​​(BEC) have binding constants (K) of approximately 0.3 μM at physiological pH. D) is a potent inhibitor of both ARG isoforms, binding with much higher affinity than the natural substrate (L-arginine Km is 5 mM) (Ash et al., 2004; Christianson et al., 2005). Human ARG1 has been crystallized bound to both inhibitors and shows catalytic activity. New mechanistic insights have been gained (Di Costanzo et al., 2005).

[0011] However, arginase-based therapies remain limited due to concerns that existing small molecule inhibitors of ARGs (NOHA and L-NMMA) inhibit both ARG1 and ARG2, potentially disrupting the urea cycle in healthy cells and causing hyperammonemia.

[0012] WO2018236828 discloses that ARG2 expression and activity are potent effectors of Treg immunosuppressive activity; impaired ARG2 is associated with abnormal autoimmune processes, whereas strong ARG2 activity is associated with deleterious suppression of immune responses to tumors. It has been proposed that modulation of Treg suppressive activity can be used to treat pathologies mediated by Treg dysfunction and Treg suppressive activity. WO2018236828 describes the inhibition of suppressive Treg activity by inhibiting or ablating ARG2. The use of ARG2 inhibitors for the treatment of cancer has been disclosed; promising ARG2 inhibitors include CB1158, 2(S)-amino-6-boronohexanoic acid (ABH), (2S)-5,29,59-trihydroxy-7,8-dimethoxyflavanone, R)-2-amino-6-borono-2-(2-(piperidin-1-yl)ethyl)hexanoic acid, and piceatannol-3'-O-β-d-glucopyranoside, antibodies, compositions that promote selective ubiquitination and proteolysis of ARG2 or reduce ARG2 gene expression, and CRISPR / Cas9 constructs for selective inhibition of the ARG2 gene. WO 2018236828 discloses a preference for ARG2 inhibitors that are selective for ARG2 and do not substantially inhibit Arginase 1 activity. The ARG2 inhibitors that selectively bind to and inactivate ARG2 may be compositions of matter that selectively bind to and inactivate ARG2, antibodies, intrabodies, molecules that promote selective ubiquitination and proteolysis of ARG2, compositions of matter that reduce ARG2 gene expression, or inhibitors of ARG2 gene expression, including, for example, small interfering RNAs, hairpin RNAs, zinc finger nucleases, transcription activator-like effector nucleases, or CRISPR / Cas9 constructs. The only example of ARG2 inhibition is the deletion of ARG2 in primary human Tregs using Crispr-Cas9 ribonucleoprotein (RNP) technology. WO 2018236828 does not exemplify antibodies that inhibit ARG2 activity. Summary of the Invention [Problem to be solved by the invention]

[0013] Antibodies against ARGs are known in the art and these bind to ARG2, but to date no antibodies have been identified that inhibit the activity of ARG2.

[0014] Therefore, there is a need for a method to target and specifically inhibit ARG2. do. [Means for solving the problem]

[0015] Summary of the Invention The use of antigen-binding proteins, such as antibodies, e.g., monoclonal antibodies, to target ARG2 offers several advantages over small molecule approaches, as they can both inhibit and delete ARG2 (through Fc-mediated clearance of antibody-antigen (Ab-Ag) complexes). Antibodies that specifically target extracellular ARG2 may alleviate concerns about toxicity and reduce the risk of unwanted adverse effects in patients caused by inhibition of intracellular arginase. Antibodies can also distinguish between the two arginase isomers, allowing for selectivity for ARG2 over ARG1, and therefore specific inhibition of ARG2, in a way that small molecules cannot achieve due to the high similarity of the active sites of ARG1 and ARG2. Finally, antibody therapy may have superior pharmacological properties to small molecule inhibitors, including increased bioavailability and sustained action in the bloodstream of patients, e.g., AML patients.

[0016] The present invention provides the following: 1. An isolated antigen-binding protein characterized by being capable of specifically binding to human arginase II (ARG2) and inhibiting the enzymatic activity of human ARG2. 2. The antigen binding protein of paragraph 1, wherein the antigen binding protein is capable of specifically binding to and inhibiting monomeric and / or trimeric human ARG2. 3. The antigen-binding protein has a dissociation constant (K) of less than 10 nM, less than 1 nM, less than 500 pM, less than 300 pM, or less than 150 pM, as assessed by Bio-Layer Interferometry (BLI). D ) and binds to trimeric human ARG2. 5. The antigen binding protein of any one of the preceding clauses, wherein the antigen binding protein is selective for binding to and inhibiting human ARG2 over human ARG1. 6. The antigen-binding protein of paragraph 5, wherein the selectivity for binding to human ARG2 over human ARG1 is assessed by biolayer interferometry (BLI). 7. The antigen binding protein of any one of the preceding clauses, wherein the antigen binding protein does not measurably bind to human ARG1 when assessed by biolayer interferometry (BLI). 8. The antigen binding protein of any one of the preceding clauses, wherein the antigen binding protein binds to human ARG2 with a 1:1 or 3:1 stoichiometry (antigen binding protein:human ARG2). 9. The antigen binding protein of any one of the preceding clauses, wherein the antigen binding protein is capable of specifically binding to cynomolgus monkey ARG2 and inhibiting its enzymatic activity. 10. The antigen binding protein of any one of the preceding clauses, wherein the antigen binding protein inhibits ARG2 via a non-competitive mode of action. 11. The antigen binding protein of any one of the preceding clauses, wherein the antigen binding protein restores T cell proliferation in vitro in the presence of ARG2. 12. The following: (a) a VH domain comprising a set of HCDRs: HCDR1, HCDR2, and HCDR3, interspersed with framework (FW) regions (HFW1-HCDR1-HFW2-HCDR2-HFW3-HCDR3-HFW4), wherein the amino acid sequence of HCDR3 (amino acids 95-102) is LRADLGLYMDL (SEQ ID NO: 315), and optionally further comprising: (b) a VL domain comprising a set of LCDRs: LCDR1, LCDR2 and LCDR3, interspersed with framework (FW) regions (LFW1-LCDR1-LFW2-LCDR2-LFW3-LCDR3-LFW4), wherein the amino acid sequence of LCDR1 (amino acid 24-34) comprises a VL domain that is SGSSSNIGNHYVS (SEQ ID NO: 318) (the sequence is defined by the Kabat nomenclature system); Item 10. The antigen binding protein of any one of the preceding clauses. 13. The following: (a) A VH domain comprising a set of HCDRs: HCDR1, HCDR2 and HCDR3 interspersed with framework regions (HFW1-HCDR1-HFW2-HCDR2-HFW3-HCDR3-HFW4), wherein said set of HCDRs is selected from the group consisting of antibodies: C0021158fgl2 (SEQ ID NOs: 313, 314 and 315), C0021181 (SEQ ID NOs: 343, 344 and 345), C0021180 (SEQ ID NOs: 333, 334 and 335), C0021177 (SEQ ID NOs: 323, 324 and 325), C0021158 (SEQ ID NOs: 273, 274 and 275), C0021158 IgG (SEQ ID NOs: 283, 284, and 285), C0021158fgl (SEQ ID NOs: 303, 304, and 305), C0021158dr (SEQ ID NOs: 293, 294, and 295), C0021061 (SEQ ID NOs: 63, 64, and 65), C0020187 (SEQ ID NOs: 13, 14, and 15), C0021155 (SEQ ID NOs: 263, 264, and 265), C0021144 (SEQ ID NOs: 253, 254, and 255), C0021142 (SEQ ID NOs: 233, 234, and 235), C0021142IgG (SEQ ID NOs: 243, 244, and 245), C0021141 (SEQ ID NOs: 223, 224, and 225), C0021139 (SEQ ID NOs: 213, 214, and 215), C0021135 (SEQ ID NOs: 203, 204, and 205), C0021133 (SEQ ID NOs: 193, 194, and 195), C0021131 (SEQ ID NO: 18 3, 184 and 185), C0021129 (SEQ ID NOs: 173, 174 and 175), C0021128 (SEQ ID NOs: 163, 164 and 165), C0021124 (SEQ ID NOs: 153, 154 and 155), C0021118 (SEQ ID NOs: 143, 144 and 145), C0021101 (SEQ ID NOs: 133, 134 and 135), C0021098 (SEQ ID NOs: 123, 124, and 125), C0021097 (SEQ ID NOs: 113, 114, and 115), C0021096 (SEQ ID NOs: 103, 104, and 105), C0021092 (SEQ ID NOs: 93, 94, and 95), C0021089 (SEQ ID NOs: 83, 84, and 85), C0021065 (SEQ ID NOs: 73, 74, and 75) a VH domain selected from those of C0021032 (SEQ ID NOs: 53, 54, and 55), C0021022 (SEQ ID NOs: 43, 44, and 45), C0021021 (SEQ ID NOs: 33, 34, and 35), C0021017 (SEQ ID NOs: 23, 24, and 25), and C0020065 (SEQ ID NOs: 3, 4, and 5); and / or (b) A VL domain comprising a set of LCDRs: LCDR1, LCDR2 and LCDR3 interspersed with framework regions (LFW1-LCDR1-LFW2-LCDR2-LFW3-LCDR3-LFW4), wherein said set of LCDRs is selected from the group consisting of antibodies: C0021158fgl2 (SEQ ID NOs: 318, 319 and 320), C0021181 (SEQ ID NOs: 348, 349 and 350), C0021180 (SEQ ID NOs: 338, 339 and 340), C0021177 (SEQ ID NOs: 328, 329 and 330), C0021158 (SEQ ID NOs: 278, 279 and 280), C0021158 IgG (SEQ ID NOs: 288, 289, and 290), C0021158fgl (SEQ ID NOs: 308, 309, and 310), C0021158dr (SEQ ID NOs: 298, 299, and 300), C0021061 (SEQ ID NOs: 68, 69, and 70), C0020187 (SEQ ID NOs: 18, 19, and 20), C0021155 (SEQ ID NOs: 268, 269, and 270), C0021144 (SEQ ID NOs: 258, 259, and 260), C0021142 (SEQ ID NOs: 238, 239, and 240), C0021142 IgG (SEQ ID NOs: 248, 249, and 250), C0021141 (SEQ ID NOs: 228, 229, and 230), C0021139 (SEQ ID NOs: 218, 219, and 220), C0021135 (SEQ ID NOs: 208, 209, and 210), C0021133 (SEQ ID NOs: 198, 199, and 200), C0021131 (SEQ ID NOs: 188, 189, and 190), C0021129 (SEQ ID NOs: 178, 179, and 180), C0021128 (SEQ ID NOs: 168, 169, and 170), C002112 4 (SEQ ID NOs: 158, 159 and 160), C0021118 (SEQ ID NOs: 148, 149 and 150), C0021101 (SEQ ID NOs: 138, 139 and 140), C0021098 (SEQ ID NOs: 128, 129 and 130), C0021097 (SEQ ID NOs: 118, 119 and 120), C0021096 (SEQ ID NOs: 108, 109 and 110), C0021092 (SEQ ID NOs: 98, 99 and 100), C A VL domain selected from those of C0021089 (SEQ ID NOs: 88, 89 and 90), C0021065 (SEQ ID NOs: 78, 79 and 80), C0021032 (SEQ ID NOs: 58, 59 and 60), C0021022 (SEQ ID NOs: 48, 49 and 50), C0021021 (SEQ ID NOs: 38, 39 and 40), C0021017 (SEQ ID NOs: 28, 29 and 30) and C0020065 (SEQ ID NOs: 8, 9 and 10) (said sequences are defined by the Kabat nomenclature). 14. The following: a VH domain comprising HCDR1 (SEQ ID NO: 313), HCDR2 (SEQ ID NO: 314) and HCDR3 (SEQ ID NO: 315) and a VL domain comprising LCDR1 (SEQ ID NO: 318), LCDR2, (SEQ ID NO: 319) and LCDR3 (SEQ ID NO: 320) of C0021158fgl2; a VH domain comprising HCDR1 (SEQ ID NO: 343), HCDR2 (SEQ ID NO: 344) and HCDR3 (SEQ ID NO: 345) of C0021181, and a VL domain comprising LCDR1 (SEQ ID NO: 348), LCDR2, (SEQ ID NO: 349) and LCDR3 (SEQ ID NO: 350); a VH domain comprising HCDR1 (SEQ ID NO: 313), HCDR2 (SEQ ID NO: 314) and HCDR3 (SEQ ID NO: 315) of C0021180, and a VL domain comprising LCDR1 (SEQ ID NO: 318), LCDR2, (SEQ ID NO: 319) and LCDR3 (SEQ ID NO: 320); a VH domain comprising HCDR1 (SEQ ID NO: 323), HCDR2 (SEQ ID NO: 324) and HCDR3 (SEQ ID NO: 325) of C0021177, and a VL domain comprising LCDR1 (SEQ ID NO: 328), LCDR2, (SEQ ID NO: 329) and LCDR3 (SEQ ID NO: 330); a VH domain comprising HCDR1 (SEQ ID NO: 273), HCDR2 (SEQ ID NO: 274) and HCDR3 (SEQ ID NO: 275) of C0021158, and a VL domain comprising LCDR1 (SEQ ID NO: 278), LCDR2, (SEQ ID NO: 279) and LCDR3 (SEQ ID NO: 280); C0021158 IgG, a VH domain comprising HCDR1 (SEQ ID NO: 283), HCDR2 (SEQ ID NO: 284) and HCDR3 (SEQ ID NO: 285), and a VL domain comprising LCDR1 (SEQ ID NO: 288), LCDR2, (SEQ ID NO: 289) and LCDR3 (SEQ ID NO: 290); a VH domain comprising HCDR1 (SEQ ID NO: 303), HCDR2 (SEQ ID NO: 304) and HCDR3 (SEQ ID NO: 305) and a VL domain comprising LCDR1 (SEQ ID NO: 308), LCDR2, (SEQ ID NO: 309) and LCDR3 (SEQ ID NO: 310) of C0021158fgl; a VH domain comprising HCDR1 (SEQ ID NO: 293), HCDR2 (SEQ ID NO: 294) and HCDR3 (SEQ ID NO: 295) and a VL domain comprising LCDR1 (SEQ ID NO: 298), LCDR2, (SEQ ID NO: 299) and LCDR3 (SEQ ID NO: 300) of C0021158dr; C0021061, a VH domain comprising HCDR1 (SEQ ID NO: 63), HCDR2 (SEQ ID NO: 64) and HCDR3 (SEQ ID NO: 65), and a VL domain comprising LCDR1 (SEQ ID NO: 68), LCDR2, (SEQ ID NO: 69) and LCDR3 (SEQ ID NO: 70); C0020187, a VH domain comprising HCDR1 (SEQ ID NO: 13), HCDR2 (SEQ ID NO: 14) and HCDR3 (SEQ ID NO: 15), and LCDR1 (SEQ ID NO: 18), LCDR2 (SEQ ID NO: 19), a VL domain comprising LCDR2, (SEQ ID NO: 19) and LCDR3 (SEQ ID NO: 20); a VH domain comprising HCDR1 (SEQ ID NO: 263), HCDR2 (SEQ ID NO: 264) and HCDR3 (SEQ ID NO: 265) of C0021155, and a VL domain comprising LCDR1 (SEQ ID NO: 268), LCDR2, (SEQ ID NO: 269) and LCDR3 (SEQ ID NO: 270); a VH domain comprising HCDR1 (SEQ ID NO: 253), HCDR2 (SEQ ID NO: 254) and HCDR3 (SEQ ID NO: 255) and a VL domain comprising LCDR1 (SEQ ID NO: 258), LCDR2, (SEQ ID NO: 259) and LCDR3 (SEQ ID NO: 260) of C0021144; a VH domain comprising HCDR1 (SEQ ID NO: 233), HCDR2 (SEQ ID NO: 234) and HCDR3 (SEQ ID NO: 235) and a VL domain comprising LCDR1 (SEQ ID NO: 238), LCDR2, (SEQ ID NO: 239) and LCDR3 (SEQ ID NO: 240) of C0021142; C0021142 IgG, a VH domain comprising HCDR1 (SEQ ID NO: 243), HCDR2 (SEQ ID NO: 244) and HCDR3 (SEQ ID NO: 245), and a VL domain comprising LCDR1 (SEQ ID NO: 248), LCDR2, (SEQ ID NO: 249) and LCDR3 (SEQ ID NO: 250); a VH domain comprising HCDR1 (SEQ ID NO: 223), HCDR2 (SEQ ID NO: 224) and HCDR3 (SEQ ID NO: 225) and a VL domain comprising LCDR1 (SEQ ID NO: 228), LCDR2, (SEQ ID NO: 229) and LCDR3 (SEQ ID NO: 230) of C0021141; a VH domain comprising HCDR1 (SEQ ID NO: 213), HCDR2 (SEQ ID NO: 214) and HCDR3 (SEQ ID NO: 215) and a VL domain comprising LCDR1 (SEQ ID NO: 218), LCDR2, (SEQ ID NO: 219) and LCDR3 (SEQ ID NO: 220) of C0021139; a VH domain comprising HCDR1 (SEQ ID NO: 203), HCDR2 (SEQ ID NO: 204) and HCDR3 (SEQ ID NO: 205) of C0021135, and a VL domain comprising LCDR1 (SEQ ID NO: 208), LCDR2, (SEQ ID NO: 209) and LCDR3 (SEQ ID NO: 210); a VH domain comprising HCDR1 (SEQ ID NO: 193), HCDR2 (SEQ ID NO: 194) and HCDR3 (SEQ ID NO: 195) of C0021133, and a VL domain comprising LCDR1 (SEQ ID NO: 198), LCDR2, (SEQ ID NO: 199) and LCDR3 (SEQ ID NO: 200); a VH domain comprising HCDR1 (SEQ ID NO: 183), HCDR2 (SEQ ID NO: 184) and HCDR3 (SEQ ID NO: 185) and a VL domain comprising LCDR1 (SEQ ID NO: 188), LCDR2, (SEQ ID NO: 189) and LCDR3 (SEQ ID NO: 190) of C0021131; a VH domain comprising HCDR1 (SEQ ID NO: 173), HCDR2 (SEQ ID NO: 174) and HCDR3 (SEQ ID NO: 175) of C0021129, and a VL domain comprising LCDR1 (SEQ ID NO: 178), LCDR2, (SEQ ID NO: 179) and LCDR3 (SEQ ID NO: 180); a VH domain comprising HCDR1 (SEQ ID NO: 163), HCDR2 (SEQ ID NO: 164) and HCDR3 (SEQ ID NO: 165) of C0021128, and a VL domain comprising LCDR1 (SEQ ID NO: 168), LCDR2, (SEQ ID NO: 169) and LCDR3 (SEQ ID NO: 170); a VH domain comprising HCDR1 (SEQ ID NO: 153), HCDR2 (SEQ ID NO: 154) and HCDR3 (SEQ ID NO: 155) and a VL domain comprising LCDR1 (SEQ ID NO: 158), LCDR2, (SEQ ID NO: 159) and LCDR3 (SEQ ID NO: 160) of C0021124; HCDR1 (SEQ ID NO: 143), HCDR2 (SEQ ID NO: 144) and a VH domain comprising LCDR1 (SEQ ID NO: 148), LCDR2, (SEQ ID NO: 149) and LCDR3 (SEQ ID NO: 150); a VH domain comprising HCDR1 (SEQ ID NO: 133), HCDR2 (SEQ ID NO: 134) and HCDR3 (SEQ ID NO: 135) and a VL domain comprising LCDR1 (SEQ ID NO: 138), LCDR2, (SEQ ID NO: 139) and LCDR3 (SEQ ID NO: 140) of C0021101; C0021098, a VH domain comprising HCDR1 (SEQ ID NO: 123), HCDR2 (SEQ ID NO: 124) and HCDR3 (SEQ ID NO: 125), and a VL domain comprising LCDR1 (SEQ ID NO: 128), LCDR2, (SEQ ID NO: 129) and LCDR3 (SEQ ID NO: 130); C0021097, a VH domain comprising HCDR1 (SEQ ID NO: 113), HCDR2 (SEQ ID NO: 114) and HCDR3 (SEQ ID NO: 115), and a VL domain comprising LCDR1 (SEQ ID NO: 118), LCDR2, (SEQ ID NO: 119) and LCDR3 (SEQ ID NO: 120); C0021096, a VH domain comprising HCDR1 (SEQ ID NO: 103), HCDR2 (SEQ ID NO: 104) and HCDR3 (SEQ ID NO: 105), and a VL domain comprising LCDR1 (SEQ ID NO: 108), LCDR2, (SEQ ID NO: 109) and LCDR3 (SEQ ID NO: 110); C0021092, a VH domain comprising HCDR1 (SEQ ID NO: 93), HCDR2 (SEQ ID NO: 94) and HCDR3 (SEQ ID NO: 95), and a VL domain comprising LCDR1 (SEQ ID NO: 98), LCDR2, (SEQ ID NO: 99) and LCDR3 (SEQ ID NO: 100); a VH domain comprising HCDR1 (SEQ ID NO: 83), HCDR2 (SEQ ID NO: 84) and HCDR3 (SEQ ID NO: 85) of C0021089, and a VL domain comprising LCDR1 (SEQ ID NO: 88), LCDR2, (SEQ ID NO: 89) and LCDR3 (SEQ ID NO: 90); C0021065, a VH domain comprising HCDR1 (SEQ ID NO: 73), HCDR2 (SEQ ID NO: 74) and HCDR3 (SEQ ID NO: 75), and a VL domain comprising LCDR1 (SEQ ID NO: 78), LCDR2, (SEQ ID NO: 79) and LCDR3 (SEQ ID NO: 80); a VH domain comprising HCDR1 (SEQ ID NO: 53), HCDR2 (SEQ ID NO: 54) and HCDR3 (SEQ ID NO: 55) of C0021032, and a VL domain comprising LCDR1 (SEQ ID NO: 58), LCDR2, (SEQ ID NO: 59) and LCDR3 (SEQ ID NO: 60); a VH domain comprising HCDR1 (SEQ ID NO: 43), HCDR2 (SEQ ID NO: 44) and HCDR3 (SEQ ID NO: 45) of C0021022, and a VL domain comprising LCDR1 (SEQ ID NO: 48), LCDR2, (SEQ ID NO: 49) and LCDR3 (SEQ ID NO: 50); C0021021, a VH domain comprising HCDR1 (SEQ ID NO: 33), HCDR2 (SEQ ID NO: 34) and HCDR3 (SEQ ID NO: 35), and a VL domain comprising LCDR1 (SEQ ID NO: 38), LCDR2, (SEQ ID NO: 39) and LCDR3 (SEQ ID NO: 40); a VH domain comprising HCDR1 (SEQ ID NO: 23), HCDR2 (SEQ ID NO: 24) and HCDR3 (SEQ ID NO: 25) of C0021017, and a VL domain comprising LCDR1 (SEQ ID NO: 28), LCDR2, (SEQ ID NO: 29) and LCDR3 (SEQ ID NO: 30); or C0020065, a VH domain comprising HCDR1 (SEQ ID NO: 3), HCDR2 (SEQ ID NO: 4) and HCDR3 (SEQ ID NO: 5), and a VL domain comprising LCDR1 (SEQ ID NO: 8), LCDR2, (SEQ ID NO: 9) and LCDR3 (SEQ ID NO: 10). (said sequences are defined by the Kabat nomenclature). 15. The following: (a) (i) a VH domain comprising the set of HCDRs of C0021158fgl2 (HCDR1 SEQ ID NO: 313, HCDR2 SEQ ID NO: 314, and HCDR3 SEQ ID NO: 315), and / or (ii) a VH domain comprising the set of LCDRs of C0021158fgl2 (LCDR1 SEQ ID NO: 318, LCDR2 SEQ ID NO: 319, and LCDR3 SEQ ID NO: 320). VL domain; (b) (i) a VH domain comprising the set of HCDRs of C0021133 (HCDR1 SEQ ID NO: 193, HCDR2 SEQ ID NO: 194 and HCDR3 SEQ ID NO: 195), and / or (ii) a VL domain comprising the set of LCDRs of C0021133 (LCDR1 SEQ ID NO: 198, LCDR2 SEQ ID NO: 199 and LCDR3 SEQ ID NO: 200); or (c) (i) a VH domain comprising the set of HCDRs of C0020187 (HCDR1 SEQ ID NO: 13, HCDR2 SEQ ID NO: 14 and HCDR3 SEQ ID NO: 15), and / or (ii) a VL domain comprising the set of LCDRs of C0020187 (LCDR1 SEQ ID NO: 18, LCDR2 SEQ ID NO: 199). a VL domain comprising SEQ ID NO: 19, and LCDR3 SEQ ID NO: 20); (d) (i) a VH domain comprising the set of HCDRs of C0020065 (HCDR1 SEQ ID NO: 3, HCDR2 SEQ ID NO: 4, and HCDR3 SEQ ID NO: 5), and / or (ii) a VL domain comprising the set of LCDRs of C0020187 (LCDR1 SEQ ID NO: 8, LCDR2 SEQ ID NO: 9, and LCDR3 SEQ ID NO: 10). (said sequences are defined by the Kabat nomenclature). 16. The following: (a) Antibodies: C0021158 fgl2 (SEQ ID NO: 312), C0021181 (SEQ ID NO: 342), C0021180 (SEQ ID NO: 332), C0021177 (SEQ ID NO: 322), C0021158 (SEQ ID NO: 272), C0021158 IgG (SEQ ID NO: 282), C0021158fgl (SEQ ID NO: 302), C0021158dr (SEQ ID NO: 292), C0021061 (SEQ ID NO: 62), C0020187 (SEQ ID NO: 12), C0021155 (SEQ ID NO: 262), C0021144 (SEQ ID NO: 252), C0021142 (SEQ ID NO: 232), C0021142 IgG (SEQ ID NO: 242), C0021141 (SEQ ID NO: 227), C0021139 (SEQ ID NO: 217), C0021135 (SEQ ID NO: 207), C0021133 (SEQ ID NO: 197), C0021131 (SEQ ID NO: 187), C0021129 (SEQ ID NO: 177), C0021128 (SEQ ID NO: 167), C0021124 (SEQ ID NO: 157), C0021118 (SEQ ID NO: 147), C0021101 (SEQ ID NO: 137), C0021098 (SEQ ID NO: 127), C0021097 (SEQ ID NO: 117), C0021096 (SEQ ID NO: 107), C0021092 (SEQ ID NO: 97), C0021 a VH domain selected from the VH domains of C0021065 (SEQ ID NO:77), C0021032 (SEQ ID NO:57), C0021022 (SEQ ID NO:47), C0021021 (SEQ ID NO:37), C0021017 (SEQ ID NO:27), and C0020065 (SEQ ID NO:7), or a germlined version thereof, or a VH domain with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology thereto; and / or (b) Antibodies: C0021158 fgl2 (SEQ ID NO: 317), C0021181 (SEQ ID NO: 347), C0021180 (SEQ ID NO: 337), C0021177 (SEQ ID NO: 327), C0021158 (SEQ ID NO: 277), C0021158 IgG (SEQ ID NO: 287), C0021158fgl (SEQ ID NO: 307), C0021158dr (SEQ ID NO: 297), C0021061 (SEQ ID NO: 67), C0020187 (SEQ ID NO: 17), C0021155 (SEQ ID NO: 267), C0021144 (SEQ ID NO: 257), C0021142 (SEQ ID NO: 237), C0021142 IgG (SEQ ID NO: 247), C0021141 (SEQ ID NO: 227), C0021139 (SEQ ID NO: 217), C0021135 (SEQ ID NO: 207), C0021133 (SEQ ID NO: 197), C0021131 (SEQ ID NO: 187), C0021129 (SEQ ID NO: 177), C0021128 (SEQ ID NO: 167), C0021124 (SEQ ID NO: 187), C0021135 ... Column number 157), C0021118 (SEQ ID NO: 147), C0021101 (SEQ ID NO: 137), C0021098 (SEQ ID NO: 127), C0021097 (SEQ ID NO: 117), C0021096 (SEQ ID NO: 107), C0021092 (SEQ ID NO: 97), C0021089 (SEQ ID NO: 87), C0021065 (SEQ ID NO: 77), C a VL domain selected from the VL domains of C0021032 (SEQ ID NO:57), C0021022 (SEQ ID NO:47), C0021021 (SEQ ID NO:37), C0021017 (SEQ ID NO:27), and C0020065 (SEQ ID NO:7), or a germlined version thereof, or a VL domain with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology thereto. (said sequences are defined by the Kabat nomenclature) 17. The following: C0021158 fgl2 VH domain (SEQ ID NO: 312) and VL domain (SEQ ID NO: 317), The VH domain (SEQ ID NO: 342) and VL domain (SEQ ID NO: 347) of C0021181; The VH domain (SEQ ID NO: 332) and VL domain (SEQ ID NO: 337) of C0021180; The VH domain (SEQ ID NO: 322) and VL domain (SEQ ID NO: 327) of C0021177; The VH domain (SEQ ID NO: 272) and VL domain (SEQ ID NO: 277) of C0021158; C0021158 IgG VH domain (SEQ ID NO: 282) and VL domain (SEQ ID NO: 287), The VH domain (SEQ ID NO: 302) and VL domain (SEQ ID NO: 307) of C0021158fgl; The VH domain (SEQ ID NO: 292) and VL domain (SEQ ID NO: 297) of C0021158dr; The VH domain (SEQ ID NO: 62) and VL domain (SEQ ID NO: 67) of C0021061, the VH domain (SEQ ID NO: 12) and VL domain (SEQ ID NO: 17) of C0020187, the VH domain (SEQ ID NO: 262) and VL domain (SEQ ID NO: 267) of C0021155, The VH domain (SEQ ID NO: 252) and VL domain (SEQ ID NO: 257) of C0021144; The VH domain (SEQ ID NO: 232) and VL domain (SEQ ID NO: 237) of C0021142; C0021142 IgG VH domain (SEQ ID NO: 242) and VL domain (SEQ ID NO: 247), the VH domain (SEQ ID NO: 222) and VL domain (SEQ ID NO: 227) of C0021141; the VH domain (SEQ ID NO: 212) and VL domain (SEQ ID NO: 217) of C0021139; The VH domain (SEQ ID NO: 202) and VL domain (SEQ ID NO: 207) of C0021135; the VH domain (SEQ ID NO: 192) and VL domain (SEQ ID NO: 197) of C0021133; the VH domain (SEQ ID NO: 182) and VL domain (SEQ ID NO: 187) of C0021131; the VH domain (SEQ ID NO: 172) and VL domain (SEQ ID NO: 177) of C0021129; The VH domain (SEQ ID NO: 162) and VL domain (SEQ ID NO: 167) of C0021128; The VH domain (SEQ ID NO: 152) and VL domain (SEQ ID NO: 157) of C0021124 ), the VH domain (SEQ ID NO: 142) and VL domain (SEQ ID NO: 147) of C0021118; the VH domain (SEQ ID NO: 132) and VL domain (SEQ ID NO: 137) of C0021101; The VH domain (SEQ ID NO: 122) and VL domain (SEQ ID NO: 127) of C0021098; the VH domain (SEQ ID NO: 112) and VL domain (SEQ ID NO: 117) of C0021097; the VH domain (SEQ ID NO: 102) and VL domain (SEQ ID NO: 107) of C0021096; the VH domain (SEQ ID NO: 92) and VL domain (SEQ ID NO: 97) of C0021092, the VH domain (SEQ ID NO: 82) and VL domain (SEQ ID NO: 87) of C0021089, the VH domain (SEQ ID NO: 72) and VL domain (SEQ ID NO: 77) of C0021065, the VH domain (SEQ ID NO: 52) and VL domain (SEQ ID NO: 57) of C0021032, the VH domain (SEQ ID NO: 42) and VL domain (SEQ ID NO: 47) of C0021022, the VH domain (SEQ ID NO: 32) and VL domain (SEQ ID NO: 37) of C0021021, the VH domain (SEQ ID NO: 22) and VL domain (SEQ ID NO: 27) of C0021017, or VH domain (SEQ ID NO: 2) and VL domain (SEQ ID NO: 7) of C0020065 9. The antigen binding protein of any one of the preceding clauses, comprising a VH domain and a VL domain that are at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to 18. The following: C0021158 fgl2 VH domain (SEQ ID NO: 312) and VL domain (SEQ ID NO: 317), The VH domain (SEQ ID NO: 342) and VL domain (SEQ ID NO: 347) of C0021181; The VH domain (SEQ ID NO: 332) and VL domain (SEQ ID NO: 337) of C0021180; The VH domain (SEQ ID NO: 322) and VL domain (SEQ ID NO: 327) of C0021177; The VH domain (SEQ ID NO: 272) and VL domain (SEQ ID NO: 277) of C0021158; C0021158 IgG VH domain (SEQ ID NO: 282) and VL domain (SEQ ID NO: 287), The VH domain (SEQ ID NO: 302) and VL domain (SEQ ID NO: 307) of C0021158fgl; The VH domain (SEQ ID NO: 292) and VL domain (SEQ ID NO: 297) of C0021158dr; The VH domain (SEQ ID NO: 62) and VL domain (SEQ ID NO: 67) of C0021061, the VH domain (SEQ ID NO: 12) and VL domain (SEQ ID NO: 17) of C0020187, the VH domain (SEQ ID NO: 262) and VL domain (SEQ ID NO: 267) of C0021155, The VH domain (SEQ ID NO: 252) and VL domain (SEQ ID NO: 257) of C0021144; The VH domain (SEQ ID NO: 232) and VL domain (SEQ ID NO: 237) of C0021142; C0021142 IgG VH domain (SEQ ID NO: 242) and VL domain (SEQ ID NO: 247), the VH domain (SEQ ID NO: 222) and VL domain (SEQ ID NO: 227) of C0021141; the VH domain (SEQ ID NO: 212) and VL domain (SEQ ID NO: 217) of C0021139; The VH domain (SEQ ID NO: 202) and VL domain (SEQ ID NO: 207) of C0021135; the VH domain (SEQ ID NO: 192) and VL domain (SEQ ID NO: 197) of C0021133; the VH domain (SEQ ID NO: 182) and VL domain (SEQ ID NO: 187) of C0021131; the VH domain (SEQ ID NO: 172) and VL domain (SEQ ID NO: 177) of C0021129; The VH domain (SEQ ID NO: 162) and VL domain (SEQ ID NO: 167) of C0021128; The VH domain (SEQ ID NO: 152) and VL domain (SEQ ID NO: 157) of C0021124; the VH domain (SEQ ID NO: 142) and VL domain (SEQ ID NO: 147) of C0021118; the VH domain (SEQ ID NO: 132) and VL domain (SEQ ID NO: 137) of C0021101; The VH domain (SEQ ID NO: 122) and VL domain (SEQ ID NO: 127) of C0021098; the VH domain (SEQ ID NO: 112) and VL domain (SEQ ID NO: 117) of C0021097; the VH domain (SEQ ID NO: 102) and VL domain (SEQ ID NO: 107) of C0021096; the VH domain (SEQ ID NO: 92) and VL domain (SEQ ID NO: 97) of C0021092, the VH domain (SEQ ID NO: 82) and VL domain (SEQ ID NO: 87) of C0021089, the VH domain (SEQ ID NO: 72) and VL domain (SEQ ID NO: 77) of C0021065, the VH domain (SEQ ID NO: 52) and VL domain (SEQ ID NO: 57) of C0021032, the VH domain (SEQ ID NO: 42) and VL domain (SEQ ID NO: 47) of C0021022, the VH domain (SEQ ID NO: 32) and VL domain (SEQ ID NO: 37) of C0021021, the VH domain (SEQ ID NO: 22) and VL domain (SEQ ID NO: 27) of C0021017, or Item 1. The antigen binding protein of any one of the preceding clauses, comprising the VH domain (SEQ ID NO:2) and VL domain (SEQ ID NO:7) of C0020065; or germlined versions thereof (said sequences are defined by the Kabat nomenclature). 19. The following: (a) C0021158 fgl2 VH domain amino acid sequence (SEQ ID NO: 312) and C0021158 fgl2 VL domain amino acid sequence (SEQ ID NO: 317); (b) C0021133 VH domain amino acid sequence (SEQ ID NO: 192) and C0021133 VL domain amino acid sequence (SEQ ID NO: 197); (c) C0020187 VH domain amino acid sequence (SEQ ID NO: 12) and C0020187 VL domain amino acid sequence (SEQ ID NO: 17); (d) C0020065 VH domain amino acid sequence (SEQ ID NO: 2) and C0020065 VL domain amino acid sequence (SEQ ID NO: 7) 3. The antigen binding protein of any one of the preceding clauses, comprising: 20. When tested in an epitope competition assay: (a) a VH domain of SEQ ID NO: 312 and a VL domain of SEQ ID NO: 317; (b) a VH domain of SEQ ID NO: 192 and a VL domain of SEQ ID NO: 197; (c) a VH domain of SEQ ID NO: 12 and a VL domain of SEQ ID NO: 17, or (d) a VH domain of SEQ ID NO: 2 and a VL domain of SEQ ID NO: 7 An antigen-binding protein that competes for binding to human ARG2 with an antigen-binding protein comprising: 21. An antigen-binding protein according to any one of the preceding clauses, characterized in that it specifically binds to an epitope of human ARG2, thereby being able to inhibit the enzymatic activity of human ARG2 by an allosteric mechanism. 22. Any of the preceding paragraphs, characterized in that it specifically binds to an epitope of human ARG2, thereby inducing structural remodeling of residues 33-40 in human ARG2, wherein the sequence numbering is that of the human ARG2 sequence of Uniprot ID#P78540. 10. The isolated antigen-binding protein according to any one of claims 1 to 9. 23. A single antibody according to any one of the preceding paragraphs, characterized in that it specifically binds to an epitope on human ARG2, thereby being able to inhibit the enzymatic activity of human ARG2 by an allosteric mechanism, wherein the antigen-binding protein binds to an epitope on human ARG2, which induces a structural or biophysical change to His160 of human ARG2, which results in a decrease in the enzymatic activity of ARG2 or its ability to process a substrate, wherein the sequence numbering is that of the human ARG2 sequence of Uniprot ID#P78540. Isolated antigen-binding proteins. 24. The antigen-binding protein binds to an epitope on human ARG2, which induces a conformational change such that Arg39 moves closer to His160 of human ARG2, thereby resulting in a decrease in the enzymatic activity of ARG2 or its ability to process a substrate, wherein the sequence numbering is the human ARG2 sequence of Uniprot ID#P78540. Item 10. The isolated antigen binding protein of any one of the preceding items, wherein: 25. The isolated antigen binding protein of any one of the preceding clauses, wherein the antigen binding protein binds to an epitope on human ARG2 that induces a conformational change that impairs the ability of His160 to act as a proton donor / acceptor and / or stabilize a catalytically competent binding orientation of a substrate, thereby resulting in reduced ARG2 enzymatic activity. 26. Binds to an epitope on human ARG2, including a conformational epitope comprising residues Gln35 to Arg39, residues Lys78 to Ile86, and / or residues Leu152 to Pro179 (sequence numbering is based on the human ARG2 sequence in Uniprot ID#P78540). 2. The antigen-binding protein of any one of the preceding claims. 27. The antigen binding protein of any one of the preceding clauses, which binds to an epitope on human ARG2 which comprises one or more residues selected from Gln35, Gly36, Gln37, Lys38, Arg39, Lys78, Asp79, Asp80, Leu81, Tyr82, Asn84, Leu85, Ile86, Leu152, Thr153 Thr154, Ser155, Ser156, Gly157, Leu178 and Pro179. 28. The antigen of paragraph 27, wherein the epitope is defined as any residue in human ARG2 with a predicted change in solvent accessibility upon Fab complex formation resulting from direct protection by the binding antibody, as obtained from the X-ray structure data of ARG2 inhibitory Fab C0020187, wherein the sequence numbering is that of the human ARG2 sequence in Uniprot ID#P78540. Binding proteins. 29. Binds to an epitope on human ARG2, including a conformational epitope comprising residues Pro32 to Glu51, residues Asp70 to Ile86, and / or residues Pro299 to Ala308 (sequence numbering is based on the human ARG2 sequence in Uniprot ID#P78540). 26. The antigen-binding protein of any one of items 1 to 25, wherein 30.Pro32, Gln37, Lys38, Lys40, Gly41, Glu43, H 30. The antigen-binding protein of clause 29, which binds to an epitope on human ARG2 which comprises one or more residues selected from is44, Ala47, Ala48, Glu51, Asp70, Ser72, Phe73, Thr74, Pro75, Lys78, Asp79, Asp80, Leu81, Tyr82, Asn84, Leu85, Ile86, Pro299, Gln300, Glu305 and Ala308. 31. Item 29 or 30, wherein the epitope is defined as any residue within human ARG2 with a predicted change in solvent accessibility upon Fab complex formation resulting from direct protection by the binding antibody, as obtained from the X-ray structural data of ARG2 inhibitory Fab C0021158, wherein the sequence numbering is that of the human ARG2 sequence in Uniprot ID#P78540. 1. An antigen-binding protein according to claim 1. 32. Binds to an epitope on human ARG2, including a conformational epitope comprising residues Gln37 to Glu51, residues Asp79 to Ile86, and / or residues Pro299 to Ala308 (sequence numbering is based on the human ARG2 sequence in Uniprot ID#P78540). 32. The antigen-binding protein of claim 31, wherein 33. The antigen-binding protein of any one of clauses 1 to 25, which binds to an epitope on recombinant human ARG2 which comprises one or more residues selected from Gln37, Lys38, Lys40, Gly41, His44, Ala47, Ala48, Glu51, Asp79, Asp80, Leu81, Tyr82, Asn84, Leu85, Ile86, Pro299, Gln300, Ala302, Thr303, Ser304, Glu305 and Ala308. 34. The antibody of claim 33, wherein the epitope is defined as any residue in human ARG2 with a predicted change in solvent accessibility upon Fab complex formation resulting from direct protection by the binding antibody, as obtained from the X-ray structure data of ARG2 inhibitory Fab C0021181, wherein the sequence numbering is that of the human ARG2 sequence of Uniprot ID#P78540. Protein binding protein. 35.Pro32, Gln35, Gly36, Gln37, Lys38, Arg39, Lys40, Gly41, Glu43, His44, Ala47, Ala48, Glu51, Asp70 , Ser72, Phe73, Thr74, Pro75, Lys78, Asp79, Asp80, Leu81, Tyr82, Asn84, Leu85, Ile86, Leu152, Thr153 35. The antigen-binding protein of clause 33 or clause 34, which binds to an epitope on human ARG2 which comprises one or more residues selected from Thr154, Ser155, Ser156, Gly157, Leu178, Pro179, Pro299, Gln300, Ala302, Thr303, Ser304, Glu305 and Ala308. 36. The epitope is defined as any residue in human ARG2 with a predicted change in solvent accessibility upon Fab complex formation as obtained from X-ray structural data of an ARG2 inhibitory Fab selected from C0020187, C0021158, and C0021181, wherein the sequence numbering is that of the human ARG2 sequence in Uniprot ID#P78540. 36. The antigen-binding protein of claim 35. 37. The antigen-binding protein of any one of the preceding clauses, wherein said antigen-binding protein is an antibody or fragment thereof, a domain antibody, a protein scaffold, or an aptamer. 38. The antigen-binding protein of any one of the preceding clauses, wherein said antigen-binding protein is human IgG or modified human IgG. 39. The antigen-binding protein of clause 38, wherein said antigen-binding protein is human IgG1, IgG2, IgG4, or a modified version thereof. 40. The antigen-binding protein of clause 38 or clause 39, wherein said antigen-binding protein is human IgG1 or IgG1-YTE. 41. The antigen binding protein of any one of the preceding clauses, wherein the antigen binding protein has a modified Fc to provide enhanced effector function and / or extended half-life. 42. A composition comprising an antigen-binding protein according to any one of the preceding clauses and a pharmaceutically acceptable excipient. 43. An antigen-binding protein or composition according to any one of the preceding clauses for use in a method of treatment of the human or animal body. 44. An antigen-binding protein or composition according to any one of the preceding clauses for use in treating an individual for the purposes of restoring immune competence, alleviating inflammation-triggered immune dysfunction, inflammation-associated immunosuppression, promoting T cell-mediated immune responses, or preventing tumor immune evasion, fibrosis, and immune disorders of infectious diseases. 45. An antigen-binding protein or composition according to any one of the preceding clauses for use in treating an individual for the purpose of restoring T-cell proliferation in the presence of ARG2. 46. ​​An antigen-binding protein or composition according to any one of the preceding clauses for use in the treatment of cancer, immune cell dysfunction, autoimmunity or unwanted immune deviation. 47. The antigen binding protein or composition of any one of the preceding clauses for use in the treatment of acute myeloid leukemia (AML), osteosarcoma, HCMV-driven GBM, pancreatic cancer, head and neck squamous cell carcinoma, thyroid cancer, prostate cancer, breast cancer, neuroblastoma or ovarian cancer. 48. The antigen binding protein or composition of any one of the preceding clauses for use in the treatment of infection (e.g. neonatal infection), endothelial dysfunction (e.g. erectile dysfunction), vascular disease, cardiovascular disease, ageing and cellular senescence, CNS disease and injury; diabetes-related disease or cystic fibrosis or an infection associated with cystic fibrosis. 49. A method of treating an individual comprising administering to the individual an antigen-binding protein or composition according to any one of paragraphs 1 to 48. 50. An isolated nucleic acid encoding an antigen-binding protein according to any one of paragraphs 1 to 41. 51. A host cell transformed in vitro with a nucleic acid according to paragraph 50. 52. A method for producing an antigen binding protein according to any one of clauses 1 to 41, comprising culturing a host cell according to clause 51 under conditions for the production of the antigen binding protein. 53. The method of claim 52, further comprising isolating and / or purifying said antigen-binding protein. 54. The method of claim 53, further comprising formulating the antigen-binding protein into a composition comprising at least one additional component. 55. A method for producing an antigen-binding protein that specifically binds to and inhibits human ARG, the method comprising: producing a variant VH domain that is an amino acid sequence variant of a parent VH domain by adding, deleting, substituting, or inserting one or more amino acids in the amino acid sequence of the parent VH domain comprising HCDR1, HCDR2, and HCDR3 (the parent VH domain HCDR1, HCDR2, and HCDR3 are selected from the group consisting of C0021158fgl2, C0021181, C0021180, C0021177, C0021158, C0021158 IgG, C0021158fgl, C0021158dr, C0021061, C0020187, C0021155, C0021144, C0021142, C0021142 HCDR sets of IgG, C0021141, C0021139, C0021135, C0021133, C0021131, C0021129, C0021128, C0021124, C0021118, C0021101, C0021098, C0021097, C0021096, C0021092, C0021089, C0021065, C0021032, C0021022, C0021021, C0021017, and C0020065 56. A method comprising providing a variant VH domain (wherein the variant VH domain is a set of HCDRs selected from the group consisting of: C0021158fgl2, C0021181, ... 0021180, C0021177, C0021158, C0021158 IgG, C0021158fgl, C0021158dr, C0021061, C0020187, C0021155, C0021144, C0021142, C0021142 51. The method of clause 50, wherein the antibody is selected from IgG, C0021141, C0021139, C0021135, C0021133, C0021131, C0021129, C0021128, C0021124, C0021118, C0021101, C0021098, C0021097, C0021096, C0021092, C0021089, C0021065, C0021032, C0021022, C0021021, C0021017 and C0020065, or germlined versions thereof. 57. The one or more VL domains are variant VL domains provided by addition, deletion, substitution or insertion of one or more amino acids in the amino acid sequence of a parent VL domain comprising LCDR1, LCDR2 and LCDR3, wherein the parent VL domains LCDR1, LCDR2 and LCDR3 are selected from the following: C0021158fgl2, C0021181, C0021180, C0021177, C0021158, C0021158 IgG, C0021158fgl, C0021158dr, C0021061, C0020187, C0021155, C0021144, C0021142, C0021142 57. The method of claim 55 or 56, wherein the set of LCDRs is selected from the set of LCDRs of IgG, C0021141, C0021139, C0021135, C0021133, C0021131, C0021129, C0021128, C0021124, C0021118, C0021101, C0021098, C0021097, C0021096, C0021092, C0021089, C0021065, C0021032, C0021022, C0021021, C0021017 and C0020065, to produce one or more VL domains which are amino acid sequence variants of a parent VL domain. 58. The parent VL domain is selected from the group consisting of C0021158fgl2, C0021181, C0021180, C0021177, C0021158, C0021158 IgG, C0021158fgl, C0021158dr, C0021061, C0020187, C0021155, C0021144, C0021142, and C0021142 58. The method of claim 57, wherein the antibody is any of IgG, C0021141, C0021139, C0021135, C0021133, C0021131, C0021129, C0021128, C0021124, C0021118, C0021101, C0021098, C0021097, C0021096, C0021092, C0021089, C0021065, C0021032, C0021022, C0021021, C0021017 and C0020065, or germlined versions thereof. 59. The method of any of clauses 55 to 58, further comprising producing the antigen-binding protein antigen-binding domain as a component of an IgG, scFv or Fab antigen-binding protein. 60. A method for producing an antigen-binding protein that binds to and inhibits human ARG2, the method comprising: providing a starting nucleic acid encoding a VH domain, or a starting repertoire of nucleic acids each encoding a VH domain, wherein the one or more VH domains comprise the HCDR1, HCDR2 and / or HCDR3 to be replaced or lack the HCDR1, HCDR2 and / or HCDR3 coding regions; IgG, C0021158fgl, C0021158dr, C0021061, C0020187, C0021155, C0021144, C0021142, C0021142 IgG, C0021141, C0021139, C0021135, C0021133, C0021131, C0021129, C0021128, C0021124, C00211 18, C0021101, C0021098, C0021097, C0021096, C0021092, C0021089, C0021065, C0021032, C0021022 , C0021021, C0021017 and C0020065, whereby said one or more donor nucleic acids are inserted into the CDR1, CDR2 and / or CDR3 regions in said starting nucleic acid or starting repertoire to provide a product repertoire of nucleic acids encoding VH domains; expressing the nucleic acids of said product repertoire to produce product VH domains; optionally combining said product VH domains with one or more VL domains; Selecting an antigen binding protein for ARG2 (wherein the antigen binding protein comprises a product VH domain and optionally a VH domain); and recovering the antigen-binding protein or the nucleic acid encoding it. A method comprising: 61. The method according to item 60, wherein the donor nucleic acid is produced by mutation of the HCDR1 and / or HCDR2. 62. The method of claim 60 or 61, wherein the donor nucleic acid is produced by mutation of HCDR3. 63. The method of any one of items 60 to 62, comprising providing the donor nucleic acid by random mutation of the nucleic acid. 64. The method of any of clauses 60-63, further comprising combining the product VH domain contained within the recovered antigen binding protein with an antigen binding protein constant region. 65. A method according to any one of clauses 60 to 64, comprising providing an IgG, scFv or Fab antigen-binding protein comprising the product VH and VL domains.

[0017] Detailed Description The present invention relates to antigen-binding proteins, particularly antibodies and antigen-binding fragments thereof, which comprise an antigen-binding site for ARG2. The antibodies or antigen-binding fragments thereof of the present invention can be produced by recombinant means. A "recombinant antibody" is an antibody produced by recombinantly engineered host cells. The antibodies or antigen-binding fragments thereof of the present invention are optionally isolated or purified.

[0018] The term "ARG2" may refer to human ARG2 and / or cynomolgus monkey ARG2, unless the context requires otherwise. Preferably, the term "ARG2" refers to human ARG2 (Uniprot ID: P78540), unless the context requires otherwise.

[0019] The term "antibody" refers to an immunoglobulin, whether natural or partially or wholly synthetically produced. Antibodies may be human or humanized. The term "antibody" is used herein in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity. The antibody is preferably a monoclonal antibody, more preferably a human monoclonal antibody. Examples of antibodies include immunoglobulin isotypes, such as immunoglobulin G, and its isotypic subclasses, such as IgG1, IgG2, IgG3, and IgG4, as well as fragments thereof. The four human subclasses (IgG1, IgG2, IgG3, and IgG4) each contain a different heavy chain; however, they are highly homologous and differ primarily in the hinge region and the extent to which they activate the host immune system. IgG1 and IgG4 contain two interchain disulfide bonds in the hinge region, IgG2 has four, and IgG3 has 11 interchain disulfide bonds.

[0020] The terms "antibody" and "antibody molecule" as used herein refer to Fab and scFv fragments, provided that said fragments contain a CDR-based antigen-binding site against ARG2. "Antibody fragment" refers to a molecule other than an intact antibody that contains a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; and single-chain molecules (e.g., scFv). Thus, unless the context requires otherwise, the terms "antigen-binding protein," "antibody," or "antibody molecule" are synonymous with "antibody or antigen-binding fragment" as used herein.

[0021] Antibodies are immunoglobulins that share the same basic structure: two heavy and two light chains that form two Fab arms containing identical domains connected by a flexible hinge region to an Fc domain that confers the antibody stem, i.e., the classic "Y" shape. The Fab domains are composed of two variable and two constant domains, including a variable heavy (VH) domain and a constant heavy chain 1 (CH1) domain on the heavy chain and a variable light (VL) domain and a constant light (CL) domain on the light chain. The two variable domains (VH and VL) form a variable fragment (Fv), which confers the antibody's CDR-based antigen specificity, while the constant domains (CH1 and VL) act as a structural framework. Each variable domain contains three hypervariable loops known as complementarity-determining regions (CDRs). In each of VH and VL, three CDRs (CDR1, CDR2, and CDR3) are flanked by four less variable framework (FR) regions (FR1, FW2, FW3, and FW4), giving the structure: FW1-CDR1-FW2-CDR2-FW3-CDR3-FW4. The CDRs provide specific antigen recognition sites on the surface of the antibody.

[0022] In general, unless indicated otherwise (explicitly or by context), amino acid residues are numbered according to the Kabat numbering scheme (Kabat et al., 1991).

[0023] Monoclonal and other antibodies can be selected and, using recombinant DNA technology methods, produced to produce other antibodies or chimeric molecules that largely retain the specificity of the original antibody. Such techniques may involve transferring CDRs into various immunoglobulin frameworks or grafting variable regions onto different immunoglobulin constant regions. Transferring CDRs from one immunoglobulin into another is described, for example, in European Patent Application No. A-184187, British Patent Application No. 2188638A, or European Patent Application No. A-239400. Alternatively, hybridomas or other cells producing antibody molecules can be subjected to genetic mutations or other alterations that may or may not alter the binding specificity of the antibodies produced.

[0024] Antibodies can be modified in a number of ways, and the term "antibody" should be interpreted as encompassing antibody fragments, derivatives, functional equivalents and homologues of antibodies, and includes any polypeptide having an immunoglobulin binding domain, whether natural or wholly or partially synthetic.

[0025] The antigen-binding proteins of the present invention, for example, antibodies or antigen-binding fragments of the present invention, bind to and inhibit ARG2, particularly human ARG2. In this context, "bind" may refer to specific binding. The term "specific" refers to a state in which the antigen-binding protein does not exhibit any significant binding to molecules other than its specific binding partner (in this case, ARG2). The term "specific" is also applicable to cases in which an antibody molecule is specific for a particular epitope carried by several antigens, for example, an epitope on ARG2, in which case the antibody molecule will be able to bind to various antigens carrying the epitope.

[0026] An antibody that binds to the same epitope as or an overlapping epitope with a reference antibody refers to an antibody that blocks binding of a reference antibody to its binding partner (e.g., antigen) by 50% or more in a competition assay, and conversely, the reference antibody blocks binding of the antibody to its binding partner by 50% or more in a competition assay. Such antibodies are said to compete for binding to the epitope of interest.

[0027] Amino acids may be referred to by their one-letter or three-letter abbreviations or by their full names. The one-letter and three-letter abbreviations for each of the 20 standard amino acids, as well as their full names, are listed below.

[0028] [Table 1]

[0029] In a preferred embodiment, the ARG2 antibodies of the invention are any of the following: C0021158fgl2, C0021181, C0021180, C0021177, C0021158, C0021158 IgG, C0021158fgl, C0021158dr, C0021061, C0020187, C0021155, C0021144, C0021142, C0021142 IgG, C0021141, C0021139, C0021135, C00 HCDR1, HCDR2 and HCDR3 of VH and / or LCDR1, LCDR2 and LCDR3 of VL of an antibody selected from C0021065, C0021032, C0021022, C0021021, C0021017 and C0020065.

[0030] More preferably, the ARG2 antibodies of the present invention are any of the following: C0021158fgl2, C0021181, C0021180, C0021177, C0021158, C0021158 IgG, C0021158fgl, C0021158dr, C0021061, C0020187 (parental lead isolated clone), C0021155, C0021144, C0021142, C0021142 IgG, C0021141, C0021139, C0021135, C0021133, C0021131, C0021129, C0021128, C0021124, C0021118, C0 021101, C0021098, C0021097, C0021096, C0021092, C0021089, C0021065, C0021032, C0021022, C0021021, comprising a VH and / or VL having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence homology to an antibody selected from C0021017 and C0020065 (alternative lead isolated clones).

[0031] In another preferred embodiment, the ARG2 antibody of the invention comprises a VH having HCDR1, HCDR2, and HCDR3 and / or a VL having LCDR1, LCDR2, and LCDR3, wherein the VH and / or VL are selected from the following: C0021158fgl2, C0021181, C0021180, C0021177, C0021158, C0021158 IgG, C0021158fgl, C0021158dr, C0021061, C0020187, C0021155, C0021144, C0021142, C0021142 IgG, C0021141, C0021139, C0021135, C0021133, C0021131, C0021129, C0021128, C0021124, C002111 8, C0021101, C0021098, C0021097, C0021096, C0021092, C0021089, C0021065, C0021032, C0021022, C and / or C0020065. The antibody has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the VH and / or VL of an antibody selected from 0021021, C0021017, and C0020065.

[0032] In particularly preferred embodiments, the ARG2 antibodies of the invention are selected from the group consisting of: C0021158fgl2, C0021181, C0021180, C0021177, C0021158, C0021158 IgG, C0021158fgl, C0021158dr, C0021061, C0020187, C0021155, C0021144, C0021142, C0021142 The antibody comprises the VH and VL of an antibody selected from IgG, C0021141, C0021139, C0021135, C0021133, C0021131, C0021129, C0021128, C0021124, C0021118, C0021101, C0021098, C0021097, C0021096, C0021092, C0021089, C0021065, C0021032, C0021022, C0021021, C0021017 and C0020065.

[0033] Unless otherwise indicated, sequence homology is based on the Clustal W alignment (Thompson, Higgins et al. 1994).

[0034] [Table 2]

[0035] [Table 3]

[0036] In Figure 24, Table 4 shows the sequence identity across the entire VH sequence (Kabat residues 1→113) of the parental C0020187 antibody and the 33 affinity-matured antibodies described herein. All affinity-matured sequences share at least 90% identity with the parental C0020187 antibody. Percent diversity and percent divergence values ​​obtained from Clustal W alignment (Thompson, Higgins et al. 1994).

[0037] In Figure 25, Table 5 shows the sequence identity across the entire VL sequence (Kabat residues 1→107) of the parent C0020187 antibody and the 33 affinity-matured antibodies described herein. All affinity-matured sequences share at least 89.1% identity with the parent C0020187 antibody. Percent diversity and percent divergence values ​​obtained from Clustal W alignment (Thompson, Higgins et al. 1994).

[0038]

[0021] The antibodies of the invention can comprise a VH and / or VL, wherein the amino acid sequence is the amino acid sequence of C0021158fgl2, C0021133, or C0020187 at each of the following positions, or optionally the following: C0021181, C0021180, C0021177, C0021158, C0021158 IgG, C0021158 fgl, C0021158dr, C0021061, C0021155, C0021144, C0021142, C0021142 Other exemplary residues found in the clones described herein selected from IgG, C0021141, C0021139, C0021135, C0021131, C0021129, C0021128, C0021124, C0021118, C0021101, C0021098, C0021097, C0021096, C0021092, C0021089, C0021065, C0021032, C0021022, C0021021, and C0021017 may be present as specified at the given positions shown below:

[0039] [Table 4]

[0040] [Table 5]

[0041] [Table 6]

[0042] Table 7

[0043] Table 8

[0044] Table 9

[0045] Table 10

[0046] Table 11

[0047] Table 12

[0048] Table 13

[0049] Table 14

[0050] Table 15

[0051] Table 16

[0052] Antibodies of the invention may comprise one or more, for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, additional amino acid modifications in the VH and / or VL sequence, provided that the functionality of the antibody is maintained.

[0053] The modification may be an amino acid substitution, deletion or insertion. Preferably, the modification is a substitution.

[0054] In preferred embodiments, where one or more amino acids are replaced with another amino acid, the substitution is a conservative substitution. In some embodiments, variants having one or more amino acids are provided. Target sites for substitution mutagenesis include the CDRs and FW. Amino acid substitutions can be introduced into antibodies or binding polypeptides, and the products are screened for the desired activity, such as retained / improved antigen binding, higher inhibition, or immunogenicity.

[0055] Amino acids share common side chain properties: (1) Hydrophobic: Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) Residues that influence chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe They can be classified according to:

[0056] Conservative substitutions are made by exchanging members within one of these groups, whereas non-conservative substitutions are made by exchanging a member of one of these groups for a member of another group.

[0057] In some embodiments, substitutions may be functionally conservative, i.e., the substitution may not affect (or not substantially affect) one or more functional properties of an antibody containing the substitution (e.g., binding affinity to ARG2, inhibition of ARG2) compared to a comparable unsubstituted antibody.

[0058] In a preferred embodiment, the ARG2 antibodies of the invention may comprise a VH and / or VL domain sequence of the invention described herein that has one or more amino acid sequence modifications (additions, deletions, substitutions and / or insertions of amino acid residues), preferably no more than 20 modifications, no more than 15 modifications, no more than 10 modifications, no more than 5 modifications, no more than 4 modifications, no more than 3 modifications, no more than 2 modifications, or no more than 1 modification, compared to the VH and / or VL of the invention depicted herein.

[0059] In a preferred embodiment, an antibody of the invention comprises the HCDR3 sequence of C0021158 fgl2 of SEQ ID NO:315.

[0060] All clones of the present invention, relative to the parent clone C0020187 described herein, share a common HCDR3 sequence shown in SEQ ID NO: 315 (LRADLGLYMDL), which is believed to be important in determining specificity for and inhibition of the enzymatic activity of human ARG2.

[0061] Preferably, the antibody of the present invention comprises: a VH domain comprising HCDR1 (SEQ ID NO: 313), HCDR2 (SEQ ID NO: 314), and HCDR3 (SEQ ID NO: 315), and a VL domain comprising LCDR1 (SEQ ID NO: 318), LCDR2 (SEQ ID NO: 319), and LCDR3 (SEQ ID NO: 320) of C0021158fgl2; a VH domain comprising HCDR1 (SEQ ID NO: 343), HCDR2 (SEQ ID NO: 344) and HCDR3 (SEQ ID NO: 345) of C0021181, and a VL domain comprising LCDR1 (SEQ ID NO: 348), LCDR2, (SEQ ID NO: 349) and LCDR3 (SEQ ID NO: 350); a VH domain comprising HCDR1 (SEQ ID NO: 313), HCDR2 (SEQ ID NO: 314) and HCDR3 (SEQ ID NO: 315) of C0021180, and a VL domain comprising LCDR1 (SEQ ID NO: 318), LCDR2, (SEQ ID NO: 319) and LCDR3 (SEQ ID NO: 320); a VH domain comprising HCDR1 (SEQ ID NO: 323), HCDR2 (SEQ ID NO: 324) and HCDR3 (SEQ ID NO: 325) of C0021177, and a VL domain comprising LCDR1 (SEQ ID NO: 328), LCDR2, (SEQ ID NO: 329) and LCDR3 (SEQ ID NO: 330); a VH domain comprising HCDR1 (SEQ ID NO: 273), HCDR2 (SEQ ID NO: 274) and HCDR3 (SEQ ID NO: 275) of C0021158, and a VL domain comprising LCDR1 (SEQ ID NO: 278), LCDR2, (SEQ ID NO: 279) and LCDR3 (SEQ ID NO: 280); C0021158 IgG, a VH domain comprising HCDR1 (SEQ ID NO: 283), HCDR2 (SEQ ID NO: 284) and HCDR3 (SEQ ID NO: 285), and a VL domain comprising LCDR1 (SEQ ID NO: 288), LCDR2, (SEQ ID NO: 289) and LCDR3 (SEQ ID NO: 290); a VH domain comprising HCDR1 (SEQ ID NO: 303), HCDR2 (SEQ ID NO: 304) and HCDR3 (SEQ ID NO: 305) and a VL domain comprising LCDR1 (SEQ ID NO: 308), LCDR2, (SEQ ID NO: 309) and LCDR3 (SEQ ID NO: 310) of C0021158fgl; a VH domain comprising HCDR1 (SEQ ID NO: 293), HCDR2 (SEQ ID NO: 294) and HCDR3 (SEQ ID NO: 295) and a VL domain comprising LCDR1 (SEQ ID NO: 298), LCDR2, (SEQ ID NO: 299) and LCDR3 (SEQ ID NO: 300) of C0021158dr; C0021061, a VH domain comprising HCDR1 (SEQ ID NO: 63), HCDR2 (SEQ ID NO: 64) and HCDR3 (SEQ ID NO: 65), and LCDR1 (SEQ ID NO: 68), LCDR2 (SEQ ID NO: 69), R2, (SEQ ID NO: 69) and LCDR3 (SEQ ID NO: 70); C0020187, a VH domain comprising HCDR1 (SEQ ID NO: 13), HCDR2 (SEQ ID NO: 14) and HCDR3 (SEQ ID NO: 15), and a VL domain comprising LCDR1 (SEQ ID NO: 18), LCDR2, (SEQ ID NO: 19) and LCDR3 (SEQ ID NO: 20); a VH domain comprising HCDR1 (SEQ ID NO: 263), HCDR2 (SEQ ID NO: 264) and HCDR3 (SEQ ID NO: 265) of C0021155, and a VL domain comprising LCDR1 (SEQ ID NO: 268), LCDR2, (SEQ ID NO: 269) and LCDR3 (SEQ ID NO: 270); a VH domain comprising HCDR1 (SEQ ID NO: 253), HCDR2 (SEQ ID NO: 254) and HCDR3 (SEQ ID NO: 255) and a VL domain comprising LCDR1 (SEQ ID NO: 258), LCDR2, (SEQ ID NO: 259) and LCDR3 (SEQ ID NO: 260) of C0021144; a VH domain comprising HCDR1 (SEQ ID NO: 233), HCDR2 (SEQ ID NO: 234) and HCDR3 (SEQ ID NO: 235) and a VL domain comprising LCDR1 (SEQ ID NO: 238), LCDR2, (SEQ ID NO: 239) and LCDR3 (SEQ ID NO: 240) of C0021142; C0021142 IgG, a VH domain comprising HCDR1 (SEQ ID NO: 243), HCDR2 (SEQ ID NO: 244) and HCDR3 (SEQ ID NO: 245), and a VL domain comprising LCDR1 (SEQ ID NO: 248), LCDR2, (SEQ ID NO: 249) and LCDR3 (SEQ ID NO: 250); a VH domain comprising HCDR1 (SEQ ID NO: 223), HCDR2 (SEQ ID NO: 224) and HCDR3 (SEQ ID NO: 225) and a VL domain comprising LCDR1 (SEQ ID NO: 228), LCDR2, (SEQ ID NO: 229) and LCDR3 (SEQ ID NO: 230) of C0021141; a VH domain comprising HCDR1 (SEQ ID NO: 213), HCDR2 (SEQ ID NO: 214) and HCDR3 (SEQ ID NO: 215) and a VL domain comprising LCDR1 (SEQ ID NO: 218), LCDR2, (SEQ ID NO: 219) and LCDR3 (SEQ ID NO: 220) of C0021139; a VH domain comprising HCDR1 (SEQ ID NO: 203), HCDR2 (SEQ ID NO: 204) and HCDR3 (SEQ ID NO: 205) of C0021135, and a VL domain comprising LCDR1 (SEQ ID NO: 208), LCDR2, (SEQ ID NO: 209) and LCDR3 (SEQ ID NO: 210); a VH domain comprising HCDR1 (SEQ ID NO: 193), HCDR2 (SEQ ID NO: 194) and HCDR3 (SEQ ID NO: 195) of C0021133, and a VL domain comprising LCDR1 (SEQ ID NO: 198), LCDR2, (SEQ ID NO: 199) and LCDR3 (SEQ ID NO: 200); a VH domain comprising HCDR1 (SEQ ID NO: 183), HCDR2 (SEQ ID NO: 184) and HCDR3 (SEQ ID NO: 185) and a VL domain comprising LCDR1 (SEQ ID NO: 188), LCDR2, (SEQ ID NO: 189) and LCDR3 (SEQ ID NO: 190) of C0021131; a VH domain comprising HCDR1 (SEQ ID NO: 173), HCDR2 (SEQ ID NO: 174) and HCDR3 (SEQ ID NO: 175) of C0021129, and a VL domain comprising LCDR1 (SEQ ID NO: 178), LCDR2, (SEQ ID NO: 179) and LCDR3 (SEQ ID NO: 180); a VH domain comprising HCDR1 (SEQ ID NO: 163), HCDR2 (SEQ ID NO: 164) and HCDR3 (SEQ ID NO: 165) of C0021128, and a VL domain comprising LCDR1 (SEQ ID NO: 168), LCDR2, (SEQ ID NO: 169) and LCDR3 (SEQ ID NO: 170); C0021124, a VH domain comprising HCDR1 (SEQ ID NO: 153), HCDR2 (SEQ ID NO: 154) and HCDR3 (SEQ ID NO: 155), and LCDR1 (SEQ ID NO: 158). a VL domain comprising LCDR2, (SEQ ID NO: 159) and LCDR3 (SEQ ID NO: 160); a VH domain comprising HCDR1 (SEQ ID NO: 143), HCDR2 (SEQ ID NO: 144) and HCDR3 (SEQ ID NO: 145) of C0021118, and a VL domain comprising LCDR1 (SEQ ID NO: 148), LCDR2, (SEQ ID NO: 149) and LCDR3 (SEQ ID NO: 150); a VH domain comprising HCDR1 (SEQ ID NO: 133), HCDR2 (SEQ ID NO: 134) and HCDR3 (SEQ ID NO: 135) and a VL domain comprising LCDR1 (SEQ ID NO: 138), LCDR2, (SEQ ID NO: 139) and LCDR3 (SEQ ID NO: 140) of C0021101; C0021098, a VH domain comprising HCDR1 (SEQ ID NO: 123), HCDR2 (SEQ ID NO: 124) and HCDR3 (SEQ ID NO: 125), and a VL domain comprising LCDR1 (SEQ ID NO: 128), LCDR2, (SEQ ID NO: 129) and LCDR3 (SEQ ID NO: 130); C0021097, a VH domain comprising HCDR1 (SEQ ID NO: 113), HCDR2 (SEQ ID NO: 114) and HCDR3 (SEQ ID NO: 115), and a VL domain comprising LCDR1 (SEQ ID NO: 118), LCDR2, (SEQ ID NO: 119) and LCDR3 (SEQ ID NO: 120); C0021096, a VH domain comprising HCDR1 (SEQ ID NO: 103), HCDR2 (SEQ ID NO: 104) and HCDR3 (SEQ ID NO: 105), and a VL domain comprising LCDR1 (SEQ ID NO: 108), LCDR2, (SEQ ID NO: 109) and LCDR3 (SEQ ID NO: 110); C0021092, a VH domain comprising HCDR1 (SEQ ID NO: 93), HCDR2 (SEQ ID NO: 94) and HCDR3 (SEQ ID NO: 95), and a VL domain comprising LCDR1 (SEQ ID NO: 98), LCDR2, (SEQ ID NO: 99) and LCDR3 (SEQ ID NO: 100); a VH domain comprising HCDR1 (SEQ ID NO: 83), HCDR2 (SEQ ID NO: 84) and HCDR3 (SEQ ID NO: 85) of C0021089, and a VL domain comprising LCDR1 (SEQ ID NO: 88), LCDR2, (SEQ ID NO: 89) and LCDR3 (SEQ ID NO: 90); C0021065, a VH domain comprising HCDR1 (SEQ ID NO: 73), HCDR2 (SEQ ID NO: 74) and HCDR3 (SEQ ID NO: 75), and a VL domain comprising LCDR1 (SEQ ID NO: 78), LCDR2, (SEQ ID NO: 79) and LCDR3 (SEQ ID NO: 80); a VH domain comprising HCDR1 (SEQ ID NO: 53), HCDR2 (SEQ ID NO: 54) and HCDR3 (SEQ ID NO: 55) of C0021032, and a VL domain comprising LCDR1 (SEQ ID NO: 58), LCDR2, (SEQ ID NO: 59) and LCDR3 (SEQ ID NO: 60); a VH domain comprising HCDR1 (SEQ ID NO: 43), HCDR2 (SEQ ID NO: 44) and HCDR3 (SEQ ID NO: 45) of C0021022, and a VL domain comprising LCDR1 (SEQ ID NO: 48), LCDR2, (SEQ ID NO: 49) and LCDR3 (SEQ ID NO: 50); C0021021, a VH domain comprising HCDR1 (SEQ ID NO: 33), HCDR2 (SEQ ID NO: 34) and HCDR3 (SEQ ID NO: 35), and a VL domain comprising LCDR1 (SEQ ID NO: 38), LCDR2, (SEQ ID NO: 39) and LCDR3 (SEQ ID NO: 40); a VH domain comprising HCDR1 (SEQ ID NO: 23), HCDR2 (SEQ ID NO: 24) and HCDR3 (SEQ ID NO: 25) of C0021017, and a VL domain comprising LCDR1 (SEQ ID NO: 28), LCDR2, (SEQ ID NO: 29) and LCDR3 (SEQ ID NO: 30); or C0020065, a VH domain comprising HCDR1 (SEQ ID NO: 3), HCDR2 (SEQ ID NO: 4) and HCDR3 (SEQ ID NO: 5), and a VL domain comprising LCDR1 (SEQ ID NO: 8), LCDR2, (SEQ ID NO: 9) and LCDR3 (SEQ ID NO: 10).

[0062] In a preferred embodiment, the antibody of the present invention comprises a VH domain having an HCDR1 domain of C0021158fgl2 of SEQ ID NO: 313, an HCDR3 sequence of C0021158fgl2 of SEQ ID NO: 315, and an HCDR2 sequence of C0021158fgl2 of SEQ ID NO: 314. Including Inn.

[0063] In a preferred embodiment, an antibody of the invention comprises the VH domain of C0021158fgl2 of SEQ ID NO: 312 or a VH domain having an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence of SEQ ID NO: 312.

[0064] In a preferred embodiment, an antibody of the invention comprises a VH domain having the HCDR3 sequence of SEQ ID NO: 315, which VH domain has the amino acid sequence of SEQ ID NO: 312 or an amino acid sequence with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence of SEQ ID NO: 312.

[0065] In a preferred embodiment, an antibody of the invention comprises a VH domain having an HCDR3 of C0021158fgl2 of SEQ ID NO: 315 and an HCDR2 sequence of C0021158fgl2 of SEQ ID NO: 314, wherein the VH domain has an amino acid sequence of SEQ ID NO: 312 or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence of SEQ ID NO: 312.

[0066] In a preferred embodiment, an antibody of the invention comprises a VH domain having an HCDR3 of C0021158fgl2 of SEQ ID NO: 315, an HCDR2 domain of C0021158fgl2 of SEQ ID NO: 314, and an HCDR1 domain of C0021158fgl2 of SEQ ID NO: 313, wherein the VH domain has an amino acid sequence of SEQ ID NO: 312 or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence of SEQ ID NO: 312.

[0067] Preferably, the antibodies of the present invention comprise the VH domains of the antibodies: C0021158fgl2 (SEQ ID NO: 312), C0021181 (SEQ ID NO: 342), C0021180 (SEQ ID NO: 332), C0021177 (SEQ ID NO: 322), C0021158 (SEQ ID NO: 272), C0021158 IgG (SEQ ID NO: 282), C0021158fgl (SEQ ID NO: 302), C0021158dr (SEQ ID NO: 292), C0021061 (SEQ ID NO: 62), C0020187 (SEQ ID NO: 12), C0021155 (SEQ ID NO: 262), C0021144 (SEQ ID NO: 252), C0021142 (SEQ ID NO: 232), C0021142 IgG (SEQ ID NO: 242), C0021141 (SEQ ID NO: 222), C0021139 (SEQ ID NO: 212), C0021135 (SEQ ID NO: 202), C0021133 (SEQ ID NO: 192), C0021131 (SEQ ID NO: 182), C0021129 (SEQ ID NO: 32), C0021128 (SEQ ID NO: 162), C0021124 (SEQ ID NO: 152), C0021118 (SEQ ID NO: 142), C0021101 (SEQ ID NO: 132), C0021098 (SEQ ID NO: 122), C0021097 (SEQ ID NO: 112), C0021096 (SEQ ID NO: 102), C0021092 (SEQ ID NO: 92), C0021089 (SEQ ID NO: 82), C0021065 (SEQ ID NO: 72), C0021032 (SEQ ID NO: 52), C0021022 (SEQ ID NO: 42), C0021021 (SEQ ID NO: 32), C0021017 (SEQ ID NO: 22), or C0020065 (SEQ ID NO: 2).

[0068] In a preferred embodiment, an antibody of the invention comprises a VL domain having the LCDR1 sequence of SEQ ID NO:318, the LCDR2 sequence of C0021158fgl2 of SEQ ID NO:319, and the LCDR3 sequence of C0021158fgl2 of SEQ ID NO:320.

[0069] In a preferred embodiment, an antibody of the invention comprises the VL domain of C0021158fgl2 of SEQ ID NO: 317 or a VL domain having an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence of SEQ ID NO: 317.

[0070] In a preferred embodiment, an antibody of the invention comprises a VL domain having an LCDR1 sequence of SEQ ID NO: 318, an LCDR2 sequence of C0021158fgl2 of SEQ ID NO: 319, an LCDR3 sequence of C0021158fgl2 of SEQ ID NO: 320, and an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence of SEQ ID NO: 317.

[0071] In a preferred embodiment, an antibody of the invention comprises a VL domain having the VL sequence of C0021158fgl2 of SEQ ID NO:317.

[0072] Preferably, the antibodies of the present invention comprise the VL domains of the antibodies: C0021158fgl2 (SEQ ID NO: 317), C0021181 (SEQ ID NO: 347), C0021180 (SEQ ID NO: 337), C0021177 (SEQ ID NO: 327), C0021158 (SEQ ID NO: 277), C0021158 IgG (SEQ ID NO: 287), C0021158fgl (SEQ ID NO: 307), C0021158dr (SEQ ID NO: 297), C0021061 (SEQ ID NO: 67), C0020187 (SEQ ID NO: 17), C0021155 (SEQ ID NO: 267), C0021144 (SEQ ID NO: 257), C0021142 (SEQ ID NO: 237), C0021142 IgG (SEQ ID NO: 247), C0021141 (SEQ ID NO: 227), C0021139 (SEQ ID NO: 217), C0021135 (SEQ ID NO: 207), C0021133 (SEQ ID NO: 197), C0021131 (SEQ ID NO: 187), C0021129 (SEQ ID NO: 177), C0021128 (SEQ ID NO: 167), C0021124 (SEQ ID NO: 157), C0021118 (SEQ ID NO: 147), C0021101 (SEQ ID NO: 137), C0021098 (SEQ ID NO: 127), C0021097 (SEQ ID NO: 117), C0021096 (SEQ ID NO: 107), C0021092 (SEQ ID NO: 97), C0021089 (SEQ ID NO: 87), C0021065 (SEQ ID NO: 77), C0021032 (SEQ ID NO: 57), C0021022 (SEQ ID NO: 47), C0021021 (SEQ ID NO: 37), C0021017 (SEQ ID NO: 27), and C0020065 (SEQ ID NO: 7).

[0073] Preferably, the antibody of the present invention comprises: C0021158 fgl2 VH domain (SEQ ID NO: 312) and VL domain (SEQ ID NO: 317), The VH domain (SEQ ID NO: 342) and VL domain (SEQ ID NO: 347) of C0021181; The VH domain (SEQ ID NO: 332) and VL domain (SEQ ID NO: 337) of C0021180; The VH domain (SEQ ID NO: 322) and VL domain (SEQ ID NO: 327) of C0021177; The VH domain (SEQ ID NO: 272) and VL domain (SEQ ID NO: 277) of C0021158; C0021158 IgG VH domain (SEQ ID NO: 282) and VL domain (SEQ ID NO: 287), The VH domain (SEQ ID NO: 302) and VL domain (SEQ ID NO: 307) of C0021158fgl; The VH domain (SEQ ID NO: 292) and VL domain (SEQ ID NO: 297) of C0021158dr; The VH domain (SEQ ID NO: 62) and VL domain (SEQ ID NO: 67) of C0021061, the VH domain (SEQ ID NO: 12) and VL domain (SEQ ID NO: 17) of C0020187, the VH domain (SEQ ID NO: 262) and VL domain (SEQ ID NO: 267) of C0021155, The VH domain (SEQ ID NO: 252) and VL domain (SEQ ID NO: 257) of C0021144; The VH domain (SEQ ID NO: 232) and VL domain (SEQ ID NO: 237) of C0021142; C0021142 IgG VH domain (SEQ ID NO: 242) and VL domain (SEQ ID NO: 247), the VH domain (SEQ ID NO: 222) and VL domain (SEQ ID NO: 227) of C0021141; the VH domain (SEQ ID NO: 212) and VL domain (SEQ ID NO: 217) of C0021139; The VH domain (SEQ ID NO: 202) and VL domain (SEQ ID NO: 207) of C0021135; the VH domain (SEQ ID NO: 192) and VL domain (SEQ ID NO: 197) of C0021133; the VH domain (SEQ ID NO: 182) and VL domain (SEQ ID NO: 187) of C0021131; the VH domain (SEQ ID NO: 172) and VL domain (SEQ ID NO: 177) of C0021129; The VH domain (SEQ ID NO: 162) and VL domain (SEQ ID NO: 167) of C0021128; The VH domain (SEQ ID NO: 152) and VL domain (SEQ ID NO: 157) of C0021124; the VH domain (SEQ ID NO: 142) and VL domain (SEQ ID NO: 147) of C0021118; the VH domain (SEQ ID NO: 132) and VL domain (SEQ ID NO: 137) of C0021101; The VH domain (SEQ ID NO: 122) and VL domain (SEQ ID NO: 127) of C0021098; the VH domain (SEQ ID NO: 112) and VL domain (SEQ ID NO: 117) of C0021097; the VH domain (SEQ ID NO: 102) and VL domain (SEQ ID NO: 107) of C0021096; VH domain (SEQ ID NO: 92) and VL domain (SEQ ID NO: 97) of C0021092, VH domain (SEQ ID NO: 82) and VL domain (SEQ ID NO: 87) of C0021089, VH domain (SEQ ID NO: 72) and VL domain (SEQ ID NO: 77) of C0021065, VH domain (SEQ ID NO: 52) and VL domain (SEQ ID NO: 57) of C0021032, VH domain (SEQ ID NO: 42) and VL domain (SEQ ID NO: 47) of C0021022, VH domain (SEQ ID NO: 32) and VL domain (SEQ ID NO: 37) of C0021021, the VH domain (SEQ ID NO: 22) and VL domain (SEQ ID NO: 27) of C0021017, or The VH domain (SEQ ID NO: 2) and VL domain (SEQ ID NO: 7) of C0020065.

[0074] Sequence identity was determined using the Bioedit ClustalW algorithm (Thompson, JD, et al. (1994). "CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice." Nucleic Acids Res 22(22): 4673-4680).

[0075] The antibody may comprise a CH2 domain. The CH2 domain is preferably located N-terminal to the CH3 domain, as in human IgG molecules. The antibody CH2 domain is preferably the CH2 domain of human IgG1, IgG2, IgG3, or IgG4, more preferably the CH2 domain of human IgG1 or IgG2. The sequences of human IgG domains are known in the art.

[0076] The antibody may comprise an immunoglobulin hinge region, or a portion thereof, at the N-terminus of the CH2 domain. The immunoglobulin hinge region enables the two CH2-CH3 domain sequences to bind and form a dimer. Preferably, the hinge region, or a portion thereof, is an IgG1, IgG2, IgG3, or IgG4 hinge region, or a portion thereof. More preferably, the hinge region, or a portion thereof, is an IgG1 or IgG2 hinge region, or a portion thereof.

[0077] The sequence of the CH3 domain is not particularly limited. Preferably, the CH3 domain is a human immunoglobulin γ domain, such as human IgG1, IgG2, IgG3, or IgG4. A CH3 domain, most preferably a human IgG1 or IgG2 CH3 domain.

[0078] The antibodies of the present invention may comprise a human IgG1, IgG2, IgG3, or IgG4 constant region. The sequences of human IgG1, IgG2, IgG3, or IgG4 domains are known in the art. The constant region may be modified, for example, to extend half-life. The Fc domain of the antibodies of the present invention may be human IgG or modified human IgG. The Fc domain may be modified to achieve desired antibody functions. The Fc may be modified to increase or decrease effector function and / or extend the half-life of the antibody.

[0079] In a preferred embodiment, the Fc domain is a modified Fc domain with enhanced effector function. The potency of antibodies can be increased by enhancing their ability to mediate cytotoxic functions, such as antibody-dependent cellular cytotoxicity (ADCC), cell-dependent cellular phagocytosis (ADCP), and complement-dependent cell-mediated cytotoxicity (CDC). Several Fc domain modifications have been demonstrated that either directly or indirectly enhance Fc receptor binding, thereby significantly enhancing cytotoxicity, such as S239D / A330L / I332E (referred to as "3M"), F243L, or G236A. Modifications of IgG1 that enhance ADCC include F243L / R292P / Y300L / V305I / P396L, S239D / I332E, S239D / A330L / I332E, S298A / E333A / K334A, or L234Y / L235Q / G236W / S239M / H268D / D270E / S298A in one heavy chain and D270E / K326D / A330M / K334E in the opposite heavy chain; modifications of IgG1 that enhance ACDP include G236A / S239D / I332E, and modifications of IgG1 that enhance CDC include K326W / E333S or S267E / H268F / S324T. CDC (increased C1q binding) can also be enhanced by the use of an IgG1 / IgG3 cross-subclass Fc or by hexamerization using an E345R / E430G / S440Y modified IgG1.

[0080] Another approach to enhancing effector function involves modifying the glycosylation of the Fc domain. FcγR reacts with carbohydrates on the CH2 domain, and the composition of the glycan has a substantial effect on effector function activity. Defucosylated (non-fucosylated) antibodies exhibit significantly enhanced ADCC activity due to increased binding to FcγRIIIa. The ADCC activity of glycosylated IgG1 antibodies is sensitive to the fucosylation state of the Fc glycan, and ADCC activity increases both in vitro and in vivo upon fucose removal ("defucosylation"). The effect of defucosylation on the activity of IgG4 antibodies has not been clearly characterized, but it has been shown to increase the in vitro ADCC activity of IgG4 antibodies. Thus, defucosylation of the antibodies of the present invention can enhance effector function.

[0081] Modifications to the Fc of the antibodies of the invention can be performed to increase in vivo stability. IgG4 subclasses undergo Fab-arm exchange, in which heavy chains can be swapped between IgG4s in vivo. The S228P mutation has been shown to block this recombination process, allowing for more predictable design of therapeutic IgG4 antibodies.

[0082] IgG naturally persists in serum for extended periods due to FcRn-mediated recycling, giving it a typical half-life of approximately 21 days. To extend half-life, the pH-dependent interaction between the Fc domain and FcRn can be manipulated to increase affinity at pH 6.0 while maintaining minimal binding at pH 7.4. The mutation T250Q / M428L ("TM") resulted in an approximately two-fold increase in IgG half-life in rhesus monkeys. The M252Y / S254T / T256E variant ("YTE") resulted in an approximately four-fold increase in IgG half-life in cynomolgus monkeys. A longer half-life is desirable in some situations to maintain or improve the efficacy of the administered antibody while reducing the frequency of administration. The antibodies of the present invention may be provided as half-life extension mutants engineered to extend their in vivo half-life after administration, and thus may be provided as M252Y / S254T / T256E or T250Q / M428L mutants.

[0083] The present invention also provides a nucleic acid or set of nucleic acids encoding an antibody or antigen-binding fragment of the invention, as well as one or more vectors comprising such a nucleic acid or set of nucleic acids.

[0084] When a nucleic acid encodes the VH and VL domains, or the heavy and light chains, of an antibody molecule of the invention, the two domains or chains can be encoded on two separate nucleic acid molecules or on the same nucleic acid molecule.

[0085] The isolated nucleic acid can be used to express the antibody molecule of the present invention. The nucleic acid will generally be provided in the form of a recombinant vector for expression. Accordingly, another aspect of the present invention provides a vector comprising the nucleic acid, as described above. A suitable vector can be selected or constructed containing appropriate regulatory sequences, such as promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes, and other sequences as needed. Preferably, the vector contains appropriate regulatory sequences for driving expression of the nucleic acid in a host cell. The vector may be a plasmid, virus, e.g., phage, or phagemid, as appropriate.

[0086] The nucleic acid molecules or vectors described herein may be introduced into a host cell. Techniques for the introduction of nucleic acid molecules or vectors into host cells are well established in the art and any suitable technique may be used. A wide variety of host cells suitable for the production of recombinant antibody molecules are known in the art and include bacterial, yeast, insect or mammalian host cells. Preferred The host cell is a mammalian cell, such as a CHO, NS0, or HEK cell, such as a HEK293 cell.

[0087] Recombinant host cells comprising the nucleic acid molecules or vectors of the invention are also provided. Such recombinant host cells can be used to produce antibodies of the invention. Accordingly, methods of producing antibodies of the invention are also provided, comprising culturing recombinant host cells under conditions suitable for antibody production. The methods may further comprise the step of isolating and / or purifying the antibody molecules.

[0088] Thus, the present invention provides a method for producing an antibody of the invention, comprising expressing nucleic acid encoding the antibody in a host cell and, optionally, isolating and / or purifying the antibody thus produced. Methods for culturing host cells are known in the art. Techniques for purifying recombinant antibodies are known in the art and include, for example, HPLC, FPLC, or affinity chromatography using Protein A or Protein L. In some embodiments, purification may be performed using an affinity tag on the antibody. The method may also include formulating the antibody into a composition comprising an excipient, for example, a pharmaceutical composition comprising a pharmaceutically acceptable excipient.

[0089] The antibodies and compositions of the invention are expected to be useful in therapeutic and diagnostic applications, particularly in conditions associated with arginase activation and upregulation, such as in humans with cancer, immune cell dysfunction, infectious diseases, vascular disease, cardiovascular disease, endothelial dysfunction, aging and cellular senescence, CNS disease and injury; diabetes-related diseases or cystic fibrosis, e.g., infectious diseases associated with cystic fibrosis.

[0090] The present invention further provides an antibody of the present invention for use in a method of treatment. Also provided is a method of treating a patient, comprising administering to the patient a therapeutically effective amount of an antibody of the present invention. Also provided is the use of an antibody of the present invention for use in the manufacture of a medicament. As referred to herein, the patient is a human patient.

[0091] The present invention also provides an antibody of the present invention for use in a method of treating cancer in a patient. Further, a method of treating cancer in a patient is provided, comprising administering to the patient a therapeutically effective amount of an antibody of the present invention. Further, the use of an antibody molecule of the present invention for use in the manufacture of a medicament for treating cancer in a patient is provided.

[0092] In another aspect, the present invention provides a method for producing a pharmaceutical composition comprising: a) treatment of cancer, b) delay in cancer progression; c) Prolonging survival of patients suffering from cancer; d) stimulation of a cellular immune response, or e) Restoration or promotion of T cell proliferation The present invention relates to an antibody that binds to and inhibits human ARG2, for use in

[0093] The present invention also provides an antibody or composition of the present invention for use in a method for treating a disease in a patient. Furthermore, a method for treating a disease in a patient is also provided, comprising administering a therapeutically effective amount of an antibody or composition of the present invention to the patient. Furthermore, the use of an antibody molecule of the present invention for use in the manufacture of a medicament for treating a disease in a patient is also provided. In each case, the disease may be a condition associated with ARG activation and upregulation, such as cancer, immune cell dysfunction, infectious disease, vascular disease, cardiovascular disease, endothelial dysfunction, aging and cellular senescence, CNS disease and injury; diabetes-related disease, or cystic fibrosis, e.g., an infectious disease associated with cystic fibrosis.

[0094] The antibodies described herein may be intended for use in methods of treatment of the human or animal body. Related aspects of the invention provide: (i) an antibody molecule or composition as described herein for use as a medicament; (ii) an antibody molecule or composition as described herein for use in a method for treating a disease or disorder; (iii) use of an antibody molecule or composition described herein in the manufacture of a medicament for use in the treatment of a disease or disorder; (iv) A method of treating a disease or disorder in an individual, the method comprising administering to the individual a therapeutically effective amount of an antibody molecule or composition described herein.

[0095] The individual may be a patient, preferably a human patient. As used herein, the term "patient" may also refer to an animal, e.g., a mammal. Treatment may be any treatment or therapy that achieves some desired therapeutic effect, e.g., inhibiting or slowing the progression of a disease state, including reducing the rate of progression, halting the rate of progression, ameliorating the disease state, curing or remission (whether partial or complete) of the disease state, preventing, ameliorating, delaying, alleviating or arresting one or more symptoms and / or signs of the disease state, or prolonging the survival of the individual or patient beyond that expected if not treated.

[0096] Also included is treatment as a preventative measure (i.e., prevention). For example, an individual susceptible to or at risk of developing or recurring a disease, such as cancer, can be treated as described herein. Such treatment can prevent or delay the development or recurrence of the disease in the individual.

[0097] Although antibody molecules can be administered alone, they are typically administered in the form of a pharmaceutical composition, which may comprise at least one component in addition to the antibody molecule. Another aspect of the present invention therefore provides a pharmaceutical composition comprising an antibody as described herein. Methods are also provided which comprise formulating the antibody molecule into a pharmaceutical composition.

[0098] In addition to the antibody molecule, the pharmaceutical composition may include pharmaceutically acceptable excipients, carriers, buffers, stabilizers, or other materials known to those of skill in the art. As used herein, the term "pharmaceutically acceptable" refers to compounds, materials, compositions, and / or dosage forms that are suitable for use in contact with the tissues of a subject (e.g., a human) without excessive toxicity, irritation, allergic response, or other impairment or complication, within the scope of sound medical judgment, commensurate with a reasonable benefit / risk ratio. Each carrier, excipient, etc. must also be "acceptable" in the sense of being compatible with the other ingredients of the formulation. The precise nature of the carrier or other material will depend on the route of administration, which may be parenteral, e.g., subcutaneous or intravenous, by injection, or any other suitable route.

[0099] Administration may be in a "therapeutically effective amount," which is an amount sufficient to show benefit to an individual. The actual amount administered, and the pace and duration of administration, will vary depending on the nature and severity of the disease being treated, the particular individual being treated, the individual's clinical condition, the cause of the disease, the site of delivery of the composition, the type of antibody molecule, the method of administration, the administration schedule, and other factors well known to physicians. A therapeutically effective amount or appropriate dose of an antibody molecule can be determined by comparing in vitro activity with in vivo activity in animal models. Methods for extrapolating effective doses in mice and other test animals to humans are well known. The exact dose will vary depending on the size and location of the site to be treated and the specific properties of the antibody molecule.

[0100] In a preferred embodiment, the antibody molecules described herein are intended for use in the treatment of cancer. It is possible.

[0101] Cancer can be characterized by the abnormal proliferation of malignant cancer cells.When referring to a specific type of cancer, such as breast cancer, this refers to the abnormal proliferation of malignant cells in related tissues, such as breast tissue.Secondary cancers that are located in the breast but are caused by the abnormal proliferation of malignant cells in another tissue, such as ovarian tissue, are referred to herein as ovarian cancer, not breast cancer.

[0102] The cancer may be a primary or secondary cancer. Accordingly, the antibody molecules described herein may be intended for use in methods of treating cancer in an individual, where the cancer is a primary tumor and / or a secondary cancer or tumor metastasis.

[0103] Cancer tumors to be treated with the antibody molecules described herein may contain cells that express ARG2. In one embodiment, the tumor may contain cells that express ARG2. The tumor may contain cells that express and secrete ARG2. Methods for identifying the expression and / or secretion of antigens such as ARG2 are known in the art.

[0104] The cancer to be treated with the antibody molecules described herein may be selected from the group consisting of acute myeloid leukemia (AML), osteosarcoma, HCMV-driven GBM, pancreatic cancer, head and neck squamous cell carcinoma, thyroid (Sousa et al., 2010), prostate (Mumenthaler et al., 2008), neuroblastoma (Mussai et al., 2015) and breast cancer (Polat et al., 2002).

[0105] In the context of cancer, treatment can activate or enhance the immune response in an individual, improving the individual's ability to resist cancer.

[0106] The antibody molecule of the present invention is considered to be useful for detecting ARG2, and therefore the present invention relates to the use of the antibody of the present invention for detecting the presence of ARG2 in a sample.Also provided is an in vitro method for detecting ARG2, which comprises incubating the antibody of the present invention with a sample of interest, and then detecting the binding of the antibody to ARG2 in the sample.The binding of the antibody molecule can be detected, for example, by using ELISA.

[0107] In a preferred embodiment, the present invention relates to an in vitro method for detecting ARG2 in a sample, which method comprises incubating an antibody of the present invention with the sample of interest and then detecting binding of the antibody to ARG2 in the sample, wherein binding of the antibody indicates the presence of ARG2 in the sample. Methods for detecting binding of an antibody molecule to its target antigen are known in the art and include ELISA and flow cytometry.

[0108] The sample of interest may be a sample obtained from an individual, including, but not limited to, primary or cultured cells or cell lines, cell supernatants, cell lysates, platelets, serum, plasma, vitreous fluid, lymphatic fluid, synovial fluid, follicular fluid, semen, amniotic fluid, milk, whole blood, plasma, serum, blood-derived cells, urine, cerebrospinal fluid, saliva, tears, sweat, mucus, tumor lysates, and tissue culture media, tissue extracts, e.g., homogenized tissue, tumor tissue, cell extracts, and combinations thereof.

[0109] Thus, the antibody molecules of the present invention are believed to be useful for detecting or diagnosing diseases or disorders, such as pathologies associated with ARG activation and upregulation, including cancer, immune cell dysfunction, infectious diseases, vascular disease, cardiovascular disease, endothelial dysfunction, aging and cellular senescence, CNS diseases and injuries; diabetes-related diseases or cystic fibrosis, such as infectious diseases associated with cystic fibrosis. The antibody molecules of the present invention may therefore be useful, inter alia, for detecting or diagnosing cancer. Cancers that can be treated with the antibody molecules described herein include, for example, acute myeloid leukemia (AML), osteosarcoma, HCMV-driven GBM, pancreatic cancer, head and neck squamous cell carcinoma, thyroid, prostate , neuroblastoma or breast cancer.

[0110] Accordingly, related aspects of the present invention provide: (i) an antibody molecule as described herein for use in vitro as a diagnostic agent or in an in vitro diagnostic method; (ii) an antibody molecule as described herein for use in an in vitro method for detecting or diagnosing a disease or disorder, such as cancer; (iii) use of an antibody molecule described herein in the manufacture of a diagnostic preparation for use in the in vitro detection or diagnosis of a disease or disorder; (iv) an in vitro method for detecting or diagnosing a disease or disorder in an individual; and (v) A kit for use in an in vitro method for detecting or diagnosing a disease or disorder in an individual, the kit comprising an antibody molecule described herein and, optionally, further comprising instructions for use and / or one or more reagents.

[0111] Further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of this disclosure, including the following experimental exemplification.

[0112] All documents mentioned herein are incorporated by reference in their entirety.

[0113] "And / or," as used herein, should be understood as a specific disclosure of each of the two listed features or components. For example, "A and / or B" should be understood as a specific disclosure of each of (i) A, (ii) B, and (iii) A and B, just as if each were individually set forth.

[0114] Unless the context requires otherwise, the feature descriptions and definitions provided above are not limited to any particular aspect or embodiment of the present invention, but apply equally to all aspects and embodiments described.

[0115] Certain aspects and embodiments of the present invention will now be described, by way of example only, and with reference to the figures provided herein. [Brief explanation of the drawings]

[0116] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1A] The results of a direct binding HTRF™ assay between purified scFv clones C0020065 and C0020187 and a series of arginase enzymes are shown. Figure 1A shows specific binding to biotinylated recombinant human trimeric ARG2 enzyme. Both C0020065 (△) and C0020187 (×) bound to recombinant human trimeric ARG2 enzyme (Figure 1A) and cynomolgus monkey trimeric ARG2 enzyme (Figure 1C), but did not show binding to recombinant human trimeric ARG1 enzyme (Figure 1B). The negative control scFv antibody CEA6 (◆) showed no binding to any of the arginase enzymes, as expected. [Figure 1B] The results of a direct binding HTRF™ assay between purified scFv clones C0020065 and C0020187 and a series of arginase enzymes are shown. Figure 1B shows no binding to biotinylated recombinant human trimeric ARG1 enzyme. Both C0020065 (△) and C0020187 (×) bound to recombinant human trimeric ARG2 enzyme (Figure 1A) and cynomolgus monkey trimeric ARG2 enzyme (Figure 1C), but showed no binding to recombinant human trimeric ARG1 enzyme (Figure 1B). The negative control scFv antibody CEA6 (◆) showed no binding to any of the arginase enzymes, as expected. [Figure 1C]The results of a direct binding HTRF™ assay between purified scFv clones C0020065 and C0020187 and a series of arginase enzymes are shown. Figure 1C shows specific binding to biotinylated recombinant cynomolgus monkey trimeric ARG2 enzyme. Both C0020065 (△) and C0020187 (×) bound to recombinant human trimeric ARG2 enzyme (Figure 1A) and cynomolgus monkey trimeric ARG2 enzyme (Figure 1C), but did not show binding to recombinant human trimeric ARG1 enzyme (Figure 1B). The negative control scFv antibody CEA6 (◆) showed no binding to any of the arginase enzymes, as expected. [Figure 2] Inhibition of trimeric ARG2 by lead-isolated scFv antibody fragments. Figure 2 shows the results of an inhibition assay of human trimeric ARG2 enzyme for purified scFv clones C0020065 and C0020187 (n = 4 for C0020187, n = 2 for all other clones). Both clones C0020065 (△) and C0020187 (×) inhibited the activity of human trimeric ARG2. The negative control antibody CEA6 (◆) failed to inhibit the activity of human trimeric ARG2, as expected. The small molecule arginase inhibitor NG-hydroxy-L-arginine (NHLA) (◯) was included as a positive control and was able to inhibit the activity of human trimeric ARG2. [Figure 3] Inhibition of trimeric human ARG2 by lead isolated IgG. Figure 3 shows the results of a human trimeric ARG2 enzyme inhibition assay for purified recombinant human IgG1 clones C0020065 and C0020187 (n = 4 for C0020187, n = 2 for all other clones). Both clones C0020065 (△) and C0020187 (×) inhibited the activity of human trimeric ARG2. The negative control antibody NIP228 (◆) failed to inhibit the activity of human trimeric ARG2, as expected. The small molecule arginase inhibitor NG-hydroxy-L-arginine (NHLA) (◯) was included as a positive control and was able to inhibit the activity of human trimeric ARG2. [Figure 4]Figure 4 shows the restoration of T cell proliferation by ARG2 IgG in the presence of ARG2. Figure 4 shows the results of a T cell proliferation assay for purified recombinant human IgG1 clones C0020065 and C0020187. Both clones inhibited the activity of human trimeric ARG2 (exogenous), resulting in increased T cell proliferation. The negative control antibody NIP228, as expected, had no effect on T cell proliferation. The small molecule arginase inhibitor NG-hydroxy-L-arginine (NHLA), included as a positive control, was able to inhibit the activity of human trimeric ARG2, resulting in increased T cell proliferation. [Figure 5] Inhibition of ARG2 monomer by lead isolated IgG. Figure 5 shows the results of a human monomeric ARG2 enzyme inhibition assay for purified recombinant human IgG1 clones C0020065 and C0020187 (n = 4 for C0020187, n = 2 for all other clones). Both clones C0020065 (△) and C0020187 (×) inhibited the activity of human monomeric ARG2. The negative control antibody NIP228 (◆) failed to inhibit the activity of human monomeric ARG2, as expected. The small molecule arginase inhibitor NG-hydroxy-L-arginine (NHLA) (◯) was included as a positive control and was able to inhibit the activity of human monomeric ARG2. [Figure 6] Figure 6 shows the specific binding of lead isolated IgG to THP1-derived ARG2. Figure 6 shows the results of an ELISA assay for the binding of biotinylated recombinant human IgG1 (huIgG1-biot) clones to human trimeric ARG2 from THP-1 lysate. Both clones C0020065 huIgG1-biot ( [ka] ) and C0020187huIgG1-biot( [ka] ) showed strong binding to human trimeric ARG2 from THP-1 lysates. No binding was observed to lysates prepared from wild-type THP-1 cells. The negative control antibody NIP228 ( [ka] ) did not bind to the recombinant human trimeric ARG2 enzyme, as expected. [Figure 7] C0020187 does not have a competitive mode of action against homotrimeric ARG2. Figure 7 shows the results of a mode of action study on C0020187 recombinant human IgG1 to evaluate whether the antibody is a competitive, noncompetitive, or uncompetitive inhibitor. This study used a human trimeric ARG2 enzyme inhibition assay. C0020187 recombinant human IgG1 at concentrations of 10 μM (◯), 3 μM (▪), 1 μM (△), and 0.3 μM (▼) was incubated in the presence of increasing concentrations of the substrate arginine to assess the effect of each inhibitor concentration on ARG2 Km and Vmax. A no-inhibitor control (◇) was also included. Increasing concentrations of C0020187 recombinant human IgG1 had a clear effect on arginine Vmax values. [Figure 8] Figure 8 shows an epitope competition assay for affinity-matured clones as scFvs against the parental C0020187 IgG. Figure 8 shows results from an epitope competition HTRF™ assay, which measured the inhibition of complex formation between biotinylated recombinant human trimeric ARG2 enzyme and C0020187 recombinant human IgG1 by increasing concentrations of competing purified scFv antibodies. All 22 scFv antibodies tested were derived from the affinity maturation process of C0020187. The parental C0020187 scFv was also included in this analysis, along with the negative control scFv antibody CEA6. See below for a list of symbols for all scFv antibodies tested. All affinity-matured scFv antibodies tested in this experiment showed improved IC50s compared to the parental C0020187 scFv. Symbol List: [ka] [Figure 9]Figure 9 shows the enzyme inhibition assay data for the affinity matured clones as scFv versus the parental C0020187 IgG. Figure 9 shows the results of a human trimeric ARG2 enzyme inhibition assay for the parental purified scFv antibodies obtained from the affinity maturation process of C0020187. The parental C0020187 scFv was also included in this analysis along with the negative control scFv antibody CEA6. See below for a list of symbols for all scFv antibodies tested. All affinity matured scFv antibodies tested in this experiment showed improved IC50s compared to the parental C0020187 scFv. Symbol List: [ka] [Figure 10] Figure 10 shows the binding of IgG to THP-1-derived ARG2 in the presence of human plasma. Figure 10 shows the results of an ELISA assay for the binding of biotinylated recombinant human IgG1 (huIgG1-biot) clones to human trimeric ARG2 derived from THP-1 lysate. Binding to human trimeric ARG2 derived from THP-1 lysate was tested in the presence and absence of 12.5% ​​human plasma. See below for a list of all biotinylated recombinant human IgG1 symbols tested. All clones showed strong binding to human trimeric ARG2 derived from THP-1 lysate in the presence and absence of 12.5% ​​human plasma. The binding signal was slightly reduced in the presence of 12.5% ​​human plasma for all clones tested. Lysates prepared from wild-type THP-1 were tested as a negative control, but no binding was observed for any of the clones. Recombinant human trimeric ARG2 (produced in-house as described in Example 1, section 1.1) was tested as a positive control, and all clones showed strong binding. The negative control antibody R347 did not bind to human trimeric ARG2 from THP-1 lysates or recombinant human trimeric ARG2 enzyme, as expected. [ka] [Figure 11]Figure 11 shows the inhibition of THP-1-derived human ARG2 activity by affinity-matured IgG in the presence of human plasma. Figure 11 shows the results of a THP-1 lysate-derived human trimeric ARG2 enzyme inhibition assay for a population of recombinant human IgG1s obtained from the affinity maturation process of C0020187. See below for a list of all recombinant human IgG1 symbols tested. Compared to the parent recombinant human IgG1 C0020187 IgG, all clones inhibited the activity of THP-1 lysate-derived human trimeric ARG2 with improved IC50s. The negative control antibody R347, as expected, was unable to inhibit the activity of THP-1 lysate-derived human trimeric ARG2. The small molecule arginase inhibitor NG-hydroxy-L-arginine (NHLA) was included as a positive control and was able to inhibit the activity of THP-1 lysate-derived human trimeric ARG2. List of symbols: [ka] [Figure 12] Figure 12 shows the restoration of T cell proliferation by affinity-matured IgG in the presence of recombinant ARG2. Figure 12 shows the results of a T cell proliferation assay for purified recombinant human IgG1 clones C0020187 (parental), C0021139, C0021133, C0021092, C0021065, C0021061, and C0021022. All clones inhibited the activity of human trimeric ARG2 (exogenous), resulting in increased T cell proliferation. The negative control antibody R347, as expected, had no effect on T cell proliferation. The first-generation affinity-matured IgG1 clones showed improved potency in the 7- to 10-fold range compared to the parental IgG1 clones. [Figure 13]Figure 13 shows the inhibition of THP-1-derived human ARG2 activity by second-generation affinity-matured IgGs. Figure 13 shows the results of a THP-1 lysate-derived human trimeric ARG2 enzyme inhibition assay for a population of recombinant human IgG1s obtained from the second-generation affinity maturation process. See below for a list of symbols for all recombinant human IgG1s tested. Compared to the first-generation lead recombinant human IgG1 C0021061, all clones inhibited the activity of THP-1 lysate-derived human trimeric ARG2 with improved IC50s. The negative control antibody R347, as expected, was unable to inhibit the activity of THP-1 lysate-derived human trimeric ARG2. The small molecule arginase inhibitor NG-hydroxy-L-arginine (NHLA) was included as a positive control and was able to inhibit the activity of THP-1 lysate-derived human trimeric ARG2. List of symbols: [ka] [Figure 14] Figure 14 shows the restoration of T cell proliferation by second-generation affinity-matured ARG2-specific IgG in the presence of ARG2. Figure 14 shows the results of a T cell proliferation assay for purified recombinant human IgG1 clones C0020187 (parent), C0021061, C0021158, C0021177, and C0021180. All clones inhibited the activity of human trimeric ARG2 (exogenous), resulting in increased T cell proliferation. The negative control antibody R347, as expected, had no effect on T cell proliferation. The second-generation affinity-matured IgG1 clones showed improved potency compared to the parental IgG1, C0020187. [Figure 15]The second-generation affinity maturation lead C0021158 has no competitive MOA. (A) Effect on C0021158-ARG2 Vmax and Km. (B) Isothermal titration calorimetry of the ARG2 small molecule inhibitor S-(2-boronoethyl)-L-cysteine ​​hydrochloride (BEC) with ARG2 alone and the ARG2-1158 Fab complex in the top and bottom panels, respectively. Figure 15 shows that the second-generation affinity maturation lead C0021158 has no competitive MOA. (A) Effect on C0021158-ARG2 Vmax and Km shows the results of a mode-of-action study on C0021158 recombinant human IgG1 to assess whether the antibody is competitive, non-competitive, or uncompetitive. This study used a human trimeric ARG2 enzyme inhibition assay. C0020187 recombinant human IgG1 was incubated at concentrations of 1 μM (◯), 100 μM (□), 30 nM (△), and 10 nM (▽) in the presence of increasing concentrations of the substrate arginine to assess the effect of each inhibitor concentration on ARG2 Km and Vmax. A no-inhibitor control (◇) was also included. Increasing concentrations of C0021158 recombinant human IgG1 had a clear effect on arginine Vmax values. The effect on arginine Km was less clear. (B) Isothermal titration calorimetry of the ARG2 small molecule inhibitor S-(2-boronoethyl)-L-cysteine ​​hydrochloride (BEC) with ARG2 alone and the ARG2-1158 Fab complex, respectively, in the upper and lower panels. Upper panel: BEC at a concentration of 0.75 mM was titrated against a solution containing 20 μM human ARG2 (monomer concentration). Data were fit to a one-to-one binding model using a 68.3% confidence interval for the error. Bottom panel: BEC at a concentration of 0.50 mM was titrated against a solution containing 20 μM human ARG2 (monomer concentration) and 20 μM C0021158 fab. The presence of C0021158 fab prevents BEC from binding. [Figure 16]Inhibition of THP-1-derived human ARG2 activity by second-generation affinity-matured lead C0021158 and its germline- and de-risked form C0021158-fgl2 shows the results of a THP-1 lysate-derived human trimeric ARG2 enzyme inhibition assay for recombinant human IgG1 C0021158 (□) and its germline- and de-risked form C0021158-fgl2 (△). The process of de-risking and germlining C0021158 did not affect the IC50 in this assay. The parent recombinant human IgG1 C0020187 and the small molecule arginase inhibitor NG-hydroxy-L-arginine (NHLA) were included in this assay as positive controls, and both were able to inhibit the activity of THP-1 lysate-derived human trimeric ARG2 with the expected IC50. [Figure 17] Figure 1 shows immunoprecipitation (IP) of ARG2 from cell culture medium (CCM) of a population of prostate, sarcoma, and ovarian cancer cell lines. ARG2 was detected in pulldowns from LNCaP, A673, and A2780-Cis cell lines, demonstrating that these cell lines release ARG2 into CCM. PNT2 was obtained from normal prostate epithelium. The cell viability (%) determined before IP is also shown. This shows the results of immunoprecipitation of ARG2 from CCM of a population of prostate, sarcoma, and ovarian cancer cell lines. The blots demonstrate that ARG2 was detected in pulldowns from LNCaP, A673, and A2780-Cis cell lines. Each blot was verified by detecting recombinant ARG2 (positive control) and non-recombinant ARG1 (negative control) in the pulldowns. This result demonstrates that ARG2 is released into CCM from LNCaP, A673, and A2780-Cis cell lines. The high cell viability counts below the blot indicate that ARG2 is released from viable cells. [Figure 18]Figure 1 shows the binding of first-generation IgG C0021061, second-generation IgG C0021158, PAD antibody (positive control), and irrelevant IgG control R347 (negative control) to ARG2 from CCM of LNCaP, A673, and A2780-Cis, as measured using ELISA. Each bar represents the mean + SD of absorbance (A460 nm) from duplicate samples. This shows the binding of first-generation IgG C0021061 and second-generation IgG C0021158 to ARG2 from CCM of LNCaP, A673, and A2780-Cis, as measured using ELISA. ARG2PAD antibody was added as a positive control, and R347 was added as an irrelevant IgG negative control. The significantly higher absorbance of the three CCM samples compared to the R347 and medium-only controls clearly demonstrates the binding of C0021061 and C0021158 to ARG2 from LNCaP, A673, and A2780-Cis cell lines. [Figure 19] Panels (A) and (B) show the front and side views, respectively, of the X-ray crystal structure of the ARG2 C0020187 Fab complex, with the Fab colored light gray and ARG2 colored dark gray. (C) Diagram of the epitope:paratope interface within the ARG complex with C0020187. For comparison purposes, ARG2 is colored dark gray and the bound Fab is colored medium gray (bottom right corner of the panel). The complex is overlaid with unbound ARG2 (from PDB entry: 4HZE) in light gray. [Figure 20] Panels (A) and (B) show the front and side views, respectively, of the X-ray crystal structure of the ARG2 C0021158 Fab complex, where the Fab is colored light gray and ARG2 is colored dark gray. (C) Diagram of the epitope:paratope interface within the ARG complex with C0021158. For comparison purposes, ARG2 is colored dark gray and the bound Fab is colored medium gray (bottom right corner of the panel). The complex is overlaid with unbound ARG2 (from PDB entry: 4HZE) in light gray. [Figure 21]Panels (A) and (B) show the front and side views, respectively, of the X-ray crystal structure of the ARG2 C0021181 Fab complex; here, the Fab is colored light gray and ARG2 is colored dark gray. (C) Diagram of the epitope:paratope interface within the ARG complex with C0021181. For comparison purposes, ARG2 is colored dark gray, the bound Fab is colored medium gray (bottom right corner of the panel), and the complex is overlaid with unbound ARG2 (from PDB entry: 4HZE) in light gray. [Figure 22]

[0023] Figure 1 shows a representation of the ARG2 active site from the X-ray crystal structure of the ARG2 C0021158 Fab complex. The ARG2 amino acid side chains as well as key interactions are shown. Residues are numbered according to human ARG2 Uniprot ID: P78540. [Figure 23] As analyzed by size-exclusion chromatography (A) and dynamic light scattering (B), Fab C0021139 occupies all three promoters in ARG2, whereas the Fab of the initial lead C0020187 binds with high affinity to only one ARG2 subunit. (A) The size-exclusion profile (open circles) of a 1:1 molar mixture of ARG2 and C0020187 Fab exhibits a peak at 150 kDa, indicating the association of one ARG2 trimer (approximately 120 kDa) with one Fab (approximately 50 kDa). Consistent with this, the ARG2-C0020187 Fab mixture contains a significant amount of unbound Fab (peak at approximately 19.2 ml). The size exclusion profile of a 1:1 molar mixture of ARG2 and LO C0021139 Fab (open triangles) exhibits a peak at 275 kDa, indicating the association of one ARG2 trimer (approximately 120 kDa) with three Fabs (approximately 150 kDa). Therefore, the ARG2-C0021139 Fab mixture contains traces of unbound Fab. (B) Dynamic light scattering demonstrates an increase of approximately 1.5 nm in the mean hydrodynamic radius (Rh) of the ARG2-C0021139 Fab complex (open triangles) compared to the Rh of the ARG2-C0020187 Fab complex (open circles). [ka] [Figure 24] Table 4 shows the sequence identity across the entire VH sequence (Kabat residues 1-113) of the parental C0020187 antibody and the 33 affinity-matured antibodies described herein. All affinity-matured sequences share at least 90% identity with the parental C0020187 antibody. Percent diversity and percent divergence values ​​obtained from Clustal W alignment (Thompson, Higgins et al. 1994). [Figure 25] Table 5 shows the sequence identity across the entire VL sequence (Kabat residues 1-107) of the parent C0020187 antibody and the 33 affinity-matured antibodies described herein. All affinity-matured sequences share at least 89.1% identity with the parent C0020187 antibody. Percent diversity and percent divergence values ​​obtained from Clustal W alignment (Thompson, Higgins et al. 1994). [Figure 26] 1 shows the heavy and light chain amino acid sequence alignment of clones C0020065 and C0020187. [Figure 27] Clones: C0020187, C0021017, C0021021, C0021022, C0021032, C0021061, C0021065, C0021089, C0021092, C0021096, C0021097, C0021098, C0021101, C0021118, C0021124, C0021128, C0021129, C0021131, C0021133, C0021135, C0021139, C0021141, C0021142, C0021142 Figure 1 shows the heavy and light chain amino acid sequence alignment of IgG, C0021144, C0021155, C0021158, C0021158dr, C0021158fgl, C0021158 fgl2, C0021158 IgG, C0021177, C0021180 and C0021181. DETAILED DESCRIPTION OF THE INVENTION

[0117] Description of the embodiment Example Example 1. Anti-ARG2-specific antibody isolation and lead selection 1.1 Production of trimeric recombinant ARG2 and ARG1 Codon-optimized human ARG2 (aa23-3) for expression in E. coli Expression vectors containing synthetic DNA sequences encoding either cynomolgus monkey ARG2 (aa 23-354) or human ARG1 (aa 1-322) and flanking regions encoding either an N-terminal Avi tag (-AGLNDIFEAQKIEWHE- (SEQ ID NO: 351)) and / or a C-terminal His10 tag. The amplified inserts containing the gene sequence of interest and tags (SEQ ID NO: 353: Avi-huARG2-His10, SEQ ID NO: 355: huARG2-His10, and SEQ ID NO: 352: Avi-huARG1-His10) were cloned into the pET16b vector (Novagen - EMD Millipore) using the NcoI and XhoI restriction sites. The resulting vectors, pAviHuArg2His10, pHuArg2His10, and pAviHuArg1His10, were transformed into BL21(DE3) E. coli, and a single transformed colony was used to inoculate a culture of 2×TYA. This was grown overnight at 37°C and 280 rpm.

[0118] The overnight cultures were used to inoculate 2xTYA cultures in 2L shake flasks. Each culture was incubated at 37°C and 280 rpm to an OD of 0.6. The cultures were then transferred to an 18°C ​​incubator, after which IPTG (0.5 M) was added and the resulting cultures were incubated overnight at 18°C ​​and 280 rpm.

[0119] The culture was pelleted by centrifugation at 12,000 rpm for 20 minutes at room temperature. After decanting and discarding the supernatant, the pellet was frozen at -80°C overnight. The resulting frozen pellet was thawed and processed for lysis using BugBuster™ Protein Extraction Reagent with the addition of Lisonase Bioprocessing Reagent (Merck, 71370). After the pellet was completely resuspended in BugBuster Reagent at 4°C, Lisonase was added, and after the addition of Lisonase, the pellet was incubated at room temperature on a shaker platform at approximately 120 rpm. The lysis mixture was pelleted by centrifugation at 20,000 rpm for 45 minutes at 4°C.

[0120] The soluble fractions were decanted and combined in a clean bottle. Ni-NTA resin (pre-washed and equilibrated in Tris 50 mM pH 8, 300 mM NaCl) was then added to the supernatant and allowed to bind for 1 hour at 4°C with gentle mixing. After binding, the mixture was centrifuged in a Pierce Centrifuge. The Ni resin was filtered from the supernatant by gravity filtration through a column (ThermoFisher, 89898) The captured resin was washed (Ni wash buffer, Tris 50 mM pH 8, 300 mM NaCl, 40 mM imidazole) and subsequently eluted in Ni elution buffer (Tris 50 mM pH 8, 300 mM NaCl, 400 mM imidazole).

[0121] The eluted fraction was further purified on a Hiload 16 60 superdex 200 prep grade SEC column on an AKTAxpress by manual injection into a 5 ml loop. The column was equilibrated in Tris-glycerol storage buffer (25 mM Tris pH 8, 150 mM NaCl, 10% glycerol) and eluted with the same buffer, after which 1.5 ml fractions were collected over the entire elution volume. Fractions containing observable peaks were pooled and analyzed by SDS-PAGE to confirm trimer and monomer fractions by comparing the peaks with molecular weight markers.

[0122] 1.2 Production of monomeric recombinant ARG2 The ARG2 sequence in the pAvihuARG2His10, phuARG2His10 vectors was modified by site-directed mutagenesis, such that amino acid 275 was mutated from glutamic acid to glutamine (the number of arginase residues is based on human ARG2 Uniprot ID: P78540). These modified vectors were then used to express monomeric ARG2 from E. coli, following the protocol used to express trimeric ARG2, as described in section 1.1. RG2 was expressed.

[0123] 1.3 Phage display selection to isolate ARG2-specific scFv Soluble phage display selection was performed using five naive libraries (nFL, DP47, CS, BMV, and EG3) cloned into a phagemid vector based on the filamentous phage M13 (Vaughan et al., 1996). Anti-ARG2 scFv antibodies were isolated from the phage display library using a series of selection cycles against recombinant human biotinylated ARG2 (avi-Arg2-His10, produced in-house) essentially as described previously (Hawkins et al., 1992; Vaughan et al., 1996). Briefly, for the first round of solution-phase selection, purified phage particles were preincubated for 1 hour in Marvel-PBS (3% w / v) containing streptavidin-conjugated paramagnetic beads (Dynabeads® M280, Invitrogen Life Sciences, UK). Biotinylated human ARG2 in Dulbecco's phosphate-buffered saline (DPBS, pH 7) was added (final concentration: 100 nM biotinylated human ARG2). After removing the streptavidin beads, antigen was added. Phage particles bound to biotinylated human ARG2 were captured using new streptavidin-conjugated paramagnetic beads, and weakly bound phage were removed by a series of washing cycles with PBS-Tween (0.1% v / v). Bound phage particles were eluted from the beads using trypsin (10 ng / ml final concentration diluted in 0.1 M sodium phosphate buffer; pH 7) and used to infect E. coli TG1 bacteria, followed by subsequent laminar The clones were rescued for selection in a round (Vaughan et al., 1996). The next two rounds of selection were performed as previously described, but with lower concentrations of biotinylated ARG2 antigen, specifically 50 nM and 25 nM in rounds 2 and 3, respectively.

[0124] 1.4 Identification of ARG2-specific scFv fragments using a direct binding assay (unpurified scFv) Unpurified scFv from periplasmic preparations were analyzed by homogenous time-resolved fluorescence (HTRF™, CisBio Bioassays, France) using a Pherastar plate reader (BMG Labtech, Germany). ) binding assay, in which binding of the unpurified scFv to recombinant human trimeric ARG2 enzyme was detected using streptavidin cryptate and anti-c-myc-XL665 detection. Reagents (CisBio International, France; cat: 610SAKLB and 61MYCXLB, respectively) were used. The activity of the scFv was assessed by measuring fluorescence resonance energy transfer (FRET) between the c-myc tagged scFv and biotinylated enzyme. Selection products were screened as crude bacterial periplasmic extracts containing scFv prepared in 200 mM Tris buffer pH 7.4, 0.5 mM EDTA, and 0.5 M sucrose. Five microliters of crude scFv sample was added to a Greiner® 384-well assay plate (Greiner Bio-one, UK; cat: 784076). Subsequently, 5 μl of 12 nM recombinant human trimeric ARG2 enzyme, 5 μl of 12 nM EDTA, and 5 μl of 12 nM EDTA were added to a Greiner® 384-well assay plate (Greiner Bio-one, UK; cat: 784076). 1 μl of 6.67 nM streptavidin cryptate and 5 μl of 40 nM anti-c-myc-XL665 were added. A negative control unpurified scFv was used in place of the test scFv sample. Non-specific binding wells (negative controls) were defined for each plate by centrifugation. Cross-reactive scFv clones were identified by a simultaneous assay using recombinant human trimeric ARG1 enzyme. All dilutions were performed in 0.4M KF (VWR, UK; cat: 26820.236) and 0.1% bovine serum albumin. Phosphate-buffered saline containing albumin (Sigma, UK; cat: A9576) (assay buffer) Assays were performed in saline (ThermoFisher Scientific, UK; cat: 14190-094). The plates were incubated overnight at 4°C and then imaged using a Pherastar printer with an excitation wavelength of 320 nm. Time-resolved fluorescence was read using a rate reader (PerkinElmer, USA) to measure the emission at 620 nm and 665 nm (100 flashes).

[0125] Data were analyzed by calculating the % Delta F value for each sample. The % Delta F value was determined according to Equation 1. Equation 1:

number

[0126] 1.5 Identification of ARG2-inhibitory scFvs using enzyme inhibition assays (unpurified scFvs) Crude anti-ARG2 scFv fragments from periplasmic preparations were screened in an ARG2 enzyme inhibition assay using a Pherastar plate reader (BMG Labtech, Germany), which assessed the ability of crude scFv to inhibit trimeric ARG2 enzyme activity by measuring the production of urea, a product of L-arginine turnover by ARG2.

[0127] Selection products were screened as crude bacterial periplasmic extracts containing scFv prepared in 200 mM TES buffer pH 7.4, 0.5 mM EDTA and 0.5 M sucrose. 10 μl of crude scFv was added to a Greiner bio-one® 3 This was followed by the addition of 5 μl of 1.6 μg / ml trimeric human ARG2 diluted in 40 mM MnCl, 100 mM Tris-HCl, pH 7.4 (4x enzyme buffer).

[0128] After overnight pre-incubation of ARG2 with unpurified scFv at 4°C, the assay plate was allowed to reach room temperature, and the enzymatic reaction was initiated by the addition of 5 μl of 100 mM L-arginine substrate and incubated at room temperature for 60 minutes.

[0129] Urea was detected by simultaneous addition of equal volumes of O-phthaldialdehyde (1.5 mM O-phthaldialdehyde, 7% H2SO4, 0.03% Brij L23) and N-(1-naphthyl)ethylenediamine (1.66 mM N-(1-naphthyl)ethylenediamine, 81 mM boric acid, 21% H2SO4, 0.03% Brij L23), followed by 18 min of incubation at room temperature and colorimetric determination on a Pherastar by measuring absorbance at 505 nm. The measurement results were quantified.

[0130] For each plate, background control wells were defined by omitting substrate. Maximum signal control wells were defined for each plate by substituting an irrelevant unpurified scFv, CEA6, for the test scFv samples.

[0131] The data was analyzed by calculating the % max control for each sample using the 505 nm absorbance data. The % max control was determined according to Equation 2. Equation 2: %max control=100*((test sample−average max control) / (average background control−average max control))

[0132] 1.6 Identification of ARG2-specific scFv using direct binding assay (purified scFv) Periplasmic extracts of crude scFvs, which were shown to bind specifically to biotinylated recombinant human trimeric ARG2 enzyme by HTRF™ assay, were subjected to DNA sequencing (Osbourn et al., 1996; Vaughan et al., 1996). scFvs with unique protein sequences were expressed in E. coli and subsequently purified by affinity chromatography. The scFvs were purified (essentially as described (Bannister et al., 2006)). The ARG2 binding profile of each purified scFv was determined by testing a dilution series of the purified scFv in the HTRF™ assay described in section 1.4, using the purified scFv instead of the unpurified scFv periplasmic preparation. Biotinylated recombinant human trimeric ARG2 enzyme, biotin The purified scFvs were simultaneously tested for binding to biotinylated recombinant human trimeric ARG1 enzyme and recombinant cynomolgus monkey trimeric ARG2 enzyme. Data were analyzed by calculating % Delta F values ​​as described in Section 1.4. Examples of results for purified C0020187scFv and purified C0020065scFv are shown in Figure 1. These results demonstrate that C0020187scFv and C0020065scFv specifically bind to biotinylated recombinant human trimeric ARG2 enzyme and biotinylated recombinant cynomolgus monkey trimeric ARG2 enzyme, but not to human recombinant human trimeric ARG1 enzyme.

[0133] 1.7 Screening of purified scFv fragments that inhibit ARG2 in enzyme inhibition assays Purified anti-ARG2 scFv fragments were screened for their ability to inhibit the enzymatic activity of trimeric ARG2 using the assay described in section 1.5 (Figure 2).

[0134] IC was determined by testing a dilution series of purified scFv prepared in TBS in the ARG2 enzyme inhibition assay described in section 1.5, using purified scFv instead of unpurified scFv periplasmic preparation. 50 value was determined.

[0135] Background control wells were defined for each plate by omitting the substrate. Maximum signal control wells containing substrate in the absence of inhibitor were defined for each plate within wells. Data were analyzed by calculating the % Maximum (max) control for each sample. The % Max control was determined according to Equation 1 detailed in Section 1.5.

[0136] Purified scFvs were tested alongside N-hydroxyl-L-arginine (a small molecule competitive inhibitor of ARG2) as a positive control and CEA6 (an irrelevant isotype control antibody) as a negative control. Data points were plotted and IC values ​​were calculated using a four-parameter fit in GraphPad Prism. 50 It was decided that:

[0137] 1.8 Reformatting of an ARG2-specific, inhibitory antibody from scFv to IgG1 Certain clones were converted from scFv to IgG1 by subcloning the variable heavy (VH) and variable light (VL) domains into vectors expressing fully human antibody heavy and light chains, respectively. The variable heavy chain was cloned into a mammalian expression vector (pEU1.3) containing human heavy chain constant domains and regulatory elements to express the full IgG1 heavy chain in mammalian cells. Similarly, the variable light chain was cloned into a mammalian expression vector for expression of the human λ light chain constant domain (pEU4.4) or the human λκ light chain constant domain (pEU3.4) and regulatory elements to express the full IgG light chain in mammalian cells. The heavy and light chain expression vectors were originally described by Persic et al., 1997. Obtaining clones as IgG1 To achieve this, heavy and light chain IgG expression vectors were transiently transfected into ExpiCHO (ThermoScientific UK; cat. number: A29133) cells, where the antibodies were expressed and distributed in the culture medium. The collected medium was filtered and then purified. IgG was purified using Protein A chromatography (MabSelect SuRe, GE Healthcare, UK). 25 mM Tris pH 7 Culture supernatants were loaded onto appropriate Protein A columns pre-equilibrated in 0.4, 50 mM NaCl. Bound IgG was eluted from the column using 0.1 M sodium citrate pH 3.0, 100 mM NaCl. The IgG was buffer exchanged into TBS (25 mM Tris pH 7.4, 150 mM NaCl). Purified IgG was passed through a 0.2 μm filter, and the IgG concentration was determined by absorbance at 280 nm using an extinction coefficient based on the IgG amino acid sequence. Purified IgG was analyzed for aggregation or degradation using SEC-HPLC and SDS-PAGE techniques.

[0138] 1.9 Confirmation of the binding profile of ARG2-specific recombinant IgG1 to ARG2 in a direct binding assay The purified scFv fragments that specifically bound to biotinylated recombinant human trimeric ARG2 enzyme were converted to recombinant IgG. Binding of the recombinant IgG to the human trimeric ARG2 enzyme was confirmed by testing a dilution series of the recombinant IgG in the HTRF™ assay described in Section 1.4, using the recombinant IgG instead of the unpurified scFv. A further modification to the above assay was the use of anti-human Fc XL665 detection reagent (CisBio International, France; cat: 61HFCXLB) instead of anti-c-myc-XL665 detection reagent, as the recombinant IgG did not contain a c-myc tag. The % delta F values ​​described in Section 1.4 were calculated. The data was analyzed by the following.

[0139] 1.10 Confirmation of the Trimeric ARG2 Inhibition Profile of ARG2-Specific Recombinant IgG1 in Enzyme Inhibition Assays The purified scFv fragments that inhibited the enzymatic activity of the human trimeric ARG2 enzyme were converted into recombinant IgG. Using the assay described in section 1.5, ARG2-specific recombinant IgG1s were screened for their ability to inhibit the enzymatic activity of trimeric ARG2 (Figure 3).

[0140] In the ARG2 enzyme inhibition assay described in section 1.5, ARG2-specific recombinant IgG1 was used instead of the unpurified scFv periplasmic preparation, and a dilution series of ARG2-specific recombinant IgG1 prepared in TBS was tested to determine the IC 50 value was determined.

[0141] An ARG2-specific recombinant IgG1 was tested along with N-hydroxyl-L-arginine (a small molecule competitive inhibitor of ARG2) as a positive control and NIP228 (an irrelevant isotype control antibody) as a negative control. Data points were plotted and IC values ​​were calculated using a four-parameter fit in GraphPad Prism. 50 value was determined.

[0142] 1.11 Restoration of T cell proliferation by ARG2 IgG Anti-ARG2 antibodies were screened for their ability to restore T cell proliferation by inhibiting the enzymatic activity of ARG2, thus preventing the loss of L-arginine required for T cell proliferation. This assay was used to provide in vitro evidence of the functional efficacy of each antibody.

[0143] Briefly, leukocyte cones (NHS Blood and Transplant, Watford, UK) were subjected to Ficoll Plaque (GE, Sweden; Cat: 17-1440-02) gradient separation, PBMCs were extracted, and pan-T cell populations were isolated by negative selection using the Human T Cell Enrichment Kit (Stemcell Technologies, France; Cat: 19051) according to the manufacturer's instructions.

[0144] 40,000 cells / well were seeded in complete growth medium (RMPI 1640, 5% human serum albumin) in 96-well clear TC-treated microplates (Greiner-Bio One, Germany; Cat: 655180). T cells were activated by adding ImmunoCult CD3 / CD28 (Stemcell Technologies, France; Cat: 10971) directly to the wells at a dilution of 1:40, and then the plates were incubated for 45 min at 37°C, 5% CO2.

[0145] EC of test antibody 50 To determine the β-actin concentration, a dilution series was made 8x in sterile TBS and the final assay concentration was added to the assay plate containing the cells. The plate was incubated at 37°C, 5% CO2 for 30 minutes.

[0146] ARG2 was added to relevant wells of the plate at a final concentration of 15 μg / ml. Equal volumes of ARG2 vehicle and test antibody vehicle were added to relevant control wells. The total volume in each well was: The volume was 200 μL. Cells were treated with test antibodies for a total of 72 hours at 37°C, 5% CO2. Antibodies were tested alongside N-hydroxyl-L-arginine (a small molecule competitive inhibitor of ARG2) as a positive control and NIP228 (an irrelevant isotype control antibody) as a negative control (Figure 4).

[0147] T cell proliferation was assessed using a BrdU Cell Proliferation Kit (Merck Millipore, UK; Cat: QIA58). 54 hours after antibody treatment, BrdU labeling reagent was added, and plates were further incubated for 18 hours at 37°C and 5% CO2. The remainder of the assay was performed according to the manufacturer's instructions.

[0148] Calculated data were expressed as % of control (Equation 3) and graphical plots were generated using a four-parameter fit, model 205 in XLFit (IDBS, UK) to calculate EC 50 Decide did. Equation 3: All test sample data were corrected for background (average of cell-free controls). %Control = 100*((Test Sample - Average Positive Control) / (Average Negative Control - Average Positive Control)) Mean positive control = T cells in the absence of ARG2 Mean negative control = T cells in the presence of ARG2

[0149] [Table 17]

[0150] This report is an independent study. NHS Blood & Transport supported the study and provided materials. The views expressed in this application are those of the authors and not necessarily those of NHS Blood & Transport.

[0151] 1.12 Confirmation of the Monomeric ARG2 Inhibitory Profile of ARG2-Specific Recombinant IgG1 in Enzyme Inhibition Assays ARG2-specific recombinant IgG1 was screened for its ability to inhibit the enzymatic activity of monomeric ARG2. In this assay, the ability of ARG2-specific recombinant IgG1 to inhibit the enzymatic activity of monomeric ARG2 was determined by measuring the production of urea, a product of L-arginine turnover by monomeric ARG2.

[0152] IC was determined by testing a dilution series of ARG2-specific recombinant IgG1 prepared in TBS. 50 value was determined.

[0153] 10 μl of ARG2-specific recombinant IgG1 (dilution series) was added to Greiner bio-one (registered trademark) ) to a 384-well assay plate (Greiner Bio-one, UK; cat: 781901), followed by the addition of 5 μl of 1.6 μg / ml trimeric human ARG2 diluted in 40 mM MnCl, 100 mM Tris-HCl, pH 7.4 (4x enzyme buffer).

[0154] After overnight pre-incubation of ARG2 with ARG2-specific IgG1 at 4°C, the assay plate was allowed to reach room temperature, and the enzymatic reaction was initiated by the addition of 5 μl of 100 mM L-arginine substrate and incubated at room temperature for 60 minutes.

[0155] Urea was detected by simultaneous addition of equal volumes of O-phthaldialdehyde (1.5 mM O-phthaldialdehyde, 7% H2SO4, 0.03% Brij L23) and N-(1-naphthyl)ethylenediamine (1.66 mM N-(1-naphthyl)ethylenediamine, 81 mM boric acid, 21% H2SO4, 0.03% Brij L23), followed by 18 min of incubation at room temperature and colorimetric determination on a Pherastar by measuring absorbance at 505 nm. The measurement results were quantified.

[0156] An ARG2-specific recombinant IgG1 was tested along with N-hydroxyl-L-arginine (a small molecule competitive inhibitor of ARG2) as a positive control and NIP228 (an irrelevant isotype control antibody) as a negative control. Data points were plotted and IC values ​​were calculated using a four-parameter fit in GraphPad Prism. 50 values ​​were determined (Figure 5).

[0157] 1.13 Binding of human ARG2 produced by THP1 cells to IgG1 C0020187 and C0020065 IgGs were tested for their ability to bind to mammalian-expressed human ARG2 produced in transfected THP-1 cells. Lysates of human ARG2-transfected THP-1 cells were prepared, and binding to these lysates was measured using ELISA.

[0158] Cell lysates were prepared from THP-1 cells overexpressing human ARG2 and wild-type THP-1 cells. 600,000–1,000,000 cells were suspended in 1 ml of Tris-buffered saline (25 mM Tris + 150 mM sodium chloride in dH2O, pH 7.4) and lysed using QIAshredders (QIAgen, UK; cat: 79654).

[0159] For ELISA, maxisorp white plates (Nunc; cat: 437796) were filled with 1 mL of PBS. The cells were coated overnight at 4°C with μg / ml of rabbit anti-human ARG2 antibody (Abcam, ab137069). The next day, the plate was washed with PBS and then treated with casein blocker (Thermo Scientific, 37528) was blocked.

[0160] Before addition to the ELISA plate, cell lysates were pre-incubated for 45 minutes (at room temperature with gentle shaking) with biotinylated versions of the lead antibodies C0020187 and C0020065. Antibodies were at a final concentration of 50 μg / ml in the pre-incubation mixture.

[0161] The blocked ELISA plate was washed with PBS, and then 50 μl of the pre-incubation mixture was transferred to the ELISA plate and incubated at room temperature for 90 minutes. After washing the ELISA plate five times with PBS-Tween (0.1%), 50 μl of 1 / 30,000 streptavidin-HRP was added to each well. The plate was incubated at room temperature for 1 hour and then washed six times with PBS-Tween (0.1%). Next, 100 μl of PICO ELISA substrate (Thermo Scientific, 37070) was added to each well and incubated in the dark. The plates were incubated at room temperature for 1 minute, and then luminescence was read using an Envision plate reader. Data was analyzed using GraphPad Prism. The results are shown in Figure 6.

[0162] 1.14 Generation of Fab fragments to allow improved kinetic profiling of mutants The heavy chain mammalian expression vector p EU1.3 was modified to remove the heavy chain constant domains CH2 and CH3, generating a vector containing only the CH1 domain and part of the hinge region. The resulting vector is called pEU1.3fab. Vectors for the expression of heavy and light chains were first described in Persic et al., 1997. The variable heavy (VH) and variable light (VL) domains were expressed as Selected clones were converted from scFv to Fab by subcloning into vectors expressing human fab heavy and light chains. To obtain clones as IgG1, the heavy and light chain fab expression vectors were cloned into ExpiCHO (ThermoScientific UK; cat. number: A29133 ) cells, where the Fab fragments were expressed and secreted into the culture medium. The collected medium was filtered and then purified. The Fabs were purified using IgG1-CH1 chromatography (CaptureSelect™ IgG-CH1 Pre-packed Column, ThermoFisher). The appropriate CaptureSelect IgG-CH1 column was pre-equilibrated in 1x DPBS. The culture supernatant was loaded onto the column. Bound Fab fragments were eluted from the column with 0.1 M glycine pH 3.0. The Fab fragments were then purified by elution with TBS (25 mM Tris pH 7.4, 150 mM The purified Fab fragments were buffer exchanged into 0.2 μm NaCl. The purified Fab fragments were passed through a 0.2 μm filter, and the Fab concentration was determined by absorbance at 280 nm using an extinction coefficient based on the amino acid sequence of the Fab. The purified Fab fragments were analyzed for aggregation or degradation using SEC-HPLC and SDS-PAGE techniques.

[0163] 1.15 Determination of Binding Affinity of Lead Antibody C0020187 to Human ARG2, Cynomolgus ARG2, and Human ARG1 Using Bio-Layer Interferometry Using the OctetRED (Pall ForteBio) instrument, C0020187 and recombinantly produced human AR were Kinetic parameters of the interaction of G2 with cynomolgus monkey ARG2 and human ARG1 were evaluated. The OctetRED biosensor uses an optical analysis technique that analyzes the interference pattern of white light reflected from two surfaces: the immobilized protein on the sensor chip and an internal standard layer. Any change in binding at the biosensor chip causes a shift in the interference pattern, which can be measured in real time. Molecules that associate with or dissociate from the ligand at the biosensor chip shift the interference pattern, generating a response in the Octet system. , which is recorded by the acquisition software.

[0164] Typically, a defined concentration of the analyte species is contacted with the bound ligand and any binding is detected as an increase in signal (association phase). This is followed by a buffer rinse period, during which dissociation of the analyte species from the surface-immobilized ligand can be observed as a decrease in signal (dissociation phase). This can be repeated using a wide range of analyte concentrations to provide data for analysis of binding kinetics.

[0165] Typically, Octet Kinetics Buffer (0.01% BSA and 0.002% Tween 10) is used as the dilution buffer for the analyte sample and as the flow buffer during the dissociation step. PBS containing n20 is used. Experimental data are recorded as the shift (nm) of the interference pattern over time, which is directly proportional to the optical thickness at the biosensor chip, which in turn is an approximate measure of the mass of bound analyte. The proprietary Octet Data Analysis software package is then used to process the data and derive a binding model. The resulting association (k a ,M -1 s -1 ) and dissociation (k d ,s -1 ) rate constant for dissociation (K D ,M) affinity constants can be calculated.

[0166] The binding affinity of C0020187 Fab to human ARG2, cynomolgus monkey ARG2, and human ARG1 was assessed using an assay in which biotinylated ARG antigen was captured on a streptavidin sensor chip. A new sensor chip was used for each measurement without regeneration. A dilution series of C0020187 Fab (3.25–240 nM) was individually contacted with the ligand surface for a time sufficient to observe a sensorgram that could be reliably fitted to an appropriate binding model (typically 5 min), followed by an appropriate dissociation time (typically 10 min). Blank standard (0 nM Fab) data were subtracted from each data set to reduce the effects of any buffer artifacts or nonspecific binding effects. Data from each analyte titration were then simultaneously fitted to the appropriate binding model using Octet Evaluation software.

[0167] An example of the results for C0020187 is shown in Table 9, which shows the association rate constant (k a ), dissociation rate constant (k d ) and dissociation constant (K D ) These parameters were obtained from a 1:1 binding fit to the data.

[0168] [Table 18]

[0169] 1.16 Understanding the mechanism of action of ARG2-specific IgG on the enzyme ARG2 The mechanism of action of lead identified clone C0020187 IgG was verified using a human ARG2 (trimeric) enzyme inhibition assay.

[0170] The enzyme inhibition assay, described in Section 1.5, is a biochemical assay that measures the production of urea, a product of L-arginine turnover by ARG2. To examine the mechanism of action of C0020187 IgG, a four-point titration was prepared in the presence of 11 concentrations of arginine (500, 400, 300, 200, 125, 75, 50, 25, 17, 10, and 5 mM). The reaction was allowed to proceed for 80, 60, 45, 30, 15, or 5 minutes before the addition of the urea detection reagents O-phthaldialdehyde and N-(1-naphthyl)ethylenediamine.

[0171] Raw data from each individual concentration of C0020187 was plotted as a function of time for each concentration of arginine and then fitted using a linear fit (GraphPad Prism) to obtain initial velocities (slope values).

[0172] The initial velocity data was plotted against the arginine concentration for each IgG concentration. See Figure 7. Each trace was fitted to a Michaelis-Menten model (Y = V max *X / (K m + X)) to obtain the maximum velocity (V max ) and enzyme / substrate affinity (K m ) values ​​were obtained (Table 10).

[0173] [Table 19]

[0174] The mechanism of action of enzyme inhibitors can be broadly classified into competitive, uncompetitive and non-competitive. m and V max They can be so identified based on their effect on

[0175] Competitive inhibitors often bind only to the free enzyme at the active site, suppressing the maximum enzyme velocity (V maxDecrease the affinity of the enzyme for the substrate (K m (increase).

[0176] An uncompetitive inhibitor binds exclusively to the enzyme-substrate complex, resulting in an inactive enzyme-substrate-inhibitor complex. The affinity of the enzyme for the substrate increases (K m decreases), maximum enzyme velocity (V max ) decreases.

[0177] Non-competitive inhibitors bind equally well to the free enzyme and the enzyme-substrate complex. The affinity of the enzyme for the substrate (K m ) is unchanged, and the maximum enzyme velocity (V max ) decreases.

[0178] For C0020187 IgG inhibitors, V in the presence of C0020187 IgG inhibitors max decreases, excluding competitive modes of action and favoring uncompetitive or non-competitive modes of action. To distinguish non-competitive modes of action from uncompetitive modes of action, K m The effect on K must be evaluated. Unfortunately, the initial velocity / arginine concentration plot has an atypical profile, where in the presence of inhibitor, the initially predicted initial velocity increase is followed by a drop as the arginine concentration increases. This atypical profile leads to poor fitting of the data and the inability to accurately estimate K m It becomes difficult to obtain an accurate K m The absence of a β-amyloid means that C0020187 IgG cannot be assigned as uncompetitive or non-competitive. All that can be said is that this inhibitor is "non-competitive."

[0179] Example 2 - Affinity maturation of C0020187 by targeted and StEP SHUFFLE mutagenesis using phage and ribosome display 2.1 Targeted mutagenesis of antibody C0020187 by phage display The lead antibody C0020187 was optimized for improved affinity to human ARG2 using a targeted mutagenesis approach involving affinity-based phage display selection. The variable heavy chain (V) was engineered using standard molecular biology techniques as described by Clackson and Lowman ((2004) A Practical Approach, Oxford University Press). H ) and variable light chain (V L ) Oligonucleotide-directed mutagenesis of complementarity-determining regions 1, 2, and 3 (CDR1, CDR2, CDR3) resulted in the construction of 12 large scFv-phages derived from C0020187. A library was created.

[0180] The library was subjected to affinity-based phage display selection to enrich for mutants with higher affinity for human ARG2. The selection was performed essentially as previously described (Hawkins et al., 1992; Schier et al., 1996; Thompson et al., 1996). Briefly, scFv phage particles were incubated with biotinylated human ARG2 (avi-Arg2-His10, produced in-house) in solution. Antigen-bound scFv-phage were then captured on streptavidin-coated paramagnetic beads (Dynabeads® M280, Invitrogen Life Sciences, UK) according to the manufacturer's instructions. Selected scFv-phage particles were subsequently rescued as previously described (Osbourn et al., 1996). The selection process was repeated over five successive rounds in the presence of decreasing concentrations of biotinylated human ARG2 target antigen (from 30 nM antigen in the first round down to 10 pM by the fifth round).

[0181] 2.2 Recombination of suitable selection products to generate "binary" libraries and their subsequent affinity optimization Collectively, screening of the C0020187 CDR-targeted mutagenesis library products in C0020187 epitope competition (Section 2.4) and human ARG2 enzyme inhibition assays (Section 1.5) allowed the identification of individual CDR-targeted phage display selection products that showed overall improvements in huARG2 direct binding and inhibition compared to the parental C0020187 scFv. In particular, four targeted mutagenesis libraries (V H CDR1, V H CDR2, V L CDR2 and V L The V CDRs (including CDR3) were recombined pairwise to generate four "binary" recombination libraries in which two of the six CDRs were mutated. H The affinity maturation library encompassing CDR1 is L Randomly recombined in the CDR2 library to generate V H 1:V L 2 libraries were generated. The remaining libraries were V H 1:V L 3. V H 2:V L 2 and V H 2:V L 3. A subset of each recombinant library was sequenced to confirm the integrity of each library.

[0182] Soluble phage display selections using these recombinant libraries were completed as previously described (Section 1.3) in the presence of decreasing concentrations of biotinylated human ARG2 target antigen (avi-Arg2-His10, produced in-house; decreasing from 1 nM to 5 pM over three successive rounds of selection). As before, all recombinant library selections were screened in C0020187 epitope competition (Section 2.4) and human ARG2 enzyme inhibition assays (Section 1.5).

[0183] 2.3 Recombination of phage-optimized CDR blocks and selection using ribosome display Ribosome display is described in Hanes J and Plueckthun A. (1997).

[0184] Using the single CDR targeting followed by the binary CDR recombination approach described in Section 2.1, a modest (approximately 8-fold) improvement in the affinity of C0020187 was achieved. To further improve C0020187 affinity, two alternative strategies were pursued simultaneously. The first strategy involved targeting two CDRs: V H CDR2 and V L This corresponds to a targeted CDR recombination approach that focuses on CDR2. The second strategy was an unbiased approach in which products from all six CDRs were recombined. This second unbiased strategy allows for sampling the entire scFv sequence space, thereby enabling both intra- and inter-chain recombination events (Zhao et al., 1998), which would be impossible using standard targeted (biased) recombination approaches.

[0185] Optimizing phage V using recombinant PCR H CDR2 and V L Combining the CDR2 repertoire into a single population of full-length scFv clones H CDR2 / V L A CDR2 recombinant library (H2L2) was generated. For the unbiased recombinant library, V was isolated by recombinant PCR. H / V L As a template for chain shuffling, V H CDR1, V H CDR2, V H CDR3, V L CDR1, V L CDR2 and V L The phage optimization products from CDR3 were used (shuffled library). The resulting library was subjected to random recombination using the staggered extension process (StEP) in vitro DNA recombination (Zhao et al., 1998) to promote further intrastrand recombination (StEP library). Larry).

[0186] Next, using standard molecular biology methods, the library of recombinant scFv constructs was modified into a ribosome display format. The ribosome display construct contains the structural features required for ribosome display, including 5' and 3' stem-loops to prevent exonuclease degradation of the mRNA transcript, a Shine-Dalgarno sequence to promote ribosome binding to the mRNA transcript, and a gene III spacer that allows the translated scFv molecule to fold while still retaining ribosome binding (Groves et al., 2005).

[0187] The libraries were then used in affinity-based soluble ribosome display selection to enrich for variants with higher affinity for human ARG2. The selection was performed essentially as described in Hanes et al., 2000. Briefly, each recombinant library was individually transcribed into mRNA. Using the process of stalled translation, The mRNA-ribosome-scFv tertiary complexes were formed (Hanes et al., 1997). These complexes were subjected to three rounds of selection while being incubated in the presence of decreasing concentrations of synthetic biotinylated human ARG2 to select for variants with higher affinity. The antigen-bound complexes were then transferred to streptavidin-coated paramagnetic beads (Dynabeads™). Invitrogen, UK; cat: 112-05D) and non-specific ribosomal complexes were washed away. Subsequently, mRNA from the bound ribosomal complexes was isolated, reverse transcribed into cDNA, and amplified by PCR. This DNA was used for the next round of selection.

[0188] After affinity maturation, the selection products were cloned out for screening purposes. The scFvs isolated by ribosome display were cloned into the NotI / NcoI restricted nuclease of the ribosome display construct (New England BioLabs, USA; cat: R0189L, R0193L). The fragment was cloned into the phagemid vector pCANTAB6 by enzyme digestion and then ligated to NotI / NcoI-digested pCANTAB6 using T4 DNA ligase (New England BioLabs, USA; cat: M0202L) essentially as described in McCafferty et al., 1994.

[0189] 2.4 Identification of improved clones from phage and ribosome display using epitope assays 2024 scFvs selected from the third and fourth rounds of selection of the targeted mutagenesis approach described in sections 2.2, 2.3 and 2.4 were expressed in E. coli. Crude periplasmic scFvs were produced by combining the scFvs. Those scFvs capable of competing with C0020187 IgG for binding to recombinant human trimeric ARG2 enzyme were elucidated in a competition format assay using the HTRF™ platform. Specifically, fluorescence resonance energy transfer (FRET) was measured between streptavidin cryptate (biotinylated human trimeric ARG2 enzyme) and anti-human Fc XL665 (associated with C0020187 IgG) in the presence of a single concentration of each crude periplasmic test scFv. Preferred occupancy of the C0020187 IgG epitope on the enzyme by the scFv resulted in a reduction in FRET as measured by a fluorescent plate reader.

[0190] The "maximum" binding signal was determined by analyzing the binding of C0020187 IgG to biotinylated recombinant human trimeric ARG2 enzyme in the absence of competing scFv. The "sample" signal was determined from analyzing the binding of C0020187 IgG to biotinylated recombinant human trimeric ARG2 enzyme in the presence of test scFv samples. Finally, the "background" signal was determined by analyzing the fluorescence generated in the absence of C0020187 IgG.

[0191] Unpurified periplasmic scFvs were supplied in sample buffer consisting of 200 mM Tris base, pH 7.4, 0.5 mM EDTA, and 0.5 M sucrose. Using an automated pipetting robot, 5 μl of each scFv was transferred to the "sample" wells of a black, shallow, solid-bottom, non-binding 384-well assay plate. The remaining reagents (prepared in assay buffer) were added to the assay plate using a multichannel pipette in the following order: 5 μl of detection cocktail consisting of 6.6 nM streptavidin cryptate and 40 nM anti-human Fc XL665 (to all wells), 5 μl of 12 nM biotinylated recombinant human trimeric ARG2 enzyme (to all wells), 5 μl of 16 nM C0020187 IgG (to "sample" and "max" wells), and 5 μl of sample buffer (to background wells). The assay plates were sealed and incubated overnight in the dark at room temperature before measuring time-resolved fluorescence at 620 and 665 nm emission wavelengths in a fluorescent plate reader.

[0192] Data were analyzed by calculating the % Delta F value for each sample. The % Delta F value was determined according to Equation 1. Equation 1:

number

[0193] The delta F values ​​were then used to calculate normalized binding values ​​as described in Equation 4. Equation 4:

number

[0194] DNA sequencing of unpurified scFv periplasm revealed significant inhibition of C0020187 IgG binding to biotinylated recombinant human trimeric ARG2 enzyme (Osbourn et al., 1996; Vaughan et al., 1996). scFvs found to contain unique protein sequences were expressed in Escherichia coli (E. coli) and purified by affinity chromatography. After purification by HPLC, the buffer was exchanged.

[0195] The potency of each purified scFv was determined by testing a dilution series of the scFv (typically 4 pM to 1200 nM) in the epitope competition assay described above. The data were again analyzed by calculating the % delta F and % control binding values ​​for each sample.

[0196] Scientific graphing software was used to plot scFv sample concentration against % control and any concentration-dependent responses were fitted to a nonlinear regression curve. IC 50 was obtained (Figure 8, Table 11).

[0197] [Table 20]

[0198] Reagents / equipment source: Tris base (Sigma, UK; cat: RDD008), potassium fluoride (VWR chemicals, Belgium; cat: 26820.236), bovine serum albumin solution (Sigma, UK; cat: A7284), C0020187 IgG (produced in-house), biotinylated recombinant human trimeric ARG2 Enzymes (produced in-house), streptavidin cryptate (Cisbio, France; cat: 610SAKLB), anti-human-IgG-XL665 (Cisbio, France; cat: 61HFCXLB), 384-well assay plates (Greiner BioOne, Germany; cat: 784076), 384-well dilution plates (Greiner BioOne, Germany; cat: 781280), automated pipetting robot (Hamilton Star™, Hamilton, USA), fluorescent plate reader (Pherastar™, BMG Labtech, USA), HTRF technology (Cisbio International, France), graphing / statistical software (Prism, GraphPad USA).

[0199] 2.5 Identification of improved clones using enzyme inhibition assays The crude periplasmic scFv tested in the epitope competition assay described in Section 2.4 were simultaneously screened for their ability to inhibit the enzymatic activity of trimeric human ARG2 in the assay described in Section 1.5. Crude periplasmic scFv C0020187 was included in this assay and used as a benchmark against which improved clones could be identified. Improved clones were required to demonstrate a 5% or greater increase in inhibition over that observed with C0020187.

[0200] Unpurified periplasmic scFvs that showed significant improvement over C0020187 IgG were subjected to DNA sequencing (Osbourn et al., 1996; Vaughan et al., 1996). The scFvs that were found to contain unique protein sequences were expressed in Escherichia coli (E. coli) and purified. After purification by affinity chromatography, the product was buffer exchanged.

[0201] The potency of each purified scFv was determined by testing a dilution series of scFv (typically 4 pM to 1200 nM) in the enzyme inhibition assay described in section 1.5. Data were again analyzed by calculating the %Max control value for each sample.

[0202] Using scientific graphing software, scFv sample concentrations were plotted against the %Max control, and any concentration-dependent responses were fitted to a nonlinear regression curve. From these analyses, IC 50 was obtained (Figure 9, Table 12).

[0203] [Table 21]

[0204] 2.6 Reformatting of scFv with improved affinity to human IgG1 The scFv with optimized sequence was reformatted using the same procedure described for reformatting the lead clone in section 1.8.

[0205] 2.7 Binding of THP1-derived ARG2 in the presence of plasma The improved mutant antibody population was tested for binding to cell lysates from THP-1 and parental THP-1 cell lines overexpressing human ARG2 in the presence and absence of healthy human plasma (Figure 10). Cell lysates were prepared in TBS buffer and total protein concentration was verified by BCA assay (Thermo Scientific, 23227). Human plasma was obtained from NHS BT Cambridge (batch: 15 / 08 / 2016).

[0206] Six ARG2 affinity matured IgGs (C0021022, C0021061, C0021065, C0021092, C0021133 and C0021139) were tested alongside the parent C0020187, as well as the irrelevant IgG control R347 and a no IgG control. Maxisorp white plates (Nunc, 437796) were pre-coated with 1 μg / ml rabbit anti-human A in PBS. The plate was coated with RG2 antibody (Abcam, ab137069) overnight at 4°C. After washing with PBS, cells were blocked with casein blocker (Thermo Scientific, 37528). Cell lysates from the THP-1-ARG2-positive cell line and the parental THP-1 cell line were diluted to 1 mg / ml in TBS buffer and tested with or without urea-depleted human normal plasma supplemented to a final concentration of 12.5%. 60 μl of each cell lysate was aliquoted onto a Greiner PP plate (Greiner, 650201). The lysates were prepared at 1 μg / ml in TBS buffer. A recombinant human ARG2-HIS (PSPUR016) prepared in-house was used as a positive control, and TBS buffer alone or 12.5% ​​plasma in TBS was used as a negative control. In-house ARG2-specific human IgG and irrelevant R347 IgG (all biotinylated) were added to cell lysate, recombinant human ARG2, and TBS buffer to a final concentration of 50 μg / ml (a no-IgG control was also added) and incubated for 45 minutes at room temperature with gentle shaking. The blocked ELISA plate was washed with PBS, and 50 μl of the preincubated sample from the previous step was transferred to the ELISA plate and incubated statically for 90 minutes at room temperature. The plate was washed five times with PBST (0.1%). 50 μl of streptavidin-HRP (Abcam, ab7403) diluted 1 / 10,000 in casein buffer (1 / 20 dilution of stock) was added to each well. The plate was then incubated statically at RT for 1 hour. After washing five times with PBST, PICO ELISA substrate (Thermo Scientific, 37070) was prepared according to the manufacturer's instructions and 100 μl was added to each well. The plate was incubated in the dark with gentle shaking for 1 minute and luminescence was read using an Envision plate reader. Data were analyzed using GraphPad Prism. Results are shown in Figure 10 for the following:

[0207] [Table 22]

[0208] All ARG2 IgGs showed specific binding to recombinant human ARG2, including ARG2-expressing THP-1 cell lysates and affinity-matured clones, revealing a high positive signal compared to the parent C0020187.

[0209] 2.8 Inhibition of THP-1-derived ARG2 by improved mutant IgG Anti-ARG2 IgGs were tested for their ability to inhibit mammalian-expressed human ARG2 produced in transfected THP-1 cells. To do so, cell lysates were prepared from THP-1 cells overexpressing human ARG2 and wild-type THP-1 cells. 350 million cells were suspended in 3 ml of Tris-buffered saline (25 mM Tris + 150 mM sodium chloride in dH2O, pH 7.4) and passed 20 times through a 26G needle to prepare cell lysates. Protein concentrations in the lysates were measured using a BCA assay. The IC of the test antibodies was 50 To determine the antibody titer, a dilution series of the test antibody was made in TBS, and 5 μl of the dilution was added to 10 μl of a lysate / human plasma mixture (1.15 mg / ml lysate, 25% (v / v) urea-deficient human plasma, and 20 mM MnCl) and incubated for 2 hours at room temperature in a 384-well plate. The enzymatic reaction was initiated by the addition of 5 μl of 100 mM L-arginine substrate and incubated for 1 hour at room temperature. Just before use, the urea detection reagent, i.e., O-phthaldialdehyde (1.5 mM O-phthaldialdehyde, 7% H2O, HCl ... 80 μl / well of the plate was added and incubated for 2 hours at room temperature. The absorbance at 505 nm was read using a Pherastar plate reader. Antibodies were tested alongside N-hydroxyl-L-arginine (a small molecule competitive inhibitor of ARG2) as a positive control and CEA6 (an irrelevant isotype control antibody) as a negative control. Data points were plotted and IC values ​​were calculated using a four-parameter fit in GraphPad Prism. 50 values ​​were determined (Figure 11, Table 13).

[0210] [Table 23]

[0211] 2.9 Restoration of T cell proliferation by improved mutant ARG2-specific IgG in the presence of ARG2 Anti-ARG2 antibodies were screened for their ability to restore T cell proliferation by inhibiting the enzymatic activity of ARG2 and thus the loss of L-arginine (which is required for T cell proliferation). This assay was used to provide evidence of improved functional potency for each affinity-matured antibody compared to the parental IgG, C0020187 (Figure 12).

[0212] Lead-optimized clones were tested up to an endpoint time of 96 hours using the BrdU Cell Proliferation ELISA Kit (Roche, France; Cat: 11647229001) according to the manufacturer's instructions. R347 was used as an irrelevant control. All clones tested were counted in duplicate.

[0213] [Table 24]

[0214] Leukocyte cones were subjected to Ficoll Plaque (GE, Sweden; Cat: 17-1440-02) gradient separation, PBMCs were extracted, and pan-T cell populations were isolated by negative selection using the Human T Cell Enrichment Kit (Stemcell Technologies, France; Cat: 19051) according to the manufacturer's instructions.

[0215] 40,000 cells / well were seeded in complete growth medium (RMPI 1640, 5% human serum albumin) in 96-well clear TC-treated microplates (Greiner-Bio One, Germany; Cat: 655180). T cells were activated by adding ImmunoCult CD3 / CD28 (Stemcell Technologies, France; Cat: 10971) directly to the wells at a dilution of 1:40, and the plates were then incubated at 37°C, 5% CO2 for 45 min.

[0216] EC of test antibody 50 To determine the β-actin concentration, a dilution series was made at 10x in sterile TBS / NaCl buffer and the final assay concentration was added to the assay plate containing the cells. The plate was incubated at 37°C, 5% CO for 30 minutes.

[0217] ARG2 was added to relevant wells of the plate at a final concentration of 15 μg / ml. Equal volumes of ARG2 vehicle and test antibody vehicle were added to relevant control wells. The total volume in each well was 200 μl. Cells were treated with test antibody for a total of 96 hours at 37°C, 5% CO2. Antibodies were tested alongside C0020187 (parent IgG) as a positive control and R347 (irrelevant isotype control antibody) as a negative control.

[0218] T cell proliferation was assessed using the BrdU Cell Proliferation Kit (Roche, Germany; Cat: 000000011647229001). 78 hours after antibody treatment, BrdU labeling reagent was added, and plates were further incubated for 18 hours at 37°C, 5% CO2. The remainder of the assay was performed according to the manufacturer's instructions.

[0219] Calculated data were expressed as % of control (Equation 3) and graphical plots were generated using a four-parameter fit, model 205 in XLFit (IDBS, UK) to calculate EC 50 Decide did.

[0220] Equation 3: All test sample data were corrected for background (average of cell-free controls). %Control = 100*((Test Sample - Average Positive Control) / (Average Negative Control - Average Positive Control)) Mean positive control = T cells in the absence of ARG2 Mean negative control = T cells in the presence of ARG2

[0221] 2.10 Kinetic Profiling of Affinity-Improved Clones as Fabs Using Bio-Layer Interferometry (BLI) The affinity of affinity-matured antibodies for human ARG2 was measured using the streptavidin capture method on the OctetRED system described in Section 1.15. Typically, an analyte titration range of 1.8-120 nM was used. The affinities obtained for human ARG2 and cynomolgus ARG2 are shown in Tables 15 and 16, respectively.

[0222] [Table 25]

[0223] [Table 26]

[0224] 2.11 Test for binding to human AR1 Binding of affinity-matured antibodies to human ARG1 was also measured with the OctetRED system using a screening assay that stably captures His-tagged ARG2 as a ligand on an anti-His surface. Typically, Fabs were tested over the analyte range of 1.8 to 120 nM using a 5-minute association time and a 10-minute dissociation time. After each cycle, the sensor was regenerated with 10 mM glycine pH 1.5. Results are shown in Table 17.

[0225] [Table 27]

[0226] Example 3 - Generation and characterization of second generation affinity matured ARG2 leads (pooled maturation by ribosome display) 3.1 Generation of second-generation affinity maturation leads A second-generation error-prone library was generated based on the scFv constructs on the pool of top antibody candidates from the preselection cascade. Error-prone PCR was used to introduce random mutations into the scFv region of the constructs, and the resulting library was used for ribosome display selection as described in Example 2. The resulting selection products were subcloned and then screened as periplasmic preparations / crude lysates in a second-generation epitope competition assay. Hits were sequenced, and lead candidates were selected based on sequence diversity and hit value. A population of Fabs was generated and analyzed using Biolayer Interferometry (BLI) on an Octet RED96. Candidates were ranked based on binding to human ARG2 by kinetic profiling.

[0227] 3.2 Identification of improved clones from second-generation error-prone library selection (ribosome display) using epitope competition assays 1320 scFvs randomly selected from the second and third selection rounds of error-prone mutagenesis described in sections 2.2 and 2.3 were expressed in bacteria to produce crude periplasmic scFvs. scFvs capable of binding to recombinant human trimeric ARG2 enzyme via the same epitope as C0021133 IgG were identified in a competitive format assay using the HTRF™ platform. Specifically, fluorescence resonance energy transfer (FRET) was measured between streptavidin cryptate (associated with biotinylated recombinant human trimeric ARG2 enzyme) and anti-human Fc XL665 (associated with C0021133 IgG) in the presence of a single concentration of each crude periplasmic test scFv. Preferred occupancy of the C0021133 IgG epitope on the enzyme by the scFvs resulted in a reduction in FRET as measured by a fluorescent plate reader.

[0228] The "maximum" binding signal was determined by analyzing the binding of C0021133 IgG to biotinylated recombinant human trimeric ARG2 enzyme in the absence of competing scFv. The "sample" signal was determined from analyzing the binding of C0021133 IgG to biotinylated recombinant human trimeric ARG2 enzyme in the presence of test scFv samples. Finally, the "background" signal was determined by analyzing the fluorescence generated in the absence of C0021133 IgG.

[0229] Unpurified periplasmic scFvs were delivered in sample buffer consisting of 200 mM Tris base, pH 7.4, 0.5 mM EDTA, and 0.5 M sucrose. Using an automated pipetting robot, 5 μl of each scFv was transferred to the "sample" wells of a black, shallow, solid-bottom, non-binding 384-well assay plate. The remaining reagents (prepared in assay buffer) were added to the assay plate using a multichannel pipette in the following order: 5 μl of detection cocktail consisting of 6.6 nM streptavidin cryptate and 40 nM anti-human Fc XL665 (to all wells), 5 μl of 12 nM biotinylated recombinant human trimeric ARG2 enzyme (to all wells), 5 μl of 8 nM C0021133 IgG (to "sample" and "max" wells), and 5 μl of sample buffer (to background wells). The assay plates were sealed and incubated overnight in the dark at room temperature before measuring time-resolved fluorescence at 620 and 665 nm emission wavelengths in a fluorescent plate reader.

[0230] Data were analyzed by calculating the % Delta F value for each sample. The % Delta F value was determined according to Equation 1. Equation 1:

number

[0231] The delta F values ​​were then used to calculate normalized binding values ​​as described in Equation 4. Equation 4:

number

[0232] DNA sequencing of unpurified scFv periplasm revealed significant inhibition of C0021133 IgG binding to biotinylated recombinant human trimeric ARG2 enzyme (Osbourn et al., 1996; Vaughan et al., 1996). scFvs found to contain unique protein sequences were expressed as IgG and Fab.

[0233] Reagents / equipment source: Tris base (Sigma, UK; cat: RDD008), potassium fluoride (VWR chemicals, Belgium; cat: 26820.236), bovine serum albumin solution (Sigma, UK; cat: A7284), C0020187 IgG (produced in-house), biotinylated recombinant human trimeric ARG2 Enzymes (produced in-house), streptavidin cryptate (Cisbio, France; cat: 610SAKLB), anti-human-IgG-XL665 (Cisbio, France; cat: 61HFCXLB), 384-well assay plates (Greiner BioOne, Germany; cat: 784076), 384-well dilution plates (Greiner BioOne, Germany; cat: 781280), automated pipetting robot (Hamilton Star™, Hamilton, USA), fluorescent plate reader (Pherastar™, BMG Labtech, USA), HTRF technology (Cisbio International, France), graphing / statistical software (Prism, GraphPad USA).

[0234] 3.3 Identification of improved clones from second-generation error-prone library selection (ribosome display) using enzyme inhibition assays The crude periplasmic scFv tested in the epitope competition assay described in Section 2.4 were simultaneously screened for their ability to inhibit the enzymatic activity of trimeric human ARG2 in the assay described in Section 1.5. In this assay, crude periplasmic scFv C0021133 was also included and used as a benchmark against which improved clones could be identified. Improved clones were required to show a 5% or greater increase in inhibition over that observed with C0021133.

[0235] The crude periplasmic scFvs, which showed significant improvement over C0021133 IgG, were subjected to DNA sequencing (Osbourn et al., 1996; Vaughan et al., 1996). The scFvs found to have unique protein sequences were expressed as IgGs (Section 1.8).

[0236] Anti-ARG2 IgGs were tested for their ability to inhibit mammalian-expressed human ARG2 produced in transfected THP-1 cells. To do so, cell lysates were prepared from THP-1 cells overexpressing human ARG2 and wild-type THP-1 cells. Cell lysates were prepared by suspending 350 million cells in 3 ml of Tris-buffered saline (25 mM Tris + 150 mM sodium chloride in dH2O, pH 7.4) and passing the cells 20 times through a 26G needle. Protein concentrations in the lysates were measured using a BCA assay. IC of test antibody 50 To determine the activity, a dilution series of the test antibody was made in TBS, and 5 μl was added to 10 μl of lysate (1.19 mg / ml lysate in MilliQ, 20 mM MnCl, 50 mM Tris-HCl, pH 7.4) and incubated at room temperature for 2 hours in a 384-well plate. The enzymatic reaction was initiated by the addition of 5 μl of 100 mM L-arginine substrate and incubated at room temperature for 1 hour. Immediately before use, the urea detection reagent, i.e., O-phthaldialdehyde (1.5 mM O-phthaldialdehyde, 7% H2SO4, 0.03% Brij L23) and N-(1-naphthyl)ethylenediamine (1.66 mM N-(1-naphthyl)ethylenediamine, 81 mM boric acid, 21% H2SO4, 0.03% Brij L23), was mixed 1:1, 80 μl / well was added, incubated at room temperature for 1 hour, and the absorbance at 505 nm was read using a Pherastar plate reader. Antibodies were tested alongside N-hydroxyl-L-arginine (a small molecule competitive inhibitor of ARG2) as a potent control and CEA6 (an irrelevant isotype control antibody) as a negative control (Figure 13, Table 18). Data points were plotted and IC values ​​were calculated using a four-parameter fit in GraphPad Prism. 50 It was decided that:

[0237] [Table 28]

[0238] 3.4 Restoration of T cell proliferation by improved mutant ARG2-specific IgG in the presence of ARG2 Anti-ARG2 antibodies were screened for their ability to restore T cell proliferation by inhibiting the enzymatic activity of ARG2 and thus the loss of L-arginine (required for T cell proliferation). This assay was used to provide evidence of improved functional potency for each affinity-matured antibody compared to the parental IgG, C0020187 (Figure 14, Table 19). R347 was used as an irrelevant control. All clones tested n=2. See section 2.9 for methods.

[0239] [Table 29]

[0240] 3.5 Kinetic Profiling of Second-Generation Antibodies as Fabs Using Bio-Layer Interferometry (BLI) The affinity of the parental second-generation antibodies for human ARG2 was measured using the OctetRED system. A screening assay stably capturing His-tagged ARG2 as a ligand on an anti-His surface was used to screen second-generation candidates as Fabs over the analyte range of 1.8–120 nM, using a 5-minute association time and a 10-minute dissociation time. After each cycle, the sensor was regenerated using 10 mM glycine pH 1.5. Based on this assay, six candidates with predicted affinities in the subnanomolar range were identified and further profiled using an extension assay, which is more suitable for high-affinity antibodies. In the extension assay, the association and dissociation times were extended to 10 and 40 minutes, respectively, using a lower Fab analyte titration range: 0.47–30 nM, on a streptavidin surface capturing bio-ARG2 as a ligand. Binding affinities were calculated using Octet analysis software by subtracting the reference standard from the sensorgrams and fitting to a 1:1 binding model. The kinetic parameters obtained are shown in Table 20.

[0241] [Table 30]

[0242] The same assay was used to determine the affinity of some of these antibodies for cynomolgus monkey ARG2, and the resulting kinetic parameters are shown in Table 21.

[0243] [Table 31]

[0244] 3.6 Testing IgG binding to human ARG1 Binding of second-generation antibodies to human ARG1 was also measured with the OctetRED system using a screening assay that stably captures His-tagged ARG2 as a ligand on an anti-His surface. Binding was measured at concentrations up to 120 nM analyte using a 5-minute association time and a 10-minute dissociation time. The Fab was tested as a Fab. After each cycle, the sensor was regenerated with 10 mM glycine pH 1.5. The results are shown in Table 22.

[0245] [Table 32]

[0246] 3.7 Understanding the mechanism of action of ARG2-specific IgG on the enzyme ARG2 The mechanism of action of lead identified clone C0021158 IgG was verified using a human ARG2 (trimeric) enzyme inhibition assay and isothermal titration calorimetry.

[0247] The enzyme inhibition assay, described in Section 1.5, is a biochemical assay that measures the production of urea, a product of L-arginine turnover by ARG2. To examine the mechanism of action of C0021158 IgG, a four-point titration of C0021158 IgG (1000, 100, 30, and 10 nM) was prepared in the presence of 11 concentrations of arginine (250, 200, 150, 100, 62.5, 37.5, 25, 12.5, 8.5, 5, 5, and 2.5 mM). The reaction was allowed to proceed for 80, 60, 45, 30, 15, or 5 minutes, after which the urea detection reagents O-phthaldialdehyde and N-(1-naphthyl)ethylenediamine were added.

[0248] Raw data from each individual concentration of C0021158 IgG was plotted as a function of time for each concentration of arginine and then fitted using a linear fit (GraphPad Prism) to obtain the initial velocity (slope value).

[0249] The initial velocity data were plotted against the arginine concentration for each concentration of C0021158 IgG (Figure 15A). Each trace was fitted to the Michaelis-Menten model (Y = V) in GraphPad Prism. max *X / (K m + X)) to obtain the maximum velocity (V max) and enzyme / substrate affinity (K m ) values ​​were obtained (Table 23).

[0250] [Table 33]

[0251] The mechanism of action of enzyme inhibitors can be broadly classified into competitive, uncompetitive and non-competitive. m and V max They can be so identified based on their effect on

[0252] Competitive inhibitors bind only to the free enzyme, often at the active site, and inhibit the maximum enzyme velocity (V max A decrease in the affinity of the enzyme for the substrate (K m This leads to an increase in

[0253] An uncompetitive inhibitor binds exclusively to the enzyme-substrate complex, producing an inactive enzyme-substrate-inhibitor complex. The affinity of the enzyme for the substrate increases (K m decreases), maximum enzyme velocity (V max ) decreases.

[0254] Non-competitive inhibitors bind equally well to the free enzyme and the enzyme-substrate complex. The affinity of the enzyme for the substrate (K m ) is unchanged, and the maximum enzyme velocity (V max ) decreases.

[0255] For C0021158 IgG inhibitor, V in the presence of C0021158 IgG inhibitor max decreases, excluding competitive modes of action and favoring uncompetitive or non-competitive modes of action. To distinguish non-competitive modes of action from uncompetitive modes of action, K mThe effect on K must be evaluated. Unfortunately, the initial velocity / arginine concentration plot has an atypical profile, where in the presence of inhibitor, an initially expected increase in initial velocity is followed by a drop as arginine concentration increases. This atypical profile leads to poor fitting of the data and makes it difficult to determine an accurate K. m It becomes difficult to obtain an accurate K m The lack of data means that C0021158 IgG cannot be assigned as uncompetitive or non-competitive. All that can be said is that this IgG inhibitor is "non-competitive."

[0256] To assess the accessibility of the ARG2 active site, we analyzed the binding of the ARG2 small molecule inhibitor S-(2-boronoethyl)-L-cysteine ​​hydrochloride (BEC) by isothermal titration calorimetry (ITC) using a VP ITC (MicroCal) set at 25°C (Figure 15B). BEC is an arginine analog that binds to the active site of ARG2. First, a volume of 10 μl of solution containing 0.75 mM BEC was added to 20 μM ARG2 (monomer concentration). The resulting thermogram was integrated using NITPIC, and a binding isotherm was fitted with SEDPHAT using a one-binding site model. In the next experiment, a volume of 10 μl of 0.75 mM BEC was added to 20 μM ARG2 (monomer concentration). 0.50 mM BEC was added to a solution containing a pre-incubated 1:1 molar mixture of C0021158 Fab and ARG2 (both 20 μM, incubated at 20°C for 1 h). Thermograms were integrated using NITPIC, and the resulting heat of injection was within the background range, suggesting that binding of BEC to ARG2 in the presence of C0021158 Fab was significantly reduced. EIA Taken together with the data, this indicates that C0021158 fab acts as an allosteric inhibitor, preventing substrate binding to ARG2.

[0257] 3.8 Germlining and De-risking Lead Antibodies Antibodies obtained from selection products during the lead optimization stage may often contain spontaneous mutations within the framework that deviate from the germline sequence. As a measure to potentially reduce immunogenicity in vivo, these mutations can be reverted to the germline sequence in lead candidates. For antibody clone C0021142, amino acid residues within the VL framework 3 region were reverted to the germline sequence during the IgG conversion process (C0021142 IgG). Similarly, amino acid residues within the VH framework 4 region were reverted to the germline sequence during the IgG conversion process (C0021158 IgG). The IgG and Fab versions of these antibody clones will carry these sequences. Furthermore, C0021158 was further germlined within the VL framework 3 region to generate a fully germlined (fgl) version (referred to as C0021158fgl). In further molecular engineering, predicted moderate-risk deamidation sites were also engineered from C0021158 to generate a risk-free (dr) version (termed C0021158dr). Finally, a fully germlined and risk-free version of C0021158 was generated and designated C0021158fgl2.

[0258] 3.9 Inhibition of THP-1-derived ARG2 by second-generation improved mutant IgG The second-generation improved variant anti-ARG2 IgG was tested for its ability to inhibit mammalian-expressed human ARG2 produced in transfected THP-1 cells. The method for this assay is described in Section 2.8. The results are shown in Figure 16 and Table 24.

[0259] [Table 34]

[0260] 3.10 Demonstration of ARG2 release from cancer cell lines Cancer cell lines representing a variety of indications were selected for this study 25. A collection of cell lines from three indications, namely prostate cancer, sarcoma, and ovarian cancer, was selected for further evaluation based on ARG2 expression in the culture medium, as described below (Table 25). These cell lines were obtained from either ATCC, ECACC, or Essen Biosciences.

[0261] Serum starvation treatment: T175 cm in a 37°C humidified incubator containing 5% CO2 2 Cell lines were grown in flasks in 35 ml of supplier-recommended medium (see table below for details*) until cells were ≥ 70% confluent. Cells were washed with 25 ml of 1x DPBS (Gibco, ref. 14190-094) and after 48 hours were resuspended in 35 ml of serum starvation medium (i.e., serum-depleted). The medium above the cells (hereafter referred to as cell-conditioned medium or CCM) was collected in a 50 ml Falcon tube and centrifuged at 300 × g at 4°C to separate floating / dead cells. The dead cells were harvested. The CCM above the cell pellet was collected in a separate 50 ml Falcon tube and then filtered through a 0.22 μm Millipore Express® filter (Millipore, cat. SCGP00525) to remove cellular debris. The filtered CCM was stored on ice for concentration.

[0262] Adherent cells were trypsinized with 5 ml of TrypLE-Express (Gibco, cat. 12604-013) for a maximum of 5 minutes in a 37°C incubator. Cells were observed under a microscope to be rounded / detached. Once this was confirmed, 25 ml of pre-warmed medium was added and gently mixed to obtain a single cell suspension. The cells were transferred to the Falcon tube containing the cells harvested from the CCM and then gently mixed. The cells were harvested by centrifugation at 300 × g for 5 minutes at 4 °C. The supernatant was removed, and the cells were resuspended in 30 ml of 1 × DPBS. Cell number and viability were determined using Trypan Blue staining (Invitrogen, cat. T10282) and a Countess® II Automated cell counter (Life Technologies) according to the manufacturer's instructions. did.

[0263] Concentration of CCM: The filtered CCM was concentrated by centrifugation using an Amicon® Ultra-15 filter (Millipore, ref. UCF901096). The filter was pre-soaked with 10 ml of 1x DPBS and centrifuged at 4000 rpm for 8 minutes at 20°C. The filtrate collected in the receptacle tube was discarded. The CCM was added to the tube and centrifuged at 4000 rpm for 6-8 minutes at 20°C. This step was repeated, discarding the filtrate and adding CCM each time until approximately 700 μl of concentrated CCM (approximately 50x concentrated) remained on the filter. The concentrated CCM was collected in a pre-labeled microfuge tube, and 1x Protease Halt inhibitor (ThermoFisher, cat. 1861281) was added to the mixing well. The concentrated CCM was then stored at -20°C. The cells were either stored at room temperature or processed directly for immunoprecipitation.

[0264] Immunoprecipitation (IP) and Western blotting (WB): 5 x 10 6The cell-equivalent volume of concentrated CCM was determined. The CCM was diluted with 500 μl of wash buffer (1×TBS, 1.5 M NaCl, 0.25 M Tris pH 7.4, 0.05% Tween-20). Similarly, 25 ng of recombinant human ARG1 and ARG2 proteins, which could serve as negative and positive controls, were individually diluted in wash buffer. For each IP replicate, 2 μl of ARG2 PAD antibody (Cloud Clone Corp, ref. PAD796Hu01) was added to 50 μl of wash buffer. The beads were diluted with PBS and then added to the IP tubes. The IP reaction tubes were left overnight on a rotator at 11 rpm at 4°C. The next day, 25 μl of Dynabeads™ (M-280 Sheep Anti-Rabbit IgG, Invitrogen, 11203D) were washed twice with 1 ml of wash buffer. The beads were resuspended in 50 μl of wash buffer and added to each IP tube. The tubes were left on a rotator at 11 rpm at room temperature for 2 h. The liquid was removed using a magnet. The beads were washed twice with 500 μl of wash buffer.

[0265] The IP beads were suspended in 15 μl of 1× NuPAGE LDS sample loading buffer (Invitrogen, cat. NP0007) and incubated at room temperature for 20 minutes. The gels were heated to RT for 10 min and then loaded onto a NuPAGE™ 4-12% Bis-Tris gel (Invitrogen, cat. NP0322BOX). Protein markers were also loaded, consisting of a mixture of 7 μl of pre-stained protein standards (Invitrogen, LC5800) and 2 μl of MagicMark™ XP (Invitrogen, LC5602). The gels were run at 200 V, 500 mA for 35 min in 1× NuPAGE™ MES SDS running buffer (Invitrogen, cat. NP0002). The gel was run. The dissolved proteins were transferred to a PVDF membrane (Invitrogen, cat. IB24001) using iBlot® 2 (Invitrogen) at 20V for 1 minute, 23V for 4 minutes, and 25V for 2 minutes. The membrane was blocked with 5% milk-TBS-Tween 20 on a roller at 22 rpm for 1 hour at room temperature. ARG2 MAD antibody (Cloud Clone Corp, ref. MAD796Hu21) was added to 5 ml of 5% milk-TBS-Tween 20. Dilute to 1:1000 in TBS-Tween 20 and place on a roller overnight at 4°C. The membrane was then washed three times with 1x TBS-Tween 20 (approximately 5 minutes each time). Anti-mouse (Fc specific)-HRP conjugated antibody (Sigma cat. A2254) was diluted to 1:2000 in 5 ml of 5% milk-TBS-Tween 20 and then incubated with the mouse-HRP antibody at room temperature for 1 hour. The membrane was then washed three times with 1x TBS-T-Tween 20. ECL Prime Blots were developed using detection reagents (GE Healthcare, RPN2232) according to the manufacturer's instructions. Chemiluminescent images were captured using a Gel Imager (ChemiDoc-It). 2 ) The final set of images was inverted in ImageJ for colorization.

[0266] Among the various cell lines tested (Table 25), ARG2 was immunoprecipitated from various cell lines, including CCM of LNCaP, A673, and A2780-Cis cell lines (FIG. 17).

[0267] [Table 35]

[0268] [Table 36]

[0269] 3.11 Binding of first and second generation IgGs (C0021061 and C0021158) to ARG2 from cancer cell lines In this experiment, we verified the presence of ARG2 in CCM samples using a commercially available ARG2 ELISA kit (Cloud-Clone Corp., cat. SED796Hu). Furthermore, to confirm the binding of ARG2 IgG to ARG2 expressed from cancer cell lines, we modified the protocol to include the first-generation C0021061 and second-generation C0021158 as detection antibodies (Figure 18).

[0270] The commercially available kit is a 96-well plate pre-coated with anti-ARG2, which uses a sandwich ELISA to quantify ARG2 in samples. Standards, biotinylated detection antibody, detection substrate, and stop reagents are all provided in the kit. Concentrated CCM samples from LNCaP, A673, and A2780-Cis cells that showed ARG2 expression (as previously described in Figure 17) and media controls were diluted 1:1 with 1x DPBS. ELISA was performed according to the manufacturer's protocol, using 100 μl of diluted CCM / media control in each well of the kit. Each sample was performed in duplicate. In parallel, a CCM / media control was also added to assay the binding of C0021061 and C0021158 to ARG2 in the CCM samples. For this, all steps were identical to the manufacturer's protocol, except that biotinylated-C0021061 / -C0021158 / -irrelevant IgG control R347 was used at 10 μg / ml instead of the biotinylated detection antibody (PAD) provided with the kit. At the end of the assay, absorbance at 450 nm was read using a PheraStar (BMG). Standard absorbance values ​​were used to generate a standard curve, which was used to interpolate ARG2 levels in the CCM samples (in this case, the PAD detection antibody provided with the kit was used). Similarly, absorbance readings from wells that received C0021061 and C0021158 were used to infer first and second generation ARG2 IgGs (C0021061 and C0021158, respectively) that bind to ARG2 in the CCM samples (Figure 18).

[0271] 3.12. Co-crystallization of ARG2-binding Fabs C0021158 and C0021181 with Human ARG2 to Reveal Epitope / Paratope Contact Residues The construct used for crystallographic studies contained a C-terminal Gly3 linker and His 10The ARG2(23-354) complexes contained either a huARG2-His tag (ARG2+C0020187, SEQ ID NO: 355: huARG2-His10) or a simple C-terminal His6 tag (ARG2+C0021181 and ARG2+C0021158, SEQ ID NO: 357: huARG2-His6). Crystals suitable for structure determination were obtained using the sitting drop method. For ARG2+C0020187, 0.2 μl of the complex solution (2.0 mg / ml, ARG2:Fab ratio 3:1, purified by gel filtration) was mixed with 0.1 μl of crystallization solution containing 2 M (NH4)2SO4. After 2 days, round, disk-shaped crystals (60 × 60 × 20 μm) appeared. For ARG2+C0021181, 0.5 μl of the complex solution (6.3 mg / ml) was mixed with 0.5 μl of crystallization solution containing 100 mM MMT (malic acid:MES:Tris = 1:2:2) pH 5.0, 20% glycerol, 10% PEG 4000, 15 mM NaNO3, 15 mM NaHPO4, and 15 mM (NH4)2SO4. After 2 days, hexagonal disk-shaped crystals (100 x 100 x 20 μm) appeared and reached their final size after 1 week. For ARG2+C0021158, 0.25 μl of the complex solution (7.0 mg / ml, containing 20% ​​diluted microseeds of ARG2+C0021181 crystals) was mixed with 0.25 μl of crystallization solution containing 2 M (NH4)2SO4. After 2 days, irregularly shaped crystals (60 × 60 × 60 μm) appeared. For ARG2+C0020187 and ARG2+C0021158 crystals, 3M (NH4)2SO4 and 5% glycerol were added for cryoprotection, followed by cryo-cooling by immersing the crystals in liquid N2. Data sets were collected at DLS, beamline i04-1 (ARG2+C0020187) or at BESSY II, HZB, beamline MX-14-1 (ARG2+C0021181 and ARG2+C0021158). The ARG2+C0020187 crystals are in the space group P652 with unit cell parameters: a = b = 138 Å, c = 551 Å. Crystals of ARG2+C0021181 diffracted to 2.9 Å in space group P3 2 1 with unit cell parameters a = b = 150 Å, c = 111 Å and exhibited twinning at a ratio of 0.50 / 0.50. Crystals of ARG2+C0021158 diffracted to 2.4 Å in space group H3 with unit cell parameters a = b = 149 Å, c = 123 Å and exhibited twinning at a ratio of 0.80 / 0.20.

[0272] 3.13 Structural analysis and refinement The reported structure of ARG2 (PDB-ID 4HZE) and the following individual Fabs were identified by molecular replacement search: Using a previously solved high-resolution structure, the structure was solved by molecular replacement using Phaser in the CCP4 program suite. In the case of ARG2+C0020187, the asymmetric unit is C, which contains three copies each of ARG2 and Fab, each present in a different orientation L and C H In the cases of ARG2+C0021181 and ARG2+C0021158, the asymmetric unit contained one copy of ARG2 and one copy of Fab, whereas in the case of ARG2+C0021181, the V L and V H Only the domain is clear, C L and C H The domain likely existed in two orientations (reflected by the increased B-factor and overall poor electron density). Automatic and manual refinements were then performed by alternating between REFMAC5 (twin and TLS refinements driven towards the completion of the structures of ARG2+C0021181 and ARG2+C0021158) and COOT, respectively. The final R / R of the ARG2+C0020187, ARG2+C0021181, and ARG2+C0021158 structures were free The values ​​were 0.30 / 0.36, 0.27 / 0.32 and 0.25 / 0.28, respectively (Figure 19).

[0273] [Table 37]

[0274] [Table 38]

[0275] [Table 39]

[0276] 3.14 Size exclusion chromatography to analyze ARG2-Fab binding stoichiometry ARG2 and Fab were analyzed at a concentration of 8 μM (referred to as monomer) in SEC buffer containing 25 mM Tris and 150 mM NaCl at pH 7.4. ARG2-Fab complexes were formed by mixing both ARG2 and Fab at a concentration of 8 μM (referred to as monomer) in SEC buffer and incubated on ice for 1 h. For size exclusion chromatography, 500 μl of sample was injected into a 100 μl loop connected to a Superose 6 Increase 10 / 300 GL column (GE Healthcare) attached to an AKTAexpress FPLC system (GE Healthcare). The column was equilibrated with SEC buffer, and SEC was performed at a flow rate of 0.5 ml / min. Molecular weight estimates were obtained using the same FPLC system under identical conditions. Based on the retention volumes observed for β-amylase, bovine serum albumin, and carbonic anhydrase analyzed in

[0277] 3.15 Dynamic Light Scattering to Analyze ARG2-Fab Complex Size ARG2 and Fab were analyzed at a concentration of 8 μM (referred to as monomer) in a buffer containing 25 mM Tris and 150 mM NaCl at pH 7.4 (Figure 23). ARG2-Fab complexes were formed by mixing both ARG2 and Fab at a concentration of 8 μM (referred to as monomer) and incubated on ice for 1 h. Dynamic light scattering (DLS) was performed on a Zetasizer Ultra (Malvern Panalytical) using multi-angle DLS. Data were collected in a 3 × 3 mm cuvette (Hellma Analytics) using a 30 μl sample volume. The weighted particle size distribution was corrected by the instrument software for refractive index, viscosity, and buffer background scattering.

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Osbourn, J.K., et al., Generation of a panel of related human scFv antibodies with high affinities for human CEA. Immunotechnology, 1996. 2(3): p. 181-96. 43. Pandey, D., et al., OxLDL triggers retrograde translocation of arginase2 in aortic endothelial cells via ROCK and mitochondrial processing peptidase. Circ Res, 2014. 115(4): p. 450-9. 44. Persic, L., et al., An integrated vector system for the eukaryotic expression of antibodies or their fragments after selection from phage display libraries. Gene, 1997. 187(1): p. 9-18. 45. Polat, M.F., et al., Elevated serum arginase activity levels in patients with breast cancer. Surg Today, 2003. 33(9): p. 655-61. 46. Rabinovich, G.A., D. Gabrilovich, and E.M. Sotomayor, Immunosuppressive strategies that are mediated by tumor cells. Annu Rev Immunol, 2007. 25: p. 267-96.47. 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Schier, R., et al., Isolation of high-affinity monomeric human anti-c-erbB-2 single chain Fv using affinity-driven selection. J Mol Biol, 1996. 255(1): p. 28-43. 53. Setty, B.A., et al., Hypoxic Proliferation of Osteosarcoma Cells Depends on Arginase II. Cell Physiol Biochem, 2016. 39(2): p. 802-13. 54. Singh, R., et al., Proteomic identification of mitochondrial targets of arginase in human breast cancer. PLoS One, 2013. 8(11): p. e79242. 55. Sousa, M.S., et al., Arginase 2 and nitric oxide synthase: Pathways associated with the pathogenesis of thyroid tumors. Free Radic Biol Med, 2010. 49(6): p . 997-1007. 56. Steggerda, S.M., et al., Inhibition of arginase by CB-1158 blocks myeloid cell-mediated immune suppression in the tumor microenvironment. J Immunother Cancer, 2017. 5(1): p. 101. 57. 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Xu, W., et al., Increased arginase II and decreased NO synthesis in endothelial cells of patients with pulmonary arterial hypertension. FASEB J, 2004. 18(14): p. 1746-8. 63. Xue, J., L.D. Nelin, and B. Chen, Hypoxia induces arginase II expression and increases viable human pulmonary artery smooth muscle cell numbers via AMPKalpha1 signaling. Am J Physiol Lung Cell Mol Physiol, 2017. 312(4): p. L568-L578. 64. Yaiw, K.C., et al., Human cytomegalovirus induces upregulation of arginase II: possible implications for vasculopathies. Basic Res Cardiol, 2014. 109(2): p. 401. 65. Yepuri, G., et al., Positive crosstalk between arginase-II and S6K1 in vascular endothelial inflammation and aging. Aging Cell, 2012. 11(6): p. 1005-16. 66. Yu, Y., et al., p38 mitogen-activated protein kinase is involved in arginase-II-mediated eNOS-uncoupling in obesity. Cardiovasc Diabetol, 2014. 13: p. 113.67. Zaytouni, T., et al., Critical role for arginase 2 in obesity-associated pancreatic cancer. 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[0279] Table 40

[0280] Table 41

[0281] Table 42

[0282] Table 43

[0283] Table 44

[0284] Table 45

[0285] Table 46

[0286] Table 47

[0287] Table 48

[0288] Table 49

[0289] [Table 50]

[0290] [Table 51]

[0291] [Table 52]

[0292] [Table 53]

[0293] [Table 54]

[0294] ARG2 amino acid sequence SEQ ID NO: 351 N-terminal Avi tag AGLNDIFEAQKIEWHE SEQ ID NO: 352 Avi-huARG1-His10 [ka] SEQ ID NO: 353 Avi-huARG2-His10 [ka] SEQ ID NO: 354 Avi-huARG2-His10 monomer [ka] SEQ ID NO: 355 huARG2-His10 [ka] SEQ ID NO: 356 Avi-cynoARG2-His10 [ka] SEQ ID NO: 357 huARG2-His6 [ka]

Claims

1. An isolated antigen-binding protein characterized by being capable of specifically binding to human arginase II (ARG2) and inhibiting the enzymatic activity of human ARG2.

2. 2. The antigen binding protein of claim 1, wherein the antigen binding protein is capable of specifically binding to and inhibiting monomeric and / or trimeric human ARG2.

3. the antigen binding protein has a dissociation constant (K) of less than 10 nM, less than 1 nM, less than 500 pM, less than 300 pM, or less than 150 pM as assessed by biolayer interferometry (BLI). Ｄ 3. The antigen-binding protein of claim 1 or 2, which binds to trimeric human ARG2 at the nucleotide sequence:

4. 4. The antigen binding protein of any one of claims 1 to 3, wherein the antigen binding protein is selective for binding to and inhibiting human ARG2 over human ARG1.

5. 5. The antigen binding protein of claim 4, wherein the selectivity for binding to human ARG2 over human ARG1 is assessed by biolayer interferometry (BLI).

6. 6. The antigen binding protein of any one of claims 1 to 5, wherein the antigen binding protein does not measurably bind to human ARG1 when assessed by biolayer interferometry (BLI).

7. 7. The antigen-binding protein of any one of claims 1 to 6, wherein the antigen-binding protein binds to human ARG2 with a 1:1 or 3:1 stoichiometry (antigen-binding protein:human ARG2).

8. 8. The antigen-binding protein of any one of claims 1 to 7, wherein the antigen-binding protein is capable of specifically binding to cynomolgus monkey ARG2 and inhibiting its enzymatic activity.

9. 9. The antigen binding protein of any one of claims 1 to 8, wherein the antigen binding protein inhibits ARG2 by a non-competitive mode of action.

10. 10. The antigen binding protein of any one of claims 1 to 9, wherein the antigen binding protein restores T cell proliferation in vitro in the presence of ARG2.

11. below: (a) a VH domain comprising a set of HCDRs: HCDR1, HCDR2, and HCDR3, interspersed with framework (FW) regions (HFW1-HCDR1-HFW2-HCDR2-HFW3-HCDR3-HFW4), wherein the amino acid sequence of HCDR3 (amino acids 95-102) is LRADLGLYMDL (SEQ ID NO: 315); and optionally further comprising: (b) a VL domain comprising a set of LCDRs: LCDR1, LCDR2, and LCDR3, interspersed with framework (FW) regions (LFW1-LCDR1-LFW2-LCDR2-LFW3-LCDR3-LFW4), wherein the amino acid sequence of LCDR1 (amino acids 24-34) is SGSSSNIGNHYVS (SEQ ID NO: 318), said sequence being defined by the Kabat nomenclature; 11. The antigen-binding protein of any one of claims 1 to 10.

12. below: (a) a VH domain comprising a set of HCDRs: HCDR1, HCDR2, and HCDR3 interspersed with framework regions (HFW1-HCDR1-HFW2-HCDR2-HFW3-HCDR3-HFW4), wherein said set of HCDRs is selected from the group consisting of antibodies: C0021158fgl2 (SEQ ID NOs: 313, 314, and 315), C0021181 (SEQ ID NOs: 343, 344, and 345), C0021180 (SEQ ID NOs: 333, 334, and 335), C0021177 (SEQ ID NOs: 323, 324, and 325), C0021158 (SEQ ID NOs: 273, 274, and 275), C0021158 IgG (SEQ ID NOs: 283, 284, and 285), C0021158fgl (SEQ ID NOs: 303, 304, and 305), C0021158dr (SEQ ID NOs: 293, 294, and 295), C0021061 (SEQ ID NOs: 63, 64, and 65), C0020187 (SEQ ID NOs: 13, 14, and 15), C0021155 (SEQ ID NOs: 263, 264, and 265), C0021144 (SEQ ID NOs: 253, 254, and 255), C0021142 (SEQ ID NOs: 233, 234, and 235), C0021142IgG (SEQ ID NOs: 243, 244, and 245), C0021141 (SEQ ID NOs: 223, 224, and 225), C0021139 (SEQ ID NOs: 213, 214, and 215), C0021135 (SEQ ID NOs: 203, 204, and 205), C0021133 (SEQ ID NOs: 193, 194, and 195), C0021131 (SEQ ID NO: 18 3, 184 and 185), C0021129 (SEQ ID NOs: 173, 174 and 175), C0021128 (SEQ ID NOs: 163, 164 and 165), C0021124 (SEQ ID NOs: 153, 154 and 155), C0021118 (SEQ ID NOs: 143, 144 and 145), C0021101 (SEQ ID NOs: 133, 134 and 135), and / or a VH domain selected from those of C0021098 (SEQ ID NOs: 123, 124, and 125), C0021097 (SEQ ID NOs: 113, 114, and 115), C0021096 (SEQ ID NOs: 103, 104, and 105), C0021092 (SEQ ID NOs: 93, 94, and 95), C0021089 (SEQ ID NOs: 83, 84, and 85), C0021065 (SEQ ID NOs: 73, 74, and 75), C0021032 (SEQ ID NOs: 53, 54, and 55), C0021022 (SEQ ID NOs: 43, 44, and 45), C0021021 (SEQ ID NOs: 33, 34, and 35), C0021017 (SEQ ID NOs: 23, 24, and 25) and C0020065 (SEQ ID NOs: 3, 4, and 5); and / or (b) A VL domain comprising a set of LCDRs: LCDR1, LCDR2 and LCDR3 interspersed with framework regions (LFW1-LCDR1-LFW2-LCDR2-LFW3-LCDR3-LFW4), wherein said set of LCDRs is selected from the group consisting of antibodies: C0021158fgl2 (SEQ ID NOs: 318, 319 and 320), C0021181 (SEQ ID NOs: 348, 349 and 350), C0021180 (SEQ ID NOs: 338, 339 and 340), C0021177 (SEQ ID NOs: 328, 329 and 330), C0021158 (SEQ ID NOs: 278, 279 and 280), C0021158 IgG (SEQ ID NOs: 288, 289, and 290), C0021158fgl (SEQ ID NOs: 308, 309, and 310), C0021158dr (SEQ ID NOs: 298, 299, and 300), C0021061 (SEQ ID NOs: 68, 69, and 70), C0020187 (SEQ ID NOs: 18, 19, and 20), C0021155 (SEQ ID NOs: 268, 269, and 270), C0021144 (SEQ ID NOs: 258, 259, and 260), C0021142 (SEQ ID NOs: 238, 239, and 240), C0021142 IgG (SEQ ID NOs: 248, 249, and 250), C0021141 (SEQ ID NOs: 228, 229, and 230), C0021139 (SEQ ID NOs: 218, 219, and 220), C0021135 (SEQ ID NOs: 208, 209, and 210), C0021133 (SEQ ID NOs: 198, 199, and 200), C0021131 (SEQ ID NOs: 188, 189, and 190), C0021129 (SEQ ID NOs: 178, 179, and 180), C0 C0021128 (SEQ ID NOs: 168, 169 and 170), C0021124 (SEQ ID NOs: 158, 159 and 160), C0021118 (SEQ ID NOs: 148, 149 and 150), C0021101 (SEQ ID NOs: 138, 139 and 140), C0021098 (SEQ ID NOs: 128, 129 and 130), C0021097 (SEQ ID NOs: 118, 119 and 120), C0021096 (SEQ ID NOs: 108, 109 and 110) , C0021092 (SEQ ID NOs: 98, 99 and 100), C0021089 (SEQ ID NOs: 88, 89 and 90), C0021065 (SEQ ID NOs: 78, 79 and 80), C0021032 (SEQ ID NOs: 58, 59 and 60), C0021022 (SEQ ID NOs: 48, 49 and 50), C0021021 (SEQ ID NOs: 38, 39 and 40), C0021017 (SEQ ID NOs: 28, 29 and 30) and C0020065 (SEQ ID NOs: 8, 9 and 10).

12. The antigen-binding protein of any one of claims 1 to 11, comprising the sequence defined by the Kabat nomenclature.

13. below: a VH domain comprising HCDR1 (SEQ ID NO:313), HCDR2 (SEQ ID NO:314), and HCDR3 (SEQ ID NO:315), and a VL domain comprising LCDR1 (SEQ ID NO:318), LCDR2, (SEQ ID NO:319), and LCDR3 (SEQ ID NO:320) of C0021158fgl2; C0021181, a VH domain comprising HCDR1 (SEQ ID NO: 343), HCDR2 (SEQ ID NO: 344), and HCDR3 (SEQ ID NO: 345), and a VL domain comprising LCDR1 (SEQ ID NO: 348), LCDR2, (SEQ ID NO: 349), and LCDR3 (SEQ ID NO: 350); C0021180, a VH domain comprising HCDR1 (SEQ ID NO:313), HCDR2 (SEQ ID NO:314), and HCDR3 (SEQ ID NO:315), and a VL domain comprising LCDR1 (SEQ ID NO:318), LCDR2, (SEQ ID NO:319), and LCDR3 (SEQ ID NO:320); C0021177, a VH domain comprising HCDR1 (SEQ ID NO: 323), HCDR2 (SEQ ID NO: 324), and HCDR3 (SEQ ID NO: 325), and a VL domain comprising LCDR1 (SEQ ID NO: 328), LCDR2, (SEQ ID NO: 329), and LCDR3 (SEQ ID NO: 330); C0021158, a VH domain comprising HCDR1 (SEQ ID NO:273), HCDR2 (SEQ ID NO:274), and HCDR3 (SEQ ID NO:275), and a VL domain comprising LCDR1 (SEQ ID NO:278), LCDR2, (SEQ ID NO:279), and LCDR3 (SEQ ID NO:280); C0021158 IgG, a VH domain comprising HCDR1 (SEQ ID NO:283), HCDR2 (SEQ ID NO:284), and HCDR3 (SEQ ID NO:285), and a VL domain comprising LCDR1 (SEQ ID NO:288), LCDR2, (SEQ ID NO:289), and LCDR3 (SEQ ID NO:290); a VH domain comprising HCDR1 (SEQ ID NO: 303), HCDR2 (SEQ ID NO: 304), and HCDR3 (SEQ ID NO: 305), and a VL domain comprising LCDR1 (SEQ ID NO: 308), LCDR2, (SEQ ID NO: 309), and LCDR3 (SEQ ID NO: 310) of C0021158fgl; C0021158dr, a VH domain comprising HCDR1 (SEQ ID NO:293), HCDR2 (SEQ ID NO:294), and HCDR3 (SEQ ID NO:295), and a VL domain comprising LCDR1 (SEQ ID NO:298), LCDR2, (SEQ ID NO:299), and LCDR3 (SEQ ID NO:300); C0021061, a VH domain comprising HCDR1 (SEQ ID NO: 63), HCDR2 (SEQ ID NO: 64), and HCDR3 (SEQ ID NO: 65), and a VL domain comprising LCDR1 (SEQ ID NO: 68), LCDR2, (SEQ ID NO: 69), and LCDR3 (SEQ ID NO: 70); C0020187, a VH domain comprising HCDR1 (SEQ ID NO: 13), HCDR2 (SEQ ID NO: 14) and HCDR3 (SEQ ID NO: 15), and a VL domain comprising LCDR1 (SEQ ID NO: 18), LCDR2, (SEQ ID NO: 19) and LCDR3 (SEQ ID NO: 20); C0021155, a VH domain comprising HCDR1 (SEQ ID NO: 263), HCDR2 (SEQ ID NO: 264), and HCDR3 (SEQ ID NO: 265), and LCDR1 (SEQ ID NO: 268). a VL domain comprising: LCDR1, LCDR2, (SEQ ID NO:269) and LCDR3 (SEQ ID NO:270); a VH domain comprising HCDR1 (SEQ ID NO:253), HCDR2 (SEQ ID NO:254), and HCDR3 (SEQ ID NO:255), and a VL domain comprising LCDR1 (SEQ ID NO:258), LCDR2, (SEQ ID NO:259), and LCDR3 (SEQ ID NO:260) of C0021144; C0021142, a VH domain comprising HCDR1 (SEQ ID NO:233), HCDR2 (SEQ ID NO:234), and HCDR3 (SEQ ID NO:235), and a VL domain comprising LCDR1 (SEQ ID NO:238), LCDR2, (SEQ ID NO:239), and LCDR3 (SEQ ID NO:240); C0021142 IgG, a VH domain comprising HCDR1 (SEQ ID NO: 243), HCDR2 (SEQ ID NO: 244), and HCDR3 (SEQ ID NO: 245), and a VL domain comprising LCDR1 (SEQ ID NO: 248), LCDR2, (SEQ ID NO: 249), and LCDR3 (SEQ ID NO: 250); C0021141, a VH domain comprising HCDR1 (SEQ ID NO:223), HCDR2 (SEQ ID NO:224), and HCDR3 (SEQ ID NO:225), and a VL domain comprising LCDR1 (SEQ ID NO:228), LCDR2, (SEQ ID NO:229), and LCDR3 (SEQ ID NO:230); a VH domain comprising HCDR1 (SEQ ID NO:213), HCDR2 (SEQ ID NO:214), and HCDR3 (SEQ ID NO:215), and a VL domain comprising LCDR1 (SEQ ID NO:218), LCDR2, (SEQ ID NO:219), and LCDR3 (SEQ ID NO:220) of C0021139; a VH domain comprising HCDR1 (SEQ ID NO:203), HCDR2 (SEQ ID NO:204), and HCDR3 (SEQ ID NO:205), and a VL domain comprising LCDR1 (SEQ ID NO:208), LCDR2 (SEQ ID NO:209), and LCDR3 (SEQ ID NO:210) of C0021135; a VH domain comprising HCDR1 (SEQ ID NO: 193), HCDR2 (SEQ ID NO: 194), and HCDR3 (SEQ ID NO: 195), and a VL domain comprising LCDR1 (SEQ ID NO: 198), LCDR2, (SEQ ID NO: 199), and LCDR3 (SEQ ID NO: 200) of C0021133; a VH domain comprising HCDR1 (SEQ ID NO: 183), HCDR2 (SEQ ID NO: 184), and HCDR3 (SEQ ID NO: 185), and a VL domain comprising LCDR1 (SEQ ID NO: 188), LCDR2, (SEQ ID NO: 189), and LCDR3 (SEQ ID NO: 190) of C0021131; a VH domain comprising HCDR1 (SEQ ID NO: 173), HCDR2 (SEQ ID NO: 174), and HCDR3 (SEQ ID NO: 175), and a VL domain comprising LCDR1 (SEQ ID NO: 178), LCDR2, (SEQ ID NO: 179), and LCDR3 (SEQ ID NO: 180) of C0021129; C0021128, a VH domain comprising HCDR1 (SEQ ID NO: 163), HCDR2 (SEQ ID NO: 164), and HCDR3 (SEQ ID NO: 165), and a VL domain comprising LCDR1 (SEQ ID NO: 168), LCDR2, (SEQ ID NO: 169), and LCDR3 (SEQ ID NO: 170); C0021124, a VH domain comprising HCDR1 (SEQ ID NO: 153), HCDR2 (SEQ ID NO: 154), and HCDR3 (SEQ ID NO: 155), and a VL domain comprising LCDR1 (SEQ ID NO: 158), LCDR2, (SEQ ID NO: 159), and LCDR3 (SEQ ID NO: 160); a VH domain comprising HCDR1 (SEQ ID NO: 143), HCDR2 (SEQ ID NO: 144), and HCDR3 (SEQ ID NO: 145), and a VL domain comprising LCDR1 (SEQ ID NO: 148), LCDR2, (SEQ ID NO: 149), and LCDR3 (SEQ ID NO: 150) of C0021118; a VH domain comprising HCDR1 (SEQ ID NO: 133), HCDR2 (SEQ ID NO: 134), and HCDR3 (SEQ ID NO: 135), and a VL domain comprising LCDR1 (SEQ ID NO: 138), LCDR2, (SEQ ID NO: 139), and LCDR3 (SEQ ID NO: 140) of C0021101; C0021098, a VH domain comprising HCDR1 (SEQ ID NO: 123), HCDR2 (SEQ ID NO: 124), and HCDR3 (SEQ ID NO: 125), and a VL domain comprising LCDR1 (SEQ ID NO: 128), LCDR2, (SEQ ID NO: 129), and LCDR3 (SEQ ID NO: 130); C0021097, a VH domain comprising HCDR1 (SEQ ID NO: 113), HCDR2 (SEQ ID NO: 114), and HCDR3 (SEQ ID NO: 115), and a VL domain comprising LCDR1 (SEQ ID NO: 118), LCDR2, (SEQ ID NO: 119), and LCDR3 (SEQ ID NO: 120); C0021096, a VH domain comprising HCDR1 (SEQ ID NO: 103), HCDR2 (SEQ ID NO: 104), and HCDR3 (SEQ ID NO: 105), and a VL domain comprising LCDR1 (SEQ ID NO: 108), LCDR2, (SEQ ID NO: 109), and LCDR3 (SEQ ID NO: 110); C0021092, a VH domain comprising HCDR1 (SEQ ID NO: 93), HCDR2 (SEQ ID NO: 94), and HCDR3 (SEQ ID NO: 95), and a VL domain comprising LCDR1 (SEQ ID NO: 98), LCDR2, (SEQ ID NO: 99), and LCDR3 (SEQ ID NO: 100); C0021089, a VH domain comprising HCDR1 (SEQ ID NO: 83), HCDR2 (SEQ ID NO: 84), and HCDR3 (SEQ ID NO: 85), and a VL domain comprising LCDR1 (SEQ ID NO: 88), LCDR2, (SEQ ID NO: 89), and LCDR3 (SEQ ID NO: 90); C0021065, a VH domain comprising HCDR1 (SEQ ID NO:73), HCDR2 (SEQ ID NO:74) and HCDR3 (SEQ ID NO:75), and a VL domain comprising LCDR1 (SEQ ID NO:78), LCDR2, (SEQ ID NO:79) and LCDR3 (SEQ ID NO:80); C0021032, a VH domain comprising HCDR1 (SEQ ID NO: 53), HCDR2 (SEQ ID NO: 54), and HCDR3 (SEQ ID NO: 55), and a VL domain comprising LCDR1 (SEQ ID NO: 58), LCDR2, (SEQ ID NO: 59), and LCDR3 (SEQ ID NO: 60); C0021022, a VH domain comprising HCDR1 (SEQ ID NO: 43), HCDR2 (SEQ ID NO: 44) and HCDR3 (SEQ ID NO: 45), and a VL domain comprising LCDR1 (SEQ ID NO: 48), LCDR2, (SEQ ID NO: 49) and LCDR3 (SEQ ID NO: 50); C0021021, a VH domain comprising HCDR1 (SEQ ID NO: 33), HCDR2 (SEQ ID NO: 34), and HCDR3 (SEQ ID NO: 35), and a VL domain comprising LCDR1 (SEQ ID NO: 38), LCDR2, (SEQ ID NO: 39), and LCDR3 (SEQ ID NO: 40); a VH domain comprising HCDR1 (SEQ ID NO:23), HCDR2 (SEQ ID NO:24) and HCDR3 (SEQ ID NO:25) and a VL domain comprising LCDR1 (SEQ ID NO:28), LCDR2 (SEQ ID NO:29) and LCDR3 (SEQ ID NO:30) of C0021017; or C0020065, a VH domain comprising HCDR1 (SEQ ID NO: 3), HCDR2 (SEQ ID NO: 4) and HCDR3 (SEQ ID NO: 5), and a VL domain comprising LCDR1 (SEQ ID NO: 8), LCDR2 (SEQ ID NO: 9) and LCDR3 (SEQ ID NO: 10).

13. The antigen-binding protein of any one of claims 1 to 12, comprising the sequence defined by the Kabat nomenclature.

14. below: (a) (i) a VH domain comprising the set of HCDRs of C0021158fgl2 (HCDR1 SEQ ID NO:313, HCDR2 SEQ ID NO:314, and HCDR3 SEQ ID NO:315), and / or (ii) a VL domain comprising the set of LCDRs of C0021158fgl2 (LCDR1 SEQ ID NO:318, LCDR2 SEQ ID NO:319, and LCDR3 SEQ ID NO:320); (b) (i) the set of HCDRs of C0021133 (HCDR1 SEQ ID NO: 193, HC or (c) a VH domain comprising (i) a set of HCDRs of C0020187 (HCDR1 SEQ ID NO:13, HCDR2 SEQ ID NO:14 and HCDR3 SEQ ID NO:15) and / or (ii) a VL domain comprising the set of LCDRs of C0020187 (LCDR1 SEQ ID NO:18, LCDR2 SEQ ID NO:196 and LCDR3 SEQ ID NO:207); or (c) a VH domain comprising (i) a set of HCDRs of C0020187 (HCDR1 SEQ ID NO:13, HCDR2 SEQ ID NO:14 and HCDR3 SEQ ID NO:15) and / or (ii) a set of LCDRs of C0020187 (LCDR1 SEQ ID NO:18, LCDR2 SEQ ID NO:209 and LCDR3 SEQ ID NO:210). a VL domain comprising a VL domain comprising a VL domain (VL domain) of the VL domain (VL domain) ... (d) (i) a VH domain comprising the set of HCDRs of C0020065 (HCDR1 SEQ ID NO:3, HCDR2 SEQ ID NO:4, and HCDR3 SEQ ID NO:5), and / or (ii) a VL domain comprising the set of LCDRs of C0020187 (LCDR1 SEQ ID NO:8, LCDR2 SEQ ID NO:9, and LCDR3 SEQ ID NO:10).

14. The antigen-binding protein of any one of claims 1 to 13, comprising the sequence defined by the Kabat nomenclature.

15. below: (a) antibodies: C0021158 fgl2 (SEQ ID NO: 312), C0021181 (SEQ ID NO: 342), C0021180 (SEQ ID NO: 332), C0021177 (SEQ ID NO: 322), C0021158 (SEQ ID NO: 272), C0021158 IgG (SEQ ID NO: 282), C0021158fgl (SEQ ID NO: 302), C0021158dr (SEQ ID NO: 292), C0021061 (SEQ ID NO: 62), C0020187 (SEQ ID NO: 12), C0021155 (SEQ ID NO: 262), C0021144 (SEQ ID NO: 252), C0021142 (SEQ ID NO: 232), C0021142 IgG (SEQ ID NO: 242), C0021141 (SEQ ID NO: 227), C0021139 (SEQ ID NO: 217), C0021135 (SEQ ID NO: 207), C0021133 (SEQ ID NO: 197), C0021131 (SEQ ID NO: 187), C0021129 (SEQ ID NO: 177), C0021128 (SEQ ID NO: 167), C0021124 (SEQ ID NO: 157), C0021118 (SEQ ID NO: 147), C0021101 (SEQ ID NO: 137), C0021098 (SEQ ID NO: 127), C0021097 (SEQ ID NO: 117), C0021096 (SEQ ID NO: 107), C0021092 (SEQ ID NO: 97), C0021 and / or a VH domain selected from the VH domains of C0020065 (SEQ ID NO:77), C0021032 (SEQ ID NO:57), C0021022 (SEQ ID NO:47), C0021021 (SEQ ID NO:37), C0021017 (SEQ ID NO:27), and C0020065 (SEQ ID NO:7), or a germlined version thereof, or a VH domain with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology thereto; and / or (b) antibodies: C0021158 fgl2 (SEQ ID NO: 317), C0021181 (SEQ ID NO: 347), C0021180 (SEQ ID NO: 337), C0021177 (SEQ ID NO: 327), C0021158 (SEQ ID NO: 277), C0021158 IgG (SEQ ID NO: 287), C0021158fgl (SEQ ID NO: 307), C0021158dr (SEQ ID NO: 297), C0021061 (SEQ ID NO: 67), C0020187 (SEQ ID NO: 17), C0021155 (SEQ ID NO: 267), C0021144 (SEQ ID NO: 257), C0021142 (SEQ ID NO: 237), C0021142 IgG (SEQ ID NO: 247), C0021141 (SEQ ID NO: 227), C0021139 (SEQ ID NO: 217), C0021135 (SEQ ID NO: 207), C0021133 (SEQ ID NO: 197), C0021131 (SEQ ID NO: 187), C0021129 (SEQ ID NO: 177), C0021128 (SEQ ID NO: 167), C0021124 (SEQ ID NO: 157), C0021118 (SEQ ID NO: No. 147), C0021101 (SEQ ID NO: 137), C0021098 (SEQ ID NO: 127), C0021097 (SEQ ID NO: 117), C0021096 (SEQ ID NO: 107), C0021092 (SEQ ID NO: 97), C0021089 (SEQ ID NO: 87), C0021065 (SEQ ID NO: 77), C0021032 (SEQ ID NO: 57), C0021022 (SEQ ID NO: 47), C002102 1 (SEQ ID NO:37), C0021017 (SEQ ID NO:27) and C0020065 (SEQ ID NO:7), or a germlined version thereof, or a VL domain having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology thereto.

15. The isolated antigen binding protein of any one of claims 1 to 14, comprising the sequence defined by the Kabat nomenclature.

16. below: C0021158 fgl2 VH domain (SEQ ID NO: 312) and VL domain (SEQ ID NO: 317); C0021181 VH domain (SEQ ID NO: 342) and VL domain (SEQ ID NO: 347); C0021180 VH domain (SEQ ID NO: 332) and VL domain (SEQ ID NO: 337); the VH domain (SEQ ID NO: 322) and VL domain (SEQ ID NO: 327) of C0021177; C0021158 VH domain (SEQ ID NO: 272) and VL domain (SEQ ID NO: 277); C0021158 IgG VH domain (SEQ ID NO: 282) and VL domain (SEQ ID NO: 287); C0021158fgl VH domain (SEQ ID NO: 302) and VL domain (SEQ ID NO: 307); C0021158dr VH domain (SEQ ID NO: 292) and VL domain (SEQ ID NO: 297); The VH domain (SEQ ID NO: 62) and VL domain (SEQ ID NO: 67) of C0021061, the VH domain (SEQ ID NO: 12) and VL domain (SEQ ID NO: 17) of C0020187, the VH domain (SEQ ID NO: 262) and VL domain (SEQ ID NO: 267) of C0021155, the VH domain (SEQ ID NO: 252) and VL domain (SEQ ID NO: 257) of C0021144; C0021142 VH domain (SEQ ID NO: 232) and VL domain (SEQ ID NO: 237); C0021142 IgG VH domain (SEQ ID NO: 242) and VL domain (SEQ ID NO: 247); C0021141 VH domain (SEQ ID NO: 222) and VL domain (SEQ ID NO: 227); C0021139 VH domain (SEQ ID NO: 212) and VL domain (SEQ ID NO: 217); the VH domain (SEQ ID NO: 202) and VL domain (SEQ ID NO: 207) of C0021135; C0021133 VH domain (SEQ ID NO: 192) and VL domain (SEQ ID NO: 197); C0021131 VH domain (SEQ ID NO: 182) and VL domain (SEQ ID NO: 187); C0021129 VH domain (SEQ ID NO: 172) and VL domain (SEQ ID NO: 177); C0021128 VH domain (SEQ ID NO: 162) and VL domain (SEQ ID NO: 167); The VH domain (SEQ ID NO: 152) and VL domain (SEQ ID NO: 157) of C0021124 ）、 C0021118 VH domain (SEQ ID NO: 142) and VL domain (SEQ ID NO: 147); C0021101 VH domain (SEQ ID NO: 132) and VL domain (SEQ ID NO: 137); C0021098 VH domain (SEQ ID NO: 122) and VL domain (SEQ ID NO: 127); C0021097 VH domain (SEQ ID NO: 112) and VL domain (SEQ ID NO: 117); C0021096 VH domain (SEQ ID NO: 102) and VL domain (SEQ ID NO: 107); the VH domain (SEQ ID NO:92) and VL domain (SEQ ID NO:97) of C0021092, the VH domain (SEQ ID NO:82) and VL domain (SEQ ID NO:87) of C0021089, the VH domain (SEQ ID NO:72) and VL domain (SEQ ID NO:77) of C0021065, the VH domain (SEQ ID NO:52) and VL domain (SEQ ID NO:57) of C0021032, the VH domain (SEQ ID NO:42) and VL domain (SEQ ID NO:47) of C0021022, the VH domain (SEQ ID NO:32) and VL domain (SEQ ID NO:37) of C0021021, the VH domain (SEQ ID NO:22) and VL domain (SEQ ID NO:27) of C0021017, or VH domain (SEQ ID NO: 2) and VL domain (SEQ ID NO: 7) of C0020065 16. The antigen binding protein of any one of claims 1 to 15, comprising a VH domain and a VL domain that are at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to

17. below: C0021158 fgl2 VH domain (SEQ ID NO: 312) and VL domain (SEQ ID NO: 317); C0021181 VH domain (SEQ ID NO: 342) and VL domain (SEQ ID NO: 347); C0021180 VH domain (SEQ ID NO: 332) and VL domain (SEQ ID NO: 337); the VH domain (SEQ ID NO: 322) and VL domain (SEQ ID NO: 327) of C0021177; C0021158 VH domain (SEQ ID NO: 272) and VL domain (SEQ ID NO: 277); C0021158 IgG VH domain (SEQ ID NO: 282) and VL domain (SEQ ID NO: 287); C0021158fgl VH domain (SEQ ID NO: 302) and VL domain (SEQ ID NO: 307); C0021158dr VH domain (SEQ ID NO: 292) and VL domain (SEQ ID NO: 297); The VH domain (SEQ ID NO: 62) and VL domain (SEQ ID NO: 67) of C0021061, the VH domain (SEQ ID NO: 12) and VL domain (SEQ ID NO: 17) of C0020187, the VH domain (SEQ ID NO: 262) and VL domain (SEQ ID NO: 267) of C0021155, the VH domain (SEQ ID NO: 252) and VL domain (SEQ ID NO: 257) of C0021144; The VH domain (SEQ ID NO: 232) and VL domain (SEQ ID NO: 237) of C0021142 ）、 C0021142 IgG VH domain (SEQ ID NO: 242) and VL domain (SEQ ID NO: 247); C0021141 VH domain (SEQ ID NO: 222) and VL domain (SEQ ID NO: 227); C0021139 VH domain (SEQ ID NO: 212) and VL domain (SEQ ID NO: 217); the VH domain (SEQ ID NO: 202) and VL domain (SEQ ID NO: 207) of C0021135; C0021133 VH domain (SEQ ID NO: 192) and VL domain (SEQ ID NO: 197); C0021131 VH domain (SEQ ID NO: 182) and VL domain (SEQ ID NO: 187); C0021129 VH domain (SEQ ID NO: 172) and VL domain (SEQ ID NO: 177); C0021128 VH domain (SEQ ID NO: 162) and VL domain (SEQ ID NO: 167); C0021124 VH domain (SEQ ID NO: 152) and VL domain (SEQ ID NO: 157); C0021118 VH domain (SEQ ID NO: 142) and VL domain (SEQ ID NO: 147); C0021101 VH domain (SEQ ID NO: 132) and VL domain (SEQ ID NO: 137); C0021098 VH domain (SEQ ID NO: 122) and VL domain (SEQ ID NO: 127); C0021097 VH domain (SEQ ID NO: 112) and VL domain (SEQ ID NO: 117); C0021096 VH domain (SEQ ID NO: 102) and VL domain (SEQ ID NO: 107); the VH domain (SEQ ID NO:92) and VL domain (SEQ ID NO:97) of C0021092, the VH domain (SEQ ID NO:82) and VL domain (SEQ ID NO:87) of C0021089, the VH domain (SEQ ID NO:72) and VL domain (SEQ ID NO:77) of C0021065, the VH domain (SEQ ID NO:52) and VL domain (SEQ ID NO:57) of C0021032, the VH domain (SEQ ID NO:42) and VL domain (SEQ ID NO:47) of C0021022, the VH domain (SEQ ID NO:32) and VL domain (SEQ ID NO:37) of C0021021, the VH domain (SEQ ID NO:22) and VL domain (SEQ ID NO:27) of C0021017, or 17. The antigen binding protein of any one of claims 1 to 16, comprising the VH domain (SEQ ID NO:2) and VL domain (SEQ ID NO:7) of C0020065; or germlined versions thereof, said sequences defined by the Kabat nomenclature.

18. below: (a) C0021158 fgl2 VH domain amino acid sequence (SEQ ID NO: 312) and C0021158 fgl2 VL domain amino acid sequence (SEQ ID NO: 317); (b) the C0021133 VH domain amino acid sequence (SEQ ID NO: 192) and the C0021133 VL domain amino acid sequence (SEQ ID NO: 197); (c) the C0020187 VH domain amino acid sequence (SEQ ID NO: 12) and the C0020187 VL domain amino acid sequence (SEQ ID NO: 17); (d) C0020065 VH domain amino acid sequence (SEQ ID NO: 2) and C0020065 VL domain amino acid sequence (SEQ ID NO:7) 18. The antigen-binding protein of any one of claims 1 to 17, comprising:

19. When tested in an epitope competition assay: (a) a VH domain of SEQ ID NO: 312 and a VL domain of SEQ ID NO: 317; (b) a VH domain of SEQ ID NO: 192 and a VL domain of SEQ ID NO: 197; (c) a VH domain of SEQ ID NO: 12 and a VL domain of SEQ ID NO: 17, or (d) a VH domain of SEQ ID NO: 2 and a VL domain of SEQ ID NO: 7 An antigen binding protein that competes for binding to human ARG2 with an antigen binding protein comprising:

20. 20. The antigen-binding protein of any one of claims 1 to 19, characterized in that it specifically binds to an epitope of human arginase II (ARG2), thereby inhibiting the enzymatic activity of human ARG2 by an allosteric mechanism.

21. 1. A compound according to claim 1, characterized in that it specifically binds to an epitope of human arginase II (ARG2) and thereby induces structural remodeling of residues 33-40 in human ARG2, wherein the sequence numbering is that of the human ARG2 sequence of Uniprot ID#P78540.

21. The isolated antigen-binding protein of any one of claims 1 to 20.

22. 22. Any of claims 1 to 21, characterized in that it specifically binds to an epitope on human arginase II (ARG2), thereby inhibiting the enzymatic activity of human ARG2 by an allosteric mechanism, wherein the antigen binding protein binds to an epitope on human ARG2, which induces a structural or biophysical change to His160 of human ARG2, which results in a decrease in the enzymatic activity of ARG2 or its ability to process a substrate, and wherein the sequence numbering is that of the human ARG2 sequence of Uniprot ID#P78540.

10. The isolated antigen-binding protein of any one of claims 1 to 9.

23. the antigen binding protein binds to an epitope on human ARG2, which induces a conformational change such that Arg39 moves closer to His160 of human ARG2, thereby resulting in a decrease in the enzymatic activity of ARG2 or its ability to process a substrate, and the sequence numbering is that of the human ARG2 sequence of Uniprot ID#P78540.

23. The isolated antigen-binding protein of any one of claims 1 to 22.

24. 24. The isolated antigen binding protein of any one of claims 1 to 23, wherein the antigen binding protein binds to an epitope on human ARG2 that induces a conformational change that impairs the ability of His160 to act as a proton donor / acceptor and / or stabilize a catalytically competent binding orientation of a substrate, thereby resulting in reduced ARG2 enzymatic activity.

25. and binding to an epitope on human ARG2, including a conformational epitope comprising residues Gln35 to Arg39, residues Lys78 to Ile86, and / or residues Leu152 to Pro179, wherein the sequence numbering is that of the human ARG2 sequence in Uniprot ID#P78540.

25. The antigen-binding protein of any one of claims 1 to 24, wherein

26. 26. The antigen-binding protein of any one of claims 1 to 25, which binds to an epitope on human ARG2 comprising one or more residues selected from GLN35, GLY36, GLN37, LYS38, ARG39, LYS78, ASP79, ASP80, LEU81, TYR82, ASN84, LEU85, ILE86, LEU152, THR153 THR154, SER155, SER156, GLY157, LEU178 and PRO179.

27. 27. The antigen binding of claim 26, wherein the epitope is defined as any residue within human ARG2 with a predicted change in solvent accessibility upon Fab complex formation resulting from direct protection by the binding antibody as obtained from the X-ray structure data of ARG2 inhibitory Fab C0020187, and the sequence numbering is that of the human ARG2 sequence of Uniprot ID#P78540. protein.

28. and binding to an epitope on human ARG2, including a conformational epitope comprising residues Pro32 to Glu51, residues Asp70 to Ile86, and / or residues Pro299 to Ala308, wherein the sequence numbering is that of the human ARG2 sequence in Uniprot ID#P78540.

25. The antigen-binding protein of any one of claims 1 to 24, wherein

29. 29. The antigen binding protein of claim 28, which binds to an epitope on human ARG2 comprising one or more residues selected from PRO32, GLN37, LYS38, LYS40, GLY41, GLU43, HIS44, ALA47, ALA48, GLU51, ASP70, SER72, PHE73, THR74, PRO75, LYS78, ASP79, ASP80, LEU81, TYR82, ASN84, LEU85, ILE86, PRO299, GLN300, GLU305 and ALA308.

30. 30. The method of claim 28 or 29, wherein the epitope is defined as any residue within human ARG2 with a predicted change in solvent accessibility upon Fab complex formation resulting from direct protection by the binding antibody, as obtained from the X-ray structure data of ARG2 inhibitory Fab C0021158, and the sequence numbering is that of the human ARG2 sequence of Uniprot ID#P78540. antigen-binding protein.

31. and binding to an epitope on human ARG2, including a conformational epitope comprising residues Gln37 to Glu51, residues Asp79 to Ile86, and / or residues Pro299 to Ala308, wherein the sequence numbering is that of the human ARG2 sequence in Uniprot ID#P78540.

31. The antigen-binding protein of claim 30, wherein:

32. 25. The antigen binding protein of any one of claims 1 to 24, which binds to an epitope on recombinant human ARG2 comprising one or more residues selected from GLN37, LYS38, LYS40, GLY41, HIS44, ALA47, ALA48, GLU51, ASP79, ASP80, LEU81, TYR82, ASN84, LEU85, ILE86, PRO299, GLN300, ALA302, THR303, SER304, GLU305 and ALA308.

33. 33. The antigen binding protein of claim 32, wherein the epitope is defined as any residue within human ARG2 with a predicted change in solvent accessibility upon Fab complex formation resulting from direct protection by the binding antibody, as obtained from the X-ray structure data of ARG2 inhibitory Fab C0021181, and the sequence numbering is that of the human ARG2 sequence of Uniprot ID#P78540. Synthetic protein.

34. PRO32, GLN35, GLY36, GLN37, LYS38, ARG39, LYS40, GLY41, GLU43, HIS44, ALA47, ALA48, GLU51, ASP70, SER72, PHE73, THR74, PRO75, LYS78, ASP79, ASP80, LEU81, TYR82, ASN84, LEU85, ILE86, LEU152, THR153 and comprising one or more residues selected from THR154, SER155, SER156, GLY157, LEU178, PRO179, PRO299, GLN300, ALA302, THR303, SER304, GLU305, and ALA308.

34. The antigen-binding protein of claim 32 or 33, which binds to an epitope on human ARG2.

35. Claim 1 wherein the epitope is defined as any residue within human ARG2 with a predicted change in solvent accessibility upon Fab complex formation as obtained from X-ray structural data of an ARG2 inhibitory Fab selected from C0020187, C0021158, and C0021181, and the sequence numbering is that of the human ARG2 sequence of Uniprot ID#P78540.

35. The antigen-binding protein according to claim 34.

36. 36. The antigen-binding protein of any one of claims 1 to 35, wherein the antigen-binding protein is an antibody or fragment thereof, a domain antibody, a protein scaffold, or an aptamer.

37. 37. The antigen-binding protein of any one of claims 1 to 36, wherein the antigen-binding protein is human IgG or modified human IgG.

38. 38. The antigen binding protein of claim 37, wherein the antigen binding protein is human IgG1, IgG2, IgG4, or a modified version thereof.

39. 39. The antigen-binding protein of claim 37 or 38, wherein the antigen-binding protein is human IgG1 or IgG1-YTE.

40. 40. The antigen-binding protein of any one of claims 1 to 39, wherein the antigen-binding protein has a modified Fc to confer effector function and / or extended half-life.

41. 41. A composition comprising the antigen-binding protein of any one of claims 1 to 40 and a pharmaceutically acceptable excipient.

42. 42. An antigen-binding protein or composition according to any one of claims 1 to 41 for use in a method of treatment of the human or animal body.

43. 43. An antigen binding protein or composition according to any one of claims 1 to 42 for use in treating an individual for the purposes of restoring immune competence, alleviating inflammation-triggered immune dysfunction, inflammation-associated immunosuppression, promoting T cell mediated immune responses, or preventing tumor immune evasion, fibrosis, and immune disorders of infectious diseases.

44. 44. An antigen binding protein or composition according to any one of claims 1 to 43 for use in treating an individual with the aim of restoring T cell proliferation in the presence of ARG2.

45. 45. An antigen binding protein or composition according to any one of claims 1 to 44 for use in the treatment of cancer, immune cell dysfunction, autoimmunity or unwanted immune deviation.

46. 46. ​​An antigen binding protein or composition according to any one of claims 1 to 45 for use in the treatment of acute myeloid leukemia (AML), osteosarcoma, HCMV-driven GBM, pancreatic cancer, head and neck squamous cell carcinoma, thyroid cancer, prostate cancer, breast cancer or ovarian cancer.

47. 47. An antigen binding protein or composition according to any one of claims 1 to 46 for use in the treatment of infection (e.g. neonatal infection), endothelial dysfunction (e.g. erectile dysfunction), vascular disease, cardiovascular disease, ageing and cellular senescence, CNS disease and injury; diabetes-related diseases, cystic fibrosis or infections associated with cystic fibrosis.

48. 49. A method of treating an individual comprising administering to said individual an antigen binding protein or composition according to any one of claims 1 to 48.

49. 41. An isolated nucleic acid encoding the antigen-binding protein of any one of claims 1 to 40.

50. A host cell transformed in vitro with the nucleic acid of claim 49.

51. 51. A method of producing an antigen binding protein according to any one of claims 1 to 40, said method comprising culturing a host cell according to claim 50 under conditions for the production of said antigen binding protein.

52. 52. The method of claim 51, further comprising isolating and / or purifying the antigen binding protein.

53. 53. The method of claim 52, further comprising formulating said antigen binding protein into a composition comprising at least one additional component.

54. 1. A method of producing an antigen binding protein that specifically binds to and inhibits a human ARG, said method comprising producing a variant VH domain that is an amino acid sequence variant of a parent VH domain by addition, deletion, substitution or insertion of one or more amino acids in the amino acid sequence of the parent VH domain comprising HCDR1, HCDR2 and HCDR3, wherein said parent VH domains HCDR1, HCDR2 and HCDR3 are selected from the group consisting of C0021158fgl2, C0021181, C0021180, C0021177, C0021158, C0021158 IgG, C0021158fgl, C0021158dr, C0021061, C0020187, C0021155, C0021144, C0021142, C0021142 IgG, C0021141, C0021139, C0021135, C0021133, C0021131, C0021129, C0021128, C0021124, C0021118, C0021101, C0021098, C0021097, C0021096, C0021092, C0021089, C0021065, C0021032, C0021022, C0021021, C0021017 and C0020065 providing a variant VH domain, wherein the variant VH domain is a set of HCDRs selected from the set of HCDRs, and optionally combining the variant VH domain thus provided with one or more VL domains to provide one or more VH / VL combinations; and testing the variant VH domain or the variant VH / VL combinations that are amino acid sequence variants of the parent VH domain to identify an antigen-binding domain of an antigen-binding protein against human ARG2.

55. The parent VH domains are: C0021158fgl2, C0021181, C0021180, C0021177, C0021158, C0021158 IgG, C0021158fgl, C0021158dr, C0021061, C0020187, C0021155, C0021144, C0021142, C0021142 55. The method of claim 54, wherein the antibody is selected from the group consisting of IgG, C0021141, C0021139, C0021135, C0021133, C0021131, C0021129, C0021128, C0021124, C0021118, C0021101, C0021098, C0021097, C0021096, C0021092, C0021089, C0021065, C0021032, C0021022, C0021021, C0021017 and C0020065, or germlined versions thereof.

56. The one or more VL domains are variant VL domains provided by addition, deletion, substitution or insertion of one or more amino acids in the amino acid sequence of a parent VL domain comprising LCDR1, LCDR2 and LCDR3, and the parent VL domains LCDR1, LCDR2 and LCDR3 are selected from the following: C0021158fgl2, C0021181, C0021180, C0021177, C0021158, C0021158 IgG, C0021158fgl, C0021158dr, C0021061, C0020187, C0021155, C0021144, C0021142, C0021142 56. The method of claim 54 or 55, wherein the LCDRs are a set selected from the set of LCDRs of: IgG, C0021141, C0021139, C0021135, C0021133, C0021131, C0021129, C0021128, C0021124, C0021118, C0021101, C0021098, C0021097, C0021096, C0021092, C0021089, C0021065, C0021032, C0021022, C0021021, C0021017 and C0020065, each of which produces one or more variant VL domains that are amino acid sequence variants of the parent VL domain.

57. The parent VL domains are: C0021158fgl2, C0021181, C0021180, C0021177, C0021158, C0021158 IgG, C0021158fgl, C0021158dr, C0021061, C0020187, C0021155, C0021144, C0021142, C0021142 57. The method of claim 56, wherein the antibody is any of IgG, C0021141, C0021139, C0021135, C0021133, C0021131, C0021129, C0021128, C0021124, C0021118, C0021101, C0021098, C0021097, C0021096, C0021092, C0021089, C0021065, C0021032, C0021022, C0021021, C0021017 and C0020065, or germlined versions thereof.

58. 58. The method of any one of claims 54 to 57, further comprising producing the antigen-binding protein antigen-binding domain as a component of an IgG, scFv or Fab antigen-binding protein.

59. 1. A method of producing an antigen binding protein that binds to and inhibits human ARG2, said method comprising: providing a starting nucleic acid encoding a VH domain, or a starting repertoire of nucleic acids each encoding a VH domain, wherein said one or more VH domains comprise the HCDR1, HCDR2 and / or HCDR3 to be replaced or lack the HCDR1, HCDR2 and / or HCDR3 coding regions; IgG, C0021158fgl, C0021158dr, C0021061, C0020187, C0021155, C0021144, C0021142, C0021142 HCDR1, HC of IgG, C0021141, C0021139, C0021135, C0021133, C0021131, C0021129, C0021128, C0021124, C0021118, C0021101, C0021098, C0021097, C0021096, C0021092, C0021089, C0021065, C0021032, C0021022, C0021021, C0021017, and C0020065 with one or more donor nucleic acids encoding HCDR1, HCDR2 and / or HCDR3 amino acid sequences selected from HCDR1, HCDR2 and / or HCDR3, whereby said one or more donor nucleic acids are inserted into CDR1, CDR2 and / or CDR3 regions in said starting nucleic acid or starting repertoire to provide a product repertoire of nucleic acids encoding VH domains; and expressing the nucleic acids of said product repertoire to produce product VH domains. and optionally combining the product VH domain with one or more VL domains; selecting an antigen binding protein for ARG2, said antigen binding protein comprising a product VH domain and optionally a VH domain; and recovering said antigen-binding protein or nucleic acid encoding same. A method comprising:

60. 62. The method of claim 61 , wherein the donor nucleic acid is produced by mutation of the HCDR1 and / or HCDR2.

61. 61. The method of claim 59 or 60, wherein the donor nucleic acid is produced by mutation of HCDR3.

62. 62. The method of any one of claims 59 to 61, comprising providing the donor nucleic acid by random mutation of nucleic acid.

63. 63. The method of any one of claims 59 to 62, further comprising combining a product VH domain contained within said recovered antigen binding protein with an antigen binding protein constant region.

64. 64. A method according to any one of claims 59 to 63, comprising providing an IgG, scFv or Fab antigen binding protein comprising said product VH and VL domains.

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