Bioscience Reports 5, 893-899 (I 985)
Printed in Great Britain
893
The antibody paradox: Trying on a pair of genes
Julian B. FLEISCHMAN
Department of Microbiology and Immunology, Washington University School of
Medicine, St. Louis, Missouri 63110, U.S.A.
(Received 25 September 1985)
Rodney Porter's separation of antibody molecules into Fab
and Fc fragments engendered the notion that a single antibody polypeptide chain might be coded by two or more
genes. This concept profoundly influenced the development
of molecular immunology over the past 25 years. Our current
knowledge of antibody gene organization has enabled investigators to recombine antibody genes to create 'chimeric' antibodies with a number of potentially useful applications.
The idea that separate genes might contribute to the formation of an
antibody polypeptide chain had its start when Rodney Porter split antibodies into distinct fragments, later called Tab' and 'Fc' (Porter, 1958;
Porter, 1959). Porter's discovery was of fundamental importance for
immunology because it offered for the first time a molecular basis for the
'antibody paradox': an antigen-specific, possibly variable portion (Fab)
coexisting in the same protein molecule with a crystallizable common
portion (Fc) (Fig. 1). The polypeptide chain structure of the antibody
molecule was still unknown in 1959, but since a proteolytic enzyme was
required to separate Fab from Fc, it was likely (and eventually turned out to
be true) that specific and common portions of the molecule were connected
in the same polypeptide chain. This remarkable phenomenon suggested
almost at once the heretical notion that more than one structural gene might
contribute to the formation of an antibody polypeptide chain. The idea
violated the one gene/one polypeptide dogma of the time, but it excited
everyone's imagination and was often discussed informally at the Antibody
Workshops (of which Rod Porter was a co-founder) in the early 1960s.
Two Genes Make One Chain
Porter immediately recognized the disparate nature of the Fab and Fc
fragments and he was the first to test the possibility that Fab and Fc were
separate biosynthetic subunits, which might be joined to form the complete
molecule. He injected 14C-amino acids into a rabbit in vivo, but the specific
activities of the subsequently isolated Fab and Fc fragments were similar,
and showed no evidence of being formed from separate precursor pools
(Porter, 1959). While I was a post-doc in Mel Cohn's lab at Stanford, I
attempted a similar experiment in a more sensitive system: adding short
894
FLEISCHMAN
CH3
Fc
Fig. 1. Schematic diagram of an IgG molecule. Approximate sites of
cleavage by papain and pepsin are shown~ Papain produces two Fab
fragments and one Fc. Pepsin produces a disulfide-linked dimer of two
Fab-like fragments; Fc is degraded by pepsin. Fd is the portion of heavy
chain contained in Fab. VL and CL are variable and constant domains
of the light chain; VH, CH1, CH2, and CH3 are the variable and three
constant domains of the heavy chain.
pulses of tritiated leucine to suspensions of antibody-forming lymph node
cells. Again, the experilnent failed to reveal evidence for separate synthesis
o f Fab and Fc (Fleischman, 1963). The labeling experiments were premature
because Fab and Fc did not correspond to the polypeptide subunits o f
immunoglobulins, which were still to be discovered. But the idea behind
them reflected the new concept, engendered b y Porter's discovery, that
a n t i b o d y molecules might be made up of separate gene products: one for
antigen specificity and another for a c o m m o n region.
A significant by-product of the experiment at Stanford was our preparation of goat antisera specific for rabbit Fab and for Fc. The Fab and Fc
antigens used to immunize the goats had been carefully purified b y Ed
Lennox, and the antisera were highly specific. I brought the antisera to
Porter's lab at St. Mary's in 1961, and during m y subsequent year there
we pursued the isolation and characterization of heavy and light chains. The
antisera proved critical in locating part o f the heavy chain, plus the light
chain, in Fab, and the remainder o f the heavy chain in Fc, thus defining the
overall architecture o f the molecule (Fleischman et al., 1963 ; Porter, 1973).
As soon as the antibody polypeptide chains were identified, the notion
that two gene products might form a single chain became more clearly
defined. Research immediately focused on the contrast between the ~specific'
(Fd) and 'common' (Fc) portions o f the heavy chain. Todd (1963) found
that certain allotype markers, presumably coded b y one gene locus, were
895
THE ANTIBODY PARADOX
located in Fd; other (isotypic) determinants were in Fc. By 1964, there had
been frequent speculation that the Fd and Fc parts of the heavy chain in
fact might be separate chains, presumably coded by different structural
genes, which were linked in the antibody by an unusual bond (Nisonoff et
al., 1960; Todd, 1963; Cohen & Porter, 1964; Franklin, 1964).
The first amino acid sequences of light chains presented at the 1965
Antibody Workshop in Warner Springs, California (Hilschmann & Craig,
1965; Titani et al., 1965), and subsequent heavy chain sequences (Edelman
et al., 1969; Press & Hogg, 1970) dramatically confirmed that both antibody
polypeptide chains were divided into variable (V) regions specific for particular antigens and constant (C) regions common to all antibody chains of a
particular class. A major structural aspect of the antibody paradox, first
recognized by Porter's division of the molecule into specific (Fab) and
c o m m o n (Fc) regions, had been solved.
On a return visit to Porter's lab, this time in Oxford, I sequenced part of
an IgG heavy chain from a homogeneous rabbit antibody to group C streptococcal carbohydrate, which bore the a2 allotype (Fleischman, 1971 ; Fleischman, 1973). The a2-related residues were confined to the V region, which
reinforced the notion that the heavy chain could be fitted by a pair of genes.
The discovery of V and C regions in the same chain made it seem even
more likely that two genes would code for a single polypeptide. But the
sequence data gave no clue as to whether V and C genes, or their products,
were joined at the DNA, RNA, or protein level. A hypothesis was proposed
for joining V and C genes in DNA (Dreyer & Bennett, !965). In the 1960s,
most of the recombinant DNA methodology was not available, but the two
gene - o n e chain idea could be tested by searching for separately synthesized
A
B
completedfNH2
chains
~.NH2
-*****- COOH
*-*****-*****- COOH
0ecfc
activity
activity
peptide
l
variable
constant
peptide
Fig. 2. (A) Labeling patterns for poIypeptide chains growing from a
single initiation point at the NH2-terminus (Dintzis, 1961). ***are
labeled amino acids. The graph schematically illustrates the relative
specific activities of peptide fragments derived from newly completed
chains. There is a single gradient of increasing specific activity from the
NH2- to the COOH-terminus. (B) Hypothetical two-gradient pattern
expected if an antibody chain were formed by joining separately
synthesized V and C polypeptides. This pattern was not found experimentally (Fleischman, 1967b; Knopf et al., 1967). Instead, growing
antibody chains revealed a single gradient, as shown in A.
896
FLEISCHMAN
V and C polypeptide chains. I continued to be intrigued by the idea, and
tested it using a labeling technique for growing polypeptide chains developed
by Dintzis (1961). He showed that if growing chains are labeled for short
periods with radioactive amino acids, the most recently completed chains
will have incorporated the radioactive amino acids near their C-terminal
ends. Cleavage of the newly completed chains into smaller peptides should
therefore reveal a gradient of increasing specific activity of labeled amino
acid residues from the. amino to the carboxyl terminus (Fig. 2A). If antibody
chains were synthesized in two separate parts, V and C, which were subsequently joined together, one would predict two such gradients of specific
activity, corresponding to the two separately growing V and C polypeptides
in the newly completed chain (Fig. 2B). My results (Fleischman, 1967a, b)
and those of Knopf, Parkhouse and Lennox (1967) indicated that the heavy
chain grew from a single initiation point at the NH2-terminus, as shown in
Fig. 2A. An intracellular light chain fragment was later suspected of being a
separately synthesized VL region (Schubert & Cohn, 1970). Sandy Bridges
in my laboratory showed that the fragment in fact contained C region
peptides and was probably a heterogeneous group of nascent light chains
(Bridges & Fleischman, 1975). Thus there was no convincing evidence that
V and C proteins were synthesized separately. If different genes coded for
variable and constant regions, they would have to be joined in DNA or at the
RNA transcript stage.
Hozumi and Tonegawa (1976) finally established that antibody light
chains were coded by separate V and C genes, and that the genes were joined
during the differentiation of antibody-forming cells. V and C genes for heavy
chains (Early et al., 1980; Sakano et al., 1980) were identified about 20
years after Porter's Fab and Fc fragments first suggested their possible existence. A great deal of genetic analysis has revealed the organization and
rearrangement of antibody V and C genes and the participation of smaller
gene segments (D and J) which contribute to V-region structure (reviewed by
Honjo, 1983). An important consequence of these gene rearrangements for
immunology is that many of them generate variability in amino acid sequence,
which is essential for the diversity of the immune system. Antibody heavy
and light chains, and their counterparts in the T cell receptor molecule
(Hood et al., 1985), are still the only known eukaryotic proteins where
separate structural gene segments are joined to form a template for transcription and translation of a single protein chain. The antibody paradox, first
highlighted by Porter's separation of Fab and Fc fragments, is now well
resolved.
Preferential Pairing of Heavy Chain V and C Regions
Other important questions concerning the association of V and C regions
persist. The organization of heavy chain genes would predict that any heavy
chain V gene (VH) could associate with any heavy chain C gene (CH). However, in many immune responses, VH idiotypes often are found preferentially
associated with certain CH isotopes. We have recently studied preferential
combinations of VH idiotypes and IgG CH isotypes in monoclonal antibodies
to dinitrophenyl (DNP) antigens (Scott & Fleischman, 1982). Two VH idio-
THE ANTIBODYPARADOX
897
types, first identified on our monoclonal antibodies 7-17 and 8-11, dominate
the in vivo primary response to DNP-Ficoll; in IgG, these idiotypes are
preferentially associated with C73 constant regions. In secondary and
tertiary IgG responses to DNP proteins, another VH idiotype, (460), dominates the response and is mainly associated with C71 constant regions and
with x light chains.
A relatively high proportion (5/11) of the primary response IgG3 monoclonal antibodies to DNP-Ficoll bore X2 light chains, and 25-50% of plaque
forming cells stimulated by DNP-Ficoll were inhibited by antiserum to ?,2
(Scott & Fleisehman, 1982). This is surprising because ?,2 and k3 light chains
are rarely expressed in the mouse (on < 1% of normal serum immunoglobulins). Others have confirmed that ?`2 and X3 light chains are unusually
frequent in antibodies to DNP (Liu et al., 1984). The preferential combination of VH idiotypes 7-17 or 8-11 with C73 constant regions, which dominate the primary IgG response to DNP-Ficoll, may create a heavy chain
which is especially adapted to pairing with a X2 light chain.
The IgG3 primary response antibodies to DNP seemed particularly interesting, and we wished.to characterize them further. Tong-Sun Kobilka in our
laboratory measured the absolute affinities of our anti-DNP monoclonal
antibodies for 3H-e-DNP-lysine by equilibrium dialysis. She also compared
the relative affinities of each monoclonal antibody for both 14C-DNP-7aminobutyric acid and 3H-TNP-7-aminobutyric acid in a competitive binding
assay developed by Hammer and Steiner (1982). The primary response
IgG3 antibodies to DNP-Ficoll generally had low binding affinity for DNPlysine, and at bast two of them exhibited heteroclitic binding, i.e. they
bound the TNP hapten more strongly than DNP (Kobilka et al., 1984). Her
results were consistent with those previously obtained for polyclonal antisera to DNP (Hammer & Steiner, 1982). The primary response IgG3 antibodies may be products of B-cell clones expanded by previously encountered
cross-reactive environmental antigens, and re-stimulated by DNP-Ficoll.
The problem of why certain VH regions are preferentially paired with
particular CH regions has not yet been answered. The genomic arrangement
of V, D, J and CH genes in DNA would not predict such preferential pairing.
Rather, combinations of factors outside the B-cell, such as the level of
circulating antigen plus the influence of idiotype- and isotype-specific helper
T cells, may select B-cell clones bearing particular idiotype-isotype combinations for expansion and subsequent antibody production. The relative
importance of each of these factors varies in immune responses to different
antigens.
Future Prospects: Designer Genes
An intriguing benefit has emerged from the genetic solution of the antibody paradox. Immunologists have begun to create genetically engineered
('chimeric') antibodies from artificial combinations of V and C genes (reviewed
by Munro, 1984). A rearranged gene coding for a VH region that binds
azophenylarsonate has been linked to a C~ gene; the resulting hybrid chain,
when paired with an appropriate light chain, specifically bound the azophenylarsonate antigen (Sharon et al., 1984). Mouse VH and VL regions
898
FLEISCHMAN
specific for phosphocholine and for TNP have been joined to h u m a n C H and
C L regions by gene splicing (Morrison et al., 1984; Boulianne et al., 1984)
and a h u m a n VH gene has been joined to a mouse CH gene (Takeda et al.,
1985). The chimeric antibodies specifically bound their appropriate antigens.
Variable regions can also be joined to other proteins to create 'antibodies'
with specialized effector functions (Neuberger et al., 1984).
Chimeric antibodies promise to be useful in i m m u n o t h e r a p y and diagnosis.
Mouse or rat V regions coupled to human constant regions m a y minimize
allergic responses or immune elimination after passive administration in a
human host. Chimeric V-C gene constructs could also be valuable for studying the genetic elements which regulate antibody gene expression in different
ceils.
Our current understanding o f the genetic events underlying antibody
formation stemmed from Rod Porter's original discovery of the Fab and Fc
fragments o f the antibody molecule. The observation that DNA segments
from separate locations in the genome must join up to make a template for
a single a n t i b o d y or T cell receptor polypeptide chain was unprecedented,
and constituted a remarkable advance in biology.
Acknowledgements
I am deeply indebted to R o d n e y Porter for the opportunities to have
worked in his laboratories b o t h at St. Mary's Hospital and at Oxford, and for
his advice, guidance, and encouragement on b o t h occasions. I am grateful
to Donna T h u r m o n d and to Tong-Sun Kobilka for their technical assistance.
Work from our laboratory was supported in part by research grants from the
U.S. Public Health Service and from the Washington University/Mallinckrodt
and Washington University/Monsanto Hybridoma contracts.
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