WO1992020799A1

WO1992020799A1 - Amino acid sequence of anticancer human monoclonal antibody and dna base sequence coding for the same

Info

Publication number: WO1992020799A1
Application number: PCT/JP1992/000650
Authority: WO
Inventors: Hideaki Hagiwara; Yasuyuki Aotsuka
Original assignee: Hagiwara, Yoshihide
Priority date: 1991-05-22
Filing date: 1992-05-21
Publication date: 1992-11-26
Also published as: JPH04346792A; AU1875592A

Abstract

Amino acid sequences of the heavy chain and light chain variable regions of the cancer cell antigen specific human immunoglobulin CLN-IgG produced by the human/human fused cell line CLN/SUZ H11 composed of the B cell of a patient with uterine cancer and a human lymphoblast cell line; and base sequences of the genes thereof. These amino acid and base sequences are useful in the medical and pharmaceutical fields for the prevention, treatment and diagnosis of human diseases and in the pharmacological and biochemical fields as biochemical reagents and reagents for purifying biopolymers.

Description

Specification

Amino acid sequence of anti-cancer human monoclonal antibody and DNA base sequence encoding the same

The present invention relates to antigens useful in a wide range of fields such as medical and pharmaceutical fields such as prevention, treatment and diagnosis of human diseases, pharmacology such as biochemical reagents and biopolymer purification reagents, and biochemical fields. It relates to the structure of the variable region of specific human immunoglobulin. More specifically, the present invention relates to a heavy chain of a cancer cell antigen-specific human immunoglobulin produced by a human Z human fusion cell line CL NZ SUZHI 1 of a B cell of a human uterine cancer patient and a human lymphoblast cell line. And the amino acid sequence of the light chain variable region and the nucleotide sequence of the gene.

Background art

Since the development of technology for producing monoclonal antibodies by cell fusion or cell immortalization, many useful antibodies have been obtained mainly using mice and the like. Among them, monoclonal antibodies against malignant tumor cells are extremely valuable, beginning to be used not only for basic research such as analysis of tumor antigens, but also for serodiagnosis and diagnostic imaging of tumors using labeled antibodies. . However, heterologous antibodies such as mice are foreign to humans, and frequent administration to humans causes an immune response to the administered antibodies, resulting in side effects and a reduction in the therapeutic or preventive effects of the antibodies. In view of the above, it is desirable to use human-type antibodies in the clinical field where antibodies are actually administered to humans, such as prevention, treatment, and in-vivo diagnosis of human cancer. However, it is difficult to prepare human monoclonal antibodies. It is hardly practical at this time.

Under such circumstances, one of the present inventors has disclosed in Japanese Patent Application Laid-Open No. 58-201994 (Japanese Patent Publication No. 01-59878), Japanese Patent Application Laid-Open Nos. 59-135898 and 59-137497. As disclosed in detail, a cell line CLNZSUZ HI 1 (ATCC No. HB8307) producing a human monoclonal antibody having high reactivity to human cancer cells was established. The antibody (CLN-IgG) produced by this cell line has an antibody class of IgG, isotypes y1 and / c, and immunohistologically binds to the 瘙 antigen present on the surface of cancer cells. However, more interesting findings have been obtained on the effect of suppressing the growth of cancer cells.

In such a technical background, there are problems to be solved as described below.

1) Monoclonal antibody-producing cell lines are generally known to decrease their antibody productivity with passage. Also, generally speaking, human hybridomas produce less antibody than mice. When human monoclonal antibodies are used for immunotherapy or diagnosis, a large amount of antibodies is required, and the solution of this problem is essential.

2) According to current knowledge of immunology, a mechanism is known in which a monoclonal antibody binds to human cancer cells and suppresses the growth of cancer cells or kills cancer cells by the action of the antibody itself. Furthermore, it is known that with the help of traps or K-cells or macrophages, the growth of cancer cells is suppressed and the cancer cells are killed. However, these effects are not as potent as expected in practice, and further attempts are needed to further increase the anti-cancer activity of the antibody.

Means for solving the above problems, namely, improvement of antibody production and anti- One means for realizing the increase in cancer activity is a method by genetic manipulation. For example, in the case of the problem 1) above, after cloning the antibody gene, the gene may be introduced into animal cells or host cells such as Escherichia coli, the antibody gene may be expressed, and a method for obtaining a large amount of the antibody may be solved. In the case of the above 2), by artificially changing the antibody gene, various functions of the antibody, such as the binding affinity with the antigen, the anticancer activity via the immunocompetent cell, or the tissue invasiveness are increased. By adding functions that are not originally possessed by antibodies, such as cytotoxicity, enzymatic activity, and immunity-inducing activity, to antibody molecules or fragments thereof, to design molecules with higher anticancer activity. It is possible to do.

In order to achieve these objectives, it is important to separate antibody genes and elucidate their structures. However, the structures of the light chain and heavy chain constituting the CLN-IgG monoclonal antibody, and the gene structure of a variable region having a function of specifically binding to an antigen have not been known at all. Therefore, a main object of the present invention is to elucidate the gene structure of the light chain and heavy chain of the CLN-IgG monoclonal antibody.

Disclosure of the invention

The present inventors separated cDNAs encoding the light chain and heavy chain of the CLN-IgG monoclonal antibody, elucidated the DNA base sequence, and, based on the sequences, the light chain and heavy chain variable regions of the antibody. The amino acid sequence of was determined, and the present invention was completed.

Specifically, niRNA was prepared from CLNZSUZ HI1, and the cDNA A lambda phage 'library' prepared therefrom was screened by the plaque hybridization method, and the isolated phage clone was used. This was achieved by determining the nucleotide sequence of the inserted DNA. Hereinafter, the present invention will be described in more detail.

BRIEF DESCRIPTION OF THE FIGURES-FIG. 1 shows a Kabat & Wu plot of 24 variable region sequences belonging to human c-chain subgroup 1.

FIG. 2 shows a Kabat & Wu plot of 21 variable region sequences belonging to human heavy chain subgroup 3.

Detailed description of the invention

According to the present invention, the DNA sequences of the light chain variable region and heavy chain variable region of the CLN-IgG monoclonal antibody can be determined using the human antibody gene fragment amplified by PCR (polymerase chain reaction) as a probe. It is determined by cloning the monoclonal antibody light chain and heavy chain cDNA and analyzing the DNA nucleotide sequence. Hereinafter, these steps will be described in more detail.

[1] mRNA isolation and purification

The cell line used in the present invention is a human Z human hybridoma obtained by fusing human uterine cancer patient lymphocytes and human lymphoblasts. Specifically, the cell line is disclosed in Japanese Patent Application Laid-Open No. 58-201994 (Japanese Patent Publication No. 01-59878). Publication No. 2), which produces a human-type monoclonal antibody that specifically reacts with the cell surface antigen of cancer cells such as human brain tumors, lung cancer, stomach cancer, and malignant melanoma. It is. This hybridoma is registered with ATCC (American Type Culture Collection) under the registration number HB8307.

The cells are grown under appropriate conditions, for example, at 37 ° C and 5% CO2. Under these conditions, the cells are grown in a culture solution containing 5% fetal bovine serum, for example, RDF medium, and the resulting cells are collected by centrifugation and then collected from the cells by a conventional method, for example, guanidinium thiosinate of Han et al. Total RNA was extracted by the method [Han, JH, Stratowa, C., & Rutter, WJ (1987) Biochemistry, 26, 16 17-1625], and then subjected to a conventional method, for example, adsorption column chromatography using oligo dT cellulose. Alternatively, separate and purify the poly (A) ⁺ RNA fraction by the batch method.

The resulting poly (A) + RNA can be further used for preparing a cDNA library. In the present invention, specifically, total RNA is extracted from CLN-SUZH11 hybridoma cells, and poly (A) + RNA is purified from this extract using an oligo dT cellulose column, and the following cDNA is prepared. The library was used for construction.

[2] Preparation of cDNA library

The poly (A) + RNA obtained in the step [1] is type II, and the primers are dATP and dGTP, using oligo dT corresponding to poly A or a synthetic nucleotide having a base sequence considered to correspond to the constant region of the antibody as a primer. A single-stranded DNA complementary to mRNA is synthesized by reverse transcriptase in the presence of dTTP and dCTP. Next, the RNA is fragmented with Escherichia coli RNA degrading enzyme H, and then the single-stranded DNA is converted into type II to prepare a double-stranded cDNA using Escherichia coli DNA polymerase I. For example, an EcoRI linker can be ligated to the cDNA thus obtained, followed by digestion with EcoRI to introduce a sticky end. The obtained fragment is ligated to an EcoRI site of an appropriate phage vector, for example, a λ £ ΐ10 vector or an igtl1 vector, and then subjected to in vitro packaging to prepare a cDNA library. Can be made.

In a specific operation of the present invention, a cDNA was synthesized using the poly (A) + RNA obtained in the step [1] as type III, and the cDNA was ligated to an Igtl0 vector to prepare a cDNA library.

[3] Preparation of probe

As the probe, a human immunoglobulin light or heavy chain constant region or variable region gene or a fragment thereof, or a chemically synthesized oligonucleotide having a nucleotide sequence corresponding to the amino acid S sequence in that portion is used. For example, those labeled with ³² P, biotin or the like by the nick translation method can be used.

In the present invention, preferably, a fragment amplified by PCR in which the cDNA is subjected to type III and sequences corresponding to a part of the light chain and heavy chain of the antibody in a primer sequence is biotinylated by the nick translation method. Things can be used as probes.

[4] cDNA cloning

The desired clone is selected by using the cDNA library obtained in the step [2] and the probe obtained in the step [3]. For example, by infecting the E. coli strain (C600H—) with Igtl0 phage of the cDNA library obtained in the step [2], plaques are formed, and positive clones are selected by plaque hybridization. As a result, ICLN-G11 was selected as a CLN-IgG heavy chain cDNA clone, and ICLN-K411 was selected as a CLN-IgG light chain cDNA clone, and subjected to nucleotide sequencing.

[5] Determination of base sequence The cDNA clone obtained in the step [4] is recloned into a plasmid vector such as pUC18, a phage vector such as M13 phage, or a phagemid vector such as pUC118 or pBluescript SK +. The nucleotide sequence of the DNA base sequence at the insertion site can be determined using the Maxam, Gilbert method and Sanger method.

In the specific operation of the present invention, the ICLN-G111 and the EcoRI fragment of ICLN-K411 obtained in the step [4] are recloned into pBluescript SK ⁺ and then infected with helper phage R408. Single-stranded DNA was prepared and its nucleotide sequence was determined by the Sanger method.

Hereinafter, the present invention will be described more specifically with reference to examples.

Example

Example 1: Isolation and purification of mRNA (poly (A) + RNA) from Hypri-Doma CLN SUZ HI 1

The method for obtaining the mRNA fraction from the human hypride dorma CLNZSUZ HI1 is as follows.

Total RNA was prepared from CLNZSUZ HI1 cells by the guanidinium thiosinate method [Han, JH, Stratowa, C, & Rutter, WJ (1987). Biochemistry, 26, 1617-1625]. 10 ⁹ cultured cells are collected by centrifugation and washed with physiological saline. 20 ml of 5 M guanidium thiosinate containing 8% 2-mercaptoethanol, which had been ice-cooled beforehand, was added to the precipitate of the cells collected by centrifugation at 80 Orpm, and the mixture was immediately homogenized. The cell lysate was previously mixed with 7.5 ml of ethanol and cooled to 120 ° C. Mix in a pyrene centrifuge tube and immediately centrifuge at 10,000 Orpm for 5 minutes. 10 ml of 5 M guanidinium thiosinate containing 8% 2-mercaptoethanol, which had been ice-cooled beforehand, was added to the obtained precipitate and homogenized. Add cold ethanol and leave at 120 ° C overnight. The precipitate obtained by centrifugation at 10,000 rpm for 10 minutes was dissolved in 10 ml of 6M guanidine hydrochloride containing 10 mM 2-mercaptoethanol, and 0.25 ml of 1 M acetic acid and 5 ml of cold ethanol were added to the mixture. Leave for 3 hours. After centrifugation at 10,000 rpm for 10 minutes, the resulting precipitate is dissolved in 5 ml of 6 M guanidine hydrochloride, 0.125 ml of 1 MS ^ acid and 2.5 ml of cold ethanol are added, and the mixture is left at 120 for another 3 hours. . The precipitate obtained by centrifugation at 10,000 Orpra for 10 minutes is dissolved in 5 ml of sterilized pure water, added with 0.5 ml of 2 M sodium acetate and 12.5 ml of cold ethanol, and stored as a total RNA fraction at 1-20. I do.

Poly (A) + RNA was obtained from the total RNA fraction obtained by the above method by the method of Chirgwin [Chirgwin, J. Μ ·, Przybyla, A. E-, MacDonald, RJ, & Rutter, WJ ( 1979) Biochemistry, 18, 5294-5299]. First, 9 mg of total RNA of CLNZSUZ HI 1 cells is dissolved in pure water to 2.5 mg / ml. After heat treatment at 100 ° C for 5 minutes and quenching on ice, add 5M lithium chloride, 10% SDS, 1M triethanolamine hydrochloride pH 7.4 to final concentrations of 0.5M, 0.2%, 1OmM, respectively. The solution is applied to an oligo (dT) cell source column that has been equilibrated with a binding buffer (0.5 M lithium chloride, 0.2% SDS, 1 OmM triethanolamine hydrochloride, pH 7.4) in advance. Further wash with 10 column volumes of binding buffer. You. The poly (A) + RNA bound to the column is eluted with elution buffer (1 OmM triethanolamine hydrochloride, pH 7.4), and the RNA fraction is collected. The resulting poly (A) + RNA solution is heat-treated at 100 ° C for 5 minutes, and the above-mentioned chromatography using an oligo (dT) cellulose column is repeated once again using a new column. For RNA, add 2.5 volumes of ethanol and 1/10 volume of 2M sodium acetate to the eluate, and recover as a precipitate after centrifugation at 10,000 xg. Dissolve the purified poly (A) + RNA precipitate in sterile pure water and store at 2 70 g at a concentration of 2 zg / zl.

Example 2: Creation of CLN / SUZ HI 1 library

Using the poly (A) ⁺ RNA4 / g obtained in Example 1, cDNA was synthesized according to the protocol of Amersham's cDNA synthesis kit and 3.2 µg was recovered. After treating this cDNA fragment with EcoRI methylase, the EcoRI linker was ligated using T4 DNA ligase. After EcoR I digestion, a cDNA fraction having an EcoR I end was recovered using an Amersham column. After ligation of 10 ng of this cDNA to 1 / zg of Igtl 0 vector-1 (Stratagene), in vitro packaging was performed using a packaging kit (GIGA PACK GOLD; Stratagene) and CLNZSU ZHl An lcDNA library of 7.8 × 10 ⁶ pfu // zg DNA was prepared.

Example 3: Determination of amino acid sequence of CLN-IgG by protein sequencer

After reducing purified CLN-IgG, 30 µg of each of the heavy and light chains purified by gel filtration was applied to Protein Sequencer 477A (manufactured by Applied Biosystems), and about 30 amino acid sequences from the N-terminus were retained. I decided. The heavy chain was specifically cleaved with methionine using cyanogen bromide. The fragments were separated and purified by reversed-phase liquid chromatography, and a part of the amino acid sequence was similarly determined.

Example 4: Probe creation method

(1) Heavyweight probe

A sequence having a homology with the partial amino acid sequence of the CLN-IgG heavy chain determined in Example 3 was searched from the NBRF protein database (NBRF-PDB; National Biomedical Research Foundation Protein Data Base). VH26 (entry H3HU26, Accession number A02047) was found to have the highest homology. Therefore, it corresponds to the N-terminal amino acid 10 residues of the VH26 DNA sequence (ID name: HSIGHAU, Accession number M17747) in the EMBL DNA database (EMBL-GDB; European Molecular Biology Laboratory Gene data Base). 30 nucleotides (Primer No. 1) were synthesized. In addition, 30 nucleotides (primer No. 2) corresponding to 10 amino acid residues at the C-terminal side of the DNA sequence of the heavy chain 1 CHI domain (EMBL-GDB; ID name HS I GCC4, Accession number J00228) were synthesized.

Synthetic DNA (Primer No. 1)

5 '-GAGGTGCAGCTGTTGGAGTCTGGGGGAGGC-3'

Synthetic DNA (Primer No.2)

5'-AACTTTCTTGTCCACCTTGGTGTTGCTGGG-3 '

PCR (polymerase chain reaction) was performed using 4 ng of CLNZSUZ HI lcDNA prepared in Example 2 using these two types of primers as a template. went. As a result, a fragment of about 660 base pairs (PCRy C3) was amplified, and it was clarified that the nucleotide sequence determination corresponded to the antibody heavy chain y1 CHI domain and variable region. This C3 was biotinylated by the method of nick translation to obtain a probe (biotinylated PCRy C3).

(2) Light chain probe

As a result of searching the NBRF protein database for a sequence having a homology with the partial amino acid sequence of the CLN-IgG light chain determined in Example 3, human immunoglobulin derived from Dau di cells (entry name K 1 HUD I, Acces sion number A01884). Therefore, 30 nucleotides (primer No. 3) corresponding to 10 residues of the N-terminal amino acid were synthesized from the DNA sequence of the Daudi antibody light chain (ID name HSVK02, Accession number X00966) in the EMBL DNA database. 30 nucleotides (primer No. 4) corresponding to the C-terminal amino acid residue 10 of the DNA sequence of the light domain (/ c chain) C domain (EMBL-GDB; ID name HS I GK1, Accession number V00557) Was synthesized. Synthetic DNA (Primer No.3)

5 '-GACATCCAGATGACCCAGTCTCCATCCTCC-3'

Synthetic DNA (Primer No.4)

5 '-CTAACACTCTCCCCTGTTGAAGCTCTTTGT-3'

CLN / SUZ prepared in Example 2 using these two primers

PCR was performed using 4 ng of HI icDNA as a template. As a result, a fragment (PCR cA4) of about 660 base pairs was amplified, and the nucleotide sequence was determined to correspond to the antibody light chain (Λ chain) C domain and variable region. This PCR c4 is used for nick translation Then, a probe (biotinylated PC RcA4) was obtained.

Example 5: Cloning of cDNA

(1) Cloning of heavyweight cDNA

The CLNZSUZ HI lcDNA library obtained in Example 2 was subjected to plaque hybridization using the biotinylated probe obtained in Example 4 to obtain 13 positive clones. One of these clones had about 1.6K base pairs of imported DNA, and this phage was named; ICLN-G111.

(2) Cloning of light chain cDNA

The CLNZSUZ HI lcDNA library obtained in Example 2 was subjected to plaque hybridization using the biotinylated probe obtained in Example 4 to obtain 27 positive clones. Clone 411 in this contains about 1.0 K base pairs of imported DNA, and this phage was named CLN-K411.

Example 6: Determination of base sequence

The EcoR I fragment of cloned CLN-G11 and of ICLN-K411 in Example 5 was recloned into the phagemid Bluescript SK +. Escherichia coli XL1-Blue transformed with this phagemid is infected with helper phage R408, the single-stranded DNA is prepared, and the Sanger method [Sanger, F. et al. Proc. Natl. Acad. Sci. USA 74; 5463 (1977)]. The nucleotide sequence of the variable region of the resulting CLN-IgG light chain cDNA clone and the amino acid sequence predicted therefrom are shown below. Table 1: CLN-IgG heavy chain (y) variable region fragment

AGC CCA GCC CTG GGA TTT TCA GGT GTT TTC ATT TGG TGA TCA GGA CTG AAC AGA GAA CTC

-19-10-1

ACC ATG GAC TTT GGG CTG AGC TGG CTT TTT CTT GTG GCT ATT TTA AAA GGT GTC CAG TGT

Met Asp Phe Gly Leu Ser Trp Leu Phe Leu al Ala lie Leu Lys Gly Val Gin Cys

10 20

GAG GTG CAG CTG TTG GAG TCT GGG GGA GAC TTG GTA CAG CCT GGG GGG TCG CTG AGA CTC

Glu Val Gin Leu Leu Glu Ser Gly Gly Asp Leu Val Gin Pro Gly Gly Ser Leu Arg Leu

30 40

TCC TGT GCA GCC TCT GGA TTC ACC TTC AGC AAC TAT GCC ATG AGC TGG GTC CGC CAG GCT

Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr Ala Met Ser Trp Val Arg Gin Ala

50 60

CCA GGG AAG GGG CTG GAG TGG GTC TCA GCG ATT ACT CCT AGT GGT GGT AGT ACA AAT TAT

Pro Gly Lys Gly Leu Glu Trp Val Ser Ala lie Thr Pro Ser Gly Gly Ser Thr Asn Tyr

70 80

GCA GAC TCC GTG AAG GGC CGG TTC ACC ATC TCC AGA GAC AAT TCC CAG AAT ACA CTG TAT

Ala Asp Ser al Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ser Gin Asn Thr Leu Tyr

90 100

CTG CAA ATG AAC AGC CTG AGA GTC GAG GAC ACG GCC GTA TAT TAC TGT GGG AGA GTC CCA

Leu Gin Met Asn Ser Leu Arg Val Glu Asp Thr Ala Val Tyr Tyr Cys Gly Argal Pro

110 120

TAT AGA AGC ACT TGG TAC CCT TTA TAT TGG GGC CAG GGA ACC CTG GTC ACC GTC TCC TCA

Tyr Arg Ser Thr Trp Tyr Pro Leu Tyr Trp Gly Gin Gly Thr Leu Val Thr Val Ser Ser

GCC

Ala

JDd,

66 UAV

SJV sAq a ηιε) Ι¾Λ sAq Jtii Λχο VOO VW DIiV 0V0 Si〇 SW 00V OOD

ATO Ato am ani, 3X1 c <j 33W uo neq sAo JAJ, xAjj ν [ί ν ν atld Js¾ η ο VOO DDO οω ODV 0Ji¾ iYi OVO JiD 001, OVJi JIVOJ JIOV VOO JnLJo .LVOτ06vo

oad uto naq jes ana, na <j i i stfd dsv n njj AtD XBS Λχο J3S ΛΤΟ J3S a¾d titO ADD ovo ai oov ODV 0Λ0 i! V DUi JiVO ¾0W OOD J «L VOO JiOV DOD OOV OJML WO

08 0

aqii ojtd Ι¾Λ 8Λι BJCV STfH BTV εχ¾ XAjj θ ΐ na as asAi cud ε ν s¾ Λ ¾ ¾DV VDD OliO OW ODV DVD DDCL VOO J D iVJL DI.V OJiD 03Λ OW JiDO DDO Wtf000 09

Uxd sAq. 0 οε

T tWi ΙΒΛ J5av dsv jess ns ¹ !

sAo Bav SAo uto naq Λ ο naq naq βχ «BTV nsq te ΒΛ BJV aw dsv aw ώΟΛ ¾0¾ OOIJ QMD 00,1 ΟΛΟ JJDO JJOIL 0J» D 000 DJ.0 DliD DOV iLDO OiD. 0ΛΟ VOW OIiY DVO Ov

OT- 02

DOV OVO VOO ¾0i ooo ων VOO

In addition, as a result of a computer search to determine the subgroups of CLN-IgG heavy and light chains, a gene with high homology was examined. It was revealed that the variable region belongs to subgroup 1.

Example 7: Determination of hypervariable region

Comparing the amino acid sequences of the heavy and light chains of various antibodies, respectively, shows that there are regions (variable regions) in which the sequences are different from certain regions (constant regions). Among the variable regions, the regions that are particularly rich in variability are called hyper-variable regions (Hv regions), and there are three heavy chains and three light chains. From the amino terminus, they are called Hvl, Ην2, and Hv3, respectively. These hypervariable regions shape the binding site for antigen and are thought to contain amino acid residues that directly contact antigenic determinants. Therefore, the hypervariable region, also called the complementarity determining region (CDR), is the region that controls the antigen specificity of the antibody.

Kabat & Wu plots were used to determine the hypervariable region of CLN-IgG. [Kabat, EA, Wu, T. Τ., Bilofsky, Η. Variable Regions of Immunoglobulin Chains (Medical Comput. Syst eras, Bolt, Beranek & Newman, Cambridge, 1976)] Are arranged and the degree of variation is calculated for each position and plotted. The degree of mutation referred to here is the ratio of `` the number of types of amino acid appearing at any position '' to `` frequency of the most frequently occurring amino acid at that position '', and theoretically takes a value from 1 to 400 . The region where the value of the degree of mutation is large is the hypervariable region. (1) CLN—determination of hypervariable region of IgG light chain

The amino acid sequence of the CLN-IgG light chain revealed that it belongs to kappa chain subgroup 1. Therefore, 24 sequences belonging to subgroup 1 contained in the NBRF-PDB (rel. 26) were arranged together with the CLN-IgG light chain, and the mutation degree was calculated at each position to create a Kabat & Wu plot (Fig. 1) From the results, Hvl, Ην2, and Hv3 were determined as residue numbers 28 to 34, 50 to 56, and 91 to 96, respectively.

CLN—Hypervariable region of IgG light chain

H V 1: Asp lie Ser Asn Tyr Leu Ala

Hv2: Ala Ala Ser Ser Leu His Arg

Hv3: Tyr Mer Thr Tyr Pro lie

(2) Determination of the hypervariable region of CLN-IgG heavyweight

The amino acid sequence of the heavy chain of CLN-IgG revealed that it belongs to Hv subgroup 3. Therefore, the Kabat & Wu plot was prepared by calculating the mutation degree at each position by juxtaposing the 21 sequences belonging to subgroup 3 contained in the NBRF-PDB (rel. 26) together with the CLN-IgG heavy chain (Fig. 2). , Up to residue number 96). As a result, Hvl and Hv2 were determined as residue numbers 31 to 35 and 49 to 59, respectively. Regarding Hv3, in the case of heavyweight, as shown in Table 3 below, it is difficult to exactly match the positions because the length of each sequence is significantly different. Calculating the degree of mutation without considering the gap, the residue number is 1.0 for 96 cysteine, 2.1 for 97 glycine, 3.9 for 98 arginine, 29.3 for 99 valine, 18.4 for 109 tyrosine, 3.5 for 111 tributophan, 111 for 111 Glycine is 1.0. Apparently, the degree of mutation is high at residues 99 to 109, and this region is H Is equivalent to

Hyper-variable region of CLN-IgG heavy chain

H V 1: Asn Tyr Ala Met Ser

Hv2: Ser Alalie Thr Pro Ser Gly Gly Ser Thr Asn Hv3: Val Pro Tyr Arg Ser Thr Trp Tyr Pro Leu Tyr 3 Table

Kol CARDGGHGFCSSASCFGPDYWGQGTPVTVS S

Bro CARSPVSLVDGWLYYYYGSVWGQGTL

Cam CAR DRPLYGDYRAFNYWGQGTLVTVS S

Tro CAA TDDFDWSTFSLDYWGEGDLVTVS S

Tei CAR VTPAAASLTFSAV GQGTLVT

Pom CAR DAGPY VS PTFF AH YGQGTLVT

Ga CAR SGIALGSVAGTDYWGEGTLVTISS

Lay CAR DAGPYVSPTFFAHWGQGTLVT

Hil CAR DPDIIiTAFSFD YWGQGVLiVTVS S

99 i09

CLN CG-PYRST YPLWGQGTLVTVS SAS

Dob CAK GYIWNGNWFDSWGQGTLVTVS was CAR FRQ PFVQFFDVFGQGTLVT Bur CAK LIAVAG - TRDFWGQGTLVTVSL Tur CAR LSVTAVAFDVWGQGTKVS Til CAK GKVSAYYFDYWGEGTLVTVS S zap CAR TRPGGYFSDVWGQGTLVS Nie CAR IRDTAMFFAHWGQGTLVTVS S Jon CAR VWS TSMDVWGQGTPVT Gal CAR GWGGGDYWGQGTLVTVST But CAR DLAAARLFGKGTTVTVS S

WEA CAR-GWLLNWGQGTLVTVS S Industrial applicability

The elucidation of the CL N-IgG antibody and its gene structure makes it possible to introduce this gene into animal cells and host cells such as Escherichia coli and express it, thereby obtaining a large amount of the antibody. Furthermore, not only complete antibodies, but also certain antibody fragments, such as heavy chains only, light chains only, Fab fragments, F (ab) ' ₂ fragments, FV fragments, domain fragments (dAb), CDR fragments and other antibody-derived fragments Can be obtained. Further, by causing artificial mutations in the antibody gene, complete antibodies or fragments derived from various antibodies whose amino acid sequences are partially different can be obtained.

As a result of research to date, CLN-IgG acts on human cancer cells such as human stomach cancer, lung cancer, brain tumor, and malignant melanoma, and suppresses the growth of these cancer cells by its own action. Is expected to kill cancer cells and further suppress cancer cell growth with the help of traps or K-cells and macrophages, and to kill cancer cells. However, by modifying the CLN-IgG gene and partially substituting the amino acids of the antibody, it is possible to further increase the activity of the antibody. For example, it can be modified so as to increase the binding affinity to an antigen, the anticancer activity via an immunocompetent cell, or the invasiveness to a tissue. Furthermore, for example, cytotoxicity, enzymatic activity, immunity-inducing activity, etc. are added to an antibody molecule or a fragment thereof at the gene level by adding toxicity, enzyme activity, immunity-inducing activity, etc. to the antibody molecule or a fragment thereof at the gene level. It is conceivable to design a molecule having a higher anticancer activity. Specific examples include the use of cancer-specific antibodies as carriers, for example, chemotherapeutic agent binding-human monoclonal antibody, interferon binding-human monoclonal antibody, high molecular weight toxin binding It is useful as a drug that induces cancer cell growth inhibition or death in the form of a monoclonal antibody, drug-containing ribosome binding-human monoclonal antibody, and the like. It is also conceivable that radiosensitizers may be conjugated to antibodies and administered to patients, selectively accumulate in spheroid cells, and improve therapeutic and diagnostic effects. For use against such cancers, a complete antibody may be used as a human monoclonal antibody, or as described above, for example, only a heavy chain, only a light chain, a Fab fragment, an F (ab) ' ₂ fragment, Smaller fragments containing specific antigen recognition sites, such as v-fragments, domain fragments (dAbs), and CDR fragments can also be used.

Claims

The scope of the claims

1. The following amino acid sequence

H V 1: Asn Tyr Ala Met Ser

H v 2: Ser Ala lie Thr Pro Ser Gly Gly Ser Thr Asn

Hv3: an immunoglobulin heavy chain variable region fragment characterized by including at least one hypervariable region selected from the hypervariable regions Hvl, Hv2, and Hv3 having Val Pro Tyr Arg Ser Thr Trp Tyr Pro Leu Tyr.

2. The following amino acid sequence

Glu Val Gin Leu Leu Glu Ser Gly Gly Asp ¹⁰

Leu Val Gin Pro Gly Gly Ser Leu Arg Leu ²⁰

Ser Cys Ala Ala Ser Gly Plie Thr Phe Ser ³⁰

Asn Tyr Ala Met Ser Trp Val Arg Gin. Ala ⁴⁰

Pro Gly Lys Gly Leu Glu Trp Val Ser Ala ⁵⁰

Lele Thr Pro Ser Gly Gly Ser Thr Asn Tyr ⁶⁰

Ala Asp Ser Val Lys Gly Arg Phe Thr lie ⁷⁰

Ser Arg Asp Asn Ser Gin Asn Thr Leu Tyr ⁸⁰

Leu Gin Met Asn Ser Leu Arg Val Glu Asp ⁹⁰

Thr Ala al Tyr Tyr Cys Gly Arg Val Pro ¹⁰⁰

Tyr Arg Ser 1¾r Trp Tyx Pro Leu Tyr Trp ¹¹⁰

Gly Gin Gly Thr Leu Val Thr Val Ser Ser ¹²⁰

Immunoglobulin heavyweight variable region fragment with Ala.

3. The following amino acid sequence H V 1: Asn Tyr Ala Met Ser

Hv2: Ser Alalie Thr Pro Ser Gly Gly Ser Thr Asn Hv3: Val Pro Tyr Arg Ser Thr Trp Tyr Pro Leu Tyr A DNA encoding at least a part of an immunoglobulin heavy chain variable region, comprising a nucleotide sequence encoding at least one hypervariable region selected from hypervariable regions Hvl, Ην2 and Ην3 having RNA fragment.

4. DNA and RNA base sequences encoding the amino acid sequence according to claim 2.

5. The following base sequence

10 20 30 40 50 60

GAGGTGCAGCTGTTGGAGTCTGGGGGAGACTTGGTACAGCCTGGGGGGTCGCTGAGACTC

7 0 80 90 100 110 120

TCCTGTGCAGCCTCTGGAT CACCTTCAGCAACTATGCCATGAGCTGGGTCCGCCAGGCT

130 140 150 160 17 0 180

CCAGGGAAGGGGCTGGAGTGGGTCTCAGCGATTACTCCTAGTGGTGGTAGTACAAATTAT

190 200 210 220 230 240

GCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCCAGAATACACTGTAT

250 260 270 280 290 300

CTGCAAATGAACAGCCTGAGAGTCGAGGACACGGCCGTATATTACTGTGGGAGAGTCCCA

310 320 330. 340 350 360

TATAGAAGCACTTGGTACCCTTTATATTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA

GCC

5. The DNA base sequence according to claim 4, which has the following sequence: and the RNA base sequence corresponding thereto.

6. The following amino acid sequence

H V 1: Asp lie Ser Asn Tyr Leu Ala

Hv 2: Ala Ala Ser Ser Leu His Arg

Hv3: Tyr Met Thr Tyr Pro lie

An immunoglobulin light chain variable region fragment comprising at least one hypervariable region selected from hypervariable regions Hvl, Hv2 and Hv3 having the following:

7. The following amino acid sequence Asp lie Gin Met Thr Gin Ser Pro Ser Ser ¹⁰

Leu Ser Ala Ser Val Gly Asp Arg Val hr ²⁰ lie Thr Cys Arg Ala Ser Gin Asp lie Ser ³⁰ Asn Tyr Leu Ala Trp Phe Gin Gin Lys Pro ⁰ Gly Lys Ala Pro Lys Ser Leu lie Tyr Ala ⁵⁰ Ala Ser Ser Leu His Arg Lys Val Pro Thr ⁶⁰

Gin Phe Ser Gly Ser Gly Ser Gly Thr Asp ⁷⁰

Phe Thr Leu Thr lie Ser Ser Leu Gin Pro ⁸⁰

Glu Asp Phe Ala Thr Tyx Tyr Cys Leu Gin ⁹⁰

Tyr Met Thr Tyr Pro lie Thr PHe Gly Gl ¹⁰⁰

Gly Thr Lys Val Glu lie Lys Arg

An immunoglobulin light chain variable region fragment having the formula:

8. The following amino acid sequence

H V 1: Asp He Ser Asn Tyr Leu Ala

Hv2: Ala Ala Ser Ser Leu His Arg

Hv3: Thr Met Thr Tyr Pro lie

And encoding at least a part of the immunoglobulin light chain variable region, which comprises a nucleotide sequence encoding at least one hypervariable region selected from Hvl, Hv2, and Hv3. DNA and RNA fragments.

9. DNA and RNA base sequences encoding the amino acid sequence according to claim 7.

1 0. The following base sequence 10 20 30 40 50 60

GACATCCAGATGACCCAGTCTCCATCCTCACTGTCTGCATCTGTAGGAGACAGAGTCACC

7 0 80 9 0 100 110 120

ATCACTTGTCGGGCGAGTCAGGACA TAGTAATTATTTAGCCTGG T CAGCAGAAACCA

130 140 150 160 170 "180

GGGAAAGCCCCTAAGTCCCTGATCTATGCTGCATCCAGT GCACAGGAAGGTCCCAACA

19 0 200 210 22 0 230 240

CAATTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCT

250 260 '270 280 290 300

GAAGATTTTGCAACTTATTACTGCCTACAGTATATGACTTACCCTATCACCTTCGGCGGA

310 320

10. The DNA base sequence according to claim 9 having GGGACCAAGGTGGAGATCAAACGA and an RNA base sequence corresponding thereto.