Humanized anti-human neurotensin receptor 1 antibody and application thereof

Abstract

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

Description

Technical Field

[0001] The present disclosure relates to novel humanized antibodies, in particular to humanized antibodies that specifically bind to human neurotensin receptor 1 (NTSR1). The present disclosure also relates to the use of such antibodies for inhibiting tumor growth and metastasis. Background Art

[0002] The ligands of NTSR1 have been extensively studied. NTSR1 comprises 424 amino acids and has a high affinity for NTS. NTSR1 has been identified in the brain and in various cancer cells. The signal transduction pathways induced by the NTS / NTSR1 complex have been studied in different cell types, such as N1E-115, HT-29, and CHO cells overexpressing NTSR1 transfected with NTSR1. The NTS / NTSR1 complex causes the activation of phospholipase C (PLC), followed by the production of inositol triphosphate (IP3) and diacylglycerol (DAG) from membrane phospholipids. The activation of PLC results in the production of inositol triphosphate (IP3) and diacylglycerol from membrane phospholipids (PIP2). These two second messengers induce the activation of PKC and the movement of intracellular calcium, which is a key carcinogenic effector factor.

[0003] Several signaling pathways that may be 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. The NTS / NTSR1 complex has been shown to enhance cancer progression in aggressive malignant solid tumors such as mesothelioma, non-small cell lung cancer, liver cancer, breast cancer, and head and neck squamous cell carcinoma. Based on patient tissue staining, NTSR1 is a promising molecular marker for non-small cell lung cancer and prostate cancer. It has been previously shown that there is a long-term self-activation loop between neurotensin and NTSR1 as a mechanism responsible for the constitutive activation of the mitogen-activated protein kinase cell division signaling pathway and the activation of sustained target genes. In recent years, NTSR1 expression has been found to be associated with poor prognosis in patients with ductal breast cancer, and similar results have been found in head and neck squamous cell carcinoma.

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

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

[0006] Therefore, the present disclosure provides a humanized anti-NTSR1 antibody or an antigen-binding fragment thereof, comprising:

[0007] A heavy chain variable region (V L ... H );and

[0008] A light chain variable region (V L) 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 ).

[0009] In one embodiment of the present disclosure, V H comprising the sequence of SEQ ID NO: 16, and V L Comprising the sequence of SEQ ID NO: 17 or 15.

[0010] In another embodiment of the present disclosure, V H comprising the sequence of SEQ ID NO: 13, and V L Comprising the sequence of SEQ ID NO: 12 or 14.

[0011] In yet another embodiment of the present disclosure, V H comprising the sequence of SEQ ID NO: 11, and V L Comprising the sequence of SEQ ID NO: 12 or 14.

[0012] In some embodiments of the disclosure, the humanized anti-NTSR1 antibody has a heavy chain constant region selected from the group consisting of IgG1, IgG2, and IgG4 isotypes, and a light chain constant region selected from the group consisting of κ and λ isotypes.

[0013] 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, a ScFv fragment, a chimeric antibody or a nanobody.

[0014] In some embodiments of the present disclosure, the humanized anti-NTSR1 antibody or antigen-binding fragment thereof is multispecific.

[0015] The present disclosure also provides an antibody conjugate comprising:

[0016] A humanized anti-NTSR1 antibody or antigen-binding fragment thereof as disclosed herein; and

[0017] A therapeutic agent that binds to a humanized anti-NTSR1 antibody or antigen-binding fragment thereof.

[0018] Examples of therapeutic agents include, but are not limited to, antimetabolites, alkylating agents, alkylating agents, DNA minor groove alkylating agents, anthracyclines, antibiotics, calicheamicins, antimitotic agents, topoisomerase inhibitors, HDAC inhibitors, proteasome inhibitors, and radioisotopes. In some embodiments of the present disclosure, the therapeutic agent is maytansine (DM1), monomethyl auristatin E (MMAE), seco-DUBA, exactecan, deruxtecan, or monomethyl auristatin F (MMAF).

[0019] The present disclosure further provides a vector encoding the humanized anti-NTSR1 antibody or antigen-binding fragment thereof as disclosed herein.

[0020] The present disclosure provides a genetically engineered cell containing a vector disclosed herein or expressing the aforementioned humanized anti-NTSR1 antibody or antigen-binding fragment thereof.

[0021] Examples of genetically engineered cells include, but are not limited to, immune cells. In some embodiments of the present disclosure, the genetically engineered cells are T cells.

[0022] The present disclosure also provides a method for producing a humanized anti-NTSR1 antibody or an antigen-binding fragment thereof as disclosed herein, comprising: (a) introducing one or more polynucleotides encoding the humanized anti-NTSR1 antibody or the antigen-binding fragment thereof into a host cell; (b) culturing the host cell under conditions conducive to the 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 the culture medium in which the host cell is grown.

[0023] The present disclosure further provides a pharmaceutical composition comprising:

[0024] An effective amount of a humanized anti-NTSR1 antibody or an antigen-binding fragment thereof, an antibody conjugate, or a genetically engineered cell as disclosed herein; and

[0025] Pharmaceutically acceptable carrier.

[0026] In some embodiments of the present disclosure, the pharmaceutical compositions are provided for use in the treatment, prophylactic treatment, and / or prevention of a disease and / or disorder caused by or associated with NTSR1 activity and / or signaling in a subject in need of such treatment.

[0027] In some embodiments of the present disclosure, the disease is cancer. Examples of cancer include, but are not limited to, head and neck cancer, lung cancer, liver cancer, stomach cancer, pancreatic cancer, breast cancer, prostate cancer, or colorectal cancer.

[0028] 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.

[0029] The present disclosure further provides a kit for detecting NTSR1 in a sample, comprising a humanized anti-NTSR1 antibody or an antigen-binding fragment thereof as disclosed herein.

[0030] Simple diagram description

[0031] Figure 1A Display mouse V L Sequence alignment between (m7c3) and human template (4d5) (VKI). The first row shows the residue numbering according to the Kabat scheme. Differences are underlined. Kabat CDRs are shown in square brackets.

[0032] Figure 1B Display mouse V H Sequence alignment between (m7c3) and human template (4d5) (VHIII). The first row shows the residue numbering according to the Kabat scheme. Differences are underlined. Kabat CDRs are shown in square brackets.

[0033] Figure 2A Display mouse V L Sequence alignment between (m7c3) and human template (IMGT) (VKII). The first row shows the residue numbering according to the Kabat scheme. Differences are underlined. Kabat CDRs are shown in square brackets.

[0034] Figure 2B Display mouse V H Sequence alignment between (m7c3) and human template (IMGT) (VHI). The first row shows the residue numbering according to the Kabat scheme. Differences are underlined. Kabat CDRs are shown in square brackets.

[0035] Figure 3A and Figure 3B Display vectors for generating mouse-human chimeric and humanized forms of anti-NTSR1 monoclonal antibodies (mAbs) as described in Example 2 are shown.

[0036] Figure 4Depicted are 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.

[0037] Figure 5 Delineation of the K of mouse-human chimeric antibody 7C3 MM and humanized antibody 7C3 HuHu and NTSR1 d Results of binding affinity analysis. Chimeric antibody expression, purification and K d The detailed analysis procedure is described in Example 3. The results showed that the K d It is not significantly different from the mouse-human chimeric antibody 7C3 MM.

[0038] Fig. 6A , Figure 6B and Figure 6C Display of V between mouse 7C3, AKT2 and affinity-enhanced humanized antibodies AKT2HuHu and AKT2 HuHd H and V L Sequence alignment. V sequences of humanized antibodies AKT2 HuHu and AKT2HuHd H and V L The mutation process was performed as described in Example 5.

[0039] Figure 7 Depicts the results of the binding affinity analysis of mouse-human chimeric antibody AKT2, humanized antibodies AKT2 HuHu and AKT2 HuHd to NTSR1. Chimeric antibody expression, purification and K d The detailed procedures of the analysis are described in Example 5. The results showed that the K d There was no significant difference compared with the mouse-human chimeric antibody AKT2.

[0040] Figure 8 The results of in vivo pharmacokinetic analysis of mouse-human chimeric antibody AKT2, humanized antibodies AKT2 HuHu and AKT2 HuHd in MSD analysis are shown.

[0041] Fig.9A Shown are the results of internalization analysis of mouse-human chimeric AKT2, humanized antibodies AKT2 HuHu and AKT2 HuHd, and antibody-drug conjugates (ADCs) in FaDu cells using flow cytometry (before normalization). Fig. 9B exhibit Fig.9A The standardized results.

[0042] Fig.10The results of cytotoxicity analysis of mouse-human chimeric antibody AKT2, humanized antibodies AKT2 HuHu and AKT2HuHd and their ADCs in FaDu cells are shown.

[0043] Fig.11 Depicted are in vivo efficacy results of ADCs of mouse-human chimeric antibody AKT2, humanized antibodies AKT2 HuHu and AKT2 HuHd in the FaDu human head and neck cancer xenograft tumor model.

[0044] Implementation

[0045] Unless otherwise defined, scientific and technical terms used herein shall have the meanings commonly understood by those of ordinary skill in the art. In addition, unless the context otherwise requires, singular terms shall include the plural and plural terms shall include the singular. In general, the nomenclature and techniques thereof described herein for use in connection with cell and tissue culture, molecular biology, and protein and oligonucleotide or polynucleotide chemistry and hybridization are well known and commonly used in the art.

[0046] The practice of the present disclosure may employ techniques including conventional techniques of cell biology, cell culture, antibody technology and genetic engineering, which are within the ordinary skill of the art. Such techniques are fully explained in the literature.

[0047] Standard techniques are used for recombinant DNA, oligonucleotide synthesis and tissue culture and transformation (e.g., electroporation, liposome transfection). Enzymatic reactions and purification techniques are performed according to the manufacturer's instructions or as generally implemented in the art or as described herein. The aforementioned 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 this specification. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual (3rd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001)), which is incorporated herein by reference. The nomenclature used in conjunction with analytical chemistry, synthetic organic chemistry, and pharmaceutical and medicinal chemistry described herein, as well as laboratory procedures and techniques thereof, are well known and commonly used in the art. Chemical synthesis, chemical analysis, pharmaceutical preparation, formulation and delivery, and patient treatment are performed using standard techniques.

[0048] As utilized in accordance with the present disclosure, unless otherwise indicated, the following terms should be understood to have the following meanings: As used herein, the term "and / or" should be considered as specific disclosure that each of the two specified features or components has or does not have the other. For example, "A and / or B" should be considered as specific disclosure of each of (i) A, (ii) B, and (iii) A and B, as if each were individually stated herein.

[0049] It must be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the content clearly dictates otherwise.

[0050] As used herein, the term "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, namely two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, and multimers thereof (e.g., IgM). Each heavy chain comprises a VH and a heavy chain constant region. The heavy chain constant region comprises three domains, CH1, CH2, and CH3. Each light chain comprises a VL and a light chain constant region. The light chain constant region comprises one domain (CL1). VH and VL can be further subdivided into hypervariable regions, called CDRs, interspersed with more conserved regions, called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, which are arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In various embodiments of the present disclosure, the FRs of the anti-NTSR1 antibodies (or antigen-binding portions thereof) may be identical to human germline sequences, or may be naturally or artificially modified. The amino acid consensus sequence may be defined based on a side-by-side analysis of two or more CDRs.

[0051] As used herein, the term "specific for" or "specifically binds to" means that the antibody does not cross-react to a significant extent with other antigenic determinants.

[0052] As used herein, the term "antigenic determinant" refers to the site where an antibody binds to an antigen.

[0053] As used herein, the term "complementarity determining region (CDR)" refers to non-contiguous antigen combining sites found within the variable regions of heavy and light chain polypeptides. Kabat et al., J. Biol. Chem. 252: 6609-6616 (1977); Kabat et al., U.S. Department 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) have described CDRs, where definitions include overlaps or subsets of amino acid residues when compared against each other.

[0054] As used herein, the term "monoclonal antibody (mAb)" is not limited to antibodies produced via fusion tumor technology. Monoclonal antibodies are derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, by any means available or known in the art.

[0055] 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.

[0056] "Humanized" forms of non-human antibodies are chimeric immunoglobulins that contain minimal sequence derived from non-human immunoglobulins. In general, a humanized antibody will comprise substantially all of at least one and usually 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.

[0057] As used herein, the term "nanobody" refers to antibodies comprising small single variable domains (VHH of antibodies obtained from camels and dromedaries). Antibody proteins obtained from camels and dromedaries (Camelus baclrianus and Calelus dromaderius) family members (including New World members, such as llama species (Lama pacos, Lama glama and Lama vicugna)) have been characterized in terms of size, structural complexity and antigenicity to human individuals. Certain IgG antibodies from this mammalian family as found in nature lack light chains, and are therefore structurally different from the typical four-chain quaternary structure with two heavy chains and two light chains of other animal antibodies.

[0058] As used herein, the terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody and similar terms include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex.

[0059] As used in this disclosure, the term "therapeutic agent" means any compound, substance, drug, medicament, or active ingredient having a therapeutic or pharmacological effect suitable for administration to a mammal, such as a human.

[0060] As used herein, the term "immune cell" refers to a cell that plays a role in an immune response. Immune cells are of hematopoietic origin and include lymphocytes, such as B cells and T cells; natural killer cells; myeloid cells, such as monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes.

[0061] As used herein, the term "T cells" includes CD4 + T cells and CD8 + T cells. The term T cells also includes T helper type 1 T cells, T helper type 2 T cells, T helper type 17 T cells, and suppressor T cells.

[0062] As used herein, the term "vector" means a nucleic acid molecule capable of transporting another nucleic acid to which it has been connected. One type of vector is a "plastid", which refers to a circular double-stranded DNA loop in which an additional DNA segment can be joined. Another type of vector is a viral vector, in which an additional DNA segment can be joined to a viral genome. Certain vectors are capable of autonomous replication in the host cell into which they are introduced (e.g., bacterial vectors and free mammalian vectors with bacterial replication origins). Other vectors (e.g., non-free mammalian vectors) can be integrated into the genome of the host cell when introduced into the host cell, and then replicated together with the host genome. In addition, certain vectors can guide the expression of genes operably connected thereto. Such vectors are referred to herein as "recombinant expression vectors" (or, in short, "expression vectors"). In general, the expression vectors used in recombinant DNA technology are usually in the form of plastids. Since plastids are the most commonly used form of vectors, in this specification, "plastids" and "vectors" are used interchangeably. However, the disclosure 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.

[0063] The term "genetically engineered" or "genetic engineering" of a cell refers to the use of genetic material to manipulate genes to change the gene copies and / or gene expression in a cell. The genetic material can be in the form of DNA or RNA. The genetic material can be transferred to the cell by various means including viral transduction and non-viral transfection. After genetic engineering, the expression of certain genes in the cell can be permanently or temporarily changed.

[0064] As used in this disclosure, the term "pharmaceutical composition" means a mixture containing therapeutic agents that is administered to a mammal (eg, a human) for the purpose of preventing, treating, or eliminating a particular disease or pathological condition from which the mammal is suffering.

[0065] As used herein, 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.

[0066] As used herein, the terms "treatment" and "treating" and similar terms encompass any treatment of a disease in mammals, especially humans, and include: (a) preventing the disease from occurring in an individual who may be susceptible to the disease but has not yet been diagnosed with the disease; (b) inhibiting the disease, that is, curbing its development; and (c) alleviating the disease, that is, causing regression of the disease.

[0067] The terms "preventing" and "prevention" are recognized in the art and when used in conjunction with a condition, include the administration of an agent prior to the onset of the condition to reduce the incidence or severity of symptoms of a medical condition in a subject, or to delay the onset of the condition, relative to a subject not receiving the agent.

[0068] 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, cows, sheep, pigs, goats), and the like.

[0069] As used herein, the term "in need of treatment" refers to the judgment made by a caregiver (e.g., a physician, nurse, care practitioner or individual in the case of humans; a veterinarian in the case of animals (including non-human mammals)) that the individual requires or will benefit from treatment. This judgment is based on a variety of factors within the caregiver's area of ​​expertise and includes knowledge that the individual is or will become ill due to a condition that can be treated by the compounds of the present disclosure.

[0070] "Cancer", "tumor" and similar terms include precancerous, neoplastic, transformed and cancerous cells, and may refer to solid tumors, or non-solid cancers (see, e.g., Edge et al. AJCC Cancer Staging Manual (7th edition, 2009); Cibas and Ducatman Cytology: Diagnostic principles and clinical correlates (3rd edition, 2009)). Cancer includes both benign and malignant neoplasms (abnormal growth). "Transformation" refers to spontaneous or induced phenotypic changes, e.g., cell immortalization, morphological changes, abnormal cell growth, reduced contact inhibition and fixation and / or malignancy (see Freshney, Culture of Animal Cells a Manual of Basic Technique (3rd edition, 1994)). Although transformation can be caused by infection with a transformed virus and the incorporation of new genomic DNA or the uptake of exogenous DNA, it can also occur spontaneously or after exposure to a carcinogen.

[0071] As used herein, the term "sample" encompasses a variety of sample types obtained from an individual, subject, or patient, and can be used for diagnostic or monitoring analysis. The definition encompasses blood and other liquid samples of biological origin; solid tissue samples, such as biopsy specimens or tissue cultures or cells derived therefrom, and their progeny.

[0072] The present disclosure provides a humanized antibody that is specific for and has high affinity to human NTSR1. Anti-NTSR1 antibodies or antigen-binding fragments thereof can provide therapeutic benefit to an individual. Anti-NTSR1 antibodies or antigen-binding fragments thereof according to the present disclosure can be used as therapeutic agents for the treatment and / or diagnosis of a variety of disorders mediated by NTSR1, which are more fully described herein.

[0073] The antibodies or antigen-binding fragments 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 isotypes, and a light chain constant region selected from the group consisting of κ and λ isotypes), or may comprise only an antigen-binding portion (e.g., a Fab, F(ab')2, or scFv fragment), and may be modified to affect function as desired.

[0074] 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) the smallest recognition unit consisting of amino acid residues that mimics the hypervariable region of an antibody (e.g., an isolated complementary determining region (CDR), such as a CDR3 peptide), or a restricted FR3-CDR3-FR4 peptide. Other engineered molecules, such as domain-specific antibodies, single-domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, bifunctional antibodies, trifunctional antibodies, tetrafunctional antibodies, 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 herein.

[0075] Antigen-binding fragments of antibodies generally contain at least one variable domain. A variable domain can be of any size or amino acid composition and will generally contain at least one CDR adjacent to or in frame with one or more framework sequences. L Domain associated V H In the antigen-binding fragment of the domain, V H and V L The domains can be positioned relative to each other in any suitable configuration. For example, the variable region can be a dimer and contain V H -V H 、V H -V L or V L -V L Alternatively, the antigen-binding fragment of an antibody may contain a monomer V H and V L domain.

[0076] As with full antibody molecules, antigen-binding fragments can be monospecific or multispecific (e.g., bispecific). A multispecific antigen-binding fragment of an antibody will generally comprise at least two different variable domains, wherein each variable domain is capable of specifically binding to a separate antigen or to a different antigenic determinant on the same antigen. Any multispecific antibody format, including the exemplary bispecific antibody formats disclosed herein, can be adapted for use with the antigen-binding fragments of the disclosed antibodies using conventional techniques available in the art.

[0077] In some embodiments of the present disclosure, the humanized anti-NTSR1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising a 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 a sequence of SEQ ID NO: 16, 11 or 13; and a light chain variable region (V L ... L ).

[0078] The term "sequence identity" means that two polynucleotide or amino acid sequences are identical over a comparison window (i.e., on a nucleotide-by-nucleotide or residue-by-residue basis). The term "sequence identity percentage" is calculated by comparing two optimally aligned sequences over the comparison window, determining the number of positions at which the same nucleic acid base (e.g., A, T, C, G, U, or I) or amino acid residue occurs in the two 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 sequence identity percentage.

[0079] In some embodiments of the present disclosure, the humanized anti-NTSR1 antibody or antigen-binding fragment thereof comprises: a V comprising the sequence of SEQ ID NO: 16 H ; and V comprising the sequence of SEQ ID NO: 17 or 15 L .

[0080] In some embodiments of the present disclosure, the humanized anti-NTSR1 antibody or antigen-binding fragment thereof comprises: a V comprising the sequence of SEQ ID NO: 13; H ; and V comprising the sequence of SEQ ID NO: 12 or 14 L .

[0081] In some embodiments of the present disclosure, the humanized anti-NTSR1 antibody or antigen-binding fragment thereof comprises: a V comprising the sequence of SEQ ID NO: 11; H ; and V comprising the sequence of SEQ ID NO: 12 or 14 L .

[0082] The humanized anti-NTSR1 antibodies 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 are derived. Such mutations can be readily determined by comparing the amino acid sequences disclosed herein with germline sequences obtained from, for example, a public antibody sequence database. The present disclosure includes antibodies and antigen-binding fragments thereof derived from any amino acid sequence disclosed herein, wherein one or more amino acids within one or more framework regions and / or CDR regions are mutated to the corresponding residues in the germline sequence from which the antibody is derived, or to the corresponding residues in the germline sequence of another mammal, or to conservative amino acid substitutions of the corresponding germline residues (such sequence changes are collectively referred to herein as "germline mutations"). One of ordinary skill in the art can readily generate a number of antibodies and antigen-binding fragments comprising one or more individual germline mutations or combinations thereof, starting with the heavy and light chain variable region sequences disclosed herein. In certain embodiments, V H and / or V L All framework and / or CDR residues in the domain are all mutated back to the residues found in the original germline sequence from which the antibody is derived. In other embodiments, only certain residues are mutated back to the original germline sequence, such as only mutated residues found within the 8 amino acids before FR1 or the 8 amino acids after 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 residues of a different germline sequence (i.e., a germline sequence different from the germline sequence from which the antibody was originally derived). In addition, the disclosed antibodies may contain any combination of two or more germline mutations in the framework and / or CDR regions, such as where certain individual residues are mutated to the corresponding residues of a specific germline sequence, while certain other residues different from the original germline sequence are maintained or mutated to the corresponding residues of a different germline sequence. Once obtained, antibodies and 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 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 by the present disclosure.

[0083] The antibodies disclosed herein may be monospecific, bispecific or multispecific. Multispecific antibodies may be specific for different antigenic determinants of a target polypeptide or may contain antigen binding domains that are specific for more than one target polypeptide. The anti-NTSR1 antibodies disclosed herein may be linked to another functional molecule (e.g., another peptide or protein) or co-expressed with another functional molecule. For example, an antibody or fragment thereof may be functionally linked (e.g., by chemical coupling, genetic fusion, non-covalent binding or other means) to one or more other molecular entities, such as another antibody or antibody fragment, to produce a bispecific or multispecific antibody with a second binding specificity. For example, the 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 bound to a therapeutic agent.

[0084] In some embodiments of the disclosure, the antibody or antigen-binding fragment thereof is in the form of a chimeric antigen receptor.

[0085] In some embodiments of the present disclosure, a humanized anti-NTSR1 antibody or an antigen-binding fragment thereof can be used as an antibody-drug conjugate (ADC) that can specifically target NTSR1. That is, the present disclosure also provides an antibody conjugate, which includes the aforementioned humanized anti-NTSR1 antibody or antigen-binding fragment, and a therapeutic agent bound to the humanized anti-NTSR1 antibody or antigen-binding fragment thereof. The therapeutic agent or payload drug can be any therapeutic agent or payload drug commonly used in ADC. In some embodiments, the therapeutic agent or payload drug is selected from the group consisting of antimetabolites, alkylating agents, alkylating agents, DNA minor groove alkylating agents, anthracyclines, antibiotics, calicheamicins, antimitotic agents, topoisomerase inhibitors, HDAC inhibitors, proteasome inhibitors, and radioisotopes. For example, the therapeutic agent or payload drug may include maytansine (DM1), monomethyl auristatin E (MMAE), seco-DUBA, exatecan, deruxtecan, or monomethyl auristatin F (MMAF). The methods used for the binding may be those known in the art.

[0086] The humanized anti-NTSR1 antibody or antigen-binding fragment thereof may be encoded in a vector. The present disclosure also provides a vector encoding a humanized anti-NTSR1 antibody or antigen-binding fragment thereof as described herein. In one embodiment, one type of vector is a "plastid", which refers to a circular double-stranded DNA loop into which additional DNA segments can be joined. Another type of vector is a viral vector, in which additional DNA segments can be joined to the viral genome. Certain vectors are capable of autonomous replication in the host cell into which they are introduced (e.g., bacterial vectors with bacterial replication origins and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of the host cell when introduced into the host cell, and then replicated together with the host genome. In addition, certain vectors are capable of directing the expression of genes operably linked thereto. Such vectors are referred to herein as "recombinant expression vectors" (or, simply, "expression vectors"). In general, expression vectors used in recombinant DNA technology are usually in the form of plastids. Since plasmids are the most commonly used form of vectors, in this specification, "plastid" and "vector" are used interchangeably. However, the disclosure 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.

[0087] In another aspect, the disclosure provides a genetically engineered cell expressing a humanized anti-NTSR1 antibody or antigen-binding fragment thereof as described herein or containing a vector as described herein. The genetically engineered cell may be an immune cell, such as a T cell. In one embodiment of the disclosure, the antibody or antigen-binding fragment thereof is expressed on the surface of a cell. Specifically, the cell is a T cell.

[0088] In some embodiments of the disclosure, the antibody or antigen-binding fragment thereof is in the form of a chimeric antigen receptor.

[0089] 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 the CAR polypeptide construct are in the same polypeptide chain, for example, constituting a chimeric fusion protein. In some embodiments, the domains in the CAR polypeptide construct are not adjacent to each other, for example, in different polypeptide chains.

[0090] Examples of methods for producing humanized anti-NTSR1 antibodies or antigen-binding fragments thereof include: (a) introducing one or more polynucleotides encoding the antibodies or antigen-binding fragments into host cells; (b) culturing the host cells under conditions conducive to the expression of the one or more polynucleotides; and (c) optionally isolating the antibodies or antigen-binding fragments from the host cells and / or the culture medium in which the host cells are grown.

[0091] The present disclosure further provides pharmaceutical compositions comprising a humanized anti-NTSR1 antibody or antigen-binding fragment thereof, antibody conjugate, or genetically engineered cell as described herein. Pharmaceutical compositions as described herein are formulated with suitable diluents, carriers, excipients, and other agents that provide improved transfer, delivery, tolerance, and the like. Such compositions can be formulated for specific uses, such as for veterinary use or human medical use. The composition form and excipients, diluents, and / or carriers used will depend on the intended use of the antibody and the mode of administration for therapeutic use. Numerous suitable formulations can be found in the formulary collection known to all medicinal chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa. Such formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, vesicles containing lipids (cationic or anionic) (such as LIPOFECTIN.TM., Life Technologies, Carlsbad, Calif.), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of various molecular weights), semisolid gels, and semisolid mixtures containing carbowax. See also Powell et al. "Compendium of excipients for parenteral formulations" PDA (1998) J Pharm Sci Technol 52: 238-311.

[0092] The dosage of the antibody administered to the patient may vary depending on the patient's age and size, the target disease, condition, route of administration, and the like. The preferred dosage is generally calculated based on body weight or body surface area. When the antibodies of the present disclosure are used to treat NTSR1-related conditions or diseases in adult patients, intravenous administration of the antibodies of the present disclosure may be advantageous. The frequency and duration of treatment may be adjusted depending on the severity of the condition. The effective dosage and time course of administration of the antibody may be determined empirically; for example, the patient's progress may be monitored by periodic assessments, and the dosage may be adjusted accordingly. In addition, interspecies proportional adjustment of dosages may be performed using well-known methods in the art (e.g., Mordenti et al., 1991, Pharmaceut. Res. 8: 1351).

[0093] Various delivery systems are known and can be used to administer the pharmaceutical compositions of the present disclosure, such as encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, receptor-mediated internalization (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 composition may be administered by any convenient route, such as by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and may be administered with other biologically active agents. It may be administered systemically or topically.

[0094] The pharmaceutical composition of the present disclosure can be delivered subcutaneously or intravenously with a standard needle and syringe. In addition, with respect to subcutaneous delivery, a pen delivery device is easy to use when delivering the pharmaceutical composition of the present disclosure. Such pen delivery devices can be reusable or disposable. Reusable pen delivery devices generally utilize replaceable cartridges containing the pharmaceutical composition. Once all the pharmaceutical compositions in the cartridge have 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 a disposable pen delivery device, there is no replaceable cartridge. In fact, the disposable pen delivery device is pre-filled with a pharmaceutical composition stored in a reservoir within the device. Once the pharmaceutical composition in the reservoir is emptied, the entire device is discarded.

[0095] In some cases, the pharmaceutical composition 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 Pres., Boca Raton, Fla. In yet another embodiment, a controlled release system can be placed close to the target of the composition, so only a portion of the systemic dose is required (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 of Langer, 1990, Science 249: 1527-1533.

[0096] Injectable preparations may include dosage forms for intravenous, subcutaneous, intradermal and intramuscular injections; drip infusions, etc. Such injectable preparations can be prepared by well-known methods. For example, injectable preparations can be prepared, for example, by dissolving, suspending or emulsifying the antibody or salt thereof described above in a sterile aqueous medium or an oily medium conventionally used for injection. For example, physiological saline, isotonic solutions containing glucose and other adjuvants, etc. are used as aqueous media for injection, which can be used in combination with appropriate cosolvents, such as alcohols (e.g., ethanol), polyols (e.g., propylene glycol, polyethylene glycol), nonionic surfactants [e.g., polysorbate 80, HCO-50 (polyethylene oxide (50 mol) adduct of hydrogenated castor oil)], etc. For example, sesame oil, soybean oil, etc. are used as oily media, which can be used in combination with cosolvents such as benzyl benzoate, benzyl alcohol, etc. It is preferred that the injection thus prepared is filled in an appropriate ampoule.

[0097] Advantageously, the pharmaceutical composition for oral or parenteral use described above is prepared in a dosage form in a unit dose suitable for the dosage of the active ingredient. Such dosage forms in a unit dose include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc.

[0098] In some embodiments of the present disclosure, the pharmaceutical composition is used to treat, prophylactically treat, and / or prevent a disease and / or condition 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 condition caused by or associated with NTSR1 activity and / or signaling in a subject in need of such treatment, 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.

[0099] 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.

[0100] The humanized anti-NTSR1 antibodies or antigen-binding fragments thereof as described herein can also be used to detect and / or measure NTSR1 or NTSR1 expressing cells in a sample, for example for diagnostic purposes. For example, the anti-NTSR1 antibodies or antigen-binding fragments thereof can be used to diagnose a condition or disease characterized by abnormal expression of NTSR1 (e.g., overexpression, underexpression, lack of expression, etc.). 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 combined with a secondary antibody that is itself detectably labeled for diagnostic applications. The detectable label or reporter molecule can be a radioactive isotope, such as 3 H. 14 C. 32 P. 35 S or 125 I; a fluorescent or chemiluminescent moiety, such as fluorescein isothiocyanate or rhodamine; or an enzyme, such as alkaline phosphatase, β-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).

[0101] The following examples are provided to help those skilled in the art to implement the present disclosure.

[0102] Examples

[0103] The following examples illustrate the development and use of NTSR1-specific antibodies that inhibit tumor growth by inducing anti-NTSR1 immune responses.

[0104] Example 1

[0105] Humanization of anti-NTSR1 mAb 7C3

[0106] Selection of human V region framework sequences:

[0107] Mouse mAb 7C3 can induce strong immunogenicity and anti-drug antibodies in patients. Therefore, humanization of 7C3 is an essential and critical step for further drug development. H and V L Mouse monoclonal antibody 7C3 (also referred to as m7C3) was used as the parent antibody, and the 7C3 mAb CDR sequences as shown in SEQ ID NOs: 1 to 6 based on the Kabat definition are shown in Figure 1A , Figure 1B , Figure 2A and Figure 2B middle.

[0108] Table 1. List of amino acid sequences

[0109]

[0110]

[0111] For the preparation of humanized 7C3 4D5 (7C3 HdHd), an accepted human 4D5 construct was selected from a construct that has 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 the clinic and have also been used in many successful humanized antibodies.

[0112] like Figure 1A As shown in L The framework sequence of 7C3 differs from that of mouse mAb 7C3 by 25 amino acids (residues indicated by underscores), which corresponds to a 30.86% (25 / 81 total residues in the framework region) variation. Figure 1B In IGHV3 V H The framework sequence of humanized 7C3 4D5 (7C3 HdHd) differs from mouse mAb 7C3 by 35 amino acids (residues indicated by underline), which corresponds to a 42.68% (35 / 82 total residues in the framework region) variation. H and V L SEQ ID NO: 11 (7C3HdV shown in Table 1 H ) and SEQ ID NO:12(7C3 HdV L )middle.

[0113] For the preparation of humanized 7C3 IMGT (7C3 HuHu), the 7C3 IMGT (International Immunogenetics Information ) Identification of human germline V with the highest degree of homology to the mouse mAb 7C3 framework region L and V H Homology searches can be performed using BLAST or similar methods. The variable region sequence of mouse mAb 7C3 was used as the query sequence. These searches identified the human germline gene IGHV1 (V H , SEQ ID NO: 27) and IGVK2 (V L , SEQ ID NO: 26) as the V sequence most homologous to the corresponding heavy and light chain framework sequences in mouse mAb 7C3. Hand V L sequence.

[0114] like Figure 2A As shown in the figure, IGVK2 V L The framework sequence of 7C3 differs from that of mouse mAb 7C3 by 11 amino acids (residues indicated by underscores), which corresponds to a 13.58% (11 / 81 total residues in the framework region) variation. Figure 2B As shown in H The framework sequence of humanized 7C3 IMGT (7C3 HuHu) differs from mouse mAb 7C3 by 26 amino acids (residues indicated by underscores), which corresponds to a 31.70% (26 / 82 total residues in the framework region) variation. H and V L SEQ ID NO: 13 (7C3HuV H ) and SEQ ID NO:14(7C3 HuV L )middle

[0115] These two pairs of light and heavy chain sequences (humanized 7C3 4D5 and humanized 7C3 IMGT) were used as examples for constructing humanized antibodies against human NTSR1.

[0116] Example 2

[0117] Performance of full-length antibodies

[0118] In order to confirm the affinity changes of mouse antibodies after humanization, V sequences encoding humanized 7C3 4D5 and humanized 7C3 IMGT were directly generated by nucleotide synthesis. H and V L DNA segment encoding mouse 7C3, humanized 7C3 4D5 and humanized 7C3 IMGT V H and V L The DNA segments of Tables 1 and 2 were cloned into a set of human Fc chimeric antibody expression vectors pTCAEDH and pTCAEDL. Figure 3A and Figure 3B As shown in. Plasmids pTCAEDH 7C3 HUVH IgG1 and pTCAEDL7C3 HUVLκ were introduced into host cells to prepare cells expressing recombinant antibodies. TM 293 cells ( Production) as a host cell for antibody expression.

[0119] Table 2. Antibody strain names and V H / V L composition

[0120]

[0121]

[0122] The following procedure was used to transfect the vector thus constructed into a 30 ml volume of FREESTYLE TM 293 cells in suspension. TM 293 cells were cultured at 2×10 6 cells / ml into a culture flask with 15 ml FREESTYLE TM 293 expression medium. Place the flask in an incubator at 37°C with 8% CO2 and shake at 135 rpm. Then, dilute 37.5 μg of plasmid DNA in 1.5 ml sterile 150 mM NaCl for a total volume of 1.5 ml. In another tube, dilute 37.5 μl of PEI (2.0 mg / ml) in 1.5 ml sterile 150 mM NaCl. Allow the DNA and PEI solutions to stand at room temperature for 5 minutes. Gently mix the solution by inverting the tube, and then allow the tube to stand at room temperature for about 10-20 minutes. Add the DNA-PEI mixture to the FREESTYLE TM 293 cells, and the transfected cells were cultured in an incubator at 37°C, 8% CO2 for 4 hours on an orbital shaker platform rotating at 135-150 rpm. Subsequently, an equal volume of fresh medium was added to a total volume of 30 ml, and the cells were cultured for 5-7 days. Cells were then collected for antibody purification and quantification.

[0123] The collected supernatant was filtered through a 0.2 μm filter ( The culture supernatant containing the antibody was filtered using protein A (made by Affinity purification was performed using 1.5 M glycine / NaOH buffer, 3 M NaCl (pH 9.0) as an absorption buffer, and 0.2 M glycine / HCl buffer (pH 2.5) as an elution buffer. The eluate was adjusted to about pH 6.0-7.0 by adding 1 M Tris / HCl buffer (pH 9.0). A dialysis membrane (10,000 MW cutoff, manufactured by SPECTRUM The prepared antibody solution was replaced with PBS and filtered through a membrane filter with a pore size of 0.22 μm ( The purified antibody was obtained by filtration sterilization (production). The concentration of the purified antibody was determined by measuring the absorbance at 280 nm and converting the measured value based on the optimal density of 1.45 equal to 1 mg / ml.

[0124] Example 3

[0125] Determination of binding affinity of humanized antibodies by ELISA

[0126] ELISA plates were coated with 1 to 2 μg / 100 μL NTSR1 linear L2-biotin protein per well. Wells were rinsed 3 times with PBS and blocked with 300 μL 5% MPBS per well at 37°C for 2 hours. After washing with PBS, wells were incubated with serially diluted NTSR1 antibodies in 5% MPBS at 37°C for 1.5 hours. Plates were washed and goat polyclonal anti-human IgG-HRP antibody (1:10,000) (JACKSON ). Read the OD at 650nm and 450nm. 450 Subtract OD 650 Obtain readings and use Prism software The binding affinity of the antibodies was calculated using nonlinear regression.

[0127] In the presence of high variation in the framework regions, 7C3 HdHd and 7C3 HdHu generated by grafting the CDR sequence from the murine mAb 7C3 MM showed lower binding signals in the binding ELISA with NTSR1 compared to mAb 7C3 MM, 7C3 HuHu and 7C3 HuHd ( Figure 4 ).

[0128] With 7C3 HDV H In contrast, the 7C3 HuV1 generated by grafting the CDR sequences from mAb 7C3MM into the IGHV1 framework sequences showed a high degree of variation in the framework regions. H , regardless of 7C3 HuV L or 7C3HdV L Pairing, with good NTSR1 binding signal (7C3 HuHu and 7C3 HuHd relative to 7C3 HdHd and 7C3 HdHu in binding ELISA) ( Figure 4 ) and has relatively good affinity (7C3 HuHu, K D =2.94×10 -10 M, compared to mAb 7C3 MM, K D =6.76×10 -10 M)( Figure 5 , Table 3).

[0129] Table 3. ELISA K of 7C3 MM and 7C3 HuHu D

[0130] <![CDATA[ELISA K D (M)]]> 7C3 MM <![CDATA[6.76*10 -10 M]]> 7C3 HuHu <![CDATA[2.94*10 -10 M]]>

[0131] These results indicate that only the IGHV1 heavy chain framework region can tolerate a relatively high degree of variation without affecting the CDR region conformation.

[0132] Example 4

[0133] Fluorescence activated cell sorting (FACS)

[0134] Anti-NTSR1 antibodies are used to detect cells expressing NTSR1 on the cell surface, for example using FACS. Cells expressing NTSR1 were collected and resuspended in 5% PBS / FBS buffer. Cells (1×10 5 ) were reacted with anti-NTSR1 antibody (1-10 μg / ml) or negative control at 4°C for 1 hour and then stained with goat anti-human IgG FITC conjugate (1 / 1000) at 4°C for 1 hour. For each analysis, two additional control groups were prepared; one without primary antibody and the other without any antibody. All treated samples were stained with FACSVERSE TM (BECTON ) analysis and the results are provided by FACSUITE TM Software (BECTON )deal with.

[0135] The results are shown in Table 4. Chimeric anti-NTSR1 antibody (7C3 MM) and humanized anti-NTSR1 antibodies 7C3HuHu and 7C3 HuHd did not bind well to FaDu cells at a concentration of 1 μg / mL. However, at a concentration of 10 μg / mL, there was no significant difference in the binding ability of chimeric anti-NTSR1 antibody (7C3 MM) and humanized anti-NTSR1 antibodies 7C3 HuHu and 7C3HuHd to FaDu cells.

[0136] Table 4. Cellular binding analysis of humanized antibody affinities determined by FACS

[0137] Pure line name Flow cytometry binding % (10 μg / mL) Flow cytometry binding % (1 μg / mL) 7C3 MM 91 31 7C3 HuHu 98 64 7C3 H 99 73

[0138] Example 5

[0139] Affinity maturation of humanized antibodies

[0140] The binding affinity of m7C3 to NTSR1 has been improved based on an affinity maturation process, and the resulting antibody strain is called AKT2. AKT2 has two mutations in the heavy chain CDR and one mutation in the light chain CDR compared to the sequence of m7C3, and shows enhanced binding affinity to NTSR1. These three mutations are T28A (in CDRH1), Y96H (in CDRH3), and S92A (in CDRL3), which are shown as underlined residues in Figure 6. Residues are numbered according to Kabat nomenclature.

[0141] In order to further enhance the binding affinity of 7C3 humanized antibody to NTSR1, 7C3 HuHu and 7C3HuHd were mutated to AKT2 HuHu and AKT2 HuHd, respectively (Figure 6, Table 1 SEQ ID NO: 15 to SEQ ID NO: 17, Table 2). The mutation process was performed using gene synthesis technology. ELISA results showed that the affinity of AKT2 HuHu and AKT2 HuHd to NTSR1 was similar to that of the original mouse antibody AKT2 ( Figure 7 ).

[0142] Example 6

[0143] Affinity measurement and kinetic analysis

[0144] To understand the differences in binding kinetics between individual anti-NTSR1 antibodies, BIACORE was used as described previously (Karlsson and Falt, (1997) J. Immunol Methods 200:121-133). TM T200( According to the instructions of the supplier, carboxymethylated polyglucose biosensor chip (CM5, The NTSR1 protein was diluted to 5 μg / ml with 10 mM sodium acetate, pH 4.0, and then injected at a flow rate of 10 μL / min to obtain approximately 1500 reaction units (RU) of coupled protein, followed by injection of 1 M ethanolamine to block unreacted groups. For kinetic measurements, the PCR products were prepared by the manufacturer ( HBS-EP+BIACORE provided by TM Two-fold serial dilutions of anti-NTSR1 mAb (0.3125 nM to 40 nM) were injected in running buffer, and the binding response on NTSR1 protein was corrected by subtracting the response on the blank flow cell. The association rate (k) was calculated using a simple one-to-one Langmuir binding model. on or K a ) and dissociation rate (k off or K d ), using k on and k off A separate fit. BIACORE TM Insight evaluation software).

[0145] The results are shown in Table 5. The k of chimeric AKT2 mAb binding to NTSR1 on and k off They are 4.068×10 5 and 6.558×10 -4 , and K D is 1.612×10 -9 M. AKT2 HuHu (V with IMGT form H , AKT2HuV H ) and NTSR1 binding k on and k off They are 7.877×10 5 and 8.084×10 -4 , and K D 1.026×10 -9 M. AKT2 HuHd (V with IMGT form H , AKT2 HuV H ) and NTSR1 binding k on and k off They are 4.614×10 5 and 6.575×10 -4 , and K D 1.425×10 -9 M.

[0146] Table 5. Multi-cycle kinetic analysis of NTSR1 binding by anti-NTSR1 antibodies

[0147] <![CDATA[K a (1 / Ms)]]> <![CDATA[K d (1 / s)]]> <![CDATA[K D (M)]]> AKT2 <![CDATA[4.068×10 5 ]]> <![CDATA[6.558×10 -4 ]]> <![CDATA[1.612×10 -9 ]]> AKT2 HuHu <![CDATA[7.877×10 5 ]]> <![CDATA[8.084×10 -4 ]]> <![CDATA[1.026×10 -9 ]]> AKT2 HuHd <![CDATA[4.614×10 5 ]]> <![CDATA[6.575×10 -4 ]]> <![CDATA[1.425×10 -9 ]]>

[0148] like Figure 7 As shown in the figure, the humanized antibodies AKT2 HuHu and AKT2 HuHd can recognize human NTSR1 protein, and after humanization, they can L or HDV L Pairing, IMGT Form V H (AKT2HuV H ) with an affinity similar to that of the mouse antibody AKT2.

[0149] Example 7

[0150] In vivo pharmacokinetic (PK) analysis

[0151] This example uses the Meso Scale Discovery (MSD) electrochemiluminescence (ECL) method to perform PK analysis of anti-NTSR1 antibodies αNTSR1(AKT2), ​​αNTSR1(AKT2 Hu / Hu), and αNTSR1(AKT2 Hu / Hd) in BALB / c mouse samples. The MSD analysis can measure both bound and unbound antibodies. As described in this example, for total antibody analysis (including bound and unbound antibodies), the discs were coated with goat anti-human IgG, which captured all humanized antibodies (bound and unbound).

[0152] Mice were administered via the tail vein at a dose level of 5 mg / kg. Blood samples were then obtained at different time points to determine the concentrations of αNTSR1(AKT2), ​​αNTSR1(AKT2 Hu / Hu), and αNTSR1(AKT2 Hu / Hd) in mice by 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 PHOENIX TM Analysis of WinNonlin program version 6.3.

[0153] Table 6 summarizes the results of the PK study. The in vivo half-life of αNTSR1(AKT2) was 71.7±5.1 hours; the half-life of αNTSR1(AKT2 Hu / Hu) was 182±90.8 hours; and the half-life of αNTSR1(AKT2 Hu / Hd) was 156±48.6 hours ( Figure 8 ).

[0154] Table 6.

[0155]

[0156] Referring to the comparison of the total antibody pharmacokinetic profiles of αNTSR1(AKT2), ​​αNTSR1(AKT2 Hu / Hu) and αNTSR1(AKT2 Hu / Hd), the half-life of humanized antibodies αNTSR1(AKT2 Hu / Hu) and αNTSR1(AKT2 Hu / Hd) in BALB / c mice was 156 to 182 hours, which is significantly better than the 71-hour half-life of mouse antibody αNTSR1(AKT2). These properties will make humanized antibodies αNTSR1(AKT2 Hu / Hu) and αNTSR1(AKT2 Hu / Hd) more useful as therapeutic agents.

[0157] Example 7

[0158] Internalization analysis

[0159] FaDu cells were trypsinized, and then collected and resuspended in FACS buffer. Control group: Secondary Ab anti-human IgG PE (1:200) was added to FaDu cells. Cells were reacted at 4°C for periods of 0, 0.5, 2, 5, and 24 hours, and then washed with 1 mL of FACS buffer. The supernatant was discarded. Test group: FaDu cells were pre-reacted on ice for 60 minutes with trimannosyl and anti-NTSR1 antibodies αNTSR1(AKT2), ​​αNTSR1(AKT2 Hu / Hu), and αNTSR1(AKT2 Hu / Hd) ADCs in 10 μg / mL FACS buffer, washed three times with FACS buffer, and then reacted at 37°C for periods of 0, 0.5, 2, 5, and 24 hours. By flow cytometry The cells were analyzed and the results are shown in FIG9 .

[0160] Example 8

[0161] In vitro cytotoxicity studies

[0162] Human head and neck cancer cell line FaDu was cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum ( FaDu cells were grown in a humidified incubator at 37°C in an atmosphere of 5% CO2. One day before treatment, cells were harvested and seeded in 96-well plates (4,000 cells per well). The next day, cells were treated with 2-fold serial dilutions of the toxic loading drugs and ADCs listed in Table 7. Each treatment was performed in eight triplicate data points. After 72 hours of treatment, cells were plated and plated according to the manufacturer's instructions by CELLTITER- Set At the end of the incubation period, the cells were analyzed using the SpectraMax i3x Multimode Detection Platform (MOLECULAR The luminescence was measured by 5% PBS (ADC) or 0.05% DMSO (toxic loading drug) to evaluate compound cytotoxicity. The IC50 values ​​were calculated by fitting the survival rate data with a four-parameter logistic equation using Prism 5.0 software. The results are shown in Table 7 ( Fig.10 )middle.

[0163] Table 7: IC50 values ​​of ADC.

[0164] name Relative IC50(nM) αNTSR1(AKT2) - αNTSR1(AKT2)4seco-DUBA 36.9 αNTSR1(AKT2 Hu / Hu) - αNTSR1(AKT2 Hu / Hu)4seco-DUBA 12.51 αNTSR1(AKT2 Hu / Hd) - αNTSR1(AKT2 Hu / Hd)4seco-DUBA 55.05

[0165] Example 9

[0166] Codon Optimization of CHO Cell Lines

[0167] From the above examples, we expect that the aforementioned anti-NTSR1 monoclonal antibodies have the potential to be developed as therapeutic antibodies.

[0168] In order to produce large amounts of humanized therapeutic antibodies in CHO cell lines, Codon optimization was performed using the Optimize Optimizer software tool. Codon optimization was performed on the variable regions of αNTSR1(AKT2), ​​αNTSR1(AKT2 Hu / Hu) and αNTSR1(AKT2 Hu / Hd) (Table 1, SEQ ID NO:9 to SEQ ID NO:10, and SEQ ID NO:15 to SEQ ID NO:17) to obtain codon-optimized DNA segments for antibody expression in CHO cell lines. Optimization parameters included codon mass distribution (to select the most frequently used codons in the desired expression system) and GC content (to control the GC content within the desired range).

[0169] Example 10

[0170] Development of anti-NTSR1 monoclonal antibody expressed in stable CHO cell populations

[0171] V encoding αNTSR1(AKT2), ​​αNTSR1(AKT2 Hu / Hu), and αNTSR1(AKT2 Hu / Hd) H and V L The codon-optimized DNA segments were directly generated by nucleotide synthesis methods. Subsequently, the optimized DNA segments were cloned into human The antibody expression vector pCHO-NTSR1 was introduced into host cells to prepare cells expressing recombinant antibodies. CHOS cells (LIFE- Company). According to the attached instruction manual (by The vector was introduced into the host cells by lipofectamine 2000. About 2.5 μg of the antibody expression vector was linearized by restriction enzymes and the gene was introduced into 4×10 6 Cells were inoculated into 6-well culture dishes. For low concentration selection, the resulting cell pool was grown in a selection medium containing 10 μg / ml puromycin and 100 nM methotrexate or 20 μg / ml puromycin and 200 nM methotrexate. For further selection, another stage of high concentration selection was performed. The primary selection pool was further grown in a medium containing 30 μg / ml puromycin and 500 nM methotrexate or a medium containing 50 μg / ml puromycin and 1000 nM methotrexate.

[0172] The results are shown in Table 8. The yields of αNTSR1(AKT2), ​​αNTSR1(AKT2 HuHu), and αNTSR1(AKT2 HuHd) were 20.49, 53.73, and 66.96 mg / liter, respectively, after 5 days of cultivation. The results indicate that the yield of the chimeric αNTSR1(AKT2) antibody was significantly poor. However, the yields of the humanized αNTSR1(AKT2 Hu / Hu) and αNTSR1(AKT2 Hu / Hd) antibodies were significantly improved after humanization and increased by 2.5 to 3 times.

[0173] Table 8: Antibody expression from the generated CHO cell population

[0174] αNTSR1 Antibody Yield 5D (mg / L) αNTSR1(AKT2) 20.49 αNTSR1(AKT2 HuHu) 53.73 αNTSR1(AKT2 HuHd) 66.96

[0175] Example 11

[0176] Xenograft Model of Anti-NTSR1 ADC (Head and Neck Cancer)

[0177] The goal 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.

[0178] Formulations containing test article αNTSR1(AKT2)4seco-DUBA, test article αNTSR1(AKT2 Hu / Hu)4seco-DUBA, test article αNTSR1(AKT2 Hu / Hd)4seco-DUBA and Hu IgG, respectively, were prepared by diluting the stock solution with 25 mM sodium citrate buffer (pH 6.5). Each formulation was administered intravenously (IV) to mice once a week for three weeks.

[0179] 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 subcultured twice a week by trypsin-EDTA treatment in a conventional manner. Cells growing in the exponential growth phase were collected and counted for tumor inoculation.

[0180] Male NOD SCID mice, 6 to 7 weeks old, were quarantined for one week. Five mice were housed in each cage. All animals were housed in an animal facility at 19-25°C with a 12-hour light / 12-hour dark cycle. Animals had free access to rodent pelleted food and water ad libitum.

[0181] FaDu cells were implanted subcutaneously (SC) into the right flank of male NOD SCID mice (containing 4×10 6 1:1 PBS / Matrigel mixture of 10 cells, 0.1 mL per mouse). When the average tumor volume reached approximately 200 mm 3 At 4 pm, the mice were randomly divided into 7 groups (N=6 in each group). Each group was intravenously injected with the following substances: 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), twice a week for a total of 3 weeks.

[0182] Tumor volume, body weight, mortality, and overt signs of toxicity were monitored and recorded three times a week for 28 days. 3 ) was measured three times a week using a caliper and calculated according to the following formula: Tumor volume = (w 2 ×l) / 2, where w = tumor width and l = tumor diameter length (mm). The percentage of tumor growth inhibition (TGI) was calculated using the following formula: %TGI = [1-(T / C)] × 100%, where T and C represent the mean tumor volume of the treatment group and the control group, respectively. TGI (%) values ​​≥ 58% were considered to indicate significant antitumor activity. Animals were weighed three times a week until the completion of the study.

[0183] Fig.10 Tumor growth curves in male NOD SCID mice implanted with FaDu are shown. TGI values ​​are shown in Table 9. The results indicate 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.

[0184] Table 9: In vivo anticancer studies

[0185]

[0186]

Claims

1. An antibody or antigen-binding fragment thereof having specificity for an antigenic determinant in nectin cell adhesion molecule 4 (nectin-4) or a fragment thereof; comprising a complementarity determining region (CDR) of a heavy chain variable region and a CDR of a light chain variable region, wherein the CDRs of the heavy chain variable region comprise CDRH1, CDRH2 and CDRH3 regions, and the CDRs of the light chain variable region comprise CDRL1, CDRL2 and CDRL3 regions, and wherein: The CDRH1 region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 6; the CDRH2 region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 to 12; and the CDRH3 region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 14 to 18; and The CDRL1 region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 20-24; the CDRL2 region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 26-30; and the CDRL3 region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 32-36.

2. The humanized anti-NTSR1 antibody or antigen-binding fragment thereof according to claim 1, wherein The VH comprises the sequence of SEQ ID NO: 16, and The VL comprises the sequence of SEQ ID NO: 17 or 15.

3. The humanized anti-NTSR1 antibody or antigen-binding fragment thereof according to claim 1, wherein The VH comprises the sequence of SEQ ID NO: 13, and The VL comprises the sequence of SEQ ID NO: 12 or 14.

4. The humanized anti-NTSR1 antibody or antigen-binding fragment thereof according to claim 1, wherein The VH comprises the sequence of SEQ ID NO: 11, and The VL comprises the sequence of SEQ ID NO: 12 or 14 5. The humanized anti-NTSR1 antibody or antigen-binding fragment thereof 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 isotypes, and a light chain constant region selected from the group consisting of κ and λ isotypes. 6 . The humanized anti-NTSR1 antibody or antigen-binding fragment thereof as claimed in claim 1 , which is a Fab fragment, a F(ab′) 2 fragment, a ScFv fragment, a chimeric antibody or a nanobody.

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

8. An antibody conjugate comprising: The humanized anti-NTSR1 antibody or antigen-binding fragment thereof as claimed in claim 1; and A therapeutic agent bound to the humanized anti-NTSR1 antibody or the antigen-binding fragment thereof.

9. The antibody conjugate of claim 8, wherein the therapeutic agent 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 inhibitors, proteasome inhibitors, and radioisotopes.

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

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

12. A genetically engineered cell comprising the vector according to claim 11.

13. A genetically engineered cell expressing the humanized anti-NTSR1 antibody or antigen-binding fragment of claim 1.

14. The genetically engineered cell according to claim 13, which is an immune cell.

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

16. A method for producing a humanized anti-NTSR1 antibody or an antigen-binding fragment thereof as claimed in any one of claims 1 to 7, comprising: (a) introducing one or more polynucleotides encoding the humanized anti-NTSR1 antibody or the antigen-binding fragment thereof into a host cell; (b) culturing the host cell under conditions conducive to the 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 the culture medium in which the host cell is grown.

17. A pharmaceutical composition 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 engineered cell according to any one of claims 12 to 15; and Pharmaceutically acceptable carrier.

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

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

20. The pharmaceutical composition of claim 19, wherein the cancer is head and neck cancer, lung cancer, liver cancer, stomach 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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