JP2005333993A - New diabody type bispecific antibody - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a diabody type bispecific antibody which has excellent characteristics such as low immunogenicity, high property for being infiltrated into tumor tissues, mass productivity at a low cost, and easy function modifiability utilizing genetic engineering, and is excellent at the points of effect, stability after administered, and the like, even when not containing a super antigen. <P>SOLUTION: This diabody type bispecific antibody is characterized by having the first specificity against human epitheliocyte growth factor receptors and the second specificity against surface antibodies expressed in cells having a phagocytosis or a cytotoxic activity, either of two single strand polypeptides constituting the antibody or each domain contained therein, a nucleic acid encoding the same, a replicable cloning vector or expression vector containing the nucleic acid, a host cell transformed with the vector, and a medicinal composition containing the same. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a diabody-type bispecific antibody that can be used for cancer-specific immunotherapy, a single-chain polypeptide constituting the same or each region contained therein, a nucleic acid encoding the polypeptide, the antibody This invention relates to a method for producing these and their uses as pharmaceuticals.

  As a main treatment for cancer (malignant tumor), surgical removal, chemotherapy, radiation therapy, immunotherapy, and the like are used in combination. Among these, immunotherapy is still under development, but has many possibilities, and further progress is expected.

  Cancer-specific immunotherapy refers to a therapeutic method in which cytotoxic activity acts only on cancer cells. It binds an antibody and a drug exhibiting cytotoxic activity to make the drug target-targeting and is now also called missile therapy. Currently, research is being conducted to target substances that are abnormally expressed in cancer cells or substances that change somewhat as the cells become cancerous, and use antigens that can exert the ability of antibodies with minimal side effects. Is underway. Such an antigen is called a cancer-associated antigen.

Bispecific antibody (BsAb), which is one of multispecific antibodies, can specifically bind to two different antigens. Research has been actively conducted on the possibility that it can be used as a therapeutic agent having an antitumor effect. A diabody is the smallest unit of such a bispecific antibody, and is a variable region (V region) (represented as “VH”) and a light chain (H chain) derived from the same parent antibody. It was devised by utilizing the property that a variable region (V region) VL (represented as “VL”) VL of a chain (L chain) forms a heterodimer by non-covalent bonds (Hollinger,
et al., Proc. Natl. Acad. Sci. USA 90, 6444-6448, 1993).

  Such diabody-type bispecific antibodies are characterized by low molecular weight (molecular weight of about 60,000), low immunogenicity and high invasiveness to tumor tissues, and, for example, microorganisms such as E. coli. It can be mentioned that inexpensive mass production is possible, and that functional modification using genetic engineering is easy.

  Therefore, various problems in conventional treatments for cancer, particularly the problem that it is difficult to completely remove cancer cells at the site where the tissue is complicated, and there is no method for removing cancer cells remaining after surgical removal. In order to solve this problem, such a diabody-type bispecific antibody is considered promising and its development is eagerly desired.

  As an example of a diabody-type bispecific antibody, a bispecific antibody derived from an anti-CD3 antibody and an antibody against MUC1, which is a kind of cancer-associated antigen and is often found on glandular cells. Furthermore, a fusion protein has been developed in which a superantigen such as bacterial enterotoxin is contained in the same polypeptide as the variable region of the antibody (Patent Document 1).

International Publication No. WO2 / 06486 Pamphlet

  However, conventional diabody-type bispecific antibodies, which are recombinant proteins produced using microorganisms, are more effective and stable after administration compared to chemically synthesized bispecific antibodies. In order to obtain the same effect, there is a problem to be solved such as preparation of a preparation containing a high concentration of the bispecific antibody.

  On the other hand, epidermal growth factor receptor (EGFR) is a cell membrane-bound glycoprotein, and when a specific antibody against HER-2 / neu (c-erbB-2) is used alone In addition, about 20% of breast cancer patients have already reported effects such as tumor shrinkage.

  As a result of earnest research to produce a diabody-type bispecific antibody that overcomes the above-described problems and has further excellent effects, the present inventors have developed diabody-type bispecific antibodies and Compared to chemically synthesized bispecific antibodies, we succeeded in producing diabody-type bispecific antibodies that can exhibit remarkable effects even at very low concentrations, especially without superantigens. . Furthermore, this humanized antibody could also be obtained. The present invention has been completed based on such results.

That is, the present invention has, as a first aspect, a first specificity for human epidermal growth factor receptor and a second specificity for surface antigens expressed in cells having phagocytic or cytotoxic activity. The present invention relates to a diabody-type bispecific antibody.
As a second aspect, the present invention relates to a single-chain polypeptide of any one of two kinds of single-chain polypeptides constituting a diabody-type bispecific antibody or a polypeptide constituting each region included therein.
As a third aspect, the present invention relates to a nucleic acid encoding the single-chain polypeptide or each region contained therein.
A fourth aspect relates to a replicable cloning vector or expression vector containing the nucleic acid.
A fifth aspect relates to a host cell transformed with the vector.
In a sixth aspect, the transformed host cell is cultured and the nucleic acid is expressed in the host cell, and the single-chain polypeptide according to claim 13 is recovered and purified. The present invention relates to a method for producing the present polypeptide.
In a seventh aspect, the present invention provides a method for producing a diabody-type bispecific antibody, comprising associating the single-chain polypeptide and separating and recovering the formed diabody-type bispecific antibody. Related.
As an eighth aspect, it is characterized by containing, as an active ingredient, one selected from the group consisting of the diabody-type bispecific antibody, single-chain polypeptide, nucleic acid, vector, and host cell of the present invention. According to pharmaceutical composition.
As a ninth aspect, the diabody-type bispecific antibody of the present invention is added to a culture system of cells having phagocytosis or cytotoxic activity and tumor cells expressing human epidermal growth factor receptor. The present invention relates to a method for increasing the amount of cytokine production in a cell having an action or cytotoxic activity.

The diabody-type bispecific antibody of the present invention was confirmed to exhibit excellent cytotoxic activity in vivo and in vitro. In addition, the amount of cytokine produced in cells having phagocytosis or cytotoxic activity by adding the antibody to a culture system of cells having phagocytosis or cytotoxic activity and tumor cells expressing human epidermal growth factor receptor Was found to increase significantly. Furthermore, a humanized diabody-type bispecific antibody was prepared, and it was demonstrated that this antibody has an activity comparable to that of a mouse-derived diabody-type bispecific antibody.
The diabody-type bispecific antibody of the present invention was confirmed to retain such activity even after the antibody was stored frozen for several months, and was found to have excellent stability.
Therefore, a pharmaceutical composition containing these diabody-type bispecific antibodies and the like as an active ingredient has an effect of significantly eliminating, killing, or injuring tumor cells in vitro and in vivo, and is used as an antitumor agent. I can do it.

  As used herein, a “diabody-type bispecific antibody” refers to an antibody fragment having two antigen-binding sites and a small fragment, which is a variable region (VH) of the heavy chain. The same polypeptide chain (VH-VL) contains the variable region (VL) of the L chain that binds to. A typical diabody uses a linker that is too short to form a pairing between the two domains on the same strand, thereby making that domain complementary to another strand. The two domains are combined to create two antigen-binding sites. The diabody and its manufacturing technology are described in U.S. Pat. No. 4,704,692; U.S. Pat. No. 4,946,778; U.S. Pat. No. 5,990,275; U.S. Pat. No. 5,994,511; U.S. Pat. No. 6,027,725; 404,097; WO93 / 11161; Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993), the disclosure of which is incorporated herein by reference. Included in the description.

  More specifically, “diabody-type bispecific antibody” refers to, for example, (1) the variable region of the heavy chain of the first antibody as described in FIG. A first polypeptide (i) having an antigen-binding site (a) and an antigen-binding site (b) of the variable region of the L chain of the second antibody on the same peptide chain, and (2) the first antibody A second polypeptide (ii) having the antigen-binding site (c) of the variable region of the L chain and the antigen-binding site (d) of the variable region of the second antibody H chain on the same peptide chain It means a fusion protein having the ability to recognize two different types of antigens.

  Therefore, in the diabody-type bispecific antibody of the present invention, the first antibody is an antibody to human epidermal growth factor receptor, and the second antibody is expressed in cells having phagocytosis or cytotoxic activity. It is an antibody against the antigen. As shown in the examples of the present specification, the diabody-type bispecific antibody of the present invention has excellent cytotoxic activity in vivo and in vitro.

  There are no particular restrictions on the animal species that produce these antibodies, but they are usually derived from mammals such as humans, mice, and rats. Further, although there is no particular limitation on the structure of the antibody, the monoclonal antibody produced by the hybridoma recognizes a single antigen site and has high specificity to it, so that the diabody-type bispecific antibody of the present invention is used. It is suitable for manufacturing.

  Human epidermal growth factor receptor (EGF receptor) is a receptor of the EGF family and is a glycoprotein of about 170 kDa. So far, four types of HER-1 / Erb1, HER-2 / Erb2, HER-3 / Erb3 and HER-4 / Erb4 have been identified. Here, HER-2 is expressed in 30 to 40% of adenocarcinoma (lung cancer, stomach cancer, colon cancer, breast cancer, and ovarian cancer). On the other hand, HER-1 / Erb1 is 80%> in head and neck cancer, 14-91% in breast cancer, 40-80% in non-small cell lung cancer, 25-77% in colorectal cancer, 30-50% in pancreatic cancer, Expression is observed in a proportion of 40 to 80% in prostate cancer, 35 to 70% in ovarian cancer, and 33 to 74% in gastric cancer. Thus, the EGF receptor is a human cancer surface antigen or a cancer associated antigen.

  Therefore, in the present invention, the “human epidermal growth factor receptor” includes at least the four types of EGF receptors identified so far. Antibodies to human epidermal growth factor receptor are known to those skilled in the art. Among them, in the present invention, diabody type 2 having the first specificity for human epidermal growth factor receptor 1 (Her1). Bispecific antibodies are preferred. An example of an antibody that can provide such specificity is anti-human epidermal growth factor receptor antibody 528, which is a monoclonal antibody produced by mouse B cell hybridoma 528. Thus, in a preferred embodiment of the invention, the first specificity is derived from the variable regions of the heavy and light chains of the anti-human epidermal growth factor receptor antibody 528 (FIG. 1).

  Examples of “cells having phagocytosis or cytotoxic activity” in the present specification include, for example, T cells such as NK cells, macrophages, T-LAK cells, etc., preferably known as cytotoxic T cells. T-LAK cells. Examples of the surface antigen expressed in the cells include CD2, CD3, CD4, CD5, CD6, CD8, CD16, CD28, CD44, and preferably CD3 expressed in cytotoxic T cells.

  Monoclonal antibodies against such surface antigens include OKT3, T3, Leu4, T11, OKT11, Leu5b, NU-T1, T4, OKT4, Leu3a, NU-TH / I, T8, OKT8, Leu2a, known to those skilled in the art. NU-Ts / c etc. are mentioned, Preferably OKT3, T3, Leu4 etc. are mentioned. Thus, in a preferred embodiment of the invention, the second specificity is derived from the variable regions of the heavy and light chains of the anti-CD3 antibody OKT3.

  Examples of the antibody that provides the variable region of the diabody-type bispecific antibody of the present invention include human epidermal growth factor receptor, or a variety of commercially available antibodies known to those skilled in the art, Using surface antigens expressed in cells having phagocytosis or cytotoxic activity as immunogens, they can be prepared by various methods well known to those skilled in the art.

  For example, in the case of a polyclonal antibody, these immunogens and, if necessary, an adjuvant are injected into the body of an animal such as a mouse a plurality of times by an appropriate route such as subcutaneously or intraperitoneally, and then generated in the body of the animal. I can do it.

Monoclonal antibodies, on the other hand, are based on Kohler & Milstein,
Obtained from a substantially homogeneous population of antibodies using the hybridoma method first described by Nature 256: 495 (1975) or made by recombinant DNA methods (US Pat. No. 4,816,567) Can do.

In addition, alternatively, transgenic animals (eg, mice) can be made to produce human antibodies in the body (eg, Duchosal et al., Nature 355: 258 (1992); Jakobovits
et al., Proc. Natl. Acad. Sci. USA 90: 2551 (1993); Jakobovits
et al., Nature 362: 255-258 (1993); Bruggermann et al., Year in Immunol. 7: 33 (1993)). Human antibodies can also be derived from phage display libraries (Marks et al., J. Mol. Biol. 222: 581-597 (1991); Hoogenboom et al., J. Mol. Biol. 227: 381 (1991); Hoogenboom
et al., Immunol. Rev. 130: 41-68 (1992); Vaughan et al., Nature Biotech. 14: 309 (1996)).

As a further method for obtaining the above antibodies, antibodies or antibody fragments can be obtained from antibody phage libraries using techniques described in, for example, McCafferty et al., Nature, 348: 552-554 (1990). A phage library can be used for isolation (Clackson et al., Nature, 352: 624-628 (1991) and Mark et al., J, Mol. Biol. 222: 581-597 (1991) For the isolation of isolated mouse and human antibodies). In order to select an appropriate antibody or antibody fragment, it can carry out using the said antigen. In addition, chain shuffling
To produce human antibodies with high affinity (on the order of nM) (Mark et al., Bio / Technol. 10: 779-783 (1992)), or as a method for constructing extremely large phage libraries Also known are combinatorial infection and in vivo recombination (Waterhouse et al., Nuc. Acids Res., 21: 2265-2266 (1993)).

  At least one of the two polypeptides constituting the diabody-type bispecific antibody of the present invention contains a linker that links the variable region of the H chain and the variable region of the L chain. In the present specification, “linker” refers to an oligopeptide or a polypeptide that serves to bind a variable region (VH) of an H chain and a variable region (VL) of an L chain to give a single chain polypeptide. Refers to a peptide. As used herein, preferably the linker is a peptide linker. The peptide linker is not particularly limited as long as it can functionally link two polypeptides to give one single-chain polypeptide. For example, a peptide linker widely known in the art or the known It is possible to select and use a modified linker. The peptide linker may be, for example, a peptide consisting of 1 to about 50 amino acids, preferably a peptide consisting of about 2 to 30 amino acids, more preferably a peptide consisting of about 2 to 20 amino acids. . Here, “functionally linked” means that the polypeptide is appropriately folded and the original protein (the polypeptide is derived from or derived from the original protein). ) Function, for example, a binding that gives a fusion protein having a three-dimensional structure that can simulate part or all of biological activity.

  The length of the linker is not limited as long as it gives desired activity to the single-chain polypeptide (or fusion protein) to be produced, although it depends on the nature of the peptide to be bound. The length of the linker should be long enough so that the resulting single chain polypeptide is properly folded to obtain the desired biological activity. The length of the linker can also be determined experimentally by testing a series of single-chain polypeptides joined by linkers of various lengths for the desired biological activity. For the linker, reference can be made to the literature cited in relation to the diabody and its production technology.

  In addition, the arrangement of VL and VH in a single-chain polypeptide is VL on the N-terminal side, followed by a linker, followed by VH (VL-Linker-VH construct), but the N-terminal side is VH. It can be any of the linkers, followed by VL (VH-Linker-VL construct).

  The diabody-type bispecific antibody of the present invention can be a humanized antibody. These humanized antibodies have a high reaction specificity derived from, for example, a mouse antibody, while other parts are derived from a human antibody, thereby reducing the possibility of becoming an immunogenic source when administered to humans. Therefore, it is an antibody that is extremely advantageous clinically against humans.

“Humanized antibody” means a non-human such as mouse, rat, or rabbit in at least part of the complementarity-determining region (CDR) of a human immunoglobulin (recipient antibody). By antibody (donor antibody) is meant an antibody that is replaced by residues from a CDR having the desired specificity, affinity, and ability. In some cases, human immunoglobulin Fv framework (FR) residues may be replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are found neither in the recipient antibody nor in the introduced CDR or framework sequences. These modifications are made to further improve or optimize the antibody performance. For more information, see Jones et al., Nature 321, 522-525 (1986); Reichmann
See et al., Nature 332, 323-329 (1988); EP-B-239400; Presta, Curr. Op. Struct. Biol. 2, 593-596 (1992); and EP-B-451216. it can.

  Humanized antibodies can be prepared according to methods known to those skilled in the art. For example, humanized antibodies are prepared by analyzing various conceptual humanized products using a three-dimensional immunoglobulin model of recipient and donor antibodies. Three-dimensional immunoglobulin models are well known to those skilled in the art. For further details, reference can be made to WO92 / 22653.

  Therefore, as an example of the diabody-type bispecific antibody of the present invention which is a humanized antibody, an antibody in which the complementarity determining region (CDR) in the variable region is derived from a mouse antibody and the other part is derived from a human antibody. Can be mentioned. Further, as a preferred example thereof, the CDR in the variable region derived from humanized anti-CD3 antibody OKT3 has at least one CDR of CDR1, CDR2 and CDR3 of the amino acid sequence shown in FIG. 21, and humanized anti-human There is a diabody-type bispecific antibody characterized in that the CDR in the variable region derived from epidermal growth factor receptor antibody 528 has at least one CDR of CDR1, CDR2 and CDR3 of the amino acid sequence shown in FIG. . In particular, the variable regions of the H chain and L chain derived from the humanized anti-CD3 antibody OKT3 have the amino acid sequences (SEQ ID NOs: 27 and 28) shown in FIG. 21, and the humanized anti-human epidermal growth factor receptor is received. Preferred is a diabody-type bispecific antibody characterized in that the variable regions of the heavy and light chains derived from the antibody 528 have the amino acid sequences shown in FIG. 22 (SEQ ID NOs: 29 and 30).

  Furthermore, in the present invention, since the functionalization of the antibody itself may occur due to humanization, an appropriate site in the single-chain polypeptide, for example, a framework (FR) that may affect the CDR structure. The function of the humanized antibody can be improved by causing site-specific mutations in the middle part, for example, canonical sequence or veriner sequence. The antibody thus obtained is also included in the humanized diabody-type bispecific antibody of the present invention.

  As already described, the diabody-type bispecific antibody of the present invention is composed of two types of single-chain polypeptides, and the present invention includes any one single-chain polypeptide or each of the single-chain polypeptides included therein. This also relates to a polypeptide constituting a region (for example, each variable region derived from each antibody). As described above, this single-chain polypeptide is composed of the variable region of the H chain of the first (or second) antibody, the L chain of the second (or first) antibody, and a linker connecting them. However, bacterial enterotoxins represented by staphylococcal enterotoxin, E. coli enterotoxin and cholera enterotoxin or various derivatives thereof may also be included. Furthermore, for the purpose of facilitating the detection and purification of the produced single-chain polypeptide, various peptide tags known to those skilled in the art (for example, c-myc tag and His-tag) are included at the ends thereof. I can do it.

  However, it is known that these superantigens may cause cytokine-dependent toxic shock syndrome due to a strong affinity for MHC class II. On the other hand, the diabody-type bispecific antibody of the present invention, as shown in the following examples, exhibits a very high cytotoxic activity even when it does not contain such a superantigen. Can be used to avoid this potential danger of superantigens.

The nucleic acid encoding the single-chain polypeptide of the present invention or each region contained therein can be obtained by a method known to those skilled in the art, and the base sequence thereof can be determined. For example, it can be performed by using oligonucleotide probes that can specifically bind to the genes encoding the heavy and light chains of mouse antibodies (R. Orlandi et al., Proc. Natl. Acad. Sci). USA 86: 3833-3837
(1993)). The hybridoma cells producing the monoclonal antibodies described above can be used as a source of DNA in such methods.

More specifically, the nucleic acid encoding the single-chain polypeptide of the present invention is a single-chain Fv ((single-chain Fv) or “scFv”) or diabody type that has already been constructed and is known to those skilled in the art. Based on the nucleic acid encoding the bispecific antibody, it can be prepared by replacing VH or VL therein with one derived from an antibody having another specificity. Here, “scFv” refers to a substance containing the VH and VL domains of a certain antibody and having the domains in one polypeptide chain. In general, the Fv polypeptide further contains a polypeptide linker between the VH and VL domains, allowing the necessary structure for antigen binding to be provided. For scFv, see Rosenburg and Moore (Ed.), “The Pharmacology of Monoclonal Antibodies”, Vol. 113, Springer-Verlag, New York, pp.269-315.
(1994).

  Furthermore, when a nucleic acid encoding a variable region in a single-chain polypeptide of a humanized diabody-type bispecific antibody is prepared, it is designed in advance as described in the following examples. Total synthesis can be performed by the overlap PCR method based on the amino acid sequence. As long as the “nucleic acid” is a molecule that encodes a single-chain polypeptide, its chemical structure and acquisition route are not particularly limited, and include, for example, gDNA, cDNA, chemically synthesized DNA, mRNA, and the like. is there.

Specifically, it can be isolated from a cDNA library by hybridization based on the sequence described in the literature or by the polymerase chain reaction (PCR) technique. Once isolated, the DNA is placed in an expression vector, which is then placed in an E. coli cell, COS cell, Chinese hamster ovary cell (CHO cell), or myeloma cell that does not produce immunoglobulin. A host cell can be transfected and a monoclonal antibody synthesized in the recombinant host cell. The PCR reaction can be performed by a method known in the art, or a method or modification method substantially similar thereto. For example, R. Saiki, et al., Science, 230: 1350, 1985; R. Saiki, et al., Science, 239: 487, 1988; HA Erlich ed., PCR Technology, Stockton Press, 1989; DM Glover et al. ed., “DNA Cloning”, 2nd ed., Vol. 1, (The Practical Approach Series ), IRL Press, Oxford University Press (1995); MA Innis et al. Ed., “PCR Protocols:
a guide to methods and applications ", Academic Press, New York (1990)); MJ McPherson, P. Quirke and GR Taylor (Ed.), PCR: a practical approach,
IRL Press, Oxford (1991); MA Frohman et al., Proc. Natl. Acad. Sci. USA, 85, 8998-9002 (1988), etc. be able to. In addition, the PCR method can be performed using a commercially available kit suitable for the PCR method, and can also be performed according to a protocol clarified by the kit manufacturer or the kit vendor.

For hybridization, see L. Grossman et al. (Ed.), “Methods in Enzymology”, Vol. 29 (Nucleic Acids and Protein Synthesis,
Part E), Academic Press, New York (1974)
Etc. can be referred to. The sequencing of nucleic acids such as DNA can be performed, for example, by Sanger et al., Proc. Natl. Acad. Sci. USA 74: 5463-5467
(1977) can be referred to. General recombinant DNA technology is also described in J. Sambrook,
EF Fritsch & T. Maniatis (ed.), “Molecular Cloning: A Laboratory
Manual (2nd edition) ", Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989) and DM Glover et al. (Ed.)," DNA Cloning ", 2nd ed., Vol. 1 to 4, (The Practical Approach Series), IRL Press, Oxford University Press (1995).

The nucleic acid encoding the single-chain polypeptide constituting the diabody-type bispecific antibody of the present invention thus obtained or each region contained therein is appropriately selected according to the purpose by means known to those skilled in the art. It can be modified to encode a peptide or amino acid. The technology for genetically modifying or modifying DNA in this way is Mutagenesis: a Practical Approach,
MJ. Mcpherson (Ed.), (IRL Press, Oxford, UK (1991)), for example, site-directed mutagenesis (site-directed mutagenesis), cassette mutagenesis, and polymerase chain reaction (PCR). ) Mention mutagenesis.

  Here, “modification” of a nucleic acid means insertion, deletion or substitution of a base in at least one codon encoding an amino acid residue in the obtained original nucleic acid. For example, there is a method of altering the amino acid sequence itself constituting a single-chain polypeptide by replacing a codon encoding an original amino acid residue with a codon encoding another amino acid residue. In this way, a single-chain polypeptide constituting the humanized diabody-type bispecific antibody of the present invention can be obtained.

  Alternatively, as described in the Examples herein, the single-chain polypeptide is encoded so that the codon (optimum codon) suitable for the host cell is used without changing the amino acid itself. Nucleic acids can also be modified. Thus, by changing to an optimal codon, it is possible to improve the expression efficiency of the single-chain polypeptide in the host cell.

  The linker, superantigen, and the like constitute the diabody-type bispecific antibody of the present invention using any technical means known to those skilled in the art, such as genetic engineering techniques such as recombinant technology and peptide chemical synthesis. Can be appropriately introduced into the single-chain polypeptide.

  Single-chain polypeptides can be produced by methods known to those skilled in the art, for example, various means such as genetic engineering techniques or chemical synthesis. As genetic engineering techniques, for example, a replicable cloning vector or expression vector containing the above-described nucleic acid is prepared, a host cell is transformed with this vector, the transformed host cell is cultured, and the host cell is cultured. In which the nucleic acid is expressed, recovered and purified. Usually, such a vector contains a nucleic acid that encodes one of the two types of single-chain polypeptides constituting the diabody-type bispecific antibody of the present invention. Yes. In such a case, it is preferable to introduce the two types of vectors obtained into the same host cell. Alternatively, two types of nucleic acids encoding each of two types of single-chain polypeptides can be contained in the same vector.

  Here, “replicable expression vector” and “expression vector” refer to a piece of DNA (usually double-stranded), in which the DNA contains Can be inserted with foreign DNA fragments. Foreign DNA is defined as heterologous DNA, which is DNA that is not found naturally in the subject host cell. Vectors are used to carry foreign or heterologous DNA into a suitable host cell. Once in the host cell, the vector can replicate independently of the host chromosomal DNA, and several copies of the vector and its inserted (foreign) DNA can be generated. In addition, the vector contains the elements essential to allow translation of the foreign DNA into a polypeptide. Thus, many molecules of polypeptides encoded by foreign DNA can be synthesized rapidly.

Such vectors are operably linked with an appropriate control sequence so that the DNA sequence is expressed in an appropriate host (ie, so that foreign DNA can be expressed). A DNA construct (DNA
construct). Such control sequences include transcriptional promoters, arbitrary operator sequences to control such transcription, sequences encoding appropriate mRNA ribosome binding sites, enhancers, reardenylation sequences, and transcription and translation. Examples include an array that controls the end of (translation). Further, the vector can appropriately contain various sequences known to those skilled in the art, for example, restriction enzyme cleavage sites, marker genes (selection genes) such as drug resistance genes, signal sequences, leader sequences, and the like as necessary. These various sequences or elements can be appropriately selected and used by those skilled in the art depending on conditions such as the type of foreign DNA, the host cell used, the culture medium, and the like.

  The vector can be in any form such as a plasmid, a phage particle, or simply a genomic insert. Once introduced into a suitable host by transformation, the vector can replicate or function independently of the resident genome. Alternatively, the vector may be one that is integrated into the genome.

Any cell known to those skilled in the art can be used as the host cell.For example, typical host cells include prokaryotic cells such as E. coli and Chinese hamster ovary cells (CHO cells). ), Mammalian cells such as human-derived cells, and eukaryotic cells such as yeast and insect cells.

  The single-chain polypeptide obtained by such expression in a host cell is preferably a single-chain polypeptide which is generally recovered from the culture medium as a secreted polypeptide, but does not have a secretion signal. Can be recovered from the host cell lysate. If the single chain polypeptide is membrane bound, it can be released from the membrane using a suitable detergent (eg, Triton-X100).

  The purification operation can be carried out by appropriately combining methods known to those skilled in the art. For example, centrifugation, hydroxylapatite chromatography, gel electrophoresis, dialysis, fractionation on ion exchange columns, ethanol precipitation, reverse phase HPLC, chromatography on silica, chromatography on heparin sepharose, anion or cation It is suitably purified by resin chromatography (polyaspartic acid column etc.), chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, and affinity chromatography. Affinity chromatography is one of the preferred purification techniques with high efficiency utilizing the affinity with a peptide tag of a single-chain polypeptide.

  Since the recovered single-chain polypeptide is often contained in the insoluble fraction, the purification operation is preferably performed after the single-chain polypeptide is solubilized and denatured. This solubilization treatment can be performed using any agent known to those skilled in the art as a dissociating agent such as alcohols such as ethanol, guanidine hydrochloride, and urea.

  Furthermore, the diabody-type bispecific antibody of the present invention is obtained by associating (unwinding) the purified single-chain polypeptide and separating and recovering the formed diabody-type bispecific antibody. Can be manufactured.

  Association processing means returning a single single-chain polypeptide to a state having a desired biological activity by returning it to an appropriate spatial arrangement. Therefore, the association treatment also has the meaning of returning the polypeptides or domains to the associated state, so it can also be referred to as “reassociation”, and in the sense of having the desired biological activity. It can also be called reconstruction, or it can be called refolding. The association treatment can be performed by any method known to those skilled in the art. For example, the concentration of the denaturing agent (for example, guanidine hydrochloride) in the buffer solution containing the single-chain polypeptide is decreased stepwise by, for example, dialysis. The method is preferred. In this process, it is possible to promote the oxidation reaction by appropriately adding an aggregation inhibitor and an oxidizing agent to the reaction system. Separation and recovery of the formed diabody-type bispecific antibody can also be performed by any method known to those skilled in the art.

  The pharmaceutical composition of the present invention is a diabody-type bispecific antibody of the present invention, particularly a humanized diabody-type bispecific antibody, single-chain polypeptide, nucleic acid, vector, and transformed It contains as an active ingredient what was chosen from the group which consists of a host cell. As shown in the following examples, such an active ingredient has an action of significantly eliminating, killing, or damaging (positive) tumor cells expressing the epidermal growth factor receptor in vitro and in vivo. Therefore, the pharmaceutical composition of the present invention can be used as an antitumor agent against such tumor cells.

  Further, as shown in the Examples below, the diabody-type bispecific antibody of the present invention can be applied to cells having phagocytosis or cytotoxic activity and tumor cells expressing human epidermal growth factor receptor in vivo. Alternatively, by coexisting in vitro, the production amount of cytokines (eg, IFN-γ, GM-CSF, TFN-α, etc.) in cells having phagocytosis or cytotoxic activity can be increased. A body-type bispecific antibody or a pharmaceutical composition containing it as an active ingredient can also be used for this purpose. For example, in vitro, cytokine production can be increased by adding the diabody-type bispecific antibody of the present invention to a culture system containing the above two types of cells.

  The effective amount of the active ingredient of the present invention can be appropriately determined by those skilled in the art depending on, for example, the therapeutic purpose, tumor type, site and size of the subject to be administered, various conditions of the patient, administration route, and the like. A typical single dose or daily dose will depend on the above conditions and, if possible, first in vitro and then, for example, using assays known in the art for tumor cell survival or growth. In addition, appropriate dose ranges can be determined with appropriate animal models that can extrapolate dose ranges for human patients.

  The pharmaceutical composition of the present invention contains various pharmaceutically acceptable pharmacological agents well known to those skilled in the art in addition to the active ingredient, depending on various conditions such as the type of active ingredient, pharmaceutical form, administration method / purpose, and pathological condition of the administration target. Components (eg, carriers, excipients, buffers, stabilizers, etc.) can be added as appropriate.

  The pharmaceutical composition of the present invention is a tablet, solution, powder, gel, spray, or microcapsule, colloidal distribution system (liposome, microemulsion, etc.), macroemulsion, etc., depending on the above various conditions. Various drug forms are possible.

  Examples of administration methods include intravenous, intraperitoneal, intracerebral, intraspinal, intramuscular, intraocular, intraarterial, in particular intrabiliary or intralesional injection or injection, and sustained release system formulations. It is done. The active substances according to the invention can be administered continuously by infusion or by bulk injection. In addition, when administering the pharmaceutical composition of this invention, it is preferable to administer with the cell which has a phagocytosis or cytotoxic activity. Alternatively, it is preferable that the antibody is previously bound to the cell before administration by mixing an active ingredient such as the diabody-type bispecific antibody of the present invention with the cell before administration.

  Sustained release formulations are generally of a form from which the active substance of the present invention can be released for a period of time, and suitable examples of sustained release preparations include solid hydrophobic polymers containing proteins. A semipermeable carrier is included, which is in the form of a molded product, such as a film or microcapsule.

  The pharmaceutical composition of the present invention is prepared by methods known to those skilled in the art, such as the Japanese Pharmacopoeia Manual Editorial Committee, 13th revised Japanese Pharmacopoeia Manual, issued on July 10, 1996, Yodogawa Shoten Co., Ltd. In view of the description, it can be appropriately selected and manufactured from among them.

  In the specification and drawings, the terms are based on the meanings of terms commonly used in the art or according to the IUPAC-IUB Commission on Biochemical Nomenclature.

  Hereinafter, the present invention will be described in detail with reference to examples. However, the examples are merely provided for the purpose of describing the present invention and for reference to specific embodiments thereof. These exemplifications are for explaining specific specific embodiments of the present invention, but are not intended to limit or limit the scope of the invention disclosed in the present application. In the present invention, it should be understood that various embodiments based on the idea of the present specification are possible.

All examples were performed or can be performed using standard techniques, except as otherwise described in detail, and are well known and routine to those skilled in the art. .
In the following examples, unless otherwise indicated, specific operations and treatment conditions are described in J. Sambrook,
EF Fritsch & T. Maniatis, “Molecular Cloning”, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989) and DM Glover et al. Ed., “DNA Cloning”, 2nd ed., Vol. 1 to 4, (The Practical Approach Series), IRL Press,
Oxford University Press (1995); Especially for PCR, HA Erlich
ed., PCR Technology, Stockton Press, 1989; DM Glover et al. ed., “DNA Cloning”, 2nd ed., Vol. 1, (The Practical Approach Series), IRL Press,
It is performed according to the method described in Oxford University Press (1995) and MA Innis et al. Ed., “PCR Protocols”, Academic Press, New York (1990), and also uses commercially available reagents or kits. In some cases, attached instructions (protocols) and attached chemicals are used.

Example 1 Cloning of anti -epidermal growth factor receptor (EGFR) antibody Anti-EGFR antibody-producing mouse B cell hybridoma 528 (Tohoku University Institute for Aging Medicine, Medical Cell Resource Center ID: TKG0555) The mRNA was extracted using ISOGEN (Nippon Gene), and the First-Strand cDNA Synthesis Kit (Amersham
CDNA was prepared by Biosciences). This cDNA was subjected to PCR using cloning primers synthesized based on Reference Paper 1 to clarify the sequences of 528 variable regions VH (hereinafter 5H) and VL (hereinafter 5L) (FIG. 1).

○ Reference paper 1-
Krebber, A. et al. Reliable cloning of functional antibody variable domains
from hybridomas and spleen cell repertoires containing a reengineered phage
display system. J Immunol Methods 201, 35-55. (1997).

Example 2 Ex3
Preparation of diabody expression vector Ex3 diabody (hereinafter Ex3), which is a representative example of the diabody-type bispecific antibody of the present invention, is prepared from two molecules of 5HOL and OH5L. An expression vector was prepared based on an Mx3 diabody (hereinafter referred to as Mx3) expression vector targeting MUC1 and CD3 that has already been constructed by the present inventors (see Patent Document 1). In other words, 5H was amplified by PCR using an AB primer into which a restriction enzyme site had been introduced, digested with NcoI-EagI, and one of the Mx3 expression vectors pSNE4-MHOL (anti-MUC1 antibody MUSE11
PRA-5HOL was prepared by replacing MH with VH (hereinafter MH) -GGGGS (hereinafter G1) -anti-CD3 antibody OKT3 VL (OL). Similarly, 5L was amplified using CD primers and digested with EcoRV-SacII, and replaced with ML of pSNE4-OHML (OKT3 VH (hereinafter OH) -G1-MUSE11 VL (ML)) to prepare pRA-OH5L (FIG. 2). ). On the C-terminal side, a c-myc peptide tag for detection and a His-tag (Hisx6: histidine hexamer tag) for purification are introduced in parallel.

A NcoI-5H back primer 5'-nnnccatggcccaggtccagctgcagcagtctg-3 '
[SEQ ID NO: 1]
B 5H-EagI forward primer 5'-nnncggccgaggagactgtgagagtggt-3 '
[SEQ ID NO: 2]
C EcoRV-5L back primer 5'-nnngatatcctaatgacccaatctcc-3 '
[SEQ ID NO: 3]
D 5L-SacII forward primer 5'-nnnccgcggcacgtttgatttccagcttg-3 '
[SEQ ID NO: 4]

Example 3 Expression of 5HOL and OH5L using Escherichia coli E. coli BL21 (DE3) was transformed with expression vectors pRA-5HOL and pRA-OH5L, respectively, and the culture was performed at 28 ° C. using 2 × YT medium. When OD ₆₀₀ = approximately 0.8, expression was induced with IPTG at a final concentration of 1 mM and cultured overnight with shaking. After centrifuging the cells (the supernatant separated by this operation is used as the medium supernatant), ultrasonically crushing, and further centrifuging the supernatant as the intracellular soluble fraction and the precipitate as the intracellular insoluble fraction did. As a result of SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western-blotting of each fraction, it was confirmed that most of 5HOL and OH5L were expressed in the insoluble fraction in the cells. Protein was prepared from the minute (FIG. 3).

Example 4 Purification of 5HOL, OH5L, and rewinding The insoluble fraction in cells was immersed in 6M guanidine hydrochloride / PBS overnight at 4 ° C to solubilize the protein, and then a metal that specifically binds to His Tag in a denatured state. Purification was performed by metal chelate affinity chromatography using a chelate resin (TALON ^™ : CLONTECH). The yield after purification was about 5 mg each per liter of the transformation culture.

  Since the purified protein is denatured by guanidine hydrochloride, a rewinding operation is required to obtain a protein having a tertiary structure that retains its activity. After diluting each 5HOL and OH5L to 7.5μM and mixing equal amounts (total protein concentration 7.5μM: Ex3 concentration 3.75μM), add 50 times the total protein 2-mercaptoethanol (β-Me, final concentration 375μM) Then, the mixture was allowed to stand at 4 ° C. for 4 hours to carry out a reduction reaction. Subsequently, the sample is placed in a dialysis membrane, and the concentration of guanidine hydrochloride in the external solution is gradually reduced to 3M (6hr), 2M (6hr), 1M (12hr), 0.5M (12hr), and 0M. In the dialysis stage of 1M and 0.5M in the middle, oxidized glutathione (GSSG) as final oxidizing agent is 375μM, and L-arginine (L-Arg) is used as aggregation inhibitor at final concentration of 0.4M. In addition to this, the oxidation reaction was promoted by keeping the temperature at 4 ° C. (FIG. 4). The rewinding efficiency was about 30%. From the SDS-PAGE of Ex3 unwound after purification, 5HOL, and OH5L in equal amounts, it was confirmed that a highly heterogeneous heterodimer was formed (FIG. 3).

Example 5 Functional evaluation of Ex3 (1)
-Flow cytometric analysis
The binding of Ex3 to each cell was examined by flow cytometry. Add 100 μL of Ex3 as the primary antibody to the target cells, leave for 30 minutes at 4 ° C, wash twice with 0.1% NaN ₃ / PBS, then add anti-c-myc antibody as the secondary antibody and perform the same procedure Went. Finally, anti-mouse labeled with FITC as a tertiary antibody
After the antibody was added and the same operation was performed, fluorescence was measured. The negative control (hereinafter NC) is performed after the secondary antibody, and the positive control (hereinafter PC) is the secondary antibody as OKT3 IgG and TFK-1 cells (human gallbladder cancer cell line) for T-LAK cells. ) For 528
IgG was used for each. The results were found to bind to both cells (Figure 5 (top)).

  Further, after standing for 48 hours under physiological conditions (37 ° C., in RPMI 1640 medium), the same operation was performed to examine the stability. As a result, there was almost no change in the binding activity (FIG. 5 (bottom)).

  Furthermore, in order to examine whether the recognition site of Ex3 is the same as each parent antibody IgG, after adding Ex3 as a primary antibody, the same operation as PC was performed to examine the binding inhibition of the parent antibody. The results showed that the recognition sites of Ex3 and each parent antibody were the same because the binding ability was reduced compared to normal PC (FIG. 6).

Flow the supernatant after reacting Ex3 with excess T-LAK cells or TFK-1 cells to each cell.
As a result of the absorption test using cytometry, the binding ability decreased in any combination. If the molecule that binds to T-LAK cells and the molecule that binds to TFK-1 cells in the Ex3 solution are different, the binding does not decrease, so this result indicates that one Ex3 molecule binds to both cells. That is, it was found to have bispecificity (FIG. 7).

Example 6 Functional evaluation of Ex3 (2)
-Microscopic observation
After cancer cells were cultured overnight and allowed to attach, T-LAK cells alone or T-LAK cells mixed with Ex3 were added, and microscopic observation was performed over time. T-LAK alone shows little change even after 12 hours, whereas on the plate with Ex3, T-LAK starts to collect around the cancer cells after 2 hours, and almost all after 12 hours. Of T-LAK cells were concentrated in cancer cells (FIG. 8).
In the figures attached to this specification, “E / T” or “Effector / Target” means the effector (T-LAK cell) / target (TFK-1 cell) ratio.

  In addition, in the inhibition test in which various IgG were added, OKT8 (anti-CD8), which is irrelevant to the system, was observed in the plate added with OKT3, which is the parent antibody, and 528 IgG, almost 18 days after the addition. Antibody), MUSE11 (anti-MUC1 antibody) IgG-added plates showed the same concentration as when IgG was not added (FIG. 9).

Example 7 Ex3 Functional Evaluation (3)
-In vitro cytotoxicity test (MTS assay)
MTS assay was used to determine how much TFK-1 cells were damaged by T-LAK cells. Perform cell count and RPMI
It was adjusted to 10 ⁴ cells per 100 [mu] L, each 100 [mu] L in 96-well plates dispensed and allowed to stand overnight at 37 ° C.. Dilute the target protein with RPMI to the target concentration, and dispense 50 μL of the protein to the plate prepared the previous day. LAK
The cells were diluted with RPMI so as to achieve the target E / T ratio, and 50 μL was dispensed. After culturing at 37 ° C. for 48 hours, the culture medium on the plate was removed, washed with PBS, MTS, PMS, and RPMI were added, and incubated at 37 ° C. for 30 to 60 minutes. Absorbance at 490 nm was measured with a plate reader. As a result, a very strong effect was seen compared with conventional Mx3 or mSEA-Mx3 fused with mSEA. The concentration of Ex3 required to kill 100% of TFk-1 cells was as low as 1 pmol / mL (about 60 ng / mL), which is about 1/100 of the amount that mSEA-Mx3 shows an equivalent effect ( (Figure 10). (Note) MTS reagent (CellTiter
96 AQueous Non-Radioactive Cell Proliferation Assay, Promega), PMS (CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay, Promega)

OKT3
As a result of comparing the effect of Ex3 with the case of mixing equimolar amounts of scFv and 528 scFv, no scavenging activity was obtained at the high concentration even when scFv was mixed. From this, not only the effect of each antibody (the ability to activate T-LAK by CD3 stimulation of OKT3, and the inhibition of TFK-1 growth by 528 EGFR blocks), but also the physical crosslinking of lymphocytes and cancer cells, In other words, it can be seen that it is important to make it closer to the bispecific antibody (FIG. 11).

Example 8 Functional evaluation of Ex3 (4)-
In vitro cytotoxicity test ( ⁵¹ Cr
release assay)
In vitro cytotoxic activity can be easily assessed by MTS assay, which is a growth inhibition of tumor cells (growth
Inhibition) can also be used only for adherent cells. For this reason, ⁵¹ Cr is used to examine direct cytotoxicity.
A release assay was performed. As a result, PBMC assumed that significant toxic activity depending on the E / T ratio was observed by the addition of Ex3, and that lymphocytes once activated outside the body such as T-LAK cells were not used as adoptive immunotherapy. (Peripheral blood mononuclear cells;
Similarly, when peripheral blood mononuclear cells) were used as effector cells, the increase in the injury activity depending on the concentration of Ex3 was observed (FIG. 12).

Similarly, the presence of Ex3 and an increase in injury activity depending on the E / T ratio were also observed for other EGFR (Her1) positive cholangiocarcinoma cell lines (FIG. 13). In addition, when Ex3 was added to the EGFR-negative cell line, the T-LAK cell cytotoxic activity was not enhanced at all (Table 1). In Table 1 and the following figures, the meanings of the English symbols indicating the cells are as follows.
TFK-1: human gallbladder cancer;
OCUCh-LM1: Human gallbladder cancer;
HuCC-T1: Human gallbladder cancer;
OBA-LK1: human large cell lung cancer;
A549: Human lung adenocarcinoma;
CRL1500: human breast cancer;
SK-BR-3: human breast cancer;
A431: Human epidermoid cancer;
MCF-7: human breast cancer;
NCI-H69: human small cell lung cancer;
CHO: Chinese hamster ovary cells

Furthermore, in the inhibition test in which various IgGs were added, the parent antibodies OKT3 and 528
With IgG, the concentration-dependent decrease in the injury activity was observed, but addition of OKT8 and MUSE11 IgG, regardless of the system, did not show any inhibition of the activity (Fig. 14).

Example 9 Functional evaluation of Ex3 (5)-
Secretion of various cytokines
In order to identify factors that contribute to the enhancement of T-LAK cell injury activity in the experimental system described above, the production levels of various cytokines were examined. TFK-1 cells were allowed to attach to the plate and allowed to stand overnight, then T-LAK cells alone or even Ex3 was added at different concentrations, and the amount of various cytokines in the culture supernatant after 48 hours was measured using an ELISA kit (ENDOGEN ). Plates containing T-LAK cells alone, T-LAK cells and Ex3, T-LAK cells and TFK-1 cells did not show a significant increase in IFN-γ production, but T-LAK cells and TFK- Addition of Ex3 to 1 cell showed an increase in IFN-γ production depending on the concentration. The parent antibody, OKT3, or 528
Inhibition effect was seen in the plate to which IgG was further added, and IFN-γ production was not seen (FIG. 15).

  Similar results were obtained for GM-CSF and TNF-α (FIGS. 16 and 17). However, IL-2 did not increase in production depending on the concentration, but showed a slight decrease (FIG. 18).

  Subsequently, changes over time in the production amounts of various cytokines were measured using EILSA. IFN-γ and GM-CSF showed a time-dependent and concentration-dependent increase in production, but TNF-α and IL-2 peaked at 12 hours and then decreased sharply. . This is considered that the target cell TFK-1 cells died in 12 hours, and after that, production of IL-2 and the like did not occur and was taken up by being taken up by T-LAK cells, which is consistent with the result of FIG. 18 ( (Figure 19).

Example 10 Ex3 function evaluation (6)-
In vivo cytotoxicity test
Ten days after delivery of the SCID mice, 5 × 10 ⁶ TFK-1 cells were subcutaneously injected into the mice. Ten days after TFK-1 cell transplantation (tumor diameter of about 4 mm to 6 mm), 2 × 10 ⁷ T-LAK cells and each antibody were mixed, and then injected through the mouse tail vein for 4 consecutive days. The tumor diameter was measured every week, and the tumor volume was estimated from the major axis and minor axis. The experiment was terminated when the control tumor diameter exceeded 20 mm.

As a result, in the group administered with T-LAK cells alone, the tumor continued to grow as in the control group administered with PBS, whereas in the group administered with Ex3, significant growth inhibition was observed. In the group administered Ex3 at 20 μg / mouse, Ex3 prepared by chemical synthesis
The tumor degeneration effect was almost equivalent to that of bsAb (FIG. 20).

Example 11 Production of Humanized Ex3 Gene The humanized OKT3 variable region has already been reported, and it has been confirmed that it retains sufficient activity compared to mouse OKT3 (Reference Paper 2). Based on the amino acid sequence of the humanized OKT3 variable region described in Reference Paper 2, total gene synthesis was performed by the overlap PCR method. At this time, the optimal codon in E. coli was used. The increase of the expression level in E. coli by using the total synthetic gene substituted with the optimal codon has already been reported (FIG. 21).

Humanization of 528 variable region is CDR
The grafting method was used. First, VH and VL homology searches are performed, and each CDR (complementarity determining region)
FR (frame) with the highest homology considering the length of
Select human antibody sequences with work). The amino acid sequence was designed by replacing the CDR of the selected human antibody with the CDR of 528, and for the corresponding codon, the E. coli optimal codon was used as before, and the total synthesis of the gene was performed by the overlap PCR method (FIG. 22). ).

○ Reference paper 2-Adair,
JR et al. Humanization of the murine anti-human CD3 monoclonal antibody OKT3. Hum
Antibodies Hybridomas 5, 41-7. (1994).

Example 12 Production of Humanized Ex3 Expression Vector Humanized Ex3
The diabody (hExh3) is made from two molecules, h5HhOL and hOHh5L. An expression vector was prepared based on each expression vector constituting Ex3. That is, humanized 5H (hereinafter “h5H”: SEQ ID NO: 29) was amplified by PCR using an EF primer into which a restriction enzyme site was introduced, digested with NcoI-EagI, and replaced with 5H of pRA-5HOL. Subsequently, humanized OL (hereinafter “hOL”: SEQ ID NO: 28) was amplified using a GH primer and then digested with EcoRV-SacII, and replaced with OL to prepare pRA-h5HhOL. Similarly, humanized OH (hereinafter “hOH”: SEQ ID NO: 27) was amplified using IJ primer, digested with NcoI-EagI, replaced with OH of pRA-OH5L, and humanized 5L (hereinafter “h5L”) using KL primer. ; SEQ ID NO: 30) was amplified and replaced with 5 L with EcoRV-SacII to prepare pRA-hOHh5L. Like the Ex3 expression vector, a c-myc peptide tag for detection and a His-tag (Hisx6: histidine hexamer tag) for purification were introduced in parallel on the C-terminal side (FIG. 23).

E NcoI-h5H back primer 5'-nnnccatggcccaggtgcaactggttcagagc-3 '
[SEQ ID NO: 5]
F h5H-EagI forward primer 5'-nnncggccgagctcacggtaaccagcgta-3 '
[SEQ ID NO: 6]
G EcoRV-hOL back primer
5'-nnngatatccagatgacccagag-3 '
[SEQ ID NO: 7]
H hOL-SacII forward primer 5'-nnnccgcggcgcgggtaatctgc-3 '
[SEQ ID NO: 8]
I NcoI-hOH back primer 5'-nnnccatggcccaggtgcaactggtg-3 '
[SEQ ID NO: 9]
J hOH-EagI forward primer 5'-nnncggccgagctaacggtcacc-3 '
[SEQ ID NO: 10]
K EcoRV-h5L back primer
5'-nnngatatcgtgatgacccagagccc-3 '
[SEQ ID NO: 11]
L h5L-SacII forward primer 5'-nnnccgcggcgcgtttaatttccactttggtgccac-3 '

[SEQ ID NO: 12]

Example 13 Expression, purification, and unwinding expression vectors pRA-h5HhOL and pRA-hOHh5L of h5HhOL and hOHh5L using Escherichia coli were cultured in the same manner as in Example 3. As a result of SDS-PAGE and Western-blotting, expression was observed in each fraction, but both h5HhOL and hOHh5L were expressed most in the insoluble fraction in the cells. A preparation was made (Figure 24).

  Purification and rewinding were performed in the same manner as in Example 4. After purifying h5HhOL and hOHh5L individually, mixing the same amount and unwinding, it can be seen from SDS-PAGE of hExh3 that it has a very high degree of purification as Ex3 and forms a homogeneous heterodimer ( (Figure 24).

Example 14 Functional evaluation of hExh3
-Flow cytometric analysis
Flow as in Example 5
The binding of hExh3 to each cell was examined by cytometry. NC and PC were performed in the same manner as in Experimental Example 5. The results showed binding to both T-LAK cells and TFK-1 cells as in Ex3 (FIG. 25).

Example 15 Examination of functional modification by site-directed mutagenesis of hExh3 The functional modification of hExh3 by mutagenesis was examined. Since the reduction or loss of the antibody's own function due to humanization is often reported, sequences that can contribute to the structure of CDR in FR, that is, canonical structures (some immobilized CDR structures) and veriner residues There is a need for site-directed mutagenesis, in which a portion whose sequence has been changed due to humanization centering on the group is returned to the mouse sequence.

  The veriner residue is a residue that is related to antigen binding by laying the foundation of CDR among the residues located before and after the CDR in FR proposed by Reference Paper 3 and adjusting the structure of CDR. is there. However, the veriner residue involved in activity recovery differs depending on the antibody, and although it can be predicted to some extent by detailed structural analysis, it is common to actually create multiple mutants and select them by functional evaluation such as binding activity (reference) Paper 4, 5).

Each mutation was introduced using the overlap PCR method. That is, a mixture of a product obtained by PCR amplification of h5H with primer EN and a product amplified by PCR with FM is used as a template for 2nd PCR, and 2nd by primer EF.
PCR was performed, digested with NcoI-EagI, and replaced with h5H of pRA-h5HhOL. As a result, pRA-h5HhOL containing an M48I h48H mutant (hereinafter referred to as h5H-m01) is prepared. Using the same method, the following 10 mutants were prepared (Table 2, FIG. 26).

M h5H-M48I (+) 5'-gcctggaatggattggtaacatttatc-3 '
[SEQ ID NO: 13]
N h5H-M48I (-) 5'-gataaatgttaccaatccattccaggc -3 '
[SEQ ID NO: 14]
O h5H-A93T (+) 5'-tattactgcacgcgcagtggc-3 '
[SEQ ID NO: 15]
P h5H-A93T (-)
5'-gccactgcgcgtgcagtaata-3 '
[SEQ ID NO: 16]
Q h5H-R66KR71V (+) 5'-atttaagaacaaagtgaccatgacggttgataccagca-3 '
[SEQ ID NO: 17]
R h5H-R66KR71V (-) 5'-tgctggtatcaaccgtcatggtcactttgttcttaaat-3 '
[SEQ ID NO: 18]
S h5H-Y27D (+) 5'-gcctcaggcgatacctttacg-3 '
[SEQ ID NO: 19]
T h5H-Y27D (-) 5'-cgtaaaggtatcgcctgaggc-3 '
[SEQ ID NO: 20]
U h5H-M69LT73R (+) 5'-caaagtgaccctgacggttgatcgcagcatttcga-3 '
[SEQ ID NO: 21]
V h5H-M69LT73R (-) 5'-tcgaaatgctgcgatcaaccgtcagggtcactttg-3 '
[SEQ ID NO: 22]
W h5H-I75SS76RA78V (+) 5'-gataccagcagtcgcacggtctatatggaa-3 '
[SEQ ID NO: 23]
X h5H-I75SS76RA78V (-) 5'-ttccatatagaccgtgcgactgctggtatc-3 '
[SEQ ID NO: 24]

○ Reference paper 3-Foote,
J. & Winter, G. Antibody framework residues affecting the conformation of
the hypervariable loops. J Mol Biol 224, 487-99. (1992).
○ Reference paper 4-Sato,
K. et al. Reshaping a human antibody to inhibit the interleukin 6-dependent
tumor cell growth. Cancer Res 53, 851-6. (1993).
○ Reference paper 5-Sato,
K. et al. Humanization of a mouse anti-human interleukin-6 receptor antibody
comparing two methods for selecting human framework regions. Mol Immunol 31,
371-81. (1994).

Example 16 Functional evaluation of hExh3 and hExh3 mutants
-In vitro cytotoxicity test (MTS assay)
MTS as in Example 7.
The in vitro cytotoxic activity of hExh3 and hExh3 mutants was evaluated by assay. The results showed that the introduction of several mutations greatly affected the cytotoxic activity, and some mutants had a slightly stronger activity than hExh3 before the mutation introduction (FIG. 27).
Concentration-dependent cytotoxic activity was also observed, and hExh3 (left bar) and h5H-m09 (middle bar) were found to have the same effect as Ex3 (right bar) (FIG. 28).

The amino acid sequence of the variable region of antibody EGFR antibody 528 is shown. It is a schematic diagram which shows the structure of pRA-5HOL and pRA-OH5L which are Ex3 expression vectors. It is a photograph which shows the result of SDS-PAGE (upper) and Western-blotting (lower) which show the expression of 5HOL and OH5L in Escherichia coli BL21. The process of unwinding by step dialysis is shown. The results of examining the binding of Ex3 to each cell by flowcytometry are shown. The result of the inhibition test of Ex3 by Flowcytometry is shown. The result of the absorption test of Ex3 by Flowcytometry is shown. It is a microscope picture which shows the collection of the T-LAK cell to the TFK-1 cell by Ex3. It is a microscope picture which shows inhibition of the collection by IgG. The result of the in vitro cytotoxicity test (MTSassay) by various diabody type | mold bispecific antibodies, such as Ex3, is shown. A comparison of in vitro cytotoxic activity when Ex3 (left bar) and each scFv were mixed (right bar) is shown. The result of the in vitro cytotoxicity test ( ⁵¹ Crrelease assay) by various diabody type | mold bispecific antibodies of Ex3 is shown. The result of the in vitro cytotoxicity test ( ⁵¹ Crrelease assay) with respect to various cells by various diabody type | mold bispecific antibodies of Ex3 is shown. The result of the inhibition test which added IgG is shown. The increase in the production amount of IFN-γ by adding Ex3 is shown. The increase of the production amount of GM-CSF by Ex3 addition is shown. The increase of TNF-α production by Ex3 addition is shown. The change of the production amount of IL-2 by Ex3 addition is shown. The time course of each cytokine production is shown. The result of the in-vivo cytotoxicity test using a SCID mouse is shown. The amino acid sequence of the variable region sequence of the fully synthesized humanized antibody OKT3 is shown. The amino acid sequence of the variable region sequence of the fully synthesized humanized antibody 528 is shown. It is a schematic diagram which shows the structure of pRA-h5HhOL and pRA-hOHh5L which are hExh3 expression vectors. It is a photograph which shows the result of SDS-PAGE and Western-blotting which confirms the expression of h5HhOL and hOHh5L. The result of having investigated the binding with respect to the cell of hExh3 by Flowcytometry is shown. Shows the introduction of site-specific mutations into h5H. The result of the in vitro cytotoxicity test (MTS assay) of hExh3 and each variant is shown. The concentration-dependent result in the in vitro cytotoxicity test (MTSassay) of hExh3 and its each variant is shown. In the figure, hExh3 (left bar), h5H-m09 (center bar), and Ex3 (right bar).

Claims (27)

A diabody-type bispecific, characterized by having a first specificity for human epidermal growth factor receptor and a second specificity for surface antigens expressed in cells having phagocytic or cytotoxic activity Sex antibody. The diabody-type bispecific antibody according to claim 1, wherein the human epidermal growth factor receptor is human epidermal growth factor receptor 1 (HER1). The diabody-type bispecific antibody according to claim 1 or 2, wherein the cell having phagocytosis or cytotoxic activity is a cytotoxic T cell. The diabody-type bispecific antibody according to claim 1, 2, or 3, wherein the antigen expressed in cytotoxic T cells is CD3. The diabody-type bispecificity according to any one of the preceding claims, wherein the first specificity is derived from the variable regions of the heavy chain and light chain of the anti-human epidermal growth factor receptor antibody 528. antibody. The diabody-type bispecific antibody according to any one of the preceding claims, wherein the second specificity is derived from the variable regions of the heavy and light chains of the anti-CD3 antibody OKT3. The diabody-type bispecific antibody according to any one of the preceding claims, which is a humanized antibody. The diabody-type bispecific antibody according to claim 7, wherein the complementarity determining region (CDR) in the variable region is derived from a mouse antibody, and the other part is derived from a human antibody. The variable region derived from humanized anti-CD3 antibody OKT3 has at least one CDR of CDR1, CDR2 and CDR3 of the amino acid sequence shown in FIG. 21, and humanized anti-human epidermal growth factor receptor antibody 528 The diabody-type bispecific antibody according to claim 8, which has at least one CDR of CDR1, CDR2 and CDR3 of the amino acid sequence shown in Fig. 22 in the variable region derived therefrom. The variable region derived from humanized anti-CD3 antibody OKT3 has the CDR1, CDR2 and CDR3 of the amino acid sequence shown in FIG. 21, and the variable region derived from humanized anti-human epidermal growth factor receptor antibody 528. The diabody-type bispecific antibody according to claim 9, which has CDR1, CDR2, and CDR3 of the amino acid sequence shown in FIG. The variable regions of the H-chain and L-chain derived from the humanized anti-CD3 antibody OKT3 have the amino acid sequences shown in FIG. 21, and the H-chain and L-chain derived from the humanized anti-human epidermal growth factor receptor antibody 528 The diabody-type bispecific antibody according to claim 10, wherein the variable region of the chain has the amino acid sequence shown in FIG. The diabody-type bispecific antibody according to any one of claims 8 to 11, which has a site-specific mutation in a framework (FR) that may affect a CDR structure. The single-chain polypeptide of any one of the diabody-type bispecific antibodies according to any one of claims 1 to 12, or each region contained therein, comprising two types of single-chain polypeptides. Constituting polypeptide. A nucleic acid encoding the single-chain polypeptide according to claim 13 or each region contained therein. 15. The nucleic acid of claim 14, having an optimal codon in the host cell in which the nucleic acid is expressed. 16. A nucleic acid according to claim 15 having an optimal codon for E. coli. A replicable cloning vector or expression vector containing the nucleic acid according to any one of claims 14 to 16. The vector according to claim 17, which is a plasmid vector. A host cell transformed with the vector according to claim 17 or 18. The host cell according to claim 19, which is Escherichia coli. The host cell according to claim 19 is cultured to express the nucleic acid in the host cell, and the single-chain polypeptide according to claim 13 is recovered and purified. A method for producing the present polypeptide. A single-chain polypeptide obtained by the method according to claim 21 is associated, and the formed diabody-type bispecific antibody according to any one of claims 1 to 12 is separated and recovered. A method for producing a diabody-type bispecific antibody according to any one of claims 1 to 12. The diabody-type bispecific antibody according to any one of claims 1 to 12, the single-chain polypeptide according to claim 13, the nucleic acid according to any one of claims 14 to 16, A pharmaceutical composition comprising as an active ingredient a vector selected from the group consisting of the vector according to 17 or 18, and the host cell according to claim 19 or 20. 24. The pharmaceutical composition according to claim 23, which is for eliminating, killing, injuring and / or reducing tumor cells. 24. The pharmaceutical composition according to claim 23, which is for increasing the production amount of cytokines in cells having phagocytosis or cytotoxic activity. 26. The pharmaceutical composition according to claim 23, 24 or 25, which comprises a humanized diabody-type bispecific antibody as an active ingredient. The diabody-type bispecific antibody according to any one of claims 1 to 12 is added to a culture system of cells having phagocytosis or cytotoxic activity and tumor cells expressing human epidermal growth factor receptor. A method for increasing the amount of cytokine production in a cell having phagocytosis or cytotoxic activity.

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