Patent Application
20220403002
The Sequence Listing submitted herewith is an ASCII text file (file name: A00544 Sequence Listing.txt, created on Apr. 26, 2022, 34669 bytes), is filed via EFS-Web, and is hereby incorporated by reference.
The present invention relates to a novel artificial receptor (B-cell antibody receptor; BAR), an immune cell, and a regenerative medicinal product (cell medicine).
Various proteins exist in a body of an organism, and their functions maintain life activities. When a mutation occurs in a gene encoding a protein, the protein cannot be produced, or its activity is reduced, so that a disease may occur.
Hemophilia is an inherited disease due to quantitative and qualitative abnormalities of blood coagulation factors, and is classified into hemophilia A, hemophilia B, and the like, depending on the type of blood coagulation factor to be a cause. The symptoms vary depending on degree of activity of the blood coagulation factor, but if it becomes severe, intraarticular bleeding, intramuscular bleeding or the like may occur, resulting in impairment of joint function and movement. As a treatment of hemophilia, a replacement therapy for replenishing an insufficient blood coagulation factor from outside the body is performed.
Thrombotic thrombocytopenic purpura is a disease caused by dysfunction due to quantitative and qualitative abnormalities in ADAMTS13, cleavage enzyme of a von Willebrand factor (VWF), which may be congenital or acquired. This dysfunction induces excessive microthrombi, platelets are consumed and reduced by thrombogenesis, and organ failure associated with ischemic changes in various peripheral organs is caused. It has been reported that in the congenital case, it is caused by mutation of a gene encoding the protein, and in the acquired case, it is caused by autoantibody to the protein. As a treatment method, for congenital cases, the onset of the disease is prevented by infusing 10 ml/kg body weight of fresh frozen plasma every 2 weeks and replenishing ADAMTS13 enzymes. For acquired cases, plasma exchange therapy is performed.
Lysosomal storage disease is a group of diseases in which a large amount of lipid or mucopolysaccharide is accumulated in lysosome due to genetic deficiency of an acidic degradation enzyme in the lysosome which is an intracellular organelle, and various symptoms such as swelling of liver and spleen, bone degeneration and central nerve disorder are exhibited, and currently, 60 types of diseases are included (Non Patent Literature 1).
The diseases classified as lysosomal storage disease are distinguished by the type of enzyme being deficient, and examples thereof include Fabry disease, Gaucher disease, Pompe disease, and mucopolysaccharidosis.
Fabry disease, which is one of lysosomal storage diseases, is a disease that occurs when glycoproteins GL-3 and Gb3 are not degraded due to genetic deficiency or activity reduction of an enzyme called α-galactosidase (α-GAL, hereinafter also simply referred to as “GLA”), and the undegraded glycoproteins accumulate in tissues and organs. As symptoms, neurological symptoms (pain), corneal opacities, skin symptoms, cardiac dysfunction, renal dysfunction and the like are observed. As a therapeutic method, formulated α-GAL is replenished by drip infusion to degrade glycoprotein, thereby ameliorating symptoms and suppressing progression (Non Patent Literature 2).
As a treatment of a disease caused by the fact that a protein function cannot be exerted due to a genetic mutation, such as hemophilia and lysosomal storage disease, replacement therapy is administered. However, in the case of a mutation having a deletion or a large deficiency of a protein of a coagulation factor or a responsible enzyme, there are not a few cases where a coagulation factor or a responsible enzyme to be replenished from outside the body is regarded as a foreign substance by immunity in the patient's body, and a neutralizing antibody against the coagulation factor or responsible enzyme replenished from outside the body is produced. In a case where an antibody is produced in the body of a patient, the enzyme cannot sufficiently exert its function, so that its therapeutic effect is attenuated. For example, in the treatment of Fabry disease, a case has been confirmed in which a neutralizing antibody against α-GAL is produced in the body of a patient, and the antibody binds to the administered α-GAL, thereby attenuating the therapeutic effect (Non Patent Literatures 3 and 4).
As described above, when an antibody against a coagulation factor or a responsible enzyme is produced in the body of a patient, a disease occurs, or a situation in which a therapeutic method cannot exert a sufficient effect occurs, and thus a technique for controlling functions of these antibodies is required.
In order to solve the above problems, the present inventors have extensively conducted studies, and resultantly found that the problems can be solved by eliminating B cells that produce a neutralizing antibody as a cause by an immune cell expressing a novel B-cell antibody receptor. That is, the present invention is as follows.
(1) A B-cell antibody receptor wherein an antibody binding domain a), a transmembrane domain b), an intracellular domain of a costimulatory factor c) and an intracellular domain of an activated receptor d) are combined.
(2) The B-cell antibody receptor according to (1), wherein a linker is inserted into a binding moiety of said a), b), c) or d).
(3) The B-cell antibody receptor according to (1) or (2), wherein the antibody binding domain a) is a protein or peptide comprising an antibody recognition sequence specifically recognized by an antibody.
(4) The B-cell antibody receptor according to any one of (1) to (3), wherein the transmembrane domain b) is selected from a group consisting of CD28, CD3ζ, CD4, and CD8α.
(5) The B-cell antibody receptor according to any one of (1) to (4), wherein the intracellular domain of a costimulatory factor c) is selected from a group consisting of OX40, 4-1BB, CD27, CD278, and CD28.
(6) The B-cell antibody receptor according to any one of (1) to (5), wherein the activated receptor d) is selected from a group consisting of TCR and NKG2D.
(7) The B-cell antibody receptor according to any one of (1) to (6), wherein the antibody binding domain a) comprises a full-length protein of α-GAL, and a base sequence encoding the full-length protein of α-GAL comprises a base sequence set forth in SEQ ID NO: 1, or a base sequence 95% or more identical to the base sequence set forth in SEQ ID NO: 1, having an identical function.
(8) The B-cell antibody receptor according to any one of (1) to (6), wherein the antibody binding domain a) comprises an antibody recognition sequence region (D1 region) of α-GAL, and the base sequence encoding the antibody recognition sequence region (D1 region) of α-GAL comprises a base sequence set forth in SEQ ID NO: 2, or a base sequence 95% or more identical to the base sequence set forth in SEQ ID NO: 2, having an identical function.
(9) The B-cell antibody receptor according to any one of (1) to (6), wherein the antibody binding domain a) comprises a spacer domain protein of ADAMTS13, and the base sequence encoding the spacer domain protein of ADAMTS13 comprises a base sequence set forth in SEQ ID NO: 3 or 37, or a base sequence 95% or more identical to the base sequence set forth in SEQ ID NO: 3 or 37, having an identical function.
(10) The B-cell antibody receptor according to any one of (1) to (6), wherein the antibody binding domain a) comprises a spacer domain of ADAMTS13 and a cysteine-rich domain, a base sequence encoding the spacer domain of ADAMTS13 comprises a base sequence set forth in SEQ ID NO: 3 or 37 or a base sequence 95% or more identical to the base sequence set forth in SEQ ID NO: 3 or 37, having an identical function, and a base sequence encoding the cysteine-rich domain comprises a base sequence set forth in SEQ ID NO: 4 or a base sequence 95% or more identical to the base sequence set forth in SEQ ID NO: 4, having an identical function.
(11) The B-cell antibody receptor according to any one of (1) to (6), wherein the antibody binding domain a) comprises a full-length protein or an antibody recognition sequence of blood coagulation factor VIII or blood coagulation factor IX.
(12) A nucleic acid encoding the B-cell antibody receptor according to any one of (1) to (8).
(13) A vector comprising the nucleic acid according to (12).
(14) An immune cell expressing a B-cell antibody receptor that has been transfected using the nucleic acid according to (12) or the vector according to (13).
(15) The immune cell according to (14), wherein the immune cell is an αβT cell, a CD8T cell, a CD4T cell, γδT cell, or an NK cell.
(16) A regenerative medicinal product (cell medicine) comprising the immune cell according to (14) or (15) as an active ingredient.
(17) The regenerative medicinal product (cell medicine) according to (16), for treating and/or preventing lysosomal storage disease.
(18) The regenerative medicinal product (cell medicine) according to (17), wherein the lysosomal storage disease is Fabry disease.
(19) A method for treating and/or preventing lysosomal storage disease, comprising administering the immune cell according to (14) or (15) or the regenerative medicinal product (cell medicine) according to (17) to a subject in need of treatment.
(20) The method according to (19), wherein the lysosomal storage disease is Fabry disease.
(21) A nucleic acid encoding the B-cell antibody receptor according to any one of (1) to (6), (9) and (10).
(22) A vector comprising the nucleic acid according to (21).
(23) An immune cell expressing a B-cell antibody receptor that has been transfected using the nucleic acid according to (21) or the vector according to (22).
(24) The immune cell according to (23), wherein the immune cell is an αβT cell, a CD8T cell, a CD4T cell, a γδT cell, or an NK cell.
(25) A regenerative medicinal product (cell medicine) comprising the immune cell according to (23) or (24) as an active ingredient.
(26) The regenerative medicinal product (cell medicine) according to (25), for treating and/or preventing thrombotic thrombocytopenic purpura.
(27) A method for treating and/or preventing thrombotic thrombocytopenic purpura, comprising administering the immune cell according to (23) or (24) or the regenerative medicinal product (cell medicine) according to (26) to a subject in need of treatment.
(28) A nucleic acid encoding the B-cell antibody receptor according to any one of (1) to (6) and (11).
(29) A vector comprising the nucleic acid according to (28).
(30) An immune cell expressing a B-cell antibody receptor that has been transfected using the nucleic acid according to (28) or the vector according to (29).
(31) The immune cell according to (30), wherein the immune cell is an αβT cell, a CD8T cell, a CD4T cell, a γδT cell, or an NK cell.
(32) A regenerative medicinal product (cell medicine) comprising the immune cell according to (30) or (31) as an active ingredient.
(33) The regenerative medicinal product (cell medicine) according to (32), for treating and/or preventing hemophilia.
(34) A method for treating and/or preventing hemophilia, comprising administering the immune cell according to (30) or (31) or the regenerative medicinal product (cell medicine) according to (33) to a subject in need of treatment.
The present invention provides an effective means for treatment of diseases caused by antibodies produced in the body of a patient, for example, lysosomal storage disease.
The present invention provides a B-cell antibody receptor-immune cells for treating a disease that occurs when an antibody against a therapeutic enzyme or a coagulation factor or an endogenous protein replenished from outside body is produced in the body of a patient, and relates to immunotherapy using the cells. Here, the “B-cell antibody receptor-immune cell” (hereinafter, also simply referred to as “BAR-immune cell”) means an immune cell expressing a B-cell antibody receptor (BAR), and when the immune cell is a T cell, it is referred to as “B-cell antibody receptor-T cell” (hereinafter also simply referred to as “BAR-T cell”).
[1] B-Cell Antibody Receptor (BAR)
The BAR of the present invention contains an antibody binding domain a), a transmembrane domain b), an intracellular domain of a costimulatory factor c), and an intracellular domain of an activated receptor d). The BAR of the present invention has high specificity and cytotoxic activity against cells overexpressing antibodies to which the antibody binding domain a) binds.
The antibody binding domain a) is not particularly limited as long as it is a substance to which an antibody causing disease specifically binds. If B cells that inhibit therapeutic effects of enzyme replacement therapy for lysosomal storage disease and the like are damaged and eliminated, for example, α-GAL in the case of Fabry disease, glucocerebrosidase in the case of Gaucher disease, α-glucosidase in the case of Pompe disease, α-L-iduronidase in the case of mucopolysaccharidosis, a full length protein of these proteins or a peptide containing an antibody recognition sequence or the like is selected as the antibody binding domain a). The amino acid sequences or base sequences of these proteins are publicly available and can be obtained by keyword search from the National Center for Biotechnology Information (NCBI) database.
This antibody binding domain binds to and interacts with an antibody causing disease, thereby damaging and eliminating B cells that produce such an antibody.
Further, if B cells that produce an antibody that inhibits therapeutic effects by administration of a blood coagulation factor like hemophilia are damaged and eliminated, for example, a full-length protein of blood coagulation factor VIII or blood coagulation factor IX, a peptide containing an antibody recognition sequence or the like is selected.
In addition, in the case of thrombotic thrombocytopenic purpura, examples thereof include a full-length protein of ADAMTS13, or a peptide containing an antibody recognition sequence, and the like.
One embodiment of the antibody binding domain includes a full-length protein (Full Length) of α-GAL or a peptide containing an antibody recognition sequence region (D1 region), and the amino acid sequence of the full-length protein (Full Length) of α-GAL and the base sequence encoding the same are shown in SEQ ID NO: 10 and SEQ ID NO: 1, and the amino acid sequence of the antibody recognition sequence region (D1 region) and the base sequence encoding the same are shown in SEQ ID NO: 11 and SEQ ID NO: 2.
The base sequence encoding the full-length protein (Full Length) of α-GAL or the antibody recognition sequence region (D1 region) may be a substantially homologous base sequence having the identical function. Here, the term “substantially homologous” encompasses two or more biomolecular sequences which are significantly similar to each other at the primary base sequence level. For example, in the context of two or more nucleic acid sequences, the “substantially homologous” means at least about 75% identical, preferably at least about 80°/o identical, and more preferably at least about 85% identical or at least about 90% identical, and even more preferably at least about 95% identical, more preferably at least 97% identical, more preferably at least about 98% identical, and more preferably at least 99% identical.
One embodiment of the antibody binding domain includes ADAMTS13. ADAMTS13 is a protein to which a large number of domains are linked, and it is reported that most of antibodies against ADAMTS13 binds to a spacer domain, and thus it is considered that it is preferable to use the spacer domain as the antibody binding domain. An amino acid sequence of a spacer domain of ADAMTS13 and a base sequence encoding the same are shown in SEQ ID NOs: 12 and 3 (
The type of the transmembrane domain b) is not limited as long as it does not inhibit function of the B-cell antibody receptor. For example, transmembrane domains such as CD28. CD3. CD4 and CD8a expressed in T cells or the like can be used. The amino acid sequence or base sequence information of these transmembrane domains is publicly available and can be obtained by keyword search from the NCBI database. In these transmembrane domains, a mutation may be appropriately transferred as long as it does not inhibit the function of the B-cell antibody receptor. Here, the term “mutation” means any mutation including deletion, substitution or addition of one, several or a plurality of amino acids.
An oligopeptide linker or polypeptide linker can optionally be inserted into a binding moiety of the antibody binding domain a), the transmembrane domain b), the intracellular domain of the costimulatory factor c) or the intracellular domain of the activated receptor d), so as to link them to each other. Preferably, a linker composed of 2 to 10 amino acids in length can be used. For example, a flexible linker having an amino acid sequence “GSGGG” can be used.
As one embodiment of the transmembrane domain, the amino acid sequence of the transmembrane domain (TM) of CD3 and the base sequence encoding the same are shown in SEQ ID NO: 15 and SEQ ID NO: 6, respectively.
In the BAR of the present invention, a spacer sequence can be arranged between an extracellular domain and the transmembrane domain or between the intracellular domain and the transmembrane domain. The spacer sequence means any oligopeptide or polypeptide that serves to link the transmembrane domain with the extracellular domain and/or the transmembrane domain with the intracellular domain. The spacer sequence contains up to 300 amino acids, preferably 10 to 100 amino acids, and most preferably 10 to 50 amino acids.
The type of the intracellular domain of the costimulatory factor c) is not particularly limited as long as it is the intracellular domain derived from the costimulatory factor of an immune cell or the like. For example, one or more intracellular domains selected from a group consisting of costimulatory factors such as OX40, 4-1BB, CD27. CD278 and CD28 can be appropriately selected and used. The amino acid sequence or base sequence information of the intracellular domains of these costimulatory factors is publicly available and can be obtained by keyword search from the NCBI database. In the intracellular domains of these costimulatory factors, a mutation may be appropriately transferred as long as it does not inhibit the function of the B-cell antibody receptor.
One embodiment of the intracellular domain of the costimulatory factor includes 4-1BB, and the amino acid sequence thereof and the base sequence encoding the same are shown in SEQ ID NO: 16 and SEQ ID NO: 7, respectively.
The type of the intracellular domain of the activated receptor d) may be appropriately selected depending on the type of immune cells. For example, when a T cell is used as the immune cell, it is preferable to select CD3ζ which is the intracellular domain of a T cell receptor (TCR) (the intracellular domain derived from CD3, also called TCRζ chain). When an NK cell is used, it is preferable to select an intracellular domain derived from NKG2D. The amino acid sequence or base sequence information of these activated receptor domains is publicly available and can be obtained by keyword search from the NCBI database. In these intracellular domains, a mutation may be appropriately transferred as long as it does not inhibit the function of the B-cell antibody receptor.
One embodiment of the intracellular domain of the activated receptor includes CD3ζ, and the amino acid sequence thereof and the base sequence encoding the same are shown in SEQ ID NO: 17 and SEQ ID NO: 8, respectively.
The term “domain” herein is a region in a polypeptide. In the present specification, the “domain” is used as an “antibody binding domain”, a “transmembrane domain”, and an “intracellular domain”, depending on its position in a B-cell antibody receptor molecule.
A signal sequence can also be attached to support transport of BAR protein expressed in the cell to a cell membrane and the like. Examples of the signal sequence include Leader 2 derived from a receptor gene called CSF2RA (SEQ ID NOs: 5 and 14) and Leader 1 derived from CD8 (SEQ ID NOs: 24 and 25).
[2] Immune Cells
As an example of an immune cell into which the B-cell antibody receptor is transfected, an αβT cell, a CD8T cell, a CD4T cell, a γδT cell, an NK cell or the like can be used.
First, peripheral blood mononuclear cells (PBMCs) as raw materials for the αβT cell and the like are prepared. A method for collecting PBMCs is not particularly limited, but for example, PBMCs can be acquired by separating peripheral blood obtained by blood collection using density gradient centrifugation. The amount of blood collected at one time can be appropriately set by a therapeutic method, and for example, when 24 to 72 mL of blood is collected, 1×10{circumflex over ( )}7 to 3×10{circumflex over ( )}7 PBMCs can be acquired. In addition, when it is necessary to secure a large amount of cells, it is possible to collect mononuclear cell components using a blood component collection device.
In the case of producing αβT cells, collected PBMCs are suspended in a culture solution (medium), and interleukin-2 (IL-2) and an anti-CD3 antibody are added to the obtained cell suspension to perform culture.
The anti-CD3 antibody may be added to the medium or immobilized on the bottom surface of a culture vessel, but it is known that PBMCs can be more suitably cultured by seeding PBMCs in the culture vessel such as a flask in which the anti-CD3 antibody is immobilized (for example, JP-B-3056230). It is preferred that IL-2 is added so as to have a concentration of 1000 to 2000 IU/mL in the medium.
Since the αβT cells prepared as described above contain CD8T cells and CD4T cells, when CD8T cells or CD4T cells are used as immune cells, CD8T cells or CD4T cells can be obtained by positive selection or negative selection from the prepared αβT cells by a cell separation method using magnetic beads or the like.
In the case of producing NK cells, a method of culturing PBMCs in the presence of an agonist of a molecule expressed in NK cells such as an anti-CD16 antibody, IL-2 or the like is common (for example, JP-B-4275680 and the like). Any method may be used to produce NK cells used in the present invention. In addition, CD16+ and/or CD56+ cells prepared by a method described in Patent Literature WO 2009/151183 and the like may be used as the NK cells.
In the case of producing γδT cells, collected PBMCs are cultured in the presence of a phosphate antigen such as bisphosphonate and IL-2 to selectively proliferate and activate γδT cells, so that a cell group containing γδT cells with high purity can be prepared (for example. Patent Literature WO 2006/006720 and the like). Any method may be used to produce γδT cells used in the present invention.
The culture is performed at a temperature of 34 to 38° C., preferably 37° C. and a CO2 concentration of 2 to 10%, preferably 5%, and the culture period is preferably about 1 to 20 days, particularly about 1 to 2 weeks.
The medium to be used is not particularly limited, and commercially available media used for cell culture, such as AIM-V medium (Invitrogen), RPMI-1640 medium (Invitrogen), Dulbecco's modified Eagle's medium (Invitrogen), Iscove's medium (Invitrogen), KBM medium (Khojin Bio), and ALys medium (Cell Science & Technology Institute. Inc.), can be used. The medium may be a medium for human peripheral blood T cells (for example, ALys505N; Cell Science & Technology Institute, Inc.) to which IL-2 is added in advance. In addition, 5 to 20% of Fetal Bovine Serum (FBS). Betal Calf Serum (FCS), Human Serum, Human Plasma (HBP) or the like can be added as necessary.
A gene encoding the B-cell antibody receptor is transfected into the immune cells prepared as described above; αβT cells, CD8T cells, CD4T cells, γδT cells, and/or NK cells. The gene to be transfected can be selected from DNA or RNA. As the transfection method, transfect by a viral vector, transfect by an electroporation method, transfect by lipofection or the like can be appropriately selected.
The immune cells thus prepared are activated by an activation signal and a costimulatory signal by specifically binding to a B cell that produces an antibody causing disease via said antibody by their antibody binding domain a), thereby damaging and eliminating said B cells. As a result, the production of the antibodies causing diseases is suppressed, and the diseases are treated.
[3] Regenerative Medicinal Product (Cell Medicine) and Pharmaceutical Composition or Treatment and/or Prevention Method Using the Same
According to the present invention, cells expressing the B-cell antibody receptor (BAR) can be used for treating and/or preventing a disease, and typically, a regenerative medicinal product (cell medicine) and a pharmaceutical composition can be provided. Here, the “treatment” includes alleviation (moderation) of symptoms or associated symptoms characteristic of the target disease, inhibition or delay of deterioration of symptoms, and the like, and the treatment also includes amelioration of the disease. The “prevention” refers to preventing or delaying the onset/expression of disease (disorder) or symptoms thereof, or reducing risk of onset/expression.
In one embodiment, the regenerative medicinal product (cell medicine) of the present invention contains the cell of the present invention expressing BAR as an active ingredient, and may further contain a suitable excipient or the like.
In another embodiment, the pharmaceutical composition of the present invention contains an effective amount of the cells of the present invention as an active ingredient, and may further contain a suitable pharmaceutically acceptable excipient or the like. Examples of the excipient contained in the cell preparation and the pharmaceutical composition include various cell culture media, phosphate buffered saline, isotonic saline, and the like.
The term “regenerative medicinal product” in the present specification refers to:
1) cells of human or animal subjected to culture or other processing, among substances intended to be used for
(i) reconstruction, restoration or formation of a structure or function of a human or animal body; or
(ii) treatment or prevention of a human or animal disease; or
2) substances transferred into human or animal cells and containing genes expressed in these bodies, among substances intended to be used for treatment of a human or animal disease. Although not particularly limited, examples of the regenerative medicine product include:
1) human cell processing products
2) animal cell processing products
3) gene therapy products
A regenerative medicinal product containing the immune cell expressing the B-cell antibody receptor of the present invention as an active ingredient corresponds to a human somatic cell processing product.
[4] Nucleic Acid Encoding the B-Cell Antibody Receptor (BAR) of the Present Invention
According to the present invention, a nucleic acid encoding an amino acid sequence of the BAR described in the above [1] is provided. Unless otherwise specified, the “nucleic acid (or base sequence) encoding the amino acid sequence” includes all base sequences that are polycondensed each other and encode the same amino acid sequence.
The nucleic acid encoding the BAR can be easily prepared from a cDNA sequence of a specified responsible enzyme by a conventional method. For example, the nucleic acid encoding a), among a) to d) constituting BAR, can be amplified and acquired, for example, by extracting mRNA from somatic cells, preparing cDNA from the mRNA by reverse transcription, and using a PCR method or the like using the cDNA as a template. The nucleic acids encoding b), c) and d) can be amplified and acquired, for example, by extracting mRNA from immune cells, preparing cDNA from the mRNA by reverse transcription, and using a PCR method or the like using the cDNA as a template.
The nucleic acid encoding the BAR can be obtained by linking the nucleic acids encoding each of domains acquired as described above. The nucleic acids encoding each of domains can be linked using a known method such as cloning by homologous recombination, an overlap extension method, or the like.
The nucleic acid of BAR prepared can be incorporated into a plasmid or a viral vector and used, or mRNA can be synthesized and used.
[5] Method for Producing Immune Cells Expressing the B-Cell Antibody Receptor (BAR) of the Present Invention
A method for producing immune cells expressing the BAR of the present invention includes a step of transfecting a nucleic acid encoding the BAR according to the above 141 into immune cells. The step is performed by a method of transforming immune cells in vitro using a viral vector or non-viral vector containing a nucleic acid encoding the BAR, or an electroporation method.
For example, they can be produced by transforming immune cells in vitro using a viral vector or plasmid vector containing the nucleic acid encoding the BAR according to the present invention or the nucleic acid itself encoding the BAR according to the present invention.
The type and use of the vector are not particularly limited, but the vector may be a plasmid vector or a viral vector (for example, lentivirus, adenovirus, or retrovirus). Also, the vector may be, for example, a cloning vector or an expression vector. Examples of the expression vector include vectors for prokaryotic cells such as E. coli or actinomycetes, or vectors for eukaryotic cells such as yeast cells, insect cells, or mammalian cells.
Device and method used for electroporation are not particularly limited, and MaxCyte GT (MaxCyte. Inc.), Nepa Gene (Nepa Gene Co., Ltd.). Nucleofector (Lonza KK.), Gene Pulser (Bio-Rad Laboratories, Inc.) or the like can be used.
The immune cell expressing the B-cell antibody receptor (BAR) recognizes an antibody on a B cell with the antibody binding domain a), then transmits its recognition signal to the inside of a T cell or the like through a ζ chain, and activates a signal for inducing cytotoxic activity in the cell through the transmembrane domain and the costimulatory factor, thereby exerting cytotoxic activity on the B cell that produces the antibody causing disease.
[6] Immune Cells Expressing B-Cell Antibody Receptor (BAR) of the Present Invention
The immune cells expressing the BAR of the present invention is immune cells into which the nucleic acid encoding BAR of the above [4] is transfected and expressed by the production method of the above [5].
The regenerative medicinal product (cell medicine) of the present invention is an injection (cell suspension) obtained by suspending the immune cells of the present invention in a liquid that can be used as a medicine (for example, physiological saline), and can be used for treating and/or preventing a disease. This injection may be injected intravenously, intradermally, subcutaneously or the like, may be directly injected into a lesion, or may be systemically administered as a drip. The injection may contain a physiologically acceptable carrier or excipient.
The number of immune cells contained in the regenerative medicinal product (cell medicine) of the present invention can be appropriately set according to administration method, type of disease, symptom of the patient, age, sex, and weight of the patient, severity of the disease, and the like. Normally, the number may be set at 10{circumflex over ( )}8 to 10{circumflex over ( )}12 cells/person (preferably 10{circumflex over ( )}9 cells/person).
A method for producing the regenerative medicinal product (cell medicine) of the present invention is not particularly limited. For example, 1) the immune cells of the present invention are collected from the medium by centrifugation or the like; 2) the collected immune cells are washed with a washing liquid (for example, physiological saline. PBS, or the like); 3) the washed immune cells are collected from the washing liquid by centrifugation or the like; and 4) the collected immune cells are suspended in a liquid that can be used as a medicine (for example, physiological saline), whereby the regenerative medicinal product (cell medicine) can be produced as a drip infusion capable of being intravenously administered.
Examples of a disease to be a target of the regenerative medicinal product (cell medicine) of the present invention include lysosomal storage disease. The lysosomal storage disease includes Fabry disease, Gaucher disease, Pompe disease, and mucopolysaccharidosis. In addition, examples of the disease include diseases to which replacement therapy is adopted, such as hemophilia and thrombotic thrombocytopenic purpura.
A subject to be administered with the regenerative medicinal product (cell medicine) of the present invention is, for example, an animal suffering from or likely to suffer from the above disease. The animal is, for example, a mammal, preferably a human.
Hereinafter, the present invention will be described in details with reference to Examples, but the present invention is not limited to these Examples.
1. Identification of Base Sequence and Amino Acid Sequence of GLA
The base sequence and amino acid sequence of GLA are publicly available, and the inventors obtained sequence information from the National Center for Biotechnology Information NCBI database (https://www.ncbi.nlm.nih.gov/protein/4NXS_A). The base sequence of D1 domain which is an antibody recognition sequence region was identified according to Non Patent Literature (J Mol Biol. 2004 Mar. 19; 337(2):319-35). The base sequence encoding the GLA full-length protein is shown in SEQ ID NO: 1, and the base sequence encoding the antibody recognition sequence region (D1 domain) of GLA is shown in SEQ ID NO: 2. MGC Human GLA Sequence-Verified cDNA (Clone ID: 3609235) (Dharmacon Inc.) in which the sequence of GLA was inserted into a plasmid was purchased, and the plasmid was used as a template to isolate the base sequence of the full-length protein (Full Length) using primer sets shown in SEQ ID NO: 19 and SEQ ID NO: 20 and the base sequence of the antibody recognition sequence region (D1 region) using primer sets shown in SEQ ID NO: 19 and SEQ ID NO: 21.
2. Specification of Base Sequences of Two Types of BARs
As amino acid sequences of the intracellular domain of the costimulatory factor (4-1BB) and the intracellular domain of the activated receptor (CD3ζ), the transmembrane domain (TM) and the spacer sequence which constitute BAR, those that have already been demonstrated to be effective in CD19 CAR-T cells (Patent Literature; WO 2012079000 A1 SEQ ID NO: 8) were employed. The base sequence in which the transmembrane domain, 4-1BB and CD3ζ were bound was synthesized by an artificial gene synthesis contract service of Integrated DNA Technologies, Inc., and the base sequence encoding the transmembrane domain, 4-1BB and CD3ζ was acquired using the synthesized base sequence as a template and using primer sets shown in SEQ ID NO: 22 and SEQ ID NO: 23.
The base sequence of the full-length protein (Full Length) or the antibody recognition sequence region (D1 region) described above and the base sequence in which the transmembrane domain, 4-1BB and CD3ζ were bound were inserted into XhoI-EcoRI of the pMSCV plasmid by In-fusion cloning, and the base sequence of the BAR was cloned.
3. Preparation of GLA BAR-T Cell
The base sequences of the two types of BARs specified in the above item 2 are subcloned into a general-purpose plasmid, and a plurality of restriction enzyme sites are transfected into the 5′ side of Spacer of this sequence. The sequence of an antigen was recombined using this restriction enzyme site. The base sequence of the full-length protein of GLA or D1 region, which is the antibody binding domain, was amplified by PCR, and after confirming that no mutation was contained in the sequence by Sequencing, recombined into the plasmid.
Using thus obtained plasmid subjected to recombination as described above, the base sequence encoding the full-length protein (Full Length: FL) BAR of GLA and the base sequence encoding GLA BAR of the D1 region were inserted into retroviral vectors (
Using these vectors, transfection into human T cells was performed to prepare FL BAR-T cells and D1 BAR-T cells. FACS was performed using an anti-GLA antibody. The results are shown in
As another transfection method. mRNA Transfection was performed. The base sequence constituting the BAR was subcloned into a plasmid vector (pmRNAex,
4. Preparation of Anti-GLA Antibody-Overexpressed Cells (Anti-GLA scFv-Overexpressing M1 Cells)
Spleen cells of a mouse inoculated with human GLA protein and myeloma cells were fused to obtain hybridomas. This heterogeneous hybridomas population was positively selected in a HAT medium to obtain hybridomas that produce an anti-GLA monoclonal antibody (
Next, antibody sequencing was performed from the anti-GLA monoclonal antibody-producing B cells to obtain amino acid sequences of variable regions of heavy chain and light chain of the anti-GLA monoclonal antibody. Based on the obtained amino acid sequence, a linker was interposed to create a sequence of scFv of the anti-GLA antibody (
The base sequence of the scFv of the anti-GLA antibody specified above was subcloned, and recombined into the plasmid vector (
The obtained plasmid vector was subcloned, and recombined into a retroviral vector. M1 cells that are established cells derived from mouse myelogenous leukemia were infected using the obtained retroviral vector, thereby creating Transfectant in which the scFv of the anti-GLA antibody was forcibly expressed, and the Transfectant was used as a model cell of neutralizing antibody-producing B cells. It was confirmed from a binding assay of GLA-mCherry for the prepared model cells that the anti-GLA antibodies were overexpressed (
5. Preparation of CD19 CAR-T Cells
According to Material and Method of Non Patent Literature (New England Journal of Medicine 2014; 371 (16): 1507-1517), a base sequence encoding CD19 CAR-T described in Patent Literature (WO 2012079000 A 1 SEQ ID NO: 8) was inserted into a retroviral vector or a lentiviral vector. After preparing the retroviral vector or the lentiviral vector, transfection into human T cells was performed using retronectin to prepare CD19 CAR-T cells.
6. In Vitro Killing Assay
(Method)
The co-culture of the BAR-T cells and the M1 cells transfected with GFP was subjected as a transfection control, and the co-culture of the CD19 CAR-T cells and the scFv-overexpressing M1 cells of the anti-GLA antibody was subjected to eliminate a possibility of non-specific reaction.
(Results)
The results are shown in
7. Preparation of Target Cells (Anti-GLA scFv-Expressing Cells)
A retroviral vector encoding scFv of the anti-GLA antibody (
8. Preparation of GLA BAR-T Cells
A retroviral vector (
9. Confirmation of Activation of BAR-T Cells by Mixed Culture
The BAR-T cells (Jurkat-GLA-BAR2-EGFP:Effector) prepared in the above item 8 and the target cells (NALM6-scFv-GLA-mCherry:Target) prepared in the above item 7 were mixed and cultured at mixing ratios of 1:0, 2:1, 1:1 for 24 hours, and a detection test of CD69 was performed. It is known that expression of CD69 increases when Jurkat is activated, and it was considered that the expression of CD69 increases depending on the mixing ratio, if the BAR-T cell transfected into Jurkat binds to the scFv of the anti-GLA antibody of the target cells and an activation signal is received. As a result of performing the mixed culture, the expression of CD69 increased in the presence of the target cells as compared with a control in the absence of the target cells (E:T=1:0), and the expression of CD69 further increased when the target cells increased (E:T=2:1→1:1) (
10. Preparation of BAR-T Cells (ADAMTS13)
A retroviral vector encoding the amino acid sequence of ADAMTS13 (
By using the present invention, it is possible to specifically damage and eliminate B cells that produce a specific antibody, and thus it can be expected to improve patients with lysosomal storage disease or the like who produce an antibody against the enzyme and cannot obtain a sufficient therapeutic effect even though enzyme replacement therapy is performed. The present invention can also be expected as a therapeutic agent for diseases caused by antibodies.
SEQ ID NO: 1<223> DNA encoding α-GAL full-length protein
SEQ ID NO: 2<223> DNA encoding α-GAL D1 region protein
SEQ ID NO: 3<223> DNA encoding ADAMTS13 spacer domain
SEQ ID NO: 4<223> DNA encoding ADAMTS13 cysteine-rich domain
SEQ ID NO: 5<223> DNA encoding signal sequence Leader 2
SEQ ID NO: 6<223> DNA encoding transmembrane domain
SEQ ID NO: 7<223> DNA encoding 4-1BB
SEQ ID NO: 8<223> DNA encoding CD3ζ
SEQ ID NO: 9<223> DNA encoding scFv of anti-GLA antibody
SEQ ID NO: 10<223> α-GAL full-length protein
SEQ ID NO: 11<223> α-GAL D1 region protein
SEQ ID NO: 12<223> ADAMTS13 Spacer domain
SEQ ID NO: 13<223> ADAMTS13 Cysteine-rich domain
SEQ ID NO: 14<223> Signal sequence Leader 2 protein
SEQ ID NO: 15<223> Transmembrane domain protein
SEQ ID NO: 16<223>4-1BB protein
SEQ ID NO: 17<223> CD3ζ protein
SEQ ID NO: 18<223> scFv protein of anti-GLA antibody
SEQ ID NO: 19<223> α-GAL Primer (forward)
SEQ ID NO: 20<223> α-GAL Primer (reverse: full length)
SEQ ID NO: 21<223> α-GAL Primer (reverse: D1 region)
SEQ ID NO: 22<223> TM, 4-1BB. CD3ζ Primer (forward)
SEQ ID NO: 23<223> TM, 4-1BB, CD3ζ Primer (reverse)
SEQ ID NO: 24<223> DNA encoding signal sequence Leader 1
SEQ ID NO: 25<223> Signal sequence Leader 1 protein
SEQ ID NO: 26<223> DNA encoding human CD8 signal peptide
SEQ ID NO: 27<223> Human CD8 signal peptide
SEQ ID NO: 28<223> DNA encoding HA
SEQ ID NO: 29<223> HA protein
SEQ ID NO: 30<223> DNA encoding IgG4 antibody hinge region
SEQ ID NO: 31<223> IgG4 antibody hinge region protein
SEQ ID NO: 32<223> DNA encoding human IgG anti-GLA antibody L chain
SEQ ID NO: 33<223> Human IgG anti-GLA antibody L-chain protein
SEQ ID NO: 34<223> DNA encoding mCherry
SEQ ID NO: 35<223> mCherry protein
SEQ ID NO: 36<223> DNA encoding scFv of anti-GLA antibody
SEQ ID NO: 37<223> DNA encoding ADAMTS13 spacer domain
SEQ ID NO: 38<223> ADAMTS13 Spacer domain
SEQ ID NO: 39<223> DNA encoding signal peptide of human IgG anti-ADAMTS13 antibody L-chain
SEQ ID NO: 40<223> Signal peptide of human IgG anti-ADAMTS13 antibody L-chain
SEQ ID NO: 41<223> DNA encoding scFv of anti-ADAMTS13 antibody
SEQ ID NO: 42<223> scFv of anti-ADAMTS13 antibody
SEQ ID NO: 43<223> DNA encoding human CD8a transmembrane domain
SEQ ID NO: 44<223> Human CD8a transmembrane domain
| Number | Date | Country | Kind |
|---|---|---|---|
| 2019-199624 | Nov 2019 | JP | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2020/040515 | 10/28/2020 | WO |
