Anti-NAPRT Antibody and Method of Use

Abstract

The present disclosure provides an antibody and a polypeptide that specifically bind to nicotinate phosphoribosyltransferase (NAPRT). Additionally, a nucleic acid encoding such an antibody, an expression vector and a host cell for producing such an antibody, a method for detecting NAPRT in a sample from a subject requiring such an antibody using such an antibody, and a method for diagnosing a subject having cancer or sepsis using such an antibody are provided. Additionally, a composition comprising such an antibody is provided. Additionally, a composition comprising a nicotinamide phosphoribosyltransferase inhibitor (NAMPTi) and a method for treating cancer using NAMPTi after diagnosis using the NAPRT antibody are provided.

Description

Technology Field

[0001] Cross-reference regarding related applications

[0002] This application claims the benefit and priority of U.S. provisional application serial number 63 / 598,869 filed on November 14, 2023, the contents of which are incorporated herein by reference in their entirety.

[0003] field

[0004] The present disclosure relates to an antibody specific to human nicotinate phosphoribosyltransferase (NAPRT) and a method of using the same. Background Technology

[0005] The nicotinate phosphoribosyltransferase (NAPRT) gene is gaining importance in research on cancer treatment strategies due to its major role as a nicotinamide adenine dinucleotide (NAD) biosynthesizing enzyme. Given the high energy requirements of proliferating cancer cells and NAD-dependent signaling, NAD metabolism is an attractive target for the development of anticancer therapies. A lack of NAPRT expression has been observed in various cancer types and has been associated with NAPRT epigenetic silencing in some cases, such as gastric and lung cancers. Conversely, NAPRT amplification and overexpression have been reported in ovarian, breast, and pancreatic cancers. Differences in NAPRT expression among cancer subtypes—namely breast, pancreatic, lung, and gastric carcinomas—suggest that individual variability must be considered in therapeutic approaches.

[0006] Therefore, there is a need for antibodies that specifically bind to NAPRT.

[0007] The present disclosure provides antibodies and polypeptides that specifically bind to NAPRT (e.g., human NAPRT). Additionally, pharmaceutical compositions comprising such antibodies, nucleic acids encoding such antibodies, expression vectors and host cells for producing such antibodies, and methods for detecting NAPRT in samples from subjects using such antibodies are provided. The anti-NAPRT antibodies provided herein are also useful for indicating the potential for a subject with cancer to be effectively treated with a nicotinamide phosphoribosyltransferase inhibitor (NAMPTi). The anti-NAPRT antibodies provided herein are also useful for indicating the potential for a subject to have sepsis.

[0008] In one embodiment, the present invention provides an antibody that specifically binds to human NAPRT, wherein the antibody comprises VH comprising the CDRH1, CDRH2, and CDRH3 amino acid sequences of the VH amino acid sequence presented in SEQ ID NO. 1; and VL comprising the CDRL1, CDRL2, and CDRL3 amino acid sequences of the VL amino acid sequence presented in SEQ ID NO. 2.

[0009] In certain embodiments, the antibodies each comprise the CDRH1, CDRH2, and CDRH3 amino acid sequences presented in SEQ ID NOs 3, 4, and 5; 9, 10, and 5; 11, 12, and 13; 15, 16, and 5; or 17, 18, and 19.

[0010] In certain embodiments, the antibodies each comprise the CDRL1, CDRL2, and CDRL3 amino acid sequences presented in SEQ ID NOs 6, 7, and 8; 14, LM, and 8; 14, LMS, and 8; or 20, 21, and 8.

[0011] In certain embodiments, the antibodies each comprise the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences presented in SEQ ID NOs 3, 4, 5, 6, 7, and 8; 9, 10, 5, 6, 7, and 8; 11, 12, 13, 14, LM, and 8; 11, 12, 13, 14, LMS, and 8; 15, 16, 5, 6, 7, and 8; and 17, 18, 19, 20, 21, and 8.

[0012] In a specific embodiment, VH comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO. 1, and VL comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO. 2.

[0013] In certain embodiments, the antibody comprises the VH amino acid sequence and the VL amino acid sequence presented in SEQ ID NOs 1 and 2, respectively.

[0014] In certain embodiments, the antibody includes a heavy chain constant region optionally selected from the group consisting of human IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

[0015] In certain embodiments, the antibody includes a heavy chain constant region that is a variant of the wild-type heavy chain constant region, and the variant heavy chain constant region binds to FcγR with a lower affinity than the wild-type heavy chain constant region binds to FcγR.

[0016] In another aspect, the present invention provides an antibody that cross-competes for binding to NAPRT with any anti-NAPRT antibody described herein.

[0017] In another embodiment, the present invention provides an antibody that specifically binds to the amino acid sequence of SEQ ID NO. 29 or 30.

[0018] In another aspect, the present invention provides a polypeptide comprising VH comprising the CDRH1, CDRH2, and CDRH3 amino acid sequences of the VH amino acid sequence presented in SEQ ID NO. 1. In certain embodiments, VH comprises the CDRH1, CDRH2, and CDRH3 amino acid sequences presented in SEQ ID NOs. 3, 4, and 5; 9, 10, and 5; 11, 12, and 13; 15, 16, and 5; or 17, 18, and 19.

[0019] In another aspect, a polypeptide comprising VL comprising the CDRL1, CDRL2, and CDRL3 amino acid sequences of the VL amino acid sequence presented in SEQ ID NO. 2 is provided. In certain embodiments, VL comprises each of SEQ ID NOs. 6, 7, and 8; 14, LM, and 8; 14, LMS, and 8; or CDRL1, CDRL2, and CDRL3 presented in 20, 21, and 8.

[0020] In another embodiment, the present invention provides a polypeptide comprising the amino acid sequence LM, LMS, or any one of SEQ ID NOs 1 to 21.

[0021] In another embodiment, the present invention provides the VH, VL, heavy chain and / or light chain of any antibody described herein; or a polynucleotide encoding any polypeptide described herein.

[0022] In another aspect, the present invention provides a vector comprising any polynucleotide described herein.

[0023] In another aspect, the present invention provides a recombinant host cell comprising: a polynucleotide described herein, a vector described herein, a first polynucleotide encoding a heavy chain variable region or heavy chain of an antibody described herein, and a second polynucleotide encoding a light chain variable region or light chain of an antibody described herein; and / or a first vector comprising a first polynucleotide encoding a heavy chain variable region or heavy chain of an antibody described herein, and a second vector comprising a second polynucleotide encoding a light chain variable region or light chain of an antibody described herein.

[0024] In another aspect, the present invention provides a composition comprising the antibody described herein, the polypeptide described herein, the polynucleotide described herein, the vector described herein, or the host cell described herein, and a pharmaceutically acceptable carrier or excipient.

[0025] In another aspect, the present invention provides a method for producing antibodies, comprising the step of culturing the host cells described herein under appropriate conditions so that a polynucleotide is expressed and an antibody is produced.

[0026] In another aspect, the present invention provides a method for determining whether a sample containing cells from a subject is nicotinic acid phosphoribosyltransferase (NAPRT) negative, comprising the steps of administering an antibody that specifically binds to NAPRT to a sample and detecting the specific binding of the antibody to NAPRT in less than 1% of the cells in the sample to determine that the sample is NAPRT negative.

[0027] In certain embodiments, the sample comprises cancer cells. In certain embodiments, the cancer cells are solid tumor cells. In certain embodiments, the cancer cells are thyroid cancer, duodenal cancer, neuroendocrine carcinoma (NEC), uterine cancer, small cell lung cancer (SCLC), hepatocellular carcinoma, mesothelioma, breast cancer, sarcoma, ovarian cancer, renal cell carcinoma, rectal cancer, head and neck cancer, prostate cancer, pancreatic cancer, melanoma, colorectal cancer, cervical cancer, non-small cell lung cancer (NSCLC), cholangiocarcinoma, or endometrial cancer cells.

[0028] In another aspect, the present invention provides a method for treating cancer comprising the step of administering a nicotinamide phosphoribosyltransferase inhibitor (NAMPTi) to a subject requiring treatment for cancer, wherein the sample from the cancer has a lower NAPRT level than the control sample.

[0029] In a specific embodiment, the method further includes the step of administering niacin to a subject.

[0030] In certain embodiments, the cancer is NAPRT negative.

[0031] In certain embodiments, the cancer is thyroid cancer, duodenal cancer, neuroendocrine carcinoma (NEC), uterine cancer, small cell lung cancer (SCLC), hepatocellular carcinoma, mesothelioma, breast cancer, sarcoma, ovarian cancer, renal cell carcinoma, rectal cancer, head and neck cancer, prostate cancer, pancreatic cancer, melanoma, colorectal cancer, cervical cancer, non-small cell lung cancer (NSCLC), cholangiocarcinoma, or endometrial cancer.

[0032] In another aspect, the present invention provides a method for diagnosing a subject having cancer with an increased likelihood of being effectively treated with NAMPTi, comprising the steps of administering an antibody that specifically binds to NAPRT to a sample from the subject and detecting the specific binding of the antibody to NAPRT, wherein if the binding to NAPRT in the sample from the subject is less than in a control sample, the likelihood of the subject's cancer being effectively treated with NAMPTi is increased.

[0033] In certain embodiments, the likelihood that the cancer of the subject can be effectively treated with niacin and NAMPTi is increased.

[0034] In certain embodiments, the cancer is thyroid cancer, duodenal cancer, neuroendocrine carcinoma (NEC), uterine cancer, small cell lung cancer (SCLC), hepatocellular carcinoma, mesothelioma, breast cancer, sarcoma, ovarian cancer, renal cell carcinoma, rectal cancer, head and neck cancer, prostate cancer, pancreatic cancer, melanoma, colorectal cancer, cervical cancer, non-small cell lung cancer (NSCLC), cholangiocarcinoma, or endometrial cancer cells.

[0035] In another aspect, the present invention provides a method for diagnosing a patient with sepsis, comprising the steps of administering an antibody that specifically binds to NAPRT to a sample from a subject and detecting the specific binding of the antibody to NAPRT, wherein if the binding to NAPRT is greater in the sample from the subject than in the control sample, the subject is more likely to have sepsis.

[0036] In a specific embodiment, the sample from the subject is NAPRT voice.

[0037] In certain embodiments, NAPRT comprises the amino acid sequence of SEQ ID NO. 29 or 30.

[0038] In a specific embodiment, the antibody comprises VH comprising the CDRH1, CDRH2, and CDRH3 amino acid sequences of the VH amino acid sequence presented in SEQ ID NO. 1; and VL comprising the CDRL1, CDRL2, and CDRL3 amino acid sequences of the VL amino acid sequence presented in SEQ ID NO. 2.

[0039] In certain embodiments, the antibodies each comprise the CDRH1, CDRH2, and CDRH3 amino acid sequences presented in SEQ ID NOs 3, 4, and 5; 9, 10, and 5; 11, 12, and 13; 15, 16, and 5; or 17, 18, and 19.

[0040] In certain embodiments, the antibodies each comprise the CDRL1, CDRL2, and CDRL3 amino acid sequences presented in SEQ ID NOs 6, 7, and 8; 14, LM, and 8; 14, LMS, and 8; or 20, 21, and 8.

[0041] In certain embodiments, the antibodies each comprise the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences presented in SEQ ID NOs 3, 4, 5, 6, 7, and 8; 9, 10, 5, 6, 7, and 8; 11, 12, 13, 14, LM, and 8; 11, 12, 13, 14, LMS, and 8; 15, 16, 5, 6, 7, and 8; and 17, 18, 19, 20, 21, and 8.

[0042] In a specific embodiment, VH comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO. 1, and VL comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO. 2.

[0043] In certain embodiments, the antibody comprises a heavy chain constant region selected from the group consisting of human IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

[0044] In certain embodiments, the antibody includes a heavy chain constant region that is a variant of the wild-type heavy chain constant region, and the variant heavy chain constant region binds to FcγR with a lower affinity than the wild-type heavy chain constant region binds to FcγR. Brief explanation of the drawing

[0045] Fig. 1 This represents a Western blot experiment showing the combination of two hybridoma clones for human NAPRT. FIGS. 2a to 2dFigures 2a to 2c illustrate exemplary data demonstrating the efficacy of the different NAMPTis of the present application in two patient-derived colorectal adenocarcinoma in vitro models (one of which is NAPRT-positive and the other is NAPRT-negative). After resection of the transplanted tumors, the tissues were cryopreserved for future use or used immediately. Tumor samples were dissociated into single cells and smaller fragments by enzymatic digestion, filtered to remove large fragments, and then treated with increasing concentrations of NAMPTi in the presence of nicotinic acid. Figures 2a to 2c show the in vitro effects of three different NAMPTis, ATN-249 (Fig. 2a), ATN-940 (Fig. 2b), and ATN-1083 (Fig. 2c), and their respective ECs in NAPRT-negative and NAPRT-positive patient-derived colorectal cancer in vitro models. 50 This is a graph showing the values ​​(96-hour exposure, 0.1% final DMSO v / w; endpoint readings were made using Cell Titer Glow 2.0 for viability via ATP detection). Figure 2d shows the NAPRT immunohistochemical staining and negative background (Ms mAb) staining of two patient-derived in vitro colorectal cancer models (NAPRT-negative and NAPRT-positive). FIGS. 3a to 3d Figures 3 and 3 represent exemplary data showing the in vitro effects of NAMPTi ATN-249 in various patient-derived tumor models (NAPRT-positive in Figures 3a and 3b, and NAPRT-negative in Figures 3c and 3d). NAPRT status was confirmed by histochemical immunochemistry (IHC) and Western blot (WB) using anti-NAPRT clone 4A5D7. Each graph includes a dashed line representing 55% cytotoxicity, which was used to define whether the model responds to ATN-249. Fig. 4This is a graph showing exemplary in vivo data demonstrating that NAMPTi ATN-940 induced strong inhibition of patient-derived NAPRT-negative tumors, while having minimal effect on tumor growth of patient-derived NAPRT-positive tumors. Fig. 5 This is a table providing details regarding the different in vitro models used in the experiments shown in Figures 2a to 2d. Specific details for implementing the invention

[0046] The present disclosure provides an anti-NAPRT antibody and a polypeptide that specifically binds to NAPRT. Additionally, a pharmaceutical composition comprising such antibody, a nucleic acid encoding such antibody, an expression vector and a host cell for producing such antibody, and a method for detecting NAPRT in a sample from a subject using such antibody are provided. In some embodiments, the anti-NAPRT antibody provided herein indicates the likelihood that a subject with cancer will be effectively treated with a nicotinamide phosphoribosyltransferase inhibitor (NAMPTi). In some embodiments, if the cells in the sample are NAPRT negative, this indicates an increased likelihood that a subject with cancer will be effectively treated with a nicotinamide phosphoribosyltransferase inhibitor (NAMPTi). In some embodiments, the anti-NAPRT antibody indicates the likelihood that a subject will have sepsis.

[0047] definition

[0048] As used herein, the term “NAPRT” refers to nicotinic acid phosphoribosyltransferase, an enzyme that catalyzes the ATP-dependent synthesis of beta-nicotinate D-ribonucleotide from nicotinate and 5-phospho-D-ribose 1-pyrophosphate, which is the first step in the biosynthesis of NAD from nicotinic acid. As used herein, the term “human NAPRT” refers to the protein encoded by the wild-type human NAPRT gene (e.g., the gene presented in Genbank registry number NM_145201.6 or NCBI gene ID: 93100). An exemplary amino acid sequence of a human NAPRT protein is presented in NCBI reference sequence: NP_660202.3. In some embodiments, the human NAPRT protein comprises the amino acid sequence of SEQ ID NO. 31. In some embodiments, the human NAPRT comprises the sequence presented in UniProtKB registry number Q6XQN6-1. In some embodiments, the human NAPRT comprises the sequence presented in NCBI reference sequence: NP_660202.3. In some embodiments, the standard amino acid sequence human NAPRT comprises the amino acid sequence of SEQ NO. 36. In some embodiments, the human NAPRT comprises the sequence presented in UniProtKB registry number Q6XQN6-2. In some embodiments, the functional human NAPRT comprises the amino acid sequence of SEQ NO. 37. In some embodiments, the human NAPRT comprises the sequence presented in UniProtKB registry number Q6XQN6-3. In some embodiments, the human NAPRT comprises the sequence presented in NCBI reference sequence: NP_001273758.1. In some embodiments, the functional human NAPRT comprises the amino acid sequence of SEQ NO. 38. In some embodiments, the functional human NAPRT comprises the amino acid sequence of SEQ NO. 39. In some embodiments, the NAPRT comprises any one of SEQ NOs 29 through 40.

[0049] In some embodiments, NAPRT is selected from Table 1 below.

[0050]

[0051] As used herein, the term "detectable NAPRT" refers to NAPRT that can be detected in a sample using one of the anti-NAPRT antibodies or polypeptides disclosed herein.

[0052] As used herein, with respect to a sample containing cells, the term “NAPRT negative” means that less than 1% of the cells in the sample have detectable NAPRT. As used herein with respect to cancer, the term “NAPRT negative” means that less than 1% of the cells in the sample from cancer have detectable NAPRT. In some embodiments, the NAPRT detectable in the sample is by using one of the anti-NAPRT antibodies or polypeptides disclosed herein.

[0053] As used herein, the terms “antibody” and “antibodies” include full-length antibodies, antigen-binding fragments of full-length antibodies, and molecules comprising antibody CDR, VH regions, and / or VL regions. Examples of antibodies include, without limitation, monoclonal antibodies, recombinant antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies comprising two heavy chain and two light chain molecules, antibody light chain monomers, antibody heavy chain monomers, antibody light chain dimers, antibody heavy chain dimers, antibody light chain-antibody heavy chain pairs, intrabodies, heteroconjugated antibodies, antibody-drug conjugates, monodomain antibodies, monovalent antibodies, single-chain antibodies or single-chain Fv(scFv), camelization antibodies, affibodies, Fab fragments, F(ab')2 fragments, disulfide-linked Fv(sdFv), anti-idiotype (anti-Id) antibodies (e.g., including anti-anti-Id antibodies), and antigen-binding fragments of any of the above. In certain embodiments, the antibodies described herein refer to a polyclonal antibody group. Antibodies are of any type (e.g., IgG, IgE, IgM, IgD, IgA, or IgY) and any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA 1, It may be an immunoglobulin molecule of IgA2), or any subclass (e.g., IgG2a or IgG2b). In certain embodiments, the antibody described herein is an IgG antibody, or a class thereof (e.g., human IgG1 or IgG4) or a subclass thereof. In certain embodiments, the antibody is a humanized monoclonal antibody. In another specific embodiment, the antibody is a human monoclonal antibody.

[0054] "Multispecific antibodies" are antibodies that specifically bind to two or more different antigens or two or more different regions of the same antigen (e.g., bispecific antibodies). Multispecific antibodies include bispecific antibodies that have two different antigen-binding sites (excluding the Fc region). Multispecific antibodies may include, for example, recombinant antibodies, human antibodies, humanized antibodies, resurfaced antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies containing two heavy chains and two light chain molecules, antibody light chain monomers, heteroconjugated antibodies, linked single-chain antibodies or linked single-chain Fv (scFv), camelized antibodies, affibodies, linked Fab fragments, F(ab')2 fragments, chemically linked Fv, and disulfide-linked Fv (sdFv). The multispecific antibody may be an immunoglobulin molecule of any type (e.g., IgG, IgE, IgM, IgD, IgA, or IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2), or any subclass (e.g., IgG2a or IgG2b). In certain embodiments, the multispecific antibody described herein is an IgG antibody, or a class thereof (e.g., human IgG1, IgG2, or IgG4) or a subclass thereof.

[0055] As used herein, the term “CDR” or “complementary determining region” refers to discontinuous antigen-binding sites found within variable regions of heavy and light chain polypeptides. Such specific regions have been described, for example, by Kabat et al. (Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) and Kabat et al., Sequences of protein of immunological interest. (1991)), Chothia et al. (Chothia et al., J. Mol. Biol. 196: 901-917 (1987)), and MacCallum et al. (MacCallum et al., J. Mol. Biol. 262: 732-745 (1996)), all of which are incorporated herein by reference in their entirety, and the definitions include an overlap or subset of amino acid residues when compared with one another. In certain embodiments, the term “CDR” is used in MacCallum et al. (MacCallum et al., J. Mol. Biol. 262:732-745 (1996) and Martin A. “Protein Sequence and Structure Analysis of Antibody Variable Domains,” in Antibody Engineering , Kontermann and D It is a CDR as defined by Springer-Verlag, Berlin (2001), bel, eds., Chapter 31, pp. 422-439, Springer-Verlag, Berlin (2001). In certain embodiments, the term "CDR" is a CDR as defined by Kabat et al. (Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) and Kabat et al., Sequences of protein of immunological interest. (1991)). In certain embodiments, the heavy chain CDR and light chain CDR of the antibody are defined using different conventions. In certain embodiments, the heavy chain CDR and / or light chain CDR are defined by performing structural analysis of the antibody and identifying residues within variable region(s) predicted to come into contact with the epitope region of the target molecule (e.g., human NAPRT). CDRH1, CDRH2, and CDRH3 represent the heavy chain CDR, and CDRL1, CDRL2, and CDRL3 represent the light chain CDR.

[0056] As used herein, the terms “variable region” and “variable domain” are used interchangeably and are common in the art. A variable region typically refers to a portion of an antibody, generally a portion of the light or heavy chain, typically about 110 to 120 amino acids or 110 to 125 amino acids at the amino terminus of the mature heavy chain, and about 90 to 115 amino acids in the mature light chain, which vary significantly in sequence among antibodies and are used for the binding and specificity of a particular antibody to a particular antigen. Sequence variability is concentrated in regions called complementarity determining regions (CDRs), whereas a more highly conserved region within the variable region is called the framework region (FR). While not wishing to be bound by specific mechanisms or theories, it is believed that the CDRs of the light and heavy chains are primarily responsible for the interaction and specificity of the antibody and antigen. In certain embodiments, the variable region is a human variable region. In certain embodiments, the variable region includes a rodent or murine CDR and a human framework region (FR). In certain embodiments, the variable region is a primate (e.g., non-human primate) variable region. In certain embodiments, the variable region includes a rodent or murine CDR and a primate (e.g., non-human primate) framework region (FR).

[0057] As used herein, the terms “VH” and “VL” refer to the antibody heavy chain and light chain variable regions, respectively, as described in the literature (Kabat et al., (1991) Sequences of Proteins of Immunological Interest (NIH publication number 91-3242, Bethesda)), the whole of which is incorporated herein by reference).

[0058] As used herein, the term "invariant region" is common in the art. The invariant region is a portion of the antibody, e.g., the carboxyl-terminal portion of the light chain and / or heavy chain, which does not directly participate in the binding of the antibody to the antigen but may exhibit various effector functions, such as interactions with Fc receptors (e.g., Fc gamma receptors).

[0059] As used herein, when used in relation to antibodies, the term “heavy chain” may refer to any distinct type, e.g., alpha (α), delta (δ), epsilon (ε), gamma (γ) and mu (μ), which produce antibodies of the IgA, IgD, IgE, IgG, and IgM classes, respectively, including subclasses of IgG, e.g. IgG1, IgG2, IgG3, and IgG4, based on the amino acid sequence of the constant region.

[0060] As used herein, when used in relation to antibodies, the term “light chain” may refer to any distinct type, e.g., kappa (κ) or lambda (λ), based on the amino acid sequence of the invariant region. Light chain amino acid sequences are well known in the art. In certain embodiments, the light chain is a human light chain.

[0061] As used herein, the term "specifically binds" refers to the specificity of a binding molecule (e.g., an antibody) to an antigen, as understood by those skilled in the art. A binding molecule that specifically binds to an antigen is typically, when measured by, for example, an ELISA assay, surface plasmon resonance, or other suitable assay known in the art, 1×10⁻⁶ -6　 It binds to the antigen with an equilibrium dissociation constant (KD) of less than M. In certain embodiments, a skilled technician will understand that the binding molecule may specifically bind to different antigens, for example, different antigens that share a common epitope recognized by the binding molecule.

[0062] As used herein, the term “affinity” refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., antibody) and its binding partner (e.g., antigen). Unless otherwise specified, as used herein, “binding affinity” refers to the intrinsic binding affinity reflecting the 1:1 interaction between the members of a binding pair (e.g., antibody and antigen). The affinity of molecule X for partner Y can generally be expressed as a dissociation constant (KD). Affinity can be measured by conventional methods known in the art, including the methods described herein.

[0063] As used herein, the term "EU numbering system" refers to literature (Edelman, GM, etc. , Refers to the EU numbering convention for the constant regions of antibodies as described in Proc. Natl. Acad. USA, 63, 78-85 (1969) and Kabat et al., Sequences of Proteins of Immunological Interest, U.S. Dept. Health and Human Services, 5th edition, 1991, each incorporated herein by reference in its entirety).

[0064] As used herein, the terms “treat,” “treating,” and “treatment” refer to the therapeutic or preventive measures described herein. A “treatment” method includes administering antibodies to a subject who has a disease or disorder or is predisposed to such disease or disorder to prevent, cure, delay, reduce the severity, or improve one or more symptoms of the disease or disorder or a recurring disease or disorder, or to prolong the subject’s survival beyond what would have been expected without such treatment.

[0065] As used herein, "inhibitor" refers to a compound that reduces activity when compared to a control, such as a compound known to be absent or inactive (e.g., a compound described herein).

[0066] As defined herein, with respect to protein-inhibitor interactions, terms such as "inhibition," "to inhibit," and "inhibiting" mean having a negative effect (e.g., reducing) on ​​the activity or function of a protein compared to the protein activity or function in the absence of an inhibitor. In embodiments, inhibition means having a negative effect (e.g., reducing) on ​​the concentration or level of a protein compared to the protein concentration or level in the absence of an inhibitor. In embodiments, inhibition refers to a reduction in a disease or disease symptoms. In embodiments, inhibition refers to a reduction in the activity of a specific protein target. Thus, inhibition includes at least partially, partially or entirely blocking a stimulus, reducing, preventing, or delaying activation, or inactivating, desensitizing, or downregulating signal transduction or enzyme activity or the amount of a protein. In embodiments, inhibition refers to a reduction in the activity of a target protein resulting from a direct interaction (e.g., an inhibitor binding to a target protein). In embodiments, inhibition refers to a reduction in the activity of a target protein resulting from an indirect interaction (e.g., an inhibitor binding to a protein that activates the target protein to prevent target protein activation).

[0067] The terms “inhibitor,” “inhibitor,” “antagonist,” or “downregulator” are used interchangeably and refer to a substance capable of detectably reducing the expression or activity of a given gene or protein. An antagonist may reduce expression or activity by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more compared to a control group without an antagonist. In certain cases, expression or activity is 1.5, 2, 3, 4, 5, or 10 times lower than the expression or activity without an antagonist.

[0068] The term "niacin" or "vitamin B3" refers to one of the water-soluble B vitamins. Niacin is a generic name for nicotinic acid (pyridine-3-carboxylic acid), nicotinamide (niacinamide or pyridine-3-carboxamide), and related derivatives such as nicotinamide riboside. Niacin is naturally present in many foods, added to some foods, and is available as a dietary supplement.

[0069] The term "NAMPT" or "nicotinamide phosphoribosyltransferase" refers to the enzyme encoded by the wild-type human NAMPT gene (e.g., the gene presented in Genbank registry number NM_005746 or NCBI gene ID: 10135). This gene codes for a protein that catalyzes the condensation of nicotinamide and 5-phosphoribosyl-1-pyrophosphate to produce nicotinamide mononucleotide, a step in the biosynthesis of nicotinamide adenine dinucleotide. This protein belongs to the nicotinic acid phosphoribosyltransferase (NAPRTase) family and is thought to be involved in many important biological processes, including metabolism, stress response, and aging. An exemplary amino acid sequence of a human NAPRT protein is presented in NCBI reference sequence: NP_660202.3.

[0070] The term "NAMPTi" or "nicotinamide phosphoribosyltransferase inhibitor" refers to a compound capable of inhibiting or reducing the activity of NAMPT. In some embodiments, NAMPTi is FK866 (PMID: 14612543). In some embodiments, NAMPTi is GNE617 (PMID: 24403854). In some embodiments, NAMPTi is OT82 (PMID: 31896781). In some embodiments, NAMPTi is LSN3154567 (PMID: 29054982).

[0071] The terms "disease" or "pathological condition" refer to the state of existence or health of a patient or subject that can be treated by the compounds or methods provided herein. The disease may be cancer. The disease may be an autoimmune disease. The disease may be an inflammatory disease. The disease may be an infectious disease.

[0072] As used herein, the term “cancer” refers to any type of cancer, neoplasm, or malignant tumor found in mammals (e.g., humans), including thyroid cancer, duodenal cancer, neuroendocrine carcinoma (NEC), uterine cancer, small cell lung cancer (SCLC), hepatocellular carcinoma, mesothelioma, breast cancer, sarcoma, ovarian cancer, renal cell carcinoma, rectal cancer, head and neck cancer, prostate cancer, pancreatic cancer, melanoma, colorectal cancer, cervical cancer, non-small cell lung cancer (NSCLC), cholangiocarcinoma, or endometrial cancer.

[0073] In some embodiments, NAPRT is not detected in samples obtained from certain cancers or tumors using one of the antibodies or polypeptides disclosed herein. In some embodiments, NAPRT is not detected in samples from thyroid cancer, duodenal cancer, neuroendocrine carcinoma (NEC), or uterine cancer. In some embodiments, NAPRT is not detected in 50% to 100% of samples obtained from thyroid cancer, duodenal cancer, neuroendocrine carcinoma (NEC), uterine cancer, small cell lung cancer (SCLC), hepatocellular carcinoma, or mesothelioma. In some embodiments, NAPRT is not detected in 13% to 100% of samples obtained from thyroid cancer, duodenal cancer, neuroendocrine carcinoma (NEC), uterine cancer, small cell lung cancer (SCLC), hepatocellular carcinoma, mesothelioma, breast cancer, sarcoma, ovarian cancer, renal cell carcinoma, rectal cancer, head and neck cancer, prostate cancer, pancreatic cancer, melanoma, colorectal cancer, cervical cancer, non-small cell lung cancer (NSCLC), cholangiocarcinoma, or endometrial cancer.

[0074] As used herein, the terms “sepsis,” “septicemia,” “septicaemia,” or “blood poisoning” refer to a potentially life-threatening condition resulting from the presence of harmful microorganisms in the blood or other tissues and the body’s reaction to such presence, which can potentially lead to failure of various organs, shock, and death. Sepsis occurs when chemicals released into the bloodstream to fight an infection cause systemic inflammation. This triggers a series of changes that damage multiple organ systems, which can lead to failure and, in some cases, death.

[0075] As used herein, in relation to the administration of therapy to a subject, the term "effective dose" refers to the amount of therapy that achieves a desired prophylactic or therapeutic effect.

[0076] As used herein, the term “object” includes any human or non-human animal. In certain embodiments, the object is a human or non-human mammal. In certain embodiments, the object is a human.

[0077] As used herein, the term “control sample” is used in its common, ordinary sense and refers to a sample obtained from a healthy subject. In some embodiments, the control sample is obtained from a subject without cancer. In some embodiments, the control sample is obtained from a subject without sepsis. In some embodiments, NAPRT is detected in the control sample obtained from a healthy subject. In some embodiments, NAPRT is detected in the control sample obtained from a subject without cancer. In some embodiments, NAPRT is detected in the control sample obtained from a subject without sepsis.

[0078] As used herein with respect to antibodies or polynucleotides, the term “isolated” refers to an antibody or polynucleotide isolated from one or more contaminants (e.g., polypeptides, polynucleotides, lipids, or carbohydrates, etc.) present in a natural source of the antibody or polynucleotide. All instances of “isolated antibody” described herein are considered to be antibodies that may be further isolated but do not necessarily need to be isolated. All instances of “isolated polynucleotide” described herein are considered to be polynucleotides that may be further isolated but do not necessarily need to be isolated. All instances of “antibody” described herein are considered to be antibodies that may be further isolated but do not necessarily need to be isolated. All instances of “polynucleotide” described herein are considered to be polynucleotides that may be further isolated but do not necessarily need to be isolated.

[0079] The determination of the "percentage of identity" between two sequences (e.g., amino acid sequences or nucleic acid sequences) can be performed using mathematical algorithms. Specific and non-limiting examples of mathematical algorithms used for comparing two sequences are the algorithms in the literature (Karlin S & Altschul SF (1990) PNAS 87: 2264-2268), which have been modified as in the literature (Karlin S and Altschul SF (1993) PNAS 90: 5873-5877), each incorporated herein by reference in its entirety. These algorithms are included in the NBLAST and XBLAST programs in the literature (Altschul SF et al., (1990) J Mol Biol 215: 403, incorporated herein by reference in its entirety). BLAST nucleotide search may be performed with NBLAST nucleotide program parameters set, for example, Score=100 and Word Length=12, to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. BLAST protein search may be performed with XBLAST program parameters set, for example, Score=50 and Word Length=3, to obtain amino acid sequences homologous to the protein molecules described herein. To obtain gap alignment for comparison purposes, gapped BLAST may be used as described in the literature (Altschul SF et al., (1997) Nuc Acids Res 25: 3389-3402, the whole of which is incorporated herein by reference). Alternatively, PSI BLAST may be used to perform iterative searches to detect distant relationships between molecules ( Id.When using BLAST, Gap BLAST, and PSI Blast programs, the default parameters of each program (e.g., XBLAST and NBLAST) can be used (e.g., see the National Center for Biotechnology Information (NCBI) website ncbi.nlm.nih.gov). Another specific and non-limiting example of a mathematical algorithm used for sequence comparison is the algorithm from the literature (Myers and Miller, 1988, CABIOS 4: 11-17, the whole incorporated herein by reference). This algorithm is included in the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When using the ALIGN program for amino acid sequence comparison, the PAM120 weighted residue table, gap length penalty 12, and gap penalty 4 can be used.

[0080] The percentage of identity between two sequences can be determined using a technique similar to that described above, with or without a gap. When calculating the percentage of identity, typically only exact matches are counted.

[0081] Anti-NAPRT antibody

[0082] In one embodiment, the present disclosure provides an antibody that specifically binds to NAPRT (e.g., human NAPRT). The VH, VL, CDRH, and CDRL amino acid sequences of the exemplary antibody are presented in Tables 2, 3, and 4 below.

[0083]

[0084]

[0085]

[0086] The individual CDRs of the antibodies disclosed herein may be determined according to any CDR numbering system known in the art.

[0087] In certain embodiments, one or more CDRs of the antibodies disclosed herein may be determined according to the literature (Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) and Kabat et al., Sequences of protein of immunological interest (1991), each incorporated herein in its entirety by reference).

[0088] In certain embodiments, the antibody provided herein comprises CDRH1, CDRH2, and / or CDRH3 of the VH amino acid sequence presented in SEQ ID NO. 1 as determined by the Kavat numbering system. In certain embodiments, the antibody provided herein comprises CDRL1, CDRL2, and / or CDRL3 of the VL amino acid sequence presented in SEQ ID NO. 2 as determined by the Kavat numbering system.

[0089] In certain embodiments, one or more CDRs of the antibodies disclosed herein are Chothia numbering systems designating locations of immunoglobulin structural loops (e.g., literature (Chothia C & Lesk AM, (1987), J Mol Biol 196: 901-917; Al-Lazikani B et al.). , (1997) J Mol Biol 273: 927-948; Chothia C et al., (1992) J Mol Biol 227: 799-817; Tramontano A et al., (1990) J Mol Biol 215(1): 175-82; and U.S. Patent No. 7,709,226, all of which are incorporated herein by reference in their entirety.

[0090] In certain embodiments, the antibody provided herein comprises CDRH1, CDRH2, and / or CDRH3 of the VH amino acid sequence presented in SEQ ID NO. 1 as determined by the Chotia numbering system. In certain embodiments, the antibody provided herein comprises CDRL1, CDRL2, and / or CDRL3 of the VL amino acid sequence presented in SEQ ID NO. 2 as determined by the Chotia numbering system.

[0091] In certain embodiments, one or more CDRs of the antibodies disclosed herein may be determined according to the literature (MacCallum RM et al., (1996) J Mol Biol 262: 732-745, the whole incorporated herein by reference). Additionally, for example, the literature (Martin A. "Protein Sequence and Structure Analysis of Antibody Variable Domains," in Antibody Engineering , Kontermann and D See bel, eds., Chapter 31, pp. 422-439, Springer-Verlag, Berlin (2001), the whole is incorporated herein by reference.

[0092] In certain embodiments, the antibody provided herein comprises CDRH1, CDRH2, and / or CDRH3 of the VH amino acid sequence presented in SEQ ID NO. 1 as determined by the McCallum numbering system. In certain embodiments, the antibody provided herein comprises CDRL1, CDRL2, and / or CDRL3 of the VL amino acid sequence presented in SEQ ID NO. 2 as determined by the McCallum numbering system.

[0093] In certain embodiments, the CDR of the antibody disclosed herein may be determined according to the IMGT numbering scheme described in the literature (Lefranc MP, (1999) The Immunologist 7: 132-136; Lefranc MP et al., (1999) Nucleic Acids Res 27: 209-212; and Lefranc MP et al., (2009) Nucleic Acids Res 37: D1006-D1012, each incorporated herein in its entirety by reference).

[0094] In certain embodiments, the antibody provided herein comprises CDRH1, CDRH2, and / or CDRH3 of the VH amino acid sequence presented in SEQ ID NO. 1 as determined by the IMGT numbering scheme. In certain embodiments, the antibody provided herein comprises CDRL1, CDRL2, and / or CDRL3 of the VL amino acid sequence presented in SEQ ID NO. 2 as determined by the IMGT numbering scheme.

[0095] In certain embodiments, the CDR of the antibody disclosed herein represents a compromise between the Kavat CDR and the Chotia structural loop and may be determined according to an AbM numbering scheme that designates the AbM hypervariable region used by Oxford Molecular’s ​​AbM antibody modeling software (Oxford Molecular Group, Inc.).

[0096] In certain embodiments, the antibody provided herein comprises CDRH1, CDRH2, and / or CDRH3 of the VH amino acid sequence presented in SEQ ID NO. 1 as determined by the AbM numbering system. In certain embodiments, the antibody provided herein comprises CDRL1, CDRL2, and / or CDRL3 of the VL amino acid sequence presented in SEQ ID NO. 2 as determined by the AbM numbering system.

[0097] In certain embodiments, the CDR of the antibody disclosed herein is from the literature (Honegger and Pl). It may be determined according to the AHo numbering scheme as described in ckthun, A., J. Mol. Biol. 309:657-670 (2001), the whole of which is incorporated herein by reference.

[0098] In certain embodiments, the antibody provided herein comprises CDRH1, CDRH2, and / or CDRH3 of the VH amino acid sequence presented in SEQ ID NO. 1 as determined by the AHo numbering system. In certain embodiments, the antibody provided herein comprises CDRL1, CDRL2, and / or CDRL3 of the VL amino acid sequence presented in SEQ ID NO. 2 as determined by the AHo numbering system.

[0099] In certain embodiments, the individual CDRs of the antibodies disclosed herein are each independently determined according to one of the Kavat, Chotia, McCallum, IMGT, AHo, or AbM numbering schemes, or by structural analysis of the multispecific molecule, the structural analysis identifies residues within variable region(s) predicted to come into contact with the epitope region of NAPRT.

[0100] In certain embodiments, the present disclosure provides an antibody that specifically binds to NAPRT (e.g., human NAPRT), comprising VH having the CDRH1, CDRH2, and CDRH3 amino acid sequences of the VH amino acid sequence presented in SEQ ID NO. 1, and VL having the CDRL1, CDRL2, and CDRL3 amino acid sequences of the VL amino acid sequence presented in SEQ ID NO. 2, wherein each CDR is independently determined according to one of the Kavat, Chotia, McCallum, IMGT, AHo, or AbM numbering schemes, or by structural analysis of a multispecific molecule, and the structural analysis identifies residues within variable region(s) predicted to come into contact with the epitope region of NAPRT (e.g., human NAPRT).

[0101] In certain embodiments, the present disclosure provides an antibody that specifically binds to NAPRT (e.g., human NAPRT), comprising a VH having the CDRH1, CDRH2, and CDRH3 amino acid sequences of the VH amino acid sequence presented in SEQ ID NO. 1, and a VL having the CDRL1, CDRL2, and CDRL3 amino acid sequences of the VL amino acid sequence presented in SEQ ID NO. 2, wherein the VH amino acid sequence and the VL amino acid sequence are identical (i.e., both VHs have the CDRH1, CDRH2, and CDRH3 amino acid sequences of the VH amino acid sequence presented in SEQ ID NO. 1, and both VHs have the VL having the CDRL1, CDRL2, and CDRL3 amino acid sequences of the VL amino acid sequence presented in SEQ ID NO. 2), and each CDR is independently determined according to one of the Kavat, Chotia, McCallum, IMGT, AHo, or AbM numbering schemes, or by structural analysis of a multispecific molecule, wherein the structural analysis is to contact the epitope region of NAPRT (e.g., human NAPRT). Check the residue within the variable region(s) predicted to be.

[0102] In certain embodiments, the present disclosure provides an antibody that specifically binds to NAPRT (e.g., human NAPRT), comprising the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences of the VH and VL amino acid sequences presented in SEQ ID NOs 1 and 2, respectively.

[0103] In certain embodiments, the present disclosure provides an antibody that specifically binds to NAPRT (e.g., human NAPRT), comprising a VH comprising the CDRH1, CDRH2, and / or CDRH3 amino acid sequences presented in Table 3. In certain embodiments, the antibody comprises a VH comprising the CDRH1 amino acid sequence presented in any one of SEQ ID NOs 3, 9, 11, 15, or 17. In certain embodiments, the antibody comprises a VH comprising the CDRH2 amino acid sequence presented in any one of SEQ ID NOs 4, 10, 12, 16, or 18. In certain embodiments, the antibody comprises a VH comprising the CDRH3 amino acid sequence presented in any one of SEQ ID NOs 5, 13, or 19.

[0104] In certain embodiments, the present disclosure provides an antibody that specifically binds to NAPRT (e.g., human NAPRT), comprising a VH comprising the CDRH1, CDRH2, and CDRH3 amino acid sequences of any antibody of Table 3. In certain embodiments, the antibody comprises a VH comprising the CDRH1, CDRH2, and CDRH3 amino acid sequences presented in SEQ ID NOs 3, 4, and 5; 9, 10, and 5; 11, 12, and 13; 15, 16, and 5; or 17, 18, and 19.

[0105] In certain embodiments, the present disclosure provides an antibody that specifically binds to NAPRT (e.g., human NAPRT), comprising a VL comprising the CDRL1, CDRL2, and / or CDRL3 amino acid sequences presented in Table 4. In certain embodiments, the antibody comprises a VL comprising the CDRL1 amino acid sequence presented in any one of SEQ ID NOs 6, 14, or 20. In certain embodiments, the antibody comprises a VL comprising the CDRL2 amino acid sequence presented in any one of SEQ ID NOs 7, 21, LM, or LMS. In certain embodiments, the antibody comprises a VL comprising the CDRL3 amino acid sequence presented in SEQ ID NO. 8.

[0106] In certain embodiments, the present disclosure provides an antibody that specifically binds to NAPRT (e.g., human NAPRT), comprising a VL comprising the CDRL1, CDRL2, and CDRL3 amino acid sequences of any antibody of Table 4. In certain embodiments, the antibody comprises a VL comprising the CDRL1, CDRL2, and CDRL3 amino acid sequences presented in SEQ ID NOs 6, 7, and 8; 14, LM, and 8; 14, LMS, and 8; or 20, 21, and 8.

[0107] In certain embodiments, the present disclosure provides an antibody that specifically binds to NAPRT (e.g., human NAPRT), comprising a VH comprising the CDRH1, CDRH2, and / or CDRH3 amino acid sequences presented in Table 3, and a VL comprising the CDRL1, CDRL2, and / or CDRL3 amino acid sequences presented in Table 4, wherein 2, 3, 4, 5, or 6 of the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences are derived from the same antibody (i.e., as shown in Tables 3 and 4). In certain embodiments, the antibody comprises a VH comprising CDRH1, CDRH2, and CDRH3 regions, and a VL comprising CDRL1, CDRL2, and CDRL3 regions, wherein the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 regions are each SEQ ID NOs 3, 4, 5, 6, 7, and 8; Includes amino acid sequences presented in 9, 10, 5, 6, 7, and 8; 11, 12, 13, 14, LM, and 8; 11, 12, 13, 14, LMS, and 8; 15, 16, 5, 6, 7, and 8; or 17, 18, 19, 20, 21, and 8.

[0108] In certain embodiments, the present disclosure provides an antibody that specifically binds to NAPRT (e.g., human NAPRT), comprising a VH having an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to the amino acid sequence presented in SEQ ID NO. 1. In certain embodiments, the present disclosure provides an antibody that specifically binds to NAPRT (e.g., human NAPRT), comprising a VH having the amino acid sequence presented in SEQ ID NO. 1. In certain embodiments, the amino acid sequence of the VH consists of the amino acid sequence presented in SEQ ID NO. 1.

[0109] In certain embodiments, the present disclosure provides an antibody that specifically binds to NAPRT (e.g., human NAPRT), comprising a VL having an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to the amino acid sequence presented in SEQ ID NO. 2. In certain embodiments, the present disclosure provides an antibody that specifically binds to NAPRT (e.g., human NAPRT), comprising a VL having the amino acid sequence presented in SEQ ID NO. 2. In certain embodiments, the amino acid sequence of the VL consists of the amino acid sequence presented in SEQ ID NO. 2.

[0110] In certain embodiments, the present disclosure comprises a VH having an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to the amino acid sequence presented in SEQ ID NO. 1, and a VL having an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to the amino acid sequence presented in SEQ ID NO. 2, specifically for NAPRT (e.g., human NAPRT). The present disclosure provides an antibody that binds. In certain embodiments, the present disclosure provides an antibody that specifically binds to NAPRT (e.g., human NAPRT), comprising VH having the amino acid sequence of SEQ ID NO. 1 and VL having the amino acid sequence of SEQ ID NO. 2. In certain embodiments, the amino acid sequence of VH is the amino acid sequence presented in SEQ ID NO. 1, and the amino acid sequence of VL is the amino acid sequence presented in SEQ ID NO. 2.

[0111] In certain embodiments, the present disclosure comprises a VH having an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to the amino acid sequence presented in SEQ ID NO. 1, and a VL having an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to the amino acid sequence presented in SEQ ID NO. 2, specifically for NAPRT (e.g., human NAPRT). The present disclosure provides a binding antibody, wherein the amino acid sequences presented in SEQ ID NO. 1 and SEQ ID NO. 2 are derived from the same antibody (i.e., as shown in Table 2). In certain embodiments, the present disclosure provides an antibody that specifically binds to NAPRT (e.g., human NAPRT), comprising VH having the amino acid sequence of SEQ ID NO. 1 and VL having the amino acid sequence of SEQ ID NO. 2, wherein the amino acid sequences presented in SEQ ID NO. 1 and SEQ ID NO. 2 are derived from the same antibody (i.e., as shown in Table 2). In certain embodiments, the amino acid sequence of VH is composed of the amino acid sequence presented in SEQ ID NO. 1, and the amino acid sequence of VL is composed of the amino acid sequence presented in SEQ ID NO. 2, wherein the amino acid sequences presented in SEQ ID NO. 1 and SEQ ID NO. 2 are derived from the same antibody (i.e., as shown in Table 2).

[0112] In certain embodiments, the present disclosure provides an antibody that specifically binds to NAPRT (e.g., human NAPRT), comprising VH and VL, wherein VH comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO. 1 and VL comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO. 2.

[0113] In certain embodiments, the present disclosure provides an antibody that specifically binds to NAPRT (e.g., human NAPRT), comprising VH and VL having amino acid sequences as presented in SEQ ID NOs 1 and 2, respectively.

[0114] In certain embodiments, the present disclosure provides an antibody that specifically binds to NAPRT (e.g., human NAPRT), comprising VH and VL, each consisting of the amino acid sequences presented in SEQ ID NOs 1 and 2.

[0115] In certain embodiments, the present disclosure provides an antibody that cross-competes with any of the antibodies described above for binding to NAPRT (e.g., human NAPRT). In certain embodiments, the present disclosure provides an antibody that binds to the same or overlapping epitopes of the antibody described above and NAPRT (e.g., epitopes of human NAPRT).

[0116] In certain embodiments, the present disclosure provides an antibody that specifically binds to the amino acid sequence of SEQ ID NO. 29 or SEQ ID NO. 30. In certain embodiments, the present disclosure provides an antibody that specifically binds to the amino acid sequences of SEQ ID NO. 29 and SEQ ID NO. 30. In certain embodiments, the present disclosure provides an antibody that does not specifically bind to the amino acid sequence of SEQ ID NO. 28.

[0117] In certain embodiments, the epitope of the antibody is, for example, NMR spectroscopy, surface plasmon resonance (BIAcore ® ), X-ray diffraction crystallography studies, ELISA assays, hydrogen / deuterium exchange coupled with mass spectrometry (e.g., liquid chromatography electrospray mass spectrometry), array-based oligopeptide scanning assays and / or mutagenic mapping (e.g., site-specific mutagenic mapping). In the case of X-ray crystallography, crystallization can be determined by any method known in the art (e.g., Gieg This can be performed using R et al., (1994) Acta Crystallogr D Biol Crystallogr 50(Pt 4): 339-350; McPherson A (1990) Eur J Biochem 189: 1-23; Chayen NE (1997) Structure 5: 1269-1274; McPherson A (1976) J Biol Chem 251: 6300-6303 (all of which are incorporated herein by reference in their entirety). Antibody:antigen determination can be studied using well-known X-ray diffraction techniques and computers such as X-PLOR (distributed by Molecular Simulations, Inc., Yale University, 1992; see, for example, literature (Meth Enzymol (1985) volumes 114 & 115, eds. Wyckoff HW et al.; U.S. Patent Application No. 2004 / 0014194)) and BUSTER (Bricogne G (1993) Acta Crystallogr D Biol Crystallogr 49(Pt 1): 37-60; Bricogne G (1997) Meth Enzymol 276A: 361-423, eds. Carter CW; Roversi P et al., (2000) Acta Crystallogr D Biol Crystallogr 56(Pt 10): 1316-1323, all of which are incorporated herein by reference in their entirety). It may be refined using software. Mutagenic mapping studies may be performed using any method known to those skilled in the art. Descriptions of mutagenic techniques, including alanine scanning mutagenic techniques, are provided in, for example, the literature (Champe M et al.). , (1995) supra and Cunningham BC & Wells J A (1989) supraRefer to ). In certain embodiments, the epitope of the antibody is determined using an alanine scanning mutagenicity study. Additionally, antibodies that recognize and bind to the same or overlapping epitopes of NAPRT (e.g., human NAPRT) can be identified using conventional techniques such as immunoassays, for example, competitive binding assays, which demonstrate the ability of one antibody to block the binding of another antibody to a target antigen. Competitive binding assays can also be used to determine whether two antibodies have similar binding specificity for the epitope. Competitive binding can be determined by an assay in which the immunoglobulin under test inhibits the specific binding of a reference antibody to a common antigen, such as NAPRT (e.g., human NAPRT). Numerous types of competitive binding assays, e.g., solid-phase direct or indirect radioimmunoassays (RIA), solid-phase direct or indirect enzyme immunoassays (EIA), and sandwich competitive assays (Stahli C et al. , (1983) Methods Enzymol 9: 242-253); Solid-phase direct biotin-avidin EIA (Kirkland TN et al. , (1986) J Immunol 137: 3614-9); Solid-phase direct labeling assay, Solid-phase direct labeling sandwich assay (Harlow E & Lane D, (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Press); Solid-phase direct labeling RIA using I-125 labeling (Morel GA et al. , (1988) Mol Immunol 25(1): 7-15); solid-phase direct biotin-avidin EIA (Cheung RC et al. , (1990) Virology 176: 546-52); and direct labeled RIA (Moldenhauer G et al. ,(1990) Scand J Immunol 32: 77-82) is known, all of which are incorporated herein by reference in their entirety. Typically, such assays involve the use of purified antigens bound to a solid surface (e.g., NAPRT, such as human NAPRT) or cells containing either of these, unlabeled test immunoglobulin, and labeled reference immunoglobulin. Competitive inhibition can be measured by determining the amount of label bound to the solid surface or cell in the presence of the test immunoglobulin. Usually, the test immunoglobulin is present in excess. Usually, when a competing antibody is present in excess, it inhibits the specific binding of the reference antibody to the common antigen by at least 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, or more. Competitive binding assays can be constructed in a wide variety of different forms using labeled antigens or labeled antibodies. In a general version of this assay, the antigen is immobilized in a 96-well plate. Subsequently, the ability of an unlabeled antibody to block the binding of a labeled antibody to the antigen is measured using radioactive or enzymatic labeling. For additional details, refer, for example, to the literature (Wagener C et al.). , (1983) J Immunol 130: 2308-2315; Wagener C et al. , (1984) J Immunol Methods 68: 269-274; Kuroki M et al. , (1990) Cancer Res 50: 4872-4879; Kuroki M et al. , (1992) Immunol Invest 21: 523-538; Kuroki M et al. , (1992) Hybridoma 11: 391-407; and Antibodies: A Laboratory Manual, Ed Harlow E & Lane D editors. supra See , pp. 386-389 (all incorporated herein by reference in their entirety).

[0118] The anti-NAPRT antigen-binding molecules of the present disclosure may be linked to or co-expressed with another functional molecule, for example, another peptide or protein. For example, an antibody or a fragment thereof may be functionally linked (e.g., by chemical coupling, genetic fusion, non-covalent association, etc.) to one or more other molecular entities, for example, another antibody or antibody fragment, to produce a bispecific or multispecific antibody having a second or additional binding specificity.

[0119] In certain embodiments, the antibody disclosed herein is conjugated to a radionuclide or a detectable label. In certain embodiments, the radionuclide is an isotope 3 H, 14 C, 32 P, 35 S, 36 Cl, 51 Cr, 57 Co, 58 Co, 59 Fe, 67 Cu, 90 Y, 99 Tc, 111 In, 117 Lu, 121 I, 124 I, 125 I, 131 I, 198 Au, 211 At, 213 Bi, 225 Ac, and 186 It is selected from the group consisting of Re. In certain embodiments, the detectable label includes a fluorescent moiety or a click chemical handle.

[0120] Any immunoglobulin (Ig) constant region may be used in the antibodies disclosed herein. In certain embodiments, the Ig region is a human IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule, any class of immunoglobulin molecules (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or any subclass (e.g., IgG2a and IgG2b).

[0121] In certain embodiments, the present disclosure provides an antibody that specifically binds to NAPRT (e.g., human NAPRT), comprising a heavy chain constant region, optionally selected from the group consisting of human IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

[0122] In certain embodiments, the present disclosure provides an antibody that specifically binds to NAPRT (e.g., human NAPRT), comprising a heavy chain constant region that is a variant of the wild-type heavy chain constant region, wherein the variant heavy chain constant region binds to FcγR with a lower affinity than that of the wild-type heavy chain constant region.

[0123] In certain embodiments, the present disclosure provides an antibody that specifically binds to NAPRT (e.g., human NAPRT), comprising a heavy chain constant region comprising the amino acid sequence shown in Table 5. In certain embodiments, the heavy chain constant region comprises the amino acid sequence of SEQ ID NO. 24 or 25. In certain embodiments, the heavy chain constant region consists of the amino acid sequence of SEQ ID NO. 24 or 25.

[0124]

[0125] In certain embodiments, the present disclosure provides an antibody that specifically binds to NAPRT (e.g., human NAPRT), comprising a light chain constant region comprising the amino acid sequence shown in Table 6. In certain embodiments, the heavy chain constant region comprises the amino acid sequence of SEQ ID NO. 26 or 27. In certain embodiments, the heavy chain constant region consists of the amino acid sequence of SEQ ID NO. 26 or 27.

[0126]

[0127] In certain embodiments, to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and / or antigen-dependent cytotoxicity, one, two, or more mutations (e.g., amino acid substitutions) are introduced into the Fc region (e.g., the CH2 domain of human IgG1 (residues 231-340)) and / or the CH3 domain (residues 341-447 of human IgG1 numbered according to the EU numbering scheme) and / or the hinge region (residues 216-230 numbered according to the EU numbering scheme) of the antibody described herein.

[0128] In certain embodiments, one, two, or more mutations (e.g., amino acid substitutions) are introduced into the hinge region of the antibody described herein so as to change (e.g., increase or decrease) the number of cysteine ​​residues within the hinge region, for example, as described in U.S. Patent No. 5,677,425 (the whole is incorporated herein by reference). The number of cysteine ​​residues within the hinge region may be changed, for example, to facilitate the assembly of the light and heavy chains or to change the stability of the antibody (e.g., increase or decrease).

[0129] In certain embodiments, one, two, or more amino acid mutations (e.g., substitution, insertion, or deletion) are introduced into the IgG constant region or its FcRn binding fragment (preferably Fc or hinge-Fc fragment) to alter (e.g., decrease or increase) the in vivo half-life of the antibody. For examples of mutations that alter (e.g., decrease or increase) the in vivo half-life of the antibody, refer, for example, to the literature (International Publication Nos. WO 02 / 060919; WO 98 / 23289; and WO 97 / 34631; and U.S. Patent Nos. 5,869,046, 6,121,022, 6,277,375, and 6,165,745, all of which are incorporated herein by reference in their entirety). In certain embodiments, one, two, or more amino acid mutations (e.g., substitutions, insertions, or deletions) are introduced into the IgG constant region or its FcRn binding fragment (preferably Fc or hinge-Fc fragment) to reduce the in vivo half-life of the antibody. In other embodiments, one, two, or more amino acid mutations (e.g., substitutions, insertions, or deletions) are introduced into the IgG constant region or its FcRn binding fragment (preferably Fc or hinge-Fc fragment) to increase the in vivo half-life of the antibody. In certain embodiments, the antibody may have one or more amino acid mutations (e.g., substitutions) in the second constant (CH2) domain (residues 231-340 of human IgG1) and / or the third constant (CH3) domain (residues 341-447 of human IgG1) numbered according to the EU numbering scheme. In certain embodiments, the IgG1 constant region of the antibody described herein comprises a substitution of methionine (M) to tyrosine (Y) at position 252 numbered according to the EU numbering scheme, a substitution of serine (S) to threonine (T) at position 254, and a substitution of threonine (T) to glutamate (E) at position 256. See U.S. Patent No. 7,658,921 (the whole incorporated herein by reference).This type of mutant IgG, referred to as "YTE mutant," was shown to have a fourfold increase in half-life compared to the wild-type version of the same antibody (Dall'Acqua WF et al. , (2006) J Biol Chem 281: 23514-24, the whole is incorporated herein by reference). In certain embodiments, the antibody comprises an IgG constant region comprising one, two, three or more amino acid substitutions at amino acid residues at positions 251-257, 285-290, 308-314, 385-389, and 428-436 numbered according to the EU numbering scheme.

[0130] In certain embodiments, to increase or decrease the affinity of an antibody for an Fc receptor on the surface of an effector cell (e.g., an activated Fc receptor), one, two, or more mutations (e.g., amino acid substitutions) are introduced into the Fc region of the antibody described herein (e.g., the CH2 domain of human IgG1 (residues 231-340)) and / or the CH3 domain (residues 341-447 of human IgG1 numbered according to the EU numbering scheme) and / or the hinge region (residues 216-230 numbered according to the EU numbering scheme). Mutations within the Fc region of an antibody that decrease or increase the affinity of the antibody for an Fc receptor, and techniques for introducing such mutations into the Fc receptor or a fragment thereof, are known to those skilled in the art. Examples of mutations within the Fc receptor of an antibody that can be made to alter the affinity of the antibody for the Fc receptor are described, for example, in the literature (Smith P et al., (2012) PNAS 109: 6181-6186, U.S. Patent No. 6,737,056, and International Publication Nos. WO 02 / 060919; WO 98 / 23289; and WO 97 / 34631, all of which are incorporated herein by reference in their entirety).

[0131] In certain embodiments, the antibody comprises a heavy chain constant region that is a variant of the wild-type heavy chain constant region, wherein the variant heavy chain constant region binds to FcγRIIB with a higher affinity than that of the wild-type heavy chain constant region binding to FcγRIIB. In certain embodiments, the variant heavy chain constant region is a variant human heavy chain constant region, e.g., variant human IgG1, variant human IgG2, or variant human IgG4 heavy chain constant region. In certain embodiments, the variant human IgG heavy chain constant region comprises one or more amino acid mutations of G236D, P238D, S239D, S267E, L328F, and L328E according to the EU numbering scheme. In certain embodiments, the variant human IgG heavy chain constant region comprises S267E and L328F; P238D and L328E according to the EU numbering scheme; It comprises a set of amino acid mutations selected from the group consisting of one or more substitutions selected from the group consisting of P238D and E233D, G237D, H268D, P271G, and A330R; P238D, E233D, G237D, H268D, P271G, and A330R; G236D and S267E; S239D and S267E; V262E, S267E, and L328F; and V264E, S267E, and L328F. In certain embodiments, FcγRIIB is expressed on cells selected from the group consisting of macrophages, monocytes, B cells, dendritic cells, endothelial cells, and activated T cells.

[0132] In additional embodiments, one, two, or more amino acid substitutions are introduced into the IgG constant region Fc region to modify the effector function(s) of the antibody. For example, one or more amino acids selected from amino acid residues 234, 235, 236, 237, 239, 243, 267, 292, 297, 300, 318, 320, 322, 328, 330, 332, and 396 numbered according to the EU numbering scheme may be replaced with different amino acid residues so that the antibody has a modified affinity for the effector ligand but maintains the antigen-binding ability of the parent antibody. The effector ligand whose affinity is modified may be, for example, the Fc receptor or the C1 component of complement. These approaches are described in more detail in U.S. Patent Nos. 5,624,821 and 5,648,260 (each incorporated herein in its entirety by reference). In certain embodiments, deletion or inactivation of the constant region domain (via point mutation or other means) may reduce Fc receptor binding of the circulating antibody, thereby increasing tumor localization. For a description of mutations that increase tumor localization by deleting or inactivating the constant region, refer, for example, to the literature (U.S. Patent Nos. 5,585,097 and 8,591,886, each incorporated herein in its entirety by reference). In certain embodiments, one or more amino acid substitutions may be introduced into the Fc region of the antibody described herein to remove potential glycosylation sites on the Fc region that may reduce Fc receptor binding (e.g., Shields RL et al.). ,(2001) J Biol Chem 276: 6591-604, the whole incorporated herein by reference). In various embodiments, one or more of the following mutations may be made in the constant regions of the antibodies described herein, numbered according to the EU numbering scheme: N297A substitution; N297Q substitution; L234A substitution; L234F substitution; L235A substitution; L235F substitution; L235V substitution; L237A substitution; S239D substitution; E233P substitution; L234V substitution; C236 deletion; P238A substitution; F243L substitution; D265A substitution; S267E substitution; L328F substitution; R292P substitution; Y300L substitution; A327Q substitution; P329A substitution; A330L substitution; I332E substitution; or P396L substitution.

[0133] In certain embodiments, a mutation selected from the group consisting of D265A, P329A, and combinations thereof numbered according to the EU numbering system may be created in the constant region of the antibody described herein. In certain embodiments, a mutation selected from the group consisting of L235A, L237A, and combinations thereof numbered according to the EU numbering system may be created in the constant region of the antibody described herein. In certain embodiments, a mutation selected from the group consisting of S267E, L328F, and combinations thereof numbered according to the EU numbering system may be created in the constant region of the antibody described herein. In certain embodiments, a mutation selected from the group consisting of S239D, I332E, optionally A330L, and combinations thereof numbered according to the EU numbering system may be created in the constant region of the antibody described herein. In certain embodiments, a mutation selected from the group consisting of L235V, F243L, R292P, Y300L, P396L and combinations thereof numbered according to the EU numbering scheme may be formed in the constant region of the antibody described herein. In certain embodiments, a mutation selected from the group consisting of S267E, L328F and combinations thereof numbered according to the EU numbering scheme may be formed in the constant region of the antibody described herein.

[0134] In certain embodiments, the antibody described herein comprises a constant region of IgG1 having an N297Q or N297A amino acid substitution numbered according to the EU numbering system. In certain embodiments, the antibody described herein comprises a constant region of IgG1 having a mutation selected from the group consisting of D265A, P329A, and combinations thereof numbered according to the EU numbering system. In another embodiment, the antibody described herein comprises a constant region of IgG1 having a mutation selected from the group consisting of L234A, L235A, and combinations thereof numbered according to the EU numbering system. In yet another embodiment, the antibody described herein comprises a constant region of IgG1 having a mutation selected from the group consisting of L234F, L235F, N297A, and combinations thereof numbered according to the EU numbering system. In certain embodiments, the amino acid residues within the constant region of the antibody described herein at positions corresponding to the L234, L235, and D265 positions of the human IgG1 heavy chain numbered according to the EU numbering scheme are not L, L, and D, respectively. This approach is described in detail in International Publication No. WO 14 / 108483 (the whole is incorporated herein by reference). In certain embodiments, the amino acids corresponding to the L234, L235, and D265 positions of the human IgG1 heavy chain numbered according to the EU numbering scheme are F, E, and A, respectively; or A, A, and A.

[0135] In certain embodiments, by replacing one or more amino acids selected from amino acid residues 329, 331, and 322 within the constant region of the antibody described herein, numbered according to the EU numbering scheme, with different amino acid residues, the antibody may have its C1q binding modified and / or its complement-dependent cytotoxicity (CDC) reduced or lost. This approach is described in more detail in U.S. Patent No. 6,194,551 (Idusogie et al., incorporated herein by reference in its entirety). In certain embodiments, the complement-fixing ability of the antibody may be altered by modifying one or more amino acid residues within amino acid positions 231 through 238 in the N-terminal region of the CH2 domain of the antibody described herein, numbered according to the EU numbering scheme. This approach is further described in International Publication No. WO 94 / 29351 (in its entirety incorporated herein by reference). In certain embodiments, the Fc region of the antibody described herein may be modified by mutating one or more amino acids at the following positions numbered according to the EU numbering scheme to increase the ability to mediate antibody-dependent cytotoxicity (ADCC) and / or increase the affinity of the antibody for the Fcγ receptor: 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303, 305, 307, 309, 312, 315, 320, 322, 324, 326, 327, 328, 329, 330, 331, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, ​​388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438, or 439.This approach is further described in International Publication No. WO 00 / 42072 (the whole is incorporated herein by reference).

[0136] In certain embodiments, the antibody described herein comprises a modified constant region of IgG1, the modification increasing the antibody's ability to mediate antibody-dependent cytotoxicity (ADCC). In certain embodiments, as described herein and / or verified by methods known to those skilled in the art, 0.1, 1, or 10 μg / ml of the antibody can induce at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60% of apoptosis in cells expressing NAPRT within 1, 2, or 3 hours. In certain embodiments, the modified constant region of IgG1 comprises S239D and I332E substitutions numbered according to the EU numbering scheme. In certain embodiments, the modified constant region of IgG1 comprises S239D, A330L, and I332E substitutions numbered according to the EU numbering scheme. In certain embodiments, the modified constant region of IgG1 includes L235V, F243L, R292P, Y300L, and P396L substitutions numbered according to the EU numbering scheme. In certain embodiments, the antibody can induce apoptosis in effector T cells and Tregs, and the percentage of Tregs undergoing apoptosis is at least 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, or 5 times higher than the percentage of effector T cells undergoing apoptosis.

[0137] In certain embodiments, the antibody described herein comprises an invariant region of an IgG4 antibody, wherein serine at amino acid residue 228 of the heavy chain, numbered according to the EU numbering system, is substituted with proline.

[0138] In certain embodiments, any invariant region mutation or modification described herein may be introduced into one or both of the heavy chain invariant regions of the antibody described herein having two heavy chain invariant regions.

[0139] polypeptide

[0140] In another aspect, the present invention provides a polypeptide comprising one or more sequences presented in Tables 2 through 4 above. In certain embodiments, the polypeptide comprises CDRH1, CDRH2, and / or CDRH3 of the VH amino acid sequence presented in SEQ ID NO. 1 as determined by any method discussed above. In certain embodiments, the polypeptide comprises VH comprising the CDRH1, CDRH2, and CDRH3 amino acid sequences of the VH amino acid sequence presented in SEQ ID NO. 1. In certain embodiments, the polypeptide comprises the CDRH1, CDRH2, and CDRH3 amino acid sequences presented in SEQ ID NOs 3, 4, and 5; 9, 10, and 5; 11, 12, and 13; 15, 16, and 5; or 17, 18, and 19.

[0141] In certain embodiments, the polypeptide comprises CDRL1, CDRL2, and / or CDRL3 of the VL amino acid sequence presented in SEQ ID NO. 2 as determined by any method discussed above. In certain embodiments, the polypeptide comprises VL comprising the CDRL1, CDRL2, and CDRL3 amino acid sequences of the VL amino acid sequence presented in SEQ ID NO. 2. In certain embodiments, the polypeptide comprises the CDRL1, CDRL2, and CDRL3 amino acid sequences presented in SEQ ID NOs 6, 7, and 8; 14, LM, and 8; 14, LMS, and 8; or 20, 21, and 8.

[0142] In a specific embodiment, the polypeptide comprises VH comprising the CDRH1, CDRH2, and CDRH3 amino acid sequences of the VH amino acid sequence presented in SEQ ID NO. 1; and VL comprising the CDRL1, CDRL2, and CDRL3 amino acid sequences of the VL amino acid sequence presented in SEQ ID NO. 2. In a specific embodiment, the polypeptide comprises the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences of the VH and VL amino acid sequences presented in SEQ ID NOs 1 and 2, respectively.

[0143] In certain embodiments, the polypeptides provided herein each comprise the amino acid sequences of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 presented in SEQ ID NOs 3, 4, 5, 6, 7, and 8; 9, 10, 5, 6, 7, and 8; 11, 12, 13, 14, LM, and 8; 11, 12, 13, 14, LMS, and 8; 15, 16, 5, 6, 7, and 8; or CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 presented in 17, 18, 19, 20, 21, and 8.

[0144] Pharmaceutical composition

[0145] The present invention provides a composition comprising a nicotinamide phosphoribosyltransferase inhibitor (NAMPTi) and optionally niacin, which is disclosed herein as having the desired purity within a physiologically acceptable carrier, excipient, or stabilizer (e.g., see Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA). The acceptable carrier, excipient, or stabilizer is non-toxic to the recipient at the dose and concentration used and comprises buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; Preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol, etc.); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; Metal complexes (e.g., Zn-protein complexes); and / or TWEEN TM , PLURONICS TM Or includes nonionic surfactants such as polyethylene glycol (PEG).

[0146] In certain embodiments, the pharmaceutical composition comprises the nicotinamide phosphoribosyltransferase inhibitor (NAMPTi) disclosed herein in a pharmaceutically acceptable carrier, and optionally one or more additional prophylactic or therapeutic agents (e.g., niacin). In certain embodiments, the pharmaceutical composition comprises the NAMPTi disclosed herein in a pharmaceutically acceptable carrier, and optionally one or more additional prophylactic or therapeutic agents (e.g., niacin). In certain embodiments, NAMPTi is the only active ingredient included in the pharmaceutical composition. In certain embodiments, the pharmaceutical composition comprises niacin and NAMPTi. The pharmaceutical composition described herein may be useful for reducing or blocking the activity of NAMPTi (e.g., human NAMPTi) and for treating pathological conditions such as cancer. In certain embodiments, the present disclosure relates to a pharmaceutical composition of the present disclosure comprising NAMPTi for use as a medicine. In certain embodiments, the present disclosure relates to a pharmaceutical composition of the present disclosure comprising NAMPTi and optionally niacin for use as a medicine. In another embodiment, the present disclosure relates to a pharmaceutical composition of the present disclosure for use in a method for treating cancer.

[0147] Pharmaceutically acceptable carriers used in parenteral formulations include aqueous vehicles, non-aqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, local anesthetics, suspending agents and dispersants, emulsifiers, sequestering agents or chelating agents, and other pharmaceutically acceptable substances. Examples of aqueous vehicles include sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, and dextrose and lactated Ringer's injection. Non-aqueous parenteral vehicles include fixed oils of plant origin, cottonseed oil, corn oil, sesame oil, and peanut oil. Antimicrobial agents at bacterial or fungal concentrations may be added to parenteral formulations packaged in multi-dose containers, and these include phenol or cresol, mercury agents, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride, and benzethonium chloride. Isotonic agents include sodium chloride and dextrose. Buffers include phosphates and citrates. Antioxidants include sodium bisulfite. Local anesthetics include procaine hydrochloride. Suspending agents and dispersants include sodium carboxymethylcellulose, hydroxypropyl methylcellulose, and polyvinylpyrrolidone. Emulsifiers include polysorbate 80 (TWEEN ® 80) includes. The metal ion sequester or chelating agent includes EDTA. The pharmaceutical carrier also includes ethyl alcohol, polyethylene glycol, and propylene glycol for the water-miscible vehicle; and sodium hydroxide, hydrochloric acid, citric acid, or lactic acid for pH adjustment.

[0148] The pharmaceutical composition may be formulated for any route of administration to the subject. Specific examples of routes of administration include intranasal, oral, pulmonary, transdermal, intradermal, and parenteral. Parenteral administration characterized by subcutaneous, intramuscular, or intravenous injection is also considered herein. The injectable may be prepared in conventional forms such as liquid solutions or suspensions, solid forms suitable for dissolving or suspending in liquid prior to injection, or emulsions. The injectable, solution, and emulsion may also contain one or more excipients. Suitable excipients are, for example, water, saline, dextrose, glycerol, or ethanol. Additionally, if desired, the pharmaceutical composition to be administered may contain small amounts of non-toxic auxiliary substances such as humectants or emulsifiers, pH buffers, stabilizers, solubility enhancers, and other such agents, for example, sodium acetate, sorbitan monolaurate, triethanolamine oleate, and cyclodextrin.

[0149] Preparations for parenteral administration of antibodies include sterile dry soluble products (including subcutaneous tablets), such as sterile solutions ready for injection and lyophilized powders ready to be combined with a solvent immediately before use; sterile suspensions ready for injection; sterile dry insoluble products ready to be combined with a vehicle immediately before use; and sterile emulsions. Solutions may be aqueous or non-aqueous.

[0150] When administered intravenously, suitable carriers include physiological saline or phosphate-buffered saline (PBS), and solutions containing thickeners and solubilizers such as glucose, polyethylene glycol, and polypropylene glycol, and mixtures thereof.

[0151] A topical mixture containing antibodies is prepared as described for topical and systemic administration. The resulting mixture may be a solution, suspension, emulsion, etc., and may be formulated into a cream, gel, ointment, emulsion, solution, elixir, lotion, suspension, tincture, paste, foam, aerosol, wash, spray, suppository, bandage, skin patch, or any other formulation suitable for topical administration.

[0152] The NAMPTi disclosed herein may be formulated, for example, as an aerosol for topical application by inhalation (see, for example, U.S. Patent Nos. 4,044,126, 4,414,209, and 4,364,923, which describe aerosols for the delivery of steroids useful for the treatment of inflammatory diseases, particularly asthma, and are incorporated herein by reference in their entirety). Such formulations for administration into the respiratory tract may be in the form of an aerosol or solution for a nebulizer, or as a fine powder for instrumental inhalation, either alone or in combination with an inert carrier such as lactose. In such cases, the particles of the formulation have a diameter of less than 50 microns in certain embodiments and less than 10 microns in certain embodiments.

[0153] The NAMPTi disclosed herein may be formulated in the form of gels, creams, and lotions for topical application to the skin and mucous membranes such as the eyes, and for application to the eyes or into the cerebrospinal or spinal cord. Topical administration is considered for transdermal delivery as well as administration to the eyes or mucous membranes, or for inhalation therapy. Nasal solutions may also be administered as the antibody alone or in combination with other pharmaceutically acceptable excipients.

[0154] Transdermal patches comprising iontophoresis and electroporation devices are well known to those skilled in the art and can be used to administer antibodies. For example, such patches are disclosed in U.S. Patent Nos. 6,267,983, 6,261,595, 6,256,533, 6,167,301, 6,024,975, 6,010715, 5,985,317, 5,983,134, 5,948,433, and 5,860,957 (all of which are incorporated herein by reference in their entirety).

[0155] The NAMPTi disclosed herein and other compositions provided herein may also be formulated to target specific tissues, receptors, or other areas of the body of the subject to be treated. Many such targeting methods are well known to those skilled in the art. All such targeting methods are considered herein for use in the compositions. For non-limiting examples of targeting methods, refer to, for example, U.S. Patent Nos. 6,316,652, 6,274,552, 6,271,359, 6,253,872, 6,139,865, 6,131,570, 6,120,751, 6,071,495, 6,060,082, 6,048,736, 6,039,975, 6,004,534, 5,985,307, 5,972,366, 5,900,252, 5,840,674, 5,759,542, and 5,709,874 (all of which are incorporated herein by reference in their entirety). In certain embodiments, the antibody described herein targets a tumor.

[0156] Compositions used for in vivo administration can be sterilized. This is easily achieved, for example, by filtration through a sterile filter membrane.

[0157] How to use

[0158] The anti-NAPRT antibody disclosed herein is particularly useful for detecting NAPRT in a sample. In certain embodiments, NAPRT comprises any one of the amino acid sequences of SEQ ID NOs 28 to 40.

[0159] In certain embodiments, an anti-NAPRT antibody is useful for indicating NAPRT levels in a sample. In certain embodiments, an anti-NAPRT antibody is useful for indicating the likelihood that a subject with cancer will be effectively treated with a nicotinamide phosphoribosyltransferase inhibitor (NAMPTi). In certain embodiments, the sample is obtained from the subject's cancer or tumor. In certain embodiments, the subject is a human subject. In certain embodiments, if the NAPRT level from the sample is lower than that in the control sample, the likelihood that the cancer will be effectively treated with NAMPTi increases. In certain embodiments, if NAPRT is not detected in the sample, the cancer can be effectively treated with NAMPTi.

[0160] In certain embodiments, the sample comprises cancer cells. In certain embodiments, the cancer cells are solid tumor cells, for example, thyroid cancer, duodenal cancer, neuroendocrine carcinoma (NEC), uterine cancer, small cell lung cancer (SCLC), hepatocellular carcinoma, mesothelioma, breast cancer, sarcoma, ovarian cancer, renal cell carcinoma, rectal cancer, head and neck cancer, prostate cancer, pancreatic cancer, melanoma, colorectal cancer, cervical cancer, non-small cell lung cancer (NSCLC), cholangiocarcinoma, or endometrial cancer cells.

[0161] In certain embodiments, cancers with increased potential to be effectively treated with NAMPTi are thyroid cancer, duodenal cancer, neuroendocrine carcinoma (NEC), uterine cancer, small cell lung cancer (SCLC), hepatocellular carcinoma, mesothelioma, breast cancer, sarcoma, ovarian cancer, renal cell carcinoma, rectal cancer, head and neck cancer, prostate cancer, pancreatic cancer, melanoma, colorectal cancer, cervical cancer, non-small cell lung cancer (NSCLC), cholangiocarcinoma, or endometrial cancer. In certain embodiments, cancer is treated with the administration of niacin in addition to NAMPTi.

[0162] In another aspect, the present invention provides a cancer treatment comprising the step of administering a nicotinamide phosphoribosyltransferase inhibitor (NAMPTi) to a subject requiring treatment for cancer, wherein the sample from the cancer has a lower NAPRT level than a control sample. In certain embodiments, the method further comprises the step of administering niacin to the subject.

[0163] In another aspect, the present invention provides a method for diagnosing a subject having cancer with an increased likelihood of being effectively treated with NAMPTi, comprising the steps of administering an antibody that specifically binds to NAPRT to a sample from the subject and detecting the specific binding of the antibody to NAPRT, wherein if the binding to NAPRT in the sample from the subject is less than in a control sample, the likelihood of the subject's cancer being effectively treated with NAMPTi is increased.

[0164] In another aspect, the anti-NAPRT antibody disclosed herein is useful for the diagnosis of sepsis. In certain embodiments, the anti-NAPRT antibody is useful for indicating the likelihood that a subject has sepsis. In certain embodiments, if the NAPRT level detected in a sample from a subject is higher than the level detected in a control sample, the likelihood that the subject has sepsis increases.

[0165] Polynucleotide, vector, recombinant host cell, and antibody generation method

[0166] In another aspect, the present invention relates to a polynucleotide comprising a nucleotide sequence encoding an antibody described herein or a part thereof or a fragment thereof (e.g., VL and / or VH; and a light chain and / or heavy chain) that specifically binds to a NAPRT (e.g., human NAPRT) antigen, and a vector, e.g., a host cell (e.g., E. coli ( E. coliVectors containing these polynucleotides are provided for recombinant expression in ) and mammalian cells). The present invention provides polynucleotides containing nucleotide sequences encoding the heavy chain and / or light chain of any antibody provided herein, as well as vectors containing these polynucleotide sequences, such as expression vectors for efficient expression in host cells, e.g., mammalian cells.

[0167] As used herein, "isolated" polynucleotide or nucleic acid molecules are separated from other nucleic acid molecules present in natural sources of nucleic acid molecules (e.g., mouse or human). Additionally, "isolated" nucleic acid molecules, such as cDNA molecules, may be substantially free of other cellular material or culture medium when produced by recombinant technology, or substantially free of chemical precursors or other chemicals when chemically synthesized. For example, the expression "substantially free" includes a preparation of polynucleotide or nucleic acid molecules in which other materials, e.g., cellular material, culture medium, other nucleic acid molecules, chemical precursors, and / or other chemicals, are about 15%, 10%, 5%, 2%, 1%, 0.5%, or less than 0.1% (particularly about 10%). In certain embodiments, the nucleic acid molecule(s) encoding the antibody described herein are isolated or purified.

[0168] In certain embodiments, the present invention provides a polynucleotide comprising a nucleotide sequence encoding an antibody that specifically binds to a NAPRT (e.g., human NAPRT) polypeptide and comprises an amino acid sequence as described herein, as well as an antibody that competes with such antibody for binding to the NAPRT (e.g., human NAPRT) polypeptide (e.g., in a dose-dependent manner) or binds to an epitope identical to the epitope of such antibody.

[0169] In certain embodiments, the present invention provides a polynucleotide comprising a nucleotide sequence encoding a light chain or a heavy chain of the antibody described herein. The polynucleotide may comprise a nucleotide sequence encoding a light chain including VL FR and CDR of the antibody described herein (e.g., see Tables 2 and 4) or a nucleotide sequence encoding a heavy chain including VH FR and CDR of the antibody described herein (e.g., see Tables 2 and 3). In certain embodiments, the polynucleotide codes for VH, VL, a heavy chain, and / or a light chain of the antibody described herein. In another embodiment, the polynucleotide codes for a first VH and a first VL of the antibody described herein. In another embodiment, the polynucleotide codes for a second VH and a second VL of the antibody described herein. In another embodiment, the polynucleotide codes for a first heavy chain and a first light chain of the antibody described herein. In another embodiment, the polynucleotide codes for the second heavy chain and the second light chain of the antibody described herein. In another embodiment, the polynucleotide codes for the VH and / or VL, or the heavy chain and / or light chain of the antibody described herein.

[0170] Additionally, the present invention provides a polynucleotide encoding an anti-NAPRT antibody, optimized, for example, by codon / RNA optimization, substitution with a heterologous signal sequence, and removal of mRNA instability elements. Methods for generating an optimized nucleic acid encoding an anti-NAPRT antibody or a fragment thereof (e.g., light chain, heavy chain, VH domain, or VL domain) for recombinant expression by introducing codon changes and / or removing repressive regions within the mRNA may be performed by adapting the optimization methods described, for example, in U.S. Patents Nos. 5,965,726, 6,174,666, 6,291,664, 6,414,132, and 6,794,498, all of which are incorporated herein by reference in their entirety. For example, potential splice sites and instability elements (e.g., A / T or A / U enrichment elements) within the RNA may be mutated without altering the amino acids encoded by the nucleic acid sequence to increase the stability of the RNA for recombinant expression. These changes utilize the degeneracy of the genetic code, for example, by using alternative codons for the same amino acid. In certain embodiments, it may be desirable to change one or more codons to code for a conserved mutation, for example, a similar amino acid having a chemical structure and properties and / or function similar to the original amino acid. This method can increase the expression of an anti-NAPRT antibody or a fragment thereof by at least 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times or more compared to the expression of an anti-NAPRT antibody encoded by an unoptimized polynucleotide.

[0171] In certain embodiments, an optimized polynucleotide sequence encoding the anti-NAPRT antibody or its fragment (e.g., VL domain and / or VH domain) described herein may be hybridized to an antisense (e.g., complementary) polynucleotide of an unoptimized polynucleotide sequence encoding the anti-NAPRT antibody or its fragment (e.g., VL domain and / or VH domain) described herein. In certain embodiments, an optimized nucleotide sequence encoding the anti-NAPRT antibody or its fragment described herein is hybridized to an antisense polynucleotide of an unoptimized polynucleotide sequence encoding the anti-NAPRT antibody or its fragment described herein under high-strict conditions. In certain embodiments, an optimized nucleotide sequence encoding the anti-NAPRT antibody or its fragment described herein is hybridized to an antisense polynucleotide of an unoptimized nucleotide sequence encoding the anti-NAPRT antibody or its fragment described herein under high-strict, medium-strict, or low-strict hybridization conditions. Information regarding hybridization conditions is described, for example, by reference to U.S. Patent Application Publication No. US 2005 / 0048549 (e.g., paragraphs 72-73), the entirety of which is incorporated herein by reference.

[0172] Polynucleotides can be obtained by any method known in the art, and the nucleotide sequence of the polynucleotide can be determined. The nucleotide sequences encoding the antibodies described herein, for example, the antibodies described in Tables 2 through 4, and modified versions of these antibodies can be determined using methods well known in the art, namely, assembling nucleotide codons known to code for specific amino acids in a manner that generates nucleic acids encoding antibodies. These polynucleotides encoding antibodies can be assembled from chemically synthesized oligonucleotides (for example, as described in the literature (Kutmeier G et al., (1994), BioTechniques 17: 242-6, the whole incorporated herein by reference), which simply comprises the synthesis of overlapping oligonucleotides containing part of the sequence encoding the antibody, the annealing and ligation of said oligonucleotides, and then amplification of the ligated oligonucleotides by PCR.

[0173] Alternatively, the polynucleotide encoding the antigen-binding region of the antibody described herein may be generated from nucleic acids from a suitable source (e.g., hybridoma) using methods well known in the art (e.g., PCR and other molecular cloning methods). For example, PCR amplification using synthetic primers capable of hybridizing to the 3' and 5' ends of a known sequence may be performed using genomic DNA obtained from a hybridoma cell that produces the antibody of interest. Such PCR amplification methods may be used to obtain nucleic acids containing sequences encoding the light chain and / or heavy chain of the antibody. Such PCR amplification methods may be used to obtain nucleic acids containing sequences encoding the variable light chain region and / or variable heavy chain region of the antibody. The amplified nucleic acids may be cloned into a vector for expression in host cells and for further cloning.

[0174] Where a clone containing nucleic acid encoding a specific antigen-binding region or antibody is unavailable but the sequence of the antigen-binding region or antibody molecule is known, the nucleic acid encoding the immunoglobulin may be obtained by PCR amplification using synthetic primers capable of hybridizing to the 3' and 5' ends of the sequence from a suitable source (e.g., an antibody cDNA library, or a cDNA library generated from any tissue or cell expressing the antibody, such as a hybridoma cell selected to express the antibody described herein, or nucleic acid isolated therefrom, preferably poly A+ RNA), or by cloning using oligonucleotide probes specific to a specific gene sequence to identify a cDNA clone from a cDNA library encoding the antibody, for example. The amplified nucleic acid generated by PCR may be cloned into a replicatable cloning vector using any method well known in the art.

[0175] The DNA encoding the anti-NAPRT (e.g., human NAPRT) antibody described herein can be easily isolated and sequenced using conventional procedures (e.g., by using an oligonucleotide probe capable of specifically binding to the genes encoding the heavy and light chains of the anti-NAPRT (e.g., human NAPRT) antibody). Hybridoma cells can serve as a source of this DNA. Once isolated, the DNA can be placed within an expression vector, which is then used in this. E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells (e.g., CHO GS System) TM Transfected into host cells such as (Lonza-derived CHO cells), or myeloma cells that do not otherwise produce immunoglobulin proteins, it induces the synthesis of anti-NAPRT antibodies in the recombinant host cells.

[0176] To generate the entire antibody or antigen-binding region, the VH or VL sequence in the scFv clone may be amplified using PCR primers comprising a VH or VL nucleotide sequence, a restriction enzyme site, and a flanking sequence to protect the restriction enzyme site. Using cloning techniques known to those skilled in the art, the PCR-amplified VH domain may be cloned into a vector expressing a heavy chain constant region, e.g., a human gamma 1 or human gamma 4 constant region, and the PCR-amplified VL domain may be cloned into a vector expressing a light chain constant region, e.g., a human kappa or lambda constant region. In certain embodiments, a vector for expressing the VH or VL domain comprises an EF-1α promoter, a secretion signal, a cloning site for the variable region, the constant region, and a screening marker such as neomycin. The VH and VL domains may also be cloned into a single vector expressing the required constant region. Next, a heavy chain conversion vector and a light chain conversion vector are co-transfected into a cell line to produce a stable or transient cell line expressing a full-length antibody, e.g. IgG, using a technique known to those skilled in the art.

[0177] In addition, DNA can be modified, for example, by substituting the coding sequence for the human heavy and light chain constant regions in place of the murine sequence, or by covalently bonding all or part of the coding sequence for a non-immunoglobulin polypeptide to the immunoglobulin coding sequence.

[0178] Additionally, a polynucleotide is provided that hybridizes to the polynucleotide encoding the antibody described herein under high, medium, or low strict hybridization conditions. In certain embodiments, the polynucleotide described herein is hybridized to the polynucleotide encoding the VH domain and / or VL domain provided herein under high, medium, or low strict hybridization conditions.

[0179] Hybridization conditions are described in the art and are known to those skilled in the art. For example, hybridization under strict conditions may include hybridization to filter-bound DNA in 6x sodium chloride / sodium citrate (SSC) at about 45°C followed by one or more washes in 0.2xSSC / 0.1% SDS at about 50-65°C; hybridization under high strict conditions may include hybridization to filter-bound nucleic acid in 6xSSC at about 45°C followed by one or more washes in 0.1xSSC / 0.2% SDS at about 68°C. Hybridization under other strict hybridization conditions is known and described to those skilled in the art, and reference is made to the literature, for example (Ausubel FM et al., eds., (1989) Current Protocols in Molecular Biology, Vol. I, Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York, pages 6.3.1–6.3.6 and 2.10.3, the whole incorporated herein by reference).

[0180] In certain embodiments, the present invention provides a cell (e.g., a host cell) that expresses (e.g., recombinantly) the antibody described herein that specifically binds to NAPRT (e.g., human NAPRT), and an associated polynucleotide and an expression vector. The present invention provides a vector (e.g., an expression vector) comprising a polynucleotide containing a nucleotide sequence encoding an anti-NAPRT antibody or fragment for recombinant expression in a host cell, preferably a mammalian cell (e.g., a CHO cell). Additionally, the present invention provides a host cell containing such a vector for recombinantly expressing the anti-NAPRT antibody described herein (e.g., a human or humanized antibody). In certain embodiments, the present invention provides a method for producing the antibody described herein, comprising the step of expressing the antibody from a host cell.

[0181] Recombinant expression of the antibody described herein that specifically binds to NAPRT (e.g., human NAPRT) (e.g., the full-length antigen-binding region of the antibody described herein or the heavy and / or light chain of the antibody) generally involves constructing an expression vector containing a polynucleotide encoding the antibody. Once a polynucleotide encoding the antibody molecule described herein, the heavy and / or light chain of the antibody, or a fragment thereof (e.g., a variable region of the heavy and / or light chain) is obtained, a vector for the production of the antibody molecule may be produced by recombinant DNA technology using techniques well known in the art. Accordingly, a method for producing a protein by expressing a polynucleotide containing a nucleotide sequence encoding the antibody or antibody fragment (e.g., light or heavy chain) is described herein. Methods well known to those skilled in the art may be used to construct an expression vector containing a coding sequence for the antibody or antibody fragment (e.g., light or heavy chain) and appropriate transcription and translation control signals. Such methods include, for example, in vitro recombinant DNA technology, synthetic technology, and in vivo gene recombination. Additionally, a replicable vector is provided comprising a nucleotide sequence encoding the antibody molecule described herein, the heavy or light chain of the antibody, the variable region of the heavy or light chain of the antibody or a fragment thereof, or the heavy or light chain CDR, and is operably linked to a promoter. Such a vector may include, for example, a nucleotide sequence encoding the constant region of the antibody molecule (see, for example, International Publication Nos. WO 86 / 05807 and WO 89 / 01036, the whole of which is incorporated herein by reference; and U.S. Patent No. 5,122,464), and the variable region of the antibody may be cloned into such a vector for the expression of the whole heavy chain, the whole light chain, or both the whole heavy chain and the light chain.

[0182] In certain embodiments, the vector comprises a polynucleotide encoding the VH, VL, heavy chain, and / or light chain of the antibody described herein. In another embodiment, the vector comprises a polynucleotide encoding the VH and VL of the antibody described herein. In yet another embodiment, the vector comprises a polynucleotide encoding the heavy chain and light chain of the antibody described herein.

[0183] The expression vector can be delivered to a cell (e.g., a host cell) by conventional techniques, and the resulting cell can then be cultured by conventional techniques to produce a cell containing the antibody or a fragment thereof described herein. Accordingly, the present invention provides a host cell containing a polynucleotide encoding the antibody or a fragment thereof, or its heavy or light chain, or its fragment, or the short-chain antibody described herein, which is operably linked to a promoter for the expression of such sequences in the host cell.

[0184] In a specific embodiment, the host cell comprises a polynucleotide encoding VH and VL of the antibody described herein. In another embodiment, the host cell comprises a vector comprising a polynucleotide encoding VH and VL of the antibody described herein. In another embodiment, the host cell comprises a first polynucleotide encoding VH of the antibody described herein and a second polynucleotide encoding VL of the antibody described herein. In another embodiment, the host cell comprises a first vector comprising a first polynucleotide encoding VH of the antibody described herein and a second vector comprising a second polynucleotide encoding VL of the antibody described herein.

[0185] In certain embodiments, a heavy chain / heavy chain variable region expressed by a first cell associates with a light chain / light chain variable region of a second cell to form the anti-NAPRT (e.g., human NAPRT) antibody described herein. In certain embodiments, a host cell population comprising such a first host cell and such a second host cell is provided.

[0186] In certain embodiments, the present invention provides a vector group comprising a first vector comprising a polynucleotide encoding a light chain / light chain variable region of the anti-NAPRT (e.g., human NAPRT) antibody described herein, and a second vector comprising a polynucleotide encoding a heavy chain / heavy chain variable region of the anti-NAPRT (e.g., human NAPRT) antibody described herein.

[0187] Various host expression vector systems may be used to express the antibody molecules described herein (see, for example, U.S. Patent No. 5,807,715, which is incorporated herein by reference in its entirety). Such host-expression systems are not only vehicles that allow the coding sequence of interest to be generated and subsequently purified, but also cells capable of expressing the antibody molecules described herein in situ when transformed or transfected with appropriate nucleotide coding sequences. These are, for example, bacteria transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing antibody-coding sequences (e.g., E. coli and B. subtilis ( B. subtilis Microorganisms such as )); for example, yeast transformed with a recombinant yeast expression vector containing an antibody-coding sequence (e.g., Saccharomyces ( Saccharomyces)and, for example, insect cell systems infected with a recombinant viral expression vector containing an antibody-coding sequence (e.g., baculovirus); for example, plant cell systems infected with a recombinant viral expression vector (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with a recombinant plasmid expression vector containing an antibody-coding sequence (e.g., Ti plasmid) (e.g., Chlamydomonas reinharty ( Chlamydomonas reinhardtii Green algae such as ); or mammalian cell systems (e.g., COS (e.g., COS1 or COS), CHO, BHK, MDCK, HEK 293, NS0, PER.C6, VERO, CRL7O3O, HsS78Bst, HeLa, and NIH 3T3, HEK-293T, HepG2, SP210, R1.1, BW, LM, BSC1, BSC40, YB / 20 and BMT10 cells) having recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoters) or promoters derived from mammalian viruses (e.g., adenovirus late promoters; vaccinia virus 7.5K promoters), but not limited thereto. In certain embodiments, the cell for expressing the antibody described herein is a Chinese hamster ovary (CHO) cell, e.g., CHO GS System TM It is a (Lonza) derived CHO cell. In certain embodiments, the heavy chain and / or light chain of the antibody produced by the CHO cell may have an N-terminal glutamine or glutamate residue replaced with pyroglutamate. In certain embodiments, the cell for expressing the antibody described herein is a human cell, e.g., a human cell line. In certain embodiments, the mammalian expression vector is pOptiVEC TM or pcDNA3.3. In certain embodiments, Escherichia coli ( Escherichia coliBacterial cells such as ) or eukaryotic cells (e.g., mammalian cells) are used for the expression of recombinant antibody molecules, particularly for the expression of whole recombinant antibody molecules. For example, mammalian cells such as CHO cells, along with a vector such as a major intermediate early gene promoter element derived from human cytomegalovirus, are an efficient expression system for antibodies (Foecking MK & Hofstetter H (1986) Gene 45: 101-5; and Cockett MI et al. , (1990) Biotechnology 8(7): 662-7, each incorporated herein in its entirety by reference). In certain embodiments, the antibody described herein is produced by CHO cells or NS0 cells. In certain embodiments, the expression of the nucleotide sequence encoding the antibody described herein that specifically binds to NAPRT (e.g., human NAPRT) is regulated by a constitutive promoter, an inducible promoter, or a tissue-specific promoter.

[0188] In bacterial systems, multiple expression vectors may be advantageously selected depending on the intended use of the antibody molecules being expressed. For example, when such antibodies need to be produced in large quantities for the production of pharmaceutical compositions of antibody molecules, a vector that induces the expression of high-level fusion protein products that are easily purified may be desirable. Such vectors include, but are not limited to, E. coli expression vectors in which coding sequences are individually ligated within the vector into the same frame as the lac Z coding region to produce fusion proteins, such as the E. coli expression vector pUR278 (Ruether U & Mueller-Hill B (1983) EMBO J 2: 1791-1794); pIN vectors (Inouye S & Inouye M (1985) Nuc Acids Res 13: 3101-3109; Van Heeke G & Schuster SM (1989) J Biol Chem 24: 5503-5509), all of which are incorporated herein by reference in their entirety. For example, pGEX vectors can also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). Generally, these fusion proteins are soluble and can be easily purified from lysed cells by adsorption and binding to matrix glutathione agarose beads followed by elution in the presence of free glutathione. pGEX vectors are designed to include a thrombin or factor Xa protease cleavage site so that the cloned target gene product can be released from the GST moiety.

[0189] In insect systems, for example, the Autographa californica nuclear polyhedron disease virus ( Autographa californica nuclear polyhedrosis virus (AcNPV) can be used as a vector for expressing foreign genes. This virus is Spodoptera plugieferda ( Spodoptera frugiperdaIt grows in cells. The coding sequence can be individually cloned into a non-essential region of the virus (e.g., polyhedrin gene) and placed under the control of an AcNPV promoter (e.g., polyhedrin promoter).

[0190] In mammalian host cells, multiple virus-based expression systems may be utilized. When an adenovirus is used as an expression vector, the coding sequence of interest may be ligated to the adenovirus transcription / translation control complex, e.g., a late promoter and a triple leader sequence. Subsequently, this chimeric gene may be inserted into the adenovirus genome by in vitro or in vivo recombination. Insertion into a non-essential region of the viral genome (e.g., the E1 or E3 region) will produce a recombinant virus capable of viability and expressing the molecule in an infected host (see, for example, Logan J & Shenk T (1984) PNAS 81(12): 3655-9, the whole of which is incorporated herein by reference). A specific initiation signal may also be required for the efficient translation of the inserted coding sequence. Such signals include an ATG initiation codon and an adjacent sequence. Additionally, to ensure the translation of the entire insertion, the initiation codon must be in phase with the leading frame of the target coding sequence. These exogenous translation control signals and start codons can be of various origins, both natural and synthetic. Expression efficiency can be enhanced by the inclusion of appropriate transcription enhancer elements, transcription termination factors, etc. (see, e.g., Bitter G et al., (1987) Methods Enzymol. 153: 516-544, the whole incorporated herein by reference).

[0191] In addition, a host cell line can be selected to regulate the expression of the inserted sequence or to modify and process the gene product in a specific desired manner. Such modification (e.g., glycosylation) and processing (e.g., cleavage) of the protein product may be important for the function of the protein. Different host cells possess characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. An appropriate cell line or host system can be selected to ensure the precise modification and processing of the expressed foreign protein. To this end, eukaryotic host cells possessing cellular mechanisms for the appropriate processing of the primary transcript and the glycosylation and phosphorylation of the gene product can be used. These mammalian host cells include, but are not limited to, CHO, VERO, BHK, Hela, MDCK, HEK 293, NIH 3T3, W138, BT483, Hs578T, HTB2, BT2O and T47D, NS0 (a murine myeloma cell line that does not endogenously produce any immunoglobulin surge), CRL7O3O, COS (e.g., COS1 or COS), PER.C6, VERO, HsS78Bst, HEK-293T, HepG2, SP210, R1.1, BW, LM, BSC1, BSC40, YB / 20, BMT10, and HsS78Bst cells. In certain embodiments, the anti-NAPRT (e.g., human NAPRT) antibodies described herein are produced in mammalian cells such as CHO cells.

[0192] In certain embodiments, the antibodies described herein have a reduced fucose content or no fucose content. Such antibodies may be produced using techniques known to those skilled in the art. For example, the antibodies may be expressed in cells lacking or deficient in fucosylation ability. In certain examples, antibodies having a reduced fucose content may be produced using cell lines in which both alleles of α1,6-fucosyltransferase have been knocked out. Potelligent ®The system (Lonza) is an example of such a system that can be used to generate antibodies with reduced fucose content.

[0193] In certain embodiments, stable expression cells can be generated for the long-term high-yield production of recombinant proteins. For example, cell lines that stably express the anti-NAPRT (e.g., human NAPRT) antibody described herein can be engineered. In certain embodiments, the cells provided herein stably express the light chain / light chain variable region and heavy chain / heavy chain variable region that associate to form the antigen-binding region or antibody described herein.

[0194] In certain embodiments, instead of using an expression vector containing a viral replication origin, host cells may be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequence, transcription termination factor, polyadenylation site, etc.) and selection markers. After introducing the foreign DNA / polynucleotide, the engineered cells are grown in enrichment medium for 1 to 2 days and then switched to selection medium. The selection markers within the recombinant plasmid confer resistance to the selector, allowing the cells to stably incorporate the plasmid into their chromosomes and grow to form a focus, which can then be cloned and expanded into a cell line. This method can be advantageously used to engineer cell lines expressing the anti-NAPRT (e.g., human NAPRT) or fragments thereof described herein. Such engineered cell lines may be particularly useful for screening and evaluating compositions that interact directly or indirectly with antibody molecules.

[0195] Multiple screening systems may be used, including herpes simplex virus thymidine kinase in tk-, hgprt-, or aprt- cells, respectively (Wigler M et al. ,(1977) Cell 11(1): 223-32), hypoxanthine guanine phosphoribosyltransferase (Szybalska EH & Szybalski W (1962) PNAS 48(12): 2026-2034), and adenine phosphoribosyltransferase (Lowy I et al., (1980) Cell 22(3): 817-23) genes, all of which are incorporated herein by reference in their entirety. Additionally, antimetabolite resistance is conferred by the following genes: those conferring resistance to methotrexate dhfr (Wigler M et al., (1980) PNAS 77(6): 3567-70; O'Hare K et al. , (1981) PNAS 78: 1527-31); conferring resistance to mycophenolic acid gpt (Mulligan RC & Berg P (1981) PNAS 78(4): 2072-6); neo conferring resistance to aminoglycoside G-418 (Wu GY & Wu CH (1991) Biotherapy 3: 87-95; Tolstoshev P (1993) Ann Rev Pharmacol Toxicol 32: 573-596; Mulligan RC (1993) Science 260: 926-932; and Morgan RA & Anderson WF (1993) Ann Rev Biochem 62: 191-217; Nabel GJ & Felgner PL (1993) Trends Biotechnol 11(5): 211-5); and conferring resistance to hygromycin hygro (anterre RF etc.) ,(1984) Gene 30(1-3): 147-56) may be used as the basis for screening, all of which are incorporated herein by reference in their entirety. Methods generally known in the field of recombinant DNA technology may be routinely applied to screen for the desired recombinant clones, and such methods are, for example, in the literature (Ausubel FM et al., (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler M, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990); and Chapters 12 and 13, Dracopoli NC et al., (eds.), Current Protocols in Human Genetics, John Wiley & Sons, NY (1994); Colb re-Garapin F etc. , (1981) J Mol Biol 150: 1-14, all of which are incorporated herein by reference in their entirety) is described.

[0196] The expression level of antibody molecules can be increased by vector amplification (for review, see the literature (Bebbington CR & Hentschel CCG, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol. 3 (Academic Press, New York, 1987), incorporated herein by reference in its entirety)). If a marker within a vector system is amplifiable, an increase in the level of inhibitor present in the host cell culture will increase the copy of the marker gene. Since the amplified region is associated with the gene of interest, protein production will also increase (Crouse GF et al., (1983) Mol Cell Biol 3: 257-66, incorporated herein by reference in its entirety).

[0197] Host cells may be co-transfected with two or more expression vectors described herein, wherein the first vector codes for a heavy chain-derived polypeptide and the second vector codes for a light chain-derived polypeptide. Both vectors may contain the same selectable marker that enables equal expression of the heavy chain and light chain polypeptides. Host cells may be co-transfected with different amounts of two or more expression vectors. For example, host cells may be transfected with any of the following ratios of the first expression vector and the second expression vector: approximately 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:12, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, or 1:50.

[0198] Alternatively, a single vector capable of encoding and expressing both heavy and light chain polypeptides may be used. In this situation, the light chain must be placed before the heavy chain to avoid an excess of toxic free heavy chains (Proudfoot NJ (1986) Nature 322: 562-565; and G (1980) PNAS 77: 2197-2199, each incorporated herein in its entirety by reference). Coding sequences for heavy and light chains may comprise cDNA or genomic DNA. The expression vector may be a single cistron or a multiple cistron. A multiple cistron nucleic acid construct may code for 2, 3, 4, 5, 6, 7, 8, 9, 10 or more gene / nucleotide sequences, or a range of 2-5, 5-10, or 10-20 gene / nucleotide sequences. For example, a double cistron nucleic acid construct may comprise, in order, a promoter, a first gene (e.g., the heavy chain of the antibody described herein), and a second gene (e.g., the light chain of the antibody described herein). In such expression vectors, transcription of both genes can be induced by a promoter, while mRNA translation from the first gene can be induced by a cap-dependent scanning mechanism and mRNA translation from the second gene can be induced by a cap-independent mechanism, e.g., IRES.

[0199] If the antibody molecules described herein are produced by recombinant expression, they may be purified by any method known in the art for the purification of immunoglobulin molecules, e.g., chromatography (e.g., ion exchange, affinity, particularly affinity for specific antigens after protein A, and sizing column chromatography), centrifugation, differential solubility, or any other standard technique for protein purification. Additionally, the antibodies described herein may be fused to heterogeneous polypeptide sequences described herein or otherwise known in the art to facilitate purification.

[0200] In certain embodiments, the antibody described herein is isolated or purified. In certain embodiments, the isolated antibody is substantially free of other antibodies having different antigen specificity from the isolated antibody. For example, in certain embodiments, the antibody preparation described herein is substantially free of cellular material and / or chemical precursors. The expression “substantially free of cellular material” comprises an antibody preparation in which the antibody is isolated or separated from the cellular components of a cell produced in a recombinant manner. Accordingly, an antibody substantially free of cellular material comprises a preparation of an antibody having less than about 30%, 20%, 10%, 5%, 2%, 1%, 0.5%, or 0.1% (on a dry weight basis) of heterogeneous proteins (also referred to herein as “contamination proteins”) and / or variants of the antibody, e.g., different post-translational modified forms of the antibody or other different versions of the antibody (e.g., antibody fragments). When antibodies are produced by a recombinant method, there is generally no culture medium, i.e., the culture medium represents about 20%, 10%, 2%, 1%, 0.5%, or less than 0.1% of the volume of the protein preparation. When antibodies are produced by chemical synthesis, there are generally no chemical precursors or other chemicals, i.e., they are separated from chemical precursors or other chemicals involved in protein synthesis. Accordingly, such antibody preparations contain chemical precursors or compounds other than the antibody of interest in an amount of about 30%, 20%, 10%, or less than 5% (on a dry weight basis). In certain embodiments, the antibodies described herein are isolated or purified.

[0201] Anti-NAPRT (e.g., human NAPRT) antibodies or fragments thereof may be produced by any method known in the art for the synthesis of proteins or antibodies, e.g., chemical synthesis or recombinant expression techniques. Unless otherwise specified, the methods described herein utilize molecular biology, microbiology, genetic analysis, recombinant DNA, organic chemistry, biochemistry, PCR, oligonucleotide synthesis and modification, nucleic acid hybridization, and ordinary techniques in related fields within the scope of the art. These techniques are described, for example, in the references cited herein and are sufficiently explained in the literature. See, for example, Maniatis T et al., (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; Sambrook J et al., (1989), Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press; Sambrook J et al., (2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel FM et al., Current Protocols in Molecular Biology, John Wiley & Sons (1987 and annual updates); Current Protocols in Immunology, John Wiley & Sons (1987 and annual updates) Gait (ed.) (1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press; (1991) Oligonucleotides and Analogues: A Practical Approach, IRL Press;Refer to ) (1999) Genome Analysis: A Laboratory Manual, Cold Spring Harbor Laboratory Press (all incorporated herein by reference in their entirety).

[0202] In certain embodiments, the antibodies described herein are prepared, expressed, generated, or isolated by any means involving generation, e.g., synthesis, or genetic manipulation of DNA sequences. In certain embodiments, these antibodies comprise sequences (e.g., DNA sequences or amino acid sequences) that do not naturally exist in vivo within the repertoire of antibody germlines of animals or mammals (e.g., humans).

[0203] In one embodiment, the present invention provides a method for producing an anti-NAPRT (e.g., human NAPRT) antibody, comprising the step of culturing the cell or host cell described herein. In certain embodiments, the method is performed in vitro. In certain embodiments, the present invention provides a method for producing an anti-NAPRT (e.g., human NAPRT) antibody, comprising the step of expressing the antibody (e.g., in a recombinant manner) using the cell or host cell described herein (e.g., a cell or host cell comprising a polynucleotide encoding the antibody described herein). In certain embodiments, the cell is an isolated cell. In certain embodiments, an exogenous polynucleotide is introduced into the cell. In certain embodiments, the method further comprises the step of purifying the antibody obtained from the cell or host cell.

[0204] In a specific embodiment, an antibody is produced by expressing the polynucleotide in a cell under appropriate conditions such that the polynucleotide encoding the VH and VL of the antibody described herein is expressed to produce an antibody. In another embodiment, an antibody is produced by expressing the polynucleotide in a cell under appropriate conditions such that the polynucleotide encoding the heavy chain and light chain of the antibody described herein is expressed to produce an antibody. In a specific embodiment, an antibody is produced by expressing the polynucleotides in a cell under appropriate conditions such that the first polynucleotide encoding the VH of the antibody described herein and the second polynucleotide encoding the VL of the antibody described herein are expressed to produce an antibody. In a specific embodiment, an antibody is produced by expressing the polynucleotides in a cell under appropriate conditions such that the first polynucleotide encoding the heavy chain of the antibody described herein and the second polynucleotide encoding the light chain of the antibody described herein are expressed to produce an antibody.

[0205] Methods for generating polyclonal antibodies are known in the art (e.g., literature (Short Protocols in Molecular Biology, (2002) 5th Ed., Ausubel FM et al.). , See Chapter 11, John Wiley and Sons, New York (the entirety incorporated herein by reference).

[0206] Monoclonal antibodies can be produced using a wide variety of techniques known in the art, including hybridoma, recombinant and phage display techniques, or combinations thereof. For example, monoclonal antibodies are known in the art, and, for instance, in the literature (Harlow E & Lane D, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling GJ et al. ,They may be generated using hybridoma technology, including as taught in Monoclonal Antibodies and T-Cell Hybridomas 563 681 (Elsevier, NY, 1981), each incorporated herein by reference in its entirety. As used herein, the term “monoclonal antibody” is not limited to antibodies generated through hybridoma technology. For example, monoclonal antibodies may be generated in a recombinant manner from host cells exogenously expressing the antibodies or fragments thereof described herein, for example, the light chain and / or heavy chain of such antibodies.

[0207] In certain embodiments, as used herein, a “monoclonal antibody” is an antibody produced by a single cell (e.g., a hybridoma or host cell that produces a recombinant antibody), and the antibody specifically binds to NAPRT (e.g., human NAPRT) as determined, for example, by ELISA or other antigen binding or competitive binding assays known in the art or in the embodiments provided herein. In certain embodiments, the monoclonal antibody may be a chimeric antibody or a humanized antibody. In certain embodiments, the monoclonal antibody is a monovalent antibody or a multivalent (e.g., divalent) antibody. In certain embodiments, the monoclonal antibody is a monospecific or multispecific antibody (e.g., a bispecific antibody). The monoclonal antibodies described herein may be prepared by a hybridoma method, for example as described in the literature (Kohler G & Milstein C (1975) Nature 256: 495, the whole is incorporated herein by reference), or may be isolated from a phage library using techniques, for example as described herein. Other methods for preparing clonal cell lines and monoclonal antibodies expressed thereby are well known in the art (see, for example, *Short Protocols in Molecular Biology, (2002) 5th Ed., Ausubel FM et al., Chapter 11 thereof).

[0208] As used herein, when an antibody comprises at least two (e.g., two or more) monovalent binding regions, and each monovalent binding region can bind to an epitope on the antigen, the antibody binds to the antigen multivalently (e.g., divalently). Each monovalent binding region can bind to the same or different epitopes on the antigen.

[0209] Methods for generating and screening specific antibodies using hybridoma technology are common and well known in the art. For example, in the hybridoma method, mice or other suitable host animals, such as sheep, goats, rabbits, rats, hamsters, or macaque monkeys, are immunized to induce lymphocytes capable of generating antibodies that specifically bind to the protein used for immunization (e.g., NAPRT (e.g., human NAPRT)). Alternatively, lymphocytes may be immunized in vitro. The lymphocytes are then fused with myeloma cells using a suitable fusion agent, such as polyethylene glycol, to form hybridoma cells (Goding JW (Ed.), Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986), incorporated herein by reference in its entirety). Additionally, RIMMS (repeated immunization multiple sites) technology may be used to immunize animals (Kilpatrick KE et al., (1997) Hybridoma 16:381-9, the whole is incorporated herein by reference).

[0210] In certain embodiments, a mouse (or other animal such as a rat, monkey, donkey, pig, sheep, hamster, or dog) may be immunized with an antigen (e.g., NAPRT (e.g., human NAPRT)), and once an immune response is detected, for example, when antibodies specific to the antigen are detected in the mouse serum, the mouse spleen is collected and splenocytes are isolated. The splenocytes are then transferred to any suitable myeloma cells, for example, American Type Culture Collection (ATCC), by well-known techniques. ® A hybridoma is formed by fusing cells from the SP20 cell line available at (Manassas, VA). The hybridoma is selected by limiting dilution and cloned. In a specific embodiment, lymph nodes from an immunized mouse are taken and fused with NS0 myeloma cells.

[0211] Hybridoma cells prepared in this way are seeded and grown in a suitable culture medium preferably containing one or more substances that inhibit the growth or survival of unfused maternal myeloma cells. For example, if the maternal myeloma cells lack the hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT) enzyme, the culture medium for the hybridoma will typically contain hypoxanthine, aminopterin, and thymidine (HAT medium), and these substances prevent the growth of HGPRT-deficient cells.

[0212] In certain embodiments, myeloma cells sensitive to media such as HAT medium are used to support the stable high-level production of antibodies by selected antibody-generating cells that are efficiently fused. Among these myeloma cell lines are the NS0 cell line available from the Salk Institute Cell Distribution Center (San Diego, CA, USA) or murine myeloma cell lines such as those derived from MOPC-21 and MPC-11 mouse tumors, and SP-2 or X63-Ag8.653 cells available from the American Type Culture Collection (Rockville, MD, USA). Human myeloma and mouse-human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies (Kozbor D (1984) J Immunol 133: 3001-5; Brodeur et al. , Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987), each incorporated herein by reference in its entirety).

[0213] The culture medium in which hybridoma cells are growing is tested for the production of monoclonal antibodies directed against NAPRT (e.g., human NAPRT). The binding specificity of the monoclonal antibodies produced by the hybridoma cells is determined by in vitro binding assays, such as methods known in the art, e.g., immunoprecipitation, radioimmunoassay (RIA), or enzyme-linked immunosorbent assay (ELISA).

[0214] Once hybridoma cells producing antibodies of the desired specificity, affinity, and / or activity are identified, the clones can be subcloned by limiting dilution procedures and grown by standard methods (Goding JW (Ed.), Monoclonal Antibodies: Principles and Practice, supra Culture media suitable for this purpose include, for example, D-MEM or RPMI 1640 medium. Additionally, hybridoma cells can be grown in vivo as assortment tumors in animals.

[0215] Monoclonal antibodies secreted by a subclone are suitably separated from culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as, for example, protein A-cephalosporin, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0216] The antibody described herein comprises an antibody fragment that recognizes, for example, NAPRT (e.g., human NAPRT) and may be produced by any technique known to those skilled in the art. For example, the Fab and F(ab')2 fragments described herein may be produced by proteolytic cleavage of an immunoglobulin molecule using an enzyme such as papain (to produce the Fab fragment) or pepsin (to produce the F(ab')2 fragment). The Fab fragment corresponds to one of two identical arms of the antibody molecule and comprises a complete light chain paired with the VH and CH1 domains of the heavy chain. The F(ab')2 fragment contains two antigen-binding arms of the antibody molecule connected by a disulfide bond at the hinge region.

[0217] Additionally, the antibodies described herein may be generated using various phage display methods known in the art. In a phage display method, a functional antibody domain is displayed on the surface of a phage particle carrying a polynucleotide sequence encoding it. In particular, the DNA sequence encoding the VH and VL domains is amplified from an animal cDNA library (e.g., a human or murine cDNA library of an infected tissue). The DNA encoding the VH and VL domains is recombined with a scFv linker by PCR and cloned into a phagemid vector. The vector is electroporated into E. coli, and the E. coli are infected by a helper phage. The phage used in this method is typically a filamentous phage containing fd and M13, and the VH and VL domains are usually fused recombinantly to phage gene III or gene VIII. Phages expressing an antigen-binding region that binds to a specific antibody may be screened or identified using an antigen, for example, by using a labeled antibody or an antigen bound to a solid surface or bead, or by using a captured antigen.Examples of phage display methods that can be used to produce the antibodies described herein are found in the literature (Brinkman U et al., (1995) J Immunol Methods 182: 41-50; Ames RS et al., (1995) J Immunol Methods 184: 177-186; Kettleborough CA et al., (1994) Eur J Immunol 24: 952-958; Persic L et al., (1997) Gene 187: 9-18; Burton DR & Barbas CF (1994) Advan Immunol 57: 191-280; PCT Application No. PCT / GB91 / 001134; International Publication Nos. WO 90 / 02809, WO 91 / 10737, WO 92 / 01047, WO 92 / 18619, WO 93 / 11236, Includes what is disclosed in WO 95 / 15982, WO 95 / 20401, and WO 97 / 13844; and U.S. Patent Nos. 5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727, 5,733,743, and 5,969,108 (all of which are incorporated herein by reference in their entirety).

[0218] As described in the aforementioned references, after phage selection, the antibody-coding region from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen-binding fragment, which can be expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, for example, as described below. Techniques for recombinantly generating antibody fragments such as Fab, Fab', and F(ab')2 fragments are also described in the literature (PCT Publication No. WO 92 / 22324; Mullinax RL et al. ,(1992) BioTechniques 12(6): 864-9; Sawai H et al. , (1995) Am J Reprod Immunol 34: 26-34; and Better M et al. , (1988) Science 240: 1041-1043, all of which are incorporated herein by reference in their entirety) may be adopted using methods known in the art.

[0219] In certain embodiments, to generate a whole antibody, the VH or VL sequence may be amplified from a template, e.g., a scFv clone, using PCR primers comprising a VH or VL nucleotide sequence, a restriction enzyme site, and a flaking sequence to protect the restriction enzyme site. Utilizing cloning techniques known to those skilled in the art, the PCR-amplified VH domain may be cloned into a vector expressing a VH constant region, and the PCR-amplified VL domain may be cloned into a vector expressing a VL constant region, e.g., a human kappa or lambda constant region. The VH and VL domains may also be cloned into a single vector expressing the required constant region. Then, using techniques known to those skilled in the art, a stable or transient cell line expressing a full-length antibody, e.g. IgG, is generated by co-transfecting the heavy chain conversion vector and the light chain conversion vector into a cell line.

[0220] Chimeric antibodies are molecules in which different portions of the antibody are derived from different immunoglobulin molecules. For example, a chimeric antibody may contain a variable region of a mouse or rat monoclonal antibody fused to a constant region of a human antibody. Methods for generating chimeric antibodies are known in the art. For example, in the literature (Morrison SL (1985) Science 229: 1202-7; Oi VT & Morrison SL (1986) BioTechniques 4: 214-221; Gillies SD et al.) ,(1989) J Immunol Methods 125: 191-202; and U.S. Patent Nos. 5,807,715, 4,816,567, 4,816,397, and 6,331,415, all of which are incorporated herein by reference in their entirety.

[0221] The humanized antibody can bind to a predetermined antigen and comprises a framework region having substantially the amino acid sequence of human immunoglobulin and a CDR having substantially the amino acid sequence of non-human immunoglobulin (e.g., murine immunoglobulin). In certain embodiments, the humanized antibody also comprises an immunoglobulin constant region (Fc), typically at least a portion of the constant region of human immunoglobulin. The antibody may also comprise the CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. The humanized antibody may be selected from any class of immunoglobulin including IgM, IgG, IgD, IgA, and IgE, and any isotype including IgG1, IgG2, IgG3, and IgG4. Humanized antibodies are subjected to CDR grafting (European Patent No. EP 239400; International Publication No. WO 91 / 09967; and U.S. Patent Nos. 5,225,539, 5,530,101, and 5,585,089), veneering, or resurfacing (European Patent Nos. EP 592106 and EP 519596; Padlan EA (1991) Mol Immunol 28(4 / 5): 489-498; Studnicka GM et al. , (1994) Prot Engineering 7(6): 805-814; and Roguska MA et al. ,(1994) PNAS 91: 969-973), chain shuffling (U.S. Patent No. 5,565,332), and literature (U.S. Patent No. 6,407,213, U.S. Patent No. 5,766,886, International Publication No. WO 93 / 17105; Tan P et al., (2002) J Immunol 169: 1119-25; Caldas C et al., (2000) Protein Eng. 13(5): 353-60; Morea V et al., (2000) Methods 20(3): 267-79; Baca M et al., (1997) J Biol Chem 272(16): 10678-84; Roguska MA et al., (1996) Protein Eng 9(10): 895-904; Couto JR et al., (1995) Cancer It may be produced using various techniques known in the art, including but not limited to the techniques disclosed in Res. 55 (23 Supp): 5973s-5977s; Couto JR et al., (1995) Am Res 55(8): 1717-22; Sandhu JS (1994) Gene 150(2): 409-10; and Pedersen JT et al., (1994) J Mol Biol 235(3): 959-73, all of which are incorporated herein by reference in their entirety. Also refer to U.S. Application Publication No. US 2005 / 0042664 A1 (February 24, 2005), which is incorporated herein by reference in its entirety.

[0222] Methods for preparing multispecific antibodies (e.g., bispecific antibodies) are described, and reference is made to, for example, the literature (U.S. Patent Nos. 7,951,917; 7,183,076; 8,227,577; 5,837,242; 5,989,830; 5,869,620; 6,132,992; and 8,586,713, all of which are incorporated herein by reference in their entirety).

[0223] Bispecific bivalent antibodies and methods for producing the same are described, for example, in the literature (U.S. Patent Nos. 5,731,168; 5,807,706; 5,821,333; and U.S. Application Publications Nos. 2003 / 020734 and 2002 / 0155537, each incorporated herein by reference in its entirety). Bispecific saga antibodies and methods for producing the same are described, for example, in the literature (International Application Publications Nos. WO 02 / 096948 and WO 00 / 44788, both incorporated herein by reference in their entirety). Generally, reference is made to the literature (International Application Publication Nos. WO 93 / 17715, WO 92 / 08802, WO 91 / 00360, and WO 92 / 05793; Tutt et al., J. Immunol. 147:60-69 (1991); U.S. Patent Nos. 4,474,893; 4,714,681; 4,925,648; 5,573,920; and 5,601,819; and Kostelny et al., J. Immunol. 148:1547-1553 (1992); each incorporated herein by reference in its entirety).

[0224] Bispecific antibodies as described herein may be produced according to the DuoBody technology platform (Genmab A / S), for example, as described in the literature (International Publication Nos. WO 2011 / 131746, WO 2011 / 147986, WO 2008 / 119353, and WO 2013 / 060867, and Labrijn AF et al., (2013) PNAS 110(13): 5145-5150). DuoBody technology may be used to combine a first monospecific antibody containing two heavy chains and two light chains, or half of a first antigen-binding region, with a second monospecific antibody containing two heavy chains and two light chains, or half of a second antigen-binding region. The resulting heterodimer contains one heavy chain and one light chain derived from the first antibody or the first antigen-binding domain paired with one heavy chain and one light chain derived from the second antibody or the second antigen-binding domain. When the two monospecific antibodies or antigen-binding domains recognize different epitopes on different antigens, the resulting heterodimer is a bispecific antibody.

[0225] Duobody technology requires that each monospecific antibody or antigen-binding region contains a heavy chain constant region with a single point mutation within the CH3 domain. The point mutation causes the interaction between the CH3 domains in the resulting bispecific antibody to be stronger than the interaction between the CH3 domains in each monospecific antibody or antigen-binding region. The single point mutation in each monospecific antibody or antigen-binding region is located at residues 366, 368, 370, 399, 405, 407, or 409 within the CH3 domain of the heavy chain constant region, numbered according to the EU numbering scheme, for example, as described in International Publication No. WO 2011 / 131746. Additionally, the single point mutation is located at a different residue of one monospecific antibody or antigen-binding region compared to other monospecific antibody or antigen-binding regions. For example, when numbered according to the EU numbering system, one monospecific antibody or antigen-binding domain may contain the F405L mutation (i.e., a mutation from phenylalanine to leucine at residue 405), while the other monospecific antibody or antigen-binding domain may contain the K409R mutation (i.e., a mutation from lysine to arginine at residue 409). The heavy chain constant region of the monospecific antibody or antigen-binding domain may be an IgG1, IgG2, IgG3, or IgG4 isotype (e.g., human IgG1 isotype), and the bispecific antibody produced by the Duobody technology may retain Fc-mediated effector function.

[0226] Another method for generating bispecific antibodies is termed the "knop-into-hole" strategy (see, for example, Intl. Publ. WO2006 / 028936). In this technique, mispatching of the IgG heavy chain is reduced by mutating selected amino acids that form the CH3 domain interface of the IgG. At a location within the CH3 domain where the two heavy chains directly interact, an amino acid having a small side chain (hole) is introduced into the sequence of one heavy chain, and an amino acid having a large side chain (knop) is introduced at the corresponding interaction residue location of the other heavy chain. In certain embodiments, the composition of the present disclosure has an immunoglobulin chain with a modified CH3 domain by mutating selected amino acids that interact at the interface between the two polypeptides to preferentially form a bispecific antibody. The bispecific antibody may consist of immunoglobulin chains of the same subclass (e.g., IgG1 or IgG3) or different subclasses (e.g., IgG1 and IgG3, or IgG3 and IgG4).

[0227] Bispecific antibodies may include IgG4 and IgG1, IgG4 and IgG2, IgG4 and IgG3, or IgG1 and IgG3 chain heterodimers in certain cases. These heterodimeric heavy-chain antibodies can typically be engineered, for example, by modifying selected amino acids that form the CH3 domain interface of human IgG4 and IgG1 or IgG3 to promote heterodimeric heavy-chain formation.

[0228] In certain embodiments, the antibody described herein that binds to the same NAPRT (e.g., human NAPRT) epitope as the anti-NAPRT (e.g., human NAPRT) antibody described herein is a human antibody. In certain embodiments, the antibody described herein that competitively blocks any of the antibodies described herein from binding to NAPRT (e.g., human NAPRT) (e.g., in a dose-dependent manner) is a human antibody. Human antibodies may be generated using any method known in the art. For example, a transgenic mouse that cannot express functional endogenous immunoglobulins but can express human immunoglobulin genes may be used. In particular, human heavy and light chain immunoglobulin gene complexes may be introduced into mouse embryonic stem cells randomly or by homologous recombination. Alternatively, in addition to human heavy and light chain genes, human variable regions, constant regions, and diversity regions may be introduced into mouse embryonic stem cells. Mouse heavy and light chain immunoglobulin genes can be defunctionalized separately from or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. In particular, J HHomozygous deletion of the region prevents the production of endogenous antibodies. Modified embryonic stem cells are proliferated and microinjected into blastocysts to generate chimeric mice. The chimeric mice are then crossbred to produce homozygous offspring expressing human antibodies. The transgenic mice are immunized in the conventional manner with a selected antigen, for example, all or part of the antigen (e.g., NAPRT (e.g., human NAPRT)). Monoclonal antibodies directed against the antigen can be obtained from the immunized transgenic mice using conventional hybridoma technology. The human immunoglobulin transgene possessed by the transgenic mice is rearranged during B cell differentiation, followed by class switching and somatic mutation. Thus, it is possible to generate therapeutically useful IgG, IgA, IgM, and IgE antibodies using this technology. For an overview of such techniques for generating human antibodies, refer to the literature (Lonberg N & Huszar D (1995) Int Rev Immunol 13: 65-93, the whole is incorporated herein by reference). For a detailed discussion of such techniques for generating human antibodies and human monoclonal antibodies and for protocols for generating these antibodies, refer to, for example, the literature (International Publication Nos. WO 98 / 24893, WO 96 / 34096, and WO 96 / 33735; and U.S. Patent Nos. 5,413,923, 5,625,126, 5,633,425, 5,569,825, 5,661,016, 5,545,806, 5,814,318, and 5,939,598, all of which are incorporated herein by reference). An example of a mouse capable of producing human antibodies is the XenoMouse TM (Abgenix, Inc.; U.S. Patent Nos. 6,075,181 and 6,150,184), HuAb-Mouse TM(Medarex, Inc. / Gen Pharm; U.S. Patent Nos. 5,545,806 and 5,569,825), TransChromo Mouse TM (Kirin) and KM Mouse TM Includes (Medarex / Kirin), all of which are incorporated herein by reference in their entirety.

[0229] Human antibodies that specifically bind to NAPRT (e.g., human NAPRT) can be prepared by various methods known in the art, including the phage display method described above using an antibody library derived from human immunoglobulin sequences. Also refer to the literature (U.S. Patent Nos. 4,444,887, 4,716,111, and 5,885,793; and International Publication Nos. WO 98 / 46645, WO 98 / 50433, WO 98 / 24893, WO 98 / 16654, WO 96 / 34096, WO 96 / 33735, and WO 91 / 10741, all of which are incorporated herein by reference in their entirety).

[0230] In certain embodiments, human antibodies may be generated using mouse-human hybridomas. For example, human peripheral blood lymphocytes transformed with Epstein-Barr virus (EBV) may be fused with mouse myeloma cells to generate a mouse-human hybridoma that secretes human monoclonal antibodies, and such mouse-human hybridomas may be screened to determine whether they secrete human monoclonal antibodies that specifically bind to a target antigen (e.g., NAPRT (e.g., human NAPRT)). Such methods are known and described in the art, for example, in the literature (Shinmoto H et al.). , (2004) Cytotechnology 46: 19-23; Naganawa Y et al. , (2005) Human Antibodies 14: 27-31, each incorporated herein by reference in its entirety.

[0231] Kit

[0232] Additionally, a kit comprising one or more of the antibodies described herein, or a pharmaceutical composition or conjugate thereof, is provided. In certain embodiments, the present invention provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the components of the pharmaceutical composition described herein, for example, one or more antibodies provided herein. In certain embodiments, the kit contains the pharmaceutical composition described herein and any prophylactic or therapeutic agent, for example, that described herein. In certain embodiments, the kit may contain T cell mitogens such as phytohemagglutinin (PHA) and / or phorbol myristate acetate (PMA), or TCR complex-stimulating antibodies such as anti-CD3 antibodies and anti-CD28 antibodies. Optionally, these container(s) may be affixed with a notice in the form prescribed by a government agency regulating the manufacture, use, or sale of the medicine or biological product, and such notice reflects the agency's approval for manufacture, use, or sale for human administration.

[0233] Additionally, a kit that can be used in the above method is provided. In certain embodiments, the kit comprises the antibody described herein, preferably a purified antibody, in one or more containers. In certain embodiments, the kit described herein contains a substantially isolated NAPRT (e.g., human NAPRT) antigen as a control. In another specific embodiment, the kit described herein further comprises a control antibody that does not react with the NAPRT (e.g., human NAPRT) antigen. In yet another specific embodiment, the kit described herein contains one or more elements for detecting the binding of the antibody to the NAPRT (e.g., human NAPRT) antigen (e.g., the antibody may be conjugated to a detectable substrate such as a fluorescent compound, an enzyme substrate, a radioactive compound, or a luminescent compound, or a second antibody that recognizes the first antibody may be conjugated to a detectable substrate). In certain embodiments, the kit provided herein may comprise a recombinantly generated or chemically synthesized NAPRT (e.g., human NAPRT) antigen. The NAPRT (e.g., human NAPRT) antigen provided in the kit may also be attached to a solid support. In a more specific embodiment, the detection means of the above-described kit comprises a solid support to which an NAPRT (e.g., human NAPRT) antigen is attached. Such a kit may also comprise an unattached reporter-labeled anti-human antibody or anti-mouse / rat antibody. In such an embodiment, the binding of the antibody to the NAPRT (e.g., human NAPRT) antigen may be detected by the binding of the reporter-labeled antibody. In a specific embodiment, the present disclosure relates to the use of the kit of the present disclosure for testing and / or detecting NAPRT (e.g., human NAPRT) antigen in a biological sample in vitro.

[0234] Examples

[0235] The following examples are provided for illustrative purposes only and are not intended to be limiting.

[0236] Example 1: Production and Characterization of NAPRT Antibodies

[0237] A NAPRT-specific monoclonal antibody was produced in mice using human NAPRT1(e3) (sequence number 31) as an immunogen.

[0238] The affinity of NAPRT-specific antibodies to NAPRT1(e3)-1861 (SEQ No. 40) was measured using an enzyme-linked immunosorbent assay (ELISA). As summarized in Table 7 below, several antigens were used in this assay.

[0239]

[0240] The antigen was coated overnight at 4°C at a volume of 100 μg / well. Subsequently, blocking was performed overnight at 4°C using 1% BSA / PBS, 200 μL / well.

[0241] To perform the assay, the primary antibody used was obtained by centrifugation from the supernatant of the hybridoma subclone. 100 μL of the supernatant from a single subclone was added to a single well of the assay, and the assay plate was incubated at 37°C for 1 hour. A positive control containing NAPRT(e3)-1861 anti-serum (1:1000 dilution) and a negative control containing only PBS were used, both at a volume of 100 μL / well.

[0242] After incubation, the secondary antibody, HRP-labeled anti-mouse IgG (SIGMA, Cat#: A0168, Lot NO: # 068M4764V), was added to each well (dilution: 1:9,000, 50 μL / well) and incubated at 37°C for 1 hour. Finally, the substrate solution (TMB (3,3',5,5'-tetramethylbenzidine)) was added at a volume of 100 μL / well and incubated at 37°C for 10 minutes. Subsequently, the optical density (OD) was read at 450 nm. Table 8 below shows the results of the ELISA experiment.

[0243]

[0244] Based on these experiments, clones 2G11A3 and 4A5D7 showed the highest binding affinity for antigens 1, 4, and 5, and thus showed the highest affinity for human NAPRT.

[0245] These two clones exhibited effective binding to NAPRT in Western blot experiments, as shown in Figure 1. In these Western blot experiments, NAPRT was indeed detected in samples from the NAPRT-positive human colorectal carcinoma cell line HCT116, but was not detected in the NAPRT-negative U2OS cell line. The 4A5D7 antibody appeared to exhibit the highest binding affinity for human NAPRT.

[0246] Example 2: Detection of NAPRT in Different Cancer Cells

[0247] The 4A5D7 antibody was used to characterize samples obtained from subjects with different cancers regarding the presence of detectable NAPRT. Table 9 below shows the results of this study.

[0248]

[0249] Example 3: NAMPTi is effective against tumors derived from NAPRT-negative patients

[0250] Three NAMPTis, ATN-249, ATN-940, and ATN-1083, were tested for their respective efficacy in two patient-derived in vitro models of colorectal adenocarcinoma, one of which was NAPRT-positive and the other was NAPRT-negative. The NAPRT status of these models was confirmed by histoimmunochemistry (Fig. 2d). As shown in Figs. 2a to 2c, these three NAMPTis were effective in reducing survival rates in these in vitro models.

[0251] Next, the effects of ATN-249 were tested in vitro on various other NAPRT-negative and NAPRT-positive patient-derived tumors. As can be seen in Figure 3a, ATN-249 had no or limited effect on NAPRT-positive tumor models, but as shown in Figure 3b, it showed a significant effect on NAPRT-negative models.

[0252] These NAPRT-negative and NAPRT-positive models were confirmed by intrahistochemical (IHC) or Western blot (WB). The reactivity of these models to ATN-249 is shown in the table below, and a "response" to NAMPTi is indicated by an IC50 of 125 nM or less. 50 and is defined as the observation of at least a 55% reduction in survival rate ("death").

[0253]

[0254]

[0255] The values ​​in Table 10 showed a positive predictive value (PPV) of 100%, a negative predictive value (NPV) of 48%, and an overall predictive value (OPV) of 65%. However, the values ​​in Table 11 showed a PPV of 92%, an NPV of 87%, and a corresponding OPV of 88%. As a result of the investigation using WB, it was determined that a subset of NAPRT-negative responders identified by IHC alone had low levels of NAPRT expression.

[0256] For both NAPRT-negative and NAPRT-positive models, the effect of ATN-940 in inhibiting the growth of colorectal cancer in vivo was also confirmed. In this experiment, ATN-940 was administered once daily (QD) at a dose of 1 mg (mpk) per kg of body weight on days 1 to 3, days 8 to 10, days 15 to 17, and days 22 to 24, and changes in tumor volume of subjects including xenografts were recorded over a period of 30 days. As shown in Figure 4, ATN-940 inhibited the growth of NAPRT-negative tumors compared to the administration of the vehicle. However, ATN-940 did not prevent the growth of NAPRT-positive tumors.

[0257]

[0258] The scope of the invention is not limited by the specific embodiments described herein. In fact, various variations of the invention in addition to those described will become apparent to those skilled in the art from the foregoing description and the accompanying drawings. Such variations are intended to fall within the scope of the appended claims.

[0259] All references cited herein (e.g., publications or patents or patent applications) are incorporated herein by reference in their entirety for all purposes to the same extent that each individual reference (e.g., publications or patents or patent applications) is specifically and individually indicated so that the whole is incorporated by reference for all purposes.

[0260] Other embodiments are within the scope of the following claims.

[0261] Unofficial ranking list

[0262] VH amino acid sequence of antibody 4A5D7:

[0263]

[0264] VL amino acid sequence of antibody 4A5D7:

[0265]

[0266] CDRH1 amino acid sequence of sequence antibody 4A5D7 (kavat):

[0267]

[0268] CDRH2 amino acid sequence of sequence antibody 4A5D7 (kavat):

[0269]

[0270] CDRH3 amino acid sequence of sequence antibody 4A5D7 (kavat):

[0271]

[0272] CDRH1 amino acid sequence of sequence antibody 4A5D7 (Chotia):

[0273] GYTFTEY (Sequence No. 9)

[0274] CDRH2 amino acid sequence of sequence antibody 4A5D7 (Chotia):

[0275]

[0276] CDRH3 amino acid sequence of sequence antibody 4A5D7:

[0277]

[0278] CDRH1 amino acid sequence (IMGT) of sequence antibody 4A5D7:

[0279]

[0280] CDRH2 amino acid sequence (IMGT) of sequence antibody 4A5D7:

[0281]

[0282] CDRH3 amino acid sequence of sequence antibody 4A5D7 (IMGT):

[0283]

[0284] CDRH1 amino acid sequence of sequence antibody 4A5D7 (AbM):

[0285]

[0286] CDRH2 amino acid sequence of sequence antibody 4A5D7 (AbM):

[0287]

[0288] CDRH3 amino acid sequence of sequence antibody 4A5D7 (AbM):

[0289]

[0290] CDRH1 amino acid sequence of sequence antibody 4A5D7 (McCallum):

[0291]

[0292] CDRH2 amino acid sequence of sequence antibody 4A5D7 (McCallum):

[0293]

[0294] CDRH3 amino acid sequence of sequence antibody 4A5D7 (McCallum):

[0295]

[0296] CDRL1 amino acid sequence of sequence antibody 4A5D7 (kavat):

[0297]

[0298] CDRL2 amino acid sequence of sequence antibody 4A5D7 (kavat):

[0299]

[0300] CDRL3 amino acid sequence of sequence antibody 4A5D7 (kavat):

[0301]

[0302] CDRL1 amino acid sequence of sequence antibody 4A5D7 (Chothia):

[0303]

[0304] CDRL2 amino acid sequence of sequence antibody 4A5D7 (Chothia):

[0305]

[0306] CDRL3 amino acid sequence of sequence antibody 4A5D7 (Chothia):

[0307]

[0308] CDRL1 amino acid sequence of sequence antibody 4A5D7 (IMGT):

[0309]

[0310] CDRL2 amino acid sequence of sequence antibody 4A5D7 (IMGT):

[0311] LM or LMS

[0312] CDRL3 amino acid sequence of sequence antibody 4A5D7 (IMGT):

[0313]

[0314] CDRL1 amino acid sequence of sequence antibody 4A5D7 (AbM):

[0315]

[0316] CDRL2 amino acid sequence of sequence antibody 4A5D7 (AbM):

[0317]

[0318] CDRL3 amino acid sequence of sequence antibody 4A5D7 (AbM):

[0319]

[0320] CDRL1 amino acid sequence of sequence antibody 4A5D7 (McCallum):

[0321]

[0322] CDRL2 amino acid sequence of sequence antibody 4A5D7 (McCallum):

[0323]

[0324] CDRL3 amino acid sequence of sequence antibody 4A5D7 (McCallum):

[0325]

[0326] Nucleic acid of VH of antibody 4A5D7:

[0327]

[0328] Nucleic acid of VL of antibody 4A5D7:

[0329]

[0330] WT IgG1 heavy chain constant region:

[0331]

[0332] S228P IgG4 heavy chain constant region:

[0333]

[0334] Kappa light chain invariant region:

[0335]

[0336] Lambda light chain invariant region:

[0337]

[0338] Antigen 3:

[0339]

[0340] Antigen 4:

[0341]

[0342] Antigen 5:

[0343]

[0344] Amino acid sequence of human NAPRT1(e3):

[0345]

[0346] Nucleic acid of human NAPRT1(e3):

[0347]

[0348] Amino acid sequence of NAPRT encoded by transcript "NAPRT-212" (UniProtKB registration number: H0YF31):

[0349]

[0350] Amino acid sequence of NAPRT encoded by transcript "NAPRT-214" (UniProtKB registration number: H0YDA6):

[0351]

[0352] Amino acid sequence of NAPRT encoded by transcript "NAPRT-205" (UniProtKB registration number: A0A087WUT5):

[0353]

[0354] Standard sequence of human NAPRT (UniProtKB registry number: Q6XQN6-1; NCBI reference sequence: NP_660202.3; 538 AA; 57,578 daltons; NM_145201.6):

[0355]

[0356] Human NAPRT isoform 1 (UniProtKB registration number: Q6XQN6-2; 567 AA; 60,291 daltons):

[0357]

[0358] Isoform 2 of human NAPRT (UniProtKB Registry No.: Q6XQN6-3; NCBI Reference Sequence: NP_001273758.1; 525 AA; 56,099 Daltons):

[0359]

[0360] Computer-mapped isoforms of human NAPRT:

[0361]

[0362] NAPRT1(e3)-1861:

[0363]

Claims

Claim 1 An antibody that specifically binds to human nicotinic acid phosphoribosyltransferase (NAPRT), wherein the antibody comprises: VH having the CDRH1, CDRH2, and CDRH3 amino acid sequences of the VH amino acid sequence presented in SEQ ID NO. 1; and VL having the CDRL1, CDRL2, and CDRL3 amino acid sequences of the VL amino acid sequence presented in SEQ ID NO.

2. Claim 2 The antibody according to claim 1, wherein the antibody comprises the CDRH1, CDRH2, and CDRH3 amino acid sequences presented in SEQ ID NOs 3, 4, and 5; 9, 10, and 5; 11, 12, and 13; 15, 16, and 5; or 17, 18, and 19. Claim 3 In paragraph 2, the antibody comprises the amino acid sequences of CDRL1, CDRL2, and CDRL3 presented in SEQ ID NOs 6, 7, and 8; 14, LM, and 8; 14, LMS, and 8; or 20, 21, and 8. Claim 4 An antibody according to any one of claims 1 to 3, wherein the antibody comprises the amino acid sequences of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 presented in SEQ ID NOs 3, 4, 5, 6, 7, and 8; 9, 10, 5, 6, 7, and 8; 11, 12, 13, 14, LM, and 8; 11, 12, 13, 14, LMS, and 8; 15, 16, 5, 6, 7, and 8; and 17, 18, 19, 20, 21, and 8. Claim 5 An antibody according to any one of claims 1 to 4, wherein the VH comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO. 1, and the VL comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO.

2. Claim 6 An antibody according to any one of claims 1 to 5, wherein the VH comprises the amino acid sequence of SEQ ID NO. 1 and the VL comprises the amino acid sequence of SEQ ID NO.

2. Claim 7 An antibody according to any one of claims 1 to 6, wherein the antibody comprises a heavy chain constant region selected from the group consisting of human IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. Claim 8 An antibody according to any one of claims 1 to 7, wherein the antibody comprises a heavy chain constant region that is a variant of a wild-type heavy chain constant region, and the variant heavy chain constant region binds to FcγR with a lower affinity than that of the wild-type heavy chain constant region binding to FcγR. Claim 9 An antibody that cross-competes with the antibody of any one of claims 1 to 8 for binding to NAPRT. Claim 10 An antibody that specifically binds to the amino acid sequence of SEQ ID NO. 29 or 30. Claim 11 A polypeptide containing VH comprising the CDRH1, CDRH2, and CDRH3 amino acid sequences of the VH amino acid sequence presented in SEQ ID NO.

1. Claim 12 In claim 10, the above VH is a polypeptide comprising the CDRH1, CDRH2, and CDRH3 amino acid sequences presented in SEQ ID NOs 3, 4, and 5; 9, 10, and 5; 11, 12, and 13; 15, 16, and 5; or 17, 18, and 19. Claim 13 A polypeptide comprising VL including the CDRL1, CDRL2, and CDRL3 amino acid sequences of the VL amino acid sequence presented in SEQ ID NO.

2. Claim 14 In claim 12, the above VL is a polypeptide comprising, respectively, SEQ ID NOs 6, 7 and 8; 14, LM and 8; 14, LMS and 8; or CDRL1, CDRL2 and CDRL3 as presented in 20, 21 and 8. Claim 15 A polypeptide comprising the amino acid sequence LM, LMS, or any one of SEQ ID NOs 1 to 21. Claim 16 VH, VL, heavy chain and / or light chain of the antibody of any one of claims 1 to 10; or a polynucleotide encoding the polypeptide of any one of claims 11 to 15. Claim 17 A vector containing the polynucleotide of claim 16. Claim 18 A recombinant host cell comprising: (a) a polynucleotide of claim 16; (b) a vector of claim 17; (c) a first polynucleotide encoding a heavy chain variable region or heavy chain of an antibody of any one of claims 1 to 10 and a second polynucleotide encoding a light chain variable region or light chain of an antibody of any one of claims 1 to 10; and / or (d) a first vector comprising a first polynucleotide encoding a heavy chain variable region or heavy chain of an antibody of any one of claims 1 to 10 and a second vector comprising a second polynucleotide encoding a light chain variable region or light chain of an antibody of any one of claims 1 to 10. Claim 19 A composition comprising an antibody of any one of claims 1 to 10, a polypeptide of any one of claims 11 to 15, a polynucleotide of claim 16, a vector of claim 17, or a host cell of claim 18, and a pharmaceutically acceptable carrier or excipient. Claim 20 A method for generating antibodies, wherein the method comprises the step of culturing the host cells of claim 18 under conditions suitable for expressing polynucleotides and generating antibodies. Claim 21 A method for determining whether a sample containing cells from a subject is nicotinic acid phosphoribosyltransferase (NAPRT) negative, comprising the steps of administering an antibody that specifically binds to NAPRT to the sample, and detecting specific binding of the antibody to NAPRT in less than 1% of the cells in the sample to determine that the sample is NAPRT negative. Claim 22 In paragraph 21, the above sample comprises cancer cells, a method. Claim 23 In paragraph 22, the above cancer cells are solid tumor cells. Claim 24 In paragraph 23, the cancer cells are thyroid cancer, duodenal cancer, neuroendocrine carcinoma (NEC), uterine cancer, small cell lung cancer (SCLC), hepatocellular carcinoma, mesothelioma, breast cancer, sarcoma, ovarian cancer, renal cell carcinoma, rectal cancer, head and neck cancer, prostate cancer, pancreatic cancer, melanoma, colorectal cancer, cervical cancer, non-small cell lung cancer (NSCLC), cholangiocarcinoma, or endometrial cancer cells. Claim 25 A method for treating cancer comprising the step of administering a nicotinamide phosphoribosyltransferase inhibitor (NAMPTi) to a subject requiring treatment for cancer, wherein a sample from the cancer has a lower NAPRT level than a control sample. Claim 26 A method according to claim 25, further comprising the step of administering niacin to the subject. Claim 27 In paragraph 25 or 26, the above cancer is NAPRT negative, method. Claim 28 A method according to any one of claims 25 to 27, wherein the cancer is thyroid cancer, duodenal cancer, neuroendocrine carcinoma (NEC), uterine cancer, small cell lung cancer (SCLC), hepatocellular carcinoma, mesothelioma, breast cancer, sarcoma, ovarian cancer, renal cell carcinoma, rectal cancer, head and neck cancer, prostate cancer, pancreatic cancer, melanoma, colorectal cancer, cervical cancer, non-small cell lung cancer (NSCLC), cholangiocarcinoma, or endometrial cancer. Claim 29 A method for diagnosing a subject having cancer that is more likely to be effectively treated with NAMPTi, comprising the steps of administering an antibody that specifically binds to NAPRT to a sample from said subject, and detecting the specific binding of said antibody to NAPRT, wherein if the binding to NAPRT in the sample from said subject is less than in a control sample, the cancer of said subject is more likely to be effectively treated with NAMPTi. Claim 30 In claim 29, a method in which the cancer of the subject is more likely to be effectively treated with niacin and NAMPTi. Claim 31 Method according to claim 29 or 30, wherein the cancer is thyroid cancer, duodenal cancer, neuroendocrine carcinoma (NEC), uterine cancer, small cell lung cancer (SCLC), hepatocellular carcinoma, mesothelioma, breast cancer, sarcoma, ovarian cancer, renal cell carcinoma, rectal cancer, head and neck cancer, prostate cancer, pancreatic cancer, melanoma, colorectal cancer, cervical cancer, non-small cell lung cancer (NSCLC), cholangiocarcinoma, or endometrial cancer cells. Claim 32 A method according to any one of claims 29 to 31, wherein the sample from the subject is NAPRT negative. Claim 33 A method for diagnosing a patient with sepsis, comprising the steps of administering an antibody that specifically binds to NAPRT to a sample from said subject, and detecting the specific binding of said antibody to NAPRT, wherein if the binding to NAPRT in the sample from said subject is greater than in a control sample, said subject is more likely to have sepsis. Claim 34 A method according to any one of claims 21 to 33, wherein the NAPRT comprises the amino acid sequence of SEQ ID NO. 29 or 30. Claim 35 A method according to any one of claims 21 to 34, wherein the antibody comprises VH having the CDRH1, CDRH2, and CDRH3 amino acid sequences of the VH amino acid sequence presented in SEQ ID NO. 1; and VL having the CDRL1, CDRL2, and CDRL3 amino acid sequences of the VL amino acid sequence presented in SEQ ID NO.

2. Claim 36 The method of claim 35, wherein the antibody comprises the CDRH1, CDRH2, and CDRH3 amino acid sequences presented in SEQ ID NOs 3, 4, and 5; 9, 10, and 5; 11, 12, and 13; 15, 16, and 5; or 17, 18, and 19. Claim 37 A method according to claim 35 or 36, wherein the antibody comprises the amino acid sequences of CDRL1, CDRL2, and CDRL3 presented in SEQ ID NOs 6, 7, and 8; 14, LM, and 8; 14, LMS, and 8; or 20, 21, and 8. Claim 38 A method according to any one of claims 35 to 37, wherein the antibody comprises the amino acid sequences of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 presented in SEQ ID NOs 3, 4, 5, 6, 7, and 8; 9, 10, 5, 6, 7, and 8; 11, 12, 13, 14, LM, and 8; 11, 12, 13, 14, LMS, and 8; 15, 16, 5, 6, 7, and 8; and 17, 18, 19, 20, 21, and 8. Claim 39 A method according to any one of claims 35 to 38, wherein the VH comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO. 1, and the VL comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO.

2. Claim 40 An antibody according to any one of claims 35 to 39, wherein the VH comprises the amino acid sequence of SEQ ID NO. 1 and the VL comprises the amino acid sequence of SEQ ID NO.

2. Claim 41 A method according to any one of claims 35 to 40, wherein the antibody comprises a heavy chain constant region selected from the group consisting of human IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. Claim 42 An antibody according to any one of claims 35 to 41, wherein the antibody comprises a heavy chain constant region that is a variant of a wild-type heavy chain constant region, and the variant heavy chain constant region binds to FcγR with a lower affinity than that of the wild-type heavy chain constant region binding to FcγR. Claim 43 An antibody of any one of claims 1 to 10 for use in medicine; a polypeptide of any one of claims 11 to 15; a polynucleotide of claim 16; a vector of claim 17; a host cell of claim 18; or a composition of claim 19.

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