U.S. patent application number 17/067691 was filed with the patent office on 2021-01-28 for proteins modified with (amino) monosaccharide-biotin adduct.
The applicant listed for this patent is GAVISH-GALILEE BIO APPLICATIONS, LTD.. Invention is credited to Elina AIZENSHTEIN, Tal GEFEN, Soliman KHATIB, Jacob PITCOVSKI, Jacob VAYA.
Application Number | 20210023217 17/067691 |
Document ID | / |
Family ID | 1000005139142 |
Filed Date | 2021-01-28 |
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United States Patent
Application |
20210023217 |
Kind Code |
A1 |
PITCOVSKI; Jacob ; et
al. |
January 28, 2021 |
PROTEINS MODIFIED WITH (AMINO) MONOSACCHARIDE-BIOTIN ADDUCT
Abstract
A protein, e.g. an antibody, coated with a non-immunogenic
molecule selected from an amino-monosaccharide-biotin adduct or a
monosaccharide-biotin adduct is disclosed, wherein the coated
protein, which has diminished immunogenicity relative to the
uncoated protein and intact biological activity, enables, for
example, cross-species vaccination.
Inventors: |
PITCOVSKI; Jacob; (Korazim,
IL) ; VAYA; Jacob; (Mizpe Amoka, IL) ; KHATIB;
Soliman; (Kfar Rajar, IL) ; AIZENSHTEIN; Elina;
(Arad, IL) ; GEFEN; Tal; (Karmiel, IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
GAVISH-GALILEE BIO APPLICATIONS, LTD. |
KIRYAT SHMONA |
|
IL |
|
|
Family ID: |
1000005139142 |
Appl. No.: |
17/067691 |
Filed: |
October 11, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14932040 |
Nov 4, 2015 |
10821179 |
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17067691 |
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13376070 |
Mar 12, 2012 |
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PCT/IL2010/000446 |
Jun 6, 2010 |
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14932040 |
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61184113 |
Jun 4, 2009 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 47/551 20170801;
A61K 39/44 20130101; A61K 47/549 20170801 |
International
Class: |
A61K 39/44 20060101
A61K039/44; A61K 47/55 20060101 A61K047/55; A61K 47/54 20060101
A61K047/54 |
Claims
1. A method of reducing the immunogenicity of an immunogenic
protein, the method comprising linking said immunogenic protein to
a non-immunogenic protein selected from an amino-mannose-biotin
adduct and a mannose-biotin-adduct, wherein said biotin is selected
from biotin or a biotin-like molecule, thereby reducing the
immunogenicity of an immunogenic protein.
2. The method of claim 1, wherein said linking is covalent
linking.
3. The method of claim 1, wherein an amino group at position 1, 2,
3, 4 or 6 of said amino-mannose molecule of a hydroxyl group at
position 1, 2, 3, 4 or 6 of said mannose molecule is linked to a
carboxyl group of said biotin.
4. The method of claim 3, wherein said linked to a carboxyl group
is direct linkage.
5. The method of claim 3, wherein said linked to a carboxyl group
forms an amide bond or ester bond.
6. The method of claim 1, wherein said biotin-like molecule is
selected from desthiobiotin and lipoic acid.
7. The method of claim 1, wherein said immunogenic protein is
linked to an amino-mannose-biotin adduct.
8. The method of claim 7, wherein said amino-mannose-biotin adduct
is selected from a 2-amino-mannose-biotin adduct, a
2-amino-mannose-desthiobiotin adduct and a 2-amino-mannose-lipoic
acid adduct.
9. The method of claim 1, wherein said immunogenic protein is an
immunogenic antibody.
10. The method of claim 9, wherein said immunogenic antibody is
selected from the group consisting of (i) a humanized or chimeric
monoclonal IgG antibody; (ii) a mammalian monoclonal IgG antibody;
(iii) a mammalian polyclonal IgG antibody; and (iv) a chicken IgY
antibody.
11. The method of claim 10, wherein said chicken IgY antibody is an
anti-bacterium or anti-virus chicken IgY antibody.
12. The method of claim 9, wherein said immunogenic antibody is an
anti-tumor associated antigen antibody, an anti-snake venom
antibody, an anti-virus antibody or an anti-bacterium antibody.
13. The method of claim 1, wherein said immunogenic protein is
linked to a plurality of non-immunogenic proteins.
14. The method of claim 13, comprising linking said plurality of
non-immunogenic proteins to said immunogenic protein to produce a
ratio of between 4:1 and 24:1 non-immunogenic proteins: immunogenic
protein.
15. A method of producing a pharmaceutical composition with
decreased immunogenicity, the method comprising producing a protein
with reduced immunogenicity by a method of claim 1 and placing it
is a pharmaceutical composition.
16. A method of treating a condition amenable to treatment by an
immunogenic protein in a subject in need thereof, the method
comprising reducing the immunogenicity of said immunogenic protein
by a method of claim 1 and administering said protein with reduced
immunogenicity to said subject, thereby treating a condition.
17. The method of claim 16, wherein said immunogenic protein is an
immunogenic antibody.
18. The method of claim 17, wherein said immunogenic antibody is
selected from the group consisting of (i) a humanized or chimeric
monoclonal IgG antibody; (ii) a mammalian monoclonal IgG antibody;
(iii) a mammalian polyclonal IgG antibody; and (iv) a chicken IgY
antibody.
19. The method of claim 18, wherein said chicken IgY antibody is an
anti-bacterium chicken IgY antibody.
20. The method of claim 17, wherein said immunogenic antibody is an
anti-tumor associated antigen antibody, an anti-snake venom
antibody, an anti-virus antibody or an anti-bacterium antibody.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 14/932,040 filed on Nov. 4, 2015, which is a
continuation of U.S. patent application Ser. No. 13/376,070 filed
on Mar. 12, 2012, now abandoned, which is a National Phase of PCT
Patent Application No. PCT/IL2010/000446 having International
filing date of Jun. 6, 2010, which claims the benefit of priority
under 35 U.S.C. .sctn. 119(e) of U.S. Provisional Application No.
61/184,113, filed on Jun. 4, 2009. The contents of the above
applications are all incorporated by reference as if fully set
forth herein in their entirety.
TECHNICAL FIELD
[0002] The present invention relates to proteins modified with
monosaccharide-biotin adducts, and in particular to modified
antibodies with reduced antigenicity.
BACKGROUND ART
[0003] Antibodies have been used for the prevention and treatment
of infectious diseases for over a century. Antibody therapy is used
in the modern arsenal of antimicrobial therapeutics (Keller and
Stiehm, 2000; Oral et al., 2002; Buchwald and Pirofski, 2003) and
in the therapy of viral diseases (Keller ant Stiehm, 2000; Law and
Hangartner, 2008). Additionally, new applications were suggested
for passive immunization, e.g. in treatment of neurological
disorders and cancer. Furthermore, antibodies are still a superior
therapeutic choice for toxin neutralization and remain a critical
component of the treatment for diphtheria, tetanus, botulism and
snake envenomation.
[0004] Passive immunization can be achieved by intravenous (i.v.)
or intramuscular (i.m.) administration of antibodies as plasma or
serum, as pooled immunoglobulin from immunized or convalescing
donors, and as monoclonal antibodies. There are several obstacles
in utilization of passive immunization in human medicine: 1. The
treatment requires antibodies of the same species in order to avoid
anti-isotype immune reaction; 2. shortage of suitable hyperimmune
donors; 3. batch-to-batch variations; 4. the risk of pathogen
transmission; 5. production cost of sufficient quantities of high
quality antibodies; and 6. the occurrence of serum sickness.
[0005] Obviously, the cheapest and most available source to produce
antibodies is animal plasma or serum. However, passive vaccination
with antibodies extracted from animal serum is inefficient due to
their antigenicity and possible adverse effects, such as the
potentially fatal anaphylactic shock and serum sickness. In some
cases, the solution to this problem has been to use fragmented
(F(ab')2 or Fab) immunoglobulins (IgG) or humanized antibodies.
Despite significant progress in minimizing immune responses they
still occur, even against fully human antibodies. Additionally,
such modifications involve time-consuming research and development,
and are limited to the identification of monoepitopes.
[0006] Reduction of protein immunogenicity, alteration of the
protein's surface properties and increase of the plasma half-life
is presently achieved mainly by Polyethylene Glycol (PEG) (for
example Gaberc-Porekar et al., 2008) and Dextran or Dextran
derivatives of various molecular weights (for example (Kobayashi et
al., 2001; Mehvar, 2003).
[0007] The use of mannose or oleic acid and mannose for obtaining a
modified protein or viruses with maintained antigen binding and
decreased antigenicity relative to unmodified protein or viruses,
respectively, has been described previously by the inventors (WO
2006/070371). Harris et al., 2003 discloses proteins modified with
linoleic acid and linoleic dicarboxylic acid, and Ong et al., 1991
have reported galactose modified antibodies that are quickly
cleared from the blood via the asialoglycoprotein receptor.
SUMMARY OF INVENTION
[0008] The present invention provides a protein, such as an
antibody, covalently linked to a non-immunogenic molecule selected
from an amino-monosaccharide-biotin adduct or a
monosaccharide-biotin adduct. In certain embodiments, the protein
is an antibody covalently linked to 2-amino-mannose-biotin
adduct.
[0009] The invention further relates to pharmaceutical compositions
comprising said antibodies. In one embodiment, the pharmaceutical
composition comprises an anti-tumor associated antigen antibody
such as Trastuzumab. In other embodiments, the pharmaceutical
composition comprises an anti-virus antibody or anti-bacterium
antibody or an anti-snake venom antibody.
BRIEF DESCRIPTION OF DRAWINGS
[0010] FIG. 1 shows an SDS-PAGE gel of hIgG coated with different
amounts of amino-mannose-biotin adduct (MBA). Equal amounts of hIgG
(1 mg in 1 ml of 25 mM PB, pH 6 were reacted with 20 (lane 1), 80
(lane 2), 300 (lane 3) or 1000 (lane 4) .mu.g of MBA for 3 h and
changes in hIgG size after modification with MBA were determined.
Lane 5, control hIgG without modification. M, molecular weight
markers.
[0011] FIG. 2 shows detection of coated hIgG with secondary
antibodies by western blot analysis. Equal amounts of hIgG were
reacted with 80, 300 or 1000 .mu.m MBA. The samples were subjected
to 15% SDS-PAGE and recognition of the coated hIgG was determined
by applying secondary antibody on a western blot.
[0012] FIG. 3 shows the immune response in chickens to hIgG coated
with different amounts of MBA. Coated/uncoated hIgG was injected
i.m., and Ab production was examined by ELISA. 20 .mu.g, Ab against
hIgG from chickens injected with hIgG-MBA.sup.20 (20 .mu.g of
MBA/mg hIgG); 80 .mu.g, Ab against hIgG from chickens injected with
hIgG-MBA.sup.80 (80 .mu.g of MBA/mg hIgG); 300 .mu.g, Ab against
hIgG from chickens injected with hIgG-MBA.sup.300 (300 .mu.g of
MBA/mg hIgG); 1000 .mu.g, Ab against hIgG from chickens injected
with hIgG-MBA.sup.1000 (1000 .mu.g of MBA/mg of hIgG); hIgG, Ab
against hIgG from chickens injected with uncoated hIgG; Neg.
control, Ab against hIgG in non-injected chickens. Inset: serum
dilutions. Results are shown as bars.+-.SD. ***P.ltoreq.0.0003,
**P.ltoreq.0.001 or *P.ltoreq.0.02 for the difference in Ab level
against hIgG between chickens treated with coated and uncoated
hIgG.
[0013] FIGS. 4A-B shows the immune response in chickens to hIgG
coated with MBA or PEG. Injection of coated hIgG with (A) and
without Freund's adjuvant (B). Antigens were injected i.m. and Ab
production against hIgG was examined by ELISA. MBA, Ab against hIgG
from chickens injected with hIgG-MBA.sup.1000; PEG, Ab against hIgG
from chickens injected with hIgG-PEG; hIgG, Ab against hIgG from
chickens injected with uncoated hIgG; Neg. control, Ab against hIgG
in non-injected chickens. Inset: serum dilutions. Representative
results of two experiments (one repeat in experiment 4A and three
repeats in 4B) are shown as bars.+-.SD. ***P.ltoreq.0.0003 or
**P.ltoreq.0.002 for differences in Ab level against hIgG between
chickens treated with hIgG-MBA.sup.1000 and hIgG. **P.ltoreq.0.001
or *P.ltoreq.0.03 for the differences in Ab level against hIgG
between hIgG-PEG- and hIgG-treated chickens.
[0014] FIG. 5 shows the immune response in chickens to molecules
used for the various coated hIgGs. Antigen was injected i.m. and Ab
against molecules used to coat the various modified hIgGs were
examined by ELISA. MBA, Ab against MBA from chickens injected with
hIgG-MBA; PEG, Ab against PEG from chickens injected with hIgG-PEG;
hIgG, Ab against hIgG from chickens injected with uncoated hIgG;
Neg. control, Ab against coated or uncoated hIgG from non-injected
chickens. Inset: serum dilutions. **P.ltoreq.0.002 for the
differences in Ab level against hIgG between sera from chickens
injected with hIgG and sera from non-treated chickens. Results are
presented as bars.+-.SD.
[0015] FIGS. 6A-B show immunological response to IgY and IgY
modified with PEG or MBA in mice, following two IV (A) or IM (B)
injections. Neg. Con.--Non-injected mice.
[0016] FIGS. 7A-B show the ability of MBA masked molecules to
reduce humoral immune response in different mouse strains. Horse
IgG (hsIgG), hsIgG-MBA or hsIgG-PEG was injected into Balb/c (A) or
C57BL6 mice (B) i.m. or i.v. three times, at a 2-week interval.
Immunized mice sera were tested by ELISA, using unmodified hsIgG as
antigen. The titer was determined as the reciprocal value of 2 fold
serial dilution end point (.times.2 of negative control OD value),
of tested serum. Results are shown as bars.+-.SD. ***P<0.001,
**P<0.01 or *P<0.05 for the difference in antibodies titer
against horse IgG between mice treated with masked and unmasked
horse IgG.
[0017] FIG. 8 shows immune response against IgG following
sequential dose treatment. Horse IgG, IgG-MBA or horse IgG-PEG were
injected into Balb/c mouse i.m. or i.v. three times during three
days, giving a total amount of 200 .mu.g of antigen per mouse.
Immune response was evaluated after two weeks. Results are shown as
bars.+-.SD. ***P<0.001 for the difference in Ab level against
horse IgG between mice treated with masked and unmasked horse
IgG
[0018] FIG. 9 shows recognition of antigen-bound hIgG-MBA.sup.1000
by monocytic THP-1 cells through their Fc receptor. An ELISA plate
was coated with tetanus toxin antigen, followed by incubation with
anti-tetanus toxin hIgG or hIgG-MBA.sup.1000. Then, fluorescent
THP-1 cells were added to the wells for various periods of time.
The fluorescence level was determined by fluorometer. hIgG,
monocyte cell binding efficiency to uncoated hIgG;
hIgG-MBA.sup.1000, monocyte cell binding efficiency to coated hIgG
(1 mg hIgG reacted with 1000 .mu.g of MBA); Neg. control, tetanus
toxin incubated with fluorescent monocytic cells without hIgG.
Inset: incubation times. *P.ltoreq.0.05 for the difference between
monocyte binding efficiency to hIgG vs. hIgG-MBA.sup.1000.
Representative values of three independent experiments are shown as
bars.+-.SD
[0019] FIG. 10 shows a hemagglutination test in which inhibition
activity of coated IgY is demonstrated. IgY, uncoated antibodies;
PEG, IgY coated with PEG; MBA, IgY coated with MBA.
[0020] FIG. 11 shows determination of multivalent antigen detection
by coated/uncoated anti-venom. Two .mu.g of venom per lane were
subjected to Western blot, and reacted with 20 .mu.g IgG and
developed with mouse anti horse IgG-horseradish peroxidase (HRP)
conjugate (lane 1) or reacted with 20 .mu.g IgG-MBA and developed
with avidin-HRP conjugate (lane 2). MW, molecular weight (kDa).
[0021] FIG. 12 depicts the effect of masking an antibody on its
binding affinity. 10 .mu.g IgG HRP conjugate were mixed with
various concentrations (100 .mu.g/ml-97 ng/ml) (X axis) of IgG,
IgG-MBA or IgG-PEG. The mixes were incubated in an ELISA plate
coated with venom as antigen. The plate was developed with OPD and
OD monitored at 450 nm. Representative values from three
independent experiments are shown.
[0022] FIG. 13 shows in vitro venom inhibition. Inhibition of venom
proteolitic enzyme, tested by azocoll. The dye monitored by optical
density reads at 550 nm and is represented as percent of
inhibition. Pre-incubated venom with hsIgG, hsIgG-MBA or hsIgG-PEG
were added to azocoll for 2 hours incubation. Results are shown as
bars.+-.SD, for the difference between 3 independent tests.
[0023] FIG. 14 shows in vivo venom inhibition. 2.times.LD50 doses
of venom were pre-incubated with IgG or masked IgG and injected
i.v. into balb/c mice. Percent of survival represent mouse which
survived 48 hours after injection. Results are shown as means of 4
independent tests. Each symbol represent the percent survival of a
group of 6 mice.
[0024] FIG. 15 depicts an experiment measuring the effect of
coating of Trastuzumab (referred to as herceptin) with MBA on the
binding affinity of the antibody using a competitive ELISA. 10
.mu.g Trastuzumab-fluorescein conjugate were mixed with various
concentrations (100 .mu.g/ml-97 ng/ml) (X axis) of herceptin or
herceptin-MBA. 10 .mu.g herceptin-fluorescein conjugate were mixed
with various concentrations (100 .mu.g/ml-97 ng/ml) (X axis) of
herceptin, herceptin-MBA or herceptin-PEG. The mixes were incubated
in 96 wells tissue culture, optic bottom, plate coated with fixated
SKBR-3 cells as antigen. The fluorescein units were detected and
shows as Mean Fluorescent Units (MFU). Results are shown as XY
point's.+-.SD. Representative values from three independent
experiments are shown.
[0025] FIG. 16 depicts the effect of coated/uncoated anti-HER2
(referred to as herceptin) on the viability of HER2+SK-BR3 or BT
474 breast cancer cells. Cells were grown with 10 .mu.g/ml of
coated/uncoated herceptin and compared to cells alone (cells alone
correspondent to 100% viability). Results are shown as scatter
plot, each point represents a single independent experiment and
horizontal line represents mean values.
DETAILED DESCRIPTION OF THE INVENTION
[0026] One of the problems encountered in the administration of
foreign molecules to an organism is their antigenicity. Foreign
molecules, e.g., antibodies, that are produced in another species
and used for passive vaccination, induce an immune response in the
host and cannot be used repeatedly.
[0027] The present invention provides masked proteins, preferably
antibodies, with decreased or eliminated antigenicity. Modified
antibodies according to the present invention enable cross-species
vaccination. For example, therapeutic murine, chimeric or humanized
monoclonal antibodies or human monoclonal antibodies that cause an
immune response in a human host may be modified according to the
present invention, thereby preventing, diminishing or eliminating
the undesired immune responses. In another example, modified
chicken- or horse-derived polyclonal antibodies against snake
toxins can be used for vaccination of humans to treat snakebite
envenomation.
[0028] In contrast to major published studies wherein proteins were
masked using synthetic polymers or polysaccharides as masking
agents, the present application discloses small endogenous
molecules for masking the proteins or the viruses. The use of
mannose and oleic acid for obtaining a modified protein or virus
with an intact native binding site and decreased antigenicity has
been described in WO 2006/070371 by the same inventors. In the
present invention, the inventors set out to label the mannose with
biotin, another endogenous molecule, in order to be able to follow
the labeled protein, for example in order to establish the
half-life of the modified protein after injection into an animal.
It was a very surprising finding of the present invention that the
amino-mannose-biotin adduct (MBA) was much more effective in
reducing the immunogenicity than the unlabeled mannose molecule,
especially since biotin, which is extensively used in the art of
labeling proteins, has not been shown to reduce immunogenicity of
proteins. As detailed in the Examples below, the biotin was, for
technical reasons, linked to the mannose via its 2-amino group.
However, it could obviously also have been linked via one of the
hydroxyl groups.
[0029] Thus, in one aspect, the invention relates to a protein,
which is covalently linked to a non-immunogenic molecule selected
from an amino-monosaccharide-biotin adduct or a
monosaccharide-biotin adduct. The terms
"amino-monosaccharide-biotin" and "monosaccharideamine-biotin" are
used interchangeably herein.
[0030] The monosaccharide moiety of the amino-monosaccharide-biotin
adduct or the monosaccharide-biotin adduct of the invention may be,
but is not limited to, ketoses or aldoses of 3-6 carbon atoms, for
example aldoses of 5-6 carbon atoms, in particular mannose. All
mannoseamine isoforms are considered, such as a 1-amino-mannose,
2-amino-mannose, 3-amino-mannose, 4-amino-mannose, 5-amino-mannose,
and 6-amino-mannose, and more preferably 2-amino-mannose.
Similarly, the mannose-biotin adduct may be a mannose-2-biotin,
mannose-3-biotin, mannose-4-biotin, mannose-5-biotin, or
mannose-6-biotin isoform. In certain embodiments, the
amino-monosaccharide-biotin adduct is 2-aminomannose-biotin.
[0031] The masking agent is preferentially reacted with functional
groups of the protein to form covalent bonds. For example,
amino-mannose-biotin, can react with the protein free carboxylic
acid residues of aspartic or glutamic acid to form esters, or with
the free amine groups of lysine or arginine. Such options allow
controlling the desired degree of masking by choosing the type of
amino acids residues of the protein or the virus surface to be
bound, in order to reach the desired and most suitable reduction of
the degree of immunogenicity. Moreover, the agent selected for
masking the protein surface may be attached to molecules which
target specific cells or tissues, and thus the modified protein,
e.g. modified antibody, may be used to deliver this agent to the
desired specific target.
[0032] Masking of the protein with amino-mannose-biotin can be
performed, for example, in two steps: first, the free amino groups
on the protein surface are masked by reaction with the aldehyde
moiety of amino-mannose, followed by addition of a coupling reagent
such as 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), to
facilitate esterification of the free carboxyl groups on the
protein surface with the mannose hydroxyl groups (Hermanson, G. T.
(1995). Bioconjugate Techniques, Academic Press, Inc). In this way,
two functional groups of the amino acid residues of the protein or
the virus--amino and carboxyl groups--are protected to a degree
that is related to the reaction conditions such as type of solvent
used (protic, aprotic, polar, etc), type and amount of
esterification catalyst used, e.g., EDC, p-toluenesulfonic acid
(pTSA), and/or 4-(dimethylamino) pyridine (DMAP), the ratio between
the reactants (protein/monosaccharide), reaction time, etc. The
double bond of the imine group of the Schiff's base formed in the
first step can be further reduced, for example, with NaH.sub.3BCN,
in order to increase the stability of the masked molecule.
[0033] In certain embodiments, the protein that is masked with,
i.e. covalently linked to, a non-immunogenic molecule of the
invention is an antibody. In certain embodiments, the masked
antibody of the invention is (i) a humanized or chimeric monoclonal
IgG antibody; (ii) a mammalian monoclonal IgG antibody; (iii) a
mammalian polyclonal IgG antibody; or (iv) a chicken IgY
antibody.
[0034] Indeed, it has been found in accordance with the present
invention that chicken IgY, mouse, horse and human IgG and
humanized monoclonal IgG antibodies can all be coated with MBA
while retaining their binding affinity and biological activities
and eliciting reduced immunological responses in foreign species as
compared with uncoated antibodies. Thus, any antibody can be masked
with MBA for the purpose of decreasing its immunogenicity. For
example, Table 1 exhibits examples of currently FDA approved
antibodies for treatment of a variety of diseases. Certainly these
antibodies, but also other antibodies not mentioned here, can be
coated with MBA and are therefore included in the scope of the
present invention.
[0035] In particular, the antibody of the present invention is
selected from an anti-tumor associated antigen antibody, an
anti-snake venom antibody, an anti-virus antibody or an
anti-bacterium antibody. For example, the anti-tumor antibody may
be an anti-HER2 receptor HER-2/neu (human epidermal growth factor
receptor-2) antibody, for example Trastuzumab which is shown in
Example 11 to retain its HER2-binding properties when coated with
MBA. However, the anti-tumor associated antigen antibody may be
directed to any tumor associated antigen such as, but not limited
to, alpha-fetoprotein, BA-46/lactadherin, BAGE (B antigen), BCR-ABL
fusion protein, beta-catenin, CASP-8 (caspase-8), CDK4
(cyclin-dependent kinase 4), CEA (carcinoembryonic antigen),
CRIPTO-1 (teratocarcinoma-derived growth factor), elongation factor
2, ETV6-AML1 fusion protein, G250/MN/CAIX, GAGE, gp100 gp100
(glycoprotein 100)/Pmel 17, intestinal carboxyl esterase, KIAA0205,
MAGE (melanoma antigen), MART-1/Melan-A (melanoma antigen
recognized by T cells/melanoma antigen A), MUC-1 (mucin 1), N-ras,
p53, PAP (prostate acid phosphatase), PSA (prostate specific
antigen), PSMA (prostate specific membrane antigen), telomerase,
TRP-1/gp75 (tyrosinase related protein 1, or gp75), TRP-2,
tyrosinase, and uroplakin Ia, Ib, II and III.
[0036] The anti-snake venom antibody may be an antibody to C. atrox
venom or viper venom. Both antibodies, when masked with MBA, are
shown hereinafter to evoke a
TABLE-US-00001 TABLE 1 FDA-approved monoclonal antibodies for
cancer treatment Main category Name of drug Type of cancer used to
treat Anti cancer Alemtuzumab Chronic lymphocytic leukemia
(Campath) Bevacizumab Breast cancer; Colon cancer; Lung cancer
(Avastin) Cetuximab Colon cancer; Head and neck cancers (Erbitux)
Gemtuzumab Acute myelogenous leukemia (Mylotarg) Ibritumomab
Non-Hodgkin's lymphoma (Zevalin) Panitumumab Colon cancer
(Vectibix) Rituximab Non-Hodgkin's lymphoma (Rituxan) Tositumomab
Non-Hodgkin's lymphoma (Bexxar) Trastuzumab Breast cancer
(Herceptin) Anti- infliximab Rheumatoid arthritis, Crohn's disease,
inflammatory ulcerative colitis adalimumab Rheumatoid arthritis,
Crohn's disease, ulcerative colitis etanercept Rheumatoid arthritis
basiliximab Acute rejection of kidney transplants daclizumab Acute
rejection of kidney transplants omalizumab Moderate-to-severe
allergic asthma Other palivizumab Respiratory syncytial virus
infections in children abciximab Prevent coagulation in coronary
angioplasty Sources: FDA and wikipedia
diminished immunological response in a host injected with these
antibodies as compared with native antibodies, and to bind as
efficiently and neutralize snake venom as well as
[0037] unmasked antibodies in vitro and in vivo. The anti-viper
antibody studied in the present invention is directed to Vipera
palaestinae serum. An anti-influenza virus antibody coated with MBA
is shown herein in Example 8 to be as efficient as an uncoated
antibody in inhibiting hemagglutination of red blood cells.
[0038] Thus, in certain embodiments the anti-tumor associated
antigen antibody is an anti-HER2 receptor antibody, the anti-snake
venom antibody is an anti-C. atrox venom antibody or an anti-viper
venom antibody, and the anti-virus antibody is an anti-influenza
virus antibody. In particular, the anti-HER2 receptor antibody is
Trastuzumab.
[0039] It has been found in accordance with the present invention
that the immunogenicity of an IgG molecule is reduced if at least
about 4 amino-mannose-biotin adduct molecules are covalently
attached to each IgG antibody molecule. It has further been found
that an increase in the molar ratio of amino-mannose-biotin
adduct:IgG further reduces the antigenicity of the antibody, such
that about 9-10 amino-mannose-biotin adduct molecules per IgG
provide improved masking, i.e. further reduced immunogenicity, and
11-12 amino-mannose-biotin adduct molecules per IgG molecule
provide optimal masking and almost completely abolishes the
immunogenicity of the antibody. Thus, in one embodiment the ratio
of amino-mannose-biotin adduct to IgG, or monosaccharide-biotin
adduct to IgG antibody, is 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1
or 12:1, i.e. between about 4:1 and about 12:1. Since the number of
free carboxyl groups in a protein is approximately similar to the
number of amino groups in the same protein, and since carboxyl
groups may easily be transformed to amino groups as detailed above,
the number of MBA molecules that may be bound to an IgG antibody is
twice that shown herein. Therefore, the ratio of
amino-mannose-biotin adduct to IgG may be as high as about
24:1.
[0040] In certain embodiments, the molar ratio of
amino-mannose-biotin adduct to IgG antibody, or
monosaccharide-biotin adduct to IgG antibody, is between about 11:1
to about 12:1.
[0041] It is expected that the biotin moiety of the
amino-monosaccharide-biotin adduct or monosaccharide-biotin adduct
may be replaced with biotin-like molecules without affecting the
ability of the adduct to reduce immunogenicity of proteins.
Non-limiting examples of biotin-like molecules are diaminobiotin
and desthiobiotin. Also molecules comprising an ureido
(tetrahydroimidizalone) ring fused with a tetrahydrothiophene ring
which is found in biotin, or analogs thereof such as those found in
diaminobiotin and desthiobiotin, that are linked to the
monosaccharide via a spacer replacing the valeric acid moiety of
biotin, are considered. The spacer may be for example a hydrocarbyl
group of 1 to about 50 carbon atoms in length, optionally
interrupted by one or more heteroatoms selected from O, S or N, or
one or more aromatic rings, or polyethylene glycol or a peptide of
similar length. In addition biotin can be replaced by lipoic acid
or lipoic acid derivatives and linked to monosaccharide or
amino-monosaccharide.
[0042] As used herein, the term "antibodies" refers to polyclonal
and monoclonal antibodies of avian, e.g. chicken, and mammals,
including humans, and to fragments thereof such as F(ab').sub.2
fragments of polyclonal antibodies, and Fab fragments and
single-chain Fv fragments of monoclonal antibodies. The term also
refers to chimeric, humanized and dual-specific antibodies.
[0043] The present invention further relates to a pharmaceutical
composition comprising a protein according to the invention, and a
pharmaceutically acceptable carrier. This pharmaceutical
composition may be used for both prophylactic as well as
therapeutic purposes
[0044] As stated above, it has been shown herein that many kinds of
antibodies can be masked with MBA without detrimental consequences
to the binding or biological activity properties of the antibodies.
Thus, the present invention is directed to any antibody approved
for therapeutic use for treating a disease such as cancer,
neurological disorders inflammation-related disease, autoimmune
disease, an infectious disease or any other disease or disorder
(see Table 1 for non-limiting examples), and masked with MBA.
[0045] In certain embodiments, the pharmaceutical composition of
the present invention is for treating a cancer selected from breast
cancer, chronic lymphocytic leukemia, colon cancer, head and neck
cancers, lung cancer, acute myelogenous leukemia and non-Hodgkin's
lymphoma. In particular, the pharmaceutical composition is for
treating breast cancer and comprises an anti-HER2 receptor
antibody, for example Trastuzumab.
[0046] In certain embodiments, the pharmaceutical composition of
the present invention comprises an anti-virus antibody or
anti-bacterium antibody. Thus, the pharmaceutical composition, in
certain cases, comprises a vaccine for passive immunization in
humans or animals against bacteria or for toxin neutralization in
diphtheria, tetanus intoxication, botulism and snake envenomation,
and they may be also useful for passive vaccination in humans or
animals against viruses such as influenza, ebola, hepatitis
respiratory syncytial virus, and avian influenza virus. In
particular, the anti-virus antibody is an anti-influenza virus.
[0047] In another embodiment, the pharmaceutical composition
comprises an anti-snake venom antibody, in particular an anti-C.
atrox venom antibody or an anti-viper venom antibody.
[0048] The non-immunogenic molecules of the invention may also be
applied to proteins associated with the surface of a virus,
preferably with the viral capsid or with the envelope, thus
rendering the virus less immunogenic and with intact binding to its
natural binding receptors.
[0049] Adenovirus (Ad) is a group of nonenveloped double-stranded
DNA viruses associated with a range of respiratory, ocular, and
gastrointestinal infections. Entry of human Ad into human cells is
a stepwise process. The primary event in this sequence is
attachment that involves an interaction between the Ad fiber
protein and its high-affinity cellular receptor. The Ad type 5
(Ad5) fiber is a homotrimer with each subunit consisting of three
domains: the amino-terminal tail that associates with the penton
base protein; the shaft, which consists of a motif of approximately
15 residues that is repeated 22 times; and the knob, which
interacts with the cellular receptor.
[0050] A replication-defective adenovirus vector has been used for
efficient delivery of DNA and is applicable in adenovirus-mediated
gene delivery in gene targeting and gene therapy.
[0051] The present invention also contemplates a modified hormone
such as parathyroid hormone (PTH) or human growth hormone (hGH)
with a functional receptor-binding site, wherein the hormone
surface is masked with non-immunogenic molecules, such as
amino-mannose biotin, except for the protected receptor-binding
site, and said masking provides the hormone with prolonged
half-life in the body.
[0052] A further protein that may benefit from reduced
immunogenicity is an enterotoxin such as the enterotoxin of
Escherichia coli (LT) with a functional GM1 ganglioside
receptor-binding site, or the cholera toxin of Vibrio cholera (CT)
with a functional GM1 ganglioside receptor-binding site. The
modified enterotoxin may be useful for delivery of molecules into
cells via oral or skin routes.
[0053] The invention will now be illustrated by the following
non-limiting Examples.
EXAMPLES
[0054] Materials and Methods
[0055] Materials. Human (h) IgG was purified from the whole serum
of patients immunized with tetanus toxoid (TT). 2-aminomannose,
NaCNBH.sub.3, MPEG-NHS (methoxy polyethyleneglycolsuccinate
N-hydroxysuccinimide) and biotin were purchased from Sigma-Aldrich.
Biotin-NHS was purchased from Pierce. Amicon ultra-centrifugal
filter devices (MWCO 10,000 and 30,000) were purchased from
Millipore. Trastuzumab was purchased from Roche.
[0056] Synthesis of amino-mannose-biotin (MBA). 2-Aminomannose (70
mg, 0.39 mmol) was dissolved in 0.5 ml DMSO and biotin-NHS (100 mg,
0.3 mmol) was added. The solution was stirred at room temperature
(RT) for 2 h. A new peak was formed as detected by HPLC at a
retention time of 14 min (for details see HPLC analysis). The new
peak was isolated and purified by flash chromatography (silica gel,
methanol:ethyl acetate, 5:95 as solvents). Using conventional
analytical methods, the pure product was identified as MBA. The
LC-MS of the product, using positive ion monitoring mode
(ES.sup.+), revealed the expected molecular ion m/z of 406
(M+H.sup.+) and fragmentations with m/z of 364.6 and 249.5,
identical to previously reported data (Lin, Chun-Cheng et al.,
Tetrahedron lett. 1997, 38, 2649).
[0057] HPLC Analysis of MBA. The HPLC was connected to a diode
array detector (HP-1100) and equipped with a reverse-phase column
(C-18, 150 mm length; 4.6 mm diameter with 5 .mu.m particles). The
mobile phase was a mixture of acetonitrile and water which was run
at a flow rate of 1 ml/min with the following gradient:
acetonitrile from 1% to 5% over 5 min, and then to 20% for another
5 min and finally to 98% for an additional 10 min.
[0058] LC/MS/MS Analysis of MBA. The product was injected into MS
in a direct injection with scan, using the ESI method. The source
temperature of the MS was set at 150.degree. C., with a cone gas
flow of 22 l/h, a desolvation gas flow of 400 l/h and a capillary
voltage of 3.5 KV. Peak spectra were monitored between 30 and 800
m/z.
[0059] Coating of hIgG Antibody with MBA. MBA (1 mg, 2.5 .mu.mol in
60 .mu.l DMSO) was added to hIgG (1 mg, 6.6 nmol in 1 ml of 25 mM
phosphate buffer (PB), pH 6). The solution was mixed for 1 h at RT,
and then NaCNBH.sub.3 (2 mg, 32 .mu.mol) was added and the reaction
was continued for an additional 2 h at RT. Excess MBA reagent was
discarded from the reaction solution by filtration through an
Amicon ultra-centrifugal filter device with a MWCO of 10,000.
[0060] Controlling the Coating Reaction (MBA/hIgG Ratio). Various
amounts of MBA (20, 80, 300 and 1000 .mu.g) from a stock solution
of 25 mg in 1 ml DMSO were added to hIgG (1 mg hIgG in 1 ml of 25
mM (PB), pH 6) to monitor the ratio of MBA/hIgG (coating/coated
ratio). The solution was mixed for 1 h at RT and different amounts
of NaCNBH.sub.3 (30, 120, 450 and 1500 .mu.g from a stock solution
of 17 mg NaCNBH.sub.3 in 1 ml PBS) were added, respectively. The
solution was left for another 2 h at RT. The excess MBA was then
removed from the reaction mixture by filtration through an Amicon
ultra-centrifugal filter device (MWCO 10,000). This filtrate was
then taken for further analysis.
[0061] Coating hIgG with Methoxy-PEG-NHS. MPEG-NHS (7 mg) was added
to hIgG (1 mg in 1 ml PBS pH 7.4). The solution was mixed for 3 h
at RT and then excess reagent was removed by filtration through an
Amicon ultra-centrifugal filter device (MWCO 30,000). The filtrate
was then taken for further analysis.
[0062] Number of Unbound Free Amino Groups in the Coated and
Uncoated Protein. The following procedure was based on a previous
work (Vidal and Franci, 1986) with some modifications. Briefly, the
coated and uncoated hIgG were reacted with TNBSA
(trinitrobenzenesulfonic acid or picrylsulfonic acid). Under mild
conditions, this reagent reacts specifically with free amino groups
on the amino acid side chain of a protein to give trinitrophenyl
(TNP) derivatives. Thus, 50 .mu.g of hIgG (from a stock solution of
1 mg/ml in PBS) was added to 140 .mu.l sodium tetraborate buffer
(0.1 M, pH 9.3) in a 96-well plate. Aqueous TNBSA (10 .mu.l of 0.01
M) was added and the solution was incubated for 30 min at
37.degree. C. The absorption of the solution was measured at 405 nm
in an ELISA reader (Lumitron) and the amount of free amine was
calculated from a calibration curve prepared by reacting TNBSA with
a known amount of glycine.
[0063] Gel Electrophoresis and Western Blot Analysis. To determine
changes in the size of hIgG after modification with various amounts
of MBA, the samples were analyzed by SDS-PAGE. Gel electrophoresis
was performed in a 15% polyacrylamide gel prepared in 1.5 M Tris
HCl, pH 8.8. The pellets (10 .mu.l) were mixed with loading buffer
(0.5 M Tris HCl pH 6.8, 33% glycerol, 3% SDS, 5% mercaptoethanol,
0.5% bromophenol blue) and the samples were run at RT at 50 mA in
25 mM Tris base, 20 mM glycine, 0.1% SDS. The gel was stained with
Coomassie blue or transferred to nitrocellulose for detection of
modified hIgG with secondary antibody by western blot analysis. The
membrane was blocked with PBS containing 0.5% Tween 20 and 5% dry
milk (blocking buffer) for 1 h at 37.degree. C. and incubated with
a 1:5000 dilution of horseradish peroxidase (HRP)-conjugated goat
anti-hIgG (Jackson ImmunoResearch Laboratories, Inc.) for 1 h at
37.degree. C. in blocking buffer. After washes in PBS containing
0.5% Tween 20, bands were detected by enhanced chemiluminescence
(ECL) (Pierce).
[0064] In-Vivo Trials.
[0065] 1. Vaccination of birds--The effect of masking molecules was
tested on laying hens, nine birds per group. The birds were
injected i.m. with unmodified hIgG or hIgG modified with various
amounts of MBA or PEG. Each bird was injected with 50 .mu.g
protein, twice at a 2-week interval. Blood was drawn 2 weeks after
the second vaccine injection and sera were kept at -20.degree. C.
until analysis. The presence of antibodies in the sera was tested
by ELISA using unmodified or modified hIgG as the antigen.
[0066] In addition, the immune response to hIgG or modified hIgG
mixed with Freund's complete adjuvant (FCA) was examined in
chickens. The birds, nine laying hens per group, were immunized
i.m. with 50 .mu.g of hIgG or hIgG modified with MBA/FCA or PEG/FCA
mixture. Two weeks later, birds were reinjected in the same manner
with the same amount of antigen in incomplete Freund's adjuvant
(IFA). Blood was drawn 2 weeks after the second vaccination and
sera were kept at -20.degree. C. until analysis. The presence of
antibodies in the sera was tested by ELISA using unmodified hIgG as
the antigen.
[0067] 2. Vaccination of mice--The effect of masking molecules was
tested on Balb/c mouse, six rodents per group. Six mice per group
were injected i.m. or i.v. with 50 .mu.g of unmodified or modified
horse IgG (hsIgG) with MBA or with PEG three times, at 2-week
intervals. Two weeks after each injection, blood was drawn and
serum was separated and sera were kept at/-20.degree. C. until
analysis.
[0068] For deferential dose treatment, each mouse was injected with
25 .mu.g on day one, 75 .mu.g on the second day and 100 .mu.g on
the third day, giving a total amount of 200 .mu.g per mouse. Two
weeks after the third injection, bloods were drawn and sera were
kept at/-20.degree. C. until analysis.
[0069] ELISA. The presence of antibodies against hIgG in chicken or
mouse sera following i.m. injection of unmodified hIgG or hIgG
modified with different amounts of MBA or with PEG was tested by
ELISA. Each of the following steps was followed by three washes
with 0.05% Tween-20 in PBS and drying on a paper towel. ELISA
plates (Nunc) were incubated overnight at 4.degree. C. with hIgG
diluted in carbonate-coating buffer (pH 9.6) to a final
concentration of 5 to 8 .mu.g/ml (for chicken) or 1 .mu.g/ml (for
mouse). Skim milk (5%) in PBS was added for 1 h at 37.degree. C. as
a blocking step. Then different serum dilutions were incubated for
1-2 h at 37.degree. C., followed by incubation with a secondary
antibody, rabbit anti-chicken IgG conjugated to HRP (Sigma),
diluted 1:5000 in PBS for 1 h at 37.degree. C. A substrate
solution, o-phenylenediamine dihydrochloride (Sigma), was added and
the OD.sub.450 was determined by ELISA reader.
[0070] For the determination of antibodies against coated molecules
in chicken sera following i.m. injection of modified hIgG, the sera
were tested by the procedure described above, except that the
antigen coating the ELISA plate was hIgG modified with the same
molecule (coated hIgG) that was injected into the tested
chickens.
[0071] Fluorometric Assay. To determine whether MBA-modified hIgG
is recognized by the Fc receptor in monocyte cells, the binding
efficiency of THP-1 monocytic cells (ATCC: TIB-202) to
antigen-bound modified hIgG was tested. Black maxisorp 96 flat
microwell plates (Nunc) were incubated overnight at 4.degree. C.
with TT protein diluted in carbonate-coating buffer (pH 9.6) to a
final concentration of 1 .mu.g/ml, followed by three washes with
0.05% Tween-20 in PBS (wash buffer) and drying on a paper towel.
PBS with 5% skim milk was added for 1.5 h at 37.degree. C. as a
blocking step. The plate was washed three times with wash buffer
and 100 .mu.l of hIgG or MBA-modified hIgG was added at a
concentration of 40 .mu.g/ml and incubated for 2 h at 37.degree. C.
The plate was then washed three times with wash buffer and 200
.mu.l of THP-1 cells dyed with carboxyfluorescein succinimidyl
ester (CFSE) (Invitrogen) at a concentration of 2.times.10.sup.5
cells/ml were added and incubated at 37.degree. C., 5% CO.sub.2 for
10, 30, or 60 min. At the end of the incubation, the medium and 100
.mu.l of sterile PBS was added. The fluorescence level was
determined by fluorometer (Victor3, PerkinElmer Life Sciences) at
535 nm.
[0072] Viper venom-binding capability of coated hsIgG. The binding
of the coated MBA-hsIgG to viper venom as compared to that of
unmodified hsIgG by Western Blot Analysis. Electrophoresis was
performed on 8-16% gradient polyacrylamide gel (Geba gel). The
Venom (10 .mu.l) was mixed with loading buffer (0.5 M Tris HCl pH
6.8, 33% glycerol, 3% SDS, 5% mercaptoethanol, 0.5% bromophenol
blue) and the samples were run at RT at 160 Volts in running Buffer
(Amresco). The gel was transferred to nitrocellulose membrane,
Hybound C (Amersham). Each of the following steps was followed by
three washes with 0.05% Tween-20 in PBS. Blocking buffer was added
(5% Skim milk, 0.05% Tween-20 in PBS) for 1 hour at 37.degree. C.
as a blocking. 20 .mu.g of hsIgG or hsIgG-MBA diluted in blocking
buffer were applied and the membrane was incubated for 1 hour at
37.degree. C., following incubation with a 1:5000 dilution of goat
anti-hsIgG HRP-conjugated (Sigma) or 1:2000 dilution Avidin-HRP
conjugated (Sigma) respectively, for 1 hour at 37.degree. C. in
blocking buffer. Bands were detected by enhanced chemiluminescence
(ECL) (Pierce).
[0073] hsIgG HRP conjugation. The conjugation of hsIgG to HRP was
as follow; 1 mg of anti-viper venom hsIgG was diluted in phosphate
buffer (PB) (25 mM sodium phosphate pH 5.5 mM, Sigma), and mixed
with 1 mg Adipic acid dihydrazide (Sigma), and 1 mg of
N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC)
(Fluka). The solution was stirred for 3 hours at room temperature
(RT) and then excess reagent was removed by filtration through an
Amicon ultra-centrifugal filter device (MWCO 30,000). The filtrate
was then diluted in PB and 1 mg of peroxidase (Sigma) was in
addition to 1 mg of EDC. The solution was stirred for 3 hours at
room temperature (RT) and then excess reagent was removed by
filtration as before.
[0074] Competitive ELISA. To evaluate the affinity of anti-viper
venom hsIgG-MBA to the antigen, ELISA plate was coated with 1
.mu.g/ml of viper venom. Thereafter, 10 .mu.g/ml of hsIgG-HRP
conjugated (constant hsIgG-HRP concentration) mixed with hsIgG,
hsIgG-MBA or hsIgG-PEG at serial twofold dilutions (0-100 .mu.g/ml)
and added to the plate for 1 h at 37.degree. C., followed by
substrate addition. OD values were detected at 450 nm by ELISA
reader.
[0075] To evaluate the affinity of Trastuzumab-MBA to the antigen,
3*10.sup.4 of SKBR-3 cells/well incubated for 24 h in 96 wells
tissue culture, optic bottom, plate (Nunc). Each of the following
steps was followed by three washes with 0.05% Tween-20 in PBS and
drying on a paper towel. The cells were fixated to the wells with
4% formaldehyde in PBS for 20 min at R.T. 10 .mu.g/ml of
Trastuzumab-fluorescein conjugated (NHS-Fluorescein (Pierce)) mixed
with Trastuzumab or Trastuzumab-MBA at twofold serial dilutions
(0-100 .mu.g/ml) and added to the plate for 1 h at 37.degree. C.,
followed by substrate addition. Fluorescent units values were
detected at ex/em 490/530 nm by multilable counter 1420 reader
(PerkinElmer).
[0076] In vitro viper venom proteolitic activity inhibition. The
ability of the coated or uncoated hsIgG to inhibit proteolitic
activity by venom was conducted with Azo dye-impregnated collagen
(Azocoll) (Sigma). Seventy five mg of azocoll were suspended in 50
ml of PBS, stirred for 2 h at RT and centrifuge at 10,000.times.g
for 10 min, following suspension in 50 ml PBS--This washing step
was repeated twice. 200 .mu.g of hsIgG, hsIgG-MBA or hsIgG-PEG were
incubated with 50 .mu.g of viper venom for 30 min at 37.degree. C.
following addition of 400 .mu.l washed azocoll and incubation for 2
h at 37.degree. C. The reactions were centrifuge and supernatant
transferred to 96 wells plate. OD values were detected at 550 nm by
ELISA reader.
[0077] Evaluation of venom neutralization. The LD50 value for
Vipera palaestinae venom in Balb/c mice (18-20 g) by the i.v. route
was determined by challenging unprotected mice with various doses
of crude venom in saline. The results revealed an LD50 value of 1
mg/kg body weight. The neutralizing ability of coated or uncoated
hsIgG was assessed by pre-incubation of 200 .mu.g of anti-venom
with 2 LD50 doses of the venom at 37.degree. C. for 30 min before
injecting to three groups of mice (6 in each group) through the
i.v. route. The animals were kept under observation for 48 h,
afterward number of deaths occurring within 48 h was scored.
[0078] Cell culture. Human breast cancer cell lines SKBR-3 and BT
474 were purchased from the American Type Tissue Culture
Collection. BT 474 was maintained in DMEM with 4.5 g/1 glucose, and
SKBR-3 was maintained in McCoy's 5A. All cells lines were
supplemented with 10% FBS and incubated at 37.degree. C. in a 5%
humidified CO.sub.2 atmosphere.
Example 1. Coating hIgG with MBA
[0079] The reaction of MBA with the antibody was designed to
proceed through binding to the amino acid side chains of the
protein, i.e. lysine and arginine, to form an imine bond which was
then further reduced using cyanoborohydride to form a more stable,
non-reversible type of bond. The ratio between MBA and protein was
controlled by changing the amount of MBA during its reaction with
the antibody, from 20 to 1000 .mu.g of MBA/mg antibody. The
molecular ratio of MBA to antibody in the coated protein was
monitored by detecting the amount of free amino groups left on the
protein relative to uncoated hIgG (see Materials and Methods),
using a known method with some modifications for small-scale
sampling (96 wells) (Vidal, J. and C. Franci (1986) J Immunol
Methods 86(1): 155-6). The findings from these experiments are
summarized in Table 2.
TABLE-US-00002 TABLE 2 Molecular ratio of MBA to antibody Free
amino No of free Lane no. groups.sup.1 hIgG amino in FIG. (.mu.M)
(.mu.M) groups.sup.2 1 53.84 5.355 10.05 2 53.55 5.923 9.04 3 24.57
5.481 4.48 4 13.26 5.197 2.55 5 94.42 6.490 14.55 .sup.1Based on
glycine calibration curve .sup.2Calculated
[0080] The number of accessible amino groups reacting with the
chromophore reagent TNBSA in the native hIgG amounted to 14 or 15
amino residues. Reaction of the antibody with 20 .mu.g MBA/mg
blocked 4 to 5 of the protein's amino groups, leaving the other 10
groups free. At a ratio of 1000 .mu.g MBA/mg hIgG, 11 to 12 of the
protein's amino groups reacted with the MBA and only 2 or 3 amino
groups remained unattached. Glycine was reacted with TNBSA in order
to construct a calibration curve. These results were further
confirmed by gel electrophoresis of the coated/uncoated hIgG, which
showed the same trend, i.e., a gradual increase in the MW of the
modified hIgG correlated to an increase in the number of MBAs per
hIgG molecule (FIG. 1).
[0081] We then tested secondary antibody recognition of hIgG
modified with various amounts of MBA (80-1000 .mu.g MBA/mg hIgG) by
western blot analysis. Samples (5 .mu.g) were run on a 15%
polyacrylamide gel and transferred to nitrocellulose for detection
of modified hIgG with HRP-conjugated goat anti-hIgG antibodies. The
results, summarized in FIG. 2, demonstrate the correlation between
increased number of MBAs per molecule of hIgG and reduced
recognition of modified hIgG by the secondary antibody.
Example 2. Immunogenic Response of Chicken to hIgG Coated with
Different Amounts of MBA
[0082] hIgG antibody was reacted with various concentrations of MBA
to examine the immunogenic response of chickens to different levels
of MBA coating (see Materials and Methods): 20 .mu.g MBA
(hIgG-MBA.sup.20), 80 .mu.g MBA (hIgG-MBA.sup.80), 300 .mu.g MBA
(hIgG-MBA.sup.300) and 1000 .mu.g MBA (hIgG-MBA.sup.1000) Coated or
uncoated hIgG (50 .mu.g) was injected twice into chickens i.m., at
a 2-week interval. Two weeks after the second injection, blood was
drawn and serum was separated and tested by ELISA for antibody
production against hIgG. The results, shown in FIG. 3, demonstrate
the correlation between increasing number of MBAs per molecule of
hIgG and decreasing production of antibodies against hIgG in
chickens. The immunological response against hIgG was significantly
suppressed in chickens injected with hIgG-MBA.sup.80,
hIgG-MBA.sup.300 and hIgG-MBA.sup.1000 (FIG. 3). Moreover, the
level of antibodies against hIgG in the serum of chickens injected
with hIgG-MBA.sup.1000 was similar to the negative control
(non-injected chickens), suggesting abolishment of hIgG
antigenicity. Based on these results, a 1:1 ratio (initial
weight/weight) of MBA to hIgG was chosen for further
experiments.
Example 3. Immunological Response in Chickens to hIgG Coated with
MBA Vs. PEG and Injected in the Presence or Absence of Adjuvant
[0083] PEGylation is known to suppress protein immunogenicity and
antigenicity (Kubetzko et al., 2005; Veronese and Pasut, 2005;
Pasut et al., 2006; Gamez et al., 2007). To compare the masking
ability of MBA to that of PEG, 50 .mu.g of hIgG, hIgG-MBA.sup.1000
or hIgG-PEG were injected into chickens i.m. twice, in the absence
or presence of adjuvant, at a 2-week interval. In the case of
injection with adjuvant, samples were mixed with Freund's adjuvant
to induce a maximal immune response (see Materials and Methods).
Two weeks after the second injection, blood was drawn and serum
separated and tested for antibody production against hIgG by ELISA.
hIgG-PEG was used as a control for immune response suppression to
the antigen (hIgG). FIGS. 4A and 4B shows the results for injection
in the absence or presence of adjuvant, respectively. Modification
with MBA suppressed the immune response to hIgG more effectively
than PEGylation in both cases. It is important to note that hIgG
coated with biotin or mannose alone did not prevent antibody
production against hIgG (data not shown). In the sera of chickens
injected with hIgG-MBA.sup.1000 without adjuvant, the level of
antibodies against hIgG was similar to that in the non-injected
chickens (negative control). Coating with MBA reduced the
immunogenic response against hIgG by an estimated 32-fold relative
to non coated hIgG, whereas PEGylation decreased it only fourfold
in the absence of adjuvant (FIG. 4A). Results summarized in FIG. 4B
demonstrate that hIgG-PEG injected together with adjuvant produced
an antibody titer similar to that with uncoated hIgG (positive
control) at a 1:128 and 1:256 dilution; its masking effect was only
observed at a 1:1024 dilution. On the other hand, in chickens
injected with hIgG-MBA.sup.1000 in the presence of adjuvant, the
immune response against hIgG was significantly reduced, even at low
dilution (1:128). According to our estimation, injection with
hIgG-MBA.sup.1000 decreased the immunogenic response against hIgG
32-fold while injection with hIgG-PEG decreased antibody production
only eightfold, relative to controls injected with uncoated
hIgG.
Example 4. Examination of MBA Immunogenicity in Chickens
[0084] To determine whether the MBA molecule is itself immunogenic
when conjugated to the antigen or if MBA bound to antibody induces
production of antibodies against the hIgG-MBA construct, sera from
chickens injected with hIgG, hIgG-MBA.sup.1000 or hIgG-PEG in the
absence of adjuvant (see above and FIG. 4A) were analyzed by ELISA.
Serum from chickens injected with hIgG-MBA.sup.1000 was examined on
hIgG-MBA.sup.1000, whereas serum from chickens injected with
hIgG-PEG was tested on hIgG-PEG and so on. The level of antibodies
against MBA and/or the hIgG-MBA.sup.1000 construct in serum from
chickens injected with hIgG-MBA.sup.1000 was not significantly
different from the negative control (serum from non-injected
chickens on hIgG-MBA.sup.1000) (FIG. 5). These results indicate
that, similar to PEG, neither MBA molecules nor hIgG coated with
MBA are immunogenic in chickens.
Example 5. Immune Response in Mice to Chicken IgY Coated with MBA
or PEG
[0085] Chicken immunoglobulin, isolated from egg yolk (IgY) was
coated with MBA or PEG 5000, and injected to mice intravenously
(IV) or intramuscularly (IM). Antibody response to the coated
immunoglobulin, and to the coated molecules was determined by
ELISA. Following one injection the antibody response to IgY was
reduced 4-fold (IV) and 2-fold (IM) by MBA, while the response to
PEG was increased, as compared to non-coated IgY (FIGS. 6A-B).
Example 6. Antibody Response to Coated or Uncoated hsIgG
[0086] MBA masking ability was tested and compared to both PEG 5
kDa and 20 kDa following i.m. or i.v. injection of 50 .mu.g of
hsIgG, hsIgG-MBA, hsIgG-PEG to Balb/c and C57BL6 mice three times
at a 2-weeks interval.
[0087] Two weeks after each injection, blood was drawn and serum
was separated and tested for antibody production against hsIgG by
ELISA.
[0088] In Balb/c mice, MBA was found to reduce the immune response
to hsIgG significantly as compared to uncoated or PEGylated hsIgG
in both administration routes (FIG. 7A and Table 2). In this
experiment, masking ability of PEG 5 was superior to PEG 20
molecule.
[0089] A similar effect was found in C57BL6 mice; the MBA molecule
was found to be superior to PEG in its masking capabilities and,
significantly reduced the immune response
TABLE-US-00003 TABLE 3 Folds of reduction in antibody response to
antigen due to MBA masking as compared to PEG-coated or uncoated
hsIgG. Route of First injection Second injection Third injection
administration Treatment Balb/C C57BL6 Balb/C C57BL6 Balb/C C57BL6
i.m. IgG 4 20 20 45 15 51 IgG-PEG 3 10 6 2 3 7 i.v. IgG 13 22 158
62 57 47 IgG-PEG 9 21 57 2 20 2 Humoral Immune response, in
Titer-folds reduction, of MBA coated vs. PEG 5 coated or uncoated
IgG. The reduction fold depict in two different mice inbred strains
(Balb/c and C57BL6) and in two routes of administration (i.m. and
i.v.). to hsIgG in both administration routes (FIG. 7B and Table
2).
Example 7. Immunological Humoral Response Against Anti-Venom after
Sequential Dose Treatment
[0090] In order to imitate sequential anti-venom dose treatment,
mice were injected with 200 .mu.g of hsIgG anti-venom in 3 days
(i.e. 25 .mu.g, 75 .mu.g, and 100 .mu.g a day respectively). Two
weeks after the third injection, bloods were drawn and antibody
level against hsIgG was tested by ELISA. Mouse antibody titer
against anti-venom was significantly lower in mouse treated with
masked anti-venom (FIG. 8). Coating with MBA reduced the humoral
response against hsIgG by an estimated 15 and 12 fold relative to
unmodified hsIgG in i.m. route and i.v. respectively. When
comparing to PEGylated hsIgG, coating with MBA reduced the humoral
immune response against hsIgG by 4 fold, in i.m. and i.v. injection
route.
Example 8. Activity of Modified Antibodies
[0091] 8.1 Determination of Inhibition of Snake Venom Activity by
Coated Antibody.
[0092] Snake venom is composed of several enzymes. The inhibition
of the activity of two of these enzymes involved in hemolysis and
fibrinogen degradation, was determined. The coating of anti-C.
atrox venom IgY did not affect the ability of the antibody to
inhibit hemolysis and inhibit more efficiently venom influence on
clotting time. Following masking with MBA or PEG, the anti-venom
IgY antibodies inhibit the hemolysis similar to unmodified
antibodies (Table 4) and the inhibition of fibrinogen degradation
by the antibody was increased (Table 5).
[0093] 8.2 Binding of hIgG-MBA.sup.1000 to Monocytic THP-1
Cells.
[0094] To examine recognition of coated hIgG by the Fc receptor,
the coated antibodies bound to their antigen
TABLE-US-00004 TABLE 4 Effect of anti-venom coated IgY on RBC
hemolysis S. no Sample OD 405 nm 1 Control plasma 0.315 2 Plasma +
20 .mu.g venom 0.7435 3 Plasma + 20 .mu.g venom + 1 mg IgY 0.428 4
Plasma + 20 .mu.g venom + 2 mg IgY 0.26 5 Plasma + 20 .mu.g venom +
1 mg IgY-MBA 0.4215 6 Plasma + 20 .mu.g venom + 2 mg IgY-MBA 0.283
7 Plasma + 20 .mu.g venom + 1 mg IgY-PEG 0.3185 8 Plasma + 20 .mu.g
venom + 2 mg IgY-PEG 0.2805
TABLE-US-00005 TABLE 5 Effect of anti-venom coated IgY on the
clotting time of human plasma Thrombin % change S. no Sample time
from control 1 Control plasma 21 -- 2 Plasma + 20 .mu.g venom 150
614 3 Plasma + 20 .mu.g venom + 1 mg IgY 138 557 4 Plasma + 20
.mu.g venom + 2 mg IgY 138 557 5 Plasma + 20 .mu.g venom + 1 mg
IgY-MBA 82 290 6 Plasma + 20 .mu.g venom + 2 mg IgY-MBA 80 280 7
Plasma + 20 .mu.g venom + 1 mg IgY-PEG 60 185 8 Plasma + 20 .mu.g
venom + 2 mg IgY-PEG 50 138
(TT) were tested. An ELISA plate with hIgG or hIgG-MBA.sup.1000
bound to TT antigen was incubated with fluorescent THP-1 cells for
different periods of time. Fluorescence level was determined by
fluorometer after 10, 30 and 60 min of incubation with the cells.
TT without hIgG was used as a negative control. Results, summarized
in FIG. 9, showed that the fluorescence level in wells with
hIgG-MBA.sup.1000 was similar to that in wells without TT-bound
antibodies (negative control). These results indicate that coating
hIgG with MBA fully abrogates recognition of the antibody by the
monocytic cells.
[0095] 8.3 Determination of Coated Antibody Hemmaglutination
Inhibition (HI) Activity.
[0096] Inhibition of hemagglutination of red blood cells by
anti-influenza antibodies (AIA) was found to be in correlation with
virus neutralization. AIA derived from hyperimmune sera were coated
with PEG or MBA, and tested for hemagglutination inhibition (HI).
Coated anti-influenza antibodies retain the ability of HI, but MBA
and PEG coating decrease the HI 4 and 16 fold, respectively (FIG.
10).
[0097] 8.4 Binding Capability of MBA Coated hsIgG to the
Antigen:
[0098] 8.4A Multi antigen detection by coated hsIgG. Anti venom
horse IgG is a set of polyclonal Abs against a set of venom
proteins. Coating a set of polyclonal Abs may possibly affect the
detection of particular antigen by specific Abs. To test coated
antivenom antibodies with V. palaestinae venom, different amounts
of the venom were immunoblotted with hsIgG or with hsIgG-MBA.
Immunoblotting showed detection of all major protein bands in the
venom with coated or uncoated antivenoms (FIG. 11). The results
suggest that coating with MBA, did not impair the detection of any
venom antigen. The intensity variation of the bands between the
coated and uncoated antibodies cannot be compared, due to different
detection methods by the second antibody.
[0099] 8.4B Binding affinity of hsIgG coated with MBA or with PEG.
To evaluate the binding affinity of coated horse antivenom to the
venom, competitive ELISA was conducted. ELISA plate was coated with
viper venom and incubated with constant concentration of hsIgG-HRP
mixed with twofold serial dilutions of non-modified hsIgG,
hsIgG-MBA or with hsIgG-PEG. Thereafter, the plate was incubated
with HRP substrate, followed by ELISA reader detection. According
to the results, summarized in FIG. 12, the affinity of the modified
hsIgG was slightly but not significantly lower from that of the
uncoated hsIgG. Antibodies coated with MBA and PEG show identical
affinity to antigen.
[0100] 8.4C Venom protease activity inhibition by coated or
uncoated hsIgG. To examine whether coated antivenom could inhibit
protease activity similarly to the uncoated antivenom in vitro,
Azocoll protease activity assay was performed. The assay relies on
the ability of the venom enzyme to digests dye-impregnated collagen
and by that, releasing the dye to the supernatant. 50 .mu.g of the
venom were pre-incubated with 200 .mu.g of hsIgG, hsIgG-MBA or with
hsIgG-PEG and added to Azocoll reagent. Thereafter, absorbance of
supernatants was monitored. FIG. 13 shows that coating of antivenom
hsIgG by MBA does not reduce the antivenom inhibition activity of
the antibody.
Example 9. Activity of Modified Anti-Venom Horse Antibodies In
Vivo
[0101] 9.1. Venom neutralization in vivo by coated or uncoated
anti-venom. To determine the effect of coating procedure of
anti-venom to the efficacy of V. palaestinae venom neutralization,
a neutralization test of venom lethality by hsIgG was performed in
vivo. 2.times. lethal dose (30 .mu.g) of venom were pre-incubated
with 200 .mu.g of coated or uncoated anti-venom for half an hour
and injected i.v. into Balb/c mice. Neutralization ability was
calculated from the number of deaths occurring within 48 hour
subsequent to injection. Antivenom coated with MBA did not impair
venom neutralization in vivo (FIG. 14). Rather, the group treated
with MBA coated anti-venom had lower death events within the group
although not significantly, the difference is consistent compared
to uncoated or PEG coated antivenom.
[0102] 9.2. Immunological humoral response against anti-venom,
following neutralization test. To evaluate the immune response
evoked by hsIgG antivenom, in mice which
TABLE-US-00006 TABLE 6 Immune response against IgG following
neutralization test Neutralization Anti hsIgG Anti MBA treatment
titer titer IgG 2048 -- IgG-MBA <256 <128 IgG-PEG 2048 --
Immune response was evaluated in mouse which survive neutralization
test. Blood was drawn two weeks after challenge end. Results are
from two independent tests. survived the neutralization test,
bloods were drawn two weeks after the neutralization test and sera
were separated and tested for antibody production against hsIgG by
ELISA. Masking antivenom with MBA evoked at least 10 fold lower
humoral immune response then uncoated or PEG-coated hsIgG (Table
6).
Example 10. Effect of MBA Coating on Monoclonal Therapeutic
Antibodies
[0103] In order to evaluate the effect of MBA coating on monoclonal
antibodies, we chose Trastuzumab (herceptin) as a candidate.
Trastuzumab is a humanized anti-HER2 monoclonal antibody directed
against the HER2 protein (p185HER2/neu), which is the product of
the HER2 proto-oncogene (also designated as c-erbB-2 or HER2/neu).
HER2 is overexpressed in approximately 20% to 25% of breast tumors.
This alteration is associated with poor prognosis and may affect
the response to hormonal therapy and chemotherapy. Trastuzumab
demonstrated a benefit as a single agent in first- or second-line
treatment of HER2-overexpressing (HER2+) metastatic breast cancer
(MBC) (Vogel et al 2002).
[0104] Exposure of human breast cancer cell lines, which express
high levels of endogenous HER2 receptor, to Trastuzumab, inhibit
cell proliferation.
[0105] Here, we tested the effect of MBA masking of Trastuzumab on
the antibody activity in vitro.
[0106] FIG. 15 shows a competitive ELISA in which plates have been
coated with cells expressing HER2. As can be clearly seen, the
coating of Trastuzumab with MBA did not affect its binding affinity
towards its native antigen.
[0107] The efficiency of the Trastuzumab to kill cancer cells is
not adversely affected by coating with MBA. In a viability test,
cancer cells (5.times.10.sup.3 per well) were seeded onto a 96-well
plate. The survival of these cells after treatment with
coated/uncoated 10 .mu.g/ml trastuzumab for 5 days at 37.degree. C.
in a 5% humidified CO.sub.2 atmosphere was determined using the
Cell Titer-blue Luminescent Cell Viability Assay reagent (Promega)
following the supplier's instructions. Fluorescent units was
measured by DTX 880 Multimode Detector (Beackman Coulter).
[0108] As can be seen in FIG. 16, exposure of the cells to
MBA-modified Trastuzumab reduced the viability of the cells to
about 60% as compared with 70% and 80% for unmodified Trastuzumab
and pegylated Trastuzumab, respectively. Thus, Trastuzumab-MBA
coated antibody retains its activity as compared to uncoated
Trastuzumab antibody.
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