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Blood, Vol. 91 No. 8 (April 15), 1998:
pp. 3066-3078
Genetic and Immunological Properties of Phage-Displayed Human
Anti-Rh(D) Antibodies: Implications for Rh(D) Epitope Topology
By
Tylis Y. Chang and
Don L. Siegel
From the Blood Bank/Transfusion Medicine Section, Department of
Pathology & Laboratory Medicine, University of Pennsylvania School of
Medicine, Philadelphia, PA.
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ABSTRACT |
Understanding anti-Rh(D) antibodies on a molecular level would
facilitate the genetic analysis of the human immune response to Rh(D),
lead to the design of therapeutically useful reagents that modulate
antibody binding, and provide relevant information regarding the
structural organization of Rh(D) epitopes. Previously, we described a
Fab/phage display-based method for producing a large array of
anti-Rh(D) antibodies from the peripheral blood lymphocytes of a single
alloimmunized donor. In the current study, we present a detailed
analysis of 83 randomly selected clones. Sequence analysis showed the
presence of 28 unique  1 heavy chain and 41 unique light
chain gene segments. These paired to produce 53 unique Fabs that had
specificity for at least half of the major Rh(D) epitopes.
Surprisingly, despite this diversity, only 4 closely related heavy
chain germline genes were used (VH3-30, VH3-30.3, VH3-33, and VH3-21).
Similarly, nearly all V light chains (15/18) were
derived from one germline gene (DPK9).  light chains showed a more
diverse VL gene usage, but all (23/23) used the identical
J 2 gene. Several Fabs that differed in epitope specificity used identical heavy chains but different light chains. In
particular, 2 such clones differed by only 3 residues, which resulted
in a change from epD2 to epD3 specificity. These results suggest a
model in which footprints of anti-Rh(D) antibodies are essentially
identical to one another, and Rh(D) epitopes, as classically defined by
panels of Rh(D) variant cells, are not discrete entities. Furthermore,
these data imply that the epitope specificity of an anti-Rh(D) antibody
can change during the course of somatic mutation. From a clinical
perspective, this process, which we term epitope migration, has
significance for the design of agents that modulate antibody production
and for the creation of mimetics that block antibody binding in the
settings of transfusion reactions and hemolytic disease of the newborn.
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INTRODUCTION |
CLINICALLY, THE HUMAN Rh(D) antigen is
the most important red blood cell (RBC) membrane protein in transfusion
medicine. The alloimmune response against Rh(D) produces high-affinity
IgG antibodies that cause hemolytic transfusion reactions and hemolytic
disease of the newborn (HDN). The prophylactic use of Rh(D)-immune
globulin in pregnant Rh(D)-negative women has been a major advance in
the prevention of HDN,1 yet the mechanism by which the drug
exerts its immune modulatory effect is not well
understood.2 Monoclonal antibodies (MoAbs) derived from the
B cells of Rh(D)-immune globulin donors have defined several dozen
Rh(D) epitopes3; paradoxically, the Rh(D) antigen, an
approximately 30-kD transmembrane protein, has minimal extracellular
mass and presents a very limited surface area for epitope
expression.4-9 The molecular cloning of large repertoires
of anti-Rh(D) antibodies would help reconcile these observations. In
addition, it would facilitate the rational development of recombinant
formulations of Rh(D)-immune globulin and aid in the design of
therapeutic agents that block antibody binding. Furthermore, the
comprehensive genetic analysis of anti-Rh(D) antibodies within a given
alloimmunized individual would serve as a paradigm for human immune
repertoire development, an area in which limited information is
currently available.
Previously, no more than 8 IgG anti-Rh(D) human MoAbs have been derived
from a single individual.10 The primary challenge in
studying the Rh(D) immune response has been technical difficulties inherent in human B-cell immortalization. Epstein-Barr virus (EBV) transformation results in relatively low transformation
efficiencies11 that can undergo a decline in antibody
production,12-15 whereas cell fusion methods have been
hampered by the lack of good fusion partners.16,17 More
recently, molecular approaches have been developed that bypass the need
for cell transformation.18-20 Conceptually, these
techniques, referred to as repertoire cloning or Fab/phage display,
seek to immortalize Ig mRNA rather than the B cells from which they
were derived. In an earlier report, our laboratory adapted these
techniques for isolating Fab/phage antibodies directed against
conformation-dependent antigens expressed on cell
surfaces.21 Using intact human RBCs, we isolated highly
diverse 1 and 1 Fab/phage libraries
against the Rh(D) antigen from the B cells of a single Rh(D)-immune
globulin donor.22
In the following report, we present a detailed genetic and serological
analysis of 53 unique anti-Rh(D) antibodies derived from 83 randomly
chosen clones. The results complement previous reports on the genetic
and biochemical makeup of monoclonal anti-Rh(D) antibodies derived from
multiple donors.10,23-25 Significantly, our data also
demonstrate extensive genetic homology between antibodies directed
against different Rh(D) epitopes. We provide evidence that antibodies
directed against different epitopes can be clonally related. Finally,
we suggest a model that reconciles the serological diversity of
anti-Rh(D) antibodies with the topological constraints imposed by the
Rh(D) antigen.
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MATERIALS AND METHODS |
Production of Monoclonal Anti-Rh(D) Phage-Displayed and Soluble
Fab Molecules
Methods for the isolation of human anti-Rh(D)-specific antibodies from
1 and 1 Fab/phage display libraries
using the pComb3H phagemid vector and a cell-surface panning protocol
have been previously published.22 Soluble anti-Rh(D) Fab
preparations for inhibition studies were produced from bacterial
cultures transfected with plasmid DNA from which the M13 gene III coat
protein sequence had been excised as described.21,26
Cultures were grown by shaking at 300 RPM at 37°C in superbroth (30 g/L tryptone, 20 g/L yeast, 10 g/L MOPS, pH 7.00) containing 20 mmol/L
MgCl2 and 50 µg/mL carbenicillin to an OD600
of 0.5. Isopropyl- -D-thiogalactopyranoside (IPTG) was added to 1 mmol/L and cultures were shaken overnight at 30°C. Bacterial
pellets were harvested and resuspended in 1/50th of the initial culture
volume with osmotic shock buffer (500 mmol/L sucrose, 1 mmol/L EDTA,
100 mmol/L Tris, pH 8.00), incubated for 30 minutes at 4°C, and
centrifuged at 16,000g for 15 minutes at 4°C.
Fab-containing supernatants were dialyzed against phosphate-buffered saline (PBS) and used in agglutination experiments without further purification.
Anti-Rh(D) Antibody Binding Assays
The binding of anti-Rh(D) Fab/phage or soluble Fab molecules to normal
or partial Rh(D) antigens was assessed by indirect agglutination assays
as described.21,22 Briefly, 100 µL aliquots of
phage-displayed Fabs or soluble Fabs were incubated with 50 µL of a
3% suspension of RBCs. After 1 hour of incubation at 37°C, the
RBCs were washed three times with 2 mL of cold PBS to remove unbound
antibody. The resulting RBC pellets were resuspended in 100 µL of a
10 µg/mL solution of sheep anti-M13 antibody (5 Prime  3 Prime, Boulder, CO) for Fab/phage experiments or goat antihuman or
light chain antibody (Tago, Burlingame, CA) for 1
or 1 soluble Fab experiments, respectively. The RBC
suspensions were transferred to the round-bottomed wells of a 96-well
microplate and left undisturbed for 2 hours. Negative reactions show
sharp approximately 2-mm diameter RBC spots, whereas the RBCs in
agglutinated wells form a thin carpet coating the entire floor of the
well.22 Agglutination titers for recombinant antibodies
were determined by performing serial twofold dilutions in 1% bovine
serum albumin (BSA)/PBS. Typically, Fab/phage had agglutination titers
of 1/1,024 to 1/2,048 (where neat is defined as 5 × 1012 tfu/mL),22 and soluble Fabs had
agglutination titers of 1/64 to 1/128 when prepared as described above.
For determining Rh(D) epitope specificity for anti-Rh(D) Fab/phage
antibodies, the following reference Rh(D) variant cells were obtained
from the MRC Blood Group Unit (London, UK), The New York Blood Center
(New York, NY), or Gamma Biologicals, Inc (Houston, TX):
O/DIIIaCce, G positive; B/DIIIcCce;
A/DIVace; A/DIVace; O/DIVace;
O/DIVbCce; B/DIVbCce, Goa negative,
Rh32 negative; O/DVaCce; O/DVacEe,
Dw positive; O/DVICce; B/DVICce;
AB/DVICce; A/DVIcEe; O/DVIICce; and
O/DVIICce. Each Fab/phage antibody was tested on at least
three separate occasions against at least two different examples of
each variant cell type, and identical epitope assignments were obtained
each time. For antibodies that demonstrated previously undescribed patterns of reactivity or repeatedly weak reactivity against one type
of cell (see the Results), monoclonal Fab/phage were prepared on a
least four separate occasions to verify the patterns of reactivity.
For inhibition studies, the ability of antibodies with different Rh(D)
epitope specificities to compete with each other for binding was
assessed by preparing stocks of each clone in both a soluble Fab form
and a phage-displayed form. Pairwise combinations of soluble Fabs and
Fab/phage were prepared and added to Rh(D)-positive RBCs. The resulting
incubation mixes comprised 50 µL of a 3% suspension of RBCs, 100 µL of undiluted soluble Fab, and 100 µL of Fab/phage diluted to its
highest agglutinating titer. After 1 hour of incubation at 37°C,
RBCs were washed, resuspended in anti-M13 antibody, and placed in
microplate wells as described above. That the amount of soluble Fab
present in an incubation mixture was sufficient to compete away a
Fab/phage that shared the same binding site was determined by verifying
that each soluble Fab preparation could block its own Fab/phage (see
the Results).
Inhibition experiments were also performed using pairwise combinations
of soluble Fabs instead of soluble Fab and Fab/phage combinations. In
this type of experiment, pairs of soluble Fabs specific for different
epitopes were chosen such that one Fab contained a light chain and
the other a light chain. Incubations with RBCs were performed with
one Fab in excess and the other in limiting amounts. Blocking of the
latter antibody was assessed using a secondary antibody (anti- or
anti-) specific for its light chain isotype.
Nucleotide Sequencing and Analysis
Plasmid DNA for sequencing was prepared using the Qiawell system
(Qiagen, Chatsworth, CA). Double-stranded DNA was sequenced using light
chain or heavy chain Ig constant region reverse primers or a set of
unique pComb3H vector primers that anneal 5 to the respective Ig
chain26,27 and automated fluorescence sequencing (Applied
Biosystems, Foster City, CA; DNA Sequencing Facility, University of
Pennsylvania Department of Genetics and Cancer Center, Philadelphia,
PA). Sequence analysis and variable region germline assignments were
performed using DNAplot28 and the V Base Directory of Human
V Gene Sequences (March 1997 update).29 Germline
assignments were corroborated with the MacVector (v. 6.0) software
package (Oxford Molecular Group, Oxford, UK) against the same database. Multiple sequence alignments and predictions of isoelectric point were
calculated using the Pileup and Isoelectric programs of the GCG
software package (v. 8.0.1; GCG, Madison, WI). Statistical analysis was
performed with Statview (Abacus Concepts, Berkeley, CA).
Because of the large number of heavy and light chain sequences (N = 69), only alignments of the predicted amino acid sequences are
presented. Nucleotide sequences of all clones are available in Genbank.
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RESULTS |
Sequence Analysis of Anti-Rh(D) Heavy and Light Chains
We previously reported on the use of Fab/phage display and cell-surface
panning to isolate a large array of anti-Rh(D) antibodies from the
peripheral blood lymphocytes of a single hyperimmunized donor.22,30 Separate 1 and
1 Fab/phage display libraries had been constructed
and contained 7 × 107 and 3 × 108
independent transformants, respectively, based on electroporation efficiency. Each library was panned independently using a simultaneous positive/negative selection strategy with magnetically labeled Rh(D)-positive RBCs and unmodified Rh(D)-negative RBCs as described. After two rounds of panning, 32 of 36 1 and 15 of 15 1 randomly chosen clones were positive for anti-Rh(D)
activity. After the third round of panning, 24 of 24 1 and 12 of 12 1 clones were positive. Nucleotide sequencing of the 83 positive clones showed a
total of 28 unique heavy and 41 unique light chains. Because of
combinatorial effects during phage display library construction, heavy
and light chain gene segments paired to produce 53 unique Fab
antibodies.22
Anti-Rh(D) heavy chains.
All of the heavy chain sequences used VHIII family-encoded
gene products (Figs 1 and
2). Several sequences shared identical VDJ joining
regions, and 12 unique VDJ rearrangements were identified and
designated VDJ1 through VDJ12. Alignment of these sequences against the
V Base Directory of Human V Gene Sequences29 showed that
only four VHIII genes were used by these antibodies:
VH3-21, VH 3-30, VH 3-33, and VH 3-30.3. VH3-21 was used by 1 of the 12 VDJs and 2 of the 28 clones; VH3-30 by 1 VDJ and 6 clones; VH3-33 by 9 VDJs and 19 clones; and VH3-30.3 by 1 VDJ and 1 clone. Interestingly, VH3-30, VH3-33, and VH3-30.3 comprise a set of closely related genes
(>98% homology; Fig 2B) and their next nearest neighbor, VH3-07, is
only 90% homologous (Fig 2C). Hereafter, these three genes are
referred to as the VH3-33 superspecies. Heavy chain E1 differed from
VH3-21 by 6 mutations and from VH3-48 by 10 mutations; hence, it was
assigned to the former germline gene. Because there were no common
mutations among the VH3-33 clones, it is highly probable that the donor
possessed the VH3-33 germline gene. However, we could not formally rule
out gene duplication with allelic variants of VH3-33 or the existence
of variant alleles of the other germline genes in the donor. The
isolation of clones sharing multiple VDJ joining regions argues that
cloning artifacts cannot account for the VH gene
restrictions observed.
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| Fig 1.
(A) Dendrogram and (B) CDR3 alignment of anti-Rh(D) heavy
chains. The 28 unique heavy chain clones are organized by
VH family, VH germline gene, and VDJ
rearrangement. Each heavy chain clone is identified by a numeral
preceded by a letter (B through E) that denotes its germline gene. The
28 heavy chains comprised 12 distinct VDJ regions, designated
VDJ1 through VDJ12. Clones with identical VDJ joins putatively result
from intraclonal diversity of 12 original B lymphocytes.
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| Fig 2.
(A) Alignment of anti-Rh(D) heavy chains to their nearest
germline V, D, and J genes. Also shown are the putative intermediate heavy chain sequences (Ca, Cb, Da, Db, and Dc; see text and Fig 3). The
number of nucleotide differences from a germline VH is tabulated to the right of each sequence. In general, D segments showed
poor homology with known D genes, so mutations were not scored in these
regions. Key: Replacement mutations indicated with letters, silent
mutations as "*", identities as ".", and insertions as
"-". Sequences derived from the 5 VH primers
used in library construction22 are marked as ">".
CDR region designations are determined as per Kabat59;
numbering and H region designations per Chothia et al.31
(B) Alignment of the four VH3 genes used by anti-Rh(D) heavy chains and
(C) dendrogram of all human VH3 family germline genes shows relatedness
of VH3-21, VH3-30, VH3-33, and VH3-30.3 and the surprising restriction
in VH gene usage. Note that the VH3-30.5 gene is present in
only certain haplotypes and is identical to VH3-30.60
Genbank accession numbers for anti-Rh(D) heavy chains are listed in the Appendix.
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Neither JH nor D segments showed restriction. At least 9 different D segments were used and JH gene use comprised
JH6 (5 VDJs and 9 clones), JH4 (4 VDJs and 10 clones), JH3 (2 VDJs and 8 clones), and JH5 (1 VDJ and 1 clone). All four VH genes were Chothia class 1-3,31 and the CDR3s showed a narrow range of length from
15 to 19 residues.
Because rearranged heavy chain genes demonstrate extensive diversity,
clones sharing identical VDJ rearrangements are generally considered to
have arisen from the same clone. Based on nucleotide alignment with the
germline genes (data not shown), an ontogeny tree was constructed for
the 12 VDJs and 28 clones (Fig 3). By using
the most parsimonious mutation scheme (ie, postulating the minimum
number of mutations), putative intermediate antibodies were derived for
several of the VDJs and were designated Ca, Cb, Da, Db, and Dc (Figs 2A
and 3). Compared with the isolated heavy chain clones, which displayed
between 6 and 23 nucleotide differences from their germline
counterparts, these putative intermediates had between 3 and 12 mutations from germline. Based on the ontogeny tree, the number of
independent mutations could be tabulated among the clones. The most
commonly mutated residues were 52a and 58 (7 independent mutations),
followed by residues 30, 31, and 50 (6 mutations) and residue 55 (5 mutations). In the VH3-33 superspecies, residues 52a and 58 in CDR2 are
tyrosines and residue 52a was mutated to phenylalanine in 6 of the 11 VDJs derived from VH3-33 superspecies VH genes. Mutations
at residue 58 comprised glutamate (3), aspartate (2), histidine (1),
and asparagine (1). The AGY serines at residues 30, 31, and 55 were
mutated to a number of different amino acids, although the AGY serine
at 82b was conserved in all clones. The valine at residue 50 in the
VH3-33 superspecies also had a diverse set of mutations. This
distribution of hot spots is similar to that seen with nonproductive
rearrangements as previously reported by Dörner et
al.32
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| Fig 3.
Ontogenic tree of anti-Rh(D) heavy chains constructed
using nucleotide alignment data. Circles represent isolated and
sequenced clones and diamonds represent putative intermediates (see Fig 2A). The number of nucleotide mutations from its germline
VH gene is shown in parentheses below the clone name. The
distance along the horizontal axis represents the degree of mutation
(including J segments) within the constraints of the diagram.
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Anti-Rh(D) light chains.
Seventeen of the 18 light chains were from the VI
family and the remaining light chain originated from a
VII family member germline gene
(Fig 4). Only 4 V germline
genes were used (15 clones were derived from DPK9 alone), and the light chain clones had between 1 and 49 mutations from their
corresponding V germline genes. All 5 of the known
J genes were used and were each joined to the DPK9 gene
in one or more clones. Because the light chains showed considerably
less diversity in their joining regions than the heavy chains, it was
difficult to assign common clonal origins. However, an ontogeny tree
was constructed by grouping common V and J gene segments along with
common mutations (data not shown). Based on this analysis, the 18 chains comprised at least 10 different recombination events.
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| Fig 4.
(A) Alignment of anti-Rh(D) light chains to their
nearest germline V and J genes shows predominance of DPK-9 usage from the VI family. Nomenclature for clones is similar to
that for heavy chains but uses the letters F through I. (B) Alignment of the four V genes used by anti-Rh(D) light
chains. The key is the same as that used in Fig 2A. Genbank accession
numbers for anti-Rh(D) light chains are listed in the Appendix.
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light chains were restricted by their J gene usage
but showed no restriction in their use of V genes
(Fig 5). The 23 light chains all used
the J2Vasicek gene but were derived from
VI (12 clones), VIII (5),
VVII (3), VII (2), and VIV
(1) family genes. The number of mutations ranged from 2 to 41 from the
nearest germline V gene. Based on common joining regions
and mutations, these 23 light chains were derived from at least 13 different B cells.
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| Fig 5.
(A) Alignment of anti-Rh(D) light chains to their
nearest germline V and J genes and (B) alignment of the 10 V germline genes used shows the use of a diverse set of
variable region genes derived from multiple families. However, all of
the clones use the identical J gene segment.
Nomenclature for the clones is similar to that for heavy chains but
uses the letters J through S. The key is the same as that used in Fig
2A. Genbank accession numbers for anti-Rh(D) light chains are
listed in the Appendix.
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Assessment of the Diversity of the Unpanned Libraries
To determine whether the apparent restriction in gene usage of the
anti-Rh(D) antibodies could have been due to preselection factors (ie,
cloning artifacts), we assessed the diversity of the unpanned
1 and 1 Fab/phage libraries. By
sequencing 39 randomly picked clones, we determined that there were no
duplicate heavy or light chain sequences and that there was significant heterogeneity in V gene family representation before selection (Fig 6). In fact, the variable region gene
family distribution was not unlike that found by other investigators
for IgG-secreting lymphocytes in adult peripheral blood.33
Furthermore, of the 14 VHIII-encoded negative clones, only
one used a VH3-33 superspecies germline gene (VH3-30.3); the other 13 were encoded by VH3-07 (3), 3-09 (2), 3-15 (2), 3-48 (2), 3-72 (2),
3-23 (1), and DP-58 (1). Therefore, the restriction of the 83 anti-Rh(D) clones to the VH3-33, 3-30, 3-30.3, and 3-21 genes is
significant and not a result of skewed representation of certain
germline genes within the originally constructed 1
and 1 Fab/phage libraries.
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| Fig 6.
Comparison of variable region gene family usage for
anti-Rh(D)-specific clones with those used by randomly picked,
non-Rh(D)-binding clones from original 1 and
1 unselected libraries. Lightly hatched bars reveal
heterogeneity in VH (left panel), V (middle panel), and V (right panel) family representation before selection for anti-Rh(D) specificity. Numbers above bars represent absolute number of clones in that group.
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Heavy and Light Chain Contribution to Rh(D) Epitope
Specificity
Because of the conformational dependency of Rh(D) antigenicity, Rh(D)
epitopes have been classically defined through the use of RBCs obtained
from rare individuals whose cells appear to produce Rh(D) antigens
lacking certain epitopes.34 Examining the pattern of
agglutination of a particular anti-Rh(D) MoAb with such sets of partial
Rh(D) RBCs enables one to categorize that antibody's fine specificity.
Monoclonal Fab/phage preparations were prepared in triplicate for each
of the 53 anti-Rh(D) clones and tested against a panel of Rh(D)
category cells IIIa/c, IVa, IVb, Va, VI, and VII. This panel of cells
can differentiate between the Rh(D) epitope specificities as described
by Lomas et al6 (designated epitopes epD1, epD2, epD3, epD4, epD5, and epD6/7). Agglutination experiments with the
Fab/phage clones showed five different patterns of reactivity, including a new pattern that had not been described in the original study by Lomas et al6 or in the more recently described 9, 30, or 37 epitope systems (Figs 7 and
8).3,35 Although nearly all
Fab/phage gave unequivocal agglutination reactions, a few antibodies
gave repeatedly weak patterns of reactivity against one of the panel
cells. For these reactions, monoclonal Fab/phage were prepared on at
least four separate occasions to verify the patterns of reactivity.
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| Fig 7.
Determination of the Rh(D) binding epitope of anti-Rh(D)
Fab/phage clones. The fine specificities of monoclonal Fab/phage clones
were determined by their ability to agglutinate members of a panel of
six Rh(D) variant RBCs. Shown are the five different agglutination
patterns obtained from screening all of the 53 Fab/phage clones. The
particular clones shown are identified by their unique heavy
chain/light chain pairings using the nomenclature defined in Figs 1, 4,
and 5. For E1/M3, reactivity with additional Rh(D) variant cells would
be required to distinguish its specificity for epD3 versus
epD9.3 Rh(D) epitope assignments are as per Lomas et
al.6 Note that inclusion of the category IVb cell (not
available in our previous study)22 permits the
identification of a new epitope designated epDX (see text).
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| Fig 8.
Matrix illustrating the genetic composition and epitope
specificity of anti-Rh(D) antibodies. The horizontal axis represents the unique 1 heavy chains and the vertical axis
represents the unique and light chains (based on amino acid
sequence). A shaded pattern at the intersection of a heavy chain/light
chain pair indicates the Rh(D) epitope specificity observed for that Fab/phage antibody. A few clones gave mixed patterns of reactivity, as
shown (see text). The order of heavy chains (left to right) and light
chains (top to bottom) was determined by the multiple alignment of
amino acid sequences as in Figs 2, 4, and 5. Note that heavy chains D1,
D15, D16, and D17, although differing in nucleotide sequence, have the
identical amino acid sequences and thus comprise a single column.
Similarly, heavy chains C5 and C8 and light chains K1 and K2 encode
the same proteins. The pairings of these 28 heavy and 41 light chain
nucleotide gene segments, which produced 53 unique Fab transcripts,
encoded 43 different Fab proteins, as indicated in the matrix.
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The most commonly recognized epitope was epD6/7, against which 13 clones were directed. Interestingly, monoclonal anti-Rh(D) clones
isolated using conventional tissue culture methods are most often
specific for epD6/7.34 epD2, epD1, and epD3 were recognized
by 10, 7, and 2 clones, respectively. Six clones agglutinated cells of
categories IIIa/c, IVa, and VII, but not of categories IVb, Va, and VI,
and were designated anti-epDX. This pattern is identical to epD1,
except that the IVa cell is agglutinated. Three clones gave
intermediate reactions with cell IVa, but otherwise showed patterns
consistent with epDX or epD1. These clones were designated
epDX1 or epD1X, depending on whether this
reactivity against cell IVa was stronger or weaker, respectively (Fig
8). Similarly, reaction patterns for epD1 and epD2 differ by a positive
reaction with the category Va cell; therefore, one clone was given
epD21 specificity because it gave only moderate reactivity
against that cell. Such variable reactions against one or more partial Rh(D) cells have been observed for anti-Rh(D) MoAbs produced through conventional tissue culture methods.36
Because of the reassortment of heavy and light chain gene segments that
occurs during the construction of a phage display library, a number of
clones were isolated that shared either a heavy (eg, E1) or light (eg,
M3) chain sequence (Fig 8). Some heavy chains were found to have paired
with both and light chains (eg, C1, D20), and each demonstrated
anti-Rh(D) specificity. Interestingly, some heavy chains (eg, E1, D12)
mapped to different epitopes depending on the light chains with which
they were paired. In particular, the light chains of two such clones,
E1/M2 and E1/M3, differed by only 3 amino acid residues (Fig 5) and
these differences appear to confer specificity for epD2 versus epD3.
Inhibition Studies
To investigate the topological relationships among the Rh(D) epitopes,
inhibition studies were performed. Previous work by Gorick et
al37 using pairs of unlabeled and 125I-labeled
anti-Rh(D) MoAbs demonstrated that antibodies to at least 3 different
Rh(D) epitopes (subsequently identified as epD1, D6, and
D7)6 could inhibit one another. We have confirmed and extended these findings using recombinant antibodies to 5 Rh(D) epitopes (Fig 9). In one series of
experiments, we exploited the ability to express each antibody in both
a soluble Fab as well as phage-displayed form to ask whether a soluble
Fab against one epitope would inhibit the agglutination induced by an
Fab/phage directed against a different epitope. Reciprocal pairs of
soluble Fab and Fab/phage specific for epD1, epD2, epD3, epD6/7, and
epDX were tested. All 10 combinations showed mutual inhibition patterns (shown in Fig 9A for an anti-epD3/anti-epD6/7 combination). To show
that this inhibition was not due to nonspecific factors, a control with
an irrelevant RBC-binding recombinant antibody (an anti-blood group B
antibody) was performed (Fig 9B). That sufficient inhibitory amounts of
soluble Fab was present were first verified by demonstrating that each
soluble Fab could inhibit its own Fab/phage (Fig 9A and B; samples on
diagonal). Similar results were obtained using pairs of soluble Fabs
which differed in their light chain isotype composition (Fig 9C).
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| Fig 9.
Inhibition studies with recombinant anti-Rh(D)
antibodies. Panels show results of representative experiments
demonstrating the mutual inhibition of antibodies directed at 2 different Rh(D) epitopes (in this example, epD3 and epD6/7; A and C),
but not between an Rh(D) antibody and an unrelated recombinant anti-RBC antibody (an anti-blood group B antibody; B). In (A), Rh(D)-positive RBCs were incubated with soluble Fabs only, phage-displayed Fabs only,
or combinations of the two, as indicated. In (B), Rh(D)-positive RBCs
that were blood group B were used. After washing, RBCs were resuspended
in anti-M13 antibody and assessed for agglutination induced by
phage-displayed Fabs. Soluble Fabs were used full-strength, whereas
Fab/phage preparations were present in limiting amounts to increase the
sensitivity of the inhibition assay (see the Materials and Methods). In
(C), mutual inhibition of epD3 and epD6/7 anti-Rh(D) antibodies was
demonstrated with Rh(D)-positive RBCs, 1 and 1 soluble Fabs, and light chain isotype-specific
antisera (see text for details). In these examples, the anti-epD3 and
anti-epD6/7 antibodies were clones E1/M3 and D5/I3, respectively. The
anti-blood group B antibody was isolated from an IgG phage display
library made from the splenic B cells of a blood group O
donor.61
|
|
Isoelectric Point (pI) Analysis of Anti-Rh(D) Antibodies
The restriction in VH germline gene usage to only four
VHIII family members was intriguing in light of their
ability to confer specificity to a number of Rh(D) epitopes. As
suggested by Boucher et al,10 VH germline gene
segments used to encode anti-Rh(D) antibodies are among the most
cationic segments available in the human VH repertoire that
may be used to account for the relatively high pI of polyclonal
anti-Rh(D)-containing antisera.38,39 Although the cationic
nature of the antibodies may be important for binding to Rh(D), it has
also been suggested that a constitutive net positive charge may be
necessary to permeate the highly negative RBC  potential, thus permitting antibody to contact antigen.34 In either case, analysis of the predicted pI for the 28 heavy chains
and 41 light chains isolated here showed an interesting phenomenon for
the heavy versus light chains. Using the pI interval scale of Boucher
et al,10 the average pI of the 4 germline VH segments used to encode the 28 heavy chains is high (9.87 ± 0.15) and significantly higher than that of 39 randomly picked, non-Rh(D) binding clones from the original unpanned libraries (9.24 ± 0.80, P < 10 5). Similar to the results of
Boucher et al,10 the addition of D and JH
segments and the introduction of somatic mutation did not significantly
change the pI of the average anti-Rh(D) heavy chain (9.81 ± 0.33, P < .37). However, for the light chains, the average pI of
their germline counterparts was not cationic, but the light chains
became so through the addition of JL segments and somatic
mutation. Overall, for all 18 and 23 light chains, paired
t-test analyses before and after somatic mutation showed a
significant increase in net positive charge when comparing germline VL (6.63 ± 1.47) with expressed VL (7.28 ± 1.51, P < 103) or germline
VLJL (7.43 ± 1.47) with expressed
VLJL (8.55 ± 1.35, P < 107). There was no significant increase in a similar
analysis of 16 non-Rh(D) binding clones (P < .59 and
P < .19, respectively). Examination of the light chain
sequences (Figs 4 and 5) showed that this increase in pI resulted from
mutations that not only introduced positively charged residues, but
also eliminated some negatively charged residues. There were 31 such
events, 29 (91%) of which occurred in the light chain CDR regions.
| |
DISCUSSION |
Conventional and Phage-Displayed Anti-Rh(D) MoAbs
Because of differences in methodology, we were interested in comparing
our phage-display-derived anti-Rh(D) clones with those produced by
conventional tissue culture techniques (EBV transformation and cell
fusion). Despite the relatively small number of previously published
sequences for IgG anti-Rh(D) antibodies (N = 21) and the fact that they
were derived from over 10 different donors,10,23-25 there
was surprisingly good correlation between the two groups (Table 1). Both cohorts demonstrated a
predominance of VHIII-family encoded germline genes,
particularly those of the VH3-33 superspecies. CDR3 regions showed
similar lengths, ranging from 15 to 19 residues for Fab/phage
antibodies and 16 to 20 for conventional monoclonals, although one
heterohybridoma was an outlier with a CDR3 length of 28 residues. light chains were biased towards V1 family members and
light chains demonstrated the preferential use of the
J2Vasicek gene. The only qualitative discrepancy was in
V family usage, where Fab/phage clones demonstrated a slight preference for VI versus VIII
family members for conventional monoclonals. However, in both cohorts,
DPL16 was used more often than any other light chain gene.
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|
Table 1.
Comparison of Current IgG Fab/Phage Library-Derived
Anti-Rh(D) MoAbs With Those Previously Produced by Conventional
Tissue Culture Methods
|
|
It has been suggested in the literature that the VH4-34 (VH4.21)
germline gene, a gene used by many autoantibodies and cold agglutinins,40-42 may play an important role in the immune
response to Rh(D).43 However, these conclusions arose from
the analysis of IgM monoclonals and only 2 of the 21 published
anti-Rh(D) IgG sequences used VH4-34.25 In a related series
of experiments, we pooled aliquots of the 1 and
1 libraries obtained after the second and third
rounds of selection and then panned them against the VH4-34 specific
rat anti-idiotypic MoAb (9G444). Although we successfully
enriched for VH4-34 encoded antibodies, the Fab/phage were not specific
for Rh(D) and displayed serological characteristics similar to those of
cold agglutinins (data not shown). We are currently examining a µ phage display library from the same donor to compare gene usage.
Rh(D) Epitopes and Significance of Antibody Sequences
Since the initial report by Argall et al45 in 1953, it has
been recognized that rare individuals who type as Rh(D)-positive can
produce allo-anti-Rh(D) antibodies in response to Rh(D) immunization by
transfusion or pregnancy. This phenomenon was explained by hypothesizing that the Rh(D) antigen is a mosaic structure and that
these individuals were producing alloantibodies to parts of the mosaic
they lack. By systematically examining patterns of reactivity between
their cells and sera, RBCs expressing partial Rh(D) antigens were
divided into categories, each presumed to have a different abnormality
in their Rh(D) antigen. Through the subsequent use of index panels of
monoclonal anti-Rh(D) antibodies, a series of epitopes were defined of
which the number and combination varied from one Rh(D) category to
another. As new monoclonals were produced, their reactivity profiles
against these partial Rh(D) RBCs became the standard method for
determining Rh(D) antibody epitope specificity. Molecular analyses of
partial Rh(D) phenotypes have shown that the Rh(D) genes in these
individuals have either undergone intergenic recombination with the
highly homologous Rh(CE) gene or, less commonly, have sustained point
mutation(s).46
As noted earlier, to investigate the topological relationships among
Rh(D) epitopes, Gorick et al37 performed competition experiments with Rh(D) MoAbs and observed varying degrees of
inhibition. These results, when combined with those of Lomas et
al,6 suggested a model for Rh(D) in which epitopes are
spatially distinct yet demonstrate a certain degree of overlap, as
shown in Fig 10A. This model explained
how antibodies to two different Rh(D) epitopes (in this case epD2 and
epD3) could inhibit each other's binding to wild-type Rh(D) and how a
change in the structure of Rh(D) in category VI RBCs (asterisk, Fig
10A) would cause the loss of epD2. However, based on this concept of
Rh(D) epitopes as distinct domains, we would expect that antibodies
against different epitopes of Rh(D) would be structurally and
genetically distinct as well. Thus, it was surprising that our
anti-Rh(D) clones demonstrated such marked restriction in gene usage.
For example, only two superspecies of VH genes were used
despite specificities for 4 of the original 6 Rh(D) epitopes described
by Lomas et al.6 Furthermore, multiple specificities could
arise from a single heavy chain depending on the light chain with which
it was paired (eg, E1 with M2, M3, L3, or L4). In addition, other
clones repeatedly demonstrated variable weak reactivity against certain
Rh(D) category RBCs that would affect the epitope specificities to
which they were assigned (eg, C1 with O1, M1, or J5).
View larger version (28K):
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| Fig 10.
Conventional (A) and proposed (B) models for Rh(D)
antigen/antibody binding. Note that the predicted combining sites and
genetic relationships between antibodies differ between the two models. (C) If antibodies directed at different Rh(D) epitopes are clonally related, then the expressed repertoire will differ between
Rh(D)-negative and partial Rh(D) individuals (see text for
discussion).
|
|
Several hypotheses could account for these findings. The most
simplistic interpretation is that the heavy chain does not directly interact with the antigen, but rather is responsible for bringing the
antibody in close proximity with the antigen. The specific interactions
between the light chain and the antigen would then determine the
epitope specificity for that antibody. In this regard, our data are
consistent with the observations of Boucher et al10 on the
relative cationic nature of anti-Rh(D) heavy chains. However, because
we found that light chains become cationic during somatic mutation, the
charge of the entire antibody may play a role in its ability to bind,
resulting in the selection and expansion of particular B-cell clones.
A more compelling hypothesis is that Rh(D) epitopes do not differ
spatially but differ only in the number and arrangement of contact
residues presented (Fig 10B). In other words, the footprints of most,
if not all, anti-Rh(D) antibodies are essentially identical to one
another. The genetic events that produce partial Rh(D) molecules result
in the loss of certain critical key points of contact necessary for
some antibodies to bind; alternatively, they result in the formation of
new structures that interfere with the binding of other anti-Rh(D) Igs.
For example, the introduction of a ledge in Rh(D) category VI cells
(asterisk, Fig 10B) does not interfere with the binding of an anti-epD3
antibody, but does prevent the binding of anti-epD2. Therefore,
category VI RBCs are said to have epD3 but lack epD2.
This model is consistent with our inhibition experiments (Fig 9) and
with those of Gorick et al37 and offers an explanation for
the marked restriction in heavy chain gene usage. It also reconciles a
mechanism by which one heavy chain (eg, E1) can confer binding to
multiple epitopes and why some of our recombinant anti-Rh(D) antibodies, as well as some conventionally produced
monoclonals,36 display variable reactivity against certain
categories of partial Rh(D) RBCs. From the antigen's perspective, this
model explains how a single point mutation in Rh(D) can result in the
loss of multiple Rh(D) epitopes (such as T283I in category HMi
RBCs47) and how the residues associated with the expression
of some epitopes appear to be distributed among nearly all the
extracellular loops of Rh(D).48 It also provides an
understanding as to how  37 epitopes can fit on the relatively small
extracellularly exposed surface of the Rh(D) molecule.3
This concept of coincident epitopes is best exemplified by comparing
the E1/M2 and E1/M3 clones. The only difference between the reactivity
of E1/M2 and E1/M3 is the ability of the latter antibody to agglutinate
Rh(D) category VI cells (Fig 7). Hence, E1/M2 is classified as an
anti-epD2 and E1/M3 as an anti-epD3 antibody. Light chains M2 and M3
differ by only 3 residues: D82A, G95aA, and W96V (Fig 5). Therefore,
some combination of these residues confers reactivity against category
VI cells. In other words, epD2 and epD3, as seen by the E1/M2 and E1/M3
antibodies, differ by the binding constraints imposed by at most three
mutations. If the model depicted in Fig 10A were correct and the
epitopes were independent, these mutations would have to cause enough
structural alteration in the antibody combining site so that a
completely separate epitope on the same antigen would be recognized. It
would seem unlikely that these 3 mutations could cause such a change, especially given the lack of internal homology domains in Rh(D). Thus,
we conclude that it is far more plausible that the footprints of these
2 antibodies are essentially identical and that one or more of these
mutations (eg, the tryptophan in CDR3 of M2) prevent(s) the interaction
of E1/M2 with category VI RBCs. Because other clones demonstrate that
the light chain can confer specificity against epD1, epD2, or epD3
(with the E1 heavy chain); epD1 or epDX (with C5); and epD1, epD2, and
epD6/7 (with D12), we suggest that all 5 of these epitopes have similar
antibody combining sites.
Immunologic and Clinical Implications of Proposed Model
The model depicted in Fig 10B leads to additional predictions
concerning the Rh(D) immune response beyond simply clarifying what is
meant by an Rh(D) epitope. It is commonly stated in the transfusion
medicine literature that individuals whose RBCs express partial Rh(D)
antigens are free to make antibodies to the Rh(D) epitopes they
lack.34 Therefore, an individual who produces category VI
RBCs should be able to make anti-epD2 but not anti-epD3. If these
epitopes were truly independent, then the immune repertoire of the
anti-epD2 antibodies made by a category VI individual would be similar
to those produced by an Rh(D)-negative person. However, to the immune
system, epD2 and epD3 are not independent. We postulate that the
somatic mutation of an anti-epD3 antibody can change its fine
specificity to that of epD2 (or vice versa, Fig 10C). Suppose that the
preferred way of making an anti-epD2 antibody is to go through an
anti-epD3 intermediate. To an Rh(D)-negative individual, this process
can take place unimpeded. However, in a category VI individual, this
route would be unfavorable because an anti-epD3 antibody would be
self-reactive. As a result, such an individual would have to make
anti-epD2 antibodies by following alternative routes or by tolerating
some degree of autoreactivity in the process. With respect to the
latter point, it is of interest to note that a transient production of
auto-anti-Rh(D) frequently precedes or accompanies the early production
of allo-anti-Rh(D) in individuals who express partial Rh(D)
antigens.49-54 We would predict, therefore, that the
anti-epD2 antibodies from a category VI individual would be different
in composition (ie, gene usage) and quite possibly quantitatively
depressed as compared with an Rh(D)-negative individual. This may be
analogous to the antibodies of the ABO blood group system in which it
has been observed that anti-A and anti-B titers in blood group O
individuals are significantly higher than in blood group B or A
individuals, respectively.55 Blood group O individuals are
unconstrained in creating their anti-A and anti-B immune repertoires,
whereas individuals who produce A or B antigens (2 nearly identical
structures) must do so in a manner that avoids self-reactivity.
In the case of antibodies E1/M2 and E1/M3, they appear to
have arisen from a common precursor B cell rather than directly from each other (Fig 5). To test the framework of our hypothesis, ie,
somatic mutation resulting in epitope migration of an antibody, we are
constructing the precursors and potential intermediates between the M2
and M3 light chains and will then determine what Rh(D) epitope
specificities (if any) they express. This concept of epitope migration
has been previously reported for murine anti-cryptococcal56 and anti-type II collagen57 antibodies.
If the proposed model for Rh(D) epitopes is correct, then the question
of the number of epitopes may be obsolete. There may be as many
epitopes as can be differentiated by the number of cell categories, ie,
2n epitopes, where n is the number of distinct partial
Rh(D) RBCs. A more important question is the interrelationships between
the various epitopes. For example, are some epitopes further away than
others not in the topological sense, but in terms of the number of
mutational hits an antibody needs to receive to change its serologic
reactivity. Furthermore, does the humoral immune response in a partial
Rh(D) individual differ from that in an Rh(D)-negative individual in
the manner predicted by this model? One may find that allo-anti-Rh(D)
antibodies made by partial Rh(D) individuals are not as clinically
significant, ie, capable of inducing hemolysis. This may explain why
hemolytic disease of the newborn due to anti-Rh(D) produced by pregnant
individuals with partial Rh(D) phenotypes is so rare even when taking
into account the low prevalence of the partial Rh(D)
phenotypes.34 A better understanding of the immune response
to Rh(D) in these patients may alleviate concerns regarding the need to
identify such individuals to ensure that they only receive
Rh(D)-negative blood products for transfusion and Rh(D)-immune globulin
during pregnancy.58 Furthermore, with respect to the design
of recombinant Rh(D)-immune globulin for use in Rh(D)-negative
patients, it may not be necessary to formulate cocktails of MoAbs
containing multiple Rh(D) epitope specificities.
In summary, we have studied the genetic and immunological properties of
a large array of anti-Rh(D) antibodies to elucidate this clinically
significant human immune response on a molecular level. Our results
show that anti-Rh(D) antibodies display a high degree of structural
relatedness and the ability to inhibit each other's binding despite
differences in epitope specificity. These findings suggest that Rh(D)
epitopes are not spatially distinct and that Rh(D) antibodies may
undergo epitope migration as a result of somatic mutation. The end
result is that the prevalence of certain anti-Rh(D) specificities in
the immune repertoire may be a function not only of what epitopes an
individual lacks, but of the number of accessible pathways that the
individual's immune system can use that avoid self-reactivity. This
process may be a general feature of human immune responses to other
clinically significant, closely related epitopes.
| |
FOOTNOTES |
Submitted September 29, 1997;
accepted November 20, 1997.
Supported in part by March of Dimes Birth Defects Foundation Basil
O'Connor (Grant No. 5-FY94-0787) and Clinical Research (Grant No.
6-FY96-0367) Awards (D.L.S.), by a National Institutes of Health
Specialized Center of Research (SCOR) in Transfusion Medicine and
Biology Award (P50-HL54516; to D.L.S.), and by a grant from the
National Blood Foundation (T.Y.C.).
Address reprint requests to Don L. Siegel, PhD, MD, Department of
Pathology & Laboratory Medicine, 6-55 Founders Pavilion, Hospital of
the University of Pennsylvania, 3400 Spruce St, Philadelphia, PA 19104.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors thank Shari Russell and Vatinee Bunya for their excellent
technical assistance and Christine Lomas-Francis, Marion Reid, Marilyn
Moulds, and Peggy Spruell for providing samples of Rh(D) variant RBCs.
| |
APPENDIX |
Genbank accession numbers for anti-Rh(D) heavy chains are as follows:
B01, AF044419; C01, AF044420; C03, AF044421; C04, AF044422; C05,
AF044423; C08, AF044424; C10, AF044425; D01, AF044426; D03; AF044427;
D04, AF044428; D05, AF044429; D07, AF044430; D08, AF044431; D09,
AF044432; D10, AF044433; D11, AF044434; D12, AF044435; D13, AF044436;
D14, AF044437; D15, AF044438; D16, AF044439; D17, AF044440; D18,
AF044441; D20, AF044442; D30, AF044443; D31, AF044444; E01, AF044445; E03, AF044446. Genbank accession numbers for antiRh(D) light chains
are as follows: F01, AF044447; G01, AF044448; H01, AF044449; I01,
AF044450; I02, AF044451; I03, AF044452; I04, AF044453; I05, AF044454;
I06, AF044455; I07, AF044456; I08, AF044457; I09, AF044458; I10,
AF044459; I11, AF044460; I12, AF044461; I13, AF044462; I15, AF044463;
I16, AF044464. Genbank accession numbers for anti-Rh(D) light
chains are as follows: J01, AF044465; J02, AF044466; J04, AF044467; J06, AF044468; K01, AF044469; K02, AF044470; K03, AF044471; L01,
AF044472; L03, AF044473; L04, AF044474; L05, AF044475; M01, AF044476;
M02, AF044477; M03, AF044478; N01, AF044479; N02, AF044480; O01,
AF044481; O02, AF044482; O03, AF044483; P01, AF044484; Q01, AF044485;
R01, AF044486; S01, AF044487.
| |
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Abstract
Introduction
Abstract