Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/286—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against neuromediator receptors, e.g. serotonin receptor, dopamine receptor
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
  • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
  • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
  • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/77—Internalization into the cell
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
  • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

• the present disclosure relates to novel humanized antibodies, particularly to humanized antibodies which bind specifically to human neurotensin receptor 1 (NTSR1).
• the present disclosure also relates to uses of such antibodies for suppressing tumor growth and metastasis.
• the ligand of NTSR1 is widely studied.
• the NTSR1 comprises 424 amino acids and has a high affinity for NTS.
• the NTSR1 has been identified in the brain and in various cancer cells.
• the signaling pathways induced by the NTS/NTSR1 complex have been studied in different cellular types, such as N1E-115, HT-29, and NTSR1-transfected CHO overexpressing NTSR1.
• NTS/NTSR1 complex leads to phospholipase C (PLC) activation with subsequent production of inositol triphosphate (IP3) and diacylglycerol (DAG) from membrane phospholipids.
• PLC phospholipase C
• IP3 inositol triphosphate
• DAG diacylglycerol
• IP3 inositol triphosphate
• PIP2 diacylglycerol
• NTSR1 neurotensinergic receptor 1
• NTS/NTSR1 complex was demonstrated which could enhance cancer progression in aggressive malignant solid tumors such as mesothelioma, non-small-cell lung, liver, breast, and head and neck squamous carcinomas.
• NTSR1 is a promising molecular marker for non-small-cell lung and prostate cancer based on patient tissue staining.
• NTSR1 is a potential target for cancer therapy.
• the carcinogenesis and cancer recurrence may be reduced via inhibiting the activity of NTSR1.
• Embodiments of the disclosure relate to humanized anti- NTSR1 antibodies that specifically bind human NTSR1, as well as methods of using such antibodies in treatment of cancers.
• antibodies as disclosed herein are able to be used to treat cancers that overexpress NTSR1, including various epithelial cancers.
• the present disclosure provides a humanized anti-NTSR1 antibody or an antigen-binding fragment thereof, including: a heavy chain variable region (V H ) comprising the sequence of SEQ ID NO: 16, 11 or 13, or a sequence with at least about 90% identity to the sequence of SEQ ID NO: 16, 11 or 13; and a light chain variable region ( V L ) comprising the sequence of SEQ ID NO: 17, 15, 12 or 14, or a sequence with at least about 90% identity to the sequence of SEQ ID NO: 17, 15, 12 or 14.
• V H heavy chain variable region
• V L light chain variable region
• the V H comprises the sequence of SEQ ID NO: 1
• V L comprises the sequence of SEQ ID NO: 17 or 15.
• the V H comprises the sequence of SEQ
• V L comprises the sequence of SEQ ID NO: 12 or 14.
• the V H comprises the sequence of
• SEQ ID NO: 11 and the V L comprises the sequence of SEQ ID NO: 12 or 14.
• the humanized anti-NTSR1 antibody has a heavy chain constant region selected from the group consisting of IgGI, lgG2 and lgG4 isoforms. and a light chain constant region selected from the group consisting of ⁇ and ⁇ isotypes.
• the humanized anti-NTSR1 antibody or the antigen-binding fragment thereof is an Fab fragment, an F(ab’) 2 fragment, an ScFv fragment. a chimeric antibody, or a nanobody.
• the humanized anti-NTSR1 antibody or the antigen-binding fragment thereof is multi-specific.
• the present disclosure also provides an antibody conjugate, including: the humanized anti-NTSR1 antibody or the antigen-binding fragment thereof as disclosed herein; and a therapeutic agent conjugated with the humanized anti-NTSR1 antibody or the antigen- binding fragment thereof.
• the therapeutic agent examples include, but are not limited to antimetabolites. alkylating agents, alkylating-like agents, DNA minor groove alkylating agents, anthracyclines. antibiotics, calicheamicins, antimitotic agents, topoisomerase inhibitors, HDAC inhibitor, proteasome inhibitors, and radioisotopes.
• the therapeutic agent is mertansine (DM1), monomethyl auristin E (MMAE), seco-DUBA, ex act ecan. deruxtecan or monomethyl auristatin F (MMAF).
• the present disclosure further provides a vector encoding the humanized anti-NTSR1 antibody or the antigen-binding fragment thereof as disclosed herein.
• the present disclosure provides a genetically engineered cell containing the vector as disclosed herein, or expressing the aforementioned humanized anti-NTSR1 antibody or the antigen-binding fragment thereof.
• An Example of the genetically engineered cell includes, but is not limited to an immune cell.
• the genetically engineered cell is a T cell.
• NTSR1 antibody or the antigen-binding fragment thereof as disclosed herein, including: (a) introducing into a host cell one or more polynucleotides encoding the humanized anti-NTSR1 antibody or the antigen-binding fragment thereof; (b) culturing the host cell under conditions favorable to expression of the one or more polynucleotides; and (c) optionally, isolating the humanized anti-NTSR1 antibody or the antigen-binding fragment thereof from the host cell and/or a medium in which the host cell is grown.
• the present disclosure further provides a pharmaceutical composition, including: an effective amount of the humanized anti-NTSR1 antibody or the antigen-binding fragment thereof, the antibody conjugate, or the genetically engineered cell as disclosed herein; and a pharmaceutically acceptable carrier.
• the pharmaceutical composition is provided for treating, prophylactic treating and/or preventing a disease and/or disorder caused by or related to NTSR1 activity and/or signaling in a subject in need of such treatment.
• the disease is a cancer. Examples of the cancer include, but are not limited to a head and neck cancer, lung cancer, liver cancer, gastric cancer, pancreatic cancer, breast cancer, prostate cancer or colorectal cancer.
• the present disclosure also provides a method for detecting NTSR1 in a sample, including contacting the sample with the humanized anti-NTSR1 antibody or the antigen-binding fragment thereof as disclosed herein.
• the present disclosure further provides a kit for detecting NTSR1 in a sample, including the humanized anti-NTSR1 antibody or the antigen-binding fragment thereof as disclosed herein.
• FIG. 1A shows the sequence alignment between mouse V L (m7c3) and human template
• FIG. IB shows the sequence alignment between mouse V H (m7c3) and human template
• FIG. 2A shows the sequence alignment between mouse V L (m7c3) and human template
• IMGT (VKII) (VKII).
• the first line shows the residue numbers according to the Kabat scheme. The mismatches are shown with underline. The Kabat CDRs are shown in square brackets.
• FIG. 2B shows the sequence alignment between mouse V H (m7c3) and human template
• IMGT Kabat complementarity determining regions
• FIGs. 3A and 3B show expression vectors for generation of mouse-human chimera and humanized editions of anti-NTSR1 monoclonal antibody (mAb) as described in Example 2.
• FIG. 4 depicts results of binding affinity analysis of the mouse-human chimera antibody
• FIG. 5 depicts results of K d binding affinity analysis of the mouse-human chimera antibody
• FIG. 6A, 6B and 6C show the V H and V L sequence alignments between mouse 7C3, AKT2, and the humanized antibodies AKT2 HuHu and AKT2 HuHd with affinity maturation.
• the mutation process of the V H and V, of the humanized antibodies AKT2 HuHu and AKT2 HuHd were performed as described in Example 5.
• FIG. 7 depicts results of binding affinity analysis of the mouse-human chimera antibody
• AKT2 the humanized antibodies AKT2 HuHu and AKT2 HuHd toNTSR1.
• Detailed procedures of chimera antibody expression, purification and K d analysis are described in Example 5. The results show that K d of the humanized antibodies AKT2 HuHu and AKT2 HuHd are not significant different from that of the mouse-human chimera antibody AKT2.
• FIG. 8 shows results of in vivo pharmacokinetic analysis of the mouse-human chimera antibody AKT2, the humanized antibodies AKT2 HuHu and AKT2 HuHd in MSD assay.
• FIG. 9A shows internalization assay results of the mouse-human chimera AKT2, humanized antibodies AKT2 HuHu and AKT2 HuHd and the antibody-drug conjugates (ADCs) thereof in FaDu cells using Flow Cytometry (before normalization).
• FIG. 9B shows a normalized result of FIG. 9A.
• FIG. 10 shows cytotoxicity assay results of the mouse-human chimera antibody AKT2, the humanized antibodies AKT2 HuHu and AKT2 HuHd, and the ADCs thereof in FaDu cells.
• AKT2 the humanized antibodies AKT2 HuHu and AKT2 HuHd in FaDu human head and neck cancer xenografts.
• Standard techniques are used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques are performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (3rd ed..
• antibody means any antigen-binding molecule or molecular complex comprising at least one CDR that specifically binds to or interacts with a particular antigen (e.g., NTSR1).
• the term “antibody” includes immunoglobulin molecules comprising four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, as well as multimers thereof (e.g., IgM).
• Each heavy chain comprises a V H and a heavy chain constant region.
• the heavy chain constant region comprises three domains, C H1 , C H2 and C H3 .
• Each light chain comprises a V L and a light chain constant region.
• the light chain constant region comprises one domain (C L1 ).
• V H and V L can be further subdivided into regions of hypervariability, termed CDRs, interspersed with regions that are more conserved, termed framework regions (FR).
• CDRs regions of hypervariability
• FR framework regions
• Each V H and V L is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3,
• epitope refers to the site on the antigen to which an antibody binds.
• CDR complementarity determining region
• mAb monoclonal antibody
• a monoclonal antibody is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, by any means available or known in the art.
• chimeric antibody refers to an antibody having variable sequences derived from a non-human immunoglobulin and human immunoglobulin constant regions, typically chosen from a human immunoglobulin template.
• Humanized forms of non-human antibodies are chimeric immunoglobulins that contain minimal sequences derived from non-human immunoglobulin.
• a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence.
• the term “nanobody” refers to an antibody comprising the small single variable domain (VHH of antibodies obtained from camelids and dromedaries.
• VHH small single variable domain
• Antibody proteins obtained from members of the camel and dromedary (Camelus baclrianus and Calelus dromaderius) family including new world members such as llama species (Lama paccos. Lama glama and Lama vicugna) have been characterized with respect to size, structural complexity and antigenicity for human subjects.
• Certain IgG antibodies from this family of mammals as found in nature lack light chains, and are thus structurally distinct from the typical four chain quaternary structure having two heavy and two light chains, for antibodies from other animals.
• the term "therapeutic agent” means any compound. substance, drug, drug or active ingredient having a therapeutic or pharmacological effect that is suitable for administration to a mammal, for example a human.
• immune cell refers to cells that play a role in the immune response. Immune cells are of hematopoietic origin, and include lymphocytes, such as B cells and
• T cells T cells; natural killer cells; myeloid cells, such as monocytes, macrophages, eosinophils, mast cells. basophils, and granulocytes.
• myeloid cells such as monocytes, macrophages, eosinophils, mast cells. basophils, and granulocytes.
• T cell includes CD4 + T cells and CD8 + T cells.
• the term T cell also includes T helper 1 type T cells, T helper 2 type T cells, T helper 17 type T cells and inhibitory
• vector is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
• plasmid which refers to a circular double stranded ONA loop into which additional DNA segments may be ligated.
• viral vector Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome.
• Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
• vectors e.g., non-episomal mammalian vectors
• vectors can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
• certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as
• recombinant expression vectors (or simply, “expression vectors”).
• expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
• plasmid and vector may be used interchangeably as the plasmid is the most commonly used form of vector.
• the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.
• genetic engineering means manipulating genes using genetic materials for the change of gene copies and/or gene expression level in the cell.
• the genetic materials can be in the form of DNA or RNA.
• the genetic materials can be transferred into cells by various means including viral transduction and non-viral transfection.
• the expression level of certain genes in the cells can be altered permanently or temporarily.
• the term "pharmaceutical composition” means a mixture containing therapeutics administered to a mammal, for example a human, for preventing, treating, or eliminating a particular disease or pathological condition that the mammal suffers.
• the term "therapeutically effective amount” or “effective amount” refers to the amount of an antibody that, when administered to a mammal or other subject for treating a disease, is sufficient to effect such treatment for the disease.
• treatment covers any treatment of a disease in a mammal, particularly in a human, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing regression of the disease.
• preventing or “prevention” is recognized in the art, and when used in relation to a condition, it includes administering, prior to onset of the condition, an agent to reduce the frequency or severity of or to delay the onset of symptoms of a medical condition in a subject, relative to a subject which does not receive the agent.
• the terms "individual,” “subject,” “host,” and “patient,” refer to a mammal, including, but not limited to, murines (rats, mice), non-human primates, humans, canines, felines, ungulates (e.g., equines, bovines, ovines, porcines, caprines), etc.
• Cancer e.g. physician, nurse, nurse practitioner, or individual in the case of humans; veterinarian in the case of animals, including non-human mammals
• a subject requires or will benefit from treatment. This judgment is made based on a variety of factors that are in the realm of a care giver's expertise, but that includes the knowledge that the subject is ill, or will be ill, as the result of a condition that is treatable by the compounds of the present disclosure.
• "Cancer,” “tumor,” and like terms include precancerous, neoplastic, transformed, and cancerous cells, and can refer to a solid tumor, or a non-solid cancer (see, e.g.. Edge et al. AJCC
• transformation can arise from infection with a transforming virus and incorporation of new genomic DNA, or uptake of exogenous DNA, it can also arise spontaneously or following exposure to a carcinogen.
• Non-limiting examples of an antigen-binding fragment includes: (i) Fab fragments; (ii)
• CDR complementarity determining region
• Other engineered molecules such as domain-specific antibodies, single domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies. minibodies, nanobodies (e.g. monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also encompassed within the expression "antigen-binding fragment," as used
• An antigen-binding fragment of an antibody typically comprises at least one variable domain.
• the variable domain may be of any size or amino acid composition and will generally comprise at least one CDR, which is adjacent to or in frame with one or more framework sequences.
• the V H and V L domains may be situated relative to one another in any suitable arrangement.
• the variable region may be dimeric and contain V H -V H , V H -V L or V L -V L dimers.
• the antigen-binding fragment of an antibody may contain a monomeric V H or V L domain.
• an antigen-binding fragment may be monospecific or multi-specific (e.g., bispecific).
• a multi-specific antigen-binding fragment of an antibody will typically comprise at least two different variable domains, wherein each variable domain is capable of specifically binding to a separate antigen orto a different epitope on the same antigen.
• Any multi-specific antibody format including the exemplary bispecific antibody formats disclosed herein, may be adapted for use in the context of an antigen-binding fragment of an antibody of the present invention using routine techniques available in the art.
• the humanized anti-NTSR1 antibody or the antigen-binding fragment thereof includes a heavy chain variable region including the sequence of SEQ ID NO: 16, 11 or 13, or a sequence with at least about 90%, 91%, 92%, 93%, 94%,
• V L light chain variable region
• sequence identity means that two polynucleotide or amino acid sequences are identical (i.e., on a nucleotide-by-nudeotide or residue-by-residue basis) over the comparison window.
• the humanized anti-NTSR1 antibody or the antigen-binding fragment thereof includes the V H including the sequence of SEQ ID NO: 16; and the V L including the sequence of SEQ ID NO: 17 or 15.
• the humanized anti-NTSR1 antibody or the antigen-binding fragment thereof includes the V H including the sequence of SEQ ID NO: 13, and the V L including the sequence of SEQ ID NO: 12 or 14.
• the humanized anti-NTSR1 antibody or the antigen-binding fragment thereof includes the V H including the sequence of SEQ ID NO: 11, and the V L including the sequence of SEQ ID NO: 12 or 14.
• the humanized anti-NTSR1 antibody disclosed herein may comprise one or more amino acid substitutions, insertions and/or deletions in the framework and/or CDR regions of the heavy and light chain variable domains as compared to the corresponding germline sequences from which the antibodies were derived. Such mutations can be readily ascertained by comparing the amino acid sequences disclosed herein to germline sequences available from, for example, public antibody sequence databases.
• the present disclosure includes an antibody, and an antigen- binding fragment thereof, which are derived from any of the amino acid sequences disclosed herein, wherein one or more amino acids within one or more framework and/or CDR regions are mutated to the corresponding residue(s) of the germline sequence from which the antibody was derived, or to the corresponding residue(s) of another mammalian germline sequence, or to a conservative amino acid substitution of the corresponding germline residue(s) (such sequence changes are referred to herein collectively as "germline mutations").
• Germline mutations A person of ordinary skill in the art, starting with the heavy and light chain variable region sequences disclosed herein, can easily produce numerous antibodies and antigen-binding fragments which comprise one or more individual germline mutations or combinations thereof.
• all of the framework and/or CDR residues within the V H and/or V L domains are mutated back to the residues found in the original germline sequence from which the antibody was derived.
• only certain residues are mutated back to the original germline sequence, e.g.. only the mutated residues found within the first 8 amino acids of FR1 or within the last 8 amino acids of FR4, or only the mutated residues found within CDR1, CDR2 or CDR3.
• one or more of the framework and/or CDR residue(s) are mutated to the corresponding residue(s) of a different germline sequence (i.e..
• the antibodies of the present disclosure may contain any combination of two or more germline mutations within the framework and/or CDR regions, e.g., wherein certain individual residues are mutated to the corresponding residue of a particular germline sequence while certain other residues that differ from the original germline sequence are maintained or are mutated to the corresponding residue of a different germline sequence.
• antibodies and antigen-binding fragments that contain one or more germline mutations can be easily tested for one or more desired property such as, improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties (as the case may be), reduced immunogenicity, etc.
• Antibodies and antigen-binding fragments obtained in this general manner are encompassed within the present disclosure.
• the antibodies of the present disclosure may be monospecific, bi-specific, or multispecific.
• Multispecific antibodies may be specific for different epitopes of one target polypeptide or may contain antigen-binding domains specific for more than one target polypeptide.
• the anti-NTSR1 antibodies of the present disclosure can be linked to or co-expressed with another functional molecule, e.g., another peptide or protein.
• an antibody or fragment thereof can be functionally linked (e.g., by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody or antibody fragment to produce a bi-specific or a multispecific antibody with a second binding specificity.
• the present disclosure includes bi-specific antibodies wherein one arm of an immunoglobulin is specific for NTSR1 or a fragment thereof, and the other arm of the immunoglobulin is specific for a second target or is conjugated to a therapeutic agent.
• the antibody or antigen-binding fragment thereof is in a form of chimeric antigen receptor.
• the humanized anti-NTSR1 antibody or the antigen-binding fragment thereof may be used as antibody-drug conjugates (ADCs), which can specifically target NTSR1. That is, the present disclosure also provides an antibody conjugate, including the aforementioned humanized anti-NTSR1 antibody or the antigen-binding fragment. and therapeutic agent conjugated with the humanized anti-NTSR1 antibody or the antigen- binding fragment thereof.
• the therapeutic agent or payload can be any that are commonly used in ADCs.
• the therapeutic agent or payload is selected from the group consisting of antimetabolites, alkylating agents, alkylating-like agents, DNA minor groove alkylating agents, anthracyclines, antibiotics, calicheamicins, antimitotic agents, topoisomerase inhibitors, HDAC inhibitor, proteasome inhibitors, and radioisotopes.
• the therapeutic agents or payloads may include mertansine (DM1), monomethyl auristin E (MMAE), seco-DUBA, exactecan, deruxtecan or monomethyl auristatin F (MMAF).
• DM1 mertansine
• MMAE monomethyl auristin E
• seco-DUBA exactecan
• deruxtecan monomethyl auristatin F
• the humanized anti-NTSR1 antibody or the antigen-binding fragment thereof may be encoded in a vector.
• the present disclosure also provide a vector encoding the humanized anti-NTSR1 antibody or the antigen-binding fragment thereof.
• one type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated.
• Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome.
• Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non- episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
• vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply, “expression vectors”).
• expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
• plasmid and vector may be used interchangeably as the plasmid is the most commonly used form of vector.
• the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.
• the present disclosure provides a genetically engineered cell expressing the humanized anti-NTSR1 antibody or the antigen-binding fragment thereof as described herein or containing the vector as described herein.
• the genetically engineered cell may be an immune cell, such as a T cell.
• the antibody or antigen-binding fragment thereof is expressed on the surface of a cell.
• the cell is a T-cell.
• the antibody or antigen-binding fragment thereof is in a form of chimeric antigen receptor.
• chimeric antigen receptor or alternatively a “CAR” refers to a recombinant polypeptide construct comprising at least an extracellular antigen binding domain, a transmembrane domain and a cytoplasmic signaling domain (also referred to herein as "an intracellular signaling domain”) comprising a functional signaling domain derived from a stimulatory molecule as defined below.
• the domains in the CAR polypeptide construct are in the same polypeptide chain, e.g., comprise a chimeric fusion protein.
• the domains in the CAR polypeptide construct are not contiguous with each other, e.g., are in different polypeptide chains.
• An example of a method for manufacturing the humanized anti-NTSR1 antibody or the antigen-binding fragment thereof includes: (a) introducing into a host cell one or more polynucleotides encoding said antibody or antigen-binding fragment; (b) culturing the host cell under conditions favorable to expression of the one or more polynucleotides; and (c) optionally. isolating the antibody or the antigen-binding fragment from the host cell and/or a medium in which the host cell is grown.
• compositions including the humanized anti-NTSR1 antibody or antigen-binding fragment thereof, the antibody conjugate or the genetically engineered cell as described herein.
• the pharmaceutical compositions as described herein e are formulated with suitable diluents, carriers, excipients, and other agents that provide improved transfer, delivery, tolerance, and the like.
• the compositions may be formulated for specific uses, such as for veterinary uses or pharmaceutical uses in humans.
• the form of the composition and the excipients, diluents and/or carriers used will depend upon the intended uses of the antibody and, for therapeutic uses, the mode of administration. A multitude of appropriate formulations can be found in the formulary known to all pharmaceutical chemists: Remington's
• compositions include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (such as LIPOFECTIN.TM., Life Technologies, Carlsbad, Calif.), DNA conjugates. anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsions carbowax
• the dose of antibody administered to a patient may vary depending upon the age and the size of the patient, target disease, conditions, route of administration, and the like.
• the preferred dose is typically calculated according to body weight or body surface area.
• an antibody of the present disclosure is used for treating a condition or disease associated with NTSR1 in an adult patient, it may be advantageous to intravenously administer the antibody of the present disclosure.
• the frequency and the duration of the treatment can be adjusted.
• Effective dosages and schedules for administering the antibody may be determined empirically; for example, patient progress can be monitored by periodic assessment, and the dose adjusted accordingly.
• interspecies scaling of dosages can be performed using well-known methods in the art (e.g., Mordenti et al., 1991, Pharmaceut. Res.
• Administration can be systemic or local.
• the pharmaceutical composition of the present disclosure can be delivered subcutaneously or intravenously with a standard needle and syringe.
• a pen delivery device readily has applications in delivering a pharmaceutical composition of the present disclosure.
• Such a pen delivery device can be reusable or disposable.
• a reusable pen delivery device generally utilizes a replaceable cartridge that contains a pharmaceutical composition. Once all of the pharmaceutical composition within the cartridge has been administered and the cartridge is empty, the empty cartridge can readily be discarded and replaced with a new cartridge that contains the pharmaceutical composition. The pen delivery device can then be reused.
• a disposable pen delivery device there is no replaceable cartridge. Rather, the disposable pen delivery device comes prefilled with the pharmaceutical composition held in a reservoir within the device. Once the reservoir is emptied of the pharmaceutical composition, the entire device is discarded.
• the pharmaceutical composition can be delivered in a controlled release system.
• a pump may be used (see Langer, supra; Sefton, 1987, CRC
• polymeric materials can be used; see,
• a controlled release system can be placed in proximity of the composition’s target, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, 1984, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138). Other controlled release systems are discussed in the review by Langer, 1990, Science 249:1527-1533.
• the injectable preparations may include dosage forms for intravenous, subcutaneous. intracutaneous and intramuscular injections, drip infusions, etc. These injectable preparations may be prepared by methods publicly known. For example, the injectable preparations may be prepared, e.g., by dissolving, suspending or emulsifying the antibody or its salt described above in a sterile aqueous medium or an oily medium conventionally used for injections.
• aqueous medium for injections there are, for example, physiological saline, an isotonic solution containing glucose and other auxiliary agents, etc., which may be used in combination with an appropriate solubilizing agent such as an alcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol. polyethylene glycol), a nonionic surfactant [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)], etc.
• an oily medium there are employed, e.g.. sesame oil, soybean oil, etc., which may be used in combination with a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc.
• the injection thus prepared is preferably filled in an appropriate ampoule.
• compositions for oral or parenteral use described above are prepared into dosage forms in a unit dose suited to fit a dose of the active ingredients.
• Such dosage forms in a unit dose include, for example, tablets, pills, capsules, injections
• the pharmaceutical composition is for use in treating, prophylactic treating and/or preventing a disease and/or disorder caused by or related to NTSR1 activity and/or signaling.
• the present disclosure also provides a method for treating, prophylactic treating and/or preventing a disease and/or disorder caused by or related to NTSR1 activity and/or signaling in a subject in need of such treatment, comprising administering to the subject the pharmaceutical composition.
• the disease is a cancer, such as head and neck cancer, lung cancer, liver cancer, gastric cancer, pancreatic cancer, breast cancer, prostate cancer or colorectal cancer.
• the present disclosure further provides a method for detecting NTSR1 in a sample, which includes contacting a sample with the anti-NTSR1 antibody or the antigen-binding fragment thereof as described herein.
• the present disclosure also provides a kit for detecting NTSR1 in a sample, including the humanized anti-NTSR1 antibody or the antigen-binding fragment thereof.
• the humanized anti-NTSR1 antibody or the antigen-binding fragment thereof as described herein may also be used to detect and/or measure NTSR1, or NTSR1-expressing cells in a sample, e.g., for diagnostic purposes.
• an anti-NTSR1 antibody. or the antigen binding fragment thereof may be used to diagnose a condition or disease characterized by aberrant expression (e.g., over-expression, under-expression, lack of expression, etc.) of NT5R1.
• Exemplary diagnostic assays for NTSR1 may comprise, e.g., contacting a sample, obtained from a patient, with an anti-NTSR1 antibody of the disclosure, wherein the anti-NTSR1 antibody is labeled with a detectable label or reporter molecule.
• an unlabeled anti-NTSR1 antibody can be used in diagnostic applications in combination with a secondary antibody which is itself detectably labeled.
• the detectable label or reporter molecule can be a radioisotope, such as 3 H,
• NTSR1 fluorescent or chemiluminescent moiety such as fluorescein isothiocyanate. or rhodamine
• an enzyme such as alkaline phosphatase, beta-galactosidase, horseradish peroxidase, or luciferase.
• Specific exemplary assays that can be used to detect or measure NTSR1 in a sample include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence-activated cell sorting (FACS).
• ELISA enzyme-linked immunosorbent assay
• RIA radioimmunoassay
• FACS fluorescence-activated cell sorting
• Mouse mAb 7C3 may induce potent immunogenicity and anti-drug antibody in patients.
• mouse monoclonal antibody 7C3 (also referred as m7C3) comprising the V H and V L of SEQ ID NOs:7 and 8 was used as the parent antibody, and the 7C3 mAb CDR sequences as shown in SEQ ID NOs:
• FIGs. 1A, IB, 2A and 2B 1 to 6 based on the Kabat definitions are shown in FIGs. 1A, IB, 2A and 2B.
• a human acceptor framework 4D5 was selected from a framework that has been validated in the clinical trial study.
• Human heavy and light chain framework sequences in the V H subgroup III (IGHV3, SEQ ID NO: 25) and V L K subgroup were selected from a framework that has been validated in the clinical trial study.
• I (IGKV1, SEQ ID NO: 24) have been validated in the clinic and also been used in many humanized antibodies with success.
• the framework sequences of IGKV1 V L differ from those in the mouse mAb 7C3 by 25 amino acids (the underlined residues), which corresponds to a 30.86% (25/81 total residues in the framework regions) variation.
• the framework sequences of IGKV1 V L differ from those in the mouse mAb 7C3 by 25 amino acids (the underlined residues), which corresponds to a 30.86% (25/81 total residues in the framework regions) variation.
• IGHV3 V H differs from those in the mouse mAb 7C3 by 35 amino acids (the underlined residues). which corresponds to a 42.68% (35/82 total residues in the framework regions) variation.
• the V H and V L of the humanized 7C34D5 (7C3 HdHd) are shown in Table 1, SEQ ID NO: 11 (7C3 HdV H ) and
• SEQ ID NO: 12 (7C3 HdV L ).
• humanized 7C3 IMGT (7C3 HuHu) preparation human germ-line V L and V H sequences with the highest degree of homology with the mouse mAb 7C3 framework regions were identified from the IMGT database (the International Immunogenetics Information System ® ). The homology searches may be performed with BLAST or similar methods. The mouse mAb 7C3 variable region sequences were used as query sequences. These searches identified the human germline gene
• IGHV1 V H , SEQ ID NO: 27
• IGVK2 V L , SEQ ID NO: 26
• the framework sequences of IGVK2 V L differ from those in the mouse mAb 7C3 by 11 amino acids (the underlined residues), which corresponds to a 13.58% (11/81 total residues in the framework regions) variation.
• the framework sequences of IGVK2 V L differ from those in the mouse mAb 7C3 by 11 amino acids (the underlined residues), which corresponds to a 13.58% (11/81 total residues in the framework regions) variation.
• IGHV1 V H differ from those in the mouse mAb 7C3 by 26 amino acids (the underlined residues), which corresponds to a 31.70% (26/82 total residues in the framework regions) variation.
• the V H and V L of the humanized 7C3 IMGT (7C3 HuHu) are shown in Table 1, SEQ ID NO:13 (7C3 HUV H ) and SEQ ID NO:14 (7C3 HuV L )
• Plasmids pTCAEDH 7C3 HUVH IgG1 and pTCAEDL 7C3 HUVL Kappa were introduced into host cells to prepare recombinant antibody-expressing cells.
• the FREESTYLETM 293 cells manufactured by INVITROGEN ® ) were used.
• DNA was diluted into 1.5 ml sterile 150 mM NaCI to a total volume of 1.5 ml. In a separate tube.
• 37.5 pl of PEI (2.0 mg/ml) was diluted in 1.5 ml sterile 150 mM NaCI.
• the DNA and PEI solutions were allowed to sit at room temperature for 5 minutes. The solutions were mixed gently by inverting the tubes and then allowed the tubes to stand at room temperature for around 10-20 minutes.
• DNA-PEI mixture was added into FREESTYLE 293 cells and incubated the transfected cell on an orbital shaker platform rotating at 135-150 rpm at 37oC, 8% CO 2 in an incubator for 4 hours. Then, an equal volume of fresh culture medium was added to a total volume of 30 ml, and the cells were cultured for 5-7 days. Cells are then harvested for antibody purification and quantification.
• Glycine/NaOH buffer 3 M NaCI (pH 9.0), as an absorption buffer, and 0.2 M Glycine/HCI buffer
• ELISA plates were coated with 1-2 ⁇ g/100 ⁇ L per well of NTSR1 linear L2-biotin protein.
• NTSR1 in comparison with mAb 7C3 MM, 7C3 HuHu and 7C3 HuHd in binding ELISA (FIG. 4).
• HUV H generated by grafting CDR sequences from mAb 7C3 MM into IGHV1 framework sequences has good binding signal for NTSR1 whether pairing with 7C3 HuV L or 7C3 HdV L (7C3 HuHu and 7C3 HuHd vs. 7C3 HdHd and 7C3 HdHu in binding ELISA) (FIG. 4), and has relatively good affinity (7C3
• Anti-NTSRl antibodies were used to detect cells that express NTSR1 on the cell surfaces. for example using FACS. NTSR1 expressing cells, FaDu, were harvested and re-suspended in 5%
• the cells (1x10 s ) were incubated with anti-NTSRl antibody (1-10 ⁇ g/ml) or negative control, at 4oC for 1 hr, and then stained with goat anti-human IgG FITC conjugate
• AKT2 The binding affinity of m7C3 to NTSR1 has been improved based on affinity maturation process, and the obtained clone is termed as AKT2.
• AKT2 has two mutations in heavy chain CDRs and one mutation in light chain CDRs, and shows an enhanced binding affinity to NTSR1. These three mutations areT28A (in CDRH1), Y96H (in CDRH3), and S92A (in CDRL3), which are shown as underlined residues in FIG. 6. Residues are numbered according to Kabat nomenclature.
• SPR surface plasmon resonance
• Carboxymethylated dextran biosensor chips (CM5, CYTIVA ® Inc.) were activated with N-ethyl-N 1 -
• NTSR1 protein was diluted with 10 mM sodium acetate. pH 4.0, into 5 ⁇ g/ml before injection at a flow rate of 10 ⁇ L/minute to achieve approximately 1500 response units (RU) of coupled protein followed by the injection of 1 M ethanolamine to block unreacted groups.
• RU response units
• HBS-EP+BIACORETM running buffer provided by the manufacturer (CYTIVA ® Inc.) at 25oC at a flow rate of 30 ⁇ L/min, and binding responses on the
• NTSR1 protein were corrected by subtraction of responses on a blank flow cell. Association rates
• NTSR1 are 4.068x10 5 and 6.558 x10 -4 , respectively, and K D is 1.612 x10 -9 M.
• K D is 1.612 x10 -9 M.
• HuHu (with V H of IMGT edition, AKT2 HuV H ,) binding with NTSR1 are 7.877 x10 5 and 8.084 x10 -4 , respectively, and K D is 1.026 x10 -9 M.
• the k on and k off of AKT2 HuHd (with V H of IMGT edition, AKT2
• HUV H binding with NTSR1 are 4.614 x10 5 and 6.575 x10 -4 , respectively, and K D is 1.425 x10 -9 M.
• This Example used Meso Scale Discovery (MSD) Electrochemiluminescent (ECL) method to conduct the PK analysis of the anti-NTSR1 antibodies aNTSR1 (AKT2), aNTSR1 (AKT2 Hu/Hu) and aNTSR1 (AKT2 Hu/Hd) in BALB/c mice samples.
• the MSD assay can measure both conjugated and unconjugated antibodies.
• the plate is coated with goat anti-human IgG, which can capture all humanized antibodies (conjugated and unconjugated).
• mice were administered at a dose level of 5 mg/kg via the tail vein. Blood samples were then obtained at different time points for determining the concentrations of aNTSR1 (AKT2), aNTSR1 (AKT2 Hu/Hu) and aNTSR1 (AKT2 Hu/Hd) in mice by MESO QuickPlex SQ 120 method.
• PK parameters of aNTSR1 (AKT2), aNTSR1 (AKT2 Hu/Hu) and aNTSR1 (AKT2 Hu/Hd) were analyzed by noncom partmental analysis using PHOENIXTM for WinNonlin Program, version 6.3.
• FaDu cells were trypsinized, and then harvested and resuspended in FAC buffer. Controls: secondary Ab anti-human IgG PE (1:200) was added to the FaDu cells. The cells were incubated at
• FaDu cells were pre-incubated with 10 ⁇ g /ml ADCs of trimannosyl and anti-NTSR1 antibodies aNTSR1 (AKT2), aNTSR1 (AKT2 Hu/Hu) and aNTSR1
• MOLECULAR DEVICES ® Multi Mode Detection Platform
• Compound cytotoxicity was evaluated in comparison to cells treated with 0.05% PBS (ADCs) or 0.05% DMSO (toxic payload).
• IC50 values were calculated by fitting viability data with a four-parameter logistic equation using GRAPHPAD ® prism 5.0 software. The results are shown in Table 7 (FIG. 10).
• Table 7 IC50 values of ADCs.
• the optimization parameters include codon quality distribution (to select the most frequently used codon for the desired expression system) and GC content (to control the GC content within the desirable range).
• aNTSR1 (AKT2 Hu/Hu) and aNTSR1 (AKT2 Hu/Hd) were directly generated by the nucleotide synthesis method, respectively. Then, the optimized DNA segments were respectively sub-cloned into a human HERCEPTINRFC ® antibody expression vector pCHO-NTSR1, and the vectors were introduced into host cells to prepare recombinant antibody-expressing cells. As the host cells for expression, the CHOS cells (LIFE-TECHNOLOGY ® Inc.) were used. The vector was introduced into the host cells by lipofectamine 2000 in accordance with the attached instruction manual
• the antibody expression vector was linearized by restriction enzymes, the gene was introduced into 4x10 6 cells, and the cells were inoculated to a 6-well culture plate.
• the resultant cell pools were grown in the selection medium containing 10 ⁇ g/ ml of puromycin and 100 nM of methotrexate or 20 ⁇ g/ ml of puromycin and 200 nM of methotrexate.
• the other stage of high concentration selection was performed.
• the primary selection pools were further grown in the medium containing 30 ⁇ g/ml of puromycin and 500 nM of methotrexate or in the medium containing 50 ⁇ g/ml of puromycin and 1000 nM of methotrexate.
• the results are shown in Table 8.
• the productivity of aNTSR1 (AKT2), aNTSR1 (AKT2 HuHu), and aNTSR1 (AKT2 HuHd) were 20.49, 53.73, and 66.96 mg per liter after 5 days incubation. respectively.
• the result indicated that the productivity of the chimeric aNTSR1 (AKT2) antibody was significantly poor.
• the productivity of the humanized aNTSR1 (AKT2 Hu/Hu) and aNTSR1 (AKT2 Hu/Hd) antibodies were significantly improved and increased to 2.5-3 times after humanization.
• aNTSR1 (AKT2 Hu/Hu) 4seco-DUBA
• aNTSR1 (AKT2 Hu/Hd) 4seco-DUBA in FaDu human head and neck cancer xenograft model in male NOD SCID mice.
• Formulations respectively comprising test article aNTSR1 (AKT2) 4seco-DUBA, test article aNTSR1 (AKT2 Hu/Hu) 4seco-DUBA, test article aNTSR1 (AKT2 Hu/Hd) 4seco-DUBA, and Hu IgG were formulated by diluting the stock with a 25 mM sodium citrate buffer (pH 6.5). Each of the formulations was administered intravenously (IV) to the mice once weekly for three weeks.
• the FaDu cells were maintained in vitro as a monolayer culture in RPMI-1640 medium supplemented with 10% fetal bovine serum at 37°C in an atmosphere of 5% CO 2 in air.
• the tumor cells were routinely sub-cultured twice weekly by trypsin-EDTA treatment.
• the cells growing in an exponential growth phase were harvested and counted for tumor inoculation.
• mice at age of 6-7 weeks were quarantined for one week. Five mice were housed in each cage. All animals were hosted in the animal facility with a 12-h light/12-h dark cycle at 19-25°C. Animals had free access to rodent pellet foods and water ad libitum.
• FaDu cells were subcutaneously (SC) implanted (4 x 10 6 cells in 1:1 PBS/matrigel mixture at 0.1 ml per mouse) into the right flank of male NOD SCID mice. When the average tumor volume had reached about 200 mm 3 , the mice were randomly divided into 7 groups (N - 6 per group).
• Each of Hu IgG, aNTSR1 (AKT2) 4seco-DUBA (5 mg/kg), aNTSR1 (AKT2 Hu/Hu) 4seco-DUBA (5 mg/kg), and aNTSR1 (AKT2 Hu/Hd) 4seco-DUBA (5 mg/kg) was intravenously administered twice weekly for 3 weeks.
• Tumor Volume (w 2 x
• TGI tumor growth inhibition
• FIG. 10 shows the tumor growth curve in FaDu implanted male NOD SCID mice.
• the TGI values are shown in Table 9.
• the results indicate that aNTSR1 (AKT2) 4seco-DUBA (5 mg/kg).
• aNTSR1 (AKT2 Hu/Hd) 4seco-DUBA (5 mg/kg) significantly reduced FaDu tumor growth from Day 10 to Day 17.

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