Humanized Anti-CKAP4 antibody or antigen-binding fragment thereof

Abstract

The present invention addresses the problem of providing a humanized anti-CKAP4 antibody, or an antigen-binding fragment thereof, having good binding properties with respect to CKAP4 and also having an anti-tumor action. The problem is solved by a humanized anti-CKAP4 antibody, or an antigen-binding fragment thereof, comprising: a heavy chain variable region that includes a heavy chain CDR1 having the amino acid sequence represented by SEQ ID NO: 1 or 6, a heavy chain CDR2 having the amino acid sequence represented by SEQ ID NO: 2 or 7, and a heavy chain CDR3 having the amino acid sequence represented by SEQ ID NO: 3 or 8, and that includes an amino acid sequence having at least 90% identity to the amino acid sequence represented by SEQ ID NO: 4 or 9; and a light chain variable region that includes a light chain CDR1 having the amino acid sequence represented by SEQ ID NO: 11, a light chain CDR2 having the amino acid sequence represented by SEQ ID NO: 12, and a light chain CDR3 having the amino acid sequence represented by SEQ ID NO: 13, and that includes an amino acid sequence having at least 90% identity to the amino acid sequence represented by SEQ ID NO: 14.

Description

Humanized anti-CKAP4 antibody or antigen-binding fragment thereof

[0001] The present invention relates to a humanized anti-CKAP4 antibody or an antigen-binding fragment thereof.

[0002] Dickkopf1 (DKK1) is an extracellular protein that inhibits Wnt signaling. DKK1 binds to the Wnt receptor LRP5 / 6, its ligand, and inhibits Wnt signaling either competitively or by sequestering LRP5 / 6 from the cell membrane. DKK1 has been known to promote the proliferation of various cancers, but the molecular mechanism behind this has remained unclear for many years. The inventors identified cytoskeleton-associated protein 4 (CKAP4) as a DKK1 receptor. CKAP4 was originally reported to be a type II transmembrane protein primarily present in the endoplasmic reticulum (ER) and involved in maintaining ER structure. By binding to CKAP4 localized on the cell membrane, DKK1 activated the phosphatidylinositol 3-kinase (PI3K)-AKT pathway, promoting cancer cell proliferation. Co-expression of DKK1 and CKAP4 was associated with poor prognosis in pancreatic, lung, esophageal, and liver cancers.

[0003] CKAP4 knockout (KO) mice are phenotypically normal and indistinguishable from heterozygote and wild-type littermates from birth to 18 months of age. Detailed histological analysis of various organs in CKAP4 KO mice revealed no differences from wild-type mice. Therefore, it was expected that CKAP4-targeted therapy would not result in severe side effects, and a mouse anti-CKAP4 monoclonal antibody was generated (Patent Document 1). The mouse anti-CKAP4 antibody inhibited the binding of DKK1 to CKAP4 and inhibited AKT activation, cell proliferation, and migration in pancreatic, lung, esophageal, and liver cancer cell lines. Furthermore, the mouse anti-CKAP4 antibody suppressed xenograft tumor formation by these cancer cells in nude mice (T cell immunodeficient mice) and extended the survival time of mice injected intraperitoneally with pancreatic cancer cells.

[0004] Furthermore, the inventors discovered that the transmembrane protein CKAP4 is secreted by pancreatic and lung cancer cells along with extracellular vesicles (also known as exosomes). CKAP4 incorporation into exosomes is mediated by a DKK1-dependent endocytosis pathway and requires exosome biogenesis molecules. The inventors developed an ELISA using a mouse anti-CKAP4 antibody to detect CKAP4 secreted from tumors. Analysis of patient samples using the CKAP4 ELISA revealed that serum CKAP4 levels were elevated in pancreatic and lung cancer patients compared with healthy controls. Furthermore, serum CKAP4 levels were higher in patients whose cancer tissues were immunohistochemically positive for CKAP4 than in patients whose cancer tissues were immunohistochemically negative. Furthermore, comparison of CKAP4 levels in the serum of the same patient before and after surgery revealed that postoperative serum CKAP4 levels were lower than preoperative serum CKAP4 levels. Anti-CKAP4 antibodies are theoretically expected to suppress the growth of cancer cells that express CKAP4 on the cell membrane and secrete DKK1, so detecting CKAP4 and DKK1 in serum is expected to be useful in predicting patients who will respond to treatment with anti-CKAP4 antibodies.

[0005] International Publication No. 2019 / 065747

[0006] Anti-CKAP4 antibodies are promising research and diagnostic tools, but none have been approved as therapeutic antibodies for cancer. To successfully develop mouse anti-CKAP4 antibodies as therapeutics, the antibodies must be humanized, which reduces the likelihood of patients developing neutralizing antibodies against non-human molecules. However, humanization often results in loss of binding affinity or the desired in vivo activity.

[0007] An objective of the present invention is to provide a humanized anti-CKAP4 antibody or an antigen-binding fragment thereof that has good binding affinity to CKAP4 and antitumor activity.

[0008] The present inventors have conducted extensive research in light of the above-mentioned problems and have found that the above-mentioned problems can be solved by a humanized anti-CKAP4 antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1 or 6, a heavy chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 2 or 7, and a heavy chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 3 or 8, and comprising an amino acid sequence that is 90% or more identical to the amino acid sequence shown in SEQ ID NO: 4 or 9, and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 11, a light chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 12, and a light chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 13, and comprising an amino acid sequence that is 90% or more identical to the amino acid sequence shown in SEQ ID NO: 14. Based on this finding, the present inventors have conducted further research and have completed the present invention. That is, the present invention encompasses the following aspects.

[0009] Item 1. A humanized anti-CKAP4 antibody or an antigen-binding fragment thereof, comprising: a heavy chain variable region comprising: a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1 or 6, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2 or 7, and a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3 or 8, and comprising an amino acid sequence having 90% or more identity to the amino acid sequence set forth in SEQ ID NO: 4 or 9; and a light chain variable region comprising: a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13, and comprising an amino acid sequence having 90% or more identity to the amino acid sequence set forth in SEQ ID NO: 14.

[0010] Item 2. The humanized anti-CKAP4 antibody or antigen-binding fragment thereof according to Item 1, wherein the identity is 95% or more.

[0011] Item 3. The humanized anti-CKAP4 antibody or antigen-binding fragment thereof according to Item 1 or 2, wherein the identity is 100%.

[0012] Item 4. The humanized anti-CKAP4 antibody or antigen-binding fragment thereof according to any one of Items 1 to 3, comprising a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 4 or 9, and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 14.

[0013] Item 5. The humanized anti-CKAP4 antibody or antigen-binding fragment thereof according to any one of Items 1 to 4, comprising: a heavy chain constant region comprising an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 16; and a light chain constant region comprising an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 17.

[0014] Item 6. A polynucleotide comprising a coding sequence for the humanized anti-CKAP4 antibody or antigen-binding fragment thereof according to any one of Items 1 to 5.

[0015] Item 7. The polynucleotide according to Item 6, which is an expression vector.

[0016] Item 8. A cell containing the polynucleotide according to Item 6 or 7.

[0017] Item 9. A pharmaceutical composition comprising the humanized anti-CKAP4 antibody or antigen-binding fragment thereof according to any one of Items 1 to 5.

[0018] Item 10. A reagent comprising the humanized anti-CKAP4 antibody or antigen-binding fragment thereof according to any one of Items 1 to 5.

[0019] Item 11. An antitumor agent comprising the humanized anti-CKAP4 antibody or antigen-binding fragment thereof according to any one of Items 1 to 5.

[0020] Item 11A: A method for treating cancer, comprising administering the humanized anti-CKAP4 antibody or antigen-binding fragment thereof according to any one of Items 1 to 5 to a subject having cancer.

[0021] Item 11B: The humanized anti-CKAP4 antibody or antigen-binding fragment thereof according to any one of Items 1 to 5, for use in treating cancer.

[0022] Item 11C: Use of the humanized anti-CKAP4 antibody or antigen-binding fragment thereof according to any one of Items 1 to 5 for the production of an antitumor agent.

[0023] Item 11D: Use of the humanized anti-CKAP4 antibody or antigen-binding fragment thereof according to any one of Items 1 to 5 as an antitumor agent.

[0024] Item 12. The antitumor agent according to Item 11, wherein the humanized anti-CKAP4 antibody or antigen-binding fragment thereof is in a conjugate with a drug or is for use in combined administration with a drug.

[0025] Item 13. An anti-tumor immune response activator comprising the humanized anti-CKAP4 antibody or antigen-binding fragment thereof according to any one of Items 1 to 5.

[0026] Item 13A: A method for activating an anti-tumor immune response, comprising administering the humanized anti-CKAP4 antibody or antigen-binding fragment thereof according to any one of Items 1 to 5 to a subject with cancer.

[0027] Item 13B: The humanized anti-CKAP4 antibody or antigen-binding fragment thereof according to any one of Items 1 to 5, for use in activating anti-tumor immune responses.

[0028] Item 13C: Use of the humanized anti-CKAP4 antibody or antigen-binding fragment thereof according to any one of Items 1 to 5 for the production of an anti-tumor immune response activator.

[0029] Item 13D: Use of the humanized anti-CKAP4 antibody or antigen-binding fragment thereof according to any one of Items 1 to 5 as an anti-tumor immune response activator.

[0030] According to the present invention, it is possible to provide a humanized anti-CKAP4 antibody or an antigen-binding fragment thereof that has good binding affinity to CKAP4 and anti-tumor activity.

[0031] Characterization of silent Fc mouse anti-CKAP4 antibodies (mF18, mFa31) with mutations introduced into the Fc region. (A) S2-CP8 cell lysate (input) was immunoprecipitated with 1 μg / mL of anti-CKAP4 antibodies (3F11-2B10, mF18, mFa31). Input and immunoprecipitates (IP) were detected with rabbit anti-CKAP4 polyclonal antibody. (B) 2 nM GST-CKAP4-ECD (extracellular domain) was pretreated with the indicated concentrations of the labeled anti-CKAP4 antibody or control IgG, and the mixture was then incubated with 2.5 nM DKK1 for 1 hour. The protein mixture was precipitated with glutathione agarose, and the precipitate was detected with anti-DKK1 antibody. (C) Lysates from labeled cells treated with 20 μg / mL of the labeled anti-CKAP4 antibody or control IgG for 4 hours were probed with the labeled antibody. (D) Left panel: S2-CP8 cells treated with 20 μg / mL of the labeled anti-CKAP4 antibody or control IgG were cultured in 3D Matrigel for 5 days. Right panel: The total area of ​​tumor spheres in three fields of view was measured and displayed as a dot plot. Tumor sphere size is expressed as a ratio to the untreated condition (median set at 1). *, P < 0.05 (Student's t-test). (E) S2-CP8 cells were subcutaneously implanted into both dorsal flanks of immunodeficient mice. Mice were intraperitoneally injected twice weekly with 200 μg / dose of silent Fc mouse anti-CKAP4 antibody (N = 15) or control IgG (N = 11). Excised xenograft tumors (left panel), tumor volume (middle panel), and tumor weight (right panel) are shown. *, P<0.05; **, P<0.01 (Student's t-test); Scale bars, 100 μm (D), 10 mm (E). Results of an in vivo tumor growth inhibition study using the humanized anti-CKAP4 antibody Hv1Lt1 are shown. S2-CP8 cells (A and B) or MIA PaCa-2 cells (C) were subcutaneously implanted into the dorsal flanks of immunodeficient mice. The humanized anti-CKAP4 antibody Hv1Lt1 or control IgG was injected intraperitoneally (A, 200 μg / injection) or via the tail vein (B and C, 400 μg / injection) twice weekly. The excised xenograft tumors (upper panel), tumor volume (lower left panel), and tumor weight (lower right panel) are shown.N indicates the number of tumors in each assay condition. *, P < 0.05; **, P < 0.01 (Student's t-test). Scale bar, 10 mm. (A) S2-CP8 cells were subcutaneously inoculated into the dorsal flanks of immunodeficient mice and randomly assigned to one of four groups: control (control IgG + phosphate-buffered saline (PBS)), Hv1Lt1 (Hv1Lt1 + PBS), GEM (control IgG + gemcitabine), and GEM + Hv1Lt1 (Hv1Lt1 + gemcitabine). Tumor growth was monitored for 15 days while each mouse received intraperitoneal injections of 280 μg of antibody twice weekly and 100 μg of gemcitabine (or PBS) once weekly. (B) S2-CP8 cells were inoculated subcutaneously into the dorsal flanks of immunodeficient mice and randomly assigned to one of four groups: control (control IgG + saline), Hv1Lt1 (Hv1Lt1 + saline), nab-PTX (control IgG + nab-PTX), or nab-PTX + Hv1Lt1 (Hv1Lt1 + nab-PTX). Mice were intraperitoneally injected with 280 μg of antibody and 10 mg / kg of nab-PTX (or saline) twice weekly, and tumor growth was monitored for 22 days. (C) Hep3B cells were inoculated subcutaneously into the dorsal flanks of immunodeficient mice and randomly assigned to one of four groups: control (control IgG + 0.5% (wt / vol) methylcellulose 400 solution), Hv1Lt1 (Hv1Lt1 + 0.5% (wt / vol) methylcellulose 400 solution), lenvatinib (control IgG + lenvatinib), and Hv1Lt1 + lenvatinib (Hv1Lt1 + lenvatinib). Each mouse received oral administration of 1 mg / kg lenvatinib or 0.5% (wt / vol) methylcellulose 400 solution once daily, 6 days per week, and intraperitoneal administration of 280 μg of antibody twice weekly. Tumor growth was monitored for 15 days. The excised xenograft tumors (left panel), tumor volumes (left center panel), and body weights (right panel) are shown. N indicates the number of tumors for each assay condition. *, P<0.05; **, P<0.01 (ANOVA post-hoc test); scale bar, 10 mm.Hv1Lt1 inhibits the growth of pancreatic cancer organoids in vitro and in vivo. (A) Wild-type (WT) KPC organoids (organoids derived from pancreatic cancers developed in LSL-KrasG12D / +;Trp53fl / fl;Elastase-Cre mice) and KPC organoids stably expressing CKAP4 and DKK1 (KPC / CKAP4 / DKK1) were biotinylated, and cell surface proteins were precipitated using NeutrAvidin agarose beads. The precipitates (membranes) and cell lysates were probed with the indicated antibodies. (B) WT KPC organoids and KPC / CKAP4 / DKK1 organoids were dissociated into single cells, mounted in Matrigel, and cultured in complete medium. Organoids are shown on day 4 (three fields per organoid). (C) Left panel: KPC / CKAP4 organoids were cultured in 3D Matrigel with 10 ng / mL purified mouse DKK1-FLAG for 5 days. After the organoids were embedded in Matrigel, 10 μg / mL of Hv1Lt1 or control IgG was added to the culture medium. Right panel: The total area of ​​organoid spheres per field (N = 3 fields, top 50 spheres per well) was measured, and the area of ​​each sphere is displayed as a dot plot. Each bar represents the mean value. **, P < 0.01 (ANOVA post-hoc test). (D) KPC / CKAP4 / DKK1 organoids were orthotopically transplanted into the pancreatic tail of immunodeficient mice. Hv1Lt1 or control IgG was injected intraperitoneally (280 μg / body) twice a week. Tumor development was monitored for 56 days. Figures show excised tumors (upper panel), macroscopic appearance of KPC tumors (lower left panel, red arrow), hematoxylin-eosin-stained resected tumor sections (lower center), and tumor volume progression (lower right panel). N indicates the number of tumors assayed. Results are expressed as mean ± standard error. *, P < 0.05 (Student's t-test); scale bars, 500 μm (B and C) and 10 mm (D). Transcriptome analysis of pancreatic tumors treated with Hv1Lt1 is shown. (A) The heat map shows the z-score for each row (gene) of gene expression data (four control tumors and four Hv1Lt1-treated tumors used in Figure 4D).The results of hierarchical clustering are shown in a dendrogram. (B) PCA (principal component analysis) plot of the RNA-seq data. (C) Heatmap showing the z-scores of each row (gene) of gene expression data for 421 differentially expressed genes (DEGs). 88 DEGs were increased in Hv1Lt1-treated tumors, while 333 DEGs were decreased. (D) Results of GO molecular function enrichment analysis for the DEGs in Figure 5C are shown. FDR: false discovery rate; nGenes: number of DEGs included in the indicated pathway. (E) Immunohistochemical staining experiments were performed on tissue sections from 12 control tumors and 10 Hv1Lt1-treated tumors to assess the number of immune cells infiltrating the tumors. Upper panel: HE-stained images, anti-CD8 antibody stained images (for CD8-positive T cells), and anti-CD11c antibody stained images (for dendritic cells) of one representative tumor from each of the control tumor and Hv1Lt1-treated tumors are shown. Lower panel: The number of CD8+ and CD11c+ cells in the total number of cells in the tumor was measured and displayed as a dot plot. Comparisons were performed between control and Hv1Lt1-treated tumors. *, P < 0.05 (Student's t-test). Scale bar: 250 μm. (E) The tumor growth suppression effect of Hv1Lt1 in combination with standard chemotherapy is shown. (A) Left panel: S2-CP8 cells were treated with 20 μg / mL of the indicated anti-CKAP4 antibody or control IgG and 1 nM gemcitabine (GEM) or an equal volume of PBS in the medium and cultured in 3D Matrigel for 6 days. Right panel: The area of ​​the top 150 largest tumorspheres in each of three observation fields was measured and displayed as a dot plot. **, P < 0.01 (ANOVA post-hoc test). (B) Left panel: S2-CP8 cells were cultured in 3D Matrigel for 7 days after treatment with 20 μg / mL of the indicated anti-CKAP4 antibody or control IgG and 10 nM nanoparticulate albumin-bound paclitaxel (nab-PTX) or an equivalent volume of saline in the culture medium. Right panel: The areas of the top 150 tumorspheres in each of the three fields were measured and displayed as dot plots. **, P < 0.01 (ANOVA post-hoc test).(C, D) Mice were inoculated subcutaneously with S2-CP8 cells in both dorsal flanks and randomly assigned to one of four groups. They received either 2 mg / kg gemcitabine once weekly (C) or 10 mg / kg nab-PTX twice weekly (D) along with 280 μg Hv1Lt1 twice weekly. Tumor development was monitored for 21 days. Tumor volume (left panel) and body weight (right panel) are shown. N indicates the number of tumors in each assay. Results are expressed as mean ± standard error; *, P < 0.05; **, P < 0.01 (ANOVA post-hoc test); scale bar, 10 mm.

[0032] In this specification, the expressions "contain" and "comprise" include the concepts of "contain", "include", "consist essentially of" and "consist only of".

[0033] 1. Antibody or Antigen-Binding Fragment Thereof In one aspect, the present invention relates to a humanized anti-CKAP4 antibody or an antigen-binding fragment thereof (sometimes referred to herein as the "antibody of the present invention"). This is described below.

[0034] The antibody of the present invention has binding activity to CKAP4.

[0035] CKAP4 is an expression product of the CKAP4 gene, for example, CKAP4 as a protein expressed in an organism. The species from which CKAP4 is derived is not particularly limited, and includes animals, such as various mammals such as humans, monkeys, mice, rats, dogs, cats, rabbits, pigs, horses, cows, sheep, goats, and deer. Among these, humans are particularly preferred.

[0036] The amino acid sequences of CKAP4 derived from various species are known or can be easily identified by identity analysis based on known amino acid sequences. Specifically, for example, human CKAP4 includes a protein consisting of the amino acid sequence set forth in SEQ ID NO: 25.

[0037] CKAP4 may have amino acid mutations such as substitutions, deletions, additions, insertions, etc. that may exist between individuals. Mutations are preferably substitutions, more preferably conservative substitutions.

[0038] As used herein, the term "conservative substitution" refers to the substitution of an amino acid residue with an amino acid residue having a similar side chain. For example, substitution between amino acid residues having basic side chains such as lysine, arginine, and histidine constitutes a conservative substitution. Other examples of conservative substitutions include substitution between amino acid residues having acidic side chains such as aspartic acid and glutamic acid; amino acid residues having uncharged polar side chains such as glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine; amino acid residues having nonpolar side chains such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; amino acid residues having β-branched side chains such as threonine, valine, and isoleucine; and amino acid residues having aromatic side chains such as tyrosine, phenylalanine, tryptophan, and histidine.

[0039] Preferred specific examples of CKAP4 include at least one selected from the group consisting of the proteins described in (a) below and the proteins described in (b) below: (a) a protein consisting of the amino acid sequence shown in SEQ ID NO: 25, and (b) a protein consisting of an amino acid sequence having 85% or more identity with the amino acid sequence shown in SEQ ID NO: 25 and having the ability to interact with the extracellular secretory protein DKK1.

[0040] The presence or absence of the ability to interact with DKK1 can be determined using an in vitro test method that can evaluate the binding between a receptor and a ligand, based on criteria well known to those skilled in the art.

[0041] As used herein, "identity" of amino acid sequences refers to the degree of correspondence between the amino acid sequences of two or more comparable amino acid sequences. Therefore, the greater the identity between two amino acid sequences, the greater the identity or similarity between those sequences. The level of identity between amino acid sequences can be determined, for example, using the sequence analysis tool FASTA with default parameters. Alternatively, it can be determined using the BLAST algorithm by Karlin and Altschul (Karlin S, Altschul SF. "Methods for assessing the statistical significance of molecular sequence features by using general scoring schemes," Proc Natl Acad Sci USA. 87:2264-2268 (1990); Karlin S, Altschul SF. "Applications and statistics for multiple high-scoring segments in molecular sequences," Proc Natl Acad Sci USA. 90:5873-5877 (1993)). A program called BLASTX, based on the BLAST algorithm, has been developed. Specific techniques for these analysis methods are known and can be found on the National Center of Biotechnology Information (NCBI) website (http: / / www.ncbi.nlm.nih.gov / ). The "identity" of nucleotide sequences is also defined in the same manner as above.

[0042] In the above (b), the identity is more preferably 90% or more, even more preferably 95% or more, and even more preferably 98% or more.

[0043] An example of the protein described in (b) above is (b') a protein consisting of an amino acid sequence in which one or more amino acids have been substituted, deleted, added, or inserted relative to the amino acid sequence shown in SEQ ID NO: 25, and which has the ability to interact with the extracellular secretory protein DKK1.

[0044] In the above (b'), "plurality" means, for example, 2 to 20, preferably 2 to 10, more preferably 2 to 5, and even more preferably 2 or 3.

[0045] The antibody of the present invention comprises a heavy chain variable region comprising: a heavy chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1 or 6; a heavy chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 2 or 7; and a heavy chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 3 or 8, and comprising an amino acid sequence that is 90% or more (preferably 95% or more, more preferably 98% or more, even more preferably 99% or more, and particularly preferably 100%) identical to the amino acid sequence shown in SEQ ID NO: 4 or 9; and a light chain variable region comprising: a light chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 11; a light chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 12; and a light chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 13, and comprising an amino acid sequence that is 90% or more (preferably 95% or more, more preferably 98% or more, even more preferably 99% or more, and particularly preferably 100%) identical to the amino acid sequence shown in SEQ ID NO: 14.

[0046] In a particularly preferred embodiment of the present invention, the present invention relates to a humanized anti-CKAP4 antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 4 or 9, and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 14.

[0047] In one embodiment, the antibody of the present invention comprises: (a) a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, a heavy chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 2, and a heavy chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 3, and comprising an amino acid sequence that has 90% or more (preferably 95% or more, more preferably 98% or more, even more preferably 99% or more, and particularly preferably 100%) identity to the amino acid sequence shown in SEQ ID NO: 4; and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 11, a light chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 12, and a light chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 13, and comprising an amino acid sequence that has 90% or more (preferably 95% or more, more preferably 98% or more, even more preferably 99% or more, and particularly preferably 100%) identity to the amino acid sequence shown in SEQ ID NO: 14; or (b) a heavy chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 6, a heavy chain variable region comprising a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:7, and a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:8, and comprising an amino acid sequence that is 90% or more (preferably 95% or more, more preferably 98% or more, even more preferably 99% or more, and particularly preferably 100%) identical to the amino acid sequence set forth in SEQ ID NO:9; and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:11, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:12, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:13, and comprising an amino acid sequence that is 90% or more (preferably 95% or more, more preferably 98% or more, even more preferably 99% or more, and particularly preferably 100%) identical to the amino acid sequence set forth in SEQ ID NO:14.

[0048] From the viewpoint of the ability to inhibit sphere formation, the antibody of the present invention is particularly preferably antibody (a).

[0049] Since the antibody of the present invention is a humanized anti-CKAP4 antibody, the constant region has sequences derived from a human antibody.

[0050] In a preferred embodiment of the present invention, the antibody of the present invention comprises, as a heavy chain signal peptide, a heavy chain signal peptide comprising an amino acid sequence that has 90% or more identity (preferably 95% or more, more preferably 98% or more, even more preferably 99% or more, and particularly preferably 100%) to the amino acid sequence shown in SEQ ID NO: 30.

[0051] In a preferred embodiment of the present invention, the antibody of the present invention comprises, as a light chain signal peptide, a light chain signal peptide comprising an amino acid sequence that has 90% or more identity (preferably 95% or more, more preferably 98% or more, even more preferably 99% or more, and particularly preferably 100%) to the amino acid sequence shown in SEQ ID NO: 31.

[0052] It is particularly preferred that the signal peptide is located at the N-terminus of the heavy / light chain.

[0053] In a preferred embodiment of the present invention, the antibody of the present invention has a human IgG1-derived heavy chain constant region as its heavy chain constant region and a human IgK-derived light chain constant region as its light chain constant region. In a more preferred embodiment of the present invention, the antibody of the present invention comprises: a heavy chain constant region comprising an amino acid sequence that is 90% or more (preferably 95% or more, more preferably 98% or more, even more preferably 99% or more, and particularly preferably 100%) identical to the amino acid sequence set forth in SEQ ID NO: 16; and a light chain constant region comprising an amino acid sequence that is 90% or more (preferably 95% or more, more preferably 98% or more, even more preferably 99% or more, and particularly preferably 100%) identical to the amino acid sequence set forth in SEQ ID NO: 17.

[0054] The constant region is usually located adjacent to the C-terminus of the variable region.

[0055] The term "antibody of the present invention" is used to encompass both monoclonal and polyclonal antibodies. The antibody of the present invention can be of any isotype, such as IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgA (e.g., IgA1, IgA2), IgD, IgE, IgM, etc. The antibody of the present invention is preferably a monoclonal antibody.

[0056] As used herein, the antigen-binding fragment of an antibody is not particularly limited as long as it comprises a heavy chain variable region and a light chain variable region, and examples include Fab, F(ab'), minibody, scFv-Fc, Fv, scFv, diabody, triabody, tetrabody, etc.

[0057] Fab comprises a heavy chain fragment containing a heavy chain variable region and a CH1 fragment in the heavy chain constant region, and a light chain fragment containing a light chain variable region and a light chain constant region (CL), and has a structure in which the heavy chain variable region and the light chain variable region are associated by the above-mentioned noncovalent intermolecular interaction or linked by a disulfide bond. In Fab, CH1 and CL may be disulfide-bonded via the thiol groups of cysteine ​​residues present in each.

[0058] F(ab')2 has two pairs of the above-mentioned Fab, and has a structure in which the CH1s are disulfide-bonded together via the thiol groups of the cysteine ​​residues contained therein.

[0059] A minibody is a structure in which two fragments, each consisting of a heavy chain variable region constituting the scFV described below and a CH3 linked thereto, are associated via non-covalent intermolecular interactions between the CH3s.

[0060] scFv-Fc is a structure in which two antibody fragments containing the scFv, CH2, and CH3 described below are associated by non-covalent intermolecular interactions between the CH3s, similar to the minibody described above, and the thiol groups of the cysteine ​​residues contained in each CH3 are disulfide-bonded.

[0061] Fv, also known as the smallest structural unit of an antibody, is a structure in which the heavy chain variable region and the light chain variable region are associated through non-covalent intermolecular interactions. In Fv, the thiol groups of cysteine ​​residues present in the heavy chain variable region and the light chain variable region may be disulfide-bonded.

[0062] An scFv has a structure in which the C-terminus of a heavy chain variable region and the N-terminus of a light chain variable region are linked by a linker, or a structure in which the N-terminus of a heavy chain variable region and the C-terminus of a light chain variable region are linked by a linker, and is also called a single-chain antibody.

[0063] Diabodies, triabodies, and tetrabodies are structures in which the above-mentioned scFvs form dimers, trimers, and tetramers, respectively, and associate in a structurally stable state through non-covalent intermolecular interactions between the variable regions, similar to Fvs.

[0064] The antibody of the present invention may be bound to or fused with another peptide, oligopeptide, or protein. Examples of other peptides, oligopeptides, or proteins include albumin (e.g., serum albumin), protein tags (e.g., biotin, His tag, FLAG tag, Halo tag, MBP tag, HA tag, Myc tag, V5 tag, and PA tag), fluorescent proteins (e.g., GFP, Azami-Green, ZsGreen, GFP2, HyperPer, Sirius, BFP, CFP, Turquoise, Cyan, TFP1, YFP, Venus, ZsYellow, Banana, KusabiraOrange, RFP, DsRed, AsRed, Strawberry, JRed, KillerRed, and Cherry). y, HcRed, mPlum), luminescent proteins (e.g., luciferase, β-galactosidase, chloramphenicol acetyltransferase, β-glucuronidase), secretory signal sequences (e.g., Igκ signal sequence), protease recognition sequences (e.g., TEV protease recognition sequence), expression-enhancing sequences, solubility sequences, multimerization domains (e.g., cartilage oligomeric matrix protein domain, leucine zipper domain, collagen-like domain, cholera toxin B subunit domain, tetrabrachion coiled core domain, reovirus σ1 protein domain, Hepatitis delta antigen domain), etc. When the antibody of the present invention is bound to or fused with a multimerization domain, an antibody or an antigen-binding fragment thereof of the same or different species as the antibody of the present invention may be further bound to or fused to the multimerization domain to form a multimer (homomultimer or heteromultimer).

[0065] The antibodies of the present invention may be chemically modified as long as their binding ability to CKAP4 is not significantly impaired.

[0066] The antibodies of the present invention may have a C-terminal carboxyl group (-COOH) or a carboxylate group (-COO － ), amide group (-CONH ２ Q in the ester group may be, for example, a C group such as methyl, ethyl, n-propyl, isopropyl, or n-butyl. １－６ Alkyl groups; for example, C groups such as cyclopentyl and cyclohexyl ３－８ Cycloalkyl groups such as phenyl and α-naphthyl ６－１２ Aryl groups; for example, phenyl-C such as benzyl and phenethyl １－２ Alkyl group: α-naphthyl-C such as α-naphthylmethyl １－２ C such as alkyl group ７－１４ Aralkyl groups, pivaloyloxymethyl groups, etc. are used.

[0067] In the antibody of the present invention, a carboxyl group (or carboxylate) other than that at the C-terminus may be amidated or esterified. In this case, the ester used may be, for example, the C-terminal ester described above.

[0068] The antibody of the present invention is a nucleotide sequence in which the amino group of the N-terminal amino acid residue is protected by a protecting group (e.g., a C group such as a formyl group or an acetyl group). １－６ C such as alkanoyl １－６ those in which the N-terminal glutamine residue that can be generated by cleavage in vivo is pyroglutamated; those in which the substituents on the side chains of amino acids in the molecule (e.g., -OH, -SH, amino group, imidazole group, indole group, guanidino group, etc.) are protected by an appropriate protecting group (e.g., C group such as formyl group, acetyl group, etc.); １－６ C such as alkanoyl group １－６ Also included are those protected by an acyl group or other suitable glycan, or conjugated proteins such as glycoproteins to which sugar chains are attached.

[0069] The antibody of the present invention also includes a conjugate to which a drug and / or label is linked. The drug is not particularly limited as long as it has physiological activity. Examples of the drug include proteins, peptides, low-molecular-weight compounds, nucleic acids, sugar chains, etc., and more specifically, examples of the drug include the antitumor agents described below. Examples of the label include radioactive labels, fluorescent labels, etc.

[0070] 2. Methods for Producing Antibodies or Antigen-Binding Fragments Thereof The antibodies of the present invention can be produced by various methods.

[0071] The antibody of the present invention can be produced, for example, by a method comprising the steps of culturing a host transformed with a polynucleotide comprising the coding sequence of the antibody of the present invention (the polynucleotide of the present invention) and recovering a fraction containing the antibody of the present invention.

[0072] The polynucleotide of the present invention is not particularly limited as long as it comprises the coding sequence of the antibody of the present invention. Preferably, the polynucleotide of the present invention comprises the above coding sequence in a state capable of expressing the antibody of the present invention. The polynucleotide of the present invention may comprise other sequences in addition to the above coding sequence. Examples of such other sequences include a secretory signal peptide coding sequence, a promoter sequence, an enhancer sequence, a repressor sequence, an insulator sequence, an origin of replication, and a drug resistance gene coding sequence located adjacent to the antibody coding sequence of the present invention. Furthermore, the polynucleotide of the present invention may be a linear polynucleotide or a circular polynucleotide. In one embodiment, the polynucleotide of the present invention is an expression vector.

[0073] Specific examples of the polynucleotide of the present invention include (I) a polynucleotide comprising a nucleotide sequence encoding at least one selected from the group consisting of the heavy chain and heavy chain variable region of the antibody of the present invention, (II) a polynucleotide comprising a nucleotide sequence encoding at least one selected from the group consisting of the light chain and light chain variable region of the antibody of the present invention, and (III) a polynucleotide comprising a nucleotide sequence encoding at least one selected from the group consisting of the heavy chain and heavy chain variable region of the antibody of the present invention, and a polynucleotide comprising a nucleotide sequence encoding at least one selected from the group consisting of the light chain and light chain variable region of the antibody of the present invention.

[0074] The host is not particularly limited, and examples thereof include prokaryotic cells and eukaryotic cells. Among these, eukaryotic mammalian cells such as HEK293 cells, CHO cells, NS0 cells, and SP2 / O cells are preferred from the viewpoint of more efficient antibody expression. The methods for transformation, culture, and recovery are not particularly limited, and known methods for antibody production can be used. After recovery, the antibody of the present invention may be purified as needed. Purification can be carried out by known methods for antibody production, such as chromatography and dialysis.

[0075] Polynucleotides such as DNA and RNA may be chemically modified, as exemplified below. To prevent degradation by hydrolases such as nucleases, the phosphate residues of each nucleotide may be substituted with chemically modified phosphate residues, such as phosphorothioate (PS), methylphosphonate, or phosphorodithioate. The hydroxyl group at the 2-position of the sugar (ribose) of each ribonucleotide may be substituted with -OR (where R represents, for example, -CH3, -CH2CHOCH3, -CH2CH2NHC(NH)NH2, -CH2CONHCH3, or -CH2CH2CN). Furthermore, the base moiety (pyrimidine or purine) may be chemically modified, such as by introducing a methyl group or a cationic functional group into the 5-position of the pyrimidine base, or by substituting a thiocarbonyl group for the carbonyl group at the 2-position. Modifications of the phosphate moiety or hydroxyl moiety with, for example, biotin, an amino group, a lower alkylamine group, or an acetyl group are also possible, but are not limited to these.

[0076] 3. Cells In one aspect, the present invention relates to cells containing the polynucleotides of the present invention.

[0077] The cells include, for example, cells into which a polynucleotide of the present invention has been introduced.

[0078] The cells are preferably isolated cells. The cells may be cultured cells, and may be primary cultured cells or subcultured cells. The cells may be established cell lines. The cells may be iPS cells.

[0079] Examples of the cells include Escherichia coli K12 and other Escherichia coli, Bacillus bacteria such as Bacillus subtilis MI114, yeast such as Saccharomyces cerevisiae AH22, insect cells such as the Sf cell line derived from Spodoptera frugiperda or the HighFive cell line derived from Trichoplusia ni, animal cells, etc. Preferred animal cells include cultured cells derived from mammals, specifically COS7 cells, CHO cells, HEK293 cells, HEK293FT cells, HeLa cells, PC12 cells, N1E-115 cells, SH-SY5Y cells, etc.

[0080] 4. Uses In one aspect, the present invention relates to pharmaceutical compositions, reagents, antitumor agents, antitumor immune response activators, and the like, which contain the antibodies of the present invention (hereinafter sometimes referred to as "active ingredients").

[0081] (Uses) As disclosed in Patent Document 1, CKAP4 is highly expressed in cancer cells and promotes their proliferation. Therefore, the antitumor agent and antitumor immune response activator of the present invention can inhibit cancer cell proliferation and activate antitumor immune responses by suppressing the expression or function of CKAP4. Therefore, the antitumor agent and antitumor immune response activator of the present invention can be used for the prevention or treatment of cancer. When the antibody of the present invention is used as an antitumor agent or antitumor immune response activator, the cancer to which it can be applied is not particularly limited, and specific examples include solid cancers such as lung cancer, pancreatic cancer, esophageal cancer, colorectal cancer, colon cancer, gastric cancer, rectal cancer, liver cancer, breast cancer, bladder cancer, prostate cancer, cervical cancer, head and neck cancer, bile duct cancer, gallbladder cancer, oral cancer, tongue cancer, pharyngeal cancer, laryngeal cancer, brain tumor, glioma, glioblastoma, hepatoblastoma, glioblastoma multiforme, and peritoneal dissemination; and blood cancers such as leukemia and malignant lymphoma.

[0082] Furthermore, as disclosed in Patent Document 1, anti-CKAP4 antibodies effectively inhibit the proliferation of cancer cells expressing CKAP4, particularly cancer cells expressing both CKAP4 and DKK1, and therefore, cancers expressing CKAP4, particularly cancers expressing both CKAP4 and DKK1, are suitable targets for the antibody of the present invention. Among cancers, lung cancer, pancreatic cancer, esophageal cancer, liver cancer, and hepatoblastoma frequently express high levels of CKAP4 and DKK1, making them particularly suitable targets for the antibody of the present invention.

[0083] The expression of CKAP4 in cancer can be confirmed by immunohistochemical staining of collected cancer tissue. Specifically, collected cancer tissue is immunostained with an anti-CKAP4 antibody, and if the area in which CKAP4 expression is observed is 5% or more in the tumor area, CKAP4 is determined to be expressed. Suitable examples of cancers to which the antibody of the present invention can be applied include cancers in which CKAP4 expression is present in 5% or more of the tumor area, more preferably cancers in which CKAP4 expression is present in 20% or more of the tumor area, and particularly preferably cancers in which CKAP4 expression is present in 50% or more of the tumor area.

[0084] CKAP4 expression in cancer can be confirmed by recovering RNA from collected cancer tissues or body fluids such as serum (test samples) and measuring it by quantitative PCR or immunostaining the cancer tissue. Alternatively, CKAP4 in serum can be measured by ELISA. In this case, the presence or absence of CKAP4 expression can be determined using non-cancerous tissues / body fluids of non-cancer patients (control samples) from the same case as an indicator. Specifically, if the amount of CKAP4 in the test sample (e.g., immunostained cancer tissue) is higher than the amount of CKAP4 in the control sample (e.g., cell lysate of non-cancerous tissue from the same case), it is determined that CKAP4 is highly expressed in the cancer. Furthermore, when using body fluids, it is possible to purify the body fluid (e.g., concentrate extracellular vesicles such as exosomes) before use in the measurement to increase sensitivity.

[0085] CKAP4 expression in lung adenocarcinoma can be evaluated based on specific values, for example, as follows: When serum CKAP4 levels were measured by sandwich ELISA using mouse anti-CKAP4 antibodies, approximately 25% of immunohistologically CKAP4-positive cases had positive serum CKAP4 levels (0.1 ng / ml or higher), whereas 4.5% of CKAP4-negative cases had positive serum CKAP4 levels. Therefore, it can be inferred that CKAP4 is highly expressed in serum CKAP4-positive cases.

[0086] CKAP4 expression in pancreatic cancer can be evaluated based on specific values, for example, as follows: When serum CKAP4 levels were measured by sandwich ELISA using mouse anti-CKAP4 antibodies, approximately 64% of immunohistologically CKAP4-positive cases had positive serum CKAP4 levels (1 ng / ml or higher), whereas no CKAP4-negative cases had positive serum CKAP4 levels. Therefore, it can be inferred that CKAP4 is highly expressed in serum CKAP4-positive cases.

[0087] The expression of DKK1 in cancer can be confirmed in the same manner as in the case of CKAP4, for example, by tissue immunization of collected cancer tissue, or by recovering RNA from collected cancer tissue and measuring it by quantitative PCR. Specifically, for example, when collected cancer tissue is immunostained using an anti-DKK1 antibody, DKK1 is determined to be expressed if DKK1 expression is observed in 5% or more of the tumor area. Suitable examples of cancers to which the antibodies of the present invention can be applied include cancers in which DKK1 expression is observed in 5% or more of the tumor area, more preferably cancers in which DKK1 expression is observed in 20% or more of the tumor area, and particularly preferably cancers in which DKK1 expression is observed in 50% or more of the tumor area.

[0088] When RNA is extracted from collected cancer tissue and DKK1 is measured, if the amount of DKK1 in the cell lysate of the cancer tissue is greater than the amount of DKK1 in the cell lysate of non-cancerous tissue from the same case, it is determined that DKK1 is expressed in the cancer.

[0089] (Administration Form) The antibody of the present invention is administered parenterally, as long as an antitumor effect is obtained. Specific examples of the administration form of the antibody of the present invention include injection (intravenous injection, subcutaneous injection, intramuscular injection, peritoneal injection, local injection into the affected area, etc.).

[0090] The dosage of the antibody of the present invention may be appropriately determined depending on the administration form, the type of cancer to be treated, the severity of the patient's symptoms, etc. For example, a single dose of the antibody of the present invention is typically about 0.1 mg to 20 mg / kg body weight, and may be administered approximately once every 1 to 12 weeks.

[0091] The antibodies of the present invention may be used alone or in combination with one or more other drugs having antitumor activity and / or radiation therapy. Examples of other drugs that may be used in combination with the antibodies of the present invention include antitumor agents used in chemotherapy. Specific examples of such antitumor agents include antimetabolites, alkylating agents, microtubule-active agents, anticancer antibiotics, topoisomerase inhibitors, platinum compounds, and kinase inhibitors. Among these, gemcitabine, paclitaxel, lenvatinib, and the like are particularly preferred.

[0092] (Formulations) The pharmaceutical composition of the present invention, the reagent of the present invention, the antitumor agent of the present invention, and the antitumor immune response activator of the present invention are prepared into formulations according to the administration route. Examples of the formulations of the antitumor agent and antitumor immune response activator of the present invention include liquid formulations such as solutions, suspensions, emulsions, and injections.

[0093] Furthermore, the pharmaceutical composition of the present invention, the reagent of the present invention, the antitumor agent of the present invention, and the antitumor immune response activator of the present invention are formulated by adding pharmaceutically acceptable carriers and additives depending on the formulation form. For example, in the case of a liquid formulation, it can be formulated using physiological saline, a buffer solution, etc.

[0094] The reagent of the present invention may be in the form of a kit containing an active ingredient. The kit may also include instruments, reagents, etc. Examples of such instruments and reagents include test tubes, microtiter plates, purification columns, labeled antibodies, and standard samples (positive controls, negative controls).

[0095] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

[0096] 1. Test Method 1-1. Preparation of Mouse Anti-CKAP4 Antibody (3F11-2B10) Mouse anti-CKAP4 antibody was prepared as follows. Specifically, a hybridoma expressing the mouse anti-CKAP4 antibody 3F11-2B10 (Patent Document 1) was cultured in a 100 mm diameter culture dish in 10% FCS RPMI-1640 medium containing recombinant human IL-6 protein (1 ng / ml, R&D). The hybridoma was collected and resuspended in ice-cold PBS (1 x 10 cells / mL). 7 The hybridomas were suspended in a 1000-well plate (1000 x g for 10 minutes, 10,000 x g for 10 minutes, and 100,000 x g for 10 minutes) and kept on ice until transplantation into mice. The hybridomas were then intraperitoneally transplanted into 6-8-week-old male BALB / cAnNCrj-nu immunodeficient mice. After abdominal distension was observed, the mice were euthanized by carbon dioxide treatment, and the ascites fluid was collected by puncture. After stepwise centrifugation (first: 2,000 x g for 10 minutes, second: 10,000 x g for 10 minutes, third: 100,000 x g for 10 minutes), the supernatant was subjected to antibody purification. Protein A-R28 Sepharose (bed volume 1 ml: Ab-Capcher, ProteNova No. P-002-10) was added to the supernatant and rotated at 4°C for 12 hours. The beads were washed three times with 10 ml of ice-cold PBS and transferred to a poly-prep column (Bio-Rad No. 7311550). 3F11-2B10 was eluted with 10 ml of 0.1 M glycine (pH 2.8). Ten 1 ml fractions were collected, neutralized with 100 μl of 1 M Tris-HCl (pH 7.5), and the antibody-containing fractions were dialyzed against ice-cold PBS.

[0097] 1-2. cDNA cloning of mouse anti-human CKAP4 antibody. Total RNA was isolated from 3F11-2B10 hybridoma cells using the RNeasy Mini Kit (QIAGEN), and cDNA was synthesized using the SMARTer RACE cDNA Amplification Kit (CLONTECH Laboratories). cDNA encoding the heavy and light chain variable regions was amplified by 5'-RACE using PCR reagents (PrimeSTAR Max for the heavy chain variable region and PrimeSTAR HS for the light chain variable region) from TaKaRa Bio, with primers specifically designed for the IgG2b heavy chain and κ light chain constant regions.

[0098] All mouse anti-CKAP4 antibodies used in the examples were expressed in Expi293F or FreeStyle 293F cells (Thermo Fisher Scientific) using the expression vectors constructed as described above, following the manufacturer's instructions. After expression, mouse anti-CKAP4 antibodies were purified from the conditioned medium by dialysis against Ab-Capcher resin (ProteNova) and 50 mM Na acetate (pH 4.5). Anti-KLH antibodies were purified using MabSelect SuRe resin (Cytiva) and then by gel filtration chromatography using one of the following buffers: D-PBS(-) or 20 mM histidine, 150 mM NaCl, pH 6.0. All purified antibodies were sterile filtered and stored at -80°C.

[0099] 1-3. Production and purification of humanized antibodies. Complementarity-determining regions (CDRs) were defined using the Kabat, IMGT, and Paratome methods. The most similar human germline sequences were identified using the IMGT / DomainGapAlign database. The Kabat / IMGT / Paratome CDRs were combined and introduced into the human germline framework sequences. To purify 10 mg of humanized anti-CKAP4 antibody (Hv1Lt1), suspension CHO cells (5 × 10 8Cells (Human sarcoma cells) were transfected with pCEC4.2-3F11-2B10 hVHv1hIgG CH and pCEC4.2-3F11-2B10 hVLv1hIgK CL and cultured for 7 days in CH200 medium supplemented with L-alanyl-L-glutamine. Cells were pelleted by centrifugation at 12,000 x g for 10 minutes, and the supernatant was collected. The supernatant (approximately 500 ml) was loaded onto an Ab-Capture™ (Protein A mutant) column, and the column was washed five times with 5 ml of 1x TBS. The antibody was then eluted with 7-10 ml of Pierce™ Gentle Ag / Ab Elution Buffer at pH 6.6, dialyzed (pore size 40-50 Å) against TBS and PBS, and stored at -80°C until use.

[0100] 1-4. Measurement of binding affinity of anti-CKAP4 antibody by surface plasmon resonance (SPR) method. For the measurement, Biacore™ was used, which had been equilibrated to 25°C. TM A T200 system (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) was used. 10 μg / ml humanized anti-CKAP4 and mouse anti-CKAP4 antibodies were captured on an anti-human IgG- or anti-mouse IgG-immobilized Biacore Series S sensor chip CM5 (GE Healthcare Bio-Sciences AB) for 2 min. Serially diluted human CKAP4 extracellular domain (amino acids 465–602) was injected at a flow rate of 10 μl / min for 2 min, followed by a 2-min buffer flow. Sensorgrams were fitted to a global 1:1 Langmuir binding model using Biacore T200 Evaluation Software (GE Healthcare Life Sciences, Chicago, IL, USA). Binding affinity (using the equilibrium dissociation constant [KD]) was calculated by dividing the dissociation constant by the association constant (kD / ka).

[0101] 1-5. Cells and Materials. S2-CP8 pancreatic cancer cells were purchased from the Cell Resource Center, Institute of Development, Aging and Cancer, Tohoku University. Hep3B cells were purchased from the American Type Culture Collection (ATCC) (Rockville, MD, USA). HuH-7 cells were purchased from the Japanese Collection of Research Bioresources. TE-5 and TE-8 cells were obtained from the RIKEN BioResource Center Cell Bank. S2-CP8, MIA PaCa-2, HuH-7, HeLa S3, and MDCK I cells were maintained in DMEM. TE-5 and TE-8 were maintained in RPMI-1640 supplemented with 10% fetal bovine serum (FBS). Hep3B cells were maintained in E-MEM (Fujifilm Wako Pure Chemical Industries, Ltd.; catalog number 051-07615). All cells were authenticated using short tandem repeat analysis and mycoplasma testing (MycoStrip, InvivoGen) in July 2022.

[0102] Lenvatinib (mesylate) was obtained from Chemscene (New Jersey, USA) and dissolved in 0.5% (wt / vol) methylcellulose 400 solution (Fujifilm Wako Pure Chemical Industries, Ltd.) for in vivo experiments in mice. Nanoparticle albumin-based paclitaxel (nab-PTX; Abraxane) was obtained from Taiho Pharmaceutical Co., Ltd. and dissolved in saline for in vivo experiments in mice. Coomassie brilliant blue staining was performed using Bullet CBB Stain One (Nacalai Tesque). Other materials and chemicals were commercially available.

[0103] Animal Experiments Mice were housed in a standardized, well-ventilated environment with a 12:12-hour light / dark cycle and free access to food and water. Sample size was selected to optimize the maximum number of samples per independent experiment, taking into account the 3Rs (reduction, refinement, and replacement) of animal experiments.

[0104] Xenograft tumorigenesis assay was performed using a modified version of a previously reported method. BALB / cAJcl-nu / nu mice (8-week-old male nude mice; CLEA Japan) were anesthetized with a combination of medetomidine (0.3 mg / kg body weight), midazolam (4 mg / kg), and butorphanol (5 mg / kg). S2-CP8 (2.5 × 10 cells) suspended in 120 μl of PBS was then injected into the anesthetized mice. 6 cells) or MIA PaCa-2 (5 × 10 6 cells), or Hep3B cells (5 × 10 ) suspended in 150 μl Matrigel. 6 The mice were injected subcutaneously into the backs of both sides of the mice until the tumor volume reached 50 mm 3 Antibody administration was initiated after the

[0105] To examine the antitumor effects of anti-CKAP4 antibodies, anti-CKAP4 antibodies (3F11-2B10, Fc-silenced antibody, or humanized antibody Hv1Lt1, 200-400 μg / injection) or control IgG were injected intraperitoneally or intravenously twice weekly. Tumor formation continued for an additional 21 days (S2-CP8 cells or Hep3B cells) or 35 days (MIA PaCa-2 cells). Xenograft tumor volumes <50 mm at the start of treatment were excluded. 3 were excluded from the analysis. The excised tumors were measured and weighed. Tumor volume was calculated using the formula: (length) × (width) × (width) / 2.

[0106] 1-8. Combination Therapy Assay of Humanized Anti-CKAP4 Antibody and Other Therapeutic Agents. To investigate the antitumor effects of the humanized anti-CKAP4 antibody Hv1Lt1 in combination with other drugs, such as gemcitabine, nanoparticle albumin-based paclitaxel (nab-PTX), and lenvatinib, mice subcutaneously implanted with S2-CP8 or Hep3B cells were randomly divided into four groups (control IgG + PBS or saline, Hv1Lt1 + PBS or saline, control IgG + drug, and Hv1Lt1 + drug, respectively). Hv1Lt1 antibody or control IgG (280 μg / dose) was administered intraperitoneally twice weekly. Gemcitabine (100 μg / dose, once weekly) or nab-PTX (10 mg / kg, twice weekly) was administered intraperitoneally. Lenvatinib (1 mg / kg) or 0.5% (wt / vol) methylcellulose 400 solution was orally administered once daily, 6 days a week, and tumor formation continued for an additional 21 days. Mice were then euthanized, and the volume and weight of the excised tumors were measured.

[0107] 1-9. In vivo toxicity evaluation of the humanized anti-CKAP4 antibody Hv1Lt1. To evaluate the in vivo toxicity of long-term administration of the humanized anti-CKAP4 antibody, C57BL / 6 mice (8-week-old male nude mice; CLEA Japan) were intraperitoneally injected with Hv1Lt1 or control IgG (200 μg / injection) twice weekly for 3 months. Afterward, the mice were weighed, and blood samples (whole blood and serum) were collected. Furthermore, the heart, lungs, liver, stomach, small intestine, spleen, pancreas, and kidneys were collected for histological evaluation. Blood testing was outsourced to Oriental Yeast.

[0108] 1-10. Histological Analysis. Organs excised for histological analysis were fixed in 10% formalin and embedded in paraffin. 5 μm sections were stained with hematoxylin and eosin (H&E) according to standard protocols. Bright-field images were obtained using an automated imaging platform (NanoZoomer) (Hamamatsu Photonics, Japan).

[0109] Immunoprecipitation assays were performed as previously described. Cells (60 mm diameter dishes) were lysed in 500 μl of Nonidet P-40 (NP-40) buffer (20 mM Tris-HCl [pH 8.0], 10% glycerol, 137 mM NaCl, 1% NP-40) supplemented with protease inhibitors (10 μg / ml leupeptin, 20 μg / ml aprotinin, 1 mM phenylmethanesulfonyl fluoride). After centrifugation, the lysates were incubated with primary antibodies and 40 μl of a 50% slurry of protein G-Sepharose beads (GE Healthcare Bio-Sciences, Buckinghamshire, UK) for 1 h at 4°C. The precipitates were washed three times with NP-40 buffer and then probed with the indicated antibodies.

[0110] 1-12. Immunocytochemical Analysis. For fluorescent immunostaining, cultured cells were fixed with 4% paraformaldehyde (PFA) in PBS and permeabilized for 10 minutes with 0.2% (wt / vol) Triton X-100 and 2 mg / ml bovine serum albumin (BSA) in PBS. Cells were then blocked with blocking buffer (2 mg / ml BSA in PBS). Cells were then incubated with primary antibodies diluted in PBS for 1 hour or overnight. After three washes with PBS, cells were stained with secondary antibodies (Alexa Fluor 488-conjugated, Invitrogen) diluted in PBS for 1 hour. Stained cells were observed using an LSM810 laser scanning microscope. For immunostaining of cell surface-localized CKAP4 protein, S2-CP8 cells were incubated with anti-CKAP4 antibodies for 1 hour under non-permeabilized conditions. After washing, cells were fixed and stained with secondary antibodies. For immunohistochemical staining, tumor masses removed from animals were fixed in 4% paraformaldehyde (PFA) in PBS, paraffin blocks were prepared, and 5 μm-thick tissue sections were prepared. After deparaffinization, the tissue sections were treated in EDTA buffer (pH 9) at 110°C for 15 minutes for antigen retrieval. The tissue sections were blocked with G-block solution (Genostaff) to retrieve endogenous HRP. After treatment with primary antibodies for 1 hour, the sections were washed and then treated with HRP polymer-conjugated secondary antibodies for 1 hour. Target antigens were stained with 3,3'Diaminobenzidine (DAB) substrate, and cell nuclei were stained with hematoxylin solution and observed under a light microscope.

[0111] Two-dimensional (2D) cell proliferation assay. Cell proliferation assays were performed using the CyQUANT NF Cell Proliferation Assay Kit (Thermo Fisher Scientific) according to the manufacturer's instructions. The relative fluorescence intensity of counted cells was expressed in arbitrary units.

[0112] To examine the cytotoxicity of the humanized anti-CKAP4 antibody, HeLa S3 cells (1 × 10 3The cells were seeded in a 96-well plate, and 200 μl of DMEM containing 10% FBS and 10 μg / ml Hv1Lt1 or control IgG was added and cultured for 5 days. Cell proliferation was measured as described above.

[0113] Three-dimensional (3D) cell proliferation assay. 3D Matrigel culture assay was performed as previously described with modifications. S2-CP8 cells (2 x 10 cells) were cultured in DMEM containing 0.1% bovine serum albumin and 2% Matrigel (BD Biosciences, San Jose, CA). 4 The cells were suspended, and 20 μg / ml of anti-CKAP4 antibody or control IgG was added, followed by culturing on solidified Matrigel for 5 to 7 days.

[0114] 1-15. In Vitro DKK1-CKAP4 Binding Assay. The DKK1-CKAP4 binding assay was performed as previously described with modifications. To examine the effect of anti-CKAP4 antibodies on DKK1 binding to CKAP4, various concentrations (0-3 μg / ml) of anti-CKAP4 antibodies were mixed with 0.5 nM GST-CKAP4-extracellular domain (ECD) in 500 μl of NP40 buffer with rotation for 1 h at 4°C. The mixture was then mixed with 2.5 nM DKK1-FLAG for 1 h, after which the GST-CKAP4-ECD was precipitated with glutathione Sepharose and washed three times with NP40 buffer. The precipitate was detected with anti-DKK1 antibodies and analyzed using ImageJ software (National Institutes of Health, Bethesda, MD).

[0115] 1-16. Quantitative PCR Quantitative PCR was performed as previously reported.

[0116] 1-17. Establishment of Organoid Cultures from a Mouse Model of Pancreatic Cancer. Primary pancreatic ductal adenocarcinoma tissue was obtained from a genetically engineered mouse model of pancreatic cancer: the KPC model (a mouse model that develops pancreatic cancer, genotype LSL-KrasG12D / +;Trp53fl / fl;Elastase-Cre) was provided by the Department of Gastroenterology, Osaka University. Fresh tumor tissue from the KPC model was minced, and the resulting cells were dissociated in DMEM / F12 medium containing 0.25% Trypsin-EDTA and 100 U DNase I (Sigma, D4527) with agitation at 37°C for 30 minutes. After passing the cells through a 40-mm sterile filter (BD Biosciences, 352340), 5,000 cells were suspended in 20 mL of ice-cold Matrigel solution. The Matrigel-containing pancreatic cancer cells were placed as droplets into a 24-well tissue culture plate, mixed for 20 minutes at 37°C to solidify, and then cultured in complete growth medium. Established mouse pancreatic cancer organoids (KPC organoids) were passaged weekly, and complete growth medium was prepared immediately before use. Its composition is as follows: DMEM / F12, 1 x N-2 (Life Technologies, 17502048), 1 x B-27 Supplements (Life Technologies, 17504044), 10 mmol / L HEPES / NaOH (pH 7.4), 50 ng / mL EGF (R&D Systems, 236-EG), 100 ng / mL Noggin (R&D Systems, 1967-NG), 100 ng / mL R-Spondin 1 (R&D Systems, 7150-RS), 100 ng / mL FGF10 (Pepro-Tech, 100-26), 10 ng / mL Gastrin I (Sigma, G9145), 10 mmol / L Y27632 (WAKO, 253-00513), 500 mM N-acetylcysteine ​​(Sigma, A9165), 10 mM Nicotinamide (Sigma, N0636), and 500 nM A83-01 (Tocris, #2939).

[0117] 1-18. Orthotopic transplantation of KPC organoids into allogeneic immunodeficient mice. 5 x 10 KPC organoids prepared from KPC organoids stably expressing human CKAP4 and human DKK1 (KPC / CKAP4 / DKK1) were transplanted into immunodeficient mice. 4 Single cells were suspended in 50 μl of Matrigel and stored on ice until transplantation. A 1.5 cm incision was made in the left flank of C57BL / 6 mice, exposing the spleen from the abdominal cavity. The spleen was fixed with sterile cotton, and the underlying pancreas was exposed. The Matrigel cell suspension was injected into the pancreatic tail using a 29G syringe. The pancreas and spleen were then returned to the abdominal cavity, and the incision was sutured. The size of the transplanted organoids was measured weekly by ultrasound. Upon detection of tumor lesions, mice were randomly divided into two groups and administered intraperitoneally either the humanized anti-CKAP4 antibody Hv1Lt1 (280 μg / injection, twice weekly) or control IgG. After 56 days, mice were euthanized, and tumors located in the pancreatic tail were excised and weighed. Tumor volume was calculated using the following formula: (length) × (width) × (width) / 2 [Mitsuishi, 2012 #4024].

[0118] 1-19. RNA Sequencing. Bulk RNA-seq analysis was performed on RNA samples excised from pancreatic tumors orthotopically implanted with KPC organoids in an immunodeficient mouse model (Figure 4D). Four control tumors and four Hv1Lt1-treated tumors were randomly selected from each group. Libraries were prepared using the TruSeq stranded mRNA sample prep kit (Illumina) according to the manufacturer's instructions. mRNA samples were subjected to whole-transcriptome sequencing in 100-base paired-end mode using the DNBSEQ sequencing platform. Sequencing reads were preprocessed with Trim Galore! v0.6.7 and quantified with Salmon v1.4.0 using the gcBias and validateMappings flags. GENCODE vM26 (GRCm39) annotations were used as a reference for transcripts. All procedures were performed using the RNA-sequencing pipeline ikra v2.0.1 (http: / / doi.org / 10.5281 / zenodo.4718200) with default parameters. Quantified transcript-level scaled TPMs were summarized into gene-level scaled TPMs using tximport v1.22.0. Downstream pathway analysis was performed using iDEP2.0 (BMC Bioinformatics 2018;19:534, doi:10.1186 / s12859-018-2486-6). Low-expression genes with <0.5 counts per million (CPM) in at least one sample were removed, leaving 15,238 candidate genes out of 22,216 genes in eight samples. After normalization using VST, principal component analysis was performed. Hierarchical clustering of the top 2,000 genes was performed based on standard deviation. Differential expression analysis by DESeq2 was performed in iDEP with the following parameters: false discovery rate <0.1, minimum fold change >1.5.

[0119] 1-20. Statistical Analysis: Student's t-test or Mann-Whitney U test was used to determine statistical differences between the means of two groups. Analysis of variance (ANOVA) with Dunnett's post-hoc test was used to compare the means of three or more groups. P < 0.05 was considered statistically significant. Statistical analysis was performed using GraphPad Prism 9 (GraphPad Software, La Jolla, CA, USA) or JMP software (SAS Institute, Inc., Cary, NC, USA).

[0120] 2. Results 2-1. cDNA cloning and purification of mouse anti-CKAP4 antibody 3F11-2B10 cDNA was synthesized using RACE cDNA Amplification technology [PMID: 2461560] from total RNA isolated from 3F11-2B10 hybridoma cells (Patent Document 1). The nucleotide sequences of the heavy and light chain variable regions of the cloned 3F11-2B10 were determined, and the cDNA contained open reading frames encoding proteins of 141 and 127 amino acids.

[0121] Two silent Fc mouse anti-CKAP4 antibodies, mF18 (based on mIgG1) and mFa31 (based on mIgG2a), were constructed by introducing mutations including P235K / S239K into the Fc domain, which is the Fc receptor binding site. The murine IgG1-type silent Fc (mF18) had P235K / S239K mutations, and the murine IgG2-type silent Fc (mFa31) had L235R / G236R / S239K mutations. The molecular weights of these recombinant antibodies were similar to those of the original antibodies under reducing conditions.

[0122] 2-2. Characterization of Silent Fc Mouse Anti-CKAP4 Antibodies Silent Fc mouse anti-CKAP4 antibodies, like the original mouse anti-CKAP4 antibody 3F11-2B10, detected and immunoprecipitated human endogenous CKAP4 from S2-CP8 cell lysates (Figure 1A). Both silent Fc mouse anti-CKAP4 antibodies dose-dependently inhibited the binding of DKK1 to CKAP4 in vitro (Figure 1B). Treatment of cancer cell lines (cells with activated DKK1-CKAP4 signaling), including pancreatic cancer cells S2-CP8, hepatocellular carcinoma cells (HuH7 and Hep3B), and esophageal squamous cell carcinoma cells (TE-5 and TE-8) with silent Fc mouse anti-CKAP4 antibodies inhibited AKT activity (Figure 1C). Tumorsphere formation of S2-CP8 and Huh7 cells was also inhibited by silent Fc mouse anti-CKAP4 antibodies (Figure 1D).

[0123] Furthermore, a silent Fc mouse anti-CKAP4 antibody (mF18) suppressed xenograft tumorigenesis induced by S2-CP8 cells (Figure 1E). Because the silent Fc mouse anti-CKAP4 antibody lacks the Fc receptor binding and complement binding sites required for antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), these results suggest that its in vivo antitumor effect and pharmacological activity are due to inhibition of DKK1 binding to CKAP4, rather than through ADCC activity.

[0124] 2-3. Construction of humanized anti-CKAP4 antibodies The complementarity-determining regions (CDRs) were defined by the Kabat, IMGT, and Paratome methods. The nucleotide and amino acid sequences of the variable regions of the 3F11-2B10 antibody are shown in Tables 1 and 2. The tables show the CDRs determined by the Kabat, IMGT, and Paratome methods, respectively.

[0125]

[0126]

[0127] The most similar human germline sequences were identified using the IMGT / DomainGapAlign database. The CDRs identified by the three methods were slightly different. Therefore, when creating humanized antibodies, a region covering all CDRs determined by the three CDR determination methods (broad CDR, designated H1) and a region overlapping the three CDR determination methods (narrow CDR, designated H2) were identified. Humanization was performed based on these two CDR patterns. The framework used in this example was selected from human germline-derived antibody sequences in the database to be similar to the mouse 3F11-2B10 sequence, and the Kabat / IMGT / Paratome CDR combination was grafted onto the human germline-derived framework sequence. The one with the broad CDRs was designated v1, and the one with the narrow CDRs was designated v2.

[0128] The amino acid sequences of the two resulting humanized antibodies (Hv1Lt1 and Hv2Lt1) are shown in Tables 3 to 5. Table 3 shows the amino acid sequence of the heavy chain of Hv1Lt1, Table 4 shows the amino acid sequence of the heavy chain of Hv2Lt1, and Table 5 shows the amino acid sequences of the light chains of Hv1Lt1 and Hv2Lt1 (the light chains of Hv1Lt1 and Hv2Lt1 have the same sequence).

[0129]

[0130]

[0131]

[0132] The nucleotide sequences of the two resulting humanized anti-CKAP4 antibodies (Hv1Lt1 and Hv2Lt1) are shown in Tables 6 to 8. Table 6 shows the nucleotide sequence of the heavy chain of Hv1Lt1, Table 7 shows the nucleotide sequence of the heavy chain of Hv2Lt1, and Table 8 shows the nucleotide sequences of the light chains of Hv1Lt1 and Hv2Lt1 (the light chains of Hv1Lt1 and Hv2Lt1 have the same sequence).

[0133]

[0134]

[0135]

[0136] 2-4. In vitro characterization of humanized anti-CKAP4 antibodies The affinity of humanized anti-CKAP4 antibodies to the extracellular domain of CKAP4 was measured using a Biacore assay. a : 1.25 × 10 for 3F11-2B10 5 , 1.50 × 10 for Hv1Lt1 4 , and 3.79 × 10 for Hv2Lt1. 4 (Table 9). d The value is 1.80 × 10 for 3F11-2B10. -3 , 1.14 × 10 for Hv1Lt1 -5 , 1.70 × 10 for Hv2Lt1 -6 The average KD values ​​of these antibodies were 14.5 nM for 3F11-2B10, 0.76 nM for Hv1Lt1, and 0.05 nM for Hv2Lt1, suggesting that the humanized anti-CKAP4 antibodies have higher (<10-fold) binding affinity for the extracellular domain of CKAP4 compared to the mouse anti-CKAP4 antibody.

[0137]

[0138] Furthermore, two humanized anti-CKAP4 antibodies (hCKAP4 Abs), Hv1Lt1 and Hv2Lt1, detected CKAP4 with comparable efficiency in lysates from human S2-CP8 cells, canine MDCK I kidney cells, and mouse Lewis Lung Carcinoma (LLC) lung cancer cells. Both humanized CKAP4 Abs were able to immunoprecipitate endogenous CKAP4 from S2-CP8 cell lysates and efficiently detected CKAP4 in the endoplasmic reticulum (ER) by immunocytochemistry. No background signal was detected in immunostaining of S2-CP8 cells in which CKAP4 had been knocked out using the Crispr-Cas9 system (S2-CP8 / CKAP4 KO), confirming that the two humanized anti-CKAP4 Abs specifically recognized CKAP4. Furthermore, Hv1Lt1 detected CKAP4 on the cell surface. Both humanized anti-CKAP4 antibodies inhibited the binding of DKK1 to CKAP4 in vitro in a dose-dependent manner.When S2-CP8 cells were treated with humanized anti-CKAP4 antibodies, AKT activity was inhibited by both antibodies.

[0139] 2-5. In vivo tumor-suppressive effect of humanized anti-CKAP4 antibody To evaluate the in vivo tumor-suppressive effect of the humanized anti-CKAP4 antibody Hv1Lt1, two human pancreatic cancer cell lines, S2-CP8 and MIA PaCa-2, which express high levels of DKK1 and plasma membrane CKAP4, were transplanted into immunodeficient mice. Hv1Lt1 was injected intraperitoneally (Figure 2A) or intravenously (Figures 2B and 2C) and inhibited the in vivo xenograft tumor formation induced by both cell lines. These results confirmed that the humanized anti-CKAP4 antibody Hv1Lt1 exhibits tumor-suppressive effects in vivo against various tumor cell lines, regardless of the route of administration.

[0140] To evaluate the nonspecific toxicity of Hv1Lt1, human cervical cancer HeLa S3 cells, which express CKAP4 but not DKK1 on the cell surface, were treated with Hv1Lt1. Hv1Lt1 did not inhibit HeLa S3 cell proliferation in vitro. Furthermore, while HeLa S3 cells treated with staurosporine, a known apoptosis inducer, showed strong nuclear staining with propidium iodide, HeLa S3 cells treated with Hv1Lt1 showed no nuclear staining with propidium iodide, indicating no apoptosis. Furthermore, to evaluate the long-term toxicity of the humanized anti-CKAP4 antibody, wild-type immunodeficient C57BL / 6 mice were intraperitoneally injected with Hv1Lt1 or control IgG for 3 months. Two mice received a therapeutic dose of Hv1Lt1 (280 μg / dose), and three mice received the same amount of IgG. After 3 months of treatment, no significant changes were observed in macroscopic appearance (e.g., coat condition, activity level) or body weight. Similarly, no abnormalities were observed in the macroscopic appearance or organ weight of the resected major organs, including the heart, lungs, liver, spleen, pancreas, stomach, small intestine, and kidneys. Histological evaluation of resected organ sections revealed no pathological abnormalities. To examine the possibility that administered Hv1Lt1 might deposit endogenous CKAP4 in non-tumor tissues, tissue sections prepared from the resected organs were stained with a secondary antibody specifically recognizing human IgG. Since no human IgG deposition was detected in the tissues, it was suggested that Hv1Lt1 did not bind to endogenous CKAP4 in the individual. Blood samples (both whole blood samples and serum) were collected at the time of euthanasia for blood testing. Results of complete blood counts and clinical examinations were within the normal range, with no significant abnormalities compared with healthy mice and the control group (treated with control IgG) (Table 10). These results confirmed that the novel humanized CKAP4 antibody has no toxicity or side effects in vitro or in vivo.

[0141]

[0142] 2-6. Inhibitory Effect of Hv1Lt1 on Pancreatic Cancer Organoid Growth in Vitro and in Vivo. Accumulating evidence suggests that DKK1 may regulate antitumor immunity in the tumor microenvironment (TME). To clarify the pharmacological activity of Hv1Lt1 in the TME, which contains mesenchymal cell components, we established a mouse model using immunodeficient mice. This model uses human pancreatic ductal-like adenocarcinoma (KPC tumor) developed in genetically engineered mice (LSL-KrasG12D / +;Trp53fl / fl;Elastase-Cre (KPC mice, which express an active mutant KRAS allele and harbor Trp53 deficiency specifically in the pancreas). Organoids established from KPC tumors (KPC organoids) can be continuously cultured and transplanted either subcutaneously or orthotopically into C57BL / 6 immunodeficient mice. DKK1 and CKAP4 were ectopically expressed in KPC / DKK1 / CKAP4 organoids (Figure 4A). Activated DKK1-CKAP4 signaling in organoids promoted the growth of organoid spheres (Fig. 4B). Furthermore, in organoids expressing CKAP4 but not DKK1 (KPC / CKAP4), DKK1 promoted organoid sphere growth, while Hv1Lt1 inhibited this growth (Fig. 4C). KPC / CKAP4 / DKK1 organoids were surgically transplanted into the pancreatic tail of wild-type C57BL / 6 mice to form pancreatic tumors, successfully recapitulating the histological features of human pancreatic cancer (Fig. 4D). Hv1Lt1 suppressed the growth of KPC organoid-derived tumors by 60% (Fig. 4D).

[0143] 2-7. Transcriptome Analysis of Hv1Lt1-Treated Pancreatic Tumors. Bulk RNA-seq analysis of resected tumors revealed clear separation between the Hv1Lt1-treated and control groups by hierarchical clustering and principal component analysis (Fig. 5A and 5B). Genetic analysis revealed 421 differentially expressed genes (DEGs) (88 upregulated and 333 downregulated genes in Hv1Lt1-treated tumors) (Fig. 5C). Enrichment analysis revealed significantly decreased expression of genes in immune response-related signaling pathways in the Hv1Lt1-treated group (Fig. 5D). Furthermore, immunohistochemical analysis of tissue sections from tumor samples excised from the control and Hv1Lt1 antibody-treated groups (including those subjected to bulk RNA-seq analysis) demonstrated enhanced infiltration of CD8+ T cells and dendritic cells (DCs), which are responsible for the anti-tumor immune response, in Hv1Lt1-treated tumors (Fig. 5E). Taken together, these results suggest that Hv1Lt1 administration not only suppresses the autonomous proliferation of tumor cells but also contributes to the TME-mediated antitumor immune control in pancreatic cancer.

[0144] 2-8. Tumor-Suppressing Effects of Humanized Anti-CKAP4 Antibody in Combination with Other Therapeutics in Vitro and in Vivo In recent years, various molecularly targeted therapeutics, including antibody drugs, have been developed. Some of these new drugs, when combined with existing drugs, have shown synergistic therapeutic effects and improved the prognosis of cancer patients. Therefore, in vitro cell sphere formation experiments using S2-CP8 cells were performed using Hv1Lt1 in liquid medium. Addition of Hv1Lt1 and gemcitabine (Figure 6A) or nanoparticle albumin-based paclitaxel (nab-PTX) (Figure 6B) alone or in combination resulted in additive inhibition of sphere formation. Furthermore, Hv1Lt1 was administered in combination with gemcitabine (Figures 3A and 6C) or nanoparticle albumin-based paclitaxel (nab-PTX) (Figures 3B and 6D) to mouse models bearing xenograft tumors derived from S2-CP8 cells. Although all of these drugs have been used as standard treatments for human pancreatic cancer, Hv1Lt1, gemcitabine, and nab-PTX all demonstrated in vivo antitumor activity as single agents, and their combination therapy demonstrated stronger tumor growth suppression effects against S2-CP8 cell-induced xenograft tumors than either monotherapy (Figures 3A, 3B, 6C, and 6D). Furthermore, the combination of Hv1Lt1 and lenvatinib also demonstrated significant synergistic antitumor effects against Hep3B cell-induced xenograft tumors (Figure 3C). These results strongly support the potential for future clinical application of humanized anti-CKAP4 antibody combination therapy against human pancreatic and liver cancer.

Claims

1. A humanized anti-CKAP4 antibody or an antigen-binding fragment thereof, comprising: a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1 or 6, a heavy chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 2 or 7, and a heavy chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 3 or 8, and comprising an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 4 or 9; and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 11, a light chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 12, and a light chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 13, and comprising an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO:

14.

2. The humanized anti-CKAP4 antibody or its antigen-binding fragment described in claim 1, wherein the identity is 95% or more.

3. The humanized anti-CKAP4 antibody or its antigen-binding fragment described in claim 1, wherein the identity is 100%.

4. A humanized anti-CKAP4 antibody or its antigen-binding fragment described in claim 1, comprising a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 4 or 9, and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:

14.

5. The humanized anti-CKAP4 antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, comprising: a heavy chain constant region comprising an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO:16; and a light chain constant region comprising an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO:

17.

6. A polynucleotide comprising a coding sequence for the humanized anti-CKAP4 antibody or antigen-binding fragment thereof according to any one of claims 1 to 4.

7. The polynucleotide of claim 6, which is an expression vector.

8. A cell containing the polynucleotide described in claim 6.

9. A pharmaceutical composition comprising the humanized anti-CKAP4 antibody or its antigen-binding fragment according to any one of claims 1 to 4.

10. A reagent comprising the humanized anti-CKAP4 antibody or its antigen-binding fragment described in any one of claims 1 to 4.

11. An antitumor agent comprising the humanized anti-CKAP4 antibody or its antigen-binding fragment according to any one of claims 1 to 4.

12. The antitumor agent according to claim 11, wherein the humanized anti-CKAP4 antibody or antigen-binding fragment thereof is conjugated with a drug or is for use in combination administration with a drug.

13. An anti-tumor immune response activator comprising a humanized anti-CKAP4 antibody or an antigen-binding fragment thereof according to any one of claims 1 to 4.

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