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Description  |
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BACKGROUND OF THE INVENTION
This invention relates to a method of linking primary aromatic amines to
carrier proteins by photochemical reactions in order to produce antibodies
against the amine haptens.
More specifically, the invention relates to a method for coupling aromatic
nitro- or amine-containing drugs or other compounds to carrier proteins
for the purpose of raising antibodies against the coupled compound. The
conjugated proteins can then be used to produce antibodies specific for
the coupled haptenic group or, if the drugs are coupled to antibodies,
these may then be used to increase the efficacy of drug delivery to
targeted sites and decrease toxicity. The coupling reaction is rapid and
occurs under mild conditions which do not result in denaturation or loss
of function of the carrier proteins. The coupling procedure provides for
high labeling densities which can be obtained without loss of protein
function.
Two methods are primarily used to conjugate primary aromatic amines to
carrier proteins. They are the diazocoupling method (Inman, et al., 1973,
Immunochem. 10, 153-163) and the isocyanate method (Spragg, et al., 1966,
J. Immunol. 96, 865-871). However, there are some disadvantages to the use
of these methods. The conjugated proteins prepared by these methods may
sometimes fail to elicit an anti-hapten antibody response, which may be
attributed to the highly alkaline conditions required for coupling. It is
also difficult to prevent side reactions associated with diazonium
coupling, which may result in extensive precipitation. Furthermore, the
bonds formed by the diazonium salts are easily cleaved. Finally, the
extent of the diazocoupling is mainly dependent on the presence of
tyrosine and histidine residues in the carrier protein, which further
restricts the applicability of this approach. The isocyanate method of
Creech, H. J. (1952) Cancer Res., 12, 557-564 has been used for
conjugating carcinogenic aromatic primary amines to carrier proteins. This
method involves the derivatization of the primary aromatic amine to form
the isocyanate followed by coupling to the .SIGMA.-amino group of a lysine
residue. Fraenkel-Conrat, H. L. (1944) J. Biol. Chem. 152, 385-389
reported that isocyanates react with the --SH groups of cysteine and
Miller, et al. (1941) J. Biol. Chem. 141, 905-920 have reported that
isocyanates also react with --OH groups of tyrosine. However, Creech, et
al. (1941) J. Am. Chem. Soc. 63, 1670-1673 were unable to conjugate zein
with isocyanates even under favorable experimental conditions.
Additionally, attempts by Creech et al. (1941) J. Am. Chem. Soc. 63,
1661-1669 to increase the epitope density (the number of haptenic groups
attached per molecule carrier) beyond a certain level resulted in
denaturation of the protein. The coupling conditions for this method,
stirring the hapten and carrier protein at alkaline pH and 4.degree. C.
overnight, are similar to those for the diazocoupling method and present
the same problems. The specific functional group requirements of the
isocyanate coupling procedure also restrict the applicability of this
method.
Photolabeling techniques have previously not been used to conjugate primary
aromatic amines with carrier proteins in order to elicit antibodies
against the hapten. Primary aromatic amines may be easily derivatized to
azido compounds which are reasonably stable in the dark and at low
temperatures. The resultant azido derivatives are photolabile when
irradiated with ultraviolet (UV) light in ethanolic or aqueous solutions,
giving rise to highly reactive nitrene radicals which can undergo
insertion reactions.
There is a need to conjugate aromatic nitro- or amine-containing compounds
to carrier proteins in a relatively short period of time at physiological
pH.
SUMMARY OF THE INVENTION
The present invention is directed to a novel approach for raising
antibodies against aromatic nitro- or amine-containing drugs, carcinogens,
carbohydrates, or other compounds by using photolabeling methods to
produce hapten-carrier conjugates. The conjugation to carrier proteins
involves mild reaction conditions which do not result in denaturation or
loss of function of the carrier protein and which results in high
labelling densities. These conjugates can then can be used in the
production of antibodies against the conjugated haptens. These antibodies
could be used for monitoring drug levels in body fluids using RIA or ELISA
methods. Further, the antibodies could also be used to estimate the
amounts of drugs, chemical carcinogens, and other xenobiotics bound to
deoxyribonucleic acid (DNA), ribonucleic acid (RNA) or protein so that
levels of these adducts could be determined in any tissue of interest.
Additionally, the coupling process can be utilized to improve the efficacy
of drug delivery to target tissues. For example, chemotherapeutic agents,
radiolabeled compounds, etc. can be conjugated to polyclonal or monoclonal
antibodies against tumor-specific antigens and these can then be used to
target the conjugated compounds for delivery to the appropriate target
tissue (i.e., in this case, the tumor). This method of selective targeting
could lead to a marked improvement of therapeutic efficacy with a
significant decrease in general toxicity.
Briefly, aromatic nitro compounds may be reduced to give the aromatic
amine. The amine can then be converted to the azido compound by
diazotization with nitrous acid followed by treatment with sodium azide.
Aromatic azides can also be produced by various other synthetic routes
such as displacement of halide ion by azide. The aromatic azide can then
be conjugated to a protein or polypeptide by irradiation with ultraviolet
light. The aryl azides are photolabile and upon photolysis give rise to
highly reactive nitrene radicals which can then undergo insertion
reactions with the amino acid side chains on proteins or polypeptides to
form covalent adducts. The conjugated protein can then be separated from
the unbound photolysis products by gel exclusion chromatography and the
conjugated protein can then be used for antibody production against the
conjugated aromatic compound, or delivery of a drug conjugated to the
protein, etc.
Therefore, it is an object of the present invention to provide a method for
the production of antibodies against a specific hapten.
It is another object of the present invention to provide a drug delivery
system.
DETAILED DESCRIPTION OF THE INVENTION
The photolysis of aryl azides is well known. The use of nitrene insertion
in the case of most biological molecules will result in a wide range of
undefined linkage positions on the molecule. Although the spectrum of
linkages may be reduced somewhat by electrostatic or other interactions,
there should be, in most cases, a large number of possible coupling sites.
Probability considerations alone dictate that coupling at most of these
sites will have little if any effect on the biological activity of the
macromolecule. Some of these insertion sites on proteins are C.dbd.S,
C--H, C.dbd.O, S--H, or N--H bonds. Under proper conditions, all proteins
can be labeled by using nitrene insertion since the functional residues
required for insertion of nitrenes are routinely found in the backbones of
all proteins. The structure of the linkage region between hapten and
carrier protein would be R--N.dbd.N-carrier for azo coupling,
R--NH--CO--NH-carrier for the isocyanate method, and R--NH-carrier for the
photolabeling method. The linkage bond obtained by photolabeling is more
similar to the parent amine, resulting in the production of antibodies of
greater specificity for the haptenic group.
The method of the present invention is applicable to the coupling of any
nitro- or amino-containing compounds to a protein or polypeptide. The
carrier protein influences the antibody response to a hapten. Due to the
general nature of the photolabeling method, it is possible to study the
effectiveness of various carriers in eliciting antibody response either
with different proteins conjugated to similar extents or for determining
optimum epitope density for antibody response by using the same protein
with varying epitope densities. In accordance with the present invention,
even at high epitope densities, the protein carriers are not denatured in
contrast to conventional procedures where high epitope densities appear to
lead to denaturation of the carrier molecule. Examples of carrier proteins
and peptides that can be utilized in the present invention include bovine
serum albumin (BSA), human transferrin (TR), thyroglobulin (TH), poly
(lysine.tyrosine), and poly (lysine.phenylalanine). This listing is not
intended to be limiting, for any protein or peptide may be used in the
process of the present invention such as various protein components
isolated from human and animal serum tissue and cellular extracts and
protein components from pathogenic parasites. The conjugation of the azido
analog of the nitro- or amino-containing compound to the carrier protein
by photoirradiation generally occurs in about 1 second to thirty minutes
at physiological pH. Preferably, the pH may range from about 6.8 to about
7.8. Irridation may be by ultraviolet or visible light for about 1 second
to about thirty minutes. The conjugated protein can be purified by
conventional techniques and isolated in about 2 to about 3 hours.
Typically, aryl azides are conjugated to bovine serum albumin using
standard photolabeling procedures. After photolysis, the conjugated
protein is separated from the unbound photolysis products of the aryl
azide on a gel exclusion column. The following examples illustrate a
preferred embodiment of the present invention but are not to be construed
as a limitation thereon.
EXAMPLE I
Hapten Carrier Conjugation
A 24 ml. volume of a 500 .mu.g/ml solution of bovine serum albumin (BSA) in
100 mM potassium phosphate buffer, pH 7.4, was stirred at 4.degree. C. in
a 50 ml beaker. To this solution, 1.0 ml of 5 mM 3-azido-N-ethylcarbazole
(ANEC) in ethanol was added and the sample was photoirradiated for 6
minutes from the top by placing a UVP model B-100A long UV lamp at a
distance of 6 cm as measured from the bottom of the beaker. The photolyzed
sample was passed through a Sephadex G-10 column (4.times.0.8 cm) and
equilibrated with 100 mM potassium phosphate, pH 7.4. The protein
containing fraction from the column was reprocessed using the same
sequence (addition of 1.0 ml of 5 mM ANEC dissolved in ethanol, photolysis
for 6 minutes, and chromatography on a Sephadex G-10 column) 3 more times.
After the last photolysis and gel filtration on the Sephadex G-10 column,
the proteins containing eluate was used for injection of rabbits as
described in Example II. Hapten-thyroglobulin (TH) and hapten-human
transferrin (TR) conjugates were also prepared in the same way. For
labeling the poly (Lys.HBr, Tyr) 1:1, and the poly(Lys.HBr, Phe) 1:1, 500
.mu.g/ml solutions of the polypeptides were made in distilled water and
the conjugations carried out as previously described.
Labeling
ANEC was photolyzed in 100 mM potassium phosphate buffer, pH 7.4, in the
absence of protein and passed through a Sephadex G-10 column. The
photolyzed ANEC remained at the top of the column and did not elute, even
after washing the column with 50 column volumes of 15% ethanol in 100 mM
phosphate buffer, pH 7.4. When the column was washed with 95% ethanol, the
photolyzed ANEC eluted quantitatively as a yellow band. In order to
estimate the number of ANEC groups bound to the carrier proteins, this
observation was utilized. The Sephadex G-10 column used to separate the
unbound ANEC and photolysis products from protein-bound ANEC, was first
washed with 10 column volumes of 15% ethanol in 100 mM phosphate buffer,
pH 7.4. The washing buffer was then changed to 95% ethanol and the unbound
photolysis products of ANEC eluted. The 95% ethanol eluate was flash
evaporated and the residue weighed. The differences between the total
amount of ANEC added in four cycles and the residue which eluted from the
column gave an approximation of the amount of ANEC bound to the carrier
protein. Calculations for this procedure are given in Table 1.
TABLE 1
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Amount of Epitope
ANEC Recovered
Amount Bound to Density
(.mu.moles) from
Carrier Protein per 1000
Carrier Protein
G-10 Column
(.mu.moles)
Epitope Density
daltons
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BSA 4.6 15.4 (77).sup.b
85 1.28
Transferrin
4.0 16.0 (80)
107 1.33
Thyroglobulin
5.6 14.4 (72)
800 1.20
Poly Lys--Phe
5.0 15.0 (75)
50 1.25
Poly Lys--Tyr
3.8 16.2 (81)
120 1.35
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.sup.a Calculated Using the following molecular weights: BSA, 66,000;
Transferrin, 80,000; Thyroglobulin, 669,000; poly (Lys--Phe), 40,000; and
poly (Lys--Tyr), 90,000.
.sup.b Percent of total ANEC bound to the carrier protein.
EXAMPLE II
Immunization Protocol
New Zealand white female rabbits weighing 1-1.5 kg were used for
immunization. They were obtained from Lesser's Rabbitory, Union Grove,
Wis. Preimmune sera were obtained from blood removed from the marginal ear
vein of each rabbit. Each antigen (600 .mu.g in 1.5 ml of 100 mM potassium
phosphate, pH 7.4) was mixed with an equal volume of Freund's complete
adjuvant and the emulsified preparation injected at multiple sites
subcutaneously and intramuscularly. Three weeks after the first injection,
the rabbits were bled and a booster dose (100 .mu.g of antigen in 1.0 ml
of 100 mM potassium phosphate, pH 7.4, mixed with equal amount of
incomplete Freund's adjuvant) was injected in multiple subcutaneous,
intramuscular and intraperitoneal sites and repeated at three to four week
intervals with specimens of blood obtained 7-10 days after each series of
injections. The blood specimens were allowed to clot at 4.degree. C. and
the collected serum was stored at -20.degree. C.
Evaluation of Antisera Titers
The titers of the antisera were measured using a solid-phase,
hapten-specific, non-competitive enzyme-linked immunosorbent assay
(ELISA). Antigen titrations (0.01, 0.05, 0.1, 0.5, 1.0, 5.0, 10.0 and 50
.mu.g/ml of ANEC-protein were used for coating the wells) and antisera
titrations (1/500, 1/1000, 1/2,500, 1/5,000, 1/10,000, 1/15,000, 1/20,000,
and 1/30,000 dilutions) were performed using Dynatech Immunolon II 96 well
microtiter plates. From the linear portions of the titration curves a
serum dilution of 1:1,000 and a coating antigen concentration of 1.0
.mu.g/ml were selected for titer evaluation studies. The wells of the
Immunolon II plates were coated with 200 .mu.l of 1 .mu.g/ml ANEC-protein
conjugate in 0.1M NaHCO.sub.3, pH 9.0, at ambient temperature for 2 hr.,
backcoated with 1% ovalbumin in PBS (0.01M sodium phosphate buffer, pH
7.3, containing 0.15M sodium chloride) for 1 hr. at ambient temperature,
and then washed three times with wash buffer (1% BSA, 0.02% sodium azide,
0.1% Tween-20 in PBS, pH 7.3). Various dilutions of immune serum were made
up in the wash buffer and 200 .mu.l aliquots of each dilution added to
duplicate wells. The plates were incubated at ambient temperature for 2
hr., washed three times with wash buffer and 200 .mu.l aliquots of
affinity purified IgG fraction of goat anti-rabbit IgG conjugated with
.beta.-galactosidase prepared by the method of Boraker et al. (1981 J.
Clin. Med. 14, 396-403) were added. The plate was incubated at 4.degree.
C. overnight, washed three times with wash buffer and then freshly made
substrate solution (4.0 mg/ml o-nitrophenyl-.beta.-D-galactopyranoside,
5.0 mM MgCl.sub.2, and 0.1M .beta.-mercaptoethanol in PBS, pH 7.3) was
added (200 .mu.l). The plates were incubated for 3 hr. at ambient
temperature and the optical density in each well was measured at 405 nm
with a Titertek Multiskan Instrument (Flow Labs., McClean, VA).
Inhibition studies were conducted by preincubating diluted antisera
(1:1000) with various concentrations of ANEC-BSA, ANEC-TH, ANEC-TR or free
hapten for 2 hours prior to addition to ANEC-BSA (1 .mu.g/ml) coated
wells, with the rest of the assay being performed as described above. The
percent inhibitions of antibody binding were plotted against the log of
the solution phase inhibitor concentrations. In a similar manner,
inhibition by the hapten, 3-amino-N-ethylcarbazole, was also investigated.
Results
Antibody responses were observed in antisera collected from two of the
rabbits three weeks after the first immunization injections using the
ELISA assay. The titers of the various sera were compared at 1:2000
dilution of the anti-ANEC-BSA and anti-ANEC-TR sera using ANEC-BSA coated
wells (1 .mu.g/ml coating concentration). Further immunization did not
increase the titer nor did the titers decrease. The rabbit immunized with
ANEC-TH conjugate failed to demonstrate anti-ANEC antibody reactivity.
This response was presumed to be due to a function of the TH carrier as
the other two rabbit antisera employing heterologous immunogen carriers
reacted well with the ANEC-TH conjugate. Also, when this rabbit was
further immunized with ANEC-TR conjugate, anti-ANEC antibodies were
elicited. No other rabbits were immunized to determine whether ANEC-TH
would act a as immunogen.
All of the appropriate controls in the ELISA were essentially negative. The
secondary galactosidase conjugated anti-rabbit immunoglobulin reagent did
not react with ANEC-conjugate coated wells and the immunoglobulins of the
antisera did not react with wells which were only backcoated with
ovalbumin.
The anti-ANEC-BSA serum (diluted 1:1000) was equally well inhibited by
ANEC-BSA, and by the ANEC-TR and -TH conjugates. Inhibition levels of 50%
were obtained for all ANEC conjugates at about 1 .mu.M concentration
calculated on the basis of the ANEC concentrations of the ANEC conjugates.
Using ANEC-BSA coated wells and the anti-ANEC-TR serum, the three
different ANEC carrier conjugates resulted in inhibition curves showing
50% inhibitions at a tenfold lower concentration (0.1 .mu.M) in comparison
to the anti-ANEC-BSA serum (1.0 .mu.M concentration yielding 50%
inhibition). The results indicate that the anti-ANEC-TR antibodies are of
slightly higher affinity than the anti-ANEC-BSA antibodies. The
ANEC-conjugates of poly-lys-phe and poly-lys-tyr also yielded similar
inhibition curves. The approximately equal inhibitions obtained with the
three ANEC proteins and two ANEC polypeptide conjugates (data not
included) indicated that the assay conditions measured only antibodies to
the ANEC haptenic group and were not apparently greatly dependent on the
amino acid sequence adjacent to the ANEC adduct.
The anti ANEC-BSA sera were also inhibited by the hapten
3-amino-N-ethyl-carbazole. Inhibition of 50% was obtained at a
concentration of 10 .mu.M and 70% inhibition was observed a 1 mM
concentration. The insolubility of 3-amino-N-ethyl-carbazole in aqueous
buffer at concentrations above 1 mM did not allow experiments in which
complete inhibition was observed.
When the anti ANEC-BSA or -TR sera were tested against BSA- or TR- coated
wells, respectively, substantial antibody binding was observed
demonstrating that the antisera also contained antibodies to the carriers
(BSA or TR). These results indicate that the carrier proteins did not lose
all their antigenicity as a result of photolabeling. The anti-BSA carrier
reactivities were effectively blocked in the assays of the anti-ANEC
specificity by the high concentrations of BSA in the buffer used for
dilution.
The results of the foregoing examples show that the photolabeling technique
of the present invention may be used to couple derivatized primary
aromatic amines to carrier proteins in order to elicit an antibody
response against the hapten. Further, the results illustrate the
simplicity and speed with which a haptenic group can be attached to a
carrier protein using the procedure of the present invention. Conventional
methods, for example the diazocoupling and isocyanate methods for
conjugating proteins involve alkaline pH conditions and longer reaction
times for coupling, whereas the photolabeling procedure of the present
invention may be carried out at physiological pH and the procedures
including isolation of the conjugates can be completed in a matter of a
few hours. Thus, the method of the present invention eliminates keeping
the hapten and protein at alkaline pH overnight for completion of the
reaction, which is one of the major disadvantages of the conventional
methods. Further, the conventional methods exhibit specific functional
group limitations which may be difficult to overcome. Additionally, the
method of the present invention produces small amounts of waste products
when compared to the diazocoupling and isocyanate coupling procedure and
is therefore useful for producing antibodies against primary aromatic
amine carcinogens.
It will be apparent to those skilled in the art that while only certain
embodiments are set forth herein, alternative embodiments and various
modifications, both of materials and methods, are apparent from the above
description and examples and are considered equivalents.
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