Note: Descriptions are shown in the official language in which they were submitted.
~ 3~75~3
ENHANCEMENT OF ANTIGEN IMMUNOGENICITY
The present invention relates to a novel procedure
for immunization against disease, and/or the generation
of antibody responses ln vivo without the use of adju-
vants and to novel vaccine materials and conjugates usedin the procedure.
In the conventional method of protection against
disease by vaccination, the central principle involves
the injection or ingestion of a material that will
elicit antibodies against said material, such that, when
challenged later with a pathogenic organism containing
homologous material, the individual to whom the material
has been administered is protected against the patho-
genic disease. The materials injected or ingested ~hat
have this property of eliciting antibodies are called
antig~ns.
It ha~ been known for many years that the immun-
ogenicity (that is, its ability to elicit antibodies) of
an antigen can be improved by the addition of so-called
adjuvants. In some cases, materials that apparently
have little or no immunogenicity have been made to make
high titxes of antibody in in vivo systems by the
addition of an adjuvant. Some of these adjuvants are,
-i however, highly toxic and can cause ~andesirabl~ side
effects or lesions. Aluminum hydroxide or aluminum
phosphate are routinely used as adjuvants for human
vaccines, but have been known to cause lumps at the site
of injection. Freund's complete adjuvant (FCA), a
mixture of a light petroleum oil and killed Myobacter-
ium tuberçulosis, can often produce excellent titres for
materials which do not normally give antibodies ln vivo
under any other conditions. Unfortunately lesions will
often develop at the site
,
~.,
~: , ., : ..
.,.. ~, . , . ~ .
.
~.
2 1 327523
of injection when using this material, making the
procedure unacceptable for human use.
There are a number of other materials which have
been investigated for use as adjuvants. In published
European Patent Application No. 0149581, the use of
muramyl di-peptide is disclosed. In published United
Kingdom Patent Application No. 2,053,231, the use of a
synthetic adjuvant consisting of tetrapeptides or
pentapeptides is disclosed. In earlier patents, the
use of oil emulsions is disclosed as having an
adjuvanting effectO Unfortunately, it appears that
the better a material is at behaving as an adjuvant to
antigen immunogenicity, the worse are the side
effects. For example, it has been shown ~hat muramyl
dipeptide is an excellent adjuvant but appears to have
a number of undesirable properties which prevent its
use in human vaccines.
In yeneral, all the adjuvants presently in use in
mammals fall into two distinct types. The first type
involves the so-called "depot" effect and the second
type depends on general immunological stimulation of
I the system under study. 'rhe adjuvants which rely on
! the depot effect are believed to bring the immune
cells to the antigen site, where the depot effect
relies on the injected antigen being trapped or
insolubilized in a medium, giving sustained
circulating levels. The second type of adjuvant,
involving general stimulation of the immune system,
appears to rely on an inflammatory reaction resulting
in a series of cells being stimulated, such that any
; antigen has an improved chance of eliciting
antibodies.
Current theories of immunology suggest that, in
order to provide a potent antibody response, an
antigen must be seen by both B cells, which
; subsequently develop into the antibody producing
cells, and also by helper T-cells, which provide
growth and differentiation signals to the antigen
specific B-cells. Helper T-cells recognize the
,: , , ' '
. .
.
: - , . , ~ ' , , ' ' . , , :
1 327523
antigen on the surface of antigen presenting cells
(APC) in association with Class II major histocom-
patibility complex (MHC) gene products.
The prasent invention involves a new method of
- 5 immunization which does not involve the use of unaccep-
table adjuvants. The present invention, in one aspect,
provides a novel immunization method and takes
advantage of the fact that both B-cells and ~PC of the
macrophage lineage express Class II ~C gene products.
Instead of injecting antigen in the presence of
;adiuvant/ the antigen is coupled to a monoclonal
antibody specific for a surface structure of antigen-
presenting cells, including Class II MHC determinants
and is injected in this form. By coupling it to the
antigen, the monoclonal antibody is converted into a
"vector" or "delivery vehicle" for targeting the
antig~n onto khe relevant cells in the immune system.
In effect, the method brings the antigen to the
antigen-presenting cells rather than the other way
around, as is thought to be the case when antigen is
injected in the presence of Freundts complete adjuvant.
,The present invention also provides, in another
'~aspect, a nov~l vaccine for utilization in the novel
imm~nization method comprising a conjugate of an antigen
~25 and a monoclonal antibody specific for a gene product of
;the major histocompatibility complex or other surface
ructure of antigen~presenting cells in a suitable
carrier medium. The present invention, in a further
aspect, provides the novel conjugate.
The present invention provides a new method of
enhancing the immunogenicity of weak antigens, which
-~ does not involve the use of unacceptable adjuvants, is
therefore much safer and can utilize materials that are
not normally very antigenicO An example of such
materials are small peptides which are epitopes of
larger proteins or are protein subunits of the pathogens
:~'
:... ~'
1 327523
themselves. Epitopes are parts of a larger antig~n
which represent the immunologic~l recognition sites
within khe structurQ. The vaccination method of the
invention, th~refore, can avoid the injection o killed
or attenuated organisms, which can have unfortunate side
effects, by the use of such epitopes or protein subunits
in the ~orm of conjugates with targeting monoclonal
antibodiesO
In the present invention, an antigen, against which
it is desired to raise antibodies in the body, is
coupled or bonded to a monoclonal antibody, which is
specific for a particular surfacP structure of antigen-
presenting cells, including a genP product of th~ major
histocompatibility complex (MHC~. This arrangement
~ 15 allows for a concentration of the antigen to the
; relevant cells in the immune system upon injection of
the coupled material. The derivatis~d antibody,
. there~ore, act~ as a "vector" or '9delivery vehicle" for
targeting antigenic determillants onto the macrophage
cells or B-cells, thereby facilitating their recogni-
tion by the T-helper cells. The presentiny cells
; possess a variety of specific cell surface structures or
markers which are targeted by the particular monoclonal
antibody, including Cla~s I and Class II hi~tocompati-
bility gene products. Thus, antigens may be coupl~d to
a monoclonal antibody specific for any of the surface
structures on the antigen presenting cells, including
Class I and Class II MHC gene produc~s.
-. The term "conjugate" as used herein is employed in
its normal s nse that the two molecules are joined
together in some convenient manner to provide a stable
structure suitable for in vivo administration.
The molecules may be joined together to ~orm the
conjugate by bonding u~ing t~chniques known to those
skilled ih the art, with the bonds being of any
, ,
, ' '
5 1 3275~3
suitable structure, including covalent bonding or
af f inity bondingO
One specific example of a conjugate use~ul in the
present invention is that of the antigenic protein
avidin and the monoclonal antibody specific for the I-Ak
Class II major histocompatibility determinants. It is
under-stood that this Pxample is but sne application of
the general principle of the present invention discussed
abov~O The preparative procedures describ~d below are
illustrative for the specific conjugate. The general
principles of such procedures, however, may be employed
to provide conjugates of any desired antigenic molecule
and any desired monoclonal antibody to provide the
desired antigen-antibody conjugate molecule for in vivo
administration to achieve the desired adjuvant-
independent serological response.
In this speci~ic illust:rative example, monoclonal
antibodies specific for the I-Ak Class II major
histocompatibility dete~minants may be prepared from a
suitable hybridoma cell line. To permit easy
conjugation with other p:roteins, the monoclonal
, antibodies preferably are biotinylated, prior to
exposure to the appropriate protein antigen, in this
example avidin. Avidin binds with high affinity to the
~5 biotinylat~d antibody, ~nd has previously been charac-
terized as an antigen. Varying doses o~ this conjugate
were used to treat a strain of mic~ expressing the I-Ak
determinants. Mice of a dif~erent strain expressing
only I-Ab determinants were used as controls. The
re~ults o~ the~e expariments, given in detail in the
Examples below, showed that avidin, when presented as an
antigen in this manner (i.e., conjugated to tha Mab oP
I-A~ Class II MHC) and in very low ~uantities can be
: made highly immunogenic in the absence of a standard
adjuvantO I~ ~quivalent quantities oP free avidin were
mixed with non-biotinylated antibody (so that no conju-:
1 327523
gation occurred), no antibody response was elicited,
indicating that the result was conjugate dependant.
Recognition of the I-Ak determinants was essential,
since avidin conjugated to an antibody of irrelevant
specificity was not e~fective in eliciting an antibody
response. In other experiments, alsu detailed in the
Example below, a monoclonal antibody that is speci~ic
~or the Class I MHC conjugated to avidin also showed an
immunogenic response and generated antibodies to avidin.
The conjugates which are employed in the present
invention are not limited to conjugates of a single
antigen with the mono lonal antibody but include
conjugates where two or mor~ antigenic proteins or
peptides are conjugated to the monoclonal antibody, to
provide a multivalent conjugate which can elicit an
antibody response to each of the conjugated antigens.
As specific examples, biotinylated monoclonal
antibody may be conjugated to avidin and bovine serum
albumin or to avidin and a Herpes synthetic peptide.
These conjugates were formed and used to immunize mice.
The Herpes peptide is part of the Herpes simplex virus
glycoprotein ~. In these experiments, reported in
-detail in the Examples below, it was found that anti-
bodies were raised against bovine serum albumen and the
Herpes peptide, as well as against the avidin when the
;lrespective conjugates were tested. Replacement of the
;~avidin by another protein, namely streptavidin, which
also has high binding affinity to biotin, provided a
~good response, indicating that the targeting e~fect is
;30 not restricted to avidin and that there was no
serological cros~-reaction of anti~streptavidin
antibodies with avidin.
It has also been shown in experiments reported in
the Examples below that the response of mice to cumula-
tive injections of a conjugate consisting of antibody-
avidin-peptide is equivalent to the response using
' ' .
. ' '. ' ' ., ' '
:
~'
1 327523
6 a
Freund's complete adjuvant. This latter experiment
indicates that vaccines can be constructed using peptide
antig~ns that elicit antibodi2s that can be neutralising
and protective again~t the pathogen from which the
5 peptide i~ extracted, without the necessity for
adjuvants.
It is evident from the abo~e that the invention may
be used as a method of immunization in vivo and in vitro
for making hybridomas, and thus specific monoclonal
antibodiesO Such specific antibodies have a wide-range
of use both in medicine and immunology.
In the following specific Examples, the efficacy of
the invention is demonstrated for three immunologically-
unrelated protein antigens, namely avidin, streptavidin
and bo~ine serum albumin. In addition, the use o~ the
m~thod resulted in generation o~ IgG antibodies against
a synthetic peptide from the glycoprotein D of herpes-
;, simplex virus. In addition, an immunogenic r~sponse has
been achieved using MAbs against Class I and Class II
determinants of MHC. This data demonstrates the gener-
ality of the invention in application to the use of any
conjugate of an antigen and a monoclonal antibody
specific for surface structures on APC.
The inve~tion is illustrated by the following
Examples. In the Examples below, reference is made to
the accompanying drawings, wherein:
:
,i .
~` 1 327523
Figure 1 contains graphical representations,
labelled A to D, of the anti-avidin response~ in mice in
one series of experiments described in the Examples
below;
Figure 2 is a graphical representation of secondary
anti-avidin responses in mice in another series of
experiments described in the Examples below;
Figure 3 contains graphical representations of
anti-bovine serum albumin responses in mice in a further
series of experiments described in the Examples below;
Figure 4 contains graphical representations of
secondary antibody responses to a synthetic Herpes
peptide in mice in another series of experiments
described in the Exa-mples below;
Figure 5 conkains graphical representations of
secondary antibody responses to streptavidin and avidin
in mice in a yet further series of experiments described
in the Examples below; and
Figure 6 contains graphical representations of
antibody responses to avidin in mice in onP more series
of experiments described in the Examples below.
` Example I~
This Example illustrates the preparation of
biotinylat~d monoclonal antibodies and conjugates
; 25 thereof with avidin.
Hybridoma cell lines TIB-92 ~anti-I-Ak) and HB-65
(anti-influenza A NP, the control antibody) were
obtained from the American Type Culture Collection
(ATCC~, RocXville, Maryland, U.S.A. and were cultured
30 in RPMI 1640 culture medium, supplemented with 10% fetal
calf serum, L-glutamine and antibiotics. The antibodies
produced by these cell lines are both of the IgG2a
subclass, and were purified from culture supernatants by
con~entional protein A-Sepharose chromatography.
The purified antibodies then were biotinylated
using the general method described by Goding in
~ 3275~3
"Monoclonal Antibodies: Principles and Practice"
(1983) Academic Press, London. For this method, the
antibodies were dialysed against O.lM sodium bicarbonate
solution at p~ 8.2, and concentrated to
. ~
i;
`''
;,
.; .
''
; ~ , ;,
:~ :.
: ~ .. : .,.: , , .. :
;. ' -- : i .
.. . ...
~` 8 1 327523
1.0 mg/ml immediately before use. Biotin succinimide
ester (Sigma Chemical Co~) was dissolved in dime~hyl
sulphoxide to a concentration of lmg/ml, immediately
before use. 60 ~1 of this ester solution were added
per ml of antibody solution at room temperature and
left for 1 hour. The biotinylated protein was
dialysed overnight against phosphate buffered saline
(PBS) and stored at 4C.
; The biotinylated antibody prepared as just
described was mixed with avidin in a ratio of 2 moles
of antibody to 1 mole of avidin to form a biotinylated
antibody-avidin conjugate for each of the antibodiesO
Exam~le II:
This Example illustrates the immunogenic response
of the conjugates prepared as described in Example I
in mice.
Two different strains of mice (namely (B6 x
C3H)Fl and B6 in groups of three were injected with
the conjugates prepared as described in Example I as
well as a mixture of antibody and avidin and of avidin
alone according to the schedule contained in the
following Table I:
'
~,'
~:'
.
~,
.
'',
- 9 1 327523
TABLE I Immunization Protocol
Priming avidin Adjuvant
GroupPriming Antigendose ~ug) (FCA)
k
5 I ~bio-anti~I-A )- 5
Avidin*
. - do - 50
. k
IIanti-I-A + Avidin 5
- do - 50
~, III (bio-anti-NP)- 5
: Avidin
- do - 50
.,' 15
:. IV Avidin 5 +/
, - do ~ 50 +/
. ; . ..... _ . .
. Notes: * = Bio~inylated
: 20~ = In Group III, antigen was injected only
into (B6 x C3HjFl mice
NP = nucleoprotein
'~ FCA = Freund's complete adjuvant
-, The injections consisted of subcutaneous
i 2S inoculations of 0.2ml of the various solutions and
^, after 21 days mice in all the groups were boosted with
an intraperitoneal injection of 5~g of avidin in 0.2ml
. of PBS. Nine days later the mice were bled from the
retro-bulbar sinus and their sera stored at 4C.
30The immune sera were tested for anti-avidin
activity as follows: Avidin solution (50ul) at a
concentration of 20ug/ml was added to the wells of a
polyvinyl microtitre plate and incubated for 1 hourr
~: after which the plates were washed. The plates then
were incubated with a 1~ solution of bovine serum
albumin (BSA) in PBS for an additional 1 hour. The
BSA-buffer was removed and 50~1 of serial dilutions of
the immune sera in 1~ BSA-PBS were added to each well
; and incubated for 1 hour. The plates were then washed
' ''
.~ .
,
o 1 327523
three times with 1~ BSA PBS and then each well treated
with 50ul of I-protein A for 1 hour. The plates
then were washed twice with buffer and the individual
wells counted in a gamma counter.
The anti-avidin response of immune sera were
plotted graphically and appear as Figure 1. In this
Figure 1, the anti-avidin responses of the immune sera
from groups I, II, III and IV (Table I~ respectively
are shown in A, B, C and D. The open circle line is
the response in (B6xC3H)Fl mice while the closed
circle line is the response in B6 mice.
As may be seen from the data presented in Figure
1, at the 5 ~ug dose of avidin, a significant response
was observed in (B6xC3H)Fl mice injec~ed with
(anti~I-A )-avidin conjugate (Fig. lA, open circles)
whereas the B6 mice (Fig. lA, closed sircles), which
do not have the particular surface antigens for which
the antibody was made, were not appreciably sensitized
Isee Fig. lA~. This result cannot be attributed to an
~ immuno-stimulating effect of the antibody alone, since
the mixture of 5 ~g of avidin with unmodified
k
anti-I-A MAb did not elicit a response (Fig. lB). An
equal amount of avidin coupled to the control anti-NP
Ab also failed to generate an appreciable response
~`~ 25 (Fig. lC), indicating that the positive response shown
in Fig lA is due to more than a simple conjugation of
avidin to an antibody. As expected 5 ~g of avidin
injected with Freund's complete adjuvant induced a
strong serological response ~Fig. lD). At the 50 ~g
of avidin dose, free avidin in the absence of adjuvant
failed to stimulate a response (Fig. lB), but in the
form of (bio-anti-I-Ak)-avidin, the conjugate
sensitized both (B6xC3H)Fl and B6 mice (Fig. lA).
Responses in the B6 mice are likely a reflection of
the elevated reactivity of the avidin-MAb conjugate on
~ the B6 targets and may be attributed either to
- cross-reactivity of the conjugated MAb or more
efficient APC uptake of the MAb-avidin complex.
,,
.:
.
, . .
: ~ :
~.
-~-`" 11 1 327523
These results demonstrate that a low dose of
avidin (i.e., 5 ~g) can be made immunogenic in the
absence of adjuvant when presented to the immune
system coupled to a monoclonal antibody specific for
recipient class II major hisocompatibility
determinants, namely the anti-I-Ak MAb.
The test procedure was repeated using a different
anti-Class II MAb, in this case an anti-I-E as the
targeting MAb. The results obtained were plotted
graphically and are reproduced in Figure 2. As may be
seen ther~in, individual (B6xC3H)Fl mice (open
squares) immunized with the (anti-I-Ek)-avidin
conjugate gave a 7 to 9 fold higher response than the
B6 mice (closed squares) that received the same dosage
of conjugate. Unconjugated mixtures of avidin and the
MAb gave only background levels of reactivity and this
data is not presented.
Example III-
This Example illustrates the use of
antibody-biotin-avidin con~ugates to raise antibodies
to a third party protein.
A conjugate of anti~ Ak antibody, avidin and
bovine serum albumin (B';A) was formed by mixing
equimolar amounts of biotinylated monoclonal antibody
(prepared as described in Example 1), avidin and
biotinylated bovine serum for 20 minutes at 20C. The
mixture then was centrifuged for five minutes at
12,000g to remove any precipatable aggregates. Mice
were injected subcutaneously with this complex
(approx. 30 ~g BSA) in a similar regime to those in
Example II, and, after three weeks, were boosted with
an intraperitoneal injection of lO~g of free BSA in
PBS Sera from the mice were obtained nine days later
and assayed in a manner similar to the samples in
Example II. The blocking buffer in the radioimmune
assay contained 0.1~ ovalbumin in PBS and 65,000 cpm
of 125I-Protein A were added to each assay well.
l'he results obtained were plotted graphically and
; appear as Figure 3. The open circle lines define the
.j
~'
,:
.,.; .
,: ~ , . .
': ~ . - - `:`
,~ ~, , , : .. .. .
-` -
. .,
12 1 327523
response in individual (B6xC3H)Fl mice while the
closed circle lines represent the response in B6 mice.
No response was observed in mice injected with an
equivalent dose of avidin in an unconjugated mixture
of non-biotinylated Mab and avidin and this data is
not presented. As may be seen in Figure 3~ a
considerable response to bovine serum albumin was
observed. These data indicate the practicality of
obtaining a serological response to any third party
protein simply by coupling it to the MAb-avidin
complex. This would allow for the preparation of any
number of adjuvant free subunit protein vaccines, that
would avoid problems connected with the parent
organism of the subunit proteinD
lS Example IV~
This Example illustrates the use of
antibody-biotin-avidin conjugates to raise antibodies
to a small peptide.
A biotin mediated conjugate was made between
2~ anti-I-Ak and avidin to which the Herpes synthetic
peptide was coupled. The Herpes peptide used was an
~,N~terminal peptide (12-20) from glycoprotein D of
;,Herpes simplex virus and was photocoupled to avidin
using the benzoyl-benzoyl-glycine linker moiety with a
molar ratio of the peptide to avidin of 8:1. The
peptide was synthesized and consisted ~f nine amino
acids. After a course of three weekly injections of
30 lug of the anti-I-A -avidin peptide conjugate into
(B6x3H)Fl mice/ the mice were boosted once with
avidin-(herpes peptide) intraperitoneally and then
sera were collected nine days after the booster
injection. The results are shown in Fig. 4. The
illustrated results represent the net antibody
`response to Herpes peptide after subtracting the
;~35 binding on the control samples and compared to the
~`~response obtained with a single priming in Freund's
complete adjuvant ~FCA) followed by the same booster
-' injection.
,~:
~:,
,~ . .
,- ::: ,. ..
~ 3~7523
13
Example V
This Example illustrates the use of antibody-
biotin-streptavidin complexes to raise antibodies to
streptavidin.
Streptavidin is a protein which was extracted from
StrePtomyces avidinii. This protein material was
conjugated with the anti-I-Ak biotinylated antibody
following the procedure generally described above in
Example I and the conjugate was injected into (B6xC3H~F
and B6 mice, in the manner described in Example II.
Se~a from these mice responded to streptavidin but were
negative when assayed against avidin. These results
appear graphically for individual mice in Figure 5.
These results show that the targeting effect is not
restricted to avidin, but is obtained with another
serologically non-cross biotin-binding active protein.
Examle VI:
This Example illustrates the enhancement of avidin
immunogenicity using monoclonal antibodies specific ~or
Class I MHC's.
Using a method similar to Example I, avidin was
coupled to biotinylated anti-Kk monoclonal antibody.
This m~terial ~30 ~y) was used to prime female C3H
(H-2k~ or B6 (H-2b) mice. Three weeks later the mice
were bled to obtain preboost sera and then subsequently
were boosted with avidin. The results appear in Figure
6. As may be seen therein the C3H mice (open triangles)
gave a higher anti-avidin response than B6 mice (closed
triangles), indicating that there was specific delivery
of avidin only in mice expressing the Kk determinants.
These results illustrate that it is possible that any
monoclonal antibodies directed against APC determinants
can mediate antigen delivery even when these
determinants are not restricted only to APC.
In summary o~ this disclosure, the present
invention provides ~ novel method of vaccinating
~`
'... ~
: ' .
:
1 3~7523
14
ma~mals by the conjugation of antigens, which may be in
the form of synthetic epitopes or protein subuni~s, to
monoclonal antibodies specific for antigen presenting
cells, such that these antigen-antibody con~ugates may
be used to elicit a beneficial antibody response without
needing to use deleterious adjuvants. Modifications are
possible within the scope of this inYentiOn.
~,
~,
,,
:,
~`
~'`'
~,~. ,.: ' ,
.. . . . . .
:~." " ' , ' ' '~
.
~"'~" ' ' ',
1 327523
SUPPLEME TARY DISCLOSURE
In the parent disclosure, there is described a new
method o~ enhancing the immunogenicity of weak antigens
by conjugation to a monoclonal an~ibody specific for a
particular surface structure of antigen presenting
cells.
The surface structures on the antigen presenting
cells o~ the immune system which can he recognized and
targeted by the monoclonal antibody portion of the
immunoconjugate are numerous and the specific such
surface antigen structure targeted by the monoclonal
antibody depends on the specific monoclonal antibody.
The monoclonal antibody may be provided specific
for a gene product of the MHC, and, in particular, may
be specific ~or class I molecules of MHC or for class II
molecules of MHC, as specifically described in the
principal disclosure. However, the invention is not
limited to such ~pecific surface structures and the
conjugates containing the corresponding monoclonal
antibodies but rather, as de~cribed in the principal
disclosure and zs will be apparent to those skilled in
the art, the invention is applicable to any other
convenient surface structure of antigen presenting
cells which can be recognized and targeted by a
specific monoclonal antibody to which an immunogenic
molecule is conjugated.
In accordance with this Supplementary Disclosure,
and as set forth in the additional Examples below,
strong adjuvant-independent serological responses to a
delivered antigen have been obtained with conjugates
formed with dendritic cell-specific monoclonal antibody
and CD4 cell-specific monoclonal antibody. In fact,
for the latter m4noclonal antibodies responses to the
delivered antigen of about 10 to 20 fold greater than
observed for the anti-class II MHC anti~odies.
:
'
;
, ~
, L~
,~
: '- , .
1 3~75~3
16
In the present invention, the monoclonal antibody
specific for the target structure is provided in the
form of a conjugate with an antigen against which it is
desired to elicit an immune respons~. The invention is
particularly useful with antigen molecules which
normally possess a weakly-immunogenic response, since
that response is potentiated by the present invention.
The antigen molecule may be in the form of a peptide or
protPin, as discussed above, in the principal
disclosure7 but is not limited to such materials.
Antigenic carbohydrates and other antigenic materials
may be employed in accordance with this Supplement~ry
Disclosure.
In the principal ~isclosure, there is described the
antibody response of a conjugate of avidin and the MAb
to I-Ak Class II MHC. As d~monstrated in th further
Examples below, considerably further enhanced antibody
response was obtained using a dendritic cell-specific
monoclonal antibody and a CD4-specific monoclonal
antibody.
The invention is illustrated further by the
following additional Example:
Example VII:
This Example illustrates the enhancement of avidin
immunogenicity using monoclonal antibodies specific for
differing antigen-presenting cell surface makers.
FQ110Wing the procedures generally described in the
above Examples, experiments were conducted to compare
the adjuvant-independent serological responses obtained
; 30 to the protein antigen avidin when conjugated to
;different biotinylated monoclonal antibodies and
injected in saline into mice~
Monoclonal antibodies specific for different cell
surface maker~ were purified by affinity chromatography
on Protein G-Sepharose ~trademark) from their respective
:,
, ~ .
. . . , ~,
: : ., ~
,~ '
~ 3275~3
17
cell culture supernatants (all cell lines were obtained
from ATCC).
The purified antibodies were biotinylated by
reaction with an N-hydroxysuccinimide dexivative of
biotin and then conjugated to monovalent avidin, made
functionally monoval~nt by adding biotin to fill three
out of four biotin binding sites in the tetramer. The
con~ugates between the different biotinylated
antibodies and monovalent avidin were formed by mixing
the two components at equimolar ratios.
Three different mice strains, namely C3H(H-2k),
C57B1/6(H-2b) and (C3HxC57Bl/6~Fl~H-2bxH-2k), were
injected with the different immunoconjugates according
to an immunization protocol of a single suhcutaneous
injection with the immunoconjugate (containing 10 ug of
avidin), followed 3 weeks later by a single intraperi-
toneal boost with 10 ug of avidin only in saline. Mice
were bled 1 week post-boost and the sera analyzed for
avidin-specific IgG responses~
The responses observed for the different immuno-
targ~ting antibodies are set forth in the following
Table II, as arbitrary units of response in an enz~me-
linked immunoassay relativ~ to a standard curve
established with a monorlonal anti-avidin antibody.
:i
,, .,
.,
',
,
~" ,~
., ~, ~_,,
.
., ,,, . ~,... .
. . .
1 3275~3
18
.~
Z I . ~ o ~a
O ~ a
~_ V ~ Z ~
~ ~ ~. t~
~ ~ ~ ~ ~ a~ 3 2~ ~r
6- ~ ~ t~ ~ ~ ~
~ ~ o ~ o ~ o
æ ~ ~
, _ 1~i9 ~ ~ g~9 N t` S`J C~ J
I_ IL ~U 0 ~~t O ~ 1~ ~ S~
Z ~ .,~ ~ ~ ~ ~ ~ o
CC ~ ~ . qt
~ ~~ ~
t~ ~ a.
U __ Z
. ~, 3~ ~ Qc~ ~c!~ ~ .~
:1 ~ O ~ ,o ~ . ~ ~
@ @ ~ r I ~ k I -c c
, . . .
'
:
" ~ . , ", , ~, . . ........ ..
1 327523
19
As expected from the findings reported in the
previous Examples, the two anti-class II major histo-
compatibility complex antibsdies (anti-I-Ak and anti-I-
Ab) promote significant responses in the (C3H x B6~ F1
animals, reflecting allele specific immunotargeting.
In addition, two other antibodies promoted strong
adjuvant-independent serological responses, namely the
dendritic specific monoclonal antibody TlB 227 and the
monoclonal antibody TlB 207 recognizing the CD4 marker
in the ~ouse. The immune response observed for these
two ~ntibodies is approximately 10 to 20 fold greater
than that observed for the anti-Class II MHc antibodies.
The data presented in this Example serve to
illustrate that immunospecific targeting of a protein
antigen to both MHC and non-~HC sur~ace markers or cells
in the immune system promotes strong adjuvant
independent serological responses to the delivered
antigen.
.,
.
:1
,
.`i`
;'
: '
,
,. 1
.,
~,
B
