CN116897207A

CN116897207A - Stable multispecific molecules and uses thereof

Info

Publication number: CN116897207A
Application number: CN202280016551.XA
Authority: CN
Inventors: 中山麻纪子; 中村健介; 古薗信二; 松元亮太
Original assignee: Daiichi Sankyo Co Ltd
Current assignee: Daiichi Sankyo Co Ltd
Priority date: 2021-03-29
Filing date: 2022-03-28
Publication date: 2023-10-17

Abstract

The present invention provides stable multispecific molecules. A Fab which specifically binds to CD3 and comprises the following CDRH1 to 3 and CDRL1 to 3: CDRH1 consisting of the amino acid sequence shown in SEQ ID NO. 32; CDRH2 consisting of the amino acid sequence shown in SEQ ID NO. 33; CDRH3 composed of the amino acid sequence shown in SEQ ID NO. 34; CDRL1 composed of the amino acid sequence shown in SEQ ID NO. 35; CDRL2 consisting of the amino acid sequence shown in SEQ ID NO. 36; and CDRL3 consisting of the amino acid sequence shown in SEQ ID NO. 37.

Description

Stable multispecific molecules and uses thereof

Technical Field

The present invention relates to a multispecific antibody, multispecific molecule, constituent element thereof, or the like that recognizes 2 or more antigens.

Background

As molecules binding to more than one target, related studies on antibodies (bispecific antibodies and multispecific antibodies) binding to 2 or more different antigens through 1 molecule are underway.

As an example of application of the bispecific antibody, there is a T cell-redirecting bispecific antibody that recognizes CD3 on T cells and a target antigen expressed on the surface of cancer cells by 1 molecule, thereby crosslinking T cells with cancer cells and inducing cytotoxicity of T cells to cancer cells. The representative example of the bleb-emetic antibody (Blinatumomab) is a bispecific antibody composed of BiTE (trademark) type (scFv-scFv type) that binds to CD3 and CD19, and is approved as a therapeutic agent for recurrent or refractory B-cell non-hodgkin lymphoma and acute lymphoblastic leukemia in adults and infants in the united states. However, it is pointed out that the bolamitraz antibody has a short half-life because of its BiTE (trademark) type, and has a large number of high molecular weight species (high molecular weights species, HMWS) under certain experimental conditions as compared with other antibodies obtained as a drug, and has a problem in terms of stability (non-patent document 1).

Preclinical testing of drugs requires the use of animals, particularly higher primate cynomolgus monkeys. In the case of using a candidate drug of an anti-CD 3 antibody, an anti-CD 3 antibody that can bind to CD3 of both human and cynomolgus monkey can be used. An anti-CD 3 antibody that binds to CD3 of human and cynomolgus monkey and recognizes a novel epitope identified by X-ray structural analysis was created, and a bispecific antibody obtained by combining the anti-CD 3 antibody with an antibody against each target antigen exhibited cytotoxic activity against cancer cells expressing the target antigen (patent documents 1 and 2). Further, a modified form or the like was created for a part of the anti-CD 3 antibody (patent document 3). In order to create multispecific antibodies and multispecific molecules composed of multiple configurations as drugs, it is necessary to create various anti-CD 3 antibody fragments.

Prior art literature

Patent literature

Patent document 1: WO 2018/117237

Patent document 2: WO 2018/147245

Patent document 3: WO 2019/244107

Non-patent literature

Non-patent document 1: journal of Chromatography B1065-1066 (2017) 35-43

Disclosure of Invention

Problems to be solved by the invention

The purpose of the present invention is to provide a bispecific molecule that has excellent solution stability and can reduce the number of dimer-containing High Molecular Weight Species (HMWS) when stored in a solution state.

Means for solving the problems

The present inventors have made intensive studies to solve the above problems, and as a result, have created novel bispecific molecules containing anti-CD 3 scFv or anti-CD 3 Fab, thereby completing the present invention.

Namely, the present invention includes the following.

[1] A Fab which specifically binds to CD3 and comprises the following CDRH1 to 3 and CDRL1 to 3:

CDRH1 consisting of the amino acid sequence shown in SEQ ID NO. 32;

CDRH2 consisting of the amino acid sequence shown in SEQ ID NO. 33;

CDRH3 composed of the amino acid sequence shown in SEQ ID NO. 34;

CDRL1 composed of the amino acid sequence shown in SEQ ID NO. 35;

CDRL2 consisting of the amino acid sequence shown in SEQ ID NO. 36; the method comprises the steps of,

CDRL3 consisting of the amino acid sequence shown in SEQ ID NO. 37.

[2] The Fab of [1], which comprises 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.

[3] The Fab of [1] or [2], which comprises a polypeptide consisting of the amino acid sequence shown in SEQ ID NO. 1 and a light chain consisting of the amino acid sequence shown in SEQ ID NO. 2.

[4] A multispecific molecule comprising the Fab of any one of [1] to [3] which specifically binds to a target molecule which is not a complex of a cancer testis antigenic peptide and a Major Histocompatibility Complex (MHC).

[5] The multi-specific molecule according to [4], wherein the MHC is Human Leukocyte Antigen (HLA).

[6] The multispecific molecule of [4] or [5], which contains an antibody or antigen-binding fragment thereof that specifically binds to the target molecule.

[7] The multispecific molecule of any one of [4] to [6], which is a multispecific antibody.

[8] The multispecific molecule of any one of [4] to [7], which contains scFv-Fab-heterodimeric Fc.

[9] The multi-specific molecule according to any one of [4] to [8], which is a trispecific antibody.

[10] The multi-specific molecule according to any one of [4] to [8], which is a bispecific antibody.

[11] The multi-specific molecule according to any one of [4] to [10], wherein the target molecule is GPRC5D, CD98 or Trop2.

[12] The multispecific molecule of any one of [4] to [11], which contains 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 obtained by deleting 1 or 2 amino acids from the carboxy terminus of the amino acid sequence; a polypeptide comprising an amino acid sequence consisting of amino acid residues 20 to 246 of SEQ ID NO. 9, or an amino acid sequence obtained by deleting 1 or 2 amino acids from the carboxy terminus of the amino acid sequence; and a polypeptide comprising an amino acid sequence consisting of amino acid residues 21 to 233 of SEQ ID NO. 12;

(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 obtained by deleting 1 or 2 amino acids from the carboxy terminus of the amino acid sequence; a polypeptide comprising an amino acid sequence consisting of amino acid residues 20 to 246 of SEQ ID NO. 9, or an amino acid sequence obtained by deleting 1 or 2 amino acids from the carboxy terminus of the amino acid sequence; and a polypeptide comprising an amino acid sequence consisting of amino acid residues 21 to 233 of SEQ ID NO. 12;

(iii) A polypeptide comprising an amino acid sequence consisting of amino acid residues 20 to 713 of SEQ ID NO. 31, or an amino acid sequence obtained by deleting 1 or 2 amino acids from the carboxy terminus of the amino acid sequence; a polypeptide comprising an amino acid sequence consisting of amino acid residues 20 to 246 of SEQ ID NO. 9, or an amino acid sequence obtained by deleting 1 or 2 amino acids from the carboxy terminus of the amino acid sequence; and a polypeptide comprising an amino acid sequence consisting of amino acid residues 21 to 233 of SEQ ID NO. 12.

[13] A polynucleotide comprising a base sequence encoding an amino acid sequence contained in the multispecific molecule of any one of [4] to [12 ].

[14] A vector comprising the polynucleotide of [13 ].

[15] A cell comprising the polynucleotide of [13] or the vector of [14], or producing the multispecific molecule of any one of [4] to [12 ].

[16] A method for producing a multispecific molecule which is the multispecific molecule of any one of [4] to [12], the method comprising the steps of: culturing the cell of [15 ].

[17] A multispecific molecule obtainable by the method of [16 ].

[18] A composition comprising: [1] the Fab of any one of [3 ]; [4] a multispecific molecule of any one of [12] or [17 ]; [13] the polynucleotide; [14] the carrier; alternatively, the cell of [15 ].

[19] An scFv formed by disulfide bonding of a heavy chain variable region and a light chain variable region, specifically binding to CD3, comprising the following CDRH1 to 3 and CDRL1 to 3:

CDRH1 consisting of the amino acid sequence shown in SEQ ID NO. 32;

CDRH2 consisting of the amino acid sequence shown in SEQ ID NO. 33;

CDRH3 composed of the amino acid sequence shown in SEQ ID NO. 34;

CDRL1 composed of the amino acid sequence shown in SEQ ID NO. 35;

CDRL3 consisting of the amino acid sequence shown in SEQ ID NO. 37.

[20] The scFv according to [19], which comprises a heavy chain variable region composed of amino acid numbers 1 to 118 of the amino acid sequence shown in SEQ ID NO. 3, and a light chain variable region composed of amino acid numbers 134 to 240 of the amino acid sequence shown in SEQ ID NO. 3.

[21] The scFv of [19] or [20], which consists of the amino acid sequence shown in SEQ ID NO: 3.

[22] The scFv according to [19], which comprises a heavy chain variable region composed of amino acid numbers 1 to 118 of the amino acid sequence shown in SEQ ID NO. 39, and a light chain variable region composed of amino acid numbers 134 to 240 of the amino acid sequence shown in SEQ ID NO. 39.

[23] The scFv of [19] or [22], which consists of the amino acid sequence shown in SEQ ID NO: 39.

[24] A multispecific molecule comprising the scFv of any one of [19] to [23] which specifically binds to a target molecule which is not a complex of a cancer testis antigenic peptide and a Major Histocompatibility Complex (MHC).

[25] The multispecific molecule of [24], wherein the MHC is Human Leukocyte Antigen (HLA).

[26] The multispecific molecule of [24] or [25], which contains an antibody or antigen-binding fragment thereof that specifically binds to the target molecule.

[27] The multispecific molecule of any one of [24] to [26], which is a multispecific antibody.

[28] The multispecific molecule of any one of [24] to [27], which contains taFv-heterodimeric Fc.

[29] The multispecific molecule of any one of [24] to [28], which is a trispecific antibody.

[30] The multi-specific molecule according to any one of [24] to [28], which is a bispecific antibody.

[31] The multi-specific molecule according to any one of [24] to [30], wherein the target molecule is GPRC5D, CD98 or Trop2.

[32] The multispecific molecule of any one of [24] to [31], which comprises 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 obtained by deleting 1 or 2 amino acids from the carboxy terminus of the amino acid sequence; and a polypeptide comprising an amino acid sequence consisting of amino acid residues 20 to 246 of SEQ ID NO. 9, or an amino acid sequence obtained by deleting 1 or 2 amino acids from the carboxy terminus of the 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 obtained by deleting 1 or 2 amino acids from the carboxy terminus of the amino acid sequence; and a polypeptide comprising an amino acid sequence consisting of amino acid residues 20 to 246 of SEQ ID NO. 9, or an amino acid sequence obtained by deleting 1 or 2 amino acids from the carboxy terminus of the amino acid sequence;

(iii) A polypeptide comprising an amino acid sequence consisting of amino acid residues 20 to 739 of SEQ ID NO. 25 or an amino acid sequence obtained by deleting 1 or 2 amino acids from the carboxy terminus of the amino acid sequence; and a polypeptide comprising an amino acid sequence consisting of amino acid residues 20 to 246 of SEQ ID NO. 9, or an amino acid sequence obtained by deleting 1 or 2 amino acids from the carboxyl terminus of the amino acid sequence.

[33] A polynucleotide comprising a base sequence encoding an amino acid sequence contained in the multispecific molecule of any one of [24] to [32 ].

[34] A vector comprising the polynucleotide of [33 ].

[35] A cell comprising the polynucleotide of [33] or the vector of [34], or producing the multispecific molecule of any one of [24] to [32 ].

[36] A method for producing a multispecific molecule which is the multispecific molecule of any one of [24] to [32], the method comprising the steps of: culturing the cell of [35 ].

[37] A multispecific molecule obtainable by the method of [36 ].

[38] A composition comprising: [19] the scFv of any one of [23 ]; [24] a multispecific molecule of any one of [32] or [37 ]; [33] the polynucleotide; [34] the carrier; alternatively, the cell of [35 ].

[39] A Fab which specifically binds to CD3 and comprises the following CDRH1 to 3 and CDRL1 to 3:

CDRH1 composed of the amino acid sequence shown in SEQ ID NO. 40;

CDRH2 composed of the amino acid sequence shown in SEQ ID NO. 41;

CDRH3 composed of the amino acid sequence shown in SEQ ID NO. 42;

CDRL1 composed of the amino acid sequence shown in SEQ ID NO. 43;

CDRL2 consisting of the amino acid sequence shown in SEQ ID NO. 44; the method comprises the steps of,

CDRL3 consisting of the amino acid sequence shown in SEQ ID NO. 45.

[40] The Fab of [39], which comprises 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.

[41] The Fab of [39] or [40], which comprises a polypeptide consisting of the amino acid sequence shown in SEQ ID NO. 46 and a light chain consisting of the amino acid sequence shown in SEQ ID NO. 47.

[42] A multispecific molecule comprising the Fab of any one of [39] to [41] which specifically binds to a target molecule which is not a complex of a cancer testis antigenic peptide and a Major Histocompatibility Complex (MHC).

[43] The multispecific molecule of [42], wherein the MHC is Human Leukocyte Antigen (HLA).

[44] The multispecific molecule of [42] or [43], which contains an antibody or antigen-binding fragment thereof that specifically binds to the target molecule.

[45] The multispecific molecule of any one of [42] to [44], which is a multispecific antibody.

[46] The multi-specific molecule according to any one of [42] to [45], which is a bispecific antibody.

[47] A polynucleotide comprising a base sequence encoding an amino acid sequence contained in the multispecific molecule of any one of [42] to [46 ].

[48] A vector comprising the polynucleotide of [47 ].

[49] A cell comprising the polynucleotide of [47] or the vector of [48], or producing the multispecific molecule of any one of [42] to [46 ].

[50] A method of preparing a multispecific molecule, the multispecific molecule being any one of [34] to [38], the method comprising the steps of: culturing the cell of [49 ].

[51] A multispecific molecule obtainable by the method of [50 ].

[52] A composition comprising: [39] the Fab of any one of [41 ]; [42] a multispecific molecule of any one of [46 ]; [47] the polynucleotide; [48] the carrier; alternatively, the cell of [49 ].

[53] An scFv formed by disulfide bonding of a heavy chain variable region and a light chain variable region, specifically binding to CD3, comprising the following CDRH1 to 3 and CDRL1 to 3:

CDRH1 composed of the amino acid sequence shown in SEQ ID NO. 40;

CDRH2 composed of the amino acid sequence shown in SEQ ID NO. 41;

CDRH3 composed of the amino acid sequence shown in SEQ ID NO. 42;

CDRL1 composed of the amino acid sequence shown in SEQ ID NO. 43;

CDRL3 consisting of the amino acid sequence shown in SEQ ID NO. 45.

[54] The scFv according to [53], which comprises a heavy chain variable region composed of amino acid numbers 1 to 118 of the amino acid sequence shown in SEQ ID NO. 48, and a light chain variable region composed of amino acid numbers 134 to 242 of the amino acid sequence shown in SEQ ID NO. 48.

[55] The scFv of [53] or [54], which consists of the amino acid sequence shown in SEQ ID NO: 48.

[56] The scFv according to [53], which comprises a heavy chain variable region composed of amino acid numbers 1 to 118 of the amino acid sequence shown in SEQ ID NO. 49 and a light chain variable region composed of amino acid numbers 134 to 242 of the amino acid sequence shown in SEQ ID NO. 49.

[57] The scFv of [53] or [56], which consists of the amino acid sequence shown in SEQ ID NO: 49.

[58] A multispecific molecule comprising the scFv of any one of [53] to [57] which specifically binds to a target molecule which is not a complex of a cancer testis antigenic peptide and a Major Histocompatibility Complex (MHC).

[59] The multispecific molecule of [58], wherein the MHC is Human Leukocyte Antigen (HLA).

[60] The multispecific molecule of [58] or [59], which contains an antibody or antigen-binding fragment thereof that specifically binds to the target molecule.

[61] The multispecific molecule of any one of [58] to [60], which is a multispecific antibody.

[62] The multi-specific molecule according to any one of [58] to [61], which is a bispecific antibody.

[63] A polynucleotide comprising a base sequence encoding an amino acid sequence contained in the multispecific molecule of any one of [58] to [62 ].

[64] A vector comprising the polynucleotide of [63 ].

[65] A cell comprising the polynucleotide of [63] or the vector of [64], or producing the multispecific molecule of any one of [58] to [62 ].

[66] A method of preparing a multispecific molecule, the multispecific molecule being any one of [58] to [62], the method comprising the steps of: culturing the cell of [65 ].

[67] A multispecific molecule obtainable by the method of [66 ].

[68] A composition comprising: [53] the scFv of any one of [57 ]; [58] a multispecific molecule of any one of [62 ]; [63] the polynucleotide; [64] the carrier; alternatively, the cell of [65 ].

[69] A pharmaceutical composition comprising the multispecific molecule of any one of [4] to [12], [24] to [32], [42] to [46] and [58] to [62 ].

The present specification contains disclosures of Japanese patent application Nos. 2021-055375, 2021-157580, which are the basis of priority of the present application.

Effects of the invention

According to the present invention, a novel bispecific antibody (bispecific molecule) that binds to a target molecule that is not a complex of a cancer testis antigen peptide and a Human Leukocyte Antigen (HLA), or a Fab that binds to CD3, or a scFv that is formed by binding a heavy chain variable region and a light chain variable region to CD3, can be obtained. The antibody has high solution stability, and can be stored in solution without aging.

Brief Description of Drawings

FIG. 1 is a graph showing the change over time of HMWS (%) of C3E-0002 to 0004.

Fig. 2: FIG. 2A is a graph showing the change over time of HMWS (%) due to oscillatory aging of C3E-0002-0004; FIG. 2B is a graph showing the change over time of the peak area due to the oscillatory aging of C3E-0002 to 0004.

Fig. 3: FIG. 3A is a graph showing the change over time of HMWS (%) of RX3-0001 to 0003; FIG. 3B is a graph showing changes over time in HMWS (%) of CX 3-0001-0003; FIG. 3C is a graph showing the change over time of HMWS (%) of TX 3-0001-0003.

Fig. 4-1: FIG. 4-1A is a graph showing the change over time of HMWS (%) caused by oscillation aging of RX3-0001 to 0003; FIG. 4-1B is a graph showing the change over time of peak areas caused by the oscillation aging of RX3-0001 to 0003.

Fig. 4-2: FIG. 4-2A is a graph showing changes over time in HMWS (%) caused by oscillatory aging of CX 3-0001-0003; FIG. 4-2B is a graph showing the change over time of peak areas caused by the oscillatory aging of CX 3-0001-0003.

Fig. 4-3: FIG. 4-3A is a graph showing the change over time of HMWS (%) caused by oscillating aging of TX 3-0001-0003; fig. 4-3B are graphs showing changes over time in peak areas caused by oscillation aging of TX3-0001 to 0003.

FIG. 5 is the amino acid sequence of C3E-7085-Fab (SEQ ID NO: 1).

FIG. 6 is the amino acid sequence of C3E-7085-LC (SEQ ID NO: 2).

FIG. 7 is the amino acid sequence of C3E-7096 (SEQ ID NO: 3).

FIG. 8 is the full length nucleotide sequence of HC_h (SEQ ID NO: 4).

FIG. 9 is the full-length nucleotide sequence of C3E-7085-HC-k (SEQ ID NO: 5).

FIG. 10 is the full length nucleotide sequence of C3E-7085-Fab-HC-k (SEQ ID NO: 6).

FIG. 11 is the full-length nucleotide sequence of C3E-7085-LC (SEQ ID NO: 7).

FIG. 12 is the full-length nucleotide sequence of C3E-7096-HC-k (SEQ ID NO: 8).

FIG. 13 is the full-length amino acid sequence of HC_h (SEQ ID NO: 9).

FIG. 14 is the full-length amino acid sequence of C3E-7085-HC-k (SEQ ID NO: 10).

FIG. 15 is the full-length amino acid sequence of C3E-7085-Fab-HC-k (SEQ ID NO: 11).

FIG. 16 is the full-length amino acid sequence of C3E-7085-LC (SEQ ID NO: 12).

FIG. 17 is the full-length amino acid sequence of C3E-7096-HC-k (SEQ ID NO: 13).

FIG. 18 is the full length nucleotide sequence of TX3-0001-HC_k (SEQ ID NO: 14).

FIG. 19 is the full length nucleotide sequence of TX3-0003-HC_k (SEQ ID NO: 15).

FIG. 20 is the full length nucleotide sequence of CX3-0001-HC_k (SEQ ID NO: 16).

FIG. 21 is the full length nucleotide sequence of CX3-0003-HC_k (SEQ ID NO: 17).

FIG. 22 is the full length nucleotide sequence of RX3-0001-HC_k (SEQ ID NO: 18).

FIG. 23 is the full-length nucleotide sequence of CX3-0001-HC_k (SEQ ID NO: 19).

FIG. 24 is the amino acid sequence of TX3-0001-HC_k (SEQ ID NO: 20).

FIG. 25 is the amino acid sequence of TX3-0003-HC_k over its full length (SEQ ID NO: 21).

FIG. 26 shows the amino acid sequence of CX3-0001-HC_k (SEQ ID NO: 22).

FIG. 27 is the full-length amino acid sequence of CX3-0003-HC_k (SEQ ID NO: 23).

FIG. 28 is the amino acid sequence of RX3-0001-HC_k (SEQ ID NO: 24).

FIG. 29 is the amino acid sequence of RX3-0003-HC_k full length (SEQ ID NO: 25).

FIG. 30 is the full length nucleotide sequence of TX3-0002-HC_k (SEQ ID NO: 26).

FIG. 31 is the full length nucleotide sequence of CX3-0002-HC_k (SEQ ID NO: 27).

FIG. 32 is the full length nucleotide sequence of RX3-0002-HC_k (SEQ ID NO: 28).

FIG. 33 is the amino acid sequence of TX3-0002-HC_k over its full length (SEQ ID NO: 29).

FIG. 34 is the full-length amino acid sequence of CX3-0002-HC_k (SEQ ID NO: 30).

FIG. 35 is the amino acid sequence of RX3-0002-HC_k full length (SEQ ID NO: 31).

FIG. 36 shows the amino acid sequences of the heavy chain CDRH1 to CDRH3 and the light chain CDRL1 to L3 (SEQ ID NO:32 to SEQ ID NO: 37) of C3E-7085.

FIG. 37 is the amino acid sequence of a peptide linker (SEQ ID NO: 38).

FIG. 38A is a scFv that binds CD3 and is formed by disulfide bonding of the heavy chain variable region and the light chain variable region (the black bars between VH and VL indicate that both are bonded via disulfide bonding. Hereinafter).

Fig. 38B is a Fab that binds to CD 3.

FIG. 38C is a diagram showing a bispecific molecule comprising scFv (upper right diagonal line) bound to CD3 and formed by disulfide bond binding of a heavy chain variable region and a light chain variable region, and X ("other binding moiety") bound to an antigen other than CD 3. The orientation of scFv shown by upper right diagonal lines may be reversed by changing the order of connection of the VH and VL domains. The scFv formed by disulfide bonding may be formed by VH and VL not via a linker.

FIG. 38D is a diagram showing a bispecific molecule comprising a Fab which binds to CD3 and X ("other binding moiety") which binds to an antigen other than CD 3.

FIG. 38E is a diagram showing tandem scFv (taFv) formed by binding a scFv binding to CD3 and a scFv binding to an antigen other than CD3 via a linker. The orientation of each scFv may be reversed by changing the order of connection of the VH and VL domains.

FIG. 38F is a diagram showing scFv-Fab formed by binding a Fab which binds to CD3 and an scFv which binds to an antigen other than CD3 via a linker. The orientation of each scFv may be reversed by changing the order of connection of the VH and VL domains.

FIG. 39A is a diagram showing a multispecific molecule comprising scFv that binds to CD3, X ("other binding moiety") and Y ("other binding moiety" or "other group") that bind to antigens other than CD 3. The orientation of scFv shown by upper right diagonal lines may be reversed by changing the order of connection of the VH and VL domains. X and Y may be any of various antibody fragments such as scFv, VHH, fab and complete antibodies. The scFv formed by disulfide bonding may be formed by VH and VL not via a linker. When Y is the "other binding moiety", the multispecific molecule is a trispecific molecule.

FIG. 39B is a diagram showing a multispecific molecule comprising Fab which binds to CD3, X ("other binding moiety") and Y ("other binding moiety" or "other group") which bind to antigens other than CD 3. X and Y (when "other binding moiety") may be any of various antibody fragments such as scFv, VHH, fab, complete antibodies, and molecules other than immunoglobulins. When Y is the "other binding moiety", the multispecific molecule is a trispecific molecule.

FIG. 40A is a diagram showing an Fc-added bispecific molecule comprising scFv (upper right diagonal line) bound to CD3 and formed by disulfide bond between a heavy chain variable region and a light chain variable region, and X bound to an antigen other than CD3 (other binding moiety). Fc is a configuration formed by association of a first polypeptide shown by upper left diagonal lines and a second polypeptide shown by solid black patterns (Fc may be any of wild-type and mutant type). The orientation of scFv shown by upper right diagonal lines may be reversed by changing the order of connection of the VH and VL domains. The positions of X and scFv in each figure may be changed.

FIG. 40B is a diagram showing an Fc-added bispecific molecule comprising an scFv (upper right diagonal line) bound to CD3 and formed by disulfide bond between a heavy chain variable region and a light chain variable region, and a Fab or scFv bound to an antigen other than CD 3. Fc is a configuration formed by association of a first polypeptide shown by upper left diagonal lines and a second polypeptide shown by solid black patterns (Fc may be any of wild-type and mutant type). The orientation of each scFv may be reversed by changing the order of connection of the VH and VL domains. The lower left panel also shows taFv-heterodimer Fc type (in the case of heterodimers).

FIG. 41A is a diagram showing an Fc-added multispecific molecule comprising scFv (upper right diagonal line) bound to CD3 and formed by disulfide bond between a heavy chain variable region and a light chain variable region, X ("other binding moiety") and Y ("other binding moiety" or "other group") bound to an antigen other than CD 3. Fc is a configuration formed by association of a first polypeptide shown by upper left diagonal lines and a second polypeptide shown by solid black patterns (Fc may be any of wild-type and mutant type). The orientation of scFv shown by upper right diagonal lines may be reversed by changing the order of connection of the VH and VL domains. When Y is the "other binding moiety", the multispecific molecule is a trispecific molecule. The positions of X, Y and scFv in the respective figures may be changed.

FIG. 41B is a diagram showing an Fc-added trispecific molecule comprising an scFv (upper right diagonal line) bound to CD3 and formed by disulfide bond between a heavy chain variable region and a light chain variable region, and 2 antibody fragments (Fab or scFv) bound to antigens other than CD 3. Fc is a configuration formed by association of a first polypeptide shown by upper left diagonal lines and a second polypeptide shown by solid black patterns (Fc may be any of wild-type and mutant type). The orientation of each scFv may be reversed by changing the order of connection of the VH and VL domains.

FIG. 42 is a diagram showing an Fc-added multispecific molecule comprising an scFv that binds to CD3, an scFv formed by disulfide bonding of a heavy chain variable region and a light chain variable region (upper right diagonal line), and an "other binding moiety" or "other group" (denoted by X, Y and Z, respectively) that binds to an antigen other than CD 3. Fc is a configuration formed by association of a first polypeptide shown by upper left diagonal lines and a second polypeptide shown by solid black patterns (Fc may be any of wild-type and mutant type). The orientation of scFv shown by upper right diagonal lines may be reversed by changing the order of connection of the VH and VL domains. If 2 or 3 of X, Y and Z are "other binding moieties," then they are trispecific or tetraspecific molecules, respectively. The positions of X, Y, Z and scFv in the respective figures may be changed.

FIG. 43A is a diagram showing an Fc-added bispecific molecule comprising a Fab which binds to CD3 and X (other binding moiety) which binds to an antigen other than CD 3. Fc is a configuration formed by association of a first polypeptide shown by upper left diagonal lines and a second polypeptide shown by solid black patterns (Fc may be any of wild-type and mutant type).

FIG. 43B is a diagram showing an Fc-added bispecific molecule comprising a Fab which binds to CD3 and an antibody fragment (scFv or Fab) which binds to an antigen other than CD 3. Fc is a configuration formed by association of a first polypeptide shown by upper left diagonal lines and a second polypeptide shown by solid black patterns (Fc may be any of wild-type and mutant type). The upper left panel also shows the Fc type of scFv-Fab-heterodimers (in the case of heterodimers). The orientation of each scFv may be reversed by changing the order of connection of the VH and VL domains.

FIG. 44A is a diagram showing Fc-added multispecific molecules each comprising Fab which binds to CD3, X ("other binding moiety") and Y ("other binding moiety" or "other group") which bind to antigens other than CD 3. Fc is a configuration formed by association of a first polypeptide shown by upper left diagonal lines and a second polypeptide shown by solid black patterns (Fc may be any of wild-type and mutant type). When Y is the "other binding moiety", the multispecific molecule is a trispecific molecule.

FIG. 44B is a diagram showing an Fc additive type trispecific molecule (bispecific molecule if the 2 antibody fragments are identical) comprising a Fab which binds to CD3 and 2 antibody fragments (scFv or Fab) which bind to antigens other than CD 3. Fc is a configuration formed by association of a first polypeptide shown by upper left diagonal lines and a second polypeptide shown by solid black patterns (Fc may be any of wild-type and mutant type). The orientation of each scFv may be reversed by changing the order of connection of the VH and VL domains.

FIG. 45 is a diagram showing Fc-added multispecific molecules each comprising a Fab which binds to CD3 and an "other binding moiety" or "other group" (at least one of. X, Y and Z is an "other binding moiety" as represented by X, Y and Z, respectively) which binds to an antigen other than CD 3. Fc is a configuration formed by association of a first polypeptide shown by upper left diagonal lines and a second polypeptide shown by solid black patterns (Fc may be any of wild-type and mutant type). If 2 or 3 of X, Y and Z are "other binding moieties," then they are trispecific or tetraspecific molecules, respectively.

FIG. 46 is the amino acid sequence of C3E-7097 (SEQ ID NO: 39).

FIG. 47 shows the amino acid sequences of the heavy chain CDRH1 to CDRH3 and the light chain CDRL1 to L3 (SEQ ID NO:40 to SEQ ID NO: 45) of C3E-7093.

FIG. 48 is the amino acid sequence of C3E-7093-Fab (SEQ ID NO: 46).

FIG. 49 is the amino acid sequence of C3E-7093-LC (SEQ ID NO: 47).

FIG. 50 is the amino acid sequence of C3E-7098 (SEQ ID NO: 48).

FIG. 51 is the amino acid sequence of C3E-7099 (SEQ ID NO: 49).

As can be seen from fig. 38-45, X, Y and Z are attached to the ends of adjacent sections, but binding or fusion to the ends is not necessary. For example, when X, Y and Z are linked to Fc (appear), the X, Y and Z can be any of the following: binding or fusing to the N-terminus or C-terminus of Fc; binding to a moiety other than the N-terminus or C-terminus of Fc; or, a sugar chain added to an amino acid contained in an Fc, or a fusion thereof. Examples of not being bonded or fused to the ends of the adjacent portions are not limited thereto.

Detailed Description

The present invention will be described in detail below.

1. Definition of the definition

In the present invention, the term "gene" refers to a nucleotide chain or a complementary strand thereof contained in a base sequence encoding an amino acid of a protein. For example, a polynucleotide, an oligonucleotide, DNA, mRNA, cDNA, cRNA, or the like, which is a nucleotide chain or a complementary strand thereof contained in a base sequence of an amino acid encoding a protein, is included in the meaning of "gene". The gene is a single-stranded, double-stranded or triple-stranded nucleotide, an association of a DNA strand and an RNA strand, a nucleotide in which a Ribonucleotide (RNA) and a Deoxyribonucleotide (DNA) are mixed on one nucleotide strand, and a double-stranded or triple-stranded nucleotide containing such a nucleotide strand are also included in the meaning of "gene". In the present invention, the base sequence and the nucleotide sequence have the same meaning.

In the present invention, "polynucleotide", "nucleotide chain", "nucleic acid" and "nucleic acid molecule" are the same. For example, DNA, RNA, probes, oligonucleotides, primers, and the like are also included in the meaning of "polynucleotide". The polynucleotide is a polynucleotide comprising a single strand, a double strand or three or more strands, an association of a DNA strand and an RNA strand, a polynucleotide in which a Ribonucleotide (RNA) and a Deoxyribonucleotide (DNA) are mixed in one polynucleotide strand, and an association of a double strand or three or more strands containing such a polynucleotide strand are also included in the meaning of "polynucleotide".

In the present invention, "polypeptide", "peptide" and "protein" are the same.

In the present invention, an "antigen" is sometimes used in the sense of an "immunogen".

In the present invention, "cells" also include various cells derived from an animal subject, secondary cells, primary cells, cell lines, recombinant cells, microorganisms, and the like.

In the present invention, "antibody" is used as an immunoglobulin having a constant region and a variable region. The antibody is a natural antibody or an immunoglobulin prepared by partial synthesis or complete synthesis, and is not particularly limited in the present invention.

The basic structure of a four-chain antibody is composed of two identical light chains (L chains) and two identical heavy chains (H chains). The light chain is linked to the heavy chain by a disulfide bond. The two heavy chains are bound to each other by one or more disulfide bonds, depending on the isotype of the heavy chains. Each light chain, heavy chain has regularly spaced intrachain disulfide bonds. In the heavy and light chains, there are constant regions in which the amino acid sequences exhibit very high similarity, and variable regions in which the amino acid sequences have low similarity. The light chain has a variable region (VL) followed by a constant region (CL) at the amino terminus. The heavy chain has a variable region (VH) at the amino terminus and 3 constant regions (CH 1/CH2/CH 3) later. VL and VH pair, CL juxtaposed with the first constant region (CH 1) of the heavy chain. VL pairs with VH to form a single antigen binding site.

The constant region of the antibody of the present invention is not particularly limited, but as the antibody of the present invention for treating or preventing human diseases, the constant region of a human antibody is preferably used. Examples of the heavy chain constant region of the human antibody include cγ1, cγ2, cγ3, cγ4, cμ, cδ, cα1, cα2, and cε. Examples of the light chain constant region of the human antibody include cκ and cλ.

Fab consists of VH and then CH1 of heavy chain, and VL and then CL of light chain in sequence. VH and VL comprise Complementarity Determining Regions (CDRs). There may be a linker or linkage between VH and CH1 and VL and CL.

Fc (also referred to as Fc region) is the carboxy-terminal region of the constant region of the heavy chain, contains 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 a mutation occurs in a native sequence (referred to as "mutant Fc"). In the multispecific and bispecific molecules of the present invention, the preferred Fc region is a mutant Fc, more preferably a set of fcs capable of forming heterodimers. As one group of Fc, a combination of Fc (i) contained in a first polypeptide and Fc (ii) contained in a second polypeptide described below is exemplified. There is no limitation as long as a group of fcs is capable of associating (forming heterodimers).

Examples of the mutant Fc include, but are not limited to: modified Fc regions (comprising heterodimeric Fc regions) comprised by the heteromultimers with improved stability disclosed in WO 2013/063702; fc comprising CH3 region of immunoglobulin induced from IgG antibody with "protuberance" and "void" contained in heteromultimer disclosed in WO 96/27011; an Fc disclosed in WO2009/089004 comprising a CH3 region comprised by a heterodimer that comprises more than one amino acid residue substituted to a charged amino acid to be electrostatically advantageous; heterodimer Fc regions comprised by heterodimers disclosed in WO2014/110601 using conformational mutations and/or pI (isoelectric point) mutations; fc disclosed in WO2010/151792, or the like, comprising a heterodimer comprising a CH3 region modified to eliminate or reduce binding to protein a.

The variable region consists of two regions: a region of extreme variability, known as the hypervariable region (HVR, hypervariable region), and a relatively constant region, known as the Framework Region (FR), separated from the region. The variable regions of the natural heavy and light chains comprise 4 FRs joined by 3 hypervariable regions, the hypervariable regions of each chain being held very close together with the hypervariable regions of the other chains by the FRs, contributing to the formation of the antigen binding site of the antibody.

The heavy and light chains of antibody molecules are known to have 3 complementarity determining regions (CDR: complementarity determining region), respectively. Complementarity determining regions, also known as hypervariable regions, are sites within the variable regions of the heavy and light chains of antibodies at which the primary structure is particularly highly mutable, and are typically separated into 3 parts on the primary structure of the polypeptide chains of the heavy and light chains. In the present invention, the complementarity determining regions of the heavy chain are denoted as CDRH1, CDRH2 and CDRH3 in this order from the amino terminal side of the heavy chain amino acid sequence; the complementarity determining regions of the light chain are designated as CDRL1, CDRL2 and CDRL3 in this order from the amino terminal side of the light chain amino acid sequence. These sites are conformationally close to each other, determining the specificity for the bound antigen.

In the present invention, the location and length of the CDRs are determined by the definition of IMGT [ Developmental and Comparative Immunology 27 (2003) 55-77 ].

FR is a variable region other than CDR residues. The variable region generally has four FRs, FR1, FR2, FR3 and FR 4.

The CDRs and FRs comprised in the heavy and light chains are sequentially arranged from the amino terminus to the carboxy terminus as FRH1-CDRH1-FRH2-CDRH2-FRH3-CDRH3-FRH4, and FRL1-CDRL1-FRL2-CDRL2-FRL3-CDRL3-FRL4, respectively.

The positions of the CDRs and FR may also be determined by various definitions known in the art, such as the definition of Kabat, chothia, abM, contact, other than IMGT, etc.

In the present invention, the "site" to which an antibody binds, i.e., the "site" recognized by an antibody, refers to a part of a peptide or a part of a higher structure on an antigen to which an antibody binds or recognizes.

In the present invention, this site is also called an epitope or a binding site for an antibody. In the present invention, the term "mutant antibody" refers to a polypeptide which has an amino acid sequence in which amino acids are substituted, deleted, added (addition includes insertion) (hereinafter, collectively referred to as "mutation") in the amino acid sequence of the original antibody, and which binds to an antigen as a target of the present invention. The number of mutated amino acids in the mutated antibody is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 40 or 50. Such mutant antibodies are also encompassed by the "antibodies" of the invention.

In the present invention, "1 or" a plurality of "means 2 to 10.

In the present specification, the term "molecule" refers to a molecule including the antibody or an antigen-binding fragment of the antibody, and also includes a multispecific molecule formed from an antibody or a plurality of antigen-binding fragments derived from such an antibody.

In the present specification, "multispecific molecule" and "multispecific molecule" mean the same, and are not particularly limited as long as they are molecules capable of binding to a plurality of epitopes different from each other on one molecule and/or to epitopes different from each other on two or more molecules. As multispecific molecules, antibodies comprising heavy chain variable regions (VH) and light chain variable regions (VL) are also included. In the present invention, such a multispecific molecule is sometimes referred to as "multispecific antibody", or the like. "recognizing 2 or more antigens" means "binding to a plurality of distinct epitopes on 1 molecule" or "binding to distinct epitopes on 2 or more molecules".

In the present specification, aggregates refer to substances produced by concentration of proteins, and are different in analytical methods by which detection can be performed by particle size. Aggregates having a particle size of about 100nm or less can be evaluated by HPLC or other size exclusion chromatography. The sub-visible particles (particle size 0.1 to 10 μm) and insoluble particles (particle size 10 μm or more) having a particle size of 100nm or more can be evaluated by a microfluidic imaging method or the like.

In the present specification, "HMWS" is a short term for high molecular weight species (high molecular weight species) and is a term for collectively referring to protein aggregate partitions having a size larger than the main peak partition of a target protein in an obtained map by size division by size exclusion chromatography or the like. "HMWS (%)" means a value calculated by a percentage method by monitoring an elution pattern at a detection wavelength of 280nm using a solution sample containing a target protein in HPLC and the like, and by using the ratio of the total peak area in the obtained spectrum to the area of the peak partition displayed on the polymer side with respect to the main peak partition of the target protein.

2. Antigens

2-1.CD3 antigen

The term "CD3" as used in the present invention has the same meaning as CD3 protein.

CD3 is expressed on T cells as part of a multi-molecule T cell receptor complex, and is a complex of 5 polypeptides (molecular weights: 25000 to 28000, 21000, 20000, 16000, 22000) of gamma chain, delta chain, epsilon chain, zeta chain, and eta chain.

Examples of the CD3 complex include γ, δ, ε, ζ, and η chains. These chains are also referred to as subunits. anti-CD 3 antibodies bind to T cells, thereby inducing cytotoxicity through T cell activation. Many anti-CD 3 antibodies bind to CD3 epsilon.

The nucleotide sequence of the cDNA encoding human CD3 epsilon was registered in NCBI/GenBank under accession number NM-000733 (NM-000733.3); the amino acid sequence of human CD3 epsilon was registered at NCBI/GenPept under the registration number NP-000724 (NM-000724.1). The nucleotide sequence of the cDNA encoding cynomolgus monkey CD3 was registered in GenBank under accession number NM-001283615.1.

2-2. Target molecules

In the present invention, the target molecule is not particularly limited as long as it is a molecule other than CD3 present in a living body, and is preferably a molecule which is expressed on the surface of a therapeutic target cell (for example, but not limited to, a cancer cell, a mesenchymal cell, and an inhibitory immune cell) and which causes the cancer cell to exert cytotoxic activity by redirecting CD 3-expressing T cells, and more preferably is not a complex of a cancer testis antigen peptide and a Major Histocompatibility Complex (MHC).

The cancer testis antigen peptide is one of the antigen peptides on the surface of cancer cells, and examples thereof include MAGE peptides, XAGE peptides, and LAGE family peptides. MHC includes Human Leukocyte Antigen (HLA), mouse H-2, and the like. HLA is classified into class I and class II. Class I includes HLA-A, HLA-B and HLA-C, class II includes HLA-DR, HLA-DP and HLA-DQ, etc. As the complex of the cancer testis antigen peptide and the HUMAN Leukocyte Antigen (HLA), for example, there may be mentioned three complexes of (1) HLA-A2/MAGE-A3, A4, A10 or LAGE peptide [ for example, amino acid numbers 157 to 165 of UniProtKB-P78358 (CTG 1B HUMAN) ], namely HLA-A2/MAGE-A3, A4, A10 or HLA-A2/LAGE.

In the present invention, as a suitable target molecule which is not a complex of a cancer testis antigen peptide and a Human Leukocyte Antigen (HLA), a cancer antigen peptide or a cancer antigen is preferably exemplified, and specifically examples thereof include 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. Examples of the target molecule for the mesenchymal cells include FAP, CDH17, LRRC15, and the like. As a target molecule for bone marrow-derived immunosuppressive cells (MDSCs), CD33 may be mentioned.

2-3 preparation of antigen

The antigen protein, CD3, and other antigen proteins which are target molecules other than a complex of a cancer testis antigen peptide and a Human Leukocyte Antigen (HLA) used in the present invention can be prepared by: purification and isolation from animal tissue (including body fluids), cells derived from the tissue, or from the cell culture; gene recombination; in vitro translation; chemical synthesis, and the like.

The cDNA of the antigen protein can be obtained, for example, by a so-called PCR method in which a cDNA library of an organ expressing mRNA of the antigen protein is used as a template, and a polymerase chain reaction (hereinafter, referred to as "PCR") is performed using a primer for amplifying the cDNA of the antigen protein [ Saiki, R.K., et al, science (1988) 239, 487-49].

And a polynucleotide consisting of a nucleotide sequence complementary to a nucleotide sequence encoding the antigen protein expressed in human or rat under stringent conditions, and a polynucleotide encoding a protein having a biological activity equivalent to that of the antigen protein is also included in the cDNA of the antigen protein. In addition, as a polynucleotide that hybridizes with a splice mutant transcribed from the locus of the antigen protein expressed in human or rat or with the splice mutant under stringent conditions, a polynucleotide encoding a protein having biological activity equivalent to that of the antigen protein is also included in the cDNA of the antigen protein.

The nucleotide sequence encoding a protein having the same biological activity as the antigen protein, which is composed of the amino acid sequence of the antigen protein of human or rat, or the amino acid sequence obtained by removing the signal sequence from these sequences, wherein 1 or several amino acids are substituted, deleted or added, is also included in the nucleotide sequence of the antigen protein gene.

Proteins consisting of an amino acid sequence encoded by a splice mutant transcribed from the locus of the antigen protein of human or rat, or consisting of an amino acid sequence in which 1 or several amino acids have been substituted, deleted or added, and having biological activity equivalent to that of the antigen protein are also included in the antigen protein.

Target molecules that are not complexes of cancer testis antigen peptide and Human Leukocyte Antigen (HLA) can also be prepared by the same method.

2-4 binding specificity for antigen proteins

The anti-CD 3 antibodies, binding fragments thereof, and the like, comprised by the multispecific molecules of the present invention recognize the CD3 antigen, i.e., bind to the CD3 antigen. The anti-CD 3 antibody and the like preferably bind to human CD3, monkey CD3 and the like, more preferably bind to human CD3 and cynomolgus monkey CD 3. On the other hand, the preferred anti-CD 3 antibodies do not bind to rat and/or mouse CD 3.

In addition, antibodies and binding fragments thereof and the like directed against a target molecule [ preferably a target molecule that is not a complex of cancer testis antigen peptide and Human Leukocyte Antigen (HLA) ] contained in the multispecific molecule of the present invention recognize, i.e., bind to, the target molecule.

In the present invention, "recognize" or "bind" refers to a binding that is not a nonspecific adsorption. As a criterion for determining whether to recognize, i.e., whether to bind, there is, for example, a dissociation constant (Dissociation Constant, hereinafter referred to as "KD"). The invention is thatPreferred antibodies and the like have a KD value of 1×10 for antigen proteins ^-5 M is less than or equal to 5×10 ^-6 M is less than or equal to 2X 10 ^-6 M is less than or equal to 1X 10 ^-6 M is less than or equal to M.

The binding between the antigen and the antibody in the present invention can be measured or determined by an analysis system for interactions between biomolecules such as SPR method and BLI method, ELISA method, RIA method, or the like. The binding of the antigen and antibody expressed on the cell surface can be measured by flow cytometry analysis or the like.

The SPR method (surface plasmon resonance analysis) was used as the following analysis method: the dissociation constant (Kd value) and the like, which are affinity indices, are determined by measuring the binding rate constant (Ka value) and dissociation rate constant (Kd value) by chemical kinetics (kinetics) analysis. As an apparatus for SPR analysis, there may be mentioned: biacore (trademark) (GE HEALTHCARE), proteon (trademark) (BioRad), SPR-Navi (trademark) (BioNavis), spreta (trademark) (Texas Instruments), SPRi-plexII (trademark) (horiba), autolab SPR (trademark) (Metrohm), etc.

The BLI method (BioLayer Interferometry, biofilm layer interference) is a method of measuring biomolecular interactions using biofilm interference. Examples of the equipment used for interaction analysis by the BLI method include the Octet system (produced by Pall ForteBio).

ELISA (Enzyme-linked ImmunoSorbent Assay ) is a method in which a target antigen or antibody contained in a sample solution is captured by a specific antibody or antigen, and detection and quantification are performed by an Enzyme reaction. Enzyme activity is detected by integrating an enzyme-labeled antigen or antibody into the reaction system. The detection of the enzyme activity uses a substrate whose absorbance spectrum changes by the reaction, and is converted into a numerical value by absorbance measurement.

Cell-ELISA is a method of capturing a measurement object of each Cell, which is located on the Cell surface, and detecting and quantifying the measurement object by an enzyme reaction.

The RIA method (Radio Immunoassay ) uses a radioactive substance to label an antibody and measures radioactivity derived from the antibody, thereby enabling the antibody to be quantified.

Flow cytometry is a method of dispersing cells in a fluid, finely flowing the fluid, and optically analyzing the individual cells. The antibody labeled with a fluorescent dye is bound to a cell surface antigen by an antigen-antibody reaction, and the fluorescence intensity of the labeled antibody bound to the cell is measured, thereby quantifying the antigen binding of the antibody.

3. Antibodies or binding fragments thereof that specifically bind to antigen proteins

3-1 antibodies or binding fragments thereof that specifically bind CD3

The anti-CD 3 antibody specifically binding to CD3 and the antigen-binding fragment of the antibody (hereinafter also referred to as the antibody of the present invention or the like) of the present invention may be any of monoclonal antibodies and polyclonal antibodies. The isotype of the monoclonal antibody of the present invention is not particularly limited, and examples thereof include IgG, igM, igA1 such as IgG1, igG2, igG3, and IgG4, igA, igD, ig such as IgA2, and the like. The isotype and subclass of monoclonal antibodies can be determined, for example, by the agar two-way immunodiffusion method, ELISA method, RIA method, and the like. The monoclonal antibodies of the present invention include: antibodies derived from non-human animals (non-human animal antibodies), human antibodies, chimeric antibodies (also called "chimeric antibodies"), humanized antibodies, and the like are preferably used. The scope of the antibody of the present invention also includes mutants of the antibody (hereinafter referred to as "mutant antibodies"), and for example, the scope of the human antibody also includes human mutant antibodies.

Examples of the non-human animal antibody include antibodies derived from vertebrates such as mammals and birds. Examples of the mammal-derived antibody include rodent-derived antibodies such as mouse antibodies and rat antibodies. Examples of the avian antibody include chicken antibodies.

Examples of chimeric antibodies include, but are not limited to: and antibodies in which a variable region derived from a non-human animal antibody is bound to a human antibody (human immunoglobulin) constant region.

Examples of humanized antibodies include, but are not limited to: antibodies formed by grafting CDRs in the variable region of a non-human animal antibody to a human antibody (variable region of a human immunoglobulin); an antibody formed by grafting a partial sequence of a framework region of a non-human animal antibody to a human antibody in addition to a CDR; an antibody obtained by substituting one or more amino acids derived from any of the above non-human animal antibodies with a human amino acid.

Antibodies can be produced by various known methods. The known methods may be those using hybridomas, cellular immunity methods, and the like, or those using genetic recombination. In addition, methods for obtaining phage display-derived human antibodies screened from a human antibody library are also known. For example, phage display methods in which the variable region of a human antibody is expressed on the phage surface as an scFv, and phages that bind to an antigen are selected can be used. By analyzing the genes of phage selected according to binding to antigen, the DNA sequence encoding the variable region of human antibody binding to antigen can be determined. When the DNA sequence of the scFv binding to the antigen is clarified, an expression vector having the sequence can be prepared, and the vector can be introduced into an appropriate host and expressed, whereby human antibodies [ WO92/01047, WO92/20791, WO93/06213, WO93/11236, WO93/19172, WO95/01438, WO95/15388, annu. Rev. Immunol (1994) 12, 433-455] can be obtained.

The antibody having high activity thus obtained may be used as a leader antibody, and a gene encoding the leader antibody may be mutated to produce a mutant having higher activity (hereinafter referred to as "mutant antibody").

As one embodiment of the present invention, an antigen-binding fragment (hereinafter, simply referred to as "binding fragment") of the antibody of the present invention is provided. The anti-CD 3 antibody of the present invention or the binding fragment of an antibody against a target molecule that is not a complex of a cancer testis antigen peptide and a Human Leukocyte Antigen (HLA) includes a chimeric antibody, a humanized antibody or a binding fragment of a human antibody. The binding fragment of an antibody refers to a fragment or a modification thereof that retains at least antigen binding properties among the functions possessed by the antibody. The functions of the antibody are generally as follows: antigen binding activity, activity that modulates antigen activity (e.g., agonist activity), activity that internalizes an antigen in a cell, activity that inhibits or promotes interaction with a substance that interacts with an antigen, and the like.

The antigen-binding fragment of an antibody is not particularly limited as long as it is a fragment of the antibody that retains at least the antigen-binding property in the activity possessed by the antibody, and examples thereof include, but are not limited to: fab, fab ', F (ab') ₂ Fv, single chain Fv (scFv) in which Fv, heavy chain and light chain are linked by an appropriate linker, single domain antibodies (sdabs), and the like. Like scFv having a linker moiety, molecules comprising a moiety other than the antigen-binding fragment of the antibody of the invention are also included in the meaning of the antigen-binding fragment of the antibody of the invention.

Among them, fab and scFv are preferred.

Fab is an antigen binding fragment containing the heavy chain variable and constant regions CH1, and the light chain variable and constant regions CL, as shown in FIG. 38B. Fab can be obtained from complete antibodies by enzymatic digestion treatment with papain or the like.

scFv is an antigen-binding fragment of an antibody obtained by ligating the heavy chain variable region and the light chain variable region of the antibody with a linker of a polypeptide [ plurkthun a.the Pharmacology of Monoclonal Antibodies, code from Rosenburg and Moore, springer Verlag, new York,269-315 (1994), nature Biotechnology (2005), 23, 1126-1136]. In addition, tandem scfvs produced by conjugating two scfvs with a polypeptide linker can also be used as bispecific molecules. In addition, a trisomy (triabody) or the like composed of three or more scfvs can be used as the multispecific molecule.

The scFv can be obtained by using a phage display method in which a variable region of an antibody is expressed on a phage surface as a single chain antibody (scFv), and phage binding to an antigen is selected [ Nature Biotechnology (2005), 23, (9), p.1105-1116]. By analyzing the genes of phage selected according to binding to antigen, the DNA sequence encoding the variable region of human antibody binding to antigen can be determined. When the DNA sequence of the scFv binding to the antigen is clarified, an expression vector having the sequence can be prepared, and the vector can be introduced into an appropriate host and expressed, whereby a human antibody [ WO92/01047, WO92/20791, WO93/06213, WO93/11236, WO93/19172, WO95/01438, WO95/15388, annu.Rev.Immunol (1994) 12, p.433-455, nature Biotechnology (2005) 23 (9), p.1105-1116] can be obtained.

In the anti-CD 3 scFv of the invention, the heavy chain variable region and the light chain variable region are bound by disulfide bonds. Such scfvs can be expressed using mammalian cultured cells as in scfvs in which the heavy and light chain variable regions are not disulfide bonded.

Regarding the position and length of the light chain variable region, if determined using a definition different from IMGT (e.g., kabat, chothia, abM, contact, etc.) as compared to the case determined according to the definition of IMGT, one or more amino acids such as arginine and glycine may sometimes be contained at the carboxy terminus of the light chain variable region amino acid sequence determined according to the definition of IMGT. Antibodies or binding fragments thereof having such light chain variable regions are also encompassed by the antibodies or binding fragments thereof of the invention.

The anti-CD 3 antibody or antigen-binding fragment thereof is not particularly limited as long as it binds to human CD3, and preferably also binds to CD3 of a non-human primate such as a cynomolgus monkey. More preferred anti-CD 3 antibodies or antigen-binding fragments thereof include antibodies or antigen-binding fragments thereof comprising the following heavy chain variable regions and light chain variable regions: a heavy chain variable region CDRH1 consisting of the amino acid sequence shown in SEQ ID NO. 32; a heavy chain variable region CDRH2 consisting of the amino acid sequence shown in SEQ ID NO. 33; a heavy chain variable region CDRH3 consisting of the amino acid sequence shown in SEQ ID NO. 34; a light chain variable region CDRL1 composed of the amino acid sequence shown in SEQ ID NO. 35; a light chain variable region CDRL2 consisting of the amino acid sequence shown in SEQ ID NO. 36; and a light chain variable region CDRL3 composed of the amino acid sequence shown in SEQ ID NO. 37 (see FIG. 36, above).

As a more preferred antibody or antigen-binding fragment thereof comprising CDRH1 to CDRH3 and CDRL1 to CDRL3 shown in fig. 36, an antibody or antigen-binding fragment thereof comprising the following heavy chain variable region: a C3E-7085 heavy chain variable region consisting of the amino acid sequences of amino acid numbers 1 to 118 of the amino acid sequence shown in SEQ ID NO. 1 (FIG. 5); a C3E-7096 heavy chain variable region consisting of the amino acid sequences of amino acid numbers 1 to 118 of the amino acid sequence shown in SEQ ID NO. 3 (FIG. 7); a C3E-7097 heavy chain variable region composed of the amino acid sequences of amino acid numbers 1 to 118 of the amino acid sequence shown in SEQ ID NO. 39 (FIG. 46). It should be noted that, cysteine was additionally 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; cysteine was additionally added at amino acid number 44 of the 7097 heavy chain variable region to form an S-S bond with amino acid number 233 of the C3E-7097 light chain variable region.

Further, as a more preferable antibody or antigen-binding fragment thereof containing CDRH1 to CDRH3 and CDRL1 to CDRL3 shown in fig. 36, an antibody or antigen-binding fragment thereof containing the following light chain variable region can be exemplified: a C3E-7085 light chain variable region consisting of the amino acid sequence of amino acid numbers 1 to 107 of the amino acid sequence shown in SEQ ID NO. 2 (FIG. 6); a C3E-7096 light chain variable region consisting of the amino acid sequence of amino acid numbers 134 to 240 of the amino acid sequence shown in SEQ ID NO. 3 (FIG. 7); 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 in SEQ ID NO. 39 (FIG. 46). It should be noted that, cysteine was additionally added to amino acid number 177 of the 7096 light chain variable region to form an s—s bond with amino acid number 110 of the 7096 heavy chain variable region; cysteine was additionally added at amino acid number 233 of the 7097 light chain variable region to form an S-S bond with amino acid number 44 of the C3E-7097 heavy chain variable region.

Further, as a more preferable antibody or antigen-binding fragment thereof containing CDRH1 to CDRH3 and CDRL1 to CDRL3 shown in fig. 36, an antibody or antigen-binding fragment thereof containing a combination of the following heavy chain variable region and light chain variable region can be exemplified: a combination of a C3E-7085 heavy chain variable region and a light chain variable region consisting of amino acid numbers 1 to 118 shown in SEQ ID NO. 1 (FIG. 5) and amino acid numbers 1 to 107 shown in SEQ ID NO. 2 (FIG. 6); a combination of a C3E-7096 heavy chain variable region and a light chain variable region consisting of amino acid numbers 1 to 118 and 134 to 240 shown in SEQ ID NO. 3 (FIG. 7); a combination of the heavy chain variable region and the light chain variable region of C3E-7097 consisting of amino acid numbers 1 to 118 and 134 to 240 shown in SEQ ID No. 39 (fig. 46).

Further, as the more preferable antibody or antigen binding fragment thereof containing CDRH1 to CDRH3 and CDRL1 to CDRL3 shown in fig. 36, there can be exemplified: C3E-7085Fab composed of the amino acid sequence of amino acid sequence 1-216 shown in SEQ ID NO. 1 (figure 5) and the amino acid sequence of amino acid sequence 1-213 shown in SEQ ID NO. 2 (figure 6).

Further, as the more preferable antibody or antigen binding fragment thereof containing CDRH1 to CDRH3 and CDRL1 to CDRL3 shown in fig. 36, there can be exemplified: C3E-7096scFv composed of the amino acid sequences of amino acid numbers 1 to 240 of the amino acid sequence shown in SEQ ID NO. 3 (FIG. 7), and C3E-7097scFv composed of the amino acid sequences of amino acid numbers 1 to 240 of the amino acid sequence shown in SEQ ID NO. 39 (FIG. 46).

Further, as another preferred anti-CD 3 antibody or antigen-binding fragment thereof, there may be mentioned an antibody or antigen-binding fragment thereof comprising the following heavy chain variable region and light chain variable region: a heavy chain variable region CDRH1 consisting of the amino acid sequence shown in SEQ ID NO. 40; a heavy chain variable region CDRH2 composed of the amino acid sequence shown in SEQ ID NO. 41; a heavy chain variable region CDRH3 consisting of the amino acid sequence shown in SEQ ID NO. 42; a light chain variable region CDRL1 consisting of the amino acid sequence shown in SEQ ID NO. 43; a light chain variable region CDRL2 consisting of the amino acid sequence shown in SEQ ID NO. 44; and a light chain variable region CDRL3 composed of the amino acid sequence shown in SEQ ID NO. 45 (see FIG. 47, above).

The preferred antibody or antigen-binding fragment thereof comprising the CDRH1 to CDRH3 and CDRL1 to CDRL3 shown in fig. 47 may be an antibody or antigen-binding fragment thereof comprising the following heavy chain variable region: a C3E-7093 heavy chain variable region consisting of the amino acid sequences of amino acid numbers 1 to 118 of the amino acid sequence shown in SEQ ID NO. 46 (FIG. 48); a C3E-7098 heavy chain variable region consisting of the amino acid sequences of amino acid numbers 1 to 118 of the amino acid sequence shown in SEQ ID NO. 48 (FIG. 50); a C3E-7099 heavy chain variable region consisting of the amino acid sequences of amino acid numbers 1 to 118 of the amino acid sequence shown in SEQ ID NO. 49 (FIG. 51). It should be noted that, cysteine was additionally 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; cysteine was additionally added at amino acid number 44 of the C3E-7099 heavy chain variable region to form an S-S bond with amino acid number 235 of the C3E-7099 light chain variable region.

Further, as a preferred antibody or antigen-binding fragment thereof comprising the CDRH1 to CDRH3 and CDRL1 to CDRL3 shown in fig. 47, an antibody or antigen-binding fragment thereof comprising the following light chain variable region may be exemplified: a C3E-7093 light chain variable region consisting of the amino acid sequences of amino acid numbers 1 to 118 of the amino acid sequence shown in SEQ ID NO. 47 (FIG. 49); a C3E-7098 light chain variable region consisting of the amino acid sequence of amino acid numbers 134 to 242 of the amino acid sequence shown in SEQ ID NO. 48 (FIG. 50); 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 SEQ ID NO. 49 (FIG. 51). It should be noted that, cysteine was additionally added to amino acid number 177 of the C3E-7098 light chain variable region to form an S-S bond with amino acid number 110 of the C3E-7098 heavy chain variable region; cysteine was additionally added at amino acid number 235 of the C3E-7099 light chain variable region to form an S-S bond with amino acid number 44 of the C3E-7099 heavy chain variable region.

The preferred antibody or antigen-binding fragment thereof comprising the CDRH1 to CDRH3 and CDRL1 to CDRL3 shown in fig. 47 may be an antibody or antigen-binding fragment thereof comprising a combination of the following heavy chain variable regions and light chain variable regions: a combination of a C3E-7093 heavy chain variable region and a light chain variable region consisting of amino acid numbers 1 to 118 shown in SEQ ID NO. 46 (FIG. 48) and amino acid numbers 1 to 109 shown in SEQ ID NO. 47 (FIG. 49); a combination of a heavy chain variable region and a light chain variable region of C3E-7098 consisting of amino acid numbers 1 to 118 and 134 to 242 shown in SEQ ID NO. 48 (FIG. 50); a combination of the heavy chain variable region and the light chain variable region of C3E-7099 consisting of amino acid numbers 1 to 118 and 134 to 242 shown in SEQ ID NO. 49 (FIG. 51).

Further, as a more preferable antibody or antigen-binding fragment thereof containing the CDRH1 to CDRH3 and CDRL1 to CDRL3 shown in fig. 46, there can be exemplified: C3E-7093Fab which consists of the amino acid sequence shown in SEQ ID NO. 46 (figure 48) and the amino acid sequence of the amino acid sequence shown in SEQ ID NO. 47 (figure 49) and the amino acid sequence of 1 to 215.

In addition, an scFv that binds to CD3 and binds VH and VL via disulfide bonds may contain a linker, linker or any other moiety (moiety) between VH and VL, even if the scFv is comprised in a multispecific molecule of the invention. For example, the anti-CD 3scFv may include "other binding moiety", "other group", or a part thereof, which will be described later, between VH and VL.

3-2 antibodies or binding fragments thereof that bind to target molecules

The antibody or binding fragment thereof that binds to the "2-2. Target molecule" may be any of monoclonal and polyclonal antibodies. The isotype of the monoclonal antibody of the present invention is not particularly limited, and examples thereof include IgG, igM, igA1 such as IgG1, igG2, igG3, and IgG4, igA, igD, ig such as IgA2, and the like. The isotype and subclass of monoclonal antibodies can be determined, for example, by the agar two-way immunodiffusion method, ELISA method, RIA method, and the like. The monoclonal antibodies of the present invention include: antibodies derived from non-human animals (non-human animal antibodies), human antibodies, chimeric antibodies (also called "chimeric antibodies"), humanized antibodies, and the like are preferably used. The scope of the antibody of the present invention also includes mutants of the antibody (hereinafter referred to as "mutant antibodies"), and for example, the scope of the human antibody also includes human mutant antibodies.

scFv against a target molecule that is not a complex of cancer testis antigen peptide and Human Leukocyte Antigen (HLA), its heavy chain variable region and light chain variable region can be bound by disulfide bonds.

3-3 mutants of antibodies or antigen binding fragments thereof

The mutant of the antibody or antigen-binding fragment thereof of the present invention may preferably have the following functions: reducing susceptibility to protein breakdown or oxidation; maintaining, improving, or inhibiting the decline and change in biological activity and function; improving or modulating antigen binding capacity, or imparting physicochemical or functional properties. It is known that the specific amino acid side chains on the surface of a protein vary, and that the function and activity of the protein vary, and such examples include deamidation of asparagine side chains, isomerization of aspartic acid side chains, and the like. Mutants substituted with other amino acids in order to prevent such changes in the amino acid side chains are also included in the scope of the mutants of the present invention.

Examples of the mutant of the present invention include: an antibody or an antigen-binding fragment thereof having an amino acid sequence obtained by substituting a preservable amino acid in the amino acid sequence of the antibody or the antigen-binding fragment thereof. The conservative amino acid substitution means a substitution occurring in an amino acid group associated with an amino acid side chain.

Preferred amino acid groups are as follows: acid group = aspartic acid, glutamic acid; basic group = lysine, arginine, histidine; nonpolar group = alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; uncharged polar groups = glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Other preferred amino acid groups are as follows: aliphatic hydroxyl group = serine and threonine; amide group = asparagine and glutamine; aliphatic group = alanine, valine, leucine, and isoleucine; aromatic group = phenylalanine, tryptophan and tyrosine. The amino acid substitution in the mutant antibody is preferably performed within a range that does not reduce the antigen binding activity possessed by the original antibody.

3-4 modifications, complexes of antibodies or binding fragments thereof

The present invention provides modifications of antibodies or binding fragments thereof. The modified substance of the antibody or the binding fragment thereof of the present invention means a modified substance obtained by chemically or biologically modifying the antibody or the binding fragment thereof of the present invention. The chemical modifier comprises: chemical modifications of chemical bonds, N-bonds or O-bond carbohydrate chains leading to chemical moieties of the amino acid backbone, and the like. Biological modifiers include: modifications that are post-translational modifications (e.g., addition of sugar chains to the N-or O-linkages, processing of the amino-or carboxy-terminal regions, deamidation, isomerization of aspartic acid, oxidation of methionine), modifications that are expressed using a prokaryotic host cell to add a methionine residue at the amino-terminus, and the like. In addition, substances labeled to enable detection or separation of the antibody or antigen of the present invention, such as enzyme labels, fluorescent labels, affinity labels, are also included in the meaning of the modification. Such a modified product of the antibody or the binding fragment thereof of the present invention can be used for improving the stability and blood retention of the original antibody or the binding fragment thereof of the present invention, reducing antigenicity, detecting or isolating the antibody or antigen, and the like.

Examples of the chemical moiety included in the chemical modifier include: polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymer, carboxymethyl cellulose, dextran, polyvinyl alcohol, and other water-soluble polymers.

As the biological modifier, there may be mentioned: modifications by enzyme treatment, cell treatment, etc., fusions of other peptides such as tags added by gene recombination, and modifications in which cells expressing an endogenous or exogenous sugar chain-modifying enzyme are prepared as hosts.

The modification may be performed at any position in the antibody or a binding fragment thereof, or at a desired position, or the same modification or two or more different modifications may be performed at one or two or more positions.

However, the deletion of these heavy chain sequences, or modification of heavy or light chain sequences, has less impact on the antigen binding capacity and effector functions of antibodies (complement activation, antibody-dependent cellular cytotoxicity, etc.).

Thus, the invention also encompasses antibodies that are subject to such deletions or modifications. For example, there may be mentioned: a deletion of one or two amino acids at the carboxy terminus of the heavy chain [ Journal of Chromatography A;705;129-134 (1995) ]; two amino acid residues glycine and lysine at the carboxy terminus of the heavy chain are deleted, whereas proline residues that are relocated at the carboxy terminus are amidated as deletions [ Analytical Biochemistry,360:75-83 (2007) ]; antibodies in which the amino-terminal glutamine or glutamic acid residue of the heavy chain or light chain of the antibody is pyroglutamyl-modified (international patent publication No. WO 2013/147153), and the like (these are collectively referred to as "deletants"). However, the carboxy-terminal deletions of the heavy and light chains of the antibodies of the present invention are not limited to the above-described species as long as the antigen binding ability and effector function are maintained. When the antibody of the present invention contains two or more chains (for example, heavy chains), the two or more chains (for example, heavy chains) may be either a heavy chain having a complete length or a heavy chain selected from the group consisting of the above deletions, or a combination of any two of them. The ratio of the amount of each deleter or the ratio of the number of molecules may be affected by the type of cultured mammalian cell and the culture conditions under which the antibody of the present invention is produced, but as the main component of the antibody of the present invention, there is a case where both heavy chains lack one amino acid residue at the carboxyl terminus.

In addition, even if the antibody or antigen-binding fragment thereof of the present invention (the antibody or antigen-binding fragment thereof contained in the molecule, multispecific molecule, bispecific molecule, etc.) is added with one or several amino acids (and some or all of them are modified as described above) derived from an expression vector and/or a signal sequence, etc. at the amino-terminus and/or the carboxyl-terminus, the molecule containing the modification of the antibody or antigen-binding fragment thereof is also included in the scope of the molecule of the present invention as long as the desired antigen-binding activity can be maintained.

In the present invention, "antibody or binding fragment thereof" also includes the meaning of "modification of antibody or antigen binding fragment thereof". The term "antibody or antigen-binding fragment thereof" included in the molecule, multispecific molecule, bispecific molecule, etc. of the present invention also includes the meaning of "modification of antibody or antigen-binding fragment thereof".

In addition, by regulating sugar chain modification (glycosylation, defucosylation, etc.) bound to the antibody of the present invention, antibody-dependent cellular cytotoxic activity can be enhanced. Examples of the antibody sugar chain modification regulation technique include, but are not limited to: international patent publication Nos. WO99/54342, WO00/61739, WO02/31140, etc.

The present invention also includes a complex (Immunoconjugate) formed by connecting the antibody and another molecule via a linker. As an example of an Antibody-Drug complex in which the Antibody is bound to a radioactive substance and a compound (Drug) having a pharmacological action, ADC (Antibody-Drug Conjugate) [ Methods Mol biol. (2013) 1045:1-27; nature Biotechnology (2005) 23, p.1137-1146].

The present invention also includes complexes of these antibodies linked to other functional polypeptides. As an example of such an antibody-peptide complex, there is given a complex of the antibody and an albumin binding polypeptide [ Protein Eng Des sel (2012) (2): 81-8].

The modified product of the antibody, the antibody having modified sugar chains, and the complex are included in the antibody of the present invention; the modified product of the antibody, the antibody having modified sugar chains, and the binding fragment of the complex are included in the binding fragment of the antibody of the present invention.

4. Method for producing antibody or antigen-binding fragment thereof

The antibody or antigen-binding fragment thereof of the present invention can be produced as a recombinant antibody by, for example, inserting a DNA encoding a heavy chain variable region or a DNA encoding a light chain variable region into an expression vector, transforming a host cell for expression with the vector, and culturing the host cell, thereby allowing the cell to produce the antibody of the present invention.

Regarding the DNA encoding an antibody or antigen-binding fragment thereof, a DNA encoding a heavy chain can be obtained by ligating a DNA encoding a heavy chain variable region with a DNA encoding a heavy chain constant region; in addition, DNA encoding a light chain can be obtained by ligating DNA encoding a light chain variable region with DNA encoding a light chain constant region.

Regarding the antibody or antigen-binding fragment thereof of the present invention, the above-described DNA encoding a heavy chain and DNA encoding a light chain may be inserted into an expression vector, the vector is used to transform a host cell, and the host cell is cultured, thereby producing the antibody or antigen-binding fragment thereof of the present invention. In this case, the above-described heavy chain-encoding DNA and light chain-encoding DNA may be introduced into the same expression vector, and the host cell may be transformed with the vector, or the heavy chain-encoding DNA and light chain-encoding DNA may be introduced into different expression vectors, and the host cell may be transformed with both vectors. In this case, the DNA 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 introduced in advance. Alternatively, the vector may comprise DNA encoding a signal peptide that facilitates secretion of antibodies by the host cell. In this case, the DNA encoding the signal peptide is linked in frame with the DNA encoding the antibody or antigen binding fragment thereof. The signal peptide is removed after the production of the antibody or antigen-binding fragment thereof, whereby an antibody or antigen-binding fragment thereof as a mature protein can be obtained.

In this case, the DNA encoding the heavy chain variable region, the DNA encoding the light chain variable region, the DNA obtained by ligating the DNA encoding the heavy chain variable region with the DNA encoding the heavy chain constant region, and the DNA obtained by ligating the DNA encoding the light chain variable region with the DNA encoding the light chain constant region may be functionally linked to a promoter, an enhancer, a polyadenylation signal or the like. Here, functionally connected means that connection is made so that the elements fulfill their functions.

The expression vector is not particularly limited as long as it can replicate in a host such as an animal cell, a bacterium, or a yeast, and examples thereof include a known plasmid and phage. Examples of the vector used for constructing the expression vector include: pcDNA (trademark) (Semer Feichi technologies), flexi (registered trademark) vector (Promega), pUC19, pUEX2 (Amersham), pGEX-4T, pKK233-2 (Pharmacia), pMAM-neo (Clontech), etc. As the host cell, a prokaryotic cell such as E.coli or Bacillus subtilis, or a eukaryotic cell such as yeast or animal cell may be used, and eukaryotic cells are preferable. For example, as animal cells, human embryonic kidney cell line HEK293 cells, chinese Hamster Ovary (CHO) cells, and the like can be used. The 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 produced antibody can be purified by using a separation and purification method used for a general protein. For example, it may be suitably selected and combined: affinity chromatography, other chromatography, filtration membranes, ultrafiltration, salting out, dialysis, etc.

5. Multispecific molecules

The multispecific molecule of the present invention includes the scFv binding to CD3, i.e., the scFv binding to the heavy chain variable region and the light chain variable region of the present invention via disulfide bonds, or the Fab binding to CD3 (hereinafter referred to as "CD 3 antibody of the present invention"), and is not particularly limited as long as it includes a moiety (moeity) (hereinafter referred to as "other binding moiety") that binds to a target molecule other than CD3, preferably a target molecule that is not a complex of a cancer testis antigen peptide and a Major Histocompatibility Complex (MHC). In addition to the "CD 3 antibodies of the invention" and "other binding moieties", other groups (hereinafter referred to as "other groups") may be included. As the "other binding moiety", there can be exemplified: antibodies or binding fragments thereof, proteins or peptides derived from non-immunoglobulins, nucleic acid molecules such as nucleic acid aptamers, low molecular weight compounds, T cell receptors, and the like. As "other group", there may be exemplified: compounds (including proteins, peptides, nucleic acid molecules, low molecular compounds, compounds of radioactive isotopes, compounds containing a labeling substance, artificially synthesized substances, pharmaceutical ingredients, antitumor agents, etc.) which exert biological activity, pharmacological action, therapeutic effect, prophylactic effect, adjuvant effect, in vivo power regulating function, labeling function, etc.; precursors (prodrugs, etc.) thereof; and a substance formed by binding or fusing a linker or a connecting part in the above compound. The "CD 3 antibodies" and "other binding moieties" of the invention, and any "other groups" may be bound or fused directly to each other, or indirectly via linkers and linkers. The number of "other binding moieties" may be 1 or more, and may be 2, 3 or more. The "other group" may be arbitrarily 1, 2, or more. They may be combined or fused in a straight line or branched, and the order of combination or fusion, the position of branching, and the like are not limited at all. Such a multispecific molecule of the present invention may be in the configuration illustrated in fig. 38A-38F, fig. 39A and 39B, fig. 40A and 40B, fig. 41A and 41B, fig. 42, fig. 43A and 43B, fig. 44A and 44B, and fig. 45, or comprise the illustrated configuration, but is not limited thereto.

Preferred multispecific molecules of the present invention include multispecific proteins (molecules composed entirely of polypeptides and/or peptides), such as multispecific antibodies. Examples of the multispecific antibody include bispecific antibodies, trispecific antibodies, and tetraspecific antibodies. "bispecific" (or "trispecific", "tetraspecific", etc.) refers to an antibody or antigen-binding fragment comprising such bispecific that is capable of binding to two (or three, four, etc.) different epitopes of the same molecule or to different epitopes on two molecules (or three molecules, four molecules, etc.) from each other. The bispecific molecules of the invention will bind to CD3 and will also bind to target molecules, preferably target molecules that are not complexes of cancer testis antigen peptides and Human Leukocyte Antigens (HLA).

Examples of the multispecific molecule of the present invention include molecules having the following structures (configurations).

Examples of the bispecific molecule of the present invention include bispecific molecules in which a first antibody Fab and a second antibody scFv are bound to one Fc of a dimer via a linker. In this case, either Fab or scFv may be bound to Fc, or Fab or scFv may be bound to Fc. Preferably, the Fab is conjugated to Fc and the scFv is conjugated to the Fab. Binding of Fab to scFv the scFv may bind the scFv to the variable region of the Fab via a linker. The bispecific molecule is in a configuration in which a mutant Fc is introduced, wherein the introduced mutation is such that a heterodimer is formed downstream of the attachment of the scFv to the Fab via a linker. Such bispecific molecules are called scFv-Fab-heterodimer Fc type bispecific molecules, or scFv-Fab-heterodimer Fc type (FIG. 43B top left).

In the present invention, for example, an scFv-Fab-heterodimer Fc type composed of an anti-CD 3 Fab and an scFv against a target molecule that is not a complex of a cancer testis antigen peptide and a Human Leukocyte Antigen (HLA) can be used.

In addition, with respect to the anti-CD 3scFv comprised by the bispecific molecule of the present invention, the heavy chain variable region and the light chain variable region thereof are bound by disulfide bonds. In addition, scFv against a target molecule that is not a complex of cancer testis antigen peptide and Human Leukocyte Antigen (HLA), its heavy chain variable region and light chain variable region can be bound by disulfide bonds.

In addition, there may be mentioned a bispecific molecule in which taFv (fig. 38E) having a configuration in which two scFv of the first antibody and the second antibody are connected via a linker is bound to one Fc of a dimer via a linker or directly bound to one Fc of a dimer without via a linker. Such bispecific molecules are called taFv-heterodimeric Fc-type bispecific molecules, or taFv-heterodimeric Fc-types (FIG. 40B bottom left). The bispecific molecule is a configuration in which a mutant Fc is introduced in association with a hybrid, wherein the introduced mutation is the formation of a heterodimer downstream of taFv. the order of attachment of the first antibody and the second antibody in taFv is not limited.

The structure of the scFv-Fab-heterodimer Fc type bispecific molecule is shown in FIG. 43B, top left. In addition, fig. 38A shows the structure of scFv, fig. 38B shows the structure of Fab, fig. 38E shows the structure of taFv, fig. 38F shows the structure of scFv-Fab, fig. 40B shows the structure of taFv-heterodimeric Fc-type bispecific molecule at the bottom left, and fig. 41B shows the structure of taFv-Fab-heterodimeric Fc-type bispecific molecule at the top right.

The bispecific molecule of the present invention has a structure in which a plurality of polypeptides are associated.

In the present invention, as taFv, for example, taFv of anti-CD 3scFv and scFv against a target molecule [ preferably a target molecule other than a complex of a cancer testis antigen peptide and a Human Leukocyte Antigen (HLA) ] can be used. Regarding taFv-heterodimeric Fc-type bispecific molecules, preferably, (a) a first polypeptide comprising, in order from the N-terminus to the C-terminus, an scFv that specifically binds to a target molecule [ preferably a target molecule that is not a complex of a cancer testis antigen peptide and a Human Leukocyte Antigen (HLA) ], an scFv that specifically binds to CD3, and an immunoglobulin Fc region (i); the second polypeptide comprises a hinge region and an Fc region of an immunoglobulin (ii); more preferably, (b) the first polypeptide and the second polypeptide associate in Fc region (i) and Fc region (ii). The Fc regions of the first and second polypeptides contain heterodimer-forming mutations. An example of a taFv-heterodimeric Fc-type bispecific molecule is shown in the lower left of figure 40B. As shown in the lower left of fig. 40B, the Fc region (i) portion of the first polypeptide binds to the Fc region (ii) portion of the second polypeptide, indicated by darkening, and the first polypeptide associates with the second polypeptide. For example, in fig. 40B, the lower left-hand, white-colored scFv is the scFv against a target molecule, preferably a target molecule that is not a complex of a cancer testis antigen peptide and a Human Leukocyte Antigen (HLA), and the upper right-hand, diagonal scFv is the anti-CD 3scFv (the black bar between VH and VL indicates that both bind via disulfide bonds).

A more preferred taFv-heterodimeric Fc-type bispecific molecule of the present invention comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO. 3. The preferred taFv-heterodimeric Fc-type bispecific molecules of the invention comprise a hinge region of human antibody origin and a wild-type or mutant Fc.

Regarding the scFv-Fab-heterodimeric Fc-type bispecific molecule, preferably, (a) comprises a first polypeptide comprising, in order from the N-terminus to the C-terminus, an scFv that specifically binds to a target molecule that is not a complex of a cancer testis antigen peptide and a Human Leukocyte Antigen (HLA), a variable region and a constant region CH1 of an antibody heavy chain that specifically binds to CD3, and an immunoglobulin Fc region (i); the second polypeptide comprises a hinge region and an Fc region of an immunoglobulin (ii); the third polypeptide comprises an antibody light chain consisting of a variable region and a constant region; more preferably, (b) the first polypeptide and the second polypeptide are associated in the Fc region (i) and the Fc region (ii), the first polypeptide being associated with (the antibody light chain of) the third polypeptide in the variable region and the constant region CH1 of the antibody heavy chain. The Fc regions of the first and second polypeptides may be wild-type or may contain heterodimer-forming mutations. An example of a scFv-Fab-heterodimeric Fc-type bispecific molecule is shown in the upper left of fig. 43B. The upper left right half of fig. 43B is the first polypeptide and the third polypeptide, and the left half is the second polypeptide. As shown in the upper left of fig. 43B, the Fc region (i) portion of the first polypeptide associates with the Fc region (ii) portion of the second polypeptide, indicated by darkening, and the first polypeptide associates with the third polypeptide. For example, in the upper left of fig. 43B, the scFv shown in black is the scFv against a target molecule, preferably a target molecule that is not a complex of a cancer testis antigen peptide and a Human Leukocyte Antigen (HLA), and the Fab shown in white and checkered pattern and in horizontal lines is an anti-CD 3 Fab.

The amino acid sequence included in the first polypeptide included in the preferred scFv-Fab-heterodimer Fc type bispecific molecule can be exemplified by the amino acid sequence shown in SEQ ID NO. 1.

The preferred scFv-Fab-heterodimeric Fc-type bispecific molecules of the invention comprise a hinge region of human antibody origin and a mutant Fc, and the more preferred scFv-Fab-heterodimeric Fc-type bispecific molecules comprise a hinge region of human antibody origin and a wild-type or mutant Fc.

The third polypeptide comprised by the preferred scFv-Fab-heterodimeric Fc-type bispecific molecule of the present invention comprises a light chain derived from a human antibody. The third polypeptide contained in the more preferred scFv-Fab-heterodimeric Fc bispecific molecule can be exemplified by the amino acid sequence shown in SEQ ID NO. 2.

The bispecific molecule of the invention may comprise one, two or more than three peptides which are "deletions" as described above, i.e. mutations (including deletions) of one or two (or more than two) amino acids at its carboxy-terminus, in particular from the carboxy-terminus of the antibody heavy chain. For example, the first polypeptide comprising RX3-0002, which is one of the preferred scFv-Fab-heterodimer Fc-type bispecific molecules of the invention, may have the carboxyl terminus of the amino acid sequence, which may be any of Lys at position 713 of SEQ ID NO. 31, gly at position 712, where one amino acid is deleted, or a mixture comprising them. Similarly, the second polypeptide contained in the preferred scFv-Fab-heterodimer Fc-type bispecific molecule of the present invention may have the carboxyl-terminus of the amino acid sequence of any of Lys at position 246, gly at position 245, deleted one amino acid of SEQ ID NO. 9, and mixtures thereof.

The scFv and Fab comprised by the bispecific molecule of the invention are preferably the scFv and Fab of a humanized or human antibody, and the Fc is preferably the Fc of a human antibody.

The variable regions included in the bispecific molecule of the present invention may be bound in the order of the heavy chain variable region and the light chain variable region from the amino terminal side of the antibody, or may be bound in the order of the light chain variable region and the heavy chain variable region. A linker (optional) may also be present between the two variable regions. In addition, glycine residues (optional) may also be present at the amino terminus of the amino-terminal side variable region. Bispecific molecules of tandem scFv type may also bind a linker, FLAG tag, and/or His tag (optional) at the carboxy terminus of the variable region on the carboxy-terminal side. One of the preferred forms is exemplified by a heavy chain variable region, a first linker, a light chain variable region, a second linker, a FLAG tag, and a His tag, which are sequentially bound from the amino terminus.

The linker includes single-chain polypeptides or single-chain oligopeptides, or also includes compositions of PEG, nucleotides, sugar chains, compounds, and the like. In addition, there is no particular limitation as long as two polypeptides are combined, and a known linker can be used.

The length of the linker is, for example, 5 to 30 amino acids. When the bispecific molecule contains multiple linkers, either peptide linkers of the same length or peptide linkers of different lengths may be used.

The peptide linker may be, for example, a loop of (Gly. Ser) (SEQ ID NO: 38), but may be added with one or more amino acid residues different from Gly and Ser.

In summary, the multispecific antibodies of the present invention can comprise structures (configurations), particularly those that can be employed by bispecific antibodies, preferably taFv-heterodimeric Fc-type and scFv-Fab-heterodimeric Fc-type; more preferably of the scFv-Fab-heterodimeric Fc type. More preferred as the taFv-heterodimer Fc type are those of scFv and anti-CD 3 scFv directed against a target molecule [ preferably a target molecule which is not a complex of a cancer testis antigen peptide and a Human Leukocyte Antigen (HLA) ] in this order from the N-terminus to the C-terminus (taFv-heterodimer Fc type: FIG. 40B, left-hand bottom). In addition, as the scFv-Fab-heterodimer Fc type, an scFv-Fab-heterodimer Fc type (upper left in fig. 43B) containing at the N-terminus an scFv against a target molecule, preferably a target molecule that is not a complex of a cancer testis antigen peptide and a Human Leukocyte Antigen (HLA), is also a more preferred configuration that can be employed by the bispecific antibody of the present invention.

Among the above-described multispecific antibodies of the present invention, particularly bispecific antibodies, preferred antibodies comprising scFv-Fab-heterodimeric Fc-type comprise a polypeptide set selected from the group consisting of: (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 obtained by deleting 1 or 2 amino acids from the carboxy terminus of the amino acid sequence; a polypeptide comprising an amino acid sequence consisting of amino acid residues 20 to 246 of SEQ ID NO. 9, or an amino acid sequence obtained by deleting 1 or 2 amino acids from the carboxy terminus of the amino acid sequence; and a polypeptide comprising an amino acid sequence consisting of amino acid residues 21 to 233 of SEQ ID NO. 12; (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 obtained by deleting 1 or 2 amino acids from the carboxy terminus of the amino acid sequence; a polypeptide comprising an amino acid sequence consisting of amino acid residues 20 to 246 of SEQ ID NO. 9, or an amino acid sequence obtained by deleting 1 or 2 amino acids from the carboxy terminus of the amino acid sequence; and a polypeptide comprising an amino acid sequence consisting of amino acid residues 21 to 233 of SEQ ID NO. 12; (iii) A polypeptide comprising an amino acid sequence consisting of amino acid residues 20 to 713 of SEQ ID NO. 31, or an amino acid sequence obtained by deleting 1 or 2 amino acids from the carboxy terminus of the amino acid sequence; a polypeptide comprising an amino acid sequence consisting of amino acid residues 20 to 246 of SEQ ID NO. 9, or an amino acid sequence obtained by deleting 1 or 2 amino acids from the carboxy terminus of the amino acid sequence; and a polypeptide comprising an amino acid sequence consisting of amino acid residues 21 to 233 of SEQ ID NO. 12.

Among the above-described multispecific antibodies of the present invention, particularly bispecific antibodies, preferred antibodies comprising taFv-heterodimer Fc-type comprise one polypeptide pair selected from the group consisting of: (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 obtained by deleting 1 or 2 amino acids from the carboxy terminus of the amino acid sequence; and a polypeptide comprising an amino acid sequence consisting of amino acid residues 20 to 246 of SEQ ID NO. 9, or an amino acid sequence obtained by deleting 1 or 2 amino acids from the carboxy terminus of the 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 obtained by deleting 1 or 2 amino acids from the carboxy terminus of the amino acid sequence; and a polypeptide comprising an amino acid sequence consisting of amino acid residues 20 to 246 of SEQ ID NO. 9, or an amino acid sequence obtained by deleting 1 or 2 amino acids from the carboxy terminus of the amino acid sequence; (iii) A polypeptide comprising an amino acid sequence consisting of amino acid residues 20 to 739 of SEQ ID NO. 25 or an amino acid sequence obtained by deleting 1 or 2 amino acids from the carboxy terminus of the amino acid sequence; and a polypeptide comprising an amino acid sequence consisting of amino acid residues 20 to 246 of SEQ ID NO. 9, or an amino acid sequence obtained by deleting 1 or 2 amino acids from the carboxyl terminus of the amino acid sequence.

In addition, examples of the configuration that can be obtained by the multispecific molecule of the present application include the configurations illustrated in FIGS. 38 to 45 and other configurations disclosed in the present application, and various configurations described by Godar et al, volume Expert Opinion on Therapeutic Patents (3 rd edition), pages 251 to 276 (2018), brinkmann and Knotelmann MABS, volume 9 (2 nd edition), pages 182 to 212 (2017), and Wu and Demarest Methods, volume 154, pages 3 to 9 (2019). All of these documents are also included in the present disclosure.

The application also includes the following molecules: a molecule comprising an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more identity to the amino acid sequence contained in the molecule of the present application, and binding to CD3 and also binding to a target molecule which is not a complex of a cancer testis antigen peptide and a Human Leukocyte Antigen (HLA).

The multispecific antibody of the present application has excellent biological activity, physicochemical properties (hereinafter referred to as "physical properties"), safety, in vivo kinetics, and the like. Since impurities contained in biopharmaceuticals are related to safety of the drugs, proper specification setting and management of whether there is an increase in preparation and preservation are required. Among them, aggregates are one of the main impurities, which leads to a risk of immunogenicity, a decrease in efficacy, and the like, and therefore the project should be extremely strictly controlled. [ Guidance for Industry Immunogenecity Assessment for Therapeutic Protein Products, U.S. Pat. No. 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, p.15-16]. The management of impurities requires evaluation not only during production but also during production steps and after production, including the stability with time (whether or not there is an increase). Since the period of validity of the drug is set according to the results of the long-term stability test, a longer period of validity can be set for an antibody that is stable over time. Therefore, as an index for screening an appropriate antibody for use in a biopharmaceutical, that is, physicochemical properties of the present application, there are solution stability, shaking stability, and the like. When a multispecific antibody having a concern about solution stability, shaking stability, or the like is selected as a drug, a large amount of resources and costs are required to be added additionally to a purification step for reducing the amount of aggregates (thus inevitably reducing the yield), a treatment method for establishing aggregates which are less likely to occur, or the like, and therefore, an antibody having less concern about these aspects is preferably selected.

Solution stability can be evaluated, for example, by: samples were prepared at 20mg/mL and HMWS content (%) was determined by size exclusion chromatography after 1 day, 7 days, and 14 days of storage at 40 ℃. Samples with relatively low increase rates of HMWS (%) can be judged as relatively high solution stability.

The oscillation stability can be evaluated, for example, by: samples were prepared at 2mg/mL, and were subjected to size exclusion chromatography with shaking at 2500rpm for 1 to 2 hours, and the peak area and HMWS (%) of each sample were calculated. However, in this method, the diameter of the aggregates detected as HMWS is about 10 to 100nm, while sub-visible particles (particle size 0.1 to 10 μm) and insoluble particles (particle size 10 μm or more) having particle diameters of 100nm or more are not detected. To confirm whether particles of this size are formed, for example, using a sample treated before and after shaking for 2 hours, the number of particles is evaluated by a microflow imaging method using FlowCAM8100 (manufactured by DKSH Japan) or the like. If the number of particles increases before and after shaking, it can be stated that shaking aging results in the formation of particles via HMWS starting from the monomers of the protein of interest. Therefore, in the evaluation of the oscillation stability, explanation and evaluation will be made based on the results of the following points: (1) rate of increase of HMWS (%); (2) a reduction rate of peak area; and/or (3) increase or decrease in particle count by microfluidic imaging. For example, if the increase rate of HMWS (%) of a certain sample is greater than that of other samples, but the decrease rate of peak area of the sample and/or the increase in the number of particles obtained by the microfluidic imaging method is smaller than that of the other samples, it can be evaluated that the oscillation stability of the sample is higher than that of the other samples. The reason for this is that the increase (%) of HMWS in this other sample is low because aggregates corresponding to HMWS associate and form large polymers, which cannot be detected by size exclusion chromatography. The aggregate consisted of the target protein, and it was confirmed that the aggregate which was hardly contained before the shaking was increased after the shaking.

The multispecific molecules of the present invention, for example, bispecific antibodies in which taFv-heterodimer Fc type, i.e., the heavy chain variable region and the light chain variable region of anti-CD 3scFv, are bound by disulfide bonds, and bispecific antibodies in scFv-Fab-heterodimer Fc type, in solution, are less prone to dimer-containing HMWS for a long period of time, and have high solution stability. For example, in the method for evaluating solution stability exemplified in paragraph 0137, the solution stability is preferably higher than that of a taFv-heterodimer Fc type, i.e., a bispecific antibody in which the heavy chain variable region and the light chain variable region of the anti-CD 3scFv are not bonded by disulfide bonds.

In addition, the multispecific molecules of the present invention, for example, bispecific antibodies in which taFv-heterodimer Fc type, i.e., the heavy chain variable region and the light chain variable region of anti-CD 3scFv, are bound by disulfide bonds, and scFv-Fab-heterodimer Fc type bispecific antibodies are interpreted according to the results of (1) the rate of increase in HMWS, (2) the rate of decrease in peak area, and/or (3) the increase or decrease in particle count by microfluidic imaging methods, which contain dimers, and have high oscillation stability. For example, in the method for evaluating oscillation stability exemplified in paragraph 0138, the oscillation stability is preferably higher than that of a taFv-heterodimer Fc type, i.e., a bispecific antibody in which the heavy chain variable region and the light chain variable region of the anti-CD 3scFv are not bonded by disulfide bonds.

The present invention provides pharmaceutical compositions containing anti-CD 3 antibodies, antigen-binding fragments thereof, or modifications thereof, and/or multispecific molecules (hereinafter referred to as "anti-CD 3 antibodies, etc.) comprising the same.

In the present invention, the treatment and/or prevention of diseases includes, but is not limited to: preventing the onset, inhibiting or impeding the progression or development of the disease; alleviating one or more symptoms exhibited by an individual suffering from the disease, inhibiting or alleviating exacerbations or progression; treatment or prevention of secondary diseases, and the like. In the case of a pharmaceutical composition containing a multispecific molecule, examples of diseases include cancer.

The pharmaceutical composition of the present invention may contain pharmaceutically acceptable diluents, carriers, solubilizers, emulsifiers, preservatives and/or adjuvants in addition to the therapeutically or prophylactically effective amount of anti-CD 3 antibodies and the like.

"therapeutically or prophylactically effective amount" refers to an amount that exerts a therapeutic or prophylactic effect, depending on the particular disease, mode of administration, and route of administration.

The pharmaceutical composition of the present invention may contain a substance for changing, maintaining or maintaining the following properties (hereinafter referred to as "a substance for a preparation"): pH, osmotic pressure, viscosity, transparency, color, isotonicity, sterility, stability, solubility, sustained release, absorption, permeability, dosage form, strength, character, shape, and the like of the composition or the antibody contained therein. The substance for preparation is not particularly limited as long as it is pharmaceutically acceptable. For example, non-toxic or low toxicity is a property that is preferred for materials for formulation.

Examples of the preparation include, but are not limited to, the following: amino acids such as glycine, alanine, glutamine, asparagine, histidine, arginine, or lysine; antioxidants such as antibacterial agents, ascorbic acid, sodium sulfate or sodium bisulphite; buffers such as phosphoric acid, citric acid, boric acid buffer, sodium bicarbonate, tris hydrochloric acid (Tris-HCl) solution, and the like; fillers such as mannitol and glycine; chelating agents such as ethylenediamine tetraacetic acid (EDTA); complexing agents such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin and hydroxypropyl-beta-cyclodextrin; bulking agents such as glucose, mannose, or dextrin; monosaccharides, disaccharides, and other carbohydrates such as glucose, mannose, and dextrins; hydrophilic polymers such as colorants, fragrances, diluents, emulsifiers, and polyvinyl pyrrolidone; low molecular weight polypeptides, salt forming counterions, benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methyl parahydroxybenzoate, propyl parahydroxybenzoate, loratadine, sorbic acid, or hydrogen peroxide; glycerol, propylene glycol or polyethylene glycol and other solvents; sugar alcohols such as mannitol and sorbitol; a suspending agent; PEG; sorbitan esters; polysorbates such as polysorbate 20 and polysorbate 80; surfactants such as Triton, tromethamine, lecithin, and cholesterol; stability enhancers such as sucrose and sorbitol; elasticity enhancers such as sodium chloride, potassium chloride, mannitol, and sorbitol; a delivery agent; a diluent; an excipient; and/or pharmaceutically acceptable adjuvants.

The amount of these agents 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-CD 3 antibody or the like.

Pharmaceutical compositions containing immunoliposome formed by incorporating anti-CD 3 antibody into liposome and antibody-modified product formed by binding antibody to liposome (U.S. patent No. 6214388, etc.) are also included in the pharmaceutical compositions of the present invention.

The excipient and carrier are usually liquid or solid, and are not particularly limited as long as they are used in water for injection, physiological saline, artificial cerebrospinal fluid, and other preparations for oral administration or non-oral administration. Examples of the physiological saline include neutral physiological saline and physiological saline containing serum albumin.

Examples of the buffering agent include: preparing a final pH of the pharmaceutical composition into a Tris buffer of 7.0 to 8.5; preparing the final pH of the pharmaceutical composition into an acetate buffer of 4.0 to 5.5; preparing a final pH of the pharmaceutical composition to a citric acid buffer of 5.0 to 8.0; the final pH of the pharmaceutical composition is prepared as a histidine buffer of 5.0 to 8.0, etc.

The pharmaceutical compositions of the present invention are solid, liquid, suspension, and the like. There may be mentioned freeze-dried preparations. For shaping the lyophilized preparation, excipients such as sucrose can be used.

The administration route of the pharmaceutical composition of the present invention may be any of enteral administration, topical administration and parenteral administration, and may be selected according to the subject disease. Specific examples include: intravenous administration, intra-arterial administration, intramuscular administration, intradermal administration, subcutaneous administration, intraperitoneal administration, transdermal administration, intraosseous administration, intra-articular administration, and the like.

The composition of the pharmaceutical composition may be determined according to the administration method, the CD3 protein binding affinity of the antibody, and the like.

The amount of the anti-CD 3 antibody or the like to be administered may be appropriately determined depending on the kind of the individual, the kind of the disease, the symptom, the sex, the age, the disease, the binding affinity of the CD3 protein of the antibody or its biological activity, and other factors, and usually 0.01 to 1000mg/kg, preferably 0.1 to 100mg/kg, may be administered 1 time within 1 to 180 days, or 2 times or 3 times or more within 1 day. As the form of the pharmaceutical composition, there can be exemplified: injections (including freeze-dried preparations, drops), suppositories, nasal-absorption preparations, percutaneous-absorption preparations, sublingual preparations, capsules, tablets, ointments, granules, aerosols, pills, powders, suspensions, emulsions, eye-drops, in vivo implantable preparations, and the like.

anti-CD 3 antibodies and the like may be used in combination with other agents. The anti-CD 3 antibody or the like or the pharmaceutical composition containing the anti-CD 3 antibody or the like as an active ingredient may be administered simultaneously or separately with other agents, i.e., pharmaceutical compositions containing agents other than the anti-CD 3 antibody or the like as an active ingredient. For example, a pharmaceutical composition containing an anti-CD 3 antibody or the like as an active ingredient may be administered after administration of other agents, or another agent may be administered after administration of a pharmaceutical composition containing an anti-CD 3 antibody or the like as an active ingredient, or a pharmaceutical composition containing an anti-CD 3 antibody or the like as an active ingredient and other agents may be administered simultaneously. In the case where two active ingredients, such as an anti-CD 3 antibody and other agents, are contained in a single pharmaceutical composition, and in the case where two active ingredients are contained separately in a plurality of pharmaceutical compositions, the "pharmaceutical composition containing an anti-CD 3 antibody and other agents" is referred to in the present invention. In the present invention, the term "pharmaceutical composition" is defined as "pharmaceutical composition for combined administration of an anti-CD 3 antibody or the like and other agents".

In the present invention, the term "combination administration" of an anti-CD 3 antibody or the like and other agents means that the anti-CD 3 antibody or the like and other agents are taken up in the body of the administration subject within a certain period of time. The anti-CD 3 antibody and other agents may be administered as a single preparation, or may be formulated and administered separately. In the case of separately preparing the preparations, the administration time is not particularly limited, and administration may be performed simultaneously or at different times or on different dates. In the case where an anti-CD 3 antibody or the like and other agents are administered separately at different times or at different days, the order of administration is not particularly limited. Each formulation is usually administered according to a respective administration method, and thus the number of administrations is sometimes the same and sometimes different. In the case of preparing each formulation, the administration methods (administration routes) of the respective formulations may be the same or may be different. The anti-CD 3 antibody and the other agent need not be present in the body at the same time, and may be taken into the body for a certain period (for example, one month, preferably 1 week, more preferably several days, still more preferably 1 day), and when one of them is administered, the other active ingredient may disappear in the body.

Examples of the administration form of the "pharmaceutical composition for combined administration of an anti-CD 3 antibody or the like and other agent" include: 1) Administering a single formulation comprising an anti-CD 3 antibody or the like and other agents; 2) 2 kinds of preparations obtained by formulating an anti-CD 3 antibody and the like with other agents respectively by the same administration route and simultaneously; 3) 2 kinds of preparations each prepared from an anti-CD 3 antibody and other agents by the same administration route with a time difference therebetween; 4) 2 kinds of preparations obtained by formulating an anti-CD 3 antibody and the like with other agents respectively by different administration routes; 5) 2 kinds of formulations obtained by formulating an anti-CD 3 antibody and the like and other agents separately by different administration routes with time difference, and the like. The dosage, interval, mode of administration, preparation, etc. of the "pharmaceutical composition for combined administration of an anti-CD 3 antibody, etc. and other agents" are as defined above with respect to the pharmaceutical composition containing an anti-CD 3 antibody, but are not limited thereto.

The pharmaceutical compositions may also be kits containing them, in the case of being formulated into 2 different formulations.

In the present invention, "combination" of an anti-CD 3 antibody or the like and other agent means "administration of a combination" of an anti-CD 3 antibody or the like and other agent.

Other drugs may also be used in the combination or pharmaceutical composition of the invention.

The invention also provides a method of treatment or prophylaxis of a disease associated with CD3, use of an antibody of the invention in the manufacture of a pharmaceutical composition for the treatment or prophylaxis of the disease, use of an antibody of the invention in the treatment or prophylaxis of the disease. Therapeutic or prophylactic kits comprising the antibodies of the invention are also encompassed by the invention.

Examples (example)

The present invention will be specifically illustrated by the following examples, but the present invention is not limited by these examples.

Evaluation of solution stability of C3E-7085 (reference example 1)

Solution stability was evaluated using the C3E-7085 purified protein described in International publication No. 2018/117237 (scFv having the amino acid sequence shown in SEQ ID NO:64, FIG. 72 of the sequence Listing of International publication No. 2018/117237). C3E-7085 was concentrated by centrifugation using Amicon Ultra-4 (Millipore Co.) and buffer-substituted with 25mM sodium acetate and 5% sorbitol to bring the pH to 5.5, and then the concentration was adjusted to 10mg/mL, and stored at 4℃for 2 weeks or 40℃for 2 weeks. Size Exclusion Chromatography (SEC) was performed on the preserved samples using TSKgel UP-SW3000 μm 4.6X300 mm. The mobile phase was analyzed using 3 XPBS at a flow rate of 0.2mL/min (detection wavelength 280 nm). The peak analysis was performed on HMWS (%) contained in each sample, and the calculation was performed by the area percentage method, and the results are shown in table 1.

TABLE 1

C3E-7085 preservation conditions	HMWS(％)
		40 ℃ for 2 weeks	24
4 ℃ for 2 weeks	22

After 2 weeks of storage at both 4℃and 40℃C3E-7085, the dimer peak in HMWS in particular increased to 20% (data not shown). In addition, the formed dimer has a reversible property that returns to the monomer by dilution (data not shown), and thus there is a problem in terms of solution stability in that HMWS containing the dimer is produced.

Example 1 evaluation of solution stability of anti-CD 3 antibody fragment-heterodimeric Fc-type molecules

2) Design of the amino acid sequence of the anti-CD 3 antibody fragment

In order to improve the physical properties of C3E-7085 in terms of solution stability, C3E-7085-Fab (SEQ ID NO:1, FIG. 5) and C3E-7085-LC (SEQ ID NO:2, FIG. 6) were designed as amino acid sequences of the configuration-altered C3E-7085Fab configuration. In addition, reference [ Proteins,1994, may;19 (1): 35-47] designed scFv incorporating disulfide linkage to introduce binding, designated C3E-7096 (SEQ ID NO:3, FIG. 7).

2) Preparation of-2 anti-CD 3 antibody fragment-heterodimeric Fc expression vector

Various anti-CD 3 antibody fragment-heterodimer Fc expression vectors were designed, which had pcDNA3.1, pcDNA3.3 or pcDNA3.4 (Semermer Feiche technologies Co.) as the backbone. The anti-CD 3 antibody fragment used was C3E-7085 (WO 2018/117237) and the anti-CD 3 antibody fragment designed in 2) -1. Heterodimeric Fc sequences (hereinafter referred to as hc_h and hc_k) were described in literature [ Nat biotechnol 1998jul;16 (7) 677-81.).

An expression vector for mammalian cells, which has been introduced with a DNA fragment encoding an amino acid sequence contained in a polypeptide consisting of Fc (HC_h) into which a heteromultimer-forming mutation has been introduced, was prepared and designated as "p_HC_h".

An expression vector for mammalian cells was prepared into which a DNA fragment encoding an amino acid sequence contained in a polypeptide formed by adding an Fc region into which a heteromultimeric mutation was introduced at the carboxyl terminus of C3E-7085 was introduced, and the expression vector was designated as "p-C3E-7085-HC-k".

An expression vector for mammalian cells was prepared by introducing a DNA fragment encoding an amino acid sequence contained in a polypeptide formed by adding, in this order, a heavy chain variable region of C3E-7085, a CH1 region derived from human IgG, and an Fc region having a heteromultimeric mutation, and designated as "p-C3E-7085-Fab-HC-k". In addition, an expression vector for mammalian cells was prepared into which a DNA fragment encoding an amino acid sequence contained in a polypeptide formed by adding a CL region derived from human Ig lambda to the carboxyl terminus of the C3E-7085 light chain variable region was introduced, and the expression vector was designated "p-C3E-7085-LC".

An expression vector for mammalian cells was prepared into which a DNA fragment encoding an amino acid sequence contained in a polypeptide formed by adding an Fc region into which a heteromultimeric mutation was introduced at the carboxyl terminus of C3E-7096 was introduced, and the expression vector was designated as "p-C3E-7096-HC-k".

The nucleotide sequence of p_HC_h was re-analyzed, and it was confirmed that the full-length nucleotide sequence of HC_h was the nucleotide sequence shown in SEQ ID NO:4 (FIG. 8) of the sequence Listing.

The nucleotide sequence of p_C3E-7085-HC-k was re-analyzed, and it was confirmed that the full-length nucleotide sequence of C3E-7085-HC-k was the nucleotide sequence shown in SEQ ID NO:5 (FIG. 9) of the sequence Listing.

The nucleotide sequence of p_C3E-7085-Fab-HC-k was re-analyzed, and it was confirmed that the full-length nucleotide sequence of C3E-7085-Fab-HC-k was the nucleotide sequence shown in SEQ ID NO. 6 (FIG. 10) of the sequence Listing.

The 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) of the sequence Listing.

The nucleotide sequence of p_C3E-7096-HC-k was re-analyzed, and it was confirmed that the full-length nucleotide sequence of C3E-7096-HC-k was the nucleotide sequence shown in SEQ ID NO:8 (FIG. 12) of the sequence Listing.

In addition, the full-length amino acid sequences of HC_h, C3E-7085-HC-k, C3E-7085-Fab-HC-k, C3E-7085-LC and C3E-7096-HC-k encoded by the above nucleotide sequences were confirmed.

The full-length amino acid sequence of HC_h is the amino acid sequence shown in SEQ ID NO 9 (FIG. 13) of the sequence Listing. In this sequence, the amino acid sequences at positions 1 to 19 are signal sequences, and the amino acid sequences at positions 20 to 246 are HC_h.

The full-length amino acid sequence of C3E-7085-HC-k is the amino acid sequence shown in SEQ ID NO 10 (FIG. 14) of the sequence Listing. In the sequence, the amino acid sequences from 1 st to 19 th are signal sequences, the amino acid sequences from 21 st to 260 th are C3E-7085, the amino acid sequences from 261 st to 262 th are linkers, and the amino acid sequences from 263 rd to 494 th are HC-k.

The full-length amino acid sequence of C3E-7085-Fab-HC-k is the amino acid sequence shown in SEQ ID NO. 11 (FIG. 15) of the sequence Listing. In the sequence, the amino acid sequence from 1 st to 19 th is a signal sequence, and the amino acid sequence from 20 th to 137 th is a C3E-7085-heavy chain variable region. In addition, the amino acid sequences at positions 138 to 467 are constant regions.

The full-length amino acid sequence of the C3E-7085-LC is the amino acid sequence shown in SEQ ID NO:12 (FIG. 16) of the sequence Listing. The amino acid sequences at positions 1 to 20 are signal sequences, the amino acid sequences at positions 21 to 127 are variable regions, and the amino acid sequences at positions 128 to 233 are constant regions.

The full-length amino acid sequence of C3E-7096-HC-k is the amino acid sequence shown in SEQ ID NO:13 (FIG. 17) of the sequence Listing. In the sequence, the amino acid sequences from 1 st to 19 th are signal sequences, the amino acid sequences from 21 st to 260 th are C3E-7096, the amino acid sequences from 261 st to 262 th are linkers, and the amino acid sequences from 263 rd to 494 th are HC-k.

2) Expression of-3 anti-CD 3 antibody fragment-heterodimeric Fc

The Expi293F cells (Semerle Feicher technologies) were subcultured according to the manual. Expi293F cell culture broth in log-proliferation phase was diluted to 2.5X10 with Expi293 expression Medium (Sesameifeier technologies Co.) ⁶ cells/mL for the production of various anti-CD 3 antibody fragment-heterodimeric Fc. In the case of 300mL of culture, 1.44mg of polyethyleneimine (Polyscience # 24765) was dissolved in 5mL of Opti-Pro SFM (Semerle Feishmania technology Co.), and then 0.3mg of a vector mixture in which vectors p_C3E-7085-HC-k, p_HC_h prepared using NucleoBond Xtra Midi kit (Takara Shuzo) were mixed was added to 5mL of Opti-Pro SFM (Invitrogen Co.). To 5mL of the polyethylenimine/Opti-Pro SFM mixture, 5mL of the expression vector/Opti-Pro SFM mixture was added and stirred slowly, and left for another 5 minutes, and then added to the Expi293F cells. The membrane was subjected to a 0.2 μm filtration (Sesameisier technologies) at 37℃with 8% CO ₂ Filtering the culture supernatant obtained by shaking culture at 135rpm for 6 days in an incubator to obtain anti-CD 3 antibody fragment-heterodimer Fc (C3E-0002)And (3) supernatant. Each vector constituting C3E-0002 was expressed, and the sequence of the resulting amino acid was shown in SEQ ID NO 9 (FIG. 13) and SEQ ID NO 10 (FIG. 14) of the sequence Listing.

By the same method, culture supernatants of the anti-CD 3 antibody fragment heterodimeric Fc (C3E-0003) were expressed and prepared using p-C3E-7085-Fab-HC-k, p-HC_h and p-C3E-7085-LC. Each vector constituting C3E-0003 was expressed, and the sequence of the resulting amino acid was shown in SEQ ID NO 9 (FIG. 13), SEQ ID NO 11 (FIG. 15) and SEQ ID NO 12 (FIG. 16) of the sequence Listing.

By the same method, using p_C3E-7096-HC-k, p_HC_h, culture supernatants of anti-CD 3 antibody fragment-heterodimer Fc (C3E-0004) were expressed and prepared. Each vector constituting C3E-0004 was expressed, and the sequence of the resulting amino acid was shown in SEQ ID NO 9 (FIG. 13) and SEQ ID NO 13 (FIG. 17) of the sequence Listing.

2) Purification of-4 anti-CD 3 antibody fragment-heterodimeric Fc

The anti-CD 3 antibody fragment-heterodimeric Fc was purified from the culture supernatants obtained in 2) -3 by a two-step method of protein a affinity chromatography and gel filtration chromatography.

The culture supernatant was loaded on a MabSelectSuRe column (Cytiva) equilibrated with PBS at pH 7.4 and the target anti-CD 3 antibody fragment-heterodimer Fc adsorbed. After removing the non-adsorbed components with PBS, the adsorbed components were eluted with an acetic acid buffer having a pH of 3.5. After the pH of the eluted fraction was adjusted to neutral with Tris buffer of pH 9.5, the eluted fraction was concentrated for use in a gel filtration column Superdex 200/300 (Cytiva Co.) equilibrated with 25mM histidine, 300mM NaCl, 5% sorbitol, pH 5.5 in advance. From the peak fractions obtained by gel filtration chromatography, fractions corresponding to the target heterodimer were recovered, and it was confirmed by capillary electrophoresis (LabChip GX Touch, perkinelmer Co.) that the target anti-CD 3 antibody fragment-heterodimer Fc had formed. Protein samples were purified by SEC for analysis and, after determination of purity and concentration, for evaluation.

2) Evaluation of solution stability of-5 anti-CD 3 antibody fragment-heterodimer Fc

Each of the samples purified in 2) to 4 was concentrated by centrifugation through Amicon Ultra-4 (Millipore Co.), the buffer was replaced with PBS, and then PBS was added thereto to adjust the sample concentration to 20mg/mL, thereby obtaining an evaluation sample. Further, HMWS (%) at the start of each evaluation sample was calculated by size exclusion chromatography using advanced Bio SEC 300A 2.7 μm 4.6X150 mm (Agilent Corp.) by area percent method. The mobile phase was analyzed (detection wavelength 280 nm) using a 0.2M Ki/200mM KCl/pH 7.0 at a flow rate of 0.2 mL/min. For the accelerated aging test of each sample, after storage at 40℃for 1 day, 7 days, and 14 days, size exclusion chromatography was performed under the same conditions as described above, and HMWS (%) of each sample was calculated by the area percentage method.

The results showing the time-dependent storage of each sample at 40℃are shown in FIG. 1. The amount of HMWS (%) of C3E-0003 after 14 days of storage was 3%, which significantly decreased with the lapse of storage time, as compared with C3E-0002, in which HMWS (%) was 15%. HMWS (%) of C3E-0004 after 14 days of storage was 13%, increased compared to C3E-0003, and tended to decrease compared to C3E-0002.

The above results indicate that the heterodimeric Fc type using the C3E-7085Fab configuration and the heterodimeric Fc type using the C3E-7096 molecule are excellent in solution stability as compared to the heterodimeric Fc type molecule using the C3E-7085.

Example 2 evaluation of the Oscillating stability of anti-CD 3 antibody fragment-heterodimer Fc

Each of the samples purified in 2) to 4 was concentrated by centrifugation through Amicon Ultra-4 (Millipore Co.) and adjusted to 2mg/mL to give an evaluation sample. In addition, the peak area and HMWS (%) at the start of each sample for evaluation were calculated by size exclusion chromatography using advanced bio SEC 300a 2.7 μm 4.6x150 mm (Agilent corporation) using area percent method. The mobile phase was analyzed (detection wavelength 280 nm) using a 0.2M Ki/200mM KCl/pH 7.0 at a flow rate of 0.2 mL/min. The oscillation aging test was performed as follows: each sample was shaken at room temperature at 2500rpm with the lapse of time for 1 hour and 2 hours using a general-purpose high-speed stirrer (manufactured by Waken), and then size-exclusion chromatography was performed under the same conditions as described above, to calculate the peak area and HMWS (%) of each sample.

The peak area and HMWS (%) after each sample was oscillated are shown in fig. 2A and 2B. C3E-0002 increased significantly at the 1 hour post-oscillation stage HMWS (%), and 99% of the peak area in the treated samples disappeared at 2 hours post-oscillation. The decrease in peak area and the increase in HMWS (%) with the lapse of oscillation time were significantly smaller in C3E-0003 than in C3E-0002. C3E-0004 the HMWS (%) in the 1-hour treatment sample was lower than that of C3E-0002, and the disappearance of the peak area of the 2-hour treatment sample was also at 74%. In order to confirm whether particles of a size which could not be detected by size exclusion chromatography were formed before and after shaking, the number of particles was evaluated by using a sample treated before shaking and after shaking for 2 hours using FlowCAM8100 (manufactured by DKSH Japan), and from the increase in the number of particles before and after shaking, it was estimated that each sample passed through HMWS from the beginning of the monomer by shaking aging to form particles. Therefore, it can be said that the sample having a low increase rate of HMWS (%) and a low decrease rate of peak area has high stability against vibration aging.

The above results indicate that the heterodimeric Fc type using the C3E-7085Fab configuration and the heterodimeric Fc type using the C3E-7096 have excellent oscillation stability compared to the heterodimeric Fc type using the C3E-7085.

Example 3 preparation of Tafv-heterodimeric Fc-type and scFv-Fab-heterodimeric Fc-type bispecific molecules

4) Preparation of vector for expression of 1 taFv-heterodimer Fc bispecific molecule

A taFv-heterodimer Fc-type bispecific molecule expression vector was designed with pcDNA3.1, pcDNA3.3, or pcDNA3.4 (Semer Feicher technologies Co.) as the backbone. Regarding the anti-CD 3 antibody fragment, a heterodimeric Fc sequence of C3E-7085 (WO 2018/117237) or C3E-7096 (hereinafter referred to as HC_h and HC_k) was used [ Nat Biotechnol.1998Jul;16 (7) 677-81.).

As a vector for expressing the anti-Trop 2-CD3 bispecific molecule, an expression vector for mammalian cells was prepared into which a DNA fragment encoding an amino acid sequence contained in a polypeptide formed by adding an Fc region into which a heteromultimeric mutation was introduced to the carboxyl terminus of taFv, in which HT1-11scFv and C3E-7085 described in International publication No. 2018/117237 were linked via a GGGGS linker, was introduced, and designated as "p_TX 3-0001-HC_k". In addition, an expression vector for mammalian cells was prepared by introducing a DNA fragment encoding an amino acid sequence contained in a polypeptide formed by adding an Fc region into which a heteromultimeric mutation was introduced to the carboxyl terminus of taFv, in which HT1-11scFv and C3E-7096 were linked via a GGGGS linker, and designated "p_TX 3-0003-HC_k".

As a vector for expressing an anti-CD 98-CD3 bispecific molecule, an expression vector for mammalian cells was prepared into which a DNA fragment encoding an amino acid sequence contained in a polypeptide comprising an anti-CD 98scFv (hereinafter referred to as hM23-H1L1 scFv) and a C3E-7085 fragment, wherein the amino acid sequence was obtained by adding glycine to the N-terminus of the hM23-H1 heavy chain variable region described in Japanese patent application laid-open No. 2017-114763 and an hM23-L1 light chain variable region was ligated by a polypeptide linker composed of a sequence obtained by repeating 3 times (GGGGS), wherein the Fc region into which a heteromultimeric mutation was introduced was added to the carboxyl terminus of the taFv obtained by ligating the anti-CD 98scFv (hereinafter referred to as hM23-H1L1 scFv), and the amino acid sequence was designated as "p_CX3-0001-HC_K". In addition, an expression vector for mammalian cells was prepared into which a DNA fragment encoding an amino acid sequence contained in a polypeptide formed by adding an Fc region into which a heteromultimeric mutation was introduced to the carboxyl terminus of taFv, in which hM23-H1L1scFv and C3E-7096 were linked via a GGGGS linker, was introduced, and designated "p-CX 3-0003-HC_k".

As a vector for expressing an anti-GPRC 5D-CD3 bispecific molecule, an expression vector for mammalian cells was prepared into which a DNA fragment encoding an amino acid sequence contained in a polypeptide formed by adding an Fc region into which a heteromultimeric mutation was introduced to the carboxyl terminus of taFv, in which C3022scFv and C3E-7085 were linked via a GGGGS linker as described in International publication No. 2018/147245, was introduced, and designated as "p_RX 3-0001-HC_k". In addition, an expression vector for mammalian cells was prepared by introducing a DNA fragment encoding an amino acid sequence contained in a polypeptide formed by adding an Fc region into which a heteromultimeric mutation was introduced to the carboxyl terminus of taFv, in which C3022scFv and C3E-7096 were linked via a GGGGS linker, and designated "p_RX 3-0003-HC_k".

The nucleotide sequence of p_TX3-0001-HC_k was re-analyzed, and it was confirmed that the full-length nucleotide sequence of TX3-0001-HC_k was the nucleotide sequence shown in SEQ ID NO:14 (FIG. 18) of the sequence Listing.

The nucleotide sequence of p_TX3-0003-HC_k was re-analyzed, and it was confirmed that the full-length nucleotide sequence of TX3-0003-HC_k was the nucleotide sequence shown in SEQ ID NO. 15 (FIG. 19) of the sequence Listing.

The nucleotide sequence of p-CX 3-0001-HC_k was re-analyzed to confirm that the full-length nucleotide sequence of CX3-0001-HC_k is the nucleotide sequence shown in SEQ ID NO:16 (FIG. 20) of the sequence Listing.

The nucleotide sequence of p-CX 3-0003-HC_k was re-analyzed to confirm that the full-length nucleotide sequence of CX3-0003-HC_k is the nucleotide sequence shown in SEQ ID NO:17 (FIG. 21) of the sequence Listing.

The nucleotide sequence of p_RX3-0001-HC_k was re-analyzed, and it was confirmed that the full-length nucleotide sequence of RX3-0001-HC_k was the nucleotide sequence shown as SEQ ID NO:18 (FIG. 22) of the sequence Listing.

The nucleotide sequence of p_RX3-0003-HC_k was re-analyzed, and it was confirmed that the full-length nucleotide sequence of RX3-0003-HC_k was the nucleotide sequence shown as SEQ ID NO:19 (FIG. 23) of the sequence Listing.

In addition, the full-length amino acid sequences of TX3-0001-HC_k, TX3-0003-HC_k, CX3-0001-HC_k, CX3-0003-HC_k, RX3-0001-HC_k, and RX3-0003-HC_k encoded by the above nucleotide sequences were confirmed.

The TX3-0001-HC_k full-length amino acid sequence is the amino acid sequence shown in SEQ ID NO:20 (FIG. 24) of the sequence Listing. In the sequence, the amino acid sequences from 1 st to 19 th are signal sequences, the amino acid sequences from 21 st to 510 st are taFv formed by connecting HT1-11scFv and C3E-7085 through GGGGS linker, the amino acid sequences from 511 st to 512 th are linker, and the amino acid sequences from 513 st to 744 st are HC_k.

The TX3-0003-HC_k full-length amino acid sequence is the amino acid sequence shown in SEQ ID NO:21 (FIG. 25) of the sequence Listing. In the sequence, the amino acid sequences from 1 st to 19 th are signal sequences, the amino acid sequences from 21 st to 510 st are taFv formed by connecting HT1-11scFv and C3E-7096 through GGGGS linker, the amino acid sequences from 511 st to 512 th are linker, and the amino acid sequences from 513 st to 744 st are HC_k.

The CX3-0001-HC_k full-length amino acid sequence is the amino acid sequence shown in SEQ ID NO. 22 (FIG. 26) of the sequence Listing. In the sequence, the amino acid sequences from 1 st to 19 th are signal sequences, the amino acid sequences from 21 st to 509 th are taFv formed by connecting hM23-H1L1scFv and C3E-7085 through GGGGS linker, the amino acid sequences from 510 st to 511 th are linker, and the amino acid sequences from 512 th to 743 th are HC_k.

The CX3-0003-HC_k full-length amino acid sequence is the amino acid sequence shown in SEQ ID NO. 23 (FIG. 27) of the sequence Listing. In the sequence, the amino acid sequences from 1 st to 19 th are signal sequences, the amino acid sequences from 21 st to 509 th are taFv formed by connecting hM23-H1L1scFv and C3E-7096 through GGGGS linker, the amino acid sequences from 510 st to 511 th are linker, and the amino acid sequences from 512 th to 743 th are HC_k.

The RX3-0001-HC_k full-length amino acid sequence is the amino acid sequence shown in SEQ ID NO:24 (FIG. 28) of the sequence Listing. In the sequence, the amino acid sequences from 1 st to 19 th are signal sequences, the amino acid sequences from 21 st to 505 st are taFv formed by connecting C3022scFv and C3E-7085 through GGGGS linker, the amino acid sequences from 506 th to 507 th are linker, and the amino acid sequences from 508 th to 739 th are HC_k.

The RX3-0003-HC_k full-length amino acid sequence is the amino acid sequence shown in SEQ ID NO:25 (FIG. 29) of the sequence Listing. In the sequence, the amino acid sequences from 1 st to 19 th are signal sequences, the amino acid sequences from 21 st to 505 st are taFv formed by connecting C3022scFv and C3E-7096 through GGGGS linker, the amino acid sequences from 506 th to 507 th are linker, and the amino acid sequences from 508 th to 739 th are HC_k.

4) Expression of-2 taFv-heterodimeric Fc bispecific molecules

Culture supernatants of anti-Trop 2-CD3 taFv-heterodimer Fc (TX 3-0001) were expressed and prepared by the same method as 2) -3 using p_tx3-0001-hc_k, p_hc_h. Each vector constituting TX3-0001 was expressed, and the sequence of the obtained amino acid was shown in SEQ ID NO 9 (FIG. 13) and SEQ ID NO 20 (FIG. 24) of the sequence Listing.

By the same method, culture supernatants of anti-Trop 2-CD3 taFv-heterodimer Fc (TX 3-0003) were expressed and prepared using p_tx3-0003-hc_k, p_hc_h. Each vector constituting TX3-0003 was expressed, and the sequence of the obtained amino acid was shown in SEQ ID NO 9 (FIG. 13) and SEQ ID NO 21 (FIG. 25) of the sequence Listing.

By the same method, using p_CX3-0001-HC_k, p_HC_h, the culture supernatant of anti-CD 98-CD3 taFv-heterodimer Fc (CX 3-0001) was expressed and prepared. Each vector constituting CX3-0001 was expressed, and the resulting amino acid sequences were shown in SEQ ID NO 9 (FIG. 13) and SEQ ID NO 22 (FIG. 26) of the sequence Listing.

By the same method, using p_CX3-0003-HC_k, p_HC_h, culture supernatants of anti-CD 98-CD3 taFv-heterodimer Fc (CX 3-0003) were expressed and prepared. Each vector constituting CX3-0003 was expressed, and the resulting amino acid sequences were shown in SEQ ID NO 9 (FIG. 13) and SEQ ID NO 23 (FIG. 27) of the sequence Listing.

By the same method, using p_RX3-0001-HC_k, p_HC_h, the culture supernatant of anti-GPRC 5D-CD3 taFv-heterodimer Fc (RX 3-0001) was expressed and prepared. Each vector constituting RX3-0001 was expressed, and the sequence of the obtained amino acid was shown in SEQ ID NO 9 (FIG. 13) and SEQ ID NO 24 (FIG. 28) of the sequence Listing.

By the same method, culture supernatants of anti-CD 98-CD3 taFv-heterodimer Fc (RX 3-0003) were expressed and prepared using p_RX3-0003-HC_k, p_HC_h. Each vector constituting RX3-0003 was expressed, and the sequence of the obtained amino acid was shown in SEQ ID NO 9 (FIG. 13) and SEQ ID NO 25 (FIG. 29) of the sequence Listing.

4) Purification of the 3 taFv-heterodimeric Fc bispecific molecules

Each culture supernatant obtained in 4) -2 was purified by the same method as in 2) -4. Purified protein samples were used for analysis SEC, after determination of purity and concentration, for various evaluations.

4) Preparation of vector for expression of 4 scFv-Fab-heterodimer Fc-type bispecific molecule

A scFv-Fab-heterodimer Fc type bispecific molecule expression vector was designed with pcDNA3.1, pcDNA3.3, or pcDNA3.4 (Semer Feiche technologies Co.) as the backbone. The anti-CD 3 antibody fragment was used with C3E-7085 (WO 2018/117237) changed to the Fab configuration. Heterodimeric Fc sequences (hereinafter referred to as hc_h and hc_k) were used [ Nat biotechnol 1998jul;16 (7) 677-81.).

As a vector for expressing the anti-Trop 2-CD3 bispecific molecule, an expression vector for mammalian cells was prepared into which a DNA fragment encoding an amino acid sequence contained in a polypeptide comprising HT1-11scFv, a heavy chain variable region of C3E-7085, a CH1 region derived from human IgG, and an Fc region into which a heteromultimeric mutation was introduced, and designated as "p_TX 3-0002-HC_k".

As a vector for expressing an anti-CD 98-CD3 bispecific molecule, an expression vector for mammalian cells was prepared into which a DNA fragment encoding an amino acid sequence contained in a polypeptide comprising hM23-H1L1scFv, a heavy chain variable region of C3E-7085, a CH1 region derived from human IgG, and a Fc region into which a heteromultimeric mutation was introduced, and the vector was designated "p-CX 3-0002-HC_k".

As a vector for expressing the anti-GPRC 5D-CD3 bispecific molecule, an expression vector for mammalian cells was prepared into which a DNA fragment encoding the amino acid sequence contained in a polypeptide comprising C3022scFv, the heavy chain variable region of C3E-7085, the CH1 region derived from human IgG, and the Fc region into which a heteromultimeric mutation was introduced, and designated as "p_RX 3-0002-HC_k".

The 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 was the nucleotide sequence shown in SEQ ID NO. 26 (FIG. 30) of the sequence Listing.

The nucleotide sequence of p-CX 3-0002-HC_k was re-analyzed to confirm that the full-length nucleotide sequence of CX3-0002-HC_k is the nucleotide sequence shown in SEQ ID NO:27 (FIG. 31) of the sequence Listing.

The nucleotide sequence of p_RX3-0002-HC_k was re-analyzed, and it was confirmed that the full-length nucleotide sequence of RX3-0002-HC_k was the nucleotide sequence shown as SEQ ID NO. 28 (FIG. 32) of the sequence Listing.

In addition, the nucleotide sequences were confirmed to encode the full-length amino acid sequences of TX3-0002-HC_k, CX3-0002-HC_k, and RX 3-0002-HC_k.

The TX3-0002-HC_k full-length amino acid sequence is the amino acid sequence shown in SEQ ID NO:29 (FIG. 33) of the sequence Listing. In this sequence, the amino acid sequence from position 1 to position 19 is a signal sequence, the amino acid sequence from position 21 to position 265 is HT1-11scFv, the amino acid sequence from position 266 to position 270 is a linker, and the amino acid sequence from position 271 to position 388 is a C3E-7085-heavy chain variable region. In addition, the amino acid sequences 389 to 718 are constant regions.

The CX3-0002-HC_k full-length amino acid sequence is the amino acid sequence shown in SEQ ID NO:30 (FIG. 34) of the sequence Listing. In this sequence, the amino acid sequence from position 1 to position 19 is a signal sequence, the amino acid sequence from position 21 to position 264 is hM23-H1L1scFv, the amino acid sequence from position 265 to position 269 is a linker, and the amino acid sequence from position 270 to position 387 is a C3E-7085-heavy chain variable region. In addition, the amino acid sequences at positions 388 to 717 are constant regions.

The RX3-0002-HC_k full-length amino acid sequence is the amino acid sequence shown in SEQ ID NO:31 (FIG. 35) of the sequence Listing. In this sequence, the amino acid sequence from position 1 to 19 is a signal sequence, the amino acid sequence from position 21 to 260 is C3022scFv, the amino acid sequence from position 261 to 265 is a linker, and the amino acid sequence from position 266 to 383 is a C3E-7085-heavy chain variable region. In addition, the amino acid sequences at positions 384 to 713 are constant regions.

4) Expression of a bispecific molecule of the Fc type of 5 scFv-Fab-heterodimers

Culture supernatants of anti-Trop 2scFv-CD3 Fab-heterodimer Fc (TX 3-0002) were expressed and prepared by the same method as 2) -3 using p_tx3-0002-hc_k, p_hc_h and p_c3e-7085-LC. Each vector constituting TX3-0002 was expressed, and the sequence of the obtained amino acid was shown in SEQ ID NO 9 (FIG. 13), SEQ ID NO 12 (FIG. 16) and SEQ ID NO 29 (FIG. 33) of the sequence Listing.

By the same method, culture supernatants of anti-CD 98scFv-CD3 Fab-heterodimer Fc (TX 3-0002) were expressed and prepared using p-CX3-0002-HC_k, p-HC_h and p-C3E-7085-LC. Each vector constituting CX3-0002 was expressed, and the resulting amino acid sequences were shown in SEQ ID NO 9 (FIG. 13), SEQ ID NO 12 (FIG. 16) and SEQ ID NO 30 (FIG. 34) of the sequence Listing.

By the same method, culture supernatants of anti-GPRC 5DscFv-CD3 Fab-heterodimer Fc (RX 3-0002) were expressed and prepared using p_RX3-0002-HC_k, p_HC_h and p_C3E-7085-LC. Each vector constituting RX3-0002 was expressed, and the sequence of the obtained amino acid was shown in SEQ ID NO 9 (FIG. 13), SEQ ID NO 12 (FIG. 16) and SEQ ID NO 31 (FIG. 35) of the sequence Listing.

4) Purification of the 6 scFv-Fab-heterodimeric Fc bispecific molecule

Each of the culture supernatants obtained in 4) to 5 was purified by the same method as in 2) to 4. Purified protein samples were used for analysis SEC, after determination of purity and concentration, for various evaluations.

Example 4 evaluation of solution stability of taFv-heterodimeric Fc-type and scFv-Fab-heterodimeric Fc-type bispecific molecules

Using each sample prepared in example 3, centrifugation and concentration were performed by Amicon Ultra-4 (Millipore Co.), the buffer was replaced with PBS, and then PBS was added thereto to adjust the sample concentration to 20mg/mL, thereby obtaining a sample for evaluation. Further, HMWS (%) at the start of each evaluation sample was calculated by size exclusion chromatography using advanced Bio SEC 300A2.7 μm 4.6X150 mm (Agilent). The mobile phase was analyzed (detection wavelength 280 nm) using a 0.2M Ki/200mM KCl/pH 7.0 at a flow rate of 0.2 mL/min. For the accelerated aging test of each sample, after storage at 40℃for 1 day, 7 days, and 14 days, size exclusion chromatography was performed under the same conditions as described above, and HMWS (%) of each sample was calculated by the area percentage method.

The results showing the time-dependent storage of each sample at 40℃are shown in FIGS. 3A to 3C.

The amount of HMWS (%) as a function of shelf life of RX3-0001 increased to 13%. On the other hand, the amounts of HMWS (%) in RX3-0002 and RX3-0003, respectively, varied with the lapse of the storage time were reduced to 6%, 8% (FIG. 3A).

The amount of HMWS (%) as a function of the lapse of the retention time of TX3-0001 was increased to 23%. On the other hand, the amounts of HMWS (%) varying with the lapse of the retention time of TX3-0002 and TX3-0003 were reduced to 10% and 11%, respectively (FIG. 3B).

The amount of HMWS (%) as a function of shelf life of CX3-0001 increased to 46% reflecting the effect of solution stability of hM23-H1L1 scFv. On the other hand, the amount of HMWS (%) in CX3-0002 was reduced to 28% as the storage time elapsed, and the amount of HMWS (%) in CX3-0003 was 43%, which was slightly reduced as compared with CX3-0001 (FIG. 3C).

The above results indicate that the solution stability of scFv-Fab-heterodimer Fc type using the C3E-7085Fab configuration and taFv-heterodimer Fc type molecule using the C3E-7096 is superior to that of taFv-heterodimer Fc type molecule using the C3E-7085.

Example 5 evaluation of the Oscillating stability of taFv-heterodimeric Fc-type and scFv-Fab-heterodimeric Fc-type bispecific molecules

RX3-0001 to 0003, CX3-0001 to 0003 and TX3-0001 to 0003 in each sample purified in example 3 were concentrated by centrifugation through Amicon Ultra-4 (Millipore Co.) and then adjusted to 2mg/mL to prepare samples for evaluation. In addition, the peak area and HMWS (%) at the start of each sample for evaluation were calculated by size exclusion chromatography using advanced bio SEC 300A2.7 μm 4.6x150 mm (Agilent corporation) using the area percentage method. The mobile phase was analyzed (detection wavelength 280 nm) using a 0.2M Ki/200mM KCl/pH 7.0 at a flow rate of 0.2 mL/min. The oscillation aging test was performed as follows: each sample was shaken at room temperature at 2500rpm with the lapse of time for 1 hour and 2 hours using a general-purpose high-speed stirrer (manufactured by Waken), and then size-exclusion chromatography was performed under the same conditions as described above, to calculate the peak area and HMWS (%) of each sample.

The peak areas and HMWS (%) after shaking of each sample are shown in FIGS. 4-1A, 4-1B, FIGS. 4-2A, 4-2B and FIGS. 4-3A, 4-3B. The residual ratio of the peak area of the RX3-0001 oscillation for 2 hours was 2%. On the other hand, the residual ratios of peak areas of RX3-0002 and RX3-0003 were 15% and 17%, respectively, which were good compared with RX 3-0001. In addition, HMWS (%) of the RX3-0001 oscillation 2 hours treated sample was 30%, while HMWS (%) of the RX3-0002 and RX3-0003 oscillation 2 hours treated sample was 5% or less, and the HMWS (%) of RX3-0002 and RX3-0003 showed lower results than RX 3-0001.

The residual ratio of the peak area of the CX3-0001 treated sample was 6% after 2 hours of shaking. On the other hand, the residual ratios of the peak areas of CX3-0002 and CX3-0003 were 11% and 26%, respectively, and the results were good compared with CX 3-0001. In addition, the HMWS (%) of the samples treated with CX3-0001, CX3-0002 and CX3-0003 for 2 hours were 54%, 17% and 41%, respectively, and the results were lower in the HMWS (%) of CX3-0002 and CX3-0003 compared with CX 3-0001.

The residual ratio of the peak area of the TX3-0001 oscillation for 2 hours was 22%. The residual ratios of the peak areas of the samples subjected to the 2-hour oscillation of TX3-0002 and TX3-0003 were 81% and 41%, respectively, and good results were obtained in terms of the residual ratio of the peak areas as compared with TX 3-0001. On the other hand, the HMWS (%) of the samples treated with TX3-0001, TX3-0002 and TX3-0003 for 2 hours were 7%, 21% and 13%, respectively, and the HMWS (%) of TX3-0001 was the lowest, showing a different trend from the other antibody groups (RX 3 and CX 3).

Therefore, in TX 3-0001-0003, in order to confirm whether large-sized particles which cannot be detected by size exclusion chromatography are formed, the particle count was evaluated by using FlowCAM8100 (manufactured by DKSH Japan) using a sample treated before and after shaking for 2 hours. The change in the number of particles of each sample with time is shown in Table 2.

TABLE 2

	TX3-0001	TX3-0002	TX3-0003
				Initiation	63	66	332
1 hour	171340	7107	78992
				For 2 hours	1272905	55243	562921

(particle/mL)

The particle count of the TX3-0001 treated sample was 1272905 particles/mL for 2 hours with shaking. The numbers of particles in the sample subjected to the treatment of TX3-0002 and TX3-0003 for 2 hours were 55243 and 562921/mL, respectively, and the number of particles formed by aggregation was smaller than that of TX3-0001. The diameter of the aggregates detected as HMWS is about 10 to 100nm, whereas large aggregates of 1 μm or more are detected by FlowCAM. The particle count of TX3-0001 is significantly greater than TX3-0002,0003, so it is inferred that TX3-0001 has a low HMWS (%) because aggregates corresponding to HMWS associate to form larger polymers. As shown above, the oscillation stability of TX3-0002 and TX3-0003 is better than TX3-0001.

The above results indicate that the stability of oscillation of scFv-Fab-heterodimeric Fc-type molecules using the C3E-7085Fab configuration and taFv-heterodimeric Fc-type molecules using the C3E-7096 is superior to those of taFv-heterodimeric Fc-type molecules using the C3E-7085.

In addition, in the above examples, the anti-CD 3scFv may be replaced with C3E-7097 (SEQ ID NO:39 having the amino acid sequence shown in FIG. 46) from C3E-7096, and preparation, test and evaluation of a sample may be performed.

Example 6 evaluation of solution stability of anti-CD 3 antibody fragment-heterodimeric Fc-type bispecific molecules

According to examples 1) -1 to 2) -4, in order to improve C3E-7093 (SEQ ID NO:80, scFv of the amino acid sequence shown in fig. 88), the amino acid sequence of C3E-7093Fab, with the configuration changed to Fab (SEQ ID NO:46, the amino acid sequence of the C3E-7093-Fab shown in fig. 48 and SEQ ID NO:47, amino acid sequence of C3E-7093-LC shown in fig. 49) and the amino acid sequence of C3E-7098, which is scFv having disulfide bonds introduced (SEQ ID NO:48, the amino acid sequence shown in fig. 50). Then, each expression vector for preparing an anti-CD 3 antibody fragment-heterodimeric Fc-type molecule containing the Fab and scFv was prepared, and both the molecules were expressed and purified.

Each of the obtained samples was concentrated by centrifugation using Amicon Ultra-4 (Millipore Co.), the buffer was replaced with PBS, and then PBS was added thereto to adjust the sample concentration to 20mg/mL, thereby obtaining an evaluation sample. Further, HMWS (%) at the start of each evaluation sample was calculated by size exclusion chromatography using advanced Bio SEC 300A2.7 μm 4.6X150 mm (Agilent). The mobile phase was analyzed (detection wavelength 280 nm) using a 0.2M Ki/200mM KCl/pH 7.0 at a flow rate of 0.2 mL/min. For the accelerated aging test of each sample, after storage at 40℃for 1 day, 7 days, and 14 days, size exclusion chromatography was performed under the same conditions as described above, and HMWS (%) of each sample was calculated by the area percentage method.

By comparing the changes over time of each sample when stored at 40 ℃, the results showed that the solution stability of the heterodimeric Fc type using the C3E-7093Fab configuration and the heterodimeric Fc type molecule using the scFv with disulfide introduced, i.e., C3E-7098, was superior to that of the heterodimeric Fc type molecule using C3E-7093.

Example 7 evaluation of the Oscillating stability of anti-CD 3 antibody fragment-heterodimeric Fc-type bispecific molecules

Each of the samples purified in example 6 was concentrated by centrifugation through Amicon Ultra-4 (Millipore Co.) and adjusted to 2mg/mL to give an evaluation sample. In addition, the peak area and HMWS (%) at the start of each sample for evaluation were calculated by size exclusion chromatography using advanced bio SEC 300A2.7 μm 4.6x150 mm (Agilent corporation) using the area percentage method. The mobile phase was analyzed (detection wavelength 280 nm) using a 0.2M Ki/200mM KCl/pH 7.0 at a flow rate of 0.2 mL/min. The oscillation aging test was performed as follows: each sample was shaken at room temperature at 2500rpm with the lapse of time for 1 hour and 2 hours using a general-purpose high-speed stirrer (manufactured by Waken), and then size-exclusion chromatography was performed under the same conditions as described above, to calculate the peak area and HMWS (%) of each sample.

Comparing the peak area after shaking with HMWS (%) of each sample, it was shown that the shaking stability of the heterodimeric Fc type using the C3E-7093Fab configuration and the heterodimeric Fc type molecule using the disulfide-introduced C3E-7098 was superior to those of the heterodimeric Fc type molecule using the C3E-7093.

Example 8 evaluation of solution stability of taFv-heterodimeric Fc-type and scFv-Fab-heterodimeric Fc-type bispecific molecules

anti-Trop 2-CD3 taFv-heterodimer Fc, anti-CD 98-CD3 taFv-heterodimer Fc and anti-GPRC 5D-CD3 taFv-heterodimer Fc containing C3E-7098 as anti-CD 3scFv, and anti-Trop 2scFv-CD3 Fab-heterodimer Fc, anti-CD 98scFv-CD3 Fab-heterodimer Fc and anti-GPRC 5 scFv-CD3 Fab-heterodimer Fc containing C3E-7093Fab as anti-CD 3Fab were prepared as per example 3.

Using each of the prepared samples, concentration was performed by centrifugation using Amicon Ultra-4 (Millipore Co.), the buffer was replaced with PBS, and then PBS was added thereto to adjust the sample concentration to 20mg/mL, thereby obtaining an evaluation sample. Further, HMWS (%) at the start of each evaluation sample was calculated by size exclusion chromatography using advanced Bio SEC 300A 2.7 μm 4.6X150 mm (Agilent Corp.) by area percent method. The mobile phase was analyzed (detection wavelength 280 nm) using a 0.2M Ki/200mM KCl/pH 7.0 at a flow rate of 0.2 mL/min. For the accelerated aging test of each sample, after storage at 40℃for 1 day, 7 days, and 14 days, size exclusion chromatography was performed under the same conditions as described above, and HMWS (%) of each sample was calculated by the area percentage method.

The results of comparing the changes over time of each sample when stored at 40℃show that the solution stability of the scFv-Fab-heterodimer Fc type using the C3E-7093Fab configuration and the taFv-heterodimer Fc type molecule using the C3E-7098 is superior to those of the taFv-heterodimer Fc type molecule using the C3E-7093.

Example 9 evaluation of the Oscillating stability of taFv-heterodimeric Fc-type and scFv-Fab-heterodimeric Fc-type bispecific molecules

The sample prepared in example 8 was concentrated by centrifugation through Amicon Ultra-4 (Millipore Co.) and adjusted to 2mg/mL to give an evaluation sample. In addition, the peak area and HMWS (%) at the start of each sample for evaluation were calculated by size exclusion chromatography using advanced bio SEC 300a 2.7 μm 4.6x150 mm (Agilent corporation) using area percent method. The mobile phase was analyzed (detection wavelength 280 nm) using a 0.2M Ki/200mM KCl/pH 7.0 at a flow rate of 0.2 mL/min. The oscillation aging test was performed as follows: each sample was shaken at room temperature at 2500rpm with the lapse of time for 1 hour and 2 hours using a general-purpose high-speed stirrer (manufactured by Waken), and then size-exclusion chromatography was performed under the same conditions as described above, to calculate the peak area and HMWS (%) of each sample.

Comparing peak areas after shaking with HMWS (%) of each sample, it was revealed that the stability of shaking was superior in the scFv-Fab-heterodimer Fc type using the C3E-7093Fab configuration and the taFv-heterodimer Fc type molecule using the C3E-7098, compared to the taFv-heterodimer Fc type molecule using the C3E-7093.

In examples 6 to 9, the anti-CD 3scFv was replaced with C3E-7098 to C3E-7099 (having the amino acid sequence shown in SEQ ID NO:49, FIG. 51), and preparation, test and evaluation of a sample were performed.

Industrial applicability

The bispecific antibody of the present invention can be used as a therapeutic or prophylactic agent for cancer and the like.

Free text of sequence Listing

SEQ ID NO. 1: amino acid sequence of C3E-7085-Fab (FIG. 5)

SEQ ID NO. 2: amino acid sequence of C3E-7085-LC (FIG. 6)

SEQ ID NO. 3: amino acid sequence of C3E-7096 (FIG. 7)

SEQ ID NO. 4: HC_h full-length nucleotide sequence (FIG. 8)

SEQ ID NO. 5: C3E-7085-HC-k full-length nucleotide sequence (FIG. 9)

SEQ ID NO. 6: C3E-7085-Fab-HC-k full-length nucleotide sequence (FIG. 10)

SEQ ID NO. 7: full-length nucleotide sequence of C3E-7085-LC (FIG. 11)

SEQ ID NO. 8: C3E-7096-HC-k full-length nucleotide sequence (FIG. 12)

SEQ ID NO. 9: HC_h full-length amino acid sequence (FIG. 13)

SEQ ID NO. 10: C3E-7085-HC-k full-length amino acid sequence (FIG. 14)

SEQ ID NO. 11: C3E-7085-Fab-HC-k full-length amino acid sequence (FIG. 15)

SEQ ID NO. 12: C3E-7085-LC full-length amino acid sequence (FIG. 16)

SEQ ID NO. 13: C3E-7096-HC-k full-length amino acid sequence (FIG. 17)

SEQ ID NO. 14: TX3-0001-HC_k full-length nucleotide sequence (FIG. 18)

SEQ ID NO. 15: TX3-0003-HC_k full-length nucleotide sequence (FIG. 19)

SEQ ID NO. 16: CX3-0001-HC_k full-length nucleotide sequence (FIG. 20)

SEQ ID NO. 17: CX3-0003-HC_k full-length nucleotide sequence (FIG. 21)

SEQ ID NO. 18: RX3-0001-HC_k full-length nucleotide sequence (FIG. 22)

SEQ ID NO. 19: CX3-0001-HC_k full-length nucleotide sequence (FIG. 23)

SEQ ID NO. 20: TX3-0001-HC_k full-length amino acid sequence (FIG. 24)

SEQ ID NO. 21: TX3-0003-HC_k full-length amino acid sequence (FIG. 25)

SEQ ID NO. 22: CX3-0001-HC_k full-length amino acid sequence (FIG. 26)

SEQ ID NO. 23: CX3-0003-HC_k full-length amino acid sequence (FIG. 27)

SEQ ID NO. 24: RX3-0001-HC_k full-length amino acid sequence (FIG. 28)

SEQ ID NO. 25: RX3-0003-HC_k full-length amino acid sequence (FIG. 29)

SEQ ID NO. 26: TX3-0002-HC_k full-length nucleotide sequence (FIG. 30)

SEQ ID NO. 27: CX3-0002-HC_k full-length nucleotide sequence (FIG. 31)

SEQ ID NO. 28: RX3-0002-HC_k full-length nucleotide sequence (FIG. 32)

SEQ ID NO. 29: TX3-0002-HC_k full-length amino acid sequence (FIG. 33)

SEQ ID NO. 30: CX3-0002-HC_k full-length amino acid sequence (FIG. 34)

SEQ ID NO. 31: RX3-0002-HC_k full-length amino acid sequence (FIG. 35)

SEQ ID NO. 32: C3E-7085 heavy chain CDRH1 amino acid sequence (FIG. 36)

SEQ ID NO. 33: C3E-7085 heavy chain CDRH2 amino acid sequence (FIG. 36)

SEQ ID NO. 34: C3E-7085 heavy chain CDRH3 amino acid sequence (FIG. 36)

SEQ ID NO. 35: C3E-7085 light chain CDRL1 amino acid sequence (FIG. 36)

SEQ ID NO. 36: C3E-7085 light chain CDRL2 amino acid sequence (FIG. 36)

SEQ ID NO. 37: C3E-7085 light chain CDRL3 amino acid sequence (FIG. 36)

SEQ ID NO. 38: amino acid sequence of peptide linker (FIG. 37)

SEQ ID NO. 39: amino acid sequence of C3E-7097 (FIG. 46)

SEQ ID NO. 40: C3E-7093 heavy chain CDRH1 amino acid sequence (FIG. 47)

SEQ ID NO. 41: C3E-7093 heavy chain CDRH2 amino acid sequence (FIG. 47)

SEQ ID NO. 42: C3E-7093 heavy chain CDRH3 amino acid sequence (FIG. 47)

SEQ ID NO. 43: C3E-7093 light chain CDRL1 amino acid sequence (FIG. 47)

SEQ ID NO. 44: C3E-7093 light chain CDRL2 amino acid sequence (FIG. 47)

SEQ ID NO. 45: C3E-7093 light chain CDRL3 amino acid sequence (FIG. 47)

SEQ ID NO. 46: amino acid sequence of C3E-7093-Fab (FIG. 48)

SEQ ID NO. 47: amino acid sequence of C3E-7093-LC (FIG. 49)

SEQ ID NO. 48: amino acid sequence of C3E-7098 (FIG. 50)

SEQ ID NO. 49: amino acid sequence of C3E-7099 (FIG. 51)

All publications, patents, and patent applications cited in this specification are incorporated herein by reference.

Sequence listing

<110> first Sanyo Co., ltd

<120> stable multispecific molecules and uses thereof

<130> PH-9341-PCT

<150> JP 2021-055375

<151> 2021-03-29

<150> JP 2021-157580

<151> 2021-09-28

<160> 49

<170> PatentIn version 3.5

<210> 1

<211> 216

<212> PRT

<213> Artificial (Artifical)

<220>

<223> amino acid sequence of C3E-7085-Fab

<400> 1

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Val Thr Phe Asn Tyr Tyr

20 25 30

Gly Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Ser Ile Thr Arg Ser Gly Gly Arg Ile Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Glu Asn Thr Gln Lys Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Leu Asp Gly Arg Asp Gly Trp Val Ala Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Arg Val

210 215

<210> 2

<211> 213

<212> PRT

<213> Artificial (Artifical)

<220>

<223> amino acid sequence of C3E-7085-LC

<400> 2

Asn Phe Met Leu Thr Gln Pro Ser Ser Val Ser Gly Val Pro Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Thr Gly Asn Thr Gly Asn Ile Gly Ser Asn

20 25 30

Tyr Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Arg Asp Asp Lys Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Phe Gln

65 70 75 80

Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Ser Ser Gly Phe

85 90 95

Ile Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro Lys Ala

100 105 110

Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln Ala

115 120 125

Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly Ala

130 135 140

Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala Gly Val

145 150 155 160

Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala Ser

165 170 175

Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser Tyr

180 185 190

Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val Ala

195 200 205

Pro Thr Glu Cys Ser

210

<210> 3

<211> 240

<212> PRT

<213> Artificial (Artifical)

<220>

<223> amino acid sequence of C3E-7096

<400> 3

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Val Thr Phe Asn Tyr Tyr

20 25 30

Gly Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Ser Ile Thr Arg Ser Gly Gly Arg Ile Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Glu Asn Thr Gln Lys Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Leu Asp Gly Arg Asp Gly Trp Val Ala Tyr Trp Gly Cys Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Gly Gly Gly Gly Ser Asn Phe Met Leu Thr Gln Pro Ser Ser Val Ser

130 135 140

Gly Val Pro Gly Gln Arg Val Thr Ile Ser Cys Thr Gly Asn Thr Gly

145 150 155 160

Asn Ile Gly Ser Asn Tyr Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr

165 170 175

Cys Pro Lys Leu Leu Ile Tyr Arg Asp Asp Lys Arg Pro Ser Gly Val

180 185 190

Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala

195 200 205

Ile Thr Gly Phe Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser

210 215 220

Tyr Ser Ser Gly Phe Ile Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

225 230 235 240

<210> 4

<211> 738

<212> DNA

<213> Artificial (Artifical)

<220>

<223> full-length nucleotide sequence of HC_h

<400> 4

atgaagcacc tctggttctt tctcctgttg gtggctgcac caagatgggt cctgagcgac 60

aagacgcaca cttgccctcc gtgtccagct cccgaagctg caggaggtcc cagcgttttc 120

ctgtttcctc caaagcccaa agacaccctg atgatctcca ggactcccga ggtgacctgt 180

gtagttgtcg acgttagcca tgaggatccc gaggtcaagt ttaactggta tgtcgacgga 240

gttgaagtcc acaatgccaa gaccaaacca cgagaagagc agtacaatag cacatatcgc 300

gtagtgtctg tactgaccgt gttgcatcag gattggctta atgggaagga gtacaagtgc 360

aaagtgagca acaaagccct tccggcgccc atagagaaaa ccatcagcaa ggcaaagggg 420

caacctcggg aacctcaggt ctgcacactg ccaccttctc gtgatgagct gactaagaac 480

caggtgtcac tgtcctgtgc cgtgaaaggc ttttacccct ccgacattgc cgtggaatgg 540

gagtcaaatg gtcagcctga gaacaactac aagactacac cgccagtgct cgattcagat 600

ggctctttct tcctggtttc caagctgaca gtggacaaat ccagatggca acagggcaat 660

gtgttcagct gcagtgtgat gcatgaagcc cttcacaacc actatacgca gaaaagtctg 720

agtctctctc ctggaaaa 738

<210> 5

<211> 1482

<212> DNA

<213> Artificial (Artifical)

<220>

<223> full-length nucleotide sequence of C3E-7085-HC-k

<400> 5

atgaagcatc tctggttttt cttgctgctc gtcgctgcac ctcgatgggt gctgtcaggc 60

gaagtccagc tggttgaatc tggcggtggc ctggtgcaac ctggagggtc tttgagactg 120

agttgtgccg catcaggagt caccttcaac tactatggca tgtcatggat acgccaagct 180

cccgggaaag ggctcgaatg ggtcgcatca atcactcggt ctggggggag aatctactat 240

ccagactcag tcaagggccg ctttaccatc agccgtgaga atacccagaa aacgctgtac 300

ctacagatga actcccttcg ggcagaggac actgccgtgt actactgtac actggatggg 360

cgagatgggt gggttgccta ttggggccaa ggtactcttg tgactgtgtc ttcgggaggc 420

ggcggttccg gtggtggtgg ttccggagga ggaggaagca acttcatgct gacacagccg 480

tcttccgtat ctggcgtacc aggtcaacgc gtgaccattt cttgcaccgg caatacaggg 540

aacattggca gcaactatgt gaactggtat cagcagcttc ccggaactgc tcctaaactc 600

ctgatataca gggatgataa gcggccttct ggggtccctg atcggtttag tggctccaaa 660

agcggaacct cggcgagcct ggcaatcaca ggctttcagg ctgaggatga ggccgactat 720

tattgccaga gttatagtag cggattcatc tttggcgggg gcacgaagtt aaccgtgctt 780

ggtggagagc ccaaaagcgc cgacaagacc cacacctgtc caccttgtcc cgctccagaa 840

gccgctgggg ggccttccgt ctttctcttt cccccgaaac ccaaagacac actgatgatc 900

tccagaacac ccgaagtaac ctgcgttgtg gttgacgtat cccacgaaga tcccgaagtg 960

aagttcaatt ggtacgtgga cggcgttgag gtgcacaatg ccaaaacaaa gcctagggag 1020

gaacagtaca attcgacata tagggtagtg tcagtcctga cagtgttaca ccaggactgg 1080

ttgaacggga aagagtacaa gtgcaaggtg agcaacaaag cgcttccagc gccaattgag 1140

aagacgattt ccaaagccaa aggacagccg agggaaccgc aggtctacac cttaccccca 1200

tgcagagacg agttgaccaa gaatcaggtg agtctatggt gcctcgtgaa gggcttctat 1260

cccagcgata ttgccgtcga gtgggaatct aatggccaac cagagaacaa ctacaagact 1320

acgccacccg ttctggacag tgatgggagc ttcttcctat actccaagct cactgtggac 1380

aagtcacgtt ggcaacaggg aaatgttttc agttgtagcg tgatgcatga ggccttgcat 1440

aatcactaca ctcagaaaag cctgtccctg tcacctggaa ag 1482

<210> 6

<211> 1401

<212> DNA

<213> Artificial (Artifical)

<220>

<223> full-length nucleotide sequence of C3E-7085-Fab-HC-k

<400> 6

atgaaacatc tctggttctt tctgctgctg gtagccgccc ctagatgggt cctcagtgag 60

gttcagcttg tagagtccgg cggcggcttg gtgcagcccg gaggttccct ccggctttct 120

tgcgctgctt caggagttac gttcaattac tatgggatgt cctggataag gcaggctccc 180

ggtaaggggt tggaatgggt ggccagtatt acccgcagtg gaggaagaat ctactatccg 240

gattctgtga aaggacggtt cactatcagc agagaaaata cacagaaaac cctttatctc 300

cagatgaact cccttcgtgc cgaggatacc gctgtgtact actgcacact ggatggccga 360

gatgggtggg tagcctactg gggtcaagga acgcttgtga cagtttcctc agccagcact 420

aagggaccaa gcgtctttcc gttggcaccc agcagcaaaa gtactagcgg cggcacagct 480

gcactgggct gcctggtgaa ggactatttc cctgaacccg tcaccgtgtc ctggaactcc 540

ggtgccttga cttccggtgt tcacacattc cccgcagtgc tccagagttc cgggctctac 600

tcactgtcat ctgtggtcac cgtgcctagc tcaagtctgg ggacccaaac ttacatctgc 660

aatgtgaacc ataagcctag taacaccaaa gtcgacaaac gcgtcgagcc caagtcatgc 720

gacaagaccc acacatgtcc accttgtccc gcacctgagg cggctggtgg cccatccgtg 780

tttctgtttc caccaaagcc taaagacacc ctgatgatta gtcgaacacc agaggtgact 840

tgcgttgtcg tcgatgtgtc acacgaagat ccggaggtaa agttcaactg gtacgtcgat 900

ggggtggagg ttcataacgc caagactaag ccaagagaag agcagtacaa ctctacgtat 960

agggtggtaa gcgtactgac agtgttgcac caggattggc tcaatggcaa ggaatacaag 1020

tgtaaggtgt ctaataaggc gctgcctgcg ccaattgaga aaacgatctc taaggccaaa 1080

gggcaaccaa gggagcccca agtctatacc ctgcctccgt gtcgggacga actgactaag 1140

aaccaagtga gcctctggtg cctggtcaaa ggcttttatc ccagcgacat agcagtggaa 1200

tgggagagca atgggcagcc agagaacaac tacaagacca cacctcccgt tctggactct 1260

gacggatcat tctttctgta ttccaaactg acagttgaca agtctcgctg gcagcaggga 1320

aatgtgttca gctgtagcgt gatgcatgaa gccctgcaca atcactatac ccagaaaagc 1380

ctgagcctgt ctcccggcaa a 1401

<210> 7

<211> 699

<212> DNA

<213> Artificial (Artifical)

<220>

<223> full-Length nucleotide sequence of C3E-7085-LC

<400> 7

atggttctgc agactcaggt gttcatttcc cttctgctgt ggattagtgg ggcatatggg 60

aatttcatgc tgactcagcc aagctccgta agcggagtac ccggccaaag agtcacaatc 120

agttgcactg gtaacaccgg taatatcggc tctaactacg tgaattggta ccagcagttg 180

ccaggaacag cacccaaact cctgatctac cgcgatgaca agcgaccatc tggcgtgcct 240

gatcggttta gcgggagcaa aagtggcaca agtgcctcat tggccataac ggggttccag 300

gcggaggatg aagcagacta ctattgccag tcctactcct ctggcttcat ctttggcgga 360

gggaccaagc tcaccgtgct tggacaaccc aaagcagctc catcagtgac cctgtttcct 420

ccttcttctg aggagctgca ggccaataag gccacactgg tgtgtctcat tagcgacttt 480

tatccgggtg cggtcactgt ggcctggaaa gccgactcaa gtccggttaa ggctggagtt 540

gagacaacca caccctctaa gcagagcaac aacaagtatg ctgcctcaag ctacctctca 600

ctgacccctg agcagtggaa atcccatagg agctattcct gccaagtcac ccacgaaggc 660

tccaccgtcg aaaagactgt ggctcccacg gaatgtagc 699

<210> 8

<211> 1482

<212> DNA

<213> Artificial (Artifical)

<220>

<223> full-length nucleotide sequence of C3E-7096-HC-k

<400> 8

atgaagcacc tgtggttttt tctgttactc gttgctgctc ccagatgggt cttgagcgga 60

gaagtacagc ttgtagagtc aggaggagga ctggtacaac caggtggctc gttacggctc 120

agttgcgcag cctccggtgt gactttcaac tactacggca tgagctggat acggcaagcg 180

cctggaaagg ggctcgaatg ggtagcctcc ataacacgga gtgggggtcg catttactac 240

cccgattctg tcaaaggccg cttcacaatc agcagagaaa acacgcagaa aacattgtac 300

ctccagatga atagccttag ggccgaagat acggcagtct actattgcac cctggatggc 360

agggacggtt gggttgccta ttggggttgt ggcaccttag tgaccgtatc ctctggagga 420

ggcggcagtg gaggaggggg atcagggggt ggagggagta acttcatgct tacacagcca 480

agttctgttt caggtgttcc tgggcagagg gttaccatct cttgcacagg gaatacaggc 540

aacattggca gcaactatgt gaattggtat cagcagctac caggcacgtg tccgaaactg 600

ttgatctatc gggatgacaa aagaccgtcc ggagtgcctg atcgcttttc cggctctaaa 660

tccgggactt cagcgagttt ggccattact gggtttcagg cagaagatga ggccgactac 720

tactgccaga gctactctag cgggttcatc tttggcggtg ggaccaagct gactgtgctg 780

ggcggcgaac ccaaatccgc cgacaaaacc cacacttgtc cgccatgtcc tgctccagag 840

gcagctggtg gcccctctgt ctttctgttt ccccctaagc ccaaggacac actgatgatt 900

tcgcgtactc cagaggtgac gtgtgtggtg gtggatgtgt ctcatgaaga tcccgaggtg 960

aagttcaatt ggtatgtgga cggagtggaa gtccacaacg ctaagacaaa gcctagggaa 1020

gagcagtata actccaccta cagagtcgtt agcgtgctga ccgtgctcca tcaagactgg 1080

ctcaatggca aggagtacaa gtgcaaagtc tccaataagg cccttcctgc tcccatcgag 1140

aaaaccatct caaaggcgaa aggacaaccc cgagaacctc aggtctatac cctaccaccc 1200

tgtcgtgacg agctgactaa aaatcaggtg tcactgtggt gcctagtcaa agggttctat 1260

ccctctgaca ttgccgtgga gtgggagagc aatggccagc cagagaacaa ctacaagacc 1320

acacctccag tcctggactc ggatggctcc ttcttcctgt acagcaagct caccgttgac 1380

aagtcacgat ggcagcaagg gaatgtgttc tcatgcagcg tgatgcatga ggcacttcac 1440

aaccactata ctcagaagtc cctgagcttg agtccgggga aa 1482

<210> 9

<211> 246

<212> PRT

<213> Artificial (Artifical)

<220>

<223> full-Length amino acid sequence of HC_h

<400> 9

Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp

1 5 10 15

Val Leu Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu

20 25 30

Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

35 40 45

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

50 55 60

Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly

65 70 75 80

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn

85 90 95

Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp

100 105 110

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro

115 120 125

Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu

130 135 140

Pro Gln Val Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn

145 150 155 160

Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile

165 170 175

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

180 185 190

Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys

195 200 205

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

210 215 220

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

225 230 235 240

Ser Leu Ser Pro Gly Lys

245

<210> 10

<211> 494

<212> PRT

<213> Artificial (Artifical)

<220>

<223> full-length amino acid sequence of C3E-7085-HC-k

<400> 10

Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp

1 5 10 15

Val Leu Ser Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val

20 25 30

Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Val Thr

35 40 45

Phe Asn Tyr Tyr Gly Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly

50 55 60

Leu Glu Trp Val Ala Ser Ile Thr Arg Ser Gly Gly Arg Ile Tyr Tyr

65 70 75 80

Pro Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Glu Asn Thr Gln

85 90 95

Lys Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala

100 105 110

Val Tyr Tyr Cys Thr Leu Asp Gly Arg Asp Gly Trp Val Ala Tyr Trp

115 120 125

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly

130 135 140

Gly Gly Gly Ser Gly Gly Gly Gly Ser Asn Phe Met Leu Thr Gln Pro

145 150 155 160

Ser Ser Val Ser Gly Val Pro Gly Gln Arg Val Thr Ile Ser Cys Thr

165 170 175

Gly Asn Thr Gly Asn Ile Gly Ser Asn Tyr Val Asn Trp Tyr Gln Gln

180 185 190

Leu Pro Gly Thr Ala Pro Lys Leu Leu Ile Tyr Arg Asp Asp Lys Arg

195 200 205

Pro Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser

210 215 220

Ala Ser Leu Ala Ile Thr Gly Phe Gln Ala Glu Asp Glu Ala Asp Tyr

225 230 235 240

Tyr Cys Gln Ser Tyr Ser Ser Gly Phe Ile Phe Gly Gly Gly Thr Lys

245 250 255

Leu Thr Val Leu Gly Gly Glu Pro Lys Ser Ala Asp Lys Thr His Thr

260 265 270

Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe

275 280 285

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

290 295 300

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

305 310 315 320

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

325 330 335

Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val

340 345 350

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

355 360 365

Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser

370 375 380

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

385 390 395 400

Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val

405 410 415

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

420 425 430

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

435 440 445

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

450 455 460

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

465 470 475 480

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

485 490

<210> 11

<211> 467

<212> PRT

<213> Artificial (Artifical)

<220>

<223> full-length amino acid sequence of C3E-7085-Fab-HC-k

<400> 11

Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp

1 5 10 15

Val Leu Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln

20 25 30

Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Val Thr Phe

35 40 45

Asn Tyr Tyr Gly Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu

50 55 60

Glu Trp Val Ala Ser Ile Thr Arg Ser Gly Gly Arg Ile Tyr Tyr Pro

65 70 75 80

Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Glu Asn Thr Gln Lys

85 90 95

Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Thr Leu Asp Gly Arg Asp Gly Trp Val Ala Tyr Trp Gly

115 120 125

Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

130 135 140

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

145 150 155 160

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

165 170 175

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

180 185 190

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

195 200 205

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

210 215 220

Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys

225 230 235 240

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

245 250 255

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

260 265 270

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

275 280 285

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

290 295 300

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

305 310 315 320

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

325 330 335

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

340 345 350

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

355 360 365

Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

370 375 380

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

385 390 395 400

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

405 410 415

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

420 425 430

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

435 440 445

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

450 455 460

Pro Gly Lys

465

<210> 12

<211> 233

<212> PRT

<213> Artificial (Artifical)

<220>

<223> full-Length amino acid sequence of C3E-7085-LC

<400> 12

Met Val Leu Gln Thr Gln Val Phe Ile Ser Leu Leu Leu Trp Ile Ser

1 5 10 15

Gly Ala Tyr Gly Asn Phe Met Leu Thr Gln Pro Ser Ser Val Ser Gly

20 25 30

Val Pro Gly Gln Arg Val Thr Ile Ser Cys Thr Gly Asn Thr Gly Asn

35 40 45

Ile Gly Ser Asn Tyr Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala

50 55 60

Pro Lys Leu Leu Ile Tyr Arg Asp Asp Lys Arg Pro Ser Gly Val Pro

65 70 75 80

Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile

85 90 95

Thr Gly Phe Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr

100 105 110

Ser Ser Gly Phe Ile Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly

115 120 125

Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu

130 135 140

Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe

145 150 155 160

Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val

165 170 175

Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys

180 185 190

Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser

195 200 205

His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu

210 215 220

Lys Thr Val Ala Pro Thr Glu Cys Ser

225 230

<210> 13

<211> 494

<212> PRT

<213> Artificial (Artifical)

<220>

<223> full-length amino acid sequence of C3E-7096-HC-k

<400> 13

Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp

1 5 10 15

Val Leu Ser Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val

20 25 30

Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Val Thr

35 40 45

Phe Asn Tyr Tyr Gly Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly

50 55 60

Leu Glu Trp Val Ala Ser Ile Thr Arg Ser Gly Gly Arg Ile Tyr Tyr

65 70 75 80

Pro Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Glu Asn Thr Gln

85 90 95

Lys Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala

100 105 110

Val Tyr Tyr Cys Thr Leu Asp Gly Arg Asp Gly Trp Val Ala Tyr Trp

115 120 125

Gly Cys Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly

130 135 140

Gly Gly Gly Ser Gly Gly Gly Gly Ser Asn Phe Met Leu Thr Gln Pro

145 150 155 160

Ser Ser Val Ser Gly Val Pro Gly Gln Arg Val Thr Ile Ser Cys Thr

165 170 175

Gly Asn Thr Gly Asn Ile Gly Ser Asn Tyr Val Asn Trp Tyr Gln Gln

180 185 190

Leu Pro Gly Thr Cys Pro Lys Leu Leu Ile Tyr Arg Asp Asp Lys Arg

195 200 205

Pro Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser

210 215 220

Ala Ser Leu Ala Ile Thr Gly Phe Gln Ala Glu Asp Glu Ala Asp Tyr

225 230 235 240

Tyr Cys Gln Ser Tyr Ser Ser Gly Phe Ile Phe Gly Gly Gly Thr Lys

245 250 255

Leu Thr Val Leu Gly Gly Glu Pro Lys Ser Ala Asp Lys Thr His Thr

260 265 270

Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe

275 280 285

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

290 295 300

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

305 310 315 320

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

325 330 335

Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val

340 345 350

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

355 360 365

Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser

370 375 380

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

385 390 395 400

Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val

405 410 415

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

420 425 430

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

435 440 445

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

450 455 460

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

465 470 475 480

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

485 490

<210> 14

<211> 2232

<212> DNA

<213> Artificial (Artifical)

<220>

<223> full-length nucleotide sequence of TX3-0001-HC_k

<400> 14

atgaagcacc tgtggttctt tctgctgctt gtggccgcac cacggtgggt gctgtcaggt 60

caggtgcagt tagtgcagtc aggcgcagaa gtaaagaaac caggagcttc agtgaaggtc 120

agttgcaagg cttctgggta tacgttcact accgcaggga tgcagtgggt gcgccaagct 180

ccagggcagg gactggagtg gatggggtgg attaataccc atagcggggt cccaaagtac 240

gcggaagatt tcaaaggcag agtcaccatt tctgccgata cgagcacatc gacagcatac 300

ctccagctta gttccttgaa gagtgaggac acagctgtat actactgcgc cagatccggt 360

tttggtagca gttactggta cttcgacgtg tggggccaag gcacactcgt gactgtatcc 420

tcaggagggg gaggttccgg tggcggcggc tctggaggag gcggaagcga cattcagatg 480

acccagtctc ccagtagcct tagcgccagt gttggcgata gagtgacaat cacctgtaaa 540

gcctctcagg acgtgtcgac agcagtggca tggtatcagc agaaacccgg aaaggctccc 600

aagctactga tatattccgc gtcgtatcgg tacacagggg taccatctcg cttctcgggt 660

tccggcagcg gcaccgactt taccctcaca attagctctt tacagcccga agacttcgca 720

gtgtattatt gccagcaaca ctatatcacc ccgctgactt ttgggcaggg tacgaagctt 780

gagatcaaac ggactggcgg gggtgggtct gaggtccagc tggttgaatc cggcggggga 840

ttagtgcagc caggcggttc actgaggttg agctgtgcag cctctggagt cacgtttaac 900

tactatggca tgtcctggat tagacaagcc ccaggcaaag gcctggagtg ggtagcctct 960

atcacccgaa gcggtggacg catctattac ccagatagcg tgaagggccg gttcacaatc 1020

agtagagaga acacccagaa gacactgtac ctacaaatga acagcctaag agccgaggat 1080

accgcggtgt actactgtac actcgatgga agggatgggt gggtggctta ctgggggcag 1140

gggactctcg tcaccgttag tagtggcggt ggcggtagcg gaggaggagg tagcggcgga 1200

ggtgggtcaa atttcatgct cacacagccc agctcagtct ccggggtacc tggccagcgt 1260

gtgactatct cctgcactgg caataccgga aacataggaa gcaattacgt aaactggtat 1320

caacagcttc cgggcactgc ccctaaactc ctgatttacc gcgatgataa aaggccttcc 1380

ggcgtcccag acaggtttag tggctcgaaa tccgggacaa gcgccagttt ggcaattacc 1440

ggatttcaag ccgaagatga agctgactac tactgccagt cctattcatc agggttcata 1500

tttggcggtg gaactaaact gaccgtcctt ggaggggaac ccaagagcgc cgacaaaaca 1560

cacacttgtc ctccgtgtcc cgcgcccgaa gctgctgggg ggcccagtgt gttcctattt 1620

ccccccaagc caaaggacac gttgatgatc tctcgtaccc ctgaggtcac gtgcgttgtg 1680

gttgacgtct cccacgaaga tcccgaggtg aaattcaact ggtatgtgga cggcgttgag 1740

gtgcacaatg ccaaaactaa gccgagggaa gagcagtaca actctaccta tcgggtggtg 1800

tctgttttga cagttctcca tcaggactgg ctgaatggaa aagagtacaa gtgcaaggtg 1860

agtaacaaag ctctgccagc ccctatcgag aaaaccatct ccaaggccaa aggccaacca 1920

cgagagcctc aagtttacac cctccctcct tgtcgagacg agctgaccaa gaaccaagtc 1980

tccttatggt gtctcgtgaa gggattttat ccctcagaca ttgccgtgga gtgggaatct 2040

aatggccagc ctgagaacaa ttataagaca actcctcctg tccttgattc cgatggctcc 2100

tttttcctgt actctaagtt gactgtcgac aagtcacgct ggcagcaggg gaatgttttc 2160

tcttgcagcg ttatgcatga agctctgcat aaccactata ctcagaaaag cctgtcactg 2220

tcaccgggga ag 2232

<210> 15

<211> 2232

<212> DNA

<213> Artificial (Artifical)

<220>

<223> full-length nucleotide sequence of TX3-0003-HC_k

<400> 15

atgaagcatc tctggttttt cctgctcctg gtggcggctc caagatgggt gttgagcggt 60

caggtgcaac tggtgcagtc aggagcggag gtcaagaaac ccggcgcgag tgtcaaagtg 120

agttgcaaag cttccggata tactttcact accgctggaa tgcaatgggt gagacaagca 180

ccagggcaag gcttggaatg gatgggctgg atcaacacac actctggggt tcccaagtat 240

gccgaagact ttaagggtag ggtcaccata tcagccgata catcaacctc gaccgcatac 300

ctccagctgt cctcccttaa gagcgaggat actgctgttt actattgcgc taggtcagga 360

ttcggctctt cttactggta ctttgatgtg tgggggcagg gcacacttgt gacggtttcc 420

tctggcggcg gcgggtcagg aggtggtggc tctggagggg gtggttcaga catccagatg 480

acccagagtc cttcctcctt gagtgccagt gtaggcgaca gagtcactat tacctgcaag 540

gcctctcagg acgtgtcgac agcggttgca tggtatcagc agaaacctgg gaaggctcca 600

aagctcctga tctactcagc cagctatcgg tacactgggg tcccttctcg ctttagcggc 660

tctggtagcg ggaccgactt cacgcttacc atcagttccc tgcaaccgga ggactttgca 720

gtgtactact gtcagcagca ttacatcacc ccactgacat ttgggcaggg gacaaagctt 780

gagattaaac ggacaggcgg cggaggaagc gaggtgcagt tagtggagag cggagggggt 840

ttagtccaac cgggcgggag cctccgtctg agctgtgctg cttccggcgt taccttcaac 900

tattatggaa tgagctggat caggcaagcc cctggcaaag gtcttgaatg ggttgcatct 960

atcactcgat cagggggtcg catttattac ccggatagcg ttaaaggcag gttcacaatc 1020

tctcgggaga acactcagaa aaccctctat ctacagatga actctttacg ggccgaagat 1080

acagccgtct attattgcac tctagatggt cgagatggct gggtcgccta ttgggggtgt 1140

ggaaccttgg tcactgttag cagtggagga ggaggatcag gagggggcgg gtccggcggt 1200

gggggtagca atttcatgct gacacaacct tcttcagtat ctggcgtgcc aggacagcgc 1260

gtcaccatct cttgtaccgg caatacaggt aacattggct ccaattacgt gaattggtac 1320

cagcagcttc ctggcacctg ccctaagctg ctgatttacc gggacgataa gaggccctcg 1380

ggggtgcccg accgtttctc gggaagcaag tccgggacat ccgcctcact tgccattacg 1440

gggtttcagg ccgaggatga agctgattac tactgccagt cttactccag tggcttcatt 1500

tttggcggag gcaccaagct gacagtgcta ggcggagaac ccaaaagtgc agacaagacg 1560

catacttgcc ccccgtgtcc tgctccagaa gcagcaggag gaccctctgt tttcctcttt 1620

ccacctaaac ccaaggacac tctgatgata agcagaacac ctgaagtgac gtgtgttgtg 1680

gtagatgtct cccatgaaga tcccgaagtc aagttcaact ggtacgtgga cggcgtggag 1740

gtgcacaatg ccaaaacgaa gcccagagag gagcagtata actccacata cagagttgtg 1800

agcgtactga ctgtattgca ccaagactgg ctgaatggta aggagtacaa gtgcaaggtg 1860

tcaaacaaag ctctgcctgc acccatagag aaaaccatca gtaaggccaa agggcagcca 1920

cgcgagccac aggtgtatac tctgccacca tgccgagacg agctcactaa gaatcaggtg 1980

agcttgtggt gtctcgtaaa gggcttttac ccctcggaca ttgccgtaga gtgggaatcc 2040

aatgggcaac ccgagaacaa ttacaaaacc acaccgccag tgttagacag cgatggaagc 2100

ttctttctct atagcaagct gaccgtcgac aaaagtcgct ggcagcaggg gaacgtcttc 2160

agttgttccg tgatgcacga agccctacac aaccactata cccagaaatc cctgtcactg 2220

agtcctggca aa 2232

<210> 16

<211> 2229

<212> DNA

<213> Artificial (Artifical)

<220>

<223> full-length nucleotide sequence of CX3-0001-HC_k

<400> 16

atgaagcacc tgtggttctt tctgctgctg gtggccgctc ctagatgggt gctgtctgga 60

caggttcagc tggttcagtc tggcgccgaa gtgaagaaac ctggcgcctc tgtgaaggtg 120

tcctgcaagg cctctggcta cgccttcagc aactacctga tcgagtgggt ccgacaggcc 180

cctggacaag gacttgaatg gatgggcgtg atcaaccctg gcagcggcgt gaccaactac 240

aacgagaagt tcaagggcag agtgaccatc accgccgaca ccagcacaag caccgcctac 300

atggaactga gcagcctgag aagcgaggac accgccgtgt actattgtgc cagagccgag 360

gcttggtttg cctattgggg acagggcacc ctggtcaccg ttagctctgg tggaggcggt 420

tcaggcggag gtggcagcgg cggtggcggg agtgatattg tgatgacaca gagccccgac 480

agcctggccg tgtctcttgg agaaagagcc accatcaact gcaagagcag ccagagcctg 540

ctgtactcca gcaaccagaa gaactacctg gcctggtatc agcagaagcc cggccagcct 600

cctaagctgc tgatctactg ggccagcacc agagaaagcg gcgtgcccga tagattttct 660

ggctctggca gcggcaccga cttcaccctg acaatttcta gcctgcaagc cgaggacgtg 720

gccgtgtact actgccagag atattacggc tacccctgga cctttggcca gggcaccaag 780

gtggaaatca agggaggcgg aggatctgaa gtgcagctgg tggaatccgg ggggggcctg 840

gtgcagcctg gggggagcct gagactgagt tgtgccgcct ctggggtgac atttaactac 900

tatggcatgt cttggatccg ccaggcacct ggaaagggcc tggagtgggt ggccagcatc 960

actaggtccg gcgggcgaat ctactatccc gacagcgtca agggcaggtt cacaatttcc 1020

cgcgagaaca cacagaaaac tctgtacctg cagatgaata gcctgagagc cgaagataca 1080

gctgtgtact attgcactct ggacggcagg gatgggtggg tcgcctattg ggggcaggga 1140

accctggtga cagtcagctc cggaggagga ggatctggcg gaggaggcag tgggggaggc 1200

gggtcaaact ttatgctcac tcagccgtcc tctgtttctg gcgtacctgg ccaacgggtg 1260

accattagct gtacgggtaa taccgggaat atcgggtcta actacgtgaa ctggtatcag 1320

cagcttccag ggacagctcc caagttgctg atctatcgcg acgacaaaag accctcaggg 1380

gtccctgacc gatttagtgg cagcaaaagc ggtacttccg cttccctggc gataaccggc 1440

tttcaggccg aagatgaggc agactactat tgccagtcat attccagcgg cttcatcttc 1500

ggaggcggaa ctaagctgac agtgttgggc ggagaaccca agagcgcaga caagacccac 1560

acctgtcctc catgtcctgc tccagaagct gcaggcggcc cttccgtgtt tctgttccct 1620

ccaaagccta aggacaccct gatgatcagc cggacacctg aagtgacctg cgtggtggtg 1680

gatgtgtccc acgaggatcc cgaagtgaag ttcaattggt acgtggacgg cgtggaagtg 1740

cacaacgcca agaccaagcc tagagaggaa cagtacaaca gcacctacag agtggtgtct 1800

gtgctgacag tgctgcacca ggactggctg aacggcaaag agtacaagtg caaggtgtcc 1860

aacaaggccc tgcctgctcc tatcgagaaa accatcagca aggccaaggg ccagcctagg 1920

gaaccccagg tttacaccct gcctccatgc agggatgagc tgaccaagaa ccaggtgtcc 1980

ctgtggtgcc tggttaaggg cttctacccc tccgatatcg ccgtggaatg ggagagcaat 2040

ggccagccag agaacaacta caagacaacc cctcctgtgc tggactccga cggctcattc 2100

ttcctgtaca gcaagctgac cgtggacaag agcagatggc agcagggcaa cgtgttcagc 2160

tgcagcgtga tgcacgaggc cctgcacaac cactacacac agaagtccct gtctctgagc 2220

cccggcaaa 2229

<210> 17

<211> 2229

<212> DNA

<213> Artificial (Artifical)

<220>

<223> full-length nucleotide sequence of p_CX3-0003-HC_k

<400> 17

atgaagcacc tgtggttctt tctgctgctg gtggccgctc ctagatgggt gctgtctgga 60

caggttcagc tggttcagtc tggcgccgaa gtgaagaaac ctggcgcctc tgtgaaggtg 120

tcctgcaagg cctctggcta cgccttcagc aactacctga tcgagtgggt ccgacaggcc 180

cctggacaag gacttgaatg gatgggcgtg atcaaccctg gcagcggcgt gaccaactac 240

aacgagaagt tcaagggcag agtgaccatc accgccgaca ccagcacaag caccgcctac 300

atggaactga gcagcctgag aagcgaggac accgccgtgt actattgtgc cagagccgag 360

gcttggtttg cctattgggg acagggcacc ctggtcaccg ttagctctgg tggaggcggt 420

tcaggcggag gtggcagcgg cggtggcggg agtgatattg tgatgacaca gagccccgac 480

agcctggccg tgtctcttgg agaaagagcc accatcaact gcaagagcag ccagagcctg 540

ctgtactcca gcaaccagaa gaactacctg gcctggtatc agcagaagcc cggccagcct 600

cctaagctgc tgatctactg ggccagcacc agagaaagcg gcgtgcccga tagattttct 660

ggctctggca gcggcaccga cttcaccctg acaatttcta gcctgcaagc cgaggacgtg 720

gccgtgtact actgccagag atattacggc tacccctgga cctttggcca gggcaccaag 780

gtggaaatca agggaggcgg aggatctgaa gtgcagctgg tggaatccgg ggggggcctg 840

gtgcagcctg gggggagcct gagactgagt tgtgccgcct ctggggtgac atttaactac 900

tatggcatgt cttggatccg ccaggcacct ggaaagggcc tggagtgggt ggccagcatc 960

actaggtccg gcgggcgaat ctactatccc gacagcgtca agggcaggtt cacaatttcc 1020

cgcgagaaca cacagaaaac tctgtacctg cagatgaata gcctgagagc cgaagataca 1080

gctgtgtact attgcactct ggacggcagg gatgggtggg tcgcctattg ggggtgtgga 1140

accctggtga cagtcagctc cggaggagga ggatctggcg gaggaggcag tgggggaggc 1200

gggtcaaact ttatgctcac tcagccgtcc tctgtttctg gcgtacctgg ccaacgggtg 1260

accattagct gtacgggtaa taccgggaat atcgggtcta actacgtgaa ctggtatcag 1320

cagcttccag ggacatgtcc caagttgctg atctatcgcg acgacaaaag accctcaggg 1380

gtccctgacc gatttagtgg cagcaaaagc ggtacttccg cttccctggc gataaccggc 1440

tttcaggccg aagatgaggc agactactat tgccagtcat attccagcgg cttcatcttc 1500

ggaggcggaa ctaagctgac agtgttgggc ggagaaccca agagcgcaga caagacccac 1560

acctgtcctc catgtcctgc tccagaagct gcaggcggcc cttccgtgtt tctgttccct 1620

ccaaagccta aggacaccct gatgatcagc cggacacctg aagtgacctg cgtggtggtg 1680

gatgtgtccc acgaggatcc cgaagtgaag ttcaattggt acgtggacgg cgtggaagtg 1740

cacaacgcca agaccaagcc tagagaggaa cagtacaaca gcacctacag agtggtgtct 1800

gtgctgacag tgctgcacca ggactggctg aacggcaaag agtacaagtg caaggtgtcc 1860

aacaaggccc tgcctgctcc tatcgagaaa accatcagca aggccaaggg ccagcctagg 1920

gaaccccagg tttacaccct gcctccatgc agggatgagc tgaccaagaa ccaggtgtcc 1980

ctgtggtgcc tggttaaggg cttctacccc tccgatatcg ccgtggaatg ggagagcaat 2040

ggccagccag agaacaacta caagacaacc cctcctgtgc tggactccga cggctcattc 2100

ttcctgtaca gcaagctgac cgtggacaag agcagatggc agcagggcaa cgtgttcagc 2160

tgcagcgtga tgcacgaggc cctgcacaac cactacacac agaagtccct gtctctgagc 2220

cccggcaaa 2229

<210> 18

<211> 2217

<212> DNA

<213> Artificial (Artifical)

<220>

<223> full-length nucleotide sequence of RX3-0001-HC_k

<400> 18

atgaagcacc tgtggttctt tctgctgctg gtggccgctc ctagatgggt gctgtctgga 60

caggtgcagc tggtgcagtc tggcgccgaa gtgaagaaac caggcgccag cgtgaaggtg 120

tcctgcaagg ccagcggcta cacctttacc ggctactaca tgcactgggt gcgccaggct 180

ccaggccagg gactggaatg gatgggccgg atcaacccca atagcggcgg caccaactac 240

gcccagaaat tccagggcag agtgaccatg acccgggaca ccagcatcag caccgcctac 300

atggaactga gcggcctgag aagcgacgac accgccgtgt actactgtgc ctctggctct 360

gtgcggcacc cttggggaca gggaacactc gtgaccgtgt ccagcggtgg aggcggttca 420

ggcggaggtg gcagcggcgg tggcgggagt cagcctgtgc tgacacagcc tccaagcgcc 480

tctggcacac ctggccagag agtgacaatc agctgcagcg gcagcagaag caacatcggc 540

ggcaacatcg tgtcctggta tcagcagttc cccggcaccg cccctagact gctgacctac 600

gccgacaacc agaggcctag cggcgtgccc gatagattca gcggctctaa gagcggcacc 660

agcgccagcc tggccatctc tggcctgcag tctgaggacg aggccgacta ctattgcgcc 720

gcctgggacg acagcctgaa cggcgtggtg tttggcggag gcaccaagct gacagtgctg 780

ggaggcggag gatctgaagt gcagctggtg gaatccgggg ggggcctggt gcagcctggg 840

gggagcctga gactgagttg tgccgcctct ggggtgacat ttaactacta tggcatgtct 900

tggatccgcc aggcacctgg aaagggcctg gagtgggtgg ccagcatcac taggtccggc 960

gggcgaatct actatcccga cagcgtcaag ggcaggttca caatttcccg cgagaacaca 1020

cagaaaactc tgtacctgca gatgaatagc ctgagagccg aagatacagc tgtgtactat 1080

tgcactctgg acggcaggga tgggtgggtc gcctattggg ggcagggaac cctggtgaca 1140

gtcagctccg gaggaggagg atctggcgga ggaggcagtg ggggaggcgg gtcaaacttt 1200

atgctcactc agccgtcctc tgtttctggc gtacctggcc aacgggtgac cattagctgt 1260

acgggtaata ccgggaatat cgggtctaac tacgtgaact ggtatcagca gcttccaggg 1320

acagctccca agttgctgat ctatcgcgac gacaaaagac cctcaggggt ccctgaccga 1380

tttagtggca gcaaaagcgg tacttccgct tccctggcga taaccggctt tcaggccgaa 1440

gatgaggcag actactattg ccagtcatat tccagcggct tcatcttcgg aggcggaact 1500

aagctgacag tgttgggcgg agaacccaag agcgcagaca agacccacac ctgtcctcca 1560

tgtcctgctc cagaagctgc aggcggccct tccgtgtttc tgttccctcc aaagcctaag 1620

gacaccctga tgatcagccg gacacctgaa gtgacctgcg tggtggtgga tgtgtcccac 1680

gaggatcccg aagtgaagtt caattggtac gtggacggcg tggaagtgca caacgccaag 1740

accaagccta gagaggaaca gtacaacagc acctacagag tggtgtctgt gctgacagtg 1800

ctgcaccagg actggctgaa cggcaaagag tacaagtgca aggtgtccaa caaggccctg 1860

cctgctccta tcgagaaaac catcagcaag gccaagggcc agcctaggga accccaggtt 1920

tacaccctgc ctccatgcag ggatgagctg accaagaacc aggtgtccct gtggtgcctg 1980

gttaagggct tctacccctc cgatatcgcc gtggaatggg agagcaatgg ccagccagag 2040

aacaactaca agacaacccc tcctgtgctg gactccgacg gctcattctt cctgtacagc 2100

aagctgaccg tggacaagag cagatggcag cagggcaacg tgttcagctg cagcgtgatg 2160

cacgaggccc tgcacaacca ctacacacag aagtccctgt ctctgagccc cggcaaa 2217

<210> 19

<211> 2217

<212> DNA

<213> Artificial (Artifical)

<220>

<223> full-length nucleotide sequence of RX3-0003-HC_k

<400> 19

atgaagcacc tgtggttctt tctgctgctg gtggccgctc ctagatgggt gctgtctgga 60

caggtgcagc tggtgcagtc tggcgccgaa gtgaagaaac caggcgccag cgtgaaggtg 120

tcctgcaagg ccagcggcta cacctttacc ggctactaca tgcactgggt gcgccaggct 180

ccaggccagg gactggaatg gatgggccgg atcaacccca atagcggcgg caccaactac 240

gcccagaaat tccagggcag agtgaccatg acccgggaca ccagcatcag caccgcctac 300

atggaactga gcggcctgag aagcgacgac accgccgtgt actactgtgc ctctggctct 360

gtgcggcacc cttggggaca gggaacactc gtgaccgtgt ccagcggtgg aggcggttca 420

ggcggaggtg gcagcggcgg tggcgggagt cagcctgtgc tgacacagcc tccaagcgcc 480

tctggcacac ctggccagag agtgacaatc agctgcagcg gcagcagaag caacatcggc 540

ggcaacatcg tgtcctggta tcagcagttc cccggcaccg cccctagact gctgacctac 600

gccgacaacc agaggcctag cggcgtgccc gatagattca gcggctctaa gagcggcacc 660

agcgccagcc tggccatctc tggcctgcag tctgaggacg aggccgacta ctattgcgcc 720

gcctgggacg acagcctgaa cggcgtggtg tttggcggag gcaccaagct gacagtgctg 780

ggaggcggag gatctgaagt gcagctggtg gaatccgggg ggggcctggt gcagcctggg 840

gggagcctga gactgagttg tgccgcctct ggggtgacat ttaactacta tggcatgtct 900

tggatccgcc aggcacctgg aaagggcctg gagtgggtgg ccagcatcac taggtccggc 960

gggcgaatct actatcccga cagcgtcaag ggcaggttca caatttcccg cgagaacaca 1020

cagaaaactc tgtacctgca gatgaatagc ctgagagccg aagatacagc tgtgtactat 1080

tgcactctgg acggcaggga tgggtgggtc gcctattggg ggtgtggaac cctggtgaca 1140

gtcagctccg gaggaggagg atctggcgga ggaggcagtg ggggaggcgg gtcaaacttt 1200

atgctcactc agccgtcctc tgtttctggc gtacctggcc aacgggtgac cattagctgt 1260

acgggtaata ccgggaatat cgggtctaac tacgtgaact ggtatcagca gcttccaggg 1320

acatgtccca agttgctgat ctatcgcgac gacaaaagac cctcaggggt ccctgaccga 1380

tttagtggca gcaaaagcgg tacttccgct tccctggcga taaccggctt tcaggccgaa 1440

gatgaggcag actactattg ccagtcatat tccagcggct tcatcttcgg aggcggaact 1500

aagctgacag tgttgggcgg agaacccaag agcgcagaca agacccacac ctgtcctcca 1560

tgtcctgctc cagaagctgc aggcggccct tccgtgtttc tgttccctcc aaagcctaag 1620

gacaccctga tgatcagccg gacacctgaa gtgacctgcg tggtggtgga tgtgtcccac 1680

gaggatcccg aagtgaagtt caattggtac gtggacggcg tggaagtgca caacgccaag 1740

accaagccta gagaggaaca gtacaacagc acctacagag tggtgtctgt gctgacagtg 1800

ctgcaccagg actggctgaa cggcaaagag tacaagtgca aggtgtccaa caaggccctg 1860

cctgctccta tcgagaaaac catcagcaag gccaagggcc agcctaggga accccaggtt 1920

tacaccctgc ctccatgcag ggatgagctg accaagaacc aggtgtccct gtggtgcctg 1980

gttaagggct tctacccctc cgatatcgcc gtggaatggg agagcaatgg ccagccagag 2040

aacaactaca agacaacccc tcctgtgctg gactccgacg gctcattctt cctgtacagc 2100

aagctgaccg tggacaagag cagatggcag cagggcaacg tgttcagctg cagcgtgatg 2160

cacgaggccc tgcacaacca ctacacacag aagtccctgt ctctgagccc cggcaaa 2217

<210> 20

<211> 744

<212> PRT

<213> Artificial (Artifical)

<220>

<223> full-Length amino acid sequence of TX3-0001-HC_k

<400> 20

Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp

1 5 10 15

Val Leu Ser Gly Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys

20 25 30

Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr

35 40 45

Phe Thr Thr Ala Gly Met Gln Trp Val Arg Gln Ala Pro Gly Gln Gly

50 55 60

Leu Glu Trp Met Gly Trp Ile Asn Thr His Ser Gly Val Pro Lys Tyr

65 70 75 80

Ala Glu Asp Phe Lys Gly Arg Val Thr Ile Ser Ala Asp Thr Ser Thr

85 90 95

Ser Thr Ala Tyr Leu Gln Leu Ser Ser Leu Lys Ser Glu Asp Thr Ala

100 105 110

Val Tyr Tyr Cys Ala Arg Ser Gly Phe Gly Ser Ser Tyr Trp Tyr Phe

115 120 125

Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly

130 135 140

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met

145 150 155 160

Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr

165 170 175

Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Thr Ala Val Ala Trp Tyr

180 185 190

Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser

195 200 205

Tyr Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly

210 215 220

Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala

225 230 235 240

Val Tyr Tyr Cys Gln Gln His Tyr Ile Thr Pro Leu Thr Phe Gly Gln

245 250 255

Gly Thr Lys Leu Glu Ile Lys Arg Thr Gly Gly Gly Gly Ser Glu Val

260 265 270

Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu

275 280 285

Arg Leu Ser Cys Ala Ala Ser Gly Val Thr Phe Asn Tyr Tyr Gly Met

290 295 300

Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Ser

305 310 315 320

Ile Thr Arg Ser Gly Gly Arg Ile Tyr Tyr Pro Asp Ser Val Lys Gly

325 330 335

Arg Phe Thr Ile Ser Arg Glu Asn Thr Gln Lys Thr Leu Tyr Leu Gln

340 345 350

Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Thr Leu

355 360 365

Asp Gly Arg Asp Gly Trp Val Ala Tyr Trp Gly Gln Gly Thr Leu Val

370 375 380

Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

385 390 395 400

Gly Gly Ser Asn Phe Met Leu Thr Gln Pro Ser Ser Val Ser Gly Val

405 410 415

Pro Gly Gln Arg Val Thr Ile Ser Cys Thr Gly Asn Thr Gly Asn Ile

420 425 430

Gly Ser Asn Tyr Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro

435 440 445

Lys Leu Leu Ile Tyr Arg Asp Asp Lys Arg Pro Ser Gly Val Pro Asp

450 455 460

Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr

465 470 475 480

Gly Phe Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Ser

485 490 495

Ser Gly Phe Ile Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gly

500 505 510

Glu Pro Lys Ser Ala Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

515 520 525

Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

530 535 540

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

545 550 555 560

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

565 570 575

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

580 585 590

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

595 600 605

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

610 615 620

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

625 630 635 640

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr

645 650 655

Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser

660 665 670

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

675 680 685

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

690 695 700

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

705 710 715 720

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

725 730 735

Ser Leu Ser Leu Ser Pro Gly Lys

740

<210> 21

<211> 744

<212> PRT

<213> Artificial (Artifical)

<220>

<223> full-length amino acid sequence of TX3-0003-HC_k

<400> 21

Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp

1 5 10 15

Val Leu Ser Gly Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys

20 25 30

Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr

35 40 45

Phe Thr Thr Ala Gly Met Gln Trp Val Arg Gln Ala Pro Gly Gln Gly

50 55 60

Leu Glu Trp Met Gly Trp Ile Asn Thr His Ser Gly Val Pro Lys Tyr

65 70 75 80

Ala Glu Asp Phe Lys Gly Arg Val Thr Ile Ser Ala Asp Thr Ser Thr

85 90 95

Ser Thr Ala Tyr Leu Gln Leu Ser Ser Leu Lys Ser Glu Asp Thr Ala

100 105 110

Val Tyr Tyr Cys Ala Arg Ser Gly Phe Gly Ser Ser Tyr Trp Tyr Phe

115 120 125

Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly

130 135 140

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met

145 150 155 160

Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr

165 170 175

Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Thr Ala Val Ala Trp Tyr

180 185 190

Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser

195 200 205

Tyr Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly

210 215 220

Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala

225 230 235 240

Val Tyr Tyr Cys Gln Gln His Tyr Ile Thr Pro Leu Thr Phe Gly Gln

245 250 255

Gly Thr Lys Leu Glu Ile Lys Arg Thr Gly Gly Gly Gly Ser Glu Val

260 265 270

Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu

275 280 285

Arg Leu Ser Cys Ala Ala Ser Gly Val Thr Phe Asn Tyr Tyr Gly Met

290 295 300

Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Ser

305 310 315 320

Ile Thr Arg Ser Gly Gly Arg Ile Tyr Tyr Pro Asp Ser Val Lys Gly

325 330 335

Arg Phe Thr Ile Ser Arg Glu Asn Thr Gln Lys Thr Leu Tyr Leu Gln

340 345 350

Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Thr Leu

355 360 365

Asp Gly Arg Asp Gly Trp Val Ala Tyr Trp Gly Cys Gly Thr Leu Val

370 375 380

Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

385 390 395 400

Gly Gly Ser Asn Phe Met Leu Thr Gln Pro Ser Ser Val Ser Gly Val

405 410 415

Pro Gly Gln Arg Val Thr Ile Ser Cys Thr Gly Asn Thr Gly Asn Ile

420 425 430

Gly Ser Asn Tyr Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Cys Pro

435 440 445

Lys Leu Leu Ile Tyr Arg Asp Asp Lys Arg Pro Ser Gly Val Pro Asp

450 455 460

Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr

465 470 475 480

Gly Phe Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Ser

485 490 495

Ser Gly Phe Ile Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gly

500 505 510

Glu Pro Lys Ser Ala Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

515 520 525

Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

530 535 540

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

545 550 555 560

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

565 570 575

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

580 585 590

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

595 600 605

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

610 615 620

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

625 630 635 640

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr

645 650 655

Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser

660 665 670

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

675 680 685

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

690 695 700

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

705 710 715 720

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

725 730 735

Ser Leu Ser Leu Ser Pro Gly Lys

740

<210> 22

<211> 743

<212> PRT

<213> Artificial (Artifical)

<220>

<223> full-length amino acid sequence of CX3-0001-HC_k

<400> 22

Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp

1 5 10 15

Val Leu Ser Gly Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys

20 25 30

Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala

35 40 45

Phe Ser Asn Tyr Leu Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly

50 55 60

Leu Glu Trp Met Gly Val Ile Asn Pro Gly Ser Gly Val Thr Asn Tyr

65 70 75 80

Asn Glu Lys Phe Lys Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr

85 90 95

Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala

100 105 110

Val Tyr Tyr Cys Ala Arg Ala Glu Ala Trp Phe Ala Tyr Trp Gly Gln

115 120 125

Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly

130 135 140

Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro Asp

145 150 155 160

Ser Leu Ala Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ser

165 170 175

Ser Gln Ser Leu Leu Tyr Ser Ser Asn Gln Lys Asn Tyr Leu Ala Trp

180 185 190

Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala

195 200 205

Ser Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser

210 215 220

Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val

225 230 235 240

Ala Val Tyr Tyr Cys Gln Arg Tyr Tyr Gly Tyr Pro Trp Thr Phe Gly

245 250 255

Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser Glu Val Gln

260 265 270

Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg

275 280 285

Leu Ser Cys Ala Ala Ser Gly Val Thr Phe Asn Tyr Tyr Gly Met Ser

290 295 300

Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Ser Ile

305 310 315 320

Thr Arg Ser Gly Gly Arg Ile Tyr Tyr Pro Asp Ser Val Lys Gly Arg

325 330 335

Phe Thr Ile Ser Arg Glu Asn Thr Gln Lys Thr Leu Tyr Leu Gln Met

340 345 350

Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Thr Leu Asp

355 360 365

Gly Arg Asp Gly Trp Val Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr

370 375 380

Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

385 390 395 400

Gly Ser Asn Phe Met Leu Thr Gln Pro Ser Ser Val Ser Gly Val Pro

405 410 415

Gly Gln Arg Val Thr Ile Ser Cys Thr Gly Asn Thr Gly Asn Ile Gly

420 425 430

Ser Asn Tyr Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys

435 440 445

Leu Leu Ile Tyr Arg Asp Asp Lys Arg Pro Ser Gly Val Pro Asp Arg

450 455 460

Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly

465 470 475 480

Phe Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Ser Ser

485 490 495

Gly Phe Ile Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gly Glu

500 505 510

Pro Lys Ser Ala Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

515 520 525

Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

530 535 540

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

545 550 555 560

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

565 570 575

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

580 585 590

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

595 600 605

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

610 615 620

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

625 630 635 640

Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys

645 650 655

Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

660 665 670

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

675 680 685

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

690 695 700

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

705 710 715 720

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

725 730 735

Leu Ser Leu Ser Pro Gly Lys

740

<210> 23

<211> 743

<212> PRT

<213> Artificial (Artifical)

<220>

<223> full-length amino acid sequence of CX3-0003-HC_k

<400> 23

Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp

1 5 10 15

Val Leu Ser Gly Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys

20 25 30

Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala

35 40 45

Phe Ser Asn Tyr Leu Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly

50 55 60

Leu Glu Trp Met Gly Val Ile Asn Pro Gly Ser Gly Val Thr Asn Tyr

65 70 75 80

Asn Glu Lys Phe Lys Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr

85 90 95

Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala

100 105 110

Val Tyr Tyr Cys Ala Arg Ala Glu Ala Trp Phe Ala Tyr Trp Gly Gln

115 120 125

Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly

130 135 140

Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro Asp

145 150 155 160

Ser Leu Ala Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ser

165 170 175

Ser Gln Ser Leu Leu Tyr Ser Ser Asn Gln Lys Asn Tyr Leu Ala Trp

180 185 190

Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala

195 200 205

Ser Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser

210 215 220

Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val

225 230 235 240

Ala Val Tyr Tyr Cys Gln Arg Tyr Tyr Gly Tyr Pro Trp Thr Phe Gly

245 250 255

Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser Glu Val Gln

260 265 270

Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg

275 280 285

Leu Ser Cys Ala Ala Ser Gly Val Thr Phe Asn Tyr Tyr Gly Met Ser

290 295 300

Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Ser Ile

305 310 315 320

Thr Arg Ser Gly Gly Arg Ile Tyr Tyr Pro Asp Ser Val Lys Gly Arg

325 330 335

Phe Thr Ile Ser Arg Glu Asn Thr Gln Lys Thr Leu Tyr Leu Gln Met

340 345 350

Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Thr Leu Asp

355 360 365

Gly Arg Asp Gly Trp Val Ala Tyr Trp Gly Cys Gly Thr Leu Val Thr

370 375 380

Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

385 390 395 400

Gly Ser Asn Phe Met Leu Thr Gln Pro Ser Ser Val Ser Gly Val Pro

405 410 415

Gly Gln Arg Val Thr Ile Ser Cys Thr Gly Asn Thr Gly Asn Ile Gly

420 425 430

Ser Asn Tyr Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Cys Pro Lys

435 440 445

Leu Leu Ile Tyr Arg Asp Asp Lys Arg Pro Ser Gly Val Pro Asp Arg

450 455 460

Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly

465 470 475 480

Phe Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Ser Ser

485 490 495

Gly Phe Ile Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gly Glu

500 505 510

Pro Lys Ser Ala Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

515 520 525

Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

530 535 540

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

545 550 555 560

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

565 570 575

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

580 585 590

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

595 600 605

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

610 615 620

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

625 630 635 640

Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys

645 650 655

Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

660 665 670

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

675 680 685

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

690 695 700

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

705 710 715 720

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

725 730 735

Leu Ser Leu Ser Pro Gly Lys

740

<210> 24

<211> 739

<212> PRT

<213> Artificial (Artifical)

<220>

<223> full Length amino acid sequence of RX3-0001-HC_k

<400> 24

Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp

1 5 10 15

Val Leu Ser Gly Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys

20 25 30

Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr

35 40 45

Phe Thr Gly Tyr Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly

50 55 60

Leu Glu Trp Met Gly Arg Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr

65 70 75 80

Ala Gln Lys Phe Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile

85 90 95

Ser Thr Ala Tyr Met Glu Leu Ser Gly Leu Arg Ser Asp Asp Thr Ala

100 105 110

Val Tyr Tyr Cys Ala Ser Gly Ser Val Arg His Pro Trp Gly Gln Gly

115 120 125

Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

130 135 140

Ser Gly Gly Gly Gly Ser Gln Pro Val Leu Thr Gln Pro Pro Ser Ala

145 150 155 160

Ser Gly Thr Pro Gly Gln Arg Val Thr Ile Ser Cys Ser Gly Ser Arg

165 170 175

Ser Asn Ile Gly Gly Asn Ile Val Ser Trp Tyr Gln Gln Phe Pro Gly

180 185 190

Thr Ala Pro Arg Leu Leu Thr Tyr Ala Asp Asn Gln Arg Pro Ser Gly

195 200 205

Val Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu

210 215 220

Ala Ile Ser Gly Leu Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala

225 230 235 240

Ala Trp Asp Asp Ser Leu Asn Gly Val Val Phe Gly Gly Gly Thr Lys

245 250 255

Leu Thr Val Leu Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser

260 265 270

Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala

275 280 285

Ala Ser Gly Val Thr Phe Asn Tyr Tyr Gly Met Ser Trp Ile Arg Gln

290 295 300

Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Ser Ile Thr Arg Ser Gly

305 310 315 320

Gly Arg Ile Tyr Tyr Pro Asp Ser Val Lys Gly Arg Phe Thr Ile Ser

325 330 335

Arg Glu Asn Thr Gln Lys Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg

340 345 350

Ala Glu Asp Thr Ala Val Tyr Tyr Cys Thr Leu Asp Gly Arg Asp Gly

355 360 365

Trp Val Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly

370 375 380

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asn Phe

385 390 395 400

Met Leu Thr Gln Pro Ser Ser Val Ser Gly Val Pro Gly Gln Arg Val

405 410 415

Thr Ile Ser Cys Thr Gly Asn Thr Gly Asn Ile Gly Ser Asn Tyr Val

420 425 430

Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu Ile Tyr

435 440 445

Arg Asp Asp Lys Arg Pro Ser Gly Val Pro Asp Arg Phe Ser Gly Ser

450 455 460

Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Phe Gln Ala Glu

465 470 475 480

Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Ser Ser Gly Phe Ile Phe

485 490 495

Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gly Glu Pro Lys Ser Ala

500 505 510

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

515 520 525

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

530 535 540

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

545 550 555 560

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

565 570 575

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

580 585 590

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

595 600 605

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

610 615 620

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

625 630 635 640

Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

645 650 655

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

660 665 670

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

675 680 685

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

690 695 700

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

705 710 715 720

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

725 730 735

Pro Gly Lys

<210> 25

<211> 739

<212> PRT

<213> Artificial (Artifical)

<220>

<223> full Length amino acid sequence of RX3-0003-HC_k

<400> 25

Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp

1 5 10 15

Val Leu Ser Gly Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys

20 25 30

Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr

35 40 45

Phe Thr Gly Tyr Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly

50 55 60

Leu Glu Trp Met Gly Arg Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr

65 70 75 80

Ala Gln Lys Phe Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile

85 90 95

Ser Thr Ala Tyr Met Glu Leu Ser Gly Leu Arg Ser Asp Asp Thr Ala

100 105 110

Val Tyr Tyr Cys Ala Ser Gly Ser Val Arg His Pro Trp Gly Gln Gly

115 120 125

Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

130 135 140

Ser Gly Gly Gly Gly Ser Gln Pro Val Leu Thr Gln Pro Pro Ser Ala

145 150 155 160

Ser Gly Thr Pro Gly Gln Arg Val Thr Ile Ser Cys Ser Gly Ser Arg

165 170 175

Ser Asn Ile Gly Gly Asn Ile Val Ser Trp Tyr Gln Gln Phe Pro Gly

180 185 190

Thr Ala Pro Arg Leu Leu Thr Tyr Ala Asp Asn Gln Arg Pro Ser Gly

195 200 205

Val Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu

210 215 220

Ala Ile Ser Gly Leu Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala

225 230 235 240

Ala Trp Asp Asp Ser Leu Asn Gly Val Val Phe Gly Gly Gly Thr Lys

245 250 255

Leu Thr Val Leu Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser

260 265 270

Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala

275 280 285

Ala Ser Gly Val Thr Phe Asn Tyr Tyr Gly Met Ser Trp Ile Arg Gln

290 295 300

Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Ser Ile Thr Arg Ser Gly

305 310 315 320

Gly Arg Ile Tyr Tyr Pro Asp Ser Val Lys Gly Arg Phe Thr Ile Ser

325 330 335

Arg Glu Asn Thr Gln Lys Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg

340 345 350

Ala Glu Asp Thr Ala Val Tyr Tyr Cys Thr Leu Asp Gly Arg Asp Gly

355 360 365

Trp Val Ala Tyr Trp Gly Cys Gly Thr Leu Val Thr Val Ser Ser Gly

370 375 380

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asn Phe

385 390 395 400

Met Leu Thr Gln Pro Ser Ser Val Ser Gly Val Pro Gly Gln Arg Val

405 410 415

Thr Ile Ser Cys Thr Gly Asn Thr Gly Asn Ile Gly Ser Asn Tyr Val

420 425 430

Asn Trp Tyr Gln Gln Leu Pro Gly Thr Cys Pro Lys Leu Leu Ile Tyr

435 440 445

Arg Asp Asp Lys Arg Pro Ser Gly Val Pro Asp Arg Phe Ser Gly Ser

450 455 460

Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Phe Gln Ala Glu

465 470 475 480

Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Ser Ser Gly Phe Ile Phe

485 490 495

Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gly Glu Pro Lys Ser Ala

500 505 510

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

515 520 525

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

530 535 540

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

545 550 555 560

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

565 570 575

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

580 585 590

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

595 600 605

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

610 615 620

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

625 630 635 640

Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

645 650 655

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

660 665 670

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

675 680 685

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

690 695 700

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

705 710 715 720

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

725 730 735

Pro Gly Lys

<210> 26

<211> 2154

<212> DNA

<213> Artificial (Artifical)

<220>

<223> full-length nucleotide sequence of TX3-0002-HC_k

<400> 26

atgaagcacc tgtggttctt tctgctgctg gtggccgctc ctagatgggt gctgtctgga 60

caggttcagc tggtgcagtc tggcgccgaa gtgaaaaaac ctggcgcctc cgtgaaggtg 120

tcctgcaagg ctagcggcta cacctttacc acagccggca tgcagtgggt ccgacaggct 180

cctggacaag gactcgagtg gatgggctgg atcaataccc acagcggcgt gcccaaatac 240

gccgaggatt tcaagggcag agtgaccatc agcgccgaca ccagcacaag cacagcctac 300

ctgcagctga gcagcctgaa gtctgaggac accgccgtgt actactgtgc cagaagcggc 360

tttggcagca gctactggta cttcgatgtg tggggccagg gcaccctggt cacagtttct 420

agcggaggcg gaggatctgg tggcggagga agtggcggag gcggttctga tatccagatg 480

acacagagcc ccagcagcct gtctgcctct gtgggagaca gagtgacaat tacatgcaag 540

gccagccagg acgtgtccac agcagttgct tggtaccagc agaagcctgg caaggcccct 600

aagctgctga tctacagcgc cagctacaga tacaccggcg tgccaagcag attttccggc 660

tctggcagcg gcaccgattt caccctgacc atatctagcc tgcagccaga ggacttcgcc 720

gtgtattatt gccagcagca ctacatcacc cctctgacct ttggccaggg gaccaagctc 780

gagatcaaga gaacaggtgg cggcggatct gaggtgcagc tggttgaaag cggcggagga 840

cttgttcagc ctggcggatc tctgagactg agctgtgctg ctagcggcgt gaccttcaac 900

tactacggca tgagctggat ccgccaagct ccaggcaagg gcctcgagtg ggttgccagc 960

attacaagat ctggcggccg catctactac cccgactctg tgaagggcag attcacaatc 1020

agccgcgaga acacccagaa aaccctgtac ctgcagatga actccctgag agccgaggat 1080

acagctgtgt actattgcac cctggacggc agagatggct gggttgcata ttggggacag 1140

ggaaccctcg tgaccgtgtc ctctgcctct acaaagggcc ctagcgtgtt ccctctggct 1200

cctagcagca agagcacaag cggaggaaca gccgctctgg gctgtctggt caaggactac 1260

tttcccgagc ctgtgacagt gtcctggaac tctggcgctc tgacatccgg cgtgcacaca 1320

tttccagccg tgctgcaaag cagcggcctg tactctctga gcagcgtcgt gacagtgcct 1380

agcagctctc tgggcaccca gacctacatc tgcaatgtga accacaagcc tagcaacacc 1440

aaggtggaca agagagtgga acccaagagc tgcgacaaga cccacacctg tcctccatgt 1500

cctgcacctg aagctgctgg cggcccttcc gtgtttctgt tccctccaaa gcctaaggac 1560

accctgatga tcagcagaac ccctgaagtg acctgcgtgg tggtggatgt gtctcacgag 1620

gacccagaag tgaagttcaa ttggtacgtg gacggcgtgg aagtgcacaa cgccaagacc 1680

aagcctagag aggaacagta caacagcacc tacagagtgg tgtccgtgct gacagtgctg 1740

caccaggatt ggctgaacgg caaagagtac aagtgcaagg tgtccaacaa ggccctgcct 1800

gctcctatcg agaaaaccat cagcaaggcc aagggccagc ctagggaacc ccaggtttac 1860

acccttcctc catgccggga tgagctgacc aagaaccagg tgtccctgtg gtgtctcgtg 1920

aagggcttct acccctccga tatcgccgtg gaatgggaga gcaatggcca gcctgagaac 1980

aactacaaga caacccctcc tgtgctggac agcgacggct cattcttcct gtacagcaag 2040

ctgaccgtgg acaagtccag atggcagcag ggcaacgtgt tcagctgctc cgtgatgcac 2100

gaggccctgc acaaccacta cacacagaag tccctgtctc tgagccccgg caaa 2154

<210> 27

<211> 2151

<212> DNA

<213> Artificial (Artifical)

<220>

<223> full-length nucleotide sequence of CX3-0002-HC_k

<400> 27

atgaagcacc tgtggttctt tctgctgctg gtggccgctc ctagatgggt gctgtctgga 60

caggttcagc tggttcagtc tggcgccgaa gtgaagaaac ctggcgcctc tgtgaaggtg 120

tcctgcaagg cctctggcta cgccttcagc aactacctga tcgagtgggt ccgacaggcc 180

cctggacaag gacttgaatg gatgggcgtg atcaaccctg gcagcggcgt gaccaactac 240

aacgagaagt tcaagggcag agtgaccatc accgccgaca ccagcacaag caccgcctac 300

atggaactga gcagcctgag aagcgaggac accgccgtgt actattgtgc cagagccgag 360

gcttggtttg cctattgggg acagggcacc ctggtcaccg ttagctctgg tggaggcggt 420

tcaggcggag gtggcagcgg cggtggcggg agtgatattg tgatgacaca gagccccgac 480

agcctggccg tgtctcttgg agaaagagcc accatcaact gcaagagcag ccagagcctg 540

ctgtactcca gcaaccagaa gaactacctg gcctggtatc agcagaagcc cggccagcct 600

cctaagctgc tgatctactg ggccagcacc agagaaagcg gcgtgcccga tagattttct 660

ggctctggca gcggcaccga cttcaccctg acaatttcta gcctgcaagc cgaggacgtg 720

gccgtgtact actgccagag atattacggc tacccctgga cctttggcca gggcaccaag 780

gtggaaatca agggaggcgg aggatctgag gtgcagcttg ttgaatcagg tggcggactt 840

gtgcagcctg gcggaagcct cagacttagc tgtgctgctt ccggcgtgac cttcaactac 900

tacggcatga gctggatcag gcaggcacct ggaaagggcc tcgagtgggt tgcctctatc 960

acaagaagcg gcggcagaat ctactacccc gactctgtga aaggccggtt caccatctcc 1020

agagagaaca cccagaaaac actctatctc cagatgaact ctctgcgcgc cgaagataca 1080

gccgtctatt actgcaccct ggacggcaga gatggctggg ttgcatattg gggacaggga 1140

accctcgtga ccgtgtcctc tgcctctaca aagggcccta gcgtgttccc tctggctcct 1200

agcagcaagt ctaccagcgg aggaacagcc gctctgggct gtctggtcaa ggactacttt 1260

cccgagccag tgacagtgtc ctggaactct ggcgctctga catccggcgt gcacacattt 1320

ccagccgtgc tgcaaagctc cggcctgtac tctctgagca gcgtggtcac agtgccaagc 1380

tctagcctgg gcacccagac ctacatctgc aatgtgaacc acaagcctag caacaccaag 1440

gtcgacaaga gagtggaacc caagagctgc gacaagaccc acacctgtcc tccatgtcct 1500

gctccagaag ctgcaggcgg cccttccgtg tttctgttcc ctccaaagcc taaggacacc 1560

ctgatgatca gccggacacc tgaagtgacc tgcgtggtgg tggatgtgtc ccacgaggat 1620

cccgaagtga agttcaattg gtacgtggac ggcgtggaag tgcacaacgc caagaccaag 1680

cctagagagg aacagtacaa cagcacctac agagtggtgt ctgtgctgac agtgctgcac 1740

caggactggc tgaacggcaa agagtacaag tgcaaggtgt ccaacaaggc cctgcctgct 1800

cctatcgaga aaaccatcag caaggccaag ggccagccta gggaacccca ggtttacacc 1860

ctgcctccat gcagggatga gctgaccaag aaccaggtgt ccctgtggtg cctggttaag 1920

ggcttctacc cctccgatat cgccgtggaa tgggagagca atggccagcc agagaacaac 1980

tacaagacaa cccctcctgt gctggactcc gacggctcat tcttcctgta cagcaagctg 2040

accgtggaca agagcagatg gcagcagggc aacgtgttca gctgcagcgt gatgcacgag 2100

gccctgcaca accactacac acagaagtcc ctgtctctga gccccggcaa a 2151

<210> 28

<211> 2139

<212> DNA

<213> Artificial (Artifical)

<220>

<223> full-length nucleotide sequence of RX3-0002-HC_k

<400> 28

atgaagcacc tgtggttctt tctgctgctg gtggccgctc ctagatgggt gctgtctgga 60

caggtgcagc tggtgcagtc tggcgccgaa gtgaagaaac caggcgccag cgtgaaggtg 120

tcctgcaagg ccagcggcta cacctttacc ggctactaca tgcactgggt gcgccaggct 180

ccaggccagg gactggaatg gatgggccgg atcaacccca atagcggcgg caccaactac 240

gcccagaaat tccagggcag agtgaccatg acccgggaca ccagcatcag caccgcctac 300

atggaactga gcggcctgag aagcgacgac accgccgtgt actactgtgc ctctggctct 360

gtgcggcacc cttggggaca gggaacactc gtgaccgtgt ccagcggtgg aggcggttca 420

ggcggaggtg gcagcggcgg tggcgggagt cagcctgtgc tgacacagcc tccaagcgcc 480

tctggcacac ctggccagag agtgacaatc agctgcagcg gcagcagaag caacatcggc 540

ggcaacatcg tgtcctggta tcagcagttc cccggcaccg cccctagact gctgacctac 600

gccgacaacc agaggcctag cggcgtgccc gatagattca gcggctctaa gagcggcacc 660

agcgccagcc tggccatctc tggcctgcag tctgaggacg aggccgacta ctattgcgcc 720

gcctgggacg acagcctgaa cggcgtggtg tttggcggag gcaccaagct gacagtgctg 780

ggaggcggag gatctgaggt gcagcttgtt gaatcaggtg gcggacttgt gcagcctggc 840

ggaagcctca gacttagctg tgctgcttcc ggcgtgacct tcaactacta cggcatgagc 900

tggatcaggc aggcacctgg aaagggcctc gagtgggttg cctctatcac aagaagcggc 960

ggcagaatct actaccccga ctctgtgaaa ggccggttca ccatctccag agagaacacc 1020

cagaaaacac tctatctcca gatgaactct ctgcgcgccg aagatacagc cgtctattac 1080

tgcaccctgg acggcagaga tggctgggtt gcatattggg gacagggaac cctcgtgacc 1140

gtgtcctctg cctctacaaa gggccctagc gtgttccctc tggctcctag cagcaagtct 1200

accagcggag gaacagccgc tctgggctgt ctggtcaagg actactttcc cgagccagtg 1260

acagtgtcct ggaactctgg cgctctgaca tccggcgtgc acacatttcc agccgtgctg 1320

caaagctccg gcctgtactc tctgagcagc gtggtcacag tgccaagctc tagcctgggc 1380

acccagacct acatctgcaa tgtgaaccac aagcctagca acaccaaggt cgacaagaga 1440

gtggaaccca agagctgcga caagacccac acctgtcctc catgtcctgc tccagaagct 1500

gcaggcggcc cttccgtgtt tctgttccct ccaaagccta aggacaccct gatgatcagc 1560

cggacacctg aagtgacctg cgtggtggtg gatgtgtccc acgaggatcc cgaagtgaag 1620

ttcaattggt acgtggacgg cgtggaagtg cacaacgcca agaccaagcc tagagaggaa 1680

cagtacaaca gcacctacag agtggtgtct gtgctgacag tgctgcacca ggactggctg 1740

aacggcaaag agtacaagtg caaggtgtcc aacaaggccc tgcctgctcc tatcgagaaa 1800

accatcagca aggccaaggg ccagcctagg gaaccccagg tttacaccct gcctccatgc 1860

agggatgagc tgaccaagaa ccaggtgtcc ctgtggtgcc tggttaaggg cttctacccc 1920

tccgatatcg ccgtggaatg ggagagcaat ggccagccag agaacaacta caagacaacc 1980

cctcctgtgc tggactccga cggctcattc ttcctgtaca gcaagctgac cgtggacaag 2040

agcagatggc agcagggcaa cgtgttcagc tgcagcgtga tgcacgaggc cctgcacaac 2100

cactacacac agaagtccct gtctctgagc cccggcaaa 2139

<210> 29

<211> 718

<212> PRT

<213> Artificial (Artifical)

<220>

<223> full-length amino acid sequence of TX3-0002-HC_k

<400> 29

Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp

1 5 10 15

Val Leu Ser Gly Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys

20 25 30

Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr

35 40 45

Phe Thr Thr Ala Gly Met Gln Trp Val Arg Gln Ala Pro Gly Gln Gly

50 55 60

Leu Glu Trp Met Gly Trp Ile Asn Thr His Ser Gly Val Pro Lys Tyr

65 70 75 80

Ala Glu Asp Phe Lys Gly Arg Val Thr Ile Ser Ala Asp Thr Ser Thr

85 90 95

Ser Thr Ala Tyr Leu Gln Leu Ser Ser Leu Lys Ser Glu Asp Thr Ala

100 105 110

Val Tyr Tyr Cys Ala Arg Ser Gly Phe Gly Ser Ser Tyr Trp Tyr Phe

115 120 125

Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly

130 135 140

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met

145 150 155 160

Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr

165 170 175

Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Thr Ala Val Ala Trp Tyr

180 185 190

Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser

195 200 205

Tyr Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly

210 215 220

Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala

225 230 235 240

Val Tyr Tyr Cys Gln Gln His Tyr Ile Thr Pro Leu Thr Phe Gly Gln

245 250 255

Gly Thr Lys Leu Glu Ile Lys Arg Thr Gly Gly Gly Gly Ser Glu Val

260 265 270

Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu

275 280 285

Arg Leu Ser Cys Ala Ala Ser Gly Val Thr Phe Asn Tyr Tyr Gly Met

290 295 300

Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Ser

305 310 315 320

Ile Thr Arg Ser Gly Gly Arg Ile Tyr Tyr Pro Asp Ser Val Lys Gly

325 330 335

Arg Phe Thr Ile Ser Arg Glu Asn Thr Gln Lys Thr Leu Tyr Leu Gln

340 345 350

Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Thr Leu

355 360 365

Asp Gly Arg Asp Gly Trp Val Ala Tyr Trp Gly Gln Gly Thr Leu Val

370 375 380

Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala

385 390 395 400

Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu

405 410 415

Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly

420 425 430

Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser

435 440 445

Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu

450 455 460

Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr

465 470 475 480

Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr

485 490 495

Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe

500 505 510

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

515 520 525

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

530 535 540

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

545 550 555 560

Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val

565 570 575

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

580 585 590

Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser

595 600 605

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

610 615 620

Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val

625 630 635 640

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

645 650 655

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

660 665 670

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

675 680 685

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

690 695 700

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

705 710 715

<210> 30

<211> 717

<212> PRT

<213> Artificial (Artifical)

<220>

<223> full-length amino acid sequence of CX3-0002-HC_k

<400> 30

Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp

1 5 10 15

Val Leu Ser Gly Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys

20 25 30

Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala

35 40 45

Phe Ser Asn Tyr Leu Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly

50 55 60

Leu Glu Trp Met Gly Val Ile Asn Pro Gly Ser Gly Val Thr Asn Tyr

65 70 75 80

Asn Glu Lys Phe Lys Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr

85 90 95

Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala

100 105 110

Val Tyr Tyr Cys Ala Arg Ala Glu Ala Trp Phe Ala Tyr Trp Gly Gln

115 120 125

Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly

130 135 140

Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro Asp

145 150 155 160

Ser Leu Ala Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ser

165 170 175

Ser Gln Ser Leu Leu Tyr Ser Ser Asn Gln Lys Asn Tyr Leu Ala Trp

180 185 190

Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala

195 200 205

Ser Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser

210 215 220

Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val

225 230 235 240

Ala Val Tyr Tyr Cys Gln Arg Tyr Tyr Gly Tyr Pro Trp Thr Phe Gly

245 250 255

Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser Glu Val Gln

260 265 270

Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg

275 280 285

Leu Ser Cys Ala Ala Ser Gly Val Thr Phe Asn Tyr Tyr Gly Met Ser

290 295 300

Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Ser Ile

305 310 315 320

Thr Arg Ser Gly Gly Arg Ile Tyr Tyr Pro Asp Ser Val Lys Gly Arg

325 330 335

Phe Thr Ile Ser Arg Glu Asn Thr Gln Lys Thr Leu Tyr Leu Gln Met

340 345 350

Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Thr Leu Asp

355 360 365

Gly Arg Asp Gly Trp Val Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr

370 375 380

Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro

385 390 395 400

Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val

405 410 415

Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala

420 425 430

Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly

435 440 445

Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly

450 455 460

Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys

465 470 475 480

Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys

485 490 495

Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu

500 505 510

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

515 520 525

Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys

530 535 540

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

545 550 555 560

Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu

565 570 575

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

580 585 590

Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys

595 600 605

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys

610 615 620

Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys

625 630 635 640

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

645 650 655

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

660 665 670

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

675 680 685

Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

690 695 700

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

705 710 715

<210> 31

<211> 713

<212> PRT

<213> Artificial (Artifical)

<220>

<223> full Length amino acid sequence of RX3-0002-HC_k

<400> 31

Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp

1 5 10 15

Val Leu Ser Gly Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys

20 25 30

Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr

35 40 45

Phe Thr Gly Tyr Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly

50 55 60

Leu Glu Trp Met Gly Arg Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr

65 70 75 80

Ala Gln Lys Phe Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile

85 90 95

Ser Thr Ala Tyr Met Glu Leu Ser Gly Leu Arg Ser Asp Asp Thr Ala

100 105 110

Val Tyr Tyr Cys Ala Ser Gly Ser Val Arg His Pro Trp Gly Gln Gly

115 120 125

Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

130 135 140

Ser Gly Gly Gly Gly Ser Gln Pro Val Leu Thr Gln Pro Pro Ser Ala

145 150 155 160

Ser Gly Thr Pro Gly Gln Arg Val Thr Ile Ser Cys Ser Gly Ser Arg

165 170 175

Ser Asn Ile Gly Gly Asn Ile Val Ser Trp Tyr Gln Gln Phe Pro Gly

180 185 190

Thr Ala Pro Arg Leu Leu Thr Tyr Ala Asp Asn Gln Arg Pro Ser Gly

195 200 205

Val Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu

210 215 220

Ala Ile Ser Gly Leu Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala

225 230 235 240

Ala Trp Asp Asp Ser Leu Asn Gly Val Val Phe Gly Gly Gly Thr Lys

245 250 255

Leu Thr Val Leu Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser

260 265 270

Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala

275 280 285

Ala Ser Gly Val Thr Phe Asn Tyr Tyr Gly Met Ser Trp Ile Arg Gln

290 295 300

Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Ser Ile Thr Arg Ser Gly

305 310 315 320

Gly Arg Ile Tyr Tyr Pro Asp Ser Val Lys Gly Arg Phe Thr Ile Ser

325 330 335

Arg Glu Asn Thr Gln Lys Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg

340 345 350

Ala Glu Asp Thr Ala Val Tyr Tyr Cys Thr Leu Asp Gly Arg Asp Gly

355 360 365

Trp Val Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala

370 375 380

Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser

385 390 395 400

Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe

405 410 415

Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly

420 425 430

Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu

435 440 445

Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr

450 455 460

Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg

465 470 475 480

Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro

485 490 495

Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

500 505 510

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

515 520 525

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

530 535 540

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

545 550 555 560

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

565 570 575

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

580 585 590

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

595 600 605

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu

610 615 620

Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro

625 630 635 640

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

645 650 655

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

660 665 670

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val

675 680 685

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

690 695 700

Lys Ser Leu Ser Leu Ser Pro Gly Lys

705 710

<210> 32

<211> 8

<212> PRT

<213> Artificial (Artifical)

<220>

<223> amino acid sequence of C3E-7085 heavy chain CDR1

<400> 32

Gly Val Thr Phe Asn Tyr Tyr Gly

1 5

<210> 33

<211> 8

<212> PRT

<213> Artificial (Artifical)

<220>

<223> amino acid sequence of C3E-7085 heavy chain CDR2

<400> 33

Ile Thr Arg Ser Gly Gly Arg Ile

1 5

<210> 34

<211> 11

<212> PRT

<213> Artificial (Artifical)

<220>

<223> amino acid sequence of C3E-7085 heavy chain CDR3

<400> 34

Thr Leu Asp Gly Arg Asp Gly Trp Val Ala Tyr

1 5 10

<210> 35

<211> 8

<212> PRT

<213> Artificial (Artifical)

<220>

<223> amino acid sequence of C3E-7085 light chain CDR1

<400> 35

Thr Gly Asn Ile Gly Ser Asn Tyr

1 5

<210> 36

<211> 3

<212> PRT

<213> Artificial (Artifical)

<220>

<223> amino acid sequence of C3E-7085 light chain CDR2

<400> 36

Arg Asp Asp

1

<210> 37

<211> 8

<212> PRT

<213> Artificial (Artifical)

<220>

<223> amino acid sequence of C3E-7085 light chain CDR3

<400> 37

Gln Ser Tyr Ser Ser Gly Phe Ile

1 5

<210> 38

<211> 5

<212> PRT

<213> Artificial (Artifical)

<220>

<223> peptide linker

<400> 38

Gly Gly Gly Gly Ser

1 5

<210> 39

<211> 240

<212> PRT

<213> Artificial (Artifical)

<220>

<223> amino acid sequence of C3E-7097

<400> 39

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Val Thr Phe Asn Tyr Tyr

20 25 30

Gly Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Cys Leu Glu Trp Val

35 40 45

Ala Ser Ile Thr Arg Ser Gly Gly Arg Ile Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Glu Asn Thr Gln Lys Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Leu Asp Gly Arg Asp Gly Trp Val Ala Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Gly Gly Gly Gly Ser Asn Phe Met Leu Thr Gln Pro Ser Ser Val Ser

130 135 140

Gly Val Pro Gly Gln Arg Val Thr Ile Ser Cys Thr Gly Asn Thr Gly

145 150 155 160

Asn Ile Gly Ser Asn Tyr Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr

165 170 175

Ala Pro Lys Leu Leu Ile Tyr Arg Asp Asp Lys Arg Pro Ser Gly Val

180 185 190

Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala

195 200 205

Ile Thr Gly Phe Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser

210 215 220

Tyr Ser Ser Gly Phe Ile Phe Gly Cys Gly Thr Lys Leu Thr Val Leu

225 230 235 240

<210> 40

<211> 8

<212> PRT

<213> Artificial (Artifical)

<220>

<223> amino acid sequence of C3E-7093 heavy chain CDR1

<400> 40

Gly Val Thr Phe Asn Tyr Tyr Gly

1 5

<210> 41

<211> 8

<212> PRT

<213> Artificial (Artifical)

<220>

<223> amino acid sequence of C3E-7093 heavy chain CDR2

<400> 41

Ile Thr Ser Ser Gly Gly Arg Ile

1 5

<210> 42

<211> 11

<212> PRT

<213> Artificial (Artifical)

<220>

<223> amino acid sequence of C3E-7093 heavy chain CDR3

<400> 42

Thr Leu Asp Gly Arg Asp Gly Trp Val Ala Tyr

1 5 10

<210> 43

<211> 8

<212> PRT

<213> Artificial (Artifical)

<220>

<223> amino acid sequence of C3E-7093 light chain CDR1

<400> 43

Thr Gly Asn Ile Gly Ser Asn Tyr

1 5

<210> 44

<211> 3

<212> PRT

<213> Artificial (Artifical)

<220>

<223> amino acid sequence of C3E-7093 light chain CDR2

<400> 44

Arg Asn Asp

1

<210> 45

<211> 8

<212> PRT

<213> Artificial (Artifical)

<220>

<223> amino acid sequence of C3E-7093 light chain CDR3

<400> 45

Gln Ser Tyr Ser Ser Gly Phe Ile

1 5

<210> 46

<211> 216

<212> PRT

<213> Artificial (Artifical)

<220>

<223> amino acid sequence of C3E-7093-Fab

<400> 46

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Val Thr Phe Asn Tyr Tyr

20 25 30

Gly Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Ser Ile Thr Ser Ser Gly Gly Arg Ile Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Glu Asn Thr Gln Lys Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Leu Asp Gly Arg Asp Gly Trp Val Ala Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Arg Val

210 215

<210> 47

<211> 215

<212> PRT

<213> Artificial (Artifical)

<220>

<223> amino acid sequence of C3E-7093-LC

<400> 47

Asn Phe Met Leu Thr Gln Pro His Ser Val Ser Glu Ser Pro Gly Lys

1 5 10 15

Thr Val Thr Ile Ser Cys Lys Arg Asn Thr Gly Asn Ile Gly Ser Asn

20 25 30

Tyr Val Asn Trp Tyr Gln Gln His Glu Gly Ser Ser Pro Thr Thr Ile

35 40 45

Ile Tyr Arg Asn Asp Lys Arg Pro Asp Gly Val Ser Asp Arg Phe Ser

50 55 60

Gly Ser Ile Asp Arg Ser Ser Lys Ser Ala Ser Leu Thr Ile Ser Asn

65 70 75 80

Leu Lys Thr Glu Asp Glu Ala Asp Tyr Phe Cys Gln Ser Tyr Ser Ser

85 90 95

Gly Phe Ile Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro

100 105 110

Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu

115 120 125

Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro

130 135 140

Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala

145 150 155 160

Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala

165 170 175

Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg

180 185 190

Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr

195 200 205

Val Ala Pro Thr Glu Cys Ser

210 215

<210> 48

<211> 242

<212> PRT

<213> Artificial (Artifical)

<220>

<223> amino acid sequence of C3E-7098

<400> 48

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Val Thr Phe Asn Tyr Tyr

20 25 30

Gly Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Ser Ile Thr Ser Ser Gly Gly Arg Ile Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Glu Asn Thr Gln Lys Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Leu Asp Gly Arg Asp Gly Trp Val Ala Tyr Trp Gly Cys Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Gly Gly Gly Gly Ser Asn Phe Met Leu Thr Gln Pro His Ser Val Ser

130 135 140

Glu Ser Pro Gly Lys Thr Val Thr Ile Ser Cys Lys Arg Asn Thr Gly

145 150 155 160

Asn Ile Gly Ser Asn Tyr Val Asn Trp Tyr Gln Gln His Glu Gly Ser

165 170 175

Cys Pro Thr Thr Ile Ile Tyr Arg Asn Asp Lys Arg Pro Asp Gly Val

180 185 190

Ser Asp Arg Phe Ser Gly Ser Ile Asp Arg Ser Ser Lys Ser Ala Ser

195 200 205

Leu Thr Ile Ser Asn Leu Lys Thr Glu Asp Glu Ala Asp Tyr Phe Cys

210 215 220

Gln Ser Tyr Ser Ser Gly Phe Ile Phe Gly Gly Gly Thr Lys Leu Thr

225 230 235 240

Val Leu

<210> 49

<211> 242

<212> PRT

<213> Artificial (Artifical)

<220>

<223> amino acid sequence of C3E-7099

<400> 49

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Val Thr Phe Asn Tyr Tyr

20 25 30

Gly Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Cys Leu Glu Trp Val

35 40 45

Ala Ser Ile Thr Ser Ser Gly Gly Arg Ile Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Glu Asn Thr Gln Lys Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Leu Asp Gly Arg Asp Gly Trp Val Ala Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Gly Gly Gly Gly Ser Asn Phe Met Leu Thr Gln Pro His Ser Val Ser

130 135 140

Glu Ser Pro Gly Lys Thr Val Thr Ile Ser Cys Lys Arg Asn Thr Gly

145 150 155 160

Asn Ile Gly Ser Asn Tyr Val Asn Trp Tyr Gln Gln His Glu Gly Ser

165 170 175

Ser Pro Thr Thr Ile Ile Tyr Arg Asn Asp Lys Arg Pro Asp Gly Val

180 185 190

Ser Asp Arg Phe Ser Gly Ser Ile Asp Arg Ser Ser Lys Ser Ala Ser

195 200 205

Leu Thr Ile Ser Asn Leu Lys Thr Glu Asp Glu Ala Asp Tyr Phe Cys

210 215 220

Gln Ser Tyr Ser Ser Gly Phe Ile Phe Gly Cys Gly Thr Lys Leu Thr

225 230 235 240

Val Leu

Claims

1. A Fab which specifically binds to CD3 and comprises the following CDRH1 to 3 and CDRL1 to 3:

CDRH1 consisting of the amino acid sequence shown in SEQ ID NO. 32;

CDRH2 consisting of the amino acid sequence shown in SEQ ID NO. 33;

CDRH3 composed of the amino acid sequence shown in SEQ ID NO. 34;

CDRL1 composed of the amino acid sequence shown in SEQ ID NO. 35;

CDRL3 consisting of the amino acid sequence shown in SEQ ID NO. 37.

2. The Fab of claim 1, which comprises 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.

3. The Fab of claim 1 or 2, which contains a polypeptide consisting of the amino acid sequence shown in SEQ ID No. 1, and a light chain consisting of the amino acid sequence shown in SEQ ID No. 2.

4. A multispecific molecule comprising a Fab according to any one of claims 1 to 3 which specifically binds to a target molecule which is not a complex of a cancer testis antigenic peptide and a Major Histocompatibility Complex (MHC).

5. The multispecific molecule of claim 4, wherein the MHC is a Human Leukocyte Antigen (HLA).

6. The multispecific molecule of claim 4 or 5, which contains an antibody or antigen-binding fragment thereof that specifically binds to the target molecule.

7. The multispecific molecule according to any one of claims 4 to 6 which is a multispecific antibody.

8. The multispecific molecule according to any one of claims 4 to 7 which contains scFv-Fab-heterodimeric Fc.

9. The multispecific molecule according to any one of claims 4 to 8 which is a trispecific antibody.

10. The multispecific molecule according to any one of claims 4 to 8 which is a bispecific antibody.

11. The multi-specific molecule according to any one of claims 4 to 10, wherein the target molecule is GPRC5D, CD or Trop2.

12. The multispecific molecule according to any one of claims 4 to 11 which contains one polypeptide group selected from the following groups (i) to (iii):

13. A polynucleotide comprising a base sequence encoding an amino acid sequence contained in the multispecific molecule of any one of claims 4 to 12.

14. A vector comprising the polynucleotide of claim 13.

15. A cell comprising the polynucleotide of claim 13 or the vector of claim 14, or producing the multispecific molecule of any one of claims 4 to 12.

16. A method of preparing a multispecific molecule, the multispecific molecule being a multispecific molecule according to any one of claims 4 to 12, the method comprising the steps of: culturing the cell of claim 15.

17. A multispecific molecule obtainable by the method of claim 16.

18. A composition comprising: the Fab of any one of claims 1 to 3; a multispecific molecule according to any one of claims 4 to 12 or claim 17; the polynucleotide of claim 13; the vector of claim 14; alternatively, the cell of claim 15.

19. An scFv formed by disulfide bonding of a heavy chain variable region and a light chain variable region, specifically binding to CD3, comprising the following CDRH1 to 3 and CDRL1 to 3:

CDRH1 consisting of the amino acid sequence shown in SEQ ID NO. 32;

CDRH2 consisting of the amino acid sequence shown in SEQ ID NO. 33;

CDRH3 composed of the amino acid sequence shown in SEQ ID NO. 34;

CDRL1 composed of the amino acid sequence shown in SEQ ID NO. 35;

CDRL3 consisting of the amino acid sequence shown in SEQ ID NO. 37.

20. The scFv according to claim 19 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.

21. The scFv according to claim 19 or 20 consisting of the amino acid sequence shown in SEQ ID No. 3.

22. The scFv according to claim 19 comprising a heavy chain variable region comprising amino acid numbers 1 to 118 of the amino acid sequence shown in SEQ ID No. 39 and a light chain variable region comprising amino acid numbers 134 to 240 of the amino acid sequence shown in SEQ ID No. 39.

23. The scFv according to claim 19 or 22 consisting of the amino acid sequence shown in SEQ ID No. 39.

24. A multispecific molecule comprising the scFv of any one of claims 19-23 that specifically binds to a target molecule that is not a complex of a cancer testis antigenic peptide and a Major Histocompatibility Complex (MHC).

25. The multispecific molecule of claim 24, wherein the MHC is a Human Leukocyte Antigen (HLA).

26. The multispecific molecule of claim 24 or 25, which contains an antibody or antigen-binding fragment thereof that specifically binds to the target molecule.

27. The multispecific molecule according to any one of claims 24 to 26 which is a multispecific antibody.

28. The multispecific molecule according to any one of claims 24 to 27 which contains taFv-heterodimeric Fc.

29. The multispecific molecule according to any one of claims 24 to 28 which is a trispecific antibody.

30. The multispecific molecule according to any one of claims 24 to 28 which is a bispecific antibody.

31. The multispecific molecule of any one of claims 24 to 30, wherein the target molecule is GPRC5D, CD or Trop2.

32. The multispecific molecule according to any one of claims 24 to 31 which comprises one polypeptide pair selected from the following (i) to (iii):

33. A polynucleotide comprising a base sequence encoding an amino acid sequence contained in the multispecific molecule of any one of claims 24 to 32.

34. A vector comprising the polynucleotide of claim 33.

35. A cell comprising the polynucleotide of claim 33 or the vector of claim 34, or producing the multispecific molecule of any one of claims 24 to 32.

36. A method of preparing a multispecific molecule, the multispecific molecule being a multispecific molecule according to any one of claims 24 to 32, the method comprising the steps of: culturing the cell of claim 35.

37. A multispecific molecule obtainable by the method of claim 36.

38. A composition comprising: the scFv of any one of claims 19-23; a multispecific molecule according to any one of claims 24 to 32 or claim 37; the polynucleotide of claim 33; the vector of claim 34; alternatively, the cell of claim 35.

39. A Fab which specifically binds to CD3 and comprises the following CDRH1 to 3 and CDRL1 to 3:

CDRH1 composed of the amino acid sequence shown in SEQ ID NO. 40;

CDRH2 composed of the amino acid sequence shown in SEQ ID NO. 41;

CDRH3 composed of the amino acid sequence shown in SEQ ID NO. 42;

CDRL1 composed of the amino acid sequence shown in SEQ ID NO. 43;

CDRL3 consisting of the amino acid sequence shown in SEQ ID NO. 45.

40. The Fab of claim 39, which comprises 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.

41. The Fab according to claim 39 or 40, which comprises a polypeptide consisting of the amino acid sequence shown in SEQ ID NO. 46 and a light chain consisting of the amino acid sequence shown in SEQ ID NO. 47.

42. A multispecific molecule comprising the Fab of any one of claims 39-41 which specifically binds to a target molecule which is not a complex of a cancer testis antigenic peptide and a Major Histocompatibility Complex (MHC).

43. The multispecific molecule of claim 42 wherein the MHC is a Human Leukocyte Antigen (HLA).

44. The multispecific molecule of claim 42 or 43, which comprises an antibody or antigen-binding fragment thereof that specifically binds to the target molecule.

45. The multispecific molecule of any one of claims 42 to 44 which is a multispecific antibody.

46. The multispecific molecule of any one of claims 42-45 which is a bispecific antibody.

47. A polynucleotide comprising a nucleotide sequence encoding an amino acid sequence contained in the multispecific molecule according to any one of claims 42 to 46.

48. A vector comprising the polynucleotide of claim 47.

49. A cell comprising the polynucleotide of claim 47 or the vector of claim 48, or producing the multispecific molecule of any one of claims 42 to 46.

50. A method of preparing a multispecific molecule, the multispecific molecule being a multispecific molecule according to any one of claims 34 to 38, the method comprising the steps of: the cell of claim 49 is cultured.

51. A multi-specific molecule obtainable by the method of claim 50.

52. A composition comprising: the Fab of any one of claims 39-41; the multispecific molecule of any one of claims 42 to 46; the polynucleotide of claim 47; the vector of claim 48; alternatively, the cell of claim 49.

53. An scFv formed by disulfide bonding of a heavy chain variable region and a light chain variable region, specifically binding to CD3, comprising the following CDRH1 to 3 and CDRL1 to 3:

CDRH1 composed of the amino acid sequence shown in SEQ ID NO. 40;

CDRH2 composed of the amino acid sequence shown in SEQ ID NO. 41;

CDRH3 composed of the amino acid sequence shown in SEQ ID NO. 42;

CDRL1 composed of the amino acid sequence shown in SEQ ID NO. 43;

CDRL3 consisting of the amino acid sequence shown in SEQ ID NO. 45.

54. The scFv according to claim 53, which comprises a heavy chain variable region comprising amino acid numbers 1 to 118 of the amino acid sequence shown in SEQ ID NO. 48, and a light chain variable region comprising amino acid numbers 134 to 242 of the amino acid sequence shown in SEQ ID NO. 48.

55. The scFv according to claim 53 or 54, which consists of the amino acid sequence shown in SEQ ID NO. 48.

56. The scFv according to claim 53, which comprises a heavy chain variable region comprising amino acid numbers 1 to 118 of the amino acid sequence shown in SEQ ID NO. 49 and a light chain variable region comprising amino acid numbers 134 to 242 of the amino acid sequence shown in SEQ ID NO. 49.

57. The scFv according to claim 53 or 56, which consists of the amino acid sequence shown in SEQ ID NO. 49.

58. A multispecific molecule comprising the scFv of any one of claims 53-57 that specifically binds to a target molecule that is not a complex of a cancer testis antigenic peptide and a Major Histocompatibility Complex (MHC).

59. The multispecific molecule of claim 58, wherein the MHC is a Human Leukocyte Antigen (HLA).

60. The multispecific molecule of claim 58 or 59, which contains an antibody or antigen-binding fragment thereof that specifically binds to the target molecule.

61. The multispecific molecule of any one of claims 58 to 60 which is a multispecific antibody.

62. The multispecific molecule of any one of claims 58 to 61 which is a bispecific antibody.

63. A polynucleotide comprising a nucleotide sequence encoding an amino acid sequence contained in the multispecific molecule of any one of claims 58 to 62.

64. A vector comprising the polynucleotide of claim 63.

65. A cell comprising the polynucleotide of claim 63 or the vector of claim 64, or producing the multispecific molecule of any one of claims 58-62.

66. A method of preparing a multispecific molecule which is a multispecific molecule according to any one of claims 58 to 62, the method comprising the steps of: the cell of claim 65 is cultured.

67. A multispecific molecule obtainable by the method of claim 66.

68. A composition comprising: the scFv of any one of claims 53-57; a multispecific molecule according to any one of claims 58 to 62; the polynucleotide of claim 63; the vector of claim 64; alternatively, the cell of claim 65.

69. A pharmaceutical composition comprising a multispecific molecule according to any one of claims 4 to 12, 24 to 32, 42 to 46 and 58 to 62.