Patent Grant
9427464
The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jan. 23, 2015, is named 086268-0136_SL.txt and is 130,893 bytes in size.
The present invention relates to an anti-human TROP-2 antibody having anti-tumor activity, and particularly, to an anti-human TROP-2 antibody having anti-tumor activity in vivo. In addition, the present invention relates to a hybridoma, which produces the aforementioned antibody, and a use of the aforementioned antibody.
Human TROP-2 (Tacstd2, GA733-1 and EGP-1) (hereinafter also referred to as “hTROP-2”) is a single transmembrane, type 1 cell membrane protein consisting of 323 amino acid residues (see SEQ ID NO: 2), and this protein has been known to be overexpressed in various types of epidermal cell carcinomas. The presence of a cell membrane protein associated with immunological resistance, which is commonly expressed in both human trophoblasts and cancer cells, had been long suggested (Non-Patent Document 1). An antigen molecule recognized by mouse monoclonal antibodies (162-25.3, 162-46.2) reacting with the cell membrane protein of a human choriocarcinoma cell line BeWo was identified. This antigen molecule was considered as one of the molecules expressed in human trophoblasts, and was named as Trop-2 (Non-Patent Document 2). Thereafter, the same molecule was discovered by other researchers. That is to say, a tumor antigen recognized by a mouse monoclonal antibody GA733 which is obtained by immunization with stomach cancer cells SW948 was named as GA733-1 (Non-Patent Document 3), and an epithelial glycoprotein recognized by a mouse monoclonal antibody RS7-3G11 which is obtained by immunization with non-small cell lung cancer cells was named as an epithelial/carcinoma antigen, EGP-1 (Non-Patent Document 4). In 1995, the Trop-2 gene was cloned, and as a result, it was confirmed that these are the same molecules (Non-Patent Document 5). Moreover, it was clarified that the molecule has a function to amplify intracellular calcium signals in cancer cells (Non-Patent Document 6), and therefore, it is also referred to as a tumor-associated calcium signal transducer 2 (TACSTD2).
The hTROP-2 gene is mapped on chromosome 1p32, and it constitutes a TACSTD gene family together with GA733-2 having a homology of approximately 50% therewith (which has been known as “TACSTD1,” “epithelial glycoprotein EGP-2,” “EpCAM” or “Trop-1”) (Non-Patent Document 7). The hTROP-2 protein (323 amino acid residues; SEQ ID NO: 2) has a molecular weight of approximately 36K Dalton, and this protein consists of a hydrophilic signal peptide (1st to 26th amino acids), an extracellular domain (27th to 274th amino acids), a transmembrane domain (275th to 297th amino acids) and an intracellular domain (298th to 323rd amino acids). The extracellular domain has four heterogeneous N-linked glycosylation sites, and its apparent molecular weight is increased by 11 to 13K Dalton due to addition of sugar chains (Non-Patent Document 5). It is considered that TACSTD gene family has a characteristic thyroglobulin (TY) sequence in the extracellular domain and is associated with the proliferation, invasion and metastasis of cancer cells.
To date, a physiological ligand of hTROP-2 has not been identified, and the molecular function thereof has not been clarified. However, it has been described that hTROP-2 transmits a calcium signal in tumor cells (Non-Patent Document 6). In addition, from the facts that intracellular serine 303 is phosphorylated by protein kinase C (PKC) that is Ca2+-dependent kinase (Non-Patent Document 4) and that hTROP-2 has a PIP2-binding sequence in its intracellular domain, it has been suggested that hTROP-2 has a signaling function in tumor cells (Non-Patent Document 8).
As a result of analyses such as immunohistochemistry (IHC) and flow cytometry, overexpression of hTROP-2 in many types of epithelium-derived carcinomas such as stomach cancer, lung cancer, colorectal cancer, ovarian cancer, breast cancer, prostate cancer, pancreatic cancer, liver cancer and esophagus cancer has been reported. In contrast, the expression of hTROP-2 in normal tissues is limited to cells in the epithelial region, and the expression level of hTROP-2 in normal cells is lower than that in cancer cells. Thus, the association of TROP-2 with tumor formation is suggested (Patent Documents 1-3 and 9).
Moreover, it has been demonstrated that the expression of hTROP-2 used as a biomarker in clinical samples correlates with the malignancy of colorectal cancer (Non-Patent Documents 10 and 11), pancreatic cancer (Non-Patent Document 12) or oral cancer (Non-Patent Document 13), and that when hTROP-2 is overexpressed, the possibility of metastasis or recurrence of such cancer is significantly high. Furthermore, in a large-scale gene expression analysis using a cDNA microarray technique, hTROP-2 has been identified as a gene cluster, which is overexpressed at the highest level in severe papillary adenocarcinoma of the ovary, in comparison with in normal ovary epithelium (Non-Patent Document 14).
Still further, in recent years, an important role of hTROP-2 in tumor formation has been demonstrated in the models by using colon cancer cells (Non-Patent Document 15). Since the expression of hTROP-2 promotes the anchorage-independent cell proliferation of tumor cells and is required for the tumor formation and proliferation of cancer cells subcutaneously transplanted in immunodeficient mice, it raised the possibility that hTROP-2 would act as a functional tumor antigen and would be used as a new therapeutic target.
To date, studies regarding the anti-tumor effects of several anti-hTROP-2 antibodies have been reported. An RS7 antibody (Patent Document 1) has been examined by employing in vivo models, in which radioactive substance-labeled antibodies were used, and anti-tumor activity was demonstrated in nude mouse xenograft models. However, the anti-tumor effects by antibody alone (a naked antibody) have not been reported.
In addition, the cytotoxicity of a cytotoxin-attached anti-hTROP-2 monoclonal antibody BR110 (Patent Document 2) on human cancer cell lines H3619, H2987, MCF-7, H3396 and H2981 in in vitro experiments has been reported. However, the cytotoxicity of a naked antibody or an immunoconjugate of BR110 in vivo has not been disclosed.
In recent years, it has been reported that an isolated monoclonal antibody, which was produced from a hybridoma cell line AR47A6.4.2 or AR52A301.5 obtained by immunizing mice with human ovarian cancer tissues, bound to hTROP-2, and that, for the first time, it exhibited, as a naked antibody, anti-tumor activity on nude mouse xenograft models, as well as cytotoxicity in vitro (Patent Documents 3 and 4). In these patent documents, the aforementioned antibody exhibited anti-tumor effects by treatment with antibody alone in mouse xenograft models, into which pancreatic cancer cell lines BxPC-3 and PL45, a prostate cancer cell line PC-3, a breast cancer cell line MCF-7 and a colon cancer cell line Colo205 had been transplanted. The therapeutic effects of the antibody have appeared in the models, into which BxPC-3 cells had been transplanted. Other than this, tumor formation and proliferation were only partially (approximately 40% to 60%) suppressed by the preventive administration of the antibody, and an extremely large amount (approximately 20 mg/kg) of the antibody was necessary for such suppression of tumor formation and proliferation.
Based on the above-mentioned previous findings, the potential use of the anti-hTROP-2 antibody as an anti-tumor antibody has been suggested. However, not all of the anti-hTROP-2 antibodies exhibit anti-tumor effects by treatment with antibody alone as naked antibodies in vivo. The antibodies exhibit different actions on hTROP-2, depending on a binding site, affinity and the properties of a monoclonal antibody.
Under the aforementioned circumstances, it has been desired to develop an anti-hTROP-2 antibody (an anti-hTROP-2 monoclonal antibody) having high anti-tumor activity in vivo, and specifically, an anti-hTROP-2 antibody or the like, which has an anti-tumor effect as a naked antibody alone in vivo and further, which has the anti-tumor effect at a low dose, and particularly, such an anti-hTROP-2 antibody, which is a humanized antibody.
The present invention has been completed, while taking into consideration the aforementioned circumstances. The present invention provides an anti-hTROP-2 antibody (an anti-hTROP-2 monoclonal antibody), a hybridoma, which produces the antibody, a fragment of the antibody, a conjugate (an immunoconjugate) of the antibody or the like and a drug, a pharmaceutical composition for diagnosing or treating a tumor, a method for detecting a tumor, a kit for detecting or diagnosing a tumor, and the like, which will be described below.
(1) An antibody against human TROP-2 in which an H chain V region of the antibody consists of the amino acid sequence shown in SEQ ID NO: 92 or 98, and an L chain V region of the antibody consists of the amino acid sequence shown in SEQ ID NO: 93.
In the antibody according to (1) above, the amino acid sequences of CDR 1 to 3 of the H chain V region of the antibody are shown in SEQ ID NOS: 36 to 38, respectively, and/or the amino acid sequences of CDR 1 to 3 of the L chain V region of the antibody are shown in SEQ ID NOS: 41 to 43, respectively.
(2) An antibody against human TROP-2 in which an H chain V region of the antibody consists of the amino acid sequence shown in SEQ ID NO: 94 or 95, and an L chain V region of the antibody consists of the amino acid sequence shown in SEQ ID NO: 96.
In the antibody according to (2) above, the amino acid sequences of CDR 1 to 3 of the H chain V region of the antibody are shown in SEQ ID NOS: 66 to 68, respectively, and/or the amino acid sequences of CDR 1 to 3 of the L chain V region of the antibody are shown in SEQ ID NOS: 71 to 73, respectively.
An example of the antibody according to (1) and (2) above is a humanized antibody.
An example of the antibody according to (1) and (2) above is an antibody having anti-tumor activity in vivo.
An example of the antibody according to (1) and (2) above is an antibody exhibiting 50% or more of tumor growth inhibitory activity at a dosage of 5 to 20 mg/kg body weight. Herein, the frequency of administration for exhibiting the tumor growth inhibitory activity is, for example, at most once a week.
An example of the antibody according to (1) and (2) above is an antibody exhibiting 50% or more of the tumor growth inhibitory activity by a single administration of the antibody at a dosage of 10 mg/kg body weight.
An example of the antibody according to (1) and (2) above is an antibody having anti-tumor activity on two or more types of human tumor cell lines.
An example of the antibody according to (1) and (2) above is an antibody having a dissociation constant (Kd value) of 1.0×10−10 M or less.
An example of the antibody according to (1) and (2) above is a monoclonal antibody.
Herein, the tumor is at least one type selected from the group consisting of human pancreatic cancer, human prostate cancer, human colorectal cancer, human breast cancer, human ovarian cancer, human lung cancer and human bile duct cancer. The tumor is preferably at least one type selected from the group consisting of human pancreatic cancer, human colorectal cancer, human breast cancer, human lung cancer and human ovarian cancer.
In addition, the tumor is, for example, a recurrent cancer or a metastatic cancer. Moreover, the tumor cell lines are, for example, at least two types selected from the group consisting of a human pancreatic cancer cell line PK-59, a human pancreatic cancer cell line BxPC-3, a human pancreatic cancer cell line KP-3L, a human pancreatic cancer cell line KP-2, a human pancreatic cancer cell line PK-1, a human pancreatic cancer cell line PK-45H, a human pancreatic cancer cell line PK-45P, a human pancreatic cancer cell line TCC-PAN2, a human pancreatic cancer cell line SUIT-2, a human colon cancer cell line CACO-2, a human colon cancer cell line SW480, a human colon cancer cell line DLD-1, a human colon cancer cell line HCT 116, a human breast cancer cell line JIMT-1, a human breast cancer cell line HCC1143, a human breast cancer cell line MCF-7, a human breast cancer cell line MDA-MB-468, a human prostate cancer cell line DU145, a human prostate cancer cell line PC-3, a human lung cancer cell line Calu-3, a human ovarian cancer cell line SK-OV-3 and a human bile duct cancer cell line TFK-1. Among others, the tumor cell lines are preferably at least two types selected from the group consisting of a human pancreatic cancer cell line PK-59, a human pancreatic cancer cell line BxPC-3, a human colon cancer cell line SW480, a human lung cancer cell line Calu-3, a human breast cancer cell line MDA-MB-468 and a human ovarian cancer cell line SK-OV-3.
(3) An antibody fragment derived from the antibody according to (1) and (2) above.
Examples of the antibody fragment according to (3) above include an antibody fragment comprising the amino acid sequence shown in SEQ ID NO: 92 or 98 and/or the amino acid sequence shown in SEQ ID NO: 93, or an antibody fragment comprising the amino acid sequence shown in SEQ ID NO: 94 or 95 and/or the amino acid sequence shown in SEQ ID NO: 96.
(4) An antibody-drug conjugate, which comprises the antibody according to (1) and (2) above and a substance having anti-tumor activity and/or cell-killing activity. (5) An antibody fragment-drug conjugate, which comprises the antibody fragment according to (3) above and a substance having anti-tumor activity and/or cell-killing activity.
In the conjugate according to (4) and (5) above, the tumor is, for example, at least one type selected from the group consisting of human pancreatic cancer, human prostate cancer, human colorectal cancer, human breast cancer, human ovarian cancer, human lung cancer and human bile duct cancer. Among others, the tumor is preferably at least one type selected from the group consisting of human pancreatic cancer, human colorectal cancer, human breast cancer, human lung cancer and human ovarian cancer. Moreover, the tumor is, for example, a recurrent cancer or a metastatic cancer.
(6) A pharmaceutical composition, which comprises at least one type selected from the group consisting of the antibody according to (1) and (2) above, the antibody fragment according to (3) above and the conjugate according to (4) and (5) above.
Examples of the composition according to (6) above include a composition which is used in the treatment of tumor, a composition which does not cause weight reduction as a side effect and a composition which is used in the diagnosis of tumor. Herein, the tumor is, for example, at least one type selected from the group consisting of human pancreatic cancer, human prostate cancer, human colorectal cancer, human breast cancer, human ovarian cancer, human lung cancer and human bile duct cancer. Among others, the tumor is preferably at least one selected from the group consisting of human pancreatic cancer, human colorectal cancer, human breast cancer, human lung cancer and human ovarian cancer. Moreover, the tumor is, for example, a recurrent cancer or a metastatic cancer
(7) A tumor therapeutic agent, which comprises at least one type selected from the group consisting of the antibody according to (1) and (2) above, the antibody fragment according to (3) above and the conjugate according to (4) and (5) above.
An example of the tumor therapeutic agent according to (7) above is a tumor therapeutic agent which does not cause weight reduction as a side effect. Herein, the tumor is, for example, at least one type selected from the group consisting of human pancreatic cancer, human prostate cancer, human colorectal cancer, human breast cancer, human ovarian cancer, human lung cancer and human bile duct cancer. Among others, the tumor is preferably at least one selected from the group consisting of human pancreatic cancer, human colorectal cancer, human breast cancer, human lung cancer and human ovarian cancer.
(8) A tumor diagnostic agent, which comprises at least one type selected from the group consisting of the antibody according to (1) and (2) above, the antibody fragment according to (3) above and the conjugate according to (4) and (5) above.
In the tumor diagnostic agent according to (8) above, the tumor is, for example, at least one type selected from the group consisting of human pancreatic cancer, human prostate cancer, human colorectal cancer, human breast cancer, human ovarian cancer, human lung cancer and human bile duct cancer. Among others, the tumor is preferably at least one selected from the group consisting of human pancreatic cancer, human colorectal cancer, human breast cancer, human lung cancer and human ovarian cancer.
(9) A method for detecting a tumor, which comprises: allowing at least one type selected from the group consisting of the antibody according to (1) and (2) above, the antibody fragment according to (3) above and the conjugate according to (4) and (5) above, to react with a sample collected from a living body; and then detecting a signal(s) of the reacted antibody and/or antibody fragment.
In the method for detecting a tumor according to (9) above, the tumor is, for example, at least one type selected from the group consisting of human pancreatic cancer, human prostate cancer, human colorectal cancer, human breast cancer, human ovarian cancer, human lung cancer and human bile duct cancer. Among others, the tumor is preferably at least one selected from the group consisting of human pancreatic cancer, human colorectal cancer, human breast cancer, human lung cancer and human ovarian cancer.
(10) A kit for treating, diagnosing or detecting a tumor, which comprises at least one type selected from the group consisting of the antibody according to (1) and (2) above, the antibody fragment according to (3) above and the conjugate according to (4) and (5) above.
In the kit for treating, diagnosing or detecting a tumor according to (10) above, the tumor is, for example, at least one type selected from the group consisting of human pancreatic cancer, human prostate cancer, human colorectal cancer, human breast cancer, human ovarian cancer, human lung cancer and human bile duct cancer. Among others, the tumor is preferably at least one selected from the group consisting of human pancreatic cancer, human colorectal cancer, human breast cancer, human lung cancer and human ovarian cancer.
(11) A polynucleotide encoding the antibody according to (1) and (2) above.
(12) A polynucleotide encoding the antibody fragment according to (3) above.
(13) A recombinant vector comprising the polynucleotide according to (11) or (12) above.
(14) A transformant comprising the recombinant vector according to (13) above.
*P<0.05, **P<0.01 (by Student's t-test).
An hTROP-2 gene and each human/mouse chimeric TROP-2 gene were introduced into HEK293 cells, and FACS analysis was then carried out using the cells, in which the genes were transiently expressed. In
The amino acid sequence underlined in the K5-70 VH indicates a CDR sequence determined in accordance with the definitions of Kabat et al. (as described above; U.S. Department of Health and Human Services, 1991). In addition, the number in the upper position of the amino acid sequence indicates the position number of an amino acid determined in accordance with the aforementioned definitions of Kabat et al. Each CDR sequence in the DA980102 VH is expressed with the symbol “---,” and thus the description is omitted. Since the amino acid underlined in the HuK5-70 VH was assumed to be important for the maintenance of the structure of CDR, the sequence of the K5-70 VH was maintained. In addition, with regard to the amino acid double-underlined in the HuK5-70 VH, the amino acid of the corresponding DA980102 VH (methionine (M)) is rarely found at this position. Hence, for the purpose of decreasing antigenicity, the amino acid double-underlined in the HuK5-70 VH was substituted with leucine (L) as a representative amino acid belonging to the same subgroup.
The amino acid sequence underlined in the K5-70 VL indicates a CDR sequence determined in accordance with the definitions of Kabat et al. (as described above; U.S. Department of Health and Human Services, 1991). In addition, the number in the upper position of the amino acid sequence indicates the position number of an amino acid determined in accordance with the aforementioned definitions of Kabat et al. Each CDR sequence in the L41174 VL is expressed with the symbol “---,” and thus the description is omitted. Since the amino acid underlined in the HuK5-70 VL was assumed to be important for the maintenance of the structure of CDR, the sequence of the K5-70 VL was maintained.
The amino acid sequence underlined in the HuT6-16 VH indicates a CDR sequence determined in accordance with the definitions of Kabat et al. (as described above; U.S. Department of Health and Human Services, 1991). In addition, the number in the upper position of the amino acid sequence indicates the position number of an amino acid determined in accordance with the aforementioned definitions of Kabat et al. Each CDR sequence in the DA935238 VH is expressed with the symbol “---,” and thus the description is omitted. Since the amino acids underlined in the HuT6-16 VH1 and HuT6-16 VH2 were assumed to be important for the maintenance of the structure of CDR, the sequence of the T6-16 VH was maintained. In addition, the lysine (K) at position 73 in the HuT6-16 VH1 was substituted with a threonine (T) derived from DA935238 as an acceptor sequence in the HuT6-16 VH2.
The amino acid sequence underlined in the T6-16 VL indicates a CDR sequence determined in accordance with the definitions of Kabat et al. (as described above; U.S.
Department of Health and Human Services, 1991). In addition, the number in the upper position of the amino acid sequence indicates the position number of an amino acid determined in accordance with the aforementioned definitions of Kabat et al. Each CDR sequence in the M99608 VL is expressed with the symbol “---,” and thus the description is omitted.
The upper position of each line indicates the gene sequence (cDNA sequence) and the lower position thereof indicates the amino acid sequence. In the amino acid sequence, a signal peptide portion is underlined with a dashed line and each CDR sequence (CDR 1 to 3) is underlined with a solid line (the amino acid sequence of only the mature protein portion, from which the signal peptide portion is removed, is shown in SEQ ID NO: 92). An EcoRI site (GAA TTC) and a Kozak sequence (ACC ACC) were added to the 5′ end of the HuK5-70 VH gene, and an NheI site (GCT AGC) was added to the 3′ end thereof.
The upper position of each line indicates the gene sequence (cDNA sequence) and the lower position thereof indicates the amino acid sequence. In the amino acid sequence, a signal peptide portion is underlined with a dashed line and each CDR sequence (CDR 1 to 3) is underlined with a solid line (the amino acid sequence of only the mature protein portion, from which the signal peptide portion is removed, is shown in SEQ ID NO: 93). An AgeI site (ACC GGT) and a Kozak sequence (ACC ACC) were added to the 5′ end of the HuK5-70 VL gene, and a BsiWI site (CGT ACG) was added to the 3′ end thereof.
The upper position of each line indicates the gene sequence (cDNA sequence) and the lower position thereof indicates the amino acid sequence. In the amino acid sequence, a signal peptide portion is underlined with a dashed line and each CDR sequence (CDR 1 to 3) is underlined with a solid line (the amino acid sequence of only the mature protein portion, from which the signal peptide portion is removed, is shown in SEQ ID NO: 94). An EcoRI site (GAA TTC) and a Kozak sequence (ACC ACC) were added to the 5′ end of the HuT6-16 VH1 gene, and an NheI site (GCT AGC) was added to the 3′ end thereof.
The upper position of each line indicates the gene sequence (cDNA sequence) and the lower position thereof indicates the amino acid sequence. In the amino acid sequence, a signal peptide portion is underlined with a dashed line and each CDR sequence (CDR 1 to 3) is underlined with a solid line (the amino acid sequence of only the mature protein portion, from which the signal peptide portion is removed, is shown in SEQ ID NO: 95). An EcoRI site (GAA TTC) and a Kozak sequence (ACC ACC) were added to the 5′ end of the HuT6-16 VH2 gene, and an NheI site (GCT AGC) was added to the 3′ end thereof.
The upper position of each line indicates the gene sequence (cDNA sequence) and the lower position thereof indicates the amino acid sequence. In the amino acid sequence, a signal peptide portion is underlined with a dashed line and each CDR sequence (CDR 1 to 3) is underlined with a solid line (the amino acid sequence of only the mature protein portion, from which the signal peptide portion is removed, is shown in SEQ ID NO: 96). An AgeI site (ACC GGT) and a Kozak sequence (ACC ACC) were added to the 5′ end of the HuT6-16 VL gene, and a BsiWI site (CGT ACG) was added to the 3′ end thereof.
The purified HuK5-70 antibody (lane 1), HuT6-16-1 antibody (lane 2) and HuT6-16-2 antibody (lane 3) were loaded in amounts of 1 μg each on SDS-PAGE, and were then stained with Coomassie.
The reactivity of each antibody shown in the figure with HEK293-hTROP-2 cells (
The antigen-binding ability of the K5-70 antibody and HuK5-70 antibody was analyzed according to an antigen-coated ELISA method. The symbol ▴ indicates the measurement results of the K5-70 antibody, and the symbol ● indicates the measurement results of the HuK5-70 antibody.
The antigen-binding ability of the T6-16 antibody, HuT6-16-1 antibody and HuT6-16-2 was analyzed according to an antigen-coated ELISA method. The symbol ▴ indicates the measurement results of the T6-16 antibody, the symbol ● indicates the measurement results of the HuT6-16-1 antibody, and the symbol ▪ indicates the measurement results of the HuT6-16-2 antibody.
(▴) K5-70 antibody and (●) HuK5-70 antibody.
Hereinafter, the present invention will be described in detail. The following descriptions are not intended to limit the scope of the present invention. Other than the following examples, the present invention may be modified and may be carried out, as appropriate, within a range that does not impair the intention of the present invention
The present specification includes all of the contents as disclosed in the specification of U.S. Provisional Patent Application No. 61/562,672 (filed on Nov. 22, 2011), which is a priority document of the present application.
In addition, all publications cited in the present specification, which include prior art documents and patent documents such as laid-open application publications and patent publications, are incorporated herein by reference in their entirety.
1. Summary of the Present Invention
As mentioned above, human TROP-2 (hTROP-2) is a single transmembrane, type 1 membrane protein having a full length of 323 amino acid residues. It has been known that an hTROP-2 gene and a gene product thereof are expressed in various types of cancer cells.
As mentioned above, it has been desired to develop an anti-hTROP-2 antibody (an anti-hTROP-2 monoclonal antibody) or the like having high anti-tumor activity in vivo. Under such circumstances, the present inventor performed a screening through an extremely large number of clones, and as a result, the inventor succeeded in obtaining a clone having high anti-tumor activity in vivo. Specifically, the present invention provides a monoclonal antibody, which specifically recognizes the extracellular region of hTROP-2 in vivo, and particularly, a monoclonal antibody exhibiting high affinity at a picomole (pM) order. The antibody of the present invention is extremely useful in that it is an anti-hTROP-2 monoclonal antibody (particularly, a humanized antibody), which exhibits significant tumor growth inhibitory activity at a lower dose than that of the existing anti-hTROP-2 antibody (for example, at a dosage of 1/20) when it is administered singly as a naked antibody, and which also exhibits significant tumor growth inhibitory activity on tumor-bearing mouse treatment models, in which multiple types of human cancer cells are used.
2. Production of Anti-hTROP-2 Antibody
(1) Preparation of Antigen
Information regarding the amino acid sequence (SEQ ID NO: 2) of hTROP-2 is disclosed under “Accession number NP_002344” in the website of, for example, NCBI (GenBank). Information regarding a nucleotide sequence (SEQ ID NO: 1) encoding the amino acid sequence of hTROP-2 is disclosed under “Accession number NM_002353” in the same website as described above.
As an antigen, a polypeptide or peptide (which is also simply referred to as a peptide) comprising at least a portion (the entire or a part) of the amino acid sequence of hTROP-2 can be used, and preferably, a peptide comprising at least a portion (the entire or a part) of the amino acid sequence of the extracellular region of hTROP-2 can be used. The extracellular region (including a signal peptide) of hTROP-2 indicates a region comprising the amino acids at positions 1 to 274 from the amino acid sequence shown in SEQ ID NO: 2 (the signal peptide: the amino acids at positions 1 to 26). Herein, with regard to a peptide used as an antigen, the above description “at least a portion of the amino acid sequence” is not particularly limited in terms of length. For example, a region comprising the amino acids at positions 1 to 145 from the amino acid sequence shown in SEQ ID NO: 2, a region comprising the amino acids at positions 146 to 274 from the same amino acid sequence as described above, and the like are preferable.
A peptide used as an antigen may be produced either by chemical synthesis, or by synthesis according to a genetic engineering method using Escherichia coli or the like. A method well known to persons skilled in the art may be applied.
When a peptide is produced by chemical synthesis, it can be synthesized by a well known peptide synthesis method. In addition, either a solid-phase synthesis method or a liquid-phase synthesis method can be applied to the peptide synthesis. A commercially available peptide synthesizer (for example, PSSM-8 manufactured by Shimadzu Corporation, etc.) may also be used.
When a peptide is synthesized by a genetic engineering method, first, DNA encoding the peptide is designed and synthesized. The design and synthesis of such DNA can be carried out according to a PCR method, using a vector comprising an entire-length hTROP-2 gene or the like as a template, and also using primers designed to be able to synthesize a desired DNA region. Thereafter, the DNA is ligated to a suitable vector to obtain a recombinant vector used for protein expression, and this recombinant vector is then introduced into a host so that a gene of interest can be expressed therein, thereby obtaining a transformant (Sambrook J. et al., Molecular Cloning, A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press, 2001).
As a vector, a phage or a plasmid capable of autonomously replicating in a host microorganism is used. Further, an animal virus or an insect virus vector can also be used. To produce a recombinant vector, a purified DNA may be cleaved with suitable restriction enzymes, and the thus cleaved DNA portion may be then inserted into the restriction enzyme site or the like of a suitable vector DNA, so as to ligate it to the vector. The type of a host used in transformation is not particularly limited, as long as it is able to express a gene of interest. Examples of such a host include bacteria (Escherichia coli, Bacillus subtilis, etc.), yeasts, animal cells (COS cells, CHO cells, etc.), insect cells, and insects. A mammal such as a goat may also be used as such a host. A method of introducing a recombinant vector into a host is publicly known.
The above-described transformant is cultured, and a peptide used as an antigen is then collected from the culture. The term “culture” is used herein to mean both (a) a culture supernatant, and (b) cultured cells, a cultured cell mass or a disintegrated product thereof.
After completion of the culture, when a peptide of interest is produced in a cell mass or in cells, the peptide is extracted by disintegrating the cell mass or the cells. On the other hand, when a peptide of interest is produced outside a cell mass or outside cells, a culture solution is directly used, or the cell mass or the cells are removed from the culture solution by centrifugation or the like. Thereafter, the peptide of interest can be isolated and purified by a single use of biochemical methods commonly used in the isolation and purification of peptides, such as ammonium sulfate precipitation, gel filtration, ion exchange chromatography and affinity chromatography, or by appropriately combining the aforementioned biochemical methods.
In the present invention, a peptide used as an antigen can also be obtained by in vitro translation using a cell-free synthesis system. In this case, two methods, namely, a method using RNA as a template and a method using DNA as a template (transcription/translation) can be applied. As such a cell-free synthesis system, a commercially available system, such as EXPRESSWAY™ system (Invitrogen), PURESYSTEM® (Post Genome Institute) or TNT® system (Promega) can be used.
The thus obtained peptide may bind to a suitable carrier protein such as bovine serum albumin (BSA), keyhole limpet hemocyanin (KLH), human thyroglobulin or chicken γ-globulin.
Moreover, the antigen may be a peptide consisting of an amino acid sequence comprising a deletion, substitution or addition of one or more amino acids with respect to the amino acid sequence of hTROP-2 (SEQ ID NO: 2) or a partial sequence thereof as described above. There can be used, for example, a peptide consisting of an amino acid sequence, in which one or more (preferably one or several (for example, 1 to 10, and more preferably 1 to 5)) amino acids are deleted, or one or more (preferably one or several (for example, 1 to 10, and more preferably 1 to 5)) amino acids are substituted with other amino acids, or one or more (preferably one or several (for example, 1 to 10, and more preferably 1 to 5)) other amino acids are added, with respect to the amino acid sequence of hTROP-2 or a partial sequence thereof.
In the present invention, a gene to be introduced into a cell or the like is a gene encoding an hTROP-2 protein or a partial fragment thereof, or a mutant protein thereof or a fragment thereof. As such a gene, a gene having the nucleotide sequence shown in SEQ ID NO: 1 or a partial sequence thereof can be used, for example.
Furthermore, as a gene to be introduced into a cell or the like, there may also be used a nucleotide sequence hybridizing under stringent conditions with a sequence complementary to the nucleotide sequence shown in SEQ ID NO: 1 and encoding a protein having hTROP-2 activity, or a partial sequence thereof.
The description “stringent conditions” is used herein to mean washing conditions after completion of the hybridization. As such stringent conditions, a salt (sodium) concentration in buffer is 10 to 500 mM and a temperature is 42° C. to 72° C., and preferably, the aforementioned salt condition is 50 to 300 mM and a temperature is 55° C. to 68° C.
Mutation can be introduced into a gene according to a known method such as a Kunkel method or a Gapped duplex method, using, for example, a mutation introduction kit which utilizes site-directed mutagenesis, such as GeneTailor™ Site-Directed Mutagenesis System (manufactured by Invitrogen) or TaKaRa Site-Directed Mutagenesis System (Mutan-K, Mutan-Super Express Km, etc.; manufactured by Takara Bio Inc.).
(2) Production of Polyclonal Antibody
The prepared antigen is administered to a mammal for immunization. The type of such a mammal is not particularly limited. For example, a rat, a mouse and a rabbit can be used, and among them, a mouse is preferable.
The dosage of the antigen per animal can be determined, as appropriate, depending on the presence or absence of an adjuvant. Examples of such an adjuvant include a Freund's complete adjuvant (FCA), a Freund's incomplete adjuvant (FIA) and an aluminum hydroxide adjuvant. Immunization can be carried out mainly by injecting the antigen into the vein, footpad, subcutis or abdominal cavity of an animal. In addition, immunization interval is not particularly limited, and immunization is carried out at intervals of several days to several weeks, preferably at intervals of 1 week, 1 to 10 times, and preferably 2 or 3 times. Three to seven days after the final immunization day, an antibody titer is measured by enzyme immunoassay (ELISA or EIA), radioimmunoassay (RIA) or other methods. On the date at which a desired antibody titer is obtained, blood can be collected to obtain antiserum. In the above-described method of collecting an antibody, if it is necessary to purify the antibody, the antibody can be purified by selecting a suitable method from known methods such as ammonium sulfate precipitation, ion exchange chromatography, gel filtration chromatography and affinity chromatography, or by combining the above-mentioned methods, as appropriate. Thereafter, the reactivity of a polyclonal antibody in the antiserum is measured by the ELISA method or the like.
(3) Production of Monoclonal Antibody
(3-1) Collection of Antibody-Producing Cells
The anti-hTROP-2 antibody of the present invention is not limited, but it is preferably a monoclonal antibody.
The prepared antigen is administered to a mammal, such as a rat, a mouse or a rabbit, for immunization. The dosage of the antigen per animal can be determined, as appropriate, depending on the presence or absence of an adjuvant. The same adjuvants as described above can be used herein. Also, the same immunization methods as described above can be applied herein. One to sixty days, and preferably, one to fourteen days after the final immunization day, antibody-producing cells are collected. Examples of such antibody-producing cells include splenic cells, lymph node cells and peripheral blood cells. Of these, lymph node cells and splenic cells are preferable.
(3-2) Cell Fusion
In order to obtain hybridomas (an antibody-producing cell line), the cell fusion of antibody-producing cells with myeloma cells is carried out. As myeloma cells to be fused with antibody-producing cells, commonly available established cells from animals such as mice can be used. The cell line used herein is preferably a cell line, which has drug selectivity, cannot survive in an unfused state in a HAT selective medium (containing hypoxanthine, aminopterin and thymidine), and can survive only in a state fused with antibody-producing cells.
Examples of myeloma cells include mouse myeloma cell lines such as P3-X63-Ag8.653, P3-X63-Ag8(X63), P3-X63-Ag8.U1(P3U1), P3/NS I/1-Ag4-1(NS1) and Sp2/0-Ag14(Sp2/0). Myeloma cells can be selected, while taking into consideration the compatibility with antibody-producing cells, as appropriate.
Subsequently, myeloma cells are fused with antibody-producing cells. For such cell fusion, antibody-producing cells (1×106 to 1×107 cells/mL) are mixed with myeloma cells (2×105 to 2×106 cells/mL) in a medium for animal cells, such as DMEM or an RPMI-1640 medium containing no serum. The cell ratio between the antibody-producing cells and the myeloma cells (antibody-producing cells: myeloma cells) is not limited. In general, the cell ratio is preferably 1:1 to 10:1, and more preferably 3:1. Thereafter, a fusion reaction is carried out in the presence of a cell fusion promoter. As such a cell fusion promoter, polyethylene glycol having a mean molecular weight of 1,000 to 6,000 Dalton (D) or the like can be used. In addition, it is also possible to fuse antibody-producing cells with myeloma cells, employing a commercially available cell fusion apparatus which utilizes electrical stimulation (for example, electroporation).
(3-3) Selection and Cloning of Hybridomas
Hybridomas of interest are selected from the cells after the cell fusion treatment. As a method of selecting hybridomas, the cell suspension is appropriately diluted with, for example, a fetal bovine serum-containing RPMI-1640 medium, and the diluted solution is then dispersed on a microtiter plate. Thereafter, a selective medium is added to each well. While the selective medium is appropriately exchanged with a fresh one, culture is carried out. As a result, approximately 14 days after initiation of the culture on the selective medium, cells growing from the selective medium can be obtained as hybridomas.
Subsequently, screening is carried out to examine whether or not an antibody reacting with hTROP-2 is present in a culture supernatant of the growing hybridomas. The screening of hybridomas may be carried out according to an ordinary method, and thus, the screening method is not particularly limited. For example, an aliquot is collected from the culture supernatant of the growing hybridomas contained in the well, and screening is then carried out by ELISA, EIA, RIA or the like.
The cloning of the fused cells can be carried out by a limiting dilution method or the like. An antibody showing high reactivity with hTROP-2 is determined by flow cytometry or the like, and a hybridoma producing this antibody is then selected. The selected hybridoma is established as a clone.
(3-4) Collection of Monoclonal Antibody
As a method of culturing the established hybridoma and then collecting a monoclonal antibody from the obtained culture, a common cell culture method, an ascites formation method or the like can be adopted. The term “culture” is used herein to mean allowing hybridomas to grow in a culture dish or a culture bottle, or allowing hybridomas to grow in the abdominal cavity of an animal, as described below.
In the case of the cell culture method, hybridomas are cultured in a medium for animal cells, such as a 10% fetal bovine serum-containing RPMI-1640 medium, an MEM medium or a serum-free medium, under common culture conditions (for example, 37° C., 5% CO2 concentration) for 7 to 14 days, and thereafter, an antibody can be obtained from the culture supernatant.
In the case of the ascites formation method, approximately 1×107 hybridomas are administered into the abdominal cavity of an animal of the same species as the mammal from which myeloma cells are derived, so that large quantities of hybridomas are allowed to proliferate. Thereafter, 2 to 3 weeks later, ascites is preferably collected.
In the above-described antibody collection methods, if it is necessary to purify the antibody, the antibody can be purified by appropriately selecting a suitable method from known methods such as ammonium sulfate precipitation, ion exchange chromatography, gel filtration and affinity chromatography, or by combining the above-mentioned methods.
(3-5) Selection of Clone Having Anti-Tumor Activity
The anti-hTROP-2 antibody of the present invention is an antibody having anti-tumor activity in vivo.
The term “anti-tumor activity” is used herein to mean activity of killing tumor cells (cancer cells) or activity of inhibiting tumor growth. Preferred examples of such anti-tumor activity include activity of inhibiting the growth of cancer cells and activity of inhibiting tumor angiogenesis. The type of human tumor (tumor cells), on which the antibody of the present invention is able to exhibit anti-tumor activity, includes various types of known human tumors, in which the expression of hTROP-2 has been confirmed. The type of such human tumor is not particularly limited. For example, one or two or more types selected from various human tumors such as human pancreatic cancer, human prostate cancer, human colorectal cancer, human breast cancer, human ovarian cancer, human lung cancer and human bile duct cancer are preferable; and one or two or more types selected from human pancreatic cancer, human colorectal cancer, human prostate cancer, human colorectal cancer, human breast cancer, human ovarian cancer and human lung cancer are more preferable. Human pancreatic cancer and/or human colorectal cancer are further preferable.
Moreover, the above-described tumor may be a recurrent cancer or a metastatic cancer. The antibody of the present invention is also able to exhibit excellent anti-tumor activity on these types of tumors.
The presence of anti-tumor activity in vivo can be confirmed, for example, by employing a tumor-bearing mouse (a mouse xenograft model), into the subcutis of which desired tumor cells have been transplanted, and then by administering the antibody as obtained above to the mouse. In this case, the antibody may be administered immediately after the transplantation of tumor cells (prevention models). Alternatively, it may be administered after confirming that the transplanted tumor has reached a predetermined volume (treatment models). The administration method is not limited. For example, the antibody may be administered once every three days, every one week, every ten days, or every two weeks, or by a single administration (only one time), at a dosage of 5 to 20 mg/kg body weight, via intraperitoneal administration or the like. In the case of prevention models, the presence or absence of anti-tumor activity and the level thereof can be evaluated based on tumor formation frequency and tumor volume. In the case of treatment models, the presence or absence of anti-tumor activity and the level thereof can be evaluated based on tumor volume.
In the present invention, a preferred example of the anti-hTROP-2 antibody having anti-tumor activity in vivo is an antibody in which the amino acid sequences of CDR 1 to 3 of the H chain V region thereof are shown in SEQ ID NOS: 36 to 38, respectively, and/or the amino acid sequences of CDR 1 to 3 of the L chain V region thereof are shown in SEQ ID NOS: 41 to 43, respectively. A preferred example of the H chain V region is an H chain V region consisting of the amino acid sequence shown in SEQ ID NO: 35. A preferred example of the L chain V region is an L chain V region consisting of the amino acid sequence shown in SEQ ID NO: 40.
As another embodiment of the anti-hTROP-2 antibody of the present invention, a preferred example is an antibody in which the amino acid sequences of CDR 1 to 3 of the H chain V region thereof are shown in SEQ ID NOS: 46 to 48, respectively, and/or the amino acid sequences of CDR 1 to 3 of the L chain V region thereof are shown in SEQ ID NOS: 51 to 53, respectively. A preferred example of the H chain V region is an H chain V region consisting of the amino acid sequence shown in SEQ ID NO: 45. A preferred example of the L chain V region is an L chain V region consisting of the amino acid sequence shown in SEQ ID NO: 50.
Likewise, as a further embodiment of the anti-hTROP-2 antibody of the present invention, a preferred example is an antibody in which the amino acid sequences of CDR 1 to 3 of the H chain V region thereof are shown in SEQ ID NOS: 56 to 58, respectively, and/or the amino acid sequences of CDR 1 to 3 of the L chain V region thereof are shown in SEQ ID NOS: 61 to 63, respectively. A preferred example of the H chain V region is an H chain V region consisting of the amino acid sequence shown in SEQ ID NO: 55. A preferred example of the L chain V region is an L chain V region consisting of the amino acid sequence shown in SEQ ID NO: 60.
Likewise, as a further embodiment of the anti-hTROP-2 antibody of the present invention, a preferred example is an antibody in which the amino acid sequences of CDR 1 to 3 of the H chain V region thereof are shown in SEQ ID NOS: 66 to 68, respectively, and/or the amino acid sequences of CDR 1 to 3 of the L chain V region thereof are shown in SEQ ID NOS: 71 to 73, respectively. A preferred example of the H chain V region is an H chain V region consisting of the amino acid sequence shown in SEQ ID NO: 65. A preferred example of the L chain V region is an L chain V region consisting of the amino acid sequence shown in SEQ ID NO: 70.
In the present invention, more specifically, preferred examples of an anti-hTROP-2 antibody having anti-tumor activity in vivo include: an anti-hTROP-2 monoclonal antibody (clone name: K5-70) produced by a hybridoma with Accession number FERM BP-11251; an anti-hTROP-2 monoclonal antibody (clone name: K5-107) produced by a hybridoma with Accession number FERM BP-11252; an anti-hTROP-2 monoclonal antibody (clone name: K5-116-2-1) produced by a hybridoma with Accession number FERM BP-11253; an anti-hTROP-2 monoclonal antibody (clone name: T6-16) produced by a hybridoma with Accession number FERM BP-11346; and an anti-hTROP-2 monoclonal antibody (clone name: T5-86) produced by a hybridoma with Accession number FERM BP-11254.
Herein, the hybridoma with Accession number FERM BP-11251 was named as “Mouse-Mouse Hybridoma K5-70” and was deposited on May 12, 2010; the hybridoma with Accession number FERM BP-11252 was named as “Mouse-Mouse Hybridoma K5-107” and was deposited on May 12, 2010; the hybridoma with Accession number FERM BP-11253 was named as “Mouse-Mouse Hybridoma K5-116-2-1” and was deposited on May 12, 2010; the hybridoma with Accession number FERM BP-11346 was named as “Mouse-Mouse Hybridoma T6-16” and was deposited on Mar. 1, 2011; and the hybridoma with Accession number FERM BP-11254 was named as “Mouse-Mouse Hybridoma T5-86” and was deposited on May 12, 2010. All of these hybridomas were deposited with the International Patent Organism Depositary (IPOD), the National Institute of Advanced Industrial Science and Technology, an Independent Administrative Institution under the Ministry of Economy, Trade and Industry (the AIST, Tsukuba Central 6, Higashi 1-1-1, Tsukuba, Ibaraki, Japan, postal code: 305-8566).
Still further, another preferred example of the anti-hTROP-2 antibody of the present invention is an anti-hTROP-2 antibody that binds to a site (e.g. an epitope), to which a monoclonal antibody produced by the hybridoma having accession No. FERM BP-11251, FERM BP-11252, FERM BP-11253, FERM BP-11346 or FERM BP-11254 binds (recognizes).
Preferred examples of such an epitope will be given in (3-6) below.
(3-6) Epitope of Anti-hTROP-2 Antibody
The type of an epitope (an antigenic determinant) of the anti-hTROP-2 antibody of the present invention is not limited, as long as it is at least a portion of hTROP-2 as an antigen. For example, such an epitope is preferably at least a portion of a region formed by removing a region consisting of amino acids at positions 252 to 260 from the amino acid sequence of hTROP-2 shown in SEQ ID NO: 2, more preferably at least a portion of a region consisting of amino acids at positions 1 to 69 or at least a portion of a region consisting of amino acids at positions 100 to 274 (except for a region consisting of amino acids at position 252 to 260), and further preferably at least a portion of a region consisting of amino acids at positions 27 to 69 or a region consisting of amino acids at positions 109 to 206. Particularly preferred examples of the above-described epitope include a region consisting of amino acids at positions 43 to 65, a region consisting of amino acids at positions 152 to 165, a region consisting of amino acids at positions 171 to 183, a region consisting of amino acids at positions 109 to 120, a region consisting of amino acids at positions 194 to 207, a region consisting of amino acids at positions 43 to 56, and a portion comprising such a region, in the amino acid sequence of hTROP-2 shown in SEQ ID NO: 2. Further particularly preferred examples include a region consisting of amino acids at positions 43 to 65, a region consisting of amino acids at positions 152 to 165, a region consisting of amino acids at positions 171 to 183, a region consisting of amino acids at positions 109 to 120, and a portion comprising such a region. An anti-hTROP-2 antibody, which recognizes the above-described regions (binds to the above-described regions or portions comprising such regions), has high internalization activity into tumor cells, for example, and thus it is extremely useful as an immunoconjugate as described later.
(3-7) Characteristics of Anti-hTROP-2 Antibody
As described above, the anti-hTROP-2 antibody of the present invention is an antibody having high anti-tumor activity in vivo at a low dose. Specifically, it is preferable that the present anti-hTROP-2 antibody exhibits tumor growth inhibitory activity of 50% or more (preferably 80% or more, more preferably 90% or more, further preferably 95% or more, and particularly preferably almost 100% (for example, 98% or more, or 99% or more)) at a dose (as a naked antibody) of 20 mg/kg body weight or less (preferably 10 mg/kg body weight or less, more preferably 5 mg/kg body weight or less, and further preferably 1 mg/kg body weight or less) with respect to a tumor-bearing animal model.
Herein, the tumor growth inhibitory activity (%) can be calculated, for example, by the following formula:
Tumor growth inhibitory activity (%)=100−[(tumor volume or tumor weight of antibody administration group)/(tumor volume or tumor weight of control group)]×100
In addition, the anti-hTROP-2 antibody of the present invention preferably has anti-tumor activity on two or more types of human tumor cell lines. The type of such a human tumor cell line is not limited. For example, such human tumor cell lines are at least two types selected from the group consisting of various types of human pancreatic cancer cell lines, human prostate cancer cell lines, human colorectal cancer cell lines, human breast cancer cell lines, human ovarian cancer cell lines, human lung cancer cell lines and human bile duct cancer cell lines. Specifically, preferred examples of such human tumor cell lines include at least two types selected from the group consisting of a human pancreatic cancer cell line PK-59, a human pancreatic cancer cell line BxPC-3, a human pancreatic cancer cell line KP-3L, a human pancreatic cancer cell line KP-2, a human pancreatic cancer cell line PK-1, a human pancreatic cancer cell line PK-45H, a human pancreatic cancer cell line PK-45P, a human pancreatic cancer cell line TCC-PAN2, a human pancreatic cancer cell line SUIT-2, a human colon cancer cell line CACO-2, a human colon cancer cell line SW480, a human colon cancer cell line DLD-1, a human colon cancer cell line HCT 116, a human breast cancer cell line JIMT-1, a human breast cancer cell line HCC 1143, a human breast cancer cell line MCF-7, a human breast cancer cell line MDA-MB-468, a human prostate cancer cell line DU145, a human prostate cancer cell line PC-3, a human ovarian cancer cell line SK-OV-3, a human lung cancer cell line Calu-3 and a human bile duct cancer cell line TFK-1. Of these, as the above-described two or more types of human tumor cell lines, at least two types selected from the group consisting of the human pancreatic cancer cell line PK-59, the human pancreatic cancer cell line BxPC-3, the human colon cancer cell line SW480, the human lung cancer cell line Calu-3, the human breast cancer cell line MDA-MB-468 and the human ovarian cancer cell line SK-OV-3 are more preferable.
Moreover, the anti-hTROP-2 antibody of the present invention has a dissociation constant (Kd value) of preferably 1.0×10−10 M or less, more preferably 1.0×10−11 M or less, and further preferably 1.0×10−12 M or less. Herein, the binding ability (affinity) of the antibody can be measured in the form of a dissociation constant (Kd value), a dissociation rate constant (Kdiss [1/Sec]) or an association rate constant (Kass [1/M.Sec]), for example, by Scatchard analysis or surface plasmon resonance sensor called Biacore. As such Biacore apparatuses, Biacore 3000, Biacore 2000, Biacore X, Biacore J and Biacore Q (all of which were manufactured by Biacore) may be used, for example. It is preferable that the antibody have a dissociation constant (Kd value) that is as small as possible because it could have high binding ability (affinity). The Kd value is determined based on the two parameters of Kdiss and Kass, and it can be expressed in the formula: Kd[M]=Kdiss/Kass. As a method of calculating the Kd value, the method described in the Examples as described later (specifically, Example 10) can be preferably adopted.
(4) Genetically Recombinant Antibody and Antibody Fragment
(4-1) Genetically Recombinant Antibody
In a preferred embodiment of the anti-hTROP-2 antibody of the present invention, there is provided a genetically recombinant antibody. The type of such a genetically recombinant antibody is not limited. Examples include a chimeric antibody, a humanized antibody, and a human antibody.
A chimeric antibody (that is, a humanized chimeric antibody) is an antibody formed by ligating (conjugating) the variable region of a mouse-derived antibody to the constant region of a human-derived antibody (please refer to Proc. Natl. Acad. Sci. U.S.A. 81, 6851-6855, (1984), etc.). When such a chimeric antibody is produced, the thus ligated antibody can be easily constructed by a genetic recombination technique.
When a humanized antibody is produced, a complementarity determining region (CDR) is transplanted from the variable region of a mouse antibody into the variable region of a human antibody, so as to produce a reconstructed variable region, in which a framework region (FR) is derived from the human and CDR is derived from the mouse (what is called CDR grafting (CDR transplantation)). Subsequently, the thus humanized, reconstructed human variable region is ligated to a human constant region. Such a method for producing a humanized antibody is well known in the present technical field (please refer to see Nature, 321, 522-525 (1986); J. Mol. Biol., 196, 901-917 (1987); Queen C et al., Proc. Natl. Acad. Sci. USA, 86: 10029-10033 (1989); J P Patent Publication (Kohyo) No. 4-502408 A (1992) (Japanese Patent No. 2828340; Queen et al.), etc.).
Herein, the type of a CDR sequence derived from the mouse, which can be used in the humanized anti-hTROP-2 antibody of the present invention, is not particularly limited. For example, the amino acid sequences shown in SEQ ID NOS: 36 to 38 or the amino acid sequences shown in SEQ ID NO: 66 to 68 are preferably used as the CDR 1 to 3 of the H chain V region (VH), respectively. The amino acid sequences shown in SEQ ID NOS: 41 to 43 or the amino acid sequences shown in SEQ ID NO: 71 to 73 are preferably used as the CDR 1 to 3 of the L chain V region (VL), respectively.
In addition, preferred examples of the amino acid sequence of an H chain V region in a humanized reconstructed human variable region include: the amino acid sequence shown in SEQ ID NO: 92 (comprising CDR 1 to 3 consisting of the amino acid sequences shown in SEQ ID NOS: 36 to 38; the amino acid sequence further comprising a signal peptide is shown in SEQ ID NO: 75); the amino acid sequence shown in SEQ ID NO: 98 (comprising CDR 1 to 3 consisting of the amino acid sequences shown in SEQ ID NOS: 36 to 38; the amino acid sequence further comprising a signal peptide is shown in SEQ ID NO: 97); the amino acid sequence shown in SEQ ID NO: 94 (comprising CDR 1 to 3 consisting of the amino acid sequences shown in SEQ ID NOS: 66 to 68; the amino acid sequence further comprising a signal peptide is shown in SEQ ID NO: 79); and the amino acid sequence shown in SEQ ID NO: 95 (comprising CDR 1 to 3 consisting of the amino acid sequences shown in SEQ ID NOS: 66 to 68; the amino acid sequence further comprising a signal peptide is shown in SEQ ID NO: 81). Herein, the above-mentioned amino acid sequence of the H chain V region shown in SEQ ID NO: 98 is a modified amino acid sequence in which the arginine (R) at position 44 of the above-mentioned amino acid sequence of the H chain V region shown in SEQ ID NO: 92 is substituted with a glycine (G).
Likewise, preferred examples of the amino acid sequence of an L chain V region in a humanized reconstructed human variable region include: the amino acid sequence shown in SEQ ID NO: 93 (comprising CDR 1 to 3 consisting of the amino acid sequences shown in SEQ ID NOS: 41 to 43; and the amino acid sequence further comprising a signal peptide is shown in SEQ ID NO: 77); and the amino acid sequence shown in SEQ ID NO: 96 (comprising CDR 1 to 3 consisting of the amino acid sequences shown in SEQ ID NOS: 71 to 73; the amino acid sequence further comprising a signal peptide is shown in SEQ ID NO: 83).
Herein, preferred examples of the humanized anti-hTROP-2 antibody of the present invention include: (i) a humanized anti-hTROP-2 antibody, in which the amino acid sequence of the H chain V region is shown in SEQ ID NO: 92 and the amino acid sequence of the L chain V region is shown in SEQ ID NO: 93; and (ii) a humanized anti-hTROP-2 antibody, in which the amino acid sequence of the H chain V region is shown in SEQ ID NO: 98 and the amino acid sequence of the L chain V region is shown in SEQ ID NO: 93. In particular, the humanized anti-hTROP-2 antibody described in (ii) above, in which the amino acid sequence of the H chain V region is shown in SEQ ID NO: 98, has an improved avidity (that is flexibility of the movement of two antigen-binding arms) and has high antigen-binding activity, and thus, this antibody is particularly preferable.
Also, other preferred examples of the humanized anti-hTROP-2 antibody of the present invention include: (iii) a humanized anti-hTROP-2 antibody, in which the amino acid sequence of the H chain V region is shown in SEQ ID NO: 94 and the amino acid sequence of the L chain V region is shown in SEQ ID NO: 96; and (ii) a humanized anti-hTROP-2 antibody, in which the amino acid sequence of the H chain V region is shown in SEQ ID NO: 95 and the amino acid sequence of the L chain V region is shown in SEQ ID NO: 96.
In general, in the case of a human antibody (a complete human antibody), its structure comprising a Hyper Variable region that is the antigen-binding site of a V region, other parts of the V region, and a constant region is the same as the structure of the antibody of a human. However, such a Hyper Variable site may also be derived from other animals. A technique of producing a human antibody is publicly known, and a method for producing gene sequences that are common in humans by genetic engineering has been established. A human antibody can be obtained, for example, by a method using a human antibody-producing mouse that has human chromosomal fragments comprising the genes of the H chain and L chain of the human antibody (please refer to Tomizuka, K. et al., Nature Genetics, (1977) 16, 133-143; Kuroiwa, Y. et. al., Nuc. Acids Res., (1998) 26, 3447-3448; Yoshida, H. et. al., Animal Cell Technology: Basic and Applied Aspects, (1999) 10, 69-73 (Kitagawa, Y., Matsuda, T. and lijima, S. eds.), Kluwer Academic Publishers; Tomizuka, K. et. al., Proc. Natl. Acad. Sci. USA, (2000) 97, 722-727, etc.), or by a method of obtaining a phage display-derived human antibody selected from a human antibody library (please refer to Wormstone, I. M. et. al, Investigative Ophthalmology & Visual Science., (2002) 43 (7), 2301-8; Carmen, S. et. al., Briefings in Functional Genomics and Proteomics, (2002) 1 (2), 189-203; Siriwardena, D. et. al., Opthalmology, (2002) 109 (3), 427-431, etc.).
In the case of the aforementioned chimeric antibody, humanized antibody and human antibody, the N-glycoside-linked sugar chain in the antibody Fc region is preferably a sugar chain, in which fucose does not bind to N-acetylglucosamine at the reducing terminal thereof. A specific example is an antibody consisting of genetically recombinant antibody molecules, which has, in the Fc region of the antibody molecules, a sugar chain in which the position 1 of the fucose does not bind to the position 6 of the N-acetylglucosamine at the reducing terminal of the N-glycoside-linked sugar chain via an a bond. Such an antibody is able to significantly improve ADCC activity. This point (the characteristics of the N-glycoside-linked sugar chain in the antibody Fc region) is preferable also for the aforementioned polyclonal antibody and monoclonal antibody.
(4-2) Antibody Fragment
The anti-hTROP-2 antibody fragment (partial fragment) of the present invention is included in the antibody of the present invention. Herein, the antibody fragment of the present invention has binding activity to hTROP-2 (namely, it is able to bind to hTROP-2) and also has anti-tumor activity in vivo, as in the case of the anti-hTROP-2 antibody of the present invention.
The fragment of the antibody means a region of a portion of an anti-hTROP-2 polyclonal antibody or anti-hTROP-2 monoclonal antibody (namely, an antibody fragment derived from the anti-hTROP-2 antibody of the present invention). Examples of such an antibody fragment include peptides comprising, as at least a portion thereof, Fab, Fab′, F(ab′)2, Fv (variable fragment of antibody), a single-stranded antibody (an H chain, an L chain, an H chain V region, and an L chain V region, etc.), scFv, diabody (scFv dimer), dsFv (a disulfide-stabilized V region), and a complementarity determining region (CDR).
Fab is an antibody fragment with a molecular weight of approximately 50,000 having antigen-binding activity, which is formed by binding about a half on the N-terminal side of the H chain to the entire L chain via a disulfide bond, among fragments obtained by treating antibody molecules with a protease, papain. In addition, it is also possible to produce such Fab by inserting DNA encoding the Fab of an antibody into a prokaryote expression vector or a eukaryote expression vector, and then introducing the vector into a prokaryote or a eukaryote so as to allow the DNA to express therein.
F(ab′)2 is an antibody fragment with a molecular weight of approximately 100,000 having antigen-binding activity, whose size is slightly greater than Fab that binds to Fab via disulfide bond in the hinge region, among fragments obtained by treating antibody molecules with a protease, pepsin. In addition, it is also possible to produce such F(ab′)2 by the thioether bond or disulfide bond of Fab, as described later.
Fab′ is an antibody fragment with a molecular weight of approximately 50,000 having antigen-binding activity, which is formed by cleaving the disulfide bond in the hinge region of the aforementioned F(ab′)2. In addition, it is also possible to produce such Fab′ by inserting DNA encoding the Fab′ fragment of an antibody into a prokaryote expression vector or a eukaryote expression vector, and then introducing the vector into a prokaryote or a eukaryote so as to allow the DNA to express therein.
scFv is an antibody fragment having antigen-binding activity, which is a VH-P-VL or VL-P-VH polypeptide formed by ligating a single H chain V region (VH) to a single L chain V region (VL) using a suitable peptide linker (P). Such scFv can be produced by obtaining cDNA encoding the VH and VL of an antibody, constructing DNA encoding scFv, inserting the DNA into a prokaryote expression vector or a eukaryote expression vector, and then introducing the vector into a prokaryote or a eukaryote so as to allow the DNA to express therein.
Diabody is an antibody fragment formed by dimerization of scFv, which has divalent antigen-binding activities. Such divalent antigen-binding activities may be identical to each other, or they may also be different from each other. Such diabody can be produced by obtaining cDNA encoding the VH and VL of an antibody, constructing DNA encoding scFv such that the length of the amino acid sequence of P is 8 residues or less, inserting the DNA into a prokaryote expression vector or a eukaryote expression vector, and then introducing the vector into a prokaryote or a eukaryote so as to allow the DNA to express therein.
dsFv is an antibody fragment formed by binding polypeptides, in which one amino acid residue in each of VH and VL has been substituted with a cysteine residue, to each other via a disulfide bond between the cysteine residues. The amino acid residue to be substituted with cysteine residues can be selected based on estimation of the three-dimensional structure of the antibody according to the method of Reiter et al. (Protein Engineering, 7, 697-704, 1994). Such dsFv can be produced by obtaining cDNA encoding the VH and VL of an antibody, constructing DNA encoding dsFv, inserting the DNA into a prokaryote expression vector or a eukaryote expression vector, and then introducing the vector into a prokaryote or a eukaryote so as to allow the DNA to express therein.
A peptide comprising a CDR comprises at least one region of CDRs of VH (CDR 1 to 3) and CDRs of VL (CDR 1 to 3). More preferred examples of such a peptide include a peptide comprising all of the CDRs of VH and a peptide comprising all of the CDRs of VL. A particularly preferred example of the peptide is a peptide comprising all of the CDRs of VH and VL (total 6 regions). Preferred examples of the amino acid sequence of such a CDR include the amino acid sequences shown in SEQ ID NOS: 36 to 38, 41 to 43, 46 to 48, 51 to 53, 56 to 58, 61 to 63, 66 to 68, and 71 to 73, as described above. A peptide comprising multiple CDRs can be bound to one another, directly or via a suitable peptide linker. Such a peptide comprising CDR can be produced by constructing DNA encoding the VH and VL of an antibody, inserting the DNA into a prokaryote expression vector or a eukaryote expression vector, and then introducing the expression vector into a prokaryote or a eukaryote so as to allow the DNA to express therein. Moreover, such a peptide comprising CDR can also be produced by chemical synthesis methods such as a Fmoc method (a fluorenylmethyloxycarbonyl method) and a tBoc method (a t-butyloxycarbonyl method).
The antibody fragment of the present invention, as is, may be an antibody fragment, which comprises a part of or the entire antibody Fc region in which fucose does not bind to N-acetylglucosamine at the reducing terminal of an N-glycoside-linked sugar chain. Otherwise, the antibody fragment of the present invention may also be a fusion protein, in which the aforementioned antibody fragment is fused with a part of or the entire antibody Fc region in which fucose does not bind to N-acetylglucosamine at the reducing terminal of an N-glycoside-linked sugar chain. Such an antibody fragment is able to significantly improve ADCC activity, and thus it is preferable.
Hereinafter, in the descriptions of the present specification, the aforementioned antibody fragments are also included in the anti-hTROP-2 antibody of the present invention.
3. Polynucleotide, Recombinant Vector and Transformant
In the present invention, a polynucleotide (a gene, DNA) encoding the above-described anti-hTROP-2 antibody of the present invention or an antibody fragment thereof can also be provided. Specifically, the present polynucleotide is preferably a polynucleotide which comprises a nucleotide sequence encoding each amino acid sequence exemplified as the above-described anti-hTROP-2 antibody of the present invention or an antibody fragment thereof. Moreover, the polynucleotide of the present invention may be either a polynucleotide consisting of a polynucleotide alone encoding the anti-hTROP-2 antibody of the present invention or an antibody fragment thereof, or a polynucleotide which comprises the present polynucleotide as a portion thereof and also comprises known nucleotide sequences necessary for gene expression (e.g. a transcriptional promoter, an SD sequence, a Kozak sequence, a terminator, etc.). Thus, the type of the present polynucleotide is not limited.
With regard to the polynucleotide encoding the anti-hTROP-2 antibody of the present invention or an antibody fragment thereof, the codon corresponding to individual amino acids after translation is not particularly limited. The polynucleotide may comprise nucleotide DNA showing a codon commonly used in mammals such as a human (preferably, a frequently-used codon), or may also comprise nucleotide DNA showing a codon commonly used in microorganisms such as Escherichia coli or yeast, plants and the like (preferably, a frequently-used codon).
In the present invention, a recombinant vector comprising the above-described polynucleotide of the present invention, or a transformant comprising the recombinant vector, can also be provided.
A transcriptional promoter, an SD sequence (in a case in which the host is a prokaryotic cell) and a Kozak sequence (in a case in which the host is a eukaryotic cell) may previously be ligated to the upstream of a polynucleotide (a gene, DNA) to be incorporated into an expression vector used as a recombinant vector. Moreover, a terminator may be ligated to the downstream of the polynucleotide. Furthermore, an enhancer, a splicing signal, a poly(A) additional signal, a selective marker and the like may also be ligated to the polynucleotide. Individual elements necessary for gene expression, such as the above-described transcriptional promoter, may be comprised in the present polynucleotide from the beginning. When these elements are originally comprised in an expression vector, they may be used. Thus, the usage of individual elements is not particularly limited.
As methods of incorporating the present polynucleotide into an expression vector, various types of methods utilizing known genetic recombination techniques, such as a method using restriction enzymes or a method using topoisomerase, can be adopted. The type of an expression vector is not limited, as long as it is able to retain a polynucleotide (a gene, DNA) encoding the anti-hTROP-2 antibody of the present invention or an antibody fragment thereof, and a vector suitable for host cells to be used can be selected, as appropriate, and can be used. Examples of such an expression vector include plasmid DNA, bacteriophage DNA, retrotransposon DNA, a retrovirus vector and artificial chromosomal DNA.
Subsequently, the thus constructed recombinant vector is introduced into a host to obtain a transformant, and the obtained transformant is then cultured, so that the anti-hTROP-2 antibody of the present invention or an antibody fragment thereof can be expressed. It is to be noted that the term “transformant” is used in the present invention to mean a host into which a foreign gene has been introduced. Examples of such a transformant include: a host into which a foreign gene has been introduced by introduction of plasmid DNA or the like (transformation); and a host into which a foreign gene has been introduced by infecting the host with various types of viruses and phages (transduction).
The type of a host is not limited, as long as it is able to express the anti-hTROP-2 antibody of the present invention or an antibody fragment thereof, after the above-described recombinant vector has been introduced into the host. Thus, a host can be selected, as appropriate. Examples of such a host include known hosts such as various types of animal cells such as a human or a mouse, and various types of plant cells, bacteria, yeasts and plant cells.
When animal cells are used as host cells, examples of such animal cells include: human fibroblasts, human embryonic kidney cells, HEK293 cells, 293F cells, CHO cells, monkey COS-7 cells, Vero, mouse L cells, rat GH3 and human FL cells. Moreover, insect cells such as Sf9 cells or Sf21 cells may also be used as host cells.
When bacteria are used as hosts, examples of such bacteria include Escherichia coli and Bacillus subtilis.
When yeast is used as a host, examples of such yeast include Saccharomyces cerevisiae and Schizosaccharomyces pombe.
When plant cells are used as host cells, tobacco BY-2 cells are used for example. The method of obtaining a transformant is not limited, and it can be selected, as appropriate, while taking into consideration a combination of the type of a host and the type of an expression vector. Preferred examples of the method of obtaining a transformant include electroporation, lipofection, a heat shock method, PEG, a calcium phosphate method, a DEAE dextran method, and a method of infecting a host with various types of viruses such as DNA virus or RNA virus.
In the obtained transformant, the codon type of a polynucleotide contained in a recombinant vector comprised may be identical to or different from the codon type of a host used. Thus, the codon type is not limited.
4. Preparation of Antibody-Drug Conjugate
As an immunoconjugate prepared using the aforementioned anti-hTROP-2 antibody of the present invention, there can be provided an antibody-drug conjugate, which comprises the aforementioned antibody and a substance (a compound, etc.) having anti-tumor activity and/or cell-killing activity. It is to be noted that a conjugate formed by previously preparing each of the aforementioned antibody molecule and the aforementioned substance having anti-tumor activity and/or cell-killing activity, separately, and then combining them, is generally referred to as an immunoconjugate. On the other hand, a conjugate obtained by ligating a protein toxin used as such a substance having anti-tumor activity and/or cell-killing activity to an antibody gene on a gene according to a genetic recombination technique, so as to allow it to express as a single protein (a fusion protein), is generally referred to as an immunotoxin.
Examples of a substance having anti-tumor activity include doxorubicin, calicheamicin, mitomycin C, Auristatin E and radioactive isotope (RI). Examples of a substance having cell-killing activity include saporin, lysine, pseudomonas exotoxin, diphtheria toxin and radioactive isotope (RI). Of these, saporin and pseudomonas exotoxin are preferably used. The type of RI having anti-tumor activity and/or cell-killing activity is not particularly limited, and examples of such RI include 90Y, 111In, 125I, 3H, 35S, 14C, 186Re, 188Re, 189Re, 177Lu, 67Cu, 212Bi, 213Bi, 211At, 198Au, 224Ac, 126I, 133I, 77Br, 113mIn, 95Ru, 97Ru, 103Ru, 105Ru, 107Hg, 203Hg, 94mTc, 121mTe, 122mTe, 125mTe 165Tm, 167Tm, 168Tm, 111Ag, 197Pt, 109Pd, 32P, 33P, 47Sc, 153Sm, 177Lu, 105Rh, 142Pr, 143Pr, 161Tb, 166Ho, 199Au, 57Co, 58Co, 51Cr, 59Fe, 18F, 75Se, 201Tl, 225Ac, 76Br, 86Y, 169Yb, 166Dy, 212Pb and 223Ra.
A method for producing an antibody-drug conjugate is not limited. For example, a method of coupling an antibody with a drug via a disulfide bond or a hydrazone bond is applied.
The aforementioned anti-hTROP-2 antibody of the present invention is excellent in terms of internalization activity into target tumor cells that express hTROP-2. Thus, by previously combining a substance having anti-tumor activity and cell-killing activity with the anti-hTROP-2 antibody, it becomes possible to allow such a substance to directly and highly selectively act on the tumor cells. The antibody-drug conjugate of the present invention is extremely excellent in terms of ability to deliver the agent to the target tumor cells.
The internalization activity into cells can be evaluated by fluorescently labeling an antibody with rhodamine or the like and then observing the migratory behavior and localization of the antibody using a fluorescence microscope or the like.
Moreover, in the present invention, in addition to the aforementioned antibody-drug conjugate, there can also be provided an antibody fragment-drug conjugate, in which the aforementioned antibody fragment is used instead of an antibody. With regard to the details of such an antibody fragment-drug conjugate, the descriptions of the aforementioned antibody-drug conjugate can be applied, as appropriate.
Hereinafter, in the descriptions of the present specification, such an antibody fragment-drug conjugate is also included in the antibody-drug conjugate of the present invention.
5. Pharmaceutical Composition
The anti-hTROP-2 antibody and antibody-drug conjugate of the present invention are useful as active ingredients contained in a pharmaceutical composition.
The pharmaceutical composition is useful as a pharmaceutical composition for treating and/or diagnosing a tumor. In particular, since the anti-hTROP-2 antibody of the present invention and an antibody-drug conjugate comprising the aforementioned antibody have excellent tumor growth inhibitory activity as such anti-tumor activity, they are preferably used in the treatment of tumor. That is to say, the anti-hTROP-2 antibody and antibody-drug conjugate of the present invention are useful as active ingredients contained in a tumor therapeutic agent and a tumor diagnostic agent. It is to be noted that the above-described treatment of tumor includes inhibition of tumor growth and suppression of tumor growth. Specifically, if it is a tumor therapeutic agent, examples of the tumor therapeutic agent include a tumor growth inhibitor and a tumor growth suppressor.
It is preferable to provide the pharmaceutical composition of the present invention in the form of a pharmaceutical composition comprising the anti-hTROP-2 antibody and/or antibody-drug conjugate of the present invention as active ingredient(s), and further comprising a pharmacologically acceptable carrier. In addition, the pharmaceutical composition of the present invention can be used in combination with known anti-tumor agents. By such a combined use, a higher anti-tumor effect can be obtained.
Target diseases (tumors), to which the pharmaceutical composition of the present invention is applied, include: the aforementioned various types of known human tumors, in which the expression of hTROP-2 has previously been confirmed. Among others, one or two or more types selected from various types of human tumors such as human pancreatic cancer, human prostate cancer, human colorectal cancer, human breast cancer, human ovarian cancer, human lung cancer and human bile duct cancer are particularly preferable. Such target disease may be a single disease, or two or more diseases may be developed in combination. Moreover, the target tumor may be a recurrent cancer or a metastatic cancer. The pharmaceutical composition of the present invention (further, the anti-hTROP-2 antibody and/or antibody-drug conjugate of the present invention) can be effectively used as a therapeutic agent and a diagnostic agent for a recurrent cancer or a metastatic cancer.
Examples of the “pharmacologically acceptable carrier” include an excipient, a diluent, an extender, a disintegrator, a stabilizer, a preservative, a buffer, an emulsifier, an aromatic, a coloring agent, a sweetener, a thickener, a corrigent, a solubilizer and other additives. Using one or more types of such carriers, a pharmaceutical composition can be prepared in the form of an injection, a liquid agent, a capsule, a suspension, an emulsion, a syrup, etc. These pharmaceutical compositions can be administered orally or parenterally. Another form for parenteral administration is, for example, an injection comprising one or more active ingredients, which is prepared by an ordinary method. Such an injection can be produced by dissolving or suspending the present antibody in a pharmacologically acceptable carrier such as a normal saline solution or a commercially available distilled water used for injection.
In particular, when an antibody fragment derived from the anti-hTROP-2 antibody of the present invention (particularly, an antibody fragment with a low molecular weight) is administered into a living body, a colloidal dispersion system can be used in addition to the aforementioned components. Such a colloidal dispersion system is anticipated to have an effect of enhancing the stability of a compound (an antibody fragment) in a living body or an effect of efficiently transporting such a compound to a specific organ, tissue, or cell. The type of such a colloidal dispersion system is not limited, as long as it is commonly used. Examples of such a colloidal dispersion system include dispersion systems comprising, as bases, polyethylene glycol, a macromolecular conjugate, a macromolecular aggregate, a nanocapsule, microsphere, beads, and lipids including an oil in water emulsifier, micelle, mixed micelle and liposome. Preferred examples of such a colloidal dispersion system include multiple liposomes and the vesicles of artificial membrane, which have an effect of efficiently transporting such a compound to a specific organ, tissue, or cell (Mannino et al., Biotechniques, 1988, 6, 682; Blume and Cevc, Biochem. et Biophys. Acta, 1990, 1029, 91; Lappalainen et al., Antiviral Res., 1994, 23, 119; Chonn and Cullis, Current Op. Biotech., 1995, 6, 698).
The dosage of the pharmaceutical composition of the present invention differs depending on the age, sex, body weight and symptoms of a patient, therapeutic effects, an administration method, a treatment time, the types of the anti-hTROP-2 antibody and antibody-drug conjugate of the present invention contained in the pharmaceutical composition, etc. In general, the present pharmaceutical composition may be administered within the range between 600 μg and 6,000 mg per adult per administration. However, the dose is not limited to the aforementioned range.
In a case in which the pharmaceutical composition is administered in the form of an injection, for example, it may be administered at a dosage of 100 μg to 100 mg, per administration, per body weight of a human patient, once or divided over several administrations, as an average daily dose. Preferably, the pharmaceutical composition may be administered once every three days, once a week, once every ten days, or once every two weeks, or by a single administration (wherein the total number of administrations is 1). Examples of the dosage form include intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection and intraperitoneal injection. Of these, intravenous injection is preferable. In addition, such an injection may be prepared in the form of a nonaqueous diluent (e.g. polyethylene glycol, vegetable oil such as olive oil, alcohols such as ethanol, etc.), a suspension, or an emulsion. Such an injection can be sterilized by mechanical sterilization using a filter, the mixing of a microbicide, etc. The injection can be produced in the form of an injection to be prepared before using. That is, a sterilized solid composition is prepared by a freeze-drying method or the like, and the composition is then dissolved in sterilized distilled water used for injection or other solvents before it is used, so that it can be then used.
The present invention provides a use of the aforementioned anti-hTROP-2 antibody and/or antibody-drug conjugate of the present invention in the production of a pharmaceutical agent (a drug) for treating and/or diagnosing a tumor. In addition, the present invention provides the aforementioned anti-hTROP-2 antibody and/or antibody-drug conjugate of the present invention, which are used for treating and/or diagnosing a tumor.
Moreover, the present invention provides a method for treating and/or diagnosing a tumor, which is characterized in that it comprises using (namely, administering to patients) the aforementioned anti-hTROP-2 antibody and/or antibody-drug conjugate of the present invention. Furthermore, the present invention also provides the use of the aforementioned anti-hTROP-2 antibody and/or antibody-drug conjugate of the present invention in the treatment and/or diagnosis of tumor.
6. Method for Detecting Tumor
The method for detecting a tumor of the present invention is characterized in that it comprises allowing the aforementioned anti-hTROP-2 antibody of the present invention to react with a sample collected from a living body (hereinafter referred to as a biological sample), and detecting a signal(s) of the reacted antibody.
As described above, hTROP-2 has been confirmed to be specifically expressed in various types of tumor cells. Thus, hTROP-2, and particularly, free hTROP-2 (an extracellular region portion of hTROP-2) can be used as a marker for various types of tumors. In particular, such hTROP-2 can be preferably used as a marker for human pancreatic cancer, human prostate cancer, human colorectal cancer and human breast cancer.
Hence, the anti-hTROP-2 antibody of the present invention is allowed to react with a biological sample, and a signal of the reacted antibody is then detected, so as to detect a tumor. The obtained antibody signal can be used as an indicator of the amount of an antigen in the biological sample (that is, an hTROP-2 amount or a free hTROP-2 amount). In detection of a tumor using the antibody of the present invention, first, a biological sample collected as an analyte from a subject, such as a tissue section or blood used as a test target, is allowed to bind to the antibody of the present invention by an antigen-antibody reaction. Subsequently, based on the measurement results of the amount of the bound antibody, the amount of an antigen of interest contained in the biological sample is measured. This measurement may be carried out in accordance with known immunoassay methods. For example, an immunoprecipitation method, an immunoagglutination method, radioimmunoassay, immunonephelometry, a Western blot method, flow cytometry and the like can be used. In radioimmunoassay, a labeled antibody is used, and thus an antibody signal is expressed as the amount of the labeled antibody that is directly detected. Otherwise, an antibody whose concentration or antibody titer has been known may be used as a standard solution, and thus a signal of the target antibody may be expressed as a relative value. That is, both the standard solution and the analyte may be measured using a measurement device, and an antibody signal in a biological sample may be expressed as a value relative to the value of the standard solution used as a criterion. Examples of such radioimmunoassay include the ELISA method, the EI method, the RIA method, fluorescence immunoassay (FIA), and luminescence immunoassay. Of these, the ELISA method is particularly preferable in that it is simple and highly sensitive.
In the present invention, the state of a tumor can be evaluated or diagnosed, using the detection result obtained by the aforementioned detection method as an indicator. For example, when the detection result exceeds a predetermined standard value, the state of a tumor is defined as tumor positive, and when the detection result is less than the predetermined standard value, it is defined as tumor negative. In the case of tumor positive, it is determined that a certain type of tumor could have been developed, and thus the tumor state can be evaluated. The term “the state of a tumor” is used herein to mean the presence or absence of the development of a tumor, or the progression degree thereof. Thus, specific examples of the state of a tumor include the presence or absence of the development of a tumor, the progression degree thereof, the degree of malignancy, the presence or absence of metastasis, and the presence or absence of recurrence.
In the aforementioned evaluation, as a state of a tumor to be evaluated, only one state may be selected from the aforementioned examples, or multiple examples may be combined and selected. The presence or absence of a tumor can be evaluated by determining whether or not the tumor has been developed, with reference to the predetermined standard value used as a boundary, based on the obtained detection result. The degree of malignancy is used as an indicator that indicates the progression degree of a cancer. Based on the detection result, the target tumor can be classified into a certain disease stage and it can be evaluated. Otherwise, an early cancer and an advanced cancer can be distinguished from each other, and then they can be evaluated. For example, it is also possible to determine the target tumor as an early cancer or an advanced cancer, using the detection result as an indicator. The metastasis of tumor can be evaluated by determining whether or not neoplasm has appeared at a site apart from the position of the initial lesion, using the detection result as an indicator. The recurrence can be evaluated by determining whether or not the detection result has exceeded the predetermined standard value again after interval stage or remission.
7. Kit for Detecting or Diagnosing Tumor
The anti-hTROP-2 antibody of the present invention can be provided in the form of a kit for detecting or diagnosing a tumor. The kit of the present invention comprises a labeling substance, a solid-phase reagent on which the antibody or the labeled antibody has been immobilized, etc., as well as the aforementioned antibody. A labeling substance that labels the antibody means a substance labeled with an enzyme, a radioisotope, a fluorescent compound, a chemiluminescent compound, etc. The kit of the present invention may also comprise other reagents used for carrying out the detection of the present invention, in addition to the aforementioned constitutional elements. For example, when such a labeling substance is an enzyme labeling substance, the kit of the present invention may comprise an enzyme substrate (a chromogenic substrate, etc.), an enzyme substrate-solving solution, an enzyme reaction stop solution, a diluent used for analytes, etc. Moreover, the present kit may further comprise various types of buffers, sterilized water, various types of cell culture vessels, various types of reactors (an Eppendorf tube, etc.), a blocking agent (a serum component such as bovine serum albumin (BSA), skim milk, or goat serum), a washing agent, a surfactant, various types of plates, an antiseptic such as sodium azide, an experimental operation manual (instruction), etc.
The kit of the present invention can be effectively used to carry out the aforementioned detection method of the present invention, and thus it is extremely useful.
Hereinafter, the present invention will be more specifically described in the following examples. However, these examples are not intended to limit the scope of the present invention.
A full-length hTROP-2 gene was isolated from human fetal liver (10-week-old embryo) according to an RT-PCR method. First, the following PCR primers were designed based on the sequence of an hTROP-2 gene (Genbank accession No. NM— 002353).
When these primers were designed, a XhoI restriction enzyme-digested sequence except for a stop codon was added to the reverse primer. cDNA was synthesized from total RNA (TAKARA) prepared from human fetal liver (10-week-old embryo). Using this cDNA as a template, a PCR reaction was carried out with the aforementioned primers. Thereafter, development by agarose gel electrophoresis and extraction of a band of interest were carried out, and it was then cloned into a pCRII vector (Invitrogen) (pCRII-hTROP-2). The cloned hTROP-2 cDNA was confirmed by sequencing.
An expression vector was constructed by cleaving a EcoRI/XhoI fragment comprising an hTROP-2 gene from pCRII-hTROP-2, and then inserting the fragment into the EcoRI/XhoI site of a pcDNA4/myc-His© A vector (Invitrogen) (pcDNA4-hTROP-2-myc/His). Moreover, a HindIII/PmeI fragment comprising an hTROP-2 gene was cut out of pcDNA4-hTROP-2-myc/His (wherein the HindIII cleavage portion was blunt-ended), and the fragment was then inserted into a PmeI site of a pcDNA3.1(+) vector (Invitrogen), so as to construct an expression vector comprising a neomycin resistance gene (pcDNA3.1-hTROP-2-myc/His).
The expression vector (pcDNA3.1-hTROP-2-myc/His) encoding the full-length cDNA of hTROP-2, which had been produced by the above-described method, was introduced into HEK293 cells (RIKEN), HuH-7 cells (HSRRB), 7E2-C cells (described in WO 2005/052156) and CHO-K1 cells (HSRRB), using a lipofectamine 2000 reagent (Invitrogen), and selection was then carried out using an antibiotic G418 (geneticin; GIBCO BRL). Thereafter, a cell line, which stably expressed hTROP-2, was established and obtained.
A gene fragment encoding a portion of the extracellular region of hTROP-2 (specifically, a region consisting of amino acids at positions 1 to 263 from the amino acid sequence shown in SEQ ID NO: 2) was amplified by a PCR method. The following primers were used in the amplification.
In this operation, a XhoI restriction enzyme-digested sequence was added to the reverse primer. The DNA fragment amplified by the PCR method was developed by agarose gel electrophoresis, and it was then purified using QIAquick® Gel Extraction Kit (QIAGEN®). The purified DNA fragment was subcloned into a pCR®-Blunt vector (Invitrogen) (pCRB-hTROP-2 EC), and the gene sequence was confirmed. Subsequently, a EcoRI/XhoI fragment comprising the gene fragment encoding the extracellular region of hTROP-2 was cut out of the pCRB-hTROP-2 EC, and it was then inserted into the EcoRI/XhoI site of a pcDNA4/myc-His© A vector (Invitrogen) (pcDNA4mH-hTROP-2 EC). Further, in order to produce a NruI restriction enzyme cleavage site, the following oligonucleotides were associated and inserted into the BamHI/EcoRI site of the pcDNA4mH-hTROP-2 EC.
Likewise, a pBgl II linker (TAKARA) was inserted into the PmeI site of the pcDNA4mH-hTROP-2 EC (pcDNA4mH-NB-hTROP-2 EC). In order to produce a recombinant protein using baculovirus, a NruI/BglII fragment comprising the gene fragment encoding the extracellular region of hTROP-2 was cut out of the pcDNA4mH-NB-hTROP-2 EC, and it was then inserted into the NruI/BglII site of a pPSC8 vector (Nosan Corporation) (pPSC8-hTROP-2 EC). The production of the recombinant protein of the extracellular region of hTROP-2 using baculovirus was delegated to Nosan Corporation.
The recombinant protein of the extracellular region of hTROP-2 was purified as follows. Ni Sepharose 6 Fast Flow (GE Healthcare Biosciences) was added to a culture supernatant comprising the recombinant protein, so that they were allowed to bind to each other at 4° C. for 2 hours. Thereafter, the resultant was washed with a phosphate buffer containing 20 mM imidazole, employing EconoColumn (BIO RAD), and it was then eluted with a phosphate buffer containing 300 mM imidazole, so that it was purified.
A full-length human EpCAM gene was isolated from human fetal liver (10-week-old embryo) according to an RT-PCR method. First, the following PCR primers were designed based on the sequence of a human EpCAM gene (Genbank accession No. NM_002354).
When these primers were designed, a XhoI restriction enzyme-digested sequence except for a stop codon was added to the reverse primer. cDNA was synthesized from total RNA (TAKARA) from human fetal liver (10-week-old embryo). Using this cDNA as a template, a PCR reaction was carried out with the aforementioned primers. Thereafter, development by agarose gel electrophoresis and extraction of a band of interest were carried out, and it was then cloned into a pCRII vector (Invitrogen) (pCRII-hEpCAM). The cloned human EpCAM cDNA was confirmed by sequencing.
An expression vector was constructed by cleaving a EcoRI/XhoI fragment comprising a human EpCAM gene from pCRII-hEpCAM, and then inserting the fragment into the EcoRI/XhoI site of a pcDNA4/myc-His© A vector (Invitrogen) (pcDNA4-hEpCAM-myc/His). Moreover, a HindIII/PmeI fragment comprising a human EpCAM gene was cut out of pcDNA4-hEpCAM-myc/His (wherein the HindIII cleavage portion was blunt-ended), and the fragment was then inserted into the PmeI site of a pcDNA3.1(+) vector (Invitrogen), so as to construct an expression vector comprising a neomycin resistance gene (pcDNA3.1-hEpCAM-myc/His).
As immunogens, there were used cell lines capable of stably expressing hTROP-2 (HEK293-hTROP-2 cells, CHO-K1-hTROP-2 cells and 7E2-C-hTROP-2 cells); human pancreatic cancer cell line endogenously expressing an hTROP-2 protein on the cell surface (PK-59, RCB1901; purchased from RIKEN cell bank); and the recombinant protein of the extracellular region of hTROP-2 produced by the above-described method.
In the case of the cell lines capable of stably expressing hTROP-2, 1×107 cells were used, and in the case of the recombinant hTROP-2 protein, 20 μg of the protein was used. The cell lines or the recombinant protein was mixed with an adjuvant TiterMax Gold (Funakoshi Corporation) at a mixing ratio of 1:1, so as to prepare an emulsion. The emulsion was then injected into the two footpads or abdominal cavity of a mouse (C57/BL6, Balb/c) (initial immunization). When immunization was carried out by injection into the two footpads for a short period of time, booster was carried out three to ten days after the initial immunization. On the day following the final immunization, lymph nodes were collected from both knees, and lymphocytes were then prepared. When immunization was carried out by injection into the abdominal cavity for a long period of time, boosters were carried out at intervals of once a week after the initial immunization (wherein boosters were carried out for 1 to 2 months). Thereafter, B cells were isolated from the spleen according to an ordinary method. In the case of immunization using cells as immunogens, a cell suspension which was PBS containing 5×106 cells was used for boosters. In the case of using a protein as an immunogen, 5 μg of a PBS solution was used.
The prepared lymphocytes were mixed with a mouse myeloma cell line (P3-X63-Ag8.653) at a mixing ratio of 3:1, and cell fusion was then carried out according to a polyethylene glycol method. Thereafter, the fused cells were cultured for 7 to 28 days in a methyl cellulose medium (trade name: ClonaCell-HY Cloning Medium D; Stem Cell), which contained HAT (hypoxanthine, aminopterin and thymidine). Single colonies of growing hybridomas were each picked up and placed on a 96-well flat-bottom plate, and using a liquid selective medium containing HAT, the hybridomas were cultured in a 5% CO2 incubator. A culture supernatant of growing hybridomas from single colonies was subjected to a primary screening via Cell ELISA (described later) and then to a secondary screening via FACS analysis using HuH-7-hTROP-2 cells, PK-59, thereby establishing 300 types of hybridomas, which produce anti-hTROP-2 monoclonal antibodies recognizing hTROP-2 proteins expressed on the cell surface of living cells.
CHO-K1 cells (hTROP-2 negative control; purchased from Japan Health Sciences Foundation) and CHO-K1-hTROP-2 cells (or HUH-7 cells (hTROP-2 negative control; purchased from Japan Health Sciences Foundation) and HuH-7-hTROP-2 cells) were alternately inoculated on a 96-well culture plate (BD Falcon) at a cell density of 3×104 cells/well, and the cells were then cultured in a 5% CO2 atmosphere at 37° C. for 1 to 2 days. The cell culture medium was removed by decantation. Thereafter, the cells were washed with ice-cold PBS, and were then treated with 4% paraformaldehyde-PBS for 5 minutes, so that the cells were immobilized. The cells were washed with PBS which had been cooled on ice, and an ELISA plate was then prepared. Thereafter, ELISA was carried out according to an ordinary method. Specific procedures will be described below.
First, blocking with a 2% skim milk-PBS solution was carried out at room temperature for 30 minutes to 1 hour. Subsequently, the hybridoma culture supernatant was added thereto, and they were then reacted at room temperature for 1 hour. Thereafter, the resultant was washed with a 0.1% Tween20-PBS solution three times. As a secondary antibody, Horseradish peroxidase (HRP)-labeled anti-mouse IgG (GE Healthcare Biosciences), which had been 1000 times diluted with a blocking solution, was added to the resultant, and they were then reacted at room temperature for 1 hour. Thereafter, the resultant was washed with a 0.1% Tween20-PBS solution three times. A TMB (3,3′,5,5′-tetramethylbenzidine: SIGMA) substrate solution was added to the reaction solution to carry out a color reaction, and the reaction was then terminated by adding 1 M sulfuric acid. Thereafter, absorbance (405 nm) was measured using Microplate reader Model 550 (BIO RAD). Hybridomas corresponding to a hybridoma culture supernatant exhibiting a high absorbance value to the negative control were subjected to a large-scale culture on a 24-well flat-bottom plate, and were then subjected to a secondary screening using FACS analysis.
Hybridomas, which were found positive in the above-described primary screening using Cell ELISA, were subjected to a secondary screening using FACS analysis. In the evaluation of Hybridoma cells, HuH-7 cells, which were human liver cancer cells which did not express hTROP-2, were used as negative control cells and the reactivity with HuH-7-hTROP-2 cells, which were stably expressing hTROP-2, was used as an indicator. Then, the evaluation was carried out based on the reactivity with PK-59 cells (RCB1901; purchased from RIKEN cell bank), which were human pancreatic cancer cells endogenously expressing an hTROP-2 protein on the cell surface.
The cells were removed from the culture dish by a trypsin treatment, and a cell suspension was then prepared (cell density: 2×106 cells/mL). The hybridoma culture supernatant, which exhibited positive in the primary screening using Cell ELISA, was reacted with 100 μL of the cell suspension at 4° C. for 20 minutes. The reaction mixture was washed with PBS, and it was then reacted with PE-labeled mouse IgG (BD Pharmingen) (0.1 μg) (4° C., 30 minutes). Thereafter, the reaction mixture was analyzed using FACSCalibur (Becton, Dickinson and Company).
Eventually, approximately 300 types of hybridomas, which produce an anti-hTROP-2 monoclonal antibody recognizing an hTROP-2 protein expressed on the cell surface of living cells, were established.
The isotype of the produced anti-hTROP-2 monoclonal antibody was identified using MOUSE MONOCLONAL ANTIBODY ISOTYPING TEST KIT (Serotec) in accordance with a method included with the above-mentioned kit.
The hybridoma clones produced by the above-described method were administered at a density of 3×106 clones into the abdominal cavity of a BALB/c nude mouse, to which 2,6,10,14-tetramethylpentadecane (pristane) had previously (seven days before) been administered. Two weeks later, ascites was collected. Moreover, this ascites was subjected to caprylic acid precipitation, and then to affinity purification using a protein G column (HiTrap protein G; GE Healthcare Biosciences) or a protein A column (HiTrap protein A; GE Healthcare Biosciences), so as to obtain anti-hTROP-2 monoclonal antibodies from individual hybridoma clones.
The antigen binding affinity (Kd value) of the generated anti-hTROP-2 monoclonal antibody was calculated by a method using ELISA (Djavadi-Ohaniance L. et al (1996), In Antibody Engineering, Chapter 4, pp. 77-97. IRL Press, Oxford).
Specifically, the purified recombinant hTROP-2 protein (0.1 μg/mL) was added to a 96-well culture plate (Corning) so that the plate was coated with the antigen (at room temperature for 1 hour, or at 4° C. overnight). Subsequently, the resultant was washed with PBS three times, and 2% skim milk (PBS solution) was then added thereto to block it (at room temperature for 1 hour). The resultant was washed with PBS twice. Thereafter, an antigen-antibody complex which had previously been formed by mixing an antigen solution (a purified hTROP-2 protein; 50, 25, 12.5, 6.25, or 3.125 nM) with each clone (0.5 nM) of the anti-hTROP-2 monoclonal antibody and then equilibrating the mixture, was added to the above-described ELISA plate, and they were reacted (at room temperature for 1 hour). The reaction product was washed with PBS three times, and it was then reacted with HRP-labeled anti-mouse IgG (final concentration: 1 μg/mL) (GE Healthcare Biosciences) diluted with a blocking solution (at room temperature for 1 hour). Subsequently, the reaction product was washed with a 0.1% Tween20-PBS solution three times, and a TMB (3,3′,5,5′-tetramethylbenzidine: SIGMA) substrate solution was then added to the resultant to carry out a color reaction. Then, 1 M sulfuric acid was added to the reaction product to terminate the reaction. Using Microplate reader Model 550 (BIO RAD), absorbance was measured.
The following calculation expressions were used to measure dissociation constant (Kd).
In accordance with the law of mass action, an antigen-antibody reaction is represented by the following expressions.
In the expression (2), Agf represents the concentration of a free antigen, Abf represents the concentration of a free antibody, and Ag-Ab represents the concentration of an antigen-antibody complex. If Ag-Ab=x, the free antibody concentration is represented by the following expression.
Abf=Abt−x (3)
The above expression (2) therefore can be
Kd=Agf×(Abt−x)/x (4)
If both terms of the expression (4) are multiplied by x/Kd×Abt,
x/Abt=Agf×(1−x/Abt)×1/Kd
x/Abt×1/Agf=(1−x/Abt)×1/Kd (5)
If X=x/Abt and Y=x/Abt×Agf in the expression (5),
Y=(1−X)×1/Kd (6)
Based on the expression (6), the Kd value was calculated.
The Kd values of the generated 300 anti-hTROP-2 monoclonal antibody clones were measured by the above-described method. As a result, there were 133 clones exhibiting a Kd value of 1×10−10 (M) or less, 59 clones exhibiting a Kd value of 1×10−11(M) or less, and 2 clones exhibiting a Kd value of 1×10−12 (M) or less.
Among the anti-hTROP-2 monoclonal antibodies, which exhibited tumor growth inhibitory activity in vivo, the Kd values of K5-70 (mouse IgG2a), T6-16 (mouse IgG2a), K5-107 (mouse IgG1), K5-116-2-1 (mouse IgG2a) and T5-86 (mouse IgG1) were found to be 6.8×10−12(M), 4.3×10−12 (M), 4.7×10−12 (M), 2.69×1041 (M) and 8.49×10−11 (M), respectively (
The human cancer cell lines (human tumor cell lines) used in this studies were acquired from Health Science Research Resources Bank (HSRRB), RIKEN cell bank (RIKEN), ATCC (American Type Culture Collection), ECACC (European Collection of Cell Cultures) and DSMZ (German Collection of Microorganisms and Cell Cultures). Specifically, the following cancer cell lines were used.
huH-1 (HSRRB), HUH-6 (HSRRB), HuH-7 (HSRRB), JHH-5 (HSRRB), JHH-6 (HSRRB), JHH-7(HSRRB), HLE (HSRRB), HLF (HSRRB), HepG2 (HSRRB), Alexander (HSRRB), KP-1N (HSRRB), KP-1NL (HSRRB), KP-2 (HSRRB), KP-3 (HSRRB), KP-3L (HSRRB), PK-1 (RIKEN), PANC-1 (RIKEN), MIA PaCa-2 (HSRRB), PK-59 (RIKEN), PK-45H (RIKEN), PK-45P (RIKEN), BxPC-3 (ATCC), SUIT-2 (HSRRB), TCC-PAN2 (HSRRB), SW480 (ATCC), DLD-1 (HSRRB), LoVo (HSRRB), COLO-320 (RIKEN), CACO-2 (RIKEN), CW-2 (RIKEN), HCT 116 (ATCC), HCC-56 (HSRRB), MCF-7 (HSRRB), JIMT-1 (DSMZ), HCC1143 (ATCC), A549 (HSRRB), DU145 (RIKEN) and PC-3 (HSRRB).
Cancer cells were removed from a culture dish by a trypsin treatment, and a cell suspension was then prepared (cell density: 2×106 cells/mL). An anti-hTROP-2 monoclonal antibody (0.1 μg) was added to 100 μL of the cell suspension, and they were then reacted at 4° C. for 20 minutes. The reaction solution was washed with PBS, and it was then reacted with PE-labeled anti-mouse IgG (BD Biosciences Pharmingen) (0.1 μg) (at 4° C. for 30 minutes). Thereafter, the resultant was analyzed by FACSCalibur (Becton, Dickinson and Company).
All of the generated anti-hTROP-2 antibodies did not bind to a human liver cancer cell line HuH-7, which did not endogenously express hTROP-2. On the other hand, the anti-hTROP-2 antibodies bound to HuH-7-hTROP-2 cells, in which an hTROP-2 gene was stably expressed (
With regard to human cancer cell lines other than PK-59 and BxPC-3, among 12 types of pancreatic cancer cell lines, the anti-hTROP-2 monoclonal antibodies bound to KP-2, KP-3L, PK-1, PK-45H, SUIT-2 and TCC-PAN2, and did not bind to KP-1N, KP-1NL, KP-3, PANC-1 and MIA-PaCa2 (
For the purpose of examining the specificity of the generated anti-hTROP-2 monoclonal antibodies, the reactivity of the antibodies with a mouse TROP-2 protein showing homology of 80% at the amino acid sequence level with the hTROP-2 protein, and with a human TROP-1/EpCAM protein showing homology of 50% at the amino acid sequence level with the hTROP-2 protein, was examined by FACS analysis.
Specifically, each of an expression vector (mouse TROP-2-pcDNA3.1(+), furnished by the Institute of Molecular and Cellular Biosciences, the University of Tokyo) comprising the full-length cDNA of a mouse TROP-2 gene (GenBank accession No. NM_020047, Y08830), and an expression vector (pcDNA3.1-hEpCAM-myc/His) comprising the full-length cDNA of a human TROP-1/EpCAM gene (GenBank accession No. NM_002354), was transiently introduced into CHO-K1 cells, using Lipofectamine2000 reagent (Invitrogen). Thereafter, 24 to 48 hours later, the cells were removed from a culture dish by treating them with trypsin, and a cell suspension was then prepared. The thus prepared cell suspension was successively reacted with the produced anti-hTROP-2 monoclonal antibody (0.1 μg) and with PE-labeled anti-mouse IgG, and it was then analyzed by FACSCalibur.
A T2-102 antibody (mouse IgG1) used as a positive control, which showed cross-reactivity with mouse TROP-2, exhibited high binding ability to the CHO-K1 cells in which the mouse TROP-2 gene was transiently expressed. On the other hand, K5-70, T6-16, K5-107, K5-116-2-1 and T5-86 antibodies did not show such cross-reactivity with mouse TROP-2 (
Similarly, an anti-human EpCAM monoclonal antibody (BD Biosciences Pharmingen) used as a positive control exhibited high binding ability to the CHO-K1 cells in which the human EpCAM/TROP-1 was transiently expressed. On the other hand, K5-70, T6-16, K5-107, K5-116-2-1 and T5-86 antibodies did not show such cross-reactivity with human EpCAM/TROP-1 (
The aforementioned results demonstrated that the generated anti-hTROP-2 monoclonal antibodies, and in particular, K5-70, T6-16, K5-107, K5-116-2-1 and T5-86 antibodies, which exhibited anti-tumor activity in vivo, specifically bound to hTROP-2.
As a method of examining the activity of the anti-hTROP-2 monoclonal antibody to inhibit the function of hTROP-2, the influence of the antibody on the cell growth of human cancer cells, which endogenously express hTROP-2 on the cell surface, was evaluated by measuring the number of living cells using TetraColor ONE (Seikagaku Corporation). Specifically, PK-59 cells were suspended in an RPM11640 medium containing 0.5% fetal bovine serum (manufactured by BioWest) at a cell concentration of 2×105 cells/mL, and 100 μL of the prepared cell suspension was then added to each well of a 96-well culture plate. Subsequently, mouse IgG (negative control) and anti-hTROP-2 monoclonal antibodies (final concentrations: 0.1 and 1 μg/mL) were added to the wells, and the mixtures were then cultured at 37° C. in a 5% CO2 incubator for 72 hours. As a control, a commercially available anti-hTROP-2 monoclonal antibody (clone YY01, Santa Cruz) was used. TetraColor ONE (Seikagaku Corporation) was added to the wells, and they were then reacted in a 5% CO2 incubator for 1 to 2 hours. After completion of the reaction, the 96-well culture plate was directly subjected to the measurement of absorbance at a wavelength of 490 nm (control wavelength: 655 nm), using Microplate Reader. The experiment was carried out using 3 wells for each group. A significant difference test was carried out according to Student's t-test, and P<0.05 was determined to be statistically significant.
Among anti-hTROP-2 monoclonal antibodies, which had been generated by our own company so far, approximately 160 clones were examined by the above-described method, in terms of their effect on the cell growth of PK-59 cells. As a result, T6-16, T5-86, K5-70 and K5-107, which had exhibited tumor growth inhibitory activity in vivo, were confirmed to have cell growth inhibitory activity of 20% to 40%, when compared with mouse IgG (negative control). It became clear that these anti-hTROP-2 antibodies have activity to bind to hTROP-2 proteins, which were expressed on the surface of human cancer cells, to neutralize the hTROP-2 proteins, and to inhibit the growth of the cancer cells (
The effect of an anti-hTROP-2 monoclonal antibody on the migratory ability of human cancer cells was evaluated by a scratch assay. PK-59 cells were suspended in an RPMI1640 medium containing 10% fetal bovine serum at a cell concentration of 3×105 cells/mL, and 100 μL of the prepared cell suspension was then added to each well of a 96-well culture plate. When the cells became confluent, a portion of the monolayer-cultured cells was peeled, such that the plate was scratched in a longitudinal direction with the end of a tip. An anti-hTROP-2 monoclonal antibody and mouse IgG used as a negative control were added to the medium to final concentrations of 0.1 and 1 μg/mL, respectively, and culture was then carried out for 24 hours. Before addition of the antibody (Day 0) and 24 hours after the culture (Day 1), the cell peeled region was photographed, and the distance between the cells was then measured. Moreover, the area of such a peeled region was quantified using Scion Image software. The experiment was carried out using 8 wells for each group. A significant difference test was carried out according to Student's t-test, and P<0.05 was determined to be statistically significant.
The effect of an hTROP-2 antibody on the migratory ability of the cells invading the scratch region was examined. As with the cell growth inhibition assay, antibodies having beneficial effects were evaluated. As an evaluation method, the cells were photographed on Day 0 (when the antibody was added) and on Day 1 (24 hours after the addition of the antibody), and the migratory distance (m) and the area of a scratch region were determined by image analysis. As a result, as shown in
Prevention Model
Pancreatic cancer cell lines (PK-59 and BxPC-3), which expressed hTROP-2, were harvested by treatment with trypsin, and PBS was added to them to prepare a cell suspension having a concentration of 1×108 cells/mL. The thus prepared cell suspension was mixed with an equal amount of Matrigel (BD Biosciences Pharmingen) on ice. Using a 26 G syringe, 100 μL of the obtained mixture (5×106 cells) was injected into the subcutis of the right flank of each of 6-week-old female nude mouse (Balb/c, nu/nu). On the day of the transplantation of the cancer cells (Day 1), the mice were divided into groups, and administration of the antibody (1, 5 or 10 mg/kg body weight, intraperitoneal administration) was initiated. Thereafter, administration of the antibody was continued at intervals of once every three days. Anti-tumor activity was evaluated based on tumor formation frequency and tumor volume. The tumor volume was calculated by the following formula.
Tumor volume (mm3)=(minor axis)2×(major axis)×π/6
Treatment Model
Pancreatic cancer cell lines (PK-59 and BxPC-3), which expressed hTROP-2, were harvested by treatment with trypsin, and PBS was added to them to prepare a cell suspension having a concentration of 1×108 cells/mL. The thus prepared cell suspension was mixed with an equal amount of Matrigel (BD Biosciences Pharmingen) on ice. Using a 26 G syringe, 100 μL of the obtained mixture (5×106 cells) was injected into the subcutis of the right flank of each of 6-week-old female nude mouse (Balb/c, nu/nu). Five to six days after the transplantation of the cancer cells, mice whose tumor volume had increased to 50 to 150 mm3 (mean value: approximately 100 mm3) were divided into groups. The day on which the mice were divided into groups was defined as a first day (Day 1), and administration of the antibody was initiated. The antibody was intraperitoneally administered at intervals of once every three days (10 mg/kg body weight). Anti-tumor activity was evaluated by measuring tumor volume. A significant difference test was carried out according to Student's t-test, and P<0.05 was determined to be statistically significant.
It is essential for an antibody used for the treatment of cancer, which targets hTROP-2, to have the activity of specifically killing tumor tissues expressing hTROP-2 or inhibiting the growth of tumor in a xenograft model.
Anti-hTROP-2 monoclonal antibodies (approximately 160 clones), which were newly produced in the present invention, were evaluated using the xenograft treatment models of a pancreatic cancer cell line PK-59. The PK-59 cells express, on the surface thereof, EpCAM (
Most of the newly generated approximately 160 clones did not exhibit beneficial effects on the xenograft treatment models of PK-59 cells. Among such clones, clones exhibiting significant tumor growth inhibitory activity, namely clones K5-70, T6-16, K5-107, T5-86 and K5-116-2-1 could be obtained.
In a clone K5-70 (mouse IgG2a) administration group, tumor growth rate is statistically significantly inhibited. On the 21st day after initiation of the administration (day 21), the tumor volume of a control group (N=14) was 1200.8±377.3 mm3, whereas the tumor volume of the clone K5-70 administration group was 748.7±175.0 mm3 (P<0.01 by Student's t-test) (
Similarly, tumor growth rate was statistically significantly inhibited even in a clone K5-107 (mouse IgG1) administration group (N=8), a clone T6-16 (mouse IgG2a) administration group (N=8), a clone T5-86 (mouse IgG1) administration group and a clone K5-116-2-1 (mouse IgG2a) administration group (N=8). On the 17th day after initiation of the administration (Day 17), the tumor volumes of the clone K5-107 administration group (N=8) and the clone T6-16 administration group (N=8) were 698.2±175.9 mm3 (P<0.05 by Student's t-test) and 707.2±254.5 mm3 (P<0.05 by Student's t-test), respectively, whereas the tumor volume of the control group was 1039.3±271.6 mm3. Likewise, on the 16th day after initiation of the administration (Day 16), the tumor volume of the clone K5-116-2-1 administration group (N=8) was 508.5±225.2 mm3 (P<0.05 by Student's t-test), whereas the tumor volume of the control group (N=8) was 797.0±172.9 mm3 (
On the other hand, in the case of the clone T5-86, on the 15th day after initiation of the administration (Day 15), the tumor of the clone T5-86 administration group (N=8) was 744.1±289.1 mm3, whereas the tumor volume of the control group (N=8) was 1033.2±319.4 mm3. Thus, there was found no significant difference in terms of tumor volume. However, in the comparison of tumor weight, which was performed on the same day, the tumor weight of the clone T5-86 administration group was 0.44±0.13 g (P<0.05 by Student's t-test), whereas the tumor weight of the control group was 0.62±0.14 g. Thus, the clone T5-86 exhibited significant inhibitory activity.
Moreover, in terms of both tumor volume and tumor weight, the ratio (T/C) of each clone antibody administration group to the control group on the final day of experiment is shown in Table 2 below. As shown in Table 2, each clone antibody exhibited significant inhibitory activity (T/C=62% to 72%) on each clone antibody administration group.
Furthermore, the anti-tumor activity of each of the clones K5-70, T6-16 and K5-116-2-1 on the xenograft prevention models of the pancreatic cancer cell line PK-59 was analyzed. After completion of the administration of each antibody clone, tumor growth was inhibited in all individuals (N=8). On the 18th day after initiation of the administration (Day 18), the tumor volume of the clone K5-70 administration group (10 mg/kg body weight) was 62.4±80.4 mm3 (P<0.01 by Student's t-test), whereas the tumor volume of the control group (N=8) was 880.8±206.4 mm3. Thus, the clone K5-70 exhibited tumor growth inhibitory activity of 92.9%. On the 28th day after initiation of the administration (Day 28), the tumor volume of the clone T6-16 administration group (10 mg/kg body weight) was 152.14±122.3 mm3 (P<0.01 by Student's t-test), whereas the tumor volume of the control group (N=8) was 992.3±250.8 mm3. Thus, the clone T6-16 exhibited tumor growth inhibitory activity of 84.6%. On the 20th day after initiation of the administration (Day 20), the tumor volume of the clone K5-116-2-1 administration group (10 mg/kg body weight) was 207.7±319.2 mm3 (P<0.01 by Student's t-test), whereas the tumor volume of the control group (N=8) was 1159.4±413.3 mm3. Thus, the clone K5-116-2-1 exhibited tumor growth inhibitory activity of 82.1% (
In terms of both tumor volume and tumor weight, the ratio (T/C) of each clone antibody administration group to the control group on the final day of experiment is shown in Table 3 below. As shown in Table 3, significant tumor growth inhibition was observed in each clone antibody administration group, and in particular, a significant effect such as T/C=10% or less was confirmed in the clone K5-70 administration group.
The known anti-TROP-2 antibody AR47A6.4.2 (U.S. Pat. No. 7,420,041) has exhibited the effect of inhibiting tumor growth, at a dosage of 20 mg/kg, on xenograft prevention models using various human cancer cell lines. This anti-TROP-2 antibody AR47A6.4.2 has inhibited the tumor growth of a human pancreatic cancer cell line PL45 at a percentage of almost 100%. However, this antibody has had the effect of inhibiting tumor on a pancreatic cancer cell line BxPC-3 at a percentage of approximately 50%, on a prostate cancer cell line PC-3 at a percentage of approximately 40%, on a breast cancer cell line MCF-7 at a percentage of approximately 60%, and on a colon cancer cell line Colo205 at a percentage of approximately 40%. In contrast, the anti-hTROP-2 antibody of the invention of the present application has exhibited a higher tumor growth inhibitory effect at a dosage of half the aforementioned dose (10 mg/kg body weight).
As in the case of using the above-described xenograft treatment models of the human pancreatic cancer cell line PK-59, the anti-tumor activity of the clone K5-70 on xenograft prevention models and xenograft treatment models of a human pancreatic cancer cell line BxPC-3 was analyzed.
When compared with a control group (N=8), the tumor growth of the clone K-70 administration group was significantly inhibited. On the 52nd day (Day 52), the tumor volume of the clone K5-70 administration group (N=8) was 236.0±136.4 mm3, whereas the tumor volume of the control group (N=8) was 616.3±266.8 mm3. Thus, the clone K-70 exhibited a tumor growth inhibitory effect of 61.7% (P<0.01 by Student's t-test) (
From the aforementioned results, it became clear that the anti-hTROP-2 monoclonal antibody exhibits significant tumor growth inhibitory activity in vivo on at least two cancer cell species.
For the purpose of analyzing more in detail the tumor growth inhibitory activity in vivo of the anti-hTROP-2 antibody, a dose-dependent test was carried out. As shown in
Preparation of Human/Mouse Chimeric TROP-2 Protein
A human/mouse TROP-2 gene was prepared according to a PCR method. PCR primers as shown below were designed based on a human TROP-2 gene sequence and a mouse TROP-2 gene sequence (Genbank accession No. NM 020047).
To the mouse TROP-2 reverse primer, a XhoI restriction enzyme-digested sequence except for a stop codon was added. A schematic view of the prepared human/mouse chimeric TROP-2 proteins is shown in
The hmTROP-2-A chimeric protein is a chimeric protein, which consists of a polypeptide ranging from the N-terminus to the amino acid at position 69 of the hTROP-2 protein and a polypeptide ranging from the amino acid at position 64 to the C-terminus of the mouse TROP-2 protein. The hmTROP-2-B chimeric protein is a chimeric protein, which consists of a polypeptide ranging from the N-terminus to the amino acid at position 101 of the hTROP-2 protein and a polypeptide ranging from the amino acid at position 96 to the C-terminus of the mouse TROP-2 protein. The hmTROP-2-C chimeric protein is a chimeric protein, which consists of a polypeptide ranging from the N-terminus to the amino acid at position 145 of the hTROP-2 protein and a polypeptide ranging from the amino acid at position 140 to the C-terminus of the mouse TROP-2 protein. The mhTROP-2-D chimeric protein is a chimeric protein, which consists of a polypeptide ranging from the N-terminus to the amino acid at position 139 of the mouse TROP-2 protein and a polypeptide ranging from the amino acid at position 146 to the C-terminus of the hTROP-2 protein. The mhTROP-2-E chimeric protein is a chimeric protein, which consists of a polypeptide ranging from the N-terminus to the amino acid at position 187 of the mouse TROP-2 protein and a polypeptide ranging from the amino acid at position 194 to the C-terminus of the hTROP-2 protein. The mhTROP-2-F chimeric protein is a chimeric protein, which consists of a polypeptide ranging from the N-terminus to the amino acid at position 227 of the mouse TROP-2 protein and a polypeptide ranging from the amino acid at position 234 to the C-terminus of the hTROP-2 protein.
Expression vectors used in the preparation of the above-described chimeric proteins were specifically constructed by the following methods. In order to prepare an hmTROP-2-A chimeric gene, the hTROP-2 gene was used as a template, and PCR was carried out using the hTROP-2 forward primer and the human/mouse TROP-2-A primer Y613. Likewise, the mouse TROP-2 gene was used as a template, and PCR was carried out using the human/mouse TROP-2-A primer Y612 and the mouse TROP-2 reverse primer. A DNA fragment amplified by the PCR was developed using acrylamide gel, and a band of interest was then recovered by extraction. Subsequently, the extracted two types of DNA fragments were mixed to prepare a template, and PCR was then carried out using the hTROP-2 forward primer and the mouse TROP-2 reverse primer. A PCR product was developed by agarose gel electrophoresis, and a DNA fragment of interest was then extracted. The extracted DNA fragment was cloned into a pCR®-Blunt vector (Invitrogen) (pCRB-hmTROP-2-A), and a gene sequence was then confirmed. An expression vector for animal cells was produced by removing the hTROP-2 gene from pcDNA3.1-hTROP-2-myc/His by EcoRI/XhoI digestion, and then inserting therein an EcoRI/XhoI fragment containing an hmTROP-2-A chimeric gene prepared from pCRB-hmTROP-2-A (pcDNA3.1-hmTROP-2-A-myc/His). Additionally, the following chimeric genes were prepared by the same method as described above, and expression vectors were constructed: hmTROP-2-B (using a human TROP-2 forward primer, a human/mouse TROP-2-B primer Y615, a human/mouse TROP-2-B primer Y614 and a mouse TROP-2 reverse primer), hmTROP-2-C (using a human TROP-2 forward primer, a human/mouse TROP-2-C primer Y607, a human/mouse TROP-2-C primer Y606 and a mouse TROP-2 reverse primer), mhTROP-2-D (using a mouse TROP-2 forward primer, a mouse/human TROP-2-D primer Y609, a mouse/human TROP-2-D primer Y608 and a human TROP-2 reverse primer), mhTROP-2-E (using a mouse TROP-2 forward primer, a mouse/human TROP-2-E primer Y617, a mouse/human TROP-2-E primer Y616 and a human TROP-2 reverse primer), mhTROP-2-F (using a mouse TROP-2 forward primer, a mouse/human TROP-2-F primer Y619, a mouse/human TROP-2-F primer Y618 and a human TROP-2 reverse primer) (pcDNA3.1-hmTROP-2-B-myc/His, pcDNA3.1-hmTROP-2-C-myc/His, pcDNA3.1-mhTROP-2-D-myc/His, pcDNA3.1-mhTROP-2-E-myc/His, and pcDNA3.1-mhTROP-2-F-myc/His).
Establishment of HEK293 Cell Lines, which Constitutively Express hTROP-2, Human/Mouse TROP-2-C and Mouse/Human TROP-2-D Chimeric Proteins
The above-described expression vectors pcDNA3.1-hTROP-2-myc/His, pcDNA3.1-hmTROP-2-C-myc/His and pcDNA3.1-mhTROP-2-D-myc/His were each introduced into HEK293 cells. Selection was carried out using an antibiotic G418 (Calbiochem), and HEK293 cell lines constitutively expressing the hTROP-2 protein, the hmTROP-2-C chimeric protein and the mhTROP-2-D chimeric protein were established.
The binding regions of the anti-hTROP-2 monoclonal antibodies K5-70, T5-86, K5-107, T6-4, T6-16 and K5-116-2-1, which had exhibited beneficial effects on the xenograft treatment models of the pancreatic cancer cell line PK-59, were identified. First, the reactivity of the anti-hTROP-2 monoclonal antibodies exhibiting beneficial effects with HEK293 cells, which constantly express the chimeric proteins hmTROP-2-C and mhTROP-2-D, was examined by FACS analysis (
In order to analyze the binding regions more in detail, vectors used in the expression of hmTROP-2-A, hmTROP-2-B, mhTROP-2-E and mhTROP-2-F chimeric proteins were prepared, and the reactivity of the chimeric proteins with the anti-hTROP-2 monoclonal antibodies exhibiting beneficial effects was analyzed (
<Materials/Method>
The following normal and cancer tissue arrays were used in immunohistostaining.
Human Normal Tissue Arrays:
Human, normal organs in duplicates (Catalog No.: AB1, Super Bio Chips)
Normal tissues more than single spots (Catalog No.: A103(VI), ISU ABXIS)
Lung Cancer Tissue Arrays:
Human lung cancer-metastasis-normal (Catalog No.: CCA3, Super Bio Chips)
Human lung carcinoma tissue with margin tissue, 2 location cores (Catalog No.: OD-CT-RsLug03-002, Shanghai Outdo Biotech) Pancreatic cancer tissue array:
Human pancreas carcinoma tissue with mono-pathological type from 60 cases, 2 location cores (Catalog No.: OD-CT-DgPan03-001, Shanghai Outdo Biotech)
Liver Cancer Tissue Arrays:
Hepatocellular carcinoma, grades I to III with normal tissue controls, 63 cases tissue arrays (Catalog No.: CS03-01-002U, Cybrdi)
Human liver carcinoma tissue with mono-pathological type from 30 cases, 2 location cores (Catalog No.: OD-CT-DgLiv02-002, Shanghai Outdo Biotech)
Colorectal Cancer Tissue Arrays:
Human, colorectal cancer (Catalog No.: CD3, Super Bio Chips)
Human colon carcinoma with margin tissue, 2 location cores (Catalog No.: OD-CT-DgCo103-002, Shanghai Outdo Biotech)
Colorectal Cancer Lymph Node Metastasis and Liver Metastasis Tissue Arrays:
Colorectal (colon and rectum) cancer with matched lymph node metastasis tissue array, 44 cases/99 cores, trial slide (Catalog No.: C0991t, Biomax us)
Colorectal (colon and rectum) cancer with matched lymph node metastasis and normal adjacent tissue array, 43 cases/99 cores (Catalog No.: C0992, Biomax us) Colon cancer tissues Jiver metastasis (Catalog No.: A203(IV), ISU ABXIS)
Breast Cancer Tissue Arrays:
Human, breast cancer-metastasis-normal (Catalog No.: CBA3, Super Bio Chips)
Human breast carcinoma with margin tissue, 2 location cores (Catalog No.: OD-CT-RpBre03-002, Shanghai Outdo Biotech)
Stomach Cancer Tissue Arrays:
Human, stomach cancer (Catalog No.: CQ1, Super Bio Chips)
Human gastric carcinoma with margin tissue, 2 location cores (Catalog No.:
OD-CT-DgStm03-002, Shanghai Outdo Biotech)
Esophagus Cancer Tissue Array:
Human, esophagus cancer (Catalog No.: CR1, Super Bio Chips) Human esophagus carcinoma with margin tissue, 2 location cores (Catalog No.: OD-CT-DgEso03-002, Shanghai Outdo Biotech)
Ovarian Cancer Tissue Array:
Human, ovary cancer (Catalog No.: CJ1, Super Bio Chips)
Prostate Cancer Tissue Array:
Human, prostate cancer-normal (Catalog No.: CA3, Super Bio Chips)
Bladder Cancer Tissue Array:
Bladder carcinoma/transitional cell carcinoma, grades I to III with normal tissue arrays (Catalog No.: CC12-01-001U, Cybrdi)
Patient information and clinical information regarding the above-described tissue arrays were obtained from data sheets attached herewith and the homepages of individual companies.
Immunohistostaining Method
After completion of a deparaffinization treatment, the tissue array slides of human normal tissues and cancer tissues were subjected to a protease treatment with pepsin at 37° C. for 5 minutes. Thereafter, the sections were used in immunostaining using an anti-hTROP-2 monoclonal antibody. A color reaction was carried out using DAB(3,3′-diaminobenzidine) as a substrate, and as a counter staining, nuclear staining was then carried out using hematoxylin.
More specifically, these operations were carried out as follows. A paraffin-embedded section was subjected to a deparaffinization treatment, and was then subjected to a protease treatment with pepsin (DAKO) at 37° C. for 5 minutes. After activation of the antigen, the section was treated at room temperature for 20 minutes using a solution prepared by adding a hydrogen peroxide solution to methanol to a final concentration of 0.3%, so that endogenous peroxidase activity was eliminated. The resultant was washed with PBS at room temperature for 5 minutes twice, and it was then blocked at room temperature for 30 minutes using a PBS solution containing 1.5% normal horse serum (DAKO), so as to carry out an operation to block non-specific binding in the tissues. Subsequently, the resultant was reacted with anti-hTROP-2 monoclonal antibody clone K5-63-17 (final concentration: 10 μg/ml), which had been diluted with a PBS solution containing 1.5% normal horse serum, at room temperature for 1 hour, and was then washed with PBS at room temperature for 5 minutes three times. Thereafter, a biotinylated anti-mouse IgG antibody (Vector), which had been 200 times diluted with a PBS solution containing 1.5% normal horse serum, was reacted at room temperature for 30 minutes. The reaction product was washed with PBS at room temperature for 5 minutes three times, and a reagent of Vectastain ABC kit (Vector) was mixed in accordance with the instruction manual included therewith, so as to prepare an ABC complex. This ABC complex was reacted at room temperature for 30 minutes. The reaction product was washed with PBS at room temperature for 5 minutes three times, and color development was then carried out using Histofine Peroxidase Substrate Simple Stain DAB solution (Nichirei Biosciences). After completion of the color development, the reaction product was washed with deionized water for 5 minutes, and the nucleus was stained with Mayer's hematoxylin solution (Wako Pure Chemical Industries, Ltd.). Thereafter, dehydration was carried out with alcohol, followed by penetration with xylene and mounting in Entellan New (Merck Japan).
<Results>
Expression of hTROP-2 in Human Normal Tissues
The expression pattern of hTROP-2 in human normal tissues was analyzed using the anti-hTROP-2 monoclonal antibody clone K5-63-17. A human normal tissue array (Catalog No.: AB1, Super Bio Chips) was deparaffinized, and was then subjected to a hydrophilic treatment. Thereafter, the antigen was activated with a protease, pepsin, and immunostaining was then carried out using the anti-hTROP-2 monoclonal antibody clone K5-63-17 (
Expression of hTROP-2 in Human Cancer Tissues
In order to examine the expression of hTROP-2 (hTROP-2-positive rate) in human cancer tissues, the cancer tissue arrays of various human cancer species were immunostained using the anti-hTROP-2 monoclonal antibody clone K5-63-17. A tissue section, in which 10% or more of cancer cells were stained, was defined as hTROP-2-positive. The staining results are shown in Table 4.
Representative stained images are shown in
The hTROP-2-positive rate in pancreatic cancer was 41.9%. The relationship between this hTROP-2-positive rate and the grade (degree of differentiation) of pancreatic cancer was analyzed. As a result, hTROP-2 was expressed at high frequency in pancreatic cancer with a high grade, namely, with a low degree of differentiation (Table 5).
Strong anti-tumor activity in vivo of a clone K5-70 (mouse IgG2a) was exhibited even by a single administration of K5-70 at a dosage of 10 mg/kg body weight to xenograft prevention models using a human pancreatic cancer cell line PK-59. In a control group (mouse IgG, 10 mg/kg body weight, N=3), tumor formation was observed in all of the individuals, and the tumor volume on the 28th day after cell transplantation (Day 28) was 781.7±74.5 mm3. On the other hand, in a group in which the K5-70 antibody was administered only once on the day of transplantation of the cancer cells (Day 1) (10 mg/kg body weight, N=3), the tumor volume on Day 28 was 144.4±176.9 mm3 (P<0.05 by Student's t-test), showing tumor growth inhibitory activity of 81.5% (
The anti-tumor activity of each of anti-hTROP-2 monoclonal antibodies (clones K5-70, K5-116-2-1, and T6-16) was examined with xenograft treatment models using a human colon cancer cell line SW480. SW480 cells (5×106 cells) were subcutaneously transplanted into the right flank of each of 6-week-old female NOD-scid mouse (Day 1). When the mean tumor volume reached 100 mm3, grouping was carried out (Day 7 or Day 10). From Day 7 or Day 10, intraperitoneal administration of the antibody was carried out at administration intervals of once every three days. The anti-tumor activity of the clone K5-70 antibody and the anti-tumor activities of the clone K5-116-2-1 antibody and the clone T6-16 antibody were evaluated by independent studies, separately. In the study of evaluating the anti-tumor activity of the K5-70 antibody, the tumor volume of a control group (mouse IgG (10 mg/kg body weight), N=8) on the 44th day after cancer cell transplantation (Day 44) was 365.4±214.6 mm3. On the other hand, the tumor volume of a K5-70 antibody (10 mg/kg body weight) administration group was 27.4±29.4 mm3 (P<0.01 by Student's t-test), and thus, tumor formation was significantly inhibited in the K5-70 administration group (inhibitory rate: 92.5%) (
In the study of evaluating the anti-tumor activities of the K5-116-2-1 antibody and the T6-16 antibody, which was carried out separately, the tumor volume of the control group on Day 42 was 713.8±354.5 mm3 (N=8). In contrast, the tumor volume of the K5-116-2-1 antibody administration group (10 mg/kg body weight) was 188.9±97.4 mm3 (N=8, P<0.01 by Student's t-test) (
Subsequently, the dose-dependent anti-tumor activity of the K5-70 antibody was examined with xenograft treatment models using a human colon cancer cell line SW480. SW480 cells (5×106 cells) were subcutaneously transplanted into the right flank of each of 6-week-old female NOD-scid mouse. Ten days after the transplantation (Day 10) at which the mean tumor volume reached 100 mm3, the mice were divided into a control group (mouse IgG, 10 mg/kg body weight administration group, N=8, 104.4±17.6 mm3), a K5-70 antibody (1 mg/kg body weight) administration group (N=8, 104.3±16.1 mm3), a K5-70 antibody (5 mg/kg body weight) administration group (N=8, 104.6±15.9 mm3), and a K5-70 antibody (10 mg/kg body weight) administration group (N=8, 104.8±14.9 mm3). Then, intraperitoneal administration was carried out at administration intervals of once every three days. On Day 42, the tumor volume of the control group was 713.8±354.5 mm3. On the other hand, in the K5-70 antibody administration groups, dose-dependent tumor formation inhibitory activity was observed. That is, the tumor volume of the 1 mg/kg body weight administration group was 485.0±207.3 mm3 (inhibitory rate: 32.1%), the tumor volume of the 5 mg/kg body weight administration group was 339.5±253.2 mm3 (inhibitory rate: 52.4%), and the tumor volume of the 10 mg/kg body weight administration group was 355.4±202.8 mm3 (inhibitory rate: 50.2%, P<0.05 by Student's t-test) (
Subsequently, in order to analyze optimal administration intervals of the K5-70 antibody, the anti-tumor activity of the clone K5-70 when it was administered once a week (once every 7 days) was examined with xenograft treatment models using a human colon cancer cell line SW480. SW480 cells (5×106 cells) were subcutaneously transplanted into the right flank of each of 6-week-old female NOD-scid mouse. Ten days after the transplantation (Day 10) at which the mean tumor volume reached 100 mm3, the mice were divided into a control group (mouse IgG, 10 mg/kg body weight, N=8, 104.42±15.1 mm3) and a K5-70 antibody administration group (10 mg/kg body weight, N=8, 104.3±16.1 mm3) Then, intraperitoneal administration was carried out once every 7 days. On Day 42, the tumor volume of the control group was 713.8±354.5 mm3, whereas the tumor volume of the K5-70 antibody administration group (administered once a week) was 332.3±239.9 mm3 (inhibitory rate: 55%, P<0.05 by Student's t-test) (
Subsequently, the dose-dependent anti-tumor activity of the T6-16 antibody was examined with xenograft treatment models using a human colon cancer cell line SW480. SW480 cells (5×106 cells) were subcutaneously transplanted into the right flank of each of 6-week-old female NOD-scid mice. Ten days after the transplantation (Day 10) at which the mean tumor volume reached 100 mm3, the mice were divided into a control group (mouse IgG, 10 mg/kg body weight, N=8, 105.8±9.9 mm3), a T6-16 antibody administration group (1 mg/kg body weight, N=8, 104.4±13.3 mm3), a T6-16 antibody administration group (5 mg/kg body weight, N=8, 104.7±13.0 mm3), and a T6-16 antibody administration group (10 mg/kg body weight, N=8, 104.8±12.4 mm3). Then, intraperitoneal administration was carried out at administration intervals of once every three days. On Day 43, the tumor volume of the control group was 473.5±137.0 mm3. On the other hand, in the T6-16 antibody administration groups, dose-dependent tumor formation inhibitory activity was observed. That is, the tumor volume of the 1 mg/kg body weight administration group was 397.9±97.5 mm3 (inhibitory rate: 16.0%), the tumor volume of the 5 mg/kg body weight administration group was 195.9±89.7 mm3 (inhibitory rate: 58.7%, P<0.01 by Student's t-test), and the tumor volume of the 10 mg/kg body weight administration group was 190.2±56.5 mm3 (inhibitory rate: 59.8%, P<0.01 by Student's t-test) (
Subsequently, in order to analyze optimal administration intervals of the T6-16 antibody, the anti-tumor activity of the clone T6-16 when it was administered at administration intervals of once a week (once every 7 days) and once every 10 days was examined with xenograft treatment models using a human colon cancer cell line SW480. SW480 cells (5×106 cells) were subcutaneously transplanted into the right flank of each of 6-week-old female NOD-scid mouse. Ten days after the transplantation (Day 10) at which the mean tumor volume reached 100 mm3, the mice were divided into a control group (mouse IgG, 10 mg/kg body weight, N=8, 105.8±9.9 mm3), a T6-16 antibody (once a week) administration group (10 mg/kg body weight, N=8, 105.0±11.6 mm3), and a T6-16 antibody (once every 10 days) administration group (10 mg/kg body weight, N=5, 130.8±2.4 mm3). Then, administration was initiated. On Day 43, the tumor volume of the control group was 473.5±137.0 mm3. On the other hand, the tumor volume of the T6-16 antibody (once a week) administration group was 243.7±65.3 mm3 (inhibitory rate: 48.5%, P<0.01 by Student's t-test), and the tumor volume of the T6-16 antibody (once every 10 days) administration group was 297.8±54.4 mm3 (inhibitory rate: 37.1%, P<0.05 by Student's t-test) (
The anti-tumor activity of clone K5-70 on human prostate cancer was evaluated with xenograft prevention models using DU-145 cells (RIKEN Cell Bank, RCB2143). DU-145 cells (5×106 cells) were subcutaneously transplanted into each of 6-week-old female nude mouse (Balb/c, nu/nu). The day on which the transplantation was carried out was defined as Day 1. The mice were divided into a control group (mouse IgG) (N=8) and a K5-70 antibody administration group (N=8). From Day 1, the K-70 antibody and the control antibody were intraperitoneally administered to the mice at a frequency of once every 3 days at a dosage of 10 mg/kg body weight. On Day 40, the tumor volume of the control group was 368.2±307.8 mm3. On the other hand, the tumor volume of the K5-70 antibody administration group was 30.6±29.6 mm3 (P<0.05 by Student's t-test), showing a tumor formation inhibitory activity of approximately 90% (
Cancer metastasis is an important factor that influences clinical prognosis in the treatment of gastrointestinal cancer. The control of metastasis is therapeutically significantly important. If not only tumor formation but also the metastasis of cancer to other organs could be suppressed by administering an antibody for use in cancer therapy that targets to TROP-2, high clinical usefulness would be anticipated. Thus, this is a desired property as a cancer therapeutic antibody.
The expression of TROP-2 was confirmed in many types of carcinomas. It was reported that TROP-2 was expressed at a high level particularly in metastatic foci (Br. J. Cancer (2008); 99: 1290-1295, Clin. Cancer Res. (2006); 12: 3057-3063, Mod. Pathol. (2008); 21: 186-191). Moreover, it was also reported that, when Trop-2-gene-introduced cancer cells were transplanted into nude mice via transsplenic or transpancreastic administration, the incidence of liver metastasis increased (WO 2010/089782, Molecular Cancer (2010); 9: 253), and thus, the report suggested the importance of TROP-2 in the cancer metastasis process. However, to date, there have been no reports specifically describing that an antibody that targets TROP-2 has metastasis-inhibitory action in vivo.
The anti-hTROP-2 mouse monoclonal antibody K5-70, which was discovered by the present invention, exhibits high therapeutic effects on xenograft models, into the subcutis of which human pancreatic cancer cells had been transplanted. It was demonstrated by a scratch assay performed in vitro that the antibody K5-70 is able to suppress the migration ability of a human pancreatic cancer cell line PK-59, in addition to the effect of suppressing the growth of cancer cells. Thus, it was considered that the antibody K5-70 could inhibit cancer metastasis in vivo. Hence, the metastasis-inhibitory effect of an anti-hTROP-2 mouse monoclonal antibody was examined, using models in which the PK-59 cells were injected into the spleen of nude mice so that liver metastasis was developed.
On the day before cancer cell transplantation, the mice were divided into groups. Then, an anti-hTROP-2 monoclonal antibody (K5-70) or a control antibody (purified mouse IgG) was intraperitoneally administered to the mice at a dosage of 10 mg/kg body weight. On the following day, a human pancreatic cancer cell line (PK-59) endogenously expressing hTROP-2 was harvested by treatment with trypsin, and a 2×107 cells/mL cell suspension was then prepared with PBS. The cell suspension was preserved on ice until transplantation. Each of 6- or 7-week-old female nude mouse (Balb/c, nu/nu) was anesthetized by intraperitoneal administration of pentobarbital, and 10 to 15 mm of the left flank thereof was excised under anesthesia. The spleen was taken out of the abdominal cavity, and 50 μL of the cell suspension (1×106 cells) was then injected into the spleen using a 27G syringe. Four minutes after injection of the cells, the hilum of spleen was ligated with 5-0 silk sutures, and the spleen was then excised. The cut peritoneum was closed with 4-0 silk sutures, and the surgical site was then closed with Wound Clips (AUTOCLIP 9 mm, Becton Dickinson). In addition, seven days after the cancer cell transplantation, the K-70 antibody and the control antibody were administered to the mice at a dosage of 10 mg/kg body weight. Four to six weeks after the cancer cell transplantation, the mice were subjected to euthanasia by cervical dislocation. Then, the liver was excised from each mouse, and the presence or absence of metastatic foci was confirmed.
In the control group in which the mouse IgG was administered to the mice, in 4 out of the 6 mice into which PK-59 cells had been transplanted, apparent metastatic foci (2 to 7 foci) were observed around the liver lobe four to six weeks after the transplantation (
From these results, it became clear that the anti-hTROP-2 antibody K5-70 has extremely strong inhibitory action on the liver metastasis of the human pancreatic cancer cell line PK-59.
In recent years, many chemotherapeutical drugs for suppressing the growth of cancer cells have been developed as cancer therapeutic drugs. These drugs have achieved certain treatment results. However, these chemotherapeutical drugs have been problematic in terms of side effects associated with the growth suppressive action thereof on normal cells other than cancer cells and the recurrence of cancer after suspension of the treatment. Accordingly, if tumor recurrence after completion of the treatment with chemotherapeutical drugs could be suppressed by administration of a cancer therapeutic antibody targeting to TROP-2, high clinical usefulness would be anticipated. Thus, this is a desired property as a cancer therapeutic antibody.
As therapeutic agents for colorectal cancer, in addition to 5-FU and, platinum-containing drugs, irinotecan hydrochloride (Topotecin, Daiichi Sankyo Co., Ltd.) having a topoisomerase inhibitory effect has been recently applied to clinical sites. With regard to animal models as well, the anti-tumor effect of irinotecan hydrochloride on mouse models, into which various types of human tumor cells including colon cancer as a typical example had been transplanted, has been reported (Cancer Chemother Pharmacol. (1991); 28(3):192-8). Thus, the recurrence-preventing effect of the anti-hTROP-2 antibody clone K5-70 (mouse IgG2a) on recurrent tumor after administration of irinotecan hydrochloride has been examined with xenograft models using a human colon cancer cell line SW480. SW480 cells (5×106 cells) were subcutaneously transplanted into the right flank of 8-week-old female NOD-scid mice. Eleven days after the transplantation (Day 11) at which the mean tumor volume reached 100 mm3, the mice were divided into a non-treat group (normal saline administration group, N=8, 130.7±16.2 mm3) and an irinotecan hydrochloride (CPT-11, Topotecin, Daiichi Sankyo Co., Ltd.) administration group (N=16, 123.0±21.4 mm3). Thereafter, irinotecan hydrochloride was intraperitoneally administered to the mice at a dosage of 40 mg/kg body weight, once every 3 days, total 3 times (Days 11, 14, and 17). On the third day after the final administration of irinotecan hydrochloride (Day 20), the tumor volume of the non-treat group reached 232.1±21.1 mm3. On the other hand, the tumor volume of the irinotecan hydrochloride administration group was 126.6±26.6 mm3 (P<0.01 by Student's t-test), and thus, an apparent tumor-suppressing effect was observed. At this stage, the irinotecan hydrochloride administration group was divided into two groups based on tumor size. One group was defined as a K5-70 antibody administration group (10 mg/kg body weight, N=8, tumor volume on Day 20: 126.0±28.0 mm3), and the other group was defined as a mouse IgG administration group (10 mg/kg body weight, N=8, tumor volume on Day 20: 127.2±27.0 mm3). Intraperitoneal administration of the antibodies and the measurement of a tumor volume were carried out on each group once every 3 days, so that the recurrence of a tumor was evaluated (
<Materials and Methods>
Peptide Synthesis
15-mer and 30-mer of linear peptides derived from TROP-2 extracellular domains, which were used in the present experiment, were obtained by solid-phase synthesis according to a Fmoc (9-Fluorenylmethoxycarbonyl) method. In addition, for discontinuous epitope analysis, a 17-mer peptides derived from a TROP-2 extracellular domain, to both ends of which cysteine residues had been added, was synthesized, and a conformation having one or two loop structures was reconstructed by CLIPS technology (Chemically Linked Peptides on Scaffolds technology). When another cysteine residue was present close to the added cysteine residue, it was substituted with alanine.
Epitope Screening ELISA
5034 types of the synthesized peptides were covalently bound to PEPSCAN cards (455 peptides/card), and the binding of the synthesized peptides to antibodies was then analyzed by the ELISA method. The PEPSCAN cards were allowed to react with anti-human TROP-2 monoclonal antibodies (K5-70, K5-107, K5-116-2-1, T5-86, and T6-16) that had been diluted to a concentration of 1 μg/mL with a blocking buffer (a phosphate buffer containing 4% horse serum, 5% ovalbumin, and 1% Tween). After washing, the resultant was allowed to react with a 1000-fold diluted peroxidase-secondary antibody complex at 25° C. for 1 hour. After washing, a substrate solution (a solution containing 2,2′-azino-di-3-ethylbenzthiazoline sulfonate (ABTS) and 2 μL of 3% hydrogen peroxide solution) was added to the reaction solution, followed by a chromogenic reaction for 1 hour. The binding activity of the antibodies was quantified by photographing with a CCD camera and then performing an image analysis.
<Results>
The anti-hTROP-2 monoclonal antibodies K5-70, K5-107, K5-116-2-1, T5-86 and T6-16, which exhibited beneficial effects, were subjected to epitope analysis using CLIPS (Chemically Linked Peptides on Scaffolds) technology. It is to be noted that the term “amino acid number” is used in the present examples to mean the amino acid number in the amino acid sequence shown in SEQ ID NO: 2 (hTROP-2 protein (323 amino acid residues)).
The result of analysis for the K5-70 antibody is shown in Table 7 below. As a result, it was found that 33 peptides exhibit strong binding activity to the K5-70 antibody. In these 33 peptides, a sequence comprising VCSPDGPGGRCQCRALGSGMAVD (amino acid numbers 43-65 of SEQ ID NO: 2) (peptide Nos. 1-7 and 9 shown in Table 7), a sequence comprising HHILIDLRHRPTAG (amino acid numbers 152-165 of SEQ ID NO: 2) (peptide Nos. 14, 22-24 and 28 shown in Table 7), a sequence comprising VHYEQPTIQIELRQ (amino acid numbers 194-207 of SEQ ID NO: 2) (peptide Nos. 10, 12, 13, 18, 20, 21, 23, 26-28, 30 and 32 shown in Table 7), and a sequence comprising DLDLDAELRRLFRER (amino acid numbers 171-183 of SEQ ID NO: 2) (peptide Nos. 11, 16, 18, 19, 21, 22, 29, 31 and 33 shown in Table 7) appeared repeatedly. The K5-70 antibody particularly strongly bound to the sequence comprising VCSPDGPGGRCQCRALGSGMAVD (amino acid numbers 43-65 of SEQ ID NO: 2). From these results, it was suggested that, in the hTROP-2 protein, the aforementioned 4 types of peptide sequence regions are likely to be epitopes of the K5-70 antibody.
The result of analysis for the K5-107 antibody is shown in Table 8 below. As a result, it was found that a sequence comprising VCSPDGPGGRCQCRALGSGMAVD (amino acid numbers 43-65 of SEQ ID NO: 2) was comprised in 9 out of the 20 peptides (peptide Nos. 1-6, 8, 9 and 17 shown in Table 8) (Table 8).
Accordingly, it was suggested that, in the hTROP-2 protein, the aforementioned peptide sequence region consisting of VCSPDGPGGRCQCRALGSGMAVD (amino acid numbers 43-65 of SEQ ID NO: 2) may be an epitope of the K5-107 antibody.
The result of analysis for the K5-116-2-1 antibody is shown in Table 9 below. In this analysis, three types of peptide sequences, namely, a sequence comprising VCSPDGPGGRCQCRALGSGMAVD (amino acid numbers 43-65 of SEQ ID NO: 2) (peptide Nos. 1-7, and 15 shown in Table 9), a sequence comprising HHILIDLRHRPTAG (amino acid numbers 152-165 of SEQ ID NO: 2) (peptide Nos. 8-11, 16, 20, 22, 24, and 27-28 shown in Table 9), and a sequence comprising DLDAELRRLFRER (amino acid numbers 171-183 of SEQ ID NO: 2) (peptide Nos. 11-13, 17, 21, and 29 shown in Table 9) appeared several times (Table 9). Accordingly, it was suggested that, in the hTROP-2 protein, these three types of peptide sequence regions may be epitopes of the K5-116-2-1 antibody.
The results of analysis for the T5-86 and T6-16 antibodies are shown in Table 10 and Table 11 below, respectively. In these analyses, the antibodies strongly bound to a peptide comprising a sequence consisting of DPEGRFKARQCN (amino acid numbers 109-120 of SEQ ID NO: 2). The above-mentioned peptide sequence was comprised in several peptides binding to the T5-86 antibody as shown in Table 10, and it was comprised in several peptides binding to the T6-16 antibody as shown in Table 11. Moreover, in the analysis regarding the T5-86 antibody, other than the sequence comprising DPEGRFKARQCN (amino acid numbers 109-120 of SEQ ID NO: 2), a sequence comprising VCSPDGPGGRCQCRA (amino acid numbers 43-57 of SEQ ID NO: 2) appeared several times (as shown in Table 10). Furthermore, in the analysis regarding the T6-16 antibody as well, another sequence comprising HHILIDLRHRPTAG (amino acid numbers 152-165 of SEQ ID NO: 2) (shown in Table 11) was found several times. Accordingly, it was suggested that, in the hTROP-2 protein, two types of peptide sequence regions, namely, DPEGRFKARQCN (amino acid numbers 109-120 of SEQ ID NO: 2) and VCSPDGPGGRCQCRA (amino acid numbers 43-57 of SEQ ID NO: 2), may be epitopes of the K5-86 antibody. It was also suggested that, in the hTROP-2 protein, two types of peptide sequence regions, namely, DPEGRFKARQCN (amino acid numbers 109-120 of SEQ ID NO: 2) and HHILIDLRHRPTAG (amino acid numbers 152-165 of SEQ ID NO: 2), may be epitopes of the T6-16 antibody.
Total RNA was extracted from 3×106 mouse anti-TROP-2 monoclonal antibody-producing hybridomas, using TRIzol reagent (Invitrogen). With regard to the clone K5-70, clone K5-107 and clone K5-116-2-1, cDNA was synthesized employing SMARTer™ RACE cDNA Amplification kit (Clontech) according to the method included with the kit, using a mouse IgG H chain-specific primer (5′-TCCAKAGTTCCA-3′ (SEQ ID NO: 24)) and a mouse IgG L chain-specific primer (5′-GCTGTCCTGATC-3′ (SEQ ID NO: 25)). With regard to the clone T6-16, cDNA was synthesized employing GeneRacer kit (Invitrogen) according to the method included with the kit, using an oligo dT primer. Genes encoding the variable regions (VH, VL) of the H and L chains of clone K5-70 (mouse IgG2a), clone K5-107 (mouse IgG1) and clone K5-116-2-1 (mouse IgG2a) were each cloned by a PCR method using the above-synthesized cDNA as a template. In this operation, 10× Universal Primer A Mix (UPM) included with SMARTer™ RACE cDNA Amplification kit was used as a 5′-primer. On the other hand, as a 3′-primer for VH amplification, a primer having a sequence specific to the mouse IgG H chain was used, and as a 3′-primer for VL amplification, a primer having a sequence specific to the mouse IgG L chain was used.
Using each of the above-described primers, PCR was carried out under the following composition of reaction solution and reaction conditions. In addition, a R primer for amplification of VH cDNA was prepared by mixing the two above sequences with each other at an equimolar ratio and was then used.
<Composition of Reaction Solution>
<Reaction Conditions>
A reaction was carried out at 94° C. (10 sec), and thereafter, a cycle consisting of “heat denaturation/dissociation at 98° C. (10 sec)→annealing at 60° C. (5 sec)→synthesis/elongation at 72° C. (60 sec)” was carried out 30 times in total. Finally, a reaction was carried out at 72° C. (3 min).
The synthesized VH and VL cDNAs were subcloned into a pMD20-T vector (Takara Bio Inc.), and the nucleotide sequences thereof were determined. The nucleotide sequences of a plurality of VH clones and VL clones were decoded, and nucleotide sequences specific to the variable regions of mouse H chain and L chain were identified.
Genes encoding the variable regions (VH, VL) of the H and L chains of clone T6-16 were cloned by a PCR method using the above-synthesized cDNA as a template. In this operation, a primer included with GeneRacer kit was used as a 5′-primer. On the other hand, as a 3′-primer for VH amplification, a primer having a sequence specific to the mouse IgG H chain was used, and as a 3′-primer for VL amplification, a primer having a sequence specific to the mouse IgG L chain was used.
Using each of the above-described primers, PCR was carried out under the following composition of reaction solution and reaction conditions.
<Composition of Reaction Solution>
<Reaction Conditions>
A cycle consisting of “heat denaturation/dissociation at 98° C. (10 sec)→annealing at 57° C. (10 sec)→synthesis/elongation at 72° C. (60 sec)” was carried out 35 times in total.
The synthesized VH and VL cDNAs were subcloned into a pCR™4Blunt-TOPO® vector (Invitrogen), and the nucleotide sequences thereof were determined. The nucleotide sequences of a plurality of VH clones and VL clones were decoded, and nucleotide sequences specific to the variable regions of mouse H chain and L chain were identified.
Humanization of the variable regions (VH, VL) of the K5-70 antibody prepared in the previous Examples was carried out as follows according to the method of Queen et al. (Proc. Natl. Acad. Sci. USA 86: 10029-10033, 1989). First, the molecular modeling of the three-dimensional structure of each K5-70 antibody variable region was carried out using a computer. Subsequently, homology search was performed with the variable region sequences of human antibody genes. As a result, a cDNA sequence (DA980102 VH) with GenBank accession number of DA980102 was selected as an acceptor that provides a framework region (FR) necessary for humanization of the K5-70 VH (Genome Res. 16:55-65, 2006). Likewise, a cDNA sequence (L41174 VL) with GenBank accession number of L41174 was selected as an acceptor that provides a framework region (FR) necessary for humanization of the K5-70 VL (J. Biol. Chem. 270:12457-12465, 1995).
For humanization of the K5-70 VH, the CDR sequence of the K5-70 VH was substituted with the corresponding position of the DA980102 VH used as an acceptor. As a result of the analysis of the three-dimensional structure according to computer modeling, with regard to amino acid residues (isoleucine (I) at position 48, lysine (K) at position 66, alanine (A) at position 67, valine (V) at position 71 and threonine (T) at position 93) that are adjacent to the CDR of the K5-70 VH and are assumed to play important roles for the maintenance of the structure, those of the K5-70 VH were retained, and the residual FR region was substituted with the acceptor sequence. The amino acid residue position numbers in VH and VL were used in accordance with the definitions of Kabat et al. (Sequences of Proteins of Immunological Interests, Fifth edition, NIH Publication No. 91-3242, U.S. Department of Health and Human Services, 1991).
Moreover, the methionine (M) as an amino acid residue at position 82 of the acceptor sequence DA980102 VH was highly likely to be a special amino acid residue caused by somatic hypermutation. Thus, in order to decrease potential antigenicity, the aforementioned methionine was substituted with leucine (L) that is most common as an amino acid residue at position 82. An alignment of the amino acid sequences of the thus designed humanized K5-70 VH (HuK5-70 VH), K5-70 VH and DA980102 VH is shown in
With regard to the design of humanized K5-70 VL as well, the same transplantation of a CDR sequence as described above was carried out. As an amino acid residue (lysine (K) at position 49) important for the maintenance of the structure of CDR, that of the K5-70 VL was retained, and the residual FR region was substituted with the acceptor sequence (HuK5-70 VL). An alignment of the amino acid sequences of the HuK5-70 VL, K5-70 VL and L41174 VL is shown in
Humanization of the variable regions (VH, VL) of the T6-16 antibody prepared in the previous Examples was carried out as follows according to the method of Queen et al. (Proc. Natl. Acad. Sci. USA 86: 10029-10033, 1989). First, the molecular modeling of the three-dimensional structure of each T6-16 antibody variable region was carried out using a computer. Subsequently, homology search was performed with the variable region sequences of human antibody genes. As a result, a cDNA sequence (DA935238 VH) with GenBank accession number of DA935238 was selected as an acceptor that provides a framework region (FR) necessary for humanization of the T6-16 VH (Genome Res. 16:55-65, 2006). Likewise, a cDNA sequence (M99608 VL) with GenBank accession number of M99608 was selected as an acceptor that provides a framework region (FR) necessary for humanization of the T6-16 VL (J. Immunol. 149:2518-2529, 1992).
For humanization of the T6-16 VH, the CDR sequence of the T6-16 VH was substituted with the corresponding position of the DA935238 VH used as an acceptor. As a result of the analysis of the three-dimensional structure according to computer modeling, with regard to amino acid residues (isoleucine (I) at position 48, alanine (A) at position 67 and lysine (K) at position 73) that are adjacent to the CDR of the T6-16 VH and are assumed to play important roles for the maintenance of the structure, those of the T6-16 VH were retained, and the residual FR region was substituted with the acceptor sequence (HuT6-16 VH1). In addition, the lysine residue (K) at position 73 was unlikely to have influence on appropriate formation of an antigen-binding site, and thus, an amino acid sequence, in which the lysine residue of the HuT6-16 VH1 was substituted with threonine (T) as a more generous amino acid residue, was designed, separately (HuT6-16 VH2). An alignment of the amino acid sequences of the thus designed humanized T6-16 VH (HuT6-16 VH1 and HuT6-16 VH2), T6-16 VH and DA935238 VH is shown in
With regard to the design of humanized T6-16 VL as well, the same transplantation of a CDR sequence as described above was carried out. Amino acid residues important for the maintenance of the structure of CDR were retained also in the acceptor sequence, and as a FR sequence, the same sequence as the acceptor sequence was used (HuT6-16 VL). An alignment of the amino acid sequences of the HuT6-16 VL, T6-16 VL and L41174 VL is shown in
Genes encoding HuK5-70 VH and HuK5-70 VL were prepared as follows. The gene were synthesized based on an amino acid sequence in which a signal peptide sequence derived from K5-70 VH or VL had been added to the N-terminal side of each of the above-designed HuK5-70 VH and VL (Operon). In this gene synthetic operation, a Kozak sequence (ACC ACC) was added to the 5′-terminal side of the gene sequence of each of the synthesized HuK5-70 VH and HuK5-70 VL. In addition, an EcoRI site (GAA TTC) was added as a restriction enzyme site to the 5′ end of the HuK5-70 VH, and an NheI site (GCT AGC) was added to the 3′ end thereof. Likewise, an AgeI site (ACC GGT) was added as a restriction enzyme site to the 5′ end of the HuK5-70 VL, and a BsiWI site (CGT ACG) was added to the 3′ end thereof. The synthesized HuK5-70 VH gene and HuK5-70 VL gene were incorporated into a pCR2.1 vector (Invitrogen) according to TA cloning. The gene sequences of the HuK5-70 VH and VL prepared by the gene synthesis are shown in
Genes encoding HuT6-16 VH1, HuT6-16 VH2 and HuT6-16 VL were prepared as follows. The gene were synthesized, based on an amino acid sequence in which a signal peptide sequence derived from T6-16 VH had been added to the N-terminal side of each of the above-designed HuT6-16 VH1 and HuT6-16 VH2, and an amino acid sequence in which a signal peptide sequence derived from T6-16 VL had been added to the N-terminal side of the HuT6-16 VL (Operon). In this gene synthetic operation, a Kozak sequence (ACC ACC) was added to the 5′-terminal side of the gene sequence of each of the synthesized HuT6-16 VH1, HuT6-16 VH2 and HuT6-16 VL. In addition, an EcoRI site (GAA TTC) was added as a restriction enzyme site to the 5′ end of each of the HuT6-16 VH1 and HuT6-16 VH2, and an NheI site (GCT AGC) was added to the 3′ end thereof. Likewise, an AgeI site (ACC GGT) was added as a restriction enzyme site to the 5′ end of the humanized T6-16VL, and a BsiWI site (CGT ACG) was added to the 3′ end thereof. The synthesized HuT6-16 VH1 gene, HuT6-16 VH2 gene and HuT6-16 VL gene were incorporated into a pCR2.1 vector (Invitrogen) according to TA cloning.
The gene sequences of the HuT6-16 VH1, HuT6-16 VH2 and HuT6-16 VL prepared by the gene synthesis are shown in
The HuK5-70 VH and VL genes, which had been each incorporated into the pCR2.1 vector (Invitrogen), were digested with the restriction enzymes EcoRI and NheI, and AgeI and BsiWI, respectively, and gene fragments were then recovered. Subsequently, the cleaved HuK5-70 VH gene was inserted into the EcoRI/NheI site of a pFUSE-CHIg-hG1 vector (InvivoGen) as an animal cell expression vector for the expression of a human IgG1 form (pFUSE-CHIg-HuK5-70), whereas the HuK5-70 VL gene was inserted into the AgeI/BsiWI site of a pFUSE2-CLIg-hk vector (InvivoGen) as a human Igκ form expression vector (pFUSE2-CLIg-HuK5-70). Thus, each construct was completed.
The T6-16 VH1 and T6-16 VH2 genes, which had been each incorporated into the pCR2.1 vector (Invitrogen), were digested with the restriction enzymes EcoRI and NheI, and the T6-16 VL gene was digested with the restriction enzymes AgeI and BsiWI. Then, gene fragments were recovered. Subsequently, the cleaved T6-16 VH1 gene was inserted into the EcoRI/NheI site of a pFUSE-CHIg-hG1 vector (InvivoGen) (pFUSE-CHIg-HuT6-16-1), and the T6-16 VH2 gene was also inserted into the EcoRI/NheI site of a pFUSE-CHIg-hG1 vector (pFUSE-CHIg-HuT6-16-2). The T6-16 VL gene was inserted into the AgeI/BsiWI site of a pFUSE2-CLIg-hk vector (InvivoGen) (pFUSE2-CLIg-HuT6-16). Thus, each construct was completed.
293F cells (Invitrogen) were maintained and cultured in FreeStyle 293 Expression Medium (Invitrogen). Genes were introduced into the 293F cells using a 293 fectin reagent (Invitrogen) in accordance with protocols included therewith. That is, pFUSE-CHIg-HuK5-70 and pFUSE2-CLIg-HuK5-70 were both introduced into the 293F cells, and drug selection was then carried out using Zeocin (InvivoGen) and Blasticidin (InvivoGen), so as to establish a cell line capable of stably expressing a HuK5-70 antibody. Also, pFUSE-CHIg-HuT6-16-1 and pFUSE2-CLIg-HuT6-16 were both introduced into the 293F cells, and the above-mentioned drug selection was then carried out, so as to establish a cell line capable of stably expressing a HuT6-16-1 antibody. Also, pFUSE-CHIg-HuT6-16-2 and pFUSE2-CLIg-HuT6-16 were both introduced into the 293F cells, and the above-mentioned drug selection was then carried out, so as to establish a cell line capable of stably expressing a HuT6-16-2 antibody.
The established antibody-expressing cell lines were each inoculated on FreeStyle 293 Expression Medium (Invitrogen) at a cell density of 1 to 2.5×105 cells/ml, and thereafter, roller bottle culture was carried out for 6 to 8 days. Thereafter, a culture supernatant was recovered, and each humanized antibody was then purified using rProtein A Sepharose Fast Flow (GE Healthcare) according to an ordinary method.
Lane 1 indicates a culture supernatant of 293F cells into which no genes had been introduced (negative control), and lane 2 indicates a culture supernatant of 293F cells into which pFUSE-CHIg-HuK5-70 and pFUSE2-CLIg-HuK5-70 had been introduced. For detection of the heavy chain and light chain proteins of the HuK5-70 antibody, a biotin-labeled anti-human IgG F(ab′)2 antibody (Rockland) was used. Lane 3 indicates a culture supernatant of 293F cells into which pFUSE-CHIg-HuT6-16-1 and pFUSE2-CLIg-HuT6-16 had been introduced, and lane 4 indicates a culture supernatant of 293F cells into which pFUSE-CHIg-HuT6-16-2 and pFUSE2-CLIg-HuT6-16 had been introduced. The heavy chain proteins of the HuT6-16-1 antibody and the HuT6-16-2 antibody were detected with a biotin-labeled anti-human IgG Fc antibody (Rockland), whereas the light chain proteins of the HuT6-16-1 antibody and the HuT6-16-2 antibody were detected with a biotin-labeled anti-human IgG F(ab′)2 antibody (Rockland).
As a result, in all cases of the HuK5-70 antibody, the HuT6-16-1 antibody and the HuT6-16-2 antibody, the expression of heavy chain and light chain proteins was confirmed in each culture supernatant.
Moreover,
The antigen affinity of the purified HuK5-70 antibody, HuT6-16-1 antibody and HuT6-16-2 antibody was examined by methods using FACS and ELISA.
FACS was carried out, using HEK293-hTROP-2 cells in which a full-length human TROP-2 gene was stably expressed in HEK293 cells, and a pancreatic cancer cell line PK-59 which endogenously expressed a human TROP-2 protein on the cell surface. 100 ml of an antibody solution used as a primary antibody, which had been diluted to 1 μg/ml with a medium containing 10% FCS, was added to a suspension (5×105 cells) of cells (HEK293-hTROP-2 cells or PK-59 cells), which had been removed from a culture dish by treatment with trypsin, and the obtained mixture was then incubated at 4° C. for 20 minutes. Thereafter, the resultant was washed with 1 ml of a medium containing 10% FCS. Then, secondary antibody (100 μl each), in which a phycoerythrin (PE)-labeled anti-mouse IgG antibody (BD Pharmingen) or a biotin-labeled anti-human IgG Fc antibody (Rockland) was diluted up to 200 times or 2000 times, respectively, was added to the resultant. The obtained mixture was incubated at 4° C. for 20 minutes, and was then washed with 1 ml of a medium containing 10% FCS again. In a case in which the biotin-labeled anti-human IgG Fc antibody was used as a secondary antibody, 100 μl of a labeling solution, in which streptavidin-labeled PE (BD Pharmingen) was diluted up to 400 times, was added thereto as a fluorescence labeling reagent. Thereafter, the obtained mixture was incubated at 4° C. for 20 minutes, and was then washed with 1 ml of a medium containing 10% FCS. Subsequently, the sample containing labeled cells was suspended in 1 ml of PBS containing 1% FCS and 2 mM EDTA, and the obtained suspension was then analyzed using FACSCalibur (Becton Dickinson). As a result, it was found that the HuK5-70 antibody showed reactivity equivalent to that of a mouse K5-70 antibody both in the HEK293-hTROP-2 cells and in the PK-59 cells. Likewise, the HuT6-16-1 antibody and the HuT6-16-2 antibody showed reactivity equivalent to that of a T6-16 antibody (
Furthermore, antigen affinity was also examined by an ELISA method. ELISA was carried out using an ELISA plate which was coated with the recombinant protein of the hTROP-2 extracellular region as described in Example 3. Specifically, a 96-well plate (BD FALCON) was coated with a 50 μl/well recombinant protein of a hTROP-2 extracellular region that had been diluted with PBS to 0.5 μg/ml (at 4° C. overnight). Thereafter, the resultant was washed with a washing buffer (PBS containing 0.05% Tween 20), and a blocking buffer (PBS containing 2% skim milk and 0.05% Tween 20) was then added thereto (200 μl/well) to block it (at room temperature for 1 hour). The resultant was washed with a washing buffer. Thereafter, a HuK5-70 antibody, a HuT6-16-1 antibody, a HuT6-16-2 antibody, a K5-70 antibody and a T6-16 antibody were diluted with an ELISA buffer (PBS containing 1% skim milk and 0.025% Tween 20) in a concentration range from 3.05×10−4 to 5 μg/ml, so as to prepare a series of two-fold dilution samples. The obtained dilution samples were each added in an amount of 50 μl/well to the above-described ELISA plate (at room temperature for 2 hours). The reaction product was washed with a washing buffer, and thereafter, a HRP-labeled goat anti-human κ chain antibody (SouthernBiotech) or a HRP-labeled sheep anti-mouse IgG antibody (GE Healthcare), each of which had been diluted to 2000 times with an ELISA buffer, was added (50 μl/well) as a detection antibody to the reaction product (at room temperature for 1 hour). After the mixture had been washed with a washing buffer, a TMB (3,3′,5,5′-tetramethylbenzidine: SIGMA) substrate solution was added (50 μl/well) to the resultant to carry out a color reaction. Then, 1 M sulfuric acid (25 μl/well) was added to the reaction product to terminate the reaction. Using Microplate reader Model 550 (BioRad), an absorbance at 450 nm was measured with an absorbance at 655 nm used as a reference. As a result, the reaction curves of K5-70 and HuK5-70 were almost overlapped with each other, and the EC50 values thereof were 27 ng/ml and 22 ng/ml, respectively (
Subsequently, the anti-tumor activity of a HuK5-70 antibody in vivo was examined with xenograft treatment models using a human colon cancer cell line SW480, which endogenously expresses human TROP-2 on the cell surface. SW480 cells (5×106 cells) were subcutaneously transplanted into the right flank of each of 7-week-old female NOD-scid mice (Day 1). When the mean tumor volume reached 100 mm3, grouping was carried out (Day 9). From Day 9, intraperitoneal administration of the antibody was carried out at administration intervals of once every three days. On the 39th day after the cancer cell transplantation (Day 39), the tumor volume of a control group (PBS administration, N=8) was 824.3±188.8 mm3. On the other hand, the tumor volume of a HuK5-70 antibody administration group (10 mg/kg body weight, N=8) was 455.5±208.6 mm3 (P<0.01 by Student's t-test), and thus, tumor formation was significantly inhibited in the HuK5-70 antibody administration group (inhibitory rate: 44.7%) (
The dose-dependent anti-tumor activity of HuK5-70 and HuT6-16-2 antibodies was examined with xenograft treatment models using a human colon cancer cell line SW480. SW480 cells (5×106 cells) were subcutaneously transplanted into the right flank of each of 7-week-old female NOD-scid mice (Day 1). On the 9th day after the cancer cell transplantation (Day 9) at which the mean tumor volume reached 100 mm3, the mice were divided into a control group (PBS administration group, N=8, 101.65±8.35 mm3), a 1 mg/kg body weight HuK5-70 antibody administration group (N=8, 103.18±9.86 mm3), a 5 mg/kg body weight HuK5-70 antibody administration group (N=8, 101.34±8.94 mm3), a 10 mg/kg body weight HuK5-70 antibody administration group (N=8, 101.53±8.98 mm3), a 1 mg/kg body weight HuT6-16-2 antibody administration group (N=8, 103.18±9.86 mm3), a 5 mg/kg body weight HuT6-16-2 antibody administration group (N=8, 101.34±8.94 mm3), and a 10 mg/kg body weight HuT6-16-2 antibody administration group (N=8, 101.53±8.98 mm3) Then, from Day 9, intraperitoneal administration of each antibody was carried out at administration intervals of once every three days. On the 48th day after the cancer cell transplantation (Day 48), the tumor volume of the control group was 754.67±276.05 mm3. On the other hand, in the HuK5-70 antibody administration groups, the tumor volume of the 1 mg/kg body weight administration group was 521.81±183.45 mm3 (inhibitory rate: 30.9%), the body volume of the 5 mg/kg body weight administration group was 258.78±137.02 mm3 (inhibitory rate: 65.7%, P<0.01 by Student's t-test), and the tumor volume of the 10 mg/kg body weight administration group was 314.60±152.89 mm3 (inhibitory rate: 58.3%, P<0.01 by Student's t-test) (
The anti-tumor activity of K5-70 and T6-16 antibodies as parent antibodies in vivo was examined with xenograft treatment models using a human ovarian cancer cell line SK-OV-3, which endogenously expresses hTROP-2 on the cell surface. SK-OV-3 cells (5×106 cells) were subcutaneously transplanted into the right flank of each of 7-week-old female nude mice (Day 1). On the 11th day after the cancer cell transplantation (Day 11), individuals mice, in which clear tumor formation was observed (mean tumor volume: approximately 50 mm3), were divided into groups. Then, from Day 11, intraperitoneal administration of each antibody was carried out at administration intervals of twice a week. On the 56th day after the cancer cell transplantation (Day 56), the tumor volume of a control group (PBS administration, N=8) was 652.6±349.1 mm3. On the other hand, the tumor volume of a K5-70 antibody administration group (10 mg/kg body weight, N=8) was 253.7±137.3 mm3 (P<0.01 by Student's t-test), and thus, tumor formation was significantly inhibited (inhibitory rate: 61.1%); and the tumor volume of a T6-16 antibody administration group (10 mg/kg body weight, N=8) was 214.6±98.6 mm3 (P<0.01 by Student's t-test), and thus, tumor formation was significantly inhibited (inhibitory rate: 67.1%) (
Likewise, the anti-tumor activity of K5-70 and T6-16 antibodies in vivo was examined with xenograft treatment models using a human breast cancer cell line MDA-MB-468, which endogenously expresses hTROP-2 on the cell surface. MDA-MB-468 cells (5×106 cells) were subcutaneously transplanted into the right flank of each of 7-week-old female nude mice (Day 1). On the 12th day after the cancer cell transplantation (Day 12), individuals mice, in which clear tumor formation was observed (mean tumor volume: approximately 50 mm3), were divided into groups. Then, from Day 12, intraperitoneal administration of each antibody was carried out at administration intervals of twice a week. On the 54th day after the cancer cell transplantation (Day 54), the tumor volume of a control group (PBS administration, N=8) was 218.6±75.5 mm3. On the other hand, the tumor volume of a K5-70 antibody administration group (10 mg/kg body weight, N=8) was 70.2±37.4 mm3 (P<0.01 by Student's t-test), and thus, tumor formation was significantly inhibited (inhibitory rate: 67.9%); and the tumor volume of a T6-16 antibody administration group (10 mg/kg body weight, N=8) was 88.3±42.9 mm3 (P<0.01 by Student's t-test), and thus, tumor formation was significantly inhibited (inhibitory rate: 59.6%) (
Likewise, the anti-tumor activity of K5-70 and T6-16 antibodies in vivo was examined with xenograft treatment models using a human lung cancer cell line Calu-3, which endogenously expresses hTROP-2 on the cell surface. Calu-3 cells (5×106 cells) were subcutaneously transplanted into the right flank of each of 7-week-old female nude mice (Day 1). On the 9th day after the cancer cell transplantation (Day 9), individuals mice, in which clear tumor formation was observed (mean tumor volume: approximately 100 mm3), were divided into groups. Then, from Day 9, intraperitoneal administration of each antibody was carried out at administration intervals of twice a week. On the 41st day after the cancer cell transplantation (Day 41), the tumor volume of a control group (PBS administration, N=8) was 395.7±221.2 mm3. On the other hand, the tumor volume of a K5-70 antibody administration group (10 mg/kg body weight, N=8) was 120.7±125.6 mm3 (P<0.01 by Student's t-test), and thus, tumor formation was significantly inhibited (inhibitory rate: 69.5%); and the tumor volume of a T6-16 antibody administration group (10 mg/kg body weight, N=8) was 146.3+128.4 mm3 (P<0.05 by Student's t-test), and thus, tumor formation was significantly inhibited (inhibitory rate: 63.0%) (
Likewise, the anti-tumor activity of a K5-70 antibody in vivo was examined with xenograft prevention models using a human bile duct cancer cell line TFK-1, which endogenously expresses hTROP-2 on the cell surface. TFK-1 cells (5×106 cells) were subcutaneously transplanted into the right flank of each of 7-week-old female nude mice (Day 1), and from the same day, intraperitoneal administration of the antibody was initiated at administration intervals of twice a week. On the 31st day after the cancer cell transplantation (Day 31), the tumor volume of a control group (PBS administration, N=5) was 1000.4±268.9 mm3. On the other hand, the tumor volume of a K5-70 antibody administration group (10 mg/kg body weight, N=5) was 197.2±215.5 mm3 (P<0.01 by Student's t-test), and thus, tumor formation was significantly inhibited (inhibitory rate: 80.3%) (
The antigen-binding activity of HuK5-70 and HuT6-16-2 antibodies was examined according to an ELISA method (low-density antigen-coated ELISA) using a 96-well plate on which a low-density antigen has been coated. A recombinant hTACSTD2-Fc-His protein (Creative BioMart), which had been prepared to a concentration of 0.1 μg/mL with a 0.1 M acetate buffer (pH 5.3), was added in an amount of 50 μL/well to a 96-well plate, and coating was carried out at 4° C. overnight. Thereafter, the same analysis as that in Example 40 was carried out. Test antibodies were diluted with an ELISA buffer (PBS containing 1% skim milk and 0.025% Tween 20), so as to prepare samples with a concentration range from 20 μg/mL to a series of two-fold dilutions (15 samples) and to use them. As a result, it was found that the HuT6-16-2 antibody had binding activity that was almost equivalent to that of the T6-16 antibody (wherein their EC50 values were 49 ng/mL and 41 ng/mL, respectively), but that the EC50 value of the HuK5-70 antibody was approximately 20 times higher than that of the K5-70 antibody (wherein their EC50 values were 222 ng/mL and 12 ng/mL, respectively;
Subsequently, the antigen-binding activity of HuK5-70 and K5-70 antibodies was examined by ELISA for analyzing a monovalent antigen-antibody reaction. Goat anti-human IgG (Fcγ specific) (Southern Biotech) which had been diluted to 1 μg/mL with a 0.1 M acetate buffer (pH 5.3) and Goat anti-mouse IgG (γ chain specific) (Southern Biotech) which had been diluted to 3 μg/mL were added in each amount of 50 μL/well to a 96-well plate. Thereafter, coating was carried out at 4° C. overnight. Thereafter, the reaction product was washed with a washing buffer (PBS containing 0.05% Tween 20), and a blocking buffer (PBS containing 2% skim milk and 0.05% Tween 20) was then added thereto (200 μL/well) to block it (at room temperature for 1 hour). The resultant was washed with a washing buffer. Thereafter, the test antibody, which had been diluted to 1 μg/mL with an ELISA buffer (PBS containing 1% skim milk and 0.025% Tween 20), was added in an amount of 50 μL/well to the above-described plate. During this operation, a HuK5-70 antibody was added to a well coated with Goat anti-human IgG (Fcγ specific), and a K5-70 antibody was added to a well coated with Goat anti-mouse IgG (γ chain specific). The mixture was left at rest at room temperature for 1 hour, and the reaction product was then washed with a washing buffer. The recombinant protein of the hTROP-2 extracellular region described in Example 3 (hTROP-2 EC) was diluted with an ELISA buffer to prepare samples with a concentration range from 5 μg/mL to a series of three-fold dilutions (10 samples). The thus obtained sample was added in each amount of 50 μL/well to the plate. The mixture was left at rest at room temperature for 1 hour, and the reaction product was then washed with a washing buffer. Then, anti-His (G-18) (Santa Cruz), which had been diluted to 2 μg/mL with an ELISA buffer, was added as a primary antibody to the reaction product (50 μL/well). The obtained mixture was left at rest at room temperature for 1 hour, and it was then washed with a washing buffer. Thereafter, HRP-labeled anti-rabbit IgG (GE Healthcare), which had been diluted to 1000 times with an ELISA buffer, was added as a secondary antibody to the reaction product (50 μL/well). The obtained mixture was left at rest at room temperature for 1 hour, and it was then washed with a washing buffer. Thereafter, a TMB (3,3′,5,5′-tetramethylbenzidine: SIGMA) substrate solution was added in an amount of 50 μL/well to the resultant to carry out a color reaction. Then, 1 M sulfuric acid (25 μL/well) was added to the reaction product to terminate the reaction. Using Microplate reader Model 550 (BioRad), an absorbance at 450 nm was measured with an absorbance at 655 nm used as a reference. As a result, the EC50 values calculated from the binding curves of the hTROP-2EC protein with the K5-70 and HuK5-70 antibodies were 7 ng/mL and 6 ng/mL, respectively (
As described in Example 40, in an ELISA method using a 96-well plate on which a high-density antigen (0.5 μg/mL) had been coated (antigen-coated ELISA), the antigen affinity of the K5-70 antibody was equivalent to the antigen affinity of the HuK5-70 antibody (
For the purpose of improving the “avidity” of a HuK5-70 antibody, the following experiment was carried out.
Whether the above-mentioned relatively low “avidity” of the HuK5-70 antibody is caused by VH or by VL was examined by the following experiment. First, genes encoding the H chain variable region (K5-70 VH) and L chain variable region (K5-70 VL) of a K5-70 antibody as a parent antibody were prepared by gene synthesis (Operon). During the gene synthesis, a Kozak sequence (ACC ACC) was added to the 5′-terminal side of the gene sequence of each of the K5-70 VH and the K5-70 VL. Further, an EcoRI site (GAA TTC) was added as a restriction enzyme site to the 5′ end of the K5-70 VH, and an NheI site (GCT AGC) was added as a restriction enzyme site to the 3′ end thereof. Likewise, an AgeI site (ACC GGT) was added as a restriction enzyme site to the 5′ end of the K5-70 VL, and a BsiWI site (CGT ACG) was added as a restriction enzyme site to the 3′ end thereof. The thus synthesized K5-70 VH gene and K5-70 VL gene were each incorporated into a pCR2.1 vector (Invitrogen). The gene sequences of the K5-70 VH and VL prepared by the gene synthesis are shown in
The thus prepared constructs, and the HuK5-70 H chain expression vector (pFUSE-CHIg-HuK5-70) and the HuK5-70 L chain expression vector (pFUSE2-CLIg-HuK5-70), which had been prepared in Example 36, were allowed to co-express in 293F cells (Invitrogen) by combinations 1 to 4 in the following table.
Transfection of the expression vectors described above into 293F cells (Invitrogen) was carried out using NeoFection reagent (Astec) in accordance with the method described in instructions included therewith. After completion of the transfection, the resultant was cultured for 5 days using FreeStyle 293 Expression Medium (Invitrogen) at 37° C. in a CO2 incubator with a CO2 concentration of 8%. Thereafter, a culture supernatant was recovered. The antibody concentration in the culture supernatant was measured by a sandwich ELISA method. Specifically, Goat anti-human IgG (Fcγ specific) (Southern Biotech), which had been diluted to 1 μg/mL with PBS, was added in an amount of 50 μL/well to a 96-well plate. Thereafter, coating was carried out at 4° C. overnight. Thereafter, the reaction product was washed with a washing buffer (PBS containing 0.05% Tween 20), and a blocking buffer (PBS containing 2% skim milk and 0.05% Tween 20) was then added thereto (200 μL/well) to block it at room temperature for 1 hour. The resultant was washed with a washing buffer. Thereafter, a culture supernatant, which had been diluted to an appropriate dilution magnification with an ELISA buffer (PBS containing 1% skim milk and 0.025% Tween 20), was added in an amount of 50 μL/well to the above-described plate, and a reaction was then carried out at room temperature for 2 hours. As a standard preparation, a HuK5-70 antibody was used. The reaction product was then washed with a washing buffer. As a detection antibody, HRP-labeled Goat anti-human kappa (κ chain specific) (Southern Biotech), which had been diluted to 1,000 times with an ELISA buffer, was added in an amount of 50 μL/well to the reaction product, and a reaction was then carried out at room temperature for 1 hour. The reaction product was washed with a washing buffer, and thereafter, a TMB (3,3′,5,5′-tetramethylbenzidine: SIGMA) substrate solution was added in an amount of 50 μL/well to the resultant to carry out a color reaction. Then, 1 M sulfuric acid (25 μL/well) was added to the reaction product to terminate the reaction. Using iMark Microplate reader (BioRad), an absorbance at 450 nm was measured with an absorbance at 655 nm used as a reference. The binding of the 4 types of antibodies contained in the culture supernatants to hTROP-2 was measured by the above-described low-density antigen-coated ELISA. As a result, it was found that the binding activity of a MuVH/HuVL antibody constituted with mouse K5-70 VH and HuK5-70 VL to hTROP-2 was equivalent to that of a ChK5-70 antibody, but that the binding activity of a HuVH/MuVL antibody constituted with HuK5-70 VH and mouse K5-70 VL was relatively lower than that of the ChK5-70 (
The prepared 18 types of HuK5-70 VH mutants (expression vectors for pFUSE-CHIg-HuK5-70 mutants) were each combined with a HuK5-70 L chain expression vector (pFUSE2-CLIg-HuK5-70), and the thus obtained expression vectors were each transfected into HEK293 cells. Then, using the obtained culture supernatant, the binding activity of each amino acid substitution antibody to hTROP-2 was examined by low-density antigen-coated ELISA. As a result, among the 18 types of HuK5-70 VH mutants, an R44G mutant, in which the R (arginine) at position 44 of HuK5-70 VH had been substituted with G (glycine) (HuK5-70 R44G; which is hereafter referred to as HuK5-70-2), was observed to have an apparently improved antigen-binding activity (
The HuK5-70-2 antibody that is a HuK5-70 R44G mutant antibody was purified as follows. That is, pFUSE-CHIg-HuK5-70 R44G and pFUSE2-CLIg-HuK5-70 were transfected into 293F cells, and the resultant was then cultured for 5 days. Thereafter, a culture supernatant was recovered. The HuK5-70-2 antibody was purified from the recovered culture supernatant, using rProtein A sepharose Fast Flow (GE Healthcare). The purified HuK5-70-2 antibody was loaded on SDS-PAGE under reducing conditions. As a result, an H chain of approximately 50 kDa and an L chain of approximately 25 kDa were found. The purity of each antibody was 95% or more (
The binding activity of the purified HuK5-70-2 and HuK5-70 antibodies to hTROP-2 was examined by high-density antigen-coated ELISA and low-density antigen-coated ELISA. The affinity of the antibodies was examined by the high-density antigen-coated ELISA (using a 96-well plate which was coated with 1 μg/mL hTROP-2). As a result, it was found that the binding curve of the HuK5-70 antibody was almost overlapped with the binding curve of the HuK5-70-2 antibody, and that their affinity was equivalent to each other. Subsequently, the avidity of the antibodies was examined by the low-density antigen-coated ELISA (using a 96-well plate which was coated with 0.1 μg/mL hTROP-2). As a result, it was found that, as same the case with using a culture supernatant, the antigen-binding activity of the HuK5-70-2 antibody was clearly higher than the antigen-binding activity of the HuK5-70 antibody (
(1) Preparation of Target Cell Solution
As target cells, a human colon cancer cell line SW480, a human pancreatic cancer cell line PK-59, and a human prostate cancer cell line PC-3, each of which endogenously expresses hTROP-2, were used. Target cells cultured on a 10-cm cell culture dish were harvested from the plate by treatment with trypsin, and were then suspended in an assay medium. A Leivovitz L-15 medium (for SW480 cells) or an RPMI-1640 medium (for PK-59 and PC-3 cells), to which 0.5% FBS had been added, was used for ADCC assay. After completion of centrifugation (at 1000 rpm for 3 minutes at room temperature), pellets were prepared at a cell density of 2×105 cells/mL with the same medium as that used above, and thus a target cell solution was prepared.
(2) Separation of Human Peripheral Blood Mononuclear Cells
Healthy venous blood was collected with heparin, and was then diluted to 2 times with PBS. Thereafter, the diluted blood was layered on Lymphoprep (Daiichi Kagaku Yakuhin K. K.) and was then centrifuged (at room temperature at 750 rpm for 5 minutes, and then at 2000 rpm for 20 minutes). After completion of the centrifugation, mononuclear cells (healthy peripheral blood monocytes) were recovered from an intermediate layer fraction, and were then washed with PBS three times. Thereafter, a cell suspension was prepared with an assay medium, and the prepared cells were used as effector cells.
(3) ADCC Activity of Humanized Anti-hTROP-2 Antibodies (HuK5-70, HuK5-70-2 and HuT6-16-2 Antibodies)
100 μL (2×104 cells/well) of the prepared target cell solution was dispensed in a 96-well flat bottom plate (manufactured by FALCON). Subsequently, human peripheral blood mononuclear cells (effector cells) were added to the plate, so that the ratio between the effector cells and the target cells could be 40:1. Thereafter, humanized anti-hTROP-2 antibodies (HuK5-70, HuK5-70-2 and HuT6-16-2 antibodies) were each added as test antibodies to the plate to a final concentration of 0.1 to 30 μg/mL. The mixture was adjusted to a total amount of 200 μL, and it was then cultured in a CO2 incubator (at 37° C. in 5% CO2) for 6 hours. After completion of the culture, the activity of lactate dehydrogenase released from the cytoplasm of the target cells damaged by the effector cells was measured using Cytotoxicity Detection Kit (LDH) (Roche, Cat. No. 11 644 793 001) in accordance with protocols included with the kit, and ADCC activity was then evaluated using the measurement result as an indicator.
As shown in
The present invention is able to provide an antibody, which specifically reacts with hTROP-2 and has high anti-tumor activity in vivo, and specifically, a monoclonal antibody having high anti-tumor activity in vivo at a low dose, and particularly, such an antibody, which is a humanized antibody. In addition, the present invention is able to provide a hybridoma, which produces the antibody, a fragment of the antibody, a complex of the antibody or the like and various types of drugs, a pharmaceutical composition for diagnosing or treating a tumor, a method for detecting a tumor, and a kit for detecting or diagnosing a tumor.
SEQ ID NO: 3 Synthetic DNA
SEQ ID NO: 4 Synthetic DNA
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SEQ ID NO: 33 Synthetic DNA
SEQ ID NO: 74 Recombinant DNA
SEQ ID NO: 75 Synthetic construct (recombinant protein)
SEQ ID NO: 76 Recombinant DNA
SEQ ID NO: 77 Synthetic construct (recombinant protein)
SEQ ID NO: 78 Recombinant DNA
SEQ ID NO: 79 Synthetic construct (recombinant protein)
SEQ ID NO: 80 Recombinant DNA
SEQ ID NO: 81 Synthetic construct (recombinant protein)
SEQ ID NO: 82 Recombinant DNA
SEQ ID NO: 83 Synthetic construct (recombinant protein)
SEQ ID NO: 92 Recombinant protein
SEQ ID NO: 93 Recombinant protein
SEQ ID NO: 94 Recombinant protein
SEQ ID NO: 95 Recombinant protein
SEQ ID NO: 96 Recombinant protein
SEQ ID NO: 97 Recombinant protein
SEQ ID NO: 98 Recombinant protein
SEQ ID NO: 99 Recombinant DNA
SEQ ID NO: 100 Recombinant DNA
The present application claims priority under 35 U.S.C. §119 to U.S. Provisional Patent Application No. 61/562,672, filed Nov. 22, 2011, which is incorporated herein by reference.
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