From the
A human monoclonal antibody (HuA) specific for blood group A
substance with two fucose groups was found to be immunochemically
almost identical with that of a previously characterized mouse
monoclonal anti-A, AC-1001. The V
The cloned HuA V
The ABO blood group antigens are carbohydrate structures present
in many tissues and synthesized under the control of several recently
characterized and highly related genes coding for glycosyltransferases
(1, 2). They may be expressed in different forms: as oligosaccharides
in urine, as glycoproteins in body secretions and tissue fluids, and as
glycolipids in membranes
(3) . Their expression varies during
differentiation and in different tissues, and they may be present at
abnormal levels or in altered forms in malignant and premalignant
lesions (reviewed in Ref. 4). Human antibodies of defined specificity
to blood group antigens are thus of potential importance in the
diagnosis, prognosis, and treatment of certain malignancies. Although
human monoclonal antibodies have been very difficult to produce in
amounts necessary for clinical use, the technology is rapidly advancing
(5). For such purposes, as well as for gaining a fundamental
understanding of the expression of these antigens in differentiation
and malignant transformation, antibodies with carefully defined
specificities will be necessary.
The smallest determinant showing A
specificity is the trisaccharide: GalNAc(
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy Type 1 and type 2 determinants can be further modified by the
addition of a second fucose linked either (
We have characterized the fine specificity and immunochemical
properties of HuA, a human anti-A produced by cloned Epstein Barr
virus-transformed lymphocytes that were stabilized by fusion with a
human-mouse fusion partner
(21) . This antibody is specific for
difucosyl A antigens of either type 1 or type 2 and is virtually
identical with a previously characterized mouse monoclonal antibody,
AC-1001
(8, 22) , in its pattern of inhibition by
oligosaccharides from blood group A substances. This gave us a unique
opportunity to compare the sequences of heavy and light chain genes
from two different species that encode antibodies with specificity for
the same carbohydrate epitope.
cDNA from the
HuA cell line and its parental fusion partner were subjected to PCR in
parallel. Aliquots of PCR products were electrophoresed in 2% agarose
gels. PCR products with bands of the expected size (approximately 450
base pairs) present in the anti-A cDNA but not in the parental cell
line's cDNA were digested with HindIII and
EcoRI, electrophoresed in polyacrylamide gel electrophoresis
gels, and appropriate sized bands were cut out, electroeluted, and
ligated into pUC 18. Dideoxynucleotide chain termination sequencing of
both strands was carried out using a modified T7 DNA Polymerase
(Sequenase) according to the manufacturer's protocol. Two
separate clones with the heavy chain were sequenced and were found to
be identical.
The immunochemical properties and fine specificity of HuA
were remarkably similar to those of a previously characterized mouse
monoclonal, AC-1001, more similar in fact than any two previosuly
characterized mouse blood group antibodies
(8) . Despite this
similarity, they use radically different heavy and light chain genes.
The human V
Although the two
V
The data
from the panel of mouse monoclonal anti-A antibodies reported
previously
(22) do not shed much light on the mechanism by which
the human and mouse antibodies achieve their similar binding
specificities. The mouse monoclonals AC-1001 and A003 = 40/5G7
were similar immunochemically and used very similar heavy chains but
different light chains. This suggested that the similarity in binding
was conferred by the heavy chain. Based on that conclusion, one might
expect that the HuA and AC-1001 heavy chains might be closely related.
Our results, however, demonstrate quite the opposite, and our
transfection experiments show that for HuA both V
Chothia et
al.(43) have begun to improve modelling techniques for
immunoglobulins. They have started to define critical residues both
within the CDRs and outside of the CDRs that influence the conformation
of the amino acid backbone of the CDR loops. Using the residues that
they define as canonical structures of the CDRs, the mouse AC-1001 and
human anti-A antibodies may share similar conformations in the light
chain CDRs but appear to differ markedly in the conformation of the
heavy chain CDRs. Most likely, the two antibodies have significant
differences both in the conformation of their CDRs and in the amino
acid side chains used in antigen contact.
In the
In other
systems it is clear that different amino acid sequences can be used to
create molecules with similar three-dimensional structures and binding
capacities. For example, hemoglobin and myoglobin molecules from
different species vary in up to 80-89% of the amino acids used
and yet maintain remarkably similar three-dimensional structures and
similar functions
(58, 59) . Other examples of molecular
mimicry exist as well: for example, an anti-idiotypic antibody can
mimic the steroid hormone, aldosterone, and displace it from its
receptor with similar kinetics
(60) . What is being recognized is
not a primary structure, but the three-dimensional arrangement of
nuclei and electrons constructed in one case by a steroid hormone and
in the other from a polypeptide.
The remarkably similar
immunochemical behavior of the human and mouse anti-A antibodies is not
readily explained by similarities in either their primary structures or
in crude predictions of the tertiary conformation of their CDRs. Either
the two antibodies form similar antigen binding sites in very different
ways or, alternatively, the two antibodies see the antigen in
topographically distinct ways. Only x-ray crystallography or much more
sophisticated molecular modeling techniques than are currently
available will resolve this question. The comparison of the primary
nucleotide and amino acid sequences of the human and mouse antibodies
demonstrates how completely different primary heavy and light chain
structures can be used to recognize the same well-defined epitope, a
possibility predicted by Wu and Kabat (61). These data suggest that
germline antibody genes are not selected through evolution on the basis
of their ability to bind one specific antigen but rather that the
repertoire as a whole has evolved to ensure maximum range of binding
activities. Redundancy, or the ability to solve particular topographic
problems in multiple ways, is probably a part of this kind of broad
repertoire and ensures that the organism is protected against
catastrophic loss of germline V
NT, not tested; R = 3-hexene-1,2,5,6-tetrol. The following mono- and
oligosaccharides at the amounts tested gave no significant inhibition
with the monoclonal anti-A: &cjs2111;, D-GalNAc; ,
L-Fuc; ◊, D-Gal; , 04 [Fuc
On-line formulae not verified for accuracy ); &cjs0383;, B-penta (Gal
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy ).
The nucleotide sequence(s) reported in this paper has been
submitted to the GenBank
We are indebted to Dr. Denong Wang for reviewing the
manuscript.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
and V
chain
cDNAs of HuA were sequenced and compared with those of AC-1001. The
human and mouse antibodies used V
and V
genes
that came from different families and shared minimal nucleotide and
amino acid sequence identity. Thus, two antibodies from two different
species can use evolutionarily unrelated sequences to bind the same
carbohydrate epitope.
and V
genes were then transfected into a mouse myeloma cell line and
re-expressed, together, and each separately with an irrelevant V
or V
. Only the original HuA V
and V
had anti-A activity, demonstrating that both the heavy and light
chains contributed to specificity.
1
3)[Fuc(
1
2)]Gal
1, but A-specific antisera
and monoclonal antibodies may vary in their reactivity, depending on
additional adjacent sugars and the ways in which they are
linked
(3, 6, 7) . The A trisaccharide can be
linked either
1
3 or
1
4 to GlcNAc in type 1
and type 2 A antigens, or it may be linked
1
3 to
GalNAc
or GalNAc
in type 3 and type 4 structures
1
4) or (
1
3) to the GlcNAc
(2) . These various A structures differ
immunochemically because the adjacent sugars may actually participate
in the antigenic site or they may influence the orientation of the A
trisaccharide itself
(7) . Monoclonal antibodies have been very
important in defining these
structures
(8, 9, 10, 11, 12, 13, 14) ,
and then, subsequently, in characterizing their expression in various
tissues, at different stages of differentiation, and in malignant
transformation
(15, 16, 17, 18, 19, 20) .
Cell Line
The human-mouse trioma cell line
secreting HuA was described previously
(21) . It is a cloned
Epstein Barr virus-transformed B cell line stabilized by fusion with
the human-mouse fusion partner SBC-H20. In tissue culture, it secretes
substantial amounts of anti-A monoclonal antibody (1-5
mg/liter)
(21) .
Immunochemical Assays
Quantitative precipitin
assays, quantitative inhibition ELISAs,(
)
and
ELISAs to detect anti-blood group activity from transfectoma
supernatants were carried out using blood group substances and
oligosaccharides as described previously
(8) . In quantitative
precipitin assays, 5-8 µg of affinity-purified antibody N
(nitrogen) was used per determination in a total volume of 200 µl.
Antibody N in washed precipitates was determined by the ninhydrin
method
(23) . For the quantitative inhibition ELISAs, blood group
A substance (Hog 4 10%) at 1 ng/ml in pH 8 borate-buffered saline was
used to coat polystyrene plates. Assays were carried out as
described
(8) , and the free energy of binding,
G
, relative to the A-trisaccharide was
calculated according to the standard formula:
G
=273R ln(x/y)
(8) , where x is the
amount of inhibitor in nanomoles giving 50% inhibition at 0 °C, and
y is the amount of A-trisaccharide giving 50% inhibition. This
permits comparison of antibody binding of the various oligosaccharides
to that of the simple trisaccharide structure.
Isolation and Sequencing of Anti-A Heavy and Light Chain
cDNAs
Before extracting RNA, culture supernatants from the HuA
cell line and the parental fusion partner were retested for anti-A
activity. The supernatant from the anti-A cell line specifically
agglutinated human type A red blood cells and had anti-A activity by
Ouchterlony diffusion and ELISA, while the supernatant of the parental
cell line did not. Total RNA was isolated from 10 cells
using guanidinium thiocyanate and cesium chloride gradient
centrifugation, first strand cDNA was prepared, and heavy and light
chain cDNAs were amplified using the polymerase chain reaction (PCR) as
described
(24) . We used the 5` and 3` primers described by
Larrick et al.(24) . For the heavy chain, oligo(dT)
priming was used to make first strand cDNA. For PCR, a mixture of three
degenerate 5` primers based on the leader sequences of each of the
three subgroups of human V
genes (as defined by Kabat
et al.(25) ) were used along with the 3` IgM primer.
For the light chain, the 3`
constant region primer was used to
make first strand cDNA, and a degenerate 5` primer based on
leader sequences was used with it in PCR. The 5` primers all have
EcoRI restriction sites, and the 3` primers have
HindIII sites to facilitate subcloning into vectors for
sequencing. The sequences of the primers used are as follows with the
bases shown within parentheses having been present in equimolar amounts
during synthesis of a particular position. V
,
5`-gggaattcatggactggacctggagg(ag)tc(ct)tct(gt)c-3`,
5`-gggaattcatggag(ct)ttgggctga(cg)ctgg(cg)tt-3`,
5`-gggaattcatg(ag)a(ac)(ac)(at)act(gt)tg(gt)(at)(cgt)c(at)(ct)(cg)ct(ct)ctg-3`;
C
, 5`-ccaagcttagacgagggggaaaagggtt; V
,
5`-gggaattcatggacatg(ag)(ag)(ag)(agt)(ct)cc(act)(acg)g(ct)(gt)ca(cg)ctt-3`;
C
, 5`-ccaagctttcatcagatggcgggaagat.
Transfection of HuA V
In order to determine if
either chain retained anti-A activity when coupled with an irrelevant
heavy or light chain, transfection experiments were carried out as
described (26). The coding sequences of HuA Vand V
Chain Shuffling Experiments
and V
were amplified by PCR using upstream primers containing an
NcoI site overlapping the translation start codon ATG and
downstream primers containing KpnI sites located in the J
segments. The NcoI-KpnI fragments were gel-purified,
resequenced, and inserted into the Ig heavy and light chain expression
vectors pSV2-
Hgpt and
pSV2-
Hneo(26) , modified by the addition of
NcoI and KpnI sites. The expression vectors
containing the V
and V
genes from a human
B-cell lymphoma (RF) were synthesized likewise. The heavy chain vector
contains human Ig
1 constant region genomic sequence and the light
chain vector contains the Ig k constant region sequence. The heavy
chain vector was then co-transfected with the light chain vector into
an Ig-nonproducing plasmacytoma cell line, P3X63Ag8.653, by
electroporation as described
(26) , cloned in medium containing
the antibiotic, G418, and screened for antibody production. Three cell
lines were made, AJ-1 expressing HuA V
and V
,
AK-6 expressing HuA V
and RF V
, and AL-23
expressing RF V
and HuA V
. Supernatants from
the three cell lines were used in ELISA assays.
Quantitative Precipitin Assays
Fig. 1
shows the quantitative precipitin curves obtained with
various blood group substances and HuA. HuA reacted well with all the
preparations of blood group A substance. With Hog 4 10%, a maximum of
6.5 g was precipitated, and, with other preparations, 5.6 to 6.2 µg
of N were precipitated. Human saliva blood group A substance, W.G. phenol insoluble, showed the lowest activity with
the antibody with a maximum of 5.6 µg of N precipitated, and about
twice as much antigen being required for 50% of maximum precipitation
(1.9 µg compared to 0.7-1.1 µg). This is most likely due
to the presence of fewer A determinants on A
substances
(27) . HuA did not react with blood group
substances B, H, Le
, Le
, and precursor Ii. The
specificity of HuA for blood group A substances and its uniform
reactivity with all those tested was very similar to the quantitative
precipitin data for the mouse anti-A, AC-1001
(8) .
Figure 1:
Quantitative precipitin
curves of human monoclonal anti-A with various blood group substances.
, Hog 4 10% (A); &cjs0800;, Cyst 9, phenol-insoluble
(A
); , Cyst 14, phenol-insoluble (A
);
,
MSM 10% (A
); , Hog 75 10% (A);
, MSS 10% 2X
(A
);
, McDon 15% (A
); &cjs0383;, Hog 67
4% (A); , W. G., phenol-insoluble (A
);
, Cyst
Beach, phenol-insoluble (B); , N-1, phenol-insoluble
(Le
); , JS, phenol insoluble (HLe
);
, Tij
II, phenol-insoluble (BI);
, Tighe, phenol-insoluble (H);
▾, Cyst OG 10% from 20%
(I
).
Quantitative Inhibition ELISAs
Inhibition of
anti-A binding to Hog 4 10% by various monosaccharides and blood group
oligosaccharides is shown in Fig. 2. The oligosaccharide
structures, amounts required for 50% inhibition, the free energies of
binding (G
), and comparable results for
the previously characterized mouse monoclonal anti-A, AC-1001, are
shown in . HuA is most specific for difucosyl
oligosaccharides. The best inhibitors were the A-hepta and the A-penta.
The human antibody did not distinguish between the type 1 (
1
3) linkage to GlcNAc of the A-hepta and the type 2 (
1
4) linkage of the A-penta. The A-hexa differs from the A-hepta
only by lacking the second fucose linked to GlcNAc, but it was less
efficient at inhibition by 10-fold. As shown, this fine specificity was
nearly identical with that of AC-1001, the mouse monoclonal. For both
the human and mouse antibodies, the oligosaccharides in their order of
ability to inhibit binding of antibody to A blood group substance was
the hepta > penta > hexa > tetra and
MSMA
R
O.56 > tri and 01. For both, the
difucosyl containing hepta- and pentasaccharides were the strongest
inhibitors indicating the specificity of both antibodies for the
difucosyl portions of the oligosaccharides. The only difference was
that the mouse monoclonal was more effectively inhibited by the
monofucosyl A-hexa. This could be explained if the Gal(
1
4)GlcNAc contributes somewhat more to the mouse anti-A binding site
than to the human
(28) .
Figure 2:
Inhibition by various oligosaccharides of
binding of human monoclonal anti-A to blood group A Hog 4 10%. Symbols
and corresponding oligosaccharide structures are shown in Table
I.
Nucleotide and Derived Amino Acid Sequence Comparisons of
the Human and Mouse Anti-A Heavy and Light Chains
Fig. 3
and 4 show the complete nucleotide and derived amino acid
sequences of the HuA V and V
. The sequences of
the V
and V
chains of the murine AC-1001
(22) are shown for comparison. The human anti-A heavy chain uses
a member of the V
3 family
(29) with a D segment
which does not correspond to a previously sequenced germline D segment,
and J
4 with 3 nucleotide changes and 1 amino acid change
from the reported germline sequence
(30) . The most closely
related previously characterized human V
gene is 56P1
(31) obtained from a cDNA library from human fetal liver. Three
other cDNAs, M72, M74, and M49, from a second fetal liver cDNA use the
same V
gene
(32) . For codons 1-94, HuA and
56P1 are 91% identical at the nucleotide level and 86% at the amino
acid level. The mouse heavy chain of AC-1001 is a member of the J558
family in the classification of Brodeur et al. (33) and
Dildrop et al.(34) and of subgroup IIb in that of
Kabat et al.(25) . The mouse and human V
sequences have only 56% identity at the nucleotide level and 43%
identity at the amino acid level.
Figure 3:
Nucleotide
and derived amino acid sequence of the HuA heavy chain. For comparison,
the nucleotide and amino acid sequences of the mouse anti-A, AC-1001,
are also shown. Nucleotide identities are represented by dots.
Only amino acid differences are shown. Nucleotides of the HuA which
differ from the germline J are indicated with capital
letters.
As shown in Fig. 4, the
human V is a member of the V
IIIa subgroup
(25) and has only one silent nucleotide difference (t instead of
c in the third position of codon 36) from a previously published
germline V
, V-g
(35) . The leader sequence of V-g
and HuA are identical except for five nucleotide differences in the
region of the 5` primer used in PCR. Thus, V-g is probably the germline
counterpart of the HuA V
with the differences in the
leader having been introduced by mismatchs during the initial cycles of
PCR priming. The anti-A V
is rearranged to
J
1 with three nucleotide differences resulting in two
amino acid changes from the reported germline sequence
(36) .
Very similar light chains, probably using the same germline V
gene, have been found in a number of other antibodies, including
a panel of cold agglutinins specific for sialic acid glycoproteins on
human erythrocytes
(37) , a human IgG rheumatoid factor from
synovial fluid
(38) , and an autoantibody specific for myelin and
denatured DNA from a patient with chronic lymphoblastic leukemia and
peripheral neuropathy
(39) . These antibodies all use V
that differ from HuA. The cold agglutinin uses a V
1
gene, the rheumatoid factor uses a V
3 gene that is 82%
identical at the nucleotide level and 72% identical at the amino acid
level for codons 1-94, and the anti-myelin/DNA uses a member of
V
3 which is 79% identical with the human anti-A at the
nucleotide level and 70% identical at the amino acid level.
Figure 4:
Nucleotide and derived amino acid
sequences of HuA light chain. For comparison, the nucleotide and amino
acid sequences of the mouse anti-A, AC-1001, the light chain from an
antibody with anti-PR activity, LS-1 (37), and the light
chain from a human IgG rheumatoid factor from synovial fluid, ka3dl
(38), are shown. Nucleotide identities are represented by
dots. Only amino acid differences are shown. Nucleotides of
the HuA which differ from the germline sequences of V-g (35) and
J
1 (36) are indicated with capital letters.
The leader sequences for these V
genes are also shown
along with the 5` primer used in PCR. Positions where more than one
base was used in synthesis of the primer are
shown.
Comparing the HuA light chains to that of the mouse, AC-1001, there
is 68% nucleotide and 57% amino acid identity with 64% identity of the
amino acids in the framework and 37% in the CDRs. Like the heavy chain
there are shared residues in the CDRs, but all are residues most
frequently used ( Fig. 5and ). Of the residues in
common, the arginine, alanine, serine, and glutamine at positions
24-27 in both antibodies appears to be outside the region of
antigen contact according to the limited data available on antibody
structures
(40) . Tyr-32 in CDR 1, Ser-52 in CDR 2, and Gln-90
and Pro-95 in CDR 3 may be at positions of antigen contact, but these
few similarities are unlikely to account for the specificity of the two
antibodies.
Figure 5:
HuA
and mouse AC-1001 heavy and light chain CDRs. Amino acid identities are
underlined, and the percent of all sequenced antibodies (25)
with those amino acids is indicated. In CDRs 2 and 3 of the heavy
chain, there are similar motifs in the human and mouse sequences which
are displaced by one amino acid. These residues are also underlined,
and their frequency among all V sequences (25) is indicated
separately for the human and mouse
sequences.
Assays of Transfectoma Cell Lines
Supernatants
from the three transfectoma cell lines were assayed by ELISA for total
IgG and for specificity against various blood group antigens including
Cyst 14 (A), Beach (phenol insoluble) (B), J.S. (phenol insoluble) (H),
OG (I), and Le. Only AJ-1 with both the HuA heavy and light
chain bound to blood group A. There was no binding of any of the three
supernatants to the B, H, I, or Le
blood group substances
(not shown). These data confirmed that the correct anti-A binding heavy
and light chain genes had been identified, and that both the heavy and
light chain together contributed to specificity. Because AL-23, with
the HuA V
paired with an irrelevant heavy chain, secreted
somewhat lower amounts of Ig than either AJ-1 or AK-6, we could not
completely exclude the possibility that the HuA V
could
have some small amount of anti-A activity independent of the heavy
chain. Multiple attempts at identifying a clone making higher
concentrations of IgG failed, probably because our method of selection
was designed to identify clones with both the heavy and light chain,
but did not specifically select for high levels of immunoglobulin
secretion.
3 gene used by HuA and the murine J558 gene
used by AC-1001 are as distantly related as any mouse and human V
can be, sharing only 56% of nucleotides and 43% of amino acids.
Schroeder and co-workers
(41, 42) have used framework
codons to define evolutionary relationships among mammalian V
genes, and, in this analysis, these two gene families most likely
arose from separate progenitor V
elements or clans. The two
V
genes also come from different families and share
only 68% nucleotide and 57% amino acid identity.
and V
are not highly related in their
framework structures nor in evolutionary origin, their similar binding
properties might be explained if there were shared residues in the
CDRs. As shown in Fig. 5and , however, there are
only a few residues in common between the mouse and human CDRs, and
most of them occur most commonly in these positions. The heavy chain
CDRs have only 15% amino acid identity, the two CDR3s differ in length,
and the light chain CDRS have 37% only amino acid identity.
and
V
must be present for anti-A activity.
(1
6)
dextran system, 43 monoclonal antibodies have been sequenced and
defined
immunochemically
(44, 45, 46, 47, 48, 49, 50) .
Four of these recognize the terminal nonreducing end of dextran and are
all quite similar in their V
and V
usage
(45-48); however, members of five different V
gene
families and three V
gene families are used in 39
antibodies that have groove type
sites
(44, 46, 47, 49, 50) .
Despite this apparent diversity, there are some remarkable patterns of
similarity. A set of 21 groove type antibodies using two different
V
genes all use the same D segment with very similar
V
-D and D-J
joints to form CDR3s which differ
from one another by only one or two amino acids. All twenty one of
these groove type antibodies use the same V
OX1 gene
(49, 50). These 21 antibodies which are extremely similar in CDR3 of
the heavy chain and CDR1, -2, and -3 of the light chain vary by up to
200-fold in their affinity for (
1
6)
dextran
(49, 50) . These data and others
(51, 52) have emphasized the importance of CDR3 of the heavy chain
in defining specificity. In comparison, the human and mouse anti-A
antibodies are much more similar immunochemically and much more
different in their primary structure. In other antigen-antibody
systems, diversity in V
and V
usage has
been documented
(53, 54, 55, 56) , but in
most of these systems the antigenic determinants are far less precisely
defined. Andria et al. (57) showed that diverse V
and V
genes are used in antibodies to a well defined
decapeptide of tobacco mosaic virus. However, these antibodies also
differed in affinity and in their patterns of reactivity from a panel
of synthetic decapeptides with amino acid substitutions.
and V
genes
(62) .
Table:
Oligosaccharide inhibition studies on human and
mouse antibodies to blood group A substance
1
2Gal
1
3GlcNAc
-O(CH
)
-COOCH
];
▾, B-Tri (Gal
1
1
1
4
Table:
Nucleotide and amino acid identity of AC-1001
and human anti-A
/EMBL Data Bank with accession number(s)
L41174 (Hu V
) and L41175 (Hu V
).
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.