JP5614606B2 - Multimerized small molecule antibody - Google Patents

Description

  The present invention relates to a multimerized low-molecular antibody that is a multimer composed of a single-chain antibody (scFv) against anti-epidermal growth factor receptor (EGFR), a method for producing the same, and the multimerized low-molecular antibody. The present invention relates to a pharmaceutical composition characterized by containing as an active ingredient.

  In recent years, immunotherapy has been used as a safe treatment for cancer (malignant tumor) and rheumatism. In immunotherapy for cancer, an antibody that specifically shows cytotoxic activity against cancer is used. Antibody drugs composed of such antibodies are recognized as safe and secure with high side effects, low side effects, and high therapeutic effects. On the other hand, it is necessary to produce them using established animal cells. .

  For this reason, the production of low molecular weight antibodies such as single chain antibodies (scFv) in which the VH and VL domains of a certain antibody are in a single polypeptide chain has become a global trend. Such low-molecular-weight antibodies can be produced even in inexpensive E. coli, but there is a concern that the half-life in the body decreases due to the decrease in molecular weight and the duration of drug efficacy decreases. In addition, a complete antibody such as IgG binds to a target antigen in a multivalent amount, whereas a low-molecular antibody usually has a monovalent valence, so that a decrease in affinity is also a problem. Furthermore, since the main mechanism of action of antibody drugs is also antibody-dependent cytotoxic activity (ADCC) via the Fc region, scFv not having the Fc region is concerned with low efficacy. For scFv, Non-Patent Document 1 can be referred to.

On the other hand, there are also antibodies that exert their growth-inhibiting effect by binding to antigens on the surface of cancer cells in a multivalent manner. An example of such an antibody is “Panitumumab”, which is an antibody against epidermal growth factor receptor (EGFR), which is a cancer-related antigen that has been recently launched and is highly expressed as cells become cancerous. it can. “Panitumumab” has a type of Fc that hardly induces ADCC, and its main mechanism of action is an inhibitory effect on cancer cell growth due to its strong binding ability to EGFR. Furthermore, “Cetuximab” has already been marketed in the United States as an anti-EGFR antibody drug using EGFR as a therapeutic target molecule.

Anti-human epidermal growth factor receptor 1 (Her1) antibody 528, which is the parent antibody of the humanized anti-EGFR antibody described herein, is a clone established at the same time as parent antibody 225 of “Cetuximab” Has almost the same growth inhibitory effect on cancer cells. However, as already described, it is known that when the valence to EGFR is monovalent, the effect is hardly seen. In fact, the humanized 528 scFv did not show an inhibitory effect on cancer cell growth. In order to overcome such drawbacks of scFv, attempts have been made to multimerize scFv by modifying a linker (Non-patent Document 2). In recent years, scFv dimers that induce apoptosis in lymphoma have also been reported (Non-patent Document 3). However, no examples of scFv multimers that exert growth inhibitory effects on solid cancers and EGFR positive carcinomas have been reported.
International Publication No. WO2007 / 108152 Pamphlet JP 2004-242638 A Rosenburg and Moore (Ed.), “The Pharmacology of Monoclonal Antibodies”, Vol. 113, Springer-Verlag, New York, pp.269-315 (1994) Biomol Eng. 2001 18 (3): 95-108 Biochem Biophys Res Commun. 2004 315 (4): 912-8

Therefore, the main object of the present invention is to construct a multimerized scFv by modifying the linker of a single-chain antibody (scFv) against the anti-epidermal growth factor receptor (EGFR), and to regulate the pharmacokinetics by increasing the molecular weight. To improve the affinity and to enhance the antitumor effect by the multivalent effect, and to provide a new inexpensive drug molecule.

That is, the present invention relates to each aspect shown below.
[Aspect 1] Consists of a single chain antibody (scFv) comprising the humanized variable region (h5H) of the H chain and the humanized variable region (h5L) of the L chain of the anti-human epidermal growth factor receptor 1 antibody 528 A multimerized small molecule antibody comprising:
H5H and h5L are the amino acid sequences shown in SEQ ID NO: 2 and SEQ ID NO: 4, respectively, or an amino acid sequence in which one or several amino acids are substituted, deleted, inserted or added in the amino acid sequence, The multimerized small molecule antibody having an amino acid sequence having substantially the same antigen binding property as the variable region.
[Aspect 2] The multimerized small molecule antibody according to Aspect 1, wherein 2 to 4 single chain antibodies (scFv) are associated.
[Aspect 3] The multimerization according to Aspect 1 or 2, wherein each single-chain antibody includes a peptide linker consisting of 1 to 9 amino acids linking the variable region of the H chain and the variable region of the L chain. Small molecule antibody.
[Aspect 4] The multimerized small molecule antibody according to aspect 3, wherein the peptide linker is composed of a peptide consisting of 5 amino acids.
[Aspect 5] The multimerized small molecule antibody according to any one of Aspects 1 to 3, wherein each single-chain antibody (scFv) has the amino acid sequence represented by SEQ ID NO: 6.
[Aspect 6] The multimerized small molecule antibody according to Aspect 1 or 2, wherein each single-chain antibody does not contain a peptide linker.
[Aspect 7] In each single-chain antibody, 1 to several amino acids at the C-terminal of the humanized variable region at the N-terminal side, or 1 to several amino acids at the N-terminal of the humanized variable region at the C-terminal side The multimerized small molecule antibody according to aspect 6, wherein one amino acid has been removed.
[Aspect 8] The multimerized small molecule antibody according to any one of Aspects 1 to 7, wherein in each single-chain antibody, the humanized variable region (h5H) of the H chain is on the N-terminal side.
[Aspect 9] A nucleic acid molecule encoding a polypeptide of a single chain antibody constituting the multimerized low molecular weight antibody according to any one of Aspects 1 to 8.
[Aspect 10] A replicable cloning vector or expression vector containing the nucleic acid molecule according to Aspect 9.
[Aspect 11] The vector according to Aspect 10, which is a plasmid vector.
[Aspect 12] A host cell transformed with the vector according to Aspect 10 or 11.
[Aspect 13] A host cell according to Aspect 12, which is a prokaryotic cell.
[Aspect 14] A host cell according to Aspect 12 or 13 is cultured, and a polypeptide of a single-chain antibody constituting the multimerized low-molecular antibody according to any one of Aspects 1 to 8 is expressed. The method for producing a multimerized low-molecular antibody according to any one of aspects 1 to 8, which comprises collecting and purifying a peptide and associating the single-chain antibody.
[Aspect 15] A large quantity according to aspect 14, wherein the prokaryotic cell is Escherichia coli, and the multimerized low molecular weight antibody is recovered from the culture medium supernatant, perizuma fraction, intracellular soluble fraction, or intracellular insoluble fraction of Escherichia coli. A method for producing an immobilized low molecular antibody.
[Aspect 16] A pharmaceutical composition comprising the multimerized low molecular weight antibody according to any one of Aspects 1 to 8 as an active ingredient.
[Aspect 17] A pharmaceutical composition according to Aspect 16, which is for eliminating, killing, injuring and / or reducing tumor cells.

In the present invention, the tetramer was successfully prepared from the dimer of scFv by modifying the scFv linker derived from the anti-human epidermal growth factor receptor 1 antibody 528. Each of these multimerized low-molecular-weight antibodies alone showed a cancer cell growth inhibitory effect equivalent to or higher than that of Cetuximab. Furthermore, under certain conditions, it has an effect that surpasses Panitumumab. Such an excellent effect is not found in the scFv monomer.

  Anti-EGFR antibody-producing mouse B cell hybridoma 528 (ID: TKG0555) was distributed from Tohoku University Research Center for Aging Medicine, and mRNA was extracted using ISOGEN (Nippon Gene), First-Strand cDNA Synthesis Kit CDNA was prepared by (Amersham Biosciences). This cDNA was synthesized based on a reference paper (Krebber, A. et al. Reliable cloning of functional antibody variable domains from hybridomas and spleen cell repertoires featuring a reengineered phage display system. J Immunol Methods 201, 35-55. (1997)). PCR was performed using cloning primers to clarify the sequences of 528 variable regions VH (hereinafter 5H) and VL (hereinafter 5L). The hybridoma producing the 528 antibody is stored as ATCC No. HB-8509 in the American Type Culture Collection (ATCC) and can be easily obtained from such depository.

  “Humanization” means at least part of the complementarity-determining region (CDR) residues in the variable region of a human immunoglobulin (recipient antibody), such as mouse, rat, or rabbit. By non-human animal (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 et al., Nature 332, 323-329 (1988); EP-B-239400; Presta, Curr. Op. Struct. Biol. 2, 593-596 (1992); and EP-B-451216.

  Such antibody variable regions can be humanized 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.

  Accordingly, examples of humanized variable regions include antibodies 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.

  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. It is possible to improve the function of the humanized antibody by causing site-specific mutations in the middle part, for example, canonical sequence or vernier sequence.

  Specifically, humanization of the 528 variable region was performed by CDR grafting. First, VH and VL homology searches are performed, and the human antibody sequence having the highest homology FR (frame work) is selected in consideration of the length of each CDR (complementarity determining region). It is preferable to design an amino acid sequence in which the CDR of the selected human antibody is replaced with the CDR of 528, and for the corresponding codon, it is preferable to use the E. coli optimal codon as before, and to perform total synthesis of the gene by overlap PCR method I can do it.

  Specific amino acid sequences of the humanized variable region (h5H) and L chain humanized variable region (h5L) contained in the single-chain antibody that is a component of the multimerized low-molecular antibody of the present invention and its base Examples of sequences are shown in SEQ ID NO: 2 and SEQ ID NO: 1, and SEQ ID NO: 4 and SEQ ID NO: 3, respectively.

  The number of single-chain antibodies constituting the multimerized low-molecular antibody of the present invention is not particularly limited. For example, 2 to 4 single-chain antibodies are associated with each other.

In the present specification, the “peptide linker” refers to an oligopeptide or polypeptide that functions to give a single-chain polypeptide by binding the variable region (VH) of the H chain and the variable region (VL) of the L chain. pointing. The peptide linker makes it difficult for single-chain antibodies to interact with each other in the molecule, and allows the formation of multimers by a plurality of single-chain antibodies, and as a result, VHs derived from different single-chain antibodies. And VL are appropriately associated, so that the function of the original protein (the polypeptide is derived from the original protein or derived from the original protein), for example, part of biological activity or It is not particularly limited as long as it can take a structure that simulates or promotes all of them, and for example, it is selected from those widely known in the art or modified from the known linker. Is possible.

  In order to facilitate the formation of the multimerized small molecule antibody of the present invention by associating a plurality of single chain antibodies, the humanized variable region of the H chain and the humanized variable region of the L chain in each single chain antibody It is preferable that the peptide linker which connects and is 1 to 9, more preferably 1 to 7 amino acids in length. An amino acid sequence of a specific example (Example 1) of a single chain antibody constituting the multimerized low molecular weight antibody of the present invention containing a peptide linker consisting of 5 amino acids, and an example of its base sequence, SEQ ID NO: 6 and Sequence The number 5 is shown.

Each single chain antibody has a structure in which the humanized variable region (h5H) of the H chain is on the N-terminal side (VH-VL type) or a structure in which the humanized variable region of the L chain (h5L) is on the N-terminal side. (VL-VH type) may be used.

Furthermore, each single-chain antibody may not include the above peptide linker, and two variable regions may be directly bonded. In such a case, in order to increase the three-dimensional degree of freedom of each single-chain antibody and promote multimerization, the C-terminal of the humanized variable region on the N-terminal side in each single-chain antibody It is preferable that 1 to several amino acids or 1 to several amino acids at the N-terminal of the humanized variable region on the C-terminal side have been removed. An example of such a type of single chain antibody is a single chain antibody in which one or two amino acids at the C-terminal end of VH have been removed from the VH-VL type of VH. Such single chain antibodies can associate to form trimers and tetramers.

Furthermore, in each amino acid sequence represented by each of the above SEQ ID Nos., An amino acid sequence in which one or several amino acids are substituted, deleted, inserted or added, and the function / activity of the polypeptide comprising the original amino acid sequence For example, an amino acid sequence that substantially retains the antigen specificity of the variable region can also be used as the polypeptide of the single chain antibody constituting the multimerized low-molecular antibody of the present invention. The amino acids that are deleted, substituted, inserted or added are preferably substituted for homologous amino acids (polar / nonpolar amino acids, hydrophobic / hydrophilic amino acids, positive / negative charged amino acids, aromatic amino acids, etc.), Alternatively, it is preferable that amino acid deletion or addition does not cause a significant change in the three-dimensional structure and / or local charge state of the protein or that they are not substantially affected. Polypeptides having such deleted, substituted or added amino acids include, for example, site-directed mutagenesis methods (such as point mutagenesis and cassette mutagenesis), gene homologous recombination methods, primer extension methods, and It can be easily prepared by appropriately combining methods known to those skilled in the art such as PCR. The amino acid sequence in which one or several amino acids are substituted, deleted, inserted or added is 90% or more, preferably 95% or more, more preferably 99% or more, with respect to the total length of the original amino acid sequence. It can also be said to show sequence homology.

A typical example of a nucleic acid molecule (oligonucleotide) encoding each region or sequence contained in each single-chain antibody of the present invention has the base sequence shown in each of the above SEQ ID NOs. In addition, a nucleic acid molecule comprising such a base sequence that exhibits a sequence homology of 90% or more, preferably 95% or more, more preferably 99% or more with the entire length of the base sequence described in each SEQ ID NO. Alternatively, these nucleic acid molecules are also included in the above-described nucleic acids of the present invention because they are considered to encode polypeptides having substantially the same activity or function as the sequences.

  In order to determine sequence homology between two amino acid sequences or base sequences, the sequences are pre-processed to the optimum for comparison. For example, by making a gap in one sequence, the alignment with the other sequence is optimized. Thereafter, the amino acid residues or bases at each site are compared. When the same amino acid residue or base as the corresponding site in the second sequence is present at a site in the first sequence, the sequences are identical at that site. Sequence homology between the two sequences is expressed as a percentage of the total number of sites (all amino acids or all bases) of the number of sites that are identical between the sequences.

Here, “homology” refers to each amino acid residue or each base constituting the chain between two chains in a polypeptide sequence (or amino acid sequence) or polynucleotide sequence (or base sequence). It means the amount (number) of things that can be determined to be identical in the mutual relationship of each other, and means the degree of sequence correlation between two polypeptide sequences or two polynucleotide sequences. Homology can be easily calculated. Many methods for measuring homology between two polynucleotide or polypeptide sequences are known, and the term “homology” (also referred to as “identity”) is well known to those skilled in the art (eg, , Lesk, AM (Ed.), Computational Molecular Biology, Oxford University Press, New York, (1988); Smith, DW (Ed.), Biocomputing: Informatics and Genome Projects, Academic Press, New York, (1993); Grifin , AM & Grifin, HG (Ed.), Computer Analysis of Sequence Data: Part I, Human Press, New Jersey, (1994); von Heinje, G., Sequence Analysis in Molecular Biology, Academic Press, New York, (1987 Gribskov, M. & Devereux, J. (Ed.), Sequence Analysis Primer, M-Stockton Press, New York, (1991), etc.). Common methods used to measure the homology of two sequences include Martin, J. Bishop (Ed.), Guide to Huge Computers, Academic Press, San Diego, (1994); Carillo, H. & Lipman , D., SIAM J. Applied Math., 48: 1073 (1988), etc., but are not limited thereto. Preferred methods for measuring homology include those designed to obtain the largest match between the two sequences being tested. An example of such a method is one assembled as a computer program. Preferred computer programming methods for measuring homology between two sequences include the GCG program package (Devereux, J. et al., Nucleic Acids Research, 12 (1): 387 (1984)), BLASTP, BLASTN, FASTA (Atschul, SF et al., J. Molec. Biol., 215: 403 (1990)) and the like, but are not limited thereto, and methods known in the art can be used. .

  Furthermore, each nucleic acid molecule described above hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the DNA consisting of the base sequence represented by each SEQ ID NO. It includes DNA encoding a polypeptide having substantially the same function and activity as the polypeptide.

  Here, hybridization can be performed according to a method known in the art such as the method described in Molecular cloning third. Ed. (Cold Spring Harbor Lab. Press, 2001) or a method analogous thereto. Moreover, when using a commercially available library, it can carry out according to the method as described in an attached instruction manual.

  Hybridization may be performed by a method known in the art, such as, for example, the method described in Current protocols in molecular biology (edited by Frederick M. Ausubel et al., 1987). It can carry out according to the method according to it. Moreover, when using a commercially available library, it can carry out according to the method as described in an attached instruction manual.

As used herein, “stringent conditions” in DNA hybridization are defined by an appropriate combination of salt concentration, organic solvent (eg, formamide), temperature, and other known conditions. That is, stringency increases depending on whether the salt concentration is reduced, the organic solvent concentration is increased, or the hybridization temperature is increased. In addition, washing conditions after hybridization also affect stringency. This wash condition is also defined by salt concentration and temperature, and the stringency of the wash increases with decreasing salt concentration and increasing temperature.

Therefore, “stringent conditions” means that the degree of homology between each base sequence is, for example, about 80% or more, preferably about 90% or more, more preferably about 95% or more on the average on the whole. It means that the hybrid is specifically formed only between base sequences having high homology. Specifically, for example, the conditions include a sodium concentration of 150 to 900 mM, preferably 600 to 900 mM, and a pH of 6 to 8 at a temperature of 60 to 68 ° C. As a specific example of stringent conditions, hybridization is performed under the conditions of 5 x SSC (750 mM NaCl, 75 mM trisodium citrate), 1% SDS, 5 x Denhardt's solution 50% formaldehyde, and 42 ° C. Washing is performed under conditions of 0.1 × SSC (15 mM NaCl, 1.5 mM trisodium citrate), 0.1% SDS, and 55 ° C.

  Furthermore, when a nucleic acid encoding a humanized variable region in each single-chain antibody polypeptide is prepared, it can be totally synthesized by an overlap PCR method based on a previously designed 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) Alternatively, it can be performed according to a modified or modified method. 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 example, Sanger et al., Proc. Natl. Acad. Sci. USA 74: 5463-5467 (1977) can be referred to for sequencing of nucleic acids such as DNA. 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), etc. .

  The nucleic acid encoding the single-chain antibody constituting the multimerized low-molecular antibody of the present invention obtained in this way or each region contained in the antibody is appropriately obtained by a means known to those skilled in the art depending on the purpose. Can be modified to code. A technique for genetically modifying or modifying DNA in this way is described in 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 antibody polypeptide by replacing a codon encoding an original amino acid residue with a codon encoding another amino acid residue.

Alternatively, as described in the examples of the present specification, a single-chain polypeptide is used so that a codon (optimum codon) suitable for a host cell such as E. coli is used without changing the amino acid itself. The encoding nucleic acid can also be modified. By changing to the optimal codon in this way, it is possible to improve the expression efficiency of the single-chain antibody in the host cell.

  The multimerized low-molecular antibody of the present invention 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 a genetic engineering technique, for example, a replicable cloning vector or expression vector containing a nucleic acid encoding a polypeptide of a single-chain antibody constituting a multimerized low-molecular antibody is prepared, and a host cell is used with this vector. Produced by transforming, culturing the transformed host cell, expressing the polypeptide of the single chain antibody in the host cell, recovering and purifying the polypeptide, and associating the single chain antibody I can do it.

  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). It means a DNA construct containing the determined DNA sequence. 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. Furthermore, for the purpose of facilitating the detection and purification of the produced single-chain polypeptide, a sequence encoding various peptide tags known to those skilled in the art (for example, c-myc tag and His-tag) is added. It can be included at the end of the sequence corresponding to the polypeptide of the present chain antibody.

  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.

  A single-chain polypeptide obtained by such expression in a host cell is generally recovered from the culture medium as a secreted polypeptide, but if it is produced directly without a secretion signal, the host It can be recovered from cell lysates. 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 multimeric low-molecular antibody of the present invention can be produced by associating (unwinding) the two kinds of single-chain polypeptides thus purified, and separating and recovering the formed antibody molecules. I can do it.

  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 multimerized low-molecular antibody can also be performed by any method known to those skilled in the art.

As described above, the multimerized low-molecular antibody of the present invention is prepared from, for example, a culture medium supernatant, a periplasma fraction, a cell-soluble fraction, or a cell-insoluble fraction of cultured host cells such as E. coli. Is possible.

  The pharmaceutical composition of the present invention contains, as an active ingredient, one selected from the group consisting of the multimerized low-molecular antibody, single-chain polypeptide, nucleic acid, vector, and transformed host cell of the present invention. It is characterized by. 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.

  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, and administration route. 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 subject 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, the antibody may be preliminarily bound to the cell before administration by mixing the active ingredient such as the humanized highly functional bispecific antibody of the present invention with the cell before administration. preferable.

  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 reference examples and examples. However, these 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 reference examples and examples were made using or can be performed using standard techniques, except as otherwise described in detail, which are well known and routine to those skilled in the art. Is. In the following examples, unless otherwise indicated, specific procedures and treatment conditions are as follows for DNA cloning 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 in 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, Oxford University Press (1995) and MA Innis et al. Ed., “PCR Protocols”, Academic Press, New York (1990), and use commercially available reagents or kits. If so, they use attached protocols (protocols) and attached chemicals.

Production of multimerized small molecule antibody (528scFv multimer) (1)
The multimerized small molecule antibody of the present invention was prepared in the following outline.
The expression vector was prepared based on the humanized diabody expression vector (pRA-h5HhOL, pRA-hOHh5L: Patent No. 380790 (Patent Document 2)) targeting EGFR and CD3 already constructed in this course. . That is, each vector was digested with restriction enzymes NcoI and EagI, and the humanized 528scFv expression vector pRA-h5Hh5L (G1) having a 5-amino acid linker (GGGGS) was prepared by exchanging the h5H site and the hOH site. 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.
The results prepared from the E. coli culture medium supernatant and the E. coli intracellular insoluble fraction are shown in FIG.

Evaluation of binding ability of multimerized low molecular antibody The surface plasmon resonance was conducted under the conditions shown below for the 528scFv dimer and monomer, which are the multimerized low molecule of the present invention prepared in Example 1. It was used to evaluate binding (FIG. 2).

Sensor chip: CM5
Running Buffer: PBS
Flow rate: 20 ml / min
Analyte addition time: 2 min (addition amount 40 ml)
Flow cell 2: Blocking
Flow cell 3: EGFR (fixed loading 3550 RU)

As a result, as shown in Table 1, the dissociation rate of the dimer was delayed compared to that of the monomer, and the enhancement of binding due to multimerization was shown.

Cell growth inhibition test of multimerized small molecule antibody The 528scFv dimer (VH-VL type) and monomer, which are the multimerized small molecule of the present invention prepared in Example 1, are summarized as follows. The cell growth inhibition test (MTS assay) was performed.

By MTS assay, how much the growth of A431 cell (ATCC No. CRL-1555), a human squamous cell carcinoma cell line, was inhibited by 528scFv dimer was compared with 528scFv monomer, Cetuximab and 528IgG. The cells were counted, adjusted to 2 × 10 ³ cells per 100 μL of RPMI (0.5% FBS), dispensed 100 μL each into a 96-well plate, and allowed to stand overnight at 37 ° C. Dilute the target protein with RPMI to the target concentration, dispense 200 μL of the protein to the plate prepared the previous day, and incubate at 37 ° C for 96 hours. The culture medium on the plate was removed, washed with PBS, MTS, PMS, and RPMI were added. After incubation at 37 ° C. for 30 to 60 minutes, the absorbance at 490 nm was measured with a plate reader. The results show that the dimer of 528scFv exerts cytotoxic activity not seen with the monomer, which is superior to commercially available Cetuximab and 528 IgG (FIG. 3).
(Note) MTS reagent (CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay, Promega), PMS (CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay, Promega)

Production of multimerized small molecule antibody (528scFv multimer) (2)
A multimerized small molecule antibody consisting of a single-chain antibody from which the peptide linker contained in the single-chain peptide that is a component of the 528scFv dimer prepared in Example 1 was removed was prepared in the following outline.

After amplifying humanized 5H (h5H) by PCR using AB primer and humanized 5L (h5L) using CD primer, mix the obtained PCR products, and further PCR amplification using AD primer went. The obtained PCR product was digested with restriction enzymes NcoI and SacII and inserted into a pRA vector to prepare a VH-VL humanized 528scFv expression vector pRA-h5Hh5L (HLG0) that does not contain a linker.
On the other hand, after amplification of h5L by PCR using EF primer and h5H using GH primer, the obtained PCR products were mixed, and further PCR amplification was performed using EH primer. The obtained PCR product was digested with restriction enzymes NcoI and SacII and inserted into a pRA vector to prepare a VL-VH type humanized 528scFv expression vector pRA-h5Hh5L (LHG0) that does not contain a linker.
In each case, a c-myc peptide tag for detection and a His-tag for purification are introduced in parallel on the C-terminal side.

A: NcoI-5H: 5'-NNNCCATGGCCCAGGTGCAACTGGTTCA-3 '[SEQ ID NO: 7]
B: 5H-5L-inverse: 5'-GGGCTCTGGGTCATCACGATATCCGAGCTCACGGTAACCAGCG-3 '[SEQ ID NO: 8]
C: 5H-5L: 5'-GATATCGTGATGACCCAGAGCCC-3 '[SEQ ID NO: 9]
D: 5L-SacII: 5'-NNNCCGCGGCGCGTTTAATTTCCACTTT-3 '[SEQ ID NO: 10]
E: NcoI-5L: 5'-NNNCCATGGATATTGTGATGACCCAGAG-3 '[SEQ ID NO: 11]
F: 5L-5H-inverse: 5'-CGCTCTGAACCAGTTGCACCTGTTTAATTTCCACTTTGGTGCCCTGGCC-3 '[SEQ ID NO: 12]
G: 5L-5H: 5′-CAGGTGCAACTGGTTCAGAGCG-3 ′ [SEQ ID NO: 13]
H: 5H-SacII: 5'-NNNCCGCGGAGCTCACGGTAACCAGCGT-3 '[SEQ ID NO: 14]

As a result, the VH-VL type was expressed in each fraction, and the VL-VH type was expressed only in the insoluble fraction (FIG. 4). As a result of gel filtration, it was confirmed that the multimerized low molecular weight antibody prepared from the culture supernatant was a trimer of a single chain antibody (FIG. 5).

Evaluation of binding property of 528scFv trimer The binding property of 528scFv trimer, which is a multimerized small molecule of the present invention prepared in Example 4, was compared with the binding property of dimer and monomer. This was performed using surface plasmon resonance under the following conditions (FIG. 6).

Sensor chip: CM5
Running Buffer: PBS
Flow rate: 20 ml / min
Analyte addition time: 2 min (addition amount 40 ml)
Flow cell 1: EGFR (fixed loading 4066 RU)
Flow cell 2: Blocking

As a result, as shown in Table 2, the dissociation rate was delayed and the dissociation equilibrium constant was decreased in the dimer and trimer as compared with the monomer, and the enhancement of binding due to multimerization was shown. It was.

Cell proliferation inhibition test of multimerized low molecular antibody Example 3 of the 528scFv trimer and dimer (VH-VL type), which are the multimerized low molecules of the present invention prepared in Example 4, were used. The cell growth inhibition test (MTS assay) was carried out in the same manner as above. As a result, it was confirmed that the cytotoxic activity was further improved by the trimer of 528scFv as compared with the dimer (FIG. 7).

Production of multimerized small molecule antibody (528scFv multimer) (3)
From the single-chain antibody (VH-VL type) from which the peptide linker contained in the single-chain peptide that is a component of the 528scFv dimer prepared in Example 4 was removed, the VH C-terminal was further removed. Multimerized small molecule antibodies from which one or two amino acids have been removed were prepared as outlined below.

After amplifying h5H by PCR using AI primer, the resulting PCR product and pRA-hOHh5L were digested with NcoI and EcoRV, respectively, and then the hOH site was replaced to delete one C-terminal amino acid of h5H. A humanized 528scFv expression vector pRA-h5Hh5L (Δ1) was prepared.
On the other hand, after amplification of h5H by PCR using AJ primer, the resulting PCR product and pRA-hOHh5L were digested with NcoI and EcoRV, respectively, and the hOH site was replaced, thereby deleting two C-terminal amino acids of h5H A humanized 528scFv expression vector pRA-h5Hh5L (Δ2) was prepared.
In each case, a c-myc peptide tag for detection and a His-tag for purification are introduced in parallel on the C-terminal side.

I: 5HΔ1-EcoRV: 5′-NNNGATATCGCTCACGGTAACCAGCGTACCCT-3 ′ [SEQ ID NO: 15]
J: 5HΔ2-EcoRV: 5′-NNNGATATCCACGGTAACCAGCGTACCCTGG-3 ′ [SEQ ID NO: 16]

As a result of gel filtration, a single chain antibody (528scFv (Δ1)) from which one of the C-terminals of VH prepared from the soluble fraction in the fungus was removed formed a trimer, and 2 of the C-terminal of VH. It was confirmed that the single-chain antibody (528scFv (Δ2)) from which the individual had been removed formed a trimer, and the multimerized low-molecular-weight antibody produced a dimer and a tetramer of a single-chain antibody ( 8 and 9).

Cell growth inhibition test of multimerized low molecular antibody 528scFv dimer, trimer and tetramer (VH-VL type) which are the multimerized low molecules of the present invention prepared in each of the above examples ) Was subjected to a cell proliferation inhibition test (MTS assay) in the same manner as in Example 3. As a result, the same results as above were obtained for the 528 scFv monomer, dimer and trimer. Furthermore, the trimer 528scFv (Δ1) has almost the same activity as the scFv dimer, and the dimer 528scFv (Δ2) and the tetramer 528scFv (Δ2) are both narrow and inaccurate. The activity was confirmed (FIG. 10).

IgG antibody has a molecular weight of about 150,000, while scFv is less than 30,000. Compared to scFv, which has a very short half-life in the body, IgG is several days long, but due to its large molecular size, its permeability to tumor tissue is low, and as a result, the tumor / blood ratio is not so high in both cases. In contrast, the multimerized scFv of the present invention having an intermediate size is also expected as a molecule having an optimal half-life in the body and permeability. Furthermore, by using the multimerized scFv of the present invention, it has optimal pharmacokinetics alone and can exhibit sufficient effects without co-administration of activated lymphocytes (T-LAK). Development of inexpensive production of antibody drugs becomes possible.

The result of preparing a 528scFv dimer from an E. coli culture medium supernatant and an E. coli intracellular insoluble fraction is shown. The result of the binding evaluation using the surface plasmon resonance about a 528scFv dimer and a monomer is shown. The result of the cell growth inhibition test about 528scFv dimer, a monomer, etc. is shown. The scFv was calculated as a single molecule. The result of the expression of the multimerized small molecule antibody not containing the peptide linker is shown. The result of carrying out gel filtration of the multimerization low molecular antibody which does not contain a peptide linker is shown. The result of the binding evaluation using the surface plasmon resonance about a 528scFv trimer, a dimer, and a monomer is shown. The result of the cell growth inhibition test about 528scFv trimer, a monomer, etc. is shown. The scFv was calculated as a single molecule. The result of the expression of the multimerized small molecule antibody which does not contain a peptide linker and further has one or two C-terminal amino acids removed from VH is shown. The results of gel filtration of a multimerized low molecular weight antibody that does not contain a peptide linker and from which one or two amino acids at the C-terminal end of VH have been removed are shown. The results of cell growth inhibition tests for various types of multimerized small molecule antibodies are shown. The scFv was calculated as a single molecule.

Claims (13)

The anti-human epidermal growth factor receptor 1 antibody 528 comprises a single chain antibody (scFv) comprising an H chain humanized variable region (h5H) and an L chain humanized variable region (h5L) associated with each other. A multimerized small molecule antibody that is a mer, trimer, or tetramer, wherein 1 to 5 linking each H chain variable region and L chain variable region to each single chain antibody A peptide linker consisting of amino acids is included, or the peptide linker is not included, and h5H and h5L are the amino acid sequences shown in SEQ ID NO: 2 and SEQ ID NO: 4, respectively, The multimerized low-molecular antibody, which is an amino acid sequence in which one or several amino acids are substituted, deleted, inserted or added and has an antigen binding property substantially equivalent to the variable region. The multimerized small molecule antibody according to claim 1, wherein each single chain antibody (scFv) has the amino acid sequence shown in SEQ ID NO: 6. In each single-chain antibody, 1 to several amino acids at the C-terminal of the humanized variable region on the N-terminal side, or 1 to several amino acids at the N-terminal of the humanized variable region on the C-terminal side The multimerized low-molecular antibody according to claim 1 or 2, which has been removed. The multimerized small molecule antibody according to any one of claims 1 to 3, wherein in each single chain antibody, the humanized variable region (h5H) of the H chain is on the N-terminal side. A nucleic acid molecule encoding a polypeptide of a single-chain antibody that constitutes the multimerized small molecule antibody according to any one of claims 1 to 4. A replicable cloning vector or expression vector containing the nucleic acid molecule of claim 5. The vector according to claim 6, which is a plasmid vector. A host cell transformed with the vector according to claim 6 or 7. The host cell according to claim 8, which is a prokaryotic cell. A host cell according to claim 8 or 9 is cultured, a polypeptide of a single-chain antibody constituting the multimerized low-molecular antibody according to any one of claims 1 to 4 is expressed, and the polypeptide is The method for producing a multimerized low-molecular antibody according to any one of claims 1 to 4, which comprises collecting and purifying and associating the single-chain antibody. 11. The multimerization according to claim 10, wherein the prokaryotic cell is Escherichia coli, and the multimerized low molecular weight antibody is prepared from a culture medium supernatant, periplasma fraction , intracellular soluble fraction or intracellular insoluble fraction of Escherichia coli. A method for producing a low molecular antibody. A pharmaceutical composition comprising the multimerized low-molecular antibody according to any one of claims 1 to 4 as an active ingredient. 13. The pharmaceutical composition according to claim 12, wherein the composition is for eliminating, killing, injuring and / or reducing tumor cells.