Patent Application
20240190992
The present invention relates to an antibody against pancreatic cancer stem cells.
Cancer is the top cause of death for Japanese people and is the greatest threat for human beings. In cancer, pancreatic cancer results in a particularly poor prognosis, and about 70 to 80% of patients in which pancreatic cancer has been found have progressed to a state where surgery cannot be already performed. Furthermore, since the pancreas is surrounded by other organs, pancreatic cancer is likely to metastasize to the lymph nodes and the liver, and is difficult to treat. While improvement in surgical treatment technology and development of new drugs have progressed, the 5-year survival rate of pancreatic cancer patients is significantly lower than the average of those for all cancers, which indicates the difficulty of treatment.
Accordingly, improvement of the therapeutic effect on pancreatic cancer is an extremely serious challenge. Current cancer treatment includes surgical therapy including surgically removing a cancer tumor, chemotherapy using an anticancer agent, and radiation therapy. However, regarding the surgical therapy, a decrease in postoperative QOL of the patient is considered as a disadvantage. In addition, it has been revealed that chemotherapy and radiation therapy bring about resistance to cancer stem cells, and their mechanisms have been studied (Non-Patent Documents 1 to 5). Furthermore, a hypothesis that chemotherapy triggers reactivation of dormant cancer stem cells to induce recurrence of cancer tumors is being supported (Non-Patent Document 6).
Although there are many unknown parts in cancer stem cells, development of therapies targeting cancer stem cells has been variously advanced. For example, cancer stem cell markers or pharmaceutical products targeting signal pathways (Wnt, Notch) specifically activated in cancer stem cells have been developed (Non-Patent Document 7). In addition, gemtuzumab ozogamicin, an antibody-drug conjugate targeting the peripheral blood monocyte marker CD33, was approved by the FDA (U.S. Food and Drug Administration) for acute myeloid leukemia, but was discontinued because toxicity was found (Non-Patent Document 8).
As there is a case where a pharmaceutical product is discontinued even when approved by the FDA, because toxicity is found, pharmaceutical products targeting cancer stem cells have a risk of affecting normal stem cells. Accordingly, pharmaceutical products are being developed but many of them remain at the stage of clinical trials. An object of the present invention is to provide an antibody against pancreatic cancer stem cells.
The present inventor has succeeded in obtaining a new antibody having a binding property to pancreatic cancer stem cells by injecting a suspension prepared by mixing KMC07 cultured using a human pancreatic cancer stem cell line KMC07 established from a patient with advanced pancreatic ductal adenocarcinoma resistant to gemcitabine chemotherapy as an antigen and a mouse fibroblast cell line PA6 as a feeder cell with an adjuvant to provide cell immunization, and screening antibodies produced by hybridomas prepared and selected using lymphocytes collected after the immunization. The present invention has been completed by further conducting studies based on this finding.
In other words, the present invention provides an invention of the aspects described below.
According to the present invention, a new antibody having a binding property to pancreatic cancer stem cells can be provided.
The present invention is an antibody against pancreatic cancer stem cells. The antibody against pancreatic cancer stem cells refers to an antibody having at least a binding property to pancreatic cancer stem cells, and the binding property at least needs to have binding ability to pancreatic cancer stem cells but not have binding ability to normal cells, which binding property includes both of a binding property specific to pancreatic cancer stem cells and a binding property not specific to pancreatic cancer stem cells (in other words, a property of binding not only to pancreatic cancer but also to cancer cells other than pancreatic cancer). The antibody of the present invention includes the following antibody I, antibody II, and antibody III.
The CDRs (complementarity determining regions) of the antibody of the present invention include CDRs defined by all combinations (hereinafter, also referred to as “Kabat/IMGT/Paratome”.) of the Kabat numbering program (Bioinformatics, 32, 298-300 (2016)), the IMGT system (Dev Comp Immunol. 27, 55-77 (2003)), and the Paratome algorithm (Nucleic Acids Res. 40 (Web Server issue): W521-4 (2012). doi: 10.1093/nar/gks 480 and PLOS Comput Biol. 8(2): e 1002388 (2012)), and CDRs defined by any of the Kabat numbering program, the IMGT system, and the Paratome algorithm. The sequences of CDRs as defined by Kabat/IMGT/Paratome encompass all of the sequences of CDRs as defined by the Kabat numbering program, the sequences of CDRs as defined by the IMGT system, and the sequences of CDRs as defined by the Paratome algorithm.
Antibody I is an antibody against pancreatic cancer stem cells containing a heavy chain variable region including heavy chain CDRs as depicted in (I-1) HCDR1 to (I-3) HCDR3 below, and a light chain variable region including light chain CDRs as depicted in (I-4) LCDR1 to (I-6) LCDR3 below.
In the following, SEQ ID NO: 1 represents an amino acid sequence of the heavy chain variable region of an example of antibody I (derived from rat), and SEQ ID NO: 2 represents an amino acid sequence of the light chain variable region of an example of antibody I (derived from rat).
In the above (I-1) to (I-6), an antibody containing a heavy chain and/or light chain complementarity determining region (hereinafter also collectively referred to as “CDR”) composed of the amino acid sequence in which one to several amino acids are substituted, deleted, added, and/or inserted only needs to have a binding property to pancreatic cancer stem cells. The number of amino acids to be substituted, deleted, added, and/or inserted is not particularly limited, but is preferably 1 to 3, more preferably 1 to 2, particularly preferably 1 per CDR. In the antibody of the present invention, substitution, deletion, addition, and/or insertion may be made in the amino acid sequence of one CDR, or substitution, deletion, addition, and/or insertion may be made in the amino acid sequence of two or more CDRs in the CDR sequence.
In addition, with respect to amino acid substitutions in the amino acid sequences of CDRs, substitutions with similar amino acids (that is, conservative amino acid substitutions), except for the 52nd position of SEQ ID NO: 1, are suitable because it is predicted that these substitutions do not adversely affect a binding property to pancreatic cancer stem cells. Specifically, the following grouping has been established based on the properties of amino acid side chains, and amino acids belonging to the same group are similar to each other.
Furthermore, the neutral amino acids can also be grouped into those having a polar side chain (asparagine, glutamine, serine, threonine, tyrosine, cysteine), those having a non-polar side chain (glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), those having an amide-containing side chain (asparagine, glutamine), those having a sulfur-containing side chain (methionine, cysteine), those having an aromatic side chain (phenylalanine, tryptophan, tyrosine), those having a hydroxyl group-containing side chain (serine, threonine, tyrosine), those having an aliphatic side chain (alanine, leucine, isoleucine, valine), and the like. The 52nd amino acid residue (aspartic acid residue) of SEQ ID NO: 1 can be substituted with an amino acid other than the similar amino acid, for example, an asparagine residue.
As a method for substituting one or several amino acid residues with another amino acid of interest, for example, a site-directed mutagenesis method (Hashimoto-Gotoh T. et al., Gene, Vol. 152, p. 271-275 (1995): Zoller M J. 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 T A., Proc Natl Acad Sci USA., Vol. 82, p. 488-492 (1985), etc.) is known, and amino acid substitutions can be made on the amino acid sequence of CDRs using the site-directed mutagenesis method. The method of substitution with another amino acid includes the library technique described in WO 2005/080432.
In the above (I-1) to (I-6), an antibody containing CDRs each composed of the amino acid sequence having 85% or more sequence identity only needs to have a binding property to pancreatic cancer stem cells. The sequence identity only needs to be 85% or more, but is preferably 87% or more, more preferably 92% or more, 95% or more, 96% or more, 97% or more, or 98% or more, particularly preferably 99% or more. Here, for example, the sequence identity to a predetermined amino acid sequence is sequence identity calculated by comparing with the predetermined amino acid sequence.
In addition, the “sequence identity” indicates a value of identity of an amino acid sequence obtained by b12seq program (Tatiana A. Tatsusova, Thomas L. Madden, FEMS Microbiol. Lett., Vol. 174, p 247-250, 1999) of BLAST PACKAGE [sgi32 bit edition, Version 2.0.12; available from National Center for Biotechnology Information (NCBI)]. The parameters may be set to Gap insertion Cost value: 11 and Gap extension Cost value: 1.
In the above (I-1) to (I-6), the “under stringent conditions” refer to conditions of incubation at 50° C. to 65° C. for 4 hours to overnight in 6×SSC (1×SSC is 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0) containing 0.5% SDS, 5× Denhartz's [0.1% Bovine Serum Albumin (BSA), 0.1% Polyvinylpyrrolidone, 0.1% Ficoll 400], and 100 μg/ml salmon sperm DNA.
Hybridization under stringent conditions is specifically performed by the following technique. That is, a nylon membrane on which a DNA library or a cDNA library is immobilized is prepared, and the nylon membrane is blocked at 65° C. in a prehybridization solution containing 6×SSC, 0.5% SDS, 5×Denhartz's, and 100 μg/ml salmon sperm DNA. Each probe labeled 32P is then added, followed by incubation at 65° C. overnight. This nylon membrane is washed in 6×SSC for 10 minutes at room temperature, in 2×SSC containing 0.1% SDS for 10 minutes at room temperature, and in 0.2×SSC containing 0.1% SDS for 30 minutes at 45° C., followed by autoradiography, by which DNA specifically hybridized with the probe can be detected.
<Suitable examples of antibody I>
Suitable examples of antibody I of the present invention include antibodies containing a heavy chain variable region including heavy chain CDRs as depicted in (I-1′) HCDR1 to (I-3′) HCDR3 below, and a light chain variable region including light chain CDRs as depicted in (1-4′) LCDR1 to (I-6′) LCDR3 below.
In the following, SEQ ID NO: 3 represents an amino acid sequence of the heavy chain variable region of another example of antibody I (derived from rat), and SEQ ID NO: 4 represents an amino acid sequence of the light chain variable region of another example of antibody I (derived from rat). SEQ ID NO: 3 represents an amino acid sequence of the amino acid sequence of SEQ ID NO: 1 in which the 60th amino acid residue (asparagine residue) is substituted with a cysteine residue and the 62nd amino acid residue (alanine residue) is substituted with a proline residue. SEQ ID NO: 4 is an amino acid sequence of the amino acid sequence of SEQ ID NO: 2 in which the 32nd amino acid residue (isoleucine residue) is substituted with an asparagine residue, the 46th amino acid residue (leucine residue) is substituted with a phenylalanine residue, and the 52nd amino acid residue (aspartic acid residue) is substituted with an asparagine residue.
In antibody I of the present invention, the framework regions of the heavy chain variable region and the light chain variable region are not particularly limited as long as they do not substantially affect the binding activity to pancreatic cancer stem cells. Another suitable example of antibody I of the present invention includes antibodies containing a heavy chain variable region and a light chain variable region below.
Heavy chain variable region composed of the amino acid sequence of SEQ ID NO: 1: an amino acid sequence having the amino acid sequence of SEQ ID NO: 1 in which one to several amino acids are substituted, deleted, added, and/or inserted: an amino acid sequence having 85% or more sequence identity to the amino acid sequence of SEQ ID NO: 1: or an amino acid sequence encoded by a polynucleotide that specifically hybridizes to a polynucleotide composed of a base sequence complementary to a base sequence encoding the amino acid sequence of SEQ ID NO: 1 under stringent conditions, and
In another suitable example of the above antibody I, an antibody containing a heavy chain and/or light chain variable region (hereinafter, also collectively referred to as a “variable region”) composed of the amino acid sequence in which one to several amino acids are substituted, deleted, added, and/or inserted only needs to have a binding property to pancreatic cancer stem cells. The number of amino acids to be substituted, deleted, added, and/or inserted is not particularly limited, but is preferably 1 to 15, more preferably 1 to 10, still more preferably 1 to 5, still more preferably 1 to 4, still more preferably 1 to 3, particularly preferably 1 to 2, most preferably 1 for each variable region. Details of the matter related to amino acid substitution (conservative amino acid substitution, method of substitution) are the same as those described in (I-1) to (I-6) above.
In another suitable example of the above antibody I, an antibody containing variable regions each composed of the amino acid sequence having 85% or more sequence identity only needs to have a binding property to pancreatic cancer stem cells. Details of sequence identity and stringent conditions are the same as those described in (I-1) to (I-6) above.
Particularly suitable examples of antibody I of the present invention include antibodies containing a heavy chain variable region composed of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3 and a light chain variable region composed of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4.
Antibody II is an antibody against pancreatic cancer stem cells containing a heavy chain variable region including heavy chain CDRs as depicted in (II-1) HCDR1 to (II-3) HCDR3 below, and a light chain variable region including light chain CDRs as depicted in (II-4) LCDR1 to (II-6) LCDR3 below.
In the above (II-1) to (II-6), an antibody containing CDRs each composed of the amino acid sequence in which one to several amino acids are substituted, deleted, added, and/or inserted only needs to have a binding property to pancreatic cancer stem cells. Details of the number of amino acids to be substituted, deleted, added, and/or inserted and the like are the same as those described in (I-1) to (I-6) of antibody I above. With respect to amino acid substitutions in the amino acid sequences of the CDRs, substitutions with similar amino acids (that is, conservative amino acid substitutions) are suitable because it is predicted that these substitutions do not adversely affect a binding property to pancreatic cancer stem cells. Details of the amino acid substitution are the same as those described in (I-1) to (I-6) of antibody I above.
In the above (II-1) to (II-6), an antibody containing CDRs each composed of the amino acid sequence having 85% or more sequence identity only needs to have a binding property to pancreatic cancer stem cells. Details of sequence identity and stringent conditions are the same as those described in (I-1) to (1-6) related to antibody I above.
In antibody II of the present invention, the framework regions of the variable region are not particularly limited as long as they do not substantially affect the binding activity to pancreatic cancer stem cells. Accordingly, suitable examples of antibody II of the present invention includes antibodies containing a heavy chain variable region and a light chain variable region below.
In the following, SEQ ID NO: 5 represents an amino acid sequence of the heavy chain variable region of an example of antibody II (derived from rat), and SEQ ID NO: 6 represents an amino acid sequence of the light chain variable region of an example of antibody II (derived from rat).
Heavy chain variable region composed of the amino acid sequence of SEQ ID NO: 5; an amino acid sequence having the amino acid sequence of SEQ ID NO: 5 in which one to several amino acids are substituted, deleted, added, and/or inserted: an amino acid sequence having 85% or more sequence identity to the amino acid sequence of SEQ ID NO: 5; or an amino acid sequence encoded by a polynucleotide that specifically hybridizes to a polynucleotide composed of a base sequence complementary to a base sequence encoding the amino acid sequence of SEQ ID NO: 5 under stringent conditions, and
In suitable examples of the above antibody II, an antibody containing a variable region composed of the amino acid sequence in which one to several amino acids are substituted, deleted, added, and/or inserted only needs to have a binding property to pancreatic cancer stem cells. Details of the number of amino acids to be substituted, deleted, added, and/or inserted are the same as those described in the other suitable examples of antibody I above, and details of the matter related to amino acid substitution (conservative amino acid substitution, method of substitution) are the same as those described in (I-1) to (1-6) related to antibody I above.
In suitable examples of the above antibody II, an antibody containing variable regions each composed of the amino acid sequence having 85% or more sequence identity only needs to have a binding property to pancreatic cancer stem cells. Details of sequence identity and stringent conditions are the same as those described in (I-1) to (I-6) related to antibody I above.
Particularly preferred examples of antibody II of the present invention include antibodies containing a heavy chain variable region composed of the amino acid sequence of SEQ ID NO: 5 and a light chain variable region composed of the amino acid sequence of SEQ ID NO: 6.
Antibody III is an anti-EpCAM antibody containing a heavy chain variable region including heavy chain CDRs as depicted in (III-1) HCDR1 to (III-3) HCDR3 below, and a light chain variable region including light chain CDRs as depicted in (III-4) LCDR1 to (III-6) LCDR3 below. EpCAM (epithelial cell adhesion molecule) is a transmembrane glycoprotein composed of 314 amino acids present on a cell membrane surface, and is overexpressed in various cancer tissues in addition to pancreatic cancer stem cells. Accordingly, antibody III is also an antibody against pancreatic cancer stem cells.
In the above (III-1) to (III-6), an antibody containing CDRs each composed of the amino acid sequence in which one to several amino acids are substituted, deleted, added, and/or inserted only needs to have a binding property to pancreatic cancer stem cells. Details of the number of amino acids to be substituted, deleted, added, and/or inserted and the like are the same as those described in (I-1) to (I-6) of antibody I above. With respect to amino acid substitutions in the amino acid sequences of the CDRs, substitutions with similar amino acids (that is, conservative amino acid substitutions) are suitable because it is predicted that these substitutions do not adversely affect a binding property to pancreatic cancer stem cells. Details of the amino acid substitution are the same as those described in (I-1) to (I-6) of antibody I above.
In the above (III-1) to (III-6), an antibody containing CDRs each composed of the amino acid sequence having 85% or more sequence identity only needs to a have binding property to pancreatic cancer stem cells. Details of sequence identity and stringent conditions are the same as those described in (I-1) to (1-6) related to antibody I above.
In antibody III of the present invention, the framework regions of the variable region are not particularly limited as long as they do not substantially affect the binding activity to pancreatic cancer stem cells. Accordingly, suitable examples of antibody III of the present invention includes antibodies containing a heavy chain variable region and a light chain variable region below.
In the following, SEQ ID NO: 7 represents an amino acid sequence of the heavy chain variable region of an example of antibody III (derived from rat), and SEQ ID NO: 8 represents an amino acid sequence of the light chain variable region of an example of antibody III (derived from rat).
Heavy chain variable region composed of the amino acid sequence of SEQ ID NO: 7; an amino acid sequence having the amino acid sequence of SEQ ID NO: 7 in which one to several amino acids are substituted, deleted, added, and/or inserted: an amino acid sequence having 85% or more sequence identity to the amino acid sequence of SEQ ID NO: 7; or an amino acid sequence encoded by a polynucleotide that specifically hybridizes to a polynucleotide composed of a base sequence complementary to a base sequence encoding the amino acid sequence of SEQ ID NO: 7 under stringent conditions, and
In suitable examples of the above antibody III, an antibody containing a heavy chain and/or light chain variable region composed of the amino acid sequence in which one to several amino acids are substituted, deleted, added, and/or inserted only needs to have a binding property to pancreatic cancer stem cells. Details of the number of amino acids to be substituted, deleted, added, and/or inserted are the same as those described in the other suitable examples of antibody I above, and details of the matter related to amino acid substitution (conservative amino acid substitution, method of substitution) are the same as those described in (I-1) to (1-6) related to antibody I above.
In suitable examples of the above antibody III, an antibody containing variable regions each composed of the amino acid sequence having 85% or more sequence identity only needs to have a binding property to pancreatic cancer stem cells. Details of sequence identity and stringent conditions are the same as those described in (I-1) to (I-6) related to antibody I above.
Particularly preferred examples of antibody III of the present invention include antibodies containing a heavy chain variable region composed of the amino acid sequence of SEQ ID NO: 7 and a light chain variable region composed of the amino acid sequence of SEQ ID NO: 8.
In the antibody of the present invention, the amino acid sequence of the constant region is also not particularly limited as long as it does not substantially affect the binding activity to pancreatic cancer stem cells.
The amino acid sequences of the variable regions set forth in SEQ ID NOs: 1 to 8 are derived from rats. Accordingly, examples of the antibody of the present invention include a rat antibody having a rat-derived constant region.
The antibody of the present invention can contain any signal region. For example, examples of the sequence of the heavy chain signal region include amino acid sequences set forth in SEQ ID NO: 17 or 21, SEQ ID NO: 25, and SEQ ID NO: 29, and examples of the sequence of the light chain signal region include amino acid sequences set forth in SEQ ID NO: 19 or 23, and SEQ ID NO: 27.
Regarding the signal region contained in antibody I, examples of the sequence of the heavy chain signal region include an amino acid sequence set forth in SEQ ID NO: 17 or 21, and examples of the sequence of the light chain signal region include an amino acid sequence set forth in SEQ ID NO: 19 or 23, preferably an amino acid sequence set forth in SEQ ID NO: 19. Regarding the signal region contained in antibody II, examples of the sequence of the heavy chain signal region include an amino acid sequence set forth in SEQ ID NO: 25, and examples of the sequence of the light chain signal region include an amino acid sequence set forth in SEQ ID NO: 27. Regarding the signal region contained in antibody III, examples of the sequence of the heavy chain signal region include an amino acid sequence set forth in SEQ ID NO: 29, and examples of the sequence of the light chain signal region include an amino acid sequence set forth in SEQ ID NO: 19.
On the other hand, preferred examples of the antibody of the present invention include chimeric antibodies (mouse chimerized antibody, human chimerized antibody, etc.) and humanized antibodies.
The chimeric antibody is an antibody in which regions whose origins are different from each other are linked to each other. When used as an active ingredient of a therapeutic pharmaceutical composition, the chimeric antibody is preferably a rat-human chimeric antibody (human chimerized antibody) composed of a variable region derived from a rat antibody and a constant region derived from a human antibody. In addition, when used as a diagnostic agent, the chimeric antibody is preferably a rat-human chimeric antibody (human chimerized antibody) or a rat-mouse chimeric antibody (mouse chimerized antibody) composed of a variable region derived from a rat antibody and a constant region derived from a mouse antibody.
In addition, the humanized antibody is obtained by grafting a non-human-derived CDR sequence onto a framework region of a human antibody, and is an antibody composed of a CDR of a non-human-derived antibody, a framework region derived from a human antibody, and a constant region derived from a human antibody. Since the humanized antibody has reduced antigenicity in the human body, the antibody of the present invention is suitable when used as an active ingredient of a therapeutic pharmaceutical composition.
The isotype of the antibody of the present invention is not particularly limited, but examples thereof include IgG (specific examples thereof include IgG1, IgG2, IgG3, and IgG4), IgA (specific examples thereof include IgA1 and IgA2), IgM, IgD, and IgE. Among them, IgG is preferable, and IgG2 is more preferable.
The light chain of the antibody of the present invention may be either a κ chain or a λ chain, and is preferably a κ chain.
The most preferred isotype of the antibody of the present invention includes IgG2 whose light chain is a κ chain.
The form of the antibody of the present invention may be a polyclonal antibody or a monoclonal antibody, but is preferably a monoclonal antibody. The method for producing the monoclonal antibody is not particularly limited, but for example, the monoclonal antibody can be prepared by a hybridoma method as described in “Kohler G, Milstein C., Nature. 1975 Aug. 7: 256(5517): 495-497.”; a recombinant method as described in U.S. Pat. No. 4,816,567; a method of 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.”, a method described in “Protein Experiment Handbook, YODOSHA CO., LTD. (2003): 92-96.”, and the like.
The antibody of the present invention includes not only a full-length antibody (an antibody having a Fab region and an Fc region) but also a fragment antibody as long as the antibody includes an antigen binding site composed of the CDRs. Examples of such a fragment antibody include Fv antibodies, Fab antibodies, Fab′ antibodies, F(ab′)2 antibodies, scFv antibodies, dsFv antibodies, diabodies, and nanobodies. The antibody of the present invention also includes a multivalent specific antibody (for example, a bispecific antibody) as long as the antibody includes the antigen binding site. These fragment antibodies and multispecific antibodies can be produced according to conventionally known methods.
The antibody of the present invention may be a conjugated antibody or a conjugated antibody fragment bound to various compounds such as polyethylene glycol, a radioactive substance, and a toxin. In addition, in the antibody of the present invention, the sugar chain bound to the antibody may be altered, or another protein may be fused, as necessary.
The DNA of the present invention is a DNA encoding the “1. Antibodies against pancreatic cancer stem cells”. The DNA of the present invention can be obtained, for example, by obtaining at least a region encoding the antibody by PCR or the like using a DNA encoding the antibody as a template. The DNA of the present invention can also be artificially synthesized by a gene synthesis method.
In addition, when a specific mutation is introduced into a specific site of the base sequence of DNA, a mutation-introducing method is known, and for example, a site-directed mutation-introducing method for DNA or the like can be used. As a specific method for converting the base in DNA, for example, a commercially available kit can also be used.
The base sequence of DNA into which a mutation has been introduced can be determined using a DNA sequencer. Once the base sequence is determined, DNA encoding the antibody of the present invention can be obtained by chemical synthesis, PCR using a cloned probe as a template, or hybridization using a DNA fragment having the base sequence as a probe.
In addition, a mutant form of DNA encoding the antibody of the present invention, which has a function equivalent to that before mutation, can be synthesized by a site-directed mutagenesis method or the like. In order to introduce a mutation into a DNA encoding the antibody of the present invention, a known technique such as a Kunkel method, a Gapped duplex method, or a megaprimer PCR method can be used.
The base sequence of the DNA of the present invention can be appropriately designed by those skilled in the art according to the sequence of the CDRs of the antibody of the present invention.
Specifically, the heavy chain CDRs of antibody I can be designed according to the sequences of the heavy chain CDRs of (I-1) to (1-3) or (I-1′) to (I-3′) above based on SEQ ID NO: 9 that is the base sequence of DNA encoding SEQ ID NO: 1 (for example, the DNA encoding the amino acid sequence from position 26 to position 35 of SEQ ID NO: 1 is composed of the base sequence from position 76 to position 105 of SEQ ID NO: 9) or based on SEQ ID NO: 11 that is the base sequence of DNA encoding SEQ ID NO: 3 (for example, the DNA encoding the amino acid sequence from position 26 to position 35 of SEQ ID NO: 3 is composed of the base sequence from position 76 to position 105 of SEQ ID NO: 11). The light chain CDRs of antibody I can be designed according to the sequences of the light chain CDRs of (I-4) to (1-6) or (I-4′) to (I-6′) above based on SEQ ID NO: 10 that is the base sequence of DNA encoding SEQ ID NO: 2 (for example, the DNA encoding the amino acid sequence from position 24 to position 34 of SEQ ID NO: 2 is composed of the base sequence from position 70 to position 102 of SEQ ID NO: 10) or based on SEQ ID NO: 12 that is the base sequence of DNA encoding SEQ ID NO: 4 (for example, the DNA encoding the amino acid sequence from position 24 to position 34 of SEQ ID NO: 4 is composed of the base sequence from position 70 to position 102 of SEQ ID NO: 12).
The heavy chain CDRs of antibody II can be designed according to the sequences of the heavy chain CDRs of (II-1) to (II-3) above based on SEQ ID NO: 13 that is the base sequence of DNA encoding SEQ ID NO: 5 (for example, the DNA encoding the amino acid sequence from position 26 to position 35 of SEQ ID NO: 5 is composed of the base sequence from position 76 to position 105 of SEQ ID NO: 13). The light chain CDRs of antibody II can be designed according to the sequences of the light chain CDRs of (II-4) to (II-6) above based on SEQ ID NO: 14 that is the base sequence of DNA encoding SEQ ID NO: 6 (for example, the DNA encoding the amino acid sequence from position 24 to position 34 of SEQ ID NO: 6 is composed of the base sequence from position 70 to position 102 of SEQ ID NO: 14).
The heavy chain CDRs of antibody III can be designed according to the sequences of the heavy chain CDRs of (III-1) to (III-3) above based on SEQ ID NO: 15 that is the base sequence of DNA encoding SEQ ID NO: 7 (for example, the DNA encoding the amino acid sequence from position 26 to position 35 of SEQ ID NO: 7 is composed of the base sequence from position 76 to position 105 of SEQ ID NO: 15). The light chain CDRs of antibody III can be designed according to the sequences of the light chain CDRs of (III-4) to (III-6) above based on SEQ ID NO: 16 that is the base sequence of DNA encoding SEQ ID NO: 8 (for example, the DNA encoding the amino acid sequence from position 24 to position 34 of SEQ ID NO: 8 is composed of the base sequence from position 70 to position 103 of SEQ ID NO: 16).
The base sequence encoding the variable region contained in the DNA of the present invention is not particularly limited as long as a base sequence encoding the CDRs is contained, and the base sequence encoding the framework region is not particularly limited as long as the framework region does not substantially affect the binding activity to pancreatic cancer stem cells. Specific examples of the base sequence encoding the variable region contained in the DNA of the present invention include the following.
Examples of the base sequence encoding the heavy chain variable region of antibody I include SEQ ID NO: 9, and examples of the base sequence encoding the light chain variable region thereof include SEQ ID NO: 10. Examples of the base sequence encoding the heavy chain variable region of antibody II include SEQ ID NO: 13, and examples of the base sequence encoding the light chain variable region thereof include SEQ ID NO: 14. Examples of the base sequence encoding the heavy chain variable region of antibody III include SEQ ID NO: 15, and examples of the base sequence encoding the light chain variable region thereof include SEQ ID NO: 16.
Another example of the base sequence encoding the variable region of each of antibodies I to III includes a base sequence that encodes the variable region containing the CDRs and that has 85% or more sequence identity to the base sequences set forth in SEQ ID NOs: 9, 10, and 13 to 16. The sequence identity is preferably 87% or more, more preferably 92% or more, 95% or more, 96% or more, 97% or more, or 98% or more, particularly preferably 99% or more. The “sequence identity” is as described above.
Still another example of the base sequence encoding the variable region of each of antibodies I to III includes a base sequence of DNA that encodes the heavy chain variable region containing the CDRs and that hybridizes under stringent conditions with DNA containing a base sequence complementary to DNA composed of the base sequences set forth in SEQ ID NOs: 9, 10, and 13 to 16. The “stringent conditions” are as described above.
More specific examples corresponding to the other example or the still other example of the base sequences encoding the variable region of antibody I include SEQ ID NO: 11 that is a more specific example of the base sequence encoding the heavy chain variable region and SEQ ID NO: 12 that is a more specific example of the base sequence encoding the light chain variable region.
The DNA of the present invention is preferably one in which the codon usage is optimized for a host. For example, when E. coli is used as a host, a DNA in which the codon usage is optimized for E. coli is suitable.
The recombinant vector of the present invention includes the DNA of the present invention described in the “2. DNA”. The recombinant vector of the present invention can be obtained by inserting the DNA of the present invention into an expression vector.
As the expression vector, those constructed for genetic recombination from phage, plasmid, or virus capable of autonomously growing in a host are suitable. Specific examples include plasmids derived from E. coli (for example, pET-Blue), plasmids derived from Bacillus subtilis (for example, pUB110), plasmids derived from yeast (for example, pSH19), animal cell expression plasmids (for example, pA1-11, pcDNA 3.1-V5/His-TOPO), bacteriophages such as lambda phage, and virus-derived vectors.
Recombinant vectors contain a regulator operably linked to the DNA of the invention. Examples of the regulator include a promoter, a signal sequence, an origin of replication, a marker gene, an enhancer, a CCAAT box, a TATA box, an SPI site, and a transcription termination sequence, and one or more of these can be used. In addition, operably linked means that various regulators for regulating the DNA of the present invention and the DNA of the present invention are linked in a state where they can operate in a host cell.
The signal sequence is not particularly limited, but examples thereof include a sequence encoding the signal region described above. For example, examples of the heavy chain signal sequence include base sequences set forth in SEQ ID NO: 18 or 22 (encoding SEQ ID NO: 17 or 21, respectively), SEQ ID NO: 26 (encoding SEQ ID NO: 25), and SEQ ID NO: 30 (encoding SEQ ID NO: 29), and examples of the light chain signal sequence include base sequences set forth in SEQ ID NO: 20 or 24 (encoding SEQ ID NO: 19 or 23, respectively) and SEQ ID NO: 28 (encoding SEQ ID NO: 27).
Regarding the signal sequence in the recombinant vector for antibody I, examples of the heavy chain signal sequence include a base sequence set forth in SEQ ID NO: 18 or 22, and examples of the light chain signal sequence include a base sequence set forth in SEQ ID NO: 20 or 24, preferably an amino acid sequence set forth in SEQ ID NO: 20 from the viewpoint of improving antibody production efficiency. Regarding the signal sequence in the recombinant vector for antibody II, examples of the heavy chain signal sequence include the base sequence set forth in SEQ ID NO: 26, and examples of the light chain signal sequence include a base sequence set forth in SEQ ID NO: 28. Regarding the signal sequence contained in the recombinant vector for antibody III, examples of the heavy chain signal sequence include a base sequence set forth in SEQ ID NO: 30, and examples of the light chain signal sequence include an amino acid sequence set forth in SEQ ID NO: 20 from the viewpoint of improving antibody production efficiency.
The recombinant vector for a chimeric antibody can be produced, for example, in the case of a recombinant vector for a human chimerized antibody, by substituting the constant region of a rat antibody having a variable region having amino acid sequences of the respective CDRs with the constant region of a human antibody. Similarly, the recombinant vector for a chimeric antibody can be produced, in the case of a recombinant vector for a mouse chimerized antibody, by substituting the constant region of a rat antibody having a variable region having amino acid sequences of the respective CDRs with the constant region of a mouse antibody. As the constant region of a human antibody, a known constant region can be used.
Specifically, a recombinant vector for the human chimerized antibody can be produced by the following method. First, a DNA encoding a rat heavy chain variable region containing CDRs each having a predetermined amino acid sequence is produced by chemical synthesis, biochemical cleavage, recombination, or the like. A heavy chain expression vector is produced by ligating the resulting DNA encoding the heavy chain variable region with a DNA encoding the human heavy chain constant region, followed by incorporation into an expression vector. A light chain expression vector is produced in the same manner.
In addition, amino acids in the framework region in the variable region of the antibody may be substituted so that the CDRs of the rat-human chimeric antibody form an appropriate antigen binding site (Sato, K. et al., Cancer Research, Vol. 53, p. 851-856 (1993)). A recombinant vector for a mouse chimerized antibody can also be produced in the same manner by using a DNA encoding a mouse constant region instead of a DNA encoding a human constant region.
In addition, a recombinant vector for the humanized antibody can be produced, for example, by transplanting CDRs containing the respective amino acid sequences described above on the framework region of a human antibody (for example, Jones et al., Nature, Vol. 321, p. 522-525 (1986): Riechmann et al., Nature, Vol. 332, p. 323-327 (1988): Verhoeven et al., Science, Vol. 239, p. 1534-1536 (1988)).
Specifically, a recombinant vector for the humanized antibody can be produced by the following method. A DNA encoding a heavy chain variable region in which CDRs each having a predetermined amino acid sequence are linked to framework regions derived from 4 human antibodies in a predetermined order is produced by chemical synthesis, biochemical cleavage, recombination, or the like. Here, mutations such as substitution, deletion, and/or addition of an amino acid in the framework region may be added so that the respective CDRs of the humanized antibody can form an appropriate antigen binding site (Sato, K. et al., Cancer Research, Vol. 53, p. 851-856 (1993)). A heavy chain expression vector is produced by ligating the resulting DNA encoding the heavy chain variable region with a DNA encoding the human heavy chain constant region, followed by incorporation into an expression vector. Similarly, a light chain-heavy chain expression vector is produced. As the constant region of a human antibody, a known constant region can be used.
The transformant is obtained by transforming a host using the DNA of the present invention or the recombinant vector.
The host for use in production of the transformant is not particularly limited as long as the host can introduce a gene, can autonomously grow, and can express the trait of the gene of the present invention, but examples thereof include cells of humans and mammals excluding humans (for example, rat, mouse, guinea pig, rabbit, cow, monkey etc.), more specifically, Chinese hamster ovary cells (CHO cells), monkey cells COS-7, human embryonic kidney cells (for example, HEK 293 cells (Expi293F, 293-F, HEK293, 293T, etc.)), mouse myeloma cells (for example, Sp2/0 or NS0); insect cells; plant cells: Bacteria belonging to genus Escherichia such as Escherichia coli, genus Bacillus such as Bacillus subtilis, genus Pseudomonas such as Pseudomonas putida, etc.: actinomycetes, etc.: yeast etc.: and filamentous fungi etc.
The transformant can be obtained by introducing the DNA of the present invention or the recombinant vector into a host. The method for introducing the DNA of the present invention or the recombinant vector is not particularly limited as long as the gene of interest can be introduced into a host. In addition, the site into which the DNA is introduced is not particularly limited as long as the gene of interest can be expressed, but may be on a plasmid or on a genome. Specific examples of the method for introducing the DNA of the present invention or the recombinant vector include a recombinant vector method and a genome editing method.
Conditions for introducing the DNA of the present invention or the recombinant vector into a host may be appropriately set according to the introduction method, the type of host, and the like. When the host is bacteria, examples of the method include a method using a competent cell with calcium ion treatment and an electroporation method. When the host is a yeast, examples of the method include an electroporation method, a spheroplast method, and a lithium acetate method. When the host is an animal cell, examples of the method include an electroporation method, a calcium phosphate method, and a lipofection method. When the host is an insect cell, examples of the method include a calcium phosphate method, a lipofection method, and an electroporation method. When the host is a plant, examples of the method include an electroporation method, an Agrobacterium method, a particle gun method, and a PEG method.
The antibody of the present invention described in the “1. Antibodies against pancreatic cancer stem cells” can be produced by culturing the transformant described in the “4. Transformant”. The culture conditions of the transformant may be appropriately set taking into consideration of the nutritional physiological properties of a host, but liquid culture is preferable. From the obtained culture, the antibody of the present invention of interest can be collected and purified.
The present invention provides a pharmaceutical composition containing the antibody of the present invention described in the “1. Antibodies against pancreatic cancer stem cells”. The pharmaceutical composition of the present invention can exhibit a pharmacological effect based on the binding ability to pancreatic cancer stem cells using the antibody of the present invention as an active ingredient.
The pharmaceutical composition of the present invention only needs to contain an effective amount of the antibody of the present invention, and may further contain a pharmaceutically acceptable carrier or additive. Examples of such a carrier or additive include a surfactant, an excipient, a colorant, an aromatizing agent, a preservative, a stabilizer, a buffer, a pH buffer, a disintegrant, a solubilizing agent, a solubilizing aid, an isotonizing agent, a binder, a disintegrant, a lubricant, a diluent, and a flavoring agent.
In addition, the administration form of the pharmaceutical composition of the present invention may be either oral or parenteral, and specific examples thereof 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.
Regarding the formulation form of the pharmaceutical composition of the present invention, the formulation form can be appropriately set according to the employed administration form. For example, when used in oral administration, the pharmaceutical composition of the present invention may be prepared in a formulation form such as a powder, a granule, a capsule, a syrup, or a suspension, and when used in parenteral administration, the pharmaceutical composition of the present invention may be prepared in a formulation form such as a liquid, a suspension, an emulsion, a spray, a suppository, or an eye drop.
The pharmaceutical composition of the present invention can be used for treatment of pancreatic cancer by utilizing the antitumor effect of the antibody of the present invention.
The dose and the number of times of administration of the pharmaceutical composition of the present invention vary depending on the administration form, the age and body weight of a patient, the type of disease and the degree of symptom, and the like, and cannot be uniformly defined. For example, in the case of human, an amount corresponding to 120 mg to 480 mg in terms of the weight of the antibody of the present invention may be administered at a frequency of about once every 7 to 30 days per administration. In order to reduce the number of times of administration, a sustained-release preparation can be used, or administration can be carried out in small amounts over a long period of time with an osmotic pump or the like.
The present invention provides an extracorporeal diagnostic agent composed of the antibody of the present invention described in the “1. Antibodies against pancreatic cancer stem cells”. The extracorporeal diagnostic agent of the present invention can exhibit a diagnostic effect based on the binding ability to pancreatic cancer stem cells using the antibody of the present invention as a tumor marker.
Since the antibody of the present invention has a binding property to pancreatic cancer stem cells for any of antibody I, antibody II and antibody III, the extracorporeal diagnostic agent of the present invention can be used for diagnosis of pancreatic cancer. Among the antibodies of the present invention, since antibody I and antibody II specifically bind to pancreatic cancer stem cells, the extracorporeal diagnostic agent of the present invention is more preferably composed of antibody I and antibody II. On the other hand, since antibody III is an anti-EpCAM antibody and has binding properties to not only pancreatic cancer stem cells but also cancer cells having EpCAM, when composed of antibody III, the extracorporeal diagnostic agent of the present invention can be used for diagnosis of cancer expressing EpCAM. The cancer expressing EpCAM is not particularly limited, but examples thereof include renal cell cancer, hepatocellular cancer, urothelial cancer, breast cancer, squamous cell cancer, and lung cancer.
Examples of the detection target of the extracorporeal diagnostic agent of the present invention include cancer cells such as pancreatic cancer stem cells collected from a living body to be diagnosed, or extracellular vesicles (such as exosomes) derived from cancer cells considered to reflect the surface state of the cancer cells. When used as a detection target, the extracellular vesicle derived from a cancer cell is preferable from the viewpoint that a non-invasive diagnosis can be expected without performing biopsy, and an improved diagnostic sensitivity can be expected in pancreatic cancer diagnosis. Specific examples of the biological sample including the detection target include a biopsy sample, a blood collection sample, and a urine collection sample.
The method for analyzing a biological sample for diagnosis of cancer using the extracorporeal diagnostic agent of the present invention includes a step of bringing the extracorporeal diagnostic agent of the present invention (that is, the antibody of the present invention) into contact with a biological sample to immunomeasure an antigen-antibody reaction.
The immunoassay may be based on any method such as a sandwich method, a competition method, or an agglutination method, but is preferably based on a sandwich method. In addition, examples of the immunoassay include an enzyme-linked immuno-sorbent assay (ELISA), fluorescence immunoassay, and radioisotope immunoassay, depending on the type of label, but from the viewpoint of simplicity and/or rapidity of measurement, an enzyme-linked immuno-sorbent assay is preferable.
Examples of specific aspects of the extracorporeal diagnostic agent of the present invention include an aspect immobilized on a substrate. That is, the present invention also provides an antibody substrate containing the above-described extracorporeal diagnostic agent and a substrate on which the extracorporeal diagnostic agent is immobilized. The material of the substrate is not particularly limited as long as it is in a solid phase, but examples thereof include polystyrene, polyethylene, polypropylene, polyester, polyacrylonitrile, polyvinyl chloride, fluororesin, crosslinked dextran, paper, silicon, glass, metal, and agarose, and examples of the form thereof include a plate and a film. As a method for immobilizing the extracorporeal diagnostic agent of the present invention (that is, the antibody of the present invention) on a substrate, a method known in the technical field of producing antibody substrates is used without particular limitation. In the use of the antibody substrate, immunoassay based on a sandwich method can be performed using the extracorporeal diagnostic agent of the present invention as a capture antibody.
Another example of specific aspects of the extracorporeal diagnostic agent of the present invention includes an aspect contained in an extracorporeal diagnostic composition. That is, the present invention also provides an extracorporeal diagnostic composition containing the extracorporeal diagnostic agent. The extracorporeal diagnostic agent contained in the extracorporeal diagnostic agent composition may be in the form of the antibody itself or in the form in which the antibody is immobilized on insoluble particles. In addition to the extracorporeal diagnostic agent, the extracorporeal diagnostic composition can contain one or more components selected from salts, sugars, polyhydric alcohols, surfactants, proteins (other than the extracorporeal diagnostic agent), stabilizers, preservatives, buffers, and the like.
The antibody substrate and the extracorporeal diagnostic composition may be provided in the form of a kit containing another reagent and/or an instrument according to the measurement principle of immunoassay and the type of label. For example, when the enzyme-linked immuno-sorbent assay is selected, the kit can further contain one or more items selected from a reaction vessel or a reaction plate, a chromogenic substrate solution, a reaction stop solution, a washing solution, a standard solution, and the like, in addition to the antibody substrate and the extracorporeal diagnostic composition.
Hereinafter, a more detailed description is made of the present invention with reference to Examples, but the present invention is not to be construed as being limited to these Examples.
The flow of antibody production performed in this Test Example is shown in
For BxPC-3 (human pancreatic adenocarcinoma cell lines) and MCF-7 (human breast cancer cell lines) was used 10% FBS [SIGMA, F7524]-containing D-MEM [Wako, 044-29765], for KP-3 (human pancreatic adenosquamous carcinoma cell lines), HARA (human lung cancer cell lines), HARA-B2 (human lung cancer cell bone metastasis lines), PC-3 (human prostate cancer cell lines), and HeLa (human cervical carcinoma cell lines) was used 10% FBS-containing RPMI-1640 [Wako, 189-02025], for NHDF (human fibroblast cell lines) was used 20% FBS-containing D-MEM, and for Sp2 (mouse myeloma cell lines) was used 10% FBS-containing Hybridoma-SFM [Gibco, 12300-067], followed by culture at 37° C. under 5% by volume CO2.
KMC07 is a human pancreatic tumor progenitor cell line (human pancreatic cancer stem cell) established by the present inventor from pancreatic cancer tissues isolated from a human pancreatic cancer patient (age: 69 years old, sex: male, pathological subtype: tubular adenocarcinoma, metastasis:
In order to produce antibodies against KMC07, one WKY/Izm rat [SLC, 8-weeks old female] was immunized twice using KMC07 as an antigen. In the initial immunization, a mixed PBS suspension of about 1.0×107 cells of KMC07 and PA6 was mixed with Titer Max Gold [CYT, G-1X5] as an adjuvant at a volume ratio of 1:1, followed by sufficient suspension using a sonicator. Into the respective foot soles of the WKY/Izm rat, 100 μl was subcutaneously injected. The booster was performed 15 days after the immunization. In the booster, only a mixed PBS suspension of about 6.7×105 cells of KMC07 and PA6 was subcutaneously injected into the respective left and right buttocks in an amount of 100 μl. Four days after the booster, lymphocytes isolated from the iliac lymph node and Sp2 were mixed at a ratio of 3:1, and cells (hybridomas) fused by a PEG method were seeded on a 96 well plate. Selection was performed using HAT medium (Hybridoma-SFM, 10% FBS, 100 mM hypoxanthine [SIGMA, H9377-1G], 0.4 mM aminopterin [SIGMA, A3411-10MG], 16 mM thymidine [Wako, 205-08091], 1 ng/ml mouse IL-6, 100 U/ml penicillin [Wako, 161-23181], 100 μg/ml streptomycin [Wako, 161-23181], 0.25 μg/ml amphotericin B [Wako, 161-23181]) as a medium.
When using KMC07: PA6 was first seeded on a 1.5 μg/ml poly-L-ornithine [SIGMA, P3655-50MG]-coated 96 well immunostaining plate [Thermo Scientific, 164588]. PA6 was cultured in 10% FBS-containing MEMα for 2 days. After 2 days, the culture supernatant of PA6 was removed, followed by rinsing with PBS. Subsequently, KMC07 and PA6 were suspended in KMC Medium so as to be about 1,500 cells/well in combination, followed by seeding on each well. After a further 3 days, the culture supernatant was removed, followed by addition of the hybridoma culture supernatant diluted 10 times as a primary antibody, and incubation for 1 hour. After 1 hour, cells were fixed by treatment with a 3.7% solution of formaldehyde [Wako, 064-00406] in PBS for 15 minutes. Finally, a solution of a secondary antibody (Alexa 488 anti-rat IgG (H+L) [Invitrogen™, A11006]) in PBS containing Hoechst (registered trademark) 33342 [Invitrogen™, H3570] was added, followed by incubation for 30 minutes. Observation was performed with a fluorescence microscope (OLYMPUS, IX71).
When using other cells: Two days before immunostaining, cells were seeded on an 8-well immunostaining plate [MP Biomedicals, 6040805E]. The hybridoma culture supernatant was directly used as a primary antibody, followed by incubation for 1 hour. After 1 hour, cells were fixed by treatment with a 3.7% solution of formaldehyde in PBS for 15 minutes. Furthermore, incubation was performed with a solution of a secondary antibody (Alexa 488 anti-rat IgG (H+L)) in PBS containing Hoechst (registered trademark) 33342 for 30 minutes. Finally, a discoloration inhibitor DABCO [SIGMA, D-2522] was added, followed by observation with a fluorescence microscope.
Cloning of the hybridoma was performed by seeding on a 96 well plate the hybridoma diluted so as to be 1 cell/well using a HAT-free medium (Hybridoma-SFM, 10% FBS, 1 ng/ml mouse IL-6, 100 U/ml penicillin, 100 μg/ml streptomycin, 0.25 μg/ml amphotericin B). After several days, it was confirmed by a microscope that single colonies were formed.
Hybridomas were cultured in a bovine serum-free medium (Hybridoma-SFM, 1 ng/ml mouse IL-6), and from the culture supernatant, antibodies were purified. For the purification, an ammonium sulfate precipitation method was used, and a solution of the purified antibodies was finally substituted with PBS. The concentration of the purified antibodies was determined using Pierce™ BCA Protein Assay Kit [Thermo Scientific, 23227]. In addition, isotyping of the respective monoclonal antibodies was performed using Rat Immunoglobulin Isotyping ELISA Kit [BD Biosciences, 557081].
Monoclonal antibodies specifically recognizing the human pancreatic cancer stem cell line KMC07 was produced using a rat iliac lymph node method. In this study, it is an object to produce an antibody targeting the cell membrane of KMC07. Here, KMC07 was mixed with PA6 for immunization of rats. For the first immunization, a mixed solution of a cell suspension and an adjuvant was used. After 15 days from the first immunization, only the cell suspension was used for booster. Four days after the booster, 6.0×108 lymphocytes were isolated from the iliac lymph node, and 1.5×108 lymphocytes among them were subjected to cell fusion with Sp2 by a PEG method. The produced hybridomas were seeded on 10 96 well plates. The hybridoma culture supernatant was screened by immunostaining. As a result of immunostaining for KMC07, about 40% of the wells were positive for KMC07. Among them, hybridomas in wells with particularly strong signals were selected for cloning using a limiting dilution method. Thereafter, immunostaining of KMC07 and PA6 was performed using the hybridoma culture supernatant forming a single colony. As a result, 5 clones of KMC07-positive/PA6-negative antibody (1B4G4: corresponding to antibody I (IgG2b, κ), 1E2D3 (IgG2a, λ), 1E7G5: corresponding to antibody I (IgG2b, κ), 1E9F7: corresponding to antibody II (IgG2a, κ), 1F9D4: corresponding to antibody III (IgG2b, κ)) were successfully established. In addition, it was confirmed that each antibody did not recognize normal human cells by performing immunostaining using NHDF (human fibroblast cell lines).
In order to examine the binding property and specificity of the purified antibodies 1B4G4, 1E2D3, 1E7G5, 1E9F7, and 1F9D4 to KMC07, immunostaining was performed using various human-derived cancer cell lines. As the human cancer cell lines, BxPC-3 (pancreatic adenocarcinoma cell lines), KP-3 (pancreatic adenosquamous carcinoma cell lines), HARA (lung cancer cell lines), HARA-B2 (lung cancer cell bone metastasis lines), MCF-7 (breast cancer cell lines), HeLa (cervical carcinoma cell lines), and PC-3 (prostate cancer cell lines) were used. The intensity of a stain signal was evaluated in the following five stages. Incidentally, KP-3 is a pancreatic cancer cell line that has been established, but since it has recently become clear that a small number of cancer stem cell-like cells are also contained in the established cancer cell line (Christine M F. & Charlotte K. (2008). Human breast cancer cell lines contain stem-like cells that self-renew, give rise to phenotypically diverse progeny and survive chemotherapy. Breast Cancer Research. 10, R 25.), it is considered that pancreatic cancer stem cell-like cells are also present in KP-3. The results are shown in Table 1.
As shown in Table 1, all antibodies exhibited a binding property to pancreatic cancer stem cells (exhibited positive).
In addition, 1B4G4 (antibody I), 1E7G5 (antibody I), and 1E9F7 (antibody II) exhibited a specific binding property to pancreatic cancer stem cells. Among them, as to the point that 1B4G4 (antibody I) and 1E7G5 (antibody I) exhibited positive for KP-3, it is considered that pancreatic cancer stem cell-like cells in KP-3 were specifically recognized. In addition, these antibodies were also tentatively positive for PC-3, but since the stain signal intensity was extremely weaker than that for KMC07, they can be evaluated as specific to pancreatic cancer stem cells.
On the other hand, since 1E2D3 and 1F9D4 (antibody III) were positive for all cells except HeLa, it is considered that they exhibit binding properties to general cancer cells including pancreatic cancer stem cells.
All antibodies were negative for HeLa. It is considered that HeLa is repeatedly passaged so that the character is changed, and various proteins are expressed. Accordingly, a stain signal is often obtained for HeLa when an antibody that recognizes cancer cells is used. However, since the antibodies established in the Test Example were negative for HeLa, it was suggested that the antibodies established in the Test Example recognized a protein highly expressed in non-established cancer cells.
Gene cloning and production of recombinant chimeric antibodies were performed for 1B4G4 (antibody I), 1E7G5 (antibody I), 1E9F7 (antibody II), and 1F9D4 (antibody III). A rough flow of the antibody gene cloning performed in the Test Example is shown in
For HEK 293 T (human embryonic kidney cell lines) was used 10% FBS-containing D-MEM, followed by culture at 37° C. under 5% by volume CO2. For Expi293F (suspension system 293 cell lines) was used 2 mmol/l L-alanyl-L-glutamine solution [Wako, 016-21841] HE200-containing [gmep (registered trademark), HE200-0010], followed by shaking culture at 37° C. under 5% by volume CO2 at 125 rpm. The method for culturing KMC07 was according to “Culture of KMC07” in “1-1. Materials and methods” of Test Example 1.
RNA Extraction from Hybridoma and Synthesis of Antibody Variable Region cDNA
Total RNA was extracted from half the amount of hybridomas cultured in a P10 dish until it became confluent. First, the hybridomas were centrifuged at 1,800 rpm for 5 minutes. To the pellet, 1 ml of TRIzol [ambion, 15596-026] was added, and the pellet was allowed to stand at room temperature for 5 minutes. Then, 200 μl of chloroform [SIGMA, 319988-500ML] was added, and the mixture was allowed to stand at room temperature for 2 to 3 minutes. The mixture was centrifuged at 12,000 g for 15 minutes at 4° C. so that the mixture separated into 2 layers. The upper layer was collected, and 500 μl of isopropanol [Wako, 166-04836] was added to and mixed with the collected upper layer. Then, the mixture was allowed to stand at room temperature for 10 minutes. After centrifugation at 12,000 g for 10 min at 4° C., the supernatant was removed, followed by addition of 1 ml of 75% ethanol solution. After centrifugation at 7,500 g for 5 min at 4° C., the supernatant was removed. After air-drying, dissolution was performed in 20 μl of RNase free water [TaKaRa, 9012]. Based on the extracted RNA, cDNA of each of the heavy chain and the light chain was synthesized using SuperScript (trademark) III Reverse Transcriptase [Invitrogen, 18080-093]. Reverse transcription PCR solution 1 (1 μg of RNA, 4 μl of 2.5 mM dNTPs, and 0.4 μl of 5 μM Primer, diluted to 13 μl with autoclaved, distilled water) was reacted at 65° C. for 5 minutes, followed by cooling to 25° C. A reverse transcription PCR solution 2 (4 μl of 5×First-Strand Buffer, 1 μl 0.1 M DTT, 1 μl of 40 U/μl Recombinant RNasin (registered trademark) Ribonuclease Inhibitor [Promega, N251A], 1 μl of 200 U/μl SuperScript (trademark) IIIRT) was added, followed by reaction at 55° C. for 30 minutes at 70° C. for 15 minutes. At this time, primer 1 was used for the heavy chain, and primer 2 was used for the light chain.
Using cDNA as a template, PCR was performed using primer sets 4 to 9 and primer 1 for the heavy chain, and primer sets 10 to 16 and primer 2 for the clone of the light chain k to amplify the heavy chain variable region (VH) and the light chain variable region (VL), respectively. The obtained PCR product was inserted into pGEM (registered trademark)-T-easy vector [Promega, A1360]. At this time, the colonies selected by the blue-white selection were subjected to PCR using primer 17 and primer 18 (in the case of light chain, 3 types with addition of primer 19), which confirmed that the hybridoma-derived gene was correctly inserted. Then, the VH and VL gene fragments of each antibody were amplified using the primers shown in Table 3. The obtained PCR products were inserted into animal expression vectors pCEC2.1-rIgG2a and pCEC2.1-rIgK incorporating heavy chain and light chain constant region genes in advance by NE builder [NEB, E2621S].
Introduction of Antibody Expression Vector into Cell
When using HEK293T: To 221 μl of FBS-free D-MEM were added 1.3 μg each of the heavy chain and light chain expression vectors. To this, 4 μl of 1 mg/ml PEI [Polysciences, 23966] was added, and the mixture was allowed to stand at room temperature for 20 minutes. Then, 1.3 ml of 2% FBS-containing D-MEM was added. The culture supernatant of HEK293T cultured in a 12 well plate was removed, and a prepared solution was added. Incubation was performed at 37° C. under 5% by volume CO2 for 4 days, and the supernatant was collected.
When using Expi 293 F: To 1.5 ml of Opti-MEM [Gibco, 31985-062], 120 μl of 1 mg/ml PEI MAX [Polysciences, 24765] was added, and the mixture was allowed to stand at room temperature for 5 minutes. Separately, 15 μg each of the heavy chain and light chain expression vectors were added to 1.5 ml of Opti-MEM to prepare a plasmid solution. The prepared solution was added to and mixed with a PEI MAX solution, and the mixture was allowed to stand at room temperature for 20 minutes. The prepared solution was added to 7.5×107 cells of Expi293F suspended in 27 ml of Expi293 Expression Medium [Gibco, A14351-01], followed by shaking culture at 37° C. under 5% by volume CO2 at 125 rpm. After 20 h, 75 μl of 0.5 M sodium valproate [TCI, S0894] and 120 μl of 1 M sodium propionate [Wako, 194-03012] were added. Seven days after transfection, the supernatant was collected.
This was according to “Immunostaining” in “1-1. Materials and methods” of Test Example 1. When using as a primary antibody a culture supernatant of HEK293T into which an anti-KMC07 antibody expression vector had been introduced, the culture supernatant was used without dilution.
The antibody was forcibly expressed in Expi293F, and the antibody was purified from the culture supernatant using Protein A. To 30 ml of the culture supernatant from which the cells had been removed, 1.5 ml of 1 M Tris-HCl and 0.2 ml of a solution of Protein A (rProtein A Sepharose (trademark) FastFlow [GE Healthcare, 17-1279-01]) in PBS were added, followed by shaking at 4° C. overnight. The next day, a column was packed with Protein A reacted with the antibody, followed by replacement with a TBS solution. The antibody solution was collected into 5 fractions by applying to the column 300 μl of Gentle Ag/Ab Elution Buffer, pH 6.6 [Thermo Scientific, 21027] at a time. For each fraction, the absorbance at 280 nm was measured, and the solution having the peak was collected in a dialysis tube. By dialysis, the purified antibody solution was finally replaced with PBS. The concentration of the purified antibodies was determined using Pierce (trademark) BCA Protein Assay Kit.
Only CH2 and CH3 corresponding to the Fc region of the rat antibody were humanized and murinized. A rat heavy chain gene fragment (VH to Hinge) of each antibody was amplified using a rat heavy chain expression vector as a template and primers shown in Table 4. The obtained PCR products were inserted into animal expression vectors pCEC2.1-hIgG1 and pUC19-mIgG2a incorporating human and mouse Fc region genes in advance by NE builder.
Production of 1E7SP-1F9D4 light chain expression vector: The 1E7G5 light chain SP sequence gene was amplified by PCR using a 1E7G5 light chain expression vector as a template, and primer 23 and primer 35, and the 1F9D4 VL gene was amplified by PCR using a 1F9D4 light chain expression vector as a template, and primer 36 and primer 2. Next, PCR was performed using the two obtained PCR products as templates, and primer 23 and primer 2 to connect the respective gene fragments. Finally, the obtained PCR product was inserted into an animal expression vector pCEC 2.1-rIgK incorporating a light chain constant region gene in advance.
Production of 1B4SP-1E7G5, 1B4SP-1F9D4 light chain expression vector: The 1B4G4 light chain SP sequence gene was amplified by PCR using a 1B4G4 light chain expression vector as a template, and primer 21 and primer 37. The 1E7G5 and 1F9D4 VL genes were amplified by PCR using 1E7G5 and 1F9D4 light chain expression vectors as templates, respectively, and primer 38 and primer 2. Next, PCR was performed using the two obtained PCR products (1B4G4 light chain SP gene and each antibody VL gene) as templates, and primer 21 and primer 2 to connect the respective gene fragments. Finally, the obtained PCR product was inserted into an animal expression vector pCEC 2.1-rIgK incorporating a light chain constant region gene in advance.
On a membrane (Hybond™-C Extra [GE Healthcare, BPN303E]) was blotted 0.5 μl of culture supernatant of Expi293F by which each antibody had been forcibly expressed. At this time, a stock solution, and 10 times, 20 times to 640 times serially diluted solutions of the culture supernatant were used. The membrane was air-dried and then immersed in a 3% solution of skim milk in TBS-T for 30 minutes. After blocking, the membrane was incubated for 30 minutes with a secondary antibody (Anti-Rat IgGAP, antibody produced in rabbit) diluted with Can Get Signal Solution II. Finally, color development was performed using a BCIP/NBT solution.
First, total RNA was extracted from 4 clones of hybridoma for the anti-KMC07 antibody, and using the extracted total RNA as a template, cDNA of each of the heavy chain and light chain variable regions VH and VL was synthesized. Next, PCR was performed using the cDNA as a template to amplify VH and VL genes of each clone. The results of agarose electrophoresis of the variable regions are shown in
Based on the cloning results, the amino acid sequences and base sequences of the variable regions in the heavy chain and the light chain of each anti-KMC07 antibody are shown in the following Tables 5A to 5D. In the following Tables, the amino acid sequence and the base sequence of the signal region (SP) in each of the heavy chain and the light chain are also shown.
Variable region genes of 4 clones, 1B4G4 (antibody I), 1E7G5 (antibody I), 1E9F7 (antibody II), and 1F9D4 (antibody III), were amplified by PCR using, as a template, a cloning vector incorporating a polynucleotide composed of the base sequences of the variable regions and the signal regions (SP) shown in Tables 5A to 5D. Thereafter, the amplified genes were inserted into animal expression vectors pCEC2.1-rIgG2a and pCEC2.1-rIgK incorporating heavy chain and light chain constant region genes using NE builder. After confirming that there was no problem in the gene sequence by sequencing, each of the produced anti-KMC07 antibody expression vectors was introduced into HEK293T. Immunostaining of KMC07 was performed using the culture supernatant of each cell. In this immunostaining, cell nuclei are stained blue and each antibody is stained green. The results are shown in
On the other hand, no signal was obtained with 1F9D4 (antibody III). Since the SP sequence of 1F9D4VL was not recognized as an SP sequence on the amino acid domain database SMART, it was considered that there was some problem in the SP sequence. Accordingly, a 1E7SP-1F9D4 light chain expression vector was produced in which the SP sequence of the light chain of 1E7G5 (antibody I) (1E7SP: SEQ ID NO: 24) had been recombined with the light chain of 1F9D4 (antibody III). The flow of construction for the 1E7SP-1F9D4 light chain expression vector is shown in
PCR using an anti-KMC07 antibody heavy chain expression vector as a template was performed to amplify the VHCH1-Hinge gene fragment of each clone. Thereafter, the amplified gene fragments were inserted into animal expression vectors pCEC2.1-hIgG1 and pUC19-mIgG2a incorporating human and mouse Fc-region genes using NE builder. After confirming that there was no problem in the gene sequence by sequencing, each of the produced anti-KMC07 antibody human chimeric heavy chain expression vector and mouse chimeric heavy chain expression vector was introduced into Exp293F together with a light chain expression vector. When the chimeric antibody was purified from the culture supernatant, chimeric antibodies of 1B4G4 (antibody I) and 1E9F7 (antibody II) could be obtained in a sufficient antibody amount.
On the other hand, chimeric antibodies of 1E7G5 (antibody I) and 1F9D4 (antibody III) were hardly obtained. Accordingly, among the above 2 clones from which a sufficient antibody amount was obtained, a 1B4SP-1E7G5 light chain expression vector and a 1B4SP-1F9D4 light chain expression vector (hereinafter, also collectively referred to as “1B4SP-added light chain vector”) were produced in which the light chain SP sequence of 1B4G4 (antibody I) sequence (1B4SP: SEQ ID NO: 20) similar to the light chain SP sequence (SEQ ID NO: 24) of 1E7G5 (antibody I) had been recombined with the light chains of 1E7G5 (antibody I) and 1F9D4 (antibody III), respectively. The flow of construction for the 1B4SP-added light chain vector is shown in
Immunostaining of KMC07 was performed using the produced antibody. As a positive control, an antibody purified from the hybridoma culture supernatant was used. In this immunostaining, cell nuclei are stained blue, and each antibody is stained green (Alexa 488) or red (Alexa 568). The results are shown in
As NK92/CD16a (NK92 (human natural killer cell line) by which CD16a had been forcibly expressed), 100 U/ml human IL-2 [Shionogi & Co., Ltd., Immunace (registered trademark)]-containing MyeloCult™ H5100 [STEMCELL, 05150] was used, followed by culture at 37° C. under 5% by volume CO2. The method for culturing other cells was according to “Cell culture” and “Culture of KMC07” in “1-1. Materials and methods” of Test Example 1.
To an extract (protein amount: 600 μg) prepared using a membrane protein extract preparation kit (Mem-PER™ Plus Kit [Thermo fisher, 89842Y]) were added 25 μl of beads, followed by stirring at 4° C. for 30 minutes. After removing the supernatant, washing was performed 5 times with RIPA buffer, followed by addition of 20 μl of a 2×Sample buffer (0.125 M Tris-HCl, 4% SDS, 10% sucrose, 0.1% bromophenol blue, 10% 2-mercaptoethanol) solution, and then incubation at 98° C. for 5 minutes. The eluted proteins were subjected to SDS gel electrophoresis (SDS-PAGE) using Hybrid Gel, and then evaluated by silver staining (2D-silver staining reagent [Cosmo Bio, 423413]).
A membrane was prepared using the immunoprecipitate as an antigen. The other methods are according to the “Western blotting analysis” in “1-1. Materials and methods” of Test Example 1.
A band obtained by silver staining the immunoprecipitate was cut out and then subjected to silver removal for subsequent mass spectrometry.
The purified anti-KMC07 antibody was coupled to 1 ml of HiTrap NHSactivated HP [GE Healthcare, 17-0716-01]. Next, KMC07 in 30 P10 dishes cultured until it became confluent was collected by a scraper. After centrifugation at 1,200 rpm for 10 min at 4° C., the supernatant was removed and KMC07 cell suspension was prepared in RIPA-buffer (1 tablet of ULTRARIPAR Kit for Lipid Raft [BDL, F015] per 20 ml of 5 μg/ml Aprotinin, 5 μg/ml Leupeptin, 20 μg/ml APMSF, complete (trademark) Protease Inhibitor Cocktail [Roche, 04693116001]). The KMC07 cell suspension was applied to an antibody coupling column by a peristaltic pump, followed by circulation at 4° C. for 1 hour. The solution flowed out at this time was applied to a column coupled with another anti-KMC07 antibody, followed by circulation at 4° C. for 1 hour. This was repeated for 5 clones of the anti-KMC07 antibody for antibody and protein reaction. Finally, proteins were eluted with an elution buffer (pH 3.0, 1% Glycine, 1% n-octyl-β-D-glucoside [Dojindo, 346-05033]) for SDS-PAGE using 12.5% polyacrylamide gel, followed by evaluation by silver staining (ProteoSilver (trademark) plus silver staining kit [SIGMA, PROT-SIL2]).
Introduction of Protein Expression Vector into HEK293T and Confirmation of Introduction
To 80 μl of FBS-free D-MEM was added 1 μg of the protein expression vector. To this, 3 μl of 1 mg/ml PEI MAX was added, and the mixture was allowed to stand at room temperature for 20 minutes. Then, 1 ml of 2% FBS-containing D-MEM was added. The culture supernatant of HEK293T cultured in a 12 well plate was removed, and a prepared solution was added. One day after incubation at 37° C. under 5% CO2, assessment was made by immunostaining. The immunostaining was according to “Immunostaining” in “1-1. Materials and methods” of Test Example 1. An anti-EpCAM rabbit polyclonal antibody [proteintech, 21050-1-AP] was diluted 100 times for use as a positive control antibody.
Case where cell membrane is perforated for immunostaining: Cells were fixed by treatment with a 3.7% solution of formaldehyde in PBS for 15 minutes. The cell was treated with a 0.5% solution of Triton X-100 in PBS for 5 minutes, and then the cell membrane was perforated. Blocking was performed in a solution of NGS blocking (100 mg/ml BSA, Goat Serum [Gibco, 16210-064], 37.5 mg/ml glycine [Wako, 077-00735], 0.1% sodium azide [Wako, 197-11091]) in PBS for 30 minutes. After removal of the solution, a primary antibody was added, followed by incubation for 1 hour. Furthermore, incubation was performed with a solution of a secondary antibody (Alexa 488 anti-rat IgG (H+L)) in PBS containing Hoechst (registered trademark) 33342 for 30 minutes. Finally, a discoloration inhibitor DABCO was added, followed by observation with a fluorescence microscope.
Assessment of ADCC Activity (LDH Assay Method, Vs. BxPC3)
In order to measure the ADCC (Antibody Dependent Cellular Cytotoxicity) activity of the anti-KMC07 antibody 1F9D4 (antibody III) against BxPC3 (pancreatic adenocarcinoma cell line) in the presence of effector cells, the activity of LDH (lactate dehydrogenase) released from the cells was measured using Cytotoxicity LDH Assay Kit-WST [DOJINDO, CK12]. The medium for effector cells (NK92/CD16a) was changed 18 hours before the start of the assay. Target cells (BxPC3) were seeded at 5.0×103 cells/well in a 96 well plate 16 hours before the start of the assay, followed by culture at 37° C. under 5% by volume CO2. On the day of the assay, the effector cells and the anti-KMC07 rat/human chimerized antibody 1F9D4 were diluted with Assay Buffer (1% FBS-containing RPMI-1640) so as to be 2 times thicker than the final concentration, and seeded at 50 μl/well in a 96 well plate from which the culture supernatant had been removed, followed by incubation at 37° C. for 4 hours under 5% by volume CO2 (n=3). At this time, as negative controls, wells for culturing only target cells, only effector cells, and wells of only 100 μl of Assay Buffer, only Lysis solution (91 μl of Assay Buffer, 9 μl of Lysis Solution) were made. Furthermore, 45 minutes before the end of incubation, 9 μl of Lysis solution was added to the target cells cultured in 91 μl of Assay buffer as a positive control. After 4 hours, the 96 well plate was centrifuged at 200 g for 4 minutes, and 50 μl each of the culture supernatants was transferred to a new 96 well plate. To this was added 50 μl each of a substrate solution (Dye Mixture-containing Assay Buffer), followed by reaction at room temperature for 15 to 30 minutes under a light-shielded condition. Finally, 50 μl each of Stop Solution was added to stop the reaction, and then the absorbance at 490 nm was measured. The absorbance for a well in which effector cells and an antibody were added to target cells was denoted as A, the absorbance for a well containing only effector cells was denoted as B, the absorbance for a well containing only target cells was denoted as C, and the absorbance for a well in which Lysis Solution was added to kill target cells was denoted as D, and the ADCC activity was calculated from the following calculation formula. For each absorbance, a value obtained by subtracting the background absorbance of each solution alone was used.
ADCC (%)={(A−B−C)/(D−C)}×100 [Mathematical formula 1]
Assessment of ADCC Activity (Colony Assay Method, Vs. KMC07)
In order to measure the ADCC activity of the anti-KMC07 antibody against KMC07 in the presence of effector cells, the number of colonies of KMC07 was counted over time. Hereinafter, this experimental system is referred to as a colony assay. In a 12 well plate in which glass coated with 1.5 μg/ml poly-L-ornithine 3 days before the start of the assay was immersed, PA6 (mouse fibroblast cell line) was seeded at 5.0×104 cells/well and cultured at 37° C. under 5% by volume CO2. The medium for effector cells (NK92/CD16a) was changed 18 hours before the start of the assay. Target cells (KMC07) were seeded at 5.0×104 cells/well in a 12 well plate 16 hours before the start of the assay, followed by culture at 37° C. under 5% by volume CO2. On the day of the assay, effector cells were diluted with KMC Medium (whose composition is described in “Culture of KMC07” in “1-1. Materials and methods” of Test Example 1) to an appropriate concentration, and anti-KMC07 rat/human chimerized antibodies (1B4G4 (antibody I) and 1F9D4 (antibody III)) were diluted therewith to 1 μg/well, and seeded at 500 μl/well in a 12 well plate from which the culture supernatant had been removed, followed by incubation at 37° C. under 5% by volume CO2 (n=2). At this time, assuming that KMC07 was present at about 5.0×104 cells/well, which was half the amount of PA6, effector cells were seeded at 2.5×105 cells/well in order to set the E:T ratio=5:1. After 6 hours, 12 hours, and 24 hours, immunostaining was performed to confirm colonies of KMC07. After treatment with a 3.7% solution of formaldehyde in PBS for 15 minutes to fix the cells, an anti-KMC07 rat antibody 1B4G4 (antibody I) (1 μg/ml) was used as a primary antibody for incubation for 1 hour. Furthermore, incubation was performed with a solution of a secondary antibody (Alexa 488 anti-rat IgG (H+L)) in PBS containing Hoechst (registered trademark) 33342 for 30 minutes. Finally, a discoloration inhibitor DABCO was added, followed by observation with a fluorescence microscope. For each glass, 9 sites were randomly selected, and the number of KMC07 colonies was measured.
On the backs of 15 BALB/cSlc-nu/nu nude mice [SLC, 6-weeks old female], 100 μl each of a KMC07+PA6 cell suspension (3.5×107 cells/ml) suspended in 10% FBS-containing MEMα was subcutaneously injected.
Mice were grouped into groups of 3 per test antibody depending on tumor size 3 weeks after production of tumor-bearing mouse (n=3). Each of the anti-KMC07 antibodies 1E7G5 (antibody I), 1F9D4 (antibody III), and the negative control antibody 4G8B4 that is not an anti-KMC07 antibody was injected into the tail vein once a week in a volume of 200 μl of a solution of 0.25 mg/ml anti-KMC07 rat/mouse chimeric antibody in PBS. This operation was performed six times in total. The body weight and tumor diameter of each mouse were measured by caliper twice a week. The tumor volume was estimated from the following calculation formula.
Tumor volume (mm3)={major axis×(minor axis)2}/2 [Mathematical formula 2]
All statistical analyses were performed by two-way ANOVA.
Immunoprecipitation using a membrane protein extract was performed on solubilized proteins with denaturation suppressed as much as possible. The results of performing silver staining on the KMC07 membrane protein extract using 5 clones of the KMC07 antibody and 10A7 as a control IgG antibody are shown in
For 1B4G4 and 1E7G5 (antibody I), broad bands (indicated by the arrow in the figure) were obtained between 75 and 100 kDa, and for 1F9D4 (antibody III), bands (indicated by the arrow in the figure) were obtained near 140 kDa and 37 kDa. The band pattern of silver staining and the result of “1-2-2. Reactivity against various human cancer cell line” in “1-2. Result” of Test Example 1 suggested that 1B4G4 and 1E7G5 (antibody I) may recognize the same antigen.
To an affinity column, 5 clones of the anti-KMC07 antibody were bound and the KMC07 whole cell extract was flown to react the antibody with the antigen. Thereafter, the antibody was eluted and evaluated by silver staining. The results are shown in
As shown in Table 6, 14-3-3 protein beta/alpha (14-3-3) was hit for 1B4G4 and 1E7G5 (antibody I). However, since 14-3-3 is a protein involved in signal transduction in a cell and is known to interact with various proteins, it is considered that 14-3-3 non-specifically bound to the antibody. On the other hand, Olfactomedin-4 (OLFM4) detected with 1B4G4 (antibody I), which is a secreted protein and has a molecular weight of about 57 kDa, is used as a candidate antigen of 1B4G4 (antibody I) from the fact that the Olfactomedin is (1) known as an intestinal epithelial stem cell marker, (2) known to be widely expressed in gastric cancer and colorectal cancer, and be involved in the progression of cancer, (3) known that the molecular weight becomes 72 kDa when the sugar chain is modified, and (4) known to interact with cadherin present in a cell membrane. It is likely that OLFM4 is subjected to special modification such as specific sugar chain modification by KMC07 and is anchored to the cell membrane. Furthermore, EpCAM detected by 1F9D4 (antibody III) is a type I transmembrane glycoprotein, is known to be involved in not only cell adhesion but also signal transduction and cell migration, is expressed in many cancer types, and has recently attracted attention as a cancer stem cell marker. Considering that 1F9D4 (antibody III) also reacts with cancer cell lines other than KMC07, it is determined that 1F9D4 (antibody III) is likely to recognize EpCAM, and thus EpCAM was used as a candidate antigen of 1F9D4 (antibody III).
HA-OLFM4 and EpCAM expression vectors were each introduced into HEK293T, and the reactivity of 5 clones of the anti-KMC07 antibody was evaluated by immunostaining. Commercially available anti-HA and anti-EpCAM antibodies were used as positive control antibodies, and each antibody was reacted with HEK293T untreated as a negative control. As a result, only for 1F9D4 (antibody III), a signal was obtained in HEK293T by which EpCAM had been forcibly expressed. Therefore, the antigen of 1F9D4 (antibody III) was more likely EpCAM.
Since it was considered that EpCAM was so specially modified with KMC07 that its expression pattern was changed, expression of EpCAM was confirmed using alive or fixed Triton X-100-treated KMC07 by immunostaining. At this time, the anti-KMC07 antibody 1F9D4 and a commercially available EpCAM antibody were used as primary antibodies. When KMC07 was immunostained as living cells, signals were obtained on the cell membrane of KMC07 and the cell membrane of PA6 with the anti-EpCAM antibody (indicated by the arrow in
The cytotoxicity was measured by measuring the LDH (lactate dehydrogenase) released from cells. Specifically, ADCC activity against BxPC3 was evaluated for 1F9D4 that also reacts with cancer cells other than KMC07. Here, the human chimeric antibody 1F9D4 produced in Test Example 2 was used.
First, it was examined at what ratio (E:T ratio) NK92/CD16a as an effector cell was reacted with BxPC3 as a target cell to determine whether 1F9D4 exhibits ADCC activity. 1F9D4 was used at an antibody concentration of 1 μg/ml. As a result, it was confirmed that the cytotoxicity of about 40% was exhibited at the E:T ratio of 5:1, and the dead cell percentage did not increase even when the E:T ratio was increased any more. That is, since it was found that 1F9D4 exhibited sufficient ADCC activity at an E:T ratio of 5:1, the subsequent experiments were performed at an E:T ratio of 5:1. Next, the test was performed by changing the concentration condition of 1F9D4. NK92/CD16a was used as an effector cell. The results are shown in
ADCC activity was evaluated by measuring the number of colonies of KMC07 at 6, 12 and 24 hours after addition of effector cells and an anti-KMC07 rat/human chimeric antibody. As the antibody, 1B4G4 (antibody I) and 1F9D4 (antibody III) diluted to 1 μg/ml were used, and the E:T ratio was 5:1. The results are shown in
Since KMC07 proliferates while forming a colony, it is considered that the antibody hardly infiltrates the inside of the colony. Therefore, by increasing the reaction time of the antibody, it is considered that the antibody has succeeded in invading the inside while damaging KMC07 outside the colony, and dramatically reducing the colony. This was confirmed, since in the immunostained image of KMC07 24 hours after the reaction of 1F9D4 (antibody III) (
The anti-KMC07 antibody was administered to a tumor-bearing mouse produced using KMC07, and the tumor volume was measured over time to evaluate the antitumor effect. At this time, 2 clones of the anti-KMC07 mouse chimeric antibody 1E7G5 (antibody I) and 1F9D4 (antibody III) produced in Test Example 2 and an anti-Septin 7 mouse monoclonal antibody 4G8B4 produced in this laboratory were used as negative controls (hereinafter, described as control antibodies). Since 4G8B4 is an antibody against a protein expressed in cytoplasm, 4G8B4 is considered to be less toxic to mice. Each antibody was administered to 3 tumor-bearing mice by tail vein injection once a week for a total of six times (n=3). The body weight and the tumor volume were measured twice a week, and the temporal change of the rate of weight gain/loss based on the start date of the clinical trial was shown in
Furthermore, for each antibody, when multiple comparison of the tumor volume ratios for the respective measurement days was made by two-way ANOVA, for the anti-KMC07 antibodies 1E7G5 and 1F9D4, no significant difference in tumor volume ratio was found for the respective measurement days, while for the control antibody, a significant difference (P<0.05) in that the tumor was enlarged between the measurement days as shown in Table 7 was found. Therefore, it was suggested that for the mice to which the anti-KMC07 antibodies 1E7G5 (antibody I) and 1F9D4 (antibody III) had been administered, the enlargement of tumor was suppressed from the initial stage of the clinical trial, whereas for the mice to which the control antibody had been administered, the tumor continued to be enlarged.
From the above in vitro and in vivo results, it can be said that the produced antibody exhibited an antitumor effect.
The method for culturing cells was according to “Cell culture” and “Culture of KMC07” in “1-1. Materials and methods” of Test Example 1.
Extracellular vesicles were purified from culture supernatants of KMC07+PA6 and PA6 using ultracentrifugation. Ultracentrifugation was performed on 30 ml of culture supernatant at 110,000 rpm for 70 min at 4° C. The supernatant was removed, followed by suspension in 500 μl of PBS to prepare an extracellular vesicle fraction.
On a membrane (Hybond™-C Extra) was blotted 1 μl of a solution of extracellular vesicle in PBS. The membrane was air-dried and then immersed in a 3% solution of skim milk in TBS-T for 30 minutes. After blocking, a primary antibody diluted to 1 μg/ml with Can Get Signal Solution I was added, followed by incubation for 1 hour. After washing with TBS-T, the membrane was incubated for 30 minutes with a secondary antibody (Anti-Rat IgG-Peroxidase, antibody produced in rabbit [SIGMA, A5795-1ML]) diluted with Can Get Signal Solution II. Finally, color development was performed using KPL TMB Membrane Peroxidase Substrate [SeraCare, 5420-0027].
As HRP labeling of the antibody, Ab-10 Rapid Peroxidase Labeling Kit [Dojindo, LK33] was used. After labeling, the antibody concentration was 0.83 mg/ml.
On an ELISA plate [Thermo Scientific, 439454] was seeded 100 μl of the anti-KMC07 antibody prepared to be 10 μg/ml as a capture antibody, followed by incubation at 4° C. overnight. After rinsing with Wash Buffer (50 mM Tris-HCl (pH 8.0), 140 mM NaCl, 0.05% Tween 20) 3 times, 200 μl of 5% BSA Wash Buffer was added, followed by incubation at room temperature for 1 hour. After rinsing with Wash Buffer 3 times, 100 μl of a solution of extracellular vesicle in PBS prepared to be 2 μg/ml was added, followed by shaking at room temperature for 2 hours. After rinsing with Wash Buffer 3 times, 100 μl of a 2000-fold diluted HRP-labeled anti-CD9 antibody/anti-CD 63 antibody solution was added as a detection antibody, followed by incubation at room temperature for 1 hour. After rinsing with Wash Buffer 5 times, 100 μl of TMB microwell peroxidase substrate [KPL, 5120-0053] was added, followed by reaction at room temperature for 20 minutes. The reaction was stopped by adding 50 μl of 2% H2SO4, and the absorbance at 450 nm was measured.
The culture supernatant of KMC07 cultured for 9 days and the culture supernatant of PA6 cultured in KMC Medium for 7 days were collected. First, the collected culture supernatant was centrifuged at 300 g for 5 minutes. The supernatant was then centrifuged at 1,200 g for 20 minutes. Furthermore, the supernatant was centrifuged at 10,000 g for 30 minutes. Finally, the supernatant was collected for use in the subsequent operations.
Biotin Labeling Kit-SH [Dojindo, LK10] was used for biotin labeling of the antibody.
In an ELISA plate for fluorescence detection [Greiner, 655077] was seeded 100 μl each of the anti-KMC07 antibody prepared to be 5 μg/ml as a capture antibody and a positive control anti-CD9 antibody 2H12-B11, followed by incubation at 37° C. for 1 hour. After rinsing with PBS once, 200 μl of a blocking solution (a 1% solution of BSA in PBS) was added, followed by incubation at room temperature for 30 minutes. After removal of the solution, 100 μl of cell culture supernatant was added, followed by incubation at room temperature for 1 hour. After rinsing with PBS three times, 100 μl of biotin-labeled anti-CD9 antibody 1G10-C11 prepared to be 1 μg/ml was added, followed by incubation at room temperature for 1 hour. After rinsing 3 times with PBS, 50 μl of a streptavidin-β-galactosidase solution [Roche, 11112481001] diluted with a blocking solution to 0.05 U/ml was added, followed by incubation at room temperature for 1 hour. After rinsing with PBS 3 times, 50 μl of a substrate solution (0.2 mM 4-MU [SIGMA, M-1633], 0.2 M sodium phosphate buffer, 1 mM magnesium chloride, 100 mM sodium chloride, 0.4% sodium azide, 0.1% BSA) was added, followed by reaction at 37° C. for 2 hours under a light-shielded condition. Finally, 50 μl of a reaction stop solution (0.1 M glycine (pH 10.2)) was added, and then fluorescence at a wavelength of 445 nm was measured at an excitation light wavelength of 372 nm.
Using extracellular vesicles purified from the culture supernatants of KMC07+PA6 and PA6, dot blot was performed on 4 clones of the established anti-KMC07 antibody 1B4G4 (antibody I), 1E7G5 (antibody I), 1E9F7 (antibody II), and 1F9D4 (antibody III). As a negative control, PBS was spotted. The results are shown in
In order to evaluate the usefulness of the antibody as a diagnostic agent targeting extracellular vesicles, the binding force of each antibody to the extracellular vesicles was examined in a state where the structure of the extracellular vesicles was maintained. Using extracellular vesicles purified from the culture supernatants of KMC07+PA6 and PA6, sandwich ELISA was performed using 4 clones of the established anti-KMC07 antibody as a capture antibody, and HRP-labeled antibodies against CD9 and CD63, which are extracellular vesicle common markers, as detection antibodies. A schematic diagram of the experimental system is shown in
It was aimed to detect a trace amount of extracellular vesicles contained in the cell culture supernatant by a high sensitivity fluorescence detection sandwich ELISA. In a preliminary experiment, it was confirmed that the fluorescence detection sandwich ELISA showed approximately 100 times the sensitivity as compared to color development. Using culture supernatants of KMC07+PA6 and PA6, 4 clones of the established anti-KMC07 antibody 1B4G4 (antibody I), 1E7G5 (antibody I), 1E9F7 (antibody II), and 1F9D4 (antibody III) were used as a capture antibody, and biotin-labeled anti-CD9 antibody 1G10-C11 was used as a detection antibody for fluorescence detection sandwich ELISA. A schematic diagram of the experimental system is shown in
CDR sequencing was performed for 1B4G4 (antibody I), 1E7G5 (antibody I), 1E9F7 (antibody II), and 1F9D4 (antibody III). Tables 8A to 8C show the CDRs defined by all combinations of the Kabat numbering program (Bioinformatics, 32, 298-300 (2016)), the IMGT system (Dev Comp Immunol. 27, 55-77 (2003)), and the Paratome algorithm (Nucleic Acids Res. 40 (Web Server issue): W521-4 (2012). doi: 10.1093/nar/gks 480 and PLOS Comput Biol. 8(2): e1002388 (2012)) (Kabat/IMGT/Paratome), the CDRs defined by the Kabat numbering program, the CDRs defined by the IMGT system, and the CDRs defined by the Paratome algorithm.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2021-064090 | Apr 2021 | JP | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2022/017037 | 4/4/2022 | WO |
