Autoantibodies which enhance the rate of a chemical reaction

Autoantibodies which enhance the rate of a chemical reaction of a substrate, processes for their preparation, their use and compositions thereof are disclosed. In particular, an autoantibody capable of catalyzing the hydrolysis of the peptide bond between amino acid residues 16 and 17 in the neurotransmitter vasoactive intestinal peptide (VIP) is disclosed.

FIELD OF THE INVENTION 
This invention pertains generally to antibodies and more particularly to 
naturally occurring antibodies capable of enhancing the rate of a chemical 
reaction. 
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 nature of 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). This has come to 
be known as the transition state theory of enzymatic catalysis. Other 
factors that may faciliate enzymatic catalysis are 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. Subsequently, antibodies have been demonstrated to catalyze or 
facilitate chemical reactions, including acyl transfer (3), pericyclic (4) 
and redox reactions (5). 
It is generally believed that these antibodies obtain their catalytic 
properties, like enzymes, from their ability to bind the transition state 
of the ligand better than its ground state. Antibodies with enzymatic 
activity offer the possibility of specific, high efficiency catalytic 
chemical conversion of ligands. Many biological mediators are peptides or 
proteins, including the antigens of pathogenic organisms, hormones, 
neurotransmitters and tumor specific antigens. It should be possible to 
utilize the vast repertoire of specificities that the immune system 
encompasses to catalyze chemical reactions not within the scope of 
naturally occurring enzymes. The combination of antibody specificity with 
the catalytic power of enzymes has the potential of generating potent 
therapeutic agents, e.g., catalytic antibodies capable of specifically 
hydrolyzing key viral coat proteins, tumor specific proteins, or 
endogeneous proteins involved in disease. Hitherto, antibody mediated 
cleavage of peptide bonds has not been demonstrated and, thus, the search 
for antibodies capable of cleaving specific peptide bonds is of 
considerable interest. Compared to the type of antibody-mediated chemical 
transformations achieved thus far, the cleavage of peptide bonds is more 
energy-demanding. 
It was also not known that naturally occurring antibodies, 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, could enhance the rate of a chemical reaction, e.g., the 
cleavage of a peptide bond. These so-called autoantibodies, which may be 
found in autoimmune disease are important in a number of therapeutic 
strategies. 
OBJECTS OF THE INVENTION 
It is therefore a general object of the invention to provide autoantibodies 
which enhance the rate of a chemical reaction. 
It is a further object of the invention to provide autoantibodies which 
catalytically enhance the rate of a chemical reaction. 
It is another object of the invention to provide antibodies which enhance 
the rate of cleavage of a peptide bond. 
It is yet another object of the invention to provide a method for preparing 
autoantibodies which enhance the rate of a chemical reaction. 
It is another object of the invention to provide autoantibodies which can 
be used as therapeutic agents in the treatment of cancer and microbial 
infection. 
It is still another object of the invention to provide methods to diagnose 
and to treat autoimmune diseases associated with autoantibodies. 
These and other objects, features and advantages of the invention will 
become readily apparent from the ensuing description, and the novel 
features will be particularly pointed out in the appended claims. 
SUMMARY OF THE INVENTION 
The invention is broadly directed to an extract of blood serum comprising 
an autoantibody which enhances the rate of a chemical reaction of a 
substrate. The autoantibody is prepared by identifying an animal with 
autoantibodies to a self-antigen of the animal, isolating the 
autoantibodies and screening the autoantibodies to identify an 
autoantibody which enhances the rate of the chemical reaction. The rate 
enhancement can be catalytic or stoichiometric. In an embodiment, the 
chemical reaction is the cleavage of a peptide bond in the substrate. 
The invention is also directed to an extract of blood serum comprising an 
autoantibody which enhances the rate of hydrolysis of a peptide bond in 
the neuropeptide vasoactive intestinal peptide (VIP). 
In still another aspect, the invention is directed to a method for 
preparing an autoantibody which enhances the rate of a chemical reaction 
of a substrate by identifying an animal with autoantibodies to a 
self-antigen of the animal, isolating the autoantibodies and screening the 
auto-antibodies to identify an autoantibody which enhances the rate of the 
chemical reaction. The autoantibodies are polyclonal antibodies. 
Monoclonal antibodies are prepared by isolating lymphocytes from the 
so-identified animals, producing a plurality of hybridomas from the 
lymphocytes and screening the monoclonal antibodies produced by the 
hybridomas to identify monoclonal antibodies which enhance the rate of the 
chemical reaction. 
In still another aspect, the invention is directed to a composition 
comprising an extract as described above and an inert carrier, said 
extract being present in an amount effective to enhance the rate of a 
chemical reaction of a substrate. 
In yet another aspect, the invention is directed to a method for enhancing 
the rate of a chemical reaction of a substrate which comprises contacting 
the substrate with an autoantibody which enhances the rate of the chemical 
reaction and which is prepared by the process described above under 
conditions sufficient for the chemical reaction to take place. 
In another aspect, the invention is directed to a method for enhancing the 
rate of hydrolysis of a peptide bond between amino acid residues 16 and 17 
of vasoactive intestinal peptide which comprises the steps of identifying 
animals with autoantibodies to vasoactive intestinal peptide, isolating 
the autoantibodies, screening the autoantibodies to identify an 
autoantibody which enhances the rate of hydrolysis and contacting an 
autoantibody so identified with vasoactive intestinal peptide under 
conditions sufficient for the hydrolysis to take place. 
In another aspect, the invention is directed to an autoantibody which 
enhances the rate of a chemical reaction of a substrate, which 
autoantibody is prepared by identifying an animal with autoantibodies to a 
self-antigen of the animal, isolating the autoantibodies and screening the 
autoantibodies to identify an autoantibody which enhances the rate of the 
chemical reaction. In one embodiment, the chemical reaction is cleavage of 
a peptide bond. 
In another aspect, the invention is a method for preparing an autoantibody 
which enhances the rate of a chemical reaction of a substrate. The method 
comprises identifying an animal with autoantibodies to a self-antigen of 
the animal, isolating the autoantibodies and ultrafiltering said 
autoantibodies to identify an autoantibody which enhances the rate of the 
chemical reaction. 
In yet another aspect, the invention is directed to a method for enhancing 
the rate of cleavage of a peptide bond in a substrate which comprises 
contacting the substrate with an autoantibody under conditions sufficient 
for the cleavage to take place, the autoantibody having been prepared by 
the process defined above. 
In another aspect, the invention is a method for treating a disease 
condition in an animal caused by an autoantibody which enhances the rate 
of a chemical reaction of a self-antigen of the animal. The method 
comprises preparing an inhibitor which is capable of binding to the 
autoantibody and administering to the animal an amount of the inhibitor 
effective to decrease the rate of said chemical reaction. 
In still another aspect, the invention is directed to a method for 
diagnosing an autoimmune disease in an animal which is caused by an 
autoantibody enhanced chemical conversion of a self-antigen of the animal. 
The method comprises contacting the self-antigen with an extract of blood 
from the animal and screening the autoantibodies to identify an 
autoantibody which enhances the rate of the chemical conversion. 
In another aspect, the invention is a method for treating a disease 
condition in an animal caused by a substrate which comprises administering 
to the animal an autoantibody capable of enhancing the rate of cleavage of 
a peptide or other bond in the substrate in an amount effective to enhance 
the rate of cleavage of the target bond.

DETAILED DESCRIPTION OF THE INVENTION 
The invention is broadly directed to an extract of blood serum comprising 
an autoantibody which enhances the rate of a chemical reaction of a 
substrate. The autoantibody is prepared by identifying an animal with 
autoantibodies to a self-antigen of the animal, isolating the 
autoantibodies and screening the autoantibodies to identify an 
autoantibody which enhances the rate of the chemical reaction. The extract 
can be blood plasma, purified immunoglobulins (IgG, IgM, IgA, IgD or IgE) 
or antibody fragments, such as, Fab, F(ab').sub.2, Fv, etc., of 
immunoglobulins. Chemical reaction refers to a reaction wherein at least 
one reactant is converted to at least one product. Such chemical reactions 
include chemical reactions which can be catalyzed by enzymes such as, for 
example, oxoreductases, transferases, hydrolases, lyases, isomerases and 
ligases as well as chemical reactions for which no catalytic enzymes are 
known, such as, for example, oxidations, reductions, additions, 
condensations, eliminations, substitutions, cleavages and rearrangements. 
The term "animal" as used herein refers to any organism with an immune 
system and includes mammalian and non-mammalian animals. The term 
"substrate" is synonymous with the reactant in the chemical reaction and 
can be any of a number of molecules and biomolecules including but not 
limited to proteins, phospholipids, carbohydrates (e.g., glycogen, 
glucose, etc.), drugs (including abused substances and drugs from 
exogenous sources). 
Autoantibodies in accordance with the invention are naturally occurring 
antibodies produced by the immune system of an animal which bind to the 
animal's own cellular components and which are not elicited by specific 
immunization against a target antigen. Autoantibodies recognize a 
self-antigen, i.e., any antigen which the body makes using its own genetic 
code. Thus, self-antigens are distinguished from foreign antigens (e.g., 
bacterial, viral antigens). The term "substrate" as defined herein can be 
the same as or different from the self-antigen. 
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 containing the peptide bond or bonds to be cleaved 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 viral 
proteins (e.g., human immunodeficiency viral(HIV) gp 120, influenza 
glycoproteins, etc.). 
An animal with autoantibodies to a target self-antigen of the animal is 
identified by measuring, in plasma samples or purified IgG from the 
animal, the saturable binding of the autoantibodies to the self-antigen of 
the animal itself, to a self-antigen of a different animal species which 
is identical or substantially identical to the self-antigen of the animal 
or to a synthetic self-antigen which is identical or substantially 
identical to the self-antigen of the animal, using methods well known in 
the art. Autoantibodies which bind to the self-antigen are isolated by 
standard methods. 
In an embodiment of the invention, the isolated autoantibodies are purified 
by standard methods and then ultrafiltered. The term "ultrafiltration" as 
used herein refers to a filtering process employing a membrane having 
pores with an average cut off molecular weight ranging from 1,000 to 
10,000 Daltons. Thus, for example, ultrafiltering an immunoglobulin with a 
molecular weight of 150,000 Daltons on a membrane with pores having an 
average cut off molecular weight of 10,000 Daltons will cause molecules 
with molecular weights smaller than 10,000 Daltons to pass through the 
membrane while the immunoglobulin will remain on the membrane. 
The isolated autoantibodies are then screened for rate enhancement 
activity. Screening can be conveniently accomplished by treating a 
standardized solution of the reactant/substrate with an aliquot of medium 
containing the autoantibodies and measuring the presence of the desired 
product by conventional instrumental methods. This measurement can be 
readily conducted, for example, by spectrophotometric methods or by 
gas-liquid or high pressure liquid chromatography. By comparison with 
standardized samples of the desired product or reactant/substrate, rates 
of reaction can be quantified. 
The rate enhancement achieved by the antibodies according to the invention 
is either catalytic or stoichiometric. Thus, antibodies in accordance with 
the invention which catalytically enhance the rate of the reaction are 
"catalytic antibodies" and antibodies which stoichiometrically enhance the 
rate of the chemical reaction are "stoichiometric 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; which, from a mechanistic viewpoint, 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. 
A stoichiometric antibody in accordance with the invention enhances the 
rate of the chemical reaction stoichiometrically. In other words, it 
enhances the rate of the reaction but, unlike a catalytic antibody, is 
stoichiometrically consumed during the reaction. Thus, the term 
"stoichiometric enhancement" implies that the antibody causing the 
observed rate enhancement enters into the reaction as a reactant and is 
consumed in the process. 
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. 
The invention is also directed to a method for preparing an autoantibody 
which enhances the rate of a chemical reaction of a substrate. The method 
comprises identifying an animal with autoantibodies to a self-antigen of 
the animal, isolating the autoantibodies and screening the autoantibodies 
to identify one or more antibodies which enhance the rate of the chemical 
reaction. Screening in order to detect antibodies with the desired rate 
enhancement activity can be achieved by, for example, high performance 
liquid chromatography (HPLC), immunoassays (e.g., radioimmunoassay and 
nonisotopic immunoassays) or electrophoresis. 
The antibodies in accordance with the invention can be monoclonal or 
polyclonal. If monoclonal antibodies are desired, they can be prepared by 
isolating lymphocytes from animals identified as having autoantibodies to 
a particular self-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 the chemical reaction. 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 series of monoclonal antibodies being 
produced by the hybridoma cells is screened under appropriate conditions 
to identify monoclonal antibodies which enhance the rate of the reaction 
under appropriate conditions. The identification can be 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 
rate enhancing monoclonal antibodies are identified. The selected 
hybridoma cells are then cultured to yield colonies. 
These colonies can be 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 are 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 rate 
enhancing autoantibodies 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, rate enhancing autoantibodies 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 rate enhancing 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 autoantibody can be 
expressed in prokaryotic and eucaryotic cells using recombinant DNA 
methodologies well known in the art. Thus, the prokaryotic and eucaryotic 
cells are used to propagate the variable region of the autoantibody. 
In accordance with an embodiment of the invention, the separately recovered 
antibodies are contacted with a molecule (e.g., a substrate, self-antigen, 
etc.) under suitable conditions permitting the formation of a complex 
between the antibody and the molecule in order to achieve rate enhancement 
of a chemical reaction of the molecule. In the case of stoichiometric rate 
enhancement, the concentration of the stoichiometric antibodies is 
equivalent to the concentration of the target molecules. 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.sup.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 method of this 
invention can be carried out at reduced or elevated pressure, but 
advantageously is 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 extract of blood 
serum comprising an autoantibody 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 acid sequence of 
VIP is as follows: 
##STR2## 
It has been discovered that VIP binding antibodies exist in human 
circulation (6-8). Immunoprecipitation with anti-human IgG as well as 
chromatography on DEAE-cellulose, gel filtration columns and immobilized 
protein-G indicate that the plasma VIP binding activity is largely due to 
IgG antibodies. The antibodies to VIP are present in the blood of 18% of 
asthma patients and 30% of healthy subjects with a history of habitual 
muscular exercise, compared to only 2% of healthy subjects with no such 
history. The antibodies are highly specific for VIP, judged by their poor 
reaction with peptides related to VIP (i.e., GRF, PHI and secretin). A 
clear difference in the VIP binding affinity of the antibodies from asthma 
patients (mean K.sub.bind =0.13 nM) and healthy subjects (mean K.sub.bind 
=7.7 nM) was observed-the antibodies from the asthmatics exhibiting a 
60-fold greater binding affinity. The immune IgG from asthma patients 
reduces the binding of VIP by lung receptors as well as the VIP-responsive 
synthesis of cyclic AMP in lung membranes. Thus, the antibodies can be 
directed against an epitope(s) that binds the receptor or maintains the 
receptor-binding epitope in an active conformation. 
These antibodies are detected by measuring their binding to porcine 
.sup.125 I-VIP. Human and porcine VIP are structurally identical (9). 
Thus, the porcine VIP-reactive antibodies found in asthma patients are 
autoantibodies. It had been observed that diabetics positive for plasma 
VIP-antibodies had been treated with insulin contaminated with VIP, 
suggesting that the formation of antibodies was related to the VIP 
contaminant (10). However, the VIP antibodies in accordance with the 
invention are naturally occurring, i.e., not elicited by specific 
immunization against a target antigen. 
The antigenic stimulus leading to formation of these autoantibodies cannot 
be identified with certainty. Candidate stimuli include exposure to viral 
determinants similar in sequence to VIP[e.g., Peptide-T, an epitope found 
on the human immunodeficiency virus] and dietary ingestion of avian, fish 
and turtle VIP known to be structurally different from human VIP. Muscular 
exercise, which results in increased plasma VIP immunoreactivity (7), 
could also be a potential stimulus for VIP autoantibody formation. Indeed, 
asthma and muscular exercise appear to be associated with an increased 
incidence of autoantibodies directed against VIP. 
Irrespective of the type of antigenic stimulation leading to 
VIP-autoantibody formation, these antibodies may produce important 
biologic changes. The range of K.sub.a values observed for the 
autoantibodies of asthma patients is similar to that reported for VIP 
receptors present in the lung and other tissues (11), and these antibodies 
neutralize VIP receptor binding. It is possible that VIP-autoantibodies 
found in asthmatics neutralize the effect of VIP in the airways. 
It has now been discovered that these VIP-autoantibodies catalyze the 
hydrolysis of VIP between amino acid residues 16 and 17, i.e. between 
glutamine and methionine. Kinetic data (FIG. 1) obtained by measuring 
antibody mediated degradation of mono (.sup.125 I-Tyr.sup.10)-VIP as a 
function of increasing concentration of unlabeled VIP indicate (i) the 
degradation conforms to Michaelis-Menten kinetics, and (ii) the K.sub.m is 
in the nanomolar range (37.9 nM). A turnover of 0.26 sec.sup.-1 (i.e., 
about 16 molecules of VIP are hydrolyzed by one molecule of antibody per 
minute) was calculated. This calculation is based on the total number of 
antibodies which are capable of binding to VIP. However, in reality, not 
all antibodies capable of binding to VIP are necessarily capable of 
catalytic hydrolysis of VIP. Therefore, the actual turnover number is 
probably greater than that calculated. Mono(.sup.125 I-Tyr.sup.10)-VIP 
binding studies by the IgG at 4.degree. C. in radioimmunoassay buffer 
indicated that hydrolysis of the peptide is undetectable under these 
conditions. A linear Scatchard plot (FIG. 2A) and a Hill slope close to 
unity (FIG. 2B) suggested a single type of antibody with K.sub.d 0.4 nM 
and concentration of 73.4 fmoles/mg IgG, or about 0.001% of the total IgG 
(assuming antibody bivalency). 
The k.sub.cat and k.sub.cat /k.sub.m values for the hydrolysis were 0.26 
sec.sup.-1 and 6.9.times.10.sup.6 M.sup.-1, indicating that anti-VIP acts 
catalytically to hydrolyse VIP. The VIP hydrolytic activity in the IgG 
fraction is precipitated by ammonium sulfate, is inhibited by antiserum 
against human IgG, and exhibits the characteristic of authentic IgG when 
chromatographed on DEAE-cellulose, immobilized protein-G and high 
performance gel filtration columns. 
That the hydrolysis of VIP is caused by anti-VIP autoantibodies and not by 
a contaminating protease is clear from the findings that (i) the IgG did 
not contain non-immunoglobulin material; (ii) the Fab fragment of IgG 
exhibited a molecular mass close to 50 kDa and it hydrolysed VIP; (iii) 
the hydrolytic activity of intact IgG was retained on immobilized protein 
G, and then released by low pH treatment; (iv) the IgG revealed a single 
peak of hydrolytic activity with a molecular mass close to 150 kDa; (v) of 
the original VIP hydrolytic activity present in IgG purified by 
DEAE-cellulose chromatography, 78% and 80% was preserved in the retentate 
after ultrafiltration on a 100 kDa cutoff filter and in the ammonium 
sulfate precipitable fraction, respectively; (vi) treatment of the IgG 
preparation with anti-human IgG and removal of the immunoprecipitate 
decreased the hydrolytic activity by 75%; (vii) only two of six immune IgG 
preparations showed hydrolytic activity, and nonimmune IgG was without 
activity, (viii) the observed K.sub.m value suggests hydrolysis of VIP by 
a relatively tight binding agent, such as an antibody; (ix) the Gln-Met 
bond hydrolyzed by the antibody has not been described as a target for 
enzymatic (peptidase) hydrolysis; (x) it is believed that tight 
association of protease with IgG has not been described; and (xi) the only 
IgG binding factors in literature are the cell surface receptors for 
immunoglobilins and intracellular regulators of IgG secretion. 
Peripheral blood lymphocytes from a subject positive for hydrolytic 
anti-VIP antibodies can be transformed with Epstein-Barr Virus (EBV). The 
culture supernatant of these EBV transformed lymphoblastoid cells cause 
hydrolysis of VIP that may be greater than that by supernatants obtained 
from a control cell line. Thus, it is believed that the hydrolytic VIP 
antibodies may be produced by the transformed cells. 
It is well known that certain diseases are associated with autoantibodies 
directed against hormones and cell surface antigens. Examples of these 
diseases and associated autoantibodies are: 
______________________________________ 
Disease Autoantibody to 
______________________________________ 
Diabetes Insulin, Insulin receptor 
Myasthenia gravis acetylcholine receptor 
Graves disease thyroid stimulating hormone 
receptor 
Systemic lupus erythematous 
small nuclear RNA, DNA, 
histones 
Pernicious anemia Intrinsic factor of Castle, 
gastric parietal cell 
antibodies 
______________________________________ 
Since catalytic autoantibodies are likely to cause more harm than 
non-catalytic antibodies, it is possible that the autoimmune diseases may 
be caused by catalytic autoantibodies directed against nucleic acids, key 
regulatory peptides and proteins (e.g., insulin, glucagon, prolactin, VIP, 
substance P, blood clotting factors) and the cell surface receptors for 
these agents. Thus, the methods of the invention provide diagnostic tests 
which may be used to evaluate whether autoimmune diseases are associated 
with catalytic autoantibodies directed against specific proteins. For 
example, asthma may be caused by a deficiency of VIP. Catalytic anti-VIP 
antibodies could bring about this deficiency. If the presence of catalytic 
anti-VIP antibodies is detected and established, using the methodologies 
described herein, in individual asthma subjects, this would help determine 
the best way to treat the asthma in such subjects. 
Autoimmune diseases can be treated in accordance with the invention by 
administering to an afflicted animal an inhibitor capable of binding to 
the autoantibody, thereby preventing the autoantibody from catalyzing a 
chemical reaction of the self-antigen, in particular, cleavage of a 
peptide bond. The inhibitor can be the self-antigen, an analog of the 
self-antigen, a small peptide containing an epitope of the self-antigen at 
which epitope the chemical reaction takes place, an analog of a small 
peptide containing the epitope, or a small peptide containing an analog of 
the epitope. The inhibitor is administered, in combination with a suitable 
pharmaceutical carrier, either orally or by injection (I.V. or I.M.). 
In addition, autoantibody catalyzed cleavage of peptide bonds is likely to 
inactivate the target protein substrate, the peptide bond cleavage is 
likely to be highly specific, and, by definition, a single catalytic 
autoantibody molecule inactivates multiple substrate molecules, 
Inactivation by catalytic autoantibodies of proteins important in cancer, 
infectious diseases and hormonal or neural disorders forms the basis for 
catalytic autoantibody based therapies in accordance with the invention. 
For example, such target proteins include molecules that are found in or 
stimulate the growth of cancer cells (oncogene products, growth factors, 
carcinoembryonic antigens). Many tumors produce VIP and this peptide 
stimulates growth of some tumors. Catalytic anti-VIP autoantibodies may 
provide a cure for these tumors. A segment of the gp120 coat protein of 
HIV shares structural similarity with VIP(7-11) (Peptide-T). Catalytic 
anti-VIP antibodies directed against Peptide-T may be effective in 
treating HIV infections. 
The invention will be more fully described and understood with reference to 
the following examples which are given by way of illustration. 
EXAMPLE 1 
Preparation of Mono (.sup.125 I-Tyr.sup.10)-VIP 
Purified porcine VIP (Bachem) was labeled with .sup.125 iodine by the 
chloramine-T method (12). The resulting mono (.sup.125 I-Tyr.sup.10)-VIP 
was purified on a Seppak C18 cartridge followed by reverse phase HPLC with 
a gradient of acetonitrile in trifluoroacetic acid. Two major peaks of 
radioactivity were obtained (FIG. 3), corresponding to compounds that 
reacted with rabbit anti-VIP antiserum in radioimmunoassay. In order to 
obtain sufficient peptide for sequencing, VIP was iodinated with .sup.125 
I diluted with .sup.127 I to reduce the specific activity, and 
purification performed as before. Analysis of the peak with retention time 
25.3 min on an Applied Biosystems sequenator with on-line 
phenylthiohydantoin amino acid detection showed radioactivity mainly in 
cycle 10, with HPLC characteristics similar to those of monoiodotyrosine 
(purchased from Calbiochem), indicating that this material was 
mono(.sup.125 I-Tyr.sup.10)-VIP. The second peak of radioactivity 
(retention time 27.8 min) was identified as di(.sup.125 
I-Tyr.sup.10,Tyr.sup.22)-VIP by similar methods. The di(.sup.125 
I-Tyr.sup.10,Tyr.sup.22)-VIP and mono(.sup.125 I-Tyr.sup.10)-VIP performed 
nearly equivalently in a radioimmunoassay test. Since native VIP, VIP 
oxidized with chloramine-T without Na.sup.125 I(CT-VIP) and mono (.sup.125 
I,Tyr.sup.10)-VIP were well separated, it was concluded that the .sup.125 
I-VIP was free of unlabeled peptide. 
EXAMPLE 2 
Demonstration of VIP Autoantibodies In Human Subjects 
The antibodies were measured in plasma samples from asthma patients and 
healthy subjects, subdivided into high exercise (Hx) and low exercise (Lx) 
subgroups (7). Asthma was diagnosed on the basis of patient history and 
typical clinical indicators. The healthy Hx subjects had a history of 
habitual muscular exercise, and the healthy Lx subjects did not. Human 
blood samples were collected in a mixture of peptide hydrolase inhibitors 
(aprotinin, phenylmethylsulfonyl fluoride, pepstatin, ethylene diamine 
tetracetic acid) (8). The immunoglobulin G (IgG) fraction from blood was 
prepared by sequential chromatography (6, 8) on DEAE-cellulose (Whatman) 
and protein G-Sepharose (Pharmacia). The IgG (4 mg/ml) was ultrafiltered 
on a YM-10 membrane having an average cut off molecular weight of 10,000 
Daltons using an Amicon Model 8 MC apparatus to 27 mg/ml, diluted back to 
0.8 mg/ml and then subjected to a second cycle of ultrafiltration. The 
final concentration of IgG prepared in this manner was about 20 mg/ml. 
Electrophoretic analysis and staining of nitrocellulose blots with 
anti-human IgG conjugated to peroxidase did not reveal presence of 
non-immunoglobulin material in this preparation (Example 7). The presence 
of VIP-antibodies was established by measuring saturable binding of 
mono(.sup.125 I-Tyr.sup.10)-VIP (binding inhibited by excess unlabelled 
VIP) in plasma samples or purified IgG. The monoidinated form of VIP was 
used because it is more likely to reproduce the interactions of native VIP 
with the antibodies. Bound and free VIP were separated by precipitation 
with polyethylene glycol or specific sheep antibodies against human IgG 
(8). Plasma samples from some asthma patients and healthy subjects were 
observed to exhibit saturable .sup.125 I-VIP binding activity (up to 67.5% 
of total .sup.125 I-VIP). The VIP-antibodies were found in 18% of asthma 
patients (N=74), 30% of healthy Hx subjects (N=51), 2% of healthy Lx 
subjects (N=44). The mean .sup.125 I-VIP binding values calculated as the 
bound .sup.125 I-VIP divided by the total .sup.125 I-VIP multiplied times 
100 ("%B/T") with SEM in parenthesis in the antibody positive asthma and 
Hx subjects were 23.4 (5.3) and 20.4 (3.2). The lone antibody positive 
subject in the Lx group showed a % B/T value of 12.1%. 
EXAMPLE 3 
Determination That VIP-Antibodies Are Predominantly Of The IgG Class 
A specific goat anti-human IgG serum precipitated 83.7%.+-.5.1% 
(mean.+-.S.E.M.) and 79.0%.+-.4.5% of the VIP binding activity present in 
the 13 asthma and 16 non-asthmatic subjects, respectively. Goat anti-human 
IgM antibodies did not precipitate the VIP-binding activity in any of the 
plasma samples tested (N=16). The plasma VIP binding activity coeluted 
with authentic human IgG from DEAE-cellulose and gel filtration columns, 
and, pepsin treatment produced a F(ab).sub.2 fragment with VIP-binding 
activity. The binding activity was bound by immobilized protein G, an 
agent that binds IgG via the Fc portion of the molecule, and was released 
by treatment at low pH. 
EXAMPLE 4 
Determination Of Autoantibody Specificity For VIP 
PHI, GRF and secretin, peptides partially identical to VIP in their amino 
acid sequence, were employed to examine the specificity of the antibodies. 
These peptides (1 .mu.M) did not significantly displace the .sup.125 I-VIP 
binding by plasma from six asthma patients and four non-asthmatic subjects 
(8). The plasma antibodies in one asthmatic and one nonasthmatic subject 
showed partial reactivity with PHI, GRF and secretin (21.9% to 33.4%). The 
poor reaction of the antibodies with PHI, GRF and secretin suggests their 
high level of specificity for VIP. 
EXAMPLE 5 
Hydrolysis Of VIP By Anti-VIP Autoantibodies 
To compare antibody mediated hydrolysis and spontaneous hydrolysis of the 
peptide VIP, mono(.sup.125 I-Tyr.sup.10)-VIP was incubated with (i) immune 
and (ii) nonimmune IgG for increasing lengths of time. IgG from a 
nonimmune human subject and a VIP antibody positive subject was prepared 
by chromatography on DEAE cellulose followed by ultrafiltration as 
described in Example 2. The IgG or assay diluent (final volume of 200 
.mu.l in 50 mM Tris-HCl, 100 mM glycine, 0.025% Tween-20 and 0.1% bovine 
serum albumin, pH 8.0) was incubated with mono (.sup.125 I,Tyr.sup.10)-VIP 
(approximately 30 pM) for increasing lengths of time at 38.degree. C. 
Bovine serum albumin and Tween-20 were included in these incubations to 
prevent adsorptive loss of the mono (.sup.125 I,Tyr.sup.10)-VIP on glass 
and plastic surfaces. Precipitation with trichloroacetic acid (TCA) (13) 
was used as the initial criterion of mono(.sup.125 I,Tyr.sup.10)-VIP 
degradation. Accordingly, 1 ml of TCA (final concentration 10% v/v) was 
added to the reaction mixtures which were then centrifuged at 3000 xg. The 
supernatants were aspirated and the radioactivity was measured in the 
pellets (Beckman model 5500 spectrometer). At this TCA concentration, 
greater than 90% of intact mono (.sup.125 I,Tyr.sup.10)-VIP was 
precipitated (i.e., found to appear in the TCA-insoluble pellet). Values 
for VIP hydrolysis were computed from the radioactivity observed as counts 
per minute (CPM) in the TCA-precipitable fractions as: 
(CPM.sub.assay buffer -CPM.sub.antibody).times.100/CPM.sub.assay buffer. 
Compared to 8% hydrolysis of the mono (.sup.125 I,Tyr.sup.10)-VIP 
incubated with nonimmune IgG, 73% of the peptide was hydrolyzed by 
treatment with immune IgG. 
The ability of the IgG to hydrolyze mono(.sup.125 I,Tyr.sup.10)-VIP was not 
lost by precipitation with 50% saturated ammonium sulfate or 
ultrafiltration on a 100 kDa membrane filter. Treatment of the IgG with 
rabbit anti-human IgG or treatment at 100.degree. C. (10 min) prior to 
incubation with mono(.sup.125 I,Tyr.sup.10)-VIP destroyed the hydrolytic 
activity of the IgG as indicated by a reduction in the amount of 
radioactivity in the peak with RT of 10 min. 
Treatment of mono (.sup.125 I,Tyr.sup.10)-VIP with immune IgG for 
increasing time periods progressively reduced the amount of radioactivity 
precipitated by 10% TCA, as shown in FIG. 4. After incubation with immune 
IgG for 6 h, 73% of the starting mono (.sup.125 I,Tyr.sup.10)-VIP was no 
longer precipitated by TCA, compared to only 8% of the mono (.sup.125 
I,Tyr.sup.10)-VIP incubated with nonimmune IgG. The degradation of mono 
(.sup.125 I-Tyr.sup.10)-VIP was pH dependent, with an optimum pH of 
8.0-8.5. 
Kinetic data were obtained by incubating IgG with increasing concentrations 
of unlabeled VIP mixed with a fixed concentration of mono (.sup.125 
I,Tyr.sup.10)-VIP as trace for 2 h at 38.degree. C. The hydrolysis was 
saturable with increasing VIP concentrations and a plot of 1/velocity vs. 
1/substrate concentration was linear, as shown in FIG. 1, indicating that 
the reaction conformed to Michaelis-Menten kinetics. A K.sub.m for the 
reaction of 37.9 nM, determined from the slope of the linear plot in FIG. 
1, indicated relatively stable antibody-VIP binding. A Scatchard plot of 
VIP binding by the antibody, under conditions that did not lead to VIP 
hydrolysis (see Example 8), was linear, as shown in FIG. 2A. The slope for 
the Hill plot, shown in FIG. 2B, was close to unity (1.02). These data 
indicated a single antibody class with K.sub.d 0.4 nM and concentration 
73.4 fmol/mg IgG (assuming antibody bivalency). The k.sub.cat and 
k.sub.cat /K.sub.m values for the hydrolysis, computed on the basis of the 
kinetics of hydrolysis and the antibody concentrations obtained from the 
binding data, were 0.26 sec.sup.-1 and 6.9.times.10.sup.6 M.sup.-1 
sec.sup.-1. These values indicated that the anti-VIP acts catalytically to 
hydrolyse VIP. A turnover of 0.26 sec.sup.-1 (i.e., about 16 molecules of 
VIP are hydrolyzed by one molecule of antibody per minute) was calculated. 
This calculation was based on the total number of antibodies which were 
capable of binding to VIP. However, in reality, not all antibodies capable 
of binding to VIP are necessarily capable of catalytic hydrolysis of VIP. 
Therefore, the actual turnover number is probably greater than that 
calculated. 
EXAMPLE 6 
Identification of Peptide Fragments Resulting From Hydrolysis of VIP 
Catalyzed by Anti-VIP Autoantibodies 
Reverse phase HPLC of mono (.sup.125 I-Tyr.sup.10)-VIP treated with the 
immune IgG revealed a reduction in the amount of intact mono (.sup.125 
I,Tyr.sup.10)-VIP (retention time (RT): 25 min.) and the appearance of an 
early eluting peak of radioactivity (RT: 10.0 min) that was well separated 
from intact mono (.sup.125 I-Tyr.sup.10)-VIP and free .sup.125 I (RT: 
65-7.0 min) (FIG. 5). Heat treatment of the IgG prior to incubation with 
mono(Tyr.sup.10,.sup.125 I)-VIP resulted in a reduction in the amount of 
radioactivity in the peak with RT 10 min. When mono (.sup.125 I, 
Tyr.sup.10)-VIP was incubated in buffer instead of the IgG, the bulk of 
the radioactivity was recovered in the form of intact peptide and only 
13.9% in the peak with RT of 10 min. In order to purify the fragments of 
VIP, unlabelled VIP (50 .mu.g) was treated with 525 .mu.g immune IgG or 
nonimmune IgG as before, except that bovine serum albumin was omitted from 
the reaction mixture. The reaction mixtures were extracted on Extract 
Clean C18 cartridges (Alltech), and then subjected to reverse phase HPLC 
on a Novapak-C18 column (Waters), eluting with a gradient of acetonitrile 
in trifluoroacetic acid. The absorbance of the eluate was monitored at 214 
nM. Two A.sub.214 nm absorbing peaks (labeled 1 and 2 in FIG. 6), noted 
after treatment of the VIP with immune IgG, were absent in peptide 
preparations treated with nonimmune IgG or assay buffer. These peaks were 
purified by a second round of reverse phase HPLC using shallower gradients 
for elution (FIG. 7 and 8). The peptide fractions purified by reverse 
phase HPLC were dried, and sequenced using an Applied Biosystems pulsed 
liquid phase sequenator (model 477A) with on-line 
phenylthiohydantoin-amino acid detection. This procedure demonstrated 
unequivocally that the major A.sub.214 absorbing peaks identified as 1 and 
2 in FIG. 7 and 8, respectively, were VIP [1-16] and VIP [17-28]. Fast 
atom bombardment (f.a.b.)-mass spectrometry of peptide 2 in FIG. 8 and 
intact VIP [1-28] was performed in the positive ion mode on a VG 
Analytical ZAB-2SE spectrometer (acceleration potential: 8 kV) (M-Scan) 
using peptides dissolved in 5% acetic acid and thioglycerol/glycerol or 
m-nitrobenzyl alcohol matrices. Mass calibration was performed with cesium 
iodide or cesium iodide/glycerol. The F.a.b.-mass spectrometric analysis 
(FIG. 9) suggested that the molecular mass of peptide 2 was 1393 daltons 
corresponding to the molecular ion of VIP [17-28]. It is believed that the 
additional peak observed with mass of 1415 daltons probably represented 
the sodium adduct of VIP [17-28]. Analysis of VIP [1-28] resulted in a 
signal at 3325 daltons that corresponded well to the molecular ion of the 
peptide. 
EXAMPLE 7 
Determination that Anti-VIP Autoantibody (IgG) And Not A Contaminating 
Peptidase Caused Hydrolysis of VIP 
IgG Did Not Contain Non-Immunoglobulin Material 
Overloaded IgG (50 .mu.g) was subjected to electrophoresis in 12-20% 
polyacrylamide gels. Silver staining revealed one major IgG band and a 
minor light chain band with molecular mass 150 kDa and 25 kDa, 
respectively. A nitrocellulose blot of the gel was treated with rabbit 
anti-human IgG conjugated to peroxidase (Accurate) and stained with 
diaminobenzidine and hydrogen peroxide. Both bands were reactive with the 
anti-human IgG, indicating that the IgG did not contain non-immunoglobulin 
material. 
VIP-Cleaving Activity Resided In The Fab Fragment 
Th IgG (10 mg in 0.5 ml) was treated with papain conjugated to agarose 
(Pierce) (1.5 ml in 20 mM sodium phosphate, pH 7.0, 10 mM EDTA and 20 mM 
cysteine) for 5 h at 38.degree. C. with vigorous shaking. The mixture was 
centrifuged, and Fab in the supernatant was purified by chromatography on 
protein A conjugated to agarose (2.2 ml gel; Pierce). The column was 
washed with 10 mM Tris-HCl, pH 7.5 to recover unretained Fab. This 
fraction was concentrated by ultrafiltration on an Amicon YM-10 filter. 
The Fab fragment prepared in this manner exhibited a molecular mass of 50 
kDa. The retentate, the parent IgG fraction and marker proteins (right 
lane) were electrophoresed on a 8-25% gradient polyacrylamide gel using a 
Phast system (Pharmacia) and the gel was stained with Coomassie blue, as 
shown in FIG. 10A. Increasing concentrations of the Fab preparation were 
incubated with mono (.sup.125 I, Tyr.sup.10)-VIP in 0.05M Tris-HCl, 0.1M 
glycine, pH 8.0, containing 0.025% Tween 20 (assay diluent) for 3 h at 
38.degree. C. Trichloroacetic acid (TCA) was added to 10% (v/v), the tubes 
centrifuged (5800.times.g; 20 min), the supernatants aspirated and 
radioactivity in the pellets determined as described in Example 5 above. 
FIG. 10B indicated that the Fab fragment caused dose dependent cleavage of 
VIP. Thus, the catalytic activity resided in the Fab fragment. 
Hydrolytic Activity Of Intact IgG Was Retained On Immobilized Protein G And 
Was Released By Low pH Treatment 
IgG purified on a DEAE-cellulose column was chromatographed on protein G 
conjugated to Sepharose (Pharmacia) in 50 mM Tris-HCl, pH 7.3. Protein G 
is an agent which binds immunoglobulin at its F.sub.c region. All of the 
A.sub.280 was retained by the protein G, and then released upon 
application of a low pH buffer (0.1M glycine-HCl, pH 2.7). The eluate 
fractions were made to pH 8 with 1M Tris-HCl, pH 9, pooled and assayed for 
VIP hydrolytic activity (inset) as in FIG. 11. 
IgG Exhibited A Single Peak Of Hydrolytic Activity With A Molecular Mass 
Close To 150 kDa 
To determine that hydrolysis took place at a single site on the antibody, 
the protein G purified IgG was gel filtered on a Superose-12 column 
(Pharmacia) in 50 mM Tris-HCl, pH 8 buffer at a rate of 0.5 ml/min. Marker 
proteins used for comparative molecular mass determination were ferritin, 
catalose, bovine serum albumia and chymotrypsinogen. Gel filtration 
chromatography indicated a single peak of hydrolytic activity with a 
molecular mass close to 150 kDa, determined by comparison with the marker 
proteins. 
Greater Than 75% of VIP Hydrolytic Activity Preserved In DEAE-Cellulose 
Chromatographed IgG After Ultrafiltration 
The IgG was diluted to 0.1 mg/ml and ultrafiltered on an Amicon YM-100 
filter to 2.2 mg/ml. IgG precipitated with 50% saturated ammonium sulfate 
was centrifuged, redissolved in assay diluent, dialyzed and then assayed 
for hydrolytic activity as described in Example 5. Of the original VIP 
hydrolytic activity present in IgG purified by DEAE-cellulose 
chromatography, 78% and 80% was preserved in the retentate after 
ultrafiltration on a 100 kDa cutoff filter and in the ammonium sulfate 
precipitable fraction, respectively. 
Treatment of IgG with Anti-Human IgG And Removal Of Immunoprecipitate 
Decreased Hydrolytic Activity By 75% 
Goat anti-human IgG (Antibodies Inc.) was purified further by 
chromatography on immobilized protein G as above. The human IgG (450 
.mu.g; 100 .mu.l) was incubated with 700 .mu.l of the anti-human IgG 
(diluted 13.5-fold) or assay diluent for 45 min at 4.degree. C., the 
precipitate removed by centrifugation and the supernatants tested for VIP 
hydrolytic activity. Treatment of the IgG preparation with anti-human IgG 
and removal of the immunoprecipitate decreased the hydrolytic activity by 
75%. The retention of a small proportion (25%) of the starting VIP 
hydrolytic activity in the supernatant was likely due to imcomplete IgG 
precipitation. 
EXAMPLE 8 
Induction of Catalytic Activity In VIP-Autoantibody By Ultrafiltration 
IgG was prepared by chromatography on (i)protein G conjugated to Sepharose, 
as described in Example 7 or (ii) DEAE-cellulose, as described in Example 
2. The IgG (4 mg/ml) was ultrafiltered on a YM-10 membrane having an 
average cut off molecular weight of 10,000 Daltons using an Amicon Model 8 
MC apparatus to 27 mg/ml, diluted back to 0.8 mg/ml and then subjected to 
a second cycle of ultrafiltration. The final concentration of IgG prepared 
in this manner was 20 mg/ml. IgG purified as above but without 
ultrafiltration and IgG purified and with ultrafiltration as above were 
each incubated with mono(.sup.125 I-Tyr.sup.10)-VIP in radioimmunoassay 
buffer at 4.degree. C. for two hours in the presence of increasing 
unlabeled VIP concentrations and the TCA soluble radioactivity was 
determined as described in Example 5. 
FIG. 12 indicates that treatment of mono(.sup.125 I, Tyr.sup.10)-VIP with 
IgG that had not been subjected to ultrafiltration resulted in a 
dose-dependent, but low-level degradation of the peptide, judged by the 
increase in TCA-soluble radioactivity over the value obtained with assay 
buffer. IgG subjected to ultrafiltration degraded VIP better than IgG that 
which had not been ultrafiltered. 
EXAMPLE 9 
VIP Hydrolytic Activity of Supernatant From EBV Transformed Lymphocytes 
Peripheral blood lymphocytes of an individual positive for hydrolytic VIP 
antibodies were transformed with Epstein-Barr virus by established 
procedures (14-19). The culture The culture supernatants from these cells 
appeared to cause 53% hydrolysis of mono(.sup.125 I-Tyr.sup.10)-VIP, 
judged by the TCA precipitation method. Control fluid (RPMI with 10% fetal 
bovine serum) did not appear to cause significant hydrolysis and the 
culture supernatant of an irrelevant EBV transformed cell line appeared to 
cause 21% hydrolysis. A low level of saturable VIP binding activity (about 
CPM/100 ul) was detected in the supernatant from EBV transformed cells of 
the VIP antibody positive subject, but not from the irrelevant cell line. 
EXAMPLE 10 
Preparation of Human Hybridoma Cell Lines Producing Catalytic Anti-VIP 
Antibodies 
Lymphocytes are isolated by density gradient centrifugation of 
Ficoll-Hypaque. VIP-specific B lymphocytes are enriched by attachment to 
Petri dishes containing immobilized producing cell line) for 2 hours, 
washed and then cultured at 1.times.10.sup.6 /ml in RPMI-1640 supplemented 
with 10% fetal bovine serum, L-glutamine and antibiotics. The cultures of 
transformed cells are examined daily and fed twice weekly. After about two 
weeks immunoglobulins from the culture supernatants are examined for VIP 
binding and hydrolytic activity by the methods described above. The 
resulting EBV cell lines are cloned in 96 well cell culture plates by 
limiting dilution using 0.5 cells/well with 10% ORIGEN cloning factor 
(IGEN) in place of feeder layers. Positive growing clones are assayed for 
immunoglobulin production and screened for VIP hydrolytic activity. The 
cells producing the hydrolytic antibodies are recloned to ensure their 
monoclonal status. The positive cell cultures are expanded for 
immunoglobulin production and for hybridization with human or mouse 
myeloma cell lines. Since EBV transformed cell lines are often low 
immunoglobulin producers, transformed cells are hybridized with either 
mouse myelomas or a mouse-human heteromyeloma in order to obtain stable 
hybrids which produce the unique catalytic antibodies. Three different 
partners are used including the mouse myeloma: SP2/0-Ag14, the human 
plasmacytoma: SKO-007 and a mouse x human heteromyeloma: SHM-D33 grown in 
the presence of the antibiotic G-418 to stabilize the human chromosomes. 
The fusion is done in the same way as mouse x mouse fusions (20). 
REFERENCES 
1. Pauling, L. Nature 161:707, 1948. 
2. Kohen, F., Kim., J. B., Lindner, H. R., Eshhar, Z., Green, B. Antibody 
enhanced hydrolysis of steroid esters FEBS. 
3. S. J. Pollack, J. W. Jacobs, P. G. Schultz, Science 234, 1570 (1986); A. 
Tramontano, A. A. Amman, R. A. Lerner, J. Am. Chem Soc. 110, 2282 (1988); 
K. D. Janda, D. Schloeder, S. J. Benkovic, R. A. Lerner, Science 241, 1188 
(1988); C. N. Durfor, R. J. Bolin, R. J. Sugasawara, R. J. Massey, J. W. 
Jacobs, P. G. Schultz, J. Am. Chem. Soc. 110, 8713 (1988). 
4. D. Y. Jackson, J. W. Jacobs, R. Sugasawara, S. H. Reich, P. A. Bartlett, 
P. G. Schultz, J. Am. Chem. Soc. 110, 4841 (1988); D. Hilvert, S. H. 
Carpenter, K. D. Nared, N. T. Auditor, Proc. Natl. Acad. Sci. USA 85, 4953 
(1988). 
5. K. Shokat, C. H. Leumann, R. Sugasawara, P. G. Schultz, Angew. Chem. 
Int. Ed. Engl. 27, 1172 (1988). 
6. Paul, S., H. Erian, P., Said, S. I. Autoantibody to vasoactive 
intestinal peptide in human circulation. Biochem. Biophys. Res. Commun. 
130:479-485, 1985. 
7. Paul, S., Said, S. I. Human autoantibody to vasoactive intestinal 
peptide: Increased incidence in muscular exercise. Life Sciences 
43:1079-1084, 1988. 
8. Paul, S., Said, S. I., Thompson, A., Volle, D. J., Agrawal, D. K., Foda, 
H., De la Rocha, S.: Characterization of autoantibodies to VIP in asthma 
J. Neuroimmunol., 133-142 1989. 
9. Itoh, N., Obata, K. -I., Yanaihara N., Okamoto, H. Human 
preprovasoactive intestinal polypeptide contains a novel PHI-27-like 
peptide, PHM-27, Nature 304:547-549, 1983. 
10. Bloom, S. R., Barnes, A. J., Adrian, T. E., Polak, J. M. Autoimmunity 
in diabetics induced by hormonal contaminants of insulin. Lancet i:14-17, 
1979. 
11. Paul, S., Said, S. I. Characterization of receptors for vasoactive 
intestinal peptide from the lung. J. Biol. Chem. 262:158-162, 1987. 
12. Paul, S., Wood, K., Said, S. I. Purification of [.sup.125 I]-Vasoactive 
intestinal peptide by reverse-phase HPLC. Peptides 5:1085-1087, 1984. 
13. Turner, J. T., Bylund, D. B. Characterization of the VIP receptor in 
rat submandibular bland: Radioligand binding assay in membrane 
preparations J. Pharmacol Exp. Therap. 242:873-881, 1987. 
14. Steinitz, M., Klein, G., Koskimies, S., Makela, O., EB Virus induced B 
lymphocyte lines producing specific antibodies. Nature 269:420-422, 1977. 
15. Steinitz, M., Seppala, I., Eichmann, K., Klein, G. Establishment of a 
Human Lymphoblastoid Cell Line with Specific antibody production against 
group A streptococcal carbohydrate. Immunobiology 156:41-47, 1979. 
16. Steinitz, M., Izak, G., Cohen, S., Ehrenfeld, M., Flechner, I. 
Continuous production of monoclonal rheumatoid Factor by EBV-transformed 
lymphocytes. Nature 287:443-445, 1980. 
17. Kozbor, D., Steinitz, M., Klein, G., Koskimies, S., Maketa, O. 
Establishment of anti-TNP antibody-producing human lymphoid lines by 
preselection for hapten binding followed by EBV transformation. Scand. J. 
Immunol. 10:187-194, 1979. 
18. Kozbor, D. & Roder, J. The production of monoclonal antibodies from 
human lymphocytes. Immunology Today 4:72-79, 1983. 
19. Roder, J., Cole, D., Kozbor, D. The EBV-Hybridoma Technique, Methods in 
Enzymology 121:140-167, 1986. 
20. Kohler G., Milstein C. Continuous Cultures of Fused Cells Secreting 
Antibody of Predefined Specificity. Nature 256:445-497 (1975)