WO2018225732A1 - Mage-a4由来ペプチドを認識する抗原結合性タンパク質 - Google Patents
Mage-a4由来ペプチドを認識する抗原結合性タンパク質 Download PDFInfo
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Definitions
- the present invention relates to an antigen-binding protein that recognizes a MAGE-A4-derived peptide.
- Immunotherapy is mainly divided into three groups: vaccine therapy, antibody therapy, and cell infusion therapy.
- TCR T cell receptor
- CAR chimeric antigen receptor
- TCR gene infusion therapy is a system that recognizes peptide MHC complexes (pMHC) and kills cancer, it is a safe and effective treatment method, and is being developed worldwide. However, there is a problem that it is extremely difficult to isolate a killer T cell clone that can be used for it.
- the following three points can be cited as the advantages of CAR infusion therapy.
- (1) Unlike original T cells, T cells into which a single chain antibody (scFv), T cell receptor (TCR), or a signal transduction domain of a costimulatory molecule has been introduced are cancers that are independent of MHC. Because it can recognize and destroy antigens, it has the advantage that it can be applied to a wide range of patients.
- CD19 molecule is also expressed on B cells, but disappearance of B cells can be dealt with by supplementation with immunoglobulins. In general, it is ideal to use an antigen that is expressed only in cancer cells. However, there is a problem that such cell surface antigens are not found at present.
- Non-patent Document 2 If an antibody that specifically recognizes a peptide MHC complex is obtained and CAR-T cells that specifically kill cancer cells can be produced, it can be a breakthrough therapy. CARs that recognize peptide MHC complexes can be expected to proliferate by antigen-presenting cells by injecting CAR-T cells into the body and administering peptides that are specifically recognized by CAR-T cells. .
- the present invention has been made in view of the above-described problems, and an object thereof is to provide an antigen-binding protein that specifically recognizes a MAGE-A4-derived peptide / HLA-A2 complex.
- MAGE-A4 molecule was used as an antigen presented to MHC.
- MAGE-A4 is expressed in solid cancers including malignant melanoma (Non-patent Document 3), and is a CTA (cancer testis antigen) as in NY-ESO-1. These molecules do not show protein expression in normal tissues other than testis and placenta.
- MAGA-A4 is expressed in cells and then undergoes proteasomal degradation. Part of it becomes a 10-amino acid MAGE-A4 peptide that associates with HLA-A * 0201 and beta-2 microglobulin in the endoplasmic reticulum and is presented on the cell surface as a cancer-specific antigen.
- P230-239 (GVYDGREHTV: SEQ ID NO: 1) was selected as a peptide presented on HLA-A * 0201 (Non-patent Document 4).
- a method of applying CAR to human cell infusion therapy by introducing CAR-T that specifically recognizes A2-MAGE-A4 into human lymphocytes and generating CAR-T cells that show a specific response to the target cancer.
- (1) Isolate multiple antibodies with high affinity that recognize MAGE-A4 derived peptide and HLA-A2 complex, (2) investigate the specificity of the isolated antibody, High antibodies were selected, and (4) CAR-T cells using the antibodies were prepared, and it was confirmed that the CAR-T cells were activated by the specific cancer cell of the target and caused cytotoxic activity.
- CAR-T cells specifically proliferated in the mouse body and tumor invasion was observed.
- the present invention has been basically completed.
- the antigen-binding protein according to the present invention includes the following polypeptide (A) or (B), and recognizes an HLA-A2-MAGE-A4 complex as an antigen, that is, (A) a VH of SEQ ID NO: 36 A polypeptide comprising the amino acid sequence of (heavy chain variable region) and the amino acid sequence of VL (light chain variable region) of SEQ ID NO: 38, or (B) the amino acid sequence of VH (heavy chain variable region) of SEQ ID NO: 36 A polypeptide comprising an amino acid sequence having 90% or more homology, an amino acid sequence of VL (light chain variable region) of SEQ ID NO: 38, and an amino acid sequence having 90% or more homology.
- the following polypeptide (C) or (D) (C) a polypeptide comprising the sc (single chain) amino acid sequence of SEQ ID NO: 37, or (D) It is preferable to include a polypeptide comprising an amino acid sequence having 90% or more homology with the amino acid sequence of sc (single chain) of SEQ ID NO: 37. Further, the polypeptide is preferably a polypeptide comprising the amino acid sequence of SEQ ID NO: 32 or an amino acid sequence having 90% or more homology with the amino acid sequence of SEQ ID NO: 32.
- the antigen-binding protein is preferably Fab, Fab ′, F (ab ′) 2 , Fv, or single chain Fv (scFv). In the present specification, the antigen-binding protein includes an antibody itself or an antibody derivative.
- homology means a relationship determined by comparing two (or three or more) amino acid sequences or base sequences.
- Homology means the degree of correlation by alignment between amino acid sequences or base sequences or between a series of partial sequences. Specifically, it is determined by sequence identity and retention (substitution that maintains specific amino acids in the sequence or physicochemical properties in the sequence).
- the method for determining homology is preferably a method that allows the longest alignment between the sequences to be compared. In order to determine the homology, it is obtained using a program available via the Internet, for example, BLAST (Basic Local Alignment Search Tool) ⁇ https://blast.ncbi.nlm.nih.gov/Blast.cgi>. It is done.
- a nucleic acid according to another invention encodes the antigen-binding protein according to the first invention.
- a vector according to another invention includes the nucleic acid.
- the nucleic acid includes DNA or RNA.
- the nucleic acid may be either single-stranded or double-stranded.
- Vectors include plasmid vectors, viral vectors, and the like.
- a chimeric antigen receptor according to another invention includes the intracellular domain of the antigen-binding protein and a signal transduction protein.
- the signal transduction protein is preferably either a CD3zeta (CD3 ⁇ ) chain or a costimulatory molecule CD (GITR). Further, it preferably further contains either the intracellular domain of CD28 or the intracellular domain of GITR.
- a nucleic acid according to another invention encodes the chimeric antigen receptor.
- the vector according to another invention includes this nucleic acid.
- a cell according to another invention expresses the chimeric antigen receptor.
- the pharmaceutical composition which concerns on another invention contains this cell as an active ingredient.
- an antigen-binding protein that specifically recognizes a MAGE-A4-derived peptide / HLA-A2 complex, a nucleic acid encoding the antigen-binding protein, a vector containing the nucleic acid, and a chimera containing the antigen-binding protein
- an antigen receptor a nucleic acid encoding a chimeric antigen receptor
- a vector containing the nucleic acid a cell expressing the chimeric antigen receptor
- a pharmaceutical composition containing the cell containing the cell.
- Antigen-binding proteins that specifically recognize MAGE-A4-derived peptides / HLA-A2 complexes can be used in the fields of cell medicine and gene therapy.
- the CAR-T cell of the present invention has very few side effects, it can provide a very effective cancer therapy.
- the arrow indicates the treatment day (day 3) of whole body radiation (TBI) on the left side, and the day of lymphocyte administration (day 4) on the right side.
- antibody and “antigen-binding fragment” mean an antigen-binding protein in the immune system.
- An antibody having an antigen-binding region is a glycoprotein containing two heavy chains (H chains) and two light chains (L chains) linked together by disulfide bonds.
- Each heavy chain includes a heavy chain variable region (VH) and a heavy chain constant region (CH).
- the heavy chain constant region is composed of three domains, CH1, CH2 and CH3.
- Each light chain includes a light chain variable region (VL) and a light chain constant region (CL).
- VL is composed of CL domains.
- the VH and VL regions can be further subdivided into regions having hypervariability called complementarity determining regions (CDR) and regions that are conserved to some extent, framework regions (FR).
- CDR complementarity determining regions
- FR framework regions
- three CDRs and four FRs are arranged in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 from the amino terminus to the carboxy terminus, respectively. It is known that CDR3 is important for binding to an antigen.
- VH and VL contain binding domains that interact with antigens.
- HLA Human Leukocyte Antigen (human leukocyte antigen), meaning human major histocompatibility complex (MHC), which is a cell surface molecule required for antigen presentation to immune cells.
- MHC major histocompatibility complex
- HLA can be classified into class I and class II.
- Class I HLA is composed of ⁇ -chain and ⁇ 2-microglobulin.
- Class I HLA is expressed on almost all nucleated cells and functions in antigen presentation to CD8 positive T cells.
- Class I HLA can be further classified into HLA-A, HLA-B and HLA-C.
- chimeric antigen receptor means an extracellular domain that binds to an antigen, a transmembrane domain derived from a polypeptide different from the extracellular domain, and at least one intracellular domain. Means. CAR is sometimes referred to as “chimeric receptor”, “T-body”, or “chimeric immune receptor (CIR)”. “Extracellular domain that binds to an antigen” means any oligopeptide or polypeptide that binds to an antigen, and “intracellular domain” refers to a signal that activates or inhibits a biological process in a cell. By any oligopeptide or polypeptide known to function as a transducing domain. As used herein, “antigen-binding protein” means a fragment of a part or all of an antibody that binds to an antigen and confers specificity to the antibody.
- Antigen-binding fragments of the present invention and nucleic acids encoding them Anti-MAGE-A4-derived peptide / HLA-A2 (HLA-A2-MAGE-A4) complex antibody is derived from HLA-A2-restricted MAGE-A4 It is an antibody that specifically recognizes and binds to a complex of peptide P230-239 (SEQ ID NO: 1) and HLA-A2.
- the antibody of the present invention has high specificity and does not bind to a complex of a peptide other than HLA-A2 and HLA-A2 and to a complex of P230-239 peptide and HLA other than HLA-A2 (or extremely low binding activity). ).
- the antigen-binding fragment of the present invention can specifically detect or target the HLA-A2-MAGE-A4 complex.
- the antigen-binding fragment of the present invention comprises (A) a polypeptide comprising the amino acid sequence of VH (heavy chain variable region) of SEQ ID NO: 36 and the amino acid sequence of VL (light chain variable region) of SEQ ID NO: 38, or ( B) Amino acid sequence having 90% or more homology with the amino acid sequence of VH (heavy chain variable region) of SEQ ID NO: 36, and 90% or more homology with the amino acid sequence of VL (light chain variable region) of SEQ ID NO: 38 And a polypeptide comprising an amino acid sequence having Further, between VH and VL, (C) a polypeptide comprising the amino acid sequence of sc (single chain) of SEQ ID NO: 37, or (D) the amino acid sequence of sc (single chain) of SEQ ID NO: 37 and 90 Polypeptides containing amino acid sequences with%
- Antigen-binding fragments include one or more fragments that specifically bind to an antigen.
- Antibody fragments that contain the antigen-binding region of an antibody specifically recognize the antigen, as do full-length antibodies. Examples of antibodies or fragments thereof contained in the antigen-binding fragment include Fab, Fab ′, F (ab ′) 2 , Fv, and single chain Fv (scFv).
- Fab is a monovalent antibody fragment composed of VL, VH, CL and CH1 domains.
- Fab ′ is a monovalent antibody fragment composed of a VL, VH, CL, CH1 domain and a hinge region.
- F (ab ′) 2 is a divalent antibody fragment containing two Fab fragments linked by a disulfide bond at the hinge region.
- Fv is a monovalent antibody fragment composed of VL and VH of a single arm of an antibody.
- the two domains VL and VH of the Fv fragment are encoded by separate genes, and these domains can be linked with a linker by a gene recombination technique to produce scFv, which is a single molecule protein.
- scFv includes a linker (single chain: sc) between VH and VL.
- This sc is a peptide commonly used in the art to link VH and VL and stabilize the antigen binding ability of the scFv antibody (for example, Huston et al., Methods in Enzymology, 203: 46- 88 (1991), Whitlow et al., Protein Eng., 6: 989 (1993)).
- sc generally comprises glycine and serine, and its length is 15-18 amino acids.
- One aspect of the invention includes combinations of antigen binding fragments.
- antigen binding fragments include Fab3, Diabody, Triabody, Tetrabody, Minibody, Bis-scFv, (scFv) 2-Fc, and intact-IgG (Holliger et al., Nature Biotechnology, 23 (9), p. 1126-36 (2005)).
- An antigen-binding fragment can be modified by one or several amino acids as long as it does not substantially affect its properties. For example, amino acids can be substituted, deleted, added or inserted in the constant region or FR region. This modification can be easily performed by combining known methods such as site-specific mutagenesis (point mutagenesis, cassette mutagenesis, etc.), gene homologous recombination, primer extension, and PCR.
- the present invention includes a nucleic acid encoding the antigen-binding fragment. Further, the nucleic acid of the present invention includes the base sequences shown in SEQ ID NOs: 31, 33 to 35 in the sequence listing. These nucleic acids can be modified into codons suitable for host cells (optimal codons) without changing the encoded amino acid sequence. By changing to an optimal codon, the expression efficiency of the polypeptide in the host cell can be improved.
- the antigen-binding fragment of the present invention can be produced using a known genetic engineering technique or a method such as chemical synthesis.
- a cloning vector or expression vector containing the nucleic acid of the present invention is prepared, this vector is introduced into a host cell, the host cell is cultured to express the nucleic acid, and the resulting polypeptide is recovered.
- -It can be manufactured by purification.
- the nucleic acid of the present invention comprises a plurality of nucleic acid molecules
- a combination of a plurality of vectors containing each nucleic acid molecule can be introduced into the host cell, or one vector containing a plurality of nucleic acids can be introduced into the host cell.
- a peptide tag can be linked and recovered and purified using this peptide tag.
- the vector of the present invention is operably linked to appropriate control sequences so that the nucleic acid is expressed in an appropriate host.
- control sequences include a promoter that transcribes nucleic acid, an operator sequence that controls transcription, a sequence that encodes a ribosome binding site, an enhancer, a polyadenylation sequence, and a sequence that controls the termination of transcription / translation.
- Various known sequences for example, restriction enzyme cleavage sites, marker genes (selection genes) such as drug resistance genes, signal sequences, leader sequences, etc.
- sequences can be appropriately selected and used depending on conditions such as the type of polypeptide to be expressed, host cells, culture medium and the like.
- a vector that is integrated into the host genome, a vector that is not integrated, an episomal vector that exists in the cytoplasm and replicates autonomously, and the like can be used.
- examples of such vectors include retrovirus vectors (including oncoretrovirus vectors, lentivirus vectors, pseudotype vectors), adenovirus vectors, adeno-associated virus (AAV) vectors, simian virus vectors, vaccinia virus vectors, Sendai virus.
- retrovirus vectors including oncoretrovirus vectors, lentivirus vectors, pseudotype vectors
- AAV adeno-associated virus vectors
- simian virus vectors simian virus vectors
- vaccinia virus vectors Sendai virus.
- Sendai virus Sendai virus.
- examples thereof include viral vectors such as vectors, Epstein-Barr virus (EBV) vectors, and HSV vectors.
- ESV Epstein-Barr virus
- HSV vectors a vector that is deficient
- Non-viral vectors can also be used in combination with condensing agents such as liposomes and cationic lipids.
- nucleic acids can be introduced into cells by calcium phosphate transfection, DEAE-dextran, electroporation, particle bombardment.
- Known cells can be used as host cells.
- prokaryotic cells such as E. coli, Chinese hamster ovary cells (CHO cells), mammalian cells such as human-derived cells, and eukaryotic cells such as yeast and insect cells.
- An antigen-binding fragment of the invention expressed in a host cell can be purified from the host cell medium, host cell extracts and / or lysates. The purification method can be performed by appropriately combining known methods.
- centrifugation hydroxyapatite chromatography, gel electrophoresis, dialysis, fractionation on ion exchange columns, ethanol precipitation, reverse phase HPLC, chromatography on silica, chromatography on heparin sepharose, anion or cation Resin chromatography (polyaspartic acid column, etc.), chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, affinity chromatography and the like can be used as appropriate.
- the present invention includes a composition comprising the antibody or antigen-binding fragment of the present invention described in (1) above, and a composition comprising a nucleic acid encoding the antibody or antigen-binding fragment of the present invention.
- the antibody or antigen-binding fragment of the present invention can be used as a probe for a complex of MAGE-A4-derived peptide and HLA-A2.
- MAGE-A4 is expressed inside cancer cells and has been shown to be presented by HLA, and T cells attack the cancer cells based on antigen recognition. Therefore, the antigen-binding fragment of the present invention can be used as a probe for cancer cells, that is, a detection composition or a diagnostic composition.
- samples to be diagnosed or detected include biological samples (for example, body fluids such as tissues, cells, blood, serum, plasma, lymph, urine, and the like at the site of disease). These samples are pre-purified, homogenized, centrifuged, or diluted as necessary, and then contacted with the antigen-binding fragment of the present invention in an appropriate buffer to detect the antigen / antibody complex. To do.
- the antigen-binding fragment of the present invention may be labeled, or a labeled secondary antibody may be used.
- Labels include enzymes (eg, horseradish peroxidase, alkaline phosphatase, etc.), radioisotopes (eg, 32 P, 35 S, 3 H, 125 I, etc.), fluorescent substances (eg, rhodamine, fluorescamine, dansyl chloride, derivatives thereof) Etc.), tag peptides and the like can be used.
- the antigen-binding fragments of the invention can be used to deliver a medicament to a target.
- the antibody or antigen-binding fragment of the invention linked to a medicament specifically recognizes the complex of WT1 peptide and HLA-A24 and delivers cytokines (IL2, TNF, IFN- ⁇ ), their receptor proteins, cytotoxins (such as ricin, diphtheria, gelonin), radionuclides (such as 90 Y, 131 I, 225 Ac, 213 Bi, 223 Ra and 227 Th), cells (T cells) And NK cells) or low molecular compounds (calicheamicin, doxorubicin, etc.).
- cytokines IL2, TNF, IFN- ⁇
- their receptor proteins such as ricin, diphtheria, gelonin
- radionuclides such as 90 Y, 131 I, 225 Ac, 213 Bi, 223 Ra and 227 Th
- T cells T cells
- NK cells NK cells
- low molecular compounds
- the effective amount of the active ingredient of the pharmaceutical composition of the present invention is appropriately determined according to the purpose, the condition of the tumor to be administered, such as the type, site and size of the tumor, the condition of the patient, the route of administration and the like.
- various known pharmaceutically acceptable ingredients for example, carriers, excipients, buffers, stabilizers, etc.
- the pharmaceutical composition can take the form of drugs such as tablets, solutions, powders, gels, sprays, or microcapsules, colloidal distribution systems (liposomes, microemulsions, etc.) and macroemulsions.
- Examples of the method for administering the pharmaceutical composition include intravenous, intraperitoneal, intracerebral, intraspinal, intramuscular, intraocular, intraarterial, intrabiliary, intralesional injection, injection, and sustained release system preparation. It is done.
- the pharmaceutical composition can be administered continuously by infusion or by injection.
- the pharmaceutical composition of the present invention includes hematopoietic tumors such as chronic myeloid leukemia, acute lymphoblastic leukemia (ALL), and acute myeloid leukemia (AML), stomach cancer, colon cancer, lung cancer, breast cancer, germ cell cancer, liver cancer, skin It is used for the treatment of solid cancers such as cancer, bladder cancer, prostate cancer, uterine cancer, cervical cancer, ovarian cancer, mesothelioma, and diagnosis / detection of each tumor / solid cancer. In particular, it is useful for the treatment, diagnosis and detection of MAGE-A4 positive and HLA-A2 positive hematopoietic tumors and solid cancers.
- ALL acute lymphoblastic leukemia
- AML acute myeloid leukemia
- the present invention includes a chimeric antigen receptor (CAR) comprising the antigen-binding fragment described in (1) above and the intracellular domain of a signal transduction protein.
- CAR specifically recognizes and binds to the HLA-A2-MAGE-A4-derived peptide complex and can signal to cells that express CAR.
- CAR includes an extracellular domain, a transmembrane domain, and an intracellular domain.
- the extracellular domain includes an antigen-binding fragment of the present invention.
- Stimulated cells produce cytokines and the like, can exert cytotoxicity against target cells expressing the complex, or can induce cytotoxicity of other immune cells.
- an intracellular domain of CAR one that can signal inside the cell when an extracellular domain existing in the same molecule interacts (bonds) with the MAGE-A4-derived peptide / HLA-A2 complex can be used.
- signal transduction proteins include proteins selected from membrane proteins, signal receptors, and cytokine receptors.
- Examples of the intracellular domain of CAR include an intracellular domain containing a primary cytoplasmic signal transduction sequence derived from CD3zeta (CD3 ⁇ ), FCR ⁇ , FcR ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD5, CD22, CD79a, CD79b and CD66d.
- cells containing secondary cytoplasmic signal (costimulatory signal) transduction sequences derived from CD2, CD4, CD5, CD8 ⁇ , CD8 ⁇ , CD28, CD137 (also referred to as “4-1BB”), CD134, ICOS, GITR and CD154 Domains are illustrated.
- an intracellular domain containing a tertiary cytoplasmic signal transduction sequence derived from IL-2 receptor and IL-21 receptor is exemplified.
- the transmembrane domain of CAR is, for example, the ⁇ chain, ⁇ chain, CD3 ⁇ chain, CD28, CD3 ⁇ , CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86 of the T cell receptor.
- CD134, CD137 (also referred to as “4-1BB”), ICOS, CD154, and GITR transmembrane domains can be used.
- an artificially designed arrangement may be used.
- the present invention includes a nucleic acid encoding CAR.
- a nucleic acid encoding CAR can be linked to another nucleic acid so that it is expressed under the control of a suitable promoter.
- any of those that constitutively promote expression and those that are induced by drugs or the like can be used.
- phosphoglycerate kinase (PGK) promoter Xist promoter, ⁇ -actin promoter, RNA polymerase II promoter and other mammalian promoters
- SV40 early promoter SV40 early promoter
- cytomegalovirus promoter herpes simplex virus thymidine kinase promoter
- various retroviruses Virus-derived promoters such as the LTR promoter can be used.
- nucleic acids containing promoters or other regulatory elements that cooperate with the transcription initiation site may be linked.
- a gene that can serve as a marker for confirming nucleic acid expression for example, a drug resistance gene, a gene encoding a reporter enzyme, or a gene encoding a fluorescent protein may be incorporated.
- a nucleic acid encoding CAR can be introduced into a cell using the vector described above. Furthermore, the nucleic acid encoding CAR can be used as an active ingredient in a pharmaceutical composition. A pharmaceutical composition containing a nucleic acid encoding CAR can be produced and used based on the description in (2) above.
- cells expressing CAR are included. The cell can be prepared by introducing a nucleic acid encoding the CAR of the present invention into the cell. A cell expressing CAR is activated by binding a signal to the HLA-A2-MAGE-A4 complex via CAR, thereby transmitting a signal into the cell.
- CAR-expressing cells are useful in adoptive immunotherapy, particularly in adoptive immunotherapy for MAGE-A4-positive, HLA-A2-positive tumors or cancer.
- the step of introducing a nucleic acid encoding CAR into a cell is performed ex vivo (in vivo) or in vivo (in vivo).
- a cell into which a nucleic acid is introduced a mammal, for example, a cell derived from a human or a cell derived from a non-human mammal such as monkey, mouse, rat, pig, cow or dog can be used.
- the cell type for example, cells collected, isolated, purified and induced from blood (peripheral blood, umbilical cord blood, etc.), body fluid such as bone marrow, tissue or organ can be used.
- PBMC immune cells
- B cells hematopoietic stem cells, macrophages, monocytes or NK cells, hematopoietic cells (neutrophils, basophils, monocytes)]
- hematopoietic stem cells cord blood mononuclear cells, fibers Blast cells, preadipocytes, hepatocytes, blood cells, skin keratinocytes, mesenchymal stem cells, hematopoietic stem cells, adipose stem cells, pluripotent stem cells, various cancer cell lines or neural stem cells can be used.
- T cells include CD8 positive T cells or CD4 positive T cells, regulatory T cells, cytotoxic T cells, and tumor infiltrating lymphocytes.
- Cell populations containing T cells and T cell progenitor cells include PBMC.
- the cells used in the present invention may be any of those collected from a living body, those obtained by culturing the cells, or those established as a cell line. When it is desired to transplant a cell into which a nucleic acid is introduced or a cell differentiated from the cell, it is preferable to introduce the nucleic acid into a cell collected from the living body itself or the same kind of living body.
- the cells expressing the CAR of the present invention may be cultured and / or stimulated using an appropriate medium and / or stimulating molecule prior to administration to a subject.
- Stimulatory molecules include cytokines, appropriate proteins, and other components.
- cytokines include IL-2, IL-7, IL-12, IL-15, IFN- ⁇ and the like, and preferably IL-2.
- the concentration of IL-2 in the medium is not particularly limited, and is, for example, 0.01 to 1 ⁇ 10 5 U / mL, and more preferably 1 to 1 ⁇ 10 4 U / mL.
- suitable proteins include CD3 ligand, CD28 ligand, and anti-IL-4 antibody.
- a lymphocyte stimulating factor such as lectin can also be added.
- serum or plasma may be added to the medium.
- the amount added to these media is not particularly limited, but is 0 to 20% by volume, and the amount of serum or plasma used can be changed depending on the culture stage.
- Serum or plasma concentrations can be used in a step-wise manner.
- the origin of serum or plasma may be either self (meaning that the origin is the same as the cell being cultured) or non-self (meaning that the origin is different from the cell being cultured), but is preferably safe Self-derived ones are used from the viewpoint of sex.
- the cell culture equipment used for cell culture is not particularly limited, and for example, a petri dish, a flask, a bag, a large culture tank, a bioreactor, or the like can be used.
- a CO 2 gas permeable bag for cell culture can be used.
- a large culture tank can be used.
- cultivation can be implemented by any of an open system and a closed system, it is preferable to culture
- the present invention includes a pharmaceutical composition containing a cell expressing CAR as an active ingredient.
- the pharmaceutical composition of the present invention is administered parenterally.
- Parenteral administration methods include methods such as intravenous, intraarterial, intramuscular, intraperitoneal, and subcutaneous administration. To enhance antitumor activity, it can be administered to meningiomas or nearby tissues, such as subcutaneously.
- the dosage is appropriately selected according to the condition, weight, age, etc. of the subject. Usually, the number of cells is about 10 7 to 10 9 cells, preferably 5 ⁇ 10 7 to 5 ⁇ 10 8 cells, per 60 kg body weight.
- the pharmaceutical composition can be administered once or multiple times.
- the pharmaceutical composition can be in a form suitable for parenteral administration, such as an injection or infusion.
- the pharmaceutical composition may optionally contain a pharmacologically acceptable excipient.
- the pharmaceutical composition may contain physiological saline, phosphate buffered saline (PBS), a medium and the like in order to stably maintain the cells.
- PBS phosphate buffered saline
- Media such as RPMI, AIM-V, and X-VIVO10, are generally mentioned.
- Antibody screening using a human antibody phage library (1) Preparation of pMHC beads 20 ⁇ g of A2-MAGE-A4 as pMHC and 200 mg of magnetic beads bound with streptavidin are mixed in a 0.05% Tween / PBS ( ⁇ ) solution. And reacted at 4 ° C overnight. 3 ⁇ L of 2 mM biotin / PBS ( ⁇ ) was added to this and allowed to react for 1 hour. This was washed twice with 0.05% Tween / PBS ( ⁇ ) solution and then suspended in 400 ⁇ L of 0.05% Tween / PBS ( ⁇ ) solution. This was designated as an antigen bead.
- Human antibody library VH and VL were amplified by PCR based on tonsils, umbilical cord blood, peripheral blood, and bone marrow derived from dozens of healthy individuals, and this was combined on the phagemid vector pUC119 to give 3.4 ⁇ 10
- a human antibody library was prepared by scFv of 12 repertoires and displayed on cp3 of M13 phage.
- the magnetic beads were trapped with a magnetic trapper (Toyobo), washed 5 times with 1% Triton X100 / PBS, and then washed once with PBS. Add this to cultured XL1-blue and infect at 37 ° C for 1 hour, then centrifuge and resuspend in 600 ⁇ L 2 ⁇ YTAG medium (200 ⁇ g / mL ampicillin, 1% glucose / 2 ⁇ YT), and YTAG agar medium (200 ⁇ g / mL ampicillin, 2% glucose / normal agar medium (Nissui)) and cultured at 37 ° C. for 15 hours.
- a magnetic trapper Toyobo
- Colonies were recovered with 30 mL of 2 ⁇ YTAG medium, 200 ⁇ L of which was added to 20 mL of 2 ⁇ YTAG medium, and 30 ⁇ L of helper phage VCSM was added. After 30 minutes of infection at 37 ° C, the cells were cultured at 37 ° C for 90 minutes. To the culture solution, 80 mL of 2 ⁇ YTAG medium, 50 ⁇ g / mL kanamycin 60 ⁇ L, and 1 MIPTG 50 ⁇ L were added and cultured at 28 ° C. for 20 hours.
- the supernatant obtained by centrifuging this solution is mixed with 25 mL of PEG solution (20% PEG # 600, 2.5 M NaCl), centrifuged, and the precipitate is recovered, suspended in 1 mL PBS, and then filtered. Sterilized. The above operation was repeated twice. After the second time, the recovered E. coli was spread on a YTAG agar medium. After culturing at 30 ° C., the obtained colonies were cultured in LB medium at 30 ° C. overnight, and a portion thereof was used to extract DNA with a miniprep DNA kit (QIAGEN), followed by sequencing.
- PEG solution 20% PEG # 600, 2.5 M NaCl
- cp3 supernatant This was used for ELISA.
- Enzyme-linked immunosorbent assay (1) MHC-peptide complex (monomer) used MAGE-A4 p230, MAGE-A4 p286, MAGE-A3 p195, CMV, MelanA, GP3, HTLV-1, NY-ESO-1 p157, NY-ESO-1 p159, Foxp3 p69, Foxp3 p127, Foxp3 p390, IDO p41 , IDO p41, IDO p195, and IDOp199 were combined with HLA-A * 0201.
- Ni Sepharose excel GE Healthcare
- 1 mL of Ni Sepharose excel GE Healthcare
- PBS phosphatidylcholine
- the sample was packed in a column, washed with 0.5 M NaCl PBS, 20 mM imidazole / 0.5 M NaCl Cl PBS, and eluted with 250 mM imidazol / 0.5 M NaCl Cl PBS.
- Dialysis was performed with PBS, and after completion of dialysis, the mixture was concentrated with Amicon Ultra 10K (Millipore).
- KD value dissociation constant
- the KD value of the purified antibody was measured using Biacore X100 (GE Healthcare). Biotion capture kit (GE Healthcare) was used to immobilize the ligand.
- Immobilization of ligand HLA-A2-MAGE-A4 p230 was immobilized on a sensor chip CAP. It was adjusted to 200 nM with HBS buffer, and solid-phased so as to be an appropriate RU (resonance unit).
- (2) Preparation of analyte solution The purified antibody was adjusted to a concentration of 12.5 nM, 25 nM, 50 nM, 100 nM, and 200 nM with HBS EP + buffer.
- FACS flow cytometry
- Tetramer PE Tetramer-PE means that four biotinylated monomers are bound to streptavidin-PE and was used for detection of antigen-specific T cells.
- the tetramers used were A2-MAGE-A4 p230 tetramer and A2-NY-ESO-1 p157 tetramer.
- T2 cells HLA-A * 02 + human BT hybrid lymphoblast cell line
- T2 cells are a Transporter Associated with Antigen Processing (TAP) deficient strain and have the characteristic that intracellular proteins are not presented to HLA.
- TIP Antigen Processing
- Peptide pulse to T2 cells In human RPMI1640, a peptide was added to a final concentration of 10 ⁇ M, left at room temperature for 15 minutes, and 20% FCS human RPMI1640 was added so that FCS was 10%. After incubating at 37 ° C. for 45 min in a CO 2 incubator, the plate was washed twice and then used for assay.
- HLA stability assay Anti-HLA-A2 Alexa Fluor488 was reacted with peptide pulsed T2 cells for 1 hour and detected by FACS to measure the amount of HLA stabilized on the T cell surface.
- MHC IC50 is a theoretical value calculated by IEDB (http://tools.immuneepitope.org/processing/). The lower the number, the stronger the binding between HLA-A * 0201 and the peptide.
- Table 2 shows the amino acid sequence and IC50 of the 1 amino acid substituted peptide.
- Table 3 shows the amino acid sequences and IC50 values of risk peptides extracted by BLAST search from amino acids involved in recognition of MAGE family peptides and MAGE # 17 antibodies.
- FIG. 1 shows a sequence image of the CAR construct. Leader, VH, VL, CL, CD28TM, CD28ICD (CD28 intracellular domain) and CD3zeta (CD3 ⁇ ) chains are linked to this construct from the 5 ′ side. Among these, VH and VL are MAGE # 17 scFv.
- CAR Construction FIG. 2 schematically shows the CAR construction steps. Each step will be described as follows. First, Leader having an EcoRI recognition site on the 5 ′ side of VH-VL and CL having an AscI recognition site on the 3 ′ side were added by PCR using scFv-cp3 as a template. Next, a fragment obtained by treating this PCR product with EcoRI and AscI and a fragment obtained by treating the 28z CAR vector with EcoRI and AscI were reacted with ligase to obtain CAR28z.
- Human Lymphocyte Adjustment Human lymphocytes were obtained by separating PBMC (Peripheral Blood Mononuclear cells) from blood donated from healthy donors using Ficoll-Paque TM PLUS (17-1440-03, GE Healthcare). The collection and analysis of human peripheral blood and other samples used in this study was conducted in accordance with the Declaration of Helsinki, and all were performed with the written consent of the subject in accordance with the protocol approved by the Mie University School of Medicine Research Ethics Committee. It was. The collected specimens were stored in a refrigerator and liquid nitrogen tank that were encrypted so that they could not be identified, and were subjected to anti-theft measures. Subject personal information was anonymized, and strict caution and treatment were implemented to prevent personal privacy and genetic analysis results from leaking out.
- PBMC Peripheral Blood Mononuclear cells
- CM caluture medium
- human IL-2 600IU
- 300- ⁇ L lymphocyte donor plasma were added to 50 mL of the lymphocyte medium GT-T503.
- PBMC culture medium 50-mL human IL-2 (600IU), 400- ⁇ L albumin (25% HSA), and 300- ⁇ L lymphocyte donor plasma were added to 50 mL of the lymphocyte medium GT-T503.
- PBMC culture medium Separation and culture of PBMC Peripheral blood mononuclear cells (PBMC) separated from healthy human blood using Ficoll were suspended in CM so as to be 2 ⁇ 10 5 cells / mL, and 2.5 mL. was placed in a plate and cultured at 37 ° C. for 4 days in a CO 2 incubator.
- Electroporation The prepared mRNA was introduced into PBMC 10 days after separation by electroporation. It was used for assay within 24 hours after introduction.
- RetroNectin was diluted with ACD-A solution and adjusted to a final concentration of 20 ⁇ g / mL. 500 ⁇ L of this was added to each well of a 24-well plate and allowed to stand at 4 ° C. with O / N. Before placing the virus solution, the plate was washed twice with ACD-A.
- Retrovirus plate adjustment for infection, infection of cells 10.
- 1 mL of the prepared virus solution was placed on a plate for retrovirus infection and centrifuged at 2000 ⁇ g for 2 hours at 32 ° C. to coat the virus on the plate. Subsequently, this was washed twice with 1.5% albumin / PBS.
- PBMC retrovirus-infected CAR-T cells Collect PBMC cultured in (2), dilute with CM to 4 ⁇ 10 5 cells / mL, place 950 ⁇ l on a plate for retrovirus infection, and centrifuge at 1000 ⁇ g for 10 minutes at 32 ° C. After that, the cells were cultured at 37 ° C. in a CO 2 incubator. The prepared CAR-T cells were used for the assay on the 12th to 14th days from the separation of the PBMC.
- (6) Preparation of control cells A part of the cells cultured in (2) was used as control cells (cells without gene transfer operation).
- ICS intracellular cytokine staining
- the cells in each well were transferred to a V-type 96-well plate, centrifuged at 1200 rpm for 5 minutes at 4 ° C., and then washed twice with 0.5% BSA / PBS.
- 0.5 ⁇ L of APC-cy7 Anti-Human CD4 and FITC Anti-human CD8 were added to each well, left on ice for 20 minutes while protected from light, and washed twice with 0.5% BSA / PBS. .
- 100 ⁇ L of cell fixative cytofixl cytoperm: BD
- BD perm / wash
- MAGE-A4 long peptide The amino acid sequence of MAGE-A4 long peptide was NYKRCFPVIF GKASEGVYDG REHTVYGEPR KAETSYVKVL EHVVRVNARV RI (SEQ ID NO: 28), and the molecular weight was 6006.811.
- This long peptide links A24 MAGE-A4 143-151 epitope (NYKRCFPVI: SEQ ID NO: 29), A2 MAGE-A4 230-239 epitope (GVYDGREHTV: SEQ ID NO: 1) and helper epitope (AETSYVKVLE HVVRVNARVR I: SEQ ID NO: 30) And designed.
- CHP-Long Peptide means a drug delivery system in which CHP nanogel is wrapped with MAGE-A4 long peptide. By using this substance, induction of cellular immunity can be expected (Non-patent Document 5).
- (3) CD3 negative beads selection Human lymphocytes were suspended in 80 ⁇ L of 2% FCS / PBS per 1 ⁇ 10 7 cells. 20 ⁇ L of CD3 Micro Beads (Miltenyi Biotec) was added thereto and mixed well. After light-shielded and allowed to react at 4 ° C. for 15 minutes, it was washed with 10 volumes of 2% FCS / PBS. Cells were suspended in 3 mL of 2% FCS / PBS to form a cell suspension. The cell suspension was passed through a MACS Separator with a MACS column set. The fraction that passed through the column was defined as a CD3 negative fraction and used as APC.
- Cytotoxicity assay (1) Cr release assay Target cells were collected by centrifugation, and 1 ⁇ 10 6 cells were suspended in 50 ⁇ L with 100% FCS. To this, 50 ⁇ Ci (3.7 ⁇ 10 6 Bq) of 51 Cr was added and cultured at 37 ° C. for 1 hour in a CO 2 incubator. The number of effector cells was adjusted with 10% FCS • RPMI1640 according to ETratio, and seeded on a U-shaped 96-well plate. After completion of the culture, the cells were washed 3 times with 10% FCS / RPMI1640.
- the cells were suspended in 10% FCS ⁇ RPMI1640 so as to be 1 ⁇ 10 5 cells / mL, and seeded on a U-shaped 96-well plate so as to be 1 ⁇ 10 4 cells / 100 ⁇ L / well.
- the cells were cultured at 37 ° C. for 8 hours in a CO 2 incubator. After completion of the culture, centrifugation at 1200 rpm for 5 minutes at 4 ° C. was performed, and 30 ⁇ L of the supernatant was transferred to a reading plate and dried overnight by air drying.
- the amount of 51 Cr was measured with a scintillation counter MicroBeta 2 (Perkin Elmer). The calculation of the cytotoxic activity was obtained by the following formula.
- the transfused CAR-T cells and human lymphocytes were 1 ⁇ 10 7 cells / mouse. Each cell was transfused into NOG mice via the tail vein. Body weight was measured every 2-3 days. Tumor transplantation into NOG mice was performed 7 days before CAR-T cell transfusion, and NW-MEL-38 (A2 positive MAGE-A4 positive) 2.5 ⁇ 10 6 mice / mouse, mouse left body side HCT116 (A2 positive MAGE-A4 negative) was subcutaneously injected at 2.5 ⁇ 10 6 cells / mouse. Tumor diameter was measured every 2-3 days.
- CAR-T cells for infusion were prepared according to the method described in “10. Gene transfer into cells by retrovirus” above.
- Isolation of PBMC in peripheral blood of mice by orbital blood collection Peripheral blood was collected from the orbits of mice and PBMC was isolated using Ficoll. Cells stained with A2-MAGE-A4 tetramer and human CD4CD8 fluorescent antibody were confirmed by FACS infusion.
- CAR-T cells In vitro test using zG-type CAR-introduced CD8-positive T cells and 4-1BBz-type CAR-introduced CD8-positive T cells The effect of CAR-T cells due to differences in the ICD part was confirmed. Specifically, GITR (zG) and CD137 (4-1BBz) were prepared from CD28 (28z), and the effects were confirmed. As shown in FIG. 21, CAR zG used GITR (glucocorticoid-induced tumor necrosis factor receptor) instead of CD28 as ICD. Similarly, CD137 (4-1BBz) was used instead of CD28. As a control, CAR8 non-introduced CD8 positive T cells (NGMC) were used.
- GITR GITR
- CD137 glucocorticoid-induced tumor necrosis factor receptor
- HLA-A2-MAGE-A4 MAGE-A4 is known to have high expression in melanoma. Therefore, a plurality of antibodies recognizing MAGE-A4 were isolated by screening using a human antibody library. Phage display method was used for antibody isolation. In this method, scFv is displayed as part of the phage coat protein cp3, and clones having high binding ability to the antigen can be narrowed down by ELISA. Approximately 1500 clones were picked up and subjected to ELISA in the state of scFv-CP3.
- ELISA was performed using a plurality of peptide-MHC complexes (pMHC).
- FIG. 3 shows an example of the result of ELISA.
- the dissociation rate constant (Kd) and the association rate constant (Ka) were obtained from the dynamic change curve, and the association constant was obtained from the ratio of the two constants.
- the binding constant of MAGE # 17 was 22 nM (FIG. 4).
- MAGE-A4 p230 Only in T2 cells pulsed with MAGE-A4 p230, changes in the shift amount were confirmed in all of the obtained antibody clones MAGE # 17, # 86, # 209, # 345, and # 672 (FIG. 5).
- a peptide was prepared by substituting the amino acids of MAGE-A4 p230 (GVYDGREHTV: SEQ ID NO: 1) one by one with another amino acid (mainly alanine), and HLA-A *
- IEDB Immunune Epitope Database
- T2 cells were pulsed with an alanine-substituted peptide and reacted with MAGE # 17 to examine amino acids involved in antibody recognition (FIG. 7). Since MFI values decreased in 1A, 2A, 3A, 4A, 5A, 6A, and 8A, it was judged that these 7 amino acids are involved in recognition.
- a BLAST search was performed with the amino acid sequence of GVYDGRxHxx, and peptides at risk were extracted (Table 3). The risk peptide was pulsed into T2 cells and the amount of HLA on the T2 cell surface was measured by FACS. Similar to the results shown in FIG. 6, the MFI values corresponded to the MHC IC50 values (FIG. 8).
- the base sequence (SEQ ID NO: 31) and peptide sequence (SEQ ID NO: 32) of MAGE # 17 are shown.
- MAGE # 17 The amino acid sequence of MAGE # 17 (SEQ ID NO: 32): QVQLVQSGAE VKKPGSSVKV SCKASGGTFS SYAISWVRQA PGQGLEWMGG IIPIFGTANY AQKFQGRVTI TADKSTSTAY MELSSLRSED TAVYYCARSP RRAYHDAFDI WGQGTMVTVS SGGGGSGGGG SGGGGSSYEL TQPPSMSVAP GKTASITCGG DHIGSKSVHW YQQKPGQAPV LVVYDDSDRP SGIPERFSGS NSGNTATLTI SGTQAMDEAD YYCLAWDSST AIFGGGTKLT VL.
- Each of the above sequences is represented by the nucleotide sequences of VH (SEQ ID NO: 33), sc (SEQ ID NO: 34) and VL (SEQ ID NO: 35), and VH (SEQ ID NO: 36), sc (SEQ ID NO: 37)
- the amino acid sequence of VL (SEQ ID NO: 38) is combined.
- VH nucleotide sequence (SEQ ID NO 33): CAGGTCCAGC TGGTACAGTC TGGGGCTGAG GTGAAGAAGC CTGGGTCCTC GGTGAAGGTC TCCTGCAAGG CTTCTGGAGG CACCTTCAGC AGCTATGCTA TCAGCTGGGT GCGACAGGCC CCTGGACAAG GGCTTGAGTG GATGGGAGGG ATCATCCCTA TCTTTGGTAC AGCAAACTAC GCACAGAAGT TCCAGGGCAG AGTCACGATT ACCGCGGACA AATCCACGAG CACAGCCTAC ATGGAGCTGA GCAGCCTGAG ATCTGAGGAC ACGGCCGTGT ATTACTGTGC GAGATCCCCC CGGCGGGCAT ATCATGATGC TTTTGATATC TGGGGCCAAG GGACAATGGT CACCGTCTCT TCA.
- Base sequence of sc part (SEQ ID NO: 34): GGTGGAGGCG GTTCAGGCGG AGGTGGCAGC GGCGGTGGCG GGAGTTCCTA T.
- Nucleotide sequence of the VL portion (SEQ ID NO 35): GAGCTGACTC AGCCACCCTC GATGTCAGTG GCCCCAGGAA AGACGGCCAG CATTACCTGT GGCGGAGACC ATATTGGAAG TAAAAGTGTT CACTGGTACC AGCAGAAGCC AGGCCAGGCC CCTGTACTGG TCGTCTATGA TGATAGCGAC CGGCCCTCAG GGATCCCTGA GCGATTCTCT GGCTCCAACT CTGGGAACAC AGCCACTCTG ACCATCAGCG GGACCCAGGC TATGGATGAG GCTGACTATT ACTGTCTGGC GTGGGACAGC AGCACTGCGA TCTTCGGCGG AGGGACCAAG CTGACCGTCC TC.
- Amino acid sequence of the VH part (SEQ ID NO: 36): QVQLVQSGAE VKKPGSSVKV SCKASGGTFS SYAISWVRQA PGQGLEWMGG IIPIFGTANY AQKFQGRVTI TADKSTSTAY MELSSLRSED TAVYYCARSP RRAYHDAFDI WGQGTMVTVS S.
- Amino acid sequence of sc part (SEQ ID NO: 37): GGGGSGGGGS GGGGSSY.
- VL amino acid sequence (SEQ ID NO: 38): ELTQPPSMSV APGKTASITC GGDHIGSKSV HWYQQKPGQA PVLVVYDDSD RPSGIPERFS GSNSGNTATL TISGTQAMDE ADYYCLAWDS STAIFGGGTK LTVL.
- MAGE # 17 CAR specifically recognized the target cancer cells and increased the production of IFN- ⁇ . The specificity of antibody clone MAGE # 17 was confirmed, so MAGE # 17 was transferred to the CAR vector. .
- mRNA was prepared and confirmed with a bioanalyzer, and then the CAR gene was introduced into human peripheral blood lymphocytes (PBMC) by electroporation. Whether or not the introduced CAR was normally expressed was confirmed by tetramer staining. As a result, the CAR positive rate of CAR-T cells was 98.8% for CD8 and 98.5% for CD4 (FIG. 10).
- CAR expression by mRNA is transient, gene transfer by retrovirus was performed for constant CAR expression.
- a retrovirus vector for CAR introduction was constructed, a retrovirus was prepared by packaging cell plat-A, and PBMC was infected to prepare CAR-T cells. Gene transfer was performed according to the method described in “10. Gene transfer into cells by retrovirus” described above. CAR expression was confirmed by tetramer staining. As a result, the CAR positive rate of CAR-T cells was 59.3% for CD8 and 49.5% for CD4 (FIG. 12). CAR-T cells transfected with retrovirus and T2 cells pulsed with peptide were co-cultured for 24 hours, and IFN- ⁇ contained in the culture supernatant was measured.
- CAR-T cells transfected with MAGE # 17 CAR specifically recognize target cancer cells and show cytotoxic activity
- CAR-T cells expressed on cancer cells are A2-MAGE -A4 was recognized as an antigen and activated, and it was expected to exert cytotoxic activity and damage target cells.
- Molecules known as indicators of T cell activation include IFN- ⁇ , TNF- ⁇ , CD107a and the like, and their expression increases with T cell activation. Therefore, intracellular cytokine staining (ICS) of MAGE # 17 CAR co-cultured with peptide-pulsed T2 cells and target cells was performed.
- ICS intracellular cytokine staining
- the ratio of CAR-T cells stained with IFN- ⁇ , TNF- ⁇ , and CD107a increased in co-culture with T2 cells pulsed with MAGE-A4p230 (FIG. 14).
- the ratio of CAR-T cells stained with IFN- ⁇ , TNF- ⁇ , and CD107a in coculture with A2 positive MAGE-A4 positive NW-MEL-38 and LB-23 Increased ( Figure 15).
- Antigen presenting cell that has incorporated MAGE-A4 Long peptide has increased the production of IFN- ⁇ in CAR-T cells by separating the PBMCs from the peripheral blood of healthy individuals.
- the CD3- fraction was used as an antigen presenting cell (APC).
- A2-APC was used as APC for displaying MAGE-A4p230, and A24-APC was used as APC for negative control.
- MAGE-A4 Long peptide and CHP-MAGE-A4 Long peptide were added to a final concentration of 10 ⁇ M, cultured overnight, and co-cultured with CAR-T for 24 hours.
- Gene transfer into CAR-T cells was performed according to the method described in “10. Gene transfer into cells by retrovirus” described above.
- CAR-T cells transfected with MAGE # 17 CAR were examined for whether or not the specific cytotoxic activity of MAGE # 17CAR occurs by 51 Cr release assay that specifically injured the target cancer cells.
- the ratio of CAR-T cells to target cancer cells is defined as ET ratio (effector / target ratio), and target cancer cells and CAR-T cells that have incorporated chromium are ET ratios of 10: 1, 3: 1, 1
- the cells were co-cultured for 8 hours, and the cytotoxicity (% lysis) was determined from the radiation dose value of 51 Cr contained in the supernatant.
- Gene transfer into CAR-T cells was performed according to the method described in “10. Gene transfer into cells by retrovirus” described above.
- Mouse cancer treatment model (1) Model using NW-MEL-38 and HCT116 NOG mice lack the common ⁇ receptor, so there are no NK cells. For this reason, the engraftability of human cells and human tissues is significantly higher than that of NOD-SCID mice, and human cancers and normal human tissues can be engrafted at a high rate.
- human T cells are differentiated after human hematopoietic stem cell transplantation, the demand for human immune system model mice is increasing.
- the characteristics of NOG mice are T cell and B cell deletion, natural killer (NK) cell deletion, dendritic cell function decrease, macrophage function decrease, complement activity deletion, senescence T cell and It is mentioned that B cell leakage (leakiness) is not observed.
- CAR zG The gene sequence image of CAR zG is shown in FIG.
- ICD intracellular domain
- GITR means a glucocorticoid-induced tumor necrosis factor receptor, and it is known that CAR production using this ICR can increase the amount of cytokine production relative to the expression level of CAR (WO2013051718).
- CAR zG is obtained by changing the CD28 ICD part to GITR ICD, which is another ICD, in CAR 28z in FIG. 2, and changing the order of CD3 ⁇ and ICD domain in CAR 28z.
- CAR-T cells (4 ⁇ 10 4 cells) transfected with retroviruses and T2 cells subjected to peptide pulses, and various cell lines NW-MEL-38, SK-MEL-37, HCT116 (2 ⁇ each) 10 5 cells) were co-cultured for 18 hours, and IFN- ⁇ contained in the culture supernatant was measured.
- IFN- ⁇ contained in the culture supernatant was measured.
- FIG. 24 in CAR-T cells into which zG type CAR and 28z type CAR were introduced, IFN was specifically expressed in T2 cells pulsed with MAGE-A4, NW-MEL-38 cells, and SK-MEL-37 cells. -Y production increased.
- a similar test was performed using control cells (cells without gene transfer).
- the antigen-binding protein that specifically recognizes the MAGE-A4-derived peptide / HLA-A2 complex the nucleic acid that encodes the antigen-binding protein, the vector containing this nucleic acid, the antigen binding A chimeric antigen receptor containing a sex protein, a nucleic acid encoding the chimeric antigen receptor, a vector containing the nucleic acid, a cell expressing the chimeric antigen receptor and a pharmaceutical composition comprising the cell are provided.
- Antigen-binding proteins that specifically recognize MAGE-A4-derived peptides / HLA-A2 complexes can be used in the fields of cell medicine and gene therapy.
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Abstract
Description
近年、免疫療法と呼ばれる第四のがん治療法が進展し、その非侵襲性と効果の高さが注目されている。免疫療法は、主としてワクチン療法、抗体療法、細胞輸注療法の3つに大別される。細胞輸注療法には、がん特異的に反応する細胞傷害性T細胞由来のT細胞受容体(T cell receptor : TCR)の遺伝子を患者のリンパ球に体外で導入した後に輸注するT細胞受容体遺伝子導入リンパ球輸注療法と、抗原認識部位としてのscFvと細胞内シグナル伝達分子としてのCD3ζを結合させたキメラ抗原受容体(chimeric antigen receptor : CAR )を患者末梢血リンパ球に導入して輸注する治療法がある。これらの治療法は、抗原特異性を持つリンパ球を受容体遺伝子導入という方法により短期間に大量に作製できるため多様ながん種の患者に対する特異的T細胞治療を可能とする。
一方、CAR輸注療法の利点として次の3点が挙げられる。(1)単鎖抗体(scFv)とT細胞受容体(TCR)や副刺激分子のシグナル伝達ドメインからなるCARを導入したT細胞は、本来のT細胞とは異なり、MHC非依存性にがん抗原を認識し破壊できることから、広範囲の患者に適応可能となる長所を持つ、(2)CD8陽性T細胞だけでなく、CD4陽性T細胞や非T細胞にも機能を与え得る、(3)抗体の反応性を受け継ぐためTCRと比較して、親和性が高い。実際、CAR治療法は、抗CD19抗体を使用したCAR治療法として白血病及びリンパ腫の患者に対して極めて高い臨床効果が報告されている(非特許文献1:文献については、末尾にまとめて示す)。CD19分子はB細胞にも発現されるが、B細胞の消失は免疫グロブリンの補充等で対処可能である。一般的には、がん細胞でのみ発現する抗原を用いることが理想的である。しかし、そのような細胞表面抗原は、現状では見あたらないという課題がある。
このような状況に鑑み、本発明者は、MHCと細胞内抗原由来ペプチドとの複合体を認識する抗体を単離し、その抗体を用いて、CAR免疫療法に応用する方法を考えた。ペプチドMHC複合体を特異的に認識する抗体は、限られた数の成功例しか報告されていない(非特許文献2)。ペプチドMHC複合体を特異的に認識する抗体を取得し、がん細胞を特異的に殺傷するCAR-T細胞を作製することができれば、画期的な治療法と成り得る。ペプチドMHC複合体を認識するCARは、体内にCAR-T細胞を輸注すると同時にCAR-T細胞が特異的に認識するペプチドを投与することで、抗原提示細胞によるCAR-T細胞の増殖が期待できる。
A2-MAGE-A4を特異的に認識するCARをヒトリンパ球に遺伝子導入し、ターゲットのがんに特異的な反応を示すCAR-T細胞を作製し、それをヒトの細胞輸注療法に応用する方法を確立した。すなわち、(1)MAGE-A4由来ペプチドとHLA-A2複合体を認識する親和性の高い複数個の抗体を単離し、(2)単離した抗体の特異性を調べ、(3)特異性の高い抗体を選別し、(4)これを使用したCAR-T細胞を作製し、そのCAR-T細胞がターゲットのがん細胞特異的によって活性化され、細胞傷害活性を引き起こすことを確認した。CAR-T細胞を輸注したヒト腫瘍移植マウスによるin vivoがん治療モデルにおいて、マウス体内でCAR-T細胞が特異的に増殖し、腫瘍への浸潤が観察された。こうして、基本的には本発明を完成するに至った。
また、別の発明に係る核酸は、第1の発明に記載の抗原結合性タンパク質をコードする。
別の発明に係るベクターは、上記核酸を含む。ここで、核酸とは、DNAまたはRNAが含まれる。また、核酸は、一本鎖または二本鎖のいずれでもよい。ベクターには、プラスミドベクター、ウイルスベクターなどが含まれる。
別の発明に係るキメラ抗原受容体は、上記抗原結合性タンパク質とシグナル伝達タンパク質の細胞内ドメインを含む。このとき、シグナル伝達タンパク質は、CD3zeta(CD3ζ)鎖または共刺激分子CD(GITR)のいずれかであることが好ましい。また、更にCD28の細胞内ドメインまたはGITRの細胞内ドメインのいずれかを含むことが好ましい。
また、別の発明に係る核酸は、上記キメラ抗原受容体をコードする。別の発明に係るベクターは、この核酸を含む。
別の発明に係る細胞は、上記キメラ抗原受容体を発現するものである。また、別の発明に係る医薬組成物は、この細胞を有効成分として含有する。
本明細書中において、「抗体」、「抗原結合性フラグメント」とは、免疫系における抗原結合性タンパク質を意味する。抗原結合性領域を有する抗体は、ジスルフィド結合により相互に連結された2つの重鎖(H鎖)及び2つの軽鎖(L鎖)を含む糖タンパク質である。各重鎖には、重鎖可変領域(VH)及び重鎖定常領域(CH)が含まれる。重鎖定常領域は、CH1、CH2及びCH3の3つのドメインから構成される。各軽鎖には、軽鎖可変領域(VL)及び軽鎖定常領域(CL)が含まれる。VLは、CLドメインから構成される。VH及びVL領域はさらに、相補性決定領域(CDR)という超可変性を有する領域及びフレームワーク領域(FR)というある程度保存されている領域に細分できる。VH及びVLは、アミノ末端からカルボキシ末端に向かって、FR1、CDR1、FR2、CDR2、FR3、CDR3及びFR4の順に、3つのCDR及び4つのFRが、それぞれ配列されている。抗原との結合には、CDR3が重要であることが知られている。VH及びVLは、抗原と相互作用する結合ドメインを含む。
本明細書において「キメラ抗原受容体(CAR)」とは、抗原に結合する細胞外ドメイン、この細胞外ドメインとは異なるポリペプチドに由来する膜貫通ドメイン及び少なくとも1つの細胞内ドメインを含む融合タンパク質を意味する。CARは、「キメラ受容体」、「T-body」、「キメラ免疫受容体(CIR)」とも呼ばれることがある。「抗原に結合する細胞外ドメイン」は、ある抗原に結合する任意のオリゴペプチド又はポリペプチドを意味し、「細胞内ドメイン」は、細胞内で生物学的プロセスを活性化又は阻害するすシグナルを伝達するドメインとして機能することが知られている任意のオリゴペプチド又はポリペプチドを意味する。
本明細書において「抗原結合性タンパク質」は、抗原に結合し、抗原に対する特異性を抗体に付与する抗体の一部又は全部のタンパク質の断片を意味する。
(1)本発明の抗原結合性フラグメント及びこれらをコードする核酸
抗MAGE-A4由来ペプチド/HLA-A2(HLA-A2-MAGE-A4)複合体抗体は、HLA-A2拘束性のMAGE-A4由来ペプチドであるP230-239(配列番号1)と、HLA-A2との複合体を特異的に認識・結合する抗体である。本発明の抗体は、特異性が高く、HLA-A2以外のペプチドとHLA-A2の複合体及びP230-239ペプチドとHLA-A2以外のHLAの複合体とは結合しない(または結合活性が極めて低い)。このため、本発明の抗原結合性フラグメントは、HLA-A2-MAGE-A4複合体を特異的に検出又は標的とできる。
本発明の抗原結合性フラグメントは、(A)配列番号36のVH(重鎖可変領域)のアミノ酸配列と、配列番号38のVL(軽鎖可変領域)のアミノ酸配列とを含むポリペプチド、または(B)配列番号36のVH(重鎖可変領域)のアミノ酸配列と90%以上の相同性を持つアミノ酸配列と、配列番号38のVL(軽鎖可変領域)のアミノ酸配列と90%以上の相同性を持つアミノ酸配列とを含むポリペプチドを含む。また、VHとVLとの間に、(C)配列番号37のsc(一本鎖)のアミノ酸配列を含むポリペプチド、または(D)配列番号37のsc(一本鎖)のアミノ酸配列と90%以上の相同性を持つアミノ酸配列を含むポリペプチドを含む。
抗原結合性フラグメントは、その特性に実質的な影響を及ぼさない限り、1つまたは数個のアミノ酸が改変でき得る。例えば、定常領域やFR領域において、アミノ酸が置換、欠失、付加又は挿入され得る。この改変は、部位特異的変異導入法(点突然変異導入及びカセット式変異導入等)、遺伝子相同組換え法、プライマー伸長法、及びPCR法等の周知の方法を組み合わせて、容易に実施できる。
本発明は、前記抗原結合性フラグメントをコードする核酸を含む。また、本発明の核酸は、配列表の配列番号31,33~35に示される塩基配列を含む。これらの核酸は、コードするアミノ酸配列を変更させることなく、宿主細胞に適したコドン(至適コドン)に改変できる。至適コドンに改変することで、宿主細胞内におけるポリペプチドの発現効率を向上させ得る。
本発明の抗原結合性フラグメントは、公知の遺伝子工学的手法又は化学合成等の方法を用いて製造できる。遺伝子工学的手法としては、本発明の核酸を含有するクローニングベクター又は発現ベクターを作製し、このベクターを宿主細胞に導入し、宿主細胞を培養して核酸を発現させ、得られたポリペプチドを回収・精製することによって製造できる。本発明の核酸が複数の核酸分子からなる場合は、各核酸分子を含む複数ベクターの組合せを宿主細胞に導入する、又は複数の核酸を含む1つのベクターを宿主細胞に導入できる。ポリペプチドを製造する際に、ペプチドタグを連結させ、このペプチドタグを使用して回収・精製できる。
ベクターとしては、宿主ゲノムに組み込まれるベクター・組み込まれないベクター、細胞質に存在して自律的に複製するエピソーマルベクターなどが使用できる。そのようなベクターとしては、例えばレトロウイルスベクター(オンコレトロウイルスベクター、レンチウイルスベクター、シュードタイプベクターを含む)、アデノウイルスベクター、アデノ随伴ウイルス(AAV)ベクター、シミアンウイルスベクター、ワクシニアウイルスベクター、センダイウイルスベクター、エプスタイン・バーウイルス(EBV)ベクター、HSVベクターなどのウイルスベクターが例示できる。ウイルスベクターとしては、感染した細胞中でウイルスが自己複製できないように複製能を欠損させたものが好ましく使用できる。また、リポソーム及び陽イオン脂質などの縮合剤との併用により、非ウイルスベクターも使用できる。更に、リン酸カルシウム形質移入、DEAE-デキストラン、エレクトロポレーション、パーティクルボンバードメントにより細胞に核酸を導入できる。
宿主細胞で発現された本発明の抗原結合性フラグメントは、宿主細胞の培地、宿主細胞の抽出物及び/又は溶解物から精製できる。精製方法は公知の方法を適宜に組み合わせて行える。例えば、遠心分離、ヒドロキシアパタイトクロマトグラフィー、ゲル電気泳動、透析、イオン交換カラム上での分画、エタノール沈殿、逆相HPLC、シリカでのクロマトグラフィー、ヘパリンセファロースでのクロマトグラフィー、陰イオンまたは陽イオン樹脂クロマトグラフィー(ポリアスパラギン酸カラム等)、クロマトフォーカシング、SDS-PAGE、硫酸アンモニウム沈殿及びアフィニティクロマトグラフィーなどを適宜に使用できる。
本発明は、前記(1)記載の本発明の抗体又は抗原結合性フラグメントを含む組成物、ならびに本発明の抗体又は抗原結合性フラグメントをコードする核酸を含む組成物を提供する。
本発明の抗体又は抗原結合性フラグメントは、MAGE-A4由来ペプチドとHLA-A2との複合体に対するプローブとして使用できる。MAGE-A4はがん細胞の内部に発現し、HLAによって提示されることが明らかとなっており、T細胞は抗原認識に基づいて、当該がん細胞を攻撃する。そこで、本発明の抗原結合性フラグメントは、がん細胞に対するプローブ、すなわち検出用組成物又は診断用組成物として使用できる。
そのような検出方法としては、公知の検出方法、例えばELISA、蛍光抗体アッセイ、放射性免疫アッセイ、放射性免疫沈降法、ドットブロットアッセイ、阻害または競合アッセイ、サンドイッチアッセイ、ラテックスビーズ凝集アッセイなどが例示できる。
診断や検出の対象とするサンプルは、生物学的サンプル(例えば、疾患部位の組織、細胞、血液、血清、血漿、リンパ液、尿などの体液)が含まれる。これらのサンプルは、必要に応じて予備精製、均質化、遠心分離、又は希釈を行った後に、適切な緩衝液中で、本発明の抗原結合性フラグメントを接触させ、抗原・抗体複合体を検出する。この場合、本発明の抗原結合性フラグメントを標識してもよく、標識二次抗体を使用してもよい。標識としては、酵素(例えば西洋ワサビペルオキシダーゼ、アルカリホスファターゼなど)、放射性同位元素(例えば32P、35S、3H、125Iなど)、蛍光性物質(例えばローダミン、フルオレサミン、ダンシルクロリド、それらの誘導体など)、タグペプチドなどを使用できる。
本発明の医薬組成物の有効成分の有効量は、目的、腫瘍の種類、部位及び大きさ等の投与対象の病状、患者の条件及び投与経路などに応じて適宜に決められる。医薬組成物として使用する際には、有効成分に加えて、公知の薬学上許容し得る各種成分(例えば、担体、賦形剤、緩衝剤、安定化剤、等)を添加できる。医薬組成物は、条件に応じて、錠剤、液剤、粉末、ゲル、噴霧剤、或いはマイクロカプセル、コロイド状分配系(リポソーム、マイクロエマルジョン等)及びマクロエマルジョン等の薬剤形態をとり得る。医薬組成物の投与方法としては、静脈内、腹腔内、脳内、脊髄内、筋肉内、眼内、動脈内、胆管内、病変内経路による注入、注射、持続放出型システム製剤による方法が挙げられる。医薬組成物は、輸液により連続的に、または注射により投与できる。
本発明の医薬組成物は、慢性骨髄性白血病、急性リンパ性白血病(ALL)、急性骨髄性白血病(AML)などの造血器腫瘍や胃癌、大腸癌、肺癌、乳癌、胚細胞癌、肝癌、皮膚癌、膀胱癌、前立腺癌、子宮癌、子宮頸癌、卵巣癌、中皮腫等の固形癌の治療、各腫瘍・固形癌の診断・検出のために使用される。特に、MAGE-A4陽性、HLA-A2陽性の造血器腫瘍・固形癌の治療・診断・検出に有用である。
本発明は、前記(1)記載の抗原結合性フラグメントとシグナル伝達タンパク質の細胞内ドメインを含むキメラ抗原受容体(CAR)を含む。CARは、HLA-A2-MAGE-A4由来ペプチド複合体を特異的に認識・結合して、CARを発現する細胞にシグナル伝達できる。CARは、細胞外ドメイン、膜貫通ドメイン及び細胞内ドメインを含む。細胞外ドメインには、本発明の抗原結合性フラグメントが含まれる。細胞外ドメインとHLA-A2-MAGE-A4複合体が特異的に結合すると、CARの細胞内ドメインを介して、細胞内にシグナルが伝達され、CARを発現している細胞を刺激する。刺激を受けた細胞は、サイトカイン等を産生し、複合体を発現している標的細胞に対し細胞傷害性を発揮し、又は他の免疫細胞の細胞傷害性を誘導できる。
CARの細胞内ドメインとして、同一分子内に存在する細胞外ドメインがMAGE-A4由来ペプチド/HLA-A2複合体と相互作用(結合)したときに、細胞内にシグナル伝達できるものが使用できる。そのようなシグナル伝達タンパク質としては、膜タンパク質、シグナル受容体、サイトカイン受容体から選択されるタンパク質が例示される。CARの細胞内ドメインは、CD3zeta(CD3ζ)、FCRγ、FcRβ、CD3γ、CD3δ、CD3ε、CD5、CD22、CD79a、CD79b及びCD66dに由来する一次細胞質シグナル伝達配列を含む細胞内ドメインが例示される。また、CD2、CD4、CD5、CD8α、CD8β、CD28、CD137(「4-1BB」ともいう)、CD134、ICOS、GITR及びCD154に由来する二次細胞質シグナル(共刺激シグナル)伝達配列を含む細胞内ドメインが例示される。さらに、IL-2受容体、IL-21受容体に由来する三次細胞質シグナル伝達配列を含む細胞内ドメインが例示される。
本発明は、CARをコードする核酸を含む。CARをコードする核酸は、適当なプロモーターの制御下に発現されるように別の核酸と連結できる。プロモーターとしては構成的に発現を促進するもの、薬剤等(例えば、テトラサイクリン又はドキソルビシン)により誘導されるもの等のいずれも用いることができる。例えば、ホスホグリセリン酸キナーゼ(PGK)プロモーター、Xistプロモーター、β-アクチンプロモーター、RNAポリメラーゼIIプロモーター等の哺乳類由来プロモーター、SV40初期プロモーター、サイトメガロウイルスプロモーター、単純ヘルペスウイルスのチミジンキナーゼプロモーター、各種レトロウイルスのLTRプロモーター等のウイルス由来プロモーターを使用することができる。効率的な核酸の転写を達成するために、プロモーター又は転写開始部位と協同する他の調節要素(例えば、エンハンサー配列又はターミネーター配列)を含む核酸を連結してもよい。また、更に核酸の発現を確認するためのマーカーとなりうる遺伝子(例えば薬剤耐性遺伝子、レポーター酵素をコードする遺伝子、又は蛍光タンパク質をコードする遺伝子等)を組み込んでもよい。
本発明の別の態様において、CARを発現する細胞が含まれる。当該細胞は、本発明のCARをコードする核酸を細胞に導入する工程により調製できる。CARを発現する細胞は、CARを介してHLA-A2-MAGE-A4複合体と結合することにより、細胞内にシグナルが伝達され活性化される。細胞の活性化は、宿主細胞の種類や細胞内ドメインにより異なるが、サイトカインの放出、細胞増殖率の向上、細胞表面分子の変化等を指標として確認できる。例えば、活性化された細胞からの細胞傷害性のサイトカイン(腫瘍壊死因子、リンホトキシンなど)の放出は、複合体を発現する腫瘍細胞の破壊をもたらす。また、サイトカイン放出や細胞表面分子の変化により、他の免疫細胞、例えば、B細胞、樹状細胞、NK細胞、マクロファージ等を刺激する。従って、CAR発現細胞は養子免疫療法、特にMAGE-A4陽性、HLA-A2陽性の腫瘍又は癌に対する養子免疫療法において有用である。
細胞の培養に使用される細胞培養用器材としては、特に限定はないが、例えば、シャーレ、フラスコ、バッグ、大型培養槽、バイオリアクター等を使用することができる。なお、バッグとしては、細胞培養用CO2ガス透過性バッグを使用することができる。また、工業的に大量の細胞集団を製造する場合には、大型培養槽を使用することができる。また、培養は開放系、閉鎖系のいずれでも実施することができるが、好適には得られる細胞集団の安全性の観点から閉鎖系で培養を行うことが好ましい。
1.ヒト抗体ファージライブラリを用いた抗体スクリーニング
(1)pMHCビーズの作製
pMHCとしてのA2-MAGE-A4 20μgと、Streptavidinを結合させた磁気ビーズ200mgとを混合し、0.05%Tween/PBS(-)溶液にて4℃で通夜反応させた。これに3μLの2mMビオチン/PBS(-)を加え、1時間反応させた。これを0.05%Tween/PBS(-)溶液にて2回洗浄した後、0.05%Tween/PBS(-)溶液400μLに懸濁した。これを抗原ビーズとした。
(2)ヒト抗体ライブラリ
数十人の健常人由来の扁桃腺、臍帯血、末梢血、骨髄をもとにVHとVLをPCRにて増幅し、これをphagemidベクターpUC119上で組み合わせて 3.4×1012のレパトアのscFvとなるように調製し、M13 phageのcp3上にデイスプレイしたものをヒト抗体ライブラリとした。
反応液として3.4×1012cfuのヒト抗体ライブラリ溶液、 Streptavidin(Pierce) 100μg、A2-CMV tetramer 30μg、A2-Foxp69 tetramer 30μg、A2-IDOp41 tetramer 30μg、A2-IDOp195 tetramer 30μg、Gamma guard 20μg、2%BSA solution 100μL、10%TritonX100 65μL、1%TritonX100 / PBS 1000μLを作製し、室温で1時間回転混和した後、A2-MAGE-A4 tetramer を結合させた磁性ビーズ液100μlを混ぜて1時間回転混和した。その後、マグネットトラッパー(東洋紡)にて磁性ビーズをトラップしながら1%TritonX100/PBSにて5回洗浄したのちPBSにて1回洗浄した。これを培養したXL1-blueに加え、37℃にて1時間感染させた後、遠心して600μLの2×YTAG培地(200μg/mLアンピシリン、1% glucose / 2×YT)で懸濁し、YTAG寒天培地(200μg/mLアンピシリン、2% glucose / 普通寒天培地(日水))に蒔き、37℃で15時間培養した。コロニーを30mLの2×YTAG培地で回収し、そのうち200μLを20mLの2×YTAG培地に加え、30μLのヘルパーphage VCSMを加えた。37℃で30分感染させたのち、37℃で90分培養した。培養液に80mLの2×YTAG培地、50μg/mLカナマイシン60μL、1MIPTG 50μLを加え、28℃で20時間培養した。これを遠心して得られた上清に25mLのPEG溶液(20% PEG#600、2.5M NaCl)を入れて混和し、遠心して沈殿を回収し、これを1mL PBSにて懸濁したのち、フィルター滅菌した。以上の操作を2回繰り返し、2回目終了後、回収された大腸菌をYTAG寒天培地に蒔いた。30℃にて培養し、得られたコロニーをLB培地で30℃通夜培養、一部を使用してminiprep DNA kit(QIAGEN)にてDNAを抽出し、配列決定を行った。また、培養50μLを1.5mLの2×YTAI(0.5mMのIPTGを入れた2×YT培地)と混ぜ、30℃通夜培養したのち、遠心して上清をとった。これをcp3上清とした。これを用いてELISAを行った。
(1)使用したMHC-ペプチド複合体(モノマー)
MAGE-A4 p230、MAGE-A4 p286、MAGE-A3 p195、CMV、MelanA、GP3、HTLV-1、NY-ESO-1 p157、NY-ESO-1 p159、Foxp3 p69、Foxp3 p127、Foxp3 p390 、IDO p41 、IDO p41、IDO p195及びIDOp199の各ペプチドがHLA-A*0201と結合したものを用いた。
(2)pMHCの固相化
PBS 50μLに懸濁した500ng neutraavidin(PIERCE社製)をMaxisop loose(Nunc)に入れ、4℃にて通夜振盪した。溶液を捨てた後、200μLの2%BSA/PBSを入れ、通夜静置し、ブロッキングを行った。溶液を捨てた後、HLA-A2-MAGE-A4 モノマー300ng/50μl PBSを入れ、4℃にて通夜振盪した。その後、PBSにて洗浄した。
陰性コントロールであるHLA-A2-CMV の固相化も同様にして行った。
(3)ELISA反応測定
固相化した抗原プレートに100μlのcp3培養上清を入れ、室温にて1時間振盪したのち、プレートをPBSにて洗浄し、0.05%Tween20/PBSにて2000倍希釈した抗cp3ウサギ抗体100μlを入れ、室温にて1時間振盪した。PBSにて洗浄後、0.05%Tween20/PBSにて4000倍希釈したHRP標識抗ウサギIgG(MBL社製) 100μlを入れ、室温にて1時間振盪した。PBSにて洗浄後、0.01%H2O2、 0.1M Na2PO4、 0.1M citric acid(pH5.1)にて懸濁させたOPD(WAKO社製)を反応させ、発色を確認したのち、2N H2SO4を添加して反応を停止させ、SpectraMaxM2(モレキュラーデバイス社製)にて490nmの波長にて吸光度を測定した。
(1)精製用ベクターへの乗せ換え
scFv-FLAG/Hisの形態とするため、His/FLAG発現ベクターに乗せ換えた。FLAG tagはFACSでの検出用に、His-tagはカラムでの精製用に、それぞれ用いた。
(2)形質転換
単離した抗体クローンDNAをSalI(タカラバイオ社製)にて消化後、T4 DNA ligaseにて連結反応を行ったのち、コンピテントセルDH5α( TOYOBO社製)にて形質転換を行い、これをLBGA プレートに蒔いた。30℃にて通夜培養し、得られたコロニーを2×YTAGにて培養した後、一部を2×YTAIにて培養、遠心して上清を調整した。
(3)scFv-FLAG/His発現の確認
抗原プレートに培養上清100μlを入れ、室温にて1時間振盪した後、PBSにて洗浄した。His-tagの発現確認の場合は、0.05%Tween20/PBSにて4000倍希釈したanti-His HRP標識抗体を反応させて室温にて1時間振盪した。FLAG-tagの発現確認の場合は、Anti-FLAG(DDDK)抗体、HRP標識抗体の順に反応させた。
抗原プレートをPBSにて洗浄した後、0.01% H2O2、 0.1M Na2HPO4、 0.1M citric acid (pH5.1)にて懸濁したOPD(WAKO社製)を反応させ、発色を確認した後、2N H2SO4を添加して反応を停止させ、SpectraMaxM2(モレキュラーデバイス社製)にて490nmの波長にて吸光度を測定した。
前培養した菌液2mLを100mLの2×YTAIに入れ、30℃にて通夜培養した。これを遠心(8000rpm、10min、4℃)して、上清を回収し、飽和硫酸100mLを入れ、攪拌した。これを遠心(8000rpm、 20min、4℃)して、得られた沈殿をPBScomplete 10.5mLに懸濁した。これを遠心(12000rpm、1h、4℃)した後、上清にPBSにて平衡化したNi Sepharose excel(GE ヘルスケア)1mLを加え、4℃にて通夜培養した。サンプルをカラムに充填し、0.5MNaCl PBS 、20mMイミダゾール・0.5MNaCl PBSで洗浄し、250mMイミダゾール・0.5MNaCl PBSで溶出した。PBSにて透析を行い、透析終了後、アミコンウルトラ10K(ミリポア社製)にて濃縮した。サンプルの一部を用いてSDS-PAGEを行い、CBB染色をしてバンドを確認し、粗精製した抗体の蛋白濃度を判定した。
(5)AKTA Prime plus を用いたHis-tagタンパク質の精製
粗精製した抗体をAKTA Prime plus(GE ヘルスケア)によってHisTRAP カラムを用いて更に精製を行った。手順は付属のHis-tag精製プロトコルに従った。ピークが現れたフラクションを回収して、SDS-PAGEで確認した。
精製した抗体のKD値は、BiacoreX100(GEヘルスケア)を用いて測定した。リガンドの固相化にはBiotion capture kit(GEヘルスケア)を用いた。
(1)リガンドの固相化
センサーチップCAPにHLA-A2-MAGE-A4 p230 を固相化した。HBS bufferで200nMに調整し、適切なRU(resonance unit)となるよう固相化した。
(2)アナライト溶液の調整
精製した抗体をHBS EP + bufferにて12.5nM、25nM、50nM、100nM、200nMの濃度となるよう調整した。
(3)KD値の測定
センサーチップのフローセルにアナライト溶液を流し、180秒間の結合反応を測定した。次にHBS bufferに切り替えて300秒間の解離反応を測定した。測定はアナライトを低い濃度から連続して流すシングルサイクル法のプログラムを用いて行った。
全部の濃度について反応が終了したら、カーブフィッティングのプログラムを用いて結合定数を計算した。
(1)使用機器
フローサイトメトリーの測定には、FACS Calibur/FACS Cant /FACS CantII(BD)を用いた。
(2)使用した蛍光標識抗体
蛍光標識抗体として、表1に記載のものを用いた。
テトラマーPE(Tetramer-PE)は、ストレプトアビジン-PEにビオチン化されたモノマーが4つ結合したものを意味しており、抗原特異的T細胞の検出に用いた。使用したテトラマーは、A2-MAGE-A4 p230テトラマーとA2-NY-ESO-1 p157テトラマーであった。
(1)T2細胞(HLA-A*02+ヒトB-Tハイブリッドリンパ芽球細胞株)
T2細胞は、Transporter Associated with Antigen Processing(TAP)欠損株であり、細胞内タンパクがHLAに提示されないという特徴を持つ。
(2)T2細胞へのペプチドパルス
ヒトRPMI1640中で、終濃度10μMとなるようペプチドを加え、室温にて15分間置き、20%FCSヒトRPMI1640を、FCSが10%となるように加えた。CO2インキュベーターにて、37℃で45minインキュベートしたのち、二回洗浄を行った後、assayに用いた。
(3)HLA stability assay
ペプチドパルスを行ったT2細胞に、抗HLA-A2 Alexa Fluor488を1時間反応させ、FACSで検出することで、T細胞表面上で安定化したHLAの量を測定した。
ペプチドパルスを行ったT2細胞に、取得した抗体を1時間反応させ、抗FLAG抗体を1時間、抗マウスFITC抗体を1時間反応させ、FACSで検出した。
(5)1アミノ酸置換ペプチドとリスクペプチド
表2に示すように、MAGE-A4p230-239の10個のアミノ酸配列のうち、1箇所のアミノ酸をアラニン(A)、メチオニン(M)、フェニルアラニン(F)またはトリプトファン(W)で置換した1アミノ酸置換ペプチドを合成し、各ペプチドのIC50を調べた。各ペプチドはDMSOで終濃度10mMとなるように調整した。
MHC IC50はIEDB (http://tools.immuneepitope.org/processing/)により計算される理論値で、数字が低いほどHLA-A*0201とペプチドとの結合が強いことを示している。
表2には、1アミノ酸置換ペプチドのアミノ酸配列とIC50を示した。
(1)MAGE#17 28z CAR
図1には、CARのコンストラクトの配列イメージを示した。このコンストラクトには、5'側から、Leader、VH、VL、CL、CD28TM、CD28ICD(CD28の細胞内ドメイン)及びCD3zeta(CD3ζ)鎖が連結されている。このうち、VH、VLがMAGE♯17 scFvである。
(2)CARの構築
図2には、CARの構築ステップを模式的に示した。各ステップを説明すると、次の通りである。
まず、scFv-cp3を鋳型としてPCR反応により、VH-VLの5'側にEcoRI認識部位を持つLeaderを、3'側にAscI認識部位を持つCLをそれぞれ付加した。次に、このPCR産物をEcoRIとAscIで処理したフラグメントと、28z CARベクターをEcoRIとAscIで処理したフラグメントとをリガーゼで反応し、CAR 28zを得た。
ヒトリンパ球は、健康なドナーから提供を受けた血液より、Ficoll-PaqueTM PLUS(17-1440-03、GE Healthcare)を用いてPBMC(Peripheral Blood Mononuclear cells)を分離することによって得た。本研究に用いたヒト末梢血等の検体の採集、解析はヘルシンキ宣言に従って行なわれ、全て三重大学医学部研究倫理委員会にて承認されたプロトコルに従い、被験者本人の書面による同意書を得て実施された。
採取した検体は、本人特定不可能な暗号化がなされ盗難防止処置を施した冷蔵庫、液体窒素タンクに保存した。被験者個人情報に関しては匿名化され、個人のプライバシー、遺伝子解析の結果が外部に漏洩されないよう厳重な注意、処置が施行された。
(1)mRNAの作製
作製したCARのDNAを一本鎖に切断し、linear DNA テンプレートを作製した。Wizard SV Gel and PCR Clean-UP System (Promega)のキットを用いてDNAの精製を行った。精製したサンプルをmMESSAGEmMACHINE T7 Ultra Kit(Life technologies)を用いてmRNAの作製を行った。
(2)バイオアナライザーによるチェック
バイオアナライザー(Agilent Technologies)を用いて、mRNAのpolyA付加を確認した。
(3)PBMC培養プレートの作製
Anti-CD3抗体(OKT-3 eBioscience)を終濃度5μg/mL、RetroNectin(タカラバイオ)を終濃度25μg/mLになるようにACD-A液(TERUMO)にて希釈したのち、12 well plate (Nunc)の各wellに対して400μLを入れ、CO2インキュベーターにて37℃、5-10時間静置した。
リンパ球用培地GT-T503 50mLに対し、human IL-2(600IU)を50μL、アルブミナー(25% HSA)400μL、リンパ球ドナーのplasma 300μLを加えたものをPBMC培養の培地とした。
(5)PBMCの分離及び培養
健常人血液よりFicollを用いて分離した末梢血単核球(peripheral blood mononuclear cells、PBMC)を2×105個/mLとなるようにCMで懸濁し、2.5mLをプレートに入れ、CO2インキュベーターにて37℃、4日間培養した。
(6)エレクトロポレーション
分離10日後のPBMCに、作製したmRNAをエレクトロポレーションで導入した。導入後、24時間以内にassayに用いた。
(1)Packaging細胞 plat-Aによるレトロウイルスの作製
Packaging細胞 plat-AにFuGENE(promega)を用いてCARの遺伝子を導入し、二日間培養したのち、上清を回収してウイルス液とした。
(2)PBMCの分離及び培養
上記9.(3)~(5)と同様の手順に従った。
(3)レトロウイルス感染用plateの作製
RetroNectinをACD-A液で希釈し、終濃度20μg/mLとなるよう調整した。これを24 well plateの各wellに対して500μL入れ、4℃にてO/Nで静置した。ウイルス液を乗せる前にACD-Aで二回洗浄した。
上記10.(1)作製したウイルス液を1mLずつレトロウイルス感染用plateに乗せ、2000×g 、2時間、32℃にて遠心し、ウイルスをプレートにコートした。続いてこれを1.5%アルブミナー/ PBSにて二回洗浄した。
(5)PBMCのレトロウイルス感染 CAR-T細胞の作製
上記10.(2)で培養したPBMCを回収し、4×105個/mLとなるようにCMで希釈したのち、950μlをレトロウイルス感染用plateに蒔き、1000×g 、10分間、32℃にて遠心した後、CO2インキュベーターにて37℃で培養した。
作製したCAR-T細胞はPBMCの分離から数えて12~14日目にassayに用いた。
(6)コントロール細胞の作製
上記10.(2)で培養したものの一部をコントロール細胞(遺伝子導入操作なし細胞)として使用した。
eBioscienceのkitを用いて、培養液中に含まれるIFN-γの量をELISAにて測定した。
10×Coating bufferを精製水にて10倍希釈し、Coating bufferを調整した。Coating buffer 12mLに対し、一次抗体を48μL添加し、96well平底プレートに100μL/wellずつ添加した。これを4℃にて、一晩静置した後、0.05% PBS-Tで5回洗浄した。5×Assay diluentsを精製水にて5倍希釈し、Assay diluentsを調整した。Assay diluents 200μL/wellずつ加えた。室温にて1時間ブロッキングした後、0.05% PBS-Tで5回洗浄した。IFN-γは最高濃度を1000pg/mLに調整し、これを2倍ずつ7段階で希釈し、スタンダードとした。サンプルとスタンダードをプレートに載せ、室温にて2時間反応させた後、0.05% PBS-Tで5回洗浄した。Assay diluents 12mLに対し、二次抗体を48μL添加し、100μLずつwellに加えた。室温にて1時間反応させた後、0.05% PBS-Tで5回洗浄した。Assay diluents 12mLにStreptavidin-HRPを48μL添加し、100μL/wellずつ加えた。暗所、室温にて30分間反応させた後、0.05% PBS-Tで7回洗浄した。TMB substrate solutionを100μLずつ加え、暗所、室温にて15分間反応させた後、0.18M H2SO4を50μLずつ加えて反応を止めた。直ちに、microplate reader Model 680(Bio-Rad)にて波長450nmにて計測した。
ターゲット細胞を1×105個/mL、エフェクター細胞を1×105個/mLとなるよう調整した。APC Anti human CD107aを1サンプルあたり0.5μL添加して、ターゲット細胞:エフェクター細胞=1:1で96well plate(U)にて、37℃にて 1時間共培養を行った。その後、各wellに対し、0.7μLずつgolgistop(Protein Transport Inhibitor)を添加し、37℃にて4時間培養した。各wellの細胞をV型96well plateに移し、1200rpm 5分間 4℃にて遠心を行ったのち、0.5%BSA/PBSにて2回洗浄した。APC-cy7 Anti-Human CD4及びFITC Anti-human CD8を各wellに対し、0.5μLずつ添加して、遮光にて氷上に20分間静置し、0.5%BSA/PBSにて2回洗浄を行った。細胞固定液(cytofixl cytoperm:BD)を100μL添加し、遮光条件にて氷上の20分間静置した後、perm/wash(BD)100μLを添加して遠心した。その後、perm/wash液にて、2回の洗浄を行った。各well 0.5μLずつV450 IFN-γ、PE-Cy7 TNF-αを添加した。遮光条件下、氷上にて30分間静置した後、0.5%BSA/PBSにて2回の洗浄を行った。その後、FACSCanto IIフローサイトメーター(BD)で測定を行い、FACS Diva Software(BD)にて解析を行った。
(1)MAGE-A4ロングペプチド
MAGE-A4ロングペプチドのアミノ酸配列は、NYKRCFPVIF GKASEGVYDG REHTVYGEPR KAETSYVKVL EHVVRVNARV RI(配列番号28)であり、分子量は6006.811であった。このロングペプチドは、A24 MAGE-A4 143-151エピトープ(NYKRCFPVI:配列番号29)、A2 MAGE-A4 230-239エピトープ(GVYDGREHTV:配列番号1)及びヘルパーエピトープ(AETSYVKVLE HVVRVNARVR I:配列番号30)を連結して設計した。
(2)CHP-ロングペプチド
CHP-ロングペプチドは、ドラックデリバリーシステムであるCHPナノゲルにMAGE-A4 ロングペプチドが包まれたものを意味する。この物質を用いることにより、細胞性免疫誘導が期待できる(非特許文献5)。
(3)CD3 negative beads selection
ヒトリンパ球を1×107個当たり2% FCS/PBS 80μLに懸濁した。ここにCD3 Micro Beads(Miltenyi Biotec)を20μL加え、よく混合した。遮光状態とし、4℃にて15分間反応させた後、10倍量の2% FCS/PBSで洗浄した。細胞を2% FCS/PBS 3mLに懸濁して細胞懸濁液とした。MACSカラムをセットしたMACS Separatorに細胞懸濁液を通した。カラムをすり抜けた分画をCD3 陰性分画とし、APCとして用いた。
(1)Cr release assay
標的細胞を遠心で集め、100% FCSにて1×106細胞が50μLに懸濁されている状態とした。これに、50μCi(3.7×106Bq)の51Crを添加し、CO2インキュベーターにて37℃にて1時間培養した。エフェクター細胞はETratioに応じて10% FCS・RPMI1640で細胞数を調整し、U字型96well plateに播種した。培養終了後、10% FCS・RPMI1640にて3回洗浄した。細胞を1×105個/mLとなるよう10% FCS・RPMI1640にて懸濁し、1×104個/100μL/wellとなるように、U字型96well plateに播種した。CO2インキュベーターにて37℃にて、8時間培養した。培養終了後、1200rpm、5分間、4℃の遠心を行い、読み取り用のプレートに上清を30μLずつ移し、一晩風乾により乾燥させた。51Crの量をシンチレーションカウンターMicroBeta2(パーキンエルマー)によって測定した。細胞傷害活性の計算は、下記式によって求めた。最大リリース値(maximam release)は、1% NP-40を100μL添加したときの測定値とした。
細胞傷害活性(%)=100×(測定値(cpm)-自発リリース値(cpm))/(最大リリース値(cpm)-自発リリース値(cpm))
(1)使用マウス
NOGマウス(NOD/Shi-scid、IL-2RγKO Jic)は日本クレア株式会社より購入した。実験には雌のマウスを用い、7-8週齢で実験に使用した。実験動物を用いたT細胞輸注療法、遺伝子免疫療法の研究は、三重大学の組換えDNA実験審査委員会、三重大学医学部研究倫理委員会、動物実験審査委員会において承認を受けた。マウスは、三重大学生命科学研究支援センター動物実験施設において飼育した。
(2)ヒト腫瘍の移植と放射線全身照射(TBI)
前処置として、細胞輸注の前日にNOGマウスに放射線全身照射(TBI)2.5Gyを行った。この前処置により、レシピエント体内のリンパ球数を減少させ、輸注したドナーリンパ球が生着しやすい状態とした。輸注したCAR-T細胞およびヒトリンパ球は1×107個/匹とした。各細胞は、尾静脈よりNOGマウスに輸注した。体重は2-3日おきに測定した。
NOGマウスへの腫瘍の移植は、CAR-T細胞を輸注する7日前に行い、マウス右体側にNW-MEL-38(A2陽性 MAGE-A4 陽性)を2.5×106個/匹、マウス左体側にHCT116(A2陽性 MAGE-A4 陰性)を2.5×106個/匹で皮下注射した。腫瘍径は2-3日おきに測定した。
輸注用のCAR-T細胞は、上記「10.レトロウイルスによる細胞への遺伝子導入」の方法に従って作製した。
(4)眼窩採血によるマウス末梢血中のPBMC分離
マウスの眼窩から末梢血を採取し、Ficollを用いてPBMCを分離した。A2-MAGE-A4テトラマー、ヒトCD4CD8蛍光抗体を用いて染色し、FACSで輸注した細胞を確認した。
(5)TIL (tumor infiltrating lymphocytes)の確認
マウスから採取した腫瘍をすりつぶし、A2-MAGE-A4テトラマー、ヒトCD4CD8蛍光抗体を用いて染色し、FACSで輸注した細胞を確認した。
(6)免疫染色
マウスから切除したがんを液体窒素で凍らせ、クライオスタットで凍結切片を作製した。DAPI、ヒトCD4-FITC、ヒトCD8-FITCによって染色を行い、蛍光顕微鏡で観察した。
ICD部分の相違によるCAR-T細胞の効果を確認した。具体的には、CD28のもの(28z)から、GITRのもの(zG)およびCD137のもの(4-1BBz)としたものを作製し、その効果を確認した。CAR zGは、図21に示すように、ICDとしてCD28のものに代えて、GITR(グルココルチコイド誘導腫瘍壊死因子受容体)を用いた。同様に、CD28のものに代えて、CD137(4-1BBz)のものを用いた。
コントロールとして、CAR非導入CD8陽性T細胞(NGMC)を使用した。
1.HLA-A2-MAGE-A4を認識する抗体の単離と評価
MAGE-A4(HLA-A2-MAGE-A4)は、メラノーマでの発現が高いことが知られている。そこで、MAGE-A4を認識する複数の抗体をヒト抗体ライブラリを使用したスクリーニングによって単離した。抗体の単離には、ファージディスプレイ法を用いた。この方法では、ファージコートタンパクcp3の一部としてscFvが提示され、抗原に対して結合性の高いクローンをELISAによって絞り込むことができる。約1500のクローンをピックアップし、scFv-CP3の状態でELISAを行った。ポジティブターゲットであるHLA-A2-MAGE-A4に反応し、ネガティブターゲットであるA2-CMVに反応しないものを選択した。選択した抗体について、結合特異性の検討を行うため、複数のペプチド-MHC複合体(pMHC)を用いてELISAを行った。HLA-A2-MAGE-A4 p230、MAGE-A4 p286、MAGE-A3 p195、CMV、MelanA、GP3、HTLV-1 p11、NY-ESO-1 p157、NY-ESO-1 p159、Foxp3 p69、Foxp3 p127、Foxp3 p390、IDO p41、IDO p41、IDO p195、IDO p199の各pMHCモノマーを固相化し、選択した抗体でELISAを行った。図3には、ELISAの結果の一例を示した。
次に、MAGE#17抗体について、BiacoreX100によるKD値の測定を行った。BiacoreでのKD値測定は、時間経過に対する検出感度(レゾナンス、チップ上の質量変化を反映する)の動的変化を測定することによって求めた。動的変化のカーブから解離速度定数(Kd)と結合速度定数(Ka)を求め、その2つの定数の比からその結合定数を求めた。測定の結果、MAGE#17の結合定数は22nMであった(図4)。
T2細胞は、Transporter Associated with Antigen Processing(TAP)欠損株であり、細胞内タンパクがHLAに提示されない。T2細胞にペプチドをパルスすると、T2上のHLAが安定化するので、蛍光標識された抗HLA抗体を用いることにより、HLA発現量を確認でき、そのペプチドがHLAにどの程度結合しているのかがわかる。
MAGE-A4p230、CMV、DMSOの各ペプチドをパルスしたT2細胞に、取得した抗体MAGE #17、#86、#209、#345、#672を反応させ、FACSにてシフト量を確認した。MAGE-A4 p230をパルスしたT2細胞においてのみ、取得した抗体クローンMAGE#17、#86、#209、#345、#672の全てにおいてシフト量の変化が確認された(図5)。
次に、抗体の認識に関与するアミノ酸を調べるため、MAGE-A4 p230(GVYDGREHTV:配列番号1)のアミノ酸を1個ずつ別のアミノ酸(主としてアラニン)に置換したペプチドを作製し、HLA-A*0201との結合力をIEDB( Immune Epitope Database)を用いて調べ、MHC IC50を求めた(表2)。MHC IC50はIEDBにより計算される理論値で、数字が低いほどHLA-A*0201とペプチドとの結合が強いことを示す。T2細胞にアラニン置換したペプチドをパルスし、T2細胞表面上のHLAの量をFACSで測定した(図6)。ペプチドが強くHLAに結合するほど、peptide-HLAの構造が安定化し、FACSのシフト量が増加するので、MFIの値はペプチドとHLAの結合力の指標となる。一部のペプチドを除き、MFIの値とMHC IC50値とは、相互に対応した。
次に、リスクペプチドをT2細胞にパルスし、MAGE#17を反応させ、リスクペプチドを認識しないことを確かめた(図9)。同様のアッセイを他のクローン#86、#209、#345、#672でも実施した結果、認識に関与するアミノ酸の数が一番多かったクローンが#17であった。このため、MAGE#17を最終的な候補とした。
MAGE#17の塩基配列(配列番号31):CAGGTCCAGC TGGTACAGTC TGGGGCTGAG GTGAAGAAGC CTGGGTCCTC GGTGAAGGTC TCCTGCAAGG CTTCTGGAGG CACCTTCAGC AGCTATGCTA TCAGCTGGGT GCGACAGGCC CCTGGACAAG GGCTTGAGTG GATGGGAGGG ATCATCCCTA TCTTTGGTAC AGCAAACTAC GCACAGAAGT TCCAGGGCAG AGTCACGATT ACCGCGGACA AATCCACGAG CACAGCCTAC ATGGAGCTGA GCAGCCTGAG ATCTGAGGAC ACGGCCGTGT ATTACTGTGC GAGATCCCCC CGGCGGGCAT ATCATGATGC TTTTGATATC TGGGGCCAAG GGACAATGGT CACCGTCTCT TCAGGTGGAG GCGGTTCAGG CGGAGGTGGC AGCGGCGGTG GCGGGAGTTC CTATGAGCTG ACTCAGCCAC CCTCGATGTC AGTGGCCCCA GGAAAGACGG CCAGCATTAC CTGTGGCGGA GACCATATTG GAAGTAAAAG TGTTCACTGG TACCAGCAGA AGCCAGGCCA GGCCCCTGTA CTGGTCGTCT ATGATGATAG CGACCGGCCC TCAGGGATCC CTGAGCGATT CTCTGGCTCC AACTCTGGGA ACACAGCCAC TCTGACCATC AGCGGGACCC AGGCTATGGA TGAGGCTGAC TATTACTGTC TGGCGTGGGA CAGCAGCACT GCGATCTTCG GCGGAGGGAC CAAGCTGACC GTCCTC。
上記各配列は、下記に示すように、VH(配列番号33)、sc(配列番号34)及びVL(配列番号35)の塩基配列、並びにVH(配列番号36)、sc(配列番号37)及びVL(配列番号38)のアミノ酸配列を結合したものとなっている。
VHの塩基配列(配列番号33):CAGGTCCAGC TGGTACAGTC TGGGGCTGAG GTGAAGAAGC CTGGGTCCTC GGTGAAGGTC TCCTGCAAGG CTTCTGGAGG CACCTTCAGC AGCTATGCTA TCAGCTGGGT GCGACAGGCC CCTGGACAAG GGCTTGAGTG GATGGGAGGG ATCATCCCTA TCTTTGGTAC AGCAAACTAC GCACAGAAGT TCCAGGGCAG AGTCACGATT ACCGCGGACA AATCCACGAG CACAGCCTAC ATGGAGCTGA GCAGCCTGAG ATCTGAGGAC ACGGCCGTGT ATTACTGTGC GAGATCCCCC CGGCGGGCAT ATCATGATGC TTTTGATATC TGGGGCCAAG GGACAATGGT CACCGTCTCT TCA。
VL部分の塩基配列(配列番号35):GAGCTGACTC AGCCACCCTC GATGTCAGTG GCCCCAGGAA AGACGGCCAG CATTACCTGT GGCGGAGACC ATATTGGAAG TAAAAGTGTT CACTGGTACC AGCAGAAGCC AGGCCAGGCC CCTGTACTGG TCGTCTATGA TGATAGCGAC CGGCCCTCAG GGATCCCTGA GCGATTCTCT GGCTCCAACT CTGGGAACAC AGCCACTCTG ACCATCAGCG GGACCCAGGC TATGGATGAG GCTGACTATT ACTGTCTGGC GTGGGACAGC AGCACTGCGA TCTTCGGCGG AGGGACCAAG CTGACCGTCC TC。
VH部分のアミノ酸配列(配列番号36):QVQLVQSGAE VKKPGSSVKV SCKASGGTFS SYAISWVRQA PGQGLEWMGG IIPIFGTANY AQKFQGRVTI TADKSTSTAY MELSSLRSED TAVYYCARSP RRAYHDAFDI WGQGTMVTVS S。
sc部分のアミノ酸配列(配列番号37):GGGGSGGGGS GGGGSSY。
VL部分のアミノ酸配列(配列番号38):ELTQPPSMSV APGKTASITC GGDHIGSKSV HWYQQKPGQA PVLVVYDDSD RPSGIPERFS GSNSGNTATL TISGTQAMDE ADYYCLAWDS STAIFGGGTK LTVL。
抗体クローンMAGE#17の特異性が確認できたので、MAGE#17をCARのベクターへと乗せ換えた。次に、mRNAを調整しバイオアナライザーで確認したのち、エレクトロポーレーション法により、ヒト末梢血リンパ球(PBMC)にCARの遺伝子を導入した。導入したCARが正常に発現しているか否かをテトラマー染色によって確認した。その結果、CAR-T細胞のCAR陽性率はCD8で98.8%、CD4で98.5%であった(図10)。CAR-T細胞とペプチドパルスを行ったT2細胞を24時間共培養し、培養上清中に含まれるIFN-γを測定したところ、MAGE-A4をパルスしたT2細胞特異的にIFN-γの産出が増大した。コントロール細胞(遺伝子導入していない細胞)を用いて、同様の試験を行った。また、ターゲット細胞であるA2陽性MAGE-A4陽性のNW-MEL-38、LB-23では、オフターゲットであるLC-1/sq、MEL72と比較して、腫瘍特異的なIFN-γの産出が見られた(図11)。
レトロウイルスにより遺伝子導入したCAR-T細胞とペプチドパルスを行ったT2細胞を24時間共培養し、培養上清中に含まれるIFN-γを測定したところ、MAGE-A4をパルスしたT2細胞特異的にIFN-γの産出が増大した。コントロール細胞(遺伝子導入していない細胞)を用いて、同様の試験を行った。また、ターゲット細胞であるA2陽性MAGE-A4陽性のNW-MEL-38、LB-23では、オフターゲットであるLC-1/sq、MEL72と比較して、腫瘍特異的なIFN-γの産出が見られた(図13)。mRNA、レトロウイルスベクターによるCAR遺伝子の導入で、両者ともにCARが発現し、特異的に腫瘍を認識し、IFN-γを産出することが確認できた。
CAR-T細胞は、がん細胞上に発現しているA2-MAGE-A4を抗原として認識して活性化し、細胞傷害活性を発揮して標的細胞を傷害することが期待された。T細胞の活性化の指標として知られている分子として、IFN-γ、 TNF-α、 CD107aなどがあり、これらはT細胞の活性化に伴って発現が亢進する。そこで、ペプチドパルスしたT2細胞、ターゲット細胞と共培養したMAGE#17CARの細胞内サイトカイン染色(intracellular cytokine staining : ICS)を行った。
その結果、MAGE-A4p230をパルスしたT2細胞との共培養で、IFN-γ、 TNF-α、 CD107aで染まるCAR-T細胞の割合が増加した(図14)。また、ターゲット細胞との共培養では、A2陽性MAGE-A4陽性のNW-MEL-38、LB-23との共培養において、IFN-γ、 TNF-α、 CD107aで染まるCAR-T細胞の割合が増加した(図15)。
健常人の末梢血からPBMCを分離し、CD3 magnet beadsを用いて、ネガティブセレクションを行い、CD3-の分画を抗原提示細胞(antigen presenting cell : APC)として用いた。MAGE-A4p230が提示されるAPCとしてA2-APC、ネガティブコントロールのAPCとしてA24-APCを用いた。MAGE-A4 Longペプチド、CHP-MAGE-A4 Longペプチドを終濃度10μMとなるよう加え、一晩培養を行い、CAR-Tと24時間共培養した。CAR-T細胞への遺伝子導入は、上記「10.レトロウイルスによる細胞への遺伝子導入」の方法に従った。その結果、A2-APCにMAGE-A4 Longペプチドをパルスしたものとの共培養で、培養上清中に含まれるIFN-γの値が上昇した(図16)。
MAGE-A4 LongペプチドがAPCに取り込まれ、MAGE-A4p230がMHC-class Iにクロスプレゼンテーションされ、それを認識したCAR-TのIFN-γ産生が特異的に増大することが確認された。一方、ドラッグデリバリーシステムであるCHPナノゲルに包まれたCHP-MAGEA4ロングペプチドにおいてはIFN-γの特異的な産生は見られなかった。
51Cr release assayによりMAGE#17CARの特異的な細胞障害活性が起こるか否かを確認した。CAR-T細胞とターゲットのがん細胞の比率をET ratio(effector/ターゲット ratio)とし、クロムを取り込ませたターゲットのがん細胞とCAR-T細胞をET ratio 10:1、3:1、1:1で8時間共培養し、上澄みに含まれる51Crの放射線量の値から、細胞障害率(%lysis)を求めた。CAR-T細胞への遺伝子導入は、上記「10.レトロウイルスによる細胞への遺伝子導入」の方法に従った。
その結果、MAGE-A4 p230 ペプチドをパルスしたT2細胞でMAGE-A4特異的な細胞障害が確認された(図17)。また、ターゲット細胞においても、A2陽性MAGE-A4陽性のNW-MEL-38に特異的な細胞障害が確認された(図18)。
(1)NW-MEL-38及びHCT116を用いたモデル
NOGマウスはコモンγレセプターを欠損しているので、NK細胞が存在しない。このため、ヒト細胞やヒト組織の生着性がNOD-SCIDマウスと比べて著しく高く、ヒトのがんやヒト正常組織を高率に生着させられる。また、ヒト造血幹細胞移植後に、ヒトT細胞の分化が認められることから、ヒト免疫系モデルマウスとしての需要が高まっている。NOGマウスの特徴として、T細胞及びB細胞の欠失、ナチュラルキラー(NK)細胞の欠失、樹状細胞機能の低下、マクロファージ機能の低下、補体活性の欠失、老化に伴うT細胞及びB細胞の漏出(leakiness)が認められないことが挙げられる。
NOGマウスについて、右体側にNW-MEL-38(A2陽性MAGE-A4陽性)を2.5×106個/匹、左体側にHCT116(A2陽性 MAGE-A4陰性)を2.5×106個/匹で皮下注射した。腫瘍移植後6日目に、前処置として、放射線全身照射(TBI)2.5Gyを行った。TBIにより、レシピエント体内でのリンパ球数を減少させ、輸注したドナーリンパ球が生着しやすい状態とした。7日目にMAGE#17 CAR-T細胞およびヒトリンパ球を細胞数1×107個/匹で、尾静脈より輸注した。CAR-T細胞への遺伝子導入は、上記「10.レトロウイルスによる細胞への遺伝子導入」の方法に従った。CAR陽性率はCD8 36.3%、CD4 38.5%であった(図19)。
CAR-T細胞輸注群(n=2)、ヒトリンパ球(CAR非導入)輸注群(n=2)、 PBS群(n=2)で実験を行い、腫瘍径と体重を2-3日おきに測定した。測定は腫瘍移植後42日目まで行った。
その結果、CAR-T細胞輸注群では、マウス#1とマウス#2でNW-MEL-38(A2陽性MAGE-A4陽性)の増殖抑制が認められた。一方、HCT116(A2陽性 MAGE-A4陰性)の増殖抑制は認められなかった。マウス#1は腫瘍移植34日後に死亡した。死亡原因は腫瘍死ではなく、感染の可能性が高いと考えられた(図20)。
次に、ICD部分の相違によるCAR-T細胞の効果を確認した。CAR zGの遺伝子配列イメージを図21に示した。CAR zGでは、ICDとして、CD28のものに代えて、GITRのものを用いた。GITRは、グルココルチコイド誘導腫瘍壊死因子受容体のことを意味しており、このICRを用いたCARでは、CARの発現量に対するサイトカイン産生量が高く成り得ることが知られている(WO2013051718)。CAR zGは、図2のCAR 28zにおいて、CD28 ICD部分を別のICDであるGITR ICDに変更し、CAR 28zにおけるCD3ζとICDドメインの順序を変更したものである。その他の部分は、CAR 28zと同様の配列を持ったものを用いた。
NOGマウスについて、NW-MEL-38(A2陽性MAGE-A4陽性)を2.5×106個/匹で皮下注射した。腫瘍移植後3日目に、前処置として、放射線全身照射(TBI)2.0Gyを行った。4日目に、下記に示すA群~C群(n=4)のそれぞれに細胞またはPBSと、ヒトリンパ球を細胞数4×106個/匹で、尾静脈より輸注した。CAR-T細胞への遺伝子導入は、上記「10.レトロウイルスによる細胞への遺伝子導入」の方法に従った。A群にはPBS、B群にはMAGE#17 CAR-T細胞(28z)、C群にはCAR-T細胞(zG)を投与した。
A群~C群について、腫瘍径と体重を2-3日おきに測定した。測定は腫瘍移植後24日目まで行った。
その結果、ICHが28z及びzGのいずれのCAR-T細胞を輸注した場合にもNW-MEL-38(A2陽性MAGE-A4陽性)の増殖抑制が認められた(図22)。このように、本実施形態では、細胞内ドメインを変更した場合にも同様の効果が得られることが分かった。
(1)zG型CAR及び28z型CARを導入したレトロウイルスベクターを構築し、packaging 細胞 plat-Aによりレトロウイルスを作製し、PBMCに感染させることで、CAR-T細胞を作製した。遺伝子導入は、上記「10.レトロウイルスによる細胞への遺伝子導入」の方法に従った。CARの発現を、テトラマー染色によって確認した。その結果、zG型CAR-T細胞のCAR陽性率はCD8で99.1%、28z型CAR-T細胞のCAR陽性率はCD8で99.8%であった(図23)。
(2)レトロウイルスにより遺伝子導入したCAR-T細胞(4×104個)とペプチドパルスを行ったT2細胞、及び各種細胞株NW-MEL-38、SK-MEL-37、HCT116(それぞれ2×105個)を18時間共培養し、培養上清中に含まれるIFN-γを測定した。図24に示すように、zG型CARおよび28z型CARを導入したCAR-T細胞では、MAGE-A4をパルスしたT2細胞とNW-MEL-38細胞及びSK-MEL-37細胞に特異的にIFN-γの産出が増大した。
コントロール細胞(遺伝子導入していない細胞)を用いて、同様の試験を行った。ターゲット細胞であるA2陽性MAGE-A4陰性のHCT116ではIFN-γの産出が見られなかった。なお、エフェクターのみ(Effector-only)および標的のみ(Target-only)では、IFN-γの産出は見られなかった。
このように、zG型CAR導入CD8陽性T細胞はin vitro系において、IFN-γの産出を誘導した。この結果は、in vivo系の結果を上手く説明できるものであった。
(3)次に、上記(1)および(2)と同様の試験を4-1BBz型CAR導入CD8陽性T細胞の特性について調べた。図25に示すように、4-1BBz型CARを導入したCAR-T細胞では、MAGE-A4をパルスしたT2細胞に特異的にIFN-γの産出が増大した。
コントロールペプチドで刺激したT2細胞では、IFN-γの産出が見られなかった。なお、エフェクターのみ(Effector-only)および標的のみ(Target-only)では、IFN-γの産出は見られなかった。
このように、ICDを4-1BBz型に変更した場合にも同様の効果が認められた。このin vitro系のデータは、in vivo系の結果を容易に説明できるものである。
こうして、ICDを変更した場合にも、zG型と同様の効果が認められた。
MAGE-A4由来ペプチド/HLA-A2複合体を特異的に認識する抗原結合性タンパク質は、細胞医療、遺伝子治療の分野で使用できる。また、MAGE-A4由来ペプチド/HLA-A2複合体を発現する腫瘍細胞の検出、腫瘍の治療及びその研究・試験に極めて有用である。本発明のCAR-T細胞は、副作用が非常に少ないので、非常に有効ながん治療法を提供できた。
Claims (13)
- 以下の(A)または(B)のポリペプチドを含み、HLA-A2-MAGE-A4複合体を抗原として認識する抗原結合性タンパク質:
(A)配列番号36のVH(重鎖可変領域)のアミノ酸配列と、配列番号38のVL(軽鎖可変領域)のアミノ酸配列とを含むポリペプチド;
(B)配列番号36のVH(重鎖可変領域)のアミノ酸配列と90%以上の相同性を持つアミノ酸配列と、配列番号38のVL(軽鎖可変領域)のアミノ酸配列と90%以上の相同性を持つアミノ酸配列とを含むポリペプチド。 - VHとVLとの間に、以下の(C)または(D)のポリペプチドを含む請求項1に記載の抗原結合性タンパク質:
(C)配列番号37のsc(一本鎖)のアミノ酸配列を含むポリペプチド;
(D)配列番号37のsc(一本鎖)のアミノ酸配列と90%以上の相同性を持つアミノ酸配列を含むポリペプチド。 - 請求項1または2に記載のものであって、配列番号32のアミノ酸配列、または配列番号32のアミノ酸配列と90%以上の相同性を持つアミノ酸配列を含むポリペプチドである抗原結合性タンパク質。
- 抗原結合性タンパク質が、Fab、Fab'、F(ab')2、Fvまたは一本鎖Fv(scFv)である請求項1~3のいずれか一つに記載の抗原結合性タンパク質。
- 請求項1~4のいずれか一つに記載の抗原結合性タンパク質をコードする核酸。
- 請求項5に記載の核酸を含むベクター。
- 請求項1~4のいずれか一つに記載の抗原結合性タンパク質とシグナル伝達タンパク質の細胞内ドメインを含むキメラ抗原受容体。
- シグナル伝達タンパク質がCD3zeta(CD3ζ)鎖または共刺激分子CD(GITR)のいずれかである請求項7に記載のキメラ抗原受容体。
- 更にCD28の細胞内ドメインまたはGITRの細胞内ドメインのいずれかを含む請求項8に記載のキメラ抗原受容体。
- 請求項7~9のいずれか一つに記載のキメラ抗原受容体をコードする核酸。
- 請求項10に記載の核酸を含むベクター。
- 請求項7~9のいずれか一つに記載のキメラ抗原受容体を発現する細胞。
- 請求項12に記載の細胞を有効成分として含有する医薬組成物。
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JP7202512B2 (ja) | 2023-01-12 |
RU2019139538A3 (ja) | 2021-08-26 |
CN110709516B (zh) | 2023-06-27 |
RU2019139538A (ru) | 2021-07-12 |
US20200276237A1 (en) | 2020-09-03 |
KR20200015567A (ko) | 2020-02-12 |
CN110709516A (zh) | 2020-01-17 |
EP3636761A4 (en) | 2021-03-03 |
KR102647696B1 (ko) | 2024-03-13 |
US11497768B2 (en) | 2022-11-15 |
EP3636761B1 (en) | 2024-07-31 |
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EP3636761A1 (en) | 2020-04-15 |
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