Anti-nampt antibodies and uses thereof

Abstract

Anti-nicotinamide phosphoribosyltransferase (NAMPT) antibodies, or antigen binding fragments thereof, and methods for treating subjects having (NAMPT)-associated local and / or systemic inflammatory disorders are described.

Description

[0001] Federally sponsored research or development

[0002] This application was made with government support under Grant Numbers R41 HL110707 STTR and R42 HL152888 awarded by the National Institutes of Health (NIH). The government has certain rights in the application.

[0003] Related Applications

[0004] This application claims priority to U.S. Provisional Application No. 62 / 883,952, filed August 7, 2019. The entire contents of the foregoing priority application are incorporated herein by reference.

[0005] SEQUENCE LISTING

[0006] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on July 1, 2020, is named A105818_1020WO_SL.txt and is 53,767 bytes in size. BACKGROUND

[0007] Nicotinamide phosphoribosyltransferase (NAMPT) gene encodes a protein that catalyzes the condensation of nicotinamide with 5-phosphoribosyl-l-pyrophosphate to produce nicotinamide mononucleotide. The protein belongs to the nicotinate phosphoribosyltransferase (NAPRTase) family and is thought to be involved in many important biological processes, including metabolism, stress response, and aging.

[0008] Nicotinamide phosphoribosyltransferase (NAMPT) exists as both an intracellular NAMPT and an extracellular NAMPT (eNAMPT) protein. eNAMPT is secreted into the blood and functions as a cytokine / enzyme (cytozyme) that activates NF-κΒ signaling through ligation of Toll-like receptor 4 (TLR4), further serving as a biomarker for inflammatory lung disorders such as acute respiratory distress syndrome. SUMMARY

[0009] Provided herein are anti-NAMPT antibodies, including humanized antibodies useful for therapeutic purposes. Currently known therapies for clinical treatment of acute and chronic inflammatory lung disorders (e.g., ARDS, VILI) are merely supportive. Thus, there is an unmet need in the art for prophylactic and therapeutic agents that reduce morbidity and mortality of inflammatory disorders and are effective in the morbidity and mortality observed in patients, particularly severe patients, with these disorders.

[0010] In a first aspect, the present application provides an isolated antibody or antigen binding fragment thereof that specifically binds to human nicotinamide phosphoribosyltransferase (NAMPT), the antibody or antigen binding fragment thereof comprising: (i) a heavy chain variable region comprising a region having a CDR1 domain of the amino acid sequence set forth in SEQ ID NO: 3; a CDR2 domain having the amino acid sequence set forth in SEQ ID NO: 4 or 29; and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 5; and (ii) a light chain variable region comprising a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 6 or 11; a CDR2 domain having the amino acid sequence set forth in SEQ ID NO: 7, 12, 14, 33, 35, or 37; and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 8.

[0011] In some embodiments of the above aspect, the heavy chain variable region of the isolated antibody or antigen binding fragment thereof comprises: (a) a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 3, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO: 4, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 5; or (b) a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 3, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO: 29, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 5.

[0012] In some embodiments of the foregoing aspects, the light chain variable region of the isolated antibody or its antigen-binding fragment comprises: (a) a CDR1 domain having the amino acid sequence shown in SEQ ID NO:6, a CDR2 domain having the amino acid sequence shown in SEQ ID NO:7, and a CDR3 domain having the amino acid sequence shown in SEQ ID NO:8; (b) a CDR1 domain having the amino acid sequence shown in SEQ ID NO:6, a CDR2 domain having the amino acid sequence shown in SEQ ID NO:12, and a CDR3 domain having the amino acid sequence shown in SEQ ID NO:8; (c) a CDR1 domain having the amino acid sequence shown in SEQ ID NO:6, a CDR2 domain having the amino acid sequence shown in SEQ ID NO:14, and a CDR3 domain having the amino acid sequence shown in SEQ ID NO:8; (d) a CDR1 domain having the amino acid sequence shown in SEQ ID NO:6, a CDR2 domain having the amino acid sequence shown in SEQ ID NO:33, and a CDR3 domain having the amino acid sequence shown in SEQ ID NO:8. (e) Having a CDR3 domain of the amino acid sequence shown in SEQ ID NO: 8; (f) Having a CDR1 domain of the amino acid sequence shown in SEQ ID NO: 6, a CDR2 domain of the amino acid sequence shown in SEQ ID NO: 35, and a CDR3 domain of the amino acid sequence shown in SEQ ID NO: 8; (g) Having a CDR1 domain of the amino acid sequence shown in SEQ ID NO: 6, a CDR2 domain of the amino acid sequence shown in SEQ ID NO: 37, and a CDR3 domain of the amino acid sequence shown in SEQ ID NO: 8; (g) Having a CDR1 domain of the amino acid sequence shown in SEQ ID NO: 11, a CDR2 domain of the amino acid sequence shown in SEQ ID NO: 7, and a CDR3 domain of the amino acid sequence shown in SEQ ID NO: 8; (h) Having a CDR1 domain of the amino acid sequence shown in SEQ ID NO: 11, a CDR2 domain of the amino acid sequence shown in SEQ ID NO: 12, and a CDR3 domain of the amino acid sequence shown in SEQ ID NO: 8; (i) Having a CDR1 domain of the amino acid sequence shown in SEQ ID NO: 6, a CDR2 domain of the amino acid sequence shown in SEQ ID NO: 12, and a CDR3 domain of the amino acid sequence shown in SEQ ID NO: 8; (j) A CDR1 domain having the amino acid sequence shown in SEQ ID NO:11; a CDR2 domain having the amino acid sequence shown in SEQ ID NO:14; and a CDR3 domain having the amino acid sequence shown in SEQ ID NO:8; (j) A CDR1 domain having the amino acid sequence shown in SEQ ID NO:11; a CDR2 domain having the amino acid sequence shown in SEQ ID NO:33; and a CDR3 domain having the amino acid sequence shown in SEQ ID NO:8;(k) a CDR1 domain having an amino acid sequence as set forth in SEQ ID NO: 11; a CDR2 domain having an amino acid sequence as set forth in SEQ ID NO: 35; and a CDR3 domain having an amino acid sequence as set forth in SEQ ID NO: 8; or (1) a CDR1 domain having an amino acid sequence as set forth in SEQ ID NO: 11; a CDR2 domain having an amino acid sequence as set forth in SEQ ID NO: 37; and a CDR3 domain having an amino acid sequence as set forth in SEQ ID NO: 8.

[0013] In another aspect, the present application provides an isolated antibody, or antigen binding fragment thereof, that specifically binds to human NAMPT, the antibody or antigen binding fragment thereof comprising: (a) a heavy chain variable region comprising a CDR1 domain having the amino acid sequence of SEQ ID NO:3, a CDR2 domain having the amino acid sequence of SEQ ID NO:4, and a CDR3 domain having the amino acid sequence of SEQ ID NO:5; and a light chain variable region comprising a CDR1 domain having the amino acid sequence of SEQ ID NO:6, a CDR2 domain having the amino acid sequence of SEQ ID NO:7, and a CDR3 domain having the amino acid sequence of SEQ ID NO:8; (b) a heavy chain variable region comprising a CDR1 domain having the amino acid sequence of SEQ ID NO:3, a CDR2 domain having the amino acid sequence of SEQ ID NO:4, and a CDR3 domain having the amino acid sequence of SEQ ID NO:5; and a light chain variable region comprising a CDR1 domain having the amino acid sequence of SEQ ID NO: 11, a CDR2 domain having the amino acid sequence of SEQ ID NO: 12, and a CDR3 domain having the amino acid sequence of SEQ ID NO:8; (c) a heavy chain variable region comprising a CDR1 domain having the amino acid sequence of SEQ ID NO:3, a CDR2 domain having the amino acid sequence of SEQ ID NO:4, and a CDR3 domain having the amino acid sequence of SEQ ID NO:5; and a light chain variable region comprising a CDR1 domain having the amino acid sequence of SEQ ID NO: 11, a CDR2 domain having the amino acid sequence of SEQ ID NO: 14, and a CDR3 domain having the amino acid sequence of SEQ ID NO:8; (d) a heavy chain variable region comprising a CDR1 domain having the amino acid sequence of SEQ ID NO:3, a CDR2 domain having the amino acid sequence of SEQ ID NO:29, and a CDR3 domain having the amino acid sequence of SEQ ID NO:5; and a light chain variable region comprising a CDR1 domain having the amino acid sequence of SEQ ID NO: 11, a CDR2 domain having the amino acid sequence of SEQ ID NO: 14, and a CDR3 domain having the amino acid sequence of SEQ ID NO:8; (e) a heavy chain variable region comprising a CDR1 domain having the amino acid sequence of SEQ ID NO:3, a CDR2 domain having the amino acid sequence of SEQ ID NO:29, and a CDR3 domain having the amino acid sequence of SEQ ID NO:5;(a) a heavy chain variable region comprising a CDR1 domain having the amino acid sequence set forth in SEQ ID NO:3, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO:4, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO:5; and a light chain variable region having a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 11, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO:33, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO:8; (b) a heavy chain variable region comprising a CDR1 domain having the amino acid sequence set forth in SEQ ID NO:3, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO:4, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO:5; and a light chain variable region having a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 11, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO:35, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO:8; (c) a heavy chain variable region comprising a CDR1 domain having the amino acid sequence set forth in SEQ ID NO:3, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO:29, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO:5; and a light chain variable region having a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 11, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO:33, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO:8; (d) a heavy chain variable region comprising a CDR1 domain having the amino acid sequence set forth in SEQ ID NO:3, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO:29, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO:5; and a light chain variable region having a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 11, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO:35, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO:8; (e) a heavy chain variable region comprising a CDR1 domain having the amino acid sequence set forth in SEQ ID NO:3, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO:29, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO:5; and a light chain variable region having a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 11, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO:37, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO:8; (f) a heavy chain variable region comprising a CDR1 domain having the amino acid sequence set forth in SEQ ID NO:3, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO:29, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO:5; and a light chain variable region having a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 11, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO:33, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO:8; (g) a heavy chain variable region comprising a CDR1 domain having the amino acid sequence set forth in SEQ ID NO:3, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO:29, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO:5; and a light chain variable region having a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 11, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO:37, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO:8; (h) a heavy chain variable region comprising a CDR1 domain having the amino acid sequence set forth in SEQ ID NO:3, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO:4, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO:5; and a light chain variable region having a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 11, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO:33, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO:8; (i) a heavy chain variable region comprising a CDR1 domain having the amino acid sequence set forth in SEQ ID NO:3, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO:4, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO:5; and a light chain variable region having a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 11, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO:35, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO:8;(j) a heavy chain variable region comprising a CDR1 domain having an amino acid sequence as set forth in SEQ ID NO: 3, a CDR2 domain having an amino acid sequence as set forth in SEQ ID NO: 4, and a CDR3 domain having an amino acid sequence as set forth in SEQ ID NO: 5; and a light chain variable region comprising a CDR1 domain having an amino acid sequence as set forth in SEQ ID NO: 11, a CDR2 domain having an amino acid sequence as set forth in SEQ ID NO: 37, and a CDR3 domain having an amino acid sequence as set forth in SEQ ID NO: 8.

[0014] In some embodiments of any of the above aspects, the isolated antibody, or antigen binding fragment thereof, is humanized.

[0015] In some embodiments of any of the above aspects, the isolated antibody, or antigen binding fragment thereof, comprises a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 2, 10, 13, 30, 31, 32, 34, or 36. In some embodiments, the isolated antibody, or antigen binding fragment thereof, comprises a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 1, 9, 15, 16, or 28.

[0016] In another aspect, the application features an isolated antibody, or antigen binding fragment thereof, that specifically binds human NAMPT, the antibody or antigen binding fragment thereof comprising a heavy chain comprising a variable region comprising an amino acid sequence as set forth in SEQ ID NO: 15 and a light chain comprising a variable region comprising an amino acid sequence as set forth in SEQ ID NO: 13.

[0017] In a different aspect, the application provides an isolated antibody, or antigen binding fragment thereof, that specifically binds human NAMPT, the antibody or antigen binding fragment thereof comprising a heavy chain comprising a variable region comprising an amino acid sequence as set forth in SEQ ID NO: 26 and a light chain comprising a variable region comprising an amino acid sequence as set forth in SEQ ID NO: 18.

[0018] In another aspect, the application features an isolated antibody, or antigen binding fragment thereof, that specifically binds human NAMPT, the antibody or antigen binding fragment thereof comprising a heavy chain comprising a variable region as set forth in Table 17 and a light chain comprising a variable region as set forth in Table 17.

[0019] In yet another aspect, the application features an isolated antibody, or antigen-binding fragment thereof, that specifically binds human NAMPT, the antibody or antigen-binding fragment thereof comprising a heavy chain comprising a heavy chain CDR1, CDR2, and CDR3 as set forth in Table 17, and a light chain comprising a light chain CDR1, CDR2, and CDR3 as set forth in Table 17.

[0020] In different aspects, the application provides an isolated anti-NAMPT antibody comprising the heavy chain variable region and the light chain variable region of antibody AL-303.

[0021] In another aspect, the application features an isolated anti-NAMPT antibody comprising the heavy chain variable region and the light chain variable region of antibody AL-310.

[0022] In yet another aspect, the application features an isolated humanized anti-NAMPT antibody comprising a humanized heavy chain variable region derived from mouse antibody AL-303 or antibody AL-310, and a humanized light chain variable region derived from mouse antibody AL-303 or antibody AL-310.

[0023] In various aspects, the application features an isolated anti-NAMPT antibody that specifically binds to human NAMPT in a homodimeric conformation, wherein the antibody binds to an epitope on human NAMPT comprising: (a) at least one amino acid in amino acid residues 17-44 of SEQ ID NO: 60; at least one amino acid in amino acid residues 117-127 of SEQ ID NO: 60; at least one amino acid in amino acid residues 162-170 of SEQ ID NO: 60; at least one amino acid in amino acid residues 242-261 of SEQ ID NO: 60; at least one amino acid in amino acid residues 262-273 of SEQ ID NO: 60; at least one amino acid in amino acid residues 289-305 of SEQ ID NO: 60; at least one amino acid in amino acid residues 332-342 of SEQ ID NO: 60; at least one amino acid in amino acid residues 374-389 of SEQ ID NO: 60; at least one amino acid in amino acid residues 418-425 of SEQ ID NO: 60; at least one amino acid in amino acid residues 453-466 of SEQ ID NO: 60; and at least one amino acid in amino acid residues 408-416 of SEQ ID NO: 60; or (b) at least one amino acid in amino acid residues 29-51 of SEQ ID NO: 60; at least one amino acid in amino acid residues 61-72 of SEQ ID NO: 60; at least one amino acid in amino acid residues 156-170 of SEQ ID NO: 60; at least one amino acid in amino acid residues 216-234 of SEQ ID NO: 60; at least one amino acid in amino acid residues 316-331 of SEQ ID NO: 60; at least one amino acid in amino acid residues 332-342 of SEQ ID NO: 60; at least one amino acid in amino acid residues 373-389 of SEQ ID NO: 60; at least one amino acid in amino acid residues 417-431 of SEQ ID NO: 60; at least one amino acid in amino acid residues 454-469 of SEQ ID NO: 60; and at least one amino acid in amino acid residues 470-478 of SEQ ID NO: 60;

[0024] In some embodiments of any of the above aspects, the antibody or antigen-binding fragment thereof comprises a Fc domain.

[0025] In some embodiments of any of the above aspects, the isolated antibody or antigen-binding fragment thereof is a monoclonal antibody.

[0026] In some embodiments of any of the above aspects, the isolated antibody or antigen binding fragment thereof is an IgG. In some embodiments, the isolated antibody or antigen binding fragment thereof is an IgGl. In some embodiments, the isolated antibody or antigen binding fragment thereof is an IgG4.

[0027] In some embodiments, the application includes a nucleic acid encoding any of the isolated antibodies or antigen binding fragments thereof described herein, a vector comprising the nucleic acid, and / or a host cell comprising the nucleic acid or vector.

[0028] In certain embodiments, the application provides a pharmaceutical composition comprising any of the isolated antibodies or antigen binding fragments thereof described herein and a pharmaceutically acceptable carrier.

[0029] In some embodiments, the application includes a method of treating a disease associated with detrimental NAMPT activity in a subject in need thereof by administering to the subject an effective amount of any of the isolated antibodies or antigen binding fragments thereof described herein.

[0030] Also featured herein are methods of treating a subject having an inflammatory disorder, the method comprising administering to the subject an effective amount of an isolated antibody or antigen binding fragment described herein. In some embodiments, the inflammatory disorder is idiopathic pulmonary fibrosis (IPF), pulmonary arterial hypertension, acute lung injury (ALI), acute respiratory distress syndrome (ARDS), ventilator-induced lung injury (VILI), ARDS / VILI-induced ALI, trauma-induced acute lung injury (TIALI), and brain injury, or radiation-induced lung injury (e.g., radiation-induced lung injury caused by radiation associated with cancer treatment).

[0031] In some embodiments, the application features a method for treating prostate cancer (PCa) in a subject in need thereof, the method comprising administering to the subject an effective amount of any of the isolated antibodies or antigen binding fragments described herein.

[0032] In some embodiments, the subject has recurrent PCa. In some embodiments, the subject is at risk of developing metastatic PCa. In certain embodiments, the PCa is resistant to androgen deprivation therapy (ADT). In some embodiments, the method further comprises administering ADT to the subject. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1 is a Western blot assay depicting detection of human and mouse NAMPT by murine anti-NAMPT antibodies AL-303, AL-304, AL-305, AL-309, and AL-310.

[0034] Figure 2This is a Western blot assay that describes the ability of mouse anti-NAMPT antibodies AL-303, AL-304, AL-305, AL-309, and AL-310 mAbs to reduce hNAMPT-induced NFκB phosphorylation.

[0035] Figure 3 This is a schematic diagram of an experimental protocol used in in vivo studies to evaluate the effects of mouse anti-NAMPT antibodies AL-303, AL-304, and AL-305 on NAMPT-induced lung injury in mice.

[0036] Figure 4 is a schematic diagram of the effects of mouse anti-NAMPT antibodies AL-303, AL-304, and AL-305 on NAMPT-induced lung injury in mice. Figure 4A This is a graph showing the effect of mouse anti-NAMPT antibody on NAMPT-induced protein levels in bronchoalveolar lavage fluid (BAL), and Figure 4B This figure shows the effect of mouse anti-NAMPT antibody on NAMPT-induced polymorphonuclear neutrophil (PMN) counts expressing BAL (n = 3–6 mice, *P < 0.05).

[0037] Figure 5 This is a schematic diagram of an experimental protocol used in an in vivo study to evaluate the effect of mouse anti-NAMPT antibody AL-310 on LPS-induced lung injury in mice.

[0038] Figure 6 This figure shows the effect of mouse anti-NAMPT antibody AL-310 on LPS-induced BAL protein levels.

[0039] Figures 7A-7B This is a graphical representation of the effects of humanized 1076 and 1093 anti-hNAMPT antibodies. Figure 7A This is a graphical representation of the effect of humanized 1076 and 1093 anti-hNAMPT antibodies on hNAMPT-induced NFκB phosphorylation. Figure 7B This is a graphical representation of the effects of humanized 1076 and 1093 anti-hNAMPT antibodies on hNAMPT-induced decrease in endothelial cell (EC) barrier integrity.

[0040] Figures 8A-8D The effects of humanized 1076 and 1093 anti-hNAMPT antibodies on lung injury models in mice and rats were described. Figure 8A The effects of anti-hNAMPT antibodies V-1076, N-1076, K-1076, and P-1076 on BAL protein levels in a mouse lung injury model ( Figure 8A (left figure) and BAL PMN count ( Figure 8A The function of (see right figure) is illustrated graphically.Figure 8B is a graphical representation of the effect of 1093 anti-hNAMPT antibodies SS-1093, CC-1093, XX-1093, and UU-1093 on BAL protein levels (left panel) and BAL PMN counts (right panel) in a mouse model of lung injury. Figure 8B Figure 8B Figure 8C is a graphical representation of the effect of 1076 anti-hNAMPT antibody P-1076 on BAL protein levels (left panel) and BAL PMN counts (right panel) in a rat model of lung injury. Figure 8C Figure 8C Figure 8D depicts a photomicrograph of H&E staining showing the effect of 1093 anti-hNAMPT antibody UU-1093 on cellular infiltration and edema in a mouse model of lung injury.

[0041] Figures 9A-9D shows immunohistochemical (IHC) staining for NAMPT in normal, minimally invasive, and highly invasive prostate cancer (PCa). Figure 9A is a photomicrograph showing very low NAMPT expression in normal prostate tissue. Figure 9B is a photomicrograph showing moderately increased NAMPT expression in prostate cancer that is confined to the gland. Figure 9C is a photomicrograph showing strong NAMPT expression within tumor cells in three prostate adenocarcinomas with smooth muscle capsule penetration and invasion into periprostatic adipose tissue. Figure 9D is a graph showing cumulative analysis of NAMPT expression in 26 PCa patients with organ-confined (n=12) and capsule-infiltrating disease (n=14).

[0042] Figures 10A-10D depicts results from analysis of acute and subacute post-radiation murine and human tissues. Figure 10A provides a photomicrograph of H&E staining showing evidence of inflammation and damage in murine lung tissue 1 week after radiation exposure (lower panel) and prior to exposure to a single dose of thoracic radiation (upper panel). Figure 10A Figure 10A Figure 10B provides a photograph of IHC staining showing NAMPT expression in murine lung tissue 1 week after radiation exposure (lower panel) and prior to exposure to radiation (upper panel). Figure 10B Figure 10B Figure 10C is a high magnification image of a photomicrograph showing NAMPT expression in alveolar macrophages (long arrows) and lung cells (short arrows). Figure 10D depicts a photomicrograph showing NAMPT expression in alveolar macrophages (long arrows) and lung cells (short arrows) in murine lung tissue exposed to 8 Gy of radiation for 24 hr ("irradiated")​​​​​​​​Figure 10D (see image below) or not exposed to radiation (“unexposed”) Figure 10D The above image shows the expression of NAMPT in normal and postoperative human tonsil epithelial tissue.

[0043] Figures 11A-11B Results of an in vitro assay depicting the migration of human DU-145PCa cells through human smooth muscle cells were described. Figure 11A This shows the number of DU-145PCa cells that invaded in the absence of NAMPT (“medium only”) or in the presence of NAMPT (“NAMPT”); the medium without PCa cells (“PCa cells-free”) was used as a negative control. Figure 11B Photomicrographs of wells containing PCa cells cultured in the absence of NAMPT (“medium only”) or in the presence of NAMPT (“NAMPT”) are provided; wells without PCa cells (“PCa cells-free”) are used as negative controls.

[0044] Figures 12A-12C The results of in vivo tests of 1076 humanized anti-hNAMPT antibodies (N-1076, K-1076, and P-1076) and 1093 humanized anti-hNAMPT antibodies (SS-1093, XX-11093, and UU-1093) on inflammation and injury in a mouse lung injury model were described. Figure 12A This figure shows the effect of humanized anti-hNAMPT antibody on lung injury score in an LPS-induced "one-hit" lung injury model. Figure 12B This is a graph showing the effect of humanized anti-hNAMPT antibody on lung injury score in an LPS / VILI-induced "double-click" lung injury model. Figure 12C Micrographs were provided showing the effects of humanized anti-hNAMPT antibody P-1076 on an LPS-induced "one-hit" lung injury model. Figure 12C (See above image) and the LPS / VILI-induced "double-click" lung injury model ( Figure 12C Histological indicators of the role of lung injury (see figure below).

[0045] Figures 13A-13C The results of in vivo assays depicting the invasiveness of PCa cells through the diaphragmatic smooth muscle were described. Figure 13A These are micrographs showing severe peritoneal studding with invasion through the smooth muscle layer in SCID mice 6 weeks after intraperitoneal (IP) injection of PC3 (a highly metastatic human PCa cell). Figure 13A The image below shows a magnified version of the photomicrograph. Figure 13B These are photomicrographs showing the inhibition of PC3 cell invasion in mice that received injections of humanized anti-hNAMPT antibody P-1076 into PC3 cells; Figure 13B, the lower panel provides a magnified image of the photomicrograph. Figure 13C is a graph showing the percentage of PC3 cells invading the septa in mice treated with anti-hNAMPT antibody P-1076 or vehicle alone.

[0046] Figures 14A-14E Depicted is the effect of NAMPT neutralizing antibodies on lung inflammation (evaluated by H&E staining), amount of BAL protein, and count of BAL expressing cells, as evaluated in a murine RILI model. Figure 14A Representative photomicrographs are provided showing H&E staining of lung tissue from non-irradiated control mice ( Figure 14A , left panel) or irradiated RILI mice injected with vehicle control ( Figure 14A , left panel), anti-NAMPT polyclonal antibody (pAb) ( Figure 14A , middle panel), or anti-NAMPT monoclonal antibody (mAb) ( Figure 14A , right panel) following radiation exposure. Figure 14B is a graphical representation of H&E staining (% area) in lung tissue from non-irradiated control mice or irradiated RILI mice injected with vehicle control, anti-NAMPT pAb, or anti-NAMPT mAb. Figure 14C is a graphical representation of BAL protein levels (pg / ml) in lung tissue from non-irradiated control mice or irradiated RILI mice injected with vehicle control, anti-NAMPT pAb, or anti-NAMPT mAb. Figure 14D is a graphical representation of the number of BAL expressing cells in lung tissue from non-irradiated control mice or irradiated RILI mice injected with vehicle control, anti-NAMPT pAb, or anti-NAMPT mAb. Figure 14E is a graphical representation of ALI severity score from non-irradiated control mice or irradiated RILI mice injected with vehicle control, anti-NAMPT pAb, or anti-NAMPT mAb. * indicates p<0.05.

[0047] Figures 15A-15D Depicted is the detection of NAMPT expression by 99m Tc-labeled anti-NAMPT mAb probe. Figure 15A Representative autoradiographic images are provided depicting the detection of NAMPT expression by 99m Tc-labeled anti-NAMPT mAb probe in non-irradiated control mice ( Figure 15A , left panel) or irradiated (RILI) mice exposed to 8 Gy partial body radiation (PBI) ( Figure 15A , right panel). Figure 15B Representative autoradiographic images are provided depicting the detection of NAMPT expression by 99m Tc-labeled anti-NAMPT mAb probe in non-irradiated control mice ( Figure 15B , left panel) or irradiated (RILI) miceFigure 15B The lower panel shows the ratio of lung activity to tissue background in the left lung and right lung of non-irradiated control mice or irradiated (RILI) mice. 99m Tc-labeled anti-NAMPT mAb probe detection of NAMPT expression. Figure 15C is a graphical representation of the ratio of lung activity to tissue background in the left lung and right lung of non-irradiated control mice or irradiated (RILI) mice. Figure 15D is a graphical representation of radioactivity (%ID / g) in lung tissue of non-irradiated control mice or irradiated (RILI) mice. * indicates p<0.05.

[0048] Figures 16A-16C depicts the effect of humanized anti-NAMPT mAb on BAL cell counts, collagen deposition, and expression of smooth muscle actin (SMA) in lung tissue, as evaluated in a murine RILI model 18 weeks after 20 Gy radiation exposure. Figure 16A is a graph depicting the number of BAL-expressed cells in lung tissue of irradiated RILI mice injected intraperitoneally with anti-NAMPT mAb or vehicle control. Figure 16B Representative images from Western blot analysis showing SMA expression in lung tissue homogenates of irradiated RILI mice injected intraperitoneally with anti-NAMPT mAb or vehicle control are provided. Figure 16C Representative photomicrographs showing collagen deposition in lung tissue of irradiated RILI mice injected intraperitoneally with anti-NAMPT mAb or vehicle control, as detected by Trichrome staining, are provided. * indicates p<0.05.

[0049] Figures 17A-17C depicts the effect of humanized anti-NAMPT mAb on inflammatory cell infiltration, edema, and lung injury score, as evaluated in a rat model of trauma (burst) / ventilation-induced lung injury (VILI). Figure 17A Representative images and photomicrographs of lungs or lung tissue sections of trauma / VILI-challenged rats injected with vehicle control are provided. Figure 17A The left panel of provides representative images from lungs of trauma / VILI-challenged rats injected with vehicle control. Figure 17A The middle and right panels of provide representative photomicrographs showing inflammatory cell infiltration and edema in trauma / VILI-challenged rats injected with vehicle control, as evaluated by H&E staining. Figure 17A The inset of the far right panel provides a representative photomicrograph showing H&E staining in lung tissue of a rat that was not challenged with trauma / VILI. Figure 17B Representative images and photomicrographs of lungs or lung tissue sections of trauma / VILI-challenged rats injected with anti-NAMPT mAb are provided. Figure 17BThe left image provides a representative image of the lungs from traumatic / VILI challenged rats injected with anti-NAMPT mAb. Figure 17B The middle and right images provide representative micrographs showing inflammatory cell infiltration and edema in trauma / VILI challenged rats injected with anti-NAMPT mAb, as evaluated by H&E staining. Figure 17C This is a graph depicting the lung injury scores of rats subjected to the trauma / VILI challenge after injection of anti-NAMPT mAb or a mediator control.

[0050] Figures 18A-18C The effects of NAMPT neutralizing antibodies on inflammatory cell infiltration, edema, and lung injury scores were described, as evaluated in a mouse LPS / VILI lung injury model. Figure 18A Representative micrographs are provided, showing inflammatory cell infiltration and edema in LPS / VILI challenged mice with injected media controls, as assessed by H&E staining. Figure 18A The illustrations provide representative photomicrographs showing H&E staining in lung tissue from mice that were not challenged with LPS / VILI. Figure 18B Representative micrographs are provided, showing inflammatory cell infiltration and edema in LPS / VILI challenged mice injected with anti-NAMPT mAb, as assessed by H&E staining. Figure 18C This is a graph depicting the ALI severity scores assessed in LPS / VILI challenged mice injected with anti-NAMPT mAb, anti-NAMPT pAb, or mediator control (PBS). Figure 18C The figure also depicts the ALI severity scores of control mice that were not challenged with LPS / VILI. * indicates p < 0.05; *** indicates p < 0.001.

[0051] Figures 19A-19D Depicting through 99m NAMPT expression was detected using a Tc-labeled anti-NAMPT mAb probe. Figure 19A Representative autoradiographic images are provided, depicting the effects of total lung radiation (WTLI) on mice exposed to 20 Gy of total lung radiation (WTLI). 99m Tc-labeled anti-NAMPT mAb probe (PRONAMPTOR) Figure 19A (See right figure) or radiolabeled IgG control Ab ( Figure 19A (Left figure) to detect NAMPT expression. Figure 19B Representative autoradiographic images are provided, depicting mice undergoing the LPS challenge 3 hours post-challenge. Figure 19B (right figure) or in non-challenge control mice ( Figure 19B (Left figure) through 99mTc-labeled anti-NAMPT mAb probe to detect NAMPT expression. Figure 19C Representative autoradiographic images depicting NAMPT expression in the lungs of LPS-challenged mice 3 hours post-LPS challenge Figure 19C (lower panel) or in the lungs of non-challenged control mice Figure 18C (upper panel) as detected by a Tc-labeled anti-NAMPT mAb probe. 99m Tc-labeled anti-NAMPT mAb probe to detect NAMPT expression. Figure 19D Graphical representation of radiolabeled anti-NAMPT mAb probe uptake (as assessed by radioactivity (%ID / g)) in lung tissue of LPS-challenged mice 3 and 18 hours post-LPS challenge or in lung tissue of non-challenged control mice. * indicates p<0.05.

[0052] Figures 20A-20B Graphical representation of the effect of humanized anti-NAMPT mAb on right ventricular systolic pressure (RVSP) and pulmonary artery thickness as assessed in a rat model of PAH using the model of monocrotaline (MCT). Figure 20A Graphical representation of RVSP in MCT-challenged rats injected with anti-NAMPT mAb or vehicle control (control MCT mice). Figure 20B Representative photomicrographs showing pulmonary artery thickness in MCT-challenged rats injected with anti-NAMPT mAb Figure 20B (right panel) and vehicle control Figure 20B (left panel) as assessed by H&E staining. * indicates p<0.05. DETAILED DESCRIPTION

[0053] The present application can take many different forms. Non-limiting illustrative embodiments of the present application are disclosed herein which exemplify its principles. Any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. For purposes of this disclosure, unless otherwise specified, all identification sequence accession numbers are findable in the NCBI Reference Sequence (REFSEQ) database and / or the NCBI Archived Sequence database.

[0054] Various aspects of the present application relate to anti-NAMPT antibodies and antibody fragments, and pharmaceutical compositions thereof, as well as nucleic acids, recombinant expression vectors, and host cells for making such antibodies and fragments. Methods of using the antibodies described herein to detect human NAMPT, to inhibit human NAMPT activity (in vitro or in vivo), and to treat NAMPT-associated diseases, including but not limited to, pulmonary fibrosis (IPF), pulmonary arterial hypertension, acute lung injury (ALI), acute respiratory distress syndrome (ARDS), ventilator-induced lung injury (VILI), ARDS / VILI-induced ALI, trauma-induced acute lung injury (TIALI), and brain injury, radiation-induced lung injury, and cancer (e.g., prostate cancer (PCa)), are also encompassed by the present application.

[0055] Definitions

[0056] For the purposes of the present application, certain terms are defined below. Furthermore, whenever a value or a range of values is described, it is intended to encompass the intermediate values and ranges as well.

[0057] The terms "NAMPT" or "eNAMPT", used interchangeably herein, refer to the secreted form of nicotinamide phosphoribosyltransferase, unless specifically mentioned to refer to the non-secreted form (e.g., intracellular NAMPT or NAMPT nucleic acid). The amino acid sequence of secreted human NAMPT (also referred to as human eNAMPT) is provided below as SEQ ID NO: 60 (see also NCBI Gene Reference Number NC_000007.14 and Protein Reference Number NP_005737.1).

[0058]

[0059] NAMPT is also known as pre-B-cell colony-enhancing factor (PBEF) or visfatin.

[0060] The terms "NAMPT antibody" or "anti-NAMPT antibody", used interchangeably herein, refer to an antibody that specifically binds to the secreted form of NAMPT (also referred to herein as eNAMPT). An antibody that "binds" a target antigen (i.e., NAMPT) is one that is capable of binding that antigen with sufficient affinity so as to be useful in targeting cells expressing that antigen. In preferred embodiments, the antibody specifically binds to human NAMPT (hNAMPT), in particular extracellular human NAMPT (human eNAMPT). Examples of anti-eNAMPT antibodies are disclosed in the Examples and Sequence Listing provided below.

[0061] As used herein, "biological activity of NAMPT" refers to all intrinsic biological properties of NAMPT, including but not limited to binding to TLR4.

[0062] The term "specifically binds" or "binds specifically to" or the like refers to an antibody or antigen-binding fragment thereof that forms a complex with an antigen (e.g., NAMPT) that is relatively stable under physiologic conditions. Specific binding can be characterized by an equilibrium dissociation constant of M or less (e.g., the smaller the K -8 M or less (e.g., the smaller the K D represents tighter binding). Methods for determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like.

[0063] The term "antibody" broadly refers to an immunoglobulin (Ig) molecule, typically composed of four polypeptide chains, two heavy (H) chains and two light (L) chains, or any functional fragment, mutant, variant, or derivative thereof that retains the essential target binding features of an Ig molecule. Such mutant, variant, or derivative antibody forms are known in the art. Non-limiting embodiments thereof are discussed below.

[0064] In a full-length antibody, each heavy chain is comprised of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region.

[0065] Both light and heavy chains are composed of structural and functional domains. The terms "constant" and "variable" are used in the functional sense. In this regard, it is understood that the variable light (VL) and variable heavy (VH) chain portions determine antigen recognition and specificity. Conversely, the constant domains of the light chain (CL) and heavy chain (CH1, CH2, or CH3) confer biological properties such as secretion, transplacental mobility, Fc receptor binding, complement binding, etc. By convention, the numbering of constant region domains increases as they become further away from the antigen binding site or amino-terminal end of the antibody. The N-terminal portion is the variable region, while the C-terminal portion is the constant region; the CH3 (or CH4 in the case of IgM) and CL domains actually comprise the carboxy-terminal end of the heavy and light chains, respectively.

[0066] The heavy chain constant region is composed of three domains, CH1, CH2 and CH3. Each light chain is composed of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region is composed of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each of VHand VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Immunoglobulin molecules can be of any class (e.g., IgG, IgE, IgM, IgD, IgA, and IgY) and isotype (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass. The light chains can be of either kappa or lambda (k, l) class.

[0067] The“complementarity determining regions” or“CDRs” present in the antibody antigen binding domain are each short, non-contiguous amino acid sequences that, when the antibody assumes its three-dimensional configuration in an aqueous environment, are specifically positioned to form the binding domain. The remaining amino acids in the antibody binding domain, termed“framework” or“FW” regions, show less inter-molecular variability. The binding domain formed by the positioned CDRs defines a surface that is complementary to an epitope on an immunoreactive antigen. This complementary surface facilitates the non-covalent binding of the antibody to its cognate epitope. For any given heavy or light chain variable region, the amino acids that make up the CDRs and framework regions, respectively, can be readily identified by one of ordinary skill in the art, as they have been variously defined as follows. Exemplary CDRs are provided herein. However, CDRs can also be defined according to Kabat, Chothia, Martin, PyIgClassify, or IMGT. Exemplary CDR definitions are described in Chiu et al.,“Antibody Structure and Function: The Basis for Engineering Therapeutics,” Antibodies, 8(55): 1-80 (2019), which is incorporated by reference herein in its entirety.

[0068] For heavy chain constant region amino acid positions discussed in the present application, numbering is according to the EU index first described in Edelman et al., Proc. Natl. Acad. Sci. USA 63(1): 78-85, which describes the amino acid sequence of myeloma protein Eu, reported to be the first sequenced human IgGl. The EU index of Edelman is also presented in Kabat et al., 1991, supra. Thus, the term "EU index as shown in Kabat" or "EU index of Kabat" or "EU index" or "EU numbering" in the context of the heavy chain refers to the residue numbering system based on the human IgGl Eu antibody of Edelman et al., as shown in Kabat et al., 1991, supra. The numbering system for light chain constant region amino acid sequences is similarly set out in Kabat et al., supra. An exemplary kappa light chain constant region amino acid sequence compatible with the present disclosure is set out below:

[0069]

[0070] Similarly, an exemplary IgGl heavy chain constant region amino acid sequence compatible with the present application is set out below:

[0071]

[0072] As used herein, the term "antigen binding portion" of an antibody, or "binding fragment," refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., hNAMPT). It has been shown that the antigen binding function of an antibody can be performed by fragments of a full-length antibody. Such antibody embodiments can also be in the form of a bispecific, bispecific, or multispecific format; specifically binding to two or more different antigens. Examples of binding fragments encompassed within the term "antigen binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al. (1989) Nature 341 :544-546, Winter et al., PCT Publication WO 90 / 05144 Al, incorporated herein by reference), which includes a single variable domain; and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see, e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chain antibodies are also intended to be encompassed within the term "antigen binding portion" of an antibody. In certain embodiments of the application, scFv molecules can be incorporated into fusion proteins. Other formats of single chain antibodies are also included, such as diabodies. Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (see, e.g., Holliger P. et al., (1993) Proc. Natl. Acad. Sci. USA 90 :6444-6448; Poljak R.J. et al., Structure 2: 1121-1123 (1994)). Such antibody binding portions are known in the art (Kontermann and Dubel eds., Antibody Engineering (2001) Springer-Verlag. New York. 790 pp. (ISBN 3-540-41354-5)).

[0073] “Fully human” antibodies include antibodies having variable domains with sequences derived from human immunoglobulin (e.g., obtained from human immunoglobulin encoding sequences). The term “human antibody” includes, for example, antibodies having variable and constant regions (if present) derived from human germline immunoglobulin sequences. The term “human” as applied to antibodies or fragments (e.g., variable domains) as used herein does not encompass antibodies that have been “humanized” by grafting of human constant region sequences onto antibody polypeptides (i.e., replacement of non-human constant regions with human constant regions) or by grafting of human V region framework sequences onto immunoglobulin variable domains from non-human mammals (i.e., replacement of non-human framework regions of V domains with human framework regions). Methods for humanizing immunoglobulin variable regions by rational modification of complementarity determining residues have been described (US 2006 / 0258852).

[0074] The term “humanized antibody” refers to an antibody from a non-human species having one or more complementarity determining regions (CDRs) from the non-human species and a framework region from a human immunoglobulin molecule. A humanized antibody can optionally further comprise one or more framework residues derived from the non-human species from which the CDRs are derived. Such framework sequences can be obtained from public DNA databases covering germline antibody gene sequences or from published references. For example, germline DNA sequences for human heavy and light chain variable region genes can be found in the “Vbase” human germline sequence database (available on the website www.mrccpe.com.ac.uk / vbase) and in Kabat, E A et al., 1991 Sequences of Proteins of Immunological Interest, 5th Ed. To avoid activity reduction in the process of reduced immunogenicity, the variable region framework sequences of the human antibody are subjected to minimal back-mutations to maintain activity.

[0075] Humanized antibodies can be selected from any class of immunoglobulin including IgM, IgG, IgD, IgA, and IgE, and any isotype, including, but not limited to, IgGl, IgG2, IgG3, and IgG4. Humanized antibodies can comprise sequences from more than one class or isotype, and the particular constant domain selected can be optimized for a desired effector function using techniques well known in the art.

[0076] The term "multispecific" antibody refers to an antibody having binding domains for two or more different epitopes within a single antibody molecule. In addition to the canonical antibody structure, other binding molecules having two binding specificities can be constructed. Epitope binding by bispecific or multispecific antibodies can be simultaneous or sequential. Triomas and hybrid hybridomas are two examples of cell lines that can secrete bispecific antibodies. Bispecific antibodies can also be constructed by recombinant means. (Strohlein and Heiss, Future Oncol. 6: 1387-94 (2010); Mabry and Snavely, IDrugs. 13:543-9 (2010)). Bispecific antibodies can also be diabodies.

[0077] As used herein, the term "labeled antibody" refers to an antibody or antigen- binding portion thereof having a binding label for identifying the binding protein (e.g., antibody). Preferably, the label is a detectable marker, such as a polypeptide incorporating a radiolabeled amino acid or a biotinylated moiety that can be detected by labeled avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or colorimetric methods). Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes or radionuclides (e.g.,3H,14C,35S,125I,99Tc, 3 H, 14 C, 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131 I, 177 Lu, 166 Ho, or 153 Sm); fluorescent markers (e.g., FITC, rhodamine, lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase, luciferase, alkaline phosphatase); chemiluminescent markers; biotinyl groups; predetermined polypeptide epitopes (e.g., leucine zipper pair sequences, binding sites for second antibodies, metal binding domains, epitope tags); and magnetic agents, such as gadolinium chelates.

[0078] A "conservative amino acid substitution" is one in which one amino acid residue is replaced with another amino acid residue having a similar side chain. Families of amino acids having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta- branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). For example, substitution of a phenylalanine for a tyrosine is a conservative substitution. In certain embodiments, conservative substitutions in the sequences of the polypeptides and antibodies of the present disclosure do not eliminate binding of the polypeptide or antibody containing the amino acid sequence to the antigen to which the binding molecule binds. Methods for identifying nucleotide and amino acid conservative substitutions that do not eliminate antigen binding are well known in the art (see, e.g., Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10):879-884 (1999); and Burks et al., Proc. Natl. Acad. Sci. USA 94:412-417 (1997)).

[0079] The term "polynucleotide" is intended to encompass a single nucleic acid as well as multiple nucleic acids, and refers to an isolated nucleic acid molecule or construct, such as messenger RNA (mRNA), cDNA, or plasmid DNA (pDNA). A polynucleotide can comprise conventional phosphodiester bonds or non-conventional bonds (e.g., amide bonds, as found in peptide nucleic acids (PNAs)). The term "nucleic acid" or "nucleic acid sequence" refers to any one or more nucleic acid segments, such as DNA or RNA fragments, present in a polynucleotide.

[0080] An "isolated" nucleic acid or polynucleotide means any form of nucleic acid or polynucleotide that is separated from the natural environment of the nucleic acid. For example, a gel-purified polynucleotide or a recombinant polynucleotide encoding a polypeptide contained in a vector would be considered "isolated." In addition, a segment of a polynucleotide that has been engineered to have restriction sites for cloning, for example, a PCR product, is considered "isolated." Other examples of isolated polynucleotides include a recombinant polynucleotide contained in a host cell, or a polynucleotide purified or partially purified from a natural or non-natural solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of a polynucleotide, where the transcript is not the transcript that would be found in nature. An isolated polynucleotide or nucleic acid also includes such molecules that are synthetically produced. Furthermore, a polynucleotide or nucleic acid can be or can include regulatory elements, such as a promoter, ribosome binding site, or transcription terminator.

[0081] The term "expression" as used herein refers to the process by which a gene produces a biochemical (e.g., a polypeptide). The process includes any manifestation of the functional presence of a gene within a cell, including but not limited to gene knockdown as well as transient and stable expression. It includes, but is not limited to, the transcription of a gene into messenger RNA (mRNA), and the translation of such mRNA into a polypeptide. If the end product desired is a biochemical, then expression includes the production of that biochemical and any precursors. Expression of a gene produces a "gene product." As used herein, a gene product can be a nucleic acid, such as messenger RNA produced by transcription of a gene, or a polypeptide translated from a transcript. Gene products described herein also include nucleic acids with post-transcriptional modifications (e.g., polyadenylation), or polypeptides with post-translational modifications (e.g., methylation, glycosylation, addition of lipids, association with other protein subunits, proteolytic cleavage, etc.).

[0082] As used herein, a "neutralizing antibody" (e.g., an "antibody that inhibits NAMPT activity") is intended to include antibodies whose binding to NAMPT results in inhibition of NAMPT biological activity. A neutralizing antibody will substantially inhibit the binding of NAMPT to its ligand or substrate when an excess of the antibody reduces the amount of binding partner that binds to the determinant by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99% or more, as measured, for example, by target molecule activity or in an in vitro competitive binding assay. It will be appreciated that the altered activity can be measured directly using art- recognized techniques, or can be measured by the effect of the altered activity on downstream effects. Such inhibition of NAMPT or its ligand biological activity can be assessed by measuring one or more indicators of NAMPT biological activity, such as the amount of extracellular NAMPT (in vitro or in vivo), NAMPT-induced cellular activation (e.g., NFKB phosphorylation), and NAMPT binding to NAMPT ligand. These indicators of NAMPT biological activity can be assessed by one or more of several standard in vitro or in vivo assays known in the art (see Examples). For example, in some embodiments, the ability of an antibody to inhibit NAMPT activity is assessed by inhibition of NAMPT-induced endothelial cell activation. As an additional or alternative parameter of NAMPT activity, the ability of an antibody to inhibit NAMPT-induced transcriptional activity by NFKB, which is a measure of NAMPT-induced cellular activation, can be assessed.

[0083] The terms "treating" or "treatment" or "treat" or "alleviating" or "alleviate" refer to therapeutic measures that cure, slow down, lessen the symptoms of, and / or halt or slow the progression of a diagnosed pathologic condition or disorder. Such treatment can include, but does not need to, completely eliminate all symptoms or cure the disease. The terms "prevent," "prevention," "avoid," "deterrence," and the like refer to prophylactic or preventative measures that prevent the development of a targeted pathologic condition or disorder that has not yet been diagnosed. Thus, "those in need of treatment" can include those already diagnosed with the disorder; those predisposed to have the disorder; those in whom the disorder is to be developed; and those in which the disorder is to be prevented.

[0084] "Subject" or "individual" or "animal" or "patient" or "mammal" refers to any subject, particularly a mammalian subject, in need of diagnosis, prognosis, or treatment. Mammalian subjects include humans, domestic animals, farm animals, and zoo, sport, or pet animals, such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, pigs, cows, bears, and the like.

[0085] As used herein, phrases such as "subject who would benefit from therapy" and "animal in need of treatment" include a subject, such as a mammalian subject, who would benefit from administration of an anti-NAMPT antibody (e.g., a humanized anti-NAMPT antibody). Such antibodies can be used, for example, in diagnostic procedures and / or for treating or preventing a disease, such as an inflammatory lung disease or a cancer (e.g., prostate cancer).

[0086] The terms "effective amount" and "therapeutically effective amount" are used interchangeably herein and refer to an amount of a compound, formulation, material, or composition, as described herein, effective to achieve a particular biological result. Such results can include, but are not limited to, inhibition of NAMPT expression or activity, or expression or activity of signaling molecules downstream of NAMPT as determined by any suitable method in the art. For example, NAMPT activity includes, but is not limited to, cytokine activity, nicotinamide phosphoribosyltransferase activity, chemokine, NF-κΒ signaling activity, redox signaling activity, and / or roles in mitochondrial function and apoptosis. It is emphasized that a therapeutically effective amount is not always effective in treating a disorder, even if such a dose is considered a therapeutically effective amount by those of skill in the art. Exemplary doses, drug delivery amounts, therapeutically effective amounts, and therapeutic levels are provided herein with reference to adult human subjects for convenience only. Those of skill in the art can adjust such amounts according to standard practices required to treat a particular subject and / or disorder.

[0087] Anti-NAMPT antibodies

[0088] As provided herein, the present disclosure relates to anti-nicotinamide phosphoribosyltransferase (NAMPT) antibodies or antigen-binding fragments thereof, including compositions comprising the same, methods and articles of manufacture (e.g., kits, systems) for prophylactic and therapeutic uses in patients with NAMPT-associated local and systemic inflammatory disorders. Nucleic acids comprising polynucleotide sequences encoding such antibodies are also described. In particular embodiments, the monoclonal antibodies provided herein bind to extracellular NAMPT (eNAMPT) and prevent Toll-like receptor 4 (TLR4) activation, thereby reducing or blocking one or more downstream signaling pathways and concomitant systemic and pulmonary inflammation in certain respiratory disorders.

[0089] The NAMPT gene product is a rate-limiting enzyme in the nicotinamide adenine dinucleotide (NAD+) salvage pathway that converts nicotinamide to nicotinamide mononucleotide to enable NAD+ biosynthesis in mammals. The mature form of the extracellular NAMPT protein is a homodimer of approximately 120 kDa (Takahashi et al., J. Biochem. 147:95-107 (2010)). Mutations that reduce or inhibit NAMPT enzyme function have been determined to reduce pathophysiological processes that cause disorders such as leukemia and pulmonary arterial hypertension (PAH).

[0090] The human NAMPT gene (NAMPT) is located on chromosome 7 (segment 7q22.3; base pairs 106,248,285 to 106,286,326). The nucleic acid sequence of the human NAMPT gene product is known in the art. See, e.g., NCBI Reference Sequence: NM_005746.2, Nicotinamide phosphoribosyltransferase (NAMPT), mRNA, Homo sapiens (also see Samal et al., Mol. Cell. Biol. 14(2), 1431-1437 (1994)). The amino acid sequence of the human NAMPT enzyme is known in the art. See, e.g., GenBank Accession No. NP_005737.1. NAMPT has been shown to increase CD14 + IL-6, TNF-a, and IL-1 β production in monocytes, macrophages, and dendritic cells, enhances T cell effectiveness, and is involved in the development of B and T lymphocytes (Sun et al., Cytokine & Growth Factor Reviews 24(5):433-442 (2013)). NAMPT enzyme crystal structure is described in detail in Kim et al., J. Mol. Biol. 362:66-77 (2006).

[0091] The receptor for NAMPT is Toll-like Receptor 4 (TLR4), a protein encoded in humans by the TLR4 gene. TLR4 is a transmembrane protein and a member of the Toll-like receptor family, which belongs to the family of pattern recognition receptors (PRRs). Its activation leads to intracellular NF-κΒ signaling pathways and inflammatory cytokine production, which are responsible for activating the innate immune system. It is best known for recognizing lipopolysaccharide (LPS), a component found in many Gram-negative bacteria (e.g., Neisseria spp.) and selected Gram-positive bacteria. Its ligands also include several viral proteins, polysaccharides, and various endogenous proteins, such as low-density lipoprotein, beta-defensins, and heat shock proteins. The human TLR4 gene (TLR4) is located on chromosome 9 (segment 9q32-q33) (Georgel et al., PLoS One 4(11): e7803 (2009)). The nucleic acid sequence of the human TLR4 gene product is known in the art. See, e.g., NCBI Reference Sequence: AAY82268.1, Homo sapiens Toll-like receptor 4 (TLR4), mRNA. The amino acid sequence of human TLR4 is known in the art. See, e.g., GenBank Accession No. AAY82268.

[0092] In certain aspects, the present disclosure provides humanized 1076 anti-hNAMPT antibodies or antigen-binding fragments thereof. Examples of anti-NAMPT antibodies are provided below.

[0093] Anti-NAMPT antibody D-1076: D-1076 has (i) a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 1, which contains a CDR1 domain having an amino acid sequence as set forth in SEQ ID NO: 3, a CDR2 domain having an amino acid sequence as set forth in SEQ ID NO: 4, and a CDR3 domain having an amino acid sequence as set forth in SEQ ID NO: 5; and (ii) a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 2, which contains a CDR1 domain having an amino acid sequence as set forth in SEQ ID NO: 6, a CDR2 domain having an amino acid sequence as set forth in SEQ ID NO: 7, and a CDR3 domain having an amino acid sequence as set forth in SEQ ID NO: 8.

[0094] Anti-NAMPT antibody G-1076: G-1076 has (i) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 9, which contains a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 3, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO: 4, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 5; and (ii) a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 10, which contains a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 11, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO: 12, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 8.

[0095] Anti-NAMPT antibody K-1076: K-1076 has (i) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 9, which contains a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 3, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO: 4, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 5; and (ii) a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 13, which contains a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 11, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO: 14, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 8.

[0096] Anti-NAMPT antibody N-1076: N-1076 has (i) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 15, which contains a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 3, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO: 4, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 5; and (ii) a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 2, which contains a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 6, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO: 7, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 8;

[0097] Anti-NAMPT antibody P-1076: P-1076 has (i) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 15, which contains a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 3, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO: 4, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 5; and (ii) a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 13, which contains a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 11, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO: 14, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 8.

[0098] Anti-NAMPT antibody V-1076: V-1076 has (i) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 16, which contains a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 3, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO: 4, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 5; and (ii) a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 10, which contains a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 11, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO: 12, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 8.

[0099] Anti-NAMPT antibody X-1076: X-1076 has (i) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 16, which contains a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 3, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO: 4, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 5; and (ii) a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 2, which contains a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 6, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO: 7, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 8.

[0100] Anti-NAMPT antibody P-1076-modl : P-1076-modl has (i) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 28, which contains a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 3, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO: 29, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 5; and (ii) a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 30, which contains a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 11, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO: 14, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 8.

[0101] Anti-NAMPT antibody P-1076-mod2: P-1076-mod2 has (i) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 28, which contains a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 3, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO: 29, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 5; and (ii) a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 31, which contains a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 11, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO: 14, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 8.

[0102] Anti-NAMPT antibody P-1076-mod3: P-1076-mod3 has (i) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 28, which contains a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 3, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO: 29, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 5; and (ii) a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 32, which contains a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 11, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO: 33, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 8.

[0103] Anti-NAMPT antibody P-1076-mod4: P-1076-mod4 has (i) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:28, which contains a CDR1 domain having the amino acid sequence set forth in SEQ ID NO:3, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO:29, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO:5; and (ii) a light chain variable region having the amino acid sequence set forth in SEQ ID NO:34, which contains a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 11, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO:35, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO:8.

[0104] Anti-NAMPT antibody P-1076-mod5: P-1076-mod5 has (i) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:28, which contains a CDR1 domain having the amino acid sequence set forth in SEQ ID NO:3, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO:29, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO:5; and (ii) a light chain variable region having the amino acid sequence set forth in SEQ ID NO:36, which contains a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 11, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO:37, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO:8.

[0105] Anti-NAMPT antibody P-1076-mod6: P-1076-mod6 has (i) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 15, which contains a CDR1 domain having the amino acid sequence set forth in SEQ ID NO:3, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO:4, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO:5; and (ii) a light chain variable region having the amino acid sequence set forth in SEQ ID NO:30, which contains a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 11, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO: 14, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO:8.

[0106] Anti-NAMPT antibody P-1076-mod7: P-1076-mod7 has (i) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 15, which contains a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 3, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO: 4, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 5; and (ii) a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 31, which contains a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 11, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO: 14, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 8.

[0107] Anti-NAMPT antibody P-1076-mod8: P-1076-mod8 has (i) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 15, which contains a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 3, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO: 4, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 5; and (ii) a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 32, which contains a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 11, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO: 33, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 8.

[0108] Anti-NAMPT antibody P-1076-mod9: P-1076-mod9 has (i) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 15, which contains a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 3, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO: 4, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 5; and (ii) a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 34, which contains a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 11, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO: 35, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 8.

[0109] Anti-NAMPT antibody P-1076-mod10: P-1076-mod10 has (i) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 15, which contains a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 3, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO: 4, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 5; and (ii) a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 36, which contains a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 11, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO: 37, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 8.

[0110] Anti-NAMPT antibody P-1076-mod11: P-1076-mod11 has (i) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 28, which contains a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 3, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO: 29, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 5; and (ii) a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 13, which contains a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 11, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO: 14, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 8.

[0111] In certain aspects, the present disclosure provides a humanized 1093 anti-hNAMPT antibody or antigen binding fragment thereof. Examples of a1093 anti-NAMPT antibodies are provided below.

[0112] Anti-NAMPT antibody FF-1093: FF-1093 has (i) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 17, which contains a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 19, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO: 20, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 21; and (ii) a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 18, which contains a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 6, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO: 7, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 22.

[0113] Anti-NAMPT antibody II-1093: II-1093 has (i) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 23, which contains a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 19, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO: 20, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 21, and (ii) a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 24, which contains a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 6, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO: 7, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 22.

[0114] Anti-NAMPT antibody NN-1093: NN-1093 has (i) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 25, which contains a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 19, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO: 20, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 21, and (ii) a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 24, which contains a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 6, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO: 7, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 22.

[0115] Anti-NAMPT antibody PP-1093: PP-1093 has (i) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 25, which contains a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 19, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO: 20, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 21, and (ii) a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 18, which contains a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 6, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO: 7, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 22.

[0116] Anti-NAMPT antibody SS-1093: SS-1093 has (i) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 26, which contains a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 19, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO: 20, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 21; and (ii) a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 24, which contains a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 6, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO: 7, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 22.

[0117] Anti-NAMPT antibody UU-1093: UU-1093 has (i) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 26, which contains a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 19, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO: 20, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 21; and (ii) a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 18, which contains a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 6, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO: 7, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 22.

[0118] Anti-NAMPT antibody XX-1093: XX-1093 has (i) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 27, which contains a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 19, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO: 20, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 21; and (ii) a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 24, which contains a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 6, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO: 7, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 22.

[0119] Anti-NAMPT antibody ZZ-1093: ZZ-1093 has (i) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 27, which contains a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 19, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO: 20, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 21; and (ii) a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 18, which contains a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 6, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO: 7, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 22.

[0120] Anti-NAMPT antibody UU-1093-mod1: UU-1093-mod1 has (i) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 38, which contains a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 19, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO: 39, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 21; and (ii) a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 47, which contains a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 11, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO: 7, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 22.

[0121] Anti-NAMPT antibody UU-1093-mod2: UU-1093-mod2 has (i) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 40, which contains a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 19, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO: 39, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 41; and (ii) a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 47, which contains a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 11, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO: 7, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 22.

[0122] Anti-NAMPT antibody UU-1093-mod3: UU-1093-mod3 has (i) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 42, which contains a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 43, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO: 39, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 44; and (ii) a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 47, which contains a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 11, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO: 7, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 22.

[0123] Anti-NAMPT antibody UU-1093-mod4: UU-1093-mod4 has (i) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 45, which contains a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 46, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO: 39, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 41; and (ii) a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 47, which contains a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 11, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO: 7, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 22.

[0124] Anti-NAMPT antibody UU-1093-mod5: UU-1093-mod5 has (i) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 38, which contains a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 19, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO: 39, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 21; and (ii) a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 48, which contains a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 49, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO: 50, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 22.

[0125] Anti-NAMPT antibody UU-1093-mod6: UU-1093-mod6 has (i) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:40, which contains a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 19, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO:39, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO:41; and (ii) a light chain variable region having the amino acid sequence set forth in SEQ ID NO:48, which contains a CDR1 domain having the amino acid sequence set forth in SEQ ID NO:49, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO:50, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO:22.

[0126] Anti-NAMPT antibody UU-1093-mod7: UU-1093-mod7 has (i) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:42, which contains a CDR1 domain having the amino acid sequence set forth in SEQ ID NO:43, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO:39, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO:44; and (ii) a light chain variable region having the amino acid sequence set forth in SEQ ID NO:48, which contains a CDR1 domain having the amino acid sequence set forth in SEQ ID NO:49, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO:50, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO:22.

[0127] Anti-NAMPT antibody UU-1093-mod8: UU-1093-mod8 has (i) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:45, which contains a CDR1 domain having the amino acid sequence set forth in SEQ ID NO:46, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO:39, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO:41; and (ii) a light chain variable region having the amino acid sequence set forth in SEQ ID NO:48, which contains a CDR1 domain having the amino acid sequence set forth in SEQ ID NO:49, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO:50, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO:22.

[0128] Anti-NAMPT antibody UU-1093-mod9: UU-1093-mod9 has (i) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 38, which contains a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 19, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO: 39, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 21; and (ii) a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 51, which contains a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 11, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO: 52, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 53.

[0129] Anti-NAMPT antibody UU-1093-mod10: UU-1093-mod10 has (i) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 40, which contains a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 19, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO: 39, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 41; and (ii) a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 51, which contains a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 11, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO: 52, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 53.

[0130] Anti-NAMPT antibody UU-1093-mod11: UU-1093-mod11 has (i) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 42, which contains a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 43, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO: 39, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 44; and (ii) a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 51, which contains a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 11, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO: 52, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 53.

[0131] Anti-NAMPT antibody UU-1093-mod12: UU-1093-mod12 has (i) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 45, which contains a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 46, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO: 39, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 41; and (ii) a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 51, which contains a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 11, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO: 52, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 53.

[0132] Anti-NAMPT antibody UU-1093-mod13: UU-1093-mod13 has (i) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 38, which contains a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 19, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO: 39, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 21; and (ii) a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 18, which contains a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 6, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO: 7, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 22.

[0133] Anti-NAMPT antibody UU-1093-mod14: UU-1093-mod14 has (i) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 40, which contains a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 19, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO: 39, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 41; and (ii) a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 18, which contains a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 6, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO: 7, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 22.

[0134] Anti-NAMPT antibody UU-1093-mod15: UU-1093-mod15 has (i) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 42, which contains a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 43, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO: 39, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 44; and (ii) a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 18, which contains a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 6, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO: 7, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 22.

[0135] Anti-NAMPT antibody UU-1093-mod16: UU-1093-mod16 has (i) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 45, which contains a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 46, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO: 39, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 41; and (ii) a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 18, which contains a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 6, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO: 7, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 22.

[0136] Anti-NAMPT antibody UU-1093-mod17: UU-1093-mod17 has (i) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 26, which contains a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 19, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO: 20, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 21; and (ii) a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 47, which contains a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 11, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO: 7, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 22.

[0137] Anti-NAMPT antibody UU-1093-mod18: UU-1093-mod18 has (i) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 26, which contains a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 19, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO: 20, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 21; and (ii) a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 48, which contains a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 49, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO: 50, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 22.

[0138] Anti-NAMPT antibody UU-1093-mod19: UU-1093-mod19 has (i) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 26, which contains a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 19, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO: 20, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 21; and (ii) a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 51, which contains a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 11, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO: 52, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 53.

[0139] In some embodiments, the present application includes anti-NAMPT antibodies that bind to human NAMPT in the NAMPT homodimeric conformation, bind to a discontinuous epitope on human NAMPT. In some embodiments, the antibody or antigen-binding portion thereof binds to an epitope on human NAMPT comprising at least one amino acid of amino acid residues 17-44 of SEQ ID NO: 60; at least one amino acid of amino acid residues 117-127 of SEQ ID NO: 60; at least one amino acid of amino acid residues 162-170 of SEQ ID NO: 60; at least one amino acid of amino acid residues 242-261 of SEQ ID NO: 60; at least one amino acid of amino acid residues 262-273 of SEQ ID NO: 60; at least one amino acid of amino acid residues 289-305 of SEQ ID NO: 60; at least one amino acid of amino acid residues 332-342 of SEQ ID NO: 60; at least one amino acid of amino acid residues 374-389 of SEQ ID NO: 60; at least one amino acid of amino acid residues 418-425 of SEQ ID NO: 60; at least one amino acid of amino acid residues 453-466 of SEQ ID NO: 60; and / or at least one amino acid of amino acid residues 408-416 of SEQ ID NO: 60. In some embodiments, the antibody or antigen-binding portion thereof binds to an epitope on human NAMPT comprising at least one amino acid of amino acid residues 29-51 of SEQ ID NO: 60; at least one amino acid of amino acid residues 61-72 of SEQ ID NO: 60; at least one amino acid of amino acid residues 156-170 of SEQ ID NO: 60; at least one amino acid of amino acid residues 216-234 of SEQ ID NO: 60; at least one amino acid of amino acid residues 316-331 of SEQ ID NO: 60; at least one amino acid of amino acid residues 332-342 of SEQ ID NO: 60; at least one amino acid of amino acid residues 373-389 of SEQ ID NO: 60; at least one amino acid of amino acid residues 417-431 of SEQ ID NO: 60; at least one amino acid of amino acid residues 454-469 of SEQ ID NO: 60; and / or at least one amino acid of amino acid residues 470-478 of SEQ ID NO: 60.

[0140] In certain aspects, the provided antibodies or fragments thereof can have a binding affinity (K D ) of about 3 nM to about 20 nM for human NAMPT expressed on a human cell, as measured by surface plasmon resonance.

[0141] In certain embodiments, an antibody or antigen-binding fragment thereof described herein binds NAMPT, e.g., human NAMPT, with an off-rate (koff) of less than or equal to, e.g., 5 x 10 -2 seconds -1 , 10 -2 seconds -1 , 5 x 10 -3 seconds -1 , 10 -3 seconds-1, 5 x 10 -4 seconds -1 , 10 -4 seconds -1 , 5 x 10 -5 seconds -1 , or 10 -5 seconds -1 , 5 x 10 -6 seconds -1 , 10 -6 seconds -1 , 5 x 10 -7 seconds -1 , or 10 -7 seconds -1 . (off)

[0142] In certain embodiments, an antibody or antigen-binding fragment thereof described herein binds NAMPT, e.g., human NAMPT, with an on-rate (kon) of greater than or equal to, e.g., 10 3 M - 1 sec -1 , 5 x 10 3 M -1 sec -1 , 10 4 M -1 sec -1 , 5 x 10 4 M -1 sec -1 , 10 5 M -1 sec -1 , 5 x 10 5 M -1 sec -1 , 10 6 M -1 sec -1 , or 5 x 10 6 M -1 sec -1 , or 10 7 M -1 sec -1 . (on)

[0143] ​​In some embodiments, the antibody or its antigen-binding fragment described herein is equal to or greater than, for example, 5 x 10⁻⁶. - 7 M, 10 -7 M, 5x10 -8 M, 10 -8 M, 5x10 -9 M, 10 -9 M, 5x10 -10 M, 10 -10 M, 5x10 -11 M, 10 -11 M, 5x10 -12 M, 10 -12 M or 5x10 -13 The dissociation constant of M or K D The binding affinity can be determined using various techniques known in the art, such as surface plasmon resonance (SPR), biolayer interference, dual polarization interference, static light scattering, dynamic light scattering, isothermal titration calorimetry, ELISA, analytical ultracentrifugation, and flow cytometry.

[0144] In some aspects, anti-NAMPT antibodies or antigen-binding fragments thereof, as provided herein, may also include heterologous reagents, such as stabilizers, immunomodulators, or detectable reagents. In some aspects, the heterologous reagent comprises one or more additional polypeptide sequences fused to a polypeptide subunit via peptide bonds, such as signal sequences (e.g., secretion signal sequences), adaptor sequences, amino acid tags or labels, or peptide or polypeptide sequences that facilitate purification. In some aspects, the heterologous polypeptide may be fused to the N-terminus or C-terminus of a heavy or light chain antibody subunit or fragment thereof, provided that the functional characteristics of the domain are preserved.

[0145] In some respects, the heterologous reagent may be chemically conjugated to the anti-NAMPT antibody or its antigen-binding fragment provided herein. Exemplary heterologous reagents that can be chemically conjugated to peptide subunits include, but are not limited to, linkers, pharmaceuticals, toxins, imaging agents, radioactive compounds, organic and inorganic polymers, and any other composition that can provide the desired activity not provided by the peptide subunit itself. Specific reagents include, but are not limited to, polyethylene glycol (PEG), cytotoxic agents, radionuclides, imaging agents, and biotin.

[0146] In some embodiments, anti-NAMPT antibodies or fragments are radiolabeled for in vitro or in vivo detection. Examples of radioisotopes that can be used to label the antibodies disclosed herein include, but are not limited to, At. 211 I 131 I 125 Y 90 Re 186 Re 188, Sm 153 , Bi 212 , P 32 , Pb 212 or Lu. In some embodiments, the present application includes an anti-NAMPT antibody as described herein conjugated to a radioactive atom to form a radioconjugate. A variety of radioactive isotopes can be used to produce radioconjugates. Examples include At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212 and Lu. When the radioconjugate is used for detection, it can contain a radioactive atom for scintigraphic studies, for example tc99m or I123, or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), for example iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.

[0147] In certain embodiments, any of the anti-NAMPT antibodies provided herein can be used to detect the presence of NAMPT in a biological sample. The term "detecting" as used herein encompasses quantitative or qualitative detection. In certain embodiments, the biological sample comprises cells or tissues, such as cells or tissues of the cerebrospinal fluid, lung, or blood.

[0148] In some embodiments, an anti-NAMPT antibody is provided for use in a diagnostic or detection method. In another aspect, a method of detecting the presence of NAMPT in a biological sample is provided. In certain embodiments, the method comprises contacting a biological sample with an anti-NAMPT antibody as described herein under conditions that allow the anti-NAMPT antibody to bind to NAMPT, and detecting whether a complex is formed between the anti-NAMPT antibody and NAMPT. Such a method can be an in vitro or in vivo method. Further, the complex formed between the anti-NAMPT antibody and NAMPT in the test biological sample can be compared to the complex formed in a control biological sample (e.g., a biological sample from one or more healthy subjects). The amount of complex formed between the anti-NAMPT antibody and NAMPT in the test biological sample can also be quantified and compared to the amount of complex formed in a control biological sample (e.g., a biological sample from one or more healthy subjects) or to the average amount of complex formed in healthy subjects.

[0149] The anti-NAMPT antibodies and fragments disclosed herein can also be used as reagents for detecting human NAMPT, and in some embodiments, for detecting murine NAMPT. For example, the anti-NAMPT antibodies described herein can be used in ELISA assays. Detecting the presence of NAMPT can be accomplished using the antibodies and fragments disclosed herein in a variety of ways, such as by Western blotting (with or without immunoprecipitation), immunoprecipitation, fluorescence-activated cell sorting (FACS), flow cytometry, and ELISA procedures, for assaying a wide variety of tissues and samples, including plasma or serum. A wide range of immunoassay techniques are available using this assay format, which can include the antibodies disclosed herein, see, e.g., U.S. Pat. Nos. 4,016,043, 4,424,279, and 4,018,653. These include non-competitive types of both single and two-site or "sandwich" assays, as well as the traditional competitive binding assays. These assays also include the direct binding of a labeled anti-NAMPT antibody to the target biomarker.

[0150] Sandwich assays are the most useful and most commonly used assays. There are many variations of sandwich assay techniques, and all of these variations are intended to be encompassed by the present application. Briefly, in a typical forward assay, an unlabelled antibody is immobilized on a solid substrate, and the sample to be tested is brought into contact with the bound molecules. After an incubation period sufficient to allow antibody-antigen complex formation, a second antibody specific for the antigen is added, the antibody being labeled with a reporter molecule capable of producing a detectable signal, and incubated, allowing sufficient time for the formation of another complex of antibody-antigen-labeled antibody. Any unreacted material is washed away, and the presence of the antigen is determined by observing the signal produced by the reporter molecule. The result can be qualitative, by simple observation of the signal, or can be quantified by comparison with control samples containing known amounts of the biomarker.

[0151] To recombinantly produce an anti-NAMPT antibody, the nucleic acid encoding, for example, an antibody as described above is isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acid can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are specific to genes encoding the heavy and light chains of an antibody).

[0152] Suitable host cells for cloning or expression of antibody-encoding vectors include prokaryotic or eukaryotic cells described herein. For example, antibodies can be produced in bacteria, in particular when glycosylation and Fc effector functions are not needed. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, N.J., 2003), pp. 245-254, describing expression of antibody fragments in E. coli.) After expression, the antibody can be isolated from the bacterial cell paste in a soluble fraction and can be further purified.

[0153] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are also suitable hosts for cloning or expression of antibody-encoding vectors, including fungal and yeast strains in which the glycosylation pathway has been “humanized,” resulting in production of antibodies having a partially or fully human glycosylation pattern. See Gerngross, Nat. Biotech. 22:1409-1414 (2004) and Li et al., Nat. Biotech. 24:210-215 (2006).

[0154] Suitable host cells for the expression of glycosylated antibody are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains have been identified which can be used in combination with insect cells, particularly for transfection of Spodoptera frugiperda cells.

[0155] Vertebrate cells can also be used as hosts. For example, mammalian cell lines that are adapted to grow in suspension can be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 line (COS-7); human embryonic kidney line (293 or 293 cells, as described, e.g., in Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse Sertoli cells (TM4 cells as described, e.g., in Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK); buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, as described, e.g., in Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982); MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR- CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines, such as Y0, NS0 and Sp2 / 0. For a review of certain mammalian host cell lines suitable for antibody production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed. Humana Press, Totowa, N.J.), pp. 255-268 (2003).

[0156] Anti-NAMPT antibody pharmaceutical compositions and therapeutic uses

[0157] Methods of treating conditions associated with inflammation are also provided. Some embodiments include reducing inflammation, injury, the amount of protein in BAL fluid, and / or BAL PMNs in a dose-dependent manner by administering an anti-NAMPT antibody or antigen-binding fragment thereof. Some embodiments include reducing eNAMPT-, lipopolysaccharide-, and / or ventilator-induced lung injury. Conditions treatable with an anti-NAMPT antibody or antigen-binding fragment thereof include, but are not limited to, inflammatory conditions such as pulmonary fibrosis (IPF), pulmonary hypertension, acute lung injury (ALI), acute respiratory distress syndrome (ARDS), ventilator-induced lung injury (VILI), ARDS / VILI-induced ALI, trauma-induced acute lung injury (TIALI), brain injury (including traumatic brain injury), or radiation-induced lung injury (RILI); prostate cancer; lung cancer; cancers associated with inflammation; pregnant women with chorioamnionitis (e.g., subjects at risk for preterm birth and maternal / neonatal complications); nonalcoholic steatohepatitis (NASH); liver fibrosis; myocardial ischemia; and cardiac fibrosis.

[0158] Compositions (e.g., pharmaceutical compositions) comprising an anti-NAMPT antibody or antigen-binding fragment thereof described herein, optionally further comprising one or more carriers, diluents, excipients, or other additives, are also provided. Some embodiments comprise compositions (e.g., pharmaceutical compositions) comprising a polynucleotide or vector, optionally further comprising one or more carriers, diluents, excipients, or other additives.

[0159] Methods of making and administering an anti-NAMPT antibody or antigen-binding fragment thereof to a subject in need thereof, e.g., to reduce symptoms, morbidity, or mortality associated with NAMPT-related acute and chronic inflammatory conditions (e.g., ARDS, VILI, and trauma-induced inflammatory lung injury), are also provided and are well known to those of skill in the art or can be readily ascertained by those of skill in the art. An anti-NAMPT antibody or antigen-binding fragment thereof can be administered to a subject, e.g., intravenously, intraarterially, intraperitoneally, intrapleurally, intratracheally, topically, subcutaneously, mucosally, intrapericardially, orally, locally, by inhalation, by injection, by infusion, by continuous infusion, by direct soaking of target cells via local perfusion, by catheter, by aerosol, by nebulizer, and / or by lavage. In some embodiments, a composition comprising an anti-NAMPT antibody or antigen-binding fragment thereof is administered directly to a tissue or organ that is inflamed or showing signs of inflammation. Generally, a suitable pharmaceutical composition can comprise, but is not limited to, a buffer (e.g., acetate, phosphate, or citrate buffer), a surfactant (e.g., polysorbate), a stabilizer (e.g., human albumin), and the like.

[0160] Some of the pharmaceutical compositions described herein can be administered orally in acceptable dosage forms, including, for example, capsules, tablets, aqueous suspensions, or solutions. Some pharmaceutical compositions can also be administered via nasal aerosol or inhalation. Such compositions can be prepared as solutions in saline solution using benzyl alcohol or other suitable preservatives, bioavailability enhancers, and / or other conventional solubilizers or dispersants.

[0161] The amount of anti-NAMPT antibody or its antigen-binding fragment that can be combined with carrier materials to produce a single dosage form will vary depending on the subject being treated and the specific route of administration. The composition can be administered as a single dose, multiple doses, or by infusion over a defined time period. Dosing regimens can also be adjusted to provide the optimal desired response (e.g., therapeutic or preventative response). For example, the antibody can be administered intravenously to / can be administered to patients at a rate of about 0.1 mg / kg to about 20 mg / kg at a frequency of about weekly to monthly.

[0162] In some implementations, the intended objective is to prevent or treat an inflammatory condition or disease, meaning that an effective amount is an amount intended to achieve some form of prevention or treatment of the inflammatory condition or disease. In some implementations, it refers to the prevention or relief of symptoms and / or cellular processes associated with a specific disease or condition, including but not limited to VILI, ALI, or ARDS; such symptoms may be vascular permeability or elevated BAL protein secretion. An inflammatory disease or condition is one characterized by inflammation. Inflammation can affect any of the following tissues or organs: heart, lungs, kidneys, liver, bone marrow, pancreas, brain, skin, bone, veins, arteries, cornea, ear, eye, nasopharyngeal tissue, stomach, joints, cartilage, vascular tissue or cells, blood, small intestine, large intestine, larynx, brain, spinal cord, smooth muscle, nerves, skeletal muscle, breast, ovary, testis, uterus, and umbilical cord. In addition, the tissue may contain one or more of the following cell types: platelets, myeloid cells, erythrocytes, lymphocytes, adipocytes, fibroblasts, epithelial cells, endothelial cells, smooth muscle cells, skeletal muscle cells, endocrine cells, glial cells, neurons, secretory cells, barrier function cells, contractile cells, absorptive cells, mucosal cells, limbal cells (derived from the cornea), stem cells (totipotent, pluripotent, or multipotent), unfertilized or fertilized oocytes, or sperm.

[0163] In some respects, one or more medications may have been administered to the patient, administered concurrently / may be administered, or administered / may be administered later. Exemplary medications include azathioprine, bortezomib, carfilzomib, cyclophosphamide, dexamethasone, doxorubicin, lenalidomide, melphalan, pomalidomide, prednisolone, thalidomide, and vincristine. Exemplary medications also include antibiotics. Exemplary medications may also be administered / may be administered as part of a treatment regimen.

[0164] The present disclosure also provides pharmaceutical packs and kits comprising one or more containers containing one or more dosages of an anti-NAMPT antibody or antigen binding fragment, including compositions useful for carrying out the methods described herein. In certain embodiments, the kits comprise at least one purified anti-NAMPT antibody or antigen binding fragment thereof. Those of skill in the art will readily recognize that the disclosed anti-NAMPT antibodies can be readily incorporated into one of the established kit formats well known in the art.

[0165] In certain aspects, the present disclosure provides prophylactic and / or therapeutic methods for preventing, reducing, and / or reversing pathophysiological processes that contribute to the onset and progression of NAMPT-associated acute and chronic inflammatory diseases in a subject in need thereof, the methods comprising administering an effective amount of an anti-NAMPT antibody or antigen binding fragment thereof, including the compositions and pharmaceutical compositions provided herein. As used herein, a "NAMPT-associated acute and chronic inflammatory disease or disorder" includes any inflammatory disorder or biological process that contributes to inflammation involving NAMPT, e.g., elevated expression and / or activity of NAMPT (e.g., eNAMPT), including downstream signaling molecules, such as inflammatory cytokines. In particular embodiments, the compositions comprise a neutralizing humanized anti-NAMPT antibody. In particular embodiments, the subject is a mammal, including but not limited to a human. The therapeutic methods and related uses taught herein can delay, prevent, or reverse one or more symptoms associated with the disease, thereby improving the quality of life of the subject and / or prolonging the life of the patient.

[0166] In some embodiments, the anti-NAMPT antibodies or fragments thereof described herein are used to treat a patient having a symptom of, or at risk of developing, an acute or chronic inflammatory disorder or disease (e.g., an inflammatory pulmonary disorder or disease).

[0167] Subjects in need of the methods provided by the present disclosure include, but are not limited to, critically ill subjects; critically ill patients with respiratory failure; critically ill patients exposed to infection, trauma, and / or sepsis; subjects with radiation exposure; subjects diagnosed with pulmonary fibrosis (IPF); subjects with pulmonary hypertension; subjects with acute respiratory distress syndrome (ARDS); subjects with ventilator-induced lung injury (VILI); intensive care unit (ICU) subjects with respiratory failure or at risk for VILI and / or ARDS / VILI-induced ALI; subjects with pancreatitis; subjects with smoke inhalation injury, blast injury, and / or trauma-induced acute lung injury (TIALI); subjects with traumatic brain injury; subjects with hemorrhagic shock and resuscitation; subjects with radiation-induced lung injury, including cancer treatment-related radiation-induced lung injury; pregnant subjects with chorioamnionitis (e.g., at risk for preterm birth and maternal / neonatal complications or preterm birth complications); and subjects with primary and metastatic cancer. Also provided are subjects at risk for developing any of the conditions disclosed herein.

[0168] In certain aspects, the present disclosure provides a method for treating a patient having or at risk for an inflammatory lung condition or disease, comprising administering to the patient an effective amount of an anti-NAMPT antibody or antigen-binding fragment thereof (including the compositions and pharmaceutical compositions provided herein). This can be based on the patient's symptoms, medical history, or the results of one or more tests. In some cases, the patient has been diagnosed with an inflammatory condition or disease of the lung at the time the anti-NAMPT antibody or antigen-binding fragment thereof (including the compositions and pharmaceutical compositions provided herein) is administered to the patient. In other cases, the patient has not been diagnosed with an inflammatory condition or disease of the lung, but the patient is at risk for such a disease or condition. Such patients include patients who have been or will be placed on a ventilator, patients with pneumonia, patients who have experienced physical trauma, patients with severe bleeding, patients who have aspirated vomit, patients who have inhaled chemicals, patients who smoke heavily, and / or patients who drink heavily. For example, the patient can have been or will be placed on a ventilator for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 30, 36, 42, 48, 54, 60, 66, 72, 78, 84, 90, 96, 102, 108, 114, or 120 hours (or any range derivable therein) and / or 1, 2, 3, 4, 5, 6, or 7 days (or any range derivable therein).

[0169] In certain aspects, the present disclosure provides a method for treating a patient having, or at risk of having, acute lung injury (ALI), ventilator-induced lung injury (VILI), or respiratory distress syndrome (ARDS), comprising administering to the patient an effective amount of an anti-NAMPT antibody or antigen-binding fragment thereof (including the compositions and pharmaceutical compositions provided herein). Patients at risk include, but are not limited to, patients with sepsis or symptoms of sepsis, patients with pneumonia or symptoms of pneumonia, patients with severe bleeding due to physical injury, patients with severe injury to the chest or head, patients who have inhaled harmful smoke or mist, patients who have inhaled vomit, patients who have received multiple or large blood transfusions, patients who have sustained a fracture of a long bone (e.g., femur), patients who have nearly drowned, patients who have had an adverse reaction to a cancer drug or other drug, patients who have taken a drug overdose, patients who have pancreatitis, patients who smoke heavily, patients who drink heavily, patients who have inflammatory bowel disease, patients who have rheumatoid arthritis, patients who have colorectal cancer, and patients who have obesity-related insulin resistance, or any combination thereof.

[0170] In certain aspects, the present disclosure provides a method for preventing ventilator-induced lung injury (VILI) in a patient, comprising administering an effective amount of an anti-NAMPT antibody or antigen-binding fragment thereof (including the compositions and pharmaceutical compositions provided herein). In particular embodiments, the anti-NAMPT antibody is a neutralizing antibody. In particular embodiments, the administration is performed prior to placing the patient on a ventilator. In particular embodiments, the administration is performed after placing the patient on a ventilator.

[0171] In certain aspects, the present disclosure provides a method of reducing the level of one or more cytokines (e.g., IL-6, TNF-a, IL-1b, IL-8) in a subject at risk of having, or having, a NAMPT-associated acute and / or chronic inflammatory disorder.

[0172] In certain aspects, the present disclosure provides a method for reducing the level of eNAMPT in a subject at risk of having, or having, a NAMPT-associated acute and / or chronic inflammatory disorder.

[0173] In some embodiments, the anti-NAMPT antibodies disclosed herein can be used to treat cancer. In some embodiments, the cancer is prostate cancer (PCa). In particular aspects, the disclosure provides a method of treating PCa in a subject in need thereof by administering to the subject a therapeutically effective amount of an anti-NAMPT antibody or antigen-binding fragment described herein. In some embodiments, the subject having prostate cancer has recurrent PCa, aggressive PCa, or metastatic PCa. In some embodiments, the subject has aggressive PCa that is resistant to androgen deprivation therapy (ADT).

[0174] Alternatively, in some cases, the anti-NAMPT antibody or antigen-binding fragment can be administered to the subject in combination with ADT. The ADT can be administered to the subject prior to, concurrently with, or after administration of the anti-NAMPT antibody or antigen-binding fragment.

[0175] The ADT that can be administered to the subject in combination with the anti-NAMPT antibody or antigen-binding fragment can include one or more of the following: a luteinizing hormone-releasing hormone (LHRH) agonist; an LHRH antagonist; a CYP17 inhibitor; an anti-androgen; and / or an androgen-inhibiting drug. In particular embodiments, the LHRH agonist can be leuprolide (e.g., LUPRON®, TM , ELIGARD® TM , etc.), goserelin (e.g., ZOLADEX® TM ), triptorelin (e.g., TRELSTAR® TM ), and / or histrelin (e.g., VANTAS® TM ); the LHRH antagonist can be degarelix (e.g., FIRMAGON® TM ); the CYP17 inhibitor can be abiraterone (e.g., ZYTIGA® TM ); the anti-androgen can be flutamide (e.g., EULEXIN® TM ), bicalutamide (e.g., CASODEX® TM ), nilutamide (e.g., NILANDRON® TM ), enzalutamide (e.g., XTANDI® TM ), and / or apalutamide (e.g., ERLEADA® TM ); and / or the androgen-inhibiting drug can be estrogen and / or ketoconazole (e.g., NIZORAL® TM ).

[0176] Some embodiments include treating coronavirus disease 2019 (COVID-19) with one or more NAMPT antibodies. COVID-19 is a severe acute respiratory syndrome caused by coronavirus 2 (SARS-CoV-2). SARS-CoV-2 is 60-140 nm in diameter and has a unique spike of 9-12 nm, giving the virion the appearance of a solar corona. Through genetic recombination and mutation, coronaviruses can adapt and infect new hosts. SARS-CoV-2 infection can be asymptomatic, or it can cause a wide range of symptoms. Exemplary symptoms include fever, cough, shortness of breath, weakness, fatigue, nausea, vomiting, and changes in taste and smell. Adverse outcomes include diffuse intravascular coagulation; inflamed lung tissue and lung endothelial cells; deep vein thrombosis; pulmonary embolism; thrombotic arterial complications (e.g., limb ischemia; ischemic stroke; myocardial infarction); sepsis; and multi-organ failure. SARS-CoV-2 infection is discussed in more detail in Wiersinga et al. “Pathophysiology, Transmission, Diagnosis, and Treatment of Coronavirus Disease 2019 (COVID-2019): A Review,” JAMA, doi:10.1001 / jama.2020.12839 (published online July 10, 2020), the entire contents of which are incorporated herein by reference. In some embodiments, the present disclosure provides methods of treating a subject having COVID-19 (e.g., a subject diagnosed with COVID-19 and / or a subject showing one or more symptoms of COVID-19) by administering to the subject an effective amount of an anti-NAMPT antibody or antigen-binding fragment thereof.

[0177] EMBODIMENTS

[0178] The following examples are included for illustrative purposes only and are not intended to limit the disclosure.

[0179] Example 1: Generation of Anti-Human NAMPT Monoclonal Antibodies

[0180] Three mice were immunized with recombinant extracellular human NAMPT (hNAMPT; MBL International) to develop anti-NAMPT neutralizing murine antibodies. Mouse 4C6 and mouse 589 together produced 52 parental clone anti-NAMPT antibodies. Murine antibodies were evaluated for binding to hNAMPT by ELISA. Murine antibodies were also tested for in vitro neutralization by analyzing their effect on hNAMPT-induced NFkB phosphorylation. Anti-hNAMPT antibodies AL-303 and AL-310 were selected for their ability to bind recombinant hNAMPT as well as hNAMPT in lysates from human pulmonary artery endothelial cells (HPAEC). AL-303 and AL-310 were also able to inhibit hNAMPT-induced NFkB phosphorylation. AL-303 was further able to cross-react with recombinant mouse NAMPT (mNAMPT), while AL-310 was not. In addition, these antibodies were tested in vivo as described in Examples 4 and 5 below.

[0181] AL-303

[0182] Murine anti-hNAMPT antibody AL-303 has a heavy chain variable region with an amino acid sequence as set forth in SEQ ID NO: 54 and a light chain variable region with an amino acid sequence as set forth in SEQ ID NO: 55. The heavy chain variable region of AL-303 contains a CDR1 domain having an amino acid sequence as set forth in SEQ ID NO: 3, a CDR2 domain having an amino acid sequence as set forth in SEQ ID NO: 4, and a CDR3 domain having an amino acid sequence as set forth in SEQ ID NO: 5. The light chain variable region of AL-303 contains a CDR1 domain having an amino acid sequence as set forth in SEQ ID NO: 6, a CDR2 domain having an amino acid sequence as set forth in SEQ ID NO: 7, and a CDR3 domain having an amino acid sequence as set forth in SEQ ID NO: 8.

[0183] AL-310

[0184] Mouse anti-NAMPT antibody AL-310 has a heavy chain variable region with an amino acid sequence as set forth in SEQ ID NO: 56 and a light chain variable region with an amino acid sequence as set forth in SEQ ID NO: 57. The heavy chain variable region of AL-310 contains a CDR1 domain having an amino acid sequence as set forth in SEQ ID NO: 19, a CDR2 domain having an amino acid sequence as set forth in SEQ ID NO: 20, and a CDR3 domain having an amino acid sequence as set forth in SEQ ID NO: 21. The light chain variable region of AL-310 contains a CDR1 domain having an amino acid sequence as set forth in SEQ ID NO: 6, a CDR2 domain having an amino acid sequence as set forth in SEQ ID NO: 7, and a CDR3 domain having an amino acid sequence as set forth in SEQ ID NO: 22.

[0185] The amino acid sequences of the heavy and light chain variable regions and CDRs of AL-303 and AL-310 are provided in Table 1.

[0186] Table 1. Sequences of murine anti-NAMPT antibodies

[0187]

[0188]

[0189] Example 2. Detection of human and mouse NAMPT by mouse anti-NAMPT antibodies

[0190] To assess the immunoreactivity of mouse anti-NAMPT antibodies AL-303 and AL-310 to human and mouse NAMPT, these antibodies were tested for detection of hNAMPT and mNAMPT by Western blot analysis. The immunoreactivity of AL-303 and AL-310 to hNAMPT was also assessed by evaluating the ability of these antibodies to detect recombinant hNAMPT and hNAMPT in HPAEC cell lysates. The immunoreactivity of AL-303 and AL-310 to mNAMPT was further tested by evaluating the ability of these antibodies to detect recombinant mNAMPT, mNAMPT in lysates from spontaneously breathing mice (“SB”), mNAMPT in lysates from LPS-exposed murine lungs, and mNAMPT in lysates from a murine ventilator-induced lung injury (VILI) model.

[0191] Figure 1Results from the corresponding Western blot analysis are provided in FIG. 3. As shown, AL-303 and AL-310 had strong immunoreactivity with hNAMPT (compared to the other antibodies). In contrast, the immunoreactivity of these antibodies with mNAMPT was significantly weaker. Of the five murine antibodies tested, only AL-303 and AL-304 (which contains the same sequence as AL-303) had immunoreactivity with recombinant mNAMPT, while all of the antibodies were reactive with recombinant hNAMPT and hNAMPT in HPAEC cells.

[0192] Example 3. Effect of murine anti-NAMPT antibodies on hNAMPT-induced NFkB phosphorylation

[0193] The effect of the murine anti-NAMPT antibodies on hNAMPT-induced NFkB phosphorylation was evaluated by Western blot analysis of phospho-NFkB (p-NFkB) expression in cells exposed to hNAMPT in the absence or presence of the murine anti-NAMPT antibodies AL-303, AL-304, AL-305, AL-309, and AL-310.

[0194] Recombinant hNAMPT (1 μg / mL) was pre-mixed with media, 100 μg / ml anti-NAMPT polyclonal (pAb), or 100 μg / mL of murine anti-NAMPT antibodies (AL-303, AL-304, AL-305, AL-309, or AL-310) for 30 min. HPAEC cells were stimulated by exposing the cells to the hNAMPT mix for 1 hr. Unstimulated cells ("Unstim") exposed to media, 100 μg / mL anti-NAMPT pAb, or 100 μg / mL of murine anti-NAMPT antibodies (AL-303, AL-304, AL-305, AL-309, or AL-310) alone were used as negative controls. HPAEC cells exposed to TNF-α were used as a positive control. p-NFkB expression in lysates from unstimulated cells, hNAMPT-stimulated cells, and TNF-α-stimulated cells was evaluated by Western blot analysis using a p-NFkB specific antibody. Results of the Western blot analysis are provided in FIG. 4. Figure 2

[0195] As Figure 2 ​As shown, hNAMPT stimulated NFkB phosphorylation in cells induced by hNAMPT was significantly attenuated in the presence of AL-303, AL-304, AL-305, AL-309, or AL-310. While all five antibodies were effective in reducing hNAMPT induced NFkB phosphorylation, the most significant effects were observed with AL-303 and AL-310. AL-304, AL-304, AL-305, and AL-309 are anti-hNAMPT antibodies also obtained in the initial mouse screen.

[0196] Example 4. In vivo testing of AL303, AL304, and AL305 in a model of lung injury

[0197] The ability of the mouse anti-NAMPT antibodies AL-303, AL-304, and AL-305 to treat lung injury was tested in vivo using a mouse model.

[0198] As Figure 3 described in the Example 3, C57 / B6 mice were injected intravenously with 50 μg, 100 μg, or 200 μg of the mouse anti-NAMPT antibodies AL-303, AL-304, or AL-305, or media alone. One hour after injection of the anti-NAMPT antibodies, the mice were exposed to 40 μg / mL of intratracheal hNAMPT (Peprotech). Eight hours after hNAMPT exposure, the anti-NAMPT antibodies were again administered at the same concentration. Control mice were injected with media and exposed to intratracheal hNAMPT, or injected with media and not exposed to intratracheal hNAMPT. All animals were sacrificed 24 hr after hNAMPT exposure, and lung injury was assessed by analyzing the expression of BAL proteins and the count of polymorphonuclear neutrophils (PMNs) expressing BAL.

[0199] The results of the assessment are provided in Figure 4. As described in Figure 4, the expression of BAL proteins Figure 4A ) and the count of PMNs expressing BAL Figure 4B ) were induced in mice exposed to intratracheal hNAMPT. The mouse anti-NAMPT antibodies AL-303, AL-304, and AL-305 were effective in reducing the induction of BAL PMN count mediated by hNAMPT, and the increase in BAL protein levels induced by hNAMPT was significantly attenuated by AL-303. Thus, as described in Figure 4, AL-303, AL-304, and AL-305 were each effective in reducing hNAMPT induced lung injury in mice, with the most significant effects observed with AL-303. Accordingly, AL-303 was selected for subsequent humanization.

[0200] Example 5. In vivo testing of AL-310 in lung injury

[0201] The ability of the murine anti-NAMPT antibody AL-310 to treat lung injury was tested in vivo using a mouse model.

[0202] As Figure 5 described in Example 1, C57 / B6 mice were injected intravenously with 10 μg, 25 μg, 50 μg, 100 μg, or 200 μg of the murine anti-NAMPT antibody AL-310, 100 μg of an anti-NAMPT pAb, or media alone. At the same time as the antibody injection, the mice were exposed to 1 mg / kg of intratracheal LPS. Mice exposed to intratracheal LPS and injected with media or pAb were used as positive controls. Mice injected with media and not exposed to intratracheal LPS were used as negative controls. All animals were sacrificed 6 hr after LPS exposure, and lung injury was assessed by analyzing the expression of BAL protein levels.

[0203] The results of the assessment are provided in Figure 6 As Figure 6 shown in Example 1, the expression of BAL proteins was induced in mice exposed to intratracheal LPS. The murine anti-NAMPT antibody AL-310 effectively reduced the LPS-mediated induction of BAL protein levels, and this effect was substantially more significant than that observed with the positive control. Thus, Figure 6 shows that AL-310 effectively reduced LPS-induced lung injury in mice. Accordingly, AL-310 was selected for subsequent humanization.

[0204] Example 6. Humanization of Anti-NAMPT Antibody AL-303

[0205] Based on the results of the in vitro and in vivo testing described in Examples 2-4, the murine anti-NAMPT antibody AL-303 was selected for humanization.

[0206] 1076 Humanized Anti-hNAMPT Antibodies

[0207] The term "1076" generally refers to the humanized versions of the anti-hNAMPT antibody AL-303. Specifically, the CDRs of the VH and VL chains of AL-303 were grafted onto human heavy and light chain acceptor sequences.

[0208] Following grafting, various framework back mutations were introduced by de novo synthesis of the variable domains, or mutagenic oligonucleotide primers and polymerase chain reaction, or both, by methods well known in the art. Different combinations of back mutations and other mutations were constructed for the heavy and / or light chains of each CDR graft to produce the 1076 humanized anti-hNMAPT antibodies D-1076, G-1076, K-1076, N-1076, P-1076, V-1076, and X-1076.

[0209] The amino acid sequences of the heavy and light chain variable regions of 1076 humanized anti-hNMAPT antibody are provided in Table 2. Table 3 provides an alignment of the amino acid sequences of the heavy chain CDRs of 1076 humanized anti-hNMAPT antibodies D-1076, G-1076, K-1076, N-1076, P-1076, V-1076, and X-1076 compared to murine anti-NAMPT antibody AL-303. Table 4 provides an alignment of the amino acid sequences of the light chain CDRs of 1076 humanized anti-hNMAPT antibodies D-1076, G-1076, K-1076, N-1076, P-1076, V-1076, and X-1076 compared to murine anti-NAMPT antibody AL-303. Blank spaces in Tables 3 and 4 indicate that the residue is identical to AL-303.

[0210] D-1076

[0211] 1076 humanized anti-hNMAPT antibody D-1076 has a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 1 and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 2. The heavy chain variable region of D-1076 contains a CDR1 domain having an amino acid sequence as set forth in SEQ ID NO: 3, a CDR2 domain having an amino acid sequence as set forth in SEQ ID NO: 4, and a CDR3 domain having an amino acid sequence as set forth in SEQ ID NO: 5. One or more back mutations and other mutations were introduced for the heavy chain of D-1076. The light chain variable region of D-1076 contains a CDR1 domain having an amino acid sequence as set forth in SEQ ID NO: 6, a CDR2 domain having an amino acid sequence as set forth in SEQ ID NO: 7, and a CDR3 domain having an amino acid sequence as set forth in SEQ ID NO: 8.

[0212] G-1076

[0213] 1076 humanized anti-hNMAPT antibody G-1076 has a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 9 and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 10. The heavy chain variable region of G-1076 contains a CDR1 domain having an amino acid sequence as set forth in SEQ ID NO: 3, a CDR2 domain having an amino acid sequence as set forth in SEQ ID NO: 4, and a CDR3 domain having an amino acid sequence as set forth in SEQ ID NO: 5. One or more back mutations and other mutations were introduced to the heavy chain of G-1076. The light chain variable region of G-1076 contains a CDR1 domain having an amino acid sequence as set forth in SEQ ID NO: 11, a CDR2 domain having an amino acid sequence as set forth in SEQ ID NO: 12, and a CDR3 domain having an amino acid sequence as set forth in SEQ ID NO: 8.

[0214] K-1076

[0215] 1076 humanized anti-hNMAPT antibody K-1076 has a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 9 and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 13. The heavy chain variable region of K-1076 contains a CDR1 domain having an amino acid sequence as set forth in SEQ ID NO: 3, a CDR2 domain having an amino acid sequence as set forth in SEQ ID NO: 4, and a CDR3 domain having an amino acid sequence as set forth in SEQ ID NO: 5. One or more back mutations and other mutations were introduced to the heavy chain of K-1076. The light chain variable region of K-1076 contains a CDR1 domain having an amino acid sequence as set forth in SEQ ID NO: 11, a CDR2 domain having an amino acid sequence as set forth in SEQ ID NO: 14, and a CDR3 domain having an amino acid sequence as set forth in SEQ ID NO: 8.

[0216] N-1076

[0217] 1076 humanized anti-hNMAPT antibody N-1076 has a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 15 and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 2. The heavy chain variable region of N-1076 contains a CDR1 domain having an amino acid sequence as set forth in SEQ ID NO: 3, a CDR2 domain having an amino acid sequence as set forth in SEQ ID NO: 4, and a CDR3 domain having an amino acid sequence as set forth in SEQ ID NO: 5. One or more back mutations and other mutations were introduced to the heavy chain of N-1076. The light chain variable region of N-1076 contains a CDR1 domain having an amino acid sequence as set forth in SEQ ID NO: 6, a CDR2 domain having an amino acid sequence as set forth in SEQ ID NO: 7, and a CDR3 domain having an amino acid sequence as set forth in SEQ ID NO: 8.

[0218] P-1076

[0219] 1076 humanized anti-hNMAPT antibody P-1076 has a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 15 and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 13. The heavy chain variable region of P-1076 contains a CDR1 domain having an amino acid sequence as set forth in SEQ ID NO: 3, a CDR2 domain having an amino acid sequence as set forth in SEQ ID NO: 4, and a CDR3 domain having an amino acid sequence as set forth in SEQ ID NO: 5. One or more back mutations and other mutations were introduced to the heavy chain of P-1076. The light chain variable region of P-1076 contains a CDR1 domain having an amino acid sequence as set forth in SEQ ID NO: 11, a CDR2 domain having an amino acid sequence as set forth in SEQ ID NO: 14, and a CDR3 domain having an amino acid sequence as set forth in SEQ ID NO: 8.

[0220] V-1076

[0221] 1076 Humanized anti-hNMAPT antibody V-1076 has a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 16 and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 10. The heavy chain variable region of V-1076 contains a CDR1 domain having an amino acid sequence as set forth in SEQ ID NO: 3, a CDR2 domain having an amino acid sequence as set forth in SEQ ID NO: 4, and a CDR3 domain having an amino acid sequence as set forth in SEQ ID NO: 5. One or more back mutations and other mutations were introduced to the heavy chain of V-1076. The light chain variable region of V-1076 contains a CDR1 domain having an amino acid sequence as set forth in SEQ ID NO: 11, a CDR2 domain having an amino acid sequence as set forth in SEQ ID NO: 12, and a CDR3 domain having an amino acid sequence as set forth in SEQ ID NO: 8.

[0222] X-1076

[0223] 1076 Humanized anti-hNMAPT antibody X-1076 has a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 16 and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 2. The heavy chain variable region of X-1076 contains a CDR1 domain having an amino acid sequence as set forth in SEQ ID NO: 3, a CDR2 domain having an amino acid sequence as set forth in SEQ ID NO: 4, and a CDR3 domain having an amino acid sequence as set forth in SEQ ID NO: 5. One or more back mutations and other mutations were introduced to the heavy chain of X-1076. The light chain variable region of X-1076 contains a CDR1 domain having an amino acid sequence as set forth in SEQ ID NO: 6, a CDR2 domain having an amino acid sequence as set forth in SEQ ID NO: 7, and a CDR3 domain having an amino acid sequence as set forth in SEQ ID NO: 8.

[0224] Table 2. Sequences of 1076 humanized anti-hNAMPT antibodies

[0225]

[0226]

[0227]

[0228]

[0229]

[0230]

[0231]

[0232] Example 7. Humanization of anti-hNAMPT antibody AL-310

[0233] Based on the results of the in vitro and in vivo tests described in Examples 2-5, murine anti-NAMPT antibody AL-310 was selected for humanization.

[0234] "1093" refers to a group of humanized antibodies derived from murine anti-NAMPT antibody AL-310. By applying humanization methods, the CDR sequences of the VH and VL chains of AL-310 were grafted onto different human heavy and light chain acceptor sequences.

[0235] By grafting the respective VH and VL CDRs of AL-310 into these acceptor sequences, CDR-grafted, humanized and modified VH and VL sequences were prepared.

[0236] To generate humanized antibodies with potential framework back mutations, mutations were identified and introduced into the CDR-grafted antibody sequences by de novo synthesis of the variable domains or mutagenic oligonucleotide primers and polymerase chain reaction, or both, by methods well known in the art. Different combinations of back mutations and other mutations were constructed for the heavy and / or light chain of each CDR graft to generate the 1093 humanized anti-hNMAPT antibodies FF-1093, II-1093, NN-1093, PP-1093, SS-1093, UU-1093, XX-1093, and ZZ-1093.

[0237] The amino acid sequences of the heavy and light chain variable regions of the 1093 humanized anti-hNMAPT antibodies are provided in Table 5. Table 6 provides an alignment of the amino acid sequences of the heavy chain CDRs of the 1093 humanized anti-hNMAPT antibodies FF-1093, II-1093, NN-1093, PP-1093, SS-1093, UU-1093, XX-1093, and ZZ-1093 compared to murine anti-NAMPT antibody AL-310. Table 7 provides an alignment of the amino acid sequences of the light chain CDRs of the 1093 humanized anti-hNMAPT antibodies FF-1093, II-1093, NN-1093, PP-1093, SS-1093, UU-1093, XX-1093, and ZZ-1093 compared to murine anti-NAMPT antibody AL-310. Blank spaces in Tables 6 and 7 indicate that the residue is identical to AL-310.

[0238] FF-1093

[0239] 1093 Humanized anti-hNMAPT antibody FF-1093 has a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 17 and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 18. The heavy chain variable region of FF-1093 contains a CDR1 domain having an amino acid sequence as set forth in SEQ ID NO: 19, a CDR2 domain having an amino acid sequence as set forth in SEQ ID NO: 20, and a CDR3 domain having an amino acid sequence as set forth in SEQ ID NO: 21. One or more back mutations and other mutations were introduced to the heavy chain of FF-1093. The light chain variable region of FF-1093 contains a CDR1 domain having an amino acid sequence as set forth in SEQ ID NO: 6, a CDR2 domain having an amino acid sequence as set forth in SEQ ID NO: 7, and a CDR3 domain having an amino acid sequence as set forth in SEQ ID NO: 22.

[0240] II-1093

[0241] 1093 Humanized anti-hNMAPT antibody II-1093 has a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 23 and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 24. The heavy chain variable region of II-1093 contains a CDR1 domain having an amino acid sequence as set forth in SEQ ID NO: 19, a CDR2 domain having an amino acid sequence as set forth in SEQ ID NO: 20, and a CDR3 domain having an amino acid sequence as set forth in SEQ ID NO: 21. One or more back mutations and other mutations were introduced to the heavy chain of II-1093. The light chain variable region of II-1093 contains a CDR1 domain having an amino acid sequence as set forth in SEQ ID NO: 6, a CDR2 domain having an amino acid sequence as set forth in SEQ ID NO: 7, and a CDR3 domain having an amino acid sequence as set forth in SEQ ID NO: 22.

[0242] NN-1093

[0243] 1093 Humanized anti-hNMAPT antibody NN-1093 has a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO:25 and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO:24. The heavy chain variable region of NN-1093 contains a CDR1 domain having an amino acid sequence as set forth in SEQ ID NO: 19, a CDR2 domain having an amino acid sequence as set forth in SEQ ID NO:20, and a CDR3 domain having an amino acid sequence as set forth in SEQ ID NO:21. One or more back mutations and other mutations were introduced to the heavy chain of NN-1093. The light chain variable region of NN-1093 contains a CDR1 domain having an amino acid sequence as set forth in SEQ ID NO:6, a CDR2 domain having an amino acid sequence as set forth in SEQ ID NO:7, and a CDR3 domain having an amino acid sequence as set forth in SEQ ID NO:22.

[0244] PP-1093

[0245] 1093 Humanized anti-hNMAPT antibody NN-1093 has a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO:25 and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO:24. The heavy chain variable region of NN-1093 contains a CDR1 domain having an amino acid sequence as set forth in SEQ ID NO: 19, a CDR2 domain having an amino acid sequence as set forth in SEQ ID NO:20, and a CDR3 domain having an amino acid sequence as set forth in SEQ ID NO:21. One or more back mutations and other mutations were introduced to the heavy chain of NN-1093. The light chain variable region of NN-1093 contains a CDR1 domain having an amino acid sequence as set forth in SEQ ID NO:6, a CDR2 domain having an amino acid sequence as set forth in SEQ ID NO:7, and a CDR3 domain having an amino acid sequence as set forth in SEQ ID NO:22.

[0246] SS-1093

[0247] 1093 humanized anti-hNMAPT antibody SS-1093 has a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 26, and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 24. The heavy chain variable region of SS-1093 contains a CDR1 domain having an amino acid sequence as set forth in SEQ ID NO: 19, a CDR2 domain having an amino acid sequence as set forth in SEQ ID NO: 20, and a CDR3 domain having an amino acid sequence as set forth in SEQ ID NO: 21. One or more back mutations and other mutations were introduced to the heavy chain of SS-1093. The light chain variable region of SS-1093 contains a CDR1 domain having an amino acid sequence as set forth in SEQ ID NO: 6, a CDR2 domain having an amino acid sequence as set forth in SEQ ID NO: 7, and a CDR3 domain having an amino acid sequence as set forth in SEQ ID NO: 22.

[0248] UU-1093

[0249] 1093 humanized anti-hNMAPT antibody UU-1093 has a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 26, and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 18. The heavy chain variable region of UU-1093 contains a CDR1 domain having an amino acid sequence as set forth in SEQ ID NO: 19, a CDR2 domain having an amino acid sequence as set forth in SEQ ID NO: 20, and a CDR3 domain having an amino acid sequence as set forth in SEQ ID NO: 21. One or more back mutations and other mutations were introduced to the heavy chain of UU-1093. The light chain variable region of UU-1093 contains a CDR1 domain having an amino acid sequence as set forth in SEQ ID NO: 6, a CDR2 domain having an amino acid sequence as set forth in SEQ ID NO: 7, and a CDR3 domain having an amino acid sequence as set forth in SEQ ID NO: 22.

[0250] XX-1093

[0251] 1093 Humanized anti-hNMAPT antibody XX-1093 has a heavy chain variable region having an amino acid sequence set forth in SEQ ID NO: 27 and a light chain variable region having an amino acid sequence set forth in SEQ ID NO: 24. The heavy chain variable region of XX-1093 contains a CDR1 domain having an amino acid sequence set forth in SEQ ID NO: 19, a CDR2 domain having an amino acid sequence set forth in SEQ ID NO: 20, and a CDR3 domain having an amino acid sequence set forth in SEQ ID NO: 21. One or more back mutations and other mutations were introduced to the heavy chain of XX-1093. The light chain variable region of XX-1093 contains a CDR1 domain having an amino acid sequence set forth in SEQ ID NO: 6, a CDR2 domain having an amino acid sequence set forth in SEQ ID NO: 7, and a CDR3 domain having an amino acid sequence set forth in SEQ ID NO: 22.

[0252] ZZ-1093

[0253] 1093 Humanized anti-hNMAPT antibody ZZ-1093 has a heavy chain variable region having an amino acid sequence set forth in SEQ ID NO: 27 and a light chain variable region having an amino acid sequence set forth in SEQ ID NO: 18. The heavy chain variable region of ZZ-1093 contains a CDR1 domain having an amino acid sequence set forth in SEQ ID NO: 19, a CDR2 domain having an amino acid sequence set forth in SEQ ID NO: 20, and a CDR3 domain having an amino acid sequence set forth in SEQ ID NO: 21. One or more back mutations and other mutations were introduced to the heavy chain of ZZ-1093. The light chain variable region of ZZ-1093 contains a CDR1 domain having an amino acid sequence set forth in SEQ ID NO: 6, a CDR2 domain having an amino acid sequence set forth in SEQ ID NO: 7, and a CDR3 domain having an amino acid sequence set forth in SEQ ID NO: 22.

[0254] Table 5. Sequences of 1093 humanized anti-hNAMPT antibodies

[0255]

[0256]

[0257]

[0258]

[0259]

[0260]

[0261]

[0262] Example 8. Epitope mapping of humanized anti-hNAMPT antibodies

[0263] Epitope mapping of humanized anti-NAMPT antibody K-1076 (described in Example 6) and humanized anti-NAMPT antibody NN-1093 (described in Example 7) was performed using both linear and conformational epitope mapping.

[0264] A library of peptide-based peptidomimetics was synthesized using Fmoc-based solid phase peptide synthesis. To generate a linear peptidomimetic library, the amino acid sequence of human NAMPT (SEQ ID NO: 60) was divided into overlapping fragments in silico and then synthesized on a solid support. Aminofunctionalized polypropylene supports were obtained by grafting with a proprietary hydrophilic polymer formulation, followed by reaction with tert-butyloxycarbonyl-hexamethylenediamine (BocHMDA) using dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide (HOBt), and subsequent cleavage of the Boc-group using trifluoroacetic acid (TFA). Peptides were synthesized on the aminofunctionalized solid support using standard Fmoc-peptide synthesis by means of a custom-modified JANUS liquid handling station (Perkin Elmer).

[0265] Synthesis of structural mimics was performed using the proprietary scaffold chemistry peptide (CLIPS) technology of Pepscan. The CLIPS technology allows structuring of peptides into single-, double-, triple-, sheet-, and helix-like folds and combinations thereof. CLIPS templates are coupled to cysteine residues. The side chains of multiple cysteines in a peptide are coupled to one or two CLIPS templates. For example, a 0.5 mM solution of P2 CLIPS (2,6-bis(bromomethyl)pyridine) was dissolved in ammonium bicarbonate (20 mM, pH 7.8) / acetonitrile (1 :3 (v / v)). This solution was added to the peptide array. The CLIPS templates bind to the side chains of two cysteines present in the solid-phase bound peptides of the peptide array (455-well plate with 3 μL wells). The peptide array was gently shaken in solution for 30 to 60 minutes while completely covered in solution. Finally, the peptide array was extensively washed with excess H2O and sonicated for 30 minutes in a disruption buffer containing 1% SDS / 0.1% 2,2'-(ethylenedioxy)diethanethiol in PBS (pH 7.2) at 70°C, followed by an additional 45 minutes in H2O. Peptide-bearing T3 CLIPS were prepared in a similar manner but with three cysteines.

[0266] Antibodies were tested for binding to each synthetic peptide in a Pepscan-based ELISA. Peptide arrays were incubated with primary antibody solution (overnight at 4°C). After washing, the peptide arrays were incubated with a 1 / 1000 dilution of the appropriate detection antibody (antibody peroxidase conjugate (SBA)) for 1 hour at 25°C. As detection antibody, HRP-conjugated goat anti-human antibody (Southern Biotech; Cat. No. 2010-05) was used. After washing, the peroxidase substrate 2,2'-azino-di-3-ethylbenzthiazoline sulfonate (ABTS) and 20 μl / ml of 3% H2O2 were added. After one hour, the color development was measured. Color development was quantified with a charge-coupled device (CCD) camera and image processing system.

[0267] Values obtained from the CCD camera were in the range of 0 to 3000 mAU, similar to standard 96-well plate ELISA readers. Results were quantified and stored into the Peplab database. Occasionally, wells containing air bubbles led to false positive values; therefore, the cards were manually checked and any values caused by air bubbles were scored as 0.

[0268] To verify the quality of the synthetic peptides, a separate set of positive and negative control peptides was synthesized in parallel. They were screened with the commercial antibodies 3C9A and 57.9 (Posthumus et al. (J Virol 64:3304-3309, 1990)).

[0269] Data analysis and interpretation were performed using box-and-whisker plots, linear intensity distributions, and heat maps analysis.

[0270] Ten different sets of peptides (Set 1 - Set 10) ranging in size from 9 to 30 amino acids were synthesized as described below.

[0271] Set 1 (LIN 15)

[0272] Set 1 peptides, also referred to herein as LIN15 peptides, are linear peptides 15 amino acids long derived from the target sequence of NAMPT with a one-residue offset.

[0273] Set 2 (LIN 15.AA)

[0274] Set 2 peptides, also referred to herein as LIN15.AA peptides, are linear peptides 15 amino acids long similar to Set 1 or LIN15 peptides, but with the 10thand 11thresidues replaced by Ala. Where a native Ala would occur, it is replaced by Gly.

[0275] Set 3 (LIN 30)

[0276] Set 3 peptide, also referred to herein as LIN30 peptide, is a 30 amino acid long linear peptide derived from the target sequence of NAMPT with a one residue offset.

[0277] Set 4 (LOOP 7)

[0278] Set 4 peptide, also referred to herein as LOOP7 peptide, is a 9 amino acid long constrained peptide. Positions 2-8 are a 7-mer peptide derived from the target sequence of NAMPT with a one residue offset. Cys residues are inserted in positions 1 and 9 and connected by mP2 CLIPS to create a cyclic mimetic. Native Cys is replaced by Cys-acm (denoted as "2").

[0279] Set 5 (LOOP 15)

[0280] Set 5 peptide, also referred to herein as LOOP 15 peptide, is a 17 amino acid long constrained peptide. Positions 2-16 are a 15-mer peptide derived from the target sequence of NAMPT (SEQ ID NO: 60) with a one residue offset. Cys residues are inserted in positions 1 and 17 and connected by mP2 CLIPS to create a cyclic mimetic. Native Cys is replaced by Cys-acm (denoted as "2").

[0281] Set 6 (LOOP 15.AA)

[0282] Set 6 peptide, also referred to herein as LOOP15.AA peptide, is a 17 amino acid long constrained peptide which is similar to Set 5 or LOOP15 peptide, but the residues in positions 10 and 11 are replaced by Ala. Where a native Ala would occur in either position, it is replaced by Gly.

[0283] Set 7 (LOOP 25)

[0284] Set 7 peptide, also referred to herein as LOOP 25 peptide, is a 27 amino acid long constrained peptide. Positions 2-26 are a 25-mer peptide derived from the target sequence of NAMPT (SEQ ID NO: 60) with a one residue offset. Cys residues are inserted in positions 1 and 27 and connected by mP2 CLIPS to create a cyclic mimetic. Native Cys is replaced by Cys-acm (denoted as "2").

[0285] Set 8 (BET)

[0286] The Set 8 peptide, also referred to herein as the BET peptide, is a 22 amino acid long beta-turn peptidomimetic. Positions 2-21 are a 20-mer peptide derived from the target sequence of NAMPT (SEQ ID NO: 60) with a one residue offset. The residues at positions 11 and 12 are replaced by a "PG" motif to induce beta-turn formation. Cys residues are inserted at positions 1 and 22 and linked by mP2 CLIPIS to stabilize the mimetic. Native Cys is replaced by Cys-acm (denoted as "2").

[0287] Set 9 (HEL.CC)

[0288] The Set 9 peptide, also referred to herein as the HEL.CC peptide, is a 22 amino acid long alpha-helix peptidomimetic derived from the target sequence (NAMPT, SEQ ID NO: 60) residues with a one residue offset. Cys residues are inserted at positions 1 and 5 and linked by mP2 CLIPIS to nucleate the alpha-helix structure. Native Cys is replaced by Cys-acm (denoted as "2").

[0289] Set 10 (HEL.IL)

[0290] The Set 10 group, also referred to herein as the HEL.IL peptide, is a 26 amino acid long alpha-helix peptidomimetic derived from the target sequence (NAMPT, SEQ ID NO: 60) residues with a one residue offset of Leu and Ile residues inserted in the sequence to promote the helical secondary structure without covalent constraints.

[0291] Screening details

[0292] Antibody binding depends on a combination of factors, including antibody concentration and the amount and nature of competing proteins in the ELISA buffer. In addition, pre-coating conditions (a specific treatment of the peptide array prior to incubation with experimental samples) influence binding. These details are summarized in Table 8. For the Pepscan buffer and pre-conditioning (SQ), the numbers indicate the relative amount of competing proteins (a combination of horse serum and egg white proteins).

[0293] Table 8. Screening details

[0294] Label Dilution Sample buffer Preconditioning K-1076 (Ab-1076-HC2-LC5) 5 ug / mL 10% SQ 10% SQ NN-1093 (NN-1093-HC3-LC3) 2 ug / mL 1% SQ 1% SQ

[0295] For both antibodies in this study, we observed binding signals in various regions throughout the NAMPT sequence. This indicates that the epitopes of both antibodies are discontinuous epitopes. All signals observed were specific for the antibody, as incubation with the secondary antibody alone did not produce any signal on the array. For Ab-1076-HC2-LC5, the core sequence derived from the overlapping peptide sequences appears to form a binding interface spanning both monomers within the NAMPT dimer. In particular in the plot of the LIN30 mimic, a dominant putative binding site can be observed, which is surrounded by other sequences derived from lower intensity peaks in the structure. The tentative core epitope of the antibody is listed below. Both antibodies bind to the dimeric form of NAMPT.

[0296] The identified epitope candidates of both sequences show significant amounts of recurring sequence similarity (e.g. 17 SYKVTHYKQYPPNTSKVYSYFEC REKK T 44 (SEQ ID NO: 61) vs. 162 ATNS REQ KK 170 (SEQ ID NO: 63), 29 NT SKVYS YFECREKKTENSKLRK 51 (SEQ ID NO: 72) vs. 332 FPVTEN SKGY K 342 (SEQ ID NO: 67), and 216 KGT DTV AGLALIKKYY GTK 234 (SEQ ID NO: 75) vs. 316 NPL DTV LKVLEIL GKK 331 (SEQ ID NO: 76).

[0297] Table 9. List of top epitope candidates found in this study. For linear and conformational peptide arrays, the best candidates were based on intensity of median intensity values of at least 2X. Figure 7A

[0298]

[0299]

[0300] Example 9. Characterization of humanized anti-hNAMPT antibodies

[0301] The 1076 humanized anti-hNAMPT antibodies described in Example 6 and the 1093 humanized anti-hNAMPT antibodies described in Example 7 were tested in vitro and in vivo to select a lead humanized anti-hNAMPT antibody.

[0302] Effect of humanized anti-hNAMPT antibodies on hNAMPT-induced inflammatory signaling

[0303] The effect of the 1093 humanized anti-hNAMPT antibodies CC-1093, KK-1093, RR-1093, UU-1093, and XX-1093 on hNAMPT-induced inflammatory signaling was assessed by evaluating NFKB activation in cells exposed to hNAMPT in the absence or presence of these antibodies.

[0304] Recombinant hNAMPT (1.5 pg / mL) was pre-mixed with medium or 100 pg / mL of the 1093 humanized anti-hNAMPT antibodies (CC-1093, KK-1093, RR-1093, UU-1093, or XX-1093). Human lung endothelial cells (ECs) were stimulated by exposing the cells to the hNAMPT mix for 1 hr. Unstimulated cells not exposed to the hNAMPT mix were used as a negative control (“NC”). Stimulated cells exposed to hNAMPT pre-mixed with medium alone (“—”) were used as a positive control. NFKB activation in the cells was assessed by evaluating NFKB luciferase activity (NFKB-Sec Nanoluc) (n = 3-4 for each mAb). The results of the luciferase assay are provided in Figure 7A .

[0305] As shown in Figure 7B , hNAMPT induced NFKB activation in hNAMPT-stimulated cells, which was significantly attenuated in the presence of the humanized anti-hNAMPT antibodies CC-1093, KK-1093, RR-1093, UU-1093, or XX-1093. While all five antibodies were effective in reducing hHNAMPT-induced NFKB activation, the most significant effects were observed with UU-1093 and XX-1093.

[0306] Effect of humanized anti-hNAMPT antibodies on hNAMPT-induced decline in EC barrier integrity

[0307] EC resistance is a reflection of lung EC barrier integrity. Therefore, the effects of the 1093 humanized anti-hNAMPT antibody UU-1093 and the 1076 humanized anti-hNAMPT antibodies H-1076, P-1076, N-1076, and D-1076 on hNAMPT-induced decrease in EC barrier integrity were assessed by evaluating resistance in ECs exposed to hNAMPT in the absence or presence of the following antibodies.

[0308] Recombinant hNAMPT (1.5 μg / mL) was premixed with a single vector, an anti-NAMPT pAb, or a humanized anti-hNAMPT antibody (UU-1093, H-1076, P-1076, N-1076, or D-1076). Human lung ECs were stimulated by exposing cells to the hNAMPT mixture for 1 hour. Cells stimulated with hNAMPT premixed with the single vector or with hNAMPT premixed with the pAb served as controls. EC resistance was assessed as a reading of EC barrier integrity. Figure 7B The test results are provided in the document.

[0309] like Figure 8A As shown, ECs exposed to hNAMPT premixed with each humanized anti-hNAMPT antibody exhibited substantial induction of EC barrier activity relative to control cells. Therefore, in the presence of the 1076 and 1093 humanized anti-hNAMPT antibodies, the hNAMPT-mediated decrease in EC barrier activity was effectively reversed.

[0310] In vivo testing of humanized anti-hNAMPT antibody in lung injury

[0311] The efficacy of 1076-humanized anti-hNAMPT antibodies (V-1076, N-1076, K-1076, and P-1076) and 1093-humanized anti-hNAMPT antibodies (SS-1093, CC-1093, XX-1093, and UU-1093) in treating lung injury was tested in vivo using mouse and rat lung injury models.

[0312] To evaluate the effect of humanized anti-hNAMPT antibody on a mouse model of lung injury, mice exposed to LPS for 8 hours and VILI for the last 4 hours were intravenously injected with V-1076, N-1076, K-1076, P-1076, SS-1093, CC-1093, XX-1093, or UU-1093 at a dose of 0.4 mg / kg. Mice injected with the mediator and exposed to hNAMPT and VILI served as controls. Lung injury in mice was assessed by analyzing BAL protein expression and the count of BAL-expressing cells. Furthermore, H&E staining was used to assess edema and inflammatory cell infiltration in lung tissue as readings of lung injury. Figure 8A ,8B Results from this "double hit" model of lung injury are provided in Figures 8A-8D.

[0313] As shown in Figure 8A , BAL protein levels ( Figure 8A , left panel) and counts of BAL expressing cells ( Figure 8B , right panel) were effectively reduced in mice injected with any of the 1076 humanized anti-hNAMPT antibodies compared to control mice. Although all of the 1076 humanized anti-hNAMPT antibodies tested effectively reduced lung injury in this "double hit" mouse model, the most significant effects were observed using P-1076.

[0314] As shown in Figure 8B , BAL protein levels ( Figure 8B , left panel) and counts of BAL expressing cells ( Figure 8D , right panel) were effectively reduced in mice injected with any of the 1093 humanized anti-hNAMPT antibodies compared to control mice. Although all of the 1093 humanized anti-hNAMPT antibodies tested effectively reduced lung injury in this "double hit" mouse model, the most significant effects were observed using UU-1093. Furthermore, as shown in Figure 8C , the protective effects of UU-1093 in the double hit lung injury model were further reflected by reduced inflammatory cell infiltration and reduced edema in lung tissue from mice injected with UU-1093.

[0315] To assess the effects of humanized anti-hNAMPT antibodies on a rat model of lung injury, P-1076 was injected intravenously at 40 mg / kg, 80 mg / kg, or 160 mg / kg to Sprague Dawley rats exposed to LPS. Rats injected with vehicle and exposed to LPS were used as a positive control. Rats injected with media alone and not exposed to LPS were used as a negative control. Lung injury in the rats was assessed by analyzing BAL protein expression and counts of BAL expressing cells. Figure 8C Results from this rat model of lung injury are provided in Figure 9.

[0316] As shown in Figure 8C , BAL protein levels ( Figure 8C , left panel) and counts of BAL expressing cells ( Table 10. Sequences of P-1076-mod anti-hNAMPT antibodies , right panel) were effectively reduced in mice injected with humanized anti-hNAMPT antibody P-1076 compared to control mice (injected with media and exposed to LPS).

[0317] Thus, as described in Figure 8, although all of the tested humanized anti-hNAMPT antibodies effectively reduced lung injury in the in vivo lung injury model, the most significant effects were observed using P-1076 and UU-1093. Accordingly, P-1076 and UU-1093 were selected as lead humanized and anti-hNAMPT antibodies, and further modified to generate improved anti-hNAMPT antibodies.

[0318] Example 10. Modification of humanized anti-hNAMPT antibody P-1076

[0319] Based on the results of the in vitro and in vivo tests described in Example 9, anti-hNAMPT antibody P-1076 was selected for further modification to generate improved 1076 anti-hNAMPT antibodies.

[0320] P-1076-mod humanized anti-hNAMPT antibodies

[0321] P-1076-mod refers to humanized anti-hNAMPT antibodies generated by modification of 1076 humanized anti-hNAMPT antibody P-1076.

[0322] To generate improved P-1076-mod anti-hNAMPT antibodies, one or more mutations were introduced into the P-1076 sequence by de novo synthesis of the variable domains, or mutagenic oligonucleotide primers and polymerase chain reactions, or both, by methods well known in the art. These mutations were introduced to remove oxidation sites, reduce or remove deamidation, remove potential cleavage or fragmentation sites, remove potential T cell epitopes, and / or reduce binding of potential T cell epitopes. Different combinations of back mutations and other mutations were constructed for the heavy and / or light chains of P-1076 to generate P-1076-mod anti-hNAMPT antibodies P-1076-modl, P-1076-mod2, P-1076-mod3, P-1076-mod4, P-1076-mod5, P-1076-mod6, P-1076-mod7, P-1076-mod8, P-1076-mod9, P-1076-modlO, and P-1076-modl l.

[0323] The amino acid sequences of the heavy and light chain variable regions of the modified P-1076-mod anti-hNAMPT antibodies are provided in Table 10. In Table 10, mutated residues are in bold. Table 11 provides an alignment of the amino acid sequences of the heavy chain CDRs of the modified P-1076-mod anti-hNAMPT antibodies P-1076-modl, P-1076-mod2, P-1076-mod3, P-1076-mod4, P-1076-mod5, P-1076-mod6, P-1076-mod7, P-1076-mod8, P-1076-mod9, P-1076-modlO, and P-1076-modl l compared to P-1076. Table 12 provides an alignment of the amino acid sequences of the light chain CDRs of the modified P-1076-mod anti-hNAMPT antibodies P-1076-modl, P-1076-mod2, P-1076-mod3, P-1076-mod4, P-1076-mod5, P-1076-mod6, P-1076-mod7, P-1076-mod8, P-1076-mod9, P-1076-modlO, and P-1076-modl l compared to P-1076. Blank spaces in Tables 11 and 12 indicate that the residue is the same as P-1076. A summary of the biophysical characteristics of the modified P-1076-mod anti-hNAMPT antibodies is provided in Table 13.

[0324] P-1076-modl

[0325] The P-1076-modl anti-hNAMPT antibody has a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 28 and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 30. The heavy chain variable region of P-1076-modl contains a CDR1 domain having an amino acid sequence as set forth in SEQ ID NO: 3, a CDR2 domain having an amino acid sequence as set forth in SEQ ID NO: 29, and a CDR3 domain having an amino acid sequence as set forth in SEQ ID NO: 5. For the heavy chain of P-1076-modl, a D to E mutation was introduced into the P-1076 VH CDR2 to remove a potential cleavage or fragmentation site. The light chain variable region of P-1076-modl comprises a CDR1 domain having an amino acid sequence as set forth in SEQ ID NO: 11, a CDR2 domain having an amino acid sequence as set forth in SEQ ID NO: 14, and a CDR3 domain having an amino acid sequence as set forth in SEQ ID NO: 8. For the light chain of P-1076-modl, a L to V mutation was introduced into the framework region of P-1076 VL to remove a potential T cell epitope.

[0326] P-1076-mod2

[0327] P-1076-mod2 has a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 28 and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 31. The heavy chain variable region of P-1076-mod2 contains a CDR1 domain having an amino acid sequence as set forth in SEQ ID NO: 3, a CDR2 domain having an amino acid sequence as set forth in SEQ ID NO: 29, and a CDR3 domain having an amino acid sequence as set forth in SEQ ID NO: 5. For the heavy chain of P-1076-mod2, a D to E mutation was introduced into the P-1076 VH CDR2 to remove a potential cleavage or fragmentation site. The light chain variable region of P-1076-mod2 contains a CDR1 domain having an amino acid sequence as set forth in SEQ ID NO: 11, a CDR2 domain having an amino acid sequence as set forth in SEQ ID NO: 14, and a CDR3 domain having an amino acid sequence as set forth in SEQ ID NO: 8. For the light chain of P-1076-mod2, an L to I mutation was introduced into the framework region of P-1076 VL to remove a potential T cell epitope.

[0328] P-1076-mod3

[0329] P-1076-mod3 has a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 28 and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 32. The heavy chain variable region of P-1076-mod3 contains a CDR1 domain having an amino acid sequence as set forth in SEQ ID NO: 3, a CDR2 domain having an amino acid sequence as set forth in SEQ ID NO: 29, and a CDR3 domain having an amino acid sequence as set forth in SEQ ID NO: 5. For the heavy chain of P-1076-mod3, a D to E mutation was introduced into P-1076 VH CDR2 to remove a potential cleavage or fragmentation site. The light chain variable region of P-1076-mod3 contains a CDR1 domain having an amino acid sequence as set forth in SEQ ID NO: 11, a CDR2 domain having an amino acid sequence as set forth in SEQ ID NO: 33, and a CDR3 domain having an amino acid sequence as set forth in SEQ ID NO: 8. For the light chain of P-1076-mod3, a L to V mutation was introduced into the framework region of P-1076 VL and a L to G mutation was introduced into P-1076 VL CDR2 to remove potential T cell epitopes.

[0330] P-1076-mod4

[0331] P-1076-mod3 has a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 28 and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 32. The heavy chain variable region of P-1076-mod3 contains a CDR1 domain having an amino acid sequence as set forth in SEQ ID NO: 3, a CDR2 domain having an amino acid sequence as set forth in SEQ ID NO: 29, and a CDR3 domain having an amino acid sequence as set forth in SEQ ID NO: 5. For the heavy chain of P-1076-mod3, a D to E mutation was introduced into P-1076 VH CDR2 to remove a potential cleavage or fragmentation site. The light chain variable region of P-1076-mod3 contains a CDR1 domain having an amino acid sequence as set forth in SEQ ID NO: 11, a CDR2 domain having an amino acid sequence as set forth in SEQ ID NO: 33, and a CDR3 domain having an amino acid sequence as set forth in SEQ ID NO: 8. For the light chain of P-1076-mod3, a L to V mutation was introduced into the framework region of P-1076 VL and a L to G mutation was introduced into P-1076 VL CDR2 to remove potential T cell epitopes.

[0332] P-1076-mod5

[0333] P-1076-mod5 is an anti-hNAMPT antibody having a heavy chain variable region having an amino acid sequence set forth in SEQ ID NO: 28 and a light chain variable region having an amino acid sequence set forth in SEQ ID NO: 36. The heavy chain variable region of P-1076-mod5 contains a CDR1 domain having an amino acid sequence set forth in SEQ ID NO: 3, a CDR2 domain having an amino acid sequence set forth in SEQ ID NO: 29, and a CDR3 domain having an amino acid sequence set forth in SEQ ID NO: 5. For the heavy chain of P-1076-mod5, a D to E mutation was introduced into P-1076 VH CDR2 to remove a potential cleavage or fragmentation site. The light chain variable region of P-1076-mod5 contains a CDR1 domain having an amino acid sequence set forth in SEQ ID NO: 11, a CDR2 domain having an amino acid sequence set forth in SEQ ID NO: 37, and a CDR3 domain having an amino acid sequence set forth in SEQ ID NO: 8. For the light chain of P-1076-mod5, a L to I mutation was introduced into the framework region of P-1076 VL to remove a potential T cell epitope, and a L to E mutation was introduced into P-1076 VL CDR2 to reduce binding of potential T cell epitopes.

[0334] P-1076-mod6

[0335] P-1076-mod6 is an anti-hNAMPT antibody having a heavy chain variable region having an amino acid sequence set forth in SEQ ID NO: 15 and a light chain variable region having an amino acid sequence set forth in SEQ ID NO: 30. The heavy chain variable region of P-1076-mod6 contains a CDR1 domain having an amino acid sequence set forth in SEQ ID NO: 3, a CDR2 domain having an amino acid sequence set forth in SEQ ID NO: 4, and a CDR3 domain having an amino acid sequence set forth in SEQ ID NO: 5. The heavy chain of P-1076-mod6 is identical to P-1076 VH. The light chain variable region of P-1076-mod6 contains a CDR1 domain having an amino acid sequence set forth in SEQ ID NO: 11, a CDR2 domain having an amino acid sequence set forth in SEQ ID NO: 14, and a CDR3 domain having an amino acid sequence set forth in SEQ ID NO: 8. For the light chain of P-1076-mod5, a L to V mutation was introduced into the framework region of P-1076 VL to remove a potential T cell epitope.

[0336] P-1076-mod7

[0337] P-1076-mod7 is an anti-hNAMPT antibody having a heavy chain variable region having an amino acid sequence set forth in SEQ ID NO: 15 and a light chain variable region having an amino acid sequence set forth in SEQ ID NO: 31. The heavy chain variable region of P-1076-mod7 contains a CDR1 domain having an amino acid sequence set forth in SEQ ID NO: 3, a CDR2 domain having an amino acid sequence set forth in SEQ ID NO: 4, and a CDR3 domain having an amino acid sequence set forth in SEQ ID NO: 5. The heavy chain of P-1076-mod7 is identical to P-1076 VH. The light chain variable region of P-1076-mod7 contains a CDR1 domain having an amino acid sequence set forth in SEQ ID NO: 11, a CDR2 domain having an amino acid sequence set forth in SEQ ID NO: 14, and a CDR3 domain having an amino acid sequence set forth in SEQ ID NO: 8. For the light chain of P-1076-mod7, a L to I mutation was introduced into the framework region of P-1076 VL to remove a potential T cell epitope.

[0338] P-1076-mod8

[0339] P-1076-mod8 is an anti-hNAMPT antibody having a heavy chain variable region having an amino acid sequence set forth in SEQ ID NO: 15 and a light chain variable region having an amino acid sequence set forth in SEQ ID NO: 32. The heavy chain variable region of P-1076-mod8 contains a CDR1 domain having an amino acid sequence set forth in SEQ ID NO: 3, a CDR2 domain having an amino acid sequence set forth in SEQ ID NO: 4, and a CDR3 domain having an amino acid sequence set forth in SEQ ID NO: 5. The heavy chain of P-1076-mod8 is identical to P-1076 VH. The light chain variable region of P-1076-mod8 contains a CDR1 domain having an amino acid sequence set forth in SEQ ID NO: 11, a CDR2 domain having an amino acid sequence set forth in SEQ ID NO: 33, and a CDR3 domain having an amino acid sequence set forth in SEQ ID NO: 8. For the light chain of P-1076-mod8, a L to V mutation was introduced into the framework region of P-1076 VL and a L to G mutation was introduced into the P-1076 VL CDR2 to remove potential T cell epitopes.

[0340] P-1076-mod9

[0341] P-1076-mod9 has a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 15 and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 34. The heavy chain variable region of P-1076-Mod9 contains a CDR1 domain having an amino acid sequence as set forth in SEQ ID NO: 3, a CDR2 domain having an amino acid sequence as set forth in SEQ ID NO: 4, and a CDR3 domain having an amino acid sequence as set forth in SEQ ID NO: 5. The heavy chain of P-1076-mod9 is identical to P-1076 VH. The light chain variable region of P-1076-Mod9 contains a CDR1 domain having an amino acid sequence as set forth in SEQ ID NO: 11, a CDR2 domain having an amino acid sequence as set forth in SEQ ID NO: 35, and a CDR3 domain having an amino acid sequence as set forth in SEQ ID NO: 8. For the light chain of P-1076-mod9, a L to V mutation was introduced into the framework region of P-1076 VL and a L to E mutation was introduced into P-1076 VL CDR2 to remove potential T-cell epitopes.

[0342] P-1076-mod10

[0343] P-1076-mod9 has a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 15 and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 34. The heavy chain variable region of P-1076-Mod9 contains a CDR1 domain having an amino acid sequence as set forth in SEQ ID NO: 3, a CDR2 domain having an amino acid sequence as set forth in SEQ ID NO: 4, and a CDR3 domain having an amino acid sequence as set forth in SEQ ID NO: 5. The heavy chain of P-1076-mod9 is identical to P-1076 VH. The light chain variable region of P-1076-Mod9 contains a CDR1 domain having an amino acid sequence as set forth in SEQ ID NO: 11, a CDR2 domain having an amino acid sequence as set forth in SEQ ID NO: 35, and a CDR3 domain having an amino acid sequence as set forth in SEQ ID NO: 8. For the light chain of P-1076-mod9, a L to V mutation was introduced into the framework region of P-1076 VL and a L to E mutation was introduced into P-1076 VL CDR2 to remove potential T-cell epitopes.

[0344] P-1076-mod11

[0345] Modified P-1076-mod anti-hNAMPT antibody P-1076-mod 11 has a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 28 and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 13. The heavy chain variable region of P-1076-mod 11 contains a CDR1 domain having an amino acid sequence as set forth in SEQ ID NO: 3, a CDR2 domain having an amino acid sequence as set forth in SEQ ID NO: 29, and a CDR3 domain having an amino acid sequence as set forth in SEQ ID NO: 5. For the heavy chain of P-1076-mod 11, a D to E mutation was introduced into the P-1076 VH CDR2 to remove a potential cleavage or fragmentation site. The light chain variable region of P-1076-mod 11 contains a CDR1 domain having an amino acid sequence as set forth in SEQ ID NO: 11, a CDR2 domain having an amino acid sequence as set forth in SEQ ID NO: 14, and a CDR3 domain having an amino acid sequence as set forth in SEQ ID NO: 8. The light chain of P-1076-mod 11 is identical to the P-1076 VL.

[0346] Table 13. Biophysical characteristics of P-1076-mod anti-hNAMPT antibodies

[0347]

[0348]

[0349]

[0350]

[0351]

[0352]

[0353] In Table 10, mutated residues are in bold.

[0354]

[0355]

[0356] Table 14. Amino acid sequences of improved UU-1093-mod anti-hNAMPT antibodies

[0357]

[0358] Example 11. Modifications of humanized anti-hNAMPT antibody UU-1093

[0359] Based on the results of the in vitro and in vivo testing described in Example 9, the anti-hNAMPT antibody UU-1093 was selected for further modification to generate modified 1093 anti-hNAMPT antibodies.

[0360] UU-1093-mod humanized anti-hNAMPT antibodies

[0361] UU-1093-mod refers to humanized anti-hNAMPT antibodies generated by modification of the 1093 humanized anti-hNAMPT antibody UU-1093.

[0362] To generate improved UU-1093-mod anti-hNAMPT antibodies, one or more mutations were introduced into the UU-1093 amino acid sequence by de novo synthesis of the variable domains, or mutagenic oligonucleotide primers and polymerase chain reactions, or both, by methods well known in the art. These mutations were introduced to remove oxidation sites, reduce or remove deamidation, remove potential cleavage or fragmentation sites, remove potential T cell epitopes, and / or reduce binding of potential T cell epitopes. Different combinations of back mutations and other mutations were constructed for the heavy and / or light chains of UU-1093 to generate modified UU-1093-mod anti-hNAMPT antibodies UU-1093-modl, UU-1093-mod2, UU-1093-mod3, UU-1093-mod4, UU-1093-mod5, UU-1093-mod6, UU-1093-mod7, UU-1093-mod8, UU-1093-mod9, UU-1093-modlO, UU-1093-modl l, UU-1093-modl2, UU-1093-modl3, UU-1093-modl4, UU-1093-modl5, UU-1093-modl6, UU-1093-modl7, UU-1093-modl8, and UU-1093-modl9.

[0363] The amino acid sequences of the heavy and light chain variable regions of the modified P-1076-mod anti-hNAMPT antibodies are provided in Table 14. In Table 14, mutated residues are in bold. Table 15 provides an alignment of the amino acid sequences of the heavy chain CDRs of the modified UU-1093-mod anti-hNAMPT antibodies UU-1093-modl, UU-1093-mod2, UU-1093-mod3, UU-1093-mod4, UU-1093-mod5, UU-1093-mod6, UU-1093-mod7, UU-1093-mod8, UU-1093-mod9, UU-1093-modlO, UU-1093-modl l, UU-1093-modl2, UU-1093-modl3, UU-1093-modl4, UU-1093-modl5, UU-1093-modl6, UU-1093-modl7, UU-1093-modl8, and UU-1093-modl9 compared to UU-1093. Table 16 provides an alignment of the amino acid sequences of the light chain CDRs of the modified UU-1093-mod anti-hNAMPT antibodies UU-1093-modl, UU-1093-mod2, UU-1093-mod3, UU-1093-mod4, UU-1093-mod5, UU-1093-mod6, UU-1093-mod7, UU-1093-mod8, UU-1093-mod9, UU-1093-modlO, UU-1093-modl l, UU-1093-modl2, UU-1093-modl3, UU-1093-modl4, UU-1093-modl5, UU-1093-modl6, UU-1093-modl7, UU-1093-modl8, and UU-1093-modl9 compared to UU-1093. The blank spaces in Tables 15 and 16 indicate that the residue is the same as UU-1093.

[0364] UU-1093-modl

[0365] The UU-1093-mod anti-hNAMPT antibody UU-1093-modl has a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 38 and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 47. The heavy chain variable region of UU-1093-modl contains a CDR1 domain having an amino acid sequence as set forth in SEQ ID NO: 19, a CDR2 domain having an amino acid sequence as set forth in SEQ ID NO: 39, and a CDR3 domain having an amino acid sequence as set forth in SEQ ID NO: 21. For the heavy chain of UU-1093-modl, a D to E mutation was introduced into the UU-1093 VH CDR2 to remove a potential cleavage or fragmentation site. The light chain variable region of UU-1093-modl contains a CDR1 domain having an amino acid sequence as set forth in SEQ ID NO: 11, a CDR2 domain having an amino acid sequence as set forth in SEQ ID NO: 7, and a CDR3 domain having an amino acid sequence as set forth in SEQ ID NO: 22. For the light chain of UU-1093-modl, an N to Q mutation was introduced into the UU-1093 VL CDR1 to reduce or remove deamidation.

[0366] UU-1093-mod2

[0367] The UU-1093-mod anti-hNAMPT antibody UU-1093-mod2 has a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 40 and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 47. The heavy chain variable region of UU-1093-mod2 contains a CDR1 domain having an amino acid sequence as set forth in SEQ ID NO: 19, a CDR2 domain having an amino acid sequence as set forth in SEQ ID NO: 39, and a CDR3 domain having an amino acid sequence as set forth in SEQ ID NO: 41. For the heavy chain of UU-1093-mod2, a D to E mutation was introduced into the UU-1093 VH CDR2 to remove a potential cleavage or fragmentation site and an S to T mutation was introduced into the UU-1093 VH CDR3 to remove a potential T cell epitope. The light chain variable region of UU-1093-mod2 contains a CDR1 domain having an amino acid sequence as set forth in SEQ ID NO: 11, a CDR2 domain having an amino acid sequence as set forth in SEQ ID NO: 7, and a CDR3 domain having an amino acid sequence as set forth in SEQ ID NO: 22. For the light chain of UU-1093-mod2, an N to Q mutation was introduced into the UU-1093 VL CDR1 to reduce or remove deamidation.

[0368] UU-1093-mod3

[0369] The anti-hNAMPT antibody UU-1093-mod3 has a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 42, and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 47. The heavy chain variable region of UU-1093-mod3 contains a CDR1 domain having an amino acid sequence as set forth in SEQ ID NO: 43, a CDR2 domain having an amino acid sequence as set forth in SEQ ID NO: 39, and a CDR3 domain having an amino acid sequence as set forth in SEQ ID NO: 44. For the heavy chain of UU-1093-mod3, a M to I mutation was introduced into UU-1093 VH CDR1 to remove an oxidation site; a M to V mutation was introduced into UU-1093 VH framework region 2 to remove an oxidation site and potential T cell epitope; a D to E mutation was introduced into UU-1093 VH CDR2 to remove a potential cleavage or fragmentation site; a M to V mutation was introduced into UU-1093 VH framework region 3 to remove an oxidation site; and a K to R mutation was introduced into UU-1093 VH CDR3 to reduce binding of potential T cell epitopes. The light chain variable region of UU-1093-mod3 contains a CDR1 domain having an amino acid sequence as set forth in SEQ ID NO: 11, a CDR2 domain having an amino acid sequence as set forth in SEQ ID NO: 7, and a CDR3 domain having an amino acid sequence as set forth in SEQ ID NO: 22. For the light chain of UU-1093-mod3, a N to Q mutation was introduced into UU-1093 VL CDR1 to reduce or remove deamidation.

[0370] UU-1093-mod4

[0371] UU-1093-mod4 has a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO:45 and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO:47. The heavy chain variable region of UU-1093-mod4 contains a CDR1 domain having an amino acid sequence as set forth in SEQ ID NO:46, a CDR2 domain having an amino acid sequence as set forth in SEQ ID NO:39, and a CDR3 domain having an amino acid sequence as set forth in SEQ ID NO:41. For the heavy chain of UU-1093-mod4, a W to F mutation and a M to I mutation were introduced into UU-1093 VH CDR1 to remove an oxidation site; a M to V mutation was introduced into UU-1093 VH framework region 2 to remove an oxidation site and a potential T cell epitope; a D to E mutation was introduced into UU-1093 VH CDR2 to remove a potential cleavage or fragmentation site; a M to V mutation was introduced into UU-1093 VH framework region 3 to remove an oxidation site; and a S to T mutation was introduced into UU-1093 VH CDR3 to remove a potential T cell epitope. The light chain variable region of UU-1093-mod4 contains a CDR1 domain having an amino acid sequence as set forth in SEQ ID NO: 11, a CDR2 domain having an amino acid sequence as set forth in SEQ ID NO:7, and a CDR3 domain having an amino acid sequence as set forth in SEQ ID NO:22. For the light chain of UU-1093-mod4, a N to Q mutation was introduced into UU-1093 VL CDR1 to reduce or remove deamidation.

[0372] UU-1093-mod5

[0373] UU-1093-mod5 has a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 38 and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 48. The heavy chain variable region of UU-1093-mod5 contains a CDR1 domain having an amino acid sequence as set forth in SEQ ID NO: 19, a CDR2 domain having an amino acid sequence as set forth in SEQ ID NO: 39, and a CDR3 domain having an amino acid sequence as set forth in SEQ ID NO: 21. For the heavy chain of UU-1093-mod5, a D to E mutation was introduced into the UU-1093 VH CDR2 to remove a potential cleavage or fragmentation site. The light chain variable region of UU-1093-mod5 contains a CDR1 domain having an amino acid sequence as set forth in SEQ ID NO: 49, a CDR2 domain having an amino acid sequence as set forth in SEQ ID NO: 50, and a CDR3 domain having an amino acid sequence as set forth in SEQ ID NO: 22. For the light chain of UU-1093-mod5, a G to A mutation was introduced into the UU-1093 VL CDR1 to reduce or remove deamidation; an I to V mutation was introduced into the UU-1093 VL framework region 2 to reduce binding of potential T cell epitopes; an M to V mutation was introduced into the UU-1093 VL CDR2 to reduce binding of potential T cell epitopes and remove an oxidation site; and an I to V mutation was introduced into the UU-1093 VH framework region 3 to reduce binding of potential T cell epitopes.

[0374] UU-1093-mod6

[0375] UU-1093-mod6 has a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 40 and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 48. The heavy chain variable region of UU-1093-mod6 contains a CDR1 domain having an amino acid sequence as set forth in SEQ ID NO: 19, a CDR2 domain having an amino acid sequence as set forth in SEQ ID NO: 39, and a CDR3 domain having an amino acid sequence as set forth in SEQ ID NO: 41. For the heavy chain of UU-1093-mod6, a D to E mutation was introduced into UU-1093 VH CDR2 to remove a potential cleavage or fragmentation site, and a S to T mutation was introduced into UU-1093 VH CDR3 to remove a potential T cell epitope. The light chain variable region of UU-1093-mod6 contains a CDR1 domain having an amino acid sequence as set forth in SEQ ID NO: 49, a CDR2 domain having an amino acid sequence as set forth in SEQ ID NO: 50, and a CDR3 domain having an amino acid sequence as set forth in SEQ ID NO: 22. For the light chain of UU-1093-mod6, a G to A mutation was introduced into UU-1093 VL CDR1 to reduce or remove deamidation; a I to V mutation was introduced into UU-1093 VL framework region 2 to reduce binding of potential T cell epitopes; a M to V mutation was introduced into UU-1093 VL CDR2 to reduce binding of potential T cell epitopes and remove an oxidation site; and a I to V mutation was introduced into UU-1093 VH framework region 3 to reduce binding of potential T cell epitopes.

[0376] UU-1093-mod7

[0377] UU-1093-mod7 has a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 42, and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 48. The heavy chain variable region of UU-1093-mod7 contains a CDR1 domain having an amino acid sequence as set forth in SEQ ID NO: 43, a CDR2 domain having an amino acid sequence as set forth in SEQ ID NO: 39, and a CDR3 domain having an amino acid sequence as set forth in SEQ ID NO: 44. For the heavy chain of UU-1093-mod7, a M to I mutation was introduced into UU-1093 VH CDR1 to remove an oxidation site; a M to V mutation was introduced into UU-1093 VH framework region 2 to remove an oxidation site and potential T cell epitope; a D to E mutation was introduced into UU-1093 VH CDR2 to remove a potential cleavage or fragmentation site; a M to V mutation was introduced into UU-1093 VH framework region 3 to remove an oxidation site; and a K to R mutation was introduced into UU-1093 VH CDR3 to reduce binding of potential T cell epitopes. The light chain variable region of UU-1093-mod7 contains a CDR1 domain having an amino acid sequence as set forth in SEQ ID NO: 49, a CDR2 domain having an amino acid sequence as set forth in SEQ ID NO: 50, and a CDR3 domain having an amino acid sequence as set forth in SEQ ID NO: 22. For the light chain of UU-1093-mod7, a G to A mutation was introduced into UU-1093 VL CDR1 to reduce or remove deamidation; a I to V mutation was introduced into UU-1093 VL framework region 2 to reduce binding of potential T cell epitopes; a M to V mutation was introduced into UU-1093 VL CDR2 to reduce binding of potential T cell epitopes and remove an oxidation site; and a I to V mutation was introduced into UU-1093 VH framework region 3 to reduce binding of potential T cell epitopes.

[0378] UU-1093-mod8

[0379] UU-1093-mod8 has a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO:45 and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO:48. The heavy chain variable region of UU-1093-mod8 contains a CDR1 domain having an amino acid sequence as set forth in SEQ ID NO:46, a CDR2 domain having an amino acid sequence as set forth in SEQ ID NO:39, and a CDR3 domain having an amino acid sequence as set forth in SEQ ID NO:41. For the heavy chain of UU-1093-mod8, a W to F mutation and a M to I mutation were introduced into UU-1093 VH CDR1 to remove an oxidation site; a M to V mutation was introduced into UU-1093 VH framework region 2 to remove an oxidation site and a potential T cell epitope; a D to E mutation was introduced into UU-1093 VH CDR2 to remove a potential cleavage or fragmentation site; a M to V mutation was introduced into UU-1093 VH framework region 3 to remove an oxidation site; and a S to T mutation was introduced into UU-1093 VH CDR3 to remove a potential T cell epitope. The light chain variable region of UU-1093-mod8 contains a CDR1 domain having an amino acid sequence as set forth in SEQ ID NO:49, a CDR2 domain having an amino acid sequence as set forth in SEQ ID NO:50, and a CDR3 domain having an amino acid sequence as set forth in SEQ ID NO:22. For the light chain of UU-1093-mod8, a G to A mutation was introduced into UU-1093 VL CDR1 to reduce or remove deamidation; a I to V mutation was introduced into UU-1093 VL framework region 2 to reduce binding of potential T cell epitopes; a M to V mutation was introduced into UU-1093 VL CDR2 to reduce binding of potential T cell epitopes and remove an oxidation site; and a I to V mutation was introduced into UU-1093 VH framework region 3 to reduce binding of potential T cell epitopes.

[0380] UU-1093-mod9

[0381] UU-1093-mod9 has a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 38 and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 51. The heavy chain variable region of UU-1093-mod9 contains a CDR1 domain having an amino acid sequence as set forth in SEQ ID NO: 19, a CDR2 domain having an amino acid sequence as set forth in SEQ ID NO: 39, and a CDR3 domain having an amino acid sequence as set forth in SEQ ID NO: 21. For the heavy chain of UU-1093-mod9, a D to E mutation was introduced into the UU-1093 VH CDR2 to remove a potential cleavage or fragmentation site. The light chain variable region of UU-1093-mod9 contains a CDR1 domain having an amino acid sequence as set forth in SEQ ID NO: 11, a CDR2 domain having an amino acid sequence as set forth in SEQ ID NO: 52, and a CDR3 domain having an amino acid sequence as set forth in SEQ ID NO: 53. For the light chain of UU-1093-mod9, an N to Q mutation was introduced into the UU-1093 VL CDR1 to reduce or remove deamidation; an I to V mutation was introduced into the UU-1093 VL framework region 2 to remove a potential T cell epitope; an M to V mutation was introduced into the UU-1093 VL CDR2 to remove a potential T cell epitope and remove an oxidation site; an A to G mutation was introduced into the UU-1093 VL CDR2 to remove a potential T cell epitope; and a W to F mutation was introduced into the UU-1093 VL CDR3 to remove an oxidation site.

[0382] UU-1093-mod10

[0383] UU-1093-mod10 has a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 40 and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 51. The heavy chain variable region of UU-1093-Mod10 contains a CDR1 domain having an amino acid sequence as set forth in SEQ ID NO: 19, a CDR2 domain having an amino acid sequence as set forth in SEQ ID NO: 39, and a CDR3 domain having an amino acid sequence as set forth in SEQ ID NO: 41. For the heavy chain of UU-1093-mod10, a D to E mutation was introduced into UU-1093 VH CDR2 to remove a potential cleavage or fragmentation site, and a S to T mutation was introduced into UU-1093 VH CDR3 to remove a potential T cell epitope. The light chain variable region of UU-1093-Mod10 contains a CDR1 domain having an amino acid sequence as set forth in SEQ ID NO: 11, a CDR2 domain having an amino acid sequence as set forth in SEQ ID NO: 52, and a CDR3 domain having an amino acid sequence as set forth in SEQ ID NO: 53. For the light chain of UU-1093-mod10, a N to Q mutation was introduced into UU-1093 VL CDR1 to reduce or remove deamidation; a I to V mutation was introduced into UU-1093 VL framework region 2 to remove a potential T cell epitope; a M to V mutation was introduced into UU-1093 VL CDR2 to remove a potential T cell epitope and remove an oxidation site; a A to G mutation was introduced into UU-1093 VL CDR2 to remove a potential T cell epitope; and a W to F mutation was introduced into UU-1093 VL CDR3 to remove an oxidation site.

[0384] UU-1093-mod11

[0385] UU-1093-mod antibody UU-1093-mod11 has a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 42, and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 51. The heavy chain variable region of UU-1093-mod11 contains a CDR1 domain having an amino acid sequence as set forth in SEQ ID NO: 43, a CDR2 domain having an amino acid sequence as set forth in SEQ ID NO: 39, and a CDR3 domain having an amino acid sequence as set forth in SEQ ID NO: 44. For the heavy chain of UU-1093-mod11, a M to I mutation was introduced into UU-1093 VH CDR1 to remove an oxidation site; a M to V mutation was introduced into UU-1093 VH framework region 2 to remove an oxidation site and potential T cell epitope; a D to E mutation was introduced into UU-1093 VH CDR2 to remove a potential cleavage or fragmentation site; a M to V mutation was introduced into UU-1093 VH framework region 3 to remove an oxidation site; and a K to R mutation was introduced into UU-1093 VH CDR3 to reduce binding of potential T cell epitopes. The light chain variable region of UU-1093-mod11 contains a CDR1 domain having an amino acid sequence as set forth in SEQ ID NO: 11, a CDR2 domain having an amino acid sequence as set forth in SEQ ID NO: 52, and a CDR3 domain having an amino acid sequence as set forth in SEQ ID NO: 53. For the light chain of UU-1093-mod11, a N to Q mutation was introduced into UU-1093 VL CDR1 to reduce or remove deamidation; a I to V mutation was introduced into UU-1093 VL framework region 2 to remove a potential T cell epitope; a M to V mutation was introduced into UU-1093 VL CDR2 to remove a potential T cell epitope and remove an oxidation site; a A to G mutation was introduced into UU-1093 VL CDR2 to remove a potential T cell epitope; and a W to F mutation was introduced into UU-1093 VL CDR3 to remove an oxidation site.

[0386] UU-1093-mod12

[0387] UU-1093-mod12 has a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 45, and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 51. The heavy chain variable region of UU-1093-mod12 contains a CDR1 domain having an amino acid sequence as set forth in SEQ ID NO: 46, a CDR2 domain having an amino acid sequence as set forth in SEQ ID NO: 39, and a CDR3 domain having an amino acid sequence as set forth in SEQ ID NO: 41. For the heavy chain of UU-1093-mod12, a W to F mutation and a M to I mutation were introduced into UU-1093 VH CDR1 to remove an oxidation site; a M to V mutation was introduced into UU-1093 VH framework region 2 to remove an oxidation site and a potential T cell epitope; a D to E mutation was introduced into UU-1093 VH CDR2 to remove a potential cleavage or fragmentation site; a M to V mutation was introduced into UU-1093 VH framework region 3 to remove an oxidation site; and a S to T mutation was introduced into UU-1093 VH CDR3 to remove a potential T cell epitope. The light chain variable region of UU-1093-mod12 contains a CDR1 domain having an amino acid sequence as set forth in SEQ ID NO: 11, a CDR2 domain having an amino acid sequence as set forth in SEQ ID NO: 52, and a CDR3 domain having an amino acid sequence as set forth in SEQ ID NO: 53. For the light chain of UU-1093-mod12, a N to Q mutation was introduced into UU-1093 VL CDR1 to reduce or remove deamidation; a I to V mutation was introduced into UU-1093 VL framework region 2 to remove a potential T cell epitope; a M to V mutation was introduced into UU-1093 VL CDR2 to remove a potential T cell epitope and remove an oxidation site; a A to G mutation was introduced into UU-1093 VL CDR2 to remove a potential T cell epitope; and a W to F mutation was introduced into UU-1093 VL CDR3 to remove an oxidation site.

[0388] UU-1093-mod13

[0389] The UU-1093-mod 13 anti-hNAMPT antibody has a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 38 and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 18. The heavy chain variable region of UU-1093-mod 13 contains a CDR1 domain having an amino acid sequence as set forth in SEQ ID NO: 19, a CDR2 domain having an amino acid sequence as set forth in SEQ ID NO: 39, and a CDR3 domain having an amino acid sequence as set forth in SEQ ID NO: 21. For the heavy chain of UU-1093-mod 13, a D to E mutation was introduced into the UU-1093 VH CDR2 to remove a potential cleavage or fragmentation site. The light chain variable region of UU-1093-mod 13 contains a CDR1 domain having an amino acid sequence as set forth in SEQ ID NO: 6, a CDR2 domain having an amino acid sequence as set forth in SEQ ID NO: 7, and a CDR3 domain having an amino acid sequence as set forth in SEQ ID NO: 22. The light chain of UU-1093-mod 13 is the same as the UU-1093 VL.

[0390] UU-1093-mod 14

[0391] The UU-1093-mod 14 anti-hNAMPT antibody has a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 40 and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 18. The heavy chain variable region of UU-1093-Mod 14 contains a CDR1 domain having an amino acid sequence as set forth in SEQ ID NO: 19, a CDR2 domain having an amino acid sequence as set forth in SEQ ID NO: 39, and a CDR3 domain having an amino acid sequence as set forth in SEQ ID NO: 41. For the heavy chain of UU-1093-mod 14, a D to E mutation was introduced into the UU-1093 VH CDR2 to remove a potential cleavage or fragmentation site, and a S to T mutation was introduced into the UU-1093 VH CDR3 to remove a potential T cell epitope. The light chain variable region of UU-1093-mod 14 contains a CDR1 domain having an amino acid sequence as set forth in SEQ ID NO: 6, a CDR2 domain having an amino acid sequence as set forth in SEQ ID NO: 7, and a CDR3 domain having an amino acid sequence as set forth in SEQ ID NO: 22. The light chain of UU-1093-mod 14 is the same as the UU-1093 VL.

[0392] UU-1093-mod 15

[0393] UU-1093-mod15 has a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 42, and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 18. The heavy chain variable region of UU-1093-mod15 contains a CDR1 domain having an amino acid sequence as set forth in SEQ ID NO: 43, a CDR2 domain having an amino acid sequence as set forth in SEQ ID NO: 39, and a CDR3 domain having an amino acid sequence as set forth in SEQ ID NO: 44. For the heavy chain of UU-1093-mod15, a M to I mutation was introduced into UU-1093 VH CDR1 to remove an oxidation site; a M to V mutation was introduced into UU-1093 VH framework region 2 to remove an oxidation site and potential T cell epitope; a D to E mutation was introduced into UU-1093 VH CDR2 to remove a potential cleavage or fragmentation site; a M to V mutation was introduced into UU-1093 VH framework region 3 to remove an oxidation site; and a K to R mutation was introduced into UU-1093 VH CDR3 to reduce binding of potential T cell epitopes. The light chain variable region of UU-1093-mod15 contains a CDR1 domain having an amino acid sequence as set forth in SEQ ID NO: 6, a CDR2 domain having an amino acid sequence as set forth in SEQ ID NO: 7, and a CDR3 domain having an amino acid sequence as set forth in SEQ ID NO: 22. The light chain of UU-1093-mod15 is the same as UU-1093 VL.

[0394] UU-1093-mod16

[0395] UU-1093-mod16 has a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO:45 and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 18. The heavy chain variable region of UU-1093-Mod16 contains a CDR1 domain having an amino acid sequence as set forth in SEQ ID NO:46, a CDR2 domain having an amino acid sequence as set forth in SEQ ID NO:39, and a CDR3 domain having an amino acid sequence as set forth in SEQ ID NO:41. For the heavy chain of UU-1093-mod16, a W to F mutation and a M to I mutation were introduced into UU-1093 VH CDR1 to remove an oxidation site; a M to V mutation was introduced into UU-1093 VH framework region 2 to remove an oxidation site and a potential T cell epitope; a D to E mutation was introduced into UU-1093 VH CDR2 to remove a potential cleavage or fragmentation site; a M to V mutation was introduced into UU-1093 VH framework region 3 to remove an oxidation site; and a S to T mutation was introduced into UU-1093 VH CDR3 to remove a potential T cell epitope. The light chain variable region of UU-1093-Mod16 contains a CDR1 domain having an amino acid sequence as set forth in SEQ ID NO:6, a CDR2 domain having an amino acid sequence as set forth in SEQ ID NO:7, and a CDR3 domain having an amino acid sequence as set forth in SEQ ID NO:22. The light chain of UU-1093-mod16 is the same as UU-1093 VL.

[0396] UU-1093-mod17

[0397] The UU-1093-mod17 anti-hNAMPT antibody has a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 26, and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 47. The heavy chain variable region of UU-1093-mod17 contains a CDR1 domain having an amino acid sequence as set forth in SEQ ID NO: 19, a CDR2 domain having an amino acid sequence as set forth in SEQ ID NO: 20, and a CDR3 domain having an amino acid sequence as set forth in SEQ ID NO: 21. The heavy chain of UU-1093-mod17 is identical to UU-1093 VH. The light chain variable region of UU-1093-mod17 contains a CDR1 domain having an amino acid sequence as set forth in SEQ ID NO: 11, a CDR2 domain having an amino acid sequence as set forth in SEQ ID NO: 7, and a CDR3 domain having an amino acid sequence as set forth in SEQ ID NO: 22. For the light chain of UU-1093-mod17, a N to Q mutation was introduced into the UU-1093 VL CDR1 to reduce or eliminate deamidation.

[0398] UU-1093-mod18

[0399] UU-1093-mod18 has a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 26 and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 48. The heavy chain variable region of UU-1093-mod18 contains a CDR1 domain having an amino acid sequence as set forth in SEQ ID NO: 19, a CDR2 domain having an amino acid sequence as set forth in SEQ ID NO: 20, and a CDR3 domain having an amino acid sequence as set forth in SEQ ID NO: 21. The heavy chain of UU-1093-mod18 is identical to UU-1093 VH. The light chain variable region of UU-1093-mod18 contains a CDR1 domain having an amino acid sequence as set forth in SEQ ID NO: 49, a CDR2 domain having an amino acid sequence as set forth in SEQ ID NO: 50, and a CDR3 domain having an amino acid sequence as set forth in SEQ ID NO: 22. For the light chain of UU-1093-mod18, a G to A mutation was introduced into the UU-1093 VL CDR1 to reduce or remove deamidization; an I to V mutation was introduced into the UU-1093 VL framework region 2 to reduce binding of potential T cell epitopes; an M to V mutation was introduced into the UU-1093 VL CDR2 to reduce binding of potential T cell epitopes and remove an oxidation site; and an I to V mutation was introduced into the UU-1093 VH framework region 3 to reduce binding of potential T cell epitopes.

[0400] UU-1093-mod19

[0401] UU-1093-mod anti-hNAMPT antibody UU-1093-mod19 has a heavy chain variable region having an amino acid sequence set forth in SEQ ID NO: 26 and a light chain variable region having an amino acid sequence set forth in SEQ ID NO: 51. The heavy chain variable region of UU-1093-mod19 contains a CDR1 domain having an amino acid sequence set forth in SEQ ID NO: 19, a CDR2 domain having an amino acid sequence set forth in SEQ ID NO: 20, and a CDR3 domain having an amino acid sequence set forth in SEQ ID NO: 21. The heavy chain of UU-1093-mod17 is identical to UU-1093 VH. The light chain variable region of UU-1093-mod19 contains a CDR1 domain having an amino acid sequence set forth in SEQ ID NO: 11, a CDR2 domain having an amino acid sequence set forth in SEQ ID NO: 52, and a CDR3 domain having an amino acid sequence set forth in SEQ ID NO: 53. For the light chain of UU-1093-mod19, a N to Q mutation was introduced into the UU-1093 VL CDR1 to reduce or remove deamidation; an I to V mutation was introduced into the UU-1093 VL framework region 2 to remove a potential T cell epitope; an M to V mutation was introduced into the UU-1093 VL CDR2 to remove a potential T cell epitope and remove an oxidation site; an A to G mutation was introduced into the UU-1093 VL CDR2 to remove a potential T cell epitope; and a W to F mutation was introduced into the UU-1093 VL CDR3 to remove an oxidation site.

[0402] Figures 9A-9C

[0403]

[0404]

[0405]

[0406]

[0407]

[0408]

[0409]

[0410]

[0411]

[0412]

[0413]

[0414]

[0415] In Table 14, mutated residues are in bold.

[0416]

[0417]

[0418]

[0419] Example 12. NAMPT tissue expression in human aggressive PCa

[0420] To assess the role of NAMPT in prostate cancer (PCa) aggressiveness and progression, NAMPT expression was studied in PCa tissues.

[0421] NAMPT expression was assessed by immunohistochemistry (IHC) staining in normal prostate tissue, prostate adenocarcinoma confined to the prostate and without capsular invasion, and prostate adenocarcinoma with capsular invasion into the periprostatic adipose tissue. Representative micrographs are provided in Figure 9D Additionally, NAMPT expression was assessed by IHC staining in benign prostate tissue, tissues from PCa patients with organ-confined disease (T2 disease, n=12), and tissues from PCa patients with capsular invasion disease (T3 disease, n=14); cumulative analysis is provided in Figure 9A

[0422] As shown in Figure 9, IHC analysis of normal and PCa tissues showed essentially no NAMPT expression in normal prostate tissue Figure 9B ), and minimal expression of NAMPT in prostate adenocarcinoma confined to the prostate and without capsular invasion Figure 9C . In contrast, prostate adenocarcinoma with capsular invasion into the periprostatic adipose tissue showed a significant steady NAMPT staining Figure 9D . Moreover, comparative analysis of benign prostate tissue and tissues from 26 PCa patients with T2 and T3 disease showed an increase in NAMPT expression and an increase in PCa aggressiveness Figure 10A . Thus, an increase in NAMPT expression was observed in human aggressive PCa.

[0423] Example 13. Effect of radiation exposure on NAMPT expression

[0424] ​Radiation therapy is a cornerstone of PCA therapy. Since extracellular NAMPT (eNAMPT) functions as a damage-associated molecular pattern protein (DAMP) in innate immunity, the effect of radiation on NAMPT expression was evaluated by assessing NAMPT expression in radiation-exposed mouse and human tissues.

[0425] To assess the effects of radiation-induced tissue damage and impairment on NAMPT expression in mouse tissues, C57 / B6 mice were exposed to a single dose of thoracic radiation (20) for one week. The effects of radiation on inflammation, vascular leakage, and inflammatory lung injury were assessed by H&E staining of mouse lung tissues after radiation exposure. Representative micrographs are provided in [reference needed]. Figure 10B NAMPT expression in lung tissues before and after radiation was assessed by IHC staining. Representative micrographs are provided. Figure 10D and 10C To assess the effect of radiation on NAMPT expression in human tissues, normal human epithelial tissue (tonsils) was exposed to radiation (8 Gy) for 24 hours, and NAMPT expression was assessed by IHC staining. Representative micrographs are provided. Figure 10A middle.

[0426] As shown in Figure 10, increased inflammation, vascular leakage, and inflammatory damage were observed in mouse lung tissue one week after radiation exposure. Figure 10B Radiation-induced lung injury is accompanied by significant NAMPT expression. Figure 10C ), especially in alveolar macrophages and epithelial cells ( Figure 10D Consistent with radiation as a stimulus for NAMPT tissue expression in mice, a significant increase in NAMPT expression was observed in normal human epithelial tissue (tonsils) 24 hours after radiation exposure. ​ Therefore, radiation-induced tissue damage significantly induces NAMPT expression.

[0427] Example 14. The role of NAMPT in PCa cell migration

[0428] The effect of NAMPT on PCa cell migration was evaluated using an in vitro assay of human smooth muscle cell migration.

[0429] To evaluate the role of NAMPT in PCa cell migration, NAMPT (100 ng / ml) was added to a culture of human DU-145 PCa cells. PCa cell migration was assessed 24 hours after NAMPT exposure. An overview of the observations is provided below. Figure 11A In China, and representative photomicrographs are provided. Figure 11B middle.

[0430] As shown in Figure 11, NAMPT was used as an effective chemical attractant for human DU-145PCa cells and induced PCa cell migration within 24 hours.

[0431] Example 15. Characterization of humanized anti-hNAMPT antibody

[0432] Two mouse lung injury models were used: a “single-click” model of lung injury developed by intratracheal delivery of LPS to mice, and a “double-click” model of lung injury developed by exposing mice to LPS and mechanical VILI. The ability of 1076-humanized anti-hNAMPT antibodies (N-1076, K-1076, and P-1076) and 1093-humanized anti-hNAMPT antibodies (SS-1093, XX-1093, and UU-1093) to treat lung injury was tested in vivo. These mice were administered either of the humanized anti-hNAMPT antibodies to assess their ability to reduce acute inflammation and injury. The test results are presented in Figure 12.

[0433] like Figure 12A As shown in the figure, the analysis of the comprehensive lung injury score revealed that all tested anti-hNAMPT antibodies effectively reduced lung injury in the LPS-induced "click" model. However, the most significant effect was observed with the anti-hNAMPT antibody P-1076. Figure 12B As described above, the analysis of the comprehensive lung injury score showed that all tested anti-hNAMPT antibodies effectively reduced lung injury in the LPS / vILI-induced "double-click" model. However, the most significant effect was observed with the anti-hNAMPT antibody P-1076. Figure 12C As described above, the anti-hNAMPT antibody P-1076 effectively reduced the histological damage index in the "double-click" model of LPS / vILI-induced acute inflammatory injury.

[0434] Example 16. Effect of humanized anti-hNAMPT antibody P-1076 on PCa cell invasion

[0435] To evaluate the effect of humanized anti-hNAMPT antibody P-1076 on PCa cell invasion, peritoneal invasion of human PCa cells was assessed in severely combined immunodeficient (SCID) mice.

[0436] To evaluate the effect of humanized anti-hNAMPT antibody P-1076 on PCa cell invasion, metastatic human PCa cells (PC3) were intraperitoneally (ip) injected into SCID mice. Mice were injected twice weekly with 2 μg of humanized anti-hNAMPT antibody P-1076 or the medium alone. Peritoneal invasion of PC3 cells was assessed 6 weeks post-injection. Representative micrographs and a summary of results are provided in Figure 13.

[0437] As shown in Figure 13, injection of the human PCA cell line resulted in substantial peritoneal muscle invasion Figure 13A ), whereas SCID mice challenged with PC3 that received humanized anti-hNAMPT antibody P-1076 exhibited significantly reduced PC3 invasion of the smooth muscle peritoneum Figure 13B .

[0438] Thus, as summarized in Figure 13C , the observations from this study strongly suggest a role for NAMPT in PCa cell invasiveness and the critical potential of humanized anti-hNAMPT antibody P-1076 to delay this invasive behavior.

[0439] Example 17. In vivo treatment of RILI using anti-NAMPT antibodies

[0440] An in vivo experiment in C57 / B6 mice was used to assess the ability of anti-NAMPT administration to affect RILI. Mice were divided into four groups: mice that received 20 Gy thoracic radiation and intraperitoneal injection of polyclonal NAMPT neutralizing antibody (pAb); mice that received 20 Gy thoracic radiation and intraperitoneal injection of monoclonal anti-NAMPT antibody (mAb) (P-1076-modl); and unirradiated mice injected with vehicle alone ("Ctrl"); and irradiated mice injected with vehicle alone ("Ctrl"). The amount of BAL protein was measured and counts of BAL expressing cells were obtained. Lung tissue was also stained with H&E to assess lung inflammation. In addition, acute lung injury (ALI) severity score was assessed based on BAL indices and H&E staining. Results from the corresponding analyses are provided in Figure 14A -E.

[0441] As shown in Figure 14A , H&E staining of lung tissue from radiated control mice (injected with vehicle alone) showed diffuse alveolar damage 4 weeks after radiation exposure Figure 14A , left panel) compared to lung tissue from unirradiated control mice Figure 14A , left panel). In contrast, lung tissue from mice injected with either anti-NAMPT pAb Figure 14A , middle panel) or anti-NAMPT mAb Figure 14A , right panel) showed reduced H&E staining, indicating less alveolar damage in anti-NAMPT Ab treated mice after radiation exposure. Figure 14B H&E staining in lung tissue of unirradiated control mice, irradiated control mice, and irradiated mice injected with either anti-NAMPT pAb or mAb was summarized. As Figure 14BAs shown, the H&E staining area was increased in lung tissue from irradiated control mice compared to lung tissue from unirradiated control mice. However, a significant reduction in H&E staining area was observed in lung tissue from mice injected with anti-NAMPT pAb or mAb compared to irradiated control mice (p<0.05), indicating the role of NAMPT in the development of RILI. Figure 14C The levels of BAL protein in the lung tissues of unirradiated control mice, irradiated control mice, and irradiated mice injected with anti-NAMPT pAb or mAb are shown. Figure 14C As shown, mice exposed to radiation exhibited increased BAL protein levels compared to unirradiated control mice. However, irradiated mice injected with anti-NAMPT pAb or mAb showed significantly reduced BAL protein levels (p<0.05) compared to irradiated control mice, with a more significant reduction observed in irradiated mice treated with anti-NAMPT mAb. Similarly, BAL cell counts were increased in irradiated mice, although irradiated mice injected with anti-NAMPT pAb or mAb showed significantly reduced BAL cell counts compared to irradiated control mice (p<0.05), with a more significant reduction observed in irradiated mice treated with anti-NAMPT mAb. Furthermore, as... Figure 14E As shown, compared with control mice, mice exposed to radiation exhibited increased ALI severity scores; however, compared with irradiated control mice, irradiated mice injected with anti-NAMPT pAb or mAb showed significantly reduced ALI severity scores, with a more significant reduction observed in irradiated mice treated with anti-NAMPT mAb. Therefore, the results demonstrate a reduction in RILI following treatment with anti-NAMPT Ab, highlighting NAMPT as a potential therapeutic target for RILI.

[0442] Example 18. Radiolabeled anti-NAMPT antibody identifies increased NAMPT expression in inflamed lung tissue.

[0443] The development of radiolabeled anti-NAMPT antibodies aims to non-invasively detect NAMPT signaling pathways and NAMPT expression in different tissues in vivo. Imaging a mouse model of RILI using a radiolabeled anti-NAMPT mAb (P-1076-mod1) can define the optimal timing for deploying the anti-NAMPT mAb as a therapeutic intervention and, following total body irradiation (TBI) or partial body irradiation (PBI) (e.g., in nuclear events), investigate inflammation and apoptosis in major organs using other specific radiolabels. To test the detection of NAMPT expression via radiolabeled anti-NAMPT antibodies, [the following will be performed]. 99mTc-labeled anti-NAMPT mAb probes were injected into control mice and mice exposed to 8 Gy PBI and rapid autoradiographic imaging was performed. Results of the analysis are depicted in Figure 15A - D.

[0444] As shown in Figure 15A -B, higher radioactivity uptake was observed in the lungs of irradiated mice compared to non-irradiated control mice, indicating higher NAMPT expression induced by RILI. In addition, the uptake of radiolabeled anti-NAMPT antibody served as a measure of lung activity in irradiated mice or non-irradiated control mice. As shown in Figure 15C -B, a significant increase in lung activity relative to tissue background was observed in both the right and left lung from irradiated mice compared to those from non-irradiated control mice (p<0.05). In addition, the level of radioactivity in irradiated mice or non-irradiated control mice was determined to assess the uptake of radiolabeled anti-NAMPT mAb. As shown in Figure 15D -B, a significant increase in radioactivity was observed in irradiated mice compared to non-irradiated control mice (p<0.05), thus confirming the increased uptake of radiolabeled anti-NAMPT mAb in irradiated mice.

[0445] Thus, radiolabeled anti-NAMPT antibody is effective in detecting increased NAMPT expression in inflamed lung tissue. This underscores the potential of utilizing radiolabeled anti-NAMPT antibody as a tool to detect NAMPT, which can be critical in using NAMPT as a biomarker in RILI.

[0446] Example 19. Validation of NAMPT as a therapeutic target in RILI using an in vivo model of radiation-induced lung fibrosis.

[0447] To further validate NAMPT as a therapeutic target in RILI, WT C57 / B6 mice were exposed to 20 Gy WTLI. Irradiated mice were injected intraperitoneally with 10 μg of anti-NAMPT mAb (P-1076-modl) or vehicle control. At 18 weeks post-radiation exposure, the mice were assessed for radiation-induced lung fibrosis (RILF) by evaluating BAL cell counts, collagen deposition, and expression of smooth muscle actin (SMA) in lung tissue, which is reflective of myofibroblast transformation and fibrosis. Results are shown in Figure 16A -C.

[0448] As shown in Figure 16A -C, anti-NAMPT mAb significantly reduced IR-induced RILI, as evidenced by a decrease in BAL cell counts in Ab-treated mice compared to vehicle-treated control mice (p<0.05). In addition, anti-NAMPT mAb significantly reduced collagen deposition in lung tissue of IR-exposed mice compared to vehicle-treated control mice (p<0.05). Furthermore, anti-NAMPT mAb significantly reduced the expression of SMA in lung tissue of IR-exposed mice compared to vehicle-treated control mice (p<0.05). Figure 16A), reduced lung tissue SMA expression (as detected by Western blot analysis, as shown in Figure 16B and reduced collagen deposition (as detected by lung tissue trichrome staining, as shown in Figure 16C .

[0449] Accordingly, the results highlight the role of anti-NAMPT Ab in attenuating RILI, further validating NAMPT as a therapeutic target in RILI.

[0450] Example 20. Evaluation of anti-NAMPT mAb efficacy in preclinical model of lung injury

[0451] The efficacy of anti-NAMPT mAb was validated in a rat model of trauma (blast) / ventilator-induced lung injury (VILI). Sprague Dawley rats were challenged with trauma (blast) and then mechanically ventilated for 4 hours. Anti-NAMPT mAb (P-1076-modl, 100 μg, intravenously (IV)) was injected into the rats 30 minutes post-blast. Rats exposed to trauma (blast) / VILI and injected with vehicle were used as controls. Lungs were removed from the rats ~5 hours post-blast and evaluated for injury. In addition, lung tissue was evaluated for edema and inflammatory cell infiltration by hematoxylin and eosin (H&E) staining as a readout for lung injury. Figure 17A Results from this trauma (blast) / VILI lung injury model are provided in

[0452] As shown in Figure 17A , lung tissue from control trauma / VILI rats injected with vehicle showed inflammatory cell infiltration and edema, which are indicative of trauma / VILI-induced lung injury, as compared to non-challenged rats Figure 17A , the inset in the far right panel). In contrast, as shown in Figure 17B , lung tissue from trauma / VILI rats treated with anti-NAMPT mAb showed significantly reduced inflammatory cell infiltration and edema, thus indicating that anti-NAMPT mAb attenuated trauma / VILI-induced lung injury. The effect of anti-NAMPT mAb on trauma / VILI-induced lung injury is summarized in Figure 17C , which shows lung injury scores for the rats as assessed from H&E staining index. As shown in Figure 17C , lung injury scores for rats treated with anti-NAMPT mAb were significantly reduced (p<0.05) as compared to vehicle-injected control rats. Accordingly, Figure 17A Results outlined in

[0453] Next, the efficacy of anti-NAMPT mAbs was validated in a murine model of LPS / VILI. Mice were challenged with LPS for 18 hours, followed by mechanical ventilation for 4 hours. Mice were injected with anti-NAMPT mAbs (P-1076-modl, 10 μg, IV), anti-NAMPT polyclonal antibody (pAb), or vehicle control (PBS). Mice not exposed to LPS / VILI were used as controls. Lung tissue from mice was then evaluated for edema and inflammatory cell infiltration by H&E staining as a readout for lung injury. Results from this LPS / VILI lung injury model are provided in Figure 18A -C.

[0454] As shown in Figure 18A , lung tissue from control mice injected with vehicle showed inflammatory cell infiltration and edema, indicative of LPS / VILI-induced lung injury, compared to non-challenged mice Figure 18A , inset). In contrast, as shown in Figure 18B , lung tissue from anti-NAMPT mAb-treated mice showed a significant reduction in inflammatory cell infiltration and edema, thus indicating that anti-NAMPT mAbs reduced LPS / VILI-induced lung injury. The effect of anti-NAMPT mAbs on trauma / VILI-induced lung injury is summarized in Figure 18C , which shows the Acute Lung Injury (ALI) severity score in mice as assessed from the H&E staining index. As shown in Figure 18C , ALI was significantly reduced in mice treated with anti-NAMPT pAb or mAbs compared to mice injected with vehicle, with the strongest reduction in ALI severity score observed in mice treated with anti-NAMPT mAbs (p<0.001). Thus, Figure 18A , the results listed in -C show the efficacy of NAMPT neutralizing mAbs in reducing trauma / VILI-induced lung injury.

[0455] Thus, the results demonstrate the effectiveness of anti-NAMPT mAbs in reducing lung injury in a preclinical in vivo lung injury model.

[0456] Example 21. Radiolabeled anti-NAMPT antibodies identify increased NAMPT expression in inflamed lung tissue

[0457] Radiolabeled humanized anti-NAMPT mAb (K-1076) was developed as an imaging probe that can non-invasively detect NAMPT signaling pathway and NAMPT expression in different tissues in vivo. Given the potential of NAMPT as a diagnostic and / or prognostic biomarker for acute inflammatory conditions (e.g., COVID-19, ARDS, and lung injury), the radiolabeled anti-NAMPT mAb can be used as a diagnostic tool for subjects at risk of developing such conditions, or for selecting subjects who can respond to treatment of such inflammatory conditions with anti-NAMPT mAb. This example describes the use of radiolabeled anti-NAMPT mAb to detect NAMPT expression in inflamed tissues, such as lungs subjected to LPS challenge and ionizing radiation exposure.

[0458] First, to test the detection of NAMPT expression by radiolabeled anti-NAMPT antibody, a 99m Tc-labeled anti-NAMPT mAb probe or radiolabeled IgG control Ab was injected into mice exposed to 20 Gy whole thorax lung irradiation (WTLI) and fast radioautographic imaging was performed.

[0459] As shown in Figure 19A , significantly higher radioactivity uptake was observed in irradiated mice injected with radiolabeled anti-NAMPT mAb (PRONAMPTOR) ( Figure 19A , right panel) compared to irradiated mice injected with radiolabeled IgG control ( Figure 19A , left panel). Thus, the results shown in Figure 19A demonstrate the ability of radiolabeled anti-NAMPT imaging probe to detect radiation-induced NAMPT expression.

[0460] To further evaluate the detection of NAMPT expression by radiolabeled anti-NAMPT imaging probe, a 99m Tc-labeled anti-NAMPT mAb was injected into control mice or LPS-challenged mice at 3 hours or 18 hours post LPS challenge and fast radioautographic imaging was performed. The results of the analysis are shown in Figure 19B -D.

[0461] As shown in Figure 19B , LPS-challenged mice showed significantly higher uptake of radiolabeled anti-NAMPT imaging probe at 3 hours post LPS challenge ( Figure 19B , right panel) compared to control mice ( Figure 19B , left panel). Radioautographic imaging of lungs from LPS-challenged mice or control mice further confirmed this observation; the lungs from LPS-challenged mice showed significantly higher radioactivity uptake compared to control mice ( Figure 19C, left panel), the lungs of LPS-challenged mice showed significantly higher uptake of the radiolabeled anti-NAMPT imaging probe 3 hours post LPS challenge Figure 19C , right panel). In addition, as shown in Figure 19D , LPS-challenged mice showed significantly higher radioactivity (p<0.05) 3 hours and 18 hours post LPS challenge compared to control mice, indicating higher uptake of the radiolabeled anti-NAMPT imaging probe. Thus, Figure 19B The results described in Section -D demonstrate the ability of the radiolabeled anti-NAMPT imaging probe to detect LPS-induced NAMPT expression.

[0462] Accordingly, the radiolabeled anti-NAMPT antibody is effective in detecting increased NAMPT expression in inflamed tissue. This underscores the potential of utilizing the radiolabeled anti-NAMPT antibody as a tool to detect NAMPT, which can be critical in using NAMPT as a diagnostic and / or prognostic biomarker in acute inflammatory conditions. Furthermore, by detecting increased NAMPT expression in inflamed tissue, this radiolabeled anti-NAMPT imaging probe can be used to select subjects that can respond to treatment of acute inflammatory conditions with neutralizing anti-NAMPT mAbs.

[0463] Example 22. Anti-NAMPT antibody reduces PAH manifestation in a rat model

[0464] To explore the potential of NAMPT as a therapeutic target in PAH, a rat model of PAH using the monocrotaline (MCT) model was used. A dose of MCT (60 mg / kg body weight) was injected subcutaneously into Sprague-Dawley rats (190-200 g). The MCT-challenged rats were then injected twice a week with anti-NAMPT mAb (P-1076-modl, i.p., 100 μg / rat) or vehicle control (control MCT rats). The rats were then evaluated for right ventricular systolic pressure (RVS) and pulmonary artery remodeling. The results are shown in Figure 20A and 20B .

[0465] RVSP was determined in anti-NAMPT Ab-treated MCT rats or control MCT rats using Millar pressure sensor catheterization through right heart catheterization. As shown in Figure 20A , a significant reduction in RVSP was observed in MCT rats treated with anti-NAMPT mAb compared to control MCT rats (p<0.05).

[0466] After staining the lungs from anti-NAMPT Ab-treated MCT rats or control MCT rats with H&E, pulmonary artery remodeling was evaluated using APERIO IMAGESCOPE software. As shown inFigure 20B Significant reduction in pulmonary artery thickness was observed in anti-NAMPT mAb treated MCT rats compared to control MCT rats.

[0467] The results demonstrate that NAMPT neutralization by anti-NAMPT mAb reversed vascular remodeling and RV dysfunction in a PAH rat model, thus demonstrating the effectiveness of NAMPT as a therapeutic target in PAH.

[0468] The disclosures of each patent, patent application, and publication cited herein are hereby incorporated herein by reference to the extent that the cited references are consistent with the information disclosed herein. Although the present application has been disclosed with reference to particular means, materials and embodiments, it is to be understood that various other changes and modifications can be suggested to one skilled in the art, and that the present application extends to combinations, permutations, and sub-combinations of the disclosed features, and to adaptations and modifications of the application such as would be obvious to one skilled in the art. The scope of the application is defined by the appended claims and their equivalents.

[0469] The sequences disclosed herein and / or relevant to the present application are listed in the following sequence summary table (Table 17).

[0470] Table 17: Sequence Summary Table

[0471]

[0472]

[0473]

[0474]

[0475]

[0476]

[0477]

[0478]

[0479]

Claims

1. An isolated antibody, or an antigen-binding fragment thereof, which binds to human nicotinamide phosphoribosyltransferase (NAMPT), said antibody or antigen-binding fragment comprising: (i) a heavy chain variable region comprising a CDR1 domain consisting of the amino acid sequence shown in SEQ ID NO:3; a CDR2 domain consisting of the amino acid sequence shown in SEQ ID NO:4; and a CDR3 domain consisting of the amino acid sequence shown in SEQ ID NO:5; and (ii) A light chain variable region comprising a CDR1 domain consisting of an amino acid sequence as shown in SEQ ID NO:6, a CDR2 domain consisting of an amino acid sequence as shown in SEQ ID NO:7, and a CDR3 domain consisting of an amino acid sequence as shown in SEQ ID NO:

8.

2. An isolated antibody, or an antigen-binding fragment thereof, which binds to human NAMPT, said antibody or antigen-binding fragment comprising: The heavy chain variable region comprises a CDR1 domain consisting of the amino acid sequence shown in SEQ ID NO:3, a CDR2 domain consisting of the amino acid sequence shown in SEQ ID NO:4, and a CDR3 domain consisting of the amino acid sequence shown in SEQ ID NO:5; and the light chain variable region comprises a CDR1 domain consisting of the amino acid sequence shown in SEQ ID NO:11, a CDR2 domain consisting of the amino acid sequence shown in SEQ ID NO:14, and a CDR3 domain consisting of the amino acid sequence shown in SEQ ID NO:

8.

3. The isolated antibody, or antigen-binding fragment thereof, according to claim 1 or 2, wherein the antibody, or antigen-binding fragment thereof, is humanized.

4. The isolated antibody or its antigen-binding fragment according to claim 1 or 2, wherein the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 2, 13, 30 or 31.

5. The isolated antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 1, 9, 15 or 16.

6. An isolated antibody, or an antigen-binding fragment thereof, which binds to human nicotinamide phosphoribosyltransferase (NAMPT), said antibody or antigen-binding fragment comprising a heavy chain and a light chain, said heavy chain comprising a variable region consisting of an amino acid sequence as shown in SEQ ID NO:15, and said light chain comprising a variable region consisting of an amino acid sequence as shown in SEQ ID NO:

30.

7. An isolated anti-NAMPT antibody comprising a heavy chain variable region as shown in SEQ ID NO:54 and a light chain variable region as shown in SEQ ID NO:

55.

8. The isolated antibody or antigen-binding fragment thereof according to any one of claims 1, 2, 6 or 7, wherein the antibody or antigen-binding fragment thereof comprises an Fc domain.

9. The isolated antibody or antigen-binding fragment thereof according to any one of claims 1, 2, 6 or 7, wherein the antibody or antigen-binding fragment thereof is a monoclonal antibody.

10. The isolated antibody, or antigen-binding fragment thereof, according to any one of claims 1, 2, 6 or 7, wherein the antibody is an IgG antibody.

11. The isolated antibody or antigen-binding fragment thereof according to claim 10, wherein the antibody is an IgG1 or IgG4 antibody.

12. A nucleic acid encoding an isolated antibody or an antigen-binding fragment thereof as claimed in any one of claims 1-11.

13. A vector comprising the nucleic acid of claim 12.

14. A host cell comprising the nucleic acid of claim 12 or the vector of claim 13.

15. A pharmaceutical composition comprising an isolated antibody of any one of claims 1-11, or an antigen-binding fragment thereof, and a pharmaceutically acceptable carrier.

16. Use of the isolated antibody or antigen-binding fragment of any one of claims 1-11 in the preparation of a medicament for treating an inflammatory condition in a subject of need, wherein the inflammatory condition is pulmonary fibrosis (IPF), pulmonary hypertension, acute lung injury (ALI), acute respiratory distress syndrome (ARDS), traumatic brain injury, or radiation-induced lung injury.

17. The use according to claim 16, wherein the ALI is ventilator-induced lung injury (VILI) or traumatic acute lung injury (TIALI).

18. The use according to claim 16, wherein the inflammatory condition is ARDS or ALI.

19. The use according to claim 18, wherein the ALI is VILI.

20. The use according to claim 16, wherein the radiation-induced lung injury is caused by radiation associated with cancer treatment.

21. Use of the isolated antibody or antigen-binding fragment thereof of any one of claims 1-11 in the preparation of a medicament for treating prostate cancer (PCa) in a subject of need.

22. The use according to claim 21, wherein the subject suffers from recurrent PCa.

23. The use according to claim 21, wherein the subject is at risk of developing metastatic PCa.

24. The use according to any one of claims 21-23, wherein the PCa is resistant to androgen deprivation therapy (ADT).

25. The use according to any one of claims 21-23, further comprising a combination with ADT.

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