Monoclonal antibody and antibody components elicited to a polypeptide antigen ground state

What is disclosed is a catalytic monoclonal antibody able to cleave a polypeptide and in particular, a catalytic monoclonal antibody elicited by a ground state antigen is disclosed. Antigens include, among others, the polypeptide, a derivative of the polypeptide, a fragment of the polypeptide or any of these bound to a carrier molecule. Methods of making and using the catalytic monoclonal antibody are also disclosed.

FIELD OF THE INVENTION 
This invention relates to antibodies able to enhance the rate of chemical 
reaction of reactants and in particular of peptides and polypeptides. More 
particularly it relates to monoclonal antibodies elicited to a polypeptide 
antigen and methods of making and using these antibodies. 
Several publications are referenced in this application by Arabic numerals 
within parentheses in order to more fully describe the state of the art to 
which this invention pertains as well as to more fully describe the 
invention itself. Full citations for these references are found at the end 
of the specification immediately preceding the claims. 
BACKGROUND OF THE INVENTION 
The forces involved in ligand binding by antibodies and substrate binding 
by enzymes is similar, viz., hydrogen bonding, electrostatic interaction 
and hydrophobic effect. The energy obtained from enzyme-substrate binding 
may be visualized to force electronic strain in the substrate and 
facilitate the formation of a transition state. There is strong evidence 
for the theory that enzymes bind the transition state of the reaction they 
catalyze better than the ground state, resulting in a reduced free energy 
of activation for the reaction (1). The transition state theory is based 
upon the observation that the reactants for a chemical reaction normally 
exist at a ground state energy level. In order for a reaction to proceed, 
the energy level of the reactants must be raised to that required to form 
transition state intermediate(s). A successful catalyst may function to 
reduce the energy requirement for the formation of such transition state 
intermediate compound(s). This has come to be known as the transition 
state theory of enzymatic catalysis. 
Other factors that may facilitate enzymatic catalysis include the proximity 
and orientation effects-apposition of correctly oriented reactants within 
the active site of the enzyme would reduce the requirement for a large 
number of random collisions prior to a productive reactant interaction. In 
principle, antibodies could catalyze chemical reactions by similar means. 
The first report of chemical conversion of a ligand by an antibody appeared 
in 1980 (2), but the steroid ester hydrolysis by a rabbit polyclonal 
antiserum described in this report was stoichiometric rather than 
catalytic. 
Massey et al, U.S. Pat. No. 4,888,281 were the first to report catalyzing a 
chemical reaction by means of an antibody elicited to a reactant, a 
reactant bound to a peptide or other carrier molecule, a reaction 
intermediate or analogs of the reactant, product or a reaction 
intermediate. 
Subsequently, antibodies have been demonstrated to catalyze or facilitate 
chemical reactions, including acyl transfer (3-6), pericyclic (7-8) and 
redox reactions (9). 
It is generally believed that reported antibodies (3-9) obtain their 
catalytic properties, like enzymes, from their ability to bind the 
transition state of the reactant better than its ground state. 
Various analogs of the transition state of reactants have been used as 
antigens in the elicitation of immune responses (10). 
The requirement for a hapten which antigenically mimics the transition 
state complicates efforts to obtain desirable catalytic antibodies. It 
would be advantageous if catalytic antibodies could be elicited to a 
ground state, as an antigen. Suckling et al. (11) have reported the use of 
a ground state antigen comprising a hapten related in structure to the 
substrate to elicit an antibody able to catalyze a Dieis-Alder reaction 
(the addition of acetoxybutadiene to N-substituted maleimides) and an 
antibody able to cleave beta lactam rings). Cleavage of peptide bonds by 
means of antibodies elicited to a selected peptide-metal complex has been 
demonstrated with the assistance of metal cofactors by Iverson et al. 
(12). Iverson et al. utilized a Co(III)triethylenetetramine 
(trien)-peptide hapten in order to elicit an antibody able to accept a 
metal complex with chemical reactivity into the binding pocket. 
The discovery, isolation and characterization of naturally occurring 
autoantibodies, i.e., antibodies produced by an animal's immune system to 
the animal's own cellular component (self-antigen), as opposed to an 
antigen introduced by immunization, which enhance the rate of a chemical 
reaction, is disclosed in copending U.S. patent application Ser. No. 
343,081, filed Apr. 4, 1989. These autoantibodies have been shown to 
enhance the rate of cleavage of one or more peptide bonds in vasoactive 
intestinal peptide (VIP). The natural occurrence of these catalytic 
autoantibodies in multiple humans suggests that there is a common, 
naturally occurring antigen capable of eliciting these autoantibodies. 
Classical catalytic antibody theory suggests that the naturally occurring 
autoantibody must be formed in response to a high energy transition state 
intermediate or in response to an unusually charged analog of the peptide. 
It would therefore not be expected that the antigen is the ground state of 
a peptide, or is a large precursor protein that is eventually digested to 
yield the peptide, for example, pro-VIP. 
It is also known that antibody binding is energetically most favored by the 
presence of the entire H-chain and L-chain binding site (13). The V.sub.H 
fragments of anti-lysozyme antibodies bind the antigen with an affinity of 
only 10% of the intact antibody (14). L-chains are also likely to 
participate in antigen binding interactions, although most studies suggest 
that the contribution of L-chains is smaller than that of H-chains 
(15-17). It would not be expected that an antibody component smaller than 
an intact catalytic antibody would possess the favorable steric 
conformation provided by the intact catalytic antibody to permit the 
catalysis of a peptide bond without the assistance of a metal trien 
cofactor as taught by Iverson et al. 
Improved antibodies and methods for selectively eliciting antibodies able 
to catalyze a chemical reaction of a peptide of interest are of singular 
interest for therapeutic products and other purposes. 
The use of a ground state antigen would eliminate the necessity for 
preparing stable analogues of transition state intermediates or haptens 
complexed with metal co-factors so that catalytic antibodies specific for 
a polypeptide of interest may be elicited as needed. 
There are obvious advantages that single chain proteins offer over 
multichain proteins (antibodies), both from the point of view of 
structure-function analysis as well as pharmacological and therapeutic 
stability. It would be advantageous if the binding and catalytic domains 
on an antibody were either the same or closely positioned to one another 
such that the benefits of catalytic activity could be achieved by a simple 
protein as opposed to a multichain antibody. Heretofore, the art has not 
demonstrated the capability of using such components of an antibody for 
catalytic purposes. Similar advantages are offered by dimers formed of the 
several combinations of light and heavy chains. Nor has the art 
demonstrated that an antibody light chain elicited by a ground state 
reactant will have catalytic activity. 
OBJECTS OF THE INVENTION 
It is thus a primary object of this invention to provide catalytic 
antibodies and catalytic antibody fragments for reactions of interest 
using ground state antigens rather than transition state analogs to elicit 
an immune response. 
It is a further and related object of this invention to provide catalytic 
antibodies and catalytic antibody fragments for polypeptide reactions of 
interest using ground state antigen to elicit an immune response. 
It is still a further object of this invention to provide catalytic 
antibodies and catalytic antibody fragments for the hydrolytic cleavages 
of polypeptides of interest. 
It is still a further specific object of this invention to provide antibody 
catalyst and antibody fragment catalysts for the cleavage of specific 
polypeptides, e.g. vasointestinal peptide. 
SUMMARY OF THE INVENTION 
These and other objects of the invention are achieved by catalytic 
antibodies elicited by a ground state polypeptide antigen. In one 
embodiment the invention is directed to a method for catalyzing a chemical 
reaction of a polypeptide by a catalytic antibody wherein the catalytic 
antibody is elicited by an antigen comprising the polypeptide, a 
derivative of the polypeptide or a fragment of the polypeptide. In yet 
another embodiment the invention is directed to a method for catalyzing a 
chemical reaction of a reactant by a catalytic antibody light chain 
wherein the catalytic antibody light chain is derived from an antibody 
elicited by an antigen comprising the reactant, a derivative of the 
reactant or a fragment of the reactant.

DETAILED DESCRIPTION OF THE INVENTION 
Definitions 
The term "chemical reaction" refers to a reaction wherein at least one 
reactant is converted to at least one product. The chemical reaction of a 
polypeptide may proceed by a number of different pathways, such as, for 
example, hydrolysis of one or more peptide bonds. 
The term "animal" as used herein refers to any organism with an immune 
system and includes mammalian and non-mammalian animals. 
Antibodies in accordance with the invention are elicited by presenting an 
antigen to immune cells. The antigen may be a protein, a polypeptide or a 
fragment or derivative of a protein or polypeptide either alone or coupled 
to a carrier. 
In one embodiment, the chemical reaction is the cleavage of a peptide bond. 
Peptide bond as used herein refers to an amide bond linking two adjacent 
amino acid residues and is generically represented by the following 
formula wherein the peptide bond is shown within the box: 
##STR1## 
An amino acid consists of a carbon atom to which is bonded an amino group, 
a carboxyl group, a hydrogen atom and a distinctive group referred to as a 
"side chain" (R.sub.1 and R.sub.2 in the formula above). Amino acid as 
used herein includes the twenty naturally occurring amino acids which 
comprise the building blocks of proteins. It is understood by those 
skilled in the art that when either of the adjacent amino acids is 
proline, the respective side chains R.sub.1 or R.sub.2 are bonded to the 
adjacent nitrogen atoms to form the characteristic 5-membered proline 
ring. 
The substrate can be any proteinaceous molecule such as, for example, a 
regulatory protein or a structural protein, and includes, but is not 
limited to, peptide hormones (e.g., insulin, growth hormone, secretin, 
etc.), peptide neurotransmitters and neuromodulators (e.g., vasoactive 
intestinal peptide, endorphins, enkephlins, bradykinins, substance P etc.) 
tumor proteins (e.g., oncogene products, carcinoembryonic antigens, etc.), 
bacterial proteins and vital proteins (e.g., human immunodeficiency 
viral(HIV) gp 120, influenza glycoproteins, etc.). 
Rate enhancing antibodies may in general provide rate enhancement by either 
catalytic or stoichiometric mechanisms. The antibodies in accordance with 
the invention catalytically enhance the rate of the reaction and are 
therefore described as "catalytic antibodies". 
A catalytic antibody in accordance with the invention is a substance which 
is capable of changing the rate of a chemical reaction, all other 
conditions (e.g., temperature, reactant/substrate concentration, etc.) 
being the same and which does not enter into the chemical reaction and 
therefore is not consumed in the reaction. It is also a substance which 
exhibits the capability of converting multiple moles of reactant/substrate 
per mole of catalytic antibody. From a mechanistic viewpoint the antibody 
binds the reactant/substrate, effects the accelerated conversion of the 
reactant/substrate to the product and then releases the product; and which 
changes the rate of the chemical reaction without shifting the position of 
the equilibrium. The aforementioned definitions are characteristics of 
ideal catalysts. However, in practice, even the best of catalysts become 
poisoned or deactivated by contamination in the reaction system or as a 
result of chemical or physical destruction during the reaction process. 
For reasons well known in the art, the true operation of a catalyst may be 
obscured by components of the reaction system or by the condition of the 
reaction environment. 
The art has adopted certain working definitions to express catalytic 
activity. These expressions are [1]k.sub.cat, or "turnover" and [2] 
k.sub.cat /k.sub.uncat, the "rate enhancement factor". Turnover indicates 
the number of molecules of reactant/substrate which can be converted to 
product per mole of catalytic antibody per unit time. For example, if a 
molecule exhibits a turnover of 10.sup.3 molecules of substrate per 
minute and the molecule maintains its catalytic activity for 24 hours at 
room temperature and at its optimal pH, each molecule of catalyst would 
then make a total of 1.4.times.10.sup.6 conversions, indicating its 
catalytic behavior. This total conversion is to be distinguished from the 
total conversion in a stoichiometric reaction, which will never exceed 
1.0, no matter how long the reaction is carried out. The rate enhancement 
factor is a dimensionless number which expresses the rate of reaction in 
the presence of catalyst to the rate of reaction in the absence of 
catalyst, all other reaction conditions (e.g., reactant concentration, 
temperature, etc.) being equal. 
Antibody and immunoglobulin refer to any of several classes of structurally 
related proteins that function as part of the immune response of an 
animal, which proteins include IgG, IgD, IgE, IgA, and IgM and related 
proteins. Antibodies are found in plasma and other body fluids and in the 
membrane of certain cells. Under normal physiological conditions (e.g. 
absent immunological dysfunction or human intervention) antibodies are 
produced by B cells (or the functional equivalent) of an animal in 
reaction to the entry of proteins or other chemical substances which that 
animal is not immunologically tolerant of into the tissue or body fluids 
of that animal. 
Antibody and immunoglobulin as used herein refer to any component part of 
an antibody. These component parts may be any fragment or other portion of 
a classical antibody molecule (IgG, IgA, IgE, IgD, IgM and related classes 
and subclasses) which retains the desired antibody function and with which 
the invention may be practiced. The catalytic domain is the minimum 
peptide sequence which retains the catalytic property inherent in that 
amino acid sequence. Catalytic antibodies as used herein thus include any 
fragment or domain in which a binding region is involved in the catalysis. 
Embodiments of the Invention 
The invention provides an antibody able to catalyze a chemical reaction of 
a polypeptide, methods for eliciting an antibody able to catalyze a 
chemical reaction of a polypeptide, and methods of using the antibody to 
cause a selected polypeptide to undergo a chemical reaction. The antibody 
is elicited to a ground state antigen which, depending on the 
immunogenicity of the antigen, is a selected peptide, peptide derivative 
or peptide fragment, or is formed by coupling a selected peptide, peptide 
derivative or peptide fragment to any useful immunological carrier known 
to the art. 
The invention also provides an antibody light chain able to catalyze a 
chemical reaction of a reactant including polypeptides and other 
substrates, methods for eliciting an antibody from which that light chain 
able to catalyze a chemical reaction may be derived, and methods for using 
the antibody to cause a selected reactant to undergo a chemical reaction. 
The antibody is elicited to a ground state antigen formed by coupling a 
selected reactant, reactant derivative or reactant fragment to any useful 
immunological carrier known to the art. The antibody thus elicited is then 
caused to separate into its component heavy and light chains, and the 
light chains are then isolated. 
The antibodies of the invention can be monoclonal or polyclonal. Monoclonal 
antibodies are prepared by isolating lymphocytes from animals identified 
as having antibodies to a particular antigen, producing a plurality of 
hybridomas from the isolated lymphocytes and then screening the monoclonal 
antibodies produced by the hybridomas to identify monoclonal antibodies 
which enhance the rate of chemical reaction of the substrate. The 
antibody-producing lymphocytes are hybridized with myeloma cells, such as, 
for example SP2/0 or NS1 cells, to produce hybridoma cells. These 
hybridoma cells are then plated in the wells of microtiter plates. The 
monoclonal antibodies produced by the hybridoma cells are screened under 
appropriate conditions to identify monoclonal antibodies which enhance the 
rate of the reaction under appropriate conditions. The identification is 
made by treating a standardized solution of the reactant/substrate with an 
aliquot withdrawn from a microtiter well and screening for the presence of 
the desired product, as described above. By comparison with standardized 
samples of the desired product or reactant/substrate, rates of reaction 
can be quantified. In this manner, wells containing hybridoma cells 
producing catalytic monoclonal antibodies can be identified. The selected 
hybridoma cells were then cultured to yield colonies. 
These colonies are further propagated in in vitro or in vivo systems. In 
the latter case, mice such as syngeneic BALB/C mice are inoculated 
intraperitoneally with the selected hybridoma cells and produce tumors, 
generally within two or three weeks. These tumors were accompanied by the 
production of ascites fluid which contains the desired monoclonal 
antibodies. The monoclonal antibodies are then separately recovered from 
the ascites fluid by conventional methods such as ultrafiltration, 
ultracentrifugation, dialysis and immunoaffinity chromatography. 
Peripheral blood lymphocytes of an animal identified as having catalytic 
antibodies for a particular substrate can be stimulated to grow in culture 
and, therefore, can be immortalized using methodologies well known in the 
art. For example, the lymphocytes can be so stimulated using a virus, a 
chemical agent or a nucleic acid (e.g., an oncogene). A particularly 
advantageous virus for immortalization is Epstein Barr virus (EBV). Thus, 
catalytic antibodies can be produced by the transformed cells. The so 
transformed cells can then be cloned using known methods to provide a 
reliable source of large amounts of monoclonal antibodies having catalytic 
activity for a given substrate. 
One skilled in the art will appreciate that the genes, or fragments 
thereof, coding for the variable region of the antibody can be expressed 
in prokaryotic and eukaryotic cells using recombinant DNA methodologies 
well known in the art. 
In accordance with an embodiment of the invention, the separately recovered 
antibodies are contacted with a molecule (e.g., a substrate, antigen, 
etc.) under suitable conditions permitting the formation of a complex 
between the antibody and the molecule in order to achieve catalysis of a 
chemical reaction of the molecule. The skilled artisan will appreciate 
that the conditions suitable for complex formation can vary depending on 
the particular molecule and antibody under consideration. Accordingly, the 
methods of this invention may be practiced under a variety of reaction 
conditions, in vivo and in vitro, as long as the antibodies are not 
prevented from complexing with the molecules or otherwise rendered 
inactive. More specifically, suitable conditions for complex formation 
encompass solution phase and emulsion reaction systems including a variety 
of solvents and solvent systems, maintained at a pH value between about 
6.0 and about 9.0, preferably between about 6.0 and about 8.5 and at a 
temperature from about 4.degree. C. to about 50.degree. C., preferably 
from about 20.degree. C. to about 45.degree. C. One of ordinary skill in 
the art will realize that the choice of solvent will depend on the type of 
reaction. For example, aqueous solvents are desirable for peptide bond 
cleavage while non-aqueous solvents can be used to achieve peptide bond 
formation. The ionic strength, =1/2 .SIGMA. c.sub.i z.sub.i 2, where c is 
the concentration and z is the electronic charge of an ionic solute, 
should be maintained at a value below about 2.0 (ionic strength units), 
preferably between 0.1 and 1.5. The methods of the invention can be 
carried out at reduced or elevated pressure, but advantageously are 
practiced at ambient pressure. In addition to solution phase and emulsion 
reaction systems, suitable conditions also include the use of solid 
support materials to which the antibody is attached. Such solid support 
materials are well-known to those of ordinary skill in the art as are 
methods for attaching antibodies to them. 
A specific embodiment of the invention is directed to an antibody to 
vasoactive intestinal peptide (VIP). VIP is a 28 amino acid peptide 
originally isolated from the intestine but now recognized to be a 
neuropeptide widely distributed in the central and peripheral nervous 
systems. There is evidence that VIP is a neurotransmitter in its own 
right. In addition, VIP may modulate neurotransmission by classical 
transmitters and has been implicated in regulation of blood pressure, 
bronchial tone, neuroendocrine activity and exocrine secretion. VIP 
appears to be the major neurobronchodilator in humans and a diminished 
influence of VIP on the airways may permit a dominance of constrictor 
influences, and may underlie airway hyperactivity in asthma. 
VIP belongs to a family of structurally related peptides, other prominent 
members of which are peptide histidine isolucine (PHI), growth hormone 
releasing factor (GRF) and secretin. Like the peptides themselves, there 
is evidence that the receptors for VIP, GRF, PHI and secretin are related. 
Receptors for VIP are found in lung, vascular smooth muscle, brain, 
pancreas, skin, intestine and other tissues. The amino acids sequence (Seq 
ID No: 11) of VIP is as follows: 
EQU H S D A V F T D N Y T R L R K Q M A V K K W L N S I L N--CO--NH.sub.2 
Monoclonal antibodies to VIP were prepared by methods well known to the art 
(19-25). In one embodiment of this invention, a series of mice were 
immunized by an antigen comprising vasoactive intestinal peptide (VIP) 
coupled to keyhole limpet hemocyanin (KLH). The sera of the mice was 
screened for the saturable (Y.sup.10 -.sup.125 I)VIP binding titer, and 
splenocytes from the mouse showing the greatest saturable (Y.sup.10 
-.sup.125 I)VIP binding titer were used to prepare hybridomas. Hybridoma 
clones were grown as ascites in host mice and the resulting antibodies 
were purified from the ascites fluid. Antibodies c23.5 and c23.4 were 
purified and screened for VIP hydrolytic activity. Both antibodies 
hydrolyzed (Y.sup.10 -.sup.125 I)VIP, whereas irrelevant antibodies used 
as controls did not. An Fab fragment of c23.5 also demonstrated VIP 
hydrolytic activity. 
While VIP is the exemplified substrate and hapten, it will be understood 
that any peptide may be used as a substrate and hapten. 
It is well known that VIP is a natural polypeptide composed of the same 
amino acids found in other peptides and proteins. Several naturally 
occurring neurotransmitters and hormones are closely related in primary 
structure to VIP. These include: secretin, glucagon, growth hormone 
releasing factor, and helodermin (26). While VIP is the exemplified 
substrate and hapten, it will be understood that any peptide may be used 
as a substrate and hapten. 
Many different bonds in VIP can be cleaved by the VIP autoantibodies 
disclosed in patent application U.S. Ser. No. 343,081, filed Apr. 4, 1989. 
(see also, Paul, S., et al. (27). Thus antibody-mediated cleavage is not 
restricted to a unique peptide bond. The scissile peptide bonds in VIP 
(e.g., Gln-Met, Ala-Val, Met-Ala, Lys-Lys) (Paul et al. supra) are also 
present in other polypeptides. 
The catalytic antibodies cleave not only full-length VIP, but also a 
fragment of VIP [VIP(15-28)] (23). This shows that the N-terminal region 
of rIP is not required for antibody catalytic activity. This is important 
because the possibility has previously been considered that the N-terminal 
residues of VIP may facilitate autocleavage of this peptide (28). 
Since the recognition of VIP by antibodies occurs by the same pattern as 
for other polypeptide antigens, viz., by recognition of linear or 
conformational epitopes composed of discrete peptide subsequences found in 
the parent antigen (29), it is likely that antibodies recognizing other 
polypeptide antigens will display catalytic activity, provided the 
conditions described below are met. The recognition sequence is a more 
important determinant than the type of bond to be cleaved. This conclusion 
is supported by studies showing that VIP(22-28) inhibits cleavage of VIP 
at residues 16-17 by antibodies (23). Therefore, there is nothing unique 
about the VIP cleavage point. 
Antibodies possess hypervariable antigen binding sites (30). The 
hypervariability is brought about by V-D-J junctional rearrangement and 
somatic hypermutation, and 10.sup.12 different antibodies can be 
synthesized by B-lymphocytes. Antibody molecules already fulfill the first 
criterion required of a catalyst, i.e., the ability to bind substrate. 
Catalysis by enzymes is brought about by positioning of specific amino 
acid residues (e.g., His, Set, Asp, Cys) or cofactors (e.g., metals) in 
the substrate binding site (31). Enzymes which cleave polypeptides, i.e. 
proteases, are inhibited by diisopropyl fluorophosphate (DFP), which 
blocks the residues having protease activity. Given that a large number of 
antibody binding sites are created by the immune system by natural 
mutagenesis, it is most probable that some antibodies directed against 
polypeptide antigens would contain protease activity in their binding 
sites. The presence of the protease activity in the binding site can be 
independent of the type of antigen. The only requirements that must be met 
are specific binding and presence of suitably positioned catalytic amino 
acids in the antibody active site. 
Therefore, the skilled artisan will appreciate that the following 
description of anti-VIP catalytic antibodies and catalytic antibody light 
chains is by way of example only, and is not intended to limit the scope 
of the invention. 
EXAMPLE 1 
Murine Monoclonal Catalytic Antibodies 
Conjugates of VIP and keyhole limpet hemocyanin (VIP-KLH) were prepared via 
coupling to primary NH.sub.2 groups on VIP and to -SH groups on thiolated 
KLH using the crosslinking reagent gammamaleimidobutyryloxysuccinimide 
(GMBS). KLH (17 mg) was thiolated with iminothiolane (33) the thiolated 
KHL was fractionated on a gel filtration column (Biorad Econo-Pak 10 DG) 
and conjugated with 3 mg synthetic VIP that had previously been treated 
for 2 h with GMBS. The molar stoichiometry of the VIP:KLH conjunction was 
360, estimated on the basis of depletion of thiol groups reactive with 
Ellman's reagent. Five mice were immunized with the resulting VIP-KLH. 
Primary immunization (in Freunds complete adjuvant) followed by two 
booster immunizations (in incomplete Freunds adjuvant) was at 2-week 
intervals using 10-50 .mu.g VIP equivalents of the antigen conjugate, 
administered intraperitoneally. The final injection was an intrasplenic 
one (17 .mu.g VIP equivalents). Splenocytes from the mouse with the 
greatest saturable (Y.sup.10 -.sup.125 I)VIP binding titer (10% binding, 
1:200 serum dilution; assayed according to Paul et al. (34) were fused 
with NS-1 myeloma cells by the PEG method (35). Cells from three culture 
wells (c23.1, c23.4 and c23.5) positive for VIP binding antibody were 
cloned twice by limiting dilution and then grown as ascites in 
pristane-treated mice. Antibodies c23.5 and c23.4 (both identified to be 
IgG.sub.2a, kappa by ELISA) were purified from tissue culture fluid or 
ascites fluid by ammonium sulfate precipitation and Protein G-Sepharose 
chromatography as described by Paul et al. (36). Both antibodies 
hydrolyzed (Y.sup.10 -.sup.125 I)VIP and the hydrolysis products were 
assayed by separation of intact and degraded peptides by trichloroacetic 
acid (TCA) precipitation. See Paul et al. (36) for the assay method. The 
detection of hydrolytic activity was a function of the concentration of 
the antibody. Surprisingly, no hydrolytic activity was observed at high 
antibody concentrations (&gt;10 nM). The properties of one of the antibodies 
(c23.5) were analyzed further. The strategies utilized to characterize the 
monoclonal antibodies were similar to those used for human antibodies 
(19-25). 
The hydrolytic activity of this antibody was confirmed by reverse phase 
HPLC of the reaction mixture. Treatment of (Y.sup.10 -.sup.125 I)VIP with 
this antibody resulted in the appearance of two early eluting peaks of 
radioactivity, corresponding to VIP fragments (retention times 46 and 50 
min. compared to 66 min. of intact (Y.sup.10-125 I)VIP. 
Other monoclonal antibodies to VIP [c23.1 (lgG.sub.1, kappa) and c.5162 
(lgG.sub.1, kappa); obtained from Dr. J. Porter, University of Texas 
Health Science Center, Dallas)] were shown to be without VIP hydrolytic 
activity. Irrelevant monoclonal antibodies purified from hybridoma tissue 
culture fluids (directed against insulin-degrading enzyme, LH-receptors, 
CD8 or whole T-lymphocytes; obtained from Monoclonal Antibody Core 
Facility, University of Nebraska) were examined for (Y.sup.10 -.sup.125 
I)VIP cleaving activity, and were observed to be without such activity as 
were irrelevant antibodies purified from commercially available ascites 
fluid (Sigma Chemical Company, St. Louis, Mo.). 
Fab was prepared by digestion of protein G-purified IgG with immobilized 
papain (Pierce; 16 mg protein per ml gel in 20 mM sodium phosphate, 10 mM 
EDTA. ph 7 for 2 h) and chromatography on an anion-exchange column (Mono 
Q; Pharmacia, Piscataway, N.J.); 0-1 NaCl in 20 mM Tris-HCl, pH 8, in 45 
min). Fab, recovered in the unbound fraction, displayed a mass of 50 kD 
estimated by SDS-polyacrylamide gel electrophoresis (FIG. 1). With 
increasing Fab concentration, progressively increasing hydrolysis of VIP 
was observed (FIG. 2). 
Evaluation using TCA precipitation to separate intact VIP from fragments 
indicates that hydrolysis of VIP by antibody c23.5 follows 
Michaelis-Menten kinetics (K.sub.m 2 nM, kcat 8.times.10.sup.-5 /min) 
(FIG. 3). Binding studies under conditions that did not permit VIP 
hydrolysis (high ionic strength) yielded a K.sub.d estimate of 3.4 nM 
(37). 
The specificity of the observed antibody hydrolytic activity was confirmed 
by the failure of the antibody to hydrolyze unrelated tripeptide 
methylcoumarinamides (Boc-L-R-R-MCA; and, Z-R-R-MCA), assayed as the 
fluorescence of the leaving group [methylcoumarinamide (MCA)] (Excitation 
wavelength, 370 mm; Emission wavelength, 460 nm). In this experiment, 100 
nM c23.5 antibody was incubated with 10 .mu.g/ml substrate in microtiter 
plated (reaction volume 50 .mu.l) for 14 h in 100 nM Tris-HCl, 50 mM 
glycine, 0,025% Tween-20 pH 8 (Buffer A). Fluorescence values with the 
antibody were always&lt;1 unit. In comparison, bovine pancreatic trypsin 
yielded values of 263, 194 and 179 units using Boc-L-R-R-MCA, P-F-R-MCA 
and Z-R-R-MCA, respectively. 
EXAMPLE 2 
Monoclonal L-Chain Mediated VIP Hydrolysis 
c23.4 antibody purified by protein G-Sepharose chromatography (Ex. 1) (0.76 
mg) was reduced with 2-mercaptoethanol 0.2M for 3 h and then alkylated 
with iodoacetamide (0.3M) for 15 min, while the pH was maintained at 8 by 
addition of 1M Tris base. The reduced and alkylated antibody was then 
treated with 6M guanidinium chloride for 2 h, followed by fractionation by 
gel filtration in 6M guanidinium chloride on two Superose-12 columns 
attached in tandem at a flow rate of 0.4 ml/min (Pharmacia, Piscataway, 
N.J.). The column fractions were dialyzed extensively against Buffer A 
(see Example 1 for composition) and then fractionated by filtration 
(Superdex). This purification protocol permitted preparation of a 25 kD 
L-chain fraction (retention time 60 min) apparently free of H-chain 
contamination, judged by SDS-polyacrylamide gel electrophoresis. Earlier 
eluting fractions from the column appeared to be enriched in H-chains, but 
also contained significant amounts of L-chains (26 Kd) and 90 kD 
constituents which may be H-L dimers. The pure L-chain fraction exhibited 
VIP binding (9.times.10.sup.3 CPM/.mu.g protein) and hydrolysis (FIG. 4) 
when assayed according to Paul etal. (19). 
In a second experiment, c23.5 antibody was treated with 143 mM 
2-mercaptoethanol, and 0.4% SDS in a Tris-HCL buffer, pH 6.8 and 
electrophoresed on a 7.5% SDS-polyacrylamide gel using a Schleicher and 
Schuellminielectrophoresis system (22-30 mA/gel). The bands corresponding 
to the H-chain (60 kD) and L-chain (25 kD) were excised, electroeluted 
(Biorad Model 422 eluter; 8-10 mA/elution tube), the eluate was 
electrodialyzed against buffer that did not contain SDS and subjected to 
Extractigel chromatography (Pierce, Rockford, Ill.) in 50 mM Tris-HCl, pH 
9, to remove residual SDS. The samples were then brought into Buffer A by 
2-cycles of ultrafiltration on Centriprep-10 ultrafilters (Amicon). The 
L-chain preparation displayed dose-dependent. hydrolysis (FIG. 5). 
EXAMPLE 3 
Cloning and Sequencing of c23.5 L-Chain and H-Chain cDNA 
Total RNA was prepared from 10.sup.8 cells (c23.5) by the one-step 
guanidine-isothiocyanate method. cDNA was made from the c23.5 RNA using 
reverse transcriptase. Forward primers used for the reverse transcriptase 
reaction and forward and back primers used to amplify the cDNA for 
V.sub.L, L-chain (V.sub.L +C.sub.L), V.sub.H and Fd-chain (V.sub.H 
+C.sub.H L) are shown in Table 1. The primers were designed based on 
consensus sequences present in L-chain (kappa) and heavy chains 
(lgG.sub.2a) (38-42). BamHI or XbaI restriction sites were incorporated 
into the backward primers and EcoR1 sites into the forward primers to 
facilitate cloning. The cDNA was then amplified by polymerase chain 
reaction using Taq DNA polymerase (30 cycles; 1 min at 95.degree. C., 2 
min at 55.degree. C. and 3 min at 72.degree. C. for each cycle). The 
amplified cDNA exhibited the expected sizes determined by agarose gel 
electrophoresis. The L-chain variable region cDNA was then cloned into a 
pBluescriptllKS.sup.+ vector (Stratagene, La Jolla, Calif.) via the 
EcoRl and Xbal restriction sites. The ligated vector was introduced into 
Escherichia coli and transformed colonies (white) were selected in LB agar 
supplemented with X-gal and ampicillin. Plasmid DNA prepared from these 
colonies and digested with EcoRI exhibited a larger size (3.35 kb) than 
the pBluescript (3.0 kb), corresponding to the length of the DNA insert. 
The DNA insert was sequenced by the dideoxy chain Germination method using 
.sup.35 S-ATP precursor and T3 and T7 sequencing primers. Ten colonies 
containing inserts were sequenced and the deduced sequence (Table 2) in 
each case was identical. The H-chain variable region cDNA was cloned and 
sequenced in a similar manner. 
TABLE 1 
______________________________________ 
PCR PRIMERS FOR pBluescript II 
______________________________________ 
L-Chain Primers 
(SEQ ID NO:1) 
EcoRI 
L-v 5' 5' GGAATTC GAC ATT GTG CTG ACC 
3' 
CAR TCT CC 
(SEQ ID NO:2) 
BamHI 
L-v 3' 5' cgggatcc cag ctt ggt ccc ccc icc gaa cg 
3' 
(SEQ ID NO:3) 
XbaI 
L-c 3' 5' gctctaga ctc att cct gtt gaa gct ctt gac 
3' 
H-Chain Primers 
(SEQ ID NO:4) 
EcoRI 
H-v 5' 5' GGAATTC GAG GTI CAG CTT CAG 
3' 
SAG TCW GG 
(SEQ ID NO:5) 
BamHI 
H-v 3' 5' cgggatcc ggt gas crk ggt icc tkk gcc cca g 
3' 
(SEQ ID NO:6) 
XbaI 
H-c 3' 5' gctctaga tgt trt ggg cac tct ggg ctc 
3' 
______________________________________ 
Synthetic oligonucleotide primers used to amplify c23.5 L-chains (SEQ ID 
NO:1 and SEQ ID NO:3), V.sub.L (SEQ ID NO:1 and SEQ ID NO:2), Fd (SEQ ID 
NO:4 and SEQ ID NO:6), and V.sub.H (SEQ ID NO:4 and SEQ ID NO:5). Forward 
primers (5') are shown in upper case and backward primers (3') in lower 
case. R is G or A; K is G or T; S is G or C; W is A or T and I is inosine. 
TABLE 2 
__________________________________________________________________________ 
Sequence of c-23:5 VL (SEQ ID NOS: 7 and 8) 
##STR2## 
##STR3## 
##STR4## 
##STR5## 
##STR6## 
##STR7## 
##STR8## 
Sequence of c-23:5 VH (SEQ ID NOS: 9 and 10) 
##STR9## 
##STR10## 
##STR11## 
##STR12## 
##STR13## 
##STR14## 
##STR15## 
##STR16## 
__________________________________________________________________________ 
Underlining indicates complementarity determining regions (CDR's) of the 
antibody chain. 
EXAMPLE 4 
Evidence for Subunit Rearrangement 
Antibodies are composed of four subunits held by disulfide bonds. Reduction 
and reformation of disulfide bonds could conceivably result in formation 
of a variety of antibody oligomers (e.g., H-H, L-L, H-L). Analogous 
disulfide bond exchange reactions are shown to occur in other oligomeric 
proteins (32). The importance of such rearrangements for antibody binding 
activity is probably minimal since dissociated antibody subunits display 
low binding activity, and variant molecules composed of incorrectly paired 
subunits are also likely to possess low binding activity. In regard to 
antibody catalysis, however, it has been observed that purified L-chains 
hydrolyze VIP faster than undissociated antibody preparations. Formation 
of rearranged antibody oligomers may well be accompanied by increased 
hydrolytic activity, if homologous H-chains are better inhibitors of 
L-chain catalytic activity than heterologous H-chains. Evidence that 
subunit rearrangement in VIP antibodies may form new catalytic species is 
as follows. 
Anion exchange chromatography (Mono Q column, Pharmacia) (0-1M NaCl in 40 
min; 1 min fractions were collected) of 0.55 mg c23.5 antibody that had 
previously been purified by affinity chromatography on immobilized Protein 
G produced a sharply eluting protein peak, corresponding to fractions 32 
and 33 (FIG. 6). Following dialysis to remove NaCl, the effluent fractions 
were assayed for VIP hydrolysis. Fractions corresponding to the main 
protein peak (fractions 32-33) displayed VIP hydrolytic activity. In 
addition, VIP hydrolytic activity was observed in fractions 34-38, located 
at the trailing edge of the protein peak was evident. To determine whether 
the hydrolytic activity was due to antibodies, the fractions (0.03 ml) 
were treated with 0.22 ml gel of anti-mouse IgG conjugated to Sepharose 
(Organon Teknika, Durham, N.C.) or an equivalent amount of control 
anti-rabbit IgG conjugated to Sepharose (Jackson Immuno Research, West 
Grove, Pa.) for 13 h in a final volume of 5 ml Buffer A (see Ex. 1), and 
the supernatants (0.05 ml) of the gels were assayed for VIP hydrolysis. 
The hydrolytic activity in all fractions was nearly completely adsorbed by 
immobilized anti-mouse IgG, but not by immobilized anti-rabbit IgG (FIG. 
7). SDS-polyacrylamide gel electrophoresis under reducing conditions and 
immunoblotting using a specific anti-mouse IgG antibody showed that all 
fractions were composed of 60 kD H- and 25 kD L-chains. The late eluting 
fractions also contained a 90 kD band that was stainable with the 
anti-mouse IgG and represents an H-L dimer. Electrophoresis under 
non-reducing conditions revealed that the main protein peak was composed 
of intact 150 kD antibodies while the trailing peak was composed of 90-120 
kD bands, representing antibody oligomers (FIG. 8). 
EXAMPLE 5 
Characterization of Anti-VIP Antibody 
A. Kinetic Data and Binding Effects 
VIP has been previously observed to undergo cleavage by human 
autoantibodies at several bonds located between residues 14 and 22 (36). A 
synthetic peptide mimic [ABz-RKQMAVKKY(NO2)D] of VIP(14-22) (RKQMAVKKY 
(SEQ ID No. 12); designated pep2) was tested as substrate for the 
monoclonal antibody. The anthraniloyl group at the N-terminus of this 
synthetic peptide in combination with the Y(NO.sub.2) residue close to the 
C-terminus functions as an intramolecularly quenched fluorescence reporter 
system for hydrolysis at peptide bonds located between the fluorophore 
(ABz) and quencher [Y(NO2)]. Synthesis of this peptide was according to 
Meldal, M. et al. (43). Treatment of pep2 with the increasing 
concentrations of c23.5 antibody monoclonal VIP antibody resulted in 
progressive hydrolysis, determined by an increase in fluorescence emission 
at 420 nm (excitation wavelength, 370 nm) (FIG. 9). 
Initial rate data obtained by varying the substrate concentrations were 
consistent with Michaelis-Menten kinetics. Reversed-phase HPLC analysis on 
a C-18 column using trifluoroacetic acid in acetonitrile for elution, 
showed the appearance of two new peptide peaks following treatment of pep1 
(20 .mu.M) with c23.5 antibody (20 .mu.M) for 100 h in MOPS buffer (FIG. 
10). These were sequenced on an automated Applied Biosystems peptide 
liquide phase sequenator equipped with online PTH-amino acid detection. 
The two peptides were identified to be RKQMAVK (SEQ ID NO: 13) and 
KY(NO2)D, indicating that the scissile bond is a K--K bond, corresponding 
to K.sup.20 -K.sup.21 in intact VIP. 
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__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 11 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 30 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ix) FEATURE: 
(A) NAME/KEY: miscfeature 
(B) LOCATION: 25 
(D) OTHER INFORMATION: /note="Location 25 (R) represents 
G or A" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
GGAATTCGACATTGTGCTGACCCARTCTCC30 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 31 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ix) FEATURE: 
(A) NAME/KEY: modifiedbase 
(B) LOCATION: 24 
(D) OTHER INFORMATION: /modbase=i 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
CGGGATCCCAGCTTGGTCCCCCCNCCGAACG31 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 32 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
GCTCTAGACTCATTCCTGTTGAAGCTCTTGAC32 
(2) INFORMATION FOR SEQ ID NO:4: 
( i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 30 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ix) FEATURE: 
(A) NAME/KEY: miscfeature 
(B) LOCATION: 13..28 
(D) OTHER INFORMATION: /note="Location 23 (S) represents 
G or C; Location 28 (W) represents A 
or T." 
(ix) FEATURE: 
(A) NAME/KEY: modifiedbase 
(B) LOCATION: 13 
(D) OTHER INFORMATION: /modbase=i 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
GGAATTCGAGGTNCAGCTTCAGSAGTCWGG30 
(2) INFORMATION FOR SEQ ID NO:5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 33 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ix) FEATURE: 
(A) NAME/KEY: miscfeature 
(B) LOCATION: 14..26 
(D) OTHER INFORMATION: /note="Location 14 (S) represents 
G or C, Location 16 (R) represents G 
or A, Locations 17,25,26 (K) 
represent G or T." 
(ix) FEATURE: 
(A) NAME/KEY: modifiedbase 
(B) LOCATION: 21 
(D) OTHER INFORMATION: /modbase=i 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
CGGGATCCGGTGASCRKGGTNCCTKKGCCCCAG33 
(2) INFORMATION FOR SEQ ID NO:6: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 29 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ix) FEATURE: 
(A) NAME/KEY: miscfeature 
(B) LOCATION: 13 
(D) OTHER INFORMATION: /note="Location 13 (R) represents 
G or A" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
GCTCTAGATGTTRTGGGCACTCTGGGCTC 29 
(2) INFORMATION FOR SEQ ID NO:7: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 312 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
GACATTGTGCTGACCCAGTCTCCTGCCTCCCAGTCTGCATCTCTGGGAGAAAGTGTCACC60 
ATCACATGCCTGGCAAGTCAGACCATTGGTACATGGTTACCATGGTATCAGCAGAAACTA120 
GGGAAATCTCCTCAGCTCCTGATATATGCTGCAACCAGCTTGGCAGATGGGGTCCCATCA180 
AGGTTCAGTGGTAGTGGATCTGCCACAAAATTTTCTTTCAAGAT CAGCAGCCTACAGGCT240 
GAAGATTTTGTAAGTTATAACTGTCAACATCTTTACAGTACTCCGCTCACGTTCGGCGGG300 
GGGACCAAGCTG312 
(2) INFORMATION FOR SEQ ID NO:8: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 104 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 
AspIleValLeuThrGlnSerProAlaSerGlnSerAlaSerLeuGly 
15 1015 
GluSerValThrIleThrCysLeuAlaSerGlnThrIleGlyThrTrp 
202530 
LeuProTrpTyrGlnGlnLysLeuGlyLy sSerProGlnLeuLeuIle 
354045 
TyrAlaAlaThrSerLeuAlaAspGlyValProSerArgPheSerGly 
5055 60 
SerGlySerAlaThrLysPheSerPheLysIleSerSerLeuGlnAla 
65707580 
GluAspPheValSerTyrAsnCysGlnHisLeuTy rSerThrProLeu 
859095 
ThrPheGlyGlyGlyThrLysLeu 
100 
(2) INFORMATION FOR SEQ ID NO:9: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 339 base pairs 
( B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: 
GAGGTGCAGCTTCAGGAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTGAAACTC60 
TCCTGTGCAGCCTCTGGATTCACTTTCAGTATCTATGGCATGTCTTGGTTTCGCCAGACT 120 
CCGGAGAAGAGGCTGGAGTGGGTCGCAACAATTAGTGGTGGTGATACTTACACCTACTAT180 
CCAGACAGTGTGAAGGGGCGATTCACCATCTCCAGAGACAATGCCAAGAACAACCTGTTC240 
CTGCAAATGAGCAGTCTGAGGTCTGAGGACACGGCCTTG TATTTCTGTGGAAGAGGGATT300 
GCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA339 
(2) INFORMATION FOR SEQ ID NO:10: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 113 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: 
GluValGlnLeuGlnGluSerGlyGlyGlyLeuValLysProGlyGly 
151015 
SerLeuLysLeuSerCysAlaAlaSerGly PheThrPheSerIleTyr 
202530 
GlyMetSerTrpPheArgGlnThrProGluLysArgLeuGluTrpVal 
3540 45 
AlaThrIleSerGlyGlyAspThrTyrThrTyrTyrProAspSerVal 
505560 
LysGlyArgPheThrIleSerArgAspAsnAlaLysAsnAsn LeuPhe 
65707580 
LeuGlnMetSerSerLeuArgSerGluAspThrAlaLeuTyrPheCys 
8590 95 
GlyArgGlyIleAlaTyrTrpGlyGlnGlyThrLeuValThrValSer 
100105110 
Ala 
(2) INFORMATION FOR SEQ ID NO:11: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 28 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: 
HisSerAspAlaValPheThrAspAsnTyrThrArgLeuArgLysGln 
1510 15 
MetAlaValLysLysTrpLeuAsnSerIleLeuAsn 
2025