Anti-human neurotensin receptor 1 antibody and its uses

Abstract

An isolated antibody comprising: a heavy chain variable region (V H ), which is at least 75% identical to the amino acid sequence of SEQ ID NO: 1; and a light chain variable region (V L ), which is at least 75% identical to the amino acid sequence of SEQ ID NO: 2; wherein the antibody specifically binds to the human neurotensin receptor 1 (hNTSR1).

Description

Technical Field

[0001] The present invention relates to an anti-human neurotensin receptor 1 antibody and its use. Background Art

[0002] This application claims priority to U.S. Provisional Application No. 63 / 036,740, filed on Jun. 9, 2020, the entire content of which is incorporated herein by reference.

[0003] Monoclonal antibodies (mAbs) have become important tools for treating diseases such as cancer and infectious diseases, as well as in multiple fields such as molecular biology, pharmaceutical, and medical research. See Baert et al., N Engl J Med, 2003. 348(7): p. 601-8; Cavalli-Bjorkman et al., Med Oncol, 2002. 19(4): p. 277-80; and Plosker and Figgitt, Drugs, 2003. 63(8): p. 803-43. In the past decade, monospecific antibodies against specific antigens or cell surface receptors in different tumor types have achieved great success and have been at the forefront of cancer treatment.

[0004] Antibody-drug conjugates (ADCs) are a novel class of highly effective biopharmaceutical drugs composed of antibodies linked to cytotoxic compounds via a chemical linker. These novel targeted drugs exhibit unique antibody targeting capabilities, enabling the selective sensitization of cytotoxic drugs to healthy and cancerous tissues. ADCs represent an innovative therapeutic tool composed of: mAbs with high specificity, characteristics, and anti-tumor activity that are cancer-specific but not cytotoxic enough; and cytotoxic small molecule drugs with strong cell killing activity that are too toxic to be used alone. At least five ADCs have been approved for the market, and currently, more than 80 ADCs are in clinical trials. See Abdollahpour-Alitappeh et al., Antibody-drug conjugates (ADCs) for cancer therapy: Strategies, challenges, and successes. J Cell Physiol, 2018. With the advancement of cutting-edge technologies in the future, it is expected that ADCs will dominate the anti-cancer therapy market.

[0005] The ligand of neuromedin receptor 1 (NTSR1), neuromedin (NTS), is a short peptide that exists in the nervous system and peripheral tissues. See Carraway and Leeman, J Biol Chem, 1973. 248(19): p. 6854-61. NTS exhibits a wide range of biological activities and plays important roles in the pathogenesis of Parkinson's disease and schizophrenia, the regulation of dopamine neurotransmission, hypothermia, antinociception, and the promotion of cancer cell growth. See Bissette, G. et al., Nature, 1976. 262(5569): p. 607-9; Carraway and Plona, Peptides, 2006. 27(10): p. 2445-60; Griebel and Holsboer, Nat Rev Drug Discov, 2012. 11(6): p. 462-78; Kitabgi, Curr Opin Drug Discov Devel, 2002. 5(5): p. 764-76; and Schimpff et al., J Neurol Neurosurg Psychiatry, 2001. 70(6): p. 784-6. Three neuromedin receptors (NTSR) have been identified. NTSR1 and NTSR2 belong to the class A GPCR family, while NTSR3 (also known as SORT1) is a member of the sortilin family with a single transmembrane domain. See Tanaka et al., Neuron, 1990. 4(6): p. 847-54; Chalon et al., FEBS Lett, 1996. 386(2-3): p. 91-4; and Mazella, Cell Signal, 2001. 13(1): p. 1-6. Most of the known effects of NTS are mediated through NTSR1, which preferentially signals through the Gq protein. See Kitabgi, Curr Opin Drug Discov Devel, 2002. 5(5): p. 764-76.

[0006] Neurotensin and its cognate receptors are neuropeptide-receptor complexes that are frequently dysregulated during tumorigenesis. Neurotensin receptor 1 (NTSR1) has been reported to promote cancer progression in invasive 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): p.164-70; Alifano et al., Clin Cancer Res, 2010.16(17): p.4401-10; Wu, Z. et al., Cancer Lett, 2017.388: p.73-84; Dupouy et al., PLoS One, 2009.4(1): p.e4223; and Shimizu et al., Int J Cancer, 2008.123(8): p.1816-23. Based on patient tissue staining, NTSR1 is a promising molecular marker for non-small cell lung cancer and prostate cancer. See He et al., Eur J Nucl Med MolImaging.2019 46(10):2199-2207; and Alifano et al., Clin Cancer Res, 2010.16(17): p.4401-10. NTSR1 activation also transactivates the EGFR receptor in colon, prostate, and pancreatic cancer cell lines. See Amorino et al., Oncogene, 2007.26(5): p.745-56; and Muller et al., BMCCancer, 2011.11: p.421. Recently, a genome-wide association analysis also 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): p.663-673. NTS and NTSR1 are aberrantly expressed in more than 50% of hepatocellular carcinoma (HCC). Elevated expression of NTS or NTSR1 mRNA is associated with poor patient prognosis. Wu et al., Cancer Lett, 2017.388: p.73-84.

[0007] Accordingly, NTSR1 is a potential target for cancer therapy. Inhibiting the activity of NTSR1 can reduce cancer formation and cancer recurrence. SUMMARY OF THE INVENTION

[0008] In one aspect, described herein is an isolated antibody comprising: a heavy chain variable region (V H ), which is at least 75% identical to the amino acid sequence of SEQ ID NO:1; and a light chain variable region (V L) that is at least 75% identical to the amino acid sequence of SEQ ID NO:2; wherein the antibody specifically binds to human neurotensin receptor 1 (hNTSR1). For example, the antibody can bind to the second extracellular loop of hNTSR1.

[0009] In some embodiments, the antibody has one or more substitutions within SEQ ID NO:1 at positions 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.

[0010] In some embodiments, the antibody has one or more substitutions within SEQ ID NO:2 at positions selected from G96, S97, H98, and P100.

[0011] In some embodiments, 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).

[0012] In some embodiments, 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).

[0013] In some embodiments, 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).

[0014] In some embodiments, 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).

[0015] In some embodiments, 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).

[0016] In some embodiments, 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).

[0017] In some embodiments, the antibody comprises a V H sequence that is the sequence of SEQ ID NO: 13; and a V L sequence that is the sequence of SEQ ID NO: 14.

[0018] In some embodiments, the antibody comprises a V H sequence that is the sequence of SEQ ID NO: 15; and a V L sequence that is the sequence of SEQ ID NO: 16.

[0019] In some 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.

[0020] In another aspect, provided herein is an antibody complex that comprises any anti-hNTSR1 antibody described herein and a non-antibody molecule.

[0021] In some embodiments, the non-antibody molecule is a polypeptide, a polymer, an oligosaccharide, a lipid, a glycolipid, a solid support, a small molecule drug, biotin, a nucleic acid molecule, a carrier protein, or a detectable label.

[0022] In some embodiments, the antibody complex is an antibody-drug complex and the non-antibody molecule is an anti-cancer drug for inhibiting cancer cells or treating tumors expressing hNTSR1. For example, the anti-cancer drug can be monomethyl auristatin E (MMAE).

[0023] 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.

[0024] In yet another aspect, described herein is a pharmaceutical composition that comprises any antibody or antibody-drug complex described herein and a pharmaceutical carrier.

[0025] In one aspect, described herein is a method of treating a tumor in a subject, comprising administering to a subject in need thereof any antibody-drug complex or pharmaceutical composition described herein. In some embodiments, the tumor expresses hNTSR1.

[0026] 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.

[0027] In another aspect, methods for detecting NTSR1 or cells expressing NTSR1 in a sample or tissue are described herein. The method includes contacting a sample, tissue, or cell with any of the antibodies or antibody complexes described herein and determining the binding of the antibody or antibody complex to a target in the sample or to the tissue or cell.

[0028] In yet another aspect, described herein is an isolated nucleic acid molecule encoding any of the antibodies or components thereof described herein.

[0029] Details of one or more embodiments are set forth in the accompanying drawings and the following description. Other features, objects, and advantages of the embodiments will be apparent from the description and drawings, and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 is a set of graphs showing the characterization analysis of 7C3 mAb. (A) Binding of 7C3 mAb to the NTSR1 loop ECL2 peptide in ELISA. (B) Binding of 7C3 (10 μg / ml) to NTSR1 on A549 cells, measured by flow cytometry.

[0031] 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).

[0032] Figure 3 is a set of graphs showing the internalization of anti-NTSR1 mAbs in various cancer cell lines overexpressing NTSR1. Flow cytometric analysis of (A) A549, (B) H1299, (C) HA22T, and (D) Mahlavu cells treated with different antibodies (10 μg / ml) at 4°C or 37°C, respectively.

[0033] Figure 4 Includes tables and graphs showing the expression of the h7C3-4 variant antibody. (A) Isoelectric point (pI) and binding affinity of h7C3-3 or h7C3-4. Binding of h7C3-3 or h7C3-4 to A549 cells expressing NTSR1 was measured by flow cytometry at a concentration of 0.5 μg / ml. (B) Pharmacokinetics of the antibodies in BLTW:CD1 (ICR) mice. Serum antibody concentration relative to time after a single intravenous administration (10 mg / kg). The concentration of h7C3-3 or h7C3-4 antibodies in mouse serum was analyzed by ELISA.

[0034] Figure 5Characterization analysis of humanized h7C3-4 and h7C3-5 antibodies. Alignment of the amino acid sequences of the heavy chains (A) and light chains (B) of h7C3-4 and h7C3-5. Mutated residues are shown in bold, and CDR sequences are defined according to Kabat. (C) Binding of h7C3-4 and h7C3-5 to NTSR1 on A549 cells was 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); h7C3-5 light chain (SEQ ID NO:16).

[0035] Figure 6 Panel showing hydrophobic interaction chromatography (HIC) analysis of (A) h7C3-3-MMAE and (B) h7C3-4-MMAE conjugates. "DAR:0", "DAR:2", "DAR:4", "DAR:6", and "DAR:8" refer to the isomers of the conjugates of each antibody conjugated with 0, 2, 4, 6, and 8 MMAE molecules, respectively.

[0036] Figure 7 Panel showing the internalization efficiency of h7C3-3 and h7C3-3-MMAE on A549 cells. (A) Binding affinity of h7C3-3 and h7C3-3-MMAE to A549 cancer cells was measured by flow cytometry after culturing at 4°C for one hour. Cell distribution is shown on the right. (B and C) Internalization efficiency of h7C3-3 and h7C3-3-MMAE on A549 cancer cells expressing NTSR1 at a concentration of 1 μg / ml was evaluated by flow cytometry analysis. Cells were incubated with the antibody on ice for 1 hour. Unbound antibody was washed off. Then the experimental groups were cultured at 37°C for 90 minutes. After collection, cells were stained with FITC-conjugated anti-human IgG and analyzed by flow cytometry. Internalization of cell distribution is shown on the left.

[0037] Figure 8 Panel showing the in vitro cytotoxicity of h7C3-3-MMAE in various cancer cell lines.

[0038] Figure 9 Includes charts and tables showing the in vitro cytotoxicity of h7C3-4-MMAE in various cancer cell lines.

[0039] Figure 10 Includes charts and tables showing the in vitro cytotoxicity of h7C3-5-MMAE in various cancer cell lines.

[0040] Figure 11Is a set of figures showing the efficacy of 10 mg / kg doses of h7C3-3-MMAE, h7C3-4, and h7C3-4-MMAE compared to a 7 mg / kg dose of cisplatin in the H1299 lung 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) No weight loss was observed in either group.

[0041] Figure 12 Is a set of figures showing the efficacy of 10 mg / kg doses of anti-NTSR1 h7C3-3-MMAE and h7C3-4-MMAE 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) No weight loss was observed in either group.

[0042] Figure 13 Is a set of figures showing the efficacy of 10 mg / kg dose of anti-NTSR1 h7C3-4-MMAE in the PC3 prostate tumor xenograft model. (A) The anti-tumor efficacy of h7C3-4-MMAE is shown by the change in tumor volume. (B) Treatment did not cause weight loss.

[0043] Figure 14 Is a set of images showing immunohistochemical staining of xenograft PC-3 prostate cancer tissue with 2 μg / ml of B-12, 7C3, h7C3-2, or h7C3-3 antibodies. Solid tumors were excised from untreated PC-3 xenograft mice and then immunohistochemically stained using an antibody specific for NTSR1. Images were taken at 50X and 100X magnifications. Detailed Description

[0044] This document describes novel antibodies and their conjugates that bind to human NTSR1. Each anti-NTSR1 antibody may include a heavy chain variable region (V H ), which 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, and a light chain variable region (V L ), which 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.

[0045] SEQ ID NO:1

[0046] QVQLQQPGSVLVRPGASVKLSCKASGYTFTSSWIHWAKQRPGQGLEWIGQIRPNSGNTYYNEKFKVKATLTVDTSSSTAYVDLSSLTSEDSAVYYCARYYYGFDYWGQGTLVTVSS

[0047] SEQ ID NO:2

[0048] DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHLPWTFGGGTKLEIKR

[0049] Each antibody may have one or more substitutions at positions 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 within SEQ ID NO:1. Additionally or alternatively, the antibody may have one or more substitutions at positions selected from G96, S97, H98, and P100 within SEQ ID NO:2. The amino acid substitutions can be any amino acid (e.g., Ala), provided that the substitution does not significantly reduce the binding affinity of the antibody for NTSR1 (e.g., by no more than 25%, 20%, 15%, 10%, or 5%) compared to the antibody with the sequences of SEQ ID NO:1 and 2. The binding affinity of the antibody for NTSR1 can be determined using various methods known in the art or described herein, e.g., binding to NTSR1 on cells or the ECL2 peptide.

[0050] In some embodiments, the anti-NTSR1 antibody has a heavy chain CDR1 having the sequence GYTFTSSWIH (SEQ ID NO: 3) or GYAFTSSWIH (SEQ ID NO: 4); a heavy chain CDR2 having the sequence QIRPNSGNTYYNEKFKV (SEQ ID NO: 5); a heavy chain CDR3 having the sequence ARYYYGFDY (SEQ ID NO: 6), ARYHYGFDY (SEQ ID NO: 7), or ARYRYGFDY (SEQ ID NO: 8); a light chain CDR1 having the sequence RSSQSIVHSNGNTYLE (SEQ ID NO: 9); a light chain CDR2 having the sequence KVSNRFS (SEQ ID NO: 10); and a light chain CDR3 having the sequence FQGSHLPWT (SEQ ID NO: 11) or FQGAHLPWT (SEQ ID NO: 12).

[0051] 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).

[0052] In some embodiments, the antibody (e.g., h7C3-2) 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 FQGAHLPWT (SEQ ID NO: 12).

[0053] In some embodiments, an antibody (e.g., h7C3-3, h7C3-4, or h7C3-5) includes 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).

[0054] In some embodiments, an antibody (e.g., h7C3) has 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). In some embodiments, the antibody includes a V having the sequence of SEQ ID NO: 13 or 15 H and a V having the sequence of SEQ ID NO: 14 or 16 L .

[0055] SEQ ID NO:13

[0056] QVQLQQPGTVLVRPGASVKLSCKASGYAFTSSWIHWAKQRPGQGLEWIGQIRPNSGNTYYNEKFKVKATLTVDTSSSTAYVELSSLTSEDSAVYYCARYHYGFDYWGQGTLVTVSS

[0057] SEQ ID NO:14

[0058] DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLTISRVEAEDLGVYYCFQGAHLPWTFGGGTKLEIKR

[0059] SEQ ID NO:15

[0060] QVQLVQSGAEVKKPGASVKVSCKASGYAFTSSWIHWVRQAPGQRLEWMGQIRPNSGNTYYNEKFKVRVTITRDTSASTAYMELSSLRSEDTAVYYCARYHYGFDYWGQGTLVTVSS

[0061] SEQ ID NO:16

[0062] DIVMTQTPLSLSVTPGQPASISCRSSQSIVHSNGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGAHLPWTFGGGTKVEIKR

[0063] 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, single-chain variable region fragment multimers (scFv multimer), monovalent antibodies, multivalent antibodies, humanized antibodies, and chimeric antibodies.

[0064] Based on the antibody CDR sequences disclosed herein, those skilled in the art will be able to generate various forms of anti-NTSR1 antibodies using methods known in the art (such as recombinant methods).

[0065] Also contemplated herein are isolated nucleic acid molecules (e.g., expression vectors) encoding one or more of the anti-NTSR1 antibodies described herein or components thereof. Host cells containing such nucleic acids are also provided. The nucleic acid molecules and host cells can be used to produce anti-NTSR1 antibodies.

[0066] Any anti-NTSR1 antibody described herein can be conjugated to a non-antibody molecule using methods known in the art to form an antibody conjugate. Non-antibody molecules can be exemplified by polypeptides, polymers, oligosaccharides, lipids, glycolipids, solid supports (such as beads or plates), small molecule drugs (such as cytotoxic drugs), biotin, nucleic acid molecules, carrier proteins, or detectable labels (such as fluorescent labels). The non-antibody molecule can be linked to the antibody through a cleavable linker (such as valine-citrulline) or an uncleavable linker (such as 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.

[0067] In some embodiments, the antibody conjugate is an antibody-drug conjugate, where the drug is used to inhibit cancer cells or treat tumors, such as cancer cells or tumors that express NTSR1. In some embodiments, the drug is monomethyl auristatin E (MMAE).

[0068] Any antibody or antibody conjugate described herein can be used to inhibit the binding between NTSR1 and its ligand, inhibit NTSR1 function, detect the NTSR1 protein or its fragments in a sample (e.g., in an immunoassay), bind to tissues or cells that express NTSR1 (e.g., identify cells or isolate cells that express NTSR1), inhibit the growth of cancer cells or tumors, or treat cancer in a subject.

[0069] The term "sample" can be any biological sample, such as a body fluid sample, a blood sample, a cell sample, a urine sample, a saliva sample, or a tissue sample.

[0070] Tumors that express NTSR1 can be potential targets for anti-NTSR1 antibodies or their conjugates. Such tumors include, but are not limited to, mesothelioma, lung tumors, breast tumors, head and neck squamous cell carcinomas, colon tumors, pancreatic tumors, prostate tumors, or liver tumors. Optionally, before administering any anti-NTSR1 antibody or conjugate to a subject, it can be determined whether the tumors in the subject express NTSR1. The treatment method can be carried out alone or in combination with other drugs or therapies.

[0071] "Subject" refers to a human or non-human animal. "Treating" or "treatment" refers to administering a compound or composition to a subject having a disorder, with the purpose of curing, alleviating, mitigating, treating, delaying the onset of the disorder or improving the disorder, the symptoms of the disorder, the disease state secondary to the disease, or the predisposition to the disease. "Effective amount" refers to the amount of a compound or composition that is capable of producing a medically desirable result in a subject being treated.

[0072] Any anti-NTSR1 antibody and antibody complex described herein can be formulated into a pharmaceutical composition suitable for various routes of administration, such as intravenous, intra-articular, intra-conjunctival, intracranial, intraperitoneal, intrapleural, intramuscular, intraspinal or subcutaneous routes of administration. The pharmaceutical composition can be an aqueous solution or a lyophilized preparation. It can contain pharmaceutically acceptable carriers, such as buffers, excipients, stabilizers or preservatives. The pharmaceutical composition can include other active ingredients that act together with the antibody or antibody complex, for example, another therapeutic agent or adjuvant.

[0073] The following specific examples will be construed as illustrative only and not limiting in any way the remainder of the disclosure. Without further elaboration, it is believed that one skilled in the art can fully utilize this disclosure based on the description herein. All publications cited herein are hereby incorporated by reference in their entirety.

[0074] Example 1: Materials and Methods

[0075] Cell Lines

[0076] A549 (NSCLC) cells were cultured in F-12K medium (Gibco, Grand Island, NY, USA) containing 10% fetal bovine serum (Gibco, Grand Island, NY, USA). H1299 (NSCLC) cells were cultured in RPMI medium supplemented with 10% FBS and 2 mM L-glutamine. HA22T (HCC) was cultured in DMEM medium supplemented with 10% FBS, 0.1 mM non-essential amino acids and 2 mM L-glutamine (Gibco). Mahlavu was cultured in DMEM medium supplemented with 10% FBS. PC-3 (PCa) cells were cultured in RPMI1640 medium (Gibco, SH30027) supplemented with 10 mM HEPES, 1 mM sodium pyruvate (Gibco), 4.5 g / L glucose and 10% FBS.

[0077] Mouse Immunization and RNA Purification

[0078] Five female BALB / c mice (4 - 6 weeks old) were intraperitoneally injected with 0.1 mg of ovalbumin-conjugated ECL2 peptide every two weeks for 12 weeks. Blood samples were collected 1 week after each immunization and titrated by indirect enzyme-linked immunosorbent assay (ELISA). After a total of six boosts, the mice were sacrificed to harvest the spleens. The 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.

[0079] Heavy chain RT-PCR amplification and Kappa chain purification

[0080] Total RNA from the harvested mouse spleens was extracted using Trizol reagent according to the manufacturer's protocol (Invitrogen, USA). The 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 a mixture of reverse transcriptase (Roche), 1 μl of RNaseOut (40 U / μl, Invitrogen), and the MuJH (or MuJK)-FOR primer.

[0081] Construction of the phage display scFv gene library

[0082] Briefly, the gene library construction process involves three steps: (i) amplifying the V H and V L domains using framework region family-specific primers, (ii) re-amplifying each fragment with primers containing linker fragments, and (iii) assembling the two fragments (V H and V L ) by overlap extension polymerase chain reaction (PCR). All PCR reactions were performed using TaKaRa Ex Taq polymerase (RR001A) and primers specific for murine heavy and Kappa light chains. See Benhar and Reiter, CurrProtoc Immunol, 2002. Chapter 10: p. Unit 10 19B.

[0083] The PCR product containing the scFv was digested with an excess of the restriction enzymes EcoRI and NcoI (NEB). Approximately 10.0 μg was ligated overnight at 16 °C with 40.0 μg of the EcoRI / NcoI-linearized pHEN2 vector (purified by agarose gel extraction) in a total volume of 100 μl containing 2400 units of T4 DNA ligase (NEB). After ligation, the recombinant DNA was precipitated, washed and dissolved in 20 μl of distilled water. 1 μl of the recombinant DNA was transformed into 25 μl of Escherichia coli TG1 (Lucigen) by electroporation each time. After transformation, 20 ml of recovery medium (Lucigen) was added and the culture was shaken at 37 °C for 1 hour, then 200 ml of 2YT containing 100 μg / ml ampicillin was placed on a shaker at 37 °C and cultured for a further 16 - 18 hours. At this point, aliquots of the culture were plated on 2YT agar / ampicillin to titer the gene library size, which was calculated by counting the number of ampicillin-resistant colonies. A phagemid containing the scFv was prepared from this overnight culture. Approximately M13K07 (1012 pfu) helper phage was added to the TG1 sample containing the scFv gene library and the culture was shaken at 37 °C for 2 - 3 hours. 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 and incubated on ice for 60 minutes, then the phage was precipitated by centrifugation at 8000 rpm for 20 minutes at 4 °C. The supernatant was discarded and the pellet was drained. The phage was 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 for the next round of biopanning.

[0084] Affinity selection (panning) of phage

[0085] Pre-wash streptavidin-coated magnetic beads with 10 volumes of TBS containing 2% (wt / vol) BSA and 0.02% (wt / vol) NaN3. The washed magnetic beads are suspended in TBS containing 2% (wt / vol) BSA, 5 mM DTT and 0.02% (wt / vol) NaN3. Non-specific binding of the scFv phage library is depleted by streptavidin beads with a final volume of 500 μl in a 1.5 ml microcentrifuge tube (mix 2×1012 phage particles in 500 μl of 4% (wt / vol) BSA in TBS supplemented with 1% (vol / vol) Tween-20 and 10 mM DTT), then add 60 μl of pre-washed streptavidin beads and incubate overnight at 4°C. Next, add 0.4 μg of biotinylated 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 to 2 ml of pre-adsorbed phage particles (2x10 12 phage particles), and incubate the mixture in an immunotube at room temperature (RT) for 4 hours. After two rounds of panning, single colonies of cells carrying the phagemid are used to prepare monoclonal phages in a sterile 96-well plate (Nunc, Sweden) and screened by ELISA.

[0086] Phage ELISA

[0087] Load 100 μl of wash buffer (25 mM Tris, 150 mM NaCl, 0.1% BSA, 0.05% Tween 20, pH 7.2) containing 5 mg / ml biotinylated human NTSR1 linear ECL2 peptide into a 96-well NeutrAvidin Coated Plate (Pierce). The next day, wash the well plate with wash buffer and block it with 5% non-fat milk in wash buffer for 1 hour at room temperature. After additional washing with wash buffer, add 100 μl of freshly prepared phage to each well and incubate for 1 hour at room temperature. Wash the well plate again with wash buffer and place a 1:5000 dilution of anti-M13 antibody (ab24229) in each well. Incubate at room temperature for 1 hour, then perform additional washing with wash buffer. Place a 1:5000 dilution of goat anti-mouse IgG-HRP (Jackson, 115-035-003) in each well for 1 hour at room temperature, then perform additional washing with wash buffer. Finally, add 100 μl of 3,3',4,4'-tetramethylbenzidine (TMB) (HRP substrate) solution (Pierce) to each well and incubate at room temperature. Add 100 μl of 1 M H2SO4 to each well to terminate the reaction. Analyze the well plate at 450 nm using a plate reader (Discover X plate reader).

[0088] Construction and Expression of Antibodies

[0089] The sequences of the heavy chain (V H ) and light chain (V L ) variable regions of 7C3 were ligated to the sequences of the constant regions of human IgG1 heavy chain and human IgG (κ) light chain encoded in the pFUSE-CHIg-hG1 and pFUSE2-CLIg-hK vectors (from InvivoGen), respectively. Expression of the human-mouse chimeric antibody was achieved by transfecting the vectors into Expi293 cells. Then, the secreted antibody was purified from the culture supernatant by protein A affinity chromatography and tested to determine whether antigen-binding ability was retained after chimerization.

[0090] Affinity Maturation by Site-Directed Mutagenesis

[0091] All mutations were introduced into the variable regions of the heavy chain (V H ) or light chain (V L ) using overlap extension PCR mutagenesis. The V H and V LThe mutant coding regions. The PCR products were annealed through their common overlap and amplified in a second PCR reaction, then purified separately and ligated into the pFUSE-CHIg-hG1 or pFUSE2-CLIg-hK vectors. After transformation of Escherichia coli JM109 (ECOSTM 9-5), individual colonies were screened by digesting each individual mutant with the appropriate restriction enzyme. Analytical restriction enzyme digestion was determined by sequencing to identify putative mutations.

[0092] NTSR1 Binding Affinity Assay

[0093] 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 at room temperature for 1 hour. The next day, the coating solution was removed, and the plates were blocked with 5% (v / v) non-fat milk in phosphate-buffered saline (PBS) for 1 hour. After washing with PBST, serial dilutions of anti-NTSR1 antibodies were added in triplicate. After incubation for 1 hour, the plates were washed 3 times with PBST, and then horseradish peroxidase (HRP)-conjugated goat anti-human IgG Fc antibody (100 μL) was added to each well and incubated for another hour at room temperature. Finally, 100 μL of tetramethylbenzidine (TMB) substrate was added to each well to generate a visible color. After incubation for 2 minutes, the reaction was stopped using 100 μL of 2 M H2SO4. The absorbance of each well was read at 450 nm using a plate reader.

[0094] Flow Cytometry

[0095] A total of 1x10 6 cells were reacted 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. Secondary FITC (BD PharmingenTM)-conjugated mouse anti-human IgG antibody was incubated on ice for another 60 minutes. The cells were washed again and then incubated with 1 ml of PBS containing 0.5 mg / ml propidium iodide at 4 °C for 10 minutes. Data from 10,000 live cells were collected by FACS (Bio-Red S3e cell sorter). The fluorescence intensity of channel FL-1 was calculated using Flow Jo flow cytometry analysis software.

[0096] Surface Plasmon Resonance

[0097] The interaction between the NTSR1-specific mAb and the extracellular loop 2 (ECL2) peptide of NTSR1 was measured by surface plasmon resonance using an Open SPR (Nicoya, Canada) instrument. The biotin-conjugated ECL2 peptide was immobilized on a streptavidin-coated sensor chip by injecting the peptide diluted in HBS-EP5 buffer saline (20 mM HEPES, 250 mM NaCl, 3 mM EDTA, 0.05% Tween 20, and 0.5% BSA, pH 7.4) at a concentration of 10 μg / ml (flow rate of 30 μl / min). For kinetic experiments, the NTSR1-specific mAb diluted in HBS-EP5 (concentration range from 24 to 1.5 nM) was injected onto the immobilized ECL2 peptide (flow rate of 30 μl / min). The binding of different concentrations of the NTSR1-specific mAb to the immobilized ECL2 peptide was analyzed using TraceDrawer software (Nicoya, Canada). The association rate (ka), dissociation rate (kd), and affinity (KD) were calculated using global analysis, and the data were fitted to a simple 1:1 binding model.

[0098] Internalization of NTSR-1 antibody in different cell lines expressing NTSR1 (flow cytometry)

[0099] The NTSR-1 antibody was used for flow cytometry analysis to compare the expression levels of NTSR1 in different lung and HCC cell lines, including A549, H1299, HA22T, and Mahlavu. A total of 1x10 6 cells were reacted with the h7C3-3 antibody in FACS buffer at 4°C for 60 minutes and washed twice to remove excess antibody. Then the cells were transferred to 4°C or 37°C for 0, 0.5, 2, 5, 24 hours, and then incubated with a secondary anti-human IgG Fc-PE (BioLegend) antibody at 4°C for another 60 minutes. The cells were washed again, and data were collected by a BD LSRFortessa. The fluorescence intensity of channel FL-1 was calculated using Flow Jo flow cytometry analysis software.

[0100] Internalization of h7C3-3 or h7C3-3-ADC antibody in A549 cells (flow cytometry)

[0101] A total of 1x10 6Cells were reacted with the indicated antibody or ADC in 100 μL of FACS buffer at 4 °C for 60 minutes and then washed to remove excess antibody or ADC. The cells were then transferred for 90 minutes at 4 °C or 37 °C. To determine the relative number of h7C3-3 or h7C3-3-ADC on the cell surface, the cells were stained with a saturating amount of FITC (BD Pharmingen™)-conjugated mouse anti-human IgG antibody and incubated for an additional 60 minutes on ice. The cells were washed again and then incubated with 1 mL of PBS containing 0.5 mg / mL propidium iodide at 4 °C for 10 minutes. Data from 10,000 live cells were collected via FACS (Bio-Red S3e cell sorter). The fluorescence intensity of channel FL-1 was calculated using Flow Jo flow cytometry analysis software.

[0102] Preparation of Anti-NTSR1 Antibody-Drug Conjugate (ADC)

[0103] 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) at 37 °C for 2 hours. The reduced antibody was reacted with vcMMAE (Achemblock, Q70231) at room temperature for 60 minutes. Unreacted vcMMAE was quenched with 1 mM N-acetyl-L-cysteine (Sigma-Aldrich) and incubated at room temperature for 30 minutes. The reaction mixture was then buffer-exchanged into PBS (pH 6.9) using an Amicon Ultrafree centrifugal filtration device (Millipore).

[0104] Hydrophobic Interaction Chromatography (HIC) Analysis

[0105] The characteristics of h7C3-3 and h7C3-3 ADC were analyzed using HIC, which is described as follows: 1200 HPLC (Agilent Technologies); TSKgel Butyl-NPR column (4.6 x 35 mm, particle size 2.5 μm; TOSOH); Solvent A, 1.5 mol L -1 ammonium sulfate and 25 mM phosphate (pH = 6.95); Solvent B, 75% (V / V) 25 mM phosphate, 25% (V / V) isopropanol (pH = 6.95); Gradient from 100% A to 100% B within 15 minutes; 0.5 mL min -1 flow rate; Column temperature was 25 °C; UV detection wavelength was 280 nm.

[0106] In vitro cytotoxicity assay

[0107] Use Luminescent Cell Viability Assay (Promega G7571) to determine the number of viable cells. Cells were seeded in 96-well opaque Costar plates (Cat. No. 136101). Briefly, 500 - 2500 cells in 90 μL of medium were added in quadruplicate to each well and allowed to adhere for 16 hours. 10 μl of anti-NTSR1 (dose range: 0 - 500 nM), anti-NTSR1-MMAE (dose range: 0 - 500 nM), or paclitaxel (dose range: 0 - 100 nM) was added. After 72 to 120 hours, the medium was removed, and 200 μL of medium containing the active reagent (1:1) was added to each well and placed on an orbital shaker in the dark for 2 minutes. Before reading with the Explorer Multimode Microplate Reader (GM3500), the plate was allowed to stand at room temperature for 10 minutes.

[0108] Pharmacokinetics

[0109] The pharmacokinetics of h7C3-3 and h7C3-4 were evaluated in B6 mice. A test material (based on the antibody component) at 10 mg / kg was administered to B6 mice (n = 6) via tail vein injection. Blood samples were collected from each mouse via the saphenous vein at 1, 2, 6 hours and on days 1, 2, 3, 4, 7, 9, 11, 14, 16, 18, 21, 23, 25, 35, and 42 days after injection, and serum samples were collected. The plasma concentrations of h7C3-3 and h7C3-4 were measured via an IgG (total) human ELISA kit (Invitrogen, BMS2091).

[0110] Mouse Xenograft Tumor Model and Antibody-Drug Conjugate (ADC) Therapy

[0111] Seven-week-old immunodeficient male NU / NU nude mice (purchased from BioLASCO for H1299 and PC-3 xenograft studies) and male Fox Chase mice (for HA22T-Luc xenograft studies) were housed in sterile cages equipped with filters and sterile bedding materials at the Laboratory Animal Center of the Taiwan Institute of Health, an AAALAC International-accredited institution. Throughout the study, all mice were maintained on a 12-hour light / 12-hour dark cycle with sterile water and ad libitum food. On the day of tumor cell inoculation, viable cell counts were determined using a hemocytometer stained with trypan blue under an optical microscope. The cells were suspended at a 1:1 ratio in phenol red-free medium [RPMI1640 (H1299) or DMEM (HA22T-Luc)] or PBS (PC-3) and Matrigel TM (356237, Matrix, Corning, MA, United States). H1299 (1 × 10 6 cells), HA22T-Luc (5 × 10 6 cells), and PC-3 (1 × 10 6 cells) were subcutaneously implanted into the left flank of nude or SCID mice. Tumor size was measured using a digital caliper, and tumor volume (mm 3 ) was calculated according to the formula: volume = (length × width^ 2 ) / 2. Tumor-bearing mice were grouped (n = 5 - 8 per group), and when the average tumor volume was approximately 200 mm 3Administration was carried out at the specified time. In three separate xenograft studies, human IgG (HU-GF-ED, Lot#120916DG, Molecular Innovations, MI, USA), h7C3-4, h7C3-3-MMAE, and h7C3-4-MMAE were diluted in 1X Dulbecco's Phosphate Buffered Saline (02-023-5A, Biological Industries, CT, USA) just before administration at a concentration of 2.5 mg / ml. Commercially available cisplatin (KEMOPLAT, 1 mg / mL, 87200009AA, Fresenius Kabi, Bad Homburg, Germany) was used. In the H1299 xenograft study, tumor-bearing mice were divided into 5 groups (6 mice per group): human IgG-10 mg / Kg group (negative control), cisplatin-7 mg / Kg group (positive control), h7C3-3-MMAE-10 mg / Kg group, h7C3-4 -10 mg / Kg group, and h7C3-4-MMAE-10 mg / Kg group. Except that the cisplatin group was administered once a week for three weeks, the mice were injected via the tail vein twice a week for three weeks at a dose volume of 4 mL / Kg. In the HA22T-Luc xenograft study, tumor-bearing mice were divided into 3 groups (8 mice per group): human IgG-10 mg / Kg group (negative control), h7C3-3-MMAE-10 mg / Kg group, and h7C3 -4-MMAE (ADC)-10 mg / Kg group. The mice were injected intravenously twice a week via the tail vein at a dose volume of 4 mL / Kg for 2 weeks. In the PC-3 xenograft study, tumor-bearing mice were divided into 2 groups: untreated group (5 mice) and h7C3-4-MMAE (ADC)-10 mg / Kg (8 mice). The mice in the h7C3-4-MMAE-10 mg / Kg group were injected intravenously twice a week via the tail vein at a dose volume of 4 mL / Kg for 3 weeks. Each treatment was based on body weight. Body weight and tumor size were measured twice a week.

[0112] Immunohistochemical staining

[0113] The dewaxed 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 the primary antibody including 2 μg / ml anti-NTSR1-B12 (SC-376958, Santa Cruz Biotechnology) and different anti-NTSR1 antibody strains (7C3, h7C3-2, and h7C3-3) for 1 hour. Then the slides were incubated with the appropriate secondary antibody; OmniMap anti-mouse HRP was used for anti-NTSR1-B12, and anti-human HRP was used for 7C3, h7C3-2, and h7C3-3, and treated at room temperature for 30 minutes. Staining was performed with diaminobenzidine tetrahydrochloride.

[0114] Example 2: Characterization of 7C3 Antibody

[0115] Antibodies against human NTSR1 were generated by peptide immunization and screening of a phage display gene library. Use of the extracellular loop 2 (ECL2) peptide of human NTSR1 led to the isolation of 7C3 scFv. The unique 7C3 scFv linker was converted to full-length IgG, and the binding affinity to the loop ECL2 peptide was tested. See Figure 1 A. Binding of 7C3 IgG to cell surface NTSR1 was measured on lung cancer cell line A549 cells by flow cytometry. The 7C3 antibody showed strong binding to A549 cells. See Figure 1 B.

[0116] Example 3: Affinity Maturation of Anti-NTSR1 Antibodies

[0117] To increase the affinity of the 7C3 antibody (having SEQ ID NO: 1 and 2), variants were tested using a computer model. The basis of this model was a large library of discrete antibodies and antigens with three-dimensional structures that exhibited properties similar to real populations. Three variants were created, including h7C3-1 (carrying the Y100H mutation in CDR H3), h7C3-2 (carrying the Y100H mutation in CDR H3 and the S97A mutation in CDR L3), and h7C3-3 (carrying the T28A mutation in CDR H1, the Y100H mutation in CDR H3, and the S97A mutation in CDR L3). To study the activity of the variants in affinity enhancement, FACS analysis was performed on these antibodies. Among these variants, h7C3-3 showed a lower KD value of 0.3 nM ( Figure 2) and an EC50 value of 1.1 μg / ml (Table 1), indicating that these amino acid substitutions enhance the affinity. Table 2 shows the CDR sequences of h7C3-3.

[0118] Table 1. Affinities of various anti-NTSR1 antibodies after affinity maturation

[0119]

[0120] a Dose-dependent binding ability of anti-NTSR1 antibodies (1 μg / ml and 10 μg / ml) to A549 cells.

[0121] b Effective concentrations (EC50) of anti-NTSR1 antibodies on A549 cells relative to mean fluorescence intensity (MFI).

[0122] Table 2. CDR sequences of h7C3-3 (Kabat definition)

[0123]

[0124] Example 4: Alanine scanning of the CDR regions of anti-NTSR1 antibodies To identify permissive sites in CDR H3 for antigen binding, variants were tested using experimental alanine scanning mutagenesis analysis at 8 sites in CDR H3 of h7C3. The ability of these variants to bind to the cyclic ECL2 peptide was compared 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 had no effect on the affinity of h7C3, including D104A and Y105A. Based on the results of computer modeling, the effect of replacing Y100 with H on the binding activity of h7C3 was investigated. h7C3 with the Y100H mutation exhibited higher affinity.

[0125] The results showed that compared with parental h7C3, the h7C3-1 antibody (h7C3 containing the Y100H mutation in CDR H3) exhibited higher affinity. Variants at 9 positions in CDR L3 of h7C3-1 were tested by alanine scanning mutagenesis. Several residues significantly reduced the affinity of h7C3-1, especially F94A and W101A (30% to 40% reduction), and also L99A (70% reduction). Substituting A for G91 greatly increased the affinity of h7C3-1 for the peptide. Other substitutions had no effect on the affinity of h7C3-1, including G96A, S97A, H98A, and P100A. To further confirm the binding of the variants to cell surface NTSR1, FACS analysis was performed on h7C3-1 with G96A, S97A, H98A, or P100A. Compared with h7C3-1, the G96A or P100A mutations significantly reduced the binding affinity of h7C3-1 to cell surface NTSR1, while the H98A slightly affected its binding to NTSR1. Surprisingly, h7C3-1 carrying the S97A mutation showed higher affinity.

[0126] To identify the key residues 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 (about 50% or more). 19 out of 28 mutants retained >80% of the binding activity of h7C3-2, including 5 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). h7C3-2 variants with the T28A or N54A mutation showed higher binding affinity for the peptide and were subjected to FACS analysis. Interestingly, h7C3-2 carrying the T28A mutation in CDR H1 showed higher affinity for NTSR1 on A549 cells.

[0127] Example 5: h7C3-3 internalization To examine whether h7C3-3 is internalized after binding to the cell surface and thus becomes a candidate for representing a cytotoxic drug complex, A549, H1299, HA22T, or Mahlavu cells were exposed to the antibody (10 μg / ml) and analyzed by FACS. As Figure 3As shown in A, as the culture time extended, the magnitude of the mean fluorescence intensity (MFI) gradually decreased when the cells were transferred from 4 °C to 37 °C, which represented an increase in the number of h7C3-3 antibodies internalized by A549 cells. In H1299 cells ( Figure 3 B), HA22T cells ( Figure 3 C), and Mahlavu cells ( Figure 3 D), similar results were observed. These results indicated that h7C3-3 was rapidly and efficiently internalized in cells expressing NTSR1.

[0128] Example 6: Characterization of h7C3-4 and h7C3-5 To improve the pharmacokinetic properties of h7C3-3, h7C3-4 (bearing S9T and D82E mutations in the V H region and K79T mutation in the V L region compared to h7C3-3) was created using a computer model. As Figure 4 shown in A, h7C3-4, which had a lower isoelectric point (pI) value compared to h7C3-3, exhibited similar binding affinity. In addition, compared to h7C3-3, h7C3-4 significantly improved the clearance after subcutaneous injection. See Figure 4 B. These results indicated that h7C3-4 had better pharmacokinetics.

[0129] Another variant, h7C3-5, was also created. See Figure 5 , A and B. h7C3-5 exhibited similar binding affinity to NTSR1 on A549 cells compared to h7C3-4. See Figure 5 C.

[0130] Example 7: Characterization of the anti-NTSR1 antibody complex MMAE After achieving high-level production of h7C3-3 and h7C3- in Expi293 cells, the conjugation of the anti-NTSR1 antibody with the anti-mitotic microtubule inhibitor monomethyl auristatin (MMAE) was explored via a cleavable chemical linker. The ADCs were prepared by partially reducing the intermolecular disulfide bonds and then conjugating with a thiol-reactive, maleimide-containing drug linker. The identity of these two ADCs was confirmed by HIC. HIC analysis allowed the disconnection to be split into five main peaks corresponding to 0, 2, 4, 6, or 8 drug molecules per anti-NTSR1 antibody ( Figure 6 , A and B), with an average DAR of approximately 3.8 to 4.

[0131] Example 8: Internalization efficiency of the anti-NTSR1 antibody-drug complex

[0132] The anti-NTSR1 ADC consists of a peptidase-cleavable maleimidocaproyl-valine citruline-p-aminobenzyloxycarbonyl (vc) linker, which is introduced into target cells and subsequently released by lysosomal proteases, which is important for cytotoxic drug delivery. The internalization of h7C3-3-MMAE ADC was measured on the NTSR1-expressing NSCLC cell line A549 and compared with human IgG Ab (negative control) and parental unbound Ab (positive control). Both h7C3-3-MMAE and unbound parental Ab (h7C3-3) have binding efficiency on the cell surface, and the cell distribution is on the right side ( Figure 7 A). To initiate the internalization of h7C3-3-MMAE or h7C3-3, the primary antibody-treated cells were suspended in 100 μL of FACS buffer and incubated at 37 °C for 90 minutes, followed by incubation with the secondary detection antibody. When the primary antibody is internalized into the cells at 37 °C, the cell distribution shifts to the left. Both h7C3-3-MMAE and unbound parental antibody (h7C3-3) are effectively internalized. See Figure 7 , B and C.

[0133] Example 9: Cytotoxic effect of anti-NTSR1 antibody-drug conjugate

[0134] NTSR1-specific ADCs were generated by conjugating h7C3-3, h7C3-4, and h7C3-5 with the dolastatin analogue MMAE. Auristatin is a potent cytotoxic agent that causes cell death by disrupting microtubules. h7C3-3-MMAE, h7C3-4-MMAE, and h7C3-5-MMAE contain a protease-sensitive valine-citrulline dipeptide sequence designed to achieve optimal stability in human plasma and efficient cleavage by human cathepsin B. After internalization, lysosomal proteases metabolize both the antibody and the linker to release the active drug. The NTSR1-specific ADCs are conjugated with an average of 3.8 - 4 MMAEs per antibody, and this ratio provides the best therapeutic index for brentuximab vedotin, polatuzumab vedotin, and enfortumab vedotin.

[0135] Table 3. Cytotoxic effects of h7C3-3 and h7C3-3-MMAE on various NTSR1-expressing cancer cell lines

[0136]

[0137] The direct comparison of h7C3-3 and h7C3-3-MMAE in vitro confirmed that the ADC retained the characteristics of binding to and internalizing the target. See Table 3 and Figure 8 . The NTSR1-specific ADC showed excellent in vitro cytotoxicity.

[0138] The comparison of h7C3-4 and h7C3-4-MMAE in the in vitro cytotoxicity assay also showed that the ADC exhibited binding to, internalizing, and cytotoxicity against the target. See Table 4 and Figure 9 .

[0139] h7C3-5 and h7C3-5-MMAE were compared in the in vitro cytotoxicity assay. The results showed that the ADC also exhibited excellent cytotoxicity. See Table 5 and Figure 10 .

[0140] As shown in Table 6, h7C3-4-MMAE and h7C3-5-MMAE exhibited comparable in vitro cytotoxic effects in the tested cell lines.

[0141] Table 4. Cytotoxic effects of h7C3-4 and h7C3-4-MMAE on various cancer cell lines expressing NTSR1

[0142]

[0143]

[0144] Table 5. Cytotoxic effects of h7C3-5 and h7C3-5-MMAE on various cancer cell lines expressing NTSR1

[0145]

[0146] Table 6. Cytotoxic effects of h7C3-4-MMAE and h7C3-5-MMAE on various cancer cell lines expressing NTSR1

[0147]

[0148] Example 10: In vivo antitumor activity The in vivo effects of anti-NTSR1 ADC on target cells were evaluated in several NTSR1-positive xenograft models.

[0149] Nude mice were subcutaneously inoculated with NTSR1-expressing cells H1299 (1×10 6 cells per mouse). When the tumor volume reached 200 mm 3At that time, the tumor-bearing mice were grouped (n = 6) and intravenously injected with human IgG (negative control), h7C3-3-MMAE, h7C3-4, and h7C3-4-MMAE, respectively, twice a week, 10 mg / Kg each time, for a total of six doses. Cisplatin (positive control) was intravenously administered at a dose of 7 mg / Kg per week for a total of three doses. As Figure 11 shown in A, on the 18th day, 10 mg / kg of h7C3-4-MMAE more effectively inhibited tumor growth than the control human IgG (10 mg / kg) group and the cisplatin (7 mg / kg) group. Compared with cisplatin, until the 35th day, an extended regression of the tumor was observed in the h7C3-4-MMAE treatment. Cisplatin is a commonly used positive control in the clinical treatment of non-small cell lung cancer. In addition, on the 35th day, 10 mg / kg of h7C3-3-MMAE showed partial inhibition of tumor growth, indicating that h7C3-4 was more effectively internalized than h7C3-3 in the lung xenograft model.

[0150] To compare the anti-tumor efficacy of h7C3-3-MMAE and h7C3-4 MMAE, 5×10 6 HA22T-luc hepatoma cells were subcutaneously inoculated into SCID mice for experiments. The tumor-bearing mice were divided into 3 groups (n = 8). When the tumor volume reached 200 mm 3 3, 10 mg / Kg of human IgG, h7C3-3-MMAE, and h7C3-4 MMAE were intravenously administered, respectively, twice a week for a total of four doses. On the 15th day, both h7C3-3-MMAE and h7C3-4 MMAE showed tumor regression ( Figure 12 A), and an extended regression of the tumor was observed until the 36th day in both ADC treatments.

[0151] For the PC3 prostate cancer cell line (1×10 6 6 cells) xenograft model, h7C3-4-MMAE treatment was used for additional experiments ( Figure 13 A). When the tumor volume reached 200 mm 3 3, the tumor-bearing mice were divided into an untreated group (n = 5) and an h7C3-4-MMAE (n = 8) group. 10 mg / kg of h7C3-4-MMAE was intravenously administered twice a week for a total of six doses. h7C3-4-MMAE was also proven to be very effective in the PC3 prostate cancer model. An extended regression of the tumor was observed until the 60th day in the h7C3-4-MMAE treatment. The tumor regression after drug administration did not cause any abnormalities in the general physiology or body weight of the mice ( Figure 8 B, 9B, 10B), indicating that h7C3-3-MMAE or h7C3-4-MMAE is an effective drug for treating NTSR1-positive malignancies.

[0152] Example 11: Immunohistochemical Staining

[0153] To evaluate the NTSR1 expression on prostate cancer cells, PC-3 xenograft tumor tissues were subjected to NTSR1 immunohistochemical staining with 2 μg / ml B-12 (anti-NTSR1-B12; SC-376958, Santa Cruz Biotechnology), 7C3, h7C3-2, or h7C3-3 antibodies. See Figure 14 . Immunohistochemical staining showed strong NTR1 expression on PC-3 xenograft tumor tissues. Among these antibodies, the staining of h7C3-3 was in clusters and had higher intensity, indicating that h7C3-3 was suitable for immunohistochemical staining.

[0154] Other Embodiments

[0155] 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. From the above description, those skilled in the art can readily ascertain the essential characteristics of the described embodiments, and without departing from their spirit and scope, can make various changes and modifications to adapt the embodiments to various uses and conditions. Accordingly, other embodiments are within the scope of the claims.

Claims

1. An isolated antibody, comprising: A heavy chain variable region (V H ), which is at least 75% identical to the amino acids sequence of SEQ ID NO: 1; and a light chain variable region (V L ), which is at least 75% identical to the sequence of SEQ ID NO: 2; wherein the antibody specifically binds to human neurotensin receptor 1 (hNTSR1), and the antibody comprises: amino acids sequence; 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).

2. The isolated antibody according to claim 1, wherein 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).

3. The isolated antibody according to claim 2, wherein 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); 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).

4. The isolated antibody of claim 3, wherein the antibody comprises a V H sequence which is the sequence of SEQ ID NO: 13; and a V L sequence which is the sequence of SEQ ID NO:

14.

5. The isolated antibody according to claim 3, wherein the antibody comprises a V H sequence that is the sequence of SEQ ID NO: 15; and a V L sequence that is the sequence of SEQ ID NO:

16.

6. The isolated antibody according to claim 1, wherein 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).

7. The isolated antibody according to claim 1, wherein 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).

8. The isolated antibody according to claim 1, wherein 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).

9. The isolated antibody according to any one of claims 1 to 8, wherein 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.

10. The isolated antibody according to any one of claims 1 to 8, wherein the antibody binds to the second extracellular loop of hNTSR1.

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

12. The antibody complex according to claim 11, wherein the non-antibody molecule is a polypeptide, a polymer, an oligosaccharide, a lipid, a small molecule drug, a nucleic acid molecule, a carrier protein or a detectable label.

13. The antibody complex according to claim 12, wherein the complex is an antibody-drug complex.

14. The antibody complex according to claim 13, wherein the non-antibody molecule is an anti-cancer drug for treating tumors expressing hNTSR1.

15. The antibody complex according to 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. The antibody complex according to claim 15, wherein the anti-cancer drug is monomethyl auristatin E (MMAE).

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

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

19. Use of a pharmaceutical composition according to claim 18 for the preparation of a medicament for treating a tumor, wherein the tumor expresses hNTSR1.

20. The use according to 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.

21. A method for detecting hNTSR1 for non-diagnostic purposes, comprising: Contacting a sample, tissue or cell with the antibody according to any one of claims 1 to 10 or with the antibody complex of claim 11 or 12; And determining the binding of the antibody or the antibody complex to a target in the sample or to the tissue or the cell.

22. The method according to claim 21, wherein the antibody complex comprises a detectable label.

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