JPH03206886A - Mouse-human chimera aio antibody having specificity against human tumor cell antigen - Google Patents

Abstract

PURPOSE: To improve therapy and diagnosis of human cancers by bringing to contain a specific sequence having specificity to the antigen bound by A10 murine monoclonal antibody (mAb).

CONSTITUTION: A chimeric mouse-humane A10 antibody having specificity to the human tumor cell antigen is produced by obtaining a messenger RNA (mRNA) (a) by separating hybridoma cell lines of mAb-producing murine B cells, treating a complete length-cDNA library obtained by treating the component (a) to obtain DNA (b) in a variable (V) region containing light (L) chains and heavy (H) chains of immunoglobulin, while cloning the cDNA obtained from the component (a) to obtain a gene part module (c) of a constant (C) region, then, bonding the component (b) to the component (c) to obtain a plasmid pING2254 (d)-coding for the H chain or the L chain, succeedingly culturing a host obtained by introducing the component (d) into a desired procaryote and extracting the culture solution.

COPYRIGHT: (C)1991,JPO

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to antibodies and derivatives thereof having specificity for human tumor antigens, nucleotide and protein sequences encoding them, and the obtaining of such sequences. , and a method for recombining it.

[Prior art]

Monoclonal antibody (mAb) technology has had a profound impact on current thinking about cancer treatment and diagnosis. To produce mAbs, Kohler and Milstein applied cell-to-cell fusion technology in Nature (Londo).
n) Distance [, 495 (1975)], which brought about a biological revolution comparable to that of recombinant DNA cloning. Hybridoma-produced mAbs are already widely used in the fields of clinical diagnostics and basic research, with potential efficacy in treating human diseases, including cancer, viral and microbial infections, and other diseases and disorders of the immune system.

Although mAbs exhibit excellent specificity for target factors that influence the progression of human diseases, murine mAbs have inherent limitations in their application to the treatment of human diseases. Mouse mAbs are obtained from mouse cells, and when used in humans, they are recognized as foreign proteins by the recipient organism and cause an immune response.

In addition, such proteins are distinguished from human proteins,
It may be immediately cleared from the human circulatory system.

Human mAbs have the potential to avoid these problems, but there have been various technical obstacles to their actual development.

Particularly difficult is the development of rnAbs that can identify human tumor antigens for the diagnosis and treatment of cancer. Since the majority of tumor antigens are not recognized as foreign antigens by the human immune system, these antigens appear to lack immunogenicity in humans. On the other hand, for human tumor antigens, using a mouse system in which the human antigen is the immunogen makes it possible to produce a mouse mAb that specifically recognizes the human antigen. Therefore, these mouse-derived antibodies can be expected to have therapeutic effects on humans. Furthermore, many human mAbs obtained in vitro are available at 1gM, which is not very efficient.
type or its isotype. Therefore, if it is necessary to obtain a human monoclonal antibody of the IgG isotype, which is a more effective antibody, a complex technique (i.e., cell sorting) must be used to identify and isolate a small number of cells that produce antibodies of the IgG type. I needed to.

Therefore, an efficient method is expected to switch antibody classes in order to obtain antibodies with predetermined antigen specificity or required antigen specificity.

The development of monoclonal antibodies is rapidly transitioning from mouse monoclonal antibodies to human monoclonal antibodies. However, there are still many difficulties at the stage of antigen sensitization in order to freely produce human monoclonal antibodies through cell fusion.

Therefore, recently, research has been actively conducted on the production of chimeric monoclonal antibodies that combine the variable region (antigen recognition site) of a mouse monoclonal antibody and the constant region of a human antibody using genetic engineering techniques (Japanese Patent Application Laid-Open No. 1983-1999- 1551
32 and others).

Chimeric antibodies, in which the constant region that occupies most of the antibody molecule is human protein, are expected to be easier to produce specific antibodies and to exhibit higher safety in terms of antigenicity and the like than mouse antibodies. Therefore, it is highly useful as a diagnostic and therapeutic agent. From this perspective, chimerism is already underway with the aim of developing imaging diagnostic and therapeutic agents for cancer.

[Problems to be Solved by the Present Invention] The chimeric antibodies described in the present invention bridge both hybridoma technology and genetic engineering, thereby providing products and test drugs that are effective in the treatment and diagnosis of human cancer. It is something to do.

[Means to solve the problem]

The present invention provides genetic engineering that combines the specificity of a desired variable (V) region and the characteristics of a constant (c) region, which is produced by gene cloning and expression of immunoglobulin light (L) chains and heavy (H) chains. Discloses a chimeric antibody that targets the target.

This chimeric antibody and its derivatives specific for human tumor antigens have applicability in human cancer treatment and diagnosis. Cloned immunoglobulin gene products and their derivatives can be produced in mammalian or microbial cells.

The present invention discloses cDNA sequences encoding immunoglobulin heavy and light chains consisting of a human C region and a non-human V region.

The present invention discloses such sequences present in a recombinant DNA molecule capable of being expressed by a desired prokaryotic or eukaryotic host and incorporated into a vehicle such as a plasmid vector.

The present invention provides a host capable of producing a chimeric antibody,
Methods of using these hosts are disclosed.

The present invention also provides for complete chimeric immunoglobulin chains having individual chimeric immunoglobulin chains and murine V regions with specificity for human C regions and human tumor cell antigens, yet with both mirror V regions having identical binding properties. It discloses a molecule assembled into a molecule.

Other immunoglobulin chains and/or molecules disclosed by the present invention include:

(i) An antibody with monovalent specificity for a tumor cell antigen, e.g. a complete functional immunoglobulin molecule composed of (a) two different chimeric heavy chains, one of which is It has a V region with tumor cell specificity.

(b) Two different tail chains, which have a specificity comparable to the V region of a heavy chain. As a result, the heterobifunctional antibodies produced exhibit monovalent binding specificity for human tumor cell antigens.

(ii) Antibody fragments such as Fab, Fab', F(ab')2 Gene sequences, particularly cDNA sequences, encoding combinations of the above chimeric chains are also provided in the present invention.

The present invention also provides a gene sequence encoding a V region with specificity to be marked in the H chain and/or L chain of an antibody molecule,
In particular, cDNA sequences are also disclosed that are linked to sequences encoding polypeptides (eg, enzymes) different from immunoglobulin chains.

These sequences are assembled by the method of the invention, which can be expressed to produce molecules with mixed functionality.

The use of cDNA sequences is equivalent to genomic sequences (including introns)
The advantage compared to
This is because it can be expressed in bacteria or other hosts that lack a splicing system.

(1) Gene generation process and product The present invention provides antibodies that can be used alone or in combination with other drugs in the treatment and diagnosis of human cancer. The antigen is one that binds to a mAb designated A10. In addition, the properties of AlomAb are disclosed in Japanese Patent Application Laid-Open No. 63-25499.
As stated in No. 2.

The production method combines the following five processes.

■ Messenger RNA (mRNA) from a mouse B cell hybridoma line producing mAb 0) Isolation, cloning and cDNA preparation: ■ Prepare a full-length cDNA library from purified mRNA. From this library, select appropriate L chain and H chain gene V region parts (
a) identified with appropriate probes, (b) sequenced, and (c) fused to the C region gene.

■ Preparation of C region gene partial module by cDNA preparation and cloning ■ Specific immunoglobulin V cloned from (■) above
Construction of a sequence encoding a complete H chain or L chain, which consists of combining the region gene part with the cloned human C region gene part module described in (1) above.

■ Chimera L within the host, including prokaryotic and eukaryotic cells.
The chain and heavy chain are expressed and produced.

A feature common to all immunoglobulin light chain genes, heavy chain genes, and the Messenger RNAs that encode them is related to the so-called J region. Although the HjlJ region and the L81 J region (J□ and JL) have different sequences, a high degree of sequence homology of over 80% is observed between each group, especially in the vicinity of the C region. . This homology is exploited in the present invention, and J. and J.L.
The consensus sequence of was used to design oligonucleotides to be used as primers or probes to introduce restriction enzyme sites into the J region that are useful for joining the V region portion and the human C region portion.

A C region cDNA module vector prepared from human cells and having a restriction enzyme cleavage site introduced into the human sequence at a similar site by a modification method using cytodirected mutagenesis (site-directed mutagenesis) was used. For example, a fully human chain C region (c) and a fully human gamma I chain C region (c10) can be cloned.

The alternative approach of using genomic C region clones as a source of C region module vectors is that expression of such genes may be difficult to achieve in systems such as bacteria, where the enzymes required to remove intervening sequences may not be present. Not allowed.

The cloned V region portion is excised and ligated to the cL or CH module vector. Furthermore, by modifying the human gamma 1 region by introducing a termination codon, it is possible to create a gene sequence encoding the H chain of the FaI molecule.

The sequences encoding the combined V and C regions are inserted into a suitable vehicle for expression in a suitable host, such as a prokaryote or a eukaryote.

Two coding DNA sequences are "operably linked" if the combination results in a gene sequence that is continuously translatable without altering or disturbing the triplet reading frame. It is said that "it has been done". A DNA coding sequence and a gene expression element are operably linked when the linkage allows the gene expression element elements to function properly to effect expression of the coding sequence.

Vehicles for expression include plasmids or other vectors. Among these vehicles, those that are preferably used are those that carry a functionally complete human C1I or CL chain sequence by engineering appropriate restriction enzyme sites. The engineering approach here is to introduce appropriate cohesive ends, such as VH or V
, a chain sequence that can be easily inserted into a vector. Therefore, a medium containing human CH or CL chain sequences is an important factor in carrying out the present invention. These vehicles can be used as intermediaries in expressing the desired complete H or L chain in a suitable host.

One suitable host is yeast.

Yeast offers substantial advantages when used to produce immunoglobulin light and heavy chains. Yeasts are capable of post-translational peptide modifications, including glycosylation. Currently, many DNA recombinant techniques use strong promoter sequences and high copy number plasmids to produce target proteins within yeast cells. Yeast recognizes the leader sequence of the cloned mammalian gene product and
secretes a peptide with a J-dar sequence (i.e., a prepeptide) (Hitzman, et al., 11
th International Conference
nce on Yeast, Genetics
andMolecular Biology, M
ontpellier, France, Septe
mber 13-17.1982).

Gene expression systems in yeast can be routinely evaluated in terms of production amount, secretion, and stability of chimeric antibodies assembled with chimeric H chain and L#1 protein. It encodes a glycolytic enzyme that is produced in large quantities when yeast is grown in a glucose-rich medium, and incorporates a promoter and transcription termination element (terminator) obtained from an actively expressed gene. A series of yeast gene expression systems are utilized. Genes for known glycolytic enzymes can also be used as highly effective transcriptional regulatory signals. For example, iso-1-cytochrome C (, CYC
-1) Gene promoters and transcription termination signals can also be used. A number of approaches can be taken to evaluate the optimal expression plasmid for expression of cloned immunoglobulin cDNA in yeast.

Bacterial strains can also be used as transformation hosts to produce the antibody molecules or antibody fragments described in this invention. E. coli W3110 (ATCC27325
) and other E. coli strains, and Salmonella enterica Salmonell.
a typhimurium and Serratia fungi 5err
Other intestinal bacteria, such as atia marcescens, and even various types of Pseudomonas aeruginosa, can also be used.

Plasmid vectors containing replicon and control sequences derived from strains compatible with host cells are used in connection with these bacterial hosts.

This vector carries not only replication sites but also special genes capable of displaying phenotypic selection within the transformed cells. There are a number of alternative approaches to evaluating expression plasmids in bacteria for the production of chimeric antibodies or antibody chains encoded by cloned immunoglobulin cDNAs.

Other suitable hosts are mammalian cells grown in vitro or invIVO.

Mammalian cells provide post-translational modifications to immunoglobulin protein molecules. These modifications include leader peptide removal, heavy and light chain folding.
and assembly, glycosylation of antibody molecules, and secretion of functional antibody proteins.

Mammalian cells useful as hosts for antibody protein production include hybridoma 5p210-Ag14 (AT
CCCRL 1581 ) or bone marrow tumor cells P3X
Lymphoblast-derived cells such as 63 A g 8 (ATCCTIB 9) and its derivatives are included. Other cells include Vero (ATCC CRL 81) or C
There are fibroblast cells such as HO-Kl (ATCC CRL 61).

There are a number of vector systems for expression of cloned heavy and light chain genes in mammalian cells. complete H
Different approaches can be taken to obtain t L2 antibodies. Complete intracellular association and complete tetrameric H2L
In order to combine H and L chains into two antibodies, H and L chains are co-expressed in the same cell.
on) is possible. This co-expression (co-e
xpression) can be achieved in the same host using either the same plasmid or a different plasmid. Both the H chain and LH genes are located within the same plasmid and subsequently transfected into cells, thus allowing direct selection of cells expressing both H and L chains. An alternative method is to first transfect the cells with a plasmid encoding one chain, e.g. the light chain, and then transfect the resulting cell line second with the heavy chain plasmid containing the selectable marker. It takes a process of effecting. A cell line producing H2L2 molecules obtained via either step is transfected with a plasmid encoding a heavy chain, light chain, or heavy heavy chain coupled with another selection marker.

The transfected cell line has enhanced properties such as increased production of assembled (H2L2) antibody molecules and increased stability of the cell line.

(I[) Polypeptide Products The present invention provides heavy or light chain "chimeric" immunoglobulin chains with specificity for human tumor cells.

The chimeric chain contains a C region that is essentially similar to that present in natural human immunoglobulins and a V region that has the anti-tumor specificity that is the object of the present invention.

The present invention also provides immunoglobulins in which the heavy and light chains are associated, thereby exerting desired binding and recognition properties as a whole molecule. Various types of immunoglobulin molecules are provided. That is, a monovalent, divalent or molecule having the V region binding domain of the present invention bound to a site having a desired function is provided. The present invention also provides Fab, F
(ab') and F(ab')2 molecules. The present invention also provides chimeric immunoglobulin "derivatives," which include proteins encoded by truncated or modified genes that produce species that are functionally similar to immunoglobulin fragments. . This modification includes, but is not limited to, gene sequences encoding plant and bacterial toxins and cytotoxic proteins such as tumor necrosis factor (TNF).

Fragments and derivatives can also be produced from any host of the invention.

Chimera H with the same or different binding specificity of the ■ region
Antibodies, fragments or derivatives having a chain and a light chain, e.g.
5ears, D.W., et al. [Proc, Nat.
1. Acad.

SciυSA 72:353-357 (1975)]
can be provided by appropriate association of the individual polypeptide chains, as disclosed by. By this approach, a host expressing a chimeric heavy chain (or its derivative) is cultured separately from a host expressing a chimeric light chain (or its derivative), and the immunoglobulin chains are separately collected and associated. Alternatively, the hosts can be co-cultured, allowing both mirrors to associate simultaneously in the medium.
Recovery of the assembled immunoglobulin, fragment or derivative may then occur.

[Action/Effect]

The antibodies of the present invention exhibit antibody-dependent cytotoxicity (ADCC) and/or
Alternatively, they can be applied for therapeutic efficacy against tumor cells by virtue of their ability to mediate complement-dependent cytotoxicity (cDC). These activities involve effector cells (
(for ADCC) or complement components (for cDC)
Whether it is of endogenous or exogenous origin, it can be used.

The chimeric antibodies, fragments and derivatives thereof of the present invention are immunoconjugates [Dillman, R,0, Ann, [nt, M
ed, 111:592-603 (1989) Reference 1. They are not limited, but
It can be conjugated to cytotoxic proteins including ricin A, Pseudomonas toxin, diphtheria toxin, and TNF. Toxins conjugated to antibodies, or other ligands, can be prepared using conventional techniques [0
1snes.

S. et al., 1mmuno1. TodaY 1
0: 291-295 (1989) reference 1. Plant and bacterial toxins kill cells by disrupting the protein synthesis machinery.

The chimeric antibodies, fragments and derivatives thereof of the present invention can be conjugated to adducted forms of most therapeutic agents, including but not limited to radionuclides and cytotoxic agents, for the purpose of treating cancer patients.

Examples of radionuclides that bind to antibodies and partition to antigenic sites in vivo include 213i, 13+[, +
Examples include 8SR and 90Y, but these are not intended to be consumed.

Radionuclides exert their cytotoxic effect by locally irradiating cells, thereby inducing intracellular damage, as is already known in the art of radiotherapy.

Cytotoxic agents that can be conjugated to antibodies and subsequently used for in vivo therapy include, but are not limited to, daunorubicin, doxorubicin, methotrexate, and mitomycin C. Cytotoxic drugs interfere with important cellular processes including DNA, RNA and protein synthesis. A full disclosure of these drug classes known in the prior art and their mechanisms of action can be found in Goodm
an, A, G,, et al, Goodman an
d Gliman's TH[E PHARMACO
LOGICAL BASISOF THE RAPEU
TIC3, 7th Ed, Macmillan Pu
See blishing Co., +985.

Treatment for individuals with tumors bearing antigens recognized by the antibodies, fragments and derivatives of the invention consists of parenteral administration of the antibodies, fragments and derivatives alone or in combination. Effective doses range from about 10 ng/kg to about 100 mg/kg body weight, depending on the function of the individual chimeric antibody, the presence and nature of the conjugated therapeutic agent (see above), the patient and his or her clinical condition.
kg body weight.

Routes of administration include intratumoral, intravenous, subcutaneous, intramuscular, intrapulmonary, intraperitoneal, intranasal, intrathecal, transdermal, or other routes.

The chimeric antibodies, fragments, or derivatives of the present invention can be effectively used in combination with other chimeric antibodies, lymphocytes, hematopoietic growth factors, etc. to help increase the number and activity of effector cells that interact with the antibody. Sometimes.

The antibodies of the present invention having all or part of the human C region are more susceptible to serum diseases and anaphylaxis than antibodies derived from mice.
The incidence of adverse immune reactions, such as shock, is low and in
It can be introduced in vivo, especially in humans. The antibody can also be used with detectable labeling methods (e.g., enzymes, 125I,
14C1 fluorescent labeling, etc.) or in a single quantification method (on polymer tubes, beads) for use in immunodiagnostic assays. Such labeled or immobilized antibodies, fragments or derivatives may also be supplied in kits.

These antibodies, fragments or derivatives may also be used in vivo.

Labeled forms are also available for imaging purposes, where the label is a radioactive substance, or a nuclear magnetic resonance contrast agent, such as a heavy metal nucleus, or an X-ray contrast agent, such as a heavy metal nucleus. The antibody can also be used to determine the in vitro localization of the tumor cell antigen it recognizes by appropriate labeling.
zation). Antibody-enzyme conjugates can be used in immunodiagnostic methods such as ELISA.

In particular, the chimeric antibody of the present invention is derived from murine A10mA.
In all applications in which chimeric molecules are used, they can be used with the distinct advantage of superior compatibility with the human body.

〔Example〕

Hereinafter, the present invention will be explained in more detail with reference to Examples.
These examples are not intended to limit the invention.

Example 1 Preparation of mouse-derived monoclonal antibody A10 1 Preparation of antigen 30 μg of carcinoembryonic antigen (cEA) obtained from ascites of a cancer patient
and purified mAb BD6 (Midori Jujisha Co., Ltd., Osaka, Japan) 120Ig were mixed to block the antigen-determining part of CEA. Add this mixture to an equal amount of Freund.
emulsified with complete adjuvant, 4-week-old male BALB/C
Mice were inoculated three times at 10 day intervals. After another 10 days, CEA was administered intraperitoneally as a final immunization without Freund's complete adjuvant. Three days after the final immunization, the spleen was removed from the mouse for use in the cell fusion described below.

2. Cell fusion and cloning (a) Fusion immune splenic cells and mouse myeloma P 3 U l (cu
rr.

Top, Microbiol, Immunol,
81:1 (1979)] with some modifications.
er et al.
.

Academic, Press, New York
, p., 391 (1979)]. That is, the immunized mouse spleen taken out above was washed with a DMEM culture solution not containing horse serum, and then ground to collect cells, which were then placed in a centrifuge tube to remove large clumps. By centrifuging the supernatant at 1200 rpm for 10 minutes,
Washed twice.

Next, 5.0 J of Tris/ammonium chloride solution was added to the precipitate to suspend the cells, and the mixture was left at room temperature for 10 minutes.

4 ml of DMEM culture solution was added to this and centrifuged, and this operation was repeated twice using DMEM culture solution for washing.

Finally, the same wave 5. Cells were suspended in Oml and counted. In addition, after washing mouse myeloma twice with DMEM culture solution, the number of cells was counted in the same manner. The spleen cells and mouse myeloma thus obtained were separated at 2:,l (3,3X
After mixing at a ratio of 107:1.6 X107), the mixture was centrifuged to obtain a precipitate (+200 rpm x 10 minutes).

After loosening the precipitate manually, 1 ml of 45% polyethylene glycol (average molecular weight: 4.000) was gradually added and treated for 1.5 to 2 minutes while rotating the reaction vessel. Thereafter, a total of 30 ml of DMEM culture solution was gradually added in 1° ml portions while rotating. After centrifuging the mixture, discard the supernatant, leave for 5 minutes, suspend the cells in HAT culture medium (see below), and inoculate them into a 96-well plate microplate (0.1 ml per well) under 7.5% CO2. It was cultured in

(b) Selective medium (1) Dulbecco's modified Eagle medium (DM
EM) was obtained from Nissui Pharmaceutical Company.

(2) HAT medium was prepared by adding the following additives to D-MEM: horse serum (10%, Flow Labor
atories), Shi-Glutamine (300/Sh),
Sodium pyruvate (110 mg/L), penicillin (+0010/ml), streptomycin (100 mg/ml),
μg/ml>, glucose (3,5 g/L), NaHCO
z (3,7g/L), Hyboxanthin (I Xl
0-'M), aminopterin (4X 10-7M), thymidine (1,6X 10-5M).

3. Screening method The anti-CEA activity of each hybridoma culture supernatant was determined by C
Screening was performed by the PHA method using sheep red blood cells that bind to EA. For all wells that produced anti-CEA antibodies, anti-MKN--45 mAb,
It was further screened by the PHA method using sheep red blood cells that bind CEA via BD6. Selected hybridomas were cloned two or three times and culture supernatants tested for reactivity with established cell lines derived from various cancers.

The hybridoma producing mAb A10 is Fer
mentation In5titude of 0s
aka ([FO), Osaka, Japan April 28, 1989
The date receipt was deposited with the deposit number IF 050188.

4 Characteristics of A10 A10 is an IgG1 that is expressed on the surface of many human cancer cells, including colon cancer, and binds to an antigen that exists in extremely small amounts in normal adult cells.

Example 2 Mouse-human chimeric immunoglobulin with human tumor specificity produced from mammalian cells 16 pBR322 with recombinant plasmids and bacteriophage DNA oligo-dG tails, 1) UCl 8, pUCI 9.
Ml 3mp 18. and M13mp+9 BRL
(Gaithersburg, Maryland). Plasmid DNA by buoyant density centrifugation
DNA manipulation methods, including purification of F. coli, restriction endonuclease digestion, purification of DNA fragments by agarose gel electrophoresis, DNA ligation and transformation of F. coli, are described by Maniatis, T. et al., Mol.
ecularCloning: A Laborat
Ory Manual (1982), or other standard operating procedures. Restriction endonucleases and other DNA/RNA modifying enzymes are described by Boehr
inger-Mannheim (lndianapol)
is, IN), BRL.

and New England Biolabs (B
Everly, MA).

2. RNA purification and cDNA library construction approximately 1 x 10
1 liter of A10 hybridoma cells/ml were collected by centrifugation and dissolved in PBS (8 g of NaCl, KH2P
O40,2g, Na2HPO,1,15g and KCI
0.2 g/L). Cells were centrifuged again and the cell pellet was suspended in guanidine thiocyanate solution, containing total cellular RNA and poly(A).
RNA was prepared from tissue culture cells according to the method described by Maniijis, T. et al.

From poly(A)'' RNA, a cDNA library using oligo-dT as a primer was generated by Gubler et al.
, [Gene 25: 263 (1983)]. Add a dC tail to the CDNA using terminal deoxynucleotide transferase and anneal with pBR322, which has a dG tail.
L/ta.

cDNA library by nick translation2
The 2p-labeled DNA fragments, i.e. clones, were screened with a mouse C region probe, and the H chain clones were screened with a mouse IgGIC region probe using the hybridization method (Maniatis, T., supra). .

Full-length cDNA clones, pKAlo-16 and pγA
The L chain and H chain V region fragments obtained from each of 10-16 were inserted into an M13 bacteriophage vector, and nucleotide sequence analysis was performed. The complete nucleotide sequences of the V regions of these clones were determined using the dideoxy chain termination method (Did
eoxy Chain TerminationMet
hod) (Figures 1 and 2). The amino acid composition of the V region predicted from these sequences matched well with the observed amino acid composition of the region. In addition, the peptide sequence of the region (■), which has already been demonstrated by direct amino acid sequencing, and the predicted peptide sequence also matched well.

The nucleotide sequence of the cDNA clone consists of the amino acid residues characteristic of the domain (Kabat et al.
sequences of proteins of i
mmunological interest; U, S
, Dept of HH3, 1983), indicating that they are immunoglobulin V region clones.

A I OV, i;! J, has 2 sequences, and Al0v has 2 sequences in J.

3. Construction of chimera expression plasmid ■ To express chimera A10. An expression vector suitable for insertion of the and vL gene module was constructed. L chain Hector plNG+712 was first extracted with plasmid DNA containing the test chimeric L chain gene (p I NC; 212
2) and added regions to the mouse genome on the 3' side of the mouse Abelson LT'R promoter, splice region, and polyadenylation signal. M 13 M8 alpha RXl 2 (
Robinson, R.R., et al., PcT.
Mouse H chain enhancer-0,7 kb Xba I to Ec derived from US 86102269).

M13m cut R1 fragment with Xba I and EcoRl
It was inserted into p19. Enhancer-containing HindI [[~
BglI [fragment, unique Xhol, BglI
I and HindI [E,c containing the I sites in this order
Bgl of oli recombinant plasmid DNA (pSH6)
Inserted into the I[~Hindn[ region. Next, the enhancer-containing Xba I to -Xho I fragment was added to plNG21
It was inserted into the enhancer-Xba I-Xho I region of 21b. This plNG2121b has 28
L6VL area [Liu, A, Y
, etal, , Proc, Natl, Acad,
Sci, USA 84:3439 (1987)].
,A,Y.

et al., J., [mmunology 139
:3251 (1987)). The plasmid thus created is plNG2122.

Mouse Abelson virus L'TR is pelin
2 (leaf.

Obtained from Owen Witte, UCLA). pelin 2 is B[! at virus position 4623. I
EcoR [oligonucleotide linker GGA
3LTR of the P120 virus except that it was modified by inserting ATTCC [(Reddy, E.
P,et al,,Proc,Natl,Acad
.

Sci, USA 80:3623 (1983)]. p1203' A 0.8 kb E-parallel R1-Ikkuchu I fragment of Kidan n2 carrying the LTR promoter.
It was inserted into pUCI8, which had been cut at R1 mark R1. LT
R was excised as EcoR[-Sal I fragment and Eco
It was ligated to pING2+22 cut with Rl and 5alI to obtain a plasmid in which the LTR bromoter was adjacent to the L6 L chain gene (plNG2+26).

The Xho I=Sal I fragment containing the SV4016s splice donor and acceptor sites was added to pUC12.
/p L I (Robinson et al.
(same as above), inserted into the Sat 1 site of pING2126, and screened for splice donor or orientation between LTR and L chain genes (plNG2133).

In addition, the polyadenylation/transcription termination region of the current vector was also modified. The first step is plNG2121
To construct a-delta H, plNG2+
21a is digested with HindIII and recombined. 1) ING2121a is plNG2 except that L6VL is used instead of 2H7V, region
+08a (Liu, A. Y. et al., supra). 1 of the mouse genomic DNA behind the polyadenylation site. I Kb BglII ~Bam
Hl fragment (Xu, M, etal, J, Bio
l, Chem, 261+3838 (1986)]
However, pS107A (Kano, Randolph Wal
PING2
Plasmids that were inserted into the ancient site of 12+a-delta H and in a homologous orientation to the native gene were screened. 3.3 including this modified 3' region
The BglII~5stl fragment of Kb was pI N G21
plNG17o3 was prepared by ligating the 5.2 kb BglII~'danI fragment of 21b. “Rainbow” of plNG1703 with modified 3゛ region and chimeric coding sequence■
~zu bearer'' fragment of plNG2+33 BglII~5al
The 9.1 kb expression vector plNG1712 in FIG. 4 was prepared by ligating it to the large fragment of plNG1712.

The Abelson LTR promoter was also used in the chimeric heavy chain expression vector plNG1714. piNG2
111 (Robinson, R, R, et al.
, ibid.) was modified by inserting an AatII oligonucleotide linker at the Xba1 site, then A
plNG1707 was prepared by atII cleavage and religation. Aat containing Abelson LTR promoter
The Aat II to Sal I fragment was excised from plNG2133 and the Aat II to Sal I fragment of plNG1707.
plNC;1711 was prepared by ligating the large fragment. Ec
H chain enhancer was extracted from plNG+711 by oRI digestion.
7.7 kb expression vector plNG17 was removed from the 7.7 kb expression vector plNG17 by treatment with T4 polymerase, ligation to an AatII oligonucleotide linker, cleavage with AatlI and religation.
I got 14.

A similar plasmid, plNG2227, carries two additional regulatory elements, the IgH enhancer and the human genomic TgG polyadenylation sequence. IgH enhancer-1A
BglII with belson LTR promoter
~ In the 5alI region and the 16S slice toner and acceptor site, plNG222°7 and plNc;
It is homologous to 1712.

Human genome Ig03° end sequence 1185 bpXma
It was obtained as an IDNA fragment and ligated to the Xma■ site located 6 bp behind the stop codon of the H chain gene in pTNG1714. 1300bp including genome gamma 3' end
XmalI [fragment isolated from a derivative of pH03A (Ellison et at, Nucleic
acidsRe5earch10:4071(19
82)).

4. Construction of AlOH chain and L chain expression plasmids Site-directed mutagenesis (Kramer, W, et al, Nuc
l.

Ac1ds Res, +2: 9441), the AlOH chain, pγA 1
A cDNA clone containing 0-16 was adapted for expression in mammalian cells (Figure 3). Oligonucleotides were transferred to a cyclone DNA synthesizer (New Brunswi
ckScientific Co.) and purified by acrylamide gel electrophoresis. BstEII
J region mutagenesis (Mutagen) was used to insert the site into pTK3-generated M13 subclone pTKI.
esis ) Primer 5'-CTGAGGAGA
CGGTGACCGTGGTGCCTT-3° was used.

Also, oligonucleotide 5'-ATGTCTGT
A 5all restriction site was inserted upstream of the initiation codon ATG of pTK3 to generate pTK7 by using GTCGACCACTGAAGAGA-3'. Next, the restriction enzyme digested fragment between 5alI and BstE■ containing the A10V region was cloned into the expression vector plNG2240. This plasmid was modified in two steps to prepare the final chimeric AlOH chain vector, plNG2254, as shown in Figure 3. pl except that it contains the area
Same as NG2227.

cDNA clone containing AI OL chain, pKA10
-16 was adapted for expression in mammalian cells in a similar manner (Figure 4). J region mutagenesis (Mutage
nesis) Bly7-5' -GTTTTATTT
A HindnI site was inserted into the M13 subclone pTK21 to generate pTK4 by using CAAGCTTGGTCC-3°. Additionally, the oligonucleotide 5°-GAACTTTGGTCGACAGCAAC
GGC; -3°''11 restriction site was inserted upstream of the start codon ATG of subclone pTK4 to generate pTK5. 5al containing Al0VL region
Expression vector pl containing the restriction enzyme fragment between I and old ndIII
NG1712 to generate the final chimeric AI OL chain expression vector, pING2252.

5 Stable Transfection of Mouse Lymphoid Cells for Chimeric Antibody Production Cell Line Sp2/O (American Type
e CultureCollection #CRLI
581) were grown in DMEM prepared to contain 4.5 g #glucose (G[BCo) and 10% fetal bovine serum. The medium contained glutamine, penicillin and streptomycin (GPS) (Irvine 5ci
entific, 1rvine, CA) was added. Electroporation method ([Electroporation] by Potter, H. et al.
Proc, Natl, AcadSci, USA
81 Near 161 (1984)] was used. After transfection, cells were left to recover for 24 hours in complete DMEM and then plated at 10,000-50 cells per well in 96-well culture plates containing selective medium.
．． 000 cells were seeded. G418 (GIBCO
) selection is 0.8■/mL and mycophenolic acid (c
albiochem) concentration is 61 tg/ml plus 0.
25 mg/J xanthine. This electroboration technique is suitable for 5l) 210 cells.
-' transfection frequency was given.

Chimeric Al0L chain expression plasmid pING2252
It was linearized by digestion with vu I restriction endonuclease and transfected into 5p210 cells to obtain mycophenolic acid resistant clones.

These cells were screened for L chain synthesis. The best producing cells were grown, subcloned, and then linearized by Pvu I digestion.
The expression plasmid plNG2254 containing the chain gene was transfected.

After selection with 6418, clone 5p210-22523C3, 4D2-22 produced the largest amount of L chain plus H chain.
546C4 secreted antibody at approximately 6-7 μg/μl.

6. Purification of chimeric A10 antibody secreted in tissue culture 5p210-2252.3C3,4D2-2254.6
C4 (c813) cells were treated with 10 mM HEPE.
S, I XGPS (Irvine 5cient
culture solution HB10I (H
ANA Biology) was grown in 11% fetal bovine serum. The cultured medium was heated at approximately 14,000×g for 20
Centrifuge for 1 minute and transfer the supernatant to 0.45μ Millipor.
e It was filtered with a nitrocellulose membrane filter and stored frozen. Antibody levels were measured by EL[SA. Approximately 17 L of cell culture supernatant was transferred to a DC10 concentrator (Amicon Co.
rp, ) and concentrated 10-fold on a S I 0Y30 cartridge. The supernatant containing approximately 40 μl of chimeric antibody was diluted with 2 ml Protein in PBS, pH 7.4.
A-Sepharose column (Pharmacia or equivalent) was run several times. Step PI (Gradient (pH5
, 6, 3, 5, 2, 2), chimera A1
0 antibody was found to elute at pH 3.5.

Antibody-containing fractions (70% yield) were mixed and 10
Buffer exchange with mM ME 5S pH 6,0, 1
ml mono-8FPLC column (Pharmacia) several times.

When eluted with a NaCl gradient (0-300mM),
Chimeric A10 antibody eluted at 120mM. Antibody-containing fractions (22% yield) were mixed and ultrafiltrated (cen
triCO mouth 30. Amlco Corp.) was concentrated 80-fold, diluted 5-fold with PBS, re-concentrated 2-fold, and finally re-diluted 16-fold with PBS. Antibodies were stored in 1 ml aliquots at -20°C.

7. Analysis of chimeric A10 antibody (a) Inhibition of binding: mouse A10 mAb and chimeric A
Ten antibodies were compared in a binding inhibition assay. This inhibition assay is used to confirm the identity of antigen recognition. Mouse A10 mAb was labeled with biotin. Then purified unlabeled chimeric A10 antibody and mouse A
For the 10 antibodies, labeled A10 target cells (LS174
The binding inhibitory activity against T colon tumor) was investigated. Chimera A
10 antibody and mouse A10 antibody are labeled A10 antibody LS1
The binding inhibitory activity against 74T tumor cells was equivalent (Table 1).

(b) Functional assay: Effector cells (ADCC
Ability of chimeric A10 and murine A10 antibodies to lyse human tumor cells in the presence of human peripheral blood leukocytes as a mediator of complement (mediated complement-dependent cytolysis, CDC) or human serum as a complement (mediate of complement-dependent cytolysis, CDC). compared. Table 2 shows that neither mouse nor chimeric A10 antibodies are able to mediate ADCC. Similarly, both mouse and chimeric A10 target LS I 74 in the presence of human serum.
It was not possible to detectably lyse T cells.

[Margin below]

Table 1 A10 antibody binding to LS l 74T tumor cells 1
．． 005. 918. 786. 7+
90.015. 977. 831.
7B40.046 1. Q49. 854
．． 7640 +4. 970. 784
．． 7580.41. 85+, 7
78. 726+, 24. 68+
, 614. 7873.70. 477
．． 444. 79711.11. 354
．． 316. 77633.33. 246
．． 242. 737100.0. 241
．． 210. 707300.0. 15
3. 151. 688a: Biotin-labeled mouse A10 antibody was incubated with LS 174T at 4°C in the presence of a competing antibody.
incubated with tumor cells. Cells were washed to eliminate bound antibodies and incubated with avidin oxidase. The fall in absorbance at 490 nm (A4°) was used as an indicator of relative inhibition of binding by biotin-labeled A10.

b, Human IgG was used as a non-specific antibody control.

Table 2. Antibody dependence 50 mediated by chimeric A10 antibody 1
33 305,
32 26
0, 5 24 27
0,05 27 2
80,005 27
280.0005 27
280 29
29a: Ls174T tumor cells labeled with 6'Cr,
Washed and incubated with fresh peripheral blood leukocytes in the presence of 17% human serum for 4 hours at 37°C. Culture was carried out at a ratio of 50 leukocytes per tumor cell.

Percent cell lysis was calculated by comparing the amount of SICr released into the medium and cells lysed by addition of 1% NP40.

[Margin below]

Example 3 Mouse-Human Chimeric Fab Produced in Escherichia coli with Human Tumor Cell Specificity Since the entire coding sequence is expressed from a well-studied promoter, chimeric antibodies expressed from mammalian cDNA are suitable for production.

Since a lot of genetic information has been obtained for optimizing the gene expression of E. coli, the production of foreign proteins (H
olland et al, BioTechnol
ogy 4: 427 (1986)].

E. coli can be used to produce foreign proteins within the bacterial body or to secrete proteins from the cytoplasm to the outside of the bacterial cell, in which case they accumulate in the periplasmic space [Gray
et al., Gene 39: 247 (19
85); 0kaet al, Proc, Na
tl, Acad, Sci, tJsA 82 Near 212 (1985)). Secretion from the E. coli cytoplasm has been observed for many proteins, but this requires a signal sequence. Proteins produced within bacterial cells are often not folded properly [Choner et al., BioTech
Nology 3:151 (1985)]. However, proteins secreted by bacteria are often properly folded and assume their original secondary and tertiary structure (
Hsiunget al.

BioTechno+ogy 4: 991 (+9
86)).

A Fab molecule consists of two non-homologous protein chains linked by a disulfide bridge, forming a complete antibody chain and
Consists of the V, J, and C81 portions (Fd) of the antibody H chain. Appropriate cDNA of A10 chimeric L chain and Fd gene
Clones have already been identified. In this example, these cDNA clones are Erwinia caroto
vora [Lei, et al., J. Bacteri
ol, 1694379 (+987)] to the pectate lyase (2view↓B) gene leader sequence, organized into one bacterial operon (dicistronic message), and expressed from a promoter with strong regulatory action. This is a method for simultaneously expressing two protein chains in E. coli, thereby producing an immunologically active, appropriately assembled F10 chimeric antibody.
ab is secreted.

In the next section, chimeric Al0Fab by E. coli
Let's talk about the secretion of

1. Synthesis of pelB leader sequence cassette Erwini
a carotovora (EC) encodes several betatate lyases (polygalacturonic acid transeliminases) (Lei et al., Ge
ne 35:63 (+985)). Three vector lyase genes have been cloned and the DNA sequences of these genes have been determined. When cloned into E. coli under the control of a strong promoter, the I]elB gene was expressed and large amounts of vectate lyase were accumulated in the periplasmic space and culture supernatant. The pelB signal sequence is E, c
In this example, it was used as a secretion signal for the antibody gene (other signal sequences could also be used for this purpose). The nucleotide sequence of the peripheral region of the pel B gene, including the sogenic sequence, has been published (Lei et al., 1987, supra).

The pelB signal sequence has a signal peptidase cleavage site: Hae at amino acid position 22 adjacent to ala-ala.
■It has a restriction enzyme site. p U C8 [Vier
ra and Messig 1982, Gene 19
: 259), a plasmid containing the pel B gene, pSS 1004 (Lei et al.,
1987. ) was digested with HaelI and EcoRI. An 8 base pair 5stl linker was added to this DNA, and pB R32 was cut with 5spI and EcoR.
Connected to 2. The plasmid thus created is pel B
The 22 amino acid leader sequence of E, car
300b containing approximately 230 bp of atovora DNA
p fragment.

This plasmid, plNG173, was digested with 5stl,
Upon treatment with T4 DNA polymerase, any gene can be directly ligated with the blunted DNA fragment at the first amino acid of the mature coding sequence, resulting in a fusion with a functional bacterial leader sequence in-frame with the entry gene. p
5stl-Ec of lNG173.

The R1 fragment was pUCC18 (Yanich-Perro
n et al., Gene 33:102 (1985
)) to obtain pRR175. This vector contains the pelB leader sequence and adjacent upstream non-coding sequences (including the lyphosome binding site) downstream of the lac promoter sequence. The plasmid plNG I500 derived from pRR175 contains -48 of the pIB gene.
It contains only the region from to the Xho 1 site downstream of the IB leader sequence, and has a 5stl site at the junction.

2 in-vitro mutagenesis (M
Site-specific in vitro mutagenesis was performed according to the method of Kramer et al., supra, to insert oligonucleotide primer 5 at the junction of the mature coding region of the A10 strand cDNA sequence and the leader peptide in pTK4 (see Figure 4). ° −GAG
AACAATTTGAGCTCTGGACAGT-3″
5stl restriction enzyme site (see Figure 2), pTK
6 was constructed.

Similarly, oligonucleotide primer 5AAGC
TTCACTTCTGCAGGGACACCATTT-
A PstI site (
(see Figure 1) and constructed pTKII (Figure 2).

3. Preparation of the chain for bacterial expression The chimeric AlovL region of pTK6 containing a 5stl restriction enzyme site at the signal sequence processing site and a unique HindI[ site in the J region is a starting point for bacterial expression. It served as a point. Plasmid pTK6 is 5st
5a and treated with T4 polymerase, resulting in 5a
was digested with lI (Fig. 5A).

(The Sal 1 site is located outside the antibody gene segment adjacent to the MI3 multiple cloning region).

An approximately 380 bp fragment containing the VL region was purified, which was cut with 5stI, treated with T4 polymerase, and 5a
ligated to plNG1500 cut with lI (Figure 5B),
pTK8 was prepared. It was confirmed by sea fencing that the plasmid containing the pel-containing plasmid pTK8:pelB:A10V L region fusion thus created had a correct in-frame fusion. The HindII fragment of pTK8 containing the leader sequence was cloned into pS2D, and the plasmid pTK9 containing the human C region was cloned into pS2D.
was prepared (Fig. 5C).

4. Preparation of Fd for bacterial expression Pstl restriction site in signal sequence processing site,
and pT K with BstEI [restriction site in the J region
The complete A10 chimeric Fd gene of 11 served as a starting point for expression in bacteria. Plasmid pTKll was cut with PstI, treated with T4 polymerase, and Bst
An approximately 340 bp fragment containing the FdV region was purified, digested with EIrI (Fig. 5D), which was cut with 5stI, treated with T4 polymerase, and ligated into plNG+500 cut with XhoI (Fig. 5E). Similarly, plasmid pTK11 was digested with BstEII (partial digestion) and ligated with a restriction fragment in Xho I containing the human FdC region and 3 nucleotides following the stop codon of pF3D (Fig. 5 F), (R,R, et a
ly, the C,1 region of pF3D was previously prepared by introducing a stop codon in the hinge). The pelB-containing plasmid thus produced::A
Plasmids containing the 10Fd fusion were seafenced to confirm that the correct in-frame fusion was formed. This plasmid was named pTKI3.

5. To produce a multicistronic expression bacterial Fab for L chain and Fd genes, both L chain and Fd need to be produced simultaneously within the cell. Plasmids constructed with each of these genes separately were used to construct a series of vectors in which both genes were sequenced so that transcription from a single promoter specified both genes. This was done in a way to minimize non-coding DNA between the two genes. Each gene has a liposome binding site necessary for translation initiation and a homologous DNA sequence from -48 to the pelB leader:, antibody gene junction. Plasmid pTK9 was cut with sph I, treated with T4 polymerase, and cut with EcoRl. The vector fragment was purified (Fig. 5G). Similarly, pTK13 was cut with Xho 1, treated with T4 polymerase, cut with EcoRI, and F
The fragment containing the d gene was purified (Fig. 5H).

These two purified DNA fragments were ligated to create pTK15.
It was created. It contains two closely linked A10 chimeric gene fusions. The two gene cistrons were placed under the control of the ara B promoter' in pING3104. Plasmid pTK15 was cut with sph I, treated with T4 polymerase, cut with Xho I, and the fragment containing the gene in Fd was purified (Figure 51).
. This DNA fragment was cut with EcoR[I, treated with T4 polymerase, and treated with Xh.

This was ligated to the vector fragment obtained from plNG3104, which had been cut with (Fig. 5J), to create pING3204. This vector possesses all the features necessary for expression of the A10 chimeric Fab in E. coli.

6. Production of chimeric Al0Fab in bacteria E, col
A10 chimera Fab from plNG3204 in i
Expression of is under the inducible control of the ara B promoter. When arabinose induction was performed, Fab secreted into the medium increased more than 10 times. Non-induced bacterial colonies harboring pING3204 are plNG3104 from the viewpoint of traits.
Comprehensive E and coli J could not be distinguished. plNG32
The 04 endophytic strain was cultured at 32°C in 10 L of minimal medium supplemented with 1.7% glycerol as a carbon source. The amount of dissolved oxygen was maintained at 20% of the saturation amount by adding minimal medium supplemented with 5 times the concentration of glycerol. O.D. :-
At the age of 50, the cells were induced with 0.05% arabinose and cultured in a 14L culture machine (chemap) for over 16 hours. Pa
b was detected in fermentation broth by ELISA.

Approximately 71 μl of the culture supernatant was transferred to a 510Y10 cartridge (DC1
0 concentrator, Amlcon Corp.).

The concentrate was dissolved in 10 mM sodium phosphate buffer pHg,
The sample was applied to a 500 g DεAE cellulose type DE52 type (Whatman) column pre-equilibrated at 0. Add sufficient amount of 0.2M sodium phosphate to pH
6 and 8, and the sample was coated with a Y2O film (Stirred C
e1l 2000. Amjcon). The sample was then diluted with sufficient water and reconcentrated to 200 rnl, and the conductivity was 1.1 mS/cm. The total amount of protein was evaluated using a colorimetric assay, and the sample was prepared using a carboxymethyl cellulose type column (cM52, Whatman
, Hg pre-equilibrated in 10 mM linthorium buffer, pH 6.8, was applied to the column at a rate of 10.0 kg of total protein. CM52 column with concentration linear gradient 0
Elution was performed with the same phosphate buffer solution containing ~0.1N Na1l. Fab-containing fractions (assessed by ELISA) were further analyzed by 5OS-PAGE and pooled. The collected Fab fraction has a Fab concentration of approximately 1.
It was concentrated on a YM10 membrane to a volume of /ml and stored frozen. AloFab molecules purified from ε, coli are
As evaluated by SDS gel electrophoresis, it has the same molecular weight as other Fab molecules produced from E. coli. Other A10 Fabs produced by bacteria include and F
It shows a positive reaction in ELISA that can detect molecules having both determinants of d, and it also reacts with antigen-positive cell lines, indicating that +Fd chain two-chain monomer is accurately assembled.

7. Binding properties of Fab proteins secreted by E. coli A I OF a b was shown to bind to antigen-positive cells by performing direct and competitive binding assays with the human tumor cell line LS174T. . In direct binding assays, the Fab bound to the same target cancer cells as the mouse A10 antibody, but not to a control cell line lacking antigen. l1ls l-labeled mouse A
In competitive assays using 10 antibodies, E, col
i The chimeric A10 antibody Fab derived from radiolabeled mouse A
The 10m Ab antibody inhibited binding to the human tumor cell line LS174T. Yeast-derived Fab is approximately lug/m
Table 3), which suppressed AlomAb binding by 50% at a concentration of
A10 mouse mAb also showed 50% inhibition of binding at the same concentration. Yeast-derived AI 0Fab inhibited the binding of mouse A10 antibody and mouse AI 0Fab to LSI 74T tumor cells.

Mouse Fab was prepared by papain digestion of mouse whole 939 antibody.

[Margin 1 below] Example 4 Mouse-human chimeric Fab produced in yeast with specificity for human tumor cells Yeast i is capable of expressing and secreting foreign proteins. In this example, yeast plays the role of host during mouse-human chimeric Fab production. The present invention can be used in cancer diagnosis by in vivo imaging using appropriately labeled Fabs, and in cancer therapy by administering Fabs as immunoconjugates with drugs, radionuclides, or toxins. It appears to be.

■, Yeast strains and growth conditions Saccharomyce grown in INGBN [E.
S cerevisiae strain P S 6 (ura
31eu2'MATa) by Ito et al.

J, Bacteriol, 153: 163-1
68 (1983) as a host for yeast transformation. Transfer yeast transformed cells to SD agar [
2% glucose, 0.67% Yeastnitrogen base, 2% agar] and 50mM sodium succinate, pH 5.
, 5 in buffered SD broth.

2. Construction of yeast expression plasmid containing antibody gene Encoding the mature Al0VL region and inserting Hind■ in the J region
site, and Sst at the signal sequence processing site.
The gene sequence into which the I site was introduced was ligated to the human C region. Plasmid pTK6 (Figure 5) was transformed into Sst I
After treatment with T4 polymerase, the product was cut with Hind■. A region fragment was obtained as an Xho I fragment from Hindll [, and this was used as a human CL fragment prepared from pMI D (same fragment containing pS2D, see Example 2 and FIG. 5).
region, yielding p[NG1149. Plasmid plNG1149 was produced under the control of the yeast PGK promoter [Hitzeman, R.A., et al.
Nucl, Ac1d, Res.

10: 7791-7800 (February 1980, gene sequence CTau encoding yeast invertase signal sequence)
ssig, R.

et al, Nucl, Ac1ds, Res,
11: 1943-1954 (1983)].

The resulting plasmid, pTK12 (Fig. 6a), was
Contains a PGK promoter (P) with an invertase signal sequence (S) aligned with the IO coding sequence. As a result of this conjugation, the gene sequence encoding the mature A10 chimeric strand was fused in frame with the gene sequence encoding the yeast invertase signal sequence. P.G.K.
The promoter invertase signal sequence-chimeric light chain (in V, C) conjugate was cloned into the ura yeast expression vector containing part of the 2μ ring and the PGK polyadenylation signal (Tm) to prepare pTKi6 (Figure 6c). .

Encodes the A10 mature vH region and contains BstE in the J region.
The gene sequence in which the PstI site was introduced into the signal sequence processing site of pTKll (Figure 5) was
Human C81 region of F3D (see Example 2 and Figure 5)
This was previously generated by introducing a stop codon into the hinge. Robinson, R.
R, et al., PCTU586102269), cut with PstI, treated with T4 polymerase, and incorporated into pING1149 cut with XhoI.

This prepared plasmid pTK+4 (Figure 6b).

PGK promoter invertase signal sequence-chimeric Fd chain (V, C, 1) conjugate with part of the 2μ ring and PGK
It was cloned into an expression vector containing a polyadenylation signal (Tm) to prepare pTK17 (Fig. 6d).

A single yeast expression vector containing both the chimeric L chain and chimeric Fd chain genes and their expression signals
Constructed from TK17 and pTKI6.

This final vector, plNG3203 (Fig. 6e), is 2μ
It contains part of the ring (or iY, REP3) and two selection markers 1eu2d and ura3.

3. Yeast chimera AIOF, ab secretion plasmid pING3203, S, cerevisiaePS6
and the transformed cells were grown in broth under selection conditions as described above. ELISA of culture supernatant
As a result of the assay, the concentration of Fab contained was approximately 100 ng.
/ It was me. This is believed to have the same properties as the Fab purified from fermentation broth and produced as described in Example 2, ie, the yeast Al0Fab has binding properties to LSI 74T cells.

Conclusion The above examples demonstrate various important properties of the chimeric A10 antibodies and genetically engineered AI 0Fab proteins of the present invention. First, the chimeric A10 antibody and its Fab derivative bind to human tumor cell lines with approximately the same affinity as the mouse A10 antibody. The chimeric A10 antibody is important because it binds to the surface of human tumor cells. A.J.O.
The reactivity of mAbs with normal cells such as fibroblasts, endothelial cells, or epithelial cells of major organs is minimal. Therefore, chimeric AlOmAbs can reveal in vitro antigens that are useful for distinguishing between human tumor cells and non-tumor cells.

Although the future potential of implementing mAb-based tumor therapy is of interest, so far, mAb-based
Therapeutic success stories are limited (Houghton et
al,, Proc, Natl, Acad, S
ci, 82: 1242~+246 (1985
) ). The therapeutic efficacy of the original, unmodified mouse mAb appears to be too low to be of practical use. Chimera A
The 10 antibody is an improved therapeutic agent over the murine A 10m Ab for in vivo treatment of human tumors. First, the chimeric A10 antibody combines high biological activity against human tumor cell lines with minimal reactivity against normal tissue, suggesting that this antibody may mediate selective destruction of malignant tumor tissue. ing. Second, human-derived J chimeric A10 antibodies will exhibit greater resistance to rapid release from the body than murine A10 antibodies. : The human component of a chimeric antibody may improve its ability to mediate targeted destruction in conjunction with human effector cells or complement. Third, improved resistance to clearance makes chimeric A10 antibodies and their derivatives advantageous for tumor diagnosis and treatment in the form of immunoconjugates with drugs, toxins, immunomodulators, radionuclides, etc. It means. Such immunoconjugates and techniques for making them are well known to those skilled in the art and can be used to modify chimeric A10 antibodies within the scope of the present invention.

Furthermore, by using the methods disclosed in this invention, any desired antibody isotype can be bound to any specific antigen binding site.

The present invention also allows for the direct production of one or more domains of an antibody molecule in a functionally active form.

Deposit Two illustrative chimeric A10 antibody-secreting cell lines have been deposited with the ATCC, Rockville MD, prior to filing in the United States. They are: 1° transfected hyperidoma 5p210
(plNG2252 and plNG2254), C
Strain 813, designated ATCC #HB I 0195.

2. E. coli MC1061 (plNG3204
), strain G268, designated ATCC #68071.

[Brief explanation of drawings]

Figure 1: Base sequence encoding the mouse V region of the AlOH chain. The base sequence from the end of the oligo dc tail to the J, 2-C, 1 junction is shown in the figure. The figure also shows the amino acid sequence that can be inferred from the base sequence. The oligonucleotide used for site-directed mutagenesis (site-directed mutagenesis) and the site where the restriction enzyme cleavage site was introduced are indicated by thick lines. Figure 2: Base sequence encoding the A10 mouse kappa V region (V). The base sequence from the end of the oligo dc tail to the J to 2-C junction is shown in the figure. The figure also shows the amino acid sequence that can be inferred from the base sequence. The oligonucleotide used for site-directed mutagenesis (site-directed mutagenesis) and the site where the restriction enzyme cleavage site was introduced are indicated by thick lines. Figure 3: Construction of plNG2254, a salivary mammalian expression plasmid for expressing mouse-human chimeric AlOH chain. An engineering procedure was performed so that the ■ region of cDNA clone p7A10-16 could be fused with a eukaryotic expression plasmid, I) ING2227. Plasmid I) ING222
7 contains the following gene control sites useful for expression in mammalian cells. : (1) IgG H chain enhancer element, (2) Abelson LTR promoter (
3) SV40 16S splice site, and (
4) IgGH chain polyazonylation signal sequence. This plasmid pING2227 is also pG
It also contains the complete human IgG 1-C region from MH-6 (Liu, A, Y. et al., Proc. Natl., Acad.
Sci, USA 84:3439-3443, (1
987) ). plNG2227 contains a neomycin phosphotransferase gene that is subject to G418 selection in transfected cells. Figure 4 - Construction of plNG2252, a mammalian expression plasmid used for mouse-human chimeric AI OL chain expression. cD
An engineering procedure was performed so that the V region of NA clone pKA10-16 could be fused with eukaryotic expression plasmid plNG1712. Plasmid pING1712 contains the following gene control sites useful for expression in mammalian cells. (11IgH enhancer element, (2) Abelson LTR promoter, (3) SV40 163 splice site, and (4) human polyazonylation signal sequence. This plasmid plNG1712 also contains a completely human constant region (Liu, A, Y, et al.
, supra) and a GPT gene that confers mycophenolic acid resistance in transformed cells. Figure 5: Construction of a bacterial chimeric At 0Fab expression plasmid, pING3204. Plasmid pING32
04 contains the following sites useful for expression in E. coli. : (1) araC gene, (2) inducible araB promoter, (A10 gene with dicistronic Pd fused to 31pe1B leader sequence, f4) trpA transcription terminator sequence, and (5) in E. coli tetR gene useful for selection. Figure 6: Yeast expression plasmid for Fab expression. The illustration is as follows. :

Claims (33)

[Claims] (1) A polynucleotide molecule comprising a sequence encoding a variable region of an immunoglobulin chain having specificity for an antigen that binds to the mouse A10 monoclonal antibody. (2) The molecule according to claim (1), wherein the chain is a heavy chain. (3) The molecule according to claim (1), wherein the chain is a light chain. (4) further comprising an additional sequence encoding at least a portion of a constant region of a human immunoglobulin chain, both of which sequences are operably linked to each other.
) term numerator. (5) Claim No. 1, wherein the additional sequence is a cDNA sequence.
4) The numerator of the term. (6) The molecule of claim (1) which is a recombinant DNA molecule. (7) The molecule of claim (6) which is in the form of double-stranded DNA. (8) The molecule of claim (7) which is an expressible vehicle. (9) Claim No. (8) in which the vehicle is a plasmid
numerator of the term. (10) A prokaryotic host transformed with the molecule of claim (4). (11) The host according to claim (10), which is a bacterium. (12) A eukaryotic host transfected with the molecule according to claim (4). (13) The host according to claim (12), which is a yeast cell or a mammalian cell. (14) A chimeric immunoglobulin heavy chain comprising at least a portion of a human constant region and at least a portion of a variable region having specificity for an antigen that binds to the A10 mouse monoclonal antibody. (15) A chimeric immunoglobulin light chain comprising at least a portion of a human constant region and at least a portion of a variable region having specificity for an antigen that binds to the A10 mouse monoclonal antibody. (16) comprising two light chains and two heavy chains, each of which includes at least a portion of a human constant region and at least a portion of a variable region that has specificity for the antigen that binds to the A10 mouse monoclonal antibody; or a chimeric antibody fragment or derivative thereof. (17) Claim No. (16) in the form of a detectable label
Antibodies, fragments or derivatives of. (18) Claim No. (17) in which the label is a radionuclide
Antibodies, fragments or derivatives of. (19) The antibody, fragment, or derivative of claim (16) conjugated to a cytotoxic drug. (20) The antibody, fragment, or derivative of claim (16) that binds to a cytotoxic protein. (21) Claim No. 1 (1) immobilized on a water-insoluble solid layer
6) Antibodies, fragments or derivatives. (22) Production of a chimeric immunoglobulin heavy chain having at least a portion of a human constant region and at least a portion of a variable region having specificity for an antigen that binds to the A10 mouse monoclonal antibody, comprising the following steps: Law. (a) Cultivation of a host capable of expressing the heavy chain under culture conditions; (b) Expression of the heavy chain; and (c) Recovery of the heavy chain from the culture medium. (23) A method for producing a chimeric immunoglobulin light chain having at least a portion of a human constant region and at least a portion of a variable region having specificity for an antigen that binds to the A10 mouse monoclonal antibody, comprising the following step: . (a) Cultivation of a host capable of expressing the light chain under culture conditions; (b) Expression of the light chain; and (c) Recovery of the light chain from the culture medium. (24) containing a heavy chain and a light chain, comprising the steps of:
Each of the heavy chain and light chain comprises at least a portion of a human constant region,
and a method for producing a chimeric immunoglobulin, fragment or derivative having at least a portion of a variable region having specificity for an antigen that binds to the A10 mouse monoclonal antibody. : (a) Cultivation of a host capable of expressing the light chain under culture conditions, expression of the light chain, and recovery of the light chain from the culture medium; (b) Culture of a host capable of expressing the heavy chain under culture conditions. Cultivating the host separately, expressing the heavy chain, and recovering the heavy chain from the culture medium:
and (c) production of said chimeric immunoglobulin, fragment or derivative by association of said recovered heavy and light chains. (25) containing a heavy chain and a light chain, comprising the steps of:
Each of the heavy chain and light chain comprises at least a portion of a human constant region,
and a method for producing a chimeric immunoglobulin, fragment or derivative having at least a portion of a variable region having specificity for an antigen that binds to the A10 mouse monoclonal antibody. : (a) co-culture of a host capable of expressing the light chain and a host capable of expressing the heavy chain under culture conditions; (b) expression of the heavy chain and the light chain; (c) the heavy chain. and preparation of said chimeric immunoglobulin, fragment or derivative by association of said light chains: (d) recovery of said chimeric immunoglobulin, fragment or derivative from said culture medium. (26) containing a heavy chain and a light chain, comprising the steps of:
Each of the heavy chain and light chain comprises at least a portion of a human constant region,
and a method for producing a chimeric immunoglobulin, fragment or derivative having at least a portion of a variable region having specificity for an antigen that binds to the A10 mouse monoclonal antibody. (a) culturing a host capable of expressing the heavy chain and the light chain under culture conditions; (b) expressing the chimeric immunoglobulin, fragment or derivative; (c) culturing the chimeric immunoglobulin, fragment from the culture medium. or recovery of derivatives. (27) Claims Nos. (22) to 3, wherein the host is a prokaryote.
Any step of (26). (28) Claims Nos. (22) to 3, wherein the host is a eukaryote.
Any step of (26). (29) A method of immunoassay for detecting an antigen capable of binding to the A10 mouse monoclonal antibody in a sample comprising: (a) using the sample in claims (16), (17), (18);
), or (21); and (b) detecting the antigen by binding of the antibody, fragment, or derivative to the antigen. (30) An imaging method for detecting an antigen capable of binding to A10 mouse monoclonal antibody in an animal comprising: (a) a labeled antibody, fragment or derivative according to claim (17); contacting the animal; and (b) detecting the antigen. (31) A method for killing cells having an antigen capable of binding to the A10 mouse monoclonal antibody, comprising: (a) killing the cells according to claims (16) to (20); (b) causing said killing. (32) The method of claim (31), wherein said killing occurs by antibody-dependent cytotoxicity, complement-dependent cytotoxicity, radiocytotoxicity, cytotoxic drug action, or proteotoxic action. (33) Subjects suspected of having a tumor carrying an antigen that can bind to the A10 mouse monoclonal antibody,
A method of treating a subject comprising administering an effective amount of the antibody, fragment or derivative according to any one of claims (16) to (20).