Humanized anti-human neurotensin receptor 1 antibodies and their uses

Abstract

The present disclosure relates to a humanized anti-neurotensin receptor 1 (NTSR1) antibody or antigen-binding fragment thereof. The present disclosure also relates to methods for treating, prophylactically treating, and / or preventing diseases and / or disorders caused by or associated with NTSR1 activity and / or signaling, and methods or kits for detecting NTSR1 in a sample.

Description

[Technical Field]

[0001] [Field of the Invention] This disclosure relates to novel humanized antibodies, particularly humanized antibodies that specifically bind to human neurotensin receptor 1 (NTSR1). This disclosure also relates to the use of such antibodies to inhibit tumor growth and metastasis.

[0002] [Background of the invention]

[0002] Ligands for NTSR1 have been extensively studied. NTSR1 contains 424 amino acids and has high affinity for NTS. NTSR1 has been identified in brain and various cancer cells. The signaling pathway induced by the NTS / NTSR1 complex has been studied in various cell types, including N1E-115, HT-29, and NTSR1-transfected CHO cells overexpressing NTSR1. The NTS / NTSR1 complex activates phospholipase C (PLC), subsequently producing inositol triphosphate (IP3) and diacylglycerol (DAG) from membrane phospholipids. PLC activation leads to the production of inositol triphosphate (IP3) and diacylglycerol (DAG) from membrane phospholipids (PIP2). These two second messengers induce the activation of PKC, a key oncogenic effector, and the mobilization of intracellular calcium.

[0003]

[0003] Several signaling pathways potentially involved in cell proliferation, survival, migration, and invasion have been described following NTSR1 stimulation. The signaling mechanisms mediating the effects of the neurotensinergic system involve multiple pathways and are cell-dependent. It has been demonstrated that the NTS / NTSR1 complex can enhance cancer progression in aggressive malignant solid tumors, such as mesothelioma, non-small cell lung cancer, liver cancer, breast cancer, and head and neck squamous cell carcinoma. NTSR1 is a promising molecular marker for non-small cell lung cancer and prostate cancer based on patient tissue staining. The existence of a chronic autoactivation loop between neurotensin and NTSR1 has previously been shown to be one mechanism responsible for the constitutive activation of mitogen-activated protein kinases in the mitogenic signaling pathway, along with sustained target gene activation. Recently, NTSR1 expression levels have been found to be associated with poor prognosis in patients with breast ductal carcinoma, and similar results have been found in head and neck squamous cell carcinoma.

[0004]

[0004] Therefore, NTSR1 is a potential target for cancer therapy. Carcinogenesis and cancer recurrence may be reduced by inhibiting the activity of NTSR1.

[0005] [Summary of the Invention]

[0005] Embodiments of the present disclosure relate to humanized anti-NTSR1 antibodies that specifically bind human NTSR1, as well as methods of using such antibodies in the treatment of cancer. By specifically binding NTSR1, antibodies as disclosed herein can be used to treat cancers that overexpress NTSR1, including various epithelial cancers.

[0006]

[0006] Accordingly, the present disclosure provides a humanized anti-NTSR1 antibody or antigen-binding fragment thereof, including: a heavy chain variable region (V) comprising the sequence of SEQ ID NO: 16, 11, or 13, or a sequence having at least about 90% identity to the sequence of SEQ ID NO: 16, 11, or 13; H ); and a light chain variable region (V) comprising the sequence of SEQ ID NO: 17, 15, 12, or 14, or a sequence having at least about 90% identity to the sequence of SEQ ID NO: 17, 15, 12, or 14; L ).

[0007] In one embodiment of the present disclosure, V H comprises the sequence of SEQ ID NO: 16, and V L comprises the sequence of SEQ ID NO: 17 or 15.

[0008] In another embodiment of the present disclosure, V H comprises the sequence of SEQ ID NO: 13, and V L comprises the sequence of SEQ ID NO: 12 or 14.

[0009] In yet another embodiment of the present disclosure, V H comprises the sequence of SEQ ID NO: 11, and V L comprises the sequence of SEQ ID NO: 12 or 14.

[0010]

[0010] In some embodiments of the present disclosure, the humanized anti-NTSR1 antibody has a heavy chain constant region selected from the group consisting of IgG1, IgG2, and IgG4 isoforms, and a light chain constant region selected from the group consisting of κ and λ isotypes.

[0011]

[0011] In some embodiments of the present disclosure, the humanized anti-NTSR1 antibody or antigen-binding fragment thereof is a Fab fragment, a F(ab')2 fragment, an ScFv fragment, a chimeric antibody, or a nanobody.

[0012]

[0012] In some embodiments of the present disclosure, the humanized anti-NTSR1 antibody or antigen-binding fragment thereof is multispecific.

[0013] The present disclosure also provides an antibody conjugate comprising: a humanized anti-NTSR1 antibody or antigen-binding fragment thereof as disclosed herein; and A therapeutic agent conjugated with the humanized anti-NTSR1 antibody or an antigen-binding fragment thereof.

[0013]

[0014] Examples of therapeutic agents include, but are not limited to, antimetabolites, alkylating agents, alkylating agent-like agents, DNA minor groove alkylating agents, anthracyclines, antibiotics, calicheamicin, mitotic inhibitors, topoisomerase inhibitors, HDAC inhibitors, proteasome inhibitors, and radioisotopes. In some embodiments of the present disclosure, the therapeutic agent is mertansine (DM1), monomethyl auristatin E (MMAE), seco-DUBA, exatecan, deruxtecan, or monomethyl auristatin F (MMAF).

[0014]

[0015] The present disclosure further provides a vector encoding a humanized anti-NTSR1 antibody or antigen-binding fragment thereof as disclosed herein.

[0015]

[0016] The present disclosure provides genetically modified cells that contain a vector as disclosed herein or that express the aforementioned humanized anti-NTSR1 antibody or antigen-binding fragment thereof.

[0016]

[0017] Examples of genetically modified cells include, but are not limited to, immune cells. In some embodiments of the present disclosure, the genetically modified cells are T cells.

[0017]

[0018] The present disclosure also provides methods for producing a humanized anti-NTSR1 antibody or antigen-binding fragment thereof as disclosed herein, comprising the steps of: (a) introducing one or more polynucleotides encoding the humanized anti-NTSR1 antibody or antigen-binding fragment thereof into a host cell; (b) culturing the host cell under conditions suitable for expression of the one or more polynucleotides; and (c) optionally, isolating the humanized anti-NTSR1 antibody or antigen-binding fragment thereof from the host cell and / or the medium in which the host cell is grown.

[0018]

[0019] The present disclosure further provides a pharmaceutical composition comprising: an effective amount of a humanized anti-NTSR1 antibody or antigen-binding fragment thereof, antibody conjugate, or genetically modified cell as disclosed herein, and A pharmaceutically acceptable carrier.

[0019]

[0020] In some embodiments of the present disclosure, pharmaceutical compositions are provided for treating, prophylactically treating, and / or preventing diseases and / or disorders caused by or associated with NTSR1 activity and / or signaling in a subject in need thereof.

[0020]

[0021] In some embodiments of the present disclosure, the disease is cancer, including but not limited to head and neck cancer, lung cancer, liver cancer, gastric cancer, pancreatic cancer, breast cancer, prostate cancer, or colorectal cancer.

[0021]

[0022] The present disclosure also provides a method for detecting NTSR1 in a sample, comprising contacting the sample with a humanized anti-NTSR1 antibody or antigen-binding fragment thereof as disclosed herein.

[0022]

[0023] The present disclosure further provides a kit for detecting NTSR1 in a sample, comprising a humanized anti-NTSR1 antibody or antigen-binding fragment thereof as disclosed herein. [Brief explanation of the drawings]

[0023] [Figure 1A] Figure 1 shows a sequence alignment between mouse VL (m7c3) and human template (4d5) (VKI). Column 1 shows residue numbers according to the Kabat scheme. Mismatches are underlined. Kabat CDRs are indicated by square brackets. [Figure 1B] Figure 1 shows the sequence alignment between mouse VH (m7c3) and human template (4d5) (VHIII). Column 1 shows residue numbers according to the Kabat scheme. Mismatches are underlined. Kabat CDRs are indicated by square brackets. [Figure 2A] Figure 1 shows the sequence alignment between mouse VL (m7c3) and human template (IMGT) (VKII). Column 1 shows residue numbers according to the Kabat scheme. Mismatches are underlined. Kabat CDRs are indicated by square brackets. [Figure 2B] Figure 1 shows the sequence alignment between mouse VH (m7c3) and human template (IMGT) (VHI). Column 1 shows residue numbers according to the Kabat scheme. Mismatches are underlined. Kabat complementarity-determining regions (CDRs) are indicated in square brackets. [Figure 3A] FIG. 1 shows expression vectors for the production of mouse-human chimeric and humanized versions of anti-NTSR1 monoclonal antibodies (mAbs), as described in Example 2. [Figure 3B] FIG. 1 shows expression vectors for the production of mouse-human chimeric and humanized versions of anti-NTSR1 monoclonal antibodies (mAbs), as described in Example 2. [Figure 4] FIG. 1 shows the results of binding affinity analysis of mouse-human chimeric antibody 7C3 MM, humanized antibodies 7C3 HdHd, 7C3 HuHu, 7C3 HdHu, and 7C3 HuHd to NTSR1, as described in Example 3. [Figure 5]

[0023] Figure 1 shows the results of Kd binding affinity analysis of mouse-human chimeric antibody 7C3 MM and humanized antibody 7C3 HuHu to NTSR1. Detailed procedures for chimeric antibody expression, purification, and Kd analysis are described in Example 3. The results show that the Kd of humanized antibody 7C3 HuHu is not significantly different from the Kd of mouse-human chimeric antibody 7C3 MM. [Figure 6A] Figure 1 shows the VH and VL sequence alignment between mouse 7C3, AKT2, and affinity-matured humanized antibodies AKT2 HuHu and AKT2 HuHd. The VH and VL mutation process of humanized antibodies AKT2 HuHu and AKT2 HuHd was performed as described in Example 5. [Figure 6B]Figure 1 shows the VH and VL sequence alignment between mouse 7C3, AKT2, and affinity-matured humanized antibodies AKT2 HuHu and AKT2 HuHd. The VH and VL mutation process of humanized antibodies AKT2 HuHu and AKT2 HuHd was performed as described in Example 5. [Figure 6C] Figure 1 shows the VH and VL sequence alignment between mouse 7C3, AKT2, and affinity-matured humanized antibodies AKT2 HuHu and AKT2 HuHd. The VH and VL mutation process of humanized antibodies AKT2 HuHu and AKT2 HuHd was performed as described in Example 5. [Figure 7]

[0023] Figure 1 shows the results of binding affinity analysis of mouse-human chimeric antibody AKT2, humanized antibodies AKT2 HuHu, and AKT2 HuHd to NTSR1. Detailed procedures for chimeric antibody expression, purification, and Kd analysis are described in Example 5. The results show that the Kd of humanized antibodies AKT2 HuHu and AKT2 HuHd is not significantly different from the Kd of mouse-human chimeric antibody AKT2. [Figure 8] FIG. 1 shows the results of in vivo pharmacokinetic analysis of mouse-human chimeric antibody AKT2, humanized antibodies AKT2 HuHu and AKT2 HuHd in an MSD assay. [Figure 9A] FIG. 1 shows the results of an internalization assay (before normalization) of mouse-human chimeric AKT2, humanized antibodies AKT2 HuHu and AKT2 HuHd, and their antibody-drug conjugates (ADCs) in FaDu cells using flow cytometry. [Figure 9B] FIG. 9B shows the normalization result of FIG. 9A. [Figure 10] FIG. 1 shows the results of cytotoxicity assays of mouse-human chimeric antibody AKT2, humanized antibodies AKT2 HuHu and AKT2 HuHd, and their ADCs in FaDu cells. [Figure 11] Figure 1 shows in vivo efficacy results of ADCs of mouse-human chimeric antibody AKT2, humanized antibodies AKT2 HuHu and AKT2 HuHd in FaDu human head and neck cancer xenografts.

[0024] [Detailed Description of the Invention]

[0037] Unless otherwise specified, scientific and technical terms used herein shall have the meanings commonly understood by those of ordinary skill in the art. Further, unless the context otherwise requires, singular terms shall include the plural and plural terms shall include the singular. Generally, the nomenclature used in connection with, and techniques of, cell and tissue culture, molecular biology, and protein chemistry and oligo- or polynucleotide chemistry and hybridization described herein are those well known and commonly used in the art.

[0025]

[0038] The practice of the present disclosure may employ technologies within the capabilities of those skilled in the art, including conventional techniques of cell biology, cell culture, antibody technology, and genetic engineering, such techniques being fully explained in the literature.

[0026]

[0039] 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 cited and discussed throughout the specification. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual (3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001)), which is incorporated herein by reference. The nomenclature utilized in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

[0027]

[0040] As utilized in accordance with the present disclosure, the following terms shall be understood to have the following meanings, unless otherwise indicated: As used herein, the term "and / or" may be interpreted as a specific disclosure of each of the two specified features or components, either together with or without the other. For example, "A and / or B" may be interpreted as a specific disclosure of (i) A, (ii) B, and (iii) each of A and B, as if each were individually set forth herein.

[0028]

[0041] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0029]

[0042] As used herein, the term "antibody" refers to any antigen-binding molecule or molecular complex that contains at least one CDR that specifically binds to or interacts with a particular antigen (e.g., NTSR1). The term "antibody" includes immunoglobulin molecules that contain four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, as well as multimers thereof (e.g., IgM). Each heavy chain contains a V H and a heavy chain constant region. The heavy chain constant region is comprised of three domains: C H1 , C H2 , and C H3 Each light chain contains V L and a light chain constant region. The light chain constant region comprises one domain (C L1 ) included. V H and V L can be further subdivided into regions of hypervariability called CDRs, interspersed with more highly conserved regions termed framework regions (FRs). 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, FR4. In different embodiments of the present disclosure, the FRs of an anti-NTSR1 antibody (or antigen-binding portion thereof) may be identical to human germline sequences or may be naturally or artificially modified. An amino acid consensus sequence may be defined based on a parallel analysis of two or more CDRs.

[0030]

[0043] As used herein, the terms "specific for" or "specifically binds to" mean that the antibody does not cross-react to a significant extent with other epitopes.

[0031]

[0044] As used herein, the term "epitope" refers to a site on an antigen to which an antibody binds.

[0032]

[0045] As used herein, the term "complementarity-determining region (CDR)" refers to the noncontiguous antigen-binding sites found within the variable regions of both heavy and light chain polypeptides. CDRs are described in Kabat et al., J. Biol. Chem. 252:6609-6616 (1977); Kabat et al., US Dept. of Health and Human Services, "Sequences of proteins of immunological interest" (1991); Chothia et al., J. Mol. Biol. 196:901-917 (1987); and MacCallum et al., J. Mol. Biol. 262:732-745 (1996), where the definitions include overlapping or subsets of amino acid residues when compared with each other.

[0033]

[0046] As used herein, the term "monoclonal antibody (mAb)" is not limited to antibodies produced through hybridoma technology. Monoclonal antibodies may be derived from a single clone by any means available or known in the art, including any eukaryotic, prokaryotic, or phage clone.

[0034]

[0047] As used herein, the term "chimeric" antibody refers to an antibody having variable sequences derived from a non-human immunoglobulin and a human immunoglobulin constant region, typically selected from a human immunoglobulin template.

[0035]

[0048] "Humanized" forms of non-human antibodies are chimeric immunoglobulins that contain minimal sequence derived from non-human immunoglobulin. Generally, 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 the CDRs of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence.

[0036]

[0049] As used herein, the term "nanobody" refers to antibodies containing a small single variable domain (VHH of antibodies from camelids and dromedaries). Antibody proteins from members of the camel and dromedary families (Camelus baclrianus and Calelus dromaderius), including New World members such as llama species (alpacas, Lama glama, and Lama vicugna), have been characterized with respect to size, structural complexity, and antigenicity to human subjects. Certain IgG antibodies from mammals of this family, as found in nature, lack light chains and are therefore structurally distinct from the typical four-chain quaternary structure with two heavy chains and two light chains for antibodies from other animals.

[0037]

[0050] As used herein, the terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, and the like, include any naturally occurring, enzymatically derived, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex.

[0038]

[0051] As used in this disclosure, the term "therapeutic agent" means any compound, substance, drug, drug component, or active ingredient that produces a therapeutic or pharmacological effect and is suitable for administration to a mammal, e.g., a human.

[0039]

[0052] As used herein, the term "immune cell" refers to a cell that plays a role in the immune response. Immune cells are of hematopoietic origin and include lymphocytes such as B cells and T cells; natural killer cells; and myeloid cells such as monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes.

[0040]

[0053] As used herein, the term "T cells" refers to CD4 + T cells and CD8 +Includes T cells. The term T cells includes type 1 helper T cells, type 2 helper T cells, type 17 helper T cells, and suppressor T cells.

[0041]

[0054] As used herein, the term "vector" is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, into which additional DNA segments can 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. Moreover, certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). In general, expression vectors useful in recombinant DNA techniques are often in the form of plasmids. As the plasmid is the most commonly used form of vector, "plasmid" and "vector" can be used interchangeably herein. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (eg, replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

[0042]

[0055] The term "genetically modified" cell or "genetic modification" of a cell refers to the manipulation of genes using genetic material to alter gene copies and / or gene expression levels in the cell. Genetic material can be in the form of DNA or RNA. Genetic material can be transferred to cells by various means, including viral transduction and non-viral transfection. After being genetically modified, the expression levels of certain genes in the cell can be altered permanently or temporarily.

[0043]

[0056] As used in this disclosure, the term "pharmaceutical composition" means a mixture containing therapeutic substances that is administered to a mammal, such as a human, to prevent, treat, or eliminate a particular disease or pathological condition from which the mammal is suffering.

[0044]

[0057] As used herein, the terms "therapeutically effective amount" or "effective amount" refer 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.

[0045]

[0058] As used herein, the terms "treatment," "treating," and the like, encompass any treatment of disease in mammals, particularly humans, and include (a) preventing the disease from occurring in a subject who may be predisposed to the disease but has not yet been diagnosed with the disease; (b) arresting the disease, i.e., suppressing the development of the disease; and (c) relieving the disease, i.e., causing regression of the disease.

[0046]

[0059] The terms "preventing" or "prevention" are art-recognized and when used with respect to a condition include administering an agent prior to the onset of a condition to reduce the frequency or severity of, or delay the onset of, symptoms of a medical condition in a subject compared to a subject not receiving the agent.

[0047]

[0060] As used interchangeably herein, the terms "individual," "subject," "host," and "patient" refer to mammals, including, but not limited to, murines (rats, mice), non-human primates, humans, dogs, cats, ungulates (e.g., horses, cattle, sheep, pigs, goats), and the like.

[0048]

[0061] As used herein, the term "in need of treatment" refers to a judgment made by a caregiver (e.g., in the case of humans, a physician, nurse, nurse practitioner, or individual; in the case of animals, including non-human mammals, a veterinarian) that a subject requires or would benefit from treatment. This judgment is made based on a variety of factors within the caregiver's area of ​​expertise, but includes knowledge that the subject is ill or will be ill as a result of a condition treatable with the compounds of the present disclosure.

[0049]

[0062] "Cancer," "tumor," and similar terms include precancerous cells, neoplastic cells, transformed cells, and cancerous cells and can refer to solid tumors or non-solid cancers (see, e.g., Edge et al., AJCC Cancer Staging Manual (7th ed. 2009); Cibas and Ducatman, Cytology: Diagnostic principles and clinical correlates (3rd ed. 2009)). Cancer includes both benign and malignant neoplasms (abnormal growth). "Transformation" refers to spontaneous or induced phenotypic changes, such as cellular immortalization, morphological changes, abnormal cell proliferation, loss of contact inhibition and anchorage dependence, and / or malignant lesions (see Freshney, Culture of Animal Cells: A Manual of Basic Technique (3rd ed. 1994)). Transformation can result from infection with transforming viruses and the incorporation of new genomic DNA or the uptake of exogenous DNA, but transformation can also occur spontaneously or after exposure to carcinogens.

[0050]

[0063] As used herein, the term "sample" encompasses a variety of sample types obtained from an individual, subject, or patient and can be used in a diagnostic or monitoring assay. The definition includes blood and other liquid samples of biological origin, solid tissue samples such as biopsy specimens or tissue cultures, or cells derived from such tissues and the progeny of such cells.

[0051]

[0064] The present disclosure provides humanized antibodies that are specific to and have high affinity for human NTSR1. The anti-NTSR1 antibodies or antigen-binding fragments thereof can deliver therapeutic benefits to a subject. The anti-NTSR1 antibodies or antigen-binding fragments thereof according to the present disclosure can be used as therapeutic agents for treating and / or diagnosing various disorders mediated by NTSR1, which are more fully described herein.

[0052]

[0065] Antibodies or antigen-binding portions thereof according to embodiments of the present disclosure may be full-length (e.g., having a heavy chain constant region selected from the group consisting of IgG1, IgG2, and IgG4 isoforms and a light chain constant region selected from the group consisting of Kappa and Lambda isotypes) or may comprise only an antigen-binding fragment (e.g., a Fab, F(ab')2, or scFv fragment), and may be modified, as needed, to affect functionality.

[0053]

[0066] Non-limiting examples of antigen-binding fragments include (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of amino acid residues mimicking the hypervariable regions of an antibody (e.g., isolated complementarity-determining regions (CDRs) such as CDR3 peptides), or constrained FR3-CDR3-FR4 peptides. 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 term "antigen-binding fragment" as used herein.

[0054]

[0067] Antigen-binding fragments of antibodies typically contain at least one variable domain, which may be of any size or amino acid composition and generally contains at least one CDR, which is adjacent to or in-frame with one or more framework sequences. L V associated with the domain H In an antigen-binding fragment having a domain, V H and V L The domains can be positioned in any suitable arrangement relative to each other. For example, the variable region can be a dimer, with the V H -V H , V H -V L , or V L -V L Alternatively, the antigen-binding fragment of an antibody may comprise a dimer of monomeric V H or V L It may contain domains.

[0055]

[0068] Like intact antibody molecules, antigen-binding fragments can be monospecific or multispecific (e.g., bispecific). Multispecific antigen-binding fragments of antibodies typically comprise at least two different variable domains, each capable of specifically binding to a separate antigen or to a different epitope on the same antigen. Any multispecific antibody format, including the exemplary bispecific antibody formats disclosed herein, can be constructed for use in the context of the antigen-binding fragments of antibodies of the present invention using routine techniques available in the art.

[0056]

[0069] In some embodiments of the present disclosure, the humanized anti-NTSR1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 16, 11, or 13, or a sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the sequence of SEQ ID NO: 16, 11, or 13; and a light chain variable region (V) comprising the sequence of SEQ ID NO: 17, 15, 12, or 14, or a sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the sequence of SEQ ID NO: 17, 15, 12, or 14. L ) is included.

[0057]

[0070] The term "sequence identity" means that two polynucleotide or amino acid sequences are identical (i.e., on a nucleotide-by-nucleotide or residue-by-residue basis) over a comparison window. The term "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over the comparison window, determining the number of positions where the same nucleic acid base (e.g., A, T, C, G, U, or I) or amino acid residue is present in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window (i.e., the window size), and multiplying the result by 100 to obtain the percentage of sequence identity.

[0058]

[0071] In some embodiments of the present disclosure, the humanized anti-NTSR1 antibody or antigen-binding fragment thereof comprises a V H and V comprising the sequence of SEQ ID NO: 17 or 15 L Includes.

[0059]

[0072] In some embodiments of the present disclosure, the humanized anti-NTSR1 antibody or antigen-binding fragment thereof comprises a V H and V comprising the sequence of SEQ ID NO: 12 or 14 L Includes.

[0060]

[0073] In some embodiments of the present disclosure, the humanized anti-NTSR1 antibody or antigen-binding fragment thereof comprises a V H and V comprising the sequence of SEQ ID NO: 12 or 14 L Includes.

[0061]

[0074] The humanized anti-NTSR1 antibodies disclosed herein may contain 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 compared to the corresponding germline sequences from which the antibody was derived. Such mutations can be readily ascertained by comparing the amino acid sequences disclosed herein to germline sequences available, for example, from public antibody sequence databases. The present disclosure includes antibodies or antigen-binding fragments thereof derived from any of the amino acid sequences disclosed herein, in which one or more amino acids in one or more framework and / or CDR regions have been mutated to the corresponding residue(s) in the germline sequence from which the antibody was derived, or to the corresponding residue(s) in another mammalian germline sequence, or to a conservative amino acid substitution of the corresponding germline residue(s) (such sequence variations are collectively referred to herein as "germline mutations"). Starting with the heavy and light chain variable region sequences disclosed herein, one of skill in the art can readily generate numerous antibodies and antigen-binding fragments containing one or more individual germline mutations or combinations thereof. In certain embodiments, V Hand / or V L All of the framework and / or CDR residues within a domain are mutated back to the residue found in the original germline sequence from which the antibody was derived. In other embodiments, only certain residues are mutated back to the original germline sequence, for example, only mutated residues found within the first 8 amino acids of FR1 or the last 8 amino acids of FR4, or only mutated residues found in CDR1, CDR2, or CDR3. In other embodiments, one or more of the framework and / or CDR residues are mutated to the corresponding residue(s) in a different germline sequence (i.e., a germline sequence that differs from the germline sequence from which the antibody was originally derived). Furthermore, 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., certain individual residues are mutated to the corresponding residue in a particular germline sequence, while certain other residues that differ from the original germline sequence are maintained or mutated to the corresponding residue in a different germline sequence. Once obtained, antibodies or antigen-binding fragments containing one or more germline mutations can be readily tested for one or more desired properties, such as improved binding specificity, increased binding affinity, improved or enhanced antagonist or agonist biological properties (as the case may be), reduced immunogenicity, etc. Antibodies and antigen-binding fragments obtained by this general method are encompassed within the present disclosure.

[0062]

[0075] Antibodies of the present disclosure may be monospecific, bispecific, or multispecific. Multispecific antibodies may be specific for different epitopes of a single target polypeptide or may contain antigen-binding domains specific for more than one target polypeptide. The anti-NTSR1 antibodies of the present disclosure may be linked to or co-expressed with another functional molecule, such as another peptide or protein. For example, an antibody or fragment thereof may be operatively 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 generate a bispecific or multispecific antibody having a second binding specificity. For example, the present disclosure includes bispecific antibodies in which one arm of the 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.

[0063]

[0076] In some embodiments of the present disclosure, the antibody or antigen-binding fragment thereof is in the form of a chimeric antigen receptor.

[0064]

[0077] In some embodiments of the present disclosure, a humanized anti-NTSR1 antibody or antigen-binding fragment thereof can be used as an antibody-drug conjugate (ADC) capable of specifically targeting NTSR1. That is, the present disclosure also provides an antibody conjugate comprising the aforementioned humanized anti-NTSR1 antibody or antigen-binding fragment thereof and a therapeutic agent conjugated to the anti-NTSR1 antibody or antigen-binding fragment thereof. The therapeutic agent or payload can be any agent commonly used in ADCs. In some embodiments, the therapeutic agent or payload is selected from the group consisting of antimetabolites, alkylating agents, alkylating agent-like agents, DNA minor groove alkylating agents, anthracyclines, antibiotics, calicheamicin, mitotic inhibitors, topoisomerase inhibitors, HDAC inhibitors, proteasome inhibitors, and radioisotopes. For example, the therapeutic agent or payload can include mertansine (DM1), monomethyl auristatin E (MMAE), seco-DUBA, exatecan, deruxtecan, or monomethyl auristatin F (MMAF). Methods for conjugation can be methods known in the art.

[0065]

[0078] The humanized anti-NTSR1 antibody or antigen-binding fragment thereof can be encoded within a vector. The present disclosure also provides a vector encoding the humanized anti-NTSR1 antibody or antigen-binding fragment thereof as described herein. In one embodiment, one type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, into which additional DNA segments can be ligated. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors containing 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. Moreover, certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). In general, expression vectors useful in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" can be used interchangeably as the plasmid is the most commonly used form of vector. However, 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.

[0066]

[0079] In another aspect, the present disclosure provides a genetically modified cell that expresses a humanized anti-NTSR1 antibody or antigen-binding fragment thereof as described herein or contains a vector as described herein. The genetically modified cell can be an immune cell, such as a T cell. In one embodiment of the present disclosure, the antibody or antigen-binding fragment thereof is expressed on the surface of the cell. In particular, the cell is a T cell.

[0067]

[0080] In some embodiments of the present disclosure, the antibody or antigen-binding fragment thereof is in the form of a chimeric antigen receptor.

[0068]

[0081] The term "chimeric antigen receptor" or alternatively "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. In some embodiments, the domains in a CAR polypeptide construct are in the same polypeptide chain, including, for example, a chimeric fusion protein. In some embodiments, the domains in a CAR polypeptide construct are not adjacent to each other, for example, are in different polypeptide chains.

[0069]

[0082] An example of a method for producing a humanized anti-NTSR1 antibody or antigen-binding fragment thereof includes the steps of: (a) introducing one or more polynucleotides encoding the antibody or antigen-binding fragment thereof into a host cell; (b) culturing the host cell under conditions suitable for expression of the one or more polynucleotides; and (c) optionally isolating the antibody or antigen-binding fragment thereof from the host cell and / or the medium in which the host cell is grown.

[0070]

[0083] The present disclosure further provides pharmaceutical compositions comprising the humanized anti-NTSR1 antibodies or antigen-binding fragments thereof, antibody conjugates, or genetically modified cells as described herein. Pharmaceutical compositions as described herein are formulated with suitable diluents, carriers, excipients, and other agents that provide improved entry, delivery, tolerance, and the like. The compositions may be formulated for a particular use, such as for veterinary use or human pharmaceutical use. The form of the composition, as well as the excipients, diluents, and / or carriers used, will depend on the intended use of the antibody and, for therapeutic use, the mode of administration. Many suitable formulations can be found in a formulary known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipids (cationic or anionic) containing vehicles (e.g., LIPOFECTIN™, Life Technologies, Carlsbad, Calif.), DNA conjugates, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, emulsions of carbowax (polyethylene glycol of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. See also Powell et al., "Compendium of excipients for parenteral formulations," PDA (1998) J Pharm Sci Technol 52:238-311.

[0071]

[0084] The dose of an antibody administered to a patient may vary depending on the patient's age and size, the target disease, condition, route of administration, and the like. A preferred dose is typically calculated according to body weight or body surface area. When the antibody of the present disclosure is used to treat an NTSR1-related condition or disease in an adult patient, it may be advantageous to administer the antibody of the present disclosure intravenously. Depending on the severity of the condition, the frequency and duration of treatment can be adjusted. Effective dosages and schedules for administering antibodies can be empirically determined; for example, patient progress can be monitored by periodic assessment, and the dose can be adjusted accordingly. Furthermore, interspecies scaling of dosages can be performed using methods well known in the art (e.g., Mordenti et al., 1991, Pharmaceut. Res. 8:1351).

[0072]

[0085] Various delivery systems, such as liposomes, microparticles, encapsulation in microcapsules, recombinant cells capable of expressing mutant viruses, and receptor-mediated endocytosis, are known and can be used to administer the pharmaceutical compositions of the present disclosure (see, e.g., Wu et al., 1987, J. Biol. Chem. 262:4429-4432). Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compositions can be administered by any convenient route, such as infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral, rectal, and intestinal mucosa, etc.), and can be administered together with other biologically active substances. Administration can be systemic or local.

[0073]

[0086] The pharmaceutical compositions of the present disclosure can be delivered subcutaneously or intravenously with a standard needle and syringe. In addition, for subcutaneous delivery, pen delivery devices are easily adapted to deliver the pharmaceutical compositions of the present disclosure. Such pen delivery devices can be reusable or disposable. Reusable pen delivery devices generally utilize a replaceable cartridge containing the pharmaceutical composition. Once all of the pharmaceutical composition in the cartridge has been administered and the cartridge is empty, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen delivery device can then be reused. In disposable pen delivery devices, there is no replaceable cartridge. Rather, the disposable pen delivery device comes pre-filled with the pharmaceutical composition held in a reservoir within the device. Once the reservoir is depleted of pharmaceutical composition, the entire device is discarded.

[0074]

[0087] In certain circumstances, pharmaceutical compositions can be delivered in a controlled-release system. In one embodiment, a pump can be used (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201). In another embodiment, a polymeric material can be used; see Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Press, Boca Raton, Fla. In yet another embodiment, a controlled-release system can be placed near the target of the composition, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, 1984, 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.

[0075]

[0088] Injectable preparations may include dosage forms for intravenous, subcutaneous, intradermal, and intramuscular injections, drip infusions, and the like. These injectable preparations may be prepared by known methods. For example, injectable preparations may be prepared by dissolving, suspending, or emulsifying the above-described antibody or a salt thereof in a sterile aqueous or oily medium commonly used for injections. Aqueous media for injection include, for example, saline, isotonic solutions containing glucose and other auxiliary agents, and the like, which may be used in combination with appropriate solubilizers such as alcohols (e.g., ethanol), polyalcohols (e.g., propylene glycol, polyethylene glycol), and nonionic surfactants [e.g., polysorbate 80, HCO-50 (a polyoxyethylene (50 mol) adduct of hydrogenated castor oil)]. Oily media include, for example, sesame oil and soybean oil, which may be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol. The injectable preparations prepared in this manner are preferably filled into appropriate ampoules.

[0076]

[0089] Preferably, the above-mentioned pharmaceutical compositions for oral or parenteral use are prepared into dosage forms with unit doses suitable for containing a dose of the active ingredient, such as tablets, pills, capsules, injections (ampoules), suppositories, etc.

[0077]

[0090] In some embodiments of the present disclosure, the pharmaceutical composition is used for treating, prophylactically treating, and / or preventing a disease and / or disorder caused by or associated with NTSR1 activity and / or signaling. Alternatively, the present disclosure also provides a method for treating, prophylactically treating, and / or preventing a disease and / or disorder caused by or associated with NTSR1 activity and / or signaling in a subject in need thereof, the method comprising administering a pharmaceutical composition to the subject. In some embodiments, the disease is cancer, such as head and neck cancer, lung cancer, liver cancer, gastric cancer, pancreatic cancer, breast cancer, prostate cancer, or colorectal cancer.

[0078]

[0091] The present disclosure further provides a method for detecting NTSR1 in a sample, comprising contacting the sample with an anti-NTSR1 antibody or antigen-binding fragment thereof as described herein. The present disclosure also provides a kit for detecting NTSR1 in a sample, comprising a humanized anti-NTSR1 antibody or antigen-binding fragment thereof.

[0079]

[0092] The humanized anti-NTSR1 antibodies or antigen-binding fragments thereof described herein can also be used to detect and / or measure NTSR1 or NTSR1-expressing cells in a sample, e.g., for diagnostic purposes. For example, the anti-NTSR1 antibodies or antigen-binding fragments thereof can be used to diagnose conditions or diseases characterized by abnormal expression (e.g., overexpression, underexpression, lack of expression, etc.) of NTSR1. An exemplary diagnostic assay for NTSR1 can include, for example, contacting a sample obtained from a patient with an anti-NTSR1 antibody of the present disclosure, wherein the anti-NTSR1 antibody is labeled with a detectable label or reporter molecule. Alternatively, an unlabeled anti-NTSR1 antibody can be used for diagnostic applications in combination with a secondary antibody that is itself detectably labeled. The detectable label or reporter molecule can be 3 H, 14 C. 32 P, 35 S, or 125 The NTSR1 may be a radioisotope such as I; a fluorescent or chemiluminescent moiety such as fluorescein isothiocyanate or rhodamine; or 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), or radioimmunoassay (RIA), and fluorescence-activated cell sorting (FACS).

[0080]

[0093] The following examples are provided to aid those of ordinary skill in the art in practicing the present disclosure.

[0081]

[0094] [Example]

[0095] The following examples illustrate the development and use of NTSR1-specific antibodies to inhibit tumor growth by inducing an anti-NTSR1 immune response.

[0082]

[0096] Example 1

[0097] Humanization of anti-NTSR1 mAb 7C3

[0098] Selection of human V region framework sequences

[0099] Murine mAb 7C3 is capable of inducing potent immunogenicity and anti-drug antibodies in patients. Therefore, humanization of 7C3 is an essential and important step for further drug development. V of SEQ ID NOs: 7 and 8 H and V L The murine monoclonal antibody 7C3 (also referred to as m7C3), comprising the CDR sequences shown in SEQ ID NOS: 1-6 based on the Kabat definition, was used as the parent antibody, and the 7C3 mAb CDR sequences are shown in Figures 1A, 1B, 2A, and 2B.

[0083]

[0100] [Table 1] TIFF2025529623000003.tif153149

[0084]

[0101] For the preparation of humanized 7C3 4D5 (7C3 HdHd), the human receptor framework 4D5 was selected from frameworks that have been validated in clinical trial studies. H Subgroup III (IGHV3, SEQ ID NO: 25) and V L Human heavy and light chain framework sequences in kappa subgroup I (IGKV1, SEQ ID NO: 24) have been validated in clinical trial studies and have been used successfully in many humanized antibodies.

[0085]

[0102] As shown in Figure 1A, IGKV1 V L The framework sequence of IGHV3 V differs from that of mouse mAb 7C3 by 25 amino acids (underlined residues), which corresponds to a variability of 30.86% (25 of the total 81 residues in the framework region). H The framework sequence of humanized 7C3 4D5 (7C3 HdHd) differs from that of murine mAb 7C3 by 35 amino acids (underlined residues), which corresponds to a percent change of 42.68% (35 of the total 82 residues in the framework region). H and V L is SEQ ID NO: 11 (7C3 HdV) in Table 1 H ) and SEQ ID NO: 12 (7C3 HdV L ) is shown.

[0086]

[0103] For the preparation of humanized 7C3 IMGT (7C3 HuHu), the human germline V domain with the highest homology to the framework region of mouse mAb 7C3 was selected. L and V H Sequences were identified from the IMGT database (International Immunogenetics Information System®). Homology searches can be performed using BLAST or similar methods. The murine mAb 7C3 variable region sequence was used as the query sequence. These searches identified the V sequences with the highest homology to the corresponding heavy and light chain framework sequences in murine mAb 7C3. H and V L The sequences are the human germline gene IGHV1 (V H , SEQ ID NO: 27) and IGVK2 (V L , SEQ ID NO: 26) was identified.

[0087]

[0104] As shown in Figure 2A, IGVK2 V LThe framework sequence of IGHV1 V differs from that of murine mAb 7C3 by 11 amino acids (underlined residues), which corresponds to a percent change of 13.58% (11 of the total 81 residues in the framework region). As shown in Figure 2B, IGHV1 V H The framework sequence of humanized 7C3 IMGT (7C3 HuHu) differs from that of murine mAb 7C3 by 26 amino acids (underlined residues), which corresponds to a percent change of 31.70% (26 of the total 82 residues in the framework region). H and V L is SEQ ID NO: 13 in Table 1 (7C3 HuV H ) and SEQ ID NO: 14 (7C3 HuV L ) is shown.

[0088]

[0105] These two pairs of light and heavy chain sequences (humanized 7C3 4D5 and humanized 7C3 IMGT) are used as examples for the construction of humanized antibodies against human NTSR1.

[0089]

[0106] Example 2

[0107] Full-length antibody expression

[0108] To confirm the change in affinity of the mouse antibody after humanization, V H and V L The DNA segments encoding the V of mouse 7C3, humanized 7C3 4D5, and humanized 7C3 IMGT were directly generated by nucleotide synthesis. As shown in Figures 3A and 3B, H and V LThe DNA segments encoding the above (Tables 1 and 2) were subcloned into a set of human Fc chimeric antibody expression vectors, pTCAEDH and pTCAEDL. The plasmids pTCAEDH 7C3 HUVH IgG1 and pTCAEDL 7C3 HUVL kappa were introduced into host cells to prepare recombinant antibody-expressing cells. FREESTYLE™ 293 cells (Invitrogen®) were used as host cells for antibody expression.

[0090]

[0109] [Table 2]

[0091]

[0110] The vectors were transfected and assembled into a suspension of FreeStyle™ 293 cells in a 30 ml volume using the following procedure: FreeStyle™ 293 cells were grown at 2 x 10 per 15 ml of FreeStyle™ 293 Expression Medium in a culture flask. 6 The DNA-PEI mixture was added to the FreeStyle™ 293 cells, and the transfected cells were incubated at 37°C with 8% CO2 and 135 rpm shaking in an incubator. Then, 37.5 μg of plasmid DNA was diluted in 1.5 ml of sterile 150 mM NaCl to a total volume of 1.5 ml. In a separate tube, 37.5 μl of PEI (2.0 mg / ml) was diluted in 1.5 ml of sterile 150 mM NaCl. The DNA and PEI solutions were allowed to stand at room temperature for 5 minutes. The solution was gently mixed by inverting the tube, and the tube was then allowed to stand at room temperature for approximately 10-20 minutes. The DNA-PEI mixture was added to the FreeStyle™ 293 cells, and the transfected cells were incubated in an incubator at 37°C with 8% CO2 for 4 hours on an orbital shaker platform rotating at 135-150 rpm. An equal volume of fresh culture medium was then added to a total volume of 30 ml, and the cells were cultured for 5-7 days, after which the cells were harvested for antibody purification and quantification.

[0092]

[0111] The collected supernatant was filtered through a 0.2 μm filter (MILLIPORE®) to remove any post-culture particulates. The antibody-containing culture supernatant was affinity purified using Protein A (MILLIPORE®), 1.5 M glycine / NaOH buffer, 3 M NaCl (pH 9.0) as the absorption buffer, and 0.2 M glycine / HCl buffer (pH 2.5) as the elution buffer. The pH of the eluted fraction was adjusted to approximately 6.0-7.0 by adding 1 M Tris / HCl buffer (pH 9.0). The prepared antibody solution was replaced with PBS using a dialysis membrane (10,000 MW cutoff, SPECTRUM LABORATORIES®) and filter-sterilized through a 0.22 μm pore membrane filter (MILLIPORE®) to obtain purified antibody. The concentration of purified antibody was determined by measuring the absorbance at 280 nm and converting the reading based on an optical density of 1.45, which is equivalent to 1 mg / ml.

[0093]

[0112] Example 3

[0113] Determination of binding affinity of humanized antibodies by ELISA

[0114] ELISA plates were coated with 1-2 μg / 100 μL of NTSR1 linear L2-biotin protein per well. The wells were rinsed three times with PBS and blocked with 300 μL of 5% MPBS per well at 37°C for 2 hours. After washing with PBS, the wells were incubated with serially diluted NTSR1 antibodies in 5% MPBS at 37°C for 1.5 hours. The plates were washed, and goat polyclonal anti-human IgG-HRP antibody (1:10,000) (Jackson ImmunoResearch®) was added to each well. OD readings were taken at 650 nm and 450 nm. OD readings were taken at 650 nm and 450 nm. 450 From OD 650and the binding affinity of the antibody was calculated by nonlinear regression in Prism software (GraphPad®).

[0094]

[0115] Due to the high degree of variation in the framework regions, 7C3 HdHd and 7C3 HdHu, which were generated by grafting CDR sequences from mAb 7C3 MM, exhibited much lower binding signals to NTSR1 (compared to mAbs 7C3 MM, 7C3 HuHu, and 7C3 HuHd in binding ELISA) (Figure 4).

[0095]

[0116] 7C3 HdV H In contrast to the 7C3 HuV1 gene, which was generated by grafting CDR sequences from mAb 7C3 MM onto IGHV1 framework sequences, with a high degree of variation in the framework regions. H is 7C3 HuV L Trying to pair with 7C3 HdV L Regardless of whether they were paired with mAB 7C3 HuHu and 7C3 HuHd versus 7C3 HdHd and 7C3 HdHu in binding ELISA (Figure 4), they produced good binding signals to NTSR1 and had relatively good affinity (mAB 7C3 MM, K D =6.76×10 -10 Compared to M, 7C3 HuHu, K D =2.94×10 -10 M) (Figure 5, Table 3).

[0096]

[0117] [Table 3]

[0097]

[0118] These results suggest that only the IGHV1 heavy chain framework regions can tolerate a relatively high rate of change without affecting the conformation of the CDR regions.

[0098]

[0119] Example 4

[0120] Fluorescence-activated cell sorting (FACS)

[0121] Using an anti-NTSR1 antibody, cells expressing NTSR1 on the cell surface were detected using, for example, FACS. NTSR1-expressing cells, FaDu, were collected and resuspended in 5% PBS / FBS buffer. The cells (1 × 10 5 ) were incubated with anti-NTSR1 antibody (1-10 μg / ml) or a negative control for 1 hour at 4°C, and then stained with goat anti-human IgG FITC conjugate (1 / 1000) for 1 hour at 4°C. Two additional controls were prepared for each assay: a control without primary antibody and another control without any antibody. All processed samples were analyzed using FACSVERSE™ (BECTON DICKINSON®), and the results were processed using FACSUITE™ software (BECTON DICKINSON®).

[0099]

[0122] The results are shown in Table 4. The chimeric anti-NTSR1 antibody (7C3 MM) and the humanized anti-NTSR1 antibodies 7C3 HuHu and 7C3 HuHd did not bind sufficiently to FaDu cells at a concentration of 1 μg / mL. However, the chimeric anti-NTSR1 antibody (7C3 MM) and the humanized anti-NTSR1 antibodies 7C3 HuHu and 7C3 HuHd did not significantly bind to FaDu cells at a concentration of 10 μg / mL.

[0100] [Table 4]

[0123] Table 4. FACS-based cell binding assay to determine affinity of humanized antibodies

[0101]

[0124] Example 5

[0125] Affinity maturation of humanized antibodies

[0126] The binding affinity of m7C3 to NTSR1 was improved through an affinity maturation process, and the resulting clone was named AKT2. Compared with the sequence of m7C3, AKT2 has two mutations in the heavy chain CDR and one mutation in the light chain CDR, which shows enhanced binding affinity to NTSR1. These three mutations are T28A (in iCDRH1), Y96H (in CDRH3), and S92A (in CDRL3), which are shown as underlined residues in Figure 6. Residues are numbered according to the Kabat nomenclature.

[0102]

[0127] To further enhance the binding affinity of the 7C3 humanized antibody to NTSR1, 7C3 HuHu and 7C3 HuHd were mutated to AKT2 HuHu and AKT2 HuHd, respectively (Figure 6, Table 1, SEQ ID NOs: 15 to 17, Table 2). The mutation process was carried out using gene synthesis technology. ELISA results show that the affinity of AKT2 HuHu and AKT2 HuHd to NTSR1 is similar to that of the original murine antibody, AKT2 (Figure 7).

[0103]

[0128] Example 6

[0129] Affinity measurements and kinetic analysis

[0130] To investigate the binding kinetics of individual anti-NTSR1 antibodies, surface plasmon resonance (SPR) measurements were performed on a BIACORE™ T200 (CYTIVA Inc.) as previously described (Karlsson and Falt, (1997) J. Immunol. Methods 200:121-133). Carboxymethylated dextran biosensor chips (CM5, CYTIVA Inc.) were activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. NTSR1 protein was injected at a flow rate of 10 μL / min to achieve approximately 1500 response units (RU) of coupled protein, followed by dilution to 5 μg / ml in 10 mM sodium acetate, pH 4.0, before injecting 1 M ethanolamine to block unreacted groups. For kinetic measurements, two-fold serial dilutions of anti-NTSR1 mAb (0.3125 nM to 40 nM) were injected at a flow rate of 30 μL / min at 25°C in HBS-EP + Biacore™ running buffer provided by the manufacturer (Cytiva®). The binding response to NTSR1 protein was corrected by subtracting the response from a blank flow cell. The association rate (k on or K a ) and dissociation rate (k off or K d ) to k on and k off The binding kinetics was calculated using a simple one-to-one Langmuir binding model with separate fitting of (Cytiva® Biacore™ Insight Evaluation Software).

[0104]

[0131] The results are shown in Table 5. k of chimeric AKT2 mAb binding to NTSR1 on and k off are 4.068×10 5 and 6.558 x 10 -4 and KD is 1.612 x 10 -9 M. AKT2 HuHu (IMGT version V H , AKT2 HuV H The k of binding of α-glucan to NTSR1 on and k off are 7.877×10 5 and 8.084 x 10 -4 and K D is 1.026 x 10 -9 M. AKT2 HuHd (IMGT version V H , AKT2 HuV H The k of binding of α-glucan to NTSR1 on and k off are 4.614×10 5 and 6.575 x 10 -4 and K D is 1.425 x 10 -9 It's M.

[0105]

[0132] [Table 5]

[0106]

[0133] As shown in Figure 7, the humanized antibodies AKT2 HuHu and AKT2 HuHd can recognize the human NTSR1 protein, and after humanization, the IMGT version V H (AKT2 HuV H ) but HuV L HdV trying to pair with L These results suggest that the murine antibody AKT2 produces an affinity similar to that of the murine antibody AKT2, regardless of the antibody pairing.

[0107]

[0134] Example 7

[0135] In vivo pharmacokinetic (PK) analysis

[0136] This example uses the Meso Scale Discovery (MSD) electrochemiluminescence (ECL) method to perform PK analysis of the anti-NTSR1 antibodies αNTSR1(AKT2), ​​αNTSR1(AKT2 Hu / Hu), and αNTSR1(AKT2 Hu / Hd) in BALB / c mouse samples. The MSD assay can measure both conjugated and unconjugated antibodies. As illustrated in this example, for the total antibody assay (including conjugated and unconjugated antibodies), the plate is coated with goat anti-human IgG, which can capture all humanized antibodies (conjugated and unconjugated).

[0108]

[0137] Mice were administered 5 mg / kg via the tail vein. Blood samples were then taken at different time points to determine the concentrations of αNTSR1 (AKT2), ​​αNTSR1 (AKT2 Hu / Hu), and αNTSR1 (AKT2 Hu / Hd) in mice using the MESO QuickPlex SQ 120 method. The PK parameters of αNTSR1 (AKT2), ​​αNTSR1 (AKT2 Hu / Hu), and αNTSR1 (AKT2 Hu / Hd) were analyzed by non-compartmental analysis using the Phoenix™ WinNonlin program, version 6.3.

[0109]

[0138] Table 6 summarizes the results of the PK study. The in vivo half-life of αNTSR1(AKT2) is 71.7±5.1 hours; the half-life of αNTSR1(AKT2 Hu / Hu) is 182±90.8 hours; and the half-life of αNTSR1(AKT2 Hu / Hd) is 156±48.6 hours (FIG. 8).

[0110]

[0139] [Table 6]

[0111]

[0140] Comparing the pharmacokinetic profiles of the whole antibodies αNTSR1(AKT2), ​​αNTSR1(AKT2 Hu / Hu), and αNTSR1(AKT2 Hu / Hd), the half-lives of the humanized antibodies αNTSR1(AKT2 Hu / Hu) and αNTSR1(AKT2 Hu / Hd) in BALB / c mice were 156-182 hours, which is significantly better than the half-life of the murine antibody αNTSR1(AKT2), ​​which was 71 hours. These properties may make the humanized antibodies αNTSR1(AKT2 Hu / Hu) and αNTSR1(AKT2 Hu / Hd) more useful as therapeutic agents.

[0112]

[0141] Example 7

[0142] Internalization assay

[0143] FaDu cells were trypsinized, then collected and resuspended in FACS buffer. Control: Secondary Ab anti-human IgG PE (1:200) was added to FaDu cells. The cells were incubated at 4°C for 0, 0.5, 2, 5, and 24 hours, then washed with 1 mL of FACS buffer. The supernatant was discarded. Test group: FaDu cells were preincubated with 10 μg / mL ADCs of trimannosyl and anti-NTSR1 antibodies, αNTSR1 (AKT2), ​​αNTSR1 (AKT2 Hu / Hu), and αNTSR1 (AKT2 Hu / Hd), in FACS buffer on ice for 60 minutes, washed three times with FACS buffer, and then incubated at 37°C for 0, 0.5, 2, 5, and 24 hours. The cells were analyzed by flow cytometry (BD LSRFORTESSA®), the results of which are shown in FIG.

[0113]

[0144] Example 8

[0145] In vitro cytotoxicity studies

[0146] The human head and neck cancer cell line, FaDu, was grown in RPMI-1640 medium (ATCC® modified) supplemented with 10% fetal bovine serum. The FaDu cell line was maintained in a humidified 37°C incubator in a 5% CO2 atmosphere. The day before treatment, cells were harvested and seeded into 96-well plates (4,000 cells per well). On day 2, cells were treated with 2-fold serial dilutions of the toxic payloads and ADCs listed in Table 7. Each treatment was performed in eight triplicate data points. After 72 hours of treatment, cell viability was assessed using a CELLTITER-GLO® kit (PROMEGA®) according to the manufacturer's instructions. At the end of the incubation, luminescence was measured using a SpectraMax i3x Multi Mode Detection Platform (MOLECULAR DEVICES®). Compound cytotoxicity was assessed relative to cells treated with 0.05% PBS (ADC) or 0.05% DMSO (toxic payload). IC50 values ​​were calculated by fitting the viability data to a four-parameter logistic equation using GRAPHPAD® Prism 5.0 software. The results are shown in Table 7 (Figure 10).

[0114]

[0147] [Table 7]

[0115]

[0148] Example 9

[0149] Codon optimization for CHO cell lines

[0150] From the above examples, the present inventors predict that the aforementioned anti-NTSR1 monoclonal antibodies have potential for development as therapeutic antibodies.

[0116]

[0151] To mass-produce humanized therapeutic antibodies in CHO cell lines, codon optimization was performed using the GENEOPTIMIZER® software tool. For antibody expression in CHO cell lines, the variable regions of αNTSR1(AKT2), ​​αNTSR1(AKT2 Hu / Hu), and αNTSR1(AKT2 Hu / Hd) (Table 1, SEQ ID NOS: 9-10 and 15-17) were subjected to codon optimization to obtain codon-optimized DNA segments. Optimization parameters included codon quality distribution (to select the most frequently used codons for the desired expression system) and GC content (to adjust the GC content within a desired range).

[0117]

[0152] Example 10

[0153] Development of anti-NTSR1 monoclonal antibody expression in stable CHO cell pools

[0154] V of αNTSR1(AKT2), ​​αNTSR1(AKT2 Hu / Hu), and αNTSR1(AKT2 Hu / Hd) H and V L Codon-optimized DNA segments encoding the following were directly prepared by nucleotide synthesis. Each optimized DNA segment was then subcloned into the human HERCEPTIN RFC (registered trademark) antibody expression vector pCHO-NTSR1, and the vector was introduced into host cells to prepare recombinant antibody-expressing cells. CHOS cells (LIFE-TECHNOLOGY Inc.) were used as host cells for expression. The vector was introduced into the host cells using Lipofectamine 2000 (INVITROGEN (registered trademark)) according to the attached instructions. Approximately 2.5 μg of the antibody expression vector was linearized with a restriction enzyme, and the gene was ligated to 4 × 10 6The cells were transfected with the ATP and seeded into 6-well culture plates. For low-concentration selection, the resulting cell pool was grown in selection medium containing 10 μg / ml puromycin and 100 nM methotrexate, or 20 μg / ml puromycin and 200 nM methotrexate. For further selection, another round of high-concentration selection was performed. The primary selection pool was further grown in medium containing 30 μg / ml puromycin and 500 nM methotrexate, or 50 μg / ml puromycin and 1000 nM methotrexate.

[0118]

[0155] The results are shown in Table 8. The productivities of αNTSR1(AKT2), ​​αNTSR1(AKT2 HuHu), and αNTSR1(AKT2 HuHd) were 20.49 mg per liter, 53.73 mg per liter, and 66.96 mg per liter, respectively, after 5 days of incubation. The results showed that the productivity of the chimeric αNTSR1(AKT2) antibody was significantly poor. However, the productivity of the humanized αNTSR1(AKT2 Hu / Hu) and αNTSR1(AKT2 Hu / Hd) antibodies was significantly improved, increasing 2.5- to 3-fold after humanization.

[0119]

[0156] [Table 8]

[0120]

[0157] Example 11

[0158] Xenograft model of anti-NTSR1 ADC (head and neck cancer)

[0159] The objective of this study was to evaluate the in vivo antitumor efficacy of αNTSR1(AKT2) 4seco-DUBA, αNTSR1(AKT2 Hu / Hu) 4seco-DUBA, and αNTSR1(AKT2 Hu / Hd) 4seco-DUBA in the FaDu human head and neck cancer xenograft model in male NOD SCID mice.

[0121]

[0160] Formulations containing the test substances αNTSR1(AKT2) 4seco-DUBA, αNTSR1(AKT2 Hu / Hu) 4seco-DUBA, αNTSR1(AKT2 Hu / Hd) 4seco-DUBA, and Hu IgG were prepared by diluting the stocks with 25 mM sodium citrate buffer (pH 6.5). Each formulation was administered intravenously to mice once a week for 3 weeks.

[0122]

[0161] FaDu cells were maintained in vitro as monolayer cultures in RPMI-1640 medium supplemented with 10% fetal bovine serum at 37°C in an atmosphere of 5% CO2 in air. Tumor cells were routinely passaged twice weekly by trypsin-EDTA treatment. For tumor inoculation, cells growing in exponential growth phase were collected and counted.

[0123]

[0162] Male NOD SCID mice, 6-7 weeks old, were quarantined for one week. Five mice were housed per cage. All animals were housed in an animal facility under a 12-hour light / 12-hour dark cycle at 19-25°C. Animals were provided with rodent pellet food and water ad libitum.

[0124]

[0163] FaDu cells were implanted subcutaneously (SC) into the right flank of male NOD SCID mice (4 × 10 cells in a 1:1 PBS / Matrigel mixture in 0.1 mL per mouse). 6 cells). The average tumor volume was approximately 200 mm 3 When the mice reached 100% CI, they were randomly divided into seven groups (N = 6 per group). Hu IgG, αNTSR1(AKT2) 4seco-DUBA (5 mg / kg), αNTSR1(AKT2 Hu / Hu) 4seco-DUBA (5 mg / kg), and αNTSR1(AKT2 Hu / Hd) 4seco-DUBA (5 mg / kg) were intravenously administered twice a week for 3 weeks.

[0125]

[0164] Tumor volume, body weight, mortality, and signs of overt toxicity were monitored and recorded three times a week for 28 days. 3) was measured three times a week using a caliper and calculated using the formula: tumor volume = (w 2 The tumor growth inhibition (TGI) was calculated according to the following formula: %TGI = [1-(T / C)] x 100% (where T and C represent the mean tumor volumes of the treatment and control groups, respectively). A TGI (%) value of ≥ 58% was considered to indicate significant antitumor activity. Animals were weighed three times a week until the end of the study.

[0126]

[0165] Figure 10 shows tumor growth curves in male NOD SCID mice implanted with FaDu. TGI values ​​are shown in Table 9. The results show that αNTSR1(AKT2) 4seco-DUBA (5 mg / kg), αNTSR1(AKT2 Hu / Hu) 4seco-DUBA (5 mg / kg), and αNTSR1(AKT2 Hu / Hd) 4seco-DUBA (5 mg / kg) significantly reduced FaDu tumor growth from day 10 to day 17.

[0127]

[0166] [Table 9]

Claims

1. a heavy chain variable region (V) comprising the sequence of SEQ ID NO: 16, 11, or 13, or a sequence having at least about 90% identity to the sequence of SEQ ID NO: 16, 11, or 13; Ｈ ); and a light chain variable region (V) comprising the sequence of SEQ ID NO: 17, 15, 12, or 14, or a sequence having at least about 90% identity to the sequence of SEQ ID NO: 17, 15, 12, or 14; Ｌ ) A humanized anti-neurotensin receptor 1 (NTSR1) antibody or an antigen-binding fragment thereof, comprising:

2. V Ｈ comprises the sequence of SEQ ID NO: 16, V Ｌ comprises the sequence of SEQ ID NO: 17 or 15; The humanized anti-NTSR1 antibody or its antigen-binding fragment according to claim 1.

3. V Ｈ comprises the sequence of SEQ ID NO: 13, V Ｌ comprises the sequence of SEQ ID NO: 12 or 14; The humanized anti-NTSR1 antibody or its antigen-binding fragment according to claim 1.

4. V Ｈ comprises the sequence of SEQ ID NO: 11, V Ｌ comprises the sequence of SEQ ID NO: 12 or 14; The humanized anti-NTSR1 antibody or its antigen-binding fragment according to claim 1.

5. The humanized anti-NTSR1 antibody or its antigen-binding fragment according to claim 1, wherein the humanized anti-NTSR1 antibody has a heavy chain constant region selected from the group consisting of IgG1, IgG2, and IgG4 isoforms, and a light chain constant region selected from the group consisting of κ and λ isotypes.

6. Fab fragment, F(ab') ２ The humanized anti-NTSR1 antibody or antigen-binding fragment thereof of claim 1, which is a fragment, an ScFv fragment, a chimeric antibody, or a nanobody.

7. The humanized anti-NTSR1 antibody or antigen-binding fragment thereof of claim 1, which is multispecific.

8. The humanized anti-NTSR1 antibody or antigen-binding fragment thereof according to claim 1; and A therapeutic agent conjugated with the humanized anti-NTSR1 antibody or an antigen-binding fragment thereof. An antibody conjugate comprising:

9. The antibody conjugate of claim 8, wherein the therapeutic agent is selected from the group consisting of antimetabolites, alkylating agents, alkylating agent-like agents, DNA minor groove alkylating agents, anthracyclines, antibiotics, calicheamicin, mitotic inhibitors, topoisomerase inhibitors, HDAC inhibitors, proteasome inhibitors, and radioactive isotopes.

10. 9. The antibody conjugate of claim 8, wherein the therapeutic agent is mertansine (DM1), monomethyl auristatin E (MMAE), seco-DUBA, exatecan, deruxtecan, or monomethyl auristatin F (MMAF).

11. A vector encoding the humanized anti-NTSR1 antibody or its antigen-binding fragment according to claim 1.

12. A genetically modified cell containing the vector of claim 11.

13. A genetically modified cell expressing the humanized anti-NTSR1 antibody or its antigen-binding fragment according to claim 1.

14. The genetically modified cell of claim 13, which is an immune cell.

15. The genetically modified cell of claim 13, which is a T cell.

16. A method for producing the humanized anti-NTSR1 antibody or antigen-binding fragment thereof described in any one of claims 1 to 7, comprising the steps of: (a) introducing one or more polynucleotides encoding the humanized anti-NTSR1 antibody or antigen-binding fragment thereof into a host cell; (b) culturing the host cell under conditions suitable for expression of the one or more polynucleotides; and (c) optionally isolating the humanized anti-NTSR1 antibody or antigen-binding fragment thereof from the host cell and / or the medium in which the host cell is grown.

17. an effective amount of the humanized anti-NTSR1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, the antibody conjugate according to any one of claims 8 to 10, or the genetically modified cell according to any one of claims 12 to 15; a pharmaceutically acceptable carrier; A pharmaceutical composition comprising:

18. 1. A pharmaceutical composition for treating, prophylactically treating, and / or preventing a disease and / or disorder caused by or associated with NTSR1 activity and / or signaling in a subject in need thereof, comprising: an effective amount of the humanized anti-NTSR1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, the antibody conjugate according to any one of claims 8 to 10, or the genetically modified cell according to any one of claims 12 to 15; a pharmaceutically acceptable carrier; A pharmaceutical composition comprising:

19. 19. The pharmaceutical composition of claim 18, wherein the disease is cancer.

20. 20. The pharmaceutical composition of claim 19, wherein the cancer is head and neck cancer, lung cancer, liver cancer, gastric cancer, pancreatic cancer, breast cancer, prostate cancer, or colorectal cancer.

21. A method for detecting NTSR1 in a sample, comprising contacting the sample with the humanized anti-NTSR1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 7.

22. A kit for detecting NTSR1 in a sample, comprising the humanized anti-NTSR1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 7.

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