Antibody or antigen-binding fragment thereof, kit for treatment of cancer, and composition for treatment of cancer

Abstract

The main purpose of the present disclosure is to provide a technique capable of exerting an antitumor effect on various types of cancer including malignant melanoma. The present inventors have found that a certain antibody or an antigen-binding fragment thereof can exert an antitumor effect on various types of cancer including malignant melanoma. On the basis of this finding, the present disclosure has been accomplished.

Description

Antibody or antigen-binding fragment thereof, cancer treatment kit, and cancer treatment composition

[0001] The present disclosure relates to an antibody or an antigen-binding fragment thereof, a composition for treating cancer, a kit for treating cancer, and methods for treating a subject with these.

[0002] CPI-613 (6,8-bis(benzylthio)octanoic acid) is an analog of α-lipoic acid coenzyme and a groundbreaking (first-in-class) drug that targets alterations in energy metabolism common to many cancer cells. Specifically, CPI-613 potently inhibits mitochondrial metabolism by inhibiting the TCA cycle in cancer cells. CPI-613 can also induce a burst of mitochondrial reactive oxygen species in cancer cells. Therefore, CPI-613 is a molecular drug that can specifically kill cancer cells without exhibiting toxicity to normal cells.

[0003] It has been reported that while CPI-613 alone showed little antitumor effect against clear cell sarcoma, its combination with hydroxychloroquine demonstrated significant antitumor effects (Non-Patent Document 1, Figure 1). Furthermore, a Phase I / II clinical trial of a combination of CPI-613 and hydroxychloroquine for recurrent and refractory clear cell sarcoma has been ongoing in the United States since November 2021. Furthermore, a Phase II clinical trial of a chemotherapy regimen centered around the combination of CPI-613 and hydroxychloroquine for solid tumors has been ongoing since February 2023.

[0004] Egawa Y, Saigo C, Kito Y, Moriki T, Takeuchi T. "Therapeutic potential of CPI-613 for targeting tumorous mitochondrial energy metabolism and inhibiting autophagy in clear cell sarcoma." PLoS One. 2018 Jun 7;13(6):e0198940. doi: 10.1371 / journal.pone.0198940. Ugawa Hisashi "Detection of BRAF V600 mutation in melanoma" Modern Media, Vol. 61, No. 8, 2015, pp.238-242 Montes de Oca Balderas P. "Mitochondria-plasma membrane interactions and communication." J Biol Chem. 2021 Oct;297(4):101164. doi: 10.1016 / j.jbc.2021.101164. Giacomello M, et al., "The cell biology of mitochondrial membrane dynamics." Nat Rev Mol Cell Biol. 2020 Apr;21(4):204-224. doi: 10.1038 / s41580-020-0210-7. Kohler, G. & Milstein, C. "Continuous cultures of fused cells secreting antibody of predefined specificity." Nature 256, 495-497 (1975)

[0005] Clear cell sarcoma (CSC) is a soft tissue tumor that commonly occurs in young adults and adolescents. It is sometimes called melanoma of soft parts, and is known to share biological similarities with malignant melanoma. Therefore, we investigated the potential of CPI-613 and hydroxychloroquine combination therapy, which exhibits significant antitumor effects against clear cell sarcoma, to be effective against malignant melanoma. However, contrary to our expectations, the combination of CPI-613 and hydroxychloroquine did not exhibit significant antitumor effects in vivo or in vitro (Figures 2–4).

[0006] In view of the above circumstances, the present inventors aimed to develop a technology that can exert an antitumor effect not only on clear cell sarcoma but also on various cancers including malignant melanoma.

[0007] The present inventors have discovered that certain antibodies or antigen-binding fragments thereof may provide a technology that can exert antitumor effects against various cancers, including malignant melanoma, and have made further improvements to this technology, leading to the completion of the present disclosure.

[0008] The present disclosure includes, for example, the subject matter described in the following items: Item 1. An antibody or an antigen-binding fragment thereof capable of binding to a PDIA3 protein, represented by the general formula (1): (In the formula, R 1 and R 2 are independent of each other, R 3 Acyl groups represented by C(O)-, C n H 2n＋1 an alkyl group represented by C m H 2m－1 an alkenyl group represented by C m H 2m－3 Alkynyl groups, aryl groups, heteroaryl groups, CH3 (CH2) n Sulfurized alkyl group represented by -S-, R 3 an imidoyl group represented by C(=NH)-, R 4 and hydrogen, and a hemiacetal group represented by CH(OH)-S- (provided that R 1 and R 2 wherein at least one of R is not hydrogen;1 and R 2 may be unsubstituted or substituted; R 3 is hydrogen, an alkyl group, an alkenyl group, an alkynyl group, a cycloalkyl group, an aryl group, an alkylaryl group, a heteroaryl group, or a heterocyclyl group, any of which may be substituted or unsubstituted; R 4 Item 1. A cancer treatment kit comprising a compound represented by the formula (I) or (II) above, wherein x is CCl3 or COOH; and x is an integer of 0 to 16, n is an integer of 0 to 10, and m is an integer of 2 to 10, or a salt thereof, and an autophagy inhibitor. Item 2. The kit according to Item 1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising a complementarity-determining region (CDR) 1 comprising the amino acid sequence set forth in SEQ ID NO: 2, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 3, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 4, and a light chain variable region comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 5, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 6, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 7. Item 3. Item 2. The kit according to Item 2, wherein the heavy chain variable region comprises (i) the amino acid sequence shown in SEQ ID NO: 8, or (ii) an amino acid sequence that has 85% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 8, and the light chain variable region comprises (iii) the amino acid sequence shown in SEQ ID NO: 9, or (iv) an amino acid sequence that has 85% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 9. Item 4. The kit according to any one of Items 1 to 3, wherein the autophagy inhibitor is a chloroquine compound. Item 5. The kit according to any one of Items 1 to 4, wherein the autophagy inhibitor is hydroxychloroquine or a salt thereof. Item 6. In the compound represented by general formula (1), R 1 and R 2 Item 7. The kit according to any one of Items 1 to 5, wherein each of the formulas is a benzyl group. or a salt thereof. Item 8. The kit according to any one of Items 1 to 6, wherein the cancer is at least one type selected from the group consisting of malignant melanoma, bladder cancer, and kidney cancer. Item 9. A composition for use in cancer treatment, comprising an antibody or an antigen-binding fragment thereof capable of binding to PDIA3 protein, wherein the treatment is performed using a compound represented by general formula (1): (In the formula, R 1 and R 2 are independent of each other, R 3 Acyl groups represented by C(O)-, C n H 2n＋1 an alkyl group represented by C m H 2m－1 an alkenyl group represented by C m H 2m－3 Alkynyl groups, aryl groups, heteroaryl groups, CH3 (CH2) n Sulfurized alkyl group represented by -S-, R 3 an imidoyl group represented by C(=NH)-, R 4 and hydrogen, and a hemiacetal group represented by CH(OH)-S- (provided that R 1 and R 2 wherein at least one of R is not hydrogen; 1 and R 2 may be unsubstituted or substituted; R 3 is hydrogen, an alkyl group, an alkenyl group, an alkynyl group, a cycloalkyl group, an aryl group, an alkylaryl group, a heteroaryl group, or a heterocyclyl group, any of which may be substituted or unsubstituted; R 4 is CCl3 or COOH; and x is an integer of 0 to 16, n is an integer of 0 to 10, and m is an integer of 2 to 10) or a salt thereof, and administration of an autophagy inhibitor. Item 10. A composition for use in the treatment of cancer, comprising: (In the formula, R 1 and R 2 are independent of each other, R 3 Acyl groups represented by C(O)-, C n H 2n＋1an alkyl group represented by C m H 2m－1 an alkenyl group represented by C m H 2m－3 Alkynyl groups, aryl groups, heteroaryl groups, CH3 (CH2) n Sulfurized alkyl group represented by -S-, R 3 an imidoyl group represented by C(=NH)-, R 4 and hydrogen, and a hemiacetal group represented by CH(OH)-S- (provided that R 1 and R 2 wherein at least one of R is not hydrogen; 1 and R 2 may be unsubstituted or substituted; R 3 is hydrogen, an alkyl group, an alkenyl group, an alkynyl group, a cycloalkyl group, an aryl group, an alkylaryl group, a heteroaryl group, or a heterocyclyl group, any of which may be substituted or unsubstituted; R 4 Item 11. A composition for use in the treatment of cancer, comprising a compound represented by the general formula (1): (In the formula, R 1 and R 2 are independent of each other, R 3 Acyl groups represented by C(O)-, C n H 2n＋1 an alkyl group represented by C m H 2m－1 an alkenyl group represented by C m H 2m－3 Alkynyl groups, aryl groups, heteroaryl groups, CH3 (CH2) n Sulfurized alkyl group represented by -S-, R 3 an imidoyl group represented by C(=NH)-, R 4and hydrogen, and a hemiacetal group represented by CH(OH)-S- (provided that R 1 and R 2 wherein at least one of R is not hydrogen; 1 and R 2 may be unsubstituted or substituted; R 3 is hydrogen, an alkyl group, an alkenyl group, an alkynyl group, a cycloalkyl group, an aryl group, an alkylaryl group, a heteroaryl group, or a heterocyclyl group, any of which may be substituted or unsubstituted; R 4Item 11. A composition comprising administering a compound represented by the following formula (I): (x is CCl3 or COOH; and x is an integer of 0 to 16, n is an integer of 0 to 10, and m is an integer of 2 to 10) or a salt thereof. Item 12. An antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising a complementarity-determining region (CDR) 1 comprising the amino acid sequence set forth in SEQ ID NO: 2, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 3, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 4, and a light chain variable region comprising CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 5, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 6, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 7. Item 13. Aspect 13. The antibody or antigen-binding fragment thereof according to Aspect 12, wherein the heavy chain variable region comprises (i) the amino acid sequence set forth in SEQ ID NO: 8, or (ii) an amino acid sequence that has 85% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 8, and the light chain variable region comprises (iii) the amino acid sequence set forth in SEQ ID NO: 9, or (iv) an amino acid sequence that has 85% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 9. Aspect 14. The antibody or antigen-binding fragment thereof according to Aspect 12 or 13, which has the ability to bind to a PDIA3 protein. Aspect 15. The composition of any one of Aspects 9 to 11, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising a complementarity-determining region (CDR) 1 comprising the amino acid sequence set forth in SEQ ID NO: 2, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 3, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 4, and a light chain variable region comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 5, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 6, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 7. Item 16. The composition of any one of Items 9 to 11 and 15, wherein the heavy chain variable region comprises (i) the amino acid sequence set forth in SEQ ID NO: 8, or (ii) an amino acid sequence that has 85% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 8, and the light chain variable region comprises (iii) the amino acid sequence set forth in SEQ ID NO: 9, or (iv) an amino acid sequence that has 85% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 9.Item 17. The composition according to any one of Items 9 to 11, 15, and 16, wherein the autophagy inhibitor is a chloroquine compound. Item 18. The composition according to any one of Items 9 to 11, and 15 to 17, wherein the autophagy inhibitor is hydroxychloroquine or a salt thereof. Item 19. In the compound represented by general formula (1), R. 1 and R 2 Item 20. The composition according to any one of items 9 to 11 and items 15 to 18, wherein each of the formulas is a benzyl group. Item 21. The composition according to any one of items 9 to 11 and items 15 to 18, wherein the compound represented by general formula (1) is a compound represented by general formula (2): or a salt thereof. Item 21. The composition according to any one of items 9 to 11 and 15 to 19, wherein the cancer is at least one type selected from the group consisting of malignant melanoma, bladder cancer, and kidney cancer. Item 22. The antibody according to any one of items 12 to 14, for use as a pharmaceutical. Item 23. The antibody according to any one of items 12 to 14, for treating cancer. Item 24. The antibody according to item 23, wherein the cancer is at least one type selected from the group consisting of malignant melanoma, bladder cancer, and kidney cancer. Item 25. A method for treating cancer in a subject, comprising the steps of administering an antibody or an antigen-binding fragment thereof capable of binding to PDIA3 protein, (In the formula, R 1 and R 2 are independent of each other, R 3 Acyl groups represented by C(O)-, C n H 2n＋1 an alkyl group represented by C m H 2m－1 an alkenyl group represented by C m H 2m－3 Alkynyl groups, aryl groups, heteroaryl groups, CH3 (CH2) n Sulfurized alkyl group represented by -S-, R 3 an imidoyl group represented by C(=NH)-, R 4 and hydrogen, and a hemiacetal group represented by CH(OH)-S- (provided that R 1 and R 2 wherein at least one of R is not hydrogen;1 and R 2 may be unsubstituted or substituted; R 3 is hydrogen, an alkyl group, an alkenyl group, an alkynyl group, a cycloalkyl group, an aryl group, an alkylaryl group, a heteroaryl group, or a heterocyclyl group, any of which may be substituted or unsubstituted; R 4 Item 26. A method according to Item 25, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising a complementarity-determining region (CDR) 1 comprising the amino acid sequence set forth in SEQ ID NO: 2, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 3, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 4, and a light chain variable region comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 5, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 6, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 7. Item 27. Item 25 or 26. The method of Item 25 or 26, wherein the heavy chain variable region comprises (i) the amino acid sequence shown in SEQ ID NO: 8, or (ii) an amino acid sequence that has 85% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 8, and the light chain variable region comprises (iii) the amino acid sequence shown in SEQ ID NO: 9, or (iv) an amino acid sequence that has 85% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 9. Item 28. The method of any one of Items 25 to 27, wherein the autophagy inhibitor is a chloroquine compound. Item 29. The method of any one of Items 25 to 28, wherein the autophagy inhibitor is hydroxychloroquine or a salt thereof. Item 30. In the compound represented by general formula (1), R 1 and R 2 Item 31. The method according to any one of Items 25 to 29, wherein each of the formulas is a benzyl group. Item 32. The method according to any one of Items 25 to 29, wherein the compound represented by General Formula (1) is a compound represented by General Formula (2): Item 32. The method according to any one of Items 25 to 31, wherein the cancer is at least one type selected from the group consisting of malignant melanoma, bladder cancer, and kidney cancer. Item 33. A cancer treatment kit comprising: an antibody or an antigen-binding fragment thereof capable of binding to a PDIA3 protein, a compound represented by general formula (2): or a salt thereof, and hydroxychloroquine or a salt thereof, wherein the antibody or antigen-binding fragment thereof is an antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising a complementarity-determining region (CDR) 1 comprising the amino acid sequence set forth in SEQ ID NO: 2, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 3, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 4, and a light chain variable region comprising CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 5, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 6, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 7. Item 34. The kit according to Item 33, wherein the cancer is at least one type selected from the group consisting of malignant melanoma, bladder cancer, and kidney cancer. Item 35. The kit according to Item 33, wherein the cancer is malignant melanoma. Item 36. The kit according to any one of Items 1 to 8 and 33 to 35, wherein the antibody is a humanized antibody. Item 37. The composition according to any one of Items 9 to 11 and Items 15 to 21, wherein the antibody is a humanized antibody. Item 38. The antibody according to any one of Items 13 to 14 and Items 22 to 24, wherein the antibody is a humanized antibody. Item 39. The method according to any one of Items 25 to 32, wherein the antibody is a humanized antibody.

[0009] According to the present disclosure, a technology is provided that can exert an antitumor effect not only on clear cell sarcoma but also on various cancers including malignant melanoma.

[0010] In clear cell sarcoma, CPI-613 alone showed little antitumor effect, whereas its combined use with hydroxychloroquine demonstrated a significant antitumor effect. Left: Tumor volume; Right: Total tumor weight (Egawa et al., 2018: Non-Patent Document 1). The following shows the results of analysis using the Guava® EasyCyte cell analyzer (Luminex) from Test Example 1-1. The vertical axis represents PI intensity, and the horizontal axis represents FITC-labeled annexin V intensity. Of the four regions in each graph, the plots in the lower right region represent early apoptotic cells, the plots in the upper right region represent late apoptotic cells, and the plots in the lower left region represent viable cells. Photographs of cells taken with an SP8 LIGHTNING confocal microscope (Leica) from Test Example 1-1 are shown. The bar in the figure represents 20 μm. The following shows the results of Test Example 1-2. The vertical axis represents tumor volume (mm 3) and the horizontal axis indicates the number of days since Mewo cell inoculation (the day of inoculation is considered day 1). The line graph shows the average value for each group, and the error bars indicate the standard deviation. The dashed line indicates the results for the Mock group, and the solid line indicates the results for the CPI+HCQ group. The timing of drug or PBS administration is indicated by downward arrows in the figure. The results of Test Example 2-2 are shown. The left figure shows non-neoplastic epidermal basal layer melanocyte tissue, and the right figure shows malignant melanoma tissue. The areas bound by the ICT antibody are stained red. The results of Test Example 2-3 are shown. The figure shows the amino acid sequence of human PDIA3 (SEQ ID NO: 1), and the sequence matching the protein fragment detected by MALDI-TOF MS is shown in red. The results of Test Example 2-4 are shown. For the heavy and light chains, CDR1 is indicated by a yellow marker and underline, CDR2 by a magenta marker and underline, and CDR3 by a green marker and underline. The results of Test Example 2-5 are shown. Lane 1 was applied with a protein extract from Mewo cells that had not been treated with Glycopeptidase F, lane 2 with a protein extract from Mewo cells that had been treated with Glycopeptidase F, lane 3 with a protein extract from G361 cells that had not been treated with Glycopeptidase F, and lane 4 with a protein extract from G361 cells that had been treated with Glycopeptidase F. The results of Test Example 3-1-1-1 are shown. The vertical axis represents PI intensity, and the horizontal axis represents FITC-labeled annexin V intensity. Of the four regions in each graph, plots in the lower right region represent early apoptotic cells, plots in the upper right region represent late apoptotic cells, and plots in the lower left region represent viable cells. The results of Test Example 3-1-1-2 are shown. The vertical axis represents PI intensity, and the horizontal axis represents FITC-labeled annexin V intensity. Of the four regions in each graph, the plots in the lower right region represent early apoptotic cells, the plots in the upper right region represent late apoptotic cells, and the plots in the lower left region represent viable cells. The results of Test Example 3-1-2 are shown. The vertical axis represents tumor volume (mm 3) and the horizontal axis indicates the number of days since Mewo cell inoculation (the day of inoculation is considered day 1). The line graphs show the average values ​​for each group, and the error bars indicate the standard deviation. The dashed line indicates the results for the Mock group, the dotted line indicates the results for the CPI+HCQ group, and the solid line indicates the results for the CPI+HCQ+ICT group. The timing of drug or PBS administration is indicated by downward arrows in the figures. The results of Test Example 3-2 are shown. The vertical axis indicates PI intensity, and the horizontal axis indicates FITC-labeled annexin V intensity. Of the four regions in each graph, the plots in the lower right region indicate early apoptotic cells, the plots in the upper right region indicate late apoptotic cells, and the plots in the lower left region indicate viable cells. The results of Test Example 4-1 are shown. The vertical axis of each graph indicates the cell count, and the horizontal axis indicates fluorescence intensity. The numerical values ​​(%) in the graphs indicate the percentage of cells in which fluorescence was observed among the analyzed cells. The results of Test Example 5 are shown. a: Immunoblotting results using normal human IgG antibody and humanized ICT antibody. Lane 1 was used for normal human IgG antibody, and lane 2 for humanized ICT antibody. b: Apoptosis induction was assessed for the CPI + HCQ, CPI + HCQ + ICT, and CPI + HCQ + rICT groups using a method using FITC-labeled annexin V and propidium iodide (PI). The vertical axis represents PI intensity, and the horizontal axis represents FITC-labeled annexin V intensity. In each graph, plots in the lower right region represent early apoptotic cells, those in the upper right region represent late apoptotic cells, and those in the lower left region represent viable cells. c: In vitro cell proliferation assay results for the mock group and the CPI + HCQ + rICT group. Each assay was performed with n = 5. Error bars indicate SD.

[0011] Each embodiment of the present disclosure is described in more detail below. The present disclosure preferably includes, but is not limited to, an antibody or an antigen-binding fragment thereof, a cancer treatment kit, a cancer treatment composition, and a method for treating a subject with the same. The present disclosure includes everything disclosed herein that would be recognizable to a person skilled in the art.

[0012] 1. Antibodies or antigen-binding fragments thereof Although not particularly limited, antibodies or antigen-binding fragments thereof encompassed by the present disclosure preferably have the ability to bind to PDIA3 protein. Hereinafter, such antibodies or antigen-binding fragments encompassed by the present disclosure may be referred to as "antibodies or fragments thereof of the present disclosure."

[0013] PDIA3 (protein disulfide-isomerase A3) is a disulfide isomerase involved in protein folding in the endoplasmic reticulum. It has been reported to be a multifunctional protein. It is originally present in the endoplasmic reticulum, but in malnutritioned cells, it couples with the cell surface membrane and is exposed on the surface or outside of the cell membrane. It is thought to be a dead-cell-associated antigen. An example of the amino acid sequence of human PDIA3 (SEQ ID NO: 1) is shown in Figure 6.

[0014] PDIA3 proteins of the present disclosure include proteins comprising the amino acid sequence set forth in SEQ ID NO: 1 and proteins comprising an amino acid sequence that has 70% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 1. The sequence identity may be 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% or more, preferably 80% or more, more preferably 85% or more, even more preferably 90% or more, and particularly preferably 95% or more.

[0015] The "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. 90:5873-7 (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.

[0016] An amino acid sequence that does not have 100% sequence identity to a certain amino acid sequence X includes, for example, an amino acid sequence in which one or more amino acids have been substituted, deleted, added, or inserted (preferably by substitution, more preferably by conservative substitution) relative to the amino acid sequence X. Here, "multiple" means, for example, 2 to 20, preferably 2 to 10, more preferably 2 to 5, and even more preferably 2 or 3. The upper or lower limit of the range may be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0017] "Conservative substitution" means that an amino acid residue is substituted with an amino acid residue having a similar side chain. For example, substitutions between amino acid residues having basic side chains such as lysine, arginine, and histidine are conservative substitutions. Other conservative substitutions include substitutions 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.

[0018] As described above, the antibody or fragment thereof of the present disclosure preferably has the ability to bind to PDIA3 protein. In the present disclosure, an antibody or fragment thereof having the ability to bind to PDIA3 protein refers to the ability of the antibody or fragment thereof to specifically interact with PDIA3 protein. Whether an antibody or fragment thereof has the ability to bind to PDIA3 protein can be confirmed by methods known in the art. Examples of such methods include co-immunoprecipitation (Co-IP), pull-down assay, Western blotting, cross-linking, label transfer, interaction mapping, surface plasmon resonance, and FRET (Fluorescence Resonance Energy Transfer). Furthermore, whether an antibody or fragment thereof has the ability to bind to PDIA3 protein can be predicted by computer simulation based on the amino acid sequences of the antibody or fragment thereof and the PDIA3 protein. In one embodiment, whether an antibody or fragment thereof of the present disclosure has the ability to bind to PDIA3 protein can be confirmed by Western blotting according to "2-5" in Test Example 2, described below.

[0019] Furthermore, although not particularly limited, it is preferred that the antibody or fragment thereof of the present disclosure exhibits stronger binding affinity to PDIA3 with less glycosylation than normal PDIA3. While PDIA3 is typically detected as a 57 kDa band in Western blotting, PDIA3 with less glycosylation will exhibit a band at a lower molecular weight. For example, glycosylation can be removed from PDIA3 by treatment with Glycopeptidase F. In one embodiment, the antibody or fragment thereof of the present disclosure exhibits stronger binding affinity to a Glycopeptidase F-treated product of PDIA3 in cells (e.g., cancer cells) than to PDIA3 in the cells (e.g., cancer cells) (e.g., densitometry measurement of the band detected by Western blotting is 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold or more). In general, it has been reported that tumor cells may differ from normal cells in terms of glycosylation, particularly on the cell surface.

[0020] The antibody or fragment thereof of the present disclosure may be, for example, an antibody or fragment thereof comprising a heavy chain variable region comprising a complementarity-determining region (CDR) 1 comprising the amino acid sequence set forth in SEQ ID NO: 2, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 3, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 4, and a light chain variable region comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 5, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 6, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 7; or an antibody or fragment thereof comprising a heavy chain variable region comprising a CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 2, a CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 3, and a CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 4, and a light chain variable region comprising a CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 5, a CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 6, and a CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 7. The amino acid sequences set forth in SEQ ID NOs: 2 to 7 are shown in FIG. 7.

[0021] Furthermore, the antibody or fragment thereof of the present disclosure may be an antibody or fragment thereof comprising a heavy chain variable region comprising an amino acid sequence that has 85% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 8, and a light chain variable region comprising an amino acid sequence that has 85% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 9; an antibody or fragment thereof comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 8, and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 9 is preferred; and an antibody or fragment thereof comprising a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 8, and a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 9 is more preferred. The amino acid sequences set forth in SEQ ID NOs: 8 and 9 are shown in FIG. 7.

[0022] The sequence identity to the amino acid sequence shown in SEQ ID NO: 8 is preferably 90% or more, more preferably 95% or more, and particularly preferably 99% or more. The sequence identity to the amino acid sequence shown in SEQ ID NO: 8 may be 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% or more.

[0023] The sequence identity to the amino acid sequence shown in SEQ ID NO: 9 is preferably 90% or more, more preferably 95% or more, and particularly preferably 99% or more. The sequence identity to the amino acid sequence shown in SEQ ID NO: 9 may be 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% or more.

[0024] The antibody or fragment thereof of the present disclosure is preferably an antibody or fragment thereof comprising a heavy chain variable region comprising an amino acid sequence that has 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 8, and a light chain variable region comprising an amino acid sequence that has 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 9; more preferably an antibody or fragment thereof comprising a heavy chain variable region comprising an amino acid sequence that has 95% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 8, and a light chain variable region comprising an amino acid sequence that has 95% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 9; and particularly preferably an antibody or fragment thereof comprising a heavy chain variable region comprising an amino acid sequence that has 99% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 8, and a light chain variable region comprising an amino acid sequence that has 99% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 9.

[0025] In the present disclosure, the term "antibody" is used to encompass both monoclonal and polyclonal antibodies. The antibody of the present disclosure may be of any isotype, such as IgG (e.g., IgG1, IgG2, IgG3, IgG4, etc.), IgA (e.g., IgA1, IgA2, etc.), IgD, IgE, IgM, etc. The antibody may be a human antibody or a non-human antibody. Non-human antibodies include, but are not limited to, mouse antibodies, rabbit antibodies, rat antibodies, hamster antibodies, guinea pig antibodies, chicken antibodies, goat antibodies, sheep antibodies, donkey antibodies, camel antibodies, alpaca antibodies, llama antibodies, monkey antibodies, chimpanzee antibodies, etc. The antibody of the present disclosure may also be a chimeric antibody, such as a mouse-human chimeric antibody. The antibody may be a partially or fully humanized antibody. Preferably, the antibody of the present disclosure is a monoclonal antibody.

[0026] As used herein, the antigen-binding fragment of an antibody is not particularly limited as long as it comprises heavy chain CDR1 to 3 and light chain CDR1 to 3, and may be, for example, Fab, F(ab')2, minibody, scFv-Fc, Fv, scFv, diabody, triabody, tetrabody, etc.

[0027] 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 are linked by a disulfide bond. In Fab, CH1 and CL may be disulfide-bonded via the thiol groups of cysteine ​​residues present in each.

[0028] 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.

[0029] 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.

[0030] 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.

[0031] Fv is also known as the smallest structural unit of an antibody, and is a structure in which a heavy chain variable region and a 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.

[0032] 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.

[0033] Diabodies, triabodies, and tetrabodies are structures in which the above-mentioned scFvs form dimers, trimers, and tetramers, respectively, and are associated in a structurally stable state through non-covalent intermolecular interactions between the variable regions, similar to Fvs and the like.

[0034] The antibody or fragment thereof of the present disclosure may be conjugated or fused to 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, PA tag), fluorescent proteins (e.g., GFP, BFP, CFP, YFP, RFP), luminescent proteins (e.g., luciferase), secretory signal sequences (e.g., Igκ signal sequence), protease recognition sequences (e.g., TEV protease recognition sequence), expression-enhancing sequences, solubilizing sequences, and multimerization domains. When the antibody or fragment thereof of the present disclosure is conjugated or fused to a multimerization domain, an antibody or antigen-binding fragment thereof of the same or different species as the antibody or fragment thereof of the present disclosure may be further conjugated or fused to the multimerization domain to form a multimer (homomultimer or heteromultimer).

[0035] The antibody or fragment thereof of the present disclosure may be chemically modified, as long as the desired effect is achieved.

[0036] The antibody or fragment thereof of the present disclosure may have a C-terminus that is a carboxyl group (—COOH), a carboxylate (—COO － The Q in the ester group may be any of C, methyl, ethyl, n-propyl, isopropyl, n-butyl, etc. 1－6 Alkyl group: C such as cyclopentyl, cyclohexyl 3－8 Cycloalkyl groups such as phenyl and α-naphthyl 6－12 Aryl group: phenyl-C such as benzyl, phenethyl 1－2 Alkyl groups include pivaloyloxymethyl groups.

[0037] In the antibody or fragment thereof of the present disclosure, a carboxyl group (or carboxylate) other than that at the C-terminus may be amidated or esterified. In this case, the above-mentioned C-terminal esters, etc., may be used as the ester.

[0038] In the antibody or fragment thereof of the present disclosure, 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 Alkanoyl etc. C 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 bound.

[0039] 2. Method for Producing Antibodies or Antigen-Binding Fragments Thereof The antibodies or fragments thereof of the present disclosure can be produced by methods known in the art or methods that can be easily derived by a person skilled in the art from methods known in the art.

[0040] For example, the antibody may be produced by a method comprising the steps of immunizing animals such as mice, rabbits, rats, hamsters, guinea pigs, chickens, goats, sheep, donkeys, camels, alpacas, llamas, monkeys, and chimpanzees with the PDIA3 protein to produce antibodies. Alternatively, the antibody may be produced by a method comprising the steps of establishing hybridomas from animals immunized with the PDIA3 protein. Alternatively, the antibody may be produced by a method comprising the steps of immunizing an animal with cancer cells (preferably melanoma cells) and selecting antibodies from hybridomas established from the animal based on their ability to bind to the PDIA3 protein. Furthermore, the antibody may be produced by a method comprising the steps of immunizing an animal with cancer cells (preferably melanoma cells) and selecting antibodies from hybridomas established from the animal based on their ability to more strongly induce apoptosis in cancer cells (preferably melanoma cells) when used in combination with an autophagy inhibitor and CPI-613. Immunization of animals, establishment and culture of hybridomas, etc. can be carried out according to known methods (for example, Current protocols in Molecular Biology, ed., Ausubel et al. (1987) Publish. John Wiley and Sons. Sections 11.4 to 11.11).

[0041] As another example, an antibody or fragment thereof of the present disclosure can be produced, for example, by a method comprising the steps of culturing a host transformed with a polynucleotide comprising a coding sequence for an antibody or fragment thereof of the present disclosure (a polynucleotide of the present disclosure), and recovering a fraction comprising an antibody or fragment thereof of the present disclosure.

[0042] The polynucleotide of the present disclosure is not particularly limited as long as it comprises a coding sequence for an antibody or fragment thereof of the present disclosure. Preferably, the polynucleotide of the present disclosure comprises the coding sequence in a state capable of expressing the antibody or fragment thereof of the present disclosure. The polynucleotide of the present disclosure may comprise other sequences in addition to the 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, a replication origin, and a drug resistance gene coding sequence located adjacent to the coding sequence. Furthermore, the polynucleotide of the present disclosure may be a linear polynucleotide or a circular polynucleotide (e.g., a vector).

[0043] Specific examples of polynucleotides of the present disclosure include: (I) a polynucleotide comprising a nucleotide sequence encoding at least one selected from the group consisting of the heavy chain, heavy chain variable region, and heavy chain CDR1 to 3 of an antibody or fragment thereof of the present disclosure; (II) a polynucleotide comprising a nucleotide sequence encoding at least one selected from the group consisting of the light chain, light chain variable region, and light chain CDR1 to 3 of an antibody or fragment thereof of the present disclosure; (III) a polynucleotide comprising a nucleotide sequence encoding at least one selected from the group consisting of the heavy chain, heavy chain variable region, and heavy chain CDR1 to 3 of an antibody or fragment thereof of the present disclosure; and a polynucleotide comprising a nucleotide sequence encoding at least one selected from the group consisting of the light chain, light chain variable region, and light chain CDR1 to 3 of an antibody or fragment thereof of the present disclosure.

[0044] The host is not particularly limited, and examples thereof include Escherichia coli, eukaryotic cells, insect cells, mammalian cells, etc. Among these, mammalian cells such as HEK cells, CHO cells, NS0 cells, and SP2 / O cells are preferred from the viewpoint of more efficient antibody expression.

[0045] The methods for transformation, culture, and recovery are not particularly limited, and known methods for antibody production can be used.

[0046] After recovery, the antibody of the present disclosure may be purified as needed. Purification can be carried out by known methods for antibody production, such as chromatography, dialysis, etc.

[0047] 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.

[0048] 3. Uses The antibody or fragment thereof of the present disclosure has the general formula (1): (In the formula, R 1 and R 2 are independent of each other, R 3 Acyl groups represented by C(O)-, C n H 2n＋1 an alkyl group represented by C m H 2m－1 an alkenyl group represented by C m H 2m－3 Alkynyl groups, aryl groups, heteroaryl groups, CH3 (CH2) n Sulfurized alkyl group represented by -S-, R 3 an imidoyl group represented by C(=NH)-, R 4 and hydrogen, and a hemiacetal group represented by CH(OH)-S- (wherein R 1 and R 2 wherein at least one of R is not hydrogen; 1 and R 2 may be unsubstituted or substituted; R 3is hydrogen, an alkyl group, an alkenyl group, an alkynyl group, a cycloalkyl group, an aryl group, an alkylaryl group, a heteroaryl group, or a heterocyclyl group, any of which may be substituted or unsubstituted; R 4 is CCl3 or COOH; and x is an integer of 0 to 16, n is an integer of 0 to 10, and m is an integer of 2 to 10) or a salt thereof, when used in combination with an autophagy inhibitor, can exhibit excellent antitumor effects against various cancers, including malignant melanoma. In this disclosure, the compound represented by the above general formula (1) or a salt thereof may be referred to as the "compound of the present disclosure or a salt thereof."

[0049] As described above, the technology of the present disclosure, which can exert excellent antitumor effects, is suitable for use in the prevention, amelioration, treatment, etc. of cancer. In the present disclosure, the term "cancer prevention" includes preventing the onset of cancer by applying it to a subject before the cancer is diagnosed, and preventing cancer metastasis by applying it to tissues in a subject diagnosed with cancer where cancer has not yet been found. In the present disclosure, the terms "cancer improvement" or "cancer treatment" include suppressing the progression of cancer, halting the progression of cancer, suppressing the proliferation of cancer cells, killing cancer cells, reducing the number of cancer cells, reducing tumor volume, and preventing cancer metastasis by applying it to a subject diagnosed with cancer. Furthermore, in the present disclosure, the term "anticancer effect" includes the effect of preventing, ameliorating, and treating cancer.

[0050] 3-1. Compounds of the present disclosure or salts thereof In the above general formula (1), the acyl group is R 3 It is represented by C(O)-. Examples of the acyl group include acetyl, benzoyl, benzoyl derivatives, 4-fluorobenzoyl, and 1-methylpyrrole-2-carboxyl.

[0051] In the above general formula (1), the alkyl group is C n H 2n＋1where n is 1 to 10, preferably 1 to 6, and particularly preferably 1 to 4. The alkyl group may be straight-chain, branched-chain, or alicyclic, and in the case of an alicyclic group, any carbon atom may be attached, or any carbon atom may be substituted to form a heterocycle. The alkyl group may have at least one heteroatom such as N, O, or S. Any carbon atom in the alkyl group may be substituted or unsubstituted. Examples of the alkyl group include methyl, ethyl, butyl, decanyl, cyclopropyl, 4-pyridinemethyl, 2-anthraquinonemethyl, N-phenylacetamido, phenylethyl, 2-ethanoic acid, 2-acetamido, 4-(2-acetamido-pyridinyl)methyl, N-[(2-fluorophenyl)methyl]acetamido, N-[(6-methoxy-3-pyridyl)methyl]acetamido, 5-(acetylamino)pyridine-2-carboxyamido, 5-(6,8-diaza-7-oxo-3-thiabicyclo[3.3.0]oct-2-yl)-N-(2-carbonylaminoethyl)pentanamide, and 5-(6,8-diaza-7-oxo-3-thiabicyclo[3.3.0]oct-2-yl)pentacarboxyl.

[0052] In the above general formula (1), the alkenyl group is C m H 2m－1 where m is 2 to 10. The alkenyl group may be straight-chain, branched-chain, or alicyclic, and in the case of an alicyclic group, any carbon atom may be attached, or any carbon atom may be substituted to form a heterocycle. The alkenyl group may contain at least one heteroatom such as N, O, or S. Any carbon atom in the alkenyl group may be substituted or unsubstituted. Examples of alkenyl groups include propenyl, 2,3-dimethyl-2-butenyl, heptenyl, and cyclopentenyl.

[0053] In the above general formula (1), the alkynyl group is C m H 2m－3where m is 2 to 10. The alkynyl group may be straight-chain, branched-chain, or alicyclic, and in the case of an alicyclic group, any carbon atom may be attached, or any carbon atom may be substituted to form a heterocycle. The alkynyl group may contain at least one heteroatom such as N, O, or S. Any carbon atom in the alkynyl group may be substituted or unsubstituted. Examples of alkynyl groups include acetylenyl, propynyl, and octynyl.

[0054] In the above general formula (1), the aryl group is an aromatic or aryl group attached to the carbon backbone of general formula (I) by a thio-ether bond. The aryl group is preferably an unsaturated ring system having 6 to 10 carbon atoms. Aryl groups include organometallic aryl groups such as ferrocene. Aryl groups may be substituted or unsubstituted at any carbon atom. Examples of aryl groups include benzyl (-CH2C6H5), benzyl derivatives such as methylbenzyl and aminobenzyl, (1,2,3,4,5-pentafluorophenyl)methyl, triphenylmethyl, 4-methylbenzoic acid, ferrocenemethyl, 2-naphthylmethyl, 4,4-biphenylmethyl, and stilbene (i.e., 1-((1E)-2-phenylvinyl)-4-methylbenzene). Of these, benzyl is particularly preferred.

[0055] In the above general formula (1), the heteroaryl group refers to a 5- or 6-membered aromatic heterocyclic ring system (monocyclic or bicyclic) in which the heteroaryl moiety contains 1 to 4 heteroatoms selected from the group consisting of S, N, and O, and the heteroaryl group is bonded to the carbon backbone of general formula (I) by a thio-ether bond. Any atom of the heteroaryl group, particularly the carbon atom, may or may not be substituted. Examples of heteroaryl groups include benzothiazole, quinoline, 7-chloroquinoline, furan, thiophene, indole, azaindole, oxazole, thiazole, isoxazole, isothiazole, imidazole, N-methylimidazole, pyridine, pyrimidine, pyrazine, pyrrole, N-methylpyrrole, pyrazole, N-methylpyrazole, 1,3,4-oxadiazole, 1,2,4-triazole, 1-methyl-1,2,4-triazole, 1H-tetrazole, 1-methyltetrazole, benzoxazole, benzofuran, benzisoxazole, benzimidazole, N-methylbenzimidazole, azabenzimidazole, indazole, quinazoline, and pyrrolidinyl.

[0056] In the above general formula (1), the alkyl sulfide group is CH3(CH2) n -S-, where n is 0 to 9. Alkyl groups (i.e., CH3(CH2) n ) any carbon atom of which may be substituted or unsubstituted, C n H 2n＋1 It has the same characteristics as those described above for alkyl groups.

[0057] In the above general formula (1), the imidoyl group is R 3 C(=NH)-, where R 3 may be, but is not limited to, hydrogen, an alkyl group, an alkenyl group, an alkynyl group, a cycloalkyl group, an aryl group, an alkylaryl group, a heteroaryl group, or a heterocyclyl group, any of which may be substituted or unsubstituted.

[0058] In the above general formula (1), the hemiacetal group is R 4CH(OH)-S-, where R 4 includes, but is not limited to, CF3, CCl 3、 It may also be a compound having a strong electron-withdrawing substituent such as COOH.

[0059] Any of the above groups may be unsubstituted or substituted. Examples of the substituents include alkyl, alkenyl, alkynyl, aryl, heteroaryl, acyl, alkoxycarbonyl, alkoxy, alkoxyalkyl, alkoxyalkoxy, cyano, halogen, hydroxy, nitro, oxo, trifluoromethyl, trifluoromethoxy, trifluoropropyl, amino, amido, alkylamino, dialkylamino, dialkylaminoalkyl, hydroxyalkyl, alkoxyalkyl, alkylthio, -SO3H, -SON2NH2, -SONHalkyl, -SON(alkyl)2, -CO2H, CO2NH2, CO2NHalkyl, and -CO2N(alkyl)2. Any of the above groups may have any number of substituents. In other words, substituted R groups such as mono-, di-, and tri- are also possible. 1 group or R 2 The substituent may have a group, and the substituent may itself be substituted. 1 Groups and R 2 Any of the groups may be substituted with either a carbohydrate, lipid, nucleic acid, amino acid or any polymer thereof, or a single-chain or branched synthetic polymer (having a molecular weight ranging from about 350 to about 40,000).

[0060] R 1 and R 2 The thio-ester or thio-ether bond connecting R to the backbone can be oxidized to form a sulfoxide or sulfone. In other words, the -S- in the bond can become -S(O)- or -S(O)2. 1 and R 2 The thio-ester or thio-ether bond connecting to the backbone may further contain a disulfide that is oxidized to a thiosulfinic or thiosulfonic acid. In other words, instead of -S- in the bond, the bond can be -S(O)-S- or -S(O)2-S-.

[0061] In the above general formula (1), R 1 and R 2 Preferably, at least one of R is a benzyl group. 1 and R 2 are particularly preferably benzyl groups.

[0062] In the above general formula (1), x is an integer of 0 to 16, preferably 1 to 10, more preferably 1 to 6, and particularly preferably 4. The upper or lower limit of the range may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16.

[0063] The compound represented by the above general formula (1) or a salt thereof is represented by the following general formula (2): The compound represented by general formula (2) (6,8-bis(benzylthio)octanoic acid) is generally called CPI-613 and is known as a cancer metabolism inhibitor.

[0064] The salt of the compound represented by the general formula (1) is not particularly limited as long as it is a pharmaceutically acceptable salt. Examples of such salts include salts with inorganic bases such as aluminum salts, ammonium salts, calcium salts, copper salts, iron salts, ferrous salts, lithium salts, magnesium salts, manganese salts, potassium salts, sodium salts, and zinc salts, and potassium salts or sodium salts are particularly preferred. Furthermore, examples of salts with organic bases include salts of primary, secondary, or tertiary amines, and specific examples of amines include arginine, betaine, caffeine, choline-N-N'-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, aminoethanol, ethanolamine-N-ethylmorpholine-N-ethylpiperidine, glucosamine, histidine, hydroxocobalamin, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, cinnamic acid, polyamine resin, procaine, purine, theobromine, triethylamine, trimethylamine, tripropylamine, and tromethamine.

[0065] 3-2. Autophagy Inhibitors In the present disclosure, the autophagy inhibitor is not particularly limited as long as it can inhibit autophagy, and any autophagy inhibitor can be used. Specific examples of autophagy inhibitors include chloroquine compounds, 4-aminoquinoline, 3-methyladenine, MHY1485, 3-methyl-6-(3-methylpiperidin-l-yl)-3H-purine, 6-chloro-N-(l-ethylpiperidin-4-yl)-l,2,3,4-tetrahydroacridin-9-amine, 4-(((l-(2-fluorophenyl)cyclopentyl)-amino)methyl)-2-((4-methylpiperazin-1-yl)methyl)phenol, 6-fluoro-N-[4-fluorobenzyl]quinazolin-4-amine, N-acetyl-L-cysteine, L-asparagine, N2,N4-dibenzylquinazoline-2,4-diamine, (2S,3S)-trans-epoxysuccinyl-L-leucylamide-3-methylbutane ethyl ester, N-[6-(4-chlorophenoxy)hexyl]-N'-cyano-N''-4-pyridinyl-guanidine, leupeptin, 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one, 4,6-di-4-morpholinyl-N-(4-nitrophenyl)-l,3,5-triazin-2-amine, pepstatin A, 2-((5-bromo-2-((3,4,5-trimethoxyphenyl)amino)pyrimidin-4-yl)oxy)-N-methylbenzamide, Examples include 6-fluoro-N-[(4-fluorophenyl)methyl]-4-quinazolinamine, thapsigargin, amodiaquine, artemisinin, mefloquine, primaquine, piperaquine, quinacrine, U0126, bafilomycin A1, LY294002, SB202190, SB203580, SC79, and wortmannin.

[0066] In the technology of the present disclosure, the autophagy inhibitor is preferably a chloroquine compound. In this disclosure, the term "chloroquine compound" is used as a general term for chloroquine, its derivatives, and pharmaceutically acceptable salts thereof. The structure of chloroquine is shown below.

[0067] Specific examples of chloroquine compounds include chloroquine and hydroxychloroquine, as well as pharmaceutically acceptable salts thereof. The structure of hydroxychloroquine is shown below.

[0068] Salts of chloroquine and hydroxychloroquine include salts with inorganic or organic acids such as hydrochloric acid, phosphoric acid, sulfuric acid, orotic acid, chondroitin sulfate, hydrobromic acid, nitric acid, perchloric acid, fumaric acid, maleic acid, glycolic acid, lactic acid, salicylic acid, succinic acid, toluene-p-sulfonic acid, tartaric acid, acetic acid, citric acid, methanesulfonic acid, ethanesulfonic acid, formic acid, benzoic acid, malonic acid, naphthalene-2-sulfonic acid, and benzenesulfonic acid.

[0069] In the technology of the present disclosure, the chloroquine compound is particularly preferably hydroxychloroquine or a salt thereof.

[0070] As described above, the antibody or fragment thereof of the present disclosure can exhibit excellent antitumor effects against various cancers, including malignant melanoma, when used in combination with the compound represented by general formula (1) above or a salt thereof and an autophagy inhibitor.

[0071] In the present disclosure, the term "cancer" encompasses cancer, neoplasms, and malignant tumors. Examples of cancer include malignant melanoma, kidney cancer, breast cancer, mesothelioma, bladder cancer, intraperitoneal disseminated cancer, lung cancer, colorectal cancer, gastric cancer, soft tissue sarcoma, osteosarcoma, brain tumor, neuroblastoma, leukemia, lymphoma, liver cancer, prostate cancer, retinoblastoma, ciliary body tumor, basal cell carcinoma, squamous cell carcinoma, malignant soft tissue tumor, chondrosarcoma, ovarian cancer, uterine cancer, and cervical cancer. While the cancer to which the technology of the present disclosure is applicable is not particularly limited, the technology is preferably used for at least one type selected from the group consisting of malignant melanoma, bladder cancer, and kidney cancer, and is particularly preferably used for malignant melanoma.

[0072] In particular, malignant melanoma is known to have a type in which BRAF, one of the signal transduction factors for cell proliferation, is mutated. Combination therapy with a BRAF inhibitor and a MEK inhibitor is known to be effective against BRAF-mutant malignant melanoma, with a reported response rate of approximately 75.9% (Non-Patent Document 2). However, it is believed that at least half of malignant melanoma patients do not have BRAF mutations (BRAF wild-type), and combination therapy with a BRAF inhibitor and a MEK inhibitor is ineffective against BRAF wild-type malignant melanoma. The technology disclosed herein is presumed to have a different mechanism of action from BRAF inhibition, and therefore can be suitably used for BRAF wild-type malignant melanoma, for which combination therapy with a BRAF inhibitor and a MEK inhibitor is ineffective.

[0073] Furthermore, it has been reported that the progression-free survival period for patients with BRAF-mutant melanoma treated with a combination therapy of a BRAF inhibitor and a MEK inhibitor was 9.4 to 11.4 months (Non-Patent Document 2), and the therapeutic effect is still insufficient. The technology disclosed herein is presumed to have a mechanism of action different from that of BRAF inhibition, and therefore can be said to be useful from the perspective of providing a new treatment option even for patients with BRAF-mutant melanoma for whom combination therapy of a BRAF inhibitor and a MEK inhibitor is effective.

[0074] The technology of the present disclosure can be applied to, for example, humans and non-human mammals (e.g., rats, mice, rabbits, cows, pigs, dogs, cats, sheep, monkeys, etc.), with humans being preferred.

[0075] 3-4. Application Method The application method of the technology of the present disclosure is not particularly limited as long as the desired effect is achieved. For example, the antibody or fragment thereof of the present disclosure, or a composition containing the same may be administered directly into a lesion by injection, or may be administered intravenously, intraarterially, intraperitoneally, intramuscularly, subcutaneously, intrapleurally, or by injection or infusion, or may be administered by perfusion via a catheter.

[0076] The frequency of application of the technology of the present disclosure is not particularly limited. For example, it may be applied once or multiple times a day, once or multiple times a week, once or multiple times a month, or once or multiple times a year. The application period of the technology of the present disclosure is also not particularly limited, so long as the effects of the present disclosure are achieved. Furthermore, the dose of the antibody or fragment thereof, the compound, and the autophagy inhibitor of the present disclosure is also not particularly limited, so long as the effects of the present disclosure are achieved. The application frequency, application period, and dose can be adjusted appropriately by those skilled in the art depending on the condition of the subject, the course of treatment, etc.

[0077] The technology of the present disclosure may be optionally combined with other pharmaceutical compositions and / or treatment methods, etc., which are applied to a subject with cancer. When the technology of the present disclosure is combined with other pharmaceutical compositions and / or treatment methods, etc., they may be applied to a subject simultaneously, or may be applied separately at any time.

[0078] In the technology of the present disclosure, at least one selected from the group consisting of the antibody and fragment thereof of the present disclosure, the compound and salt thereof of the present disclosure, and an autophagy inhibitor may be mixed with a pharmaceutically acceptable base, carrier, excipient, diluent, solubilizer, emulsifier, preservative, pH adjuster, adjuvant, chelating agent, etc. These components may be used alone or in combination of two or more.

[0079] Examples of preservatives include parabens such as methylparaben, ethylparaben, propylparaben, and butylparaben, sodium benzoate, phenoxyethanol, alkyldiaminoethylglycine hydrochloride, etc. The preservatives may be used alone or in combination of two or more.

[0080] Examples of pH adjusters include citric acid, phosphoric acid, malic acid, pyrophosphoric acid, lactic acid, tartaric acid, glycerophosphoric acid, acetic acid, nitric acid, and chemically possible salts thereof, as well as sodium hydroxide, potassium hydroxide, etc. pH adjusters can be used alone or in combination of two or more types so that the pH of a composition containing at least one selected from the group consisting of an antibody and a fragment thereof of the present disclosure, a compound and a salt thereof of the present disclosure, and an autophagy inhibitor is in the range of 4 to 8, preferably 5 to 7.

[0081] The method for preparing a composition comprising at least one selected from the group consisting of the antibody and fragment thereof of the present disclosure, the compound and salt thereof of the present disclosure, and an autophagy inhibitor is not particularly limited, as long as the effects of the present disclosure are achieved. For example, the composition can be prepared according to a method known in the art. More specifically, the composition can be prepared by mixing at least one selected from the group consisting of the antibody and fragment thereof of the present disclosure, the compound and salt thereof of the present disclosure, and an autophagy inhibitor with other components, with sterile distilled water.

[0082] 4. Cancer Treatment Kit The present disclosure also encompasses a cancer treatment kit comprising an antibody or antigen-binding fragment thereof capable of binding to PDIA3 protein, a compound or salt thereof of the present disclosure, and an autophagy inhibitor. This cancer treatment kit may be referred to as the "kit of the present disclosure." The items described in "1. Antibody or Antigen-Binding Fragment Thereof" to "3. Use" are incorporated by reference into the kit of the present disclosure.

[0083] In the present disclosure, the term "for treating cancer" encompasses applications such as suppressing the progression of cancer, halting the progression of cancer, suppressing the proliferation of cancer cells, killing cancer cells, reducing the number of cancer cells, reducing tumor volume, and preventing cancer metastasis, when applied to a subject diagnosed with cancer.

[0084] The timing of administration of the antibody or antigen-binding fragment thereof capable of binding to PDIA3 protein, the compound or salt thereof of the present disclosure, and the autophagy inhibitor contained in the kit of the present disclosure is not particularly limited as long as the desired effect is achieved. For example, all elements may be administered simultaneously, or each may be administered at a different time, or two may be administered simultaneously and the remaining may be administered at a different time. When not all elements are administered simultaneously, the interval between each administration is not limited as long as the desired effect is achieved, and the order of administration is also not limited. Furthermore, administration of any of the elements may be repeated until the desired effect is achieved. Furthermore, the administration route of each element may be the same or different.

[0085] 5. Compositions The present disclosure also encompasses (I) a composition for use in the treatment of cancer, comprising an antibody or antigen-binding fragment thereof capable of binding to PDIA3 protein, wherein the treatment comprises administering a compound or a salt thereof of the present disclosure and an autophagy inhibitor; (II) a composition for use in the treatment of cancer, comprising a compound or a salt thereof of the present disclosure, wherein the treatment comprises administering an antibody or an antigen-binding fragment thereof capable of binding to PDIA3 protein and an autophagy inhibitor; and (III) a composition for use in the treatment of cancer, comprising an autophagy inhibitor, wherein the treatment comprises administering an antibody or an antigen-binding fragment thereof capable of binding to PDIA3 protein and a compound or a salt thereof of the present disclosure. The compositions (I) to (III) above are collectively referred to as "compositions of the present disclosure." The items described in "1. Antibody or Antigen-Binding Fragment Thereof" to "4. Kit" are incorporated by reference into the compositions of the present disclosure.

[0086] In this specification, the term "comprising" includes "essentially consisting of" and "consisting of" in addition to "containing." Furthermore, the present disclosure includes any and all combinations of the constituent elements described in this specification.

[0087] Furthermore, the various characteristics (properties, values, functions, etc.) described in each embodiment of the present disclosure above may be combined in any way to identify the subject matter encompassed by the present disclosure. In other words, the present disclosure encompasses all subject matter consisting of any combination of the combinable characteristics described herein.

[0088] Hereinafter, the embodiments of the present disclosure will be described more specifically with reference to examples, but the embodiments of the present disclosure are not limited to the following examples.

[0089] Test Example 1. Examination of the Effect of Combination Therapy of CPI-613 and Hydroxychloroquine on Malignant Melanoma 1-1. In Vitro Examination First, the present inventors examined whether combination therapy of CPI-613 and hydroxychloroquine, which has been shown to have antitumor effects on clear cell sarcoma, is also effective against malignant melanoma.

[0090] Specifically, 1x10 BRAF wild-type melanoma Mewo cells 5 1x10 cells were cultured for 48 hours in a medium containing 1 μg / ml CPI-613 and 10 μg / ml hydroxychloroquine (CPI+HCQ group). 5 Mewo cells were cultured for 48 hours (Mock group).

[0091] After incubation, the cells were stained with FITC-labeled annexin V and propidium iodide (PI) (PromoCell GmbH). Apoptosis induction was then assessed using a Guava® EasyCyte cell analyzer (Luminex). The results are shown in Figure 2. The vertical axis represents PI intensity, and the horizontal axis represents FITC-labeled annexin V intensity. In each of the four graphs in Figure 2, the plots in the lower right region represent early apoptotic cells, the plots in the upper right region represent late apoptotic cells, and the plots in the lower left region represent viable cells.

[0092] As shown in Figure 2, the percentage of melanoma cells treated with CPI-613 and hydroxychloroquine increased from 7.75% to 12.69% in early apoptosis compared to mock-treated cells, but approximately 80% of the cells did not undergo apoptosis.

[0093] Mock-treated cells and cells treated with both CPI-613 and hydroxychloroquine were stained with DAPI (4',6-diamidino-2-phenylindole) and FITC-labeled annexin V, respectively, and then photographed with an SP8 LIGHTNING confocal microscope (Leica). The results are shown in Figure 3. The bar in Figure 3 indicates 20 μm.

[0094] The green fluorescence in Figure 3 comes from FITC-labeled annexin V and indicates Mewo cells that underwent early apoptosis. As is clear from Figure 3, in melanoma cells treated with CPI-613 and hydroxychloroquine in combination, some cells underwent early apoptosis, but apoptosis was not induced in the majority of cells.

[0095] 1-2. In vivo study BALB / c nu / nu mice (BALB / c nude mice in which the nu gene was introduced into inbred BALB / c mice by backcrossing) were subcutaneously injected with 1x10 Mewo cells at the right flank. 6 Mice in the CPI-613 and hydroxychloroquine group (CPI + HCQ) were intraperitoneally injected with 500 μg of CPI-613 and 1.0 mg of hydroxychloroquine per mouse on days 18, 25, and 32, respectively, starting with the day of Mewo cell inoculation (day 1). Mice in the mock group were intraperitoneally injected with 0.5 ml of PBS per mouse on days 18, 25, and 32, starting with the day of Mewo cell inoculation (day 1). Note that day 18 after Mewo cell inoculation marks the time when tumor engraftment was confirmed in this test example. Thereafter, the drug or PBS was administered three times, every week. Each group consisted of five mice.

[0096] After inoculation of Mewo cells, the major axis (a) and minor axis (b) of the tumor were measured using a caliper (vernier caliper) on days 18, 25, 32, and 39. The measurements of tumor diameter on days 18, 25, and 32 were carried out before the administration. The tumor volume was calculated from the measured major and minor axes using the following formula: Tumor volume (mm 3 ) = 4 / 3π×[a / 2]×[b / 2] 2

[0097] The results are shown in Figure 4. The vertical axis represents tumor volume (mm 3 ) are shown. The horizontal axis shows the number of days since Mewo cell inoculation (the day of inoculation is considered day 1). The line graph shows the average value for each group, and the error bars show the standard deviation. The dashed line shows the results for the Mock group, and the solid line shows the results for the CPI+HCQ group. The timing of drug administration is indicated by downward arrows in the figure. As is clear from Figure 4, the group treated with a combination of CPI-613 and hydroxychloroquine did not show a significant antitumor effect compared to the Mock group.

[0098] 1-3. Summary The results of Test Example 1 suggest that the combination therapy of CPI-613 and hydroxychloroquine, which has been shown to have antitumor effects against clear cell sarcoma, does not exert the expected effect against malignant melanoma.

[0099] Test Example 2: Investigation of Antibodies That Exert Antitumor Effects in Combination with CPI-613 and Hydroxychloroquine Based on the results of Test Example 1, the inventors aimed to develop a technology that could exert antitumor effects not only on clear cell sarcoma but also on various cancers, including malignant melanoma. Here, the inventors focused on the recent proposal of interactions between cell surface membrane proteins and mitochondria (Non-Patent Documents 3 and 4). As described above, CPI-613 strongly inhibits mitochondrial metabolism by inhibiting the TCA cycle in cancer cells. Furthermore, CPI-613 can induce a burst of mitochondrial reactive oxygen species in cancer cells. Because CPI-613 exerts its antitumor effect via mitochondria, the inventors attempted to enhance the efficacy of CPI-613 and hydroxychloroquine combination therapy by using a cell surface antibody.

[0100] 2-1. Antibody production: 1x10 6 Mewo cells were intraperitoneally administered at 2-week intervals for a total of four immunizations. Three days after the fourth immunization, the spleen was removed from the mouse and spleen cells were obtained. 7 The spleen cells were cultured at a concentration of 1.1 x 10 6 The cells were fused with P3U1 myeloma cells using a polyethylene glycol solution (product name: Hybri-Max™ 50% (w / v), Sigma-Aldrich, Cat. No. P7181) according to the method described in Non-Patent Document 5 (note that Non-Patent Document 5 uses Sendai virus for cell fusion).

[0101] The fused cells were cultured in HAT medium for 1 week by standard methods, and hybridomas were selected. They were then cultured in HT medium for 14 days to obtain a culture supernatant. The culture supernatant, 1 μg / ml CPI-613, and 10 μg / ml hydroxychloroquine were added to 1x10 Mewo cells. 5The cells were cultured for 16 hours (supernatant + CPI-613 + hydroxychloroquine group). Similarly, 1 μg / ml CPI-613 and 10 μg / ml hydroxychloroquine were added to 1x10 Mewo cells without the addition of culture supernatant. 5 The cells were cultured for 16 hours (CPI-613 + hydroxychloroquine group). Hybridomas were screened based on the finding that the supernatant + CPI-613 + hydroxychloroquine group induced more apoptosis than the CPI-613 + hydroxychloroquine group. Apoptosis induction was evaluated using the method described in Test Example 1-1, using FITC-labeled annexin V and propidium iodide (PI).

[0102] A single hybridoma clone was isolated from the hybridomas obtained by the above screening by performing limiting dilution twice. Analysis using the Antagen ISO-M8A kit (Antagen Biosciences) revealed that the hybridoma produced a monoclonal antibody with a μ heavy chain and a κ light chain. In this example, this monoclonal antibody is sometimes referred to as the ICT antibody or simply ICT. The ICT antibody was purified from the culture supernatant of the hybridoma using ImmunoAssist MG-PP Re (Kanto Chemical Co., Ltd., Cat. No. 20352-33) according to the attached protocol.

[0103] 2-2. Immunohistochemical Staining Non-neoplastic epidermal basal layer melanocyte tissue and malignant melanoma tissue were incubated with purified ICT antibody at 10 μg / ml for 1 hour at room temperature. After incubation, ImmPRESS™ Reagent, Anti-Mouse IgG (Vector Laboratories, Cat. No. MP-7402) was added according to the attached protocol and incubated at room temperature for 30 minutes. Subsequently, Vector™ NovaRED (Vector Laboratories, Cat. No. SK-4800), a chromogenic substrate, was added and stained.

[0104] The results are shown in Figure 5. The left side of Figure 5 shows tissue from non-neoplastic epidermal basal layer melanocytes, and the right side shows tissue from malignant melanoma, with the areas bound by the ICT antibody stained red. As is clear from Figure 5, the ICT antibody exhibited binding to malignant melanoma. However, no binding of the ICT antibody to non-neoplastic epidermal basal layer melanocytes was observed.

[0105] 2-3. Analysis of the antigen of the ICT antibody Following the protocol attached to the kit, 0.5 mg of the ICT antibody was coupled to the M-270 epoxy magnetic beads (hereinafter simply referred to as "beads") included in the Dynabeads™ Antibody Coupling Kit (Life Technologies). 7 Mewo cells were lysed in RIPA buffer, then mixed with the ICT antibody-coupled beads and incubated at 4°C for 2 hours. After the reaction, the beads were washed with RIPA buffer and the bound proteins were eluted by boiling at 95°C for 5 minutes. The eluted proteins were subjected to SDS-PAGE, and the gel after electrophoresis was stained with CBB. A band of approximately 50 kDa was excised from the gel and subjected to limited digestion with trypsin to obtain protein fragments.

[0106] The protein fragments were analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). The estimated weights of the fragments obtained by MALDI-TOF MS were analyzed using the analysis software MASCOT (Matrix Science; http: / / www.matrixscience.com / ). The results were consistent with the fragments being human PDIA3 (protein disulfide-isomerase A3). Figure 6 shows the amino acid sequence of human PDIA3 (SEQ ID NO: 1), with the sequence matching the protein fragment detected by MALDI-TOF MS highlighted in red.

[0107] The above analysis suggested that the ICT antibody recognizes PDIA3.

[0108] 2-4. Analysis of Amino Acid Sequences of the Variable Regions of the ICT Antibody cDNA was obtained from the hybridoma producing the ICT antibody obtained in Test Example 2-1 using the SMARTer™ RACE 5' / 3' Kit (Takara Bio). The base sequence was obtained from the cDNA by DNA sequencing, and the predicted amino acid sequence was obtained using ORF finder (https: / / www.ncbi.nlm.nih.gov / orffinder / ). The amino acid sequence was compared with the variable regions of antibodies registered in the database using Blast (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi), and the variable regions and complementarity determining regions (CDRs) of the heavy and light chains of the ICT antibody were identified. The results are shown in Figure 7. Both the heavy and light chains contained novel sequences not registered in the database.

[0109] 2-5. Analysis of binding of ICT antibody to PDIA3 protein Proteins were extracted from BRAF wild-type melanoma Mewo cells and BRAF mutant (V600E) melanoma G361 cells using standard methods. Each protein extract was treated with Glycopeptidase F. Glycopeptidase F is an enzyme that specifically cleaves the bond between N-glycosidic glycans and proteins (GlcNAc-Asn bond). Western blotting was performed using ICT antibody as the primary antibody for each protein extract treated with and without Glycopeptidase F. The results are shown in Figure 8.

[0110] In Figure 8, lane 1 was used to apply a protein extract derived from Mewo cells that had not been treated with glycopeptidase F; lane 2 was used to apply a protein extract derived from Mewo cells that had been treated with glycopeptidase F; lane 3 was used to apply a protein extract derived from G361 cells that had not been treated with glycopeptidase F; and lane 4 was used to apply a protein extract derived from G361 cells that had been treated with glycopeptidase F.

[0111] PDIA3, also known as ERp57, is typically detected as a 57 kDa band in Western blotting. As shown in Figure 8, the band shifted to lower molecular weight in the samples treated with Glycopeptidase F (lanes 2 and 4). This reflects the deglycosylation of the protein by Glycopeptidase F treatment. Furthermore, as evident from comparisons between lanes 1 and 2 and between lanes 3 and 4, the band was more intense in the Glycopeptidase F-treated samples. These results suggest that deglycosylated PDIA3 has higher binding affinity to ICT antibodies. This suggests that an antigenic determinant for ICT antibodies may exist near the glycan-binding site in the amino acid sequence of PDIA3. It has been reported that tumor cells, particularly the cell surface, may exhibit different glycosylation from normal cells.

[0112] Test Example 3. Study of the antitumor effect of combined therapy with ICT antibody, CPI-613, and hydroxychloroquine 3-1. Study of the antitumor effect on malignant melanoma 3-1-1. In vitro study 3-1-1-1. BRAF mutant (V600E) malignant melanoma A2058 cells and G361 cells, and BRAF wild-type malignant melanoma Mewo cells were each treated with 1x10 51x10 cells were cultured for 48 hours in a medium containing 1 μg / ml ICT antibody, 1 μg / ml CPI-613, and 10 μg / ml hydroxychloroquine (CPI + HCQ + ICT group). 1x10 cells were also cultured for 48 hours in a medium containing 1 μg / ml CPI-613 and 10 μg / ml hydroxychloroquine without ICT antibody (CPI + HCQ + ICT group). 5 The cells were cultured for 48 hours (CPI+HCQ group). After the culture, induction of apoptosis was evaluated by the method using FITC-labeled annexin V and propidium iodide (PI) described in Test Example 1-1.

[0113] The results are shown in Figure 9. The vertical axis represents the intensity of PI, and the horizontal axis represents the intensity of FITC-labeled annexin V. Of the four regions in each graph in Figure 9, the plots in the lower right region represent early apoptotic cells, the plots in the upper right region represent late apoptotic cells, and the plots in the lower left region represent viable cells.

[0114] As shown in Figure 9, in both BRAF mutant and BRAF wild-type melanoma, the number of apoptotic cells was significantly increased when ICT antibody was combined with CPI-613 and hydroxychloroquine compared to when ICT antibody was not used. This suggests that the combination of ICT antibody, CPI-613, and hydroxychloroquine can exert a significant antitumor effect on melanoma, regardless of the presence or absence of BRAF mutation.

[0115] 3-1-1-2. BRAF mutant (V600E) melanoma A2058 cells and G361 cells, and BRAF wild-type melanoma Mewo cells were each treated with 1x10 5The cells were cultured for 16 hours in each of the following media: medium containing no CPI-613, hydroxychloroquine, or ICT antibody (Mock group); medium containing 100 ng / ml CPI-613 and 1 μg / ml hydroxychloroquine but no ICT antibody (CPI + HCQ group); medium containing 1 μg / ml ICT antibody but no CPI-613 or hydroxychloroquine (ICT group); medium containing 100 ng / ml CPI-613 and 1 μg / ml ICT antibody but no hydroxychloroquine (CPI + ICT group); medium containing 1 μg / ml hydroxychloroquine and 1 μg / ml ICT antibody but no CPI-613 (HCQ + ICT group); or medium containing 100 ng / ml CPI-613, 1 μg / ml hydroxychloroquine, and 1 μg / ml ICT antibody (CPI + HCQ + ICT group).

[0116] After the culture, induction of apoptosis was evaluated by the method using FITC-labeled annexin V and propidium iodide (PI) described in Test Example 1-1.

[0117] The results are shown in Figure 10. The vertical axis represents the intensity of PI, and the horizontal axis represents the intensity of FITC-labeled annexin V. Of the four regions in each graph in Figure 10, the plots in the lower right region represent early apoptotic cells, the plots in the upper right region represent late apoptotic cells, and the plots in the lower left region represent viable cells.

[0118] As shown in Figure 10, for both BRAF mutant and BRAF wild-type melanoma, the CPI + HCQ, ICT, CPI + ICT, and HCQ + ICT groups did not show a significant apoptosis-inducing effect compared to the Mock group. However, only the CPI + HCQ + ICT group showed a significant increase in apoptotic cells.

[0119] In other words, it was suggested that ICT antibody alone or a combination of only two of the following elements did not show a significant antitumor effect in melanoma, whereas a combination of the three elements of ICT antibody, CPI-613, and hydroxychloroquine could specifically exert a significant antitumor effect against melanoma.

[0120] 3-1-2. In vivo study 5x10 Mewo cells were injected subcutaneously into the right flank of BALB / c nu / nu mice. 5 Mice in the CPI-613, hydroxychloroquine, and ICT antibody group (CPI + HCQ + ICT) were intraperitoneally injected with 500 μg of CPI-613, 1.0 mg of hydroxychloroquine, and 0.1 mg of ICT antibody per mouse on days 16, 23, and 30, respectively, starting with the day of Mewo cell inoculation (day 1). Mice in the CPI-613 and hydroxychloroquine group (CPI + HCQ) were intraperitoneally injected with 500 μg of CPI-613 and 1.0 mg of hydroxychloroquine per mouse on days 16, 23, and 30, respectively, starting with the day of Mewo cell inoculation (day 1). Mice in the mock group were intraperitoneally injected with 0.5 ml of PBS per mouse on days 16, 23, and 30, respectively, starting with the day of Mewo cell inoculation (day 1). In this test, the 16th day after inoculation of Mewo cells was the time point when tumor engraftment was confirmed, and thereafter, the drug or PBS was administered three times, every other week. The test was conducted with n=5 for each group.

[0121] After inoculation of Mewo cells, the major axis (a) and minor axis (b) of the tumor were measured using a caliper (vernier caliper) on days 16, 23, 30, and 37. The measurements of tumor diameter on days 16, 23, and 30 were performed before the administration. The tumor volume was calculated from the measured major and minor axes using the following formula: Tumor volume (mm 3 ) = 4 / 3π×[a / 2]×[b / 2] 2

[0122] The results are shown in Figure 11. The vertical axis represents tumor volume (mm 3), and the horizontal axis indicates the number of days since Mewo cell inoculation (the day of inoculation is considered day 1). The line graph shows the average value for each group, and the error bars indicate the standard deviation. The dashed line indicates the results for the Mock group, the dotted line indicates the results for the CPI+HCQ group, and the solid line indicates the results for the CPI+HCQ+ICT group. The timing of drug or PBS administration is indicated by downward arrows in the figure. As is clear from Figure 11, the CPI+HCQ group did not exhibit a significant antitumor effect compared to the Mock group, while the CPI+HCQ+ICT group exhibited a significant antitumor effect.

[0123] 3-2. Examination of antitumor effects against various cancers Next, the inventors examined the antitumor effects of combined therapy with ICT antibody, CPI-613, and hydroxychloroquine against cancers other than malignant melanoma.

[0124] 1x10 for bladder cancer T24 cells and renal clear cell carcinoma KMRC-1 cells 5 1x10 cells were cultured for 16 hours in a medium containing 1 μg / ml ICT antibody, 1 μg / ml CPI-613, and 10 μg / ml hydroxychloroquine (CPI + HCQ + ICT group). 1x10 cells were also cultured in a medium containing 1 μg / ml CPI-613 and 10 μg / ml hydroxychloroquine without ICT antibody (CPI + HCQ + ICT group). 5 1x10 cells were cultured for 16 hours (CPI+HCQ group). Furthermore, 1x10 cells were cultured for 16 hours in a medium containing no CPI-613, no hydroxychloroquine, and no ICT antibody (1x10 cells / mL). 5 The cells were cultured for 16 hours (Mock group). After the culture, induction of apoptosis was evaluated by the method using FITC-labeled annexin V and propidium iodide (PI) described in Test Example 1-1.

[0125] The results are shown in Figure 12. The vertical axis represents the intensity of PI, and the horizontal axis represents the intensity of FITC-labeled annexin V. Of the four regions in each graph in Figure 12, the plots in the lower right region represent early apoptotic cells, the plots in the upper right region represent late apoptotic cells, and the plots in the lower left region represent viable cells.

[0126] As shown in Figure 12, the CPI + HCQ group did not show a significant antitumor effect compared to the Mock group for both bladder cancer T24 cells and renal clear cell carcinoma KMRC-1 cells, whereas the CPI + HCQ + ICT group showed a significant antitumor effect for both bladder cancer T24 cells and renal clear cell carcinoma KMRC-1 cells. This suggests that the combination therapy of ICT antibody, CPI-613, and hydroxychloroquine may have a significant antitumor effect not only against malignant melanoma but also against other cancer types.

[0127] Test Example 4. Analysis of PDIA3 expression 4-1. BRAF mutant (V600E) malignant melanoma A2058 cells and G361 cells, and BRAF wild-type malignant melanoma Mewo cells were each treated with 1x10 5 The cells were cultured for 16 hours in one of the following media: medium containing no CPI-613, hydroxychloroquine, or ICT antibody (Mock group); medium containing 100 ng / ml CPI-613 and 1 μg / ml hydroxychloroquine but no ICT antibody (CPI + HCQ group); or medium containing 100 ng / ml CPI-613, 1 μg / ml hydroxychloroquine, and 1 μg / ml ICT antibody (CPI + HCQ + ICT group).

[0128] After incubation, a commercially available anti-PDAI3 antibody, ERp57 / ERp60 Polyclonal antibody (Proteintech, Cat. No. 15967-1-AP), was added to the culture medium at a dilution of 1:200 and incubated for 30 minutes at 4°C. Subsequently, a commercially available secondary antibody, Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ Plus 488 (Thermo Fisher Scientific, Cat. No. A32723), was added to the culture medium at a dilution of 1:200 and incubated for 30 minutes at 4°C.

[0129] After the above reaction, the cells were analyzed using a Guava® EasyCyte cell analyzer (Luminex). The results are shown in Figure 13. The vertical axis of each graph represents the cell count, and the horizontal axis represents the fluorescence intensity. The numerical values ​​(%) in the graphs represent the proportion of cells in which fluorescence was observed (i.e., cells with PDIA3 present on the cell surface) among the analyzed cells.

[0130] As shown in Figure 13, for both BRAF mutant and BRAF wild-type melanoma, the percentage of cells expressing PDIA3 on the cell surface was significantly higher in the CPI + HCQ group compared to the Mock group. These results suggest that CPI-613 and hydroxychloroquine increase the amount of PDIA3 present on the cell surface.

[0131] On the other hand, for both BRAF mutant and BRAF wild-type melanoma, the percentage of cells expressing PDIA3 on the cell surface was significantly lower in the CPI + HCQ + ICT group compared to the CPI + HCQ group.

[0132] 4-2. Discussion PDIA3 expression is known to increase in response to metabolic stress. It has also been reported that PDIA3 expression on the cell surface membrane is promoted in cancer cells in a glucose-starved state. Furthermore, as mentioned above, CPI-613 has a cancer cell-specific metabolic inhibitory effect. Based on these facts and the result of increased PDIA3 expression on the cell surface in the CPI + HCQ group in Test Example 4-1, it is possible that CPI-613 and hydroxychloroquine cause metabolic stress in cancer cells, which in turn increases PDIA3 expression and promotes the migration of PDIA3 to the cell surface.

[0133] Furthermore, Test Example 2-3 suggested that the ICT antibody is an antibody that recognizes PDIA3, Test Example 3 suggested that combination therapy of the ICT antibody, CPI-613, and hydroxychloroquine can exhibit significant antitumor effects, and Test Example 4-1 showed that the proportion of cells expressing PDIA3 on the cell surface was significantly lower in the CPI + HCQ + ICT group compared to the CPI + HCQ group. Therefore, it is possible that the ICT antibody directly induces apoptosis by binding to PDIA3 whose expression on the cell surface has been increased by CPI-613 and hydroxychloroquine, or indirectly induces apoptosis by removing PDIA3 whose expression on the cell surface has been increased by CPI-613 and hydroxychloroquine.

[0134] Test Example 5. Study of humanized antibodies Using known gene recombination techniques, a construct was prepared by joining the Fab region of the ICT antibody prepared in Test Example 2 to the Fc region of human IgG1. The construct was then introduced into CHO (Chinese Hamster Ovary) cells to express the protein. The recombinant IgG antibody was purified from the CHO cell culture supernatant using a Protein G column. This antibody may be referred to as a humanized ICT antibody or rICT (recombinant ICT) antibody. The humanized ICT antibody retains all of the heavy chain CDRs 1-3 (SEQ ID NOs: 2-4) and light chain CDRs 1-3 (SEQ ID NOs: 5-7) of the original (pre-humanization) ICT antibody.

[0135] Figure 14a shows the results of immunoblotting using normal human IgG antibody and humanized ICT antibody. Normal human IgG antibody was applied to lane 1, and humanized ICT antibody to lane 2. Detection was performed using anti-human light chain-specific antibody and anti-human heavy chain-specific antibody. As shown in Figure 14a, for both normal human IgG antibody and humanized ICT antibody, bands corresponding to the light chain and heavy chain generated by cleavage of disulfide bonds within the antibody upon reduction were detected. These results confirmed that the humanized ICT antibody we prepared has a human light chain and human heavy chain structure.

[0136] 1x10 51x10 Mewo cells were cultured for 14 hours in a medium containing 5 μg / ml humanized ICT antibody, 1 μg / ml CPI-613, and 10 μg / ml hydroxychloroquine (CPI + HCQ + rICT group). 1x10 Mewo cells were also cultured in a medium containing 1 μg / ml CPI-613 and 10 μg / ml hydroxychloroquine without ICT antibody (CPI + HCQ + rICT group). 5 1x10 Mewo cells were cultured for 14 hours (CPI+HCQ group). Furthermore, 1x10 Mewo cells were cultured in a medium containing 5 μg / ml ICT antibody (the pre-humanized antibody prepared in Test Example 2), 1 μg / ml CPI-613, and 10 μg / ml hydroxychloroquine. 5 Mewo cells were cultured for 14 hours (CPI + HCQ + ICT group). After culture, apoptosis induction was assessed using the FITC-labeled annexin V and propidium iodide (PI) method described in Test Example 1-1. The results are shown in Figure 14b. The vertical axis represents PI intensity, and the horizontal axis represents FITC-labeled annexin V intensity. Of the four regions in each graph, the plots in the lower right region represent early apoptotic cells, the plots in the upper right region represent late apoptotic cells, and the plots in the lower left region represent viable cells. The percentages at the top of the graph represent the percentage of cells plotted in the upper right region (i.e., the percentage of cells that reached late apoptosis). As shown in Figure 14b, when the humanized ICT antibody was used, 23.24% of cells reached late apoptosis. This percentage was similar to the percentage (21.34%) when the non-humanized ICT antibody was used.

[0137] 1x10 4 Mewo cells were cultured in a medium containing 5 μg / ml humanized ICT antibody, 1 μg / ml CPI-613, and 10 μg / ml hydroxychloroquine, and the number of viable cells was counted at 24 and 49 hours after the start of culture (CPI + HCQ + rICT group). 1x10 Mewo cells were also cultured in a medium without humanized ICT antibody, CPI-613, or hydroxychloroquine. 4Mewo cells were cultured, and the number of viable cells was counted at 24 and 49 hours after the start of culture (Mock group). The results are shown in Figure 14c. As shown in Figure 14c, a significant cell proliferation inhibitory effect was observed in the CPI + HCQ + rICT group.

[0138] These results suggest that humanization of the ICT antibody does not impair its antitumor effect.

Claims

1. An antibody or an antigen-binding fragment thereof having binding ability to PDIA3 protein, represented by the general formula (1): (In the formula, R 1 and R 2 are independent of each other, R 3 Acyl groups represented by C(O)-, C n H 2n＋1 an alkyl group represented by C m H 2m－1 an alkenyl group represented by the formula: m H 2m－3 Alkynyl groups, aryl groups, heteroaryl groups, CH3 (CH2) n Sulfurized alkyl group represented by -S-, R 3 An imidoyl group represented by C(=NH)-, R 4 and hydrogen, wherein R 1 and R 2 at least one of R is not hydrogen; 1 and R 2 may be unsubstituted or substituted; R 3 is hydrogen, an alkyl group, an alkenyl group, an alkynyl group, a cycloalkyl group, an aryl group, an alkylaryl group, a heteroaryl group, or a heterocyclyl group, any of which may be substituted or unsubstituted; R 4 is CCl3 or COOH; and x is an integer of 0 to 16, n is an integer of 0 to 10, and m is an integer of 2 to 10) or a salt thereof, and an autophagy inhibitor.

2. The kit described in claim 1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising a complementarity determining region (CDR) 1 comprising the amino acid sequence shown in SEQ ID NO:2, a CDR2 comprising the amino acid sequence shown in SEQ ID NO:3, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO:4, and a light chain variable region comprising a CDR1 comprising the amino acid sequence shown in SEQ ID NO:5, a CDR2 comprising the amino acid sequence shown in SEQ ID NO:6, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO:

7.

3. The kit according to claim 2, wherein the heavy chain variable region comprises: (i) the amino acid sequence shown in SEQ ID NO: 8; or (ii) an amino acid sequence that has 85% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 8; and the light chain variable region comprises: (iii) the amino acid sequence shown in SEQ ID NO: 9; or (iv) an amino acid sequence that has 85% or more sequence identity to the amino acid sequence shown in SEQ ID NO:

9.

4. The kit according to any one of claims 1 to 3, wherein the autophagy inhibitor is a chloroquine compound.

5. The kit according to any one of claims 1 to 3, wherein the autophagy inhibitor is hydroxychloroquine or a salt thereof.

6. In the compound represented by the general formula (1), R 1 and R 2 The kit according to any one of claims 1 to 3, wherein each of 7. The compound represented by the general formula (1) is a compound represented by the general formula (2): The kit according to any one of claims 1 to 3, wherein the compound is represented by the following formula (I):

8. The kit according to any one of claims 1 to 3, wherein the cancer is at least one selected from the group consisting of malignant melanoma, bladder cancer, and kidney cancer.

9. A composition for use in the treatment of cancer, comprising an antibody or an antigen-binding fragment thereof capable of binding to a PDIA3 protein, wherein the treatment comprises administering to a patient a compound represented by the general formula (1): (In the formula, R 1 and R 2 are independent of each other, R 3 Acyl groups represented by C(O)-, C n H 2n＋1 an alkyl group represented by C m H 2m－1 an alkenyl group represented by the formula: m H 2m－3 Alkynyl groups, aryl groups, heteroaryl groups, CH3 (CH2) n Sulfurized alkyl group represented by -S-, R 3 An imidoyl group represented by C(=NH)-, R 4 and hydrogen, wherein R 1 and R 2 at least one of R is not hydrogen; 1 and R 2 may be unsubstituted or substituted; R 3 is hydrogen, an alkyl group, an alkenyl group, an alkynyl group, a cycloalkyl group, an aryl group, an alkylaryl group, a heteroaryl group, or a heterocyclyl group, any of which may be substituted or unsubstituted; R 4 is CCl3 or COOH; and x is an integer of 0 to 16, n is an integer of 0 to 10, and m is an integer of 2 to 10) or a salt thereof, and administration of an autophagy inhibitor.

10. A composition for use in the treatment of cancer, comprising a compound represented by the general formula (1): (In the formula, R 1 and R 2 are independent of each other, R 3 Acyl groups represented by C(O)-, C n H 2n＋1 an alkyl group represented by C m H 2m－1 an alkenyl group represented by the formula: m H 2m－3 Alkynyl groups, aryl groups, heteroaryl groups, CH3 (CH2) n Sulfurized alkyl group represented by -S-, R 3 An imidoyl group represented by C(=NH)-, R 4 and hydrogen, wherein R 1 and R 2 at least one of R is not hydrogen; 1 and R 2 may be unsubstituted or substituted; R 3 is hydrogen, an alkyl group, an alkenyl group, an alkynyl group, a cycloalkyl group, an aryl group, an alkylaryl group, a heteroaryl group, or a heterocyclyl group, any of which may be substituted or unsubstituted; R 4 is CCl3 or COOH; and x is an integer of 0 to 16, n is an integer of 0 to 10, and m is an integer of 2 to 10) or a salt thereof, wherein the treatment comprises administration of an antibody or an antigen-binding fragment thereof capable of binding to PDIA3 protein, and administration of an autophagy inhibitor.

11. A composition for use in the treatment of cancer, comprising an autophagy inhibitor, the treatment comprising administering an antibody or an antigen-binding fragment thereof capable of binding to PDIA3 protein, and a compound represented by the general formula (1): (In the formula, R 1 and R 2 are independent of each other, R 3 Acyl groups represented by C(O)-, C n H 2n＋1 an alkyl group represented by C m H 2m－1 an alkenyl group represented by the formula: m H 2m－3 Alkynyl groups, aryl groups, heteroaryl groups, CH3 (CH2) n Sulfurized alkyl group represented by -S-, R 3 An imidoyl group represented by C(=NH)-, R 4 and hydrogen, wherein R 1 and R 2 at least one of R is not hydrogen; 1 and R 2 may be unsubstituted or substituted; R 3 is hydrogen, an alkyl group, an alkenyl group, an alkynyl group, a cycloalkyl group, an aryl group, an alkylaryl group, a heteroaryl group, or a heterocyclyl group, any of which may be substituted or unsubstituted; R 4 is CCl3 or COOH; and x is an integer from 0 to 16, n is an integer from 0 to 10, and m is an integer from 2 to 10, or a salt thereof.

12. An antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising complementarity determining region (CDR) 1 comprising the amino acid sequence set forth in SEQ ID NO: 2, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 3, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 4, and a light chain variable region comprising CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 5, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 6, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:

7.

13. The antibody or antigen-binding fragment thereof according to claim 12, wherein the heavy chain variable region comprises: (i) an amino acid sequence set forth in SEQ ID NO: 8; or (ii) an amino acid sequence having 85% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 8; and the light chain variable region comprises: (iii) an amino acid sequence set forth in SEQ ID NO: 9; or (iv) an amino acid sequence having 85% or more sequence identity to the amino acid sequence set forth in SEQ ID NO:

9.

14. An antibody or an antigen-binding fragment thereof according to claim 12 or 13, which has the ability to bind to PDIA3 protein.

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