U.S. patent application number 10/653706 was filed with the patent office on 2005-04-14 for compounds having reduced immunogenicity and a method of reducing the immunogenicity of compounds.
This patent application is currently assigned to Centocor, Inc.. Invention is credited to Jordan, Robert E., Knight, David M., Wagner, Carrie Lynne.
Application Number | 20050079183 10/653706 |
Document ID | / |
Family ID | 25525432 |
Filed Date | 2005-04-14 |
United States Patent
Application |
20050079183 |
Kind Code |
A1 |
Jordan, Robert E. ; et
al. |
April 14, 2005 |
Compounds having reduced immunogenicity and a method of reducing
the immunogenicity of compounds
Abstract
Reduced-immunogenic fusion compounds are disclosed. The fusion
compounds of the invention comprise immunogenic compounds linked to
auto-antigenic sequences which render the compound less
immunogenic. In addition, a method of reducing the immunogenicity
of an immunogenic compound is disclosed. The method comprises
linking an auto-antigenic sequence to an otherwise immunogenic
compound. Recombinant nucleotide sequence encoding auto-antigenic
sequences are also disclosed.
Inventors: |
Jordan, Robert E.; (Malvern,
PA) ; Wagner, Carrie Lynne; (Malvern, PA) ;
Knight, David M.; (Berwyn, PA) |
Correspondence
Address: |
HAMILTON, BROOK, SMITH & REYNOLDS, P.C.
530 VIRGINIA ROAD
P.O. BOX 9133
CONCORD
MA
01742-9133
US
|
Assignee: |
Centocor, Inc.
Malvern
PA
|
Family ID: |
25525432 |
Appl. No.: |
10/653706 |
Filed: |
September 2, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10653706 |
Sep 2, 2003 |
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08433441 |
Jul 6, 1995 |
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6652863 |
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08433441 |
Jul 6, 1995 |
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PCT/US93/11148 |
Nov 16, 1993 |
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10653706 |
Sep 2, 2003 |
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07977705 |
Nov 16, 1992 |
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Current U.S.
Class: |
424/178.1 ;
424/132.1; 530/387.3 |
Current CPC
Class: |
C07K 14/4713 20130101;
C07K 16/00 20130101; C07K 2319/00 20130101; C07K 14/3153 20130101;
A61K 38/00 20130101; C07K 16/46 20130101 |
Class at
Publication: |
424/178.1 ;
424/132.1; 530/387.3 |
International
Class: |
A61K 039/395; C07K
016/44 |
Claims
1. A fusion compound comprising an immunogenic compound linked to
the amino terminal of an auto-antigenic sequence, the carboxy
terminal of the auto-antigenic sequence being unlinked, with the
proviso that the fusion compound is not c7E3 Fab.
2. The fusion compound of claim 1 wherein said auto-antigenic
sequence is an IgG amino acid sequence.
3. The fusion compound of claim 1 wherein said auto-antigenic
sequence is an IgG heavy chain amino acid sequence.
4. The fusion compound of claim 1 wherein said auto-antigenic
sequence comprises amino acid sequences selected from the group
consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO: 4
r SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO:8, SEQ ID
NO:9 and SEQ ID NO:10.
5. The fusion compound of claim 1 wherein said auto-antigenic
sequence comprises SEQ ID NO:1.
6. The fusion compound of claim 1 wherein said immunogenic compound
is a protein.
7. The fusion compound of claim 1 wherein said immunogenic compound
is a non-human protein.
8. The fusion compound of claim 1 wherein said immunogenic compound
is a murine monoclonal antibody.
9. The fusion compound of claim 1 wherein said immunogenic compound
is a chimeric monoclonal antibody.
10. The fusion compound of claim 1 wherein said immunogenic
compound is Streptokinase.
11. The fusion compound of claim 1 wherein said immunogenic
compound is a monoclonal antibody and said auto-antigenic sequence
is SEQ ID NO:1.
12. A method of reducing the immunogenicity of an immunogenic
compound comprising the step of linking said compound to the amino
terminal of an auto-antigenic sequence, the carboxy terminal of the
auto-antigenic sequence being unlinked, with the proviso that the
fusion compound is not c7E3 Fab.
13. The method of claim 12 wherein said auto-antigenic sequence is
an IgG amino acid sequence.
14. The method of claim 12 wherein said auto-antigenic sequence is
an IgG heavy chain amino acid sequence.
15. The method of claim 12 wherein said auto-antigenic sequence
comprises amino acid sequences selected from the group consisting
of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID
NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID
NO:10.
16. The method of claim 12 wherein said auto-antigenic sequence
comprises SEQ ID NO:1.
17. The method of claim 12 wherein said immunogenic compound is a
protein.
18. The method of claim 12 wherein said immunogenic compound is a
non-human protein.
19. The method of claim 12 wherein said immunogenic compound is a
murine monoclonal antibody.
20. The method of claim 12 wherein said immunogenic compound is a
chimeric monoclonal antibody.
21. The method of claim 12 wherein said immunogenic compound is
Streptokinase.
22. The method of claim 12 wherein said immunogenic compound is a
monoclonal antibody and said auto-antigenic sequence is SEQ ID
NO:1.
23. The method of claim 12 wherein said auto-antigenic sequence is
linked to said immunogenic compound by chemically binding a peptide
comprising auto-antigenic sequence to said immunogenic
compound.
24. The method of claim 12 wherein said immunogenic compound is a
protein and said auto-antigenic sequence is linked to said
immunogenic protein by binding a nucleotide sequence that encodes
said auto-antigenic sequence to the 3' terminal end of nucleotide
sequence that encodes said immunogenic protein to form a chimeric
gene, expression of said chimeric gene produces a fusion protein
having said auto-antigenic sequence linked to said immunogenic
protein.
25. A recombinant nucleic acid molecule consisting a nucleotide
sequence encoding an auto-antigenic sequence.
26. The recombinant nucleotide sequence of claim 25 wherein said
auto-antigenic sequence comprises SEQ ID NO:1.
27. The recombinant nucleotide sequence of claim 25 further
comprising a nucleotide sequence encoding an immunogenic protein
wherein the 5' end of said nucleotide sequence that encodes an
auto-antigenic sequence is linked to the 3' end of said nucleotide
sequence that encodes said immunogenic protein, the 3' end of said
nucleotide sequence that encodes the auto-antigenic sequence being
linked to a termination sequence without any intervening coding
sequences, said immunogenic protein is a monoclonal antibody and
said auto-antigenic sequence is SEQ ID NO:1.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of application Ser. No.
08/433,441, filed Jul. 6, 1995, which is the U.S. National Stage of
International Application No. PCT/US93/11148, filed on Nov. 16,
1993, which is a Continuation-in-part of application Ser. No.
07/977,705, filed Nov. 16, 1992 (abandoned). The entire teachings
of the above applications are incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The present invention relates to fusion compounds having
reduced immunogenicity resulting from the addition to an
immunogenic compound of an amino acid sequence that renders the
compound less immunogenic. The amino acid sequence is found in
human proteins. The present invention relates to a method of
reducing the immunogenicity of compounds by the incorporation of an
amino acid sequence, the presence of which results in a reduced
immune response against the compound.
BACKGROUND OF THE INVENTION
[0003] The pharmaceutical use of immunogenic compounds, such as
proteins and carbohydrates, for diagnosis or therapy in humans has
enormous potential. A major concern, however, is that immunogenic
compounds often elicit immune responses which could limit their
effectiveness and, in some cases, cause dangerous allergic
reactions. This is particularly true of non-human proteins. In
addition, it is possible that even proteins with human amino acid
sequences could be immunogenic, as in the cases where the protein
is altered in structure or conformation as a consequence of
manufacturing or where the protein is produced in foreign hosts due
to inappropriate post-translational modification or improper
folding. Moreover, many non-protein compounds elicit an immune
response.
[0004] The immune system of the human to whom the immunogenic
compound is administered recognizes the compound as "foreign" and
mounts an immune response to remove it. The immune response
includes the production of specific, high affinity antibodies which
bind to and effect elimination of the immunogenic compound.
[0005] Monoclonal antibodies (Mabs) provide examples of the
therapeutic uses of foreign proteins. Most Mabs are of murine
origin, and have generally been found to be immunogenic when
injected into humans. Attempts have been made to reduce the
immunogenicity of murine Mabs by substituting human constant
regions for the analogous murine regions to form chimeric
antibodies or chimeric Mabs, or by going one step further and
substituting human framework sequences for the murine counterparts
in the variable regions of the antibodies (humanized antibodies or
humanized Mabs). These approaches may reduce the immune response
elicited by murine constant regions or frameworks, but may be
ineffective in reducing immune responses directed against the
variable regions or idiotypes of the Mabs. Indeed, there are
several examples of chimeric Mabs eliciting immune responses
directed against the variable regions (for example, B72.3 reported
by Meredith, et al., (1992) J. Nucl. Medicine 33:23-29, and ch14.18
reported by Saleh, et al., (1992) Hum. Antibody Hybridoma 3:19-24).
In fact, immune responses to the anti-variable region may be the
rule rather than the exception. In these cases, another approach is
required.
[0006] There is a need for therapeutic or diagnostic compounds
which do not elicit either an immune response or which elicit a
reduced immune response. There is a need for a method of reducing
or eliminating the immunogenicity of therapeutic and diagnostic
compounds.
[0007] The present invention provides reduced-immunogenic compounds
which elicit either a reduced immune response or essentially no
immune response in humans and a method of reducing the
immunogenicity of compounds. Reduced-immunogenic compounds
according to the present invention comprise an auto-antigenic amino
acid sequence linked to an otherwise immunogenic protein. By
associating an auto-antigenic amino acid sequence with an
immunogenic protein, the human immune system mounts a reduced
immune response against the compound or does not mount an
immunogenic response against it all. Accordingly, these compounds
can be administered as therapeutics or diagnostics with a reduction
or elimination of the problems associated with the administration
of immunogenic compounds.
SUMMARY OF THE INVENTION
[0008] The present invention relates to reduced-immunogenic fusion
compounds which comprise immunogenic compounds linked to
auto-antigenic sequences. The presence of the auto-antigenic
sequence renders the compound less immunogenic. In addition, the
present invention relates to a method of reducing the
immunogenicity of an immunogenic compound by linking an
auto-antigenic sequence to an otherwise immunogenic compound. The
present invention also relates to recombinant nucleotide sequence
that encoding auto-antigenic sequences and to essentially pure
auto-antigenic peptides.
DETAILED DESCRIPTION OF THE INVENTION
[0009] The presence of antibodies in normal human sera which are
specific for portions of degraded proteins, such as portions of
endogenous proteins degraded by proteolytic enzymes, has been
observed. There are many reports in the literature that refer to
observed endogenous immunoreactivity to cleaved antibody fragments.
This endogenous immunoreactivity to cleaved endogenous proteins is
referred to herein as "preimmunity". The antibodies involved in
preimmunity immunoreactivity were initially described as
"agglutinators" or "anti-Fab antibodies" (".alpha.FABA"). It is
reported that preimmunity antibodies 1) are present in most
individuals, 2) have varying titers across a population, 3) are not
IgM or rheumatoid factors, 4) are fragment specific, and 5) are
generally of low affinity. These antibodies can be generally
described as a heterogenous group of antibodies that share the
characteristic of recognizing endogenous protein fragments, usually
the terminal portions of antibody-fragments, which are exposed by
protein degradation, usually proteolytic degradation.
[0010] Osterland, C. K. et al. (1963) Vos Sang 8:133, report a
serum activity capable of "agglutinating" Fab- or F(ab').sub.2
coated human erythrocytes. The reactivity is directed to epitopes
that only become exposed after an immunoglobulin is cleaved by a
proteolytic enzyme. That is, these antibodies recognize the
degraded protein but not the intact protein.
[0011] Waller, M. et al., (1969) Immunochemistry 6:207-214, report
that "natural antibodies" in human sera were able to differentiate
Fab fragments produced by different enzymes. Different antibodies
of this group were specific to different epitopes on Fab fragments
which were generated i.e., exposed, by the specific cleavage that a
specific Fab fragment underwent.
[0012] Ling, N. R. and P. Drysdale, (1981) Int. Archs. Allergy
Appl. Immun. 66:459, report that F(ab').sub.2 fragments of human,
bovine, and rabbit polyclonal and of human IgG paraproteins of
different subclass and light-chain type were coupled to human red
cells and used to detect "agglutinator antibodies" in normal and
pathological human sera. Such antibodies were reported to commonly
occur and demonstrate specificity heterogeneity.
[0013] Persselin, J. E. and R. H. Stevens, (1985) J. Clin. Invest.
76:723, report that sera from rheumatoid arthritis patients
contained two populations of antibodies directed against the Fab
portion of pooled human IgG.
[0014] Heimer R., et al., (May 1985) Arthritis and Rheumatism
28(5):562, report an examination of the specificity of IgG
anti-F(ab').sub.2 antibodies in unfractionated sera of patients
with rheumatoid arthritis and from affinity purified antibody
preparations.
[0015] Persselin, J. E. and R. H. Stevens (1989) Mongr. Allergy
26:74, report a group of "autologous antibodies" that are directed
against the Fab and F(ab').sub.2 portions of human IgG. This group,
which was reported to be prevalent in normal individuals and
patients suffering a variety of disorders, was characterized to be
a heterogenous group of antibodies with diverse biological
properties and target specificities.
[0016] Although many of the reports of "natural antibodies" relate
to the existence of such antibodies that specifically bind to IgG
fragments, it is believed that groups these type of antibodies
exist which bind to degraded portions of other endogenous
proteins.
[0017] As used herein, the terms "agglutinators", "agglutinating
antibodies", "natural antibodies", "autologous antibodies",
"preimmunity antibodies" and "preimmune serum antibodies" are used
interchangeably and are meant to refer to antibodies that are
normally present in an individual. Preimmunity antibodies are a
heterologous group of antibodies which bind to degraded but usually
do not bind to intact endogenous proteins. They exist at low levels
and generally bind to the terminal portion at a cleavage site of a
cleaved endogenous protein. There are some preimmunity antibodies
which react to intact proteins. However, many such antibodies
recognize fragments but do not bind to the intact protein. In cases
in which they do not cross-react with intact proteins, a
preimmunity antibody generally recognizes an epitope that occurs at
the terminal portion of a protein following cleavage. This epitope
usually occurs at the C-terminus. By occurring at these positions,
the epitope is extremely specific such that the epitope is only
accessible and recognizable when it appears at an end of the
protein.
[0018] It has been discovered that the presence, on an otherwise
immunogenic compound, of an amino acid sequence which forms the
epitope for a preimmunity antibody reduces or eliminates the
immunogenicity of that compound. The inclusion of such an amino
acid sequence allows one to convert an immunogenic compound into a
reduced-immunogenic compound.
[0019] As used herein, the terms "auto-antigenic sequence",
"auto-antigenic peptide", "preimmunity sequence", "preimmunity
amino acid sequence" and "preimmunity peptide" are used
interchangeably and are meant to refer to an amino acid sequence
that is an epitope recognized by preimmunity antibody and that,
when associated with an immunogenic compound, renders the
immunogenic compound less immunogenic.
[0020] As used herein, the terms "fusion compounds",
"reduced-immunogenic compound" and "less-immunogenic compounds" are
used interchangeably and refer to compounds which comprise an
otherwise immunogenic compound linked to an auto-antigenic sequence
whereby the presence of the auto-antigenic sequences results in the
reduction or elimination of the immunogenicity of the otherwise
immunogenic compound. When an immunogenic compound that is a
compound that normally elicits an immune response when administered
to a patient, is linked with an auto-antigenic sequence to form a
fusion compound, the fusion compound elicits no immune response or
a reduced immune response when administered to a patient compared
to the immune response elicited by the immunogenic compound by
itself.
[0021] As used herein, the term "less immunogenic" refers to the
comparatively reduced-immunogenicity exhibited by an immunogenic
compounds linked to an auto-antigenic sequence relative to the
immunogenicity exhibited by the same immunogenic compound which is
not linked to an auto-antigenic compound.
[0022] As used herein, the term "immunogenic" refers to the ability
of a compound to elicit an immune response.
[0023] As used herein, the term "antigenic" refers to the ability
of a compound to react with the immune system, i.e. antibodies.
[0024] Auto-antigenic sequences can be identified by a variety
methods which can be readily performed by those having ordinary
skill in the art. Endogenous proteins, such as antibodies,
cytokines, growth factors, receptors, enzymes and structural
proteins, can be cleaved by a panel of proteolytic enzymes. The
fragments produced can be exposed to human sera and those fragments
that bind to preimmunity antibodies in the sera can be readily
identified. The amino acid sequence of the epitope that is involved
in the preimmunity antibody can be determined. This epitope
represents an auto-antigenic sequence. Alternatively, peptide
libraries that contain a random assortment of peptides of about 5
or more amino acid residues can be produced. These libraries can be
used in a screen with normal human sera to identify peptides that
are epitopes recognized by endogenous preimmunity antibodies. These
peptides can be identified and used tested as auto-antigenic
sequences.
[0025] One example of a heterogeneous group of preimmunity
antibodies specific for protein degradation products are antibodies
which recognize epitopes that occur on the heavy chain C-terminal
sequence when IgG antibodies are degraded by proteolytic cleavage.
Accordingly, some embodiments of the present invention relate to a
method of rendering an otherwise immunogenic compound
less-immunogenic by linking to it one of a variety of
auto-antigenic sequences found at the heavy chain C-terminus of
human or chimeric Fab or F(ab').sub.2 molecules.
[0026] Amino acid sequences that are the epitopes for anti-IgG
fragment preimmunity antibodies are often found at the hinge region
of the heavy chain. Cleavage of the heavy chain at the hinge region
generates an amino acid sequence at the C-terminus which may be
recognized by specific preimmunity antibodies that do not react
with intact IgG molecules. Depending upon where cleavage occurs, a
different epitope is exposed and thus a different set of
preimmunity antibodies may bind to it. Thus, individual antibodies
of this group recognize the discreet epitopes produced by cleavage
at various sites.
[0027] In some embodiments, the auto-antigenic sequence is derived
from the hinge region of IgG. In some preferred embodiments, the
auto-antigenic sequences is derived from the hinge region of
IgG.sub.1.
[0028] The hinge region refers to various structural segments of
the heavy chain of IgG molecules. Different classes of IgG
molecules have different amino acid sequences at their respective
hinge regions. The hinge region is a particularly variable element
of immunoglobulin structure. Table 1 provides a listing of the
different amino acid sequences of the respective hinge regions of
the various classes of IgG molecules.
[0029] In order to identify whether a particular amino acid
sequence is an auto-antigenic sequence, IgG molecules can, for
example, be cleaved with one of a panel of proteases to provide IgG
fragments that contain different hinge region sequences at the
terminal end. Alternatively, peptides and polypeptides can be
produced by peptide synthesis or recombinant DNA technology which
are modelled upon the sequence of the hinge region. In either case,
human sera can be screened to determine whether preimmunity
antibodies are present which bind to a particular exposed terminal
sequence or synthetic peptide, respectively.
[0030] It has been observed that the amino acid sequence near the
papain cleavage site of human Fab molecules is reactive with the
endogenous human "anti-Fab" preimmunity antibodies. This sequence
can instruct the immune system to ignore a molecule that includes
it such that no further immune response is elicited. This
auto-antigenic sequence prevents an immune response to a linked
compound that would be otherwise immunogenic.
[0031] A Fab-derived preimmunity sequence having the C-terminal
sequence CDKTH (SEQ ID NO:1) was identified from observations made
concerning the nature of preexisting human immunity and induced
immune responses to murine and chimeric 7E3 Fab fragments. Both the
light and heavy chains of the chimeric 7E3 Fab comprises of murine
variable regions and human constant regions. This sequence mimics a
natural fragment or conformation of human IgG found in human serum,
and therefore, a typical high affinity immune response is not
mounted against the molecule, or against the related Fab sequence.
A reduced immunogenicity of the murine 7E3 variable region when
linked to this sequence in the c7E3 molecule was observed.
According to the invention, an immunogenic compound, such as a
foreign protein, may be rendered non-immunogenic or less
immunogenic by linking an auto-antigenic sequence, such as those
found at the C-terminus of papain generated human Fab molecules
derived from IgG.sub.1, to a foreign compound. Thus, association of
an auto-antigenic sequence with an immunogenic therapeutic or
diagnostic agent is useful for reducing the immunogenicity of the
therapeutic or diagnostic agent, thereby preventing or reducing a
significant immune response to the agent when administered to a
patient. Similarly, papain generated Fab fragments of chimeric
antibody c128 which is specific for CD4 and papain generated Fab
fragments of chimeric antibody c168 which is specific for tumor
necrosis factor have the preimmunity sequence, CDKTH (SEQ ID NO:1),
at their the C-termini.
[0032] In addition, cleavage of antibodies with human constant
regions such as c128, c168 or c7E3 with elastase exposes a
preimmunity sequence at the C termini of the antibody fragment thus
generated.
[0033] The F(ab').sub.2 fragment of chimeric 7E3 has also been
found to exhibit a similar characteristic to the Fab molecule, such
as showing a natural immunity, or preimmunity, in normal human
sera. Therefore, the auto-antigenic sequence at its C-terminal
sequence may also be useful for reducing the immunogenicity of
foreign compounds in humans.
[0034] Preferred auto-antigenic sequences may comprise amino acid
sequences selected from the group consisting of: CDKTH (SEQ ID
NO:1), PKSCD (SEQ ID NO:2), KSCDK (SEQ ID NO:3), SCDKT (SEQ ID
NO:4), DKTHT (SEQ ID NO:5), KTHTC (SEQ ID NO:6), THTCP (SEQ ID
NO:7), HTCPP (SEQ ID NO:8), TCPPC (SEQ ID NO:9) and CPPCP (SEQ ID
NO:10). These sequences can be associated with the otherwise
immunogenic compound in such a way as to ensure that the last
residue of each particular sequence represents a C-terminal amino
acid residue.
[0035] In order to determine whether an amino acid sequence will be
useful as an auto-antigenic sequence, peptide constructs comprising
the specific sequence to be tested are exposed to human sera to
determine whether antibodies are present in the sera which
recognize and specifically bind to the sequence.
[0036] The most preferred auto-antigenic sequence comprises the
amino acid sequence CDKTH (SEQ ID NO:1). As noted above, the H
residue is the C-terminal residue of any construction which
contains this peptide. When associated with a compound and present
at the C-terminal region, this peptide reduces or eliminates the
immunogenicity of the compound, thus rendering the compound
less-immunogenic.
[0037] Once identified, an auto-antigenic sequence can be linked to
an immunogenic compound by a variety of means that can be readily
practiced by those having ordinary skill in the art. If the
immunogenic compound is a protein, a fusion protein comprising the
auto-antigenic sequence at the terminal portion of the immunogenic
protein can be produced using recombinant DNA technology. A
nucleotide sequence that encodes an auto-antigenic sequence can be
linked to the nucleotide sequence that encodes the immunogenic
protein to form a chimeric gene that encodes a fusion protein. The
auto-antigenic sequence will appear at the terminal portion of the
resulting fusion protein when the chimeric gene is expressed. If
the immunogenic compound is not a protein, synthetic peptides that
comprise the auto-antigenic sequence at a terminus can be produced
by standard methodology. These peptides can be chemically linked to
the immunogenic compound using well known techniques.
Auto-antigenic sequences can also be linked to proteins by chemical
means. Regardless of the method of linking an auto-antigenic
sequence to an otherwise immunogenic compound, the immunogenic
compound is converted to a reduced-immunogenic compound by the
incorporation of an auto-antigenic sequence which serves as an
epitope for preimmunity antibodies. One having ordinary skill in
the art can accomplish linkage of an auto-antigenic sequence to an
immunogenic compound by well known techniques. Standard coupling
techniques, for example, include but are not limited to: coupling
through free sulfhydryl of cysteine, coupling through
.epsilon.-amino group of lysine and coupling through any free
amine. Techniques for engineering antibodies are well known and
described in Winter and Millstein (1991) Nature 349:293, and
Larrich and Fry (1991) Hum. Antibod. and Hybridomas 2:17, both of
which are incorporated herein by reference.
[0038] According to the invention, an auto-antigenic sequence can
be attached to an immunogenic compound in order to convert the
immunogenic to a reduced-immunogenic compound. As used herein, the
terms "reduced-immunogenic compounds"; "less-immunogenic
compounds", "non-immunogenic compounds" and "fusion compounds" are
used interchangeably and meant to refer to compounds which comprise
an auto-antigenic sequence linked to an otherwise immunogenic
compound. The presence of the auto-antigenic sequence linked to the
otherwise immunogenic compound causes a reduced immune response in
individuals administered such compounds relative to the immune
response elicited by the immunogenic compound absent the
auto-antigenic sequence. An auto-antigenic sequence may be added to
non-human proteins, processed or recombinantly produced human
proteins or non-protein immunogenic compounds.
[0039] Examples of non-human proteins include, but are not limited
to, Mabs, Fabs, F(ab').sub.2s, non-human cytokines, non-human
growth factors, non-human receptors, non-human structural proteins
and non-human enzymes such as Streptokinase.
[0040] Examples of processed or recombinantly produced human
proteins include, but are not limited to, human and chimeric
antibodies and fragments thereof, human cytokines, human growth
factors, human receptors, human structural proteins and human
enzymes such as coagulation and fibrinolytic agents.
[0041] Examples of non-protein immunogenic compounds include, but
are not limited to, carbohydrates such as heparin.
[0042] A preferred immunogenic compound to be converted to a
reduced-immunogenic compound according to the present invention is
a murine IgG molecule. Ordinarily, a murine IgG will elicit an
immune response when administered to a human. This response can
render it ineffective or less effective because the IgG molecule is
neutralized and/or removed prior to reaching and binding to its
target antigen. By rendering the murine IgG less-immunogenic, it
becomes more effective as a therapeutic or diagnostic since it is
less deterred by the patient's immune system.
[0043] A preferred embodiment of the present invention is a murine
IgG molecule having an auto-antigenic sequence comprising CDKTH
(SEQ ID NO:1) linked such that the H residue is a C-terminal
residue. According to the invention, such a molecule can be
produced by standard recombinant DNA techniques used to produce
antibodies. For example, a nucleotide sequence encoding the
auto-antigenic sequence can be inserted at the 3' end of a gene
encoding a C-terminal portion of the IgG molecule, preferably the
C-terminal portion of the heavy chain. The nucleotide sequence is
inserted in the proper reading frame such that the residues encoded
by it will occur at the very end of the resulting protein.
[0044] Clinical results demonstrate a reduction the immunogenicity
to foreign antigens containing an auto-antigenic sequence that is
exposed by proteolytic cleavage of an IgG heavy chain. Several
observations have been made during the development of the
therapeutic anti-platelet Mab 7E3 which led to the discovery that
the immunogenicity of a normally immunogenic compound may be
reduced by associating it with an amino acid sequence which
represents an epitope recognized by a preimmunity antibody. These
experiments are reported in Example 1.
[0045] Briefly, Mab 7E3 was injected into humans both as a murine
Fab fragment and as a chimeric Fab fragment (c7E3). The
immunogenicity of both fragments were analyzed. There was a
fundamental difference in the nature of the immune responses
elicited by the murine and chimeric Fabs. The murine 7E3 Fab
elicited an immune response in patients which was directed almost
entirely against the 7E3 variable region. In contrast, even though
the c7E3 Fab contained the identical variable region as murine 7E3
Fab, c7E3 did not elicit comparable immune responses, indicating
that the human constant region of the c7E3 Fab rendered the
variable region less immunogenic.
[0046] Direct-coated, affinity-independent EIA analyses indicated
that 50-80% of the normal human population has preimmune serum
antibodies that react with chimeric Fab fragments. This preimmunity
reactivity is not specific for the variable regions of these
molecules since various monoclonal chimeric Fab fragments as well
as bulk Fab fragments prepared from total human serum Ig were
recognized by the endogenous anti-Fab antibodies. This
anti-fragment reactivity appeared to be of low affinity, and was
detectable only by relatively sensitive, solid phase EIA
assays.
[0047] The location of the reactive epitopes of the monoclonal
chimeric (or human) Fabs was at the C-terminus of the heavy chain
of the Fab fragment generated by papain digestion of the intact IgG
molecule. These endogenous anti-chimeric 7E3 Fab preimmunity
antibodies were readily neutralized using the Fab fragment of
another IgG.sub.1 chimeric antibody but not by other proteins.
These observations indicated that the reactive epitope is probably
a short sequence of amino acids which must be-present at or near
the C-terminus of the Fab fragment.
[0048] A human or chimeric Fab fragment mimics a molecule which is
normally found in serum and which elicits a normal low affinity
antibody response. It appears that the immune response against
molecules that contain the auto-antigenic sequence as an accessible
epitope may be regulated to preclude a high affinity secondary
response. In effect, the immune system may be desensitized or
tolerized to antigen challenge with molecules bearing the epitope.
Further challenge, therefore, with a similar molecule would not
lead to a typical high affinity immune response.
[0049] Preimmunity to particular epitopes appears to be
species-specific. When the immune responses from primates treated
with chimeric 7E3 Fab were analyzed, pretreatment sera from these
monkeys demonstrated little or no immunoreactivity to chimeric 7E3
Fab but showed a significant reactivity to Fab fragments generated
from monkey IgG. Conversely, human sera showed no preexisting
immunoreactivity to monkey Fab. In addition, monkeys have
demonstrated a significantly greater induced immunogenicity to
chimeric 7E3 Fab than have the humans enrolled Phase I clinical
trials.
[0050] Other species were also examined for preexisting
immunoreactivity to their autologous Fab and F(ab').sub.2
fragments. Autologous panels of sera from rabbits and goats were
screened for reactivity to polyclonal Fab and F(ab').sub.2
fragments from IgG of their respective species. Again, it was
observed that there was significant IgG reactivity to both of these
antibody fragments from their own species. The same observation,
however, was not made for murine antibodies to murine 7E3 Fab.
[0051] Although comparative experiments on humans have not been
performed using murine and chimeric 7E3 F(ab').sub.2 fragments or
other murine and chimeric Fab and F(ab').sub.2 fragments, specific
antibodies which bind to human Fab and F(ab').sub.2 fragments have
been observed. The individuals with high anti-chimeric Fab
reactivity do not necessarily have high anti-1-5 chimeric
F(ab').sub.2 reactivity, and vice versa. Furthermore, the immune
recognition of these chimeric fragments is apparently specific,
since the binding of anti-Fab antibodies generally cannot be
blocked by chimeric Fab' or F(ab').sub.2.
[0052] The description of the present invention is generally
presented herein as relating to compounds which are non-immunogenic
as a result of linking a human auto-antigenic sequence to an
otherwise immunogenic compound, and to a method of reducing the
immunogenicity of compounds by linking the immunogenic compound
with a human auto-antigenic sequence. It is contemplated that the
present invention can be applied to other species. The scope of the
present invention is intended to include to compounds which are
less-immunogenic in a particular species as a result of linking an
auto-antigenic sequence form the species to an otherwise
immunogenic compound and to a method of reducing the immunogenicity
of compounds in a particular species by linking the immunogenic
compound with an auto-antigenic sequence from that species.
[0053] Furthermore, the description of the present invention is
presented as relating to compounds in which an auto-antigenic
sequence is physically attached to an otherwise immunogenic
compound. However, it is possible that physical linkage is not
required. It is contemplated that reduced immunogenicity of a
protein can be achieved by co-injection of an auto antigenic
peptide sequence or a non-specific human Fab fragments.
EXAMPLES
Example 1
[0054] The 7E3 monoclonal antibody has been developed for human
therapy as a Fab fragment of the antibody molecule. The reagent was
produced in two versions; one derived from the murine monoclonal
antibody (murine gamma 1 isotype) by papain digestion (m7E3), and
one derived from a mouse/human chimeric monoclonal antibody (human
gamma 1 isotype) also by papain digestion (c7E3). The chimeric Mab
was produced using standard cloning techniques to obtain the
variable region genes from the 7E3 hybridoma and fuse them to
previously cloned human constant region genes in vitro. The
chimeric genes were introduced into appropriate mammalian cells for
expression.
[0055] The two Fab molecules have the same variable region, but
different constant regions. The C-terminal amino acid sequences
exposed after papain digestion are different. Papain clips the
antibody molecules in the hinge region of the heavy chain between
the CH1 and CH2 domain. The hinge amino acid sequences of the human
gamma 1 and the mouse gamma 1 are very different (See Table 1).
After cleavage of c7E3 with papain, the C-terminus of the heavy
chain ends with the amino acids CDKTH (SEQ ID NO:1). The exact
papain cleavage point in the mouse gamma 1 hinge is unknown, but
must produce a very different C-terminus from the human sequence,
since the mouse hinge does not contain a similar sequence to CDKTH
(SEQ ID NO:1).
[0056] The m7E3 and c7E3 Fab fragments were tested to determine
whether human sera contains antibodies which react with the
fragments. A solid phase ELISA assay was used in which either m7E3
Fab or c7E3 Fab was immobilized directly on plastic assay plate and
exposed to human serum. Bound human antibodies were detected using
goat anti-human antibodies conjugated to an enzyme that will
produce a colored product when incubated with the appropriate
substrate. This assay is very sensitive and capable of detecting
low affinity interactions. A large number of human sera were
tested, and the m7E3 Fab was generally non-reactive, whereas c7E3
Fab reacted with at least 60% of the human serum samples.
[0057] The specificity of the reactive antibodies in human serum
was assessed by including an excess of various molecules in the
assay as competitive inhibitors. If a given molecule inhibits
binding to c7E3, then it must also display the reactive epitope(s).
As expected, soluble c7E3 could inhibit the binding of the human
antibodies to immobilized c7E3. Other molecules that could inhibit
the binding included Fab fragments of other chimeric gamma 1
antibodies, and polyclonal human Fab derived from serum. In
contrast, none of the following molecules could inhibit the
binding: 1) m7E3 Fab; 2) whole c7E3 IgG; 3) Fab fragment derived
from IgG.sub.4 version of 7E3 (contains different hinge region from
gamma 1; 4) F(ab').sub.2 fragment of c7E3; and 5) Fab' fragment of
c7E3 Fab fragment is extremely specific for the C-terminus exposed
after papain digestion of the c7E3 antibody.
[0058] The immunogenicities of m7E3 Fab and c7E3 Fab were tested in
humans in Phase I clinical trials. Using an assay format that
minimizes the low-affinity preimmune reactivity so that actual
treatment-related responses can be easily seen, it was found that
17/86 or about 20% of the subjects receiving m7E3 Fab exhibited
immune responses at titers ranging from 1:50-1:1600. In contrast,
only 1/67 or 1.5% of the subjects receiving c7E3 Fab exhibited an
immune response (titer=1:50). The c7E3 Fab, which reacts with
endogenous human antibodies, was therefore shown to be must less
immunogenic than m7E3 Fab, which does not react with endogenous
antibodies.
Example 2
[0059] The possible sequences derived from the hinge region of
human gamma 1 Fab (and F(ab').sub.2) reactive with endogenous
"anti-fragment" preimmunity antibodies can be identified by
screening and competition experiments using synthetic peptides.
Synthetic peptides of about 5 amino acids or more in length are
produced which contain various sequences found in the amino acid
sequence of the hinge region.
[0060] The most reactive sequences may then be linked to an
immunogenic compound of interest to reduce immunogenicity. Linkage
can be accomplished in several ways. If the immunogenic compound is
a protein, the natural C-terminus of the immunogenic protein can be
converted to the desired sequence by site-directed mutagenesis of
the gene, most easily accomplished by using appropriately designed
PCR primers to delete the natural termination codon and add the
desired sequence. These techniques could be used to add or
substitute the sequence at any position in any gene. Alternatively,
a synthetic peptide can be constructed corresponding to the minimal
reactive-sequence and could linked to the immunogenic compound by
chemical means.
Example 3
[0061] Amino acid sequences that are reactive with antibodies in
normal human serum, which may be used as auto-antigenic sequences
to render foreign or normally immunogenic molecules
less-immunogenic, may be identified by screening synthetic peptides
with human sera. This is a general method which does not require
identification of a protease-specific cleavage site of an
endogenous protein, and does not even require that the actual
sequence occur naturally.
[0062] Briefly, a library of random peptides of at least about 5 or
more, and preferably about 5-10 amino acids in length is generated.
These peptides are screened for reactivity with antibodies in human
sera. This is accomplished, for example, by immobilizing the
peptides on a solid phase and performing a standard ELISA assay to
detect bound human antibodies after exposure to sera.
[0063] Positive peptides are then evaluated further to establish if
they are reactive with human sera only and not with other species,
and to determine the affinity of the interactions. Peptides that
react with the majority of human sera but not sera of other
species, are then studied to determine whether they are capable of
conferring reduced immunogenicity on otherwise immunogenic
compounds. Whether or not a given peptide elicits an immune
response requires empirical immunogenicity data.
[0064] There are many possible ways to generate and screen
libraries or random peptide sequences. One method is to synthesize
collections of peptides (Schoofs, P. G. et al. (1988) Immun.
140:611) and assay pools for reactivity against human serum.
Positive pools are subdivided and reassayed multiple times so that
the active species would eventually be identified. Another method
is to generate DNA sequence that codes for random amino acid
segments and fuse the DNA sequences to a bacteriophage gene
(McCafferty et al., (1990) Nature 348:552). The random amino acid
sequences are-displayed on the outside of the bacteriophage
particle as an artificial C-terminus of a phage protein. The phage
is immobilized and screened for reactivity to endogenous human
antibodies. Positive phage are isolated and the DNA extracted and
sequenced to determine the amino acid sequence of the reactive
peptide segment.
Example 4
[0065] In general, it is practically difficult to test
auto-antigenic sequence candidates in humans to determine whether
or not their presence renders immunogenic compounds less
immunogenic because it requires experimental testing in humans
which raises ethical concerns in many circumstances. Because of
this difficulty in testing the immunogenicity of candidate
molecules in humans, a rabbit model is used to mimic the human
system and demonstrate that epitopes of preimmunity antibodies can
be identified and linked to immunogenic compounds to reduce the
immunogenicity of the compounds.
[0066] Epitopes of preimmunity antibodies can be identified as
described in Example 3 but substituting rabbit sera for the human
sera used in the protocols described therein. Amino acid sequences
that react with the endogenous antibodies can be identified and
linked to various compounds by well known methods. Comparative data
can be generated from experiments in which the compounds linked to
the suspected auto-antigenic sequences and compounds alone are
administered to rabbits. The immune response against each of the
compounds can be measured.
[0067] Rabbits have been shown to exhibit the same type of normal
reactivity to homologous Fab fragments; that is, preimmunity to Fab
fragments. The appropriate region of rabbit DNA from the
immunoglobulin heavy chain locus is cloned and the sequence of the
hinge region is determined. This allows the construction of fusion
compound which comprise a rabbit auto-antigenic sequence linked to
the chimeric 7E3 IgG at the C-terminus of a Fab molecule and
evaluating its immunogenicity compared to the immunogenicity of
chimeric 7E3 Fab in rabbits.
Example 5
[0068] Streptokinase is a thrombolytic protein which has been
approved as a drug for heart attack patients suffering coronary
blockages. One major disadvantage of Streptokinase is that it is
highly immunogenic in humans. Accordingly, its usefulness is
greatly limited. Particularly, once a patient has been administered
Streptokinase, there is the chance of dangerous immune reaction to
any subsequent administration.
[0069] The present invention provides a method of reducing the
immunogenicity of Streptokinase, thereby reducing the inherent
shortcomings of the molecule as a therapeutic which forms the basis
for safety concerns that are associated with its use.
[0070] Streptokinase production using recombinant DNA technology is
well known and can be performed by those having ordinary skill in
the art using readily available starting materials. Similarly, well
known techniques may be performed to produce a Streptokinase
derivative that has the auto-antigenic sequence CDKTH (SEQ ID NO:1)
at the C-terminus of the protein. One having ordinary skill in the
art can produce such a Streptokinase derivative without undue
experimentation.
[0071] Streptokinase derivative can be tested in vitro and in
animal models to ensure that it retains Streptokinase activity.
Comparative clinical experiments can then be performed to measure
the immunogenicity of Streptokinase versus the immunogenicity of
Streptokinase derivative. The techniques to perform these
experiments are well known and can be readily performed by those
having ordinary skill in the art.
Example 6
[0072] There are several examples of chimeric Mabs which elicit
immune responses directed against the variable regions. These
include B72.3 (Meredith et al., (1992) J. Nucl. Medicine 33:23-29)
and ch14.18 (Saleh, et al., (1992) Hum. Antibody Hybridoma
3:19-24). The fact that these antibodies elicit immune responses
represents a major obstacle in their effectiveness and their
usefulness is greatly limited by their immunogenicity.
Particularly, patients develop antibodies against the antibodies
and neutralize their activity.
[0073] The present invention provides a method of reducing the
immunogenicity of chimeric Mabs, thereby reducing the basis for
safety concerns that are associated with its use and increasing
their utility. Techniques for engineering antibodies are described
in Winter and Millstein (1991) Nature 349:293, and Larrich and Fry
(1991) Hum. Antibod. and Hybridomas 2:17. Production of B72.3 and
ch14.18 can be accomplished from readily available starting
materials using recombinant DNA technology well known by those
having ordinary skill in the art. Likewise, well known techniques
may be performed to produce chimeric Mab derivatives dB72.3 and
dch14.18 in which the auto-antigenic sequence CDKTH (SEQ ID NO:1)
is present at the C-terminus of each antibody respectively,
preferably the heavy chain. One having ordinary skill in the art
can produce such a chimeric Mabs and chimeric Mab derivatives
without undue experimentation.
[0074] Chimeric Mab derivatives dB72.3 and dch14.18 can be tested
in vitro and in animal models to ensure that they retain their
specificity and activity. Comparative clinical experiments can then
be performed to measure the immunogenicity of chimeric Mabs B72.3
and ch14.18 versus the immunogenicity of chimeric Mab derivatives
dB72.3 and dch14.18, respectively. The techniques to perform these
experiments are well known and can be readily performed by those
having ordinary skill in the art.
1TABLE 1 Hinge Sequences of Human and Mouse Immunoglobulins
Antibody type Hinge Sequence Human IgG.sub.1 EPKSCDKTHTCPPCP (SEQ
ID NO:11) Human IgG.sub.2 ERKCCVECPPCP (SEQ ID NO:12) Human
IgG.sub.3 ELKTPLGDTTHTCPRCP (SEQ ID NO:13) Human IgG.sub.3M15
EPKSCDTPPPCPRCP (SEQ ID NO:14) Human IgG.sub.4 ESKYGPPVPRDCG (SEQ
ID NO:15) Mouse IgG.sub.1 EPRGPTIKPGPSCP (SEQ ID NO:16) Mouse
IgG.sub.2a CKPCICTCPPCKCP (SEQ ID NO:17)
[0075]
Sequence CWU 1
1
* * * * *