Anti-Hsp70 antibody

Abstract

The objective of this invention is to provide a novel useful antibody against Hsp70. [Solution] An anti-Hsp70 antibody having an antibody that recognizes a region corresponding to the 453rd to 499th amino acid sequence of a certain specific amino acid sequence in the Hsp70 protein, or an anti-Hsp70 antibody having, as a heavy chain complementarity determining region, HCDR1 consisting of amino acid sequences at positions 45 to 56, 50 to 56, or 45 to 54 of another specific amino acid sequence, HCDR2 consisting of amino acid sequences at positions 71 to 86, or 72 to 78, and HCDR3 consisting of amino acid sequences at positions 117 to 128, or 119 to 128, and as a light chain complementarity determining region, LCDR1 consisting of amino acid sequences at positions 44 to 54, or 47 to 52 of yet another specific amino acid sequence, LCDR2 consisting of amino acid sequences at positions 70 to 76, or 70 to 71, and LCDR3 consisting of amino acid sequences at positions 109 to 117, has excellent antitumor activity.

Description

Technical Field

[0001] The present invention relates to an anti-Hsp70 antibody.

Background Art

[0002] Heat shock proteins (Hsps) are a highly conserved protein family that function as molecular chaperones playing important roles in intracellular protein homeostasis, regulation of apoptosis, and protection from various stress factors (hypoxia, heat stress, oxidative stress, etc.). Among Hsps, proteins such as HSP40, HSP60, HSP70, and HSP90 are known to localize to the plasma membrane of malignantly transformed cells. (Non-Patent Document 1)

[0003] Currently, an anti-Cell Membrane HSP70 antibody is commercially available as a useful antibody against Hsp70 (Non-Patent Document 1). This antibody hardly binds to intracellular HSP70, and has the property of specifically binding to cell membrane HSP70, and is used in cancer research.

Prior Art Literature

Non-Patent Literature

[0004]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

Problem to be Solved by the Invention

[0005] Although proteins such as Hsp70 are promising targets for the diagnosis or treatment of cancer, there is a lack of suitable antibodies to address them. Therefore, the present invention aims to provide novel useful antibodies against Hsp70. [Means for solving the problem]

[0006] The inventors diligently screened for anti-Hsp70 antibodies from 2700 hybridoma clones obtained by the lymph node method, and as a result, selected an antibody that barely showed a slight antitumor effect in vitro. Furthermore, when the antitumor effect of this selected antibody was examined in vivo, it was confirmed to exhibit a remarkable antitumor effect. This invention was completed by further investigation based on this finding.

[0007] In other words, the present invention provides inventions in the following embodiments. Item 1. An anti-Hsp70 antibody that recognizes the region corresponding to positions 453-499 of SEQ ID NO: 1 in the Hsp70 protein. Item 2. As heavy chain complementarity determining regions, HCDR1 consists of amino acid sequences 45-56, 50-56, or 45-54 of SEQ ID NO: 2, HCDR2 consists of amino acid sequences 71-86, or 72-78 of SEQ ID NO: 2, and HCDR3 consists of amino acid sequences 117-128, or 119-128 of SEQ ID NO: 2, An anti-Hsp70 antibody having LCDR1 consisting of amino acid sequences 44-54 or 47-52 of SEQ ID NO: 3, LCDR2 consisting of amino acid sequences 70-76 or 70-71 of SEQ ID NO: 3, and LCDR3 consisting of amino acid sequences 109-117 of SEQ ID NO: 3 as light chain complementarity determining regions. Item 3. The anti-Hsp70 antibody described in Item 2, wherein the heavy chain variable region consists of the amino acid sequence from positions 20 to 139 of SEQ ID NO: 2, and the light chain variable region consists of the amino acid sequence from positions 21 to 127 of SEQ ID NO: 3. Item 4. An antitumor agent comprising an anti-Hsp70 antibody as described in any of Items 1 to 3. Item 5. An antitumor agent as described in Item 4, applicable to liver cancer. Item 6. An antitumor agent as described in item 4 or 5, not used in combination with other pharmacotherapy agents. Item 7. An antigen test kit containing the anti-Hsp70 antibody described in any of Items 1-3. Item 8. A nucleic acid encoding the anti-Hsp70 antibody described in Item 2 or 3. Item 9. An expression cassette or recombinant vector containing the nucleic acid described in Item 8. Item 10. A transformant obtained by transforming a host using the expression cassette or recombinant vector described in Item 9. Item 11. A method for producing an anti-Hsp70 antibody, comprising the step of culturing the transformant described in Item 10. [Effects of the Invention]

[0008] According to the present invention, a novel useful antibody against Hsp70 is provided. [Brief explanation of the drawing]

[0009] [Figure 1] This shows the changes in luciferase activity derived from pancreatic cancer cells when mice carrying human pancreatic cancer cells PANC-1 expressing the luciferase gene were administered 20 mg / kg of mouse IgG or 7H8 antibody (the anti-Hsp70 antibody of the present invention). [Figure 2] Figure 1 shows the results of in vivo imaging of the luciferase activity of the primary tumor four weeks after antibody administration. [Figure 3] Figure 1 shows the tumor weight at 4 weeks after antibody administration. [Figure 4] The results of immunostaining of human pancreatic ductal adenocarcinoma tissue using the 7H8 antibody (anti-Hsp70 antibody of the present invention) are shown. [Figure 5] The results of immunostaining of human lung squamous cell carcinoma tissue using the 7H8 antibody (anti-Hsp70 antibody of the present invention) are shown. [Modes for carrying out the invention]

[0010] 1. Anti-Hsp70 antibody The anti-Hsp70 antibody of the present invention can be specified as an antibody that recognizes a predetermined epitope in Hsp70 protein. The Hsp70 targeted by the anti-Hsp70 antibody of the present invention is not particularly limited as long as it has the predetermined epitope, and may be either constitutive or inducible. Specific examples of the target Hsp70 include the proteins shown in the following table (represented by representative names: Hsp70-1a, Hsp70-1b, Hsp70-1t, Hsp70-2, Hsp70-5, Hsp70-6, HSC70, Hsp70-9), preferably Hsp70-1a and Hsp70-1b.

[0011]

Table 1

[0012] The predetermined epitope recognized by the anti-Hsp70 antibody of the present invention is a region corresponding to positions 453 to 499 of SEQ ID NO: 1. SEQ ID NO: 1 is the amino acid sequence of Hsp70-1a. In the present invention, as long as the predetermined epitope is a region corresponding to positions 453 to 499 of SEQ ID NO: 1, it includes not only the amino acid sequence consisting of positions 453 to 499 of SEQ ID NO: 1, but also an amino acid sequence having 70% or more sequence identity with said sequence. The region corresponding to positions 453 to 499 of SEQ ID NO: 1 can be easily identified by aligning the amino acid sequence of Hsp70-1a (SEQ ID NO: 1) with the amino acid sequences of, for example, Hsp70-1b, Hsp70-1t, Hsp70-2, Hsp70-5, Hsp70-6, HSC70, Hsp70-9, etc.

[0013] Specific examples of the region (predetermined epitope) corresponding to positions 453 to 499 of SEQ ID NO: 1 include the following [a] to [h]. [a] A region consisting of the amino acid sequence at positions 453 to 499 of Hsp70-1a (SEQ ID NO: 1) [b] A region consisting of the amino acid sequence at positions 453 to 499 of Hsp70-1b (SEQ ID NO: 4) [c] A region consisting of the amino acid sequence at positions 455 to 501 of Hsp70-1t (SEQ ID NO: 5) [d] A region consisting of the amino acid sequence at positions 456 to 502 of Hsp70-2 (SEQ ID NO: 6) [e] A region consisting of the amino acid sequence at positions 476 to 522 of Hsp70-5 (SEQ ID NO: 7) [f] A region consisting of the amino acid sequence at positions 455 to 501 of Hsp70-6 (SEQ ID NO: 8) [g] A region consisting of the amino acid sequence at positions 453 to 499 of HSC70 (SEQ ID NO: 9) [h] A region consisting of the amino acid sequence at positions 497 to 543 of Hsp70-9 (SEQ ID NO: 10)

[0014] The regions of [a] and [b] above are identical in sequence to the amino acid sequence consisting of positions 453 to 499 of SEQ ID NO: 1. The regions of [c] to [h] above have 70% or higher sequence identity with the amino acid sequence consisting of positions 453 to 499 of SEQ ID NO: 1. In the present invention, preferred examples of the region corresponding to positions 453 to 499 of SEQ ID NO: 1 (a predetermined epitope) include the regions of [a] and [b] above.

[0015] An example of the anti-Hsp70 antibody of the present invention includes an antibody comprising a heavy chain containing the amino acid sequence set forth in SEQ ID NO: 2 and a light chain containing the amino acid sequence set forth in SEQ ID NO: 3.

Table 2

[0016] SEQ ID NO: 2 consists of a signal sequence at positions 1 to 19 and a heavy chain variable region (VH) at positions 20 to 139, and SEQ ID NO: 3 consists of a signal sequence at positions 1 to 20 and a light chain variable region (VL) at positions 21 to 127.

[0017]

Table 3

[0018] The heavy chain complementarity determination regions in the heavy chain variable region (VH) and the light chain complementarity determination regions in the light chain variable region (VL) are shown below in terms of the complementarity determination regions (CDRs) defined by all combinations (Kabat / IMGT) of the Kabat numbering program (Bioinformatics, 32, 298-300 (2016)) and the IMGT system (Dev Comp Immunol. 27, 55-77 (2003)), the CDRs defined by the Kabat numbering program, and the CDRs defined by the IMGT system.

[0019] [Table 4]

[0020] Therefore, the anti-Hsp70 antibody of the present invention may be identified as an antibody having the following complementarity-determining region (CDR). [HCDR] Heavy Chain Complementarity Determination Region HCDR1 consists of amino acid sequences 45-56, 50-56, or 45-54 of SEQ ID NO: 2; HCDR2 consists of amino acid sequences 71-86, or 72-78 of SEQ ID NO: 2; and HCDR3 consists of amino acid sequences 117-128, or 119-128 of SEQ ID NO: 2. [LCDR] Light chain complementarity determination region LCDR1 consists of amino acid sequences 44-54 or 47-52 of SEQ ID NO: 3, LCDR2 consists of amino acid sequences 70-76 or 70-71 of SEQ ID NO: 3, and LCDR3 consists of amino acid sequences 109-117 of SEQ ID NO: 3.

[0021] In the anti-Hsp70 antibody of the present invention, the regions other than the CDR (signal sequence and framework sequence) are not particularly limited as long as they do not impair the binding activity of Hsp70 to a predetermined epitope, preferably in addition to said binding activity, antitumor activity. Specific amino acid sequences that constitute the regions other than the CDR include amino acid sequences that have 80% or more sequence identity with the signal sequence (amino acid sequences 1 to 19 of SEQ ID NO: 2, amino acid sequences 1 to 20 of SEQ ID NO: 3) and framework sequence (amino acid sequences connecting HCDR1 and HCDR2 of SEQ ID NO: 2, amino acid sequences connecting HCDR2 and HCDR3 of SEQ ID NO: 2, amino acid sequences connecting LCDR1 and LCDR2 of SEQ ID NO: 3, and amino acid sequences connecting LCDR2 and L of SEQ ID NO: 3), which are the regions other than the CDR in SEQ ID NO: 2 and 3.

[0022] Preferred examples of sequence identity of 80% or more may vary depending on the total length of the reference sequence, but preferably include 85% or more, more preferably 90% or more, even more preferably 92% or more, even more preferably 94% or more or 95% or more, even more preferably 96% or more or 97% or more, and most preferably 100%.

[0023] Note that "sequence identity" refers to the value of amino acid sequence identity obtained by the bl2seq program (Tatiana A. Tatsusova, Thomas L. Madden, FEMS Microbiol. Lett., Vol. 174, p247-250, 1999) from BLAST PACKAGE [sgi32 bit edition, Version 2.0.12; available from National Center for Biotechnology Information (NCBI)]. The parameters should be set to Gap insertion Cost value: 11 and Gap extension Cost value: 1.

[0024] In amino acid sequences with 80% or more and less than 100% sequence identity, if a predetermined amino acid is substituted from the amino acid sequences in the signal sequences shown in SEQ ID NOs: 2 and 3 (amino acid sequences 1 to 19 of SEQ ID NO: 2, and amino acid sequences 1 to 20 of SEQ ID NO: 3), and the framework sequences shown in SEQ ID NOs: 2 and 3 (amino acid sequences connecting HCDR1 and HCDR2 of SEQ ID NO: 2, amino acid sequences connecting HCDR2 and HCDR3 of SEQ ID NO: 2, amino acid sequences connecting LCDR1 and LCDR2 of SEQ ID NO: 3, and amino acid sequences connecting LCDR2 and L of SEQ ID NO: 3), then substitution with similar amino acids (i.e., conservative amino acid substitutions) is preferred because it is predicted that it will not cause a change in the neutralizing activity of the antibody. Specifically, based on the properties of the amino acid side chains, the following classification has been established, and it is preferable to substitute with amino acids belonging to the same classification. Basic amino acids: lysine, arginine, histidine Acidic amino acids: glutamic acid, aspartic acid Neutral amino acids: Glycine, Alanine, Serine, Threonine, Methionine, Cysteine, Phenylalanine, Tryptophan, Tyrosine, Leucine, Isoleucine, Valine, Glutamine, Asparagine, Proline Furthermore, the aforementioned neutral amino acids can also be classified into those having polar side chains (asparagine, glutamine, serine, threonine, tyrosine, cysteine), those having nonpolar side chains (glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), those having amide-containing side chains (asparagine, glutamine), those having sulfur-containing side chains (methionine, cysteine), those having aromatic side chains (phenylalanine, tryptophan, tyrosine), those having hydroxyl-containing side chains (serine, threonine, tyrosine), and those having aliphatic side chains (alanine, leucine, isoleucine, valine), etc.

[0025] Regarding methods for substituting specific amino acids in an amino acid sequence with other amino acids, site-directed mutagenesis (SMS) is known, for example (Hashimoto-Gotoh T. et al., Gene, Vol.152, p.271-275 (1995); Zoller MJ. et al., Methods Enzymol. Vol.100, p.468-500 (1983), Kramer W. et al., Nucleic Acids Res. Vol.12, p.9441-9456 (1984); Kramer W. et al., Methods Enzymol. Vol.154, p.350-367 (1987); Kunkel TA., Proc Natl Acad Sci USA., Vol.82, p.488-492 (1985), etc.), and amino acid substitutions can be performed in the amino acid sequence of CDR using this SMS method. Another method for substituting with other amino acids is the library technique described in WO2005 / 080432.

[0026] The isotype of the anti-Hsp70 antibody of the present invention is not particularly limited and includes, for example, IgG (IgG1, IgG2, IgG3, IgG4), IgA (IgA1, IgA2), IgM, IgD, and IgE.

[0027] The anti-Hsp70 antibody of the present invention is typically an isolated monoclonal antibody. The method for producing the monoclonal antibody is not particularly limited, but it can be produced by, for example, the hybridoma method as described in "Kohler G, Milstein C., Nature. 1975 Aug 7;256(5517):495-497."; the recombinant method as described in U.S. Patent No. 4816567; isolation from a phage antibody library as described in "Clackson et al., Nature. 1991 Aug 15;352(6336):624-628." or "Marks et al., J Mol Biol. 1991 Dec 5;222(3):581-597."; or by the method described in "Protein Experiment Handbook, Yodosha (2003):92-96.".

[0028] Furthermore, the anti-Hsp70 antibody of the present invention includes not only full-length antibodies (antibodies having a Fab region and an Fc region) but also fragment antibodies. Such fragment antibodies include Fv antibodies, Fab antibodies, Fab' antibodies, and F(ab') antibodies. 2 Examples include antibodies, scFv antibodies, dsFv antibodies, diabodies, nanobodies, etc. Furthermore, the anti-Hsp70 antibody of the present invention also includes multivalent specific antibodies (e.g., bispecific antibodies) insofar as they contain the aforementioned variable region. These fragment antibodies and multispecific antibodies can be produced according to conventionally known methods.

[0029] The anti-Hsp70 antibody of the present invention may be a conjugate antibody or conjugate antibody fragment conjugated with various compounds such as polyethylene glycol, radioactive substances, or toxins. Furthermore, the anti-Hsp70 antibody of the present invention may have its conjugated sugar chain modified or may be fused with other proteins, as needed.

[0030] 2. Nucleic acid The present invention also provides nucleic acids encoding the anti-Hsp70 antibody described above. The nucleic acids of the present invention can be obtained, for example, by using the nucleic acids encoding the anti-Hsp70 antibody described above as a template and obtaining at least the region encoding the anti-Hsp70 antibody by PCR or the like. Furthermore, the nucleic acids of the present invention can also be artificially synthesized by gene synthesis methods.

[0031] Those skilled in the art can appropriately design the nucleic acid sequence of the present invention according to the CDR sequence of the anti-Hsp70 antibody of the present invention. For example, SEQ ID NO: 11 is an example of the nucleotide sequence encoding SEQ ID NO: 2, and SEQ ID NO: 12 is an example of the nucleotide sequence encoding SEQ ID NO: 3. Therefore, the nucleotide sequence of the nucleic acid of the present invention can be designed using the regions encoding each CDR contained in these nucleotide sequences.

[0032] 3. Expression cassette or recombinant vector The expression cassette or recombinant vector of the present invention comprises the nucleic acid of the present invention as described in "2. Nucleic Acids" above. The expression cassette or recombinant vector of the present invention can be obtained by linking a promoter and a terminator to the nucleic acid of the present invention, or by inserting the expression cassette or nucleic acid of the present invention into an expression vector.

[0033] The expression cassette or recombinant vector of the present invention includes regulatory factors such as promoters operably linked to the nucleic acid of the present invention. Typical regulatory factors include promoters, but may also include, as needed, enhancers, CCAAT boxes, TATA boxes, SPI sites, and other transcription elements. Furthermore, "operably linked" means that various regulatory factors such as promoters and enhancers that regulate the nucleic acid of the present invention are linked to the nucleic acid of the present invention in a manner that allows them to function within the host cell.

[0034] Preferred expression vectors are those constructed for genetic recombination from phages, plasmids, or viruses that can autonomously proliferate within a host. Specifically, examples include plasmids derived from E. coli (e.g., pET-Blue), plasmids derived from Bacillus subtilis (e.g., pUB110), plasmids derived from yeast (e.g., pSH19), animal cell expression plasmids (e.g., pA1-11, pcDNA3.1-V5 / His-TOPO), bacteriophages such as λ phage, and virus-derived vectors.

[0035] 4. Transformed organism The transformant is obtained by transforming the host using the expression cassette of the present invention described in "3. Expression Cassette or Recombinant Vector" above or the recombinant vector described above.

[0036] The host used to produce the transformant is not particularly limited as long as it is capable of gene introduction, autonomous proliferation, and expression of the gene traits of the present invention. Examples include human and non-human mammal cells (e.g., rats, mice, guinea pigs, rabbits, cattle, monkeys, etc.), more specifically, Chinese hamster ovary cells (CHO cells), monkey cells COS-7, human fetal kidney cells (e.g., HEK293 cells); insect cells; plant cells; bacteria belonging to the Escherichia genus such as Escherichia coli, the Bacillus genus such as Bacillus subtilis, the Pseudomonas genus such as Pseudomonas putida, etc.; actinomycetes, etc.; yeast, etc.; filamentous fungi, etc.

[0037] Transformants can be obtained by introducing the expression cassette or recombinant vector of the present invention into a host. The site on which the nucleic acid of the present invention is introduced is not particularly limited as long as the target gene can be expressed, and may be on a plasmid or on the genome. Specific methods for introducing the expression cassette or recombinant vector of the present invention include, for example, recombinant vector methods and genome editing methods.

[0038] The conditions for introducing an expression cassette or recombinant vector into the host can be appropriately set according to the type of host, etc. If the host is bacteria, for example, methods using competent cells treated with calcium ions and electroporation can be used. If the host is yeast, for example, electroporation, spheroplast method and lithium acetate method can be used. If the host is animal cells, for example, electroporation, calcium phosphate method and lipofection method can be used. If the host is insect cells, for example, calcium phosphate method, lipofection method and electroporation method can be used. If the host is plant cells, for example, electroporation, Agrobacterium method, particle gun method and PEG method can be used.

[0039] 5. Method for producing anti-Hsp70 antibodies The present invention provides a method for producing an anti-Hsp70 antibody, as described in "1. Anti-Hsp70 Antibody" above, and includes the step of culturing the transformant of the present invention as described in "4. Transformant" above. The culture conditions for the transformant can be appropriately set considering the nutritional and physiological properties of the host, but liquid culture is preferred. The anti-Hsp70 antibody can be appropriately recovered and purified from the obtained culture.

[0040] 6. Antitumor agents The present invention also provides an antitumor agent comprising the anti-Hsp70 antibody described in "1. Anti-Hsp70 antibody" above.

[0041] The antitumor agent of the present invention is typically prepared in the form of a pharmaceutical composition containing the above-mentioned anti-Hsp70 antibody as an active ingredient. The antitumor agent of the present invention only needs to contain an effective amount of the above-mentioned anti-Hsp70 antibody and may also contain other pharmaceutically acceptable carriers or additives. Examples of such carriers or additives include surfactants, excipients, colorants, flavorings, preservatives, stabilizers, buffers, pH buffers, disintegrants, solubilizers, solubilizers, isotonic agents, binders, disintegrants, lubricants, diluents, and flavorings.

[0042] The antitumor agent of the present invention may contain other antitumor agents besides the anti-Hsp70 antibody. However, since the anti-Hsp70 antibody has excellent antitumor effects, a preferred embodiment of the antitumor agent of the present invention does not contain other antitumor agents.

[0043] Furthermore, the method of administering the antitumor agent of the present invention may be either oral or parenteral. Examples of specific administration methods include oral administration; and parenteral administration such as intravenous administration, intramuscular administration, intraperitoneal administration, subcutaneous administration, nasal administration, pulmonary administration, transdermal administration, transmucosal administration, and intraocular administration.

[0044] The formulation form of the antitumor agent of the present invention can be appropriately determined depending on the method of administration used. For example, when used for oral administration, it can be prepared in the form of a powder, granules, capsules, syrup, suspension, etc., and when used for parenteral administration, it can be prepared in the form of a liquid, suspension, emulsion, spray, suppository, eye drops, etc.

[0045] The antitumor agent of the present invention can preferably be applied to humans or non-human animals (typically mammals).

[0046] The antitumor agent of the present invention can be used to treat any tumor disease. Specific tumor diseases include liver cancer, gastric cancer, bladder cancer, pancreatic cancer, lung cancer, and gallbladder cancer.

[0047] The dosage of the antitumor agent of the present invention is not particularly limited, but a human dose may be, for example, 0.5 to 5 mg / kg, preferably 0.8 to 3 mg / kg, and more preferably 1 to 2 mg / kg per dose.

[0048] The antitumor agent of the present invention may be used in combination with other drug therapies (chemotherapy, endocrine therapy (hormone therapy), molecular targeted therapy, etc.) when used to treat any tumor disease. However, because the anti-Hsp70 antibody has excellent antitumor effects, in a preferred embodiment of the antitumor agent of the present invention, it is used as a single active ingredient without being used in combination with other drug therapies.

[0049] 7. Antigen test kit The present invention also provides an antigen test kit containing the anti-Hsp70 antibody described in "1. Anti-Hsp70 antibody" above.

[0050] The above anti-Hsp70 antibody may be immobilized on a water-insoluble carrier. The water-insoluble carrier may be particles or a substrate.

[0051] The specimen is not particularly limited, as long as it is required to detect Hsp70 having a predetermined epitope. Specifically, examples include bodily fluid samples such as blood and urine, and tissue samples such as tumor tissue.

[0052] The antigen test kit of the present invention can be constructed based on an immunoassay (a typical example being enzyme immunosorbent assay (ELISA)) that measures the antigen-antibody reaction between the anti-Hsp70 antibody and the antigen test in the sample.

[0053] In addition to the anti-Hsp70 antibody described above, the antigen test kit of the present invention may also contain other antibodies and / or other reagents and / or instruments corresponding to the measurement method. [Examples]

[0054] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0055] [Test Example 1] (1) Acquisition of antibody 7H8 From 2700 hybridoma clones obtained by lymph node biopsy using GST-Hsp70 as the antigen, screening for anti-Hsp70 antibodies yielded the anti-Hsp70 monoclonal antibody 7H8(IgM), which showed slight antitumor effects in vitro.

[0056] (2) Epitope mapping of antibody 7H8 A fragment of human Hsp70 (HSPA1A) having the amino acid sequence shown in Sequence ID No. 1 (Table 5) was inserted into pGEX-2TK to create a GST fusion protein. [Table 5]

[0057] The following ELISA was performed for epitope mapping. Each Hsp70 fragment, full-length Hsp70, or full-length Hsc70 (a related molecule with 86% homology to Hsp70 (HspA8)) GST fusion protein from Table 5 was diluted to 1 μL in 20 mM sodium phosphate buffer (pH 7.2) and immobilized on a 96-well plate. After blocking with 1% BSA / TBS-T, hybridoma culture supernatant was added and antibody reaction was carried out. After washing, the mixture was reacted with HRP-conjugated anti-mouse IgM + IgG antibody. After washing, substrate was added and color development was carried out, and the absorbances at OD450 and OD570 were measured. The results are shown in Table 6.

[0058] [Table 6]

[0059] Fr5 showed high absorbance, while Fr4 and Fr6 showed low absorbance. However, the regions of both fragments overlapping with Fr5 were judged to be non-antigenic, and the region 453-499 of Fr5 (396-550) was identified as the epitope of antibody 7H8. Furthermore, antibody 7H8 cross-reacted with the entire length of Hsp70. In addition, a reaction was observed with Hsc70 (gene HSPA8), which has 86% homology at the amino acid level, suggesting that antibody 7H8 may also cross-react with HSC70.

[0060] (3) Sequence analysis of antibody 7H8 Sequence analysis of the heavy and light chains of antibody 7H8 yielded Sequence ID No. 2 and Sequence ID No. 3, respectively. Sequence ID No. 2 consisted of signal sequences 1-19 followed by the heavy chain variable region (VH), and Sequence ID No. 3 consisted of signal sequences 1-20 followed by the light chain variable region (VL). The sequence analysis results for Sequence ID Nos. 2, 3, the heavy chain variable region (VH), the light chain variable region (VL), and each CDR in these regions are as follows. Note that the CDR sequences are shown for all combinations of the Kabat numbering program (Bioinformatics, 32, 298-300 (2016)) and the IMGT system (Dev Comp Immunol. 27, 55-77 (2003)) (Kabat / IMGT), as well as for CDRs defined by the Kabat numbering program and CDRs defined by the IMGT system.

[0061] [Table 7A]

[0062] [Table 7B]

[0063] [Table 7C]

[0064] [Test Example 2] Four 6-week-old female BALB / c Slc-nu / nu mice were used. 10,000,000 human pancreatic cancer cells, PANC-1, which stably express the luciferase gene, were transplanted subcutaneously on the right dorsal side of each mouse. Tumor growth was evaluated once a week using IVIS (PerkinElmer) starting one week after transplantation. IVIS measurements were taken 15 minutes after intraperitoneal administration of luciferin, with the primary tumor imaged in the prone position, followed immediately by a supine position image to check for metastasis. Four weeks after transplantation, two mice with similar tumor sizes were selected, and mouse IgG or 7H8 antibody was administered at a dose of 20 mg / kg (400 μg / mouse / administration, as the mouse body weight was 20 g) once a week for three weeks. Four weeks after antibody administration, the mice were sacrificed, the tumors were excised, and the tumor weight was measured.

[0065] The results of measuring luciferase derived from pancreatic cancer cells are shown in Figure 1 (changes in luciferase activity) and Figure 2 (measurement results at 4 weeks after antibody administration), and the results of measuring the weight of the excised tumor are shown in Figure 3.

[0066] As shown in Figures 1 and 2, mice administered with the 7H8 antibody showed a remarkable reduction in tumor size in one group and complete disappearance of tumors in the other. This was also reflected in the results in Figure 3. In other words, the 7H8 antibody exhibited extremely high antitumor activity on its own.

[0067] [Test Example 3] The staining properties for human pancreatic ductal adenocarcinoma and lung squamous cell carcinoma were evaluated. Paraffin-embedded human cancer tissue blocks were sectioned, deparaffinized, and the antigens were inactivated using microwave and 3% hydrogen peroxide. VECSTAIN ABC Kit Mouse IgM (VECTOR, PK-4010) was used for staining. After blocking, antibody 7H8 was diluted 100-fold and the antibody reaction was carried out four times overnight. After washing, biotinylated secondary antibody was reacted for 30 minutes, followed by reaction with ABC reagent and then with DAB (3,3′-diaminobenzidine). The color development was performed. After stopping the reaction, the samples were washed, stained with hematoxylin, dehydrated, cleared, and mounted. Positive or negative results were determined based on the presence or absence of staining.

[0068] Pancreatic ductal adenocarcinoma was positive in 4 out of 28 cases (positive rate 14%), and lung squamous cell carcinoma was positive in 1 out of 30 cases (positive rate 3%). An example of staining results for pancreatic ductal adenocarcinoma is shown in Figure 4, and an example of staining results for lung squamous cell carcinoma is shown in Figure 5.

Claims

1. An anti-Hsp70 antibody that recognizes the region of the Hsp70 protein corresponding to positions 453-499 of Sequence ID No.

1.

2. The heavy chain complementarity determining regions are HCDR1, consisting of amino acid sequences 45-56, 50-56, or 45-54 of SEQ ID NO: 2; HCDR2, consisting of amino acid sequences 71-86, or 72-78 of SEQ ID NO: 2; and HCDR3, consisting of amino acid sequences 117-128, or 119-128 of SEQ ID NO:

2. An anti-Hsp70 antibody having, as a light chain complementarity determining region, LCDR1 consisting of amino acid sequences 44-54 or 47-52 of SEQ ID NO: 3, LCDR2 consisting of amino acid sequences 70-76 or 70-71 of SEQ ID NO: 3, and LCDR3 consisting of amino acid sequences 109-117 of SEQ ID NO:

3.

3. The anti-Hsp70 antibody according to claim 2, wherein the heavy chain variable region consists of the amino acid sequence from position 20 to 139 of SEQ ID NO: 2, and the light chain variable region consists of the amino acid sequence from position 21 to 127 of SEQ ID NO:

3.

4. An antitumor agent comprising the anti-Hsp70 antibody according to claim 1 or 2.

5. The antitumor agent according to claim 4, applicable to liver cancer.

6. The antitumor agent according to claim 4, which is not used in combination with other drug therapy agents.

7. An antigen test kit comprising the anti-Hsp70 antibody according to claim 1 or 2.

8. A nucleic acid encoding the anti-Hsp70 antibody according to claim 2.

9. An expression cassette or recombinant vector comprising the nucleic acid described in claim 8.

10. A transformant obtained by transforming a host using the expression cassette or recombinant vector described in claim 9.

11. A method for producing an anti-Hsp70 antibody, comprising the step of culturing the transformant described in claim 10.

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