JP2019104699A - Bispecific antibody - Google Patents

Abstract

To provide bispecific antibodies having cytotoxic activity which comprise an immunoglobulin variable region of epidermal growth factor receptor (EGFR) specific antibody as a cancer cell specific antibody, and a CD3 specific antibody immunoglobulin variable region as a T cell specific antibody, in one polypeptide chain.SOLUTION: The present invention relates to a bispecific antibody comprising an antibody polypeptide comprising a structure represented by VH1-L1-VL1-L2-VH2-L3-VL2 or the like from the N-terminal side to the C-terminal side, where VH1 represents an amino acid sequence of a heavy chain variable region of anti-EGFR immunoglobulin, VL1 represents an amino acid sequence of a light chain variable region of anti-EGFR immunoglobulin, VH2 represents an amino acid sequence of a heavy chain variable region of anti-CD3 immunoglobulin, VL2 represents an amino acid sequence of a light chain variable region of anti-CD3 immunoglobulin, as well as L1, L2 and L3 each independently represent an amino acid sequence of a linker.SELECTED DRAWING: Figure 3

Description

  The present invention is bispecific having the immunoglobulin variable region of epidermal growth factor receptor (EGFR) specific antibody as a cancer cell specific antibody and the immunoglobulin variable region of CD3 specific antibody as a T cell specific antibody. It relates to an antibody. The present invention also relates to a vector containing a nucleotide sequence encoding the bispecific antibody, a cell containing the vector, and a method for producing a bispecific antibody using the cell.

  In antibody drugs, research and development of low molecular weight antibodies with relatively small molecular weights while maintaining high specificity have been conducted in recent years. For example, a diabody (Diabody) type composed of only variable regions, a single chain Diabody (scDb) type, and a tandem scFv (taFv) in which two single chain Fv (scFv) are linked Low-molecular-type bispecific antibodies, such as type), have been reported (Non-patent Document 1). Bispecific antibodies can be used to cross-link two types of cells, for example, one on an antigen on a cancer tumor cell and the other on a surface antigen on an effector cell (eg, T cell) having strong cytotoxic activity. The combination of variable regions that recognize and activate H. can have a stronger antitumor effect in cancer treatment than existing antibody pharmaceuticals (Non-patent Document 2).

  In general, preparation of bispecific antibodies using two types of antibodies, such as Diabody, results in the formation of byproducts other than the target complex (heterodimer) due to the association of heavy and light chains of different antibodies. . On the other hand, taFv, which is a single-chain bispecific antibody containing variable regions of two types of antibodies, is expected to be less affected by byproducts. Amgen BiTE (bispecific T-cell engager) antibody Blinatumomab links the variable region of the antibody to the CD19 antigen of target B cells and the variable region of the antibody to the CD3 antigen of cytotoxic T cells It is characterized by potent T cell activation via CD3 and is FDA approved (non-patent documents 3 to 5).

H. Byrne et al., Trends in Biotechnology, Nov; 31 (11): 621-32 (2013) R. Bargou et al., Science, 321, 974 (2008) M. Mack et al., PNAS, 92, 7021 (1995) P. A. Baeuerle and C. Reinhardt, Cancer Res., 69, 4941 (2009) M. Klinger et al., Immunol. Rev., 270, 193 (2016)

  The present invention provides a single polypeptide chain of an immunoglobulin variable region of epidermal growth factor receptor (EGFR) specific antibody as a cancer cell specific antibody and a CD3 specific antibody immunoglobulin variable region as a T cell specific antibody. It is an object of the present invention to provide bispecific antibodies having cytotoxic activity, including

  The present inventors have prepared an immunoglobulin variable region of epidermal growth factor receptor (EGFR) specific antibody as a cancer cell specific antibody and an immunoglobulin variable region of a CD3 specific antibody as a T cell specific antibody. A bispecific antibody (taFv) contained in a polypeptide chain was produced and found to have cytotoxic activity, and the present invention has been completed.

That is, the present invention includes the following aspects.
(1) from the amino terminal side to the carboxy terminal side,
Formula 1: VH1-L1-VL1-L2-VH2-L3-VL2,
Formula 2: VH1-L1-VL1-L2-VL2-L3-VH2,
Formula 3: VL1-L1-VH1-L2-VH2-L3-VL2,
Formula 4: VL1-L1-VH1-L2-VL2-L3-VH2,
Formula 5: VH2-L1-VL2-L2-VH1-L3-VL1,
Formula 6: VH2-L1-VL2-L2-VL1-L3-VH1,
Formula 7: VL2-L1-VH2-L2-VH1-L3-VL1 or Formula 8: VL2-L1-VH2-L2-VL1-L3-VH1
Comprising an antibody polypeptide comprising a structure represented by
VH1 represents the amino acid sequence of the heavy chain variable region of anti-EGFR immunoglobulin,
VL1 represents the amino acid sequence of the light chain variable region of anti-EGFR immunoglobulin,
VH2 represents the amino acid sequence of the heavy chain variable region of anti-CD3 immunoglobulin,
VL2 represents the amino acid sequence of the light chain variable region of anti-CD3 immunoglobulin;
A bispecific antibody, wherein L1, L2 and L3 each independently represent the amino acid sequence of a linker.
(2) The bispecific antibody according to (1), wherein the antibody polypeptide comprises a structure represented by Formula 1, Formula 3 or Formula 5.
(3) VH1 is any one of the following amino acid sequences (a) to (c):
VL1 is an amino acid sequence of any one of the following (d) to (f),
VH2 is an amino acid sequence of any of the following (g) to (i),
The bispecific antibody according to (1) or (2), wherein VL2 is an amino acid sequence of any one of the following (j) to (l):
(a) the amino acid sequence shown in SEQ ID NO: 1 or 2;
(b) An amino acid sequence in which one or more amino acids are substituted, deleted and / or added in the amino acid sequence shown in (a) above,
(c) an amino acid sequence having a sequence identity of 85% or more with the amino acid sequence shown in (a) above;
(d) the amino acid sequence shown in SEQ ID NO: 3 or 4;
(e) An amino acid sequence in which one or more amino acids are substituted, deleted and / or added in the amino acid sequence shown in (d) above,
(f) an amino acid sequence having a sequence identity of 85% or more with the amino acid sequence shown in (d) above;
(g) the amino acid sequence shown in SEQ ID NO: 5 or 6;
(h) An amino acid sequence in which one or more amino acids are substituted, deleted and / or added in the amino acid sequence shown in (g) above,
(i) an amino acid sequence having a sequence identity of 85% or more with the amino acid sequence shown in (g) above;
(j) the amino acid sequence shown in SEQ ID NO: 7 or 8;
(k) An amino acid sequence in which one or more amino acids are substituted, deleted and / or added in the amino acid sequence shown in (j) above,
(l) An amino acid sequence having a sequence identity of 85% or more with the amino acid sequence shown in (j) above.
(4) The VH1 contains one or more of the following (VH1-1CDR1), (VH1-1CDR2) and (VH1-1CDR3), and the VL1 has the following (VL1-1CDR1), (VL1-1CDR2) and Or one or more of (V L-1 CDR3) or
VH1 contains one or more of the following (VH1-2 CDR1), (VH1-2 CDR2) and (VH1-2 CDR3), and the VL1 has the following (VL1-2 CDR1), (VL1-2 CDR2) and (VL1-) 2) including one or more of 2) CDR3),
Satisfy the condition 1 and
The VH2 contains one or more of the following (VH2-1 CDR1), (VH2-1 CDR2) and (VH2-1 CDR3), and the VL2 is the following (VL2-1 CDR1), (VL2-1 CDR2) and (VL2-) 1) including one or more of
The VH2 contains one or more of the following (VH2-2 CDR1), (VH2-2 CDR2) and (VH2-2 CDR3), and the VL2 has the following (VL2-2 CDR1), (VL2-2 CDR2) and (VL2-) 2) including one or more of 2) CDR3),
The bispecific antibody according to any one of (1) to (3), which satisfies the following condition 2:
(VH1-1 CDR1) amino acid sequence of positions 31 to 37 of SEQ ID NO: 1,
(VH1-1CDR2) amino acid sequence of the 52nd position to the 67th position of SEQ ID NO: 1,
(VH1-1CDR3) amino acid sequence of positions 100 to 110 of SEQ ID NO: 1,
(VH1-2CDR1) amino acid sequence of positions 29 to 33 of SEQ ID NO: 2,
(VH1-2 CDR2) The amino acid sequence of the 50th to the 59th positions of SEQ ID NO: 2,
(VH1-2CDR3) amino acid sequence of positions 99 to 110 of SEQ ID NO: 2,
(VL1-1 CDR1) amino acid sequence of positions 24 to 34 of SEQ ID NO: 3,
(VL1-1 CDR2) the amino acid sequence of the 50th to 56th positions of SEQ ID NO: 3,
(VL1-1CDR3) amino acid sequence of positions 89 to 97 of SEQ ID NO: 3,
(VL1-2 CDR1) amino acid sequence of positions 28 to 33 of SEQ ID NO: 4,
(VL1-2 CDR2) the amino acid sequence of the 50th to the 53rd positions of SEQ ID NO: 4,
(VL1-2 CDR3) amino acid sequence of positions 91 to 96 of SEQ ID NO: 4,
(VH2-1 CDR1) amino acid sequence of positions 23 to 35 of SEQ ID NO: 5,
(VH2-1 CDR2) The amino acid sequence of the 50th to 66th positions of SEQ ID NO: 5,
(VH2-1 CDR3) amino acid sequence of positions 99 to 108 of SEQ ID NO: 5,
(VH2-2 CDR1) amino acid sequence of positions 31 to 35 of SEQ ID NO: 6,
(VH2-2 CDR2) the amino acid sequence of positions 50 to 66 of SEQ ID NO: 6,
(VH2-2 CDR3) amino acid sequence of positions 99 to 108 of SEQ ID NO: 6,
(VL2-1 CDR1) amino acid sequence of positions 24 to 33 of SEQ ID NO: 7,
(VL2-1 CDR2) the amino acid sequence of the 49th to 55th positions of SEQ ID NO: 7,
(VL2-1 CDR3) the amino acid sequence of positions 88 to 96 of SEQ ID NO: 7,
(VL2-2 CDR1) the amino acid sequence of positions 24 to 33 of SEQ ID NO: 8,
(VL2-2 CDR2) the amino acid sequence of positions 49 to 55 of SEQ ID NO: 8,
(VL2-2 CDR3) The amino acid sequence of positions 88 to 96 of SEQ ID NO: 8.
(5) The bispecific antibody according to any one of (1) to (4), wherein at least one of L1, L2 and L3 is independently any of the following (m) to (o): :
(m) the amino acid sequence shown in SEQ ID NO: 9, 10 or 11;
(n) An amino acid sequence in which one or more amino acids are substituted, deleted and / or added in the amino acid sequence shown in (m) above,
(o) An amino acid sequence having 85% or more of sequence identity with the amino acid sequence shown in (m) above.
(6) The bispecific antibody according to any of (1) to (5), further comprising one or more tags linked to the carboxy terminus of the antibody polypeptide.
(7) The polypeptide is a polypeptide comprising an amino acid sequence of any one of the following (p1) to (r1), and / or a polypeptide comprising an amino acid sequence of any of (p2) to (r2): The bispecific antibody according to (6):
(p1) the amino acid sequence shown in SEQ ID NO: 12,
(q1) An amino acid sequence in which one or more amino acids are substituted, deleted and / or added in the amino acid sequence shown in the above (p1),
(r1) an amino acid sequence having a sequence identity of 85% or more with the amino acid sequence shown in (p1) above,
(p2) the amino acid sequence shown in SEQ ID NO: 13,
(q2) an amino acid sequence in which one or more amino acids are substituted, deleted and / or added in the amino acid sequence shown in the above (p2),
(r2) An amino acid sequence having a sequence identity of 85% or more with the amino acid sequence shown in (p2) above.
(8) The bispecific antibody according to any one of (1) to (7), wherein the antibody polypeptide is a polypeptide comprising an amino acid sequence of any one of the following (s) to (u):
(s) amino acid sequence of positions 3 to 497 of SEQ ID NO: 18; amino acid sequence of positions 3 to 497 of SEQ ID NO: 19; amino acid sequence of positions 3 to 497 of SEQ ID NO: 20; The amino acid sequence of positions 3 to 501 of 21, the amino acid sequence of positions 3 to 501 of SEQ ID NO: 22, the amino acid sequence of positions 3 to 501 of SEQ ID NO: 23, the third position of SEQ ID NO: 24 The amino acid sequence of SEQ ID NO: 50, the amino acid sequence of positions 3 to 501 of SEQ ID NO: 25 or the amino acid sequence of positions 3 to 501 of SEQ ID NO: 26,
(t) An amino acid sequence in which one or more amino acids are substituted, deleted and / or added in the amino acid sequence shown in the above (s),
(u) An amino acid sequence having 85% or more sequence identity with the amino acid sequence shown in (s) above.
(9) The bispecific antibody according to any of (1) to (7), further comprising a secretion signal linked to the amino terminus of the antibody polypeptide.
(10) The bispecific antibody according to (9), which is a polypeptide whose secretion signal comprises the amino acid sequence of any one of the following (v) to (x):
(v) the amino acid sequence shown in SEQ ID NO: 14 or 15;
(w) an amino acid sequence in which one or more amino acids are substituted, deleted and / or added in the amino acid sequence shown in (v) above,
(x) An amino acid sequence having a sequence identity of 85% or more with the amino acid sequence shown in (v) above.
(11) A nucleic acid comprising a base sequence encoding the bispecific antibody according to any one of (1) to (10).
(12) A vector comprising the nucleic acid according to (11).
(13) A cell comprising the vector according to (12).
(14) The cell according to (13), which is a yeast cell, a bacterial cell, a fungal cell, an insect cell, an animal cell or a plant cell.
(15) The cell according to (14), which is a yeast cell that is a methanol-utilizing yeast cell or an animal cell that is a CHO cell.
(16) a culture step of culturing the cell according to any one of (13) to (15), and
A method for producing a bispecific antibody, comprising a recovery step of recovering the bispecific antibody from the culture obtained in the culture step.

  According to the present invention, a single polypeptide chain of an immunoglobulin variable region of epidermal growth factor receptor (EGFR) specific antibody as a cancer cell specific antibody and a CD3 specific antibody immunoglobulin variable region as a T cell specific antibody And a bispecific antibody (taFv) having cytotoxic activity.

FIG. 1 shows the constitution of the amino acid sequence of an unsecreted bispecific antibody polypeptide which is expressed and translated from the vector pUC_Pgap_MFα_ # 218_Taox1_T38473_G418 constructed in Example 1 under the control of a GAP promoter in a host yeast cell. FIG. 2 is a photograph showing the results of SDS-PAGE of the bispecific antibody isolated and purified in Example 4. FIG. 3 is a graph showing the relationship between the concentration of nine constructed bispecific antibodies and the cytotoxicity.

  Hereinafter, the present invention will be described in detail using preferred embodiments.

  In the present invention, the "anti-EGFR immunoglobulin" and the "anti-CD3 immunoglobulin" are respectively immunoglobulin G (IgG), immunoglobulin M (IgM), immunoglobulin D (IgD), immunoglobulin E (IgE), immunoglobulin It may be of any class, such as A (IgA), but is typically immunoglobulin G.

  Immunoglobulins, in their full-length state, are heterotetrameric proteins comprising two heavy chains and two light chains. The heavy and light chains are each composed of a variable region and a constant region involved in antigen binding. In the present specification, the variable region of the light chain may be referred to as VL, and the variable region of the heavy chain may be referred to as VH.

  The source organism of "anti-EGFR immunoglobulin" and "anti-CD3 immunoglobulin" is not particularly limited, but human, chimpanzee, monkey, sheep, goat, pig, rabbit, hamster, guinea pig, mouse, rat, shark, ostrich, chicken, Frogs are preferred, with humans, chimpanzees, monkeys, hamsters, mice and rats being particularly preferred. The amino acid sequences of the heavy chain variable region and light chain variable region of each immunoglobulin are not limited to naturally occurring amino acid sequences, and may be amino acid sequences modified from naturally occurring amino acid sequences.

  In the present invention, a "bispecific antibody" is an antibody having the ability to bind to two types of specific antigens, and specifically, two single-chain Fv (scFv) are linked to It is a tandem scFv (taFv) type antibody that has been denatured.

  In the present invention, "polypeptide" refers to peptide bonds of two or more amino acids, and includes proteins, as well as short chain length peptides called peptides and oligopeptides.

  In the present invention, a polypeptide that functions as an antibody that specifically binds to two antigens, which is included in a bispecific antibody, may be referred to as an "antibody polypeptide".

  "Amino terminal" refers to the terminal end of the polypeptide where the terminal is the amino group, also referred to as the N terminal.

  The term "carboxy terminus" refers to the terminus at which the terminus of the polypeptide is a carboxyl group, also referred to as the C terminus.

  In the present invention, "EGFR" refers to an epidermal growth factor receptor, a membrane protein that recognizes and / or binds to epidermal growth factor (EGF) and / or induces intracellular signal transduction by binding, and / or Receptor proteins and / or their soluble proteins.

  In the present invention, “CD3” refers to cluster of differentiation 3 and is a monomer and / or multimeric protein complex composed of multiple types of polypeptide chains.

  EGFR and CD3 serving as antigens are not limited to EGFR and CD3 of a specific species, but are preferably mammalian EGFR and CD3 such as human, chimpanzee, monkey, hamster, mouse, rat etc., more preferably human EGFR and CD3.

  In the present invention, “linker” refers to a polypeptide that links between adjacent pairs of multiple variable regions having the ability to bind to a specific antigen that constitutes an antibody.

  In the present invention, "single-stranded" refers to a state in which a protein is not in a complex with a plurality of polypeptide chains but in a structure with a single polypeptide chain.

In the bispecific antibody of the present invention, from the amino terminal side to the carboxy terminal side,
Formula 1: VH1-L1-VL1-L2-VH2-L3-VL2,
Formula 2: VH1-L1-VL1-L2-VL2-L3-VH2,
Formula 3: VL1-L1-VH1-L2-VH2-L3-VL2,
Formula 4: VL1-L1-VH1-L2-VL2-L3-VH2,
Formula 5: VH2-L1-VL2-L2-VH1-L3-VL1,
Formula 6: VH2-L1-VL2-L2-VL1-L3-VH1,
Formula 7: VL2-L1-VH2-L2-VH1-L3-VL1 or Formula 8: VL2-L1-VH2-L2-VL1-L3-VH1
Comprising a single-chain antibody polypeptide comprising a structure represented by
VH1 represents the amino acid sequence of the heavy chain variable region of anti-EGFR immunoglobulin,
VL1 represents the amino acid sequence of the light chain variable region of anti-EGFR immunoglobulin,
VH2 represents the amino acid sequence of the heavy chain variable region of anti-CD3 immunoglobulin,
VL2 represents the amino acid sequence of the light chain variable region of anti-CD3 immunoglobulin;
L1, L2 and L3 are each a bispecific antibody which independently represents the amino acid sequence of the linker.

  The bispecific antibody may comprise the antibody polypeptide, and the antibody polypeptide may further have another substance linked and integrated, for example, the amino terminus and / or carboxy of the antibody polypeptide. Another polypeptide may be linked to the end to constitute a single polypeptide chain as a whole. As other polypeptide, the tag mentioned later, a secretion signal, etc. can be illustrated. In addition, a pharmaceutical compound may be further linked to the antibody polypeptide. As a pharmaceutical compound, an anticancer drug compound etc. can be illustrated.

  It is particularly preferred that the antibody polypeptide comprises a structure represented by Formula 1, 3 or 5 and most preferably a structure represented by Formula 3 or 5. The antibody polypeptide comprising the structure represented by Formula 1, Formula 3 or Formula 5 has particularly high cytotoxic activity, and the antibody polypeptide comprising the structure represented by Formula 3 or 5 has the highest cytotoxic activity.

  VH1 is not particularly limited as long as it has an ability to bind to EGFR when combined with VL1, but is particularly preferably the amino acid sequence of any of the above (a) to (c), most preferably It is an amino acid sequence of said (a).

  There are no particular limitations on VL1 as long as it has the ability to bind to EGFR when combined with VH1, but is particularly preferably the amino acid sequence of any of the above (d) to (f), most preferably It is an amino acid sequence of said (d).

  Here, when the “amino acid sequence shown in SEQ ID NO: 1 or 2” in (a) is “the amino acid sequence shown in SEQ ID NO: 1”, the “amino acid sequence shown in SEQ ID NO: 3 or 4” in (d) above. Is preferably “the amino acid sequence shown in SEQ ID NO: 3”, and when “the amino acid sequence shown in SEQ ID NO: 1 or 2” in (a) is “the amino acid sequence shown in SEQ ID NO: 2” Preferably, the “amino acid sequence shown in SEQ ID NO: 3 or 4” is the “amino acid sequence shown in SEQ ID NO: 4”.

  The above (a) is most preferably the amino acid sequence shown in SEQ ID NO: 1, and the above (d) is most preferably the amino acid sequence shown in SEQ ID NO: 3.

  VH2 is not particularly limited as long as it has the ability to bind to CD3 when combined with VL2, but is particularly preferably the amino acid sequence of any of (g) to (i) above, most preferably It is an amino acid sequence of said (g).

  The VL2 is not particularly limited as long as it has the ability to bind to CD3 when combined with VH2, but is particularly preferably the amino acid sequence of any of the above (j) to (l), most preferably It is the amino acid sequence of said (j).

  Here, when the “amino acid sequence shown in SEQ ID NO: 5 or 6” in (g) above is “the amino acid sequence shown in SEQ ID NO: 5”, the “amino acid sequence shown in SEQ ID NO: 7 or 8” in (j) above Is preferably “the amino acid sequence shown in SEQ ID NO: 7”, and when “the amino acid sequence shown in SEQ ID NO: 5 or 6” in (g) above is “the amino acid sequence shown in SEQ ID NO: 6” Preferably, the “amino acid sequence shown in SEQ ID NO: 7 or 8” is the “amino acid sequence shown in SEQ ID NO: 8”.

  In the above (b), (e), (h) and (k), each of “one or more” is preferably 1 to 20, more preferably 1 to 10, more preferably 1 to 2. There are five, more preferably one to four, more preferably one to three, more preferably one or two, and most preferably one.

  In the above (c), (f), (i) and (l), each independently, the “sequence identity of 85% or more” is more preferably 90% or more, still more preferably 95% or more, still more preferably Preferably, the sequence identity is 98% or more, most preferably 99% or more.

  In the present invention, the sequence identity of the nucleotide sequence or the amino acid sequence can be determined using methods well known to those skilled in the art, sequence analysis software and the like. For example, blastn program and blastp program of BLAST algorithm and fasta program of FASTA algorithm can be mentioned. In the present invention, “sequence identity” of an amino acid sequence to be evaluated with an amino acid sequence X of a certain amino acid sequence to be evaluated means aligning the amino acid sequence X with the amino acid sequence to be evaluated and introducing gaps as necessary. It is a value in which the frequency at which the same amino acid appears at the same site in the amino acid sequence including the gap portion when the amino acid identity between the two is maximized is represented by%.

  Immunoglobulins have three sites in the heavy chain and light chain variable regions, respectively, called complementarity determining regions (CDRs) or hypervariable regions, which are particularly highly variable in primary structure. The complementarity determining region is important in determining the specificity of the immunoglobulin for an antigen. The complementarity determining regions in the amino acid sequence of the heavy chain variable region (VH1-1) of anti-EGFR immunoglobulin 1 shown in SEQ ID NO: 1 are the above (VH1-1 CDR1), (VH1-1 CDR2) and (VH1-1 CDR3). It is. The complementarity determining regions in the amino acid sequence of the heavy chain variable region (VH1-2) of anti-EGFR immunoglobulin 2 shown in SEQ ID NO: 2 are the above (VH1-2 CDR1), (VH1-2 CDR2) and (VH1-2 CDR3). It is. The complementarity determining regions in the amino acid sequence of the light chain variable region (VL1-1) of anti-EGFR immunoglobulin 1 shown in SEQ ID NO: 3 are the aforementioned (VL1-1 CDR1), (VL1-1 CDR2) and (VL1-1 CDR3) It is. The complementarity determining regions in the amino acid sequence of the light chain variable region (VL1-2) of anti-EGFR immunoglobulin 2 shown in SEQ ID NO: 4 are the aforementioned (VL1-2 CDR1), (VL1-2 CDR2) and (VL1-2 CDR3) It is. The complementarity determining regions in the amino acid sequence of the heavy chain variable region (VH2-1) of anti-CD3 immunoglobulin 1 shown in SEQ ID NO: 5 are the above (VH2-1 CDR1), (VH2-1 CDR2) and (VH2-1 CDR3) It is. The complementarity determining regions in the amino acid sequence of the heavy chain variable region (VH2-2) of anti-CD3 immunoglobulin 2 shown in SEQ ID NO: 6 are the above (VH2-2 CDR1), (VH2-2 CDR2) and (VH2-2 CDR3). It is. The complementarity determining regions in the amino acid sequence of the light chain variable region (VL2-1) of anti-CD3 immunoglobulin 1 shown in SEQ ID NO: 7 are the aforementioned (VL2-1 CDR1), (VL2-1 CDR2) and (VL2-1 CDR3) It is. The complementarity determining regions in the amino acid sequence of the light chain variable region (VL2-2) of anti-CD3 immunoglobulin 2 shown in SEQ ID NO: 8 are the above (VL2-2 CDR1), (VL2-2 CDR2) and (VL2-2 CDR3). It is.

In another preferred embodiment of the above antibody polypeptide:
The VH1 contains one or more, preferably two or more, most preferably three of the (VH1-1CDR1), (VH1-1CDR2) and (VH1-1CDR3), and the V L1 is the above (V L1-1CDR1) , (VL1-1 CDR2) and (VL1-1 CDR3), preferably containing one or more, preferably two or more, most preferably three or
The VH1 comprises one or more, preferably two or more, most preferably three of the (VH1-2CDR1), (VH1-2CDR2) and (VH1-2CDR3), and the VL1 is the above (VL1-2CDR1) , (VL1-2 CDR2) and (VL1-2 CDR3), including one or more, preferably two or more, most preferably three,
Satisfy the condition 1 and
The VH2 contains one or more, preferably two or more, most preferably three of the (VH2-1CDR1), (VH2-1CDR2) and (VH2-1CDR3), and the VL2 is the above (VL2-1CDR1) , (VL2-1CDR2) and (VL2-1CDR3), preferably containing one or more, preferably two or more, most preferably three, or
The VH2 comprises one or more, preferably two or more, most preferably three of the (VH2-2 CDR1), (VH2-2 CDR2) and (VH2-2 CDR3), and the VL2 is the above (VL2-2 CDR1) , (VL2-2CDR2) and (VL2-2CDR3), containing one or more, preferably two or more, most preferably three,
The condition 2 is satisfied.

In another preferred embodiment of the above antibody polypeptide:
When VH1 is the amino acid sequence of (b) or (c) and VL1 is the amino acid sequence of (e) or (f), VH1 and VL1 satisfy the condition 1 and
When VH2 is the amino acid sequence of (h) or (i) and VL2 is the amino acid sequence of (k) or (l), VH2 and VL2 satisfy the condition 2.

  Each of L1, L2 and L3 may be any one as long as adjacent variable regions can be linked to form a single-chain antibody polypeptide, and the specific sequence is not particularly limited, but preferred embodiments Is preferably an amino acid sequence having 3 to 36, more preferably 6 to 18, more preferably 50% or more of the constituent amino acids belongs to the group consisting of glycine, alanine and serine, particularly preferably Is an amino acid sequence in which at least 50% of the constituent amino acids are glycine.

  In a preferred embodiment, at least one of L 1, L 2 and L 3, more preferably at least two of L 1, L 2 and L 3, more preferably L 1, L 2 and L 3 are independently said (m) to (o) Or the amino acid sequence of (m).

  In the above (n), “one or more” is preferably 1 to 5, more preferably 1 to 4, more preferably 1 to 3, more preferably 1 or 2, and most preferably 1 It is.

  In the above (o), “sequence identity of 85% or more” is more preferably 90% or more, still more preferably 95% or more, still more preferably 98% or more, most preferably 99% or more. It is. The method of calculating sequence identity is as described above.

  Preferably, the bispecific antibody of an embodiment of the present invention further comprises one or more tags linked to the carboxy terminus of the antibody polypeptide. Particularly preferably, the tag is a tag consisting of a polypeptide, which is linked to the carboxy terminus of the antibody polypeptide to form a single-stranded polypeptide as a whole. The carboxy terminus of the antibody polypeptide and the tag may be directly linked without any other amino acid sequence, or indirectly linked with another amino acid sequence. Other amino acid sequences include the amino acid sequences described for L1, L2 and L3, and amino acid sequences consisting of 1 to 5 alanine.

  Here, “tag” refers to a polypeptide that can be a marker of a target protein, and can be used for purification and detection of protein. The tag is not particularly limited as long as it can be a mark of the target protein, preferably a polypeptide comprising the amino acid sequence of any of (p1) to (r1) above, particularly preferably the amino acid sequence of (p1) above, And one or both of the polypeptides comprising the amino acid sequence of any of (p2) to (r2), particularly preferably the amino acid sequence of (p2). When the tag contains a polypeptide comprising the amino acid sequence of any of (p1) to (r1) and a polypeptide comprising the amino acid sequence of any of (p2) to (r2), either one is on the amino terminal side It may be located at Between two adjacent pairs of two or more polypeptides used as a tag, a polypeptide consisting of another amino acid sequence may be interposed. As the other amino acid sequences, those described above can be used.

  In the above (q1) and (q2), “one or more” is preferably 1 to 5, more preferably 1 to 4, more preferably 1 to 3, more preferably 1 or 2, most Preferably it is one.

  In the above (r1) and (r2), “sequence identity of 85% or more” is more preferably 90% or more, still more preferably 95% or more, still more preferably 98% or more, most preferably 99% or more Sequence identity of The method of calculating sequence identity is as described above.

  A specific embodiment of the antibody polypeptide is preferably a polypeptide comprising the amino acid sequence of any of (s) to (u), and a polypeptide comprising the amino acid sequence of (s). Is most preferred.

  Among the amino acid sequences of (s), particularly, the amino acid sequence of positions 3 to 497 of SEQ ID NO: 19; the amino acid sequence of positions 3 to 497 of SEQ ID NO: 20; The amino acid sequence at position 501, the amino acid sequence at positions 3 to 501 of SEQ ID NO: 23, the amino acid sequence at position 3 to 501 of SEQ ID NO: 25, or positions 3 to 501 of SEQ ID NO: 26 Amino acid sequence of SEQ ID NO: 19, amino acid sequence of SEQ ID NO: 20, amino acid sequence of SEQ ID NO: 20, amino acid sequence of SEQ ID NO: 20, amino acid sequence of SEQ ID NO: 22, amino acid sequence of SEQ ID NO: 22 The amino acid sequence of positions 3 to 501 of SEQ ID NO: 23 or the amino acid sequence of positions 3 to 501 of SEQ ID NO: 26 is more preferable, and the amino acid sequence of positions 3 to 501 of SEQ ID NO: 23 Alternatively, the amino acid sequence of positions 3 to 501 of SEQ ID NO: 26 is most preferable.

  In the above (t), “one or more” is preferably 1 to 20, more preferably 1 to 10, more preferably 1 to 5, more preferably 1 to 4, more preferably 1 to Three, more preferably one or two, most preferably one.

  In the above (u), “sequence identity of 85% or more” is more preferably 90% or more, still more preferably 95% or more, still more preferably 98% or more, most preferably 99% or more. It is. The method of calculating sequence identity is as described above.

  A polypeptide consisting of another amino acid sequence may be linked to the amino terminus and carboxy terminus of the antibody polypeptide comprising the amino acid sequence of (s) above to form a single-stranded polypeptide as a whole. As a polypeptide consisting of another amino acid sequence, Leu-Glu (an amino acid sequence encoded by a nucleotide sequence cleaved by restriction enzyme XhoI) which can be linked to the amino terminus of the antibody polypeptide containing the amino acid sequence of (s) above, The above-mentioned tag which can be linked to the carboxy terminus of the antibody polypeptide containing the amino acid sequence of (s) above can be exemplified. An amino acid sequence of SEQ ID NO: 19 is a single-chain polypeptide in which Leu-Glu is linked to the amino terminus of the antibody polypeptide containing the amino acid sequence of (s) and a tag is linked to the carboxy terminus. Amino acid sequence SEQ ID NO: 20 amino acid sequence SEQ ID NO: 21 amino acid sequence SEQ ID NO: 22 amino acid sequence SEQ ID NO: 23 amino acid sequence SEQ ID NO: 24 amino acid sequence SEQ ID NO 25 amino acid sequence SEQ ID NO: 26 The amino acid sequence of is particularly preferred. Among these amino acid sequences, particularly the amino acid sequence of SEQ ID NO: 19, the amino acid sequence of SEQ ID NO: 20, the amino acid sequence of SEQ ID NO: 22, the amino acid sequence of SEQ ID NO: 23, the amino acid sequence of SEQ ID NO: 25 or the amino acid of SEQ ID NO: 26 Preferably, the amino acid sequence of SEQ ID NO: 19, the amino acid sequence of SEQ ID NO: 20, the amino acid sequence of SEQ ID NO: 22, the amino acid sequence of SEQ ID NO: 23, or the amino acid sequence of SEQ ID NO: 26; Alternatively, the amino acid sequence of SEQ ID NO: 26 is most preferred.

  The bispecific antibody of another embodiment of the invention further comprises a secretion signal linked to the amino terminus of the antibody polypeptide. The antibody polypeptide and the secretory signal combine to form a single chain polypeptide.

  In the present invention, the term "secretory signal" refers to a polypeptide for extracellularly secreting a target protein which is not secretory, and a base sequence encoding the amino acid sequence of the secretion signal is introduced upstream of the target protein gene. That is, in host cells, bispecific antibodies are preferably present as linked to the secretion signal at the amino terminus of the antibody polypeptide. Another amino acid sequence may be interposed between the secretory signal and the antibody polypeptide, and another amino acid sequence may be, for example, Leu-Glu.

  The secretory signal is not particularly limited as long as it is a polypeptide that can be secreted and expressed by cells, but there is no particular limitation on Mating factor α (MFα) of Saccharomyces cerevisiae, acid phosphatase (PHO1) of Komagataella pastoris, invertase (SUC2) of Saccharomyces cerevisiae, Globulin secretion signal, CHO cell secretion signal, preferably a polypeptide comprising the amino acid sequence of any of (v) to (x) above, and most preferably a poly comprising the amino acid sequence of (v) above It is a peptide.

  In the above (w), “one or more” is preferably 1 to 5, more preferably 1 to 4, more preferably 1 to 3, more preferably 1 or 2, and most preferably 1 It is.

  In the above (x), “sequence identity of 85% or more” is more preferably 90% or more, still more preferably 95% or more, still more preferably 98% or more, most preferably 99% or more. It is. The method of calculating sequence identity is as described above.

  In the present invention, “a base sequence encoding a bispecific antibody” refers to a base sequence designed based on a codon table for a polypeptide consisting of an amino acid sequence constituting a bispecific antibody, The nucleotide sequence results in the formation of a polypeptide by transcription and translation.

  In the present invention, "nucleic acid" refers to DNA or RNA, preferably DNA. The DNA may be single stranded DNA or double stranded DNA.

  One embodiment of the present invention relates to a vector comprising a nucleic acid comprising a nucleotide sequence encoding a bispecific antibody.

  The vector of the present invention can be a circular vector, a linear vector, a plasmid, an artificial chromosome and the like.

  In the present invention, "vector" is a nucleic acid molecule artificially constructed. The nucleic acid molecule constituting the vector of the present invention is usually DNA, preferably double-stranded DNA, and may be circular or linear. The vector of the present invention is usually a cloning site containing one or more restriction enzyme recognition sites, an overlapping region for using Clontech's In-Fusion cloning system, New England Biolabs' Gibson Assembly system, etc., endogenous It can contain a nucleotide sequence of a sex gene, a nucleotide sequence encoding a polypeptide consisting of an amino acid sequence of a target protein, a nucleotide sequence of a selectable marker gene (auxotrophic complementation gene, a drug resistance gene, etc.), and the like. Examples of linear vectors include auxotrophic complementation genes such as URA3 gene, LEU2 gene, ADE1 gene, HIS4 gene, ARG4 gene, G418 resistance gene, Zeocin (trade mark) resistance gene, hygromycin resistance gene, Clone NAT resistance A gene, a PCR product having a nucleotide sequence of a drug resistant gene such as blasticidin S resistant gene, or one obtained by cutting a circular vector or a plasmid with an appropriate restriction enzyme to make it linear may be mentioned.

  The vector of the present invention is preferably introduced into cells. In the present invention, "cell" refers to a basic unit that constitutes an organism, and is not particularly limited as long as it contains a vector, but yeast cells, bacterial cells, fungal cells, insect cells, animal cells, or plant cells, etc. And yeast cells are preferable, and as the yeast cells, cells of methanol-utilizing yeast are more preferable, and cells of Pleurotus spp. Yeast are particularly preferable. Animal cells are also preferred, and CHO (Chinese Hamster Ovary) cells are more preferred as animal cells.

  Although the methanol-utilizing yeast in the present invention is defined as a yeast which can be cultured using methanol as a sole carbon source, it was originally a methanol-utilizing yeast, but it is methanol by artificial modification or mutation. Yeasts that have lost assimilation performance are also included in the methanol-utilizing yeast of the present invention.

  Examples of the methanol-utilizing yeast include yeasts belonging to the genus Pichia, genus Ogataea, genus Komagataella, and the like. Pichia methanolica (Pichia), Ogataea angusta (Ogataea angusta), Ogataea polimorpha (Ogataea polymorpha), Ogataea parapolymorpha (Ogataea parapolymorpha), Oitaea In the genus Komagataella, preferred examples thereof include Komagataella pastoris, Komagataella phaffii, and the like.

  As the Komagata genus yeast, Komagataella pastoris (Komagataella pastoris) and Komagataella phaffii are preferable. Both Komagataella pastoris and Komagataella fafy have the alias Pichia pastoris.

  Specific examples of strains that can be used as a host include strains such as Komagataella pastoris ATCC 76273 (Y-11430, CBS 7435) and Komagataella pastoris X-33. These strains can be obtained from American Type Culture Collection, Thermo Fisher Scientific Co., etc.

  Moreover, in the present invention, derivatives derived from these Komagataella yeast strains can also be used, and examples thereof include Histidine auxotrophic Komagataella pastoris GS115 strain (available from Thermo Fisher Scientific Co., Ltd.). In the present invention, derivatives from these strains can also be used.

Animal cells include CHO cells, mouse myeloma cells, COS cells and the like.
CHO cells include CHO-K1 cells, CHO / dhfr- cells and the like. Specific examples of cells that can be used include CHO-K1 cells (ATCC CCL-61), CHO / dhfr- cells (ATCC CRL-9096) and the like, and can be obtained from the American Type Culture Collection and the like.

Another embodiment of the present invention relates to a culture step of culturing the above cells, and
The present invention relates to a method for producing a bispecific antibody, which comprises a recovery step of recovering the bispecific antibody from the culture obtained in the culture step.

  Here, the “culture obtained in the culture step” may be a culture that can contain a bispecific antibody, such as culture supernatant, cultured cells, disrupted cultured cells, and the like. Therefore, as a method for producing the bispecific antibody of the present invention, a method of culturing the cell described above and accumulating the bispecific antibody in the cell, or bispecificity in the culture supernatant The method includes a method of secreting and accumulating an antibody, preferably a method of secreting and accumulating a bispecific antibody in a culture supernatant.

  The culture conditions of the cells are not particularly limited, and may be appropriately selected depending on the cells. In the culture, any medium can be used as long as it contains a nutrient source that can be used by cells. In addition, culture can be either batch culture or continuous culture.

  Hereinafter, the present invention will be described in detail by way of production examples, comparative examples, and examples, but the present invention is not limited thereto.

  The features of the amino acid sequence and base sequence shown in the sequence listing are summarized in the following table. In the column of remarks in the table, Fw means forward primer and Re means reverse primer.

Production Example 1: Preparation of Various Genes Used for Preparation of Vectors
Detailed procedures for recombinant DNA techniques used in the following examples are described in the following paper: Molecular Cloning 2nd Edition (Cold Spring Harbor Laboratory Press, 1989), Current Protocols in Molecular Biology (Greene) Publishing Associates and Wiley-Interscience.

  Moreover, in the following examples, the plasmid used for transformation of yeast was introduced by introducing the constructed vector into E. coli E. coli DH5α competent cells (manufactured by Takara Bio Inc.), and culturing and amplifying the obtained transformants. Prepared by Preparation of the plasmid from the plasmid holding strain was performed using FastGene Plasmid Mini Kit (manufactured by Japan Genetics).

  The GAP promoter (SEQ ID NO: 16), AOX1 terminator (SEQ ID NO: 27), and CCA 38473 terminator (SEQ ID NO: 28) used in the construction of the vector are chromosomal DNAs of the Coccataella pastoris strain ATCC 76273 (the base sequence is EMBL (The European Molecular Biology Laboratory) ACCESSION No. FR839628 to FR839631) The mixture was used as a template and prepared by PCR. GAP promoter is primer 1 (SEQ ID NO: 29) and primer 2 (SEQ ID NO: 30), AOX1 terminator is primer 3 (SEQ ID NO: 31) and primer 4 (SEQ ID NO: 32), CCA 38473 terminator is primer 5 (SEQ ID NO: 33) and primer Prepared by PCR using 6 (SEQ ID NO: 34).

  The secretion signal MFα gene (SEQ ID NO: 17) used in construction of the vector is the chromosomal DNA of Saccharomyces cerevisiae strain BY4741 (the base sequence is described in ACCESSION No. BK006934 to BK006949) Primer 7 (SEQ ID NO: 35) with the mixture as a template It prepared by PCR using primer 8 (sequence number 36).

  The promoter-controlled G418 resistance gene (SEQ ID NO: 37) used in the construction of the vector was prepared by PCR using synthetic DNA as a template.

  The nucleotide sequence (SEQ ID NO: 38) encoding the # 218 bispecific antibody used in construction of the vector was prepared by PCR using synthetic DNA as a template.

  The nucleotide sequence (SEQ ID NO: 39) encoding the # 219 bispecific antibody used in the construction of the vector was prepared by PCR using synthetic DNA as a template.

  The nucleotide sequence (SEQ ID NO: 40) encoding the # 220 bispecific antibody used in construction of the vector was prepared by PCR using synthetic DNA as a template.

  The nucleotide sequence (SEQ ID NO: 41) encoding the # 221 bispecific antibody used in the construction of the vector was prepared by PCR using synthetic DNA as a template.

  The nucleotide sequence (SEQ ID NO: 42) encoding the # 222 bispecific antibody used in the construction of the vector was prepared by PCR using synthetic DNA as a template.

  The nucleotide sequence (SEQ ID NO: 43) encoding the # 223 bispecific antibody used in construction of the vector was prepared by PCR using synthetic DNA as a template.

  The nucleotide sequence (SEQ ID NO: 44) encoding the # 224 bispecific antibody used in construction of the vector was prepared by PCR using synthetic DNA as a template.

  The nucleotide sequence (SEQ ID NO: 45) encoding the # 225 bispecific antibody used in the construction of the vector was prepared by PCR using synthetic DNA as a template.

  The nucleotide sequence (SEQ ID NO: 46) encoding the # 226 bispecific antibody used in construction of the vector was prepared by PCR using synthetic DNA as a template.

  The PCR was performed using Prime STAR HS DNA Polymerase (manufactured by Takara Bio Inc.) or the like under the reaction conditions described in the attached manual. Preparation of chromosomal DNA was carried out using the Kaneka simplified DNA extraction kit version 2 (manufactured by Kaneka Co., Ltd.) or the like from Komagataella pastoris strain ATCC 76273 or Saccharomyces cerevisiae BY4741 strain under the conditions described therein.

Production Example 2: Construction of Basic Vector
A nucleic acid fragment in which HindIII recognition sequence and EcoRI recognition sequence are added to the end of the promoter-controlled G418 resistance gene (SEQ ID NO: 37) is PCR by using Primer 9 (SEQ ID NO: 47) and Primer 10 (SEQ ID NO: 48). It was prepared and inserted between HindIII and EcoRI sites of pUC19 (Takara Bio, Code No. 3219) after HindIII and EcoRI treatment to construct pUC_G418.

  Next, a nucleic acid fragment of CCA38473 terminator (SEQ ID NO: 28) is prepared by PCR using Primer 5 (SEQ ID NO: 33) and Primer 6 (SEQ ID NO: 34), and the above pUC_G418 is mixed with the XbaI-treated nucleic acid fragment. pUC_T38473_G418 was constructed by joining together using a fusion HD Cloning Kit (manufactured by Clontech Inc.).

  Next, a nucleic acid fragment in which BamHI recognition sequence and SpeI recognition sequence were added to the end of GAP promoter (SEQ ID NO: 16) was prepared by PCR using Primer 1 (SEQ ID NO: 29) and Primer 2 (SEQ ID NO: 30) A nucleic acid fragment in which SpeI recognition sequence and BglII recognition sequence were added to the end of secretion signal MFα gene (SEQ ID NO: 17) was prepared by PCR using Primer 7 (SEQ ID NO: 35) and Primer 8 (SEQ ID NO: 36). The nucleic acid fragment was treated with BamHI and SpeI, and the secretion signal MFα gene nucleic acid fragment was treated with SpeI and BglII and then inserted between the BamHI and BglII sites of pUC_T38473_G418 to construct pUC_Pgap_MFα_T38473_G418.

  Next, a nucleic acid fragment having MluI recognition sequence and BglII recognition sequence added to the end of AOX1 terminator (SEQ ID NO: 27) is prepared by PCR using Primer 3 (SEQ ID NO: 31) and Primer 4 (SEQ ID NO: 32) PUC_Pgap_MFα_T38473_G418 was constructed by inserting between MluI-BglII sites of pUC_Pgap_MFα_T38473_G418 after MluI and BglII treatment.

Example 1 Construction of Bispecific Antibody Expression Vector
The nucleotide sequence encoding the bispecific antibody described in Table 2 was prepared by PCR using synthetic DNA as a template. ID of bispecific antibody, amino acid sequence of bispecific antibody, base sequence encoding bispecific antibody, primers used for amplification of nucleic acid fragment (forward (Fw) side and reverse (Re) side) The numbers and sequences are shown in Table 2 below.

  In each of these nucleic acid fragments, the downstream end region of the secretion signal MFα gene sequence is added as an overlapping region upstream of the nucleotide sequence encoding the bispecific antibody, and the upstream terminal region of the AOX1 terminator sequence is added downstream as an overlapping region. There is. In addition, between the base sequence encoding the bispecific antibody and the upstream end region of the AOX1 terminator sequence, a base sequence encoding a C-myc tag (SEQ ID NO: 12) and a His tag (SEQ ID NO: 13) The nucleotide sequence is added.

A nucleic acid fragment is prepared after XhoI and MluI treatment of pUC_Pgap_MFα_Taox1_T38473_G418, and mixed with the nucleic acid fragment of the base sequence encoding the bispecific antibody prepared by the above PCR, using In-fusion HD Cloning Kit (manufactured by Clontech) Connect them together,
pUC_Pgap_MFα_ # 218_Taox1_T38473_G418, pUC_Pgap_MFα_ # 219_Taox1_T38473_G418, pUC_Pgap_MFα_ # 220_Taox1_T38473_G418, pUC_Pgap_MFα_ # 221_Taox1_T38473_G418, pUC_Pgap_MFα_ # 222_Taox1_T38473_G418, pUC_Pgap_MFα_ # 223_Taox1_T38473_G418, pUC_Pgap_MFα_ # 224_Taox1_T38473_G418, pUC_Pgap_MFα_ # 225_Taox1_T38473_G418, it was constructed pUC_Pgap_MFα_ # 226_Taox1_T38473_G418. These vectors are designed such that each bispecific antibody is expressed under the control of the GAP promoter.

The structure of each bispecific antibody is a structure as shown in Table 4 from the amino terminal side to the carboxy terminal side,
VH1: heavy chain variable region of anti-EGFR immunoglobulin,
VL1: light chain variable region of anti-EGFR immunoglobulin,
VH2: heavy chain variable region of anti-CD3 immunoglobulin,
VL2: light chain variable region of anti-CD3 immunoglobulin,
L1, L2, L3: A single-stranded bispecific antibody comprising a polypeptide chain having a linker.

  Anti-EGFR immunoglobulin 1 and anti-EGFR immunoglobulin 2 were used as anti-EGFR immunoglobulins. The heavy chain variable region of anti-EGFR immunoglobulin 1 was designated VH1-1, and the light chain variable region was designated VL1-1. The heavy chain variable region of anti-EGFR immunoglobulin 2 was designated VH1-2, and the light chain variable region was designated VL1-2.

Anti-CD3 immunoglobulin 1 and anti-CD3 immunoglobulin 2 were used as anti-CD3 immunoglobulin. The heavy chain variable region of anti-CD3 immunoglobulin 1 was designated VH2-1, and the light chain variable region was designated VL2-1. The heavy chain variable region of anti-CD3 immunoglobulin 2 was VH2-2 and the light chain variable region was VL2-2.
The amino acid sequences of each heavy chain variable region and light chain variable region are shown in the following table.

  The constitution of the amino acid sequence of the unsecreted bispecific antibody polypeptide produced and expressed by translation under the control of a GAP promoter from the above-mentioned pUC_Pgap_MFα_ # 218_Taox1_T38473_G418 in the host yeast cell described later is shown in FIG. The bispecific antibody polypeptide secreted from the host yeast cell has the amino terminal secretion signal MFα (SEQ ID NO: 14) excised from the unsecreted bispecific antibody polypeptide shown in FIG. 1, SEQ ID NO: 18 It consists of the amino acid sequence shown.

  An unsecreted bispecific antibody polypeptide generated through transcription and translation from pUC_Pgap_MFα_ # 219_Taox1_T38473_G418 in a host yeast cell has a secretion signal MFα (sequence) at the amino terminus of the amino acid sequence of the bispecific antibody polypeptide shown in SEQ ID NO: 19 It consists of an amino acid sequence to which No. 14) was added.

  An unsecreted bispecific antibody polypeptide generated through transcription and translation from pUC_Pgap_MFα_ # 220_Taox1_T38473_G418 in a host yeast cell has a secretion signal MFα (sequence) at the amino terminus of the amino acid sequence of the bispecific antibody polypeptide shown in SEQ ID NO: 20 It consists of an amino acid sequence to which No. 14) was added.

  An unsecreted bispecific antibody polypeptide generated through transcription and translation from pUC_Pgap_MFα_ # 221_Taox1_T38473_G418 in a host yeast cell has a secretion signal MFα (sequence) at the amino terminus of the amino acid sequence of the bispecific antibody polypeptide shown in SEQ ID NO: 21 It consists of an amino acid sequence to which No. 14) was added.

  An unsecreted bispecific antibody polypeptide generated through transcription and translation from pUC_Pgap_MFα_ # 222_Taox1_T38473_G418 in a host yeast cell has a secretion signal MFα (sequence) at the amino terminus of the amino acid sequence of the bispecific antibody polypeptide shown in SEQ ID NO: 22 It consists of an amino acid sequence to which No. 14) was added.

  An unsecreted bispecific antibody polypeptide generated through transcription and translation from pUC_Pgap_MFα_ # 223_Taox1_T38473_G418 in a host yeast cell has a secretion signal MFα (sequence) at the amino terminus of the amino acid sequence of the bispecific antibody polypeptide shown in SEQ ID NO: 23 It consists of an amino acid sequence to which No. 14) was added.

  An unsecreted bispecific antibody polypeptide generated through transcription and translation from pUC_Pgap_MFα_ # 224_Taox1_T38473_G418 in a host yeast cell has a secretion signal MFα (sequence) at the amino terminus of the amino acid sequence of the bispecific antibody polypeptide shown in SEQ ID NO: 24 It consists of an amino acid sequence to which No. 14) was added.

  An unsecreted bispecific antibody polypeptide generated through transcription and translation from pUC_Pgap_MFα_ # 225_Taox1_T38473_G418 in a host yeast cell has a secretion signal MFα (sequence) at the amino terminus of the amino acid sequence of the bispecific antibody polypeptide shown in SEQ ID NO: 25 It consists of an amino acid sequence to which No. 14) was added.

  An unsecreted bispecific antibody polypeptide generated through transcription and translation from pUC_Pgap_MFα_ # 226_Taox1_T38473_G418 in a host yeast cell has a secretion signal MFα (sequence) at the amino terminus of the amino acid sequence of the bispecific antibody polypeptide shown in SEQ ID NO: 26 It consists of an amino acid sequence to which No. 14) was added.

<Example 2: Acquisition of transformed yeast>
Using the bispecific antibody expression vector constructed in Example 1, Komagataella pastoris was transformed as follows.

  A wild-type strain of Komagataella pastoris ATCC 76273 is cultured with shaking in YPD medium (1% dry yeast extract (Nacalai Tesque), 2% Bacto Pepton (Becton Dickinson), 2% glucose) 2 ml at 30 ° C. for 16 hours. Then, the cells were transplanted into fresh YPD medium at 10-fold dilution, and shake cultured at 30 ° C. for 4 hours. The cultured yeast cells are collected by centrifugation, and after washing the yeast cells (suspending by adding 6 ml of sterile water, suspending the cells by centrifugation, recovering the yeast cells by centrifugation), resuspend the yeast cells with sterile water remaining on the test tube wall. It became turbid, and this was made into the competent cell solution.

  E. coli was transformed with the nine bispecific antibody expression vectors constructed in Example 1, and the transformant obtained was treated with 5 ml of ampicillin-containing LB medium (1% tryptone (manufactured by Nacalai Tesque), 0.5% The cells are cultured with dry yeast extract (manufactured by Nacalai Tesque), 1% sodium chloride, 0.01% ampicillin sodium (manufactured by Nacalai Tesque), and the cells obtained are obtained using FastGene Plasmid Mini Kit (manufactured by Japan Genetics). , Obtained the plasmid. This plasmid was linearized by EcoRV treatment using the EcoRV recognition sequence in the CCA38473 terminator.

  42 μl of the competent cell solution and 20 μg of linear bispecific antibody expression vector, 8 μl of 10 mg / ml carrier DNA (Takara Bio) solution, 8 μl of 1 M DTT solution, 4 μl of 4 M lithium acetate solution, 60% polyethylene glycol 100 μl of the solution was mixed and allowed to stand at 42 ° C. for 20 minutes. After standing for 20 minutes, the yeast cells are recovered and suspended in 500 μl of YPD medium (1% dry yeast extract (Nacalai Tesque), 2% Bacto Pepton (Becton Dickinson), 2% glucose), and 30 Let stand at 2 ° C for 2 hours. After standing for 2 hours, YPDG 418 selective agar plate (1% dried yeast extract (manufactured by Nacalai Tesque), 2% Bacto Pepton (manufactured by Becton Dickinson), 2% glucose, 2% agarose, 0.05% G418 disulfate A salt (manufactured by Nacalai Tesque) was applied, and a strain grown by stationary culture at 30 ° C. for 3 days was selected to obtain a bispecific antibody-expressing yeast.

Example 3 Culture of Transformed Yeast
The bispecific antibody-expressing yeast obtained in Example 2 was used in 5 ml of BMGY medium (1% yeast extract bacto (Becton Dickinson), 2% Hippolypeton (Nippon Pharmaceutical Co., Ltd.), 0.34% yeast nitrogen base Without Amino Acid and Ammonium Sulfate (Becton Dickinson), 1% Ammonium Sulfate, 0.4 mg / l Biotin, 100 mM potassium phosphate (pH 6.0), 2% Glycerol) and incubate it at 30 ° C., 170 rpm for 24 hours. did. After pre-incubation, the pre-culture was inoculated into 250 ml of BMGY medium and cultured with shaking at 30 ° C., 180 rpm for 48 hours. After the culture, the culture supernatant was recovered by centrifugation (3000 g, 10 minutes, 4 ° C.).

Example 4: Purification of bispecific antibody from culture fluid
The culture supernatant obtained in Example 3 was adjusted to pH 6.5 with 1 M Tris-HCl (pH 8.0), and was used for 2 mL of nickel sepharose resin (Ni Sepharose excel (manufactured by GE Healthcare)). The resin was washed with 10 mL of Buffer A (20 mM sodium phosphate, 0.5 M sodium chloride, pH 7.4) and 10 mL of Buffer B (20 mM sodium phosphate, 0.5 M sodium chloride, 10 mM imidazole, pH 7.4). The bispecific antibody bound to the resin was eluted with 5 mL of Buffer C (20 mM sodium phosphate, 0.5 M sodium chloride, 500 mM imidazole, pH 7.4). After elution, the bispecific antibody monomer was separated and purified by gel filtration chromatography using a Prepack HiLoad 26/600 Superdex 200 pg column (manufactured by GE Healthcare).

  The separated and purified bispecific antibody was subjected to SDS-PAGE using 12.5% polyacrylamide gel (e-PAGEL, manufactured by ATTO), and stained with Coomassie blue (manufactured by Bio-Rad) is shown in FIG.

Example 5 Evaluation of Cytotoxic Activity
T-LAK cells used for cytotoxic activity measurement (MTS assay) were prepared from peripheral blood mononuclear cells (PBMC) (manufactured by CTL Co.). 1.5 mL of PBS (manufactured by Nacalai Tesque) and 15 μL of OKT3 IgG (1 mg / mL) were added to a T-flask (25 cm ² ), and incubated at 37 ° C. and 5% CO ₂ for 2 hours. After aspiration with an aspirator, thawed PBMC (manufactured by CTL), 5 mL RPMI (10% FBS (manufactured by Thermo Fisher Scientific), 1% penicillin-streptomycin mixed solution) medium, 2 μL of IL-2 (350,000 IU) / ml, Shionogi & Co., Ltd.) put 2 days, 37 ° C., and incubated at 5% CO _2. Then, change to a 75 cm ² T-flask, add 15 mL of RPMI medium and 6 μL of IL-2 and incubate at 37 ° C, 5% CO ₂ for 2 days, with T-LAK cell suspension did. When cells started to aggregate in T-LAK cell suspension, they were used for MTS assay. On the first day of MTS assay, TFK-1 cells (Medical Cell Resource Center, Aging Medicine Institute, Tohoku University) are seeded in 10 ⁴ cells / 100 μL in 96 well plates in RPMI medium for 24 hours at 37 ° C., 5% It was incubated in CO _2. On the second day, T-LAK cells were prepared at 5 × 10 ⁴ cells / 50 μL, and dilution series of antibodies were also prepared at 50 μL each. The culture supernatant of TFK-1 cells was removed, and 50 μL each of T-LAK cell suspension and antibody solution were added and incubated for 24 hours at 37 ° C., 5% CO ₂ . The culture supernatant was removed on the third day and washed once with 100 μL / well of PBS. A mixture of 15 μL of CellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega) and 85 μL of RPMI medium was added, and incubated at 37 ° C., 5% CO ₂ for 10 minutes. Absorbance was measured using a plate reader (490 nm) to calculate cell viability. The measurement results of the cytotoxic activity of the bispecific antibody are shown in FIG. The effect was confirmed with all nine constructed bispecific antibodies.

From the results shown in FIG. 3, among the nine bispecific antibodies,
# 220 (VL1-1- (L1) -VH1-1- (L2) -VH2-1- (L3) -VL2-1),
# 223 (VH2-1- (L1) -VL2-1- (L2) -VH1-1- (L3) -VL1-1),
# 226 (VH1-1- (L1) -VL1-1- (L2) -VH2-1- (L3) -VL2-1),
# 219 (VL1-1- (L1) -VH1-1- (L2) -VH2-2- (L3) -VL2-2),
# 222 (VH2-2- (L1) -VL2-2- (L2) -VH1-1- (L3) -VL1-1),
# 225 (VH1-1- (L1) -VL1-1- (L2) -VH2-2- (L3) -VL2-2)
Especially the cytotoxic activity of
# 220 (VL1-1- (L1) -VH1-1- (L2) -VH2-1- (L3) -VL2-1),
# 223 (VH2-1- (L1) -VL2-1- (L2) -VH1-1- (L3) -VL1-1),
# 226 (VH1-1- (L1) -VL1-1- (L2) -VH2-1- (L3) -VL2-1),
# 219 (VL1-1- (L1) -VH1-1- (L2) -VH2-2- (L3) -VL2-2),
# 222 (VH2-2- (L1) -VL2-2- (L2) -VH1-1- (L3) -VL1-1),
Higher cytotoxic activity,
# 223 (VH2-1- (L1) -VL2-1- (L2) -VH1-1- (L3) -VL1-1),
# 226 (VH1-1- (L1) -VL1-1- (L2) -VH2-1- (L3) -VL2-1),
It was confirmed that the cytotoxic activity of

Claims (16)

From the amino terminal side to the carboxy terminal side,
Formula 1: VH1-L1-VL1-L2-VH2-L3-VL2,
Formula 2: VH1-L1-VL1-L2-VL2-L3-VH2,
Formula 3: VL1-L1-VH1-L2-VH2-L3-VL2,
Formula 4: VL1-L1-VH1-L2-VL2-L3-VH2,
Formula 5: VH2-L1-VL2-L2-VH1-L3-VL1,
Formula 6: VH2-L1-VL2-L2-VL1-L3-VH1,
Formula 7: VL2-L1-VH2-L2-VH1-L3-VL1 or Formula 8: VL2-L1-VH2-L2-VL1-L3-VH1
Comprising an antibody polypeptide comprising a structure represented by
VH1 represents the amino acid sequence of the heavy chain variable region of anti-EGFR immunoglobulin,
VL1 represents the amino acid sequence of the light chain variable region of anti-EGFR immunoglobulin,
VH2 represents the amino acid sequence of the heavy chain variable region of anti-CD3 immunoglobulin,
VL2 represents the amino acid sequence of the light chain variable region of anti-CD3 immunoglobulin;
A bispecific antibody, wherein L1, L2 and L3 each independently represent the amino acid sequence of a linker.   The bispecific antibody of claim 1, wherein the antibody polypeptide comprises a structure represented by Formula 1, Formula 3 or Formula 5. VH1 is an amino acid sequence of any one of the following (a) to (c),
VL1 is an amino acid sequence of any one of the following (d) to (f),
VH2 is an amino acid sequence of any of the following (g) to (i),
The bispecific antibody according to claim 1 or 2, wherein VL2 is an amino acid sequence of any one of the following (j) to (l):
(a) the amino acid sequence shown in SEQ ID NO: 1 or 2;
(b) An amino acid sequence in which one or more amino acids are substituted, deleted and / or added in the amino acid sequence shown in (a) above,
(c) an amino acid sequence having a sequence identity of 85% or more with the amino acid sequence shown in (a) above;
(d) the amino acid sequence shown in SEQ ID NO: 3 or 4;
(e) An amino acid sequence in which one or more amino acids are substituted, deleted and / or added in the amino acid sequence shown in (d) above,
(f) an amino acid sequence having a sequence identity of 85% or more with the amino acid sequence shown in (d) above;
(g) the amino acid sequence shown in SEQ ID NO: 5 or 6;
(h) An amino acid sequence in which one or more amino acids are substituted, deleted and / or added in the amino acid sequence shown in (g) above,
(i) an amino acid sequence having a sequence identity of 85% or more with the amino acid sequence shown in (g) above;
(j) the amino acid sequence shown in SEQ ID NO: 7 or 8;
(k) An amino acid sequence in which one or more amino acids are substituted, deleted and / or added in the amino acid sequence shown in (j) above,
(l) An amino acid sequence having a sequence identity of 85% or more with the amino acid sequence shown in (j) above. The VH1 contains one or more of the following (VH1-1CDR1), (VH1-1CDR2) and (VH1-1CDR3), and the V L1 is the following (V L-1 -CDR1), (V L-1 -CDR2) and (V L1- 1) including one or more of
VH1 contains one or more of the following (VH1-2 CDR1), (VH1-2 CDR2) and (VH1-2 CDR3), and the VL1 has the following (VL1-2 CDR1), (VL1-2 CDR2) and (VL1-) 2) including one or more of 2) CDR3),
Satisfy the condition 1 and
The VH2 contains one or more of the following (VH2-1 CDR1), (VH2-1 CDR2) and (VH2-1 CDR3), and the VL2 is the following (VL2-1 CDR1), (VL2-1 CDR2) and (VL2-) 1) including one or more of
The VH2 contains one or more of the following (VH2-2 CDR1), (VH2-2 CDR2) and (VH2-2 CDR3), and the VL2 has the following (VL2-2 CDR1), (VL2-2 CDR2) and (VL2-) 2) including one or more of 2) CDR3),
The bispecific antibody according to any one of claims 1 to 3, wherein the condition 2 is satisfied:
(VH1-1 CDR1) amino acid sequence of positions 31 to 37 of SEQ ID NO: 1,
(VH1-1CDR2) amino acid sequence of the 52nd position to the 67th position of SEQ ID NO: 1,
(VH1-1CDR3) amino acid sequence of positions 100 to 110 of SEQ ID NO: 1,
(VH1-2CDR1) amino acid sequence of positions 29 to 33 of SEQ ID NO: 2,
(VH1-2 CDR2) The amino acid sequence of the 50th to the 59th positions of SEQ ID NO: 2,
(VH1-2CDR3) amino acid sequence of positions 99 to 110 of SEQ ID NO: 2,
(VL1-1 CDR1) amino acid sequence of positions 24 to 34 of SEQ ID NO: 3,
(VL1-1 CDR2) the amino acid sequence of the 50th to 56th positions of SEQ ID NO: 3,
(VL1-1CDR3) amino acid sequence of positions 89 to 97 of SEQ ID NO: 3,
(VL1-2 CDR1) amino acid sequence of positions 28 to 33 of SEQ ID NO: 4,
(VL1-2 CDR2) the amino acid sequence of the 50th to the 53rd positions of SEQ ID NO: 4,
(VL1-2 CDR3) amino acid sequence of positions 91 to 96 of SEQ ID NO: 4,
(VH2-1 CDR1) amino acid sequence of positions 23 to 35 of SEQ ID NO: 5,
(VH2-1 CDR2) The amino acid sequence of the 50th to 66th positions of SEQ ID NO: 5,
(VH2-1 CDR3) amino acid sequence of positions 99 to 108 of SEQ ID NO: 5,
(VH2-2 CDR1) amino acid sequence of positions 31 to 35 of SEQ ID NO: 6,
(VH2-2 CDR2) the amino acid sequence of positions 50 to 66 of SEQ ID NO: 6,
(VH2-2 CDR3) amino acid sequence of positions 99 to 108 of SEQ ID NO: 6,
(VL2-1 CDR1) amino acid sequence of positions 24 to 33 of SEQ ID NO: 7,
(VL2-1 CDR2) the amino acid sequence of the 49th to 55th positions of SEQ ID NO: 7,
(VL2-1 CDR3) the amino acid sequence of positions 88 to 96 of SEQ ID NO: 7,
(VL2-2 CDR1) the amino acid sequence of positions 24 to 33 of SEQ ID NO: 8,
(VL2-2 CDR2) the amino acid sequence of positions 49 to 55 of SEQ ID NO: 8,
(VL2-2 CDR3) The amino acid sequence of positions 88 to 96 of SEQ ID NO: 8. The bispecific antibody according to any one of claims 1 to 4, wherein at least one of L1, L2 and L3 is independently any one of the following (m) to (o):
(m) the amino acid sequence shown in SEQ ID NO: 9, 10 or 11;
(n) An amino acid sequence in which one or more amino acids are substituted, deleted and / or added in the amino acid sequence shown in (m) above,
(o) An amino acid sequence having 85% or more of sequence identity with the amino acid sequence shown in (m) above.   6. The bispecific antibody of any one of claims 1-5, further comprising one or more tags linked to the carboxy terminus of the antibody polypeptide. The tag is a polypeptide comprising an amino acid sequence of any one of the following (p1) to (r1), and / or a polypeptide comprising an amino acid sequence of any of (p2) to (r2): Bispecific antibodies as described in:
(p1) the amino acid sequence shown in SEQ ID NO: 12,
(q1) An amino acid sequence in which one or more amino acids are substituted, deleted and / or added in the amino acid sequence shown in the above (p1),
(r1) an amino acid sequence having a sequence identity of 85% or more with the amino acid sequence shown in (p1) above,
(p2) the amino acid sequence shown in SEQ ID NO: 13,
(q2) an amino acid sequence in which one or more amino acids are substituted, deleted and / or added in the amino acid sequence shown in the above (p2),
(r2) An amino acid sequence having a sequence identity of 85% or more with the amino acid sequence shown in (p2) above. The bispecific antibody according to any one of claims 1 to 7, wherein the antibody polypeptide is a polypeptide comprising an amino acid sequence of any one of the following (s) to (u):
(s) amino acid sequence of positions 3 to 497 of SEQ ID NO: 18; amino acid sequence of positions 3 to 497 of SEQ ID NO: 19; amino acid sequence of positions 3 to 497 of SEQ ID NO: 20; The amino acid sequence of positions 3 to 501 of 21, the amino acid sequence of positions 3 to 501 of SEQ ID NO: 22, the amino acid sequence of positions 3 to 501 of SEQ ID NO: 23, the third position of SEQ ID NO: 24 The amino acid sequence of SEQ ID NO: 50, the amino acid sequence of positions 3 to 501 of SEQ ID NO: 25 or the amino acid sequence of positions 3 to 501 of SEQ ID NO: 26,
(t) An amino acid sequence in which one or more amino acids are substituted, deleted and / or added in the amino acid sequence shown in the above (s),
(u) An amino acid sequence having 85% or more sequence identity with the amino acid sequence shown in (s) above.   9. The bispecific antibody of any one of claims 1 to 8, further comprising a secretion signal linked to the amino terminus of the antibody polypeptide. The bispecific antibody according to claim 9, wherein the secretory signal is a polypeptide comprising an amino acid sequence of any one of the following (v) to (x):
(v) the amino acid sequence shown in SEQ ID NO: 14 or 15;
(w) an amino acid sequence in which one or more amino acids are substituted, deleted and / or added in the amino acid sequence shown in (v) above,
(x) An amino acid sequence having a sequence identity of 85% or more with the amino acid sequence shown in (v) above.   A nucleic acid comprising a base sequence encoding the bispecific antibody according to any one of claims 1 to 10.   A vector comprising the nucleic acid according to claim 11.   A cell comprising the vector of claim 12.   14. The cell according to claim 13, which is a yeast cell, a bacterial cell, a fungal cell, an insect cell, an animal cell or a plant cell.   The cell according to claim 14, which is a yeast cell which is a methanol-utilizing yeast cell or an animal cell which is a CHO cell. A culture step of culturing the cell according to any one of claims 13 to 15, and
A method for producing a bispecific antibody, comprising a recovery step of recovering the bispecific antibody from the culture obtained in the culture step.

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