Anti-NAMPT antibodies and their uses

Abstract

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

Description

[Technical Field]

[0001] Federally sponsored research or development This invention was made with U.S. government support under grant numbers R41 HL110707 STTR and R42 HL152888 from the National Institutes of Health (NIH). The U.S. government has certain rights in this invention.

[0002] Related Applications This application claims priority to U.S. Provisional Application No. 62 / 883,952, filed August 7, 2019. The entire contents of the aforementioned priority application are incorporated herein by reference.

[0003] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy, created on July 1, 2020, is named A105818_1020WO_SL.txt and is 53767 bytes in size. [Background technology]

[0004] The nicotinamide phosphoribosyltransferase (NAMPT) gene encodes a protein that catalyzes the condensation of nicotinamide with 5-phosphoribosyl-1-diphosphate to produce nicotinamide mononucleotide. This 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.

[0005] Nicotinamide phosphoribosyltransferase (NAMPT) exists as both intracellular and extracellular NAMPT (eNAMPT) proteins. eNAMPT is secreted into the blood and functions as a cytokine / enzyme (cytozyme) that activates NF-κB signaling via ligation of Toll-like receptor 4 (TLR4), and further functions as a biomarker for inflammatory lung disorders such as acute respiratory distress syndrome. Summary of the Invention [Problem to be solved by the invention]

[0006] According to the present specification, anti-NAMPT antibodies, including humanized antibodies, are provided that are useful for therapeutic purposes.Currently known therapies for the clinical treatment of acute and chronic inflammatory lung disorders (e.g., ARDS, VILI) are merely supportive.Therefore, there is an unmet need in the art for effective preventive and therapeutic agents to reduce the morbidity and mortality observed in inflammatory disorders and in patients suffering from these disorders, especially in critically ill patients. [Means for solving the problem]

[0007] In a first aspect, the isolated antibody or antigen-binding fragment thereof of the present invention that specifically binds to human nicotinamide phosphoribosyltransferase (NAMPT) comprises: (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 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.

[0008] In some embodiments of the above aspects, 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.

[0009] In some embodiments of the above aspects, the light 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: 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; (b) 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: 12, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 8; (c) 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: 14, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 8; (d) 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: 33, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 8; (e) 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: 35, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: (f) 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: 37, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 8, (g) 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: 8, (h) 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, (i) 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, (j) 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,(k) 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: 35, and a CDR3 domain having the amino acid sequence shown in SEQ ID NO: 8, or (l) 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: 37, and a CDR3 domain having the amino acid sequence shown in SEQ ID NO: 8.

[0010] In another aspect, the isolated antibody or antigen-binding fragment thereof according to the present invention, which specifically binds to human NAMPT, comprises: (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 comprising 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; (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 comprising 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; (c) 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: 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 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 CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 11;(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 comprising 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; (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 comprising 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; 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 comprising 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 comprising 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; or (j) 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;a light chain variable region comprising 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.

[0011] In some embodiments of the above aspects, the isolated antibody or antigen-binding fragment thereof is humanized.

[0012] In some embodiments of any of the foregoing aspects, the isolated antibody or antigen-binding fragment thereof comprises a light chain variable region having the amino acid sequence 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 the amino acid sequence set forth in SEQ ID NO: 1, 9, 15, 16, or 28.

[0013] In another aspect, the invention features an isolated antibody or antigen-binding fragment thereof that specifically binds to human NAMPT, the isolated antibody or antigen-binding fragment thereof comprising a heavy chain comprising a variable region comprising the amino acid sequence set forth in SEQ ID NO: 15, and a light chain comprising a variable region comprising the amino acid sequence set forth in SEQ ID NO: 13.

[0014] In a different aspect, the isolated antibody or antigen-binding fragment thereof of the present invention that specifically binds to human NAMPT comprises a heavy chain comprising a variable region comprising the amino acid sequence set forth in SEQ ID NO: 26 and a light chain comprising a variable region comprising the amino acid sequence set forth in SEQ ID NO: 18.

[0015] In another aspect, the invention features an isolated antibody or antigen-binding fragment thereof that specifically binds to human NAMPT, the antibody or antigen-binding fragment thereof comprising a heavy chain comprising a variable region as described in Table 17, and a light chain comprising a variable region as described in Table 17.

[0016] In yet another aspect, the invention features an isolated antibody or antigen-binding fragment thereof that specifically binds to human NAMPT, the antibody or antigen-binding fragment thereof comprising a heavy chain comprising a heavy chain CDR1, CDR2, and CDR3 as described in Table 17, and a light chain comprising a light chain CDR1, CDR2, and CDR3 as described in Table 17.

[0017] In a different embodiment, the isolated anti-NAMPT antibody of the present invention comprises the heavy chain variable region and the light chain variable region of the antibody AL-303.

[0018] In another aspect, the invention features an isolated anti-NAMPT antibody, the isolated anti-NAMPT antibody comprising the heavy chain variable region and the light chain variable region of antibody AL-310.

[0019] In yet another aspect, the invention features an isolated humanized anti-NAMPT antibody, the isolated anti-NAMPT antibody comprising a humanized heavy chain variable region derived from murine antibody AL-303 or AL-310 and a humanized light chain variable region derived from murine antibody AL-303 or AL-310.

[0020] In a different aspect, the present invention provides an isolated anti-NAMPT antibody that specifically binds to human NAMPT in a homodimeric structure, comprising: (a) at least one amino acid selected from the group consisting of at least one amino acid selected from the group consisting of amino acid residues 17 to 44 of SEQ ID NO: 60, at least one amino acid selected from the group consisting of amino acid residues 117 to 127 of SEQ ID NO: 60, at least one amino acid selected from the group consisting of amino acid residues 162 to 170 of SEQ ID NO: 60, at least one amino acid selected from the group consisting of amino acid residues 242 to 261 of SEQ ID NO: 60, at least one amino acid selected from the group consisting of amino acid residues 262 to 273 of SEQ ID NO: 60, at least one amino acid selected from the group consisting of amino acid residues 289 to 305 of SEQ ID NO: 60, at least one amino acid selected from the group consisting of amino acid residues 332 to 342 of SEQ ID NO: 60, at least one amino acid selected from the group consisting of amino acid residues 374 to 389 of SEQ ID NO: 60, at least one amino acid selected from the group consisting of amino acid residues 418 to 425 of SEQ ID NO: 60, at least one amino acid selected from the group consisting of amino acid residues 453 to 466 of SEQ ID NO: 60, and at least one amino acid selected from the group consisting of amino acid residues 408 to 416 of SEQ ID NO: 60; The present invention features an isolated anti-NAMPT antibody that binds to an epitope on human NAMPT comprising either (a) at least one amino acid among amino acid residues 29 to 51 of SEQ ID NO: 60, at least one amino acid among amino acid residues 61 to 72 of SEQ ID NO: 60, at least one amino acid among amino acid residues 156 to 170 of SEQ ID NO: 60, at least one amino acid among amino acid residues 216 to 234 of SEQ ID NO: 60, at least one amino acid among amino acid residues 316 to 331 of SEQ ID NO: 60, at least one amino acid among amino acid residues 332 to 342 of SEQ ID NO: 60, at least one amino acid among amino acid residues 373 to 389 of SEQ ID NO: 60, at least one amino acid among amino acid residues 417 to 431 of SEQ ID NO: 60, at least one amino acid among amino acid residues 454 to 469 of SEQ ID NO: 60, and at least one amino acid among amino acid residues 470 to 478 of SEQ ID NO: 60.

[0021] In some embodiments of any of the above aspects, the antibody or antigen-binding fragment thereof comprises an Fc domain.

[0022] In some embodiments of any of the preceding aspects, the isolated antibody or antigen-binding fragment thereof is a monoclonal antibody.

[0023] In some embodiments of any of the foregoing 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 IgG1. In some embodiments, the isolated antibody or antigen-binding fragment thereof is an IgG4.

[0024] In some embodiments, the invention includes a nucleic acid encoding any of the isolated antibodies or antigen-binding fragments thereof featured herein, a vector comprising the nucleic acid, and / or a host cell comprising the nucleic acid or the vector.

[0025] In certain embodiments, the invention provides a pharmaceutical composition comprising any of the isolated antibodies or antigen-binding fragments thereof featured herein and a pharmaceutically acceptable carrier.

[0026] In some embodiments, the present invention includes a method of treating a disorder associated with deleterious 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 featured herein.

[0027] Also featured herein are methods of treating a subject having an inflammatory condition, comprising administering to the subject an effective amount of an isolated antibody or antigen-binding fragment described herein. In some embodiments, the inflammatory condition is 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), and brain injury or radiation-induced lung injury (e.g., radiation-induced lung injury caused by radiation associated with cancer therapy).

[0028] In some embodiments, the present invention features a method of treating prostate cancer (PCa) in a subject in need thereof, comprising administering to the subject an effective amount of an isolated antibody or antigen-binding fragment thereof described herein. 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 explanation of the drawings]

[0029] [Figure 1] 1 is a Western blot assay showing the detection of human NAMPT and mouse NAMPT by mouse anti-NAMPT antibodies AL-303, AL-304, AL-305, AL-309, and AL-310. [Figure 2] 1 is a Western blot assay showing 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. [Figure 3] FIG. 1 is a schematic diagram of the experimental protocol for an in vivo study to evaluate the effects of mouse anti-NAMPT antibodies AL-303, AL-304, and AL-305 on NAMPT-induced mouse lung injury. [Figure 4] Figure 4A shows the effect of mouse anti-NAMPT antibodies AL-303, AL-304, and AL-305 on NAMPT-induced mouse lung injury. Figure 4A shows the effect of mouse anti-NAMPT antibodies on NAMPT-induced bronchoalveolar lavage (BAL) protein levels. Figure 4B shows the effect of mouse anti-NAMPT antibodies on NAMPT-induced BAL-expressing polymorphonuclear neutrophil (PMN) counts (n = 3-6 mice, *P < 0.05). [Figure 5] FIG. 1 is a schematic diagram of the experimental protocol for an in vivo study to evaluate the effect of the murine anti-NAMPT antibody AL-310 on LPS-induced mouse lung injury. [Figure 6] 1 is a graph showing the effect of mouse anti-NAMPT antibody AL-310 on LPS-induced BAL protein levels. [Figures 7A-7B] Figure 7A is a graphical representation of the effect of humanized 1076 and 1093 anti-hNAMPT antibodies on hNAMPT-induced NFκB phosphorylation. Figure 7B is a graphical representation of the effect of humanized 1076 and 1093 anti-hNAMPT antibodies on hNAMPT-induced reduction in endothelial cell (EC) barrier integrity. [Figures 8A-8D]Figure 8 shows the effects of humanized 1076 and 1093 anti-hNAMPT antibodies on mouse and rat models of lung injury. Figure 8A is a graphical representation of the effects of 1076 anti-hNAMPT antibodies V-1076, N-1076, K-1076, and P-1076 on BAL protein levels (left panel of Figure 8A) and BAL PMN counts (right panel of Figure 8A) in a mouse model of lung injury. Figure 8B is a graphical representation of the effects of 1093 anti-hNAMPT antibodies SS-1093, CC-1093, XX-1093, and UU-1093 on BAL protein levels (left panel of Figure 8B) and BAL PMN counts (right panel of Figure 8B) in a mouse model of lung injury. Figure 8C is a graphical representation of the effect of the 1076 anti-hNAMPT antibody P-1076 on BAL protein levels (left panel of Figure 8C) and BAL PMN counts (right panel of Figure 8C) in a rat model of lung injury. Figure 8D shows H&E stained photomicrographs showing the effect of the 1093 anti-hNAMPT antibody UU-1093 on cellular infiltration and edema in a mouse model of lung injury. [Figures 9A-9D] Immunohistochemical (IHC) staining of NAMPT in normal, minimally invasive, and highly invasive prostate cancer (PCa) is shown. Figure 9A is a photomicrograph showing very low NAMPT expression in normal prostate tissue. Figure 9B is a photomicrograph showing modest but significantly increased NAMPT expression in gland-confined prostate adenocarcinoma. Figure 9C is a photomicrograph showing strong NAMPT expression within tumor cells in three separate prostate adenocarcinomas with penetration into the smooth muscle capsule and invasion into periprostatic adipose tissue. Figure 9D is a graph showing cumulative analysis of NAMPT expression in 26 PCa patients with organ-confined disease (n = 12) and capsular-invasive disease (n = 14). [Figures 10A-10D]Results from analysis of mouse and human tissues after acute and subacute radiation exposure are shown. Figure 10A provides photomicrographs of H&E staining showing evidence of inflammation and damage in mouse lung tissue one week after radiation exposure (lower panel of Figure 10A) and before exposure to a single dose of thoracic radiation (upper panel of Figure 10A). Figure 10B provides photomicrographs of IHC staining showing NAMPT expression in mouse lung tissue one week after radiation exposure (lower panel of Figure 10B) and before exposure to radiation (upper panel of Figure 10B). Figure 10C is a high-magnification image of the photomicrograph showing NAMPT expression in alveolar macrophages (long arrows) and pneumocytes (short arrows). Figure 10D shows photomicrographs showing NAMPT expression in normal, post-operative human tonsillar epithelial tissue that was exposed to 8 Gy of radiation for 24 hours ("irradiated") (lower panel of Figure 10D) or not exposed to radiation ("non-irradiated") (upper panel of Figure 10D). [Figures 11A-11B]

[0049] Figure 11A shows results from an in vitro assay of human DU-145 PCa cell migration through human smooth muscle cells. Figure 11A is a graph showing the number of invaded DU-145 PCa cells in the absence ("medium only") or presence ("NAMPT") of NAMPT, with medium without PCa cells ("No PCa cells") serving as a negative control. Figure 11B provides photomicrographs of wells with PCa cells cultured in the absence ("medium only") or presence ("NAMPT") of NAMPT, with wells without PCa cells ("No PCa cells") serving as a negative control. [Figures 12A-12C]Figure 12 shows results from in vivo testing 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) on inflammation and injury in a mouse lung injury model. Figure 12A is a graph showing the effect of humanized anti-hNAMPT antibodies on lung injury scores in an LPS-induced "one-hit" lung injury model. Figure 12B is a graph showing the effect of humanized anti-hNAMPT antibodies on lung injury scores in an LPS / VILI-induced "two-hit" lung injury model. Figure 12C provides photomicrographs showing histological indices of the effect of humanized anti-hNAMPT antibody P-1076 on lung injury in an LPS-induced "one-hit" lung injury model (upper panel of Figure 12C) and an LPS / VILI-induced "two-hit" lung injury model (lower panel of Figure 12C). [Figures 13A-13C] Figure 13 shows results from an in vivo assay of PCa cell invasion through diaphragm smooth muscle. Figure 13A is a photomicrograph showing severe peritoneal dissemination with invasion through the smooth muscle layer of a SCID mouse 6 weeks after intraperitoneal (IP) injection of highly metastatic human PCa cells, PC3; a magnified image of the photomicrograph is provided in the bottom panel of Figure 13A. Figure 13B is a photomicrograph showing inhibition of PC3 cell invasion in PC3-injected mice that received the humanized anti-hNAMPT antibody P-1076; a magnified image of the photomicrograph is provided in the bottom panel of Figure 13B. Figure 13C is a graph showing the percentage of PC3 cells invading the diaphragm of mice treated with the anti-hNAMPT antibody P-1076 or vehicle alone. [Figures 14A-14E]The effects of NAMPT neutralizing antibodies on lung inflammation (assessed by H&E staining), the amount of BAL protein, and the number of BAL-expressing cells, as assessed in a mouse model of RILI, are shown. Figure 14A provides representative photomicrographs showing H&E staining in lung tissue from vehicle control (left panel of Figure 14A), non-irradiated control mice (left panel of the inset in Figure 14A), or irradiated RILI mice injected with a vehicle control (left panel of Figure 14A), an anti-NAMPT polyclonal antibody (pAb) (middle panel of Figure 14A), or an anti-NAMPT monoclonal antibody (mAb) (right panel of Figure 14A) after radiation exposure. Figure 14B is a graphical representation of H&E staining (% area) in lung tissue from vehicle control, non-irradiated control mice, or irradiated RILI mice injected with an anti-NAMPT pAb or anti-NAMPT mAb. Figure 14C is a graphical representation of BAL protein levels (μg / ml) in the lung tissue of non-irradiated control mice or irradiated RILI mice injected with a vehicle control, anti-NAMPT pAb, or anti-NAMPT mAb. Figure 14D is a graphical representation of the number of BAL-expressing cells in the lung tissue of non-irradiated control mice or irradiated RILI mice injected with a vehicle control, anti-NAMPT pAb, or anti-NAMPT mAb. Figure 14E is a graphical representation of the ALI severity score of non-irradiated control mice or irradiated RILI mice injected with a vehicle control, anti-NAMPT pAb, or anti-NAMPT mAb. * indicates p<0.05. [Figures 15A-15D]Figure 15A shows the detection of NAMPT expression with a 99mTc-labeled anti-NAMPT mAb probe in a non-irradiated control mouse (left panel of Figure 15A) or an irradiated (RILI) mouse (PBI) exposed to 8 Gy partial body irradiation (right panel of Figure 15A). Figure 15B shows the detection of NAMPT expression with a 99mTc-labeled anti-NAMPT mAb probe in a non-irradiated control mouse (top panel of Figure 15B) or an irradiated (RILI) mouse (bottom panel of Figure 15B). Figure 15C shows a graphical representation of the ratio of lung activity to tissue background from the left and right lungs of a non-irradiated control mouse or an irradiated (RILI) mouse. Figure 15D shows a graphical representation of the radioactivity (%ID / g) in the lung tissue of a non-irradiated control mouse or an irradiated (RILI) mouse. * indicates p<0.05. [Figures 16A-16C] Figure 16 shows the effects of humanized anti-NAMPT mAb on BAL cell counts, collagen deposition, and lung tissue smooth muscle actin (SMA) expression, as assessed in a mouse model of RILI 18 weeks after 20 Gy radiation exposure. Figure 16A is a graph showing the number of BAL-expressing cells in the lung tissue of irradiated RILI mice intraperitoneally injected with anti-NAMPT mAb or vehicle control. Figure 16B provides representative images from Western blot analysis showing SMA expression in lung tissue homogenates of irradiated RILI mice intraperitoneally injected with anti-NAMPT mAb or vehicle control. Figure 16C provides representative photomicrographs showing collagen deposition detected by trichrome staining in the lung tissue of irradiated RILI mice intraperitoneally injected with anti-NAMPT mAb or vehicle control. * indicates p<0.05. [Figures 17A-17C]Figure 17 shows the effects of humanized anti-NAMPT mAb on inflammatory cell infiltration, edema, and lung injury score, as assessed in a rat model of trauma (blast injury) / ventilation-induced lung injury (VILI). Figure 17A provides representative images and photomicrographs showing lungs or lung tissue sections from trauma / VILI-challenged rats injected with vehicle control. The left panel of Figure 17A provides representative images of lungs from trauma / VILI-challenged rats injected with vehicle control. The middle and right panels of Figure 17A provide representative photomicrographs showing inflammatory cell infiltration and edema, as assessed by H&E staining, in trauma / VILI-challenged rats injected with vehicle control. The inset in the right panel of Figure 17A provides representative photomicrographs showing H&E staining in lung tissue from a rat not challenged with trauma / VILI. Figure 17B provides representative images and photomicrographs showing lungs or lung tissue sections from trauma / VILI-challenged rats injected with anti-NAMPT mAb. The left panel of Figure 17B provides representative images of lungs from trauma / VILI-challenged rats injected with anti-NAMPT mAb. The middle and right panels of Figure 17B provide representative photomicrographs showing inflammatory cell infiltration and edema, as assessed by H&E staining, in trauma / VILI-challenged rats injected with anti-NAMPT mAb. Figure 17C is a graph showing lung injury scores in trauma / VILI-challenged rats injected with either anti-NAMPT mAb or vehicle control. [Figures 18A-18C]Figure 18 shows the effects of NAMPT neutralizing antibodies on inflammatory cell infiltration, edema, and lung injury scores, as assessed in a rat model of murine LPS / VILI lung injury. Figure 18A provides representative photomicrographs showing inflammatory cell infiltration and edema, as assessed by H&E staining, in LPS / VILI-challenged mice injected with a vehicle control. The inset in Figure 18A provides representative photomicrographs showing H&E staining in lung tissue from mice not challenged with LPS / VILI. Figure 18B provides representative photomicrographs showing inflammatory cell infiltration and edema, as assessed by H&E staining, in LPS / VILI-challenged mice injected with an anti-NAMPT mAb. Figure 18C is a graph showing ALI severity scores, as assessed in LPS / VILI-challenged mice injected with an anti-NAMPT mAb, an anti-NAMPT pAb, or a vehicle control (PBS). The graph in Figure 18C also shows the ALI severity scores in control mice not challenged with LPS / VILI. * indicates p<0.05. *** indicates p<0.001. [Figures 19A-19D]Figure 19A shows the detection of NAMPT expression by a 99mTc-labeled anti-NAMPT mAb probe. Figure 19A provides representative autoradiographic images showing the detection of NAMPT expression by a 99mTc-labeled anti-NAMPT mAb probe (PRONAMPTOR) (right panel of Figure 19A) or a radiolabeled IgG control Ab (left panel of Figure 19A) in mice exposed to 20 Gy of whole lung irradiation (WTLI). Figure 19B provides representative autoradiographic images showing the detection of NAMPT expression by a 99mTc-labeled anti-NAMPT mAb probe in LPS-challenged mice (right panel of Figure 19B) or unchallenged control mice (left panel of Figure 19B) 3 hours after LPS challenge. Figure 19C provides representative autoradiographic images showing detection of NAMPT expression by a 99mTc-labeled anti-NAMPT mAb probe in the lungs of LPS-challenged mice (bottom panel of Figure 19C) or unchallenged control mice (top panel of Figure 18C) 3 hours after LPS challenge. Figure 19D is a graphical representation of the uptake of the radiolabeled anti-NAMPT mAb probe, as assessed by radioactivity (%ID / g), in the lung tissues of LPS-challenged or unchallenged control mice 3 and 18 hours after LPS challenge. * indicates p<0.05. [Figures 20A-20B] Figure 20 shows the effect of humanized anti-NAMPT mAb on right ventricular systolic pressure (RVSP) and pulmonary artery thickness as assessed in the rat monocrotaline (MCT) model of PAH. Figure 20A is a graphical representation of RVSP in MCT-challenged rats injected with either anti-NAMPT mAb or vehicle control (control MCT mice). Figure 20B provides representative photomicrographs showing pulmonary artery thickness as assessed by H&E staining in MCT-challenged rats injected with either anti-NAMPT mAb (right panel of Figure 20B) or vehicle control (left panel of Figure 20B). * indicates p<0.05. DETAILED DESCRIPTION OF THE INVENTION

[0030] The present invention can be embodied in many different forms. Disclosed herein are non-limiting exemplary embodiments of the present invention that illustrate the principles of the present invention. Any section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described. For purposes of this disclosure, all identifying sequence accession numbers can be found in the NCBI Reference Sequence (REFSEQ) database and / or the NCBI GenBank® archive sequence database, unless otherwise specified.

[0031] Various aspects of the present invention relate to anti-NAMPT antibodies and antibody fragments thereof and pharmaceutical compositions, as well as nucleic acids, recombinant expression vectors, and host cells for producing such antibodies and fragments. Also included in the present invention are methods of using the antibodies described herein to detect human NAMPT, inhibit human NAMPT activity (in vitro or in vivo), and treat NAMPT-related diseases, including, but not limited to, 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), and brain injury, radiation-induced lung injury, and cancer (e.g., prostate cancer (PCa)).

[0032] definition In order that the present invention may be more readily understood, certain terms are first defined. It should also be noted that whenever a value or range of values ​​for a parameter is listed, it is intended that values ​​and ranges intermediate to the listed values ​​are also part of the invention.

[0033] The terms "NAMPT" or "eNAMPT," used interchangeably herein, refer to the secreted form of nicotinamide phosphoribosyltransferase unless specifically stated to be related to a 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).

[0034] MNPAAEAEFN ILLATDSYKV THYKQYPPNT SKVYSYFECR EKKTENSKLR KVKYEETVFY GLQYILNKYL KGKVVTKEKI QEAKDVYKEH FQDDVFNEKG WNYILEKYDG HLPIEIKAVP EGFVIPRGNV LFTVENTDPE CYWLTNWIET ILVQSWYPIT VATNSREQKK ILAKYLLETS GNLDGLEYKL HDFGYRGVSS QETAGIGASA HLVNFKGTDT VAGLALIKKY YGTKDPVPGY SVPAAEHSTI TAWGKDHEKD AFEHIVTQFS SVPVSVVSDS YDIYNACEKI WGEDLRHLIV SRSTQAPLII RPDSGNPLDT VLKVLEILGK KFPVTENSKG YKLLPPYLRV IQGDGVDINT LQEIVEGMKQ KMWSIENIAF GSGGGLLQKL TRDLLNCSFK CSYVVTNGLG INVFKDPVAD PNKRSKKGRL SLHRTPAGNF VTLEEGKGDL EEYGQDLLHT VFKNGKVTKS YSFDEIRKNA QLNIELEAAHH (SEQ ID NO: 60) NAMPT is also called pre-B cell colony-enhancing factor (PBEF) or visfatin.

[0035] 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" to an antigen of interest, i.e., NAMPT, is one that can bind to the antigen with sufficient affinity so that the antibody is useful for targeting cells that express the antigen. In preferred embodiments, the antibody specifically binds to human NAMPT (hNAMPT), particularly extracellular human NAMPT (human eNAMPT). Examples of anti-eNAMPT antibodies are disclosed in the Examples and Sequence Listing provided below.

[0036] As used herein, "biological activity of NAMPT" refers to all inherent biological properties of NAMPT, including but not limited to binding to TLR4.

[0037] The terms "specifically bind" or "specifically binds to" and the like mean that an antibody or antigen-binding fragment thereof forms a complex with an antigen, e.g., NAMPT, that is relatively stable under physiological conditions. Specific binding is at least about 1 x 10 -8 can be characterized by an equilibrium dissociation constant less than or equal to M (e.g., K D (The smaller the , the stronger the 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.

[0038] The term "antibody" broadly refers to an immunoglobulin (Ig) molecule, generally composed of four polypeptide chains, two heavy (H) chains and two light (L) chains, or any functional fragment, mutant, variant, or derivative of those chains that retains the essential target-binding function of an Ig molecule. Such mutant, variant, or derivative antibody formats are known in the art. Non-limiting embodiments thereof are discussed below.

[0039] 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.

[0040] Both light and heavy chains are divided into regions of structural and functional homology. The terms "constant" and "variable" are used functionally. In this regard, it is understood that the variable domains of both the variable light (VL) and variable heavy (VH) chain portions determine antigen recognition and specificity. Conversely, the constant domains of the light (CL) and heavy chains (CH1, CH2, or CH3) confer biological properties such as secretion, transplacental transfer, Fc receptor binding, and complement binding. By convention, the numbering of constant region domains increases with increasing distance from the antigen-binding site or amino terminus of the antibody. The N-terminal portion is the variable region, the C-terminal portion is the constant region, and the CH3 (or CH4 in the case of IgM) and CL domains actually comprise the carboxy termini of the heavy and light chains, respectively.

[0041] 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 called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from the amino terminus to the 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., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or subclass. Light chains are classified as either kappa or lambda (κ, λ).

[0042] The "complementarity-determining regions" or "CDRs" present in the antigen-binding domain of an antibody are short, noncontiguous sequences of amino acids that are specifically positioned to form a binding domain when the antibody assumes its three-dimensional structure in an aqueous environment. The remaining amino acids in the binding domain of an antibody, called the "framework" or "FW" regions, exhibit lower intermolecular variability. The binding domain formed by the positioned CDRs defines a surface complementary to the epitope on the immunoreactive antigen. This complementary surface promotes noncovalent binding of the antibody to its cognate epitope. The amino acids that constitute the CDRs and framework regions, respectively, have been defined in a variety of different ways, as described below, and therefore can be easily identified by those skilled in the art for any given heavy or light chain variable region. Exemplary CDRs are provided herein. However, CDRs can also be defined according to Kabat, Chothia, Martin, PyIgClassify, or IMGT. Exemplary CDR definitions are provided in Chiu et al., "Antibody Structure and Function: The Basis for Engineering Therapeutics," Antibodies, 8(55):1-80 (2019), which is incorporated by reference in its entirety.

[0043] For the amino acid positions of the heavy chain constant regions discussed in this invention, numbering is done according to the EU index first described in Edelman et al., 1969, Proc. Natl. Acad. Sci. USA 63(1):78-85, which describes the amino acid sequence of the myeloma protein Eu, reported to be the first sequenced human IgG1. Edelman's EU index is also described in Kabat et al., 1991 (supra). Thus, the term "EU index as described in Kabat" or "Kabat's EU index" or "EU index" or "EU numbering" in the context of heavy chains refers to the residue numbering system based on the human IgG1 Eu antibody of Edelman et al., as described in Kabat et al., 1991 (supra). The numbering system used for the amino acid sequences of the light chain constant regions is similarly described in Kabat et al. (supra). An exemplary amino acid sequence of a kappa light chain constant region compatible with the present disclosure is set forth immediately below. RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 59) Similarly, exemplary IgG1 heavy chain constant region amino acid sequences compatible with the present invention are set forth immediately below. ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 58)

[0044] As used herein, the term "antigen-binding portion" or "binding fragment" of an antibody 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 may also be bispecific, bifunctional, or multispecific formats that specifically bind to two or more different antigens. Examples of binding fragments encompassed by the term "antigen-binding portion" of an antibody include: (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) an F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (v) a dAb fragment (Ward et al. (1989) Nature 341:544-546; PCT application WO 90 / 05144 A1 to Winter et al., which is incorporated herein by reference), which comprises 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 encoded by separate genes, they can be joined by a synthetic linker, which can be produced using recombinant techniques as a single protein chain in which the VL and VH regions pair to form a monovalent molecule (known as a 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 by the term "antigen-binding portion" of an antibody. In certain embodiments of the invention, scFv molecules may be incorporated into fusion proteins. Other forms of single-chain antibodies, such as diabodies, are also included.Diabodies are bivalent, bispecific antibodies in which a VH domain and a VL domain are expressed on a single polypeptide chain, but using a linker that is too short to allow the two domains to pair on the same chain, forcing the domains to pair with the complementary domains on another chain and creating two antigen-binding sites (see, e.g., Holliger, P. et al. (1993) Proc. Natl. Acad. Sci. USA). 90 (See Poljak, RJ et al. (1994) Structure 2:1121-1123.) Such antibody binding moieties are known in the art (Kontermann and Dubel, eds., Antibody Engineering (2001) Springer-Verlag New York, 790 pp (ISBN 3-540-41354-5)).

[0045] A "fully human" antibody comprises an antibody variable domain having a sequence derived from a human immunoglobulin (e.g., obtained from a human immunoglobulin coding sequence). The term "human antibody" includes, for example, antibodies having variable and constant regions (if present) derived from human germline immunoglobulin sequences. As used herein, the term "human" as applied to antibodies or fragments such as variable domains does not include antibodies from another species, e.g., a mouse, that have been "humanized" by grafting human constant region sequences onto an antibody polypeptide (i.e., replacing non-human constant regions with human constant regions) or by grafting human V region framework sequences from a non-human mammal onto an immunoglobulin variable domain (i.e., replacing non-human framework regions of a V domain with human framework regions). Methods for humanizing immunoglobulin variable regions through rational modification of complementarity-determining residues have been described (US2006 / 0258852).

[0046] The term "humanized antibody" refers to an antibody from a non-human species having one or more complementarity-determining regions (CDRs) from said non-human species and framework regions from a human immunoglobulin molecule. A humanized antibody may optionally further comprise one or more framework residues 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 web at www.mrccpe.com.ac.uk / vbase) and in Kabat, EA et al., 1991, Sequences of Proteins of Immunological Interest, 5th Edition. To avoid loss of activity during immunogenicity reduction, the variable region framework sequences of human antibodies undergo minimal backmutations to maintain activity.

[0047] The humanized antibody can be selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA, and IgE, and any isotype, including, but not limited to, IgG1, IgG2, IgG3, and IgG4. The humanized antibody may comprise sequences from multiple classes or isotypes, and particular constant domains may be selected to optimize desired effector functions using techniques well known in the art.

[0048] The term "multispecific" antibody refers to an antibody having binding domains for two or more different epitopes in a single antibody molecule. In addition to standard antibody structures, other binding molecules can be constructed with two binding specificities. 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.

[0049] As used herein, the term "labeled antibody" refers to an antibody or antigen-binding portion thereof incorporating a label that provides for the identification of the binding protein, e.g., antibody. Preferably, the label is the incorporation of a detectable marker, e.g., a radiolabeled amino acid, or the attachment of a biotinyl moiety to the polypeptide that can be detected by marked 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 radioisotopes or radionuclides (e.g., 3 H, 14 C. 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131 I, 177 Lu, 166 Ho or 153 Sm), fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase, luciferase, alkaline phosphatase), chemiluminescent markers, biotinyl groups, predetermined polypeptide epitopes recognized by secondary reporters (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags), and magnetic agents such as gadolinium chelates.

[0050] A "conservative amino acid substitution" is one in which one amino acid is replaced with another amino acid having a similar side chain. Families of amino acids with similar side chains have been defined in the art and include amino acids with 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, substituting phenylalanine for tyrosine is a conservative substitution. In certain embodiments, conservative substitutions in the sequences of the polypeptides and antibodies of the present disclosure do not abrogate binding of an antibody comprising the polypeptide or amino acid sequence to an antigen to which the binding molecule binds. Methods for identifying conservative nucleotide and amino acid 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 Burkset et al., Proc. Natl. Acad. Sci. USA 94:412-417 (1997)).

[0051] The term "polynucleotide" is intended to include single nucleic acids and multiple nucleic acids and refers to an isolated nucleic acid molecule or construct, e.g., messenger RNA (mRNA), cDNA, or plasmid DNA (pDNA). A polynucleotide can contain conventional phosphodiester bonds or non-conventional bonds (e.g., amide bonds as found in peptide nucleic acids (PNA)). The term "nucleic acid" or "nucleic acid sequence" refers to any one or more nucleic acid segments, e.g., DNA or RNA fragments, present in a polynucleotide.

[0052] By "isolated" nucleic acid or polynucleotide, we mean any form of nucleic acid or polynucleotide that has been separated from its native environment. For example, a gel-purified polynucleotide or a recombinant polynucleotide encoding a polypeptide contained in a vector is considered "isolated." Also, a polynucleotide segment engineered to contain restriction sites for cloning, e.g., a PCR product, is considered "isolated." Further examples of isolated polynucleotides include recombinant polynucleotides maintained in heterologous host cells or purified (partially or substantially) polynucleotides in a non-native solution, such as a buffer or saline solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of polynucleotides, where the transcript is not found in nature. Isolated polynucleotides or nucleic acids further include such molecules produced synthetically. Additionally, a polynucleotide or nucleic acid may be or include regulatory elements, such as a promoter, ribosome binding site, or transcription terminator.

[0053] As used herein, the term "expression" refers to the process by which a gene produces a biochemical, e.g., a polypeptide. This process includes any manifestation of the functional presence of a gene in a cell, including, but not limited to, gene knockdown and both transient and stable expression. This process includes, but is not limited to, transcription of a gene into messenger RNA (mRNA) and translation of such mRNA into a polypeptide. When the final desired product is a biochemical, 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 either a nucleic acid, e.g., a messenger RNA produced by transcription of a gene, or a polypeptide translated from a transcript. Gene products as described herein further include nucleic acids with post-transcriptional modifications, e.g., polyadenylation, or polypeptides with post-translational modifications, e.g., methylation, glycosylation, addition of lipids, conjugation to other protein subunits, proteolytic cleavage, etc.

[0054] As used herein, a "neutralizing antibody" (e.g., an "antibody that inhibits NAMPT activity") is intended to include an antibody whose binding to NAMPT inhibits the biological activity of NAMPT. A neutralizing antibody substantially inhibits the binding of NAMPT to its ligand or substrate if the excess antibody reduces the amount of binding partner bound 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. As will be understood, modified activity may be measured directly using art-recognized techniques or may be measured by the downstream effects of altered activity. This inhibition of the biological activity of NAMPT or its ligand can be assessed by measuring one or more indicators of NAMPT biological activity, such as the amount of extracellular NAMPT (either in vitro or in vivo), NAMPT-induced cellular activation (e.g., NFκB phosphorylation), and NAMPT binding to an NAMPT ligand. These indicators of NAMPT biological activity can be assessed by one or more 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 activation of endothelial cells. As an additional or alternative parameter of NAMPT activity, the ability of an antibody to inhibit NAMPT-induced transcriptional activity via NFκB can be assessed as a measure of NAMPT-induced cellular activation.

[0055] Terms such as "treat" or "treatment" or "to treat" or "alleviate" or "to alleviate" refer to therapeutic measures that cure, slow, or relieve symptoms and / or halt or slow the progression of an existing diagnosed pathological condition or disorder. Such treatment includes, but does not require, the complete elimination of all symptoms or curing the disease. Terms such as "prevent," "prevention," "avoid," and "inhibit" refer to prophylactic or preventative measures that prevent the onset of an undiagnosed targeted pathological condition or disorder. Thus, a "person in need of treatment" can include a person already suffering from a disorder, a person susceptible to a disorder, a person at risk of developing a disorder, or a person in whom a disorder is to be prevented.

[0056] "Subject" or "individual" or "animal" or "patient" or "mammal" means any subject for whom diagnosis, prognosis, or treatment is desired, particularly a mammalian subject. Mammalian subjects include humans, livestock, farm animals, or zoo, athletic, or pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, pigs, cows, bears, etc.

[0057] As used herein, phrases such as "subject benefiting from treatment" and "animal in need of treatment" include subjects, such as mammalian subjects, who would benefit from the administration of an anti-NAMPT antibody (e.g., a humanized anti-NAMPT antibody). Such antibodies can be used, for example, for diagnostic procedures and / or treatment or prevention of diseases, such as inflammatory lung disorders or cancer (e.g., prostate cancer).

[0058] The terms "effective amount" and "therapeutically effective amount" are used interchangeably herein and refer to an amount of a compound, formulation, material, or composition described herein that is effective to achieve a particular biological result. Such results may include, but are not limited to, inhibition of NAMPT expression or activity, or the expression or activity of signaling molecules downstream of NAMPT, as determined by any means suitable in the art. For example, NAMPT activity includes, but is not limited to, cytokine activity, nicotinamide phosphoribosyltransferase activity, chemotactic activity, NF-κB signaling activity, redox signaling activity, and / or its role in mitochondrial function and apoptosis. It should be emphasized that, even if a skilled artisan would consider such a dosage to be therapeutically effective, it may not always be effective in treating a disease. For convenience, exemplary dosages, drug delivery amounts, therapeutically effective amounts, and therapeutic levels are provided herein for adult human subjects. Those skilled in the art can adjust such amounts according to standard practices as needed to treat a particular subject and / or disease.

[0059] Anti-NAMPT antibody As provided herein, the present disclosure relates to anti-nicotinamide phosphoribosyltransferase (NAMPT) antibodies or antigen-binding fragments thereof, including compositions, methods, and articles of manufacture (e.g., kits, systems) comprising the antibodies or antigen-binding fragments thereof for prophylactic and therapeutic use in patients suffering from NAMPT-associated local and systemic inflammatory disorders. Nucleic acids comprising polynucleotide sequences encoding such antibodies are also described. In certain embodiments, the monoclonal antibodies provided herein bind to extracellular NAMPT (eNAMPT) and prevent activation of Toll-like receptor 4 (TLR4), thereby reducing or blocking one or more downstream signaling pathways and resulting systemic and pulmonary inflammation in certain respiratory disorders.

[0060] The NAMPT gene product is the rate-limiting enzyme of the mammalian NAD+ salvage pathway, which converts nicotinamide to nicotinamide mononucleotide, enabling nicotinamide adenine dinucleotide (NAD+) biosynthesis. The mature form of the extracellular NAMPT protein is a homodimer of approximately 120 kDa (Takahashi et al., J. Biochem. 147:95-107 (2010)). It has been established that mutations that reduce or inhibit the function of the NAMPT enzyme may impair pathophysiological processes that cause disorders such as leukemia and pulmonary arterial hypertension (PAH).

[0061] The human NAMPT gene (NAMPT) is located on chromosome 7 (segment 7q22.3, base pairs 106248285 to 106286326). The nucleic acid sequence of the human NAMPT gene product is known in the art. See, for example, NCBI Reference Sequence: NM_005746.2, Homo sapiens nicotinamide phosphoribosyltransferase (NAMPT), mRNA (see also 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, for example, GenBank Accession No. NP005737.1. NAMPT is a member of the CD14 + It has been shown to increase the production of IL-6, TNF-α, and IL-1β in monocytes, macrophages, and dendritic cells, improve T cell efficacy, and participate in the growth of both B and T lymphocytes (Sun et al., Cytokine & Growth Factor Reviews 24(5):433-442 (2013)). The crystal structure of the NAMPT enzyme is described in detail in Kim et al., J. Mol. Biol., 362:66-77 (2006).

[0062] The receptor for NAMPT is Toll-like receptor 4 (TLR4), a protein encoded by the TLR4 gene in humans. TLR4 is a transmembrane protein and a member of the Toll-like receptor family, a group of pattern recognition receptors (PRRs). Activation of this receptor leads to the intracellular NF-κB signaling pathway and the production of proinflammatory cytokines involved in the activation of the innate immune system. It is best known for recognizing lipopolysaccharide (LPS), a component present in many Gram-negative bacteria (e.g., Neisseria species), and for its selective activity against Gram-positive bacteria. Ligands for this receptor also include various endogenous proteins, such as several viral proteins, polysaccharides and low-density lipoproteins, 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, for example, 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, for example, GenBank Accession No. AAY82268.

[0063] In certain aspects, the present disclosure provides a humanized 1076 anti-hNAMPT antibody or antigen-binding fragment thereof. Examples of anti-NAMPT antibodies are shown below.

[0064] Regarding the anti-NAMPT antibody D-1076, D-1076 has (i) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 1, which includes a CDR1 domain having the amino acid sequence shown in SEQ ID NO: 3, a CDR2 domain having the amino acid sequence shown in SEQ ID NO: 4, and a CDR3 domain having the amino acid sequence shown in SEQ ID NO: 5, and (ii) a light chain variable region having the amino acid sequence shown in SEQ ID NO: 2, which includes 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.

[0065] Regarding the anti-NAMPT antibody G-1076, G-1076 has (i) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 9, which includes a CDR1 domain having the amino acid sequence shown in SEQ ID NO: 3, a CDR2 domain having the amino acid sequence shown in SEQ ID NO: 4, and a CDR3 domain having the amino acid sequence shown in SEQ ID NO: 5, and (ii) a light chain variable region having the amino acid sequence shown in SEQ ID NO: 10, which includes 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: 12, and a CDR3 domain having the amino acid sequence shown in SEQ ID NO: 8.

[0066] Regarding the anti-NAMPT antibody K-1076, K-1076 has (i) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 9, which includes a CDR1 domain having the amino acid sequence shown in SEQ ID NO: 3, a CDR2 domain having the amino acid sequence shown in SEQ ID NO: 4, and a CDR3 domain having the amino acid sequence shown in SEQ ID NO: 5, and (ii) a light chain variable region having the amino acid sequence shown in SEQ ID NO: 13, which includes 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.

[0067] Regarding the anti-NAMPT antibody N-1076, N-1076 has (i) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 15, which includes a CDR1 domain having the amino acid sequence shown in SEQ ID NO: 3, a CDR2 domain having the amino acid sequence shown in SEQ ID NO: 4, and a CDR3 domain having the amino acid sequence shown in SEQ ID NO: 5, and (ii) a light chain variable region having the amino acid sequence shown in SEQ ID NO: 2, which includes 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.

[0068] Regarding the anti-NAMPT antibody P-1076, P-1076 has (i) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 15, which includes a CDR1 domain having the amino acid sequence shown in SEQ ID NO: 3, a CDR2 domain having the amino acid sequence shown in SEQ ID NO: 4, and a CDR3 domain having the amino acid sequence shown in SEQ ID NO: 5, and (ii) a light chain variable region having the amino acid sequence shown in SEQ ID NO: 13, which includes 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.

[0069] Regarding the 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 includes 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 includes 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.

[0070] Regarding the anti-NAMPT antibody X-1076, X-1076 has (i) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 16, which includes a CDR1 domain having the amino acid sequence shown in SEQ ID NO: 3, a CDR2 domain having the amino acid sequence shown in SEQ ID NO: 4, and a CDR3 domain having the amino acid sequence shown in SEQ ID NO: 5, and (ii) a light chain variable region having the amino acid sequence shown in SEQ ID NO: 2, which includes 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.

[0071] Regarding the anti-NAMPT antibody P-1076-mod1, P-1076-mod1 has (i) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 28, which includes 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 includes 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.

[0072] Regarding the 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 includes 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: 31, which includes 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.

[0073] Regarding the 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 includes 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 includes 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.

[0074] Regarding the 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 includes 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 includes 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.

[0075] Regarding the 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 includes 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 includes 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.

[0076] Regarding the anti-NAMPT antibody P-1076-mod6, P-1076-mod6 has (i) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 15, which includes a CDR1 domain having the amino acid sequence shown in SEQ ID NO: 3, a CDR2 domain having the amino acid sequence shown in SEQ ID NO: 4, and a CDR3 domain having the amino acid sequence shown in SEQ ID NO: 5, and (ii) a light chain variable region having the amino acid sequence shown in SEQ ID NO: 30, which includes 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.

[0077] Regarding the anti-NAMPT antibody P-1076-mod7, P-1076-mod7 has (i) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 15, which includes a CDR1 domain having the amino acid sequence shown in SEQ ID NO: 3, a CDR2 domain having the amino acid sequence shown in SEQ ID NO: 4, and a CDR3 domain having the amino acid sequence shown in SEQ ID NO: 5, and (ii) a light chain variable region having the amino acid sequence shown in SEQ ID NO: 31, which includes 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.

[0078] Regarding the anti-NAMPT antibody P-1076-mod8, P-1076-mod8 has (i) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 15, which includes a CDR1 domain having the amino acid sequence shown in SEQ ID NO: 3, a CDR2 domain having the amino acid sequence shown in SEQ ID NO: 4, and a CDR3 domain having the amino acid sequence shown in SEQ ID NO: 5, and (ii) a light chain variable region having the amino acid sequence shown in SEQ ID NO: 32, which includes 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.

[0079] Regarding the anti-NAMPT antibody P-1076-mod9, P-1076-mod9 has (i) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 15, which includes a CDR1 domain having the amino acid sequence shown in SEQ ID NO: 3, a CDR2 domain having the amino acid sequence shown in SEQ ID NO: 4, and a CDR3 domain having the amino acid sequence shown in SEQ ID NO: 5, and (ii) a light chain variable region having the amino acid sequence shown in SEQ ID NO: 34, which includes 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: 35, and a CDR3 domain having the amino acid sequence shown in SEQ ID NO: 8.

[0080] Regarding the 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 includes 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 includes 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.

[0081] Regarding the anti-NAMPT antibody P-1076-mod11, P-1076-mod11 has (i) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 28, which includes a CDR1 domain having the amino acid sequence shown in SEQ ID NO: 3, a CDR2 domain having the amino acid sequence shown in SEQ ID NO: 29, and a CDR3 domain having the amino acid sequence shown in SEQ ID NO: 5, and (ii) a light chain variable region having the amino acid sequence shown in SEQ ID NO: 13, which includes 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.

[0082] In certain aspects, the present disclosure provides a humanized 1093 anti-hNAMPT antibody or antigen-binding fragment thereof. Examples of 1093 anti-NAMPT antibodies are shown below.

[0083] Regarding the 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 includes 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 includes 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.

[0084] Regarding the anti-NAMPT antibody II-1093, II-1093 has (i) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 23, which includes a CDR1 domain having the amino acid sequence shown in SEQ ID NO: 19, a CDR2 domain having the amino acid sequence shown in SEQ ID NO: 20, and a CDR3 domain having the amino acid sequence shown in SEQ ID NO: 21, and (ii) a light chain variable region having the amino acid sequence shown in SEQ ID NO: 24, which includes 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: 22.

[0085] Regarding the 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 includes 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 includes 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.

[0086] Regarding the 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 includes 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 includes 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.

[0087] Regarding the 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 includes 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 includes 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.

[0088] Regarding the anti-NAMPT antibody UU-1093, UU-1093 has (i) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 26, which includes a CDR1 domain having the amino acid sequence shown in SEQ ID NO: 19, a CDR2 domain having the amino acid sequence shown in SEQ ID NO: 20, and a CDR3 domain having the amino acid sequence shown in SEQ ID NO: 21, and (ii) a light chain variable region having the amino acid sequence shown in SEQ ID NO: 18, which includes 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: 22.

[0089] Regarding the 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 includes 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 includes 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.

[0090] Regarding the anti-NAMPT antibody ZZ-1093, ZZ-1093 has (i) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 27, which includes a CDR1 domain having the amino acid sequence shown in SEQ ID NO: 19, a CDR2 domain having the amino acid sequence shown in SEQ ID NO: 20, and a CDR3 domain having the amino acid sequence shown in SEQ ID NO: 21, and (ii) a light chain variable region having the amino acid sequence shown in SEQ ID NO: 18, which includes 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: 22.

[0091] Regarding the 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 includes 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 includes 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.

[0092] Regarding the 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 includes 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 includes 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.

[0093] Regarding the 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 includes 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 includes 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.

[0094] Regarding the 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 includes 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 includes 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.

[0095] Regarding the 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 includes 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 includes 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.

[0096] Regarding the 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 includes 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 includes 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.

[0097] Regarding the 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 includes 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 includes 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.

[0098] Regarding the 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 includes 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 includes 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.

[0099] Regarding the 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 includes 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 includes 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.

[0100] Regarding the 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 includes 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 includes 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.

[0101] Regarding the 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 includes 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 includes 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.

[0102] Regarding the 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 includes 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 includes 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.

[0103] Regarding the 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 includes 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 includes 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.

[0104] Regarding the 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 includes 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 includes 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.

[0105] Regarding the 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 includes 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 includes 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.

[0106] Regarding the 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 includes 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 includes 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.

[0107] Regarding the 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 includes 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 includes 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.

[0108] Regarding the 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 includes 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 includes 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.

[0109] Regarding the 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 includes 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 includes 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.

[0110] In some embodiments, the present invention comprises an anti-NAMPT antibody that binds to a discontinuous epitope on human NAMPT in the NAMPT homodimer structure. In some embodiments, the antibody or antigen-binding portion thereof binds to an epitope on human NAMPT that includes at least one amino acid selected from amino acid residues 17-44 of SEQ ID NO:60, at least one amino acid selected from amino acid residues 117-127 of SEQ ID NO:60, at least one amino acid selected from amino acid residues 162-170 of SEQ ID NO:60, at least one amino acid selected from amino acid residues 242-261 of SEQ ID NO:60, at least one amino acid selected from amino acid residues 262-273 of SEQ ID NO:60, at least one amino acid selected from amino acid residues 289-305 of SEQ ID NO:60, at least one amino acid selected from amino acid residues 332-342 of SEQ ID NO:60, at least one amino acid selected from amino acid residues 374-389 of SEQ ID NO:60, at least one amino acid selected from amino acid residues 418-425 of SEQ ID NO:60, at least one amino acid selected from amino acid residues 453-466 of SEQ ID NO:60, and / or at least one amino acid selected from 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 that includes at least one amino acid selected from amino acid residues 29-51 of SEQ ID NO:60, at least one amino acid selected from amino acid residues 61-72 of SEQ ID NO:60, at least one amino acid selected from amino acid residues 156-170 of SEQ ID NO:60, at least one amino acid selected from amino acid residues 216-234 of SEQ ID NO:60, at least one amino acid selected from amino acid residues 316-331 of SEQ ID NO:60, at least one amino acid selected from amino acid residues 332-342 of SEQ ID NO:60, at least one amino acid selected from amino acid residues 373-389 of SEQ ID NO:60, at least one amino acid selected from amino acid residues 417-431 of SEQ ID NO:60, at least one amino acid selected from amino acid residues 454-469 of SEQ ID NO:60, and / or at least one amino acid selected from amino acid residues 470-478 of SEQ ID NO:60.

[0111] In certain embodiments, the provided antibodies or fragments thereof have a binding affinity (K) of about 3 nM to about 20 nM for human NAMPT expressed on human cells, as measured by surface plasmon resonance. D ) can be included.

[0112] In certain embodiments, the antibodies or antigen-binding fragments thereof described herein are administered in a concentration of, for example, 5×10 -2 seconds -1 , 10 -2 seconds -1 , 5×10 -3 seconds -1 , 10 -3 seconds -1 , 5×10 -4 seconds -1 , 10 -4 seconds -1 , 5×10 -5 seconds -1 or 10 -5 seconds -1 , 5×10 -6 seconds -1 , 10 -6 seconds -1 , 5×10 -7 seconds -1 or 10 -7 seconds -1 The dissociation rate (k (off) ) and binds to NAMPT, for example, human NAMPT.

[0113] In certain embodiments, the antibodies or antigen-binding fragments thereof described herein are administered intracellularly, e.g., at 10 3 M -1 seconds -1 , 5×10 3 M -1 seconds -1 , 10 4 M -1 seconds -1 , 5×10 4 M -1 seconds -1 , 10 5 M -1 seconds -1 , 5×10 5 M -1 seconds -1 , 10 6 M -1seconds -1 or 5 x 10 6 M -1 seconds -1 or 10 7 M -1 seconds -1 The association rate (k (on) ) and binds to NAMPT, for example, human NAMPT.

[0114] In certain embodiments, the antibodies or antigen-binding fragments thereof described herein are administered in amounts of, for example, 5×10 -7 M, 10 -7 M, 5 x 10 -8 M, 10 -8 M, 5 x 10 -9 M, 10 -9 M, 5 x 10 -10 M, 10 -10 M, 5 x 10 -11 M, 10 -11 M, 5 x 10 -12 M, 10 -12 M or 5 x 10 -13 Dissociation constant or K D The binding affinity may be determined using various techniques known in the art, such as surface plasmon resonance (SPR), biolayer interferometry, dual polarization interferometry, static light scattering, dynamic light scattering, isothermal titration calorimetry, ELISA, analytical ultracentrifugation, and flow cytometry.

[0115] In certain embodiments, the anti-NAMPT antibodies or antigen-binding fragments thereof provided herein may further comprise a heterologous agent, such as a stabilizer, immune response modifier, or detectable agent. In certain embodiments, the heterologous agent comprises one or more additional polypeptide sequences fused to the polypeptide subunit via a peptide bond, such as a signal sequence (e.g., a secretory signal sequence), a linker sequence, an amino acid tag or label, or a peptide or polypeptide sequence that facilitates purification. In certain embodiments, the heterologous polypeptide can be fused to the N-terminus or C-terminus of either the heavy or light chain antibody subunit or a fragment thereof, as long as the functional characteristics of the domain are maintained.

[0116] In certain embodiments, heterologous agents can be chemically conjugated to the anti-NAMPT antibody or antigen-binding fragment thereof, as provided herein. Exemplary heterologous agents that can be chemically conjugated to a polypeptide subunit include, but are not limited to, linkers, drugs, toxins, imaging agents, radioactive compounds, organic and inorganic polymers, and any other composition that can provide a desired activity not provided by the polypeptide subunit itself. Specific agents include, but are not limited to, polyethylene glycol (PEG), cytotoxic agents, radionuclides, imaging agents, and biotin.

[0117] In some embodiments, the anti-NAMPT antibody or fragment is labeled with a radioactive label for use in in vitro or in vivo detection. Examples of radioisotopes that may be used to label the antibodies disclosed herein include At 211 , I 131 , I 125 , Y 90 ,Re 186 ,Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212 In some embodiments, the present invention includes an anti-NAMPT antibody described herein that is bound to a radioactive atom to form a radioconjugate. A variety of radioisotopes are available for producing radioconjugates. Examples include At 211 , I 131 , I 125 , Y 90 ,Re 186 ,Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212, radioactive isotopes of Lu, etc. When used for detection, the radioconjugates may contain radioactive atoms for scintigraphic studies, such as tc99m or I123, or spin labels for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), such as iodine-123 again, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.

[0118] In certain embodiments, any of the anti-NAMPT antibodies provided herein is useful for detecting the presence of NAMPT in a biological sample. As used herein, the term "detection" includes quantitative detection or qualitative detection. In certain embodiments, the biological sample includes cells or tissues such as cerebrospinal fluid, lung cells or tissues, or blood.

[0119] In some embodiments, an anti-NAMPT antibody is provided for use in a diagnostic or detection method. In a further aspect, a method for detecting the presence of NAMPT in a biological sample is provided. In certain embodiments, the method comprises contacting the biological sample with an anti-NAMPT antibody as described herein under conditions that allow binding of the anti-NAMPT antibody to NAMPT, and detecting whether a complex is formed between the anti-NAMPT antibody and NAMPT. Such a method may be an in vitro or in vivo method. Furthermore, the complex formed between the anti-NAMPT antibody and NAMPT in the test biological sample can be compared with the complex formed in a control biological sample (e.g., a biological sample from a healthy subject). The amount of complex formed between the anti-NAMPT antibody and NAMPT in the test biological sample can also be quantified and compared with the amount of complex formed in the control biological sample (e.g., a biological sample from a healthy subject) or the average amount of complex known to form in healthy subjects.

[0120] The anti-NAMPT antibodies and fragments disclosed herein can also be used as reagents for detecting human NAMPT, and in some embodiments, mouse NAMPT. For example, the anti-NAMPT antibodies described herein can be used in ELISA assays. Detection of the presence of NAMPT can be achieved in a number of ways using the antibodies and fragments disclosed herein, including 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 immunological assay techniques using such assay formats, which can include the antibodies disclosed herein, are available; see, for example, U.S. Patent Nos. 4,016,043, 4,424,279, and 4,018,653. These assays include both non-competitive single-site and two-site or "sandwich" assays, as well as traditional competitive binding assays. These assays also include direct binding of labeled anti-NAMPT antibodies to target biomarkers.

[0121] Sandwich assays are the most useful and commonly used assays. Many variations of the sandwich assay technique exist, and all are intended to be encompassed by the present invention. Briefly, in a typical forward assay, an unlabeled antibody is immobilized on a solid substrate, and the sample to be tested is contacted with the bound molecule. After incubation for a time sufficient to allow the formation of an antibody-antigen complex, a second antibody specific for the antigen, labeled with a reporter molecule capable of producing a detectable signal, is added and incubated for a time sufficient to form another antibody-antigen-labeled antibody complex. Unreacted material is washed away, and the presence of the antigen is determined by observing the signal produced by the reporter molecule. Results may be qualitative, by simply observing the visible signal, or quantitated by comparing with a control sample containing a known amount of biomarker.

[0122] For recombinant production of an anti-NAMPT antibody, for example, as described above, nucleic acid encoding the antibody is isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acid may be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the antibody heavy and light chains).

[0123] Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies may be produced in bacteria, particularly if glycosylation and Fc effector functions are not required. 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 (ed. BKC Lo, Humana Press, Totowa, NJ, 2003), pp. 245-254, which describes the expression of antibody fragments in E. coli.) Following expression, the antibody may be isolated from the bacterial cell paste in a soluble fraction and further purified.

[0124] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors, including fungal and yeast strains in which the glycosylation pathway has been "humanized" to result in the production of antibodies with partially or fully human glycosylation patterns. See Gerngross, Nat. Biotech. 22:1409-1414 (2004) and Li et al., Nat. Biotech. 24:210-215 (2006).

[0125] Suitable host cells for the expression of glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. Numerous baculovirus strains have been identified that can be used in conjunction with insect cells, particularly for transfection of fall armyworm cells.

[0126] Vertebrate cells may also be used as hosts, for example, mammalian cell lines adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines are the SV40-transformed monkey kidney CV1 line (COS-7), human embryonic kidney lines (e.g., 293 or 293 cells as described in Graham et al., J. GenVirol. 36:59 (1977)), baby hamster kidney cells (BHK), mouse Sertoli cells (e.g., TM4 cells as described 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 hepatocytes (BRL 3A), human lung cells (W138), human hepatocytes (Hep G2), mouse mammary tumor (MMT 060562), and other cells as described, for example, in Mather et al., Annals Examples of useful mammalian host cell lines include TRI cells, MRC5 cells, and FS4 cells, as described in NYAcad. Sci. 383:44-68 (1982). 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 specific mammalian host cell lines suitable for antibody production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (eds. BKC Lo, Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0127] Pharmaceutical compositions and therapeutic uses of anti-NAMTP antibodies Methods for treating inflammation-related conditions are also provided. Some embodiments involve dose-dependently reducing inflammation, injury, the amount of proteins in BAL fluid, and / or BAL PMNs via administration of an anti-NAMPT antibody or antigen-binding fragment thereof. Some embodiments involve reducing eNAMPT-induced lung injury, lipopolysaccharide-induced lung injury, and / or ventilator-induced lung injury. Diseases that can be treated with an anti-NAMPT antibody or a binding fragment thereof include, but are not limited to, inflammatory diseases 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 of preterm birth and maternal / neonatal complications), non-alcoholic steatohepatitis (NASH), liver fibrosis, cardiac ischemia, and cardiac fibrosis.

[0128] Also provided are compositions (e.g., pharmaceutical compositions) comprising an anti-NAMPT antibody or antigen-binding fragment thereof, and optionally further comprising one or more carriers, diluents, excipients, or other additives. Some embodiments include compositions (e.g., pharmaceutical compositions) comprising a polynucleotide or vector, and optionally further comprising one or more carriers, diluents, excipients, or other additives.

[0129] Also provided are methods for producing and administering anti-NAMPT antibodies or antigen-binding fragments thereof to a subject in need thereof to reduce and / or treat symptoms, morbidity, or mortality associated with NAMPT-related acute and chronic inflammatory disorders (e.g., ARDS, VILI, and trauma-induced inflammatory lung injury), for example, as is well known or easily determined by one of skill in the art. Anti-NAMPT antibodies or antigen-binding fragments thereof can be administered to a subject, for example, intravenously, intraarterially, intraperitoneally, intrapleurally, intratracheally, topically, subcutaneously, mucosally, intrapericardially, orally, locally, by inhalation, by injection, by infusion, by continuous infusion, by local perfusion bath directly into target cells, via a catheter, via aerosol, via a nebulizer, and / or via 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. In general, suitable pharmaceutical compositions may include, but are not limited to, buffers (e.g., acetate, phosphate, or citrate buffers), surfactants (e.g., polysorbates), stabilizers (e.g., human albumin), and the like.

[0130] Certain pharmaceutical compositions provided herein can be orally administered in acceptable dosage forms, including, for example, capsules, tablets, aqueous suspensions, or solutions. Certain pharmaceutical compositions can also be administered by nasal aerosol or inhalation. Such compositions can be prepared as solutions in saline using benzyl alcohol or other suitable preservatives, absorption enhancers to enhance bioavailability, and / or other conventional solubilizers or dispersions.

[0131] The amount of anti-NAMPT antibody or antigen-binding fragment thereof that can be combined with the carrier materials to produce a single dosage form varies depending on the subject being treated and the particular mode of administration. The composition can be administered as a single dose, multiple doses, or over an established period of time in an infusion. The dosing regimen can also be adjusted to provide the optimum desired response (e.g., a therapeutic or prophylactic response). As an example, the antibody can be administered / administered intravenously to a patient in an amount of about 0.1 mg / kg to about 20 mg / kg at a frequency of about weekly to monthly.

[0132] In some embodiments, the intended goal is to prevent or treat an inflammatory disease or disorder, meaning that an effective amount is an amount that is expected to achieve some prevention or treatment of the inflammatory disease or disorder. In some embodiments, the goal refers to preventing or alleviating symptoms and / or cellular processes associated with a particular disease or disorder, including, but not limited to, vasculitis, pulmonary edema, atrial fibrillation (ALI), or acute respiratory distress syndrome (ARDS). Such symptoms may be vascular permeability or elevated BAL protein secretion. An inflammatory disease or disorder refers to a disease or disorder characterized by inflammation. Inflammation can affect the following tissues or organs: heart, lung, kidney, 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, ovaries, testes, uterus, and umbilical cord. Additionally, the tissue may comprise one or more cell types, such as platelets, myelocytes, 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, marginal cells (from the cornea), stem cells (totipotent, pluripotent or multipotent), unfertilized or fertilized egg cells or sperm.

[0133] In some embodiments, one or more drugs may be administered to a patient, either simultaneously or subsequently. Exemplary drugs include azathioprine, bortezomib, carfilzomib, cyclophosphamide, dexamethasone, doxorubicin, lenalidomide, melphalan, pomalidomide, prednisolone, thalidomide, and vincristine. Exemplary drugs also include antibiotics. Exemplary drugs may also be administered or may be administered as part of a treatment plan.

[0134] The present disclosure further provides pharmaceutical packs and kits comprising one or more containers, which may contain one or more doses of an anti-NAMPT antibody or antigen-binding fragment, including compositions that can be used to practice the methods described herein. In certain embodiments, the kits comprise at least one purified anti-NAMPT antibody or antigen-binding fragment thereof. Those skilled in the art will readily recognize that the disclosed anti-NAMPT antibodies can be easily incorporated into one of the established kit formats well known to those skilled in the art.

[0135] In certain aspects, the present disclosure provides prophylactic and / or therapeutic methods for preventing, reducing, and / or reversing pathophysiological processes leading to the development and progression of NAMPT-associated acute and chronic inflammation 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, "NAMPT-associated acute and chronic inflammatory disorders or conditions" includes any inflammatory disease or biological process that results in inflammation involving NAMPT, e.g., increased expression and / or activity of NAMPT (e.g., eNAMPT), including downstream signaling molecules such as inflammatory cytokines. In certain embodiments, the composition comprises a neutralizing humanized anti-NAMPT antibody. In certain embodiments, the subject is a mammal, including, but not limited to, a human. The therapeutic methods and corresponding uses according to the teachings herein can improve the quality of life of a subject and / or extend the patient's lifespan by slowing, halting, or reversing one or more symptoms associated with the disorder.

[0136] In some embodiments, the anti-NAMPT antibodies or fragments thereof described herein are used to treat patients who have symptoms of or are at risk of developing an acute or chronic inflammatory disease or disorder (e.g., an inflammatory lung condition or disorder).

[0137] Subjects who should benefit from the methods provided by the present disclosure include critically ill subjects, critically ill subjects with respiratory failure, critically ill subjects exposed to infection, trauma and / or sepsis, subjects with radiation exposure, subjects diagnosed with pulmonary fibrosis (IPF), subjects with pulmonary hypertension, subjects suffering from acute respiratory distress syndrome (ARDS), subjects with ventilator-induced lung injury (VILI), and subjects with respiratory failure or at risk of ALI induced by VILI and / or ARDS / VILI. The present invention provides a method for treating a range of conditions, including, but not limited to, intensive care unit (ICU) subjects, 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 treatments associated with radiation-induced lung injury), pregnant subjects with chorioamnionitis (e.g., those at risk for preterm birth and maternal / neonatal complications, or complications in the preterm infant), and subjects with primary and metastatic cancer. Also provided are subjects at risk of developing any of the conditions disclosed herein.

[0138] In certain aspects, the present disclosure provides a method for treating a patient suffering from or at risk of an inflammatory lung disease or disorder, the method 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 administration may be based on the patient's symptoms, medical history, or the results of one or more tests. In some cases, the patient has already been diagnosed with a lung inflammatory disease or disorder when the patient is administered an anti-NAMPT antibody or antigen-binding fragment thereof (including the compositions and pharmaceutical compositions provided herein). In other cases, the patient has not been diagnosed with a lung inflammatory disease or disorder but is at risk for such a disease or disorder. Such patients include patients who are or will be placed on a ventilator, patients with pneumonia, patients who have experienced physical trauma, patients with severe bleeding, patients who have inhaled vomit, patients who have inhaled chemicals, patients who are heavy smokers, and / or patients who are heavy drinkers. For example, the patient may be placed on a ventilator or have been placed on a ventilator within 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).

[0139] In certain aspects, the present disclosure provides methods for treating a patient suffering from or at risk of acute lung injury (ALI), ventilator-induced lung injury (VILI), or acute respiratory distress syndrome (ARDS), the methods 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, pneumonia or symptoms of pneumonia, patients with severe bleeding due to a physical injury, patients with severe chest or head injuries, patients who have inhaled harmful fumes or smoke, patients who have inhaled vomit, patients who have received multiple or large amounts of blood transfusions, patients with fractures of long bones (such as the femur), patients who have near-drowned, patients who have had adverse reactions to anti-cancer drugs or other medications, patients who have overdosed on drugs, patients with pancreatitis, patients who are heavy smokers, patients who are heavy alcohol drinkers, patients with inflammatory bowel disease, patients with rheumatoid arthritis, patients with colon cancer, and patients with obesity-related insulin resistance, or any combination thereof.

[0140] In certain aspects, the present disclosure provides a method for preventing ventilator-induced lung injury (VILI) in a patient, the method comprising administering an effective amount of an anti-NAMPT antibody or antigen-binding fragment thereof (including the compositions and pharmaceutical compositions provided herein). In certain embodiments, the anti-NAMPT antibody comprises a neutralizing antibody. In certain embodiments, administration occurs before the patient is placed on a ventilator. In certain embodiments, administration occurs after the patient is placed on a ventilator.

[0141] In certain aspects, the present disclosure provides methods for reducing the levels of or decreasing one or more cytokines (e.g., IL-6, TNF-α, IL-1β, IL-8) in a subject at risk of suffering from or suffering from a NAMPT-associated acute and / or chronic inflammatory disorder.

[0142] In certain aspects, the present disclosure provides methods for reducing eNAMPT levels in a subject at risk of suffering from or suffering from an NAMPT-associated acute and / or chronic inflammatory disorder.

[0143] 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 certain aspects, the present disclosure provides a method for treating PCa in a subject by administering a therapeutically effective amount of an anti-NAMPT antibody or antigen-binding fragment thereof described herein to a subject in need thereof. In some embodiments, the subject with 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).

[0144] Alternatively, in some cases, the anti-NAMPT antibody or antigen-binding fragment can be administered to the subject in combination with ADT, which can be administered to the subject before, simultaneously with, or after the administration of the anti-NAMPT antibody or antigen-binding fragment.

[0145] The ADT that can be administered to a subject in combination with the anti-NAMPT antibody or antigen-binding fragment may include one or more of a luteinizing hormone-releasing hormone (LHRH) agonist, an LHRH antagonist, a CYP17 inhibitor, an antiandrogen, and / or an androgen suppressant. In certain embodiments, the LHRH agonist may be leuprorelin (e.g., LUPRON®, ELIGARD®, etc.), goserelin (e.g., ZOLADEX®), triptorelin (e.g., TRELSTAR®), and / or histrelin (e.g., VANTAS®), the LHRH antagonist may be degarelix (e.g., FIRMAGON®), and the CYP17 inhibitor may be abiraterone (e.g., ZYTIGA®). The antiandrogen may be flutamide (e.g., EULEXIN®), bicalutamide (e.g., CASODEX®), nilutamide (e.g., NILANDRON®), enzalutamide (e.g., XTANDI®), and / or apalutamide (e.g., ERLEADA®), and / or the androgen suppressant may be estrogen and / or ketoconazole (e.g., NIZORAL®).

[0146] Some embodiments involve treating coronavirus disease 2019 (COVID-19) with one or more NAMPT antibodies. COVID-19 is a severe acute respiratory syndrome caused by SARS-CoV-2. SARS-CoV-2 is 60-140 nm in diameter with characteristic spikes ranging from 9-12 nm, giving the virion a solar corona appearance. Coronaviruses can adapt and infect new hosts through genetic recombination and mutation. SARS-CoV-2 infection can be asymptomatic or 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 consequences include disseminated intravascular coagulation, inflamed lung tissue and pulmonary endothelial cells, deep vein thrombosis, pulmonary embolism, thrombotic arterial complications (e.g., limb ischemia, ischemic stroke, myocardial infarction), sepsis, and multiple organ failure. SARS-CoV-2 infection is described in 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), which is incorporated herein by reference in its entirety. In some embodiments, the present disclosure provides methods for treating a subject having COVID-19 (e.g., a subject diagnosed with COVID-19 and / or a subject exhibiting 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. [Example]

[0147] The following examples are included for illustrative purposes only and are not intended to be limiting.

[0148] Example 1. Generation of anti-human NAMPT monoclonal antibodies Neutralizing anti-NAMPT mouse antibodies were developed by immunizing three mice with recombinant extracellular human NAMPT (hNAMPT, MBL International). Mouse 4C6 and mouse 589 jointly generated 52 parental clone anti-NAMPT antibodies. Binding of the mouse antibodies to hNAMPT was assessed by ELISA. The mouse antibodies were also tested for in vitro neutralization by analyzing their effect on hNAMPT-induced NFκB phosphorylation. Anti-hNAMPT antibodies AL-303 and AL-310 were selected because of their ability to bind to recombinant hNAMPT and hNAMPT in lysates from human pulmonary artery endothelial cells (HPAEC). Both AL-303 and AL-310 were able to inhibit hNAMPT-induced NFκB phosphorylation. AL-303, but not AL-310, was also able to cross-react with recombinant mouse NAMPT (mNAMPT). These antibodies were further subjected to in vivo testing, as described in Examples 4 and 5 below.

[0149] AL-303 The mouse anti-hNAMPT antibody AL-303 has a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 54 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 55. The heavy chain variable region of AL-303 comprises 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. The light chain variable region of AL-303 comprises 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.

[0150] AL-310 The murine anti-NAMPT antibody AL-310 has a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 56 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 57. The heavy chain variable region of AL-310 comprises 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. The light chain variable region of AL-310 comprises 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.

[0151] The amino acid sequences of the heavy and light chain variable regions and CDRs of AL-303 and AL-310 are shown in Table 1.

[0152] [Table 1] [Table 2]

[0153] Example 2. Detection of human NAMPT and mouse NAMPT using mouse anti-NAMPT antibody To evaluate the immunoreactivity of mouse anti-NAMPT antibodies AL-303 and AL-310 with human and mouse NAMPT, we tested their detection of hNAMPT and mNAMPT by Western blot analysis. The immunoreactivity of AL-303 and AL-310 with 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 with 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 mouse lungs exposed to LPS, and mNAMPT in lysates from a mouse ventilator-induced lung injury (VILI) model.

[0154] The corresponding results of Western blot analysis are provided in Figure 1. As shown, AL-303 and AL-310 strongly immunoreacted with hNAMPT (compared to the other antibodies). In comparison, the immunoreactivity of these antibodies with mNAMPT was substantially weaker. Of the five murine antibodies tested, only AL-303 and AL-304 (which contain the same sequence as AL-303) immunoreacted with recombinant mNAMPT, whereas all antibodies reacted with recombinant hNAMPT and hNAMPT in HPAEC cells.

[0155] Example 3. Effect of mouse anti-NAMPT antibody on hNAMPT-induced NFκB phosphorylation The effects of mouse anti-NAMPT antibodies AL-303, AL-304, AL-305, AL-309, and AL-310 on hNAMPT-induced NFκB phosphorylation were assessed by Western blot analysis of phospho-NFκB (p-NFκB) expression in cells exposed to hNAMPT in the absence or presence of these antibodies.

[0156] Recombinant hNAMPT (1 μg / ml) was premixed with vehicle, 100 μg / ml anti-NAMPT polyclonal antibody (pAb), or 100 μg / ml mouse anti-NAMPT antibody (AL-303, AL-304, AL-305, AL-309, or AL-310) for 30 minutes. HPAEC cells were stimulated by exposure to the hNAMPT mixture for 1 hour. Unstimulated cells ("Unstim") exposed to vehicle, 100 μg / ml anti-NAMPT pAb, or 100 μg / ml mouse anti-NAMPT antibody (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 positive controls. Expression of p-NFκB in lysates from unstimulated, hNAMPT-stimulated, and TNF-α-stimulated cells was assessed by Western blot analysis using a p-NFκB-specific antibody. The results of the Western blot analysis are provided in Figure 2.

[0157] As shown in Figure 2, hNAMPT induced NFκB phosphorylation in hNAMPT-stimulated cells, which was substantially attenuated in the presence of AL-303, AL-304, AL-305, AL-309, or AL-310. All five antibodies effectively reduced hNAMPT-induced NFκB phosphorylation, but the most substantial effect was observed with AL-303 and AL-310. AL-304, AL-304, AL-305, and AL-309 were anti-hNAMPT antibodies also obtained in the initial mouse screening.

[0158] Example 4. In vivo testing of AL-303, AL-304, and AL-305 in a lung injury model The ability of murine anti-NAMPT antibodies AL-303, AL-304, and AL-305 to treat lung injury was tested in vivo using a mouse model.

[0159] As shown in Figure 3, C57 / B6 mice were intravenously injected with 50 μg, 100 μg, or 200 μg of mouse anti-NAMPT antibodies, AL-303, AL-304, or AL-305, or vehicle alone. One hour after anti-NAMPT antibody injection, mice were challenged with 40 μg / ml intratracheal hNAMPT (Peprotech). Eight hours after hNAMPT challenge, anti-NAMPT antibodies were administered again at the same concentration. Control mice were either injected with vehicle and challenged with intratracheal hNAMPT, or injected with vehicle and not challenged with intratracheal hNAMPT. Twenty-four hours after hNAMPT challenge, all animals were sacrificed, and lung injury was assessed by analyzing bronchoalveolar lavage (BAL) protein expression and the number of BAL-expressing polymorphonuclear neutrophils (PMNs).

[0160] The results of the evaluation are presented in Figure 4. As shown in Figure 4, mice exposed to intratracheal hNAMPT showed an increase in BAL protein expression (Figure 4A) and the number of BAL-expressing PMNs (Figure 4B). The mouse anti-NAMPT antibodies AL-303, AL-304, and AL-305 effectively reduced the hNAMPT-mediated increase in BAL PMN numbers, and the hNAMPT-induced increase in BAL protein levels was significantly attenuated by AL-303. Thus, as shown in Figure 4, AL-303, AL-304, and AL-305 all effectively reduced hNAMPT-induced mouse lung injury, but the most substantial effect was observed with AL-303. Therefore, AL-303 was selected for subsequent humanization.

[0161] Example 5. In vivo testing of AL-310 in lung injury The ability of the murine anti-NAMPT antibody AL-310 to treat lung injury was tested in vivo using a mouse model.

[0162] As shown in Figure 5, C57 / B6 mice were intravenously injected with 10 μg, 25 μg, 50 μg, 100 μg, or 200 μg of the mouse anti-NAMPT antibody AL-310, 100 μg of anti-NAMPT pAb, or vehicle alone. Simultaneously with antibody injection, mice were exposed to 1 mg / kg of intratracheal LPS. Mice exposed to intratracheal LPS and injected with vehicle or pAb served as positive controls. Mice injected with vehicle but not exposed to intratracheal LPS served as negative controls. Six hours after LPS exposure, all animals were sacrificed, and lung injury was assessed by analyzing BAL protein levels.

[0163] The results of the evaluation are provided in Figure 6. As shown in Figure 6, BAL protein expression was induced in mice exposed to intratracheal LPS. The mouse anti-NAMPT antibody AL-310 effectively reduced the LPS-mediated induction of BAL protein levels, and the effect was substantially more pronounced compared to the effect observed with the positive control. Thus, Figure 6 shows that AL-310 effectively alleviated LPS-induced mouse lung injury. Therefore, AL-310 was selected for subsequent humanization.

[0164] Example 6. Humanization of anti-NAMPT antibody AL-303 Based on the results of the in vitro and in vivo studies described in Examples 2-4, the murine anti-NAMPT antibody AL-303 was selected for humanization.

[0165] 1076 Humanized anti-hNAMPT antibody The term "1076" collectively refers to a humanized version 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 receptor sequences.

[0166] Following grafting, various framework backmutations were introduced by methods well known in the art, either by de novo synthesis of the variable domains or by mutagenic oligonucleotide primers and polymerase chain reaction, or both. Various combinations of backmutations and other mutations were constructed in the heavy and / or light chains of each CDR graft to generate 1076 humanized anti-hNMAPT antibodies D-1076, G-1076, K-1076, N-1076, P-1076, V-1076, and X-1076.

[0167] The amino acid sequences of the heavy and light chain variable regions of the 1076 humanized anti-hNMAPT antibody are shown in Table 2. Table 3 provides an alignment of the amino acid sequences of the heavy chain CDRs of the 1076 humanized anti-hNMAPT antibodies D-1076, G-1076, K-1076, N-1076, P-1076, V-1076, and X-1076 when compared with the murine anti-NAMPT antibody AL-303. Table 4 provides an alignment of the amino acid sequences of the light chain CDRs of the 1076 humanized anti-hNMAPT antibodies D-1076, G-1076, K-1076, N-1076, P-1076, V-1076, and X-1076 when compared with the murine anti-NAMPT antibody AL-303. Blank spaces in Tables 3 and 4 indicate residues that are the same as those in AL-303.

[0168] D-1076 The 1076 humanized anti-hNMAPT antibody D-1076 has a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 1 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 2. The heavy chain variable region of D-1076 comprises 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. One or more back mutations and other mutations were introduced into the heavy chain of D-1076. The light chain variable region of D-1076 comprises 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.

[0169] G-1076 The 1076 humanized anti-hNMAPT antibody G-1076 has a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:9 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO:10. The heavy chain variable region of G-1076 comprises 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. One or more back mutations and other mutations were introduced into the heavy chain of G-1076. The light chain variable region of G-1076 comprises 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.

[0170] K-1076 The 1076 humanized anti-hNMAPT antibody K-1076 has a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:9 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO:13. The heavy chain variable region of K-1076 comprises 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. One or more back mutations and other mutations were introduced into the heavy chain of K-1076. The light chain variable region of K-1076 comprises 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.

[0171] N-1076 The 1076 humanized anti-hNMAPT antibody N-1076 has a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 15 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 2. The heavy chain variable region of N-1076 comprises 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. One or more back mutations and other mutations were introduced into the heavy chain of N-1076. The light chain variable region of N-1076 comprises 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.

[0172] P-1076 The 1076 humanized anti-hNMAPT antibody P-1076 has a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 15 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 13. The heavy chain variable region of P-1076 comprises 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. One or more back mutations and other mutations were introduced into the heavy chain of P-1076. The light chain variable region of P-1076 comprises 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.

[0173] V-1076 The 1076 humanized anti-hNMAPT antibody V-1076 has a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 16 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 10. The heavy chain variable region of V-1076 comprises 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. One or more back mutations and other mutations were introduced into the heavy chain of V-1076. The light chain variable region of V-1076 comprises 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.

[0174] X-1076 The 1076 humanized anti-hNMAPT antibody X-1076 has a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 16 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 2. The heavy chain variable region of X-1076 comprises 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. One or more back mutations and other mutations were introduced into the heavy chain of X-1076. The light chain variable region of X-1076 comprises 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.

[0175] [Table 3] [Table 4] [Table 5]

[0176] [Table 6] [Table 7]

[0177] [Table 8] [Table 9]

[0178] Example 7. Humanization of anti-NAMPT antibody AL-310 Based on the results of the in vitro and in vivo studies described in Examples 2-5, the murine anti-NAMPT antibody AL-310 was selected for humanization.

[0179] "1093" refers to a set of humanized antibodies derived from the murine anti-NAMPT antibody AL-310. By applying humanization methodology, the CDR sequences of the VH and VL chains of AL-310 were grafted onto different human heavy and light chain receptor sequences.

[0180] The corresponding VH and VL CDRs of AL-310 were grafted onto these receptor sequences to generate CDR-grafted, humanized and modified VH and VL sequences.

[0181] To generate humanized antibodies with potential framework backmutations, mutations were identified and introduced into the CDR-grafted antibody sequence by methods well known in the art, such as de novo synthesis of variable domains or by mutagenic oligonucleotide primers and polymerase chain reaction, or both. Various combinations of backmutations and other mutations were constructed for the heavy and / or light chains 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.

[0182] The amino acid sequences of the heavy and light chain variable regions of the 1093 humanized anti-hNMAPT antibody are shown 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 when compared with the 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 when compared with the murine anti-NAMPT antibody AL-310. Blank spaces in Tables 6 and 7 indicate residues that are the same as in AL-310.

[0183] FF-1093 The 1093 humanized anti-hNMAPT antibody FF-1093 has a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 17 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 18. The heavy chain variable region of FF-1093 comprises 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. One or more back mutations and other mutations were introduced into the heavy chain of FF-1093. The light chain variable region of FF-1093 comprises 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.

[0184] II-1093 The 1093 humanized anti-hNMAPT antibody II-1093 has a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:23 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO:24. The heavy chain variable region of II-1093 comprises 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. One or more back mutations and other mutations were introduced into the heavy chain of II-1093. The light chain variable region of II-1093 comprises 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.

[0185] NN-1093 The 1093 humanized anti-hNMAPT antibody NN-1093 has a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:25 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO:24. The heavy chain variable region of NN-1093 comprises 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. One or more back mutations and other mutations were introduced into the heavy chain of NN-1093. The light chain variable region of NN-1093 comprises 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.

[0186] PP-1093 The 1093 humanized anti-hNMAPT antibody PP-1093 has a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:25 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO:18. The heavy chain variable region of PP-1093 comprises 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. One or more back mutations and other mutations were introduced into the heavy chain of PP-1093. The light chain variable region of PP-1093 comprises 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.

[0187] SS-1093 The 1093 humanized anti-hNMAPT antibody SS-1093 has a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:26 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO:24. The heavy chain variable region of SS-1093 comprises 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. One or more back mutations and other mutations were introduced into the heavy chain of SS-1093. The light chain variable region of SS-1093 comprises 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.

[0188] UU-1093 The 1093 humanized anti-hNMAPT antibody UU-1093 has a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:26 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO:18. The heavy chain variable region of UU-1093 comprises 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. One or more back mutations and other mutations were introduced into the heavy chain of UU-1093. The light chain variable region of UU-1093 comprises 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.

[0189] XX-1093 The 1093 humanized anti-hNMAPT antibody XX-1093 has a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:27 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO:24. The heavy chain variable region of XX-1093 comprises 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. One or more back mutations and other mutations were introduced into the heavy chain of XX-1093. The light chain variable region of XX-1093 comprises 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.

[0190] ZZ-1093 The 1093 humanized anti-hNMAPT antibody ZZ-1093 has a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:27 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO:18. The heavy chain variable region of ZZ-1093 comprises 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. One or more back mutations and other mutations were introduced into the heavy chain of ZZ-1093. The light chain variable region of ZZ-1093 comprises 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.

[0191] [Table 10] [Table 11] [Table 12] [Table 13]

[0192] [Table 14] [Table 15]

[0193] [Table 16] [Table 17]

[0194] Example 8. Epitope mapping of humanized anti-hNAMPT antibodies Both linear and conformational epitope mapping were used to map the epitopes of humanized anti-NAMPT antibody K-1076 (described in Example 6) and humanized anti-NAMPT antibody NN-1093 (described in Example 7).

[0195] A library of peptide-based peptidomimetics was synthesized using Fmoc-based solid-phase peptide synthesis. To generate a library of linear peptidomimetics, the amino acid sequence of human NAMPT (SEQ ID NO: 60) was split into overlapping fragments in silico and then synthesized on solid supports. Amino-functionalized polypropylene supports were obtained by grafting with a proprietary hydrophilic polymer formulation, followed by reaction with t-butyloxycarbonyl-hexamethylenediamine (BocHMDA) using dicyclohexylcarbodiimide (DCC) and N-hydroxybenzotriazole (HOBt), followed by cleavage of the Boc group using trifluoroacetic acid (TFA). Peptides were synthesized on the amino-functionalized solid supports using standard Fmoc-peptide synthesis on a custom-modified JANUS liquid handling station (Perkin Elmer).

[0196] Structural mimetics were synthesized using Pepscan's proprietary Chemically Ligated Peptides on Scaffolds (CLIPS) technology. CLIPS technology allows peptides to be constructed into single loops, double loops, triple loops, sheet-like folds, helix-like folds, and combinations thereof. CLIPS templates are attached to cysteine ​​residues. Multiple cysteine ​​side chains in a peptide were attached 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 then added to the peptide array. The CLIPS templates were attached to the side chains of two cysteines present in the solid-phase-bound peptides of the peptide array (455-well plate with 3 μL of each well). The peptide array was left completely covered in the solution and gently shaken for 30–60 minutes. Finally, the peptide array was washed extensively with excess HO and sonicated for 30 min at 70°C in disruption buffer containing 1% SDS / 0.1% 2,2'-(ethylenedioxy)diethanethiol in PBS (pH 7.2), followed by an additional 45 min in HO. The T3 CLIPS-retained peptide was generated in a similar manner, but with three cysteines.

[0197] Antibody binding to each of the synthesized peptides was tested using a Pepscan-based ELISA. The peptide array was incubated with the primary antibody solution (overnight at 4°C). After washing, the peptide array was incubated with the appropriate detection antibody (antibody peroxidase conjugate (SBA)) at a 1 / 1000 dilution for 1 hour at 25°C. HRP-conjugated goat anti-human antibody (Southern Biotech, catalog number 2010-05) was used as the detection antibody. After washing, the peroxidase substrate 2,2'-azino-di-3-ethylbenzthiazoline sulfonate (ABTS) and 20 μl / ml of 3 percent H2O2 were added. After 1 hour, color development was measured. Color development was quantified using a charge-coupled device (CCD) camera and image processing system.

[0198] The values ​​obtained from the CCD camera ranged from 0 to 3000 mAU, similar to a standard 96-well plate ELISA reader. Results were quantified and stored in a Peplab database. Occasionally, wells contained air bubbles, resulting in false positive values; therefore, cards were manually inspected and any values ​​resulting from air bubbles were scored as 0.

[0199] To verify the quality of the synthesized peptides, another set of positive and negative control peptides was synthesized in parallel and screened using the commercial antibodies 3C9 and 57.9 (Posthumus et al., J Virol, 64:3304-3309, 1990).

[0200] Data analysis and interpretation was performed using box plots, linear intensity profiles and heat map analysis.

[0201] Ten different sets of peptides (Set 1 to Set 10) ranging in size from 9 to 30 amino acids were synthesized as described below.

[0202] Set 1 (LIN15) The peptides of Set 1, also referred to herein as LIN15 peptides, are 15 amino acid long linear peptides derived from the target sequence of NAMPT with a one residue offset.

[0203] Set 2 (LIN15.AA) Set 2 peptides, also referred to herein as LIN15.AA peptides, are 15 amino acid long linear peptides similar to Set 1 or LIN15 peptides, but with residues at positions 10 and 11 replaced by Ala. The native Ala, when occurring at either position, was replaced by Gly.

[0204] Set 3 (LIN30) The peptides of Set 3, also referred to herein as LIN30 peptides, are 30 amino acid long linear peptides derived from the target sequence of NAMPT with a one residue offset.

[0205] Set 4 (LOOP7) The peptides in Set 4, also referred to herein as LOOP7 peptides, are 9 amino acid long constrained peptides. Positions 2-8 contain a 7-mer peptide derived from the target sequence of NAMPT with a one-residue offset. To create a loop mimic, Cys residues were inserted at positions 1 and 9 and attached with mP2 CLIPS. The native Cys was replaced by Cys-acm (labeled "2").

[0206] Set 5 (LOOP15) The peptides of Set 5, also referred to herein as LOOP15 peptides, are 17 amino acid long constrained peptides. Positions 2-16 contain a 15-mer peptide derived from the target sequence of NAMPT (SEQ ID NO: 60) with a one-residue offset. To create a loop mimic, Cys residues were inserted at positions 1 and 17 and attached with mP2 CLIPS. The native Cys was replaced by Cys-acm (denoted "2").

[0207] Set 6 (LOOP15.AA) Set 6 peptides, also referred to herein as LOOP15.AA peptides, are 17 amino acid long constrained peptides similar to Set 5 or LOOP15 peptides, but with residues at positions 10 and 11 replaced by Ala. The native Ala, when occurring at either position, was replaced by Gly.

[0208] Set 7 (LOOP25) The peptides of Set 7, also referred to herein as LOOP25 peptides, are 27 amino acid long constrained peptides. Positions 2-26 contain a 25-mer peptide derived from the target sequence of NAMPT (SEQ ID NO: 60) with a one-residue offset. To create a loop mimic, Cys residues were inserted at positions 1 and 27 and attached with mP2 CLIPS. The native Cys was replaced by Cys-acm (denoted "2").

[0209] Set 8 (BET) The peptides in Set 8, also referred to herein as BET peptides, are 22-amino acid-long β-turn peptidomimetics. Positions 2-21 contain a 20-mer peptide derived from the target sequence of NAMPT (SEQ ID NO: 60) with a one-residue offset. Residues at positions 11 and 12 were replaced by a "PG" motif to induce β-turn formation. To stabilize the mimic, Cys residues were inserted at positions 1 and 22 and attached with mP2 CLIPS. The native Cys was replaced by Cys-acm (denoted "2").

[0210] Set 9 (HEL.CC) The Set 9 peptides, also referred to herein as HEL.CC peptides, are 22 amino acid long α-helical peptidomimetics derived from residues of the target sequence (NAMPT, SEQ ID NO: 60) with a one residue offset. To nucleate the α-helical structure, Cys residues were inserted at positions 1 and 5 and attached with mP2 CLIPS. The native Cys was replaced by Cys-acm (denoted "2").

[0211] Set 10 (HEL.IL) The peptides of Set 10, also referred to herein as HEL.IL peptides, are 26 amino acid long α-helical peptidomimetics derived from residues of the target sequence (NAMPT, SEQ ID NO: 60) with a one residue offset. Leu and Ile residues were inserted into the sequence to promote helical secondary structure without covalent constraints.

[0212] Screening Details Antibody binding depends on a combination of factors, including antibody concentration and the amount and nature of the competing protein in the ELISA buffer. Precoat conditions (specific treatment of the peptide array prior to incubation with the experimental sample) also affected binding. These details are summarized in Table 8. For Pepscan buffer and preconditioning (SQ), the numbers indicate the relative amount of competing protein (combination of horse serum and ovalbumin).

[0213] [Table 18]

[0214] For both antibodies in this study, binding signals were observed in various distinct regions throughout the NAMPT sequence. This suggests that the epitopes of both antibodies are discontinuous. All observed signals were specific to the antibody, as incubation with the secondary antibody alone did not produce any signal on the array. In the case of Ab-1076-HC2-LC5, a core sequence derived from the overlapping peptide sequence appears to form a binding interface spanning both monomers within the NAMPT dimer. In particular, in the plot for the LIN30 mimetic, a dominant putative binding site can be observed, surrounded within the structure by additional sequences derived from lower intensity peaks. The tentative core epitopes of the antibodies are shown below. Both antibodies bound to the dimeric form of NAMPT.

[0215] The epitope candidates identified for both sequences showed a significant amount of repeated sequence similarity (e.g., 17 SYKVTHYKQYPPNTSKVYSYFEC REKKT 44 (SEQ ID NO: 61) pair 16 2ATNS REQK K 170 (SEQ ID NO: 63), 29 NT SKVYS YFECREKKTENSKLRK 51 (SEQ ID NO: 72) pair 332 FPVTEN SKGY K 342 (SEQ ID NO: 67), and 216 KGT DTV AGLALIKKYY GTK 234 (SEQ ID NO: 75) pair 316 NPL DTV LKVLEIL GKK 331 (SEQ ID NO: 76).

[0216] [Table 19]

[0217] Example 9. Characterization of humanized anti-hNAMPT antibodies The 1076 humanized anti-hNAMPT antibody described in Example 6 and the 1093 humanized anti-hNAMPT antibody described in Example 7 were subjected to in vitro and in vivo testing for the selection of lead humanized anti-hNAMPT antibodies.

[0218] Effect of humanized anti-hNAMPT antibody on hNAMPT-induced inflammatory signaling The effects of the 1093 humanized anti-hNAMPT antibodies CC-1093, KK-1093, RR-1093, UU-1093, and XX-1093 on hNAMPT-induced inflammatory signaling were evaluated by assessing NFκB activation in cells exposed to hNAMPT in the presence or absence of these antibodies.

[0219] Recombinant hNAMPT (1.5 μg / ml) was premixed with vehicle or 100 μg / ml of 1093 humanized anti-hNAMPT antibody (CC-1093, KK-1093, RR-1093, UU-1093, or XX-1093). Human pulmonary endothelial cells (ECs) were stimulated by exposure to the hNAMPT mixture for 1 hour. Unstimulated cells not exposed to the hNAMPT mixture served as a negative control ("NC"). Stimulated cells exposed to hNAMPT premixed with vehicle alone ("--") served as a positive control. NFκB activation in cells was assessed by assessing NFκB luciferase activity (NFκB-SecNanoLuc) (n = 3–4 for each mAb). The results of the luciferase assay are provided in Figure 7A.

[0220] As shown in Figure 7A, hNAMPT induced NFκB activation in hNAMPT-stimulated cells, which was substantially attenuated in the presence of humanized anti-hNAMPT antibodies CC-1093, KK-1093, RR-1093, UU-1093, or XX-1093. All five antibodies effectively reduced hNAMPT-induced NFκB activation, but the most substantial effect was observed with UU-1093 and XX-1093.

[0221] Effect of humanized anti-hNAMPT antibody on hNAMPT-induced decrease in EC barrier integrity EC electrical resistance reflects 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 reduction in EC barrier integrity were evaluated by assessing the electrical resistance of ECs exposed to hNAMPT in the absence or presence of these antibodies.

[0222] Recombinant hNAMPT (1.5 μg / ml) was premixed with vehicle alone, anti-NAMPT pAb, or humanized anti-hNAMPT antibody (UU-1093, H-1076, P-1076, N-1076, or D-1076). Human lung ECs were stimulated by exposing the cells to the hNAMPT mixture for 1 hour. Stimulated cells exposed to hNAMPT premixed with vehicle alone or hNAMPT premixed with pAb served as controls. EC electrical resistance was assessed as a readout of EC barrier integrity. The results of the study are provided in Figure 7B.

[0223] As shown in Figure 7B, ECs exposed to hNAMPT premixed with each humanized anti-hNAMPT antibody showed substantial induction of EC barrier activity relative to control cells. Thus, the hNAMPT-mediated decrease in EC barrier activity was effectively reversed in the presence of the 1076 and 1093 humanized anti-hNAMPT antibodies.

[0224] In vivo testing of humanized anti-hNAMPT antibody in lung injury The ability 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) to treat lung injury was tested in vivo using mouse and rat lung injury models.

[0225] To evaluate the effects of humanized anti-hNAMPT antibodies on a mouse model of lung injury, V-1076, N-1076, K-1076, P-1076, SS-1093, CC-1093, XX-1093, or UU-1093 were intravenously injected at a dose of 0.4 mg / kg into mice exposed to LPS for 8 hours and VILI for the final 4 hours. Mice injected with vehicle and exposed to hNAMPT and VILI served as controls. Lung injury in mice was assessed by analyzing the expression of BAL proteins and the number of BAL-expressing cells. Edema and inflammatory cell infiltration in lung tissue were also assessed by H&E staining as a readout of lung injury. The results of this "two-hit" model of lung injury are provided in Figures 8A, 8B, and 8D.

[0226] As shown in Figure 8A, compared with control mice, BAL protein levels (left panel of Figure 8A) and the number of BAL-expressing cells (right panel of Figure 8A) were effectively reduced in mice injected with any of the 1076 humanized anti-hNAMPT antibodies. All tested 1076 humanized anti-hNAMPT antibodies effectively reduced lung injury in this "two-hit" mouse model, but the most substantial effect was observed with P-1076.

[0227] As shown in Figure 8B, compared with control mice, BAL protein levels (left panel of Figure 8B) and the number of BAL-expressing cells (right panel of Figure 8B) were effectively reduced in mice injected with any of the 1093 humanized anti-hNAMPT antibodies. All tested 1093 humanized anti-hNAMPT antibodies effectively reduced lung injury in this "two-hit" mouse model, but the most substantial effect was observed with UU-1093. Furthermore, as shown in Figure 8D, the protective effect of UU-1093 in this two-hit lung injury model was further reflected by reduced inflammatory cell infiltration and reduced edema of lung tissue in UU-1093-injected mice.

[0228] To evaluate the effect of humanized anti-hNAMPT antibodies on a rat model of lung injury, Sprague-Dawley rats exposed to LPS were intravenously injected with 40 mg / kg, 80 mg / kg, or 160 mg / kg of P-1076. Rats injected with vehicle and exposed to LPS served as positive controls. Rats injected with vehicle alone and not exposed to LPS served as negative controls. Lung injury in the rats was assessed by analyzing the expression of BAL proteins and the number of BAL-expressing cells. The results of this rat model of lung injury are provided in Figure 8C.

[0229] As shown in Figure 8C, compared with control mice (vehicle-injected and exposed to LPS), BAL protein levels (left panel of Figure 8C) and the number of BAL-expressing cells (right panel of Figure 8C) were effectively reduced in mice injected with humanized anti-hNAMPT antibody P-1076.

[0230] Thus, as shown in Figure 8, all tested humanized anti-hNAMPT antibodies effectively reduced lung injury in an in vivo lung injury model, but the most substantial effect was observed with P-1076 and UU-1093. Therefore, P-1076 and UU-1093 were selected as lead humanized anti-hNAMPT antibodies and further modified to generate improved anti-hNAMPT antibodies.

[0231] Example 10. Modification of humanized anti-hNAMPT antibody P-1076 Based on the results of the in vitro and in vivo studies described in Example 9, anti-NAMPT antibody P-1076 was selected for further modification to generate an improved 1076 anti-hNAMPT antibody.

[0232] P-1076-mod humanized anti-hNAMPT antibody P-1076-mod refers to a humanized anti-hNAMPT antibody generated by modifying the 1076 humanized anti-hNAMPT antibody P-1076.

[0233] To generate the improved P-1076-mod anti-hNAMPT antibody, one or more mutations were introduced into the P-1076 sequence by de novo synthesis of the variable domains or by mutagenic oligonucleotide primers and polymerase chain reaction, or both, using methods well known in the art. These mutations were introduced to remove oxidation sites, reduce or eliminate deamidation, remove potential cleavage or fragmentation sites, remove potential T-cell epitopes, and / or reduce binding of potential T-cell epitopes. Various combinations of back mutations and other mutations were constructed in the heavy and / or light chains of P-1076 to generate the P-1076-mod anti-hNAMPT antibodies P-1076-mod1, 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-mod10, and P-1076-mod11.

[0234] 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 shown in bold. Table 11 shows an alignment of the amino acid sequences of the heavy chain CDRs of the modified P-1076-mod anti-hNAMPT antibodies P-1076-mod1, 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-mod10, and P-1076-mod11, compared to P-1076. Table 12 shows an alignment of the amino acid sequences of the light chain CDRs of the modified P-1076-mod anti-hNAMPT antibodies P-1076-mod1, 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-mod10, and P-1076-mod11, compared to P-1076. Blanks in Tables 11 and 12 indicate residues that are the same as P-1076. A summary of the biophysical properties of the modified P-1076-mod anti-hNAMPT antibodies is provided in Table 13.

[0235] P-1076-mod1 The P-1076-mod anti-hNAMPT antibody P-1076-mod1 has a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:28 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO:30. The heavy chain variable region of P-1076-mod1 comprises 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. For the heavy chain of P-1076-mod1, a D-to-E mutation was introduced into the P-1076 VH CDR2 to remove potential cleavage or fragmentation sites. The light chain variable region of P-1076-mod1 comprises 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. For the light chain of P-1076-mod1, L-to-V mutations were introduced into the framework region of P-1076 VL to remove potential T-cell epitopes.

[0236] P-1076-mod2 The P-1076-mod anti-hNAMPT antibody P-1076-mod2 has a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:28 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO:31. The heavy chain variable region of P-1076-mod2 comprises 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. 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 comprises 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. 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 potential T cell epitopes.

[0237] P-1076-mod3 The P-1076-mod anti-hNAMPT antibody P-1076-mod3 has a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:28 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO:32. The heavy chain variable region of P-1076-mod3 comprises 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. For the heavy chain of P-1076-mod3, 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-mod3 comprises 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. For the light chain of P-1076-mod3, L-to-V mutations were introduced into the framework region of P-1076 VL, and L-to-G mutations were introduced into the P-1076 VL CDR2 framework region to remove potential T cell epitopes.

[0238] P-1076-mod4 The P-1076-mod anti-hNAMPT antibody P-1076-mod4 has a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:28 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO:34. The heavy chain variable region of P-1076-mod4 comprises 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. For the heavy chain of P-1076-mod4, 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-mod4 comprises 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. For the light chain of P-1076-mod4, L-to-V mutations were introduced into the framework region of P-1076 VL, and L-to-E mutations were introduced into the P-1076 VL CDR2 framework region to remove potential T cell epitopes.

[0239] P-1076-mod5 The P-1076-mod anti-hNAMPT antibody P-1076-mod5 has a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:28 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO:36. The heavy chain variable region of P-1076-mod5 comprises 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. For the heavy chain of P-1076-mod5, 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-mod5 comprises 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. For the light chain of P-1076-mod5, an L-to-I mutation was introduced into the framework region of P-1076 VL to eliminate potential T cell epitopes, and an L-to-E mutation was introduced into the P-1076 VL CDR2 framework region to eliminate potential T cell epitope binding.

[0240] P-1076-mod6 The P-1076-mod anti-hNAMPT antibody P-1076-mod6 has a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 15 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 30. The heavy chain variable region of P-1076-mod6 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. 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 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. For the light chain of P-1076-mod5, an L-to-V mutation was introduced into the framework region of P-1076 VL to remove potential T cell epitopes.

[0241] P-1076-mod7 The P-1076-mod anti-hNAMPT antibody P-1076-mod7 has a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 15 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 31. The heavy chain variable region of P-1076-mod7 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. 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 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. For the light chain of P-1076-mod7, an L-to-I mutation was introduced into the framework region of P-1076 VL to remove potential T cell epitopes.

[0242] P-1076-mod8 The P-1076-mod anti-hNAMPT antibody P-1076-mod8 has a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 15 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 32. The heavy chain variable region of P-1076-mod8 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. 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 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. For the light chain of P-1076-mod8, L-to-V mutations were introduced into the framework region of P-1076 VL, and L-to-G mutations were introduced into the P-1076 VL CDR2 framework region to remove potential T cell epitopes.

[0243] P-1076-mod9 The P-1076-mod anti-hNAMPT antibody P-1076-mod9 has a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 15 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 34. The heavy chain variable region of P-1076-mod9 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. 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 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. For the light chain of P-1076-mod9, L-to-V mutations were introduced into the framework region of P-1076 VL, and L-to-E mutations were introduced into the P-1076 VL CDR2 framework region to remove potential T cell epitopes.

[0244] P-1076-mod10 The P-1076-mod anti-hNAMPT antibody P-1076-mod10 has a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 15 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 36. The heavy chain variable region of P-1076-mod10 comprises 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. The heavy chain of P-1076-mod10 is identical to P-1076 VH. The light chain variable region of P-1076-mod10 comprises 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. For the light chain of P-1076-mod10, an L-to-I mutation was introduced into the framework region of P-1076 VL to eliminate potential T-cell epitopes, and an L-to-V mutation was introduced into P-1076 VL CDR2 to eliminate potential T-cell epitope binding.

[0245] P-1076-mod11 The modified P-1076-mod anti-hNAMPT antibody P-1076-mod11 has a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:28 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO:13. The heavy chain variable region of P-1076-mod11 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. For the heavy chain of P-1076-mod11, a D-to-E mutation was introduced into the P-1076 VH CDR2 to remove potential cleavage or fragmentation sites. The light chain variable region of P-1076-mod11 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. The light chain of P-1076-mod11 is identical to P-1076 VL.

[0246] [Table 20] [Table 21] [Table 22] [Table 23] [Table 24] [Table 25] [Table 26] *In Table 10, the mutated residues are shown in bold.

[0247] [Table 27] [Table 28]

[0248] [Table 29] [Table 30]

[0249] [Table 31]

[0250] Example 11. Modification of humanized anti-hNAMPT antibody UU-1093 Based on the results of the in vitro and in vivo studies described in Example 9, the anti-NAMPT antibody UU-1093 was selected for further modification to generate the modified 1093 anti-hNAMPT antibody.

[0251] UU-1093-mod humanized anti-hNAMPT antibody UU-1093-mod refers to a humanized anti-hNAMPT antibody generated by modifying the 1093 humanized anti-hNAMPT antibody UU-1093.

[0252] To generate the improved UU-1093-mod anti-hNAMPT antibody, one or more mutations were introduced into the UU-1093 amino acid sequence by methods well known in the art, such as de novo synthesis of variable domains or mutagenic oligonucleotide primers and polymerase chain reaction, or both, to remove oxidation sites, reduce or eliminate deamidation, remove potential cleavage or fragmentation sites, remove potential T-cell epitopes, and / or reduce binding of potential T-cell epitopes. Various combinations of back mutations and other mutations were constructed in the heavy and / or light chains of UU-1093 to produce modified UU-1093-mod anti-hNAMPT antibodies UU-1093-mod1, 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-mod10, UU-1093-mod11, UU-1093-mod12, UU-1093-mod13, UU-1093-mod14, UU-1093-mod15, UU-1093-mod16, UU-1093-mod17, UU-1093-mod18, UU-1093-mod19 ...9, UU-1093-mod19, UU-1093-mod19, UU-1093-mod19, UU-1093-mod19, UU-1093-mod19, UU-1093-mod19, UU-1093-mod19, UU- UU-1093-mod8, UU-1093-mod9, UU-1093-mod10, UU-1093-mod11, UU-1093-mod12, UU-1093-mod13, UU-1093-mod14, UU-1093-mod15, UU-1093-mod16, UU-1093-mod17, UU-1093-mod18 and UU-1093-mod19 were generated.

[0253] 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, the mutated residues are shown in bold. Table 15 shows the amino acid sequences of the modified UU-1093-mod anti-hNAMPT antibodies UU-1093-mod1, 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-mod10, UU-1093-mod20, UU-1093-mod31, UU-1093-mod42, UU-1093-mod53, UU-1093-mod64, UU-1093-mod75, UU-1093-mod86, UU-1093-mod97, UU-1093-mod10, UU-1093-mod11, UU-1093-mod12, UU-1093-mod13, UU-1093-mod14, UU-1093-mod15, UU-1093-mod16, UU-1093-mod17, UU-1093-mod18, UU-1093-mod19, UU-1093-mod19, UU-1093-mod10, UU-1093-mod11, UU-1093-mod12, UU-1093-mod13, UU-1093-mod14, UU-1093-mod15, UU-1093-mod16, UU-10 Figure 1 shows an alignment of the amino acid sequences of the heavy chain CDRs of U-1093-mod10, UU-1093-mod11, UU-1093-mod12, UU-1093-mod13, UU-1093-mod14, UU-1093-mod15, UU-1093-mod16, UU-1093-mod17, UU-1093-mod18 and UU-1093-mod19. Table 16 shows the results of the modified UU-1093-mod anti-hNAMPT antibodies UU-1093-mod1, 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-mod10, UU-1093-mod20, UU-1093-mod31, UU-1093-mod42, UU-1093-mod53, UU-1093-mod64, UU-1093-mod75, UU-1093-mod86, UU-1093-mod97, UU-1093-mod10, UU-1093-mod11, UU-1093-mod12, UU-1093-mod13, UU-1093-mod14, UU-1093-mod15, UU-1093-mod16, UU-1093-mod17, UU-1093-mod18, UU-1093-mod19 ...9, UU The amino acid sequence alignment of the light chain CDRs of U-1093-mod10, UU-1093-mod11, UU-1093-mod12, UU-1093-mod13, UU-1093-mod14, UU-1093-mod15, UU-1093-mod16, UU-1093-mod17, UU-1093-mod18, and UU-1093-mod19 is shown. Blanks in Tables 15 and 16 indicate residues that are identical to UU-1093.

[0254] UU-1093-mod1 The UU-1093-mod anti-hNAMPT antibody UU-1093-mod1 has a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:38 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO:47. The heavy chain variable region of UU-1093-mod1 comprises 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. For the heavy chain of UU-1093-mod1, 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-mod1 comprises 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. For the light chain of UU-1093-mod1, an N to Q mutation was introduced into UU-1093 VL CDR1 to reduce or eliminate deamidation.

[0255] UU-1093-mod2 The UU-1093-mod anti-hNAMPT antibody UU-1093-mod2 has a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 40 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 47. The heavy chain variable region of UU-1093-mod2 comprises 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. For the heavy chain of UU-1093-mod2, a D to E mutation was introduced into UU-1093 VH CDR2 to eliminate a potential cleavage or fragmentation site, and an S to T mutation was introduced into UU-1093 VH CDR3 to eliminate a potential T cell epitope. The light chain variable region of UU-1093-mod2 comprises 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. For the light chain of UU-1093-mod2, an N to Q mutation was introduced into UU-1093 VL CDR1 to reduce or eliminate deamidation.

[0256] UU-1093-mod3 The UU-1093-mod anti-hNAMPT antibody UU-1093-mod3 has a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 42 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 47. The heavy chain variable region of UU-1093-mod3 comprises 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. For the heavy chain of UU-1093-mod3, M to I mutations were introduced into UU-1093 VH CDR1 to remove an oxidation site, M to V mutations were introduced into UU-1093 VH framework region 2 to remove an oxidation site and a potential T cell epitope, D to E mutations were introduced into UU-1093 VH CDR2 to remove a potential cleavage or fragmentation site, M to V mutations were introduced into UU-1093 VH framework region 3 to remove an oxidation site, and K to R mutations were introduced into UU-1093 VH CDR3 to reduce binding of a potential T cell epitope. The light chain variable region of UU-1093-mod3 comprises 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. For the light chain of UU-1093-mod3, an N to Q mutation was introduced into UU-1093 VL CDR1 to reduce or eliminate deamidation.

[0257] UU-1093-mod4 The UU-1093-mod anti-hNAMPT antibody UU-1093-mod4 has a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 45 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 47. The heavy chain variable region of UU-1093-mod4 includes 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. For the heavy chain of UU-1093-mod4, W-to-F and M-to-I mutations were introduced into UU-1093 VH CDR1 to remove an oxidation site, M-to-V mutations were introduced into UU-1093 VH framework region 2 to remove an oxidation site and a potential T-cell epitope, D-to-E mutations were introduced into UU-1093 VH CDR2 to remove a potential cleavage or fragmentation site, M-to-V mutations were introduced into UU-1093 VH framework region 3 to remove an oxidation site, and S-to-T mutations were introduced into UU-1093 VH CDR3 to remove a potential T-cell epitope. The light chain variable region of UU-1093-mod4 comprises 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. For the light chain of UU-1093-mod4, an N to Q mutation was introduced into UU-1093 VL CDR1 to reduce or eliminate deamidation.

[0258] UU-1093-mod5 The UU-1093-mod anti-hNAMPT antibody UU-1093-mod5 has a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:38 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO:48. The heavy chain variable region of UU-1093-mod5 comprises 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. For the heavy chain of UU-1093-mod5, a D-to-E mutation was introduced into the UU-1093 VH CDR2 to remove potential cleavage or fragmentation sites. The light chain variable region of UU-1093-mod5 comprises 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. For the light chain of UU-1093-mod5, G to A mutations were introduced into UU-1093 VL CDR1 to reduce or eliminate deamidation, I to V mutations were introduced into UU-1093 VL framework region 2 to reduce binding of potential T cell epitopes, M to V mutations were introduced into UU-1093 VL CDR2 to reduce binding of potential T cell epitopes and remove oxidation sites, and I to V mutations were introduced into UU-1093 VH framework region 3 to reduce binding of potential T cell epitopes.

[0259] UU-1093-mod6 The UU-1093-mod anti-hNAMPT antibody UU-1093-mod6 has a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 40 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 48. The heavy chain variable region of UU-1093-mod6 comprises 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. For the heavy chain of UU-1093-mod6, a D to E mutation was introduced into UU-1093 VH CDR2 to eliminate a potential cleavage or fragmentation site, and an S to T mutation was introduced into UU-1093 VH CDR3 to eliminate a potential T cell epitope. The light chain variable region of UU-1093-mod6 comprises 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. For the light chain of UU-1093-mod6, G-to-A mutations were introduced into UU-1093 VL CDR1 to reduce or eliminate deamidation, I-to-V mutations were introduced into UU-1093 VL framework region 2 to reduce binding of potential T-cell epitopes, M-to-V mutations were introduced into UU-1093 VL CDR2 to reduce binding of potential T-cell epitopes and remove oxidation sites, and I-to-V mutations were introduced into UU-1093 VH framework region 3 to reduce binding of potential T-cell epitopes.

[0260] UU-1093-mod7 The UU-1093-mod anti-hNAMPT antibody UU-1093-mod7 has a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 42 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 48. The heavy chain variable region of UU-1093-mod7 includes 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. For the heavy chain of UU-1093-mod7, an M to I mutation was introduced into UU-1093 VH CDR1 to remove an oxidation site, an 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, an 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 a potential T cell epitope. The light chain variable region of UU-1093-mod7 comprises 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. For the light chain of UU-1093-mod7, G to A mutations were introduced into UU-1093 VL CDR1 to reduce or eliminate deamidation, I to V mutations were introduced into UU-1093 VL framework region 2 to reduce binding of potential T cell epitopes, M to V mutations were introduced into UU-1093 VL CDR2 to reduce binding of potential T cell epitopes and remove oxidation sites, and I to V mutations were introduced into UU-1093 VH framework region 3 to reduce binding of potential T cell epitopes.

[0261] UU-1093-mod8 The UU-1093-mod anti-hNAMPT antibody UU-1093-mod8 has a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 45 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 48. The heavy chain variable region of UU-1093-mod8 includes 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. For the heavy chain of UU-1093-mod8, W-to-F and M-to-I mutations were introduced into UU-1093 VH CDR1 to remove an oxidation site, M-to-V mutations were introduced into UU-1093 VH framework region 2 to remove an oxidation site and a potential T-cell epitope, D-to-E mutations were introduced into UU-1093 VH CDR2 to remove a potential cleavage or fragmentation site, M-to-V mutations were introduced into UU-1093 VH framework region 3 to remove an oxidation site, and S-to-T mutations were introduced into UU-1093 VH CDR3 to remove a potential T-cell epitope. The light chain variable region of UU-1093-mod8 comprises 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. For the light chain of UU-1093-mod8, G to A mutations were introduced into UU-1093 VL CDR1 to reduce or eliminate deamidation, I to V mutations were introduced into UU-1093 VL framework region 2 to reduce binding of potential T cell epitopes, M to V mutations were introduced into UU-1093 VL CDR2 to reduce binding of potential T cell epitopes and remove oxidation sites, and I to V mutations were introduced into UU-1093 VH framework region 3 to reduce binding of potential T cell epitopes.

[0262] UU-1093-mod9 The UU-1093-mod anti-hNAMPT antibody UU-1093-mod9 has a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:38 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO:51. The heavy chain variable region of UU-1093-mod9 comprises 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. 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 comprises 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. For the light chain of UU-1093-mod9, an N to Q mutation was introduced into UU-1093 VL CDR1 to reduce or eliminate deamidation, an I to V mutation was introduced into UU-1093 VL framework region 2 to eliminate a potential T cell epitope, an M to V mutation was introduced into UU-1093 VL CDR2 to eliminate a potential T cell epitope and eliminate an oxidation site, an A to G mutation was introduced into UU-1093 VL CDR2 to eliminate a potential T cell epitope, and a W to F mutation was introduced into UU-1093 VL CDR3 to eliminate an oxidation site.

[0263] UU-1093-mod10 The UU-1093-mod anti-hNAMPT antibody UU-1093-mod10 has a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 40 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 51. The heavy chain variable region of UU-1093-mod10 comprises 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. For the heavy chain of UU-1093-mod10, a D to E mutation was introduced into UU-1093 VH CDR2 to eliminate a potential cleavage or fragmentation site, and an S to T mutation was introduced into UU-1093 VH CDR3 to eliminate a potential T cell epitope. The light chain variable region of UU-1093-mod10 comprises 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. For the light chain of UU-1093-mod10, an N to Q mutation was introduced into UU-1093 VL CDR1 to reduce or eliminate deamidation, an I to V mutation was introduced into UU-1093 VL framework region 2 to eliminate a potential T cell epitope, an M to V mutation was introduced into UU-1093 VL CDR2 to eliminate a potential T cell epitope and to remove an oxidation site, an A to G mutation was introduced into UU-1093 VL CDR2 to eliminate a potential T cell epitope, and a W to F mutation was introduced into UU-1093 VL CDR3 to remove an oxidation site.

[0264] UU-1093-mod11 The UU-1093-mod anti-hNAMPT antibody UU-1093-mod11 has a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 42 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 51. The heavy chain variable region of UU-1093-mod11 comprises 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. For the heavy chain of UU-1093-mod11, an M to I mutation was introduced into UU-1093 VH CDR1 to remove an oxidation site, an 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, an 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 a potential T cell epitope. The light chain variable region of UU-1093-mod11 comprises 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. For the light chain of UU-1093-mod11, an N to Q mutation was introduced into UU-1093 VL CDR1 to reduce or eliminate deamidation, an I to V mutation was introduced into UU-1093 VL framework region 2 to eliminate a potential T cell epitope, an M to V mutation was introduced into UU-1093 VL CDR2 to eliminate a potential T cell epitope and eliminate an oxidation site, an A to G mutation was introduced into UU-1093 VL CDR2 to eliminate a potential T cell epitope, and a W to F mutation was introduced into UU-1093 VL CDR3 to eliminate an oxidation site.

[0265] UU-1093-mod12 The UU-1093-mod anti-hNAMPT antibody UU-1093-mod12 has a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 45 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 51. The heavy chain variable region of UU-1093-mod12 comprises 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. For the heavy chain of UU-1093-mod12, W-to-F and M-to-I mutations were introduced in UU-1093 VH CDR1 to remove an oxidation site, M-to-V mutations were introduced in UU-1093 VH framework region 2 to remove an oxidation site and a potential T-cell epitope, D-to-E mutations were introduced in UU-1093 VH CDR2 to remove a potential cleavage or fragmentation site, M-to-V mutations were introduced in UU-1093 VH framework region 3 to remove an oxidation site, and S-to-T mutations were introduced in UU-1093 VH CDR3 to remove a potential T-cell epitope. The light chain variable region of UU-1093-mod12 comprises 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. For the light chain of UU-1093-mod12, an N to Q mutation was introduced into UU-1093 VL CDR1 to reduce or eliminate deamidation, an I to V mutation was introduced into UU-1093 VL framework region 2 to eliminate a potential T cell epitope, an M to V mutation was introduced into UU-1093 VL CDR2 to eliminate a potential T cell epitope and eliminate an oxidation site, an A to G mutation was introduced into UU-1093 VL CDR2 to eliminate a potential T cell epitope, and a W to F mutation was introduced into UU-1093 VL CDR3 to eliminate an oxidation site.

[0266] UU-1093-mod13 The UU-1093-mod anti-hNAMPT antibody UU-1093-mod13 has a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:38 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO:18. The heavy chain variable region of UU-1093-mod13 comprises 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. For the heavy chain of UU-1093-mod13, a D-to-E mutation was introduced into the UU-1093 VH CDR2 to eliminate a potential cleavage or fragmentation site. The light chain variable region of UU-1093-mod13 comprises 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. The light chain of UU-1093-mod13 is identical to UU-1093 VL.

[0267] UU-1093-mod14 The UU-1093-mod anti-hNAMPT antibody UU-1093-mod14 has a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 40 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 18. The heavy chain variable region of UU-1093-mod14 comprises 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. For the heavy chain of UU-1093-mod14, a D to E mutation was introduced into UU-1093 VH CDR2 to eliminate a potential cleavage or fragmentation site, and an S to T mutation was introduced into UU-1093 VH CDR3 to eliminate a potential T cell epitope. The light chain variable region of UU-1093-mod14 comprises 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. The light chain of UU-1093-mod14 is identical to that of UU-1093 VL.

[0268] UU-1093-mod15 The UU-1093-mod anti-hNAMPT antibody UU-1093-mod15 has a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 42 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 18. The heavy chain variable region of UU-1093-mod15 comprises 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. For the heavy chain of UU-1093-mod15, an M to I mutation was introduced into UU-1093 VH CDR1 to remove an oxidation site, an 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, an 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 a potential T cell epitope. The light chain variable region of UU-1093-mod15 comprises 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. The light chain of UU-1093-mod15 is identical to UU-1093 VL.

[0269] UU-1093-mod16 The UU-1093-mod anti-hNAMPT antibody UU-1093-mod16 has a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 45 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 18. The heavy chain variable region of UU-1093-mod16 comprises 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. For the heavy chain of UU-1093-mod16, W-to-F and M-to-I mutations were introduced in UU-1093 VH CDR1 to remove an oxidation site, M-to-V mutations were introduced in UU-1093 VH framework region 2 to remove an oxidation site and a potential T-cell epitope, D-to-E mutations were introduced in UU-1093 VH CDR2 to remove a potential cleavage or fragmentation site, M-to-V mutations were introduced in UU-1093 VH framework region 3 to remove an oxidation site, and S-to-T mutations were introduced in UU-1093 VH CDR3 to remove a potential T-cell epitope. The light chain variable region of UU-1093-mod16 comprises 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. The light chain of UU-1093-mod16 is identical to UU-1093 VL.

[0270] UU-1093-mod17 The UU-1093-mod anti-hNAMPT antibody UU-1093-mod17 has a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:26 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO:47. The heavy chain variable region of UU-1093-mod17 comprises 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. The heavy chain of UU-1093-mod17 is identical to UU-1093 VH. The light chain variable region of UU-1093-mod17 comprises 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. For the light chain of UU-1093-mod17, an N to Q mutation was introduced into UU-1093 VL CDR1 to reduce or eliminate deamidation.

[0271] UU-1093-mod18 The UU-1093-mod anti-hNAMPT antibody UU-1093-mod18 has a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:26 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO:48. The heavy chain variable region of UU-1093-mod18 comprises 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. The heavy chain of UU-1093-mod18 is identical to UU-1093 VH. The light chain variable region of UU-1093-mod18 comprises 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. For the light chain of UU-1093-mod18, G to A mutations were introduced into UU-1093 VL CDR1 to reduce or eliminate deamidation, I to V mutations were introduced into UU-1093 VL framework region 2 to reduce binding of potential T cell epitopes, M to V mutations were introduced into UU-1093 VL CDR2 to reduce binding of potential T cell epitopes and remove oxidation sites, and I to V mutations were introduced into UU-1093 VH framework region 3 to reduce binding of potential T cell epitopes.

[0272] UU-1093-mod19 The UU-1093-mod anti-hNAMPT antibody UU-1093-mod19 has a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:26 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO:51. The heavy chain variable region of UU-1093-mod19 comprises 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. The heavy chain of UU-1093-mod17 is identical to the UU-1093 VH. The light chain variable region of UU-1093-mod19 comprises 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. For the light chain of UU-1093-mod19, an N to Q mutation was introduced into UU-1093 VL CDR1 to reduce or eliminate deamidation, an I to V mutation was introduced into UU-1093 VL framework region 2 to eliminate a potential T cell epitope, an M to V mutation was introduced into UU-1093 VL CDR2 to eliminate a potential T cell epitope and eliminate an oxidation site, an A to G mutation was introduced into UU-1093 VL CDR2 to eliminate a potential T cell epitope, and a W to F mutation was introduced into UU-1093 VL CDR3 to eliminate an oxidation site.

[0273] [Table 32] [Table 33] [Table 34] [Table 35] [Table 36] [Table 37] [Table 38] [Table 39] [Table 40] [Table 41] [Table 42] [Table 43] In Table 14, the mutated residues are shown in bold.

[0274] [Table 44] [Table 45] [Table 46] [Table 47]

[0275] [Table 48] [Table 49] [Table 50] [Table 51]

[0276] Example 12. Tissue expression of NAMPT in human invasive PCa To evaluate the role of NAMPT on the invasiveness and progression of prostate cancer (PCa), the expression of NAMPT was studied in PCa tissues.

[0277] NAMPT expression was assessed by immunohistochemical (IHC) staining in normal prostate tissue, prostate adenocarcinoma confined to the prostate but without capsular invasion, and prostate adenocarcinoma with capsular invasion into the periprostatic adipose tissue. Representative photomicrographs are provided in Figures 9A-9C. Furthermore, NAMPT expression was assessed by IHC staining in benign prostate tissue, tissue from PCa patients with organ-confined disease (T2 disease, n=12), and tissue from PCa patients with capsular invasive disease (T3 disease, n=14). The cumulative analysis is provided in Figure 9D.

[0278] As shown in Figure 9, IHC analysis of normal and PCa tissues revealed virtually no NAMPT expression in normal prostate tissue (Figure 9A), and minimal NAMPT expression in prostate adenocarcinomas confined to the prostate but without capsular invasion (Figure 9B). In contrast, prostate adenocarcinomas with capsular invasion into periprostatic adipose tissue exhibit significantly stronger NAMPT staining (Figure 9C). Furthermore, comparative analysis of benign prostate tissues and tissues from 26 PCa patients with T2 and T3 disease showed increased NAMPT expression with increasing PCa invasiveness (Figure 9D). Thus, increased NAMPT expression was observed in invasive PCa in humans.

[0279] Example 13. Effect of radiation exposure on NAMPT expression Radiation therapy is the mainstay of PCa therapy. Because extracellular NAMPT (eNAMPT) functions as a damage-associated molecular pattern protein (DAMP) in innate immunity, we evaluated the effect of radiation on NAMPT expression by assessing its expression in radiation-exposed mouse and human tissues.

[0280] To evaluate the effect of radiation-induced tissue injury and damage on NAMPT expression in mouse tissues, C57 / B6 mice were exposed to a single dose of thoracic radiation (20 Gy) for one week. The effects of radiation on inflammation, blood leakage, and inflammatory lung injury were evaluated by H&E staining of mouse lung tissue after radiation exposure. Representative photomicrographs are provided in Figure 10A. NAMPT expression was evaluated in lung tissue before and after radiation exposure by IHC staining. Representative photomicrographs are provided in Figures 10B and 10C. To evaluate the effect of radiation on NAMPT expression in human tissues, normal human epithelial tissue (tonsil) was exposed to radiation (8 Gy) for 24 hours, and NAMPT expression was evaluated by IHC staining. Representative photomicrographs are provided in Figure 10D.

[0281] As shown in Figure 10, after one week of radiation exposure, increased inflammation, blood leakage, and inflammatory damage were observed in mouse lung tissue (Figure 10A). Radiation-induced lung injury was accompanied by significant NAMPT expression (Figure 10B), particularly in alveolar macrophages and epithelial cells (Figure 10C). 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 (tonsil) 24 hours after radiation exposure (Figure 10D). Thus, radiation-induced tissue damage significantly induced NAMPT expression.

[0282] Example 14. Role of NAMPT in PCa cell migration An in vitro assay of PCa cell migration through human smooth muscle cells was used to evaluate the role of NAMPT in PCa cell migration.

[0283] To evaluate the role of NAMPT in PCa cell migration, NAMPT (100 ng / mL) was added to cultures of human DU-145 PCa cells. After 24 hours of NAMPT exposure, PCa cell migration was assessed. A summary of the observations is provided in Figure 11A, and representative photomicrographs are provided in Figure 11B.

[0284] As observed in FIG. 11, NAMPT functioned as a potent chemoattractant for human DU-145 PCa cells and induced PCa cell migration over a 24-hour period.

[0285] Example 15. Characterization of humanized anti-hNAMPT antibodies The ability of the 1076 humanized anti-hNAMPT antibodies (N-1076, K-1076, and P-1076) and the 1093 humanized anti-hNAMPT antibodies (SS-1093, XX-1093, and UU-1093) to treat lung injury was tested in vivo using two mouse lung injury models: a "one-hit" model of lung injury caused by intratracheal delivery of LPS to mice, and a "two-hit" model of lung injury caused by exposure of mice to LPS and mechanical VILI. To evaluate the ability of the antibodies to reduce acute inflammation and injury, the mice were administered one of the humanized anti-hNAMPT antibodies. The results of the study are provided in Figure 12.

[0286] As shown in Figure 12A, analysis of the integrated lung injury scores indicated that all tested anti-hNAMPT antibodies were effective in reducing lung injury in the LPS-induced "one-hit" model. However, the most substantial effect was observed with the anti-hNAMPT antibody P-1076. As shown in Figure 12B, analysis of the integrated lung injury scores indicated that all tested anti-hNAMPT antibodies were effective in reducing lung injury in the LPS / VILI-induced "two-hit" model. However, the most substantial effect was observed with the anti-hNAMPT antibody P-1076. As shown in Figure 12C, the anti-hNAMPT antibody P-1076 was effective in reducing the histological injury index in the LPS / VILI-induced "two-hit" model of acute inflammatory injury.

[0287] Example 16. Effect of humanized anti-hNAMPT antibody P-1076 on PCa cell invasion To evaluate the effect of the humanized anti-hNAMPT antibody P-1076 on PCa cell invasion, we assessed peritoneal invasion of human PCa cells in mice with severe combined immunodeficiency (SCID).

[0288] To evaluate the role of the 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 the humanized anti-hNAMPT antibody P-1076 or vehicle alone. Peritoneal invasion of PC3 cells was evaluated 6 weeks after PC3 cell injection. Representative photomicrographs and a summary of the results are provided in Figure 13.

[0289] As shown in Figure 13, injection of human PCa cell lines resulted in substantial peritoneal muscle infiltration (Figure 13A), whereas PC3-challenged SCID mice administered the humanized anti-hNAMPT antibody P-1076 showed a significant reduction in PC3 infiltration into the smooth muscle peritoneum (Figure 13B).

[0290] Thus, as summarized in Figure 13C , the observations from this study strongly suggest a role for NAMPT in the invasiveness of PCa cells and the important potential of the humanized anti-hNAMPT antibody P-1076 to transientize this invasive behavior.

[0291] Example 17. In vivo treatment of RILI using anti-NAMPT antibodies The ability of anti-NAMPT administration to affect RILI was evaluated using an in vivo experiment in C57 / B6 mice. Mice were divided into four groups: mice that received 20 Gy of thoracic radiation and were intraperitoneally injected with a polyclonal NAMPT neutralizing antibody (pAb); mice that received 20 Gy of thoracic radiation and were intraperitoneally injected with a monoclonal anti-NAMPT antibody (mAb) (P-1076-mod1); non-irradiated mice that were injected with vehicle only ("Ctrl"); and irradiated mice that were injected with vehicle only ("Ctrl"). The amount of BAL protein was measured, and the number of BAL-expressing cells was obtained. Lung tissues were also subjected to H&E staining to evaluate lung inflammation. Furthermore, acute lung injury (ALI) severity scores were assessed based on the BAL index and H&E staining. The results of the corresponding analyses are provided in Figures 14A–E.

[0292] As shown in Figure 14A, H&E staining of lung tissue from irradiated control mice (injected with vehicle only) showed diffuse alveolar damage 4 weeks after radiation exposure (left panel of Figure 14A) compared with lung tissue from non-irradiated control mice (inset in the left panel of Figure 14A). In contrast, lung tissue from mice injected with anti-NAMPT pAb (middle panel of Figure 14A) or anti-NAMPT mAb (right panel of Figure 14A) showed reduced H&E staining, indicating less alveolar damage in mice treated with anti-NAMPT Ab after radiation exposure. Figure 14B summarizes H&E staining in lung tissue from non-irradiated control mice, irradiated control mice, and irradiated mice injected with anti-NAMPT pAb or mAb. As shown in Figure 14B, the H&E stained area increased in lung tissue from irradiated control mice compared with non-irradiated control mice. However, a significant decrease in H&E-stained area was observed in lung tissue from mice injected with anti-NAMPT pAb or mAb compared with irradiated control mice (p<0.05), suggesting a role for NAMPT in the development of RILI. Figure 14C summarizes the BAL protein levels in lung tissue from non-irradiated control mice, irradiated control mice, and irradiated mice injected with anti-NAMPT pAb or mAb. As shown in Figure 14C, compared with non-irradiated control mice, mice exposed to radiation showed an increase in BAL protein levels. However, irradiated mice injected with anti-NAMPT pAb or mAb showed a significant decrease in BAL protein levels compared with irradiated control mice (p<0.05), with a more pronounced decrease observed in irradiated mice treated with anti-NAMPT mAb. Similarly, the number of BAL cells was increased in mice exposed to irradiation, but irradiated mice injected with anti-NAMPT pAb or mAb showed a significant decrease in BAL cell number compared to irradiated control mice (p<0.05), with a more pronounced decrease observed in irradiated mice treated with anti-NAMPT mAb.Furthermore, as shown in Figure 14E, compared with control mice, radiation-exposed mice showed an increase in ALI severity scores, whereas irradiated mice injected with anti-NAMPT pAb or mAb showed a significant decrease in ALI severity scores compared with irradiated control mice, with a more pronounced decrease observed in irradiated mice treated with anti-NAMPT mAb. Thus, these results demonstrate the alleviation of RILI after treatment with anti-NAMPT Ab and highlight NAMPT as a potential therapeutic target for RILI.

[0293] Example 18. Increased NAMPT expression in inflamed lung tissue identified by radiolabeled anti-NAMPT antibody Radiolabeled anti-NAMPT antibodies have been developed for the purpose of noninvasively detecting the NAMPT signaling pathway and NAMPT expression in various tissues in vivo. Imaging a mouse model with RILI using radiolabeled anti-NAMPT mAb (P-1076-mod1) will define the optimal time for developing anti-NAMPT mAb as a therapeutic intervention, and other specific radiolabels can be used to investigate major organs for inflammation and cell apoptosis after total body irradiation (TBI) or partial body irradiation (PBI), such as in a nuclear accident. To test the detection of NAMPT expression with radiolabeled anti-NAMPT antibodies, 99m Tc-labeled anti-NAMPT mAb probe was injected into control mice and mice exposed to 8 Gy of PBI, and rapid autoradiographic imaging was performed. The results of the analysis are shown in Figures 15A-D.

[0294] As shown in Figures 15A-B, higher radioactivity uptake was observed in the lungs of irradiated mice compared with non-irradiated control mice, indicating higher NAMPT expression induced by RILI. Furthermore, uptake of radiolabeled anti-NAMPT antibody was used as a measure of lung activity in irradiated or non-irradiated control mice. As shown in Figure 15C, a significant increase in lung activity above tissue background was observed in both the right and left lungs from irradiated mice compared with the right and left lungs from non-irradiated control mice (p<0.05). Furthermore, the level of radioactivity in irradiated or non-irradiated control mice was determined to assess the uptake of radiolabeled anti-NAMPT mAb. As shown in Figure 15D, a significant increase in radioactivity was observed in irradiated mice compared with non-irradiated control mice (p<0.05), confirming the increased uptake of radiolabeled anti-NAMPT mAb in irradiated mice.

[0295] Therefore, radiolabeled anti-NAMPT antibodies were effective in detecting increased NAMPT expression in inflamed lung tissue, highlighting the potential of radiolabeled anti-NAMPT antibodies as a detection tool for NAMPT and potentially crucial for the use of NAMPT as a biomarker for RILI.

[0296] Example 19. Validation of NAMPT as a therapeutic target for RILI using an in vivo model of radiation-induced pulmonary fibrosis To further validate NAMPT as a therapeutic target for RILI, WT C57 / B6 mice were exposed to 20 Gy WT LI. Irradiated mice were intraperitoneally injected with 10 μg of anti-NAMPT mAb (P-1076-mod1) or vehicle control. Mice were evaluated for radiation-induced pulmonary fibrosis (RILF) 18 weeks after radiation exposure by assessing BAL cell counts, collagen deposition, and lung tissue smooth muscle actin (SMA) expression, a reflection of myofibroblast migration and fibrosis. Results are shown in Figure 16A-C.

[0297] As shown in Figures 16A-C, anti-NAMPT mAb significantly reduced IR-induced RILI in Ab-treated mice compared with vehicle-treated control mice, reflected by reduced BAL cell counts (Figure 16A), reduced lung tissue SMA expression (detected by Western blot analysis and shown in Figure 16B), and reduced collagen deposition (detected by trichrome staining of lung tissue and shown in Figure 16C).

[0298] Thus, the results highlight the role of anti-NAMPT Abs in alleviating RILF and further validate NAMPT as a therapeutic target for RILF.

[0299] Example 20. Evaluation of the efficacy of anti-NAMPT mAb in preclinical models of lung injury The efficacy of anti-NAMPT mAb was tested in a rat model of trauma (blast injury) / ventilator-induced lung injury (VILI). Sprague-Dawley rats were challenged with trauma (blast injury) and then mechanically ventilated for 4 hours. Thirty minutes after blast injury, rats were injected intravenously with 100 μg of anti-NAMPT mAb (P-1076-mod1). Rats exposed to trauma (blast injury) / VILI and injected with vehicle served as controls. Approximately 5 hours after the onset of blast injury, lungs were removed from the rats and evaluated for injury. Edema and inflammatory cell infiltration in lung tissue were also assessed by hematoxylin and eosin (H&E) staining as a readout of lung injury. The results of this trauma (blast injury) / VILI lung injury model are presented in Figure 17A-C.

[0300] As shown in Figure 17A, compared with non-challenged rats (inset in the rightmost box in Figure 17A), lung tissue from vehicle-injected control trauma / VILI rats exhibited inflammatory cell infiltration and edema, indicating trauma / VILI-induced lung injury. In contrast, as shown in Figure 17B, lung tissue from trauma / VILI rats treated with anti-NAMPT mAb exhibited a significant reduction in inflammatory cell infiltration and edema, indicating the attenuation of trauma / VILI-induced lung injury by anti-NAMPT mAb. The effect of anti-NAMPT mAb on trauma / VILI-induced lung injury is summarized in Figure 17C, which shows the lung injury scores of rats as assessed by H&E staining index. As shown in Figure 17C, the lung injury score was significantly reduced in rats treated with anti-NAMPT mAb compared with rats injected with vehicle control (p<0.05). Thus, the results outlined in Figures 17A-C demonstrate the efficacy of NAMPT-neutralizing mAbs in attenuating trauma / VILI-induced lung injury.

[0301] Next, the efficacy of the anti-NAMPT mAb was tested in a mouse model of LPS / VILI. Mice were challenged with LPS for 18 hours and then mechanically ventilated for 4 hours. Mice were injected with anti-NAMPT mAb (P-1076-mod1, 10 μg, IV), anti-NAMPT polyclonal antibody (pAb), or vehicle control (PBS). Mice not exposed to LPS / VILI served as controls. Next, edema and inflammatory cell infiltration in lung tissue from mice were assessed by H&E staining as a readout of lung injury. The results of this LPS / VILI lung injury model are presented in Figures 18A-C.

[0302] As shown in Figure 18A, compared with unchallenged mice (inset in Figure 18A), lung tissue from vehicle-injected control mice showed inflammatory cell infiltration and edema, indicating LPS / VILI-induced lung injury. In contrast, as shown in Figure 18B, lung tissue from mice treated with anti-NAMPT mAb showed a significant reduction in inflammatory cell infiltration and edema, indicating the attenuation of LPS / VILI-induced lung injury by anti-NAMPT mAb. The effect of anti-NAMPT mAb on trauma / VILI-induced lung injury is summarized in Figure 18C, which shows the acute lung injury (ALI) severity scores of mice as assessed by H&E staining index. As shown in Figure 18C, compared with vehicle-injected mice, ALI was significantly reduced in mice treated with anti-NAMPT pAb or mAb, with the strongest reduction in ALI severity score observed in mice treated with anti-NAMPT mAb (p<0.001). Thus, the results outlined in Figures 18A-C demonstrate the efficacy of NAMPT-neutralizing mAbs in attenuating trauma / VILI-induced lung injury.

[0303] Thus, these results demonstrate the efficacy of anti-NAMPT mAb in reducing lung injury in a preclinical in vivo lung injury model.

[0304] Example 21. Increased NAMPT expression in inflamed lung tissue identified by radiolabeled anti-NAMPT antibody We radiolabeled a humanized anti-NAMPT mAb (K-1076) to develop an imaging probe capable of noninvasively detecting the NAMPT signaling pathway and NAMPT expression in various tissues in vivo. Given the potential of NAMPT as a diagnostic and / or prognostic biomarker in acute inflammatory diseases (e.g., COVID-19, ARDS, and lung injury), radiolabeled anti-NAMPT mAb can be used as a diagnostic tool in subjects at risk of developing such diseases or to select subjects likely to respond to treatment with anti-NAMPT mAb. In this example, we describe the use of radiolabeled anti-NAMPT mAb to detect NAMPT expression in inflamed tissues, such as lungs, after LPS challenge and ionizing radiation exposure.

[0305] First, to test the detection of NAMPT expression using radiolabeled anti-NAMPT antibody, 99m Tc-labeled anti-NAMPT mAb probe or radiolabeled IgG control Ab was injected into mice exposed to 20 Gy whole chest lung irradiation (WTLI), and rapid autoradiographic imaging was performed.

[0306] As shown in Figure 19A, significantly higher radioactive uptake was observed in irradiated mice injected with radiolabeled anti-NAMPT mAb (PRONAMPTOR) (right panel of Figure 19A) compared with irradiated mice injected with radiolabeled IgG control (left panel of Figure 19A). Thus, the results shown in Figure 19A demonstrate the ability of the radiolabeled anti-NAMPT imaging probe in detecting radiation-induced NAMPT expression.

[0307] To further evaluate the detection of NAMPT expression by radiolabeled anti-NAMPT imaging probes, 99m Tc-labeled anti-NAMPT mAb was injected into vehicle-challenged control mice or LPS-challenged mice 3 or 18 hours after LPS challenge, and rapid autoradiographic imaging was performed. The results of the analysis are shown in Figure 19B-D.

[0308] As shown in Figure 19B, compared with control mice (left panel of Figure 19B), LPS-challenged mice showed significantly higher uptake of the radiolabeled anti-NAMPT imaging probe 3 hours after LPS challenge (right panel of Figure 19B). Autoradiographic imaging of lungs from LPS-challenged or control mice further confirmed this observation; compared with control mice (left panel of Figure 19C), the lungs of LPS-challenged mice showed significantly higher uptake of the radiolabeled anti-NAMPT imaging probe 3 hours after LPS challenge (right panel of Figure 19C). Furthermore, as shown in Figure 19D, compared with control mice, LPS-challenged mice showed significantly higher radioactivity 3 and 18 hours after LPS challenge (p<0.05), indicating higher uptake of the radiolabeled anti-NAMPT imaging probe. Therefore, the results depicted in Figures 19B–D demonstrate the ability of the radiolabeled anti-NAMPT imaging probe in detecting LPS-induced NAMPT expression.

[0309] Thus, radiolabeled anti-NAMPT antibodies were effective in detecting increased NAMPT expression in inflamed tissues. This highlights the potential of radiolabeled anti-NAMPT antibodies as a tool for detecting NAMPT, which may be crucial for using NAMPT as a diagnostic and / or prognostic biomarker in acute inflammatory diseases. Furthermore, by detecting increased NAMPT expression in inflamed tissues, this radiolabeled anti-NAMPT imaging probe can be used to select subjects likely to respond to treatment of acute inflammatory diseases with neutralizing anti-NAMPT mAbs.

[0310] Example 22. Reduction of PAH manifestations in a rat model by anti-NAMPT antibodies To explore the potential of NAMPT as a therapeutic target for PAH, we used the rat monocrotaline (MCT) model of PAH. A single dose (60 mg / kg body weight) of MCT was subcutaneously injected into Sprague-Dawley rats (190–200 g). The MCT-challenged rats were then injected twice weekly with either anti-NAMPT mAb (P-1076-mod1, i.p., 100 μg / rat) or vehicle control (control MCT rats). Next, the rats were evaluated for right ventricular systolic pressure (RVSP) and pulmonary artery remodeling. The results are shown in Figures 20A and 20B.

[0311] RVSP was measured in MCT rats treated with anti-NAMPT mAb or control MCT rats by right heart catheterization using a Millar pressure transducer catheter. As shown in Figure 20A, a significant decrease in RVSP was observed in MCT rats treated with anti-NAMPT mAb compared with control MCT rats (p<0.05).

[0312] Lungs from anti-NAMPT Ab-treated or control MCT rats were stained with H&E, and pulmonary artery remodeling was evaluated using APERIO IMAGESCOPE software. As shown in Figure 20B, a significant decrease in pulmonary artery thickness was observed in MCT rats treated with anti-NAMPT mAb compared with control MCT rats.

[0313] The results show that neutralization of NAMPT with anti-NAMPT mAb reverses vascular remodeling and RV dysfunction in a rat model of PAH, thus demonstrating the efficacy of NAMPT as a therapeutic target for PAH.

[0314] Unless otherwise stated, the disclosures of all patents, patent applications, and publications cited herein are incorporated herein by reference to the extent that the reference describes information relevant to the present disclosure. While the present invention has been disclosed with reference to particular embodiments, it will be apparent that several embodiments and variations of the present invention may be devised by those skilled in the art without departing from the true spirit and scope of the present invention. The appended claims include all such embodiments and equivalent variations.

[0315] The sequences disclosed herein and / or related to the present invention are set out in the sequence summary table below (Table 17).

[0316] [Table 52] [Table 53] [Table 54] [Table 55] [Table 56] [Table 57] [Table 58] [Table 59] [Table 60] [Table 61] [Table 62]

Claims

1. 1. An isolated antibody or antigen-binding fragment thereof that binds to human nicotinamide phosphoribosyltransferase (NAMPT), comprising: (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 comprising 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; (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 comprising 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; (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: 4, and a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 5; and a light chain variable region comprising 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; (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 comprising 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; (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 comprising 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; (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 comprising 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; (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 comprising 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 comprising 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 comprising 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; or (j) An isolated antibody or antigen-binding fragment thereof, comprising 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 comprising 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.

2. 2. The isolated antibody or antigen-binding fragment thereof of claim 1, which is humanized.

3. The isolated antibody or antigen-binding fragment thereof of claim 1 or 2, wherein the light chain variable region has the amino acid sequence set forth in SEQ ID NO: 2, 10, 13, 30, 31, 32, 34 or 36.

4. The isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 3, wherein the heavy chain variable region has the amino acid sequence set forth in SEQ ID NO: 1, 9, 15, 16 or 28.

5. An isolated antibody or antigen-binding fragment thereof that binds to human NAMPT, comprising a heavy chain comprising a variable region comprising the amino acid sequence set forth in SEQ ID NO: 15, and a light chain comprising a variable region comprising the amino acid sequence set forth in SEQ ID NO:

13.

6. An isolated antibody or antigen-binding fragment thereof that binds to human NAMPT, comprising a heavy chain comprising a variable region comprising the amino acid sequence set forth in SEQ ID NO: 26, and a light chain comprising a variable region comprising the amino acid sequence set forth in SEQ ID NO:

18.

7. 1. An isolated antibody or antigen-binding fragment thereof that binds to human NAMPT, a heavy chain comprising a variable region comprising the amino acid sequence set forth in SEQ ID NO: 17, and a light chain comprising a variable region comprising the amino acid sequence set forth in SEQ ID NO: 18; a heavy chain comprising a variable region comprising the amino acid sequence set forth in SEQ ID NO: 23, and a light chain comprising a variable region comprising the amino acid sequence set forth in SEQ ID NO: 24; a heavy chain comprising a variable region comprising the amino acid sequence set forth in SEQ ID NO:25, and a light chain comprising a variable region comprising the amino acid sequence set forth in SEQ ID NO:24; a heavy chain comprising a variable region comprising the amino acid sequence set forth in SEQ ID NO: 25, and a light chain comprising a variable region comprising the amino acid sequence set forth in SEQ ID NO: 18; a heavy chain comprising a variable region comprising the amino acid sequence set forth in SEQ ID NO:26, and a light chain comprising a variable region comprising the amino acid sequence set forth in SEQ ID NO:24; a heavy chain comprising a variable region comprising the amino acid sequence set forth in SEQ ID NO: 27, and a light chain comprising a variable region comprising the amino acid sequence set forth in SEQ ID NO: 24; a heavy chain comprising a variable region comprising the amino acid sequence set forth in SEQ ID NO: 27, and a light chain comprising a variable region comprising the amino acid sequence set forth in SEQ ID NO: 18; a heavy chain comprising a variable region comprising the amino acid sequence set forth in SEQ ID NO: 38, and a light chain comprising a variable region comprising the amino acid sequence set forth in SEQ ID NO: 47; a heavy chain comprising a variable region comprising the amino acid sequence set forth in SEQ ID NO: 40, and a light chain comprising a variable region comprising the amino acid sequence set forth in SEQ ID NO: 47; a heavy chain comprising a variable region comprising the amino acid sequence set forth in SEQ ID NO: 42, and a light chain comprising a variable region comprising the amino acid sequence set forth in SEQ ID NO: 47; a heavy chain comprising a variable region comprising the amino acid sequence set forth in SEQ ID NO:45, and a light chain comprising a variable region comprising the amino acid sequence set forth in SEQ ID NO:47; a heavy chain comprising a variable region comprising the amino acid sequence set forth in SEQ ID NO: 38, and a light chain comprising a variable region comprising the amino acid sequence set forth in SEQ ID NO: 48; a heavy chain comprising a variable region comprising the amino acid sequence set forth in SEQ ID NO: 40, and a light chain comprising a variable region comprising the amino acid sequence set forth in SEQ ID NO: 48; a heavy chain comprising a variable region comprising the amino acid sequence set forth in SEQ ID NO: 42, and a light chain comprising a variable region comprising the amino acid sequence set forth in SEQ ID NO: 48; a heavy chain comprising a variable region comprising the amino acid sequence set forth in SEQ ID NO:45, and a light chain comprising a variable region comprising the amino acid sequence set forth in SEQ ID NO:48; a heavy chain comprising a variable region comprising the amino acid sequence set forth in SEQ ID NO: 38, and a light chain comprising a variable region comprising the amino acid sequence set forth in SEQ ID NO: 51; a heavy chain comprising a variable region comprising the amino acid sequence set forth in SEQ ID NO: 40, and a light chain comprising a variable region comprising the amino acid sequence set forth in SEQ ID NO: 51; a heavy chain comprising a variable region comprising the amino acid sequence set forth in SEQ ID NO: 42, and a light chain comprising a variable region comprising the amino acid sequence set forth in SEQ ID NO: 51; a heavy chain comprising a variable region comprising the amino acid sequence set forth in SEQ ID NO: 45, and a light chain comprising a variable region comprising the amino acid sequence set forth in SEQ ID NO: 51; a heavy chain comprising a variable region comprising the amino acid sequence set forth in SEQ ID NO: 38, and a light chain comprising a variable region comprising the amino acid sequence set forth in SEQ ID NO: 18; a heavy chain comprising a variable region comprising the amino acid sequence set forth in SEQ ID NO: 40, and a light chain comprising a variable region comprising the amino acid sequence set forth in SEQ ID NO: 18; a heavy chain comprising a variable region comprising the amino acid sequence set forth in SEQ ID NO: 42, and a light chain comprising a variable region comprising the amino acid sequence set forth in SEQ ID NO: 18; a heavy chain comprising a variable region comprising the amino acid sequence set forth in SEQ ID NO:45, and a light chain comprising a variable region comprising the amino acid sequence set forth in SEQ ID NO:18; a heavy chain comprising a variable region comprising the amino acid sequence set forth in SEQ ID NO:26, and a light chain comprising a variable region comprising the amino acid sequence set forth in SEQ ID NO:47; a heavy chain comprising a variable region comprising the amino acid sequence set forth in SEQ ID NO:26 and a light chain comprising a variable region comprising the amino acid sequence set forth in SEQ ID NO:48; or a heavy chain comprising a variable region comprising the amino acid sequence set forth in SEQ ID NO: 26, and a light chain comprising a variable region comprising the amino acid sequence set forth in SEQ ID NO: 51; 1. An isolated antibody or antigen-binding fragment thereof comprising:

8. 1. An isolated antibody or antigen-binding fragment thereof that binds to human NAMPT, a heavy chain comprising a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 19, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 20, and a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 21, and a light chain comprising a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 6, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 7, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 22; a heavy chain comprising a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 19, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 39, and a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 21, and a light chain comprising a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 7, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 22; a heavy chain comprising a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 19, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 39, and a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 41, and a light chain comprising a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 7, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 22; a heavy chain comprising a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:43, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:39, and a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:44, and a light chain comprising a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:11, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:7, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:22; a heavy chain comprising a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:46, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:39, and a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:41, and a light chain comprising a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:11, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:7, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:22; a heavy chain comprising a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 19, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 39, and a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 21, and a light chain comprising a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 49, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 50, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 22; a heavy chain comprising a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 19, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 39, and a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 41, and a light chain comprising a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 49, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 50, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 22; a heavy chain comprising a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:43, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:39, and a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:44, and a light chain comprising a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:49, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:50, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:22; a heavy chain comprising a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:46, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:39, and a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:41, and a light chain comprising a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:49, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:50, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:22; a heavy chain comprising a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 19, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 39, and a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 21, and a light chain comprising a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 52, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 53; a heavy chain comprising a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 19, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 39, and a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 41, and a light chain comprising a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 52, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 53; a heavy chain comprising a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:43, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:39, and a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:44, and a light chain comprising a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:11, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:52, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:53; a heavy chain comprising a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:46, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:39, and a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:41, and a light chain comprising a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:11, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:52, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:53; a heavy chain comprising a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 19, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 39, and a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 21, and a light chain comprising a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 6, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 7, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 22; a heavy chain comprising a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 19, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 39, and a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 41, and a light chain comprising a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 6, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 7, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 22; a heavy chain comprising a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:43, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:39, and a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:44, and a light chain comprising a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:6, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:7, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:22; a heavy chain comprising a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:46, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:39, and a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:41, and a light chain comprising a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:6, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:7, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:22; a heavy chain comprising a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 19, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 20, and a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 21, and a light chain comprising a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 7, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 22; a heavy chain comprising a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 19, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 20, and a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 21, and a light chain comprising a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 49, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 50, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 22; or a heavy chain comprising a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 19, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 20, and a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 21, and a light chain comprising a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 52, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 53; 1. An isolated antibody or antigen-binding fragment thereof comprising:

9. An isolated anti-NAMPT antibody comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:54 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:

55.

10. An isolated anti-NAMPT antibody comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:56 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:

57.

11. a humanized heavy chain variable region derived from a mouse antibody and a humanized light chain variable region derived from the mouse antibody, The mouse antibody a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 54 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 55; or A heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 56 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 57, An isolated humanized anti-NAMPT antibody.

12. 12. The isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 11, comprising an Fc domain.

13. The isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 12, which is a monoclonal antibody.

14. The isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 13, which is an IgG antibody.

15. 15. The isolated antibody or antigen-binding fragment thereof of claim 14, which is an IgG1 or IgG4 antibody.

16. A nucleic acid encoding the isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 15.

17. A vector comprising the nucleic acid of claim 16.

18. 18. A host cell comprising the nucleic acid of claim 16 or the vector of claim 17.

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

20. A medicament for treating a disorder associated with deleterious NAMPT activity, comprising the isolated antibody or antigen-binding fragment of any one of claims 1 to 15.

21. A medicament for treating an inflammatory disease comprising the isolated antibody or antigen-binding fragment of any one of claims 1 to 15.

22. The pharmaceutical of claim 21, wherein the inflammatory disease is 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, or radiation-induced lung injury.

23. The pharmaceutical of claim 22, wherein the radiation-induced lung damage is caused by radiation associated with cancer treatment.

24. A medicament for treating prostate cancer (PCa), comprising the isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 15.

25. The pharmaceutical composition of claim 24, for administration to a subject with recurrent PCa.

26. The pharmaceutical composition of claim 24, for administration to a subject at risk of developing metastatic PCa.

27. The pharmaceutical composition of any one of claims 24 to 26, wherein the PCa is resistant to androgen deprivation therapy (ADT).

28. The pharmaceutical agent according to any one of claims 24 to 27, for administration to a subject in combination with ADT.

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