1 INTRODUCTION

In 1966 two researchers, an Immunologist and a Biochemist, met at Karolinska Institute in Stockholm and collaborated on the application of fluorochromasia on HLA (Human Leucocyte Antigen) Typing. The two also discussed the possibility of restoring the catalytic activity of a mutant enzyme molecule by exposing it to an anti-wild type enzyme antibody. The scheme looked like a shot in the dark, nevertheless in June 1967 the two friends convened again at Brown University, Providence (RI, USA) and set up an experiment where antibodies elicited against the β-galactosidase (GZ) enzyme from wild type Escherichia coli were added to defective (almost inactive) betaGal molecules from E coli strains carrying missense point mutations in the lacZ region. The result was a clear success: the defective betaGal from one of the mutant strains reached a 500-fold enhancement of activity. This phenomenon was called AMEF, acronym of antibody-mediated enzyme formation.

Fifty-three years later, a combination of two improbabilities, the free time suddenly allotted to the hostages of the coronavirus pandemic coupled with the persistent curiosity in aging finders and witnesses of the discovery of AMEF, prompted the same Immunologist, Franco Celada, together with a somewhat younger Biochemist, Roberto Strom (who had participated in most of the refinement studies during the decade following the first result), to undertake a re-visitation of AMEF history and of its significance. The aim of this review is to commemorate the birth of a new use of specific antibodies as TOOLS to re-activate the betaGal enzyme made defective by a missense point mutation and to illustrate the new developments triggered by AMEF as well as the brilliant and unsuspected inventions and unveilings, which come as late cherries to the AMEF saga.

As insider reviewers, we had to pledge total objectivity, and our advantage was the enjoyment of a constant flow of personal memories about experiments, results, facts, and other events, which, if allowed to enter the text, would decrease the distance between Life and Science, and would lighten the scientific reading.

2 INTERDISCIPLINARITY

At a Nobel Symposium on SYMMETRY (in Stockholm in the 60's), Celada heard two Nobel laureates exchanging these spirited remarks:

“I - said Monod - speak to the Immunologists!”

“I - said Crick - do not speak to the Immunologists, but the Immunologists speak to me!”

Celada laughed with the audience at this exchange, but he also took it as a precious advice to be an interdisciplinarian, working with Biochemists, Geneticists, Enzymologists, Semiologists, Astrophysicists, and cooperating with mathematicians to build a computational modeling of the immune system.

3 THE ORIGIN

1966, Stockholm. Franco Celada, a recent Docent at Karolinska Institutet, is looking for the highest sensitivity method that would facilitate his work1 on adoptive memory cell cultures. He is hoping that, with a method sensitive to single molecules, he may succeed to disprove the idea that long-term Memory should require the presence of antigen in the memory cell. Cinader,2, 3 had recently examined several enzymes, whose activity was modified by specific antibodies; only few of them, as described, for example, by Pollock,4 exhibited a small increase of activity, certainly not sufficient for a precision measuring. To clear his mind, Celada has taken an appointment with Boris Rotman, a Chilean Biochemist who is spending a sabbatical at Karolinska before moving, as a newly appointed professor, to Brown University (R.I., U.S.A.). Celada and Rotman are already collaborating: a manuscript5 proposing the use of fluorochromasia in HLA typing has just been submitted.

Here is an extract of their lunch conversation:

FC: Congratulations for your seminar last week: those droplets containing one molecule of enzyme were fantastic! Now, regarding my adoptive memory cultures, my question is, how can one measure a single antibody molecule, or a single Ab+Ag event?

BR: Only if the antibody has enzymatic activity! Have you heard about such antibodies?

FC: No, I have not. Let me now consider enzyme molecules that are inactivated by specific antibodies, by picking one of Cinader‘s enzymes and using it as antigen. The antibodies will be measured by the decrease of enzyme activity … but most of the time the signal will be calculated against a high background. I would greatly prefer a quite opposite design, where the free antigen, untouched, would have no activity – and where the antibody would light up the enzyme activity, by its specific binding! A firefly in a dark forest, or a star in a moonless night!

Tell me, Boris, is this feasible, or incurably crazy?

BR Perhaps one could modify the Ab to introduce β-galactosidase activity. But I do not know how that can be done. It is not feasible at present. Perhaps one could find a mutant of β-galactosidase without enzymatic activity that will become active in the presence of antibody.

FC: How difficult is it to find such a mutant?

BR: It can be extremely difficult, but Van Niels said: “If a reaction can exist in nature, one should find a bacterium that carries it.” There are several bacterial strains with mutated inactive β-galactosidase, and I can obtain some samples from Lederberg's lab in Stanford, CA.

FC: “Aren't we forgetting something? What are the probabilities of finding an activable mutant?”

Boris answered:

BR: “Nearly zero, but not zero. Nothing is impossible in biology. Anyway, I like nearly impossible projects.”

FC: Let's try it! If we succeed, we shall spot fireflies in a moonless night.

Boris remained skeptical about the feasibility of the experiment, because there was no precedent for any similar effect of antibodies (at the time he was not aware of Pollock's experiments) - but Celada's enthusiasm convinced him to give it a try.

4 THE ABDUCTION

A review should make order and help connect with the object presented. The AMEF legacy begins with the formulation of a hypothesis (called abduction here, in Peirce's mode) sometime in the autumn of 1966 in Stockholm and continues as a two-month-collaboration leading to a discovery in Providence, conveniently located in the Narragansett Bay, in full sailing season. The nine-month interval phase was not used for any preparation except the immediate adoption of Rotman's suggestion to use point-mutant, inactive betaGal as target. This completed their abduction: it was never actually written, but I can transcribe it from memories and talks of the two.

Imagine two doctors standing in front of the patient, a “mutant,” with a tentative diagnosis and only one therapy, never tried before. Here is the abduction:

“A single mutation hits a spot and alters an unknown number of equilibria of the betaGal conformation, causing the enzyme to become active.”

The two doctors say:

We bet our money that this specific antibody is a cure for the mutant. If it manages to bind to the mutant, the antibody will do its best to improve its grasp by convincing the epitope to come back to its original wild-type (wt) shape: the steps in this direction are energetically virtuous, and it is feasible for the entire conformation to snap back to wt, and thus, to function.

5 THE 9-MONTHS HIATUS

A 9-months interval was used by Rotman to set up his new Lab at Brown University, and by Celada to publish his paper1 on Memory Cell Adoptive Transfers and, immediately afterward, to take his wife and their three children to the Arlanda Airport, to wave them off to a vacation in the Alps, ship his Volvo to the harbor of New York, from where he would pick it up and drive to Rhode Island on June 2. The shipping of a two-year-old car on the Volvo-cargo vessel is part of a Swedish scheme to avoid taxes, cost 100 dollars and it worked great.

6 DISCLAIMER

We will not discuss why the elder of two colleagues arranged a meeting with Geneticist Seymour Lederberg of Brown University the day before the experiment, to evaluate the sanity of our project. For the record the answer was: “If you two guys can think of it, the Phenomenon exists. To find it, depends on Probability.” (cf. Saint Anselm's proof of the existence of God: “If the greatest being exists in the mind, it must also exist in reality”).

7 THE 1967 EXPERIMENT

The newly built laboratory in Brown University, Rhode Island, was large and had working rooms at −20°C, +5°C, and +37°C. Franco Celada joined Boris Rotman and his assistants, Rosario Guzman (from Chile) and John Ellis. We were going to test 47 different E coli strains carrying point mutations in the lac Z gene (and produced, therefore, mutated betaGal proteins with a very low level of enzymatic activity), strains that Boris had just received by mail from Esther Lederberg in California. We planned to grow the cultures overnight, centrifuge, separate the sediments, pass them in the French press (characteristically noisy but efficient) to break all bacterial cells, distribute them into large tubes in 10x4 racks, then add rabbit serum that contained anti-betaGal antibodies (or control serum, or no serum), incubate for 1 hour, add o-nitrophenyl galactoside (ONPG), and measure, in a colorimeter, the orange color produced by the betaGal catalysis.

The work was relatively simple, but it had to be re-planned because of the exceedingly high number of tests to be performed on the 47 different bacterial strains. Fortunately, none of the members of the team was in a hurry to leave. We decided to prolong our stay without changing the rhythm: a sustainable number of three tests per day, three testing days per week; the testing went on for 6 weeks, allowing some re-testing and more necessary controls. By the end of July, we were done with the experiments, the figures needed for an article, and many, enthusiastic discussions. During all week-ends of July and August, there were seven-class regattas in Narraganset Bay: Boris was tactician on a noble Herreeschof-S, and Franco was crewing on an Ensign.

8 THE DISCOVERY: FIRST EXPERIMENTAL RESULTS AND THE FULFILLMENT OF THE ABDUCTION

Thirty-seven bacterial strains (out of 47) showed no enhancement of the basal enzymatic activities of their betaGal defective enzymes. In six of them, the enhancement factor was between 2% and 14%, two had enhancement factors 9 and 43, one was over 500. None of us, who were in the lab on that June 13, will ever forget the moment when strain W1601 produced an explosion of color and was named “AMEF” (it will be become, later on, “AMEF#6101”).

This first experiment in a new field was yielding thrilling results, but needed refinements. Anti-wt.GZ antibodies had caused a strong activation of betaGal activity in the AMEF extract, and the team knew, before the third week expired, that their ABDUCTION was confirmed. The relatively low (but measurable) basal betaGal activity contained in freshly prepared AMEF extracts appeared to be associated, when examined in a sucrose gradient, to a macromolecule with a sedimentation pattern very similar to that of the tetrameric wtGZ enzyme. A reasonable point to be investigated that was raised in our early discussions, would have been to check whether anti-AMEF antibodies exerted any effect on the betaGal activity of the W1601 strain - but 3 months were needed to raise these antibodies. In addition, other points, in our opinion, needed further clarification: (a) the size of the minimal protein unit (monomer, dimer, or tetramer?) susceptible of being activated upon interaction with anti-wtGZ, (b) the effective minimal paratope-to-protein ratio that could, in our AMEF preparation, lead to full activation of its latent enzymatic capability. The results of our first experiment seemed to us, anyhow, to be worth, per se, of more detailed examination and evaluation.

Figure 1 is the first image of the results, obtained in June 1967 at Brown University upon measurement of the levels of betaGal enzymatic activity in pairs of AMEF-containing parallel tubes after addition (at time 0, but alternatively also after 30 or 60 minutes) of anti-wtGZ antibody or of control serum). All tubes were measured for enzyme activity at 5 or 10 minutes intervals.

Development of betaGal enzymatic activity in the extract of AMEF#6101 after addition of anti-wtGZ antibodies (full circles) or of normal rabbit serum (fig. 3 by Rotman and Celada6) Celada remembers how, just by watching what happened in the assay tubes after addition of the ONPG betaGal substrate, he had two iconic impressions, one being the sheer magnitude of the antibody-dependent activation of ONPG hydrolysis, the other being, in controls, the surprisingly high background activity at time 0 that decayed to traces in about 1 hour, This experiment led in fact to two distinct findings: Anti-wtGZ antibodies activated the mutant betaGal, a reaction that reached its apex in 30 minutes. The extent of the activation process was surprisingly high – about 10-fold in the first set of tubes and becoming 550-fold when AMEF had been pre-incubated at 37°C for 30 minutes. The “second result” was seen in the tubes that were considered controls. These tubes, taken from the refrigerator and transferred to 37°C, showed a relatively high level of basal catalytic activity that, however, rapidly decreased upon more prolonged exposure to 37°C and dropped to about 1% of the initial value after 60 minutes. This peculiar temperature dependence of the basal catalytic activity suggested (without, however, proving it) that the AMEF protein could undergo some conformational change that severely decreased its basal enzymatic activity without, however, modifying the ability of anti-wtGZ to stimulate a full activation of that defective lacZ gene product. 9 WAITING FOR PUBLICATION 9.1 The “kidnap” to West-Berlin

In late August 1967, Franco Celada had flown from Boston to London in order to catch the connection to Stockholm. In the Heathrow waiting lounge, he met another Immunologist, Fritz Melchers. The two had met once in California, where Fritz was staging at the Salk Institute, and Fritz gave the impression of high competence. In the enthusiasm of his recent discovery, Celada told him the entire story of AMEF. This, however, increased, instead of appeasing, Fritz's curiosity. He said: “Can't you fly with me to West-Berlin today? This afternoon you will give a talk to my group, mostly students: they will be thrilled to hear it from the horse's mouth, and by tomorrow midday, you will land in Arlanda !" Franco was trying to raise some objections, but Fritz was unstoppable, he immediately got the tickets changed through his office. It was nevertheless past 7 pm when they arrived at West-Berlin University. Celada was wondering about his oncoming seminar. Fritz introduced him to the audience, who timely informed him that all of them had skipped their suppers. All the students were taking notes, but many of them (obviously those educated in East Germany), were not fluent in English. Celada felt almost obliged to give the seminar in his broken German, although it was for him a real effort.

9.2 The Roma connection

A few months later Franco Celada contacted his close friend Valerio Monesi, a brilliant Cell Biologist that he had met in 1959-61 when both of them had spent two very fruitful years in the Biology Division of the Oak Ridge National Laboratory. Monesi had returned a few years before to Italy at the Casaccia Laboratory (near Rome) of the Italian National Center for Nuclear Energy, but in 1968 he was on the verge of being nominated as full professor of Histology and Human Embryology at La Sapienza University of Rome. Upon learning about Celada's results on AMEF, he presumed that they could be of interest to Professor Alessandro Rossi Fanelli, who was well known, with the members of his team - among whom, in particular, Eraldo Antonini - for their scientific contributions to the structure-function problems of hemoglobin and of enzymatically active proteins. Celada was, therefore, invited, in the first months of year 1969, to give a seminar at the Institute of Biochemistry of La Sapienza University of Rome.

Before the seminar, he got a tour of the laboratories of that Institute, particularly of the students' benches, where he met a young frizzy-redhead post-doc, Roberto Strom, who showed a very high interest in the discussion about AMEF.

The seminar was a success. As well as the members of the Institute of Biochemistry, the audience included one of its a frequent guests, the well-known expert of Protein Physical Chemistry Jeffries Wyman Jr., who, 3 years earlier, had elaborated, with Monod and Changeux, the allosteric theory of ligand binding to multimeric proteins. Here is his dialogue with Celada:

JW: Nice finding, congrats, what is next in your Karolinska lab?

FC: My anti-AMEF rabbit sera are almost ready to be tested: will they activate AMEF, or not? This is the question! Would you care to guess?

JW: No need to guess. Anti-AMEF will not activate, by pure reason.

As soon as Celada returned to Stockholm, he verified this prediction, finding that it was fully confirmed.

As a consequence of this seminar, Roberto Strom, the young post-doc of the Rome Institute of Biochemistry, joined Franco Celada in June 1968 at the Karolinska Institute in Stockholm: a long friendship began.

10 THE AMEF LABS

Experimental work on AMEF continued for almost 15 years in three different laboratories. While Boris Rotman and his staff (Rosario Guzman and John Ellis) invited Alberto Macario and his wife Everly Conway de Macario to come to Brown University in Providence, Rhode Island, Franco Celada remained at Karolinska Institute in Stockholm for two more years, working with Roberto Strom and Kerstin Bodlund. He then decided to spend 1 year, starting from autumn 1970, at the Pasteur Institute in Paris, collaborating with Agnès Ullmann and Jacques Monod on the effects of anti-wtGZ antibodies on the ω-complementation of β-galactosidase. In 1971, he returned to Italy as “chief Immunologist” at the Italian Research Council in Rome; he worked at the Cell Biology Laboratory with Roberto Strom, Roberto Tosi, Roberto Accolla and Birgitta Åsjö for 5 years, then took a sabbatical year at University of California in Los Angeles (UCLA) working with Irving Zabin and with Eli Sercarz. In 1977, he returned to Italy, and settled in Genova University as full Professor of Immunology. In this lab, his collaborators were Jasna Radojkovic, Fabrizio Manca, Annalisa Kunkl, Antonio Lanzavecchia, Giuseppina LaPira, Caterina Cambiaggi, and Renata Cinà. During his wandering time, his lab changed addresses but kept the focus on AMEF alive.

The third independent group, located in Berlin University, was formed by Walter Messer (a bacterial Geneticist) and Fritz Melchers (an immunologist, who became, a few years later, Director of the Basel Institute). They had been directly informed about AMEF activation, through Celada's seminar in West Berlin, before the publication of the first paper, and joined enthusiastically and effectively the AMEF history.

The articles published on AMEF and mentioned in this review span from 1968 to 2014 for a total of 27; they are listed as “Amef-related” references, 24 of these articles (i.e. until year 1992) were directly aimed to the study of AMEF, while the last ones – namely references 36 (dated 1998), 37 (dated 2002) and 38 (dated 2014) reached the same goal by utilizing AMEF as a tool or as a marker promoting original ideas and advances in modern Biotechnology or in the highest levels of Crystallography.

11 FURTHER INVESTIGATIONS, IN THE AMEF LABS, ON THE ACTIVATION PROCESS OF DEFECTIVE BETAGAL GENE PRODUCTS BY anti-wtGZ POLYCLONAL ANTIBODIES

In a meeting on The Lactose Operon, held in the late Spring of 1969 at the Cold Spring Harbor Laboratory (with written reports published in 1970 as CSH Monograph vol.1), identical activating effects on AMEF#6101 could be exerted, in parallel experiments, by preparations of divalent anti-wtGZ and by their Fab monovalent fragments obtained by digestion with papain. Upon centrifugation that caused selective sedimentation of the immune complexes cross-linked by the divalent antibodies, all the enzymatic activity of the samples treated with monovalent Fab fragments remained in the supernatant (Figure 2).

Dependence of enzymatic activity of AMEF#6101 upon addition of anti-GZ antibodies concentrations of divalent anti-GZ antibodies (full circles) or of their monovalent Fab fragments (empty circles) (fig. 2 by Celada et al7)

It was thus possible to discard the hypothesis that, in AMEF, the defective betaGal protein be activated through an antibody-induced cross-linking mechanism acting on its subunits. When increasing concentrations of AMEF#6101 are exposed to a fixed amount of anti-wtGZ antibodies (or of their Fab fragments), the final levels of activity follow a hyperbolic shape toward a horizontal asymptote (that can be converted to a straight line in a double reciprocal plot). The activation process seems to occur as a consequence of a basically simple 1:1 epitope-paratope interaction, but its kinetics is consistent with the presence of a monomolecular rate-limiting step occurring within the AMEF molecules.

In fact, shortly after the end of the Lactose Operon Meeting, Celada and Rotman could even show, by a similar procedure that their anti-AMEF antibodies were unable to activate AMEF#6101, and also inhibited, through a competition involving the same AMEF epitope, the activating effect of anti-GZ antibodies (Figure 3).

Anti-AMEF sera competition against activation of AMEF#6101 by anti-wtGZ antibodies. The enzyme activity is plotted, in a double reciprocal plot, against the AMEF concentration in the presence of various concentrations of an anti-AMEF antibody preparation (fig. 2 by Celada et al8)

Roth and Rotman9 showed, some years later that these anti-AMEF antibodies were also non-competitive inhibitors of the catalytic activity of the wild-type β-galactosidase enzyme.

In the same Lactose Operon Meeting, the Berlin group (the immunologist Fritz Melchers and the bacterial geneticist Walter Messer) showed10 that the AMEF concept could be extended to several other defective lacZ gene products - produced by as many as 11 other missense point mutant E coli strains, whose genetic map is shown in Figure 4.

Genetic map of the Berlin defective point mutants (fig. 1 by Messer and Melchers10). The position of the lacz gene in the E.coli genome is shown on the top line, followed in the top line, followed by a scheme of its deletion mapping. The central part of the figure indicates the positions of of the various Berlin defective point mutants estimated from their relative recombination frequencies in PI(lac+) assays

These other antibody-activable defective enzymes (that we will also call AMEFs) exhibited11 non-uniform susceptibility to activation by anti-wtGZ antibodies: in AMEF#40 and AMEF#918 - products of strains carrying their point mutations at the distal end of the lacZ gene (the so-called “MM.group 2”) - the level of the “activation factor” was around 50×, while it was definitely higher (over 250×) for all the other AMEFs (except for AMEF#950). It was later shown by the same research group12 that all their 11 AMEFs had, in their native state, a tetrameric structure similar to that of wtGZ, that is, formed by association of 4 subunits whose molecular mass of 130 ± 15 kDa.

These AMEF protomers could even combine with wt-GZ, forming hybrids (Figure 5) whose enzymatic activity was, however, found13, 14 to be proportional to the number of wt-GZ protomers.

Enzymatic activity of wtGZ-AMEF#645 hybrids having different GZ/AMEF ratios. Empty circles and full circles indicate the situations immediately after hybrid formation and 7 days, after, respectively (fig. 1 by Melchers and Messer14)

Association of betaGal monomers (wt-GZ as well as AMEFs) in tetrameric structures appears, therefore, to be a necessary but not sufficient condition for the acquisition of a high level of enzymatic activity by each protomer. In the very first 1967 experiment,6 in fact, the freshly prepared (but enzymatically defective) AMEF#6101 had already been shown to be in a tetrameric form that lost, however, most of its basal activity upon incubation at 37°C. A later sucrose gradient centrifugation analysis by Rotman's group15 at Brown University in Rhode Island showed in fact (Figure 6) that these almost inactive AMEF#6101 macromolecules were essentially in a dimeric form that could be reverted to a tetrameric one upon interaction with anti-wtGZ Fab fragments.

Sucrose gradient centrifugation of a 14C-labeled AMEF#6101 preparation in the absence (empty circles) or presence (full circles) of antibody Fab fragments. The Fab fragments had been prepared from anti-wtGZ antibodies (panel A) or from normal immunoglobulins (panel B) (fig. 4 by Conway de Macario et al15)

Most of this inactivation process could, however, be prevented, or reversed,16 by adding to this AMEF a betaGal substrate analog. As already shown in Figure 1, anti-wtGZ antibodies produced almost identical activation levels when they interacted with either form (tetrameric or dimeric) of AMEF#6101.6 In both cases, this interaction definitely stabilized a tetrameric structure of the enzymologically activated AMEF.15

The anti-wtGZ antibodies obtained at various times after immunization of a donor rabbit differ in terms of their ability to activate a same AMEF. By assuming a Sips distribution for the samples containing non-homogeneous populations of anti-wtGZ antibodies, the mean values of their association constants for AMEF#6101 vary (Table 1) from 4.83 × 105 M−1 for the very early antibodies to over 32 × 105 M−1 for those taken 8 months after immunization.17

TABLE 1. Variation in activating antibody affinity during the primary immune response in vivo Time after primary immunization Activating titer (Enzyme units/μL) Ko × 105 M−1 Heterogeneity index of the Sips distribution 11 days 3.01 4.83 0.9 68 days 6.50 9.30 0.8 8 months 0.75 32.60 1 Source: Modified from table 3 by Celada et al.17

Although the unimodal Sips heterogeneity index has been shown by Bruni et al18 to be inadequate for a full characterization of the affinity distribution of antibody populations raised during an immune response, these results indicate anyhow that most of the early antibodies have, toward AMEF#6101, an affinity constant much lower than those that are synthesized at later times.

In our early experiments,7 the kinetics of the activation process of AMEF#6101 elicited by addition of anti-wtGZ antibodies was, as previously mentioned, consistent with the presence of a monomolecular rate-limiting step. This was found to be true also for AMEF#645,19 with first-order rate constants having always the same value – around 1.0 hour−1 at 30°C – even upon wide variations of AMEF concentrations or of anti-wtGZ antiserum dilutions. In a later detailed analysis20 of AMEF#6101 activation, we could instead detect a relatively wide range of these first-order rate values that anyhow asymptotically converged, when activation was highest, toward a value somewhat higher than 2.0 hours−1 at 25°C.

The monomolecular rate-limiting step of the antibody-induced activation process may, however, be generated by AMEF interaction with anti-GZ paratope or pre-exist in AMEF itself.   Experiments with Sepharose-bound activating antibodies have indeed shown20,21 that both pathways do exist simultaneously, at different extent depending on the experimental conditions (Figure 7).

The two different pathways of antibody-mediated enzyme activation (fig. 3 by Celada and Strom21)

12 EFFECTS OF anti-wtGZ ANTIBODIES ON BETAGAL COMPLEMENTATION

In 1963, during a Cold Spring Harbor Symposium on “Synthesis and Structure of Macromolecules,” Perrin22 had shown that, in E coli mutants characterized by partial deletions of their β-galactosidase structural gene, the betaGal enzymatic activity could be restored in the “acceptor” (inactive) protein by “complementation” with some “donor” peptide sequences encoded by the deleted segments of the lacZ gene. Two distinct α- and Ω-complementation systems were indeed described as occurring, respectively, at the N-terminal23 or at the C-terminal24 regions of the betaGal protein.

Accolla and Celada25 found that, upon addition of moderate concentrations of anti-wtGZ antibodies to the “α-acceptor” delM15 protein (produced by an E coli strain with a large deletion of the operator-proximal portion of its lacZ gene), this protein acquired a rather relevant β-galactosidase activity (Figure 8A). As shown in Figure 8B and in Table 2, this effect was, however, at his maximum, only 20% of the enzymatic activation produced23 by the α-complementing “donor” peptide - while addition of higher amounts of anti-wtGZ antibodies caused a loss of enzymatic activity even in the presence of the α-complementing peptide.

Panel A reported the enzymatic activation of delM15 by increasing concentrations of divalent anti-wt GZ antibody (empty circles) or of the corresponding Fab monovalent fragments (full circles). Panel B shows the activation curve obtained upon addition, to delM15, of the α-complementing peptide at increasing concentrations (fig. 1 by Accolla and Celada25)

TABLE 2. Effect of different combinations of anti-wtGZ antibodies and of α-complementing peptide on the activity of a fixed quantity of the delM15 (α-acceptor) protein Relative antibody concentration Relative peptide concentration 0 1 16 128 0 <1 5 120 25 1 18 28 140 23 8 220 250 340 170 128 2000 2000 1600 700 Source: From table 1 by Accolla and Celada.25

At low saturation values of delM15 by the α-complementing peptide, addition of anti-wtGZ antibodies causes an increase of betaGal enzymatic activity, which is not only larger than the sum of the effects caused by each of these “activators,” but is also characterized by an increase in the rate of activation (Figure 9). Anti-wtGZ antibodies are, therefore, capable not only of “facilitating” the activation of delM15 by low amounts of the α-complementing peptide, but also of “accelerating” this process.

Acceleration by anti-wtGZ of the kinetics of α-complementation. The enzymatic activity acquired by a fixed quantity of delM15 was measured at different times after: (a) addition of an amount of anti-wtGZ antibody (empty circles) capable of producing optimal activation; (b) addition of a limited (non-saturating) amount of α-complementing peptide (open squares); (c) simultaneous addition (full triangles) of anti-wtGZ antibody and of α-complementing peptide in the same amounts as in (a) and (b). The broken curve is the sum of the effects produced separately by (a) and (b) (fig. 2 by Accolla and Celada25)

In the Ω-complementation system described in detail by Ullmann et al,24 preliminary experiments had shown26 that anti-wtGZ antibodies failed to exert any effect on the β-galactosidase activity induced by limiting amounts of an Ω-donor added to an excess of Ω-acceptor. Unexpectedly, however, in the presence of an excess of Ω-donor,27 the same antibodies markedly increased (possibly through an “Ω-donor recruitment effect”) the overall yield of Ω-complementation (Figure 10) - whose time course remained, however, unchanged.

Ω-donor recruitment effect by anti-wtGZ antibodies on Ω-complementation of betaGal truncated mutants. The Ω-complementation reaction was performed by adding the “Ω-donor” protein from E coli strain W4680 to the “Ω-acceptor” protein from E coli strain S9080. In panel A, β-galactosidase activity was measured, in the presence (full circles) or absence (empty circles) of anti-wtGZ antibodies, starting immediately after addition of the Ω-acceptor and of a 6-fold excess of Ω-donor. Alternatively, the antibody was added, 2 hours after the onset of Ω-complementation reaction (crosses) (fig. 3 by Celada et al27) 13 ENZYMATIC ACTIVATION OF DEFECTIVE BETAGAL PROTEINS BY MONOCLONAL anti-wtGZ ANTIBODIES

In October 1981, a European Molecular Biology Organization (EMBO)-sponsored meeting on “Protein Conformation as an Immunological Signal,” was convened by Franco Celada in collaboration with Verne Schumaker and Eli Sercarz. Within a topic focused on the changes in antigen conformation induced by specific antibodies, Roberto Accolla reported the results28, 29 that, in collaboration with R. Cinà, E. Montesoro, and F. Celada, had been obtained by using anti-wtGZ monoclonal antibodies present in the culture fluids of three different hybridoma clones (generated by somatic cell fusion a myeloma cell line with spleen cells from mice immunized with wtGZ). These three different monoclonals that had been generated by somatic cell fusion on a myeloma cell line with spleen cells from mice immunized with wtGZ, had been selected for their ability to “activate” AMEF#6101, but the level of activation was quite different under comparable experimental conditions: less than 2-fold for ZL.1-1b, almost 4-fold for ZL.2-1b, and over 15-fold for ZL.2-2.

When the three monoclonals (previously labeled by adding 3H-leucine to their hybridoma cultures) were tested for their ability to compete with each other for wtGZ-coated wells of a polyvinyl plate, the antibodies from clone ZL.2-2 were found to exert a very strong competition toward the antibodies synthesized by the other two clones. The reciprocal competition between antibodies of clone ZL.1-1b and those from clone ZL.2-1b was instead almost negligible (Table 3).

TABLE 3. Competition, in terms of binding capacity to polyvinyl-adsorbed wtGZ, between monoclonal antibodies from different anti-wtGZ hybridoma clones % displacement by unlabeled antibodies from hybridoma clones Labeled monoclonals Unlabeled ZL.1-1b Unlabeled ZL.2-1b Unlabeled ZL.2-2 3H-ZL.1-1b −71.9% −7.8% −93.8% 3H-ZL.2-1b −14.1% −75.0% −70.3% 3H-ZL.2-2 −68.0% −40.6% −75.0% Note: Monoclonal antibodies were biosynthetically labeled by addition of 3H-leucine to the corresponding hybridoma cultures. Comparable 3H-labeled aliquots of the culture fluids from these hybridomas were added to the wells of a polyvinyl plate pre-treated with wtGZ, and allowed to react with the adsorbed GZ in the presence (or absence) of saturating amounts of homologous or nonhomologous unlabeled culture fluids. The % displacement of radioactivity by the unlabeled antibodies is expressed as % of the value obtained in the absence of any competing antibody. The displacement values occurring upon addition of homologous culture fluids are shaded. The bold values indicate, for each labeled monoclonal, the maximum displacement that can be obtained by using the same unlabeled monoclonal. Source: Data from fig. 2 by Accolla et al.28

The paratopes of antibodies ZL.1-1b and ZL.2-1b seem, therefore, able to interact with AMEF#6101 at distinct, though presumably adjacent, epitope-like regions of this defective betaGal protein. AMEF#6101 activation by ZL.2.2. appears instead to involve both these regions, with an overall binding affinity value of approximately 5 × 106 M−1. It was, however, found that this same ZL.2.2. monoclonal has an almost 200-fold higher binding affinity (Ka = 9.6 × 108 M−1) for the active wtGZ enzyme.

Since every AMEF is a defective protein generated by one missense point mutation of its coding sequence, it can hardly be the difference between these affinity values, which can hardly be ascribed to the loss, in the AMEF defective protein generated by a single missense point mutation of its coding sequence, of several epitopes present in the wild type enzyme. It could perhaps be due to the presence, in wtGZ but not in AMEF#6101, of a larger or conformationally different epitope with a higher affinity for the ZL.2.2 paratope. Alternatively, anyhow, it could be due to the conversion, upon antibody binding to AMEF#6101, of a portion of the binding energy into the conformational change leading to the increased enzymatic activity of this otherwise defective betaGal protein.

These three monoclonal antibodies were able to activate, beyond AMEF#6101, also other AMEFs, differing, however, both in the extent of their activation potency and in the preference for single AMEFs. As shown in Table 4, ZL.2-2 was very active on AMEF#959 and AMEF#918, ZL1-1b (definitely less potent) could instead induce a significant activation of AMEF#645 and AMEF#918 (but not of AMEF#959), while the preferred targets of ZL2.1b (that disdained AMEF#918) were AMEF#645 and AMEF#959.

TABLE 4. Capacity of monoclonal antibodies present in the fluids of 3 hybridoma