Antibodies and uses thereof

Abstract

An antibody or a fragment thereof in which complementarity determining regions (CDR-H1 to CDR-H3) of a heavy chain variable region (VH region) each comprise an amino acid sequence depicted in SEQ ID NO: 1 to 3, and complementarity determining regions (CDR-L1 to CDR-L3) of a light chain variable region (VL region) each comprise an amino acid sequence depicted in SEQ ID NO: 4 to 6.

Description

Technical Field

[0001] The present invention relates to antibodies and uses thereof. This application claims priority based on Japanese Patent Application No. 2022-165631 filed in Japan on October 14, 2022, and the contents are incorporated herein by reference. Background Art

[0002] Protein Tyrosine Kinase 7 (PTK7), also known as colon carcinoma kinase 4 (CCK-4), is known to be overexpressed in various diseases including cancer. Therefore, it is believed that it is possible to develop a therapeutic method for diseases by inhibiting signal transduction via PTK7.

[0003] For example, it is known that PTK7 and its degradation products control the motility and metastasis of cancer (see non-patent document 1), that the pseudokinase of PTK7 or its fragments in cancer cells is involved in the activation of cancer-related signal transduction such as Akt and c-Jun signal transduction (see non-patent document 2), that PTK7 is involved in the control of the β-catenin-independent pathway of Wnt signal transduction, is involved in diseases related to the upregulation of Wnt signal transduction, and that PTK7 is upregulated in many cancers (see non-patent document 3).

[0004] Therefore, PTK7 has been studied as a cancer marker and a therapeutic target for cancer. For example, the use of antibodies that recognize the extracellular domain of PTK7 as a marker for human colon stem cells is known (see non-patent document 4), a therapeutic method using an antibody-drug complex using a PTK7 antibody to target tumor progenitor cells of triple-negative breast cancer, ovarian cancer, and lung cancer (see non-patent document 5), and a therapeutic method using an antibody-drug complex of a PTK7 antibody and a microtubule depolymerizing agent to stimulate dendritic cells for anti-cancer immunity (see non-patent document 6).

[0005] Prior art literature

[0006] Non-patent literature

[0007] Non-patent literature 1: Golubkov V Set al., Protein-tyrosine Pseudokinase 7 (PTK7) Directs Cancer Cell Motility and Metastasis., J Biol Chem, 289(35), 24238-24249, 2014.

[0008] Non-patent literature 2: Golubkov VSand Strongin AY,Downstream Signaling and Genome-Wide Regulatory Effects of PTK7 Pseudokinase and Its ProteolyticFragments in Cancer Cells., Cell Commun Signal, l12,15,2014.

[0009] Non-patent literature 3: Berger H., et al., PTK7 Faces the Wnt in Development and Disease, Front Cell Dev Biol, 5, 31, 2017.

[0010] Non-patent document 4: Jung P., et al., Isolation of Human Colon Stem Cells Using Surface Expression of PTK7., Stem Cell Reports, 5(6), 979-987, 2015.

[0011] Non-patent document 5: Damelin M., et al., APTK7-targeted antibody-drug conjugatereduces tumor-initiating cells and induces sustained tumor regressions., SciTransl Med.9, eaag2611, 2017.

[0012] Non-patent document 6: MulleRP., et al., Microtubule-depolymerizing agents used inantibody-drug conjugates induce antitumor immunity by stimulation of dendritic cells., Cancer Immunol. Res., 2, 741-755, 2014. Summary of the invention

[0013] The object of the present invention is to provide an antibody or a fragment thereof against PTK7.

[0014] The present invention includes the following embodiments.

[0015] [1] an antibody or a fragment thereof, wherein the heavy chain variable region (V H The complementarity determining regions (CDR-H1 to CDR-H3) of the light chain variable region (V L The complementarity determining regions (CDR-L1 to CDR-L3) of the SEQ ID NOs: 4 to 6, respectively.

[0016] [2] The antibody or fragment thereof according to [1], wherein the V H The CDR-H1 to CDR-H3 of the region are composed of the amino acid sequences described in SEQ ID NOs: 1 to 3, respectively. L The regions CDR-L1 to CDR-L3 are composed of the amino acid sequences set forth in SEQ ID NOs: 4 to 6 in a different order.

[0017] [3] The antibody or fragment thereof according to [1] or [2], wherein the V H The region comprises the amino acid sequence set forth in SEQ ID NO: 7, wherein the V H CDR-H1 to CDR-H3 in the region are the amino acid sequences at positions 50 to 54, the amino acid sequences at positions 69 to 85, and the amino acid sequences at positions 118 to 122 in the amino acid sequence of SEQ ID NO: 7, respectively. L The region comprises the amino acid sequence set forth in SEQ ID NO: 8, wherein the V L CDR-L1 to CDR-L3 in the region are the amino acid sequences at positions 49 to 59, 75 to 81, and 114 to 122 of the amino acid sequence described in SEQ ID NO: 8, respectively.

[0018] [4] The antibody or fragment thereof according to any one of [1] to [3], wherein the V H The region consists of the amino acid sequence described in SEQ ID NO: 7, and the V L The region consists of the amino acid sequence described in SEQ ID NO:8.

[0019] [5] The antibody or fragment thereof according to any one of [1] to [4], wherein the V H Framework region (FR H ) relative to the FR in the continuous amino acid sequence described in SEQ ID NO: 7 H Having at least 80% sequence homology, the above V L Framework region (FR L ) relative to the FR in the continuous amino acid sequence described in SEQ ID NO: 8 L At least 80% sequence homology.

[0020] [6] The antibody or fragment thereof according to any one of [1] to [5], which binds to human PTK7.

[0021] [7] An antibody or a fragment thereof that binds to an epitope comprising the amino acid sequence at positions 310 to 331 and the amino acid sequence at positions 104 to 113 of the amino acid sequence of any one of SEQ ID NOs: 9 to 14.

[0022] [8] The antibody or fragment thereof according to [7], wherein the epitope consists of the amino acid sequence at positions 310 to 331 of the amino acid sequence of any one of SEQ ID NOs: 9 to 14.

[0023] [9] The antibody or fragment thereof according to [7], wherein the epitope consists of the amino acid sequence at positions 104 to 113 of the amino acid sequence of any one of SEQ ID NOs: 9 to 14.

[0024]

[10] The antibody or fragment thereof according to [7], which binds to an epitope comprising the amino acid sequence at positions 310 to 331 and the amino acid sequence at positions 104 to 113 of the amino acid sequence described in SEQ ID NO: 11.

[0025]

[11] The antibody or fragment thereof according to [7], wherein the epitope consists of the amino acid sequence at positions 310 to 331 of the amino acid sequence set forth in SEQ ID NO: 11.

[0026]

[12] The antibody or fragment thereof according to [7], wherein the epitope consists of the amino acid sequence at positions 104 to 113 of the amino acid sequence set forth in SEQ ID NO: 11.

[0027]

[13] An antibody or a fragment thereof which competes with the antibody or a fragment thereof according to any one of [1] to

[12] when binding to human PTK7.

[0028]

[14] The antibody or fragment thereof according to

[13] , wherein the human PTK7 is a protein consisting of the amino acid sequence of any one of SEQ ID NOs: 9 to 14.

[0029]

[15] The antibody or fragment thereof according to

[14] , wherein the human PTK7 is a protein consisting of the amino acid sequence set forth in SEQ ID NO: 11.

[0030]

[16] The antibody or fragment thereof according to any one of [1] to

[15] , which is a chimeric antibody, a humanized antibody or a human-derived antibody.

[0031]

[17] An anticancer agent comprising the antibody or fragment thereof according to any one of [1] to

[16] as an active ingredient.

[0032]

[18] An anticancer pharmaceutical composition comprising the anticancer agent described in

[17] and a pharmaceutically acceptable carrier.

[0033]

[19] A method for treating cancer, comprising the step of administering an effective amount of the anticancer agent described in

[17] or the anticancer pharmaceutical composition described in

[18] to a patient in need thereof.

[0034] According to this embodiment, an antibody or a fragment thereof against human PTK7 can be provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is a graph showing the results of evaluating the reactivity of the #19 antibody in Experimental Example 3.

[0036] Figure 2 This is a graph showing the results of evaluating the stability of antibody #19 in Experimental Example 4.

[0037] Figure 3 This is a graph showing the results of flow cytometry in which the expression of PTK7 in HCT116 cells was examined in Experimental Example 6.

[0038] Figure 4 This is a fluorescence microscope photograph showing representative results of studying the reactivity of the #19 antibody to HCT116 cells by immunofluorescence cell staining in Experimental Example 7.

[0039] Figure 5 This is a graph showing the results of evaluating the reactivity of the #19 chimeric antibody in Experimental Example 10.

[0040] Figure 6 This is a graph showing the results of flow cytometry in which the expression of PTK7 in blood cancer cells was examined in Experimental Example 11.

[0041] Figure 7 This is a graph showing the results of flow cytometry in which the expression of PTK7 in MOLT-4 cell-transplanted SCID mice was examined in Experimental Example 12.

[0042] Figure 8 This is a graph showing the results of measuring the time-dependent changes in tumor volume of HCT116 cell-transplanted humanized mice in Experimental Example 13.

[0043] Fig. 9 This is a graph showing the results of measuring the tumor volume of HCT116 cell-transplanted humanized mice in Experimental Example 13.

[0044] Fig.10 This is a graph showing the results of time-dependent observation of the survival rate of hematopoietic stem cell transplanted NOG mice in Experimental Example 13. DETAILED DESCRIPTION

[0045] In this specification, "antibody" refers to a protein that interacts with an antigen and comprises at least two heavy chains and two light chains interconnected by disulfide bonds. The heavy chain consists of a heavy chain variable region (V H region) and the heavy chain constant region (C H region), and the light chain consists of the light chain variable region (V L region) and the light chain constant region (C L Region) composition. H Area and V L The V region is composed of a framework region (FR region) and a complementary determining region (CDR) having a high frequency of variability in the region that directly contacts the antibody. H Area and V L The region is composed of three CDRs and four FRs arranged in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 from the amino terminus to the carboxyl terminus. CDR-H1 to CDR-H3 represent V H The CDR1 to CDR3 and CDR-L1 to CDR-L3 regions represent V L The CDR1 to CDR3 region. H and FR L Respectively represent V H FR area of ​​the area, V L FR area of ​​the region, FR-H1 to FR-H4 represent FR H FR1~FR4, FR-L1~FR-L4 represent FR L FR1~FR4.

[0046] In the present specification, the amino acid positions assigned to the complementarity determining regions of antibodies are defined according to Kabat (Kabat E., Wu T., Perry H., Gottesman K., Sequences of Proteins of Immunological Interest, National Institute of Health, Bethesda, Md., 1987 and 1991).

[0047] In the present specification, antibodies include isotypes such as IgG, IgE, IgM, IgD, IgA, and IgY, and classes and subclasses such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

[0048] In this specification, "antibody fragment" means one or more parts contained in an antibody, which have the ability to bind to human PTK7. Examples of antibody fragments include F(ab')2 (a fragment comprising two Fab fragments linked by a disulfide bond in the hinge region), Fab (a fragment consisting of V L 、V H , C L , and C H 1 domain), Fv (a fragment consisting of a single-arm V L and V H domain), Fd (fragment consisting of V H and C H The antibody fragments include a fragment consisting of one or more domains), CDR-H1 to H3, CDR-L1 to L3, etc. The antibody fragments also include a mixture of one or more kinds.

[0049] In this specification, the amino acid sequence shown in the sequence number includes an amino acid sequence of conservative substitutions. "Conservative substitution" refers to replacing any amino acid residue with another amino acid residue that has similar properties chemically and / or biologically to its residue. As substitutions, substitutions between acidic residues, substitutions between basic residues, substitutions between hydrophilic non-charged residues, substitutions between aliphatic non-charged residues, substitutions between non-polar non-charged residues, and substitutions between aromatic residues can be cited. As substitutions, for example, substitutions within the combination of Val, Ile, Leu and Met; Asp and Glu; Gly and Ala; Ser and Thr; Asn and Gln; Lys and Arg; Cys and Pro; and Phe and Tyr can be cited.

[0050] [Antibody or fragment thereof]

[0051] In one embodiment, the present invention provides an antibody or fragment thereof, wherein the heavy chain variable region (V H The complementarity determining regions (CDR-H1 to CDR-H3) of the light chain variable region (V L The complementarity determining regions (CDR-L1 to CDR-L3) of the SEQ ID NOs: 4 to 6, respectively. Hereinafter, this embodiment is also referred to as "Embodiment 1".

[0052] As described later in the Examples, the antibody or fragment thereof according to the present embodiment can bind to human protein tyrosine kinase 7 (human PTK7) expressed in cancer cells.

[0053] The GeanBank accession numbers and amino acid sequences of human PTK7 proteins are EAX04154.1 (SEQ ID NO: 9), EAX04155.1 (SEQ ID NO: 10), EAX04156.1 (SEQ ID NO: 11), EAX04158.1 (SEQ ID NO: 12), EAX04159.1 (SEQ ID NO: 13), and EAX04160.1 (SEQ ID NO: 14).

[0054] In the present specification, the amino acid sequence of any one of SEQ ID NOs: 9 to 14 includes not only conservatively substituted amino acid sequences but also deleted or added amino acid sequences and is an amino acid sequence having the same function as the protein composed of the amino acid sequence of any one of SEQ ID NOs: 9 to 14.

[0055] CDR-H1 comprises the 5 consecutive amino acid sequence shown in sequence number 1, CDR-H2 comprises the 17 consecutive amino acid sequence shown in sequence number 2, CDR-H3 comprises the 5 consecutive amino acid sequence shown in sequence number 3, CDR-L1 comprises the 11 consecutive amino acid sequence shown in sequence number 4, CDR-L2 comprises the 7 consecutive amino acid sequence shown in sequence number 5, and CDR-L3 comprises the 9 consecutive amino acid sequence shown in sequence number 6.

[0056] Preferably, CDR-H1 is composed of the 5 consecutive amino acid sequence shown in SEQ ID NO: 1, CDR-H2 is composed of the 17 consecutive amino acid sequence shown in SEQ ID NO: 2, CDR-H3 is composed of the 5 consecutive amino acid sequence shown in SEQ ID NO: 3, CDR-L1 is composed of the 11 consecutive amino acid sequence shown in SEQ ID NO: 4, CDR-L2 is composed of the 7 consecutive amino acid sequence shown in SEQ ID NO: 5, and CDR-L3 is composed of the 9 consecutive amino acid sequence shown in SEQ ID NO: 6. The amino acid sequences of SEQ ID NOs. 1 to 6 are amino acid sequences derived from rat-derived sequences.

[0057] In one embodiment, the present invention provides an antibody or fragment thereof, wherein V H The region comprises the amino acid sequence described in SEQ ID NO: 7, preferably V H The amino acid sequence of the region consists of the amino acid sequence described in SEQ ID NO: 7, V H CDR-H1 to CDR-H3 in the region are the amino acid sequences at positions 50 to 54, 69 to 85, and 118 to 122 in SEQ ID NO: 7, respectively. L The region comprises the amino acid sequence described in SEQ ID NO: 8, preferably V L The region consists of the amino acid sequence set forth in SEQ ID NO: 8, V LCDR-L1 to CDR-L3 in the region are respectively the amino acid sequence at positions 49 to 59, the amino acid sequence at positions 75 to 81, and the amino acid sequence at positions 114 to 122 in the amino acid sequence described in SEQ ID NO: 8. Hereinafter, this embodiment is also referred to as "Embodiment 2".

[0058] V H When the region consists of the amino acid sequence described in SEQ ID NO: 7, FR-H1 to FR-H4 are positions 1 to 49, positions 55 to 68, positions 86 to 117, and positions 123 to 133 in the amino acid sequence described in SEQ ID NO: 7, respectively.

[0059] V H When the region consists of the amino acid sequence described in SEQ ID NO: 7, FR-H1 to FR-H4 are positions 1 to 48, positions 60 to 74, positions 82 to 113, and positions 123 to 133 in the amino acid sequence described in SEQ ID NO: 7, respectively.

[0060] The antibody of embodiment 2 includes the following antibody: FR H With respect to the FR in the continuous amino acid sequence described in SEQ ID NO: 7 H have at least 80%, 85%, 90%, or 95% sequence homology, FR L With respect to the FR in the continuous amino acid sequence described in SEQ ID NO: 8 L Having at least 80%, 85%, 90%, or 95% sequence homology.

[0061] In the present specification, sequence homology refers to the percentage of the number of identical nucleotides or amino acids shared between two sequences when the two sequences are optimally aligned in amino acid sequences.

[0062] In one embodiment, the present invention provides an antibody or a fragment thereof that binds to an epitope comprising an amino acid sequence at positions 310 to 331 and an amino acid sequence at positions 104 to 113 of an amino acid sequence as described in any one of SEQ ID NOs: 9 to 14. In this case, the epitope of human PTK7 is a discontinuous epitope. Hereinafter, this embodiment is also referred to as "embodiment 3".

[0063] The amino acid sequences of SEQ ID NOs. 9 to 14 are amino acid sequences of representative variants of human PTK7. An epitope refers to an amino acid residue that can directly and specifically bind to an antibody. In addition, an epitope generally has three-dimensional structural characteristics and charge characteristics.

[0064] The epitope can be determined by appropriately combining known methods such as epitope mapping, Western blotting, and immunoprecipitation. In addition, the spatial conformation of the epitope can be determined by appropriately combining known methods such as X-ray crystal structure analysis and 2D nuclear magnetic resonance.

[0065] The epitope may consist of the amino acid sequence at positions 310 to 331 of the amino acid sequence of any one of SEQ ID NOs: 9 to 14. Alternatively, the epitope may consist of the amino acid sequence at positions 104 to 113 of the amino acid sequence of any one of SEQ ID NOs: 9 to 14.

[0066] The amino acid sequence of human PTK7 is preferably the amino acid sequence of SEQ ID NO:11.

[0067] In one embodiment, the present invention provides an antibody or a fragment thereof, which, when binding to human PTK7, competes with the antibody or a fragment thereof of Embodiments 1 to 3. Hereinafter, this embodiment is also referred to as "Embodiment 4".

[0068] "Competition" means that a certain antibody or fragment thereof has the property of being easily bound to a certain binding partner in the presence of another antibody or fragment thereof at an equal or greater level. Whether a certain antibody or fragment thereof is the antibody or fragment thereof of Embodiment 4 can be evaluated by using an antibody or fragment thereof, the antibodies or fragments thereof of Embodiments 1 to 3, and a human PTK7 antibody by ELISA analysis or FACS analysis.

[0069] The antibodies of embodiments 1 to 4 include antibodies derived from non-human animals, chimeric antibodies, humanized antibodies, and human antibodies. Chimeric antibodies have CDR-H1 to CDR-H3, CDR-L1 to CDR-L3, FR-H1 to FR-H4, and FR-L1 to FR-L4 derived from sequences derived from non-human animals, and CDR-L1 to CDR-L3 derived from sequences derived from humans. H Area and C L Humanized antibodies have CDR-H1 to CDR-H3 and CDR-L1 to CDR-L3 sequences derived from non-human animals, FR-H1 to FR-H4 and FR-L1 to FR-L4 sequences derived from humans, and CDR-H2 to CDR-H3 sequences derived from non-human animals. H Area and C L Human antibodies have CDR-L1 to CDR-L3, FR-H1 to FR-H4, and FR-L1 to FR-L4, and C H Area and C L area.

[0070] The dissociation constant (K) of the antibody or fragment thereof according to embodiments 1 to 4 D ) is usually 10 －2 M or less, 10 －3 M or less, 10 － 4 M or less, 10 －5 M or less, 10 －6 M or less, 10－7 M or less, 10 －8 M or less, 10 －9 M or less, 10 －10 M or less, 10 －11 The dissociation constant can be measured by surface plasmon resonance or isothermal titration calorimetry.

[0071] The antibodies of Embodiments 1 to 4 are preferably isolated antibodies. "Isolated antibody" means an antibody that does not substantially contain other antibodies having different antigen specificity, and is preferably a monoclonal antibody.

[0072] Antibodies derived from non-human animals can be produced by the hybridoma method, that is, cells (hybridomas) formed by fusing non-human animal-derived B cells producing monoclonal antibodies with cancer cells such as myeloma cells are prepared, and the antibody activity of the culture supernatant is examined to obtain target clones.

[0073] Chimeric antibodies are produced by analyzing the gene sequence of antibodies of mice, rats, etc. obtained by the hybridoma method and replacing at least a part of the constant region with the gene sequence of a human antibody by genetic recombination.

[0074] Humanized antibodies are produced by analyzing the gene sequences of monoclonal antibodies of mice, rats, etc. obtained by the hybridoma method and replacing the constant regions and framework regions with the gene sequences of human antibodies by genetic recombination.

[0075] Human antibodies are antibodies that are derived from humans as a whole, including CDRs. Human antibodies can be produced by screening from human antibody gene libraries using display technologies such as phage display technology, ribosome display technology, and yeast display technology, or by immunizing genetically modified mice carrying human Ig loci.

[0076] If the antibody or fragment thereof of the present embodiment is a chimeric antibody or fragment thereof, a humanized antibody or fragment thereof, or a human antibody or fragment thereof, the immunogenicity when administered to humans can be reduced, and side effects such as anaphylactic shock can be suppressed, so it can be administered to humans.

[0077] [Anticancer agents and antiancer pharmaceutical compositions]

[0078] In one embodiment, the present invention provides an anticancer agent comprising the above-mentioned antibody or a fragment thereof as an active ingredient. As described below in the Examples, cancer can be treated by administering the anticancer agent of this embodiment.

[0079] Cancers that can be treated by the anticancer agent of this embodiment include cancers in which the expression of PTK7 is upregulated. Examples of cancers in which the expression of PTK7 is upregulated include colon cancer, rectal cancer, acute lymphoblastic leukemia, T-cell leukemia, lung cancer, breast cancer, esophageal cancer, thyroid cancer, gastric cancer, oral cancer, cervical cancer, intrahepatic bile duct cancer, prostate cancer, and liposarcoma.

[0080] The anticancer agent of the present embodiment includes an antibody-drug complex in which the anti-PTK7 antibody of the present embodiment forms a complex with a drug having pharmacological activity against cancer.

[0081] The anticancer agent of this embodiment is preferably mixed with a pharmaceutically acceptable carrier and formulated as an anticancer pharmaceutical composition. Examples of pharmaceutically acceptable carriers include buffers such as neutral buffered saline and phosphate buffered saline; carbohydrates such as glucose, mannose, sucrose, dextran, and mannitol; and amino acids such as glycine, but are not limited thereto.

[0082] The anticancer pharmaceutical composition may contain additives such as antioxidants, chelating agents, preservatives, etc. The anticancer pharmaceutical composition is preferably formulated for intravenous administration.

[0083] [Other embodiments]

[0084] In one embodiment, the present invention provides a method for treating cancer, comprising the step of administering an effective amount of the above anticancer agent or the above anticancer pharmaceutical composition to a patient in need thereof.

[0085] The dosage of the above anticancer agent or the above anticancer pharmaceutical composition varies depending on the patient's symptoms, body weight, age, sex, etc. and cannot be generalized, but it is considered that 0.001 to 500 mg of the active ingredient (antibody or fragment thereof) is administered once by intravenous drip at intervals of 1 to 5 weeks.

[0086] In one embodiment, the present invention provides the above-mentioned antibody or fragment thereof for use in cancer treatment.

[0087] In one embodiment, the present invention provides use of the above-mentioned antibody or a fragment thereof for producing an anticancer agent.

[0088] Example

[0089] Hereinafter, the present embodiment will be described in more detail based on examples, but the present embodiment is not limited to these examples.

[0090] [Experimental Example 1]

[0091] (Preparation of human PTK7 expressing HEK293T cells)

[0092] The human PTK7 gene was introduced into HEK293T cells, which are human fetal kidney cell lines, to produce human PTK7-expressing HEK293 cells. (R) CRL-3216”) were inoculated in an expansion medium and cultured at 37°C for 1 day (60-70% confluence). The expansion medium used contained DMEM, fetal bovine serum (FBS) and antibiotics.

[0093] Next, the expression plasmid of human PTK7 (full length) (pcDNA3-PTK7-VSV) was introduced into HEK293T cells. (R) (a) solution consisting of 5-7 μg of pcDNA3-PTK7-VSV (Thermo Fisher Scientific, 250 μL), and OptiMEM (R) The (b) solution consisting of PEG (Thermo Fisher Scientific, 250 μL) and PEI (polyethyleneimine, 25 to 35 μL) was pipetted separately and then allowed to stand at room temperature (23° C.) for 5 minutes.

[0094] Mix solution (a) and solution (b), and let stand at room temperature for 15 minutes. Add the mixed solution to the culture medium of HEK293T cells in amplified culture, and culture at 37°C overnight. Replace the amplified culture medium, and culture at 37°C for 1 day. Recover the cultured HEK293T cells, wash them twice with phosphate-buffered saline (PBS), and obtain human PTK7-expressing HEK293T cells.

[0095] [Experimental Example 2]

[0096] (Preparation of hybridoma and acquisition of #19 antibody)

[0097] Rats (Wister rats) were immunized three times with the human PTK7-expressing HEK293T cells prepared in Experimental Example 1 to obtain B cells. B cells were fused with myeloma cells to prepare hybridomas by a conventional method.

[0098] Based on the ELISA method using a human PTK7 fragment consisting of amino acid residues 31 to 702 of genetically recombinant human PTK7 (sequence number 11), the flow cytometry study results of the reactivity to human PTK7-expressing HEK293T cells, and the reactivity of magnetic particles bound to monoclonal antibodies in the culture supernatant of hybridomas to human PTK7-expressing HEK293T cells, #19 antibody was screened as a reactive monoclonal antibody.

[0099] Hybridomas were awakened with awakening medium (Hybridoma-SFM, 10% FBS and 1% penicillin-streptomycin) and serum-free acclimation was performed with serum-free medium (Hybridoma-SFM and 1% penicillin-streptomycin). The acclimated cells were inoculated in roller bottles and cultured until the viable cell rate was less than 10%. The culture supernatant was recovered and filtered with a 0.22μm filter, and the monoclonal antibody (#19 antibody) was purified with a protein G column. Purity was confirmed by CBB staining after SDS-PAGE.

[0100] As the recombinant human PTK7, a gene of a plasmid (pSec-Tag2-PTK7(31-702)) was introduced into Expi293T cells (Thermo Fisher Scientific) using PEI, and the recombinant PTK7(31-702) secreted from the culture supernatant was purified to obtain the recombinant human PTK7.

[0101] [Experimental Example 3]

[0102] (Confirmation of the binding activity of antibody #19 to human PTK7 by ELISA)

[0103] The binding activity of antibody #19 to human PTK7 was evaluated by ELISA method. The following materials were used.

[0104] 96-well plate: Product name: "MaxiSorp surface-treated Nunc-Immuno Module, F8", Cosmo Bio Co., Ltd.

[0105] Buffer 1 (TBS-T): Tris-buffered saline containing Tween 20.

[0106] Buffer 2: Dilute the product name "Blocking One" to 5 w / v% with TBS-T.

[0107] Blocking buffer: product name "Blocking One" (Nacalai Tesque) was diluted to 20 w / v% with TBS-T.

[0108] Antigen diluent: The human PTK7 fragment of Experimental Example 2 was diluted to 1 μg / mL using Tris-buffered saline (TBS).

[0109] Primary antibody diluent: dilute the #19 antibody to 0.001-1 μg / mL using buffer 2.

[0110] Secondary antibody diluent: Donkey Anti-Rat IgG H&L (HRP) (Abcam) was diluted with buffer 2.

[0111] ELISA matrix solution: Product name "1-Step TM Ultra TMB-ELISA Substrate” (ThermoFisher Scientific).

[0112] Add antigen diluent (50 μL / well) to the wells of a 96-well plate and let stand overnight at 4°C. After washing the wells 5 times with TBS-T, add blocking buffer (200 μL / well) to the wells and let stand at 4°C for more than 2 hours. After washing the wells 5 times with TBS-T, add primary antibody diluent (50 μL / well) to the wells and shake at room temperature for 1 hour. Wash the wells 5 times with TBS-T, add secondary antibody diluent (50 μL / well) and shake at room temperature for 1 hour. Wash the wells 5 times with TBS-T, add ELISA substrate solution (50 μL / well) and let stand at room temperature for 5 minutes. Add 2N HCl aqueous solution (50 μL / well) and measure the absorbance at a wavelength of 450 nm.

[0113] Figure 1 This is a graph showing the absorbance measurement results. The vertical axis represents the absorbance, and the horizontal axis represents the antibody concentration (μg / mL). The results show that the #19 antibody recognizes the fragment of human PTK7 in a concentration-dependent manner.

[0114] [Experimental Example 4]

[0115] (Stability evaluation of #19 antibody)

[0116] The #19 antibody was stored at 37°C for 1 to 7 days, and the stability was evaluated by ELISA. The stability was evaluated by the ratio of the antigen reactivity (absorbance) of the #19 antibody stored at 37°C to the antigen reactivity (absorbance) of the #19 antibody stored at 4°C.

[0117] #19 antibody was diluted to 1 mg / mL using mouse serum (product name "Normal Mouse Serum", Fujifilm Wako Pure Chemical Industries, Ltd.). Antigen diluent (50 μL / well) was added to the wells of a 96-well plate and allowed to stand overnight at 4°C. After washing the wells 5 times with TBS-T, blocking buffer (200 μL / well) was added to the wells and allowed to stand at 4°C for more than 2 hours. After washing the wells 5 times with TBS-T, #19 antibody stored at 37°C or #19 antibody stored at 4°C was added to the wells at 50 μL / well and shaken at room temperature for 1 hour. After washing the wells 5 times with TBS-T, secondary antibody diluent (50 μL / well) was added and shaken at room temperature for 1 hour. After washing the wells 5 times with TBS-T, ELISA substrate solution (50 μL / well) was added and allowed to stand at room temperature for 5 minutes. A 2N HCl aqueous solution (50 μL / well) was added, and the absorbance at a wavelength of 450 nm was measured.

[0118] Figure 2 This is a graph showing the evaluation results of stability. The vertical axis represents stability (%) (absorbance of #19 antibody stored at 37°C / absorbance of #19 antibody stored at 4°C×100), and the horizontal axis represents storage time (days). The results show that #19 antibody is stable.

[0119] [Experimental Example 5]

[0120] (Analysis of CDR of Antibody #19)

[0121] Total RNA was extracted from the hybridoma (#19) selected in Experimental Example 2, and cDNA was synthesized using the GeneRacer kit (ThermoFisher Scientific), and gene fragments of rat IgG antibody heavy chain gene and rat κ light chain gene were amplified by PCR.

[0122] A portion of the variable region and constant region was recovered for the heavy chain, and the full length of the variable region and constant region was recovered for the light chain. CDRs were predicted based on the Kabat rule.

[0123] Sequence number 7 shows the amino acid sequence of the heavy chain variable region of antibody #19, and sequence number 8 shows the amino acid sequence of the light chain variable region of antibody #19. In addition, sequence numbers 1 to 3 show the amino acid sequences of heavy chain CDR1 to 3 of antibody #19, respectively, and sequence numbers 4 to 6 show the amino acid sequences of light chain CDR1 to 3 of antibody #19, respectively.

[0124] [Experimental Example 6]

[0125] (Confirmation of PTK7 expression in HCT116 cells using flow cytometry)

[0126] The expression of PTK7 was confirmed in HCT116 cells, a human colon adenocarcinoma cell line. 6 Add #19 antibody (concentration 10μg / mL, diluted with 100μL PBS) to the cell suspension of HCT116 cells and let it stand on ice for 15 minutes. In addition, as a control, the same operation was performed using an isotype control antibody. After washing the HCT116 cells with PBS, add Alexa Fluor 488-labeled secondary antibody (product name "anti-rat IgG Alexa Fluor488", ThermoFisher Scientific) and let it stand on ice for 15 minutes. After washing the HCT116 cells with PBS, resuspend them and filter them using a filter with a pore size of 40μm (BD Biosciences). Next, use a flow cytometer (product name "BDLSRFortessa", BD Biosciences) to measure the fluorescence of Alexa Fluor 488.

[0127] Figure 3 It is a graph showing the results of flow cytometry. The vertical axis represents the number of cells (relative value), and the horizontal axis represents the fluorescence intensity (relative value). Figure 3 In the table, "anti-PTK7#19" indicates the result of the #19 antibody, and "Isotype control" indicates the result of the isotype control antibody. As a result, it was confirmed that HCT116 cells expressed PTK7.

[0128] [Experimental Example 7]

[0129] (Study on the reactivity of #19 antibody with HCT116 cells)

[0130] Immunofluorescence cell staining was used to study the reactivity of antibody #19. A coverslip was placed in a well of a 12-well plate and 1×10 5 Then, the cells were cultured in an incubator at 37°C and 5 v / v% CO2 for 24 hours.

[0131] Add the primary antibody dilution to the wells and leave overnight in an incubator at 37°C and 5 v / v% CO2. As the primary antibody dilution, use the #19 antibody diluted to 20 μg / mL with PBS. In addition, as a control, use the rat IgG2a isotype control antibody diluted to 20 μg / mL with PBS.

[0132] HCT116 cells were washed three times with PBS, 4 w / v% polyoxymethylene was added, and the cells were fixed by standing in an incubator at 37°C for 10 minutes. Then, a blocking solution was added and the cells were left standing at room temperature for 30 minutes. As the blocking solution, skim milk (Snow Brand Milk Products Co., Ltd.) diluted to 5 w / v% with PBS was used.

[0133] HCT116 cells were washed three times with PBS. Then, a secondary antibody diluent was added and the cells were left standing at room temperature for 30 minutes. As a secondary antibody diluent, anti-rat IgG Alexa Fluor 488 (Thermo Fisher Scientific) diluted with PBS was used. HCT116 cells were washed three times with PBS. 4',6-diamidino-2-phenylindole (DAPI) was added to stain the cell nuclei, and HCT116 cells were washed three times with PBS.

[0134] The cover glass was removed and mounted on a slide glass using a mounting medium (product name: "VECTASHIELD Mounting Medium", Vector Laboratories).

[0135] The stained HCT116 cells were observed using a fluorescence microscope (product name "BZ-9000", Keyence Corporation). Figure 4 This is a fluorescence microscope photo. Figure 4 The left side shows the results using antibody #19. Figure 4 The right side shows the results using isotype control antibodies. The results show that the #19 antibody reacts with PTK7 expressed on the cell membrane of HCT116 cells.

[0136] [Experimental Example 8]

[0137] (Epitope Analysis)

[0138] The #19 antibody was immobilized on magnetic particles (product name "Magnoshere MS300 / Carboxyl", JSR Life Sciences) using a covalent bonding method based on amino coupling. The #19 antibody was reacted with an antigen (PTK7, sequence number 11) or a synthetic peptide (a peptide consisting of 38 amino acid residues of EIEDMPLFEPRVFTAGSEERVTCLPPKGLPEPSVWWEH (sequence number 15)), and then the antigen was decomposed using a digestive enzyme. After decomposition, the fragment of PTK7 bound to the #19 antibody was extracted, desalted, and then analyzed by mass spectrometry using high performance liquid chromatography (UPLC, product name "ACOQITY M-CLASS Nano LC", Waters).

[0139] The peaks inferred as epitopes were determined by analysis based on the total ion current chromatogram (Total Ion Chromatogram) for the results of mass spectrometry. In addition, the results of mass spectrometry were analyzed by PEAKS (BSI) to determine the epitopes. For the validity of the experimental results, the comprehensive computational chemistry system MOE (Molecular Operating Environment, CCG) was used for modeling analysis.

[0140] Based on these results, it was evaluated that the epitope for PTK7 against the #19 antibody included DVTGEEAR (SEQ ID NO: 16) at positions 331 to 310 of the amino acid sequence of any one of SEQ ID NOs: 9 to 14 and / or VFTAGSEER (SEQ ID NO: 17) at positions 104 to 113 of the amino acid sequence of SEQ ID NOs: 9 to 14.

[0141] [Experimental Example 9]

[0142] (Preparation of chimeric antibodies)

[0143] Chimeric antibody heavy chain plasmid DNA was prepared by inserting the gene fragment of the heavy chain variable region of the #19 antibody determined in Experimental Example 5 into the plasmid DNA of the human antibody heavy chain (IgG1). In addition, the gene fragment of the light chain variable region was inserted into the plasmid DNA of the human antibody light chain to prepare a chimeric antibody light chain plasmid DNA.

[0144] Next, the genes of the plasmid DNA of the chimeric antibody heavy chain and the chimeric antibody light chain were introduced into a Chinese hamster ovary-derived CHO cell line. The culture supernatant of the gene-introduced CHO cells was purified, and a chimeric antibody (hereinafter, sometimes referred to as "#19 chimeric antibody") was obtained from the culture supernatant.

[0145] [Experimental Example 10]

[0146] (Confirmation of the binding activity of chimeric antibody #19 to human PTK7 by ELISA)

[0147] The binding activity of the #19 chimeric antibody to human PTK7 was studied by the same operation as in Experimental Example 3, except that the primary antibody diluent used a solution prepared by diluting the #19 chimeric antibody with buffer 2 to 0.001 to 1 μg / mL, and the secondary antibody diluent used a solution prepared by diluting the product name "Goat anti-human IgG H&L (HRP)" (MBL) with buffer 2. In addition, the binding activity of the #19 antibody to human PTK7 was also measured by the same operation as in Experimental Example 3 for comparison.

[0148] Figure 5: is a graph showing the absorbance measurement results. The vertical axis represents the absorbance, and the horizontal axis represents the antibody concentration (μg / mL). Figure 5 In the figure, "rat antibody" means antibody #19, and "human chimeric antibody" means chimeric antibody #19. The results show that chimeric antibody #19 exhibits the same binding activity to human PTK7 fragment as antibody #19.

[0149] [Experimental Example 11]

[0150] (Studying PTK7 expression in blood cancer cells)

[0151] The expression of PTK7 in blood cancer cells was studied. As blood cancer cells, MOLT-4, a cell line derived from a human acute lymphoblastic leukemia patient, CCRF-CEM, a cell line derived from a human acute lymphoblastic leukemia patient, and Jurkat, a cell line derived from a human T-cell leukemia patient, were used.

[0152] In the presence of 1×10 6 In each blood cancer cell sample, the #19 antibody was used as a primary antibody and reacted with the Dylight 488-labeled secondary antibody as a secondary antibody to prepare the Dylight 488-labeled #19 antibody.

[0153] In addition, a sample reacted with Dylight 488-labeled isotype control antibody was prepared for comparison.

[0154] After washing with PBS, the cells were resuspended and filtered using a filter with a pore size of 40 μm (BD Biosciences). Next, the fluorescence of Dylight 488 was measured using a flow cytometer (product name: "BD LSRFortessa", BD Biosciences).

[0155] Figure 6 It is a graph showing the results of flow cytometry. The vertical axis represents the number of cells (relative value), and the horizontal axis represents the fluorescence intensity (relative value). Figure 6 In the table, “Isotype control” indicates the result of isotype control antibody, and “anti-PTK7#19” indicates the result of #19 antibody. Figure 6 The left side shows the results of MOLT-4 cells. Figure 6 The center is the result of CCRF-CEM cells. Figure 6 The right side shows the results of Jurkat cells.

[0156] As a result, it was confirmed that MOLT-4 cells, CCRF-CEM cells, and Jurkat cells expressed PTK7.

[0157] [Experimental Example 12]

[0158] (Confirmation of PTK7 expression in MOLT-4 cell transplanted SCID mice)

[0159] SCID mice were created and MOLT-4 cells were transplanted into the peritoneal cavity to confirm the expression of PTK7 in the peritoneal cells. 7 MOLT-4 cells were transplanted into the peritoneal cavity of SCID mice (severe combined immunodeficient mice, female, 8 weeks old), and the cells were recovered by peritoneal washing 28 days after transplantation.

[0160] In the presence of 1×10 6 Dylight 488-labeled #19 antibody, PE-labeled anti-human CD45 antibody (BioLegend), and BV421-labeled anti-mouse CD45 antibody (BD Biosciences) were added to the sample of 1000 peritoneal cells and the mixture was allowed to stand on ice for 15 minutes.

[0161] After washing with PBS, the mixture was resuspended and filtered through a filter with a pore size of 40 μm (BD Biosciences). The fluorescence of Dylight 488, PE, and BV421 was measured using a flow cytometer (product name: "BD LSRFortessa", BD Biosciences).

[0162] Figure 7 is a graph showing the results of flow cytometry. Figure 7 The center is a graph showing the expression of human CD45 and mouse CD45 in peritoneal cells derived from SCID mice into which MOLT-4 cells were transplanted. As a result, 0.74% of human CD45-positive and mouse CD45-negative cells were detected. These cells are MOLT-4 cells.

[0163] Figure 7 The left side shows Figure 7 The center is a graph of cells with a gate for human CD45-positive and mouse CD45-negative cells and Dylight 488-labeled #19 antibody. The vertical axis represents the number of cells (relative value) and the horizontal axis represents the fluorescence intensity (relative value). In addition, the staining results based on the isotype control antibody are also shown for comparison.

[0164] As a result, it was confirmed Figure 7 The human CD45-positive mouse CD45-negative cells in the center showed reactivity to Dylight 488-labeled #19 antibody and expressed PTK7.

[0165] Figure 7The right side is a graph showing the expression of human CD45 and mouse CD45 in peritoneal cells derived from SCID mice into which MOLT-4 cells were not transplanted. The results confirmed the absence of human CD45-positive mouse CD45-negative cells.

[0166] [Experimental Example 13]

[0167] (Evaluation of Anti-Cancer Activity of Antibody #19 in HCT116 Cell-Transplanted Humanized Mice)

[0168] Anti-cancer activity was evaluated by administering #19 antibody to humanized mice transplanted with HCT116 cells. As humanized mice, NOG mice transplanted with cord blood-derived CD34-positive cells (hematopoietic stem cell transplanted NOG mice) and NOG mice were used.

[0169] Each humanized mouse was anesthetized with isoflurane (Pfizer) and 1×10 6 HCT116 cells. Then, 5 mg / kg of #19 antibody or rat IgG2a isotype control antibody was administered intravenously in a once-weekly regimen. Then, the size of the viable tumor was measured, and the tumor volume was calculated by the following formula (1).

[0170] Tumor volume = (maximum diameter) × (minimum diameter) × (minimum diameter) / 2…(1)

[0171] Figure 8 This is a graph showing the results of measuring the temporal changes in tumor volume. Figure 8 The upper layer shows the results of hematopoietic stem cell transplantation in NOG mice. Figure 8 The lower panel shows the results of NOG mice. Figure 8 In the table, "anti-PTK7" indicates the result of administration of antibody #19. In addition, "isotype ctrl" indicates the result of administration of isotype control antibody. In addition, "*" indicates that there is a significant difference when p < 0.05.

[0172] Fig. 9 It is a graph showing the tumor volume of mice in each group 42 days after HCT116 cell transplantation. Fig. 9 The upper panel shows the results of each group of mice given the #19 antibody. Fig. 9 The lower panel shows the results of each group of mice administered with isotype / control antibodies. Fig. 9 In the upper layer, “*” indicates a significant difference at p < 0.05.

[0173] The results showed that when the #19 antibody was administered to NOG mice transplanted with hematopoietic stem cells, the increase in tumor volume was significantly suppressed. These results indicate that the #19 antibody has anticancer activity.

[0174] Fig.10 This is a graph showing the results of time-dependent observation of the survival rate of NOG mice transplanted with hematopoietic stem cells. Fig.10 In the graph, the vertical axis represents the survival rate (%), and the horizontal axis represents the number of days of survival (days). In addition, "anti-PTK7" shows the result of administration of the #19 antibody, and "isotype ctrl" shows the result of administration of the isotype control antibody.

[0175] The results, such as Fig.10 As shown, the survival time of mice in the group treated with #19 was prolonged. This result indicates that the #19 antibody has anticancer activity.

[0176] Industrial Applicability

[0177] According to this embodiment, an antibody or a fragment thereof against human PTK7 can be provided.

Claims

1. An antibody or fragment thereof, wherein the heavy chain variable region is V H The complementarity determining regions of the region, namely CDR-H1 to CDR-H3, respectively comprise the amino acid sequences set forth in SEQ ID NOs: 1 to 3, and the light chain variable region, namely V L The complementarity determining regions of the region, namely CDR-L1 to CDR-L3, include the amino acid sequences set forth in SEQ ID NOs: 4 to 6, respectively.

2. The antibody or fragment thereof according to claim 1, wherein The V H The CDR-H1 to CDR-H3 of the region are composed of the amino acid sequences described in SEQ ID NOs: 1 to 3, respectively, and the V L The regions CDR-L1 to CDR-L3 are composed of the amino acid sequences set forth in SEQ ID NOs: 4 to 6 in a different order.

3. The antibody or fragment thereof according to claim 1, wherein The V H The region comprises the amino acid sequence recorded in SEQ ID NO: 7, wherein the V H CDR-H1 to CDR-H3 in the region are the amino acid sequences at positions 50 to 54, the amino acid sequences at positions 69 to 85, and the amino acid sequences at positions 118 to 122, respectively, of the amino acid sequence described in SEQ ID NO:

7. The V L The region comprises the amino acid sequence set forth in SEQ ID NO: 8, wherein the V L CDR-L1 to CDR-L3 in the region are the amino acid sequences at positions 49 to 59, 75 to 81, and 114 to 122 of the amino acid sequence described in SEQ ID NO: 8, respectively.

4. The antibody or fragment thereof according to claim 3, wherein The V H The region consists of the amino acid sequence described in SEQ ID NO: 7, wherein the V L The region consists of the amino acid sequence described in SEQ ID NO:

8.

5. The antibody or fragment thereof according to claim 4, wherein The V H FR H With respect to the FR in the continuous amino acid sequence described in SEQ ID NO: 7 H have at least 80% sequence homology, The V L FR L With respect to the FR in the continuous amino acid sequence described in SEQ ID NO: 8 L Have at least 80% sequence homology. The antibody or fragment thereof according to any one of claims 1 to 5, which binds to human PTK7.

7. An antibody or a fragment thereof that binds to an epitope comprising the amino acid sequence at positions 310 to 331 and the amino acid sequence at positions 104 to 113 of the amino acid sequence described in any one of SEQ ID NOs: 9 to 14.

8. The antibody or fragment thereof according to claim 7, wherein The epitope consists of the amino acid sequence at positions 310 to 331 of the amino acid sequence described in any one of SEQ ID NOs: 9 to 14.

9. The antibody or fragment thereof according to claim 7, wherein The epitope consists of the amino acid sequence at positions 104 to 113 of the amino acid sequence described in any one of SEQ ID NOs: 9 to 14. 10 . The antibody or fragment thereof according to claim 7 , which binds to an epitope comprising the amino acid sequence at positions 310 to 331 and the amino acid sequence at positions 104 to 113 of the amino acid sequence described in SEQ ID NO:

11.

11. The antibody or fragment thereof according to claim 7, wherein The epitope is composed of the amino acid sequence at positions 310 to 331 of the amino acid sequence described in SEQ ID NO:

11.

12. The antibody or fragment thereof according to claim 7, wherein The epitope is composed of the amino acid sequence at positions 104 to 113 of the amino acid sequence described in SEQ ID NO:

11. 13 . An antibody or a fragment thereof which competes with the antibody or a fragment thereof according to any one of claims 1 to 5 when binding to human PTK7.

14. The antibody or fragment thereof according to claim 13, wherein The human PTK7 is a protein consisting of the amino acid sequence described in any one of SEQ ID NOs: 9 to 14.

15. The antibody or fragment thereof according to claim 14, wherein The human PTK7 is a protein consisting of the amino acid sequence set forth in SEQ ID NO:

11. 16 . The antibody or fragment thereof according to any one of claims 1 to 5 and 7 to 12, which is a chimeric antibody, a humanized antibody or a human antibody.

17. An anticancer agent comprising the antibody or fragment thereof according to any one of claims 1 to 5 and 7 to 12 as an active ingredient.

18. An anticancer pharmaceutical composition comprising the anticancer agent according to claim 17 and a pharmaceutically acceptable carrier.

Images