Note: Descriptions are shown in the official language in which they were submitted.
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CONTRACEPTIVE ANTIBODY VACCINES
1. FIELD OF THE INVENTION
The present invention relates to modified antibodies, and vaccine compositions
thereof, that have one or more complementary determining regions that contain
portions of
sperm antigens, in which modified antibodies one or more variable region
cysteine residues
that form intrachain disulfide bonds have been replaced with amino acid
residues that do not
contain a sulfhydryl group and, therefore, do not form disulfide bonds. The
present
invention also relates to use of the vaccine compositions of the invention as
a contraceptive.
2. BACKGROUND OF THE INVENTION
Z.1. IMMUNOGLOBULIN STRUCTURE
The basic unit of immunoglobulin structure is a complex of four polypeptides --
two identical low molecular weight or "light" chains and two identical high
molecular
weight or "heavy" chains, linked together by both noncovalent associations and
by disulfide
bonds. Each light and heavy chain of an antibody has a variable region at its
amino
terminus and a constant domain at its carboxyl terminus (Figure 1 ). The
variable regions
are distinct for each antibody and contain the antibody antigen binding site.
Each variable
domain is comprised of four relatively conserved framework regions and three
regions of
sequence hypervariabiIity termed complementarity determining regions or CDRs
(Figure 2).
For the most part, it is the CDRs that form the antigen binding site and
confer antigen
specificity. The constant regions are more highly conserved than the variable
domains, with
slight variations due to haplotypic differences.
Based on their amino acid sequences, tight chains are classified as either
kappa or
lambda. The constant region heavy chains are composed of multiple domains
(CHI, CH2,
CH3...CHx), the number depending upon the particular antibody class. The CHI
region is
separated from the CH2 region by a hinge region which aIiows flexibility in
the antibody.
The variable region of each light chain aligns with the variable region of
each heavy chain,
and the constant region of each light chain aligns with the first constant
region of each
heavy chain. The CH2-CHx domains of the constant region of a heavy chain form
an "Fc
region" which is responsible for the effector functions of the immunoglobulin
molecule,
such as complement binding and binding to the Fc receptors expressed by
lymphocytes,
granulocytes, monocyte lineage cells, killer cells, mast cells and other
immune effector
cells.
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As seen in Figure 3. the light and heavy chains of an IgG molecule form the
variabie
region domain and the constant region domain. Each domain is composed of a
sandwich of
t<vo parallel extended protein layers of about 100 amino acids in length which
are connected
by a single disulfide bond (See Roitt et al., Immunoloav, 3rd Edition, London:
Mosby,
j 1993, p4.4 (Figure 3)). Each of the two extended protein layers of the
domain, in turn,
contains two "anti-parallel" adjacent strands which adopt a beta-sheet
conformation. (See,
e.g., Stryer, 197j, Biochemistry, WH Freeman and Co., p. 9j0}. Each of the
domains has a
similar three-dimensional structure based on the immunoglobulin fold.
2.2. IMMUNOTHERAPY AND ANTI-IDIOTYPE ANTIBODIES
In modern medicine, immunotherapy or vaccination has virtually eradicated
diseases
such as polio, tetanus, tuberculosis, chicken pox, measles, hepatitis, etc.
The approach
using vaccinations has exploited the ability of the immune system to prevent
infectious
diseases.
1 j Use of immunotherapy has also been explored for cancer therapy. The era of
tumor
immunology began with experiments by Prehn and Main, who showed that antigens
on the
methylcholanthrene (MCA)-induced sarcomas were tumor specific in that
transplantation
assays could not detect these antigens in normal tissue of the mice (Prehn et
al., 1957, J.
Natl. Cancer Inst. 18:79-778). This notion was confirmed by further
experiments
demonstrating that tumor specific resistance against MCA-induced tumors could
be elicited
in the autochthonous host, that is, the mouse in which the tumor originated
(Klein et al.,
1990, Cancer Res. 20:1 j 1-1 j72).
There are many reasons why immunotherapy is desired for use in cancer
patients.
First, if cancer patients are immunosuppressed in surgery, with anesthesia and
subsequent
2j chemotherapy, it may worsen the immunosuppression, then with appropriate
immunotherapy in the preoperative period, this immunosuppression may be
prevented or
reversed. This could lead to fewer infectious complications and accelerated
wound healing.
Second, tumor bulk is minimal following surgery and immunotherapy is most
likely to be
effective in this situation. A third reason is the possibility that tumor
cells are shed into the
circulation at surgery and effective immunotherapy applied at this time can
eliminate these
cells.
There are nvo types of immunotherapy, the "active immunotherapy" and the
"passive immunotherapy". In "active immunotherapy", an antigen is administered
in the
form of a vaccine, to a patient, so as to elicit a protective immune response.
"Passive
immunotherapy" involves the administration of antibodies to a patient without
eliciting a
concommitant immune response. When a specific antibody from one animal is
injected as
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an immunogen into a suitable second animal, the injected antibody will elicit
an immune
response. Antibody therapy is conventionally characterized as passive since
the patient is
not the source of the antibodies. However, the term passive is misleading
because the
patient can produce anti-idiotypic secondary antibodies which in turn provoke
an immune
response which is cross-reactive with the original antigen. Immunotherapy
where the
patient generates secondary antibodies is often more therapeutically effective
than passive
immunotherapy because the patient's own immune system continues to fight the
cells
bearing the particular antigen well after the initial infusion of antibody.
In an anti-idiotype response, antibodies produced initially during an immune
response or introduced into an organism will carry unique new epitopes to
which the
organism is not tolerant, and therefore will elicit production of secondary
antibodies
(termed "Ab2"), some of which are directed against the idiotype (i.e., the
antigen binding
site) of the primary antibody (termed "Ab 1 "), i. e., the antibody that was
initially produced
or introduced exogenously. These secondary antibodies or Ab2 likewise will
have an
idiotype, which will induce production of tertiary antibodies (termed "Ab3"),
some of which
will recognize the antigen binding site of Ab2, and so forth. This is known as
the "network"
theory. Some of the secondary antibodies will have a binding site which is an
analog of the
original antigen, and thus will reproduce the "internal image" of the original
antigen. And,
the tertiary or Ab3 antibodies that recognize this antigen binding site of the
Ab2 antibody
will also recognize the original antigen (Figure 4).
Therefore, anti-idiotypic antibodies have binding sites that are similar in
conformation and charge to the antigen, and can elicit the same or greater
response than that
of the cancer antigen itself. Administration of an exogenous antibody that can
elicit a
strong anti-idiotypic response can thus serve as an effective vaccine, by
maintaining a
constant immune response.
To date, anti-idiotypic vaccines have comprised murine antibodies because the
anti-
idiotypic response occurs as part of the typical human anti-mouse antibody
(HAMA)
response. A strong anti-idiotypic cascade has been observed when Ab 1 has been
structurally damaged (Madiyalakan et aL, 1995, Hybridoma 14:199-203),
rendering the
antibody mare foreign. There has been direct administration to the subject of
exogenously
produced anti-idiotype antibodies that are raised against the idiotype of an
anti-tumor
antibody (U.S. Patent i~o. 4,918,14). After administration, the subject's body
will produce
anti-antibodies which not only recognize these anti-idiotype antibodies, but
also recognize
the original tumor epitope, thereby directing complement activation and other
immune
system responses to a foreign entity to attack the tumor cell that expresses
the tumor
epitope.
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PCTNS99J26671
However, while anti-idiotypic vaccines are desirable targets and several have
been
identified, the ability to deliver antibodies that reproducibly cause the
generation of such an
anti-idiotypic response is not currently possible. (Foon et al., 1995, J.
Clin. Invest. 9:334-
342; Madiyalakan et al., 1995, Hvbridoma 14:199-203). One of the reasons for
the failure
to generate an anti-idiotypic response is that, Abl, while exogenous, is still
very similar to
"self', as all antibodies have very similar structures, and anti-idiotypic
responses to self
molecules tend to be very limited. Thus, there is a need in the art for
methods of reliably
generating an anti-idiotype response to a specific antibody.
2.3. CONTRACEPTIVE METHODS
A variety of contraceptive methods are currently available. Such methods
include
barrier methods such as condoms or diaphragms, or use of spermicidal agents
such as non-
oxynol-9, hormone therapies such as birth control pills or implants, and other
methods such
as intrauterine devices. All of these methods pose problems as convenient and
effective
1 S methods of preventing conception. Some methods are inconvenient or
ineffective, some
pose health risks, while others are costly. Accordingly, there is a need in
the art for a safe,
inexpensive, and convenient method of contraception.
3. SUMMARY OF THE INVENTION
The present invention is based upon the realization of the present inventors
that an
antibody in which one or more variable region cysteine residues that form one
or more
intrachain disulfide bonds have been replaced with amino acid residues that do
not contain
sulfhydryI groups, such that the particular disulfide bonds do not fotitt,
elicit a much
stronger anti-idiotype response than an antibody in which the variable region
disulfide
bonds are intact. Additionally, the present inventors have realized that
portions of antigens
of proteins or reproductive cells, particularly sperm antigens, can be
inserted into or used to
replace portions of one or more complementarity determining regions, such that
the
modified antibody can be used as a vaccine to generate anti-idiotype
antibodies that
recognize the particular antigen.
Accordingly, the present invention provides modified immunoglobulin molecules
or
antibodies (and functionally active fragments, derivatives and analogs
thereof), and vaccine
compositions containing these immunoglobulin molecules, wherein the variable
region of
the immunoglobulin is subject to decreased conformational constraints, such
as, but not
limited to, by breaking one or more intrachain or interchain disulfide bonds.
Specifically,
the invention provides modified immunoglobulins that comprise a variable
region and are
identical, except for one or more amino acid substitutions in said variable
region, to a
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second immunoglobulin molecule, said second immunoglobulin molecule being
capable of
immunospecifically binding (i.e., specific binding of the immunoglobulin to
its antigen as
determined by any method known in the art for determining antibody-antigen
binding,
which excludes non-specific binding but not necessarily cross-reactivity with
other
antigens) an antigen or having a CDR that contains a portion of an antigen,
said one or more
amino acid substitutions being the substitution of one or more amino acid
residues that do
not have a sulfhydryl group at one or more positions corresponding to one or
more cysteine
residues that form a disulfide bond in said second immunoglobulin molecule. In
preferred
embodiments, the second immunoglobulin molecule contains a CDR that contains a
portion
of an antigen of a cell or protein involved in reproductive function,
preferably spetrn
antigens, more preferably the sperm antigens SP-10, LDH-C~, or MSA-63.
The invention further provides methods of eliciting an anti-idiotype response
in a
subject by administering the modified immunoglobulins of the invention. In
particular, the
modified immunoglobulins of the invention can be used as contraceptives,
either in males
or, preferably in females, specifically by administering an immunoglobulin
molecule of the
invention, which immunoglobulin molecule was derived (i.e., by modification
according to
the invention to replace one or more variable region cysteine residues that
form an
intrachain disulfide bond with an amino acid residue that does not contain a
sulthydryl
group) from an immunoglobulin molecule that contains a CDR that contains a
portion of an
antigen of a protein or cell associated with reproductive function, preferably
a sperm
antigen.
The invention also provides methods of producing the modified immunoglobulin
molecules of the invention and vaccine compositions containing the modified
immunoglobulin molecules of the invention.
4. DESCRIPTION OF FIGURES
Figure 1. A schematic diagram showing the structure of the light and heavy
chain of
an immunoglobulin molecule, each chain consisting of a variable region
positioned at the
amino terminal region (HZN-) and a constant region positioned at a carboxyl
terminal region
(-COOH).
Figure 2. A schematic diagram of an IgG showing the four framework regions
(FRI, FR2, FR3 and FR4) and three complementarity determining regions (CDR1,
CDR2
and CDR3} in the variable regions ofthe light and heavy chains (labeled as V~
and VH,
respectively}. The constant region domains are indicated as CL for the light
chain constant
domain and CH,, CHI and CH3 for the three domains of the heavy chain constant
region.
Fab indicates the portion of the antibody fragment which includes the variable
region
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domains of both light and heavy chains and the CL and CH, domains. Fc
indicates the
constant region fragment containing the CHI and CH3 domains.
Figure 3. A schematic diagram of an antibody structure as shown in Figure 2,
but
drawn to emphasize that each domain (the loop structures labeled as V~, ~'H,
C~, CH,, CH_,,
and CH3, respectively) is structurally defined by a disulfide bond (indicated
with darkest
lines) that maintains the three-dimensional structure (Roitt et al.,
Immunology, Second
Edition, London: Gower Medical Publishing, 1989, p 5.3).
Figure 4. A schematic diagram showing the development of internal image
bearing
anti-idiotype antibodies (Ab2) and anti-anti-idiotype antibodies (Ab3) from
idiotype
I0 antibodies (Ab 1 ) directed against an antigen of a tumor cell in an
antiidiotypic cascade.
Figure 5. Modification of the variable region of an immunoglobulin by
replacing
cysteine residues in the variable regions with alanine residues to remove an
intrachain
disulfide bond. CHI, CH2 and CH3 are constant regions. VH is the heavy chain
variable
region and V~ is the light chain variable region.
Figures 6A-C. (A). The structure of the expression vector pMRR010.1, which
contains a human kappa light chain constant region sequence. (B). The
structure of the
expression vector pGammal that contains a sequence encoding a human IgGI
constant
region (CH1, CH2, CH3) heavy chain and hinge region sequences. (C) The
structure of the
expression vector pNEPuDGV which contains a sequence encoding the kappa
constant
domain of the light chain and the constant domain and hinge region of the
heavy chain. For
all three vectors see Bebbington et al., 1991, Methods in Enzymology 2:136-
145.
Figures 7A and B. (A) The amino acid sequence and corresponding nucleotide
sequence for the consensus light chain variable region ConVLI. (B) The amino
acid and
corresponding nucleotide sequences for the consensus heavy chain variable
region
ConVHl.
Figure 8. A schematic diagram of the general steps that were followed for the
assembly of an engineered gene encoding the synthetic modified antibody
specific to human
colon cancer antigen.
Figure 9. Dot blot showing the result of an assay for the competition of
binding of
the antibody derived from mAB31.1, but not having the cysteine to alanine
changes with
the same antibody which is biotin labeled to an antigen preparation derived
from LS-174 T-
cells. The concentration of the unlabeled antibody is indicated as nM
unlabeled antibody.
The "blk" lane has no antigen.
Figures l0A-D. (A)Results of competition binding assay of the biotin-labeled
anti-
colon carcinoma cell antibody to LS-174T cells in the presence of antisera
from mice
vaccinated with vehicle alone, control antibody that binds the colon carcinoma
cell antibody
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but has not been modified, and peptides CDRI, CDR2, CDR3, CDR4, CDRS, and
CDR6,
having the CDR sequences containing the bradykinin receptor binding site
expressed as
percent of control binding to LS-174T cells. (B). Results of competition
binding assays of
the biotin-labeled anti-colon carcinoma cell antibody to LS-174T cells in the
presence of
antisera from mice vaccinated with vehicle alone, control antibody that binds
the colon
carcinoma cell antibody, but has not been modified, 2CAVHCOL1, and 2CAVLCOL1.
(C)
Diagram showing the binding of a biotin-labeled (indicated by the "b")
antibody (inverted
Y) to antigen (solid triangles). (D) Diagram showing the inhibition of binding
of the biotin-
labeled (indicated by the "b") antibody (inverted Y) by anti-idiotype
antibodies (solid
arrows) to antigen (solid triangles).
Figure 11. Nucleotide sequences of the oligonucleotides used to construct the
MSAl and MSALVAC-1 variable regions.
Figures 12A-C. (A) Nucleotide sequence for the MSA-63 epitope. (B) Amino acid
sequence of the MSA-63 epitope encoded by the nucleotide sequence of Figure
12A. (C)
MSA-63 oligonucleotides used to construct a modified variable region. Each
oligo overlaps
for five codons and transitions the entire sequence of Figure 12A.
Figures 13 A-C. (A) Nucleotide sequence for the SP-10 epitope. (B) Amino acid
sequence of the SP-10 epitope encoded by the nucleotide sequence of Figure
13A. (C)
Oligonucleotides of Sp-10 used to construct a modified variable region. SP3a
and SP4a are
modified to change the codons encoding certain cysteine residues to codons
encoding
alanine residues.
Figure 14. Oligonucleotides of LDH-C4 epitope sequence for construction of a
modified variable region gene containing a LDH-C4.
Figure 15. Nucleotide and amino acid sequence of the consensus contraceptive
light
chain variable region.
Figure 16 A-B. (A) Sequences of oiigos used in the construction of 2CAVHCOL1.
(B) Sequences of oligos used in the construction of 2CAVLCOL 1.
5. DETAILED DESCRIPTION OF THE INVENTION
The present invention provides modified immunoglobulins (particularly
antibodies
and functionally active fragments, derivatives, and analogs thereof) that can
be used as
contraceptive vaccines. Specifically, these antibodies have one or more
complementarity
determining regions (CDRs) that contain a portion of an antigen of a cell or
protein
involved in reproductive functio, preferably a sperm antigen. In addition,
these antibodies
have been engineered to elicit a stronger immune response, particuiariy a
stronger anti-
idiotypic response, than the corresponding unmodified immunoglobulins. In
particular, the
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modified immunoglobulins of the invention are immunoglobulins that are
modified to
decrease the conformational constraints on one variable region of the
immunoglobulin
molecule, preferably, such that at least one of the cysteines that
participates in forming an
intrachain disulfide bond in the variable region of the immunoglobulin has
been replaced
S with an amino acid residue that does not have a sulfhydryl group and,
therefore, does not
form a disulfide bond, thereby decreasing the conformational constraints of at
least one of
the variable regions of the immunoglobulin (Figure S).
The invention also provides vaccine compositions containing the modified
immunoglobulin molecules of the invention. Additionally, the invention
provides methods
of generating an anti-idiotvpe response in a subject by administration of the
modified
immunoglobulin molecules of the invention.
In specific embodiments, the invention provides methods of contraception by
administration of a modified immunoglobulin molecule of the invention which,
in its
unmodified state, is capable of immunaspecifically binding an antigen of a
protein or cell
I S associated with reproductive function, such as a sperm antigen.
Administration of the
modified immunoglobulin elicits an anti-idiotype reaction in the subject,
leading to the
production, by the subject, of antibodies specific for the particular antigen.
For clarity of disclosure, and not by way of limitation, the detailed
description of the
invention is divided into the subsections which follow.
5.1. MODIFIED ANTIBODIES
The modified immunoglobulins, particularly antibodies, of the invention are
immunoglobulins that, at least in the unmodified state, can immunospecifically
bind an
antigen of a cell or protein associated with reproductive function, and have
been modified to
enhance their ability to elicit an anti-idiotype response. Such
immunoglobulins are
modified to reduce the conformational constraints on a variable region of the
immunoglobulin, e.g., by removing or reducing intrachain or interchain
disulfide bonds.
Specifically, the invention provides a first immunoglobulin molecule that
comprises a
variable region and that is identical, except for one or more amino acid
substitutions in the
variable region, to a second immunoglobulin molecule, the second
immunoglobulin
molecule being capable of immunospecifically binding an antigen, the amino
acid
substitutions being the substitution of one or more amino acid residues that
do not have a
sulfhydryl group at one or more positions corresponding to one or more
cysteine residues
that form a disulfide bond in said second immunoglobulin molecule. (See, co-
pending
United States Patent Application Serial No., entitled "Modified Antibodies
With Enhanced
Ability To Elicit An Anti-Idiotype Response", filed November 13, 1998
(attorney docket
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no. 6750-015), which is incorporated by reference herein in its entirety. The
invention also
provides nucleic acids containing a nucleotide sequence encoding a modif ed
immunoglobulin of the invention.
Identifying the cysteine residues that form a disulfide bond in a variable
region of a
particular antibody can be accomplished by any method known in the art. For
example, but
not by way of limitation, it is well known in the art that the cysteine
residues that form
intrachain disulfide bonds are highly conserved among antibody classes and
across species.
Thus, the cysteine residues that participate in disulfide bond formation can
be identified by
sequence comparison with other antibody molecules in which it is know which
residues
form a disulfide bond.
Table I provides a list of the positions of disulfide bond forming cysteine
residues
for a number of antibody molecules.
Table I (derived from Kabat et al, 1991, sequences of Proteins of
Immunological Interest,
5th Ed., U.S. Department of Health and Human Services, Bethesda, Maryland).
Disulfide bond-forming
Variable domain cysteines
Species Subgroup (positions)
Htunan kappa light I 23
88
H~~ kappa light II ,
23,88
Human kappa light III 23,88
H~~ kappa light IV 23,88
Human lambda light I 23,88
Human Iambda light iI 23,88
Human lambda Iight IIII 23,88
H~~ lambda light IV 23,88
Human lambda light V 23,88
Humat'i Iambda light VI 23,88
Mouse kappa light I 23,88
Mouse kappa light II 23,88
Mouse kappa light III 23,88
Mouse kappa light IV 23,88
Mouse kappa light V 23,88
Mouse kappa Iight VI 23,88
Mouse kappa light VII 23,88
Mouse kappa light Miscellaneous 23,gg
Mouse lambda light 23,88
Chimpanzee lambda light 23,88
Rat kappa light 23,88
Rat lambda Iight 23,88
Rabbit kappa light 23,88
Rabbit lambda light 23,88
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Disulfide bond-forming
Variable domain cysteines
Species Subgroup (positions)
Dog kappa light 23,88
Pig kappa light 23 (88)
Pig lambda light 23,88
Guinea pig lambda light 23 (88)
Sheep lambda light 23,88
Chicken lambda light 23,88
Turkey lambda light 23 (88)
Ratfish lambda light 23 (88)
Shark kappa light 23,88
Human heavy I 22,92
Human heavy II 22,92
Human heavy III 22,92
Mouse heavy I (A) 22,92
Mouse heavy I (B) 22,92
Mouse heavy II (A) 22,92
I S Mouse heavy II (B) 22,92
Mouse heavy II (C) 22,92
Mouse heavy III (A) 22,92
Mouse heavy III(B) 22,92
Mouse heavy III (C) 22,92
Mouse heavy III (D) 22,92
Mouse heavy V (A) 22,92
M
ouse heavy V (B) 22,92
Mouse heavy Miscellaneous 22,92
Rat heavy 22,92
Rabbit heavy 22,92
Guinea pig heavy 22,92
Cat heavy 22 (92)
Dog heavy 22,92
Pig heavy 22 (92)
M~k heavy 22 (92)
Sea lion heavy 22 (92)
Seal heavy 22 (92)
Chicken heavy 22,92
Duck heavy 22 (92)
Goose heavy 22 (92)
Pigeon heavy 22 (92)
Turkey heavy 22 (92)
Caiman heavy 22, 92
Xenopus frog heavy 22,92
Elops heavy , 22,92
Goldfish heavy 22,92
Ratfish heavy 22 (92)
Shark heavy 22,92
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Position numbers enclosed by () indicate that the protein was not sequenced to
that position.
but the residue is inferred by comparison to known sequences.
Notably, for all of the antibody molecules listed in Table 1. the cysteine
residues
that form the intrachain disulfide bonds are the residues at positions 23 and
88 of the light
S chain variable domain and the residues at positions 22 and 92 of the heavy
chain variable
domain. The position numbers refer to the residue corresponding to that
residue in the
consensus sequences as defined in Kabat, ( 1991, Sequences of Proteins of
Immunological
Interest, 5th Ed., LT.S. Department of Health and Human Services, Bethesda,
Maryland) or
as indicated in the heavy and light chain variable region sequences depicted
in Figures 7A
and B, respectively ("corresponding" means as determined by aligning the
particular
antibody sequence with the consensus sequence or the heavy or light chain
variable region
sequence depicted in Figure 7A or B).
Accordingly, in one embodiment of the invention, the modified immunoglobulin
molecule is an antibody in which the residues at positions 23 ancfor 88 of the
light chain are
1 S substituted with an amino acid residue that does not contain a sulfhydryl
group and/or the
residues at positions 22 and/or 92 of the heavy chain are substituted with an
amino acid
residue that does not contain a sulfhydryl group.
In the modified immunoglobulin of the invention, the amino acid residue that
substitutes for the disulfide bond forming cysteine residue is any amino acid
residue that
does not contain a sulfltydryl group, e.g., alanine, arginine, asparagine,
aspartate (or aspartic
acid), glutamine, glutamate (or glutamic acid), glycine, histidine,
isoleucine, leucine, lysine,
phenylalanine, proline, serine, threonine, tryptophan, tyrosine or valine. In
a preferred
embodiment, the cysteine residue is replaced with a glycine, serine,
threonine, tyrosine,
asparagine, or glutamine residue, most preferably, with an alanine residue.
Additionally, the disulfide bond forming cysteine residue may be replaced by a
nonclassical amino acid or chemical amino acid analog that does not contain a
sulfhydryl
group (for example, but not by way of limitation, using routine protein
synthesis methods).
Non-classical amino acids include, but are not limited, to the D-isomers of
the common
amino acids, a-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-aminobutyric
acid,
y-Abu, E-Ahx, -amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino
propionic acid,
ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, t-
butylglycine, t-
butylalanine, phenylglycine, cyclohexylalanine, ~3-alanine, fluoro-amino
acids, designer
amino acids such as (3-methyl amino acids, Ca-methyl amino acids. Na-methyl
amino
acids, and amino acid analogs in general. Furthermore, the amino acid can be D
(dextrorotary) or L (levorotary). In an alternative embodiment, the disulfide
bond forming
residue is deleted.
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In specific embodiments, the substitution of the disulfide bond forming
residue is in
the heavy chain variable region or is in the light chain variable region or is
in both the heavy
chain and light chain variable regions. In other specific embodiments, one of
the residues
that forms a particular disulfide bond is replaced (or deleted) or,
alternatively, both residues
that form a particular disulfide bond may be replaced (or deleted).
In other embodiments, the invention provides immunoglobulin molecules that
have
one or more amino acid substitutions relative to the second immunoglobulin
molecule of a
disulfide bond forming residue in the variable region with an amino acid
residue that does
not contain a sulfhydryl group and that additionally have one or more other
amino acid
substitutions (i.e., that are not a replacement of a disulfide bond forming
residue with a
residue that does not contain a sulfhydryl group).
In particular, the invention provides a first immunoglobulin molecule
comprising a
variable region and which is identical, except for one or more amino acid
substitutions in
said variable region, to a second immunoglobulin molecule, said second
immunoglobulin
molecule being capable of immunospecifically binding an antigen of a cell or
protein
associated with reproductive function or that has at least one CDR that
contains a portionof
an antigen of a cell or protein associated with reproductive function, in
which at least one of
said one or more amino acid substitutions are the substitution of an amino
acid residue that
does not have a sulfhydryl group at one or more positions corresponding to one
or more
cysteine residues that form a disulfide bond in said second immunoglobulin
molecule.
In a preferred embodiment, the amino acid substitutions that are not the
substitution
of a disulfide bond forming cysteine residue with a residue that does not have
a sulfliydryl
group, are not stabilizing changes. Stabilizing changes are defined as those
amino acid
changes that increase the stability of the antibody molecule. Such stabilizing
amino acid
changes are those changes that substitute an amino acid that is not common at
that particular
position in the particular antibody molecule (e.g., as defined by the
consensus sequences for
a number of antibody molecules provided in Kabat et al., 1991, Sequences of
Proteins of
Immunological Interest, 5th Ed., U.S. Department of Health and Human Services,
Bethesda,
Maryland) with a residue that is common at that particular position, e.g., is
the amino acid
at that position in the consensus sequence for that antibody molecule (see PCT
Publication
WO 96/02574, dated February 1, 1996 by Steipe et al.).
Such other amino acid substitutions can be any amino acid substitution that
does not
alter the ability of the modified immunoglobulin to elicit the formation of
anti-anti-idiotype
antibodies, e.g., as determined, for example, as described in Section 5.5,
infra. For
example, such other amino acid substitutions include substitutions of
functionally
equivalent amino acid residues. For example, one or more amino acid residues
can be
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substituted by another amino acid of a similar polarity which acts as a
functional equivalent.
Substitutes for an amino acid within the sequence may be selected from other
members of
the class to which the amino acid belongs. For example, the nonpolar
!hydrophobic) amino
acids include alanine, Ieucine, isoleucine, valine, proline, phenylalanine.
tr<~ptophan and
methionine. The polar neutral amino acids include glycine. serine, threonine,
cysteine,
tyrosine, asparagine, and glutamine. The positively charged (basic) amino
acids include
arginine, lysine and histidine. The negatively charged (acidic) amino acids
include aspartic
acid and glutamic acid.
The modified immunoglobulin is derived from an antibody that has one or more
CDRs containing a portion of an antigen of a cell or protein associated with
reproductive
function. In specific embodiments, the antigen is a sperm antigen, preferably
SP-10. Other
antigens include lactate dehydrogenase LDH-C4, SP-17, PH-20, FA-l, FA-2, PH-
30, RSA,
HAS-63, MSA-63, or zona pellucida proteins ZPl, ZP2, and ZP3 (see, e.Q.,
Freemetman et
al., 1993, Molecular Reproduction and Development 34:140-148; Heir et al.,
1990, Biol.
Reproduction 42:181-193; O'Hern et al., 1995, Biol. Reproduction 52:331-339;
Anderson
et al., 1986, J. Reprod. Immunol. 10:231-257; Wright et al., 1990, Biology of
Reproduction
42:693-701; Lea et al., 1997, Fertility and Sterility 67:355-361; O'Hern et
al., Elsevier
Science Ltd. 16:1761-1766; Ken, 1995, Reprod. Fertil. Dev. 7:825-830; Kaul et
al., 1996,
Reprod. FertiI Dev. SO:I27-134; Liu et al., 1990, Molecular Reproduction and
Development
25:302-308; Bambra, 1992, Scand. J. Immunol. 11:1 Z 8-122) or another antigen
of a cell or
protein associated with reproductive function, for example but not limited to
gonadotropin-
releasing hormone, any gonadotropin, prostaglandin F2 alpha, oxytocin, and
gonadotropin 1
receptors.
The immunoglobulin molecules of the invention can be of any tye, class, or
subclass of immunoglobulin molecules. In a preferred embodiment, the
immunoglobulin
molecule is an antibody molecule, more preferably of a type selected from the
group
consisting of IgG, IgE, IgM, IgD and IgA, most preferably is an IgG molecule.
Alternatively, the immunoglobulin molecule is a T cell receptor, a B cell
receptor, a cell-
surface adhesion molecule such as the co-receptors CD4, CDB, or CD19, or an
invariant
domain of an MHC molecule.
The modified immunoglobulin can be derived from any naturally occurring
antibody, preferably a monoclonal antibody, or can be derived from a synthetic
or
engineered antibody. Specifically, the modified immunoglobulin molecules are
derived
from an antibody in which a portion of an antigen of a cell or protein
associated with
reproductive function is inserted into or replaces ail or a portion of one of
the CDRs in the
variable region, for example as described in co-pending United States Patent
application
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WO 00/29443 PCT/US99/26671
Serial No., entitled "Immunoglobulin Molecules Having .-~ Synthetic Variable
Region And
Modified Specificity", by Burch, filed November 13, 1998 (attorney docket no.
6750-016),
which is incorporated by reference herein in its entirety.
In particular, the synthetic antibodies are antibodies that in which at least
one of the
S CDRs of the antibody contains an antigen of a cell or protein associated
with reproductive
function. In one aspect of the invention, the amino acid sequence of the
antigen is not
found naturally within the CDR. One or more CDRs may also contain a binding
site for a
cell or protein involved in reproductive function.
The amino acid sequence of the binding site may be identified by any method
known in the art. For example, in some instances, the sequence of a member of
a binding
pair has already been determined to be directly involved in binding the other
member of the
binding pair. In this case, such a sequence can be used to construct the CDR
of a synthetic
antibody that specifically recognizes the other member of the binding pair. If
the amino
acid sequence for the binding site in the one member of the binding pair for
the other
member of the binding pair is not known, it can be determined by any method
known in the
art, for example, but not limited to, molecular modeling methods or empirical
methods, e.g.,
by assaying portions (e.g., peptides) of the member for binding to the other
member, or by
making mutations in the member and determining which mutations prevent
binding.
The binding pair can be any two molecules, including proteins, nucleic acids,
carbohydrates, or lipids, that interact with each other, although preferably
the binding
partner from which the binding site is derived is a protein molecule. In
preferred
embodiments, the modified immunoglobulin contains a binding sequence for a
cancer
antigen, an infectious disease antigen, a cellular receptor for a pathogen, or
a receptor or
ligand that participates in a receptor-ligand binding pair.
In specific embodiments, the binding pair is a protein-protein interaction
pair which
is either homotypic interaction (i.e., is the interaction between two of the
same proteins} or a
heterotypic interaction (i.e., is the interaction between two different
proteins).
The synthetic antibody may be built upon (i.e., the binding site sequences
inserted
into the CDR of) the sequence of a naturally occurring or previously existing
antibody or
may be synthesized from known antibody consensus sequences, such as the
consensus
sequences for the light and heavy chain variable regions in Figures 7A and B,
or any other
antibody consensus or germline (i.e., unrecombined genomic sequences)
sequences (e.g.,
those antibody consensus and germline sequences described in Kabat et al.,
1991,
Sequences of Proteins of Immunological Interest, S'h edition, NIH Publication
No. 9I-3242,
pp 2147-2172).
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Each antibody molecule has six CDR sequences, three on the light chain and
three
on the heavy chain, and five of these CDRs are germline CDRs (i.e., are
directly derived
from the getmline genomic sequence of the animal, without any recombination)
and one of
the CDRs is a non-germline CDR (i.e., differs in sequence from the germline
genomic
sequence of the animal and is generated by recombination of the germline
sequences).
Whether a CDR is a germline or non-germline sequence can be determined by
sequencing
the CDR and then comparing the sequence with known germline sequences, e.g.,
as listed in
Kabat et al. (1991, Sequences of Proteins of Immunological Interest, 5'"
edition, NIH
Publication No. 91-3242, pp 2147-2172). Significant variation from the known
germline
sequences indicates that the CDR is a non-germline CDR. Accordingly, the CDR
that
contains the amino acid sequence of the binding site or antigen is a germline
CDR or,
alternatively, is a non-germline CDR.
The binding site or antigen sequence can be inserted into any of the CDRs of
the
antibody, and it is within the skill in the art to insert the binding site
into different CDRs of
the antibody and then screen the resulting modified antibodies for the ability
to bind to the
particular member of the binding pair, e.g. as discussed in Section, infra, or
to elicit an
immune response against the antigenic site, e.g., as described in Section,
infra. Thus, one
can determine which CDR optimally contains the binding site or antigen. In
specif c
embodiments, a CDR of either the heavy or light chain variable region is
modified to
contain the amino acid sequence of the binding site or antigen. In another
specific
embodiment, the modified antibody contains a variable domain in which the
first, second or
third CDR of the heavy variable region or the first, second or third CDR of
the light chain
variable region contains the amino acid sequence of the binding site or
antigen. In another
embodiment of the in~~ention, more than one CDR contains the amino acid
sequence of the
binding site or antigen or more than one CDR each contains a different binding
site for the
same molecule or contains a different binding site for a different molecule.
In particular,
embodiments, two, three, four, five or six CDRs have been engineered to
contain a binding
site for the first member of the binding pair. In a preferred embodiment, one
of the CDRs
contains a portion of one sperm antigen and another CDR contains a portion of
a second
sperm antigen, more particularly, where one sperm antigen in SP-10 and the
other sperm
antigen is MSA-63 or LHD-Gs.
In specific embodiments of the invention, the binding site or antigen amino
acid
sequence is either inserted into the CDR without replacing any of the amino
acid sequence
of the CDR itself or, alternatively, the binding site or antigen amino acid
sequence replaces
all or a portion of the amino acid sequence of the CDR. In specific
embodiments, the
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binding site amino acid sequence replaces l, 2, S, 8, 10, 15, or 20 amino
acids of the CDR
sequence.
The amino acid sequence of the binding site or antigen present in the CDR can
be
the minimal binding site necessary for the binding of the member of the
binding pair or for
eliciting an immune response against the antigen(which can be determined
empirically by
any method known in the art); alternatively, the sequence can be greater than
the minimal
binding site or antigen sequence necessary for the binding of the member of
the binding pair
or eliciting of an immune response against the antigen. In particular
embodiments, the
binding site or antigen amino acid sequence is at least 4 amino acids in
length, or is at least
6, 8, 10, 15, or 20 amino acids in length. In other embodiments the binding
site amino acid
sequence is no more than 10, 15, 20, or 25 amino acids in length, or is ~-10,
5-15, 5-20, 10-
15, IO-20 or 10-25 amino acids in length.
In addition, the total length of the CDR (i.e., the combined length of the
binding site
sequence and the rest of the CDR sequence) should be of an appropriate number
of amino
acids to allow binding of the antibody to the antigen. CDRs have been observed
to have a
range of numbers of amino acid residues, and the observed size ranges for the
CDRs (as
denoted by the abbreviations indicated in figure 2) are provided in Table 1.
Table 1
CDR Number of residues
L1 10-I7
L2 7
L3 7-11
H1 5_7
H2 9-12
H3 2-25
(compiled from data in Kabat and Wu, 1971, Ann. NYAcad. Sci.
190:382-93)
While many CDR H3 regions are of 5-9 residue in length, certain CDR H3 regions
have
been observed that are much longer. In particular, a number of antiviral
antibodies have
heavy chain CDR H3 regions of 17-24 residues in length.
Accordingly, in specific embodiments of the invention, the CDR containing the
binding site or antigen portion is within the size range provided for that
particular CDR in
Table 1, i.e., if it is the first CDR of the light chain, L1, the CDR is 10 to
17 amino acid
residues: if it is the second CDR of the light chain, L2, the CDR is 7 amino
acid residues; if
it is the third CDR of the light chain, L3, the CDR is 7 to 11 amino acid
residues; if it is the
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first CDR of the heavy chain, H1, the CDR is ~ to 7 amino acid residues: if it
is the second
CDR of the heavy chain, H2, the CDR is 9 to 12 amino acid residues; and if it
is the third
CDR of the heavy chain, H3, the CDR is 2 to 25 amino acid residues. In other
specific
embodiments, the CDR containing the binding site is ~-10. ~-15, 5-20, 11-15, 1
I-20, 11-25,
or 16-25 amino acids in length. In other embodiments, the CDR containing the
binding site
is at least 5, 10, 15, or 20 amino acids or is no more than 10, 1 S, 20, 25,
or 30 amino acids
in length.
After constructing antibodies containing modified CDRs, the modified
antibodies
can be further altered and screened to select an antibody having higher
affinity or
specificity. Antibodies having higher affinity or specificity for the target
antigen may be
generated and selected by any method known in the art. For example, but not by
way of
limitation, the nucleic acid encoding the synthetic modified antibody can be
mutagenized,
either randomly, i. e., by chemical or site-directed mutagenesis, or by making
particular
mutations at specific positions in the nucleic acid encoding the modified
antibody, and then
1 S screening the antibodies exposed from the mutated nucleic acid molecules
for binding
affinity for the target antigen. Screening can be accomplished by testing the
expressed
antibody molecules individually or by screening a library of the mutated
sequences, e.g., by
phage display techniques (see, e.g., U. S. Patent Nos. 5,223,409; 5,403,484;
and 5,571,698,
all by Ladner et al; PCT Publication WO 92/01047 by McCafferty et al. or any
other phage
display technique known in the art).
In specific embodiments, the invention provides a functionally active
fragment,
derivative or analog of the modified immunoglobulin molecules of the
invention.
Functionally active means that the fragment, derivative or analog is able to
elicit anti-anti-
idiotype antibodies (i.e., tertiary antibodies or Ab3 antibodies) that
recognize the same
antigen that the antibody from which the fragment, derivative or analog is
derived
recognized (e.g., as determined by the methods described in Section 5.4,
infra).
Specifically, in a preferred embodiment, the antigenicity of the idiotype of
the
immunoglobulin molecule may be enhanced by deletion of framework and CDR
sequences
that are N-terminal to the particular CDR sequence that specifically
recognizes the antigen.
To determine which CDR sequences bind the antigen, synthetic peptides
containing the
CDR sequences can be used in binding assays with the antigen by any binding
assay
method known in the art. Accordingly, in a preferred embodiment, the invention
includes
modified immunoglobulin molecules that have one disulfide bond forming
cysteine residue
in a variable region domain replaced with an amino acid residue that does not
contain a
sulfhydryl group and in which a portion of that variable domain has been
deleted N-terminal
to the CDR sequence that recognizes the antigen.
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Other embodiments of the invention include fragments of the modified
antibodies of
the invention such as. but not limited to, F(ab')2 fragments, which contain
the variable
region, the light chain constant region and the CHI domain of the heavy chain
can be
produced by pepsin digestion of the antibody molecule, and Fab fragments,
which can be
generated by reducing the disulfide bridges of the F(ab'), fragments. The
invention also
provides heavy chain and light chain dimers of the modified antibodies of the
invention, or
any minimal fragment thereof such as Fvs or single chain antibodies (SCAB)
(e.g., as
described in U.S. Patent 4,946,778; Bird, 1988, Science 242:423-42; Huston et
al., 1988,
Proc. Natl. Acad. Sci. USA 85:879-5883; and Ward et al., 1989, Nature 334:544-
54), or
any other molecule with the same specificity as the modified antibody of the
invention.
Techniques have been developed for the production of "chimeric antibodies"
(Morrison et al., 1984, Proc. Natl. Acad. Sci. 81:851-855; Neuberger et aL,
1984, Nature
312:604-608; Takeda et al., 1985, Nature 314:452-454) by splicing the genes
from a mouse
antibody molecule of appropriate antigen specificity together with genes from
a human
antibody molecule of appropriate biological activity. A chimeric antibody is a
molecule in
which different portions are derived from different animal species, such as
those having a
variable region derived from a marine monoclonal antibody and a constant
domain from a
human immunoglobulin, e.g., humanized antibodies.
In a preferred embodiment, the modified immunogIobulin of the invention is a
hm'n~zed antibody, more preferably an antibody having a variable domain in
which the
framework regions are from a human antibody and the CDRs are from an antibody
of a non-
human animal, preferably a mouse (see, International Patent Application No.
PCT/GB8500392 by Neuberger et al. and Celltech Limited).
CDR grafting is another method of humanizing antibodies. It involves reshaping
marine antibodies in order to transfer full antigen specificity and binding
affinity to a
human framework (Winter et al. U.S. Patent No. 5,225,539). CDR-grafted
antibodies have
been successfully constructed against various antigens, for example,
antibodies against IL-2
receptor as described in Queen et al., 1989 (Proc. Natl. Acad. Sci. USA
86:I0029);
antibodies against cell surface receptors-CAMPATH as described in Riechmann et
al.
(1988, Nature, 332:323); antibodies against hepatitis B in Cole et al. (1991,
Proc. Natl.
Acad. Sci. USA 88:2869); as well as against viral antigens-respiratory
syncitial virus in
Tempest et al. (1991, Bio-Technology 9:267). CDR-grafted antibodies are
generated in
which the CDRs of the marine monoclonal antibody are grafted into a human
antibody.
Following grafting, most antibodies benefit from additional amino acid changes
in the
framework region to maintain affinity, presumably because framework residues
are
necessary to maintain CDR conformation, and some framework residues have been
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demonstrated to be part of the antigen binding site. However, in order to
preserve the
framework region so as not to introduce any antigenic site, the sequence is
compared with
established germline sequences followed by computer modeling.
In other embodiments, the invention provides fusion proteins of the modified
immunoglobulins of the invention (or functionally active fragments thereof),
for example in
which the modified immunoglobulin is fused via a covalent bond (e.g., a
peptide bond), at
either the N-terminus or the C-terminus to an amino acid sequence of another
protein (or
portion thereof, preferably an at least 10, 20 or ~0 amino acid portion of the
protein) that is
not the modified immunoglobulin. Preferably the modified immunoglobulin, or
fragment
thereof, is covalentIy linked to the other protein at the N-terminus of the
constant domain.
In preferred embodiments. the invention provides fusion proteins in which the
modified
immunoglobulin is covalently linked to IL-2, IL-4, IL-5, IL-6, IL-7, IL-10, y-
interferon,
MHC derived peptide, G-CSF, a porin, TNF, NK cell antigens, or cellular
endocytosis
receptor.
The modified immunoglobulins of the invention include analogs and derivatives
that
are either modified, i.e, by the covalent attachment of any type of molecule
as long as such
covalent attachment does not prevent the modified immunoglobulin from
generating an
anti-idiotypic response (e.g., as determined by any of the methods described
in Section 5.5,
infra). For example, but not by way of limitation, the derivatives and analogs
of the
modified immunoglobulins include those that have been further modified, e.g.,
by
glycosylation, acetylation, pegylation, phosphylation, amidation,
derivatization by known
protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand
or other
protein, etc. Any of numerous chemical modifications may be carried out by
known
techniques, including, but not limited to specif c chemical cleavage,
acetylation,
formylation, metabolic synthesis of tunicamycin, etc. Additionally, the analog
or derivative
may contain one or more non-classical amino acids, e.g., as listed above in
this Section.
Methods of producing the modified immunoglobulins, and fragments, analogs, and
derivatives thereof, are described in Section 5.4, infra.
5.2. CONTRACEPTIVE METHODS
The present in~~ention provides methods of contraception by eliciting
production of
anti-idiotype antibodies and anti-anti-idiotype antibodies in a subject by the
administration
of a therapeutic (termed herein "Therapeutic"). Such Therapeutics include the
modified
immunoglobulins of the invention. and functionally active fragments, analogs,
and
derivatives thereof (e.g.. as described in Section ~.1, supra), and nucleic
acids encoding the
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modified antibodies of the invention, and functionally active fragments and
derivatives
thereof (e.g., as described in Section 5.1, supra).
Generally. administration of products of a species origin or species
reactivity that is
the same species as that of the subject is preferred. Thus, in a preferred
embodiment, the
methods of the invention use a modified antibody that is derived from a human
antibody; in
other embodiments, the methods of the invention use a modified antibody that
is derived
from a chimeric or humanized antibody.
Specifically, vaccine compositions (e.g., as described in Section ~.3, infra)
containing the modified antibodies of the invention are administered to the
subject to elicit
the production of an antibody (i.e., the anti-idiotype antibody or Ab2) that
specifically
recognizes the idiotype of the modified antibody, the Ab2, in turn, elicits
the production
anti-anti-idiotype antibodies (Ab3) that specifically recognize the idiotype
of Ab2, such that
these Ab3 antibodies have the same or similar binding specificity as the
modified antibody.
The invention provides methods of administering the modified antibodies of the
invention to elicit an anti-idiotype response, i.e., to generate Ab2 and Ab3
type antibodies.
Alternatively, the invention provides methods of administering the modified
antibodies of
the invention to one subject to generate Ab2 antibodies, isolating the Ab2
antibodies, and
then administering the Ab2 antibodies to a second subject to generate Ab3 type
antibodies
in that second subject.
Accordingly, the invention provides a method of generating an anti-idiotype
response in a subject comprising administering an amount of first
immunoglobulin
molecule (or functionally active fragment, analog, or derivative thereof)
sufficient to induce
an anti-idiotype response, said first immunoglobulin comprising a variable
region and being
identical, except for one or more amino acid substitutions in said variable
region, to a
second immunoglobulin molecule, said second immunoglobulin molecule being
capable of
immunospecifically binding an antigen, said one or more amino acid
substitutions being the
substitution of an amino acid residue that does not have a sulfhydryl group at
one or more
positions corresponding to one or more cysteine residues that form a disulfide
bond in said
second immunoglobulin molecule. In another embodiment, the method further
provides
isolating the anti-idiotype antibody that recognizes the idiotype of said
second
immunoglobulin molecule, and administering to a second subject the anti-
idiotype antibody.
Modified immunoglobulins capable of inhibiting the gamete interaction i.e., of
eggs
and sperm are preferably employed. The key to this method of contraception is
to either
immunologically regulate molecules involved in reproduction or to inhibit
fertilization.
Such contraceptive vaccines target reproductive hormone or receptor-specific
antigens or
gamete-specific antigens. The goal is to elicit an immune response which
targets
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reproductive hormones or receptors or native gamete molecules. In prefered
embodiments,
the vaccine targets sperm by eliciting production of antibodies that recognize
sperm
antigens.
Fertility can be suppressed by immunization against a reproductive hormone or
receptor such as gonadotropin-releasing hormone, gonadotropins, prostaglandin
F2 alpha,
oxytocin and gonadotropin receptors.
Fertility can also be suppressed by immunization against gamete or embryonic
antigens. Fertilization is mediated through specific molecules of the sperm
and egg. In
mammals, the sperm and egg interact at an egg-specific extracellular matrix,
the zona
pellucida {ZP), and the sperm plasma membrane (Gupta et al., 1997, Hum.
Reprod. Update,
3(4):311-324). The zona pellucida comprises three glycoproteins ZPI, ZP2 and
ZP3 (Kaul
et al., 1997, Mol. Reprod. Dev. 47(2):140-147) which are target antigens for
designing
immunocontraceptives. Some of the sperm plasma membrane proteins which are
useful as
antigens for immunocontraception are PH-20 (Primakoff et al., 1997, Biol.
Reprod.,
56(5): I 142-1146) and PH-30 (Ken, Reprod. Fertil. Dev., 1995, 7(4):825-830).
Other sperm
proteins are SP-10 (Kurth et al., 1997, BioI. Reprod., 57(5):981-989) and SP-
17 (Adoyo et
al., 1997, Mol. Reprod. Dev., 47( 1 ):66-71 ). Other gamete proteins include
lactate
dehydrogenase-C4 (LDH-C4) (Bradley et al., Reprod. Fertil. Dev., 9(1):111-116)
and
fertilization antigen-1 (FA-1) {Zhu and Naz, Proc. Natl. Acad. Sci. USA.,
94(9):4704-
4709).
In particular, the contraceptive methods of the invention involve
administration of
modif ed immunoglobulin molecules (or functionally active fragments,
derivatives or an
analog thereof, or nucleic acids encoding the same) derived from an
immunoglobulin
molecule that specifically recognizes a molecule or cell involved in
reproductive function.
In a specific embodiment, the contraceptive methods of the invention involve
the
administration of a modified immunoglobulin molecule that is derived from an
antibody
that is capable of immunospecifically binding to gonadotropin-releasing
hormone, any
gonadotropin, prostaglandin F2 alpha, oxytocin, gonadotropin receptors, gamete
or
embryonic antigens, sperm antigens, preferably SP-10. Other antigens include,
but are not
limited to, lactate dehydrogenase LDH-C4, SP-17, PH-20, FA-l, FA-2, PH-30,
RSA, HAS-
63, MSA-63, or zona pellucida proteins ZP1, ZP2, and ZP3 (see, e.g.,
Freemerman et al.,
1993, Molecular Reproduction and Development 34:140-148; Hen et al., 1990.
Biol.
Reproduction 42:181-193; O'Hern et al., 1995, Biol. Reproduction 52:331-339;
Anderson
et al., 1986, J. Reprod. Immunol. 10:231-257; Wright et al., 1990, Biology of
Reproduction
42:693-701; Lea et al., 1997, Fertility and Sterility 67:355-361; O'Hern et
al.. Elsevier
Science Ltd. 16:1761-1766; Kerr, 1995, Reprod. Fertil. Dev. 7:825-830; Kaul et
al., 1996,
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Reprod. Fertil Dev. 50:127-134; Liu et al., 1990, Molecular Reproduction and
Development
25:302-308; Bambra, 1992, Scand. J. Immunol. 11:118-122).
The invention also includes contraceptive methods whereby a modified
immunoglobulin of the invention is administered in conjunction with use of
another
contraceptive method. such as, but not limited to, barrier methods such as the
use of
condoms or diaphragms or cervical caps, or intravaginal use of contraceptive
compounds
such as, but not limited to, non-oxynol-9, intrauterine devices, birth control
pills or
implants, etc.
The invention also includes administrations of anti-anti-idiotype antibodies
against a
modified immunoglobulin of the invention to acutely neutralize the
contraceptive activity of
the modified immunoglobulin.
The methods and vaccine compositions of the present invention may be used to
elicit a humoral and/or a cell-mediated response against a modified
immunoglobulin in a
subject. In one specific embodiment, the methods and compositions of the
invention elicit a
humoral response in a subject. In another specif c embodiment, the methods and
compositions of the invention elicit a cell-mediated response in a subject. In
a preferred
embodiment, the methods and compositions of the invention elicit both a
humoral and a
cell-mediated response.
The subjects to which the present invention is applicable may be any mammalian
or
vertebrate species, which include, but are not limited to, cows, horses,
sheep, pigs, fowl
(e.g., chickens), goats, cats, dogs, hamsters, mice, rats, monkeys, rabbits,
chimpanzees, and
humans. In a preferred embodiment, the subject is a human.
5.2.1. GENE THERAPY
Gene therapy may be used by administering a nucleic acid containing a
nucleotide
sequence encoding the modified immunoglobulin of the invention as a
contraceptive. In
this embodiment of the invention, the therapeutic nucleic acid encodes a
sequence that
produces intracellulariy (without a leader sequence) or intercellularly (with
a leader
sequence), a modified immunogIobulin.
For general reviews of the methods of gene therapy, see Goldspiel et al.,
1993,
Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy 3:87-95; Tolstoshev,
1993,
Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan, 1993, Science 260:926-932;
and
Morgan and Anderson. 1993, Ann. Rev. Biochem. 62:191-217). Methods commonly
known
in the art of recombinant DNA technology which can be used are described in
Ausubel et al.
(eds.), 1993, Current Protocols in Molecular Biology, John Wiley & Sons, NY;
Kriegler,
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1990, Gene Transj"er and E.tpression. A Laboratory Manual, Stockton Press, NY;
and in
Chapters I2 and 13, Dracopoli et al. (eds), 1994, Current Protocols in Human
Genetics,
John Wiley & Sons. NY).
In one aspect, the therapeutic nucleic acid comprises an expression vector
that
expresses the modified immunoglobulin molecule.
Delivery of the nucleic acid into a patient may be either direct, in which
case the
patient is directly exposed to the nucleic acid or nucleic acid-carrying
vector or a delivery
complex, or indirect. in which case. cells are first transformed with the
nucleic acid in vitro,
then transplanted into the patient. These two approaches are known,
respectively, as in vivo
or ex vivo gene therapy.
In a specific embodiment, the nucleic acid is directly administered in vivo,
where it
is expressed to produce the antibodies. This can be accomplished by any of
numerous
methods known in the art, e.g., by constructing it as part of an appropriate
nucleic acid
expression vector and administering it so that it becomes intracellular, e.g.,
by infection
using a defective or attenuated retroviral or other viral vector (see U.S.
Patent No.
4,980,286}, or by direct injection of naked DNA, or by use of microparticle
bombardment
(e.g., a gene gun; Biolistic, Dupont), or coating with lipids or cell-surface
receptors or
transfecting agents, encapsulation in biopolymers (e.g., poly-13-1->4-N-
acetylglucosamine
polysaccharide; see U.S. Patent No. 5,635,493), encapsulation in liposomes,
microparticles,
or microcapsules, or by administering it in linkage to a peptide which is
known to enter the
nucleus, by administering it in linkage to a ligand which is known to enter
the nucleus, by
administering it in linkage to a ligand subject to receptor-mediated
endocytosis (see e.g.,
Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432), etc. In another embodiment, a
nucleic
acid-ligand complex can be formed in which the ligand comprises a fusogenic
viral peptide
to disrupt endosomes, allowing the nucleic acid to avoid lysosomal
degradation. In yet
another embodiment. the nucleic acid can be targeted in vivo for cell specific
uptake and
expression, by targeting a specific receptor (see, e.g., PCT Publications WO
92/06180 dated
April 16, 1992 (Wu et al.); WO 92/22635 dated December 23, 1992 (Wilson et
al.);
W092/20316 dated November 26, 1992 (Findeis et al.); W093/14188 dated July 22,
1993
(Young). Alternatively, the nucleic acid can be introduced intracellularly and
incorporated
within host cell DNA for expression, by homologous recombination (Koller and
Smithies,
1989, Proc. Natl. Acad. Sci. USA 86:8932-8935; Zijlstra et al., 1989, Nature
342:435-438).
Alternatively, single chain antibodies, such as neutralizing antibodies, which
bind to
intracellular epitopes can also be administered. Such single chain antibodies
can be
administered, for example, by expressing nucleotide sequences encoding single-
chain
antibodies within the target cell population by utilizing, for example,
techniques such as
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those described in Marasco et al. (Marasco et al., 1993, Proc. Natl. Acad.
Sci. USA 90:7889-
7893). Adenoviruses are other viral vectors that can be used in gene therapy.
Adenoviruses
are especially attractive vehicles for delivering genes to respiratory
epithelia where they
cause a mild disease. Other targets for adenovirus-based delivery systems are
liver, the
central nervous system, endothelial cells, and muscle. Adenoviruses have the
advantage of
being capable of infecting non-dividing cells. Kozarsky and Wilson. 1993,
Current
Opinion in Genetics and Development 3:499-503 present a review of adenovirus-
based gene
therapy. Bout et al., 1994, Human Gene Therapy 5:3-10 demonstrated the use of
adenovirus vectors to transfer genes to the respiratory epithelia of rhesus
monkeys. Other
instances of the use of adenoviruses in gene therapy can be found in Rosenfeld
et al., 1991,
Science 252:431-434; Rosenfeld et al., 1992, Cell 68:143-155; and Mastrangeli
et al., 1993,
J. Clin. Invest. 91:225-234. Adeno-associated virus (AAV) has also been
proposed for use
in gene therapy (Walsh et al., 1993, Proc. Soc. Exp. Biol. Med. 204:289-300).
The fornn and amount of therapeutic nucleic acid envisioned for use depends on
the
type of disease and the severity of its desired effect, patient state, etc.,
and can be
determined by one skilled in the art.
5.3. VACCINE FORMULATIONS AND ADMINISTRATION
The invention also provides vaccine formulations containing Therapeutics of
the
invention, which vaccine formulations are suitable for administration to
elicit a protective
immune (humoraI and/or cell mediated) response against certain antigens, e.g.,
for the
contraceptive uses described herein.
Suitable preparations of such vaccines include injectables, either as liquid
solutions
or suspensions; solid forms suitable for solution in, or suspension in, liquid
prior to
injection, may also be prepared. The preparation may also be emulsified, or
the
polypeptides encapsulated in liposomes. The active immunogenic ingredients are
often
mixed with excipients which are pharmaceutically acceptable and compatible
with the
active ingredient. Suitable excipients are, far example, water, saline,
buffered saline,
dextrose, glycerol, ethanol, sterile isotonic aqueous buffer or the like and
combinations
thereof. In addition, if desired, the vaccine preparation may also include
minor amounts of
auxiliary substances such as wetting or emulsifying agents, pH buffering
agents, and/or
adjuvants which enhance the effectiveness of the vaccine.
Examples of adjuvants which may be effective, include, but are not limited to:
aluminim hydroxide, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-
acetyl-
nor-muramyl-L-alanyl-D-isoglutamine, N-acetylmuramyl-L-alanyl-D-isogIutaminyl-
L-
alanine-2-( 1'-2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine.
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The effectiveness of an adjuvant may be determined by measuring the induction
of
anti-idiotype antibodies directed against the injected immunoglobulin
formulated with the
particular adjuvant.
The composition can be a liquid solution, suspension. emulsion, tablet, pill,
capsule,
sustained release formulation, or powder. Oral formulation can include
standard carriers
such as pharmaceutical grades of mannitol, lactose, starch. magnesium
stearate, sodium
saccharine, cellulose, magnesium carbonate; etc.
Generally, the ingredients are supplied either separately or mixed together in
unit
dosage form, for example, as a dry lyophilized powder or water-free
concentrate in a
hermetically sealed container such as an ampoule or sachette indicating the
quantity of
active agent. Where the composition is administered by injection, an ampoule
of sterile
diluent can be provided so that the ingredients may be mixed prior to
administration.
In a specific embodiment, the lyophilized modified immunoglobulin of the
invention is provided in a first container; a second container comprises
diluent consisting of
an aqueous solution of 50% glycerin, 0.25% phenol, and an antiseptic (e.g.,
0.005% brilliant
green).
The invention also provides a pharmaceutical pack or kit comprising one or
more
containers filled with one or more of the ingredients of the vaccine
formulations of the
invention. Associated with such containers) can be a notice in the form
prescribed by a
governmental agency regulating the manufacture, use or sale of pharmaceuticals
or
biological products, which notice reflects approval by the agency of
manufacture, use or
sale for human administration.
The compositions may, if desired, be presented in a pack or dispenser device
which
may contain one or more unit dosage forms containing the active ingredient.
The pack may
for example comprise metal or plastic foil, such as a blister pack. The pack
or dispenser
device may be accompanied by instructions for administration. Composition
comprising a
compound of the invention formulated in a compatible pharmaceutical carrier
may also he
prepared, placed in an appropriate container, and labeled for treatment of an
indicated
condition.
The subject to which the vaccine is administered is preferably a mammal, most
preferably a human, but can also be a non-human animal including but not
limited to cows,
horses, sheep, pigs, fowl (e.g., chickens), goats, cats, dogs, hamsters, mice
and rats.
Many methods may be used to introduce the vaccine formulations of the
invention;
these include but are not limited to oral, intracerebral, intradermal,
intramuscular,
intraperitoneal, intravenous, subcutaneous, intranasal routes. and via
scarification
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(scratching through the top layers of skin, e.g., using a bifurcated needle)
or any other
standard routes of immunization. In a specific embodiment, scarification is
employed.
The precise dose of the modif ed immunoglobulin molecule to be employed in the
formulation will also depend on the route of administration, and the nature of
the patient,
and should be decided according to the judgment of the practitioner and each
patient's
circumstances according to standard clinical techniques. An effective
immunizing amount
is that amount sufficient to produce an immune response to the modified
immunoglobulin
molecule in the host (i.e., an anti-idiotype reaction) to which the vaccine
preparation is
administered. Effective doses may also be extrapolated from dose-response
curves derived
from animal model test systems.
5.4. METHOD OF PRODUCING THE MODIFIED IMMUNOGLOBULINS
The modified immunoglobulins of the invention can be produced by any method
known in the art for the synthesis of immunoglobulins, in particular, by
chemical synthesis
or by recombinant expression, and is preferably produced by recombinant
expression
techniques.
Recombinant expression of the modified immunoglobulin of the invention, or
fragment, derivative or analog thereof, requires construction of a nucleic
acid that encodes
the modified immunoglobulin. If the nucleotide sequence of the modified
immunoglobulin
is known, a nucleic acid encoding the modified immunoglobulin may be assembled
from
chemically synthesized oligonucleotides (e.g., as described in Kutmeier et
al., 1994,
BioTechniques 17:242), which, briefly, involves the synthesis of overlapping
oligonucleotides containing portions of the sequence encoding the modified
immunoglobulin, annealing and Iigation of those oligonucleotides, and then
amplification of
the ligated oligonucleotides by PCR, e.g., as exemplified in Section 6, infra.
Alternatively, the nucleic acid encoding the modified immunoglobulin may be
generated from a nucleic acid encoding the immunoglobulin from which the
modified
immunoglobulin was derived. If a clone containing the nucleic acid encoding
the particular
immunoglobulin is not available, but the sequence of the immunoglobulin
molecule is
known, a nucleic acid encoding the immunoglobulin may be obtained from a
suitable
source (e.g., an antibody cDNA library, or cDNA library generated from any
tissue or cells
expressing the immunoglobulin) by PCR amplification using synthetic primers
hybridizable
to the 3' and 5' ends of the sequence or by hybridization using an
oligonucIeotide probe
specific for the particular gene sequence.
If an immunoglobulin molecule that specifically recognizes a particular
antigen is
not available (or a source for a cDNA library for cloning a nucleic acid
encoding such an
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WO 00/29443 PCT/US99/26671
immunoglobulin is not available), immunoglobulins specific for a particular
antigen may be
generated by any method known in the art. for example, by immunizing an
animal, such as
a rabbit, to generate polyclonal antibodies or, more preferably, by generating
monoclonal
antibodies, e.g., as described by Kohler and Milstein ( 1975, Nature 256:495-
497) or, as
S described by Kozbon et al. ( 1983, Immunology Todav 4:72) or Cole et al.
(1985 in
Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).
Alternatively,
a clone encoding at least the Fab portion of the immunoglobulin can be
obtained by
screening Fab expression libraries (e.g., as described in Huse et al., 1989,
Science
246:1275-1281 ) for clones of Fab fragments that bind the specific antigen or
by screening
antibody libraries (see, e.g.. Clackson et al., 1991, Nature 352:624; Hane et
al., 1997 Proc.
Natl. Acad. Sci. USA 94:4937).
Once a nucleic acid encoding at least the variable domain of the
immunoglobulin
molecule is obtained. it may be introduced into any available cloning vector,
and may be
introduced into a vector containing the nucleotide sequence encoding the
constant region of
the immunoglobuIin molecule (see, e.g., PCT Publication WO 86/05807; PCT
Publication
WO 89/01036; U.S. Patent No. 5,122,464; and Bebbington, 1991, Methods in
Enzymology
2:136-145). Vectors containing the complete light or heavy chain for co-
expression with
the nucleic acid to allow the expression of a complete antibody molecule are
also available,
see Id Then, the nucleic acid encoding the immunoglobulin can be modified to
introduce
the nucleotide substitutions or deletion necessary to substitute (or delete)
the one or more
variable region cysteine residues participating in an intrachain disulfide
bond with an amino
acid residue that does not contain a sulfhydyl group, along with any other
desired amino
acid substitutions, deletions or insertions. Such modifications can be carried
out by any
method known in the art for the introduction of specific mutations or
deletions in a
nucleotide sequence, for example, but not limited to, chemical muagenesis, in
vitro site
directed mutagenesis (Hutchinson et al., 1978, J. Biol. Chem. 253:6551 ), PCR
based
methods, etc.
In addition, techniques developed for the production of chimeric antibodies
(Morrison et al., 1984, Proc. Natl. Acad Sci. 81:851-855; Neuberger et al.,
1984, Nature
312:604-608; Takeda et al., 1985, Nature 314:452-454) by splicing genes from a
mouse
antibody molecule of appropriate antigen specificity together with genes from
a human
antibody molecule of appropriate biological activity can also be used. As
described supra, a
chimeric antibody is a molecule in which different portions are derived from
different
animal species, such as those having a variable region derived from a murine
monoclonal
antibody and a consmat region derived from a human immunoglobulin, e.g.,
humanized
antibodies.
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Alternatively, techniques described for the production of sinele chain
antibodies
(U.S. Patent 4,694,778; Bird, 1988, Science 242:423-42; Huston et al., 1988,
Proc. Natl.
Acad. Sci. USA 85:5879-5883; and Ward et al., 1989, Nature 334:544-~:1) can be
adapted to
produce single chain antibodies. Single chain antibodies are formed by linking
the heavy
and light chain fragments of the Fv region via an amino acid bridge, resulting
in a single
chain polypeptide. Techniques for the assembly of functional Fv fragments in
E. coli may
also be used (Skerra et al., 1988, Science 242:1038-1041).
Antibody fragments which recognize specific epitopes may be Qenerated by known
techniques. For example, such fragments include but are not limited to: the
F(ab')~
fragments which can be produced by pepsin digestion of the antibody molecule
and the Fab
fragments which can be generated by reducing the disulfide bridges of the
F(ab')Z
fragments.
Once a nucleic acid encoding the modified immunogIobulin molecule of the
invention has been obtained. the vector for the production of the
immunoglobulin molecule
may be produced by recombinant DNA technology using techniques well known in
the art.
The modified immunoglobulin molecule can then be recombinantly expressed and
isolated
by any method known in the art, for example, using the method described in
Section 6,
supra, (see also Bebbington, 1991, Methods in Enzymology 2:136-145). Briefly,
COS
cells, or any other appropriate cultured cells, can be transiently or non-
transiently
transfected with the expression vector encoding the modified immunoglobulin,
cultured for
an appropriate period of time to permit immunoglobulin expression, and then
the supernatan
can be harvested from the COS cells, which supernatant contains the secreted,
expressed
modified immunoglobulin.
Methods which are well known to those skilled in the art can be used to
construct
expression vectors containing the immunoglobulin molecule coding sequences and
appropriate transcriptional and translational control signals. These methods
include, for
example, in vitro recombinant DNA techniques, synthetic techniques, and in
vivo genetic
recombination. See, for example, the techniques described in Sambrook et a1.
(1990,
Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory,
Cold
Spring Harbor, NY) and Ausubel et aI. (eds., 1998, Current Protocols in
Molecular
Biology, John Wiley & Sons, NY).
The expression vector is transferred to a host cell by conventional techniques
and
the transfected cells are then cultured by conventional techniques to produce
the
immunoglobulin of the invention.
The host cells used to express the recombinant antibody of the invention may
be
either bacterial cells such as Escherichia coli, or, preferably, eukaryotic
cells, especially for
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the expression of whole recombinant immunoglobulin molecules. In particular,
mammalian
cells such as Chinese hamster ovary cells (CHO), in conjunction with a vector
such as the
major intermediate early gene promoter element from human cytomegalovirus is
an
effective expression system for immunoglobulins (Foeckin~ et al., 198, Gene
45:101;
Cockett et al., 1990, BiolTechnology 8:2).
A variety of host-expression vector systems may be utilized to express the
modified
immunoglobulin molecules of the invention. Such host-expression systems
represent
vehicles by which the coding sequences of interest may be produced and
subsequently
purified, but also represent cells which may, when transformed or transfected
with the
appropriate nucleotide coding sequences, express the immunoglobulin molecule
of the
invention in situ. These include but are not limited to microorganisms such as
bacteria
(e.g., E. coli, B. subtilis) transformed with recombinant bacteriophage DNA,
plasmid DNA
or cosmid DNA expression vectors containing immunoglobulin coding sequences;
yeast
(e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression
vectors
containing immunoglobulin coding sequences; insect cell systems infected with
recombinant virus expression vectors (e.g., baculovirus) containing the
immunoglobulin
coding sequences; plant cell systems infected with recombinant virus
expression vectors
(e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or
transformed with
recombinant plasmid expression vectors (e.g., Ti plasmid) containing
immunoglobulin
coding sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3
cells)
harboring recombinant expression constructs containing promoters derived from
the
genome of mammalian cells {e.g., metallothionein promoter) or from mammalian
viruses
(e.g., the adenovirus late promoter; the vaccinia virus 7.SK promoter).
In bacterial systems, a number of expression vectors may be advantageously
selected depending upon the use intended for the immunoglobulin molecule being
expressed. For example, when a large quantity of such a protein is to be
produced, for the
generation of pharmaceutical compositions of an immunoglobulin molecule,
vectors which
direct the expression of high levels of fusion protein products that are
readily purified may
be desirable. Such vectors include, but are not limited, to the E. coli
expression vector
p~78 (Ruther et al., 1983, EMBO J. 2:1791 ), in which the immunoglobulin
coding
sequence may be ligated individually into the vector in frame with the lac Z
coding region
so that a fusion protein is produced; pIN vectors (Inouye & Inouye, 1985,
Nucleic Acids
Res. 13:3101-3109; Van Heeke & Schuster, 1989. J. Biol. Chem. 24:5503-5509);
and the
like. pGEX vectors may also be used to express foreign polypeptides as fusion
proteins
with glutathione S-transferase (GST). In general, such fusion proteins are
soluble and can
easily be purified from lysed cells by adsorption and binding to a matrix
glutathione
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agarose beads followed by elution in the presence of free glutathione. The
pGEX vectors
are designed to include thrombin or factor Xa protease cleavage sites so that
the cloned
target gene product can be released from the GST moiety.
In an insect system. Aurographa californica nuclear polyhedrosis virus (AcNPV)
is
used as a vector to express foreign genes. The virus grows in Spodoptera
frugiperda cells.
The immunoglobulin coding sequence may be cloned individually into non-
essential
regions (for example the polyhedrin gene) of the virus and placed under
control of an
AcNPV promoter (for example the polyhedrin promoter}.
In mammalian host cells, a number of viral-based expression systems may be
utilized. In cases where an adenovirus is used as an expression vector, the
immunoglobulin
coding sequence of interest may be ligated to an adenovirus
transcription/translation control
complex, e.g., the late promoter and tripartite leader sequence. This chimeric
gene may
then be inserted in the adenovirus genome by in vitro or in vivo
recombination. Insertion in
a non-essential region of the viral genome (e.g., region E1 or E3) will result
in a
recombinant virus that is viable and capable of expressing the immunoglobulin
molecule in
infected hosts (e.g., see Logan & Shenk, 1984, Proc. Natl. Acad. Sci. USA
81:355-359).
Specific initiation signals may also be required for efficient translation of
inserted
immunoglobulin coding sequences. These signals include the ATG initiation
codon and
adjacent sequences. Furthermore, the initiation codon must be in phase with
the reading
frame of the desired coding sequence to ensure translation of the entire
insert. These
exogenous translationai control signals and initiation codons can be of a
variety of origins,
both natural and synthetic. The efficiency of expression may be enhanced by
the inclusion
of appropriate transcription enhancer elements, transcription terminators,
etc. (see Bittner et
al., 1987, Methods in Enrymol. 253:51-544).
In addition, a host cell strain may be chosen which modulates the expression
of the
inserted sequences, or modifies and processes the gene product in the specific
fashion
desired. Such modifications (e.g., glycosylation) and processing (e.g.,
cleavage) of protein
products may be important for the function of the protein. Different host
cells have charac-
teristic and specific mechanisms for the post-translational processing and
modification of
proteins and gene products. Appropriate cell lines or host systems can be
chosen to ensure
the correct modification and processing of the foreign protein expressed. To
this end,
eukaryotic host cells which possess the cellular machinery for proper
processing of the
primary transcript, glycosylation, and phosphorylation of the gene product may
be used.
Such mammalian host cells include but are not limited to CHO, VERO, BHK, HeLa,
COS,
MDCK, 293, 3T3, WI38.
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For long-term, high-yield production of recombinant proteins. stable
expression is
preferred. For example, cell lines which stably express the immunoglobulin
molecule may
be engineered. Rather than using expression vectors which contain viral
origins of
replication, host cells can be transformed with DNA controlled by appropriate
expression
control elements (e.g., promoter, enhancer, sequences, transcription
terminators,
polyadenylation sites, etc.), and a selectable marker. Following the
introduction of the
foreign DNA, engineered cells may be allowed to grow for 1-2 days in an
enriched media,
and then are switched to a selective media. The selectable marker in the
recombinant
plasmid confers resistance to the selection and allows cells to stably
integrate the plasmid
into their chromosomes and grow to form foci which in turn can be cloned and
expanded
into cell lines. This method may advantageously be used to engineer cell lines
which
express the immunoglobulin molecule. Such engineered cell lines may be
particularly
useful in screening and evaluation of compounds that interact directly or
indirectly with the
immunoglobulin molecule.
A number of selection systems may be used, including but not limited to the
herpes
simplex virus thymidine kinase (Wigler et al., 1977, Cell 11:223),
hypoxanthine-guanine
phosphoribosyltransferase (Szybalska & Szybalski, 192, Proc. Natl. Acad Sci.
USA
48:202), and adenine phosphoribosyltransferase (Lowy et al., 1980, Cell
22:817) genes can
be employed in tk-, hgprt- or aprt~ cells, respectively. Also, antimetabolite
resistance can be
used as the basis of selection for the following genes: dhfr, which confers
resistance to
methotrexate (Wigler et al., 1980, Natl. Acaa'. Sci. USA 77:357; O'Hare et
al., 1981, Froc.
Natl. Acad Sci. USA 78:I527); gpt, which confers resistance to mycophenolic
acid
(Mulligan & Berg, 1981, Proc. Natl. Acaa: Sci. USA 78:2072); neo, which
confers
resistance to the aminoglycoside G-418CIinical Pharmacy 12:488-505; Wu and Wu,
1991,
Biotherapy 3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-
596;
Mulligan, 1993, Science 260:926-932; and Morgan and Anderson, 1993, Ann. Rev.
Biochem. 62:191-2I7; May, 1993, TIBTECH 11(5):155-215). Methods commonly known
in the art of recombinant DNA technology which can be used are described in
Ausubel et aI.
(eds.), 1993, Current Protocols in Molecular Biology, John Wiley & Sons, NY;
Kriegler,
1990, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY;
and in
Chapters 12 and 13. Dracopoli et al. (eds), 1994, Current Protocols in Human
Genetics,
John Wiley & Sons. NY.; Colberre-Garapin et al., 1981, J. Mol. Biol. 150:1.
Alternatively, any fusion protein may be readily purified by utilizing an
antibody
specific for the fusion protein being expressed. For example, a system
described by
Janknecht et al. allows for the ready purification of non-denatured fusion
proteins expressed
in human cell lines (Janknecht et al., 1991, Proc. Natl. Acad. Sci. USA
88:8972-897}. In
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this system, the gene of interest is subcloned into a vaccinia recombination
plasmid such
that the open reading frame of the gene is translationally fused to an amino-
terminal tag
consisting of six histidine residues. The tag serves as a matrix binding
domain for the
fusion protein. Extracts from cells infected with recombinant vaccinia virus
are loaded onto
Ni'~'~nitriloacetic acid-agarose columns and histidine-tagged proteins are
selectively eluted
with imidazole-containing buffers.
The expression levels of the immunoglobulin molecule can be increased by
vector
amplification (for a review, see Bebbington and Hentschel, the Use of Vectors
Based on
Gene Ampliftcation for the Expression of Cloned Genes in Mammalian Cells in
DNA
Cloning, Vol.3. (Academic Press, New York, 1987)). When a marker in the vector
system
expressing immunogIobulin is amplifiable, increase in the level of inhibitor
present in
culture of host cell will increase the number of copies of the marker gene.
Since the
amplified region is associated with the immunoglobulin gene, production of the
immunoglobulin will also increase (Grouse et al., 1983, Mol. Cell. Biol.
3:257).
The host cell may be co-transfected with two expression vectors of the
invention, the
first vector encoding a heavy chain derived polypeptide and the~second vector
encoding a
light chain derived polypeptide. The two vectors may contain identical
selectable markers
which enable equal expression of heavy and light chain polypeptides.
Alternatively, a
single vector may be used which encodes both heavy and light chain
polypeptides. In such
situations, the light chain should be placed before the heavy chain to avoid
an excess of
toxic free heavy chain (Proudfoot, 1986, Nature 322:52; Kohler, 1980, Proc.
Natl. Acad.
Sci. USA 77:2197). The coding sequences for the heavy and light chains may
comprise
cDNA or genomic DNA.
Once the modified immunoglobulin molecule of the invention has been
recombinantly expressed, it may be purified by any method known in the art for
purification
of an immunoglobulin molecule, for example, by chromatography (e.g., ion
exchange,
affinity, particularly by affinity for the specific antigen after Protein A,
and sizing column
chromatography), centrifugation, differential solubility, or by any other
standard technique
for the purification of proteins.
5.5. DEMONSTRATION OF THERAPEUTIC UTILITY
The modified antibodies of the invention can be screened or assayed in a
variety of
ways for efficacy in treating or preventing a particular disease .
First, the immunopotency of a vaccine formulation containing the modified
antibody
of the invention can be determined by monitoring the anti-idiotypic response
of test animals
following immunization with the vaccine. Generation of a humoral response may
be taken
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as an indication of a generalized immune response. other components of which,
particularly
cell-mediated immunity, may also be important. Test animals may include mice,
rabbits,
chimpanzees and eventually human subjects. A vaccine made in this invention
can be made
to infect chimpanzees experimentally. However, since chimpanzees are a
protected species,
the antibody response to a vaccine of the invention can first be studied in a
number of
smaller, less expensive animals. with the goal of finding one or two best
candidate
immunogIobulin molecules or best combinations of immunoglobulin molecules to
use in
chimpanzee eff cacy studies.
The immune response of the test subjects can be analyzed by various approaches
such as the reactivity of the resultant immune serum to antibodies, as assayed
by known
techniques, e.g., enzyme linked immunosorbent assay (ELISA), immunoblots,
radioimmunoprecipitations, etc.; or protection from infection and/or
attenuation of disease
symptoms in immunized hosts.
As one example of suitable animal testing, the vaccine composition of the
invention
I5 may be tested in rabbits for the ability to induce an anti-idiotypic
response to the modified
immunoglobulin molecule. For example, male specific-pathogen-free (SPF) young
adult
New Zealand White rabbits may be used. The test group of rabbits each receives
an
effective amount of the vaccine. A control group of rabbits receives an
injection in 1 mM
Tris-HCl pH 9.0 of the vaccine containing a naturally occurring antibody.
Blood samples
may be drawn from the rabbits every one or two weeks, and serum analyzed for
anti-
idiotypic antibodies to the modified immunoglobulin molecule and anti-anti-
idiotypic
antibodies specific for the antigen against which the modified antibody was
directed using,
e.g., a radioimmunoassay (Abbott Laboratories). The presence of anti-idiotypic
antibodies
may be assayed using an ELISA. Because rabbits may give a variable response
due to their
outbred nature, it may also be useful to test the vaccines in mice.
In addition, a modified antibody of the invention may be tested by first
administering the modified antibody to a test subject, either animal or human,
and then
isolating the anti-anti-idiotypic antibodies (i.e., the Ab3 antibodies)
generated as part of the
anti-idiotype response to the injected modified antibody. The isolated Ab3 may
then be
tested for the ability to bind the particular antigen (e.g., a tumor antigen,
antigen of an
infectious disease agent by any immunoassays known in the art, for example,
but not
limited to, radioimmunoassays, ELISA, "sandwich" immunoassay, gel diffusion
precipitin
reactions, immunodiffusion assays, western blots, precipitation reactions,
agglytination
assays, complement fxation assays, immunofluorescence assays, protein A
assays,
immunoelectrophoresis assays, etc.
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Additionally, the modified antibodies of the invention may also be tested
directly in
vivo. The strength of the immune response in vivo to the modified
immunogluobulin may
be determined by any method known in the art, for example, but not limited to,
delayed
hypersensitivity skin tests and assays of the activity of cytolytic T-
lymphocytes in vitro.
Delayed hypersensitivity skin tests are of great value in the testing of the
overall
immunocompetence and cellular immunity to an antigen. Proper technique of skin
testing
requires that the antigens be stored sterile at 4°C, protected from
light and reconstituted
shortly before use. A 35- or 27-gauge need ensures intradermal, rather than
subcutaneous,
administration of antigen. Twenty-four and 48 hours after intradermal
administration of the
antigen, the largest dimensions of both erythema and induration are measured
with a ruler.
Hypoactivity to any given antigen or group of antigens is confirmed by testing
with higher
concentrations of antigen or, in ambiguous circumstances, by a repeat test
with an
intermediate test.
To test the activity of cytolytic T-lymphocytes, T-lymphocytes isolated from
the
immunized subject, e.g., by the Ficoll-Hypaque centrifugation gradient
technique, are
restimulated with cells bearing the antigen against which the modified
antibody was
directed in 3 ml R.PMI medium containing 10% fetal calf serum. In some
experiments, 33%
secondary mixed lymphocyte culture supernatant or IL-2 is included in the
culture medium
as a source of T cell growth factors. In order to measure the primary response
of cytolytic
T-lymphocytes after immunization, the isolated T cells are cultured with or
without the cells
bearing the antigen. After six days, the cultures are tested for cytotoxity in
a 4 hour 5'Cr-
release assay. The spontaneous ''Cr-release of the targets should reach a
level less than
20% if immunization was effective (Heike et al., J. Immunotherapy 15:15-174).
The efficacy of the modified antibody as a contraceptive can also be tested by
any
method known for tested contraceptive methods. For example, a vaccine
composition
containing a modified antibody of the invention specific for an antigen of a
protein or cell
involved in reproductive function. First, the level of the particular antigen
in the subject can
be measured by any method known in the art where a reduction in the level of
the antigen
compared to the level prior to administration of the modified antibody
(accounting for
normal, cyclical changes of the level of the particular antigen) indicates
that the modified
antibody may be effective. The modified antibody must then be administered to
a
population of child bearing age (and having partners of childbearing age) and
the percentage
of females that conceive over a suitable period of time is determined. If the
number of
females that conceive is significantly lower than those in a control
population, e.g., those
adrninistered a placebo or not using a contraceptive method. indicates that
the modified
antibody is effective as a contraceptive.
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Additionally, the efficacy of the contraceptive vaccine may be assayed by
administering the vaccine to a subject or animal model, allowing an
appropriate amount of
time for the production of anti-idiotype antibodies, and then testing serum
taken from the
subject or animal for the ability to bind the particular antigen (indicating
that an anti-
s idiotype reaction has occurred) and/or testing whether the serum can block
fertilization in
vitro, which can be tested by any method known in the art, for example as
described in
Brannen-Brock et al., 1985, Arch. Androl. 15:15-19. .
6. EXAMPLE: ANTI-IDIOTYPIC VACCINE INDUCER FOR COLON
CANCER
This example describes the construction of a modified antibody derived from
the
monoclonal antibody MAb3l. I (hybridoma secreting Mab31.1 is available from
the
American Type Tissue Collection as accession No. HB 12314). Mab31.1 recognizes
an
antigen expressed by human colon carcinomas. The modified antibody of the
invention,
based on Mab31.1, was engineered to have variable region cysteine residues of
both the
heavy and light chain variable regions substituted with alanine. Therefore,
the resulting
modified antibody, was missing intrachain disulfide bonds in either the heavy
and light
chain variable regions.
6.1. CONSTRUCTION OF A MODIFIED ANTIBODY
The strategy for construction of the modified antibody was to construct two
engineered genes that encoded the heavy and light chain variable regions
wherein specific
cysteine residues, known to be important in intra-chain disulfide bonding ,
were altered to
alanine. Alanine residues were substituted for the cysteine residues at
positions 22 and 92
of the heavy chain variable region of the antibody derived from Mab31.1 or at
positions 23
and 88 of the Mab3 I .1 light chain variable region of the antibody derived
from Mab31.1.
In order to construct these engineered genes, groups of olionucleotides were
assembled (as
discussed below) and inserted into an appropriate vector providing constant
regions.
In order to construct variable region genes encoding CDRs lacking intrachain
disulfide bonds, the following strategy was performed.
First, single strand oligonucleotides were annealed to create cohesive double
stranded DNA fragments (as diagramed in Figure 8, Step 1 ). Specifically,
oligonucleotides
of about 80 bases in length corresponding to the sequences of interest with 20
base
overlapping regions were synthesized using automated techniques of GenoSys
Biotech Inc.
The specific sequences of each of these oligonucleotides. The specific
sequences of these
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oligonucleotides are presented in Figures 16A and 16B. Figure 16A list the
group of ten
oligos used in engineering a heavy chain variable region gene called 2CAVHCOL
1.
2CAVHCOL 1 lacked 2 cysteine residues as compared to the consensus heavy chain
variable gene. Figure 16B lists the group of 12 oligos used in the engineering
of the light
$ chain variable region gene called 2CAVLCOL 1. 2CAVLCOL 1 lacked t<vo
cysteine
residues as compared to the consensus light chain variable region gene. In
order to combine
the oligos into the desired gene, groups of 10 or 12 oligos were combined as
described
below and as presented in Figure 8, where the identities of oligos 1 to 10
indicated in Figure
8 are provided in Table $. Prior to combining, each oligonucleotide was $'
phosphorylated
as follows: 2Sp1 of each oligo was incubated for 1 hour in the presence of T4
polynucleotide kinase and 50mM ATP at 37°C. The reactions were stopped
by heating for
$ minutes at 70°C followed by ethanol precipitation. Once
phosphorylated. complementary
oligonucleotides (oligo 1 + oligo 10, oligo 2 + oligo 9, oligo 3 + oligo 8,
oligo 4 + oligo 7,
oligo $ + oligo 6), as shown in Figure 8, were then mixed in sterile
microcentrifuge tubes
1$ and annealed by heating the tube in a water bath at 6$ °C for $
minutes followed by cooling
at room temperature for 30 minutes. Annealing resulted in short double strand
DNA
fragments with cohesive ends.
Next, the cohesive double stand DNA fragments were ligated into longer strands
(Figure 8, Steps 2-4), until the engineered variable region gene was
assembled.
Specifically, cohesive double strand DNA fragments were ligated in the
presence of T4
DNA ligase and IOmM ATP for 2 hours in a water bath maintained at 16°C.
Annealed
oligo 1/10 was mixed with annealed oligo 2/9, and annealed oligo 3/8 was mixed
with
annealed oligo 4/7. The resulting oligos were labeled oligo 1/10/2/9 and oligo
3/8/4/7.
Next, oligo 3/8/4/7 was ligated to oligo $/6. The resulting oligo 3/8/4/7/$/6
was then
2$ ligated to oligo 1/10/2/9 resulted in a full length variable region gene.
Alternatively, when groups of 12 oligos were used, the order of addition was:
1+12
= 1/12, 2+11=2/11, 3+10=3/10, 4+9=~/9, $+8=$/8, 6+7=6/7, 1/12+2/11=1/12/2/1 I,
3/10+4/9=3/10/4/9, $/8+6/7=$/8/6/7, 1/12/2/11+3/10/4/9 = 1/12/2/11/3/10/4/9,
1/12/2/11/3/10/4/9+$/8/6/7= full length variable region gene. The names of
oligonucleotides used in construction of the engineered genes are listed in
Table $. The
modified heavy chain variable region gene was denoted as 2CAVHCOL1. The
modified
light chain variable region gene was denoted as 2CAVLCOL 1.
The resulting modified variable region genes were then purified by gel
electrophoresis. To remove unligated excess of oligos and other incomplete DNA
3$ fragments, ligated product was run on 1% low melting agarose gel at
constant 110 V for 2
hours. The major band containing full length DNA product was cut out and
placed in a
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sterile l.~ ml centrifuge tube. To release the DNA from the agarose, the gel
slice was
digested with f3-Agrase I at 40 ° C for 3 hours. The DNA was recovered
by precipitation
with 0.3 M NaOAc and isopropanol at -20°C for 1 hour followed by
centrifugation at
12,000 rpm for 15 minutes. The purified DNA pellet was resuspended in 50 ul of
TE buffer.
pH 8Ø The engineered variable region gene was then amplified by PCR.
Specifically, 100
ng of the engineered variable region gene was mixed with 25mM dNTPs, 200 ng of
primers
and 5 U of high fidelity thermostable Pfu DNA polymerase in buffer. Resulting
PCR
product was analyzed on 1 % agarose gel.
Each purified DNA corresponding to the engineered variable region gene was
subsequently inserted into the pUCI9 bacterial vector. pUCl9, is a 2686 base
pair, a high
copy number E. coli plasmid vector containing a 54 base pair polylinicer
cloning site in lacZ
and an Amp selection marker. In order to prepare the vector for insertion of
the engineered
variable region gene, lOpg of pUCl9 was linearized with Hinc II (50 U) for 3
hours at
37°C resulting in a vector with blunt end sequence 5' GTC. To prevent
self re-ligation,
linear vector DNA was dephosphorylated with 25 U of calf intestine alkaline
phosphatase
(CIP) for 1 hour at 37°C. In order to insert the engineered variable
region gene into the
pUCl9 vector, approximately 0.5 pg of dephosphorylated linear vector DNA was
mixed
with 3 pg of phosphorylated variable region gene in the presence of T4 DNA
ligase ( 1000
U), and incubated at 16°C for 12 hours.
The bacterial vector containing the engineered variable region gene was then
used to
transform bacterial cells. Specifically, freshly prepared competent DHS-a
cells, 50 ~,1, were
mixed with 1 pg of pUC 19 containing the engineered variable region gene and
transferred
to an electroporation cuvette (0.2 cm gap; Bio-Rad). Each cuvette was pulsed
at 2.5 kV/200
ohm/25 pF in an electroporator (Bio-Rad Gene Pulser). Immediately thereafter,
1 ml of
SOC media was added to each cuvette and cells were allowed to recover for 1
hour at 37°C
in centrifuge tubes. An aliquot of cells from each transformation was removed,
diluted
1:100, then 100 pl plated onto LB plates containing ampicillin {Amp 40 ~g/ml).
The plates
were incubated at 37°C overnight due to the presence of the Amp marker.
Only
transformants containing pUC 19 vector grew on LB/Amp plates.
A single transformant colony was picked and grown overnight in a 3 m1 LB/Amp
sterile glass tube with constant shaking at 37°C. The plasmid DNA was
isolated using Easy
Prep columns (Pharmacia Biotech.) and suspended in 100 ul of TE buffer, pH
7.5. To
confirm the presence of gene insert in pUCl9, 25 ul of plasmid DNA from each
colony was
digested with a restriction endonuclease for 1 hour at 37°C, and was
analyzed on a 1%
agarose gel. By this method plasmid DNA containing gene insert was resistant
to enzyme
cleavage due to loss of restriction site ( 5'..GTCGAC.. 3') and migrated as
closed circular
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(CC) DNA, while those plasmids without insert were cleaved and migrated as
linear (L)
double strand DNA fragment on gel.
In order to confirm correct gene sequences of the engineered variable region
genes
and to eliminate the possibility of unwanted mutations generated during the
construction
procedure, DNA sequencing was performed using M13/pUC reverse primer
(5'AACAGCTATG ACCATG 3') for the clones as well as PCR gene products using 5'
end
20 base primer ( ~' G.AATT CATGGCTTG GGTGTG 3') on automated ABI 377 DNA
Sequencer. All clones were confirmed to contain correct sequences.
Table Construction
5. of gene encoding
modified
antibodies containing CDRs from Mab 31.1
Oligol Oligo Oligo 4 Oligo 5 Oligo Oligo 7 Oligo
2 Oligo 3 8 Oligo 9 Oligol0
2CAVHC VHC1 VHC2 VHC3 VHC4 VHCS VHC VHC7 VHC8 VHC9 VHCIO
OLI
2CAVLC VLC1 ~'LC~ VLC3 VLC4 VLCS VLC VLC7 VLC8 VLC9 VLC10
1 S OLI
6.3. INSERTION OF THE ENGINEERED VARIABLE REGION GENE
INTO :~ MAMMALIAN EXPRESSION VECTOR
A complete antibody light chain has both a variable region and a constant
region. A
complete antibody heavy chain contains a variable region, a constant region,
and a hinge
region. A modified ~~ariable region genes 2CAVHCOL1 or 2CAVLCOL1 were inserted
into vectors containing appropriate constant regions. Engineered variable
region genes
lacking cysteine residues in the light chain, were inserted into the pMRROlO.I
vector
Figure 6A. The pViRR010.1 vector contained a human kappa light chain constant
region.
Insertion of the engineered light chain variable region into this vector gave
a complete light
chain sequence. Alternatively, the engineered variable region gene lacking
cysteine
residues in the heaw~ chain, were inserted into the pGAMMAI vector Figure 6B.
The
pGAMMA 1 vector contained human and IgG I constant region and hinge region
sequences.
Insertion of the engineered heavy chain variable region gene into this vector
gave a
complete heavy chain sequence.
In order to engineer a mammalian vector comprising both heavy chain and light
chain genes, the complete light chain sequence and complete heavy chain
sequence were
inserted into mammalian expression vector pNEPuDGV as shown in Figure 6C
(Bebbington, C.R., 1991. In METHODS: A Companion to Methods in Enzymology,
vol. 2,
pp' 136-145). The resulting vector encoding both light chain and the heavy
chain of the
modified antibod~~.
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6.4. EXPRESSION OF SYNTHETIC MODIFIED ANTIBODIES IN
MAMMALIAN CELLS
To examine the production of assembled antibodies the mammalian expression
vector was transfected into COS cells. COS cells (an African green monkey
kidney cell
line, CV-1, transformed with an origin-defective SV40 virus) were used for
short-term
transient expression of the synthetic antibodies because of their capacity to
replicate circular
plasmids containing an SV40 origin of replication to very high copy number.
The antibody
expression vector was transferred to COS7 cells (obtained from the American
Type Culture
Collection). The transfected cells were grown in Dulbecco's modified Eagle's
Medium and
transfected with the expression vectors using calcium precipitation (Sullivan
et al., FEES
Lett. 285:120-123, 1991 ). The transfected cells were cultured for 72 hours
after which
supernatants were collected. Supernatants from transfected COS cells were
assayed using
ELISA method for assembled IgG. ELISA involves capture of the samples and
standards
onto a 96-well plate coated with an anti-human IgG Fc. Bound assembled IgG was
detected with an anti-human Kappa chain linked to horse radish peroxidase
(HRP) and the
substrate tetramethylbenzidine (TMB). Color development was proportional to
the amount
of assembled antibody present in the sample.
6.5. MODIFIED ANTIBODY IMMUNOSPECIFICALLY BINDS TO HUMAN
COLON CARCINOMA CELLS AND ANTIGENS PRODUCED BY THESE CELLS
The modified antibody was expressed and isolated as indicated in Section 6.4,
supra. The binding capacity and specificity were then assayed using LS-174T
cells WiDR
cells (a human colon cancer cell line) and an antigen derived from these
cells.
In order to examine the binding potency as well as specificity of MA31.1
binding, a
dot blot analysis was performed (see Figure 9). Membrane preparations from LS-
I74T cells
was applied to a nitrocellulose membrane using a Bio-Blot apparatus (Bio-Rad).
The wells
were blocked for non-specific binding using skim milk. Biotinylated antibody
derived from
Mab31.1 was incubated in all wells. Unlabelled antibody at concentrations of
0.003 to 20
nM was then applied to the nitrocellulose membrane and allowed to incubate.
Unbound
antibody was removed from the membrane by washing and a second antibody,
alkaline
phosphatase labeled antihuman IgG, was added. Finally, an alkaline phosphatase
substrate
was added which generated a dark purple precipitate, indicating the presence
of bound
labeled antibody. Figure 9 provides the results of the dot blot analysis. The
figure
demonstrated that the labeled antibody bound to the LS-174 T cells.
Additionally, the
unlabeled antibody competed with biotinylated antibody binding, indicating
specificity of
binding of the antibody derived from Mab31.1 to tumor cell antigens.
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6.6. ANTI-IDIOTYPE RESPONSE
The effect on binding of modified antibody to LS-174T cells was examined in a
competition binding assay. LS-174T cells are human colon carcinoma cells which
express
antigen recognized by the Mab31.1 antibody. Peptides containing the sequence
of one of
the CDRs of the Mab3l.1 antibody were generated. These peptides, the modified
antibody
and the control antibody derived from Mab31.1 were administered to mice in
order to
generate antisera against the CDR regions of Mab31.1 and the antibodies. Blood
samples
from mice were drawn two weeks and four weeks following injection. Antisera
from the
immuized mice were used in binding competition assays presented in Figures I
OA and B.
Antisera and biotinylated antibodies were assayed for their ability to bind LS-
174T
cells. As demonstrated in Figure l0A and B, antisera raised to the CDR3 and
CDR4
peptides dramatically competed for control antibody (antibody derived from
Mab3l.l)
binding to LS-174T cells. Additionally, antisera raised against CDR1 and CDR2
also
significantly competed for control antibody binding to LS-174T cells.
Additionally,
antisera from nice injected with the 2CAVHCOL1 and 2CAVLCOL1 antibodies (i.e.,
the
modified antibodies having the cysteine to alanine change in the variable
region) competed
for binding with the biotinylated antibody derived from Mab31. I better than
antiserum from
mice injected with the antibody derived from Mab31.1 (Figure l OB). This
result indicates
that administration of the antibodies having the cysteine to alanine change in
the variable
region elicit an anti-idiotype antibodies that recognize the colon carcinoma
cell antigen
better than antibodies with variable region infra-chain disulfide bonds.
Table 6. Biotin-Labeled Peptides Derived
from CDR Sequences of Ma_b 31.1
Peptide ID Sequence
COL31I L1 biotin-N-Thr-Ala-Lys-Ala-Ser-Gln-Ser-Val-Ser-Asn-Asp-Val-Ala
COL311 L2 biotin-N-Ile-Tyr-Tyr-Ala-Ser-Asn-Arg-Tyr-Thr
COL311 L3 biotin-N-Phe-Ala-Gln-Gln-Asp-Tyr-Ser-Ser-Pro-Leu-Thr
COL311 H1 biotin-N-Phe-Thr-Asn-Tyr-Gly-Met-Asn
COL311 H2 biotin-N-Ala-Gly-Trp-Ile-Asn-Thr-Tyr-Thr-Gly-Glu-Pro-Thr-Tyr-Ala-Asp-
Asp-Phe-Lys-Gly
COL311 H3 biotin-N-Ala-Arg-Ala-Tyr-Tyr-Gly-Lys-Tyr-Phe-Asp-Tyr
7. EXAMPLE: SPERM ANTIGEN VACCINES
The example herein describes the construction of defined epitopes that
replace the complementarity determining regions (CDR) of an antibody.
Specifically, the
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epitopes are derived from sperm antigens SP-10, LDH-C, or MSA-G3. These
constructs
express an antibody, which, when injected into an appropriate host, induces an
immune
reaction that precipitates the formation of anti-idiotype antibodies that are
active against the
sperm antigens.
The strategy for producing the antibody containing a sperm cell epitope is
outlined as follows: ( 1 ) a CDR is engineered to contain a nucleotide
sequence encoding one
or more epitopes from a sperm specific protein, {2) the engineered CDR is then
cloned into
a mammalian expression vector containing the appropriate heavy or light chain
constant
regions, (3) the vector is transfected into a cell that supports expression,
proper folding and
I O modif canon of functional antibodies, (4) the antibody is harvested from
the supernatant and
is confirmed for the epitope expression by standard assays (e.g. ELISA,
western blot, etc.),
and (5) the antibody is used as an immunogen in an appropriate host to
generate anti-sperm
antibodies, thereby inducing long lasting infertility.
15 7.1. CONSTRUCTION OF THE SPERM ANTIGEN VACCINE
The following describes the construction of a modified variable region gene
containing at least one CDR that contains a sperm antigen epitope, i.e., SP-10
or L~H-C,
epitope and/or an MSA-63 epitope.
First, an epitope is chosen and defined so that oligonucleotides may be
20 synthesized. In the following example, an SP-10 epitope from the sperm
antigen SP-10 is
used. SP-10 is a suitable epitope because it is expressed exclusively in sperm
cells. It is
also expressed on the surface of the membrane of the acrosome, thus, it is
accessible to
therapeutic antibodies. Other antibodies are produced that contain portions of
the LDH-C,
and MSA-63 antigens.
25 The nucleotide and protein sequences of the SP-10 epitope are:
GAA TTC CAG CCT TCA GGT GAA CAT GGC TCC GGT GAA CAG CCT TCT GGT
GAG CAG GCC TCG GGT GAA CAG CCT TCA GGT GAG CAC GCT TCA GGG GAA
CAG GCT TCA GGT GCA CCA ATT TCA AGC ACA TCT ACA GGC ACA ATA TTA
AAT TGC TAC ACA TGT GCT TAT ATG AAT GAT CAA GGA AAA TGT CTT CGT
GGA GAG GGA ACC TGC ATC ACT CAG AAT TC;
30 Gln Pro Ser Gly Glu His Gly Glu Gln Pro Ser Gly Glu Gln Ala Ser Gly Glu Gln
Pro Ser gly
Glu His AIa Ser GIy Glu Gln Ala Ser Gly Ala Gin Ile Ser Ser Thr Ser Thr Gly
Thr Ile Leu
Asn Cys Tyr Thr Cys Ala Tyr Met Asn Asp Gln Gly Lys Cys Leu Arg Gly Glu Gly
Thr
Cys Ile Thr Gln Asn.
The replacement of an antibody's CDR with another epitope is made easier
by the fact that the variable region sequence of antibodies are relatively
short, and are
35 ~o~,~,n. One is then able to synthetically generate a series of
complementary
oligonucleotides that, when annealed and Iigated, reconstruct the entire
coding region of
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variable region portion of the gene. In this manner, the CDR is replaced with
sequences of
the epitope of interest, in this example, SP-10. The following is a list of
the sequences of
the oligonucleotides designed for cloning SP-10 epitopes into the CDR:
Oligo SP 1:
GAA TTC CAG CCT TCA GGT GAA CAT GGC TCC GGT GA.a CAG CCT TCT GGT
GAG CAG GCC TCG GGT GAA CAG CCT TAG,
Oligo SP 2:
GTG AGC ACG CTT CAG GGG AAC AGC CTT CAG GTG CAC CAA TTT CAA GCA
CAT CTA CAG GC:~ CAA TAT TAA ATT GCT,
OIigo SP 3:
ACA CAT GTG CTT ATA TGA ATG ATC AAG GAA AAT GTC TTC GTG GAG AGG
G~ CCT GCA TCA CTC AGA ATT C,
Otigo SP 3a(3Cys-> Ala):
ACA CAG CAG CTT ATA TGA ATG ATC AAG GAA AAG CAC TTC GTG GAG AGG
GAA CCG CAA TC A CTC AGA ATT C,
Oligo SP 4:
GAA TTC TGA GTG ATG CAG GTT CCC TCT CCA CGA AGA CAT TTT CCT TGA
TCA TTC ATA TA.~ GCA CAT GTG TAG CAA TTT A,
Oligo SP 4a (3Cys->Ala):
G~ TTC TGA GTG ATT GCG GTT CCC TCT CCA CGA AGT GCT TTT TGA TGA
TCA TTC ATA TAA GCT GCT GTG TAG CAA TTT A,
Oligo SP 5:
ATA TTG TGC CTG TAG ATG TGC TTG AAA TTG GTG CAC CTG AAG CCT GTT
CCC CTG AAG CGT GCT CAC CTG AAG GCT,
Oligo SP 6:
GTT CTC CCG AGG CCT GCT CAC CAG AAG GCT GTT CAC CGG AGC CAT GTT
ZO CAC CTG AAG GCT GGA ATT C.
Antibodies containing portions of the MSA-63 antigen are also described.
To identify the optimal portion of the antigen to be introduced into the
antibody,
oligonucleotides encoding different portions of the antigen are synthesized.
Practically, the first tv~~o amino acid codons of the sperm cell specific
epitope, MSA-63, an oligonucleotide encoding residues 143 and 144 (i.e. GTC
GGC, infra),
is cloned into the immunoglobulin CDR, using the methods described infra,. The
MSA-63
DNA sequence encoding the epitope:
GTC GGC AGC CTC CGA AGC AGC CCG CTC CAG AGC CCG CTG CTC CGA CCG
CTC GTC CAG AGC AGC CTC TGC TTG CTG TTC CTC TTG CTG CGA TAC AGC
TGC GGC GAC GGC AGC TGC AGC CGA CGA TAC TGC GAC TTG ACG GTG TGC
CGG CGA ATG TAC TTG CTG CTG CGA TTC ACG GAC CCG CCG CTC CCG CAG
ACG TGC TGC GTC TTG AGC
The MSA-63 protein sequence epitope encoded by the nucleic acid sequence
above, which
starts at amino acid 1-13 and ends at 233.
Gln Pro Ser Glu Ala Ser Ser Gly Glu Val Ser Gly Asp Glu Ala Gly Glu Gln Val
Ser Ser
Glu Thr Asn Asp Lys Glu Asn Asp Ala Met Ser Thr Pro Leu Pro Ser Thr Ser Ala
Ala Ile
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Thr Leu Asn Cys His Thr Cys Ala Tyr Met Asn Asp Asp Ala Lys Cv_ s Leu Arg Gly
Glu
Gly Val Cys Thr Thr Gln Asn Ser
For the second two amino acid colons, an oligonucleotide encoding residues 144
and 145 is
utilized (i.e., GGC AGC). For the third, 145 and 146 and so on until the
entire epitope is
synthesized and inserted into the CDR, two amino acids at a time. For peptides
three amino
acids in length, an oligonucleotide encoding residues 143 to I46 is
synthesized. The second
oligonucleotide synthesized encodes residues 146 to I48. The third encodes
residues 148 to
150, and this continues until the entire epitope is covered in this fashion.
The next
oligonucleotide that is synthesized is four amino acid colons in length. It
begins with
residues 143 to 146, its second segment is equivalent to residues 145 to 148,
its third
segment corresponds to residues 147 to 1 S0, and so on until the entire
epitope is
transitioned in this fashion. The next oligonucleotide synthesized contains
five amino acid
colons with two overlapping with the previous. For example, the first
oligonucleotide
encodes residues 143 to 147, and the second residues 146 to 1 S0. This pattern
continues
until the entire epitope has been transitioned. The next construct encoding an
epitope uses
nucleotides for six amino acid colons with two overlapping with the previous
colons as
described infra.
The epitopes thereafter contain peptides of seven residues with three
overlapping. The pattern of adding one amino acid to each small peptide and
increasing the
overlap by one colon continues until an overlap of five is reached and then
the small
peptides are synthesized adding one colon each time until the full length of
the epitope is
encoded in the CDR. The overlap is never bigger than five amino acid colons
although the
entire peptide is lengthened by one amino acid in each new combination.
In a specific example, oligomers have been designed which scan the entire
length of the MSA-63 epitope and encode 15 amino acids. Each olio overlaps
with the
previous one for the equivalent of five amino acids. MSA-63 oligos encoding 15
amino
acids, with overlap of five amino acids each:
MSAl: GTC GGC AGC CTC CGA AGC AGC CCG CTC CAG AGC CCG CTG CTC
CGA
MSA2: AGC CCG CTG CTC CGA CCG CTC GTC CAG AGC AGC CTC TGC TTG
CTG
MSA3: AGC CTC TGC TTG CTG TTC CTC TTG CTG CGA TAC AGC TGC GGC
GAC
MSA4: TAC AGC TGC GGC GAC GGC AGC TGC AGC CGA CGA TAC TGC GAC
TTG
MSAS: CGA TAC TGC GAC TTG ACG GTG TGC ACG CGA ATG TAC TTG CTG
CTG
MSA6: ATG TAC TTG CTG CTG CGA TTC ACG GAC GCG CCG CTC CCG CAG
ACG
MSA7: CGA TTC ACG GAC GCG CCG CTC CCG CAG ACG TGC TGC GTC TTG
AGC
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Antibodies in which a portion of the MSA-63 antigen has been inserted into
CDR1, i.e., where residues Lys24 through A1a34 of the consensus contraceptive
light chain,
the sequence of which is in Figure 15, are replaced with the sequence Gln-Pro-
Ser-Glu-Ala-
Ser-Ser-Gly-Glu-Val-Ser-Gly-Asp-Glu-Ala-Gly-Glu. The antibody, MSA1, can be
constructed using the oligonucIeotides provided in Figure 11 in the scheme
provided in
Figure 8 and described below, where the identities of oligonucleotides 1-12
are indicated in
Table 7. The antibody MSA1 VAC can also be constructed using the oligos of
Figure 11 by
the scheme of Figure 8, as indicated in Table 7. MSA 1 VAC is the same as MSA
1 except
that the cysteine at position 23 of the light chain variable region has been
replaced with
alanine. These light chains can be expressed with the heavy chain consensus
sequence
CONVH1, the sequence of which is provided in Figure 7B, and the construction
of which
can be accomplished with the oligonucleotides as indicated in Table 4. These
single
stranded oligonucleotides sequences are annealed to create cohesive double
stranded DNA
fragments suitable for ligation as diagramed in Figure 8, along with
oligonucleotides
encoding the remainder of the consensus variable region, to construct the
variable region
gene. For the MSA-63 containing variable regions MSA1 and MSA1VAC the
oligonucIeotides corresponding to oligonucleotides 1 to 10, or 1 to 12, of
Figure 8 are
provided in Table 7, and the sequences of these oligonucleotides are provided
in Figure 11.
Specifically, oligonucleotides of about 70 bases in length corresponding to
the sequences of
interest with 20 base overlapping regions are synthesized (GenoSys Biotech
Inc.). Each
oligonucleotide is 5' phosphorylated as follows: 25p1 of each oligo is
incubated for one
hour in the presence of T4 polynucleotide kinase and 50 mM ATP in appropriate
buffer at
37°C. The enzyme is heat killed and the reaction stopped by heating for
ten minutes at 70°C
followed by ethanol precipitation with sodium acetate. The oligos are then
resuspended in
TE buffer ( 10 mM Tris, pH 8.0, 1 mM EDTA).
Complementary oligonucleotides (oligo 1 + oligo 10, oligo 2 + oligo 9, oligo
3 + oligo 8, oligo 4 + oligo 7, and oligo 5 + oligo 6) were then mixed in a
sterile
microcentrifuge tube and annealed by heating the tube in a water bath at
65°C for 5 minutes
followed by cooling at room temperature for 30 minutes. Annealing results in
double
stranded DNA with cohesive ends. The cohesive double stranded DNA fragments
are
ligated into longer strands (Figure 8, Steps 2-4), until the engineered
variable region gene
was assembled. Specifically, cohesive double stranded DNA fragments are
ligated in the
presence of T~ DNA ligase, ligase buffer and 10 mM ATP for two hours in a
water bath
maintained at 16°C. Annealed oligo 1/10 is mixed with annealed oligo
2/9, and annealed
oligo 3/8 is mixed with annealed oiigo 4/7. The resulting oli~os are 1/10/2/9
and 3/8/4/7.
Next, oligo 3/8/4/7 is ligated to oligo 5/6. The resulting oligo 3/8/4/7/5/6
is then ligated to
_q.4_
CA 02350917 2001-05-09
WO 00/29443 PCT/US99/26671
oligo 1/10/2/9 resulting in a full length variable region gene. Alternatively,
when 12 oligos
are used, the order of addition is 1+12=1/12, 2+1 I=2/11, 3+10=3/10, 4+9=4/9,
5+8=5/8,
6+7=6/7, 1/12+2/11=1/12/2/11, 3/10+4/9=3/10/419, 5/8+6/7=5/8/6/7,
1 / I 2/2/ 11 +3/ 101419= I / 12/2/ 11 /3/ 10/4/9,
//12/2/11/3/10/4/9+5/8/6/7=1 //2/2/11/3/10/4/9/5/8/6/7, which is the full
length modified
variable region gene. The names of oligonucleotides used for construction are
listed in
Table 7 and Figures 9, , 11, 12C, or 13C.
Using this method, variable region sequences in which an alanine has been
substituted for a cysteine that forms an intrachain disulfide bond can be
constructed using
oligonucleotides introducing this change. For example, in constructing the
antibody
contains the SP-10 portion, oligos SP 3a and SP 4a could be used instead of
oligo SP3 or
SP4.
The modified variable region DNA fragment is then cloned into a shuttle
vector (e.g. pUCl9, infra) for sequence analysis and upon sequence
confirmation, cloned
I 5 into an expression vector. After running the DNA for two hours at 1 I 0
volts in a I % low
melting agarose gel, DNA fragments are visualized by ethidium bromide staining
and gel
slices are cut out and placed in a sterile microfuge tube. Gel purification
removes excess
free oligomers that may interfere with future Iigations. The DNA is eluted
from the agarose
by addition with f3-Agrase I at 40°C for three hours. DNA is
precipitated using 0.3 M
sodium acetate and isopropanol at -20°C for one hour, followed by
centrifugation at high
speed in a microcentrifuge for ten minutes. Isopropanol is aspirated and the
pellet is
washed once with 70% ethanol, the sample is spun again and the ethanol is
aspirated and
the pellet air dried. The DNA pellet is quantitated by running a small
fraction of the
resuspended pellet (i.e. 1/lOth) on a gel and visually comparing to known DNA
standards,
or measuring the absorbance of UV light at 260 nM. If the quantity of DNA is
to limiting
for cloning at this point, it can be amplified by PCR techniques well known to
those skilled
in the art.
7.2 LIGATION OF THE MODIFIED CDR INTO PUC19
Purified DNA corresponding to the engineered variable region gene is
subsequently inserted into the pUC 19 vector by ligation. The pUC 19 vector is
a 2686 base
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CA 02350917 2001-05-09
WO 00/29443 PCT/US99/Z6671
x
A A
N a
~
m
0
U U
.r
a
0 0 A
U
o ~ ~ o
.-.
o ~ ~ U
~ ~;
F ~ ~1
. ~n v~
.
O
a
eo
O
a ~
U
o
e_o
O
~o
eo
o x x w
E. ~ c'~3
o x x oa
c~
o x x
M
E~0
>
~
O ~~ ~U
.-.
~
N ,
~
O D. O
~
U
o a a
_.I
~ >
4
~ Cg
U
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CA 02350917 2001-05-09
WO 00/29443 PCT/US99/26671
pair, high copy number E. coli plasmid containing a 54 base pair polylinker
cloning site in
the middle of the lacZ gene. The pUC 19 vector also contains an ampicillin
resistance
marker for selection of bacteria containing the plasmid. The pUCl9 is digested
with the
restriction enzyme Hinc II ( 10 pg plasmid in ~0 units enzyme). The resulting
blunt ends are
dephosphorylated with calf intestinal phosphatase (CIP, 2 units in alkaline
buffer, 30
minutes at 37°C), to prevent recircularization during the ligation
step. The phosphatase is
removed by extraction with phenol and chloroform, followed by precipitation
with sodium
acetate and ethanol on ice for I hour. The precipitated DNA is pelletted by
high speed
centrifugation and the ethanol is removed by aspiration, followed by a washing
step with
I O 70% ethanol to remove excess salts. The DNA pellet is air dried to
completely remove any
ethanol. The digested, phosphatased vector is then resuspended in TE buffer to
0.5 p,g/wl.
Approximately 0.1-0.5 p.g of vector is incubated with a ten fold molar excess
of the
constructed variable region containing the sperm cell epitope in the CDR
(modified variable
region) with T, Iigase ( 1000 units) in appropriate buffer and incubated at
16°C for 12 hours.
7.3 BACTERIAL TRANSFORMATION
The ligation mixture containing the engineered variable region gene cloned
into pUCl9, is transformed into competent bacterial cells. Specifically, 50 pl
of freshly
prepared competent DHS-a cells are mixed with the ligation mixture of pUC 19
and
modified variable region DNA and transferred to an electroporation cuvette
(0.2 cm gap;
Bio-Rad). Each cuvette is pulsed at 2.5 kV/200 ohm/25 pF in an electroporator
(Bio-Rad
Gene Pulser). Immediately thereafter, 1 ml of SOC media is added to each
cuvette and cells
are allowed to recover for 1 hour at 37°C in centrifuge tubes. An
aliquot of cells from each
transformation is removed. diluted 1:100, then 100 pl is plated onto LB plates
with
ampicillin (Amp 40 pg/ml). The plates are then incubated at 37°C
overnight and only cells
containing a plasmid grow.
The plasmid DNA is analyzed after isolation from single colonies picked by
sterile toothpick and grown up overnight in 3 ml LB/Amp in a sterile glass
test tube, with
constant shaking at 37°C. The plasmid DNA is isolated using Easy Prep
columns
(Phannacia Biotech) and suspended in 100 pl of TE buffer. To confirm the
presence of
insert, isolated pIasmid DNA is digested with Hinc II and the digestion
product is analyzed
by 1.2% agarose gel electrophoresis in Tris-Acetate EDTA buffer (TAE). DNA is
stained
in the gel with ethidium bromide and visualized under UV light. The colonies
that
correspond to plasmids with insert are selected for further analysis.
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7.4 DNA SEQUENCING
DNA sequencing is performed to verify the accuracy of the sequence in the
cloned fragment. Sequencing across the pUCl9 polylinker is done using the
M13/pUC
universal forward and universal reverse primers using the Sanger dideoxy chain
termination
procedure. The M 13/pUC universal primers are readily found in biotechnology
supply
catalogues. Sequencing is performed on the ABI377 DNA sequencer, and sequence
comparison is performed using standard computer alignment programs or visual
inspection.
7.5 CLONING INTO THE Va AND VL CHAIN CONSTRUCTS
Once the sequence of the modified CDR has been confirmed, it is cut out of
the pUCl9 plasmid and ligated into either the heavy or light chain antibody
expression
vectors pMRR010.1 or pGAMMAl, respectively (See Figures 6A and B).
Alternatively,
both the heavy and light chain genes are expressed on the same plasmid, and
the modified
CDR is ligated into either the heavy or light chain variable region as
appropriate.
A complete antibody light chain has both a variable region and a constant
region. A complete antibody heavy chain contains a variable region, a constant
region, and
a hinge region. The synthetic variable region genes of the invention are
inserted into
vectors containing appropriate constant regions. Engineered variable region
genes with the
sperm antigen epitope sequences are cloned into the pMRROIO.1 vector. The
pMRR010.1
vector contains a human kappa light chain constant region. Insertion of the
engineered light
chain variable region into this vector gives a complete light chain sequence.
Alternatively,
the engineered variable region gene with the sperm antigen sequence, of the
heavy chain is
inserted into the pGAMMA 1 vector. The pGAMMA 1 vector contains human and IgG
1
constant region and hinge region sequences. Insertion of the engineered heavy
chain
variable region gene into this vector gave a complete heavey chain sequence.
In order to engineer a mammalian vector comprising both heavy chain and
light chain genes, the complete light chain sequence and heavy chain sequence
were
inserted into a mammalian expression vector pNEPuDGV (Figure 6C; Bebbington,
C.,
1991, In METHODS: A Companion to Methods in Enzymology, 2_:136-145). The
resulting vector encodes both light chain and the heavy chain of the antibody.
7.6 TRANSFECTION OF EUKARYOTIC CELLS
The antibody expression plasmid, pNEPuDGV, is then transfected into a
suitable host cell for expression of the antibody of interest. COS-7 (an
African green
monkey kidney cell line, CV-1, transformed with an origin defective SV40
virus), 293, or
CHO cells are capable of being transfected and support expression of foreign
proteins.
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Transfection is performed by standard calcium phosphate precipitation
(Sullivan et al.,
1991, FEBS Lett. 25:120-123). Alternatively, cells may be transfected using
lipid vesicles
or electroporation. Transient or stable transfections are suitable depending
on how much
protein is expressed and harvested.
7.7 EXPRESSION AND PROTEIN ANALYSIS
Transfected cell supernatants are collected and analyzed for proper
expression of anti-idiotype antibodies. The antibodies are purified away from
cell debris
and growth media serum and also concentrated from the supernatant by binding
the
I O antibody Fc domain to a protein A or protein G column. The antibody is
eluted from the
column by low pH glycine and dialyzed against BSA and Tris buffer.
7.8 IN VIVO ANALYSIS OF ANTI-IDIOTYPE EFFICACY
To test the ability of the antibody to elicit an immune response or for a
15 contraceptive effect. the antibody is injected into a mouse at a
pharmaceutically significant
dose range and serum samples are taken from the mice. The production of anti-
idiotype
antibodies is confirmed by harvesting peripheral blood serum and performing
ELISAs with
the sperm antigen (or sperm), or western blots using the sperm antigen (or
sperm) as target
and the vaccinated mouse serum as probe.
20 ELISA involves capture of the samples and standards onto a 96 well plate
coated with an anti-epitope antibody. Bound antibody is detected with a
secondary
antibody crosslinked to horse radish peroxidase (HRP) and the substrate
tetramethylbenzidine (TMB) and specific to the kappa or lambda light chain of
the mouse.
Alternatively, western blots are performed using the anti-idiotype as the
target and probing
25 it with anti-epitope antibodies.
Confirmation of production of anti-idiotypes in the mice is then followed by
in vivo analysis to determine whether the mice are capable of conception.
Control mice and
test mice are mated in statistically significant groups and the number of
pregnancies are
monitored. Effective immunocontraceptive therapy will result in a significant
reduction in
30 the number of pregnancies.
Additionally, the induction of effective quantities of anti-idiotype anti-
bodies
is also assayed for prevention of in vitro fertilization. Donor sperm is mixed
in vitro with
donor eggs in the presence or absence of test serum yr negative control serum.
The failure
of sperm to fertilize the egg when test serum is added is a positive
indication that the
35 vaccine is effective.
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The present invention is not to be limited in scope by the specific
embodiments
described herein. Indeed, various modif cations of the invention in addition
to those
described herein will become apparent to those skilled in the art from the
foregoing
description and accompanying figures. Such modifications are intended to fall
within the
scope of the appended claims.
Various references are cited herein, the disclosures of which are incorporated
by
reference in their entireties.
15
25
35
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