Download Chimerization of antibodies by isolation of rearranged genomic

Gene, 106 (1991) 273-277
c 1991 Elsevier Science Publishers
GENE
B.V. All rights reserved.
273
0378-l 119/91/$03.50
06068
Chimerization of antibodies by isolation of rearranged genomic variable regions by the polymerase chain
reaction
(Hybridoma;
transfectoma;
Winfried Weissenhorn”,
<’Institut ftir Immunologie
force cloning;
Elisabeth Weiss”,
cassette
D-8122
PCR;
recombinant
DNA)
Marina Schwirzke b, Brigitte Kaluza b and Ulrich H. Weidle b
der Universitiit Miinchen,
Abteilung ftir DNS-Neukombination,
vectors;
D-8000
Penzberg
Munich
(F.R.G.)
Tel. (49~89)5996-689;
and h Boehringer
Mannheim
GmbH,
(F.R.G.)
Received by H.G. Zachau: 14 January 1991
Revised/Accepted:
14 April/IO July 1991
Received at publishers: 30 July 1991
SUMMARY
We describe a new method for amplification, by polymerase chain reaction (PCR), of rearranged segments encoding the
variable part of light and heavy chains of an antibody (Ab) from the chromosomal
DNA of hybridoma cells for the
chimerization ofAbs. A fundamental prerequisite for this is the knowledge ofthe exact sequences in the 5’-untranslated
region
of light and heavy chain mRNA, and of the joining segment used for rearrangement.
This allows the design of nondegenerated
oligodeoxyribonucleotides
for PCR. The primer design permits directional cloning of the amplified, promoterless fragments
into cassette vectors, in which they will be linked to the appropriate human constant domains and immunoglobulin
(Ig)
promoter/enhancer
elements. The method is illustrated for chimerization
of an Ab directed against the human T-lymphocyte
antigen, CD4. The chimerized Ab is secreted in abundant
quantities after transfection
of the engineered plasmids into
non-Ig-producing
myeloma cells.
INTRODUCTION
The benefits of mAbs in the treatment of human diseases,
such as autoimmune
diseases, graft rejections and cancer
Correspondence to: Dr. U.H.
Abteilung
Weidle,
Boehringer
fur DNS-Neukombination,
Nonnenwald
Mannheim
GmbH,
2, Postfach
1152,
D-8122 Penzberg (F.R.G.)
Tel. (49-8856)60-2801;
Fax (49-8856)8744.
Abbreviations:
aa, amino acid(s); Ab, antibody;
pair(s);
C, constant
diversity
region;
region; cDNA,
ELISA,
Ap, ampicillin;
DNA complementary
enzyme-linked
bp, base
to mRNA;
immunosorbent
assay;
D,
Fab
fragments, antigen-binding
fragments of Abs that are derived by papain
digestion and contain the light chain and part ofthe heavy chain (variable
region and first constant
bulin; J, joining
domain);
H, heavy chain of Ab; Ig, immunoglo-
region; kb, kilobase
or 1000 bp; L, light chain of Ab;
mAb, monoclonal Ab; nt, nucleotide(s);
oligo, oligodeoxyribonucleotide;
PA, polyacrylamide;
PCR, polymerase
chain reaction;
ss, single
strand(ed);
c/r, untranslated
variable light chain.
region;
V,,,
variable
heavy
chain;
V,
,
are now widely accepted. A major progress toward therapeutic applicability was the construction
of chimeric Abs
with variable
rodent
and constant
human
domains
(Verhoeyen and Riechmann,
1988). Substantially lower immunogenicity has been found for chimeric Abs compared to
their murine counterparts
in colon cancer patients (Lo
Buglio et al., 1989). With the advent of PCR technology
(Saiki et al., 1985; 1988) cloning of cDNAs for L and H
chains of Ig genes has been dramatically
facilitated by
making use of degenerate
oligos (LeBceuf et al., 1989;
Orlandi et al., 1989; Huse et al., 1989; Larrick et al., 1989).
For expression, the V, and I’, regions have to be cloned
into vectors carrying the appropriate
constant
human
region thus mimicking Zg genes. In contrast to Ig cDNAs,
Zg genes are well expressed in Ig nonproducer
hybridoma
cells (Weidle et al., 1987). For this purpose, the isolated V
regions have to be mutagenized to match the reading frame
of the 1g gene on the vector. Alternately, restriction sites
have to be incorporated into the primers for directed cloning
274
and expression (Orlandi et al., 1989). In this paper we
present a different approach for cloning the relevant VLJ
and V,r>J regions with non-degenerate
primers. Our technique includes PCR amplification of specific genomic DNA
fragments and their subsequent force-cloning into cassette
vectors. To our knowledge, this is the first report on the
cloning of rearranged V,_J and V,OJ regions from genomic
DNA by PCR. In contrast to the procedure described by
Orlandi et al. (1989), the expressed Ab gene retains its
authentic signal peptide sequence (including introns) and
does not differ at the N-terminal aa from the original murine
a: 5'-GATCGTCGACAAGACTCAGCCTGGACATGATGTCC
Primer
sail
Primer b:
Primer c: 5'-CATAGTCGACGTTAGTCTTAAGGCACCACTGAGCC
-............,.......*....
&/I
Primer d:
used for ampli~~ation
region, c and d for the amplification
Primers
(a) Principle of the method for chimerization
The most important prerequisite for the new method of
chimerization
is the knowledge of the exact sequence of the
5’UT region of the mRNA and of the 3 region used for the
V,.J and V,,DJ rearrangement
for the L and H chain of the
Ab. This information
allows the design of nondegenerate
primers for the isolation of the relevant V,J and V,,DJ
a. b and c are 35.mers,
nt for primers
tvere synthesized
synthesizer.
a G/I
of V, J and Y,,DJ regions ofm4b
I by PCR. Primers a and b were used for amplification
of matching
A:
5'UT+a
5'-GAGCGCGGCCGCTTAAAAATAAAGACCTGGAGAGGCCA
. . . . . .. . . .. .. . . . .. . . . . . . . .
Notl
Intron J4-,za
Fig. 1. Primers
MT15
AND DISCUSSION
.. .. . . . .. . ..*.*......
5'UT
5'-ATCAGCGGCCGCACTTAACAAGGTTAGACTTAGTG
Not,
.."..intron.~;.'......
Ab.
EXPERIMENTAL
'...
and primer d is a 3X-mer. The numbers
a, b, E and d are 25, 23. 25 and 26. Primers
on an Applied
The 5’ primers
Riosystems
contain
matching
encoding
(Foster
part corresponds
The 3’ primers
part
four random
part
23 nt corresponding
to nt in the f’C’7
contain
nt and a lYorl recognition
to the J,
gene and to the J4 intron of the H-chain
tion sites are underlined.
City, CA) oligo
in their overhanging part four nt and
re~ognitiol~ site, the matching
region of ti and the yzl, mRNA.
matching
of the V’~.I
of the V, ,DJ region of mAb MT1 5 1.
Dashed
lines represent
intron
encoding
in their nonsite, in their
of the L-chain
gene. Restric-
Ig gene sequences.
Liiht chain
CAP
Pwner 2
Noti
PCR
s
1
S ‘4
Jz
Sal I
5’UT
B:
lntron
lntron
Light chain
q lg promoter
n Signal sequence
q Variable region
q Joining region
.
Enhancer
H
Mouse constant region
@ 5’ untranslated region
q lntr0rl
Heavy chain
CAP
Heavy chain
q lg promoter
n Signal sequence
q Variable ragion
q Divarsity ragion
•$ Joining region
of the chimerization method. The procedure
to the 5’UT region of the mRNA and to intron
Fig. 2. Schematic representation
with primers
complementary
Enhancer
q Exons of mouse
constant region
q 5’ untranslated
0
region
lntron
is shown for the L chain (A) and for the H chain (B). PCR amplification
sequences 3’ to the J segment used for rearrangement
leads to V, J and
V,,DJ fragments with a .%/I site at their 5’ ends and a N<J!I site at their 3’ ends for directed
and the appropriate
human constant regions. A general outline of the procedure is described
mRNA; I), diversity region; E, and E,,, enhancers
region of L and H Ig genes.
e
cloning into cassette vectors with Ig promoter elements
in section a. C, constant region: CAP, initiation site for
for light and heavy chain; H. hinge region; J, joining region;
S, signal sequence:
V, and V,,, variable
275
regions from DNA of the hybridoma cell line by PCR. The
primers (Fig. 1) are designed as follows: (a) and (c) 5’
primers matching to the 5’ UT regions of L and H chain
mRNA. They include at their 5’ end a restriction enzyme
cleavage site (SalI) useful for force cloning. Primers (b) and
(d) are the 3’ primers matching the intron sequences downstream from the J region used for the rearrangement.
This
is possible because the J loci for the L and H chains of the
ABCDEFGH
kb
mouse are completely
sequenced
(Max, 1981; Sakano
et al., 1980). The 3’ primers include a Not1 cleavage site at
their 5’ ends for force cloning of the amplified fragments.
As a result of the amplification procedure promoterless I’,__!
and V,OJ fragments are obtained including its intron, the
V,J or V,OJ region and intron sequences 3’ from the J
region used for rearrangement
including the splice donor
consensus
GT (Fig. 2). The resulting amplified V,J and
V,OJ fragments are endowed with a SafI site at their 5’
ends and a Not1 site at their 3’ ends for directed cloning into
a cassette vector containing Ig promoter elements and appropriate
human
constant
regions.
Fig. 3. Amplification
by PCR. Lanes:
of V,J and V,,DJ regions
A and H, marker
(minus DNA); C, amplification
tion with yprimers
with K primers
(b) Application of the technique to the chimerization of an
anti-CD4 antibody
The mAb MT1 5 1 (IgG,,) binds to an epitope in the first
extracellular loop of the human T lymphocyte surface antigen CD4 (Reinherz et al., 1986). The murine Ab has been
successfully used in the treatment of patients with rheumatoid arthritis (Herzog et al., 1987). Therefore, a chimerized, less immunogenic version of this Ab is of great importance for clinical applications. The sequence of the 5’ UT
and the V regions of the K and 1/2, chains of mAb MT151
was determined
as described in the legend to Fig. 3. We
identified rearrangement
of V, with the J2 segment of the L
chains and of V, with J4 of the H chains. This information
enabled us to synthesize 5’ and 3’ primers for amplification
of the rearranged
VJ and VDJ segment by PCR (Fig. 1).
For the light chain, a 600-bp V,J fragment, and for the
heavy chain, an approx. 700-bp VDJ fragment was amplified (Fig. 3). The fragments with Sal1 and Not1 sites were
subcloned and the sequences of live isolates for each chain
were determined
(Sanger et al., 1977). Compared to the
cDNA sequence we detected an exchange in one of the five
V,J isolates as well as in one of the five V,DJ isolates due
to the high error rate of Taq polymerase. We have applied
the described technique to isolate the V,J and V,DJ segments of the two additional mAbs, one directed against an
epitope on human CD4 different from that of mAb MT 15 1
and another binding to the cc-chain of the human interleukin-2 receptor. Also for these two mAbs fragments in the
range of 600-700 bp were amplified (data not shown). Sequencing of live isolates of subcloned
V,J and V,DJ
regions for each Ab revealed that the error rate of Taq
polymerase
was comparable
to that obtained for mAb
of primers
(250 pg) was dissolved
of cDNA
labelled primers
fragments
visualized
residues
with
y primer: 5’-GGCCAGTGGATAGAmatch the 5’ part of the constant regions of
acetate
precipitation,
the ssDNAs
precipitated,
and tailed with G
deoxynucleotidyl-transferase
in 10 ~1 H,O.
(Maniatis
and ethanol precipitation,
3 ~1 were used for the following
HCl pH 8.4/50 mM KCl/l mM MgClJ0.1
polymerase.
PCR reaction.
CCCCCCCCCCC
(contains
end and matches
3’
primer
72°C
K:
sites for SUCH, SphI, Nor1 and SJI;
Loh
contains
an EcoRI cleavage
site near the 5’
regions
of K and
5’-GGGAATTCTGGTGCAGCATCAGCCC.
out in a Perkin-Elmer
5 min at 94°C
1min at 94°C
10 min at 72°C. An aliquot
with EcoRI +Notl,
fication
with primers
KQl.5
mM MgCI,/O.l
five cycles (1 min each)
gel, the amplified
and
The
with the following
20 cycles at 72°C
of the PCR products
subcloned
dideoxy method (Sanger
cycler
2 min at 42°C
1 min at 55’C,
on a 1 y0 low-melting-agarose
3’ primer
Taq
7: 5’-GGGAATTCGATAGACAGATGGGGGTG.
was carried
conditions:
and
the nt of the 5’ part of the constant
3’ primer
reaction
in 10 mM
mM dNTPs
5’ primer: 5’-GCATGCGCGCGGGCCGCGGAGGCCC-
et al., 1989). The 3’ primer
y mRNA.
et al.,
the ss DNA was
10 pmol of 5’ and 3’ oligos were used in a 20~1 reaction
Tris
were
of the Vt, and I’,,
The bands were excised, DNA
at 37°C
1982). After phenol extraction
dissolved
of the ss DNA with 50 pg RNase
and ethanol
by autoradiography.
terminal
synthesis
k primer: 5’-GAA-
on a 5% PA gel and the position
eluted with 2 M NH,.
in H,O.
buffer of the BRL cDNA
ofthe manufacturer.
the K and the y chain. After treatment
A for 30 min, phenolization
and dissolved
with 200 pg RNA and 20 ng “P-
with the first-strand
GATGGATACAGTTGGTGC.
CAGATGG.
These primers
RNA
with 375 ~1 dimethylsul-
ethanol-precipitated
was performed
determination
et al., 1979). The RNA pellet
in 12.5 ~1 H,O, incubated
to the instructions
size-fractionated
PCR
a, b, c and d are shown
For cDNA cloning and sequence
from 2 x 10’ cells (Chirgwin
kit according
mAb MT151
different from those shown in Fig. 1; G, amplification
foxide for 20 min at 45°C
Synthesis
encoding
B and F, control
with primers c and d; D and E, amplifica-
a and b. The sequences
in Fig. 1. Methods.
was isolated
fragments;
the
was size-fractionated
DNA was eluted, cleaved
sequence
et al., 1977). Reaction
at
(1 min each),
determined
conditions
by the
for PCR ampli-
a, b, c and d: 10 mM Tris. HCI pH 8.3/50 mM
% (w/v) gelatine/l
1 ng each/dATP,
dGTP,
pg chromosomal
DNA/s’
dCTP, dTTP 200 PM each/Taq
and
poly-
merase (Boehringer-Mannheim)
2.5 units in a reaction volume of 100 ~1.
The reaction mixture was overlayered
with 100 ~1 mineral oil (Sigma).
The reaction mixture was kept at 92°C for 5 min, then for 2 min at 55°C
and 2 min at 72°C. Before the following
26 cycles at 72°C (2 min each)
the mixture was kept for 2 min at the temperatures
of the mix was size-fractionated
(0.5 ng ethidium
bromide/ml),
indicated
on a 1.5 y0 low-melting-point
the amplified
with Sal1 + Not1 and ethanol-precipitated.
above. 10%
agarose
DNA was isolated,
gel
cleaved
276
MT151. The size of the intron which separates the signal
sequences was in the range between 60 and 120 nt, when the
six isolates of the three mAbs were compared (data not
shown). All three mAbs make use of different J segments.
From these observations we conclude that the method is of
general applicability. After sequencing, the amplified S&INot1 fragments (V,J and V’,OJ) of mAb MT151 were
force-cloned into cassette vectors ~UHWK- (with a human
K constant region) and pUHWyl
(with a human ~1 constant region). The vectors are displayed in Fig. 4. Both
vectors make use of heavy-chain enhancer and promoter
elements at the 5’ end of the V,,J and V, DJ fragments to
be inserted by force-cloning and can be linearized at their
unique Paul sites in the ApK gene before transfection
into
hybridoma
cells.
Plasmids
~UHWICCD~
and
pUHWyCD4
obtained after ligation of the appropriate V,J
and V,DJ fragments into ~UHWK and pUHWy1 were
transfected into Sp2/0 cells (Ochi et al., 1983) and stable
transformants
were isolated by G418 selection. Transformants expressing up to 10 pg/ml of chimerized Ab per 10h
cells and 24 h were obtained.
‘?‘I
Knnl
human intron K
AP
pUHWK:
1
u40,
B
Sal
Kpnl ,I,,,
I
3’UT
mouse intron
Not1
heavy chain
pUHW),i
C ~‘1 (human)
CHz
Fig. 4. Expression
cells. Amplified
vectors for chimeric Ab in non-Ig producer
V,J and VHDJ fragments
were cloned as SalI-Not1
and pUHWy1
vectors
moter
pUHW-CD~K
vectors
Plasmid
contain
including
contains
the constant
(Southern
hybrid
intron
light-chain
contains
EcoRI-Asp700
with
the mouse
fragment
from
by forced
cloning.
(Picard
pC1 (Klobeck
As pro-
of a heavy-chain
unique KpnI, Sal1 and Not1 sites.
region of a human
K gene and is
and Berg, 1982). The plasmid
mouse
and
light-chain
human
elements.
enhancer
region
and Schaffner,
human light chain intron, the human
fragment
pUHW ti (A),
to give rise to
(PPr) (Weidle et al., 1987). Both
a polylinker
~UHWK
vectors
a combination
(E,,), and a p gene promoter
contain
hybridoma
mAb MT15 1 (Fig. 3)
with Sal1 +NotI)
and pUHW-CD4yl
both vectors
based on pSV2neo
~UHWK
into cassette
(B) (which were cleaved
elements
enhancer
fragments
encoding
contains
a
Plasmid
as a 2-kb
1984) and a part of the
K exon and 3’ (iTregion
et al., 1984). Plasmid
as a 2.8-kb
pUHWy1
is the
cassette vector for the insertion of amplified V,,DJ fragments. It contains
the mouse heavy-chain enhancer region as a 1.6-kb HindIII-EcoRI
fragment (Neuberger.
1983) and part of the human heavy-chain
four exons and three introns and 3’ UT region of the human
a 7-kb Hind111 fragment
(Takahashi
CD~K and pUHW-CD4yl
were linearized
an equimolar
mixture ofboth
plasmids
(c) Conclusions
(I) We demonstrated
for the first time the feasibility of
cloning rearranged genomic V,J and V,DJ segments of Ab
with PCR and nondegenerate
oligos.
(2) After the PCR, the amplified promoterless
fragments can be directionally
cloned into cassette vectors
carrying the appropriate human constant regions.
(3) The method seems to be of general applicability
because it has been applied successfully to the chimerization of Ab from three different hybridoma cell lines.
(4) Contrary to previous approaches
making use of
cDNA cloning, mutation and insertion into expression vectors, authentic
V,J and V,DJ
genomic segments are
isolated by our method.
(5) High-level synthesis of chimerized Ab was monitored after transfection of expression vectors with human K
and yl constant regions and the V,J and V,DJ regions of
the murine Ab into non-Ig-producer
myeloma cells.
et al., 1984). Plasmids
intron, the
~1 gene as
pUHW-
at their unique PvuI sites and
(5 pg per 10h cells) was transfected
ACKNOWLEDGEMENTS
Our work was partly supported by BMFT (Bundesministerium fiir Forschung und Technologie) and Genzentrum Miinchen. Thanks are due to Dr. Sandro Rusconi for
reading this paper instead of a dull magazine in the train and
Brigitte Kindermann
for help with the preparation
of the
into Sp2iO cells (Ochi et al., 1983) by electroporation.
Stable transfectants were isolated by selection with G418 (1000 pgjml) as described
(Lenz and Weidle, 1990) and reconstituted
Ab was quantified
by ELISA.
PPr, promoter of a mouse /.I gene; E, and E,,, enhancers
of light and
heavy chains of the mouse. respectively:
3’ UT, 3’ untranslated
region;
A,,(SVJO), polyadenylation
signal derived from simian virus 40; H, exon
coding for hinge region; Ch,, CH,, CH,, exons of the human ;‘l gene.
Arrows
indicate
the direction
of transcription.
277
manuscript.
Zachau
We are indebted
for providing
to Drs. T. Honjo
and H.G.
Neuberger,
M.: Expression
chain
us with plasmids.
gene
transfected
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of immunoglobulin
into
ceils.
lymphoid
EMBO
heavy
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1373-1378.
Ochi,
A., Hawley,
Kohler,
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