Patent Application
20110091896
The present invention relates to a method for analyzing/identifying an antibody gene at one cell level in human B cells, a method for producing an antibody derived from one B cell, and a method for preparing an antibody gene derived from one B cell.
In the body of higher vertebrate animals such as human, there is an acquired immune system for protection of life from foreign bodies including externally invading pathogens such as bacteria and viruses, hazardous substances, and cancer cells. An antibody is a protein, referred to as immunoglobulin, capable of specifically distinguishing among substances such as proteins analogous to one another and is responsible for the antigen-specific humoral immune system in a living body. Since the antibody recognizes numerous foreign bodies, genes encoding a part (the variable region) of antibody undergo rearrangement at the DNA level, resulting in the formation of a population of B lymphocytes having diversified antibody gene sequences. The mechanism of gene diversification through the DNA rearrangement is also found in the sexual reproduction process during which a living organism diversifies genes of its progeny and thereby attempts to make the progeny adapted to environmental changes for survival. The individual single B lymphocyte is known to produce always one type of immunoglobulin encoded by one type of antibody gene.
Conventionally known methods for obtaining an antibody gene include a method for obtaining an antigen-specific antibody gene which involves separating human peripheral blood lymphocytes, removing CD11c-specific cells therefrom using a CD11c-positive antibody and magnetic beads, performing in vitro immunization to induce an antigen-specific human antibody production response, using Epstein-Barr virus to immortalize the peripheral blood lymphocyte cells in which the antigen-specific antibody production response has been induced, isolating antigen-specific B cells, extracting RNA from the antigen-specific antibody-producing B cells, synthesizing cDNA from the extracted RNA, and amplifying an antibody variable region gene by PCR using the synthesized cDNA as a template and primers specific to VH and VL (see, e.g., Patent Document 1); and an antibody production method which involves contacting a labeled antigen in which the antigen recognized by a desired antibody is labeled, with a cell population containing target cells producing the antibody to cause the binding of labeled antigen to the target cells, separating the resultant labeled target cells, using the separated labeled target cells to prepare an antibody gene possessed thereby, and expressing the prepared antibody gene using an expression vector (see, e.g., Patent Document 2).
Japanese Patent Laid-Open No. 2004-121237
Japanese Patent Laid-Open No. 2006-180708
Attention has been given in recent years to abnormal clones of immune cells in pathological conditions such as cancer and autoimmune diseases. Given the dynamic adaptation mechanism of immunity, it is easily conceivable that individual cells have different functional characters. From the viewpoint of immune response also, variation in a T-cell receptor or an antibody gene is a phenomenon originally starting from a single cell; functional studies of immune cells are in an era in which studies at one-cell level can no longer be skirted. Particularly, dynamics of individual immune cells under the pathological condition such as cancer have not yet been studied in detail and can be an important study subject in considering a new therapy. The present inventors carried out clinical trials of a dendritic cell vaccine treated with an HLA-A2 or A24 peptide cocktail for cases of metastatic melanoma and have already proposed the analysis and identification of a melanoma peptide-specific antibody gene at one cell level using B cells derived from cancer patients with a confirmed immune response to the cancer-specific antigen peptide (Japanese Patent Application No. 2007-147525). However, this method targeted particular cancer patients in whom a vaccine therapy was established because it was essential in the method to increase B cells producing an antibody to a particular antigen such as a cancer-specific peptide to a certain percentage or more by vaccine administration.
An object of the present invention is to provide a technique for exhaustive analysis of an antibody gene intended for not only samples after vaccine administration but also B cells derived from any cancer-bearing patient, and more specifically to provide a method for analyzing/identifying an antibody gene of one B cell in human and a technique for producing an antibody derived from the one B cell identified, and others.
The present inventors analyzed and identified a melanoma peptide-specific antibody gene at one cell level using immortalized B cells prepared from peripheral blood monocytes derived from melanoma patients before vaccine administration. Specifically, the present inventors have found that the immortalized B cells can be stained with a GST-labeled melanoma-specific cancer antigen MAGE1, an Alexa-labeled anti-GST antibody, and a PE-labeled anti-human IgG antibody and subjected to single cell sorting to separate B cells producing a particular antibody on one cell by one cell basis, and have established a practical technique where a specific antibody gene is efficiently cloned after extracting total RNA from the separated one B cell. In addition, the present inventors considered it important to be able to amplify antibody genes even in normal B cells in view of the diversity of antibody induction, developed a more sensitive technique by improving an RT-PCR technique, and established a practical technique where an antibody gene is efficiently cloned from one B cell using non-immortalized B cells, thereby accomplishing the present invention.
Thus, the present invention relates to [1] a method for analyzing/identifying a gene for an antibody in one B cell derived from a human, successively comprising the steps of (A), (B), (C), (D), (E), (F) and (G): (A) harvesting peripheral blood mononuclear cells from peripheral blood obtained from a human; (B) producing an immortalized B cell (EBV-B cell) line from the obtained peripheral blood mononuclear cells using Epstein-Barr virus (EBV); (C) labeling the EBV-B cells with a marker-labeled antigen and with an antibody which is capable of recognizing a human antibody and is labeled with a marker different from the above marker; (D) separating EBV-B cells, that express an antibody recognizing the antigen on the cell membrane, on one cell by one cell basis; (E) extracting total RNA from the one cell and synthesizing cDNA by reverse transcription reaction; (F) using the synthesized cDNA as a template to perform a PCR reaction using a pair of primers specific for a human antibody heavy chain region gene, a PCR reaction using a pair of primers specific for a human antibody light chain κ region gene, or a PCR reaction using a pair of primers specific for a human antibody light chain λ region gene to amplify each of the region gene fragments; and (G) analyzing/determining the base sequence of the amplified gene fragment, [2] a method for analyzing/identifying a gene for an antibody in one B cell derived from a human, successively comprising the steps of (a), (c), (d), (e), (f) and (g): (a) harvesting peripheral blood mononuclear cells from peripheral blood obtained from a human; (c) labeling B cells included in the obtained peripheral blood mononuclear cells with a marker-labeled antigen and with an antibody which is capable of recognizing a human antibody and is labeled with a marker different from the above marker; (d) separating B cells, that express an antibody recognizing the antigen on the cell membrane, on one cell by one cell basis; (e) extracting total RNA from the one cell and synthesizing cDNA by reverse transcription reaction; (f) using the synthesized cDNA as a template to perform a FOR reaction using a pair of primers specific for a human antibody heavy chain region gene, a FOR reaction using a pair of primers specific for a human antibody light chain κ region gene, or a FOR reaction using a pair of primers specific for a human antibody light chain λ region gene to amplify each of the region gene fragments; and (G) analyzing/determining the base sequence of the amplified gene fragment, and [3] the analyzing/identifying method according to the above [1] or [2], wherein the human is a cancer-bearing patient, [4] the analyzing/identifying method according to any one of the above [1] to [3], wherein the antigen is a cancer-specific antigen peptide or cancer-specific antigen protein, and [5] the analyzing/identifying method according to the above [4], wherein the cancer-specific antigen peptide or cancer-specific antigen protein is MAGE1, MAGE2, MAGE3, MART1, tyrosinase, or gp100.
The present invention also relates to [6] a method for producing an antibody of one B cell derived from a human, successively comprising the steps of (A), (B), (C), (D), (E), (F) and (H): (A) harvesting peripheral blood mononuclear cells from peripheral blood obtained from a human; (B) producing an immortalized B cell (EBV-B cell) line from the obtained peripheral blood mononuclear cells using Epstein-Barr virus (EBV); (C) labeling the EBV-B cells with a marker-labeled antigen and with an antibody which is capable of recognizing a human antibody and is labeled with a marker different from the above marker; (D) separating EBV-B cells, that express an antibody recognizing the antigen on the cell membrane, on one cell by one cell basis; (E) extracting total RNA from the one cell and synthesizing cDNA by reverse transcription reaction; (F) using the synthesized cDNA as a template to perform a PCR reaction using a pair of primers specific for a human antibody heavy chain region gene, a PCR reaction using a pair of primers specific for a human antibody light chain κ region gene, or a PCR reaction using a pair of primers specific for a human antibody light chain λ region gene to amplify each of the region gene fragments; and (H) expressing the amplified gene fragment using an expression vector, [7] a method for producing an antibody of one B cell derived from a human, successively comprising the steps of (a), (c), (d), (e), (f) and (h): (a) harvesting peripheral blood mononuclear cells from peripheral blood obtained from a human; (c) labeling B cells included in the obtained peripheral blood mononuclear cells with a marker-labeled antigen and with an antibody capable of recognizing a human antibody and is labeled with a marker different from the above marker; (d) separating B cells, that express an antibody recognizing the antigen on the cell membrane, on one cell by one cell basis; (e) extracting total RNA from the one cell and synthesizing cDNA by reverse transcription reaction; (f) using the synthesized cDNA as a template to perform a PCR reaction using a pair of primers specific for a human antibody heavy chain region gene, a PCR reaction using a pair of primers specific for a human antibody light chain κ region gene, or a PCR reaction using a pair of primers specific for a human antibody light chain λ region gene to amplify each of the region gene fragments; and (h) expressing the amplified gene fragment using an expression vector, [8] the method for producing an antibody according to the above [6] or [7], wherein the human is a cancer-bearing patient, [9] the method for producing an antibody according to any one of the above [6] to [8], wherein the antigen is a cancer-specific antigen peptide or cancer-specific antigen protein, and [10] the method for producing an antibody according to the above [9], wherein the cancer-specific antigen peptide or cancer-specific antigen protein is MAGE1, MAGE2, MAGE3, MART1, tyrosinase, or gp100.
The present invention further relates to [11] a method for preparing an antibody gene of one B cell derived from a human, successively comprising the steps of (A), (B), (C), (D), (B), (F) and (G): (A) harvesting peripheral blood mononuclear cells from peripheral blood obtained from a human; (B) producing an immortalized B cell (EBV-B cell) line from the obtained peripheral blood mononuclear cells using Epstein-Barr virus (EBV); (C) labeling the EBV-B cells with a marker-labeled antigen and with an antibody which is capable of recognizing a human antibody and is labeled with a marker different from the above marker; (D) separating EBV-B cells, that express an antibody recognizing the antigen on the cell membrane, on one cell by one cell basis; (B) extracting total RNA from the one cell and synthesizing cDNA by reverse transcription reaction; and (F) using the synthesized cDNA as a template to perform a PCR reaction using a pair of primers specific for a human antibody heavy chain region gene, a PCR reaction using a pair of primers specific for a human antibody light chain κ region gene, or a PCR reaction using a pair of primers specific for a human antibody light chain λ region gene to amplify each of the region gene fragments, [12] a method for preparing an antibody gene of one B cell derived from a human, successively comprising the steps of (a), (c), (d), (e) and (f): (a) harvesting peripheral blood mononuclear cells from peripheral blood obtained from a human; (c) labeling B cells included in the obtained peripheral blood mononuclear cells with a marker-labeled antigen and with an antibody which is capable of recognizing a human antibody and is labeled with a marker different from the above marker; (d) separating B cells, that express an antibody recognizing the antigen on the cell membrane, on one cell by one cell basis; (e) extracting total RNA from the one cell and synthesizing cDNA by reverse transcription reaction; and (f) using the synthesized cDNA as a template to perform a PCR reaction using a pair of primers specific for a human antibody heavy chain region gene, a PCR reaction using a pair of primers specific for a human antibody light chain κ region gene, or a PCR reaction using a pair of primers specific for a human antibody light chain λ region gene to amplify each of the region gene fragments, [13] the method for preparing an antibody gene according to the above [11] or [12], wherein the human is a cancer-bearing patient, [14] the method for preparing an antibody gene according to any one of the above [11] to [13], wherein the antigen is a cancer-specific antigen peptide or cancer-specific antigen protein, and [15] the method for preparing an antibody gene according to the above [14], wherein the cancer-specific antigen peptide or cancer-specific antigen protein is MAGE1, MAGE2, MAGE3, MART1, tyrosinase, or gp100.
According to the present invention, genes of functional antibodies can be analyzed and identified for human B cells at one B cell level and genes of antibodies to specific immune epitopes and tumor antigens can be exhaustively analyzed and identified; thus, the present invention is extremely useful for the screening of a new cancer treatment target, the development of a cancer therapeutic agent, and further for the future tailor-made medicine and diagnosis for cancer.
The analyzing/identifying method for an antibody gene of a single B cell according to the present invention is not particularly limited provided that it is a method successively comprising the steps of (A), (B), (C), (D), (E), (F) and (G) described below; the method for producing an antibody of a single B cell according to the present invention is not particularly limited provided that it is a method successively comprising the steps of (A), (B), (C), (D), (E), (F) and (H) described below; and the method for preparing an antibody gene of a single B cell according to the present invention is not particularly limited provided that it is a method successively comprising the steps of (A), (B) (C), (D), (E) and (F) described below.
(A) harvesting peripheral blood mononuclear cells from peripheral blood derived from a human;
(C) labeling B cells with a marker-labeled antigen and with an antibody which is capable of recognizing a human antibody and is labeled with a marker different from the above marker;
(D) separating B cells, that express an antibody recognizing the antigen on the cell membrane, on one cell by one cell basis;
(E) extracting total RNA from the one cell and synthesizing cDNA by reverse transcription reaction;
(F) using the synthesized cDNA as a template to perform a PCR reaction using a pair of primers specific for a human antibody heavy chain region gene, a PCR reaction using a pair of primers specific for a human antibody light chain κ region gene, or a PCR reaction using a pair of primers specific for a human antibody light chain λ region gene to amplify each of the region gene fragments; and
(G) analyzing/determining the base sequence of the amplified gene fragment.
The method of the present invention may not comprise the above step (B) of preparing immortalized B cells, in which case the analyzing/identifying method for an antibody gene of a single B cell according to the present invention is not particularly limited provided that it is a method successively comprising the steps of (a), (c), (d), (e), (f) and (g) described below; the method for producing an antibody of a single B cell according to the present invention is not particularly limited provided that it is a method successively comprising the steps of (a), (c), (d), (e) (f) and (h) described below; and the method for preparing an antibody gene of a single B cell according to the present invention is not particularly limited provided that it is a method successively comprising the steps of (a), (c), (d), (e) and (f) described below.
(a) harvesting peripheral blood mononuclear cells from peripheral blood derived from a human;
(c) labeling B cells included in the obtained peripheral blood mononuclear cells with a marker-labeled antigen and with an antibody which is capable of recognizing a human antibody and is labeled with a marker different from the above marker;
(d) separating B cells, that express an antibody recognizing the antigen on the cell membrane, on one cell by one cell basis;
(e) extracting total RNA from the one cell and synthesizing cDNA by reverse transcription reaction;
(f) using the synthesized cDNA as a template to perform a PCR reaction using a pair of primers specific for a human antibody heavy chain region gene, a PCR reaction using a pair of primers specific for a human antibody light chain κ region gene, or a PCR reaction using a pair of primers specific for a human antibody light chain λ region gene to amplify each of the region gene fragments;
(g) analyzing/determining the base sequence of the amplified gene fragment; and
(h) expressing the amplified gene fragment using an expression vector.
The human from which the peripheral blood is harvested in the steps (A) and (a) is not particularly limited; however, preferred examples thereof can include a cancer patient being in a cancer-bearing state. The cancer patient is not particularly limited provided that the patient is in a cancer-bearing state, and may be a unvaccinated cancer patient or a vaccinated cancer patient with a confirmed immune response to a particular antigen; the cancer may be a solid cancer or a blood cancer. Here, the solid cancer includes a sarcoma and a carcinoma; specific examples include melanoma, fibrosarcoma, mucosal sarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesoepithelioma, Ewing tumor, leiomyosarcoma, rhabdomyosarcoma, stomach cancer, esophageal cancer, large bowel cancer, colon cancer, rectal cancer, pancreas cancer, breast cancer, ovarian cancer, prostatic cancer, squamous cell carcinoma, basal cell cancer, adenocarcinoma, sweat gland carcinoma, sebaceous carcinoma, papillary cancer, papillary adenocarcinoma, cystadenocarcinoma, bone marrow cancer, bronchogenic cancer, renal cell cancer, ureter cancer, liver cancer, bile duct cancer, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, endometrial cancer, testicular cancer, small-cell lung cancer, non-small-cell lung cancer, bladder cancer, epithelial cancer, neuroglioma, astrocytoma, myeloblastoma, craniopharyngeal cancer, laryngeal cancer, tongue cancer, astroependymoma, pinealoma, angioblastoma, acoustic nerve tumor, oligodendroglioma, meningioma, peritoneal dissemination, teratoma, neuroblastoma, medulloblastoma, and retinoblastoma. The above blood cancer includes a myeloma and a lymphoma; specific examples thereof can include acute myelocytic leukemia, acute myelogenous leukemia, chronic myelocytic leukemia, acute lymphatic leukemia, chronic lymphatic leukemia, Hodgkin disease, non-Hodgkin disease, adult T cell leukemia, and multiple myeloma.
To produce an immortalized B cell (EBV-B cell) line from peripheral blood mononuclear cells using Epstein-Barr virus (EBV) in the above step (B), the peripheral blood mononuclear cells may be cultured with EBV in the presence of feeder cells to immortalize the peripheral blood mononuclear cells; a labeled antigen peptide may also be used in addition to an anti-CD19 antibody and an anti-human IgG antibody, markers for B lymphocytes, to identify the presence of a desired antigen-specific antibody-producing EBV-B cell line.
The antigen in the above steps (C) and (c) is not particularly limited provided that it is a molecule specifically recognized by the antibody; examples thereof can include peptides or proteins and nucleic acids such as DNA and RNA. Among others, preferred examples thereof can include peptides or proteins highly expressed specifically in cancer cells (cancer-specific peptides or cancer-specific proteins). More specifically, preferred examples thereof can include cancer-specific peptides and cancer-specific proteins such as MAGE1, MAGE2, MAGE3, MART1, tyrosinase, and gp100. The antigen may be variously modified provided that it has a function as an antigen; for example, it may be a so-called fused protein in which another peptide or protein moiety is added to a functional portion as antigen (an epitope) or in which a sugar chain or an aliphatic chain is added. Examples of the marker in the above step (C) can include fluorescent substances such as Alexa Fluor 488, green fluorescent protein (GFP), fluorescein isothiocyanate (FITC), phycoerythrin (PE), and tetramethylrhodamine isothiocyanate (TRITC), chemiluminescent substances such as luminol, isoluminol, and acridinium derivatives, biotin, and magnet beads. In addition, the above step (C) may be a step of labeling EBV-B cells with a marker-labeled antigen, an antibody to the marker, and an antibody which is capable of recognizing a human antibody and is labeled with a marker different from the above marker; examples of the marker here can include epitope tags such as glutathione S-transferase (GST), c-Myc, HA, and FLAG. An antibody specifically recognizing any of these epitope tags can be used as an antibody to a marker. The antibody specifically recognizing any of these epitope tags may use one labeled with a marker such as the above fluorescent substance and chemiluminescent substance. These labeling can be carried out by a conventional method. See, for example, Molecular Cloning, Third Edition, Cold Spring Harbor Laboratory Press, New York. Preferred examples of the antibody capable of recognizing a human antibody in the step (C) can include an anti-human anti-human IgG antibody. In the step (C), for example, the labeling is preferably carried out under conditions not causing the dropping off of a cell membrane-bound antibody of cancer antigen-specific antibody-producing B cells, such as using GST-labeled cancer specific antigen protein as a marker-labeled cancer-specific antigen protein, an Alexa-labeled anti-GST antibody, and a PE-labeled anti-human anti-human IgG antibody as a different marker-labeled antibody capable of recognizing a human antibody.
In the above steps (D) and (d), EBV-B cells or non-immortalized primary B cells expressing an antibody recognizing an antigen such as a cancer-specific antigen peptide and cancer-specific protein on the cell membrane are separated on one cell by one cell basis. To separate B cells on one cell by one cell basis, a suitable technique is used depending on the type of the marker employed. For example, when a fluorescent substance is used as a marker, B cells are preferably separated on one cell by one cell basis by flow cytometry (single cell sorter) using fluorescence as an indicator. Flow cytometry can efficiently separate cells with high accuracy. The adoption of biotin as a marker also enables the separation thereof on one cell by one cell basis using a binding reaction with avidin. Similarly, when magnet beads are used, good separation is also possible using the magnet. Separation may also be performed on one cell by one cell basis using cell microarray, a micromanipulator, or a micro mesh filter.
In the above steps (E) and (e), total RNA is extracted from one antibody-producing B cell separated on one cell by one cell basis and cDNA are synthesized by reverse transcription reaction. The separation of total RNA, the separation and purification of mRNA, and the obtaining, cloning of cDNA and the like may be all carried out according to ordinary methods (see, for example, Molecular Cloning: A laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989).
In the above steps (F) and (f), the synthesized cDNA are used as a template to perform a PCR reaction using a pair of primers specific for a human antibody heavy chain region gene, a PCR reaction using a pair of primers specific for a human antibody light chain κ region gene, or a PCR reaction using a pair of primers specific for a human antibody light chain λ region gene to amplify each of the region gene fragments. The pair of primers specific for a human antibody heavy chain region gene, a human antibody light chain κ region gene or a human antibody light chain λ region gene is not particularly limited provided that it is a pair of primers specific for the sequence of each region gene. However, examples of the pair of primers specific for a human antibody heavy chain region gene can include a pair of primers consisting of one or two or more sequences of the base sequences represented by SEQ ID NOS: 1 to 24 and the base sequence represented by SEQ ID NO: 25 or 26; examples of the pair of primers specific for a human antibody light chain κ region gene can include a pair of primers consisting of one or two or more sequences of the base sequences represented by SEQ ID NOS: 27 to 37 and the base sequence represented by SEQ ID NO: 38; and examples of the pair of primers specific for a human antibody light chain λ region gene can include a pair of primers consisting of one or two or more sequences of the base sequences represented by SEQ ID NOS: 39 to 61 and one or two sequences of the base sequences represented by SEQ ID NOS: 62 and 63. The base sequences of the amplified gene fragments are analyzed and determined by an ordinary method in the above step (G). Examples of the type of the antibody can include IgA, IgD, IgE, and IgM in addition to IgG.
The gene fragments amplified in the steps (F) and (f) can be used to prepare an antibody gene derived from one B cell. Specifically, a human antibody heavy chain gene can be prepared by PCR reaction using a pair of primers specific for a human antibody heavy chain region gene. A human antibody light chain gene can also be prepared by PCR reaction using a pair of primers specific for a human antibody light chain κ region gene or PCR reaction using a pair of primers specific for a human IgG light chain λ region gene. In addition, a human antibody heavy chain variable region gene fragment can be prepared by PCR reaction using a pair of primers specific for the human antibody heavy chain variable region gene fragment. A human light chain variable region gene fragment can be prepared by FOR reaction using a pair of primers specific for a human antibody light chain κ variable region gene fragment or PCR reaction using a pair of primers specific for a human IgG light chain λ variable region gene fragment. These prepared human antibody genes can also be subcloned for amplification. However, when a genomic DNA is used as a template, their effective amplification cannot be expected because exons of are separated.
The use of the following method of the present inventors (Japanese Patent Application No. 2007-92968) enables the efficient and abundant production of a human ScFv fragment. The human antibody heavy chain variable region gene fragment (heavy chain fragment) and the human light chain variable region gene fragment (light chain fragment) are amplified by the PCR method. These heavy chain and light chain fragments are amplified by the PCR method in the forms of a heavy chain conjugate fragment containing the heavy chain fragment-heavy chain linker sequence-restriction enzyme XbaI recognition sequence and a light chain conjugate fragment containing a restriction enzyme NheI recognition sequence-light chain linker sequence-light chain fragment, respectively. The heavy chain conjugate fragment and the light chain conjugate fragment are digested with the restriction enzyme XbaI and the restriction enzyme NheI, respectively and then linked by ligation. The ligation product is digested with the restriction enzyme XbaI and the restriction enzyme NheI and then amplified by the PCR method in the form of a human single-chain antibody gene (ScFv) fragment consisting of a heavy chain fragment-linker sequence-light chain fragment.
In the steps (H) and (h), the human antibody heavy chain gene and the human antibody light chain gene, which are the gene fragments amplified in the steps (F) and (f), can be expressed using an expression vector to produce an antibody derived from the one B cell. The expression vector is not particularly limited provided that it is suitable for the expression of an antibody gene; however, examples thereof can include an SV40 virus vector, an EB virus vector, and a papilloma virus vector in addition to an adenovirus vector used for transient expression in all cells (except hematocytic cells) including non-dividing cells (Science, 252, 431-434, 1991), a retroviral vector used for long-term expression in dividing cells (Microbiology and Immunology, 158, 1-23, 1992), and an adeno-associated virus vector also capable of being introduced into non-pathogenic and non-dividing cells and used for long-term expression (Curr. Top. Microbiol. Immunol., 158, 97-129, 1992). To increase the expression efficiency, a selection marker gene may also be introduced into these virus vectors in addition to control sequences such as a promoter sequence and an enhancer sequence. The introduction of the antibody genes into the expression vector can be carried out by a well-known method using restriction enzymes and a DNA ligase (see, e.g., Molecular Cloning, Third Edition, 1.84, Cold Spring Harbor Laboratory Press, New York).
Preferably, the human antibody heavy chain gene and the human antibody light chain gene are typically inserted into separate expression vectors and a host is co-transformed with these two recombinant vectors to express the heavy chain and the light chain in the same cells. The host is not particularly limited provided that it can be transformed with the recombinant vectors to hold the introduced antibody genes in a state capable of expression; examples thereof can include Vero cells, Hela cells, CHO cells, W138 cells, BHK cells, COS-7 cells, and MDCK cells. Methods for transforming the host with the recombinant vector can include, for example, a ripofectin method, an electroporation method, and a calcium phosphate method. In this manner, a monoclonal antibody can be produced from one B cell without using a hybridoma. Escherichia coli can also be transformed with a phagemid vector or phage vector having the human ScFv fragment incorporated to produce a phage-displayed human single stranded antibody using the transformed Escherichia coli.
An antibody gene of a cancer patient can be analyzed and identified by the analyzing/identifying method for an antibody gene derived from one B cell according to the present invention to provide information on an overview of types of cancer antigen-specific antibodies produced in the body of an individual patient. In addition, the method for producing an antibody derived from one B cell according to the present invention can provide a cancer antigen-specific antibody in large quantities, enabling the tailor-made diagnosis and treatment of an individual patient. The antibody gene obtained by the method for preparing an antibody gene derived from one B cell according to the present invention is advantageously used in producing a cancer antigen-specific antibody in large quantities and also utilized for the tailor-made diagnosis of an individual patient. Particularly, the method of the present invention has a large advantage of being capable of providing a human antibody not in the form of a partial human antibody as conventionally obtained by immunizing mice and producing a hybridoma but in the form of a 100% human antibody formed by actual amplification in the human body.
The present invention will be described more specifically below with reference to Examples. However, these Examples are not intended to limit the technical scope of the present invention.
Twelve samples of peripheral blood mononuclear cells (PBMC) were harvested from peripheral bloods derived from melanoma patients before and/or after dendritic cell vaccine administration (including the same patient before and after the vaccine administration). The dendritic cell vaccine was one treated with 5 types of peptide: HLA-A24 restriction MAGE1135-143 (the amino acid sequence of MAGE1135-143 is shown in SEQ ID NO: 64), MAGE2, MAGE3, gp100 and tyrosinase or 5 types of peptide: HLA-A2 restriction MAGE2, MAGE3, gp100, MART1 and tyrosinase.
[Production of Immortalized B Cell Line]
Using Epstein-Barr virus (EBV), an immortalized B cell line (an EBV-transformed B cell line; hereinafter referred to as an EBV-B cell line) was produced from the harvested PBMC. Specifically, a human fibroblast cell line (MRC-5; ATCC cat. CCL-171) as a feeder was propagated to 90% confluency in a 25-cm2 flask (medium: MEM+10% FBS) and then subjected to 30 to 40 Gy irradiation. Twenty-four hours after, PBMC harvested in Example 1 were suspended in a medium (IMDM+20% FES) to 1 to 2×107 cells/4 ml, which was then added to the above culture MRC-5. Thereafter, 1 ml of an EBV solution (EBV strain B95-8, ATCC cat. VR-1492) was added to a 25-ml flask and cultured under gas conditions of 37° C. and 5% CO2. The medium was exchanged 48 hours after the start of culture and then exchanged every 4 days. The propagation of B cells was identified after 3 to 4 weeks, and B cells immortalized by EBV (EBV-B cells) were recovered to prepare 12 samples of EBV-B cells in total (MEL-001post, MEL-006post, MEL-014, MEL-016, MEL-017, MEL-018, MEL-018post, MEL-021, MEL-022, MEL-023, MEL-SCC004, and MEL-SCC005). Of the 12 cell lines prepared, MEL-014, MEL-016, MEL-017, MEL-018, MEL-021, MEL-022, MEL-023, MEL-SCC004, and MEL-SCC005 are derived from patients before vaccine administration and MEL-001post, MEL-006post, and MEL-018post are derived from patients after vaccine administration. MEL-018 and MEL-018post are EBV-B cells produced from PBMC before vaccine administration (MEL-018) and after vaccine administration (MEL-018post) derived from the same patient.
EB-B cells were stained with a GST-labeled melanoma-associated recombinant protein, an Alexa Fluor 488-labeled anti-GST antibody, and a PE-labeled anti-human IgG antibody and analyzed by flow cytometry. The schematic of this experiment is shown in
The staining was carried out by the following procedure. First, EBV-B cells were washed with a sorter buffer (PBS 2% FBS 0.1% NaN3) and then adjusted to an amount of 20 μl/tube.
A solution of GST or each GST-labeled protein adjusted to a concentration of 100 ng/20 μl with PBS (0.2% BSA) was added to the EBV-B cells, which was then reacted at 4° C. for 30 minutes. Next, 20 μl (10 μg/ml) of Alexa-anti-GST antibody was added thereto, which was then reacted at 4° C. for 30 minutes, followed by adding 20 μl of PE-hIgG antibody for reaction at 4° C. for 30 minutes. The stained EBV-B cells were analyzed by a flow cytometer (FAGS-CANTO, BD). For identification of live cells, PI staining was performed immediately before measurement.
The analysis results of flow cytometry are shown in
Green fluorescence protein (GFP)-labeled MAGE-1 protein (hereinafter referred to as GFP-MAGE1) was synthesized and used to perform the immunohistochemical staining of EBV-B cells. GFP-MAGE1 was made using a production system of baculovirus. As shown in
Single cell sorting was carried out for the purpose of separating GFP-MAGE1+/PE-anti-hIgG+ EBV-B cells on one cell by one cell basis. For sorting, BD FACSAria™ cell sorter (from ED Science) equipped with a module for single cell sorting was used. For sorting, cells were separated into a 96-well plate (MicroAmp (R) Optical 96-well Reaction Plate; from Applied Biosystem) on one cell by one cell basis using 100 μm nozzle and the conditions of sort setup: low, flow rate: 5,000 events/sec and drop delay: 25.73.
The EBV-B cells separated on one cell by one cell basis in Example 4 were subjected to single cell RT-PCR cloning. The experimental outline of cloning is shown in
First, RNA was extracted from the single cell separated, and cDNA was synthesized by reverse transcription reaction. Super Script™ III Cells Direct cDNA Synthesis System (cat. 18080-300, from Invitrogen) was used for synthesis thereof. A resuspension buffer (10 μl) and a lysis enhancer solution (1 pa) were added thereto, which was then treated at 75° C. for 10 minutes using a thermal cycler. Subsequently, 5 μl of DNase I (1 U/μl) and 1.6 μl of 10×DNase I buffer were added thereto, which was then mixed by pipetting and incubated at room temperature for 5 minutes. The plate was slightly centrifuged, to which 1.2 of 25 mM EDTA was added, followed by incubation at 70° C. for 5 minutes using a thermal cycler. The plate was then slightly centrifuged, and 2 μl of 50 mM Oligo (dT)20 and 1 μl of 10 mM dNTP mix were added to each well on ice. After mixing by pipetting, the plate was treated at 70° C. for 5 minutes using a thermal cycler and incubated for 2 minutes on ice. The plate was slightly centrifuged, to which 6 μl of 5×RT buffer, 1 μl of RNase OUT™ (40 U/μl), 1 μl of Super Script™ III RT (200 U/μl), and 1 μl of 0.1 M DTT were then added again on ice, followed by mixing by pipetting. The plate was slightly centrifuged and incubated at 50° C. for 50 minutes and at 85° C. for 5 minutes using a thermal cycler to synthesize cDNA.
Next, cDNA prepared from one cell was subjected to PCR using primers for cloning. One μl of cDNA was dispensed into each of four 0.2-ml PCR tubes (#1 to #4) for each sample, and 1 μl of 10×PCR buffer II (Mg+), 1 μl of 2.5 mM dNTP mix, 5.9 μl of dH2O, 0.1 μl of LA-Tag polymerase (TaKaRa LA-Tag(R) Hot Start version; from Takara Bio Inc.), 0.5 μl of a forward primer (10 μM), and 0.5 μl of a reverse primer (10 μM) were added to each tube, which was then subjected to PCR reaction using a thermal cycler (Gene AmpR PCR System 9700; from Applied Biosystems). Tubes #1, #2, #3 and #4 were used for the PCR reaction of the β-actin gene, the human IgG heavy chain region gene, the human IgG light chain κ region gene and the human IgG light chain κ region gene, respectively. The PCR reaction conditions for each tube were as follows:
Tube #1: 94° C. for 5 minutes, (94° C. for 15 seconds, 68° C. for 2 minutes)×55 cycles, 72° C. for 5 minutes;
Tube #2 and tube #3: 94° C. for 5 minutes, (94° C. for 15 seconds, 68° C. for 1 minutes)×55 cycles, 72° C. for 5 minutes; and
Tube #4: 94° C. for 5 minutes, (94° C. for 15 seconds, 60° C. for 30 seconds)×40 cycles, 72° C. for 5 minutes.
After PCR reaction, the resultant reaction solution was subjected to electrophoresis using 1.5% agarose gel, and a desired band was identified with ethidium bromide staining. The size of bands of PCR products amplified with primer sets for human IgG region gene region amplification was identified, and 2 samples of DNA for each primer set were extracted and purified from gel. Specifically, gels of amplification band portions for the PCR reaction tubes #2, #3 and #4 were cut out using a scalpel and transferred to 1.5-ml sample tubes, and the cut-out gels were weighed. Converting at the rate of 1 μl to 1 mg, a 3-fold volume of Buffer Q×1 and 17.2 μl of QIAEX II Suspension were added to each cut-out gel. This was mixed thoroughly with a vortex, placed on a heat block set at 50° C. in advance and subjected to vortex mixing every 2 minutes, resulting in treatment for 10 minutes in total. Centrifugation was then carried out at 10,000×g and room temperature for one minute and the supernatant was removed. Again, 500 μl of Buffer Q×1 was added to the precipitate, which was then mixed using a vortex and centrifuged at 10,000×g and room temperature for one minute. The supernatant was removed, and 500.1 of Buffer PE to which ethanol was added in advance was added to the precipitate, which was then mixed using a vortex and centrifuged at 10,000×g and room temperature for one minute, followed by removing the supernatant. Again, 10,000×g was applied to the precipitate, which was then mixed using a vortex and centrifuged at 10,000×g and room temperature for one minute, followed by removing the supernatant. Each sample tube was placed while opening the lid thereof in a clean bench for 15 minutes to dry the precipitate. Buffer PE (Min Elute™ Reaction Cleanup Kit; from QIAGEN) (20 μl) was added to the precipitate, which was then mixed using a vortex and placed at room temperature for 5 minutes. This was centrifuged at 10,000×g and room temperature for one minute, the supernatant was recovered into another 1.5-ml sample tube, and 20 μl of Buffer PE was again added to the precipitate. This was mixed using a vortex, placed at room temperature for 5 minutes, centrifuged at 10,000×g and room temperature for one minute, and added to the supernatant previously recovered.
To a total 40 μl of the recovered solution was added 300 μl of Buffer ERC (MinElute™ Reaction Cleanup Kit; from QIAGEN) which was then mixed using a vortex. A total amount of the mixed solution after being identified to have a yellow color was applied into MinElute column (MinElute™ Reaction Cleanup Kit; from QIAGEN) set in a 2-ml collection tube (MinElute™ Reaction Cleanup Kit; from QIAGEN) and centrifuged at 10,000×g and room temperature for one minute. After discarding the effluent, the column was again returned to the collection tube, and 750 μl of Buffer PE to which ethanol was added in advance was added thereto, which was centrifuged at 10,000×g and room temperature for one minute. After discarding the effluent, the column was again returned to the collection tube, and centrifugation was carried out at room temperature and 22,000×g for one minute. Droplets attached to the brim of the column were removed using a micropipette, and the column was set in a new 1.5-ml sample tube. Thereto was added 10 μl of Buffer EB (NinElute™ Reaction Cleanup Kit; from QIAGEN), which was then placed at room temperature for one minute and then centrifuged at 10,000×g and room temperature for one minute, followed by recovering a purified DNA fragment.
The PCR fragment was inserted into pCR4-TOPO-TA Plasmid vector to produce a plasmid DNA. Salt Solution (TOPO TA CloningR Kit for Sequencing; from Invitrogen) of 1 μl of the DNA fragment and 1 μl of TOPOR Vector (TOPO TA CloningR Kit for Sequencing; from Invitrogen) were mixed on ice. The mixture was reacted at room temperature for 5 minutes, again returned onto ice, and used for transformation. The plasmid DNA was introduced into DH5a competent cells (Competent high DH5a; from Toyobo Co., Ltd.). The plasmid DNA was added 2 μl by 2 μl to 20 μl of thawed DH5a competent cells, which was gently mixed using an end of a tip. The mixture was placed on ice for 30 minutes and then treated at 42° C. for 30 seconds using a heat block. This was again placed on ice for 2 minutes for cooling, to which 250 μl of SOC medium was then added, followed by shaking culture at 37° C. for one hour. While the shaking culture was carried out, 50 μl each of 0.1 M IPTG (isopropyl 3-D-1-thiogalactopyranoside; from Sigma) and 0.1 M X-Gal (5-bromo-4-chloro-3-indolyl β-D-galactopyranoside; from Sigma) were coated on 2×YT solid medium containing 50 μg/ml of kanamycin. The cultured sample (100 μl) was seeded on the prepared solid medium and cultured 37° C. overnight. After identifying the appearance of colonies on the solid medium, this was allowed to stand at 4° C. for 5 hours. White colonies were marked, poked with an end of a tip, and placed and slightly rinsed in a 96-well plate containing 50 μl of sterilized water. This was treated at 95° C. for 5 minutes using a thermal cycler and then slightly centrifuged, and 2 μl thereof was placed in a fresh plate well. Subsequently, 1 μl of 10×PCR buffer II (Mg+), 0.8 μl of 2.5 mM dNTP Mix, 6.11 μl of dH2O, 0.05 μl of LA-Taq polymerase (TaKaRa LA-Tag (R) Hot Start version; from Takara Bio Inc.), 0.02 μl of 100 μM M13 forward primer, and 0.02 μl of 100 μM M13 reverse primer were added thereto, and the plate was slightly centrifuged. After thermal denaturation at 94° C. for one minute using a thermal cycler, Gene Amp(R) PCR System 9700, PCR reaction was performed under reaction conditions in which the cycle of reaction at 94° C. for 10 seconds, 50° C. for 10 seconds, and 68° C. for 2 minutes was repeated 35 times. PCR products (tube #2: 1.6 Kbp, tube #3: 0.9 Kbp, and tube #4: 0.9 Kbp) in PCR reaction solutions were identified by electrophoresis using 1.5% agarose gel.
Colonies in which the insertion of the PCR-amplified fragment into the vector was identified were selected and cultured with shaking at 37° C. overnight in a 2×YT liquid medium containing 3.5 ml of 50 μg/ml kanamycin. The cultured sample (1.8 ml) was placed in a 2-ml sample tube and centrifuged at 1,000×g for 10 minutes. The supernatant was discarded, and 250 μl of Buffer A1 (NucleoSpin(R) Multi-8 Plasmid; from Macherey-Nagel) was added to the precipitate, which was then mixed using a vortex. Subsequently, 250 μl of Buffer A2 was added thereto, which was then mixed by inversion and allowed to stand at room temperature for 5 minutes to lyse the cells. Buffer A3 (350 μl) was added thereto and mixed by inversion, which was then centrifuged at 4° C. and 14,000×g for 10 minutes. The supernatant was transferred to NucleoSipn(R) Plasmid Binding Strips set in NucleoVac vacuum manifold. The solution was passed through the silica membrane by suction at 400 mbar for one minute to produce DNA binding. The silica membrane was washed by adding 600 ml of Buffer AW and passing the solution therethrough by suction at 400 mbar for one minute and then adding 900 ml of Buffer A4 and passing the solution therethrough by suction at 400 mbar for one minute. The silica membrane was again washed by adding 900 ml of Buffer A4 and passing the solution therethrough by suction at 400 mbar for one minute. The silica membrane was dried by suction at 600 mbar for 15 minutes. The plasmid DNA was recovered by subjecting the NucleoVac vacuum manifold to replacement with NucleoSipnR MN Tube Strips for recovery, adding 120 μl of Buffer AE to the membrane, allowing the mixture to stand for one minute, and suctioning it at 400 mbar for one minute. To 3 μl of the recovered plasmid DNA solution were added 1 μl of 10×H Buffer, 5 μl of dH2O, and 1 μl of EcoRI (from Toyobo Co., Ltd.), which was then treated at 37° C. for one hour. DNA digestion fragments (tube #2: 1.4 Kbp, tube #3: 0.7 Kbp, and tube #4: 0.7 Kbp) in the enzymatic reaction solution were identified by electrophoresis using 1.5% agarose gel; absorbance for each plasmid sample was measured to calculate the DNA concentration; and 100 μg/μl of a plasmid DNA diluted solution was prepared.
The clone obtained was sequenced by the cycle sequencing method. The DNA diluted solution prepared in Example 11 was added in amounts of 6 μl/well to 3 wells for tube #2 (#2-1, #2-2 and #2-3), 2 wells each for tubes #3 and #4 (#3-1 and #3-2 and #4-1 and #4-2) in a 96-well plate placed on ice. Subsequently, 3 μl of 5×Sequencing Buffer (BigDyeR Terminator v3.1 Cycle Sequencing Kit; from Applied Biosystem), 2 μl of BigDyeR Terminator Pre mix (BigDyeR Terminator v3.1 Cycle Sequencing Kit; from Applied Biosystem), 8 μl of dH2O, and 1 μl of 3.2 μM primer were added to each well, and slightly centrifuged.
The primers used for the samples are M13 reverse primer for sample #2-1, M13 forward primer for sample #2-2, HuIGCH-seq001 for sample #2-3, M13 reverse primer for sample #3-1, M13 forward primer for sample #3-2, M13 reverse primer for sample #4-1, and M13 forward primer for sample #4-2. After thermal denaturation at 94° C. for one minute using a thermal cycler, Gene Amp(R) PCR System 9700, PCR reaction was performed under reaction conditions in which the cycle of reaction at 94° C. for 10 seconds, 50° C. for 5 seconds, and 68° C. for 4 minutes was repeated 25 times. By the time the reaction was completed, Sephadex G-50 (Sephadex G-50 Superfine; from GE Healthcare) and 300 μl of sterilized water were added to wells of MultiScreen™ HV-plate (MultiScreenRHV-plate; from Millipore) and allowed to stand at room temperature for 2 hours. After sufficient hydration, centrifugation was carried out at room temperature and 1,100×g for 5 minutes, and the effluent was discarded. The MultiScreen™ HV-plate was subjected to replacement with a fresh 96-well plate (ASSAY PLATE 96 well round bottom; from Iwaki); a total amount of the reaction solution was applied to wells; and centrifugation was carried out at room temperature and 1,100×g for 5 minutes to recover the sample.
A total amount of the purified sample was transferred to a 96-well plate for sequencing (MicroAmpR Optical 96-well Reaction Plate; from Applied Biosystem); in addition, 17.2 μl of sterilized water was added to the preceding well and a total amount thereof was added to the same sample while washing the well therewith. Using x3130/Genetic Analyzer (from Applied Biosystem), sequences of 6 to 8 clones were read for each sample, and the resultant sequences were subjected to multiple alignment analysis. For a base different between clones, the base which more clones have was regarded as correct sequence to determine the base sequence for each sample.
As a result of the above cloning, as shown in Table 1, 12 clones of IgG antibody genes were successfully isolated from cases (MEL-008, MEL-014, MEL-016, MEL-018Pre, and MEL-018Post) of melanoma patient-derived EBV-5 cell lines (at a single-cell level). For five of these clones, the expression of single-chain recombinant antibodies has been identified and the antibodies are now at a stage of purification.
Expression of MEL-018scFv: Of the IgG genes cloned in Example 5, the gene sequence for #008 (MEL018Pre) was used to incorporate the genes VH and VL of the variable regions of the IgH chain and the IgL chain into a plasmid for single-chain antibody gene expression (p0Z1, a pUC119-derived self-made vector) to express MEL-018 scFv (single chain recombinant antibody) in Escherichia coli (
Purification of MEL-018 scFv Antibody: Using His tag as a marker, Metal chelate affinity purification employing a Ni Sepharose column was carried out. Then, two-step purification was performed by anion exchange chromatography (HiTrap QFF column). In addition, for the purpose of evaluating the specificity of the finally purified MEL-018 scFv, western blotting was performed using the GST-labeled recombinant MAGE1 protein (543 aa, 59.74 kDa) as an antigen. A purified primary antibody (MEL-018 scFv) was diluted by 1/1,000 and reacted for 2 hours, and then a secondary antibody (anti-FLAG M2 monoclonal antibody) was diluted by 1/2,000 and reacted for 2 hours. A mouse anti-human MAGE1 monoclonal antibody (from Abnova) was used as a control antibody. ECLplus reagent (from GE Healthcare) was reacted for 10 minutes to detect a signal.
The results of the above experiment are shown in
The IgG antibody gene of immortalized B cells was quantified at a single-cell level by the real-time PCR method. Probe (TaqMan) primers targeting a conserved region of the Fc segment of the IgG antibody were designed, and the mRNA of a partial sequence of the Fc segment for preparing a calibration curve was synthesized in vitro (
The results of the above quantitation using the real-time PCR method are shown in
Sera were collected from cancer patients and healthy subjects, and the anti-CMVpp65 antigen-specific IgG antibody titer in each serum was measured. The results are shown in
Subsequently, B cells were stained with GST-labeled CMVpp65 antigen protein, Alexa488-labeled anti-GST antibody, and a PE-labeled anti-human IgG antibody (
As in Example 8, B cells from cases in which the serum antibody titer to CMVpp65 was increased were used to perform the stain identification and capture of CMVpp65 antigen-positive B cells with cell microarray (
Single cell RT-PCR cloning was then carried out (
1. Sterilized water (5 μl) containing yeast transfer RNA as a carrier was added to each well of a 96-well plate or each 0.2-ml tube for PCR and thereby adapted to receive the separated live cells;
2. DNase treatment and cell-lysing treatment were omitted, considering that the cell-lysing treatment was unnecessary because cell rupture by osmotic pressure and content release occurred the instant cells contacted the sterilized water.
3. A solution (6 μl ) of the synthesized cDNA was used to perform IGH-PCR. In the method of Example 5, the cDNA solution could be introduced into the PCR reaction only under conditions of 1 μl/30 μl of the total PCR reaction solution; thus, this system (6 μl/12 μl of the total PCR reaction solution) could be expected to have higher detection sensitivity than that of the previous one.
4. Nested PCR method was adopted in order to improve specificity and amplification efficiency. Both 1st-PCR and 2nd PCR used Ex-taq HotStart ver; in 1st-PCR, primers corresponding to each repertoire used the same ones as those in Example 5.
Reverse transcription reaction directly targeting a single cell was carried out as follows. Sterilized water (5 μl) containing yeast transfer RNA (from Ambion) as a carrier and RNase OUT™ (from Invitrogen) were placed in a 96-well plate (MicroAmpR Optical 96-Well Reaction Plate; from Applied Biosystem) or 0.2-ml PCR tubes, and the sorted cells were separated thereinto on one cell by one cell basis using FACS or Cell porter mini. The 96-well plate or 0.2-ml tubes receiving the cells were slightly centrifuged and placed on ice, and 10×PCR buffer II (0.80 μl), 25 mM MgCl2 (0.48 μl), 0.1 M DTT (0.40 μl), 40 U/μl RNase OUT™ (0.16 μl), 50 mM Oligo (dT)20 (from Invitrogen) (0.16 μl), 10 mM dNTPmix (0.16 μl), and dH2O (0.84 μl) were added to each well, which was then treated ashing thermal cycler at 70° C. for 90 seconds using a thermal cycler (Gene AmpR PCR System 9700; from Applied Biosystems). After thermal treatment, the 96-well plate or 0.2-ml PCR tubes were quickly transferred onto ice and allowed to stand for 2 minutes. The 96-well plate or 0.2-ml PCR tubes were slightly centrifuged and then again transferred onto ice, and RNase OUT™ (40 U/μl), 0.05 μl of Super Script™ III RT (200 U/μl; from Invitrogen), 0.40 μl and 1.35 μl of dH2O were added to each well, which was then mixed by pipetting. The 96-well plate was slightly centrifuged and then treated using thermal cycler at 50° C. for 50 minutes and 70° C. for 10 minutes to synthesize DNA. In addition, 10×PCR buffer II (0.20 μl), MgCl2 (0.12 μl), RNase H (0.30 μl; from Invitrogen), and dH2O (1.38 μl) were added thereto, which was then thoroughly mixed and treated at 37° C. for 15 minutes and 70° C. for 10 minutes to prepare 12 μl of a cDNA solution.
Using each of the synthesized cDNA solutions as a template, 1st PCR of a human antibody heavy chain region gene (IGH) was carried out as follows. Specifically, 0.2-ml tubes for PCR were provided and placed on ice, and 6 μl of the cDNA solution prepared in Example 9 was placed in each 0.2-ml tube for PCR. Subsequently, 10×Ex Taq buffer II (2.0 μl), 2.5 mM dNTP Mix (2.0 μl) dH2O (13.8 μl), Ex-Taq Hot Start version (0.2 μl; from Takara Bio Inc.), 10 μM forward primer mix [HuIGHV—1 to 24] (1.0 μl), and 10 μM reverse primer mix [HuIGHC—1 to 2] (1.0 μl) were added thereto, which was then slightly centrifuged. The base sequences of primers specific to the human IgG heavy chain genes used here are shown in SEQ ID NO: 1-26, respectively. The 0.2-ml tubes for PCR were set in a thermal cycler (Gene AmpR PCR System 9700), and reaction was performed using the program of (95° C. for 15 seconds, 68° C. for 1 minute, and 72° C. for 2 minutes)×30 cycles and 72° C. for 5 minutes.
Using each of the PCR products obtained in the above 1st PCR as a template, 2nd PCR was further performed. Specifically, 0.2-ml tubes for PCR were each placed on ice, and 0.5 μl of the 1st PCR reaction product was added to each 0.2-ml tube. Subsequently, 10×Ex Taq buffer II (2.0 μl), 2.5 mM dNTP Mix (2.0 μl) dH2O (13.8 μl), Ex-Taq Hot Start version (0.2 μl) 10 μM forward primer mix [M13-HuVH—201 to 206] (1.0 μl), and 10 μM reverse primer [M13-HuCH—401] (1.0 μl) were added thereto, which was then slightly centrifuged. The base sequences of the above primers [M13-HuVH—201 to 206 and M13-HuCH—401] are shown in
[DNA Extraction from PCR Product]
The 2nd PCR reaction solutions thus obtained were each fractionated by electrophoresis to purify a desired PCR fragment. The 2nd PCR reaction solution was subjected to electrophoresis using 1.5% agarose gel to separate bands; after electrophoresis, the gel was stained with ethidium bromide; and after identifying amplification using UV, a gel of an amplified band portion was cut out and transferred to a 1.5-ml sample tube. For the clone in which the amplification of IGH could be identified, PCR amplifications of the human antibody light chain κ and λ region genes as shown in the following Example 11 were carried out, or a PCR amplified band of a sample for which the amplification of both human antibody heavy chain region gene and human antibody light chain κ or λ region gene was identified was purified. Specifically, the cut-out gel was weighed and, converting at the rate of 1 μl to 1 mg, Buffer QX1 (QIAEXIIR Gel Extraction Kit; from QIAGEN) was added in an amount of 3 times that of the gel thereto together with 17.2 μl of QIAEXII Suspension (MinElute™ Reaction Cleanup Kit; from QIAGEN), which was then thoroughly mixed using a vortex. The mixture was placed on a heat block set at 50° C. in advance and subjected to vortex mixing every 2 minutes, resulting in treatment for 10 minutes in total to completely dissolve the gel. This solution was identified to have a yellow color and then centrifuged at room temperature and 10,000×g for one minute to remove the supernatant. Again, 500 μl of Buffer QX1 was added to the precipitate, which was then mixed using a vortex and centrifuged at room temperature and 10,000×g for one minute. After removing the supernatant, 500 μl of Buffer PE (QIAEXIIR Gel Extraction Kit; from QIAGEN) was added thereto, which was then mixed using a vortex. The mixture was centrifuged at room temperature and 10,000×g for one minute to remove the supernatant, and again, 500 μl of Buffer PE was added to the precipitate, which was then mixed using a vortex and centrifuged at room temperature and 10,000×g for one minute. After removing the supernatant, the sample tube was placed while opening the lid thereof in a clean bench for 15 minutes to dry the precipitate. Buffer EB (Min Elute™ Reaction Cleanup Kit; from QIAGEN) (20 μl) was added to the precipitate, which was then mixed using a vortex and placed at room temperature for 5 minutes. This was centrifuged at 10,000×g and room temperature for one minute, the supernatant was recovered into another 1.5-ml sample tube, and 20 μl of Buffer EB was again added to the precipitate. This was mixed using a vortex, placed at room temperature for 5 minutes, centrifuged at 10,000×g and room temperature for one minute, and the supernatant was recovered in the same tube. To a total 40 μl of the recovered solution was added 300 μl of Buffer ERC (MinElute™ Reaction Cleanup Kit; from QIAGEN), which was then mixed using a vortex. A total amount of the mixed solution was applied into MinElute column (MinElute™ Reaction Cleanup Kit; from QIAGEN) set in a 2-ml collection tube (MinElute™ Reaction Cleanup Kit; from QIAGEN) and centrifuged at 10,000×g and room temperature for one minute. After discarding the effluent, the column was again returned to the collection tube, and 750 μl of Buffer PE (Min Elute™ Reaction Cleanup Kit; from QIAGEN) to which ethanol was added in advance was added thereto, which was centrifuged at 10,000×g and room temperature for one minute. After discarding the effluent, the column was again returned to the collection tube, and centrifugation was carried out at room temperature and 22,000×g for one minute. Droplets attached to the brim of the column were removed using a micropipette, and the column was set in a new 1.5-ml sample tube. Thereto was added 10 μl of Buffer EB (MinElute™ Reaction Cleanup Kit; from QIAGEN), which was then placed at room temperature for one minute and then centrifuged at 10,000×g and room temperature for one minute, followed by recovering a purified DNA fragment.
Two 0.2-ml PCR tubes (#1 and #2) were provided for each sample and placed on ice, and 1 μl of the cDNA solution prepared in Example 10 was placed in each 0.2-ml PCR tube. Subsequently, 10×PCR buffer II (Mg+) (1 μl), 2.5 mM dNTP Mix (1 μl), dH2O (5.9 μl), LA-Taq Hot Start version (TaKaRa LA-Tae Hot Start version; from Takara Bio Inc.) (0.1 μl), 10 μM forward primer (0.5 μl), and 10 μM reverse primer (0.5 μl) were added thereto, which was then slightly centrifuged. HuIGKV—1 to 11 mix and HuIGKC—1 (SEQ ID NOS: 27 to 38) were used as primers for light chain κ region amplification in the tube #1 and HuIGLV—1 to 23 mix and HuIGLC—1 to 2 mix (SEQ ID NOS: 39 to 64) were used as primers for light chain 74 region amplification in the tube #2. The 0.2-ml PCR tubes were set in a thermal cycler, and reaction was performed using the program of 95° C. for 5 minutes, (95° C. for 30 seconds, 68° C. for 1 minute, and 72° C. for 5 minutes)×55 cycles. After the end of FOR, PCR amplification was identified by the same way as in Example 11, and DNA fragments were purified for samples in which the amplification of IGH and IGK/L derived from the same clone could be identified in a set.
It was demonstrated that the foregoing nested FOR eases the sensitivity of a Single-Cell RT-PCR method. The results of comparing the amplification efficiency of a FOR method of an antibody gene derived from one cell before and after the technological improvement depicted in
Subsequently, the base sequences of the human antibody heavy chain gene (IGH) DNA fragments obtained were identified by a PCR-Direct sequence method, and cloning samples were selected. Specifically, 2 μl of the IGH 2nd FOR fragment purified in Example 11 was added to each of two wells (#1 and #2) of a 96-well plate placed on ice. In addition, 5×Sequencing Buffer (3, BigDyeR Terminator Premix (BigDyeR Terminator v3.1 Cycle Sequencing Kit; from Applied Biosystem) (2 ml), dH2O (12 μl), and 3.2 μM of a primer (1 μl) were added thereto, which was then slightly centrifuged. An M13 reverse primer was used for the sample #1 and an M13 forward primer, for the sample #2. Then, 0.2-ml PCR tubes were set in a thermal cycler, and reaction was performed using the program of 95° C. for 1 minute, (95° C. for 10 seconds, 50° C. for 5 seconds, and 68° C. for 4 minutes)×24 cycles. By the time the reaction was completed, Sephadex G-50 (from GE Healthcare) and 300 μl of sterilized water were added to wells of MultiScreen™ HV-plate (from Millipore) and the plate was allowed to stand at room temperature for 2 hours. After sufficient hydration, centrifugation was carried out at room temperature and 1,100×g for 5 minutes, and the effluent was discarded. The MultiScreen™ HV-plate was subjected to replacement with a fresh 96-well assay plate (from Iwaki); a total amount of each of the preceding reacted samples was applied to each well; and centrifugation was carried out at room temperature and 1,100×g for 5 minutes to recover the samples. A total amount of the purified sample was transferred to a 96-well plate for sequencing. In addition, 17.2 μl of sterilized water was added to the preceding well and a total amount thereof was transferred to the 96-well plate to determine the sequence thereof using a DNA sequencer (x3130/Genetic Analyzer; from Applied Biosystem). After sequence determination, alignment analysis with the deposited repertoire was performed using V-QUEST (http://www.imgt.org/IMGT_vquest/share/textes/) to determine the IGH repertoire and CDR-3 sequence of a clone. Based on the information of the IGH repertoire and CDR-3 sequences thus determined, clones having overlapping IGH repertoire and CDR-3 sequences were excluded, and cloning was carried out from the remaining clones by a method as described below.
[Cloning]
First, a PCR fragment and pCR4.0-TA Plasmid vector were linked using TOPOR TA PCR Cloning Kit for Sequencing (from Invitrogen). Specifically, a 500-μl sample tube was placed on ice, and 4 μl of a DNA fragment obtained in Example 10, 1 μl of Salt Solution, and 1 μl of a plasmid vector were added thereto, which was then mixed by pipetting. The mixture was reacted at room temperature for 5 minutes, again returned onto ice, and used for transformation. DH5a competent cells (from Toyobo Co., Ltd.) were thawed on ice and 20 μl thereof was dispensed into each of other sample tubes. To each of these tubes was added 2 μl of the above plasmid vector reaction solution, which was then gently mixed using an end of a tip. Each tube was placed on ice for 30 minutes, and treated at 42° C. for 30 seconds using a heat block. The tube was placed on ice for 2 minutes for cooling, to which 250 μl of SOC medium was then added, followed by shaking culture at 37° C. for one hour. The cultured sample (100 μl) was seeded on 2×YT/Km plate coated with 50 μleach of 0.1 M IPTG (from Sigma) and 0.1 M X-Gal (from Sigma) and cultured at 37° C. overnight.
White colonies were selected, inoculated into 3.5 ml of 2×YT/Km liquid medium, and shaking cultured at 37° C. overnight. The culture solution (1.8 ml) after culture was placed in a 2-ml sample tube, which was then centrifuged at 1,000×g for 10 minutes. The supernatant was discarded, and 250 μl of Buffer A1 (NucleoSpinRMulti-8 Plasmid; from Macherey-Nagel) was added to the precipitate, which was then mixed using a vortex. In addition, 250 μl of Buffer A2 (NucleoSpinRMulti-8Plasmid; from Macherey-Nagel) was added thereto, which was then mixed by inversion and allowed to stand at room temperature for 5 minutes to lyse the cells. Further, 350 μl of Buffer A3 (NucleoSpinR Multi-8 Plasmid; from Macherey-Nagel) was added thereto, which was then mixed by inversion and centrifuged at 4° C. and 14,000×g for 10 minutes. The supernatant was recovered and transferred to NucleoSipnR Plasmid Binding Strips (NucleoSpinR Multi-8 Plasmid; from Macherey-Nagel) set in NucleoVac vacuum manifold. This was suctioned at 400 mbar for one minute to cause DNA to bind to a silica membrane. The silica membrane was washed by adding 600 ml of Buffer AW (NucleoSpinR Multi-8 Plasmid; from Macherey-Nagel) and passing the solution therethrough by suction at 400 mbar for one minute and then adding 900 ml of Buffer A4 (NucleoSpinR Multi-8 Plasmid; from Macherey-Nagel) and passing the solution therethrough by suction at 400 mbar for one minute. The membrane was washed again by adding 900 ml of Buffer A4 and passing solution therethrough by suction at 400 mbar. After drying the membrane, the plasmid DNA was recovered by subjecting the NucleoVac vacuum manifold to replacement with NucleoSipnR MN Tube Strips (NucleoSpinR Multi-8 Plasmid; from Macherey-Nagel) for recovery, adding 120 μl of Buffer AE (NucleoSpinR Multi-8 Plasmid; from Macherey-Nagel) to the membrane, allowing the mixture to stand for one minute, and suctioning it at 400 mbar for one minute. To 3 μl of the recovered plasmid DNA solution were added 10×H Buffer (1 μl), dH2O (5 μl), and 1 μl of EcoRI (from Toyobo Co., Ltd.), which was then treated at 37° C. for one hour. The restriction enzyme reaction solution was fractionated by electrophoresis using 1.5% agarose gel, and clones in which inserts (IGH: 0.5 kbp, IGK/L: 0.7 kbp) were identified were selected as samples for sequence analysis. Absorbance for each plasmid sample was measured to calculate the DNA concentration; and 100 ng/μl of a plasmid DNA solution was prepared.
[Sequencing of Plasmid DNA]
The base sequence of a plasmid DNA was determined by a cycle sequencing method. Specifically, the prepared plasmid DNA diluted solution was added in amounts of 6 μl/well to 3 wells (#1, #2, and #3) for IGH and 2 wells (#4 and #5) for IGK/L in a 96-well plate placed on ice. Subsequently, 5×Sequencing Buffer (3 μl) BigDyeR Terminator Pre mix (2 μl) dH2O (8 μl), and 3.2 μM primer (11 μl) were added to each well, and slightly centrifuged. The combination of 3.2 μM primer and sample # is as follows:
#1: T7p primer
#2: HuIGCH-seq001 primer
#3: T3p primer
#4: M13 reverse primer
#5: M13 forward primer
A 96-well plate was set in a thermal cycler, and reaction was performed using the program of 94° C. for 1 minute, (94° C. for 10 seconds, 50° C. for 5 seconds, and 68° C. for 4 minutes)×25 cycles. By the time the reaction was completed, Sephadex G-50 was placed in wells of MultiScreen™ HV-plate and 300 μl of sterilized water were added thereto and the plate was allowed to stand at room temperature for 2 hours. After sufficient hydration, centrifugation was carried out at room temperature and 1,100×g for 5 minutes, and the effluent was discarded. The MultiScreen™ HV-plate was subjected to replacement with a fresh 96-well assay plate (from Iwaki); a total amount of the PCR reaction solution was applied to the wells; and centrifugation was carried out at room temperature and 1,100×g for 5 minutes to recover the samples. A total amount of the purified sample was transferred to a 96-well plate for sequencer. In addition, 17.2 μl of sterilized water was added to the preceding well and used for washing and then a total amount thereof was transferred to the 96-well plate. Sequence analysis was carried out using x3130/Genetic Analyzer. Sequences of 6 to 8 clones were read for each sample, and the resultant sequences were subjected to multiple alignment analysis; for a base different between clones, the base which more clones have was regarded as correct to determine the base sequence for each sample.
As a result of the above experiment, both antibody genes of IGH/L were successfully cloned in 8 of the 57 cells captured by the single cell sorting method (
The results of sequence analysis of the repertoire, CDR-3 and full length of CMVpp65 antigen-specific IgG antibody genes are shown in
| Number | Date | Country | Kind |
|---|---|---|---|
| 2008-146964 | Jun 2008 | JP | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/JP2009/002539 | 6/4/2009 | WO | 00 | 11/30/2010 |
