JP2009055899A - Antibody with enhanced adcc activity and method for producing the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an antibody with enhanced ADCC (antibody-dependent cellular cytotoxicity) activity, and to provide a method for producing the antibody. <P>SOLUTION: The inventors studied whether the ADCC activity can be enhanced or not by mutating the amino acid of the Fc part to cysteine (Cys) of an amino acid having potential of causing a large structural change, which are not elicited from computer seach by developing the amino acid modification of the Fc part carried out by researchers of Genentech Inc or the like. As a result, the invention provides the human antibody obtained by changing the amino acid residue at the 286 or 298 position to the Cys in the constant region of the H chain. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a method for enhancing ADCC activity of an antibody and an antibody having enhanced ADCC activity.

  Chimeric antibodies in which the variable region of the antibody is mouse type and the constant region is human type, and humanized antibodies in which both the variable region and constant region are human type are expected as therapeutic agents for cancer and rheumatoid arthritis. Chemically synthesized therapeutic agents such as cisplatin conventionally used in cancer treatment have a low ability to distinguish cancer cells from normal cells and are highly toxic. Therefore, cancer chemotherapy currently has a heavy physical burden on cancer patients. On the other hand, chimerized or humanized antibodies recognize and act on specific molecules on the surface of cancer cells, so that their toxicity is low, and the physical burden is low even for patients undergoing treatment. In contrast to the treatment of rheumatoid arthritis, conventional steroid-based treatments have only slowed the progression of rheumatic symptoms, but humanized antibodies to interleukin-6 receptor are effective in bone destruction and inflammation. Because it can suppress the causative factors, it has a remarkable therapeutic effect.

  There are roughly three methods for producing these therapeutic antibodies (chimeric antibodies, humanized antibodies). The first is a method of humanization by replacing a mouse antibody with a human-type protein from a mouse antibody to a chimeric antibody or a humanized antibody by genetic recombination technology, leaving the antigen-binding site of the antibody. The second is a method using phage display. In this method, a fully human variable region capable of recognizing the target protein can be selected from a variety of human-derived antibody variable regions expressed on the phage surface. A fully humanized antibody in which a human-type constant region is further added can be produced using genetic recombination technology as a material. Finally, there is a method using TC mice genetically engineered to have human antibody-producing genes (Nature Genetics, Vol. 16, 113-114, 1997). These TC mice are genetically engineered so that when the antigen, the target protein, is immunized, all antibodies produced by the mice become human. Hybridoma cells obtained by fusing sphere cells and mouse myeloma cells produce fully humanized antibodies that recognize the immunized antigen.

  Chimeric antibodies or humanized antibodies can be prepared by the above three methods, but the problem is that the cost of producing therapeutic antibodies is much higher than that of low molecular weight therapeutic agents, regardless of which method is used. Due to its high production cost, the price of therapeutic antibodies is very high, and it is a current treatment cost for cancer and rheumatic patients.

  As one solution to solve this problem, it was considered to increase the therapeutic effect of therapeutic antibodies per unit mass on cancer cells. In other words, even if a therapeutic antibody amount is small, if the same therapeutic effect as a conventional dose can be obtained, the dosage per patient can be reduced, and the dosage cost per dosage can also be reduced. be able to. One indicator of the therapeutic effect of this therapeutic antibody is the antibody-dependent-cellular-cytotoxicity (ADCC) activity. This ADCC activity results when the Fc portion of the therapeutic antibody binds to the Fcγ receptor on the killer cell that can kill the cancer cell, and further leads to the killer cell to the cancer cell through the recognition of the variable region of the therapeutic antibody. In addition, killer cells kill cancer cells via therapeutic antibodies.

  The therapeutic antibody performance required to increase ADCC activity includes 1) that the variable region of the therapeutic antibody can strongly recognize proteins that are specifically on the surface of cancer cells, and 2) the Fcγ receptor on killer cells. The Fc portion of the therapeutic antibody can bind strongly. Many studies have been made to increase ADCC activity by resolving item 2).

  Shinkawa T et al. Focused on the structure of two sugar chains (N-Linked oligosaccharide) bound to asparagine (Asn), the 297th amino acid in the Fc region of therapeutic antibody IgG1, and the fucose of the sugar chain structure It was found that the ADCC activity increased 20 to 100 times when the sugar chain structure was deficient (J. Biol. Chem. Vol. 278, 3466-3473, 2003). The ADCC activity due to this fucose-deficient structure was also reported by Shields RL et al. (J. Biol. Chem. Vol. 277, 26733-26740, 2002). Pablo U et al. Also found that ADCC activity can be increased several times by controlling the amount of binding N-acetylglucosamine in the sugar chain structure, focusing on the same sugar chain structure (Nature Biotechnology Vol. .17, 176-180, 1999).

In addition to the method of increasing the ADCC activity of the therapeutic antibody by modifying the sugar chain structure bound to the Fc region of IgG1, researchers of US Genentech Inc. We searched for a technique that enhances binding to the Fc receptor by adding mutations to the amino acid itself, and succeeded in enhancing ADCC activity several tens of times by triple mutation of S298A, E333A, and K334A (J Biol. Chem. Vol. 276, 6591-6604, 2001).
Prior art documents related to the present invention are shown below.
WO 2004/029207 WO 2000/042072 WO 2004/063351 WO 2004/099249 WO 2006/019447 Special table 2006-512407 Special table 2003-512019 WO2006 / 104989 WO2006 / 105062 Nature Genetics, Vol. 16, 113-114, 1997 J. Biol. Chem. Vol.278, 3466-3473, 2003 J. Biol. Chem. Vol.277, 26733-26740, 2002 Nature Biotechnology Vol.17, 176-180, 1999 J. Biol. Chem. Vol.276, 6591-6604, 2001

  An object of the present invention is to provide an antibody having enhanced ADCC activity or antitumor activity and a method for producing the same.

  The present inventors further developed the amino acid mutation of the Fc region performed by researchers of Genentech Inc. and others, and cysteine, which is an amino acid that may be accompanied by a significant structural change that cannot be derived from computer search in the Fc region. It was examined whether ADCC activity or antitumor activity could be enhanced by introducing (Cys). Cysteine substitution means the introduction of a thiol group (-SH), resulting in a disulfide bond (-S-S-). Therefore, it was expected to call a drastic change in the Fc structure. In human Cγ1 (the amino acid sequence described in SEQ ID NO: 164), the present inventors have described Human IgG1 H shown by Elvin A. Kabat et al. In Sequence of proteins of Immunological Interest (NIH Publication No.91-3242, 1991). The amino acid at position 286 or 298 in the EU index number of the chain constant region (hereinafter sometimes referred to as “kabat number”) is replaced with cysteine (hereinafter sometimes abbreviated as Cys). Thus, it was examined whether ADCC activity or antitumor activity could be enhanced.

As a result, it was found that a human antibody in which the amino acid at position 286 or 298 of human Cγ1 was substituted with Cys showed very high ADCC activity compared to the wild type human antibody, and the present invention was completed. That is, the present invention provides the following [1] to [31].
[1] An antibody in which the amino acid residue at position 286 or 298 is substituted with cysteine in the heavy chain constant region.
[2] The antibody according to [1], wherein the heavy chain constant region is any constant region selected from the group consisting of human Cγ1, Cγ2, Cγ3, and Cγ4.
[3] The antibody according to any one of [1] or [2], wherein ADCC activity is increased as compared with that before substitution.
[4] The antibody according to any one of [1] to [3], which has improved stability, solubility, or binding affinity to an Fc receptor.
[5] The antibody according to any one of [1] to [4], which is provided with a sugar chain.
[6] The antibody according to claim 5, which improves an effector function.
[7] The antibody according to any one of [1] to [6], which binds to FcγRI, FcγRII, FcγRIII, or FcRn.
[8] The antibody according to any one of [1] to [7], which has specificity for a target antigen selected from the group consisting of ALCAM, ICAM1, and EGFR.
[9] The antibody according to any one of (1) to (4) below;
(1) The amino acid sequence described in SEQ ID NO: 12 as CDR1, the amino acid sequence described in SEQ ID NO: 13 as CDR2, the amino acid sequence described in SEQ ID NO: 14 as CDR3, and the amino acid described in SEQ ID NO: 91 as CH An antibody comprising an H chain having a sequence;
(2) an antibody comprising an H chain having the amino acid sequence set forth in SEQ ID NO: 95,
(3) The amino acid sequence described in SEQ ID NO: 12 as CDR1, the amino acid sequence described in SEQ ID NO: 13 as CDR2, the amino acid sequence described in SEQ ID NO: 14 as CDR3, and the amino acid sequence described in SEQ ID NO: 97 as Fc region An antibody comprising an H chain having an amino acid sequence,
(4) An antibody having a pair of the H chain according to any one of (1), (2) or (3) above and the L chain according to (i) or (ii) below,
(I) The amino acid sequence described in SEQ ID NO: 4 as CDR1, the amino acid sequence described in SEQ ID NO: 5 as CDR2, the amino acid sequence described in SEQ ID NO: 6 as CDR3, and the amino acid described in SEQ ID NO: 8 as CL A light chain having a sequence;
(Ii) An L chain having the amino acid sequence set forth in SEQ ID NO: 99.
[10] The antibody according to any one of (1) to (4) below;
(1) The amino acid sequence described in SEQ ID NO: 12 as CDR1, the amino acid sequence described in SEQ ID NO: 13 as CDR2, the amino acid sequence described in SEQ ID NO: 14 as CDR3, and the amino acid described in SEQ ID NO: 93 as CH3 An antibody comprising an H chain having a sequence;
(2) an antibody comprising an H chain having the amino acid sequence of SEQ ID NO: 101,
(3) The amino acid sequence described in SEQ ID NO: 12 as CDR1, the amino acid sequence described in SEQ ID NO: 13 as CDR2, the amino acid sequence described in SEQ ID NO: 14 as CDR3, and the amino acid sequence described in SEQ ID NO: 103 as Fc region An antibody comprising an H chain having an amino acid sequence,
(4) An antibody having a pair of the H chain according to any one of (1), (2) or (3) above and the L chain according to (i) or (ii) below,
(I) The amino acid sequence described in SEQ ID NO: 4 as CDR1, the amino acid sequence described in SEQ ID NO: 5 as CDR2, the amino acid sequence described in SEQ ID NO: 6 as CDR3, and the amino acid described in SEQ ID NO: 8 as CL A light chain having a sequence;
(Ii) An L chain having the amino acid sequence set forth in SEQ ID NO: 99.
[11] The antibody according to any one of (1) to (4) below;
(1) The amino acid sequence described in SEQ ID NO: 42 as CDR1, the amino acid sequence described in SEQ ID NO: 43 as CDR2, the amino acid sequence described in SEQ ID NO: 44 as CDR3, and the amino acid described in SEQ ID NO: 91 as CH An antibody comprising an H chain having a sequence;
(2) an antibody comprising an H chain having the amino acid sequence of SEQ ID NO: 105,
(3) The amino acid sequence described in SEQ ID NO: 42 as CDR1, the amino acid sequence described in SEQ ID NO: 43 as CDR2, the amino acid sequence described in SEQ ID NO: 44 as CDR3, and the amino acid sequence described in SEQ ID NO: 107 as Fc region An antibody comprising an H chain having an amino acid sequence,
(4) An antibody having a pair of the H chain according to any one of (1), (2) or (3) above and the L chain according to (i) or (ii) below,
(I) The amino acid sequence described in SEQ ID NO: 34 as CDR1, the amino acid sequence described in SEQ ID NO: 35 as CDR2, the amino acid sequence described in SEQ ID NO: 36 as CDR3, and the amino acid described in SEQ ID NO: 38 as CL A light chain having a sequence;
(Ii) An L chain having the amino acid sequence set forth in SEQ ID NO: 109.
[12] The antibody according to any one of (1) to (4) below;
(1) The amino acid sequence described in SEQ ID NO: 42 as CDR1, the amino acid sequence described in SEQ ID NO: 43 as CDR2, the amino acid sequence described in SEQ ID NO: 44 as CDR3, and the amino acid described in SEQ ID NO: 93 as CH3 An antibody comprising an H chain having a sequence;
(2) an antibody comprising an H chain having the amino acid sequence set forth in SEQ ID NO: 111,
(3) The amino acid sequence described in SEQ ID NO: 42 as CDR1, the amino acid sequence described in SEQ ID NO: 43 as CDR2, the amino acid sequence described in SEQ ID NO: 44 as CDR3, and the amino acid sequence described in SEQ ID NO: 113 as Fc region An antibody comprising an H chain having an amino acid sequence,
(4) An antibody having a pair of the H chain according to any one of (1), (2) or (3) above and the L chain according to (i) or (ii) below,
(I) The amino acid sequence described in SEQ ID NO: 34 as CDR1, the amino acid sequence described in SEQ ID NO: 35 as CDR2, the amino acid sequence described in SEQ ID NO: 36 as CDR3, and the amino acid described in SEQ ID NO: 38 as CL A light chain having a sequence;
(Ii) An L chain having the amino acid sequence set forth in SEQ ID NO: 109.
[13] The antibody according to any one of (1) to (4) below;
(1) The amino acid sequence described in SEQ ID NO: 57 as CDR1, the amino acid sequence described in SEQ ID NO: 58 as CDR2, the amino acid sequence described in SEQ ID NO: 59 as CDR3, and the amino acid described in SEQ ID NO: 91 as CH An antibody comprising an H chain having a sequence;
(2) an antibody comprising an H chain having the amino acid sequence set forth in SEQ ID NO: 115,
(3) The amino acid sequence described in SEQ ID NO: 57 as CDR1, the amino acid sequence described in SEQ ID NO: 58 as CDR2, the amino acid sequence described in SEQ ID NO: 59 as CDR3, and the amino acid sequence described in SEQ ID NO: 117 as Fc region An antibody comprising an H chain having an amino acid sequence,
(4) An antibody having a pair of the H chain according to any one of (1), (2) or (3) above and the L chain according to (i) or (ii) below,
(I) the amino acid sequence described in SEQ ID NO: 49 as CDR1, the amino acid sequence described in SEQ ID NO: 50 as CDR2, the amino acid sequence described in SEQ ID NO: 51 as CDR3, and the amino acid described in SEQ ID NO: 53 as CL A light chain having a sequence;
(Ii) An L chain having the amino acid sequence set forth in SEQ ID NO: 119.
[14] The antibody according to any one of (1) to (4) below;
(1) The amino acid sequence described in SEQ ID NO: 57 as CDR1, the amino acid sequence described in SEQ ID NO: 58 as CDR2, the amino acid sequence described in SEQ ID NO: 59 as CDR3, and the amino acid described in SEQ ID NO: 93 as CH An antibody comprising an H chain having a sequence;
(2) an antibody comprising an H chain having the amino acid sequence of SEQ ID NO: 121,
(3) The amino acid sequence described in SEQ ID NO: 57 as CDR1, the amino acid sequence described in SEQ ID NO: 58 as CDR2, the amino acid sequence described in SEQ ID NO: 59 as CDR3, and the amino acid sequence described in SEQ ID NO: 123 as Fc region An antibody comprising an H chain having an amino acid sequence,
(4) An antibody having a pair of the H chain according to any one of (1), (2) or (3) above and the L chain according to (i) or (ii) below,
(I) the amino acid sequence described in SEQ ID NO: 49 as CDR1, the amino acid sequence described in SEQ ID NO: 50 as CDR2, the amino acid sequence described in SEQ ID NO: 51 as CDR3, and the amino acid described in SEQ ID NO: 53 as CL A light chain having a sequence;
(Ii) An L chain having the amino acid sequence set forth in SEQ ID NO: 119.
[15] The antibody according to any one of (1) to (4) below;
(1) The amino acid sequence described in SEQ ID NO: 71 as CDR1, the amino acid sequence described in SEQ ID NO: 72 as CDR2, the amino acid sequence described in SEQ ID NO: 73 as CDR3, and the amino acid described in SEQ ID NO: 91 as CH An antibody comprising an H chain having a sequence;
(2) an antibody comprising an H chain having the amino acid sequence set forth in SEQ ID NO: 125,
(3) The amino acid sequence described in SEQ ID NO: 71 as CDR1, the amino acid sequence described in SEQ ID NO: 72 as CDR2, the amino acid sequence described in SEQ ID NO: 73 as CDR3, and the amino acid sequence described in SEQ ID NO: 127 as Fc region An antibody comprising an H chain having an amino acid sequence,
(4) An antibody having a pair of the H chain according to any one of (1), (2) or (3) above and the L chain according to (i) or (ii) below,
(I) The amino acid sequence described in SEQ ID NO: 63 as CDR1, the amino acid sequence described in SEQ ID NO: 64 as CDR2, the amino acid sequence described in SEQ ID NO: 65 as CDR3, and the amino acid described in SEQ ID NO: 67 as CL A light chain having a sequence;
(Ii) An L chain having the amino acid sequence set forth in SEQ ID NO: 129.
[16] The antibody according to any one of (1) to (4) below;
(1) The amino acid sequence described in SEQ ID NO: 71 as CDR1, the amino acid sequence described in SEQ ID NO: 72 as CDR2, the amino acid sequence described in SEQ ID NO: 73 as CDR3, and the amino acid described in SEQ ID NO: 93 as CH3 An antibody comprising an H chain having a sequence;
(2) an antibody comprising an H chain having the amino acid sequence set forth in SEQ ID NO: 131,
(3) The amino acid sequence described in SEQ ID NO: 71 as CDR1, the amino acid sequence described in SEQ ID NO: 72 as CDR2, the amino acid sequence described in SEQ ID NO: 73 as CDR3, and the amino acid sequence described in SEQ ID NO: 133 as an Fc region An antibody comprising an H chain having an amino acid sequence,
(4) An antibody having a pair of the H chain according to any one of (1), (2) or (3) above and the L chain according to (i) or (ii) below,
(I) The amino acid sequence described in SEQ ID NO: 63 as CDR1, the amino acid sequence described in SEQ ID NO: 64 as CDR2, the amino acid sequence described in SEQ ID NO: 65 as CDR3, and the amino acid described in SEQ ID NO: 67 as CL A light chain having a sequence;
(Ii) An L chain having the amino acid sequence set forth in SEQ ID NO: 129.
[17] The antibody according to any one of (1) to (4) below;
(1) The amino acid sequence described in SEQ ID NO: 86 as CDR1, the amino acid sequence described in SEQ ID NO: 87 as CDR2, the amino acid sequence described in SEQ ID NO: 88 as CDR3, and the amino acid described in SEQ ID NO: 91 as CH An antibody comprising an H chain having a sequence;
(2) an antibody comprising an H chain having the amino acid sequence set forth in SEQ ID NO: 135,
(3) The amino acid sequence described in SEQ ID NO: 86 as CDR1, the amino acid sequence described in SEQ ID NO: 87 as CDR2, the amino acid sequence described in SEQ ID NO: 88 as CDR3, and the amino acid sequence described in SEQ ID NO: 137 as an Fc region An antibody comprising an H chain having an amino acid sequence,
(4) An antibody having a pair of the H chain according to any one of (1), (2) or (3) above and the L chain according to (i) or (ii) below,
(I) The amino acid sequence set forth in SEQ ID NO: 78 as CDR1, the amino acid sequence set forth in SEQ ID NO: 79 as CDR2, the amino acid sequence set forth in SEQ ID NO: 80 as CDR3, and the amino acid set forth in SEQ ID NO: 82 as CL A light chain having a sequence;
(Ii) An L chain having the amino acid sequence set forth in SEQ ID NO: 139.
[18] The antibody according to any one of (1) to (4) below:
(1) The amino acid sequence described in SEQ ID NO: 86 as CDR1, the amino acid sequence described in SEQ ID NO: 87 as CDR2, the amino acid sequence described in SEQ ID NO: 88 as CDR3, and the amino acid described in SEQ ID NO: 93 as CH3 An antibody comprising an H chain having a sequence;
(2) an antibody comprising an H chain having the amino acid sequence set forth in SEQ ID NO: 141,
(3) The amino acid sequence described in SEQ ID NO: 86 as CDR1, the amino acid sequence described in SEQ ID NO: 87 as CDR2, the amino acid sequence described in SEQ ID NO: 88 as CDR3, and the amino acid sequence described in SEQ ID NO: 143 as an Fc region An antibody comprising an H chain having an amino acid sequence,
(4) An antibody having a pair of the H chain according to any one of (1), (2) or (3) above and the L chain according to (i) or (ii) below,
(I) The amino acid sequence set forth in SEQ ID NO: 78 as CDR1, the amino acid sequence set forth in SEQ ID NO: 79 as CDR2, the amino acid sequence set forth in SEQ ID NO: 80 as CDR3, and the amino acid set forth in SEQ ID NO: 82 as CL A light chain having a sequence;
(Ii) An L chain having the amino acid sequence set forth in SEQ ID NO: 139.
[19] A method for producing an antibody with enhanced ADCC activity or antitumor activity, comprising a step of substituting an amino acid residue at position 286 or 298 with cysteine in the heavy chain constant region.
[20] A method for producing an antibody with enhanced ADCC activity or antitumor activity, comprising the following steps (a) and (b):
(A) a step of expressing DNA encoding an H chain in which the amino acid residue at position 286 or 298 is substituted with cysteine in the H chain constant region, and DNA encoding an L chain;
(B) A step of recovering the expression product of the step (a).
[21] The method according to [19] or [20], wherein the heavy chain constant region is any constant region selected from the group consisting of human Cγ1, Cγ2, Cγ3, and Cγ4.
[22] A pharmaceutical composition comprising the antibody according to any one of [1] to [18] and a pharmaceutically acceptable carrier.
[23] A method for treating a non-human mammal, comprising a step of administering the antibody of any one of [1] to [18] to a subject.
[24] A nucleic acid encoding the antibody according to any one of [1] to [18].
[25] A vector comprising the nucleic acid according to [24].
[26] A host cell having the vector according to [25].
[27] A host organism having the vector according to [25].
[28] A method for enhancing ADCC activity or antitumor activity of an antibody, comprising a step of replacing an amino acid residue at position 286 or 298 with cysteine in the heavy chain constant region.
[29] The method according to [26], wherein the heavy chain constant region is any constant region selected from the group consisting of human Cγ1, Cγ2, Cγ3, and Cγ4.
[30] A method for determining whether the ADCC activity or antitumor activity of the antibody of step (a) is enhanced, wherein the ADCC activity or antitumor activity measured in step (b) is an antibody before substitution A method for determining that the ADCC activity or antitumor activity of the antibody of step (a) is enhanced when it is higher than the ADCC activity or antitumor activity of
(A) providing a variant of an antibody having ADCC activity or antitumor activity, wherein the amino acid residue at position 286 or 298 in the heavy chain constant region is substituted with cysteine; and
(B) A step of measuring ADCC activity or antitumor activity of the antibody of step (a).
[31] A screening method for an antibody with enhanced ADCC activity or antitumor activity, comprising the following steps (a) to (d):
(A) providing an antibody having ADCC activity or antitumor activity;
(B) replacing the amino acid residue at position 286 or 298 in the heavy chain constant region of the antibody of step (a) with cysteine,
(C) determining whether the ADCC activity or antitumor activity of the antibody obtained in step (b) has been enhanced by the method according to claim 30,
(D) A step of selecting the antibody determined to have enhanced ADCC activity or antitumor activity in step (c).
[Embodiment of the Invention]

  The present invention relates to the EU index number of the human IgG1 heavy chain constant region shown by Elvin A. Kabat et al. In Sequence of proteins of Immunological Interest (NIH Publication No.91-3242, 1991) (hereinafter referred to as Unless otherwise specified, the present invention provides an antibody in which the amino acid residue at position 286 or 298 in the amino acid position is described according to the EU index described in kabat number) is artificially substituted with cysteine. The base sequence of human Cγ1 is exemplified in SEQ ID NO: 164, and the amino acid sequence is exemplified in SEQ ID NO: 165.

As human immunoglobulins, nine types (isotype) of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, and IgM are known. The antibodies of the present invention include IgG1, IgG2, IgG3, and IgG4 among these isotypes.
The antibodies of the present invention also include those in which a part other than the Fc region of the antibody is replaced with another peptide having antigen-binding ability. Examples of the part other than the Fc region of the antibody include, but are not limited to, a variable region, a CH1 region, a hinge region, and the like. The isotype of an antibody is determined by the structure of the constant region. The isotype constant regions of IgG1, IgG2, IgG3, and IgG4 are called Cγ1, Cγ2, Cγ3, and Cγ4, respectively. The antibodies of the present invention also include antibodies in which the amino acid at position 286 or 298 in Cγ2, Cγ3, or Cγ4 is substituted with Cys. The nucleotide sequences of human Cγ2, Cγ3, and Cγ4 are exemplified in SEQ ID NOs: 166, 168, and 170. The amino acid sequences of human Cγ2, Cγ3, and Cγ4 are exemplified in SEQ ID NOs: 167, 169, and 171.

In addition, Cγ1 (base sequence SEQ ID NO: 164, amino acid sequence SEQ ID NO: 165), Cγ2 (base sequence SEQ ID NO: 166, amino acid sequence SEQ ID NO: 167), Cγ3 (base) described in the sequence listing attached to the present specification Sequence SEQ ID NO: 168, amino acid sequence SEQ ID NO: 169), and Cγ4 (base sequence SEQ ID NO: 170, amino acid sequence SEQ ID NO: 171) in each sequence, the relationship between the EU index of kabat, Shown in Tables 1-7. The codons and amino acids in Tables 1 to 7 correspond to the codons and amino acids from the N-terminal side to the C-terminal side of the sequence listing in order from top to bottom.
Examples of the sequence of the constant region include those disclosed in [Sequence of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)], but are not limited thereto. It is not done.

  Cysteine in the present invention includes derivatives thereof. As the cysteine derivative in the present invention, a compound in which a hydroxyl group, a methyl group, an ethyl group, a carboxyl group, an amino group or the like is added to the cysteine molecule, a compound in which a part of atoms or functional groups constituting the cysteine molecule is deleted, Examples include, but are not limited to, compounds in which a part of the atoms or functional groups constituting the cysteine molecule is substituted with another.

Regarding the ADCC activity evaluated by the lactate dehydrogenase release assay system, the antibody of the present invention showed an increase in ADCC activity compared to the wild type antibody (the wild type between the two points where the wild type antibody and the mutant antibody had the same concentration). The ratio of the ratio of cytotoxicity of the mutant antibody to the antibody increased). The 286Cys and 298Cys antibodies also had increased ADCC activity (the ratio of the concentration of the mutant antibody to the wild type antibody increased between the two points where the wild type antibody and the mutant antibody had the same cytotoxicity ratio). did).
The antibodies of the present invention also have improved stability, solubility, binding affinity for Fc receptors, antitumor activity or CDC activity. The antibody of the present invention preferably has unchanged or increased stability, solubility, binding affinity to Fc receptor, or CDC activity compared to wild type. However, it is sufficient that the antibody of the present invention has at least ADCC activity or antitumor activity increased, and an antibody whose stability, solubility, binding affinity to Fc receptor, or CDC activity is also decreased. Of antibodies. Examples of the Fc receptor include, but are not limited to, FcγI, FcγII, FcγIII, FcRn, protein A, and protein G. The antitumor activity is to be interpreted in the broadest sense, including a selective growth-inhibiting action or damaging action on malignant tissues or cells, and a shrinking or disappearing action of tumor tissues or cells.

  The antibodies of the present invention include both polyclonal and monoclonal antibodies. Methods for preparing and purifying monoclonal and polyclonal antibodies are known in the art and are described, for example, in Harlow and Lane, Antibodies: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1988).

  One form of the antibody of the present invention is a humanized antibody. “Humanized antibody” refers to an antibody having a structure similar to that of a human antibody. A human chimeric antibody (for example, an antibody in which a part of the antibody is humanized, an antibody in which the CH2 region is humanized, Fc Humanized antibody, constant region humanized antibody), and human-type CDR grafting (CDR-grafted) in which parts other than the CDR (complementarity determining region) present in the constant region and variable region are humanized ) Antibodies (PTJohons et al., Nature 321,522 (1986)), fully humanized antibodies and the like. In order to enhance the antigen binding activity of human CDR-grafted antibody, a method of selecting human antibody FR having high homology with mouse antibody, a method of producing humanized antibody having high homology, and further after transplanting mouse CDR to human antibody Techniques for improving the method of substituting amino acids in FR have already been developed (US Patent No. 5585089, US Patent No. 5693761, US Patent No. 5693762, US Patent No. 6180370, European Patent No. 451216, European Patent No. 682040) , Refer to Japanese Patent No. 2828340, etc.) and can also be used to prepare the human CDR-grafted antibody of the present invention.

  A human chimeric antibody can be prepared, for example, by substituting the constant region of an antibody having the structure of the H chain variable region and / or the structure of the L chain variable region with a constant region of a human antibody. Known constant regions of human antibodies can be employed. An example of a method for producing a human chimeric antibody is shown below.

  First, mRNA is extracted from a hybridoma producing a mouse antibody against a specific target antigen, and cDNA is synthesized according to a conventional method. The synthesized cDNA is incorporated into a vector to construct a cDNA library. From this cDNA library, a vector containing the H chain gene and the L chain gene is selected by using the H chain gene fragment and the L chain gene fragment as probes. By sequencing the insertion sequence of the selected vector, the sequences of the H chain variable region and L chain variable region genes are determined. Based on the sequence data thus obtained, DNA encoding the H chain variable region is prepared by chemical synthesis, biochemical cleavage / recombination, or the like. The resulting DNA encoding the H chain variable region is ligated with DNA encoding the human H chain constant region and incorporated into an expression vector to prepare an H chain expression vector. Examples of expression vectors include, but are not limited to, SV40 virus based vectors, EB virus based vectors, BPV (papilloma virus) based vectors, and the like. On the other hand, an L chain expression vector is prepared by the same method. Host cells are cotransformed with these H chain expression vector and L chain expression vector. CHO cells (Chinese hamster ovary) (A. Wright & SL Morrison, J. Immunol. 160, 3393-3402 (1998)), SP2 / 0 cells (mouse myeloma) (K. Motmans et al., Eur. J. Cancer Prev. 5, 512-519 (1996), RP Junghans et al., Cancer Res. 50, 1495-1502 (1990)) are preferably used. For transformation, the lipofectin method (RWMalone et al., Proc. Natl. Acad. Sci. USA 86, 6077 (1989), PLFelgner et al., Proc. Natl. Acad. Sci. USA 84, 7413 ( 1987), electroporation method, calcium phosphate method (FL Graham & AJvan der Eb, Virology 52, 456-467 (1973)), DEAE-Dextran method and the like are preferably used.

  After culturing the transformant, the human chimeric antibody is separated from the cells of the transformant or from the culture solution. For separation and purification of antibodies, centrifugation, ammonium sulfate fractionation, salting out, ultrafiltration, affinity chromatography, ion exchange chromatography, gel filtration chromatography, protein A column chromatography, protein G column chromatography, protein L Methods such as column chromatography can be used in appropriate combination.

  On the other hand, a human CDR-grafted antibody can be prepared, for example, by the following method. First, the amino acid sequences of the H chain variable region and the L chain variable region of an antibody against a specific antigen and the base sequence encoding the same are determined by the method described in the column of the method for producing a chimeric antibody. In addition, the amino acid sequence and base sequence of each CDR region are determined.

  Next, an FR (framework region) that exists across the CDR region is selected. There are approximately three methods for selecting FR. The first method is a method using a human antibody frame whose three-dimensional structure has already been clarified, such as NEWM and REI (Riechmann L. et al., Nature 332,323-3Z7 (1988); Tempst, PR. Et al. , Protein Engineering 7, 1501-1507 (1994); Ellis JH. Et al., J. Immunol 155, 925-937 (1995)). In the second method, a human antibody variable region having the highest homology with the mouse antibody variable region of interest is selected from a database, and its FR is used (Queen C. et al., Proc Natl Acad SciUSA 86, 10029). -10033 (1989); Rozak MJ. Et al., J Biol Chem 271, 22611-22618 (1996); Shearman CW. Et al., J. Immunol 147, 4366-4373 (1991)). The third method is to select the most commonly used amino acid in human antibody FRs (Sato K. et al., Mol Immunol 31, 371-381 (1994); Kobinger F. et al., Protein Engineering 6, 971-980 (1993); Kettleborough CA. et al., Protein Engineering 4, 773-783 (1991)). Any of these methods can be used in the present invention.

  Even if the amino acid sequence of the selected human FR is modified, it should be used as the FR amino acid sequence as long as the finally obtained human CDR-grafted antibody has a specific binding property to the target antigen. Can do. In particular, when a part of the amino acid of the selected human FR is changed to the FR amino acid of the antibody derived from CDR, it is highly possible that the characteristics of the antibody are maintained. The number of amino acids to be modified is preferably 30% or less of the entire FR, more preferably 20% or less of the entire FR, and further preferably 10% or less of the entire FR.

  Next, a DNA encoding the H chain variable region and the L chain variable region is designed by combining the FR selected by any of these methods and the CDR. Based on this design, DNA encoding the H chain variable region and DNA encoding the L chain variable region are respectively prepared by chemical synthesis, biochemical cleavage / recombination, and the like. Then, the DNA encoding the H chain variable region is incorporated into an expression vector together with the DNA encoding the human immunoglobulin H chain constant region to construct an H chain expression vector. Similarly, an L chain expression vector is constructed by incorporating DNA encoding the L chain variable region into an expression vector together with DNA encoding the human immunoglobulin L chain constant region. Examples of expression vectors include, but are not limited to, SV40 virus based vectors, EB virus based vectors, BPV (papilloma virus) based vectors, and the like.

  A host cell is cotransformed with the H chain expression vector and the L chain expression vector prepared by the above method. As host cells, CHO cells (Chinese hamster ovary) (A. Wright & SL Morrison, J. Immunol. 160, 3393-3402 (1998)), SP2 / 0 cells (mouse myeloma) (K. Motmans etal., Eur. J Cancer Prev. 5, 512-519 (1996), RP Junghans et al., Cancer Res. 50, 1495-1502 (1990)) and the like are preferably used. For transformation, the lipofectin method (RWMalone et al., Proc. Natl. Acad. Sci. USA 86, 6077 (1989), PLFelgner et al., Proc. Natl. Acad. Sci. USA 84, 7413 ( 1987), electroporation method, calcium phosphate method (FL Graham & AJvan der Eb, Virology 52, 456-467 (1973)), DEAE-Dextran method and the like are preferably used.

  After culturing the transformant, the human CDR-grafted antibody is isolated from the transformant cells or from the culture solution. Separation and purification of antibodies include centrifugation, ammonium sulfate fractionation, salting out, ultrafiltration, affinity chromatography, ion exchange chromatography, gel filtration chromatography, protein A column chromatography, protein G column chromatography, protein L column Methods such as chromatography can be appropriately combined.

  A method for obtaining a human antibody is also known. For example, human lymphocytes are sensitized with a desired antigen or a cell that expresses the desired antigen in vitro, and the sensitized lymphocyte is fused with a human myeloma cell such as U266 to have a desired human antibody having an antigen-binding activity. (See Japanese Patent Publication No. 1-59878). Further, a desired human antibody can be obtained by immunizing a transgenic animal having all repertoires of human antibody genes with a desired antigen (International Patent Application Publication Nos. WO 93/12227, WO 92/03918, WO 94/02602, WO 94/25585, WO 96/34096, WO 96/33735).

  In a further embodiment, antibodies or antibody fragments can be separated from antibody phage libraries produced using techniques described in McCafferty et al. (Nature, 348: 552-554 (1990)). Clackson et al. (Nature, 352: 624-628 (1991)) and Marks et al. (J. Mol. Biol., 222: 581-597 (1991)) describe the separation of mouse and human antibodies using phage libraries. is doing. Subsequent publications to generate high affinity (nM range) human antibodies by chain shuffling (Marks et al., Bio / Technology, 10: 779-783 [1992]), as well as to construct very large phage libraries Describes combinatorial infection and in vivo recombination (Waterhouse et al., Nuc. Acids. Res., 21: 2265-2266 [1993]). These techniques are therefore viable alternatives to traditional monoclonal antibody hybridoma methods for the separation of monoclonal antibodies.

  In this regard, bacteriophage (phage) display is well known to allow searching large oligopeptide libraries to identify those library members capable of specifically binding to a polypeptide target. Technology. Phage display is a technique by which various polypeptides are presented as fusion proteins to coat proteins on the surface of bacteriophage particles (Scott, J.K. and Smith G. P. (1990) Science 249: 386). The usefulness of phage display can quickly and effectively classify large libraries of selectively randomized protein variants (or random clone cDNAs) for those sequences that bind to target molecules with high affinity. In the point. Peptides on phage (Cwirla, SE et al. (1990) Proc. Natl. Acad. Sci. USA, 87: 6378) or proteins (Lowman, HB et al. (1991) Biochemistry, 30: 10832; Clackson, T. et al. (1991) Nature, 352: 624; Marks, JD et al. (1991), J. Mol. Biol., 222: 581; Kang, AS et al. (1991) Proc. Natl. Acad. Sci. USA, 88: 8363) Library display Has been used to screen a myriad of polypeptides or oligopeptides for those with specific binding properties (Smith, GP (1991) Current Opin. Biotechnol., 2: 668). Classification of phage libraries of random mutants requires methods to construct and propagate a large number of variants, methods of affinity purification using target receptors, and means to evaluate the results of enhanced binding (US) Nos. 5223409, 5,403,484, 5,571,689, and 5,663,143).

  Although most phage display methods used filamentous phage, the λ phage display system (WO95 / 34683; US Pat. No. 5,627,024), the T4 phage display system (RenJ. Et al. Gene 215: 439 (1998); Zhu et al. Cancer Researc, 58 (15): 3209-3214 (1998); Jiang et al. Infection & Immunity, 65 (11): 4770-4777 (1997); Ren et al., Gene, 195 (2): 303-311 (1997); Ren, Protein Sci. 5: 1833 (1996); Efimov et al. VirusGenes 10: 173 (1995) and T7 phage display system (Smith and Scott Methods in Enzymology, 217, 228-257 (1993); US Pat. No. 5,766,905) Are known.

  Currently, many improvements and variations of the basic phage display method have been developed. These improvements have improved the method of screening from peptide libraries and protein libraries based on properties, abilities, etc., such as binding to selected target molecules. Recombination reaction means for the phage display method is described in WO98 / 14277. Phage display libraries have been used to analyze and control bimolecular interactions (WO 98/20169; WO 98/20159) and restricted helix peptide properties (WO 98/20036). In WO97 / 35196, a phage display library is contacted with a first solution in which a ligand can bind to a target molecule and a second solution in which an affinity ligand does not bind to the target molecule to selectively isolate the bound ligand. A method for isolating affinity ligands is described. WO 97/46251 describes a method of biopanning a random phage display library with affinity purified antibodies, then isolating bound phage, followed by micropanning in the wells of a microplate to isolate high affinity binding phage. To do. The use of Staphlylococcus aureus protein A as an affinity tag has also been reported (Li et al., (1998) Mol Biotech., 9: 187). WO 97/47314 describes the use of a substrate subtraction library to identify enzyme specificity using a combinatorial library that may be a phage display library. A method for selecting enzymes suitable for use in detergents used for phage display is described in WO 97/09446. Additional methods for selecting proteins that specifically bind are described in US Pat. Nos. 5,498,538, 5,432,018, and WO 98/15833. Methods for preparing peptide libraries and screening these libraries are described in US Pat. Nos. 5,723,286, 5,432,018, 5,580,717, 5,427,908, 5,498,530, 5,770,434 and 5,734,018. Nos. 5,698,426, 5762192, and 5723323.

  Furthermore, a technique for obtaining a human antibody by panning using a human antibody library is also known. For example, the variable region of a human antibody is expressed as a single chain antibody (scFv) on the surface of the phage by the phage display method, and a phage that binds to the antigen can be selected. By analyzing the gene of the selected phage, the DNA sequence encoding the variable region of the human antibody that binds to the antigen can be determined. If the DNA sequence of scFv that binds to the antigen is clarified, a human antibody can be obtained by preparing an appropriate expression vector having the sequence and introducing it into an appropriate host for expression. These methods are already well known and can be carried out with reference to WO 92/01047, WO 92/20791, WO 93/06213, WO 93/11236, WO 93/19172, WO 95/01438, and WO 95/15388. it can.

  Alternatively, human antibodies and antibody fragments can be generated in vitro from the immunoglobulin variable (V) domain gene repertoire of non-immunized donors using phage display technology (McCafferty et al., Nature 348: 552-553 [1990]). Can be produced. According to this technique, antibody V domain genes are cloned in frame, either in filamentous bacteriophages, eg, M13 phage or fd coat protein genes, and displayed as functional antibody fragments on the surface of phage particles. Since the filamentous particle contains a single-stranded DNA copy of the phage genome, the selection of a gene encoding an antibody exhibiting these properties results as a result even based on selection based on the functional properties of the antibody. Thus, this phage mimics some properties of B cells. Phage display can be performed in a variety of formats; see, eg, Johnson, Kevin S. and Chiswell, David J., Current Opinion in Structural Biology 3: 564-571 (1993). Several sources of V-gene segments can be used for phage display. Clackson et al., Nature, 352: 624-628 (1991) isolated a large variety of anti-oxazolone antibodies from a small random combinatorial library of V genes derived from the spleens of immunized mice. The V gene repertoire of non-immunized human donors is configurable, and antibodies against diverse and many antigens (including self-antigens) can be found in Marks et al., J. Mol. Biol. 222: 581-597 (1991), or Griffith. Et al., EMBO J. 12: 725-734 (1993). See also U.S. Pat. Nos. 5,565,332 and 5,573,905.

  The antibody of the present invention also includes a fusion protein in which the antibody of the present invention and another peptide or protein are fused. In the method for producing a fusion protein, a polynucleotide encoding the antibody of the present invention and a polynucleotide encoding another peptide or polypeptide are linked so that the frames coincide with each other, introduced into an expression vector, and expressed in a host. Any technique known to those skilled in the art can be used. Other peptides or polypeptides to be subjected to fusion with the antibody of the present invention include, for example, FLAG (Hopp, TP et al., BioTechnology (1988) 6, 1204-1210), 6 His (histidine) residues 6 × His, 10 × His, influenza agglutinin (HA), human c-myc fragment, VSV-GP fragment, p18HIV fragment, T7-tag, HSV-tag, E-tag, SV40T antigen Known peptides such as fragments, lck tag, α-tubulin fragment, B-tag, and protein C fragment can be used. Further, examples of other polypeptides to be subjected to fusion with the antibody of the present invention include GST (glutathione-S-transferase), HA (influenza agglutinin), β-galactosidase, MBP (maltose binding protein) and the like. Can be mentioned.

In the antibody of the present invention, a sugar chain can be added. Glycosylation to the antibody can be achieved by linking the -SH group in the mutated cysteine and the bromoacetyl group introduced into the sugar chain to introduce the sugar chain into the antibody, or fucose transferase-deficient cells. Alternatively, it can be performed using fucose transfer knockdown cells. By providing a sugar chain, peptide or polypeptide, the effector function of the antibody can be improved. The effector function includes, but is not limited to, ADCC activity and CDC activity.
Furthermore, the antibody of the present invention includes an antibody in which some amino acids are chemically modified such as acetylation, PEGylation, phosphorylation, amidation. Such chemical modification can be performed by methods known to those skilled in the art.

The antibody of the present invention is characterized by binding to FcγRI, FcγRII, or FcγRIII on the surface of effector cells. Effector function is also mediated by the interaction of the Fc receptor (FcR) with the Fc region of an antibody. Fc receptors are known as differentiated cell surface receptors present on hematopoietic cells. Among Fc receptors, Fc receptor for IgG antibody is called FcγR, Fc receptor for IgE is called FcεR, and Fc receptor for IgA is called FcαR, and FcR is defined by immunoglobulin isotype. Regarding Fcγ receptors, three subclasses of FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16) have been identified. Since each FcγR subclass is encoded by two or three genes and alternative RNA splicing results in multiple transcripts, there is a wide diversity in FcγR isoforms. Three genes encoding FcγRI subclasses (FcγRIA, FcγRIB, and FcγRIC) are clustered in region 1q21.1 of the long arm of chromosome 1 and encode FcγRII isoforms (FcγRIIA, FcγRIIB, and FcγRIIC) And the two genes encoding FcγRIll (FcγRIIIA and FcγRIIIB) are all clustered in region 1q22. FcR is described in Ravetch and Kinet, Annu. Rev. Immunol 9: 457-92 (1991); Capel et al., Immunomethods 4: 25-34 (1994); and de Haas et al., J Lab. Clin. Med. 126: 330-41 (1995).
While FcγRI binds to monomeric IgG with high affinity, FcγRII and FcγRIII are low-affinity receptors that interact with complex or massive IgG. A classic method for detecting these low affinity receptors is known as “rosette formation” using red blood cells (EA) covered with antibodies sensitized by IgG (Bredius et al. Immunology 83: 624-630 (1994) and with regard to rosette assay, Tax et al. J. Immunol. 133 (3): 1185-1189 (1984); Nagarajan et al. J. Biol. Chem. 270 (43) : 25762-25770 (1995); and also Warmerdam et al. J. Immunol. 147 (4): 1338-1343 (1991)).
In the present invention, the effector cell is not particularly limited, and examples thereof include PBMC.

  Furthermore, in the present invention, deamidation is suppressed by substituting an amino acid adjacent to the amino acid deamidated or deamidated with another amino acid for the purpose of increasing the stability of the antibody. May be. Examples of amino acids to be deamidated include asparagine and glutamine, and asparagine is preferred. The amino acid adjacent to asparagine is not particularly limited and may be any amino acid. However, since the asparagine-glycine sequence is known to be particularly easily deamidated, glycine is preferable as the amino acid adjacent to asparagine. The amino acid after substitution is not particularly limited as long as it is other than asparagine and glutamine, and any amino acid may be used, but amino acids other than valine and proline are preferable. Therefore, in the present invention, when deamidation of an antibody is intended, substitution with an amino acid other than asparagine, glutamine, valine or proline is preferred. Suppression of deamidation by amino acid substitution can be performed with reference to, for example, WO03 / 057881. When amino acid substitution is performed for the purpose of suppressing deamidation, it is preferable to maintain the antigen-binding activity before substitution.

  Furthermore, as another aspect of antibody stabilization, substitution of glutamic acid with another amino acid can be mentioned. Further, in the present invention, it has been found that when the sixth antibody heavy chain is glutamic acid, the antibody can be remarkably stabilized by replacing the glutamic acid with glutamine. Therefore, the present invention relates to a method for stabilizing an antibody by replacing the sixth glutamic acid in the antibody heavy chain with glutamine. The amino acid number in the antibody is known to those skilled in the art (eg, Kabat, EA et al., “Sequences of Proteins of Immunological Interest”, US Dept. Health and Human Services 1983).

  The antibody of the present invention may be a conjugated antibody bound to various molecules such as polyethylene glycol (PEG), radioactive substances, and toxins. Such a conjugated antibody can be obtained by chemically modifying the obtained antibody. Antibody modification methods have already been established in this field. These conjugate antibodies are also included in the antibody of the present invention.

  The antibody used in the present invention may be a bispecific antibody (for example, Journal of Immunology, 1994, 152, 5368-5374, etc.). Bispecific antibodies may recognize one of ALCAM, ICAM1, and EGFR, the other may recognize other antigens, or recognize different epitopes on either ALCAM, ICAM1, or EGFR molecules. May be.

  The antibody of the present invention may be fused with another protein at its N-terminus or C-terminus (Clinical Cancer Research, 2004, 10, 1274-1281). Those skilled in the art can appropriately select the protein to be fused.

  The present invention also provides a nucleic acid encoding the antibody of the present invention, a vector into which the nucleic acid has been inserted, and a transformed cell into which the vector has been introduced. Examples of vectors include M13 vectors, pUC vectors, pBR322, pBluescript, pCR-Script, and the like. In addition, for the purpose of subcloning and excision of cDNA, in addition to the above vector, for example, pGEM-T, pDIRECT, pT7 and the like can be mentioned.

  As an expression vector, for example, for the purpose of expression in E. coli, in addition to the above characteristics that the vector is amplified in E. coli, the host is E. coli such as JM109, DH5α, HB101, XL1-Blue, etc. In some cases, promoters that can be efficiently expressed in E. coli, such as the lacZ promoter (Ward et al., Nature (1989) 341, 544-546; FASEB J. (1992) 6, 2422-2427), araB promoter (Better et al. , Science (1988) 240, 1041-1043), or the T7 promoter or the like. In addition to the above vectors, such vectors include pGEX-5X-1 (Pharmacia), “QIAexpress system” (Qiagen), pEGFP, or pET (in this case, the host expresses T7 RNA polymerase). BL21 is preferred).

  The vector may contain a signal sequence for polypeptide secretion. As a signal sequence for protein secretion, the pelB signal sequence (Lei, S. P. et al J. Bacteriol. (1987) 169, 4379) may be used when the protein is produced in the periplasm of E. coli. Introduction of a vector into a host cell can be performed using, for example, a calcium chloride method or an electroporation method.

  In addition to E. coli, for example, mammalian-derived expression vectors (for example, pcDNA3 (manufactured by Invitrogen), pEGF-BOS (Nucleic Acids. Res. 1990, 18 (17), p5322), pEF, pCDM8), insects Cell-derived expression vectors (eg, “Bac-to-BAC baculovairus expression system” (manufactured by Gibco BRL), pBacPAK8), plant-derived expression vectors (eg, pMH1, pMH2), animal virus-derived expression vectors (eg, pHSV, pMV, pAdexLcw), retrovirus-derived expression vectors (eg, pZIPneo), yeast-derived expression vectors (eg, “Pichia Expression Kit” (manufactured by Invitrogen), pNV11, SP-Q01), Bacillus subtilis-derived expression vectors (For example, pPL608, pKTH50).

  When intended for expression in animal cells such as CHO cells, COS cells, NIH3T3 cells, etc., a promoter necessary for expression in the cells, such as the SV40 promoter (Mulligan et al., Nature (1979) 277, 108), It is essential to have MMTV-LTR promoter, EF1α promoter (Mizushima et al., Nucleic Acids Res. (1990) 18, 5322), CMV promoter, etc., and genes for selecting transformation into cells (for example, More preferably, it has a drug (drug resistance gene that can be discriminated by a drug such as neomycin, G418, or puromycin). Examples of such a vector include pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, and pOP13.

  Furthermore, when the gene is stably expressed and the purpose is to amplify the copy number of the gene in the cell, a vector having a DHFR gene complementary to the CHO cell lacking the nucleic acid synthesis pathway (for example, , PCHOI, etc.) and amplifying with methotrexate (MTX), and for the purpose of transient expression of the gene, COS having a gene expressing SV40 T antigen on the chromosome An example is a method of transforming with a vector (such as pcD) having an SV40 replication origin using cells. As the replication origin, those derived from polyoma virus, adenovirus, bovine papilloma virus (BPV) and the like can also be used. Furthermore, for gene copy number amplification in host cell systems, the expression vectors are selectable markers: aminoglycoside transferase (APH) gene, thymidine kinase (TK) gene, E. coli xanthine guanine phosphoribosyltransferase (Ecogpt) gene, dihydrofolate reductase ( dhfr) gene and the like.

  The host cell into which the vector is introduced is not particularly limited, and for example, E. coli and various animal cells can be used. The host cell can be used, for example, as a production system for production and expression of the antibody of the present invention. Production systems for producing polypeptides include in vitro and in vivo production systems. Examples of in vitro production systems include production systems that use eukaryotic cells and production systems that use prokaryotic cells.

  When eukaryotic cells are used, for example, animal cells, plant cells, and fungal cells can be used as the host. Animal cells include mammalian cells such as CHO (J. Exp. Med. (1995) 108, 945), COS, 3T3, myeloma, BHK (baby hamster kidney), HeLa, Vero, amphibian cells such as Xenopus eggs Mother cells (Valle, et al., Nature (1981) 291, 358-340) or insect cells such as Sf9, Sf21, Tn5 are known. In the present invention, CHO-DG44, CHO-DXB11, COS7 cells, and BHK cells are preferably used. In animal cells, CHO cells are particularly preferred for mass expression purposes. Introduction of a vector into a host cell can be performed by, for example, a calcium phosphate method, a DEAE dextran method, a method using a cationic ribosome DOTAP (Boehringer Mannheim), an electroporation method, a lipofection method, or the like.

  As plant cells, for example, cells derived from Nicotiana tabacum are known as protein production systems, and these may be cultured in callus. Fungal cells include yeasts such as the genus Saccharomyces, such as Saccharomyces cerevisiae, Saccharomyces pombe, filamentous fungi such as the genus Aspergillus, such as Aspergillus Aspergillus niger) is known.

  When prokaryotic cells are used, there are production systems using bacterial cells. Examples of bacterial cells include E. coli, for example, JM109, DH5α, HB101, and others, and Bacillus subtilis is known. The antibody of the present invention can be obtained by culturing cells transformed with the DNA of the present invention in vitro and purifying the cells by a method commonly used by those skilled in the art.

  The present invention also provides a host organism having a vector comprising a nucleic acid encoding the antibody of the present invention. The host organisms of the present invention are useful for the production of recombinant antibodies. Examples of host organisms in the present invention include goats. For example, the transgenic goat of the present invention can be produced as follows. That is, a fusion gene is constructed in which an antibody gene is inserted in-frame into a gene encoding a protein (such as goat β-casein) that is uniquely produced in milk. When a DNA fragment containing a fusion gene into which an antibody gene has been inserted is injected into a goat embryo, the injected embryo is introduced into a female goat. The antibody of the present invention can be obtained from the milk produced by a transgenic goat born from a goat that has received the embryo or its offspring. Hormones can also be used as appropriate in transgenic goats to increase the amount of milk containing the antibody of the invention produced from the transgenic goat (Ebert, KM etal., Bio / Technology (1994) 12, 699-702).

The antibody of the present invention is preferably an antibody that recognizes a molecule present on the surface of a target cell to be damaged. The target cell from which cytotoxicity is desired is preferably human, but is not limited thereto. In addition, the target cell to be damaged is not particularly limited, for example, cancer cells, Raji, KG-1a, TL-1, HUT78, Jurkat, BALL-1, HEPG2, MKN-7, KB, or Hela However, it is not limited to these. Examples of the molecule (antigen) present on the surface of the target cell to be damaged by a cell include, but are not limited to, a target antigen selected from the group consisting of ALCAM, ICAM1, and EGFR.
Specific examples of the 286Cys anti-ALCAM (066-174) antibody include, but are not limited to, the following antibodies.
(1) The amino acid sequence described in SEQ ID NO: 12 as CDR1, the amino acid sequence described in SEQ ID NO: 13 as CDR2, the amino acid sequence described in SEQ ID NO: 14 as CDR3, and the amino acid described in SEQ ID NO: 91 as CH An antibody comprising an H chain having a sequence;
(2) an antibody comprising an H chain having the amino acid sequence set forth in SEQ ID NO: 95,
(3) The amino acid sequence described in SEQ ID NO: 12 as CDR1, the amino acid sequence described in SEQ ID NO: 13 as CDR2, the amino acid sequence described in SEQ ID NO: 14 as CDR3, and the amino acid sequence described in SEQ ID NO: 97 as Fc region An antibody comprising an H chain having an amino acid sequence,
(4) An antibody having a pair of the H chain according to any one of (1), (2) or (3) above and the L chain according to (i) or (ii) below,
(I) The amino acid sequence described in SEQ ID NO: 4 as CDR1, the amino acid sequence described in SEQ ID NO: 5 as CDR2, the amino acid sequence described in SEQ ID NO: 6 as CDR3, and the amino acid described in SEQ ID NO: 8 as CL A light chain having a sequence;
(Ii) An L chain having the amino acid sequence set forth in SEQ ID NO: 99.
Specific examples of the 298Cys-type Anti-ALCAM (066-174) antibody include, but are not limited to, the following antibodies.
(1) The amino acid sequence described in SEQ ID NO: 12 as CDR1, the amino acid sequence described in SEQ ID NO: 13 as CDR2, the amino acid sequence described in SEQ ID NO: 14 as CDR3, and the amino acid described in SEQ ID NO: 93 as CH3 An antibody comprising an H chain having a sequence;
(2) an antibody comprising an H chain having the amino acid sequence of SEQ ID NO: 101,
(3) The amino acid sequence described in SEQ ID NO: 12 as CDR1, the amino acid sequence described in SEQ ID NO: 13 as CDR2, the amino acid sequence described in SEQ ID NO: 14 as CDR3, and the amino acid sequence described in SEQ ID NO: 103 as Fc region An antibody comprising an H chain having an amino acid sequence,
(4) An antibody having a pair of the H chain according to any one of (1), (2) or (3) above and the L chain according to (i) or (ii) below,
(I) The amino acid sequence described in SEQ ID NO: 4 as CDR1, the amino acid sequence described in SEQ ID NO: 5 as CDR2, the amino acid sequence described in SEQ ID NO: 6 as CDR3, and the amino acid described in SEQ ID NO: 8 as CL A light chain having a sequence;
(Ii) An L chain having the amino acid sequence set forth in SEQ ID NO: 99.
Specific examples of the 286Cys-type Anti-ALCAM (035-234) antibody include, but are not limited to, the following antibodies.
(1) The amino acid sequence described in SEQ ID NO: 42 as CDR1, the amino acid sequence described in SEQ ID NO: 43 as CDR2, the amino acid sequence described in SEQ ID NO: 44 as CDR3, and the amino acid described in SEQ ID NO: 91 as CH An antibody comprising an H chain having a sequence;
(2) an antibody comprising an H chain having the amino acid sequence of SEQ ID NO: 105,
(3) The amino acid sequence described in SEQ ID NO: 42 as CDR1, the amino acid sequence described in SEQ ID NO: 43 as CDR2, the amino acid sequence described in SEQ ID NO: 44 as CDR3, and the amino acid sequence described in SEQ ID NO: 107 as Fc region An antibody comprising an H chain having an amino acid sequence,
(4) An antibody having a pair of the H chain according to any one of (1), (2) or (3) above and the L chain according to (i) or (ii) below,
(I) The amino acid sequence described in SEQ ID NO: 34 as CDR1, the amino acid sequence described in SEQ ID NO: 35 as CDR2, the amino acid sequence described in SEQ ID NO: 36 as CDR3, and the amino acid described in SEQ ID NO: 38 as CL A light chain having a sequence;
(Ii) An L chain having the amino acid sequence set forth in SEQ ID NO: 109.
Specific examples of the 298Cys-type Anti-ALCAM (035-234) antibody include, but are not limited to, the following antibodies.
(1) The amino acid sequence described in SEQ ID NO: 42 as CDR1, the amino acid sequence described in SEQ ID NO: 43 as CDR2, the amino acid sequence described in SEQ ID NO: 44 as CDR3, and the amino acid described in SEQ ID NO: 93 as CH3 An antibody comprising an H chain having a sequence;
(2) an antibody comprising an H chain having the amino acid sequence set forth in SEQ ID NO: 111,
(3) The amino acid sequence described in SEQ ID NO: 42 as CDR1, the amino acid sequence described in SEQ ID NO: 43 as CDR2, the amino acid sequence described in SEQ ID NO: 44 as CDR3, and the amino acid sequence described in SEQ ID NO: 113 as Fc region An antibody comprising an H chain having an amino acid sequence,
(4) An antibody having a pair of the H chain according to any one of (1), (2) or (3) above and the L chain according to (i) or (ii) below,
(I) The amino acid sequence described in SEQ ID NO: 34 as CDR1, the amino acid sequence described in SEQ ID NO: 35 as CDR2, the amino acid sequence described in SEQ ID NO: 36 as CDR3, and the amino acid described in SEQ ID NO: 38 as CL A light chain having a sequence;
(Ii) An L chain having the amino acid sequence set forth in SEQ ID NO: 109.
Specific examples of the 286 Cys-type Anti-ICAM1 (053-059) antibody include, but are not limited to, the following antibodies.
(1) The amino acid sequence described in SEQ ID NO: 57 as CDR1, the amino acid sequence described in SEQ ID NO: 58 as CDR2, the amino acid sequence described in SEQ ID NO: 59 as CDR3, and the amino acid described in SEQ ID NO: 91 as CH An antibody comprising an H chain having a sequence;
(2) an antibody comprising an H chain having the amino acid sequence set forth in SEQ ID NO: 115,
(3) The amino acid sequence described in SEQ ID NO: 57 as CDR1, the amino acid sequence described in SEQ ID NO: 58 as CDR2, the amino acid sequence described in SEQ ID NO: 59 as CDR3, and the amino acid sequence described in SEQ ID NO: 117 as Fc region An antibody comprising an H chain having an amino acid sequence,
(4) An antibody having a pair of the H chain according to any one of (1), (2) or (3) above and the L chain according to (i) or (ii) below,
(I) the amino acid sequence described in SEQ ID NO: 49 as CDR1, the amino acid sequence described in SEQ ID NO: 50 as CDR2, the amino acid sequence described in SEQ ID NO: 51 as CDR3, and the amino acid described in SEQ ID NO: 53 as CL A light chain having a sequence;
(Ii) An L chain having the amino acid sequence set forth in SEQ ID NO: 119.
Specific examples of the 298 Cys-type Anti-ICAM1 (053-059) antibody include, but are not limited to, the following antibodies.
(1) The amino acid sequence described in SEQ ID NO: 57 as CDR1, the amino acid sequence described in SEQ ID NO: 58 as CDR2, the amino acid sequence described in SEQ ID NO: 59 as CDR3, and the amino acid described in SEQ ID NO: 93 as CH An antibody comprising an H chain having a sequence;
(2) an antibody comprising an H chain having the amino acid sequence of SEQ ID NO: 121,
(3) The amino acid sequence described in SEQ ID NO: 57 as CDR1, the amino acid sequence described in SEQ ID NO: 58 as CDR2, the amino acid sequence described in SEQ ID NO: 59 as CDR3, and the amino acid sequence described in SEQ ID NO: 123 as Fc region An antibody comprising an H chain having an amino acid sequence,
(4) An antibody having a pair of the H chain according to any one of (1), (2) or (3) above and the L chain according to (i) or (ii) below,
(I) the amino acid sequence described in SEQ ID NO: 49 as CDR1, the amino acid sequence described in SEQ ID NO: 50 as CDR2, the amino acid sequence described in SEQ ID NO: 51 as CDR3, and the amino acid described in SEQ ID NO: 53 as CL A light chain having a sequence;
(Ii) An L chain having the amino acid sequence set forth in SEQ ID NO: 119.
Specific examples of the 286Cys type Anti-ICAM1 (053-085) antibody include, but are not limited to, the following antibodies.
(1) The amino acid sequence described in SEQ ID NO: 71 as CDR1, the amino acid sequence described in SEQ ID NO: 72 as CDR2, the amino acid sequence described in SEQ ID NO: 73 as CDR3, and the amino acid described in SEQ ID NO: 91 as CH An antibody comprising an H chain having a sequence;
(2) an antibody comprising an H chain having the amino acid sequence set forth in SEQ ID NO: 125,
(3) The amino acid sequence described in SEQ ID NO: 71 as CDR1, the amino acid sequence described in SEQ ID NO: 72 as CDR2, the amino acid sequence described in SEQ ID NO: 73 as CDR3, and the amino acid sequence described in SEQ ID NO: 127 as Fc region An antibody comprising an H chain having an amino acid sequence,
(4) An antibody having a pair of the H chain according to any one of (1), (2) or (3) above and the L chain according to (i) or (ii) below,
(I) The amino acid sequence described in SEQ ID NO: 63 as CDR1, the amino acid sequence described in SEQ ID NO: 64 as CDR2, the amino acid sequence described in SEQ ID NO: 65 as CDR3, and the amino acid described in SEQ ID NO: 67 as CL A light chain having a sequence;
(Ii) An L chain having the amino acid sequence set forth in SEQ ID NO: 129.
Specific examples of the 298Cys type Anti-ICAM1 (053-085) antibody include, but are not limited to, the following antibodies.
(1) The amino acid sequence described in SEQ ID NO: 71 as CDR1, the amino acid sequence described in SEQ ID NO: 72 as CDR2, the amino acid sequence described in SEQ ID NO: 73 as CDR3, and the amino acid described in SEQ ID NO: 93 as CH3 An antibody comprising an H chain having a sequence;
(2) an antibody comprising an H chain having the amino acid sequence set forth in SEQ ID NO: 131,
(3) The amino acid sequence described in SEQ ID NO: 71 as CDR1, the amino acid sequence described in SEQ ID NO: 72 as CDR2, the amino acid sequence described in SEQ ID NO: 73 as CDR3, and the amino acid sequence described in SEQ ID NO: 133 as an Fc region An antibody comprising an H chain having an amino acid sequence,
(4) An antibody having a pair of the H chain according to any one of (1), (2) or (3) above and the L chain according to (i) or (ii) below,
(I) The amino acid sequence described in SEQ ID NO: 63 as CDR1, the amino acid sequence described in SEQ ID NO: 64 as CDR2, the amino acid sequence described in SEQ ID NO: 65 as CDR3, and the amino acid described in SEQ ID NO: 67 as CL A light chain having a sequence;
(Ii) An L chain having the amino acid sequence set forth in SEQ ID NO: 129.
Specific examples of the 286Cys anti-EGFR (048-006) antibody include, but are not limited to, the following antibodies.
(1) The amino acid sequence described in SEQ ID NO: 86 as CDR1, the amino acid sequence described in SEQ ID NO: 87 as CDR2, the amino acid sequence described in SEQ ID NO: 88 as CDR3, and the amino acid described in SEQ ID NO: 91 as CH An antibody comprising an H chain having a sequence;
(2) an antibody comprising an H chain having the amino acid sequence set forth in SEQ ID NO: 135,
(3) The amino acid sequence described in SEQ ID NO: 86 as CDR1, the amino acid sequence described in SEQ ID NO: 87 as CDR2, the amino acid sequence described in SEQ ID NO: 88 as CDR3, and the amino acid sequence described in SEQ ID NO: 137 as an Fc region An antibody comprising an H chain having an amino acid sequence,
(4) An antibody having a pair of the H chain according to any one of (1), (2) or (3) above and the L chain according to (i) or (ii) below,
(I) The amino acid sequence set forth in SEQ ID NO: 78 as CDR1, the amino acid sequence set forth in SEQ ID NO: 79 as CDR2, the amino acid sequence set forth in SEQ ID NO: 80 as CDR3, and the amino acid set forth in SEQ ID NO: 82 as CL A light chain having a sequence;
(Ii) An L chain having the amino acid sequence set forth in SEQ ID NO: 139.
Specific examples of the 298 Cys-type Anti-EGFR (048-006) antibody include, but are not limited to, the following antibodies.
(1) The amino acid sequence described in SEQ ID NO: 86 as CDR1, the amino acid sequence described in SEQ ID NO: 87 as CDR2, the amino acid sequence described in SEQ ID NO: 88 as CDR3, and the amino acid described in SEQ ID NO: 93 as CH3 An antibody comprising an H chain having a sequence;
(2) an antibody comprising an H chain having the amino acid sequence set forth in SEQ ID NO: 141,
(3) The amino acid sequence described in SEQ ID NO: 86 as CDR1, the amino acid sequence described in SEQ ID NO: 87 as CDR2, the amino acid sequence described in SEQ ID NO: 88 as CDR3, and the amino acid sequence described in SEQ ID NO: 143 as an Fc region An antibody comprising an H chain having an amino acid sequence,
(4) An antibody having a pair of the H chain according to any one of (1), (2) or (3) above and the L chain according to (i) or (ii) below,
(I) The amino acid sequence set forth in SEQ ID NO: 78 as CDR1, the amino acid sequence set forth in SEQ ID NO: 79 as CDR2, the amino acid sequence set forth in SEQ ID NO: 80 as CDR3, and the amino acid set forth in SEQ ID NO: 82 as CL A light chain having a sequence;
(Ii) An L chain having the amino acid sequence set forth in SEQ ID NO: 139.

  As described above, the antibodies of the present invention also include recombinant antibodies such as human antibodies and humanized antibodies. Humanized antibodies include chimeric antibodies (particularly human chimeric antibodies) and CDR grafted antibodies (particularly human CDR grafted antibodies). Specific descriptions of these antibodies are as described above.

The present invention also provides, for example, the 286 Cys-type Anti-ALCAM (066-174) antibody described in any of (1) to (4) below.
(1) CDR1 encoded from the base sequence set forth in SEQ ID NO: 9, CDR2 encoded from the base sequence set forth in SEQ ID NO: 10, CDR3 encoded from the base sequence set forth in SEQ ID NO: 11, and sequence An antibody comprising an H chain having CH encoded by the nucleotide sequence of No. 90;
(2) an antibody comprising an H chain encoded by the base sequence set forth in SEQ ID NO: 94,
(3) CDR1 encoded from the base sequence set forth in SEQ ID NO: 9, CDR2 encoded from the base sequence set forth in SEQ ID NO: 10, CDR3 encoded from the base sequence set forth in SEQ ID NO: 11, and sequence An antibody comprising an H chain having an Fc region encoded by the nucleotide sequence of No. 96;
(4) an antibody having a pair of the H chain according to any of (1), (2) or (3) above and the L chain according to (i) or (ii) below,
(I) CDR1 encoded from the nucleotide sequence set forth in SEQ ID NO: 1, CDR2 encoded from the nucleotide sequence set forth in SEQ ID NO: 2, CDR3 encoded from the nucleotide sequence set forth in SEQ ID NO: 3, and sequence L chain having CL encoded by the nucleotide sequence of No. 7;
(Ii) An L chain encoded from the nucleotide sequence set forth in SEQ ID NO: 98.
In addition, the present invention provides, for example, the 298Cys type Anti-ALCAM (066-174) antibody described in any of (1) to (4) below.
(1) CDR1 encoded from the base sequence set forth in SEQ ID NO: 9, CDR2 encoded from the base sequence set forth in SEQ ID NO: 10, CDR3 encoded from the base sequence set forth in SEQ ID NO: 11, and sequence An antibody comprising an H chain having CH encoded by the nucleotide sequence of No. 92;
(2) an antibody comprising an H chain encoded from the base sequence set forth in SEQ ID NO: 100,
(3) CDR1 encoded from the base sequence set forth in SEQ ID NO: 9, CDR2 encoded from the base sequence set forth in SEQ ID NO: 10, CDR3 encoded from the base sequence set forth in SEQ ID NO: 11, and sequence An antibody comprising an H chain having an Fc region encoded by the nucleotide sequence of No: 102;
(4) an antibody having a pair of the H chain according to any of (1), (2) or (3) above and the L chain according to (i) or (ii) below,
(I) CDR1 encoded from the nucleotide sequence set forth in SEQ ID NO: 1, CDR2 encoded from the nucleotide sequence set forth in SEQ ID NO: 2, CDR3 encoded from the nucleotide sequence set forth in SEQ ID NO: 3, and sequence L chain having CL encoded by the nucleotide sequence of No. 7;
(Ii) An L chain encoded from the nucleotide sequence set forth in SEQ ID NO: 98.
The present invention also provides, for example, the 286 Cys-type Anti-ALCAM (035-234) antibody described in any of (1) to (4) below.
(1) CDR1 encoded from the base sequence set forth in SEQ ID NO: 39, CDR2 encoded from the base sequence set forth in SEQ ID NO: 40, CDR3 encoded from the base sequence set forth in SEQ ID NO: 41, and sequence An antibody comprising an H chain having CH encoded by the nucleotide sequence of No. 90;
(2) an antibody comprising an H chain encoded from the base sequence set forth in SEQ ID NO: 104,
(3) CDR1 encoded from the base sequence set forth in SEQ ID NO: 39, CDR2 encoded from the base sequence set forth in SEQ ID NO: 40, CDR3 encoded from the base sequence set forth in SEQ ID NO: 41, and sequence An antibody comprising an H chain having an Fc region encoded by the nucleotide sequence of No: 106,
(4) An antibody having a pair of the H chain according to any of (1), (2) or (3) above and the L chain according to (i) or (ii) below,
(I) CDR1 encoded from the nucleotide sequence set forth in SEQ ID NO: 31, CDR2 encoded from the nucleotide sequence set forth in SEQ ID NO: 32, CDR3 encoded from the nucleotide sequence set forth in SEQ ID NO: 33, and the sequence L chain having CL encoded from the nucleotide sequence of No. 37;
(Ii) An L chain encoded from the base sequence set forth in SEQ ID NO: 108.
The present invention also provides, for example, the 298Cys-type Anti-ALCAM (035-234) antibody described in any of (1) to (4) below.
(1) CDR1 encoded from the base sequence set forth in SEQ ID NO: 39, CDR2 encoded from the base sequence set forth in SEQ ID NO: 40, CDR3 encoded from the base sequence set forth in SEQ ID NO: 41, and sequence An antibody comprising an H chain having CH encoded by the nucleotide sequence of No. 92;
(2) an antibody comprising an H chain encoded from the base sequence set forth in SEQ ID NO: 110,
(3) CDR1 encoded from the base sequence set forth in SEQ ID NO: 39, CDR2 encoded from the base sequence set forth in SEQ ID NO: 40, CDR3 encoded from the base sequence set forth in SEQ ID NO: 41, and sequence An antibody comprising an H chain having an Fc region encoded by the nucleotide sequence of No. 112;
(4) An antibody having a pair of the H chain according to any of (1), (2) or (3) above and the L chain according to (i) or (ii) below,
(I) CDR1 encoded from the nucleotide sequence set forth in SEQ ID NO: 31, CDR2 encoded from the nucleotide sequence set forth in SEQ ID NO: 32, CDR3 encoded from the nucleotide sequence set forth in SEQ ID NO: 33, and the sequence L chain having CL encoded from the nucleotide sequence of No. 37;
(Ii) An L chain encoded from the base sequence set forth in SEQ ID NO: 108.
In addition, the present invention provides, for example, the 286Cys type Anti-ICAM1 (053-059) antibody described in any of (1) to (4) below.
(1) CDR1 encoded from the base sequence set forth in SEQ ID NO: 54, CDR2 encoded from the base sequence set forth in SEQ ID NO: 55, CDR3 encoded from the base sequence set forth in SEQ ID NO: 56, and sequence An antibody comprising an H chain having CH encoded by the nucleotide sequence of No. 90;
(2) an antibody comprising an H chain encoded by the base sequence set forth in SEQ ID NO: 114,
(3) CDR1 encoded from the base sequence set forth in SEQ ID NO: 54, CDR2 encoded from the base sequence set forth in SEQ ID NO: 55, CDR3 encoded from the base sequence set forth in SEQ ID NO: 56, and sequence An antibody comprising an H chain having an Fc region encoded by the nucleotide sequence of No. 116;
(4) An antibody having a pair of the H chain according to any of (1), (2) or (3) above and the L chain according to (i) or (ii) below,
(I) CDR1 encoded from the base sequence set forth in SEQ ID NO: 46, CDR2 encoded from the base sequence set forth in SEQ ID NO: 47, CDR3 encoded from the base sequence set forth in SEQ ID NO: 48, and sequence L chain having CL encoded by the nucleotide sequence of No. 52;
(Ii) An L chain encoded from the nucleotide sequence set forth in SEQ ID NO: 118.
The present invention also provides, for example, the 298Cys anti-ICAM1 (053-059) antibody described in any of (1) to (4) below.
(1) CDR1 encoded from the base sequence set forth in SEQ ID NO: 54, CDR2 encoded from the base sequence set forth in SEQ ID NO: 55, CDR3 encoded from the base sequence set forth in SEQ ID NO: 56, and sequence An antibody comprising an H chain having CH encoded by the nucleotide sequence of No. 92;
(2) an antibody comprising an H chain encoded from the nucleotide sequence set forth in SEQ ID NO: 120,
(3) CDR1 encoded from the base sequence set forth in SEQ ID NO: 54, CDR2 encoded from the base sequence set forth in SEQ ID NO: 55, CDR3 encoded from the base sequence set forth in SEQ ID NO: 56, and sequence An antibody comprising an H chain having an Fc region encoded by the nucleotide sequence of No. 122;
(4) An antibody having a pair of the H chain according to any of (1), (2) or (3) above and the L chain according to (i) or (ii) below,
(I) CDR1 encoded from the base sequence set forth in SEQ ID NO: 46, CDR2 encoded from the base sequence set forth in SEQ ID NO: 47, CDR3 encoded from the base sequence set forth in SEQ ID NO: 48, and sequence L chain having CL encoded by the nucleotide sequence of No. 52;
(Ii) An L chain encoded from the nucleotide sequence set forth in SEQ ID NO: 118.
The present invention also provides the 286Cys anti-ICAM1 (053-085) antibody described in any of (1) to (4) below, for example.
(1) CDR1 encoded from the base sequence set forth in SEQ ID NO: 68, CDR2 encoded from the base sequence set forth in SEQ ID NO: 69, CDR3 encoded from the base sequence set forth in SEQ ID NO: 70, and sequence An antibody comprising an H chain having CH encoded by the nucleotide sequence of No. 90;
(2) an antibody comprising an H chain encoded from the base sequence set forth in SEQ ID NO: 124,
(3) CDR1 encoded from the nucleotide sequence set forth in SEQ ID NO: 68, CDR2 encoded from the nucleotide sequence set forth in SEQ ID NO: 69, CDR3 encoded from the nucleotide sequence set forth in SEQ ID NO: 70, and the sequence An antibody comprising an H chain having an Fc region encoded by the nucleotide sequence of No. 126;
(4) an antibody having a pair of the H chain according to any of (1), (2) or (3) above and the L chain according to (i) or (ii) below,
(I) CDR1 encoded from the base sequence set forth in SEQ ID NO: 60, CDR2 encoded from the base sequence set forth in SEQ ID NO: 61, CDR3 encoded from the base sequence set forth in SEQ ID NO: 62, and sequence An L chain having CL encoded by the nucleotide sequence of No. 66;
(Ii) An L chain encoded from the nucleotide sequence set forth in SEQ ID NO: 128.
The present invention also provides, for example, the 298Cys anti-ICAM1 (053-085) antibody described in any of (1) to (4) below.
(1) CDR1 encoded from the base sequence set forth in SEQ ID NO: 68, CDR2 encoded from the base sequence set forth in SEQ ID NO: 69, CDR3 encoded from the base sequence set forth in SEQ ID NO: 70, and sequence An antibody comprising an H chain having CH encoded by the nucleotide sequence of No. 92;
(2) an antibody comprising an H chain encoded by the base sequence set forth in SEQ ID NO: 130,
(3) CDR1 encoded from the nucleotide sequence set forth in SEQ ID NO: 68, CDR2 encoded from the nucleotide sequence set forth in SEQ ID NO: 69, CDR3 encoded from the nucleotide sequence set forth in SEQ ID NO: 70, and the sequence An antibody comprising an H chain having an Fc region encoded by the nucleotide sequence of No. 132;
(4) An antibody having a pair of the H chain according to any of (1), (2) or (3) above and the L chain according to (i) or (ii) below,
(I) CDR1 encoded from the base sequence set forth in SEQ ID NO: 60, CDR2 encoded from the base sequence set forth in SEQ ID NO: 61, CDR3 encoded from the base sequence set forth in SEQ ID NO: 62, and sequence An L chain having CL encoded by the nucleotide sequence of No. 66;
(Ii) An L chain encoded from the nucleotide sequence set forth in SEQ ID NO: 128.
The present invention also provides, for example, the 286 Cys-type Anti-EGFR (048-006) antibody described in any of (1) to (4) below.
(1) CDR1 encoded from the base sequence set forth in SEQ ID NO: 83, CDR2 encoded from the base sequence set forth in SEQ ID NO: 84, CDR3 encoded from the base sequence set forth in SEQ ID NO: 85, and sequence An antibody comprising an H chain having CH encoded by the nucleotide sequence of No. 90;
(2) an antibody comprising an H chain encoded by the base sequence set forth in SEQ ID NO: 134,
(3) CDR1 encoded from the base sequence set forth in SEQ ID NO: 83, CDR2 encoded from the base sequence set forth in SEQ ID NO: 84, CDR3 encoded from the base sequence set forth in SEQ ID NO: 85, and sequence An antibody comprising an H chain having an Fc region encoded by the nucleotide sequence of No. 136;
(4) an antibody having a pair of the H chain according to any of (1), (2) or (3) above and the L chain according to (i) or (ii) below,
(I) CDR1 encoded from the base sequence set forth in SEQ ID NO: 75, CDR2 encoded from the base sequence set forth in SEQ ID NO: 76, CDR3 encoded from the base sequence set forth in SEQ ID NO: 77, and sequence L chain having CL encoded from the nucleotide sequence of No. 81;
(Ii) An L chain encoded from the nucleotide sequence set forth in SEQ ID NO: 138.
The present invention also provides the 298Cys anti-EGFR (048-006) antibody described in any of (1) to (4) below, for example.
(1) CDR1 encoded from the base sequence set forth in SEQ ID NO: 83, CDR2 encoded from the base sequence set forth in SEQ ID NO: 84, CDR3 encoded from the base sequence set forth in SEQ ID NO: 85, and sequence An antibody comprising an H chain having CH encoded by the nucleotide sequence of No. 92;
(2) an antibody comprising an H chain encoded from the base sequence set forth in SEQ ID NO: 140,
(3) CDR1 encoded from the base sequence set forth in SEQ ID NO: 83, CDR2 encoded from the base sequence set forth in SEQ ID NO: 84, CDR3 encoded from the base sequence set forth in SEQ ID NO: 85, and sequence An antibody comprising an H chain having an Fc region encoded by the nucleotide sequence of No. 142;
(4) An antibody having a pair of the H chain according to any of (1), (2) or (3) above and the L chain according to (i) or (ii) below,
(I) CDR1 encoded from the base sequence set forth in SEQ ID NO: 75, CDR2 encoded from the base sequence set forth in SEQ ID NO: 76, CDR3 encoded from the base sequence set forth in SEQ ID NO: 77, and sequence L chain having CL encoded from the nucleotide sequence of No. 81;
(Ii) An L chain encoded from the nucleotide sequence set forth in SEQ ID NO: 138.

  The present invention also provides Cγ1, Cγ2, Cγ3, and Cγ4 in which the amino acid at position 286 or 298 is substituted with cysteine. Such Cγ1, Cγ2, Cγ3, and Cγ4 are useful in the production of an antibody with enhanced ADCC activity.

  A person skilled in the art, for example, can add any antibody variable region with enhanced ADCC activity by combining Cγ1, Cγ2, Cγ3, or Cγ4 in which the amino acid at position 286 or 298 is replaced with Cys. It is possible to produce an antibody that recognizes an antigen. Further, those skilled in the art can enhance ADCC activity by combining, for example, the Fc region of Cγ1, Cγ2, Cγ3, or Cγ4 in which the amino acid at position 286 or 298 is substituted with Cys with any antibody Fab region. It is possible to produce an antibody that recognizes any antigen. As described above, the antibody of the present invention is preferably an antibody that recognizes a molecule present on the surface of a target cell to be damaged. Further, the origin of the target cell to be damaged is preferably human but is not limited thereto. Examples of the molecule (antigen) present on the surface of the target cell to be damaged by a cell include, but are not limited to, a target antigen selected from the group consisting of ALCAM, ICAM1, and EGFR.

  “ALCAM (Activaed leukocyte cell adhesion molecule)” is a transmembrane protein also called as CD166 antigen, KG-CAM, CD6 Ligand, Neurolin. It is an immunoglobulin superfamily molecule containing 10 N-linked glycosylation sites. It has a molecular weight of 100 to 105 kDa and is composed of 5 extracellular Ig-like domains, an intracellular terminal of 32 amino acids, and a short transmembrane region. ALCAM is an adhesion molecule that is on activated leukocytes and has been identified as a ligand molecule for the CD6 molecule, which acts as a signal receptor on T cells. ALCAM also acts as an adhesion factor in homophylic (ALCAM-ALCAM) or heterophylic (ALCAM-CD6) interactions. It has been suggested that ALCAM can form oligomers through three C2-like domains close to the membrane at the cell-cell adhesion site. The distribution of ALCAM is not limited by cell lineage and is expressed on various cell types such as hematopoietic cells, endothelial cells, thymic cortex and medullary epithelial cells, bone marrow mesenchymal stem cells, fibroblasts, and hepatocytes. ing. Peripheral blood is weakly expressed on activated T and B cells, monocytes, circulating dendritic cells, and granulocytes. While showing a wide tissue distribution, ALCAM expression is usually restricted to cell populations involved in proliferation and migration. In the thymus, ALCAM is expressed in CD6 thymocytes and thymic epithelial cells. Therefore, interaction with CD6 molecules is thought to play a role in T cell differentiation and maturation. In addition, it is suggested that ALCAM adhesion molecules may be involved in fetal hematopoiesis, hemangioblast differentiation, and capillary formation. The role of ALCAM in canceration is variously presumed (control of MMP activity, internalization and recyling occur, ADM17 and ADAM10 (abbreviation for a disintegrin and metalloprotease), protection from apoptosis and autophasy) But there is nothing definitive. The interaction of ALCAM-CD6 is considered to be unidirectional.

  “ICAM1 (Intercellular adhesion molecule-1)” is also called Intercellular Adhesion Molecule 1 or CD54 Antigen, which is a transmembrane glycoprotein and has 7 binding sites for N-linked sugar chains. The molecular weight is 90 kDa. ICAM1 belongs to the Ig-superfamily and is known to be mainly involved in leukocyte adhesion. It mediates T lymphocyte adhesion to antigen-presenting cells (APCs) and is involved in the interaction of T cells and T cells or T cells and B cells. It is also involved in the adhesion of monocytes, lymphocytes and neutrophils to activated endothelial cells. It binds to the integrin of LFA-1 (CD11a / CD18) and Mac-1 (CD11b / CD18). It is also a rhinovirus receptor. In addition to endothelial cells, it is expressed on various types of activated cells. For example, it is expressed on monocytes and is enhanced on B and T lymphocytes, thymocytes, dendritic cells, endothelial cells, fibroblasts, keratinocytes, chondrocytes and epithelial cells. Properties that need to be acquired in the process of epithelial cells becoming cancerous include the ability to invade between cells, as well as the ability to migrate and settle upon metastasis. Thus, expression of adhesion factors is thought to contribute to carcinogenesis. Adhesion factors are large and are divided into five groups: selectins (E-, P-, L-), molecules with immunoglobulin-like domains (Ig-superfamily), integrins, cadherins and CD44. It is widely recognized that the expression of E-cadherin is suppressed during canceration. Abnormal expression has been reported in several cancers.

“EGFR (Epidermal Growth Factor Receptor)” is also called erbB1, c-erbB-1, HER1, or v-erbB.
Members of the receptor family of epidermal growth factor (EGF) are particularly important growth factor receptor tyrosine kinases associated with tumorigenesis of epithelial cells. The first member of the EGF receptor family to be discovered was EGFR expressed on many types of tumor cells. EGFR has been shown to be involved in the regulation of tumor cell division and growth, repair and survival, angiogenesis, invasion, and tumor metastasis.

  EGFR is a 170 kD transmembrane glycoprotein with an extracellular ligand binding domain, a transmembrane region, and a cytoplasmic protein tyrosine kinase domain. Examples of ligands that stimulate EGFR include epidermal growth factor (EGF), transforming growth factor α (TGR-α), heparin binding growth factor (HBGF), β-cellulin, and crypto-1 (Cripto). -1). Specific ligand binding causes EGFR autophosphorylation, activation of the receptor's cytoplasmic tyrosine kinase domain, and activation of multiple signaling pathways that control tumor growth and survival. This EGFR pathway also affects the production of various other pro-angiogenic factors such as VEGF and basic fibroblast growth factor (bFGF) in tumors.

  Growth factors that activate EGFR are also thought to play a role in tumor angiogenesis. Angiogenesis, which means the formation of capillaries from pre-existing vessels in the embryo and adult organisms, is known to be an important factor in tumor growth, survival, and metastasis. EGFR-mediated stimulation of tumor cells is responsible for the expression of pro-angiogenic factors, namely vascular endothelial growth factor (VEGF), interleukin-8 (IL-8), and basic fibroblast growth factor (bFGF) It has been reported to lead to an increase, which may lead to activation of tumor-associated vascular endothelial cells. Stimulation of tumor-associated vascular endothelial cells may also occur by activation of their own EGF receptors by growth factors produced by tumors such as TGF-α and EGF.

  Many human tumors have been reported to express or overexpress EGFR. EGFR expression correlates with unsatisfactory prognosis, low survival, and / or increased metastasis. Because of this involvement in tumorigenesis, EGFR has been particularly targeted for anticancer therapy. These therapies mainly include either monoclonal antibodies that block the ligand from binding to the extracellular domain of the receptor, or synthetic tyrosine kinase inhibitors that act directly on intracellular regions to prevent signal transduction. It was out.

  For example, cetuximab MAb (ERBITUX®) is a recombinant human / mouse chimeric monoclonal antibody that specifically binds to the extracellular domain of human EGFR. Cetuximab is an EGFR antagonist that blocks ligand binding to EGFR, prevents receptor activation, and inhibits the growth of tumor cells that express EGFR. Cetuximab is approved for use in some cases in combination with irinotecan in the treatment of patients with epithelial growth factor receptor-expressing metastatic colorectal cancer that is refractory or unable to tolerate chemotherapy with irinotecan Yes. Cetuximab has also been shown to be effective in treating psoriasis.

  The anti-EGFR antibody of the present invention is effective for the treatment of patients with vascularized tumors or neoplasms or angiogenic diseases. Such tumors and neoplasms include, for example, malignant tumors and neoplasms such as blastomas, carcinomas, or sarcomas, and significant vascular tumors and neoplasms. Cancers that can be treated by the methods of the present invention include, for example, brain, urogenital tract, lymphatic system, stomach, kidney, colon, pharynx and lung, and bone cancer. Non-limiting examples include epidermoid tumors, squamous tumors such as head and neck tumors, colorectal tumors, prostate tumors, breast tumors, lung adenocarcinoma and small and non-small cell lungs These include lung tumors, including tumors, pancreatic tumors, thyroid tumors, ovarian tumors, and liver tumors. This method also includes the treatment of vascularized skin cancers, including squamous cell carcinomas, basal cell carcinomas, and skin cancers that can be treated by inhibiting the growth of malignant keratinocytes such as human malignant keratinocytes. Used for treatment. Other cancers that can be treated include Kaposi's sarcoma, CNS neoplasms (neuroblastoma, capillary blastoma, meningioma, and brain metastases), melanoma, gastrointestinal and renal carcinomas and sarcomas, rhabdomy Examples include sarcomas, glioblastoma including glioblastoma multiforme, and leiomyosarcoma.

  In still another embodiment of the present invention, the agent of the present invention contains an anti-ALCAM antibody as an active ingredient. In a preferred embodiment of the drug of the present invention, an anti-ALCAM antibody having ADCC activity is contained as an active ingredient. In the drug of this embodiment, a therapeutic effect can be obtained by cell injury utilizing ADCC activity. As anti-ALCAM antibodies having ADCC activity, the 066-174 antibody or 035-234 antibody shown in the examples below may be partially modified as long as the specific binding property to ADCAM and ADCC activity are maintained. ), Or different types of antibodies constructed based on this (for example, IgG type). This antibody has both specific binding to ALCAM and ADCC activity. Therefore, it is possible to specifically bind to cancer cells expressing ALCAM, and then exert ADCC activity to damage the cancer cells. The target cancer cells of the drug of this embodiment are not particularly limited, and target various cells such as hematopoietic cells, endothelial cells, thymic cortex and medullary epithelial cells, bone marrow mesenchymal stem cells, fibroblasts, hepatocytes, etc. can do. Particularly preferred are lung adenocarcinoma cells and the like.

The present invention also provides a method for producing an antibody with enhanced ADCC activity or antitumor activity, comprising the step of substituting cysteine for the amino acid residue at position 286 or 298 in the heavy chain constant region.
In one embodiment of the production method, the amino acid residue at position 286 or 298 is substituted with cysteine in the heavy chain constant region of an antibody having ADCC activity known to those skilled in the art. There are no limitations on the method for obtaining an antibody having ADCC activity known to those skilled in the art. For example, the person who implements the manufacturing method of the present invention can make the product himself or can purchase it from another person. As a method for substituting amino acid residues with cysteine, for example, site-directed mutagenesis (Hashimoto-Gotoh, T, Mizuno, T, Ogasahara, Y, and Nakagawa, M. (1995) An oligodeoxyribonucleotide-directed dual amber method for site-directed mutagenesis. Gene 152, 271-275, Zoller, MJ, and Smith, M. (1983) Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13 vectors.Methods Enzymol. 100, 468-500, Kramer, W, Drutsa, V, Jansen, HW, Kramer, B, Pflugfelder, M, andFritz, HJ (1984) The gapped duplex DNA approach to oligonucleotide-directed mutation construction.Nucleic Acids Res. 12, 9441-9456, Kramer W, and Fritz HJ (1987) Oligonucleotide-directed construction of mutations via gapped duplex DNA Methods.Enzymol.154, 350-367, Kunkel, TA (1985) Rapid and efficient site-specific mutagenesis without phenotypic selection.Proc Natl Acad Sci US A. 82, 488 -492). Using this method, the desired amino acid of the antibody can be replaced with cysteine.
As another embodiment of the production method, first, an antibody that binds to a desired antigen is obtained by a method well known to those skilled in the art. If the obtained antibody is a non-human animal antibody, it can be humanized. Next, it is determined by a method well known to those skilled in the art whether or not the obtained antibody has ADCC activity. The ADCC activity of the antibody can be measured by, for example, the lactate dehydrogenase release assay system described in the Examples, but is not limited to this method, and can be measured by, for example, Cr51-release assay. Next, in the heavy chain constant region of the antibody determined to have ADCC activity, the amino acid residue at position 286 or 298 is substituted with cysteine.
As an evaluation method of antitumor activity, it can be evaluated by confirming the tumor growth inhibitory effect described in the examples, but is not limited to this method, for example, can be measured using SCID Hu mice, It is also possible to evaluate the tumor metastasis inhibitory effect (Clynes RA, Towers TL, Presta LG, Ravetch JV (2000) Inhibitory Fc receptors modulate in vivo cytoxicity against tumor targets. Nat Med. 6 (4), 443-446.)
More specifically, the present invention provides a method for producing an antibody with enhanced ADCC activity or antitumor activity, which comprises the following steps (a) and (b).
(A) a step of expressing a DNA encoding an H chain in which the amino acid residue at position 286 or 298 is substituted with a cysteine in the H chain constant region, and a DNA encoding an L chain (b) step (a) In the production method of the present invention, first, DNA encoding the heavy chain of an antibody variant having ADCC activity, wherein the amino acid residue at position 286 or 298 is substituted with cysteine. DNA encoding the H chain thus prepared, and DNA encoding the L chain of an antibody having ADCC activity are expressed. In the H chain constant region, DNA encoding the H chain in which the amino acid residue at position 286 or 298 is replaced with cysteine is obtained, for example, by obtaining the H chain constant region portion of the DNA encoding the wild type H chain. The codon encoding a specific amino acid in the H chain constant region can be obtained by appropriately introducing substitution so that cysteine is encoded. The codon encoding cysteine is TGT or TGC.
In addition, by designing a DNA encoding a protein in which the amino acid in the constant region of the wild type H chain is cysteine-substituted in advance, and chemically synthesizing this DNA, It is also possible to obtain DNA encoding an H chain in which the amino acid residue at the position is substituted with cysteine.
Further, in the Hc constant region of the Fc region, DNA encoding the H chain in which the amino acid residue at position 286 or 298 is replaced with cysteine can be produced by dividing it into partial DNAs. Examples of combinations of partial DNAs include DNA encoding a variable region and DNA encoding a constant region, or DNA encoding a Fab region and DNA encoding an Fc region, but are not limited to these combinations. Absent. Similarly, the DNA encoding the L chain can also be produced by dividing it into partial DNAs.

For example, DNA encoding an H chain constant region in which the amino acid residue at position 286 or 298 is substituted with cysteine, DNA encoding an H chain variable region, DNA encoding an L chain constant region, and an H chain constant region Examples of methods for expressing the encoded DNA include the following methods. That is, a DNA encoding the H chain variable region is incorporated into an expression vector together with DNA encoding the H chain constant region to construct an H chain expression vector. Similarly, a DNA encoding an L chain variable region is incorporated into an expression vector together with DNA encoding an L chain constant region to construct an L chain expression vector. Examples of expression vectors include, but are not limited to, SV40 virus based vectors, EB virus based vectors, BPV (papilloma virus) based vectors, and the like.
As a method of introducing the above four DNAs into a vector, a method of introducing four DNAs into one vector, a method of introducing four DNAs into two vectors, and a method of introducing four DNAs into three vectors Method (introducing one DNA into one vector, introducing one DNA into another vector and introducing two DNAs into the remaining vector) method of introducing four DNAs into four vectors (A method of introducing each of the four DNAs into separate vectors). Here, when the four DNAs are divided and introduced into two vectors and when the four DNAs are divided and introduced, the grouping of the four DNAs is not limited at all.

  A host cell is cotransformed with the antibody expression vector prepared by the above method. As the host cells, the above-described cells such as CHO cells (Chinese hamster ovary) are preferably used. For transformation, the lipofectin method (RWMalone et al., Proc. Natl. Acad. Sci. USA 86, 6077 (1989), PLFelgner et al., Proc. Natl. Acad. Sci. USA 84, 7413 ( 1987), electroporation method, calcium phosphate method (FL Graham & AJvan der Eb, Virology 52, 456-467 (1973)), DEAE-Dextran method and the like are preferably used.

In the method for producing an antibody of the present invention, next, the expression product obtained in step (a) is recovered. The expression product can be collected, for example, by culturing the transformant and then separating it from the cells of the transformant or the culture solution. For separation and purification of antibodies, methods such as centrifugation, ammonium sulfate fractionation, salting out, ultrafiltration, protein A, protein G, protein L column, affinity chromatography, ion exchange chromatography, gel filtration chromatography, etc. are used as appropriate. Can be combined.
By the above method, the ADCC activity of the antibody can be enhanced. That is, the present invention also provides a method for enhancing the ADCC activity of an antibody, comprising the step of substituting the amino acid residue at position 286 or 298 with cysteine in the heavy chain constant region.

  The present invention provides a pharmaceutical composition comprising an antibody of the present invention and a pharmaceutically acceptable carrier. The present invention also provides a method for treating a mammal, comprising administering the antibody of the present invention. Mammals include humans and non-human mammals (eg, mice, rats, monkeys, etc.). Further, when the antibody is an antibody that recognizes EGFR, it is useful in the treatment of lung cancer and the like. Further, when the antibody is an antibody that recognizes Her2, it is useful in the treatment of breast cancer and the like.

  The pharmaceutical composition of the present invention can be formulated by a known method by introducing a pharmaceutically acceptable carrier in addition to the antibody. For example, it can be used parenterally in the form of a sterile solution with water or other pharmaceutically acceptable liquid, or an injection of suspension. For example, a pharmacologically acceptable carrier or medium, specifically, sterilized water, physiological saline, vegetable oil, emulsifier, suspension, surfactant, stabilizer, flavoring agent, excipient, vehicle, preservative It is conceivable to formulate by combining with a binder or the like as appropriate and mixing in a unit dosage form generally required for pharmaceutical practice. The amount of active ingredient in these preparations is such that an appropriate volume within the indicated range can be obtained.

Sterile compositions for injection can be formulated according to normal pharmaceutical practice using a vehicle such as distilled water for injection.
Examples of aqueous solutions for injection include isotonic solutions containing, for example, physiological saline, glucose and other adjuvants, such as D-sorbitol, D-mannose, D-mannitol, sodium chloride, and suitable solubilizers such as Alcohols, specifically ethanol, polyalcohols such as propylene glycol, polyethylene glycol, nonionic surfactants such as polysorbate 80 (TM), HCO-50 may be used in combination.

  Examples of the oily liquid include sesame oil and soybean oil, which may be used in combination with benzyl benzoate or benzyl alcohol as a solubilizing agent. Moreover, you may mix | blend with buffer, for example, phosphate buffer, sodium acetate buffer, a soothing agent, for example, procaine hydrochloride, stabilizer, for example, benzyl alcohol, phenol, antioxidant. The prepared injection solution is usually filled into a suitable ampoule.

  Administration is preferably parenteral administration, and specific examples include injection, nasal administration, pulmonary administration, and transdermal administration. As an example of the injection form, it can be administered systemically or locally by, for example, intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, or the like.

  The administration method can be appropriately selected depending on the age and symptoms of the patient. The dosage of the pharmaceutical composition containing the antibody or the polynucleotide encoding the antibody can be selected, for example, in the range of 0.0001 mg to 1000 mg per kg body weight. Alternatively, for example, the dose can be selected in the range of 0.001 to 100,000 mg / body per patient, but is not necessarily limited to these values. The dose and administration method vary depending on the weight, age, symptoms, etc. of the patient, but can be appropriately selected by those skilled in the art.

The present invention also provides a method for determining whether ADCC activity of a test antibody is enhanced. Specifically, provided is a method for determining that the ADCC activity of the antibody in the following step (a) is enhanced when the ADCC activity measured in the following step (b) is higher than the ADCC activity before substitution. To do.
(A) providing a variant of an antibody having ADCC activity, wherein the amino acid residue at position 286 or 298 in the heavy chain constant region is substituted with cysteine; and
(B) A step of measuring ADCC activity of the antibody in step (a).
The present invention also provides a method of screening for an antibody with enhanced ADCC activity using the determination method described above. Specifically, a method for screening an antibody with enhanced ADCC activity, comprising the following steps (a) to (d) is provided.
(A) providing an antibody having ADCC activity;
(B) replacing the amino acid residue at position 286 or 298 in the heavy chain constant region of the antibody of step (a) with cysteine,
(C) determining whether the ADCC activity of the antibody obtained in step (b) is enhanced by the above method,
(D) A step of selecting the antibody determined to have enhanced ADCC activity in step (c).
In these determination methods and screening methods, the antibody having ADCC activity is not particularly limited as long as it has ADCC activity. The method for obtaining an antibody having ADCC activity is as described above.

In addition, the base sequence and amino acid sequence of the antibody disclosed in this specification are described in the sequence listing according to the following sequence numbers.
<286Cys anti-ALCAM (066-174) antibody>
SEQ ID NO: 9: base sequence of H chain CDR1 SEQ ID NO: 12: amino acid sequence of H chain CDR1 SEQ ID NO: 10: base sequence of H chain CDR2 SEQ ID NO: 13: amino acid sequence of H chain CDR2 SEQ ID NO: 11: base sequence of H chain CDR3 SEQ ID NO: 14: Amino acid sequence of H chain CDR3 SEQ ID NO: 90: Base sequence of H chain constant region (286 Cys type)
SEQ ID NO: 91: Amino acid sequence of heavy chain constant region (286 Cys type)
SEQ ID NO: 94: base sequence of full length of H chain (286Cys type)
SEQ ID NO: 95: Full length amino acid sequence (286 Cys type)
SEQ ID NO: 96: base sequence of H chain Fc region (286 Cys type)
SEQ ID NO: 97: Amino acid sequence of H chain Fc region (286 Cys type)
SEQ ID NO: 1: Base sequence of L chain CDR1 SEQ ID NO: 4: Amino acid sequence of L chain CDR1 SEQ ID NO: 2: Base sequence of L chain CDR2 SEQ ID NO: 5: Amino acid sequence of L chain CDR2 SEQ ID NO: 3: Base sequence of L chain CDR3 SEQ ID NO: 6: L chain CDR3 amino acid sequence SEQ ID NO: 7: L chain constant region nucleotide sequence SEQ ID NO: 8: L chain constant region amino acid sequence SEQ ID NO: 98: L chain full length nucleotide sequence SEQ ID NO: 99: L chain full length Amino acid sequence

<298Cys Anti-ALCAM (066-174) antibody>
SEQ ID NO: 9: base sequence of H chain CDR1 SEQ ID NO: 12: amino acid sequence of H chain CDR1 SEQ ID NO: 10: base sequence of H chain CDR2 SEQ ID NO: 13: amino acid sequence of H chain CDR2 SEQ ID NO: 11: base sequence of H chain CDR3 SEQ ID NO: 14: amino acid sequence of H chain CDR3 SEQ ID NO: 92: base sequence of H chain constant region (298Cys type)
SEQ ID NO: 93: Amino acid sequence of heavy chain constant region (298Cys type)
SEQ ID NO: 100: Full length base sequence (298Cys type)
SEQ ID NO: 101: amino acid sequence of full length H chain (298Cys type)
SEQ ID NO: 102: base sequence of H chain Fc region (298Cys type)
SEQ ID NO: 103: amino acid sequence of H chain Fc region (298Cys type)
SEQ ID NO: 1: Base sequence of L chain CDR1 SEQ ID NO: 4: Amino acid sequence of L chain CDR1 SEQ ID NO: 2: Base sequence of L chain CDR2 SEQ ID NO: 5: Amino acid sequence of L chain CDR2 SEQ ID NO: 3: Base sequence of L chain CDR3 SEQ ID NO: 6: L chain CDR3 amino acid sequence SEQ ID NO: 7: L chain constant region nucleotide sequence SEQ ID NO: 8: L chain constant region amino acid sequence SEQ ID NO: 98: L chain full length nucleotide sequence SEQ ID NO: 99: L chain full length Amino acid sequence

<286Cys Anti-ALCAM (035-234) antibody>
SEQ ID NO: 39: base sequence of H chain CDR1 SEQ ID NO: 42: amino acid sequence of H chain CDR1 SEQ ID NO: 40: base sequence of H chain CDR2 SEQ ID NO: 43: amino acid sequence of H chain CDR2 SEQ ID NO: 41: base sequence of H chain CDR3 SEQ ID NO: 44: amino acid sequence of H chain CDR3 SEQ ID NO: 90: base sequence of H chain constant region (286 Cys type)
SEQ ID NO: 91: Amino acid sequence of heavy chain constant region (286 Cys type)
SEQ ID NO: 104: Full length base sequence (286 Cys type)
SEQ ID NO: 105: Full length amino acid sequence (286 Cys type)
SEQ ID NO: 106: base sequence of H chain Fc region (286 Cys type)
SEQ ID NO: 107: amino acid sequence of H chain Fc region (286 Cys type)
SEQ ID NO: 31: base sequence of L chain CDR1 SEQ ID NO: 34: amino acid sequence of L chain CDR1 SEQ ID NO: 32: base sequence of L chain CDR2 SEQ ID NO: 35: amino acid sequence of L chain CDR2 SEQ ID NO: 33: base sequence of L chain CDR3 SEQ ID NO: 36: L chain CDR3 amino acid sequence SEQ ID NO: 37: L chain constant region base sequence SEQ ID NO: 38: L chain constant region amino acid sequence SEQ ID NO: 108: L chain full length base sequence SEQ ID NO: 109: L chain full length Amino acid sequence

<298Cys Anti-ALCAM (035-234) antibody>
SEQ ID NO: 39: base sequence of H chain CDR1 SEQ ID NO: 42: amino acid sequence of H chain CDR1 SEQ ID NO: 40: base sequence of H chain CDR2 SEQ ID NO: 43: amino acid sequence of H chain CDR2 SEQ ID NO: 41: base sequence of H chain CDR3 SEQ ID NO: 44: amino acid sequence of H chain CDR3 SEQ ID NO: 92: base sequence of H chain constant region (298Cys type)
SEQ ID NO: 93: Amino acid sequence of heavy chain constant region (298Cys type)
SEQ ID NO: 110: Full length base sequence (298Cys type)
SEQ ID NO: 111: Full length amino acid sequence (298Cys type)
SEQ ID NO: 112: base sequence of H chain Fc region (298Cys type)
SEQ ID NO: 113: amino acid sequence of H chain Fc region (298Cys type)
SEQ ID NO: 31: base sequence of L chain CDR1 SEQ ID NO: 34: amino acid sequence of L chain CDR1 SEQ ID NO: 32: base sequence of L chain CDR2 SEQ ID NO: 35: amino acid sequence of L chain CDR2 SEQ ID NO: 33: base sequence of L chain CDR3 SEQ ID NO: 36: Amino acid sequence of L chain CDR3 SEQ ID NO: 37: Base sequence of L chain constant region SEQ ID NO: 38: Amino acid sequence of L chain constant region SEQ ID NO: 108: Base sequence of L chain full length SEQ ID NO: 109: Full length of L chain Amino acid sequence

<286Cys anti-ICAM1 (053-059) antibody>
SEQ ID NO: 54: base sequence of H chain CDR1 SEQ ID NO: 57: amino acid sequence of H chain CDR1 SEQ ID NO: 55: base sequence of H chain CDR2 SEQ ID NO: 58: amino acid sequence of H chain CDR2 SEQ ID NO: 56: base sequence of H chain CDR3 SEQ ID NO: 59: Amino acid sequence of H chain CDR3 SEQ ID NO: 90: Base sequence of H chain constant region (286 Cys type)
SEQ ID NO: 91: Amino acid sequence of heavy chain constant region (286 Cys type)
SEQ ID NO: 114: base sequence of full length of H chain (286Cys type)
SEQ ID NO: 115: amino acid sequence of full length of H chain (286Cys type)
SEQ ID NO: 116: base sequence of H chain Fc region (286 Cys type)
SEQ ID NO: 117: amino acid sequence of H chain Fc region (286 Cys type)
SEQ ID NO: 46: Base sequence of L chain CDR1 SEQ ID NO: 49: Amino acid sequence of L chain CDR1 SEQ ID NO: 47: Base sequence of L chain CDR2 SEQ ID NO: 50: Amino acid sequence of L chain CDR2 SEQ ID NO: 48: Base sequence of L chain CDR3 SEQ ID NO: 51: L chain CDR3 amino acid sequence SEQ ID NO: 52: L chain constant region nucleotide sequence SEQ ID NO: 53: L chain constant region amino acid sequence SEQ ID NO: 118: L chain full length nucleotide sequence SEQ ID NO: 119: L chain full length Amino acid sequence

<298Cys Anti-ICAM1 (053-059) antibody>
SEQ ID NO: 54: base sequence of H chain CDR1 SEQ ID NO: 57: amino acid sequence of H chain CDR1 SEQ ID NO: 55: base sequence of H chain CDR2 SEQ ID NO: 58: amino acid sequence of H chain CDR2 SEQ ID NO: 56: base sequence of H chain CDR3 SEQ ID NO: 59: Amino acid sequence of H chain CDR3 SEQ ID NO: 92: Base sequence of H chain constant region (298Cys type)
SEQ ID NO: 93: Amino acid sequence of heavy chain constant region (298Cys type)
SEQ ID NO: 120: Full length of H chain (298Cys type)
SEQ ID NO: 121: amino acid sequence of full length of H chain (298Cys type)
SEQ ID NO: 122: base sequence of H chain Fc region (298Cys type)
SEQ ID NO: 123: amino acid sequence of H chain Fc region (298Cys type)
SEQ ID NO: 46: Base sequence of L chain CDR1 SEQ ID NO: 49: Amino acid sequence of L chain CDR1 SEQ ID NO: 47: Base sequence of L chain CDR2 SEQ ID NO: 50: Amino acid sequence of L chain CDR2 SEQ ID NO: 48: Base sequence of L chain CDR3 SEQ ID NO: 51: L chain CDR3 amino acid sequence SEQ ID NO: 52: L chain constant region nucleotide sequence SEQ ID NO: 53: L chain constant region amino acid sequence SEQ ID NO: 118: L chain full length nucleotide sequence SEQ ID NO: 119: L chain full length Amino acid sequence

<286Cys anti-ICAM1 (053-085) antibody>
SEQ ID NO: 68: base sequence of H chain CDR1 SEQ ID NO: 71: amino acid sequence of H chain CDR1 SEQ ID NO: 69: base sequence of H chain CDR2 SEQ ID NO: 72: amino acid sequence of H chain CDR2 SEQ ID NO: 70: base sequence of H chain CDR3 SEQ ID NO: 73: amino acid sequence of H chain CDR3 SEQ ID NO: 90: nucleotide sequence of H chain constant region (286 Cys type)
SEQ ID NO: 91: Amino acid sequence of heavy chain constant region (286 Cys type)
SEQ ID NO: 124: Full length base sequence (286 Cys type)
SEQ ID NO: 125: Full length amino acid sequence (286 Cys type)
SEQ ID NO: 126: base sequence of H chain Fc region (286 Cys type)
SEQ ID NO: 127: Amino acid sequence of H chain Fc region (286 Cys type)
SEQ ID NO: 60: base sequence of L chain CDR1 SEQ ID NO: 63: amino acid sequence of L chain CDR1 SEQ ID NO: 61: base sequence of L chain CDR2 SEQ ID NO: 64: amino acid sequence of L chain CDR2 SEQ ID NO: 62: base sequence of L chain CDR3 SEQ ID NO: 65: L chain CDR3 amino acid sequence SEQ ID NO: 66: L chain constant region base sequence SEQ ID NO: 67: L chain constant region amino acid sequence SEQ ID NO: 128: L chain full length base sequence SEQ ID NO: 129: L chain full length Amino acid sequence

<298Cys Anti-ICAM1 (053-085) antibody>
SEQ ID NO: 68: base sequence of H chain CDR1 SEQ ID NO: 71: amino acid sequence of H chain CDR1 SEQ ID NO: 69: base sequence of H chain CDR2 SEQ ID NO: 72: amino acid sequence of H chain CDR2 SEQ ID NO: 70: base sequence of H chain CDR3 SEQ ID NO: 73: Amino acid sequence of H chain CDR3 SEQ ID NO: 92: Base sequence of H chain constant region (298Cys type)
SEQ ID NO: 93: Amino acid sequence of heavy chain constant region (298Cys type)
SEQ ID NO: 130: Full length base sequence (298Cys type)
SEQ ID NO: 131: amino acid sequence of full length of H chain (298Cys type)
SEQ ID NO: 132: base sequence of H chain Fc region (298Cys type)
SEQ ID NO: 133: Amino acid sequence of heavy chain Fc region (298Cys type)
SEQ ID NO: 60: base sequence of L chain CDR1 SEQ ID NO: 63: amino acid sequence of L chain CDR1 SEQ ID NO: 61: base sequence of L chain CDR2 SEQ ID NO: 64: amino acid sequence of L chain CDR2 SEQ ID NO: 62: base sequence of L chain CDR3 SEQ ID NO: 65: L chain CDR3 amino acid sequence SEQ ID NO: 66: L chain constant region base sequence SEQ ID NO: 67: L chain constant region amino acid sequence SEQ ID NO: 128: L chain full length base sequence SEQ ID NO: 129: L chain full length Amino acid sequence

(286Cys anti-EGFR (048-006) antibody)
SEQ ID NO: 83: base sequence of H chain CDR1 SEQ ID NO: 86: amino acid sequence of H chain CDR1 SEQ ID NO: 84: base sequence of H chain CDR2 SEQ ID NO: 87: amino acid sequence of H chain CDR2 SEQ ID NO: 85: base sequence of H chain CDR3 SEQ ID NO: 88: Amino acid sequence of H chain CDR3 SEQ ID NO: 90: Base sequence of H chain constant region (286 Cys type)
SEQ ID NO: 91: Amino acid sequence of heavy chain constant region (286 Cys type)
SEQ ID NO: 134: base sequence of full length of H chain (286Cys type)
SEQ ID NO: 135: amino acid sequence of full length of H chain (286Cys type)
SEQ ID NO: 136: Nucleotide sequence of H chain Fc region (286 Cys type)
SEQ ID NO: 137: Amino acid sequence of H chain Fc region (286 Cys type)
SEQ ID NO: 75: base sequence of L chain CDR1 SEQ ID NO: 78: amino acid sequence of L chain CDR1 SEQ ID NO: 76: base sequence of L chain CDR2 SEQ ID NO: 79: amino acid sequence of L chain CDR2 SEQ ID NO: 77: base sequence of L chain CDR3 SEQ ID NO: 80: L chain CDR3 amino acid sequence SEQ ID NO: 81: L chain constant region base sequence SEQ ID NO: 82: L chain constant region amino acid sequence SEQ ID NO: 138: L chain full length base sequence SEQ ID NO: 139: L chain full length Amino acid sequence

(298Cys anti-EGFR (048-006) antibody)
SEQ ID NO: 83: base sequence of H chain CDR1 SEQ ID NO: 86: amino acid sequence of H chain CDR1 SEQ ID NO: 84: base sequence of H chain CDR2 SEQ ID NO: 87: amino acid sequence of H chain CDR2 SEQ ID NO: 85: base sequence of H chain CDR3 SEQ ID NO: 88: Amino acid sequence of H chain CDR3 SEQ ID NO: 92: Base sequence of H chain constant region (298Cys type)
SEQ ID NO: 93: Amino acid sequence of heavy chain constant region (298Cys type)
SEQ ID NO: 140: base sequence of full length of H chain (298Cys type)
SEQ ID NO: 141: Full length amino acid sequence (298 Cys type)
SEQ ID NO: 142: base sequence of H chain Fc region (298Cys type)
SEQ ID NO: 143: Amino acid sequence of H chain Fc region (298Cys type)
SEQ ID NO: 75: Base sequence of L chain CDR1 SEQ ID NO: 78: Amino acid sequence of L chain CDR1 SEQ ID NO: 76: Base sequence of L chain CDR2 SEQ ID NO: 79: Amino acid sequence of L chain CDR2 SEQ ID NO: 77: Base sequence of L chain CDR3 SEQ ID NO: 80: amino acid sequence of L chain CDR3 SEQ ID NO: 81: base sequence of L chain constant region SEQ ID NO: 82: amino acid sequence of L chain constant region SEQ ID NO: 138: base sequence of full length of L chain SEQ ID NO: 139: full length of L chain Amino acid sequence It should be noted that all prior art documents cited herein are hereby incorporated by reference.

EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, the scope of the present invention is not limited to the aspect described in these Examples.
[Example 1]
Anti-ALCAM (066-174) human antibodies of wild type, 286 Cys type, 295 Cys type, and 298 Cys type were prepared, and ADCC activity of these Anti-ALCAM (066-174) human antibodies was evaluated. As a result, comparing the ADCC activity of the wild type with the anti-ALCAM (066-174) human antibody, the anti-ALCAM (066-174) human antibody of 286 Cys type, 295 Cys type, and 298 Cys type has high ADCC activity. showed that.
The following describes methods for preparing anti-ALCAM (066-174) human antibody wild type, 286 Cys type, 295 Cys type, and 298 Cys type and measuring ADCC activity.

1) Production of Anti-ALCAM (066-174) Human Antibody A human antibody is obtained as a purified human antibody through the following steps A) to E).
A) Mutation of Fc region gene,
B) Construction of human antibody expression vector combining Fc region and mutated Fc region gene,
C) Gene transfer of human antibody expression vector into CHO cells and screening for CHO cells that highly express human antibodies,
D) culture of CHO cells highly expressing human antibodies,
E) Column purification from culture supernatant of human antibody high expressing cells.
The steps A) to E) are described in order below.

Four genes are required to produce human antibodies. These four genes are a human antibody L chain variable region gene, a human antibody H chain variable region gene, a human IgG1 L chain constant region gene, and a human IgG1 H chain constant region gene. The L chain variable region and constant region genes and the H chain variable region and constant region genes of Anti-ALCAM (066-174), which is an antibody clone that recognizes ALCAM, were provided by Antibody Research Institute Co., Ltd. The DNA sequence of the human L chain variable region of the provided Anti-ALCAM (066-174) is shown in FIG. 1 (SEQ ID NO: 144), and the DNA sequence of the human H chain variable region is shown in FIG. 2 (SEQ ID NO: 146). .
In addition, the nucleotide sequences of CDR1, CDR2 and CDR3 of the human L chain variable region of Anti-ALCAM (066-174) are shown in SEQ ID NOs: 1 to 3. In addition, the amino acid sequences of CDR1, CDR2, and CDR3 of the human L chain variable region of Anti-ALCAM (066-174) are shown in SEQ ID NOs: 4 to 6, respectively. The provided human IgG1 L chain constant region gene is a λ chain, and its nucleotide sequence is shown in SEQ ID NO: 7 and amino acid sequence is shown in SEQ ID NO: 8.
In addition, the nucleotide sequences of CDR1, CDR2, and CDR3 of the human H chain variable region of Anti-ALCAM (066-174) are shown in SEQ ID NOs: 9-11. In addition, the amino acid sequences of CDR1, CDR2, and CDR3 of the human H chain variable region of Anti-ALCAM (066-174) are shown in SEQ ID NOs: 12-14. The nucleotide sequence of the provided human IgG1 H chain constant region is shown in SEQ ID NO: 15, and the amino acid sequence is shown in SEQ ID NO: 16.

A) Mutation of Fc region gene
In order to obtain 286 Cys human antibody, 295 Cys human antibody, and 298 Cys human antibody, mutations must be introduced into specific portions of the human IgG1 heavy chain constant region. According to the number of constant region of human IgG1 heavy chain shown by Elvin A. Kabat et al. In Sequence of proteins of Immunological Interest (NIH Publication No.91-3242, 1991), Asn at 286 and Cy at 295 are Cys. In addition, in order to change Ser No. 298 to Cys, a human IgG1 heavy chain constant region gene was inserted into a pCR2.1 vector (Invitrogen). This vector was named pCR2.1-HC (wild type).
PCR reaction conditions are as follows.
Anti-ALCAM (066-174) H chain (25 ng / μL) 2 μL
2.5 mM dNTPs 4 μL
hIgG1 HCF primer (20 μM) 2.5 μL
hIgG1 HCR primer (20 μM) 2.5 μL
DMSO 2.5 μL
× 10 pfu polymerase Buffer 5 μL
pfu polymerase 1 μL
Sterile water 30.5 μL
total 50 μL
94 ℃ 2 min
94 ℃ 1 min 55 ℃ 2min 72 ℃ 2min (30 cycles)
72 ℃ 4 min
4 ℃ unlimited time
See below for Primer DNA sequences.
hIgG1 HCF primer: GCCTCCACCAAGGGCCCATCGGTC (SEQ ID NO: 17)
hIgG1 HCR primer: TTATTTACCCGGAGACAGGGAGAGGCT (SEQ ID NO: 18)

A pCR2.1 vector having a human IgG1 H chain constant region inserted: pCR2.1-HC (wild type) was used as a template, and the mutation was sequentially introduced by PCR. Details of this mutagenesis method are described below.
286 (Asn → Cys) mutation
pCR2.1-HC (wild type) (25 ng / μL) 2 μL
2.5 mM dNTPs 4 μL
286 Cys1 primer (20 μM) 1.25 μL
286 Cys2 primer (20 μM) 1.25 μL
× 10 pfu polymerase Buffer 5 μL
pfu DNA polymerase 1 μL
Sterile water 35.5 μL
total 50 μL
(PCR amplification reaction)
95 ℃ 30 sec
95 ° C 30 sec 55 ° C 1 min 68 ° C 13 min (12 cycles)
68 ℃ 4 min
4 ℃ unlimited time
The base sequence of Primer is as follows.
286 Cys1 primer: GTGGAGGTGCATTGTGCCAAGACAAAG (SEQ ID NO: 19)
286 Cys2 primer: CTTTGTCTTGGCACAATGCACCTCCAC (SEQ ID NO: 20)
After completion of the PCR reaction, 1 μL of DpnI (New England BioLabs) was added and incubated at 37 ° C. for 2 hours. Using the solution after the incubation, E. coli JM109 was transformed, and the plasmid was purified from the transformed E. coli. By confirming the DNA sequence of the purified plasmid, it was named pCR2.1 vector pCR2.1-HC (286Cys) into which the 286 (Asn → Cys) mutated human IgG1 H chain constant region was inserted.
295 (Glu → Cys) mutation
pCR2.1-HC (wild type) (25 ng / μL) 2 μL
2.5 mM dNTPs 4 μL
295 Cys1 primer (20 μM) 1.25 μL
295 Cys2 primer (20 μM) 1.25 μL
× 10 pfu polymerase Buffer 5 μL
pfu DNA polymerase 1 μL
Sterile water 35.5 μL
total 50 μL
(PCR amplification reaction)
95 ℃ 30 sec
95 ° C 30 sec 55 ° C 1 min 68 ° C 13 min (12 cycles)
68 ℃ 4 min
4 ℃ unlimited time
The base sequence of Primer is as follows.
295 Cys1 primer: ACAAAGCCGCGTGAGGAGTGCTACAACAGCACGTACCGT (SEQ ID NO: 21)
295 Cys2 primer: ACGGTACGTGCTGTTGTAGCACTCCTCACGCGGCTTTGT (SEQ ID NO: 22)
After completion of the PCR reaction, 1 μL of DpnI (New England BioLabs) was added and incubated at 37 ° C. for 2 hours. Using the solution after the incubation, E. coli JM109 was transformed, and the plasmid was purified from the transformed E. coli. By confirming the DNA sequence of the purified plasmid, it was named pCR2.1 vector pCR2.1-HC (295Cys) into which the 295 (Glu → Cys) mutated human IgG1 H chain constant region was inserted.
298 (Ser → Ala) mutation
pCR2.1-HC (wild type) (25 ng / μL) 2 μL
2.5 mM dNTPs 4 μL
298 Cys1 primer (20 μM) 1.25 μL
298 Cys2 primer (20 μM) 1.25 μL
× 10 pfu polymerase Buffer 5 μL
pfu DNA polymerase 1 μL
Sterile water 35.5 μL
total 50 μL
(PCR amplification reaction)
95 ℃ 30 sec
95 ° C 30 sec 55 ° C 1 min 68 ° C 13 min (12 cycles)
68 ℃ 4 min
4 ℃ unlimited time
The base sequence of Primer is as follows.
298 Cys1 primer: GAGCAGTACAACTGCACGTACCGTGTG (SEQ ID NO: 23)
298 Cys2 primer: CACACGGTACGTGCAGTTGTACTGCTC (SEQ ID NO: 24)
After completion of the PCR reaction, 1 μL of DpnI (New England BioLabs) was added and incubated at 37 ° C. for 2 hours. Using the solution after the incubation, E. coli JM109 was transformed, and the plasmid was purified from the transformed E. coli. By confirming the DNA sequence of the purified plasmid, it was named pCR2.1 vector pCR2.1-HC (298Cys) into which the 298 (Ser → Cys) mutated human IgG1 H chain constant region was inserted.

B) Construction of human antibody expression vector combining Fc region and mutated Fc region gene
Anti-ALCAM (066-174) human antibody can be expressed by transfecting CHO cells with two types of expression vectors of L-chain and H-chain of Anti-ALCAM (066-174) human antibody. Here, the expression vector of the anti-ALCAM (066-174) human antibody L chain is expressed by combining the anti-ALCAM (066-174) L chain variable region gene + human IgG1 L chain constant region gene. Anti-ALCAM (066-174) H chain variable region gene + human IgG1 H chain constant region gene combined with Anti-ALCAM (066-174) H chain expression vector. Is incorporated into an expression vector. The anti-ALCAM (066-174) human antibody L chain expression vector for the production of wild-type, 286Cys-type, 295Cys-type or 298Cys-type Anti-ALCAM (066-174) antibody is the four human antibodies. The anti-ALCAM (066-174) human antibody H chain expression vector is commonly used to express wild type, 286 Cys type, 295 Cys type or 298 Cys type human antibody. Requires a dedicated heavy chain expression vector. Here, the construction of an anti-ALCAM (066-174) human antibody L chain expression vector that can be commonly used for the expression of each human antibody is described in detail, and then the 286 Cys type, 295 Cys type or 298 Cys type human antibody is described. Describes the construction of a dedicated Anti-ALCAM (066-174) human antibody heavy chain expression vector required for expression.

Construction of anti-ALCAM (066-174) human antibody L chain expression vector BCMG-neo vector is used as the expression vector, and Anti-ALCAM (066-174) human L chain variable is used at the XhoI and NotI sites of this vector. Insert a region gene + human IgG1 L chain constant region gene. Anti-ALCAM (066-174) human L chain variable region and human IgG1 L chain constant region genes are subjected to the following PCR reaction using the Anti-ALCAM (066-174) L chain gene provided by the antibody laboratory as a template. A fragment was obtained.

Anti-ALCAM (066-174) Human L chain variable region and human IgG1 L chain constant region gene amplification reaction
Anti-ALCAM (066-174) L chain (25 ng / μL) 2 μL
2.5 mM dNTPs 4 μL
EGFR-L1 primer (20μM) 2.5 μL
EGFR-L2 primer (20μM) 2.5 μL
DMSO 2.5 μL
× 10 pfu polymerase Buffer 5 μL
pfu polymerase 1 μL
Sterile water 30.5 μL
total 50 μL
94 ℃ 2 min
94 ℃ 1 min 55 ℃ 2min 72 ℃ 2min (30 cycles)
72 ℃ 4 min
4 ℃ unlimited time
See below for Primer DNA sequences.
EGFR-L1 primer: ACCGCTCGAGATGGACATGAGGGTCCCCGCTCAGCTC (SEQ ID NO: 25)
EGFR-L2 primer: ATAGTTTAGCGGCCGCTTACGAACATTCTGTAGGGGCCACTGTCTT (SEQ ID NO: 26)

  Anti-ALCAM (066-174) human L chain variable region and human IgG1 L chain constant region gene fragments obtained by the above PCR reaction were cleaved with restriction enzyme XhoI (Takara) and restriction enzyme NotI (Takara). Purified. The fragment purified by cutting the BCMG-neo vector with XhoI and NotI was mixed with the purified Anti-ALCAM (066-174) human L chain variable region and human IgG1 L chain constant region gene fragment and ligated. In the vector obtained by this ligation, it was confirmed by sequencing that the desired fragment was inserted. This anti-ALCAM (066-174) human antibody L chain expression vector was named ALCAM (066-174) LC.

Construction of Anti-ALCAM (066-174) human antibody heavy chain expression vector BCMG-neo vector is used as the expression vector, and Anti-ALCAM (066-174) human heavy chain variable region is used at the XhoI and NotI sites of this vector. Gene + H human IgG1 H chain constant region gene is inserted. The Anti-ALCAM (066-174) human heavy chain variable region gene was obtained by performing the following PCR reaction using the Anti-ALCAM (066-174) heavy chain gene provided by the antibody laboratory as a template.
The dedicated H chain expression vector for each of the wild type, 286Cys type, 295Cys type or 295Cys type human antibody has a different plasmid as a template for PCR amplification of the human IgG1 H chain constant region gene, but the operation itself is The procedure was exactly the same, and various human IgG1 H chain gene fragments were obtained. Details including the PCR reaction are described below.

Anti-ALCAM (066-174) Human antibody heavy chain variable region amplification reaction
Anti-ALCAM (066-174) H chain (25 ng / μL) 2 μL
2.5 mM dNTPs 4 μL
EGFR-H1 primer (20μM) 2.5 μL
ALCAM-heavy-variable-R primer (20μM) 2.5 μL
DMSO 2.5 μL
× 10 pfu polymerase Buffer 5 μL
pfu polymerase 1 μL
Sterile water 30.5 μL
total 50 μL
94 ℃ 2 min
94 ℃ 1 min 55 ℃ 2min 72 ℃ 2min (30 cycles)
72 ℃ 4 min
4 ℃ unlimited time
EGFR-H1 primer: ACCGCTCGAGATGAAACACCTGTGGTTCTTCCTC (SEQ ID NO: 27)
ALCAM-heavy-variable-R primer: CGACGAGACGGTGACCGTGGTCCCTTGGCC (5'-phosphorylated) (SEQ ID NO: 28)

Amplification reaction of human IgG1 H chain constant region gene # Various template plasmids (25 ng / μL) 2 μL
2.5 mM dNTPs 4 μL
H3 primer (20μM) 2.5 μL
H4 primer (20μM) 2.5 μL
DMSO 2.5 μL
× 10 pfu polymerase Buffer 5 μL
pfu polymerase 1 μL
Sterile water 30.5 μL
total 50 μL
94 ℃ 2 min
94 ℃ 1 min 55 ℃ 2min 72 ℃ 2min (30 cycles)
72 ℃ 4 min
4 ℃ unlimited time
H3 primer: GCCTCCACCAAGGGCCCATCGGTC (5'-phosphorylated) (SEQ ID NO: 29)
H4 primer: ATAGTTTAGCGGCCGCTTATTTACCCGGAGACAGGGAGAGGCTCTT (SEQ ID NO: 30)

  In order to prepare a gene fragment for a wild type Anti-ALCAM (066-174) human antibody, pCR2.1-HC (wild type) was used as a template. PCR2.1-HC (286Cys) was used as a template in order to prepare a gene fragment for 286Cys type Anti-ALCAM (066-174) human antibody. PCR2.1-HC (295Cys) was used as a template to prepare a gene fragment for 295 Cys-type Anti-ALCAM (066-174) human antibody. PCR2.1-HC (298Cys) was used as a template to prepare a gene fragment for the 298Cys-type Anti-ALCAM (066-174) human antibody.

  The Anti-ALCAM (066-174) H chain variable region gene fragment obtained by the above PCR reaction was purified after being cleaved with a restriction enzyme XhoI (Takara). Moreover, human IgG1 H chain constant region gene fragments obtained by various PCR reactions with different templates were purified after being cleaved with a restriction enzyme NotI (Takara). The BCMG-neo vector cut with XhoI and NotI was purified and mixed with the purified Anti-ALCAM (066-174) heavy chain variable region gene fragment and purified human IgG1 heavy chain constant region gene fragment. . In the vector obtained by this ligation, it was confirmed by sequencing that the desired fragment was inserted. The expression vectors of this ALCAM (066-174) human antibody H chain are p human ALCAM (066-174) HC (wild type), p human ALCAM (066-174) HC (286 Cys type), p human ALCAM (066- 174) HC (295 Cys) or p human ALCAM (066-174) HC (298Cys type), respectively.

C) Gene transfer of human antibody expression vectors to CHO cells and screening of CHO cells that highly express human antibodies Various expression vectors constructed using the TransIT-CHO Transfection kit (Mirus, MIR2170) in the following combinations: The gene was introduced into CHO cells.
Wild type human antibody: p Human Anti-ALCAM (066-174) LC + p Human Anti-ALCAM (066-174) HC (wild type)
286Cys type human antibody: p human Anti-ALCAM (066-174) LC + p human Anti-ALCAM (066-174) HC (286Cys type)
295Cys type human antibody: p human Anti-ALCAM (066-174) LC + p human Anti-ALCAM (066-174) HC (295Cys type)
298Cys type human antibody: p human Anti-ALCAM (066-174) LC + p human Anti-ALCAM (066-174) HC (298Cys type)

Transduced CHO cells were Dulbecco's Modified Eagle's Medium (Sigma, D5796) supplemented with 10% Fetal Bovine Serum (EQITEC-BIO) and 1 mg / mL Geneticin (GIBCO, 10131-035) at 37 ° C, 5% CO2. It grew by culturing under carbon conditions. CHO cells that highly express human antibodies are selected from the CHO cells that have grown and formed colonies. First, a portion of each colony was transplanted into a 96-well plate and cultured for another 10 days. . A part of the culture supernatant was taken out and subjected to ELISA measurement shown below to determine a highly expressing CHO cell clone.
(ELISA measurement)
1) The culture supernatant of Tansfectioned CHO cells was added to a 96-well plate coated with Anti-Human IgG (γ-chain) (MBL, 103AG) at 100 μL / well and left at room temperature for 1 hour.
2) The culture supernatant was discarded from the 96-well plate, and wells were sufficiently washed with PBS containing 0.05% Tween 20 (PBS-0.05% Tween 20).
3) A solution obtained by diluting Anti-Human IgG (γ-chain) Peroxidase conjugated (MBL, 208) 2000-fold with PBS-0.05% Tween20 was added at 100 μL / well and left at room temperature for 1 hour.
4) The solution was discarded from the 96-well plate, and wells were sufficiently washed with PBS-0.05% Tween20.
5) 100 μL / well of TMB Peroxidase Substrate (Moss, TMBE-1000s) was added to cause a color reaction.
6) After 10 to 15 minutes of color reaction, 1N sulfuric acid was added at 100 μL / well to stop the color reaction.
7) The absorbance at 450 nm was measured.
Colonies that show high color development values in this ELISA measurement are clones that highly express human antibodies.

D) Culture of CHO cells highly expressing human antibodies
CHO cells highly expressing human antibodies selected by ELISA measurement were grown by culturing in Dulbecco's Modified Eagle's Medium supplemented with 10% Fetal Bovine Serum and 0.1 mg / mL Geneticin under conditions of 5% carbon dioxide at 37 ° C. It was. Fully proliferated human antibody high-expression CHO cells were replaced with CHO-S-SFMII (GIBCO, 12052-098), a serum-free medium supplemented with 0.1 mg / mL Geneticin. It was cultured to some extent.

E) Culture supernatant of human antibody high-expression CHO cells cultured in CHO-S-SFMII, which is a column-purified serum-free medium, from the culture supernatant of human antibody high-expression cells. Purification of the human antibody was performed by Protein A column purification that is usually used for antibody purification.
Protein A column purification of human antibodies
1) Culture supernatant obtained by culturing CHO cells with high expression of human antibody in 1000 mL of CHO-S-SFMII is Amicon Stirred Uitrafiltration Cell (Millipore, Model 8400) with Amicon Ultrafiltration Membranes (Millipore, Code YM10). ) Was concentrated from 1000 mL to 100 mL.
2) To the culture supernatant concentrate, 8 ml of Protein A-Sepharose rProteinA Sepharose Fast Flow (Amersham Bioscience, 17-1279-03) was added and stirred at 4 ° C. for 2 days to adsorb the human antibody to ProteinA. .
3) Protein A-Sepharose adsorbed with human antibodies was packed in a column having a diameter of 1.5 cm and a length of 8 cm.
4) The column packed with Protein A-Sepharose was washed with 100 mL of PBS, and then human antibody was eluted from the column with Elution Buffer (0.17M Glycine-HCl, pH 2.3).
5) The eluted human antibody solution was immediately neutralized with pH by adding an appropriate amount of 1M Tris-HCl pH8.5.
6) The human antibody eluate was dialyzed into 5 L of PBS at 4 ° C. in Cell Sep T2 (Membrane filtration products Inc, 8030-23) as a dialysis tube. Human antibodies collected after dialysis were used as purified products.

Evaluation of ADCC activity of various human antibodies The evaluation of the antibody-dependent cellular cytotoxicity (ADCC) activity of various human antibodies was performed by a lactate dehydrogenase release assay. In this assay, NCI-H1373 (sometimes abbreviated as H1373) cells having ALCAM molecules on the cell membrane surface were used as Target cells, and Human peripheral blood mononuclear cells (PBMC) were used as Effector cells. This Human PBMC was prepared from human blood using Lymphoprep (Axis Shield).
H1373 cells (1 × 10 ⁴ cells / 50 μL) are placed in each well of a 96-well U-bottomed plate, and Human PBMC, which is an Effector cell, is 2 × 10 2 so that the E / T ratio is 20/1. ⁵ were added. Further, various anti-ALCAM (066-174) human antibodies were added to the cell solution so as to form a serial dilution series, and incubated at 37 ° C. for 20 hours. After incubation, the 96-well U-bottomed plate was centrifuged and the lactate dehydrogenase activity in the supernatant was measured using CytoTox 96 Non-Radioactive Cytotoxicity Assay Kit (Promega, G1780). Antibody-dependent and specific Cytotoxicity% was calculated using the following formula.
% Cytotoxicity = 100 × (E−S _E −S _T ) / (M−S _T )

Here, E represents Experimental release, and is the lactate dehydrogenase activity relaese from the Target cells when the antibody and Effctor cells are incubated with the Target cells. S _E is lactate dehydrogenase activity released spontaneously from Effector cells. The S _T, is spontaneously released lactate dehydrogenase activity from Target cells. M represents the lactate dehydrogenase activity released to the maximum of Target cells, which is the lactate dehydrogenase activity released from Target cells by addition of Lysis Solution (9% Triton X-100).

  FIG. 11 and Table 8 show the results of ADCC activity evaluation of wild-type, 295 Cys-type, and 298 Cys-type Anti-ALCAM (066-174) human antibodies performed using the above-described evaluation method. The 298Cys type human antibody showed higher ADCC activity than the wild type under high concentration conditions (0.0001 μg / mL or more) compared to the wild type.

Furthermore, the percentage of cytotoxicity between the wild-type human antibody and the mutant human antibody was compared by a lactate dehydrogenase release assay. As a result, the percentage of cytotoxicity when the concentration of the added antibody was 0.00001, 0.0001, 0.001, 0.01, 0.1, 1, 10 μg / mL was 2.22%, 2.65%, 4.31%, 18.68 for the wild type human antibody, respectively. %, 37.35%, 36.73%, 30.82%, 295Cys human antibodies, 1.62%, 3.14%, 8.78%, 25.46%, 42.49%, 43.69%, 44.58%, and 298Cys human antibodies, respectively, 2.13% 3.66%, 4.60%, 30.76%, 44.87%, 45.64% and 52.45%.
Therefore, the ratio of ADCC activity (ratio of the ratio of cytotoxicity of 298Cys type human antibody to wild type human antibody) should be 1.0 times, 1.4 times, 1.1 times, 1.6 times, 1.2 times, 1.2 times, and 1.7 times, respectively. (Table 8).
In addition, from Table 8, two points with the same rate of cytotoxicity between the wild type human antibody and the type 298 human antibody were selected, and comparison of ADCC activity was attempted. For example, wild type human antibody shows about 37% cytotoxicity at 0.1 μg / mL, and type 298 human antibody shows about 31% cytotoxicity at 0.01 μg / mL. When compared using these two points, the antibody concentration required to obtain the same ADCC activity was reduced to about 1/10. From this, ADCC activity increased about 10 times (the ratio of the concentration of type 298 human antibody to wild type human antibody between the two points where the wild type human antibody and type 298 human antibody showed the same cytotoxicity ratio) 1/10 times).
Those skilled in the art can calculate the increase in ADCC activity from the figures and tables following the above method.

[Example 2]
In the same manner as in Example 1, anti-ALCAM (035-234) antibodies of wild type, 286 Cys type, 295 Cys type or 298 Cys type were prepared, and these various variants of Anti-ALCAM (035-234) human antibodies were prepared. ADCC activity was evaluated. As a result, comparing the ADCC activity of the wild type and the anti-ALCAM (035-234) human antibody, the 295 Cys type and 298 Cys type Anti-ALCAM (035-234) human antibody showed high ADCC activity.
The following describes a method for producing an anti-ALCAM (035-234) human antibody wild type, 286 Cys type, 295 Cys type or 298 Cys type and measuring ADCC activity thereof.

1) Preparation of Anti-ALCAM (035-234) Antibody A human antibody is obtained as a purified human antibody through the following steps A) to E).
A) Mutation of Fc region gene,
B) Construction of human antibody expression vector combining Fc region and mutated Fc region gene,
C) Gene transfer of human antibody expression vector into CHO cells and screening for CHO cells that highly express human antibodies,
D) culture of CHO cells highly expressing human antibodies,
E) Column purification from culture supernatant of cells expressing high human antibody.
The steps A) to E) are described in order below.
Among these steps, A), B), D) and E) are the same as those in Example 1. Accordingly, in this example, step C) will be described.

Four genes are required to produce human antibodies. These four genes are a human antibody L chain variable region gene, a human antibody H chain variable region gene, a human IgG1 L chain constant region gene, and a human IgG1 H chain constant region gene. The L chain variable region and constant region genes and the H chain variable region and constant region genes of Anti-ALCAM (035-234), which is an antibody clone that recognizes ALCAM, were provided by Antibody Research Institute Co., Ltd. The DNA sequence of the provided human L chain variable region of Anti-ALCAM (035-234) is shown in FIG. 3 (SEQ ID NO: 148), and the DNA sequence of the human H chain variable region is shown in FIG. 4 (SEQ ID NO: 150). .
In addition, the nucleotide sequences of CDR1, CDR2, and CDR3 of the human L chain variable region of Anti-ALCAM (035-234) are shown in SEQ ID NOs: 31-33. In addition, the amino acid sequences of CDR1, CDR2, and CDR3 of the human L chain variable region of Anti-ALCAM (035-234) are shown in SEQ ID NOs: 34 to 36, respectively. The provided human IgG1 L chain constant region gene is a λ chain, the base sequence thereof is shown in SEQ ID NO: 37, and the amino acid sequence thereof is shown in SEQ ID NO: 38.
The nucleotide sequences of CDR1, CDR2, and CDR3 of the human H chain variable region of Anti-ALCAM (035-234) are shown in SEQ ID NOs: 39 to 41. In addition, the amino acid sequences of CDR1, CDR2, and CDR3 of the human H chain variable region of Anti-ALCAM (035-234) are shown in SEQ ID NOs: 42 to 44, respectively. The base sequence and amino acid sequence of the provided human IgG1 heavy chain constant region are the same as Anti-ALCAM (066-174).

C) Construction of human antibody expression vector combining the cloned gene and its mutated gene
Anti-ALCAM (035-234) human antibody is expressed by transfecting CHO cells with two types of expression vectors of the anti-ALCAM (035-234) human antibody, L chain and H chain. Here, the expression vector of the anti-ALCAM (035-234) human antibody L chain is expressed by combining the anti-ALCAM (035-234) L chain variable region gene + human IgG1 L chain constant region gene. Anti-ALCAM (035-234) H chain variable region gene + human IgG1 H chain constant region gene was combined with the anti-ALCAM (035-234) H chain expression vector. Is incorporated into an expression vector. In preparing anti-ALCAM (035-234) antibodies of wild type, 286Cys type, 295Cys type or 298Cys type, the anti-ALCAM (035-234) human antibody L chain expression vector is the four human antibodies. The anti-ALCAM (035-234) human antibody H chain expression vector is used for each of the wild type, 286 Cys type, 295 Cys type and 298 Cys type human antibodies. A special heavy chain expression vector is required. Here, the construction of an anti-ALCAM (035-234) human antibody L chain expression vector that can be commonly used for expression of each human antibody is described in detail, and then the 286 Cys type, 295 Cys type or 298 Cys type human antibody Describes the construction of a dedicated Anti-ALCAM (035-234) human antibody heavy chain expression vector required for expression.

Construction of the expression vector of Anti-ALCAM (035-234) human antibody L chain As the expression vector , BCMG-neo vector is used, and Anti-ALCAM (035-234) human L chain variable at the XhoI and NotI sites of this vector. Insert a region gene + human IgG1 L chain constant region gene. Anti-ALCAM (035-234) human L chain variable region and human IgG1 L chain constant region genes are subjected to the following PCR reaction using the Anti-ALCAM (035-234) L chain gene provided by the antibody laboratory as a template. A fragment was obtained.

Anti-ALCAM (035-234) Human L chain variable region and human IgG1 L chain constant region gene amplification reaction
Anti-ALCAM (035-234) L chain (25 ng / μL) 2 μL
2.5 mM dNTPs 4 μL
EGFR-L1 primer (20μM) 2.5 μL
EGFR-L2 primer (20μM) 2.5 μL
DMSO 2.5 μL
× 10 pfu polymerase Buffer 5 μL
pfu polymerase 1 μL
Sterile water 30.5 μL
total 50 μL
94 ℃ 2 min
94 ℃ 1 min 55 ℃ 2min 72 ℃ 2min (30 cycles)
72 ℃ 4 min
4 ℃ unlimited time
See below for Primer DNA sequences.
EGFR-L1 primer: ACCGCTCGAGATGGACATGAGGGTCCCCGCTCAGCTC (SEQ ID NO: 25)
EGFR-L2 primer: ATAGTTTAGCGGCCGCTTACGAACATTCTGTAGGGGCCACTGTCTT (SEQ ID NO: 26)

  Anti-ALCAM (035-234) human L chain variable region and human IgG1 L chain constant region gene fragments obtained by the above PCR reaction were cleaved with restriction enzyme XhoI (Takara) and restriction enzyme NotI (Takara). Purified. The fragment purified by cutting the BCMG-neo vector with XhoI and NotI was mixed with the purified Anti-ALCAM (035-234) human L chain variable region and human IgG1 L chain constant region gene fragment and ligated. In the vector obtained by this ligation, it was confirmed by sequencing that the desired fragment was inserted. This anti-ALCAM (035-234) human antibody L chain expression vector was named phuman ALCAM (035-234) LC.

Construction of Anti-ALCAM (035-234) human antibody heavy chain expression vector BCMG-neo vector is used as the expression vector, and Anti-ALCAM (035-234) human heavy chain variable region is used at the XhoI and NotI sites of this vector. Gene + H human IgG1 H chain constant region gene is inserted. The anti-ALCAM (035-234) human heavy chain variable region gene was obtained by performing the following PCR reaction using the anti-ALCAM (035-234) heavy chain gene provided by the antibody laboratory as a template.
The dedicated H chain expression vector for each of the wild type, 286Cys type, 295Cys type or 295Cys type human antibody has a different plasmid as a template for PCR amplification of the human IgG1 H chain constant region gene, but the operation itself is The procedure was exactly the same, and various human IgG1 H chain gene fragments were obtained. Details including the PCR reaction are described below.

Anti-ALCAM (035-234) Human antibody heavy chain variable region gene amplification reaction
Anti-ALCAM (066-174) H chain (25 ng / μL) 2 μL
2.5 mM dNTPs 4 μL
EGFR-H1 primer (20μM) 2.5 μL
ALCAM-heavy-variable-234-R primer (20μM) 2.5 μL
DMSO 2.5 μL
× 10 pfu polymerase Buffer 5 μL
pfu polymerase 1 μL
Sterile water 30.5 μL
total 50 μL
94 ℃ 2 min
94 ℃ 1 min 55 ℃ 2min 72 ℃ 2min (30 cycles)
72 ℃ 4 min
4 ℃ unlimited time
EGFR-H1 primer: ACCGCTCGAGATGAAACACCTGTGGTTCTTCCTC (SEQ ID NO: 27)
ALCAM-heavy-variable-234-R primer: CGACGAGACGGTGACCAGGGTTCCCTG (5'-phosphorylated) (SEQ ID NO: 45)

Amplification reaction of human IgG1 heavy chain constant region gene #Various template plasmids (20 ng / μL) 2 μL
2.5 mM dNTPs 4 μL
H3 primer (20μM) 2.5 μL
H4 primer (20μM) 2.5 μL
DMSO 2.5 μL
× 10 pfu polymerase Buffer 5 μL
pfu polymerase 1 μL
Sterile water 30.5 μL
total 50 μL
94 ℃ 2 min
94 ℃ 1 min 55 ℃ 2min 72 ℃ 2min (30 cycles)
72 ℃ 4 min
4 ℃ unlimited time
H3 primer: GCCTCCACCAAGGGCCCATCGGTC (5'-phosphorylated) (SEQ ID NO: 29)
H4 primer: ATAGTTTAGCGGCCGCTTATTTACCCGGAGACAGGGAGAGGCTCTT (SEQ ID NO: 30)

  In order to prepare a gene fragment for a wild type Anti-ALCAM (035-234) human antibody, pCR2.1-HC (wild type) was used as a template. PCR2.1-HC (286Cys) was used as a template in order to prepare a gene fragment for 286Cys type Anti-ALCAM (035-234) human antibody. PCR2.1-HC (295Cys) was used as a template to prepare a gene fragment for 295 Cys-type Anti-ALCAM (035-234) human antibody. PCR2.1-HC (298Cys) was used as a template to prepare a gene fragment for the 298Cys-type Anti-ALCAM (035-234) human antibody.

  The Anti-ALCAM (035-234) H chain variable region gene fragment obtained by the above PCR reaction was purified after being cleaved with a restriction enzyme XhoI (Takara). Moreover, human IgG1 H chain constant region gene fragments obtained by various PCR reactions with different templates were purified after being cleaved with a restriction enzyme NotI (Takara). Ligation was performed by mixing the purified anti-ALCAM (035-234) heavy chain variable region gene fragment and purified human IgG1 heavy chain constant region gene fragment into the fragment purified by cutting the BCMG-neo vector with XhoI and NotI. . In the vector obtained by this ligation, it was confirmed by sequencing that the desired fragment was inserted. This anti-ALCAM (035-234) human antibody H chain expression vector includes p human ALCAM (035-234) HC (wild type), p human ALCAM (035-234) HC (286 Cys type), p human ALCAM ( 035-234) HC (295 Cys) or p Human ALCAM (035-234) HC (298 Cys type), respectively.

  Anti-body-dependent cellular cytotoxicity (ADCC) activity of various human antibodies was evaluated according to the same method as described in Example 1. The ADCC activity results of wild-type, 286Cys-type, 295Cys-type, and 298Cys-type human antibodies are shown in FIG. From the figure, the 286Cys type human antibody showed higher ADCC activity than the wild type in the high concentration condition (0.1 μg / mL or more) compared to the wild type, and the 298Cys type human antibody showed higher ADCC activity in the whole concentration range.

その結果、添加した抗体の濃度が0.0001、0.001、0.01、0.1、1、10μg/mLの場合における細胞障害の割合は、野生型ヒト抗体の場合それぞれ2.36％、2.99％、4.70％、11.66％、8.99％、12.06％、286Cys型ヒト抗体の場合はそれぞれ2.59％、3.30％、4.35％、13.93％、14.97％、15.06％、であった。295型ヒト抗体の場合はそれぞれ2.97％、3.63％、5.24％、11.51％、11.77％、14.40％、であった。298型ヒト抗体の場合はそれぞれ4.46％、4.76％、6.00％、13.39％、19.48％、24.13％、であった。
よって、ADCC活性の比（野生型ヒト抗体と変異型ヒト抗体が同一濃度である2点間における、野生型ヒト抗体に対する変異型ヒト抗体の細胞障害の割合の比）は、それぞれ286C/野生型では1.1倍、1.1倍、0.9倍、1.2倍、1.7倍、1.2倍、295C/野生型では1.3倍、1.2倍、1.1倍、1.0倍、1.3倍、1.2倍、298C/野生型では1.9倍、1.6倍、1.3倍、1.1倍、2.2倍、2.0倍、となることがわかった（表９）。
また表９より、野生型ヒト抗体と298型ヒト抗体との間で細胞障害の割合が同程度である2点を選択し、ADCC活性の比較を試みた。例えば、野生型ヒト抗体は10μg/mLで約12％、298型ヒト抗体は0.1μg/mLで約13％の細胞障害を示している。この2点を用いて比較した場合、同じADCC活性を得るために必要な抗体濃度が約100分の1に減少した。このことから、ADCC活性が約100倍上昇した（野生型ヒト抗体と298型ヒト抗体が同一の細胞障害の割合を示す2点間における、野生型ヒト抗体に対する298型ヒト抗体の濃度の比が1/100倍になった）と判断することが出来る。
また表９より、野生型ヒト抗体と286型ヒト抗体との間で細胞障害の割合が同程度である2点を選択し、ADCC活性の比較を試みた。例えば、野生型ヒト抗体は10μg/mLで約12
％、286型ヒト抗体は0.1μg/mLで約13％の細胞障害を示している。この2点を用いて比較した場合、同じADCC活性を得るために必要な抗体濃度が約100分の1に減少した。このことから、ADCC活性が約100倍上昇した（野生型ヒト抗体と286型ヒト抗体が同一の細胞障害の割合を示す2点間における、野生型ヒト抗体に対する286型ヒト抗体の濃度の比が1/100倍になった）と判断することが出来る。
当業者であれば、上記の方法に倣い、図および表よりADCC活性の上昇を算出することが可能である。 Furthermore, the percentage of cytotoxicity between the wild type human antibody and the mutant human antibody was compared by a lactate dehydrogenase release assay (Table 9).

As a result, the percentage of cytotoxicity when the concentration of the added antibody was 0.0001, 0.001, 0.01, 0.1, 1, 10 μg / mL was 2.36%, 2.99%, 4.70%, 11.66% for the wild type human antibody, In the case of the 8.99%, 12.06%, and 286Cys human antibodies, they were 2.59%, 3.30%, 4.35%, 13.93%, 14.97%, and 15.06%, respectively. In the case of type 295 human antibody, they were 2.97%, 3.63%, 5.24%, 11.51%, 11.77%, and 14.40%, respectively. In the case of type 298 human antibody, they were 4.46%, 4.76%, 6.00%, 13.39%, 19.48%, and 24.13%, respectively.
Therefore, the ratio of ADCC activity (ratio of the ratio of cytotoxicity of the mutant human antibody to the wild type human antibody between the two points at the same concentration of the wild type human antibody and the mutant human antibody) is 286C / wild type, respectively. Is 1.1 times, 1.1 times, 0.9 times, 1.2 times, 1.7 times, 1.2 times, 295C / wild type is 1.3 times, 1.2 times, 1.1 times, 1.0 times, 1.3 times, 1.2 times, 298C / wild type is 1.9 times, It was found to be 1.6 times, 1.3 times, 1.1 times, 2.2 times, and 2.0 times (Table 9).
In addition, from Table 9, two points with the same rate of cytotoxicity between the wild type human antibody and the type 298 human antibody were selected, and comparison of ADCC activity was attempted. For example, the wild type human antibody shows about 12% cytotoxicity at 10 μg / mL, and the type 298 human antibody shows about 13% cytotoxicity at 0.1 μg / mL. When compared using these two points, the antibody concentration required to obtain the same ADCC activity was reduced to about 1/100. From this, ADCC activity increased by about 100 times (the ratio of the concentration of the 298 type human antibody to the wild type human antibody between the two points where the wild type human antibody and the type 298 human antibody showed the same cytotoxicity ratio) 1/100 times).
In addition, from Table 9, two points with the same rate of cytotoxicity were selected between the wild type human antibody and the type 286 human antibody, and comparison of ADCC activity was attempted. For example, the wild type human antibody is about 12 at 10 μg / mL.
%, Type 286 human antibody shows about 13% cytotoxicity at 0.1 μg / mL. When compared using these two points, the antibody concentration required to obtain the same ADCC activity was reduced to about 1/100. As a result, ADCC activity increased about 100 times (the ratio of the concentration of type 286 human antibody to wild type human antibody between the two points where the wild type human antibody and type 286 human antibody showed the same cytotoxicity ratio). 1/100 times).
Those skilled in the art can calculate the increase in ADCC activity from the figures and tables following the above method.

Example 3
In the same manner as in Example 1, anti-ICAM1 (053-059) antibodies of wild type, 286Cys type, 295Cys type or 298Cys type were prepared, and anti-ICAM1 (053-059) human antibodies of these various mutants were produced. ADCC activity was evaluated. As a result, comparing the ADCC activity of the wild type and the anti-ICAM1 (053-059) human antibody, the anti-ICAM1 (053-059) human antibody of 286Cys type, 295Cys type, and 298Cys type has high ADCC activity. showed that.
The following describes a method for producing wild-type, 286Cys, 295Cys or 298Cys type anti-ICAM1 (053-059) human antibody and measuring its ADCC activity.

1) Preparation of Anti-ICAM1 (053-059) Antibody A human antibody is obtained as a purified human antibody through the following steps A) to E).
A) Mutation of Fc region gene,
B) Construction of human antibody expression vector combining Fc region and mutated Fc region gene,
C) Gene transfer of human antibody expression vector into CHO cells and screening for CHO cells that highly express human antibodies,
D) culture of CHO cells highly expressing human antibodies,
E) Column purification from culture supernatant of cells expressing high human antibody.
The steps A) to E) are described in order below.
Among these steps, A), B), D) and E) are the same as those in Example 1. Accordingly, in this example, step C) will be described.

Four genes are required to produce human antibodies. These four genes are a human antibody L chain variable region gene, a human antibody H chain variable region gene, a human IgG1 L chain constant region gene, and a human IgG1 H chain constant region gene. The L chain variable region and constant region genes, and the H chain variable region and constant region genes of Anti-ICAM1 (053-059), which is an antibody clone that recognizes ALCAM, were provided by Antibody Research Laboratories. The DNA sequence of the human L chain variable region of the provided Anti-ICAM1 (053-059) is shown in FIG. 5 (SEQ ID NO: 152), and the DNA sequence of the human H chain variable region is shown in FIG. 6 (SEQ ID NO: 154). .
In addition, the nucleotide sequences of CDR1, CDR2, and CDR3 of the human L chain variable region of Anti-ICAM1 (053-059) are shown in SEQ ID NOs: 46 to 48, respectively. In addition, the amino acid sequences of CDR1, CDR2, and CDR3 of the human L chain variable region of Anti-ICAM1 (053-059) are shown in SEQ ID NOs: 49 to 51, respectively. The provided human IgG1 L chain constant region gene is a λ chain, the base sequence thereof is shown in SEQ ID NO: 52, and the amino acid sequence thereof is shown in SEQ ID NO: 53.
The nucleotide sequences of CDR1, CDR2, and CDR3 of the human H chain variable region of Anti-ICAM1 (053-059) are shown in SEQ ID NOs: 54 to 56, respectively. The amino acid sequences of CDR1, CDR2, and CDR3 of the human H chain variable region of Anti-ICAM1 (053-059) are shown in SEQ ID NOs: 57 to 59. The base sequence and amino acid sequence of the provided human IgG1 heavy chain constant region are the same as Anti-ALCAM (066-174).

C) Construction of human antibody expression vector combining the cloned gene and its mutated gene
Anti-ICAM1 (053-059) human antibody can be expressed by transfecting CHO cells with two types of expression vectors of L-chain and H-chain of Anti-ICAM1 (053-059) human antibody. Here, the expression vector of the anti-ICAM1 (053-059) human antibody L chain is expressed by combining Anti-ICAM1 (053-059) L chain variable region gene + human IgG1 L chain constant region gene Anti-ICAM1 (053-059) H chain expression vector combined with Anti-ICAM1 (053-059) H chain variable region gene + human IgG1 H chain constant region gene Is incorporated into an expression vector. In preparing anti-ICAM1 (053-059) antibody of wild type, 286Cys type, 295Cys type or 298Cys type, the anti-ICAM1 (053-059) human antibody L chain expression vector is these four human antibodies The anti-ICAM1 (053-059) human antibody H chain expression vector is commonly used for the expression of wild type, 286 Cys type, 295 Cys type or 298 Cys type human antibody. A special heavy chain expression vector is required. Here, the construction of an anti-ICAM1 (053-059) human antibody L chain expression vector that can be commonly used for the expression of each human antibody is described in detail, and then the 286Cys type, 295Cys type or 298Cys type human antibody is described. Describes the construction of a dedicated anti-ICAM1 (053-059) human antibody heavy chain expression vector required for expression.

Construction of expression vector of Anti-ICAM1 (053-059) human antibody L chain As the expression vector , BCMG-neo vector is used, and Anti-ICAM1 (053-059) human L chain variable at XhoI and NotI sites of this vector A region gene + human IgG1 L chain constant region gene is inserted. Anti-ICAM1 (053-059) human L chain variable region and human IgG1 L chain constant region genes are subjected to the following PCR reaction using the Anti-ICAM1 (053-174) L chain gene provided by the antibody laboratory as a template. A fragment was obtained.

Anti-ICAM1 (053-059) Human L chain variable region and human IgG1 L chain constant region gene amplification reaction
Anti-ICAM1 (053-059) L chain (25 ng / μL) 2 μL
2.5 mM dNTPs 4 μL
EGFR-L1 primer (20μM) 2.5 μL
EGFR-L2 primer (20μM) 2.5 μL
DMSO 2.5 μL
× 10 pfu polymerase Buffer 5 μL
pfu polymerase 1 μL
Sterile water 30.5 μL
total 50 μL
94 ℃ 2 min
94 ℃ 1 min 55 ℃ 2min 72 ℃ 2min (30 cycles)
72 ℃ 4 min
4 ℃ unlimited time
See below for Primer DNA sequences.
EGFR-L1 primer: ACCGCTCGAGATGGACATGAGGGTCCCCGCTCAGCTC (SEQ ID NO: 25)
EGFR-L2 primer: ATAGTTTAGCGGCCGCTTACGAACATTCTGTAGGGGCCACTGTCTT (SEQ ID NO: 26)

  Anti-ICAM1 (053-059) human L chain variable region and human IgG1 L chain constant region gene fragments obtained by the above PCR reaction are cleaved with restriction enzyme XhoI (Takara) and restriction enzyme NotI (Takara). Purified. The fragment purified by cutting the BCMG-neo vector with XhoI and NotI was mixed with the purified Anti-ICAM1 (053-059) human L chain variable region and human IgG1 L chain constant region gene fragment and ligated. In the vector obtained by this ligation, it was confirmed by sequencing that the desired fragment was inserted. This anti-ICAM1 (053-059) human antibody L chain expression vector was named phuman ICAM1 (053-059) LC.

Construction of Anti-ICAM1 (053-059) human antibody heavy chain expression vector BCMG-neo vector is used as the expression vector, and Anti-ICAM1 (053-059) human heavy chain variable region is located at the XhoI and NotI sites of this vector. Gene + H human IgG1 H chain constant region gene is inserted. The anti-ICAM1 (053-059) human heavy chain variable region gene was obtained by performing the following PCR reaction using the anti-ICAM1 (035-059) heavy chain gene provided by the antibody laboratory as a template.
The dedicated H chain expression vector for each of the wild type, 286Cys type, 295Cys type or 295Cys type human antibody has a different plasmid as a template for PCR amplification of the human IgG1 H chain constant region gene, but the operation itself is The procedure was exactly the same, and various human IgG1 H chain gene fragments were obtained. Details including the PCR reaction are described below.

Anti-ICAM1 (053-059) human antibody heavy chain variable region gene amplification reaction
Anti-ICAM1 (053-059) H chain (25 ng / μL) 2 μL
2.5 mM dNTPs 4 μL
EGFR-H1 primer (20μM) 2.5 μL
ALCAM-heavy-variable-R primer (20μM) 2.5 μL
DMSO 2.5 μL
× 10 pfu polymerase Buffer 5 μL
pfu polymerase 1 μL
Sterile water 30.5 μL
total 50 μL
94 ℃ 2 min
94 ℃ 1 min 55 ℃ 2min 72 ℃ 2min (30 cycles)
72 ℃ 4 min
4 ℃ unlimited time
EGFR-H1 primer: ACCGCTCGAGATGAAACACCTGTGGTTCTTCCTC (SEQ ID NO: 27)
ALCAM-heavy-variable-R primer: CGACGAGACGGTGACCGTGGTCCCTTGGCC (5'-phosphorylated) (SEQ ID NO: 28)

Amplification reaction of human IgG1 heavy chain constant region gene #Various template plasmids (20 ng / μL) 2 μL
2.5 mM dNTPs 4 μL
H3 primer (20μM) 2.5 μL
H4 primer (20μM) 2.5 μL
DMSO 2.5 μL
× 10 pfu polymerase Buffer 5 μL
pfu polymerase 1 μL
Sterile water 30.5 μL
total 50 μL
94 ℃ 2 min
94 ℃ 1 min 55 ℃ 2min 72 ℃ 2min (30 cycles)
72 ℃ 4 min
4 ℃ unlimited time
H3 primer: GCCTCCACCAAGGGCCCATCGGTC (5'-phosphorylated) (SEQ ID NO: 29)
H4 primer: ATAGTTTAGCGGCCGCTTATTTACCCGGAGACAGGGAGAGGCTCTT (SEQ ID NO: 30)

  In order to prepare a gene fragment for a wild type Anti-ICAM1 (053-085) human antibody, pCR2.1-HC (wild type) was used as a template. PCR2.1-HC (286Cys) was used as a template to prepare a gene fragment for the 286Cys type Anti-ICAM1 (053-059) human antibody. PCR2.1-HC (295Cys) was used as a template to prepare a gene fragment for 295 Cys-type Anti-ICAM1 (053-059) human antibody. PCR2.1-HC (298Cys) was used as a template in order to prepare a gene fragment for 298Cys-type Anti-ICAM1 (053-059) human antibody.

  The Anti-ICAM1 (053-059) H chain variable region gene fragment obtained by the above PCR reaction was purified after being cleaved with a restriction enzyme XhoI (Takara). Moreover, human IgG1 H chain constant region gene fragments obtained by various PCR reactions with different templates were purified after being cleaved with a restriction enzyme NotI (Takara). The BCMG-neo vector digested with XhoI and NotI was purified and mixed with the purified Anti-ICAM1 (053-059) heavy chain variable region gene fragment and purified human IgG1 heavy chain constant region gene fragment. . In the vector obtained by this ligation, it was confirmed by sequencing that the desired fragment was inserted. Anti-ICAM1 (053-059) human antibody H chain expression vectors are p human ICAM1 (053-059) HC (wild type), p human ICAM1 (053-059) HC (286 Cys type), p human ICAM1 ( 053-059) HC (295Cys) or p human ICAM1 (053-059) HC (298Cys type), respectively.

  Anti-body-dependent cellular cytotoxicity (ADCC) activity of various human antibodies was evaluated according to the same method as described in Example 1. FIG. 13 shows the ADCC activity results of human antibodies of wild type, 286 Cys type, 295 Cys type, and 298 Cys type. The 286Cys type and 298Cys type human antibodies showed higher ADCC activity under higher concentration conditions (0.01 μg / mL or more) than the wild type.

その結果、添加した抗体の濃度が0.0001、0.001、0.01、0.1、1、10μg/mLの場合における細胞障害の割合は、野生型ヒト抗体の場合それぞれ8.90％、8.76％、11.91％、20.70％、23.11％、22.17％、、286Cys型ヒト抗体の場合はそれぞれ7.85％、7.61％、13.55％、27.52％、32.15％、31.30％、295Cys型ヒト抗体の場合はそれぞれ3.71％、5.08％、13.15％、23.65％、25.61％、29.03％、298Cys型ヒト抗体の場合はそれぞれ8.10％、6.21％、13.89％、26.02％、27.99％、32.76％であった。
よって、ADCC活性の比（野生型ヒト抗体と変異型ヒト抗体が同一濃度である2点間における、野生型ヒト抗体に対する変異型ヒト抗体の細胞障害の割合の比）は、286C/野生型それぞれ、0.9倍、0.9倍、1.1倍、1.3倍、1.4倍、1.4倍、295C/野生型それぞれ、0.4倍、0.6倍、1.1倍、1.1倍、1.1倍、1.3倍、298C/野生型それぞれ、0.9倍、0.7倍、1.2倍、1.3倍、1.2倍、1.5倍、となることがわかった（表１０）。 Furthermore, the percentage of cytotoxicity between the wild-type human antibody and the mutant human antibody was compared by a lactate dehydrogenase release assay (Table 10).

As a result, the percentage of cytotoxicity when the concentration of the added antibody was 0.0001, 0.001, 0.01, 0.1, 1, 10 μg / mL was 8.90%, 8.76%, 11.91%, 20.70% for the wild type human antibody, 23.11%, 22.17%, 286Cys human antibodies are 7.85%, 7.61%, 13.55%, 27.52%, 32.15%, 31.30% and 295Cys human antibodies are 3.71%, 5.08%, 13.15%, respectively. In the case of 23.65%, 25.61%, 29.03%, and 298Cys type human antibodies, they were 8.10%, 6.21%, 13.89%, 26.02%, 27.99%, and 32.76%, respectively.
Therefore, the ratio of ADCC activity (ratio of the ratio of cytotoxicity of the mutant human antibody to the wild type human antibody between the two points where the wild type human antibody and the mutant human antibody are at the same concentration) is 286C / wild type, respectively. 0.9, 0.9, 1.1, 1.3, 1.4, 1.4, 295C / wild type, 0.4, 0.6, 1.1, 1.1, 1.1, 1.3, 298C / wild type, 0.9 It was found that they were double, 0.7 times, 1.2 times, 1.3 times, 1.2 times, and 1.5 times (Table 10).

In addition, from the table, two points with the same rate of cytotoxicity were selected between the wild type human antibody and the 286Cys type human antibody, and comparison of ADCC activity was attempted. For example, the wild type human antibody shows about 22% cytotoxicity at 10 μg / mL, and the 286 Cys type and 298 Cys type human antibodies show about 26% cytotoxicity at 0.1 μg / mL. When compared using these two points, the antibody concentration required to obtain the same ADCC activity was reduced to about 1/100. As a result, ADCC activity increased about 100 times (the wild-type human antibody and the 286 Cys-type or 298 Cys-type human antibody showed the same cell damage ratio between the two points of the mutant human antibody against the wild-type human antibody. It can be determined that the concentration ratio has increased 100 times.
Those skilled in the art can calculate the increase in ADCC activity from the figures and tables following the above method.

Example 4
In the same manner as in Example 1, anti-ICAM1 (053-085) antibodies of wild type, 286Cys type, 295Cys type or 298Cys type were prepared, and anti-ICAM1 (053-085) human antibodies of these various mutant types were prepared. ADCC activity was evaluated. As a result, comparing the ADCC activity of the wild type and the anti-ICAM1 (053-085) human antibody, the anti-ICAM1 (053-085) human antibody of the 286Cys type, 295Cys type, and 298Cys type has high ADCC activity. showed that.
The following describes methods for producing anti-ICAM1 (053-085) human antibody wild type, 286 Cys type, 295 Cys type or 298 Cys type and measuring ADCC activity.

1) Production of Anti-ICAM1 (053-085) Antibody A human antibody is obtained as a purified human antibody through the following steps A) to E).
A) Mutation of Fc region gene,
B) Construction of human antibody expression vector combining Fc region and mutated Fc region gene,
C) Gene transfer of human antibody expression vector into CHO cells and screening for CHO cells that highly express human antibodies,
D) culture of CHO cells highly expressing human antibodies,
E) Column purification from culture supernatant of cells expressing high human antibody.
The steps A) to E) are described in order below.
Among these steps, A), B), D) and E) are the same as those in Example 1. Accordingly, in this example, step C) will be described.

Four genes are required to produce human antibodies. These four genes are a human antibody L chain variable region gene, a human antibody H chain variable region gene, a human IgG1 L chain constant region gene, and a human IgG1 H chain constant region gene. The L chain variable region and constant region genes, and the H chain variable region and constant region genes of Anti-ICAM1 (053-085), which is an antibody clone that recognizes ALCAM, were provided by Antibody Research Laboratories. The DNA sequence of the human L chain variable region of the provided Anti-ICAM1 (053-085) is shown in FIG. 7 (SEQ ID NO: 156), and the DNA sequence of the human H chain variable region is shown in FIG. 8 (SEQ ID NO: 158). .
In addition, the nucleotide sequences of CDR1, CDR2, and CDR3 of the human L chain variable region of Anti-ICAM1 (053-085) are shown in SEQ ID NOs: 60-62. In addition, the amino acid sequences of CDR1, CDR2, and CDR3 of the human L chain variable region of Anti-ICAM1 (053-085) are shown in SEQ ID NOs: 63 to 65, respectively. The provided human IgG1 L chain constant region gene is a λ chain, the base sequence thereof is shown in SEQ ID NO: 66, and the amino acid sequence thereof is shown in SEQ ID NO: 67.
In addition, the nucleotide sequences of CDR1, CDR2, and CDR3 of the human H chain variable region of Anti-ICAM1 (053-085) are shown in SEQ ID NOs: 68-70. The amino acid sequences of CDR1, CDR2, and CDR3 of the human H chain variable region of Anti-ICAM1 (053-059) are shown in SEQ ID NOs: 71 to 73, respectively. The base sequence and amino acid sequence of the provided human IgG1 heavy chain constant region are the same as Anti-ALCAM (066-174).

C) Construction of human antibody expression vector combining the cloned gene and its mutated gene
Anti-ICAM1 (053-085) human antibody can be expressed by transfecting CHO cells with two types of expression vectors of L-chain and H-chain of Anti-ICAM1 (053-085) human antibody. Here, L-chain expression vector of Anti-ICAM1 (053-085) human antibody expresses anti-ICAM1 (053-085) L chain variable region gene + human IgG1 L chain constant region gene combined Anti-ICAM1 (053-085) H chain expression vector was linked with Anti-ICAM1 (053-085) H chain variable region gene + human IgG1 H chain constant region gene. Is incorporated into an expression vector. In preparing anti-ICAM1 (053-085) antibody of wild type, 286Cys type, 295Cys type or 298Cys type, the anti-ICAM1 (053-085) human antibody L chain expression vector is these four human antibodies The anti-ICAM1 (053-085) human antibody H chain expression vector is commonly used for the expression of wild type, 286 Cys type, 295 Cys type or 298 Cys type human antibody. A dedicated heavy chain expression vector is required. Here, the construction of an anti-ICAM1 (053-085) human antibody L chain expression vector that can be commonly used for the expression of each human antibody is described in detail, and then the 286Cys type, 295Cys type or 298Cys type human antibody is described. Describes the construction of a dedicated Anti-ICAM1 (053-085) human antibody heavy chain expression vector required for expression.

Construction of expression vector of Anti-ICAM1 (053-085) human antibody L chain As the expression vector , BCMG-neo vector is used, and Anti-ICAM1 (053-085) human L chain variable at XhoI and NotI sites of this vector A region gene + human IgG1 L chain constant region gene is inserted. Anti-ICAM1 (053-085) human L chain variable region and human IgG1 L chain constant region genes are subjected to the following PCR reaction using the Anti-AICAM1 (053-085) L chain gene provided by the antibody laboratory as a template. A fragment was obtained.

Anti-ICAM1 (053-085) Human L chain variable region and human IgG1 L chain constant region gene amplification reaction
Anti-AICAM1 (053-085) light chain (25 ng / μL) 2 μL
2.5 mM dNTPs 4 μL
EGFR-L1 primer (20μM) 2.5 μL
EGFR-L2 primer (20μM) 2.5 μL
DMSO 2.5 μL
× 10 pfu polymerase Buffer 5 μL
pfu polymerase 1 μL
Sterile water 30.5 μL
total 50 μL
94 ℃ 2 min
94 ℃ 1 min 55 ℃ 2min 72 ℃ 2min (30 cycles)
72 ℃ 4 min
4 ℃ unlimited time
See below for Primer DNA sequences.
EGFR-L1 primer: ACCGCTCGAGATGGACATGAGGGTCCCCGCTCAGCTC (SEQ ID NO: 25)
EGFR-L2 primer: ATAGTTTAGCGGCCGCTTACGAACATTCTGTAGGGGCCACTGTCTT (SEQ ID NO: 26)

  Anti-ICAM1 (053-085) human L chain variable region and human IgG1 L chain constant region gene fragments obtained by the above PCR reaction were cleaved with restriction enzyme XhoI (Takara) and restriction enzyme NotI (Takara). Purified. The fragment purified by cutting the BCMG-neo vector with XhoI and NotI was mixed with the purified anti-ICAM1 (053-059) human L chain variable region and human IgG1 L chain constant region gene fragment and ligated. In the vector obtained by this ligation, it was confirmed by sequencing that the desired fragment was inserted. The expression vector for this anti-ICAM1 (053-085) human antibody light chain was named phuman ICAM1 (053-085) LC.

Construction of Anti-ICAM1 (053-085) human antibody heavy chain expression vector BCMG-neo vector is used as the expression vector, and Anti-ICAM1 (053-085) human heavy chain variable region is located at the XhoI and NotI sites of this vector. Gene + H human IgG1 H chain constant region gene is inserted. Anti-ICAM1 (053-085) human heavy chain variable region gene was obtained by performing the following PCR reaction using the Anti-AICAM1 (053-085) heavy chain gene provided by the antibody laboratory as a template. .
The dedicated H chain expression vector for each of the wild type, 286Cys type, 295Cys type or 295Cys type human antibody has a different plasmid as a template for PCR amplification of the human IgG1 H chain constant region gene, but the operation itself is The procedure was exactly the same, and various human IgG1 H chain gene fragments were obtained. Details including the PCR reaction are described below.

Anti-ICAM1 (053-085) human antibody heavy chain variable region gene amplification reaction
Anti-ICAM1 (053-085) (25 ng / μL) 2 μL
2.5 mM dNTPs 4 μL
EGFR-H1 primer (20μM) 2.5 μL
ICAM-heavy-variable-85-R primer (20μM) 2.5 μL
DMSO 2.5 μL
× 10 pfu polymerase Buffer 5 μL
pfu polymerase 1 μL
Sterile water 30.5 μL
total 50 μL
94 ℃ 2 min
94 ℃ 1 min 55 ℃ 2min 72 ℃ 2min (30 cycles)
72 ℃ 4 min
4 ℃ unlimited time
EGFR-H1 primer: ACCGCTCGAGATGAAACACCTGTGGTTCTTCCTC (SEQ ID NO: 27)
ICAM-heavy-variable-85-R primer: CGACGAGACGGTGACCATTGTCCCTTG (5'-phosphorylated) (SEQ ID NO: 74)

Amplification reaction of human IgG1 heavy chain constant region gene #Various template plasmids (20 ng / μL) 2 μL
2.5 mM dNTPs 4 μL
H3 primer (20μM) 2.5 μL
H4 primer (20μM) 2.5 μL
DMSO 2.5 μL
× 10 pfu polymerase Buffer 5 μL
pfu polymerase 1 μL
Sterile water 30.5 μL
total 50 μL
94 ℃ 2 min
94 ℃ 1 min 55 ℃ 2min 72 ℃ 2min (30 cycles)
72 ℃ 4 min
4 ℃ unlimited time
H3 primer: GCCTCCACCAAGGGCCCATCGGTC (5'-phosphorylated) (SEQ ID NO: 29)
H4 primer: ATAGTTTAGCGGCCGCTTATTTACCCGGAGACAGGGAGAGGCTCTT (SEQ ID NO: 30)

  In order to prepare a gene fragment for a wild type Anti-ICAM1 (053-085) human antibody, pCR2.1-HC (wild type) was used as a template. PCR2.1-HC (286Cys) was used as a template in order to prepare a gene fragment for 286Cys type Anti- ICAM1 (053-085) human antibody. PCR2.1-HC (295Cys) was used as a template in order to prepare a gene fragment for 295 Cys type Anti- ICAM1 (053-085) human antibody. PCR2.1-HC (298Cys) was used as a template to prepare a gene fragment for the 298Cys type Anti-ICAM1 (053-085) human antibody.

  The Anti-ICAM1 (053-085) H chain variable region gene fragment obtained by the above PCR reaction was purified after being cleaved with a restriction enzyme XhoI (Takara). Moreover, human IgG1 H chain constant region gene fragments obtained by various PCR reactions with different templates were purified after being cleaved with a restriction enzyme NotI (Takara). The BCMG-neo vector was digested with XhoI and NotI and purified, and the purified Anti-ICAM1 (053-085) heavy chain variable region gene fragment and purified human IgG1 heavy chain constant region gene fragment were mixed and ligated. . In the vector obtained by this ligation, it was confirmed by sequencing that the desired fragment was inserted. Anti-ICAM1 (053-085) human antibody H chain expression vectors are p human ICAM1 (053-085) HC (wild type), p human ICAM1 (053-085) HC (286Cys type), p human ICAM1 ( 053-085) HC (295Cys) or p Human ICAM1 (053-085) HC (298Cys type), respectively.

Anti-body-dependent cellular cytotoxicity (ADCC) activity of various human antibodies was evaluated according to the same method as described in Example 1. The ADCC activity results of wild-type, 286Cys-type, 295Cys-type, and 298Cys-type human antibodies are shown in FIG. 14, Table 11, FIG. 15, and FIG. In FIG. 14, Table 11 and FIG. 15, Table 12, in FIG. 14, the antibody clone is the same, but the person who collected the effector cells is different.
From FIG. 14, the 286Cys type human antibody is under a high concentration condition (1 μg / mL or more) compared to the wild type, and the 298Cys type human antibody is under a high concentration condition (0.1 μg / mL or more) compared to the wild type. It showed high ADCC activity.
From FIG. 15, the 286Cys type human antibody showed higher ADCC activity than the wild type in all concentration ranges. The 298Cys human antibody showed higher ADCC activity under higher concentration conditions (0.01 μg / mL or more) than the wild type.

その結果、表１１より、添加した抗体の濃度が0.0001、0.001、0.01、0.1、1、10μg/mLの場合における細胞障害の割合は、野生型ヒト抗体の場合それぞれ7.19％、8.72％、12.84％、28.99％、27.88％、30.11％、286Cys型ヒト抗体の場合はそれぞれ6.53％、8.38％、11.37％、27.56％、43.28％、52.95％、295Cys型ヒト抗体の場合はそれぞれ8.77％、7.15％、11.38％、31.62％、37.71％、32.42％、298Cys型ヒト抗体の場合はそれぞれ8.26％、7.34％、9.23％、31.30％、53.98％、55.07％であった。
よって、ADCC活性の比（野生型ヒト抗体と変異型ヒト抗体が同一濃度である2点間における、野生型ヒト抗体に対する変異型ヒト抗体の細胞障害の割合の比）は、それぞれ286C/野生型では0.9倍、1.0倍、0.9倍、1.0倍、1.6倍、1.8倍、295C/野生型では1.2倍、0.8倍、0.9倍、1.1倍、1.4倍、1.1倍、298C/野生型では1.1倍、0.8倍、0.7倍、1.1倍、1.9倍、1.8倍となることがわかった。
また、表１２より、添加した抗体の濃度が0.001、0.01、0.1、1、10μg/mLの場合における細胞障害の割合は、野生型ヒト抗体の場合それぞれ1.29％、1.28％、1.85％、3.20％、3.11％、286Cys型ヒト抗体の場合はそれぞれ2.36％、2.68％、7.50％、33.22％、40.17％、であった。298Cys型ヒト抗体の場合はそれぞれ1.19％、3.51％、18.41％、46.16％、46.54％、であった。
よって、ADCC活性の比（野生型ヒト抗体と変異型ヒト抗体が同一濃度である2点間における、野生型ヒト抗体に対する286Cys型ヒト抗体の細胞障害の割合の比）は、それぞれ、1.8 倍、2.1倍、4.1倍、10.4倍、12.9倍、となることがわかった。野生型ヒト抗体に対する298Cys型ヒト抗体の細胞障害の割合の比）は、それぞれ、0.9倍、2.7倍、10.0倍、14.4倍、15.0倍、となることがわかった。 Furthermore, the percentage of cytotoxicity between the wild-type human antibody and the mutant human antibody was compared by a lactate dehydrogenase release assay.

As a result, from Table 11, the ratio of cytotoxicity when the concentration of the added antibody is 0.0001, 0.001, 0.01, 0.1, 1, 10 μg / mL is 7.19%, 8.72%, 12.84% for the wild type human antibody, respectively. , 28.99%, 27.88%, 30.11%, 286Cys human antibodies are 6.53%, 8.38%, 11.37%, 27.56%, 43.28%, 52.95%, 295Cys human antibodies are 8.77%, 7.15%, respectively. In the case of 11.38%, 31.62%, 37.71%, 32.42%, and 298Cys type human antibody, they were 8.26%, 7.34%, 9.23%, 31.30%, 53.98%, and 55.07%, respectively.
Therefore, the ratio of ADCC activity (ratio of the ratio of cytotoxicity of the mutant human antibody to the wild type human antibody between two points where the wild type human antibody and the mutant human antibody are at the same concentration) is 286C / wild type, respectively. 0.9 times, 1.0 times, 0.9 times, 1.0 times, 1.6 times, 1.8 times, 295C / wild type 1.2 times, 0.8 times, 0.9 times, 1.1 times, 1.4 times, 1.1 times, 298C / wild type 1.1 times, It was found to be 0.8 times, 0.7 times, 1.1 times, 1.9 times, and 1.8 times.
From Table 12, the percentage of cytotoxicity when the concentration of the added antibody is 0.001, 0.01, 0.1, 1, and 10 μg / mL is 1.29%, 1.28%, 1.85%, and 3.20% for the wild-type human antibody, respectively. 3.11% and 286Cys human antibodies were 2.36%, 2.68%, 7.50%, 33.22%, and 40.17%, respectively. In the case of the 298Cys type human antibody, they were 1.19%, 3.51%, 18.41%, 46.16%, and 46.54%, respectively.
Therefore, the ratio of ADCC activity (ratio of the ratio of cytotoxicity of 286Cys human antibody to wild type human antibody between two points where the same concentration of wild type human antibody and mutant human antibody) is 1.8 times, respectively. It was found to be 2.1 times, 4.1 times, 10.4 times, 12.9 times. It was found that the ratio of the cytotoxicity ratio of the 298Cys human antibody to the wild type human antibody was 0.9 times, 2.7 times, 10.0 times, 14.4 times, and 15.0 times, respectively.

In the experimental results shown in Table 11 and the experimental results shown in Table 12, the antibody clones are the same, but the person who provided the effector cells in the blood is different. Is hardly seen.
However, surprisingly, as a result of introducing a 286Cys mutation or a 298Cys mutation to a wild-type antibody that hardly shows ADCC activity, the ADCC activity was increased even though the same concentration of antibody was added dropwise. It was 10 times to 15 times.

  Further, from Table 11, two points having the same rate of cytotoxicity between the wild type human antibody and the type 286 human antibody were selected, and comparison of ADCC activity was attempted. For example, wild-type human antibodies show about 30% cytotoxicity at 10 μg / mL, and type 286 and 298 human antibodies show about 31% cytotoxicity at 0.1 μg / mL. When compared using these two points, the antibody concentration required to obtain the same ADCC activity was reduced to about 1/100. As a result, ADCC activity increased about 100-fold (the concentration of the human antibody relative to the wild-type human antibody between the two points where the wild-type human antibody and the 286-type or 298-type human antibody showed the same rate of cytotoxicity. It is possible to judge that the ratio of has increased 100 times.

Further, from Table 12, two points having the same rate of cytotoxicity between the wild type human antibody and the type 298 human antibody were selected, and comparison of ADCC activity was attempted. For example, the wild type human antibody is about 3 at 10 μg / mL.
%, Type 298 human antibody shows about 3% cytotoxicity at 0.01 μg / mL. When compared using these two points, the antibody concentration required to obtain the same ADCC activity was reduced to about 1/1000. As a result, ADCC activity increased about 1000 times (the ratio of the concentration of type 298 human antibody to wild type human antibody between the two points where the wild type human antibody and type 298 human antibody showed the same cytotoxicity ratio). 1 / 1,000 times).
Further, from Table 12, two points having the same rate of cytotoxicity between the wild type human antibody and the type 298 human antibody were selected, and comparison of ADCC activity was attempted. For example, the wild type human antibody is about 3 at 10 μg / mL.
%, Type 286 human antibody shows about 2% cytotoxicity at 0.01 μg / mL. When compared using these two points, the antibody concentration required to obtain the same ADCC activity was reduced to about 1/1000. From this, ADCC activity increased about 1000 times (the ratio of the concentration of type 286 human antibody to wild type human antibody between the two points where the wild type human antibody and type 286 human antibody show the same cytotoxicity ratio) 1 / 1,000 times).
Those skilled in the art can calculate the increase in ADCC activity from the figures and tables following the above method.

Example 5
In the same manner as in Example 1, anti-EGFR (048-006) antibodies of wild type, 286 Cys type, 295 Cys type or 298 Cys type were prepared, and these various mutant anti-EGFR (048-006) human antibodies were prepared. In vivo antitumor effects were evaluated.
Below, the preparation method of the anti-EGFR (048-085) human antibody wild type, 286 Cys type, 295 Cys type or 298 Cys type, its antitumor effect measurement and reactivity to the EGFR molecule are described.

1) Preparation of Anti-EGFR (048-006) Antibody A human antibody is obtained as a purified human antibody through the following steps A) to E).
A) Mutation of Fc region gene,
B) Construction of human antibody expression vector combining Fc region and mutated Fc region gene,
C) Gene transfer of human antibody expression vector into CHO cells and screening for CHO cells that highly express human antibodies,
D) culture of CHO cells highly expressing human antibodies,
E) Column purification from culture supernatant of cells expressing high human antibody.
The steps A) to E) are described in order below.
Among these steps, A), B), D) and E) are the same as those in Example 1. Accordingly, in this example, step C) will be described.

Four genes are required to produce human antibodies. These four genes are a human antibody L chain variable region gene, a human antibody H chain variable region gene, a human IgG1 L chain constant region gene, and a human IgG1 H chain constant region gene. The L chain variable region and constant region genes, and the H chain variable region and constant region genes of Anti-EGFR (048-006), which is an antibody clone that recognizes EGFR, were provided by Antibody Research Institute Co., Ltd. The DNA sequence of the provided human L chain variable region of Anti-EGFR (048-006) is shown in FIG. 9 (SEQ ID NO: 160), and the DNA sequence of the human H chain variable region is shown in FIG. 10 (SEQ ID NO: 162). .
In addition, the nucleotide sequences of CDR1, CDR2, and CDR3 of the human L chain variable region of Anti-EGFR (048-006) are shown in SEQ ID NOs: 75-77. In addition, the amino acid sequences of CDR1, CDR2, and CDR3 of the human L chain variable region of Anti-EGFR (048-006) are shown in SEQ ID NOs: 78 to 80, respectively. The provided human IgG1 L chain constant region gene is a λ chain, the base sequence thereof is shown in SEQ ID NO: 81, and the amino acid sequence thereof is shown in SEQ ID NO: 82.
In addition, the nucleotide sequences of CDR1, CDR2, and CDR3 of the human H chain variable region of Anti-EGFR (048-006) are shown in SEQ ID NOs: 83 to 85. The amino acid sequences of CDR1, CDR2, and CDR3 of the human H chain variable region of Anti-EGFR (048-006) are shown in SEQ ID NOs: 86 to 88. The base sequence and amino acid sequence of the provided human IgG1 heavy chain constant region are the same as Anti-ALCAM (066-174).

C) Construction of human antibody expression vector combining the cloned gene and its mutated gene
Anti-EGFR (048-006) human antibody can be expressed by transfecting CHO cells with two types of expression vectors of L-chain and H-chain of Anti-EGFR (048-006) human antibody. Here, L-chain expression vector of Anti-EGFR (048-006) human antibody expresses anti-EGFR (048-006) L chain variable region gene + human IgG1 L chain constant region gene combined Anti-EGFR (048-006) H chain expression vector combined with Anti-EGFR (048-006) H chain variable region gene + human IgG1 H chain constant region gene Is incorporated into an expression vector. In preparing anti-EGFR (048-006) antibody of wild type, 286Cys type, 295Cys type or 298Cys type, the anti-EGFR (048-006) human antibody L chain expression vector is these four human antibodies The anti-EGFR (048-006) human antibody H chain expression vector is used for each of wild type, 286 Cys type, 295 Cys type or 298 Cys type human antibody. A special heavy chain expression vector is required. Here, the construction of an expression vector for an anti-EGFR (048-006) human antibody L chain that can be commonly used for the expression of each human antibody is described in detail, followed by 286 Cys type, 295 Cys type or 298 Cys type human antibody. Describes the construction of a dedicated anti-EGFR (048-006) human antibody heavy chain expression vector required for expression.

Construction of expression vector of Anti-EGFR (048-006) human antibody L chain As the expression vector , BCMG-neo vector is used, and Anti-EGFR (048-006) human L chain variable at the XhoI and NotI sites of this vector Insert a region gene + human IgG1 L chain constant region gene. Anti-EGFR (048-006) human L chain variable region and human IgG1 L chain constant region genes were subjected to the following PCR reaction using the Anti-EGFR (048-006) L chain gene provided by the antibody laboratory as a template. Fragment was obtained by doing.

Anti-EGFR (048-006) human L chain variable region and human IgG1 L chain constant region gene amplification reaction
Anti-EGFR (048-006) L chain (25 ng / μL) 2 μL
2.5 mM dNTPs 4 μL
EGFR-L1 primer (20μM) 2.5 μL
EGFR-L2 primer (20μM) 2.5 μL
DMSO 2.5 μL
× 10 pfu polymerase Buffer 5 μL
pfu polymerase 1 μL
Sterile water 30.5 μL
total 50 μL
94 ℃ 2 min
94 ℃ 1 min 55 ℃ 2min 72 ℃ 2min (30 cycles)
72 ℃ 4 min
4 ℃ unlimited time
See below for Primer DNA sequences.
EGFR-L1 primer: ACCGCTCGAGATGGACATGAGGGTCCCCGCTCAGCTC (SEQ ID NO: 25)
EGFR-L2 primer: ATAGTTTAGCGGCCGCTTACGAACATTCTGTAGGGGCCACTGTCTT (SEQ ID NO: 26)

  Anti-EGFR (048-006) human L chain variable region and human IgG1 L chain constant region gene fragments obtained by the above PCR reaction were cleaved with restriction enzyme XhoI (Takara) and restriction enzyme NotI (Takara). Purified. The fragment purified by cutting the BCMG-neo vector with XhoI and NotI was mixed with the purified Anti-EGFR (048-006) human L chain variable region and human IgG1 L chain constant region gene fragment and ligated. In the vector obtained by this ligation, it was confirmed by sequencing that the desired fragment was inserted. This anti-EGFR (048-006) human antibody L chain expression vector was named phuman EGFR (048-006) LC.

Construction of anti-EGFR (048-006) human antibody heavy chain expression vector BCMG-neo vector is used as the expression vector, and the anti-EGFR (048-006) human heavy chain variable region is located at the XhoI and NotI sites of this vector. Gene + H human IgG1 H chain constant region gene is inserted. Anti-EGFR (048-006) human heavy chain variable region gene was obtained by performing the following PCR reaction using the anti-EGFR (048-006) heavy chain gene provided by the antibody laboratory as a template. .
The dedicated H chain expression vector for each of the wild type, 286Cys type, 295Cys type or 295Cys type human antibody has a different plasmid as a template for PCR amplification of the human IgG1 H chain constant region gene, but the operation itself is The procedure was exactly the same, and various human IgG1 H chain gene fragments were obtained. Details including the PCR reaction are described below.

Anti-EGFR (048-006) human antibody heavy chain variable region amplification reaction
Anti-EGFR (048-006) (25 ng / μL) 2 μL
2.5 mM dNTPs 4 μL
EGFR-H1 primer (20μM) 2.5 μL
EGFR-H2 primer (20μM) 2.5 μL
DMSO 2.5 μL
× 10 pfu polymerase Buffer 5 μL
pfu polymerase 1 μL
Sterile water 30.5 μL
total 50 μL
94 ℃ 2 min
94 ℃ 1 min 55 ℃ 2min 72 ℃ 2min (30 cycles)
72 ℃ 4 min
4 ℃ unlimited time
EGFR-H1 primer: ACCGCTCGAGATGAAACACCTGTGGTTCTTCCTC (SEQ ID NO: 27)
EGFR-H2 primer: CGAGACGGTGACCATTGTCCCTTG (5'-phosphorylated) (SEQ ID NO: 89)

Amplification reaction of human IgG1 heavy chain constant region gene #Various template plasmids (20 ng / μL) 2 μL
2.5 mM dNTPs 4 μL
H3 primer (20μM) 2.5 μL
H4 primer (20μM) 2.5 μL
DMSO 2.5 μL
× 10 pfu polymerase Buffer 5 μL
pfu polymerase 1 μL
Sterile water 30.5 μL
total 50 μL
94 ℃ 2 min
94 ℃ 1 min 55 ℃ 2min 72 ℃ 2min (30 cycles)
72 ℃ 4 min
4 ℃ unlimited time
H3 primer: GCCTCCACCAAGGGCCCATCGGTC (5'-phosphorylated) (SEQ ID NO: 29)
H4 primer: ATAGTTTAGCGGCCGCTTATTTACCCGGAGACAGGGAGAGGCTCTT (SEQ ID NO: 30)

  In order to prepare a gene fragment for a wild type Anti-EGFR (048-006) human antibody, pCR2.1-HC (wild type) was used as a template. PCR2.1-HC (286Cys) was used as a template to prepare a gene fragment for 286Cys type Anti-EGFR (048-006) human antibody. PCR2.1-HC (295Cys) was used as a template to prepare a gene fragment for 295 Cys-type Anti-EGFR (048-006) human antibody. PCR2.1-HC (298Cys) was used as a template to prepare a gene fragment for a 298Cys-type Anti-EGFR (048-006) human antibody.

  The Anti-EGFR (048-006) H chain variable region gene fragment obtained by the PCR reaction was purified after being cleaved with the restriction enzyme XhoI (Takara). Moreover, human IgG1 H chain constant region gene fragments obtained by various PCR reactions with different templates were purified after being cleaved with a restriction enzyme NotI (Takara). The BCMG-neo vector was cleaved with XhoI and NotI and purified, and then the purified Anti-EGFR (048-006) heavy chain variable region gene fragment and purified human IgG1 heavy chain constant region gene fragment were mixed and ligated. . In the vector obtained by this ligation, it was confirmed by sequencing that the desired fragment was inserted. This anti-EGFR (048-006) human antibody H chain expression vector is p human EGFR (048-006) HC (wild type), p human EGFR (048-006) HC (286 Cys type), p human EGFR ( 048-006) HC (295Cys) or p human EGFR (048-006) HC (298Cys type), respectively.

Next, using the cancer cell transplanted mouse, it was confirmed whether or not the prepared antibody exhibited antitumor activity in vivo.
Animals and cell lines used
A 4-week-old female BALB / c nude mouse (Nippon Charles River Co., Ltd.) was acclimated for 1 week and used for the experiment. The breeding was performed in an SPF environment, and sterilized water and feed were given.
Human kidney cancer cell CCF-RC-1 was used after culture and passage in RPMI 1640 medium containing 10% FBS in the presence of 5% CO _{2 at} 37 ° C.
Method of anti-tumor experiment 1 × 10 ⁷ human renal cancer cells CCF-RC-1 cells were implanted subcutaneously on the dorsal side of nude mice, and antibody administration was started 5 days later. Tumor diameter and body weight are measured twice a week, and the estimated tumor volume is W = a × b2 / 2 (W: estimated tumor volume (mm ³ ), a: major axis (mm), b: minor axis (mm)) I asked more. The experimental groups were control group (PBS administration), 048-006 (286C) IgG administration group (0.1 mg and 0.5 mg / individual), 048-006 (295C) IgG administration group (0.1 mg and 0.5 mg / individual), 048 -006 IgG (298C) administration group (0.1 mg and 0.5 mg / individual) and 048-006 (wild) IgG administration group (0.1 mg / individual) were 4 or 5 groups, and the administration route was intraperitoneal administration. The antitumor effect was examined by administering 6 times twice a week.

As a result of the 0.5 mg / individual administration group, the volume of a tumor transplanted into a mouse usually requires angiogenesis in order to grow as a cell mass and exhibits exponential growth instead of linearity. Also in this embodiment, the PBS administration group, in 15day-19day, a significant increase is observed from 380.53 (mm ³⁾ 718.20 to (mm ³⁾ (1.88-fold). However, in the mice administered 298Cys antibody, tumor volume, in 15day-19day, decreased from 294.02 (mm ³⁾ 291.76 and (mm ³⁾ (0.99 fold), the inhibition of growth is remarkably observed. In the group administered with the 286Cys antibody, the tumor volume decreased by 65% from 75.3 (mm ³ ) to 34.76 (mm ³ ) by the first administration (5day-8day). This indicates the usefulness of the 286Cys type antibody.
Next, comparing the volumes on the 19th day after administration, the volume was 718.20 (mm ³ ), 400.69 (mm ³ ) in the PBS group, 286 Cys type antibody administration group, 298 Cys type antibody administration group, and 295 Cys type antibody administration group, respectively. They are 291.76 (mm ³ ) and 532.67 (mm ³ ). When the volume ratio of the 286 Cys type antibody administration group, the 298 Cys type antibody administration group, and the 295 Cys type antibody administration group is compared with the PBS group, it is suppressed to 56%, 41%, and 74%. It is remarkable.

As a result of the 0.1 mg / individual administration group , the volume on the 19th day after administration was 807.8 (mm ³ ), 577.1 (mm ³ ), 477.0 (mm ³ ), 721.3 (mm ³ ), 718.2 (mm ³ ) Yes. When the 286 Cys type antibody administration group, 298 Cys type antibody administration group, 295 Cys type antibody administration group, and wild type antibody administration group were compared with the PBS group in volume ratio, they were suppressed to 80%, 66%, 112%, and 100%. Significant effects were obtained in the 286 Cys antibody administration group and the 298 Cys antibody administration group.

Example 6
<Flow site analysis of purified human antibodies>
In order to confirm that the reactivity of various human antibodies obtained by column purification to the ALCAM molecule was the same, flow site analysis was performed. H1373 cells are known to have many ALCAM molecules on the surface of their cell membranes. This flow site analysis verified whether various anti-ALCAM human antibodies could recognize ALCAM molecules on H1373 cells. The measurement method is described in detail below.
1) Centrifugation (1200 rpm, 5 min) of 10 mL of H1373 cells (2 × 10 ⁷ cells) cultured in RPMI-1640 (Sigma, R8758) medium supplemented with 10% Fetal Bovine Serum The supernatant was removed and the H1373 cell pellet was removed.
2) 10 mL of Wash Buffer (PBS + 10% FCS) was added to the H1373 cell pellet, suspended, and centrifuged again (1200 rpm, 5 min), and then the supernatant was removed.
3) After suspending the H1373 cell pellet with 2 mL of Wash Buffer, add 0.3 mL of the cell suspension to each tube, add 0.5 mL of Wash Buffer to each tube, suspend, and then centrifuge (2000 rpm, 5 min) and the supernatant was discarded.
4) 5 μL of various purified Anti-ALCAM human antibodies were added to the H1373 cell pellet and reacted at room temperature for 30 minutes.
(I) After 1 mL of Wash Buffer was added and suspended, the supernatant was discarded by centrifugation (2000 rpm, 5 min).
(Ii) 30 μL of Anti-Human IgG (γ-chain) FITC conjugated (MBL, code 214) diluted 50-fold with Wash Buffer was added to H1373 cells and allowed to react at room temperature for 15 minutes.
(Iii) After adding 1 mL of Wash Buffer and suspending, the supernatant was discarded by centrifugation (2000 rpm, 5 min).
(Iv) The H1373 cell pellet was suspended by adding 0.5 mL of Wash Buffer, and flow cytometric analysis was performed with FACS Calibur (Becton Dickinson).
The above flow site analysis results are shown in FIG. From this result, it can be seen that the reactivity of the wild type human antibody and the two types of Cys type human antibodies to ALCAM is the same.
Similarly to the above, it was confirmed that the anti-ICAM1 antibody and the anti-EGFR antibody did not change in the reactivity with the antigen.

The present invention provides an antibody with enhanced ADCC activity and a method for producing the same. Regarding the ADCC activity evaluated by the lactate dehydrogenase release assay system, the antibody of the present invention showed an increase in ADCC activity compared to the wild type antibody (the wild type between the two points where the wild type antibody and the mutant antibody had the same concentration). The ratio of the ratio of cytotoxicity of the mutant antibody to the antibody increased). Therefore, by using the mutant antibody of the present invention, more cell damage can be caused as compared with the wild type antibody.
The antibody of the present invention also showed increased ADCC activity, particularly at a low concentration (ratio of the concentration of the mutant antibody to the wild type antibody between two points where the wild type antibody and the mutant antibody showed the same rate of cytotoxicity). Rose). Therefore, by using the antibody of the present invention as a therapeutic antibody, it is possible to increase the therapeutic effect on cancer cells per unit mass. Moreover, since the dosage per patient can be reduced by using the antibody of the present invention, it is possible to reduce the dosage cost.

It is a figure which shows the arrangement | sequence of the L chain variable region gene of Anti-ALCAM (066-174) antibody. It is a figure which shows the arrangement | sequence of the heavy chain variable region gene of Anti-ALCAM (066-174) antibody. It is a figure which shows the arrangement | sequence of the L chain variable region gene of Anti-ALCAM (035-234) antibody. It is a figure which shows the arrangement | sequence of the heavy chain variable region gene of Anti-ALCAM (035-234) antibody. It is a figure which shows the arrangement | sequence of the L chain variable region gene of Anti-ICAM1 (053-059) antibody. It is a figure which shows the arrangement | sequence of the heavy chain variable region gene of Anti-ICAM1 (053-059) antibody. It is a figure which shows the arrangement | sequence of the L chain variable region gene of Anti-ICAM1 (053-085) antibody. It is a figure which shows the arrangement | sequence of the heavy chain variable region gene of Anti-ICAM1 (053-085) antibody. It is a figure which shows the arrangement | sequence of the L chain variable region gene of Anti-EGFR (048-006) antibody. It is a figure which shows the arrangement | sequence of the H chain variable region gene of Anti-EGFR (048-006) antibody. It is a figure which shows the ADCC activity result of an anti-ALCAM (066-174) human antibody of a wild type, 295Cys, and 298Cys. It is a figure which shows the ADCC activity result of an anti-ALCAM (035-234) human antibody of a wild type, 286Cys, 295Cys, and 298Cys. It is a figure which shows the ADCC activity result of the anti-ICAM1 (053-059) human antibody of a wild type, 286Cys, 295Cys, and 298Cys. It is a figure which shows the ADCC activity result of an anti-ICAM1 (053-085) human antibody of a wild type, 286Cys, 295Cys, and 298Cys. It is a figure which shows the ADCC activity result of an anti-ICAM1 (053-085) human antibody of a wild type, 286Cys, and 298Cys. It is a figure which shows the anti-tumor effect in vivo of the Anti-EGFR (048-006) human antibody of 286Cys, 295Cys, and 298Cys. (A) is the result of the 0.5 mg / individual administration group, and (B) is the result of the 0.1 mg / individual administration group. It is a figure which shows the result of a flow site. (A) ALCAM 066-174, (B) ALCAM 035-234, (C) ICAM1 053-059), (D) ICAM1 053-085.

Claims (31)

An antibody in which the amino acid residue at position 286 or 298 is substituted with cysteine in the heavy chain constant region. The antibody according to claim 1, wherein the heavy chain constant region is any constant region selected from the group consisting of human Cγ1, Cγ2, Cγ3, and Cγ4. The antibody according to claim 1, wherein ADCC activity is increased as compared with that before substitution. 4. An antibody according to any one of claims 1 to 3 having improved stability, solubility or binding affinity to Fc receptors. The antibody according to any one of claims 1 to 4, which is provided with a sugar chain. The antibody according to claim 5, which improves effector function. The antibody according to any one of claims 1 to 6, which binds to FcγRI, FcγRII, FcγRIII, or FcRn. The antibody according to any one of claims 1 to 7, which has specificity for a target antigen selected from the group consisting of ALCAM, ICAM1, and EGFR. The antibody according to any one of (1) to (4) below;
(1) The amino acid sequence described in SEQ ID NO: 12 as CDR1, the amino acid sequence described in SEQ ID NO: 13 as CDR2, the amino acid sequence described in SEQ ID NO: 14 as CDR3, and the amino acid described in SEQ ID NO: 91 as CH An antibody comprising an H chain having a sequence;
(2) an antibody comprising an H chain having the amino acid sequence set forth in SEQ ID NO: 95,
(3) The amino acid sequence described in SEQ ID NO: 12 as CDR1, the amino acid sequence described in SEQ ID NO: 13 as CDR2, the amino acid sequence described in SEQ ID NO: 14 as CDR3, and the amino acid sequence described in SEQ ID NO: 97 as Fc region An antibody comprising an H chain having an amino acid sequence,
(4) An antibody having a pair of the H chain according to any one of (1), (2) or (3) above and the L chain according to (i) or (ii) below,
(I) The amino acid sequence described in SEQ ID NO: 4 as CDR1, the amino acid sequence described in SEQ ID NO: 5 as CDR2, the amino acid sequence described in SEQ ID NO: 6 as CDR3, and the amino acid described in SEQ ID NO: 8 as CL A light chain having a sequence;
(Ii) An L chain having the amino acid sequence set forth in SEQ ID NO: 99. The antibody according to any one of (1) to (4) below;
(1) The amino acid sequence described in SEQ ID NO: 12 as CDR1, the amino acid sequence described in SEQ ID NO: 13 as CDR2, the amino acid sequence described in SEQ ID NO: 14 as CDR3, and the amino acid described in SEQ ID NO: 93 as CH3 An antibody comprising an H chain having a sequence;
(2) an antibody comprising an H chain having the amino acid sequence of SEQ ID NO: 101,
(3) The amino acid sequence described in SEQ ID NO: 12 as CDR1, the amino acid sequence described in SEQ ID NO: 13 as CDR2, the amino acid sequence described in SEQ ID NO: 14 as CDR3, and the amino acid sequence described in SEQ ID NO: 103 as Fc region An antibody comprising an H chain having an amino acid sequence,
(4) An antibody having a pair of the H chain according to any one of (1), (2) or (3) above and the L chain according to (i) or (ii) below,
(I) The amino acid sequence described in SEQ ID NO: 4 as CDR1, the amino acid sequence described in SEQ ID NO: 5 as CDR2, the amino acid sequence described in SEQ ID NO: 6 as CDR3, and the amino acid described in SEQ ID NO: 8 as CL A light chain having a sequence;
(Ii) An L chain having the amino acid sequence set forth in SEQ ID NO: 99. The antibody according to any one of (1) to (4) below;
(1) The amino acid sequence described in SEQ ID NO: 42 as CDR1, the amino acid sequence described in SEQ ID NO: 43 as CDR2, the amino acid sequence described in SEQ ID NO: 44 as CDR3, and the amino acid described in SEQ ID NO: 91 as CH An antibody comprising an H chain having a sequence;
(2) an antibody comprising an H chain having the amino acid sequence of SEQ ID NO: 105,
(3) The amino acid sequence described in SEQ ID NO: 42 as CDR1, the amino acid sequence described in SEQ ID NO: 43 as CDR2, the amino acid sequence described in SEQ ID NO: 44 as CDR3, and the amino acid sequence described in SEQ ID NO: 107 as Fc region An antibody comprising an H chain having an amino acid sequence,
(4) An antibody having a pair of the H chain according to any one of (1), (2) or (3) above and the L chain according to (i) or (ii) below,
(I) The amino acid sequence described in SEQ ID NO: 34 as CDR1, the amino acid sequence described in SEQ ID NO: 35 as CDR2, the amino acid sequence described in SEQ ID NO: 36 as CDR3, and the amino acid described in SEQ ID NO: 38 as CL A light chain having a sequence;
(Ii) An L chain having the amino acid sequence set forth in SEQ ID NO: 109. The antibody according to any one of (1) to (4) below;
(1) The amino acid sequence described in SEQ ID NO: 42 as CDR1, the amino acid sequence described in SEQ ID NO: 43 as CDR2, the amino acid sequence described in SEQ ID NO: 44 as CDR3, and the amino acid described in SEQ ID NO: 93 as CH3 An antibody comprising an H chain having a sequence;
(2) an antibody comprising an H chain having the amino acid sequence set forth in SEQ ID NO: 111,
(3) The amino acid sequence described in SEQ ID NO: 42 as CDR1, the amino acid sequence described in SEQ ID NO: 43 as CDR2, the amino acid sequence described in SEQ ID NO: 44 as CDR3, and the amino acid sequence described in SEQ ID NO: 113 as Fc region An antibody comprising an H chain having an amino acid sequence,
(4) An antibody having a pair of the H chain according to any one of (1), (2) or (3) above and the L chain according to (i) or (ii) below,
(I) The amino acid sequence described in SEQ ID NO: 34 as CDR1, the amino acid sequence described in SEQ ID NO: 35 as CDR2, the amino acid sequence described in SEQ ID NO: 36 as CDR3, and the amino acid described in SEQ ID NO: 38 as CL A light chain having a sequence;
(Ii) An L chain having the amino acid sequence set forth in SEQ ID NO: 109. The antibody according to any one of (1) to (4) below;
(1) The amino acid sequence described in SEQ ID NO: 57 as CDR1, the amino acid sequence described in SEQ ID NO: 58 as CDR2, the amino acid sequence described in SEQ ID NO: 59 as CDR3, and the amino acid described in SEQ ID NO: 91 as CH An antibody comprising an H chain having a sequence;
(2) an antibody comprising an H chain having the amino acid sequence set forth in SEQ ID NO: 115,
(3) The amino acid sequence described in SEQ ID NO: 57 as CDR1, the amino acid sequence described in SEQ ID NO: 58 as CDR2, the amino acid sequence described in SEQ ID NO: 59 as CDR3, and the amino acid sequence described in SEQ ID NO: 117 as Fc region An antibody comprising an H chain having an amino acid sequence,
(4) An antibody having a pair of the H chain according to any one of (1), (2) or (3) above and the L chain according to (i) or (ii) below,
(I) the amino acid sequence described in SEQ ID NO: 49 as CDR1, the amino acid sequence described in SEQ ID NO: 50 as CDR2, the amino acid sequence described in SEQ ID NO: 51 as CDR3, and the amino acid described in SEQ ID NO: 53 as CL A light chain having a sequence;
(Ii) An L chain having the amino acid sequence set forth in SEQ ID NO: 119. The antibody according to any one of (1) to (4) below;
(1) The amino acid sequence described in SEQ ID NO: 57 as CDR1, the amino acid sequence described in SEQ ID NO: 58 as CDR2, the amino acid sequence described in SEQ ID NO: 59 as CDR3, and the amino acid described in SEQ ID NO: 93 as CH An antibody comprising an H chain having a sequence;
(2) an antibody comprising an H chain having the amino acid sequence of SEQ ID NO: 121,
(3) The amino acid sequence described in SEQ ID NO: 57 as CDR1, the amino acid sequence described in SEQ ID NO: 58 as CDR2, the amino acid sequence described in SEQ ID NO: 59 as CDR3, and the amino acid sequence described in SEQ ID NO: 123 as Fc region An antibody comprising an H chain having an amino acid sequence,
(4) An antibody having a pair of the H chain according to any one of (1), (2) or (3) above and the L chain according to (i) or (ii) below,
(I) the amino acid sequence described in SEQ ID NO: 49 as CDR1, the amino acid sequence described in SEQ ID NO: 50 as CDR2, the amino acid sequence described in SEQ ID NO: 51 as CDR3, and the amino acid described in SEQ ID NO: 53 as CL A light chain having a sequence;
(Ii) An L chain having the amino acid sequence set forth in SEQ ID NO: 119. The antibody according to any one of (1) to (4) below;
(1) The amino acid sequence described in SEQ ID NO: 71 as CDR1, the amino acid sequence described in SEQ ID NO: 72 as CDR2, the amino acid sequence described in SEQ ID NO: 73 as CDR3, and the amino acid described in SEQ ID NO: 91 as CH An antibody comprising an H chain having a sequence;
(2) an antibody comprising an H chain having the amino acid sequence set forth in SEQ ID NO: 125,
(3) The amino acid sequence described in SEQ ID NO: 71 as CDR1, the amino acid sequence described in SEQ ID NO: 72 as CDR2, the amino acid sequence described in SEQ ID NO: 73 as CDR3, and the amino acid sequence described in SEQ ID NO: 127 as Fc region An antibody comprising an H chain having an amino acid sequence,
(4) An antibody having a pair of the H chain according to any one of (1), (2) or (3) above and the L chain according to (i) or (ii) below,
(I) The amino acid sequence described in SEQ ID NO: 63 as CDR1, the amino acid sequence described in SEQ ID NO: 64 as CDR2, the amino acid sequence described in SEQ ID NO: 65 as CDR3, and the amino acid described in SEQ ID NO: 67 as CL A light chain having a sequence;
(Ii) An L chain having the amino acid sequence set forth in SEQ ID NO: 129. The antibody according to any one of (1) to (4) below;
(1) The amino acid sequence described in SEQ ID NO: 71 as CDR1, the amino acid sequence described in SEQ ID NO: 72 as CDR2, the amino acid sequence described in SEQ ID NO: 73 as CDR3, and the amino acid described in SEQ ID NO: 93 as CH3 An antibody comprising an H chain having a sequence;
(2) an antibody comprising an H chain having the amino acid sequence set forth in SEQ ID NO: 131,
(3) The amino acid sequence described in SEQ ID NO: 71 as CDR1, the amino acid sequence described in SEQ ID NO: 72 as CDR2, the amino acid sequence described in SEQ ID NO: 73 as CDR3, and the amino acid sequence described in SEQ ID NO: 133 as an Fc region An antibody comprising an H chain having an amino acid sequence,
(4) An antibody having a pair of the H chain according to any one of (1), (2) or (3) above and the L chain according to (i) or (ii) below,
(I) The amino acid sequence described in SEQ ID NO: 63 as CDR1, the amino acid sequence described in SEQ ID NO: 64 as CDR2, the amino acid sequence described in SEQ ID NO: 65 as CDR3, and the amino acid described in SEQ ID NO: 67 as CL A light chain having a sequence;
(Ii) An L chain having the amino acid sequence set forth in SEQ ID NO: 129. The antibody according to any one of (1) to (4) below;
(1) The amino acid sequence described in SEQ ID NO: 86 as CDR1, the amino acid sequence described in SEQ ID NO: 87 as CDR2, the amino acid sequence described in SEQ ID NO: 88 as CDR3, and the amino acid described in SEQ ID NO: 91 as CH An antibody comprising an H chain having a sequence;
(2) an antibody comprising an H chain having the amino acid sequence set forth in SEQ ID NO: 135,
(3) The amino acid sequence described in SEQ ID NO: 86 as CDR1, the amino acid sequence described in SEQ ID NO: 87 as CDR2, the amino acid sequence described in SEQ ID NO: 88 as CDR3, and the amino acid sequence described in SEQ ID NO: 137 as an Fc region An antibody comprising an H chain having an amino acid sequence,
(4) An antibody having a pair of the H chain according to any one of (1), (2) or (3) above and the L chain according to (i) or (ii) below,
(I) The amino acid sequence set forth in SEQ ID NO: 78 as CDR1, the amino acid sequence set forth in SEQ ID NO: 79 as CDR2, the amino acid sequence set forth in SEQ ID NO: 80 as CDR3, and the amino acid set forth in SEQ ID NO: 82 as CL A light chain having a sequence;
(Ii) An L chain having the amino acid sequence set forth in SEQ ID NO: 139. The antibody according to any one of (1) to (4) below;
(1) The amino acid sequence described in SEQ ID NO: 86 as CDR1, the amino acid sequence described in SEQ ID NO: 87 as CDR2, the amino acid sequence described in SEQ ID NO: 88 as CDR3, and the amino acid described in SEQ ID NO: 93 as CH3 An antibody comprising an H chain having a sequence;
(2) an antibody comprising an H chain having the amino acid sequence set forth in SEQ ID NO: 141,
(3) The amino acid sequence described in SEQ ID NO: 86 as CDR1, the amino acid sequence described in SEQ ID NO: 87 as CDR2, the amino acid sequence described in SEQ ID NO: 88 as CDR3, and the amino acid sequence described in SEQ ID NO: 143 as an Fc region An antibody comprising an H chain having an amino acid sequence,
(4) An antibody having a pair of the H chain according to any one of (1), (2) or (3) above and the L chain according to (i) or (ii) below,
(I) The amino acid sequence set forth in SEQ ID NO: 78 as CDR1, the amino acid sequence set forth in SEQ ID NO: 79 as CDR2, the amino acid sequence set forth in SEQ ID NO: 80 as CDR3, and the amino acid set forth in SEQ ID NO: 82 as CL A light chain having a sequence;
(Ii) An L chain having the amino acid sequence set forth in SEQ ID NO: 139. A method for producing an antibody with enhanced ADCC activity or antitumor activity, comprising a step of substituting an amino acid residue at position 286 or 298 with cysteine in a heavy chain constant region. A method for producing an antibody with enhanced ADCC activity or antitumor activity, comprising the following steps (a) and (b):
(A) a step of expressing DNA encoding an H chain in which the amino acid residue at position 286 or 298 is substituted with cysteine in the H chain constant region, and DNA encoding an L chain;
(B) A step of recovering the expression product of the step (a). 21. The method according to claim 19 or 20, wherein the heavy chain constant region is any constant region selected from the group consisting of human Cγ1, Cγ2, Cγ3, and Cγ4. A pharmaceutical composition comprising the antibody according to any one of claims 1 to 18 and a pharmaceutically acceptable carrier. A method for treating a non-human mammal, comprising a step of administering the antibody of any one of claims 1 to 18 to a subject. A nucleic acid encoding the antibody of any one of claims 1 to 18. A vector comprising the nucleic acid according to claim 24. A host cell comprising the vector of claim 25. A host organism comprising the vector of claim 25. A method for enhancing ADCC activity or antitumor activity of an antibody, comprising a step of substituting an amino acid residue at position 286 or 298 with cysteine in a heavy chain constant region. 27. The method of claim 26, wherein the heavy chain constant region is any constant region selected from the group consisting of human Cγ1, Cγ2, Cγ3, and Cγ4. A method for determining whether or not the ADCC activity or antitumor activity of the antibody in step (a) is enhanced, wherein the ADCC activity or antitumor activity measured in step (b) is the ADCC activity of the antibody before substitution. Or a method for determining that the ADCC activity or antitumor activity of the antibody of step (a) is enhanced when it is higher than the antitumor activity;
(A) providing a variant of an antibody having ADCC activity or antitumor activity, wherein the amino acid residue at position 286 or 298 in the heavy chain constant region is substituted with cysteine; and
(B) A step of measuring ADCC activity or antitumor activity of the antibody of step (a). A method for screening an antibody having enhanced ADCC activity or antitumor activity, comprising the following steps (a) to (d):
(A) providing an antibody having ADCC activity or antitumor activity;
(B) replacing the amino acid residue at position 286 or 298 in the heavy chain constant region of the antibody of step (a) with cysteine,
(C) determining whether the ADCC activity or antitumor activity of the antibody obtained in step (b) has been enhanced by the method according to claim 30,
(D) A step of selecting the antibody determined to have enhanced ADCC activity or antitumor activity in step (c).

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