JPH1014570A - Antibody dna - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a cDNA that contains a specific nucleotide sequence and is useful for antitoxic therapy and prevention of toxic infectious diseases by Clostridium tetani and mass production of a human monoclonal antibody in industrial scale by a genetic engineering technique. SOLUTION: This cDNA, which codes a variable region of a heavy antibody chain, particularly codes a human monoclonal antibody containing a nucleotide sequence of the formula, is obtained by cloning cDNAs produced from mRNAs taken from a hybridoma [for example, hybridoma TTG6 (FERM P-15719)] that produces a specific antigen by subjecting lymphocytes taken from persons who have been immunized, by tetanus toxin (T.T.) and have an anti-tetanus toxin neutralizing antibody to in vitro antigen-stimulation with the T.T. and then to fusion with parent cells.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a cDNA encoding a human monoclonal antibody and a DNA encoding the CDR of the antibody. The human monoclonal antibody,
It can be used for antitoxin therapy and prevention of toxic infections caused by tetanus. In addition, the cDNA and the like can be used for mass production of the human monoclonal antibody on an industrial scale using genetic engineering techniques.

[0002]

2. Description of the Related Art Equine antitoxin serum therapy for diphtheria and tetanus has been in practical use for more than 100 years. Meanwhile, antitoxin therapy has been very effective in treating and preventing toxic infections. However, horse antitoxin is a heterologous protein for humans, and has a risk of side effects such as serum sickness. Therefore, as a safer tetanus antitoxin with fewer side effects, an anti-tetanus human immunoglobulin G preparation, which is a protein homologous to humans, prepared from human antiserum immunized with adult tetanus toxoid has been widely used in industrialized countries. I have. However, since it is obtained from human immune serum, the supply is limited, and there is a risk of viral infection such as hepatitis and AIDS. by the way,
All antitoxins that have been put into practical use so far are so-called polyclonal antibodies. In contrast, monoclonal antibodies produced by hybridomas that fuse antibody-producing lymphocytes and myeloma cells with permanent proliferation ability are:
Since the cells can be cultured in a test tube, the supply can be sufficiently performed, and the risk of virus infection such as hepatitis and AIDS can be avoided. In particular, when using a human monoclonal antibody, the problem of antigenicity can not be considered from the beginning, so it is expected to be applied not only to antitoxin therapy for toxic infections but also to immunotherapy for viral infections and cancer. ing.

As a result of vigorous studies, the present inventors have succeeded in obtaining a hybridoma producing an anti-tetanus toxin human monoclonal antibody having high toxin neutralizing activity against tetanus toxin ( Kamei M. et al.,
Eur. J. Epidemiol., Vol. 6, No. 4, p. 386-397 (199
0); and JP-A-2-57195). Since such a hybridoma can stably produce an antibody even in a serum-free medium, an inexpensive and high-purity human monoclonal antibody can be obtained. On the other hand, genetic engineering techniques have provided techniques for producing various useful proteins such as insulin in large quantities on an industrial scale. The production of monoclonal antibodies by this genetic engineering technique is based on the chromosomal DNA of the monoclonal antibodies or the complementary DN.
It begins with obtaining A (cDNA).

[0004] Among the antibodies (immunoglobulins),
The basic structure and genes of human IgG related to the present invention will be briefly described.

An IgG molecule is composed of two heavy chains of a polypeptide chain having a molecular weight of about 50,000 and two short chains of a polypeptide chain having a molecular weight of about 25,000. The heavy chain-light chain and heavy chain-heavy chain are connected by an SS bond. About 10 from the amino terminus of the heavy and light chains
The zero amino acid sequence is antigen specific and binds to the antigen by this region. This area is called a “variable area”. The region following this has a certain structure called the “constant region”. The heavy chains are called gamma chains and the light chains are called kappa or lambda chains. A specific monoclonal antibody is a group of homogeneous antibody molecules in which the variable regions of the heavy and light chains each have a specific amino acid sequence, and the amino acid sequences of the variable regions differ greatly from one monoclonal antibody to another. The variable region further includes a complementarity determining region (CDR).
Hypervariable region (termining region)
able region) and relatively common framework residues (FR: Framework region). There are three CDRs in each of the heavy and light chains, and the antigen binding site is formed by CDRs gathered by folding the heavy and light chains. For this reason, the antigen binding specificity and binding strength of the monoclonal antibody are mainly determined by the amino acid sequence of the CDR.

[0006] On the other hand, the variable region is composed of the V gene and the D gene in the heavy chain.
It is known that genes, J genes and light chains are encoded by V genes and J genes. In the human heavy chain, 100 or less V genes, 6 D genes, and 12 J genes
In human and human κ chains, there are 100 or less V genes and 5 J genes. With the differentiation of B cells, V in the heavy chain,
Arbitrary one gene from each of the gene groups D and J is adjacent by rearrangement to form a gene corresponding to a variable region. Similarly, in the light chain, a gene in the variable region is formed by rearrangement of the V and J genes. The gene coding for the constant region is located downstream of the variable region gene group. When the recombination of the variable region gene is completed, this gene is expressed in conjunction with the variable region gene, and heavy and light chain polypeptides are produced.

[0007]

This time, the present inventors have
From the complementary DNA (cDNA) prepared based on the messenger RNA (mRNA) collected from the hybridoma producing the anti-tetanus toxin human monoclonal antibody, the variable regions of the heavy and light chains constituting the human monoclonal antibody Was successfully cloned. Furthermore, we succeeded in elucidating the nucleotide sequence of these cDNAs and determining the amino acid sequence,
The present invention has been completed.

[0008]

That is, the present invention provides a cDNA encoding a human monoclonal antibody, wherein the cDNA encoding the variable region of the antibody heavy chain comprises the nucleotide sequence shown in FIG. A cDNA encoding a human monoclonal antibody, characterized in that the cDNA encoding the light chain variable region comprises the nucleotide sequence shown in FIG. 2, and the antibody heavy chain variable region consisting of the amino acid sequence shown in FIG. DN characterized by doing
A, a DNA encoding the variable region of the antibody light chain consisting of the amino acid sequence shown in FIG. 2; and CDR-1, CDR-2 and CDR-3 of the antibody heavy chain each having the nucleotide sequence shown in FIG. CDNA encoding a human monoclonal antibody, characterized by being encoded by a sequence,
CDNA encoding a human monoclonal antibody, wherein CDR-1, CDR-2 and CDR-3 of the antibody light chain are each encoded by the nucleotide sequence shown in FIG. 2; DNA encoding CDR-1, CDR-2 and CDR-3 of the antibody heavy chain comprising
CDR-1 of an antibody light chain consisting of the amino acid sequence of
DNA encoding CDR-2 and CDR-3.

As described in detail in JP-A-2-57195, a hybridoma producing the human monoclonal antibody of the present invention can be prepared, for example, as follows. Immunized with tetanus toxin (TT),
Lymphocytes collected from humans with high titers of anti-tetanus toxin neutralizing antibodies were further purified in vitro by T. T.
After the antigen stimulation, fusion with the parent cell is performed. Lymphocytes are separated from peripheral blood by, for example, density gradient centrifugation using Ficoll Pack (Pharmacia). As a parent cell, human-mouse heteromyeloma, such as RF-S1, which has a high fusion efficiency and can produce an IgG-type antibody relatively stably and can easily produce ascites in a nude mouse. Is used. Fusion with the parent cell is carried out by a known method using polyethylene glycol. T. By an EIA method (enzyme-linked immunosorbent assay) using a plate coated with. Cloning is performed by the limiting dilution method or the soft agar method. Antibodies can be easily obtained in large quantities in high purity from ascites of nude mice or from culture supernatant using a continuous culture apparatus by ammonium sulfate fractionation and / or Protein-A column chromatography. Among the human monoclonal antibodies produced by the hybridomas thus obtained, some have a neutralizing antibody titer against tetanus toxin of several IU / 100 μg IgG, and as a specific example, as described on July 4, 1996, Hybridoma TTG6 deposited at the Institute of Biotechnology, Industrial Technology Research Institute
5719).

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

[0011]

【Example】

Example 1 Preparation of mRNA (a) Cell culture Anti-tetanus toxin human monoclonal antibody-producing hybridoma cell line TTG6 [Kamei M. et al. , Eur. J. Ep, supra.
idemiol., Vol. 6, No. 4, p. 386-397 (1990), which is described as "G6"], 20% fetal bovine serum, 10 mg / ml transferrin, 0.0043 mg /
The cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing ml of sodium selenite, 1.53 mg / ml of ethanolamine, and 5 mg / ml of insulin at 37 ° C. in an environment of 5% CO ₂ . (B) is in total RNA recovered logarithmic growth phase, washed with 10 ⁷ hybridoma cell lines producing a monoclonal antibody in phosphate-buffered saline and subjected to the recovery of RNA. Recovery of total RNA
This was performed as follows using the Pharmacia QuickPrep Total RNA Extraction Kit. 2 hybridoma cells
The cells were seeded at 1 × 10 ⁵ cells / ml in a 0% FCS-DMEM medium. After culturing for 3 days, 1 × 10 ⁷ cells were obtained. The cells were washed three times with PBS (previously cooled on ice) at 4 ° C. (2,000 × g, 5 min). Transfer cells to 525 μl of Lith
Suspend in ium Chloride Solution and add 4.5 μl of β
Mercaptoethanol, 225 μl of Extraction buff
The mixture was crushed with ice-cooling using a glass-Teflon homogenizer (2 to 3 min). 750 μl of CsT
After adding FA and stirring, the crushed liquid was transferred to a 1.5 ml centrifuge tube, and centrifuged at 15,000 rpm for 30 minutes.
The precipitate is mixed with 113 μl of Extraction buffer, 263
μl Lithium Chloride Solution, 375 μl Cs
After suspending in a TFA mixture, the suspension was centrifuged at 15000 rpm for 10 minutes. Wash the sediment with 70% ethanol (1500
(0 rpm, 5 minutes), and then dissolved in 100 μl of TE buffer and used for the following cDNA synthesis.

Example 2 Synthesis and RT-PCR of first-strand cDNA
Amplification of antibody variable region cDNA by the method described in Welschof et al. (J. I) in order to obtain cDNAs encoding the variable regions of the light and heavy chains of each monoclonal antibody.
mmunol. Meth., vol. 179, p. 203-214 (1995))
RT-PCR (Reverse Transcriptase Polymerase Chai
n Reaction). For PCR, 1 to 10 amino acids of the light chain of human IgG (kappaCL primer, LambdaCL
primer) and amino acids 1 to 7 of the heavy chain (IgG primer)
An oligonucleotide containing a base sequence corresponding to the amino acid sequence was used as a forward primer. Human I
Amino acids 109 to 116 of the gGκ light chain (κ series p
rimer), an oligonucleotide containing a base sequence corresponding to the amino acid sequence of amino acids 115 to 122 of the λ light chain (λ series primer) and amino acids 115 to 121 of the heavy chain (VH series primer) was used as a back primer (Table 1). 1).

[0013]

[Table 1]

Total RNA 1 obtained from the above hybridoma strain
μg as template, random 9mer as primer and Sup
erscript II reverse transcriptase (GIBCO-BRL), 50 mM Tris-HCl (pH 8.3), 40 mM KCl, 6 mM M
gCl ₂ , 1 mM DTT, 0.75 mM dNTP, 200 units Supersc
45 in the reaction solution containing ript II reverse transcriptase
The reaction was carried out at 1 ° C. for 1 hour to synthesize a first strand. 1 in boiling water
Let stand for 0 min to inactivate reverse transcriptase,
Mix with M forward and back primer
PCR was performed using Mannheim's Expand Long Template PCR System [50 mM Tris-HCl (pH 9.2), 16 mM
In a reaction solution containing mM (NH ₄ ) ₂ SO ₄ , 1.75 mM MgCl ₂ , and 0.35 mM dNTP, 94 ° C. for 30 seconds, 55 ° C. for 60 seconds and 68 ° C. for 30 seconds, 35 cycles], heavy chain cDNA and light chain cDNA were amplified.

Example 3 Subcloning of amplified fragment The amplified cDNA was blunt-ended with T4 DNA polymerase (Takara Shuzo Co., Ltd.), and then T4 polynuc.
Phosphorylation using leotide kinase (Takara Shuzo Co., Ltd.), digestion with EcoRV (New England Biolab), and then E. coli alkaline phosphatase (Takara Shuzo)
SK- dephosphorylated with (Stratagene)
It was inserted into a vector, and Escherichia coli DH-5α strain was transformed according to a conventional method. As a result, about 85
%. When the size of these was confirmed using the alkaline rapid method, 9 out of 20 clones contained a fragment of the desired size. Then, the base sequence was determined next.

Example 4 The nucleotide sequences of the light chain and heavy chain cDNA clones subcloned into pBluescript SK- were obtained from Amersham, Thermo.
Sequenase Fluorescent labelled primer cycle seque
After performing a sequencing reaction using the ncing kit, an automatic sequencer (LI-COR, DNA sequencer model 400
It was analyzed and determined using (0). The sequence reaction is
M13 forward primer (5'-CACGACGTTTGT
AAAAACGAC-3 ') and M13 reverse prime
r (5'-GGATAACAATTTCACACAGGG
-3 ') for both sense and anti-sense strands. As a result, the nucleotide sequences of the cDNAs encoding the heavy and light chain variable regions of the antibody of the present invention as shown in FIGS. 1 and 2, respectively, were obtained. By translating this nucleotide sequence, the amino acid sequences of the variable regions of the antibody heavy and light chains were elucidated and shown in each figure in single letter notation.

The amino acid sequence shown in FIG. 1 and FIG. 2 is used as a list of amino acid sequence data of known human antibodies (Elvin
A. Kabat et al., SEQUENCES OF PROTEINS OF IMMUNOLO
GICAL INTEREST, FIFTH EDITION, US DEPARTMENT OF
HEALTH AND HUMAN SERVICES, Public Health Service, N
National Institute of Health (1991)), and further, the nucleic acid sequence and the amino acid sequence shown in FIGS. 1 and 2 were compared with DNA and protein databases (EMBL-GDB (Relele)).
ase 41), LASL-GDB (Release 86), NBRF
-PDB (Release 43) and SWISS-PROT (Release
By performing a homology search on lease 30)),
The antibody heavy chain was determined to belong to subgroup III of the subgroups I-III. In addition, three C
Since the amino acid sequences of the four FRs located at positions sandwiching the DR are substantially conserved for each subgroup, the amino acid sequence shown in FIG. CDR-
1. It was determined to contain CDR-2 and CDR-3.

The above operations were also performed on the light chain of the antibody of the present invention, and the respective sequences were determined.

[Brief description of the drawings]

FIG. 1 shows the amino acid sequence of the variable region of the heavy chain of the antibody of the present invention and the nucleotide sequence encoding the same.

FIG. 2 shows the amino acid sequence of the variable region of the light chain of the antibody of the present invention and the nucleotide sequence encoding the same.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification code Agency reference number FI Technical display location G01N 33/577 G01N 33/577 B // A61K 39/395 A61K 39/395 R ADZ ADZH C12N 1 / 21 C12N 1/21 C12P 21/08 C12P 21/08 C12Q 1/68 7823-4B C12Q 1/68 A (C12N 15/02 C12R 1:91) (C12N 1/21 C12R 1:19)

Claims (8)

[Claims] 1. A cDN encoding a variable region of an antibody heavy chain
CDNA encoding a human monoclonal antibody, wherein A comprises the nucleotide sequence shown in FIG. 2. A cDN encoding a variable region of an antibody light chain.
2. A cDNA encoding a human monoclonal antibody, wherein A comprises the nucleotide sequence shown in FIG. 3. A DNA encoding a variable region of an antibody heavy chain consisting of the amino acid sequence shown in FIG. 4. A DNA encoding a variable region of an antibody light chain consisting of the amino acid sequence shown in FIG. 5. A cDNA encoding a human monoclonal antibody, wherein CDR-1, CDR-2 and CDR-3 of the antibody heavy chain are each encoded by the nucleotide sequence shown in FIG. 6. A cDNA encoding a human monoclonal antibody, wherein CDR-1, CDR-2 and CDR-3 of the antibody light chain are each encoded by the nucleotide sequence shown in FIG. 7. CDR-1, CDR-2 and CDR- of an antibody heavy chain each consisting of the amino acid sequence shown in FIG.
DNA encoding No. 3. 8. CDR-1, CDR-2 and CDR- of an antibody light chain each having the amino acid sequence shown in FIG.
DNA encoding No. 3.