Download Genetically Engineered Antibodies

CLIN.CHEM.35/9, 1849-1853 (1989)
Genetically Engineered Antibodies
GordonP. Moore
The technologyneeded
to geneticallyengineer antibodiesis
6-8),
the possibility is now enhanced that the undoubted
of immunotherapy
can someday be realized.
evolving rapidly and the potential utility of these novel reagents is being explored with vigor. The process includes
promise
cloningof the antibodygenes, their in vitromanipulationand
mutagenesis, expression in a suitable host/vectorsystem,
and, for commercial production,scale-up, purification,and
productevaluation.At each step, significantadvances have
been achieved recently.Forexample:at first,antibodygenes
were cloned from genomic librariesby using adjacent DNA
probes;techniquesfor rapidsequencingby pnmerextension
of total mRNA allowed more specificscreeningwith synthesized oligomers;finally,antibodygenes can now be created
de novo by chemical synthesis. Moreover, such synthesis
allows total control over the antibody sequence so that
molecules of any configuration can be produced. New reagents created in this way includemunne antibodieswhose
constantregionsand variable-regionframeworkshave been
replacedwith human sequence to enhance immunocompat-
Genetic EngineerIng of AntIbodies
ibility with patients, to switch immunoglobulin class, or both.
Additional Keyphrases: monoclonal antibodies
sis
expression vector
immunotherapy
bodies
gene synthechimeric anti-
Monoclonal antibodies have been available for more than
15 years (1). Despite their great utility in the laboratory
and in diagnostic testing, and notwithstanding
their high
promise and the enormous research effort expended on
them, they have yet to make a significant impact as clinical
tools. Monoclonals are not used routinely to treat or image
tumors, to provide passive immunization
against infectious
diseases,
or for immunosuppression
after organ transplant-all
uses for which they have been intended.
Among the problems encountered
in the effort to translate the diversity and specificity of monoclonals into clinically useful reagents are the following:
#{149}
The human immune response to foreign antibodies.
#{149}
Difficulty in making physical contact between the antibody and the target antigen.
.
#{149}
Change in antigen structure
during tumor development
or through mutation, as well as antigen shedding.
#{149}
Low affinity, inappropriate
isotype, or nonoptimal systemic half-life of immunotherapeutic
antibodies.
#{149}
Difficulty in producing sufficient quantities of antibody
for therapy.
In 1983, Oi et al. reported (2) that lymphoid
cells can
express cloned, transfected
immunoglobulin
genes. This
demonstration
opened the way to a new technology
that
may provide solutions to some of the problems listed above,
i.e., the cloning, engineering,
and expression
of genes that
code for antibodies.
Because
these methods
have been
shown capable of creating new kinds of antibody molecules
(e.g., 3-5), and some of these are clinically relevant
(e.g.,
Integrated
Genetics, Inc., One Mountain Road, Framingham,
MA 01701.
Received April 13, 1989; accepted June 9, 1989.
The process by which genetically engineered antibody
variants
can be created has been reviewed extensively
elsewhere (e.g., 9, 10). The major goals of such engineering
include:
#{149}
Creating immunocompatible
reagents by joining rodent
variable regions to human constant regions.
#{149}
Altering systemic half-life by truncation or amino acid
addition.
#{149}
Isotype switching.
#{149}
Increasing
affinity
or avidity.
#{149}
Using gene fusion to generate bispecific antibodies.
#{149}
Using gene fusion to generate
molecules
that bind
antigen but also possess additional activities.
#{149}
Altering effector functions
such as F receptor binding
and complement activation.
#{149}
Reducing anti-idiotypic response to rodent antibodies
by replacing rodent variable-region
framework sequence
with
human
sequence.
For each antibody
of interest, engineering
begins
by
cloning the variable regions, which dictates antigen specificity. DNA is isolated from hybridoma
cells and cloned,
usually in Aphage vectors. The phage library is screened by
hybridization with a radioactively labeled probe. Because
the genomic rearrangement
that results in production of
functional
immunoglobulin
genes leaves common sequences between the variable (V) and constant (C) regions,
any functional
heavy-chain
gene can be isolated
with a
single (“universal”) probe.’ The same is true for the light
chain. These clones can be verified with synthesized oligomere as described below.
Once the desired V regions have been isolated, they must
be cloned into a vector appropriate for expression. If immunocompatibility
is an objective, it is desirable that the
expression vector contain a human
C region; given that
most available hybridomas are murine, the V region is
usually from mouse. Moreover, panels of expression vectors
now exist, each containing
a different human C region.
Thus, the isotype of the antibody can be chosen at will.
Expression vectors have been designed for use in bacteria, yeast, or mammalian cells (2, 11, 12). The most common hosts are murine myeloma cells, which, through
mutation, have lost the ability to make endogenous antibody. Vectors for myeloma cells usually contain heavy(H)and light(L)-chain
genes individually,
and these are cointroduced into the host (6, 7). In addition to the murine V
and human C regions, the vectors contain a bacterial origin
of replication and drug-selectable
markers to identify
which cells have taken up and expressed the vectors. The
immunoglobulin
genes are usually controlled by their own
promoters and enhancers. These constructions
may contain
‘Nonstandard abbreviations: V, variable; C, constant; H, heavy;
L, light; and CDR, complementarity-determining
region.
CLINICAL CHEMISTRY, Vol. 35, No.9, 1989 1849
other features as described below.
When antibody genes have been engineered as desired
and placed in expression vectors, they are transfected into
myeloma cell hosts. Transfection is by electroporation (13)
or protoplast
fusion (14). Although both H and L chain
vectors are individually
selectable, it is interesting that
co-transfection
is so frequent that selection with only one
drug is necessary. Transfection frequency is about one in
1000. Drug-resistant
colonies are grown, tested for secretion of antibody by enzyme-linked
immunosorbent
assay,
and high-producing lines are identified.
A
B
C
200-
92 566 2450-
Fig.2. Purification
of chimericB6.2 immunoglobulin
by (A) nonresodiumdodecyl sulfate-polyacrylamide
gelelectrophoresis;
(C) isoelectric focusing
ducingpolyacrylamidegel electrophoresis;(
Purificationof Murine V/Human C ChimericAntibodies
Once a cell line secreting the engineered antibody has
been identified, it must be scaled-up
for production. This
can be done in tissue culture or by growing the cells in mice
and collecting antibody from ascites fluid. Various purification schemes have been reported (e.g., 15), some of which
utilize the binding activity of the antibody. My colleagues
and I have used methods that rely on properties of the
human C regions that distinguish chimeric from contaminating antibodies. In the case of tissue culture media, the
major contaminant is bovine antibody found in serum. As
shown in Figure 1 (C. Shearman and D. Lawrie, Integrated
Genetics, unpublished), elution from Protein A-Sepharose
can clearly separate a murine/human
chimeric antibody
from contaminating
bovine immunoglobulins.
In ascites
fluid, murine antibodies constitute a major contaminant.
These are removed effectively (Figure 2) by binding to
Protein A-Sepharose,
elution, then passage over Sepharose
complexed with anti-murine
IgG (16). Both these methods
yield chimeric antibody of sufficient purity for subsequent
functional analysis. Moreover, these methods can be applied to any murine V/human C chimeric antibody without
regard to its antigen specificity.
A and B: lane 1, molecular mass standards; lane 2, unpurIfied ascites fluid
from mice injected with cB6.2-producing transfectoma cells; lane 3, ascites
fluid after binding and elution of Igs from Protein A-agarose; lane 4, cB6.2 after
purification from ascites fluid by binding to Protein A, followed by chromatography on Sepharose-bound sheep anti-munnelgG; lane 5, B6.2 purified by
“Fast Protein Liquid Chromatography’ (LKB, Bromma, Sweden). C: lane 1,
unpurified ascites from mice injected withB6.2-producing transfectoma cells;
lane 2, the same material after binding and elution from Protein A; lane 3, the
same material after passage over anti-murine lgG-Sepharose; lane 4, purIfied
B6.2; upper arrow, purified B6.2; lower arrow, positionof purified cB6.2. From
reference 16; used with permission
are laborious and costly, it is critical that novel variants be
screened by various in vitro assays or in animal studies.
The nature of such analysis is dependent upon the particular antibody in question. To illustrate, I refer to a murine
V/human
C chimera of the murine antibody B6.2 (6), which
has specificity for human breast, colon, and lung carcinomas (17). Comparisons
of chimeric and murine B6.2 with
Characterization of Chimeric Antibodies
As described above, a virtually limitless array of new
antibody variants can be produced by genetic engineering.
The utility of each reagent for immunotherapy
must ultimately be determined in clinical trials. Because such trials
I
3
5
7
s
II
3
IS
I?
FUC3IOI
II
21
23
23
25
27
25
21
33
33
55Usd
Fig. 1. Purificationof a murine V/human C chimeric antibody by
chromatography
on a column of ProteinA-Sepharose
About 400 mL of tissue culture supemate containingabout 5 mg of chimenc
antibodyper milliliter was passed over a 2.0-mL column. Bound antibodywas
eluted witha linearpH gradientof 0.1 mot/Lcitrate
bufferfrom pH 6.5 to 3.0.
Fractions (1.0mL) were assayed forbovine arid chimeric antibody by ELISA.
The symbols designate (11) total protein, (+) bovine, and () chimeric
antibody (Chip Shoarman and Dawson Lawrie, unpublished)
1850
CLINICAL CHEMISTRY, Vol. 35, No. 9, 1989
Fig. 3. Indirectimmunofluorescentlabeling of human tumor cells
boundto murineor chimericB6.2
Top of each pair of photographs (upper-case lettering): cells bound with cB6.2,
then stained with rtiodamine-conjugated goat anti-human K. Bottom photographs (lower.case lettering): cells of the same line bound with 86.2, then
stained with fluorescein isothiocyanate.conjugated goat anti-munne lgG. The
cell lines are A549 (A,a),9812 (B,b), LS174T (C,c), SW900(E,e), and human
fibroblasts (F,!). InD,d, A549 cells were bound with total human (A) or murine
(a) lgG, then stained as above. From reference 16; used with permission
Fig. 5. Scintigraphicimages of mice bearing human tumors,24 h
after injectionof ‘251-IabeledcB6.2
In antibody-positiveLS174 tumor (A) the radionuclideis localized in the
xenograft.In antibody-negative
HCT-15tumor(B) the radlonuclideis foundin
the blood pool. In A, a small amountoffree 1251 hasbeenIncorporatedintothe
thyroid. Tumors were implanted in the right hind limb. From reference 16;used
with premission
rig Unbeled
Competitor
general,
their properties are similar to the parent murine
Their utility in immunotherapy
is being tested
clinical
trials,
the results of which should be
available over the next few years. It is hoped that, because
of their presumed greater immunocompatability
in patients, they will outperform
the murine antibodies
from
which they are derived. Meanwhile, new kinds of antibody
variants are being produced and tested (see below).
monoclonal.
in various
Increasing the Expression Level of Engineered Antibodies
o.a
1.0
Unbdsd
Competito
#{231}sg)
Fig.4. Competitive bindingof murine and chimencB6.2 to human
tumorantigen
(A) 66.2 was purified, labeled with I, and incubated with antigen coated on
microtiterwells in the presence of increasing amounts of unlabeled 66.2 (0),
cB6.2 (to), or anti-horseradish peroxidase (anti-HRP), an unrelated antibody
(0). (B) cB6.2 was purified, labeled with 1251, and Incubated with increasing
amounts of 66.2 (0), cB6.2 (A), or anti-HRP, an unrelated antibody (0). The
membrane was prepared from LS174T human colon carcinoma cells; 60-80%
of the input counts were bound.(C) cB6.2 wasboundto intact LS174Tcellsin
the presence of increasing amounts of 66.2 (0), or OKT9 (0), and the cells
were labeled by incubation with fluorescein isothiocyanate(FITC)-conjugated
goat anti-human lgG. 86.2 was bound to LS1741 cells in the presence of
increasing amounts of 86.2 (I), and the cells were labeled by incubation with
FITC-conjugated goat anti-munne lgG. Fluorescein-labeled OKT9 was bound
toLS174Tcellsin the presence of increasing amountsof 66.2. (#{149}).
Labeling
was quantifiedby flow cytometry; 70-85% of the cells were labeled. From
reference 16; used with permission
respect to a variety of biochemical,
biological, and immunologic characteristics
(6, 16) showed that, for example, the
murine and chimeric antibodies bound to the same panel of
cell types (Figure 3). Most important, competition studies
carried
out in the cell sorter or with in-vivo-labeled antibody demonstrated
that the chimeric and murine molecules competed for binding to antigen to an approximately
equal extent (Figure 4). This important result implied that
exchange of the human and murine C regions had little or
no effect on the ability of the antibody to bind antigen. In a
further
test, chimeric
B6.2 was used successfully to image
a human tumor implanted in a mouse (Figure 5).
Murine V/human C chimeric antibodies of many specificities now have been produced in several laboratories. In
A major issue in the widespread clinical application of
genetically
engineered antibodies
will be the ability to
produce them in large amounts. This is particularly
important because many immunotherapeutic
regimens involve
repeated administration
of relatively large doses of antibodies. Unfortunately,
engineered antibodies expressed by
transfection
into eukaryotic
hosts, e.g., myeloma cells,
often are not produced efficiently. Most “transfectomas”
produce 2-10 pg/1O6 cells per day; this is only 1-10% of the
production level of the best murine hybridomas.
The probable reason is that the transfected genes integrate into the
host chromosomes at random and, therefore, at loci that are
not necessarily optimal for expression. Although antibody
genes have been expressed in yeast and bacteria, there are
problems in chain association,
secretion,
glycation,
and
biological activity.
We have attempted to increase the expression of transfected antibody genes by linking them to genes that can be
amplified by a slow increase
in the concentrations
of
various drugs (18). As the number of copies of the amplifiable gene increases in response to the drug, the linked
antibody gene(s) is also amplified in copy number. This
amplification
results in an increase in gene expression and
thus increased secretion of antibody.
A commonly utilized gene-drug combination is the gene
coding for dihydrofolate reductase, which is amplified by the
drug methotrexate. We have adapted this combination for use
in myeloma cells; a representative
vector is shown in Figure
6. Using this vector, we have achieved a large increase in the
expression of transfected immunoglobulin
genes (18). Moreover, similar ampliflable vectors are now being used to express nonimmunoglobulin
genes in myeloma cells (19, and M.
Hendricks, Integrated Genetics, unpublished).
CLINICAL CHEMISTRY, Vol. 35, No. 9, 1989 1851
B.
SV40
SVIO
SOIl
Fig. 6. Structuresof plasmidscontainingchimericIg genes (derived
from munne hybridoma B6.2) and the methotrexate (MTX)-resistant
dihydrofolatereductase (DHFR) gene
Chimencheavy chain (A) or chimeric light chain (B)wereclonedadjacentto
mDHFR and designated DHFR/H and DHFR/L, respectively. From reference
18;used with permission
The Use of DNA Synthesis In AntIbody EngIneering
The chemical synthesis of DNA of desired nucleotide
sequence is an important
new technology
in molecular
biology. The process has now been automated,
and fragments about 100 nucleotides long can be made without
great difficulty. We have been applying this technique to
antibody engineering in a variety of ways.
The process of cloning desired V regions is laborious
because genomic libraries must be constructed and the tiny
minority (perhaps one in 106) of target clones must be
purified from the background. Moreover, a serious problem
is that aberrantly rearranged, and hence nonfunctional, V
regions are often obtained from the screen (20). These
problems can be circumvented
by synthesizing the desired
V regions de novo rather than cloning them.
Obviously, the DNA sequence of the region must be
determined before synthesis. Because mRNA that specifies
immunoglobulin
is so prevalent in hybridoma cells, this
can be accomplished
directly (21). Poly A mRNA is
isolated from the hybridoma and hybridized with a short
“universal” DNA primer synthesized with the knowledge of
the sequence of the common C region. The duplex is then
extended by using reverse transcriptase,
and the product is
sequenced. This sequence can be used to make specific
probes to screen libraries.
The more innovative approach is to synthesize the entire
V region. Although the sequence is too long (400-500
nucleotides) to synthesize in one piece, several alternative
methods are available to assemble the synthesized
fragments. One technique is to synthesize pairs of opposite
strands
with restriction sites at their ends and short overlaps. The overlaps are annealed, then the structure is filled
in with DNA polymerase. The resulting double-stranded
fragments are then ligated through the restriction sites at
their ends to form the finished structure. Depending on the
length, intermediate cloning may be required to generate
sufficient material for the next step. Care must be taken
that the engineered restriction sites do not interfere with
either the desired amino acid sequence or with physiologic
codon usage. The number of known restriction sites is so
large, however, that this is rarely a problem.
The advantage
of polymerase is that it reduces the
amount of DNA synthesis required.
The disadvantage is
that the error rate is relatively high. Because even a
one-base error can render the construct nonfunctional, each
1852 CLINICAL CHEMISTRY, Vol. 35, No. 9, 1989
piece must be sequenced after synthesis and after each
cloning step.
A second method involves synthesis of both complementary strands in their entirety. A 10- to 15-nucleotide “overhang” allows ligation of serial double-stranded fragments.
Surprisingly, one simply mixes a relatively large number
of single-stranded,
overlapping fragments. This complex
reaction results simultaneously
in opposite-strand hybridization and overlapping pair ligation to form the completed
structure in a single step. The synthesized V region is then
cloned, sequenced (for verification), and inserted into an
appropriate
expression vector.
As described above, one advantage of total V region
synthesis is ease and speed. A second advantage is avoidance of cloning aberrant
V regions. More importantly,
however, this method gives absolute control over the structure of the resulting gene. Thus, any variant can be created
easily. Further, mixing of nucleotides at nucleotide addition steps during the gene synthesis generates multiple
amino acids at particular positions. This new form of in
vitro mutagenesis is extremely powerful.
Replacement of Compiementarity-Determinlng Regions
Any antibody variant can be made by using the technique of total gene synthesis. Technical
limitations
include
possible problems with message or protein stability, chain
association, glycation, or secretion. The major limitations,
however, are the creative imagination,
the knowledge of
antibody structure/function
needed to design improved
antibodies, and the resources necessary to test them.
A major goal of antibody engineering is to make immunocompatible therapeutic agents. I have described chimeric
antibodies containing human C regions. These reagents,
however, are still capable of eliciting host immune response against portions of the rodent V regions (22). As an
example of the use of total gene synthesis, I now describe a
new antibody variant designed to reduce further, or eliminate, host response to therapeutic antibodies.
It has been known for some time that most antigen
specificity resides in defined portions of the V regions called
“hypervariable”
or “complementarity-determining”
regions (CDRs) (23). Spatially, the CDRs are looped away
from the V region framework, which consists of a pleated
sheet (24). These CDR loops form the antigen-combining
sites. Comparison
of V region sequence among immunoglobulins from a wide variety of sources shows that sequence variation is far greater in the CDRs than elsewhere
in the V region (25). Consequently,
it should be possible to
transplant the CDRS from a rodent antibody into a human
framework (3,10) and to maintain the antigen specificity of
the rodent parent antibody while eliminating host response
to the V region framework. Further, because the CDRs are
so variable, even within a species, they are unlikely to elicit
an immune response. Thus, host immune response should
be reduced greatly, or eliminated, by CDR replacement.
The three H-chain and three L-chain CDRS are interspersed with framework sequence. Thus, total gene synthesis represents the most practical way to make CDR-replaced variants. In our hands, the process is carried out as
shown in Figure 7. Poly A mRNA is isolated from the
target rodent hybridoma and sequenced as described previously. A computer search is made to identify a closely
related pair of human H and L chains. Alternatively,
one
can utilize the most related H and L chains and assume
that they will later be capable of association. The next step
Steps
__
Leader
1.
in CDR
Replacement
14 I O)IU
Isolate
poly
__
4
I
rF’
A4’ mRNA
CDR2
f
FR3
CDR3
FR4
from the target hybridoma
and sequence
by primary extension.
the H and L chain V regions
2.
Computer
search to
related human H and L chain
identify
V
3.
Design
civilized
imarine CDR
into
restriction
sites at
physiologic
codon
4.
Synthesize
S.
Ligate
civilized
V cxons
transfect and select.
6.
Test
the
civilized
engineered
CP, Hellstrom I. Chimeric mouse-human IgGi antibody that can
mediate lysis of cancer cells. Proc Nat! Acad Sci USA
1987;84:3429-43.
9. Morrison SL, Wims LA, Wallick S, Tan L, Oi VT. Genetically
engineered antibody molecules and their application [Review].
Ann NY Acad Sci 1988;507:187-98.
10. Riechmann L, Clark M, Waldman H, Winter G. Reshaping
human antibodies for therapy. Nature (London) 1988;332:323-7.
11. Boss MA, Kenton JH, Wood CR, Emtage JS. Assembly of
H and I. chain V exonswhich incorporate
a human framework.
Includeunique
the CDR/FR
usage.
V exons
boarders and maintain
and confmn
into expression
by sequencing.
rectors.
functional antibodies from immunoglobulin heavy and light
chains synthesized mE. coli. Nucleic Acids Res 1984;12:3791-806.
12. Wood CR, Boss MA, Kenton JH, Calvert JE, Roberts NA,
Emtage JS. The synthesis and in vivo assembly of functional
then
product.
Fig. 7. Procedure to generate a CDR-replaced antibody; arrows
indicatethe positionsof engineered,uniquerestrictionsites
sites rather
than by synthesis
and assembly
antibodies in yeast. Nature (London) 1985;314:44&-9.
13. Potter H, Weir L, Leder P. Enhancer dependent expression
of
human K immunoglobulin genes introduced into mouse pre-B
lymphocytes by electroporation. Proc Natl Acad Sci USA
is to design the CDR-replaced variant by incorporating the
rodent CDRS into the human frameworks. Unfortunately,
the exact borders of the CDRs are not clear and probably
vary among different antibodies. Thus, it is not always
obvious exactly which amino acids to change to human and
which to leave as rodent. The goal, of course, is maximal
antigen
binding
with minimal
host immune
response.
Much of our research
is directed towards learning
rules
that will help dictate these choices. When suitable CDRreplaced
sequences for H and L chains have been designed,
they are synthesized,
cloned, sequenced,
expressed,
and
tested. A further
feature of the scheme shown in Figure 7 is
that unique restriction
sites have been engineered
at the
CDR/framework
borders. In the future, new antibody specificities will be generated
by insertion of synthesized
CDRS
at these
possessing
novel
effector
functions.
Nature
(London)
1984;312:604-8.
4. Jones PT, Dear PH, Foote J, Neuberger MS, Winter G. Replacing the complementarity-determining
regions in a human antibody with those from a mouse. Nature (London) 1986;321:522-5.
5. Morrison SL, Johnson MJ, Herzenberg LA, Oi VT. Chimeric
human antibody molecules: mouse antigen-binding domains with
human constant region domains. Proc Nat! Acad Sci USA
1984;81:6851-5.
6. Sahagan BG, Dorai H, Saltzgaber-Muller
J, et al. A genetically
engineered murine/human chimeric antibody retains specificity
for human tumor-associated antigen. J Imunol 1986;137:1066-74.
7. Beidler CB, Ludwig TR, Cardenas HJ, et al. Cloning and high
level expression of a chimeric antibody with specificity for human
carcinoembryonic antigen. J Immunol 1988;141:4053-60.
8. Liu AY, Robinson RR, Helistrom KE, Murray Jr ED, Chang
of the
entire V regions. This will result in a significant saving of
effort.
One CDR-replaced antibody has been described by Riechmann et al. (10), and others are being evaluated in our
laboratory and elsewhere. Many other engineered antibody
variants are also being produced and studied. At the least,
this work will result in a greater understanding
of immunoglobulin structure and function. New molecules will be
produced that can be applied successfully for immunotherapy.
References
1. Kohler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined
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(London)
1975:256:495-7.
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14. Sandri-Goldin RM, Goldin AL, Levine M, Glorioso JC. Highfrequency transfer of cloned herpes simplex virus type 1 sequences
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body molecules. Clin Chem 1988;34:1668-75.
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17. Kufe DS, Nadler L, Sargent
L, et al. Biological
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hmnan breast carcinoma-associated
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cellular proliferation. Cancer Res 1983;43:851-9.
18. Dorai H, Moore GP. The effect of dihydrofolate reductasemediated gene amplification on the expression of transfected
immunoglobulin genes. J bumunol 1987;139:4232-41.
19. Hendricks MB, Banker MJ, McLaughlin A. A high efficiency
vector for expression
1988;64:43-51.
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in myeloma
cells. Gene
20. Yancopoulos GD. Alt FW. Regulation of the assembly and
expression of variable-region
genes [Review]. Annu Rev Immunol
1986;3:339-68.
21. Hanlyn PH, Brownlee GO, Cheng CC, Gait MJ, Milstein C.
Complete sequence of constant and 3’ noncoding regions of an
immunoglobulin
mRNA using the dideoxynucleotide method of
RNA sequencing. Cell 1978;15:1067-75.
22. Jerne NK. Towards a network theory of the immune system.
Ann Imniunol (Paris) 1974;125c:373-89.
23. Kabat EA, Wu T1. Attempts to locate complementaritydetermining residues in the variable positions of light and heavy
chains. Ann NY Acad Sci 1971;190:382-93.
24. Davies DR, Padlan EA. Correlations between antigen-binding
specificity and the three dimensional structure of the antibody
combining site. In: Haber E, Krause RM, eds. Antibodies in human
diagnosis and therapy. New York: Raven Press, 1977:119-28.
25. Wu ‘VF, Kabat EA. An analysis of the sequences of the
variable regions of Bence Jones proteins and myeloma light chains
and their implication for antibody complementarity.
J Exp Med
1978;132:211-50.
CLINICAL CHEMISTRY, Vol. 35, No. 9, 1989 1853