WO2012074097A1

WO2012074097A1 - Anti-cd33 antibody

Info

Publication number: WO2012074097A1
Application number: PCT/JP2011/077924
Authority: WO
Inventors: 丹羽　倫平; 知明中川; 拓也村上; 麗夫久保田
Original assignee: 協和発酵キリン株式会社
Priority date: 2010-12-03
Filing date: 2011-12-02
Publication date: 2012-06-07

Abstract

Provided are: a monoclonal antibody or a fragment thereof, said monoclonal antibody being capable of binding to the extracellular region of a human CD33 cell at a high affinity and exerting a high ADCC activity; a DNA encoding said antibody or fragment; a vector containing said DNA; a transformant obtained by transferring said vector; a method for producing the aforesaid antibody or fragment using said transformant; and a therapeutic agent and a diagnostic agent using the aforesaid antibody or fragment.

Description

Anti-CD33 antibody

The present invention relates to a monoclonal antibody or fragment thereof that binds to the human CD33 extracellular region with high affinity and exhibits high antibody-dependent cellular cytotoxicity (hereinafter sometimes referred to as ADCC activity). Hybridoma producing the antibody, DNA encoding the antibody, a vector containing the DNA, a transformant obtained by introducing the vector, a method for producing an antibody or fragment using the hybridoma or the transformant, antibody Alternatively, the present invention relates to a therapeutic agent and a diagnostic agent using the fragment.

Cluster of differentiation (hereinafter sometimes referred to as CD) 33 is a glycoprotein having a molecular weight of about 67,000 expressed in myeloid progenitor cells, monocytes and granulocytes. CD33 is a type I transmembrane protein, and there are two immunoglobulin superfamily domains (IgV and IgC2 in order from the N-terminal toward the cell membrane) outside the cell. Three cysteine residues exist in each domain. Form intradomain and interdomain disulfide bonds. There are a total of five N-linked glycosylation sequences in the CD33 extracellular region. CD33 is known to bind to sialic acid via the IgV region, and is also called as Siglec-3 or sialoadhesin, but the details of its function are unknown (Non-patent Document 1).

CD33, also known as gp67, sialoadhesin, siglec-3, was cloned in 1988 (Non-Patent Document 2), and the DNA sequence, amino acid sequence and three-dimensional structure of CD33 have been published on public databases. For example, it can be referred to from an accession number such as P20138 (SWISSPROT) or M23197 (EMBL).

L4F3 and L1B2 established for the first time as monoclonal antibodies that bind to CD33 (hereinafter sometimes referred to as mAb) were reported in 1983 (Non-patent Document 3). Since then, many monoclonal antibodies against CD33 (hereinafter sometimes referred to as anti-CD33 mAb) have been reported. Many of the reported anti-CD33 mAbs are known to recognize the same or adjacent regions (Non-patent Document 1).

CD33 is highly expressed in most acute myelogenous leukemia (acute myelogenous leukemia, hereinafter sometimes referred to as AML) cells compared to normal cells, whereas it is not expressed in hematopoietic stem cells. CD33 mAb is being clinically applied with the aim of treating AML. For example, gemtuzumab ozogamicin, a conjugate of P67.6, an anti-CD33 mAb, and a calicheamicin derivative has been used clinically as an AML therapeutic.

Examples of anti-CD33 mAb developed as a cancer therapeutic agent include gemtumumab ozogamicin (P67.6) (Non-patent Document 4), lintuzumab (HuM195) (Non-patent Document 5), and the like. These antibodies are antibodies that exert their medicinal effects by specifically damaging CD33-expressing cells that are target cells. Gemtumumab ozogamicin has the property of being bound into the cell after binding to the CD33 antigen on the cell surface. Utilizing this property, calicheamicin derivatives are sent into the cell and have cytotoxic activity in the cell. It is considered that ADCC activity is the main medicinal mechanism of lintuzumab (Non-patent Document 6).

The ADCC activity is an activity that activates immune cells (such as natural killer cells) by damaging the target cells by binding an antibody bound to an antigen on the target cells to the Fc receptor of immune cells through its Fc region. On the other hand, the complement-dependent injury activity of these antibodies (hereinafter sometimes referred to as CDC activity) is expressed only when the antigen expression level is high (Non-patent Document 6).

CDC activity is an activity in which an antibody bound to an antigen on a target cell activates a series of cascades (complement activation pathways) composed of complement-related proteins in the blood and damages the target cell. In addition, migration and activation of immune cells can be induced by protein fragments generated by complement activation.

Usually, a human antibody of IgG type has a consensus sequence in which one N-linked sugar chain binds to its constant region. However, an antibody having a consensus sequence to which an N-linked sugar chain binds also in the variable region changes the sugar chain binding, making it difficult to stably supply a uniform antibody as a pharmaceutical product. Furthermore, in some cases, it is essential for the binding between proteins. For example, LFA-3 (lymphocyte function-associated antigen 3) has been reported to require an N-linked sugar chain for binding to CD2, and antibody There is a possibility that the binding property of the antibody to the antigen is changed by binding of the sugar chain to the variable region which is the binding site of (Non-patent Document 7).

In the binding between an antibody and an Fc receptor, the importance of a sugar chain bound to the second domain of the hinge region and constant region of the antibody has been suggested (Non-patent Document 8). It is known that galactose is added to the non-reducing end of the N-glycoside-linked complex sugar chain bound to the Fc region of the antibody molecule and fucose is added to N-acetylglucosamine at the reducing end. In particular, an antibody in which fucose is not bound to N-acetylglucosamine at the reducing end of a sugar chain has been reported to exhibit high ADCC activity (Non-patent Document 10 and Patent Document 9). 1).

Many of the antibodies generally used as pharmaceuticals are produced using gene recombination techniques, and are produced using animal cells such as CHO cells derived from Chinese hamster ovary tissue as host cells. The sugar chain structure varies depending on the host cell.

By reducing or eliminating the activity of α1,6-fucosyltransferase, GDP-mannose 4,6-dehydratase or GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase in antibody-producing cells It is possible to increase the proportion of sugar chains in which fucose is not bound to N-acetylglucosamine at the reducing end of the N-glycoside-linked complex-type sugar chain that binds to the Fc region of the antibody molecule (Patent Document 2).

It is known that CDC activity varies depending on the antibody subclass. CDC activity is high in human IgG1 and IgG3, and its strength is generally in the order of IgG3 ≧ IgG1 >> IgG2≈IgG4. Furthermore, it is known that the CDC activity can be further increased as compared with IgG3 by exchanging the CH2 domain of the constant region of human IgG1 with the CH2 domain of IgG3 (Patent Document 3).

International Publication No. 00/61739 International Publication No. 02/31140 US Patent Application Publication No. 2007/0148165

An object of the present invention is to provide a monoclonal antibody or fragment thereof that binds to the human CD33 extracellular region with high affinity and exhibits high ADCC activity, DNA encoding the antibody or the fragment, a vector containing the DNA, and a vector comprising the vector It is to provide a transformant obtained by introduction, a method for producing the antibody or the fragment using the transformant, and a therapeutic agent and a diagnostic agent containing the antibody or the fragment.

The present inventors obtained mouse and rat monoclonal antibodies that bind to the human CD33 extracellular region with high affinity, and the complementarity determining region (hereinafter sometimes referred to as VH) of the antibody heavy chain variable region (hereinafter sometimes referred to as VH). Complementarity Determining Region (hereinafter, sometimes referred to as CDR) 1-3 and anti-CD33 humanized antibody having CDR 1-3 of light chain variable region (hereinafter sometimes referred to as VL), and one of the CDRs An anti-CD33 humanized antibody in which amino acid residues at certain parts are appropriately substituted was prepared, and the present invention was completed.

That is, the present invention is as follows.
1. (I) a heavy chain variable region comprising CDR1 comprising the amino acid sequence represented by SEQ ID NO: 52, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 99, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 54, and SEQ ID NO: 55 A monoclonal antibody that binds to human CD33, comprising a light chain variable region comprising CDR1 comprising the amino acid sequence represented by SEQ ID NO: 56, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 56, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 57, or A fragment of the antibody,
(Ii) a heavy chain variable region comprising CDR1 comprising the amino acid sequence represented by SEQ ID NO: 52, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 100, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 54, and SEQ ID NO: 55 A monoclonal antibody that binds to human CD33, comprising a light chain variable region comprising CDR1 comprising the amino acid sequence represented by SEQ ID NO: 56, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 56, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 57, or A fragment of the antibody,
(Iii) a heavy chain variable region comprising CDR1 comprising the amino acid sequence represented by SEQ ID NO: 52, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 101, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 54, and SEQ ID NO: 55 A monoclonal antibody that binds to human CD33, comprising a light chain variable region comprising CDR1 comprising the amino acid sequence represented by SEQ ID NO: 56, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 56, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 57, or A heavy chain variable comprising a fragment of said antibody, or (iv) CDR1 comprising the amino acid sequence represented by SEQ ID NO: 52, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 102, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 54 Region and the amino acid sequence shown in SEQ ID NO: 55. CDR1, CDR2 comprising the amino acid sequence shown in SEQ ID NO: 56 and a light chain variable region comprising a CDR3 comprising an amino acid sequence shown in SEQ ID NO: 57, a fragment of a monoclonal antibody or the antibody that binds human CD33.
2. A heavy chain variable region comprising CDR1, comprising the amino acid sequence represented by SEQ ID NO: 52, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 96, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 54, and represented by SEQ ID NO: 55 A monoclonal antibody that binds to human CD33, comprising a CDR1 comprising an amino acid sequence, a CDR2 comprising an amino acid sequence represented by SEQ ID NO: 56, and a light chain variable region having a CDR3 comprising an amino acid sequence represented by SEQ ID NO: 57 fragment.
3. The monoclonal antibody or the fragment of the antibody according to item 2, comprising a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 86 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 85.
4). The monoclonal antibody or fragment of the antibody according to item 2, comprising a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 87 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 85.
5. (I) a heavy chain variable region comprising CDR1 comprising the amino acid sequence represented by SEQ ID NO: 34, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 35, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 36, and SEQ ID NO: 37 A monoclonal antibody that binds to human CD33, comprising a light chain variable region comprising CDR1, comprising the amino acid sequence represented by SEQ ID NO: 38, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 38, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 39; A fragment of the antibody,
(Ii) a heavy chain variable region comprising CDR1 comprising the amino acid sequence represented by SEQ ID NO: 40, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 41, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 42, and SEQ ID NO: 43 A monoclonal antibody that binds to human CD33, comprising a light chain variable region comprising CDR1, comprising the amino acid sequence represented by SEQ ID NO: 44, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 44, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 45, or A fragment of the antibody,
(Iii) a heavy chain variable region comprising CDR1 comprising the amino acid sequence represented by SEQ ID NO: 46, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 47, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 48, and SEQ ID NO: 49 A monoclonal antibody that binds to human CD33, comprising a CDR1 comprising the amino acid sequence shown, a CDR2 comprising the amino acid sequence represented by SEQ ID NO: 50, and a light chain variable region comprising CDR3 comprising the amino acid sequence represented by SEQ ID NO: 51, or Antibody fragments,
(Iv) a heavy chain variable region comprising CDR1 comprising the amino acid sequence represented by SEQ ID NO: 52, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 53, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 54, and SEQ ID NO: 55 A monoclonal antibody that binds to human CD33 comprising a CDR1 comprising the amino acid sequence shown, a CDR2 comprising the amino acid sequence represented by SEQ ID NO: 56, and a light chain variable region comprising CDR3 comprising the amino acid sequence represented by SEQ ID NO: 57 or A heavy chain variable region comprising a fragment of an antibody, or (v) CDR1 comprising the amino acid sequence represented by SEQ ID NO: 58, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 59, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 60 As well as a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 61, CDR2 comprising an amino acid sequence represented by sequence number 62, and comprises a light chain variable region comprising a CDR3 comprising an amino acid sequence represented by SEQ ID NO: 63, a fragment of a monoclonal antibody or the antibody that binds human CD33.
6). (I) a monoclonal antibody that binds to human CD33 comprising a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 132 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 137;
(Ii) a monoclonal antibody that binds to human CD33 comprising a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 133 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 138;
(Iii) a monoclonal antibody that binds to human CD33 comprising a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 134 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 139,
(Iv) a monoclonal antibody that binds to human CD33 comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 135 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 140, or (v) in SEQ ID NO: 136 Binds to human CD33, which binds to an epitope on human CD33 to which a monoclonal antibody that binds to human CD33, which comprises a heavy chain variable region comprising the amino acid sequence shown and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 141 A monoclonal antibody or a fragment of said antibody.
7. (I) a monoclonal antibody that binds to human CD33 comprising a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 132 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 137;
(Ii) a monoclonal antibody that binds to human CD33 comprising a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 133 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 138;
(Iii) a monoclonal antibody that binds to human CD33 comprising a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 134 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 139,
(Iv) a monoclonal antibody that binds to human CD33 comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 135 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 140, or (v) in SEQ ID NO: 136 Monoclonal anti or a fragment of said antibody that binds to human CD33 in competition with a monoclonal antibody that binds to human CD33 comprising a heavy chain variable region comprising the amino acid sequence shown and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 141 .
8). Dissociation constant _{K D} for human CD33 is 4.0 × ^{10 -9} M or less, a monoclonal antibody or fragment of the antibody according to any one of the preceding 1 7.
9. Dissociation constant _{K D} for human CD33 is 2.0 × ^{10 -9} M or less, a monoclonal antibody or fragment of the antibody according to [8.
10. 10. The monoclonal antibody or the antibody fragment according to any one of 1 to 9 above, which comprises a heavy chain constant region, wherein the heavy chain constant region class is human IgG or a fragment of the antibody.
11. 11. The monoclonal antibody or fragment of the antibody described in 10 above, wherein the subclass of human IgG is human IgG1.
12 12. The monoclonal antibody according to any one of 1 to 11 above or a fragment of the antibody comprising a heavy chain constant region, wherein fucose is bound to an N-linked complex type sugar chain that is bound to Fc of the heavy chain constant region. Not a monoclonal antibody or a fragment of said antibody.
13. 13. The monoclonal antibody or the antibody fragment according to any one of 1 to 12 above, which comprises a light chain constant region, wherein the light chain constant region class is human κ.
14 14. The antibody fragment according to any one of 1 to 13 above, which is a peptide comprising Fab, F (ab ′) ₂ , Fab ′, scFv, diabody, dsFv, or CDR.
15. 15. A DNA encoding the monoclonal antibody or the antibody fragment according to any one of 1 to 14 above.
16. 16. A recombinant vector containing the DNA according to item 15 above.
17. (I) a recombinant vector comprising DNA encoding a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 89;
(Ii) a recombinant vector comprising DNA encoding a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 90;
(Iii) a recombinant vector comprising DNA encoding a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 91;
(Iv) a recombinant vector comprising DNA encoding a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 90 and DNA encoding a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 89;
Or (v) a recombinant vector comprising DNA encoding a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 91 and DNA encoding a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 89.
18. 18. A transformant obtained by introducing at least one recombinant vector selected from the recombinant vectors described in 16 and 17 above into a host cell.
19. 19. The transformant according to 18 above, which is obtained by introducing the following recombinant vector (i) or (ii) into a host cell.
(I) a recombinant vector comprising DNA encoding a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 89 and a recombinant vector comprising DNA encoding a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 90 (Ii) a recombinant vector comprising a DNA encoding a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 89 and a recombinant vector comprising a DNA encoding a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 91 20. The transformant according to the item 18 or 19 is cultured in a medium, and the monoclonal antibody or the fragment of the antibody according to any one of the items 1 to 14 is produced and accumulated in the culture, and the monoclonal antibody is produced from the culture. Alternatively, the method for producing a monoclonal antibody or a fragment of the antibody according to any one of items 1 to 14, wherein a fragment of the antibody is collected.
21. 15. A pharmaceutical composition comprising the monoclonal antibody or the antibody fragment according to any one of items 1 to 14 as an active ingredient.
22. 15. A therapeutic agent for a disease involving CD33 positive cells, comprising the monoclonal antibody or the antibody fragment according to any one of items 1 to 14 as an active ingredient.
23. 23. The therapeutic agent according to item 22 above, wherein the disease involving CD33 positive cells is cancer, autoimmune disease or allergic disease.
24. The preceding paragraph 23, wherein the cancer is blood cancer, breast cancer, uterine cancer, colon cancer, esophageal cancer, stomach cancer, ovarian cancer, lung cancer, kidney cancer, rectal cancer, thyroid cancer, cervical cancer, small intestine cancer, prostate cancer or pancreatic cancer. The therapeutic agent as described in.
25. 25. The therapeutic agent according to 24 above, wherein the blood cancer is acute myeloid leukemia, anaplastic large cell lymphoma, acute lymphocytic leukemia, myelodysplastic syndrome, multiple myeloma, Hodgkin lymphoma, or non-Hodgkin lymphoma.
26. 15. A method for treating a disease involving CD33-positive cells, comprising a step of administering the monoclonal antibody or the antibody fragment according to any one of items 1 to 14 to a mammal.
27. 27. The method according to item 26 above, wherein the disease involving CD33 positive cells is cancer, autoimmune disease or allergic disease.
28. The preceding paragraph 27, wherein the cancer is blood cancer, breast cancer, uterine cancer, colon cancer, esophageal cancer, stomach cancer, ovarian cancer, lung cancer, kidney cancer, rectal cancer, thyroid cancer, cervical cancer, small intestine cancer, prostate cancer or pancreatic cancer. The method described in 1.
29. 29. The method according to item 28 above, wherein the blood cancer is acute myeloid leukemia, anaplastic large cell lymphoma, acute lymphocytic leukemia, myelodysplastic syndrome, multiple myeloma, Hodgkin lymphoma, or non-Hodgkin lymphoma.
30. 15. Use of the monoclonal antibody or fragment of the antibody according to any one of 1 to 14 above for treating a disease involving CD33 positive cells.
31. 31. The use according to item 30 above, wherein the disease involving CD33 positive cells is cancer, autoimmune disease or allergic disease.
32. Paragraph 31 above, wherein the cancer is blood cancer, breast cancer, uterine cancer, colon cancer, esophageal cancer, stomach cancer, ovarian cancer, lung cancer, kidney cancer, rectal cancer, thyroid cancer, cervical cancer, small intestine cancer, prostate cancer or pancreatic cancer. Use as described in.
33. 33. The use according to item 32 above, wherein the blood cancer is acute myeloid leukemia, anaplastic large cell lymphoma, acute lymphocytic leukemia, myelodysplastic syndrome, multiple myeloma, Hodgkin lymphoma or non-Hodgkin lymphoma.

As shown in the Examples, the monoclonal antibody of the present invention binds to the human CD33 extracellular region with high affinity and exhibits high ADCC activity. Therefore, the present invention relates to the antibody or the fragment, the DNA encoding the antibody or the fragment, the vector containing the DNA, the transformant obtained by introducing the vector, the antibody using the transformant or the A method for producing a fragment, and a therapeutic agent and a diagnostic agent containing the antibody or the fragment can be provided.

FIG. 1A shows a schematic diagram of human CD33 expression vector construction. FIG. 1B shows a schematic diagram of cynomolgus monkey CD33 expression vector construction. FIG. 1C shows a schematic diagram of chimpanzee CD33 expression vector construction. FIG. 2 shows a schematic diagram of the construction of a histidine-tagged soluble human CD33 expression vector. FIG. 3 shows the evaluation (FCM) of rat and mouse anti-CD33 monoclonal antibodies. FIG. 4 shows the evaluation (ELISA) of rat and mouse anti-CD33 monoclonal antibodies. FIG. 5A shows a schematic diagram of construction of an anti-CD33 chimeric antibody expression vector. FIG. 5B shows a schematic diagram of construction of an anti-CD33 chimeric antibody expression vector. FIG. 6 shows the evaluation (FCM) of the anti-CD33 chimeric antibody. FIG. 7 shows the evaluation (ELISA) of anti-CD33 chimeric antibody. FIG. 8 shows the evaluation (ADCC) of anti-CD33 chimeric antibody. FIG. 9 shows the evaluation (CDC) of anti-CD33 chimeric antibody. FIG. 10 shows the binding amount (FIG. 10A) and residual rate (FIG. 10B) of the antibody on the cell membrane of the anti-CD33 chimeric antibody. FIG. 11 shows a schematic diagram of HM195-1, -113F antibody expression vector construction. FIG. 12 shows the anti-CD33 humanized antibody evaluation (ADCC). FIG. 13 shows the amount of antibody bound on the cell membrane of the anti-CD33 humanized antibody (FIG. 13A) and the residual ratio (FIG. 13B). FIG. 14 shows the cell removal activity of anti-CD33 humanized antibody in human peripheral blood. FIG. 15 shows the reactivity of anti-CD33 humanized antibodies to human, cynomolgus monkey and chimpanzee CD33.

The present invention relates to a monoclonal antibody or a fragment thereof that binds to the extracellular region of human CD33 with high affinity and has high ADCC activity (hereinafter simply referred to as “CD33” means human CD33). Suppose).

As CD33 in the present invention, one or more amino acids in the polypeptide having the amino acid sequence represented by SEQ ID NO: 3 or P20148 (SWISSPROT) or the amino acid sequence represented by SEQ ID NO: 3 or EMBL accession number M23197 are deleted or substituted. Or a polypeptide comprising an added amino acid sequence and having the function of CD33, and 60% or more, preferably 80% or more, more preferably 90% or more, with the amino acid sequence represented by SEQ ID NO: 3 or EMBL accession number M23197 And a polypeptide having an amino acid sequence having the homology of (2), most preferably a polypeptide having an amino acid sequence having a homology of 95% or more and having the function of CD33.

A polypeptide having an amino acid sequence in which one or more amino acids have been deleted, substituted, or added in the amino acid sequence represented by SEQ ID NO: 3 or P20148 (SWISSPROT) can be obtained by site-directed mutagenesis [Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989), Current Protocols in Molecular Biology, John Wiley & Sons (1987-1997), Nucleic 64 Proc. Natl. Acad. Sci. USA, 79, 6409 (1982), Gene, 34, 315 (1985), Nucleic Acids Research, 13, 4431 (1985), Proc. Natl. Acad. Sci. USA, 82, 488 (1985)] and the like, for example, can be obtained by introducing a site-specific mutation into DNA encoding a polypeptide having the amino acid sequence represented by SEQ ID NO: 3. The number of amino acids to be deleted, substituted or added is not particularly limited, but is preferably 1 to several tens, for example 1 to 20, more preferably 1 to several, for example 1 to 5 amino acids. It is.

Examples of the gene encoding CD33 include the nucleotide sequence represented by SEQ ID NO: 4 or EMBL accession number M23197. A gene comprising a DNA encoding a polypeptide consisting of a base sequence in which one or more bases are deleted, substituted or added in the base sequence represented by SEQ ID NO: 4 or EMBL accession number M23197, and having a CD33 function; A nucleotide sequence having at least 60% homology with the nucleotide sequence represented by SEQ ID NO: 4 or EMBL accession number M23197, preferably a nucleotide sequence having 80% or more homology, more preferably 95% or more homology It hybridizes under stringent conditions with a gene comprising a DNA comprising a nucleotide sequence having a CD33 function and a DNA encoding a polypeptide having a CD33 function, and a DNA having the nucleotide sequence represented by SEQ ID NO: 4 or EMBL accession number M23197 Consisting of DNA, and Such as a gene containing DNA encoding a polypeptide having the function of D33 is also included in the gene encoding CD33 of the present invention.

As DNA that hybridizes under stringent conditions, DNA having the base sequence represented by SEQ ID NO: 4 or EMBL accession number M23197 was used as a probe, and colony hybridization, plaque hybridization, Southern blot -It means a hybridizable DNA obtained by a hybridization method or a DNA microarray method.

Specifically, 0.7 to 1.0 mol / L of sodium chloride is present using a DNA or DNA derived from a hybridized colony or plaque, or a filter or slide glass on which a PCR product or oligo DNA having the sequence is immobilized. Hybridization at 65 ° C. [Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989), Current Protocols in BioClinology, BioClinology, BioClinology, BioClinology, BioClinicology App oach, Second Edition, Oxford University, (1995)], followed by 0.1-2 fold concentration of SSC solution (1 fold concentration of SSC solution is 150 mmol / L sodium chloride, 15 mmol / L sodium citrate And a DNA that can be identified by washing a filter or glass slide at 65 ° C.

The DNA capable of hybridizing is DNA having at least 60% homology with the base sequence represented by SEQ ID NO: 4 or EMBL accession number M23197, preferably DNA having 80% homology or more, more preferably 95 % Of DNA having a homology of at least%.

In the base sequence of a gene encoding a eukaryotic protein, a polymorphism of the gene is often observed. A gene in which a small-scale variation is caused in the nucleotide sequence due to such polymorphism in the gene used in the present invention is also included in the gene encoding CD33 of the present invention.

The numerical value of homology in the present invention may be a numerical value calculated using a homology search program known to those skilled in the art unless otherwise specified, but the base sequence may be BLAST [J. Mol. Biol. , 215, 403 (1990)], for amino acid sequences such as numerical values calculated using default parameters, BLAST2 [Nucleic Acids Res. , 25, 3389 (1997), Genome Res. , 7, 649 (1997), http: // www. ncbi. nlm. nih. gov / Education / BLASTinfo / information3. numerical values calculated using default parameters in [html].

The default parameters are 5 if G (Cost to open gap) is a base sequence, 11 if it is an amino acid sequence, 2 if -E (Cost to extend gap) is a base sequence, and 1 if it is an amino acid sequence. , -Q (Penalty for nucleotide mismatch) is -3, -r (reward for nucleotide match) is 1, -e (expect value) is 10, 11 residues when -W (wordsize) is a base sequence, amino acid sequence 3 residues, -y [Dropoff (X) for blast extensions in bits] is 20 if blastn, 7 for programs other than blastn, -X (X dropoff value f If the r aligned alignment in bits) is 15 and -Z (final X dropoff value for gapd alignment in bits) is blastn, it is 25 for programs other than blastn (http: // www. gov / blast / html / blastcgihelp.html).

A polypeptide comprising a partial sequence of the amino acid sequence represented by SEQ ID NO: 3 or P20148 (SWISSPROT) can be prepared by methods known to those skilled in the art. For example, it can be produced by culturing a transformant in which a part of DNA encoding the amino acid sequence represented by SEQ ID NO: 3 has been deleted and an expression vector containing the DNA is introduced. Further, based on the polypeptide or DNA produced by the above method, one or more amino acids are deleted or substituted in the partial sequence of the amino acid sequence represented by SEQ ID NO: 3 or P20148 (SWISSPROT) by the same method as above. Alternatively, a polypeptide having an added amino acid sequence can be obtained. Further, a polypeptide comprising a partial sequence of the amino acid sequence represented by SEQ ID NO: 3 or P20148 (SWISSPROT), or one or more amino acids in the partial sequence of the amino acid sequence represented by SEQ ID NO: 3 or P20138 (SWISSPROT) is deleted or substituted Alternatively, a polypeptide having an added amino acid sequence can also be produced by a chemical synthesis method such as a fluorenylmethyloxycarbonyl (Fmoc) method or a t-butyloxycarbonyl (tBoc) method.

As the extracellular region of CD33 in the present invention, for example, the amino acid sequence of the polypeptide represented by SEQ ID NO: 3 is converted into a known transmembrane region prediction program SOSUI (http://bp.nuap.nagoya-u.ac.jp / SOSUI / SOSUI_submit.), TMHMM ver. 2 (http://www.cbs.dtu.dk/services/TMHMM-2.0/) or ExPASy Proteomics Server (http://Ca.expasy.org/) It is done. Specifically, the 242nd from the N-terminal which is an extracellular domain predicted in SOSUI is mentioned. The extracellular domain has two immunoglobulin superfamily domains, and both domains are included.

The extracellular region of CD33 in the present invention is any as long as the extracellular region of CD33 having the amino acid sequence represented by SEQ ID NO: 3 or P20148 (SWISSPROT) has a structure equivalent to the structure that can be taken in the natural state. It may be a structure. The structure that the extracellular region of CD33 can take in the natural state refers to the natural three-dimensional structure of CD33 expressed on the cell membrane.

The details of the function of CD33 are unknown. Examples of the function of CD33 in the present invention include expression in the differentiation stage of myeloid cells, adhesion to sialic acid-containing cells such as CD34 positive stem cells, and involvement in the control of cell differentiation. Further, since CD33 is a sialic acid binding protein, the activity of binding to sialic acid molecules and the intracellular signal transduction function or the motility of cells due to the binding with sialic acid molecules can also be mentioned as functions of CD33.

The binding of the antibody of the present invention or a fragment thereof to the extracellular region of CD33 is known for cells expressing CD33 using a radioimmunoassay using a solid phase sandwich method or an enzyme immunoassay (ELISA). It can be confirmed by an immunological detection method, preferably a method capable of examining the binding ability of a cell expressing a specific antigen and an antibody to the specific antigen, such as a fluorescent cell staining method.

For example, a fluorescent antibody staining method using a FMAT8100HTS system (Applied Biosystems) or the like [Cancer Immunol. Immunother. , 36, 373 (1993)], fluorescent cell staining using flow cytometry, or surface plasmon resonance using a Biacore system (manufactured by GE Healthcare). In addition, known immunological detection methods [Monoclonal Antibodies-Principles and practices, Third edition, Academic Press (1996), Antibodies-A Laboratory Manual, ColdHordrology, ColdHordrology, ColdHorman, ColdHordrology, ColdHorman, ColdHorman (1987)] etc. can also be confirmed.

The cells expressing CD33 may be any cells as long as they express CD33. For example, cells naturally existing in the human body, cell lines established from cells naturally existing in the human body, or genetic recombination Examples include cells obtained by technology.

Examples of cells that naturally exist in the human body include normal monocytes, granulocytes, or myeloid cells, as well as myeloid leukemia, for example, cells in which the polypeptide is expressed in AML patients. For example, among the tumor cells obtained by biopsy or the like, cells expressing CD33 can be mentioned.

As a cell line established from cells naturally existing in the human body, CD33 is expressed among the cell lines obtained by stocking cells expressing CD33 obtained from the above cancer patients. Cell lines. For example, human myeloid leukemia cell line NB-4 [DSMZ number: ACC207] or HL-60 [ATCC number: CCL-240], which are cell lines established from humans, can be mentioned.

Specifically, the cells obtained by gene recombination techniques include, for example, cells expressing CD33 obtained by introducing an expression vector containing cDNA encoding CD33 into insect cells or animal cells. Etc.

The antibody of the present invention is a monoclonal antibody against human CD33 that binds to the extracellular region of human CD33 and has high ADCC activity, and a fragment of the antibody, and preferably has high affinity for human CD33. Having high affinity for human CD33 means that the dissociation constant for human CD33 is low.

Dissociation constant for human CD33 antibodies of the present invention (hereinafter sometimes referred to as _{K D)} is preferably at 4.0 × ^{10 -9} M or less, more preferably 2.0 × ^{10 -9} M It is as follows. For example, 0.5 × 10 ⁻⁹ M to 4.0 × 10 ⁻⁹ M, 1.0 × 10 ⁻⁹ M to 4.0 × 10 ⁻⁹ M, 0.5 × 10 ⁻⁹ M to 2.0 × 10 ⁻⁹ M or 1.0 × 10 ⁻⁹ M to 2.0 × 10 ⁻⁹ M.

With a dissociation constant _{K D} for human CD33 4.0 × ^{10 -9} M or less, can bind to the extracellular region of CD33 with sufficient affinity as a therapeutic antibody, and high ADC activity and antitumor activity Can have. Already lintuzumab clinical trials have been conducted (HuM195) the dissociation constant, _{K D,} is 4 × ^{10 -9} M about affinity, since the show a therapeutic effect, the antibodies of the invention, higher therapeutic effects Can be shown.

Affinity is measured by reaction kinetic analysis, and can be measured using, for example, Biacore T100 (manufactured by GE Healthcare Bioscience).

In addition, the antibody of the present invention is characterized by very slow dissociation from the antigen. Since the dissociation is slow, or the dissociation is slow and the incorporation into the cell is slow, there is an advantage that the amount of antibody bound to the cell surface and the residual ratio are high.

In the present invention, examples of the monoclonal antibody include an antibody produced by a hybridoma or a recombinant antibody produced by a transformant transformed with an expression vector containing an antibody gene.

A monoclonal antibody is an antibody that is secreted by an antibody-producing cell of a single clone, recognizes only one epitope (also referred to as an antigenic determinant), and has a uniform amino acid sequence (primary structure) constituting the monoclonal antibody. .

Epitopes include a single amino acid sequence that is recognized and bound by a monoclonal antibody, a three-dimensional structure composed of amino acid sequences, an amino acid sequence linked with sugar chains, and a three-dimensional structure composed of amino acid sequences combined with sugar chains.

The epitope that is recognized and bound by the monoclonal antibody of the present invention is preferably an epitope consisting of a plurality of amino acid residues contained in the extracellular region of CD33, the 18th to 259th amino acid sequences of the amino acid sequence shown in SEQ ID NO: 3. More preferably, an epitope consisting of a plurality of amino acid residues contained in an immunoglobulin domain far from the cell membrane of the extracellular region of CD33, or a plurality of amino acid residues contained in an immunoglobulin domain close to the cell membrane of the extracellular region of CD33 The epitope etc. which become can be mention | raise | lifted.

In the present invention, slow dissociation means that the value of the antibody dissociation rate constant kd calculated in Biacore T100 shows a smaller value. The dissociation rate constant kd is measured using, for example, Biacore T100 (manufactured by GE Healthcare Bioscience), and can be calculated using attached software Biacore T100 evaluation software (manufactured by Biacore).

In the present invention, an antibody having ADCC activity refers to a known measurement method [Cancer Immunol. Immunother. , 36, 373 (1993)], which is an antibody having detectable ADCC activity.

In the present invention, an antibody having CDC activity refers to the CDC activity of a plurality of antibodies at the same time against CD33-expressing cells using a known CDC measurement method [Cancer Immunol. Immunother. , 36, 373 (1993)], which is an antibody having detectable CDC activity.

The monoclonal antibody of the present invention specifically includes a monoclonal antibody that binds to the same epitope as that on human CD33 to which any one of the following antibodies (i) to (v) binds: Examples thereof include a monoclonal antibody that competes with any one of the antibodies (1) to (v) and binds to human CD33, and an antibody having the same reactivity as any one of the following antibodies (i) to (v).
(I) VH of an antibody is an amino acid sequence represented by SEQ ID NO: 132, and VL of an antibody is an amino acid sequence represented by SEQ ID NO: 137. (ii) VH of an antibody is represented by SEQ ID NO: 133 And the VL of the antibody is the amino acid sequence represented by SEQ ID NO: 138. The VH of the monoclonal antibody (iii) antibody is the amino acid sequence represented by SEQ ID NO: 134, and the VL of the antibody is SEQ ID NO: The monoclonal antibody (v) of the monoclonal antibody (iv) antibody having the amino acid sequence represented by 139 is the amino acid sequence represented by SEQ ID NO: 135, and the monoclonal antibody (v ) An amino acid sequence in which the VH of the antibody is an amino acid sequence represented by SEQ ID NO: 136 and the VL of the antibody is represented by SEQ ID NO: 141 Monoclonal antibody that binds to an epitope on human CD33 monoclonal antibody that is acid sequence binds

In addition, the monoclonal antibody of the present invention includes a monoclonal antibody that binds to the same epitope on human CD33 to which any one of the following antibodies (i) to (v) binds, and the following (i) to (v) Examples thereof include a monoclonal antibody that competes with any one of the antibodies and binds to human CD33, and an antibody having reactivity equivalent to any one of the following antibodies (i) to (v).
(I) VH of an antibody is an amino acid sequence represented by SEQ ID NO: 103, and VL of an antibody is an amino acid sequence represented by SEQ ID NO: 95. (ii) VH of an antibody is represented by SEQ ID NO: 104 And the VL of the antibody is the amino acid sequence represented by SEQ ID NO: 95. The VH of the monoclonal antibody (iii) antibody is the amino acid sequence represented by SEQ ID NO: 105, and the VL of the antibody is SEQ ID NO: The monoclonal antibody (v) of the monoclonal antibody (iv) having the amino acid sequence represented by 95 is the amino acid sequence represented by SEQ ID NO: 93, and the monoclonal antibody (v ) The VH of the antibody is the amino acid sequence represented by SEQ ID NO: 106, and the VL of the antibody is the amino acid sequence represented by SEQ ID NO: 95 Monoclonal antibody

Furthermore, the monoclonal antibodies of the present invention include the following monoclonal antibodies that bind to human CD33 (i) to (v).
(I) a heavy chain variable region comprising CDR1 comprising the amino acid sequence represented by SEQ ID NO: 34, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 35, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 36, and SEQ ID NO: 37 A monoclonal antibody that binds to human CD33, comprising a light chain variable region comprising CDR1 comprising the amino acid sequence represented by SEQ ID NO: 38, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 38, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 39. ii) a heavy chain variable region comprising CDR1, comprising the amino acid sequence represented by SEQ ID NO: 40, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 41, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 42, and SEQ ID NO: 43 CDR1, comprising the amino acid sequence shown, represented by SEQ ID NO: 44 A monoclonal antibody (iii) that binds human CD33, comprising a light chain variable region comprising CDR2 comprising a amino acid sequence and CDR3 comprising an amino acid sequence represented by SEQ ID NO: 45, CDR1 comprising an amino acid sequence represented by SEQ ID NO: 46, A heavy chain variable region comprising CDR2 comprising the amino acid sequence represented by SEQ ID NO: 47, and a CDR3 comprising the amino acid sequence represented by SEQ ID NO: 48, and a CDR1 comprising the amino acid sequence represented by SEQ ID NO: 49, the amino acid represented by SEQ ID NO: 50 A monoclonal antibody that binds human CD33 (iv) comprising a CDR2 comprising a sequence and a light chain variable region comprising a CDR3 comprising an amino acid sequence represented by SEQ ID NO: 51, CDR1 comprising an amino acid sequence represented by SEQ ID NO: 52, SEQ ID NO: CDR2, comprising the amino acid sequence represented by 53, And a heavy chain variable region comprising CDR3 comprising the amino acid sequence represented by SEQ ID NO: 54, and CDR1 comprising the amino acid sequence represented by SEQ ID NO: 55, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 56, and SEQ ID NO: 57. A monoclonal antibody that binds to human CD33 comprising a light chain variable region comprising CDR3 comprising an amino acid sequence (v) a CDR1 comprising an amino acid sequence represented by SEQ ID NO: 58, a CDR2 comprising an amino acid sequence represented by SEQ ID NO: 59, and a sequence A heavy chain variable region comprising CDR3 comprising the amino acid sequence represented by number 60, and CDR1 comprising the amino acid sequence represented by SEQ ID NO: 61, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 62, and an amino acid sequence represented by SEQ ID NO: 63 A human CD comprising a light chain variable region comprising CDR3 comprising Monoclonal antibody binding to 33

Furthermore, the monoclonal antibody of the present invention comprises a CDR1 comprising the amino acid sequence represented by SEQ ID NO: 52, a CDR2 comprising the amino acid sequence represented by SEQ ID NO: 53, and a CDR3 comprising the amino acid sequence represented by SEQ ID NO: 54. A chain variable region, and a light chain variable region comprising CDR1 comprising the amino acid sequence represented by SEQ ID NO: 55, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 56, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 57, An antibody modified so as not to have a consensus sequence for binding an N-linked sugar chain to the V region of a monoclonal antibody that binds to human CD33 (hereinafter sometimes referred to as a modified antibody) is also included.

Specifically, as the monoclonal antibody of the present invention, CDR1 and CDR3 of VH of the antibody include the amino acid sequences shown by SEQ ID NOs: 52 and 54, respectively, and CDR1, CDR2 and CDR3 of VL of the antibody are respectively And monoclonal antibodies comprising the amino acid sequences shown in SEQ ID NOs: 55, 56 and 57.

The VH CDR2 of the above antibody is the CDR2 of the modified antibody VH, for example, a modification in which the third Asn in the amino acid sequence represented by SEQ ID NO: 53 is substituted with another amino acid residue, and the fifth CDR2 containing an amino acid sequence in which at least one modification is introduced among the modifications that substitute Ser in the other amino acid residues.

Specific examples include the following amino acid sequences (1) to (5).
(1) The amino acid sequence represented by SEQ ID NO: 99 in which the third Asn of the amino acid sequence represented by SEQ ID NO: 53 is substituted with Tyr (2) The third Asn of the amino acid sequence represented by SEQ ID NO: 53 is substituted with Ser The amino acid sequence represented by SEQ ID NO: 100 (3) The amino acid sequence represented by SEQ ID NO: 101 in which the third Asn of the amino acid sequence represented by SEQ ID NO: 53 was replaced with Gln (4) The amino acid represented by SEQ ID NO: 53 An amino acid sequence represented by SEQ ID NO: 96 in which the fifth Ser of the sequence is substituted with Ala (5) An amino acid sequence represented by SEQ ID NO: 102 in which the fifth Ser of the amino acid sequence represented by SEQ ID NO: 53 is substituted with Gly

The hybridoma is prepared by using, for example, cells expressing the above-mentioned CD33 as an antigen, inducing antibody-producing cells having antigen specificity from an animal immunized with the antigen, and further fusing the antibody-producing cells and myeloma cells. Can be prepared. The anti-CD33 monoclonal antibody can be obtained by culturing the hybridoma or administering the hybridoma to an animal to cause the animal to undergo ascites cancer, and separating and purifying the culture medium or ascites.

Any animal can be used as the animal to immunize with the antigen as long as it can produce a hybridoma, but mice, rats, hamsters, chickens, rabbits, cows and the like are preferably used. An antibody produced by a hybridoma prepared by obtaining a cell capable of producing an antibody from such an animal, immunizing the cell in vitro, and then fusing it with a myeloma cell is also an antibody of the present invention. Is included.

Examples of the recombinant antibody in the present invention include antibodies produced by genetic recombination, such as human chimeric antibodies, human CDR-grafted antibodies (sometimes referred to as humanized antibodies), human antibodies or antibody fragments. .

A recombinant antibody having characteristics of a monoclonal antibody, low antigenicity, and extended blood half-life is preferable as a therapeutic agent. Examples of the recombinant antibody include those obtained by modifying the monoclonal antibody of the present invention using a gene recombination technique.

The human chimeric antibody is sometimes referred to as VH and VL of a non-human animal antibody, heavy chain constant region of human antibody (hereinafter sometimes referred to as CH) and light chain constant region (hereinafter referred to as CL). ).

The human chimeric antibody of the present invention obtains cDNAs encoding VH and VL from a hybridoma that specifically recognizes CD33 and produces a monoclonal antibody that binds to the extracellular region of CD33. A human chimeric antibody expression vector can be constructed by inserting into an expression vector for animal cells having a gene encoding CL, and introduced into animal cells for expression and production.

The CH of the human chimeric antibody may be any as long as it belongs to human immunoglobulin (hereinafter sometimes referred to as hIg), but preferably the hIgG class is used, and hIgG1, which belongs to the hIgG class, Any of the subclasses such as hIgG2, hIgG3 or hIgG4 can be used. The CL of the human chimeric antibody may be any as long as it belongs to hIg, and those of κ class or λ class can be used.

Furthermore, the chimeric antibody of the present invention is the same as the chimeric antibody that binds to the extracellular region of CD33 and the epitope present on the extracellular region of CD33 to which the chimeric antibody binds by competing with the monoclonal antibody described above. Mention may be made of chimeric antibodies that bind to an epitope.

The human CDR-grafted antibody refers to an antibody obtained by grafting the VH and VL CDR amino acid sequences of non-human animal antibodies to appropriate positions of the human antibody VH and VL. The human CDR-grafted antibody of the present invention specifically recognizes V33 of a non-human animal antibody produced from a hybridoma that specifically recognizes CD33 and produces a monoclonal antibody of a non-human animal that binds to the extracellular region. A cDNA encoding the V region in which the amino acid sequence of the CDR of the VL was grafted to the VH and VL framework regions of the human antibody (hereinafter sometimes referred to as FR) was constructed, and the CH and CL of the human antibody were A human CDR-grafted antibody expression vector can be constructed by inserting it into an expression vector for animal cells having a gene to be encoded, and introduced into animal cells for expression and production.

The CH of the human CDR-grafted antibody may be any as long as it belongs to hIg, but preferably the hIgG class is used, and any subclass such as hIgG1, hIgG2, hIgG3, or hIgG4 belonging to the hIgG class is used. be able to. The CL of the human CDR-grafted antibody may be any CL as long as it belongs to hIg, and those of κ class or λ class can be used.

As the human CDR-grafted antibody of the present invention, specifically, for example, the VH of the antibody has the amino acid sequence represented by SEQ ID NO: 84 or the 25th Ser in the amino acid sequence represented by SEQ ID NO: 84, 27th Gly, 28th Ser, 29th Val, 30th Ser, 40th Gln, 45th Gly, 46th Leu, 49th Ile, 72nd Val, 93th Val, An amino acid sequence in which at least one amino acid residue selected from the 95th Tyr, the 97th Ala and the 106th Thr is substituted with another amino acid residue, and / or the VL of the antibody is SEQ ID NO: 85; Or the second Ile in the amino acid sequence represented by SEQ ID NO: 85, the 8th Pro, the 11th Leu, the 15th And a humanized antibody including an amino acid sequence in which at least one amino acid residue selected from Pro and 90th Val is substituted with another amino acid residue, but the number of modifications to be introduced is not limited .

For example, the following humanized antibodies can be mentioned.
The VH of the antibody is the 25th Ser, 27th Gly, 28th Ser, 29th Val, 30th Ser, 40th Gln, 45th Gly in the amino acid sequence represented by SEQ ID NO: 84, At least one amino acid residue selected from 46th Leu, 49th Ile, 72nd Val, 93th Val, 95th Tyr, 97th Ala and 106th Thr is another amino acid residue A humanized antibody comprising an amino acid sequence substituted with.

Among the humanized antibodies, the following (1) to (3) are preferable.
(1) 27th Gly, 40th Gln, 45th Gly, 46th Leu, 49th Ile, 72nd Val, and 97 in the amino acid sequence represented by SEQ ID NO: 84 as the VH of the antibody The humanized antibody comprising the amino acid sequence in which the No. Ala is substituted with another amino acid residue (2) 27th Gly, 28th Ser, 29 in the amino acid sequence in which the VH of the antibody is represented by SEQ ID NO: 84 The VH of the humanized antibody (3) antibody comprising the amino acid sequence in which the No. Val, the 30th Ser, the 72nd Val, and the 97th Ala are substituted with other amino acid residues is represented by SEQ ID NO: 84 Humanized antibody comprising an amino acid sequence in which the 27th Gly and the 72nd Val in the amino acid sequence are substituted with other amino acid residues

As a result of the above amino acid modification, the amino acid sequence of the VH of the obtained antibody is, for example, the 25th Ser in the amino acid sequence represented by SEQ ID NO: 84, the 27th Gly in the Tyr, and the 28th in the amino acid sequence. Ser to Thr, 29th Val to Ile, 30th Ser to Thr, 40th Gln to Lys, 45th Gly to Arg, 46th Leu to Met, 49th Ile Met, at least selected from a modification in which the 72nd Val is replaced with Arg, the 93rd Val is replaced with Thr, the 95th Tyr is replaced with Phe, the 97th Ala is replaced with Thr, and the 106th Thr is replaced with Val. An amino acid sequence into which one modification has been introduced is mentioned.

Specific examples of the amino acid sequence of VH into which 14 modifications have been introduced include, for example, 25th Ser in the amino acid sequence represented by SEQ ID NO: 84, Thr, 27th Gly in Tyr, 28 The th Ser, Thr, the 29th Val to Ile, the 30th Ser to Thr, the 40th Gln to Lys, the 45th Gly to Arg, the 46th Leu to Met, and the 49th An amino acid sequence in which Ile is replaced by Met, 72nd Val is replaced by Arg, 93rd Val is replaced by Thr, 95th Tyr is replaced by Phe, 97th Ala is replaced by Thr, and 106th Thr is replaced by Val. Can be mentioned.

Specific examples of the amino acid sequence of VH into which 13 modifications have been introduced include the following amino acid sequences (1) to (14).
(1) The 27th Gly in the amino acid sequence represented by SEQ ID NO: 84 is Tyr, the 28th Ser is Thr, the 29th Val is Ile, the 30th Ser is Thr, and the 40th Gln To Lys, 45th Gly to Arg, 46th Leu to Met, 49th Ile to Met, 72nd Val to Arg, 93rd Val to Thr, 95th Tyr to Phe In the amino acid sequence represented by SEQ ID NO: 84, the 97th Ala is replaced with Thr and the 106th Thr is replaced with Val. The 25th Ser in the amino acid sequence represented by SEQ ID NO: 84 is Thr, and the 28th Ser is Thr. 29th Val to Ile, 30th Ser to Thr, 40th Gln to Lys, 45th Gly to Arg, 46th Leu to M t, 49th Ile to Met, 72nd Val to Arg, 93rd Val to Thr, 95th Tyr to Phe, 97th Ala to Thr, and 106th Thr to Val (3) In the amino acid sequence represented by SEQ ID NO: 84, the 25th Ser in Thr, the 27th Gly in Tyr, the 29th Val in Ile, and the 30th Ser in Thr 40th Gln to Lys, 45th Gly to Arg, 46th Leu to Met, 49th Ile to Met, 72nd Val to Arg, 93th Val to Thr, 95 An amino acid sequence in which the thirr is replaced with Phe, the 97th Ala with Thr, and the 106th thr with Val (4) The 25th Ser in the no acid sequence is Thr, the 27th Gly is Tyr, the 28th Ser is Thr, the 30th Ser is Thr, the 40th Gln is Lys, and the 45th Gly is Arg, 46th Leu to Met, 49th Ile to Met, 72nd Val to Arg, 93rd Val to Thr, 95th Tyr to Phe, 97th Ala to Thr , And the amino acid sequence in which the 106th Thr is substituted with Val (5), the 25th Ser in the amino acid sequence represented by SEQ ID NO: 84, the 27th Gly in the Tyr, and the 28th Ser in the Thr 29th Val to Ile, 40th Gln to Lys, 45th Gly to Arg, 46th Leu to Met, 49th Ile to Met The amino acid sequence (6) in which the 72nd Val is replaced with Arg, the 93rd Val is replaced with Thr, the 95th Tyr is replaced with Phe, the 97th Ala is replaced with Thr, and the 106th Thr is replaced with Val. In the amino acid sequence represented by number 84, the 25th Ser is set to Thr, the 27th Gly to Tyr, the 28th Ser to Thr, the 29th Val to Ile, the 30th Ser to Thr, 45th Gly to Arg, 46th Leu to Met, 49th Ile to Met, 72nd Val to Arg, 93rd Val to Thr, 95th Tyr to Phe, 97th Amino acid sequence in which Ala is replaced with Thr, and Thr at 106th is replaced with Val (7) The 25th Ser in the amino acid sequence represented by SEQ ID NO: 84 is hr, 27th Gly to Tyr, 28th Ser to Thr, 29th Val to Ile, 30th Ser to Thr, 40th Gln to Lys, 46th Leu to Met The 49th Ile is replaced by Met, the 72nd Val is replaced by Arg, the 93rd Val is replaced by Thr, the 95th Tyr is replaced by Phe, the 97th Ala is replaced by Thr, and the 106th Thr is replaced by Val. In the amino acid sequence represented by SEQ ID NO: 84, the 25th Ser in Thr, the 27th Gly in Tyr, the 28th Ser in Thr, the 29th Val in Ile, 30 The 41st Ser to Thr, the 40th Gln to Lys, the 45th Gly to Arg, the 49th Ile to Met, and the 72nd Val to Arg An amino acid sequence in which the 93rd Val is replaced with Thr, the 95th Tyr is replaced with Phe, the 97th Ala is replaced with Thr, and the 106th Thr is replaced with Val (9) in the amino acid sequence represented by SEQ ID NO: 84 25th Ser to Thr, 27th Gly to Tyr, 28th Ser to Thr, 29th Val to Ile, 30th Ser to Thr, 40th Gln to Lys, 45th Gly in Arg, 46th Leu in Met, 72nd Val in Arg, 93rd Val in Thr, 95th Tyr in Phe, 97th Ala in Thr, and 106th Amino acid sequence in which Thr is replaced with Val (10) The 25th Ser in the amino acid sequence represented by SEQ ID NO: 84 is Thr, and the 27th Gly is T yr, 28th Ser to Thr, 29th Val to Ile, 30th Ser to Thr, 40th Gln to Lys, 45th Gly to Arg, 46th Leu to Met Amino acid sequence in which 49th Ile is replaced by Met, 93rd Val is replaced by Thr, 95th Tyr is replaced by Phe, 97th Ala is replaced by Thr, and 106th Thr is replaced by Val (11) In the amino acid sequence represented by 84, the 25th Ser is Thr, the 27th Gly is Tyr, the 28th Ser is Thr, the 29th Val is Ile, the 30th Ser is Thr, 40 The 41st Gln is Lys, the 45th Gly is Arg, the 46th Leu is Met, the 49th Ile is Met, and the 72nd Val is Arg. The amino acid sequence in which the 95th Tyr is substituted with Phe, the 97th Ala with Thr, and the 106th Thr with Val (12), the 25th Ser in the amino acid sequence represented by SEQ ID NO: 84, with Thr, 27th Gly as Tyr, 28th Ser as Thr, 29th Val as Ile, 30th Ser as Thr, 40th Gln as Lys, 45th Gly as Arg, 46th Amino acid sequence in which Leu is replaced with Met, 49th Ile is replaced with Met, 72nd Val is replaced with Arg, 93rd Val is replaced with Thr, 97th Ala is replaced with Thr, and 106th Thr is replaced with Val. (13) The 25th Ser in the amino acid sequence represented by SEQ ID NO: 84 is Thr, the 27th Gly is Tyr, and the 28th Ser Thr, 29th Val to Ile, 30th Ser to Thr, 40th Gln to Lys, 45th Gly to Arg, 46th Leu to Met, 49th Ile to Met An amino acid sequence in which the 72nd Val is replaced with Arg, the 93rd Val is replaced with Thr, the 95th Tyr is replaced with Phe, and the 106th Thr is replaced with Val (14) in the amino acid sequence represented by SEQ ID NO: 84 25th Ser to Thr, 27th Gly to Tyr, 28th Ser to Thr, 29th Val to Ile, 30th Ser to Thr, 40th Gln to Lys, 45 The 41st Gly is Arg, the 46th Leu is Met, the 49th Ile is Met, the 72nd Val is Arg, and the 93rd Val is Thr. Amino acid sequence in which the 95th Tyr is substituted with Phe and the 97th Ala with Thr

Specific examples of the amino acid sequence of VH into which 10 modifications have been introduced include the following amino acid sequences (1) to (3).
(1) The 25th Ser in the amino acid sequence represented by SEQ ID NO: 84 is Thr, the 27th Gly is Tyr, the 28th Ser is Thr, the 29th Val is Ile, and the 30th Ser Is replaced with Thr, 72nd Val with Arg, 93rd Val with Thr, 95th Tyr with Phe, 97th Ala with Thr, and 106th Thr with Val. ) In the amino acid sequence represented by SEQ ID NO: 84, the 27th Gly is Tyr, the 28th Ser is Thr, the 29th Val is Ile, the 30th Ser is Thr, and the 40th Gln is Lys. 45th Gly to Arg, 46th Leu to Met, 49th Ile to Met, 72nd Val to Arg, and 97th Al In the amino acid sequence represented by SEQ ID NO: 84, the 25th Ser in Thr, the 27th Gly in Tyr, the 40th Gln in Lys, and the 45th Gly in Arg, 46th Leu to Met, 49th Ile to Met, 72nd Val to Arg, 93rd Val to Thr, 95th Tyr to Phe, and 97th Ala to Thr Amino acid sequence replaced with

Specifically, the amino acid sequence of VH into which 8 modifications have been introduced is, for example, 28th Ser in the amino acid sequence represented by SEQ ID NO: 84, Thr, 29th Val in Ile, 30 An amino acid in which the th Ser is replaced with Thr, the 40th Gln with Lys, the 45th Gly with Arg, the 46th Leu with Met, the 49th Ile with Met, and the 97th Ala with Thr Examples include sequences

Specific examples of the amino acid sequence of VH into which seven modifications have been introduced include, for example, the 27th Gly in the amino acid sequence represented by SEQ ID NO: 84 as Tyr, the 40th Gln as Lys, and 45 Examples include amino acid sequences in which the first Gly is replaced by Arg, the 46th Leu is replaced by Met, the 49th Ile is replaced by Met, the 72nd Val is replaced by Arg, and the 97th Ala is replaced by Thr.

Specifically, the amino acid sequence of VH into which six modifications are introduced is, for example, the 27th Gly in the amino acid sequence represented by SEQ ID NO: 84 as Tyr, the 28th Ser as Thr, 29 Examples include amino acid sequences in which the 1st Val is replaced with Ile, the 30th Ser with Thr, the 72nd Val with Arg, and the 97th Ala with Thr.

Specifically, the amino acid sequence of VH into which five modifications are introduced is, for example, that the 40th Gln in the amino acid sequence represented by SEQ ID NO: 84 is Lys, the 45th Gly is Arg, Examples include amino acid sequences in which the first Leu is replaced by Met, the 49th Ile is replaced by Met, and the 97th Ala is replaced by Thr.

Specific examples of the amino acid sequence of VH into which four modifications have been introduced include the following amino acid sequences (1) to (6).
(1) An amino acid sequence in which the 40th Gln in the amino acid sequence represented by SEQ ID NO: 84 is substituted with Lys, the 45th Gly with Arg, the 46th Leu with Met, and the 49th Ile with Met (2) An amino acid sequence in which the 27th Gly in the amino acid sequence represented by SEQ ID NO: 84 is substituted with Tyr, the 28th Ser with Thr, the 29th Val with Ile, and the 30th Ser with Thr. (3) An amino acid sequence in which the 40th Gln in the amino acid sequence represented by SEQ ID NO: 84 is substituted with Lys, the 45th Gly with Arg, the 46th Leu with Met, and the 97th Ala with Thr (4) The 40th Gln in the amino acid sequence represented by SEQ ID NO: 84 is Lys, the 45th Gly is Arg, and the 49th Ile is Met. And amino acid sequence in which 97th Ala is replaced with Thr (5) In the amino acid sequence represented by SEQ ID NO: 84, 40th Gln is Lys, 46th Leu is Met, 49th Ile is Met, In the amino acid sequence represented by SEQ ID NO: 84, the 45th Gly is Arg, the 46th Leu is Met, the 49th Ile is Met, and the 97th Ala is replaced with Thr. And amino acid sequence in which 97th Ala is replaced with Thr

Specific examples of the amino acid sequence of VH into which two modifications have been introduced include the following amino acid sequences (1) to (12).
(1) An amino acid sequence in which the 27th Gly in the amino acid sequence represented by SEQ ID NO: 84 is substituted with Tyr and the 72nd Val is substituted with Arg (2) The 27th amino acid in the amino acid sequence represented by SEQ ID NO: 84 In the amino acid sequence represented by SEQ ID NO: 84, the 40th Gln in the amino acid sequence represented by SEQ ID NO: 84 was replaced with Lys, and the 45th Gly was replaced with Arg. Amino acid sequence (4) In the amino acid sequence represented by SEQ ID NO: 84, the 40th Gln in the amino acid sequence was replaced with Lys, and the 46th Leu was replaced with Met (5) in the amino acid sequence represented by SEQ ID NO: 84 An amino acid sequence in which the 40th Gln is replaced with Lys and the 49th Ile is replaced with Met (6) 4 in the amino acid sequence represented by SEQ ID NO: 84 The amino acid sequence in which the Ginth Gln is replaced with Lys and the 97th Ala is replaced with Thr (7) The 45th Gly in the amino acid sequence represented by SEQ ID NO: 84 is replaced with Arg, and the 46th Leu is replaced with Met Amino acid sequence (8) In the amino acid sequence represented by SEQ ID NO: 84, the 45th Gly in the amino acid sequence represented by Arg and the 49th Ile substituted with Met (9) In the amino acid sequence represented by SEQ ID NO: 84 In the amino acid sequence represented by SEQ ID NO: 84, the 45th Gly in Arg was replaced with Arg, and the 97th Ala was replaced with Thr. The 46th Leu in the amino acid sequence represented by SEQ ID NO: 84 was designated as Met, and the 49th Ile was designated as Met. The amino acid sequence substituted with (11) the 46th Leu in the amino acid sequence represented by SEQ ID NO: 84, and the 97th la amino acid sequence is substituted with Thr (12) with Met 49th Ile in the amino acid sequence represented by SEQ ID NO: 84, and 97 amino acid sequence was replaced with Thr to Ala

Specifically, the amino acid sequence of VH into which one modification is introduced is, for example, an amino acid sequence in which the 25th Ser in the amino acid sequence represented by SEQ ID NO: 84 is replaced with Thr, and the 27th Gly is Amino acid sequence substituted with Tyr, amino acid sequence substituted with 28th Ser with Thr, amino acid sequence substituted with 29th Val with Ile, amino acid sequence substituted with 30th Ser with Thr, 40th Gln with Lys Substituted amino acid sequence, amino acid sequence in which the 45th Gly was replaced with Arg, amino acid sequence in which the 46th Leu was replaced with Met, amino acid sequence in which the 49th Ile was replaced with Met, and the 72nd Val was replaced with Arg Amino acid sequence, amino acid sequence in which 93rd Val is replaced with Thr, 95th Tyr is placed in Phe Amino acid sequence, the 97th Ala amino acid sequence is substituted with Thr, or the amino acid sequence obtained by replacing 106 th Thr in Val, and the like.

For antibody VL, for example, at least one amino acid residue selected from the second Ile, the eighth Pro, the eleventh Leu, the fifteenth Pro, and the 90th Val in the amino acid sequence of SEQ ID NO: 85 Are humanized antibodies comprising amino acid sequences substituted with other amino acid residues. *

Among the humanized antibodies, the following (1) to (4) are preferable.
(1) A humanized antibody comprising an amino acid sequence in which the second Ile, the 11th Leu, the 15th Pro, and the 90th Val in the amino acid sequence of SEQ ID NO: 85 are substituted with other amino acid residues ( 2) A humanized antibody comprising an amino acid sequence in which the second Ile, 15th Pro, and 90th Val in the amino acid sequence of SEQ ID NO: 85 are substituted with other amino acid residues.
(3) A humanized antibody comprising an amino acid sequence in which the second Ile, 11th Leu, and 15th Pro in the amino acid sequence of SEQ ID NO: 85 are substituted with other amino acid residues.
(4) A humanized antibody comprising an amino acid sequence in which the second Ile and 15th Pro in the amino acid sequence of SEQ ID NO: 85 are substituted with other amino acid residues

The amino acid sequence of the antibody VL obtained as a result of the above amino acid modification is as follows. The second Ile in the amino acid sequence of SEQ ID NO: 85 is Val, the 8th Pro is Leu, the 11th Leu is Gln, 15 An amino acid sequence in which at least one modification selected from the modification in which the 1st Pro is replaced with Leu and the 90th Val is replaced with Leu is introduced.

Specifically, as the amino acid sequence of VL into which five modifications are introduced, for example, the second Ile in the amino acid sequence of SEQ ID NO: 85 is Val, the 8th Pro is Leu, and the 11th Leu Amino acid sequence in which is replaced with Gln, 15th Pro is replaced with Leu, and 90th Val is replaced with Leu.

Specific examples of the amino acid sequence of VL into which four modifications have been introduced include the following amino acid sequences (1) to (5).
(1) Amino acid sequence in which the 8th Pro in the amino acid sequence of SEQ ID NO: 85 is replaced with Leu, the 11th Leu is replaced with Gln, the 15th Pro is replaced with Leu, and the 90th Val is replaced with Leu (2) The amino acid sequence in which the second Ile in the amino acid sequence of SEQ ID NO: 85 is replaced with Val, the 11th Leu is replaced with Gln, the 15th Pro is replaced with Leu, and the 90th Val is replaced with Leu (3) SEQ ID NO: 85 The amino acid sequence of SEQ ID NO: 85 in which the second Ile is replaced with Val, the 8th Pro is replaced with Leu, the 15th Pro is replaced with Leu, and the 90th Val is replaced with Leu. The second Ile is replaced with Val, the 8th Pro is replaced with Leu, the 11th Leu is replaced with Gln, and the 90th Val is replaced with Leu. Acid sequence of (5) the second Ile in the amino acid sequence of SEQ ID NO: 85 to Val, a Leu 8th Pro, 11 th Leu to Gln, and the amino acid sequence obtained by replacing the 15th Pro to Leu

Specific examples of the amino acid sequence of VL into which three modifications have been introduced include the following amino acid sequences (1) to (3).
(1) Amino acid sequence in which the second Ile in the amino acid sequence of SEQ ID NO: 85 is replaced with Val, the 8th Pro is replaced with Leu, and the 15th Pro is replaced with Leu (2) in the amino acid sequence of SEQ ID NO: 85 The amino acid sequence in which the second Ile is replaced with Val, the 11th Leu is replaced with Gln, and the 15th Pro is replaced with Leu. (3) The second Ile in the amino acid sequence of SEQ ID NO: 85 is Val, and the 15th Amino acid sequence in which Pro is replaced with Leu and 90th Val is replaced with Leu

Specific examples of the amino acid sequence of VL into which two modifications are introduced include the following amino acid sequences (1) to (7).
(1) An amino acid sequence in which the second Ile in the amino acid sequence of SEQ ID NO: 85 is substituted with Val, and the 8th Pro is substituted with Leu. (2) The second Ile in the amino acid sequence of SEQ ID NO: 85 is Val. And the amino acid sequence (4) of SEQ ID NO: 85 in which the second Ile in the amino acid sequence of SEQ ID NO: 85 is replaced with Val, and the 15th Pro is replaced with Leu. The amino acid sequence in which the second Ile in the amino acid sequence is substituted with Val and the 90th Val is substituted with Leu (5) The 8th Pro in the amino acid sequence of SEQ ID NO: 85 is Leu, and the 15th Pro is Leu In the amino acid sequence of SEQ ID NO: 85, the 11th Leu in Gln and the 15th Pro in Leu Was the 15 th Pro amino acid sequence (7) in the amino acid sequence of SEQ ID NO: 85 to Leu, and 90 amino acid sequence Val has been substituted with Leu

Specifically, the amino acid sequence of VL into which one modification is introduced is, for example, an amino acid sequence in which the second Ile in the amino acid sequence of SEQ ID NO: 85 is substituted with Val, and the eighth Pro is substituted with Leu. An amino acid sequence in which the 11th Leu is replaced with Gln, an amino acid sequence in which the 15th Pro is replaced with Leu, or an amino acid sequence in which the 90th Val is replaced with Leu.

A human antibody originally refers to an antibody that naturally exists in the human body, but a human antibody phage library and a human antibody-producing transgene prepared by recent advances in genetic engineering, cell engineering, and developmental engineering techniques. Also included are antibodies obtained from transgenic animals.

The antibody naturally present in the human body can be cultured by, for example, isolating human peripheral blood lymphocytes, infecting and immortalizing EB virus, etc., and cloning the lymphocytes that produce the antibody. The antibody can be further purified.

The human antibody phage library is a library in which antibody fragments such as Fab or scFv are expressed on the phage surface by inserting antibody genes prepared from human B cells into the phage genes. From the library, phages expressing an antibody fragment having a desired antigen-binding activity on the surface can be collected using the binding activity to the substrate on which the antigen is immobilized as an index. The antibody fragment can be further converted into a human antibody molecule comprising two complete heavy chains and two complete light chains by genetic engineering techniques.

A human antibody-producing transgenic animal means an animal in which a human antibody gene is incorporated into cells. Specifically, for example, a human antibody-producing transgenic mouse can be produced by introducing a human antibody gene into a mouse ES cell, transplanting the ES cell into an early mouse embryo, and generating it. For human antibodies from human antibody-producing transgenic animals, human antibody-producing hybridomas are obtained and cultured using normal hybridoma production methods performed in non-human animals to produce human antibodies in the culture supernatant. It can be produced by accumulating.

A monoclonal antibody or fragment thereof in which one or more amino acids are deleted, added, substituted or inserted in the amino acid sequence constituting the above-described antibody or antibody fragment and has the same activity as the above-described antibody or fragment thereof, It is included in the monoclonal antibody of the invention or a fragment thereof.

The number of amino acids to be deleted, substituted, inserted and / or added is one or more, and the number is not particularly limited, but site-specific mutagenesis [Molecular Cloning, 2nd Edition, Cold Spring Harbor Laboratory Press (1989), Current protocols in Molecular Biology, John Wiley & Sons (1987-1997), Nucleic Acids Research, 10, 6487 (1982), Proc. Natl. Acad. Sci. USA, 79, 6409 (1982), Gene, 34, 315 (1985), Nucleic Acids Research, 13, 4431 (1985), Proc. Natl. Acad. Sci USA, 82, 488 (1985)], etc., and the number that can be deleted, substituted, or added. For example, the number is 1 to several tens, preferably 1 to 20, more preferably 1 to 10, and still more preferably 1 to 5.

The deletion, substitution, insertion or addition of one or more amino acid residues in the amino acid sequence of the above antibody indicates the following. That is, it means that there is a deletion, substitution, insertion or addition of one or a plurality of amino acid residues in any one and a plurality of amino acid sequences in the same sequence. In addition, deletion, substitution, insertion or addition may occur simultaneously, and the amino acid residue to be substituted, inserted or added may be either a natural type or a non-natural type.

Examples of natural amino acid residues include L-alanine, L-asparagine, L-aspartic acid, L-glutamine, L-glutamic acid, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L -Methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, or L-cysteine.

The preferred examples of amino acid residues that can be substituted with each other are shown below. Amino acid residues contained in the same group can be substituted for each other.
Group A: leucine, isoleucine, norleucine, valine, norvaline, alanine, 2-aminobutanoic acid, methionine, O-methylserine, t-butylglycine, t-butylalanine, cyclohexylalanine Group B: aspartic acid, glutamic acid, isoaspartic acid, Isoglutamic acid, 2-aminoadipic acid, 2-aminosuberic acid Group C: asparagine, glutamine Group D: lysine, arginine, ornithine, 2,4-diaminobutanoic acid, 2,3-diaminopropionic acid Group E: proline, 3 -Hydroxyproline, 4-hydroxyproline Group F: serine, threonine, homoserine Group G: phenylalanine, tyrosine

In the present invention, antibody fragments include, for example, peptides including Fab, F (ab ′) ₂ , Fab ′, scFv, diabody, dsFv and CDR.

Fab is a fragment obtained by treating IgG with papain, a proteolytic enzyme (cleaved at the 224th amino acid residue of the H chain), about half of the N chain side of the H chain and the entire L chain are disulfides. It is an antibody fragment having an antigen binding activity with a molecular weight of about 50,000 bound by binding.

The Fab of the present invention can be obtained by treating a monoclonal antibody that specifically recognizes CD33 and binds to the extracellular region with papain. Alternatively, a Fab may be produced by inserting a DNA encoding the Fab of the antibody into a prokaryotic expression vector or a eukaryotic expression vector, and expressing the vector by introducing the vector into a prokaryotic or eukaryotic organism. it can.

F (ab ′) ₂ was obtained by decomposing the lower part of two disulfide bonds in the hinge region of IgG with pepsin, a proteolytic enzyme, and was constructed by binding two Fab regions at the hinge portion. This fragment has an antigen binding activity with a molecular weight of about 100,000. The F (ab ′) ₂ of the present invention can be obtained by treating a monoclonal antibody specifically recognizing CD33 and binding to its extracellular region with pepsin. Alternatively, Fab ′ described below can be produced by thioether bond or disulfide bond.

Fab ′ is an antibody fragment having an antigen-binding activity having a molecular weight of about 50,000, which is obtained by cleaving the disulfide bond in the hinge region of F (ab ′) ₂ . Fab ′ of the present invention can be obtained by treating F (ab ′) ₂ that specifically recognizes CD33 of the present invention and binds to the extracellular region with a reducing agent such as dithiothreitol. Alternatively, Fab ′ may be produced by inserting a DNA encoding a Fab ′ fragment into a prokaryotic expression vector or a eukaryotic expression vector and introducing the vector into a prokaryotic or eukaryotic organism to express the Fab ′ fragment. it can.

scFv is a VH-P-VL or VL-P-VH polypeptide in which one VH and one VL are linked using an appropriate peptide linker (hereinafter referred to as P), and has antigen-binding activity. It is an antibody fragment having

The scFv of the present invention specifically recognizes the CD33 of the present invention and obtains cDNA encoding the VH and VL of a monoclonal antibody that binds to the extracellular region, constructs a DNA encoding scFv, Can be expressed and produced by inserting the expression vector into a prokaryotic expression vector or eukaryotic expression vector and introducing the expression vector into a prokaryotic or eukaryotic organism.

Diabody is an antibody fragment obtained by dimerizing scFv and is an antibody fragment having a bivalent antigen-binding activity. The bivalent antigen binding activity can be the same, or one can be a different antigen binding activity.

The diabody of the present invention obtains cDNA encoding VH and VL of a monoclonal antibody that specifically recognizes CD33 of the present invention and binds to the extracellular region, and converts the DNA encoding scFv into the amino acid sequence of the peptide linker. Is constructed so that its length is 8 residues or less, and the DNA is inserted into a prokaryotic expression vector or a eukaryotic expression vector, and the expression vector is introduced into a prokaryotic or eukaryotic expression. And can be manufactured.

DsFv refers to a polypeptide in which one amino acid residue in each of VH and VL is substituted with a cysteine residue and bonded via a disulfide bond between the cysteine residues. The amino acid residue to be substituted for the cysteine residue can be selected based on the three-dimensional structure prediction of the antibody according to a known method [Protein Engineering, 7, 697 (1994)].

The dsFv of the present invention specifically recognizes the CD33 of the present invention and obtains cDNA encoding the VH and VL of the monoclonal antibody that binds to the extracellular region, constructs a DNA encoding dsFv, Can be expressed and produced by inserting the expression vector into a prokaryotic expression vector or eukaryotic expression vector and introducing the expression vector into a prokaryotic or eukaryotic organism.

The peptide containing CDR is configured to contain at least one region of CDR of VH or VL. Peptides containing multiple CDRs can be linked directly or via a suitable peptide linker. The peptide comprising the CDR of the present invention constructs DNA encoding the CDRs of VH and VL of the monoclonal antibody that specifically recognizes CD33 of the present invention and binds to the extracellular region, and uses the DNA for prokaryotes It can be expressed and produced by inserting into an expression vector or eukaryotic expression vector and introducing the expression vector into a prokaryotic or eukaryotic organism. Moreover, the peptide containing CDR can also be manufactured by chemical synthesis methods, such as Fmoc method or tBoc method.

The monoclonal antibody of the present invention specifically recognizes the extracellular region of CD33 of the present invention and binds to the extracellular region with a radioisotope, a low molecular drug, a high molecular drug, It includes a derivative of an antibody in which a protein or an antibody drug is chemically or genetically bound.

The derivative of the antibody in the present invention specifically recognizes the extracellular region of CD33 of the present invention and binds to the extracellular region, or the N-terminal side or C-terminal of the H chain or L chain thereof. On the side, an appropriate substituent or side chain in the antibody or fragment thereof, or a sugar chain in the monoclonal antibody or fragment thereof, etc., radioisotope, low molecular weight drug, high molecular weight drug, immunostimulant, protein or It can be produced by combining antibody drugs and the like by chemical methods [Introduction to Antibody Engineering, Jinshokan (1994)].

Further, a DNA encoding a monoclonal antibody or a fragment thereof that specifically recognizes the extracellular region of CD33 of the present invention and binds to the extracellular region is linked to a DNA encoding a protein or antibody drug to be bound. And then inserted into an expression vector, introduced into an appropriate host cell, and expressed by a genetic engineering technique.

Examples of the radioisotope include ¹³¹ I, ¹²⁵ I, ⁹⁰ Y, ⁶⁴ Cu, ⁹⁹ Tc, ⁷⁷ Lu, and ²¹¹ At. The radioisotope can be directly bound to the antibody by the chloramine T method or the like. Further, a substance that chelates a radioisotope may be bound to the antibody. Examples of the chelating agent include 1-isothiocyanate benzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA).

Examples of low molecular weight drugs include alkylating agents, nitrosourea agents, antimetabolites, antibiotics, plant alkaloids, topoisomerase inhibitors, hormone therapy agents, hormone antagonists, aromatase inhibitors, P glycoprotein inhibitors, platinum Anticancer agents such as complex derivatives, M phase inhibitors or kinase inhibitors [Clinical Oncology, Cancer and Chemotherapy (1996)], steroidal agents such as hydrocortisone or prednisone, non-steroidal agents such as aspirin or indomethacin, gold thiomalate or Anti-inflammatory agents such as penicillamine, immunosuppressive agents such as cyclophosphamide or azathioprine, or antihistamines such as chlorpheniramine maleate or clemacitine [Inflammation and anti-inflammatory therapy, Ishiyaku Publishing Co., Ltd. ( (1982) And the like.

Examples of anticancer agents include amifostine (ethiol), cisplatin, dacarbazine (DTIC), dactinomycin, mechlorethamine (nitrogen mustard), streptozocin, cyclophosphamide, ifosfamide, carmustine (BCNU), lomustine (CCNU), doxorubicin (Adriamycin), epirubicin, gemcitabine (gemzar), daunorubicin, procarbazine, mitomycin, cytarabine, etoposide, methotrexate, 5-fluorouracil, fluorouracil, vinblastine, vincristine, bleomycin, daunomycin, pepromycin, estramustine, paclitaxel, paclitaxel, paclitaxel Taxotea), aldesleukin, asparaginase, buoy Ruphan, carboplatin, oxaliplatin, nedaplatin, cladribine, camptothecin, 10-hydroxy-7-ethyl-camptothecin (SN38), floxuridine, fludarabine, hydroxyurea, ifosfamide, idarubicin, mesna, irinotecan (CPT-11), nogitane, Mitoxantrone, topotecan, leuprolide, megestrol, melphalan, mercaptopurine, hydroxycarbamide, pricamycin, mitotan, pegsparagase, pentostatin, pipobroman, streptozocin, tamoxifen, goserelin, leuprorenin, flutamide, teniposide, test Lactone, thioguanine, thiotepa, uracil mustard, vinorelbine, chlorambucil, hydrocortisone, Donisolone, methylprednisolone, vindesine, nimustine, semstin, capecitabine, tomdex, azacitidine, UFT, oxaloplatin, gefitinib (Iressa), imatinib (STI571), erlotinib, FMS-like tyrosinek inhibitor facotr receptor (VEGFR) inhibitor, fibroblast growth factor receptor (FGFR) inhibitor, epidermal growth factor receptor (EGFR) inhibitors such as Iressa, Tarceva, methoxyamino 17 A Musacrine, all-trans retinoic acid, thalidomide, anastrozole, fadrozole, letrozole, exemestane, gold thiomalate, D-penicillamine, bucillamine, azathioprine, mizoribine, cyclosporine, rapamycin, hydrocortisone, bexarotene (targretin), tamoxifen, dexamethasone , Estrogens, anastrozole (Arimidex), leupurine, aspirin, indomethacin, celecoxib, azathioprine, penicillamine, gold thiomalate, chlorpheniramine maleate, chloropheniramine, clemacitine, tretinoin, bexarotene, arsenic, bortezomib, allopurinol, cali Keamycin, Ibritumomab tiuxetan, Targretin, Ozoga Emissions, clarithromycin machine, leucovorin, Ifasufamido, ketoconazole, aminoglutethimide, suramin, methotrexate, maytansinoid, or derivatives, and the like.

Examples of a method for binding a low molecular weight drug and an antibody include, for example, a method of binding a drug and an amino group of an antibody via glutaraldehyde, or a drug amino group and an antibody carboxyl group via a water-soluble carbodiimide. And the like.

Examples of the polymer drug include polyethylene glycol (hereinafter sometimes referred to as PEG), albumin, dextran, polyoxyethylene, styrene maleic acid copolymer, polyvinyl pyrrolidone, pyran copolymer, or hydroxypropyl methacrylamide. It is done. By binding these macromolecular compounds to antibodies or fragments thereof, (1) improved stability against various chemical, physical or biological factors, (2) significant increase in blood half-life, ( 3) Effects such as loss of immunogenicity or suppression of antibody production are expected [Bioconjugate Pharmaceuticals, Yodogawa Shoten (1993)]. For example, as a method of linking PEG and an antibody, a method of reacting with a PEGylation modifying reagent and the like can be mentioned [Bioconjugate Pharmaceutical, Yodogawa Shoten (1993)].

Examples of the PEGylation modifying reagent include a modifying agent for ε-amino group of lysine (Japanese Unexamined Patent Publication No. 61-178926), a modifying agent for carboxyl group of aspartic acid and glutamic acid (Japanese Unexamined Patent Publication No. Sho 56). No. -23587), or a modifier for guanidino group of arginine (Japanese Patent Laid-Open No. 2-117920).

The immunostimulant may be a natural product known as an immunoadjuvant, and specific examples thereof include β (1 → 3) glucan (lentinan, schizophyllan), α galactosylceramide and the like. It is done.

Examples of proteins include cytokines or growth factors that activate immunocompetent cells such as NK cells, macrophages, or neutrophils, or toxin proteins.

Examples of cytokines or growth factors include interferon (hereinafter referred to as INF) -α, INF-β, INF-γ, interleukin (hereinafter referred to as IL) -2, IL-12, IL-15, IL- 18, IL-21, IL-23, granulocyte colony stimulating factor (G-CSF), granulocyte / macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF) and the like.

Examples of the toxin protein include ricin and diphtheria toxin, and also include protein toxins in which mutations are introduced into the protein in order to regulate toxicity.

Examples of the antibody drug include an antigen against which apoptosis is induced by antibody binding, an antigen associated with tumor pathogenesis or an antigen that regulates immune function, and an antibody against an antigen involved in angiogenesis of a lesion site.

Antigens that induce apoptosis by antibody binding include, for example, CD4, CD19, CD20, CD21, CD22, CD23, CD24, CD37, CD53, CD72, CD73, CD74, CDw75, CDw76, CD77, CDw78, CD79a, CD79b , CD80 (B7.1), CD81, CD82, CD83, CDw84, CD85, CD86 (B7.2), human leukocyte antigen (HLA) -Class II, or Epidermal Growth Factor Receptor (EGFR).

Examples of antigens involved in tumor pathogenesis or antibodies that regulate immune functions include CD4, CD40, CD40 ligand, B7 family molecules (CD80, CD86, CD274, B7-DC, B7-H2, B7-H3, Or B7-H4), a ligand of a B7 family molecule (CD28, CTLA-4, ICOS, PD-1, or BTLA), OX-40, OX-40 ligand, CD137, a tumor necrosis factor (TNF) receptor family molecule ( DR4, DR5, TNFR1, or TNFR2), TNF-related apoptosis-inducing ligand receptor (TRAIL) family molecule, TRAIL family molecule receptor family (TRAIL-R1, RAIL-R2, TRAIL-R3, or TRAIL-R4), receptor activator of nuclear factor kappa B ligand (RANK), RANK ligand, CD25, folate receptor 4, cytokine [IL-1α, IL-1β, IL-4, IL-5, IL-6, IL-10, IL-13, transforming growth factor (TGF) β, or TNFα, etc.], receptors for these cytokines, chemokines (SLC, ELC, I-309, TARC, MDC, Or CTACK), or receptors for these chemokines.

Examples of antigens for antibodies that inhibit angiogenesis of a lesion site include, for example, vascular electrical growth factor (VEGF), anangiopoietin, fibroblast growth factor (FGF), EGF, and platelet-derived GF. IGF), erythropoietin (EPO), TGFβ, IL-8, Ephilin, SDF-1, or a receptor thereof.

A fusion antibody of a monoclonal antibody or a fragment thereof that binds to CD33 and a protein or antibody drug is linked to a cDNA encoding the monoclonal antibody or the fragment, and a cDNA encoding the protein or the antibody drug is linked to encode the fusion antibody. The fusion antibody can be produced by constructing a DNA to be expressed, inserting the DNA into a prokaryotic or eukaryotic expression vector, and introducing the expression vector into a prokaryotic or eukaryotic organism.

When the derivative of the antibody is used as a detection method, a quantification method, a detection reagent, a quantification reagent or a diagnostic agent, the extracellular region of CD33 of the present invention is specifically recognized and binds to the extracellular region. Examples of the drug that binds to the monoclonal antibody or a fragment thereof include a label used in a usual immunological detection or measurement method.

Examples of the label include an enzyme such as alkaline phosphatase, peroxidase or luciferase, a luminescent substance such as acridinium ester or lophine, or a fluorescent substance such as fluorescein isothiocyanate (FITC) or tetramethylrhodamine isothiocyanate (RITC). Can be mentioned.

The present invention also relates to a therapeutic agent for a disease involving CD33-positive cells that specifically recognize the extracellular region of CD33 and contain a monoclonal antibody or fragment thereof that binds to the extracellular region as an active ingredient.

The disease involving CD33-positive cells may be any disease involving cells expressing CD33, and examples thereof include cancer, autoimmune diseases, and allergic diseases.

Examples of CD33 positive cells include monocytes, macrophages, granulocytes and the like which are CD33 positive cells. Since monocytes and neutrophils expressing CD33 are cells involved in the removal of foreign substances such as microorganisms, diseases involving foreign body phagocytosis can be mentioned.

Examples of cancer include blood cancer, breast cancer, uterine cancer, colon cancer, esophageal cancer, stomach cancer, ovarian cancer, lung cancer, kidney cancer, rectal cancer, thyroid cancer, cervical cancer, small intestine cancer, prostate cancer or pancreatic cancer. Preferably, hematological cancer, esophageal cancer, stomach cancer, colon cancer, liver cancer or prostate cancer can be mentioned.

Examples of hematological cancers include AML, anaplastic large cell lymphoma (ALCL), acute lymphocytic leukemia (ALL), myelodysplastic syndrome, MD, and other myelodysplastic syndromes. , Multiple myeloma, Hodgkin lymphoma, or non-Hodgkin lymphoma.

Examples of the autoimmune disease include rheumatoid arthritis, psoriasis, Crohn's disease, ankylosing spondylitis, multiple sclerosis, type I diabetes, hepatitis, myocarditis, Sjogren's syndrome, or rejection after transplantation.

Examples of allergic diseases include acute or chronic airway hypersensitivity, bronchial asthma, atopic dermatitis, or allergic rhinitis.

The therapeutic agent of the present invention contains the above-described monoclonal antibody of the present invention or a fragment thereof as an active ingredient.

The therapeutic agent containing the antibody of the present invention or a fragment thereof may contain only the antibody or the fragment as an active ingredient, but usually together with one or more pharmacologically acceptable carriers. It is desirable to mix and provide as a pharmaceutical formulation produced by any method known in the art of pharmaceutical sciences.

It is preferable to use the administration route that is most effective for treatment, and examples include oral administration, oral administration, intratracheal, rectal, subcutaneous, intramuscular or intravenous administration, preferably Intravenous administration is mentioned.

Examples of administration forms include sprays, capsules, tablets, powders, granules, syrups, emulsions, suppositories, injections, ointments, or tapes.

The dose or frequency of administration varies depending on the intended therapeutic effect, administration method, treatment period, age, weight, etc., but is usually 10 μg / kg to 20 mg / kg per day for an adult.

Furthermore, the present invention relates to a method for immunological detection or measurement of CD33, which contains as an active ingredient a monoclonal antibody or a fragment thereof that specifically recognizes the extracellular region of CD33 and binds to the extracellular region. The present invention relates to a reagent for immunological detection or measurement, an immunological detection or measurement method for cells expressing CD33, and a diagnostic agent for a disease involving CD33-positive cells.

In the present invention, any known method may be used as a method for detecting or measuring the amount of CD33. Examples include immunological detection or measurement methods.

The immunological detection or measurement method is a method of detecting or measuring the amount of antibody or the amount of antigen using a labeled antigen or antibody. Examples of immunological detection or measurement methods include radiolabeled immunoassay (RIA), enzyme immunoassay (EIA or ELISA), fluorescence immunoassay (FIA), luminescence immunoassay (Western immunoassay), Western Examples include blotting or physicochemical techniques.

By detecting or measuring cells expressing CD33 using the monoclonal antibody of the present invention or a fragment thereof, a disease associated with CD33 can be diagnosed.

Known immunological detection methods can be used for detection of cells expressing the polypeptide, but immunoprecipitation, fluorescent cell staining, immunohistochemical staining, immunohistological staining, etc. Preferably used. Moreover, fluorescent antibody staining methods such as FMAT8100HTS system (Applied Biosystems) can also be used.

Examples of biological samples to be detected or measured for CD33 in the present invention include CD33 expression such as tissue cells, bone marrow cells, bone marrow fluid, blood, plasma, serum, pancreatic juice, urine, stool, tissue fluid, or culture fluid. The cell is not particularly limited as long as it may contain the cells.

The diagnostic agent containing the monoclonal antibody or fragment thereof of the present invention may contain a reagent for conducting an antigen-antibody reaction and a reagent for detecting the reaction, depending on the target diagnostic method. Examples of the reagent for performing the antigen-antibody reaction include a buffer or a salt. Examples of the reagent for detection include a labeled secondary antibody that recognizes the monoclonal antibody or a fragment thereof, or a reagent used for usual immunological detection or measurement methods such as a substrate corresponding to the label.

The antibody production method, disease treatment method, and disease diagnosis method of the present invention will be specifically described below.

1. Method for Producing Monoclonal Antibody (1) Preparation of antigen Cells expressing CD33 or CD33 as an antigen are expressed in E. coli, yeast, insect cells, or animal cells using an expression vector containing cDNA encoding CD33 full length or a partial length thereof. It can be obtained by introducing into the above. In addition, CD33 can be purified and obtained from cultured human tumor cells, human tissues, etc. that express CD33 in large amounts. Further, the cultured tumor cells or the tissue can be used as an antigen as it is. Furthermore, a synthetic peptide having a partial sequence of CD33 can be prepared by a chemical synthesis method such as the Fmoc method or the tBoc method and used as an antigen.

The CD33 used in the present invention is described in Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989) or Current Protocols in MolecularJoule7, 198 For example, the DNA encoding CD33 can be expressed in a host cell and produced by the following method.

First, a recombinant vector is prepared by inserting a full-length cDNA containing a portion encoding CD33 downstream of a promoter of an appropriate expression vector. Instead of the full-length cDNA, a DNA fragment of an appropriate length containing a polypeptide-encoding portion prepared based on the full-length cDNA may be used. Next, a transformant producing a polypeptide can be obtained by introducing the obtained recombinant vector into a host cell suitable for the recombinant vector.

Any expression vector can be used as long as it can autonomously replicate in the host cell to be used or can be integrated into the chromosome, and contains an appropriate promoter at a position where DNA encoding the polypeptide can be transcribed. Can do.

As the host cell, any microorganism that belongs to the genus Escherichia such as Escherichia coli, yeast, insect cells, or animal cells can be used so long as it can express the target gene.

When a prokaryote such as E. coli is used as a host cell, the recombinant vector is capable of autonomous replication in a prokaryote and at the same time contains a promoter, a ribosome binding sequence, DNA containing a CD33-encoding portion, and a transcription termination sequence. It is preferable that it is a vector containing. The recombinant vector does not necessarily require a transcription termination sequence, but it is preferable to place the transcription termination sequence immediately below the structural gene. Furthermore, the recombinant vector may contain a gene that controls the promoter.

As the recombinant vector, it is preferable to use a plasmid in which the distance between the Shine-Dalgarno sequence (also referred to as SD sequence), which is a ribosome binding sequence, and the start codon is adjusted to an appropriate distance (eg, 6 to 18 bases).

In addition, the base sequence of the DNA encoding CD33 can be substituted so that the codon is optimal for expression in the host, thereby improving the production rate of the target CD33. .

Any expression vector can be used as long as it can function in the host cell to be used. For example, pBTrp2, pBTac1, pBTac2 (above, Roche Diagnostics), pKK233-2 (Pharmacia), pSE280 (Invitrogen), pGEMEX-1 (Promega), pQE-8 (Qiagen) PKYP10 (Japanese Unexamined Patent Publication No. 58-110600), pKYP200 [Agricultural Biological Chemistry, 48, 669 (1984)], pLSA1 [Agric. Biol. Chem. , 53, 277 (1989)], pGEL1 [Proc. Natl. Acad. Sci. USA, 82, 4306 (1985)], pBluescript II SK (-) (manufactured by Stratagene), pTrs30 [prepared from E. coli JM109 / pTrS30 (FERM BP-5407)], pTrs32 [E. coli JM109 / pTrS32 (FERM BP-5408). ) Prepared], pGHA2 [prepared from E. coli IGHA2 (FERM BP-400), Japanese Unexamined Patent Publication No. 60-221091], pGKA2 [prepared from E. coli IGKA2 (FERM BP-6798), Japanese Unexamined Patent Publication 60- 221091], pTerm2 (US Pat. No. 4,686,191, US Pat. No. 4,939,094, US Pat. No. 5,160,735), pSupex, pUB110, pTP5, pC194, pEG400 [J. Bacteriol. 172, 2392 (1990)], pGEX (Pharmacia), pET system (Novagen), or pME18SFL3.

As the promoter, any promoter can be used as long as it can function in the host cell to be used. For example, promoters derived from Escherichia coli or phage, such as trp promoter (Ptrp), lac promoter, PL promoter, PR promoter, or T7 promoter can be mentioned. In addition, artificially designed and modified promoters such as a tandem promoter, tac promoter, lacT7 promoter, or let I promoter in which two Ptrps are connected in series can also be used.

Examples of host cells include E. coli XL1-Blue, E. coli XL2-Blue, E. coli DH1, E. coli MC1000, E. coli KY3276, E. coli W1485, E. coli JM109, E. coli HB101, E. coli No. 49, E. coli W3110, E. coli NY49, or E. coli DH5α.

Any method can be used for introducing a recombinant vector into a host cell as long as it is a method for introducing DNA into the host cell to be used. For example, a method using calcium ions [Proc. Natl. Acad. Sci. USA, 69, 2110 (1972), Gene, 17, 107 (1982), Molecular & General Genetics, 168, 111 (1979)].

When animal cells are used as a host, any expression vector can be used as long as it can function in animal cells. For example, pcDNAI, pcDM8 (manufactured by Funakoshi), pAGE107 [Japanese Unexamined Patent Publication No. 3-22979; Cytotechnology, 3, 133 (1990)], pAS3-3 (Japanese Unexamined Patent Publication No. 2227705), pCDM8 [ Nature, 329, 840 (1987)], pcDNAI / Amp (Invitrogen), pcDNA3.1 (Invitrogen), pREP4 (Invitrogen), pAGE103 [J. Biochemistry, 101, 1307 (1987)], pAGE210, pME18SFL3, or pKANTEX93 (International Publication No. 97/10354).

Any promoter can be used as long as it can function in animal cells. Examples include cytomegalovirus (CMV) immediate early (IE) gene promoter, SV40 early promoter, retroviral promoter, metallothionein promoter, heat shock promoter, SRα promoter, or Moloney murine leukemia virus promoter or enhancer. . In addition, an enhancer of human CMV IE gene may be used together with a promoter.

Examples of host cells include human leukemia cells Namalwa cells, monkey cells COS cells, Chinese hamster ovary cells CHO cells (Journal of Experimental Medicine, 108, 945 (1958); Proc. Natl. Acad. Sci. USA, 60, 1275 (1968); Genetics, 55, 513 (1968); Chromoma, 41, 129 (1973); Methods in Cell Science, 18, 115 (1996); Radiation Research, 148, 260 (1997); Proc. Natl. Sci.USA, 77, 4216 (1980); Proc.Natl.Acad.Sci., 60,127 (1968); Cell, 6, 121 (1975); Molecular Cell Genetics, Appendix I, II (pp. 883-900);), CHO / DG44, CHO-K1 (ATCC CCL-61), DUkXB11 (ATCC CCL- 9096), Pro-5 (ATCC CCL-1781), CHO-S (Life Technologies, Cat # 11619), Pro-3, rat myeloma cells YB2 / 3HL. P2. G11.16 Ag. 20 (also referred to as YB2 / 0), mouse myeloma cell NSO, mouse myeloma cell SP2 / 0-Ag14, Syrian hamster cell BHK or HBT5637 (Japanese Unexamined Patent Publication No. 63-000299), and the like.

As a method for introducing a recombinant vector into a host cell, any method can be used as long as it introduces DNA into animal cells. For example, electroporation method [Cytotechnology, 3, 133 (1990)], calcium phosphate method (Japanese Patent Laid-Open No. 2-227075), or lipofection method [Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)].

Culturing a transformant derived from a microorganism or animal cell having a recombinant vector into which DNA encoding CD33 obtained as described above is incorporated in a medium, and producing and accumulating the CD33 in the culture, By collecting from the culture, CD33 can be produced. The method of culturing the transformant in a medium can be performed according to a usual method used for culturing a host.

When expressed in cells derived from eukaryotes, CD33 with an added sugar or sugar chain can be obtained.

When culturing a microorganism transformed with a recombinant vector using an inducible promoter, an inducer may be added to the medium as necessary. For example, when cultivating a microorganism transformed with a recombinant vector using the lac promoter, cultivate a microorganism transformed with isopropyl-β-D-thiogalactopyranoside or the like using a recombinant vector using the trp promoter. In this case, indole acrylic acid or the like may be added to the medium.

Examples of a medium for culturing a transformant obtained using an animal cell as a host include, for example, a commonly used RPMI 1640 medium [The Journal of the American Medical Association, 199, 519 (1967)], Eagle's MEM medium [Science]. , 122, 501 (1952)], Dulbecco's modified MEM medium [Virology, 8, 396 (1959)], 199 medium [Proc. Soc. Exp. Biol. Med. 73, 1 (1950)], Iscove's Modified Dulbecco's Medium (IMDM) medium, or a medium obtained by adding fetal bovine serum (FBS) or the like to these mediums. The culture is usually carried out for 1 to 7 days under conditions such as pH 6 to 8, 30 to 40 ° C., and 5% CO ₂ . Moreover, you may add antibiotics, such as kanamycin and penicillin, to a culture medium as needed during culture | cultivation.

As an expression method of the gene encoding CD33, for example, methods such as secretory production or fusion protein expression other than direct expression [Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989)] can be mentioned. .

Examples of the production method of CD33 include a method of producing in a host cell, a method of secreting it outside the host cell, and a method of producing it on the host cell outer membrane. The host cell to be used and the structure of CD33 to be produced are exemplified. By changing, an appropriate method can be selected.

When CD33 is produced in the host cell or on the host cell outer membrane, the method of Paulson et al. [J. Biol. Chem. , 264, 17619 (1989)], the method of Lowe et al. [Proc. Natl. Acad. Sci. USA, 86, 8227 (1989), Genes Develop. , 4, 1288 (1990)], and actively secreting CD33 outside the host cell by using the method described in Japanese Patent Application Laid-Open No. 05-336963 or International Publication No. 94/23021. Can do.

In addition, the production amount of CD33 can be increased by using a gene amplification system using a dihydrofolate reductase gene or the like (Japanese Patent Laid-Open No. 2-227075).

The obtained CD33 can be isolated and purified as follows, for example.

When CD33 is expressed in a dissolved state in the cells, the cells are collected by centrifugation after culturing, suspended in an aqueous buffer solution, and then used with an ultrasonic crusher, a French press, a Manton Gaurin homogenizer, or a dynomill. The cells are disrupted to obtain a cell-free extract. From the supernatant obtained by centrifuging the cell-free extract, an ordinary protein isolation and purification method, that is, a solvent extraction method, a salting-out method using ammonium sulfate, a desalting method, a precipitation method using an organic solvent, diethylamino Anion exchange chromatography using a resin such as ethyl (DEAE) -Sepharose, DIAION HPA-75 (manufactured by Mitsubishi Chemical), cation exchange chromatography using a resin such as S-Sepharose FF (manufactured by Pharmacia) , Methods such as hydrophobic chromatography using resins such as butyl sepharose and phenyl sepharose, gel filtration using molecular sieve, affinity chromatography, chromatofocusing, or electrophoresis such as isoelectric focusing Can be used alone or in combination to obtain purified preparations. Can.

When CD33 is expressed by forming an insoluble substance in the cell, the cell is recovered and crushed in the same manner as described above, and centrifuged to collect the insoluble substance of CD33 as a precipitate fraction. The recovered insoluble matter of CD33 is solubilized with a protein denaturant. The solubilized solution is diluted or dialyzed to return the CD33 to a normal three-dimensional structure, and then a purified polypeptide preparation can be obtained by the same isolation and purification method as described above.

When a derivative such as CD33 or a modified sugar thereof is secreted extracellularly, the derivative such as CD33 or a modified sugar thereof can be recovered from the culture supernatant. A soluble fraction can be obtained by treating the culture by a method such as centrifugation as described above, and a purified preparation can be obtained from the soluble fraction by using the same isolation and purification method as described above. it can.

Purification can be easily performed by adding an appropriate peptide to CD33. For example, by adding the Fc region of immunoglobulin to CD33 and using CD33-Fc fusion protein secreted extracellularly using protein A or protein G, a purified preparation can be obtained.

Further, CD33 used in the present invention can also be produced by a chemical synthesis method such as the Fmoc method or the tBoc method. In addition, chemical synthesis using peptide synthesizers such as Advanced Chemtech, Perkin Elmer, Pharmacia, Protein Technology Instrument, Synthecel-Vega, Perceptive, or Shimadzu It can also be synthesized.

(2) Immunization of animals and preparation of antibody-producing cells for fusion 3 to 20-week-old animals such as mice, rats or hamsters are immunized with the antigen obtained in (1), and the spleen, lymph nodes, Collect antibody-producing cells in peripheral blood. In addition, CD33 knockout mice can be used as immunized animals when the immunogenicity is low and the above-mentioned animals do not have a sufficient increase in antibody titer.

Immunization is performed by administering the antigen subcutaneously, intravenously or intraperitoneally to the animal together with an appropriate adjuvant such as Freund's complete adjuvant or aluminum hydroxide gel and pertussis vaccine. When the antigen is a partial peptide, a conjugate with a carrier protein such as BSA (bovine serum albumin) or KLH (Keyhole limpet hemocyanin) is prepared and used as an immunogen.

The antigen is administered 5 to 10 times every 1 to 2 weeks after the first administration. Three to seven days after each administration, blood is collected from the fundus venous plexus, and the antibody titer of the serum is measured using an enzyme immunoassay [Antibodies-A Laboratory Manual, Cold Spring Harbor Laboratory (1988)]. An animal whose serum shows a sufficient antibody titer against the antigen used for immunization is used as a source of antibody producing cells for fusion.

3 to 7 days after the final administration of the antigen, tissues containing antibody-producing cells such as the spleen are removed from the immunized animal, and antibody-producing cells are collected. When spleen cells are used, the spleen is shredded and loosened, and then centrifuged, and the erythrocytes are removed to obtain antibody producing cells for fusion.

(3) Preparation of myeloma cells As myeloma cells, cell lines obtained from mice are used. For example, 8-azaguanine resistant mouse (BALB / c-derived) myeloma cell line P3-X63Ag8-U1 (P3-U1) [Current Topics in Microbiology and Immunology, 18, 1 (1978)], P3-NS1 / 1-Ag41 (NS-1) [European J. et al. Immunology, 6, 511 (1976)], SP2 / 0-Ag14 (SP-2) [Nature, 276, 269 (1978)], P3-X63-Ag8653 (653) [J. Immunology, 123, 1548 (1979)], or P3-X63-Ag8 (X63) [Nature, 256, 495 (1975)].

The myeloma cells are passaged in normal medium [RPMI 1640 medium supplemented with glutamine, 2-mercaptoethanol, gentamicin, FBS, and 8-azaguanine] and passaged to normal medium 3-4 days prior to cell fusion. On the day of fusion, secure a cell count of 2 × 10 ⁷ or more.

(4) Cell fusion and preparation of monoclonal antibody-producing hybridoma The antibody-producing cells for fusion obtained in (2) and the myeloma cells obtained in (3) were treated with Minimum Essential Medium (MEM) medium or PBS (disodium phosphate 1. 83 g, monopotassium phosphate 0.21 g, sodium chloride 7.65 g, distilled water 1 liter, pH 7.2), and the number of cells becomes fusion antibody-producing cells: myeloma cells = 5 to 10: 1 After mixing and centrifuging, remove the supernatant.

After loosening the precipitated cells, a mixture of polyethylene glycol-1000 (PEG-1000), MEM medium and dimethyl sulfoxide is added at 37 ° C. with stirring. Add 1-2 mL of MEM medium several times every 1-2 minutes, and then add MEM medium to a total volume of 50 mL. After centrifugation, remove the supernatant. After loosening the precipitated cell group, the antibody-producing cells for fusion are gently suspended in a HAT medium [normal medium supplemented with hypoxanthine, thymidine, and aminopterin]. This suspension is cultured for 7-14 days at 37 ° C. in a 5% CO ₂ incubator.

After culturing, a part of the culture supernatant is extracted, and a cell group that reacts with an antigen containing CD33 and does not react with an antigen not containing CD33 is selected by a hybridoma selection method such as a binding assay described later. Next, cloning was repeated twice by the limiting dilution method (first time was HT medium (medium obtained by removing aminopterin from HAT medium), second time normal medium was used), and a stable and strong antibody titer was observed. One is selected as a monoclonal antibody-producing hybridoma.

(5) Preparation of Purified Monoclonal Antibody 8- to 10-week-old mice or nude mice treated with pristane [2,6,10,14-tetramethylpentadecane (Pristane) 0.5 ml intraperitoneally and bred for 2 weeks] Then, the monoclonal antibody-producing hybridoma obtained in (4) is injected intraperitoneally. In 10 to 21 days, the hybridoma becomes ascites tumor. Ascites was collected from this mouse, centrifuged to remove solids, salted out with 40-50% ammonium sulfate, and purified by caprylic acid precipitation, DEAE-Sepharose column, Protein A-column or gel filtration column. The IgG or IgM fractions are collected and used as a purified monoclonal antibody.

In addition, the monoclonal antibody-producing hybridoma obtained in (4) is cultured in an RPMI1640 medium supplemented with 10% FBS, the supernatant is removed by centrifugation, and the suspension is suspended in a Hybridoma SFM medium and cultured for 3 to 7 days. . The obtained cell suspension is centrifuged, and purified using a protein A-column or protein G-column from the resulting supernatant, and the IgG fraction is collected to obtain a purified monoclonal antibody. In addition, 5% Digo GF21 can also be added to the Hybridoma SFM medium.

The antibody subclass is determined by enzyme immunoassay using a sub-clustering kit. The amount of protein is calculated from the Raleigh method or absorbance at 280 nm.

(6) Selection of monoclonal antibody The monoclonal antibody is selected by a binding assay by the enzyme immunoassay shown below and a kinetic analysis by Biacore.

(6-a) Binding assay As an antigen, for example, a gene-transferred cell obtained by introducing an expression vector containing cDNA encoding CD33 obtained in (1) into Escherichia coli, yeast, insect cells or animal cells, Recombinant protein, or purified polypeptide or partial peptide obtained from human tissue. When the antigen is a partial peptide, a conjugate with a carrier protein such as BSA or KLH is prepared and used.

After dispensing the antigen to a plate such as a 96-well plate and immobilizing the antigen, a test substance such as serum, hybridoma culture supernatant or purified monoclonal antibody is dispensed as the first antibody and allowed to react. After thoroughly washing with PBS or PBS-Tween or the like, an anti-immunoglobulin antibody labeled with biotin, an enzyme, a chemiluminescent substance, a radiation compound or the like is dispensed and reacted as a second antibody. After thoroughly washing with PBS-Tween, a reaction is performed according to the labeling substance of the second antibody, and a monoclonal antibody that specifically reacts with the immunogen is selected.

In addition, a monoclonal antibody that competes with the anti-CD33 monoclonal antibody of the present invention and binds to CD33 can be obtained by adding a test antibody to the above-described binding assay system for reaction. That is, by screening for an antibody that inhibits the binding of the monoclonal antibody when the test antibody is added, a monoclonal antibody that competes with the acquired monoclonal antibody for binding to the CD33 extracellular region can be obtained.

Furthermore, the monoclonal antibody that binds to the extracellular region of CD33 of the present invention recognizes and binds to the same epitope as the epitope that identifies the epitope of the antibody obtained by the above-described binding assay system, A partial synthetic peptide of the identified epitope, or a synthetic peptide mimicking the three-dimensional structure of the epitope can be prepared and immunized.

(6-b) Kinetics analysis by Biacore Using Biacore T100, kinetics in the binding between an antigen and a test substance is measured, and the result is analyzed by analysis software attached to the instrument. After immobilizing the anti-mouse IgG antibody on the sensor chip CM5 by the amine coupling method, a test substance such as a hybridoma culture supernatant or a purified monoclonal antibody is allowed to flow, and an appropriate amount is bound. Measure dissociation. Using the software attached to the device, the obtained data is subjected to kinetic analysis using a 1: 1 binding model, and various parameters are acquired.

Alternatively, human CD33 is immobilized on a sensor chip by, for example, an amine coupling method, and then purified monoclonal antibodies having a plurality of known concentrations are flowed to measure binding and dissociation. The obtained data is subjected to kinetic analysis using a software that is included with the device, using a binding binding model, and various parameters are acquired.

2. Production of Recombinant Antibody As an example of producing a recombinant antibody, a method for producing a human chimeric antibody and a human CDR-grafted antibody is shown below.
(1) Construction of Recombinant Antibody Expression Vector A recombinant antibody expression vector is an animal cell expression vector in which DNAs encoding human antibodies CH and CL are incorporated, and the animal cell expression vector is human. It can be constructed by cloning DNAs encoding antibody CH and CL, respectively.

Any human antibody CH and CL can be used for the C region of a human antibody. For example, γ1 subclass CH and κ class CL of human antibodies are used. Although cDNA is used for DNA encoding CH and CL of human antibodies, chromosomal DNA consisting of exons and introns can also be used. Any animal cell expression vector can be used as long as it can incorporate and express a gene encoding the C region of a human antibody.

For example, pAGE107 [Cytotechnol. , 3, 133 (1990)], pAGE103 [J. Biochem. , 101, 1307 (1987)], pHSG274 [Gene, 27, 223 (1984)], pKCR [Proc. Natl. Acad. Sci. USA, 78, 1527 (1981)], pSG1bd2-4 [Cytotechnol. , 4, 173 (1990)], or pSE1UK1Sed1-3 [Cytotechnol. , 13, 79 (1993)].

Among the expression vectors for animal cells, examples of promoters and enhancers include SV40 early promoter [J. Biochem. , 101, 1307 (1987)], Moloney murine leukemia virus LTR [Biochem. Biophys. Res. Commun. , 149, 960 (1987)], or an immunoglobulin heavy chain promoter [Cell, 41, 479 (1985)] and an enhancer [Cell, 33, 717 (1983)].

Recombinant antibody expression vectors balance the ease of construction of recombinant antibody expression vectors, the ease of introduction into animal cells, and the balance of expression levels of antibody H and L chains in animal cells. In view of the above, a vector for expressing a recombinant antibody of a type (tandem type) in which the antibody H chain and L chain are present on the same vector [J. Immunol. Methods, 167, 271 (1994)], but it is also possible to use a type in which the antibody H chain and L chain are present on separate vectors. As a tandem recombinant antibody expression vector, pKANTEX93 (International Publication No. 97/10354), pEE18 [Hybridoma, 17, 559 (1998)] or the like is used.

(2) Obtaining cDNA encoding the V region of an antibody derived from a non-human animal and analyzing the amino acid sequence Obtaining cDNA encoding the VH and VL of the non-human antibody and analyzing the amino acid sequence should be performed as follows. Can do.

MRNA is extracted from hybridoma cells producing non-human antibodies, and cDNA is synthesized. The synthesized cDNA is cloned into a vector such as a phage or a plasmid to prepare a cDNA library. Recombinant phages or recombinant plasmids having cDNA encoding VH or VL are isolated from the library using DNA encoding the C region portion or V region portion of the mouse antibody as a probe. The entire base sequence of VH or VL of the target mouse antibody on the recombinant phage or recombinant plasmid is determined, respectively, and the total amino acid sequence of VH or VL is estimated from the base sequence.

As a non-human animal for producing a hybridoma cell producing a non-human antibody, a mouse, rat, hamster, rabbit or the like is used, but any animal can be used as long as it can produce a hybridoma cell. .

For preparation of total RNA from hybridoma cells, the guanidine thiocyanate-cesium trifluoroacetate method [Methods in Enzymol. , 154, 3 (1987)], or a kit such as RNA easy kit (manufactured by Qiagen).

For preparation of mRNA from total RNA, oligo (dT) -immobilized cellulose column method [Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Press (1989)] or Oligo-dT> SurPiSpR> <SpiK> Use a kit such as (Takara Bio). Alternatively, mRNA can be prepared from hybridoma cells using a kit such as Fast Track mRNA Isolation Kit (manufactured by Invitrogen) or QuickPrep mRNA Purification Kit (manufactured by Pharmacia).

For the synthesis of cDNA and preparation of cDNA library, known methods [Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989), Current Protocols in BioProsthetics, BioProsthetics, BioProsthetics, BioProsthetics, BioProsthetics, BioProsthetics. )], Or a kit such as SuperScript Plasmid System for cDNA Synthesis and Plasmid Cloning (manufactured by Invitrogen), or ZAP-cDNA Synthesis Kit (manufactured by Stratagene) Used.

When preparing a cDNA library, any vector can be used as a vector into which cDNA synthesized using mRNA extracted from a hybridoma cell as a template is incorporated. For example, ZAP Express [Stratesies, 5, 58 (1992)], pBluescript II SK (+) [Nucleic Acids Research, 17, 9494 (1989)], λZAPIII (manufactured by Stratagene), λgt10, Clgt11A: Clgt11 DNA Approach, I, 49 (1985)], Lambda BlueMid (Clontech), λExCell, pT7T3-18U (Pharmacia), pcD2 [Mol. Cell. Biol. 3, 280 (1983)], or pUC18 [Gene, 33, 103 (1985)].

Any Escherichia coli into which a cDNA library constructed by a phage or plasmid vector is introduced can be used as long as the cDNA library can be introduced, expressed and maintained. For example, XL1-Blue MRF '[Stratesies, 5, 81 (1992)], C600 [Genetics, 39, 440 (1954)], Y1088, Y1090 [Science, 222, 778 (1983)], NM522 [J. Mol. Biol. , 166, 1 (1983)], K802 [J. Mol. Biol. 16, 118 (1966)] or JM105 [Gene, 38, 275 (1985)].

As a method for selecting a cDNA clone encoding VH or VL of a non-human antibody from a cDNA library, for example, a colony hybridization method using an isotope or fluorescently labeled probe, or a plaque hybridization method [Molecular Cloning] , A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989)].

In addition, using primers prepared and cDNA synthesized from mRNA or cDNA library as a template, Polymerase Chain Reaction method (hereinafter referred to as PCR method, Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Labor 198) ), Current Protocols in Molecular Biology, Supplement 1, John Wiley & Sons (1987-1997)], cDNA encoding VH or VL can also be prepared.

The selected cDNA is cleaved with an appropriate restriction enzyme and then cloned into a plasmid such as pBluescript SK (-) (Stratagene), and the nucleotide sequence of the cDNA is determined by a commonly used nucleotide sequence analysis method. . Examples of the base sequence analysis method include the dideoxy method [Proc. Natl. Acad. Sci. USA, 74, 5463 (1977)], etc., followed by ABI PRISM 3700 (manufactured by PE Biosystems) or A.P. L. F. An automatic base sequence analyzer such as a DNA sequencer (Pharmacia) is used.

From the determined base sequence, all amino acid sequences of VH and VL are estimated, respectively, and all amino acid sequences of known antibodies VH and VL [Sequences of Proteins of Immunological Interest, US Dept. Comparison with Health and Human Services (1991)] confirms whether the obtained cDNA encodes the complete amino acid sequence of VH and VL of the antibody including the secretory signal sequence.

Regarding the complete amino acid sequence of VH and VL of an antibody including a secretory signal sequence, all amino acid sequences of known antibodies VH and VL [Sequences of Proteins of Immunological Interest, US Dept. By comparing with Health and Human Services (1991)], the length of the secretory signal sequence and the N-terminal amino acid sequence can be estimated, and further, the subgroup to which they belong can be known.

Also, regarding the amino acid sequences of CDRs of VH and VL, the VH and VL amino acid sequences of known antibodies [Sequences of Proteins of Immunological Interest, US Dept. It can be found by comparing with Health and Human Services (1991)].

Also, using the obtained VH and VL complete amino acid sequences, for example, BLAST method [J. Mol. Biol. , 215, 403 (1990)], and the like, it can be confirmed whether the complete amino acid sequences of VH and VL are novel.

(3) Production of recombinant antibody having no consensus sequence for binding N-linked sugar chain to V region Consensus sequence (Asn-Xaa-Ser / Thr) for binding N-linked sugar chain to V region The V region of an antibody that does not have (modified antibody) can be obtained by substituting other amino acid residues for the Asn residue and / or the Ser residue / Thr residue of the consensus sequence to which the N-linked sugar chain binds. Can be produced.

Antibody V region, in particular CDR, is an important region that regulates antibody binding to antigen. Therefore, substitution to any amino acid residue in the V region of an antibody, particularly in a CDR, may change the binding property of the antibody to an antigen. Therefore, when producing a V region that does not have the consensus sequence, it is necessary to modify the amino acid sequence so that the binding property of the antibody to the antigen does not change. The specific method is shown below.

To change the Asn residue in the consensus sequence to which the N-linked sugar chain of the V region binds, as well as the Ser residue or Thr residue, to an amino acid sequence that does not change the binding property of the antibody to the antigen, It is important to predict amino acid residues that are less likely to affect binding. Therefore, X-ray crystal structure analysis [J. Mol. Biol. 112, 535 (1977)] or computer modeling [Protein Engineering, 7, 1501 (1994)], etc., to construct and analyze the three-dimensional structure of the antibody.

However, even based on the information on the three-dimensional structure of these antibodies, introduction of mutations into the V region of the antibody, particularly the CDR, may change the binding property of the antibody to the antigen. Therefore, trials and errors, such as preparing several types of modified products and examining the correlation between the amino acid modification and the antigen binding activity, are necessary for introducing the mutation.

In this way, after estimating site-specific mutations of amino acid residues that are less likely to affect the binding of an antibody to an antigen, a DNA sequence encoding the amino acid sequence of the antibody V region into which the mutation has been introduced is obtained. design. Based on the designed DNA sequence, several synthetic DNAs having a length of about 100 bases are synthesized, and PCR reaction is performed using them. In this case, preferably 6 synthetic DNAs are designed for both the H chain and the L chain from the reaction efficiency in the PCR reaction and the length of the synthesizable DNA.

Alternatively, a known site-directed mutagenesis method such as Kunkel method [Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989) The amino acid sequence of the portion containing a consensus sequence to which an N-linked sugar chain binds using a restriction enzyme recognition site present in the cDNA encoding the V region of the antibody from which the mutation was introduced A plasmid containing a cDNA having a designed DNA sequence may be prepared by introducing a mutation into DNA encoding DNA by PCR or the like and substituting it with the cDNA encoding the V region of the antibody from which the mutation was introduced. it can.

The method according to (2) above, wherein the amino acid residue of the V region is a base sequence of a plasmid containing cDNA having a DNA sequence encoding the VH or VL amino acid sequence of the recombinant antibody obtained as described above. Confirm that the target modification has been made.

(4) Construction of human chimeric antibody expression vector The non-human antibody VH or VL is encoded upstream of each gene encoding the human antibody CH or CL of the recombinant antibody expression vector obtained in (1). A human chimeric antibody expression vector can be constructed by cloning each of the cDNAs.

In order to link the 3 ′ end of cDNA encoding VH or VL of a non-human antibody and the 5 ′ end of CH or CL of a human antibody, the base sequence of the linking portion encodes an appropriate amino acid, and VH and VL cDNAs designed to be appropriate restriction enzyme recognition sequences are prepared. The prepared VH and VL cDNAs are expressed in an appropriate form upstream of each gene encoding the human antibody CH or CL of the human CDR-grafted antibody expression vector obtained in (1). Each is cloned to construct a human chimeric antibody expression vector.

Further, a cDNA encoding the non-human antibody VH or VL is amplified by a PCR method using a synthetic DNA having a recognition sequence of an appropriate restriction enzyme at both ends, and the recombinant antibody expression vector obtained in (1) Can also be cloned.

(5) Construction of cDNA encoding V region of human CDR-grafted antibody A cDNA encoding VH or VL of human CDR-grafted antibody can be constructed as follows.

The amino acid sequence of the VH or VL FR of the human antibody to be grafted with the VH or VL CDR amino acid sequence of the non-human antibody is selected. Any amino acid sequence can be used as long as it is derived from a human antibody. For example, the FR amino acid sequence of human antibodies registered in databases such as Protein Data Bank, or the common amino acid sequence of each subgroup of FRs of human antibodies [Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services (1991)] are used. In order to suppress a decrease in the binding activity of the antibody, an FR amino acid sequence having the highest homology (at least 60% or more) with the FR amino acid sequence of the VH or VL of the original antibody is selected.

Next, the amino acid sequence of the CDR of the original antibody is transplanted to the amino acid sequence of VH or VL of the selected human antibody, respectively, and the amino acid sequence of VH or VL of the human CDR-grafted antibody is designed. Frequency of codon usage of the designed amino acid sequence in the nucleotide sequence of the antibody gene [Sequences of Proteins of Immunological Interest, US Dept. Considering Health and Human Services (1991)], the DNA sequence is converted into a DNA sequence, and the DNA sequence encoding the VH or VL amino acid sequence of the human CDR-grafted antibody is designed.

Based on the designed DNA sequence, several synthetic DNAs having a length of about 100 bases are synthesized, and PCR reaction is performed using them. In this case, preferably 6 synthetic DNAs are designed for both the H chain and the L chain from the reaction efficiency in the PCR reaction and the length of the synthesizable DNA.

In addition, by introducing an appropriate restriction enzyme recognition sequence into the 5 ′ end of the synthetic DNA located at both ends, the human CDR-grafted antibody VH can be easily added to the human CDR-grafted antibody expression vector obtained in (1). Alternatively, cDNA encoding VL can be cloned.

Alternatively, it can be carried out by using each H chain and L chain full-length synthetic DNA synthesized as one DNA based on the designed DNA sequence.

After the PCR reaction, the amplified product is cloned into a plasmid such as pBluescript SK (-) (manufactured by Stratagene), the base sequence is determined by the same method as described in (2), and the desired human CDR is obtained. A plasmid having a DNA sequence encoding the amino acid sequence of the VH or VL of the transplanted antibody is obtained.

(6) Modification of amino acid sequence of V region of human CDR-grafted antibody A human CDR-grafted antibody can be obtained by transplanting only the non-human antibody VH and VL CDRs into the human antibody VH and VL FRs. The binding activity is reduced compared to the original non-human antibody [BIO / TECHNOLOGY, 9, 266 (1991)]. In the human CDR-grafted antibody, among the amino acid sequences of human antibody VH and VL FRs, amino acid residues that are directly involved in antigen binding, amino acid residues that interact with CDR amino acid residues, and Reduced by maintaining the conformation of the antibody, identifying amino acid residues that are indirectly involved in antigen binding, and substituting those amino acid residues with the amino acid residues of the original non-human antibody Antigen binding activity can be increased.

In order to identify FR amino acid residues involved in antigen binding activity, X-ray crystallography [J. Mol. Biol. , 112, 535 (1977)] or computer modeling [Protein Engineering, 7, 1501 (1994)] or the like, the three-dimensional structure of the antibody can be constructed and analyzed. In addition, several types of variants are prepared for each antibody, and the correlation with each antigen binding activity is repeatedly studied. By trial and error, a modified human CDR-grafted antibody having the necessary antigen binding activity is obtained. You can get it.

The amino acid residues of FR of human antibody VH and VL can be modified by performing the PCR reaction described in (4) using the synthetic DNA for modification. For the amplified product after the PCR reaction, the base sequence is determined by the method described in (2) and it is confirmed that the target modification has been performed.

(7) Construction of human CDR-grafted antibody expression vector VH of the constructed recombinant antibody upstream of each gene encoding the human antibody CH or CL of the recombinant antibody expression vector obtained in (1) Alternatively, cDNA encoding VL can be cloned, and a human CDR-grafted antibody expression vector can be constructed.

For example, among the synthetic DNAs used in constructing the VH or VL of the human CDR-grafted antibody obtained in (4) and (5), a recognition sequence for an appropriate restriction enzyme at the 5 ′ end of the synthetic DNA located at both ends Are introduced into the human CDR-grafted antibody expression vector obtained in (1), and cloned so as to be expressed in an appropriate form upstream of each gene encoding CH or CL of the human antibody.

(8) Transient expression of recombinant antibodies Transient expression of recombinant antibodies using the recombinant antibody expression vectors obtained in (3) and (6) or modified expression vectors Thus, the antigen-binding activity of the prepared various types of human CDR-grafted antibodies can be efficiently evaluated.

As the host cell into which the expression vector is introduced, any cell can be used as long as it can express the recombinant antibody. For example, COS-7 cells [American Type Culture Collection (ATCC) number: CRL1651] [Methods in Nucleic Acids Res. , CRC press, 283 (1991)].

For the introduction of expression vectors into COS-7 cells, the DEAE-dextran method [Methods in Nucleic Acids Res. , CRC press (1991)], or lipofection method [Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)].

After the introduction of the expression vector, the expression amount and antigen binding activity of the recombinant antibody in the culture supernatant are measured by enzyme immunoantibody method [Monoclonal Antibodies-Principles and Practice, Third edition, Academic Press (1996), Antibodies-ALaboratory. Cold Spring Harbor Laboratory (1988), monoclonal antibody experiment manual, Kodansha Scientific (1987)] and the like.

(9) Obtaining a transformant that stably expresses the recombinant antibody and preparation of the recombinant antibody. Introducing the recombinant antibody expression vector obtained in (3) and (6) into an appropriate host cell. Thus, a transformed strain that stably expresses the recombinant antibody can be obtained.

For introduction of an expression vector into a host cell, an electroporation method [Japanese Patent Laid-Open No. 2-257891, Cytotechnology, 3, 133 (1990)] or the like is used.

Any host cell capable of expressing a recombinant antibody can be used as a host cell into which the recombinant antibody expression vector is introduced. For example, CHO-K1 (ATCC CCL-61), DUkXB11 (ATCC CCL-9096), Pro-5 (ATCC CCL-1781), CHO-S (Life Technologies, Cat # 11619), rat myeloma cell YB2 / 3HL. P2. G11.16 Ag. 20 (also referred to as YB2 / 0), mouse myeloma cell NSO, mouse myeloma cell SP2 / 0-Ag14 (ATCC number: CRL1581), mouse P3X63-Ag8.653 cell (ATCC number: CRL1580), dihydrofolate reductase gene ( (Hereinafter referred to as dhfr) deficient CHO cells [Proc. Natl. Acad. Sci. USA, 77, 4216 (1980)], Lec13 [Somatic Cell and Molecular genetics, 12, 55 (1986)] that has acquired lectin resistance, CHO cells deficient in the α1,6-fucose transferase gene (International Publication No. 2005 / 035586, WO 02/31140), rat YB2 / 3HL. P2. G11.16 Ag. 20 cells (ATCC number: CRL1662) and the like.

In addition, for example, a protein such as an enzyme involved in the synthesis of intracellular sugar nucleotide GDP-fucose, a sugar in which the 1-position of fucose is α-bonded to the 6-position of N-acetylglucosamine at the reducing end of the N-glycoside-linked complex sugar chain Host cells in which the activity of a protein such as an enzyme involved in chain modification or a protein involved in the transport of intracellular sugar nucleotide GDP-fucose to the Golgi apparatus is reduced or deleted, such as α1,6-fucose transferase gene Deficient CHO cells (WO 2005/035586, WO 02/31140) and the like can be mentioned.

After the introduction of the expression vector, a transformant that stably expresses the recombinant antibody is selected by culturing in an animal cell culture medium containing a drug such as G418 sulfate (hereinafter referred to as G418) (Japan). (Japanese Patent Laid-Open No. 2-257891).

Examples of the animal cell culture medium include RPMI 1640 medium (manufactured by Invitrogen), GIT medium (manufactured by Nippon Pharmaceutical Co., Ltd.), EX-CELL301 medium (manufactured by JRH), IMDM medium (manufactured by Invitrogen), Hybridoma-SFM. Examples thereof include a medium (manufactured by Invitrogen) or a medium obtained by adding various additives such as FBS to these mediums. By culturing the obtained transformant in a medium, the recombinant antibody is expressed and accumulated in the culture supernatant. The expression level and antigen binding activity of the recombinant antibody in the culture supernatant can be measured by ELISA method or the like. The transformed strain can increase the expression level of the recombinant antibody using a DHFR amplification system (Japanese Patent Laid-Open No. 2-257891).

The recombinant antibody is purified from the culture supernatant of the transformant using a protein A-column [Monoclonal Antibodies-Principles and Practice, Third edition, Academic Press (1996), Antibodies-A LaboratoryLaboratoryLaboratoryLaboratoryLaboratory. (1988)]. In addition, methods used in protein purification such as gel filtration, ion exchange chromatography, and ultrafiltration can be combined.

The molecular weight of the purified recombinant antibody H chain, L chain, or whole antibody molecule is determined by polyacrylamide gel electrophoresis [Nature, 227, 680 (1970)] or Western blotting [Monoclonal Antibodies-Principles and practicies, Third]. edition, Academic Press (1996), Antibodies-A Laboratory Manual, Cold Spring Harbor Laboratory (1988)].

3. Evaluation of activity of purified monoclonal antibody or fragment thereof The activity of the purified monoclonal antibody or fragment thereof of the present invention can be evaluated as follows.

The binding activity to the CD33-expressing cell line is measured using the above-described binding assay described in 1- (6a) and the surface plasmon resonance method using the Biacore system described in (6b). In addition, the fluorescent antibody method [Cancer Immunol. Immunother. , 36, 373 (1993)].

CDC activity or ADCC activity against an antigen-positive cultured cell line is measured by a known measurement method [Cancer Immunol. Immunother. , 36, 373 (1993)].

4). Method for Controlling Effector Activity of Antibody As a method for controlling the effector activity of the anti-CD33 monoclonal antibody of the present invention, an N-linked complex type sugar chain that binds to the 297th asparagine (Asn) of the Fc region of the antibody Methods for controlling the amount of fucose (also referred to as core fucose) that binds α-1,6 to existing N-acetylglucosamine (GlcNAc) (WO 2005/035586, WO 2002/31140, WO 00) No. / 61739) and a method of controlling by modifying amino acid residues in the Fc region of an antibody are known. The effector activity can be controlled using any method for the anti-CD33 monoclonal antibody of the present invention.

The effector activity refers to antibody-dependent activity caused through the Fc region of an antibody, and includes ADCC activity, CDC activity, or antibody-dependent phagocytosis by phagocytic cells such as macrophages or dendritic cells (Antibody-dependent phagocytosis). , ADP activity) and the like.

By controlling the content of core fucose in the N-linked complex sugar chain of the Fc of the antibody, the effector activity of the antibody can be increased or decreased. As a method for reducing the content of fucose bound to the N-linked complex type sugar chain bound to the Fc of the antibody, expressing the antibody using CHO cells deficient in the α1,6-fucose transferase gene, An antibody to which fucose is not bound can be obtained.

An antibody to which fucose is not bound has high ADCC activity. On the other hand, as a method for increasing the content of fucose bound to the N-linked complex type sugar chain bound to the Fc of the antibody, the antibody is expressed using a host cell into which an α1,6-fucose transferase gene has been introduced. Thus, an antibody to which fucose is bound can be obtained. An antibody to which fucose is bound has a lower ADCC activity than an antibody to which fucose is not bound.

Also, ADCC activity or CDC activity can be increased or decreased by modifying amino acid residues in the Fc region of the antibody. For example, the CDC activity of an antibody can be increased by using the amino acid sequence of the Fc region described in US Patent Application Publication No. 2007/0148165.

Further, by performing amino acid modification described in US Pat. No. 6,737,056, US Pat. No. 7,297,775 or US Pat. No. 7,317,091, ADCC activity or CDC activity can be increased or decreased.

Furthermore, an antibody with controlled effector activity of an antibody can be obtained by using the above method in combination with one antibody.

5. Method for Treating Disease Using Anti-CD33 Monoclonal Antibody or Fragment thereof of the Present Invention The monoclonal antibody or fragment thereof of the present invention can be used for the treatment of diseases involving CD33 positive cells.

The therapeutic agent containing the monoclonal antibody or fragment thereof of the present invention may contain only the antibody or fragment as an active ingredient, but usually together with one or more pharmacologically acceptable carriers. And prepared as a pharmaceutical preparation produced by a method known in the technical field of pharmaceutics.

Examples of the administration route include oral administration and parenteral administration such as intraoral, intratracheal, rectal, subcutaneous, intramuscular or intravenous. Examples of the dosage form include sprays, capsules, tablets, powders, granules, syrups, emulsions, suppositories, injections, ointments, or tapes.

Suitable formulations for oral administration are emulsions, syrups, capsules, tablets, powders, or granules.

Liquid preparations such as emulsions or syrups include saccharides such as water, sucrose, sorbitol or fructose, glycols such as polyethylene glycol or propylene glycol, oils such as sesame oil, olive oil or soybean oil, p-hydroxybenzoic acid Manufactured using preservatives such as esters, or flavors such as strawberry flavor or peppermint as additives.

Capsules, tablets, powders or granules include excipients such as lactose, glucose, sucrose or mannitol, disintegrants such as starch or sodium alginate, lubricants such as magnesium stearate or talc, polyvinyl alcohol, hydroxy A binder such as propylcellulose or gelatin, a surfactant such as fatty acid ester, or a plasticizer such as glycerin is used as an additive.

Examples of preparations suitable for parenteral administration include injections, suppositories, and sprays.

Injection is manufactured using a carrier made of a salt solution, a glucose solution, or a mixture of both.

Suppositories are produced using a carrier such as cacao butter, hydrogenated fat or carboxylic acid.

The propellant is produced using a carrier that does not irritate the recipient's oral cavity and airway mucosa, and in which the monoclonal antibody of the present invention or a fragment thereof is dispersed as fine particles to facilitate absorption. As the carrier, for example, lactose or glycerin is used. It can also be produced as an aerosol or dry powder.

Furthermore, in the above parenteral preparations, the components exemplified as additives in preparations suitable for oral administration can also be added.

6). Method of diagnosing disease using anti-CD33 monoclonal antibody or fragment thereof of the present invention By detecting or measuring cells expressing CD33 or CD33 using the monoclonal antibody or fragment thereof of the present invention, a disease associated with CD33 can be detected. Can be diagnosed.

The diagnosis of cancer, which is one of the diseases associated with CD33, can be performed by detecting or measuring CD33 as follows, for example.

Diagnosis can be made by detecting CD33 expressed in cancer cells in a patient using an immunological technique such as a flow cytometer.

An immunological technique is a method for detecting or measuring the amount of antibody or the amount of antigen using a labeled antigen or antibody. For example, a radioactive substance-labeled immunoantibody method, an enzyme immunoassay method, a fluorescence immunoassay method, a luminescence immunoassay method, a Western blot method, a physicochemical method, or the like can be given.

The radioactive substance-labeled immunoantibody method is, for example, reacting an antigen or a cell expressing the antigen with the antibody of the present invention or a fragment thereof, and further reacting with a radiolabeled anti-immunoglobulin antibody or a binding thereof, Measure with a scintillation counter.

In the enzyme immunoassay, for example, an antigen or a cell expressing the antigen is reacted with the antibody of the present invention or a fragment thereof, and further, a labeled anti-immunoglobulin antibody or a binding fragment thereof is reacted, and then a coloring dye. Is measured with an absorptiometer. For example, a sandwich ELISA method is used.

As the label used in the enzyme immunoassay, a known enzyme label [enzyme immunoassay, Medical School (1987)] can be used. For example, alkaline phosphatase label, peroxidase label, luciferase label, biotin label or the like is used.

Sandwich ELISA is a method in which an antibody to be bound to a solid phase, an antigen to be detected or measured is trapped, and a second antibody is reacted with the trapped antigen. In the ELISA method, two types of antibodies that recognize an antigen to be detected or measured or fragments thereof and having different antigen recognition sites are prepared, and the first antibody or fragment thereof is preliminarily placed on a plate (for example, 96 Next, the second antibody or a fragment thereof is labeled with a fluorescent substance such as FITC, an enzyme such as peroxidase, or biotin.

After the cells adsorbed with the antibody are reacted with cells or a lysate thereof, tissue or a lysate thereof, cell culture supernatant, serum, pleural effusion, ascites or ocular fluid, the label is obtained. The detected monoclonal antibody or a fragment thereof is reacted, and a detection reaction corresponding to the labeling substance is performed. The antigen concentration in the test sample is calculated from a calibration curve prepared by diluting antigens with known concentrations stepwise.

As an antibody used in the sandwich ELISA method, either a polyclonal antibody or a monoclonal antibody may be used, and an antibody fragment such as Fab, Fab ′, or F (ab) ₂ may be used. The combination of the two types of antibodies used in the sandwich ELISA method may be a combination of a monoclonal antibody that recognizes different epitopes or a fragment thereof, or a combination of a polyclonal antibody and a monoclonal antibody or a fragment thereof.

The fluorescence immunoassay is measured by the method described in the literature [Monoclonal Antibodies-Principles and practices, Third edition, Academic Press (1996), Monoclonal Antibody Experiment Manual, Kodansha Scientific (1987)]. As a label used in the fluorescence immunoassay, a fluorescent label known in the art [fluorescent antibody method, Soft Science (1983)] can be used. For example, FITC or RITC is used.

The luminescent immunoassay is measured by the method described in the literature [Bioluminescence and chemiluminescence, clinical examination 42, Yodogawa Shoten (1998)]. Examples of the label used in the luminescent immunoassay include known luminescent labels, such as acridinium ester or lophine.

In Western blotting, an antigen or cells expressing the antigen are fractionated with SDS (sodium dodecyl sulfate) -PAGE [Antibodies-A Laboratory Manual Spring Spring Laboratory (1988)], and the gel is then polyvinylidene fluoride (PVDF). B) blotting on a membrane or a nitrocellulose membrane, reacting an antibody that recognizes the antigen or a fragment thereof on the membrane, and further applying an anti-mouse IgG antibody or a fluorescent substance such as FITC, an enzyme label such as peroxidase, or a biotin label After the bound fragment is reacted, the label is measured by visualization. An example is shown below.

A cell or tissue expressing a polypeptide containing the amino acid sequence represented by SEQ ID NO: 3 is lysed, and 0.1 to 30 μg of protein per lane is electrophoresed by SDS-PAGE under reducing conditions. The migrated protein is transferred to a PVDF membrane and reacted with PBS containing 1 to 10% BSA (hereinafter sometimes referred to as BSA-PBS) at room temperature for 30 minutes to perform a blocking operation. Here, the monoclonal antibody of the present invention was reacted, washed with PBS containing 0.05 to 0.1% Tween-20 (hereinafter sometimes referred to as Tween-PBS), and peroxidase-labeled goat anti-mouse IgG. For 2 hours at room temperature.

By washing with Tween-PBS and detecting the band to which the monoclonal antibody is bound using ECL Western Blotting Detection Reagents (manufactured by Amersham), a polypeptide containing the amino acid sequence represented by SEQ ID NO: 3 is detected. As an antibody used for detection by Western blotting, an antibody capable of binding to a polypeptide that does not have a natural three-dimensional structure is used.

The physicochemical method is performed by, for example, forming an aggregate by binding the antigen CD33 and the monoclonal antibody of the present invention or a fragment thereof, and detecting the aggregate. In addition, examples of the physicochemical method include a capillary method, a one-dimensional immunodiffusion method, an immunoturbidimetric method, or a latex immunoturbidimetric method [Proposal for Clinical Laboratory Methods, Kanbara Publishing (1998)].

Latex immunoturbidimetry is a method in which an antibody or antigen-sensitized carrier such as polystyrene latex having a particle size of about 0.1 to 1 μm is used to cause an antigen-antibody reaction with the corresponding antigen or antibody. Scattered light increases and transmitted light decreases. By detecting this change as absorbance or integrating sphere turbidity, the antigen concentration or the like in the test sample is measured.

On the other hand, for the detection or measurement of cells expressing CD33, a known immunological detection method can be used. Preferably, immunoprecipitation method, immune cell staining method, immunohistochemical staining method, fluorescent antibody staining method, etc. Is used.

In the immunoprecipitation method, a cell expressing CD33 or the like is reacted with the monoclonal antibody of the present invention or a fragment thereof, and then a carrier having a specific binding ability to immunoglobulin such as protein G-sepharose is added to the antigen-antibody complex. To settle. Alternatively, the following method can be used.

The above-described monoclonal antibody of the present invention or a fragment thereof is immobilized on a 96-well plate for ELISA, and then blocked with BSA-PBS. When the antibody is in an unpurified state, such as a hybridoma culture supernatant, anti-mouse immunoglobulin, anti-rat immunoglobulin, protein-A or protein-G is immobilized on a 96-well plate for ELISA in advance. After blocking with BSA-PBS, the hybridoma culture supernatant is dispensed and bound. Next, after discarding BSA-PBS and washing well with PBS, a lysate of cells and tissues expressing CD33 is reacted. Immunoprecipitates are extracted from the well-washed plate with SDS-PAGE sample buffer and detected by Western blotting as described above.

The immune cell staining method or the immunohistochemical staining method is a method in which cells or tissues expressing an antigen are treated with a surfactant or methanol in order to improve antibody passage, and then reacted with the monoclonal antibody of the present invention. And then reacting with a fluorescent label such as FITC, an enzyme label such as peroxidase or a biotin label, or an anti-immunoglobulin antibody or a binding fragment thereof, then the label is visualized and microscopically observed .

In addition, a fluorescent antibody staining method in which a fluorescently labeled antibody is reacted with a cell and analyzed with a flow cytometer [Monoclonal Antibodies-Principles and Practice, Academic Press (1996), Monoclonal Antibody Experiment Manual, Kodansha Scientific Fick (1987)]. In particular, the monoclonal antibody of the present invention or a fragment thereof that binds to the extracellular region of CD33 can detect cells expressing the natural three-dimensional structure by fluorescent antibody staining.

In addition, among the fluorescent antibody staining methods, when the FMAT8100HTS system (Applied Biosystems) or the like is used, the formed antibody-antigen complex and the free that is not involved in the formation of the antibody-antigen complex The amount of antigen or antibody can be measured without separating the antibody or antigen.

Hereinafter, the present invention will be specifically described by way of examples. However, the present invention is not limited to the following examples.

[Example 1]
Production of CD33 transfectant
(1) Preparation of CD33 expression vector From a vector in which the human CD33 gene is incorporated into the vector pCMV / SPORT6 (Open Biosystems, Clone No. 5217182, hereinafter sometimes referred to as pCMV / hCD33), human CD33 is prepared as follows. A vector incorporating the gene sequence was prepared.

The human CD33 gene was amplified by a normal PCR method using pCMV / hCD33 as a template and primers having the nucleotide sequences represented by SEQ ID NOs: 1 and 2. The PCR product was purified by PCR Purification Kit (manufactured by QIAGEN) and treated with restriction enzymes with SacI and HindIII. The vector pBluescript II sk (-) (Stratagene) (hereinafter sometimes referred to as pBS) is similarly treated with SacI and HindIII, and these two fragments are ligated high (TOYOBO). Used and ligated according to the attached instructions.

The obtained recombinant plasmid DNA solution was used to transform E. coli DH5α strain (manufactured by TOYOBO). A plasmid pBS / hCD33 into which the human CD33 gene has been inserted is obtained by preparing plasmid DNA from a clone of the transformant and confirming the size of the fragment excised by restriction enzyme treatment by agarose gel electrophoresis. did.

The pBS / hCD33 into which the human CD33 gene thus obtained was integrated and the animal cell expression vector pKANTEX93 (International Publication No. 97/10354) were subjected to restriction enzyme treatment with BsiWI and SpeI. The reaction mixture was fractionated by agarose gel electrophoresis, and the BsiWI-SpeI fragment of hCD33 and the BsiWI-SpeI fragment of pKANTEX93 were recovered.

These two types of fragments were ligated using Ligation high according to the attached instructions. The resulting recombinant plasmid DNA solution was used to transform E. coli DH5α strain. Plasmid DNA was prepared from the clone of the transformant and confirmed by comparing the size of the fragment excised by the restriction enzyme treatment by agarose gel electrophoresis.

Further, the base sequence of the plasmid was analyzed using the BigDye Terminator Cycle Sequencing FS Ready Reaction Kit (Applied Biosystems) according to the attached instructions, and then analyzed by the sequencer ABI PRISM 3700 of the company.

As a result, a vector pKANTEX / hCD33 in which cDNA encoding the human CD33 gene was cloned was obtained. A schematic diagram of vector construction is shown in FIG. 1A. The amino acid sequence of human CD33 is shown in SEQ ID NO: 3, and the nucleotide sequence of CD33 is shown in SEQ ID NO: 4.

An expression vector pKANTEX / cynoCD33 incorporating a cynomolgus monkey CD33 gene is a base represented by SEQ ID NOs: 115 and 116 from mRNA extracted from a cynomolgus monkey peripheral blood mononuclear cell (Peripheral blood mononuclear cell: hereinafter sometimes referred to as PBMC). A human CD33 gene was amplified by a normal PCR method using a primer having a sequence, and prepared by the same method as described above. A schematic diagram of vector construction is shown in FIG. 1B. The base sequence confirmed by the above analysis is shown in SEQ ID NO: 118, and the amino acid sequence of cynomolgus CD33 predicted from the base sequence is shown in SEQ ID NO: 117.

The chimpanzee CD33 gene is a plasmid in which the base sequence represented by SEQ ID NO: 119 is designed based on the base sequence of the chimpanzee CD33 gene registered in the NCBI database as XM — 5128850.2, and the base sequence is incorporated into the cloning vector pUC53. Was purchased using a gene synthesis contract service of GenScript.

Using the plasmid, an expression vector pKANTEX / chimpCD33 incorporating the chimpanzee CD33 gene was prepared by the same method as described above. A schematic diagram of vector construction is shown in FIG. 1C. The amino acid sequence of chimpanzee CD33 is shown in SEQ ID NO: 120, and the base sequence of chimpanzee CD33 is shown in SEQ ID NO: 121.

(2) Expression of CD33 in CHO / DG44 cells The CD33 expression vectors pKANTEX / hCD33, pKANTEX / cynoCD33, and pKANTEX / chimpCD33 prepared in (1) above were prepared by electroporation [Cytotechnology, 3, 133 (1990)]. Then, it was introduced into CHO / DG44 cells [Somatic Cell and Molecular Genetics, 12, 555 (1986)] as follows.

Cells were added to IMDM medium (Invitrogen) (hereinafter sometimes referred to as A3 medium) supplemented with 10% fetal calf serum (Life Technologies) and Gentamin (Nacalai Tesque, 50 μg / mL). 1 × HT supplement (manufactured by Invitrogen) was used in a subculture medium.

CHO / DG44 cells contain 137 nmol / L potassium chloride, 2.7 nmol / L sodium chloride, 8.1 mmol / L disodium monohydrogen phosphate, 1.5 nmol / L monosodium dihydrogen phosphate and 4 mmol / L magnesium chloride Suspend in a buffer solution (hereinafter sometimes referred to as K-PBS) to 8 × 10 ⁶ cells / mL, and express 200 μL (1.6 × 10 ⁶ cells) of the resulting cell suspension for each expression. Mixed with vector (8 μg).

The mixture was transferred to a cuvette (distance between electrodes: 2 mm), and gene introduction was performed using GenePulser II (Bio-Rad) under conditions of a pulse voltage of 0.35 kV and an electric capacity of 250 μF. After leaving the cuvette on ice, the cell suspension in the cuvette was suspended in a cell culture vessel containing A3 medium, and cultured in a 37 ° C., 5% carbon dioxide incubator.

Thereafter, the cells were cultured in a medium supplemented with G418 (manufactured by Invitrogen, 0.5 mg / mL) to obtain a transformed cell line resistant to G418. Further, methotrexate-resistant clones were selected by adding methotrexate to the medium and gradually increasing the concentration.

Furthermore, according to the method of Example 1 (3), these cells were fluorescently immunostained, and the fluorescence intensity was measured with a flow cytometer (manufactured by Beckman Coulter) to select a clone that highly expresses CD33. A CD33-expressing cell line, a cynomolgus monkey CD33-expressing cell line, and a chimpanzee CD33-expressing cell line were obtained.

(3) Comparison of CD33 expression level on cells by flow cytometry (FCM) 1 to 5 × 10 ⁵ CD33-expressing cells were blocked by adding 1 mg / mL human IgG (manufactured by Sigma), and anti-mouse CD33 antibody P67.6 (Santa Cruze Biosystems) or WM-53 (Ancell) 1% bovine serum albumin (BSA) -PBS (hereinafter referred to as 0.02% EDTA and 0.05% sodium azide) The total volume was adjusted to 100 μL.

These cell suspensions were reacted for 60 minutes on ice, and then washed twice with Dulbecco's phosphate buffer (hereinafter sometimes referred to as PBS). 30 μL of Alexa-488-labeled goat anti-mouse IgG (H + L) (manufactured by Invitrogen) diluted with FCM buffer was added to the cells and reacted on ice for 40 minutes. After washing 3 times with PBS, it was suspended in FCM buffer and the fluorescence intensity was measured with a flow cytometer.

[Example 2]
Preparation of histidine-tagged soluble human CD33 (hCD33-His) (1) Preparation of hCD33-His expression vector Using plasmid pBS / hCD33 inserted with human CD33 gene obtained in Example 1 (1) as a template, A gene sequence (hereinafter referred to as a his sequence) encoding a 6 × histidine sequence (hereinafter sometimes referred to as a His sequence) by a normal PCR method using a primer having a base sequence represented by SEQ ID NOs: 1 and 5 The hCD33 gene (hereinafter sometimes referred to as hCD33-his) added to the 3 ′ end side was amplified.

The PCR product was purified by PCR Purification Kit and subjected to restriction enzyme treatment with BsiWI and SpeI. The vector pKANTEX93 was similarly digested with BsiWI and SpeI. The reaction solution was fractionated by agarose gel electrophoresis, and the BsiWI-SpeI fragment of hCD33-his and the BsiWI-SpeI fragment of pKANTEX93 were recovered. These two types of fragments were ligated using Ligation high according to the attached instructions.

The obtained recombinant plasmid DNA solution was used to transform E. coli DH5α strain. Plasmid DNA was prepared from the clone of the transformant and confirmed by restriction enzyme treatment. Further, the plasmid was reacted using the BigDye Terminator Cycle Sequencing FS Ready Reaction Kit according to the attached instruction, and the base sequence was analyzed by the sequencer ABI PRISM 3700.

As a result, a vector pKANTEX / hCD33-his in which cDNA encoding hCD33-his was cloned was obtained. A schematic diagram of vector construction is shown in FIG. The amino acid sequence of hCD33-His is shown in SEQ ID NO: 6, and the nucleotide sequence of hCD33-his is shown in SEQ ID NO: 7.

(2) Production of hCD33-His in CHO / DG44 cells By the following method, hCD33-His to which a His sequence, which is a sequence of six histidines at the C-terminus of the human CD33 extracellular region, was added. The hCD33-His expression vector pKANTEX / hCD33-his prepared in (1) above was introduced into CHO / DG44 cells by electroporation as follows.

The cells used were subcultured with a medium supplemented with 1 × HT supplement to A3 medium. CHO / DG44 cells were suspended in K-PBS to 8 × 10 ⁶ cells / mL, and 200 μL (1.6 × 10 ⁶ cells) of the obtained cell suspension was used as an expression vector pKANTEX / hCD33-his (8 μg). ).

The mixture was transferred to a cuvette (distance between electrodes: 2 mm), and gene introduction was performed using GenePulser II under conditions of a pulse voltage of 0.35 kV and an electric capacity of 250 μF. After leaving the cuvette on ice, the cell suspension in the cuvette was suspended in a cell culture vessel containing A3 medium, and cultured in a 37 ° C., 5% carbon dioxide incubator.

Thereafter, the cells were cultured in a medium supplemented with G418 to obtain a transformed cell line having resistance to G418. Further, methotrexate-resistant clones were selected by adding methotrexate to the medium and gradually increasing the concentration. Further, according to the method (3) below, by comparing the concentration of hCD33-His in the culture supernatant of these cells, a clone producing high hCD33-His was selected, and an hCD33-His-producing cell line was obtained.

(3) Comparison of hCD33-His concentration in culture supernatant using ELISA A 96-well ELISA plate (Greiner) was used for the assay. P67.6 diluted to 2 microgram / mL with PBS was dispensed at 50 μL / well, and allowed to stand overnight at 4 ° C. for adsorption. The plate was washed with PBS, 1% BSA-PBS was added at 100 μL / well, and left at room temperature for 1 hour to block remaining active groups.

Thereafter, 1% BSA-PBS was discarded, and the cell culture supernatant containing hCD33-His was appropriately diluted on the plate, dispensed at 50 μL / well, and allowed to stand for 1 hour. The plate was washed with 0.05% polyoxyethylene (20) sorbitan monolaurate (ICI trademark Tween 20 equivalent: manufactured by Wako Pure Chemical Industries, Ltd.) / PBS (hereinafter sometimes referred to as Tween / PBS). Thereafter, a peroxidase-labeled mouse anti-Penta-His antibody (manufactured by QIAGEN) was added as a secondary antibody at 50 μL / well and allowed to stand at room temperature for 1 hour.

The plate was washed with Tween / PBS, ABTS [2.2-azinobis (3-ethylbenzothiazole-6-sulfonic acid) ammonium] substrate solution [1 mmoL / L ABTS / 0.1 moL / L citrate buffer (pH 4. 2), 0.1% H ₂ O ₂ ] was added at 50 μL / well for color development, and 5% SDS (Sodium Lauryl Sulfate) solution was added at 50 μL / well to stop the reaction, and the sample wavelength was 415 nm and the reference wavelength was 490 nm. Absorbance (OD415-OD490) was measured using a plate reader (Emax microplate reader, Molecular Devices).

[Example 3]
Preparation of anti-CD33 monoclonal antibody (1) Preparation of immunogen Recombinant human Siglec-3 (CD33) / Fc Chimera (catalog number 1137-SL) manufactured by R & D Systems (hereinafter sometimes referred to as recombinant human CD33-Fc) The freeze-dried product, human CD33-expressing cell line obtained by the method described in Example 1, or human acute monocytic leukemia cell line THP-1 positive for CD33 was dissolved in PBS and used as an immunogen.

(2) Immunization of animals and preparation of antibody-producing cells Recombinant human CD33-Fc 20 μg prepared in (1) above, aluminum hydroxide adjuvant [Antibodies-A Laboratory Manual, Cold Spring Harbor Laboratory, p99, 1988] 2 mg pertussis vaccine It was administered to SD rats (Japan SLC) together with 1 × 10 ⁹ cells (manufactured by Serological Research Institute, Chiba Prefecture). From 2 weeks after administration, 20 μg of CD33 and pertussis vaccine 1 × 10 ⁹ cells alone were administered once a week for a total of 3 times.

Alternatively, the human CD33-expressing cell line 1 × 10 ⁷ cells prepared in (1) above was administered to SD rats (Japan SLC) four times in total, once per week, with pertussis vaccine 1 × 10 ⁹ cells. Alternatively, THP-1 1 × 10 ⁷ cells prepared in (1) above and pertussis vaccine 1 × 10 ⁹ cells once a week for a total of 4 times, Balb / c mice (Japan SLC), BXSB mice (Japan SLC) ).

Partial blood was collected from the fundus vein of the rat or mouse, the serum antibody titer was confirmed by the enzyme immunoassay shown below, and the spleen was removed 3 days after the final immunization from the rat or mouse showing a sufficient antibody titer. The spleen was shredded in MEM (Minimum Essential Medium) medium (manufactured by Nissui Pharmaceutical), ground and centrifuged (1200 rpm, 5 minutes).

To the resulting precipitate fraction, tris-ammonium chloride buffer (pH 7.6) was added and treated at 37 ° C. for 1 minute to remove erythrocytes. The obtained precipitate fraction (cell fraction) was washed 3 times with MEM medium and used for cell fusion.

(3) Enzyme immunoassay (binding ELISA)
A 96-well ELISA plate (Greiner) was used for the assay. Recombinant human CD33-Fc, anti-rat immunoglobulin antibody (DAKO) or anti-mouse immunoglobulin antibody (DAKO) was dispensed at 50 μL / well and allowed to stand overnight at 4 ° C. for adsorption. Using. The plate was washed with PBS, 1% bovine serum albumin (BSA) -PBS was added at 100 μL / well, and the plate was allowed to stand at room temperature for 1 hour to block remaining active groups.

Thereafter, 1% BSA-PBS was discarded, and immunized rat or mouse serum was appropriately diluted as a primary antibody on the plate, dispensed at 50 μL / well, and allowed to stand for 1 hour. After washing the plate with Tween-PBS, 50 μL / peroxidase-labeled goat anti-rat IgG (H + L chain) antibody (ZYMED) or peroxidase-labeled goat anti-mouse IgG (γ) antibody (manufactured by KPL) was used as the secondary antibody. The wells were added and left at room temperature for 1 hour.

The plate was washed with Tween-PBS, ABTS [2.2-azinobis (3-ethylbenzothiazole-6-sulfonic acid) ammonium] substrate solution [1 mmoL / L ABTS / 0.1 moL / L citrate buffer (pH 4. 2), 0.1% H ₂ O ₂ ] was added at 50 μL / well for color development, and 5% SDS (Sodium Lauryl Sulfate) solution was added at 50 μL / well to stop the reaction, and the sample wavelength was 415 nm and the reference wavelength was 490 nm. Absorbance (OD415-OD490) was measured using a plate reader (Emax microplate reader, manufactured by Molecular Devices).

(4) Preparation of mouse myeloma cells 8-Azaguanine resistant mouse myeloma cell line P3X63Ag8U. 1 (P3-U1: purchased from ATCC) was cultured in RPMI 1640 supplemented with 10% fetal bovine serum (manufactured by Invitrogen), 2 × 10 ⁷ cells or more were secured at the time of cell fusion, and used as a parent strain for cell fusion.

(5) Production of hybridoma The mouse spleen cells obtained in (2) above and the myeloma cells obtained in (4) above were mixed at 10: 1 and centrifuged (1200 rpm, 5 minutes). After thoroughly disaggregating the cells of the obtained precipitate fraction, a mixed solution of 1 g of polyethylene glycol-1000 (PEG-1000), 1 mL of MEM medium, and 0.35 mL of dimethyl sulfoxide (DMSO) is stirred at 37 ° C. 0.5 mL per 1 × 10 ⁸ mouse spleen cells was added, and 1 mL of MEM medium was added several times to the suspension every minute, and then the MEM medium was added to make the total volume 50 mL.

The suspension was centrifuged (900 rpm, 5 minutes), and the cells of the obtained precipitate fraction were loosely loosened. Medium gently added to 100 mL. The suspension was dispensed into a 96-well culture plate at 200 μL / well and cultured at 37 ° C. for 10-14 days in a 5% CO ₂ incubator.

After culture, the culture supernatant was measured by the FMAT method described in (6) below, and a well that reacted with the human CD33-expressing cell line but did not react with CHO / DG44 cells was selected. Cloning by limiting dilution was repeated twice from cells contained therein to establish anti-CD33 monoclonal antibody-producing hybridoma strains KM4071, KM4072, KM4073, KM4074, and KM4075 (Table 1). For example, the expression “KM4071” refers to a hybridoma or a monoclonal antibody produced by the hybridoma (the same applies to other hybridomas).

(6) Measurement of culture supernatant by FMAT method The human CD33-expressing cell line prepared by the method described in Example 1 was placed in a 96-well FMAT plate (Applied Biosystems) at 1 × 10 ⁴ cells / 50 μL / well. Then, the cells were cultured overnight in a 5% CO ₂ incubator at 37 ° C. 10 μL / well of hybridoma culture supernatant was added to the plate, and immediately Alexa-647-labeled goat anti-rat IgG (H + L) antibody (manufactured by Invitrogen) diluted with 0.05% sodium azide and 1% BSA-PBS. Was added at 50 μL / well and allowed to stand at room temperature for 3 hours in the dark. Thereafter, the wavelength of 650 nm to 685 nm excited by laser light 633 nm He / Ne was measured with 8200 Cellular Detection System (Applied Biosystems). Hereinafter, this measurement method is referred to as FMAT method.

[Example 4]
Activity evaluation of rat or mouse anti-CD33 monoclonal antibody against human CD33 (1) Reactivity with human CD33-positive cell line in FCM 1-5 × 10 ⁵ cells of THP-1 with human IgG (Sigma) KM4071 to KM4075 and P67.6 [Biocomjugate Chem, 13, 47 (2002)] were appropriately diluted with FCM buffer to make a total volume of 100 μL. These cell suspensions were reacted for 60 minutes on ice and then washed twice with PBS.

30 μL of Allexa-488-labeled goat anti-rat IgG (H + L) antibody (manufactured by Invitrogen) or Allexa-488-labeled goat anti-mouse IgG (H + L) diluted with FCM buffer was added to the cells and allowed to react on ice for 40 minutes. It was. After washing 3 times with PBS, it was suspended in FCM buffer and the fluorescence intensity was measured using a flow cytometer (Beckman Coulter).

FIG. 3 shows the average fluorescence intensity when the anti-CD33 monoclonal antibody obtained in Example 3 was reacted stepwise diluted from 3 μg / mL at a 4-fold dilution (hereinafter sometimes referred to as MFI). Indicates. As shown in FIG. 3, it was confirmed that KM4071 to KM4075 and p67.6 all bind to the CD33 positive cell line in a concentration-dependent manner.

(2) Binding activity with recombinant human CD33-Fc in ELISA This was performed according to the method described in Example 3 (3). FIG. 4 shows the results of using KM4071 to KM4075 and P67.6 obtained in Example 3 stepwise diluted from 2 μg / mL at a 5-fold dilution as the primary antibody.

(3) Binding activity to recombinant human CD33-Fc in Biacore In order to analyze the binding activity of KM4071 to KM4075 and P67.6 to recombinant human CD33-Fc, the surface plasmon resonance method (SPR method) was used. The binding activity was measured. The following operations were all performed using Biacore T-100 (manufactured by GE Healthcare Bioscience).

Using Mouse Antibody Capture Kit (manufactured by GE Healthcare Bioscience), the anti-mouse IgG antibody was solidified on a CM5 sensor chip (manufactured by GE Healthcare Bioscience) by the amine coupling method according to the attached protocol. Measurement samples (KM4071 to KM4075 and P67.6 were added to a chip on which an anti-mouse IgG antibody was solidified, and the sample was captured so as to be about 200 RU (resonance unit).

Thereafter, recombinant human CD33-Fc diluted in 10 stages from 10 μg / mL to 2 stages was flowed on the chip at a rate of 30 μL / min, and sensorgrams at each concentration were obtained. Using the analysis software attached to the apparatus, analysis was performed using a 1: 1 binding model, and the binding rate constant ka and the dissociation rate constant kd of each antibody for human CD33 were calculated.

The resulting ka of each antibody, indicating a kd and dissociation constant, K _D, in Table 2.

As shown in Table 2, KM4071, 4073, 4074, and 4075 showed higher affinity for CD33-Fc than P67.6.

[Example 5]
Isolation and Analysis of cDNA Encoding Variable Region of Anti-CD33 Monoclonal Antibody (1) Preparation of mRNA from Anti-CD33 Monoclonal Antibody-Producing Hybridoma Cells From KM4071 to KM4075 described in Example 3, RNAeasy Mini kit (QIAGEN) Using Oligotex ™ -dT30 <Super> mRNA Purification Kit (TaKaRa), mRNA was prepared from hybridoma 5 × 10 ⁷ to 1 × 10 ⁸ cells according to the attached instruction manual.

(2) Gene cloning of H chain and L chain variable regions of anti-CD33 monoclonal antibody From 0.5 μg of the monoclonal antibody mRNA obtained in (1) above, using SMART RACE cDNA Amplification Kit (Clontech), the attached CDNA was obtained according to the instructions for use.

Using the cDNA as a template, a universal primer A mix attached to the kit, a primer specific to rat IgG1 (SEQ ID NO: 8), a primer specific to rat IgG2a (SEQ ID NO: 9), or a primer specific to mouse IgG1 (SEQ ID NO: 10) or PCR reaction using a primer specific to mouse IgG2a (SEQ ID NO: 11) to amplify the cDNA fragment of each antibody heavy chain variable region (hereinafter sometimes referred to as VH) did.

PCR was performed using a rat Ig (κ) -specific primer (SEQ ID NO: 12) or a mouse Ig (κ) -specific primer (SEQ ID NO: 13) instead of each antibody subclass-specific primer. A cDNA fragment of the light chain variable region (hereinafter sometimes referred to as VL) was amplified. PCR is a reaction consisting of 5 reaction cycles consisting of 94 ° C for 15 seconds and 72 ° C for 3 minutes after heating at 94 ° C for 5 minutes, 94 ° C for 15 seconds, 70 ° C for 30 seconds, and 72 ° C for 3 minutes. The reaction was repeated 30 times, consisting of 5 cycles, 94 ° C for 15 seconds, 68 ° C for 30 seconds, 72 ° C for 3 minutes, and then reacted at 72 ° C for 10 minutes. PCR was performed using PTC-200 DNA Engine (manufactured by BioRad).

The obtained PCR products were separated by agarose gel electrophoresis in order to clone and determine the base sequence, and the H chain and L chain were extracted using Gel Extraction Kit (manufactured by QIAGEN). Target Clone Plus (manufactured by TOYOBO) was used for the extracted fragment, and after the dA addition reaction according to the attached instruction, VH or VL of each antibody was incorporated into the pTA2 vector (manufactured by TOYOBO).

Using the vector thus obtained, Escherichia coli DH5α strain was transformed according to the instruction manual attached to Competent Quick (manufactured by TOYOBO). A plasmid was extracted from the obtained transformant using an automatic plasmid extractor (manufactured by Kurabo Industries), and reacted according to the attached instructions using BigDye Terminator Cycle Sequencing FS Ready Reaction Kit (manufactured by PE Biosystems). The base sequence of the PCR product cloned by the sequencer ABI PRISM 3700 was analyzed.

As a result, a plasmid containing a full-length H chain cDNA and a plasmid containing an L chain cDNA in which an ATG sequence presumed to be the initiation codon was present at the 5 'end of the cDNA were obtained.

(3) Analysis of gene sequence of V region of anti-CD33 monoclonal antibody The entire nucleotide sequence of VH of hybridomas KM4071 to KM4075 contained in the plasmid obtained in (2) above is represented by SEQ ID NOs: 14 to 18, The estimated entire amino acid sequence of VH including the signal sequence is represented by SEQ ID NOs: 19 to 23, the entire nucleotide sequence of VL is represented by SEQ ID NOs: 24-28, and the entire VL including the signal sequence is estimated from the sequences. Amino acid sequences are shown in SEQ ID NOs: 29 to 33, respectively.

Further, the base sequence obtained by removing the signal sequence from the base sequence shown in SEQ ID NOs: 14 to 18 is shown in SEQ ID NO: 122-126, and the base sequence obtained by removing the signal sequence from the base sequences shown in SEQ ID NOs: 24-28 is shown in SEQ ID NO: 127 -131, the amino acid sequence obtained by removing the signal sequence from the amino acid sequence shown in SEQ ID NOs: 19-23, and the amino acid sequence obtained by removing the signal sequence from the amino acid sequences shown in SEQ ID NOs: 29-33, The numbers 137 to 141 are shown respectively.

Sequence data of known rat and mouse antibodies [SEQUENCES of Proteins of Immunological Interest, US Dept. According to comparison with Health and Human Services (1991)], each isolated cDNA is a full-length cDNA encoding KM4071 to KM4075 including a secretory signal sequence, and the KM4071 heavy chain is represented by SEQ ID NO: 19. The 1st to 19th amino acid sequence of the sequence is the secretion signal sequence for the KM4071 L chain, the 1st to 19th amino acid sequence of the amino acid sequence shown in SEQ ID NO: 29, and the KM4072 H chain is represented by SEQ ID NO: 20 The amino acid sequence from 1 to 18 of the amino acid sequence shown is the secretory signal sequence for the L chain of KM4072 and the amino acid sequence of 1 to 22 of the amino acid sequence shown in SEQ ID NO: 30 is the sequence for the H chain of KM4073 Ami shown in number 21 The 1st to 18th amino acid sequence of the acid sequence is the secretory signal sequence for the L chain of KM4073, the 1st to 20th amino acid sequence of the amino acid sequence shown in SEQ ID NO: 31, and the SEQ ID NO: 22 for the H chain of KM4074 The amino acid sequence from 1 to 20 of the amino acid sequence shown in Fig. 4 is the secretory signal sequence for the KM4074 L chain, the amino acid sequence from 1 to 19 of the amino acid sequence shown in SEQ ID NO: 32, and the KM4075 H chain is It is clear that the 1st to 20th amino acid sequence of the amino acid sequence shown in SEQ ID NO: 23 is the secretory signal sequence, and the 1st to 19th amino acid sequence of the amino acid sequence shown in SEQ ID NO: 33 for the KM4075 L chain is the secretory signal sequence. became.

The CDRs of VH and VL of each monoclonal antibody were identified by comparing with the amino acid sequences of known antibodies.

The amino acid sequences of CDR1, CDR2, and CDR3 of VH of KM4071 are shown in SEQ ID NOs: 34, 35, and 36, and the amino acid sequences of CDR1, CDR2, and CDR3 of VL are shown in SEQ ID NOs: 37, 38, and 39, respectively.

The amino acid sequences of CDR1, CDR2 and CDR3 of VH of KM4072 are shown in SEQ ID NOs: 40, 41 and 42, and the amino acid sequences of CDR1, CDR2 and CDR3 of VL are shown in SEQ ID NOs: 43, 44 and 45, respectively.

The amino acid sequences of CDR1, CDR2 and CDR3 of VH of KM4073 are shown in SEQ ID NOs: 46, 47 and 48, and the amino acid sequences of CDR1, CDR2 and CDR3 of VL are shown in SEQ ID NOs: 49, 50 and 51, respectively.

The amino acid sequences of CDR1, CDR2, and CDR3 of VH of KM4074 are shown in SEQ ID NOs: 52, 53, and 54, and the amino acid sequences of CDR1, CDR2, and CDR3 of VL are shown in SEQ ID NOs: 55, 56, and 57, respectively.

The amino acid sequences of CDR1, CDR2 and CDR3 of VH of KM4075 are shown in SEQ ID NOs: 58, 59 and 60, and the amino acid sequences of CDR1, CDR2 and CDR3 of VL are shown in SEQ ID NOs: 61, 62 and 63, respectively.

[Example 6]
Preparation of anti-CD33 chimeric antibody In an evaluation method using immunostaining such as ELISA, FCM, etc., the activity between antibodies of different species, or antibodies of different subclasses of the same species, is the reactivity of the detection antibody. It cannot be compared because it is different. Therefore, a chimeric antibody in which the constant regions KM4071 to KM4075 established in Example 3 were replaced with human constant region sequences was prepared according to the following method. Thus, by aligning the constant region with human-derived sequences, various activities depending on the constant region can be measured and compared.

(1) Construction of anti-CD33 chimeric antibody expression vector The chimeric antibody produced this time is a strong complement-dependent cytotoxic activity consisting of a mixed sequence of human IgG1 and human IgG3 (hereinafter sometimes referred to as CDC activity). A chimeric antibody comprising a constant region containing (hereinafter, an antibody containing the constant region may be referred to as a 113F antibody) and the variable region of the monoclonal antibody obtained in Example 5 (2).

As a method, high CDC type chimeric antibody expression vector PKTX93 / 113F (described in US Patent Application Publication No. 2007/0148165) containing the variable part of anti-CD20 antibody and obtained in Example 5 (2) were used. Using a plasmid in which the VH or VL of each monoclonal antibody was incorporated into the pTA2 vector, an anti-CD33 chimeric antibody expression vector was constructed as follows.

Using 100 ng of pTA2 vector incorporating VH or VH prepared in Example 5 (2) as a template for VH or VL of each monoclonal antibody, 5 × L of 10 × KOD Plus buffer, 2 μL of dNTP 5 μL, 25 mmol / L A solution consisting of 2 μL of magnesium chloride, 2.5 μL of DMSO, 1 μL of KOD plus polymerase (manufactured by TOYOBO), and a total volume of 50 μL containing 1 μL of each 10 μmol / L primer specific for VH and VL of each monoclonal antibody, 94 After heating at ° C for 2 minutes, the reaction was carried out for 30 cycles of 94 ° C for 15 seconds, 60.6 ° C for 30 seconds, and 68 ° C for 1 minute.

The base sequence of the KM4071 VH primer is SEQ ID NOs: 64 and 65, the base sequence of the VL primer is SEQ ID NOs: 66 and 67, the base sequence of the KM4072 VH primer is SEQ ID NOs: 68 and 69, and the base sequence of the VL primer is SEQ ID NO: 70 and 71, KM4073 VH primer base sequence is SEQ ID NO: 72 and 73, VL primer base sequence SEQ ID NO: 74 and 75, KM4074 VH primer base sequence is SEQ ID NO: 76 and 77, VL SEQ ID NOs: 78 and 79 are shown in SEQ ID NOs: 78 and 79, SEQ ID NOs: 80 and 81 are shown in the sequence of VH primers in KM 4075, and SEQ ID NOS: 82 and 83 are used in the base sequences of VL primers.

By this reaction, a gene sequence encoding VH or VL of each antibody to which a restriction enzyme recognition sequence for insertion into the vector pKANTEX93 (described in WO 97/10354) was added was synthesized. Each PCR reaction product was purified using PCR Purification Kit (manufactured by QIAGEN).

The VH of each antibody thus obtained was subjected to restriction enzyme treatment with ApaI (manufactured by New England Biolabs) and NotI (manufactured by New England Biolabs) to obtain a NotI-ApaI fragment of VH.

Further, the VL of each antibody was subjected to restriction enzyme treatment with BsiWI (manufactured by New England Biolabs) and EcoRI (manufactured by New England Biolabs) to obtain an EcoRI-BsiWI fragment of VL. Similarly, the vector PKTX93 / 113F was subjected to restriction enzyme treatment with NotI and ApaI, or EcoRI and BsiWI.

The obtained VH or VL fragment of each monoclonal antibody was ligated with a restriction enzyme-treated vector and Ligation high (manufactured by TOYOBO) according to the attached instructions, and the resulting recombinant plasmid DNA solution was used. E. coli DH5α strain (manufactured by TOYOBO) was transformed. Each plasmid DNA was prepared from the clone of the transformant and confirmed by restriction enzyme treatment to obtain a plasmid into which the ApaI-NotI fragment of the target VH or the EcoRI-BSIWI fragment of VL was inserted.

The plasmid was reacted using the BigDye Terminator Cycle Sequencing FS Ready Reaction Kit (manufactured by PE Biosystems) according to the attached instructions, and the base sequence was analyzed by the sequencer ABI PRISM 3700 of the company.

As a result, a vector in which a cDNA encoding KM4071 to KM4075 VH was cloned and a vector in which a cDNA encoding KM4071 to KM4075 VL was cloned were obtained. The thus obtained PKTX93 / 113F vector incorporating VH or VL of each antibody was subjected to restriction enzyme treatment by adding EcoRI and NotI, and the EcoRI-NotI fragment of VL and the PKTX93 / 113F vector containing VH were incorporated. A NotI-EcoRI fragment was obtained.

These two kinds of fragments were ligated using Ligation high (manufactured by TOYOBO), and Escherichia coli DH5α strain (manufactured by TOYOBO) was transformed with the obtained recombinant plasmid DNA solution. Each plasmid DNA was prepared from the clone of the transformant and confirmed by restriction enzyme treatment to obtain a plasmid into which the EcoRI-NotI fragment of the target VL was inserted.

The plasmid was reacted using the BigDye Terminator Cycle Sequencing FS Ready Reaction Kit (manufactured by PE Biosystems) according to the attached instructions, and the base sequence was analyzed by the sequencer ABI PRISM 3700 of the company. As a result, an anti-CD33 chimeric antibody expression vector in which cDNAs encoding VH and VL of KM4071 to KM4075 were cloned was obtained. A schematic diagram of vector construction is shown in FIG.

(2) Expression of anti-CD33 chimeric antibody in animal cells Using the anti-CD33 chimeric antibody expression vector obtained in (1) above, the expression of anti-CD33 chimeric antibody in animal cells was determined using conventional methods [Antibody Engineering, A Practical. Guide, W.M. H. Freeman and Company (1992)], transformants KM4051 to KM4055 producing anti-CD33 chimeric antibodies were obtained (Table 3).

As an animal cell line to be expressed, a CHO / DG44 cell line (hereinafter sometimes referred to as FUT8 knockout CHO cell or CHO / Ms704) in which the α1,6-fucosyltransferase (FUT8) gene was double knocked out was used. It is known that fucose is not added to the core part of the N-linked complex sugar chain of the antibody expressed in this host cell line (WO 2002/31140).

Further, it is known that the human IgG1 antibody to which fucose is not added has higher antibody-dependent cytotoxic activity (hereinafter sometimes referred to as ADCC activity) than the case where it is added. Hereinafter, an antibody to which fucose is not added may be referred to as a high ADCC type antibody.

(3) Acquisition of purified chimeric antibody After KM4051 to KM4055 obtained in (2) above were cultured by a normal culture method, the cell suspension was recovered and centrifuged for 20 minutes at 3000 rpm and 4 ° C. After collecting the culture supernatant, the solution was sterilized by filtration through a 0.22 μm pore size Millex GV filter (manufactured by Millipore). KM4051 to KM4055 were purified from the obtained culture supernatant using a Protein A High-capacity resin (Millipore) column according to the attached instructions.

(4) Measurement of fucose content of anti-CD33 chimeric antibody According to the method described in International Publication No. 2002/31140, fucose at position 6 of N-acetylglucosamine at the reducing end of the N-linked sugar chain that binds to Fc of each chimeric antibody The proportion of sugar chains that were not α-bonded at the 1-position was examined. The results are shown in Table 4.

As shown in Table 4, it was confirmed that fucose was not added to the chimeric antibody obtained in (3) above.

[Example 7]
Evaluation of Activity of Anti-CD33 Chimeric Antibody In the following (1) to (6), KM4051 to KM4055 obtained in Example 6 and the anti-CD33 antibody HM195 IgG1 type antibody (hereinafter referred to as HM195) prepared in Example 8 HM195 high ADCC type antibody (hereinafter sometimes referred to as HM195-P), HM195 type 113F and high ADCC type antibody (hereinafter referred to as HM195-113F). Activity evaluation was performed.

(1) Reactivity with human CD33-positive cell line in FCM 1-5 × 10 ⁵ cells of THP-1 were diluted with serum from a diluted mouse serum (CEDARLANE LABORATORIES) and blocked, and KM4051 to KM4055 and HM195-113F Was appropriately diluted with the above FCM buffer to make a total volume of 100 μL. These cell suspensions were reacted for 60 minutes on ice and washed 3 times with PBS.

30 μL of FITC-labeled goat anti-human IgG (Fc) antibody (manufactured by Acris Antibodies) diluted with FCM buffer was added to the cells and reacted on ice for 40 minutes. After washing with PBS three times, the suspension was suspended in FCM buffer and the fluorescence intensity was measured using a flow cytometer (manufactured by Beckman Coulter).

FIG. 6 shows the MFI when the KM4051 to KM4055 obtained in Example 6 are reacted stepwise diluted from 2 μg / mL at a 4-fold dilution at each concentration. It was confirmed that KM4051 to KM4055 and HM195-113F all bind to THP-1 in a concentration-dependent manner.

(2) Evaluation of binding activity to recombinant human CD33-Fc by binding ELISA was performed according to the method described in Example 3 (3). Recombinant human CD33-Fc or goat anti-human IgG (H + L) antibody (American Qualex) was adsorbed on the plate. As the primary antibody, KM4051 to KM4055 obtained in Example 6 and HM195-113F obtained in Example 8 were diluted stepwise from 2 μg / mL at a 5-fold dilution.

Peroxidase-labeled goat anti-human IgG (H + L chain) antibody (American Qualex) or peroxidase-labeled goat anti-human κ chain (Southern Biotech) was used as the secondary antibody. The results of recombinant human CD33-Fc and goat anti-human IgG (H + L) antibody are shown in FIGS. 7 (A) and (B), respectively.

As shown in FIG. 7, KM4051 to KM4055 and HM195-113F all showed reactivity to recombinant human CD33-Fc.

(3) Binding activity to recombinant human CD33-Fc in Biacore In order to analyze the binding activity of KM4051 to KM4055 and HM195-113F to hCD33-His, binding was performed using the surface plasmon resonance method (SPR method). Activity measurement was performed.

The following operations were all performed using Biacore T-100 (GE Healthcare Bioscience). Using an Human Antibody Capture Kit (manufactured by GE Healthcare Bioscience), the anti-human IgG antibody was solidified on a CM5 sensor chip (manufactured by GE Healthcare Bioscience) by the amine coupling method according to the attached protocol.

Samples (KM4051 to KM4055 and HM195-113F) were added to the chip on which the anti-human IgG antibody was solidified, and captured to about 300 RU (resonance unit), and then diluted from 1.25 μg / mL in 5 steps The hCD33-His was flowed over the chip at a rate of 30 μL / min, and sensorgrams at each concentration were obtained, analyzed using a 1: 1 binding model using analysis software attached to the device, and each antibody against human CD33 The association rate constant ka and the dissociation rate constant kd were calculated.

The resulting ka of each antibody, the kd and dissociation constant, K _D, shown in Table 5.

As shown in Table 5, all of the chimeric antibodies showed high affinity exceeding HM195-113F.

(4) Evaluation of ADCC activity of anti-CD33 chimeric antibody against human hematological cancer cell line ADCC activity of KM4051 to KM4055, HM195-1, and HM195-113F was measured according to the following method.

(4) -1 Preparation of target cell solution After washing CD33-positive human acute promyelocytic leukemia cell line NB-4 with PBS, phenol red-free RPMI 1640 medium containing 5% fetal bovine serum (dFBS) , Sometimes referred to as ADCC medium), and adjusted to an optimal concentration with the same medium to obtain a target cell solution.

(4) -2 Preparation of effector cell solution PBMCs were separated from healthy human peripheral blood by the method described below. 50 mL of healthy human peripheral blood was collected with a syringe containing 0.5 mL of heparin sodium injection N “Shimizu” (manufactured by Shimizu Pharmaceutical Co., Ltd.). The collected peripheral blood was diluted by adding the same amount of physiological saline (manufactured by Otsuka Pharmaceutical) and stirred well. On a Lymphoprep (Axis-Shield) dispensed 4.7 mL into a 15 mL tube (Greiner), 10 mL of diluted peripheral blood is gently layered, and then 20 minutes at 2000 rpm, brake off, and room temperature. Keep in mind to separate the mononuclear cell layer. The thus obtained mononuclear cell fraction was washed twice with an ADCC medium, adjusted to an optimal concentration with the same medium, and used as an effector cell solution.

(4) -3 Measurement of ADCC activity CytoTox96 Non-Radioactive Cytotoxicity Assay (manufactured by Promega) was used and measured according to the following procedure.

50 μL of antibody solution diluted to an appropriate concentration is dispensed into each well of a 96-well U-bottom plate (manufactured by FALCON), and 50 μL of the target cell solution prepared in (4) -1 is prepared in (4) -2. 50 μL of the effector cell solution was added to a total volume of 150 μL, and reacted at 37 ° C. for 4 hours. ADCC activity was determined by the following formula.

(formula)
ADCC activity (%) = {([Absorbance of specimen]-[Absorbance of spontaneous release of target cell]-[Absorption of spontaneous release of effector cell]) / ([Absorbance of total release of target cell]-[Absorbance of spontaneous release of target cell] ]) X 100

The results are shown in FIG. The target cells were 1.0 × 10 ⁴ cells / well, and the effector cells were 2 × 10 ⁵ cells / well (effector cells (E): target cells (T) = 20: 1). The antibody concentration was diluted 10-fold in steps from 1 μg / mL.

As shown in FIG. 8, KM4051 to KM4055, HM195-1, and HM195-113F showed antibody concentration-dependent ADCC activity against NB-4. In addition, KM4051 to KM4055 showed stronger ADCC activity than HM195-113F.

(5) Evaluation of CDC activity of CD33 transfectant of anti-CD33 chimeric antibody The CDC activity of KM4051 to KM4055 was measured according to the method shown below.

After washing the CD33 transfectant with PBS, the CD33 transfectant was washed with RPMI 1640 medium (Wako Pure Chemical Industries, Ltd.) containing 10% fetal bovine serum (FBS). . The target cell solution is added to a 96-well flat bottom plate (manufactured by Sumitomo Bakelite Co., Ltd.) so that the number of target cell solutions is 5 × 10 ⁴ per well, and the anti-CD33 chimeric antibody solution prepared to an appropriate concentration and the final concentration is diluted 4-fold. Human complement (manufactured by SIGMA) was added to prepare 100 μL per well.

Also, a reaction well containing no antibody (0% Lysis well) was prepared as a control when CDC was not induced, and a reaction well containing no cells (100% Lysis well) was prepared as a control when CDC was induced. The reaction was performed in a 37 ° C. incubator for 3 hours.

After completion of the reaction, 10 μL of WST-1 reagent (manufactured by ROCHE) is added to each reaction well, reacted at 37 ° C. for about 3 hours, OD450 in each well is measured, and the following formula is used from the absorbance of each well. CDC activity (cytotoxic activity [%]) was calculated. The result is shown in FIG.

(formula)
CDC activity (cytotoxic activity [%]) = 100 × {1- (absorbance of reaction well−absorbance of 100% Lysis well) / (absorbance of 0% Lysis well−absorbance of 100% Lysis well)}

As a result, as shown in FIG. 9, KM4051 to KM4055 all exhibited CDC activity.

(6) Antibody binding amount on cell membrane of CD33-expressing cell line of anti-CD33 chimeric antibody The amount of antibody binding over time of anti-CD33 chimeric antibody and HM195-113F on the cell membrane of THP-1 was measured according to the following method. .

Diluted mouse serum (CEDARLANE LABORATORIES) was added to 1-5 × 10 ⁵ THP-1 for blocking, and KM4051-KM4055 and HM195-113F shown in Example 8 were appropriately diluted with 10% FCS-RPMI. Was added to make a total volume of 100 μL.

These cell suspensions were reacted on ice for 40 minutes and then incubated at 37 ° C. for 30, 60 and 120 minutes. Thereafter, the cells were washed 3 times with cold PBS, and 30 μL of FITC-labeled goat anti-human IgG (Fc) antibody (manufactured by Acris Antibodies) diluted with the above FCM buffer was added to the cells and reacted on ice for 40 minutes. After washing with PBS three times, the suspension was suspended in FCM buffer and the fluorescence intensity was measured using a flow cytometer (manufactured by Beckman Coulter).

The obtained results are shown in FIG. 10 ((A) and (B) in FIG. 10 show the amount of antibody binding and the antibody remaining rate, respectively). As shown in FIG. 10, KM4051 to KM4055 and HM195-113F decreased the amount of binding on the cell surface in a time-dependent manner. In each clone, KM4051 to KM4055 exceeded both HM195-113F and the amount of antibody bound to the cell surface and the residual ratio at each time.

[Example 8]
Production of HM195 IgG1-type and 113F-type antibodies (1) Production of HM195-1 and HM195-113F expression vectors Human antibodies that specifically recognize human CD33 based on the sequence information described in US Pat. No. 7,022,500 Genes encoding HM195 VH and VL were constructed by the method described in Example 6 (1) and incorporated into vector pKANTEX93 and vector PKTX93 / 113F. A schematic diagram of vector construction is shown in FIG.

(2) Expression of HM195-1 and HM195-113F in animal cells The vector obtained in (1) above was used and the method described in Example 6 (2).
As animal cells to express HM195-1, CHO / DG44 cells and CHO / Ms704 cells were used to express normal and non-fucose-added antibodies. CHO / Ms704 cells were used as animal cells that express HM195-113F.

(3) Obtaining HM195-1, HM195-P, and HM195-113F Purified Antibody The animal cell line prepared in (2) above was used and the method described in Example 6 (3) was performed.

[Example 9]
Preparation of anti-CD33 chimeric antibody having no consensus sequence for binding N-linked sugar chain to variable region Anti-CD33 chimeric antibody having no consensus sequence for binding N-linked sugar chain to variable region (hereinafter referred to as modified anti-antibody) CD33 chimeric antibody) was prepared as described below in Example 3 based on KM4074. The antibody had a consensus sequence to which an N-linked sugar chain was bound in the amino acid sequence of CDR2 of VH represented by SEQ ID NO: 53.

(1) Design of amino acid sequence of recombinant antibody having no consensus sequence to which N-linked sugar chain binds to V region The consensus sequence to which the N-linked sugar chain contained in VH of KM4074 binds is SEQ ID NO: 22 Is a sequence consisting of 71st Asn, 72nd Ser, and 73rd Ser of the amino acid sequence shown in FIG. In the amino acid sequence shown in SEQ ID NO: 22, CDR2 has the amino acid sequence shown in SEQ ID NO: 53. In the amino acid sequence represented by SEQ ID NO: 53, the consensus sequence to which the N-linked sugar chain binds is a sequence composed of the third Asn, the fourth Ser, and the fifth Ser.

The consensus sequence to which the N-linked sugar chain binds is that the fourth amino acid is arbitrary in the amino acid sequence represented by SEQ ID NO: 53, and therefore the third Asn and the fifth Ser were used as candidate modification residues. Existing antibody sequences [Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services (1991)] and the amino acid modification and side chain structure in consideration of the amino acid modification and the third Asn are considered as Tyr, Ser, or Gln. The amino acid modification which substitutes either of these, or the amino acid modification which substitutes 5th Ser to either Ala or Gly was selected.

Specifically, ver.3 in which the third Asn is replaced with Tyr. 1 and 3 where Asn is replaced with Ser. The second and third Asn were replaced with Gln ver. 3 and 5 where Ser was replaced with Ala. 4th and 5th Ser were replaced with Gly. Five VHs of 5 types of modified CD33 chimeric antibodies were designed. Ver. 1 to ver. The amino acid sequences of each VH of 5 are shown in SEQ ID NOs: 103, 104, 105, 93, and 106, respectively. Each Ver. The amino acid sequences of CDR2 of VH are shown in SEQ ID NOs: 99, 100, 101, 96 and 102, respectively. Hereinafter, ver. Antibody with 1 is simply ver. Sometimes referred to as 1.

Thus, the VH of the antibody is an amino acid sequence represented by any one of SEQ ID NOs: 103, 104, 105, 93, and 106, and the VL of the antibody is SEQ ID NO: 95 (represented by SEQ ID NO: 32). A modified CD33 chimeric antibody having an amino acid sequence represented by the amino acid sequence minus the signal sequence was designed.

(2) Production of expression vector of modified CD33 chimeric antibody Amino acid modification was introduced using KM4054 expression vector having the same V region as KM4074 produced in Example 6 as a template. For introducing amino acid modifications,
Ver. For the preparation of 1, primers having the base sequences shown in SEQ ID NOs: 107 and 108 are used.
Ver. 2 is prepared by using primers having the nucleotide sequences represented by SEQ ID NOs: 109 and 110,
Ver. 3 is prepared by using primers having the nucleotide sequences represented by SEQ ID NOs: 111 and 112,
Ver. For the preparation of 4, primers having the base sequences shown in SEQ ID NOs: 97 and 98 are used.
Ver. 5 was prepared using primers having the nucleotide sequences shown in SEQ ID NOs: 113 and 114, and using the QuickChange Site-Directed Mutagenesis Kit (manufactured by Stratagene) according to the attached instruction manual. The obtained plasmid DNA was obtained.

The obtained vector was analyzed using the BigDye Terminator Cycle Sequencing FS Ready Reaction Kit (manufactured by PE Biosystems) according to the attached instructions, and the base sequence of the PCR product cloned by its sequencer ABI PRISM3700 was analyzed. It was confirmed that a modified antibody expression vector having been modified was obtained.

(3) Expression of modified CD33 chimeric antibody in animal cells Using the modified antibody expression vector obtained in (2) above, a transformant was obtained by the same method as in Example 6- (2).

(4) Obtaining purified modified CD33 chimeric antibody Using each transformed strain obtained in (3) above, purifying the modified CD33 chimeric antibody purified in the same manner as in Example 6- (3). I got it.

[Example 10]
Activity evaluation of the modified CD33 chimeric antibody The activity of the KM4054 obtained in Example 6 and the modified CD33 chimeric antibody prepared in Example 9 were evaluated.

(1) Binding activity with hCD33-His in Biacore In order to analyze the binding activity of the modified CD33 chimeric antibody produced in KM4054 and Example 9 to hCD33-His kinetically, surface plasmon resonance method (SPR method) ) Was used to measure the binding activity by the same method as in Example 7- (3).

The resulting ka of each antibody, the kd and dissociation constant, K _D, shown in Table 6.

As shown in Table 6, modified CD33 chimeric antibody ver. 1 to 5 have an affinity equivalent to that of KM4054. 4 was named KM4084.

The amino acid sequences of CDR1, CDR2 and CDR3 of VH of KM4084 are shown in SEQ ID NOs: 52, 96 and 54, respectively, and the amino acid sequences of CDR1, CDR2 and CDR3 of VL are shown in SEQ ID NOs: 55, 56 and 57, respectively.

In addition, the nucleotide sequence of the DNA encoding the amino acid sequence of KM4084 represented by SEQ ID NO: 93 is represented by SEQ ID NO: 92, and the nucleotide sequence of the DNA encoding the amino acid sequence of VL of KM4084 represented by SEQ ID NO: 95 is sequenced. The number 94 is shown.

[Example 11]
Preparation of anti-CD33 humanized antibody (1) Design of amino acid sequences of VH and VL of anti-CD33 humanized antibody The amino acid sequence of VH of anti-CD33 humanized antibody was designed as follows. First, in order to transplant the amino acid sequence of CDR1 to 3 of VH of KM4084 (SEQ ID NO: 52, 96, 54), the amino acid sequence of the VH framework region of human antibody (hereinafter sometimes referred to as FR) Selected.

Kabat et al. Classify VHs of various known human antibodies into subgroups (HSG I-III) based on their amino acid sequence homology, and report a common sequence for each subgroup [Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services (1991)]. Therefore, a homology search was performed between the FR amino acid sequence of the consensus sequence of VH subgroups I to III of the human antibody and the FR amino acid sequence of KM4084VH.

As a result of searching for homology, the homology of HSGI, HSGII, and HSGIII was 50.6%, 67.1%, and 54.1%, respectively. Therefore, the FR amino acid sequence of KM4084VH had the highest homology with subgroup II.

Based on the above results, the amino acid sequence of CDRs 1 to 3 (SEQ ID NOs: 52, 96, and 54) of KM4084VH was transplanted to the appropriate position of the FR amino acid sequence of the consensus sequence of VH subgroup II of the human antibody. In this way, the amino acid sequence HV0 (SEQ ID NO: 84) of VH of the KM4084 humanized antibody was designed.

Next, the amino acid sequence of VL of the KM4084 humanized antibody was designed as follows. In order to graft the amino acid sequence of CDR1-3 of VL of KM4084 (SEQ ID NOs: 55-57), the amino acid sequence of FR of VL of human antibody was selected. Kabat et al. Classify VL of various known human antibodies into subgroups (HSG I-IV) based on their amino acid sequence homology, and report common sequences for each subgroup [Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services (1991)]. Therefore, a homology search was performed between the FR amino acid sequence of the consensus sequence of subgroups I to IV of human antibody VL and the FR amino acid sequence of KM4084VL.

As a result of searching for homology, the homology of HSGI, HSGII, HSGIII, and HSGIV was 60.0%, 77.5%, 65.0%, and 65.0%, respectively. Therefore, the amino acid sequence of FR of KM4084VL had the highest homology with subgroup II.

Based on the above results, the amino acid sequence of CDRs 1 to 3 (SEQ ID NOs: 55 to 57) of KM4084VL was transplanted to the appropriate position of the FR amino acid sequence of the consensus sequence of subgroup II of the VL of the human antibody. However, the 4th Leu and 109th Leu in the amino acid sequence of KM4084VL (SEQ ID NO: 95) are the most frequently used amino acid residues at the site corresponding to the amino acid sequence of human antibody FR mentioned by Kabat et al. Although it is not a group, since it is an amino acid residue that is used at a relatively high frequency, the amino acid residue recognized in the amino acid sequence of KM4084 is used. In this way, the amino acid sequence LV0 (SEQ ID NO: 85) of the VL of the KM4084 humanized antibody was designed.

The VH amino acid sequence HV0 and VL amino acid sequence LV0 of the KM4084 humanized antibody designed above are sequences in which only the amino acid sequence of the CDR of KM4084 is grafted to the FR amino acid sequence of the selected human antibody. However, in general, when a humanized antibody is prepared, the binding activity often decreases only by transplanting the amino acid sequence of the CDR of the mouse antibody to the FR of the human antibody.

For this reason, in order to avoid a decrease in binding activity, along with the grafting of CDR amino acid sequences, among amino acid residues of FR that differ between human and mouse antibodies, amino acid residues that are thought to affect binding activity Modifications are being made. Therefore, FR amino acid residues that are thought to affect the binding activity were identified as follows.

First, using a computer modeling technique, the three-dimensional structure of the antibody V region (hereinafter sometimes referred to as HV0LV0) consisting of the VH amino acid sequence HV0 and the VL amino acid sequence LV0 of the KM4084 humanized antibody designed above is used. It was constructed. Discovery Studio (manufactured by Accelrys Co., Ltd.) was used according to the attached instruction manual for preparing the three-dimensional structure coordinates and displaying the three-dimensional structure.

The computer model of the three-dimensional structure of the V region of KM4084 was also constructed in the same manner. Furthermore, in the amino acid sequence of HV0LV0 VH and VL FR, an amino acid residue different from KM4084 is selected, an amino acid sequence modified to KM4084 amino acid residue is prepared, and a three-dimensional structure model is constructed in the same manner. did. The three-dimensional structures of the V regions of these prepared KM4084, HV0LV0 and the variant were compared, and amino acid residues predicted to affect the binding activity of the antibody were identified.

As a result, among the amino acid residues of FR of HV0LV0, the three-dimensional structure of the antigen binding site is changed, and the amino acid residues that are thought to affect the binding activity of the antibody are 25th Ser, 27th in HV0. Gly, 28th Ser, 29th Val, 30th Ser, 40th Gln, 45th Gly, 46th Leu, 49th Ile, 72nd Val, 93th Val, 95th Tyr, 97th Ala, and 106th Thr were selected for LV0 as 2nd Ile, 8th Pro, 11th Leu, 15th Pro, and 90th Val, respectively.

Among these selected amino acid residues, at least one amino acid sequence was modified to an amino acid residue present at the same site of KM4084, and VH and VL of humanized antibodies having various modifications were designed. Specifically, for VH, the 25th Ser of the amino acid sequence of SEQ ID NO: 84 is Thr, the 27th Gly is Tyr, the 28th Ser is Thr, the 29th Val is Ile, and the 30th Ser to Thr, 40th Gln to Lys, 45th Gly to Arg, 46th Leu to Met, 49th Ile to Met, 72nd Val to Arg, 93th Val At least one of the amino acid modifications was introduced into Thr, replacing 95th Tyr with Phe, 97th Ala with Thr, and 106th Thr with Val.

For VL, the second Ile of the amino acid sequence of SEQ ID NO: 85 is Val, the 8th Pro is Leu, the 11th Leu is Gln, the 15th Pro is Leu, and the 90th Val. At least one modification among the amino acid modifications in which is substituted for Leu was introduced.

HV0LV0, HV7LV0, and HV14LV0 were each designed as an antibody V region of an anti-CD33 humanized antibody in which at least one amino acid residue was present in the FR of HV0LV0. The amino acid sequences of the heavy chain variable regions HV7 and HV14 are shown in SEQ ID NOs: 86 and 87, respectively.

(2) Preparation of anti-CD33 humanized antibody The DNA encoding the variable region amino acid sequence of the anti-CD33 humanized antibody is the codon used in the DNA encoding the amino acid sequences of KM4084VH and KM4084VL (SEQ ID NOs: 92 and 94). When amino acid modification is carried out using a codon, it was prepared using codons frequently used in mammalian cells.

The DNA sequences encoding the amino acid sequences of HV0 and LV0 of the anti-CD33 humanized antibody are shown in SEQ ID NOs: 88 and 89, respectively, and the DNA sequences encoding the amino acid sequences of the variable regions HV7 and HV14 subjected to amino acid modification are The numbers 90 and 91 are shown respectively.

In the same manner as in Example 6, DNA encoding each variable region was prepared and inserted into the antibody expression vector pKANTEX93 to prepare an anti-CD33 humanized antibody expression vector. That is, according to the method of Example 6 (2) and (3), the prepared anti-CD33 humanized antibody expression vector is introduced into animal cells to express the humanized antibody as a high ADCC type antibody, and anti-CD33 having hIgG1 as CH. Humanized antibodies HV0LV0, HV7LV0, and HV14LV0 were generated.

(3) Binding activity with hCD33-His in Biacore In order to analyze the binding activity of each anti-CD33 humanized antibody obtained in (2) above with respect to hCD33-His, the surface plasmon resonance method (SPR method) is used. The binding activity was measured by the same method as in Example 7- (3).

The resulting ka of each antibody, the kd and dissociation constant, K _D, shown in Table 7.

As shown in Table 7, binding activity was not recognized in HV0LV0 among the prepared anti-CD33 humanized antibodies. On the other hand, both HV7LV0 and HV14LV0 had high affinity for hCD33-His. Further, the affinity was equal to or higher than that of KM4054. Therefore, an anti-CD33 humanized antibody having high binding activity was produced.

[Example 12]
Evaluation of activity of anti-CD33 humanized antibody In the following (1) to (4), KM8084 which is a high ADCC type antibody of HV7LV0 of KM4084 produced in Example 11 and HM195-1, HM1955-P produced in Example 8 The activity was evaluated. As a negative control antibody, a high ADCC type antibody (anti-DNP) against dinitrophenol (DNP) was used. In addition, the antibody-free sample was shown as -Ab.

(1) Evaluation of ADCC activity against human blood cancer cell line It was performed according to the method described in Example 7 (4). As target cells, THP-1, NB-4, human erythroleukemia cell line TF-1, and human acute megakaryocytic leukemia cell line CMK11-5 were used. Target cells were 1.0 × 10 ⁴ cells / well, and effector cells were 2.5 × 10 ⁵ cells / well (effector cells (E): target cells (T) = 25: 1). The antibody concentration was diluted 4-fold stepwise from 0.5 μg / mL. The results are shown in FIG.

As shown in FIG. 12, KM8084, HM195-1, and HM195-P exhibited an antibody concentration-dependent ADCC activity against each cell line. KM8084 showed stronger ADCC activity than HM195-P.

(2) Amount of antibody binding on cell membrane in CD33-expressing cell line This was performed according to the method of Example 7 (6). THP-1 was used as the cell. KM8084 and HM195-P were used as primary antibodies.

The obtained results are shown in FIG. 13 [(A) and (B) of FIG. 13 show the amount of antibody binding and the antibody remaining rate, respectively]. As shown in FIG. 13, KM8084 and HM195-P decreased the amount of binding on the cell surface in a time-dependent manner. In addition, KM8084 exceeded both HM195-P and the amount of antibody bound to the cell surface and the residual rate at each time.

(3) Cell removal activity in human peripheral blood Cell removal activity of KM8084, HM195-1, and HM195-P against NB-4 and TF-1 in the peripheral blood was measured by the following method.

NB-4 and TF-1 were fluorescently labeled according to the instructions attached to PKH26 Red Fluorescent Cell Linker Kit (manufactured by Sigma) and diluted with RPMI 1640 (manufactured by Invitrogen).

Healthy human peripheral blood containing 1% of heparin sodium injection N (manufactured by Shimizu Pharmaceutical Co., Ltd.) in a 24-well plate (manufactured by NUNC) was dispensed at 500 μL / well. 50 μL of each antibody diluted with RPMI1640 and 50 μL of the fluorescently labeled cell solution were added to make a total volume of 600 μL, and the reaction was carried out with shaking and stirring at 37 ° C. for 16 hours in a 5% CO ₂ incubator.

After the reaction, 100 μL / well of Flow-Count (manufactured by Beckman Coulter) was added as an internal standard particle Flow Beas (FB), and after mixing, 400 μL of each well was transferred to a 15 mL centrifuge tube (manufactured by NUNC). . After centrifuging the tube at 400 × g for 5 minutes, the supernatant was removed, 1 mL of ACK Lysing Buffer (manufactured by Lonza) was added to each tube, and the mixture was reacted by shaking at 37 ° C. for 10 minutes.

The tube was washed twice with 1% BSA-PBS, suspended in 500 μL of 1% BSA-PBS, and the number of FB and the number of fluorescently labeled cells were measured using a flow cytometer (manufactured by Beckman Coulter). . From the obtained results, the number of fluorescently labeled cells per 1000 FBs was calculated.

The obtained results are shown in FIG. As shown in FIG. 14, HM195-P, which is a high ADCC type antibody of the same antibody, showed about 10 times higher activity than HM195-1. In addition, KM8084 exhibited a cell removal activity 100 times or more stronger than HM195-1, and several tens of times stronger than HM195-P.

This evaluation system in which leukemia cells are added to human peripheral blood is a system that models the environment in the blood of an acute myeloid leukemia (AML) patient, and is more compared to the ADCC evaluation system of Example 12 (1). It is thought to reflect clinical efficacy. For this reason, it is suggested that KM8084 exhibits an effect higher than that of HM195 for blood cancer such as acute myeloid leukemia and myelodysplastic syndrome accompanying bone marrow cell lesions.

(4) Reactivity to human, cynomolgus monkey and chimpanzee CD33 The reaction was performed according to the method described in Example 7 (1). The cells used were the human CD33-expressing cell line, cynomolgus monkey CD33-expressing cell line, and chimpanzee CD33-expressing cell line prepared in Example 1. As primary antibodies, KM8084, HM195-1, and HM195-P were diluted serially. A FITC-labeled goat anti-human IgG (Fc) antibody (manufactured by Acris Antibodies) was used as the secondary antibody.

FIG. 15 shows the MFI when KM8084, HM195-1, and HM195-P are reacted stepwise diluted from 50 μg / mL at 10-fold dilutions. As shown in FIG. 15, KM8084, HM195-1, and HM195-P all reacted with human CD33-expressing cell lines in a concentration-dependent manner, and there was no difference in the strength of reactivity. On the other hand, KM8084 reacted in a concentration-dependent manner to cynomolgus monkey CD33-expressing cell line and chimpanzee CD33-expressing cell line, but HM195-1 and HM195-P hardly reacted.

The sequence of each SEQ ID NO is as follows.
SEQ ID NO: 1: nucleotide sequence of primer for preparing human CD33 expression vector SEQ ID NO: 2: nucleotide sequence of primer for preparing human CD33 expression vector SEQ ID NO: 3: amino acid sequence of human CD33 SEQ ID NO: 4: nucleotide sequence of human CD33 gene SEQ ID NO: 5 : Nucleotide sequence of primer for preparing hCD33-His expression vector SEQ ID NO: 6: amino acid sequence of hCD33-His SEQ ID NO: 7: nucleotide sequence of hCD33-his sequence SEQ ID NO: 8: nucleotide sequence of rat IgG1-specific primer SEQ ID NO: 9: rat Base sequence of IgG2a-specific primer SEQ ID NO: 10: Base sequence of mouse IgG1-specific primer SEQ ID NO: 11: Base sequence of mouse IgG2a-specific primer SEQ ID NO: 12: Base sequence of rat Ig (κ) -specific primer SEQ ID NO: 13: Mouse Ig (κ) specific Primer nucleotide sequence SEQ ID NO: 14: KM4071 VH nucleotide sequence SEQ ID NO: 15: KM4072 VH nucleotide sequence SEQ ID NO: 16: KM4073 VH nucleotide sequence SEQ ID NO: 17: KM4074 VH nucleotide sequence SEQ ID NO: 18: KM4075 VH nucleotide sequence No. 19: KM4071 VH amino acid sequence SEQ ID NO: 20: KM4072 VH amino acid sequence SEQ ID NO: 21: KM4073 VH amino acid sequence SEQ ID NO: 22: KM4074 VH amino acid sequence SEQ ID NO: 23: KM4075 VH amino acid sequence SEQ ID NO: 24: KM4071 VL Nucleotide sequence SEQ ID NO: 25: KM4072 VL nucleotide sequence SEQ ID NO: 26: KM4073 VL nucleotide sequence SEQ ID NO: 27: KM4074 VL nucleotide sequence SEQ ID NO: 28: KM4075 VL nucleotide sequence SEQ ID NO: 29: K 4071 VL amino acid sequence SEQ ID NO: 30: KM4072 VL amino acid sequence SEQ ID NO: 31: KM4073 VL amino acid sequence SEQ ID NO: 32: KM4074 VL amino acid sequence SEQ ID NO: 33: KM4075 VL amino acid sequence SEQ ID NO: 34: KM4071 VH CDR1 amino acid SEQ ID NO: 35: amino acid sequence of KM4071 VH CDR2 SEQ ID NO: 36: amino acid sequence of KM4071 VH CDR3 SEQ ID NO: 37: amino acid sequence of KM4071 VL CDR1 SEQ ID NO: 38: amino acid sequence of KM4071 VL CDR2 SEQ ID NO: 39: amino acid of KM4071 VL CDR3 SEQ ID NO: 40: KM4072 VH CDR1 amino acid sequence SEQ ID NO: 41: KM4072 VH CDR2 amino acid sequence SEQ ID NO: 42: KM4072 VHC DR3 amino acid sequence SEQ ID NO: 43: KM4072 VL CDR1 amino acid sequence SEQ ID NO: 44: KM4072 VL CDR2 amino acid sequence SEQ ID NO: 45: KM4072 VL CDR3 amino acid sequence SEQ ID NO: 46: KM4073 VH CDR1 amino acid sequence SEQ ID NO: 47: KM4073 VH CDR2 amino acid sequence SEQ ID NO: 48: KM4073 VH CDR3 amino acid sequence SEQ ID NO: 49: KM4073 VL CDR1 amino acid sequence SEQ ID NO: 50: KM4073 VL CDR2 amino acid sequence SEQ ID NO: 51: KM4073 VL CDR3 amino acid sequence SEQ ID NO: 52: KM4074 and Amino acid sequence of KM4084 VH CDR1 SEQ ID NO: 53: Amino acid sequence of KM4074 VH CDR2 SEQ ID NO: 54: KM4074 and KM408 4 VH CDR3 amino acid sequence SEQ ID NO: 55: KM4074 and KM4084 VL CDR1 amino acid sequence SEQ ID NO: 56: KM4074 and KM4084 VL CDR2 amino acid sequence SEQ ID NO: 57: KM4074 and KM4084 VL CDR3 amino acid sequence SEQ ID NO: 58: KM4075 VH CDR1 Amino acid sequence SEQ ID NO: 59: KM4075 VH CDR2 amino acid sequence SEQ ID NO: 60: KM4075 VH CDR3 amino acid sequence SEQ ID NO: 61: KM4075 VL CDR1 amino acid sequence SEQ ID NO: 62: KM4075 VL CDR2 amino acid sequence SEQ ID NO: 63: KM4075 VL CDR3 Amino acid sequence SEQ ID NO: 64: KM4071 VH chimerization primer base sequence SEQ ID NO: 65: KM4071 VH chimerization sequence Limer base sequence SEQ ID NO: 66: chimerization primer of KM4071 VL SEQ ID NO: 67: base sequence of chimerization primer of KM4071 VL SEQ ID NO: 68: base sequence of chimerization primer of KM4072 VH SEQ ID NO: 69: chimerization primer of KM4072 VH Nucleotide sequence SEQ ID NO: 70: nucleotide sequence of KM4072 VL chimerization primer SEQ ID NO: 71: nucleotide sequence of KM4072 VL chimerization primer SEQ ID NO: 72: nucleotide sequence of KM4073 VH chimerization primer SEQ ID NO: 73: chimera of KM4073 VH SEQ ID NO: 74: nucleotide sequence of chimerized primer of KM4073 VL SEQ ID NO: 75: nucleotide sequence of chimerized primer of KM4073 VL SEQ ID NO: 76: chimerized primer of KM4074 VH Base sequence SEQ ID NO: 77: Base sequence of chimerization primer of KM4074 VH SEQ ID NO: 78: Base sequence of chimerization primer of KM4074 VL SEQ ID NO: 79: Base sequence of chimerization primer of KM4074 VL SEQ ID NO: 80: Chimerization of KM4075 VH Primer base sequence SEQ ID NO: 81: KM4075 VH chimerized primer base sequence SEQ ID NO: 82: KM4075 VL chimerized primer base sequence SEQ ID NO: 83: KM4075 VL chimerized primer base sequence SEQ ID NO: 84: KM4084 humanized Antibody VH amino acid sequence HV0
SEQ ID NO: 85: VL amino acid sequence LV0 of the KM4084 humanized antibody
SEQ ID NO: 86: Amino acid sequence HV7 of the VH of the KM4084 humanized antibody
SEQ ID NO: 87: VH amino acid sequence HV14 of KM4084 humanized antibody
SEQ ID NO: 88: base sequence of KM4084 humanized antibody HV0
SEQ ID NO: 89: KM4084 humanized antibody LV0 nucleotide sequence SEQ ID NO: 90: KM4084 humanized antibody HV7 nucleotide sequence SEQ ID NO: 91: KM4084 humanized antibody HV14 nucleotide sequence SEQ ID NO: 92: Ver. 4 VH base sequence / KM4084 VH base acid sequence SEQ ID NO: 93: Ver. 4 VH amino acid sequence / KM4084 VH amino acid sequence
SEQ ID NO: 94: Base sequence of KM4084 VL SEQ ID NO: 95: Amino acid sequence of KM4084 VL SEQ ID NO: 96: Amino acid-modified antibody without the N-linked sugar chain binding consensus sequence Ver. 4 CDR2 sequence / KM4084 VH CDR2 amino acid sequence SEQ ID NO: 97: VH CDR2 amino acid modified antibody ver. 4. Base sequence of primer for preparation (sense strand)
SEQ ID NO: 98: VH CDR2 amino acid modified antibody ver. 4. Base sequence of primer for preparation (antisense strand)
SEQ ID NO: 99: Amino acid-modified antibody Ver. 1 amino acid sequence of CDR2 SEQ ID NO: 100: amino acid-modified antibody Ver. 2 CDR2 amino acid sequence SEQ ID NO: 101: Amino acid-modified antibody Ver. 3 amino acid sequence of CDR2 SEQ ID NO: 102: amino acid-modified antibody Ver.1 having no N-linked sugar chain-binding consensus sequence 5 amino acid sequence of CDR2 SEQ ID NO: 103: Ver. 1 VH amino acid sequence
SEQ ID NO: 104: Ver. 2 VH amino acid sequence
SEQ ID NO: 105: Ver. 3 VH amino acid sequence
SEQ ID NO: 106: Ver. 5 VH amino acid sequence
SEQ ID NO: 107: VH CDR2 amino acid modified antibody ver. 1. Base sequence of primer for preparation (sense strand) SEQ ID NO: 108: VH CDR2 amino acid-modified antibody ver. 1 Base sequence of primer for preparation (antisense strand) SEQ ID NO: 109: VH CDR2 amino acid modified antibody ver. 2 Base sequence of primer for preparation (sense strand)
SEQ ID NO: 110: VH CDR2 amino acid modified antibody ver. 2 Base sequence of primer for preparation (antisense strand)
SEQ ID NO: 111: VH CDR2 amino acid modified antibody ver. 3. Base sequence of primer for preparation (sense strand)
SEQ ID NO: 112: VH CDR2 amino acid modified antibody ver. 3 Base sequence of primer for preparation (antisense strand)
SEQ ID NO: 113: VH CDR2 amino acid modified antibody ver. 5 Base sequence of primer for preparation (sense strand)
SEQ ID NO: 114: VH CDR2 amino acid modified antibody ver. 5 Base sequence of primer for preparation (antisense strand)
SEQ ID NO: 115: base sequence of primer for preparing cynomolgus monkey CD33 expression vector SEQ ID NO: 116: base sequence of primer for preparing cynomolgus monkey CD33 expression vector SEQ ID NO: 117: amino acid sequence of cynomolgus monkey CD33 SEQ ID NO: 118: base sequence of cynomolgus monkey CD33 Base sequence of chimpanzee CD33 total synthetic gene SEQ ID NO: 120: amino acid sequence of chimpanzee CD33 SEQ ID NO: 121: base sequence of chimpanzee CD33 SEQ ID NO: 122: base sequence of KM4071 VH (no signal sequence)
SEQ ID NO: 123: KM4072 VH base sequence (no signal sequence)
SEQ ID NO: 124: Base sequence of KM4073 VH (no signal sequence)
SEQ ID NO: 125: KM4074 VH base sequence (no signal sequence)
SEQ ID NO: 126: KM4075 VH base sequence (no signal sequence)
SEQ ID NO: 127: KM4071 VL base sequence (no signal sequence)
SEQ ID NO: 128: KM4072 VL base sequence (no signal sequence)
SEQ ID NO: 129: Base sequence of KM4073 VL (no signal sequence)
SEQ ID NO: 130: KM4074 VL base sequence (no signal sequence)
SEQ ID NO: 131: KM4075 VL base sequence (no signal sequence)
SEQ ID NO: 132: amino acid sequence of KM4071 VH (no signal sequence)
SEQ ID NO: 133: KM4072 VH amino acid sequence (no signal sequence)
SEQ ID NO: 134: amino acid sequence of KM4073 VH (no signal sequence)
SEQ ID NO: 135: amino acid sequence of KM4074 VH (no signal sequence)
SEQ ID NO: 136: KM4075 VH amino acid sequence (no signal sequence)
SEQ ID NO: 137: Amino acid sequence of KM4071 VL (no signal sequence)
SEQ ID NO: 138: Amino acid sequence of KM4072 VL (no signal sequence)
SEQ ID NO: 139: amino acid sequence of KM4073 VL (no signal sequence)
SEQ ID NO: 140: amino acid sequence of KM4074 VL (no signal sequence)
SEQ ID NO: 141: amino acid sequence of KM4075 VL (no signal sequence)

Although the present invention has been described in detail using specific embodiments, it will be apparent to those skilled in the art that various modifications and variations can be made without departing from the spirit and scope of the invention. In addition, this application is based on the US provisional application (61/419342) for which it applied on December 3, 2010, The whole is used by reference.

Claims

(I) a heavy chain variable region comprising CDR1 comprising the amino acid sequence represented by SEQ ID NO: 52, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 99, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 54, and SEQ ID NO: 55 A monoclonal antibody that binds to human CD33, comprising a light chain variable region comprising CDR1 comprising the amino acid sequence represented by SEQ ID NO: 56, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 56, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 57, or A fragment of the antibody,
(Ii) a heavy chain variable region comprising CDR1 comprising the amino acid sequence represented by SEQ ID NO: 52, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 100, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 54, and SEQ ID NO: 55 A monoclonal antibody that binds to human CD33, comprising a light chain variable region comprising CDR1 comprising the amino acid sequence represented by SEQ ID NO: 56, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 56, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 57, or A fragment of the antibody,
(Iii) a heavy chain variable region comprising CDR1 comprising the amino acid sequence represented by SEQ ID NO: 52, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 101, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 54, and SEQ ID NO: 55 A monoclonal antibody that binds to human CD33, comprising a light chain variable region comprising CDR1 comprising the amino acid sequence represented by SEQ ID NO: 56, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 56, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 57, or A heavy chain variable comprising a fragment of said antibody, or (iv) CDR1 comprising the amino acid sequence represented by SEQ ID NO: 52, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 102, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 54 Region and the amino acid sequence shown in SEQ ID NO: 55. CDR1, CDR2 comprising the amino acid sequence shown in SEQ ID NO: 56 and a light chain variable region comprising a CDR3 comprising an amino acid sequence shown in SEQ ID NO: 57, a fragment of a monoclonal antibody or the antibody that binds human CD33.
A heavy chain variable region comprising CDR1, comprising the amino acid sequence represented by SEQ ID NO: 52, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 96, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 54, and represented by SEQ ID NO: 55 A monoclonal antibody that binds to human CD33, comprising a CDR1 comprising an amino acid sequence, a CDR2 comprising an amino acid sequence represented by SEQ ID NO: 56, and a light chain variable region having a CDR3 comprising an amino acid sequence represented by SEQ ID NO: 57 fragment.
The monoclonal antibody or fragment of the antibody according to claim 2, comprising a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 86 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 85.
The monoclonal antibody or fragment of the antibody according to claim 2, comprising a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 87 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 85.
(I) a heavy chain variable region comprising CDR1 comprising the amino acid sequence represented by SEQ ID NO: 34, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 35, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 36, and SEQ ID NO: 37 A monoclonal antibody that binds to human CD33, comprising a light chain variable region comprising CDR1, comprising the amino acid sequence represented by SEQ ID NO: 38, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 38, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 39; A fragment of the antibody,
(Ii) a heavy chain variable region comprising CDR1 comprising the amino acid sequence represented by SEQ ID NO: 40, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 41, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 42, and SEQ ID NO: 43 A monoclonal antibody that binds to human CD33, comprising a light chain variable region comprising CDR1, comprising the amino acid sequence represented by SEQ ID NO: 44, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 44, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 45, or A fragment of the antibody,
(Iii) a heavy chain variable region comprising CDR1 comprising the amino acid sequence represented by SEQ ID NO: 46, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 47, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 48, and SEQ ID NO: 49 A monoclonal antibody that binds to human CD33, comprising a CDR1 comprising the amino acid sequence shown, a CDR2 comprising the amino acid sequence represented by SEQ ID NO: 50, and a light chain variable region comprising CDR3 comprising the amino acid sequence represented by SEQ ID NO: 51, or Antibody fragments,
(Iv) a heavy chain variable region comprising CDR1 comprising the amino acid sequence represented by SEQ ID NO: 52, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 53, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 54, and SEQ ID NO: 55 A monoclonal antibody that binds to human CD33 comprising a CDR1 comprising the amino acid sequence shown, a CDR2 comprising the amino acid sequence represented by SEQ ID NO: 56, and a light chain variable region comprising CDR3 comprising the amino acid sequence represented by SEQ ID NO: 57 or A heavy chain variable region comprising a fragment of an antibody, or (v) CDR1 comprising the amino acid sequence represented by SEQ ID NO: 58, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 59, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 60 As well as a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 61, CDR2 comprising an amino acid sequence represented by sequence number 62, and comprises a light chain variable region comprising a CDR3 comprising an amino acid sequence represented by SEQ ID NO: 63, a fragment of a monoclonal antibody or the antibody that binds human CD33.
(I) a monoclonal antibody that binds to human CD33 comprising a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 132 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 137;
(Ii) a monoclonal antibody that binds to human CD33 comprising a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 133 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 138;
(Iii) a monoclonal antibody that binds to human CD33 comprising a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 134 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 139,
(Iv) a monoclonal antibody that binds to human CD33 comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 135 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 140, or (v) in SEQ ID NO: 136 Binds to human CD33, which binds to an epitope on human CD33 to which a monoclonal antibody that binds to human CD33, which comprises a heavy chain variable region comprising the amino acid sequence shown and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 141 A monoclonal antibody or a fragment of said antibody.
(I) a monoclonal antibody that binds to human CD33 comprising a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 132 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 137;
(Ii) a monoclonal antibody that binds to human CD33 comprising a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 133 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 138;
(Iii) a monoclonal antibody that binds to human CD33 comprising a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 134 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 139,
(Iv) a monoclonal antibody that binds to human CD33 comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 135 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 140, or (v) in SEQ ID NO: 136 Monoclonal anti or a fragment of said antibody that binds to human CD33 in competition with a monoclonal antibody that binds to human CD33 comprising a heavy chain variable region comprising the amino acid sequence shown and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 141 .
Dissociation constant K D for human CD33 is 4.0 × 10 -9 M or less, a monoclonal antibody or fragment of the antibody according to any one of claims 1 to 7.
Dissociation constant K D for human CD33 is the 2.0 × 10 -9 M or less, a monoclonal antibody or fragment of the antibody of claim 8.
The monoclonal antibody or fragment of the antibody according to any one of claims 1 to 9, comprising a heavy chain constant region, wherein the heavy chain constant region class is human IgG or a fragment of the antibody.
The monoclonal antibody or fragment of the antibody according to claim 10, wherein the subclass of human IgG is human IgG1.
The monoclonal antibody according to any one of claims 1 to 11, or a fragment of the antibody, comprising a heavy chain constant region, wherein fucose is present in an N-linked complex type sugar chain that is bound to Fc of the heavy chain constant region. Unbound monoclonal antibody or fragment of the antibody.
The monoclonal antibody or fragment of the antibody according to any one of claims 1 to 12, which comprises a light chain constant region, wherein the light chain constant region class is human κ.
14. The antibody fragment according to any one of claims 1 to 13, which is a peptide comprising Fab, F (ab ') 2 , Fab', scFv, diabody, dsFv or CDR.
DNA encoding the monoclonal antibody or fragment of the antibody according to any one of claims 1 to 14.
A recombinant vector containing the DNA according to claim 15.
(I) a recombinant vector comprising DNA encoding a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 89;
(Ii) a recombinant vector comprising DNA encoding a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 90;
(Iii) a recombinant vector comprising DNA encoding a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 91;
(Iv) a recombinant vector comprising DNA encoding a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 90 and DNA encoding a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 89;
Or (v) a recombinant vector comprising DNA encoding a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 91 and DNA encoding a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 89.
A transformant obtained by introducing at least one recombinant vector selected from the recombinant vectors according to claims 16 and 17 into a host cell.
The transformant according to claim 18, which is obtained by introducing the following recombinant vector (i) or (ii) into a host cell.
(I) a recombinant vector comprising a DNA encoding a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 89 and a recombinant vector comprising a DNA encoding a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 90 (Ii) a recombinant vector comprising a DNA encoding a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 89 and a recombinant vector comprising a DNA encoding a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 91
The transformant according to claim 18 or 19 is cultured in a medium, the monoclonal antibody according to any one of claims 1 to 14 or a fragment of the antibody is produced and accumulated in the culture, and the culture is used. The method for producing a monoclonal antibody or a fragment of the antibody according to any one of claims 1 to 14, wherein the monoclonal antibody or a fragment of the antibody is collected.
A pharmaceutical composition comprising the monoclonal antibody according to any one of claims 1 to 14 or a fragment of the antibody as an active ingredient.
A therapeutic agent for a disease involving CD33-positive cells, comprising the monoclonal antibody according to any one of claims 1 to 14 or a fragment of the antibody as an active ingredient.
The therapeutic agent according to claim 22, wherein the disease involving CD33 positive cells is cancer, autoimmune disease or allergic disease.
The cancer is blood cancer, breast cancer, uterine cancer, colon cancer, esophageal cancer, stomach cancer, ovarian cancer, lung cancer, kidney cancer, rectal cancer, thyroid cancer, cervical cancer, small intestine cancer, prostate cancer or pancreatic cancer. 24. The therapeutic agent according to 23.
The therapeutic agent according to claim 24, wherein the blood cancer is acute myeloid leukemia, anaplastic large cell lymphoma, acute lymphocytic leukemia, myelodysplastic syndrome, multiple myeloma, Hodgkin lymphoma, or non-Hodgkin lymphoma.
A method for treating a disease involving CD33-positive cells, comprising a step of administering the monoclonal antibody or fragment of the antibody according to any one of claims 1 to 14 to a mammal.
The method according to claim 26, wherein the disease involving CD33-positive cells is cancer, autoimmune disease or allergic disease.
The cancer is blood cancer, breast cancer, uterine cancer, colon cancer, esophageal cancer, stomach cancer, ovarian cancer, lung cancer, kidney cancer, rectal cancer, thyroid cancer, cervical cancer, small intestine cancer, prostate cancer or pancreatic cancer. 28. The method according to 27.
The method according to claim 28, wherein the blood cancer is acute myeloid leukemia, anaplastic large cell lymphoma, acute lymphocytic leukemia, myelodysplastic syndrome, multiple myeloma, Hodgkin lymphoma, or non-Hodgkin lymphoma.
Use of the monoclonal antibody according to any one of claims 1 to 14 or a fragment of the antibody for treating a disease involving CD33-positive cells.
The use according to claim 30, wherein the disease involving CD33 positive cells is cancer, autoimmune disease or allergic disease.
The cancer is blood cancer, breast cancer, uterine cancer, colon cancer, esophageal cancer, stomach cancer, ovarian cancer, lung cancer, kidney cancer, rectal cancer, thyroid cancer, cervical cancer, small intestine cancer, prostate cancer or pancreatic cancer. 31. Use according to 31.
The use according to claim 32, wherein the blood cancer is acute myeloid leukemia, anaplastic large cell lymphoma, acute lymphocytic leukemia, myelodysplastic syndrome, multiple myeloma, Hodgkin lymphoma or non-Hodgkin lymphoma.