Anti-human neurotensin receptor 1 antibodies and uses thereof

Abstract

The isolated antibody comprises a heavy chain variable domain (V) that is at least 75% identical to the amino acid sequence of SEQ ID NO: 1. H ) and a light chain variable domain (V) that is at least 75% identical to the amino acid sequence of SEQ ID NO:2. L ) wherein the antibody specifically binds to human neurotensin receptor 1 (hNTSR1).

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 036,740, filed June 9, 2020, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] Monoclonal antibodies (mAbs) have become important tools in the treatment of diseases such as cancer and infectious diseases, as well as in several fields, including molecular biology, pharmaceutical science, and medical research. See Baert et al., N Engl J Med, 2003, 348(7):601-8; Cavalli-Bjorkman et al., Med Oncol, 2002, 19(4):277-80; and Plosker and Figgitt, Drugs, 2003, 63(8):803-43. Over the past decade, monospecific antibodies targeting specific antigens or cell surface receptors in various tumor types have achieved substantial success and are at the forefront of cancer therapy.

[0003] Antibody-drug conjugates (ADCs) are a new class of highly potent biopharmaceuticals consisting of antibodies linked to cytotoxic compounds by chemical linkers. These novel targeted agents demonstrate the unique targeting capabilities of antibodies, enabling highly sensitive differentiation between healthy and cancerous tissues by cytotoxic drugs. ADCs are innovative therapeutic tools that combine the highly specific, specific, and antitumor activity of cancer-specific but insufficiently cytotoxic mAbs with the potent cell-killing activity of cytotoxic small molecule drugs that are too toxic when used alone. At least five ADCs have achieved market approval, and more than 80 ADCs are currently in clinical trials. See Abdollahpour-Alitappeh et al., Antibody-drug conjugates (ADCs) for cancer therapy: Strategies, challenges, and successes. J Cell Physiol, 2018. With advances in cutting-edge technology, ADCs are highly anticipated to dominate the anticancer therapeutic market in the future.

[0004] Neurotensin (NTS), the ligand for neurotensin receptor 1 (NTSR1), is a short peptide found in the nervous system and in peripheral tissues. See Carraway and Leeman, J Biol Chem, 1973, 248(19):6854-61. NTS exhibits a wide range of biological activities and plays an important role in the pathogenesis of Parkinson's disease and schizophrenia, in the regulation of dopamine neurotransmission, hypothermia, antinociception, and in promoting the growth of cancer cells. See Bissette, G. et al., Nature, 1976, 262(5569):607-9; Carraway and Plona, ​​Peptides, 2006, 27(10):2445-60; Griebel and Holsboer, Nat Rev Drug Discov, 2012, 11(6):462-78; Kitabgi, Curr Opin Drug Discov Devel, 2002, 5(5):764-76; and Schimpff et al., J Neurol Neurosurg Psychiatry, 2001, 70(6):784-6. Three neurotensin receptors (NTSRs) have been identified. NTSR1 and NTSR2 belong to the class A GPCR family, while NTSR3 (also called SORT1) is a member of the sortilin family with a single transmembrane domain. See Tanaka et al., Neuron, 1990, 4(6):847-54; Chalon et al., FEBS Lett, 1996, 386(2-3):91-4; and Mazella, Cell Signal, 2001, 13(1):1-6. Most of the known effects of NTS are mediated through NTSR1, which signals preferentially through Gq proteins. See Kitabgi, Curr Opin Drug Discov Devel, 2002, 5(5):764-76.

[0005] Neurotensin and its cognate receptor are neuropeptide-receptor complexes that are frequently deregulated during tumorigenesis. Neurotensin receptor 1 (NTSR1) has been reported to enhance cancer progression in aggressive malignant solid tumors, such as mesothelioma, non-small cell lung cancer, liver cancer, breast cancer, and head and neck squamous cell carcinoma. See Alifano et al., Biochimie, 2010, 92(2):164-70; Alifano et al., Clin Cancer Res, 2010, 16(17):4401-10; Wu, Z. et al., Cancer Lett, 2017, 388:73-84; Dupouy et al., PLoS One, 2009, 4(1):e4223; and Shimizu et al., Int J Cancer, 2008, 123(8):1816-23. Based on staining of patient tissues, NTSR1 has become a promising molecular marker for non-small cell lung cancer and prostate cancer. See He et al., Eur J Nucl Med Mol Imaging, 2019, 46(10):2199-2207, and Alifano et al., Clin Cancer Res, 2010, 16(17):4401-10. Activation of NTSR1 also transactivates the EGFR receptor in colon, prostate, and pancreatic cancer cell lines. See Amorino et al., Oncogene, 2007, 26(5):745-56, and Muller et al., BMC Cancer, 2011, 11:421. Recently, genome-wide association studies have revealed that NTSR1 may play a role in the prognosis of patients with non-small cell lung cancer (NSCLC). See Chang et al., Am J Respir Crit Care Med, 2017, 195(5):663-673. NTS and NTSR1 are aberrantly expressed in over 50% of hepatocellular carcinomas (HCCs). Elevated expression of NTS or NTSR1 mRNA correlates with poor patient outcome. Wu et al., Cancer Lett, 2017, 388:73-84.

[0006] Therefore, NTSR1 is a potential target for cancer therapy. Carcinogenesis and cancer recurrence can be reduced through inhibiting the activity of NTSR1. Summary of the Invention

[0007] In one embodiment, a heavy chain variable domain (V) that is at least 75% identical to the amino acid sequence of SEQ ID NO: 1. H ) and a light chain variable domain (V) that is at least 75% identical to the amino acid sequence of SEQ ID NO:2 L ), wherein the isolated antibody specifically binds to human neurotensin receptor 1 (hNTSR1). For example, the antibody can bind to the second extracellular loop of hNTSR1.

[0008] In one embodiment, the antibody has one or more substitutions within SEQ ID NO: 1 at a position selected from T28, F29, T30, S31, S32A, I51, P53A, N54, S55, G56, N57, T58, Y60, N61, E62, K63, F64, K65, V66A, Y100, D104 and Y105.

[0009] In certain embodiments, the antibody comprises one or more substitutions in SEQ ID NO:2 at a position selected from G96, S97, H98, and P100.

[0010] In one embodiment, the antibody comprises heavy chain CDR1: GYTFTSSWIH (SEQ ID NO: 3) or GYAFTSSWIH (SEQ ID NO: 4), heavy chain CDR2: QIRPNSGNTYYNEKFKV (SEQ ID NO: 5), heavy chain CDR3: ARYYYGFDY (SEQ ID NO: 6), ARYHYGFDY (SEQ ID NO: 7), or ARYRYGFDY (SEQ ID NO: 8), light chain CDR1: RSSQSIVHSNGNTYLE (SEQ ID NO: 9), light chain CDR2: KVSNRFS (SEQ ID NO: 10), and light chain CDR3: FQGSHLPWT (SEQ ID NO: 11) or FQGAHLPWT (SEQ ID NO: 12).

[0011] In one embodiment, the antibody comprises heavy chain CDR1: GYTFTSSWIH (SEQ ID NO: 3), heavy chain CDR2: QIRPNSGNTYYNEKFKV (SEQ ID NO: 5), heavy chain CDR3: ARYHYGFDY (SEQ ID NO: 7) or ARYRYGFDY (SEQ ID NO: 8), light chain CDR1: RSSQSIVHSNGNTYLE (SEQ ID NO: 9), light chain CDR2: KVSNRFS (SEQ ID NO: 10), and light chain CDR3: FQGSHLPWT (SEQ ID NO: 11).

[0012] In one embodiment, the antibody comprises heavy chain CDR1: GYTFTSSWIH (SEQ ID NO: 3), heavy chain CDR2: QIRPNSGNTYYNEKFKV (SEQ ID NO: 5), heavy chain CDR3: ARYHYGFDY (SEQ ID NO: 7) or ARYRYGFDY (SEQ ID NO: 8), light chain CDR1: RSSQSIVHSNGNTYLE (SEQ ID NO: 9), light chain CDR2: KVSNRFS (SEQ ID NO: 10), and light chain CDR3: FQGAHLPWT (SEQ ID NO: 12).

[0013] In one embodiment, the antibody comprises heavy chain CDR1: GYAFTSSWIH (SEQ ID NO: 4), heavy chain CDR2: QIRPNSGNTYYNEKFKV (SEQ ID NO: 5), heavy chain CDR3: ARYHYGFDY (SEQ ID NO: 7) or ARYRYGFDY (SEQ ID NO: 8), light chain CDR1: RSSQSIVHSNGNTYLE (SEQ ID NO: 9), light chain CDR2: KVSNRFS (SEQ ID NO: 10), and light chain CDR3: FQGAHLPWT (SEQ ID NO: 12).

[0014] In one embodiment, the antibody comprises heavy chain CDR1: GYTFTSSWIH (SEQ ID NO: 3), heavy chain CDR2: QIRPNSGNTYYNEKFKV (SEQ ID NO: 5), heavy chain CDR3: ARYYYGFDY (SEQ ID NO: 6), light chain CDR1: RSSQSIVHSNGNTYLE (SEQ ID NO: 9), light chain CDR2: KVSNRFS (SEQ ID NO: 10), and light chain CDR3: FQGSHLPWT (SEQ ID NO: 11).

[0015] In one embodiment, the isolated antibody comprises heavy chain CDR1: GYAFTSSWIH (SEQ ID NO: 4), heavy chain CDR2: QIRPNSGNTYYNEKFKV (SEQ ID NO: 5), heavy chain CDR3: ARYHYGFDY (SEQ ID NO: 7), light chain CDR1: RSSQSIVHSNGNTYLE (SEQ ID NO: 9), light chain CDR2: KVSNRFS (SEQ ID NO: 10), and light chain CDR3: FQGAHLPWT (SEQ ID NO: 12).

[0016] In one embodiment, the antibody comprises a V H and V, which is the sequence of SEQ ID NO: 14 L Contains arrays.

[0017] In one embodiment, the antibody comprises a V H and V, which is the sequence of SEQ ID NO: 16 L Contains arrays.

[0018] In certain embodiments, the antibody is a recombinant antibody, a monoclonal antibody, a chimeric antibody, a humanized antibody, an IgG1 antibody, or an antibody fragment comprising an antigen-binding site.

[0019] In another aspect, provided herein is an antibody conjugate comprising any of the anti-hNTSR1 antibodies described herein and a non-antibody molecule.

[0020] In certain embodiments, the non-antibody molecule is a polypeptide, polymer, oligosaccharide, lipid, glycolipid, solid support, small molecule drug, biotin, a nucleic acid molecule, a carrier protein, or a detectable label.

[0021] In some embodiments, the antibody conjugate is an antibody-drug conjugate and the non-antibody molecule is an anti-cancer drug that inhibits cancer cells or treats tumors that express hNTSR1. For example, the anti-cancer drug can be monomethyl auristatin E (MMAE).

[0022] In some embodiments, the tumor is mesothelioma, lung tumor, breast tumor, head and neck squamous cell carcinoma, colon tumor, pancreatic tumor, prostate tumor, or liver tumor.

[0023] In yet another aspect, described herein are pharmaceutical compositions comprising any of the antibodies or antibody-drug conjugates described herein and a pharmaceutical carrier.

[0024] In one aspect, described herein is a method of treating a tumor in a subject in need thereof, comprising administering to the subject any of the antibody-drug conjugates or pharmaceutical compositions described herein. In certain embodiments, the tumor expresses hNTSR1.

[0025] In some embodiments, the tumor is mesothelioma, lung tumor, breast tumor, head and neck squamous cell carcinoma, colon tumor, pancreatic tumor, prostate tumor, or liver tumor.

[0026] In another aspect, described herein are methods for detecting NTSR1 in a sample or in tissues or cells that express NTSR1, the methods comprising contacting the sample, tissue, or cells with any of the antibodies or antibody conjugates described herein, and determining binding of the antibody or antibody conjugate to a target in the sample or to the tissue or cells.

[0027] In yet another aspect, described herein is an isolated nucleic acid molecule encoding any of the antibodies or components thereof described herein.

[0028] The details of one or more embodiments are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the embodiments will be apparent from the description and drawings, and from the claims. [Brief explanation of the drawings]

[0029] [Figure 1] Figure 1 is a set of graphs showing the characterization of 7C3 mAb. (A) Binding of 7C3 mAb to the cyclic ECL2 peptide of NTSR1 in ELISA. (B) Binding of 7C3 (10 μg / ml) to NTSR1 in A549 cells measured by flow cytometry. [Figure 2]Figure 2 is a graph showing the affinity and kinetic data of h7C3-3. The binding affinity between h7C3-3 and the NTSR1 ECL2 peptide was determined by surface plasmon resonance (SPR). [Figure 3] Figure 3 is a set of graphs showing the internalization of anti-NTSR1 mAbs in various cancer cell lines overexpressing NTSR1. Flow cytometry analysis of (A) A549, (B) H1299, (C) HA22T, and (D) Mahlavu cells treated with various antibodies (10 μg / ml) at 4°C or 37°C, respectively. [Figure 4] Figure 4 includes tables and graphs showing the characterization of h7C3-4 mutant antibodies. (A) Isoelectric point (pI) and binding affinity of h7C3-3 or h7C3-4. Binding of h7C3-3 or h7C3-4 to NTSR1-expressing A549 cells was measured by flow cytometry at a concentration of 0.5 μg / ml. (B) Pharmacokinetics of the antibody in BLTW:CD1(ICR) mice. Serum concentration of the antibody versus time profile after a single intravenous administration (10 mg / kg). The concentration of h7C3-3 or h7C3-4 antibody in mouse serum was analyzed by ELISA. [Figure 5] Figure 5 shows the characterization of humanized h7C3-4 and h7C3-5 antibodies. Alignment of (A) the heavy chain amino acid sequence and (B) the light chain amino acid sequence of h7C3-4 and h7C3-5. Mutated residues are shown in bold, and CDR sequences are defined according to Kabat's definition. (C) Binding of h7C3-4 and h7C3-5 to NTSR1 in A549 cells measured by flow cytometry. h7C3-4 heavy chain (SEQ ID NO: 13), h7C3-4 light chain (SEQ ID NO: 14), h7C3-5 heavy chain (SEQ ID NO: 15), and h7C3-5 light chain (SEQ ID NO: 16). [Figure 6]6 is a set of graphs showing hydrophobic interaction chromatography (HIC) analysis of (A) h7C3-3-MMAE conjugate and (B) h7C3-4-MMAE conjugate. "DAR:0," "DAR:2," "DAR:4," "DAR:6," and "DAR:8" refer to isomers of the conjugate with 0, 2, 4, 6, and 8 MMAE molecules attached per antibody, respectively. [Figure 7] Figure 7 is a set of graphs showing the internalization efficiency of h7C3-3 and h7C3-3-MMAE in A549 cells. (A) The binding affinity of h7C3-3 and h7C3-3-MMAE in A549 cancer cells was measured by flow cytometry after 1 hour of incubation at 4°C. The cellular distribution is shown on the right. (B and C) Flow cytometry analysis was applied to evaluate the internalization efficiency of h7C3-3 and h7C3-3-MMAE at a concentration of 1 μg / ml in NTSR1-expressing A549 cancer cells. Cells were incubated with antibody on ice for 1 hour. Unbound antibody was washed away. The experimental group was then incubated at 37°C for 90 minutes. After harvesting, the cells were stained with FITC-conjugated anti-human IgG and analyzed by flow cytometry. The cellular distribution of internalization is shown on the left. [Figure 8] FIG. 8 is a set of graphs showing the in vitro cytotoxic effects of h7C3-3-MMAE in various cancer cell lines. [Figure 9] FIG. 9 contains graphs and tables showing the in vitro cytotoxic effects of h7C3-4-MMAE in various cancer cell lines. [Figure 10] FIG. 10 contains graphs and tables showing the in vitro cytotoxic effects of h7C3-5-MMAE in various cancer cell lines. [Figure 11] Figure 11 is a set of graphs showing the efficacy of h7C3-3-MMAE, h7C3-4, and h7C3-4-MMAE at a dose of 10 mg / kg compared to cisplatin at a dose of 7 mg / kg in an H1299 lung tumor xenograft model. (A) The antitumor efficacy of h7C3-3-MMAE and h7C3-4-MMAE is shown by the change in tumor volume. (B) Neither group showed weight loss. [Figure 12] Figure 12 is a set of graphs showing the efficacy of anti-NTSR1 h7C3-3-MMAE and h7C3-4-MMAE at a dose of 10 mg / kg in the HA22T-Luc liver tumor xenograft model. (A) The anti-tumor efficacy of h7C3-3-MMAE and h7C3-4-MMAE is shown by the change in tumor volume. (B) Neither group showed weight loss. [Figure 13] Figure 13 is a set of graphs showing the efficacy of anti-NTSR1 h7C3-4-MMAE at a dose of 10 mg / kg in a PC3 prostate tumor xenograft model. (A) The antitumor efficacy of h7C3-4-MMAE is shown by the change in tumor volume. (B) Treatment did not result in weight loss. [Figure 14] Figure 14 is a set of images showing immunohistochemical staining of xenografted PC-3 prostate cancer tissues with 2 μg / ml of B-12, 7C3, h7C3-2, or h7C3-3 antibodies. Solid tumors were excised from untreated PC-3 xenografted mice and subsequently subjected to immunohistochemical staining using an antibody specific for NTSR1. Images were taken at 50x and 100x magnification. DETAILED DESCRIPTION OF THE INVENTION

[0030] Described herein are novel antibodies and conjugates thereof that bind to human NTSR1.

[0031] Each of the anti-NTSR1 antibodies comprises a heavy chain variable domain (V) that is at least 75% (e.g., at least 78%, at least 79%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%) identical to the amino acid sequence of SEQ ID NO:1. H ), and a light chain variable domain (V) that is at least 75% (e.g., at least 78%, at least 79%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%) identical to the amino acid sequence of SEQ ID NO:2. L ). SEQ ID NO: 1 QVQLQQPGSVLVRPGASVKLSCKASGYTFTSSWIHWAKQRPGQGLEWIGQIRPNSGNTYYNEKFKVKATLTVDTSSSTAYVDLSSLTSEDSAVYYCARYYYGFDYWGQGTLVTVSS SEQ ID NO: 2 DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHLPWTFGGGTKLEIKR

[0032] The antibodies may each have one or more substitutions within SEQ ID NO: 1 at a position selected from T28, F29, T30, S31, S32A, I51, P53A, N54, S55, G56, N57, T58, Y60, N61, E62, K63, F64, K65, V66A, Y100, D104, and Y105. Additionally or alternatively, the antibodies may have one or more substitutions within SEQ ID NO: 2 at a position selected from G96, S97, H98, and P100. The amino acid substitutions may be any amino acid (e.g., Ala), as long as the substitution does not significantly reduce the binding affinity of the antibody to NTSR1 (e.g., by no more than 25%, 20%, 15%, 10%, or 5%) compared to an antibody having the sequence of SEQ ID NO: 1 or 2. The binding affinity of an antibody to NTSR1 can be determined using various methods known in the art or described herein, such as binding to NTSR1 in cells or to ECL2 peptides.

[0033] In one embodiment, the anti-NTSR1 antibody has a heavy chain CDR1 having the sequence of GYTFTSSWIH (SEQ ID NO: 3) or GYAFTSSWIH (SEQ ID NO: 4), a heavy chain CDR2 having the sequence of QIRPNSGNTYYNEKFKV (SEQ ID NO: 5), a heavy chain CDR3 having the sequence of ARYYYGFDY (SEQ ID NO: 6), ARYHYGFDY (SEQ ID NO: 7), or ARYRYGFDY (SEQ ID NO: 8), a light chain CDR1 having the sequence of RSSQSIVHSNGNTYLE (SEQ ID NO: 9), a light chain CDR2 having the sequence of KVSNRFS (SEQ ID NO: 10), and a light chain CDR3 having the sequence of FQGSHLPWT (SEQ ID NO: 11) or FQGAHLPWT (SEQ ID NO: 12).

[0034] In some embodiments, the antibody (e.g., h7C3-1) has GYTFTSSWIH (SEQ ID NO: 3), QIRPNSGNTYYNEKFKV (SEQ ID NO: 5), ARYHYGFDY (SEQ ID NO: 7) or ARYRYGFDY (SEQ ID NO: 8), RSSQSIVHSNGNTYLE (SEQ ID NO: 9), KVSNRFS (SEQ ID NO: 10), and light chain CDR3: FQGSHLPWT (SEQ ID NO: 11).

[0035] In some embodiments, the antibody (e.g., h7C3-2) has the sequences GYTFTSSWIH (SEQ ID NO: 3), QIRPNSGNTYYNEKFKV (SEQ ID NO: 5), ARYHYGFDY (SEQ ID NO: 7) or ARYRYGFDY (SEQ ID NO: 8), RSSQSIVHSNGNTYLE (SEQ ID NO: 9), KVSNRFS (SEQ ID NO: 10), and FQGAHLPWT (SEQ ID NO: 12).

[0036] In some embodiments, the antibody (e.g., h7C3-3, h7C3-4, or h7C3-5) comprises GYAFTSSWIH (SEQ ID NO: 4), QIRPNSGNTYYNEKFKV (SEQ ID NO: 5), ARYHYGFDY (SEQ ID NO: 7) or ARYRYGFDY (SEQ ID NO: 8), RSSQSIVHSNGNTYLE (SEQ ID NO: 9), KVSNRFS (SEQ ID NO: 10), and FQGAHLPWT (SEQ ID NO: 12).

[0037] In some embodiments, the antibody (e.g., h7C3) has the sequences GYTFTSSWIH (SEQ ID NO: 3), QIRPNSGNTYYNEKFKV (SEQ ID NO: 5), ARYYYGFDY (SEQ ID NO: 6), RSSQSIVHSNGNTYLE (SEQ ID NO: 9), KVSNRFS (SEQ ID NO: 10), and FQGSHLPWT (SEQ ID NO: 11).

[0038] In one embodiment, the antibody comprises a V having the sequence of SEQ ID NO: 13 or 15. H and V having the sequence of SEQ ID NO: 14 or 16 L Includes: SEQ ID NO: 13 QVQLQQPGTVLVRPGASVKLSCKASGYAFTSSWIHWAKQRPGQGLEWIGQIRPNSGNTYYNEKFKVKATLTVDTSSSTAYVELSSLTSEDSAVYYCARYHYGFDYWGQGTLVTVSS SEQ ID NO: 14 DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLTISRVEAEDLGVYYCFQGAHLPWTFGGGTKLEIKR SEQ ID NO: 15 QVQLVQSGAEVKKPGASVKVSCKASGYAFTSSWIHWVRQAPGQRLEWMGQIRPNSGNTYYNEKFKVRVTITRDTSASTAYMELSSLRSEDTAVYYCARYHYGFDYWGQGTLVTVSS SEQ ID NO: 16 DIVMTQTPLSLSVTPGQPASISCRSSQSIVHSNGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGAHLPWTFGGGTKVEIKR

[0039] As used herein, the term "antibody" includes various antibody structures having antigen-binding activity, including, but not limited to, monoclonal antibodies, polyclonal antibodies, full-length antibodies or fragments thereof, antibodies containing an Fc region, Fab fragments, Fab' fragments, F(ab')2 fragments, single-chain antibodies, scFV multimers, univalent antibodies, multivalent antibodies, humanized antibodies, and chimeric antibodies.

[0040] Based on the antibody CDR sequences disclosed herein, a skilled practitioner can generate anti-NTSR1 antibodies in a variety of forms using methods well known in the art, such as recombinant methods.

[0041] Also contemplated herein is an isolated nucleic acid molecule (e.g., an expression vector) or component thereof encoding one of the anti-NTSR1 antibodies described herein. Host cells containing the nucleic acid are also provided herein. The nucleic acid molecules and host cells can be used to produce anti-NTSR1 antibodies.

[0042] Any of the anti-NTSR1 antibodies described herein can be conjugated to a non-antibody molecule to form an antibody conjugate using methods well known in the art. The non-antibody molecule can be, for example, a polypeptide, polymer, oligosaccharide, lipid, glycolipid, solid support (e.g., beads or plates), small molecule drug (e.g., cytotoxic drug), biotin, nucleic acid molecule, carrier protein, or detectable label (e.g., fluorescent label). The non-antibody molecule can be linked to the antibody via a cleavable linker (e.g., valine-citrulline) or a non-cleavable linker (e.g., N-maleimidomethylcyclohexane-1-carboxylate (MCC) or maleimidocaproyl, mercaptoacetamidocaproyl). Such antibody conjugates can be used for various purposes, such as treating cancer or detecting NTSR1 in a sample.

[0043] In some embodiments, the antibody conjugate is an antibody-drug conjugate in which the drug is for inhibiting cancer cells or treating tumors, e.g., for cancer cells or tumors that express NTSR1. In some embodiments, the drug is monomethyl auristatin E (MMAE).

[0044] Any of the antibodies or antibody conjugates described herein can be used to inhibit binding between NTSR1 and its ligand, inhibit the function of NTSR1, detect NTSR1 protein or fragments thereof in a sample (e.g., in an immunoassay), bind to tissues or cells that express NTSR1 (e.g., to identify cells or isolate NTSR1-expressing cells), inhibit the growth of cancer cells or tumors, or treat cancer in a subject.

[0045] The term "sample" can be any biological sample, for example a body fluid sample, a blood sample, a cell sample, a urine sample, a saliva sample or a tissue sample.

[0046] Tumors that express NTSR1 may be potential targets for anti-NTSR1 antibodies or conjugates thereof. Such tumors include, but are not limited to, mesothelioma, lung tumors, breast tumors, head and neck squamous cell carcinoma, colon tumors, pancreatic tumors, prostate tumors, or liver cancer. Optionally, before administering an anti-NTSR1 antibody or conjugate to a subject, the tumor in the subject may be identified as expressing NTSR1. The treatment method may be performed alone or in combination with other drugs or therapies.

[0047] "Subject" refers to a human or non-human animal. "Treating" or "treatment" refers to the administration of a compound or composition to a subject having a disorder with the intent to cure, alleviate, relieve, repair, delay the onset of, or ameliorate the disorder, the symptoms of the disorder, a disease state secondary to the disorder, or the predisposition to the disorder. "Effective amount" refers to the amount of a compound or composition that can produce a medically desirable result in the treated subject.

[0048] Any of the anti-NTSR1 antibodies and antibody conjugates described herein can be formulated as pharmaceutical compositions suitable for various routes of administration, for example, intravenous, intraarticular, conjunctival, intracranial, intraperitoneal, intrapleural, intramuscular, intrathecal, or subcutaneous. The pharmaceutical composition can be an aqueous solution or a lyophilized formulation. It can include a pharmaceutically acceptable carrier, for example, a buffer, excipient, stabilizer, or preservative. The pharmaceutical composition can include other active ingredients that act in conjunction with the antibody or antibody conjugate, for example, other therapeutic agents or adjuvants.

[0049] The following specific examples are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way. Without further elaboration, it is believed that one skilled in the art can, based on the description herein, utilize the present disclosure to its fullest extent. All publications cited herein are incorporated by reference in their entirety. [Example]

[0050] Example 1 Materials and Methods cell line A549 (NSCLC) cells were maintained in F-12K medium (Gibco, Grand Island, NY, USA) with 10% fetal bovine serum (Gibco, Grand Island, NY, USA). H1299 (NSCLC) cells were maintained in RPMI medium supplemented with 10% FBS and 2 mM L-glutamine. HA22T (HCC) cells were maintained in DMEM medium supplemented with 10% FBS, 0.1 mM non-essential amino acids, and 2 mM L-glutamine (Gibco). Mahlavu cells were maintained in DMEM medium supplemented with 10% FBS. PC-3 (PCa) cells were maintained in RPMI 1640 medium (Gibco, SH30027) supplemented with 10 mM HEPES, 1 mM sodium pyruvate (Gibco), 4.5 g / L glucose, and 10% FBS.

[0051] Mouse immunization and RNA purification Five female BALB / c mice (4–6 weeks old) were given intraperitoneal injections of 0.1 mg of ovalbumin-conjugated ECL2 peptide every two weeks for 12 weeks. Blood samples were collected one week after each immunization and titrated by indirect enzyme-linked immunosorbent assay (ELISA). After a total of six booster immunizations, mice were sacrificed and their spleens were removed. Cells were lysed, and total mRNA was generated from the mice using the RNeasy protect midi kit (Qiagen, Germany) according to the manufacturer's instructions.

[0052] RT-PCR amplification of heavy chain and kappa chain purification Total RNA from harvested mouse spleens was extracted using Trizol Reagent according to the manufacturer's protocol (Invitrogen, USA). Purity and concentration were determined by measuring the absorbance (A) at 260 nm and 280 nm (A260 / A280). First-strand cDNA was generated from 350 ng of mRNA (Oligotex mRNA Mini kit, QIAGEN) using reverse transcriptase (Roche) with 1 μl of RNaseOut (40 U / μl, Invitrogen) and a mixture of MuJH (or MuJK)-FOR primers.

[0053] Construction of phage display scFv library Briefly, the library construction process consists of (i) V amplification using framework region family-specific primers; H and V L (ii) reamplification of each segment with primers containing a linker segment; and (iii) amplification of both segments (V H and V L ) and assembly of the nucleotide sequence. All PCR reactions were performed using TaKaRa Ex Taq polymerase (RR001A) and primers specific for mouse heavy chain and kappa light chain. See Benhar and Reiter, Curr Protoc Immunol, 2002, Chapter 10: p. Unit 10, 19B.

[0054] The PCR product containing the scFv was digested with excess restriction enzymes EcoRI and NcoI (NEB), and approximately 10.0 μg of the product was ligated with EcoRI / NcoI-linearized pHEN2 vector (purified by agarose gel extraction) in a total volume of 100 μl using 2400 units of T4 DNA ligase (NEB) overnight at 16°C. Following ligation, the recombinant DNA was precipitated, washed, and dissolved in 20 μl of distilled water. Each 1 μl of recombinant DNA was transformed into 25 μl of E. coli TG1 (Lucigen) by electroporation. After transformation, 20 ml of Recover medium (Lucigen) was added, and the culture was shaken at 37°C for 1 hour, followed by incubation in 200 ml of 2YT containing 100 μg / ml ampicillin on a shaker at 37°C for an additional 16–18 hours. At this point, an aliquot of the culture was plated onto 2YT agar / ampicillin to titer the library size calculated by counting the number of ampicillin-resistant colonies. Phagemids containing scFvs were prepared from this overnight culture. M13K07 (approximately 10 12 pfu) of helper phage were added to the TG1 sample containing the scFv gene library and incubated at 37°C for 2-3 hours with shaking. 50 μg / ml kanamycin was added, and the culture was shaken overnight at 30°C. The cells were centrifuged at 4000 rpm for 20 minutes at 4°C. The supernatant was mixed with 50 ml of 20% PEG8000 / 2.5 M NaCl, incubated on ice for 60 minutes, and then centrifuged at 8000 rpm for 20 minutes at 4°C to precipitate the phage. The supernatant was discarded, and the pellet was drained. The phage were resuspended in 1 ml of PBS, vortexed, and centrifuged at 13000 rpm for 10 minutes to pellet debris. The supernatant was stored at 4°C or used directly in the next biopanning step.

[0055] Phage affinity selection (panning) Streptavidin-coated magnetic beads were prewashed with 10 volumes of TBS containing 2% (wt / vol) BSA and 0.02% (wt / vol) NaN3, and the washed beads were suspended in TBS containing 2% (wt / vol) BSA, 5 mM DTT, and 0.02% (wt / vol) NaN3. Nonspecific binding of the scFv phage library was depleted with streptavidin beads in a final volume of 500 μl in a 1.5 ml microcentrifuge tube (2 × 10 in 500 μl of 4% (wt / vol) BSA, 1% (vol / vol) Tween-20, and 10 mM DTT in TBS). 12 The phage particles were mixed), and 60 μl of prewashed streptavidin beads were added and incubated overnight at 4° C. Next, 0.4 μg of biotin-human NTSR1 linear ECL2 peptide in 3 ml of 1× TBS, 2% (wt / vol) BSA, 0.5% (vol / vol) Tween-20, and 5 mM DTT was added to 2 ml of preadsorbed phage particles (2 × 10 12 The mixture was incubated in an immunotube at room temperature (RT) for 4 hours. After two panning cycles, individual colonies of phagemid-bearing cells were used to prepare monoclonal phages in sterile 96-well plates (Nunc, Sweden) that were screened by ELISA.

[0056] Phage ELISA A 96-well NeutrAvidin-coated plate (Pierce) was filled with 100 ml of 5 mg / ml biotin-human NTSR1 linear ECL2 peptide in wash buffer (25 mM Tris, 150 mM NaCl, 0.1% BSA, 0.05% Tween 20 pH 7.2). The next day, the plate was washed with wash buffer and blocked with 5% nonfat milk in wash buffer for 1 hour at room temperature. After a further wash with wash buffer, 100 μl of freshly prepared phage was added per well and incubated for 1 hour at room temperature. The plate was washed again with wash buffer, and a 1:5000 diluted anti-M13 antibody (ab24229) was placed in each well for 1 hour at room temperature, followed by another wash with wash buffer. A 1:5000 dilution of goat anti-mouse IgG-HRP (Jackson, 115-035-003) was placed in each well for 1 hour at room temperature, followed by further washing with wash buffer. Finally, an additional 100 μl of 3,3',4,4'-tetramethylbenzidine (TMB) (HRP substrate) solution (Pierce) was added to each well and incubated at room temperature. The reaction was stopped by adding 100 μl of 1 M H2SO4 to each well. The plate was analyzed at 450 nm using a plate reader (DiscoverX reader).

[0057] Antibody construction and expression The heavy chain of 7C3 (V H ) and light chain (V L The variable domain sequences of human IgG1 heavy chain and human IgG(κ) light chain were fused to the constant domain sequences of human IgG1 heavy chain and human IgG(κ) light chain encoded by the pFUSE-CHIg-hG1 and pFUSE2-CLIg-hK vectors (InvivoGen), respectively. Expression of the human-mouse chimeric antibody was achieved by transfecting the vectors into Expi293 cells. The secreted antibody was purified from the culture supernatant by Protein A affinity chromatography and tested to determine whether it retained its antigen-binding ability after chimerization.

[0058] Affinity maturation via site-directed mutagenesis All mutations were generated using overlap extension PCR mutagenesis in the heavy chain (V H ) or light chain (V L ) into the variable domain of V H and V L The mutated coding regions of the mutated genes were amplified by PCR using specific primers. The PCR products were annealed via their common overlap, amplified in a second PCR reaction, purified, and ligated into the pFUSE-CHIg-hG1 or pFUSE2-CLIg-hK vector, respectively. After transformation of E. coli JM109 (ECOS™ 9-5), individual colonies were screened for each individual mutant by digestion with the appropriate restriction enzyme. Putative mutants identified by this analytical restriction enzyme digestion were confirmed by sequencing.

[0059] NTSR1-binding affinity assay The NTSR1 binding ability of different antibodies was measured by ELISA. Briefly, NeutrAvidin 96-well plates (Pierce, Cat: 15128) were coated with cyL2-biotin peptide at a final concentration of 5 μg / ml for 1 hour at room temperature. The next day, the coating solution was removed, and the plates were blocked with 5% (v / v) nonfat dry milk in phosphate-buffered saline (PBS) for 1 hour. After washing with PBST, graded concentrations of anti-NTSR1 antibodies were added in triplicate. After 1 hour of incubation, the plates were washed three times with PBST, followed by the addition of horseradish peroxidase (HRP)-conjugated goat anti-human IgG Fc antibody (100 μL) to each well for another 1 hour at room temperature. Finally, 100 μL of tetramethylbenzidine (TMB) substrate was added to each well to generate color for visualization. After 2 minutes of incubation, the reaction was stopped with 100 μL of 2 M H2SO4, and the absorbance of each well was read at 450 nm using a plate reader.

[0060] Flow cytometry Total 1 x 10 6Cells were incubated with anti-NTSR1 antibody in 100 μL of FACS buffer (PBS containing 0.5% BSA) at 4°C for 60 minutes and washed twice to remove excess antibody. FITC (BD Pharmingen™)-conjugated mouse anti-human IgG secondary antibody was incubated on ice for an additional 60 minutes. Cells were washed again and subsequently incubated with 1 ml of PBS containing 0.5 mg / mL propidium iodide for an additional 10 minutes at 4°C. Data were collected from 10,000 live cells using a FACS (Bio-Red S3e cell sorter). Fluorescence intensity in channel FL-1 was calculated using Flow Jo flow cytometry analysis software.

[0061] Surface plasmon resonance The interaction of NTSR1-specific mAb with the extracellular loop 2 (ECL2) peptide of NTSR1 was measured by surface plasmon resonance using an Open SPR (Nicoya, Canada) instrument. Biotin-conjugated ECL2 peptide was immobilized on a streptavidin-coated sensor chip by injecting the peptide diluted to a concentration of 10 μg / ml in HBS-EP5 buffered saline (20 mM HEPES, 250 mM NaCl, 3 mM EDTA, 0.05% Tween 20, and 0.5% BSA, pH 7.4) at a flow rate of 30 μl / min. For kinetic experiments, NTSR1-specific mAb diluted to concentrations ranging from 24 to 1.5 nM in HBS-EP5 was injected over the immobilized ECL2 peptide at a flow rate of 30 μl / min. Binding of NTSR1-specific mAb to immobilized ECL2 peptide at various concentrations was analyzed using TraceDrawer software (Nicoya, Canada). Association rates (k), dissociation rates (k), and affinity (K) were calculated using global analysis, fitting the data to a simple 1:1 binding model.

[0062] Internalization of NTSR1 antibody in various NTSR1-expressing cell lines (flow cytometry) NTSR1 antibody was used in flow cytometry analysis to compare NTSR1 expression levels in various lung and HCC cell lines, including A549, H1299, HA22T, and Mahlavu. A total of 1 × 10 6 Cells were incubated with h7C3-3 antibody in FACS buffer at 4°C for 60 minutes and washed twice to remove excess antibody. Cells were then transferred to 4°C or 37°C for 0, 0.5, 2, 5, or 24 hours, followed by further incubation with anti-human IgG Fc-PE (BioLegend) secondary antibody at 4°C for 60 minutes. Cells were washed again, and data were collected using a BD LSRFortessa. Fluorescence intensity in channel FL-1 was calculated using Flow Jo flow cytometry analysis software.

[0063] Internalization of h7C3-3 or h7C3-3-ADC antibodies in A549 cells (flow cytometry) Total 1 x 10 6 Cells were reacted with the indicated antibodies or ADCs in 100 μL of FACS buffer at 4°C for 60 minutes and washed to remove excess antibody or ADC. The cells were then transferred to 4°C or 37°C for 90 minutes. To determine the relative number of h7C3-3 or h7C3-3-ADCs on the cell surface, the cells were stained with a saturating amount of FITC (BD Pharmingen™)-conjugated mouse anti-human IgG antibody and incubated on ice for an additional 60 minutes. The cells were washed again, followed by an additional 10-minute incubation with 1 ml of PBS containing 0.5 mg / mL propidium iodide at 4°C. Data were collected on 10,000 live cells by FACS (Bio-Red S3e cell sorter). Fluorescence intensity in channel FL-1 was calculated using Flow Jo flow cytometry analysis software.

[0064] Preparation of anti-NTSR1 antibody-drug conjugates (ADCs) h7C3-3 or h7C3-4 was treated with 0.1 molar equivalent of TCEP-HCl (tris(2-carboxyethyl)phosphine hydrochloride, Sigma-Aldrich) in 50 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer, Sigma-Aldrich) pH 6.9 and 1 mM ethylenediaminetetraacetic acid (EDTA, Sigma-Aldrich) for 2 hours at 37°C. The reduced antibody was reacted with vcMMAE (Achemblock, Q70231) for 60 minutes at room temperature. Unreacted vcMMAE was quenched with 1 mM N-acetyl-L-cysteine ​​(Sigma-Aldrich) and incubated for 30 minutes at room temperature. The reaction mixture was then buffer-exchanged into PBS (pH 6.9) using an Amicon Ultrafree centrifugal filter unit (Millipore).

[0065] Hydrophobic Interaction Chromatography (HIC) Analysis h7C3-3 and h7C3-3 ADC were characterized using the following: 1200 HPLC (Agilent Technologies), TSKgel Butyl-NPR column (4.6 × 35 mm, particle size 2.5 μm, TOSOH), 1.5 mol / L -1 Solvent A was 75% (V / V) ammonium sulfate and 25 mM phosphate (pH = 6.95), solvent B was 75% (V / V) 25 mM phosphate and 25% (V / V) isopropanol (pH = 6.95), the gradient was 100% A to 100% B over 15 min, and the flow rate was 0.5 mL min -1 The analysis was carried out using HIC with the following conditions: column temperature 25°C, UV detection wavelength 280 nm.

[0066] In vitro cytotoxicity assay Viable cell numbers were determined using the CellTiter-Glo® Luminescent Cell Viability Assay (Promega, G7571). Cells were seeded into 96-well opaque Costar plates (catalog no. 136101). Briefly, 500–2500 cells in 90 μL of medium were added to each well in quadruplicate and allowed to adhere for 16 hours. Anti-NTSR1 (dose range: 0–500 nM), anti-NTSR1-MMAE (dose range: 0–500 nM), or paclitaxel (dose range: 0–100 nM) was added in 10 μL. After 72–120 hours, the medium was removed, and 200 μL of CellTiter-Glo® Viability Reagent-containing culture medium (1:1) was added to each well and placed on an orbital shaker in the dark for 2 minutes. Plates were allowed to rest at room temperature for 10 minutes before being read on a GloMax® Explorer Multimode Microplate Reader (GM3500).

[0067] Pharmacokinetics The pharmacokinetics of h7C3-3 and h7C3-4 were evaluated in B6 mice. B6 mice (n=6) were administered 10 mg / kg (based on antibody components) of test material via tail vein injection. Blood samples were collected from each mouse via the saphenous vein at 1, 2, and 6 hours after injection and on days 1, 2, 3, 4, 7, 9, 11, 14, 16, 18, 21, 23, 25, 35, and 42, and serum samples were collected. Plasma concentrations of h7C3-3 and h7C3-4 were measured using an IgG (Total) Human ELISA Kit (Invitrogen, BMS2091).

[0068] Mouse xenograft tumor model and antibody drug conjugate (ADC) treatment Seven-week-old immunodeficient male NU / NU nude mice (for H1299 and PC-3 xenograft studies) and male Fox Chase SCID® mice (for HA22T-Luc xenograft studies) purchased from BioLASCO were housed in sterile cages equipped with air filters and sterile bedding at the Laboratory Animal Center of the National Institutes of Health Research (NHRI), an AAALAC International accredited facility. All mice were provided with sterile water and food ad libitum under a 12-hour light / 12-hour dark cycle throughout the study. On the day of tumor cell inoculation, viable cells were counted under a light microscope using a hemocytometer with trypan blue staining. Cells were suspended in a 1:1 ratio in phenol red-free medium [RPMI 1640 (H1299) or DMEM (HA22T-Luc)] or PBS (PC-3) and Matrigel™ (356237, Matrigel® Matrix, Corning, MA, USA). H1299(1×10 6 cells), HA22T-Luc (5 × 10 6 cells) and PC-3 (1 × 10 6 Cells (1000 cells) were subcutaneously implanted into the left flank of nude or SCID mice using a 1 mL syringe (24G x 1 inch needle). Tumor dimensions were measured with a digital caliper, and tumor volume (mm 3 ) and volume = (length x width ∧2 The tumor-bearing mice were divided into groups (n = 5-8 per group), and the mean tumor volume was approximately 200 mm. 3In three separate xenograft studies, human IgG (HU-GF-ED, lot number 120916DG, Molecular Innovations, MO, USA), h7C3-4, h7C3-3-MMAE, and h7C3-4-MMAE were freshly diluted in 1× Dulbecco's phosphate-buffered saline (02-023-5A, Biological Industries, CT, USA) before administration at a concentration of 2.5 mg / ml. Cisplatin (KEMOPLAT, 1 mg / mL, 87200009AA, Fresenius Kabi, Bad Homburg, Germany) was purchased from the manufacturer. For the H1299 xenograft study, tumor-bearing mice were divided into five groups (six mice per group): human IgG-10 mg / kg (negative control), cisplatin-7 mg / kg (positive control), h7C3-3-MMAE-10 mg / kg, h7C3-4-10 mg / kg, and h7C3-4-MMAE-10 mg / kg. Mice were intravenously injected via the tail vein with a dose volume of 4 mL / kg twice per week for three weeks, except for the cisplatin group, which received the injection once per week for three weeks. For the HA22T-Luc xenograft study, tumor-bearing mice were divided into three groups (eight mice per group): human IgG-10 mg / kg (negative control), h7C3-3-MMAE-10 mg / kg, and h7C3-4-MMAE (ADC)-10 mg / kg. Mice were intravenously injected via the tail vein with a dose volume of 4 mL / Kg twice per week for two weeks. In the PC-3 xenograft study, tumor-bearing mice were divided into two groups: an untreated group (5 mice) and h7C3-4-MMAE (ADC)-10 mg / Kg (8 mice). Mice in the h7C3-4-MMAE-10 mg / Kg group were intravenously injected via the tail vein with a dose volume of 4 mL / Kg twice per week for three weeks. Each treatment was based on body weight. Body weight and tumor size were measured twice per week.

[0069] immunohistochemistry Deparaffinized tissue sections (4 μm) were subjected to heat-induced epitope retrieval in Tris-EDTA buffer (pH 9) at 95°C for 30 minutes. The sections were blocked with inhibitor CM at 37°C for 4 minutes. The slides were incubated with primary antibodies, including 2 μg / ml anti-NTSR1-B12 (SC-376958, Santa Cruz Biotechnology) and various anti-NTSR1 antibody clones (7C3, h7C3-2, and h7C3-3), for 1 hour. The slides were then incubated with the appropriate secondary antibodies, OmniMap anti-mouse-HRP for anti-NTSR1-B12 and anti-human-HRP for 7C3, h7C3-2, and h7C3-3, at room temperature for 30 minutes. Staining was performed with diaminobenzidine tetrahydrochloride. [Example]

[0070] Example 2 Characterization of the 7C3 antibody An antibody against human NTSR1 was generated by peptide immunization and phage display library screening using the extracellular loop 2 (ECL2) peptide of human NTSR1, resulting in the isolation of 7C3scFv. Unique 7C3scFv binders were converted to full-length IgG and tested for binding affinity to the cyclic ECL2 peptide (see Figure 1A). Binding of 7C3 IgG to cell surface NTSR1 was measured by flow cytometry in lung cancer cell line A549 cells. The 7C3 antibody showed strong binding to A549 cells (see Figure 1B). [Example]

[0071] Example 3 Affinity maturation of anti-NTSR1 antibodies To increase the affinity of the 7C3 antibody (having SEQ ID NOs: 1 and 2), mutants were tested through the use of a computer model. The model is based on a large repertoire of individual antibodies and antigens with three-dimensional structures that exhibit properties similar to those of the actual population. Three mutants were created, including h7C3-1 (having a Y100H mutation in CDR H3), h7C3-2 (having a Y100H mutation in CDR H3 and a S97A mutation in CDR L3), and h7C3-3 (having a T28A mutation in CDR H1, a Y100H mutation in CDR H3, and a S97A mutation in CDR L3). To investigate the activity of the mutants in enhancing affinity, the antibodies were analyzed by FACS. Among these mutants, h7C3-3 had a lower K of 0.3 nM. D The CDR sequences of h7C3-3 were shown in Table 2. Table 1. Affinity of various anti-NTSR1 antibodies after affinity maturation [Table 1] a Dose-dependent binding activity of anti-NTSR1 antibodies (1 μg / ml and 10 μg / ml) in A549 cells b Effective concentration (EC50) of anti-NTSR1 antibodies on A549 cells for mean fluorescence intensity (MFI) Table 2. CDR sequences of h7C3-3 (Kabat definition) [Table 2] [Example]

[0072] Example 4 Alanine scanning of the CDR regions of anti-NTSR1 antibodies To identify permissive sites in CDR H3 involved in antigen binding, we tested mutants at eight sites in CDR H3 of h7C3 using experimental alanine scanning mutagenesis. The mutants were compared for their ability to bind to the cyclic ECL2 peptide in an enzyme-linked immunosorbent assay (ELISA). R98A, Y99A, Y100A, Y101A, G102A, and F103A significantly reduced the apparent affinity of h7C3 for the peptide by approximately 30-70%. Other substitutions, including D104A and Y105A, did not affect h7C3 affinity. Based on the results of computer modeling, we investigated the effect of replacing Y100 with H on h7C3 binding activity. h7C3 with the Y100H mutation exhibited higher affinity.

[0073] The results showed that the h7C3-1 antibody (h7C3 containing a Y100H mutation in CDR H3) exhibited higher affinity compared to the parent h7C3. Mutations at nine positions in CDR L3 of h7C3-1 were tested via alanine-scanning mutagenesis. Several residues, particularly F94A and W101A (30%-40% reduction), and even L99A (70% reduction), significantly reduced h7C3-1 affinity. Substitution of G91 with A significantly increased the affinity of h7C3-1 for the peptide. Other substitutions, including G96A, S97A, H98A, and P100A, had no effect on h7C3-1 affinity. To further confirm the binding of the mutants to cell surface NTSR1, h7C3-1 with G96A, S97A, H98A, or P100A were analyzed by FACS. The G96A or P100A mutation significantly reduced the binding affinity of h7C3-1 to cell surface NTSR1 compared with that of h7C3-1, while H98A only slightly affected its binding to NTSR1. Surprisingly, h7C3-1 with the S97A mutation showed higher affinity.

[0074] To identify residues important for antigen-antibody interaction, alanine-scanning mutagenesis was performed at 10 and 18 positions in CDR H1 and CDR H2 of h7C3-2, respectively. G26A, Y27A, W33A, I34A, H35A, Q50A, R52A, and Y59A significantly reduced the apparent affinity of h7C3-2 for the peptide by approximately 50% or more. Nineteen of the 28 mutants, including five in CDR H1 (T28A, F29A, T30A, S31A, and S32A) and 14 in CDR H2 (I51A, P53A, N54A, S55A, G56A, N57A, T58A, Y60A, N61A, E62A, K63A, F64A, K65A, and V66A), retained greater than 80% h7C3-2 binding activity. h7C3-2 mutants with the T28A or N54A mutations showed higher binding affinity to the peptide as analyzed by FACS. Interestingly, h7C3-2 with the T28A mutation in CDR H1 showed higher affinity for NTSR1 in A549 cells. [Example]

[0075] Example 5 h7C3-3 Internalization To test whether h7C3-3 is internalized upon binding to the cell surface and thus represents a potential cytotoxic payload conjugate, A549, H1299, HA22T, or Mahlavu cells were exposed to the antibody (10 μg / ml) and analyzed by FACS. As shown in Figure 3A, with increasing incubation time, the magnitude of the mean fluorescence intensity (MFI) gradually decreased as the cells were shifted from 4°C to 37°C, indicating an increase in the number of h7C3-3 antibodies internalized by A549 cells. Similar results were observed in H1299 cells (Figure 3B), HA22T cells (Figure 3C), and Mahlavu cells (Figure 3D). These results indicated that h7C3-3 was rapidly and efficiently internalized into NTSR1-expressing cells. [Example]

[0076] Example 6 Characterization of h7C3-4 and h7C3-5 To improve the pharmacokinetic properties of h7C3-3 (V H S9T and D82E mutations in the V domain L h7C3-4 (having a K79T mutation in the α-terminal domain) was created through the use of computer modeling. As shown in Figure 4A, h7C3-4, which has a lower isoelectric point (pI), exhibited similar binding affinity compared to h7C3-3. Furthermore, h7C3-4 exhibited significantly improved clearance after saphenous vein injection compared to h7C3-3 (see Figure 4B). These results indicated that h7C3-4 has better pharmacokinetics.

[0077] An additional mutant, h7C3-5, was also constructed (see Figure 5A and B). h7C3-5 showed similar binding affinity to NTSR1 in A549 cells compared to h7C3-4 (see Figure 5C). [Example]

[0078] Example 7 Characterization of anti-NTSR1 antibody conjugated MMAE Following the achievement of highly efficient production of h7C3-3 and h7C3- in Expi293 cells, we investigated conjugation of anti-NTSR1 antibodies with the antimitotic tubulin inhibitor monomethyl auristatin E (MMAE) via a cleavable chemical linker. The ADCs were prepared by partial reduction of the intramolecular disulfide bond followed by conjugation with a thiol-reactive maleimide-containing drug linker. The identity of these two ADCs was confirmed by HIC. HIC analysis allowed resolution into five major peaks corresponding to 0, 2, 4, 6, or 8 drug molecules per conjugated anti-NTSR1 antibody (Figure 6A and B), with an average DAR of approximately 3.8–4. [Example]

[0079] Example 8 Internalization efficiency of anti-NTSR1 antibody-drug conjugates Anti-NTSR1 ADCs, consisting of a peptide-cleavable maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (vc) linker, are introduced into target cells and subsequently released, usually by lysosomal proteases, for cytotoxic drug delivery. Internalization of the h7C3-3-MMAE ADC was measured in the NTSR1-expressing NSCLC cell line A549 and compared with that of a human IgG Ab (negative control) and the unconjugated parent Ab (positive control). Both h7C3-3-MMAE and the unconjugated parent Ab (h7C3-3) exhibited cell surface binding efficiency and cellular distribution (Figure 7A). To initiate internalization of h7C3-3-MMAE or h7C3-3, primary antibody-treated cells were suspended in 100 μL of FACS buffer and incubated at 37°C for 90 minutes before incubation with the secondary detection Ab. When the primary antibody was internalized by cells at 37°C, the cellular distribution is shown on the left. Both h7C3-3-MMAE and the unconjugated parent antibody (h7C3-3) were efficiently internalized. See Figure 7B and C. [Example]

[0080] Example 9 Cytotoxic Effects of Anti-NTSR1 Antibody-Drug Conjugates NTSR1-specific ADCs were generated by conjugating h7C3-3, h7C3-4, and h7C3-5 with the dolastatin analog MMAE. Auristatins are potent cytotoxic agents that induce cell death by disrupting microtubules. h7C3-3-MMAE, h7C3-4-MMAE, and h7C3-5-MMAE contain a protease-sensitive valine-citrulline dipeptide sequence designed for optimal stability in human plasma and efficient cleavage by human cathepsin B. Following internalization, ribosomal proteases metabolize both the antibody and linker, releasing the active drug. NTSR1-specific ADCs were conjugated with an average of 3.8-4 MMAE per antibody, a ratio shown to provide optimal therapeutic indices for brentuximab vedotin, polatuzumab vedotin, and enfortumab vedotin. Table 3. Cytotoxic effects of h7C3-3 and h7C3-3-MMAE in various NTSR1-expressing cancer cell lines [Table 3]

[0081] Direct comparison of h7C3-3 and h7C3-3-MMAE in vitro confirmed that target binding and internalization characteristics were preserved in the ADCs (see Table 3 and Figure 8). The NTSR1-specific ADC demonstrated superior cytotoxicity in vitro.

[0082] Comparison of h7C3-4 and h7C3-4-MMAE in in vitro cytotoxicity assays also demonstrated that the ADCs exhibited target binding, internalization, and cytotoxicity (see Table 4 and Figure 9).

[0083] h7C3-5 and h7C3-5-MMAE were compared in an in vitro cytotoxicity assay. The results showed that this ADC also exhibited excellent cytotoxicity. See Table 5 and Figure 10.

[0084] As shown in Table 6, h7C3-4-MMAE and h7C3-5-MMAE exhibited comparable in vitro cytotoxic effects in the cell lines tested. Table 4. Cytotoxic effects of h7C3-4 and h7C3-4-MMAE in various NTSR1-expressing cancer cell lines [Table 4] Table 5. Cytotoxic effects of h7C3-5 and h7C3-5-MMAE in various NTSR1-expressing cancer cell lines [Table 5] Table 6. Cytotoxic effects of h7C3-4-MMAE and h7C3-5-MMAE in various NTSR1-expressing cancer cell lines [Table 6] [Example]

[0085] Example 10 In vivo antitumor activity The in vivo efficacy of anti-NTSR1 ADCs on target cells was evaluated in several NTSR1-positive xenograft models.

[0086] Nude mice were subcutaneously inoculated with NTSR1-expressing cells, H1299 (1 × 10 per mouse). 6 cells). The tumor is 200 mm 3 When tumor volume reached 100 μg / kg, tumor-bearing mice were divided into groups (n=6) and administered six doses of human IgG (negative control), h7C3-3-MMAE, h7C3-4, and h7C3-4-MMAE intravenously at 10 mg / kg twice weekly. Cisplatin (positive control) was administered three doses of 7 mg / kg intravenously once weekly. As shown in Figure 11A, on day 18, h7C3-4-MMAE at 10 mg / kg inhibited tumor growth more potently than the control human IgG (10 mg / kg) and cisplatin (7 mg / kg) groups. By day 35, long-term tumor regression was observed with h7C3-4-MMAE treatment compared with cisplatin, a positive control commonly used in clinical treatment of non-small cell lung cancer. Furthermore, at day 35, 10 mg / kg h7C3-3-MMAE showed partial inhibition of tumor growth, indicating that h7C3-4 is internalized more efficiently than h7C3-3 in a lung xenograft model.

[0087] To compare the antitumor efficacy of h7C3-3-MMAE and h7C3-4-MMAE, 5 × 10 6 The experiment was performed by subcutaneously inoculating HA22T-luc hepatoma cells into SCID mice. The tumor-bearing mice were divided into three groups (n=8). 3Once tumor volume reached 100 mg / kg, four doses of 10 mg / kg human IgG, h7C3-3-MMAE, and h7C3-4-MMAE were each administered intravenously twice weekly. By day 15, both h7C3-3-MMAE and h7C3-4-MMAE demonstrated tumor regression (Figure 12A), and long-term tumor regression was observed with both ADC treatments by day 36.

[0088] Further experiments were performed using the PC3 prostate cancer cell line (1 × 10 6 xenografts of 10 ... 3 When tumor volume reached 100 μg / kg, the tumor-bearing mice were divided into untreated (n=5) and h7C3-4-MMAE (n=8) groups. Six doses of 10 mg / kg h7C3-4-MMAE were administered intravenously twice weekly. h7C3-4-MMAE also demonstrated high efficacy in the PC3 prostate cancer subtype. Long-term tumor regression was observed with h7C3-4-MMAE treatment up to day 60. Treatment-induced tumor regression did not induce any abnormalities in the general physiological status or body weight of the mice (Figures 8B, 9B, and 10B), demonstrating that h7C3-3-MMAE or h7C3-4-MMAE are potent agents for the treatment of NTSR1-positive malignancies. [Example]

[0089] Example 11 Immunohistochemical staining To evaluate NTSR1 expression in prostate cancer cells, PC-3 xenograft tumor tissues were subjected to NTSR1 immunohistochemical staining using 2 μg / ml of B-12 (anti-NTSR1-B12, SC-376958, Santa Cruz Biotechnology), 7C3, h7C3-2, or h7C3-3 antibodies (see Figure 14). Immunohistochemical staining demonstrated strong NTR1 expression in PC-3 xenograft tumor tissues. Among these antibodies, the staining with h7C3-3 was clustered, suggesting that h7C3-3 was more suitable for immunohistochemical staining.

[0090] Other embodiments All features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.

[0091] From the above description, those skilled in the art can easily grasp the essential features of the described embodiments, and can make various changes and modifications to the embodiments to adapt them to various uses and conditions without departing from the spirit and scope thereof. Accordingly, other embodiments are also within the scope of the claims.

Claims

1. 1. An isolated antibody, comprising: A heavy chain variable domain (V) that is at least 75% identical to the amino acid sequence of SEQ ID NO: 1 Ｈ )and, a light chain variable domain (V) that is at least 75% identical to the amino acid sequence of SEQ ID NO:2; Ｌ )and, Equipped with Heavy chain CDR1: GYTFTSSWIH (SEQ ID NO: 3) or GYAFTSSWIH (SEQ ID NO: 4), Heavy chain CDR2: QIRPNSGNTYYNEKFKV (SEQ ID NO: 5); Heavy chain CDR3: ARYYYGFDY (SEQ ID NO: 6), ARYHYGFDY (SEQ ID NO: 7) or ARYRYGFDY (SEQ ID NO: 8), Light chain CDR1: RSSQSIVHSNGNTYLE (SEQ ID NO: 9); Light chain CDR2: KVSNRFS (SEQ ID NO: 10); Light chain CDR3: FQGSHLPWT (SEQ ID NO: 11) or FQGAHLPWT (SEQ ID NO: 12), An isolated antibody that specifically binds to human neurotensin receptor 1 (hNTSR1).

2. Heavy chain CDR1: GYAFTSSWIH (SEQ ID NO: 4), and Heavy chain CDR2: QIRPNSGNTYYNEKFKV (SEQ ID NO: 5); Heavy chain CDR3: ARYHYGFDY (SEQ ID NO: 7) or ARYRYGFDY (SEQ ID NO: 8), and Light chain CDR1: RSSQSIVHSNGNTYLE (SEQ ID NO: 9); Light chain CDR2: KVSNRFS (SEQ ID NO: 10); 2. The isolated antibody of claim 1, comprising: light chain CDR3: FQGAHLPWT (SEQ ID NO: 12).

3. Heavy chain CDR1: GYAFTSSWIH (SEQ ID NO: 4), and Heavy chain CDR2: QIRPNSGNTYYNEKFKV (SEQ ID NO: 5); Heavy chain CDR3: ARYHYGFDY (SEQ ID NO: 7), and Light chain CDR1: RSSQSIVHSNGNTYLE (SEQ ID NO: 9); Light chain CDR2: KVSNRFS (SEQ ID NO: 10); 3. The isolated antibody of claim 2, comprising: light chain CDR3: FQGAHLPWT (SEQ ID NO: 12).

4. V, which is the sequence of SEQ ID NO: 13 Ｈ V, which is the sequence of SEQ ID NO: 14 Ｌ 4. The isolated antibody of claim 3, comprising the sequence:

5. V, which is the sequence of SEQ ID NO: 15 Ｈ V, which is the sequence of SEQ ID NO: 16 Ｌ 4. The isolated antibody of claim 3, comprising the sequence:

6. Heavy chain CDR1: GYTFTSSWIH (SEQ ID NO: 3), and Heavy chain CDR2: QIRPNSGNTYYNEKFKV (SEQ ID NO: 5); Heavy chain CDR3: ARYHYGFDY (SEQ ID NO: 7) or ARYRYGFDY (SEQ ID NO: 8), and Light chain CDR1: RSSQSIVHSNGNTYLE (SEQ ID NO: 9); Light chain CDR2: KVSNRFS (SEQ ID NO: 10); 2. The isolated antibody of claim 1, comprising: light chain CDR3: FQGSHLPWT (SEQ ID NO: 11).

7. Heavy chain CDR1: GYTFTSSWIH (SEQ ID NO: 3), and Heavy chain CDR2: QIRPNSGNTYYNEKFKV (SEQ ID NO: 5); Heavy chain CDR3: ARYHYGFDY (SEQ ID NO: 7) or ARYRYGFDY (SEQ ID NO: 8), and Light chain CDR1: RSSQSIVHSNGNTYLE (SEQ ID NO: 9); Light chain CDR2: KVSNRFS (SEQ ID NO: 10); 2. The isolated antibody of claim 1, comprising: light chain CDR3: FQGAHLPWT (SEQ ID NO: 12).

8. Heavy chain CDR1: GYTFTSSWIH (SEQ ID NO: 3), and Heavy chain CDR2: QIRPNSGNTYYNEKFKV (SEQ ID NO: 5); Heavy chain CDR3: ARYYYGFDY (SEQ ID NO: 6), and Light chain CDR1: RSSQSIVHSNGNTYLE (SEQ ID NO: 9); Light chain CDR2: KVSNRFS (SEQ ID NO: 10); 2. The isolated antibody of claim 1, comprising: light chain CDR3: FQGSHLPWT (SEQ ID NO: 11).

9. 9. The isolated antibody of any one of claims 1 to 8, which is a recombinant antibody, a monoclonal antibody, a chimeric antibody, a humanized antibody, an IgG1 antibody, or an antibody fragment comprising an antigen-binding site.

10. An antibody described in any one of claims 1 to 9 that binds to the second extracellular loop of hNTSR1.

11. 1. An antibody conjugate comprising: An isolated antibody according to any one of claims 1 to 10; a non-antibody molecule; 1. An antibody conjugate comprising:

12. The antibody conjugate of claim 11, wherein the non-antibody molecule is a polypeptide, polymer, oligosaccharide, lipid, glycolipid, solid support, small molecule drug, biotin, a nucleic acid molecule, a carrier protein, or a detectable label.

13. The antibody conjugate of claim 12 , wherein the conjugate is an antibody-drug conjugate.

14. The antibody conjugate of claim 13 , wherein the non-antibody molecule is an anti-cancer drug for treating tumors that express hNTSR1.

15. 15. The antibody conjugate of claim 14, wherein the tumor is mesothelioma, lung tumor, breast tumor, head and neck squamous cell carcinoma, colon tumor, pancreatic tumor, prostate tumor, or liver tumor.

16. 16. The antibody conjugate of claim 15, wherein the anticancer drug is monomethyl auristatin E (MMAE).

17. A pharmaceutical composition comprising the isolated antibody of any one of claims 1 to 10 and a pharmaceutical carrier.

18. A pharmaceutical composition comprising the antibody-drug conjugate of any one of claims 14 to 16 and a pharmaceutical carrier.

19. The pharmaceutical composition of claim 18, for use in treating a tumor in a subject.

20. 20. The pharmaceutical composition of claim 19, wherein the tumor expresses hNTSR1.

21. 20. The pharmaceutical composition of claim 19, wherein the tumor is mesothelioma, lung tumor, breast tumor, head and neck squamous cell carcinoma, colon tumor, pancreatic tumor, prostate tumor, or liver tumor.

22. A method for detecting hNTSR1, comprising: contacting a sample, tissue or cell with an antibody according to any one of claims 1 to 10 or an antibody complex according to claim 11 or 12; identifying binding of said antibody or antibody complex to a target in said sample or to said tissue or cell; A method for providing the above.

23. 23. The method of claim 22, wherein the antibody complex comprises a detectable label.

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