U.S. patent application number 10/941768 was filed with the patent office on 2005-07-28 for chimeric antibody with specificity to human b cell surface antigen.
Invention is credited to Ledbetter, Jeffrey A., Liu, Alvin Y., Robinson, Randy R..
Application Number | 20050163708 10/941768 |
Document ID | / |
Family ID | 34577905 |
Filed Date | 2005-07-28 |
United States Patent
Application |
20050163708 |
Kind Code |
A1 |
Robinson, Randy R. ; et
al. |
July 28, 2005 |
Chimeric antibody with specificity to human B cell surface
antigen
Abstract
A chimeric antibody with human constant region and murine
variable region, having specificity to a 35 kDA polypeptide
(Bp35(CD20)) expressed on the surface of human B cells, methods of
production, and uses.
Inventors: |
Robinson, Randy R.; (Walnut
Creek, CA) ; Liu, Alvin Y.; (Seattle, WA) ;
Ledbetter, Jeffrey A.; (Seattle, WA) |
Correspondence
Address: |
GOODWIN PROCTER LLP
PATENT ADMINISTRATOR
53 STATE PLACE
BOSTON
MA
02109-2881
US
|
Family ID: |
34577905 |
Appl. No.: |
10/941768 |
Filed: |
September 15, 2004 |
Related U.S. Patent Documents
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Application
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Patent Number |
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10941768 |
Sep 15, 2004 |
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09630198 |
Aug 1, 2000 |
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6893625 |
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09630198 |
Aug 1, 2000 |
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09021934 |
Feb 12, 1998 |
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6120767 |
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09021934 |
Feb 12, 1998 |
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08471984 |
Jun 6, 1995 |
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5721108 |
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08471984 |
Jun 6, 1995 |
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07665939 |
Mar 5, 1991 |
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5500362 |
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07665939 |
Mar 5, 1991 |
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07195961 |
May 13, 1988 |
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07195961 |
May 13, 1988 |
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07016202 |
Jan 8, 1987 |
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07016202 |
Jan 8, 1987 |
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PCT/US86/02269 |
Oct 27, 1986 |
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Current U.S.
Class: |
424/1.49 ;
424/133.1; 424/9.6; 435/252.3; 435/320.1; 435/328; 435/69.1;
530/387.3; 536/23.53 |
Current CPC
Class: |
A61K 2039/505 20130101;
A61K 47/6849 20170801; C07K 2317/24 20130101; C07K 2319/40
20130101; C07K 2319/00 20130101; A61K 47/6877 20170801; C07K
2319/02 20130101; C07K 16/462 20130101; C07K 2317/732 20130101;
A61K 38/00 20130101; C07K 2319/30 20130101; C07K 16/2887 20130101;
C07K 2317/734 20130101 |
Class at
Publication: |
424/001.49 ;
424/009.6; 435/069.1; 435/328; 435/320.1; 435/252.3; 530/387.3;
424/133.1; 536/023.53 |
International
Class: |
A61K 051/00; C07H
021/04; C12P 021/04; A61K 039/395 |
Claims
What is new and intended to be covered by Letters Patent of the
United States is:
1. A polynucleotide molecule comprising a cDNA sequence coding for
the variable region of an immunoglobulin chain having specificity
to a 35-kDa polypeptide (Bp35(CD20)) expressed on the surface of B
cells.
2. The molecule of claim 1 wherein said chain is a heavy chain.
3. The molecule of claim 1 wherein said chain is a light chain.
4. The molecule of claim 1 which further comprises an additional
sequence coding for the constant C region of a human immunoglobulin
chain, both said sequences in operable linkage with each other.
5. The molecule of claim 4 wherein said additional sequence is a
cDNA sequence.
6. The molecule of claim 4 wherein said additional sequence is a
genomic sequence.
7. The molecule of claim 1 which is a recombinant DNA molecule.
8. The molecule of claim 7 which is in double-stranded DNA
form.
9. The molecule of claim 7 which is an expressible vehicle.
10. The molecule of claim 9 wherein said vehicle is a plasmid.
11. A prokaryotic host transformed with the molecule of claim
4.
12. The host of claim 11 which is a bacterium.
13. A eukaryotic host transfected with the molecule of claim 4.
14. The host of claim 13 which is yeast or a mammalian cell.
15. A heavy immunoglobulin chain comprising a constant human region
and a variable region having specificity to a 35 kDa polypeptide
(Bp35(CD20)) expressed on the surface of human B cells.
16. A light immunoglobulin chain comprising a constant human region
and a variable region having specificity to a 35 kDa polypeptide
(Bp35(CD20)) expressed on the surface of human B cells.
17. A chimeric antibody molecule comprising two light chains and
two heavy chains, each of said chains comprising a constant human
region and a variable region having specificity to a 35 kDa
polypeptide (Bp35(CD20)) expressed on the surface of human B
cells.
18. The antibody of claim 17 in detectably labelled form.
19. The antibody of claim 17 immobilized on an aqueous-insoluble
solid phase.
20. A process of preparing an immunoglobulin heavy chain having a
constant human region and a variable region having specificity to a
35 kDa polypeptide (Bp35(CD20)) expressed on the surface of human B
cells which comprises: culturing a host capable of expressing said
chain under culturing conditions and recovering from said culture
said heavy chain.
21. A process of preparing an immunoglobulin light chain having a
constant human region and a variable region with specificity to a
35 kDa polypeptide (Bp35(CD20)) expressed on the surface of human B
cells which comprises: culturing a host capable of expressing said
chain under culturing conditions; and recovering from said culture
said light chain.
22. A process of preparing a chimeric immunoglobulin containing a
heavy chain and a light chain, each of said heavy and light chains
having a constant human region and a variable region with
specificity to a 35 kDa polypeptide (Bp35(CD20)) expressed on the
surface of human B cells which comprises: culturing a host capable
of expressing said heavy chain, or said light chain, or both, under
culturing conditions; and recovering from said culture said
chimeric immunoglobulin molecule.
23. The process of any of claims 20, 21 or 22 wherein said host is
prokaryotic.
24. The process of any of claims 20, 21 or 22 wherein said host is
eukaryotic.
25. An immunoassay method for the detection of a 35 kDa polypeptide
normally expressed on the surface of B cells in a sample, which
comprises: contacting said sample with the antibody of claim 17 and
detecting whether said antibody binds to said antigen.
26. An in vivo or in vitro imaging method to detect an antigen
comprising a 35 kDa polypeptide normally expressed on the surface
of `B ` cells which comprises contacting said antigen with the
labelled antibody of claim 18 and detecting said antibody.
27. A method of killing cells carrying an antigen thereon, which
antigen comprising a 35 kDa polypeptide normally expressed on the
surface of B cells which comprises: contacting said cells with the
antibody of claim 17.
28. The method of claim 27 wherein said killing occurs by
complement mediated lysis of said cells.
29. The method of claim 27 wherein said killing occurs by ADCC.
Description
[0001] This application is a continuation in part of U.S.
application Ser. No. ______, International Application No.
PCT/US86/02269, filed Oct. 27, 1986, in the PCT receiving office of
the U.S.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] This invention relates to recombinant DNA methods of
preparing an antibody with specificity for an antigen on the
surface of human B cells, genetic sequences coding therefor, as
well as methods of obtaining such sequences.
[0004] 2. Background Art
[0005] The application of cell-to-cell fusion for the production of
monoclonal antibodies by Kohler and Milstein (Nature (London), 256:
495, 1975) spawned a revolution in biology equal in impact to that
from the invention of recombinant DNA cloning. Monoclonal
antibodies produced from hybridomas are already widely used in
clinical and basic scientific studies. Applications of human
monoclonal antibodies produced by human hybridomas hold great
promise for the treatment of cancer, viral and microbial
infections, certain immunodeficiencies with diminished antibody
production, and other diseases and disorders of the immune
system.
[0006] Unfortunately, a number of obstacles exist with respect to
the development of human monoclonal antibodies. This is especially
true when attempting to develop therapeutically useful monoclonal
antibodies which define human cell surface antigens. Many of these
human cell surface antigens are not recognized as foreign antigens
by the human immune system; therefore, these antigens are not
immunogenic in man. By contrast, human cellular antigens which are
immunogenic in mice can be used for the production of mouse
monoclonal antibodies that specifically recognize the human
antigens. Although such antibodies may be used therapeutically in
man, repeated injections of "foreign" antibodies, such as a mouse
antibody, in humans, can lead to harmful hypersensitivity reactions
as well as increased rate of clearance of the circulating antibody
molecules so that the antibodies do not reach their target site.
Furthermore, mouse monoclonal antibodies may lack the ability to
efficiently interact with human effector cells as assessed by
functional assays such as antibody-dependent cellular cytotoxicity
(ADCC) and complement-mediated cytolysis (CDC).
[0007] Another problem faced by immunologists is that most human
monoclonal antibodies obtained in cell culture are of the IgM type.
When it is desirable to obtain human monoclonals of the IgG type,
however, it has been necessary to use such techniques as cell
sorting to identify and isolate the few cells which are producing
antibodies of the IgG or other type from the majority producing
antibodies of the IgM type. A need therefore exists for an
efficient method of switching antibody classes, for any given
antibody of a predetermined or desired antigenic specificity.
[0008] The present invention bridges both the hybridoma and genetic
engineering technologies to provide a quick and efficient method,
as well as products derived therefrom, for the production of a
chimeric human/non-human antibody.
[0009] The chimeric antibodies of the present invention embody a
combination of the advantageous characteristics of monoclonal
antibodies derived from mouse-mouse hybridomas and of human
monoclonal antibodies. The chimeric monoclonal antibodies, like
mouse monoclonal antibodies, can recognize and bind to a human
target antigen; however, unlike mouse monoclonal antibodies, the
species-specific properties of the chimeric antibodies will avoid
the induction of harmful hypersensitivity reactions and may allow
for resistance to clearance when used in humans in vivo. Also, the
inclusion of appropriate human immunoglobulin sequences can result
in an antibody which efficiently interacts with human effector
cells in vivo to cause tumor cell lysis and the like. Moreover,
using the methods disclosed in the present invention, any desired
antibody isotype can be conferred upon a particular antigen
combining site.
Information Disclosure Statement*
[0010] Approaches to the problem of producing chimeric antibodies
have been published by various authors.
[0011] Morrison, S. L. et al., Proc. Natl. Acad. Sci., USA, 81:
6851-6855 (November 1984), describe the production of a mouse-human
antibody molecule of defined antigen binding specificity, produced
by joining the variable region genes of a mouse antibody-producing
myeloma cell line with known antigen binding specificity to human
immunoglobulin constant region genes using recombinant DNA
techniques. Chimeric genes were constructed, wherein the heavy
chain variable region exon from the myeloma cell line S107 well
joined to human IgG1 or IgG2 heavy chain constant region exons, and
the light chain variable region exon from the same myeloma to the
human kappa light chain exon. These genes were transfected into
mouse myeloma cell lines and. Transformed cells producing chimeric
mouse-human antiphosphocholine antibodies were thus developed.
[0012] Morrison, S. L. et al., European Patent Publication No.
173494 (published Mar. 5, 1986), disclose chimeric "receptors"
(e.g. antibodies) having variable regions derived from one species
and constant regions derived from another. Mention is made of
utilizing cDNA cloning to construct the genes, although no details
of cDNA cloning or priming are shown. (see pp 5, 7 and 8). * Note:
The present Information Disclosure Statement is subject to the
provisions of 37 C.F.R. 1.97(b). In addition, Applicants reserve
the right to demonstrate that their invention was made prior to any
one or more of the mentioned publications.
[0013] Boulianne, G. L. et al., Nature, 312: 643 (December 13,
1984), also produced antibodies consisting of mouse variable
regions joined to human constant regions. They constructed
immunoglobulin genes in which the DNA segments encoding mouse
variable regions specific for the hapten trinitrophenyl (TNP) were
joined to segments encoding human mu and kappa constant regions.
These chimeric genes were expressed as functional TNP binding
chimeric IgM. For a commentary on the work of Boulianne et al. and
Morrison et al., see Munro, Nature, 312: 597 (Dec. 13, 1984),
Dickson, Genetic Engineerinq News, 5, No. 3 (March 1985), or Marx,
Science, 229: 455 (August 1985).
[0014] Neuberger, M. S. et al., Nature, 314: 268 (Mar. 25, 1985),
also constructed a chimeric heavy chain immunoglobulin gene in
which a DNA segment encoding a mouse variable region specific for
the hapten 4-hydroxy-3-nitrophenacetyl (NP) was joined to a segment
encoding the human epsilon region. When this chimeric gene was
transfected into the J558L cell line, an antibody was produced
which bound to the NP hapten and had human IgE properties.
[0015] Neuberger, M. S. et al., have also published work showing
the preparation of cell lines that secrete hapten-specific
antibodies in which the Fc portion has been replaced either with an
active enzyme moiety (Williams, G. and Neuberger, M. S. Gene 43:
319, 1986) or with a polypeptide displaying c-myc antigenic
determinants (Nature, 312: 604, 1984).
[0016] Neuberger, M. et al., PCT Publication WO 86/01533,
(published Mar. 13, 1986) also disclose production of chimeric
antibodies (see p. 5) and suggests, among the technique's many uses
the concept of "class switching" (see p. 6).
[0017] Taniguchi, M., in European Patent Publication No. 171 496
(published Feb. 19, 1985) discloses the production of chimeric
antibodies having variable regions with tumor specificty derived
from experimental animals, and constant regions derived from human.
The corresponding heavy and light chain genes are produced in the
genomic form, and expressed in mammalian cells.
[0018] Takeda, S. et al., Nature, 314: 452 (Apr. 4, 1985) have
described a potential method for the construction of chimeric
immunoglobulin genes which have intron sequences removed by the use
of a retrovirus vector. However, an unexpected splice donor site
caused the deletion of the V region leader sequence. Thus, this
approach did not yield complete chimeric antibody molecules.
[0019] Cabilly, S. et al., Proc. Natl. Acad. Sci., USA, 81:
3273-3277 (June 1984), describe plasmids that direct the synthesis
in E. coli of heavy chains and/or light chains of
anti-carcinoembryonic antigen (CEA) antibody. Another plasmid was
constructed for expression of a truncated form of heavy chain (Fd')
fragment in E. coli. Functional CEA-binding activity was obtained
by in vitro reconstitution, in E. coli extracts, of a portion of
the heavy chain with light chain.
[0020] Cabilly, S., et al., European Patent Publication 125023
(published Nov. 14, 1984) describes chimeric immunoglobulin genes
and their presumptive products as well as other modified forms. On
pages 21, 28 and 33 it discusses cDNA cloning and priming.
[0021] Boss, M. A., European Patent Application 120694 (published
Oct. 3, 1984) shows expression in E. coli of non-chimeric
immunoglobulin chains with 4-nitrophenyl specificity. There is a
broad description of chimeric antibodies but no details (see p.
9).
[0022] Wood, C. R. et al., Nature, 314: 446 (April, 1985) describe
plasmids that direct the synthesis of mouse anti-NP antibody
proteins in yeast. Heavy chain mu antibody proteins appeared to be
glycosylated in the yeast cells. When both heavy and light chains
were synthesized in the same cell, some of the protein was
assembled into functional antibody molecules, as detected by
anti-NP binding activity in soluble protein prepared from yeast
cells.
[0023] Alexander, A. et al., Proc. Nat. Acad. Sci. USA, 79:
3260-3264 (1982), describe the preparation of a cDNA sequence
coding for an abnormally short human Ig gamma heavy chain (OMM
gamma.sup.3 HCD serum protein) containing a 19-amino acid leader
followed by the first 15 residues of the V region. An extensive
internal deletion removes the remainder of the V and the entire
C.sub.H1 domain. This is cDNA coding for an internally deleted
molecule.
[0024] Dolby, T. W. et al., Proc. Natl. Acad. Sci., USA, 77:
6027-6031 (1980), describe the preparation of a cDNA sequence and
recombinant plasmids containing the same coding for mu and kappa
human immunoglobulin polypeptides. One of the recombinant DNA
molecules contained codons for part of the CH.sub.3 constant region
domain and the entire 3' noncoding sequence.
[0025] Seno, M. et al., Nucleic Acids Research, 11: 719-726 (1983),
describe the preparation of a cDNA sequence and recombinant
plasmids containing the same coding for part of the variable region
and all of the constant region of the human IgE heavy chain
(epsilon chain).
[0026] Kurokawa, T. et al., ibid, 11: 3077-3085 (1983), show the
construction, using cDNA, of three expression plasmids coding for
the constant portion of the human IgE heavy chain.
[0027] Liu, F. T. et al., Proc. Nat. Acad. Sci., USA, 81: 5369-5373
(September 1984), describe the preparation of a cDNA sequence and
recombinant plasmids containing the same encoding about two-thirds
of the CH.sub.2, and all of the C.sub.H3 and C.sub.H4 domains of
human IgE heavy chain.
[0028] Tsujimoto, Y. et al., Nucleic Acids Res., 12: 8407-8414
(November 1984), describe the preparation of a human V lambda cDNA
sequence from an Ig lambda-producing human Burkitt lymphoma cell
line, by taking advantage of a cloned constant region gene as a
primer for cDNA synthesis.
[0029] Murphy, J., PCT Publication WO 83/03971 (published Nov. 24,
1983) discloses hybrid proteins made of fragments comprising a
toxin and a cell-specific ligand (which is suggested as possibly
being an antibody).
[0030] Tan, et al., J. Immunol. 135: 8564 (November, 1985),
obtained expression of a chimeric human-mouse immunoglobulin
genomic gene after transfection into mouse myeloma cells.
[0031] Jones, P. T., et al., Nature 321: 552 (May 1986) constructed
and expressed a genomic construct where CDR domains of variable
regions from a mouse monolonal antibody were used to substitute for
the corresponding domains in a human antibody.
[0032] Sun, L. K., et al., Hybridoma 5 suppl. 1 S17 (1986),
describes a chimeric human/mouse antibody with potential tumor
specificty. The chimeric heavy and light chain genes are genomic
constructs and expressed in mammalian cells.
[0033] Sahagan et al., J. Immun. 137-1066-1074 (August 1986)
describe a chimeric antibody with specificity to a human tumor
associated antigen, the genes for which are assembled from genomic
sequences.
[0034] For a recent review of the field see also Morrison, S. L.,
Science 229: 1202-1207 (Sep. 20, 1985) and Oi, V. T., et al.,
BioTechniques 4: 214 (1986).
[0035] The Oi, et al., paper is relevant as it argues that the
production of chimeric antibodies from cDNA constructs in yeast
and/or bacteria is not necessarily advantageous.
[0036] See also Commentary on page 835 in Biotechnology 4
(1986).
SUMMARY OF THE INVENTION
[0037] The invention provides a genetically engineered chimeric
antibody of desired variable region specificity and constant region
properties, through gene cloning and expression of light and heavy
chains. The cloned immunoglobulin gene products can be produced by
expression in genetically engineered cells.
[0038] The application of oligodeoxyribonucleotide synthesis,
recombinant DNA cloning, and production of specific immunoglobulin
chains in various prokaryotic and eukaryotic cells provides a means
for the large scale production of a chimeric human/mouse monoclonal
antibody with specificity to a human B cell surface antigen.
[0039] The invention provides cDNA sequences coding for
immunoglobulin chains comprising a constant human region and a
variable, non-human, region. The immunoglobulin chains can be
either heavy or light.
[0040] The invention provides gene sequences coding for
immunoglobulin chains comprising a cDNA variable region of the
desired specificity. These can be combined with genomic constant
regions of human origin.
[0041] The invention provides sequences as above, present in
recombinant DNA molecules in vehicles such as plasmid vectors,
capable of expression in desired prokaryotic or eukaryotic
hosts.
[0042] The invention provides hosts capable of producing, by
culture, the chimeric antibodies and methods of using these
hosts.
[0043] The invention also provides individual chimeric
immunoglobulin individual chains, as well as complete assembled
molecules having human constant regions and variable regions with a
human B cell surface antigen specificity, wherein both variable
regions have the same binding specificity.
[0044] Among other immunoglobulin chains and/or molecules provided
by the invention are:
[0045] (a) a complete functional, immunoglobulin molecule
comprising:
[0046] (i) two identical chimeric heavy chains comprising a
variable region with a human B cell surface antigen specificity and
human constant region and
[0047] (ii) two identical all (i.e. non-chimeric) human light
chains.
[0048] (b) a complete, functional, immunoglobulin molecule
comprising:
[0049] (i) two identical chimeric heavy chains comprising a
variable region as indicated, and a human constant region, and
[0050] (ii) two identical all (i.e. non-chimeric) non-human light
chains.
[0051] (c) a monovalent antibody, i.e., a complete, functional
immunoglobulin molecule comprising:
[0052] (i) two identical chimeric heavy chains comprising a
variable region as indicated, and a human constant region, and
[0053] (ii) two different light chains, only one of which has the
same specificity as the variable region of the heavy chains. The
resulting antibody molecule binds only to one end thereof and is
therefore incapable of divalent binding.
[0054] Genetic sequences, especially cDNA sequences, coding for the
aforementioned combinations of chimeric chains or of non-chimeric
chains are also provided herein.
[0055] The invention also provides for a genetic sequence,
especially a cDNA sequence, coding for the variable region of
desired specificity of an antibody molecule heavy and/or light
chain, operably linked to a sequence coding for a polypeptide
different than an immunoglobulin chain (e.g., an enzyme). These
sequences can be assembled by the methods of the invention, and
expressed to yield mixed-function molecules.
[0056] The use of cDNA sequences is particularly advantageous over
genomic sequences (which contain introns), in that cDNA sequences
can be expressed in bacteria or other hosts which lack appropriate
RNA splicing systems.
BRIEF DESCRIPTION OF THE FIGURES
[0057] FIG. 1 shows the DNA rearrangements and the expression of
immunoglobulin mu and gamma heavy chain genes. This is a schematic
representation of the human heavy chain gene complex (not shown to
scale). Heavy chain variable V region formation occurs through the
proper joining of V.sub.H, D and J.sub.H gene segments. This
generates an active mu gene. A different kind of DNA rearrangement
called "class switching" relocates the joined V.sub.H, D and
J.sub.H region from the vicinity of mu constant C region to that of
another heavy chain C region (switching to gamma is diagrammed
here).
[0058] FIG. 2 shows the known nucleotide sequences of human and
mouse J regions. Consensus sequences for the J regions are shown
below the actual sequences. The oligonucleotide sequence below the
mouse kappa J region consensus sequence is a Universal
Immunoglobulin Gene (UIG) oligonucleotide. Note that there are only
a few J regions with relatively conserved sequences, especially
near the constant regions, in each immunoglobulin gene locus.
[0059] FIG. 3 shows the nucleotide sequences of the mouse J
regions. Shown below are the oligonucleotide primers UIG-H and
UIG-K. Note that each contains a restriction enzyme site. They can
be used as primers for the synthesis of cDNA complementary to the
variable region of mRNA, and can also be used to mutagenize, in
vitro, cloned cDNA.
[0060] FIG. 4 Human Constant Domain Module. The human C gamma 1
clone, pGMH6, was isolated from the cell line GM2146. The sequence
at its J.sub.H-C.sub.H1 junction is shown. Two restriction enzyme
sites are useful as joints in recombining the C.sub.H1 gene with
different V.sub.H genes. The ApaI site is 16 nucleotide residues
into the C.sub.H1 coding domain of Human gamma 1; and is used in a
previous construction of a mouse-human chimeric heavy-chain
immunoglobulin. The BstEII site is in the J.sub.H region, and is
used in the construction described in this application.
[0061] The human C.sub.K clone, pGML60, is a composite of two cDNA
clones, one isolated from GM1500 (pK2-3), the other from GM2146
(pGML1). The J.sub.K-C.sub.K junction sequence shown comes from
pK2-3. In vitro mutagenesis using the oligonucleotide,
J.sub.KHindIII, was carried out to engineer a HindIII site 14
nucleotide residues 5' of the J-C junction. This changes a human
GTG codon into a CTT codon.
[0062] FIG. 5 shows the nucleotide sequence of the V region of the
2H7 V.sub.H cDNA clone pH2-11. The sequence was determined by the
dideoxytermination method using M13 subclones of gene fragments.
Open circles denote amino acid residues confirmed by peptide
sequence. A sequence homologous to D.sub.SP.2 in the CDR3 region is
underlined. The NcoI site at 5' end was converted to a SalI site by
using SalI linkers.
[0063] FIG. 6 shows the nucleotide sequence of the V region of the
2H7 V.sub.K cDNA clone pL2-12. The oligonucleotide primer used for
site-directed mutagenesis is shown below the J.sub.K5 segment. Open
circles denote amino acid residues confirmed by peptide
sequence.
[0064] FIG. 7 shows the construction of the light and heavy chain
expression plasmids pING2106 (panel a) and pING2101 (panel B). The
SalI to BamHI fragment from pING2100 is identical to the SalI to
BamHI fragment from pING2012E (see panel C). A linear
representation of the circular plasmid pING2012E is shown in panel
C. The 6.6 Kb SalI to BamHI fragment contains sequences from
pSV2-neo, puc12, M8alphaRX12, and pL1. The HindIII site in pSV2-neo
was destroyed before assembly of pING2012E by HindIII cleavage,
fill-in, and religation.
[0065] FIG. 8 shows the structure of several chimeric 2H7-V.sub.H
expression plasmids. pING2107 is a qpt version of the light chain
plasmid, pING2106. The larger ones are two-gene plasmids. pHL2-11
and pHL2-26 contain both H and L genes, while pLL2-25 contains two
L genes. They were constructed by joining an NdeI fragment
containing either an H or L gene to partially digested (with NdeI)
pING2106.
[0066] FIG. 9 shows a summary of the sequence alterations made in
the construction of the 2H7 chimeric antibody expression plasmids.
Residues underlined in the 5' untranslated region are derived from
the cloned mouse kappa and heavy-chain genes. Residues circled in
the V/C boundary result from mutagenesis operations to engineer
restriction enzyme sites in this region.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
Introduction
[0067] Generally, antibodies are composed of two light and two
heavy chain molecules. Light and heavy chains are divided into
domains of structural and functional homology. The variable domains
of both the light (V.sub.L) and the heavy (V.sub.H) chains
determine recognition and specificity. The constant region domains
of light (C.sub.L) and heavy (C.sub.H) chains confer important
biological properties such as antibody chain association,
secretion, transplacental mobility, complement binding, and the
like.
[0068] A complex series of events leads to immunoglobulin gene
expression in the antibody producing cells. The V region gene
sequences conferring antigen specificity and binding are located in
separate germ line gene segments called V.sub.H, D and J.sub.H; or
V.sub.L and J.sub.L. These gene segments are joined by DNA
rearrangements to form the complete V regions expressed in heavy
and light chains respectively (FIG. 1). The rearranged, joined
(V.sub.L-J.sub.L and V.sub.H-D-J.sub.H)V segments then encode the
complete variable regions or antigen binding domains of light and
heavy chains, respectively.
Definitions
[0069] Certain terms and phrases are used throughout the
specification and claims. The following definitions are provided
for purposes of clarity and consistency.
[0070] 1. Expression vector--a plasmid DNA containing necessary
regulatory signals for the synthesis of mRNA derived from any gene
sequence, inserted into the vector.
[0071] 2. Module vector--a plasmid DNA containing a constant or
variable region gene module.
[0072] 3. Expression plasmid--an expression vector that contains an
inserted gene, such as a chimeric immunoglobulin gene.
[0073] 4. Gene cloning--synthesis of a gene, insertion into DNA
vectors, identification by hybridization, sequence analysis and the
like.
[0074] 5. Transfection--the transfer of DNA into mammalian
cells.
Genetic Processes and Products
[0075] The invention provides a novel approach for the cloning and
production of a human/mouse chimeric antibody with specificity to a
human B cell surface antigen. The antigen is a polypeptide or
comprises a polypeptide bound by the 2H7 monoclonal antibody
described in Clark et al. Proc. Natl. Acad. Sci., U.S.A. 82:
1766-1770 (1985). This antigen is a phosphoprotein designated
(Bp35(CD20)) and is only expressed on cells of the B cell lineage.
Murine monoclonal antibodies to this antigen have been made before
and are described in Clark et al., supra; see also Stashenko, P.,
et al., J. Immunol. 125: 1678-1685 (1980).
[0076] The method of production combines five elements:
[0077] (1) Isolation of messenger RNA (mRNA) from the mouse
hybridoma line producing the monoclonal antibody, cloning and cDNA
production therefrom;
[0078] (2) Preparation of Universal Immunoglobulin Gene (UIG)
oligonucleotides, useful as primers and/or probes for cloning of
the variable region gene segments in the light and heavy chain mRNA
from the hybridoma cell line, and cDNA production therefrom;
[0079] (3) Preparation of constant region gene segment modules by
cDNA preparation and cloning, or genomic gene preparation and
cloning;
[0080] (4) Construction of complete heavy or light chain coding
sequences by linkage of the cloned specific immunoglobulin variable
region gene segments of part (2) above to cloned human constant
region gene segment modules;
[0081] (5) Expression and production of light and heavy chains in
selected hosts, including prokaryotic and eukaryotic cells, either
in separate fermentations followed by assembly of antibody
molecules in vitro, or through production of both chains in the
same cell.
[0082] One common feature of all immunoglobulin light and heavy
chain genes and the encoded messenger RNAs is the so-called J
region (i.e. joining region, see FIG. 1). Heavy and light chain J
regions have different sequences, but a high degree of sequence
homology exists (greater than 80%) especially near the constant
region, within the heavy J.sub.H regions or the kappa light chain J
regions. This homology is exploited in this invention and consensus
sequences of light and heavy chain J regions were used to design
oligonucleotides (designated herein as UIGs) for use as primers or
probes for cloning immunoglobulin light or heavy chain mRNAs or
genes (FIG. 3). Depending on the sequence of a particular UIG, it
may be capable of hybridizing to all immunoglobulin mRNAs or a
specific one containing a particular J sequence. Another utility of
a particular UIG probe may be hybridization to light chain or heavy
chain mRNAs of a specific constant region, such as UIG-MJK which
detects all mouse J.sub.K-containing sequences (FIG. 2).
[0083] UIG design can also include a sequence to introduce a
restriction enzyme site into the cDNA copy of an immunoglobulin
gene (see FIG. 3). The invention may, for example, utilize chemical
gene synthesis to generate the UIG probes for the cloning and
modification of V regions from immunoglobulin mRNA. On the other
hand, oligonucleotides can be synthesized to recognize
individually, the less conserved 5'-region of the J regions as a
diagnostic aid in identifying the particular J region present in
the immunoglobulin mRNA.
[0084] A multi-step procedure is utilized for generating complete
V+C region cDNA clones from the hybridoma cell light and heavy
chain mRNAs. First, the complementary strand of oligodT-primed cDNA
is synthesized, and this double-stranded cDNA is cloned in
appropriate cDNA cloning vectors such as pBR322 (Gubler and
Hoffman, Gene, 25: 263 (1983)). Clones are screened by
hybridization with UIG oligonucleotide probes. Positive heavy and
light chain clones identified by this screening procedure are
mapped and sequenced to select those containing V region and leader
coding sequences. In vitro mutagenesis including, for example, the
use of UIG oligonucleotides, is then used to engineer desired
restriction enzyme cleavage sites. We used this approach for the
chimeric 2H7 light chain.
[0085] An expedient method is to use synthetic UIG oligonucleotides
as primers for the synthesis of cDNA. This method has the advantage
of simultaneously introducing a desired restriction enzyme site,
such as BstEII (FIG. 3) into a V region cDNA clone. We used this
approach for the chimeric 2H7 heavy chain.
[0086] Second, cDNA constant region module vectors are prepared
from human cells. These cDNA clones are modified, when necessary,
by site-directed mutagenesis to place a restriction site at the
analogous position in the human sequence or at another desired
location near a boundary of the constant region. An alternative
method utilizes genomic C region clones as the source for C region
module vectors.
[0087] Third, cloned V region segments generated as above are
excised and ligated to light or heavy chain C region module
vectors. For example, one can clone the complete human kappa light
chain C region and the complete human gamma.sub.1 C region. In
addition, one can modify the human gamma.sub.1 region to introduce
a termination codon and thereby obtain a gene sequence which
encodes the heavy chain portion of an Fab molecule.
[0088] The coding sequences having operationally linked V and C
regions are then transferred into appropriate expression vehicles
for expression in appropriate hosts, prokaryotic or eukaryotic.
Operationally linked means in-frame joining of coding sequences to
derive a continuously translatable gene sequence without
alterations or interruptions of the triplet reading frame.
[0089] One particular advantage of using cDNA genetic sequences in
the present invention is the fact that they code continuously for
immunoglobulin chains, either heavy or light. By "continuously" is
meant that the sequences do not contain introns (i.e. are not
genomic sequences, but rather, since derived from mRNA by reverse
transcription, are sequences of contiguous exons). This
characteristic of the cDNA sequences provided by the invention
allows them to be expressible in prokaryotic hosts, such as
bacteria, or in lower eukaryotic hosts, such as yeast.
[0090] Another advantage of using cDNA cloning method is the ease
and simplicity of obtaining variable region gene modules.
[0091] The terms "constant" and "variable" are used functionally to
denote those regions of the immunoglobulin chain, either heavy or
light chain, which code for properties and features possessed by
the variable and constant regions in natural non-chimeric
antibodies. As noted, it is not necessary for the complete coding
region for variable or constant regions to be present, as long as a
functionally operating region is present and available.
[0092] Expression vehicles include plasmids or other vectors.
Preferred among these are vehicles carrying a functionally complete
human constant heavy or light chain sequence having appropriate
restriction sites engineered so that any variable-heavy or light
chain sequence with appropriate cohesive ends can be easily
inserted thereinto. Human constant heavy or light chain
sequence-containing vehicles are thus an important embodiment of
the invention. These vehicles can be used as intermediates for the
expression of any desired complete heavy or light chain in any
appropriate host.
[0093] One preferred host is yeast. Yeast provides substantial
advantages for the production of immunoglobulin light and heavy
chains. Yeasts carry out post-translational peptide modifications
including glycosylation. A number of recombinant DNA strategies now
exist which utilize strong promoter sequences and high copy number
plasmids which can be used for overt production of the desired
proteins in yeast. Yeast recognizes leader sequences on cloned
mammalian gene products and secretes peptides bearing leader
sequences (i.e. prepeptides) (Hitzman, et al., 11th International
Conference on Yeast, Genetics and Molecular Biology, Montpelier,
France, Sep. 13-17, 1982).
[0094] Yeast gene expression systems can be routinely evaluated for
the level of heavy and light chain production, protein stability,
and secretion. Any of a series of yeast gene expression systems
incorporating promoter and termination elements from the actively
expressed genes coding for glycolytic enzymes produced in large
quantities when yeasts are grown in mediums rich in glucose can be
utilized. Known glycolytic genes can also provide very efficient
transcription control signals. For example, the promoter and
terminator signals of the iso-1-cytochrome C (CYC-1) gene can be
utilized.
[0095] The following approach can be taken to develop and evaluate
optimal expression plasmids for the expression of cloned
immunoglobulin cDNAs in yeast.
[0096] (1) The cloned immunoglobulin DNA linking V and C regions is
attached to different transcription promoters and terminator DNA
fragments;
[0097] (2) The chimeric genes are placed on yeast plasmids (see,
for example, Broach, J. R. in Methods in Enzymology--Vol. 101: 307
ed. Wu, R. et al., 1983));
[0098] (3) Additional genetic units such as a yeast leader peptide
may be included on immunoglobulin DNA constructs to obtain antibody
secretion.
[0099] (4) A portion of the sequence, frequently the first 6 to 20
codons of the gene sequence may be modified to represent preferred
yeast codon usage.
[0100] (5) The chimeric genes are placed on plasmids used for
integration into yeast chromosomes.
[0101] The following approaches can be taken to simultaneously
express both light and heavy chain genes in yeast.
[0102] (1) The light and heavy chain genes are each attached to a
yeast promoter and a terminator sequence and placed on the same
plasmid. This plasmid can be designed for either autonomous
replication in yeast or integration at specific sites in the yeast
chromosome.
[0103] (2) The light and heavy chain genes are each attached to a
yeast promoter and terminator sequence on separate plasmids
containing different selectable markers. For example, the light
chain gene can be placed on a plasmid containing the trp1 gene as a
selectable marker, while the heavy chain gene can be placed on a
plasmid containing ura3 as a selectable marker. The plasmids can be
designed for either autonomous replication in yeast or integration
at specific sites in yeast chromosomes. A yeast strain defective
for both selectable markers is either simultaneously or
sequentially transformed with the plasmid containing the light
chain gene and with the plasmid containing the heavy chain
gene.
[0104] (3) The light and heavy chain genes are each attached to a
yeast promoter and terminator sequence on separate plasmids each
containing different selectable markers as described in (2) above.
A yeast mating type "a" strain defective in the selectable markers
found on the light and heavy chain expression plasmids (trp1 and
ura3 in the above example) is transformed with the plasmid
containing the light chain gene by selection for one of the two
selectable markers (trp1 in the above example). A yeast mating type
"alpha" strain defective in the same selectable markers as the "a"
strain (i.e. trp1 and ura3 as examples) is transformed with a
plasmid containing the heavy chain gene by selection for the
alternate selectable marker (i.e. ura3 in the above example). The
"a" strain containing the light chain plasmid (phenotype:
Trp.sup.+Ura.sup.- in the above example) and the strain containing
the heavy chain plasmid (phenotype: Trp.sup.-Ura.sup.+ in the above
example) are mated and diploids are selected which are prototrophic
for both of the above selectable markers (Trp.sup.+Ura.sup.+ in the
above example).
[0105] Among bacterial hosts which may be utilized as
transformation hosts, E. coli K12 strain 294 (ATCC 31446) is
particularly useful. Other microbial strains which may be used
include E. coli X1776 (ATCC 31537). The aforementioned strains, as
well as E. coli W3110 (ATCC 27325) and other enterobacteria such as
Salmonella typhimurium or Serratia marcescens, and various
Pseudomonas species may be used.
[0106] In general, plasmid vectors containing replicon and control
sequences which are derived from species compatible with a host
cell are used in connection with these hosts. The vector ordinarily
carries a replication site, as well as specific genes which are
capable of providing phenotypic selection in transformed cells. For
example, E. coli is readily transformed using pBR322, a plasmid
derived from an E. coli species (Bolivar, et al., Gene, 2: 95
(1977)). pBR322 contains genes for ampicillin and tetracycline
resistance, and thus provides easy means for identifying
transformed cells. The pBR322 plasmid or other microbial plasmids
must also contain, or be modified to contain, promoters which can
be used by the microbial organism for expression of its own
proteins. Those promoters most commonly used in recombinant DNA
construction include the beta-lactamase (penicillinase) and lactose
(beta-galactosidase) promoter systems (Chang et al., Nature, 275:
615 (1978); Itakura et al., Science, 198: 1056 (1977)); and
tryptophan promoter systems (Goeddel et al., Nucleic Acids
Research, 8: 4057 (1980); EPO Publication No. 0036776). While these
are the most commonly used, other microbial promoters have been
discovered and utilized.
[0107] For example, a genetic construct for any heavy or light
chimeric immunoglobulin chain can be placed under the control of
the leftward promoter of bacteriophage lambda (P.sub.L). This
promoter is one of the strongest known promoters which can be
controlled. Control is exerted by the lambda repressor, and
adjacent restriction sites are known.
[0108] The expression of the immunoglobulin chain sequence can also
be placed under control of other regulatory sequences which may be
"homologous" to the organism in its untransformed state. For
example, lactose dependent E. coli chromosomal DNA comprises a
lactose or lac operon which mediates lactose digestion by
elaborating the enzyme beta-galactosidase. The lac control elements
may be obtained from bacteriophage lambda pLAC5, which is infective
for E. coli. The lac promoter-operator system can be induced by
IPTG.
[0109] Other promoter/operator systems or portions thereof can be
employed as well. For example, arabinose, colicine E1, galactose,
alkaline phosphatase, tryptophan, xylose, tac, and the like can be
used.
[0110] Other preferred hosts are mammalian cells, grown in vitro in
tissue culture, or in vivo in animals. Mammalian cells provide
post-translational modifications to immunoglobulin protein
molecules including leader peptide removal, correct folding and
assembly of heavy and light chains, proper glycosylation at correct
sites, and secretion of functional antibody protein.
[0111] Mammalian cells which may be useful as hosts for the
production of antibody proteins include cells of lymphoid origin,
such as the hybridoma Sp2/0-Ag14 (ATCC CRL 1581) or the myleoma
P3X63Ag8 (ATCC TIB 9), and its derivatives. Others include cells of
fibroblast origin, such as Vero (ATCC CRL 81) or CHO-K1 (ATCC CRL
61).
[0112] Several possible vector systems are available for the
expression of cloned heavy chain and light chain genes in mammalian
cells. One class of vectors relies upon the integration of the
desired gene sequences into the host cell genome. Cells which have
stably integrated DNA can be selected by simultaneously introducing
drug resistance genes such as E. coli gpt (Mulligan, R. C. and
Berg, P., Proc. Natl. Acad. Sci., USA, 78: 2072 (1981)) or Tn5 neo
(Southern, P. J. and Berg, P., J. Mol. Appl. Genet., 1: 327
(1982)). The selectable marker gene can be either linked to the DNA
gene sequences to be expressed, or introduced into the same cell by
co-transfection (Wigler, M. et al., Cell, 16: 77 (1979)). A second
class of vectors utilizes DNA elements which confer autonomously
replicating capabilities to an extrachromosomal plasmid. These
vectors can be derived from animal viruses, such as bovine
papillomavirus (Sarver, N. et al., Proc. Natl. Acad. Sci., USA, 79:
7147 (1982)), polyoma virus (Deans, R. J. et al., Proc. Natl. Acad.
Sci., USA, 81: 1292 (1984)), or SV40 virus (Lusky, M. and Botchan,
M., Nature, 293: 79 (1981)).
[0113] Since an immunoglobulin cDNA is comprised only of sequences
representing the mature mRNA encoding an antibody protein
additional gene expression elements regulating transcription of the
gene and processing of the RNA are required for the synthesis of
immunoglobulin mRNA. These elements may include splice signals,
transcription promoters, including inducible promoters enhancers,
and termination signals. cDNA expression vectors incorporating such
elements include those described by Okayama, H. and Berg, P., Mol.
Cell Biol., 3: 280 (1983); Cepko, C. L. et al., Cell, 37: 1053
(1984); and Kaufman, R. J., Proc. Natl. Acad. Sci., USA, 82: 689
(1985).
[0114] An additional advantage of mammalian cells as hosts is their
ability to express chimeric immunoglobulin genes which are derived
from genomic sequences. Thus, mammalian cells may express chimeric
immunoglobulin genes which are comprised of a variable region cDNA
module plus a constant region which is composed in whole or in part
of genomic sequences. Several human constant region genomic clones
have been described (Ellison, J. W. et al., Nucl. Acids Res., 10:
4071 (1982), or Max, E. et al., Cell, 29: 691 (1982)). The use of
such genomic sequences may be convenient for the simultaneous
introduction of immunoglobulin enhancers, splice signals, and
transcription termination signals along with the constant region
gene segment.
[0115] Different approaches can be followed to obtain complete
H.sub.2L.sub.2 antibodies.
[0116] First, one can separately express the light and heavy chains
followed by in vitro assembly of purified light and heavy chains
into complete H.sub.2L.sub.2 IgG antibodies. The assembly pathways
used for generation of complete H.sub.2L.sub.2 IgG molecules in
cells have been extensively studied (see, for example, Scharff, M.,
Harvey Lectures, 69: 125 (1974)). In vitro reaction parameters for
the formation of IgG antibodies from reduced isolated light and
heavy chains have been defined by Beychok, S., Cells of
Immunoglobulin Synthesis, Academic Press, New York, page 69,
1979.
[0117] Second, it is possible to co-express light and heavy chains
in the same cells to achieve intracellular association and linkage
of heavy and light chains into complete H.sub.2L.sub.2 IgG
antibodies. The co-expression can occur by using either the same or
different plasmids in the same host.
Polypeptide Products
[0118] The invention provides "chimeric" immunoglobulin chains,
either heavy or light. A chimeric chain contains a constant region
substantially similar to that present in a natural human
immunoglobulin, and a variable region having the desired antigenic
specificity of the invention, i.e., to the specified human B cell
surface antigen.
[0119] The invention also provides immunoglobulin molecules having
heavy and light chains associated so that the overall molecule
exhibits any desired binding and recognition properties. Various
types of immunoglobulin molecules are provided: monovalent,
divalent, molecules with chimeric heavy chains and non-chimeric
light chains, or molecules with the invention's variable binding
domains attached to moieties carrying desired functions.
[0120] Antibodies having chimeric heavy chains of the same or
different variable region binding specificity and non-chimeric
(i.e., all human or all non-human) light chains, can be prepared by
appropriate association of the needed polypeptide chains. These
chains are individually prepared by the modular assembly methods of
the invention.
Uses
[0121] The antibodies of the invention having human constant region
can be utilized for passive immunization, especially in humans,
without negative immune reactions such as serum sickness or
anaphylactic shock. The antibodies can, of course, also be utilized
in prior art immunodiagnostic assays and kits in detectably
labelled form (e.g., enzymes, .sup.125I, .sup.14C, fluorescent
labels, etc.), or in immunobilized form (on polymeric tubes, beads,
etc.), in labelled form for in vivo imaging, wherein the label can
be a radioactive emitter, or an NMR contrasting agent such as a
carbon-13 nucleus, or an X-ray contrasting agent, such as a heavy
metal nucleus. The antibodies can also be used for in vitro
localization of the antigen by appropriate labelling.
[0122] The antibodies can be used for therapeutic purposes, by
themselves, in complement mediated lysis, or coupled to toxins or
therapeutic moieties, such as ricin, etc.
[0123] Mixed antibody-enzyme molecules can be used for
immunodiagnostic methods, such as ELISA. Mixed antibody-peptide
effector conjugates can be used for targeted delivery of the
effector moiety with a high degree of efficacy and specificity.
[0124] Specifically, the chimeric antibodies of this invention can
be used for any and all uses in which the murine 2H7 monoclonal
antibody can be used, with the obvious advantage that the chimeric
ones are more compatible with the human body.
[0125] Having now generally described the invention, the same will
be further understood by reference to certain specific examples
which are included herein for purposes of illustration only and are
not intended to be limiting unless otherwise specified.
Experimental
[0126] Materials and Methods
[0127] Tissue Culture Cell Lines
[0128] The human cell lines GM2146 and GM1500 were obtained from
the Human Mutant Cell Repository (Camden, N.J.) and cultured in
RPMI1640 plus 10% fetal bovine serum (M. A. Bioproducts). The cell
line Sp2/0 was obtained from the American Type Culture Collection
and grown in Dulbecco's Modified Eagle Medium (DMEM) plus 4.5 g/l
glucose (M. A. Bioproducts) plus 10% fetal bovine serum (Hyclone,
Sterile Systems, Logan, Utah). Media were supplemented with
penicillin/streptomycin (Irvine Scientific, Irvine,
California).
[0129] Recombinant Plasmid and Bacteriophage DNAs
[0130] The plasmids pBR322, pL1 and pUC12 were purchased from
Pharmacia P-L Biochemicals (Milwaukee, Wisconsin). The plasmids
pSV2-neo and pSV2-qpt were obtained from BRL (Gaithersburg, Md.),
and are available from the American Type Culture Collection
(Rockville, Md.). pHu-gamma-1 is a subclone of the 8.3 Kb HindIII
to BamHI fragment of the human IgG1 chromosomal gene. An isolation
method for of the human IgG1 chromosomal gene is described by
Ellison, J. W. et al., Nucl. Acids Res., 10: 4071 (1982).
M8alphaRX12 contains the 0.7 Kb XbaI to EcoRI fragment containing
the mouse heavy chain enhancer from the J-C intron region of the
M603 chromosomal gene (Davis, M. et al., Nature, 283: 733, 1979)
inserted into M13mp10. DNA manipulations involving purification of
plasmid DNA by buoyant density centrifugation, restriction
endonuclease digestion, purification of DNA fragments by agarose
gel electrophoresis, ligation and transformation of E. coli were as
described by Maniatis, T. et al., Molecular Cloning: A Laboratory
Manual, (1982) or other procedures. Restriction endonucleases and
other DNA/RNA modifying enzymes were purchased from
Boehringer-Mannheim (Indianapolis, Ind.), BRL, New England Biolabs
(Beverly, Mass.) and Pharmacia P-L.
[0131] Oligonucleotide Preparation
[0132] Oligonucleotides were either synthesized by the triester
method of Ito et al. (Nucl. Acids Res., 10: 1755 (1982)), or were
purchased from ELESEN, Los Angeles, Calif. Tritylated, deblocked
oligonucleotides were purified on Sephadex-G50, followed by
reverse-phase HPLC with a 0-25% gradient of acetonitrile in 10 mM
triethylamine-acetic acid, pH 7.2, on a C18 Bondapak column (Waters
Associates). Detritylation was in 80% acetic acid for 30 min.,
followed by evaporation thrice. Oligonucleotides were labeled with
[gamma-.sup.32P]ATP by T4 polynucleotide kinase.
[0133] RNA Preparation and Analysis
[0134] Total cellular RNA was prepared from tissue culture cells by
the method of Auffray, C. and Rougeon, F. (Eur. J. Biochem., 107:
303 (1980)) or Chirgwin, J. M. et al. (Biochemistry, 18: 5294
(1979)). Preparation of poly(A).sup.+ RNA, methyl-mercury agarose
gel electrophoresis, and "Northern" transfer to nitrocellulose were
as described by Maniatis, T. et al., supra. Total cellular RNA or
poly(A).sup.+ RNA was directly bound to nitrocellulose by first
treating the RNA with formaldehyde (White, B. A. and Bancroft, F.
C., J. Biol. Chem., 257: 8569 (1982)). Hybridization to filterbound
RNA was with nick-translated DNA fragments using conditions
described by Margulies, D. H. et al. (Nature, 295: 168 (1982)) or
with .sup.32P-labelled oligonucleotide using 4.times.SSC, 10.times.
Denhardt's, 100 ug/ml salmon sperm DNA at 37.degree. C. overnight,
followed by washing in 4.times.SSC at 37.degree. C.
[0135] cDNA Preparation and Cloning
[0136] Oligo-dT primed cDNA libraries were prepared from
poly(A).sup.+ RNA from GM1500 and GM2146 cells by the methods of
Land, H. et al. (Nucl. Acids Res., 9: 2251 (1981)) and Gubler, V.
and Hoffman, B. J., Gene, 25: 263 (1983), respectively. The cDNA
libraries were screened by hybridization (Maniatis, T., supra) with
.sup.32P-labelled oligonucleotides using the procedure of de Lange
et al. (Cell, 34: 891 (1983)), or with nick-translated DNA
fragments.
[0137] Oligonucleotide Primer Extension and Cloning
[0138] Poly(A).sup.+ RNA (20 ug) was mixed with 1.2 ug primer in 40
ul of 64 mM KCl. After denaturation at 90.degree. C. for 5 min. and
then chilling in ice, 3 units Human Placental Ribonuclease
Inhibitor (BRL) was added in 3 ul of 1M Tris-HCl, pH 8.3. The
oligonucleotide was annealed to the RNA at 42.degree. C. for 15
minutes, then 12 ul of 0.05M DTT, 0.05M MgCl.sub.2, and 1 mM each
of dATP, dTTP, dCTP, and dGTP was added. 2 ul of
alpha-.sup.32P-dATP (400 Ci/mmol, New England Nuclear) was added,
followed by 3 ul of AMV reverse transcriptase (19 units/ul, Life
Sciences).
[0139] After incubation at 42.degree. C. for 105 min., 2 ul 0.5 M
EDTA and 50 ul 10 mM Tris, 1 mM EDTA, pH 7.6 were added.
Unincorporated nucleotides were removed by Sephadex G-50 spin
column chromatography, and the RNA-DNA hybrid was extracted with
phenol, then with chloroform, and precipitated with ethanol. Second
strand synthesis, homopolymer tailing with dGTP or dCTP, and
insertion into homopolymer tailed vectors was as described by
Gubler and Hoffman, supra.
[0140] Site-Directed Mutagenesis
[0141] Single stranded M13 subclone DNA (1 ug) was combined with 20
ng oligonucleotide primer in 12.5 ul of Hin buffer (7 mM Tris-HCl,
pH 7.6, 7 mM MgCl.sub.2, 50 mM NaCl). After heating to 95.degree.
C. in a sealed tube, the primer was annealed to the template by
slowly cooling from 70.degree. C. to 37.degree. C. for 90 minutes.
2 ul dNTPs (1 mM each), 1 ul .sup.32P-DATP (10 uCi), 1 ul DTT (0.1
M) and 0.4 ul Klenow DNA PolI (2u, Boehringer Mannheim) were added
and chains extended at 37.degree. C. for 30 minutes. To this was
added 1 ul (10 ng) M13 reverse primer (New England Biolabs), and
the heating/annealing and chain extension steps were repeated. The
reaction was stopped with 2 ul of 0.5M EDTA, pH 8, plus 80 ul of 10
mM Tris-HCl, pH 7.6, 1 mM EDTA. The products were phenol extracted
and purified by Sephadex G-50 spun column chromatography and
ethanol precipitated prior to restriction enzyme digestion and
ligation to the appropriate vector.
[0142] Transfection of Myeloma Tissue Culture Cells
[0143] The electroporation method of Potter, H. et al. (Proc. Natl.
Acad. Sci., USA, 81: 7161 (1984)) was used. After transfection,
cells were allowed to recover in complete DMEM for 48-72 hours,
then were seeded at 10,000 to 50,000 cells per well in 96-well
culture plates in the presence of selective medium. G418 (GIBCO)
selection was at 0.8 mg/ml, and mycophenolic acid (Calbiochem) was
at 6 ug/ml plus 0.25 mg/ml xanthine.
[0144] Assays for Immunoglobulin Synthesis and Secretion
[0145] Secreted immunoglobulin was measured directly from tissue
culture cell supernatants. Cytoplasmic protein extract was prepared
by vortexing 10.sup.6 cells in 160 ul of 1% NP40, 0.15 M NaCl, 10
mM Tris, 1 mM EDTA, pH 7.6 and leaving the lysate at 0.degree. C.,
15 minutes, followed by centrifugation at 10,600.times.g to remove
insoluble debris.
[0146] A double antibody sandwich ELISA (Voller, A. et al., in
Manual of Clinical Immunology, 2nd Ed., Eds. Rose, N. and Friedman,
H., pp. 359-371, 1980) using affinity purified antisera was used to
detect specific immunoglobulins. For detection of human IgG, the
plate-bound antiserum is goat anti-human IgG (KPL, Gaithersburg,
Md.) at 1/1000 dilution, while the peroxidase-bound antiserum is
goat anti-human IgG (KPL or Tago, Burlingame) at 1/4000 dilution.
For detection of human immunoglobulin kappa, the plate-bound
antiserum is goat anti-human kappa (Tago) at 1/500 dilution, while
the peroxidase-bound antiserum is goat anti-human kappa (Cappel) at
1/1000 dilution.
EXAMPLE 1
A Chimeric Mouse-Human Immunoglobulin with Specificity for a Human
B-Cell Surface Antigen
[0147] (1) Antibody 2H7.
[0148] The 2H7 mouse monoclonal antibody (gamma 2b, kappa)
recognizes a human B-cell surface antigen, (Bp35(CD20)) Clark, E.
A., et al., Proc. Natl. Acad. Sci., U.S.A. 82: 1766 (1985)). The
(Bp35(CD20)) molecules presumably play a role in B-cell activation.
The antibody 2H7 does not react with stem cells which are
progenitors of B-cells epithelial, mesenchymal and fibroblastic
cells of other organs.
[0149] (2) Identification of J Sequences in the Immunoglobulin mRNA
of 2H7.
[0150] Frozen cells were thawed on ice for 10 minutes and then at
room temperature. The suspension was diluted with 15 ml PBS and the
cells were centrifuged down. They were resuspended, after washes in
PBS, in 16 ml 3M LiCl, 6M urea and disrupted in a polytron shear.
The preparation of mRNA and the selection of the poly(A+) fraction
were carried out according to Auffray, C. and Rougeon, F., Eur. J.
Biochem. 107: 303, 1980.
[0151] The poly (A+) RNA from 2H7 was hybridized individually with
labeled J.sub.H1, J.sub.H2, J.sub.H3 and J.sub.H4 oligonucleotides
under conditions described by Nobrega et al. Anal. Biochem 131:
141, 1983). The products were then subjected to electrophoresis in
a 1.7% agarose-TBE gel. The gel was fixed in 10% TCA, blotted dry
and exposed for autoradiography. The result showed that the 2H7
V.sub.H contains J.sub.H1, J.sub.H2, or J.sub.H4 but not J.sub.H3
sequences.
[0152] For the analysis of the V.sub.K mRNA, the dot-blot method of
White and Bancroft J. Biol. Chem. 257: 8569, (1982) was used. Poly
(A+) RNA was immobilized on nitrocellulose filters and was
hybridized to labeled probe-oligonucleotides at 400 in 4.times.SSC.
These experiments show that 2H7 contains J.sub.K5 sequences.
[0153] (3) V Region cDNA Clones.
[0154] A library primed by oligo (dT) on 2H7 poly (A+) RNA was
screened for kappa clones with a mouse C.sub.K region probe. From
the 2H7 library, several clones were isolated. A second screen with
a 5' J.sub.K5 specific probe identified the 2H7 (J.sub.K5)
light-chain clones. Heavy chain clones of 2H7 were generated by
priming the poly(A+) RNA with the UIGH(BstEII) oligonucleotide (see
FIG. 3), and identified by screening with the UIGH(BstEII)
oligonucleotide.
[0155] The heavy and light chain genes or gene fragments from the
V.sub.H and V.sub.K cDNA clones pH2-11 and pL2-12 were inserted
into M13 bacteriophage vectors for nucleotide sequence analysis.
The complete nucleotide sequences of the variable region of these
clones were determined (FIGS. 5 and 6) by the dideoxy chain
termination method. These sequences predict V region amino acid
compositions that agree well with the observed compositions, and
predict peptide sequences which have been verified by direct amino
acid sequencing of portions of the V regions.
[0156] The nucleotide sequences of the cDNA clones show that they
are immunoglobulin V region clones as they contain amino acid
residues diagnostic of V domains (Kabat et al., Sequences of
Proteins of Immunological Interest; U.S. Dept of HHS, 1983).
[0157] The 2H7 V.sub.H has the J.sub.H1 sequence. The 2H7 V.sub.L
is from the V.sub.K-KpnI family (Nishi et al. Proc. Nat. Acd. Sci.
USA 82: 6399, 1985), and uses J.sub.K5. The cloned 2H7 V.sub.L
predicts an amino acid sequence which was confirmed by amino acid
sequencing of peptides from the 2H7 light chain corresponding to
residues 81-100. The cloned 2H7 V.sub.H predicts an amino acid
sequence confirmed also by peptide sequencing, namely residues
1-12.
[0158] (4) In Vitro Mutagenesis to Engineer Restriction Enzyme
Sites in the J Region for Joining to a Human C-Module, and to
Remove Oligo (dC) Sequences 5' to the V Modules.
[0159] For the 2H7 V.sub.K, the J-region mutagenesis primer
J.sub.KHindIII, as shown in FIG. 6, was utilized. A human C.sub.K
module derived from a cDNA clone was also mutagenized to contain
the HindIII sequence (see FIG. 4). The mutagenesis reaction was
performed on M13 subclones of these genes. The frequency of mutant
clones ranged from 0.5 to 1% of the plaques obtained.
[0160] It had been previously observed that the oligo (dC) sequence
upstream of the AUG codon in a V.sub.H chimeric gene interferes
with proper splicing in one particular gene construct. It was
estimated that perhaps as much as 70% of the RNA transcripts had
undergone the mis-splicing, wherein a cryptic 3' splice acceptor in
the leader sequence was used. Therefore the oligo (dC) sequence
upstream of the initiator AUG was removed in all of the clones.
[0161] In one approach, an oligonucleotide was used which contains
a SalI restriction site to mutagenize the 2H7 V.sub.K clone. The
primer used for this oligonucleotide-directed mutagenesis is a
22-mer which introduces a SalI site between the oligo (dC) and the
initiator met codon (FIG. 6).
[0162] In a different approach, a convenient NcoI site was utilized
to delete the 5' untranslated region and oligo (dC) of the 2H7
V.sub.H clone (see FIG. 5).
[0163] The human C gamma 1 gene module is a cDNA derived from
GM2146 cells (Human Genetic Mutant Cell Repository, Newark, N.J.).
This C gamma 1 gene module was previously combined with a mouse
V.sub.H gene module to form the chimeric expression plasmid
pING2012E (FIG. 7C).
[0164] (5) Chimeric 2H7 Expression Plasmids.
[0165] A 2H7 chimeric heavy chain expression plasmid was derived
from the replacement of the V.sub.H module of pING2012E with the
V.sub.H cDNA modules to give the expression plasmid pING2101 (FIG.
7b). This plasmid directs the synthesis of chimeric 2H7 heavy chain
when transfected into mammalian cells.
[0166] For the 2H7 light chain chimeric gene, the SalI to HindIII
fragment of the mouse V.sub.K module was joined to the human C K
module by the procedure outlined in FIG. 7a, forming pING2106.
Replacement of the neo sequence with the E. coli gpt gene derived
from pSV2-gpt resulted in pING2107, which expresses 2H7 chimeric
light chain and confers mycophenolic acid resistance when
transfected into mammalian cells.
[0167] The inclusion of both heavy and light chain chimeric genes
in the same plasmid allows for the introduction into transfected
cells of a 1:1 gene ratio of heavy and light chain genes leading to
a balanced gene dosage. This may improve expression and decrease
manipulations of transfected cells for optimal chimeric antibody
expression. For this purpose, the DNA fragments derived from the
chimeric heavy and light chain genes of pING2101 and pING2106 were
combined into the expression plasmids pHL2-11 and pHL2-26 (FIG. 8).
This expression plasmid contains a selectable neoR marker and
separate transcription units for each chimeric gene, each including
a mouse heavy chain enhancer.
[0168] The modifications and V-C joint regions of the 2H7 chimeric
genes are summarized in FIG. 9.
[0169] (6) Stable Transfection of Mouse Lymphoid Cells for the
Production of Chimeric Antibody.
[0170] Electroporation was used (Potter et al. supra; Toneguzzo et
al. Mol. Cell Biol. 6: 703 1986) for the introduction of 2H7
chimeric expression plasmid DNA into mouse Sp2/0 cells. The
electroporation technique gave a transfection frequency of
10.sup.4.times.10.sup.5 for the Sp2/0 cells.
[0171] The expression plasmids, pING2101 and pING2106, were
digested with NdeI; and the DNA was introduced into Sp2/0 cells by
electroporation. Transformant 1D6 was obtained which secretes
chimeric 2H7 antibody. Antibody isolated from this cell line was
used for the functional assays done to characterize the chimeric
antibody. We have also obtained transformants from experiments
using the two-gene plasmids.
[0172] (7) Purification of Chimeric 2H7 Antibody Secreted in Tissue
Culture.
[0173] a. 1D6 (Sp2/0.pING2101/pING2106.1D6) cells were grown in
culture medium [DMEM (Gibco #320-1965), supplemented with 10% Fetal
Bovine Serum (Hyclone #A-1111-D), 10 mM HEPES, 1.times.
Glutamine-Pen-Strep (Irvine Scientific #9316) to 1.times.10.sup.6
cell/ml.
[0174] b. The cells were then centrifuged at 400.times.g and
resuspended in serum-free culture medium at 2.times.10.sup.6
cell/ml for 18-24 hr.
[0175] c. The medium was centrifuged at 4000 RPM in a JS-4.2 rotor
(3000.times.g) for 15 min.
[0176] d. 1.6 liter of supernatant was then filtered through a 0.45
micron filter and then concentrated over a YM30 (Amicon Corp.)
filter to 25 ml.
[0177] e. The conductance of the concentrated supernatant was
adjusted to 5.7-5.6 mS/cm CDM 80 radiometer and the pH was adjusted
to 8.0.
[0178] f. The supernatant was centrifuged at 2000.times.g, 5 min.,
and then loaded onto a 40 ml DEAE column, which was preequilibrated
with 10 mM sodium phosphate, pH8.0.
[0179] g. The flow through fraction was collected and loaded onto a
1 ml protein A-Sepharose (Sigma) column preequilibrated with 10 mM
sodium phosphate, pH8.0.
[0180] h. The column was washed first with 6 ml 10 mM sodium
phosphate buffer pH 8.0, followed by 8 ml 0.1M sodium citrate pH
3.5, then by 6 ml 0.1M citric acid (pH 2.2). Fractions of 0.5 ml
were collected in tubes containing 50 ul 2M Tris base (Sigma).
[0181] i. The bulk of the IgG was in the pH 3.5 elution and was
pooled and concentrated over Centricon 30 (Amicon Corp.) to
approximately 0.06 ml.
[0182] j. The buffer was changed to PBS (10 mM sodium phosphate pH
7.4, 0.15M NaCl) in Centricon 30 by repeated diluting with PBS and
reconcentrating.
[0183] k. The IgG solution was then adjusted to 0.10 ml and bovine
serum albumin (Fraction V, U.S. Biochemicals) was added to 1.0% as
a stabilizing reagent.
[0184] (9) Chimeric 2H7 Antibody, Like the Mouse 2H7 Antibody,
Specifically Binds to Human B Cells.
[0185] First, the samples were tested with a binding assay, in
which cells of both an 2H7 antigen-positive and an 2H7
antigen-negative cell line were incubated with standard mouse
monoclonal antibody 2H7 with chimeric 2H7 antibody derived from the
cell culture supernatants, followed by a second reagent,
fluoresceinisothiocyanate (FITC)-conjugated goat antibodies to
human (or mouse, for the standard) immunoglobulin.
[0186] Binding Assays. Cells from a human B cell line, T51, were
used. Cells from human colon carcinoma line C3347 were used as a
negative control, since they, according to previous testing, do not
express detectable amounts of the 2H7 antigen. The target cells
were first incubated for 30 min at 4.degree. C. with either the
chimeric 2H7 or with mouse 2H7 standard, which had been purified
from mouse ascites. This was followed by incubation with a second,
FITC-labelled, reagent, which for the chimeric antibody was
goat-anti-human immunoglobulin, obtained from TAGO (Burlingame,
Calif.), and used at a dilution of 1:50. For the mouse standard, it
was goat-anti-mouse immunoglobulin, also obtained from TAGO and
used at a dilution of 1:50. Antibody binding to the cell surface
was determined using a Coulter Model EPIC-C cell sorter.
[0187] As shown in Table I, both the chimeric and the mouse
standard 2H7 bound significantly, and to approximately the same
extent, to the positive T51 line. They did not bind above
background to the 2H7 negative C-3347 line.
[0188] Functional Assays.
[0189] In previous studies, antibody 2H7 was tested for
antibody-dependent cellular cytotoxicity (ADCC) measured by its
ability to lyse Cr-labelled human B lymphona cells in the presence
of human peripheral blood leukocytes as the source of effector
cells. It was also tested for its ability to lyse .sup.51Cr
labelled hum B cells in the presence of human serum as the source
of complement. These tests were carried out as previously described
for mouse monoclonal anti-carcinoma antibody L6, which can mediate
ADCC, as well as complement-mediated cytoxicity, CDC. The
techniques used and the data described for the L6 antibody have
been previously described. Hellstrom, et al., Proc. Natl. Acad.
Sci. U.S.A. 83: 7059-7063 (1986).
[0190] Chimeric 2H7, but not mouse 2H7 antibody, will be able to
mediate both ADCC and CDC against human B lymphoma cells. Thus a
hybridoma producing a non-functional mouse antibody can be
converted to a hybridoma producing a chimeric antibody with ADCC
and CDC activities. Such a chimeric antibody is a prime candidate
for the treatment or imaging of B-cell disorders, such as
leukemias, lymphomas, and the like.
[0191] This invention therefore provides a method for making
biologically functional antibodies when starting with a hybridoma
which produces antibody which has the desired specificity for
antigen but lacks biological effector functions such as ADCC and
CDC.
Conclusions
[0192] The results presented above demonstrate that the chimeric
2H7 antibody binds to (Bp35(CD20)) antigen positive human B cells
to approximately the same extent as the mouse 2H7 monoclonal
antibody. This is significant because the 2H7 antibody defines a
surface phosphoprotein antigen (Bp35(CD20)), of about 35,000
daltons, which is expressed on the cells of B cell lineage. The 2H7
antibody does not bind detectably to various other cells such as
fibroblasts, endothelial cells, or epithelial cells in the major
organs or the stem cell precursors which give rise to B cells.
[0193] Although the prospect of attempting tumor therapy using
monoclonal antibodies is attractive, with some partial tumor
regressions being reported, to date such monoclonal antibody
therapy has been met with limited success (Houghton et al.,
February 1985, Proc. Natl. Acad. Sci. 82: 1242-1246). Murine
monoclonal anti-(Bp35(CD20)) antibody has been used for therapy of
B cell malignancies (Press, et al.,) Blood: February 1987, in
press). The therapeutic efficacy of mouse monoclonal antibodies
(which are the ones that have been tried so far) appears to be too
low for most practical purposes. Because of the "human" properties
which may make the chimeric 2H7 monoclonal antibodies more
resistant to clearance and less immunogenic in vivo, the chimeric
2H7 monoclonal antibodies will be advantageously used not only for
therapy with unmodified chimeric antibodies, but also for
development of various immunoconjugates with drugs, toxins,
immunomodulators, isotopes, etc., as well as for diagnostic
purposes such as in vivo imaging of B-cell tumors (for example,
lymphomas and leukemias) using appropriately labelled chimeric 2H7
antibodies. Such immunoconjugation techniques are known to those
skilled in the art and can be used to modify the chimeric 2H7
antibody molecules of the present invention. The chimeric 2H7
antibody, by virtue of its having the human constant portion, will
possess biological activity in complement dependent and antibody
dependent cytotoxicity which the mouse 2H7 does not.
[0194] An illustrative cell line secreting chimeric 2H7 antibody
was deposited prior to the U.S. filing date at the ATCC, Rockville
Md. This is a transfected hybridoma (corresponds to 1D6 cells
supra) ATCC HB 9303.
[0195] The present invention is not to be limited in scope by the
cell lines deposited since the deposited embodiment is intended as
a single illustration of one aspect of the invention and all cell
lines which are functionally equivalent are within the scope of the
invention. Indeed, various modifications of the invention in
addition to those shown in the art from the foregoing description
and accompanying drawings. Such modifications are intended to fall
within the scope of the appended claims.
1TABLE 1 Binding Assays Of Chimeric 2H7 Antibody and Mouse 2H7
Monoclonal Antibody to a B cell Line Expressing (Bp35(CD20)) and a
Cell Line Not Expressing This Antigen. Antibody GAM GAH Binding
Ratio* for T51 B-Cells 2H7 Mouse 37 ND 2H7 Chimeric ND 29 L6 Mouse
1 ND Binding Ratio* for C3347 Cells 2H7 Mouse 1.4 ND 2H7 Chimeric
ND 1.3 L6 Mouse 110 ND *All assays were conducted using an antibody
concentration of 10 ug/ml. The binding ratio is the number of times
brighter a test sample is than a control sample treated with GAM
(FITC-Conjugated goat anti-mouse) or GAH (FITC conjugated goat
anti-human) alone. A ratio of 1 means that the test sample is just
as bright as the control; a ratio of 2 means the test sample is
twice as bright as the control and so on. ND--not done
[0196]
Sequence CWU 1
1
50 1 52 DNA Homo sapiens 1 gctgaatact tccagcactg gggccagggc
accctggtca ccgtctcctc ag 52 2 53 DNA Homo sapiens 2 ctactggtac
ttcgatctct ggggccgtgg caccctggtc actgtctcct cag 53 3 48 DNA Homo
sapiens 3 atgcttttga tgtctggggc caagggacaa tggtcaccgt ctcttcag 48 4
48 DNA Homo sapiens 4 actactttga ctactggggc caaggaaccc tggtcaccgt
ctcctcag 48 5 50 DNA Homo sapiens 5 acactggttc gactcctggg
gccaaggaac cctggtcacc gtctcctcag 50 6 63 DNA Homo sapiens 6
attactacta ctactacggt atggacgtct gggggcaagg gaccacggtc accgtctcct
60 cag 63 7 42 DNA Artificial Sequence Consensus of human heavy
chain J regions 7 tcgacctctg gggccaagga accctggtca ccgtctcctc ag 42
8 52 DNA mus musculus 8 tactggtact tcgatgtctg gggcgcaggg accacggtca
ccgtctcctc ag 52 9 46 DNA mus musculus 9 tactttgact actggggcca
aggcaccact ctcacagtct cctcag 46 10 48 DNA mus musculus 10
cctggtttgc ttactggggc caagggactc tggtcactgt ctctgcag 48 11 52 DNA
mus musculus 11 tactatgcta tggactactg gggtcaagga acctcagtca
ccgtctcctc ag 52 12 43 DNA Artificial Sequence Consensus of mouse
heavy chain J regions 12 tttgactact ggggccaagg gaccacggtc
accgtctcct cag 43 13 36 DNA Homo sapiens 13 ggacgttcgg ccaagggacc
aaggtggaaa tcaaac 36 14 36 DNA Homo sapiens 14 acacttttgg
ccaggggacc aagctggaga tcaaac 36 15 36 DNA Homo sapiens 15
tcactttcgg ccctgggacc aaagtggata tcaaac 36 16 36 DNA Homo sapiens
16 tcactttcgg cggagggacc aaggtggaga tcaaac 36 17 36 DNA Homo
sapiens 17 tcaccttcgg ccaagggaca cgactggaga ttaaac 36 18 31 DNA
Artificial Sequence Consensus of human Kappa J regions 18
ttcggccaag ggaccaaggt ggagatcaaa c 31 19 37 DNA mus musculus 19
tggacgttcg gtggaggcac caagctggaa atcaaac 37 20 37 DNA mus musculus
20 tacacgttcg gaggggggac caagctggaa ataaaac 37 21 37 DNA mus
musculus 21 ttcacattca gtgatgggac cagactggaa ataaaac 37 22 37 DNA
mus musculus 22 ttcacgttcg gctcggggac aaagttggaa ataaaac 37 23 37
DNA mus musculus 23 ctcacgttcg gtgctgggac caagctggag ctgaaac 37 24
31 DNA Artificial Sequence Consensus of mouse Kappa J regions 24
ttcggtgggg ggaccaagct ggaaataaaa c 31 25 19 DNA Artificial Sequence
consensus primer UIG-MJK 25 gttttatttc cagcttggt 19 26 37 DNA Homo
sapiens 26 cacatgtttg gcagcaagac ccagcccact gtcttag 37 27 37 DNA
mus musculus 27 tgggtgttcg gtggaggaac caaactgact gtcctag 37 28 37
DNA mus musculus 28 tatgttttcg gcggtggaac caaggtcact gtcctag 37 29
37 DNA mus musculus 29 tttattttcg gcagtggaac caaggtcact gtcctag 37
30 31 DNA Artificial Sequence Consensus of mouse Lambda J regions
30 ttcggcggtg gaaccaaggt cactgtccta g 31 31 51 DNA mus musculus 31
tactggtact tcgatgtctg gggcgcaggg accacggtca ccgtctcctc a 51 32 45
DNA mus musculus 32 tactttgact actggggcca aggcaccact ctcacagtct
cctca 45 33 47 DNA mus musculus 33 cctggtttgc ttactggggc caagggactc
tggtcactgt ctctgca 47 34 51 DNA mus musculus 34 tactatgcta
tggactactg gggtcaagga acctcagtca ccgtctcctc a 51 35 21 DNA
Artificial Sequence Consensus primer UIGH 35 agggaccacg gtcaccgtct
c 21 36 36 DNA mus musculus 36 tggacgttcg gtggaggcac caagctggaa
atcaaa 36 37 36 DNA mus musculus 37 tacacgttcg gaggggggac
caagctggaa ataaaa 36 38 36 DNA mus musculus 38 ttcacgttcg
gctcggggac aaagttggaa ataaaa 36 39 36 DNA mus musculus 39
ctcacgttcg gtgctgggac caagctggag ctgaaa 36 40 15 DNA Artificial
Sequence Consensus primer UIGK 40 gggaccaagc ttgag 15 41 43 DNA
Homo sapiens 41 ggtcaccgtc tcttcagcct ccaccaaggg cccatcggtc ttc 43
42 48 DNA Homo sapiens 42 gatcatctcc ctctcacttt cggcggaggg
accaaggtgg agatgaaa 48 43 459 DNA Mus musculus CDS (40)..(459) 43
cgtacctctc tacagtccct gaagacactg actctaacc atg gga ttc agc agg 54
Met Gly Phe Ser Arg 1 5 atc ttt ctc ttc ctc ctg tca gta act aca ggt
gtc cac tcc cag gct 102 Ile Phe Leu Phe Leu Leu Ser Val Thr Thr Gly
Val His Ser Gln Ala 10 15 20 tat cta cag cag tct ggg gct gag ctg
gtg agg cct ggg gcc tca gtg 150 Tyr Leu Gln Gln Ser Gly Ala Glu Leu
Val Arg Pro Gly Ala Ser Val 25 30 35 aag atg tcc tgc aag gct tct
ggc tac aca ttt acc agt tac aat atg 198 Lys Met Ser Cys Lys Ala Ser
Gly Tyr Thr Phe Thr Ser Tyr Asn Met 40 45 50 cac tgg gta aag cag
aca cct aga cag ggc ctg gaa tgg att gga gct 246 His Trp Val Lys Gln
Thr Pro Arg Gln Gly Leu Glu Trp Ile Gly Ala 55 60 65 att tat cca
gga aat ggt gat act tcc tac aat cag aag ttc aag ggc 294 Ile Tyr Pro
Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe Lys Gly 70 75 80 85 aag
gcc aca ctg act gta gac aaa tcc tcc agc aca gcc tac atg cag 342 Lys
Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln 90 95
100 ctc agc agc ctg aca tct gaa gac tct gcg gtc tat ttc tgt gca aga
390 Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys Ala Arg
105 110 115 gtg gtg tac tat agt aac tct tac tgg tac ttc gat gtc tgg
ggc aca 438 Val Val Tyr Tyr Ser Asn Ser Tyr Trp Tyr Phe Asp Val Trp
Gly Thr 120 125 130 ggg acc acg gtc acc gtc tcg 459 Gly Thr Thr Val
Thr Val Ser 135 140 44 140 PRT Mus musculus 44 Met Gly Phe Ser Arg
Ile Phe Leu Phe Leu Leu Ser Val Thr Thr Gly 1 5 10 15 Val His Ser
Gln Ala Tyr Leu Gln Gln Ser Gly Ala Glu Leu Val Arg 20 25 30 Pro
Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40
45 Thr Ser Tyr Asn Met His Trp Val Lys Gln Thr Pro Arg Gln Gly Leu
50 55 60 Glu Trp Ile Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser
Tyr Asn 65 70 75 80 Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr Val Asp
Lys Ser Ser Ser 85 90 95 Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr
Ser Glu Asp Ser Ala Val 100 105 110 Tyr Phe Cys Ala Arg Val Val Tyr
Tyr Ser Asn Ser Tyr Trp Tyr Phe 115 120 125 Asp Val Trp Gly Thr Gly
Thr Thr Val Thr Val Ser 130 135 140 45 403 DNA Mus musculus CDS
(20)..(403) 45 ccccaaaatt caaagacaa atg gat ttt caa gtg cag att ttc
agc ttc ctg 52 Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu 1 5 10
cta atc agt gct tca gtc ata att gcc aga gga caa att gtt ctc tcc 100
Leu Ile Ser Ala Ser Val Ile Ile Ala Arg Gly Gln Ile Val Leu Ser 15
20 25 cag tct cca gca atc ctg tct gca tct cca ggg gag aag gtc aca
atg 148 Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly Glu Lys Val Thr
Met 30 35 40 act tgc agg gcc agc tca agt gta agt tac atg cac tgg
tac cag cag 196 Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met His Trp
Tyr Gln Gln 45 50 55 aag cca gga tcc tcc ccc aaa ccc tgg att tat
gcc cca tcc aac ctg 244 Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr
Ala Pro Ser Asn Leu 60 65 70 75 gct tct gga gtc cct gct cgc ttc agt
ggc agt ggg tct ggg acc tct 292 Ala Ser Gly Val Pro Ala Arg Phe Ser
Gly Ser Gly Ser Gly Thr Ser 80 85 90 tac tct ctc aca atc agc aga
gtg gag gct gaa gat gct gcc act tat 340 Tyr Ser Leu Thr Ile Ser Arg
Val Glu Ala Glu Asp Ala Ala Thr Tyr 95 100 105 tac tgc cag cag tgg
agt ttt aac cca ccc acg ttc ggt gct ggg acc 388 Tyr Cys Gln Gln Trp
Ser Phe Asn Pro Pro Thr Phe Gly Ala Gly Thr 110 115 120 aag ctg gag
ctg aaa 403 Lys Leu Glu Leu Lys 125 46 128 PRT Mus musculus 46 Met
Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser 1 5 10
15 Val Ile Ile Ala Arg Gly Gln Ile Val Leu Ser Gln Ser Pro Ala Ile
20 25 30 Leu Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg
Ala Ser 35 40 45 Ser Ser Val Ser Tyr Met His Trp Tyr Gln Gln Lys
Pro Gly Ser Ser 50 55 60 Pro Lys Pro Trp Ile Tyr Ala Pro Ser Asn
Leu Ala Ser Gly Val Pro 65 70 75 80 Ala Arg Phe Ser Gly Ser Gly Ser
Gly Thr Ser Tyr Ser Leu Thr Ile 85 90 95 Ser Arg Val Glu Ala Glu
Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp 100 105 110 Ser Phe Asn Pro
Pro Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 115 120 125 47 12
DNA Artificial Sequence 5' region of VH pH2-7(JH1)Bst EII Clone 47
gtcgacatgg ga 12 48 24 DNA Artificial Sequence Joint region of VH
pH2-7(JH1)Bst EII Clone 48 acggtcaccg tctcttcagc ctcc 24 49 15 DNA
Artificial Sequence 5' region of Vk pL2-12 (Jk5) oligo(dT) clone 49
gtcgacaaaa tggat 15 50 24 DNA Artificial Sequence Joint region of
Vk pL2-12 (Jk5) oligo(dT) clone 50 accaagcttg agatgaaacg aact
24
* * * * *