WO2022210485A1 - 安定な多重特異性分子及びその利用 - Google Patents
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Definitions
- the present invention relates to multispecific antibodies, multispecific molecules, components thereof, etc. that recognize two or more antigens.
- bispecific antibodies and multispecific antibodies As molecules that bind to multiple targets, research is underway on antibodies that bind to two or more different antigens with one molecule (bispecific antibodies and multispecific antibodies).
- bispecific antibodies As an example of the use of bispecific antibodies, a single molecule recognizes CD3 on T cells and a target antigen expressed on the surface of cancer cells, thereby bridging T cells and cancer cells. and T cell-redirecting bispecific antibodies that induce cytotoxicity to.
- Blinatumomab a representative example, is a bispecific antibody that binds CD3 and CD19, consisting of BiTETM types (scFv-scFv types), used in the United States for relapsed or refractory B-cell non-Hodgkin's disease in adults and children. Approved for the treatment of lymphoma and acute lymphoblastic leukemia.
- Non-Patent Document 1 Blinatumomab has a short half-life because it is a BiTE (trademark) type, and under certain experimental conditions, there are many high molecular weights species (HMWS) compared to antibodies approved as other drugs, and stability is an issue. It was suggested (Non-Patent Document 1).
- HMWS high molecular weights species
- An object of the present invention is to provide a bispecific molecule that has excellent solution stability and can reduce high molecular weight species (HMWS) containing dimers when stored in a solution state.
- HMWS high molecular weight species
- the present inventors have conducted intensive research to solve the above problems, created a novel bispecific molecule containing anti-CD3scFv or anti-CD3Fab, and completed the present invention.
- the present invention includes the following inventions.
- [2] comprising a heavy chain variable region consisting of amino acid numbers 1 to 118 of the amino acid sequence shown in SEQ ID NO: 1 and a light chain variable region consisting of amino acid numbers 1 to 107 of the amino acid sequence shown in SEQ ID NO: 2,
- the Fab of [1] or [2], comprising a polypeptide consisting of the amino acid sequence of SEQ ID NO:1 and a light chain consisting of the amino acid sequence of SEQ ID NO:2.
- [4] A multiplex comprising any one of the Fabs of [1] to [3] and specifically binding to a target molecule that is not a complex of a cancer testis antigen peptide and a major histocompatibility gene complex (MHC) Specificity molecule.
- MHC major histocompatibility gene complex
- [5] The multispecific molecule of [4], wherein said MHC is human leukocyte antigen (HLA).
- HLA human leukocyte antigen
- the multispecific molecule of [4] or [5] comprising an antibody or antigen-binding fragment thereof that specifically binds to the target molecule.
- a multispecific molecule according to any one of [4] to [11], comprising one polypeptide group selected from the following (i) to (iii): (i) a polypeptide comprising an amino acid sequence consisting of amino acid residues 20 to 718 of SEQ ID NO: 29 or an amino acid sequence in which 1 or 2 amino acids are deleted at the carboxyl terminus of said amino acid sequence, 20 to SEQ ID NO: 9; A polypeptide comprising an amino acid sequence consisting of the 246th amino acid residue or an amino acid sequence in which 1 or 2 amino acids are deleted at the carboxyl terminus of the amino acid sequence and amino acids consisting of the 21st to 233rd amino acid residues of SEQ ID NO: 12 a polypeptide comprising a sequence; (ii) a polypeptide comprising an amino acid sequence consisting of amino acid residues 20 to 717 of SEQ ID NO: 30 or an amino acid sequence in which 1 or 2 amino acids are deleted at the carboxyl terminus of said amino acid sequence, 20 to SEQ ID
- a polynucleotide comprising a nucleotide sequence encoding an amino acid sequence contained in the multispecific molecule of any one of [4] to [12].
- a vector comprising the polynucleotide of [13].
- a method for producing the multispecific molecule of any one of [4] to [12] which comprises culturing the cell of [15].
- a multispecific molecule obtained by the method of [16].
- [20] comprising a heavy chain variable region consisting of amino acid numbers 1 to 118 of the amino acid sequence shown in SEQ ID NO:3 and a light chain variable region consisting of amino acid numbers 134 to 240 of the amino acid sequence shown in SEQ ID NO:3, [19] scFv.
- [22] comprising a heavy chain variable region consisting of amino acid numbers 1 to 118 of the amino acid sequence shown in SEQ ID NO:39 and a light chain variable region consisting of amino acid numbers 134 to 240 of the amino acid sequence shown in SEQ ID NO:39, [19] scFv.
- a multiplex comprising any one scFv of [19] to [23] and specifically binding to a target molecule that is not a complex of a cancer testis antigen peptide and major histocompatibility complex (MHC) Specificity molecule.
- MHC major histocompatibility complex
- HLA human leukocyte antigen
- the multispecific molecule of [24] or [25] comprising an antibody or antigen-binding fragment thereof that specifically binds to the target molecule.
- a multispecific molecule according to any one of [24] to [31], comprising one polypeptide group selected from the following (i) to (iii): (i) a polypeptide comprising an amino acid sequence consisting of amino acid residues 20 to 744 of SEQ ID NO: 21 or an amino acid sequence in which 1 or 2 amino acids are deleted at the carboxyl terminus of said amino acid sequence, and 20 to SEQ ID NO: 9; A polypeptide comprising an amino acid sequence consisting of the 246th amino acid residue or an amino acid sequence in which 1 or 2 amino acids are deleted at the carboxyl terminus of said amino acid sequence; (ii) a polypeptide comprising an amino acid sequence consisting of amino acid residues 20 to 743 of SEQ ID NO: 23 or an amino acid sequence in which 1 or 2 amino acids are deleted at the carboxyl terminus of said amino acid sequence, and 20 to SEQ ID NO: 9; A polypeptide comprising an amino acid sequence consisting of the 246th amino acid residue or an amino acid sequence in which 1
- a polynucleotide comprising a base sequence encoding an amino acid sequence contained in the multispecific molecule of any one of [24] to [32].
- a vector comprising the polynucleotide of [33].
- a cell comprising the polynucleotide of [33] or the vector of [34], or producing the multispecific molecule of any one of [24]-[32].
- [36] A method for producing the multispecific molecule according to any one of [24] to [32], comprising culturing the cell according to [35].
- a multispecific molecule obtained by the method of [36].
- [40] comprising a heavy chain variable region consisting of amino acid numbers 1 to 118 of the amino acid sequence shown in SEQ ID NO:46 and a light chain variable region consisting of amino acid numbers 1 to 109 of the amino acid sequence shown in SEQ ID NO:47, [39] Fab.
- a multiplex comprising any one of the Fabs of [39] to [41] and specifically binding to a target molecule that is not a complex of a cancer testis antigen peptide and major histocompatibility complex (MHC) Specificity molecule.
- MHC major histocompatibility complex
- HLA human leukocyte antigen
- a polynucleotide comprising a base sequence encoding an amino acid sequence contained in any one of the multispecific molecules of [42] to [46].
- a vector comprising the polynucleotide of [47].
- a cell comprising the polynucleotide of [47] or the vector of [48], or producing the multispecific molecule of any one of [42]-[46].
- a multispecific molecule obtained by the method of [50].
- a scFv that specifically binds to CD3 and comprises disulfide-bonded heavy and light chain variable regions including the following CDRH1-3 and CDRL1-3: CDRH1 consisting of the amino acid sequence shown in SEQ ID NO: 40; CDRH2 consisting of the amino acid sequence shown in SEQ ID NO: 41; CDRH3 consisting of the amino acid sequence shown in SEQ ID NO: 42; CDRL1 consisting of the amino acid sequence shown in SEQ ID NO: 43; CDRL2 consisting of the amino acid sequence shown in SEQ ID NO:44; and CDRL3 consisting of the amino acid sequence shown in SEQ ID NO:45.
- [54] comprising a heavy chain variable region consisting of amino acid numbers 1 to 118 of the amino acid sequence shown in SEQ ID NO:48 and a light chain variable region consisting of amino acid numbers 134 to 242 of the amino acid sequence shown in SEQ ID NO:48, [53] scFv.
- [56] comprising a heavy chain variable region consisting of amino acid numbers 1 to 118 of the amino acid sequence shown in SEQ ID NO:49 and a light chain variable region consisting of amino acid numbers 134 to 242 of the amino acid sequence shown in SEQ ID NO:49, [53] scFv.
- [57] The scFv of [53] or [56], which consists of the amino acid sequence shown in SEQ ID NO:49.
- a multiplex comprising any one scFv of [53] to [57] and specifically binding to a target molecule that is not a complex of a cancer testis antigen peptide and major histocompatibility complex (MHC) Specificity molecule.
- MHC major histocompatibility complex
- HLA human leukocyte antigen
- the multispecific molecule of [58] or [59] comprising an antibody or antigen-binding fragment thereof that specifically binds to the target molecule.
- [62] The multispecific molecule of any one of [58]-[61], which is a bispecific antibody.
- a polynucleotide comprising a nucleotide sequence encoding an amino acid sequence contained in the multispecific molecule of any one of [58] to [62].
- a vector comprising the polynucleotide of [63].
- a cell comprising the polynucleotide of [63] or the vector of [64], or producing the multispecific molecule of any one of [58]-[62].
- [66] A method for producing the multispecific molecule of any one of [58] to [62], which comprises culturing the cell of [65]. [67] A multispecific molecule obtained by the method of [66]. [68] The scFv of any one of [53] to [57], the multispecific molecule of any one of [58] to [62], the polynucleotide of [63], the vector of [64], or [ 65]. [69] A pharmaceutical composition comprising the multispecific molecule of any one of [4]-[12], [24]-[32], [42]-[46] and [58]-[62].
- a CD3-binding scFv in which a heavy chain variable region and a light chain variable region are bound by a disulfide bond or a CD3-binding Fab, a cancer testis antigen peptide and a human leukocyte antigen (HLA) Novel bispecific antibodies (bispecific molecules) are obtained that bind to target molecules that are not complexed with The antibody has high solution stability and can be stored in solution without deterioration.
- FIG. 10 is a diagram showing changes over time in HMWS (%) of C3E-0002 to 0004.
- FIG. FIG. 2A is a diagram showing changes over time in HMWS (%) due to shaking deterioration of C3E-0002 to 0004, and
- FIG. 2B is a diagram showing changes over time in peak areas due to shaking deterioration of C3E-0002 to 0004.
- FIG. 3A is a diagram showing HMWS (%) changes over time for RX3-0001 to 0003
- FIG. 3B is a diagram showing HMWS (%) changes over time for CX3-0001 to 0003
- FIG. 10 is a graph showing HMWS (%) changes over time for 0001 to 0003;
- Figure 4-1A is a diagram showing the temporal change in HMWS (%) due to shaking deterioration of RX3-0001 to 0003, and
- Figure 4-1B shows the temporal change in peak area due to shaking deterioration of RX3-0001 to 0003.
- FIG. 4 is a diagram showing;
- Figure 4-2A is a diagram showing the change in HMWS (%) over time due to shaking deterioration of CX3-0001 to 0003, and
- Figure 4-2B shows the change over time in the peak area due to shaking deterioration of CX3-0001 to 0003.
- FIG. 4 is a diagram showing;
- Figure 4-3A is a diagram showing the time-dependent change in HMWS (%) due to shaking deterioration of TX3-0001 to 0003, and
- Figure 4-3B shows the time-dependent change in the peak area due to shaking deterioration of TX3-0001 to 0003.
- FIG. 4 is a diagram showing;
- Figure 4-3A is a diagram showing the time-dependent change in HMWS (%) due to shaking deterioration of TX3-0001 to 0003, and
- Figure 4-3B shows the time-dependent change in the peak area due to shaking deterioration of TX3-0001 to 0003.
- FIG. 4 is a diagram showing; Amino acid sequence of C3E-7085-Fab (SEQ ID NO: 1) Amino acid sequence of C3E-7085-LC (SEQ ID NO: 2) Amino acid sequence of C3E-7096 (SEQ ID NO: 3) HC_h full length nucleotide sequence (SEQ ID NO: 4) C3E-7085-HC-k full length nucleotide sequence (SEQ ID NO: 5) C3E-7085-Fab-HC-k full length nucleotide sequence (SEQ ID NO: 6) C3E-7085-LC full length nucleotide sequence (SEQ ID NO: 7) C3E-7096-HC-k full length nucleotide sequence (SEQ ID NO: 8) HC_h full-length amino acid sequence (SEQ ID NO: 9) C3E-7085-HC-k full length amino acid sequence (SEQ ID NO: 10) C3E-7085-Fab-HC-k full length amino acid sequence (
- FIG. 1 shows a bispecific molecule comprising a Fab that binds to CD3 and an X that binds to something other than CD3 (“another binding moiety”).
- FIG. 10 is a diagram showing tandem scFv (taFv) in which scFv that binds to CD3 and scFv that binds to non-CD3 are bound via a linker.
- the orientation of each scFv may be reversed, with the order of linkage of the VH and VL domains reversed.
- FIG. 2 shows scFv-Fab, in which Fab that binds to CD3 and scFv that binds to other than CD3 are bound via a linker.
- the orientation of each scFv may be reversed, with the order of linkage of the VH and VL domains reversed.
- FIG. 1 shows a multispecific molecule comprising a scFv that binds CD3 and X (“other binding moieties”) and Y (“other binding moieties” or “other groups”) that binds other than CD3.
- the orientation of the scFv indicated by the upper right hatched line may be reversed, in which the order of linkage of the VH and VL domains is reversed.
- X and Y may be various antibody fragments such as scFv, VHH, Fab, or whole antibodies.
- a disulfide-bonded scFv may be composed of VH and VL without a linker.
- a trispecific molecule results when Y is the "other binding moiety".
- X and Y can be various antibody fragments such as scFv, VHH, Fab, whole antibodies, or molecules other than immunoglobulins.
- a trispecific molecule results when Y is the "other binding moiety”.
- An Fc-added bispecific molecule comprising a CD3-binding scFv in which a heavy chain variable region and a light chain variable region are disulfide-bonded (upper right hatched line) and an X (“another binding moiety”) that binds to a substance other than CD3
- Fc is a format in which the first polypeptide indicated by the hatched upper left and the second polypeptide indicated by the solid line are associated (Fc may be wild-type or mutant).
- the orientation of the scFv indicated by the upper right hatched line may be reversed, in which the order of linkage of the VH and VL domains is reversed.
- the positions of X and scFv in each figure may be interchanged.
- FIG. 3 shows an Fc-attached bispecific molecule comprising a CD3-binding scFv in which the heavy chain variable region and the light chain variable region are disulfide-bonded (upper right hatched line) and an Fab or scFv that binds to something other than CD3.
- Fc is a format in which the first polypeptide indicated by the hatched upper left and the second polypeptide indicated by the solid line are associated (Fc may be wild-type or mutant). The orientation of each scFv may be reversed, with the order of linkage of the VH and VL domains reversed.
- the lower left figure also shows the taFv-heterodimeric Fc form (if the Fc is a heterodimer).
- FIG. 1 shows an Fc-tagged multispecific molecule comprising the group of ).
- Fc is a format in which the first polypeptide indicated by the hatched upper left and the second polypeptide indicated by the solid line are associated (Fc may be wild-type or mutant).
- the orientation of the scFv indicated by the upper right hatched line may be reversed, in which the order of linkage of the VH and VL domains is reversed.
- a trispecific molecule results when Y is the "other binding moiety".
- Fc-added trispecific molecule comprising a CD3-binding scFv in which a heavy chain variable region and a light chain variable region are disulfide-bonded (upper right hatched line) and two antibody fragments (Fab or scFv) that bind to other than CD3
- Fab or scFv two antibody fragments
- Fc is a format in which the first polypeptide indicated by the hatched upper left and the second polypeptide indicated by the solid line are associated (Fc may be wild-type or mutant).
- the orientation of each scFv may be reversed, in which the order of linkage of the VH and VL domains is reversed.
- FIG. 1 shows an Fc-tagged multispecific molecule comprising ).
- Fc is a format in which the first polypeptide indicated by the hatched upper left and the second polypeptide indicated by the solid line are associated (Fc may be wild-type or mutant).
- the orientation of the scFv indicated by the upper right hatched line may be reversed, in which the order of linkage of the VH and VL domains is reversed.
- FIG. 1 shows an Fc-tagged bispecific molecule comprising a Fab that binds to CD3 and an X (another binding moiety) that binds to something other than CD3.
- Fc is a format in which the first polypeptide indicated by the hatched upper left and the second polypeptide indicated by the solid line are associated (Fc may be wild-type or mutant).
- Fc is a format in which the first polypeptide indicated by the hatched upper left and the second polypeptide indicated by the solid line are associated (Fc may be wild-type or mutant).
- the upper left figure also shows the scFv-Fab-heterodimeric Fc form (if the Fc is a heterodimer).
- the orientation of each scFv may be reversed, with the order of linkage of the VH and VL domains reversed.
- Fc-tagged multispecific molecule comprising a Fab that binds to CD3 and X (an “other binding moiety") and Y (an “other binding moiety” or “another group”) that binds to something other than CD3. It is a diagram. Fc is a format in which the first polypeptide indicated by the hatched upper left and the second polypeptide indicated by the solid line are associated (Fc may be wild-type or mutant). A trispecific molecule results when Y is the "other binding moiety".
- An Fc-tagged trispecific molecule (a bispecific molecule if the two antibody fragments are the same) comprising a CD3-binding Fab and a non-CD3-binding antibody fragment (scFV or Fab).
- Fc is a format in which the first polypeptide indicated by the hatched upper left and the second polypeptide indicated by the solid line are associated (Fc may be wild-type or mutant). The orientation of each scFv may be reversed by interchanging the order of linkage of the VH and VL domains.
- Fabs that bind to CD3 and "other binding moieties” or “other groups” that bind to non-CD3 denoted by X, Y and Z, respectively. At least one of X, Y and Z is the "other binding moiety").
- FIG. 1 shows an Fc-tagged multispecific molecule comprising ).
- Fc is a format in which the first polypeptide indicated by the hatched upper left and the second polypeptide indicated by the solid line are associated (Fc may be wild-type or mutant).
- Two or three of X, Y and Z are "other binding moieties" resulting in trispecific or tetraspecific molecules, respectively.
- Amino acid sequence of C3E-7097 (SEQ ID NO:39) Amino acid sequences of CDRH1-CDRH3 of the C3E-7093 heavy chain and CDRL1-L3 of the light chain (SEQ ID NOs:40-45) Amino acid sequence of C3E-7093-Fab (SEQ ID NO:46) Amino acid sequence of C3E-7093-LC (SEQ ID NO: 47) Amino acid sequence of C3E-7098 (SEQ ID NO:48) Amino acid sequence of C3E-7099 (SEQ ID NO:49)
- X, Y, and Z appear to abut the ends of adjacent moieties, but attachment or fusion to the ends is not essential.
- X, Y and Z are (appear to be) adjacent to Fc
- said X, Y and Z are bound or fused to the N-terminus or C-terminus of Fc, the N-terminus of Fc or It may be bound to a portion other than the C-terminus, or bound or fused to a sugar chain added to an amino acid contained in Fc.
- non-bonded or fused ends of adjacent moieties are not limited to these.
- the term "gene” means a nucleotide chain containing a nucleotide sequence encoding an amino acid of a protein or a complementary strand thereof.
- genes are included within the meaning of "gene.” Such genes are single-stranded, double-stranded, or triple- or more-stranded nucleotides, and are aggregates of DNA strands and RNA strands, and ribonucleotides (RNA) and deoxyribonucleotides (DNA) are mixed on one nucleotide strand. Also included within the meaning of “gene” are double-stranded or triple-stranded or more nucleotides containing such nucleotide chains. In the present invention, base sequence and nucleotide sequence are synonymous.
- polynucleotide In the present invention, "polynucleotide”, “nucleotide chain”, “nucleic acid” and “nucleic acid molecule” are synonymous, and for example, DNA, RNA, probes, oligonucleotides, primers, etc. are also included in the meaning of “polynucleotide”. .
- Such polynucleotides are single-stranded, double-stranded, or three or more-stranded polynucleotides, including associations of DNA strands and RNA strands, and ribonucleotides (RNA) and deoxyribonucleotides (RNA) on a single polynucleotide strand.
- RNA ribonucleotides
- RNA deoxyribonucleotides
- mixtures of DNA and double-stranded or three or more-stranded aggregates comprising such polynucleotide strands.
- polypeptide In the present invention, "polypeptide”, “peptide” and “protein” are synonymous.
- antigen may be used to mean “immunogen”.
- cells include various cells derived from individual animals, subcultured cells, primary cultured cells, cell lines, recombinant cells, microorganisms, and the like.
- antibody is used to mean an immunoglobulin having a constant region and a variable region. Antibodies are not particularly limited, whether they are naturally occurring immunoglobulins or partially or wholly synthetically produced immunoglobulins.
- the basic four-chain antibody structure consists of two identical light chains (L chains) and two identical heavy chains (H chains).
- a light chain is linked to a heavy chain by one disulfide bond.
- the two heavy chains are held together by one or more disulfide bonds depending on the heavy chain isotype.
- Each light and heavy chain has regularly spaced intrachain disulfide bonds.
- Heavy and light chains have a constant region with very high amino acid sequence similarity and a variable region with low amino acid sequence similarity.
- Light chains have at the amino terminus a variable region (VL) followed by a constant region (CL).
- the heavy chain has at the amino terminus a variable region (VH) followed by three constant regions (CH1/CH2/CH3).
- VL and VH are paired and CL is aligned with the first constant region (CH1) of the heavy chain.
- VL and VH pair to form a single antigen-binding site.
- the constant region of the antibody of the present invention is not particularly limited, but the constant region of a human antibody is preferably used as the antibody of the present invention for treating or preventing human diseases.
- Heavy chain constant regions of human antibodies include, for example, C ⁇ 1, C ⁇ 2, C ⁇ 3, C ⁇ 4, C ⁇ , C ⁇ , C ⁇ 1, C ⁇ 2, and C ⁇ .
- Light chain constant regions of human antibodies include, for example, C ⁇ and C ⁇ .
- a Fab consists of a heavy chain VH followed by CH1, and a light chain VL followed by CL.
- VH and VL contain complementarity determining regions (CDRs).
- CDRs complementarity determining regions
- Fc (also referred to as Fc region) is the carboxyl-terminal region of the heavy chain constant region, comprising CH2 and CH3, and is a dimer.
- the Fc of the present invention may be a native sequence Fc or a mutant Fc in which the native sequence is mutated (referred to as "mutant Fc"), the multispecific molecule of the present invention and In the bispecific molecule, preferred Fc regions are variant Fc's, more preferably a pair of Fc's capable of forming heterodimers.
- mutant Fc a mutant Fc in which the native sequence is mutated
- preferred Fc regions are variant Fc's, more preferably a pair of Fc's capable of forming heterodimers.
- a combination of Fc (i) contained in the first polypeptide and Fc (ii) contained in the second polypeptide, which will be described later, can be mentioned. dimer formation), it is not limited to them.
- variant Fc examples include modified Fc regions (including heterodimeric Fc regions) contained in heteromultimers with improved stability disclosed in WO2013/063702, heterodimeric multimers disclosed in WO96/27011 Substitution of one or more amino acid residues with charged amino acids as disclosed in WO 2009/089004, an Fc comprising the CH3 region of an immunoglobulin derived from an IgG antibody with "protrusions” and “cavities” contained in the body to heterodimers using conformational and/or pI (isoelectric point) mutations as disclosed in WO 2014/110601 Included heterodimeric Fc regions, heterodimeric Fc comprising a CH3 domain containing a modification that abolishes or reduces binding to protein A disclosed in WO2010/151792, and the like, but these is not limited to
- variable region consists of regions of extreme variability, called hypervariable regions (HVR), and relatively invariant regions, called framework regions (FR), separated by these regions.
- HVR hypervariable regions
- FR framework regions
- Natural heavy and light chain variable regions comprise four FRs joined by three hypervariable regions, each chain's hypervariable region being held in close proximity with the other chain's hypervariable region by the FRs; It contributes to the formation of the antigen-binding site of antibodies.
- CDRs complementary determining regions
- Complementarity-determining regions also called hypervariable regions, are sites in the heavy and light chain variable regions of an antibody that exhibit particularly high variability in the primary structure. On the primary structure, each is usually separated into three places.
- the complementarity determining region of an antibody the heavy chain complementarity determining region is denoted as CDRH1, CDRH2, and CDRH3 from the amino terminal side of the heavy chain amino acid sequence, and the light chain complementarity determining region is denoted as light chain amino acid sequence.
- CDRL1, CDRL2 and CDRL3 are designated from the amino terminal side of the sequence.
- the positions and lengths of CDRs were determined according to the IMGT definition (Developmental and Comparative Immunology 27 (2003) 55-77).
- FR is a variable region other than CDR residues.
- a variable region generally has four FRs, FR1, FR2, FR3 and FR4.
- the CDRs and FRs contained in the heavy and light chains are, from amino-terminus to carboxyl-terminus, FRH1-CDRH1-FRH2-CDRH2-FRH3-CDRH3-FRH4 and FRL1-CDRL1-FRL2-CDRL2-FRL3-CDRL3- FRL4, and so on.
- CDRs and FRs can also be determined by various definitions known in the art, such as Kabat, Chothia, AbM, contact, etc., in addition to IMGT.
- the "site" to which an antibody binds that is, the "site” recognized by an antibody means a partial peptide or partial conformation on an antigen that is bound or recognized by an antibody.
- mutant antibody refers to an amino acid that is substituted, deleted, or added (addition includes insertion) (hereinafter collectively referred to as "mutation") in the amino acid sequence of the original antibody.
- the number of mutated amino acids in such mutated antibodies is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 40 or 50.
- Such mutant antibodies are also included in the "antibody” of the present invention.
- molecule refers to the above-described antibody, a molecule comprising an antigen-binding fragment of an antibody, and further includes a multispecific molecule formed from an antibody or a plurality of antigen-binding fragments derived therefrom. .
- molecules that are multispecific are synonymous and refer to multiple different epitopes on one molecule, and/or , is not particularly limited as long as it is capable of binding to different epitopes on two or more molecules.
- Molecules that are multispecific also include antibodies comprising a heavy chain variable region (VH) and a light chain variable region (VL).
- VH heavy chain variable region
- VL light chain variable region
- multispecific molecules may also be referred to as “multispecific antibodies”, “multispecific antibodies” and the like.
- “Recognizing two or more antigens” means "binding to multiple different epitopes on one molecule” or “binding to different epitopes on two or more molecules”.
- an aggregate is formed by gathering proteins, and the analytical method that can be detected differs depending on the particle size.
- Aggregates with a particle size of about 100 nm or less can be evaluated by size exclusion chromatography such as HPLC.
- Size exclusion chromatography such as HPLC.
- Subvisible particles with a particle size of 100 nm or more (particle size of 0.1 to 10 ⁇ m) and insoluble fine particles (particle size of 10 ⁇ m or more) can be evaluated using the Microflow Imaging method or the like.
- HMWS is an abbreviation for high molecular weight species, and the chromatogram obtained by size fractionation using size exclusion chromatography or the like has a larger size than the main peak fraction of the target protein. It is a generic term for fractionated protein aggregate fractions. "HMWS (%)” refers to subjecting a solution sample containing the target protein to size exclusion chromatography such as HPLC, monitoring the elution pattern at a detection wavelength of 280 nm, and occupying the total peak area in the obtained chromatogram. It means a value obtained by calculating the ratio of the area of the peak fraction appearing on the polymer side from the main peak fraction of the target protein by the percentage method. 2. Antigen 2-1. CD3 Antigen In the present invention, "CD3" is used synonymously with CD3 protein.
- CD3 is expressed on T cells as part of the multimolecular T-cell receptor complex, and consists of five polypeptides, ⁇ -chain, ⁇ -chain, ⁇ -chain, ⁇ -chain and ⁇ -chain (with molecular weights of 25000-28000, 21000, 20000, 16000, 22000).
- the CD3 complex includes ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ chains. These are also called subunits. Binding of anti-CD3 antibodies to T cells induces cytotoxicity through T cell activation. Many anti-CD3 antibodies bind to CD3 ⁇ .
- the nucleotide sequence of the cDNA encoding human CD3 ⁇ is provided by NCBI/GenBank with accession number: NM_000733 (NM_000733.3), and the amino acid sequence of human CD3 ⁇ is provided by NCBI/GenPept with accession number: NP_000724 (NM_000724.1). Registered.
- the nucleotide sequence of cDNA encoding cynomolgus monkey CD3 is registered in GenBank under accession number: NM_001283615.1. 2-2.
- Target Molecule In the present invention, the target molecule is not particularly limited as long as it is a molecule other than CD3 that exists in a living body.
- CD3-expressing T cell It is a molecule that is expressed on the surface of a CD3-expressing T cell and causes the cancer cell to exhibit cytotoxic activity by redirection of CD3-expressing T cells, more preferably a cancer testis antigen peptide and a major It is not a histocompatibility complex (MHC) complex.
- MHC histocompatibility complex
- a cancer testis antigen peptide is one type of cancer cell surface antigen peptide, and includes, for example, MAGE peptide, XAGE peptide, LAGE family peptide, and the like.
- MHC includes human leukocyte antigen (HLA), mouse H-2, etc.
- HLA is classified into class 1 and class 2, class 1 is HLA-A, HLA-B and HLA-C, class 2 is HLA-DR , HLA-DP and HLA-DQ, respectively.
- HLA human leukocyte antigen
- HLA-A2 human leukocyte antigen
- MAGE-A3, A4 A10 or LAGE peptide (e.g., UniProtKB -P78358 (amino acid numbers 157-165 of CTG1B HUMAN)), HLA-A2/MAGE-A3, A4, A10 or HLA-A2/LAGE, which are ternary complexes.
- suitable target molecules that are not complexes of cancer testis antigen peptide and human leukocyte antigen preferably include cancer antigen peptides or cancer antigens, specifically WT1, gp100, p53, KRAS, CD19, CD20, CD33, CD37, CD38, CD98, CD123, EGFR, HER2, HER3, HER4, EpCAM, CEA, PSMA, B7-H3, Trop-2, BCMA, GPRC5D and the like.
- target molecules for stromal cells include FAP, CDH17, LRRC15, and the like.
- CD33 can be exemplified as a target molecule for bone marrow-derived immunosuppressive cells (MDSC). 2-3.
- Preparation of Antigens The above-described antigen proteins used in the present invention; It can be prepared by purification and isolation from tissue-derived cells or cell cultures, genetic recombination, in vitro translation, chemical synthesis and the like.
- the cDNA of the antigen protein can be obtained by polymerase chain reaction (hereinafter referred to as "PCR") using primers that specifically amplify the cDNA of the antigen protein, for example, using a cDNA library of an organ expressing the mRNA of the antigen protein as a template. ”) (Saiki, RK, et al., Science (1988) 239, 487-49).
- Encoding polynucleotides are also included in the cDNA of the antigen protein.
- a splicing variant transcribed from the antigen protein locus expressed in humans or rats, or a polynucleotide that hybridizes thereto under stringent conditions and has biological activity equivalent to that of the antigen protein.
- a polynucleotide encoding a protein is also included in the cDNA of the antigen protein.
- the antigen protein consists of an amino acid sequence in which one to several amino acids are substituted, deleted or added in the amino acid sequence of the human or rat antigen protein, or in the amino acid sequence obtained by removing the signal sequence from these sequences, Also included in the nucleotide sequence of the antigen protein gene is a nucleotide sequence encoding a protein having biological activity equivalent to that of .
- a target molecule that is not a complex of cancer testis antigen peptide and human leukocyte antigen (HLA) can also be prepared in a similar manner.
- 2-4. Binding Specificity to Antigen Protein The anti-CD3 antibody, binding fragment thereof, etc. contained in the multispecific molecule of the present invention recognizes, that is, binds to the CD3 antigen.
- Such anti-CD3 antibodies and the like preferably bind to human CD3, monkey CD3 and the like, more preferably to human CD3 and cynomolgus monkey CD3.
- such preferred anti-CD3 antibodies do not bind to rat and/or mouse CD3.
- a target molecule contained in the multispecific molecule of the present invention preferably an antibody against a target molecule that is not a complex of an anticancer testis antigen peptide and a human leukocyte antigen (HLA), and a binding fragment thereof Recognizes, ie binds, molecules.
- HLA human leukocyte antigen
- binding means binding other than nonspecific adsorption.
- a dissociation constant hereinafter referred to as “KD”.
- the KD value for antigen proteins such as the preferred antibodies of the present invention is 1 ⁇ 10 ⁇ 5 M or less, 5 ⁇ 10 ⁇ 6 M or less, 2 ⁇ 10 ⁇ 6 M or less, or 1 ⁇ 10 ⁇ 6 M or less.
- the binding between an antigen and an antibody in the present invention can be measured or determined by a biomolecular interaction analysis system such as the SPR method or the BLI method, or by an ELISA method, an RIA method, or the like.
- the binding between an antigen expressed on the cell surface and an antibody can be measured by flow cytometry or the like.
- the SPR method is a dissociation constant (KD value), etc.
- Instruments used for SPR analysis include Biacore (trademark) (manufactured by GE Healthcare), ProteOn (trademark) (manufactured by BioRad), SPR-Navi (trademark) (manufactured by BioNavis), and Spreeta (trademark) (Texas Instruments). ), SPRi-Plex II (trademark) (manufactured by Horiba), Autolab SPR (trademark) (manufactured by Metrohm), and the like.
- the BLI method (BioLayer Interferometry) is a method for measuring biomolecular interactions using biolayer interference.
- An Octet system (manufactured by Pall ForteBio) can be exemplified as an instrument used for interaction analysis using the BLI method.
- the ELISA (Enzyme-linked ImmunoSorbent Assay) method is a method of capturing a target antigen or antibody contained in a sample solution with a specific antibody or antigen, and detecting and quantifying using an enzymatic reaction. Enzymatic activity is detected by incorporating an enzyme-labeled antigen or antibody into the reaction system. Enzyme activity is detected using a substrate whose absorbance spectrum changes depending on the reaction, and is quantified by absorbance measurement.
- Cell-ELISA is a method that captures the measurement target on the cell surface together with the cell and detects and quantifies it using an enzymatic reaction.
- the antibody in the RIA method (Radio Immunoassay method), can be quantified by labeling the antibody with a radioactive substance and measuring the radioactivity from the antibody.
- Flow cytometry is a method of optically analyzing individual cells by dispersing cells in a fluid and slicing the fluid.
- An antibody labeled with a fluorescent dye binds to a cell surface antigen through an antigen-antibody reaction, and the antigen-binding ability of the antibody is quantified by measuring the fluorescence intensity of the labeled antibody bound to the cell.
- Antibodies that specifically bind to antigen proteins or binding fragments thereof 3-1.
- Antibody that specifically binds to CD3 or binding fragment thereof The anti-CD3 antibody that specifically binds to CD3 of the present invention and the antigen-binding fragment thereof (hereinafter also referred to as the antibody of the present invention, etc.) are monoclonal antibodies and polyclonal antibodies. may be either.
- the isotype of the monoclonal antibody of the present invention is not particularly limited, and examples thereof include IgG such as IgG1, IgG2, IgG3 and IgG4, IgA such as IgM, IgA1 and IgA2, IgD and Ig.
- the isotype and subclass of a monoclonal antibody can be determined by, for example, the Ouchterony method, ELISA method, RIA method and the like.
- the monoclonal antibodies of the present invention include non-human animal-derived antibodies (non-human animal antibodies), human antibodies, chimerized antibodies (also referred to as "chimeric antibodies"), humanized antibodies, etc., preferably human antibodies. can be used.
- the scope of antibodies of the present invention also includes antibody variants (“variant antibodies” described below), for example, the scope of human antibodies also includes human variant antibodies.
- non-human animal antibodies include antibodies derived from vertebrates such as mammals and birds.
- Antibodies derived from mammals include antibodies derived from rodents such as mouse antibodies and rat antibodies.
- Examples of antibodies derived from birds include chicken antibodies.
- chimerized antibodies include, but are not limited to, antibodies formed by combining variable regions derived from non-human animal antibodies and human antibody (human immunoglobulin) constant regions.
- Humanized antibodies include those in which the CDRs in the variable regions of non-human animal antibodies are grafted into human antibodies (variable regions of human immunoglobulins), and in addition to the CDRs, the sequences of the framework regions of non-human animal antibodies are also partly human antibodies. and those in which one or more amino acids from any of these non-human animal antibodies are replaced with human amino acids, and the like, but are not limited thereto.
- Antibodies can be produced by various known methods. As well-known methods, it can be produced by a method using a hybridoma, a cell immunization method, or the like, and can be produced by a genetic recombination technique. Also known is a method for obtaining phage-display-derived human antibodies selected from a human antibody library. For example, a phage display method can be used in which variable regions of human antibodies are expressed as scFv on the surface of phages and phages that bind to the antigen are selected. By analyzing the genes of phages selected for binding antigen, the DNA sequences encoding the variable regions of human antibodies that bind antigen can be determined.
- a human antibody can be obtained by constructing an expression vector having the sequence and introducing it into an appropriate host for expression (WO92/01047, WO92/ 20791, WO93/06213, WO93/11236, WO93/19172, WO95/01438, WO95/15388, Annu. Rev. Immunol (1994) 12, 433-455).
- An antibody having high activity thus obtained can be used as a lead antibody, and the gene encoding the lead antibody can be mutated to produce a variant with higher activity ("mutant antibody" to be described later). .
- an antigen-binding fragment (hereinafter simply referred to as "binding fragment") of the antibody of the present invention is provided.
- the binding fragment of an antibody against a target molecule that is not a complex of the anti-CD3 antibody of the present invention or cancer testis antigen peptide and human leukocyte antigen (HLA) includes a chimerized antibody, a humanized antibody, or a binding fragment of a human antibody. subsumed.
- a binding fragment of an antibody means a fragment or a modification thereof that retains at least antigen-binding among the functions of the antibody.
- antigen-binding activity activity of regulating antigen activity (e.g., agonist activity), activity of internalizing antigen into cells, and interaction with substances that interact with antigens. Inhibitory or promoting activity and the like can be mentioned.
- the antigen-binding fragment of the antibody is not particularly limited as long as it retains at least antigen-binding among the activities of the antibody.
- Examples include Fab, Fab', F(ab') 2 , Examples include, but are not limited to, Fv, single-chain Fv (scFv) in which heavy and light chain Fvs are linked with an appropriate linker, single domain antibody (sdAb), and the like.
- Molecules containing portions other than antigen-binding fragments of the antibodies of the invention, such as scFv possessing a linker portion, are also included within the meaning of antigen-binding fragments of the antibodies of the invention.
- Fab and scFv are preferred.
- Fab is an antigen-binding fragment comprising a heavy chain variable region and constant region CH1, and a light chain variable region and constant region CL, as shown in FIG. 38B.
- Fabs can also be obtained from whole antibodies by enzymatic digestion with papain or the like.
- scFv is an antigen-binding fragment of an antibody obtained by connecting the heavy chain variable region and the light chain variable region of an antibody with a polypeptide linker (Pluckthun A. The Pharmacology of Monoclonal Antibodies 113, Rosenburg and Moore eds., Springer Verlag, New York, 269-315 (1994), Nature Biotechnology (2005), 23, 1126-1136).
- a tandem scFv made by joining two scFvs with a polypeptide linker can also be used as a bispecific molecule.
- triabodies and the like consisting of three or more scFv can also be used as multispecific molecules.
- scFv is produced by a phage display method (Nature Biotechnology (2005), 23, (9), p. 1105) in which the variable region of an antibody is expressed on the surface of a phage as a single-chain antibody (scFv) to select phage that bind to an antigen. -1116).
- scFv single-chain antibody
- a human antibody can be obtained by constructing an expression vector having the sequence and introducing it into an appropriate host for expression (WO92/01047, WO92/ 20791, WO93/06213, WO93/11236, WO93/19172, WO95/01438, WO95/15388, Annu. 1105-1116).
- the heavy chain variable region and the light chain variable region are disulfide-bonded.
- Such scFv can be expressed using cultured mammalian cells, as can scFv in which the heavy and light chain variable regions are not disulfide-bonded.
- the position and length of the light chain variable region are determined by the IMGT definition when determined using a definition different from IMGT (e.g., Kabat, Chothia, AbM, contact, etc.) compared to when determined by the IMGT definition.
- the carboxyl terminus of the light chain variable region amino acid sequence may further include one or more amino acids such as arginine and glycine.
- Antibodies or binding fragments thereof having such light chain variable regions are also included in the antibodies or binding fragments thereof of the present invention.
- the anti-CD3 antibody or antigen-binding fragment thereof is not particularly limited as long as it is an antibody or binding fragment thereof that binds to human CD3, but preferably also binds to CD3 of non-human primates such as cynomolgus monkeys.
- More preferred anti-CD3 antibodies or antigen-binding fragments thereof include a heavy chain variable region CDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 32, a heavy chain variable region CDRH2 consisting of the amino acid sequence represented by SEQ ID NO: 33, and SEQ ID NO: a heavy chain variable region CDRH3 consisting of the amino acid sequence represented by 34, a light chain variable region CDRL1 consisting of the amino acid sequence represented by SEQ ID NO:35, a light chain variable region CDRL2 consisting of the amino acid sequence represented by SEQ ID NO:36, and , an antibody or an antigen-binding fragment thereof comprising a light chain variable region CDRL3 consisting of the amino acid sequence represented by SEQ ID NO: 37 (above, FIG. 36).
- a more preferred antibody or antigen-binding fragment thereof comprising the CDRH1 to CDRH3 and CDRL1 to CDRL3 shown in FIG. C3E-7085 heavy chain variable region, represented by SEQ ID NO: 3 (FIG. 46), consisting of the amino acid sequence of amino acid numbers 1-118 of the amino acid sequence represented by SEQ ID NO: 3 (FIG. 7)
- An antibody or an antigen-binding fragment thereof comprising a C3E-7097 heavy chain variable region consisting of the amino acid sequence of amino acid numbers 1 to 118 of the amino acid sequence of the above can be exemplified.
- a cysteine was added to amino acid number 110 of the 7096 heavy chain variable region to form an S—S bond with amino acid number 177 of the C3E-7096 light chain variable region, and amino acid number 44 of the 7097 heavy chain variable region has an additional cysteine to form an S—S bond with amino acid number 233 of the C3E-7097 light chain variable region.
- antibodies or antigen-binding fragments thereof comprising the CDRH1 to CDRH3 and CDRL1 to CDRL3 shown in FIG. C3E-7085 light chain variable region, consisting of the amino acid sequence of amino acid numbers 134-240 of the amino acid sequence represented by SEQ ID NO: 3 ( Figure 7), C3E-7096 light chain variable region, SEQ ID NO: 39 ( Figure 46)
- An antibody or antigen-binding fragment thereof comprising a C3E-7097 light chain variable region consisting of the amino acid sequence of amino acid numbers 134 to 240 of the amino acid sequence shown can be exemplified.
- a cysteine is added to form an S—S bond with amino acid number 110 of the 7096 heavy chain variable region, and at amino acid number 233 of the 7097 light chain variable region, A cysteine has been added to form an SS bond with amino acid number 44 of the C3E-7097 heavy chain variable region.
- antibodies or antigen-binding fragments thereof comprising the CDRH1 to CDRH3 and CDRL1 to CDRL3 shown in FIG. 6) a combination of C3E-7085 heavy chain variable region and light chain variable region consisting of 1 to 107 represented by C3E- 7096 heavy chain variable region and light chain variable region combination, C3E-7097 heavy chain variable region and light chain variable region combination consisting of amino acid numbers 1-118 and 134-240 represented by SEQ ID NO: 39 ( Figure 46) Antibodies or antigen-binding fragments thereof can be exemplified.
- antibodies or antigen-binding fragments thereof comprising the CDRH1 to CDRH3 and CDRL1 to CDRL3 shown in FIG. and SEQ ID NO: 2 (Fig. 6).
- antibodies or antigen-binding fragments thereof comprising the CDRH1 to CDRH3 and CDRL1 to CDRL3 shown in FIG. C3E-7096scFv consisting of and C3E-7097scFv consisting of the amino acid sequence of amino acid numbers 1 to 240 of the amino acid sequence represented by SEQ ID NO: 39 (Fig. 46).
- anti-CD3 antibodies or antigen-binding fragments thereof include the heavy chain variable region CDRH1 consisting of the amino acid sequence represented by SEQ ID NO:40 and the heavy chain variable region CDRH2 consisting of the amino acid sequence represented by SEQ ID NO:41. , a heavy chain variable region CDRH3 consisting of the amino acid sequence represented by SEQ ID NO: 42, a light chain variable region CDRL1 consisting of the amino acid sequence represented by SEQ ID NO: 43, and a light chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 44
- An antibody or an antigen-binding fragment thereof comprising CDRL2 and a light chain variable region CDRL3 consisting of the amino acid sequence represented by SEQ ID NO: 45 can be exemplified.
- a suitable antibody or antigen-binding fragment thereof comprising the CDRH1 to CDRH3 and CDRL1 to CDRL3 shown in FIG. 47 is C3E consisting of the amino acid sequence of amino acid numbers 1 to 118 of the amino acid sequence represented by SEQ ID NO: 46 (FIG. 48).
- C3E-7098 heavy chain variable region, represented by SEQ ID NO: 49 ( Figure 51) consisting of the amino acid sequence of amino acid numbers 1-118
- Antibodies or antigen-binding fragments thereof comprising a C3E-7099 heavy chain variable region consisting of amino acid sequences of amino acid numbers 1 to 118 can be exemplified.
- a cysteine is added to amino acid number 110 of the C3E-7098 heavy chain variable region to form an S—S bond with amino acid number 177 of the C3E-7098 light chain variable region.
- a cysteine is added at amino acid number 44 to form an SS bond with amino acid number 235 of the C3E-7099 light chain variable region.
- preferred antibodies or antigen-binding fragments thereof comprising the CDRH1 to CDRH3 and CDRL1 to CDRL3 shown in FIG. C3E-7093 light chain variable region, consisting of the amino acid sequence of amino acid numbers 134 to 242 of the amino acid sequence represented by SEQ ID NO: 48 ( Figure 50), C3E-7098 light chain variable region, represented by SEQ ID NO: 49 ( Figure 51)
- An antibody or an antigen-binding fragment thereof comprising a C3E-7099 light chain variable region consisting of the amino acid sequence of amino acid numbers 134 to 242 of the amino acid sequence shown in FIG.
- a cysteine is added to amino acid number 177 of the C3E-7098 light chain variable region so as to form an S—S bond with amino acid number 110 of the C3E-7098 heavy chain variable region.
- a cysteine is added at amino acid number 235 to form an SS bond with amino acid number 44 of the C3E-7099 heavy chain variable region.
- suitable antibodies or antigen-binding fragments thereof comprising the CDRH1 to CDRH3 and CDRL1 to CDRL3 shown in FIG. ), C3E-7098 consisting of amino acid numbers 1-118 and 134-242, represented by SEQ ID NO: 48 ( Figure 50) heavy and light chain variable region combinations, including C3E-7099 heavy and light chain variable region combinations consisting of amino acid numbers 1-118 and 134-242 represented by SEQ ID NO: 49 ( Figure 51) An antibody or an antigen-binding fragment thereof can be exemplified.
- antibodies or antigen-binding fragments thereof comprising the CDRH1 to CDRH3 and CDRL1 to CDRL3 shown in FIG. and SEQ ID NO: 47 (Fig. 49).
- the scFv in which VH and VL are disulfide-bonded, which binds to CD3, may contain a linker, a junction, or any other portion between VH and VL, and such scFv
- the "other binding portion", “other group”, or a portion thereof may be included.
- Antibody or binding fragment thereof that binds to target molecule The antibody or binding fragment thereof that binds to “2-2.
- Target molecule may be either a monoclonal antibody or a polyclonal antibody.
- the isotype of the monoclonal antibody of the present invention is not particularly limited, and examples thereof include IgG such as IgG1, IgG2, IgG3 and IgG4, IgA such as IgM, IgA1 and IgA2, IgD and Ig.
- the isotype and subclass of a monoclonal antibody can be determined by, for example, the Ouchterony method, ELISA method, RIA method and the like.
- the monoclonal antibodies of the present invention include non-human animal-derived antibodies (non-human animal antibodies), human antibodies, chimerized antibodies (also referred to as "chimeric antibodies"), humanized antibodies, etc., preferably human antibodies. can be used.
- the scope of antibodies of the present invention also includes antibody variants (“variant antibodies” described below), for example, the scope of human antibodies also includes human variant antibodies.
- non-human animal antibodies include antibodies derived from vertebrates such as mammals and birds.
- Antibodies derived from mammals include antibodies derived from rodents such as mouse antibodies and rat antibodies.
- Examples of antibodies derived from birds include chicken antibodies.
- chimerized antibodies include, but are not limited to, antibodies formed by combining variable regions derived from non-human animal antibodies and human antibody (human immunoglobulin) constant regions.
- Humanized antibodies include those in which the CDRs in the variable regions of non-human animal antibodies are grafted into human antibodies (variable regions of human immunoglobulins), and in addition to the CDRs, the sequences of the framework regions of non-human animal antibodies are also partly human antibodies. and those in which one or more amino acids from any of these non-human animal antibodies are replaced with human amino acids, and the like, but are not limited thereto.
- Antibodies can be produced by various known methods. As well-known methods, it can be produced by a method using a hybridoma, a cell immunization method, or the like, and can be produced by a genetic recombination technique. Also known is a method for obtaining phage-display-derived human antibodies selected from a human antibody library. For example, a phage display method can be used in which variable regions of human antibodies are expressed as scFv on the surface of phages and phages that bind to the antigen are selected. By analyzing the genes of phages selected for binding antigen, the DNA sequences encoding the variable regions of human antibodies that bind antigen can be determined.
- a human antibody can be obtained by constructing an expression vector having the sequence and introducing it into an appropriate host for expression (WO92/01047, WO92/ 20791, WO93/06213, WO93/11236, WO93/19172, WO95/01438, WO95/15388, Annu. Rev. Immunol (1994) 12, 433-455).
- An antibody having high activity thus obtained can be used as a lead antibody, and the gene encoding the lead antibody can be mutated to produce a variant with higher activity ("mutant antibody" to be described later). .
- the antigen-binding fragment of the antibody is not particularly limited as long as it retains at least antigen-binding among the activities of the antibody.
- Examples include Fab, Fab', F(ab') 2 , Examples include, but are not limited to, Fv, single-chain Fv (scFv) in which heavy and light chain Fvs are linked with an appropriate linker, single domain antibody (sdAb), and the like.
- Molecules containing portions other than antigen-binding fragments of the antibodies of the invention, such as scFv possessing a linker portion, are also included within the meaning of antigen-binding fragments of the antibodies of the invention.
- the scFv directed against a target molecule that is not a complex of cancer testis antigen peptide and human leukocyte antigen (HLA) may have a disulfide bond between the heavy chain variable region and the light chain variable region.
- Variants of antibodies or antigen-binding fragments thereof preferably have reduced susceptibility to protein degradation or oxidation, maintenance, improvement, reduction or change in biological activity or function. can be suppressed, the antigen-binding ability can be improved or regulated, or physicochemical or functional properties can be imparted. Proteins are known to change their function and activity by changing specific amino acid side chains on their surface. Examples include deamidation of asparagine side chains and Isomerization etc. are included. Alternative amino acid substitutions to prevent such amino acid side chain changes are also included within the scope of the variants of the present invention.
- variants of the present invention include antibodies or antigen-binding fragments thereof having amino acid sequences in which conservative amino acid substitutions are made in the amino acid sequence of antibodies or antigen-binding fragments thereof.
- Conservative amino acid substitutions are substitutions that occur within a group of amino acids that are related in their amino acid side chains.
- aliphatic hydroxy groups serine and threonine
- amide containing groups asparagine and glutamine
- aliphatic groups alanine, valine, leucine and isoleucine
- aromatic groups phenylalanine, tryptophan. and tyrosine.
- Amino acid substitutions in such mutant antibodies are preferably made within a range that does not reduce the antigen-binding activity of the original antibody.
- Modified Antibodies or Binding Fragments Thereof, Conjugates The present invention provides modified antibodies or binding fragments thereof.
- a modified antibody of the present invention or a binding fragment thereof means one obtained by chemically or biologically modifying the antibody of the present invention or a binding fragment thereof.
- Chemical modifications include attachment of chemical moieties to the amino acid backbone, chemical modifications of N-linked or O-linked carbohydrate chains, and the like.
- Biological modifications include post-translational modifications (e.g., N- or O-linkage glycosylation, amino- or carboxyl-terminal region processing, deamidation, aspartic acid isomerization, methionine oxidized), and those with a methionine residue added to the amino terminus by expression using a prokaryotic host cell.
- those labeled to enable detection or isolation of the antibody or antigen of the present invention such as enzyme labels, fluorescent labels, and affinity labels, are also included in the meaning of such modifications.
- Such modifications of the antibody or binding fragment thereof of the present invention can improve the stability and blood retention of the original antibody or binding fragment thereof, reduce antigenicity, detect such antibody or antigen, or useful for separation.
- Examples of chemical moieties included in chemical modifications include water-soluble polymers such as polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymer, carboxymethylcellulose, dextran, and polyvinyl alcohol.
- biological modifications include those modified by enzymatic or cell treatments, fusions to which other peptides such as tags are added by genetic recombination, and endogenous or exogenous glycosylation enzymes. and those prepared by using cells expressing as a host.
- Such modifications may be made at any position or desired position in the antibody or binding fragment thereof, and the same or two or more different modifications may be made at one or more positions.
- heavy chain sequence deletions or heavy or light chain sequence modifications have little effect on antibody antigen-binding ability and effector functions (such as complement activation and antibody-dependent cytotoxicity). do not affect antibody antigen-binding ability and effector functions (such as complement activation and antibody-dependent cytotoxicity). do not affect antibody antigen-binding ability and effector functions (such as complement activation and antibody-dependent cytotoxicity). do not affect antibody antigen-binding ability and effector functions (such as complement activation and antibody-dependent cytotoxicity).
- the present invention also includes the deleted or modified antibody.
- a deletion in which one or two amino acids are deleted at the heavy chain carboxyl terminus Journal of Chromatography A; 705; 129-134 (1995)
- two amino acid residues of glycine and lysine at the heavy chain carboxyl terminus are deleted.
- the deletion product in which the proline residue located at the carboxyl terminus is newly amidated (Analytical Biochemistry, 360: 75-83 (2007)), glutamine or glutamic acid at the amino terminus of the antibody heavy chain or light chain Antibodies whose residues are modified by pyroglutamylation (International Patent Publication No.
- the carboxyl-terminal truncations of the heavy and light chains of the antibody of the present invention are not limited to the above types, as long as antigen-binding ability and effector function are maintained.
- the two or more chains e.g., heavy chains
- the two or more chains are heavy chains selected from the group consisting of full-length and truncated forms described above. Any one of these may be used, or any two of them may be used in combination.
- the amount ratio or molecular number ratio of each deletion can be affected by the type and culture conditions of cultured mammalian cells that produce the antibody of the present invention. in which one amino acid residue at the carboxyl terminus is deleted.
- the antibody of the present invention or an antigen-binding fragment thereof may contain one or more derived from an expression vector and/or a signal sequence, etc. Even if one amino acid is added to the amino terminus and/or the carboxyl terminus (and some or all of them are modified as described above), the desired antigen-binding activity of the antibody of the present invention is maintained. Included within the scope of modifications or modifications of antigen-binding fragments thereof, molecules comprising such modifications of antibodies or antigen-binding fragments are also encompassed within the scope of the molecules of the present invention.
- the term "antibody or its binding fragment” also includes “modified antibody or its antigen-binding fragment”.
- the "antibody or antigen-binding fragment thereof” included in the molecules, multispecific molecules, bispecific molecules, etc. of the present invention includes such "modified antibody or antigen-binding fragment thereof” in its meaning. .
- the present invention also includes a conjugate (Immunoconjugate) in which the above-described antibody and other molecule are linked by a linker.
- a conjugate Immunoconjugate
- Examples of antibody-drug conjugates in which the antibody is bound to a radioactive substance or a compound (drug) having a pharmacological action include ADC (Antibody-Drug Conjugate) (Methods Mol Biol. (2013) 1045 : 1-27; Nature Biotechnology (2005) 23, p.1137-1146).
- the present invention also includes complexes in which these antibodies are linked to other functional polypeptides.
- antibody-peptide conjugates include those in which the antibody is conjugated with an albumin-binding polypeptide (Protein Eng Des Sel. (2012) (2): 81-8).
- the above-mentioned modified antibody, glycosylated antibody, and conjugate are encompassed by the antibodies of the present invention, and the above-described modified antibody, glycosylated antibody, and conjugate-binding fragment are , are included in the binding fragments of the antibodies of the invention.
- the antibody or antigen-binding fragment thereof of the present invention can be produced, for example, by inserting a DNA encoding a heavy chain variable region or a DNA encoding a light chain variable region into an expression vector, and producing a host for expression. By transforming a cell with the vector and culturing the host cell, the recombinant antibody can be produced in the cell.
- a DNA encoding an antibody or an antigen-binding fragment thereof is obtained by ligating a DNA encoding a heavy chain variable region and a DNA encoding a heavy chain constant region to obtain a DNA encoding a heavy chain, and further ligating a DNA encoding a light chain variable region.
- the light chain-encoding DNA is obtained by ligating the encoding DNA and the light chain constant region-encoding DNA.
- the antibody or antigen-binding fragment thereof of the present invention can be produced by inserting the above-described heavy chain-encoding DNA and light chain-encoding DNA into an expression vector, transforming host cells with the vectors, and culturing the host cells. can be produced.
- the above-described heavy chain-encoding DNA and light chain-encoding DNA may be introduced into the same expression vector, and the vector may be used to transform host cells.
- the DNA encoding the strands may be inserted into separate vectors and the two vectors used to transform a host cell.
- the DNAs encoding the heavy chain variable region and the light chain variable region may be introduced into a vector into which the DNA encoding the heavy chain constant region and the DNA encoding the light chain constant region have been previously introduced.
- the vector may also contain a DNA encoding a signal peptide that promotes antibody secretion from host cells. Connect with a frame. An antibody or antigen-binding fragment thereof can be obtained as a mature protein by removing the signal peptide after the antibody or antigen-binding fragment thereof is produced.
- a DNA encoding a heavy chain variable region a DNA encoding a light chain variable region, a DNA linking a DNA encoding a heavy chain variable region and a DNA encoding a heavy chain constant region, and a DNA encoding a light chain variable region
- the DNA obtained by linking the DNA and the DNA encoding the light chain constant region may be functionally linked to elements such as promoters, enhancers and polyadenylation signals.
- “functionally linked” means linked so that the elements perform their functions.
- Expression vectors are not particularly limited as long as they are replicable in hosts such as animal cells, bacteria and yeast, and examples thereof include known plasmids, phages and the like.
- Vectors used for construction of expression vectors include, for example, pcDNATM (Thermo Fisher Scientific), Flexi (registered trademark) vector (Promega), pUC19, pUEX2 (Amersham), pGEX-4T, pKK233- 2 (manufactured by Pharmacia), pMAM-neo (manufactured by Clontech) and the like.
- host cells prokaryotic cells such as E.
- coli and Bacillus subtilis and eukaryotic cells such as yeast and animal cells can be used, but eukaryotic cells are preferably used.
- animal cells human embryonic kidney cell line HEK293 cells, Chinese hamster ovary (CHO) cells, and the like may be used.
- An expression vector may be introduced into a host cell by a known method to transform the host cell. Examples include electroporation, calcium phosphate precipitation, DEAE-dextran transfection, and the like.
- the antibodies produced can be purified using separation and purification methods commonly used for proteins. For example, affinity chromatography, other chromatography, filter, ultrafiltration, salting out, dialysis, etc. may be appropriately selected and combined. 5.
- the multispecific molecule of the present invention is the CD3-binding scFv of the present invention in which the heavy chain variable region and the light chain variable region are linked by disulfide bonds, or the Fab that binds to CD3 (hereinafter , referred to as "the CD3 antibody of the present invention") and binds to a target molecule other than CD3, preferably a target molecule that is not a complex of cancer testis antigen peptide and major histocompatibility complex (MHC) is not particularly limited as long as it contains a moiety (hereinafter referred to as "another binding moiety"), and in addition to the "CD3 antibody of the present invention” and “other binding moiety” Groups (hereinafter referred to as "other groups”) may be included.
- “other binding moieties” include antibodies or binding fragments thereof, non-immunoglobulin-derived proteins or peptides, nucleic acid molecules such as nucleic acid aptamers, low-molecular-weight compounds, T-cell receptors, and the like.
- “Other groups” include compounds (proteins, peptides, nucleic acid molecules, low-molecular-weight compounds, radioactive compounds containing isotopes, compounds containing labeling substances, artificially synthesized products, pharmaceutical ingredients, antitumor agents, etc.), their precursors (prodrugs, etc.), and those compounds in which a linker or connecting part is bound or fused can be exemplified.
- the "CD3 antibody of the present invention” and the "other binding moiety” and optionally the “other group” may be directly conjugated or fused to each other, or may be indirectly conjugated or fused via a linker or connecting moiety. may be There may be one or more "other binding moieties", and there may be two, three, or more.
- the "other group” may optionally be one, two or more. They may be linearly linked or fused, or branched and linked or fused, and there are no restrictions on the order of bonding or fusion, the position of branching, and the like.
- Such multispecific molecules of the invention are shown in FIGS. 38A-F, FIGS. 39A-B, FIGS. 40A-B, FIGS. It may be or include, but is not limited to, the illustrated formats.
- suitable multispecific molecules of the present invention include multispecific proteins (molecules composed entirely of polypeptides and/or peptides) such as multispecific antibodies.
- Multispecific antibodies can include bispecific antibodies, trispecific antibodies, and tetraspecific antibodies.
- Bispecificity refers to two (or three, four, etc.) different epitopes on the same molecule or two (or three It means that it is capable of binding to epitopes that are different from each other (up to three molecules, up to four molecules, etc.), and includes such bispecific antibodies or antigen-binding fragments.
- the bispecific molecule of the invention binds CD3 and further binds a target molecule, preferably a target molecule that is not a complex of cancer testis antigen peptide and human leukocyte antigen (HLA).
- HLA human leukocyte antigen
- Examples of multispecific molecules of the present invention include those having the following structure (format).
- the bispecific molecule of the present invention includes a bispecific molecule in which the Fab of the first antibody and the scFv of the second antibody are bound to one of the Fc dimers via a linker.
- Fab may be bound to Fc and scFv may be bound to the Fab, or scFv may be bound to Fc and Fab may be bound to the scFv.
- the Fab is bound and the scFv is bound to the Fab. Binding of Fab and scFv can be achieved by binding scFv to the variable region of Fab via a linker.
- the bispecific molecule is an Fc-associated format in which scFv and Fab are linked by a linker and mutated to form a heterodimer downstream.
- Such bispecific molecules are referred to as scFv-Fab-heterodimeric Fc-type bispecific molecules or scFv-Fab-heterodimeric Fc-type bispecific molecules (FIG. 43B, top left).
- an scFv-Fab-heterodimeric Fc type consisting of scFv against a target molecule that is not a complex of anti-CD3 Fab, cancer testis antigen peptide and human leukocyte antigen (HLA) may be used.
- the heavy chain variable region and light chain variable region are bound by disulfide bonds.
- the scFv directed against a target molecule that is not a cancer testis antigen peptide-human leukocyte antigen (HLA) complex may have a disulfide bond between the heavy chain variable region and the light chain variable region.
- a taFv (FIG. 38E) having a format in which two types of scFv, the first antibody and the second antibody, are connected by a linker, is directly attached to one of the Fc dimers via a linker or not via a linker.
- Conjugated bispecific molecules are included. Such bispecific molecules are referred to as taFv-heterodimeric Fc-type bispecific molecules or taFv-heterodimeric Fc-type molecules (FIG. 40B bottom left).
- the bispecific molecule is a hetero-associated format of Fc with a heterodimer-forming mutation downstream of taFv.
- the order of conjugation of the first antibody and the second antibody in taFv is not limited.
- FIG. 43B The structure of the scFv-Fab-heterodimeric Fc-type bispecific molecule is shown in the upper left of FIG. 43B. Also, scFv in FIG. 38A, Fab in FIG. 38B, taFv in FIG. 38E, scFv-Fab in FIG. 38F, taFv-heterodimer Fc-type bispecific molecule in FIG. 40B lower left, taFv-Fab-heterodimer in FIG. 41B upper right Figure 2 shows the structures of Fc-type bispecific molecules.
- the bispecific molecule of the present invention has a structure in which multiple polypeptides are associated.
- the taFv may be an anti-CD3 scFv and a target molecule, preferably a taFv of a scFv against a target molecule that is not a complex of cancer testis antigen peptide and human leukocyte antigen (HLA).
- HLA human leukocyte antigen
- the taFv-heterodimeric Fc-type bispecific molecule preferably comprises (a) a complex of a target molecule, preferably a cancer testis antigen peptide, and a human leukocyte antigen (HLA) from the N-terminus to the C-terminus a scFv that specifically binds to a target molecule that is not a target molecule, a scFv that specifically binds to CD3 and an immunoglobulin Fc region (i), in that order, and an immunoglobulin hinge region and Fc region (ii ), more preferably (b) the first polypeptide and the second polypeptide are associated in the Fc region (i) and the Fc region (ii).
- a target molecule preferably a cancer testis antigen peptide, and a human leukocyte antigen (HLA) from the N-terminus to the C-terminus
- HLA human leukocyte antigen
- the Fc regions of the first and second polypeptides contain mutations that form heterodimers.
- An example of a taFv-heterodimeric Fc-type bispecific molecule is shown in FIG. 40B bottom left. As shown in the lower left of FIG. 40B, the Fc region (i) portion of the first polypeptide and the Fc region (ii) portion of the second polypeptide shown in black are bound, and the first polypeptide and the second polypeptide associate. is doing. For example, in the lower left of FIG.
- the scFv represented in white is the scFv against the target molecule, preferably a target molecule that is not a complex of the cancer testis antigen peptide and human leukocyte antigen (HLA), and is represented by the upper right hatched line.
- the scFv obtained is the anti-CD3 scFv (the black bar between VH and VL indicates that they are disulfide-bonded).
- the first polypeptide contained in the more preferred taFv-heterodimeric Fc bispecific molecule of the present invention comprises the amino acid sequence represented by SEQ ID NO:3.
- a second polypeptide comprised in a preferred taFv-heterodimeric Fc-type bispecific molecule of the invention comprises a hinge region and a wild-type or mutant Fc derived from a human antibody.
- the scFv-Fab-heterodimeric Fc-type bispecific molecule is preferably (a) a target molecule that is not a complex of a cancer testis antigen peptide and a human leukocyte antigen (HLA) from the N-terminus to the C-terminus.
- HLA human leukocyte antigen
- a scFv that specifically binds to CD3, a first polypeptide comprising, in that order, an antibody heavy chain variable region and constant region CH1 that specifically binds to CD3 and an immunoglobulin Fc region (i), an immunoglobulin hinge region and a second polypeptide comprising an Fc region (ii), and a third polypeptide comprising an antibody light chain consisting of a variable region and a constant region, more preferably (b) the first polypeptide and the Two polypeptides are associated in Fc region (i) and Fc region (ii), and the first polypeptide is associated with a third polypeptide (of the antibody light chain) in the antibody heavy chain variable region and constant region CH1 .
- the Fc regions of the first and second polypeptides may be wild-type or may contain mutations that form heterodimers.
- An example of a scFv-Fab-heterodimeric Fc-type bispecific molecule is shown in FIG. 43B upper left.
- the right half of the upper left of FIG. 43B is the first and third polypeptides, and the left half is the second polypeptide.
- the Fc region (i) portion of the first polypeptide and the Fc region (ii) portion of the second polypeptide shown in black are associated, and the first polypeptide and the third polypeptide are associated. is doing.
- the scFv shown in black is the target molecule, preferably the scFv against the target molecule that is not a complex of cancer testis antigen peptide and human leukocyte antigen (HLA), white and checkered and Fabs represented by horizontal lines are anti-CD3 Fabs.
- HLA human leukocyte antigen
- amino acid sequence represented by SEQ ID NO: 1 can be exemplified as the amino acid sequence contained in the first polypeptide contained in a suitable scFv-Fab-heterodimeric Fc-type bispecific molecule.
- the second polypeptide contained in the preferred scFv-Fab-heterodimeric Fc-type bispecific molecule of the present invention comprises a hinge region and a mutant Fc derived from a human antibody, and an even more preferred scFv-Fab-
- the second polypeptide contained in the heterodimeric Fc-type bispecific molecule comprises a hinge region and a wild-type or mutant Fc derived from a human antibody.
- a third polypeptide contained in a preferred scFv-Fab-heterodimeric Fc bispecific molecule of the present invention comprises a light chain derived from a human antibody, and a more preferred scFv-Fab-heterodimeric Fc bispecific
- the amino acid sequence shown in SEQ ID NO: 2 can be exemplified as the third polypeptide contained in the bispecific molecule.
- One, two or more of the peptides included in the bispecific molecules of the invention may be "truncated” as described above, i.
- One or two (or more) amino acids may be mutated (including deleted) at the terminus.
- the carboxyl terminal of the amino acid sequence of the first polypeptide contained in RX3-0002, which is one of the scFv-Fab-heterodimeric Fc bispecific molecules of the present invention is at position 713 of SEQ ID NO: 31. It may be Lys, 712th Gly in which one amino acid is deleted, or a mixture containing them.
- the carboxyl terminal of the amino acid sequence of the second polypeptide contained in the preferred scFv-Fab-heterodimeric Fc-type bispecific molecule of the present invention is Lys at position 246 of SEQ ID NO: 9, and 1 amino acid is deleted. 245th Gly, or a mixture containing them.
- the scFv and Fab contained in the bispecific molecule of the present invention are preferably humanized or human antibody scFv and Fab, and Fc is preferably human antibody Fc.
- the heavy chain variable region and the light chain variable region may be linked in this order from the amino terminal side of the antibody, or the light chain variable region and the heavy chain They may be linked in the order of the variable regions. There may (optionally) be a linker between both variable regions. It may also (optionally) have a glycine residue at the amino terminus of the amino terminal variable region.
- the carboxyl-terminal variable region may (optionally) have a linker, a FLAG tag and/or a His tag attached to the carboxyl terminus.
- One preferred embodiment is one in which a heavy chain variable region, a first linker, a light chain variable region, a second linker, a FLAG tag, and a His tag are linked in this order from the amino terminus. .
- Linkers include single-chain polypeptides or single-chain oligopeptides, or synthetic products such as PEG, nucleotides, sugar chains, and compounds. In addition, any known linker can be used without particular limitation as long as it binds two polypeptides.
- the length of the linker is, for example, 5-30 amino acids in the case of a peptide linker.
- all peptide linkers of the same length may be used, or peptide linkers of different lengths may be used.
- peptide linkers examples include repeats of (Gly-Gly-Gly-Gly-Ser) (SEQ ID NO: 38), to which one to several amino acid residues different from Gly and Ser are added. may be
- the structures (formats) that can be included in the multispecific antibodies of the present invention particularly the structures (formats) that can be adopted by bispecific antibodies, as described above, taFv-heterodimeric Fc type, and scFv-Fab-heterodimeric Fc type, more preferred is scFv-Fab-heterodimeric Fc type.
- taFv-heterodimeric Fc type from the N-terminus to the C-terminus, scFv against a target molecule, preferably a target molecule that is not a complex of cancer testis antigen peptide and human leukocyte antigen (HLA), anti-CD3 scFv
- a target molecule preferably a target molecule that is not a complex of cancer testis antigen peptide and human leukocyte antigen (HLA), anti-CD3 scFv
- HLA human leukocyte antigen
- the scFv-Fab-heterodimeric Fc type includes a target molecule at the N-terminus, preferably a scFv against a target molecule that is not a complex of cancer testis antigen peptide and human leukocyte antigen (HLA), scFv-Fab-
- HLA human leukocyte antigen
- scFv-Fab- The heterodimeric Fc type (Fig. 43B, upper left) is also a more preferred format that the bispecific antibodies of the invention can adopt.
- scFv-Fab-heterodimeric Fc type are (i) amino acids 20 to 718 of SEQ ID NO: 29 A polypeptide comprising an amino acid sequence consisting of residues or an amino acid sequence in which 1 or 2 amino acids are deleted at the carboxyl terminus of said amino acid sequence, an amino acid sequence consisting of amino acid residues 20 to 246 of SEQ ID NO: 9, or said amino acid (ii) a polypeptide comprising an amino acid sequence with one or two amino acids deleted at the carboxyl terminus of the sequence and a polypeptide comprising an amino acid sequence consisting of amino acid residues 21 to 233 of SEQ ID NO: 12; A polypeptide comprising an amino acid sequence consisting of amino acid residues 20 to 717 or an amino acid sequence in which 1 or 2 amino acids are deleted at the carboxyl terminus of the amino acid sequence, from amino acid residues 20 to 246 of SEQ ID NO: 9 (
- preferred ones containing taFv-heterodimeric Fc type are (i) amino acid residues 20 to 744 of SEQ ID NO: 21 or a polypeptide comprising an amino acid sequence in which 1 or 2 amino acids are deleted at the carboxyl terminus of the amino acid sequence consisting of and an amino acid sequence consisting of amino acid residues 20 to 246 of SEQ ID NO: 9, or the amino acid sequence (ii) an amino acid sequence consisting of amino acid residues 20 to 743 of SEQ ID NO: 23, or 1 or 2 amino acid sequences at the carboxyl terminus of SEQ ID NO: 23
- a polypeptide containing an amino acid sequence with amino acids deleted, an amino acid sequence consisting of amino acid residues 20 to 246 of SEQ ID NO: 9, or an amino acid sequence with 1 or 2 amino acids deleted at the carboxyl terminus of said amino acid sequence iii) a polypeptide and sequence comprising an amino acid sequence consisting of amino acid residue
- formats that the multispecific molecule of the present invention can take include those disclosed in Godar et al., Expert Opinion on Therapeutic Patents, Vol. 276 (2016), Brinkmann and Knotermann MABS 9(2) 182-212 (2017), and Wu and Demarest Methods 154 3-9 (2019). can do. The full disclosure of these documents is also included in the disclosure of this application.
- the present invention includes amino acid sequences that are 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to the amino acid sequences contained in the molecules of the present invention. and that binds to CD3 and also to a target molecule that is not a complex of cancer testis antigen peptide and human leukocyte antigen (HLA).
- HLA human leukocyte antigen
- the multispecific antibody of the present invention has excellent biological activity, physicochemical properties (hereinafter referred to as "physical properties"), safety, pharmacokinetics, and the like. Impurities contained in biopharmaceuticals are related to the safety of the drug, so it is required to establish appropriate standards and control whether they increase during manufacturing and storage. Among them, aggregates are one of the main impurities, and since they are involved in immunogenicity risk, deterioration of drug efficacy, etc., they should be controlled particularly strictly. (Guidance for Industry Immunogenecity Assessment for Therapeutic Protein Products,U.S. Department of Health and Human Services/Food and Drug Administration/Center for Drug Evaluation and Research(CDER)/Center for Biologics Evaluation and Research(CBER),August 2014, pp. 15-16).
- Impurity control should be evaluated not only during manufacturing, but also during the manufacturing process and over time after manufacturing (presence or absence of increase). Since the shelf life of a drug is set based on the results of long-term stability studies, antibodies that are stable over time can be set for a longer shelf life. Therefore, physicochemical properties in the present invention, which serve as indicators for selecting suitable antibodies for biopharmaceuticals, include solution stability, shaking stability, and the like. When a multispecific antibody with concerns about solution stability, shaking stability, etc.
- Solution stability is evaluated by, for example, preparing a sample to 20 mg/mL and measuring the content (%) of HMWS after storage at 40°C for 1 day, 7 days, and 14 days by size exclusion chromatography. can do.
- a specimen with a relatively low rate of increase in HMWS (%) can be determined to have relatively high solution stability.
- Shaking stability for example, prepare a specimen to 2 mg / mL, shake at 2500 rpm for 1 to 2 hours, perform size exclusion chromatography, and calculate the peak area and HMWS (%) of each specimen. , can be evaluated.
- aggregates detected as HMWS by this method are those with a diameter of about 10 to 100 nm, and subvisible particles with a particle size of 100 nm or more (particle size of 0.1 to 10 ⁇ m) and insoluble fine particles (particle size of 10 ⁇ m or more) are cannot be detected.
- the number of particles is counted by the MicroFlow Imaging method using a FlowCAM8100 (manufactured by DKSH Japan) or the like using a specimen that has been treated for 2 hours before shaking and after shaking. evaluate. If the number of particles increases before and after shaking, it can be assumed that particles are formed from monomers of the target protein via HMWS due to shaking degradation. Therefore, in the evaluation of shaking stability, (1) increase rate of HMWS (%), (2) decrease rate of peak area, and/or (3) increase or decrease in the number of particles by MicroFlow Imaging method. interpret and evaluate.
- the rate of increase in HMWS (%) of a specimen is greater than that of other specimens, the rate of decrease in the peak area of the specimen and/or the increase in the number of particles by the MicroFlow Imaging method is higher than that of the other specimens. is smaller, it can be evaluated that the specimen has higher shaking stability than the other specimen.
- the reason for this is that the increase rate (%) of HMWS in the other sample is considered to be low because aggregates corresponding to HMWS aggregate to form larger multimers, which cannot be detected by size exclusion chromatography.
- Multispecific molecules of the present invention for example, taFv-heterodimeric Fc-type bispecific antibodies in which the heavy chain variable region and light chain variable region of anti-CD3 scFv are linked by disulfide bonds, and scFv-Fab- A heterodimeric Fc-type bispecific antibody is less likely to form HMWS containing dimers over a long period of time in a solution state, and has high solution stability.
- the taFv-heterodimeric Fc type anti-CD3scFv heavy chain variable region and light chain variable region are not bound by disulfide bonds than the bispecific antibody Also, it is preferable that the solution stability is high.
- the multispecific molecules of the present invention for example, taFv-heterodimeric Fc-type bispecific antibody in which the heavy chain variable region and light chain variable region of anti-CD3 scFv are linked by disulfide bonds, and scFv- Bispecific antibodies that are Fab-heterodimeric Fc type contain dimers (1) HMWS increase rate, (2) peak area decrease rate, and / or (3) MicroFlow Imaging method It is interpreted based on the results of the increase and decrease in the number of particles due to the high shaking stability.
- the bispecific antibody that is a taFv-heterodimeric Fc type and the heavy chain variable region and light chain variable region of anti-CD3scFv are not bound by disulfide bonds It is preferable that the shaking stability is higher than that.
- anti-CD3 antibodies antigen-binding fragments thereof or modifications thereof, and/or multispecific molecules containing them (hereinafter referred to as "anti-CD3 antibodies, etc.”).
- the treatment and/or prevention of a disease includes prevention of onset, suppression or inhibition of exacerbation or progression of such disease, alleviation of one or more symptoms exhibited by an individual suffering from such disease, increase of It includes, but is not limited to, suppression or amelioration of exacerbation or progression, treatment or prevention of secondary diseases, and the like.
- Diseases include, for example, cancer for pharmaceutical compositions comprising multispecific molecules.
- the pharmaceutical composition of the present invention contains a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative and/or adjuvant in addition to a therapeutically or prophylactically effective amount of an anti-CD3 antibody or the like. can be squeezed.
- a therapeutically or prophylactically effective amount means an amount that exhibits a therapeutic or prophylactic effect for a specific disease, dosage form, and administration route.
- the pharmaceutical composition of the present invention has pH, osmotic pressure, viscosity, transparency, color, isotonicity, sterility, stability of the composition or the antibody contained therein, solubility, sustained release, absorbability, penetration.
- Substances for changing, maintaining, or retaining properties, dosage form, strength, properties, shape, etc. can be contained.
- Substances for formulation are not particularly limited as long as they are pharmacologically acceptable substances. For example, non-toxicity or low toxicity is a property that a pharmaceutical agent preferably possesses.
- Substances for formulation include, but are not limited to, amino acids such as glycine, alanine, glutamine, asparagine, histidine, arginine or lysine, antibacterial agents, ascorbic acid. , antioxidants such as sodium sulfate or sodium hydrogen sulfite, phosphoric acid, citric acid, borate buffer, sodium hydrogen carbonate, buffers such as Tris-hydrochloric acid (Tris-Hcl) solution, fillers such as mannitol and glycine, ethylenediamine Chelating agents such as tetraacetic acid (EDTA), complexing agents such as caffeine, polyvinylpyrrolidine, ⁇ -cyclodextrin and hydroxypropyl- ⁇ -cyclodextrin, bulking agents such as glucose, mannose or dextrin, monosaccharides, disaccharides and Glucose, other carbohydrates such as mannose and dextrins, coloring agents, flavoring agents, diluents
- sorbic acid or hydrogen peroxide preservatives solvents such as glycerin, propylene glycol or polyethylene glycol, sugar alcohols such as mannitol or sorbitol, suspending agents, PEG, sorbitan Esters, polysorbates such as polysorbitate 20 and polysorbitate 80, surfactants such as triton, tromethamine, lecithin or cholesterol, stabilization enhancers such as sucrose and sorbitol, sodium chloride, potassium chloride , elasticity enhancers such as mannitol and sorbitol, transport agents, diluents, excipients and/or pharmaceutical adjuvants.
- solvents such as glycerin, propylene glycol or polyethylene glycol
- sugar alcohols such as mannitol or sorbitol
- suspending agents PEG, sorbitan Esters
- polysorbates such as polysorbitate 20 and polysorbitate 80
- surfactants such as triton, trometh
- the amount of these formulation substances to be added is 0.001 to 1000 times, preferably 0.01 to 100 times, more preferably 0.1 to 10 times the weight of the anti-CD3 antibody or the like.
- compositions of the present invention also include immunoliposomes containing an anti-CD3 antibody and the like, and pharmaceutical compositions containing modified antibodies obtained by binding antibodies and liposomes (U.S. Pat. No. 6,214,388, etc.).
- Excipients and carriers are usually liquid or solid, and are not particularly limited as long as they are water for injection, physiological saline, artificial cerebrospinal fluid, and other substances used in formulations for oral or parenteral administration.
- physiological saline include neutral saline and serum albumin-containing saline.
- Tris buffer prepared so that the final pH of the pharmaceutical composition was 7.0 to 8.5, acetate buffer similarly prepared so that it was 4.0 to 5.5, and 5.5.
- Examples include a citrate buffer adjusted to 0 to 8.0, a histidine buffer adjusted to 5.0 to 8.0, and the like.
- compositions of the present invention are solids, liquids, suspensions, and the like. Lyophilized formulations may be mentioned. An excipient such as sucrose can be used to mold the freeze-dried formulation.
- the administration route of the pharmaceutical composition of the present invention may be any of enteral administration, topical administration and parenteral administration, and a suitable administration route may be selected depending on the target disease. Specific examples include intravenous administration, intraarterial administration, intramuscular administration, intradermal administration, subcutaneous administration, intraperitoneal administration, transdermal administration, intraosseous administration, and intraarticular administration.
- composition of such a pharmaceutical composition can be determined according to the administration method, CD3 protein binding affinity of the antibody, and the like.
- the dosage of the anti-CD3 antibody or the like can be appropriately determined according to the individual species, disease type, symptoms, sex, age, chronic disease, CD3 protein binding affinity or biological activity of the antibody, and other factors. However, usually 0.01 to 1000 mg/kg, preferably 0.1 to 100 mg/kg can be administered once every 1 to 180 days, or twice or more times a day.
- Forms of pharmaceutical compositions include injections (including freeze-dried formulations and drip infusions), suppositories, nasal absorption formulations, transdermal absorption formulations, sublingual formulations, capsules, tablets, ointments, granules, and aerosols. Examples include formulations, pills, powders, suspensions, emulsions, eye drops, bioimplantable preparations, and the like.
- Anti-CD3 antibodies and the like can be used in combination with other agents.
- An anti-CD3 antibody, etc., or a pharmaceutical composition containing it as an active ingredient can be administered simultaneously or separately with other agents, ie, pharmaceutical compositions containing a drug other than an anti-CD3 antibody, etc. as an active ingredient.
- administering a pharmaceutical composition containing an anti-CD3 antibody or the like as an active ingredient after administration of another agent, or administering a pharmaceutical composition containing an anti-CD3 antibody or the like as an active ingredient and then administering the other agent, or , an anti-CD3 antibody or the like as an active ingredient and other agents may be administered simultaneously.
- such a "pharmaceutical composition” is synonymous with "a pharmaceutical composition administered in combination with an anti-CD3 antibody or the like and another agent”.
- "to be administered in combination" with an anti-CD3 antibody or the like and another drug means that the anti-CD3 antibody or the like and the other drug are taken into the body of the subject for a certain period of time.
- the anti-CD3 antibody and the like may be administered in a single formulation, or they may be separately formulated and administered separately.
- the timing of their administration is not particularly limited, and they may be administered at the same time, or may be administered at different times or on different days.
- the anti-CD3 antibody or the like and the other agent are administered on different times or days, the order of administration is not particularly limited. Since each preparation is usually administered according to its own administration method, the administration times may be the same or different.
- each formulation when each formulation is separately formulated, the administration method (administration route) of each formulation may be the same, or may be administered by a different administration method (administration route).
- administration route there is no need for the anti-CD3 antibody or the like and the other agent to exist in the body at the same time. and the other active ingredient may disappear from the body at the time of administration of either one.
- Examples of dosage forms of the "pharmaceutical composition administered in combination with an anti-CD3 antibody or the like and other agents” include: 1) administration of a single preparation containing an anti-CD3 antibody or the like and other agents; 2) anti-CD3 antibody or the like 3) Simultaneous administration of two formulations obtained by separately formulating anti-CD3 antibody and other drugs via the same administration route 4) Simultaneous administration of two formulations obtained by separately formulating an anti-CD3 antibody or the like and another drug through different administration routes, 5) Administration of an anti-CD3 antibody or the like and the other drug Examples include the administration of two formulations obtained by separately formulating them through different administration routes at different times.
- the dosage, dosing interval, dosage form, formulation, etc. of the “pharmaceutical composition administered in combination with an anti-CD3 antibody etc. and other agents” conform to those of the pharmaceutical composition containing the anti-CD3 antibody, but are limited to them. not to be
- Such a pharmaceutical composition may be a kit containing them when it is formulated into two different formulations.
- the "combination" of an anti-CD3 antibody and the like and other agents means that the anti-CD3 antibody and the like and other agents are "administered in combination”.
- the present invention provides a method for treating or preventing a disease associated with CD3, use of the antibody of the present invention for preparing a pharmaceutical composition for treating or preventing the disease, antibody of the present invention for treating or preventing the disease
- the use of also provides.
- therapeutic or prophylactic kits containing the antibodies of the invention are also included in the invention.
- Example 1 Solution stability evaluation of anti-CD3 antibody fragment-heterodimer Fc type molecule 2)-1 Design of amino acid sequence of anti-CD3 antibody fragment Aiming to improve physical properties in solution stability of C3E-7085, the format was changed C3E-7085-Fab (SEQ ID NO: 1, Figure 5) and C3E-7085-LC (SEQ ID NO: 2, Figure 6) were designed as amino acid sequences in C3E-7085 Fab format.
- a mammalian cell expression vector incorporating a DNA fragment encoding an amino acid sequence contained in a polypeptide consisting of Fc (HC_h) introduced with a mutation that forms a heteromultimer was prepared and named "p_HC_h”. .
- a mammalian cell expression vector is prepared in which a DNA fragment encoding an amino acid sequence contained in a polypeptide obtained by adding an Fc region that has been mutated to form a heteromultimer is incorporated. , named "p_C3E-7085-HC-k”.
- the heavy chain variable region of C3E-7085, and the CH1 region derived from human IgG, the DNA fragment encoding the amino acid sequence contained in the polypeptide formed by adding the Fc region introduced with mutations that form heteromultimers in this order was incorporated.
- a mammalian cell expression vector was constructed and named "p_C3E-7085-Fab-HC-k".
- a mammalian cell expression vector was prepared in which a DNA fragment encoding an amino acid sequence contained in a polypeptide in which a CL region derived from human IgLabmda was added to the carboxyl terminus of the C3E-7085 light chain variable region was incorporated, and "p_C3E -7085-LC".
- a mammalian cell expression vector is prepared in which a DNA fragment encoding an amino acid sequence contained in a polypeptide obtained by adding an Fc region that has been mutated to form a heteromultimer is incorporated. , named “p_C3E-7096-HC-k”.
- nucleotide sequence of p_HC_h was re-analyzed, and it was confirmed that the full-length nucleotide sequence of HC_h is the nucleotide sequence shown in SEQ ID NO: 4 (Fig. 8) in the sequence listing.
- nucleotide sequence of p_C3E-7085-HC-k was reanalyzed, and the full-length nucleotide sequence of C3E-7085-HC-k was confirmed to be the nucleotide sequence shown in SEQ ID NO: 5 (Fig. 9) in the sequence listing.
- nucleotide sequence of p_C3E-7085-Fab-HC-k was re-analyzed, and the full-length nucleotide sequence of C3E-7085-Fab-HC-k was found to be the nucleotide sequence shown in SEQ ID NO: 6 (FIG. 10) in the sequence listing. confirmed.
- nucleotide sequence of p_C3E-7085-LC was re-analyzed, and it was confirmed that the full-length nucleotide sequence of C3E-7085-LC was the nucleotide sequence shown in SEQ ID NO: 7 (Fig. 11) in the sequence listing.
- nucleotide sequence of p_C3E-7096-HC-k was reanalyzed, and it was confirmed that the full-length nucleotide sequence of C3E-7096-HC-k is the nucleotide sequence shown in SEQ ID NO: 8 (Fig. 12) in the sequence listing.
- the amino acid sequence of the full-length HC_h is the amino acid sequence shown in SEQ ID NO: 9 (Fig. 13) in the sequence listing. In this sequence, the 1st to 19th amino acid sequence is the signal sequence, and the 20th to 246th amino acid sequence is HC_h.
- the amino acid sequence of the full-length C3E-7085-HC-k is the amino acid sequence shown in SEQ ID NO: 10 (Fig. 14) in the sequence listing.
- the 1st to 19th amino acid sequences are signal sequences
- the 21st to 260th amino acid sequences are C3E-7085
- the 261st to 262nd amino acid sequences are linkers
- the 263rd to 494th amino acid sequences are The amino acid sequence is HC-k.
- the amino acid sequence of the full-length C3E-7085-Fab-HC-k is the amino acid sequence shown in SEQ ID NO: 11 (Fig. 15) in the sequence listing.
- the 1st to 19th amino acid sequence is the signal sequence
- the 20th to 137th amino acid sequence is the C3E-7085-heavy chain variable region.
- the amino acid sequence from 138th to 467th is the constant region.
- the amino acid sequence of the full-length C3E-7085-LC is the amino acid sequence shown in SEQ ID NO: 12 (Fig. 16) in the sequence listing.
- the 1st to 20th amino acid sequence is the signal sequence
- the 21st to 127th amino acid sequence is the variable region
- the 128th to 233rd amino acid sequence is the constant region.
- the full-length amino acid sequence of C3E-7096-HC-k is the amino acid sequence shown in SEQ ID NO: 13 (Fig. 17) in the Sequence Listing.
- the 1st to 19th amino acid sequences are signal sequences
- the 21st to 260th amino acid sequences are C3E-7096
- the 261st to 262nd amino acid sequences are linkers
- the 263rd to 494th amino acid sequences are The amino acid sequence is HC-k.
- 2)-3 Expression of anti-CD3 antibody fragment-heterodimeric Fc Expi293F cells were passaged and cultured according to the manual.
- Expi293F cell culture medium in logarithmic growth phase was diluted to 2.5 ⁇ 10 6 cells/mL with Expi293 Expression medium (Thermo Fisher Scientific) and used for production of various anti-CD3 antibody fragment-heterodimer Fc.
- Expi293F expression medium Thermo Fisher Scientific
- For 300 mL culture dissolve 1.44 mg of Polyethyleneimine (Polyscience #24765) in 5 mL of Opti-Pro SFM (Thermo Fisher Scientific), then vector p_C3E-7085 prepared using NucleoBond Xtra Midi Kit (Takara Bio Inc.) - 0.3 mg of vector mixture containing HC-k and p_HC_h was added to 5 mL of Opti-Pro SFM (Invitrogen).
- p_C3E-7085-Fab-HC-k, p_HC_h and p_C3E-7085-LC were used to express and prepare the culture supernatant of anti-CD3 antibody fragment-heterodimer Fc (C3E-0003).
- the amino acid sequences obtained by expressing each vector constituting C3E-0003 are shown in SEQ ID NOs: 9 (FIG. 13), 11 (FIG. 15) and 12 (FIG. 16) in the sequence listing.
- p_C3E-7096-HC-k and p_HC_h were used to express and prepare the culture supernatant of anti-CD3 antibody fragment-heterodimer Fc (C3E-0004).
- the amino acid sequences obtained by expressing each vector constituting C3E-0004 are shown in SEQ ID NOs: 9 (FIG. 13) and 13 (FIG. 17) in the sequence listing.
- 2)-4 Purification of anti-CD3 antibody fragment-heterodimeric Fc 2 Purify the anti-CD3 antibody fragment-heterodimeric Fc from the culture supernatant obtained in 2)-3 in a two-step process of Protein A affinity chromatography and gel filtration chromatography did.
- the culture supernatant was applied to a MabSelectSuRe column (Cytiva) equilibrated with PBS pH 7.4 to adsorb the desired anti-CD3 antibody fragment-heterodimer Fc. After removing the non-adsorbed components with PBS, the adsorbed components were eluted with acetate buffer pH 3.5. The eluted fraction was adjusted to neutral pH with Tris buffer pH 9.5, concentrated, and applied to a gel filtration column Superdex 200 10/300 (Cytiva company).
- the HMWS (%) at the start of each evaluation sample was calculated by area percentage method using size exclusion chromatography using AdvanceBio SEC 300A 2.7 ⁇ m 4.6 ⁇ 150 mm (Agilent). A mobile phase of 0.2 M Ki/200 mM KCl/pH 7.0 was used, and analysis was performed at a flow rate of 0.2 mL/min (detection wavelength: 280 nm). In the accelerated deterioration test of each sample, after storage at 40°C for 1 day, 7 days, and 14 days, size exclusion chromatography was performed under the same conditions as above, and the HMWS (%) of each sample was calculated by the area percentage method. did.
- Fig. 1 shows the results showing changes over time when each sample was stored at 40°C.
- the HMWS (%) of C3E-0003 after storage for 14 days was 3%, which was significantly less than that of C3E-0002, which was 15%.
- the HMWS (%) of C3E-0004 after storage for 14 days was 13%, which increased compared to C3E-0003 but showed a downward trend compared to C3E-0002.
- a mobile phase of 0.2 M Ki/200 mM KCl/pH 7.0 was used, and analysis was performed at a flow rate of 0.2 mL/min (detection wavelength: 280 nm).
- each sample was shaken at room temperature at 2500 rpm for 1 hour and 2 hours using a universal high-speed mixer (manufactured by Waken), and then subjected to size exclusion chromatography under the same conditions as above. was performed to calculate the peak area and HMWS (%) of each sample.
- Figures 2A and 2B show the peak area and HMWS (%) of each sample after shaking.
- C3E-0002 markedly increased HMWS (%) 1 hour after shaking, and 99% of the peak area disappeared in samples treated 2 hours after shaking.
- C3E-0003 showed significantly less decrease in peak area and increase in HMWS (%) with the passage of shaking time.
- C3E-0004 had a lower HMWS (%) in the sample treated with shaking for 1 hour, and the peak area in the sample treated with shaking for 2 hours remained at 74% disappearance.
- heterodimeric Fc-type molecules using heterodimeric Fc-type and C3E-7096 using C3E-7085 Fab format have shaking stability. was excellent.
- Example 3 Preparation of taFv-heterodimeric Fc type and scFv-Fab-heterodimeric Fc type bispecific molecule 4)-1 Preparation of taFv-heterodimeric Fc type bispecific molecule expression vector pcDNA3.1 , pcDNA3.3 or pcDNA3.4 (Thermo Fisher Scientific) as a backbone, taFv-heterodimer Fc-type bispecific molecule expression vectors were designed.
- Anti-CD3 antibody fragments are C3E-7085 (WO2018/117237), or heterodimeric Fc sequences using C3E-7096 (HC_h and HC_k are described below), (Nat Biotechnol. 1998 Jul; 16 (7) 677-81 .) were used.
- HT1-11scFv and C3E-7085 described in International Publication No. 2018/117237 are connected to the carboxyl terminal of taFv with a GGGGS linker Mutation that forms a heteromultimer
- An expression vector for mammalian cells was prepared in which a DNA fragment encoding the amino acid sequence contained in a polypeptide to which an Fc region was introduced was incorporated, and named "p_TX3-0001-HC_k".
- a DNA fragment encoding an amino acid sequence contained in a polypeptide obtained by adding an Fc region introduced with a mutation that forms a heteromultimer to the carboxyl terminal of taFv in which HT1-11scFv and C3E-7096 are connected with a GGGGS linker was prepared and named "p_TX3-0003-HC_k".
- hM23-H1L1scFv a sequence obtained by adding glycine to the N-terminal of the hM23-H1 heavy chain variable region described in JP-A-2017-114763 and the hM23-L1 light chain variable region (GGGGS ) to the carboxyl terminus of taFv linked with anti-CD98scFv (hereinafter referred to as hM23-H1L1scFv) and C3E-7085 linked via a polypeptide linker consisting of a sequence repeated three times with a GGGGS linker, a mutation that forms a heteromultimer
- a mammalian cell expression vector was constructed in which a DNA fragment encoding the amino acid sequence contained in the polypeptide to which the introduced Fc region was added was constructed and named "p_CX3-0001-HC_k".
- a DNA encoding an amino acid sequence contained in a polypeptide obtained by adding an Fc region introduced with a mutation that forms a heteromultimer to the carboxyl terminal of taFv in which hM23-H1L1 scFv and C3E-7096 are connected with a GGGGS linker A mammalian cell expression vector incorporating the fragment was constructed and named "p_CX3-0003-HC_k".
- C3022scFv and C3E-7085 described in International Publication No. 2018/147245 are connected to the carboxyl terminus of taFv with a GGGGS linker, introducing a mutation that forms a heteromultimer
- a mammalian cell expression vector was constructed in which a DNA fragment encoding an amino acid sequence contained in a polypeptide to which the above Fc region was added was constructed and named "p_RX3-0001-HC_k".
- a DNA fragment encoding an amino acid sequence contained in a polypeptide obtained by adding an Fc region introduced with a mutation that forms a heteromultimer to the carboxyl terminus of taFv connecting C3022scFv and C3E-7096 with a GGGGS linker is incorporated.
- a mammalian cell expression vector was constructed and named "p_RX3-0003-HC_k".
- nucleotide sequence of p_TX3-0001-HC_k was reanalyzed, and it was confirmed that the full-length nucleotide sequence of TX3-0001-HC_k is the nucleotide sequence shown in SEQ ID NO: 14 (Fig. 18) in the sequence listing.
- nucleotide sequence of p_TX3-0003-HC_k was reanalyzed, and it was confirmed that the full-length nucleotide sequence of TX3-0003-HC_k is the nucleotide sequence shown in SEQ ID NO: 15 (Fig. 19) in the sequence listing.
- nucleotide sequence of p_CX3-0001-HC_k was reanalyzed, and it was confirmed that the full-length nucleotide sequence of CX3-0001-HC_k is the nucleotide sequence shown in SEQ ID NO: 16 (Fig. 20) in the sequence listing.
- nucleotide sequence of p_CX3-0003-HC_k was reanalyzed, and it was confirmed that the full-length nucleotide sequence of CX3-0003-HC_k is the nucleotide sequence shown in SEQ ID NO: 17 (Fig. 21) in the sequence listing.
- the nucleotide sequence of p_RX3-0001-HC_k was reanalyzed, and it was confirmed that the full-length RX3-0001-HC_k nucleotide sequence is the nucleotide sequence shown in SEQ ID NO: 18 (Fig. 22) in the sequence listing.
- the nucleotide sequence of p_RX3-0003-HC_k was reanalyzed, and it was confirmed that the full-length RX3-0003-HC_k nucleotide sequence is the nucleotide sequence shown in SEQ ID NO: 19 (Fig. 23) in the sequence listing.
- TX3-0001-HC_k TX3-0003-HC_k, CX3-0001-HC_k, CX3-0003-HC_k, RX3-0001-HC_k, RX3-0003-HC_k full-length amino acids encoded by the sequences Checked the sequence.
- the full-length amino acid sequence of TX3-0001-HC_k is the amino acid sequence shown in SEQ ID NO: 20 (Fig. 24) in the sequence listing.
- the amino acid sequence from 1st to 19th is a signal sequence
- the amino acid sequence from 21st to 510th is taFv connecting HT1-11scFv and C3E-7085 with a GGGGS linker
- the amino acid sequence from 511th to 512th is The amino acid sequence is the linker
- the amino acid sequence from 513th to 744th is HC_k.
- the full-length amino acid sequence of TX3-0003-HC_k is the amino acid sequence shown in SEQ ID NO: 21 (Fig. 25) in the sequence listing.
- the amino acid sequence from 1st to 19th is a signal sequence
- the amino acid sequence from 21st to 510th is taFv connecting HT1-11scFv and C3E-7096 with a GGGGS linker
- the amino acid sequence from 511th to 512th is the linker
- the amino acid sequence from 513th to 744th is HC_k.
- the full-length amino acid sequence of CX3-0001-HC_k is the amino acid sequence shown in SEQ ID NO: 22 (Fig. 26) in the sequence listing.
- the 1st to 19th amino acid sequences are signal sequences
- the 21st to 509th amino acid sequences are taFv in which hM23-H1L1scFv and C3E-7085 are linked with a GGGGS linker
- the 510th to 511th amino acid sequences are The amino acid sequence is the linker
- the amino acid sequence from 512th to 743rd is HC_k.
- the full-length amino acid sequence of CX3-0003-HC_k is the amino acid sequence shown in SEQ ID NO: 23 (Fig. 27) in the sequence listing.
- the 1st to 19th amino acid sequences are signal sequences
- the 21st to 509th amino acid sequences are taFv in which hM23-H1L1scFv and C3E-7096 are linked with a GGGGS linker
- the 510th to 511th amino acid sequences are The amino acid sequence is the linker
- the amino acid sequence from 512th to 743rd is HC_k.
- the amino acid sequence of the full-length RX3-0001-HC_k is the amino acid sequence shown in SEQ ID NO: 24 (Fig. 28) in the sequence listing.
- the 1st to 19th amino acid sequence is a signal sequence
- the 21st to 505th amino acid sequences are taFv connecting C3022scFv and C3E-7085 with a GGGGS linker
- the 506th to 507th amino acid sequences is a linker
- the amino acid sequence from 508th to 739th is HC_k.
- the amino acid sequence of the full-length RX3-0003-HC_k is the amino acid sequence shown in SEQ ID NO: 25 (Fig. 29) in the Sequence Listing.
- the 1st to 19th amino acid sequence is a signal sequence
- the 21st to 505th amino acid sequences are taFv connecting C3022scFv and C3E-7096 with a GGGGS linker
- the 506th to 507th amino acid sequences is a linker
- the amino acid sequence from 508th to 739th is HC_k.
- TX3-0001 Expression of -2 taFv-heterodimeric Fc type bispecific molecule 2
- p_TX3-0001-HC_k p_HC_h Anti-Trop2-CD3 taFv-heterodimer Fc (TX3-0001 ) was prepared for expression.
- the amino acid sequences obtained by expressing each vector constituting TX3-0001 are shown in SEQ ID NOs: 9 (FIG. 13) and 20 (FIG. 24) in the sequence listing.
- TX3-0003-HC_k and p_HC_h were used to express and prepare the culture supernatant of anti-Trop2-CD3taFv-heterodimer Fc (TX3-0003).
- the amino acid sequences obtained by expressing each vector constituting TX3-0003 are shown in SEQ ID NOs: 9 (FIG. 13) and 21 (FIG. 25) in the sequence listing.
- p_CX3-0001-HC_k and p_HC_h were used to express and prepare the culture supernatant of anti-CD98-CD3taFv-heterodimer Fc (CX3-0001).
- the amino acid sequences obtained by expressing each vector constituting CX3-0001 are shown in SEQ ID NOs: 9 (FIG. 13) and 22 (FIG. 26) in the sequence listing.
- p_CX3-0003-HC_k and p_HC_h were used to express and prepare the culture supernatant of anti-CD98-CD3taFv-heterodimer Fc (CX3-0003).
- the amino acid sequences obtained by expressing each vector constituting CX3-0003 are shown in SEQ ID NOs: 9 (FIG. 13) and 23 (FIG. 27) in the sequence listing.
- p_RX3-0001-HC_k and p_HC_h were used to express and prepare the culture supernatant of anti-GPRC5D-CD3taFv-heterodimer Fc (RX3-0001).
- the amino acid sequences obtained by expressing each vector constituting RX3-0001 are shown in SEQ ID NOs: 9 (FIG. 13) and 24 (FIG. 28) in the sequence listing.
- p_RX3-0003-HC_k and p_HC_h were used to express and prepare the culture supernatant of anti-CD98-CD3taFv-heterodimer Fc (RX3-0003).
- the amino acid sequences obtained by expressing each vector constituting RX3-0003 are shown in SEQ ID NOs: 9 (FIG. 13) and 25 (FIG. 29) in the sequence listing.
- 4)-3 Purification of taFv-heterodimeric Fc bispecific molecule Each culture supernatant obtained in 4)-2 was purified in the same manner as in 2)-4. Purified protein samples were subjected to analytical SEC to determine purity and concentration before being used for various evaluations.
- HT1-11scFv As an anti-Trop2-CD3 bispecific molecule expression vector, HT1-11scFv, the heavy chain variable region of C3E-7085, the CH1 region derived from human IgG, and the Fc region introduced with a mutation that forms a heteromultimer are added in this order.
- a mammalian cell expression vector into which a DNA fragment encoding the amino acid sequence contained in the polypeptide was inserted was constructed and named "p_TX3-0002-HC_k".
- hM23-H1L1scFv As an anti-CD98-CD3 bispecific molecule expression vector, hM23-H1L1scFv, the heavy chain variable region of C3E-7085, the CH1 region derived from human IgG, and the Fc region introduced with a mutation that forms a heteromultimer are added in this order.
- a mammalian cell expression vector into which a DNA fragment encoding the amino acid sequence contained in the polypeptide was inserted was constructed and named "p_CX3-0002-HC_k".
- nucleotide sequence of p_TX3-0002-HC_k was re-analyzed, and it was confirmed that the full-length nucleotide sequence of TX3-0002-HC_k is the nucleotide sequence shown in SEQ ID NO: 26 (Fig. 30) in the sequence listing.
- nucleotide sequence of p_CX3-0002-HC_k was re-analyzed, and it was confirmed that the full-length nucleotide sequence of CX3-0002-HC_k is the nucleotide sequence shown in SEQ ID NO: 27 (Fig. 31) in the sequence listing.
- the nucleotide sequence of p_RX3-0002-HC_k was re-analyzed, and it was confirmed that the full-length RX3-0002-HC_k nucleotide sequence is the nucleotide sequence shown in SEQ ID NO: 28 (Fig. 32) in the sequence listing.
- the amino acid sequence of the full-length TX3-0002-HC_k is the amino acid sequence shown in SEQ ID NO: 29 (Fig. 33) in the sequence listing.
- the 1st to 19th amino acid sequence is the signal sequence
- the 21st to 265th amino acid sequence is HT1-11scFv
- the 266th to 270th amino acid sequence is the linker
- the 271st to 388th amino acid sequences are
- the second amino acid sequence is C3E-7085-heavy chain variable region.
- the amino acid sequence from 389th to 718th is the constant region.
- the full-length amino acid sequence of CX3-0002-HC_k is the amino acid sequence shown in SEQ ID NO: 30 (Fig. 34) in the sequence listing.
- the 1st to 19th amino acid sequences are signal sequences
- the 21st to 264th amino acid sequences are hM23-H1L1scFv
- the 265th to 269th amino acid sequences are linkers
- the 270th to 387th amino acid sequences are
- the second amino acid sequence is C3E-7085-heavy chain variable region.
- the amino acid sequence from 388th to 717th is the constant region.
- the amino acid sequence of the full-length RX3-0002-HC_k is the amino acid sequence shown in SEQ ID NO: 31 (Fig. 35) in the Sequence Listing.
- the 1st to 19th amino acid sequences are signal sequences
- the 21st to 260th amino acid sequences are C3022scFv
- the 261st to 265th amino acid sequences are linkers
- the 266th to 383rd amino acid sequences are The amino acid sequence is C3E-7085-heavy chain variable region.
- the amino acid sequence from 384th to 713th is the constant region.
- p_CX3-0002-HC_k, p_HC_h and p_C3E-7085-LC were used to express and prepare the culture supernatant of anti-CD98scFv-CD3Fab-heterodimer Fc (TX3-0002).
- the amino acid sequences obtained by expressing each vector constituting CX3-0002 are shown in SEQ ID NOs: 9 (FIG. 13), 12 (FIG. 16) and 30 (FIG. 34) in the sequence listing.
- p_RX3-0002-HC_k, p_HC_h and p_C3E-7085-LC were used to express and prepare the culture supernatant of anti-GPRC5DscFv-CD3Fab-heterodimer Fc (RX3-0002).
- the amino acid sequences obtained by expressing each vector constituting RX3-0002 are shown in SEQ ID NOs: 9 (FIG. 13), 12 (FIG. 16) and 31 (FIG. 35) in the sequence listing.
- Each culture supernatant obtained in 4)-5 was purified in the same manner as in 2)-4.
- Example 4 Solution stability evaluation of taFv-heterodimeric Fc type and scFv-Fab-heterodimeric Fc type bispecific molecules Using each sample prepared in Example 3, Amicon Ultra-4 (Millipore ), and after buffer exchange with PBS, PBS was added to adjust the sample concentration to 20 mg/mL, which was used as an evaluation sample.
- the HMWS (%) at the start of each evaluation sample was calculated by area percentage method using size exclusion chromatography using AdvanceBio SEC 300A 2.7 ⁇ m 4.6 ⁇ 150 mm (Agilent).
- a mobile phase of 0.2 M Ki/200 mM KCl/pH 7.0 was used, and analysis was performed at a flow rate of 0.2 mL/min (detection wavelength: 280 nm).
- detection wavelength 280 nm.
- size exclusion chromatography was performed under the same conditions as above, and the HMWS (%) of each sample was calculated by the area percentage method. did.
- Figures 3A to 3C show the changes over time of each sample when stored at 40°C.
- the HMWS (%) amount of RX3-0001 increased to 13% over time.
- the HMWS (%) amounts of RX3-0002 and RX3-0003 decreased to 6% and 8%, respectively, with the passage of storage time (Fig. 3A).
- HMWS (%) due to the passage of storage time for TX3-0001 increased to 23%.
- HMWS (%) amounts of TX3-0002 and TX3-0003 decreased to 10% and 11%, respectively, with the passage of storage time (Fig. 3B).
- HMWS (%) due to the passage of storage time for CX3-0001 increased to 46%, reflecting the effect of solution stability of hM23-H1L1 scFv.
- the HMWS (%) amount of CX3-0002 was reduced to 28% with the passage of storage time, and the HMWS (%) amount of CX3-0003 was 43%, showing a slight decrease compared to CX3-0001 (Fig. 3C).
- Example 5 Shaking stability evaluation of taFv-heterodimeric Fc type and scFv-Fab-heterodimeric Fc type bispecific molecules
- RX3-0001 to 0003, CX3-0001 ⁇ 0003 and TX3-0001 ⁇ 0003 were concentrated by centrifugation using Amicon Ultra-4 (Millipore) and adjusted to 2 mg/mL to prepare samples for evaluation.
- the initial peak area and HMWS (%) of each evaluation sample were calculated by the area percentage method using size exclusion chromatography using AdvanceBio SEC 300A 2.7 ⁇ m 4.6 ⁇ 150 mm (Agilent).
- a mobile phase of 0.2 M Ki/200 mM KCl/pH 7.0 was used, and analysis was performed at a flow rate of 0.2 mL/min (detection wavelength 280 nm).
- each sample was shaken at room temperature at 2500 rpm for 1 hour and 2 hours using a universal high-speed mixer (manufactured by Waken), and then subjected to size exclusion chromatography under the same conditions as above. was performed to calculate the peak area and HMWS (%) of each sample.
- HMWS (%) after shaking of each sample are shown in Figures 4-1A and B, Figures 4-2A and B, and Figures 4-3A and B.
- RX3-0001 had a peak area retention rate of 2% in samples treated with shaking for 2 hours.
- the peak area residual rates of RX3-0002 and RX3-0003 were 15% and 17%, respectively, which were better results than RX3-0001.
- the HMWS (%) of the sample treated with shaking for 2 hours of RX3-0001 was 30%, but the HMWS (%) of the samples treated with shaking for 2 hours of RX3-0002 and RX3-0003 were both 5% or less.
- HMWS (%) of RX3-0002 and RX3-0003 were smaller than that of RX3-0001.
- CX3-0001 the residual rate of peak area in samples treated with shaking for 2 hours was 6%.
- CX3-0002 and CX3-0003 had peak area retention rates of 11% and 26%, respectively, which were better results than CX3-0001.
- the HMWS (%) of CX3-0001, CX3-0002, and CX3-0003 samples treated for 2 hours with shaking were 54%, 17%, and 41%, respectively.
- the HMWS (%) of CX3-0003 was small.
- TX3-0001 had a peak area retention rate of 22% in samples treated with shaking for 2 hours.
- TX3-0002 and TX3-0003 had a peak area retention rate of 81% and 41%, respectively, for samples treated with shaking for 2 hours, and compared with TX3-0001, good results were obtained regarding the peak area retention rate. was taken.
- the HMWS (%) of the TX3-0001, TX3-0002, and TX3-0003 samples treated with shaking for 2 hours were 7%, 21%, and 13%, respectively, and the HMWS (%) of TX3-0001 was the highest. It showed a tendency different from that of other antibody groups (RX3, CX3).
- TX3-0001 had a particle count of 1272905 particles/mL in a sample treated with shaking for 2 hours.
- TX3-0002 and TX3-0003 had 55243 particles/mL and 562921 particles/mL, respectively, in samples treated with shaking for 2 hours.
- Aggregates detected by HMWS have a diameter of about 10 to 100 nm, whereas FlowCAM detects large multimers of 1 ⁇ m or more. Since the number of particles of TX3-0001 is significantly larger than that of TX3-0002 and TX3-0003, the reason why HMWS (%) of TX3-0001 is low is that aggregates corresponding to HMWS associate to form larger multimers. This is presumed to be because From the above, it was shown that TX3-0002 and TX3-0003 have better shaking stability than TX3-0001.
- sample preparation, testing, evaluation, etc. can be carried out by replacing anti-CD3scFv with C3E-7096 to C3E-7097 (SEQ ID NO: 39, having the amino acid sequence shown in FIG. 46).
- Example 6 Solution stability evaluation of anti-CD3 antibody fragment-heterodimeric Fc-type bispecific molecule Example 1)-1 to 2)-4, C3E-7093 (International Publication No.
- HMWS % at the start of each evaluation sample is calculated by area percentage method using size exclusion chromatography using AdvanceBio SEC 300A 2.7 ⁇ m 4.6 ⁇ 150 mm (Agilent).
- a mobile phase of 0.2 M Ki/200 mM KCl/pH 7.0 is used, and analysis is performed at a flow rate of 0.2 mL/min (detection wavelength 280 nm).
- Example 7 Shaking stability evaluation of anti-CD3 antibody fragment-heterodimeric Fc type bispecific molecule Each sample purified in Example 6 was centrifuged and concentrated with Amicon Ultra-4 (Millipore) to 2 mg/ Prepare to mL and use as a sample for evaluation.
- the peak area and HMWS (%) at the start of each evaluation sample are calculated by area percentage method using size exclusion chromatography using AdvanceBio SEC 300A 2.7 ⁇ m 4.6 ⁇ 150 mm (Agilent).
- a mobile phase of 0.2 M Ki/200 mM KCl/pH 7.0 is used and analyzed at a flow rate of 0.2 mL/min (detection wavelength 280 nm).
- each sample was shaken over time at 2500 rpm for 1 hour and 2 hours at room temperature using a universal high-speed mixer (manufactured by Waken), and then subjected to size exclusion chromatography under the same conditions as above. to calculate the peak area and HMWS (%) of each sample.
- Example 8 Solution stability evaluation of taFv-heterodimeric Fc type and scFv-Fab-heterodimeric Fc type bispecific molecules
- HMWS % at the start of each evaluation sample is calculated by area percentage method using size exclusion chromatography using AdvanceBio SEC 300A 2.7 ⁇ m 4.6 ⁇ 150 mm (Agilent).
- a mobile phase of 0.2 M Ki/200 mM KCl/pH 7.0 is used, and analysis is performed at a flow rate of 0.2 mL/min (detection wavelength 280 nm).
- Example 9 Shaking stability evaluation of taFv-heterodimeric Fc type and scFv-Fab-heterodimeric Fc type bispecific molecules The sample prepared in Example 8 was subjected to Amicon Ultra-4 (Millipore).
- HMWS % at the start of each evaluation sample are calculated by area percentage method using size exclusion chromatography using AdvanceBio SEC 300A 2.7 ⁇ m 4.6 ⁇ 150 mm (Agilent).
- a mobile phase of 0.2 M Ki/200 mM KCl/pH 7.0 is used and analyzed at a flow rate of 0.2 mL/min (detection wavelength 280 nm).
- each sample was shaken at room temperature at 2500 rpm for 1 hour and 2 hours using a universal high-speed mixer (manufactured by Waken), and then subjected to size exclusion chromatography under the same conditions as above. to calculate the peak area and HMWS (%) of each sample.
- sample preparation, testing, evaluation, etc. can be performed by replacing anti-CD3scFv with C3E-7098 to C3E-7099 (SEQ ID NO: 49, having the amino acid sequence shown in Figure 51).
- the bispecific antibody of the present invention can be used as a therapeutic or preventive agent for cancer and the like.
- SEQ ID NO: 1 amino acid sequence of C3E-7085-Fab
- Figure 5 amino acid sequence of C3E-7085-LC
- Figure 6 Amino acid sequence of C3E-7085-LC
- Figure 7-9 amino acid sequence of C3E-7096
- Figure 7 amino acid sequence of C3E-7096
- Figure 8 SEQ ID NO: 5: C3E-7085-HC-k full length nucleotide sequence
- Figure 9 SEQ ID NO: 6: C3E-7085-Fab-HC-k full length nucleotide sequence
- Figure 11 SEQ ID NO: 8: C3E-7096-HC-k full length nucleotide sequence
- Figure 12 SEQ ID NO: 9: HC_h full length amino acid sequence (Fig.
- SEQ ID NO: 10 C3E-7085-HC-k full-length amino acid sequence
- Figure 14 SEQ ID NO: 11: C3E-7085-Fab-HC-k full length amino acid sequence
- SEQ ID NO: 12 C3E-7085-LC full length amino acid sequence
- Figure 16 SEQ ID NO: 13: C3E-7096-HC-k full length amino acid sequence
- Figure 17 SEQ ID NO: 14: Nucleotide sequence of TX3-0001-HC_k full length
- SEQ ID NO: 15 Nucleotide sequence of TX3-0003-HC_k full length
- Figure 19 SEQ ID NO: 16: CX3-0001-HC_k full length nucleotide sequence
- Figure 20 SEQ ID NO: 17: CX3-0003-HC_k full length nucleotide sequence
- Figure 21 SEQ ID NO: 18: RX3-0001-HC_k full length nucleotide sequence
- Figure 22 SEQ ID NO: 19: CX
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Abstract
Description
[1] 下記CDRH1~3及びCDRL1~3を含む、CD3に特異的に結合するFab:
配列番号32で示されるアミノ酸配列からなるCDRH1;
配列番号33で示されるアミノ酸配列からなるCDRH2;
配列番号34で示されるアミノ酸配列からなるCDRH3;
配列番号35で示されるアミノ酸配列からなるCDRL1;
配列番号36で示されるアミノ酸配列からなるCDRL2;及び
配列番号37で示されるアミノ酸配列からなるCDRL3。
[2] 配列番号1で示されるアミノ酸配列のアミノ酸番号1~118からなる重鎖可変領域及び配列番号2で示されるアミノ酸配列のアミノ酸番号1~107からなる軽鎖可変領域を含む、[1]のFab。
[3] 配列番号1で示されるアミノ酸配列からなるポリペプチド及び配列番号2で示されるアミノ酸配列からなる軽鎖を含む、[1]又は[2]のFab。
[4] [1]~[3]のいずれか一つのFabを含み、がん精巣抗原ペプチドと主要組織適合性遺伝子複合体(MHC)の複合体ではない標的分子に特異的に結合する、多重特異性分子。
[5] 当該MHCがヒト白血球抗原(HLA)である、[4]の多重特異性分子。
[6] 当該標的分子に特異的に結合する抗体又はその抗原結合断片を含む、[4]又は[5]の多重特異性分子。
[7] 多重特異性抗体である、[4]~[6]のいずれか一つの多重特異性分子。
[8] scFv-Fab-ヘテロダイマーFcを含む、[4]~[7]のいずれか一つの多重特異性分子。
[9] 三重特異性抗体である、[4]~[8]のいずれか一つの多重特異性分子。
[10] 二重特異性抗体である、[4]~[8]のいずれか一つの多重特異性分子。
[11] 標的分子がGPRC5D、CD98又はTrop2である、[4]~[10]のいずれか一つの多重特異性分子。
[12] 下記(i)乃至(iii)から選択される一のポリペプチド群を含む、[4]~[11]のいずれか一つの多重特異性分子:
(i)配列番号29の20~718番目のアミノ酸残基からなるアミノ酸配列又は該アミノ酸配列のカルボキシル末端において1若しくは2アミノ酸が欠失してなるアミノ酸配列を含むポリペプチド、配列番号9の20~246番目のアミノ酸残基からなるアミノ酸配列又は該アミノ酸配列のカルボキシル末端において1若しくは2アミノ酸が欠失してなるアミノ酸配列を含むポリペプチド及び配列番号12の21~233番目のアミノ酸残基からなるアミノ酸配列を含むポリペプチド;
(ii)配列番号30の20~717番目のアミノ酸残基からなるアミノ酸配列又は該アミノ酸配列のカルボキシル末端において1若しくは2アミノ酸が欠失してなるアミノ酸配列を含むポリペプチド、配列番号9の20~246番目のアミノ酸残基からなるアミノ酸配列又は該アミノ酸配列のカルボキシル末端において1若しくは2アミノ酸が欠失してなるアミノ酸配列を含むポリペプチド及び配列番号12の21~233番目のアミノ酸残基からなるアミノ酸配列を含むポリペプチド;
(iii)配列番号31の20~713番目のアミノ酸残基からなるアミノ酸配列又は該アミノ酸配列のカルボキシル末端において1若しくは2アミノ酸が欠失してなるアミノ酸配列を含むポリペプチド、配列番号9の20~246番目のアミノ酸残基からなるアミノ酸配列又は該アミノ酸配列のカルボキシル末端において1若しくは2アミノ酸が欠失してなるアミノ酸配列を含むポリペプチド及び配列番号12の21~233番目のアミノ酸残基からなるアミノ酸配列を含むポリペプチド。
[13] [4]~[12]のいずれか一つの多重特異性分子に含まれるアミノ酸配列をコードする塩基配列を含むポリヌクレオチド。
[14] [13]のポリヌクレオチドを含むベクター。
[15] [13]のポリヌクレオチド又は[14]のベクターを含むか、又は、[4]~[12]のいずれか一つの多重特異性分子を産生する細胞。
[16] [15]の細胞を培養する工程を含む、[4]~[12]のいずれか一つの多重特異性分子の製造方法。
[17] [16]の方法により得られる多重特異性分子。
[18] [1]~[3]のいずれか一つのFab、[4]~[12]若しくは[17]のいずれか一つの多重特異性分子、[13]のポリヌクレオチド、[14]のベクター、又は、[15]の細胞を含む組成物。
[19] 下記CDRH1~3及びCDRL1~3を含む、CD3に特異的に結合し、重鎖可変領域及び軽鎖可変領域がジスルフィド結合してなるscFv:
配列番号32で示されるアミノ酸配列からなるCDRH1;
配列番号33で示されるアミノ酸配列からなるCDRH2;
配列番号34で示されるアミノ酸配列からなるCDRH3;
配列番号35で示されるアミノ酸配列からなるCDRL1;
配列番号36で示されるアミノ酸配列からなるCDRL2;及び
配列番号37で示されるアミノ酸配列からなるCDRL3。
[20] 配列番号3で示されるアミノ酸配列のアミノ酸番号1~118からなる重鎖可変領域及び配列番号3で示されるアミノ酸配列のアミノ酸番号134~240からなる軽鎖可変領域を含む、[19]のscFv。
[21] 配列番号3で示されるアミノ酸配列からなる、[19]又は[20]のscFv。
[22] 配列番号39で示されるアミノ酸配列のアミノ酸番号1~118からなる重鎖可変領域及び配列番号39で示されるアミノ酸配列のアミノ酸番号134~240からなる軽鎖可変領域を含む、[19]のscFv。
[23] 配列番号39で示されるアミノ酸配列からなる、[19]又は[22]のscFv。
[24] [19]~[23]のいずれか一つのscFvを含み、がん精巣抗原ペプチドと主要組織適合性遺伝子複合体(MHC)の複合体ではない標的分子に特異的に結合する、多重特異性分子。
[25] 当該MHCがヒト白血球抗原(HLA)である、[24]の多重特異性分子。
[26] 当該標的分子に特異的に結合する抗体又はその抗原結合断片を含む、[24]又は[25]の多重特異性分子。
[27] 多重特異性抗体である、[24]~[26]のいずれか一つの多重特異性分子。
[28] taFv-ヘテロダイマーFcを含む、[24]~[27]のいずれか一つの多重特異性分子。
[29] 三重特異性抗体である、[24]~[28]のいずれか一つの多重特異性分子。
[30] 二重特異性抗体である、[24]~[28]のいずれか一つの多重特異性分子。
[31] 標的分子がGPRC5D、CD98又はTrop2である、[24]~[30]のいずれか一つの多重特異性分子。
[32] 下記(i)~(iii)から選択される一のポリペプチド群を含む、[24]~[31]のいずれか一つの多重特異性分子:
(i)配列番号21の20~744番目のアミノ酸残基からなるアミノ酸配列又は該アミノ酸配列のカルボキシル末端において1若しくは2アミノ酸が欠失してなるアミノ酸配列を含むポリペプチド及び配列番号9の20~246番目のアミノ酸残基からなるアミノ酸配列又は該アミノ酸配列のカルボキシル末端において1若しくは2アミノ酸が欠失してなるアミノ酸配列を含むポリペプチド;
(ii)配列番号23の20~743番目のアミノ酸残基からなるアミノ酸配列又は該アミノ酸配列のカルボキシル末端において1若しくは2アミノ酸が欠失してなるアミノ酸配列を含むポリペプチド及び配列番号9の20~246番目のアミノ酸残基からなるアミノ酸配列又は該アミノ酸配列のカルボキシル末端において1若しくは2アミノ酸が欠失してなるアミノ酸配列を含むポリペプチド;
(iii)配列番号25の20~739番目のアミノ酸残基からなるアミノ酸配列又は該アミノ酸配列のカルボキシル末端において1若しくは2アミノ酸が欠失してなるアミノ酸配列を含むポリペプチド及び配列番号9の20~246番目のアミノ酸残基からなるアミノ酸配列又は該アミノ酸配列のカルボキシル末端において1若しくは2アミノ酸が欠失してなるアミノ酸配列を含むポリペプチド。
[33] [24]~[32]のいずれか一つの多重特異性分子に含まれるアミノ酸配列をコードする塩基配列を含むポリヌクレオチド。
[34] [33]のポリヌクレオチドを含むベクター。
[35] [33]のポリヌクレオチド又は[34]のベクターを含むか、又は、[24]~[32]のいずれか一つの多重特異性分子を産生する細胞。
[36] [35]の細胞を培養する工程を含む、[24]~[32]のいずれか一つの多重特異性分子の製造方法。
[37] [36]の方法により得られる多重特異性分子。
[38] [19]~[23]のいずれか一つのscFv、[24]~[32]若しくは[37]のいずれか一つの多重特異性分子、[33]のポリヌクレオチド、[34]のベクター、又は、[35]の細胞を含む組成物。
[39] 下記CDRH1~3及びCDRL1~3を含む、CD3に特異的に結合するFab:
配列番号40で示されるアミノ酸配列からなるCDRH1;
配列番号41で示されるアミノ酸配列からなるCDRH2;
配列番号42で示されるアミノ酸配列からなるCDRH3;
配列番号43で示されるアミノ酸配列からなるCDRL1;
配列番号44で示されるアミノ酸配列からなるCDRL2;及び
配列番号45で示されるアミノ酸配列からなるCDRL3。
[40] 配列番号46で示されるアミノ酸配列のアミノ酸番号1~118からなる重鎖可変領域及び配列番号47で示されるアミノ酸配列のアミノ酸番号1~109からなる軽鎖可変領域を含む、[39]のFab。
[41] 配列番号46で示されるアミノ酸配列からなるポリペプチド及び配列番号47で示されるアミノ酸配列からなる軽鎖を含む、[39]又は[40]のFab。
[42] [39]~[41]のいずれか一つのFabを含み、がん精巣抗原ペプチドと主要組織適合性遺伝子複合体(MHC)の複合体ではない標的分子に特異的に結合する、多重特異性分子。
[43] 当該MHCがヒト白血球抗原(HLA)である、[42]の多重特異性分子。
[44] 当該標的分子に特異的に結合する抗体又はその抗原結合断片を含む、[42]又は[43]の多重特異性分子。
[45] 多重特異性抗体である、[42]~[44]のいずれか一つの多重特異性分子。
[46] 二重特異性抗体である、[42]~[45]のいずれか一つの多重特異性分子。
[47] [42]~[46]のいずれか一つの多重特異性分子に含まれるアミノ酸配列をコードする塩基配列を含むポリヌクレオチド。
[48] [47]のポリヌクレオチドを含むベクター。
[49] [47]のポリヌクレオチド又は[48]のベクターを含むか、又は、[42]~[46]のいずれか一つの多重特異性分子を産生する細胞。
[50] [49]の細胞を培養する工程を含む、[34]~[38]のいずれか一つの多重特異性分子の製造方法。
[51] [50]の方法により得られる多重特異性分子。
[52] [39]~[41]のいずれか一つのFab、[42]~[46]のいずれか一つの多重特異性分子、[47]のポリヌクレオチド、[48]のベクター、又は、[49]の細胞を含む組成物。
[53] 下記CDRH1~3及びCDRL1~3を含む、CD3に特異的に結合し、重鎖可変領域及び軽鎖可変領域がジスルフィド結合してなるscFv:
配列番号40で示されるアミノ酸配列からなるCDRH1;
配列番号41で示されるアミノ酸配列からなるCDRH2;
配列番号42で示されるアミノ酸配列からなるCDRH3;
配列番号43で示されるアミノ酸配列からなるCDRL1;
配列番号44で示されるアミノ酸配列からなるCDRL2;及び
配列番号45で示されるアミノ酸配列からなるCDRL3。
[54] 配列番号48で示されるアミノ酸配列のアミノ酸番号1~118からなる重鎖可変領域及び配列番号48で示されるアミノ酸配列のアミノ酸番号134~242からなる軽鎖可変領域を含む、[53]のscFv。
[55] 配列番号48で示されるアミノ酸配列からなる、[53]又は[54]のscFv。
[56] 配列番号49で示されるアミノ酸配列のアミノ酸番号1~118からなる重鎖可変領域及び配列番号49で示されるアミノ酸配列のアミノ酸番号134~242からなる軽鎖可変領域を含む、[53]のscFv。
[57] 配列番号49で示されるアミノ酸配列からなる、[53]又は[56]のscFv。
[58] [53]~[57]のいずれか一つのscFvを含み、がん精巣抗原ペプチドと主要組織適合性遺伝子複合体(MHC)の複合体ではない標的分子に特異的に結合する、多重特異性分子。
[59] 当該MHCがヒト白血球抗原(HLA)である、[58]の多重特異性分子。
[60] 当該標的分子に特異的に結合する抗体又はその抗原結合断片を含む、[58]又は[59]の多重特異性分子。
[61] 多重特異性抗体である、[58]~[60]のいずれか一つの多重特異性分子。
[62] 二重特異性抗体である、[58]~[61]のいずれか一つの多重特異性分子。
[63] [58]~[62]のいずれか一つの多重特異性分子に含まれるアミノ酸配列をコードする塩基配列を含むポリヌクレオチド。
[64] [63]のポリヌクレオチドを含むベクター。
[65] [63]のポリヌクレオチド又は[64]のベクターを含むか、又は、[58]~[62]のいずれか一つの多重特異性分子を産生する細胞。
[66] [65]の細胞を培養する工程を含む、[58]~[62]のいずれか一つの多重特異性分子の製造方法。
[67] [66]の方法により得られる多重特異性分子。
[68] [53]~[57]のいずれか一つのscFv、[58]~[62]のいずれか一つの多重特異性分子、[63]のポリヌクレオチド、[64]のベクター、又は、[65]の細胞を含む組成物。
[69] [4]~[12]、[24]~[32]、[42]~[46]及び[58]~[62]のいずれか一つの多重特異性分子を含む医薬組成物。
1.定義
本発明において、「遺伝子」とは、蛋白質のアミノ酸をコードする塩基配列が含まれるヌクレオチド鎖又はその相補鎖を意味し、例えば、蛋白質のアミノ酸をコードする塩基配列が含まれるヌクレオチド鎖又はその相補鎖であるポリヌクレオチド、オリゴヌクレオチド、DNA、mRNA、cDNA、cRNA等は「遺伝子」の意味に含まれる。かかる遺伝子は一本鎖、二本鎖又は三本鎖以上のヌクレオチドであり、DNA鎖とRNA鎖の会合体、一本のヌクレオチド鎖上にリボヌクレオチド(RNA)とデオキシリボヌクレオチド(DNA)が混在するもの及びそのようなヌクレオチド鎖を含む二本鎖又は三本鎖以上のヌクレオチドも「遺伝子」の意味に含まれる。本発明において塩基配列とヌクレオチド配列は同義である。
本発明において、「抗原」を「免疫原」の意味に用いることがある。
2.抗原
2-1.CD3抗原
本発明において、「CD3」は、CD3蛋白質と同じ意味で用いている。
2-2.標的分子
本発明において、標的分子は、生体に存在するCD3以外の分子であれば特に限定されないが、好適には、治療標的細胞(がん細胞、間質細胞、及び抑制性の免疫細胞を例示できるが、それらに限定されない)の表面に発現しており、CD3発現T細胞のリダイレクションにより当該がん細胞に細胞傷害活性を発揮させる分子であり、より好適には、がん精巣抗原ペプチドと主要組織適合性遺伝子複合体(MHC)の複合体ではない。
2-3.抗原の調製
本発明で用いる上述の抗原蛋白質;CD3及びがん精巣抗原ペプチドとヒト白血球抗原(HLA)の複合体ではない標的分子であるその他の抗原蛋白質は、動物組織(体液を含む)、該組織由来の細胞若しくは該細胞培養物からの精製及び単離、遺伝子組換え、インビトロ翻訳、化学合成等により調製することができる。
2-4.抗原蛋白質への結合特異性
本発明の多重特異性分子に含まれる抗CD3抗体及びその結合断片等は、CD3抗原を認識、すなわち、結合する。かかる抗CD3抗体等は、好適には、ヒトCD3、サルCD3等に結合し、より好適には、ヒトCD3及びカニクイザルCD3に結合する。一方、かかる好適な抗CD3抗体は、ラット及び/又はマウスのCD3には結合しない。
3.抗原蛋白質に特異的に結合する抗体又はその結合断片
3-1.CD3に特異的に結合する抗体又はその結合断片
本発明のCD3に特異的に結合する抗CD3抗体及び該抗体の抗原結合断片(以下、本発明の抗体等とも記す)は、モノクローナル抗体及びポリクローナル抗体のいずれであってもよい。本発明のモノクローナル抗体のアイソタイプは、特に限定されるものではなく、例えば、IgG1、IgG2、IgG3、IgG4等のIgG、IgM、IgA1、IgA2等のIgA、IgD、Ig等をあげることができる。モノクローナル抗体のアイソタイプ及びサブクラスは、例えば、オクテロニー法、ELISA法、RIA法等で決定することができる。本発明のモノクローナル抗体としては、非ヒト動物由来の抗体(非ヒト動物抗体)、ヒト抗体、キメラ化抗体(「キメラ抗体」ともいう)、ヒト化抗体などを挙げることができ、好ましくはヒト抗体を用いることができる。本発明の抗体の範囲には、抗体の変異体(後述の「変異抗体」)も含まれ、例えば、ヒト抗体の範囲には、ヒト変異抗体も含まれる。
3-2.標的分子に結合する抗体又はその結合断片
「2-2.標的分子」に結合する抗体又はその結合断片は、モノクローナル抗体及びポリクローナル抗体のいずれであってもよい。本発明のモノクローナル抗体のアイソタイプは、特に限定されるものではなく、例えば、IgG1、IgG2、IgG3、IgG4等のIgG、IgM、IgA1、IgA2等のIgA、IgD、Ig等をあげることができる。モノクローナル抗体のアイソタイプ及びサブクラスは、例えば、オクテロニー法、ELISA法、RIA法等で決定することができる。本発明のモノクローナル抗体としては、非ヒト動物由来の抗体(非ヒト動物抗体)、ヒト抗体、キメラ化抗体(「キメラ抗体」ともいう)、ヒト化抗体などを挙げることができ、好ましくはヒト抗体を用いることができる。本発明の抗体の範囲には、抗体の変異体(後述の「変異抗体」)も含まれ、例えば、ヒト抗体の範囲には、ヒト変異抗体も含まれる。
3-3.抗体又はその抗原結合断片の変異体
本発明の抗体又はその抗原結合断片の変異体には、好適には、蛋白質の分解若しくは酸化に対する感受性の低下、生物活性や機能の維持、改善若しくは低下や変化の抑制、抗原結合能の改善若しくは調節、又は理化学的性質若しくは機能的性質の付与等がなされ得る。蛋白質は、その表面にある特定のアミノ酸側鎖が変化して当該蛋白質の機能や活性が変化することが知られ、そのような例には、アスパラギン側鎖の脱アミド化、アスパラギン酸側鎖の異性化等が含まれる。そのようなアミノ酸側鎖の変化を防ぐために別のアミノ酸に置き換えたものも、本発明の変異体の範囲に含まれる。
3-4.抗体又はその結合断片の修飾体、複合体
本発明は、抗体又はその結合断片の修飾体を提供する。本発明の抗体又はその結合断片の修飾体とは、本発明の抗体又はその結合断片に化学的又は生物学的な修飾が施されてなるものを意味する。化学的な修飾体には、アミノ酸骨格への化学部分の結合、N-結合又はO-結合炭水化物鎖の化学修飾体等が含まれる。生物学的な修飾体には、翻訳後修飾(例えば、N-結合又はO-結合への糖鎖付加、アミノ末端領域又はカルボキシル末端領域のプロセッシング、脱アミド化、アスパラギン酸の異性化、メチオニンの酸化)されたもの、原核生物宿主細胞を用いて発現させることによりアミノ末端にメチオニン残基が付加したもの等が含まれる。また、本発明の抗体又は抗原の検出又は単離を可能にするために標識されたもの、例えば、酵素標識体、蛍光標識体、アフィニティー標識体もかかる修飾物の意味に含まれる。このような本発明の抗体又はその結合断片の修飾物は、元の本発明の抗体又はその結合断片の安定性及び血中滞留性の改善、抗原性の低減、かかる抗体又は抗原の検出又は単離等に有用である。
4.抗体又はその抗原結合断片の産生方法
本発明の抗体又はその抗原結合断片は、例えば、重鎖可変領域をコードするDNA又は軽鎖可変領域をコードするDNAを発現ベクターに挿入し、発現用の宿主細胞を該ベクターで形質転換し、宿主細胞を培養することにより、リコンビナント抗体として細胞に産生させることができる。
5.多重特異性分子
本発明の多重特異性分子は、本発明のCD3に結合するscFvであって重鎖可変領域と軽鎖可変領域がジスルフィド結合で結合したscFv、又は、CD3に結合するFab(以下、「本発明のCD3抗体」という)を含み、且つ、CD3以外の標的分子、好適には、がん精巣抗原ペプチドと主要組織適合性遺伝子複合体(MHC)の複合体ではない標的分子に結合する部分(moiety)(以下、「他の結合部分」という)を含む分子であれば特に限定されるものではなく、「本発明のCD3抗体」及び「他の結合部分」以外に、さらに他の群(group)(以下、「他の群」という)を含んでいてもよい。「他の結合部分」としては、抗体又はその結合断片、免疫グロブリン由来ではないタンパク質又はペプチド、核酸アプタマー等の核酸分子、低分子化合物、T細胞受容体等を例示することができる。「他の群」としては、生物学的活性、薬理作用、治療効果、予防効果、アジュバント作用、体内動態調節機能、又は標識機能等を奏する化合物(タンパク質、ペプチド、核酸分子、低分子化合物、放射性同位元素を含む化合物、標識物質を含む化合物、人工的に合成したもの、医薬成分、抗腫瘍剤等)、その前駆体(プロドラッグ等)、それらの化合物にリンカー又は連結部が結合又は融合してなるものを例示することができる。「本発明のCD3抗体」及び「他の結合部分」、並びに、任意で「他の群」は、互いに直接結合又は融合していても、リンカーや連結部を介して間接的に結合又は融合していてもよい。「他の結合部分」は1つ以上あればよく、2つ、3つ、又はそれ以上であってもよい。「他の群」は、任意で、1つ、2つ又はそれ以上であってもよい。それらは直線的に結合又は融合していても、分枝して結合又は融合していてもよく、結合又は融合の順序及び分枝の位置等に何ら限定はない。そのような本発明の多重特異性分子は、図38A~F、図39A及びB、図40A及びB、図41A及びB、図42、図43A及びB、図44A及びB、並びに、図45に例示されるフォーマットであるか、又は、例示されるフォーマットを含んでいてよいが、それらに限定されるものではない。
また、本発明の多重特異性分子がとり得るフォーマットとしては、図38~45に例示されるもの及びその他本願が開示するものに加え、Godar他 Expert Opinion on Therapeutic Patents 28巻(3号)251-276頁(2018年)、Brinkmann及びKnotermann MABS 9巻(2号)182-212頁(2017年)、並びに、Wu及びDemarest Methods 154巻 3-9頁(2019年)に記載された各種フォーマットも例示することができる。それらの文献の全記載も本願の開示に包含される。
(参考例1)C3E-7085の溶液安定性評価
国際公開第2018/117237号に記載のC3E-7085精製タンパク質(国際公開第2018/117237号の配列表の配列番号64、図72で示されるアミノ酸配列を有するscFvである。)を用いて、溶液安定性を評価した。C3E-7085は、Amicon Ultra-4(Millipore社)にて遠心濃縮し25mM酢酸ナトリウム、5%ソルビトール、pH5.5へバッファ交換後、濃度を10mg/mLに調製し、4℃で2週間若しくは40℃で2週間保存した。保存後のサンプルは、TSKgel UP-SW3000 2μm 4.6X 300mmを用いたサイズ排除クロマトグラフィー(SEC)を行った。移動相は3XPBSを用い、流速0.2mL/minにて分析した(検出波長280nm)。各サンプル中に含まれるHMWS(%)をピーク解析し、面積百分率法により算出、結果を表1に記載した。
(実施例1)抗CD3抗体フラグメント-ヘテロダイマーFc型分子の溶液安定性評価
2)-1 抗CD3抗体フラグメントのアミノ酸配列の設計
C3E-7085の溶液安定性における物性改善をめざし、フォーマットを変更したC3E-7085 Fabフォーマットのアミノ酸配列として、C3E-7085-Fab(配列番号1、図5)及びC3E-7085-LC(配列番号2、図6)を設計した。また、文献(Proteins,1994,May;19(1):35-47)を参考にジスルフィド導入結合を加えたscFvを設計しC3E-7096と命名した(配列番号3、図7)。
2)-2 抗CD3抗体フラグメント-ヘテロダイマーFc発現ベクターの作製
pcDNA3.1、pcDNA3.3あるいはpcDNA3.4(Thermo Fisher Scientific社)を骨格とした各種抗CD3抗体フラグメント-ヘテロダイマーFc発現用ベクターを設計した。抗CD3抗体フラグメントはC3E-7085(WO2018/117237)及び2)-1で設計した抗CD3抗体フラグメントを用いた。ヘテロダイマーFc配列(HC_h及びHC_kと以下記載する)には、文献(Nat Biotechnol.1998Jul;16(7)677-81.)に報告されたものを用いた。
2)-3 抗CD3抗体フラグメント-ヘテロダイマーFcの発現
Expi293F細胞(Thermo Fisher Scientific社)はマニュアルに従い、継代、培養をおこなった。対数増殖期にあるExpi293F細胞培養液を2.5×106 cells/mL になるようExpi293 Expression medium(Thermo Fisher Scientific社)で希釈し、各種抗CD3抗体フラグメント-ヘテロダイマーFcの生産に用いた。300mL培養する場合は、Polyethyleneimine(Polyscience #24765)1.44mgをOpti-Pro SFM(Thermo Fisher Scientific社)5mLに溶解、次にNucleoBond Xtra Midiキット(タカラバイオ社)を用いて調製したベクターp_C3E-7085-HC-k、p_HC_hを混合したベクター混合物0.3mgを、Opti-Pro SFM(Invitrogen社)5mLに添加した。Polyethyleneimine/Opti-Pro SFM混合液5mLに、発現ベクター/Opti-Pro SFM混合液5mLを加え穏やかに攪拌し、さらに5分間放置した後にExpi293F細胞に添加した。37℃、8%CO2インキュベーターで6日間、135rpmで振とう培養して得られた培養上清を0.2μm filter(Thermo Fisher Scientific社)でろ過し、抗CD3抗体フラグメント-ヘテロダイマーFc(C3E-0002)の培養上清を得た。C3E-0002を構成する各ベクターを発現させて得られるアミノ酸の配列を配列表の配列番号9(図13)及び10(図14)に示す。
2)-4 抗CD3抗体フラグメント-ヘテロダイマーFcの精製
2)-3で得られた培養上清から抗CD3抗体フラグメント-ヘテロダイマーFcをProteinAアフィニティクロマトグラフィーとゲルろ過クロマトグラフィーの2段階工程で精製した。
2)-5 抗CD3抗体フラグメント-ヘテロダイマーFcの溶液安定性評価
2)-4で精製した各サンプルは、Amicon Ultra-4(Millipore社)にて遠心濃縮しPBSへbuffer交換後、PBSを添加しサンプル濃度を20mg/mLに調製し評価用サンプルとした。また各評価用サンプルの開始時のHMWS(%)は、AdvanceBio SEC 300A 2.7μm 4.6×150mm(Agilent社)を用いたサイズ排除クロマトグラフィーを用い面積百分率法により算出した。移動相は0.2M Ki/200mM KCl/pH7.0を用い、流速0.2mL/minにて分析した(検出波長280nm)。各サンプルの加速劣化試験は、40℃で1日、7日、14日保存後、上記と同条件にてサイズ排除クロマトグラフィーを実施し、各サンプルのHMWS(%)を面積百分率法にて算出した。
(実施例2)抗CD3抗体フラグメント-ヘテロダイマーFcの振とう安定性評価
2)-4で精製した各サンプルをAmicon Ultra-4(Millipore社)にて遠心濃縮後2mg/mLに調製し、評価用サンプルとした。また各評価用サンプルの開始時のピーク面積及びHMWS(%)は、AdvanceBio SEC 300A 2.7μm 4.6×150mm(Agilent社)を用いたサイズ排除クロマトグラフィーを用い面積百分率法により算出した。移動相は0.2M Ki/200mM KCl/pH7.0を用い、流速0.2mL/minにて分析した(検出波長280nm)。振とう劣化試験は各サンプルをユニバーサル高速ミキサー(Waken製)を用いて、室温にて、2500rpmで1時間、2時間と経時的に振とうさせた後、上記と同条件にてサイズ排除クロマトグラフィーを実施し、各サンプルのピーク面積及びHMWS(%)を算出した。
(実施例3)taFv-ヘテロダイマーFc型及びscFv-Fab-ヘテロダイマーFc型二重特異性分子の作製
4)-1 taFv-ヘテロダイマーFc型二重特異性分子発現用ベクターの作製
pcDNA3.1、pcDNA3.3あるいはpcDNA3.4(Thermo Fisher Scientific社)を骨格としたtaFv-ヘテロダイマーFc型二重特異性分子発現用ベクターを設計した。抗CD3抗体フラグメントはC3E-7085(WO2018/117237)、あるいはC3E-7096を用いたヘテロダイマーFc配列(HC_h及びHC_kと以下記載する)には、(Nat Biotechnol.1998Jul;16(7)677-81.)に報告されたものを用いた。
4)-2 taFv-ヘテロダイマーFc型二重特異性分子の発現
2)-3と同様の手法にて、p_TX3-0001-HC_k、p_HC_hを用いて抗Trop2-CD3taFv-ヘテロダイマーFc(TX3-0001)の培養上清を発現調製した。TX3-0001を構成する各ベクターを発現させて得られるアミノ酸の配列を配列表の配列番号9(図13)及び20(図24)に示す。
4)-3 taFv-ヘテロダイマーFc型二重特異性分子の精製
4)-2で得られた各培養上清は、2)-4 と同様の手法で精製した。精製タンパク質サンプルは分析用SECに供し、純度と濃度を決定した後、各種評価に用いた。
4)-4 scFv-Fab-ヘテロダイマーFc型二重特異性分子発現用ベクターの作製
pcDNA3.1、pcDNA3.3あるいはpcDNA3.4(Thermo Fisher Scientific社)を骨格としたscFv-Fab-ヘテロダイマーFc型二重特異性分子発現用ベクターを設計した。抗CD3抗体フラグメントはC3E-7085(WO2018/117237)をFabフォーマットへ変更し用いた。ヘテロダイマーFc配列(HC_h及びHC_kと以下記載する)には、(Nat Biotechnol.1998Jul;16(7)677-81.)に報告されたものを用いた。
4)-5 scFv-Fab-ヘテロダイマーFc型二重特異性分子の発現
2)-3と同様の手法にて、p_TX3-0002-HC_k、p_HC_h及びp_C3E-7085-LCを用いて抗Trop2scFv-CD3Fab-ヘテロダイマーFc(TX3-0002)の培養上清を発現調製した。TX3-0002を構成する各ベクターを発現させて得られるアミノ酸の配列を配列表の配列番号9(図13)及び12(図16)及び29(図33)に示す。
4)-6 scFv-Fab-ヘテロダイマーFc型二重特異性分子の精製
4)-5で得られた各培養上清は、2)-4 と同様の手法で精製した。精製タンパク質サンプルは分析用SECに供し、純度と濃度を決定した後各種評価に用いた。
(実施例4)taFv-ヘテロダイマーFc型及びscFv-Fab-ヘテロダイマーFc型二重特異性分子の溶液安定性評価
実施例3にて調製した各サンプルを用いて、Amicon Ultra-4(Millipore社)にて遠心濃縮しPBSへbuffer交換後、PBSを添加しサンプル濃度を20mg/mLに調製し評価用サンプルとした。また各評価用サンプルの開始時のHMWS(%)は、AdvanceBio SEC 300A 2.7μm 4.6×150mm(Agilent社)を用いたサイズ排除クロマトグラフィーを用い面積百分率法により算出した。移動相は0.2M Ki/200mM KCl/pH7.0を用い、流速0.2mL/minにて分析した(検出波長280nm)。各サンプルの加速劣化試験は、40℃で1日、7日、14日保存後、上記と同条件にてサイズ排除クロマトグラフィーを実施し、各サンプルのHMWS(%)を面積百分率法にて算出した。
(実施例5)taFv-ヘテロダイマーFc型及びscFv-Fab-ヘテロダイマーFc型二重特異性分子の振とう安定性評価
実施例3で精製したサンプルのうち、RX3-0001~0003、CX3-0001~0003及びTX3-0001~0003をAmicon Ultra-4(Millipore社)にて遠心濃縮後2mg/mLに調製し、評価用サンプルとした。また各評価用サンプルの開始時のピーク面積及びHMWS(%)は、AdvanceBio SEC 300A 2.7μm 4.6×150mm(Agilent社)を用いたサイズ排除クロマトグラフィーを用い面積百分率法により算出した。移動相は0.2M Ki/200mM KCl/pH7.0を用い、流速0.2mL/min にて分析した(検出波長280nm)。振とう劣化試験は各サンプルをユニバーサル高速ミキサー(Waken製)を用いて、室温にて、2500rpmで1時間、2時間と経時的に振とうさせた後、上記と同条件にてサイズ排除クロマトグラフィーを実施し、各サンプルのピーク面積及びHMWS(%)を算出した。
(実施例6)抗CD3抗体フラグメント-ヘテロダイマーFc型二重特異性分子の溶液安定性評価
実施例1)-1~2)-4に準じて、C3E-7093(国際公開第2018/117237号の配列番号80、図88で示されるアミノ酸配列を有するscFvである。)の溶液安定性における物性改善をめざし、フォーマットがFabに変更されたものであるC3E-7093Fabのアミノ酸配列(配列番号46、図48で示されるC3E-7093-Fabのアミノ酸配列及び配列番号47、図49で示されるC3E-7093-LCのアミノ酸配列)及びジスルフィド結合が導入されたscFvであるC3E-7098のアミノ酸配列(配列番号48、図50で示されるアミノ酸配列)を設計する。次いで当該Fab及びscFvを含む抗CD3抗体フラグメント-ヘテロダイマーFc型分子を調製するための各発現ベクターを作製し、当該両分子を発現させ、精製する。
(実施例7)抗CD3抗体フラグメント-ヘテロダイマーFc型二重特異性分子の振とう安定性評価
実施例6で精製される各サンプルをAmicon Ultra-4(Millipore社)にて遠心濃縮後2mg/mLに調製し、評価用サンプルとする。また各評価用サンプルの開始時のピーク面積及びHMWS(%)は、AdvanceBio SEC 300A 2.7μm 4.6×150mm(Agilent社)を用いたサイズ排除クロマトグラフィーを用い面積百分率法により算出する。移動相は0.2M Ki/200mM KCl/pH7.0を用い、流速0.2mL/minにて分析する(検出波長280nm)。振とう劣化試験は各サンプルをユニバーサル高速ミキサー(Waken製)を用いて、室温にて、2500rpmで1時間、2時間と経時的に振とうさせた後、上記と同条件にてサイズ排除クロマトグラフィーを実施し、各サンプルのピーク面積及びHMWS(%)を算出する。
(実施例8)taFv-ヘテロダイマーFc型及びscFv-Fab-ヘテロダイマーFc型二重特異性分子の溶液安定性評価
実施例3に準じて、C3E-7098を抗CD3scFvとして含む抗Trop2-CD3taFv-ヘテロダイマーFc、抗CD98-CD3taFv-ヘテロダイマーFc及び抗GPRC5D-CD3taFv-ヘテロダイマーFc、並びに、C3E-7093 Fabを抗CD3Fabとして含む抗Trop2scFv-CD3Fab-ヘテロダイマーFc、抗CD98scFv-CD3Fab-ヘテロダイマーFc及び抗GPRC5DscFv-CD3Fab-ヘテロダイマーFcを調製する。
(実施例9)taFv-ヘテロダイマーFc型及びscFv-Fab-ヘテロダイマーFc型二重特異性分子の振とう安定性評価
実施例8で調製されるサンプルをAmicon Ultra-4(Millipore社)にて遠心濃縮後2mg/mLに調製し、評価用サンプルとする。また各評価用サンプルの開始時のピーク面積及びHMWS(%)は、AdvanceBio SEC 300A 2.7μm 4.6×150mm(Agilent社)を用いたサイズ排除クロマトグラフィーを用い面積百分率法により算出する。移動相は0.2M Ki/200mM KCl/pH7.0を用い、流速0.2mL/min にて分析する(検出波長280nm)。振とう劣化試験は各サンプルをユニバーサル高速ミキサー(Waken製)を用いて、室温にて、2500rpmで1時間、2時間と経時的に振とうさせた後、上記と同条件にてサイズ排除クロマトグラフィーを実施し、各サンプルのピーク面積及びHMWS(%)を算出する。
配列番号2:C3E-7085-LCのアミノ酸配列(図6)
配列番号3:C3E-7096のアミノ酸配列(図7)
配列番号4:HC_h全長のヌクレオチド配列(図8)
配列番号5:C3E-7085-HC-k全長のヌクレオチド配列(図9)
配列番号6:C3E-7085-Fab-HC-k全長のヌクレオチド配列(図10) 配列番号7:C3E-7085-LC全長のヌクレオチド配列(図11)
配列番号8:C3E-7096-HC-k全長のヌクレオチド配列(図12)
配列番号9:HC_h全長のアミノ酸配列(図13)
配列番号10:C3E-7085-HC-k全長のアミノ酸配列(図14)
配列番号11:C3E-7085-Fab-HC-k全長のアミノ酸配列(図15)
配列番号12:C3E-7085-LC全長のアミノ酸配列(図16)
配列番号13:C3E-7096-HC-k全長のアミノ酸配列(図17)
配列番号14:TX3-0001-HC_k全長のヌクレオチド配列(図18)
配列番号15:TX3-0003-HC_k全長のヌクレオチド配列(図19)
配列番号16:CX3-0001-HC_k全長のヌクレオチド配列(図20)
配列番号17:CX3-0003-HC_k全長のヌクレオチド配列(図21)
配列番号18:RX3-0001-HC_k全長のヌクレオチド配列(図22)
配列番号19:CX3-0001-HC_k全長のヌクレオチド配列(図23)
配列番号20:TX3-0001-HC_k全長のアミノ酸配列(図24)
配列番号21:TX3-0003-HC_k全長のアミノ酸配列(図25)
配列番号22:CX3-0001-HC_k全長のアミノ酸配列(図26)
配列番号23:CX3-0003-HC_k全長のアミノ酸配列(図27)
配列番号24:RX3-0001-HC_k全長のアミノ酸配列(図28)
配列番号25:RX3-0003-HC_k全長のアミノ酸配列(図29)
配列番号26:TX3-0002-HC_k全長のヌクレオチド配列(図30)
配列番号27:CX3-0002-HC_k全長のヌクレオチド配列(図31)
配列番号28:RX3-0002-HC_k全長のヌクレオチド配列(図32)
配列番号29:TX3-0002-HC_k全長のアミノ酸配列(図33)
配列番号30:CX3-0002-HC_k全長のアミノ酸配列(図34)
配列番号31:RX3-0002-HC_k全長のアミノ酸配列(図35)
配列番号32:C3E-7085重鎖CDRH1アミノ酸配列(図36)
配列番号33:C3E-7085重鎖CDRH2アミノ酸配列(図36)
配列番号34:C3E-7085重鎖CDRH3アミノ酸配列(図36)
配列番号35:C3E-7085軽鎖CDRL1アミノ酸配列(図36)
配列番号36:C3E-7085軽鎖CDRL2アミノ酸配列(図36)
配列番号37:C3E-7085軽鎖CDRL3アミノ酸配列(図36)
配列番号38:ペプチドリンカーのアミノ酸配列(図37)
配列番号39:C3E-7097のアミノ酸配列(図46)
配列番号40:C3E-7093重鎖CDRH1アミノ酸配列(図47)
配列番号41:C3E-7093重鎖CDRH2アミノ酸配列(図47)
配列番号42:C3E-7093重鎖CDRH3アミノ酸配列(図47)
配列番号43:C3E-7093軽鎖CDRL1アミノ酸配列(図47)
配列番号44:C3E-7093軽鎖CDRL2アミノ酸配列(図47)
配列番号45:C3E-7093軽鎖CDRL3アミノ酸配列(図47)
配列番号46:C3E-7093-Fabのアミノ酸配列(図48)
配列番号47:C3E-7093-LCのアミノ酸配列(図49)
配列番号48:C3E-7098のアミノ酸配列(図50)
配列番号49:C3E-7099のアミノ酸配列(図51)
本明細書で引用した全ての刊行物、特許及び特許出願はそのまま引用により本明細書に組み入れられるものとする。
Claims (69)
- 下記CDRH1~3及びCDRL1~3を含む、CD3に特異的に結合するFab:
配列番号32で示されるアミノ酸配列からなるCDRH1;
配列番号33で示されるアミノ酸配列からなるCDRH2;
配列番号34で示されるアミノ酸配列からなるCDRH3;
配列番号35で示されるアミノ酸配列からなるCDRL1;
配列番号36で示されるアミノ酸配列からなるCDRL2;及び
配列番号37で示されるアミノ酸配列からなるCDRL3。 - 配列番号1で示されるアミノ酸配列のアミノ酸番号1~118からなる重鎖可変領域及び配列番号2で示されるアミノ酸配列のアミノ酸番号1~107からなる軽鎖可変領域を含む、請求項1記載のFab。
- 配列番号1で示されるアミノ酸配列からなるポリペプチド及び配列番号2で示されるアミノ酸配列からなる軽鎖を含む、請求項1又は2記載のFab。
- 請求項1~3のいずれか一つに記載のFabを含み、がん精巣抗原ペプチドと主要組織適合性遺伝子複合体(MHC)の複合体ではない標的分子に特異的に結合する、多重特異性分子。
- 当該MHCがヒト白血球抗原(HLA)である、請求項4記載の多重特異性分子。
- 当該標的分子に特異的に結合する抗体又はその抗原結合断片を含む、請求項4又は5記載の多重特異性分子。
- 多重特異性抗体である、請求項4~6のいずれか一つに記載の多重特異性分子。
- scFv-Fab-ヘテロダイマーFcを含む、請求項4~7のいずれか一つに記載の多重特異性分子。
- 三重特異性抗体である、請求項4~8のいずれか一つに記載の多重特異性分子。
- 二重特異性抗体である、請求項4~8のいずれか一つに記載の多重特異性分子。
- 標的分子がGPRC5D、CD98又はTrop2である、請求項4~10のいずれか一つに記載の多重特異性分子。
- 下記(i)乃至(iii)から選択される一のポリペプチド群を含む、請求項4~11のいずれか一つに記載の多重特異性分子:
(i)配列番号29の20~718番目のアミノ酸残基からなるアミノ酸配列又は該アミノ酸配列のカルボキシル末端において1若しくは2アミノ酸が欠失してなるアミノ酸配列を含むポリペプチド、配列番号9の20~246番目のアミノ酸残基からなるアミノ酸配列又は該アミノ酸配列のカルボキシル末端において1若しくは2アミノ酸が欠失してなるアミノ酸配列を含むポリペプチド及び配列番号12の21~233番目のアミノ酸残基からなるアミノ酸配列を含むポリペプチド;
(ii)配列番号30の20~717番目のアミノ酸残基からなるアミノ酸配列又は該アミノ酸配列のカルボキシル末端において1若しくは2アミノ酸が欠失してなるアミノ酸配列を含むポリペプチド、配列番号9の20~246番目のアミノ酸残基からなるアミノ酸配列又は該アミノ酸配列のカルボキシル末端において1若しくは2アミノ酸が欠失してなるアミノ酸配列を含むポリペプチド及び配列番号12の21~233番目のアミノ酸残基からなるアミノ酸配列を含むポリペプチド;
(iii)配列番号31の20~713番目のアミノ酸残基からなるアミノ酸配列又は該アミノ酸配列のカルボキシル末端において1若しくは2アミノ酸が欠失してなるアミノ酸配列を含むポリペプチド、配列番号9の20~246番目のアミノ酸残基からなるアミノ酸配列又は該アミノ酸配列のカルボキシル末端において1若しくは2アミノ酸が欠失してなるアミノ酸配列を含むポリペプチド及び配列番号12の21~233番目のアミノ酸残基からなるアミノ酸配列を含むポリペプチド。 - 請求項4~12のいずれか一つに記載の多重特異性分子に含まれるアミノ酸配列をコードする塩基配列を含むポリヌクレオチド。
- 請求項13記載のポリヌクレオチドを含むベクター。
- 請求項13記載のポリヌクレオチド又は請求項14記載のベクターを含むか、又は、請求項4~12のいずれか一つに記載の多重特異性分子を産生する細胞。
- 請求項15記載の細胞を培養する工程を含む、請求項4~12のいずれか一つに記載の多重特異性分子の製造方法。
- 請求項16記載の方法により得られる多重特異性分子。
- 請求項1~3のいずれか一つに記載のFab、請求項4~12若しくは請求項17のいずれか一つに記載の多重特異性分子、請求項13記載のポリヌクレオチド、請求項14記載のベクター、又は、請求項15記載の細胞を含む組成物。
- 下記CDRH1~3及びCDRL1~3を含む、CD3に特異的に結合し、重鎖可変領域及び軽鎖可変領域がジスルフィド結合してなるscFv:
配列番号32で示されるアミノ酸配列からなるCDRH1;
配列番号33で示されるアミノ酸配列からなるCDRH2;
配列番号34で示されるアミノ酸配列からなるCDRH3;
配列番号35で示されるアミノ酸配列からなるCDRL1;
配列番号36で示されるアミノ酸配列からなるCDRL2;及び
配列番号37で示されるアミノ酸配列からなるCDRL3。 - 配列番号3で示されるアミノ酸配列のアミノ酸番号1~118からなる重鎖可変領域及び配列番号3で示されるアミノ酸配列のアミノ酸番号134~240からなる軽鎖可変領域を含む、請求項19記載のscFv。
- 配列番号3で示されるアミノ酸配列からなる、請求項19又は20記載のscFv。
- 配列番号39で示されるアミノ酸配列のアミノ酸番号1~118からなる重鎖可変領域及び配列番号39で示されるアミノ酸配列のアミノ酸番号134~240からなる軽鎖可変領域を含む、請求項19記載のscFv。
- 配列番号39で示されるアミノ酸配列からなる、請求項19又は22記載のscFv。
- 請求項19~23のいずれか一つに記載のscFvを含み、がん精巣抗原ペプチドと主要組織適合性遺伝子複合体(MHC)の複合体ではない標的分子に特異的に結合する、多重特異性分子。
- 当該MHCがヒト白血球抗原(HLA)である、請求項24記載の多重特異性分子。
- 当該標的分子に特異的に結合する抗体又はその抗原結合断片を含む、請求項24又は25記載の多重特異性分子。
- 多重特異性抗体である、請求項24~26のいずれか一つに記載の多重特異性分子。
- taFv-ヘテロダイマーFcを含む、請求項24~27のいずれか一つに記載の多重特異性分子。
- 三重特異性抗体である、請求項24~28のいずれか一つに記載の多重特異性分子。
- 二重特異性抗体である、請求項24~28のいずれか一つに記載の多重特異性分子。
- 標的分子がGPRC5D、CD98又はTrop2である、請求項24~30のいずれか一つに記載の多重特異性分子。
- 下記(i)~(iii)から選択される一のポリペプチド対を含む、請求項24~31のいずれか一つに記載の多重特異性分子:
(i)配列番号21の20~744番目のアミノ酸残基からなるアミノ酸配列又は該アミノ酸配列のカルボキシル末端において1若しくは2アミノ酸が欠失してなるアミノ酸配列を含むポリペプチド及び配列番号9の20~246番目のアミノ酸残基からなるアミノ酸配列又は該アミノ酸配列のカルボキシル末端において1若しくは2アミノ酸が欠失してなるアミノ酸配列を含むポリペプチド;
(ii)配列番号23の20~743番目のアミノ酸残基からなるアミノ酸配列又は該アミノ酸配列のカルボキシル末端において1若しくは2アミノ酸が欠失してなるアミノ酸配列を含むポリペプチド及び配列番号9の20~246番目のアミノ酸残基からなるアミノ酸配列又は該アミノ酸配列のカルボキシル末端において1若しくは2アミノ酸が欠失してなるアミノ酸配列を含むポリペプチド;
(iii)配列番号25の20~739番目のアミノ酸残基からなるアミノ酸配列又は該アミノ酸配列のカルボキシル末端において1若しくは2アミノ酸が欠失してなるアミノ酸配列を含むポリペプチド及び配列番号9の20~246番目のアミノ酸残基からなるアミノ酸配列又は該アミノ酸配列のカルボキシル末端において1若しくは2アミノ酸が欠失してなるアミノ酸配列を含むポリペプチド。 - 請求項24~32のいずれか一つに記載の多重特異性分子に含まれるアミノ酸配列をコードする塩基配列を含むポリヌクレオチド。
- 請求項33記載のポリヌクレオチドを含むベクター。
- 請求項33記載のポリヌクレオチド又は請求項34記載のベクターを含むか、又は、請求項24~32のいずれか一つに記載の多重特異性分子を産生する細胞。
- 請求項35記載の細胞を培養する工程を含む、請求項24~32のいずれか一つに記載の多重特異性分子の製造方法。
- 請求項36記載の方法により得られる多重特異性分子。
- 請求項19~23のいずれか一つに記載のscFv、請求項24~32若しくは請求項37のいずれか一つに記載の多重特異性分子、請求項33記載のポリヌクレオチド、請求項34記載のベクター、又は、請求項35記載の細胞を含む組成物。
- 下記CDRH1~3及びCDRL1~3を含む、CD3に特異的に結合するFab:
配列番号40で示されるアミノ酸配列からなるCDRH1;
配列番号41で示されるアミノ酸配列からなるCDRH2;
配列番号42で示されるアミノ酸配列からなるCDRH3;
配列番号43で示されるアミノ酸配列からなるCDRL1;
配列番号44で示されるアミノ酸配列からなるCDRL2;及び
配列番号45で示されるアミノ酸配列からなるCDRL3。 - 配列番号46で示されるアミノ酸配列のアミノ酸番号1~118からなる重鎖可変領域及び配列番号47で示されるアミノ酸配列のアミノ酸番号1~109からなる軽鎖可変領域を含む、請求項39記載のFab。
- 配列番号46で示されるアミノ酸配列からなるポリペプチド及び配列番号47で示されるアミノ酸配列からなる軽鎖を含む、請求項39又は40記載のFab。
- 請求項39~41のいずれか一つに記載のFabを含み、がん精巣抗原ペプチドと主要組織適合性遺伝子複合体(MHC)の複合体ではない標的分子に特異的に結合する、多重特異性分子。
- 当該MHCがヒト白血球抗原(HLA)である、請求項42記載の多重特異性分子。
- 当該標的分子に特異的に結合する抗体又はその抗原結合断片を含む、請求項42又は43記載の多重特異性分子。
- 多重特異性抗体である、請求項42~44のいずれか一つに記載の多重特異性分子。
- 二重特異性抗体である、請求項42~45のいずれか一つに記載の多重特異性分子。
- 請求項42~46のいずれか一つに記載の多重特異性分子に含まれるアミノ酸配列をコードする塩基配列を含むポリヌクレオチド。
- 請求項47記載のポリヌクレオチドを含むベクター。
- 請求項47記載のポリヌクレオチド又は請求項48記載のベクターを含むか、又は、請求項42~46のいずれか一つに記載の多重特異性分子を産生する細胞。
- 請求項49記載の細胞を培養する工程を含む、請求項34~38のいずれか一つに記載の多重特異性分子の製造方法。
- 請求項50記載の方法により得られる多重特異性分子。
- 請求項39~41のいずれか一つに記載のFab、請求項42~46のいずれか一つに記載の多重特異性分子、請求項47記載のポリヌクレオチド、請求項48記載のベクター、又は、請求項49記載の細胞を含む組成物。
- 下記CDRH1~3及びCDRL1~3を含む、CD3に特異的に結合し、重鎖可変領域及び軽鎖可変領域がジスルフィド結合してなるscFv:
配列番号40で示されるアミノ酸配列からなるCDRH1;
配列番号41で示されるアミノ酸配列からなるCDRH2;
配列番号42で示されるアミノ酸配列からなるCDRH3;
配列番号43で示されるアミノ酸配列からなるCDRL1;
配列番号44で示されるアミノ酸配列からなるCDRL2;及び
配列番号45で示されるアミノ酸配列からなるCDRL3。 - 配列番号48で示されるアミノ酸配列のアミノ酸番号1~118からなる重鎖可変領域及び配列番号48で示されるアミノ酸配列のアミノ酸番号134~242からなる軽鎖可変領域を含む、請求項53記載のscFv。
- 配列番号48で示されるアミノ酸配列からなる、請求項53又は54記載のscFv。
- 配列番号49で示されるアミノ酸配列のアミノ酸番号1~118からなる重鎖可変領域及び配列番号49で示されるアミノ酸配列のアミノ酸番号134~242からなる軽鎖可変領域を含む、請求項53記載のscFv。
- 配列番号49で示されるアミノ酸配列からなる、請求項53又は56記載のscFv。
- 請求項53~57のいずれか一つに記載のscFvを含み、がん精巣抗原ペプチドと主要組織適合性遺伝子複合体(MHC)の複合体ではない標的分子に特異的に結合する、多重特異性分子。
- 当該MHCがヒト白血球抗原(HLA)である、請求項58記載の多重特異性分子。
- 当該標的分子に特異的に結合する抗体又はその抗原結合断片を含む、請求項58又は59記載の多重特異性分子。
- 多重特異性抗体である、請求項58~60のいずれか一つに記載の多重特異性分子。
- 二重特異性抗体である、請求項58~61のいずれか一つに記載の多重特異性分子。
- 請求項58~62のいずれか一つに記載の多重特異性分子に含まれるアミノ酸配列をコードする塩基配列を含むポリヌクレオチド。
- 請求項63記載のポリヌクレオチドを含むベクター。
- 請求項63記載のポリヌクレオチド又は請求項64記載のベクターを含むか、又は、請求項58~62のいずれか一つに記載の多重特異性分子を産生する細胞。
- 請求項65記載の細胞を培養する工程を含む、請求項58~62のいずれか一つに記載の多重特異性分子の製造方法。
- 請求項66記載の方法により得られる多重特異性分子。
- 請求項53~57のいずれか一つに記載のscFv、請求項58~62のいずれか一つに記載の多重特異性分子、請求項63記載のポリヌクレオチド、請求項64記載のベクター、又は、請求項65記載の細胞を含む組成物。
- 請求項4~12、24~32、42~46及び58~62のいずれか一つに記載の多重特異性分子を含む医薬組成物。
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