method for recombinant production of a complex, complex, pharmaceutical formulation and use of the c

专利摘要:
METHOD FOR THE RECOMBINANT PRODUCTION OF A COMPLEX, COMPLEX, NUCLEIC ACID, PHARMACEUTICAL FORMULATION, USE OF THE COMPLEX, METHOD FOR THE ATTRACTION OF CYTOXIC T-CELLS AND VIRUS-SPECIFIC CELLS. A complex is disclosed in the present invention comprising as a first part an antibody-derived part that specifically binds to a target antigen, and as a second part a virus-derived peptide linked to one of the MHC class I protein. With the disclosed complex in the present invention, the virus-specific circulating cytotoxic T cells (memory T cells and / or effector T cells) of an individual can be targeted to cells that express the target antigen, to which the antibody derived from part of the covalent complex is specifically binds and targets these cells with MHC class I complexes, thus mimicking an acute viral infection. Another aspect of the present invention is a method for the recombinant production of a complex comprising: i) a (beta) 2 microglobulin fusion polypeptide and the extracellular domains (alpha) 1, (alpha) 2 and (alpha) 3 of a molecule Class I MHC; ii) a pair of polypeptide chains linked by disulfide bonds each comprising a region of (...).
公开号:BR112013029746B1
申请号:R112013029746-8
申请日:2012-06-19
公开日:2021-02-02
发明作者:Hendrik Knoetgen
申请人:F. Hoffmann-La Roche Ag；
IPC主号:

专利说明:

[001] A fusion polypeptide comprising an antibody and an MHC class I component is disclosed herein and the use thereof for the removal of tumor cells by the targeted attraction of circulating virus-specific cytotoxic T cells. BACKGROUND OF THE INVENTION
[002] The MHC class I protein consists of an α-chain (α-1 to α-3 and a transmembrane domain), and β2-microglobulin. It is polygenic (3 gene loci for MHC class I protein in the haploid genome) giving rise to six different α-chains for MHC class I protein (in humans two HLA-A, two HLA-B, and two HLA- Ç). MHC is still polymorphic. The human HLA-A A * 0201 allele is prevalent in about 30% to 50% of the Caucasian population (Player et al., J Immunother. Emphasis Tumor Immunol. 19 (1996) 357-363).
[003] The human cytomegalovirus HCMV (= human herpes virus 5, HHV-5) is one of the largest human viruses. Its genome comprises about 230,000 bp of double-stranded linear DNA and encodes more than 160 proteins (Davison, A.J., et al., J. Gen. Virol. 84 (2003) 17-28).
[004] CMV has evolved to become a sublime parasite of the human genome, and is a potent immunogen and triggers strong immune responses by all weapons of the immune system. This virus appears to be among the most immunodominant antigens known to the human immune system and stimulates responses of CD8 + T cells of unprecedented magnitude.
[005] CMV “latency” depends on chronic immunological suppression of the CMV virus rather than a change in the pattern of viral transcription (Moss and Khan, Human Immunology 65 (2004) 456-464).
[006] CD8 + T cell immune responses are not uniformly directed against all CMV proteins, but are focused. CMV proteins pp65 and IE-1 are the predominant targets (McLaughlin-Taylor, E., et al., J. Med. Chem. Virol. 43 (1994) 103-110; Khan & Moss, Human Immunology 65 (2004 ) 456-464).
[007] The true frequency of CMV-specific T-cells is very high, with the frequencies of individual peptides ranging from 1 to 2% of the total CD8 + T cells in the repertoire (Moss and Khan, Human Immunology, Wills, MR, et al. Virol. 70 (1996) 7569-7579).
[008] The response of CMV-specific CD8 + T-cells increases significantly with age and individual HLA peptide tetramers often stain in excess of 10% of the total CD8 + T cell pool (Khan, N., et al. J. Immunol.169 (2002) 1984-1992).
[009] The total CD8 + T cell response in healthy elderly donors may constitute approximately 50% of the CD8 + T cell repertoire.
[010] Huge expansions of CD8 + T cells are often clonally restricted, and CMV is estimated to be the cause of at least 30% of the clonal expansion of CD8 + T cells that are seen in peripheral blood with aging. The total CD8 T cell count is twice as high in CMV seropositive donors over the age of 60, compared to a group of CMV seronegative patients (Looney, RJ, et al. Clin. Immunol. 90 (1999) 213-219).
[011] A fusion of soluble HLA and β-2-microglobulin is disclosed by Mottez et al. (Eur. J. Immunol. 21 (1991) 467-471); Godeau et al. (J. Biol. Chem. 267 (1992) 24223-24229) and Mage et al. (Proc. Natl. Acad. Sci. 89 (1992) 10658-10662). A fusion between the virus-derived peptide with soluble HLA and β-2-microglobulin is reported by Mottez et al. (J. Exp. Med. 181 (1995) 493-502). A fusion of an immunoglobulin heavy chain with soluble HLA being coexpressed to β-2-microglobulin is disclosed by Dal Porto et al. (Proc. Natl. Acad. Sci. USA 90 (1993) 6671-6675). A tetrameric complex of the soluble HLA peptide and biotinylated β-2-microglobulin chemically coupled with streptoavidine to a Fab fragment is described by Robert et al. (Eur. J. Immun. 30 (2000) 3165-3170). A Fab chemically coupled with a fusion of a viral-derived peptide with soluble HLA and β-2-microglobulin is disclosed by Robert et al. (Cancer Immunity 1 (2001) 2). A fusion of a viral-derived peptide with soluble HLA and β-2-microglobulin to a heavy chain of a murine monoclonal antibody is disclosed by Greten et al. (J. Immunol. Methods 271 (2002) 125-135). An expression in E. coli of scFv fusions without peptide, with protein folding and peptide loading in vitro is reported by Lev et al. (J. Immunol. 169 (2002) 2988-2996), Lev et al. (Proc. Natl. Acad. Sci. 101 (2004) 9051-9056), and Novak et al. (Int. J. Cancer 120 (2006) 329-336). The use of soluble biotinylated MHC loaded with peptides and coupled to streptoavidin fused to Fab or scFv antibodies is disclosed by Mous et al. (Leukemia 20 (2006) 1096-1102).
[012] WO 2005/099361 discloses conjugates 'MHC class I peptide - -antibody' modified with beta-2-microglobulin. Examples of conjugates as disclosed in WO 2005/099361 are obtained from the in vitro conjugation of the alpha chain of the MHC complex (HLA) or by the coexpression of separate genes in the same cell.
[013] US 2004/0091488 discloses antigenic constructions of major class I histocompatibility complex antigens with specific carrier molecules. The fusion polypeptides mentioned in the present invention are disclosed without a hinge region. BRIEF DESCRIPTION OF THE INVENTION
[014] A method for recombinantly producing a complex comprising as a first part an antibody-derived part that specifically binds to a target antigen, and as a second part, a virus-derived peptide (virus-derived) is disclosed in the present invention. covalently linked to a class I MHC protein complex, as well as the complex itself.
[015] With the complex as disclosed in the present invention the virus-specific circulating cytotoxic T cells (memory T cells and / or effector T cells) of an individual can be directed to cells that express the target antigen, to which part the antibody derived from part of the complex binds specifically, addressing these cells with MHC class I complexes mimicking an acute viral infection by the peptide derived from the virus bound to the MHC class I protein complex.
[016] One aspect as disclosed in the present invention is a method for the recombinant production of a complex comprising: i) a β2-microglobulin fusion polypeptide and the α1, α2 and α3 extracellular domains of a MHC class I molecule; ii) a pair of polypeptide chains linked by disulfide bonds each comprising an antibody hinge region; and iii) at least one pair of a light chain variable domain and antibody heavy chain variable domain of a eukaryotic cell, comprising the steps of: i) culturing a eukaryotic cell comprising one or more nucleic acids encoding the complex; and ii) recovering the complex from the cell or the culture medium, where the complex comprises exactly a β2-microglobulin fusion polypeptide and the extracellular domains α1, α2 and α3 of a MHC class I molecule.
[017] In one example of an embodiment, the complex comprises exactly one polypeptide derived from MHC or exactly one polypeptide fusion comprising a molecule derived from MHC.
[018] In one embodiment, the complex is obtained with a concentration of 1 mg / ml or more in the culture medium. In one embodiment, the complex is obtained with a concentration of 4 mg / ml or more in the culture medium.
[019] In one example of an embodiment, the eukaryotic cell is a mammalian cell. In an example of an embodiment, the mammalian cell is a human embryonic kidney cell, or a Chinese hamster ovary cell, or a hamster cub kidney cell, or a mouse myeloma cell.
[020] In one embodiment, the fusion polypeptide comprises, in the N-terminal to C-terminal direction, a β2-microglobulin and the α1, α2 and α3 extracellular domains of a class I MHC molecule that has a relative frequency occurrence of less than 1%.
[021] In one embodiment, the fusion polypeptide comprises a peptide that elicits a T cell response, a β2-microglobulin and the α1, α2 and α3 extracellular domains of a class I MHC molecule that has a relative frequency of occurrence of 1% or more.
[022] In one embodiment, the polypeptides of the pair of polypeptide chains linked by disulfide bonds derived from an antibody hinge region: i) are linked by one or more disulfide bonds; ii) the first disulfide linked polypeptide chain comprises, in the N-terminal to C-terminal direction, an immunoglobulin light or heavy chain variable domain, or a heavy chain constant domain, and a polypeptide from the hinge region of the antibody heavy chain, and the second disulfide linked polypeptide chain comprises a region of the polypeptide of the hinge region of the antibody heavy chain.
[023] In one embodiment, the fusion polypeptide is; i) covalently linked at both the C-terminus and the N-terminus of one of the polypeptide chains linked by disulfide bonds; or ii) covalently linked to the N-terminus of an antibody variable domain that is the complementary cognate of the light or heavy chain variable domain that is comprised in the disulfide-linked first polypeptide chain; or iii) covalently linked to the C-terminal end of an antibody constant domain that is complementary to the light or heavy chain constant domain that is comprised in the disulfide-linked first polypeptide chain.
[024] In one embodiment, the peptide that elicits a T-cell response is a virus-derived peptide.
[025] In one embodiment, the fusion polypeptide comprises in the N- to C-terminal direction: (I) a virus-derived peptide that has an amino acid sequence selected from SEQ ID NO: 1 through SEQ ID NO: 9; (II) a first binding peptide that has an amino acid sequence selected from SEQ ID NO: 16, 17, 18, 21, 22, and 23; (III) a β2-microglobulin that has an amino acid sequence of SEQ ID NO: 10; (iv) a second binding peptide that has an amino acid sequence selected from SEQ ID NO: 16, 17, 18, 21, 22 and 23; (v) the extracellular domains α1, α2 and α3 of a MHC class I molecule that has an amino acid sequence of SEQ ID NO: 11; and (vi) a third binding peptide that has an amino acid sequence selected from SEQ ID NO: 12, 16, 17, 18, 21, 22 and 23.
[026] In one embodiment, the disulfide-linked first polypeptide chain and the disulfide-linked second polypeptide chain comprise; i) a human IgG1 CH2 domain, comprising an amino acid sequence selected from SEQ ID NO: 31, 32 and 33, and a human IgG1 CH3 domain comprising an amino acid sequence selected from SEQ ID NO: 34, 35, and 36.
[027] In an example of an embodiment, the complex comprises: i) a first binding peptide that has the amino acid sequence of SEQ ID NO: 21; and / or ii) a second binding peptide that has the amino acid sequence of SEQ ID NO: 22; and / or iii) a third binding peptide that has the amino acid sequence of SEQ ID NO: 12; and / or iv) a human IgG1 CH2 domain that has the amino acid sequence of SEQ ID NO: 32 or 33; and / or v) in the first disulfide-linked polypeptide a CH3 domain of human IgG1 that has the amino acid sequence of SEQ ID NO: 35; and in the second disulfide-linked polypeptide a CH3 domain of human IgG1 that has the amino acid sequence of SEQ ID NO: 36.
[028] One aspect as disclosed in the present invention, is a complex, characterized by comprising: - a fusion polypeptide comprising in the N- to C-terminal direction; and both: (i) a β2-microglobulin, and (ii) the extracellular domains α1, α2 and α3 of a class I MHC molecule, with a relative frequency of less than 1%; or (i) a peptide that elicits T cell response; (ii) a β2-microglobulin, and (iii) the extracellular domains α1, α2 and α3 of a class I MHC molecule with a relative frequency of 1% or more; and; - two polypeptide chains, which are linked by one or more disulfide bonds; wherein the disulfide-linked first polypeptide chain comprises, in the N-terminal to C-terminal direction: (i) an immunoglobulin light or heavy chain variable domain; (ii) an immunoglobulin light or heavy chain constant domain, and (iii) a polypeptide from the hinge region of the antibody heavy chain; and wherein the second disulfide-linked polypeptide chain comprises a polypeptide from the hinge region of the antibody heavy chain; wherein the fusion polypeptide is: - covalently linked to the C-terminal or N-terminal end of one of the polypeptide chains linked by disulfide bonds, or - covalently linked to the N-terminal of an antibody variable domain which is the light or heavy chain variable domain complementary to that comprised in the disulfide-linked first polypeptide chain, or - covalently linked to the C-terminus of an antibody constant domain which is the complementary light or heavy chain constant domain to that comprised in the first linked polypeptide chain by disulfide.
[029] In an example of carrying out all aspects, the complex is an antigen-binding complex.
[030] In an example of carrying out all aspects, the complex is a covalent complex.
[031] In an example of carrying out all aspects, the MHC class I molecule with a relative frequency of 1% or more, has a relative frequency of 10% or more. In one example, the MHC class I molecule with a relative frequency of 10% or more is HLA-A * 0201 or HLA-A * 1101 or HLA-A * 2402 or HLA-A * 340101, or HLA- C * 0304 or HLA-C * 0401 or HLA-C * 0702.
[032] In an example of carrying out all aspects, the MHC class I molecule with a relative frequency of 10% or more, is selected, depending on the region of the individual to which the complex will be administered as follows: - For a individual of European origin, the MHC class I molecule is selected from the group comprising HLA-A * 0101, HLA-A * 0201, HLA-A * 0301, HLA-B * 0702, HLA-B * 0801, HLA- B * 4402, HLA-C * 0401, HLA-C * 0501, HLA-C * 0701 and HLA-C * 0702; - For an individual of Australian origin, the MHC class I molecule is selected from the group comprising HLA-A * 0201, HLA-A * 1101, HLA-A * 2402, HLA-A * 340101, HLA-B * 1301 , HLA-B * 1521, HLA-B * 5601, HLA B * 5602, HLA-C * 0102, HLA-C * 0401, HLA-C * 0403 and HLA C * 1502; - For an individual of North American origin, the MHC class I molecule is selected from the group comprising HLA-A * 0201, HLA-A * 2402, HLA C * 0202, HLA-C * 0304, HLA-C * 0401 and HLA-C * 0702; e - For an individual of Southeast Asian origin the MHC class I molecule is selected from the group comprising HLA-A * 1101, HLA-A * 2402, HLA B * 1504, HLA-C * 0102, HLA-C * 0304, HLA-C * 0702 and HLA C * 0801.
[033] In an example of carrying out all aspects, the MHC class I molecule with a relative frequency of 10% or more, is selected, depending on the region of the individual to which the complex will be administered as follows: - For a individual of European origin, the MHC class I molecule is HLA-A * 0201; - For an individual of Australian origin the MHC class I molecule is selected from the group comprising HLA-A * 2402, HLA-B * 1301, HLA-C * 0102 and HLA-C * 0401; - For an individual of North American origin, the MHC class I molecule is selected from the group comprising HLA-A * 2402 and HLA-C * 0304; and - For an individual of Southeast Asian origin, the MHC class I molecule is HLA-A * 2402.
[034] In an example of carrying out all aspects the fusion polypeptide, which comprises in the N-terminal to C-terminal direction, a β2-microglobulin and the extracellular domains α1, α2 and α3 of a class I MHC molecule, with a relative frequency of less than 1%, it further comprises at its N-terminal end a binding peptide to the binding groove in the MHC peptide-groove.
[035] In an example of carrying out all aspects, the peptide that elicits a T cell response is a peptide that elicits a CD8 + T cell response. In one embodiment, the peptide that elicits a T-cell response is a virus-derived peptide.
[036] In an example of carrying out all aspects, the MHC class I molecule, with a relative frequency of less than 1%, is selected from the group comprising HLA-B * 4201, HLA-B * 5901, HLA- B * 6701 and HLA-B * 7802.
[037] In an example of carrying out all aspects, the fusion polypeptide comprises: (i) a virus-derived peptide; (ii) β2-microglobulin; and (iii) the soluble HLA-A of the A * 0201 allele.
[038] In an example of carrying out all aspects, the virus is selected from adenovirus, human herpesvirus 1, human herpesvirus 2, human herpesvirus 4 (Epstein-Barr virus), hepatitis-B virus, hepatitis-virus C, human cytomegalovirus, human immunodeficiency virus, human papillomavirus type 16, human papillomavirus type 18, human papillomavirus type 31, human papillomavirus type 33, human papillomavirus type 35, human papillomavirus type 39, human papillomavirus type 45, human papillomavirus type 51, human papillomavirus type 52, human papillomavirus type 56, human papillomavirus type 58, human papillomavirus type 59, human papillomavirus type 68, human papillomavirus type 73, human papillomavirus type 82, Human T-cell lymphotropic virus type 1, human influenza virus, virus human influenza B, vaccinia virus, dengue virus.
[039] In an example of carrying out all aspects, the virus-derived peptide is selected from NLVPMVATV (SEQ ID NO: 1), SLYNTVATL (SEQ ID NO: 2), GLCTLVAML (SEQ ID NO: 3), GILGFVFTL (SEQ ID NO: 4), STNRQSGRQ (SEQ ID NO: 5), LLFGYPVYV (SEQ ID NO: 6), FAEGFVRAL (SEQ ID NO: 7), LIVIGILIL (SEQ ID NO: 8), or ILHTPGCV (SEQ ID NO: 8) NO: 9), WYAQIQPHW (SEQ ID NO: 52), AFSGVSWTM (SEQ ID NO: 53), ILIGVVITW (SEQ ID NO: 54), MMIPTVVAF (SEQ ID NO: 55), PFPQSNAPI (SEQ ID NO: 56), LLLTLLATV (SEQ ID NO: 57), IVLEHGSCV (SEQ ID NO: 58), LLFKTENGV (SEQ ID NO: 59), PLNEAIMAV (SEQ ID NO: 60), NLVRLQSGV (SEQ ID NO: 61), LVISGLFPV (SEQ ID NO : 62), LLLVAHYAI (SEQ ID NO: 63), LALLAAFKV (SEQ ID NO: 64), VILAGPMPV (SEQ ID NO: 65), HVLGRLITV (SEQ ID NO: 66), VTEHDTLLY (SEQ ID NO: 67), NTDFRVLELEL (SEQ ID NO: 68), CVETMCNEY (SEQ ID NO: 69), VLEETSVML (SEQ ID NO: 70), NLVPMVATV (SEQ ID NO: 71), RIFAELEGV (SEQ ID NO: 72), IIYTRNHEV (SEQ ID NO: 73), VLAELVKQI (SEQ ID NO: 74), AVGGAVASV (SEQ ID NO: 75), TVRSH CVSK (SEQ ID NO: 76), IMREFNSYK (SEQ ID NO: 77), GPISHGHVLK (SEQ ID NO: 78), ATVQGQNLK (SEQ ID NO: 79), VYALPLKML (SEQ ID NO: 80), AYAQKIFKIL (SEQ ID NO : 81), QYDPVAALF (SEQ ID NO: 82), YVKVYLESF (SEQ ID NO: 83), DIYRIFAEL (SEQ ID NO: 84), VFETSGGLVV (SEQ ID NO: 85), KARDHLAVL (SEQ ID NO: 86), QARLTVSGL (SEQ ID NO: 87), KARAKKDEL (SEQ ID NO: 88), QIKVRVDMV (SEQ ID NO: 89), RRRHRQDAL (SEQ ID NO: 90), ARVYEIKCR (SEQ ID NO: 91), KMQVIGDQY (SEQ ID NO: 92), NVRRSWEEL (SEQ ID NO: 93), CPSQEPMSIYVY (SEQ ID NO: 94), KPGKISHIMLDVA (SEQ ID NO: 95), ELRRKMMYM (SEQ ID NO: 96), IPSINVHHY (SEQ ID NO: 97), FEQPTETPP ( SEQ ID NO: 98), YAYIYTTYL (SEQ ID NO: 99), QEFFWDANDIY (SEQ ID NO: 100), YEQHKITSY (SEQ ID NO: 101), QEPMSIYVY (SEQ ID NO: 102), SEHPTFTSQY (SEQ ID NO: 103) ), QAIRETVEL (SEQ ID NO: 104), TRATKMQVI (SEQ ID NO: 105), DALPGPCI (SEQ ID NO: 106), CEDVPSGKL (SEQ ID NO: 107), HERNGFTVL (SEQ ID NO: 108), PTFTSQYRIQGKL (SEQ ID NO: 109), QMWQARLTV (SEQ ID NO: 110), HELLVLVKKAQL (SEQ ID NO: 111), or DDYSNTHSTRYV (SEQ ID NO: 112), or a variant thereof comprising 1 to 3 changes, additions and / or deletions of amino acids.
[040] In an example of carrying out all aspects, β2-microglobulin is human β2-microglobulin. In one embodiment, β2-microglobulin is human β2-microglobulin wild type. In one embodiment, β2-microglobulin is made up of the amino acid sequence of SEQ ID NO: 10 or is a variant thereof comprising between 1 and 10 exchanges, additions and / or deletions of amino acids.
[041] In an example of carrying out all aspects, β2-microglobulin is human β2-microglobulin and the class I MHC molecule with a relative frequency of 10% or more is human HLA-A * 0201. In one embodiment, the extracellular domains α1, α2 and α3 of the MHC class I molecule consist of the amino acid sequence of SEQ ID NO: 11 or a variant thereof comprising between 1 and 10 amino acid changes, additions and / or deletions.
[042] In an example of carrying out all aspects, the virus-derived peptide is fused to β 2-microglobulin via a first binding peptide. In one embodiment, the virus-derived peptide is fused to the N-terminal end of β2-microglobulin.
[043] In an example of carrying out all aspects, β2-microglobulin is fused to the α1 extracellular domain of a class I MHC molecule through a second binding peptide.
[044] In an example of carrying out all aspects, the α3 extracellular domains of the MHC class I molecule are fused to one of the polypeptide chains linked by disulfide bonds, via a third binding peptide.
[045] In an example of carrying out all aspects, the first, second and third binding peptide is the same or is a different binding peptide.
[046] In an example of carrying out all aspects, the first binding peptide, the second binding peptide, and the third binding peptide are selected independently of each other from the amino acid sequences: GS (SEQ ID NO: 12 ), GGS (SEQ ID NO: 13), GGGS (SEQ ID NO: 14), GGGSGGGS (SEQ ID NO: 15), GGGSSGGGSGGGS (SEQ ID NO: 16), GGGSGGGSGGGSGGGS (SEQ ID NO: 17), GGGGGGGGSG ID NO: 18), GGGGS (SEQ ID NO: 19), GGGGSGGGGS (SEQ ID NO: 20), GGGGSGGGGSGGGGS (SEQ ID NO: 21), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 22), GGGS ).
[047] In an example of carrying out all aspects, the first binding peptide comprises the amino acid sequence of SEQ ID NO: 21.
[048] In an example of carrying out all aspects, the second binding peptide comprises the amino acid sequence of SEQ ID NO: 22.
[049] In an example of carrying out all aspects, the third binding peptide comprises the amino acid sequence of SEQ ID NO: 12.
[050] In an example of carrying out all aspects, the antibody heavy chain hinge region polypeptide is selected from a heavy chain hinge region polypeptide of a human IgG class or IgE class antibody.
[051] In an example of carrying out all aspects, the polypeptide of the hinge region of the antibody heavy chain is selected from a polypeptide of the hinge region of the heavy antibody of a human antibody of the subclass IgG1, or IgG2, or IgG3 or IgG4.
[052] In an example of carrying out all aspects, the polypeptide of the hinge region of the antibody heavy chain comprises or consists of the amino acid sequence EPKSCDKTHTCPPCP (SEQ ID NO: 24), EPKSADKTHTCPPCP (SEQ ID NO: 25), ERKCCVECPPCP (SEQ ID NO: 26), ERKCAVECPPCP (SEQ ID NO: 27), ERKACVECPPCP (SEQ ID NO: 28), ELKTPLGDTTHTCPRCP (EPKSCDTPPPCPRCP) 3 (SEQ ID NO: 29), ESKYGPPCPSCP (SEQ ID NO: 30), (SEQ ID NO: 47), VECPPCP (SEQ ID NO: 48), AVECPPCP (SEQ ID NO: 49), DTTHTCPRCP (SEQ ID NO: 50) or PPCPSCP (SEQ ID NO: 51).
[053] In an example of carrying out all aspects, the first disulfide-linked polypeptide and / or the second disulfide-linked polypeptide comprises a CH2 domain and / or a CH3 domain of human origin. In one exemplary embodiment, the CH2 domain and the CH3 of human origin is a human antibody of the IgG or IgE class. In one embodiment, the CH2 and CH3 domain is a human antibody of the subclass IgG1, or IgG2, or IgG3, or IgG4. In one embodiment, the CH2 domain comprises the amino acid sequence of SEQ ID NO: 31. In one embodiment, the CH2 domain is a human antibody of the IgG1 or IgG2 subclass, and comprises at least one mutation in E233, L234, L235, G236, D265, D270, N297, E318, K320, K322, A327, P329, A330 and / or P331 (numbering according to the EU Kabat index). In one embodiment, the CH2 domain is a human antibody of the IgG1 subclass or the human IgG2 subclass with the L234A and L235A mutations and / or the D265A and N297A mutations, and / or contains the PVA236 mutation, and / or contains the P329G mutation. In one embodiment, the CH2 domain is a human antibody of the IgG1 subclass with mutations L234A and L235A and / or P329G. In one embodiment, the CH2 domain is a human antibody of the IgG4 subclass with the S228P and / or L235E mutation. In one embodiment, the CH2 domain comprises the amino acid sequence of SEQ ID NO: 32 or SEQ ID NO: 33. In one embodiment, the CH3 domain comprises the amino acid sequence of SEQ ID NO: 34.
[054] In an example of carrying out all aspects, the first disulfide-linked peptide comprises the amino acid sequence of SEQ ID NO: 35, and the second disulfide-linked peptide comprises the amino acid sequence of SEQ ID NO: 36.
[055] In an example of carrying out all aspects, the first and the second polypeptide linked by disulfide bonds comprise the amino acid sequence of SEQ ID NO: 37 or SEQ ID NO: 38.
[056] In an example of carrying out all aspects, the first or second disulfide linked polypeptide consists of the amino acid sequence of SEQ ID NO: 37 or SEQ ID NO: 38.
[057] In an example of carrying out all aspects, the first disulfide-linked peptide comprises the amino acid sequence of SEQ ID NO: 39, and the second disulfide-linked peptide comprises the amino acid sequence of SEQ ID NO: 40.
[058] In an example of carrying out all aspects, the polypeptide chains, which are linked by one or more disulfide bonds, are linked by two, or three, or four disulfide bonds.
[059] In an example of carrying out all aspects, the complex is characterized by the fact that the fusion polypeptide comprises in the N- to C-terminal direction: (i) a virus-derived peptide that has a selected amino acid sequence from the group comprising SEQ ID NO: 1 to SEQ ID NO: 9; (ii) a first binding peptide that has an amino acid sequence selected from the group comprising SEQ ID NO: 16, 17, 18, 21, 22, and 23; (iii) a β2-microglobulin that has an amino acid sequence of SEQ ID NO: 10 or is a variant thereof that comprises from 1 to 10 amino acid changes, additions and / or deletions; (iv) a second binding peptide that has an amino acid sequence selected from the group comprising SEQ ID NO: 16, 17, 18, 21, 22, and 23; (v) the extracellular domains α1, α2 and α3 of the MHC class I molecule that has the amino acid sequence of SEQ ID NO: 11 or is a variant thereof comprising from 1 to 10 amino acid changes, additions and / or deletions; and (vi) a third binding peptide that has an amino acid sequence selected from the group comprising SEQ ID NO: 12, 16, 17, 18, 21, 22 and 23. (vii)
[060] In an example of carrying out all aspects, the first disulfide-linked polypeptide and the second disulfide-linked polypeptide further comprise: (viii) a CH2 domain of human IgG1, comprising an amino acid sequence selected from from SEQ ID NO: 31, 32 and 33; and (ix) a CH3 domain of human IgG1, comprising an amino acid sequence selected from SEQ ID NO: 34, 35 and 36.
[061] In an example of carrying out all aspects, the complex is characterized by the fact that it comprises: (x) a fusion polypeptide that comprises in the N- to C-terminal direction; (i) a virus-derived peptide that has an amino acid sequence selected from SEQ ID NO: 1 through SEQ ID NO: 9; (ii) a first binding peptide that has an amino acid sequence selected from SEQ ID NO: 16, 17, 18, 21, 22 and 23; (iii) a β2-microglobulin that has an amino acid sequence of SEQ ID NO: 10 or is a variant thereof that comprises from 1 to 10 amino acid changes, additions and / or deletions; (iv) a second binding peptide that has an amino acid sequence selected from SEQ ID NO: 16, 17, 18, 21, 22 and 23; (v) the extracellular domains α1, α2 and α3 of the MHC class I molecule that has the amino acid sequence of SEQ ID NO: 11 or is a variant thereof comprising from 1 to 10 amino acid changes, additions and / or deletions; and (vi) a third binding peptide that has an amino acid sequence selected from SEQ ID NO: 12, 16, 17, 18, 21, 22 and 23; and; - two polypeptide chains, which are linked by one or more disulfide bonds; wherein the disulfide-linked first polypeptide chain comprises, in the N-terminal to C-terminal direction: (i) an immunoglobulin light or heavy chain variable domain; (ii) an immunoglobulin light or heavy chain constant domain; (iii) a polypeptide from the hinge region of the antibody heavy chain comprising an amino acid sequence selected from SEQ ID NO: 24 through SEQ ID NO: 30 and SEQ ID NOs: 47-51; (iv) a human IgG1 CH2 domain comprising an amino acid sequence selected from SEQ ID NO: 31, 32 and 33; and (v) a human IgG1 CH3 domain comprising an amino acid sequence selected from SEQ ID NO: 34, 35 and 36; the second disulfide-linked polypeptide chain comprises, in the N-terminal to C-terminal direction: (i) a polypeptide from the hinge region of the antibody heavy chain comprising an amino acid sequence selected from SEQ ID NO: 24 to SEQ ID NO: 30 and SEQ ID NOs: 47-51; (ii) a human IgG1 CH2 domain comprising an amino acid sequence selected from SEQ ID NO: 31, 32 and 33; and (iii) a human IgG1 CH3 domain comprising an amino acid sequence selected from SEQ ID NO: 34, 35 and 36; wherein the fusion polypeptide is: - covalently attached to the C-terminal or N-terminal end of the second disulfide-linked polypeptide chain.
[062] In an example of carrying out all aspects, the first and second disulfide-linked polypeptide chain comprises the same polypeptide as the hinge region of the antibody heavy chain.
[063] In an example of carrying out all aspects, the virus-derived polypeptide comprises the amino acid sequence of SEQ ID NO: 1, the first binding peptide comprises the amino acid sequence of SEQ ID NO: 21, the second peptide linker comprises the amino acid sequence of SEQ ID NO: 22, the third binding peptide comprises the amino acid sequence of SEQ ID NO: 12, the CH2 domain of human IgG1 comprises the amino acid sequence of SEQ ID NO: 32 or 33 , and the CH3 domain of human IgG1 of a disulfide-linked polypeptide comprises the amino acid sequence of SEQ ID NO: 35 and the CH3 domain of human IgG1 of the other disulfide-linked polypeptide comprises the amino acid sequence of SEQ ID NO: 36.
[064] In an example of carrying out all aspects, the complex is characterized by the fact that it comprises: - a fusion polypeptide that comprises in the N- to C-terminal direction; (i) a virus-derived peptide that has an amino acid sequence selected from SEQ ID NO: 1 through SEQ ID NO: 9; (ii) a first binding peptide that has an amino acid sequence selected from SEQ ID NO: 16, 17, 18, 21, 22 and 23; (iii) a β2-microglobulin that has an amino acid sequence of SEQ ID NO: 10 or is a variant thereof that comprises from 1 to 10 amino acid changes, additions and / or deletions; (iv) a second binding peptide that has an amino acid sequence selected from SEQ ID NO: 16, 17, 18, 21, 22 and 23; (v) the extracellular domains α1, α2 and α3 of the MHC class I molecule that has the amino acid sequence of SEQ ID NO: 11 or is a variant thereof comprising from 1 to 10 amino acid changes, additions and / or deletions; and (vi) a third binding peptide that has an amino acid sequence selected from SEQ ID NO: 12, 16, 17, 18, 21, 22 and 23; and; - two polypeptide chains, which are linked by one or more disulfide bonds; wherein the disulfide-linked first polypeptide chain comprises, in the N-terminal to C-terminal direction: (i) an immunoglobulin light or heavy chain variable domain; (ii) an immunoglobulin light or heavy chain constant domain; (iii) a polypeptide from the hinge region of the antibody heavy chain comprising an amino acid sequence selected from SEQ ID NO: 24 through SEQ ID NO: 30 and SEQ ID NOs: 47-51; (iv) a human IgG1 CH2 domain comprising an amino acid sequence selected from SEQ ID NO: 31, 32 and 33; and (v) a human IgG1 CH3 domain comprising an amino acid sequence selected from SEQ ID NO: 34, 35 and 36; and the second disulfide-linked polypeptide chain comprises the hinge region of the antibody heavy chain comprising an amino acid sequence selected from SEQ ID NO: 24 through SEQ ID NO: 30 and SEQ ID NOs: 47-51; wherein the fusion polypeptide is: - covalently linked to the C-terminal or N-terminal end of the first disulfide-linked polypeptide chain, or - covalently linked to the N-terminal of an antibody variable domain which is the variable domain light or heavy chain complementary to that comprised in the disulfide-linked first polypeptide chain, or - covalently linked to the C-terminus of an antibody constant domain which is the complementary light or heavy chain constant domain to that comprised in the disulfide-linked first polypeptide chain .
[065] In an example of carrying out all aspects, the virus-derived polypeptide comprises the amino acid sequence of SEQ ID NO: 1, the first binding peptide comprises the amino acid sequence of SEQ ID NO: 21, the second peptide linker comprises the amino acid sequence of SEQ ID NO: 22, the third binding peptide comprises the amino acid sequence of SEQ ID NO: 12, the CH2 domain of human IgG1 comprises the amino acid sequence of SEQ ID NO: 32 or 33 , and the CH3 domain of human IgG1 of a disulfide-linked polypeptide comprises the amino acid sequence of SEQ ID NO: 35 and the CH3 domain of human IgG1 of the other disulfide-linked polypeptide comprises the amino acid sequence of SEQ ID NO: 36.
[066] In an example of carrying out all aspects, the complex is characterized by the fact that it comprises: - a fusion polypeptide that comprises in the N- to C-terminal direction; (i) a virus-derived peptide that has an amino acid sequence selected from SEQ ID NO: 1 through SEQ ID NO: 9; (ii) a first binding peptide that has an amino acid sequence selected from SEQ ID NO: 16, 17, 18, 21, 22 and 23; (iii) a β2-microglobulin that has an amino acid sequence of SEQ ID NO: 10 or is a variant thereof that comprises from 1 to 10 amino acid changes, additions and / or deletions; (iv) a second binding peptide that has an amino acid sequence selected from SEQ ID NO: 16, 17, 18, 21, 22 and 23; (v) the extracellular domains α1, α2 and α3 of the MHC class I molecule that has the amino acid sequence of SEQ ID NO: 11 or is a variant thereof comprising from 1 to 10 amino acid changes, additions and / or deletions; and (vi) a third binding peptide that has an amino acid sequence selected from SEQ ID NO: 12, 16, 17, 18, 21, 22 and 23; and; - two polypeptide chains, which are linked by one or more disulfide bonds; wherein the first and second disulfide-linked polypeptide chain comprises in the N-terminal to C-terminal direction: (i) an immunoglobulin light or heavy chain variable domain; (ii) an immunoglobulin light or heavy chain constant domain; (iii) a polypeptide from the hinge region of the antibody heavy chain comprising an amino acid sequence selected from SEQ ID NO: 24 through SEQ ID NO: 30 and SEQ ID NOs: 47-51; (iv) a human IgG1 CH2 domain comprising an amino acid sequence selected from SEQ ID NO: 31, 32 and 33; and; (v) a human IgG1 CH3 domain comprising an amino acid sequence selected from SEQ ID NO: 34, 35 and 36; wherein the fusion polypeptide is: - covalently linked to the C-terminal or N-terminal end of the second disulfide-linked polypeptide chain, or - covalently linked to the N-terminal of an antibody variable domain which is the variable domain light or heavy chain complementary to that comprised in the disulfide-linked first polypeptide chain, or - covalently linked to the C-terminus of an antibody constant domain which is the complementary light or heavy chain constant domain to that comprised in the disulfide-linked first polypeptide chain .
[067] In an example of carrying out all aspects, the virus-derived polypeptide comprises the amino acid sequence of SEQ ID NO: 1, the first binding peptide comprises the amino acid sequence of SEQ ID NO: 21, the second peptide linker comprises the amino acid sequence of SEQ ID NO: 22, the third binding peptide comprises the amino acid sequence of SEQ ID NO: 12, the CH2 domain of human IgG1 comprises the amino acid sequence of SEQ ID NO: 32 or 33 , and the CH3 domain of human IgG1 of a disulfide-linked polypeptide comprises the amino acid sequence of SEQ ID NO: 35 and the CH3 domain of human IgG1 of the other disulfide-linked polypeptide comprises the amino acid sequence of SEQ ID NO: 36.
[068] In an example of carrying out all aspects: - the first binding peptide comprises the amino acid sequence of SEQ ID NO: 21; and / or - the second binding peptide comprises the amino acid sequence of SEQ ID NO: 22; and / or - the third binding peptide comprises the amino acid sequence of SEQ ID NO: 12; and / or - the CH2 domain of human IgG1 comprises the amino acid sequence of SEQ ID NO: 32 or 33; and / or - the human IgG1 CH3 domain of a disulfide-linked polypeptide comprises the amino acid sequence of SEQ ID NO: 35, and the human IgG1 CH3 domain of the other disulfide-linked polypeptide comprises the amino acid sequence of SEQ ID NO: 35 : 36.
[069] In an example of carrying out all aspects, the virus-derived peptide is selected from the group comprising SEQ ID NO: 1 through SEQ ID NO: 9, SEQ ID NO: 52 through SEQ ID NO: 112, or a variant of these that comprises 1 to 3 changes, additions and / or deletions of amino acids.
[070] One aspect as disclosed in the present invention is a nucleic acid encoding the complex, as disclosed in the present invention.
[071] In one embodiment, the nucleic acid comprises two to four expression cassettes comprising the structural genes encoding polypeptides with different amino acid sequences.
[072] One aspect as disclosed in the present invention is a host cell comprising the nucleic acid as disclosed in the present invention.
[073] One aspect as disclosed in the present invention is a method for producing a complex as disclosed in the present comprising cultivating the host cell as disclosed in the present so that the complex is produced.
[074] In one example of an embodiment, the complex is recovered from the cells or from the culture medium and, in this way, the complex is produced.
[075] One aspect as disclosed in the present invention is an immunoconjugate comprising the complex, as reported in the present invention and a cytotoxic agent.
[076] One aspect as disclosed in the present invention is a pharmaceutical formulation comprising the complex, as disclosed in the present invention and, optionally, a pharmaceutically acceptable carrier.
[077] In one embodiment, the pharmaceutical formulation further comprises an additional therapeutic agent.
[078] One aspect as disclosed in the present invention is the complex, as disclosed herein, for use as a medicament.
[079] One aspect as disclosed in the present invention is the complex, as reported here, for use in the treatment of cancer or a chronic viral infection.
[080] One aspect as disclosed in the present invention is the complex, as disclosed herein, for use in attracting virus-specific cytotoxic T cells to a target in an individual.
[081] One aspect as disclosed in the present invention is the complex, as disclosed herein, for use in removing cancer cells or virus-infected cells.
[082] One aspect as disclosed in the present invention is the use of the complex, as disclosed herein, in the manufacture of a medicament.
[083] In one example, the drug is for the treatment of cancer or a chronic viral infection.
[084] In one embodiment, the drug is to attract virus-specific cytotoxic T cells to a target in an individual.
[085] In one example of an embodiment, the drug is for removing cancer cells or cells infected by viruses.
[086] One aspect as disclosed in the present invention is a method of treating an individual with cancer or chronic viral infection, the method of which comprises administering to the individual an effective amount of the complex disclosed in the present invention.
[087] In one embodiment, the method further comprises administering an additional therapeutic agent to the individual.
[088] One aspect as disclosed in the present invention is a method of attracting virus-specific cytotoxic T cells from an individual to a target in the individual, the method of which comprises administering to the individual an effective amount of the complex disclosed in the present invention to attract the virus-specific cytotoxic T cells of the individual to a target.
[089] One aspect as disclosed in the present invention is a method for removing cancer cells or virus-infected cells in an individual, the method of which comprises administering to the individual an effective amount of the complex disclosed in the present invention to remove / disintegrate cancer cells or virus-infected cells. DETAILED DESCRIPTION OF THE INVENTION BRIEF DESCRIPTION OF THE FIGURES
[090] Figure 1 Annotated scheme of the complexes, as disclosed in the present invention.
[091] Figure 2 Exemplary polypeptides comprised in the complex, as disclosed in the present invention: fusion polypeptides were fused at the N-terminus to a polypeptide comprising the antibody light chain or a hinge region of the antibody heavy chain.
[092] Figure 3 Western blot of an SDS polyacrylamide gel from the HEK 293 cell culture supernatant transfected with the corresponding expression plasmids. Staining was performed with rabbit polyclonal anti-human K-chain light conjugated to peroxidase and rabbit polyclonal anti-human IgG antibody conjugated to horseradish peroxidase.
[093] Lanes: 1: peptide-β2-microglobulin-HLA-A0201-IgG-Fc of two arms; 2: peptide-e2-microglobulin-HLA-A0201-IgG-Fc of an arm + IgG-Fc, 3: peptide β2-microglobulin-HLA-A0201-IgG-heavy chain of an arm + IgG-light chain + IgG-Fc , 4: peptide β2-microglobulin-HLA-A0201-IgG-heavy chain of one arm + IgG-heavy chain + IgG-light chain; 5: β2-microglobulin-HLA-A0201-IgG-light chain with two arms + IgG-heavy chain; 6: peptide-e2-microglobulin-HLA-A0201-IgG-light chain of two arms + IgG-heavy chain; 7: peptide-e2-microglobulin-HLA-A0201-IgG-two-arm heavy chain + IgG-light chain; 8: peptide-e2-microglobulin-HLA-A0201-IgG-Fc-scFv of two arms; 9: β2-microglobulin-HLA-A0201-IgG-Fc peptide from one arm + IgG from one arm (heavy and light chain); 10: molecular weight marker; 11: reference standard IgG1 antibody.
[094] Figure 4 Flow cytometry analysis to determine the number of cytolytic CMV-specific T cells from different donors before and after in vitro stimulation with the specific peptide: Analysis of the human donor's peripheral blood lymphocytes (PBL) 4; anti-CD8 antibody conjugated to the FITC marker (BD, Cat. No. 345,772), conjugated to Pro5 pentamer APC (ProImmune, Cat. No. F008-4A-E) stained TCR staining MHC class I (HLA-A * 0201) loaded with CMV-derived peptide (NLVPMVATV, SEQ ID NO: 1); circle: CD8 + specific CMV- T-cells.
[095] Figure 5 Flow cytometry to analyze the cytolytic capacity of CTL stimulated by lysis of MN60 tumor cells loaded with CMV peptide.
[096] Figure 6 Removal of specific T cells from CMV-pulsed T cells: Flow cytometric analysis to assess the cytolytic capacity of CTLs stimulated by lysis of MN60 tumor cells loaded with the CMV peptide depending on the effector cell / cell ratio - target; black: MN60 cells loaded with CMV peptide, white: MN60 cells not loaded with CMV peptide.
[097] Figure 7 A: SDS-PAGE gel with Coomassie staining: Lane 1: molecular weight standard, lane 2: β2-microglobulin-HLA-A0201-IgG-Fc from one arm + IgG complex from one arm ( light and heavy chain), under non-reducing conditions, Lane 3: β2-microglobulin-HLA-A0201-IgG-Fc from one arm + IgG complex from one arm (light and heavy chain), reduction conditions.
[098] B: chromatogram of size exclusion chromatography, 1: forms of high molecular weight (0.7 area%); 2: monomeric complex (99.3% area%).
[099] Figure 8 A: SDS-PAGE gel with Coomassie staining: Lane 1: molecular weight standard, lane 2: β2-microglobulin-HLA-A0201- IgG-heavy arm IgG + IgG light chain + IgG - Fc complex under non-reducing conditions, Lane 3: β2-microglobulin-HLA-A0201-IgG-one-arm heavy chain + IgG-light chain + IgG-Fc complex, reduction conditions.
[0100] B: chromatogram of size exclusion chromatography, 1: forms of high molecular weight (1.8% area); 2: monomeric complex (99.3% area%).
[0101] Figure 9 Analysis of the binding of a complex as disclosed herein to the human IGF-1R positive cell line using FACS; 1: H460M2 cells incubated with β2-microglobulin-HLA-A0201-IgG-heavy arm chain + IgG-light chain + IgG-Fc complex, unstained; 2: H460M2 cells incubated with β2-microglobulin-HLA-A0201-IgG-arm heavy chain + IgG-light chain + IgG-Fc complex at 4 ° C, stained using fluorescently labeled anti-human IgG antibody; 3: H460M2 cells incubated with β2-microglobulin-HLA-A0201-IgG-heavy chain from one arm + IgG-light chain + IgG-Fc complex at 37 ° C, stained using fluorescently labeled anti-human IgG antibody; 4: H460M2 cells incubated with peptide β2-microglobulin-HLA-A0201-IgG-heavy chain of an arm + IgG-light chain + IgG-complex, stained using fluorescently labeled antibody (anti-DIG antibody); 5: H460M2 cells stained with fluorescently labeled anti-human IgG antibody; 6: H460M2 cells stained with fluorescently labeled human anti-IGF-1R antibody.
[0102] Figure 10 Microscopy image of the antigen binding complex as disclosed in the present invention by lysis mediated by human I24 3T3 cells expressing IGF-1R (large cells growing adherently, white arrow). Lysis is mediated by CMV-specific human T cells (small round cells - white arrows, or migrating amoeboid cells, black arrow).
[0103] Figure 11 Image obtained by microscopy of I24 3T3 cells (adherent large cresenco cells, white arrow) incubated with CMV-specific human T cells (small rounded cells - white arrow, or ameboid migrating cells, black arrow) ), in the absence of a complex as disclosed in the present invention.
[0104] Figure 12 Cytotoxicity assay: antigen binding complex, as disclosed in the present invention, triggers the lysis of H460M2 tumor cells through CMV-specific human T-cells. a) (6h) Target cells: effector T cells specific for CMV 1: 1.5; b) (6h), target cells: effector T cells specific for CMV 1: 0.75; c) (6h) Target cells: effector T cells specific for CMV 1: 0.5; left bar: complex as disclosed in the present invention, right bar: IGF-1R-afucosylated mAb.
[0105] Figure 13 Cytotoxicity assay: antigen binding complex, as disclosed in the present invention triggers lysis of I24 3T3 tumor cells through CMV-specific human T cells. a) (9h) Target cells: effector T cells specific for CMV 1: 1.5; b) (9h) Target cells: CMV-specific effector T cells 1: 0.75; c) (9h) Target cells: effector T cells specific for CMV 1: 0.5; left bar: complex as disclosed in the present invention; middle bar: mAb IGF-1R-afu, right bar: mAb ed; right bar: anti-digoxigenin antibody.
[0106] Figure 14 FACS analysis of anti-IGF-1R antibody binding and complexes as disclosed in the present invention for the H460M2 lung adenocarcinoma cell line; a) secondary antibody only (goat anti-human IgG (H + L) antibody (Jackson Laboratories, Cat. # 109-116-088)); b) complex as disclosed in the present invention, wherein the fusion polypeptide is fused to the N-terminal heavy chain of an anti-IGF-1R antibody comprising only a pair of variable domains; c) anti-IGF-1R antibody.
[0107] Figure 15 Effectiveness and specificity in vitro (cytotoxicity assay) of different complexes, as disclosed in the present invention; a) complex comprising a monovalent anti-IGF1R antibody and a CMV-derived peptide, b) complex comprising a monovalent anti-IGF1R antibody and an EBV-derived peptide (control); c) complex comprising a divalent anti-IGF1R antibody and a CMV-derived peptide; d) anti-IGF-1R antibody (control); e) anti-digoxigenin antibody (control).
[0108] Figure 16 Efficacy and specificity in vitro (EC50 value) of a complex as disclosed in the present invention, where the fusion polypeptide is fused to the N-terminal end of the heavy chain of an anti-IGF-1R antibody complete determined in different target relationships (T) for effector cell (E).
[0109] Figure 17 Lysis of target cells after 6 hours of incubation with: a) a complex comprising a monovalent anti-IGF1R antibody and a fusion polypeptide comprising a CMV-derived peptide; and b) an anti-IGF-1R antibody at a target ratio for effector cells of 1: 1.5. BRIEF DESCRIPTION OF THE SEQUENCES
[0110] SEQ ID NO: 1: Peptide derived from human cytomegalovirus.
[0111] SEQ ID NO: 2: Peptide derived from the human immunodeficiency virus.
[0112] SEQ ID NO: 3: Peptide derived from human herpesvirus 4.
[0113] SEQ ID NO: 4: peptide derived from the Influenza A virus.
[0114] SEQ ID NO: 5: Hepatitis-B virus derived peptide.
[0115] SEQ ID NO: 6: Peptide derived from human T-cell lymphotropic virus type 1.
[0116] SEQ ID NO: 7: Peptide derived from the homologous oncogene of the Sarcoma virus V-jun 17.
[0117] SEQ ID NO: 8: Peptide derived from human adenovirus type 3.
[0118] SEQ ID NO: 9: Peptide derived from the Hepatitis-C virus.
[0119] SEQ ID NO: 10: Human β2-microglobulin amino acid sequence.
[0120] SEQ ID NO: 11: Human amino acid sequence of the αl- α3 HLA-A * 0201 chain.
[0121] SEQ ID NO: 12-23: Amino acid sequences of the binding peptide.
[0122] SEQ ID NO: 24: Amino acid sequence of the human IgG1 heavy chain hinge polypeptide.
[0123] SEQ ID NO: 25: Amino acid sequence of the variant polypeptide of the human IgG1 heavy chain hinge.
[0124] SEQ ID NO: 26: Amino acid sequence of the human IgG2 heavy chain hinge polypeptide.
[0125] SEQ ID NO: 27: Amino acid sequence of the variant polypeptide of the human IgG2 heavy chain hinge.
[0126] SEQ ID NO: 28: Amino acid sequence of the variant polypeptide of the human IgG2 heavy chain hinge.
[0127] SEQ ID NO: 29: Amino acid sequence of the human IgG3 heavy chain hinge polypeptide.
[0128] SEQ ID NO: 30: Amino acid sequence of the human IgG4 heavy chain hinge polypeptide.
[0129] SEQ ID NO: 31: Amino acid sequence of the CH2 domain of human IgG1.
[0130] SEQ ID NO: 32: Amino acid sequence of the CH2 domain of human IgG1 with L234A, L235A mutations.
[0131] SEQ ID NO: 33: Amino acid sequence of the CH2 domain of human IgG1 with L234A, L235A and P329G mutations.
[0132] SEQ ID NO: 34: Amino acid sequence of the CH3 domain of human IgG1.
[0133] SEQ ID NO: 35: Variant amino acid sequence of the human IgG1 CH3 knob domain.
[0134] SEQ ID NO: 36: Variant amino acid sequence of the CH3 hole domain of human IgG1.
[0135] SEQ ID NO: 37: Amino acid sequence of the human IgG1 Fc region.
[0136] SEQ ID NO: 38: Amino acid sequence of the human IgG1 Fc region with L234A, L235A mutations.
[0137] SEQ ID NO: 39: Amino acid sequence of the human IgG1 Fc region with mutations L234A, L235A and hole variant chain.
[0138] SEQ ID NO: 40: Amino acid sequence of the human IgG1 Fc region with mutations L234A, L235A and chain variant knob.
[0139] SEQ ID NO: 41: Amino acid sequence of the humanized anti-IGF-1R monoclonal antibody (kappa).
[0140] SEQ ID NO: 42: Amino acid sequence of the humanized anti-IGF-1R monoclonal antibody (mutant IgG1; L234A, L235A).
[0141] SEQ ID NO: 43: Amino acid sequence of the humanized anti-IGF-1R monoclonal antibody (mutant IgG1; L234A, L235A and knob variant).
[0142] SEQ ID NO: 44: Amino acid sequence of the humanized anti-IGF-1R monoclonal antibody (mutant IgG1; L234A, L235A and hole variant).
[0143] SEQ ID NO: 45: hinge region mutant of the human IgG1 Fc region and mutant L234A, L235A and knob variant.
[0144] SEQ ID NO: 46: Single chain Fv stabilized by disulfide bonds of the humanized anti-IGF-1R monoclonal antibody.
[0145] SEQ ID NO: 47-51: Amino acid sequence of the shortened human antibody heavy chain hinge polypeptide.
[0146] SEQ ID NO: 52-66: Peptides derived from the dengue virus.
[0147] SEQ ID NO: 67-112: Peptides derived from human cytomegalovirus. I. DEFINITIONS
[0148] "Affinity" refers, in general, to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (for example, an antibody) and its binding partner (for example, an antigen) . The "binding affinity" unless otherwise indicated, refers to the intrinsic binding affinity that reflects a 1: 1 interaction between the members of the binding pair (for example, antibody and antigen). The affinity of the molecule X for its partner Y can generally be represented by the dissociation constant (Kd). Affinity can be measured by methods known in the art, including the methods described in the present invention. Specific illustrative and exemplary embodiments to measure binding affinity are described below.
[0149] The term "amino acid" as used in the present application indicates the group of α-amino acids carboxy, which directly or in the form of a precursor can be encoded by a nucleic acid. Individual amino acids are encoded by nucleic acids that consist of three nucleotides, called codons or base triplets. Each amino acid is encoded by at least one codon. This is known as "degeneracy of the genetic code". The term "amino acid" as used in this application indicates the group of naturally occurring carboxy α-amino acids comprising alanine (three letter code: ala, one letter code: A), arginine (Arg, R), asparagine (Asn, N ), aspartic acid (Asp, D), cysteine (Cis, C), glutamine (Gln, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lis, K), methionine (Met, M), phenylalanine (Fen, F), proline (Pro, P), serine (Ser, S), threonine (Tre, T ), tryptophan (Trp, W), tyrosine (Tir, Y) and valine (Val, V).
[0150] The terms "anti-target antibody" and "an antibody that binds to the target" refer to an antibody that is capable of binding to the target with sufficient affinity for the antibody to become useful as a diagnostic agent and / or therapeutic in targeting the target. In certain embodiments, an antibody that binds to the target has a dissociation constant (Kd) <10 nM; <1 nM; <0.1 nM; <0.01 nM; or <0.001 nM (for example, 10-8 M or less; for example, from 10-8 M to 10-13 M; for example, from 10-9 M to 10-13 M).
[0151] The term "antibody" in the present invention is used in the broadest sense and encompasses several antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (for example, bispecific antibodies), and antibody fragments , as long as they exhibit the desired antigen-binding activity.
[0152] An "antibody fragment" refers to a molecule that is not an intact antibody, which comprises a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab ', Fab'-SH, F (ab') 2; diabodies, linear antibodies, single chain antibody molecules (e.g., scFv), single domain antibodies, and multispecific antibodies formed from antibody fragments.
[0153] The "class" of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five main classes of antibodies: IgA, IgD, IgE, IgG and IgM, and several of them can be divided into subclasses (isotypes), for example, IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, Y, and μ, respectively.
[0154] The term “MHC class I molecule with a relative frequency of” indicates that the respective MHC class I molecule has a frequency of occurrence in a specific population of humans or within all humans of a given relative frequency . Thus, a class I MHC molecule with a relative frequency of 10% or more, can be found in 10% or more of all humans in a specific population, for example, in 27.2% of all humans of European origin.
[0155] The "conjugation" of a complex with its conjugation partner can be accomplished by different methods, such as chemical bonding, or bonding by means of a specific bonding pair. The term "conjugation partner" indicates, for example, detectable marker polypeptides, members of specific binding pairs. In an example of an embodiment, the conjugation of a complex with its conjugation partner is carried out by chemical bonding through the N-terminal end and / or ε-amino groups (lysine), ε-amino groups of different functional groups lysine, carboxy, sulfhydryl, hydroxyl, and / or phenolics of the amino acid sequence of the complex parts, and / or alcohol sugar groups of the complex carbohydrate structure. In one embodiment, the complex is conjugated to its conjugation partner by means of a specific bonding pair.
[0156] The term "cytotoxic agent", as used herein, refers to a substance that inhibits or prevents cell function and / or causes cell destruction or death. Cytotoxic agents include, but are not limited to, radioactive isotopes (for example, At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, P212 and radioactive isotopes of Lu), drugs or chemotherapeutic agents (for example , methotrexate, adriamycin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents), growth inhibiting agents; enzymes and fragments thereof, such as nucleolytic enzymes, antibiotics and toxins such as, for example, small molecules of toxin or ezimatically active toxins of bacterial, fungal, plant or animal origin, including fragments or variants thereof; and the various anti-tumor or anti-cancer agents.
[0157] Chromogens (groups and dyes and fluorescent or luminescent), enzymes, NMR-active groups or metallic particles, haptens, for example, digoxigenin, are examples of "detectable markers". The detectable marker can also be a photoactivable cross-linking group, for example, an azide or azirine group. Metal chelates that can be detected by electrochemiluminescence are also suitable signal-emitting groups, of particular interest to ruthenium chelates, for example, a 32+ ruthenium (bispyridyl) chelate. Suitable ruthenium labeling groups are described, for example, in EP 0580979, WO 90/05301, WO 90/11511 and WO 92/14138. For the direct detection of the labeling group, any known detectable marker group can be selected from dyes, luminescent labeling groups, such as chemiluminescent groups, for example, acridinium esters or dioxetanes, or fluorescent dyes, for example, fluorescein , rhodamine, coumarin, oxazine, resorufin, cyanine and their derivatives. Other examples of labeling groups are luminescent metal complexes, such as europium complexes, enzymes, for example, such as those used in ELISA or for CEDIA (Immunoassay with Cloned Enzyme Donor, for example, EP-A-0 061 888) and radioisotopes.
[0158] "Effector functions" refer to the biological activities attributable to the Fc region of an antibody, which vary with the antibody's isotype. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (for example, B cell receptor), and activation of B cells.
[0159] An "effective amount" of an agent, for example, a pharmaceutical formulation, refers to an effective amount, at the required dosages and time periods, to achieve the desired prophylactic or therapeutic result.
[0160] The term "expression", as used herein, refers to transcription and / or translation and secretion processes that occur within a cell. The level of transcription of a nucleic acid sequence of interest in a cell can be determined based on the amount of corresponding mRNA that is present in the cell. For example, mRNA transcribed from a sequence of interest can be quantified by RT-PCR or by Northern hybridization (see Sambrook et al, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor. , New York (1989)). Polypeptides encoded by a nucleic acid can be quantified by various methods, for example, by ELISA, by assessing the biological activity of the polypeptide, or by using assays that are independent of such activity, such as radioimmunoassay or Western blotting, using immunoglobulins that recognize and bind to the polypeptide (see Sambrook, et al., (1989), supra).
[0161] An "expression cassette" indicates a construct that contains the necessary regulatory elements, such as the promoter and the polyadenylation site, for the expression of at least the nucleic acid contained in a cell.
[0162] The term “expression machinery” indicates the sum of enzymes, cofactors, etc., of a cell that is involved in the steps beginning with the stage of transcription of a gene or nucleic acid (that is, also called “machinery of gene expression ”) until post-translational modification of the polypeptide encoded by the nucleic acid. The expression machinery comprises, for example, the steps of transcribing the DNA into pre-mRNA, splicing the pre-mRNA into mature mRNA, translating the mRNA into a polypeptide, and modifying the post-translational polypeptide.
[0163] An "expression plasmid" is a nucleic acid that provides all the elements necessary for the expression of the structural gene (s) comprised in a host cell. Typically, an expression plasmid comprises a prokaryotic plasmid propagation unit, for example, for E. coli, comprising an origin of replication and a selectable marker, a eukaryotic selection marker, and one or more expression cassettes for the expression of ( s) structural gene (s) of interest, each comprising a promoter, a structural gene, and a transcription terminator, including a polyadenylation signal. The expression of the gene is normally placed under the control of a promoter, and such a structural gene is referred to as "operationally linked" to the promoter. Similarly, a regulatory (or regulatory) element and a minimum promoter (core promoter) are operationally linked if the regulatory element modulates the activity of the minimum promoter.
[0164] The term "Fc-region" is used at present to define a C-terminal region of an immunoglobulin heavy chain that contains at least one constant portion. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, a human IgG heavy chain Fc region extends from Cis226, or from Pro230, to the carboxy-terminal end of the heavy chain. However, C-terminal lysine (Lis447) from Fc-Region may or may not be present. Unless otherwise specified, the numbering of amino acid residues in the Fc-region or constant region is in accordance with the EU numbering system, also called the EU index, as described in Kabat, EA, et al., Sequences of Proteins of Immunological Interest, 5th Ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication 91-3242.
[0165] An "Fc region" is a well-known term and defined based on the cleavage of an antibody heavy chain by papain. The complexes as disclosed in the present invention may comprise, in an embodiment as a polypeptide of the hinge region of the antibody heavy chain, a human Fc region or a human-derived Fc region. In an additional embodiment example, the Fc region is an Fc region of a human antibody of the IgG4 subclass, or an Fc region of a human antibody of the IgG1, IgG2 or IgG3 subclass, which is modified in such a way that no binding to an FCY receptor (for example, FcyRIIIa) and / or no binding to C1q can be detected. In one embodiment, the Fc-region is a human Fc-region and, especially, any human IgG4 subclass or a mutated Fc-region from the human IgG1 subclass. In one embodiment, the Fc-region is from the human IgG1 subclass with mutations L234A and L235A. While IgG4 exhibits reduced binding to the FCY receptor (FcyRIIIa), antibodies to the other IgG subclasses exhibit strong binding. However residues Pro238, Asp265, Asp270, Asn297 (loss of carbohydrates Fc), Pro329, Leu234, Leu235, Gli236, Gli237, Ile253, Ser254, Lis288, Tre307, Gln311, Asn434, and / or His435 are residues that, if changed , Also provide reduced binding to the FCY receptor (Shields, RL, et al., J. Biol. Chem. 276 (2001) 6591-6604; Lund, J., et al, FASEB J. 9 (1995) 115-119; Morgan, A., et al, Immunology 86 (1995) 319-324; EP 0307434). In an exemplary embodiment, a complex as disclosed in the present invention is related to binding to the FCY receptor of the IgG4 subclass or IgG1 or IgG2 subclass, with an L234, L235, and / or D265 mutation, and / or contains the PVA236 mutation . In one embodiment, the mutations are S228P, L234A, L235A, L235E, and / or PVA236 (PVA236 means that the ELLG amino acid sequence (given as the one letter amino acid code) from amino acid position 233 to 236 IgG1 or IgG4 EFLG is replaced by PVA). In one embodiment, the mutations are IgG4 S228P, and IgG1 L234A and L235A. The Fc-region of an antibody is directly involved in ADCC (antibody-dependent cell-mediated cytotoxicity) and CDC (complement-dependent cytotoxicity). A complex that does not bind to the Fy receptor and / or the C1q factor of the complement system does not give rise to antigen-dependent cell cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC).
[0166] The terms "host cell", "host cell lineage", and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of these cells. Host cells include "transformants" and "transformed cells", which include the primary transformed cell and the progeny derived from this cell without regard to the number of passages. Descending cells (progeny) may not be completely identical in terms of the nucleic acid content of the parent cell (parental cell), and may contain mutations. Mutant progeny that have the same biological function or activity, as screened or selected in the initial transformed cell, are encompassed by the present invention.
[0167] The term "cell" includes both prokaryotic cells, which are used for the propagation of plasmids, and eukaryotic cells, which are used for the expression of a nucleic acid. In one example of an embodiment, the eukaryotic cell is a mammalian cell. In one example of an embodiment, the mammalian cell is selected from the group of mammalian cells comprising CHO cells (e.g. CHO K1, CHO DG44), BHK cells, NS0 cells, Sp2 / 0 cells, HEK 293 cells, HEK 293 EBNA cells, PER.C6® cells and COS cells.
[0168] A "human antibody" is one that has an amino acid sequence that corresponds to an antibody produced by a human or a human cell or is derived from a non-human source that uses repertoires of human antibodies or other sequences that encode human antibodies. This definition of a human antibody specifically excludes a humanized antibody, which comprises antigen-binding non-human residues.
[0169] An "immunoconjugate" indicates a conjugate complex, as disclosed in the present invention, for one or more heterologous molecule (s), including, but not limited to, a cytotoxic agent.
[0170] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domesticated animals (for example, cows, sheep, cats, dogs and horses), primates (for example, humans and non-human primates such as monkeys), and rodents (for example, mice and rats) ). In certain embodiments, the individual or subject is a human.
[0171] An “isolated” complex is one that has been separated from a component from its natural environment. In some embodiments, a complex is purified to more than 95% or 99% purity, as determined, for example, by electrophoresis (eg, SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or by chromatography ( for example, ion exchange or reverse phase HPLC).
[0172] An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that normally contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal site that is different from its natural chromosomal location.
[0173] The term "a fusion polypeptide" means exactly a fusion polypeptide as defined and excludes the presence of an additional fusion polypeptide, as defined. The term "one" indicates "exactly one" or "a single".
[0174] “Operationally linked” refers to the juxtaposition of two or more components, in which the components described are in a relationship that allows them to function in the desired way. For example, a promoter and / or facilitator (enhancer) is operationally linked to a coding sequence, whether it acts in cis to control or modulate the transcription of the sequence to which it is linked. Generally, but not necessarily, the DNA sequences that are "operationally linked" are contiguous and, when necessary to join two protein coding regions, such as a secretory leader and a polypeptide, are contiguous and within the (reading) frame . However, although an operationally linked promoter is generally located upstream of the coding sequence, it is not necessarily contiguous with it. Enhancers need not be contiguous. An enhancer is operably linked to a coding sequence if the enhancer increases the transcription of the coding sequence. Operably linked enhancers can be located upstream, in or downstream of the coding sequences, and at a considerable distance from the promoter. A polyadenylation site is operably linked to a coding sequence if it is located at the downstream end of the coding sequence in such a way that transcription continues through the coding sequence for the polyadenylation sequence. A translation termination codon is operationally linked to an exonic nucleic acid sequence if it is located at the downstream end (3 'end) of the coding sequence such that the translation proceeds through the coding sequence to the stop codon and terminates in this place. Binding is achieved by recombinant methods known in the art, for example, using the PCR methodology and / or by binding at suitable restriction sites. If suitable restriction sites do not exist, then synthetic oligonucleotide adapters or ligands are used according to conventional practice.
[0175] The term "package insert" as used herein refers to instructions usually included in commercial containers of therapeutic products, which contain information on the indications, use, dosage, administration, combination therapy, contraindications and / or warnings concerning the use of such therapeutic products.
[0176] The term "peptide ligand" denotes sequences of amino acids of natural and / or synthetic origin. They consist of a linear chain of amino acids, where the 20 naturally occurring amino acids are the monomeric building blocks. The binding peptide has a length of 1-50 amino acids, in one embodiment it has between 1 and 28 amino acids, in another example it has between 2 and 25 amino acids. The binding peptide can contain repetitive amino acid sequences or naturally occurring polypeptide sequences. The ligand has the function of ensuring that the polypeptides conjugated to each other can carry out their biological activity, allowing the polypeptides to fold up correctly and to be presented in an appropriate way. In one embodiment, the binding peptide is rich in glycine, glutamine and / or serine residues. These residues are arranged, for example, in small repetitive units of up to five amino acids, such as GS (SEQ ID NO: 12), GGS (SEQ ID NO: 13), GGGS (SEQ ID NO: 14) and GGGGS (SEQ ID NO: 19). This small repetitive unit can be repeated from one to five times. At the amino and / or carboxy-terminal ends of the multimeric unit, up to six additional naturally occurring arbitrary amino acids can be added. Other synthetic peptide linkers are made up of a single amino acid, which is repeated 10 to 20 times and can comprise at the amino-terminal and / or carboxy-terminal end up to six additional arbitrary naturally occurring amino acids. All peptide ligands can be encoded by a nucleic acid molecule and, therefore, can be expressed recombinantly. Since the linkers are themselves peptides, the polypeptide connected by the linker is linked to the linker by means of a peptide bond that is formed between two amino acids.
[0177] The term "pharmaceutical formulation" refers to a preparation that is in a form that allows the biological activity of an active ingredient contained in the present to be effective, and that does not contain additional components that are unacceptably toxic to an individual. which formulation should be administered.
[0178] A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation that is not the active ingredient, and that is not toxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer or preservative.
[0179] A "polypeptide" is a polymer consisting of amino acids joined by peptide bonds, whether produced naturally or synthetically. Polypeptides smaller than about 25 amino acid residues can be referred to as "peptides", whereas molecules composed of two or more polypeptides, or comprising a polypeptide with more than 100 amino acid residues can be referred to as "proteins". A polypeptide can also comprise non-amino acid components, such as groups of carbohydrates, metal ions, or esters of carboxylic acids. Non-amino acid components can be added by the cell in which the polypeptide is expressed, and can vary with the type of cell. Polypeptides are defined in the present invention in terms of their main or central amino acid structure or the nucleic acid that encodes it. Additions such as carbohydrate groups are generally unspecified, however, they may be present.
[0180] A "structural gene" denotes the region of a gene without a signal sequence, that is, the coding region.
[0181] The term "peptide that elicits a T cell response" means a peptide that is presented in the peptide-binding groove of a MHC class I complex and that is recognized by circulating memory effector T cells. The recognition of the peptide resulted in an immune response that performs the removal of the presenting cell of such a 'peptide - MHC class I complex'.
[0182] As used herein, the term "treatment" (and variations such as "treating" or "treating") refers to clinical interventions in an attempt to alter the natural course of the individual being treated, and can be performed for prophylaxis or during the development of clinical pathology. Desirable treatment effects include, but are not limited to, preventing the occurrence or recurrence of the disease, relieving symptoms, decreasing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or relief from sickness, and improved remission or prognosis. In some embodiments, the antibodies of the present invention are used to slow the development of a disease, or to slow the progression of a disease.
[0183] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain, which is involved in binding the antibody to the antigen. The heavy chain and light chain variable domains (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved structural regions (FR) and three hypervariable regions (HVRs). (See, for example, Kindt, T.J., et al., Kuby Immunology, 6th ed., W.H. Freeman & Co., N.Y. (2007), page 91). A single VH or VL domain may be sufficient to confer antigen binding specificity. In addition, antibodies that bind to a specific antigen can be isolated using a VH or VL domain from an antibody that binds to the antigen to search a library of complementary VL or VH domains, respectively. See, for example, Portolano, S., et al., J. Immunol. 150 (1993) 880-887; Clackson, T., et al., Nature 352 (1991) 624-628).
[0184] The term "vector", as used in the present invention, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is attached. The term includes the vector as a self-replicating nucleic acid structure, as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operationally linked. Such vectors are referred to in the present invention as "expression vectors". II. COMPOSITIONS AND METHODS A. EXAMPLE COMPLEXES
[0185] An antigen-binding complex is disclosed in the present invention comprising as the first part an antibody-derived part that specifically binds to a target antigen, and as a second part a virus-derived peptide linked to one of the MHC complex protein of class I.
[0186] With the complex as disclosed in the present invention the virus-specific circulating cytotoxic T cells (memory T cells and / or effector T cells) of an individual can be directed to cells that express the target antigen, to which part the antibody derived from part of the complex binds specifically, addressing these cells with MHC class I complexes mimicking an acute viral infection.
[0187] In one aspect, the invention is based, in part, on the finding that a complex, as disclosed in the present invention, comprising as a first part a virus-derived peptide linked to a class I MHC protein, and as second part a disulfide-linked derivative antibody molecule can be used to target an individual's existing virus-specific cytotoxic T cells to cells that express a target antigen mimicking an acute viral infection and thus removing the cells that express the target antigen can be initiated.
[0188] In certain embodiments, a complex is provided which comprises a fusion polypeptide comprising (i) a virus-derived peptide; (ii) the soluble HLA-A * 0201 allele; and (iii) beta-2-microglobulin.
[0189] The complexes of the invention are useful, for example, for the diagnosis or treatment of various diseases such as cancer or viral infections.
[0190] In one aspect, the invention provides fusion polypeptides that bind to: (i) a cell surface antigen; and (ii) cytotoxic T cells. In certain embodiments, the fusion polypeptides bind to the cell surface antigen with an affinity <. 10 nM.
[0191] The complexes as disclosed in the present invention exploit a naturally occurring antiviral immune response, highly effective in removing / disintegrating target cells, for example, tumor cells or virus-infected cells. Cell removal is accomplished by using the individual's own potent circulating T cells, which do not need any co-stimulation for activation. In addition, a small number of therapeutic molecules are needed on the cell surface for the mechanism of action (Mottez et al., 1995).
[0192] During treatment, the fusion polypeptide can trigger the individual's antiviral immune response similar to an immunization and thereby increase the effectiveness of multiple treatments / applications.
[0193] Although only a limited population of patients is allotype specific (HLA-A allotype: 30% to 50% of the population with A * 0201 allele) and the prevalence of specific viral infections is also limited (CMV infection of 60 % to 90% depending mainly on age) an immunization as a pretreatment can be used to increase effectiveness.
[0194] Alternatively, an allotype can be used whose frequency in the population is very low, in an example of realization, less than 1%. The use of such an allotype may render the use of an immunization step obsolete, as the allotype will be recognized by the individual's immune system as foreign and an immune response will be initiated.
[0195] The target antibody needs to be highly cellular or antigen-specific to limit toxicity and side effects.
[0196] Thus, by means of a complex, as disclosed in the present invention: (i) only a population of highly specific T cells is activated (specific CD8 positive effector cells / memory for a single MHC-peptide complex), all others CD3 + cells are unaffected (CD4 + T cells: TH1, TH2, TH17, regulatory T cells); (ii) the natural response of the individual's immune system is mimicked (normal removal of cells infected by viruses); and (iii) the response to the request will be low at the beginning, but may intensify during treatment (specific T cells will be activated and number expansion will occur) initially with the same infusion reactions and the initial cytokine release may be reduced.
[0197] In one embodiment, the method comprises the step of stimulating CD8-positive cytotoxic T cells by applying a virus-derived peptide selected, for example, from a peptide derived from human cytomegalovirus (HCMV).
[0198] It has been shown that specific T cells for the active CMV peptide could effectively mediate the removal of tumor cells in vitro (tumor cells loaded with the CMV-derived peptide in vitro).
[0199] Virus-infected cells present virus-derived peptides with MHC class I proteins on their cell surfaces. These are recognized by specific CD8 + T cells that remove / deplete the presenting cells. Cytolytic (cytotoxic) CD8 + T-cells (CTL) recognize peptides in MHC class I proteins by their specific T cell receptors. CTLs trigger the removal of virus-infected cells without the need for a co-stimulating signal.
[0200] Effector cells, for example, peripheral blood mononuclear cells (PBMC) or CD8 + T-cells separated by FACS, which can be pre-stimulated with the CMV-derived peptide, as comprised in the fusion polypeptide disclosed herein invention, can be used.
[0201] The HLA allotype of an individual to be treated must be recognized.
[0202] According to the NCBI, HLA allotypes with a frequency of 10% or more are distributed as shown in the following table: TABLE

[0203] Thus, an aspect as disclosed in the present invention is an antigen-binding complex, characterized by the fact that it comprises: - a single fusion polypeptide comprising in the N- to C-terminal direction; both (i) a β2-microglobulin; and (ii) the extracellular domains α1, α2 and α3 of a class I MHC molecule with a relative frequency of less than 10%; or (i) a peptide that elicits a T-cell response; (ii) a β2-microglobulin; and (iii) the extracellular domains α1, α2 and α3 of a class I MHC molecule with a relative frequency of 10% or more, and; - two polypeptide chains, which are linked by one or more disulfide bonds; wherein the disulfide-linked first polypeptide chain comprises, in the N-terminal to C-terminal direction: (i) an immunoglobulin light or heavy chain variable domain; (ii) an immunoglobulin light or heavy chain constant domain, and (iii) a polypeptide from the hinge region of the antibody heavy chain; wherein the light or heavy chain variable domain, alone or in combination with its complementary light or heavy chain variable domain, specifically binds to an antigen; and the second disulfide-linked polypeptide chain comprises a polypeptide from the hinge region of the antibody heavy chain; wherein the fusion polypeptide is: - covalently linked to the C-terminal or N-terminal end of one of the disulfide-linked polypeptide chains, or - covalently linked to the N-terminal of an antibody variable domain which is the light or heavy chain variable domain complementary to that comprised in the disulfide-linked first polypeptide chain, or - covalently linked to the C-terminus of an antibody constant domain which is the complementary light or heavy chain constant domain to that comprised in the first linked polypeptide chain by disulfide.
[0204] In one example of the fusion polypeptide comprising, in the N-terminal to C-terminal direction, a β2-microglobulin and the α1, α2 and α3 extracellular domains of a class I MHC molecule, with a relative frequency less than 1%, further comprises at its N-terminal end a peptide binding to the MHC peptide binding groove. In one embodiment, the peptide is a peptide that elicits a T-cell response.
[0205] In one example of an embodiment, the complex is characterized by the fact that it comprises: - a fusion polypeptide that comprises in the N- to C-terminal direction; both (i) a β2-microglobulin; and (ii) the extracellular domains α1, α2 and α3 of a class I MHC molecule with a relative frequency of less than 10%; or (i) a peptide that elicits T cell response; (ii) a β2-microglobulin; and (iii) the extracellular domains α1, α2 and α3 of a class I MHC molecule with a relative frequency of 10% or more; and; - two polypeptide chains, which are linked by one or more disulfide bonds; wherein the disulfide-linked first polypeptide chain comprises, in the N-terminal to C-terminal direction: (i) an immunoglobulin light or heavy chain variable domain; (ii) an immunoglobulin light or heavy chain constant domain, and (iii) a polypeptide from the hinge region of the antibody heavy chain; wherein the light or heavy chain variable domain, alone or in combination with its complementary light or heavy chain variable domain, specifically binds to an antigen; and the second disulfide-linked polypeptide chain comprises a polypeptide from the hinge region of the antibody heavy chain; wherein the fusion polypeptide is: - covalently linked to the C-terminal or N-terminal end of one of the polypeptide chains linked by disulfide bonds, or - covalently linked to the N-terminal of an antibody variable domain which is the light or heavy chain variable domain complementary to that comprised in the disulfide-linked first polypeptide chain, or - covalently linked to the C-terminus of an antibody constant domain which is the complementary light or heavy chain constant domain to that comprised in the first linked polypeptide chain by disulfide.
[0206] In one example of an embodiment, the complex is characterized by the fact that it comprises: - a fusion polypeptide comprising in the N- to C-terminal direction; (i) a peptide that elicits T cell response; (ii) a β2-microglobulin; and (iii) the extracellular domains α1, α2 and α3 of a class I MHC molecule with a relative frequency of 10% or more; and; - two polypeptide chains, which are linked by one or more disulfide bonds; wherein the first disulfide-linked polypeptide chain comprises, in the N-terminal to C-terminal direction: (I) an immunoglobulin light chain (VL) variable domain and a constant domain selected from the immunoglobulin light chain constant domain (CL) and constant domain of the first immunoglobulin heavy chain (CH1); or an immunoglobulin heavy chain (VH) variable domain and a constant domain selected from the first immunoglobulin heavy chain (CH1) constant domain and immunoglobulin light chain (CL) constant domain; and (ii) a polypeptide from the hinge region of the antibody heavy chain; - m that the light or heavy chain variable domain, alone or in combination with the respective complementary light or heavy chain variable domain, specifically binds to an antigen; - the second disulfide-linked polypeptide chain comprises a polypeptide from the hinge region of the antibody heavy chain; wherein the fusion polypeptide is: - covalently linked to the C-terminal or N-terminal end of one of the polypeptide chains linked by disulfide bonds, or - covalently linked to the N-terminal of an antibody variable domain which is the light or heavy chain variable domain complementary to that comprised in the disulfide-linked first polypeptide chain, or - covalently linked to the C-terminus of an antibody constant domain which is the complementary light or heavy chain constant domain to that comprised in the first linked polypeptide chain by disulfide.
[0207] In one example of an embodiment, the complex is characterized by the fact that it comprises: - a fusion polypeptide that comprises in the N- to C-terminal direction; (i) a peptide that elicits a T-cell response; (ii) a β2-microglobulin; and (iii) the extracellular domains α1, α2 and α3 of a class I MHC molecule with a relative frequency of 10% or more; and; - two polypeptide chains, which are linked by one or more disulfide bonds; wherein the first disulfide-linked polypeptide chain comprises, in the N-terminal to C-terminal direction: (i) an immunoglobulin light chain (VL) variable domain and a constant domain selected from the immunoglobulin light chain constant domain (CL) and immunoglobulin heavy chain (CH1) constant domain, or an immunoglobulin heavy chain (VH) variable domain and a constant domain selected from the immunoglobulin first heavy chain (CH1) constant domain and constant domain the immunoglobulin light chain (CL); and (ii) a polypeptide from the hinge region of the antibody heavy chain; and (iii) a second (CH2) and third (CH3) immunoglobulin heavy chain constant domain; wherein the light or heavy chain variable domain, alone or in combination with its complementary light or heavy chain variable domain, specifically binds to an antigen; and the second disulfide-linked polypeptide chain comprises a polypeptide from the hinge region of the antibody heavy chain; wherein the fusion polypeptide is: - covalently linked to the C-terminal or N-terminal end of one of the polypeptide chains linked by disulfide bonds, or - covalently linked to the N-terminal of an antibody variable domain which is the light or heavy chain variable domain complementary to that comprised in the disulfide-linked first polypeptide chain, or - covalently linked to the C-terminus of an antibody constant domain which is the complementary light or heavy chain constant domain to that comprised in the first linked polypeptide chain by disulfide.
[0208] In an example of an embodiment, the complex is characterized by the fact that it comprises: - a fusion polypeptide that comprises in the N- to C-terminal direction; (i) a peptide that elicits a T-cell response; (ii) a β2-microglobulin; and (iii) the extracellular domains α1, α2 and α3 of a class I MHC molecule with a relative frequency of 10% or more; and; - two polypeptide chains, which are linked by one or more disulfide bonds; wherein the first disulfide-linked polypeptide chain comprises, in the N-terminal to C-terminal direction: (i) an immunoglobulin light chain (VL) variable domain and a constant domain selected from the immunoglobulin light chain constant domain (CL) and constant domain of the first immunoglobulin heavy chain (CH1); or an immunoglobulin heavy chain (VH) variable domain and a constant domain selected from the first immunoglobulin heavy chain (CH1) constant domain and immunoglobulin light chain (CL) constant domain; and (ii) a polypeptide from the hinge region of the antibody heavy chain; and (iii) a second (CH2) and third (CH3) immunoglobulin heavy chain constant domain; wherein the light or heavy chain variable domain, alone or in combination with its complementary light or heavy chain variable domain, specifically binds to an antigen; and the second disulfide-linked polypeptide comprises, in the N- to C-terminal direction, a polypeptide from the hinge region of the antibody heavy chain, constant region of the second immunoglobulin heavy chain (CH2), and a constant region of the third heavy chain immunoglobulin (CH3); wherein the fusion polypeptide is: - covalently linked to the C-terminal or N-terminal end of one of the polypeptide chains linked by disulfide bonds, or - covalently linked to the N-terminal of an antibody variable domain which is the light or heavy chain variable domain complementary to that comprised in the disulfide-linked first polypeptide chain, or - covalently linked to the C-terminus of an antibody constant domain which is the complementary light or heavy chain constant domain to that comprised in the first linked polypeptide chain by disulfide.
[0209] In one example of an embodiment, the complex is characterized by the fact that it comprises: - a fusion polypeptide that comprises in the N- to C-terminal direction; (i) a peptide that elicits a T-cell response; (ii) a β2-microglobulin; and (iii) the extracellular domains α1, α2 and α3 of a class I MHC molecule with a relative frequency of 10% or more, and; - two polypeptide chains, which are linked by one or more disulfide bonds; wherein the first disulfide-linked polypeptide chain comprises, in the N-terminal to C-terminal direction: (i) an immunoglobulin light chain (VL) variable domain and a constant domain selected from the immunoglobulin light chain constant domain (CL) and immunoglobulin heavy chain (CH1) constant domain, or an immunoglobulin heavy chain (VH) variable domain and a constant domain selected from the immunoglobulin first heavy chain (CH1) constant domain and constant domain the immunoglobulin light chain (CL); and (ii) a polypeptide from the hinge region of the antibody heavy chain, and (iii) a constant domain of the second (CH2) and third (CH3) immunoglobulin heavy chain; wherein the light or heavy chain variable domain, alone or in combination with its complementary light or heavy chain variable domain, specifically binds to an antigen; in which the second disulfide-linked polypeptide chain comprises, in the N-terminal to C-terminal direction: (i) an immunoglobulin light chain (VL) variable domain and a constant domain selected from the immunoglobulin light chain constant domain (CL) and constant domain of the first immunoglobulin heavy chain (CH1); or an immunoglobulin heavy chain (VH) variable domain and a constant domain selected from the first immunoglobulin heavy chain (CH1) constant domain and immunoglobulin light chain (CL) constant domain; (ii) a polypeptide from the antibody heavy chain hinge region, and (iii) an immunoglobulin second heavy chain (CH2) constant region, and an immunoglobulin third heavy chain (CH3) constant region; wherein the fusion polypeptide is: - covalently linked to the C-terminal or N-terminal end of one of the polypeptide chains linked by disulfide bonds, or - covalently linked to the N-terminal of an antibody variable domain which is the light or heavy chain variable domain complementary to that comprised in the disulfide-linked first polypeptide chain, or - covalently linked to the C-terminus of an antibody constant domain which is the complementary light or heavy chain constant domain to that comprised in the first linked polypeptide chain by disulfide.
[0210] In an example of an embodiment, the complex is characterized by the fact that it comprises: - a fusion polypeptide that comprises in the N- to C-terminal direction; (i) a peptide that elicits a T-cell response; (ii) a β2-microglobulin; and (iii) the extracellular domains α1, α2 and α3 of a class I MHC molecule with a relative frequency of 10% or more; - two polypeptide chains, which are linked by one or more disulfide bonds; wherein the first disulfide-linked polypeptide chain comprises, in the N-terminal to C-terminal direction: (i) an immunoglobulin light chain (VL) variable domain and a constant domain selected from the immunoglobulin light chain constant domain (CL) and immunoglobulin heavy chain (CH1) constant domain, or an immunoglobulin heavy chain (VH) variable domain and a constant domain selected from the immunoglobulin first heavy chain (CH1) constant domain and constant domain the immunoglobulin light chain (CL); and (ii) a polypeptide from the hinge region of the antibody heavy chain; and (iii) a second (CH2) and third (CH3) immunoglobulin heavy chain constant domain; wherein the light or heavy chain variable domain, alone or in combination with its complementary light or heavy chain variable domain, specifically binds to an antigen; in which the second disulfide-linked polypeptide chain comprises, in the N-terminal to C-terminal direction: (i) an immunoglobulin light chain (VL) variable domain and a constant domain selected from the immunoglobulin light chain constant domain (CL) and immunoglobulin heavy chain (CH1) constant domain, or an immunoglobulin heavy chain (VH) variable domain and a constant domain selected from the immunoglobulin first heavy chain (CH1) constant domain and constant domain the immunoglobulin light chain (CL); (ii) a polypeptide from the hinge region of the antibody heavy chain; and (iii) an immunoglobulin second heavy chain constant region (CH2), and an immunoglobulin third heavy chain constant region (CH3); - a polypeptide chain comprising in the N- to C-terminal direction; an immunoglobulin variable domain complementary to the variable domain on the disulfide-linked first polypeptide chain and a constant domain complementary to the constant domain after the variable domain on the disulfide-linked first polypeptide chain; wherein the fusion polypeptide is: - covalently linked to the C-terminal or N-terminal end of one of the polypeptide chains linked by disulfide bonds, or - covalently linked to the N-terminal of an antibody variable domain which is the light or heavy chain variable domain complementary to that comprised in the disulfide-linked first polypeptide chain, or - covalently linked to the C-terminus of an antibody constant domain which is the complementary light or heavy chain constant domain to that comprised in the first linked polypeptide chain by disulfide.
[0211] In one example of an embodiment, the complex is characterized by the fact that it comprises: - a fusion polypeptide that comprises in the N- to C-terminal direction; (i) a peptide that elicits a T-cell response; (ii) a β2-microglobulin; (iii) the extracellular domains α1, α2 and α3 of a class I MHC molecule with a relative frequency of 10% or more; (iv) a polypeptide from the hinge region of the antibody heavy chain; (v) a constant domain of the second (CH2) and third (CH3) immunoglobulin heavy chain; and; - a first polypeptide chain comprising in the N- to C-terminal direction; (i) an immunoglobulin light chain (VL) variable domain and a constant domain selected from the immunoglobulin light chain (CL) constant domain and constant domain of the first immunoglobulin heavy chain (CH1), or a variable domain of immunoglobulin heavy chain (VH) and a constant domain selected from the constant domain of the first immunoglobulin heavy chain (CH1) and immunoglobulin light chain (CL) constant domain; and (ii) a polypeptide from the hinge region of the antibody heavy chain; and (iii) a constant domain of the second (CH2) and third (CH3) immunoglobulin heavy chain, and - a second polypeptide chain comprising in the N- to C-terminal direction; an immunoglobulin variable domain complementary to the variable domain on the first polypeptide chain and a constant domain complementary to the constant domain on the first polypeptide chain; wherein the light or heavy chain variable domain, alone or in combination with its complementary light or heavy chain variable domain, specifically binds to an antigen, and where the fusion polypeptide and the first polypeptide chain are linked by bonds disulfide.
[0212] In one example of an embodiment, the complex is characterized by the fact that it comprises: - a fusion polypeptide that comprises in the N- to C-terminal direction; (i) a peptide that elicits a T-cell response; (ii) a β2-microglobulin; (iii) the extracellular domains α1, α2 and α3 of a class I MHC molecule with a relative frequency of 10% or more; (iv) an immunoglobulin light chain (VL) variable domain and a constant domain selected from the immunoglobulin light chain (CL) constant domain and the first immunoglobulin heavy chain (CH1) constant domain; or an immunoglobulin heavy chain (VH) variable domain and a constant domain selected from the first immunoglobulin heavy chain (CH1) constant domain and immunoglobulin light chain (CL) constant domain; (v) a polypeptide from the hinge region of the antibody heavy chain; (vi) a second domain (CH2) and third (CH3) immunoglobulin heavy chain; and; - a first polypeptide chain comprising in the N- to C-terminal direction; (i) a polypeptide from the hinge region of the antibody heavy chain; and (ii) a domain of the second (CH2) and third (CH3) immunoglobulin heavy chain; and; - a second polypeptide chain comprising in the N- to C-terminal direction; an immunoglobulin variable domain complementary to the variable domain on the first polypeptide chain and a constant domain complementary to the constant domain on the first polypeptide chain; wherein the light or heavy chain variable domain, alone or in combination with its complementary light or heavy chain variable domain, specifically binds to an antigen; and wherein the fusion polypeptide and the first polypeptide chain are linked by disulfide bonds.
[0213] In one example of an embodiment, the complex is characterized by the fact that it comprises: - a fusion polypeptide that comprises in the N- to C-terminal direction; (i) a peptide that elicits a T-cell response; (ii) a β2-microglobulin; (iii) the extracellular domains α1, α2 and α3 of a class I MHC molecule with a relative frequency of 10% or more; and (iv) an immunoglobulin light chain (VL) variable domain and a constant domain selected from the immunoglobulin light chain (CL) constant domain and immunoglobulin first heavy chain (CH1) constant domain; or an immunoglobulin heavy chain (VH) variable domain and a constant domain selected from the first immunoglobulin heavy chain (CH1) constant domain and immunoglobulin light chain (CL) constant domain; and - a first polypeptide chain comprising in the N- to C-terminal direction; (i) an immunoglobulin variable domain complementary to the variable domain in the fusion polypeptide and a constant domain complementary to the constant domain in the fusion polypeptide; (ii) a polypeptide from the hinge region of the antibody heavy chain; and (iii) a second (CH2) and third (CH3) immunoglobulin heavy chain constant domain; wherein the light or heavy chain variable domain, alone or in combination with its complementary light or heavy chain variable domain, specifically binds to an antigen; and wherein the fusion polypeptide and the first polypeptide chain are linked by disulfide bonds.
[0214] In one example of an embodiment, the complex is characterized by the fact that it comprises: - a fusion polypeptide that comprises in the N- to C-terminal direction; (i) a peptide that elicits a T-cell response; (ii) a β2-microglobulin; (iii) the extracellular domains α1, α2 and α3 of a class I MHC molecule with a relative frequency of 10% or more; (iv) an immunoglobulin light chain (VL) variable domain and a constant domain selected from the immunoglobulin light chain (CL) constant domain and constant domain of the first immunoglobulin heavy chain (CH1), or a variable domain of the immunoglobulin heavy chain (VH) and a constant domain selected from the constant domain of the first immunoglobulin heavy chain (CH1) and immunoglobulin light chain (CL) constant domain; and (v) a polypeptide from the hinge region of the antibody heavy chain; (vi) a constant domain of the second (CH2) and third (CH3) immunoglobulin heavy chain, and - a first polypeptide chain comprising in the N- to C-terminal direction; (i) an immunoglobulin light chain (VL) variable domain and a constant domain selected from the immunoglobulin light chain (CL) constant domain and constant domain of the first immunoglobulin heavy chain (CH1), or a variable domain of immunoglobulin heavy chain (VH) and a constant domain selected from the constant domain of the first immunoglobulin heavy chain (CH1) and immunoglobulin light chain (CL) constant domain; (ii) a polypeptide from the hinge region of the antibody heavy chain; and (iii) a second (CH2) and third (CH3) immunoglobulin heavy chain constant domain; and; - a second polypeptide chain comprising in the N- to C-terminal direction; an immunoglobulin variable domain complementary to the variable domain in the fusion polypeptide chain and a constant domain complementary to the constant domain in the fusion polypeptide chain; and; - a third polypeptide chain comprising in the N- to C-terminal direction; an immunoglobulin variable domain complementary to the variable domain on the first polypeptide chain and a constant domain complementary to the constant domain on the first polypeptide chain; wherein the light or heavy chain variable domain of the fusion polypeptide and / or the first polypeptide chain, alone or in combination with the respective complementary light or heavy chain variable domain, specifically binds to an antigen; and wherein the fusion polypeptide and the first polypeptide chain are linked by disulfide bonds.
[0215] In one example of an embodiment, the complex is characterized by the fact that it comprises: - a fusion polypeptide that comprises in the N- to C-terminal direction; (i) a peptide that elicits a T-cell response; (ii) a β2-microglobulin; (iii) the extracellular domains α1, α2 and α3 of a class I MHC molecule with a relative frequency of 10% or more, and (iv) an immunoglobulin light chain (VL) variable domain and a constant domain selected from the constant domain of the immunoglobulin light chain (CL) and constant domain of the first immunoglobulin heavy chain (CH1); or an immunoglobulin heavy chain (VH) variable domain and a constant domain selected from the first immunoglobulin heavy chain (CH1) constant domain and immunoglobulin light chain (CL) constant domain; and; - a first polypeptide chain comprising in the N- to C-terminal direction; (i) an immunoglobulin variable domain complementary to the variable domain in the fusion polypeptide and a constant domain complementary to the constant domain in the fusion polypeptide; (ii) a polypeptide from the hinge region of the antibody heavy chain; and (iii) a second (CH2) and third (CH3) immunoglobulin heavy chain constant domain; and; - a second polypeptide chain comprising in the N- to C-terminal direction; (i) an immunoglobulin light chain (VL) variable domain and a constant domain selected from the immunoglobulin light chain (CL) constant domain and immunoglobulin first heavy chain (CH1) constant domain; or an immunoglobulin heavy chain (VH) variable domain and a constant domain selected from the first immunoglobulin heavy chain (CH1) constant domain and immunoglobulin light chain (CL) constant domain; (ii) a polypeptide from the hinge region of the antibody heavy chain; and (iii) a second (CH2) and third (CH3) immunoglobulin heavy chain constant domain; and; - a third polypeptide chain comprising in the N- to C-terminal direction; an immunoglobulin variable domain complementary to the variable domain in the second polypeptide chain and a constant domain complementary to the constant domain in the second polypeptide chain; wherein the light or heavy chain variable domain of the fusion polypeptide and / or the second polypeptide chain, isolated in combination with the respective complementary light or heavy chain variable domain, specifically binds to an antigen; and wherein the first polypeptide chain and the second polypeptide chain are linked by disulfide bonds.
[0216] In one example of an embodiment, the peptide that elicits a T-cell response is a virus-derived peptide. In one embodiment, the virus is selected from adenovirus, human herpesvirus 1, human herpesvirus 2, human herpesvirus 4 (Epstein-Barr virus), hepatitis-B virus, hepatitis-C virus, human cytomegalovirus, human immunodeficiency, human papillomavirus type 16, human papillomavirus type 18, human papillomavirus type 31, human papillomavirus type 33, human papillomavirus type 35, human papillomavirus type 39, human papillomavirus type 45, human papillomavirus type 51, human papillomavirus type 52, human papillomavirus type 56, human papillomavirus type 58, human papillomavirus type 59, human papillomavirus type 68, human papillomavirus type 73, human papillomavirus type 82, Human T cell lymphotropic virus Type 1, human flu virus, human influenza B virus or vaccinia virus .
[0217] In one embodiment, the virus-derived peptide is selected from NLVPMVATV (SEQ ID NO: 1), SLYNTVATL (SEQ ID NO: 2), GLCTLVAML (SEQ ID NO: 3), GILGFVFTL (SEQ ID NO: 3) NO: 4), STNRQSGRQ (SEQ ID NO: 5), LLFGYPVYV (SEQ ID NO: 6), FAEGFVRAL (SEQ ID NO: 7), LIVIGILIL (SEQ ID NO: 8), or ILHTPGCV (SEQ ID NO: 9) .
[0218] In one embodiment, β2-microglobulin is human β2-microglobulin. In one embodiment, β2-microglobulin consists of the amino acid sequence of SEQ ID NO: 10.
[0219] In one embodiment, the MHC class I molecule with a relative frequency of 10% or less is human HLA-A * 0201. In one embodiment, the extracellular domains α1, α2, and α3 of the MHC class I molecule consist of the amino acid sequence of SEQ ID NO: 11.
[0220] In one embodiment, the virus-derived peptide is fused to β 2-microglobulin via a first binding peptide.
[0221] In one embodiment, β2-microglobulin is fused to the α1 extracellular domain of a class I MHC molecule via a second binding peptide.
[0222] In one embodiment, the α3 extracellular domain of the MHC class I molecule is fused to the polypeptide (by a disulfide bond or a non-disulfide bond) by means of a third binding polypeptide.
[0223] In one embodiment, the first, second and third binding peptide is the same binding peptide or is a different binding peptide.
[0224] In one embodiment, the first binding peptide, the second binding peptide, and the third binding peptide are selected independently of each other from the amino acid sequences: GS (SEQ ID NO: 12), GGS ( SEQ ID NO: 13), GGGS (SEQ ID NO: 14), GGGSGGGS (SEQ ID NO: 15), GGGSGGGSGGGS (SEQ ID NO: 16), GGGSGGGSGGGSGGGS (SEQ ID NO: 17), GGGSGGGGGGGSGGS ), GGGGS (SEQ ID NO: 19), GGGGSGGGGS (SEQ ID NO: 20), GGGGGGGGGSGGGGS (SEQ ID NO: 21), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 22), and GGGGSGGGGG
[0225] In one embodiment, the first binding peptide comprises the amino acid sequence of SEQ ID NO: 21.
[0226] In one embodiment, the second binding peptide comprises the amino acid sequence of SEQ ID NO: 22.
[0227] In one embodiment, the third binding peptide comprises the amino acid sequence of SEQ ID NO: 12.
[0228] In one embodiment, the antibody heavy chain hinge region polypeptide is selected from a heavy chain hinge region polypeptide of a human IgG class or IgE class antibody.
[0229] In one embodiment, the polypeptide of the hinge region of the antibody heavy chain is selected from a polypeptide of the hinge region of the heavy antibody of a human antibody of the subclass IgG1, or IgG2, or IgG3 or IgG4.
[0230] In one embodiment, the polypeptide of the hinge region of the antibody heavy chain comprises or consists of the amino acid sequence EPKSCDKTHTCPPCP (SEQ ID NO: 24), EPKSADKTHTCPPCP (SEQ ID NO: 25), ERKCCVECPPCP (SEQ ID NO: 26), ERKCAVECPPCP (SEQ ID NO: 27), ERKACVECPPCP (SEQ ID NO: 28), ELKTPLGDTTHTCPRCP (EPKSCDTPPPCPRCP) 3 (SEQ ID NO: 29), or ESKYGPPCPSCP (SEQ ID NO: 30).
[0231] In one embodiment, the first disulfide-linked polypeptide and / or the second disulfide-linked polypeptide comprises a CH2 domain and / or a CH3 domain of human origin. In one exemplary embodiment, the CH2 domain and the CH3 of human origin is a human antibody of the IgG or IgE class. In one embodiment, the CH2 and CH3 domain is a human antibody of the subclass IgG1, or IgG2, or IgG3, or IgG4. In one embodiment, the CH2 domain comprises the amino acid sequence of SEQ ID NO: 31. In one embodiment, the CH2 domain is a human antibody of the IgG1 or IgG2 subclass, and comprises at least one E233, L234 mutation , L235, G236, D265, D270, N297, E318, K320, K322, A327, A330, P331 and / or P329 (numbering according to the EU Kabat index). In one embodiment, the CH2 domain is a human antibody of the IgG1 subclass or the human IgG2 subclass with the L234A and L235A mutations and / or the D265A and N297A mutations, and / or contains the PVA236 mutation, and / or contains the P329G mutation. In one embodiment, the CH2 domain is a human antibody of the IgG1 subclass with mutations L234A and L235A and / or P329G. In one embodiment, the CH2 domain is a human antibody of the IgG4 subclass with S228P and / or L235E mutations. In one embodiment, the CH2 domain comprises the amino acid sequence of SEQ ID NO: 32 or SEQ ID NO: 33. In one embodiment, the CH3 domain comprises the amino acid sequence of SEQ ID NO: 34.
[0232] In one embodiment, the first disulfide-linked peptide comprises the amino acid sequence of SEQ ID NO: 35, and the second disulfide-linked peptide comprises the amino acid sequence of SEQ ID NO: 36.
[0233] In one embodiment, the first and second disulfide linked polypeptide comprise the amino acid sequence of SEQ ID NO: 37 or SEQ ID NO: 38.
[0234] In one embodiment, the first or second disulfide linked polypeptide consists of the amino acid sequence of SEQ ID NO: 37 or SEQ ID NO: 38.
[0235] In one embodiment, the first disulfide-linked peptide comprises the amino acid sequence of SEQ ID NO: 39, and the second disulfide-linked peptide comprises the amino acid sequence of SEQ ID NO: 40.
[0236] In one embodiment, the polypeptide chains, which are linked by one or more disulfide bonds, are linked by two, or three, or four disulfide bonds.
[0237] In one example of an embodiment, the complex is characterized by the fact that it comprises: - a fusion polypeptide that comprises in the N- to C-terminal direction; (i) a virus-derived peptide that has an amino acid sequence of SEQ ID NO: 1; (ii) a first binding peptide that has an amino acid sequence of SEQ ID NO: 21; (iii) a β2-microglobulin that has an amino acid sequence of SEQ ID NO: 10; (iv) a second binding peptide that has an amino acid sequence of SEQ ID NO: 22; (v) the extracellular domains α1, α2 and α3 of a MHC class I molecule that has an amino acid sequence of SEQ ID NO: 11; and; (vi) a third binding peptide that has an amino acid sequence of SEQ ID NO: 12. e; - two polypeptide chains, which are linked by one or more disulfide bonds; wherein the disulfide-linked first polypeptide chain comprises, in the N-terminal to C-terminal direction: (i) an immunoglobulin light or heavy chain variable domain; (ii) an immunoglobulin light or heavy chain constant domain; (iii) a polypeptide from the hinge region of the antibody heavy chain; (iv) a human IgG1 CH2 domain comprising an amino acid sequence selected from SEQ ID NO: 32 and 33; and (v) a human IgG1 CH3 domain comprising an amino acid sequence selected from SEQ ID NO: 35 or 36; the second disulfide-linked polypeptide chain comprises, in the N-terminal to C-terminal direction: (i) a polypeptide from the hinge region of the antibody heavy chain; (ii) a human IgG1 CH2 domain comprising an amino acid sequence selected from SEQ ID NO: 32 and 33; and (iii) a human IgG1 CH3 domain comprising an amino acid sequence selected from SEQ ID NO: 36 or 35; wherein the fusion polypeptide is: - covalently attached to the C-terminal or N-terminal end of the second disulfide-linked polypeptide chain.
[0238] In one example of an embodiment, the complex is characterized by the fact that it comprises: - a fusion polypeptide that comprises in the N- to C-terminal direction; (i) a virus-derived peptide that has an amino acid sequence of SEQ ID NO: 1, (ii) a first binding peptide that has an amino acid sequence of SEQ ID NO: 21; (iii) a β2-microglobulin that has an amino acid sequence of SEQ ID NO: 10; (iv) a second binding peptide that has an amino acid sequence of SEQ ID NO: 22; (v) the extracellular domains α1, α2 and α3 of a MHC class I molecule that has an amino acid sequence of SEQ ID NO: 11; and (vi) a third binding peptide that has an amino acid sequence of SEQ ID NO: 12. e; - two polypeptide chains, which are linked by one or more disulfide bonds; wherein the disulfide-linked first polypeptide chain comprises, in the N-terminal to C-terminal direction: (i) an immunoglobulin light or heavy chain variable domain; (ii) an immunoglobulin light or heavy chain constant domain; (iii) a polypeptide from the hinge region of the antibody heavy chain; (iv) a human IgG1 CH2 domain comprising an amino acid sequence selected from SEQ ID NO: 32 and 33; and (v) a human IgG1 CH3 domain comprising an amino acid sequence selected from SEQ ID NO: 35 or 36; (vi) the second disulfide-linked polypeptide chain comprises a polypeptide from the hinge region of the antibody heavy chain; wherein the fusion polypeptide is: (vii) covalently linked to the C-terminal or N-terminal end of the first disulfide-linked polypeptide chain, or (viii) covalently linked to the N-terminal of an antibody variable domain that is the light or heavy chain variable domain complementary to that comprised in the first disulfide-linked polypeptide chain, or (ix) covalently linked to the C-terminus of an antibody constant domain which is the complementary light or heavy chain constant domain that comprised in disulfide-linked first polypeptide chain.
[0239] In one example of an embodiment, the complex is characterized by the fact that it comprises: (x) a fusion polypeptide that comprises in the N- to C-terminal direction; (i) a virus-derived peptide that has an amino acid sequence of SEQ ID NO: 1; (ii) a first binding peptide that has an amino acid sequence of SEQ ID NO: 21; (iii) a β2-microglobulin that has an amino acid sequence of SEQ ID NO: 10; (iv) a second binding peptide that has an amino acid sequence of SEQ ID NO: 22; (v) the extracellular domains α1, α2 and α3 of a MHC class I molecule that has an amino acid sequence of SEQ ID NO: 11; and (vi) a third binding peptide that has an amino acid sequence of SEQ ID NO: 12; and; - two polypeptide chains, which are linked by one or more disulfide bonds; wherein the first and second disulfide-linked polypeptide chain comprises in the N-terminal to C-terminal direction: (i) an immunoglobulin light or heavy chain variable domain; (ii) an immunoglobulin light or heavy chain constant domain; (iii) a polypeptide from the hinge region of the antibody heavy chain; (iv) a human IgG1 CH2 domain comprising an amino acid sequence selected from SEQ ID NO: 32 and 33; and (v) a human IgG1 CH3 domain comprising an amino acid sequence selected from SEQ ID NO: 35 and 36; wherein the fusion polypeptide is: - covalently linked to the C-terminal or N-terminal end of the second disulfide-linked polypeptide chain, or - covalently linked to the N-terminal of an antibody variable domain which is the variable domain light or heavy chain complementary to that comprised in the disulfide-linked first polypeptide chain, or - covalently linked to the C-terminus of an antibody constant domain which is the complementary light or heavy chain constant domain to that comprised in the disulfide-linked first polypeptide chain .
[0240] From Figure 14, it can be seen that the complex as disclosed in the present invention maintains the binding properties of the antibody to which it is fused (Figure 14 b) and c)).
[0241] In Figures 15 and 17, the in vitro efficacy and specificity of a complex as disclosed in the present invention are demonstrated.
[0242] The cytotoxicity assay was performed in the presence of CM8-specific CD8 + T cells. It can be seen that complexes comprising a peptide derived from the CMV virus induce lysis / removal / disintegration of target cells (see Figure 15-A for the monovalent antibody, and Figure 15 b for the divalent antibody). It can also be observed that the lysis of the target cells is highly specific by incubation with the complexes comprising a peptide derived from the EBV virus (Figure 15 b)); and the control antibodies (Figure 15 d) and e)) do not result in large cell lysis (spontaneous lysis is approximately 3.5%).
[0243] In Figure 17, lysis of the H460M2 cell line of IGF-1R positive lung adenocarcinoma is demonstrated.
[0244] The EC50 value for a complex comprising a CMV-derived peptide and a bivalent antibody is about 10 ng / ml, corresponding to about 50 pM. The EC50 value determined is independent of the target cell to effector cell ratio (see Figure 16; the ratio between target cell and effector cell is 1: 3 to 1: 1, corresponding to an effective ratio of 1: 0.44 to 1: 0.14 (76% of effector cells are CD8 positive cells and 19% are specific for CMV)).
[0245] Thus, in an example of an embodiment the complex as disclosed in the present invention has an EC50 value of approximately 50 pM. 1. AFFINITY
[0246] In certain embodiments, a complex as provided in the present invention comprises an antigen binding pair of antibody variable domains. In certain realization examples, the variable domain has a dissociation constant (Kd) <10 nM; <1 nM; <0.1 nM; <0.01 nM; or <0.001 nM (for example, 10-8 M or less; for example, 10-8 M to 10-13 M; for example, 10-9 M to 10-13 M) with respect to its antigen.
[0247] In one example of an embodiment, Kd is measured using surface plasmon resonance assays.
[0248] For example, this can be done using a BIACORE®-2000 or BIACORE®-3000 (BIAcore, Inc., Piscataway, NJ) instrument at 25 ° C with CM5 immobilized antigen chips at ~ 10 response units (UK ). Briefly, the carboxymethylated dextran (CM5, BIAcore Inc.) biosensor chips are activated with N-ethyl-N '- (3-dimethylaminopropyl) -carbodiimide hydrochloride (EDC) and N-hydroxysucinimide (NHS) according to the instructions in the provider. The antigen is diluted with 10 mM sodium acetate, pH 4.8, to 5 μg / mL (~ 0.2 μM) before being injected at a flow rate of 5 μL / minute to achieve approximately 10 response units (RU) of coupled protein. After injection of the antigen, 1 M ethanolamine is added to block unreacted groups. For kinetic mediation, serial dilutions of two times with each Fab pass (0.78 nM and 500 nM) are injected in PBS with 0.05% polysorbate-20 surfactant (Tween-20®) (PBST) at 25 ° C at a flow rate of about 25 μl / min. The association speed (kon) and dissociation speed (koff) are calculated using a Langmuir simple one-to-one connection model (BIAcore Evaluation Software version 3.2) by simultaneously adjusting association and dissociation sensograms. The dissociation equilibrium constant (Kd) is calculated as the koff / kon ratio. See, for example., Chen, Y., et al., J. Mol. Biol. 293 (1999): 865-881. 2. EXPRESSION
[0249] Expression of specific form of the complex as disclosed in the present invention (different ligand, different combinations of HLA and β2 microglobulin) in HEK 293 and CHO cells led to an accumulation of the complex, if detectable, within the endoplasmic reticulum, ie , the isolation and secretion of the complex were severely impaired.
[0250] No secretion of a complex into the culture medium could be detected when the complex was designed to comprise one of the polypeptides as described in the following tables: TABLE


[0251] It has been found that the expression, and especially the secretion of complexes comprising two parts of a virus-derived peptide linked to a class I MHC protein, and at least one variable domain and an antibody constant domain, it is not possible in eukaryotic cells.
[0252] Additionally, it has been found that the expression, and especially the secretion of complexes comprising two fusion polypeptides comprising a virus-derived peptide linked to a class I MHC protein, and at least one variable domain, and a pair of polypeptides derived from the disulfide-linked hinge region is not possible in eukaryotic cells.
[0253] Thus, a complex as disclosed in the present of a fusion polypeptide comprising a virus-derived peptide linked to an MHC class I protein cannot be present more than once and in at least one variable domain of antibody and an antibody constant domain must be present in order to allow the production and secretion of the complex using eukaryotic cells.
[0254] Thus, a complex comprising exactly a virus-derived peptide fusion polypeptide linked to an MHC class I protein, an antibody heavy chain hinge region, and at least one antibody variable domain and one domain antibody constant can be produced recombinantly and secreted by eukaryotic cells. In one embodiment the at least one constant domain is an antibody heavy chain constant domain (CH1) or an antibody light chain constant domain (CL).
[0255] Thus, a complex comprising exactly one hinge region of the antibody heavy chain, at least a pair of antibody variable domains, optionally an antibody constant domain, and exactly a fusion polypeptide of a peptide derived from linked virus to a class I MHC protein can be produced recombinantly and secreted by eukaryotic cells. In one embodiment the at least one constant domain is an antibody heavy chain constant domain (CH1) or an antibody light chain constant domain (CL).
[0256] Various combinations of different polypeptides have been tested. Secreted expression can be accomplished by, for example, N-terminal fusion of an immunoglobulin-derived signal peptide in complexes where the virus-derived peptide is fused at the N-terminal to the MHC class I molecule. The MHC class I molecule of heavy chain (α1-α2-α3 without the transmembrane domain and cytoplasmic domain) and the light chain (beta2-microglobulin) may have their order changed. The different fusion polypeptides were fused at the N-terminal end to a polypeptide comprising the antibody light chain or a hinge region of the antibody heavy chain. Exemplary combinations are provided in Figure 2.
[0257] As can be seen from the following table of complexes comprising fusion polypeptides, such complexes can only be expressed with the antibody variable domain and hinge region of the antibody heavy chain when a single peptide derived from virus-microglobulin- HLA-fusion polypeptide is present. TABLE



[0258] In some embodiments, the complex comprises, as disclosed in the present invention, different pairs of polypeptides. In order to allow a good pairing of the polypeptides, the knobs-into-holes technology or the cross-mAb technology can be used in order to reduce the amount of complex that is not associated correctly.
[0259] The knob modification indicates the T366W mutation in the CH3 domain of an antibody (numbered according to the EU Kabat index).
[0260] The hole modification indicates the T366S, L368A and Y407V mutation in the CH3 domain of an antibody (numbered according to the EU Kabat index).
[0261] In addition to the knob and hole modification, the S354C mutation in the CH3 domain and the Y349C mutation in the other CH3 domain may be present.
[0262] The cross-mAb technology is reported, for example, in WO 2009/080251, WO 2009/080252, WO 2009/080254, WO 2009/080253, WO 2010/112193, WO 2010/115589, WO 2010/136172, WO 2010/145792, and WO 2010/145793. 3. VARIANTS
[0263] In certain embodiments, variant amino acid sequences of the complex provided in the present invention are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the complex. Variant amino acid sequences of the complex can be prepared by introducing appropriate modifications to the nucleotide sequence encoding the complex's polypeptide chains, or by peptide synthesis. Such modifications include, for example, deletions and / or insertions and / or substitutions of residues within the amino acid sequences of the complex polypeptides. Any combination of exclusion, insertion and substitution can be done to arrive at the final construction, as long as the final construction has the desired characteristics, for example, antigen binding. A) VARIATORS BY SUBSTITUTION, INSERTION AND DELETION
[0264] In certain embodiments, variant forms of complex containing one or more amino acid substitutions in the polypeptide chains are provided. Conservative substitutions are shown in the table below under the heading “preferred substitutions”. More substantial changes are provided in the table below under the heading of “exemplary substitutions” and, as best described below in reference to the classes of amino acid side chains. Amino acid substitutions can be introduced into a complex of interest and the products selected for a desired activity, for example, maintained / improved antigen binding, decreased immunogenicity, or improved ADCC or CDC. TABLE


[0265] Amino acids can be grouped according to common side chain properties: (1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cis, Ser, Tre, Asn, Gln; (3) acid: Asp, Glu; (4) basic: His, Lis, Arg; (5) residues that influence the orientation of the chains: Gli, Pro; (6) aromatic: Trp, Tir, Fen.
[0266] Non-conservative substitutions will cause a member of one of these classes to be replaced by another class.
[0267] Amino acid sequence insertions include: carboxy- and / or amino-terminal fusions that vary in length from a residue, to polypeptides that have one hundred or more residues, as well as intrasequential insertions of single or multiple amino acid residues. Examples of terminal inserts include a complex comprising a polypeptide with an N-terminal methionyl residue. Other insertion variants include the N- or C-terminal fusion of the complex's polypeptide chains to an enzyme. B) GLYCOSILATION VARIABLES
[0268] In certain embodiments, a polypeptide of the complex provided in the present invention is altered to increase or decrease the degree to which the polypeptide is glycosylated. The addition or deletion of glycosylation sites on a polypeptide can be conveniently accomplished by changing the amino acid sequence in such a way that one or more glycosylation sites are created or removed.
[0269] When the complex comprises an antibody Fc region, the carbohydrates attached to them can be altered. Native Fc regions produced by mammalian cells generally comprise a bianternary branched oligosaccharide that is generally linked by an N bond to an Asn297 of the CH2 domain of the Fc region. See, for example, Wright, A. and Morrison, S.L., TIBTECH 15 (1997) 26-32. Oligosaccharides can include various carbohydrates, for example, mannose, N-acetyl glucosamine (GlcNAc), galactose and sialic acid, as well as a GlcNAc-linked fucose in the “trunk” of the bianternal oligosaccharide structure. In some exemplary embodiments, modifications of the oligosaccharides in an antibody of the invention can be made in order to create antibody variants with certain improved properties.
[0270] In one embodiment, complexes comprising variant polypeptides are provided having a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc region. For example, the amount of fucose in such an Fc region can be from 1% to 80%, from 1% to 65%, from 5% to 65% or from 20% to 40%. The amount of fucose is determined by calculating the average value of fucose within the sugar chain in Asn297, in relation to the sum of all glycostructures linked to Asn297 (for example, complex and hybrid structures with a high mannose content), measured by spectrometry of MALDI-TOF dough, as described in WO 2008/077546, for example. Asn297 refers to the asparagine residue located approximately at position 297 of the Fc region (numbering residues from the Fc-EU region), however, Asn297 can also be located approximately ± 3 amino acids upstream or downstream of position 297, or that is, between positions 294 and 300, due to minor variations in the antibody sequence. These fucosylation variants may have improved ADCC function. See, for example, US publications 2003/0157108; US 2004/0093621. Examples of publications related to "defucosylated" or "fucose deficient" antibody variants include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778; WO 2005/053742; WO 2002/031140; Okazaki et al. J. Mol. Biol. 336: 1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87: (2004) 614-622. Examples of cell lines that produce defucosylated antibodies include Lec13 CHO cells deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. (1986) 249: 533-545; US 2003/0157108, and WO 2004/056312, especially in Example 11), and knockout cell lines, such as for the alpha-1,6-fucosyltransferase gene, FUT8, CHO knockout cells (see, for example, Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004 ); Kanda, Y. et al., Biotechnol. Bioeng., 94 (4): 680-688 (2006); and WO2003 / 085107).
[0271] Complexes comprising variant Fc-region are additionally provided with divided oligosaccharides, for example, where a bianternal oligosaccharide attached to the Fc region is cut by GlcNAc. Such variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, for example, in WO 2003/011878; US 6,602,684 and US 2005/0123546. Variants of the Fc region with at least one galactose residue are provided in the oligosaccharide attached to the Fc region. These Fc-region variants may have improved CDC function. Corresponding variants of antibodies are described, for example, in WO 97/30087, WO 98/58964 and WO 99/22764. c) VARIATORS OF THE REGION-FC
[0272] In certain embodiments, one or more amino acid modifications can be introduced into the Fc region of a polypeptide comprised in the complex provided in this document, thereby generating a variant Fc Region. The variant Fc region can comprise a sequence from the human Fc region (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) comprising an amino acid modification (e.g., a substitution) at one or more amino acid positions.
[0273] In an example of an embodiment, the invention contemplates a variant Fc region that has some, but not all effector functions, which makes it a desirable candidate for many applications in which the in vivo half-life of the complex is important, although certain effector functions (such as add-on and ADCC) are unnecessary or harmful. An in vitro or in vivo cytotoxicity assay can be conducted to confirm the reduction / depletion of CDC and / or ADCC activities. For example, Fc receptor binding assays (FcR) can be performed to ensure that the complex is not bound to FCYR (and therefore lacks ADCC activity), but maintains the ability to bind to FcRn. The main mediating cells of ADCC, NK cells, express only FCYRIII, while monocytes express FCYRI, FCYRII and FCYRIII. FcR expression in hematopoietic cells is summarized in Table 3 on page 464 of Ravetch J.V. and Kinet, J. P., Annu. Rev. Immunol. 9 (1991) 457-92. Non-limiting examples of in vitro assays to assess the ADCC activity of a molecule of interest are described in US Patent 5,500,362 (see, for example, Hellstrom, I., et al. Proc. Nat Acad. Sci. USA 83 (1986 ) 7059-7063 and Hellstrom, I et al., Proc. Nat Acad. Sci. USA 82 (1985) 1499-1502; US patent 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166 (1987) 1351-1361) Alternatively, non-radioactive assay methods can be used (see, for example, ACTI ™ non-radioactive cytotoxicity assay by flow cytometry (CellTechnology, Inc., Mountain View, CA, and CyTotox 96® non-radioactive cytotoxicity assay (Promega, Madison, WI). Effector cells useful for such tests include peripheral blood mononuclear cells (PBMC) and natural killer cells (natural killer - NK). interest can be assessed in vivo, for example, in an animal model as described in Clynes R. et al., Proc. N at Acad. Sci. USA 95 (1998) 652-656. C1q binding assays can also be performed to confirm that the antibody is unable to bind to C1q and therefore lacks CDC activity. See, for example, the C1q and C3c binding ELISA in WO 2006/029879 and WO 2005/100402. To determine complement activation, a CDC assay can be performed (see, for example, Gazzano-Santoro, H. et al., J. Immunol. Methods 202 (1996) 163-171 and Cragg, MS et al., Blood 101 (2003) 1045-1052 and Cragg, MS and Glennie, MJ, Blood 103 (2004) 2738-2743). Determination of FcRn binding and in vivo clearance / half-life can also be performed using methods known in the art (see, for example, Petkova, SB et al., Int'l. Immunol. 18 (12): 1759 -1769 (2006)).
[0274] Fc regions with reduced effector function include those with the replacement of one or more of the waste from the Fc region: 234, 235, 238, 265, 269, 270, 297, 327 and 329 (see, for example, the US Patent 6,737,056). Such Fc mutants include Fc mutants with substitutions at two or more of the amino acid positions 265, 269, 270, 297 and 327, including the mutant Fc called “DANA” with the substitution of residues 265 and 297 for alanine (US 7,332,581) .
[0275] Certain variants of the Fc region with improved or decreased FcR binding are described. (See, for example, US 6,737,056; WO 2004/056312, and Shields, R.L., et al., J. Biol. Chem. 276 (2001) 6591-6604).
[0276] In certain embodiments, a variant polypeptide comprises an Fc region with one or more amino acid substitutions that further improve ADCC function, for example, substitutions at positions 298, 333, and / or 334 of the Fc region (residue numbering ME).
[0277] In some embodiments, changes are made to the Fc region resulting in the change (whether improved or decreased) of binding to C1q and / or complement-dependent cytotoxicity (CDC), for example, as described in US Patent 6,194 .551, WO 99/51642, and in Idusogie, EE et al. J. Immunol.164 (2000) 4178-4184.
[0278] Antibodies with increased half-life and improved neonatal Fc receptor (FcRn) binding, which are responsible for the transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol. 117: 587 (1976) and Kim et al., J. Immunol. 24: 249 (1994)), are described in US 2005 / 0014934A1. These antibodies comprise an Fc region with one or more substitutions at this site that improve the binding of the Fc region to the FcRn. Such variants of the Fc region include those with substitutions in one or more residues of the Fc region: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434, for example, replacement of residue 434 in the region - Fc (US 7,371,826).
[0279] See also Duncan, A.R. and Winter, G., Nature 322 (1988) 738-740; US 5,648,260; US 5,624,821 and WO 94/29351 for other examples of variants of the Fc-Region. D) DERIVATIVES
[0280] In certain embodiments, a complex provided herein can be further modified to contain additional non-protein portions which are known in the art and are readily available. Suitable portions for derivatizing the complex include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, pyrrolidone polychloride, poly-1, 3-dioxolane, poly-1,3 , 6-trioxane, ethylene / maleic anhydride copolymer, polyamino acids (homopolymers or random copolymers), and dextran or poly (n-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers (polyols, polyols, polyols for example, glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in the manufacturing process due to its stability in water. The polymer can be of any molecular weight, and can be branched or unbranched. The number of polymers coupled by antibody can vary, and if more than one polymer is attached, the polymers can be the same molecule or different molecules. In general, the number and / or type of polymers used for derivatization can be determined based on considerations, including, but not limited to, specific properties or functions of the antibody to be improved, whether derived antibodies will be used within the framework of a therapy, etc.
[0281] In another embodiment, conjugates of a complex and the non-proteinaceous portion are provided which can be selectively heated by exposure to radiation. In one embodiment, the non-proteinaceous portion is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. 102: 11600-11605 (2005)). The radiation can be of any wavelength, and includes, but is not limited to, wavelengths that do not harm normal cells, but that heat the non-proteinaceous cluster to a temperature at which cells close to the antibody-non-proteinaceous conjugate are killed. B. RECOMBINANT METHODS AND COMPOSITIONS
[0282] Complexes can be produced using recombinant methods and compositions, for example, as described in US Patent 4,816,567. In one embodiment, isolated nucleic acids encoding the complex polypeptides described herein are provided. In an example of a further embodiment, one or more vectors (e.g., expression vectors) comprising such nucleic acid are provided. In another embodiment, a host cell comprising such a nucleic acid is provided. In such an embodiment, the host cell comprises (for example, it has been transformed with): (1) a vector comprising a nucleic acid that encodes an amino acid sequence comprising the VL of the antibody and an amino acid sequence comprising the VH of the antibody ; or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and a second vector comprising a nucleic acid encoding an amino acid sequence comprising the VH of the antibody. In one embodiment, the eukaryotic host cell is, for example, a Chinese hamster ovary (CHO) cell or lymphoid cell (for example, Y0, NS0, Sp2 / 0 cell). In one embodiment, a method of making a complex, as disclosed in the present invention, is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the complex's polypeptides, as provided above, under suitable conditions for polypeptide expression and complex formation and, optionally, recovering the complex from the host cell (or host cell culture medium).
[0283] For recombinant production of a complex, the nucleic acid encoding the complex's polypeptides, for example, as described above, is isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such a variant nucleic acid can be easily isolated and sequenced using conventional procedures (using, for example, oligonucleotide probes that are able to specifically bind to genes encoding the heavy and light chains of the antibody).
[0284] Host cells suitable for cloning or expression vectors include prokaryotic or eukaryotic cells described in the present invention. For example, complexes can be produced in bacteria, particularly when glycosylation and the effective function of Fc are not necessary. For the expression of antibody fragments and polypeptides in bacteria, see, for example, US 5,648,237, US 5,789,199, US 5,840,523 e. (See also Charlton, KA, Em:. Methods in Molecular Biology, Vol. 248, Lo, BKC (Ed.), Humana Press, Totowa, NJ (2003), pages 245-254, which describes the expression of antibody fragments in E. coli). After expression, the complex can be isolated from the bacterial cell paste in a soluble fraction and can be further purified.
[0285] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeasts are suitable hosts for cloning or expression vectors encoding the polypeptide, including fungi and yeast strains whose glycosylation pathways have been "humanized", resulting in the production of an antibody with a glycosylation pattern partially or totally human. See, Gerngross, T.U., Nat. Biotech. 22 (2004) 1409-1414, and Li, H. et al, Nat .. Biotech.24 (2006) 210-215.
[0286] Host cells suitable for the expression of glycosylated complexes can be derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Several strains of baculovirus have been identified that can be used together with insect cells, particularly for transfection of Spodoptera frugiperda cells.
[0287] Plant cell cultures can also be used as hosts. See, for example, US Patents 5,959,177, US 6,040,498, US 6,420,548, US 7,125,978 and US 6,417,429 (describing PLANTIBODIES® technology for the production of antibodies in transgenic plants).
[0288] Vertebrate cells can also be used as hosts. For example, mammalian cell lines that are adapted to grow in suspension can be useful. Other examples of useful mammalian host cell lines are: monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney cell line (HEK 293 cells or 293 cells subcloned for growth in culture suspension, Graham et al., J. Gen Virol. 36 (1977) 59-74), baby hamster cub kidney cells ( BHK), mouse Sertoli cells (TM4 cells, as described, for example, in Mather, JP, Biol. Reprod. 23 (1980) 243-252), monkey kidney cells (CV1), monkey kidney cells African green (VERO-76), human cervical carcinoma cells (HeLa), canine kidney cells (MDCK cells) buffalo rat liver cells (BRL 3A); human lung cells (W138), human liver cells (Hep G2); mouse mammary tumor cells (MMT 060562); TRI cells, as described, for example, in Mather, J.P., et al, Annals NY Acad. Sci. 383 (1982) 44-68; MRC 5 cells and FS4 cells. Other useful mammalian host cell lines include the Chinese hamster ovary (CHO) cell, including DHFR-CHO cells (Urlaub et al, Proc Acad Nat. Sci USA 77 (1980) 4216-4220) and myeloma cell line , such as Y0, NS0 and Sp2 / 0. For a review of certain mammalian host cell lines suitable for antibody production, see, for example, Yazaki P. and Wu A. M., Methods in Molecular Biology, vol. 248 (B.K.C. Lo, Ed.), Humana Press, Fairfield, NJ, (2004), pp 255-268. C. PHARMACEUTICAL FORMULATIONS
[0289] Suitable pharmaceutical formulations of a complex as described in the present invention are prepared by mixing the complex having the desired degree of purity with one or more pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980) ), in the form of lyophilized formulations or aqueous solutions. Pharmaceutically acceptable carriers are generally non-toxic to receptors at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m- cresol); low molecular weight polypeptides (less than about 10 residues); proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as poly (vinylpyrrolidone); amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (such as, Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG). Examples of pharmaceutically acceptable carriers in the present invention further include interstitial drug dispersing agents such as soluble neutral-active hyaluronidase glycoproteins (sHASEGP), for example, human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, Baxter International, Baxter International, .). Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in US 2005/0260186 and US 2006/0104968. In one aspect, a sHASEGP is combined with one or more additional glycosaminoglycans, such as chondroitinases.
[0290] Exemplary lyophilized antibody formulations are described in US 6,267,958. Aqueous antibody formulations include those described in US 6,171,586 and WO 2006/044908, formulations of the latter include a histidine-acetate buffer.
[0291] The present formulation can also contain more than one active ingredient according to the need for the specific indication being treated, preferably active compounds with complementary activities that are not harmful to each other. Such active ingredients are suitably present in the combination in amounts that are effective for the intended purpose.
[0292] Active ingredients can be stored in prepared microcapsules, for example, through coacervation techniques or through interfacial polymerization, for example, hydroxymethylcellulose or gelatin microcapsules and poly (methylmethacrylate) microcapsules, respectively, in supply systems. colloidal drugs (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are described in Remington's Pharmaceutical Sciences, 16th Edition, Osol, A. Ed. (1980).
[0293] Controlled release preparations can be manufactured. Suitable examples of sustained release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which are in the form of molded articles, such as films or microcapsules.
[0294] The formulations to be used for in vivo administration are generally sterile. Sterilization can be easily achieved, for example, by filtering through sterile filter membranes. D. THERAPEUTIC COMPOSITIONS AND METHODS
[0295] Any complex provided in the present invention can be used in therapeutic methods.
[0296] In one aspect, a complex is provided as disclosed in the present invention for use as a medicament.
[0297] In additional aspects, a complex is provided as disclosed in the present invention for use in the treatment of cancer or viral infection.
[0298] In certain embodiments, a complex is provided as disclosed in the present invention for use in a treatment method.
[0299] In certain embodiments, the invention provides a complex as disclosed in the present invention for use in a method of treating an individual who has cancer or viral infection, the method of which comprises administering to an individual an effective amount of such a complex as disclosed in the present invention. In such an embodiment, the method further comprises administering to an individual an effective amount of at least one additional therapeutic agent, for example, as described below.
[0300] In other embodiments, the invention provides a complex as disclosed in the present invention for use in removing cancer cells or for removing virus-infected cells. In certain embodiments, the invention provides a complex as disclosed in the present invention for use in a method of removing cancer cells or removing virus-infected cells in an individual, comprising administering the complex to the individual in an effective manner as required. disclosed in the present invention to remove cancer cells or to remove virus infected cells. An "individual", according to any of the above embodiments, may be a human.
[0301] In a further aspect, the invention provides the use of a complex as disclosed in the present invention in the manufacture or preparation of a medicament. In one example, the drug is for the treatment of cancer or viral infection. In one embodiment, the drug is for use in a method for treating cancer or viral infections, comprising administering to an individual having cancer or chronic viral infection an effective amount of the drug. In such an embodiment, the method further comprises administering to an individual an effective amount of at least one additional therapeutic agent. In an example of a further embodiment, the drug is for the removal of cancer cells or for the removal of virus-infected cells. In an example of a further embodiment, the drug is for use in a method of removing cancer cells or removing virus-infected cells in an individual comprising administering to the individual an effective amount of the drug to remove the cancer cells or to remove virus-infected cells. An "individual", according to any of the above embodiments, may be a human.
[0302] In another aspect, the invention provides a method for treating cancer or viral infection. In an example of an embodiment, the method comprises administering an effective amount of the complex as disclosed in the present invention to an individual having such a cancer or viral infection. In such an embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent. An "individual", according to any of the above embodiments, may be a human.
[0303] In a further aspect, the invention provides a method for removing cancer cells or cells infected with viruses in an individual. In an example of an embodiment, the method comprises administering to the individual an effective amount of a complex as disclosed in the present invention to remove cancer cells or virus-infected cells. In an example of realization, the "individual" is a human.
[0304] In a further aspect, the invention provides pharmaceutical formulations that comprise any of the complexes provided herein, for example, for use in any of the therapeutic methods described above. In one embodiment, a pharmaceutical formulation comprises any of the complexes provided in the present invention and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical formulation comprises any of the complexes, as disclosed in the present invention, and at least one additional therapeutic agent.
[0305] The complexes of the present invention can be used alone or in combination with other agents in a therapy. For example, a complex of the invention can be co-administered with at least one additional therapeutic agent.
[0306] This combination therapy referred to above encompasses combined administration (in which two or more therapeutic agents are included in the same formulation or in different formulations), and separate administration, in which case the administration of the complex of the invention, can occur before, simultaneously and / or after administration of the additional therapeutic agent and / or adjuvant The complexes of the invention can also be used in combination with radiotherapy.
[0307] A complex of the invention (and any additional therapeutic agent) can be administered by any appropriate route, which includes parenteral, intrapulmonary and intranasal and, if desired for local treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Preferably, the dosage is provided by means of injections, such as intravenous or subcutaneous injections, depending, in part, on whether the administration is brief or chronic. Dosage schedules, including but not limited to, single or multiple administrations over various time points, bolus administration and pulsed infusion, are contemplated in the present invention.
[0308] The complexes of the invention can be formulated, dosed, and administered in a manner consistent with good medical practice. Factors that should be considered in this context include the specific disorder to be treated, the specific mammal to be treated, the clinical condition of the patient, the location of delivery of the agent, the administration schedule and other factors known to doctors. The complex need not be, but it is optionally formulated with one or more agents currently used to prevent or treat the disorders in question. The effective amount of such other agents depends on the amount of complex present in the formulation, the type of disorder or treatment, and other factors discussed above. These are generally used in the same dosages and with routes of administration as described above or about 1 to 99% of the dosages described herein, or in any dosage and by any route of administration described which is empirically / clinically determined to be appropriate.
[0309] For disease prevention or treatment, the appropriate dosage of a complex of the invention (when used alone or in combination with one or more additional therapeutic agents) will depend on the type of disease to be treated, the type of complex, the severity and the course of the disease, if the complex is administered for preventive or therapeutic purposes, if there was and what was the previous therapy, the patient's clinical history, the reaction to the complex, and the criterion of the attending physician. The complex is properly administered to the patient in one go or over a series of treatments. Depending on the type and severity of the disease, about 1 μg / kg to 15 mg / kg (for example, 0.5 mg / kg to 10 mg / kg) of complex is a possible initial candidate dose for administration to the patient, either , for example, through one or more separate administrations or through continuous infusion. A typical daily dosage can range from about 1 μg / kg to 100 mg / kg or more, depending on the factors mentioned above. For repeated administrations over several days or more, depending on the condition, treatment can be continued until the desired suppression of the symptoms of the disease occurs. An exemplary dosage of the antibody will be in the range of about 0.05 mg / kg to about 10 mg / kg. In this way, one or more doses of about 0.5 mg / kg, 2.0 mg / kg, 4.0 mg / kg or 10 mg / kg (or any combination of these) can be administered to the patient. These doses can be administered intermittently, such as weekly or every three weeks (for example, in such a way that the patient receives about two to about twenty, such as about six doses of the antibody). A higher initial loading dose may be administered, followed by one or more lower doses. However, other dosage regimens may be useful. The progress of this therapy is easily monitored using conventional techniques and trials.
[0310] It is understood that any of the above therapeutic formulations or methods can be performed using an immunoconjugate of the invention in place or in addition to a complex as disclosed herein.
[0311] One aspect as disclosed in the present invention is the complex, as disclosed, for use in a method of treating cancer or viral infection in a patient, the complex of which will be administered before, simultaneously or after the patient's immunization with the peptide derived from viruses comprised in the complex.
[0312] One aspect as disclosed in the present invention is the use of a complex as disclosed, for the manufacture of a medicament for the treatment of cancer or viral infection, in combination with immunization against the peptide derived from the virus comprised in the complex.
[0313] In the first step, the virus-derived peptide contained in the complex is first administered to the subject to be treated. In a given period of time later, that is, between 4 days and 28 days, the complex disclosed in the present invention is administered to the individual.
[0314] Through this successive and separate application of the virus-derived polypeptide, in the first step alone and in the second step with the complex disclosed in the present invention, it is possible to increase the number of T-cells specific for the virus-derived peptide and, thereby increasing the effectiveness of the treatment. III. MANUFACTURING ITEMS
[0315] In another aspect of the invention, a manufacturing article is provided that contains materials useful for the treatment, prevention and / or diagnosis of the disorders described above. The article of manufacture comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, bags for IV administration and etc. The containers can be formed from a variety of materials such as glass or plastic. The container holds a composition that is alone (it is unique), or that is combined with other composition (s) effective for the treatment, prevention and / or diagnosis of the condition; and it may have a sterile access port (for example, the container may be a bag of intravenous solution or an ampoule containing a cap that can be pierced by a hypodermic injection needle). At least one active agent in the composition is a complex of the present invention. The label or package insert indicates that the composition is used to treat the condition of choice. In addition, the article of manufacture may comprise (a) a first container with a composition contained therein, wherein the composition comprises a complex of the present invention, and (b) a second container with a second composition contained therein, wherein the second The composition comprises an additional cytotoxic agent or other therapeutic agent. The article of manufacture in this embodiment example may further comprise a package insert indicating that the composition can be used to treat a specific condition. Alternatively or additionally, the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution and dextrose solution. It may additionally include other materials desirable from a commercial and user point of view, including other buffers, thinners, filters, needles and syringes.
[0316] It is understood that any of the aforementioned articles of manufacture may include an immunoconjugate of the invention instead of or in addition to the complex disclosed herein. IV. EXAMPLES
[0317] The following describes examples of methods and compositions of the present invention. It is understood that several other achievements can be practiced, given the general description provided above. EXAMPLE 1 PROCESS FOR THE INSULATION AND STIMULATION OF SPECIFIC CD8 + POSITIVE T CELLS FOR HUMAN DONOR CMV ISOLATION OF PBLS
[0318] PBLs were isolated by Ficoll gradient centrifugation from the blood of human donors (Greiner bio-one, Cat. No. 227290). PBLs were cultured in RPMI medium supplemented with 5% human serum (Sigma cat. No. H2520), 2 mM L-glutamine (PAN Biotech, cat. No. P04-80100), 100 μg / ml Penicillin / Streptomycin (Roche cat. No. 14,001,100). PBLS STIMULATION
[0319] The cells (2 x 107 cells / ml) were grown in cell culture medium supplemented with 50 μg / ml peptide derived from pp65 CMV (SEQ ID NO: 1) for two hours under cell culture conditions (37 ° C, 5% CO2.80% humidity). Then, the cell suspension was diluted 20 times with culture medium and cultured in 96-well flat-bottom plates at a density of 2 x 105 cells per well. After 4 to 5 days, 20 U / ml of IL-2 (Roche, Cat. No. 11011456001), 25 ng / ml of IL-7 (Peprotech, cat. No. 200-01) and 25 ng / ml were added of IL-15 (Peprotech, cat. No. 200-15) and the cells were cultured for an additional 7-8 days. T-cell stimulation is visible under the microscope as clusters of cells. PBLS RESTIMULATION
[0320] T cells were co-cultured with stimulator cells, which are pulsed with autologous primary PBL peptides from the same donor (prepared fresh or derived from frozen stocks). The stimulator cells were pulsed with the peptide, as described above. After the two hours of incubation with the peptides, the PBLs were irradiated (4000 rad; STS GmbH OB29 Nr.9510-5) and washed twice with culture medium without peptide. Restimulation was performed in 96-well round-bottom plates. 8 to 104 to 1 x 105 stimulator cells were used in the 96 wells. The primary culture cells were washed twice with culture medium, resuspended in 200 μL of culture medium and 80 μL were transferred to each well of the stimulator cells. After 3 days, 20 U / ml of IL-2, 25 ng / ml of IL-7 and 25 ng / ml of IL-15 were added. The cells proliferated and expanded every 2 to 3 days in new wells with fresh medium. T-CELL ANALYSIS
[0321] The cells were stained for expression of CD8 (BD, cat. No. 345,772), and CMV-specific T cell receptors (ProImmune, cat. No. F008-4A-E) and analyzed in FACS. CELL CULTURE MEDIA
[0322] RPMI1640 (. PAN Biotech, Cat. No. P04-17500), 5% human serum (HS ,. Sigma Cat. No. H2520), 2 mM L-glutamine (PAN, Biotech, Cat. No. P04-80100), 100 μg / mL penicillin / streptomycin (Roche, Cat. No. 14001100). RESULTS
[0323] FACS analysis of human peripheral blood lymphocytes (PBLs) derived from four donors was performed. The cells were stained with a FITC-conjugated anti-CD8 antibody (BD, cat. No. 345,772) combined with the APC-conjugated Pro5 pentamer (ProImmune, cat. No. F008-4A-E) to stain T cells that carry a T cell receptor (TCR) recognizing MHC class I (HLA-A * 0201), loaded with the CMV-derived peptide (NLVPMVATV (SEQ ID NO: 1)). For results see Figure 4. On day 0, donor 1 and 4 carried low numbers of CMV-specific CD8 + T cells (0.08% and 0.1%, respectively). Donor 2 and 3 carried a greater number of CMV-specific CD8 + T cells in peripheral blood (0.25% and 3.12%, respectively). Fourteen days later, after stimulation with autologous cells pulsed with the CMV-derived peptide, only donors 2 and 3 show a significant increase in CMV-specific CD8 + T cells (6.2% and 71.2%, respectively), while donors 1 and 4 do not show an increase in the number of CMV-specific CD8 + T cells (0.01% and 0.05%, respectively). 14 days later, after a second stimulation with autologous cells pulsed with the CMV-derived peptide, donors 2 and 3 show an increase in CMV-specific CD8 + T cells (15.1% and 96.6%, respectively). EXAMPLE 2 CITOTOXICITY TEST
[0324] MN60 acute lymphoblastic leukemia cells carry the HLA-A A * 0201 allele. MN60 cells (1 x 106 cells / ml) were incubated with 50 μg / ml of CMV pp65 peptide (SEQ ID NO: 1) for 45 minutes, under cell culture conditions (37 ° C, 5% CO2, 80% humidity). The results of incubation in a peptide exchange in the HLA-A * 0201 peptide binding slit. MN60 cells with exchanged peptide were centrifuged and diluted to a density of 1 x 10 6 cells / ml with PBS (PanBiotech, cat. No. P04-36500) and stained with 1 μM of the carboxy fluorescein ester succinimidyl ester marker (CFSE, Invitrogen, No. 34554) for 15 minutes at room temperature (RT). The cells were washed once with PBS and diluted to 1 x 105 cells / ml with AIM-V medium (Gibco, cat. No. 0870112DK). For the assay, MN60 cells (target cells) were co-cultured in 96-well round-bottom plates with CMV-specific CD8 + T cells derived from human donor 3 (effector cells, see example 1), for four hours, under culture conditions of cells. The ratio of effector cells to target cells was 4: 1. Dead cells are stained with 1 μl / 100 μl propidium iodide (PI, Sigma, cat. No. P-4864) and analyzed by FACS. RESULTS
[0325] Flow Cytometry analysis was performed to analyze the cytolytic capacity of CTL stimulated by lysis of MN60 tumor cells loaded with CMV peptide.
[0326] Figure 5a shows the FACS analysis of the co-culture of MN60 cells not loaded with the CMV-derived peptide. MN60 cells are FICT-positive. Effector cells are FICT-negative. Dead cells are PI positive, living cells are PI negative. It can be seen that more than 85% of MN60 cells are alive when they are not loaded with the CMV-derived peptide (Q2 and Q4).
[0327] Figure 5b shows the FACS analysis of MN60 cells loaded with the CMV-derived peptide. It can be seen that more than 80% of MN60 cells are dead (Q2 and Q4), while the proportion of live and dead effector cells is not noticeably changed between the FACS analyzes shown in Figure 5a and Figure 5b (compare Q1 and Q3 in Figure 5a and Figure 5b), indicating a specific lysis of target cell loaded with CMV peptide.
[0328] Flow cytometry analysis to analyze the cytolytic capacity of CTLs stimulated by lysis of MN60 tumor cells loaded with CMV peptide depending on the ratio of effector cells: target cells: The cytotoxicity assay was performed as described above. Different ratios of effector cells: target cells were applied ranging from 0.5 effector cells per target cell to four effector cells per target cell (see Figure 6). The incubation time was four hours. MN60 cells that were not loaded with the CMV-derived peptide did not exhibit an increase in the number of dead cells with an increase in the ratio of effector cells / target cells, that is, varying between 8% and 13%, with a ratio 0.5: 1 to 4: 1.
[0329] Nearly 20% of MN60 cells loaded with the CMV-derived peptide are readily killed with a low effector cell / target cell ratio of 0.5: 1 within four hours. The number of dead cells increases markedly with the increase in the effector cells / target cells ratio, reaching up to 83% for the 4: 1 effector cells / target cells ratio. EXAMPLE 3 DNA PREPARATION, TRANSFECTION, EXPRESSION, PURIFICATION AND ANALYSIS DNA PREPARATION
[0330] 250 mL of bacterial LB culture grown for one night was collected and the plasmid DNA was extracted according to the manufacturer's protocol (High speed Maxi kit, Qiagen, cat. No. 12663). The resulting plasmid DNA was eluted in 1 ml of TE buffer and the DNA concentration was determined by spectrophotometry (Epoch, Biotek).
[0331] The final expression vector comprises the following elements: - HindIII, Nhel, endonucleolytic restriction sites, - A CMV promoter, - a 5'UTR 1 (derived from human CMV), - Intron A, - a 5'UTR 2, - an ampicillin resistance gene, - a BGH Poly A site (bovine growth hormone polyadenylation signal), - pUC Ori.
[0332] The amino acid sequences of complex elements: Peptide CMV pp65: SEQ ID NO: 1 NLVPMVATV linker (linker) 1: SEQ ID NO: 21 GGGGSGGGGSGGGGS β2 microglobulin: SEQ ID NO: 10 IQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDL SFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM Linker 2: SEQ ID NO: 22 GGGGSGGGGSGGGGSGGGGS HLA-a * 0201 α1 - α3: SEQ ID NO: 11 GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPW IEQEGPEYWDGETRKVKAHSQTHRVDLGTLRGYYNQSEAGSHTVQRMYGCD VGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVA EQLRAYLEGTCVEWLRRYLENGKETLQRTDAPKTHMTHHAVSDHEATLRCW ALSFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGQEQ RYTCHVQHEGLPKPLTLRW Binder 3: SEQ ID NO: 12 GS Linker 4: SEQ ID NO: 22 GGGGSGGGGSGGGGSGGGGS light Ig chain: SEQ ID NO : 41 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASK RATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSKWPPWTFGQGTKV ESKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC Ig heavy chain (IgG1 -mutante L234A, L235A): SEQ ID NO: 42 QVELVESGGGVVQPGRSQRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAII WFDGSSTYYADSVRGRFTISRDNSKNTLYLQMNSLRAEDTAVYFCARELGRR YFDLWGRGTLVSVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPS NTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVT CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Ig heavy chain (IgG1-mutant L234A, L235A knob to change): SEQ ID NO: 43 QVELVESGGGVVQPGRSQRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAII WFDGSSTYYADSVRGRFTISRDNSKNTLYLQMNSLRAEDTAVYFCARELGRR YFDLWGRGTLVSVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPS NTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVT CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVS LWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Ig heavy chain (IgG1-mutant L234A, L235A with hole variant): SEQ ID NO: 44 QVELVESGGGVVQPGRSQRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAII WFDGSSTYYADSVRGRFTISRDNSKNTLYLQMNSLRAEDTAVYFCARELGRR YFDLWGRGTLVSVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPS NTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVT CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVS LSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK heavy chain region-Fc Ig (IgG1-mutant L234A, L235A variant knob in Region -Fc): SEQ ID NO: 45 EPKSADKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGK scFv: SEQ ID NO: 46 QVELVESGGGVVQPGRSQRLSCAASGFTFSSYGMHWVRQAPGKCLEWVAII WFDGSSTYYADSVRGRF TISRDNSKNTLYLQMNSLRAEDTAVYFCARELGRR YFDLWGRGTLVSVSSGGGGSGGGGSGGGGSGGGGSEIVLTQSPATLSLSP GERATLSCRASQSVSSYLAWYQQPGQAPFTLGYGSKTGFGFGFGFGKLGFGFGGG
[0333] HEK 293 cells were diluted to 8 x 105 cells / ml the day before transfection. About 1 to 1.6 x 10 6 cells / mL were transfected according to the manufacturer's protocol. For a final transfection volume of 30 mL, 30 μg of DNA was diluted to a final volume of 1 mL with medium with reduced serum “Opti-MEM® I Reduced Serum Medium” (Gibco, cat. No. 31,985,070). 2 μL of 293fectin® Reagent (Invitrogen, cat. No. 12.347.019) to 1 μg of DNA was likewise diluted in a final volume of 1 ml with Opti-MEM® medium and incubated for 5 minutes. After incubation, the diluted DNA was added to the diluted 293fectin® reagent, mixed gently and incubated for another 20-30 minutes and then pipetted dropwise into 28 ml of HEK293 cells to obtain a final volume of 30 ml. The cells were incubated under cell culture conditions (37 ° C, 8% CO2, 80% humidity) on a rotary orbital shaker at 125 rpm and collected after 72 hours. The collected cells were centrifuged for 10 minutes at 1000 rpm, for 10 minutes at 3000 rpm and filtered through a sterile 22 μm filter (Millipore, cat. No. SCGPU05RE). WESTERN BLOTTING
[0334] 500 μL of cell culture supernatant was concentrated with a Pall Nanosep OmegaMembran 30Kd centrifuge device (Pall, cat. No. OD030C33) to a volume of 50 μL. 17.5 μL of each concentrate was diluted to a final volume of 25 μL with XT 4x sample buffer (Bio Rad, cat. No. 161-0791) and XT 20x Reducing Agent (BioRad, Cat .. No. 161-0792 ), heated to 96 ° C for 8 minutes and applied on 4-12% Criterion XT Precast Gel (Cat. No. 345-0124). Blotting was performed with a Trans-Blot SD semi-dry Transfer Cell device (Biorad) at 20 V for 30 minutes on a nitrocellulose membrane Trans-blot Pure Nitrocellulose membrane (0.45 μm) (BioRad, Cat. No. 162 -0117). Blocking the membrane was performed with blocking reagent for Western 1x (Roche, Cat. No. 11921681001) for one hour at room temperature. Staining was performed with rabbit polyclonal conjugated with anti-human K light chain peroxidase (DAKO, Cat. No. P0129, diluted 1: 3000) and polyclonal rabbit anti-human IgG antibody conjugated with HRP (DAKO, Cat. No P0214, diluted 1: 5000) for one hour at room temperature. The luminescence detection was performed with Lumi-Imager F1 (Roche). PURIFICATION
[0335] The cells were removed from the culture medium by centrifugation. The complexes were purified from the supernatants by Protein A affinity chromatography (MabSelect-Sepharose on an AKTA-Avant). Eluted complexes were concentrated with Amicon centrifuge tubes to a protein concentration of 3 mg / mL. An aliquot was analyzed by size exclusion chromatography (HPLC TSKgel GFC300 Sys89). A preparative SEC on a Superdex 200 column was performed to remove aggregates and buffer the fusion proteins in 20 mM histidine, 140 mM NaCl, pH 6.0. The eluted complexes were concentrated with an Amicon centrifuge tube to a protein concentration of 1 mg / mL and sterilized by filtration (pore size 0.2 μm). ANALYTICS
[0336] Complex samples were analyzed by OD280 using a UV spectrophotometer to determine the protein concentration in the solution. The required extinction coefficient for this was calculated from the amino acid sequence according to Pace (Pace et al., Protein Science 4 (1995) 2411-2423). A size exclusion chromatography (SE-HPLC) was performed on TSK-Gel300SWXL or Superdex 200 columns with a 0.2 M potassium phosphate buffer, comprising 0.25 M KCl, pH 7.0 as the mobile phase, in order to determine the content of monomeric species, aggregated and degraded species in the samples. An electrophoresis in sodium dodecyl sulfate (SDS) polyacrylamide gel (reduction and non-reduction conditions) was performed to analyze the purity of complex preparations with respect to product related degradation products and unrelated impurities. An electrospray ionization mass spectrometry (ESI-MS) was performed with reduced (TCEP) and deglycosylated (N-glycosidase F) samples to confirm the correct mass / identity of each chain and detect chemical changes. The ESI-MS of the deglycosylated samples was performed to analyze the nature and quality of the fully assembled protein and to detect potential side products related to the product. SDS-PAGE METHOD AND COOMASSIE COLORING Device: Invitrogen XCell Sure Lock Mini-Cell Gel: 4-20% Tris-Glycine gel, Invitrogen EC6025BOX Buffer: SDS Tris-glycine running buffer (10x), Invitrogen LC2675-5 Buffer Sample: Tris-Glycine SDS Sample Buffer (2x), Invitrogen LC2676 Reduction Buffer: NuPAGE Sample Reducing Agent (10x), Invitrogen NP0004 Molecular Weight Marker: Mark 12, PM Standard, Invitrogen LC5677 PREPARATION OF PROTEIN SAMPLES
[0337] The sample was adjusted to a protein concentration of 1 mg / mL with buffer. To reduce the sample, the following procedure was performed: - Reduction Buffer: 4 mL of sample buffer (2x) and 1 mL of reduction buffer (10x) - Dilute the sample 1: 1 with reduction buffer - Incubate for 5 minutes at 70 ° C
[0338] Gel electrophoresis was performed at 125 V for 90 minutes. The gels were stained with SimplyBlue SafeStain (Invitrogen, cat. No LC6065). TABLE RESULTS


[0339] The SDS gel with Coomassie staining and the corresponding SEC chromatograms of the selected complexes number 1 and 2a according to the table above are shown in Figures 7 and 8. It can be seen that the defined complexes can be obtained. EXAMPLE 4 CONNECTION OF THE MHC-ANTIBODY FUSION TO THE POSITIVE IGF-1R HUMAN CELL LINE
[0340] H460M2 cells were diluted to 8 x 105 cells / ml in AIM-V medium (Gibco, cat. No. 0870112DK). 500 μL of the cell suspension was stained with 10 μg of a complex as disclosed in the present invention, at 4 ° C or 37 ° C for one hour. Then the cells were washed once with ice-cold AIM-V medium and stained with a second antibody, which was goat anti-human IgG (H + L) conjugated to R-PE (Dianova, cat. No. 109-116-088, 1:50 dilution) for 30 minutes at 4 ° C. Then the cells were washed once with ice-cold AIM-V medium and fluorescence was measured using FACS Canto II (BD Bioscience) with selection using the “gating” strategy on live cells. A bispecific antibody served as an isotypic control, an anti-IGF-1R antibody (see, for example, WO 2004/087756 and WO 2007/115814) served as a positive control. RESULTS
[0341] The results are shown in Figure 9. Considering the change in the measurement of PE fluorescence (X-axis), the complex, as disclosed in the present invention, does not exhibit any visible difference in binding to the H460M2 target cells (Figure 9 -2), compared to the control antibody (Figure 9-6). There is also no difference if the incubation with the complex, as disclosed in the present invention, is carried out at 4 ° C or 37 ° C (see Figure 9-2 vs Figure 9-3). Neither incubation with the isotypic control (Figure 9-4), nor with the fluorescence-labeled secondary antibody alone (Figure 9.5) shows any change in the PE fluorescence measurement. Despite the fusion of the MHC class I molecule, the antibody variable domain of the complex, as disclosed in the present invention, still binds to the target cells H460M2.
[0342] Although the invention disclosed above has been described in detail by way of illustrations and examples for the sake of clarity of understanding, the descriptions and examples should not be construed as limiting the scope of the present invention. Disclosures of all patents and scientific literature cited herein are expressly and fully incorporated by reference. EXAMPLE 5 IN VITRO REMOVAL OF CELLS EXPRESSING THE ANTIGEN
[0343] I24 target cells (1x105 cells / ml) were seeded in cell culture medium (RPMI 1640 supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 0.1 mM NEAA, and 10% (v / p) FCS) in Willco glass bottom plates (FA. Willco Wells BV, REF GWST-3522) for 24 to 48 hours. Willco plates were pre-coated with 50 μg / mL of poly-L-lysine hydrochloride (Sigma Aldrich, Cat # P2658) per plate for 30 min. After coating the plates were carefully washed with tissue culture grade sterile water, and dried for two hours.
[0344] After the culture medium has been removed and the antigen binding complex comprising a fusion polypeptide [eptide-CMV-pp65] - [linker 1] - [β2-microglobulin] - [linker 2] - [HLA- A-α1-α2-α3] - [ligand 3] - [IgG1-L234A, L235A mutant with hole variation], a disulfide-linked polypeptide IgG1-L234A, L235A mutant with a knob variant in the Fc region and an Ig light chain, whose complex specifically binds to human IGF-1R as disclosed in the present invention (see, for example, Example 3) was added in a final concentration of 5 μg / mL in 3 mM Krebs Ringer Hepes + K + buffer, pH 7.3 (supplemented with 0.5 mM DL-dithiothreitol, 1 mM ascorbic acid, and 4 mM glutathione).
[0345] T cells were added in a 1:10 effector cell / target cell ratio. The images were obtained for 4 hours using a Zeiss Axiovert 135 microscope. RESULTS
[0346] In Figure 10 are shown microscopic images of the incubation of the antigen binding complex comprising a fusion polypeptide [eptide-CMV-pp65] - [ligand 1] - [β2-microglobulin] - [ligand 2] - [HLA -A-α1-α2-α3] - [ligand 3] - [IgG1-L234A, L235A mutant with hole variation], an IgG1-L234A, L235A disulfide-linked mutant with Fc region with knob variation and an Ig light chain, whose complex specifically binds to human IGF-1R. Lysis mediated by the complex of I24 3T3 cells expressing IGF-1R (large cells growing adherently, white arrow) can be observed. Lysis is mediated by CMV-specific human T cells (small round cells - white arrows, or migrating amoeboid cells, black arrow). I24 cells are incubated with the complex at a concentration of 5 μg / mL and human T cells specific for CMV (pre-activated with HLA-A0201 + APCs / pulsed with CMV peptide). The time lapse is indicated below the respective image. Observe the interaction of I24 cells with T-cells at 56 min and 76 min and, subsequently, the collapse of I24 cells after 125 min.
[0347] In figure 11 the microscopy image of a control shows the absence of lysis of I24 3T3 cells (large cells growing adherently, white arrowheads) incubated with CMV-specific human T cells (small rounded cells) , white arrow, or amebooid migrating cells - black arrow) in the absence of an antigen binding complex as disclosed in the present invention. I24 cells are incubated with specific cytotoxic T cells (pre-activated with HLA-A0201 + APCs / pulsed with CMV peptide). The time lapse is indicated below the respective image. EXAMPLE 6 CITOTOXICITY TEST
[0348] Cell culture medium (50 μL) was pipetted into each well of a 96-well xCELLigence plate (Roche, Cat. # 05232368001) to perform background measurement.
[0349] I24 cells were diluted to 1x106 cells / ml in cell culture medium (RPMI 1640, 2 mM L-glutamine, 1 mM sodium pyruvate, 0.1 mM NEAA, 10% (v / w) ) and 50 μl (2x104 cells per well) were pipetted into each well of a 96-well xCELLigence plate to a final volume of 100 μL and cells were cultured for 24 hours (37 ° C, 8% CO2, 80% moisture). After 24 hours the medium was removed and the cells were washed with 200 μL of T-cell medium “AIM-V” (serum-free medium (Invitrogen), (Cat: 12055-083). The antigen binding complex comprising a polypeptide [eptide-CMV-pp65] - [ligand 1] - [β2-microglobulin] - [ligand 2] - [HLA-A-α1-α2-α3] - [ligand 3] - [IgG1-L234A, L235A mutant with hole variation], a disulfide-linked mutant IgG1-L234A, L235A with a knob-altered Fc region and an Ig light chain, whose complex specifically binds to human IGF-1R, as disclosed in the present invention, was added to the washed at a final concentration of 1 μg / mL in AIM-V medium. Effector cells in a respectable proportion were added to the AIM-V medium to a final volume of 150 μl. A human IgG1 afucosylated monoclonal antibody directed against IGF-1R ( anti-IGF-1R-afucosylated antibody) and non-binding human anti-digoxigenin antibody (anti-digoxigenin antibody) served as isotypic control and specific antibody control, respectively. The measurement was carried out for 6 to 9 hours, respectively, with the xCELLigence System (Roche). RESULTS
[0350] The complex as disclosed in the present invention causes lysis of H460M2 tumor cells by means of human CMV-specific T-cells.
[0351] Tumor cells were incubated for four hours with 1 μg / mL of the complex comprising a fusion polypeptide [eptide-CMV-pp65] - [ligand 1] - [β2-microglobulin] - [ligand 2] - [HLA- A-α1-α2-α3] - [ligand 3] - [IgG1-L234A, L235A mutant with hole variation], an IgG1-L234A, L235A disulfide-linked mutant with Fc region with knob variation and an Ig light chain, in that the complex specifically binds to human IGF-1R, and specific T cells in the respective ratio (1: 1.5 to 1: 0.5) (see Figure 12). The percentage of lysis is shown above the respective bars. The human monoclonal antibody IgG1 afucosylated directed against IGF-1R (Mab IGF-1R-afu) does not trigger significant lysis of tumor cells.
[0352] The complex as disclosed in the present invention triggers the lysis of I24 3T3 target cells by means of CMV-specific T cells.
[0353] The target cells were incubated for 4 hours with 1 μg / ml of the antigen binding complex comprising a fusion polypeptide [eptide-CMV-pp65] - [ligand 1] - [β2-microglobulin] - [ligand 2 ] - [HLA-A-α1- α2-α3] - [ligand 3] - [IgG1-L234A, L235A mutant with hole variation], an IgG1-L234A, L235A disulfide mutant with Fc region with knob variation and a chain Ig light, in which the complex specifically binds to human IGF-1R, and specific T cells in the respective ratio (1: 1.5 to 1: 0.5) (see Figure 13). The percentage of lysis is shown above the respective bars. An afucosylated human IgG1 monoclonal antibody directed against IGF-1R (anti-IGF-1R-afucosylated antibody) and non-binding human anti-digoxigenin antibody (anti-digoxigenin antibody) did not trigger significant cell lysis. EXAMPLE 7 EFFECTIVENESS IN VITRO
[0354] A positive H460M2 IGF-1R lung adenocarcinoma cell line was incubated with 1 μg / ml of a complex comprising a hCMV-derived peptide and an anti-IGF-1R antibody and CMV-specific CD8 positive T cells in a relationship low from effector cell to target cells (1.5 to 0.5 specific T cells per tumor cell). The control antibody was an anti-IGF-1R antibody developed by glyco-engineering. The incubation time was 6 hours. Incubation with the complex resulted in the potent removal of H460M2 tumor cells.

权利要求:
Claims (9)
[0001]
1. METHOD FOR THE RECOMBINANT PRODUCTION OF A COMPLEX, comprising: -) a fusion polypeptide comprising in the N-terminal to C-terminal direction: - a virus-derived peptide that has an amino acid sequence selected from SEQ ID NO : 1 to SEQ ID NO: 9, - a first binding peptide that has an amino acid sequence selected from SEQ ID NO: 16, 17, 18, 21, 22, and 23; - a β2-microglobulin that has an amino acid sequence of SEQ ID NO: 10; - a second binding peptide that has an amino acid sequence selected from SEQ ID NO: 16, 17, 18, 21, 22 and 23; - the α1, α2 and α3 extracellular domains of a MHC class I molecule that has an amino acid sequence of SEQ ID NO: 11; and - a third binding peptide that has an amino acid sequence selected from SEQ ID NO: 12, 16, 17, 18, 21, 22 and 23; - i) a pair of disulfide-linked polypeptide chains derived from an antibody hinge region, wherein the first disulfide-linked polypeptide chain and the second disulfide-linked polypeptide chain comprise a human IgG1 CH2 domain, comprising an amino acid sequence selected from SEQ ID NO: 31, 32 and 33, and a CH3 domain of human IgG1 comprising an amino acid sequence selected from SEQ ID NO: 34, 35, and 36; and - ii) at least one pair of an antibody light chain variable domain and an antibody heavy chain variable domain in a eukaryotic cell, characterized by comprising the following steps: - cultivating a eukaryotic cell comprising one or more encoding nucleic acids the complex, and - recovering the complex from the cell or culture medium; wherein the complex comprises exactly one of said fusion polypeptide, and wherein the fusion polypeptide is: - covalently linked, both at the C-terminus and at the N-terminus, to one of the disulfide-linked polypeptide chains; - covalently linked to the N-terminus of an antibody variable domain that is complementary to the heavy or light chain variable domain to that comprised in the disulfide-linked first polypeptide chain; - covalently linked to the C-terminus of an antibody constant domain that is the constant domain of the heavy or light chain complementary to that comprised in the first disulfide-linked polypeptide chain.
[0002]
2. METHOD, according to claim 1, characterized in that the complex is obtained with a concentration of 1 mg / mL or more in the culture medium.
[0003]
METHOD according to any one of claims 1 to 2, characterized in that the eukaryotic cell is a mammalian cell.
[0004]
METHOD according to claim 3, characterized in that the mammalian cell is a human embryonic kidney cell, or a Chinese hamster ovary cell, or a baby hamster kidney cell, or a mouse myeloma cell.
[0005]
5. COMPLEX, characterized by comprising: - a fusion polypeptide comprising in the N-terminal to C-terminal direction: (i) a virus-derived peptide that has an amino acid sequence selected from SEQ ID NO: 1 to SEQ ID NO: 9, (ii) a first binding peptide that has an amino acid sequence selected from SEQ ID NO: 16, 17, 18, 21, 22, and 23; (iii) a β2-microglobulin that has an amino acid sequence of SEQ ID NO: 10; (iv) a second binding peptide that has an amino acid sequence selected from SEQ ID NO: 16, 17, 18, 21, 22 and 23; (v) the extracellular domains α1, α2 and α3 of a MHC class I molecule that has an amino acid sequence of SEQ ID NO: 11; and (vi) a third binding peptide that has an amino acid sequence selected from SEQ ID NO: 12, 16, 17, 18, 21, 22 and 23, and - two polypeptide chains, which are linked by one or further disulfide bonds, wherein the first disulfide-linked polypeptide chain comprises, in the N-terminal to C-terminal direction: (i) an immunoglobulin light or heavy chain variable domain, (ii) a light chain constant domain or heavy immunoglobulin, and (iii) a polypeptide from the hinge region of the antibody heavy chain that has an amino acid sequence selected from SEQ ID NO: 24-30 or 47-51, wherein the second disulfide-linked polypeptide chain comprises a polypeptide from the hinge region of the antibody heavy chain, where both the first and the second disulfide-linked polypeptide chain comprise a CH2 domain comprising an amino acid sequence selected from SEQ ID NO: 31, 32 and 33 and ad human CH3 domain comprising the amino acid sequence selected from SEQ ID NO: 34, 35 and 36; wherein the fusion polypeptide is: - covalently linked to the C-terminal or N-terminal end of one of the disulfide-linked polypeptide chains, or - covalently linked to the N-terminal of an antibody variable domain which is the light or heavy chain variable complementary to that comprised in the disulfide-linked first polypeptide chain, or - covalently attached to the C-terminus of an antibody constant domain which is the light or heavy chain constant domain complementary to that comprised in the first polypeptide chain linked by disulfide; with the proviso that the complex comprises exactly a fusion polypeptide.
[0006]
6. PHARMACEUTICAL FORMULATION, characterized in that it comprises the complex, as defined in claim 5, and a pharmaceutically acceptable carrier.
[0007]
7. USE OF THE COMPLEX, as defined in claim 5, characterized by being in the manufacture of a medication for the treatment of cancer or chronic viral infection.
[0008]
8. USE, according to claim 7, characterized in that the drug is for attracting virus-specific cytotoxic T cells from an individual to a target.
[0009]
9. USE, according to claim 7, characterized in that the drug is for the removal of cancer cells or cells infected by viruses.

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同族专利:

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JP6246711B2|2017-12-13|

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MA35604B1|2014-11-01|

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AU2012274127A1|2013-11-14|

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KR102038155B1|2019-10-29|

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US20160362500A1|2016-12-15|

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AU2012274127B2|2017-06-22|

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US20200157239A1|2020-05-21|

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TWI563087B|2016-12-21|

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WO2012175508A1|2012-12-27|

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US20130011394A1|2013-01-10|

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EP2723773A1|2014-04-30|

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CL2013003634A1|2014-07-11|

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JP6542303B2|2019-07-10|

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MX354663B|2018-03-14|

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CR20130609A|2014-01-09|

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TW201311893A|2013-03-16|

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EA201400046A1|2014-07-30|

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EP2723773B1|2018-02-28|

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PE20141212A1|2014-09-19|

---

CA2835772A1|2012-12-27|

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CO6801646A2|2013-11-29|

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JP2014519830A|2014-08-21|

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MX2013014869A|2014-03-31|

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引用文献:

---

公开号 | 申请日 | 公开日 | 申请人 | 专利标题

---

---

GB2095818B|1981-03-27|1985-10-02|Exxon Research Engineering Co|Staged adsorption/resorption heat pump|

---

US4816567A|1983-04-08|1989-03-28|Genentech, Inc.|Recombinant immunoglobin preparations|

---

US5238808A|1984-10-31|1993-08-24|Igen, Inc.|Luminescent metal chelate labels and means for detection|

---

IL85035D0|1987-01-08|1988-06-30|Int Genetic Eng|Polynucleotide molecule,a chimeric antibody with specificity for human b cell surface antigen,a process for the preparation and methods utilizing the same|

---

AU600575B2|1987-03-18|1990-08-16|Sb2, Inc.|Altered antibodies|

---

DE3825615A1|1988-07-28|1990-02-01|Behringwerke Ag|ANTIGENT CONSTRUCTS OF "MAJOR HISTOCOMPATIBILITY COMPLEX" CLASS I ANTIGENS WITH SPECIFIC CARRIER MOLECULES, THEIR PRODUCTION AND USE|

---

US5068088A|1988-11-03|1991-11-26|Igen, Inc.|Method and apparatus for conducting electrochemiluminescent measurements|

---

DE68928994T2|1988-11-03|1999-12-16|Igen Int Inc|ELECTROCHEMILUMINESCENT TEST PROCEDURE|

---

US5959177A|1989-10-27|1999-09-28|The Scripps Research Institute|Transgenic plants expressing assembled secretory antibodies|

---

IL100866A|1991-02-06|1995-10-31|Igen Inc|Method and apparatus for magnetic microparticulate based luminescence assay including plurality of magnets|

---

DE69233254T2|1991-06-14|2004-09-16|Genentech, Inc., South San Francisco|Humanized Heregulin antibody|

---

US7018809B1|1991-09-19|2006-03-28|Genentech, Inc.|Expression of functional antibody fragments|

---

AU691811B2|1993-06-16|1998-05-28|Celltech Therapeutics Limited|Antibodies|

---

US5789199A|1994-11-03|1998-08-04|Genentech, Inc.|Process for bacterial production of polypeptides|

---

US5840523A|1995-03-01|1998-11-24|Genetech, Inc.|Methods and compositions for secretion of heterologous polypeptides|

---

US6267958B1|1995-07-27|2001-07-31|Genentech, Inc.|Protein formulation|

---

GB9603256D0|1996-02-16|1996-04-17|Wellcome Found|Antibodies|

---

US6171586B1|1997-06-13|2001-01-09|Genentech, Inc.|Antibody formulation|

---

ES2244066T3|1997-06-24|2005-12-01|Genentech, Inc.|PROCEDURE AND COMPOSITIONS OF GALACTOSILATED GLICOPROTEINS.|

---

JP2001521909A|1997-10-31|2001-11-13|ジェネンテク・インコーポレイテッド|Methods and compositions comprising glycoprotein glycoforms|

---

CA2323757C|1998-04-02|2011-08-02|Genentech, Inc.|Antibody variants and fragments thereof|

---

US6194551B1|1998-04-02|2001-02-27|Genentech, Inc.|Polypeptide variants|

---

DK1071700T3|1998-04-20|2010-06-07|Glycart Biotechnology Ag|Glycosylation modification of antibodies to enhance antibody-dependent cellular cytotoxicity|

---

US7521197B2|1998-06-05|2009-04-21|Alexis Biotech Limited|Method for producing cytotoxic T-cells|

---

DE69930630T2|1998-06-10|2007-01-18|The Government Of The United States Of America As Represented By The Secretary, Department Of Health And Human Services|B2 microglobulin fusion proteins and high affinity variants|

---

US6040498A|1998-08-11|2000-03-21|North Caroline State University|Genetically engineered duckweed|

---

HU0104865A3|1999-01-15|2004-07-28|Genentech Inc|Polypeptide variants with altered effector function|

---

US6737056B1|1999-01-15|2004-05-18|Genentech, Inc.|Polypeptide variants with altered effector function|

---

EP2270147B2|1999-04-09|2020-07-22|Kyowa Kirin Co., Ltd.|Method for controlling the activity of immunologically functional molecule|

---

US7125978B1|1999-10-04|2006-10-24|Medicago Inc.|Promoter for regulating expression of foreign genes|

---

PT1222292E|1999-10-04|2005-11-30|Medicago Inc|METHOD FOR REGULATING THE TRANSCRIPTION OF EXOGENEOUS GENES IN THE PRESENCE OF NITROGEN|

---

WO2001029246A1|1999-10-19|2001-04-26|Kyowa Hakko Kogyo Co., Ltd.|Process for producing polypeptide|

---

EP2316950A1|2000-03-27|2011-05-04|Technion Research and Development Foundation, Ltd.|Single chain class I major histo-compatibility complexes, constructs encoding same and methods of generating same|

---

MXPA03002974A|2000-10-06|2004-05-05|Kyowa Hakko Kogyo Kk|Cells producing antibody compositions.|

---

US7064191B2|2000-10-06|2006-06-20|Kyowa Hakko Kogyo Co., Ltd.|Process for purifying antibody|

---

US6946292B2|2000-10-06|2005-09-20|Kyowa Hakko Kogyo Co., Ltd.|Cells producing antibody compositions with increased antibody dependent cytotoxic activity|

---

US20030017134A1|2001-06-19|2003-01-23|Technion Research And Development Foundation Ltd.|Methods and pharmaceutical compositions for immune deception, particularly useful in the treatment of cancer|

---

KR20040054669A|2001-08-03|2004-06-25|글리카트 바이오테크놀로지 아게|Antibody glycosylation variants having increased antibody-dependent cellular cytotoxicity|

---

DE60232265D1|2001-10-25|2009-06-18|Genentech Inc|GLYCOPROTEIN COMPOSITIONS|

---

US20040093621A1|2001-12-25|2004-05-13|Kyowa Hakko Kogyo Co., Ltd|Antibody composition which specifically binds to CD20|

---

WO2003084569A1|2002-04-09|2003-10-16|Kyowa Hakko Kogyo Co., Ltd.|Drug containing antibody composition|

---

US20050031613A1|2002-04-09|2005-02-10|Kazuyasu Nakamura|Therapeutic agent for patients having human FcgammaRIIIa|

---

EP1498490A4|2002-04-09|2006-11-29|Kyowa Hakko Kogyo Kk|Process for producing antibody composition|

---

AT503829T|2002-04-09|2011-04-15|Kyowa Hakko Kirin Co Ltd|CELL WITH REDUCED OR DELETED ACTIVITY OF A PROTEIN INVOLVED IN GDP FUCOSET TRANSPORT|

---

WO2003085119A1|2002-04-09|2003-10-16|Kyowa Hakko Kogyo Co., Ltd.|METHOD OF ENHANCING ACTIVITY OF ANTIBODY COMPOSITION OF BINDING TO FcϜ RECEPTOR IIIa|

---

JPWO2003085107A1|2002-04-09|2005-08-11|協和醗酵工業株式会社|Genome-modified cells|

---

US8895020B2|2002-04-19|2014-11-25|Washington University|Single chain trimers and uses therefor|

---

US7361740B2|2002-10-15|2008-04-22|Pdl Biopharma, Inc.|Alteration of FcRn binding affinities or serum half-lives of antibodies by mutagenesis|

---

HU227217B1|2002-12-16|2010-11-29|Genentech Inc|Immunoglobulin variants and uses thereof|

---

US7871607B2|2003-03-05|2011-01-18|Halozyme, Inc.|Soluble glycosaminoglycanases and methods of preparing and using soluble glycosaminoglycanases|

---

US20060104968A1|2003-03-05|2006-05-18|Halozyme, Inc.|Soluble glycosaminoglycanases and methods of preparing and using soluble glycosaminogly ycanases|

---

JP4473257B2|2003-04-02|2010-06-02|エフ．ホフマン−ラロシュアーゲー|Antibodies against insulin-like growth factor I receptor and uses thereof|

---

US7081432B2|2003-07-10|2006-07-25|General Electric Company|Alkylation catalyst and method for making alkylated phenols|

---

US20050042218A1|2003-07-10|2005-02-24|Vaccinex, Inc.|MHC class I - peptide-antibody conjugates with modified beta2-microglobulin|

---

WO2005035586A1|2003-10-08|2005-04-21|Kyowa Hakko Kogyo Co., Ltd.|Fused protein composition|

---

WO2005035778A1|2003-10-09|2005-04-21|Kyowa Hakko Kogyo Co., Ltd.|PROCESS FOR PRODUCING ANTIBODY COMPOSITION BY USING RNA INHIBITING THE FUNCTION OF α1,6-FUCOSYLTRANSFERASE|

---

AU2004287643C1|2003-11-05|2012-05-31|Roche Glycart Ag|CD20 antibodies with increased FC receptor binding affinity and effector function|

---

WO2005053742A1|2003-12-04|2005-06-16|Kyowa Hakko Kogyo Co., Ltd.|Medicine containing antibody composition|

---

ES2403055T3|2004-04-13|2013-05-13|F. Hoffmann-La Roche Ag|Anti-P-selectin antibodies|

---

TWI309240B|2004-09-17|2009-05-01|Hoffmann La Roche|Anti-ox40l antibodies|

---

JO3000B1|2004-10-20|2016-09-05|Genentech Inc|Antibody Formulations.|

---

US20080014203A1|2006-04-11|2008-01-17|Silke Hansen|Antibodies against insulin-like growth factor I receptor and uses thereof|

---

KR101442209B1|2006-05-19|2014-11-18|테크니온 리서치 엔드 디벨로프먼트 화운데이션 엘티디.|Fusion proteins, uses thereof and processes for producing same|

---

US20080226635A1|2006-12-22|2008-09-18|Hans Koll|Antibodies against insulin-like growth factor I receptor and uses thereof|

---

US20090162359A1|2007-12-21|2009-06-25|Christian Klein|Bivalent, bispecific antibodies|

---

US8242247B2|2007-12-21|2012-08-14|Hoffmann-La Roche Inc.|Bivalent, bispecific antibodies|

---

US8227577B2|2007-12-21|2012-07-24|Hoffman-La Roche Inc.|Bivalent, bispecific antibodies|

---

US9266967B2|2007-12-21|2016-02-23|Hoffmann-La Roche, Inc.|Bivalent, bispecific antibodies|

---

BRPI1014089A2|2009-04-02|2016-04-19|Roche Glycart Ag|multispecific antibodies comprising full length antibodies and single chain fab fragments|

---

EP2417156B1|2009-04-07|2015-02-11|Roche Glycart AG|Trivalent, bispecific antibodies|

---

EP2435473B1|2009-05-27|2013-10-02|F.Hoffmann-La Roche Ag|Tri- or tetraspecific antibodies|

---

US9676845B2|2009-06-16|2017-06-13|Hoffmann-La Roche, Inc.|Bispecific antigen binding proteins|

---

US8703132B2|2009-06-18|2014-04-22|Hoffmann-La Roche, Inc.|Bispecific, tetravalent antigen binding proteins|CN104781279A|2012-11-30|2015-07-15|罗切格利卡特公司|Removal of cancer cells by circulating virus-specific cytotoxic t-cells using cancer cell targeted MHC class I comprising multi-function proteins|

---

EP3640375A3|2012-12-11|2020-07-29|Albert Einstein College of Medicine|Methods for high throughput receptor/ligand identification|

---

SG11201504414UA|2012-12-21|2015-07-30|Hoffmann La Roche|Disulfide-linked multivalent mhc class i comprising multi-function proteins|

---

EA035322B1|2013-05-28|2020-05-28|ДиЭсБи-ЮЭсЭй ЛЛК|Antibody locker for the inactivation of protein drug|

---

EP3013361B1|2013-06-24|2021-10-06|NexImmune, Inc.|Compositions and methods for immunotherapy|

---

BR112016014913A8|2013-12-23|2020-06-09|Zymeworks Inc|antibody, or an antigen-binding fragment thereof, conjugate, nucleic acid, vector, host cell, pharmaceutical composition, method of preparing an antibody light chain or antigen-binding fragment, and method of preparing a conjugate|

---

KR20160144430A|2014-04-10|2016-12-16|시애틀 칠드런즈 호스피탈 디/비/에이 시애틀 칠드런즈 리서치 인스티튜트|Transgene genetic tags and methods of use|

---

WO2016141284A1|2015-03-04|2016-09-09|The Johns Hopkins University|Compositions and methods for enhancing an immune response|

---

WO2017027291A1|2015-08-07|2017-02-16|Seattle Children's Hospital |Bispecific car t-cells for solid tumor targeting|

---

US10730908B2|2016-05-11|2020-08-04|Ge Healthcare Bioprocess R&D Ab|Separation method|

---

US10889615B2|2016-05-11|2021-01-12|Cytiva Bioprocess R&D Ab|Mutated immunoglobulin-binding polypeptides|

---

US10654887B2|2016-05-11|2020-05-19|Ge Healthcare Bio-Process R&D Ab|Separation matrix|

---

US10513537B2|2016-05-11|2019-12-24|Ge Healthcare Bioprocess R&D Ab|Separation matrix|

---

US10995152B2|2016-10-26|2021-05-04|The Board Of Trustees Of The Leland Stanford Junior University|Modified immunoglobulin hinge regions to reduce hemagglutination|

---

EP3558339A4|2016-12-22|2020-08-19|Cue Biopharma, Inc.|T-cell modulatory multimeric polypeptides and methods of use thereof|

---

WO2018170168A1|2017-03-15|2018-09-20|Cue Biopharma, Inc.|Methods for modulating an immune response|

---

WO2020136060A1|2018-12-28|2020-07-02|F. Hoffmann-La Roche Ag|A peptide-mhc-i-antibody fusion protein for therapeutic use in a patient with amplified immune response|

---

WO2021027200A1|2019-08-13|2021-02-18|Cure Genetics Co., Ltd.|Genetically engineered cells and uses thereof|

---

CN112285083B|2020-10-28|2022-01-07|上海睿钰生物科技有限公司|Method for evaluating cell killing efficacy|

法律状态:
2018-01-23| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|

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2018-03-27| B15K| Others concerning applications: alteration of classification|Ipc: C07K 16/46 (2006.01), A61K 47/00 (2006.01), C07K 1 |

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2018-04-03| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2019-07-02| B07E| Notice of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|Free format text: NOTIFICACAO DE ANUENCIA RELACIONADA COM O ART 229 DA LPI |

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2019-10-29| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2020-05-05| B06A| Notification to applicant to reply to the report for non-patentability or inadequacy of the application [chapter 6.1 patent gazette]|

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2020-11-10| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2021-02-02| B16A| Patent or certificate of addition of invention granted|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 19/06/2012, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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EP11171027|2011-06-22|

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EP11171027.3|2011-06-22|

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PCT/EP2012/061734|WO2012175508A1|2011-06-22|2012-06-19|Removal of target cells by circulating virus-specific cytotoxic t-cells using mhc class i comprising complexes|

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