WO2018117237A1 - 抗cd3抗体及び該抗体を含む分子 - Google Patents
抗cd3抗体及び該抗体を含む分子 Download PDFInfo
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- WO2018117237A1 WO2018117237A1 PCT/JP2017/046006 JP2017046006W WO2018117237A1 WO 2018117237 A1 WO2018117237 A1 WO 2018117237A1 JP 2017046006 W JP2017046006 W JP 2017046006W WO 2018117237 A1 WO2018117237 A1 WO 2018117237A1
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- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
- C07K16/2809—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
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- C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
- C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
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- C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
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- C07K2317/622—Single chain antibody (scFv)
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- C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
- C12N2015/8518—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic expressing industrially exogenous proteins, e.g. for pharmaceutical use, human insulin, blood factors, immunoglobulins, pseudoparticles
Definitions
- the present invention relates to a novel antibody that binds to human CD3 and binds to cynomolgus monkey CD3, and a molecule containing the antibody.
- anti-CD3 monoclonal antibodies function with a high degree of recognition of their target molecules. Anti-CD3 antibodies recognize only a single epitope on the target CD3 molecule. The most widely used and best characterized monoclonal antibody specific for the CD3 complex is OKT3. 2) OKT3 is a mouse-derived anti-human CD3 monoclonal antibody (Non-patent Document 1). In order to prevent allograft rejection of the organ, the transplanted organ is rejected by administering an anti-CD3 monoclonal antibody, and the antibody binds to the TCR complex on human T cells and suppresses activation and proliferation of T cells. The treatment method which prevents this has been used for a long time (Non-patent Documents 2-4).
- OKT3 is the first anti-CD3 antibody used for such treatment. While OKT3 has a strong immunosuppressive effect, its clinical use has been hampered due to severe side effects associated with its immunogenicity and mitogenic potential (Non-Patent Documents 5-8). 3) OKT3 induced T cell proliferation and cytokine production in vitro, released a large amount of cytokine in vivo, and caused cytokine syndrome (Non-patent Document 5). This is because OKT3 is a divalent IgG, and thus a cross-link between T cells and Fc ⁇ receptor-expressing cells occurs, resulting in T cell proliferation (Non-patent Document 8).
- OKT3 is a mouse antibody
- heterophilic antibodies such as human anti-mouse antibody (HAMA) are produced by long-term administration
- Non-patent Document 7 The application of anti-CD3 antibody to treatment and reports of side effects are summarized below (Patent Document 1).
- OKF3 scFv Non-patent Document 9
- humanized OKT3 Non-patent Document 10
- OKT3 a bispecific antibody in which a single chain of OKT3 utilizing T cell activation ability and a single chain of an antibody against a target antigen expressed on the surface of cancer cells has been reported.
- Non-Patent Document 2 Non-Patent Document 11
- Multispecific antibodies using anti-CD3 antibodies described in the art are expected to have great therapeutic potential for the treatment of malignant diseases.
- TROP2 is known to be overexpressed in various epithelial cell carcinoma types (Non-Patent Documents 12-16).
- a bispecific antibody in which an antigen-binding fragment of an antibody specific for human TROP2 and an antigen-binding fragment of an anti-CD3 antibody are connected on a gene and expressed has not been reported so far.
- OKT3 reacts with chimpanzee CD3 but does not react with CD3 derived from other primates such as cynomolgus monkeys (Non-patent Document 17).
- UCHT-1 which is an anti-CD3 monoclonal antibody, also reacts with CD3 derived from chimpanzees but does not react with cynomolgus monkey-derived CD3 (Non-patent Document 18).
- monoclonal antibodies that recognize cynomolgus monkey antigens but not their human counterparts.
- An example of this group is FN-18, a monoclonal antibody directed against CD3 derived from cynomolgus monkeys (Non-patent Document 19).
- the limitation of OKT3 and a series of antibodies that modify OKT3 is that it specifically binds to human CD3 only. This limitation can be a serious obstacle to the development of therapeutic agents for treating human diseases.
- An object of the present invention is to provide a novel antibody that binds to human CD3 and cynomolgus CD3, an antigen-binding fragment of the antibody (hereinafter also referred to as an antibody), the antibody, and one or more additional antibodies or the It is to provide a molecule containing an antigen-binding fragment of an antibody, the multispecific molecule, the antibody or the like, or a pharmaceutical composition containing the molecule as an active ingredient and having cytotoxic activity.
- the inventors of the present invention conducted intensive research to solve the title problem, and created a novel anti-CD3 antibody and a molecule containing the antibody, thereby completing the present invention.
- the present invention includes the following inventions.
- CDRH1 whose heavy chain sequence comprises the amino acid sequence shown in SEQ ID NO: 26, CDRH2 comprising the amino acid sequence shown in SEQ ID NO: 98, and CDRH3 comprising the amino acid sequence shown in SEQ ID NO: 28; CDRL1, wherein the light chain sequence comprises the amino acid sequence shown in SEQ ID NO: 29, CDRL2 comprising the amino acid sequence shown in SEQ ID NO: 99, and CDRL3 comprising the amino acid sequence shown in SEQ ID NO: 31
- CDRL1 wherein the light chain sequence comprises the amino acid sequence shown in SEQ ID NO: 29, CDRL2 comprising the amino acid sequence shown in SEQ ID NO: 99, and CDRL3 comprising the amino acid sequence shown in SEQ ID NO: 31
- An antibody or an antigen-binding fragment of the antibody which binds to human CD3 and cynomolgus monkey CD3.
- the first X aa of CDRH2 is selected from the group consisting of (A, E, G, H, I, L, T, V, R, S) and the second X aa is S Or
- the first X aa is N
- the second X aa is selected from the group consisting of (E, R, F, Y, L, V, I, K, T)
- X aa of the CDRL2 is selected from the group consisting of (Q, A, G, S , N, D)
- the antibody or antigen-binding fragment of the antibody according to (1) which binds to human CD3 and cynomolgus monkey CD3.
- the first X aa of the CDRH2 is selected from the group consisting of (R, S), 2 th X aa is S, and X aa of the CDRL2 is (Q, A, G, Selected from the group consisting of S, N, D),
- the heavy chain sequence comprises a variable region having CDRH1, CDRH2, and CDRH3;
- CDRH1 consists of the amino acid sequence shown in SEQ ID NO: 26
- CDRH2 consists of the amino acid sequence shown in SEQ ID NO: 27
- Said CDRH3 comprises the amino acid sequence set forth in SEQ ID NO: 28;
- the light chain sequence comprises a variable region having CDRL1, CDRL2, CDRL3,
- the CDRL1 consists of the amino acid sequence shown in SEQ ID NO: 29,
- the CDRL2 consists of the amino acid sequence shown in SEQ ID NO: 30,
- Said CDRL3 comprises the amino acid sequence set forth in SEQ ID NO: 31; and
- the first X aa of the amino acid sequence shown in SEQ ID NO: 100 is selected from the group consisting of (A, E, G, H, I, L, T, V, R, S) and the second X aa is S, or The first X aa is N and the second X aa is selected from the group consisting of (E, R, F, Y, L, V, I, K, T);
- the first X aa of the amino acid sequence represented by SEQ ID NO: 100 is selected from the group consisting of (R, S), and the second X aa is S.
- the light chain variable region comprises the amino acid sequence represented by any one of SEQ ID NOs: 101, 102, and 103 The antibody or antigen-binding fragment thereof described.
- SEQ ID NO: 101, 102, and, the X aa in the amino acid sequence shown in any one of 103, (Q, A, G , S, N, D) is selected from the group consisting of the ( The antibody or antigen-binding fragment thereof according to 8).
- the light chain variable region sequence comprises the amino acid sequence shown in any one of SEQ ID NOs: 17, 20, and 23 Sex fragment.
- a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 100 and a light chain variable region comprising the amino acid sequence represented by any one of SEQ ID NOs: 101, 102, and 103
- the first X aa in the amino acid sequence shown in SEQ ID NO: 100 is selected from the group consisting of (A, E, G, H , I, L, T, V, R, S), and, second X aa is S or
- the first X aa is N and the second X aa is selected from the group consisting of (E, R, F, Y, L, V, I, K, T);
- SEQ ID NO: 101, 102 and,, X aa in the amino acid sequence shown in any one of 103, (Q, A, G, S, N, D) is selected from the group consisting of, (1) or The antibody or antigen-binding fragment thereof according to (2).
- the first X aa of SEQ ID NO: 100 is selected from the group consisting of (R, S),
- the second X aa is S, and SEQ ID NO: 101, 102, and, the X aa in the amino acid sequence shown in any one of 103, (Q, A, G , S, N, D) is selected from the group consisting of, in the (12) The antibody or antigen-binding fragment thereof described.
- an antibody comprising a heavy chain variable region comprising amino acid residues 2 to 119 of SEQ ID NO: 60 according to (13) and a light chain variable region comprising amino acid residues 135 to 243 of SEQ ID NO: 60;
- An antigen-binding fragment of the antibody An antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising amino acid residues 2 to 119 of SEQ ID NO: 64 and a light chain variable region comprising amino acid residues 135 to 241 of SEQ ID NO: 64;
- An antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising amino acid residues 2 to 119 of SEQ ID NO: 66 and a light chain variable region comprising amino acid residues 135 to 243 of SEQ ID NO: 66;
- An antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising amino acid residues 2 to 119 of SEQ ID NO: 68 and a light chain variable region comprising amino acid residues 135 to 243 of SEQ
- variable region including a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 16, a linker, and a light chain variable region comprising the amino acid sequence represented by any one of SEQ ID NOs: 17, 20, and 23
- the antibody or antigen-binding fragment of the antibody according to (1), (4), (5), (8), (10), or (11).
- the variable region is bound in the order of the heavy chain variable region and the light chain variable region from the amino terminal side of the antibody, or the light chain variable region and the heavy chain variable region are bound in this order.
- amino acid sequence comprising amino acid residues 2 to 269 of SEQ ID NO: 19 An amino acid sequence comprising amino acid residues 2 to 269 of SEQ ID NO: 22, An amino acid sequence comprising 2 to 267 amino acid residues of SEQ ID NO: 25, An amino acid sequence comprising 2 to 269 amino acid residues of SEQ ID NO: 60; An amino acid sequence comprising 2 to 267 amino acid residues of SEQ ID NO: 64, An amino acid sequence comprising 2 to 269 amino acid residues of SEQ ID NO: 66; An amino acid sequence comprising amino acid residues 2 to 269 of SEQ ID NO: 68, An amino acid sequence comprising amino acid residues 2 to 269 of SEQ ID NO: 70, An amino acid sequence comprising 2 to 269 amino acid residues of SEQ ID NO: 72; An amino acid sequence comprising 2 to 269 amino acid residues of SEQ ID NO: 74, An amino acid sequence comprising 2 to 269 amino acid residues of SEQ ID NO: 76; An amino acid sequence comprising amino acid residues
- a stringent condition and a complementary strand of a polynucleotide comprising a nucleotide sequence encoding an amino acid sequence contained in the antibody or antigen-binding fragment of the antibody according to any one of (14) to (18)
- An antibody comprising an amino acid sequence encoded by a nucleotide sequence contained in a polynucleotide that hybridizes underneath and binding to human CD3 and cynomolgus CD3, or an antigen-binding fragment thereof.
- a heavy chain comprising 90% or more of the amino acid sequence of the heavy chain contained in the antibody or the antigen-binding fragment of the antibody according to any one of (14) to (18), and A light chain comprising at least 70% amino acid sequence identical to the amino acid sequence of the light chain contained in the antibody or antigen-binding fragment of the antibody according to any one of (14) to (18), and An antibody that binds to human CD3 and cynomolgus monkey CD3, or an antigen-binding fragment thereof.
- (22) An antibody that binds to the same site on human CD3 to which the antibody or antigen-binding fragment of the antibody according to any one of (14) to (18) binds and binds to cynomolgus monkey CD3 Or an antigen-binding fragment of the antibody.
- (23) An antibody that competes with the antibody according to any one of (14) to (18) or an antigen-binding fragment of the antibody for binding on human CD3 and binds to cynomolgus monkey CD3, or the antibody An antigen-binding fragment of.
- the site on human CD3 to which the antibody or antigen-binding fragment of the antibody binds is the 55th serine (Ser), the 56th glutamic acid (Glu), 58 in the amino acid sequence represented by SEQ ID NO: 1.
- a polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of the antibody or antigen-binding fragment of the antibody according to any one of (1) to (27).
- an amino acid sequence comprising amino acid residues 2 to 243 of SEQ ID NO: 19, An amino acid sequence comprising amino acid residues 2 to 243 of SEQ ID NO: 22, An amino acid sequence comprising amino acid residues 2 to 241 of SEQ ID NO: 25;
- a vector comprising the polynucleotide according to any one of (28) or (29).
- a pharmaceutical composition comprising the antibody or the antigen-binding fragment of the antibody according to any one of (1) to (27) and (33) as an active ingredient.
- the molecule according to (35) which is multispecific.
- the antibody or antigen-binding fragment of the antibody according to any one of (1) to (27), (33), and one or more additional antibodies or antigen-binding properties of the antibody The molecule according to the above (35) or (36), comprising a fragment.
- the molecule according to (37), wherein the antigen-binding fragment of the further antibody is Fab, F (ab) ′, Fv, scFv, or sdAb.
- variable region is a heavy chain variable region, a light chain from the amino terminal side of the antibody.
- linker between both variable regions ii) having a glycine residue at the amino terminus of the variable region on the amino terminus; iii) a linker at the carboxyl terminus of the variable region on the carboxyl terminus;
- a FLAG tag and / or a His tag are bound: The molecule according to any one of (35) to (42).
- Applicable forms include hybrid and dual bispecific molecules.
- the additional antibody or antigen-binding fragment of the antibody An antibody comprising the amino acid residues 2 to 243 of SEQ ID NO: 19 or an antigen-binding fragment thereof, An antibody comprising the amino acid residues 2 to 241 of SEQ ID NO: 25 or an antigen-binding fragment thereof.
- Applicable forms include hybrid and dual bispecific molecules.
- (46) The molecule according to any one of (36) to (45), wherein the further antibody is an anticancer target antibody.
- (47) The molecule according to any one of (36) to (46), which is bispecific.
- 48) The molecule according to any one of (35) to (47), which is a polypeptide.
- (49) A polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of the molecule according to (48).
- (50) A vector comprising the polynucleotide according to (49).
- (51) A cell that produces the polynucleotide according to (49), the vector according to (50), or the molecule according to (48).
- a pharmaceutical composition comprising the molecule according to any one of (35) to (48) and (53) as an active ingredient.
- a novel anti-CD3 antibody that binds to human CD3 and binds to cynomolgus CD3, an antigen-binding fragment of the antibody, and a novel molecule having antigen-binding properties, including the antibody and the like, can be obtained.
- a novel pharmaceutical composition containing such an antibody or molecule as an active ingredient can be obtained.
- the antibody or the molecule has a T cell-dependent cytotoxic activity and is useful as a therapeutic or prophylactic agent for various diseases such as cancer.
- FIG. 3 is a view showing the nucleotide sequence of a sense primer Nhe-polyCS for heavy chain gene amplification.
- FIG. 3 shows the nucleotide sequence of the first antisense primer rIg ⁇ -AS1 for heavy chain gene amplification.
- FIG. 3 shows the nucleotide sequence of the second antisense primer rIg ⁇ -AS2 for heavy chain gene amplification.
- FIG. 3 is a view showing a nucleotide sequence of a sense primer Nhe-polyC-S2 for light chain gene amplification.
- FIG. 3 shows the nucleotide sequence of the first antisense primer rIgL-AS1 for light chain gene amplification.
- FIG. 3 shows the nucleotide sequence of the second antisense primer rIgL-AS2 for light chain gene amplification.
- FIG. 3 shows the nucleotide sequence of a heavy chain sequencing sense primer rIg ⁇ -seq.
- FIG. 4 shows a nucleotide sequence encoding the heavy chain variable region of C3-147. It is the figure which showed the amino acid sequence of the heavy chain variable region of C3-147.
- FIG. 3 shows a nucleotide sequence encoding the light chain variable region of C3-147. It is the figure which showed the amino acid sequence of the light chain variable region of C3-147. It is the figure which showed the oligonucleotide sequence of G4S linker sense strand.
- FIG. 3 shows a nucleotide sequence encoding C3E-7000. It is the figure which showed the amino acid sequence of C3E-7000. It is the figure which showed the amino acid sequence of the heavy chain variable region of C3E-7034. It is the figure which showed the amino acid sequence of the light chain variable region of C3E-7034. It is the figure which showed the amino acid sequence of CDR-H1 of C3E-7000. It is the figure which showed the amino acid sequence of CDR-H2 of C3E-7000. It is the figure which showed the amino acid sequence of CDR-H3 of C3E-7000.
- FIG. 3 shows a nucleotide sequence encoding C3E-7034.
- FIG. 3 shows a nucleotide sequence encoding C3E-7035.
- FIG. 3 shows a nucleotide sequence encoding C3E-7036. It is the figure which showed the amino acid sequence of human CD3 ⁇ . It is the figure which showed the amino acid sequence of C3E-7034.
- FIG. 3 is a view showing a complex structure of CD3 ⁇ and C3E-7034.
- FIG. 3 shows the interaction between CD3 ⁇ and the heavy chain and light chain of C3E-7034.
- A is a diagram showing the amino acid residues of CD3 ⁇ within a distance of 4 mm or less in a light stick variable region of C3E-7034 and CD3 ⁇ in a thick stick model, and other amino acids in a thin stick model.
- B is a diagram showing the heavy chain variable region of C3E-7034 and CD3 ⁇ with the amino acid residues of CD3 ⁇ within a distance of 4 mm or less displayed in a thick stick model and the other amino acids displayed in a thin stick model. It is the figure which showed the interaction site on the arrangement
- FIG. 4 shows the binding of humanized anti-CD3 scFv C3E-3007, C3E-7034, C3E-7035, and C3E-7036 to human CD3 (PBMC).
- FIG. 4 shows the binding of humanized anti-CD3 scFv C3E-3007, C3E-7034, C3E-7035, and C3E-7036 to cynomolgus monkey CD3 (PBMC).
- A is a diagram showing the activation action of humanized anti-CD3 scFv C3E-7034 and C3E-3007 on human CD8-positive cells.
- B is a figure showing the CD8 positive cell activation action of cynomolgus monkeys of humanized anti-CD3 scFv C3E-7034 and C3E-3007.
- FIG. 3 shows the amino acid sequence of HT1-11 scFv.
- FIG. 2 is a view showing a nucleotide sequence encoding HT1-11 scFv.
- FIG. 3 shows that HT1-11 scFv binds to a human TROP2-positive cell line.
- A is a view showing binding to a pharyngeal squamous cell carcinoma cell line FaDu.
- B shows the binding to pancreatic cancer cell line HPAF-II.
- FIG. 3 shows an ORF nucleotide sequence encoding T2C-0001.
- FIG. 2 shows an ORF nucleotide sequence encoding T2C-0003.
- FIG. 2 shows an ORF nucleotide sequence encoding T2C-0005.
- FIG. 2 shows an ORF nucleotide sequence encoding T2C-0006. It is the figure which showed the amino acid sequence of T2C-0001. It is the figure which showed the amino acid sequence of T2C-0003. It is the figure which showed the amino acid sequence of T2C-0005.
- FIG. 2 shows that the anti-TROP2-CD3 bispecific molecule binds to TRO2-positive cell lines of T2C-0001, T2C-0003, T2C-0005, and T2C-0006.
- A is a view showing binding to the pharyngeal squamous cell carcinoma cell line FaDu.
- B shows the binding to the pancreatic cancer cell line HPAF-II.
- FIG. 3 shows the binding of anti-TROP2-CD3 bispecific molecules, T2C-0001, T2C-0003, T2C-0005, and T2C-0006 to human CD3 (PBMC).
- PBMC human CD3
- FIG. 3 shows the binding of anti-TROP2-CD3 bispecific molecules, T2C-0001, T2C-0003, T2C-0005, and T2C-0006 to cynomolgus CD3 (PBMC).
- A is a figure showing that TROP2 is expressed in the pharyngeal squamous cell carcinoma cell line FaDu.
- B shows that TROP2 is expressed in pancreatic cancer cell line HPAF-II.
- C is a diagram showing that TROP2 is not expressed in lung cancer cell line Calu-6.
- A shows that anti-TROP2-CD3 bispecific molecules, T2C-0001, T2C-0003, T2C-0005, and T2C-0006 have cytotoxic activity against pharyngeal squamous cell carcinoma cell line FaDu in the presence of human PBMC It is the figure which showed that.
- B shows that anti-TROP2-CD3 bispecific molecules, T2C-0001, T2C-0003, T2C-0005, and T2C-0006 have cytotoxic activity against pancreatic cancer cell line HPAF-II in the presence of human PBMC It is the figure which showed that.
- FIG. 5 shows a nucleotide sequence encoding C3E-7078. It is the figure which showed the amino acid sequence of C3E-7078.
- FIG. 3 shows the nucleotide sequence encoding C3E-7079. It is the figure which showed the amino acid sequence of C3E-7079.
- FIG. 5 shows a nucleotide sequence encoding C3E-7085. It is the figure which showed the amino acid sequence of C3E-7085.
- FIG. 5 shows the nucleotide sequence encoding C3E-7086. It is the figure which showed the amino acid sequence of C3E-7086.
- FIG. 3 shows a nucleotide sequence encoding C3E-7087. It is the figure which showed the amino acid sequence of C3E-7087.
- FIG. 5 shows a nucleotide sequence encoding C3E-7088. It is the figure which showed the amino acid sequence of C3E-7088.
- FIG. 3 shows a nucleotide sequence encoding C3E-7089. It is the figure which showed the amino acid sequence of C3E-7089.
- FIG. 3 shows a nucleotide sequence encoding C3E-7090.
- FIG. 3 shows the nucleotide sequence encoding C3E-7091. It is the figure which showed the amino acid sequence of C3E-7091.
- FIG. 3 shows a nucleotide sequence encoding C3E-7092. It is the figure which showed the amino acid sequence of C3E-7092.
- FIG. 3 shows a nucleotide sequence encoding C3E-7093. It is the figure which showed the amino acid sequence of C3E-7093.
- FIG. 3 shows a nucleotide sequence encoding C3E-7094. It is the figure which showed the amino acid sequence of C3E-7094.
- FIG. 3 shows the nucleotide sequence encoding C3E-7091. It is the figure which showed the amino acid sequence of C3E-7091.
- FIG. 3 shows a nucleotide sequence encoding C3E-7092. It is the figure which showed the amino acid sequence of C3E-7092.
- FIG. 3 shows
- FIG. 3 shows a nucleotide sequence encoding C3E-7095. It is the figure which showed the amino acid sequence of C3E-7095. It is the figure which showed the nucleotide sequence which codes primer HN53R Fw. It is the figure which showed the nucleotide sequence which codes primer HN53R Rv. It is the figure which showed the nucleotide sequence which codes primer HN53S Fw. It is the figure which showed the nucleotide sequence which codes primer HN53S Rv.
- FIG. 3 shows an ORF nucleotide sequence encoding AXC-0001. It is the figure which showed the amino acid sequence of AXC-0001.
- FIG. 3 shows an ORF nucleotide sequence encoding AXC-0002.
- FIG. 2 shows an ORF nucleotide sequence encoding MGC-0001. It is the figure which showed the amino acid sequence of MGC-0001.
- FIG. 2 shows an ORF nucleotide sequence encoding MGC-0002. It is the figure which showed the amino acid sequence of MGC-0002. It is the list
- PBMC cynomolgus monkey CD3
- Human lung cancer cell line A549 A
- human pancreatic cancer cell line PANC-1 B
- human pancreatic cancer cell line MIA PaCa-2 C
- human myeloma cell line U266B1 D
- mantle cell lymphoma cell line Jeko It is the figure which showed the expression of Axl in -1 (E).
- A, B and C are anti-Axl-CD3 bispecific molecules
- AXC-0001 and AXC-0002 are Axl-expressing cell lines: human lung cancer cell line A549 (A), human pancreatic cancer cell line PANC-1 (B),
- FIG. 3 is a view showing that human pancreatic cancer cell line MIA PaCa-2 (C) has cytotoxic activity in the presence of human PBMC.
- D and E are anti-Axl-CD3 bispecific molecules
- AXC-0001 and AXC-0002 are Axl non-expressing cell lines: human myeloma cell line U266B1 (D), mantle cell lymphoma cell line Jeko-1 (E) It is the figure which showed that it did not show a cytotoxic activity in the presence of human PBMC. It is the figure which showed the binding property of MAG-032scFv in the human lymphoblast cell fusion cell line T2 cell which added MAGEC1 peptide (A) or DMSO.
- A is a diagram showing that anti-HLA-A2 / MAGEC1-CD3 bispecific molecule, MGC-0001, MGC-0002 has cytotoxic activity in the presence of human PBMC against T2 cells added with MAGEC1 peptide. is there.
- B shows that anti-HLA-A2 / MAGEC1-CD3 bispecific molecules, MGC-0001 and MGC-0002 do not show cytotoxic activity in the presence of human PBMC against T2 supplemented with DMSO. . It is the figure which showed the amino acid sequence of the MAGEC1 peptide. It is the figure which showed the amino acid sequence of CDRH2 area
- the first X aa and the second X aa are each an arbitrary natural amino acid residue. It is the figure which showed the amino acid sequence of CDRL2 area
- X aa is any natural amino acid residue. It is the figure which showed the heavy chain amino acid sequence of CDR variant of C3E-7034.
- the first X aa and the second X aa are each an arbitrary natural amino acid residue. It is the figure which showed the light chain amino acid sequence of CDR variant of C3E-7034.
- X aa of CDRL2 is the amino acid residue of any naturally occurring. It is the figure which showed the light chain amino acid sequence of CDR variant of C3E-7035.
- X aa of CDRL2 is the amino acid residue of any naturally occurring. It is the figure which showed the light chain amino acid sequence of CDR variant of C3E-7036.
- X aa of CDRL2 is the amino acid residue of any naturally occurring.
- gene means a nucleotide containing a nucleotide sequence encoding a protein amino acid or a complementary strand thereof, for example, a nucleotide containing a nucleotide sequence encoding a protein amino acid or a complementary strand thereof.
- Certain polynucleotides, oligonucleotides, DNA, mRNA, cDNA, cRNA and the like are included in the meaning of “gene”.
- Such a gene is a single-stranded, double-stranded, or triple-stranded nucleotide, and an assembly of a DNA strand and an RNA strand, and ribonucleotide (RNA) and deoxyribonucleotide (DNA) are mixed on one nucleotide strand.
- RNA ribonucleotide
- DNA deoxyribonucleotide
- gene also included within the meaning of “gene” are double-stranded or triple-stranded nucleotides comprising such nucleotide chains.
- the base sequence and the nucleotide sequence are synonymous.
- polynucleotide “nucleic acid” and “nucleic acid molecule” are synonymous, and for example, DNA, RNA, probe, oligonucleotide, primer and the like are also included in the meaning of “polynucleotide”.
- a polynucleotide is a polynucleotide composed of a single strand, a double strand, or three or more strands.
- RNA ribonucleotide
- DNA deoxyribonucleotide
- polypeptide In the present invention, “polypeptide”, “peptide” and “protein” are synonymous.
- antigen is sometimes used to mean “immunogen”.
- cell includes various cells derived from individual animals, subculture cells, primary culture cells, cell lines, recombinant cells, microorganisms, and the like.
- antibody is synonymous with immunoglobulin.
- the “antibody” in the case of the anti-CD3 antibody of the present invention is used to mean an immunoglobulin having a constant region and a variable region. It is not particularly limited whether the antibody is a natural antibody or an immunoglobulin produced by partial or complete synthesis.
- the anti-CD3 antibody of the present invention is included in the “molecule” described later.
- the basic 4-chain antibody structure is composed of two identical light (L) chains and two identical heavy (H) chains.
- the light chain is attached to the heavy chain by one covalent disulfide bond.
- the two heavy chains are linked to each other by one or more disulfide bonds, depending on the heavy chain isotype.
- Each light and heavy chain has intrachain disulfide bonds with regular intervals.
- the heavy chain and the light chain there are a constant region having very high similarity in amino acid sequence and a variable region having low similarity in amino acid sequence.
- the light chain has a variable region (VL) at the amino terminus followed by a constant region (CL).
- the heavy chain has a variable region (VH) at the amino terminus followed by three constant regions (CH1 / CH2 / CH3).
- VL and VH are paired, and CL is aligned with the first constant region (CH1) of the heavy chain. VL and VH pair to form a single antigen binding site.
- the constant region of the antibody of the present invention is not particularly limited, but a human antibody is preferably used as the antibody of the present invention for treating or preventing a human disease.
- Examples of the heavy chain constant region of a human antibody include C ⁇ 1, C ⁇ 2, C ⁇ 3, C ⁇ 4, C ⁇ , C ⁇ , C ⁇ 1, C ⁇ 2, and C ⁇ .
- Examples of the light chain constant region of a human antibody include C ⁇ and C ⁇ .
- VH and VL contain complementarity determining regions (CDRs).
- Fc is the carboxyl terminal region of the constant region of the heavy chain and contains CH2 and CH3 and is a dimer.
- the Fc of the present invention may be a natural sequence Fc or a mutant Fc in which a mutation is added to the natural sequence.
- variable region is composed of a region having extreme variability called a hypervariable region (HVR) and a relatively invariable region called a framework region (FR) separated by the region.
- HVR hypervariable region
- FR framework region
- the natural heavy and light chain variable regions comprise four FRs connected by three hypervariable regions, each chain hypervariable region being held in close proximity with the other chain hypervariable regions by FRs, It contributes to the formation of the antigen binding site of the antibody.
- CDRs complementarity determining regions
- Complementarity-determining regions also called hypervariable regions
- CDRH1, CDRH2, CDRH3 the complementarity determining region of the heavy chain
- the complementarity determining region of the light chain is defined as the light chain amino acid.
- CDRL1, CDRL2, and CDRL3 are represented from the amino terminal side of the sequence.
- the position and length of the CDR were determined according to the definition of IMGT (Developmental and Comparative Immunology 27 (2003) 55-77).
- Framework region is a variable region other than CDR residues.
- the variable region generally has four FRs, FR1, FR2, FR3, and FR4. *
- CDR and FR can be determined by various definitions well known in the art, for example, definitions of Kabat, Chothia, AbM, contact, etc. in addition to IMGT.
- the “antigen-binding fragment of an antibody” means a partial fragment of an antibody having an antigen-binding activity, comprising a heavy chain variable region and a light chain variable region.
- the “antigen-binding fragment of an antibody” include, for example, antigen-binding fragments such as Fab, F (ab ′) 2 , scFv, Fab ′, Fv, and single-domain antibody (sdAb). It is not limited.
- antigen-binding fragments of antibodies include recombinant proteins produced in appropriate host cells using recombinant genes in addition to those obtained by treating full-length antibody protein molecules with enzymes such as papain and pepsin. It may be.
- the “site” to which the antibody binds that is, the “site” recognized by the antibody means a partial peptide or a partial higher order structure on the antigen to which the antibody binds or recognizes.
- an “antibody variant” is obtained by substituting, deleting, or adding an amino acid in the amino acid sequence of the original antibody (the addition includes an insertion) (hereinafter collectively referred to as “mutation”). It means a polypeptide having an amino acid sequence and binding to CD3 of the present invention.
- the number of mutated amino acids in such antibody variants is 1 to 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 40 or 50.
- Such antibody variants are also included in the “antibody” of the present invention.
- “several” in “1 to several” refers to 2 to 10.
- the “molecule” is a molecule containing the above-mentioned antibody or antigen-binding fragment of an antibody, and a multispecific molecule formed from an antibody or a plurality of antigen-binding fragments derived therefrom. including.
- a molecule that is multispecific is synonymous, and a plurality of different epitopes on one molecule, and / or The molecule is not particularly limited as long as it can bind to different epitopes on two or more molecules.
- a molecule that is multispecific also includes an antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL).
- VH heavy chain variable region
- VL light chain variable region
- Such multispecific molecules include full-length antibody molecules having two or more different types of heavy and light chains, ie, IgG type multispecific molecules, and antigen-binding fragments having two or more types of VL and VH.
- molecules generated by genetically or chemically linking proteins having antigen-binding properties without having an immunoglobulin skeleton to antigen-binding fragments are also included as multispecific molecules.
- Examples of the activity / property exhibited by the anti-CD3 antibody of the present invention or the antigen-binding fragment of the antibody or the multispecific molecule of the present invention include biological activity, physicochemical properties and the like. Specifically, various biological activities, binding activities against antigens and epitopes, stability during production and storage, thermal stability, and the like can be mentioned.
- hybridize under stringent conditions means hybridization at 65 ° C. in a solution containing 5 ⁇ SSC, and then in an aqueous solution containing 2 ⁇ SSC-0.1% SDS. 20 minutes at 65 ° C. in an aqueous solution containing 0.5 ⁇ SSC-0.1% SDS for 20 minutes at 65 ° C. and 65 ° C. in an aqueous solution containing 0.2 ⁇ SSC-0.1% SDS Means to hybridize under the conditions of washing for 20 minutes or under equivalent conditions.
- SSC is an aqueous solution of 150 mM NaCl-15 mM sodium citrate, and nx SSC means n-fold concentration of SSC.
- cytotoxicity refers to causing a pathological change in a cell in some form, and is not limited to direct trauma, but also includes DNA breakage, base dimer formation, chromosome breakage, It means any structural or functional damage of cells, such as damage to cell division apparatus or reduction of various enzyme activities.
- cytotoxic activity means causing the above cytotoxicity.
- antibody-dependent cytotoxic activity refers to “antibody dependent cellular cytotoxicity (ADCC) activity” and means an activity of NK cells damaging target cells such as tumor cells via antibodies.
- ADCC antibody dependent cellular cytotoxicity
- cytotoxic activity by T cell redirection means causing the above cytotoxicity via a multispecific molecule including an anti-tumor antigen antibody and an anti-CD3 antibody. That is, the anti-tumor antigen antibody binds to the target tumor cell, and the anti-CD3 antibody binds to the T cell, thereby reducing the distance between the target tumor cell and the T cell and inducing cytotoxicity through T cell activation. To do.
- the molecule can be included in a pharmaceutical composition. 2.
- Antigen protein CD3 CD3 complex
- CD3 is used interchangeably with CD3 protein.
- CD3 is expressed on T cells as a part of a multimolecular T cell receptor complex, and is composed of five polypeptides of ⁇ chain, ⁇ chain, ⁇ chain, ⁇ chain, and ⁇ chain (molecular weights in order 25000-28000, 21000, 20000, 16000, 22000).
- CD3 used in the present invention can be prepared by purification and isolation from animal tissues (including body fluids), cells derived from the tissues or cell cultures, gene recombination, in vitro translation, chemical synthesis, and the like.
- the nucleotide sequence of cDNA encoding human CD3 ⁇ is registered in GenBank with accession number: NM_000733.3.
- the nucleotide sequence of cDNA encoding cynomolgus monkey CD3 is registered in GenBank with accession number: NM_0012833615.1.
- the amino acid sequence of human CD3 ⁇ is described in SEQ ID NO: 1 in the sequence listing.
- the CD3 ⁇ cDNA is, for example, a polymerase chain reaction (hereinafter referred to as “PCR”) using a cDNA library of an organ expressing CD3 ⁇ mRNA as a template and a primer that specifically amplifies the CD3 ⁇ cDNA (Saiki, R , K., et al., Science (1988) 239, 487-49).
- PCR polymerase chain reaction
- a nucleotide sequence that encodes a protein having a biological activity equivalent to that of CD3 is composed of an amino acid sequence in which one to several amino acids are substituted, deleted, or added.
- a protein having a biological activity equivalent to that of CD3, which is composed of an amino acid sequence in which one or several amino acids are substituted, deleted or added in the amino acid sequence of CD3, is also included in CD3.
- a polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising a nucleotide sequence that is complementary to a nucleotide sequence encoding human or cynomolgus monkey CD3 ⁇ , and that encodes a protein having biological activity equivalent to CD3 ⁇ is also CD3 ⁇ .
- a polynucleotide encoding a splicing variant transcribed from a human or cynomolgus monkey CD3 ⁇ locus or a stringent hybridizing condition thereto, and having a biological activity equivalent to that of CD3 ⁇ is also CD3 ⁇ cDNA.
- the anti-CD3 antibody of the present invention and the antigen-binding fragment of the antibody may be either a monoclonal antibody or a polyclonal antibody.
- the monoclonal antibody of the present invention include antibodies derived from non-human animals (non-human animal antibodies), human antibodies, chimerized antibodies (also referred to as “chimeric antibodies”), humanized antibodies, and the like.
- non-human animal antibodies include antibodies derived from vertebrates such as mammals and birds.
- antibodies derived from mammals include antibodies derived from rodents such as mouse antibodies and rat antibodies.
- avian-derived antibodies include chicken antibodies.
- the anti-human CD3 rat monoclonal antibody include C3-147 (Example 1) -7) of the present invention.
- chimerized antibody examples include, but are not limited to, antibodies formed by binding a variable region derived from a non-human animal antibody and a human antibody (human immunoglobulin) constant region.
- CDRs in the variable region of a non-human animal antibody are transplanted to a human antibody (variable region of human immunoglobulin), and in addition to the CDR, the sequence of the framework region of the non-human animal antibody is also partially human antibody And those obtained by substituting one or two or more amino acids derived from any of these non-human animal antibodies with human-type amino acids, but are not limited thereto.
- CDRs in the variable region of the non-human animal antibody include CDRH1 to CDRH3 in the heavy chain variable region derived from the rat anti-CD3 antibody C3E-7000 of the present invention, CDRL1 to CDRRL3 in the light chain variable region, and amino acids of those CDRs Examples thereof include those in which 1 or 2 amino acids are substituted with other amino acids in the sequence.
- the human antibody is not particularly limited as long as it preferably binds to human CD3 and more preferably binds to human CD3 and cynomolgus monkey CD3.
- Examples also include human antibodies that bind to the same site as the humanized antibody of the present invention.
- a human antibody that binds to the same site as C3E-7034 is exemplified.
- the preferred antibody of the present invention binds to human CD3. Furthermore, a more suitable antibody of the present invention also has a binding activity with cynomolgus monkey CD3.
- the antibody of the present invention may be an antibody composed of portions derived from a plurality of different antibodies, as long as it binds to human CD3 and also to cynomolgus monkey CD3.
- a chain and / or light chain exchanged, a heavy chain and / or light chain full length exchange, a variable region only or constant region exchange, a CDR or all or a part exchanged, etc. Can be mentioned.
- the heavy chain variable region and the light chain variable region of the chimerized antibody may be derived from different antibodies of the present invention.
- CDRH1 to CDRH3 and CDRL1 to CDRL3 in the variable regions of the heavy and light chains of the humanized antibody may be derived from two or more antibodies of the invention.
- CDRH1 to CDRH3 and CDRL1 to CDRL3 in the variable region of the heavy chain and light chain of a human antibody may be a combination of CDRs of two or more antibodies of the present invention.
- the isotype and subclass of the monoclonal antibody can be determined by, for example, an Octellony method, an Enzyme-Linked ImmunoSorbent Assay (ELISA) method, a Radio Immuassay (RIA) method, and the like.
- ELISA Enzyme-Linked ImmunoSorbent Assay
- RIA Radio Immuassay
- An ELISA Kit (BD Pharmingen, etc.) can also be used.
- the antibody of the present invention recognizes CD3. That is, the antibody of the present invention binds to CD3.
- the antibody of the present invention preferably binds to human CD3, monkey CD3, etc., and more preferably binds to human CD3 and cynomolgus CD3.
- the antibody of the present invention, the antigen-binding fragment thereof, and the variable region thereof bind to an Ig-like domain existing in the extracellular region of the ⁇ chain (FIG. 1, SEQ ID NO: 1) of the human CD3 complex. . It also binds to the Ig-like domain present in the extracellular region of the ⁇ chain of the cynomolgus monkey CD3 complex.
- the epitope present in the extracellular region of the ⁇ chain (FIG. 1, SEQ ID NO: 1) of the human CD3 complex to which the antibody of the present invention binds comprises the following amino acids; Ser55, Glu56, Leu58, Trp59, Asn65, Ile66, Ser77, Asp78, Arg101, Gly102, Ser103, Lys104, and Pro105.
- the antibody or the like of the present invention can preferably maintain the binding with human CD3 by binding to an epitope region comprising at least 7 amino acids selected from these 13 amino acids.
- an antibody When an antibody is adjacent to the above-mentioned amino acid within a distance of 4 mm or less, it can be determined that such an antibody has the same epitope specificity as the antibody of the present invention.
- Arg101, Gly102, Ser103, Lys104, and Pro105 are also epitope residues that interact with known anti-CD3 antibodies OKT3 and UCHT1 (Lars Kjer-Nielsen et al., PNAS ( 2004) (Kelly L Arnett et al., PNAS (2004))
- OKT3 and UCHT1 bind to human CD3 but not to cynomolgus CD3.
- “recognition”, that is, “binding” means binding that is not non-specific adsorption.
- a dissociation constant hereinafter referred to as “KD”.
- the KD value for CD3 of the preferred antibody of the present invention is 1 ⁇ 10 ⁇ 5 M or less, 5 ⁇ 10 ⁇ 6 M or less, 2 ⁇ 10 ⁇ 6 M or less, or 1 ⁇ 10 ⁇ 6 M or less.
- the binding between an antigen and an antibody can be measured or determined by a biomolecular interaction analysis system such as SPR method or BLI method, ELISA method, RIA method or the like.
- the binding between the antigen expressed on the cell surface and the antibody can be measured by a flow cytometry method or the like.
- the SPR method is a dissociation constant (KD) which is an index of affinity by measuring a binding rate constant (Ka value) and a dissociation rate constant (Kd value) by reaction kinetic analysis. Value) and the like.
- Biacore (trademark) (manufactured by GE Healthcare), ProteOn (trademark) (manufactured by BioRad), SPR-Navi (trademark) (manufactured by BioNavis), Spreeta (trademark) (Texas Instruments, Inc.) And SPRi-PlexII (trademark) (manufactured by Horiba), Autolab SPR (trademark) (manufactured by Metrohm), and the like.
- the BLI method (BioLayer Interferometry) is a method for measuring the interaction between biomolecules using biolayer interference.
- An example of an instrument used for interaction analysis using the BLI method is an Octet system (Pall ForteBio).
- the ELISA method is a method in which a target antigen or antibody contained in a sample solution is captured with a specific antibody or antigen, and detected and quantified using an enzyme reaction.
- the enzyme activity is detected by incorporating an enzyme-labeled antigen or antibody into the reaction system.
- a substrate whose absorption spectrum changes depending on the reaction is used, and it is digitized by measuring absorbance.
- Cell-ELISA is a method of detecting and quantifying using an enzymatic reaction while supplementing each cell with a measurement target on the cell surface.
- the antibody in the RIA method (Radio Immunoassay), can be quantified by labeling the antibody with a radioactive substance and measuring the radioactivity from the antibody.
- Flow cytometry is a technique in which fine cells are dispersed in a fluid, the fluid is flowed finely, and individual cells are optically analyzed.
- the antibody labeled with a fluorescent dye binds to a cell surface antigen by an antigen-antibody reaction, and the number of cells bound by the antibody is measured using the fluorescence intensity, thereby measuring the antigen binding property of the antibody.
- antibodies that bind to human CD3 and cynomolgus monkey CD3 can be used in various tests related to efficacy and safety using primates essential for non-clinical development (preclinical development) of pharmaceuticals, particularly cynomolgus monkeys. preferable.
- antibodies that bind to human CD3 and cynomolgus monkey CD3 have cytotoxic activity, and are useful alone or as a molecule of the present invention for the treatment or prevention of diseases such as cancer in humans and cynomolgus monkeys.
- the pharmaceutical composition will be described later.
- the antibodies and the like that bind to human CD3 and cynomolgus monkey CD3 of the present invention do not bind to mouse CD3, cells, tissues, and individuals (transgenic animals, knockout animals, knockin animals) into which a human CD3 gene has been introduced. And various assays using the antibodies, immunohistochemistry, etc. can be performed without the influence of CD3 of the mouse as a host, and mice such as drugs, animal drugs or diagnostic drugs containing the antibodies can be used. Preferred for research used and non-clinical development. (3-3) Monoclonal Antibody The present invention provides a monoclonal antibody.
- Monoclonal antibodies include rat antibodies, mouse antibodies, rabbit antibodies, chicken antibodies, monoclonal antibodies derived from non-human animals such as fish antibodies, chimerized antibodies, humanized antibodies, human antibodies, antigen-binding fragments thereof, and antibodies thereof Mutants, modifications thereof and the like are included.
- C3-147 obtained by the method described in Example 1 is an anti-CD3 rat monoclonal antibody.
- the nucleotide sequence of DNA encoding the heavy chain variable region of C3-147 is described in SEQ ID NO: 6 (FIG. 14), and the amino acid sequence is described in SEQ ID NO: 7 (FIG. 15).
- the nucleotide sequence of DNA encoding the light chain variable region of C3-147 is described in SEQ ID NO: 8 (FIG. 16), and the amino acid sequence is described in SEQ ID NO: 9 (FIG. 17).
- the antibody variants of the present invention preferably have reduced sensitivity to protein degradation or oxidation, maintenance of biological activity or function, improvement or reduction or suppression of change, improvement or regulation of antigen binding ability, or physicochemical properties Alternatively, functional properties can be imparted.
- Proteins are known to change the function and activity of a specific amino acid side chain on the surface of the protein, such as deamidation of asparagine side chains, aspartic acid side chains. Isomerization and the like are included. Those substituted with other amino acids to prevent such amino acid side chain changes are also included in the scope of the antibody variants of the present invention.
- Examples of the antibody variant of the present invention include an antibody having an amino acid sequence obtained by conservative amino acid substitution in the amino acid sequence of the antibody.
- Conservative amino acid substitutions are those that take place within a group of amino acids that are related to an amino acid side chain.
- the amino acid substitution in such an antibody variant is preferably performed within a range that does not reduce the antigen binding activity of the original antibody.
- Mouse antibodies, rat antibodies, chimerized antibodies, humanized antibodies, human antibodies, antigen-binding fragments thereof, molecules containing them, and the like containing the CDRs are also encompassed in the present invention.
- an antigen-binding fragment of the anti-CD3 antibody of the present invention is provided.
- the antigen-binding fragment of an antibody means a fragment that retains at least antigen-binding among the functions of the antibody or a modified product thereof.
- Such antibody functions generally include antigen-binding activity, activity that regulates antigen activity, antibody-dependent cytotoxic activity, complement-dependent cytotoxic activity, and the like.
- the functions of the antibody of the present invention and the like and the multispecific molecule containing the antibody of the present invention include, for example, T cell redirection, T cell activation, and cancer cell cells by activating T cells. Injury activity can be mentioned.
- the antigen-binding fragment of an antibody is not particularly limited as long as it is a fragment of the antibody that retains at least antigen-binding activity among the activities of the antibody.
- Fab, Fab ′, F (ab ′) 2 , Fv, heavy chain and light chain Fv linked by a suitable linker, single chain Fv (scFv), single domain antibody (sdAb), and the like but are not limited thereto .
- Molecules containing portions other than the antigen-binding fragment of the antibody of the present invention, such as scFv having a linker moiety, are also encompassed within the meaning of the antigen-binding fragment of the antibody of the present invention.
- a molecule in which one or more amino acid and / or carboxyl terminal amino acids of an antibody protein are deleted and which retains at least a part of the function of the antibody is also an antigen binding property of the antibody. Included in the meaning of the fragment. Modified forms of such antigen-binding fragments of antibodies are also encompassed by the antibody of the present invention or antigen-binding fragments thereof, or modified forms thereof (described later).
- scFv is obtained by linking antibody heavy chain variable region and light chain variable region with a polypeptide linker (Pluckthun A. The Pharmacology of Monoclonal Antibodies 113, Rosenburg and Moore, edited by Springer Verlag, N26 315 (1994), Nature Biotechnology (2005), 23, 1126-1136).
- a tandem scFv produced by linking two scFvs with a polypeptide linker can also be used as a bispecific molecule.
- triabodies composed of three or more scFvs can also be used as multispecific molecules.
- the antibody of the present invention may be an antibody having a single heavy chain variable region and no light chain sequence.
- Such an antibody is referred to as a single domain antibody (single domain: sdAb) or nanobody, and it has been reported that the antigen-binding ability is retained (Muyldemans S. et. Al.). , Protein Eng., (1994) 7 (9), 1129-35, Hamers-Casterman C. et al., Nature (1993) 363 (6428), 446-448).
- sdAb single domain antibody
- Protein Eng., (1994) 7 (9), 1129-35 Hamers-Casterman C. et al., Nature (1993) 363 (6428), 446-448.
- These antibodies are also included in the meaning of the antigen-binding fragment of the antibody in the present invention.
- the present invention also includes single-chain immunoglobulins in which the full-length sequences of antibody heavy and light chains are linked using an appropriate linker (Lee, HS, et al. , Molecular Immunology (1999) 36, 61-71; Shirrmann, T. et al., MAbs (2010), 2 (1), 1-4). By dimerizing such a single-chain immunoglobulin, it is possible to retain a structure and activity similar to those of an antibody that is originally a tetramer.
- the molecules of the present invention include the anti-CD3 antibody of the present invention or an antigen-binding fragment thereof.
- the molecule of the present invention further includes a signal sequence, a tag for purification, an amino-terminal Gly, an ADC drug linker moiety, an albumin-binding polypeptide, a polymer such as PEG, an antibody other than an anti-CD3 antibody, an antigen thereof, which will be described later
- a binding fragment, a protein having an antigen binding property without having an immunoglobulin skeleton, a compound (part thereof) having an anticancer activity, a cytotoxic activity, and other pharmacological activities can be included.
- the molecule of the present invention binds to human CD3 and cynomolgus monkey CD3.
- the molecule of the present invention includes multispecific molecules described later.
- the molecule of the present invention may be in a form introduced into a cell, such as CAR-T, or a form presented on the cell surface.
- the multispecific molecule of the present invention is a molecule having two or more antigen-binding sites. That is, a molecule capable of binding to two or more different epitopes on one molecule or different epitopes on two or more molecules, and wraps a plurality of different antigen-binding fragments.
- Such multispecific molecules include IgG type multispecific molecules, multispecific molecules having two or more variable regions, such as tandem scFv, single-chain diabody, diabody, and triabodies. It includes, but is not limited to, antibody fragments, antibody fragments linked by covalent or non-covalent bonds.
- Multispecific molecules may include Fc.
- the multispecific molecule of the present invention includes the anti-CD3 antibody of the present invention or an antigen-binding fragment of the antibody.
- the multispecific molecule of the present invention includes the antibody of the present invention and the like and one or more additional antibodies or antigen-binding fragments of the antibodies. Further antigen-binding fragments of antibodies include, for example, Fab, F (ab) ', Fv, scFv, and sdAb.
- the multispecific molecule of the present invention may specifically bind to CD3, or may further bind to a target such as an Fc receptor on effector cells.
- bispecific molecules means capable of binding to two different epitopes of the same molecule or different epitopes on two molecules, or antibodies having such bispecificity or Includes antigen-binding fragments.
- the bispecific molecule of the invention binds to CD3 and does not have CD3 but binds to epitopes on other antigens. More specifically, such bispecific molecules bind to (i) an epitope on CD3 (Epitope 1) and (ii) an epitope different from Epitope 1 on CD3 (Epitope 2). Or bind to an epitope on an antigen other than CD3 (Epitope 3).
- the antigen binding site of the heavy chain variable region of the first antibody and the antigen binding site of the light chain variable region of the first antibody are used as the linker.
- Linked together or directly linked without a linker to form the first polypeptide, and the antigen-binding site of the heavy chain variable region of the second antibody and the antigen of the light chain variable region of the second antibody The binding site is linked by a linker or directly linked without a linker to form a second polypeptide, and the first polypeptide and the second polypeptide are linked by a linker or a linker. Directly coupled without.
- the first polypeptide and the second polypeptide may be bound via another molecule.
- a diabody-type bispecific molecule the antigen-binding site of the heavy chain variable region of the first antibody and the antigen-binding site of the light chain variable region of the second antibody are linked by a linker or directly bonded without a linker.
- the antigen binding site of the light chain variable region of the first antibody and the antigen binding site of the heavy chain variable region of the second antibody are linked by a linker or directly linked without a linker.
- a bispecific molecule obtained by further dimerizing a diabody-type bispecific molecule can also be prepared.
- a diabody-type bispecific molecule may be linked to only one chain of Fc or both chains with a linker (diabody-Fc type bispecific molecule).
- dual scFv type bispecific molecule two types of scFv that bind to different epitopes are linked to one of the dimeric Fc's by a linker or directly linked without a linker.
- two types of scFvs that bind to different epitopes are linked to CH and CL, respectively, and further linked to one of the dimeric Fc's via a linker.
- the dual scFv type bispecific molecule is hereinafter also referred to as dual type bispecific molecule or simply dual type.
- two Fabs that bind to different epitopes are linked to one of the dimeric Fc's by a linker or directly linked without a linker.
- the IgG type bispecific molecule is hereinafter also referred to as a Full-Size Antibody (FSA) type bispecific molecule, or simply FSA type.
- FSA Full-Size Antibody
- a bispecific molecule of the present invention a bispecific antibody in which Fab and scFv that bind to different epitopes are linked to one of dimeric Fc's by a linker or directly linked without a linker, respectively. It may be.
- it may be a bispecific molecule in which a Fab of the first antibody is bound to one of the dimeric Fc and a scFv of the second antibody is bound to the other via a linker.
- Such a bispecific molecule is hereinafter also referred to as a hybrid type bispecific molecule or a hybrid type.
- the scFv and Fab contained in the bispecific molecule of the present invention are preferably a humanized antibody or a human antibody scFv and Fab, and Fc is preferably a human antibody Fc.
- the heavy chain variable region and the light chain variable region may be linked in this order from the amino terminal side of the antibody, or the light chain variable region and the heavy chain They may be combined in the order of the variable regions.
- a linker, a FLAG tag, and / or a His tag may be bonded to the carboxyl terminus of the variable region on the carboxyl terminus side (optional).
- a heavy chain variable region, a first linker, a light chain variable region, a second linker, a FLAG tag, and a His tag can be exemplified from the amino terminus. .
- Linkers include single-chain polypeptides or single-chain oligopeptides, or synthetic products such as PEG, nucleotides, sugar chains, and compounds. In addition, it will not specifically limit if two polypeptides are couple
- the length of the linker is, for example, 5 to 30 amino acids in the case of a peptide linker.
- peptide linkers having the same length may be used, or peptide linkers having different lengths may be used.
- peptide linkers include repeating (Gly, Gly, Gly, Gly, Ser), but one to several amino acid residues different from Gly and Ser may be added thereto.
- the present invention provides a humanized anti-CD3 antibody or an antigen-binding fragment thereof.
- humanized antibody of the present invention examples include an antibody (Nature (1986) 321, 522-525) in which only a complementarity determining region (CDR) is incorporated into a human-derived antibody, and CDR sequence by CDR grafting.
- CDR complementarity determining region
- an antibody International Patent Publication No. WO1990 / 007861 in which amino acid residues of some frameworks are grafted to a human antibody can be mentioned.
- the first X aa of CDRH2 is also referred to as X1
- the second X aa is also referred to as X 1 and X 2 respectively).
- CDRL1 TNIGNSY
- RX aa D RX aa D
- X 3 and X aa of CDRL2. It possesses CDRL3 (QSYSGSFI) consisting of the amino acid sequence shown in SEQ ID NO: 31 (FIG. 29).
- X 1 is selected from the group consisting of (A, E, G, H, I, L, T, V, R, S), and X 2 is S; or X 1 is N and X 2 is selected from the group consisting of (E, R, F, Y, L, V, I, K, T);
- X 3 is preferably selected from the group consisting of (Q, A, G, S, N, D).
- X 1 is selected from the group consisting of (R, S), and X 2 is S;
- X 3 is selected from the group consisting of (Q, A, G, S, N, D).
- Examples of such a heavy chain variable region contained in a suitable humanized anti-CD3 antibody of the present invention or an antigen-binding fragment of the antibody include: A heavy chain variable region comprising the amino acid residue shown in SEQ ID NO: 100 (FIG. 114) can be exemplified.
- Examples of the light chain variable region contained in the preferred humanized anti-CD3 antibody or antigen-binding fragment of the antibody of the present invention include: A light chain variable region comprising the amino acid residues shown in SEQ ID NO: 101 (FIG. 115), SEQ ID NO: 102 (FIG. 116), SEQ ID NO: 103 (FIG. 117) can be exemplified.
- CDRH1 GVTFNYYG
- IDRH2 IDRH2
- TDGRDGWVAY TDRH3
- CDRL1 TNFNIGNSY
- RDD light chain variable region having CDRL2 (RDD) consisting of the amino acid sequence shown in SEQ ID NO: 30 (FIG. 28)
- CDRL3 QSYSGSFI
- the position and length of the CDR were determined according to the definition of IMGT (Developmental and Comparative Immunology 27 (2003) 55-77).
- heavy chain variable region of the present invention include an amino acid sequence containing the amino acid residue represented by SEQ ID NO: 16.
- light chain variable region of the present invention include amino acid sequences including the amino acid residues shown in SEQ ID NOs: 17, 20, and 23. *
- Suitable specific examples contained in the humanized anti-CD3 antibody or antigen-binding fragment of the antibody of the present invention include: An antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising amino acid residues 2 to 119 of SEQ ID NO: 60 and a light chain variable region comprising amino acid residues 135 to 243 of SEQ ID NO: 60; An antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising amino acid residues 2 to 119 of SEQ ID NO: 64 and a light chain variable region comprising amino acid residues 135 to 241 of SEQ ID NO: 64; An antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising amino acid residues 2 to 119 of SEQ ID NO: 66 and a light chain variable region comprising amino acid residues 135 to 243 of SEQ ID NO: 66; An antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising amino acid residues 2 to 119 of SEQ ID NO: 68 and a
- humanized anti-CD3 antibody of the present invention or the antigen-binding fragment of the antibody include a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 16, a linker, SEQ ID NOs: 17, 20 And an antibody comprising a light chain variable region comprising the amino acid sequence shown in any one of 23, or an antigen-binding fragment of the antibody.
- variable region contained in the antibody of the present invention or the antigen-binding fragment of the antibody may be bound in the order of the heavy chain variable region and the light chain variable region from the amino terminal side of the antibody, or the light chain variable region. , And may be linked in the order of the heavy chain variable region.
- a glycine residue may be present at the amino terminus of the variable region, and a linker, FLAG, and His tag may be bound to the carboxyl terminus of the variable region.
- Preferred specific examples included in the humanized anti-CD3 antibody of the present invention or the antigen-binding fragment thereof include An antibody comprising the amino acid residues 2 to 243 of SEQ ID NO: 19 or an antigen-binding fragment thereof, An antibody comprising the amino acid residues 2 to 243 of SEQ ID NO: 22 or an antigen-binding fragment thereof, An antibody containing the 2nd to 241st amino acid residues of SEQ ID NO: 25 or an antigen-binding fragment of the antibody can be mentioned.
- an antibody comprising the amino acid residues 1 to 243 of SEQ ID NO: 60 (FIG. 68) or an antigen-binding fragment thereof
- An antibody comprising the amino acid residues 1 to 241 of SEQ ID NO: 64 (FIG. 72) or an antigen-binding fragment thereof
- An antibody comprising the amino acid residues 1 to 243 of SEQ ID NO: 66 (FIG.
- An antibody (Clone ID: C3E-7095) containing the 1st to 243rd amino acid residues of SEQ ID NO: 84 (Fig. 92) or an antigen-binding fragment thereof can be mentioned.
- the heavy chain variable region amino acid sequence and / or the light chain variable region amino acid sequence is contained in the above-described anti-CD3 antibody of the present invention or the antigen-binding fragment thereof.
- the molecule may be an antibody that is 98% or 99% or more identical and includes an antibody that binds to human CD3 and cynomolgus CD3.
- the antibody or the like of the present invention may be one in which a mutation is introduced into the above-mentioned humanized anti-CD3 antibody or an antigen-binding fragment of the antibody to optimize the binding ability to CD3.
- Specific methods for introducing mutations include random mutagenesis using error-prone PCR, site-specific amino acid mutagenesis using an NNK library, site-specific mutagenesis using structural information, and combinations thereof Can be mentioned.
- the antibody of the present invention may be one in which the constant region is substituted to reduce ADCC or CDC activity in order to reduce the effector activity of the antibody.
- the antibody has a low effector activity. It is known that effector activity varies depending on the antibody subclass. IgG4 has a low ADCC and CDC activity, and IgG2 has a CDC activity but has a low ADCC activity. From this feature, it is possible to produce an antibody with reduced ADCC and CDC activity by replacing the constant region of IgG1 with the constant region of IgG2 and 4. Further, by substituting a partial sequence of the constant region of IgG1 with reference to IgG2 and 4, it is possible to produce an IgG1 antibody with reduced ADCC and CDC activities. As an example, Marjan Hezareh et. Al.
- Antibody that binds to the same site and also binds to cynomolgus monkey CD3 is also an antibody of the present invention. Etc.
- An “antibody that binds to the same site” as an antibody means another antibody that binds to a site on an antigen molecule that the antibody recognizes. If the second antibody binds to the partial peptide or partial conformation on the antigen molecule to which the first antibody binds, it can be determined that the first antibody and the second antibody bind to the same site.
- the second antibody competes for the binding of the first antibody to the antigen, that is, the second antibody prevents the binding between the first antibody and the antigen, the peptide sequence of the specific binding site or Even if the three-dimensional structure is not determined, it can be determined that the first antibody and the second antibody bind to the same site.
- the second antibody when the first antibody and the second antibody bind to the same site and the first antibody has a characteristic effect on one embodiment of the antibody of the present invention such as cytotoxic activity, the second antibody has the same activity. The probability of having is very high.
- the second anti-CD3 antibody binds to the binding site of the first anti-CD3 antibody and the second anti-CD3 antibody binds to cynomolgus CD3, the first antibody and the second antibody are identical on the CD3 protein. It can be determined that it binds to the site.
- the second anti-CD3 antibody competes with the binding of the first anti-CD3 antibody to the CD3 protein and the second anti-CD3 antibody binds to cynomolgus CD3, the first antibody and the second antibody It can be determined that the antibody binds to the same site on the CD3 protein.
- An antibody that competes with the anti-CD3 antibody of the present invention or an antigen-binding fragment of the antibody for binding to human CD3 and binds to cynomolgus CD3 or an antigen-binding fragment of the antibody is also included in the antibody of the present invention.
- Antibody binding sites can be determined by methods well known to those skilled in the art, such as immunoassays. For example, a series of peptides are prepared by appropriately trimming the amino acid sequence of the antigen from the carboxyl terminus or amino terminus, examining the reactivity of antibodies against them, determining a rough recognition site, and then synthesizing a shorter peptide. By examining the reactivity of antibodies to those peptides, the binding site can be determined. Antigen fragment peptides can be prepared using techniques such as gene recombination and peptide synthesis.
- the antibody of the present invention recognizes and binds to a region forming a three-dimensional structure in CD3, that is, an Ig-like domain.
- the binding site (epitope) of such an antibody can be determined by specifying amino acid residues on the antigen adjacent to the antibody using X-ray structural analysis.
- the present invention provides a modified form of antibody or antigen-binding fragment thereof.
- the modified form of the antibody of the present invention or an antigen-binding fragment thereof means a product obtained by chemically or biologically modifying the antibody of the present invention or an antigen-binding fragment thereof.
- Chemical modifications include chemical modifications to the amino acid backbone, chemical modifications of N-linked or O-linked carbohydrate chains, and the like.
- Biological modifications include post-translational modifications (eg, glycosylation to N- or O-linkages, amino- or carboxyl-terminal processing, deamidation, isomerization of aspartic acid, methionine Oxidized ones, and those obtained by adding methionine residues to the amino terminus by expression using prokaryotic host cells are included.
- post-translational modifications eg, glycosylation to N- or O-linkages, amino- or carboxyl-terminal processing, deamidation, isomerization of aspartic acid, methionine Oxidized ones, and those obtained by adding methionine residues to the amino terminus by expression using prokaryotic host cells are included.
- those modified to allow detection or isolation of the antibody or antigen of the present invention for example, an enzyme label, a fluorescent label, and an affinity label are also included in the meaning of such a modified product.
- Such a modified product of the antibody of the present invention or an antigen-binding fragment thereof is improved in stability and blood retention of the original antibody of the present invention or antigen-binding fragment thereof, reduced in antigenicity, such antibody or antigen. It is useful for the detection or isolation of.
- Examples of the chemical moiety contained in the chemically modified product include water-soluble polymers such as polyethylene glycol, ethylene glycol / propylene glycol copolymer, carboxymethyl cellulose, dextran, and polyvinyl alcohol. *
- Bioly modified products include those modified by enzyme treatment or cell treatment, fusions to which other peptides such as tags have been added by genetic recombination, and endogenous or exogenous sugar chain-modifying enzymes. And the like, which are prepared using a cell that expresses as a host.
- Such modifications may be made at any position in the antibody or antigen-binding fragment thereof, or at a desired position, and the same or two or more different modifications may be made at one or more positions. .
- modified body of an antigen-binding fragment of an antibody also includes “fragment of a modified body of an antibody” in its meaning.
- antibodies produced in cultured mammalian cells are known to lack the lysine residue at the carboxyl terminus of the heavy chain (Journal of Chromatography A, 705: 129-134 (1995)).
- two amino acid residues of glycine and lysine at the heavy chain carboxyl terminus are deleted and a proline residue located at the carboxyl terminus is newly amidated (Analytical Biochemistry, 360: 75-83). (2007)).
- glutamine or glutamic acid residue at the amino terminus of the heavy or light chain of an antibody is pyroglutamylated at the time of antibody production, and the antibody of the present invention has such a modification. (International Patent Publication WO2013 / 147153).
- the present invention also includes the modified antibody and the antigen-binding fragment of the antibody, a deletion in which one or two amino acids are deleted at the heavy chain carboxyl terminus, and the amidated deletion (For example, a heavy chain in which the proline residue at the carboxyl terminal site is amidated), an antibody in which the amino terminal residue of the heavy chain or light chain is pyroglutamylated, etc. ").
- the heavy chain and light chain carboxyl-terminal deletions of the antibody according to the present invention are not limited to the above types.
- the two or more chains are heavy chains selected from the group consisting of full length and the above-mentioned deletions. Any one of these may be sufficient, and what combined any 2 or more types may be sufficient.
- the amount ratio or molecular number ratio of each deletion may be affected by the type and culture conditions of cultured mammalian cells that produce the antibody according to the present invention. A case where one amino acid residue at the carboxyl terminus is deleted from both of the two heavy chains can be mentioned. These are all encompassed within the meaning of the antibody variant of the present invention, the antigen-binding fragment of an antibody, or a modified form thereof.
- antibody-dependent cytotoxic activity by regulating the modification of the sugar chain bound to the antibody of the present invention (glycosylation, defucose, etc.).
- Known techniques for regulating antibody sugar chain modifications include, but are not limited to, International Patent Publication Nos. WO99 / 54342, WO00 / 61739, WO02 / 31140, and the like.
- the antibody of the present invention and the antigen-binding fragment of the antibody also include an antibody whose sugar chain modification is regulated and an antigen-binding fragment of the antibody.
- the range of “antibody or antigen-binding fragment thereof” includes “deleted form”, “modified form” of “antibody or antigen-binding fragment thereof”, and mixtures thereof.
- the “antibody or antigen-binding fragment thereof” included in the molecule having antigen-binding property, multispecific molecule, bispecific molecule, etc. of the present invention described in (3-5) and (3-6) The range includes “deletions” and “modifications” of “antibodies or antigen-binding fragments thereof” and mixtures thereof.
- an anti-CD3 antibody is prepared, for example, by the method of Kohler and Milstein (Kohler and Milstein, Nature (1975) 256, p. 495-497, According to Kennet, R. ed., Monoclonal Antibodies, p. 365-367, Plenum Press, NY (1980)), an anti-CD3 antibody-producing cell is isolated from the spleen of an animal immunized with the CD3 protein, And myeloma cells are fused together to establish a hybridoma, and a monoclonal antibody can be obtained from the hybridoma culture.
- An antigen for producing an anti-CD3 antibody can be obtained according to a method for preparing a natural or recombinant CD3 protein (human CD3 ⁇ single-chain antigen).
- Examples of antigens that can be prepared in this manner include CD3 protein or CD3 protein fragment, or derivatives obtained by adding any amino acid sequence or carrier thereto (hereinafter collectively referred to as “CD3”).
- Natural CD3 can be purified and isolated from, for example, human tissue-derived cells or cultures of the cells.
- Recombinant human CD3 ⁇ single-chain antigen is obtained by introducing a gene containing a base sequence encoding the amino acid sequence of human CD3 ⁇ single-chain antigen into a host cell and recovering the antigen from the cell culture. Can be prepared.
- CD3 obtained by cell-free protein synthesis of a gene containing a base sequence encoding the amino acid sequence of CD3 antigen by an in vitro translation system is also included in the “CD3 antigen” of the present invention.
- Production of anti-CD3 monoclonal antibody usually involves the following steps.
- cloning a single cell clone (cloning).
- step (A) Step of preparing antigen The CD3 protein of the present invention is purified and isolated from animal tissues (including body fluids), cells derived from the tissues or cell cultures, genetic recombination, cell-free protein synthesis, chemistry It can be prepared by synthesis or the like.
- step (B) Step of preparing antibody-producing cells The antigen obtained in step (a) is mixed with Freund's complete or incomplete adjuvant or auxiliary agent such as potassium alum, and an experimental animal is immunized as an immunogen.
- mice an animal used in a known hybridoma production method can be used without any problem. Specifically, for example, mice, rats, goats, sheep, cows, horses and the like can be used. However, from the viewpoint of easy availability of myeloma cells to be fused with the extracted antibody-producing cells, it is preferable to use mice or rats as immunized animals.
- mice for example, A, AKR, BALB / c, BALB / cAnNCrj, BDP, BA, CE, C3H, 57BL, C57BL, C57L , DBA, FL, HTH, HT1, LP, NZB, NZW, RF, R III, SJL, SWR, WB, 129, etc. are rats, for example, Wistar, Low, Lewis, Sprague-Dawley, ACI, BN, Fischer, etc. can be used.
- mice and rats can be obtained, for example, from laboratory animal breeders such as Nippon Clare and Nippon Charles River.
- the BALB / c line is particularly preferable as the immunized animal for mice and the Wistar and Low lines for rats.
- mice with reduced biological mechanisms for removing autoantibodies that is, autoimmune disease mice.
- the age at the time of immunization of these mice or rats is preferably 5 to 12 weeks, more preferably 6 to 8 weeks.
- antibody titer measurement method examples include immunoassays such as RIA method and ELISA method, but are not limited to these methods.
- Antibody-producing cells derived from spleen cells or lymphocytes isolated from immunized animals can be obtained from, for example, Kohler et al. , Nature (1975) 256, 495; Kohler et al. , Eur. J. et al. Immunol. (1977) 6,511, Milstein et al. , Nature (1977), 266, 550, and Walsh, Nature (1977) 266, 495).
- Step of preparing myeloma There is no particular limitation on the myeloma cell used for cell fusion, and it can be appropriately selected from known cell lines, but considering the convenience in selecting a hybridoma from the fused cell HGPRT (Hypoxanthine-guanine phosphoryltransferase) -deficient strains, ie, X63-Ag8 (X63), NS1-ANS / 1 (NS1), P3X63-Ag8.
- HGPRT Hypoxanthine-guanine phosphoryltransferase
- 8-azaguanine in a suitable medium such as 8-azaguanine medium [RPMI-1640 medium supplemented with glutamine, 2-mercaptoethanol, gentamicin, and fetal calf serum (hereinafter referred to as “FCS”). Added medium], Iscove's Modified Dulbecco's Medium (Iscove's Modified Dulbecco's Medium; hereinafter referred to as “IMDM”), or Dulbecco's Modified Eagle Medium (Dulbecco's Modified Eagle Medium; hereinafter referred to as “DMEM”).
- IMDM Iscove's Modified Dulbecco's Medium
- DMEM Dulbecco's Modified Eagle Medium
- Step of Fusing Antibody-Producing Cells and Myeloma Cells Fusion of antibody-producing cells and myeloma cells can be accomplished by a known method (Weir, DM, Handbook Experimental Immunology Vol. I. III., Blackwell Scientific Public , Oxford (1987), Kabat, EA and Mayer, MM, Experimental Immunochemistry, Charles C Thomas Publisher Publisher, Illinois (1964), etc.) Can be implemented.
- Step of selecting a hybridoma group producing an antibody of interest The method of selecting a hybridoma obtained by cell fusion is not particularly limited, but is usually a HAT (hypoxanthine / aminopterin / thymidine) selection method (Kohler et al. , Nature (1975) 256, 495; Milstein et al., Nature (1977) 266, 550).
- HAT hyperxanthine / aminopterin / thymidine
- Step of obtaining a single cell clone As a method for cloning a hybridoma, for example, a known method such as a methylcellulose method, a soft agarose method, a limiting dilution method, or the like can be used (for example, Barbara, B.M. and Stanley, MS: Selected Methods in).
- Hybridoma culture process breeding process of animal transplanted with hybridoma Monoclonal antibody can be produced by culturing the selected hybridoma. Preferably, the desired hybridoma is cloned before producing the antibody. To serve.
- Monoclonal antibodies produced by such hybridomas can be recovered from the hybridoma culture. Moreover, it can also be recovered as a recombinant antibody from a culture of cells into which the monoclonal antibody gene has been introduced. Further, the hybridoma can be injected into the abdominal cavity of the same strain of mice (for example, the above-mentioned BALB / cAnNCrj) or Nu / Nu mouse, and the hybridoma can be proliferated to recover from the ascites.
- H Monoclonal antibody biological activity measurement step / judgment step Various biological tests can be selected and applied according to the purpose.
- An anti-CD3 antibody is prepared by the hybridoma method described above by using cells expressing natural CD3, cells expressing recombinant CD3 or a fragment thereof, and the like as immunogens. Can do.
- Examples of cells expressing natural CD3 include human thymocytes and T lymphocytes.
- Those CD3 expressing cells are 1 ⁇ 10 5 to 1 ⁇ 10 9 cells, preferably 1 ⁇ 10 6 to 1 ⁇ 10 8 cells, more preferably 0.5 to 2 ⁇ 10 7 cells, and even more preferably.
- 1 ⁇ 10 7 cells are used for one immunization, but the number of cells to be immunized can be changed according to the expression level of CD3.
- Such an immunogen is generally administered intraperitoneally, but can also be administered intradermally or the like.
- the method described in (4-1-2) can be applied.
- the anti-CD3 antibody of the present invention can also be obtained using a DNA immunization method.
- An antigen expression plasmid is introduced into an animal individual such as a mouse or a rat, and the antigen is expressed in the individual to induce immunity to the antigen.
- Gene transfer methods include direct injection of plasmids into muscles, intravenous injection of introduction reagents such as liposomes and polyethyleneimine, methods using viral vectors, and gold particles with plasmids attached to them by Gene Gun. There are techniques, such as the Hydrodynamic method in which a large amount of plasmid solution is intravenously injected.
- C3-147 can be mentioned.
- the amino acid sequence of the light chain variable region of C3-147 is shown in SEQ ID NO: 9 (FIG. 17) in the sequence listing.
- the amino acid sequence of the heavy chain variable region of C3-147 is shown in SEQ ID NO: 7 (FIG. 15) in the sequence listing.
- the antibody of the present invention comprises a nucleotide containing a nucleotide sequence encoding its heavy chain amino acid sequence (heavy chain nucleotide) and a nucleotide containing a nucleotide sequence encoding its light chain amino acid sequence (light chain).
- a heavy chain nucleotide and a light chain nucleotide may be inserted into one vector.
- Prokaryotic cells or eukaryotic cells can be used as host cells.
- eukaryotic cells animal cells, plant cells, and eukaryotic microorganisms can be used.
- animal cells include cells derived from mammals, ie, human embryonic kidney cells HEK293F cells (Subedi GP et al., J Vis Exp. (2015) 106) monkey kidney. COS cells (Gluzman, Y. Cell). (1981) 23,175-182, ATCC CRL-1650), mouse fibroblasts NIH3T3 (ATCC No. CRL-1658), Chinese hamster ovary cells (CHO cells, ATCC CCL-61), and their dihydrofolate reductase deficiency Strains (CHOdhfr-: Urlaub, G. and Chasin, LA, PNAS (1980) 77, 4126-4220), chicken-derived cells such as chicken, insect-derived cells, and the like.
- mammals ie, human embryonic kidney cells HEK293F cells (Subedi GP et al., J Vis Exp. (2015) 106) monkey kidney.
- COS cells Gluzman, Y. Cell). (1981) 23,175-182, ATCC
- cells modified so that the biological activity of the antibody can be enhanced by modifying the sugar chain structure can be used as a host.
- the sugar chain in which fucose is not bound to N-acetylglucosamine at the sugar chain reducing end has been modified to be 20% or more.
- By using CHO cells it is possible to prepare an antibody with enhanced ADCC activity or CDC activity (International Patent Publication WO02 / 31140).
- Examples of eukaryotic microorganisms include yeast.
- Examples of prokaryotic cells include Escherichia coli and Bacillus subtilis.
- the signal peptide for secreting the antibody of the present invention is the same type, the same type and the same subtype as the antibody. It is not limited to the secretion signal of the antibody of the type, and the secretion signal of the antibody itself, but the secretion signal of other types or subtypes of antibodies, or of proteins from other eukaryotic species or prokaryotes Any secretory signal can be selected and used.
- Antibodies secreted including a signal peptide are also included in the antibody of the present invention or the molecule of the present invention.
- the antibody of the present invention can further include a human antibody.
- the human anti-CD3 antibody means an anti-CD3 antibody consisting of the amino acid sequence of a human-derived antibody.
- the human anti-CD3 antibody is obtained by a method using a human antibody-producing mouse having a human genomic DNA fragment containing heavy and light chain genes of human antibody (Tomizuka, K. et al., Nature Genetics (1997) 16, 133-143. Kuroiwa, Y. et.al., Nuc.Acids Res. (1998) 26,3447-3448; Yoshida, H. et al., Animal Cell Technology: Basic and Applied Aspects vol.10, 69-73.
- Such a human antibody-producing animal specifically destroys the endogenous immunoglobulin heavy chain and light chain loci of a non-human mammal, and instead uses a yeast artificial chromosome (YAC) vector or the like.
- YAC yeast artificial chromosome
- eukaryotic cells are transformed with cDNA encoding each of the heavy and light chains of such a human antibody, preferably a vector containing the cDNA, to produce a gene recombinant human monoclonal antibody.
- This antibody can also be obtained from the culture supernatant by culturing the transformed cells.
- eukaryotic cells preferably mammalian cells such as HEK293F cells and CHO cells can be used as the host.
- a method for obtaining a human antibody derived from phage display selected from a human antibody library is also known.
- a phage display method can be used in which a variable region of a human antibody is expressed as scFv on the surface of a phage and a phage that binds to an antigen is selected.
- the DNA sequence encoding the variable region of the human antibody that binds to the antigen can be determined.
- an expression vector having the sequence is prepared, and introduced into a suitable host for expression to obtain a human antibody (WO92 / 01047, WO92 / 20791, WO93 / 06213, WO93 / 11236, WO93 / 19172, WO95 / 01438, WO95 / 15388, Annu. Rev. Immunol (1994) 12, 433-455).
- polypeptide linker that links the variable regions for example, any single chain peptide consisting of 5 to 30 residues is used.
- the scFv-encoding DNA is a DNA encoding the heavy chain or heavy chain variable region of the antibody, and a DNA encoding the light chain or light chain variable region.
- DNA encoding the entire scFv region may be obtained by total synthesis.
- an expression vector containing the DNA and a host cell transformed with the expression vector can be prepared according to a conventional method, and by culturing the host cell, The scFv can be recovered from such culture according to the method.
- Antigen-binding fragments of other antibodies can also be obtained by obtaining a gene encoding the antigen-binding fragment according to the above method, introducing the gene into the cell, and recovering the antigen-binding fragment from the cell culture. ,Obtainable.
- the antibody or the like of the present invention may be increased in quantity and increased in affinity for the antigen.
- the antibody that multiplies may be one type of antibody or a plurality of antibodies that recognize multiple epitopes of the same antigen. Examples of the method for increasing the amount of antibody include binding of IgG CH3 domain and two scFvs, binding to streptavidin, introduction of helix-turn-helix motif, and the like.
- the antibody of the present invention may be a mixture of a plurality of types of anti-CD3 antibodies having different amino acid sequences, that is, a polyclonal antibody.
- a polyclonal antibody examples include a mixture of a plurality of types of antibodies in which part or all of the CDR set is different.
- Such a polyclonal antibody can be obtained by co-culturing cells producing different antibodies and recovering from the culture (WO 2004/061104). It is also possible to mix separately prepared antibodies.
- the antiserum which is one embodiment of the polyclonal antibody can be prepared by immunizing an animal with a desired antigen and collecting the serum from the animal according to a standard method.
- an antibody conjugated with various molecules such as polyethylene glycol (PEG) can also be used.
- PEG polyethylene glycol
- the antibody of the present invention may be a complex (Immunoconjugate) in which these antibodies and other molecules are connected by a linker.
- An example of an antibody-drug complex in which the antibody is bound to a radioactive substance or a compound having a pharmacological action (drug) is ADC (Antibody-Drug Conjugate) (Methods Mol Biol. (2013) 1045). : 1-27).
- the antibodies of the present invention may further link these antibodies to other functional polypeptides.
- the antibody can be complexed with an albumin-binding polypeptide (Protein Eng Des Sel. (2012) (2): 81-8).
- the obtained antibody and antigen-binding fragment of the antibody can be uniformly purified so as not to contain other than the antibody and the like. Separation and purification of antibodies and antigen-binding fragments of antibodies may be carried out using separation and purification methods used in ordinary proteins.
- antibodies can be separated and purified by appropriately selecting and combining chromatography columns, filters, ultrafiltration, salting out, dialysis, preparative polyacrylamide gel electrophoresis, isoelectric focusing, etc. It is not limited to.
- an expression vector is prepared by adding a DNA sequence encoding a His tag or a FLAG tag to the carboxyl terminus of an antibody variable region, and cells are transformed with this vector.
- the antibody and antigen-binding fragment of the antibody are expressed by culturing, and after culturing, the culture supernatant is extracted, metal affinity chromatography such as Ni, Co, etc., anti-FLAG tag antibody column, gel filtration, ion exchange chromatography Etc. and can be purified.
- An antibody and an antigen-binding fragment of an antibody expressed by including an amino acid sequence encoding a tag such as a His tag or a FLAG tag are also included in the antibody of the present invention or the molecule of the present invention.
- the bispecific molecule and multispecific molecule of the present invention are a method for introducing an expression plasmid into a host cell and expressing it transiently. After introducing the plasmid, select a stable expression cell line by drug selection, constitutive expression method, acellular synthesis method, each antibody or antigen-binding fragment is prepared by the above method, then synthetic peptide linker is added And a method of chemically bonding them.
- two single-chain antibodies are linked by a peptide linker (tandem scFv), and the domains of the two antibodies with different specificities are exchanged and non-covalently bound.
- Dimers diabodies
- domains of two antibodies with different specificities are exchanged to form single chains (single chain diabodies), and diabodies are made into single chains
- methods such as forming a dimer non-covalently (TandAb, US Pat. No. 7,129,330).
- the present invention includes a gene encoding the antibody of the present invention or an antigen-binding fragment of the antibody, or a modified product such as an antigen, a recombinant vector into which the gene is inserted, a cell into which the gene or vector has been introduced, and other Also provided are cells that produce the antibodies of the invention.
- composition comprising an anti-CD3 antibody, an antigen-binding fragment thereof or a modified form thereof, and / or a molecule of the present invention containing them, for example, a multispecific molecule.
- the treatment and / or prevention of a disease includes prevention of the onset of such disease, suppression or inhibition of progression or progression, reduction or increase of one or more symptoms exhibited by an individual suffering from such disease.
- Examples include, but are not limited to, suppression or remission of deterioration or progression, treatment or prevention of secondary diseases, and the like.
- Examples of the disease include cancer in the case of the molecule.
- the pharmaceutical composition of the present invention comprises a therapeutically or prophylactically effective amount of an anti-CD3 antibody or antigen-binding fragment of the antibody and a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative and / or Adjuvants can be included.
- An amount effective for treatment or prevention means an amount exhibiting a treatment or prevention effect for a specific disease, administration form and administration route.
- the pharmaceutical composition of the present invention includes pH, osmotic pressure, viscosity, transparency, color, isotonicity, sterility, stability of the composition or antibody contained therein, solubility, sustained release, absorbability, penetration.
- Substances for changing, maintaining, and maintaining properties, dosage forms, strength, properties, shapes, etc. can be included.
- the substance for the preparation is not particularly limited as long as it is a pharmacologically acceptable substance. For example, non-toxicity or low toxicity is a property that a drug substance preferably has.
- Examples of the substance for formulation include, but are not limited to, the following: amino acids such as glycine, alanine, glutamine, asparagine, histidine, arginine or lysine, antibacterial agent, ascorbic acid Antioxidants such as sodium sulfate or sodium bisulfite, phosphoric acid, citric acid, boric acid buffer, sodium hydrogen carbonate, buffer such as tris-hydrochloric acid (Tris-Hcl) solution, filler such as mannitol and glycine, ethylenediamine Chelating agents such as tetraacetic acid (EDTA), caffeine, polyvinylpyrrolidine, complexing agents such as ⁇ -cyclodextrin and hydroxypropyl- ⁇ -cyclodextrin, bulking agents such as glucose, mannose or dextrin, monosaccharides, disaccharides, Glucose, mannose and dextrin Other carbohydrates, coloring agents, flavoring agents, diluents, hydrophil
- the amount of substances for these preparations added is 0.001 to 1000 times the weight of the anti-CD3 antibody, its antigen-binding fragment or its modification, or the molecule of the present invention, for example, a multispecific molecule, Preferably it is 0.01 to 100 times, more preferably 0.1 to 10 times.
- Anti-CD3 antibody of the present invention antigen-binding fragment thereof or modified product thereof, molecule of the present invention, for example, immunoliposome containing multispecific molecule in liposome, antibody formed by binding antibody and liposome
- a pharmaceutical composition containing a modified substance is also included in the pharmaceutical composition of the present invention.
- the excipient and carrier are usually liquid or solid, and are not particularly limited as long as they are water used for injection, physiological saline, artificial cerebrospinal fluid, and other substances used for oral or parenteral administration.
- physiological saline examples include neutral ones and those containing serum albumin.
- the buffer examples include Tris buffer prepared so that the final pH of the pharmaceutical composition is 7.0 to 8.5, acetate buffer prepared so as to be 4.0 to 5.5, and 5. Examples thereof include a citrate buffer prepared to be 0 to 8.0, a histidine buffer prepared to be 5.0 to 8.0, and the like.
- the pharmaceutical composition of the present invention is a solid, liquid, suspension or the like. Mention may be made of freeze-dried preparations. An excipient such as sucrose can be used to mold the lyophilized preparation.
- the administration route of the pharmaceutical composition of the present invention may be enteral administration, local administration or parenteral administration, and a suitable administration route may be selected depending on the target disease. Specific examples include intravenous administration, intraarterial administration, intramuscular administration, intradermal administration, subcutaneous administration, intraperitoneal administration, transdermal administration, intraosseous administration, and intraarticular administration. *
- composition of such a pharmaceutical composition can be determined according to the administration method, the CD3 protein binding affinity of the antibody, and the like.
- the dose of the anti-CD3 antibody, antigen-binding fragment thereof or modified form thereof, or the molecule of the present invention depends on the species of the individual, the type of disease, symptoms, sex, age, chronicity , which can be appropriately determined according to the CD3 protein binding affinity of the antibody or its biological activity, and other factors, but is usually 0.01 to 1000 mg / kg, preferably 0.1 to 100 mg / kg. It can be administered once every 1 to 180 days, or twice or more times a day.
- the form of the pharmaceutical composition includes injections (including lyophilized preparations and infusions), suppositories, nasal absorption preparations, transdermal absorption preparations, sublingual preparations, capsules, tablets, ointments, granules, aerosols. Examples thereof include pills, pills, powders, suspensions, emulsions, eye drops, and implantable preparations.
- an anti-CD3 antibody, an antigen-binding fragment thereof or a modified form thereof, and / or a molecule of the present invention containing them, such as a multispecific molecule (hereinafter referred to as an anti-CD3 antibody) is combined with other agents.
- An anti-CD3 antibody or the like, or a pharmaceutical composition containing it as an active ingredient can be administered simultaneously or separately with a pharmaceutical composition containing another agent, ie, a drug other than an anti-CD3 antibody or the like as an active ingredient.
- a pharmaceutical composition containing an anti-CD3 antibody or the like as an active ingredient is administered, or after administering a pharmaceutical composition containing an anti-CD3 antibody or the like as an active ingredient, or the other agent is administered, or A pharmaceutical composition containing an anti-CD3 antibody or the like as an active ingredient and another agent may be administered simultaneously.
- both the case where the active ingredients of both the anti-CD3 antibody and other agents and other agents are contained in a single pharmaceutical composition, and the case where both active ingredients are separately contained in a plurality of pharmaceutical compositions are referred to as “ It is referred to as “pharmaceutical composition containing anti-CD3 antibody and other agents”.
- such a “pharmaceutical composition” is synonymous with “a pharmaceutical composition administered in combination with an anti-CD3 antibody or the like and another agent”.
- administered in combination with an anti-CD3 antibody or the like and another agent means that the anti-CD3 antibody or the like and the other agent are taken into the body of the administration target for a certain period.
- a preparation containing an anti-CD3 antibody and the like and another agent in a single preparation may be administered, or each may be formulated separately and administered separately.
- the timing of administration is not particularly limited, and may be administered at the same time, and may be administered at different times or on different days.
- the anti-CD3 antibody or the like and the other agent are administered at different times or days, the order of administration is not particularly limited. Usually, since each formulation is administered according to each administration method, the administration may be the same number of times or may be different times.
- the administration method (administration route) of each formulation may be the same, and may be administered by different administration methods (administration route).
- the anti-CD3 antibody or the like and the other agent do not need to be present in the body at the same time, and the body within a certain period of time (for example, one month, preferably one week, more preferably several days, even more preferably one day).
- the other active ingredient may disappear from the body at any time of administration.
- Examples of the administration form of “pharmaceutical composition administered in combination with anti-CD3 antibody etc.” and other agents include, for example, 1) administration of a single preparation containing anti-CD3 antibody etc. and other agents, 2) anti-CD3 antibody etc. 3) Simultaneous administration of the two preparations obtained by separately formulating the other agent with the same administration route, 3) Same administration route of the two preparations obtained by separately formulating the anti-CD3 antibody and the other agent 4) Simultaneous administration of two types of preparations obtained by separately formulating anti-CD3 antibody and other agents in different administration routes, 5) Anti-CD3 antibody and other agents
- administration of two kinds of preparations obtained by separate preparations at different time intervals in different administration routes can be mentioned.
- the dosage, administration interval, dosage form, formulation, etc. of the “pharmaceutical composition administered in combination with an anti-CD3 antibody and the like” are similar to those of the pharmaceutical composition containing the anti-CD3 antibody, but are not limited thereto. Is not to be done.
- Such pharmaceutical compositions may be kits containing them when they are made into two different formulations.
- the “combination” of an anti-CD3 antibody or the like and another agent means “administered in combination” of the anti-CD3 antibody or the like and another agent.
- the present invention relates to a method for treating or preventing a disease associated with CD3, use of the antibody of the present invention for preparing a pharmaceutical composition for treating or preventing the disease, antibody of the present invention for treating or preventing the disease Use, also provide.
- a therapeutic or prophylactic kit containing the antibody of the present invention is also included in the present invention.
- Example 1 Preparation of Rat Anti-Human CD3 Antibody 1
- pcDNA3.1-DEST modified to Destination Vector by Gateway Vector Conversion System (Thermo Fisher Scientific) was prepared.
- the cDNA encoding the human CD3 ⁇ protein (NCBI Reference Sequence: NP_000724.1) shown in FIG. -Cloned into DEST vector to construct hCD3 ⁇ -pcDNA3.1.
- the cDNA encoding the human CD3 ⁇ protein (NP — 000073.1) shown in FIG.
- SEQ ID NO: 2 (SEQ ID NO: 2) is amplified by the PCR method using human T cell-derived cDNA as a template according to a method known to those skilled in the art, and pcDNA3.1 (+ ) (Thermo Fisher Scientific) to construct an expression vector hCD3 ⁇ -pcDNA3.1. Endofree Plasmid Giga Kit (QIAGEN) was used for mass preparation of each expression vector.
- hCD3 ⁇ -pcDNA3.1 and hCD3 ⁇ -pcDNA3.1 or pcDNA3.1-DEST as a control was introduced according to the transfection procedure using Lipofectamine 2000 (Thermo Fisher Scientific), and 96-well plate (Corning) 100 ⁇ L each, and cultured overnight in a DMEM medium containing 10% FBS under conditions of 37 ° C. and 5% CO 2 . The obtained transfected cells were used for Cell-ELISA while still in an adherent state.
- OPD color solution OPD solution (0.05 M trisodium citrate, 0.1 M disodium hydrogen phosphate.12 water, pH 4.5) was added.
- OPD color solution 0.05 M trisodium citrate, 0.1 M disodium hydrogen phosphate.12 water, pH 4.5
- o-Phenylenediamine dihydrochloride Wako Pure Chemical Industries, Ltd.
- H 2 O 2 dissolved in 0.4 mg / mL and 0.6% (v / v), respectively, were added at 100 ⁇ L / well.
- the color development reaction was carried out with occasional stirring, 100 ⁇ L / well of 1M HCl was added to stop the color development reaction, and then the absorbance at 490 nm was measured with a plate reader (ENVISION: PerkinElmer).
- hCD3 ⁇ -pcDNA3.1 and hCD3 ⁇ -pcDNA3.1 expression vector-introduced HEK293 ⁇ Hybridomas producing culture supernatants with higher absorbance in the cells were selected as positive for anti-human CD3 antibody production.
- JACKSON IMMUNORESEARCH crosslinker Goat Anti-rat IgG Fc ⁇ Fragment specific
- a histogram of PE fluorescence intensity is prepared, and a hybridoma that produces a sample in which the histogram of PE fluorescence intensity is shifted to the strong fluorescence intensity side with respect to the fluorescence intensity histogram of the rat IgG isotype control antibody is positive for anti-human T cell activation ability. Selected as a human CD3 antibody-producing hybridoma.
- the expression vector introduced Lenti-X293T cells were treated with TrypLE Express (Thermo Fisher Scientific), washed with DMEM containing 10% FBS, and then adjusted to 5 ⁇ 10 6 cells / mL with PBS containing 5% FBS. Prepared. The resulting cell suspension was used for flow cytometry analysis.
- Example 1 Flow cytometry analysis of binding to human CD3
- Flow cytometry was performed to determine the binding specificity of human antibodies produced by hybridomas determined to be positive for human T cell activation in Example 1-5. Further confirmation by law.
- Example 1) The Lenti-X293T cell suspension prepared in -6-1 was seeded on a 96-well U-bottom microplate at 100 ⁇ L / well, and the supernatant was removed after centrifugation.
- Hybridoma culture supernatant was added to each of hCD3 ⁇ -pcDNA3.1 and hCD3 ⁇ -pcDNA3.1-introduced Lenti-X293T cells and pcDNA3.1-DEST-introduced Lenti-X293T cells, and the mixture was allowed to stand at 4 ° C. for 1 hour. After washing once with PBS containing 5% FBS, Anti-Rat IgG FITC conjugate (SIGMA) diluted 500 times with PBS containing 5% FBS was added and suspended, and the mixture was allowed to stand at 4 ° C. for 30 minutes.
- SIGMA Anti-Rat IgG FITC conjugate
- Lenti-X293T cells introduced with expression vectors were treated with TrypLE Express, washed with DMEM containing 10% FBS, and then adjusted to a concentration of 5 ⁇ 10 6 cells / mL with PBS containing 5% FBS. The resulting cell suspension was used for flow cytometry analysis.
- Example 1 Binding flow cytometric analysis to monkey CD3
- the binding specificity of the antibody produced by the hybridoma determined to be an antibody-producing hybridoma that binds to human CD3 in -6-2 to the monkey CD3. Further confirmation by flow cytometry.
- Lenti-X293T cell suspension prepared in Example 1) -6-4 was seeded on a 96-well U-bottom microplate at 100 ⁇ L / well, and the supernatant was removed after centrifugation.
- Hybridoma culture supernatant was added to each of cynoCD3 ⁇ -pcDNA3.1 and cynoCD3 ⁇ -pcDNA3.1-introduced Lenti-X293T cells and pcDNA3.1-DEST-introduced Lenti-X293T cells, and the mixture was allowed to stand at 4 ° C. for 1 hour. After washing once with PBS containing 5% FBS, Anti-Rat IgG FITC conjugate diluted 500 times with PBS containing 5% FBS was added and suspended, and the mixture was allowed to stand at 4 ° C. for 30 minutes.
- Lenti-X293T cells introduced with expression vectors were treated with TrypLE Express, washed with DMEM containing 10% FBS, and then adjusted to a concentration of 5 ⁇ 10 6 cells / mL with PBS containing 5% FBS. The resulting cell suspension was used for flow cytometry analysis.
- Example 1 Binding to human CD3 ⁇ Flow cytometric analysis Example 1) The binding specificity of an antibody produced by a hybridoma determined to be an antibody-producing hybridoma that binds to monkey CD3 in -6-5 to human CD3 ⁇ Further confirmation by flow cytometry.
- Example 1) The Lenti-X293T cell suspension prepared in 6-6 was seeded on a 96-well U-bottom microplate at 100 ⁇ L / well, and the supernatant was removed after centrifugation.
- Hybridoma culture supernatant was added to each of hCD3 ⁇ -pcDNA3.1-introduced Lenti-X293T cells and pcDNA3.1-DEST-introduced Lenti-X293T cells, and the suspension was allowed to stand at 4 ° C. for 1 hour. After washing once with PBS containing 5% FBS, Anti-Rat IgG FITC conjugate diluted 500 times with PBS containing 5% FBS was added and suspended, and the mixture was allowed to stand at 4 ° C. for 30 minutes.
- Cynomolgus monkey T cell line HSC-F (JCRB cell bank, No. JCRB1164) was prepared at a concentration of 5 ⁇ 10 6 cells / mL in RPMI 1640 medium containing FBS, and seeded at 100 ⁇ L in a 96-well plate.
- the supernatant of the antibody-producing hybridoma that was not excluded in Example 1) -5-7 or the rat IgG isotype control antibody was added to the HSC-F cells from which the supernatant was removed by centrifugation, and the mixture was allowed to stand at 4 ° C. for 1 hour. I put it. Thereafter, the supernatant was removed, and the cells in the well were washed once with 5% FBS-containing PBS and then added with Anti-Rat IgG FITC conjugate diluted 500-fold with 5% FBS-containing PBS, and suspended at 4 ° C. Let stand for 1 hour.
- the suspension was resuspended in PBS containing 5% FBS containing 2 ⁇ g / ml 7-aminoactinomycin D, and detection was performed with a flow cytometer. Data analysis was performed with Flowjo. 7-aminoactinomycin D-positive dead cells were excluded at the gate, and a histogram of FITC fluorescence intensity of live cells was created.
- the hybridoma producing the shifted sample was selected as an antibody producing hybridoma that also binds to the monkey T cell line.
- Example 2 Examination of binding of rat anti-CD3 monoclonal antibody (C3-147) to human CD3 2) -1 Preparation of monoclonal antibody from hybridoma supernatant 2) -1-1 Culture of hybridoma producing C3-147 Rat anti-CD3 monoclonal antibody was purified from the hybridoma culture supernatant. First, a C3-147-producing hybridoma was grown to a sufficient amount with ClonaCell-HY Selection Medium E (StemCell Technologies), and then Ultra Low IgG FBS (Thermo Fisher Scientific) was added with 20 mL of 5% / micron of Gen. The medium was changed to Hybridoma SFM (Thermo Fisher Scientific) containing Thermo Fisher Scientific) and cultured for 7 days. The main culture supernatant was collected and sterilized through a 0.22 ⁇ m filter (Corning).
- the antibody was purified from the culture supernatant of the hybridoma prepared in Example 2) -1-1 by Protein G affinity chromatography.
- the antibody was adsorbed onto a Protein G column (GE Healthcare Bioscience), and the column was washed with PBS and then eluted with 0.1 M glycine / hydrochloric acid aqueous solution (pH 2.7). 1M Tris-HCl (pH 9.0) is added to the eluate to adjust the pH to 7.0 to 7.5, and then the buffer is replaced with PBS using Centrifugal UF Filter Device VIVASPIN 20 (fractionated molecular weight UF30K, Sartorius). At the same time, the antibody was concentrated to adjust the antibody concentration to 2 mg / mL. Finally, it was filtered through a Minisart-Plus filter (Sartorius) to obtain a purified sample.
- HBS-EP + (10 mM HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.05% Surfactant P20) was used as a running buffer.
- the antibody was added to the chip on which the anti-mouse IgG antibody had been immobilized for about 1 minute to capture as a ligand, and then 100 nM antigen was added at a flow rate of 30 ⁇ l / min for 120 seconds to monitor antigen binding.
- 10 mM glycine-HCl pH 1.7 was added at a flow rate of 10 ⁇ l / min for 3 minutes.
- the binding signal after 120 seconds was 34 RU.
- Example 3 Determination of the nucleotide sequence of the cDNA encoding the variable region of the rat anti-CD3 antibody (C3-147) The nucleotide sequence of the cDNA encoding the variable region of the rat anti-CD3 antibody (C3-147) Determined by.
- Rat anti-CD3 antibody (C3-147) producing hybridoma cell lysate (50 mM Tris-HCl (pH 7.5), 250 mM LiCl, 5 mM EDTA (pH 8), 0.5% dodecyl sulfate Li (LiDS) ), 2.5 mM dithiothreitol (DTT)) was mixed with magnetic beads of Dynabeads mRNA DIRECT Kit (Thermo Fisher Scientific) to which oligo dT25 was bound, and mRNA was bound to the magnetic beads.
- Hybrids mRNA DIRECT Kit Thermo Fisher Scientific
- RNA washing solution A (10 mM Tris-HCl (pH 7.5), 0.15 M LiCl, 1 mM EDTA, 0.1% LiDS, 0.1% Triton X-100) and a solution for cDNA synthesis (50 mM Tris X-100).
- rat immunoglobulin heavy and light chain variable region gene fragments Wash the magnetic beads with TE solution (10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.1% Triton X-100). Later, rat immunoglobulin heavy chain and light chain genes were amplified using 5'-RACE PCR. Specifically, the magnetic beads were transferred to a PCR reaction solution (0.2 ⁇ M primer, 0.2 mM dNTP, 0.25 unit PrimeSTAR HS DNA Polymerase (TAKARA)), and the reaction at 94 ° C. for 30 seconds to 68 ° C. for 90 seconds was performed for 35 cycles. .
- the primer sets used are as follows.
- FIG. 5 (SEQ ID NO: 50) 1st antisense primer rIg ⁇ -AS1 5′-TCACTGAGCTGGGTGAGAGTGTAGAGCCCC-3 ′
- FIG. 6 (SEQ ID NO: 51) Second antisense primer rIg ⁇ -AS2 5′-TCACCGAGCTGCTGAGGGGTGTAGAGCCCC-3 ′
- FIG. 5 (SEQ ID NO: 50) Second antisense primer rIg ⁇ -AS2 5′-TCACCGAGCTGCTGAGGGGTGTAGAGCCCC-3 ′
- FIG. 7 (SEQ ID NO: 52) PCR primer set sense primer for light chain gene amplification Nhe-polyC-S2 5′-GCTAGCGCTACCGCGACTCAGATCCCCCCCCCCCCCCCCDN-3 ′
- FIG. 8 (SEQ ID NO: 53) 1st antisense primer rIgL-AS1 5′-TTCCACCATCACTCGGGTAGAAAATCAG-3 ′
- FIG. 9 (SEQ ID NO: 54) Second antisense primer rIg ⁇ -AS2 5′-TAACACCAGGGTAGAAATTCTTCACCAT-3 ′
- FIG. 10 (SEQ ID NO: 55) A sequence analysis of the nucleotide sequence was performed on the fragment amplified by the PCR reaction. The primers used are as follows.
- FIG. 11 (SEQ ID NO: 56), Antisense primer for light chain rIgL-seq1 5′-TCCCTGGAGCTCTCCAGT-3 ′
- FIG. 12 (SEQ ID NO: 57)
- FIG. 13 (SEQ ID NO: 58) Sequence analysis was performed using a gene sequence analyzer (“ABI PRISM 3700 DNA Analyzer; Applied Biosystems” or “Applied Biosystems 3730xl Analyzer; applied Cysystem”).
- the base sequence of the C3-147 heavy chain variable region determined by sequence analysis is shown in FIG. 14 (SEQ ID NO: 6), the amino acid sequence is shown in FIG. 15 (SEQ ID NO: 7), and the base sequence of the C3-147 light chain variable region is shown in FIG. SEQ ID NO: 8) and amino acid sequence are shown in FIG. 17 (SEQ ID NO: 9), respectively.
- Example 4 Production of rat anti-CD3 scFv (C3E-7000) and its humanized form (C3E-7034) 4) -1 Production of rat anti-CD3 antibody (C3-147) scFv 4) -1-1 Construction of rat antibody CD3 scFv expression vector (pC3E-7000) Before and after DNA sequence encoding linker inserted between C3-147 heavy chain variable region (VH) and light chain variable region (VL) 18 (SEQ ID NO: 10) of the DNA fragment having an additional sequence of 15 bases in FIG. 18 (SEQ ID NO: 10) and the antisense strand oligonucleotide FIG.
- SEQ ID NO: 11 (Sigma Aldrich Custom Oligos Custom Synthesis Service) , Adjusted to 100 pmol / ⁇ L, then mixed 20 ⁇ L each, and allowed to stand at 96 ° C. for 10 minutes, 70 ° C. for 2 minutes, 60 ° C. for 2 minutes, 40 ° C. for 2 minutes, and 30 ° C. for 2 minutes. Both were annealed to produce a linker fragment that was inserted between VH and VL. Next, a DNA fragment amplified by PCR so as to add a human IgG heavy chain signal sequence to the vector backbone derived from the animal cell expression vector pcDNA-3.3TOPO (Thermo Scientific), and the rat shown in FIG.
- pcDNA-3.3TOPO Thermo Scientific
- rat anti-CD3 scFv C3E-7000
- Expi293F cells Thermo Scientific were subcultured and cultured according to the manual.
- the scFv expression vector was introduced into Expi293F cells in the logarithmic growth phase for transient expression, filtered, and used for purification. Purification is performed in a two-step process of Ni affinity chromatography using His Trap excel (GE Healthcare) and gel filtration using Superdex 200 increment (GE Healthcare), and a peak corresponding to the scFv monomer molecular weight is recovered. A purified protein sample was used.
- an AKTA chromatography system was used, and all steps were performed at 4 ° C.
- HBSor 25 mM histidine / 5% sorbitol, pH 5.0
- the purified protein sample was subjected to analytical SEC, purity and concentration were determined, and then used for various assays.
- the amino acid sequence of C3E-7000 is set forth in FIG. 21 (SEQ ID NO: 15).
- a C3E-7034VH designed to replace valine number 23 with alanine, amino acid number 24 valine with alanine, amino acid number 88 with serine with alanine, and amino acid number 93 with threonine with valine.
- the amino acid sequence is set forth in FIG. 22 (SEQ ID NO: 16).
- the glutamine with the first amino acid number is asparagine
- the valine with the third amino acid number is methionine
- the asparagine with the eighth amino acid number is histidine
- the 12th threonine is glutamic acid
- the 13th asparagine amino acid is serine
- the 14th amino acid leucine is proline
- the 16th amino acid threonine is lysine
- the 19th amino acid glutamic acid is threonine
- the amino acid The 20th leucine is isoleucine
- the 43rd amino acid arginine is serine
- the 75th amino acid leucine is serine
- the 79th amino acid asparagine is serine
- the 81st amino acid valine is leucine
- Lysine acid number 82 th glutamine the C3E-7034VL which is accompanied designed to replace the amino acid sequence is described in FIG.
- FIG. 24 SEQ ID NO: 26
- FIG. 25 SEQ ID NO: 27
- FIG. 26 for CDR-H3, respectively.
- CDR-L1 is described in FIG. 27 (SEQ ID NO: 29)
- CDR-L2 in FIG. 28 SEQ ID NO: 30
- CDR-L3 in FIG. 29 (SEQ ID NO: 31).
- C3E-7035 The amino acid sequence of C3E-7035, which is a variant of C3E-7034, was designed.
- the asparagine at amino acid number 1 in the variable region is glutamine
- the phenylalanine at amino acid number 2 is alanine
- the methionine at amino acid number 3 is valine.
- Histidine at amino acid number 8 as serine glutamic acid at amino acid number 12 as glycine, serine at amino acid number 13 as valine, lysine at amino acid number 16 as glutamine, and threonine at amino acid number 17 as arginine.
- Histidine at amino acid number 40 is leucine
- glutamic acid at amino acid number 41 is proline
- serine at amino acid number 43 is threonine
- serine at amino acid number 44 is alanine
- threonine at amino acid number 46 is lysine.
- Amino acid number 47th threonine is leucine
- 48th amino acid isoleucine is leucine
- 57th aspartic acid is serine
- 60th amino acid serine is proline
- 67th amino acid isoleucine is lysine
- amino acid Aspartic acid at number 68 deficient at arginine at amino acid number 69
- serine at amino acid number 71 as glycine serine at amino acid number 71 as glycine
- Serine at amino acid number 79 is threonine
- asparagine at amino acid number 80 is glycine
- leucine at amino acid number 81 is phenylalanine
- lysine at amino acid number 82 is glutamine
- threonine at amino acid number 83 is allyl.
- FIG. 30 shows the amino acid sequence of the C3E-7035 light chain designed by substituting phenylalanine at amino acid number 90 for tyrosine, immediately before the variable region of the light chain.
- the full-length sequence of C3E-7035 with methionine and alanine inserted in is shown in FIG. 31 (SEQ ID NO: 22).
- C3E-7036 The amino acid sequence of C3E-7036, which is a variant of C3E-7034, was designed.
- the histidine at amino acid number 8 in the variable region is serine
- glutamic acid at amino acid number 12 is glycine
- serine at amino acid number 13 is valine.
- Lysine at amino acid number 16 is glutamine
- threonine at amino acid number 17 is arginine
- lysine at amino acid number 23 is threonine
- arginine at amino acid number 24 is glycine
- histidine at amino acid number 40 is leucine.
- the amino acid number 41 glutamic acid is proline
- the amino acid number 43 serine is threonine
- the amino acid number 44 serine is alanine
- the amino acid number 46 threonine is lysine
- the amino acid number 47 threonine is leucine.
- Amino acid number 4 The amino acid No. 57 is aspartic acid, the amino acid No. 57 aspartic acid is serine, the amino acid No. 60 serine is proline, the amino acid No. 67 isoleucine is lysine, and the amino acid No. 68 aspartic acid is deleted.
- No. 69 arginine is deleted, amino acid No. 71 serine is glycine, amino acid No.
- lysine is threonine
- amino acid No. 77 threonine is alanine
- amino acid No. 79 serine is threonine
- amino acid Asparagine at number 80 is glycine
- leucine at amino acid number 81 is phenylalanine
- lysine at amino acid number 82 is glutamine
- threonine at amino acid number 83 is alanine
- phenylalanine at amino acid number 90 is The amino acid sequence of the C3E-7036 light chain designed to be replaced with Shin is described in FIG. 32 (SEQ ID NO: 23), and the full-length amino acid sequence of C3E-7036 is described in FIG. 33 (SEQ ID NO: 25). Has been.
- C3E-7091 in which the aspartic acid at amino acid number 52 in the light chain variable region of C3E-7079 was replaced with glycine, C3E-7092 replaced with glutamine, C3E-7093 replaced with asparagine, C3E-7094 replaced with serine, and C3E-7095 replaced with alanine were designed.
- the full length amino acid sequence of C3E-7086 is shown in FIG. 74 (SEQ ID NO: 66)
- the full length amino acid sequence of C3E-7087 is shown in FIG. 76 (SEQ ID NO: 68)
- the full length amino acid sequence of C3E-7088 is shown in FIG.
- a region containing C3E-7034 and the additional sequences before and after was amplified using the PCR method to obtain an insert DNA fragment.
- the vector region excluding the scFv region was amplified using the PCR method to obtain a vector fragment.
- the respective DNA fragments were ligated using an In-Fusion HD cloning kit (CLONTECH) to prepare a humanized anti-CD3 scFv expression vector pC3E-7034 containing the nucleotide sequence of FIG. 34 (SEQ ID NO: 18) in the ORF.
- FIG. 93, 94 Construction of an expression vector for the 3-3-4 CDR-modified humanized antibody CD3 scFv FIG. 93, 94 (SEQ ID NO: 18) using pC3E-7034 containing the nucleotide sequence of C3E-7034 shown in FIG. 34 (SEQ ID NO: 18) in the ORF as a template.
- C3E-7078 in which site-directed mutagenesis was performed using PCR, and the asparagine at amino acid number 53 in the heavy chain variable region of C3E-7034 was replaced with arginine.
- a C3E-7078 expression vector containing the nucleotide sequence of The obtained expression vector was designated as “pC3E-7078”.
- site-directed mutagenesis was carried out using PCR using pC3E-7034 as a template, and FIGS. 95 and 96 (SEQ ID NOs: 87 and 88) as primers, and the heavy chain variable region of C3E-7034 had the 53rd amino acid number.
- An C3E-7079 expression vector containing the nucleotide sequence of C3E-7079 in which asparagine was replaced with serine was prepared in the ORF. The obtained expression vector was designated as “pC3E-7079”.
- site-directed mutagenesis was carried out using PCR using pC3E-7036 as a template, and FIGS.
- a C3E-7085 expression vector containing the nucleotide sequence of C3E-7085 with asparagine replaced with arginine in the ORF was constructed. The obtained expression vector was designated as “pC3E-7085”.
- C3E-7086 in which the aspartic acid at amino acid number 52 in the light chain variable region of C3E-7078 was replaced with glycine, C3E-7087 replaced with glutamine, C3E-7088 replaced with asparagine, C3E-7089 replaced with serine, alanine
- An expression vector containing each nucleotide sequence of C3E-7093 replaced with asparagine, C3E-7094 replaced with serine, and C3E-7095 replaced with alanine in the ORF was also prepared in the same manner.
- a list of the vector names, templates and primers prepared is shown in Table 1, and a primer list is shown in FIG.
- Example 5 Crystal structure analysis of humanized anti-CD3 scFv (C3E-7034) 5) -1 Preparation of humanized anti-CD3 scFv (C3E-7034) -human CD3 ⁇ single chain antigen complex
- Example 2) -2 CD3 ⁇ prepared in -1 and C3E-7034 prepared in Example 4) -3-1 were mixed at a molar ratio of 1: 2, and 10 mM Tris HCl (pH 7.5), 50 mM in Amicon Ultra 15 MWCO 10 kg (Millipore). The buffer was replaced with NaCl and concentrated to 3.5 mg / mL. This was purified by gel filtration chromatography using Superdex 200 10 / 300GL (GE Healthcare), and the fraction of the complex was concentrated to about 4.0 mg / mL using Amicon Ultra 15 MWCO 10 kg (Millipore).
- a molecular replacement method was performed to determine the phase.
- a software phaser (CCP4: Collaborative Computational Project No. 4) was used for the calculation.
- the crystal contained one complex in the asymmetric unit.
- the structure was refined using software Refmac5 (CCP4: Collaborative Computational Project No. 4), and the model was corrected using software coot. This operation was repeated to obtain a final R value of 22.1% and a free R value of 27.0% with a 3.3 resolution.
- the final model is amino acid residue 1-108 of the light chain region of C3E-7034 (FIG.
- SEQ ID NO: 17 amino acid residue 1-118 of the heavy chain region of C3E-7034 (FIG. 22, SEQ ID NO: 16), It includes amino acid residues 33-67 and 71-118 of the CD3 ⁇ region (FIG. 1, SEQ ID NO: 1), and amino acid residues 23-103 of the CD3 ⁇ region (FIG. 37, SEQ ID NO: 3). Amino acid residues 68-70 of the CD3 ⁇ region (FIG. 1, SEQ ID NO: 1), and amino terminal region (amino acid residue 1) of C3E-7034 (FIG.
- FIG. 39 shows the ribbon model and the surface of the entire composite.
- FIG. 40 shows the interaction between CD3 ⁇ and the light and heavy chains of C3E-7034.
- Panel A is a diagram in which amino acid residues of CD3 ⁇ within a distance of 4 km or less from the light chain variable region of C3E-7034 are displayed with a thick stick model, and other amino acid residues are displayed with a thin stick model.
- Ser55, Glu56, Arg101, Gly102, Ser103, Lys104, and Pro105 labeled with the residue name and residue number in the square are amino acids of CD3 ⁇ within a distance of 4 mm from the light chain variable region of C3E-7034 Each amino acid number corresponds to SEQ ID NO: 1 in the Sequence Listing.
- Panel B is a diagram in which amino acid residues of CD3 ⁇ within a distance of 4 km or less from the heavy chain variable region of C3E-7034 are displayed with a thick stick model, and other amino acids are displayed with a thin stick model.
- Ser55, Glu56, Leu58, Trp59, Asn65, Ile66, Ser77, Asp78, and Arg101 labeled with the residue name and residue number in the square are CD3 ⁇ amino acids, and each amino acid number is a sequence in the sequence listing. Corresponds to number 1. Amino acid residues that were within a distance of 4 km from C3E-7034 and were interpreted as epitopes of CD3 ⁇ for C3E-7034 were: Ser55, Glu56, Leu58, Trp59, Asn65, Ile66, Ser77, Asp78, Arg101 , Gly102, Ser103, Lys104, and Pro105.
- CD3 ⁇ epitopes for C3E-7034 Arg101, Gly102, Ser103, Lys104, and Pro105 are also common epitope residues of CD3 ⁇ for OKT3 and UCHT1 (Kjer-Nielsen et al., PNAS 101 (2004), p. 7675-80; Arnett et al., PNAS 101 (2004), p. 16268-73). It was also found that C3E-7034 does not interact with amino acid numbers 22 to 48 in SEQ ID NO: 1 corresponding to the epitope of anti-CD3 antibody including I2C and H2C described in WO2008 / 119565A2.
- FIG. 41 shows interacting residues on the CD3 ⁇ sequence. The signal sequence for CD3 ⁇ is shown in italics, and amino acids within 4 km of C3E-7034 are underlined.
- Example 6 Production of Humanized OKT3 scFv 6) -1 Construction of OKT3 scFv expression vector pC3E-3000
- Example 4 Mouse anti-CD3 monoclonal antibody OKT3 (Sgro, Toxicology 105 (1995), 23-29, Orthoclone, Janssen-Cilag) in the same manner as in 1-1. ScFv was prepared and introduced into a pcDNA3.3-derived animal cell expression vector. The obtained expression vector was designated as “pC3E-3000”.
- the amino acid number 5 glutamine of the variable region is valine
- the amino acid number 11 leucine is serine
- the amino acid number 12 alanine is lysine
- the amino acid The 13th arginine is lysine
- the 20th amino acid methionine is valine
- the 38th amino acid lysine is arginine
- the 40th arginine amino acid is alanine
- the 48th amino acid isoleucine is methionine
- the amino acid The lysine at number 67 is arginine
- alanine at amino acid number 68 is valine
- leucine at amino acid number 70 is isoleucine
- threonine at amino acid number 72 is alanine
- serine at amino acid number 76 is threonine
- amino acid number 3 valine is glutamine, amino acid number 4 leucine is methionine, amino acid number 9 alanine is serine, amino acid number
- the 10th isoleucine is serine, the 11th amino acid methionine is leucine, the 12th amino acid serine is alanine, the 13th alanine amino acid is valine, the 15th amino acid proline is leucine, the amino acid number
- the 18th lysine is arginine, the amino acid number 19 valine is alanine, the amino acid number 21 methionine is isoleucine, the amino acid number 39 serine is proline, the amino acid number 41 threonine is lysine, the amino acid number 42nd serine
- the amino acid number 59 alanine is aspartic acid
- the amino acid number 60 histidine is arginine
- the amino acid number 62 arginine is serine
- the amino acid number 59 alanine is aspartic acid
- the amino acid sequence of the C3E-3007 light chain, which was designed to replace valine, serine at amino acid number 99 with glutamine, and leucine at amino acid number 103 with valine, is shown in FIG. 45 (SEQ ID NO: 39). ing.
- C3E-3007 Expression and purification of humanized OKT3 scFv (C3E-3007) Expression and purification of C3E-3007 were performed in the same manner as in Example 4) -1-2.
- the amino acid sequence of C3E-3007 is set forth in FIG. 47 (SEQ ID NO: 35).
- Example 7 In vitro activity of humanized anti-CD3 scFv 7) -1 Binding studies of humanized anti-CD3 scFv (C3E-3007, C3E-7034, C3E-7035, C3E-7036) with human CD3 7) -1-1 Examination of binding of humanized anti-CD3 scFv (C3E-3007, C3E-7034, C3E-7035, C3E-7036) to human CD3 by flow cytometry Commercial human PBMC (CTL) containing 5% FBS An appropriate concentration was prepared with PBS, LIVE / DEAD Fixable Near-IR Dead Cell Stain Kit (Thermo Fisher Scientific) and anti-CD19 antibody (Beckman Coulter) were added, and the mixture was allowed to stand at 4 ° C.
- Example 7 7) -2-2 Examination of binding of humanized anti-CD3 scFv (C3E-3007, C3E-7034, C3E-7035, C3E-7036) to cynomolgus CD3 by flow cytometry Obtained in Example 7) -2-1
- the prepared cynomolgus monkey PBMC was adjusted to an appropriate concentration with PBS containing 5% FBS, and stained and analyzed in the same manner as in Example 7) -1-1.
- humanized anti-CD3 scFv C3E-7034, C3E-7035, C3E-7036
- Human peripheral blood mononuclear cells were obtained by concentration gradient density separation using Lymphocyte-H (Cedarlane). Isolated from a random donor's fresh buffy coat. Humanized anti-CD3 scFv (C3E-3007, C3E-7034) diluted to 100 nM with LR10 (RPMI 1640 containing 10% ultra low IgG FBS (Thermo Fisher Scientific)) and Anti-His antibody (Q3E) at the same concentration The same amount was mixed.
- LR10 RPMI 1640 containing 10% ultra low IgG FBS (Thermo Fisher Scientific)
- Q3E Anti-His antibody
- Human or monkey PBMCs were prepared to 2 ⁇ 10 5 cells with LR10, and mixed in 96-well U-bottom microplate with the same amount of humanized anti-CD3 scFv mixed with Anti-His antibody, and incubated at 37 ° C. for 24 hours. The cells were cultured under 5% CO 2 conditions.
- Example 8 Production of Humanized Anti-TROP2 scFv 8) -1 Construction of HT1-11 scFv Expression Vector pHT1-11scFv
- a DNA fragment containing the DNA sequence encoding the amino acid of HT1-11 scFv shown in FIG. 51 (SEQ ID NO: 41) was synthesized (GENEART).
- the nucleotide sequence shown in FIG. 52 (SEQ ID NO: 40) is inserted by inserting the synthesized DNA fragment into a vector derived from pcDNA-3.3 TOPO (Thermo Scientific) using an In-Fusion HD PCR cloning kit (Clontech).
- a humanized anti-TROP2 scFv expression vector pHT1-11 scFv included in the ORF was constructed.
- Example 9 Evaluation of binding of humanized anti-TROP2 scFv (HT1-11 scFv) to human TROP2 by flow cytometry
- the concentration is adjusted to 1 ⁇ 10 6 cells / mL with 5% FBS-containing PBS, seeded on a 96-well U-bottom microplate at 100 ⁇ L / well, and the supernatant after centrifugation is collected. Removed. 100 ⁇ L / well of humanized anti-TROP2 scFv (HT1-11 scFv) diluted with PBS containing 5% FBS was added, and the mixture was allowed to stand at 4 ° C. for 60 minutes.
- humanized anti-TROP2 scFv diluted with PBS containing 5% FBS was added, and the mixture was allowed to stand at 4 ° C. for 60 minutes.
- Example 10 Preparation of anti-TROP2-CD3 bispecific molecule 10) -1 Construction of anti-TROP2-CD3 bispecific molecule expression vector 10) -1-1 Construction of HT1-11 scFv / C3E-7034 bispecific molecule (T2C-0001) expression vector Example 8) -1 Using the pHT1-11 scFv prepared in step 1 as a template, PCR was performed using a primer designed to add a linker connecting HT1-11 scFv and a part of the human antibody heavy chain signal sequence on the 5 ′ side and scFv on the 3 ′ side. An insert DNA fragment was obtained.
- PCR was performed using the expression vector pC3E-7034 prepared in 4) -3-1 as a template and a primer encoding the amino terminal sequence of the signal sequence and anti-CD3 scFv, and the entire vector region containing anti-CD3 scFv was obtained.
- a vector DNA fragment containing was obtained.
- Each DNA fragment was ligated using an In-Fusion HD cloning kit (CLONTECH), and an anti-TROP2-CD3 bispecific molecule expression vector pT2C-0001 containing the nucleotide sequence of FIG. 54 (SEQ ID NO: 42) in the ORF was obtained.
- T2C-0001 is set forth in FIG. 58 (SEQ ID NO: 43).
- the amino acid sequence of T2C-0003 is set forth in FIG. 59 (SEQ ID NO: 45).
- the amino acid sequence of T2C-0005 is set forth in FIG. 60 (SEQ ID NO: 47).
- the amino acid sequence of T2C-0006 is set forth in FIG. 61 (SEQ ID NO: 49).
- Example 11 Evaluation of in vitro activity of anti-TROP2-CD3 bispecific molecule 11) -1 Evaluation of binding activity by SPR 11) -1-1
- Binding of anti-TROP2-CD3 bispecific molecule to TROP2 BIOcore 3000 was used to bind the bispecific molecule to TROP2 using an anti-human IgG antibody.
- the captured antigen was measured by a capture method in which a bispecific molecule was measured as an analyte.
- a recombinant human TROP-2 / human IgG Fc fusion R & D SYSTEMS
- An anti-human IgG (Fc) antibody (Human Antibody Capture Kit, GE Healthcare Bioscience) was covalently bound to a sensor chip CM5 (GE Healthcare Bioscience) by an amine coupling method at about 2000 RU.
- the reference cell was similarly fixed.
- HBS-P (10 mM HEPES pH 7.4, 0.15 M NaCl, 0.005% Surfactant P20) was used as a running buffer.
- Bispecific molecules were prepared from 200 nM to 1 nM at 2-fold dilution.
- each concentration of bispecific molecule is added at a flow rate of 30 ⁇ l / min for 300 seconds, followed by 600 seconds.
- the dissociation phase of was monitored.
- 3M magnesium chloride solution was added for 30 seconds.
- Bispecific molecules were prepared from a maximum concentration of 1 ⁇ M to 2 nM at 2-fold dilution, or from 200 nM to 1 nM at 2-fold dilution.
- each concentration of the bispecific molecule was added at a flow rate of 10 ⁇ l / min for 25 minutes, and the amount of binding was monitored.
- 10 mM glycine hydrochloric acid solution pH 1.5 was added for 30 seconds.
- the dissociation constant KD was calculated from the amount of binding at each concentration using analysis software (BIAevaluation software, version 4.1.1). The results are shown in Tables 2 and 3.
- the concentration is adjusted to 2 ⁇ 10 6 cells / mL with 5% FBS-containing PBS, seeded on a 96-well U-bottom microplate at 100 ⁇ L / well, and the supernatant after centrifugation is collected. Removed.
- Anti-TROP2 Alexa Fluor 488 antibody (eBioscience) and Isotype Control antibody (eBioscience) diluted with 5% FBS-containing PBS were added at 25 ⁇ L / well and allowed to stand at 4 ° C. for 30 minutes.
- Target cell preparation FaDu, HPAF-II, and Calu-6 were prepared at a concentration of 2 ⁇ 10 6 cells / mL in RPMI 1640 medium (Thermo Fisher Scientific) containing 10% FBS, and the cell suspension was prepared. 100 ⁇ L of Chromium-51 Radionuclide (PerkinElmer) was added per 1 mL of the solution, and the cells were cultured at 37 ° C. under 5% CO 2 for 2 hours. After washing twice with 10% FBS-containing RPMI 1640 medium and resuspending in 10% FBS-containing RPMI 1640 medium to 2 ⁇ 10 5 cells / mL, the target cells were used.
- FIG. 66 shows the cytotoxic activity of various TROP2-CD3 bispecific molecules against FaDu and HPAF-II). On the other hand, cytotoxic activity against Calu-6 was not observed.
- Example 12 Preparation of anti-Axl-CD3 bispecific molecule 12) -1 Construction of anti-Axl-CD3 bispecific molecule expression vector 12) -1-1 11D5-T3scFv / C3E-7034 bispecific Construction of molecular (AXC-0001) expression vector h # 11D5-T3H (described in FIG. 12 of European Patent Application No. 2270053) with glycine added to the N-terminus thereof, and h # 11D5-T3L (European patent) Designed the amino acid sequence of an anti-Axl single-chain antibody, 11D5-T3scFv, in which the sequence of Application No. 2270053 (described in FIG.
- the entire region of the vector containing anti-CD3 scFv using a primer comprising a nucleotide sequence encoding a human antibody heavy chain signal sequence and anti-CD3 scFv Amplification was performed by PCR to obtain a vector DNA fragment.
- Each DNA fragment was ligated using an In-Fusion HD cloning kit (CLONTECH), and the anti-Axl-CD3 bispecific molecule expression vector pAXC-0001 containing the nucleotide sequence shown in FIG. 97 (SEQ ID NO: 89) in the ORF. was made.
- AXC-0001 and AXC-0002 were carried out in the same manner as in Example 4) -1-2.
- the amino acid sequence of AXC-0001 is set forth in FIG. 98 (SEQ ID NO: 90).
- the amino acid sequence of AXC-0002 is set forth in FIG. 100 (SEQ ID NO: 92).
- Human lung cancer cell line A549 (ATCC), human pancreatic cancer cell line PANC-1 (ATCC) , MIA PaCa-2 (ATCC), human myeloma cell line U266B1 (ATCC), mantle cell lymphoma cell line Jeko-1 (ATCC) were prepared in 5% FBS-containing PBS to an appropriate concentration, and LIVE / DEAD Fixable Dead Cell Stain Kit (Thermo Fisher Scientific) was added and allowed to stand at 4 ° C. for 30 minutes.
- the concentration is adjusted to 2 ⁇ 10 6 cells / mL with 5% FBS-containing PBS, seeded on a 96-well U-bottom microplate at 100 ⁇ L / well, and the supernatant after centrifugation is collected. Removed.
- Anti-Axl antibody (RD-SYSTEMS) and Isotype Control antibody (RD-SYSTEMS) diluted with 5% FBS-containing PBS were added at 25 ⁇ L / well and allowed to stand at 4 ° C. for 30 minutes.
- target cells A549, PANC-1, MIA PaCa-2, U266B1, and Jeko-1 were prepared at a concentration of 2 ⁇ 10 6 cells / mL in RPMI1640 medium (Thermo Fisher Scientific) containing 10% FBS. Then, 100 ⁇ L of Chromium-51 Radiochloride (PerkinElmer) per 1 mL of the cell suspension was added, and the cells were cultured at 37 ° C. under 5% CO 2 for 2 hours. After washing twice with 10% FBS-containing RPMI 1640 medium and resuspending in 10% FBS-containing RPMI 1640 medium to 2 ⁇ 10 5 cells / mL, the target cells were used.
- RPMI1640 medium Thermo Fisher Scientific
- Cell lysis rate (%) (AB) / (CB) ⁇ 100
- A Sample well count.
- FIG. 108 As shown in FIG. 108 (A, B, C, D, E), the cytotoxic activity of various anti-Axl-CD3 bispecific molecules against A549, PANC-1, and MIA PaCa-2 was shown. On the other hand, cytotoxic activity against U266B1 and Jeko-1 was not observed.
- Example 14 Preparation of anti-HLA-A2 / MAGEC1-CD3 bispecific molecule expression vector 14) -1 Construction of anti-HLA-A2 / MAGEC1-CD3 bispecific molecule expression vector 14) -1-1 MAG -032scFv / C3E-7034 Bispecific Molecule (MGC-0001) Expression Vector Construction MAG-032 scFv binding specifically to HLA-A2 / MAGEC1 was obtained from a human antibody phage library.
- MAG-032 scFv amino acid sequence As a template, 5'-side part of human antibody heavy chain signal sequence, 3'-side linker connecting scFv, and part of C3E-7034 An insert DNA fragment containing a nucleotide sequence encoding the amino acid sequence of MAG-032 scFv was obtained by PCR using a primer designed to add a nucleotide sequence.
- a human antibody heavy chain signal sequence and a primer comprising a nucleotide sequence encoding the amino terminal sequence of anti-CD3 scFv are used to contain anti-CD3 scFv
- the entire vector region was amplified by the PCR method to obtain a vector DNA fragment.
- Each DNA fragment was ligated using an In-Fusion HD cloning kit (CLONTECH), and anti-HLA-A2 / MAGEC1-CD3 bispecific molecule expression containing the nucleotide sequence shown in FIG. 101 (SEQ ID NO: 93) in the ORF Vector pMGC-0001 was made.
- MGC-0001 and MGC-0002 were expressed and purified in the same manner as in Example 4) -1-2.
- the amino acid sequence of MGC-0001 is set forth in FIG. 102 (SEQ ID NO: 94).
- the amino acid sequence of MGC-0002 is set forth in FIG. 104 (SEQ ID NO: 96).
- Example 15 Evaluation of in vitro activity of anti-HLA-A2 / MAGEC1-CD3 bispecific molecule 15) -1 Expression analysis of HLA-A2 / MAGEC1 in target cells Human lymphoblast cell fusion cell line T2 (ATCC) cells was adjusted to an appropriate concentration with 20% FBS-containing AIM-V medium (Thermo Fisher Scientific), FIG. 106 (SEQ ID NO: 97) MAGEC1 peptide (Sigma Genosys) or DMSO was added, and incubated at 37 ° C. for 4 hours. did.
- Anti-HLA-A2 / MAGEC1 antibody (MAG032 scFv) diluted with 5% FBS-containing PBS was added at 25 ⁇ L / well, and the mixture was allowed to stand at 4 ° C. for 30 minutes. After washing twice with PBS containing 5% FBS, 25 ⁇ L / well of Penta-His Alexa Fluor 488 (QIAGEN) diluted with PBS containing 5% FBS was added, and the mixture was allowed to stand at 4 ° C. for 30 minutes. After washing twice with 5% FBS-containing PBS and resuspending in 5% FBS-containing PBS, detection was performed with a flow cytometer (Cytomics FC500, Beckman Coulter).
- T2 cells were adjusted to an appropriate concentration with 20% FBS-containing AIM-V medium (Thermo Fisher Scientific), added with MAGEC1 peptide or DMSO, and incubated at 37 ° C for 4 hours.
- FBS-containing AIM-V medium Thermo Fisher Scientific
- MAGEC1 peptide or DMSO MAGEC1 peptide or DMSO
- RPMI1640 medium containing 10% FBS Thermo Fisher Scientific
- Chromium-51 Radioionide PerkinElmer
- Cytotoxic assay T2 cells obtained in Example 15) -2 were added to a 96-well U-bottom microplate at 50 ⁇ L / well.
- Various anti-HLA-A2 / MAGEC1-CD3 bispecific molecules prepared at various concentrations were added thereto at 50 ⁇ L / well, effector cells prepared in 15) -3 were added at 100 ⁇ L / well, and 1000 rpm ⁇ 1 at room temperature. After centrifuging for 5 minutes, the cells were cultured at 37 ° C. under 5% CO 2 for 20-24 hours. 50 ⁇ L of the supernatant was collected on LumaPlate (PerkinElmer), dried at 50 ° C.
- Cell lysis rate (%) (AB) / (CB) ⁇ 100
- A Sample well count.
- FIG. 110 As shown in FIG. 110 (A, B), the cytotoxic activity of various anti-HLA-A2 / MAGEC1-CD3 bispecific molecules against T2 cells added with MAGEC1 peptide was shown. On the other hand, cytotoxic activity against T2 cells added with DMSO was not observed.
- humanized anti-CD3 antibody of the present invention binds to human CD3 as well as to cynomolgus monkey CD3, it can be suitably used for non-clinical studies for drug development.
- SEQ ID NO: 1 amino acid sequence of human CD3 ⁇
- SEQ ID NO: 2 amino acid sequence of human CD3 ⁇
- SEQ ID NO: 3 amino acid sequence of human CD3 ⁇
- SEQ ID NO: 4 nucleotide sequence encoding human CD3 ⁇ single-chain antigen
- SEQ ID NO: 5 amino acid sequence of His-scCD3 antigen
- SEQ ID NO: 6 nucleotide sequence encoding heavy chain variable region of C3-147
- SEQ ID NO: 8 (C3-147_VL DNA): Nucleotide sequence encoding the light chain variable region of C3-147 SEQ ID NO: 9: (C3-147_VL AA): Amino acid sequence of the light chain variable region of C3-147 SEQ ID NO: 10: (G4S linker sense): SEQ ID NO: 11: (G4S linker anti
- C3E-7036_VL_AA Amino acid sequence of the light chain variable region of C3E-7036
Abstract
Description
2)OKT3はマウス由来の抗ヒトCD3モノクローナル抗体である(非特許文献1)。臓器の同種移植片拒絶を防ぐために、抗CD3モノクローナル抗体を投与し、抗体がヒトT細胞上のTCR複合体に結合しT細胞の活性化と増殖を抑えることで、移植臓器が拒絶されるのを防ぐ処置法が長い間用いられてきた(非特許文献2-4)。OKT3はそのような処置に用いられた最初の抗CD3抗体である。OKT3は強い免疫抑制効果を有する一方、その免疫原性及び分裂促進の潜在能力に関連する重篤な副作用が原因で、その臨床上の使用が阻まれている(非特許文献5-8)。
3)OKT3はインビトロにおいてT細胞増殖及びサイトカイン産生を誘導し、インビボにおいて大量のサイトカインを放出し、サイトカインシンドロームを引き起こした(非特許文献5)。これはOKT3が2価のIgGであるため、T細胞とFcγ受容体発現細胞へのクロスリンクが生じ、結果としてT細胞の増殖が引き起こされることによる(非特許文献8)。またOKT3はマウス抗体であるため、長期投与によりヒト抗マウス抗体(HAMA)のような異好性抗体が生じることが知られている(非特許文献7)。抗CD3抗体の治療への応用と副作用の報告は以下にまとめられている(特許文献1)。
4)このような問題を解決するため、OKT3のscFv(非特許文献9)、あるいはヒト化OKT3(非特許文献10)等が作製されている。またOKT3の更なる応用例として、T細胞活性化能を利用したOKT3の単鎖とがん細胞表面で発現する標的抗原に対する抗体の単鎖を結合させた二重特異性抗体が報告されている(特許文献2、非特許文献11)。
5)当技術分野で説明される抗CD3抗体を用いた多重特異性抗体は、悪性疾患の治療に対して大きな治療的可能性を有することが期待される。例えば、TROP2は種々の上皮細胞癌種において過剰発現していることが知られている(非特許文献12-16)。ヒトTROP2特異的な抗体の抗原結合性断片と抗CD3抗体の抗原結合性断片を遺伝子上でつなげて発現させた二重特異性抗体は、これまで報告されていない。
6)OKT3は、チンパンジーのCD3とは反応するが、カニクイザルなど他の霊長動物由来のCD3とは反応しない(非特許文献17)。抗CD3モノクローナル抗体であるUCHT-1もまた、チンパンジーに由来するCD3とは反応するがカニクイザル由来CD3とは反応しない(非特許文献18)。他方、カニクイザル抗原は認識するが、それらのヒト対応物は認識しないモノクローナル抗体の例も見られる。この群の一例は、カニクイザルに由来するCD3を指向するモノクローナル抗体であるFN-18である(非特許文献19)。
7)OKT3及びOKT3を改変した一連の抗体の限界は、ヒトCD3のみに特異的に結合することである。この限界はヒトの疾患を治療する治療薬剤として開発するにあたり、重大な障害となり得る。なぜならば、開発候補薬品は市場の承認を得るために、前臨床試験を実施する必要があり、前臨床試験では動物、特に高等霊長類であるカニクイザルを用いて行うことが望まれているからである。したがって、抗CD3抗体を用いた候補薬品の場合、ヒトとカニクイザル両方のCD3に結合可能な抗CD3抗体を用いることが非常に望ましい。
8)ヒトとカニクイザル両方と結合する抗体は、特許文献3、4、及び非特許文献20で報告されている。またこのような抗CD3抗体の単鎖と、がん細胞表面で発現する標的抗原に対する抗体の単鎖を結合させた二重特異性抗体が報告されている(特許文献5、非特許文献21)。しかし、多様性に富む癌標的に適応可能な多重特異性抗体や多重特異性分子を利用可能とするために、上述のものとは異なるエピトープと結合し、かつヒトとカニクイザル両方に結合する抗CD3抗体が望まれている。
(1)重鎖配列が
配列番号26に示されるアミノ酸配列を含むCDRH1、
配列番号98に示されるアミノ酸配列を含むCDRH2、及び、
配列番号28に示されるアミノ酸配列を含むCDRH3;
軽鎖配列が
配列番号29に示されるアミノ酸配列を含むCDRL1、
配列番号99に示されるアミノ酸配列を含むCDRL2、及び、
配列番号31に示されるアミノ酸配列を含むCDRL3
をそれぞれ含み;且つ、
ヒトCD3及びカニクイザルCD3に結合することを特徴とする抗体又は該抗体の抗原結合性断片。
(2)前記CDRH2の
1番目のXaaは、(A、E、G、H、I、L、T、V、R、S)からなる群より選択され
且つ2番目のXaaはSであるか、又は、
1番目のXaaはNであり、
且つ2番目のXaaは、(E、R、F、Y、L、V、I、K、T)からなる群より選択され、
前記CDRL2の
Xaaは、(Q、A、G、S、N、D)からなる群より選択され、
ヒトCD3及びカニクイザルCD3に結合することを特徴とする前記(1)に記載の抗体又は該抗体の抗原結合性断片。
(3)前記CDRH2の
1番目のXaaは、(R、S)からなる群より選択され、2番目のXaaはSであり、且つ
前記CDRL2の
Xaaは、(Q、A、G、S、N、D)からなる群より選択される、
ヒトCD3及びカニクイザルCD3に結合することを特徴とする前記(1)又は(2)に記載の抗体又は該抗体の抗原結合性断片。
(4)重鎖配列がCDRH1、CDRH2、及び、CDRH3を有する可変領域を含み、
前記CDRH1は配列番号26に示されるアミノ酸配列からなり、
前記CDRH2は配列番号27に示されるアミノ酸配列からなり、
前記CDRH3は配列番号28に示されるアミノ酸配列からなること;並びに
軽鎖配列がCDRL1、CDRL2、CDRL3を有する可変領域を含み、
前記CDRL1は配列番号29に示されるアミノ酸配列からなり、
前記CDRL2は配列番号30に示されるアミノ酸配列からなり、
前記CDRL3は配列番号31に示されるアミノ酸配列からなること;並びに、
ヒトCD3及びカニクイザルCD3に結合することを特徴とする、前記(1)に記載の抗体又は該抗体の抗原結合性断片。
(5) 重鎖可変領域配列が、配列番号100に示されるアミノ酸配列を含むことを特徴とする前記(1)乃至(4)のいずれか1つに記載の抗体又は該抗体の抗原結合性断片。
(6) 配列番号100に示されるアミノ酸配列の
1番目のXaaは、(A、E、G、H、I、L、T、V、R、S)からなる群より選択され
且つ2番目のXaaはSであるか、又は、
1番目のXaaはNであり
且つ2番目のXaaは、(E、R、F、Y、L、V、I、K、T)からなる群より選択される、
前記(5)に記載の抗体又はその抗原結合性断片。
(7) 配列番号100に示されるアミノ酸配列の
1番目のXaaは、(R、S)からなる群より選択され、且つ2番目のXaaはSである、
前記(5)に記載の抗体又はその抗原結合性断片。
(8) 軽鎖可変領域が、配列番号101、102、及び、103のいずれか1つに示されるアミノ酸配列を含むことを特徴とする、前記(1)乃至(7)のいずれか1つに記載の抗体又はその抗原結合性断片。
(9) 配列番号101、102、及び、103のいずれか1つに示されるアミノ酸配列の Xaaは、(Q、A、G、S、N、D)からなる群より選択される、前記(8)に記載の抗体又その抗原結合性断片。
(10) 重鎖可変領域配列が、配列番号16に示されるアミノ酸配列を含むことを特徴とする前記(5)に記載の抗体又は該抗体の抗原結合性断片。
(11) 軽鎖可変領域配列が、配列番号17、20、及び、23のいずれか1つに示されるアミノ酸配列を含むことを特徴とする前記(8)に記載の抗体又は該抗体の抗原結合性断片。
(12) 配列番号100に示されるアミノ酸配列を含む重鎖可変領域と、配列番号101、102、及び103のいずれか1つに示されるアミノ酸配列を含む軽鎖可変領域を含み、
配列番号100で示されるアミノ酸配列の1番目のXaaは、(A、E、G、H、I、L、T、V、R、S)からなる群より選択され、且つ、2番目のXaaはSであるか、又は、
1番目のXaaはNであり、且つ、2番目のXaaは、(E、R、F、Y、L、V、I、K、T)からなる群より選択され、
配列番号101、102、及び、103のいずれか1つに示されるアミノ酸配列の
Xaaは、(Q、A、G、S、N、D)からなる群より選択される、前記(1)又は(2)に記載の抗体又はその抗原結合性断片。
(13) 配列番号100の
1番目のXaaは、(R、S)からなる群より選択され、
2番目のXaaはSであり、且つ、
配列番号101、102、及び、103のいずれか1つに示されるアミノ酸配列の
Xaaは、(Q、A、G、S、N、D)からなる群より選択される、前記(12)に記載の抗体又はその抗原結合性断片。
(14) 前記(13)に記載の
配列番号60の2乃至119のアミノ酸残基を含む重鎖可変領域と、配列番号60の135乃至243のアミノ酸残基を含む軽鎖可変領域を含む抗体又は該抗体の抗原結合性断片、
配列番号64の2乃至119のアミノ酸残基を含む重鎖可変領域と、配列番号64の135乃至241のアミノ酸残基を含む軽鎖可変領域を含む抗体又は該抗体の抗原結合性断片、
配列番号66の2乃至119のアミノ酸残基を含む重鎖可変領域と、配列番号66の135乃至243のアミノ酸残基を含む軽鎖可変領域を含む抗体又は該抗体の抗原結合性断片、
配列番号68の2乃至119のアミノ酸残基を含む重鎖可変領域と、配列番号68の135乃至243のアミノ酸残基を含む軽鎖可変領域を含む抗体又は該抗体の抗原結合性断片、
配列番号70の2乃至119のアミノ酸残基を含む重鎖可変領域と、配列番号70の135乃至243のアミノ酸残基を含む軽鎖可変領域を含む抗体又は該抗体の抗原結合性断片、
配列番号72の2乃至119のアミノ酸残基を含む重鎖可変領域と、配列番号72の135乃至243のアミノ酸残基を含む軽鎖可変領域を含む抗体又は該抗体の抗原結合性断片、
配列番号74の2乃至119のアミノ酸残基を含む重鎖可変領域と、配列番号74の135乃至243のアミノ酸残基を含む軽鎖可変領域を含む抗体又は該抗体の抗原結合性断片、
配列番号76の2乃至119のアミノ酸残基を含む重鎖可変領域と、配列番号76の135乃至243のアミノ酸残基を含む軽鎖可変領域を含む抗体又は該抗体の抗原結合性断片、
配列番号78の2乃至119のアミノ酸残基を含む重鎖可変領域と、配列番号78の135乃至243のアミノ酸残基を含む軽鎖可変領域を含む抗体又は該抗体の抗原結合性断片、
配列番号80の2乃至119のアミノ酸残基を含む重鎖可変領域と、配列番号80の135乃至243のアミノ酸残基を含む軽鎖可変領域を含む抗体又は該抗体の抗原結合性断片、
配列番号82の2乃至119のアミノ酸残基を含む重鎖可変領域と、配列番号82の135乃至243のアミノ酸残基を含む軽鎖可変領域を含む抗体又は該抗体の抗原結合性断片、
又は、
配列番号84の2乃至119のアミノ酸残基を含む重鎖可変領域と、配列番号84の135乃至243のアミノ酸残基を含む軽鎖可変領域を含む抗体又は該抗体の抗原結合性断片。
(15) 配列番号16に示されるアミノ酸配列を含む重鎖可変領域と、リンカーと、配列番号17、20、及び、23のいずれか1つに示されるアミノ酸配列を含む軽鎖可変領域とを含む前記(1)、(4)、(5)、(8)、(10)、又は(11)に記載の抗体又は該抗体の抗原結合性断片。
(16) 可変領域が、抗体のアミノ末端側から重鎖可変領域、軽鎖可変領域の順で結合しており、又は、軽鎖可変領域、重鎖可変領域の順で結合しており、任意で:i) 両可変領域の間にリンカーを有し;ii)アミノ末端側の可変領域のアミノ末端にグリシン残基を有し;iii)カルボキシル末端側の可変領域のカルボキシル末端に、リンカー、FLAGタグ、及び/又は、Hisタグが結合している:前記(1)乃至(15)のいずれか1つに記載の抗体又はその抗原結合性断片。
(17) 配列番号60の2乃至243番目のアミノ酸残基を含むアミノ酸配列、
配列番号64の2乃至241番目のアミノ酸残基を含むアミノ酸配列、
配列番号66の2乃至243番目のアミノ酸残基を含むアミノ酸配列、
配列番号68の2乃至243番目のアミノ酸残基を含むアミノ酸配列、
配列番号70の2乃至243番目のアミノ酸残基を含むアミノ酸配列、
配列番号72の2乃至243番目のアミノ酸残基を含むアミノ酸配列、
配列番号74の2乃至243番目のアミノ酸残基を含むアミノ酸配列、
配列番号76の2乃至243番目のアミノ酸残基を含むアミノ酸配列、
配列番号78の2乃至243番目のアミノ酸残基を含むアミノ酸配列、
配列番号80の2乃至243番目のアミノ酸残基を含むアミノ酸配列、
配列番号82の2乃至243番目のアミノ酸残基を含むアミノ酸配列、
又は、
配列番号84の2乃至243番目のアミノ酸残基を含むアミノ酸配列
を含む前記(16)に記載の抗原又は該抗体の抗原結合性断片。
(18) 配列番号19の2乃至269のアミノ酸残基を含むアミノ酸配列、
配列番号22の2乃至269のアミノ酸残基を含むアミノ酸配列、
配列番号25の2乃至267のアミノ酸残基を含むアミノ酸配列、
配列番号60の2乃至269のアミノ酸残基を含むアミノ酸配列、
配列番号64の2乃至267のアミノ酸残基を含むアミノ酸配列、
配列番号66の2乃至269のアミノ酸残基を含むアミノ酸配列、
配列番号68の2乃至269のアミノ酸残基を含むアミノ酸配列、
配列番号70の2乃至269のアミノ酸残基を含むアミノ酸配列、
配列番号72の2乃至269のアミノ酸残基を含むアミノ酸配列、
配列番号74の2乃至269のアミノ酸残基を含むアミノ酸配列、
配列番号76の2乃至269のアミノ酸残基を含むアミノ酸配列、
配列番号78の2乃至269のアミノ酸残基を含むアミノ酸配列、
配列番号80の2乃至269のアミノ酸残基を含むアミノ酸配列、
配列番号82の2乃至269のアミノ酸残基を含むアミノ酸配列、又は、
配列番号84の2乃至269のアミノ酸残基を含むアミノ酸配列
を含む前記(16)に記載の抗原又は該抗体の抗原結合性断片。
(20) 前記(14)乃至(18)のいずれか1つに記載の抗体又は該抗体の抗原結合性断片に含まれる重鎖のアミノ酸配列と90%以上同一なアミノ酸配列を含む重鎖、及び、前記(14)乃至(18)のいずれか1つに記載の抗体又は該抗体の抗原結合性断片に含まれる軽鎖のアミノ酸配列と70%以上同一なアミノ酸配列を含む軽鎖を含み、且つ、ヒトCD3及びカニクイザルCD3に結合する抗体又は該抗体の抗原結合性断片。
(21) 前記(14)乃至(18)のいずれか1つに記載の抗体又は該抗体の抗原結合性断片に含まれる重鎖が含むアミノ酸配列において1乃至数個のアミノ酸が置換、欠失又は付加されてなるアミノ酸配列を含む重鎖、並びに、前記(14)乃至(18)のいずれか1つに記載の抗体又は該抗体の抗原結合性断片に含まれる軽鎖が含むアミノ酸配列において1乃至数個のアミノ酸が置換、欠失又は付加されてなるアミノ酸配列を含む軽鎖を含み、且つ、ヒトCD3及びカニクイザルCD3に結合する抗体又は該抗体の抗原結合性断片。
(22) 前記(14)乃至(18)のいずれか1つに記載の抗体又は該抗体の抗原結合性断片が結合するヒトCD3上の同一の部位に結合し、且つ、カニクイザルCD3に結合する抗体又は該抗体の抗原結合性断片。
(23) 前記(14)乃至(18)のいずれか1つに記載の抗体又は該抗体の抗原結合性断片とヒトCD3上への結合において競合し、且つ、カニクイザルCD3に結合する抗体又は該抗体の抗原結合性断片。
(24) 前記抗体又は該抗体の抗原結合性断片が結合するヒトCD3上の部位が、配列番号1で示されるアミノ酸配列中の55番目のセリン(Ser)、56番目のグルタミン酸(Glu)、58番目のロイシン(Leu)、59番目のトリプトファン(Trp)、65番目のアスパラギン(Asn)、66番目のイソロイシン(Ile)、77番目のセリン(Ser)、78番目のアスパラギン酸(Asp)、101番目のアルギニン(Arg)、101番目のグリシン(Gly)、103番目のセリン(Ser)、104番目のリジン(Lys)、および105番目のプロリン(Pro)のうちの7アミノ酸以上から構成される、前記(22)に記載の抗体又は該抗体の抗原結合性断片。
(25) IgGである前記(1乃至17)、(19)乃至(24)のいずれか1つに記載の抗体又は該抗体の抗原結合性断片。
(26) Fab、F(ab)’、Fv、scFv及びsdAbからなる群から選択される前記(1)乃至(23)のいずれか1つに記載の抗体又は該抗体の抗原結合性断片。
(27) ヒト免疫グロブリン定常領域を含むヒト化抗体又はヒト抗体である、前記(1)乃至(17)、(19)乃至(25)のいずれか1つに記載の抗体又は該抗体の抗原結合性断片。
(28) 前記(1)乃至(27)のいずれか1つに記載の抗体又は該抗体の抗原結合性断片のアミノ酸配列をコードするヌクレオチド配列を含むポリヌクレオチド。
(29) 配列番号19の2乃至243番目のアミノ酸残基を含むアミノ酸配列、
配列番号22の2乃至243番目のアミノ酸残基を含むアミノ酸配列、
配列番号25の2乃至241番目のアミノ酸残基を含むアミノ酸配列、
配列番号60の2乃至243番目のアミノ酸残基を含むアミノ酸配列、
配列番号64の2乃至241番目のアミノ酸残基を含むアミノ酸配列、
配列番号66の2乃至243番目のアミノ酸残基を含むアミノ酸配列、
配列番号68の2乃至243番目のアミノ酸残基を含むアミノ酸配列、
配列番号70の2乃至243番目のアミノ酸残基を含むアミノ酸配列、
配列番号72の2乃至243番目のアミノ酸残基を含むアミノ酸配列、
配列番号74の2乃至243番目のアミノ酸残基を含むアミノ酸配列、
配列番号76の2乃至243番目のアミノ酸残基を含むアミノ酸配列、
配列番号78の2乃至243番目のアミノ酸残基を含むアミノ酸配列、
配列番号80の2乃至243番目のアミノ酸残基を含むアミノ酸配列、
配列番号82の2乃至243番目のアミノ酸残基を含むアミノ酸配列、
又は、
配列番号84の2乃至243番目のアミノ酸残基を含むアミノ酸配列
をコードするヌクレオチド配列を含む、前記(27)に記載のポリヌクレオチド。
(30) 前記(28)又は(29)のいずれか1つに記載のポリヌクレオチドを含むベクター。
(31) 前記(28)又は(29)のいずれか1つに記載のポリヌクレオチド又は前記(30)に記載のベクターを含むか、又は前記(1)乃至(27)のいずれか1つに記載の抗体又は該抗体の抗原結合性断片を産生する細胞。
(32) 前記(31)に記載の細胞を培養する工程、及び、該培養物からヒトCD3に結合する抗体又は該抗体の抗原結合性断片を回収する工程を含む、ヒトCD3及びカニクイザルCD3に結合する抗体又は該抗体の抗原結合性断片の製造方法。
(33) 前記(32)に記載の方法により得られる、ヒトCD3及びカニクイザルCD3に結合する抗体又は該抗体の抗原結合性断片。
(34) 前記(1)乃至(27)、(33)のいずれか1つに記載の抗体又は該抗体の抗原結合性断片を有効成分として含有する医薬組成物。
(35) 前記(1)乃至(27)、(33)のいずれか1つに記載の抗体又は該抗体の抗原結合性断片を含む抗原結合性を有する分子。
(36) 多重特異的である前記(35)に記載の分子。
(38) さらなる抗体の抗原結合性断片が、Fab、F(ab)’、Fv、scFv、又は、sdAbである、前記(37)に記載の分子。
(39) Fcを含む前記(38)に記載の分子。
(40) さらなる抗体が、ヒト免疫グロブリン定常領域を含むヒト化抗体又はヒト抗体である、前記(37)乃至(39)のいずれか1つに記載の分子。
(41) 該さらなる抗体又は該抗体の抗原結合性断片と、前記(1)乃至(27)、(33)のいずれか1つに記載の抗体又は該抗体の抗原結合性断片とが、リンカーにより結合してなる、あるいはリンカーなしで結合してなる前記(37)乃至(38)のいずれか1つに記載の分子。
(42) 該さらなる抗体又は該抗体の抗原結合性断片の有するアミノ酸配列のカルボキシル末端と、リンカーとが結合しており、さらに該リンカーの有するアミノ酸配列のカルボキシル末端と、前記(1)乃至(27)、(33)のいずれか1つに記載の抗体又は該抗体の抗原結合性断片とが結合してなる前記(41)に記載の分子。
(43) 該さらなる抗体又は該抗体の抗原結合性断片の有するアミノ酸配列のカルボキシル末端と、リンカーとが結合しており、さらに該リンカーの有するアミノ酸配列のカルボキシル末端と、
配列番号19の2乃至243番目のアミノ酸残基を含む抗体又は該抗体の抗原結合性断片、
配列番号22の2乃至243番目のアミノ酸残基を含む抗体又は該抗体の抗原結合性断片、
又は、
配列番号25の2乃至241番目のアミノ酸残基を含む抗体又は該抗体の抗原結合性断片が結合してなるアミノ酸配列を含む前記(42)に記載の分子。
(44) 前記(1)乃至(27)、(33)のいずれか1つに記載の抗体又は該抗体の抗原結合性断片において、可変領域が、抗体のアミノ末端側から重鎖可変領域、軽鎖可変領域の順で結合しており、あるいは、軽鎖可変領域、重鎖可変領域の順で結合しており、
任意で:i)両可変領域の間にリンカーを有し:ii)アミノ末端側の可変領域のアミノ末端にグリシン残基を有し;iii)カルボキシル末端側の可変領域のカルボキシル末端に、リンカー、FLAGタグ、及び/又は、Hisタグが結合している:前記(35)乃至(42)のいずれか1つに記載の分子。適用可能な形態には、Hybrid型、Dual型の二重特異的分子が含まれる。
(45) 該さらなる抗体又は該抗体の抗原結合性断片と、
配列番号19の2乃至243番目のアミノ酸残基を含む抗体又は該抗体の抗原結合性断片、
配列番号25の2乃至241番目のアミノ酸残基を含む抗体又は該抗体の抗原結合性断片。
配列番号60の2乃至243番目のアミノ酸残基を含む抗体又は該抗体の抗原結合性断片、配列番号64の2乃至241番目のアミノ酸残基を含む抗体又は該抗体の抗原結合性断片、配列番号66の2乃至243番目のアミノ酸残基を含む抗体又は該抗体の抗原結合性断片、
配列番号68の2乃至243番目のアミノ酸残基を含む抗体又は該抗体の抗原結合性断片、
配列番号70の2乃至243番目のアミノ酸残基を含む抗体又は該抗体の抗原結合性断片、
配列番号72の2乃至243番目のアミノ酸残基を含む抗体又は該抗体の抗原結合性断片、
配列番号74の2乃至243番目のアミノ酸残基を含む抗体又は該抗体の抗原結合性断片、
配列番号76の2乃至243番目のアミノ酸残基を含む抗体又は該抗体の抗原結合性断片、
配列番号78の2乃至243番目のアミノ酸残基を含む抗体又は該抗体の抗原結合性断片、
配列番号80の2乃至243番目のアミノ酸残基を含む抗体又は該抗体の抗原結合性断片、
配列番号82の2乃至243番目のアミノ酸残基を含む抗体又は該抗体の抗原結合性断片、
又は、
配列番号84の2乃至243番目のアミノ酸残基を含む抗体又は該抗体の抗原結合性断片
を含む前記(44)に記載の分子。適用可能な形態には、Hybrid型、Dual型の二重特異的分子が含まれる。
(46) 該さらなる抗体が抗癌標的抗体である前記(36)乃至(45)のいずれかに記載の分子。
(47) 二重特異的である前記(36)乃至(46)のいずれか1つに記載の分子。
(48) ポリペプチドである前記(35)乃至(47)のいずれか1つに記載の分子。
(49) 前記(48)に記載の分子の有するアミノ酸配列をコードするヌクレオチド配列を含むポリヌクレオチド。
(50) 前記(49)に記載のポリヌクレオチドを含むベクター。
(51) 前記(49)に記載のポリヌクレオチド若しくは前記(50に記載のベクター、又は、前記(48に記載の分子を産生する細胞。
(52) 前記(51)に記載の細胞を培養する工程、及び、該培養物からヒトCD3に結合する分子を回収する工程を含む、ヒトCD3及びカニクイザルCD3に結合する分子の製造方法。
(53) 前記(52)に記載の方法により得られる、ヒトCD3及びカニクイザルCD3に結合する分子。
(54) 前記(35)乃至(48)、(53)のいずれか1つに記載の分子を有効成分として含有する医薬組成物。
(55) 標的細胞へのT細胞リダイレクションによって、該標的細胞への細胞傷害を誘導することを特徴とする前記(54)に記載の医薬組成物。
本発明において、「遺伝子」とは、蛋白質のアミノ酸をコードする塩基配列が含まれるヌクレオチド又はその相補鎖を意味し、例えば、蛋白質のアミノ酸をコードする塩基配列が含まれるヌクレオチド又はその相補鎖であるポリヌクレオチド、オリゴヌクレオチド、DNA、mRNA、cDNA、cRNA等は「遺伝子」の意味に含まれる。かかる遺伝子は一本鎖、二本鎖又は三本鎖以上のヌクレオチドであり、DNA鎖とRNA鎖の会合体、一本のヌクレオチド鎖上にリボヌクレオチド(RNA)とデオキシリボヌクレオチド(DNA)が混在するもの及びそのようなヌクレオチド鎖を含む二本鎖又は三本鎖以上のヌクレオチドも「遺伝子」の意味に含まれる。本発明において塩基配列とヌクレオチド配列は同義である。
本発明の抗体の定常領域としては、とくに限定されるものではないが、ヒトの疾患を治療又は予防するための本発明の抗体としては、好適にはヒト抗体のものが使用される。ヒト抗体の重鎖定常領域としては、例えば、Cγ1、Cγ2、Cγ3、Cγ4、Cμ、Cδ、Cα1、Cα2、Cε等を挙げることができる。ヒト抗体の軽鎖定常領域としては、例えば、Cκ、Cλ等を挙げることができる。
本発明において「細胞傷害活性」とは上記細胞傷害を引き起こすことを意味する。
2.抗原蛋白質 CD3(CD3複合体)
本発明において、「CD3」は、CD3蛋白質と同じ意味で用いている。
3.抗CD3抗体
(3-1)抗体の分類
本発明の抗CD3抗体及び該抗体の抗原結合性断片(以下、本発明の抗体等とも記す)は、モノクローナル抗体及びポリクローナル抗体のいずれであってもよい。本発明のモノクローナル抗体としては、非ヒト動物由来の抗体(非ヒト動物抗体)、ヒト抗体、キメラ化抗体(「キメラ抗体」ともいう)、ヒト化抗体などを挙げることができる。
(3-2)抗CD3抗体の結合特異性
本発明の抗体等はCD3を認識する。すなわち、本発明の抗体等はCD3に結合する。本発明の抗体等は、好適には、ヒトCD3、サルCD3等に結合し、より好適には、ヒトCD3及びカニクイザルCD3に結合する。
Ser55、Glu56、Leu58、Trp59、Asn65、Ile66、Ser77、Asp78、Arg101、Gly102、Ser103、Lys104、及びPro105。
上述の通り、ヒトCD3及びカニクイザルCD3に結合する抗体等は、医薬品の非臨床開発(前臨床開発)に必須な霊長類、特にカニクイザルを用いた有効性や安全性に関する各種試験に供することができ好ましい。また、ヒトCD3及びカニクイザルCD3に結合する抗体等は、細胞傷害活性を有し、単独で又は本発明の分子として、ヒト及びカニクイザルにおける癌等の疾患の治療又は予防に有用である。医薬組成物については後述する。
(3-3)モノクローナル抗体
本発明はモノクローナル抗体を提供する。モノクローナル抗体には、ラット抗体、マウス抗体、ウサギ抗体、ニワトリ抗体、魚類抗体等の非ヒト動物由来のモノクローナル抗体、キメラ化抗体、ヒト化抗体、ヒト抗体、それらの抗原結合性断片、それらの抗体変異体、それらの修飾体等が含まれる。
本発明の抗体変異体には、好適には、蛋白質の分解もしくは酸化に対する感受性の低下、生物活性や機能の維持、改善もしくは低下や変化の抑制、抗原結合能の改善もしくは調節、又は理化学的性質もしくは機能的性質の付与等がなされ得る。蛋白質は、その表面にある特定のアミノ酸側鎖が変化して当該蛋白質の機能や活性が変化することが知られ、そのような例には、アスパラギン側鎖の脱アミド化、アスパラギン酸側鎖の異性化等が含まれる。そのようなアミノ酸側鎖の変化を防ぐために別のアミノ酸に置き換えたものも、本発明の抗体変異体の範囲に含まれる。
本発明の抗体変異体の例として、抗体の有するアミノ酸配列において保存的アミノ酸置換されてなるアミノ酸配列を有する抗体を挙げることができる。保存的アミノ酸置換とは、アミノ酸側鎖に関連のあるアミノ酸グループ内で生じる置換である。
(3-4)抗CD3抗体の抗原結合性断片
本発明の一つの態様として、本発明の抗CD3抗体の抗原結合性断片を提供する。抗体の抗原結合性断片とは、該抗体の有する機能のうち少なくとも抗原結合性を保持する断片又はその修飾物を意味する。かかる抗体の機能としては、一般的には、抗原結合活性、抗原の活性を調節する活性、抗体依存性細胞傷害活性及び補体依存性細胞傷害活性等を挙げることができる。本発明の抗体等及び本発明の抗体等を含む多重特異的な分子とした場合の機能としては、例えばT細胞のリダイレクション、T細胞の活性化、T細胞を活性化することによる癌細胞の細胞傷害活性を挙げることができる。
(3-5)抗原結合性を有する分子
本発明の分子は、本発明の抗CD3抗体又はその抗原結合性断片を含む。
(3-6)多重特異的な分子、二重特異的な分子
本発明の多重特異的な分子は、2以上の抗原結合部位を持つ分子である。すなわち、1つの分子上の2つ以上の互いに異なるエピトープ又は2つ以上の分子上の互いに異なるエピトープに結合することが可能な分子であり、複数の互いに異なる抗原結合性断片を包む。このような多重特異的な分子には、IgG型多重特異性分子、2種類以上の可変領域を有する多重特異性分子、例えばタンデムscFv、一本鎖ダイアボディ、ダイアボディ、及びトリアボディのような抗体断片、共有結合又は非共有結合して連結されている抗体断片を含むが、これらに限定されない。多重特異的な分子はFcを含んでいてもよい。
本発明の多重特異的な分子の好適な例として、二重特異的な分子を挙げることができる。「二重特異的」とは、同一分子の2つの互いに異なるエピトープ又は2つ分子上の互いに異なるエピトープに結合することが可能であることを意味し、このような二重特異性を有する抗体又は抗原結合性断片を包含する。本発明の二重特異的な分子は、CD3に結合し、且つCD3は有さず他の抗原にあるエピトープに結合する。より具体的には、かかる二重特異的な分子は、(i)CD3上のあるエピトープ(エピトープ1)に結合し、且つ、(ii)CD3にあるエピトープ1とは異なるエピトープ(エピトープ2)に結合するか、又は、CD3以外の抗原にあるエピトープ(エピトープ3)に結合する。
IgG型の二重特異性分子では、異なるエピトープに結合する2種のFabが、二量体のFcの一方とそれぞれリンカーにて連結され又はリンカーなしで直接結合されている。IgG型の二重特異性分子を、以下、Full-Size Antibody(FSA)型二重特異性分子、又は単にFSA型とも記す。
あるいは、本発明の二重特異性分子として、異なるエピトープに結合するFabとscFvが、二量体のFcの一方とそれぞれリンカーにて連結され又はリンカーなしで直接結合されている二重特異性抗体であってもよい。あるいは二量体のFcの一方に第一の抗体のFab,もう一方に第二の抗体のscFvをリンカーを介して結合させた二重特異性分子であっても良い。このような二重特異性分子を、以下Hybrid型二重特異性分子、又はHybrid型とも記す。
(3-7)ヒト化抗CD3抗体
本発明はその一つの態様において、ヒト化抗CD3抗体又はその抗原結合性断片を提供する。
配列番号26(図24)に示されるアミノ酸配列からなるCDRH1(GVTFNYYG)、
配列番号98(図112)に示されるアミノ酸配列からなるCDRH2(ITXaaXaaGGRI)(ここで、1番目のXaaと2番目のXaaは、それぞれ、任意の天然のアミノ酸残基である。以下、CDRH2の1番目のXaaをX1、2番目のXaaを、それぞれX1、X2とも記す。)、
及び、
配列番号28(図26)に示されるアミノ酸配列からなるCDRH3(TLDGRDGWVAY)を保有している。
配列番号29(図27)に示されるアミノ酸配列からなるCDRL1(TGNIGSNY)、
配列番号99(図113)に示されるアミノ酸配列からなるCDRL2(RXaaD)(ここで、Xaaは、任意の天然のアミノ酸残基である。以下、CDRL2のXaaをX3とも記す。)、
及び、
配列番号31(図29)に示されるアミノ酸配列からなるCDRL3(QSYSSGFI)を保有している。
上述のCDRL2(RX3D)において、好ましくは、X3は、(Q、A、G、S、N、D)からなる群より選択される。
上述のCDRL2(RX3D)において、更に好ましくは、X3は、(Q、A、G、S、N、D)からなる群より選択される。
配列番号100(図114)に示されるアミノ酸残基を含む重鎖可変領域を挙げることができる。
配列番号101(図115)、配列番号102(図116)、配列番号103(図117)に示されるアミノ酸残基を含む軽鎖可変領域を挙げることができる。
配列番号26(図24)に示されるアミノ酸配列からなるCDRH1(GVTFNYYG)、
配列番号27(図25)に示されるアミノ酸配列からなるCDRH2(ITNSGGRI)、及び
配列番号28(図26)に示されるアミノ酸配列からなるCDRH3(TLDGRDGWVAY)を保有している重鎖可変領域が挙げられる。
配列番号29(図27)に示されるアミノ酸配列からなるCDRL1(TGNIGSNY)、
配列番号30(図28)に示されるアミノ酸配列からなるCDRL2(RDD)、及び
配列番号31(図29)に示されるアミノ酸配列からなるCDRL3(QSYSSGFI)を保有している軽鎖可変領域が挙げられる。
配列番号60の2乃至119のアミノ酸残基を含む重鎖可変領域と、配列番号60の135乃至243のアミノ酸残基を含む軽鎖可変領域を含む抗体又は該抗体の抗原結合性断片、
配列番号64の2乃至119のアミノ酸残基を含む重鎖可変領域と、配列番号64の135乃至241のアミノ酸残基を含む軽鎖可変領域を含む抗体又は該抗体の抗原結合性断片、
配列番号66の2乃至119のアミノ酸残基を含む重鎖可変領域と、配列番号66の135乃至243のアミノ酸残基を含む軽鎖可変領域を含む抗体又は該抗体の抗原結合性断片、
配列番号68の2乃至119のアミノ酸残基を含む重鎖可変領域と、配列番号68の135乃至243のアミノ酸残基を含む軽鎖可変領域を含む抗体又は該抗体の抗原結合性断片、
配列番号70の2乃至119のアミノ酸残基を含む重鎖可変領域と、配列番号70の135乃至243のアミノ酸残基を含む軽鎖可変領域を含む抗体又は該抗体の抗原結合性断片、
配列番号72の2乃至119のアミノ酸残基を含む重鎖可変領域と、配列番号72の135乃至243のアミノ酸残基を含む軽鎖可変領域を含む抗体又は該抗体の抗原結合性断片、
配列番号74の2乃至119のアミノ酸残基を含む重鎖可変領域と、配列番号74の135乃至243のアミノ酸残基を含む軽鎖可変領域を含む抗体又は該抗体の抗原結合性断片、
配列番号76の2乃至119のアミノ酸残基を含む重鎖可変領域と、配列番号76の135乃至243のアミノ酸残基を含む軽鎖可変領域を含む抗体又は該抗体の抗原結合性断片、
配列番号78の2乃至119のアミノ酸残基を含む重鎖可変領域と、配列番号78の135乃至243のアミノ酸残基を含む軽鎖可変領域を含む抗体又は該抗体の抗原結合性断片、
配列番号80の2乃至119のアミノ酸残基を含む重鎖可変領域と、配列番号80の135乃至243のアミノ酸残基を含む軽鎖可変領域を含む抗体又は該抗体の抗原結合性断片、
配列番号82の2乃至119のアミノ酸残基を含む重鎖可変領域と、配列番号82の135乃至243のアミノ酸残基を含む軽鎖可変領域を含む抗体又は該抗体の抗原結合性断片、
又は、
配列番号84の2乃至119のアミノ酸残基を含む重鎖可変領域と、配列番号84の135乃至243のアミノ酸残基を含む軽鎖可変領域を含む抗体又は該抗体の抗原結合性断片を挙げることができる。
配列番号19の2乃至243番目のアミノ酸残基を含む抗体又は該抗体の抗原結合性断片、
配列番号22の2乃至243番目のアミノ酸残基を含む抗体又は該抗体の抗原結合性断片、
配列番号25の2乃至241番目のアミノ酸残基を含む抗体又は該抗体の抗原結合性断片を挙げることができる。
配列番号60(図68)の1乃至243番目のアミノ酸残基を含む抗体(Clone ID:C3E-7078)又は該抗体の抗原結合性断片、
配列番号64(図72)の1乃至241番目のアミノ酸残基を含む抗体(Clone ID:C3E-7085)又は該抗体の抗原結合性断片、
配列番号66(図74)の1乃至243番目のアミノ酸残基を含む抗体(Clone ID:C3E-7086)又は該抗体の抗原結合性断片、
配列番号68(図76)の1乃至243番目のアミノ酸残基を含む抗体(Clone ID:C3E-7087)又は該抗体の抗原結合性断片、
配列番号70(図78)の1乃至243番目のアミノ酸残基を含む抗体(Clone ID:C3E-7088)又は該抗体の抗原結合性断片、
配列番号72(図80)の1乃至243番目のアミノ酸残基を含む抗体(Clone ID:C3E-7089)又は該抗体の抗原結合性断片、
配列番号74(図82)の1乃至243番目のアミノ酸残基を含む抗体(Clone ID:C3E-7090)又は該抗体の抗原結合性断片、
配列番号76(図84)の1乃至243番目のアミノ酸残基を含む抗体(Clone ID:C3E-7091)又は該抗体の抗原結合性断片、
配列番号78(図86)の1乃至243番目のアミノ酸残基を含む抗体(Clone ID:C3E-7092)又は該抗体の抗原結合性断片、
配列番号80(図88)の1乃至243番目のアミノ酸残基を含む抗体(Clone ID:C3E-7093)又は該抗体の抗原結合性断片、
配列番号82(図90)の1乃至243番目のアミノ酸残基を含む抗体(Clone ID:C3E-7094)又は該抗体の抗原結合性断片、
配列番号84(図92)の1乃至243番目のアミノ酸残基を含む抗体(Clone ID:C3E-7095)又は該抗体の抗原結合性断片
を挙げることができる。
配列番号22の2乃至269のアミノ酸残基を含むアミノ酸配列、
配列番号25の2乃至267のアミノ酸残基を含むアミノ酸配列、
配列番号60の2乃至269のアミノ酸残基を含むアミノ酸配列、
配列番号64の2乃至267のアミノ酸残基を含むアミノ酸配列、
配列番号66の2乃至269のアミノ酸残基を含むアミノ酸配列、
配列番号68の2乃至269のアミノ酸残基を含むアミノ酸配列、
配列番号70の2乃至269のアミノ酸残基を含むアミノ酸配列、
配列番号72の2乃至269のアミノ酸残基を含むアミノ酸配列、
配列番号74の2乃至269のアミノ酸残基を含むアミノ酸配列、
配列番号76の2乃至269のアミノ酸残基を含むアミノ酸配列、
配列番号78の2乃至269のアミノ酸残基を含むアミノ酸配列、
配列番号80の2乃至269のアミノ酸残基を含むアミノ酸配列、
配列番号82の2乃至269のアミノ酸残基を含むアミノ酸配列、又は、
配列番号84の2乃至269のアミノ酸残基を含むアミノ酸配列
を含む前記(16に記載の抗原又は該抗体の抗原結合性断片が挙げられる。
本発明の提供する抗体等と「同一の部位に結合し、且つカニクイザルCD3にも結合する抗体」も本発明の抗体等に含まれる。ある抗体と「同一の部位に結合する抗体」とは、該抗体が認識する抗原分子上の部位に結合する他の抗体を意味する。第一抗体が結合する抗原分子上の部分ペプチド又は部分立体構造に第二抗体が結合すれば、第一抗体と第二抗体は同一の部位に結合すると判定することができる。
(3-9)抗CD3抗体又はその抗原結合性断片の修飾体
本発明は、抗体又はその抗原結合性断片の修飾体を提供する。本発明の抗体又はその抗原結合性断片の修飾体とは、本発明の抗体又はその抗原結合性断片に化学的又は生物学的な修飾が施されてなるものを意味する。化学的な修飾体には、アミノ酸骨格への化学部分の結合、N-結合又はO-結合炭水化物鎖の化学修飾体等が含まれる。生物学的な修飾体には、翻訳後修飾(例えば、N-結合又はO-結合への糖鎖付加、アミノ末端領域又はカルボキシル末端領域のプロセッシング、脱アミド化、アスパラギン酸の異性化、メチオニンの酸化)されたもの、原核生物宿主細胞を用いて発現させることによりアミノ末端にメチオニン残基が付加したもの等が含まれる。また、本発明の抗体又は抗原の検出又は単離を可能にするために標識されたもの、例えば、酵素標識体、蛍光標識体、アフィニティー標識体もかかる修飾物の意味に含まれる。このような本発明の抗体又はその抗原結合性断片の修飾物は、元の本発明の抗体又はその抗原結合性断片の安定性及び血中滞留性の改善、抗原性の低減、かかる抗体又は抗原の検出又は単離等に有用である。
しかし、これらの重鎖配列の欠失及び修飾は、抗体の抗原結合能及びエフェクター抗原結合性(補体の活性化や抗体依存性細胞傷害作用など)にはあまり影響を及ぼさない。従って、本発明には当該修飾を受けた抗体及び当該抗体の抗原結合性断片も含まれ、重鎖カルボキシル末端において1又は2つのアミノ酸が欠失した欠失体、及びアミド化された当該欠失体(例えば、カルボキシル末端部位のプロリン残基がアミド化された重鎖)、重鎖又は軽鎖のアミノ末端残基がピログルタミル化された抗体等も含まれる(それらをまとめて「欠失体」と呼ぶ)。但し、抗原結合能の全部又は一部が保たれている限り、本発明に係る抗体の重鎖及び軽鎖のカルボキシル末端の欠失体は上記の種類に限定されない。本発明に係る抗体が2本以上の鎖(例えば重鎖)を含む場合、当該2本以上の鎖(例えば重鎖)は、完全長及び上記の欠失体からなる群から選択される重鎖のいずれか一種であっても良いし、いずれか2種以上を組み合わせたものであっても良い。各欠失体の量比又は分子数比は本発明に係る抗体を産生する哺乳類培養細胞の種類及び培養条件等の影響を受け得るが、本発明に係る抗体の欠失体としては、例えば、2本の重鎖の双方でカルボキシル末端の1つのアミノ酸残基が欠失している場合を挙げることができる。これらも全て本発明の抗体変異体、抗体の抗原結合性断片又はそれらの修飾体の意味に包含される。
4.抗体の製造
(4-1)ハイブリドーマを用いる方法
本発明の一つの態様として、抗CD3抗体は、例えば、コーラーとミルスタインの方法(Kohler and Milstein,Nature(1975)256,p.495-497、Kennet,R.ed.,Monoclonal Antibodies,p.365-367,Plenum Press,N.Y.(1980))に従って、CD3蛋白質で免疫した動物の脾臓より抗CD3抗体の産生細胞を単離し、該細胞とミエローマ細胞とを融合させることによりハイブリドーマを樹立し、かかるハイブリドーマの培養物からモノクローナル抗体を取得することができる。
(4-1-1)抗原の調製
抗CD3抗体を作製するための抗原は、天然型又は組換え型のCD3蛋白質(ヒトCD3εγ一本鎖抗原)の調製法等に従って取得することができる。そのようにして調製し得る抗原としては、CD3蛋白質若しくはCD3蛋白質断片、又はそれらに任意のアミノ酸配列や担体が付加された誘導体等(以下、まとめて「CD3」とよぶ)を挙げることができる。
(4-1-2)抗CD3モノクローナル抗体の製造
モノクローナル抗体の製造は、通常、下記のような工程を経る。
(a)抗原を調製する工程、
(b)抗体産生細胞を調製する工程、
(c)骨髄腫細胞(以下、「ミエローマ」という)を調製する工程、
(d)抗体産生細胞とミエローマとを融合させる工程、
(e)目的とする抗体を産生するハイブリドーマ群を選別する工程、及び
(f)単一細胞クローンを得る工程(クローニング)。
(a)抗原を調製する工程
本発明のCD3蛋白質は、動物組織(体液を含む)、該組織由来の細胞もしくは該細胞培養物からの精製及び単離、遺伝子組換え、無細胞蛋白質合成、化学合成等により調製することができる。
(b)抗体産生細胞を調製する工程
工程(a)で得られた抗原と、フロインドの完全又は不完全アジュバント、又はカリミョウバンのような助剤とを混合し、免疫原として実験動物に免疫する。実験動物は公知のハイブリドーマ作製法に用いられる動物を支障なく使用することができる。具体的には、たとえばマウス、ラット、ヤギ、ヒツジ、ウシ、ウマ等を使用することができる。ただし、摘出した抗体産生細胞と融合させるミエローマ細胞の入手容易性等の観点から、マウス又はラットを被免疫動物とするのが好ましい。
(c)ミエローマを調製する工程
細胞融合に用いるミエローマ細胞には特段の限定はなく、公知の細胞株から適宜選択して用いることができるが、融合細胞からハイブリドーマを選択する際の利便性を考慮して、その選択手続が確立しているHGPRT(Hypoxanthine-guanine phosphoribosyl transferase)欠損株、すなわちマウス由来のX63-Ag8(X63)、NS1-ANS/1(NS1)、P3X63-Ag8.Ul(P3Ul)、X63-Ag8.653(X63.653)、SP2/0-Ag14(SP2/0)、MPC11-45.6TG1.7(45.6TG)、FO、S149/5XXO、BU.1等、ラット由来の210.RSY3.Ag.1.2.3(Y3)等、ヒト由来のU266AR(SKO-007)、GM1500・GTG-A12(GM1500)、UC729-6、LICR-LOW-HMy2(HMy2)、8226AR/NIP4-1(NP41)等を用いるのが好ましい。これらのHGPRT欠損株は例えば、American Type Culture Collection (ATCC)等から入手することができる。
(d)抗体産生細胞とミエローマ細胞を融合させる工程
抗体産生細胞とミエローマ細胞との融合は、公知の方法(Weir,D.M.,Handbookof Experimental Immunology Vol.I.II.III.,Blackwell Scientific Publications,Oxford(1987)、Kabat,E.A.and Mayer,M.M.,Experimental Immunochemistry,Charles C Thomas Publisher Spigfield,Illinois(1964)等)に従い、細胞の生存率を極度に低下させない程度の条件下で実施することができる。例えば、ポリエチレングリコール等の高濃度ポリマー溶液中で抗体産生細胞とミエローマ細胞とを混合する化学的方法、電気的刺激を利用する物理的方法等を用いることができる。
(e)目的とする抗体を産生するハイブリドーマ群を選別する工程
細胞融合により得られるハイブリドーマの選択方法は特に制限はないが、通常HAT(ヒポキサンチン・アミノプテリン・チミジン)選択法(Kohler et al.,Nature(1975)256,495;Milstein et al.,Nature(1977)266,550)が用いられる。この方法は、アミノプテリンで生存し得ないHGPRT欠損株のミエローマ細胞を用いてハイブリドーマを得る場合に有効である。すなわち、未融合細胞及びハイブリドーマをHAT培地で培養することにより、アミノプテリンに対する耐性を持ち合わせたハイブリドーマのみを選択的に残存させ、かつ増殖させることができる。
(f)単一細胞クローンを得る工程(クローニング)
ハイブリドーマのクローニング法としては、例えば、メチルセルロース法、軟アガロース法、限界希釈法等の公知の方法を用いることができるが(例えば、Barbara, B.M. and Stanley, M.S.:Selected Methods in Cellular Immunology,W.H. Freeman and Company,San Francisco(1980))、好適には限界希釈法である。
(g)ハイブリドーマの培養工程、ハイブリドーマを移植した動物の飼育工程
選択されたハイブリドーマを培養することにより、モノクローナル抗体を産生することができるが、好適には所望のハイブリドーマをクローニングしてから抗体の産生に供する。
(h)モノクローナル抗体の生物活性の測定工程・判定工程
各種生物試験を目的に応じて選択し、適用することができる。
(4-2)細胞免疫法
天然型のCD3を発現する細胞、組換え型CD3又はその断片を発現する細胞等を免疫原として使用することにより、前記のハイブリドーマ法により抗CD3抗体を調製することができる。
(4-3)DNA免疫法
本発明の抗CD3抗体は、DNA免疫法を使用して得ることもできる。抗原発現プラスミドをマウスやラットなどの動物個体に遺伝子導入し、抗原を個体内で発現させることによって、抗原に対する免疫を誘導する。遺伝子導入の手法には、直接プラスミドを筋肉に注射する方法や、リポソームやポリエチレンイミンなどの導入試薬を静脈注射する方法、ウイルスベクターを用いる手法、プラスミドを付着させた金粒子をGene Gunにより射ち込む手法、急速に大量のプラスミド溶液を静脈注射するHydrodynamic法などが存在する。
本発明の抗体は、その重鎖アミノ酸配列をコードするヌクレオチド配列が含まれるヌクレオチド(重鎖ヌクレオチド)及びその軽鎖アミノ酸配列をコードするヌクレオチド配列が含まれるヌクレオチド(軽鎖ヌクレオチド)、又は、かかる重鎖ヌクレオチドが挿入されたベクター及び軽鎖ヌクレオチドが挿入されたベクターを宿主細胞に導入し、該細胞を培養した後その培養物からかかる抗体を回収することにより調製することができる。一つのベクターに重鎖ヌクレオチド及び軽鎖ヌクレオチドが挿入されていてもよい。
(4-5)ヒト化抗体のデザイン法及び調製法
ヒト化抗体としては、非ヒト動物抗体のCDRのみがヒト由来の抗体に組込まれ抗体(Nature(1986)321,p.522-525参照)、CDR移植法によりCDRの配列に加え一部のフレームワークのアミノ酸残基もヒト抗体に移植した抗体(WO90/07861号、US6972323号公報参照)、それらのいずれかにおける非ヒト動物抗体の1つ又は2つ以上のアミノ酸がヒト型のアミノ酸で置換されてなる抗体等を挙げることができるが、それらに限定されるものではない。
(4-6)ヒト抗体の調製法
本発明の抗体としては、さらに、ヒト抗体を挙げることができる。ヒト抗CD3抗体とは、ヒト由来の抗体のアミノ酸配列からなる抗CD3抗体を意味する。ヒト抗CD3抗体は、ヒト抗体の重鎖と軽鎖の遺伝子を含むヒトゲノムDNA断片を有するヒト抗体産生マウスを用いた方法(Tomizuka,K.et al.,Nature Genetics(1997)16,133-143,; Kuroiwa,Y.et.al.,Nuc.Acids Res.(1998)26,3447-3448;Yoshida, H.et.al.,Animal Cell Technology:Basic and Applied Aspects vol.10,69-73(Kitagawa, Y.,Matuda,T.and Iijima,S.eds.),Kluwer Academic Publishers,1999.;Tomizuka,K.et.al.,Proc.Natl.Acad.Sci.USA(2000)97,722-727等を参照。)によって取得することができる。
(4-7)抗体の抗原結合性断片の調製法
scFvを作成する方法は当技術分野において周知である(例えば、米国特許第4,946,778号、米国特許第5,260,203号、米国特許第5,091,513号、米国特許第5,455,030号等を参照)。このscFvにおいて、重鎖可変領域と軽鎖可変領域は、コンジュゲートを作らないようなリンカー、好ましくはポリペプチドリンカーを介して連結される(Huston,J.S.et al.,PNAS(1988),85,5879-5883)。scFvにおける重鎖可変領域及び軽鎖可変領域は、同一の抗体に由来してもよく、別々の抗体に由来してもよい。
(4-8)抗体及び抗体の抗原結合性断片の精製
得られた抗体及び抗体の抗原結合性断片は、該抗体等以外を含まないよう均一に精製することができる。抗体及び抗体の抗原結合性断片の分離、精製は通常の蛋白質で使用されている分離、精製方法を使用すればよい。
(4-9)多重特異性分子、二重特異性分子
本発明の二重特異性分子及び多重特異的分子は、宿主細胞へ発現プラスミドを導入し、一過性で発現させる方法、宿主細胞にプラスミドを導入後、薬剤選択により安定発現細胞株を選び、恒常的に発現させる方法、無細胞的に合成する方法、それぞれの抗体あるいは抗原結合性断片を上記の方法で作製後、合成ペプチドリンカーを用いて化学的に結合させる方法が挙げられる。
5.医薬組成物
本発明は抗CD3抗体、その抗原結合性断片あるいはその修飾体、及び/又は、それらを含む本発明の分子、例えば、多重特異性分子を含む医薬組成物を提供する。
医薬組成物の形態としては、注射剤(凍結乾燥製剤、点滴剤を含む)、坐剤、経鼻型吸収製剤、経皮型吸収製剤、舌下剤、カプセル、錠剤、軟膏剤、顆粒剤、エアーゾル剤、丸剤、散剤、懸濁剤、乳剤、点眼剤、生体埋め込み型製剤等を例示することができる。
(実施例1)ラット抗ヒトCD3抗体の作製
1)-1 ヒトCD3εδ発現ベクターの構築
Gateway Vector Conversion System(Thermo Fisher Scientific社)によりDestination Vectorに改変したコントロールベクターpcDNA3.1-DESTを作製した。図1(配列番号1)に示すヒトCD3ε蛋白質(NCBI Reference Sequence: NP_000724.1)をコードするcDNAはSino Biologicalより購入し、Gateway LR Clonase Enzyme mix(Thermo Fisher Scientific社)を用いて、pcDNA3.1-DESTベクターにクローニングしhCD3ε-pcDNA3.1を構築した。図2(配列番号2)に示すヒトCD3δ蛋白質(NP_000723.1)をコードするcDNAは、当業者に公知な方法に従いヒトT細胞由来cDNAを鋳型としたPCR法により増幅し、pcDNA3.1(+)(Thermo Fisher Scientific社)にクローニングすることで発現ベクターhCD3δ-pcDNA3.1を構築した。各発現ベクターの大量調製には、Endofree Plasmid Giga Kit(QIAGEN社)を用いた。
1)-2 免疫
免疫にはWKY/Izmラットの雌(日本エスエルシー社)を使用した。まずラット両足下腿部をHyaluronidase(SIGMA-ALDRICH社)にて前処理後、同部位に実施例1)- 1で作製したhCD3ε-pcDNA3.1及びhCD3δ-pcDNA3.1発現ベクターを筋注した。続けて、ECM830(BTX社)を使用し、2ニードル電極を用いて、同部位にインビボエレクトロポレーションを実施した。約二週間に一度、同様のインビボエレクトロポレーションを繰り返した後、ラットのリンパ節又は脾臓を採取しハイブリドーマ作製に用いた。
1)-3 ハイブリドーマ作製
リンパ節細胞あるいは脾臓細胞とマウスミエローマSP2/0-ag14細胞(ATCC, No.CRL-1 581)とをLF301 Cell Fusion Unit(BEX社)を用いて電気細胞融合し、ClonaCell-HY Selection Medium D(StemCell Technologies社)に希釈して培養した。出現したハイブリドーマコロニーを回収することでモノクローンハイブリドーマを作製した。回収された各ハイブリドーマコロニーをClonaCell-HY Selection Medium E(StemCell Technologies社)を用いて培養し、得られたハイブリドーマ培養上清を用いて抗ヒトCD3抗体産生ハイブリドーマのスクリーニングを行った。
1)-4 Cell-ELISA法による抗体スクリーニング
1)-4-1 Cell-ELISA用抗原遺伝子発現細胞の調製
HEK293α細胞(インテグリンαv及びインテグリンβ3を発現するHEK293由来の安定発現細胞株) を10% FBS含有DMEM培地中7.5×105細胞/mLになるよう調製した。それに対し、Lipofectamine 2000(Thermo Fisher Scientific社)を用いた形質移入手順に従い、hCD3ε-pcDNA3.1及びhCD3δ-pcDNA3.1、もしくはコントロールとしてpcDNA3.1-DESTを導入し、96-well plate(Corning社)に100μLずつ分注し、10%FBS含有DMEM培地中で37℃、5%CO2の条件下で一晩培養した。得られた導入細胞を接着状態のまま、Cell-ELISAに使用した。
1)-4-2 Cell-ELISA
実施例1)- 4- 1で調製した発現ベクター導入HEK293α細胞の培養上清を除去後、hCD3ε-pcDNA3.1及びhCD3δ-pcDNA3.1、又はpcDNA3.1-DEST導入HEK293α細胞のそれぞれに対しハイブリドーマ培養上清を添加し、4℃で1時間静置した。well中の細胞を5% FBS含有PBSで1回洗浄後、5% FBS含有PBSで500倍に希釈したAnti-Rat IgG, HRP-Linked Whole Ab Goat(GE Healthcare Bioscience社)を加えて、4℃で1時間静置した。well中の細胞を5% FBS含有PBSで2回洗浄した後、OPD発色液(OPD溶解液(0.05 M クエン酸3ナトリウム、0.1M リン酸水素2ナトリウム・12水 pH4.5)にo-フェニレンジアミン二塩酸塩(和光純薬社)、H2O2をそれぞれ0.4mg/mL、0.6%(v/v)になるように溶解)を100μL/wellで添加した。時々攪拌しながら発色反応を行い、1M HClを100μL/wellを添加して発色反応を停止させた後、プレートリーダー(ENVISION:PerkinElmer社)で490nmの吸光度を測定した。細胞膜表面上に発現するヒトCD3に結合する抗体を産生するハイブリドーマを選択するため、コントロールのpcDNA3.1-DEST導入HEK293α細胞と比較し、hCD3ε-pcDNA3.1及びhCD3δ-pcDNA3.1発現ベクター導入HEK293α細胞の方でより高い吸光度を示す培養上清を産生するハイブリドーマを抗ヒトCD3抗体産生陽性として選択した。
1)-5 ヒトT細胞活性化による抗体スクリーニング
得られたハイブリドーマが産生する抗CD3抗体によるT細胞の活性化を、CD69活性化マーカーの検出を指標に評価した。ヒトT細胞株であるJurkat細胞(ATCC,No.TIB-152)を、FBS含有RPMI1640培地中5×106細胞/mLの濃度に調製して、96-well plateに100μLずつ播種した。遠心して上清を除去したJurkat細胞に、実施例1)-4のCell-ELISAで抗ヒトCD3抗体産生陽性として選択したハイブリドーマの培養上清、あるいはラットIgGアイソタイプコントロール抗体(R&D Systams社)終濃度5μg/mLを添加し、37℃で30分間静置した。その後、クロスリンカーGoat Anti-rat IgG FcγFragment specific(JACKSON IMMUNORESEARCH社)を終濃度10μg/wellで添加し37℃、5%CO2の条件下で一晩培養した。翌日、上清を除去し、well中の細胞を5% FBS含有PBSで1回洗浄後、PE Mouse Anti-Human CD69抗体(BD Bioscience社)を20μL/well添加して4℃で30分間静置した。well中の細胞を5% FBS含有PBSで2回洗浄した後、5% FBS含有PBSに再懸濁し、フローサイトメーター(FC500:BeckmanCoulter社)で検出を行った。データ解析はFlowjo(Treestar社)で行った。PE蛍光強度のヒストグラムを作成し、ラットIgGアイソタイプコントロール抗体の蛍光強度ヒストグラムに対しPEの蛍光強度のヒストグラムが強蛍光強度側にシフトしているサンプルを産生するハイブリドーマをヒトT細胞活性化能陽性抗ヒトCD3抗体産生ハイブリドーマとして選択した。
1)-6 フローサイトメトリーによるヒト又はサルCD3への選択的結合性スクリーニング
1)-6-1 ヒト抗原遺伝子発現細胞の調製
Lenti-X293T細胞(TAKARA社,Cat#632180)を5.3×104細胞/cm2になるよう225cm2フラスコに播種し、10% FBS含有DMEM培地中で37℃、5%CO2の条件下で一晩培養した。翌日、hCD3ε-pcDNA3.1及びhCD3δ-pcDNA3.1又は、コントロールとしてpcDNA3.1-DESTをそれぞれLenti-X293T細胞にLipofectamine 2000を用いて導入し、37℃、5%CO2の条件下でさらに一晩培養した。翌日、発現ベクター導入Lenti-X293T細胞をTrypLE Express(Thermo Fisher Scientific社)で処理し、10% FBS含有DMEMで細胞を洗浄した後、5% FBS含有PBSで5×106細胞/mLの濃度に調製した。得られた細胞懸濁液をフローサイトメトリー解析に使用した。
1)-6-2 ヒトCD3への結合性フローサイトメトリー解析
実施例1)-5でヒトT細胞活性化能陽性と判定されたハイブリドーマが産生する抗体のヒトCD3に対する結合特異性をフローサイトメトリー法によりさらに確認した。実施例1)-6-1で調製したLenti-X293T細胞懸濁液を100μL/wellで96-well U底マイクロプレートに播種し、遠心後上清を除去した。hCD3ε-pcDNA3.1及びhCD3δ-pcDNA3.1導入Lenti-X293T細胞及びpcDNA3.1-DEST導入Lenti-X293T細胞のそれぞれに対しハイブリドーマ培養上清を加えて懸濁し、4℃で1時間静置した。5% FBS含有PBSで1回洗浄した後、5% FBS含有PBSで500倍に希釈したAnti-Rat IgG FITC conjugate(SIGMA社)を加えて懸濁し、4℃で30分静置した。5% FBS含有PBSで2回洗浄した後、2μg/ml 7-aminoactinomycin D(Molecular Probes社)を含む5% FBS含有PBSに再懸濁し、フローサイトメーターで検出を行った。データ解析はFlowjoで行った。7-aminoactinomycin D陽性の死細胞をゲートで除外した後、生細胞のFITC蛍光強度のヒストグラムを作成した。コントロールであるpcDNA3.1-DEST導入Lenti-X293T細胞の蛍光強度ヒストグラムに対しhCD3ε-pcDNA3.1及びhCD3δ-pcDNA3.1導入Lenti-X293T細胞のヒストグラムが強蛍光強度側にシフトしているサンプルを産生するハイブリドーマをヒトCD3に結合する抗体産生ハイブリドーマとして選択した。
1)-6-3 サルCD3εδ発現ベクターの構築
サルCD3ε蛋白質(NCBI Reference Sequence: NP_001270544.1)及びサルCD3δ蛋白質(NCBI Reference Sequence: NP_001274617.1)をコードするcDNAは、当業者に公知な方法に従いサルT細胞由来cDNAを鋳型としたPCR法により増幅し、pcDNA3.1(+)(Thermo Fisher Scientific社)にクローニングすることで発現ベクターcynoCD3ε-pcDNA3.1及びcynoCD3δ-pcDNA3.1を構築した。
1)-6-4 サル抗原遺伝子発現細胞の調製
Lenti-X293T細胞を5.3×104細胞/cm2になるよう225cm2フラスコに播種し、10% FBS含有DMEM培地中で37℃、5%CO2の条件下で一晩培養した。翌日、cynoCD3ε-pcDNA3.1及びcynoCD3δ-pcDNA3.1又は、コントロールとしてpcDNA3.1-DESTをそれぞれLenti-X293T細胞にLipofectamine 2000を用いて導入し、37℃、5% CO2の条件下でさらに一晩培養した。翌日、発現ベクター導入Lenti-X293T細胞をTrypLE Expressで処理し、10% FBS含有DMEMで細胞を洗浄した後、5% FBS含有PBSで5×106細胞/mLの濃度に調製した。得られた細胞懸濁液をフローサイトメトリー解析に使用した。
1)-6-5 サルCD3への結合性フローサイトメトリー解析
実施例1)-6-2でヒトCD3に結合する抗体産生ハイブリドーマと判定されたハイブリドーマが産生する抗体のサルCD3に対する結合特異性をフローサイトメトリー法によりさらに確認した。実施例1)-6-4で調製したLenti-X293T細胞懸濁液を100μL/wellで96-well U底マイクロプレートに播種し、遠心後上清を除去した。cynoCD3ε-pcDNA3.1及びcynoCD3δ-pcDNA3.1導入Lenti-X293T細胞及びpcDNA3.1-DEST導入Lenti-X293T細胞のそれぞれに対しハイブリドーマ培養上清を加えて懸濁し、4℃で1時間静置した。5% FBS含有PBSで1回洗浄した後、5% FBS含有PBSで500倍に希釈したAnti-Rat IgG FITC conjugateを加えて懸濁し、4℃で30分静置した。5% FBS含有PBSで2回洗浄した後、2μg/ml 7-aminoactinomycin Dを含む5% FBS含有PBSに再懸濁し、フローサイトメーターで検出を行った。データ解析はFlowjoで行った。7-aminoactinomycin D陽性の死細胞をゲートで除外した後、生細胞のFITC蛍光強度のヒストグラムを作成した。コントロールであるpcDNA3.1-DEST導入Lenti-X293T細胞の蛍光強度ヒストグラムに対しcynoCD3ε-pcDNA3.1及びcynoCD3δ-pcDNA3.1導入Lenti-X293T細胞のヒストグラムが強蛍光強度側にシフトしているサンプルを産生するハイブリドーマをサルCD3に結合する抗体産生ハイブリドーマとして選択した。
1)-6-6 ヒトCD3δ遺伝子発現細胞の調製
Lenti-X293T細胞を5.3×104細胞/cm2になるよう225cm2フラスコに播種し、10% FBS含有DMEM培地中で37℃、5% CO2の条件下で一晩培養した。翌日、hCD3δ-pcDNA3.1又は、コントロールとしてpcDNA3.1-DESTをそれぞれLenti-X293T細胞にLipofectamine 2000を用いて導入し、37℃、5%CO2の条件下でさらに一晩培養した。翌日、発現ベクター導入Lenti-X293T細胞をTrypLE Expressで処理し、10% FBS含有DMEMで細胞を洗浄した後、5% FBS含有PBSで5×106細胞/mLの濃度に調製した。得られた細胞懸濁液をフローサイトメトリー解析に使用した。
1)-6-7 ヒトCD3δへの結合性フローサイトメトリー解析
実施例1)-6-5でサルCD3に結合する抗体産生ハイブリドーマと判定されたハイブリドーマが産生する抗体のヒトCD3δに対する結合特異性をフローサイトメトリー法によりさらに確認した。実施例1)-6-6で調製したLenti-X293T細胞懸濁液を100μL/wellで96-well U底マイクロプレートに播種し、遠心後上清を除去した。hCD3δ-pcDNA3.1導入Lenti-X293T細胞及びpcDNA3.1-DEST導入Lenti-X293T細胞のそれぞれに対しハイブリドーマ培養上清を加えて懸濁し、4℃で1時間静置した。5% FBS含有PBSで1回洗浄した後、5% FBS含有PBSで500倍に希釈したAnti-Rat IgG FITC conjugateを加えて懸濁し、4℃で30分静置した。5% FBS含有PBSで2回洗浄した後、2μg/ml 7-aminoactinomycin Dを含む5% FBS含有PBSに再懸濁し、フローサイトメーターで検出を行った。データ解析はFlowjoで行った。7-aminoactinomycin D陽性の死細胞をゲートで除外した後、生細胞のFITC蛍光強度のヒストグラムを作成した。コントロールであるpcDNA3.1-DEST導入Lenti-X293T細胞の蛍光強度ヒストグラムに対しhCD3δ-pcDNA3.1導入Lenti-X293T細胞のヒストグラムが強蛍光強度側にシフトしているサンプルを産生するハイブリドーマをヒトCD3δに結合する抗体産生ハイブリドーマとして除外した。
1)-6-8 サルT細胞株への結合性フローサイトメトリー解析
実施例1)-6-7で除外されなかった抗体産生ハイブリドーマが産生する抗体のサルT細胞株への結合特異性をフローサイトメトリー法によりさらに確認した。カニクイザルT細胞株であるHSC-F(JCRB細胞バンク,No.JCRB1164)を、FBS含有RPMI1640培地中5×106細胞/mLの濃度に調製して、96-well plateに100μLずつ播種した。遠心して上清を除去したHSC-F細胞に、実施例1)-5- 7で除外されなかった抗体産生ハイブリドーマの培養上清、あるいはラットIgGアイソタイプコントロール抗体を添加し、4℃で1時間静置した。その後、上清を除去し、well中の細胞を5% FBS含有PBSで1回洗浄後、5% FBS含有PBSで500倍に希釈したAnti-Rat IgG FITC conjugateを加えて懸濁し、4℃で1時間静置した。5% FBS含有PBSで3回洗浄した後、2μg/ml 7-aminoactinomycin Dを含む5% FBS含有PBSに再懸濁し、フローサイトメーターで検出を行った。データ解析はFlowjoで行った。7-aminoactinomycin D陽性の死細胞をゲートで除外した後、生細胞のFITC蛍光強度のヒストグラムを作成し、ラットIgGアイソタイプコントロール抗体の蛍光強度ヒストグラムに対しFITCの蛍光強度のヒストグラムが強蛍光強度側にシフトしているサンプルを産生するハイブリドーマをサルT細胞株にも結合する抗体産生ハイブリドーマとして選択した。
1)-7 抗体のアイソタイプ決定
実施例1)-6で取得したラット抗CD3抗体産生ハイブリドーマの中から、ヒト及びサルCD3εに結合し、サルT細胞株にも結合、ならびに高いヒトT細胞活性化能を有することが示唆されたC3-147を選抜し、抗体アイソタイプを同定した。アイソタイプは、Rat Immunoglobulin Isotyping ELISA Kit(BD Pharmingen社)により決定された。その結果、ラット抗CD3モノクローナル抗体C3-147のアイソタイプはIgG2b、λ鎖であることが確認された。
(実施例2)ラット抗CD3モノクローナル抗体(C3-147)のヒトCD3への結合性検討
2)-1 ハイブリドーマ上清からのモノクローナル抗体の調製
2)-1-1 C3-147を産生するハイブリドーマの培養
ラット抗CD3モノクローナル抗体は、ハイブリドーマ培養上清から精製した。まず、C3-147産生ハイブリドーマをClonaCell-HY Selection Medium E(StemCell Technologies社)で十分量まで増殖させた後、Ultra Low IgG FBS(Thermo Fisher Scientific社)を20%添加した、5μg/mLのゲンタマイシン(Thermo Fisher Scientific社)含有Hybridoma SFM(Thermo Fisher Scientific社)に培地交換し、7日間培養した。本培養上清を回収し、0.22μmのフィルター(Corning社)を通して滅菌した。
2)-1-2 精製
実施例2)-1-1で調製したハイブリドーマの培養上清から抗体をProtein Gアフィニティークロマトグラフィーで精製した。Protein Gカラム(GE Healthcare Bioscience社)に抗体を吸着させ、PBSでカラムを洗浄後に0.1M グリシン/塩酸水溶液(pH2.7)で溶出した。溶出液に1M Tris-HCl(pH9.0)を加えてpH7.0~7.5に調整した後に、Centrifugal UF Filter Device VIVASPIN20(分画分子量UF30K、Sartorius社)にてPBSへのバッファー置換を行うとともに抗体の濃縮を行い、抗体濃度を2mg/mLに調製した。最後にMinisart-Plus filter(Sartorius社)でろ過し、精製サンプルとした。
2)-2 取得ラット抗CD3抗体(C3-147)のヒト一本鎖抗原への結合
2)-2-1 ヒトCD3εγ一本鎖抗原の調製
CD3εあるいはCD3γをコードするアミノ酸配列は蛋白質データバンクに投稿されたOTK3-ヒトCD3εγ一本鎖抗原複合体の結晶構造(PDBID:1SY6)において用いられた配列を用いた。CD3εのカルボキシル末端とCD3γのアミノ末端をつなぐリンカーは参考文献(Kim,K.S.et al., (2000)J.Mol.Biol. 302,899-916)で報告されたものと同じ26アミノ酸から成るペプチドリンカーを用いた。図3(配列番号4)に示すヒトCD3εγ一本鎖抗原をコードする遺伝子は5‘末端、3’末端それぞれに制限酵素サイトBamHI、Hind IIIを付加させて合成した(GENEART社)。プラスミドpQE80L(Qiagen社)を制限酵素BamHI、Hind IIIで消化して得られる約4.8kbのフラグメントと、ヒトCD3εγ一本鎖抗原遺伝子をBamHI、Hind IIIで消化して得られる約0.6kbのフラグメントをLigation high(東洋紡)を用いて結合させ、大腸菌発現用プラスミドpQE80L-scCD3εγを作製した。生成するscCD3εγのアミノ酸配列は、図4(配列番号5)に記載されている。発現用大腸菌BL21(DE3)を発現用プラスミドpQE80L-scCD3εγで形質転換し、得られたコロニーを1L MagicMedia(Invitrogen社)/Ultrayieldフラスコ(Thomson社)に植菌し、30℃、250 rpmで21時間振盪培養した。培養後の菌体を回収し、1%Triton溶液を含むTris緩衝液存在下で超音波ホモジナイザーによる菌体破砕と凍結融解を繰り返し、最終的に4℃、15000 rpm、15分間遠心して、インクルージョンボディを回収した。インクルージョンボディーからの巻き戻しから精製までは参考文献(Kjer-Nielsen et al.(2004) PNAS vol. 101, no. 20,7675-7680)の手法に従って行なった。ただし、抗体カラムに用いた抗CD3抗体は文献で用いられた2C11ではなく、マウス抗CDモノクローナル抗体OKT3(Sgro、Toxicology 105(1995)、23~29、Orthoclone、Janssen-Cilag社)を用いた。
2)-2-2 SPRによるヒトCD3εγ一本鎖抗原への結合性
抗体と抗原との結合は、Biacore 3000(GE Healthcare Bioscience社)を使用し、固定化した抗マウスIgG抗体に抗体をリガンドとして捕捉(キャプチャー)し、抗原をアナライトとして測定するキャプチャー法にて測定した。抗原には、2)-2-1にて調製したヒトCD3εγを使用した。抗マウスIgG抗体(Mouse Antibody Capture Kit、GE Healthcare Bioscience社)は、センサーチップCM5(GE Healthcare Bioscience社)へアミンカップリング法にて約11000RU共有結合させた。リファレンスセルにも同様に固定化した。ランニングバッファーとしてHBS-EP+(10mM HEPES pH7.4、0.15M NaCl、3mM EDTA、0.05%Surfactant P20)を用いた。抗マウスIgG抗体を固定化したチップ上に、抗体を約1分間添加しリガンドとして捕捉した後、100nMの抗原を流速30μl/分で120秒間添加し、抗原の結合をモニターした。再生溶液として、10mM glycine-HCl pH1.7を流速10μl/分で3分間添加した。その結果、C3-147では、120秒後の結合シグナルは34RUであった。
2)-3 SPRによる取得ラット抗CD3抗体(C3-147)の抗原結合部位の確認
抗原結合部位の確認方法は、2)-2-2に準じた。その結果、C3-147では、34RUの結合シグナルを得た。一方、比較例として、公知の抗CD3抗体であるSP34(BD Pharmingen社)についても同様に抗原結合部位の確認を行った。SP34では、34RUの結合シグナルが見られなかった。従って、C3-147が認識するCD3εγ表面の結合部位は、SP34とは異なることが示された。
(実施例3) ラット抗CD3抗体(C3-147)の可変領域をコードするcDNAの塩基配列の決定
ラット抗CD3抗体(C3-147)の可変領域をコードするcDNAのヌクレオチド配列は、以下の方法により決定した。
3)-1 cDNA合成
ラット抗CD3抗体(C3-147)産生ハイブリドーマの細胞溶解液(50mM Tris-HCl(pH7.5)、250mM LiCl、5mM EDTA(pH8)、0.5%ドデシル硫酸Li(LiDS)、2.5mM dithiothreitol(DTT))を、オリゴdT25が結合したDynabeads mRNA DIRECT Kit(Thermo Fisher Scientific社)の磁気ビーズと混合し、mRNAを磁気ビーズに結合させた。次に磁気ビーズをmRNA洗浄溶液A(10mM Tris-HCl(pH7.5)、0.15M LiCl、1mM EDTA、0.1% LiDS、0.1% TritonX-100)とcDNA合成用溶液(50mM Tris-HCl(pH8.3)、75mM KCl、3mM MgCl2、5mM DTT、0.5mM dNTP、0.2% TritonX-100、1.2unit RNase inhibitor(Thermo Fisher Scientific社)で1回ずつ洗浄した後、12unit SuperScriptIII Reverse Transcriptase(Thermo Fisher Scientific社)を加えたcDNA合成用溶液でcDNA合成を行った。続いて3’テーリング反応溶液(50mM リン酸カリウム、4mM MgCl2、0.5mM dGTP、0.2% TritonX-100、1.2unit RNase inhibitor)で洗浄した後、48unit Terminal Transferase, recombinant(Roche社)を加えた反応溶液で3’テーリング反応を行った。
3)-2 ラット免疫グロブリン重、軽鎖可変領域遺伝子断片の増幅及び配列決定
磁気ビーズをTE溶液(10mM Tris-HCl(pH7.5)、1mM EDTA、0.1% TritonX-100)にて洗浄後、5’-RACE PCR法を用いてラット免疫グロブリン重鎖及び軽鎖遺伝子の増幅を行った。即ち、磁気ビーズをPCR反応溶液(0.2μM プライマー、0.2mM dNTP、0.25unit PrimeSTAR HS DNA Polymerase(TAKARA社))に移し、94℃ 30秒-68℃ 90秒の反応を35サイクル行った。用いたプライマーセットは下記の通りである。
重鎖遺伝子増幅用 PCRプライマーセット
センスプライマー Nhe-polyC-S
5’-GCTAGCGCTACCGGACTCAGATCCCCCCCCCCCCCDN-3’図5(配列番号50)
1回目アンチセンスプライマー rIgγ-AS1
5’-TCACTGAGCTGGTGAGAGTGTAGAGCCC-3’図6(配列番号51)
2回目アンチセンスプライマー rIgγ-AS2
5’-TCACCGAGCTGCTGAGGGTGTAGAGCCC-3’図7(配列番号52)
軽鎖遺伝子増幅用 PCRプライマーセット
センスプライマー Nhe-polyC-S2
5’-GCTAGCGCTACCGGACTCAGATCCCCCCCCCCCCCDN-3’図8(配列番号53)
1回目アンチセンスプライマー rIgL-AS1
5’-TTCCACATCACTCGGGTAGAAATCAG-3’図9(配列番号54)
2回目アンチセンスプライマー rIgγ-AS2
5’-TAACACCAGGGTAGAAATCTGTCACCAT-3’図10(配列番号55)
上記PCR反応により増幅した断片について、塩基配列のシーケンス解析を実施した。
用いたプライマーは下記の通りである。
重鎖シーケンス用センスプライマー rIgγ-seq
5’-CTGGCTCAGGGAAATAGCC-3’図11(配列番号56)、
軽鎖用シーケンス用アンチセンスプライマー rIgL-seq1
5’-TCCCTGGAGCTCCTCAGT-3’図12(配列番号57)
軽鎖用シーケンス用アンチセンスプライマー rIgL-seq2
5’-GCCTTGTCAGTCTTGAGC-3’図13(配列番号58)
シークエンス解析は遺伝子配列解析装置(「ABI PRISM 3700 DNA Analyzer;Applied Biosystems」あるいは「Applied Biosystems 3730xl Analyzer;Applied Biosystems」)を用いて実施し、シークエンス反応は、Dye Terminator Cycle Sequencing System with AmpliTaq DNA polymerase(Life Technologies社)及びGeneAmp 9700(Applied Biosystems社)を用いた。
シーケンス解析により決定されたC3-147重鎖可変領域の塩基配列を図14(配列番号6)、アミノ酸配列を図15(配列番号7)、C3-147軽鎖可変領域の塩基配列を図16(配列番号8)、アミノ酸配列を図17(配列番号9)にそれぞれ記す。
(実施例4) ラット抗CD3 scFv(C3E-7000)、及びそのヒト化体(C3E-7034)の作製
4)-1 ラット抗CD3抗体(C3-147) scFvの作製
4)-1-1 ラット抗体CD3 scFv発現ベクター(pC3E-7000)の構築
C3-147重鎖可変領域(VH)と軽鎖可変領域(VL)の間に挿入するリンカーをコードするDNA配列の前後に15塩基の付加配列を有するDNA断片のセンス鎖オリゴヌクレオチド図18(配列番号10)及び、そのアンチセンス鎖オリゴヌクレオチド図19(配列番号11)を合成し(Sigma Aldrich社 カスタムオリゴ受託合成サービス)、100 pmol/μLに調整した後、それぞれ20μLずつ混合し、96℃で10分、70℃で2分、60℃で2分、40℃で2分、30℃で2分静置することにより、両者をアニーリングさせVHとVLの間に挿入するリンカー断片を作製した。次に動物細胞発現ベクターpcDNA-3.3TOPO(Thermo Scientific社)由来のベクター骨格にヒトIgG重鎖シグナル配列を付加するようにPCRで増幅したDNA断片と、図14(配列番号6)に示すラット抗CD3抗体C3-147のVHをPCRで増幅したDNA断片、VHとVLの間に挿入するリンカー断片、図16(配列番号8)に示すC3-147のVLを含む領域にFLAG-HisタグをコードするDNA配列をカルボキシル末端に付加させPCRで増幅したDNA断片をIn-Fusion HD クローニングキット(CLONTECH社)を用いて結合させ、図20(配列番号14)のヌクレオチド配列をORFに含むscFv発現ベクターpC3E-7000を作製した。
4)-1-2 ラット抗CD3 scFv(C3E-7000)の発現・精製
Expi293F細胞(Thermo Scientific社)はマニュアルに従い、継代、培養を行った。対数増殖期のExpi293F細胞に上記のscFv発現ベクターを導入して一過性発現させ、フィルターろ過した後、精製に用いた。精製はHis Trap excel(GE Healthcare社)を用いたNiアフィニティークロマトグラフィーと、Superdex 200 increase (GE Healthcare社)を用いたゲルろ過の2段階工程で行い、scFvモノマー分子量に相当するピークを回収し、精製蛋白質サンプルとした。精製にはAKTAクロマトグラフィーシステムを用い、全ての工程を4℃下で行なった。精製蛋白質のバッファーはHBSor(25mM ヒスチジン/5% ソルビトール、pH5.0)を用いた。精製蛋白質サンプルは分析用SECに供し、純度と濃度を決定したのち、各種アッセイに用いた。C3E-7000のアミノ酸配列は、図21(配列番号15)に記載されている。
4)-2 ラット抗CD3 scFv(C3E-7000)のヒト化
4)-2-1 抗CD3抗体のヒト化設計
ラット抗体の可変領域の分子モデリングは、ホモロジーモデリングとして公知の方法(Methods in Enzymology,203,121-153,(1991))に従い、市販の蛋白質立体構造解析プログラムDiscovery Studio3.5(Dassault Systems社)を用いて行った。
4)-2-2 C3E-7000のヒト化アミノ酸配列の設計
実施例4)-2-1に記載の手法に基づき、ヒトのサブグループ・コンセンサス配列のγ3及びλ6をアクセプターとして、C3E-7000のヒト化体となるC3E-7034のアミノ酸配列を設計した。図15(配列番号7)に示されるC3-147VHのアミノ酸配列のうち、アミノ酸番号16番目のアルギニンをグリシンに、アミノ酸番号17番目のアラニンをセリンに、アミノ酸番号19番目のリジンをアルギニンに、アミノ酸番号23番目のバリンをアラニンに、アミノ酸番号24番目のバリンをアラニンに、アミノ酸番号88番目のセリンをアラニンに、アミノ酸番号93番目のスレオニンをバリンに、置き換えることを伴い設計されたC3E-7034VHのアミノ酸配列は、図22(配列番号16)に記載されている。
IMGTのCDR定義におけるC3E-7000及びC3E-7034のCDR配列はそれぞれ、CDR-H1は図24(配列番号26)に、CDR-H2は図25(配列番号27)に、CDR-H3は図26(配列番号28)に、CDR-L1は図27(配列番号29)に、CDR-L2は図28(配列番号30)に、CDR-L3は図29(配列番号31)に記載されている。
4)-2-3 ヒト化抗CD3 scFvC3E-7034の改変
サルCD3εへの交差性を維持しつつ、結合活性や細胞傷害性活性に差を有するバリアントを作製するために、C3E-7034のVLに対して、実施例4)-2-1に記載の手法と同様にVLのフレームワーク領域のアミノ酸を、scFvのVL(IGLV1-40*01にA2S, S8P, V13A, F80Lの4変異が入った配列)に置き換えたバリアントを設計した。
4)-2-3-1 C3E-7035のアミノ酸配列設計
C3E-7034の改変体となるC3E-7035のアミノ酸配列を設計した。図23(配列番号17)に示されるC3E-7034の軽鎖のうち可変領域のアミノ酸番号1番目のアスパラギンをグルタミンに、アミノ酸番号2番目のフェニルアラニンをアラニンに、アミノ酸番号3番目のメチオニンをバリンに、アミノ酸番号8番目のヒスチジンをセリンに、アミノ酸番号12番目のグルタミン酸をグリシンに、アミノ酸番号13番目のセリンをバリンに、アミノ酸番号16番目のリジンをグルタミンに、アミノ酸番号17番目のスレオニンをアルギニンに、アミノ酸番号40番目のヒスチジンをロイシンに、アミノ酸番号41番目のグルタミン酸をプロリンに、アミノ酸番号43番目のセリンをスレオニンに、アミノ酸番号44番目のセリンをアラニンに、アミノ酸番号46番目のスレオニンをリジンに、アミノ酸番号47番目のスレオニンをロイシンに、アミノ酸番号48番目のイソロイシンをロイシンに、アミノ酸番号57番目のアスパラギン酸をセリンに、アミノ酸番号60番目のセリンをプロリンに、アミノ酸番号67番目のイソロイシンをリジンに、アミノ酸番号68番目のアスパラギン酸を欠損に、アミノ酸番号69番目のアルギニンを欠損に、アミノ酸番号71番目のセリンをグリシンに、アミノ酸番号72番目のリジンをスレオニンに、アミノ酸番号77番目のスレオニンをアラニンに、アミノ酸番号79番目のセリンをスレオニンに、アミノ酸番号80番目のアスパラギンをグリシンに、アミノ酸番号81番目のロイシンをフェニルアラニンに、アミノ酸番号82番目のリジンをグルタミンに、アミノ酸番号83番目のスレオニンをアラニンに、アミノ酸番号90番目のフェニルアラニンをチロシンに、置き換えることを伴い設計されたC3E-7035軽鎖のアミノ酸配列は、図30(配列番号20)に記載されており、軽鎖の可変領域の直前にメチオニンとアラニンが挿入されたC3E-7035の全長配列は図31(配列番号22)に記載されている。
4)-2-3-2 C3E-7036のアミノ酸配列設計
C3E-7034の改変体となるC3E-7036のアミノ酸配列を設計した。図23(配列番号17)に示されるC3E-7034の軽鎖のうち可変領域のアミノ酸番号8番目のヒスチジンをセリンに、アミノ酸番号12番目のグルタミン酸をグリシンに、アミノ酸番号13番目のセリンをバリンに、アミノ酸番号16番目のリジンをグルタミンに、アミノ酸番号17番目のスレオニンをアルギニンに、アミノ酸番号23番目のリジンをスレオニンに、アミノ酸番号24番目のアルギニンをグリシンに、アミノ酸番号40番目のヒスチジンをロイシンに、アミノ酸番号41番目のグルタミン酸をプロリンに、アミノ酸番号43番目のセリンをスレオニンに、アミノ酸番号44番目のセリンをアラニンに、アミノ酸番号46番目のスレオニンをリジンに、アミノ酸番号47番目のスレオニンをロイシンに、アミノ酸番号48番目のイソロイシンをロイシンに、アミノ酸番号57番目のアスパラギン酸をセリンに、アミノ酸番号60番目のセリンをプロリンに、アミノ酸番号67番目のイソロイシンをリジンに、アミノ酸番号68番目のアスパラギン酸を欠損に、アミノ酸番号69番目のアルギニンを欠損に、アミノ酸番号71番目のセリンをグリシンに、アミノ酸番号72番目のリジンをスレオニンに、アミノ酸番号77番目のスレオニンをアラニンに、アミノ酸番号79番目のセリンをスレオニンに、アミノ酸番号80番目のアスパラギンをグリシンに、アミノ酸番号81番目のロイシンをフェニルアラニンに、アミノ酸番号82番目のリジンをグルタミンに、アミノ酸番号83番目のスレオニンをアラニンに、アミノ酸番号90番目のフェニルアラニンをチロシンに、置き換えることを伴い設計されたC3E-7036軽鎖のアミノ酸配列は、図32(配列番号23)に記載されており、C3E-7036の全長アミノ酸配列は図33(配列番号25)に記載されている。
4)-2-3-3 CDR改変体のアミノ酸配列設計
C3E-7034のCDRH2(図25、配列番号27)に存在する脱アミノ化部位を除去する目的で、図22(配列番号16)に示されるC3E-7034の重鎖可変領域のアミノ酸番号53番目のアスパラギンをアルギニンに置き換えたC3E-7078、およびセリンに置き換えたC3E-7079を設計した。また、C3E-7036の重鎖可変領域のアミノ酸番号53番目のアスパラギンをアルギニンに置き換えたC3E-7085を設計した。C3E-7078の全長アミノ酸配列は図_68(配列番号60)に、C3E-7079の全長アミノ酸配列は図70(配列番号62)に、C3E-7085の全長アミノ酸配列は図72(配列番号64)に記載されている。
さらにC3E-7078のヒトCD3親和性を低下させる目的で、C3E-7078の軽鎖可変領域のアミノ酸番号52番目のアスパラギン酸をグリシンに置き換えたC3E-7086、グルタミンに置き換えたC3E-7087、アスパラギンに置き換えたC3E-7088、セリンに置き換えたC3E-7089、アラニンに置き換えたC3E-7090を設計した。同様に、C3E-7079のヒトCD3親和性を低下させる目的で、C3E-7079の軽鎖可変領域のアミノ酸番号52番目のアスパラギン酸をグリシンに置き換えたC3E-7091、グルタミンに置き換えたC3E-7092、アスパラギンに置き換えたC3E-7093、セリンに置き換えたC3E-7094、アラニンに置き換えたC3E-7095を設計した。C3E-7086の全長アミノ酸配列は図74(配列番号66)に、C3E-7087の全長アミノ酸配列は図76(配列番号68)に、C3E-7088の全長アミノ酸配列は図78(配列番号70)に、C3E-7089の全長アミノ酸配列は図80(配列番号72)に、C3E-7090の全長アミノ酸配列は図82(配列番号74)に、C3E-7091の全長アミノ酸配列は図84(配列番号76)に、C3E-7092の全長アミノ酸配列は図86(配列番号78)に、C3E-7093の全長アミノ酸配列は図88(配列番号80)に、C3E-7094の全長アミノ酸配列は図90(配列番号82)に、C3E-7095の全長アミノ酸配列は図92(配列番号84)に記載されている。
4)-3 ヒト化抗CD3 scFv(C3E-7034、C3E-7035, C3E-7036)の作製
4)-3-1 ヒト化抗CD3 scFv(C3E-7034)発現ベクターpC3E-7034の構築
図22(配列番号16)に示すC3E-7034重鎖のカルボキシル末端に15アミノ酸フレキシブルリンカーを介して図23(配列番号17)に示すC3E-7034軽鎖を含む領域をつなげたscFvDNA配列、及び前後15塩基の付加配列を含むDNA断片を合成した(GENEART社)。これを鋳型としてC3E-7034と前後の付加配列を含む領域をPCR法を用いて増幅し、インサートDNA断片を得た。また実施例4)-1-1で作製した発現ベクターpC3E-7000を鋳型として、scFv領域を除いたベクター領域をPCR法を用いて増幅し、ベクター断片を得た。それぞれのDNA断片をIn-Fusion HD クローニングキット(CLONTECH社)を用いて結合させ、図34(配列番号18)のヌクレオチド配列をORFに含むヒト化抗CD3 scFv発現ベクターpC3E-7034を作製した。
4)-3-2 ヒト化抗CD3 scFv(C3E-7035)発現ベクターpC3E-7035の構築
図22(配列番号16)に示すC3E-7034重鎖のカルボキシル末端に17アミノ酸から成るフレキシブルリンカーを介して図30(配列番号20)に示すC3E-7035軽鎖をつなげたscFvDNA配列及び前後15塩基の付加配列を含むDNA断片を合成した(GENEART社)。実施例4)-3-1と同様の方法で図35(配列番号21)のヌクレオチド配列をORFに含むC3E-7035発現ベクターを構築した。得られた発現ベクターを「pC3E-7035」と命名した。
4)-3-2 ヒト化抗CD3 scFv(C3E-7036)発現ベクターpC3E-7036の構築
図22(配列番号16)に示すC3E-7034重鎖のカルボキシル末端に15アミノ酸から成るフレキシブルリンカーを介して図32(配列番号23)に示すC3E-7036軽鎖をつなげたscFvDNA配列及び前後15塩基の付加配列を含むDNA断片を合成した(GENEART社)。実施例4)-3-1と同様の方法で図36(配列番号24)のヌクレオチド配列をORFに含むC3E-7036発現ベクターを構築した。得られた発現ベクターを「pC3E-7036」と命名した。
4)-3-4CDR改変ヒト化抗体CD3 scFvの発現ベクターの構築
図34(配列番号18)に示すC3E-7034のヌクレオチド配列をORFに含むpC3E-7034を鋳型とし、図93、94(配列番号85、86)に示す塩基配列を有するプライマーを用いて、PCRを用いた部位特異的変異導入を行い、C3E-7034の重鎖可変領域のアミノ酸番号53番目のアスパラギンをアルギニンに置き換えたC3E-7078のヌクレオチド配列をORFに含むC3E-7078発現ベクターを作製した。得られた発現ベクターを「pC3E-7078」と命名した。同様にpC3E-7034を鋳型とし、図95、96(配列番号87、88)をプライマーとして、PCRを用いた部位特異的変異導入を行い、C3E-7034の重鎖可変領域のアミノ酸番号53番目のアスパラギンをセリンに置き換えたC3E-7079のヌクレオチド配列をORFに含むC3E-7079発現ベクターを作製した。得られた発現ベクターを「pC3E-7079」と命名した。同様にpC3E-7036を鋳型とし、図93,94(配列番号85、86)をプライマーとして、PCRを用いた部位特異的変異導入を行い、C3E-7036の重鎖可変領域のアミノ酸番号53番目のアスパラギンをアルギニンに置き換えたC3E-7085のヌクレオチド配列をORFに含むC3E-7085発現ベクターを作製した。得られた発現ベクターを「pC3E-7085」と命名した。
C3E-7034、C3E-7035、C3E-7036の発現・精製は、実施例4)-1-2と同様の方法で行なった。
4)-3-6 CDR改変ヒト化抗CD3 scFvの発現・精製
C3E-7078、C3E-7079、C3E-7085、C3E-7086、C3E-7087、C3E-7088、C3E-7089、C3E-7090、C3E-7091、C3E-7092、C3E-7093、C3E-7094、C3E-7095の各CDR改変体の発現・精製は、実施例4)-1-2と同様の方法で行なった。
(実施例5) ヒト化抗CD3 scFv(C3E-7034)の結晶構造解析
5)-1 ヒト化抗CD3 scFv(C3E-7034)-ヒトCD3εγ一本鎖抗原複合体の調製
実施例2)-2-1で作製したCD3εγと実施例4)-3-1で作製したC3E-7034をモル比1:2で混合し、AmiconUltra15 MWCO 10キロ(Millipore社)で10mM Tris HCl(pH7.5)、50mM NaClに緩衝液を置換し、3.5mg/mLに濃縮した。これをSuperdex200 10/300GL(GE Healthcare)を用いてゲルろ過クロマトグラフィーで精製し、複合体の画分をAmiconUltra15MWCO 10キロ(Millipore社)を用いて約4.0mg/mLに濃縮した。
5)-2 結晶化
得られたCD3εγとC3E-7034の複合体を蒸気拡散法により結晶化した。蛋白質溶液0.5μLに沈殿剤溶液(0.1M MES monohydrate(pH 6.5)、1.6M Ammonium sulfate、10% v/v 1,4-Dioxane)を等量加えた溶液を、0.05mLの沈殿剤溶液を入れた密閉容器に両溶液が触れ合わないように収め、25℃で静置した。1ヶ月後に0.1mm×0.05mm×0.05mmの棒状晶が得られた。
5)-3 X線結晶構造解析とエピトープの同定
得られた結晶をPerfluoropolyether PFO-X175/08 (Hampton Research)に浸し、続いて液体窒素で凍結した。ビームラインBL41XU (SPring-8, Hyogo)にてX線回折デ―タを収集した。得られた回折像からソフトウェアimosflm(CCP4:Collaborative Computational Project No.4)を用いて回折強度を数値化し、結晶構造因子を求めた。結晶は六方晶系で空間群はP62、結晶の単位格子はa=193.54 Å、b=193.54 Å、c=43.88 Åであった。
ソフトウェアRefmac5(CCP4:Collaborative Computational Project No.4)を用いて構造の精密化を行い、ソフトウェアcootを用いてモデルの修正を行った。この操作を繰り返し行い、3.3 Å分解能で最終のR値22.1%、free R値27.0%を得た。最終のモデルはC3E-7034の軽鎖領域(図23、配列番号17)のアミノ酸残基1-108、C3E-7034の重鎖領域(図22、配列番号16)のアミノ酸残基1-118、CD3ε領域(図1、配列番号1)のアミノ酸残基33-67及び71-118、及びCD3γ領域(図37、配列番号3)のアミノ酸残基23-103を含む。CD3ε領域(図1、配列番号1)のアミノ酸残基68-70、及びC3E-7034 (図38、配列番号19)のアミノ末端領域(アミノ酸残基1)、リンカー部分(アミノ酸残基120-134)、及びカルボキシル末端領域(アミノ酸残基243-269)はそれぞれ電子密度が明瞭でなかったためモデル構築していない。図39に複合体全体のリボンモデルと表面を示す。
図40にCD3εとC3E-7034の軽鎖及び重鎖との相互作用を示す。パネルAはC3E-7034の軽鎖可変領域から4Å以内の距離にあるCD3εのアミノ酸残基を太いスティックモデルで、それ以外のアミノ酸残基を細いスティックモデルで表示した図である。図中において四角内に残基名と残基番号が標識されたSer55、Glu56、Arg101、Gly102、Ser103、Lys104、及びPro105はC3E-7034の軽鎖可変領域から4Å以内の距離にあるCD3εのアミノ酸残基であり、各アミノ酸番号は配列表の配列番号1に対応している。パネルBはC3E-7034の重鎖可変領域から4Å以内の距離にあるCD3εのアミノ酸残基を太いスティックモデルで、それ以外のアミノ酸を細いスティックモデルで表示した図である。図中において四角内に残基名と残基番号が標識されたSer55、Glu56、Leu58、Trp59、Asn65、Ile66、Ser77、Asp78、及びArg101はCD3εのアミノ酸であり、各アミノ酸番号は配列表の配列番号1に対応している。C3E-7034から4Å以内の距離にあり、C3E-7034に対するCD3εのエピトープと解釈されるアミノ酸残基は次のとおりであった:Ser55、Glu56、Leu58、Trp59、Asn65、Ile66、Ser77、Asp78、Arg101、Gly102、Ser103、Lys104、及びPro105。
(実施例6) ヒト化OKT3 scFvの作製
6)-1 OKT3 scFv発現ベクターpC3E-3000の構築
実施例4)-1-1と同様の手法でマウス抗CD3モノクローナル抗体OKT3(Sgro、Toxicology 105(1995)、23~29、Orthoclone、Janssen-Cilag社)のscFvを作製し、pcDNA3.3由来動物細胞発現ベクターに導入した。得られた発現ベクターを「pC3E-3000」と命名した。
6)-2 OKT3 scFv(C3E-3000)のヒト化アミノ酸配列の設計
実施例4)-2-1に記載の手法に基づき、ヒトのサブグループ・コンセンサス配列のgamma1及びkappa4をアクセプターとして、OKT3のヒト化体となるC3E-3007のアミノ酸配列を設計した。なお、免疫原性スコア及び物性への影響を勘案して、一部の箇所においてはkappa1のアミノ酸を導入した。図42(配列番号36)に示されるOKT3の重鎖のうち可変領域のアミノ酸番号5番目のグルタミンをバリンに、アミノ酸番号11番目のロイシンをセリンに、アミノ酸番号12番目のアラニンをリジンに、アミノ酸番号13番目のアルギニンをリジンに、アミノ酸番号20番目のメチオニンをバリンに、アミノ酸番号38番目のリジンをアルギニンに、アミノ酸番号40番目のアルギニンをアラニンに、アミノ酸番号48番目のイソロイシンをメチオニンに、アミノ酸番号67番目のリジンをアルギニンに、アミノ酸番号68番目のアラニンをバリンに、アミノ酸番号70番目のロイシンをイソロイシンに、アミノ酸番号72番目のスレオニンをアラニンに、アミノ酸番号76番目のセリンをスレオニンに、アミノ酸番号82番目のグルタミンをグルタミン酸に、アミノ酸番号87番目のスレオニンをアルギニンに、アミノ酸番号91番目のセリンをスレオニンに、アミノ酸番号114番目のスレオニンをロイシンに、アミノ酸番号115番目のロイシンをバリンに、置き換えることを伴い設計されたC3E-3007重鎖のアミノ酸配列は、図43(配列番号38)に記載されている。
6)-3 ヒト化OKT3 scFv(C3E-3007)発現ベクターpC3E-3007の構築
図43(配列番号38)に示すC3E-3007重鎖のカルボキシル末端に15アミノ酸フレキシブルリンカーを介して図45(配列番号39)に示すC3E-3007軽鎖を含む領域をつなげたscFvDNA配列及び前後15塩基の付加配列を含むDNA断片を合成した(GENEART社)。実施例4)-3-1と同様の方法で図46(配列番号34)のヌクレオチド配列をORFに含むC3E-3007発現ベクターを構築した。得られた発現ベクターを「pC3E-3007」と命名した。
6)-4 ヒト化OKT3 scFv(C3E-3007)の発現・精製
C3E-3007の発現・精製は、実施例4)-1-2と同様の方法で行なった。C3E-3007のアミノ酸配列は、図47(配列番号35)に記載されている。
(実施例7) ヒト化抗CD3 scFvのin vitro活性7)- 1 ヒト化抗CD3 scFv(C3E-3007、C3E-7034、C3E-7035、C3E-7036)のヒトCD3との結合性検討
7)-1-1 ヒト化抗CD3 scFv(C3E-3007、C3E-7034、C3E-7035、C3E-7036)のフローサイトメトリーによるヒトCD3への結合性検討
市販ヒトPBMC(CTL社)を5% FBS含有PBSで適当な濃度に調製し、LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit(Thermo Fisher Scientific社)と抗CD19抗体(Beckman Coulter社)を添加し、4℃で30分静置した。5% FBS含有PBSで2回洗浄後、5% FBS含有PBSで1×106細胞/mLの濃度に調製し、100μL/wellで96-well U底マイクロプレートに播種し、遠心後上清を除去した。5% FBS含有PBSで希釈したヒト化抗CD3 scFv(C3E-3007、C3E-7034、C3E-7035、C3E-7036)を100μL/well添加し、4℃で60分静置した。5% FBS含有PBSで2回洗浄後、5% FBS含有PBSで希釈したPenta-His Alexa Fluor 488(QIAGEN社)を30μL/well添加し、4℃で30分静置した。5% FBS含有PBSで2回洗浄後、5% FBS含有PBSで再懸濁し、フローサイトメーター(FACSCanto(商標) II:BD社)で検出を行った。データ解析はFlowjo(Treestar社)で行い、死細胞とCD19陽性細胞を除去した画分のAlexa Fluor 488の平均蛍光強度(MFI)を算出し、scFv添加サンプルのMFI値から抗体未添加サンプルのMFI値を引いて、MFI値の相対値(rMFI)を計算した。図48に示す通り、ヒト化抗CD3 scFvはヒトCD3に結合することが示された。
7)-1-2 ヒト化抗CD3 scFv(C3E-3007、C3E-7034、C3 E-7035、C3E-7036)のSPRによるヒトCD3への結合性検討
ヒト化抗CD3 scFv(C3E-3007、C3E-7034、C3E-7035、C3E-7036)のCD3に対するアフィニティーはBIAcore T-200(GE Healthcare社)を用いた表面プラズモン共鳴法により決定した。センサーチップ上に固定化したCD3に異なる5つの濃度の該scFvを流し、得られたレスポンスからRmaxを推定して、その1/2を与える抗体濃度を該scFvのCD3に対する解離定数とした。その結果、該scFvのCD3に対する解離定数はそれぞれ400、4.5、22、25nMであった。
7)-2 ヒト化抗CD3 scFv(C3E-3007、C3E-7034、C3E-7036)のカニクイザルCD3との結合性検討
7)-2-1 カニクイザルPBMCの調製
SepMate(STEMCELL社)とLymphocyte Separation Solution(ナカライテスク社)を使用して、カニクイザルの血液からPBMCを定法に従い採取した。
7)-2-2 ヒト化抗CD3 scFv(C3E-3007、C3E-7034、C3E-7035、C3E-7036)のフローサイトメトリーによるカニクイザルCD3への結合性検討
実施例7)-2-1で取得したカニクイザルPBMCを5% FBS含有PBSで適当な濃度に調製し、実施例7)-1-1と同様の方法で染色、解析を実施した。図49に示す通り、ヒト化抗CD3 scFv(C3E-7034、C3E-7035、C3E-7036)はカニクイザルCD3に結合することが示された。
7)-3 ヒト化抗CD3 scFv(C3E-3007、C3E-7034)のT細胞活性化
ヒト末梢血単核細胞(PBMC)を、Lympholyte-H(Cedarlane社)を用いた濃度勾配密度分離法により、無作為のドナーの新鮮なバフィーコートより単離した。LR10(10% ultra low IgG FBS含有RPMI1640(Thermo Fisher Scientific社))で100nMに希釈したヒト化抗CD3 scFv(C3E-3007、C3E-7034)と、同濃度のAnti-His antibody(Qiagen社)を同量混和した。ヒトあるいはサルPBMCをLR10にて2×105細胞に調製し、96-well U底マイクロプレートに、Anti-His antibodyを混和したヒト化抗CD3 scFvと同量混和し、37℃で24時間、5%CO2条件下にて培養した。反応終了後、遠心し、sorter buffer(HBSS(―)(Thermo Fisher Scientific社)、0.1% BSA(Sigma-Aldrich社)、0.1% sodium azide(Sigma-Aldrich社))を添加し、遠心後、細胞にLIVE/DEAD Fixable Near-IR Dead Cell Stain Kit(Thermo Fisher Scientific社)を添加し、4℃で20分静置した。Sorter bufferで洗浄後、sorter bufferで希釈したPEラベル抗CD69抗体(Becton、Dickinson社)、FITCラベル抗CD8抗体(Becton、Dickinson社)を添加し、4℃で20分静置した。Sorter bufferで洗浄後、1% paraformaldehyde含有PBS(和光純薬工業社)で再懸濁し、フローサイトメーター(FACSCanto II:Becton、Dickinson社)で検出を行った。データ解析はFlowjo(Treestar社)で行い、死細胞を除去した画分から、CD8高発現かつPE高発現画分の割合を母集団からの百分率(% of parents)として算出した。図50に示す通り、ヒト化抗CD3 scFvはヒト及びサルCD8高発現細胞を活性化することが示された。
7)-4 CDR改変ヒト化抗CD3 scFvのフローサイトメトリーによるヒト及びカニクイザルCD3への結合性比較
実施例7)-1-1で取得したヒトPBMC、及び7)-2-1で取得したカニクイザルPBMCを5% FBS含有PBSで適当な濃度に調製し、実施例7)-1-1と同様の方法で染色、解析を実施した。図106で示す通り、CDR改変ヒト化抗CD3 scFvのヒト及びサルCD3に対する結合性が確認された。
(実施例8) ヒト化抗TROP2 scFvの作製
8)-1 HT1-11 scFv発現ベクターpHT1-11scFvの構築
図51(配列番号41)に示すHT1-11 scFvのアミノ酸をコードするDNA配列を含むDNA断片を合成した(GENEART社)。In-Fusion HD PCRクローニングキット(Clontech社)を用いて、pcDNA-3.3TOPO(Thermo Scientific社)由来のベクターに合成したDNA断片を挿入することにより図52(配列番号40)に示すヌクレオチド配列をORFに含むヒト化抗TROP2 scFv発現ベクターpHT1-11scFvを構築した。
8)-2 HT1-11 scFvの発現・精製
HT1-11scFvの発現・精製は、実施例4)-1-2と同様の方法で行なった。
(実施例9) ヒト化抗TROP2 scFv(HT1-11scFv)のフローサイトメトリーによるヒトTROP2への結合性評価
咽頭扁平上皮癌細胞株FaDu(ATCC)と膵臓癌細胞株HPAF-II(ATCC)を5% FBS含有PBSで適当な濃度に調製し、LIVE/DEAD Fixable Near-IR Dead Cell Stain Kitを添加し、4℃で30分静置した。5% FBS含有PBSで2回洗浄後、5% FBS含有PBSで1×106細胞/mLの濃度に調製し、100μL/wellで96-well U底マイクロプレートに播種し、遠心後上清を除去した。5% FBS含有PBSで希釈したヒト化抗TROP2 scFv(HT1-11scFv)を100μL/well添加し、4℃で60分静置した。5% FBS含有PBSで2回洗浄後、5% FBS含有PBSで希釈したPenta-His Alexa Fluor 488を30μL/well添加し、4℃で30分静置した。5% FBS含有PBSで2回洗浄後、5% FBS含有PBSで再懸濁し、フローサイトメーター(FACSCanto(商標) II)で検出を行った。データ解析はFlowjoで行い、死細胞を除去した画分のAlexa Fluor 488の平均蛍光強度(MFI)を算出し、scFv添加サンプルのMFI値から抗体未添加サンプルのMFI値を引いて、MFI値の相対値(rMFI)を計算した。図53に示す通り、ヒト化抗TROP2 scFvはヒトTROP2に結合することが示された。
(実施例10) 抗TROP2-CD3二重特異性分子の作製
10)-1 抗TROP2-CD3二重特異性分子発現ベクターの構築
10)-1-1 HT1-11scFv/C3E-7034二重特異性分子(T2C-0001)発現ベクターの構築
実施例8)-1で作製したpHT1-11scFvを鋳型として5’側にHT1-11scFvとヒト抗体重鎖シグナル配列の一部、3’側にscFv間を繋ぐリンカーが付加するようにデザインしたプライマーを用いてPCRを行い、インサートDNA断片を得た。また4)-3-1で作製した発現ベクターpC3E-7034を鋳型として、シグナル配列、及び抗CD3 scFvのアミノ末端配列をコードするプライマーを用いてPCRを行い、抗CD3 scFvを含むベクター全領域を含むベクターDNA断片を得た。それぞれのDNA断片をIn-Fusion HD クローニングキット(CLONTECH社)を用いて結合させ、図54(配列番号42)のヌクレオチド配列をORFに含む抗TROP2-CD3二重特異性分子発現ベクターpT2C-0001を作製した。
10)-1-2 HT1-11scFv/C3E-3007二重特異性分子(T2C-0003)発現ベクターの構築
実施例10)-1-1と同様の方法で図55(配列番号44)のヌクレオチド配列をORFに含む抗TROP2-CD3二重特異性分子発現ベクターを構築した。ただし、ベクター断片を作製する際の鋳型としてpC3E-3007を用いた。得られた発現ベクターを「pT2C-0003」と命名した。
10)-1-3 HT1-11scFv/C3E-7035二重特異性分子(T2C-0005)発現ベクターの構築
実施例10)-1-1と同様の方法で図56(配列番号46)のヌクレオチド配列をORFに含む抗TROP2-CD3二重特異性分子発現ベクターを構築した。ただし、ベクター断片を作製する際の鋳型としてpC3E-7035を用いた。得られた発現ベクターを「pT2C-0005」と命名した。
10)-1-4 HT1-11scFv/C3E-7036二重特異性分子(T2C-0006)発現ベクターの構築
実施例10)-1-1と同様の方法で図57(配列番号48)のヌクレオチド配列をORFに含む抗TROP2-CD3二重特異性分子発現ベクターを構築した。ただし、ベクター断片を作製する際の鋳型としてpC3E-7036を用いた。得られた発現ベクターを「pT2C-0006」と命名した。
10)-2 抗TROP2-CD3二重特異性分子の発現・精製
T2C-0001、T2C-0003、T2C-0005、T2C-0006の発現・精製は、実施例4)-1-2と同様の方法で行なった。T2C-0001のアミノ酸配列は、図58(配列番号43)に記載されている。T2C-0003のアミノ酸配列は、図59(配列番号45)に記載されている。T2C-0005のアミノ酸配列は、図60(配列番号47)に記載されている。T2C-0006のアミノ酸配列は、図61(配列番号49)に記載されている。
(実施例11) 抗TROP2-CD3二重特異性分子のin vitro活性評価
11)-1 SPRによる結合活性評価
11)-1-1 抗TROP2-CD3二重特異性分子のTROP2への結合
二重特異性分子とTROP2との結合は、BIAcore 3000(GE Healthcare Bioscience社)を使用し、抗ヒトIgG抗体にて捕捉(キャプチャー)した抗原に対して、二重特異性分子をアナライトとして測定するキャプチャー法にて測定した。抗原は、組み換えヒトTROP-2/ヒトIgG Fc融合体(R&D SYSTEMS)を使用した。抗ヒトIgG(Fc)抗体(Human Antibody Capture Kit、GE Healthcare Bioscience社)は、センサーチップCM5(GE Healthcare Bioscience社)へアミンカップリング法にて約2000RU共有結合させた。リファレンスセルにも同様に固定化した。ランニングバッファーとしてHBS-P(10mM HEPES pH7.4、0.15M NaCl,0.005% Surfactant P20)を用いた。二重特異性分子は、200nMから2倍希釈にて1nMまで調製した。抗ヒトIgG(Fc)抗体を固定化したチップ上に、1μg/mlの抗原を約30秒間添加した後、各濃度の二重特異性分子を流速30μl/分で300秒間添加し、引き続き600秒間の解離相をモニターした。再生溶液として、3M塩化マグネシウム溶液を30秒間添加した。データの解析には、分析ソフトウ ェア(BIAevaluation software, version 4.1.1)の 1:1結合モデルを用いて、結合速度定数ka、解離速度定数kd及び解離定数(KD; KD=kd/ka)を算出した。
11)-1-2 抗TROP2-CD3二重特異性分子のヒトCD3εγ一本鎖抗原への結合
二重特異性分子とCD3εγ抗原との結合は、BIAcore 3000(GE Healthcare Bioscience社)を使用し、固定化した抗原に対して、抗体をアナライトとして測定する方法にて測定した。抗原には、2)-2-1にて調製したヒトCD3εγ一本鎖抗原を使用し、センサーチップCM5(GE Healthcare Bioscience社)へアミンカップリング法にて約100RU共有結合させた。リファレンスセルには抗原蛋白を添加せず固定化処理のみ行った。ランニングバッファーとしてHBS-P(10mM HEPES pH7.4、0.15M NaCl,0.005% Surfactant P20)を用いた。二重特異性分子は、最高濃度1μMから2倍希釈にて4nMまで、あるいは200nMから2倍希釈にて1nMまで調製した。抗原を固定化したチップ上に、各濃度の二重特異性分子を流速10μl/分で25分間添加し、結合量をモニターした。再生溶液として、10mMグリシン塩酸溶液pH1.5を30秒間添加した。データの解析には、分析ソフトウ ェア(BIAevaluation software, version 4.1.1)を用いて、各濃度における結合量から解離定数KDを算出した。結果を表2、表3に記載した。
11)-2-1 抗TROP2-CD3二重特異性分子のTROP2への結合
実施例9と同様の癌細胞株を用いて、同様の方法で染色、解析を実施した。図62に示す通り、抗TROP2-CD3二重特異性分子はTROP2に結合することが示された。
11)-2-2 抗TROP2-CD3二重特異性分子のヒトCD3εγ一本鎖抗原への結合
実施例7)-1-1と同様の方法で染色、解析を実施した。図63に示す通り、抗TROP2-CD3二重特異性分子はヒトCD3εγ一本鎖抗原に結合することが示された。
11)-2-3 抗TROP2-CD3二重特異性分子のカニクイザルCD3抗原への結合
実施例7)-2-1で取得したカニクイザルPBMCを5% FBS含有PBSで適当な濃度に調製し、実施例7)-1-1と同様の方法で染色、解析を実施した。図64に示す通り、抗TROP2-CD3二重特異性分子(T2C-0001、T2C-0005、T2C-0006)はカニクイザルCD3抗原に結合することが示された。
11)-3 抗TROP2-CD3二重特異性分子の細胞傷害活性評価
11)-3-1 標的細胞におけるTROP2の発現解析
咽頭扁平上皮癌細胞株FaDu(ATCC)、膵臓癌細胞株HPAF-II(ATCC)、及びヒト肺癌細胞株Calu-6を5% FBS含有PBSで適当な濃度に調製し、LIVE/DEAD Fixable Dead Cell Stain Kit(Thermo Fisher Scientific社)を添加し、4℃で30分静置した。5% FBS含有PBSで2回洗浄後、5% FBS含有PBSで2×106細胞/mLの濃度に調製し、100μL/wellで96-well U底マイクロプレートに播種し、遠心後上清を除去した。5% FBS含有PBSで希釈した抗TROP2 Alexa Fluor 488抗体(eBioscience社)及びIsotype Control抗体(eBioscience社)を25μL/well添加し、4℃で30分静置した。5% FBS含有PBSで2回洗浄後、5% FBS含有PBSで再懸濁し、フローサイトメーター(Cytomics FC500、BeckmanCoulter社)で検出を行った。データ解析はFlowjo(Treestar社)で行い、死細胞除去画分のAlexa Fluor 488の幾何学的平均蛍光強度(geometric MFI)を算出した。図65 (A、B、C)に示す通り、FaDu、及びHPAF-IIにおいてTROP2の発現が認められ、Calu-6においては発現が認められなかった。
11)-3-2 標的細胞の調製
FaDu、HPAF-II、及びCalu-6を10% FBS含有RPMI1640培地(Thermo Fisher Scientific社)で2×106細胞/mLの濃度に調製し、細胞懸濁液1mLあたり100μLのChromium-51 Radionuclide(PerkinElmer社)を添加し、37℃、5% CO2の条件下で2時間培養した。10% FBS含有RPMI1640培地で2回洗浄した後、10% FBS含有RPMI1640培地で2×105細胞/mLになるよう再懸濁したものを標的細胞として用いた。
11)-3-3 エフェクター細胞の調製
市販の凍結PBMC(Cellular Technology Limited社)を37℃で解凍し、10% FBS含有RPMI1640培地にAnti-aggregate Wash試薬(Cellular Technology Limited社)を添加した溶液に移して2回洗浄した後に10% FBS含有RPMI1640培地で1×106細胞/mLになるよう調製し、エフェクター細胞とした。
11)-3-4 細胞傷害アッセイ
実施例11)-3-2で取得したFaDu、HPAF-II、及びCalu-6を50μL/wellで96-well U底マイクロプレートに添加した。そこに各濃度に調製した各種抗TROP2-CD3二重特異性分子を50μL/wellで添加し、11)-3-1-3で調製したエフェクター細胞を100μL/well添加し、室温で1000rpm×1分間遠心後、37℃、5% CO2の条件下で20-24時間培養した。上清50μLをLumaPlate(PerkinElmer社)に回収し、50℃で約2時間乾燥させた後、プレートリーダー(TopCount:PerkinElmer社)で測定した。細胞溶解率は次式で算出した。
細胞溶解率(%)=(A-B)/(C-B)×100
A:サンプルウェルのカウント。
B:バックグラウンド(抗体非添加ウェル)カウントの平均値(n=3)。抗体添加時にアッセイ用培地を50μL添加した。それ以外はサンプルウェルと同様の操作を行った。
C:最大放出(標的細胞を界面活性剤で溶解させたウェル)カウントの平均値(n=3)。抗体添加時にアッセイ培地を50μL添加した。界面活性剤は100μL添加し、サンプルウェルと同様に50μL分をLumaPlateに移して測定を実施した。
図66 (A、B、C)に示す通り、FaDu、及びHPAF-IIに対する各種TROP2-CD3二重特異性分子の細胞傷害活性が示された)。一方Calu-6に対する細胞傷害活性は認められなかった。
(実施例12) 抗Axl-CD3二重特異性分子の作製
12)-1 抗Axl-CD3二重特異性分子発現ベクターの構築
12)-1-1 11D5-T3scFv/C3E-7034二重特異性分子(AXC-0001)発現ベクターの構築
h#11D5-T3H(欧州特許出願公開第2270053号明細書のFigure 12に記載)のN末端にグリシンを加えた配列と、h#11D5-T3L(欧州特許出願公開第2270053号明細書のFigure 6に記載)の配列を、(GGGGS)を三回繰り返した配列からなるポリペプチドリンカーを介して連結した抗Axl一本鎖抗体、11D5-T3scFvのアミノ酸配列を設計し、これをコードするヌクレオチド配列を合成した(GENEART,Thermo Fisher Science社)。これを鋳型として5‘側にヒト抗体重鎖シグナル配列の一部、3’側にscFv間を繋ぐリンカーが付加するようにデザインしたプライマーを用いてPCRを行い、インサートDNA断片を得た。また4)-3-1で作製した発現ベクターpC3E-7034を鋳型として、ヒト抗体重鎖シグナル配列、及び抗CD3 scFvをコードするヌクレオチド配列からなるプライマーを用いて抗CD3 scFvを含むベクター全領域をPCR法により増幅し、ベクターDNA断片を得た。それぞれのDNA断片をIn-Fusion HD クローニングキット(CLONTECH社)を用いて結合させ、図97(配列番号89)に示すヌクレオチド配列をORFに含む抗Axl-CD3二重特異性分子発現ベクターpAXC-0001を作製した。
12)-1-2 11D5-T3scFv/C3E-7036二重特異性分子(AXC-0002)発現ベクターの構築
実施例10)-1-1と同様の方法で図99(配列番号91)に示すヌクレオチド配列をORFに含む抗Axl-CD3二重特異性分子発現ベクターを構築した。ただし、ベクター断片を作製する際の鋳型としてpC3E-7036を用いた。得られた発現ベクターを「pAXC-0002」と命名した。
12)-2 抗Axl-CD3二重特異性分子の発現・精製
AXC-0001、AXC-0002の発現・精製は、実施例4)-1-2と同様の方法で行なった。AXC-0001のアミノ酸配列は、図98(配列番号90)に記載されている。AXC-0002のアミノ酸配列は、図100(配列番号92)に記載されている。
(実施例13) 抗Axl-CD3二重特異性分子のin vitro活性評価
13)-1 標的細胞におけるAxlの発現解析
ヒト肺癌細胞株A549(ATCC)、ヒト膵臓癌細胞株PANC-1(ATCC)、MIA PaCa-2(ATCC)、ヒト骨髄腫細胞株U266B1(ATCC)、マントル細胞リンパ腫細胞株Jeko-1(ATCC)を5% FBS含有PBSで適当な濃度に調製し、LIVE/DEAD Fixable Dead Cell Stain Kit(Thermo Fisher Scientific社)を添加し、4℃で30分静置した。5% FBS含有PBSで2回洗浄後、5% FBS含有PBSで2×106細胞/mLの濃度に調製し、100μL/wellで96-well U底マイクロプレートに播種し、遠心後上清を除去した。5% FBS含有PBSで希釈した抗Axl抗体(RD-SYSTEMS社)及びIsotype Control抗体(RD-SYSTEMS社)を25μL/well添加し、4℃で30分静置した。5% FBS含有PBSで2回洗浄後、5% FBS含有PBSで希釈したAlexa Fluor488抗マウスIgG抗体(Thermo Fisher Scientific社)を25μL/well添加し、4℃で30分静置した。5% FBS含有PBSで2回洗浄後、5% FBS含有PBSで再懸濁し、フローサイトメーター(Cytomics FC500、BeckmanCoulter社)で検出を行った。データ解析はFlowjo(Treestar社)で行い、死細胞除去画分のAlexa Fluor 488の幾何学的平均蛍光強度(geometric MFI)を算出した。図107(A、B、C、D、E)に示す通り、A549、PANC-1、及びMIA PaCa-2においてAxlの発現が認められ、U266B1、Jeko-1においては発現が認められなかった。
13)-2 標的細胞の調製
A549、PANC-1、MIA PaCa-2、U266B1、及びJeko-1を10% FBS含有RPMI1640培地(Thermo Fisher Scientific社)で2×106細胞/mLの濃度に調製し、細胞懸濁液1mLあたり100μLのChromium-51 Radionuclide(PerkinElmer社)を添加し、37℃、5% CO2の条件下で2時間培養した。10% FBS含有RPMI1640培地で2回洗浄した後、10% FBS含有RPMI1640培地で2×105細胞/mLになるよう再懸濁したものを標的細胞として用いた。
13)-3 エフェクター細胞の調製
市販の凍結PBMC(Cellular Technology Limited社)を37℃で解凍し、10% FBS含有RPMI1640培地にAnti-aggregate Wash試薬(Cellular Technology Limited社)を添加した溶液に移して2回洗浄した後に10% FBS含有RPMI1640培地で1×106細胞/mLになるよう調製し、エフェクター細胞とした。
13)-4 細胞傷害アッセイ
実施例13)-2で取得したA549、PANC-1、MIA PaCa-2、U266B1、及びJeko-1を50μL/wellで96-well U底マイクロプレートに添加した。そこに各濃度に調製した各種抗Axl-CD3二重特異性分子を50μL/wellで添加し、13)-3で調製したエフェクター細胞を100μL/well添加し、室温で1000rpm×1分間遠心後、37℃、5% CO2の条件下で20-24時間培養した。上清50μLをLumaPlate(PerkinElmer社)に回収し、50℃で約2時間乾燥させた後、プレートリーダー(TopCount:PerkinElmer社)で測定した。細胞溶解率は次式で算出した。
細胞溶解率(%)=(A-B)/(C-B)×100
A:サンプルウェルのカウント。
B:バックグラウンド(抗体非添加ウェル)カウントの平均値(n=3)。抗体添加時にアッセイ用培地を50μL添加した。それ以外はサンプルウェルと同様の操作を行った。
C:最大放出(標的細胞を界面活性剤で溶解させたウェル)カウントの平均値(n=3)。抗体添加時にアッセイ培地を50μL添加した。界面活性剤は100μL添加し、サンプルウェルと同様に50μL分をLumaPlateに移して測定を実施した。
(実施例14) 抗HLA-A2/MAGEC1-CD3二重特異性分子発現ベクターの作製
14)-1 抗HLA-A2/MAGEC1-CD3二重特異性分子発現ベクターの構築
14)-1-1 MAG-032scFv/C3E-7034二重特異性分子(MGC-0001)発現ベクターの構築
HLA-A2/MAGEC1特異的に結合するMAG-032 scFvはヒト抗体ファージライブラリーより取得した。MAG-032 scFvのアミノ酸配列をコードするヌクレオチド配列を鋳型として、5‘側にヒト抗体重鎖シグナル配列の一部、3’側にscFv間を繋ぐリンカー、及びC3E-7034の一部をコードするヌクレオチド配列が付加するようにデザインしたプライマーを用いたPCR法により、MAG-032 scFvのアミノ酸配列をコードするヌクレオチド配列を含むインサートDNA断片を得た。また4)-3-1で作製した発現ベクターpC3E-7034を鋳型として、ヒト抗体重鎖シグナル配列、及び抗CD3 scFvのアミノ末端配列をコードするヌクレオチド配列からなるプライマーを用いて抗CD3 scFvを含むベクター全領域をPCR法により増幅し、ベクターDNA断片を得た。それぞれのDNA断片をIn-Fusion HD クローニングキット(CLONTECH社)を用いて結合させ、図101(配列番号93)に示すヌクレオチド配列をORFに含む抗HLA-A2/MAGEC1-CD3二重特異性分子発現ベクターpMGC-0001を作製した。
14)-1-2 MAG-032scFv/C3E-7036二重特異性分子(MGC-0002)発現ベクターの構築
実施例14)-1-1と同様の方法で図103(配列番号95)に示すヌクレオチド配列をORFに含む抗HLA-A2/MAGEC1-CD3二重特異性分子発現ベクターを構築した。ただし、ベクター断片を作製する際の鋳型としてpC3E-7036を用いた。得られた発現ベクターを「pMGC-0002」と命名した。
14)-2 抗HLA-A2/MAGEC1-CD3二重特異性分子の発現・精製
MGC-0001、MGC-0002の発現・精製は、実施例4)-1-2と同様の方法で行なった。MGC-0001のアミノ酸配列は、図102(配列番号94)に記載されている。MGC-0002のアミノ酸配列は、図104(配列番号96)に記載されている。
(実施例15) 抗HLA-A2/MAGEC1-CD3二重特異性分子のin vitro活性評価
15)-1 標的細胞におけるHLA-A2/MAGEC1の発現解析
ヒトリンパ芽球細胞融合細胞株T2(ATCC)細胞を20%FBS含有AIM-V培地(Thermo Fisher Scientific社)で適当な濃度に調整し、図106(配列番号97)MAGEC1ペプチド(Sigma Genosys社)、もしくはDMSOを添加し、37℃で4時間インキュベートした。20%FBS含有AIM-V培地で2回洗浄後、5% FBS含有PBSで適当な濃度に調製し、LIVE/DEAD Fixable Dead Cell Stain Kit(Thermo Fisher Scientific社)を添加し、4℃で30分静置した。5% FBS含有PBSで2回洗浄後、5% FBS含有PBSで2×106細胞/mLの濃度に調製し、100μL/wellで96-well U底マイクロプレートに播種し、遠心後上清を除去した。5% FBS含有PBSで希釈した抗HLA-A2/MAGEC1抗体(MAG032 scFv)を25μL/well添加し、4℃で30分静置した。5% FBS含有PBSで2回洗浄後、5% FBS含有PBSで希釈したPenta-His Alexa Fluor 488(QIAGEN社)を25μL/well添加し、4℃で30分静置した。5% FBS含有PBSで2回洗浄後、5% FBS含有PBSで再懸濁し、フローサイトメーター(Cytomics FC500、BeckmanCoulter社)で検出を行った。データ解析はFlowjo(Treestar社)で行い、死細胞除去画分のAlexa Fluor 488の幾何学的平均蛍光強度(geometric MFI)を算出した。図109 (A、B)に示す通り、MAGEC1ペプチドを添加したT2細胞においてHLA-A2/MAGEC1の発現が認められ、DMSOを添加したT2細胞においては発現が認められなかった。
15)-2 標的細胞の調製
T2細胞を20%FBS含有AIM-V培地(Thermo Fisher Scientific社)で適当な濃度に調整し、MAGEC1ペプチド、もしくはDMSOを添加し、37℃で4時間インキュベートした。10% FBS含有RPMI1640培地(Thermo Fisher Scientific社)で2×106細胞/mLの濃度に調製し、細胞懸濁液1mLあたり100μLのChromium-51 Radionuclide(PerkinElmer社)を添加し、37℃、5% CO2の条件下で2時間培養した。10% FBS含有RPMI1640培地で2回洗浄した後、10% FBS含有RPMI1640培地で2×105細胞/mLになるよう再懸濁したものを標的細胞として用いた。
15)-3 エフェクター細胞の調製
市販の凍結PBMC(Cellular Technology Limited社)を37℃で解凍し、10% FBS含有RPMI1640培地にAnti-aggregate Wash試薬(Cellular Technology Limited社)を添加した溶液に移して2回洗浄した後に10% FBS含有RPMI1640培地で1×106細胞/mLになるよう調製し、エフェクター細胞とした。
15)-4 細胞傷害アッセイ
実施例15)-2で取得したT2細胞を50μL/wellで96-well U底マイクロプレートに添加した。そこに各濃度に調製した各種抗HLA-A2/MAGEC1-CD3二重特異性分子を50μL/wellで添加し、15)-3で調製したエフェクター細胞を100μL/well添加し、室温で1000rpm×1分間遠心後、37℃、5% CO2の条件下で20-24時間培養した。上清50μLをLumaPlate(PerkinElmer社)に回収し、50℃で約2時間乾燥させた後、プレートリーダー(TopCount:PerkinElmer社)で測定した。細胞溶解率は次式で算出した。
細胞溶解率(%)=(A-B)/(C-B)×100
A:サンプルウェルのカウント。
B:バックグラウンド(抗体非添加ウェル)カウントの平均値(n=3)。抗体添加時にアッセイ用培地を50μL添加した。それ以外はサンプルウェルと同様の操作を行った。
C:最大放出(標的細胞を界面活性剤で溶解させたウェル)カウントの平均値(n=3)。抗体添加時にアッセイ培地を50μL添加した。界面活性剤は100μL添加し、サンプルウェルと同様に50μL分をLumaPlateに移して測定を実施した。
配列番号2:ヒトCD3δのアミノ酸配列
配列番号3:ヒトCD3γのアミノ酸配列
配列番号4:ヒトCD3εγ一本鎖抗原をコードするヌクレオチド配列
配列番号5:His-scCD3抗原のアミノ酸配列
配列番号6:C3-147の重鎖可変領域をコードするヌクレオチド配列
配列番号7:(C3-147_VH AA):C3-147の重鎖可変領域のアミノ酸配列
配列番号8:(C3-147_VL DNA):C3-147の軽鎖可変領域をコードするヌクレオチド配列
配列番号9:(C3-147_VL AA):C3-147の軽鎖可変領域のアミノ酸配列
配列番号10:(G4S linker センス):
配列番号11:(G4S linker アンチセンス):
配列番号12:(C3E-7000_VH AA):C3E-7000の重鎖可変領域のアミノ酸配列
配列番号13:(C3E-7000_VL AA):C3E-7000の軽鎖可変領域のアミノ酸配列
配列番号14:(C3E-7000 ORF):C3E-7000をコードするヌクレオチド配列
配列番号15:(C3E-7000 AA):C3E-7000のアミノ酸配列
配列番号16:(C3E-7034_VH AA):C3E-7034の重鎖可変領域のアミノ酸配列
配列番号17:C3E-7034の軽鎖可変領域のアミノ酸配列
配列番号18:(C3E-7034 ORF):C3E-7034をコードするヌクレオチド配列
配列番号19:(C3E_7034 AA):C3E-7034のアミノ酸配列
配列番号20:(C3E-7035_VL AA):C3E-7035の軽鎖可変領域のアミノ酸配列
配列番号21:(C3E-7035 ORF):C3E-7035をコードするヌクレオチド配列
配列番号22:(C3E_7035 AA):C3E-7035のアミノ酸配列
配列番号23:(C3E-7036_VL_AA):C3E-7036の軽鎖可変領域のアミノ酸配列
配列番号24:(C3E-7036 ORF):C3E-7036をコードするヌクレオチド配列
配列番号25:(C3E_7036 AA):C3E-7036のアミノ酸配列
配列番号26:(7000_CDR-H1):C3E-7000系のCDR-H1のアミノ酸配列
配列番号27:(7000_CDR-H2):C3E-7000系のCDR-H2のアミノ酸配列
配列番号28:(7000_CDR-H3):C3E-7000系のCDR-H3のアミノ酸配列
配列番号29:(7000_CDR-L1):C3E-7000系のCDR-L1のアミノ酸配列
配列番号30:(7000_CDR-L2):C3E-7000系のCDR-L2のアミノ酸配列
配列番号31:(7000_CDR-L3):C3E-7000系のCDR-L3のアミノ酸配列
配列番号32:(C3E-3000 ORF): OKT3 scFvをコードするヌクレオチド配列
配列番号33:(C3E-3000 AA): OKT3 scFvのアミノ酸配列
配列番号34:(C3E-3007 ORF): C3E-3007 scFvをコードするヌクレオチド配列
配列番号35:(C3E-3007 AA): C3E-3007 scFvのアミノ酸配列
配列番号36:(C3E-3000_VH AA): OKT3の重鎖可変領域のアミノ酸配列
配列番号37:(C3E-3000_VL AA): OKT3の軽鎖可変領域のアミノ酸配列
配列番号38:(C3E-3007_VH AA): C3E-3007の重鎖可変領域のアミノ酸配列
配列番号39:(C3E-3007_VL AA): C3E-3007の軽鎖可変領域のアミノ酸配列
配列番号40:(HT1-11 ORF):HT1-11 scFvをコードするヌクレオチド配列
配列番号41:(HT1-11 AA):HT1-11 scFvのアミノ酸配列
配列番号42:(T2C-0001 ORF):T2C-0001をコードするORFヌクレオチド配列
配列番号43:(T2C-0001 AA):T2C-0001のアミノ酸配列
配列番号44:(T2C-0003 ORF):T2C-0003をコードするORFヌクレオチド配列
配列番号45:(T2C-0003 AA):T2C-0003のアミノ酸配列
配列番号46:(T2C-0005 ORF):T2C-0005をコードするORFヌクレオチド配列
配列番号47:(T2C-0005 AA):T2C-0005のアミノ酸配列
配列番号48:(T2C-0006 ORF):T2C-0006をコードするORFヌクレオチド配列
配列番号49:(T2C-0006 AA):T2C-0006のアミノ酸配列
配列番号50:重鎖遺伝子増幅用 センスプライマーのアミノ酸配列
配列番号51:重鎖遺伝子増幅用 1回目アンチセンスプライマーのヌクレオチド配列
配列番号52:重鎖遺伝子増幅用 2回目アンチセンスプライマーのヌクレオチド配列
配列番号53:軽鎖遺伝子増幅用 センスプライマーのヌクレオチド配列
配列番号54:軽鎖遺伝子増幅用 1回目アンチセンスプライマーのヌクレオチド配列
配列番号55:軽鎖遺伝子増幅用 2回目アンチセンスプライマーのヌクレオチド配列
配列番号56:重鎖シーケンス用センスプライマーのヌクレオチド配列
配列番号57:軽鎖シーケンス用アンチセンスプライマー1のヌクレオチド配列
配列番号58:軽鎖シーケンス用 アンチセンスプライマー2のヌクレオチド配列
配列番号59:C3E-7078をコードするORFヌクレオチド配列
配列番号60:C3E-7078のアミノ酸配列
配列番号61:C3E-7079をコードするORFヌクレオチド配列
配列番号62:C3E-7079のアミノ酸配列
配列番号63:C3E-7085をコードするORFヌクレオチド配列
配列番号64:C3E-7085のアミノ酸配列
配列番号65:C3E-7086をコードするORFヌクレオチド配列
配列番号66:C3E-7086のアミノ酸配列
配列番号67:C3E-7087をコードするORFヌクレオチド配列
配列番号68:C3E-7087のアミノ酸配列
配列番号69:C3E-7088をコードするORFヌクレオチド配列
配列番号70:C3E-7088のアミノ酸配列
配列番号71:C3E-7089をコードするORFヌクレオチド配列
配列番号72:C3E-7089のアミノ酸配列
配列番号73:C3E-7090をコードするORFヌクレオチド配列
配列番号74:C3E-7090のアミノ酸配列
配列番号75:C3E-7091をコードするORFヌクレオチド配列
配列番号76:C3E-7091のアミノ酸配列
配列番号77:C3E-7092をコードするORFヌクレオチド配列
配列番号78:C3E-7092のアミノ酸配列
配列番号79:C3E-7093をコードするORFヌクレオチド配列
配列番号80:C3E-7093のアミノ酸配列
配列番号81:C3E-7094をコードするORFヌクレオチド配列
配列番号82:C3E-7094のアミノ酸配列
配列番号83:C3E-7095をコードするORFヌクレオチド配列
配列番号84:C3E-7095のアミノ酸配列
配列番号85:HN53R Fwのヌクレオチド配列
配列番号86:HN53R Rvのヌクレオチド配列
配列番号87:HN53S Fwのヌクレオチド配列
配列番号88:HN53S Rvのヌクレオチド配列
配列番号89:(AXC-0001 ORF):AXC-0001をコードするORFヌクレオチド配列
配列番号90:(AXC-0001 AA):AXC-0001のアミノ酸配列
配列番号91:(AXC-0002 ORF):AXC-0002をコードするORFヌクレオチド配列
配列番号92:(AXC-0002 AA):AXC-0002のアミノ酸配列
配列番号93:(MGC-0001 ORF):MGC-0001をコードするORFヌクレオチド配列
配列番号94:(MGC-0001 AA):MGC-0001のアミノ酸配列
配列番号95:(MGC-0002 ORF):MGC-0002をコードするORFヌクレオチド配列
配列番号96:(MGC-0002 AA):MGC-0002のアミノ酸配列
配列番号97:(MAGEC1ペプチド):MAGEC1ペプチドのアミノ酸配列
配列番号98:(CDRH2 of variants):CDR改変体CDRH2のアミノ酸配列
配列番号99:(CDRL2 of variants):CDR改変体CDRL2のアミノ酸配列
配列番号100:(VH of C3E-7034 variants):C3E-7034のCDR改変体の重鎖可変領域のアミノ酸配列
配列番号101:(VL of C3E-7034 variants):C3E-7034のCDR改変体の軽鎖可変領域のアミノ酸配列
配列番号102:(VL of C3E-7035 variants):C3E-7035のCDR改変体の軽鎖可変領域のアミノ酸配列
配列番号103:(VL of C3E-7036 variants):C3E-7036のCDR改変体の軽鎖可変領域のアミノ酸配列
Claims (55)
- 重鎖配列が
配列番号26に示されるアミノ酸配列を含むCDRH1、
配列番号98に示されるアミノ酸配列を含むCDRH2、及び、
配列番号28に示されるアミノ酸配列を含むCDRH3;
軽鎖配列が
配列番号29に示されるアミノ酸配列を含むCDRL1、
配列番号99に示されるアミノ酸配列を含むCDRL2、及び、
配列番号31に示されるアミノ酸配列を含むCDRL3
をそれぞれ含み;且つ、
ヒトCD3及びカニクイザルCD3に結合することを特徴とする抗体又は該抗体の抗原結合性断片。 - 前記CDRH2の
1番目のXaaは、(A、E、G、H、I、L、T、V、R、S)からなる群より選択され
且つ2番目のXaaはSであるか、又は、
1番目のXaaはNであり、
且つ2番目のXaaは、(E、R、F、Y、L、V、I、K、T)からなる群より選択され、
前記CDRL2の
Xaaは、(Q、A、G、S、N、D)からなる群より選択され、
ヒトCD3及びカニクイザルCD3に結合することを特徴とする請求項1に記載の抗体又は該抗体の抗原結合性断片。 - 前記CDRH2の
1番目のXaaは、(R、S)からなる群より選択され、2番目のXaaはSであり、且つ
前記CDRL2の
Xaaは、(Q、A、G、S、N、D)からなる群より選択される、
ヒトCD3及びカニクイザルCD3に結合することを特徴とする請求項1又は2に記載の抗体又は該抗体の抗原結合性断片。 - 重鎖配列がCDRH1、CDRH2、及び、CDRH3を有する可変領域を含み、
前記CDRH1は配列番号26に示されるアミノ酸配列からなり、
前記CDRH2は配列番号27に示されるアミノ酸配列からなり、
前記CDRH3は配列番号28に示されるアミノ酸配列からなること;並びに
軽鎖配列がCDRL1、CDRL2、CDRL3を有する可変領域を含み、
前記CDRL1は配列番号29に示されるアミノ酸配列からなり、
前記CDRL2は配列番号30に示されるアミノ酸配列からなり、
前記CDRL3は配列番号31に示されるアミノ酸配列からなること;並びに、
ヒトCD3及びカニクイザルCD3に結合することを特徴とする、請求項1記載の抗体又は該抗体の抗原結合性断片。 - 重鎖可変領域配列が、配列番号100に示されるアミノ酸配列を含むことを特徴とする請求項1乃至4のいずれか1つに記載の抗体又は該抗体の抗原結合性断片。
- 配列番号100に示されるアミノ酸配列の
1番目のXaaは、(A、E、G、H、I、L、T、V、R、S)からなる群より選択され
且つ2番目のXaaはSであるか、又は、
1番目のXaaはNであり
且つ2番目のXaaは、(E、R、F、Y、L、V、I、K、T)からなる群より選択される、
請求項5に記載の抗体又はその抗原結合性断片。 - 配列番号100に示されるアミノ酸配列の
1番目のXaaは、(R、S)からなる群より選択され、且つ2番目のXaaはSである、
請求項5に記載の抗体又はその抗原結合性断片。 - 軽鎖可変領域が、配列番号101、102、及び、103のいずれか1つに示されるアミノ酸配列を含むことを特徴とする、請求項1乃至7のいずれか1つに記載の抗体又はその抗原結合性断片。
- 配列番号101、102、及び、103のいずれか1つに示されるアミノ酸配列の
Xaaは、(Q、A、G、S、N、D)からなる群より選択される、
請求項8に記載の抗体又その抗原結合性断片。 - 重鎖可変領域配列が、配列番号16に示されるアミノ酸配列を含むことを特徴とする請求項5に記載の抗体又は該抗体の抗原結合性断片。
- 軽鎖可変領域配列が、配列番号17、20、及び、23のいずれか1つに示されるアミノ酸配列を含むことを特徴とする請求項8に記載の抗体又は該抗体の抗原結合性断片。
- 配列番号100に示されるアミノ酸配列を含む重鎖可変領域と、配列番号101、102、及び103のいずれか1つに示されるアミノ酸配列を含む軽鎖可変領域を含み、
配列番号100で示されるアミノ酸配列の1番目のXaaは、(A、E、G、H、I、L、T、V、R、S)からなる群より選択され、且つ、2番目のXaaはSであるか、又は、
1番目のXaaはNであり、且つ、2番目のXaaは、(E、R、F、Y、L、V、I、K、T)からなる群より選択され、
配列番号101、102、及び、103のいずれか1つに示されるアミノ酸配列の
Xaaは、(Q、A、G、S、N、D)からなる群より選択される、
請求項1又は2に記載の抗体又はその抗原結合性断片。 - 配列番号100の
1番目のXaaは、(R、S)からなる群より選択され、
2番目のXaaはSであり、且つ、
配列番号101、102、及び、103のいずれか1つに示されるアミノ酸配列の
Xaaは、(Q、A、G、S、N、D)からなる群より選択される、
請求項12に記載の抗体又はその抗原結合性断片。 - 請求項13に記載の
配列番号60の2乃至119のアミノ酸残基を含む重鎖可変領域と、配列番号60の135乃至243のアミノ酸残基を含む軽鎖可変領域を含む抗体又は該抗体の抗原結合性断片、
配列番号64の2乃至119のアミノ酸残基を含む重鎖可変領域と、配列番号64の135乃至241のアミノ酸残基を含む軽鎖可変領域を含む抗体又は該抗体の抗原結合性断片、
配列番号66の2乃至119のアミノ酸残基を含む重鎖可変領域と、配列番号66の135乃至243のアミノ酸残基を含む軽鎖可変領域を含む抗体又は該抗体の抗原結合性断片、
配列番号68の2乃至119のアミノ酸残基を含む重鎖可変領域と、配列番号68の135乃至243のアミノ酸残基を含む軽鎖可変領域を含む抗体又は該抗体の抗原結合性断片、
配列番号70の2乃至119のアミノ酸残基を含む重鎖可変領域と、配列番号70の135乃至243のアミノ酸残基を含む軽鎖可変領域を含む抗体又は該抗体の抗原結合性断片、
配列番号72の2乃至119のアミノ酸残基を含む重鎖可変領域と、配列番号72の135乃至243のアミノ酸残基を含む軽鎖可変領域を含む抗体又は該抗体の抗原結合性断片、
配列番号74の2乃至119のアミノ酸残基を含む重鎖可変領域と、配列番号74の135乃至243のアミノ酸残基を含む軽鎖可変領域を含む抗体又は該抗体の抗原結合性断片、
配列番号76の2乃至119のアミノ酸残基を含む重鎖可変領域と、配列番号76の135乃至243のアミノ酸残基を含む軽鎖可変領域を含む抗体又は該抗体の抗原結合性断片、
配列番号78の2乃至119のアミノ酸残基を含む重鎖可変領域と、配列番号78の135乃至243のアミノ酸残基を含む軽鎖可変領域を含む抗体又は該抗体の抗原結合性断片、
配列番号80の2乃至119のアミノ酸残基を含む重鎖可変領域と、配列番号80の135乃至243のアミノ酸残基を含む軽鎖可変領域を含む抗体又は該抗体の抗原結合性断片、
配列番号82の2乃至119のアミノ酸残基を含む重鎖可変領域と、配列番号82の135乃至243のアミノ酸残基を含む軽鎖可変領域を含む抗体又は該抗体の抗原結合性断片、
又は、
配列番号84の2乃至119のアミノ酸残基を含む重鎖可変領域と、配列番号84の135乃至243のアミノ酸残基を含む軽鎖可変領域を含む抗体又は該抗体の抗原結合性断片。 - 配列番号16に示されるアミノ酸配列を含む重鎖可変領域と、リンカーと、配列番号17、20、及び、23のいずれか1つに示されるアミノ酸配列を含む軽鎖可変領域とを含む請求項1、4、5、8、10、又は11に記載の抗体又は該抗体の抗原結合性断片。
- 可変領域が、抗体のアミノ末端側から重鎖可変領域、軽鎖可変領域の順で結合しており、又は、軽鎖可変領域、重鎖可変領域の順で結合しており、任意で:i) 両可変領域の間にリンカーを有し;ii)アミノ末端側の可変領域のアミノ末端にグリシン残基を有し;iii)カルボキシル末端側の可変領域のカルボキシル末端に、リンカー、FLAGタグ、及び/又は、Hisタグが結合している:請求項1乃至15のいずれか1つに記載の抗体又はその抗原結合性断片。
- 配列番号60の2乃至243番目のアミノ酸残基を含むアミノ酸配列、
配列番号64の2乃至241番目のアミノ酸残基を含むアミノ酸配列、
配列番号66の2乃至243番目のアミノ酸残基を含むアミノ酸配列、
配列番号68の2乃至243番目のアミノ酸残基を含むアミノ酸配列、
配列番号70の2乃至243番目のアミノ酸残基を含むアミノ酸配列、
配列番号72の2乃至243番目のアミノ酸残基を含むアミノ酸配列、
配列番号74の2乃至243番目のアミノ酸残基を含むアミノ酸配列、
配列番号76の2乃至243番目のアミノ酸残基を含むアミノ酸配列、
配列番号78の2乃至243番目のアミノ酸残基を含むアミノ酸配列、
配列番号80の2乃至243番目のアミノ酸残基を含むアミノ酸配列、
配列番号82の2乃至243番目のアミノ酸残基を含むアミノ酸配列、
又は、
配列番号84の2乃至243番目のアミノ酸残基を含むアミノ酸配列
を含む請求項16に記載の抗原又は該抗体の抗原結合性断片。 - 配列番号19の2乃至269のアミノ酸残基を含むアミノ酸配列、
配列番号22の2乃至269のアミノ酸残基を含むアミノ酸配列、
配列番号25の2乃至267のアミノ酸残基を含むアミノ酸配列、
配列番号60の2乃至269のアミノ酸残基を含むアミノ酸配列、
配列番号64の2乃至267のアミノ酸残基を含むアミノ酸配列、
配列番号66の2乃至269のアミノ酸残基を含むアミノ酸配列、
配列番号68の2乃至269のアミノ酸残基を含むアミノ酸配列、
配列番号70の2乃至269のアミノ酸残基を含むアミノ酸配列、
配列番号72の2乃至269のアミノ酸残基を含むアミノ酸配列、
配列番号74の2乃至269のアミノ酸残基を含むアミノ酸配列、
配列番号76の2乃至269のアミノ酸残基を含むアミノ酸配列、
配列番号78の2乃至269のアミノ酸残基を含むアミノ酸配列、
配列番号80の2乃至269のアミノ酸残基を含むアミノ酸配列、
配列番号82の2乃至269のアミノ酸残基を含むアミノ酸配列、又は、
配列番号84の2乃至269のアミノ酸残基を含むアミノ酸配列
を含む請求項16に記載の抗原又は該抗体の抗原結合性断片。 - 請求項14乃至18のいずれか1つに記載の抗体又は該抗体の抗原結合性断片に含まれるアミノ酸配列をコードするヌクレオチド配列を含むポリヌクレオチドの相補鎖とストリンジェントな条件下でハイブリダイズするポリヌクレオチドに含まれるヌクレオチド配列によりコードされるアミノ酸配列を含み、且つ、ヒトCD3及びカニクイザルCD3に結合する抗体又は該抗体の抗原結合性断片。
- 請求項14乃至18のいずれか1つに記載の抗体又は該抗体の抗原結合性断片に含まれる重鎖のアミノ酸配列と90%以上同一なアミノ酸配列を含む重鎖、及び、請求項14乃至18のいずれか1つに記載の抗体又は該抗体の抗原結合性断片に含まれる軽鎖のアミノ酸配列と70%以上同一なアミノ酸配列を含む軽鎖を含み、且つ、ヒトCD3及びカニクイザルCD3に結合する抗体又は該抗体の抗原結合性断片。
- 請求項14乃至18のいずれか1つに記載の抗体又は該抗体の抗原結合性断片に含まれる重鎖が含むアミノ酸配列において1乃至数個のアミノ酸が置換、欠失又は付加されてなるアミノ酸配列を含む重鎖、並びに、請求項14乃至18のいずれか1つに記載の抗体又は該抗体の抗原結合性断片に含まれる軽鎖が含むアミノ酸配列において1乃至数個のアミノ酸が置換、欠失又は付加されてなるアミノ酸配列を含む軽鎖を含み、且つ、ヒトCD3及びカニクイザルCD3に結合する抗体又は該抗体の抗原結合性断片。
- 請求項14乃至18のいずれか1つに記載の抗体又は該抗体の抗原結合性断片が結合するヒトCD3上の同一の部位に結合し、且つ、カニクイザルCD3に結合する抗体又は該抗体の抗原結合性断片。
- 請求項14乃至18のいずれか1つに記載の抗体又は該抗体の抗原結合性断片とヒトCD3上への結合において競合し、且つ、カニクイザルCD3に結合する抗体又は該抗体の抗原結合性断片。
- 前記抗体又は該抗体の抗原結合性断片が結合するヒトCD3上の部位が、配列番号1で示されるアミノ酸配列中の55番目のセリン(Ser)、56番目のグルタミン酸(Glu)、58番目のロイシン(Leu)、59番目のトリプトファン(Trp)、65番目のアスパラギン(Asn)、66番目のイソロイシン(Ile)、77番目のセリン(Ser)、78番目のアスパラギン酸(Asp)、101番目のアルギニン(Arg)、101番目のグリシン(Gly)、103番目のセリン(Ser)、104番目のリジン(Lys)、および105番目のプロリン(Pro)のうちの7アミノ酸以上から構成される、請求項22に記載の抗体又は該抗体の抗原結合性断片。
- IgGである請求項1乃至17、19乃至24のいずれか1つに記載の抗体又は該抗体の抗原結合性断片。
- Fab、F(ab)’、Fv、scFv及びsdAbからなる群から選択される請求項1乃至23のいずれか1つに記載の抗体又は該抗体の抗原結合性断片。
- ヒト免疫グロブリン定常領域を含むヒト化抗体又はヒト抗体である、請求項1乃至17、19乃至25のいずれか1つに記載の抗体又は該抗体の抗原結合性断片。
- 請求項1乃至27のいずれか1つに記載の抗体又は該抗体の抗原結合性断片のアミノ酸配列をコードするヌクレオチド配列を含むポリヌクレオチド。
- 配列番号19の2乃至243番目のアミノ酸残基を含むアミノ酸配列、
配列番号22の2乃至243番目のアミノ酸残基を含むアミノ酸配列、
配列番号25の2乃至241番目のアミノ酸残基を含むアミノ酸配列、
配列番号60の2乃至243番目のアミノ酸残基を含むアミノ酸配列、
配列番号64の2乃至241番目のアミノ酸残基を含むアミノ酸配列、
配列番号66の2乃至243番目のアミノ酸残基を含むアミノ酸配列、
配列番号68の2乃至243番目のアミノ酸残基を含むアミノ酸配列、
配列番号70の2乃至243番目のアミノ酸残基を含むアミノ酸配列、
配列番号72の2乃至243番目のアミノ酸残基を含むアミノ酸配列、
配列番号74の2乃至243番目のアミノ酸残基を含むアミノ酸配列、
配列番号76の2乃至243番目のアミノ酸残基を含むアミノ酸配列、
配列番号78の2乃至243番目のアミノ酸残基を含むアミノ酸配列、
配列番号80の2乃至243番目のアミノ酸残基を含むアミノ酸配列、
配列番号82の2乃至243番目のアミノ酸残基を含むアミノ酸配列、
又は、
配列番号84の2乃至243番目のアミノ酸残基を含むアミノ酸配列
をコードするヌクレオチド配列を含む、請求項27に記載のポリヌクレオチド。 - 請求項28又は29のいずれか1つに記載のポリヌクレオチドを含むベクター。
- 請求項28又は29のいずれか1つに記載のポリヌクレオチド又は請求項30に記載のベクターを含むか、又は請求項1乃至27のいずれか1つに記載の抗体又は該抗体の抗原結合性断片を産生する細胞。
- 請求項31に記載の細胞を培養する工程、及び、該培養物からヒトCD3に結合する抗体又は該抗体の抗原結合性断片を回収する工程を含む、ヒトCD3及びカニクイザルCD3に結合する抗体又は該抗体の抗原結合性断片の製造方法。
- 請求項32に記載の方法により得られる、ヒトCD3及びカニクイザルCD3に結合する抗体又は該抗体の抗原結合性断片。
- 請求項1乃至27、33のいずれか1つに記載の抗体又は該抗体の抗原結合性断片を有効成分として含有する医薬組成物。
- 請求項1乃至27、33のいずれか1つに記載の抗体又は該抗体の抗原結合性断片を含む抗原結合性を有する分子。
- 多重特異的である請求項35に記載の分子。
- 請求項1乃至27、33のいずれか1つに記載の抗体又は該抗体の抗原結合性断片と、1つ又は2つ以上のさらなる抗体又は該抗体の抗原結合性断片を含む請求項35又は36に記載の分子。
- さらなる抗体の抗原結合性断片が、Fab、F(ab)’、Fv、scFv、又は、sdAbである、請求項37に記載の分子。
- Fcを含む請求項38に記載の分子。
- さらなる抗体が、ヒト免疫グロブリン定常領域を含むヒト化抗体又はヒト抗体である、請求項37乃至39のいずれか1つに記載の分子。
- 該さらなる抗体又は該抗体の抗原結合性断片と、請求項1乃至27、33のいずれか1つに記載の抗体又は該抗体の抗原結合性断片とが、リンカーにより結合してなる、あるいはリンカーなしで結合してなる請求項37乃至38のいずれか1つに記載の分子。
- 該さらなる抗体又は該抗体の抗原結合性断片の有するアミノ酸配列のカルボキシル末端と、リンカーとが結合しており、さらに該リンカーの有するアミノ酸配列のカルボキシル末端と、請求項1乃至27、33のいずれか1つに記載の抗体又は該抗体の抗原結合性断片とが結合してなる請求項41に記載の分子。
- 該さらなる抗体又は該抗体の抗原結合性断片の有するアミノ酸配列のカルボキシル末端と、リンカーとが結合しており、さらに該リンカーの有するアミノ酸配列のカルボキシル末端と、
配列番号19の2乃至243番目のアミノ酸残基を含む抗体又は該抗体の抗原結合性断片、
配列番号22の2乃至243番目のアミノ酸残基を含む抗体又は該抗体の抗原結合性断片、
又は、
配列番号25の2乃至241番目のアミノ酸残基を含む抗体又は該抗体の抗原結合性断片が結合してなるアミノ酸配列を含む請求項42に記載の分子。 - 請求項1乃至27、33のいずれか1つに記載の抗体又は該抗体の抗原結合性断片において、可変領域が、抗体のアミノ末端側から重鎖可変領域、軽鎖可変領域の順で結合しており、あるいは、軽鎖可変領域、重鎖可変領域の順で結合しており、
任意で:i)両可変領域の間にリンカーを有し:ii)アミノ末端側の可変領域のアミノ末端にグリシン残基を有し;iii)カルボキシル末端側の可変領域のカルボキシル末端に、リンカー、FLAGタグ、及び/又は、Hisタグが結合している:請求項35乃至42のいずれか1つに記載の分子。 - 該さらなる抗体又は該抗体の抗原結合性断片と、
配列番号19の2乃至243番目のアミノ酸残基を含む抗体又は該抗体の抗原結合性断片、
配列番号25の2乃至241番目のアミノ酸残基を含む抗体又は該抗体の抗原結合性断片。
配列番号60の2乃至243番目のアミノ酸残基を含む抗体又は該抗体の抗原結合性断片、配列番号64の2乃至241番目のアミノ酸残基を含む抗体又は該抗体の抗原結合性断片、配列番号66の2乃至243番目のアミノ酸残基を含む抗体又は該抗体の抗原結合性断片、
配列番号68の2乃至243番目のアミノ酸残基を含む抗体又は該抗体の抗原結合性断片、
配列番号70の2乃至243番目のアミノ酸残基を含む抗体又は該抗体の抗原結合性断片、
配列番号72の2乃至243番目のアミノ酸残基を含む抗体又は該抗体の抗原結合性断片、
配列番号74の2乃至243番目のアミノ酸残基を含む抗体又は該抗体の抗原結合性断片、
配列番号76の2乃至243番目のアミノ酸残基を含む抗体又は該抗体の抗原結合性断片、
配列番号78の2乃至243番目のアミノ酸残基を含む抗体又は該抗体の抗原結合性断片、
配列番号80の2乃至243番目のアミノ酸残基を含む抗体又は該抗体の抗原結合性断片、
配列番号82の2乃至243番目のアミノ酸残基を含む抗体又は該抗体の抗原結合性断片、
又は、
配列番号84の2乃至243番目のアミノ酸残基を含む抗体又は該抗体の抗原結合性断片
を含む請求項44に記載の分子。 - 該さらなる抗体が抗癌標的抗体である請求項36乃至45のいずれかに記載の分子。
- 二重特異的である請求項36乃至46のいずれか1つに記載の分子。
- ポリペプチドである請求項35乃至47のいずれか1つに記載の分子。
- 請求項48に記載の分子の有するアミノ酸配列をコードするヌクレオチド配列を含むポリヌクレオチド。
- 請求項49に記載のポリヌクレオチドを含むベクター。
- 請求項49に記載のポリヌクレオチド若しくは請求項50に記載のベクター、又は、請求項48に記載の分子を産生する細胞。
- 請求項51に記載の細胞を培養する工程、及び、該培養物からヒトCD3に結合する分子を回収する工程を含む、ヒトCD3及びカニクイザルCD3に結合する分子の製造方法。
- 請求項52に記載の方法により得られる、ヒトCD3及びカニクイザルCD3に結合する分子。
- 請求項35乃至48、53のいずれか1つに記載の分子を有効成分として含有する医薬組成物。
- 標的細胞へのT細胞リダイレクションによって、該標的細胞への細胞傷害を誘導することを特徴とする請求項54に記載の医薬組成物。
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JP2018558078A JP7202185B2 (ja) | 2016-12-22 | 2017-12-21 | 抗cd3抗体及び該抗体を含む分子 |
US16/472,346 US11618785B2 (en) | 2016-12-22 | 2017-12-21 | Anti-CD3 antibody and molecules comprising the antibody |
EP17884717.4A EP3561057A4 (en) | 2016-12-22 | 2017-12-21 | ANTI-CD3 ANTIBODY AND MOLECULE CONTAINING LEDIT ANTIBODY |
BR112019012901-4A BR112019012901A2 (pt) | 2016-12-22 | 2017-12-21 | Anticorpo ou fragmento de ligação a antígeno do anticorpo, polinucleotídeo, vetor, célula, métodos para produzir um anticorpo ou um fragmento de ligação a antígeno do anticorpo e uma molécula que se ligue ao cd3 humano e ao cd3 de macaco cinomolgo, composição farmacêutica, e, molécula |
KR1020197021178A KR20190121294A (ko) | 2016-12-22 | 2017-12-21 | 항-cd3 항체, 및 항-cd3 항체를 포함하는 분자 |
MX2019007347A MX2019007347A (es) | 2016-12-22 | 2017-12-21 | Anticuerpo anti-cd3 y moleculas que comprenden el anticuerpo. |
IL311136A IL311136A (en) | 2016-12-22 | 2017-12-21 | Anti-CD3 antibody for use in the treatment or prevention of cancer and molecules containing the antibody |
RU2019122411A RU2790326C2 (ru) | 2016-12-22 | 2017-12-21 | Анти-сd3-антитело и молекула, содержащая это антитело |
CN201780079717.1A CN110431231B (zh) | 2016-12-22 | 2017-12-21 | 抗-cd3抗体和包含所述抗体的分子 |
IL267567A IL267567B1 (en) | 2016-12-22 | 2017-12-21 | Anti-CD3 antibody - for use in treating or preventing cancer and molecules containing the antibody |
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AU2017379382A AU2017379382A1 (en) | 2016-12-22 | 2017-12-21 | Anti-CD3 Antibody and Molecules Comprising the Antibody |
PH12019501387A PH12019501387A1 (en) | 2016-12-22 | 2019-06-18 | Anti-cd3 antibody and molecules comprising the antibody |
CONC2019/0007823A CO2019007823A2 (es) | 2016-12-22 | 2019-07-19 | Anticuerpo anti-cd3 y moléculas que comprenden el anticuerpo |
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WO2019244107A1 (en) * | 2018-06-21 | 2019-12-26 | Daiichi Sankyo Company, Limited | Compositions including cd3 antigen binding fragments and uses thereof |
WO2021200857A1 (ja) | 2020-03-30 | 2021-10-07 | 国立大学法人三重大学 | 二重特異的抗体 |
WO2022210485A1 (ja) | 2021-03-29 | 2022-10-06 | 第一三共株式会社 | 安定な多重特異性分子及びその利用 |
EP3973000A4 (en) * | 2019-06-07 | 2023-09-06 | Adimab, LLC | HIGH AFFINITY ANTI-CD3 ANTIBODIES AND METHODS OF GENERATION AND USE THEREOF |
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IL267567B1 (en) | 2016-12-22 | 2024-04-01 | Daiichi Sankyo Co Ltd | Anti-CD3 antibody - for use in treating or preventing cancer and molecules containing the antibody |
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WO2019244107A1 (en) * | 2018-06-21 | 2019-12-26 | Daiichi Sankyo Company, Limited | Compositions including cd3 antigen binding fragments and uses thereof |
EP3973000A4 (en) * | 2019-06-07 | 2023-09-06 | Adimab, LLC | HIGH AFFINITY ANTI-CD3 ANTIBODIES AND METHODS OF GENERATION AND USE THEREOF |
WO2021200857A1 (ja) | 2020-03-30 | 2021-10-07 | 国立大学法人三重大学 | 二重特異的抗体 |
JPWO2021200857A1 (ja) * | 2020-03-30 | 2021-10-07 | ||
CN115023500A (zh) * | 2020-03-30 | 2022-09-06 | 国立大学法人三重大学 | 双特异性抗体 |
JP7285495B2 (ja) | 2020-03-30 | 2023-06-02 | 国立大学法人三重大学 | 二重特異的抗体 |
US11952423B2 (en) | 2020-03-30 | 2024-04-09 | Mie University | Bispecific antibody |
WO2022210485A1 (ja) | 2021-03-29 | 2022-10-06 | 第一三共株式会社 | 安定な多重特異性分子及びその利用 |
KR20230162597A (ko) | 2021-03-29 | 2023-11-28 | 다이이찌 산쿄 가부시키가이샤 | 안정적인 다중 특이성 분자 및 그 이용 |
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EP3561057A1 (en) | 2019-10-30 |
AU2017379382A1 (en) | 2019-07-11 |
TW201829467A (zh) | 2018-08-16 |
PH12019501387A1 (en) | 2020-02-10 |
KR20190121294A (ko) | 2019-10-25 |
IL311136A (en) | 2024-04-01 |
IL267567B1 (en) | 2024-04-01 |
US20230287119A1 (en) | 2023-09-14 |
CO2019007823A2 (es) | 2019-10-09 |
SG10201912366YA (en) | 2020-02-27 |
RU2019122411A (ru) | 2021-01-25 |
US11618785B2 (en) | 2023-04-04 |
US20190359712A1 (en) | 2019-11-28 |
JPWO2018117237A1 (ja) | 2019-10-31 |
CN110431231A (zh) | 2019-11-08 |
CA3048174A1 (en) | 2018-06-28 |
IL267567A (ja) | 2024-04-01 |
JP7202185B2 (ja) | 2023-01-11 |
BR112019012901A2 (pt) | 2020-01-07 |
JP7478222B2 (ja) | 2024-05-02 |
JP2023027374A (ja) | 2023-03-01 |
EP3561057A4 (en) | 2020-09-16 |
CN110431231B (zh) | 2024-01-02 |
MX2019007347A (es) | 2019-12-05 |
RU2019122411A3 (ja) | 2021-03-05 |
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