Note: Descriptions are shown in the official language in which they were submitted.
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IMMUNOGLOBULIN MOLECULES HAVING A SYNTHETIC VARIABLE
REGION AND MODIFIED SPECIFICITY
('-ROSS REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of Provisional application Serial No.
60/065,716,
filed November 14, 1997, and Provisional application Serial No. 60/081,403,
filed April 10,
1998, both of which are incorporated by reference herein in their entireties.
1. FIELD OF THE INVENTION
The present invention relates to modified immunoglobulin molecules,
particularly
antibodies, that bind one member of a binding pair and have at least one
complementarity
determining region (CDR) that contains the amino acid sequence of a binding
site for that
member of the binding pair. which binding site is derived from the other
member of the
binding pair. 1'he .invention also relates to methods for treating,
diagnosing, or screening for
diseases and disorders associated with the expression of the member of the
binding pair,
particularly. cancer or infectious diseases, using the modified antibodies of
the invention.
The present invention also relates to pharmaceutical compositions and
diagnostic kits
containing the modified antibodies of the invention.
2. ~3ACKGROUND OF THE INVENTION
2.1. ANTIBOD1ES AND THE IMMUNE SYS~>~ VI
Antibodies are proteins that belong to the immuroglobulin superfamily. The
immunoglobulin superfamily includes T cell receptors, B cell receptors, cell-
surface
adhesion molecules such as the co-receptors CD4, CDB, CD 19, and the invariant
domains of
~e MHC molecules. In their soluble form, antibodies are glycoproteins produced
by mature
B cells which are also called plasma cells. Antibodies are secreted mto the
blood and other
extracellular fluids tc~ circulate throughout the body in all animals and
humans in response to
foreign antigens.
Antibodies have two principal functions. The first is to recognize or bind to
foreign
~tigens. The second is to mobilize other elements of the immune system to
destroy the
foreign entity. The receptors on the surfaces of immune effector cells are
designed for
recognition of antigens and cell surface markers on other cells. This
recognition process
imparts information as to whether the markers are self or non-self, and is an
important
element involved in modulating the immune system response to the presence of
antigens.
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The portion of an antigen to which an antibody binds is called its antigenic
determinant, or epitope. Some antigens are capable of eliciting an immune
response, while
others are recognized as self by the immune system. Antigens which can elicit
an immune
response are termed immunogens, and are usually macromolecules of at least
5000 Dalton
molecular weight, such as proteins, nucleic acids, carbohydrates, and lipids.
Smaller
nonimmunogenic molecules, termed haptens, also are capable of stimulating an
immune
response when coupled to a large carrier molecule.
2.2. STRUCTURE OF ANTIBODIES
The basic complete unit of an antibody is a four-chain Y-shaped structure
(Figure 1 ).
In the early 1970s, Wu and Kabat assembled the amino acid sequences of a large
collection
of antibodies and demonstrated that the structure of antibodies and. in fact.
all members of
the immunoglobulin superfamily, consists of a constant region and four
relatively conserved
framework regions of semi-rigid beta-sheet, with three relatively short
hypervariable
sequence regions known as complementarity determining regions (CDRs)
interspersed
among them (Vfu and Kabat, 1970, J. Exp. Med. x(2):211-250; Wu and Kabat,
1971,
Proc. Natl. Acad. Sci. USA 68(7):1501-1506). This prediction was confirmed by
crystallographic studies of antibody structure (Poljak et al., i 973, Proc
Natl Acad Sci USA
70(12):3305-3310; Diesenhofer et al., 1976, Hoppe Seylers Z Physiol Chem
(Germany,
West) 357(10):435-445: Diesenhofer et al., 1976, Hoppe Seylers ZPhysiol C:hem.
(Germany,
West) x(10):1421-1434).
Figure 1 represents the overall structure of an antibody molecule. Antibodies
are
made up of two shorter light chains linked via disulfide bonds to two longer
heavy chains,
which are themselves connected by disulfide bonds. As indicated in Figure 2.
both the
heavy and light antibody protein chains are composed of multiple domains, each
about 110
amino acid residues in length. Each light and heavy chain of an antibody has a
variable
region at its amino terminus (V~ and VH respectively); it is the variable
region of the
antibody that confers the antigen-binding specificity. A heavy chain variable
domain and a
light chain variable domain together form a single antigen-binding site, thus,
the basic
i~~oglobulin unit has two antigen-binding sites.
Diversity in the variable regions of both the light and heavy chains is
restricted to the
three "hypervariable" regions or CDRs. There are a total of six CDRs in each
antibody.
molecule (Figure 2), each of which CDR contains from about five to about ten
amino acids,
or up to about 20 amino acids when the CDR is endogenously recombined, as is
common in
some antibody classes. The three CDRs of the variable region of each light and
each heavy
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chain form loops which are clustered together and are connected to the four
remaining parts
of the variable region, called the framework regions ("FRs") which are
relatively conserved
among antibody molecules. Antibody diversity is generally created by changing
the
sequences of the CDRs.
The variable regions are distinct for each antibody, whereas the constant
regions are
more highly conserved. While the light chain has only one constant region
domain, the
heavy chain constant region is composed of multiple domains, named CHI, CH2,
CH3...CHx. The constant region domains are charged with the various antibody
effector
functions, such as complement binding and binding to the Fc receptors
expressed by
lymphocyres, granulocytes, monocyte lineage cells, killer the stimulation of B
cells to
undergo proliferation and cells, mast cells and other immune effector cells.
C)ther effector
functions are differentiation, activation of the complement cell lysis system,
opsonization,
attraction of macrophages. Antibodies of different isotypes have different
constant domains
and therefore have different effector functions. The best studied isotypes are
IgG arid IgM.
All animal species express several different classes of antibodies. Five human
antibody classes (IgG, IgA; IgM, IgD and IgB), and within these classes,
various subclasses,
are recognized on the basis of structural differences, such as the number of
immunoglobulin
units in a single antibody molecule, the disulfide bridge structure of the
individual units, and
differences in chain length and sequence. IgG antibodies arc, thus far, the
most generally
u~~l of these classes~for diagnostic and therapeutic pharmaceutical uses,
although
antibodies .from other ;;lasses may find utility ir~ certain uses.
2.3. ANTIBODY ENGINEERING
The development of monoclonal antibody technology; first disclosed-by Kohler
and
Milstein ( 1975, Nature 256:495-497), has allowed the generation of unlimited
quantities of
antibodies of precise and reproducible specificity. The Kohler and Milstein
procedure
involves the fusion of spleen cells obtained from an immunized animal, with an
im=portal
myeloma cell line to produce hybridomas. Clones which produce an antibody
having the
requisite specificity are then selected from these hybridomas. The hybridomas
produce
monoclonal antibodies which are uniform in their properties and specificity. .
To date, identification and production of suitable antibodies useful in
diagnostic and
therapeutic applications has depended on chance. The generation of antibody-
producing
hybridomas involves immunization of a mouse with an antigen, or,
alternatively, the antigen
is added to spleen cell preparations in vitro. The population of spleen cells
and, therefore, of
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potential monoclonal antibodies with a particular specificity depends upon the
animal's
immune reaction to the antigen.
Additional approaches to generating antibodies useful for diagnostic and
therapeutic
uses have been developed as an alternative to the laborious immunization
procedure
mentioned above. One approach entails the cloning of antibody genes into phage
viruses,
which will express on the virus surfaces a single variable region as described
in Clackson et
al.; 1991, Nature 352:624; Marks et al., 1992, J. Mol. Biol. 222:581; Zebedee
et al., 1992,
Proc. Natl. Acad. Sci. USA 39:3175; Gram et al., 1992, Proc. Natl. Acad. Sci.
USA 89:3576.
Using phage library techniques, one can generate large libraries that express
much of the
i~erent genetic diversity. However, such libraries are still constrained by
the antibody
repertoire from which they were derived. In yet another approach, variable
domain genes
which are randomly mutagenized and expressed. also result in the production of
large
libraries as described in Pack ( 1997, High Quality Antibody Libraries,
Abstracts of the
Eighth International Conference of Antibody Engineering). While both
approaches are
successful in generating great diversity, they are generally little more
successful in
identifying useful antibodies when compared with traditional immunization
methods
because they rely on random generation of CDR sequences. Moreover, antibodies
generated
through immunization of mice are of limited use in human therapeutics. Since
mouse
monoclonal antibodies are foreign and thus immunogenic to humans, they induce
a human
~timouse antibody (HAMA) response (Shawler etal., 1985, J. In:mureol.
135:1530;
Chatenaud et al., 1986, J; Immunol. 137:$30).
2.4. PHARMACEUTICALS BASED UPON MANIPULATION OF
INTERMOLECULAR INTERAC'T'IONS
The efficacy of a pharmaceutical is often derived from the.ability of the
pharmaceutical to enhance, antagonize or mimic the binding of one molecule to
another, for
example, a ligand to its receptor, or a pathogen to a cellular receptor,
thereby achieving
certain physiological and pharmacological activity useful for disease
prevention or
amelioration. Until recently, pharmaceuticals were limited to serendipitously
discovered
synthetic or natural products, and were small molecule effectors that mimicked
the binding
of naturally occurring ligands. Even when information is available concerning
the structure
of ligands or their binding sites, currently available methods have not
readily led to the
development of effective pharmaceuticals. Methods such as the use of molecular
modeling
to design small molecule analogs based on crystal structure data for ligand-
receptor binding
Pairs, or the screening for binding to a receptor using peptide combinatorial
libraries or.
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natural product extracts, have not proved to be reliable. Additionally, these
synthetic or
natural products do not always have the ability to discriminate in binding
affinity and
specificity for receptor subtypes, which can result in undesirable side
effects due to
insufficient control over the pharmacological effects.
There is a need for a method to more directly reproduce or inhibit the effects
of
natural interactions, and to be able to design specific pharmaceutical agents
that interact with
members of a particular binding pair and which more closely mimic the behavior
of
naturally occurring ligands.
Citation of references hereinabove shall not be construed as an admission that
such
references are prior art to the present invention.
3. SUMMARY OF THE INVENTION
The present invention is based upon the observation of the present inventors
that the
binding site contained within one member of a binding pair for another member
of the
binding pair can be -transplanted into at least one CDR of an immunoglobulin
molecule to
confer specificity on the immunoglobulin for the second member of the binding
pair.
The present invention is aimed at prow iding a method to design,
immunoglobulins,
particularly antibodies, with a particular specif city, which method
circumvents the
unpredictable immunization and scrrfning processes currently employed to
isolate specific
antibodies.~-In particular:~synthetic mudiiied antibodies that
immunospecifically bind one
member of a binding pair are engineered such that the variable region of the
modified
. antibody has.one .or more CDRs that contain the binding sequence for that
member of the
binding pair, which binding sequence is derived from the other member of the
binding pair.
his method, thus, dramatically simplifies the process of identifying suitable
antibodies and
makes available antibodies for many antigens than are inaccessible due to
immune tolerance
or cryptic expression.
Accordingly, the present invention provides modified im_munoglobulin
molecules,
particularly antibodies, that immunospecifically bind a first member of a
binding pair, which
binding pair consists of the first member and a second member, which
antibodies comprise a
variable domain which has at least one CDR containing an amino acid sequence
of the
binding site for the first member of the binding pair, which binding site is
derived from the
second member of the binding pair. 1n a preferred aspect of the invention, the
amino acid
sequence of the binding site is not found naturally within the CDR.
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The binding pair can be any two molecules that specifically interact with each
other.
In specific embodiments, the first member of the binding pair is a cancer
antigen (i.e., a
molecule expressed on the surface of a cancer cell), an antigen of an
infectious disease agent
(i.e., a molecule on the surface of an infectious disease agent) or a cellular
receptor for an
infectious disease agent. Such cancer antigens include human milk fat globule
antigen
(HMFG), an epitope of polymorphic epithelial mucin antigen (PEM), or a human
colon
carcinoma-associated protein antigen. Such acltigens of infectious disease
agents include a
Brambell receptor (FcRB), and antigens of fiSV-2, gonococcus, Treponema
pallidum,
Chlamydia trachomatis or human papillomavrus. In other specific embodiments,
the
binding pair is a receptor-ligand binding pair. for example. where the first
member of the
binding pair is a bradykinin receptor.
The invention further provides methods of treatment or prevention using the
modified immunoglobulins of the invention. For example, modified antibodies
having one
or more CDRs containing the binding site for a cancer antigen or an antigen of
an infectious
agent or a cellular receptor for an infectious disease agent can be used in
the treatment or
prevention of a cancer or an infectious disease a.5soc.iated with the
expression of the
particular cancer antigen or antigen of the infectious disease agent or the
cellular receptor for
the infectious disease agent.
The invention further provides methods for screening or detection or diagnosis
using
~e modified immunoglobulins of the invention. For example, modified antibodies
having
.. one or more CDRs containing the binding site for a cancer antigen or an
antigen of an
infectious disease agent can be used in the screening, detection and diagnosis
o° a cancer or
an infectious disease.associated. with the expression of the particular cancer
antigen or
antigen of the infectious disease agent.
The invention also provides therapeutic and diagnostic kits and pharmaceutical
compositions containing the modified immunoglobulins of the invention.
The invention further provides methods of producing a synthetic modified
immiuloglobulin of the invention.
Section 6, infra, describes the synthesis of synthetic modified antibodies in
which
one of the CDRs contains an amino acid sequence from bradykinin encompassing
the
binding sequence for the bradykinin receptor. The example demonstrates that
this synthetic
modified antibody immunospecifically binds the bradykinin receptor, and
competes with
bradykinin for binding to the bradykinin receptor. The activity of the
synthetic modified
antibody is antagonized by an antagonist of bradykinin activity.
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4. BRIEF DESCRIPTION OF THE FIGURES
Figure 1. A schematic diagram showing the structure of the light and heavy
chains
of an immunoglobulin molecule, each chain consisting of a variable region
positioned at the
amino terminal region {HZN-) of the immunoglobulin and a constant region
positioned at a
carboxyl terminal region (-COOH) of the immunoglobulin.
Figure 2. A schematic diagram of an IgG showing the four framework regions
(F'RI,
FK2, FR3 and FR4) and three complementarity determining regions {CDRI, CDR2
and
CDR3 j in the variable regions of the light and heavy chains (labeled as V~
and VH
respectively). The constant regio:~ domains are indicated as C~ 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
domains of both light and heavy chains and the C. and CH, domains. Fc
indicates the
corustant region fragment containing the CHI and CH3 domains.
Figures 3A-C. (A). The structure of the expressiol: vector pMRR010.1, which
c~n~ains 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 (CHI, CHZ, CH3) heat' chain and hinge region sequences. (C) The
structure of the
expression vector pNEPuDGV which contains a sequence encoding the kappa
constant
~0..domain of the light chain and the constant domain. and hinge region of the
heavy chain. E or
.. . .: ~ai!.three vectors see Bebbington et al., I9yl, 'l<terho~s in
Enz3:rraalngy 2_:136-145.
figures 4A-H. The~amino acid and nucleotide sequences for the heavy and light
. .....chain variable domains..that have a CDR containirg.bradykinin sequences
and corresponding
heavy and light chain variable doma.m consensus sequences of the synthetic
antilx~dies. .All
of these sequences also contain a leader sequence. (A) The amino acid sequence
and
corresponding nucleotide sequence for the consensus light chairs variable
region C'.~~nVLI.
{B) The; amino acid and corresponding nucleotide sequence for the light chain
variable
-~wregion ~BKCDR1~ in which CDR1 contains a bradykinin sequence. (C.) 'fhe
amino acid and
cctmsponding nucleotide sequences for the light chain variable region BKCDR2
in which
CDR2 contains a bradykinin sequence. (D) The amino acid and coaesponding
nucleotide
sequences for the light chain variable region BKCDR3 in which CDR3 contains a
bradykinin sequence. (E) The amino acid and corresponding nucleotide sequences
for the
consensus heavy chain variable region ConVH 1. (F) The amino acid and
corresponding
nucleotide sequences for the heavy chain variable region BKCDR4 in which CDR4
contains
a bradykinin sequence. (G) The amino acid and corresponding nucleotide
sequences of the
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heavy chain variable region BKCDRS in which the CDRS contains a bradykinin
sequence.
(H) The amino acid and corresponding nucleotide sequence of the heavy chain
variable
region BKCDR6 in which CDR6 contains a bradykinin sequence.
Figure 5. A schematic diagram of the general steps that were followed for
assembly
of an engineered gene encoding the synthetic modified antibody containing A
sequence of
bradykinin. The oligonucleotides used to assemble the gene are indicated as
"oligol" to
"oligo 10".
Figures 6A and B. (A) Nucleotide sequences of the oligonucleotides used to
assemble the consensus light chain (ConVhl j, and the bradykinin containing
light chain
variable regions, by the scheme indicated in Figure 5. (B) Nucleotide
sequences of the
oiigonucleotides used to assemble the consensus heavy chain variable region
(ConVHI; and
the bradykinin .containing heavy chain variable regions. as indicated in F
figure 5.
Figures 7A-C. (A) Stimulation of PGE, synthesis by bradykinin in SV-T2 cells
as
indicated in ng/well of PGE2 for each treatment. In the legend below the
figure a "-"
indicates that cells were incubated in the absence of the factor while "+"
indicates that the
cells were incubated in the presence of the factor, i.s., either 1 cM
bcadykinin (upper row) or
i nM HOE 140, a bradykinin antagonist (lower row). (B) Stimulation of PGEZ
synthesis by
certain synthetic modified antibodies having CDRs containing bradykinir_
sequences is
depicted as pg/well PGE2, as a function of the dilution of the synthetic
antibody BKCDR3
(lines with .solid squares), BKCDR4 (lines with solid triangles), and
RK(.'.DRS (lines with
~: ~ solid diamonds), the consensus heavy chain variable regio:~ (line with
solid circles) and
media alone (line with open circles). (C) The bar graph depicts PGE~
stimulation (in PGE,
in pg/well j in SV-T2 cells.incubated in the presence or abs~we of bradykinin
(indicated as
"+" or "-", respectively, in legend below graph) anriwith an antibody .having
the BKCDR3.
BKCDR4, or BKCDRS variable domain or an araibody having the heavy chain
consensus
variable domain (ConVH), as indicated above the b~ of the graph.
5. DETAII:ED~DESCRIPTION OF Tl~lE INVENTI
The present invention is directed to modified immunoglobulin molecules,
30. p~icularly antibodies that immunospecifically bind (e.g., as determined by
any method
known in the art for determining the binding specificity of an antibody for
its antigen, for
example, as described in section 5.7, infra, and which immunospecific binding
excludes
non-specific binding, but not necessarily the cross-reactivity often observed
with naturally
occurring antibodies) a first member of a binding pair and have at least one
complementarily
determining region {CDR) that contains an amino acid sequence from the second
member of
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the binding pair, which amino acid sequence is a binding sequence for the
first member of
the binding pair. 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 antibody contains a binding sequence for a cancer antigen
(i.e., a molecule
on the surface of a cancer or tumor cell), an infectious disease antigen,
(i.e., a molecule on
the surface of an infectious disease agent), a cellular receptor for a
pathogen, or a receptor or
ligand (preferably, a receptor or ligand of a receptor-ligand binding pair in
which the ligand
binds to the receptor and thereby elicits a physioiugical response).
The present invention~also providesfor:methods of treatment using the modified
immunoglobulins of the invention, for example, Gut wot by way of limitation, a
modified
antibody having at least one CDR containing a-binding sequence for a
particular cancer
antigen or antigen of an infectious disease agent cr a cellul;~r receptor for
an infectious
disease agent can be used to treat or present a cancer or an infectious
disease characterized
by the presence of that particular antigen by binding of the infectious
disease agent to the
particular receptor.
The present invention also provides for methods of diagnosis and screening
using the
modified immunoglabulins of the inventicrn, fo~~ example;. hut not by way of
limitation, a
modified antibody having at least one CDR vontaining a binding sequence for a
particular
o~cer antigen or antigen of an infectious di: ease agent ;;an be used to
detect a cancer or.
. : ir~fect:ous disease characterized bythat parii,ular:intigc.n or ry binding
of the infectious
disease agent to the particular receptor.
For clarity of disclosure. and not by way of limitat;on. the detailed
description of the
invention is divided into the subsections :a~~hic?: Follow.
5.1. MODIFIED IMMC1NOGLi7~$lll:f~ M~ _L_ECiJLES
'hhe invention provides for modif ed immur~oglobulin moleculrs, particularly
wantibodies, that immunospecitically bind fe.~., s~s deterrtiined by any
method known in the
art for determining the binding specificity..~f ~.n antibody for its antigen,
for example, as
described in section 5.7; infra) to a first .member of a binding pair where at
least one of the
CDRs of the antibody contains a binding site for t~'~e first member of the
binding pair, which
binding site is derived from an amino acid sequence of the othei member of the
binding pair.
1n a prefen:ed aspect of the invention, the amino acid sequence of the binding
site is not
found naturally within the CDR.
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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 far binding to the other
member, ur by
I 0 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
1 ' antigen, an infectious disease antigen, a cclleIur receptor for a
pathogen, or a receptor or
ligand that participates in a receptor-ligand binding ,r:air.
In ~pecifc embodiments, the binding pair i:: a protein-protein interaction
pair which
i.s either homotypic interaction (i.e., is the interecti.~n between two of the
same proteins) or a
heterotypic :nteractiori (i.e., is the interaction between twee different
preteins).
20 In a specific embodiment, the fiat rnembur is a meznber of a. ligarzd-
receptor binding
~~ pair,~prefer hly, of a receptor-ligand bindiz:g pair :r. which the liyud
binds to the receptor
and thereby elicits a physiological response, suc): as intracehular signaling.
By way of
.. example, an~a not by way of limitation. the liganJ or rec;,ptot can be a
horn~one, autocoid,
~. gro~~~th~.fuct«r. c5rtokine or.neurc,~transmitter, cr :ec-rpu~r fer a
t;ormnie~, autocoid, growth
v~ ~S factor, cytokine;.or.neurotransmitter, or any receptor or ligand
involved in signal
transductior. (Foz reviews of signal tr2nsduction tlathways, see, e.g .
(.,ampbell, 199?. .I.
Pediat.' 131.:S42-544: Hamilton, 1997, ,. t,euluw. 8iol. 62:145-i 55: Soede-
Bobok & Touw.,
1997, J. ?~to?. Meci 75:470-477; tieldir., 1995, l.:'eil 8U:21:3-223;
Kishimoto et al., ' 994, Cr:i
. 76:253-262; \~Iiyajima et al.., 1.992, Annu Rev. Immunol. 10:295-331; and
Cantley et al.,
30 1991, Cell 6_St28i-302.). ~ In specific embodzrrtents,~one member of the
binding pair is ~,
ligand such as, but not limited to, cholecystokinin, galanin, IL-1, IL-2, IL-
4, IL-5, IL-6, IL-
11, a chemokine, leptin, a protease, neuropeptide Y, neurokinin-1, netzrokinin-
2, neurokinin-
3, bombesin, gastrin, corticotropin releasing hormone, endothelia, melatonin,
somatostatin,
vasoactive intestinal peptide, epidermal growth factor, tumor necrosis factor,
dopamine,
35 endothelia, or a receptor for any of these ligands. In other embodiments,
one member of the
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binding pair is a receptor, such as, but not limited to, an opioid receptor, a
glucose
transporter, a glutamate receptor, an orphanin receptor, erythropoietin
receptor, insulin
receptor, tyrosine kinase (TK)-receptor, KIT stern cell factor receptor, nerve
growth factor
receptor, insulin-like growth factor receptor, granuiocyte-colony stimulating
factor receptor,
somatotropin receptor, glial-derived neurotrophic factor receptor or gp39
receptor, G-protein
receptor class or 132-adrenergic receptor, or a ligand that binds any of these
receptors. In
another embodiment, one of the members of the binding pair is a ligand gated
ion channel,
Such as but not limited to a calcium channel, a so~ium channel, or a potassium
channel. In
certain embodiments, the invention provides T~.adified imrnunoglobulins that
lQ..immunospecitically bind.a receptor and arc ru=tagonicts the ligand that
binds that receptor,
for example, but not by way of limitati.nn, are antagonists of endorphin.
enkephalin or
nociceptin. .1n other embodiments, the im~entio:~ provides synthetic modified
antibodies that
immunospec:lflcally bind a receptor and are agonists of the receptor, for
example, hut not by
way of limitation, the endorphin, enkcphalin, or n~ciceptin receptors. In a
preferred
... I S embodiment. the modified immunoglobulin. does clot bind the
iibronectin receptor. In
. another areferred embodiment, the binding sequence is not Arg-Gly -AsF. is
not a multimer
cf a binding sequence. and preferably is not a multimc;r of the se;~uence ~.rg-
Gly-.4sp.
1n otl r specific embodiments, the modified imnmnoglcbulin laas a CDR that
c:cniairm a hi'nding site for a transcription .factor. Irr a preferred 2aPect,
~he modified
. ,' '-O..immanogli~bulin does. not bind to a specific PNE1. sequence,
particular~tyioes nu!: bitad to a .
w~-~ ~ tra:lserip:ion fa:cror binding site.
. In pret~rred embodiments. tr.~ modif ad immunoglobulin nas a: :z;at one CDR
that
.,~ c~~ntairts.an.a~nino. acid sequence: of a.binding sit: for..a
ca.~cer.anti~en ~r :~ ticnor antigen
. . (e.g., as de~cribed.in detail in srctiwn 5.3.1, iiFh-u.:~:~.rftore
preierrhly Uhc anli~En is human
ZS colon carcinorna-associated antigen or epithelial muc~.n antigen. ,.Iri
ettter:emb~diments, at
ic:ast one CDR of the modified imxnnnogtohulir: contains an atnina nci.d
sequence for a
.. binding site for a human milk fat glob;rle receptor. in other
emho~yi:n~~ris, the rnodified
r~immunoglobulin.has at least~one l'.DR~ that contains as amino acid s:qttenci
of a binding site
for stn antigen .~f a t-amor of the brrast,~ovary;wiems, prostate, biad3er, t-
~tng, skin, pancreas,
30 colon; .gastrointestinal tract, B lymphocytes, or T lymphocytes.
In other preferred embodiments of the invention, at least one CDR of the
modified
antibody contains an amino acid sequence for. a binding site for an antigen of
ar_ infectious
disease agent (e.g., as described in.detail in section 5.3.2, infra.), or a
binding site for a
cellular r,°ceptor of an infectious disease agent, preferably where the
binding site is~not an
35 wino acid sequence of a Plasmodium antigen, or is not the binding site Asn-
Ala-Asn=Pro or
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Asn-Val-Asp-Pro. In additional embodiments, the modified antibody has a CDR
that
contains the binding site for a bacterial or viral enzyme.
The modified immunoglobulin molecules of the inventin can be derived from any
type of immunoglobulin molecule, for example. but not limited to, antibodies,
T cell
receptors, B cell receptors, cell-surface adhesion molecules such as the co-
receptors CD4,
CDB, CD19, and the invariant domains of MHC molecwles. In a preferred
ernbodiment of
the icivention, the modified imrizunoglobulin molecule is an antibody, which
can be any class
of antibody, e.~,~., an IgG, IgE, IgM, IgD or IgA, preferably, the antibody is
an IgG. In
addition the antibody many be of any subclass of the particular class of
antibodies. In
.. . 1 ~ another specific embodiment, the modified ir:lmunoglobulin molecule
is a T cell receptor.
. ~ 'l he immunoglobulin which is modifiedao generate the modified
immur~oglobulin
can be any available immunoglobulin molecule: ::nd is preferably a monoclonal
antibody or
. . is a synthetic arnibody. The antibody that is modified maw be a naturally
occurring or
previously existing antibody or may be synthesised from known antibody
consensus
. 1~ ~ senuel:ces, such as the consensus sequences for the fight a.~d heavy,
chain variable regions in
Fi~,Lres 4A and B, or any other antibody consensus or germline (i.c.,
um~..cumbined genornic
sequences) sequences (e.g., those antibody consensus and germline sequences
described in
Ie..l6at et. al., 1991, Sequences of Proteins of Irrnnunological lrterest,
5't' edition, N1H
Punlficatior. ~~'do. 91-3242, pp 2147-2172).
. . ~: 2lt . :. . ,~~; noted :~upru, Zach antibody molev;~:le.h~ six CDR
sequ~n~e;" three an the light
. chaixwnd t:~ree owthe heavy chain, and five of these C:DRs are germlirAe
CDF.s (i. ~., are
dircctly.dcW es from the germline genomic sequenrx of the animal, witrao'my
.recombmatior~) and one of the CDRs is a non-germIinc CDR (i.c., differs.in
sequel:ce from
the germlir..e gcnomic sequence of the anima! and ~i:,~encrat~d by~
re:.or:bination of tree
,~. aa.2~, germlir_~$equences j.x..~lVhether a CDR is a g~nnline or~nor.-
germlineacquenre can be
. determined by.~equencing the CDR and then comparing :hN ~edu~nc~ wills known
germiine
~xyuent2s, e.~:, as listed in Kabat et al. (19~)l.. Seque:~ces of Proteins cvf
hnntunological
~Interest~'~ edit'son,~i'~If~ Publication No. 91-324, gp 214'7-2172).
Significant variation
~. . from the ?mown germline sequences indicates. that the CDR is a non-
germlir..e CDR.
Accordingly, in other embodiments of the invention, the CDR that contains the
amino acid sequence of the binding site is a germline CDR or, alternatively,
is a non-
germline CDR.
The binding site can be inserted into any of thewCDRs of the antibody, and it
is
within the skill. in the art to insert the binding site into different CDP,s
of the antibody and
35 then screen the resulting modified antibodies for=the ability to bind to
the particular member
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of the binding pair, e.g. as discussed in Section 5.7, injr~a. Thus, one can
determine which
CDR optimally contains the binding site. In speci~Fc embodiments, a CDR of
either the
heavy or light chain variable region is modified to contain the amino acid
sequence of the
binding site. 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
...;Uie binding site. In another embodiment of the in~:rntion, more than one
CDR contains the
~arnino acid sequence of the binding site or more than one i:DR each contains
a different
-binding site for the same molecule or contains a different binding site for a
different
10.~-.mol~~ule..~In:particular, embodiments, tvvo, three, four, five or six
CDRs have been
engineered to contain a binding site for the first mcmber:ni the binding pair.
In a preferred
. embodiment,.one or~more .CDRs contain a binding site for the~.first member
of.a binding pair
. . and one or more other CDRs contain a binding site for.a rr:~~lecule on
the. surfare,of an
immune cell, such as, but not limited to, a T cell, F3 cell, N K cell, K cell,
TII, cell or
I' =neutrot~hil.. for example,:a modified antibody havingw oindirg site ior.a
career. antigen or
.an infc;ctlous disease antigen and a binding site for a mcalecule on the
surface ~>f an imenune
cril ,~,ai; be used to target the immune cell to a cane; r ~~eli ?gearing the
cancer antigen or to the
infectious disease agent.
In specific embodiments of the invention, the binding site amino acid sequence
is
'4.::eit.~rer.insertedintwthe.CDlt without replacing anyafthe uminc~ acid
seq~r~n~r of the CDR
Y~ ir,~c-1!-c~r,-~llenratiwly; the binding site~aminoacid~sque~n~teplaccs ~eil
or a portir~n of the
.-.amino-acid.srquence ~of. the CDR.- In specifii; emhodiruc;nts,~.tiie
binding site arninu avid
.,.sequence replaces.1, ~2, 5, 8, '10,.1 S,.Qr.2U..atnina. acids of the CDR.
sequence.
:..'fir ono avid s~uence of the bindiiy ~sitc;:~r~;sc~~t in~.'tht :C:I.yH
ean.°be~.the minimal
25 .binding site necessary for the binsiing of the. mernb~r~
ot:~the.~inding~air ~(wrhich can be
;_de;ermined empirically by any method known in the artl: alrerlaatively, the
binding site c;an
:be greatc: :han the.minimal binding site neccssary.for ihc~ binding of the
member of the
. . c~!~inding paii:~Iwparticular~embodimerits;the°bindinysit~ amino
acid sequence is arleast 4
.:amino acids in length, or is at Least 6, 8,.10, 15, ur 2U amine acids in
length. In other
3d ~ embodiments the binding site amino acidsequence is no tnorc than ..10,'
15, 2U, or 25 amino
acids in length, or is 5-10, 5-15, 5-20, 10-L 5,10-24.or 10-25 amin~.acids in
length.
In addition, the total length of the CDR (i.e., the combined length of thz
binding site
sequence. and the rest of the CDR sequence) should be of awappmpriate number
of amino
acids ~to allow binding of the. antibody.to the antigen. CDRs have been
observed to. have a
~5
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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
CSR Number of residues
L1 10-17
L2 . 7
L3 7-11
H 1 5-7
~ 9-1?.
H3 .. . 2~~25
. .(compiled~from.data in Kabat and'afu, 1971,.4nnw.NYAcad Sci.
x:382-93)
~~ .Vi~ hile.many CDR H3 regions are of 5-9 residue ir. length, certain C:DR
H3 regions have
.bin observrd that are much longer.: ln.palticular, a number cfantiviral
antibodies have
heave. chain CDR H3 xegions of 17-24 residues in length.
Accordingly, in sp~~.cific embodiments of the invention, the CDR Containing
the
binding site is within the size range provided for that particular ('.DR 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
20. second CDR of the light chain, L', the CDR~is~ 7 amino acid residues; if
it is~the third CDP:
.; .... ,~. of the light chaily.L3, 'the (:DR is 7 to .11~ amino acid
residuws; if it is the first CI)R of the
heavy chain; I-il; the. CDR i~.5.to 7 amino acid.residues: i!' it is the
second CDR of the heavy
.,~. ~..ohain, H2,.the.CUR is.9 to,l2..amino acid.residues;.and i~.it_is the
third CDR of the heavy
. chain,.I-I3, the CDR is 2 to 25~amino.acid
residuesh3noth~r_specific.rnax~dimrnts, the C:Di~
~2$~ containing the binding site is 5-1D, 5-15, 5-20,=1.~1 ~15;~:1~1=._'G.l~:l-
'~~,:or 16_.25 amino acids in
. . ~ .... . . length.: In other embodiments, the CDR containing tha birdrng
sitz is at least 5.. 10, 15, or
. : 20 amino acids or is no more than 10, 15, 20, 25, or 3f.~ am:ino acids n
length.
,. .., .:~~In~specific~embodiments the mc~difie~ immunoglobulin of the
invention contains a
portion of a .variable region, i. e. , ~'vhere either the heavy or the light
chain .contains. less than
30. ~e:framework regions and three (:DRs, for example ~burnot limited to,
where the variable
region contains one or two GDRs, and preferably, the intervening framework
regions.
In a specific embodiment, the modified antibody immunospecifically binds the
bradykinin receptor (for example, but not limited to the modified antibody
described in
section 6, infra).: In particular, the embodiment provides a~modi6ed antibody
in which at
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least one CDR contains the amino acid sequence Arg-Pro-Pro-Gly-Phe-Gly-Phe-Ser-
Pm-
Phe-Arg.
in other specific embodiments, the modified antibody immunospecifically binds
the
human milk fat globule antigen, and at least one of the CDRs of the modified
antibody
contains an amino acid sequence selected from the following: (i) Ala-Tyr-Trp-
Ile-Glu; (ii)
. . Glu-Ile-I,eu-Pro-Gly-Ser-Asn-Asn-Ser-Arg-Tyr-Asn-Glu-Lys-Fhe-Lys-Gly;
(iiil Ser-Glu-
Asp-Ser-Ala-Val-Tyr-Tyr-Gys-Ser-Arg-Ser-Tyr-Asp-Phe-Ala-'rrp-Phe-Ala-Tyr; (iv)
Lys-
Ser-Ser-Gln-Ser-Ixu-Leu-1'yr-Ser-Ser-Asn-Gln-Lys-Ile-Tyr-I:eu-Ala; (v) Trp-Ala-
Ser-Tllr-
. , ~A;~g-G1u-Ser; and (vi) Gln-Gln-Tyr-Tyr-Arg-Tyr-Pro-Arg-Thr.
,. ;,.tw.;~n a~rnoreapecific embodiment,.the-CDRs.of the heavy chain variable
region contain
the amino acid sequences (i)-(iii) above, whereas the Gi3Rs of the light
chainwariable region
.contain the amino acid sequencxs (iv)-(vi) above.
. .. , in specific embodiments, the invention provide a. mc~ified antibody
that binds
. . human colon carcinoma-associated antigen and comprises' a variable region
having at least
one CDR containing one~of the following amino acid sequences: Thr-Ala-Lys-Ala-
Se~:-cJl.l-
. : ..Ser-Val-Ser-.Asn-Asp-Va.1-Ala; Ile-Tyr-Tyr-Ala-Ser-A.sn-Arg-lyr=I'hr;
Phe =Ala-(.Lln-Gln-
Asp-'l~yr-Ser-Ser.-Pro-Leu-'Thr; Phe-Thr-Asn-'I~yr-.UIy~~Met-Asn; Ala-Gly-Txp-
Ile-Asn-Thr-
Tyr-Thr-GIy~GIu-Pro;Thr-Tyr-Ala-Asp.Asp-Phe-Lys-Gly; .~r Ala-Arg-Ala-Tyr-'Ty r-
Cily- .
Lys~Tyr-Phe-Asp-Tyr.
. . ~ v After constricting antibodies containing modified GDRs, the modif ~.d
~:~tih,~.~dir. .
:~cim.l~.:furtheraltered and screened to~sele~ct awantibodyhaving higher
af.~imty.Gr~E-~~_iilGlty.
.vAnt~bodies 1-.aving higher affinity or specificity for the target antigen
nlay be generated and
..sele~ted.by.anv methc~l.known in the art. Far..o~cample,.but.not by. way of
limitation, the
.. : :nucleic acid encoding the synthetic.modifed~arrtibbdy=car..fie
mutagenizr~cither.ralxdomly.
. 25 .i,e.; by chemical or site-directed triutagenesi~~ ~:.by rucking
particular~muttdiem at specific
.,:: positions iwthe nucleic acid encoding the sn~ified antibody, and ther.
Sc~rcxning the
,.W utibc~ie3 exposed from the mutated nucleic av;i~i molecules for binding
ri:fjr:ity for the
;~~.set.an~g~ ~;~gw~-~ocomplished ~by~. testingthe expressed antibody
molecules
. :: individual ly or by . screening a .library wf .the mutated . sequences,
e.,g: , by. phage display
.. ~~ 30 ~~~~i9y.(see; a,s; U:.S..Yaterrt Nns..5,223~,409:5;403;484; and
5,571,698, all-by Ladner
et al; PCTi"uhiication ~V(~ 92101049 by McCafferty .et al. or any other phage
display'
technique known in the art).
Accordingly, in a specific embodiment, the modified antibody has. a higher
spcxill~ity or affinity for an antigen than a naturally occurring antibody
that
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immunospecifically binds the same antigen. In another embodiment, the modified
antibody
exhibits a binding constant for an antigen of at least 2x 1 b' M.
The modified antibodies of the invention may also be further modified in any
way
know in the art for the modification of antibodies as long as the further
modification does
. . S not prevent or inhibit binding of the modified anfibody to the
particular antigen. In
particular, the modified antibodies of the invention may have one or .more
amino acid
-substitutions, deletions, insertions besides the insertion into or
replacement of CDR
sequences with the amino acid sequence of a binding sequence. Such amino acid
. substitutions, deletions or insertions can be any substitution, deletion or
insertion that does
~.10..not:prevent ~or inhibit the immunospecific binding of the modified
antibody to the target
antigen. For example, such amino acid substitutions include substitution'
of~functionally
. . . equivalent amino acid residues. For example, one or more am~ro.acid
.residues can be
. . .. .substituted by another.amino acid of a similar polarity which acts as
a functional equivalent,
resulting in a silent alteration. Substitutes for an amino acid may be
selected from other
. 1 S members of the class to which the amino acid belongs. ~ For example, the
nonpolar.
. (hydrophobic) amino acids include alan'tne, leucine, isoleucine., vulit~e,
proline,
phcnylalanine, tryptophan and rriathionine. The polar neutral amino acids
includE glycine.
:~eerine, tlueonine, cysteiiie, tyrosine; asparagine, and gluts;nine. The
positively charged
. . ..(basic) amino acids include arginine, lysine and histidine. The
negatively charged (acidic)
ZO-. amino acids include aspartic acid and glutatnic acid.
-.<.::v.~~:,4dditionally~; one or moreamino acid residues within the seque:lc4
.:an be substitui~cl
. ~. v by a nonclassicai amine acid or chemical .amino. acid analogs can be.
introduced as a
...:....substit~~tion oi,addition into_the immunoglobulin sequence. ..Non-
classical amino acids
~: _. :: include but.arewot limited to the D-isomers
of.thr~.~c~.omni:~n.ar~iino acids:.a-.amino isobulyric
. ..25 .~id,~4-aminobutyric~acid, Abu, 2-amino butyric;:acid~.~-
Abu,~.=A~xti~3minu hexanpic
., .. ; acid. Aih, ?.-amino isoblttyric acid, 3-amino propionic acid,
ornithine, norleucinc, norvalii»,
~' hydxoxyproline, sarcosine, citrulline; vysteic acid, t-butylglycine, t-
i'~atyla=anine.
::; ;: ~rphenylgiycine;wyclohexylalanine; (3-alariine, ~tluoro=amino acids,
designer amino acids su4h
..,. ...~ p_methyl amino acids,.Ca-methyl amino. acids, Na-methyl amino
acids;.and amino acid
.. 34 ~~ogs ~,g' . ~y~ore, ~e amino acid can be D~ (daxtmrotary.)'or. L
(levorotary j.
. . . In a particular embodiment of the invention, the modified
immunoglolfulin tics been
further modified to enhance its ability to elicit an anti-idiotype
reaponse,.for example; as
described in co-pending.United States Patent Application Serial hlo. ,_"-__,
entitled
"Modified Antibodies with Enhanced Ability to Elicit An Anti-Idiotype
Response" by-
35 g~~ filed November 13, 1998 (attorney docket no. 6750-O15), which is
incorporated by
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reference herein in its entirety. Such modifications are made 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 modified
immunoglobulin is
further modified such that one or more variable region cysteine residues that
form disulfide
bonds are replaced with an amino acid residue that does not have a stllfhydryl
group.
. ...Identifying.the cysteine residues that.fomi a disulfide bond :n a
variable region of a
particular antibody can be accomplished by any method known in the art. For
example, but
not~by way o;'limitation, it.is well known in the art that the cysteine
residues that form
..w intrachain disulfide bonds are highly consen~ed among antibody classes and
across species.
.:1~0 :Thus,.the cysceine residues that participate in disulfide band
formation can be identified by
sequence comparison with other antibody.molecules iw.which it is itnuwn which
residues
. . form a disulfide bond (for example the consensus-sequences provided
in:Figures. 4 A and E,
. or thOSt.df:SCrlbed in Kabat et al, 1991; sequences of Proteins of
Tmcnur~«logical Interest, 5th
W;d., L'.S. Uepartmer~t of Health and Human Services, l3ethesda, Maryland).
I S :. .. ; v.:~ :.Table i provides~awlist-t~f the positions of dise~lhd,
bond~formi7g cysteiw residues
for a nurnber.nf antibody molecules.
Table 2
(derived from Kabat et~al, 1991, sequences of Proteins of Inunucwlogical
Interest, St.h~ F:d:, w
;yl~:-.U.S._Uepartrnent of Health and Human.:Ser4ices, Bethegda; :~-i~uylandi
x....:..
...I?-isulfid~ bond-forming
.. ..Variable domain ~ ~ ~ ~ ~ rysteines
S ecies Sib rou ~ ~ w ( ositions)
25 ~~ kappa light ~ :E '.23:88
Hucnfun kappa light I: 23,88
. : ~ Human kappa light lIl 23,88
Human kappa light I~' 23,8R
Human lambda light I ~ 23,88
. rHuinan lambda light II 23,88
~~ 30 ~ H~ ~~a light . IIII 23,88
i~Iun~an lambda light IV 23,88
Human lambda light V 23,88
Human lambda light Vl 23,88
Mouse kappa light I 23,88
Mouse kappa light ~ II 23,88
Mouse kappa light III 23,88
35 Mouse kappa light IV 23,88
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WO 99/25378 PCTNS98/24302
Disulfide bond-forming
Variable domain cysteines
Species Subgroup (positions)
Mouse kappa light V 23,88
. Mouse kappa light VI 23,88
Mouse kappa light VII 23,88
.Mouse kappa light Miscellaneous 23,88
Mouse lambda light 23,88
Chimpanzee lambda light 23,88
Rat kappa light ~~ 23,88
Rat lambda light 23,88
~bbit kappa light 23,88
Rabbit lambda light ~ z:~,88
Dog kappa.light 13;8g
' Pig kappa light ~ ~ 23 (88)
Pig lambda light 23,88
C'.ruinea pig lambda light 23 ! gg)
Sheep lambda light 23,88
IS Ghickcr. lambda light . 23,8g
Turkey lambda light 23 (88)
Rattish . 23 (88)
lambda light
Shark kappa light 23,88
Human heavy t 22,92
Human heavy . . , lI 22,92
Human. heavy ~ 'III L'~,92
'.Mouse ~ wheavy I .(Aj . 22,92
. Mouse heavy I (I3) i ~.92
Mouse heavy II lA) 22,92
,Mouse heavy '.II (H) 22.92
Mouse heavy II (C) ?2,92 .
- Mouse heavy ~: II1 (A). ?2,92
Mouse 'heavy ~ ~III(~3) 22.92
Mouse heavy .,..III~(C) 22;92
Mouse heavy ~ lII (I)) 22,92
Mouse heavy V (.A) .22,92
.Mouse heavy 1,r !R) 22,92
Mouse heavy . : -.~:Iviiscellaneou~22,92
Rat heavy 22,92
Rabbit heavy ?2,92
~ca pig heavy 22,92
fat heavy ..22 (92)
Dog heavy 22,92
Pig heavy 22 (92)
Mink heavy 22 (92)
Sea lion heavy 22 (92)
Seal heavy 22 (92)
Chicken heavy 22,92
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WO 99!25378 PCTNS98/24302
. Disulfide bond-forming
Variable domain cysteines
Species Subgroup (positions)
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
C'.roldfish heavy 22,92
~~sh heavy 22 (92)
Shark ~ heavy 22,92
-.position numt~ers enclosed by ~) indicate that the protein was nca sequenced
to that position,
but the residue is inferr:;d by comparison to known sequences.
'S :w : r: Notably, tc~r all ot'the antibody molecules listed in"fable 2, the
cysteine residues that.
form the iritrachain disulfide bonds are residues at positions 2:; and 8R of
the lightwhain
variable dorrtair. and residues at positions J2 and 92 of the heavy chair..
variable:domain.
The pc~sit:on n-ambers refer to the residue corresponding to that residue in
the.ccnsensus
.. sequences a,~ defined in Kabat. (1991, Sequences of Proteins of
Immmolugi.cal Interest, 5th.
.ZO.:Ed., iJ. S.. Department.of.Health and liu~-nan Services, .Bethesda,
Maryland:! or as.iradicaaccl in
;.-,the=hcavy~and light~chain~rariable region sequur~ccs~depicted in Figures
4,A and E,
nesp~ctively. (as determined by a~igning.die particular antibody sequence with
the.consensus
:..sequetlce_:nr.the.heavy .or light chain variable region. sequence depicted
itz Figures 4:4 and
lr).
25 Accordixigly, in one embodiment of thG invention, the
modifiedunnnunoglobulin
r v ~ molecule i5 feuthPr modtf ed such that tha residues at positions 23
and~or 88 of the light
:-...chain are substituted with an amino acid residue tract does not contain o
sulti~ydryl group
yandlor~the;.-esiduses,at.positions~22vt~dlor~:y2~are substituted with
an.aminc~.acid ce'sidue trat
. . .does nut contain a sulfltydryl group.
30 ~ ~ ~ ~ - ~. 'ftte amino acid residue: that substitutes for the disulfide
bond .forming cyst~eine
. . . residue is any amino acid residue that does not contain a sulthydryl
group, v.k , alanine,
arginine, asparagine, aspartate (or aspartic acid), glutamine, glutamate (or
glutamic acid),
glycine, histiduw, isoleucint, leucine, lysine, phenylalanine, proline,
scrine, threonine;
tryptophan; tyrosine or valine. In a prefened embo~d~ment, the cysteine
residue is replaced
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VirO 99/25378 PCT/US98/24302
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, such as those listed
supra, that does
not contain a sulfhydryl group.
.. : .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
w~~~ Chain and light chain variable regions. w In other specific embodiments,
one of the residues
.. ~ that forms a panic;ular disulfide bond is replaced {or deleted) or,
alternatively, both residues
. ::1 Q that form a particular disulfide bond may he replaced (or deleted).
In other specific embodiments, the invention provides functionally aiaive
fragments
of a modified immunoglobulin. Funetionaily aetive~ fragment means: that ttte
fragment can
... immullospecifically bind the target antigen as determined by any method
known.in the art to
w ~ determine immunospecific binding {e.g. , as described in Section 5.7 ~in,
fin); r or example,
r'l~~such~.fragments include butane not limited to: F(ab')z fragments. which
contain thz variable
. ~ :.regions of both the heaiy and the light.ohains, the light constanr
region and the t;.'.HI domain
of the hear~~~~ cizain, which fragments can be generated by pepsin digestion
of the antibody.
and the Fab fragments, generated by reducing the disulfide bonds of an F(ab')2
fragment
(Figure 1; King et., 1992, Biochem. J x:317); and Fw fragments, i.e.,
fragments thi3t
...2.0 . contain tlw variable region dpmains of both the.heavy and light
chains (~Rcichntac~ci ar~cl
,aW,inter,:.14~t8, J..~Lfol.~;Bivl.,~Q~:825"King et a1.~.199a; l3i~rchem. J.
~~:7?.3~.
. . . T he invention also,includes single chain antitx~di~s (SCA) (U.3. Patent
4,94n,778;
:: : Bird. 198E, S;.icrce x:.423-426; Hucton et al., .19813, Prc~c. Natl A~a~'
.~ci. JSA x:5879-
. . 5883; and Ward et al., 1989, Nature ~;544-54f):#~Si~glo.chain:~nt3bodies
are. formed by
w' 25 linking the heavy and light chain fragmentsof-the.Fvc:~gi~n
via.an~arino.acid bridge,
:resulting in a single ~cbain polypeptide... Additionally, the invention also
prow%idc~s heavy
v.t. xhain and light chain dimers and diabodies.
r ~:a The,invention.:further~pmvides~modified..ar~tibodi~a that are also
cl:imeric or
. . ~. humanized antibodies. . A chimeric antibody. is a molecule in which
different portions of the
~, ~.,30 . antibody molecule are derived frorri different animal. species;
such as-thosehaving a variable
region derived from a murine mAb and a constant region derived from a human
immunoglobulin constant region, e.g., humanized antibodies. ~fechniques have
been
developed for the production of chimeric antibodies (Marrison et al., ~i9R4,
Proc..Natl.
Acad Sci. $1:6851-b855; Neuberger et al., 1984, Nature ~:6(~4-6D8;''Ta'keda et
al., 1985,
35 Nature X4_:452-454; International Patent Application No. PCT/GB85100392
(Neubarger et
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al. and Celltech Limited)) 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 can be used. In a specific embodiment, the sy
nthetic
modified antibody is a chimeric antibody containing the variable domain of a
non-human
antibody and the constant domain of a human antibody.
. .. . In a more preferred embodiment, the .modified antibody. is a humanized
antibody,
w particularly an antibody in which the CDRs of the antibody (except for the
one or more
CDR.s-containing the' binding sequence] are derived from an antibody of a non-
human
animal and the framework regions and constant region are from a human antibody
(U.S.
;10:-,patent No.:5,225,539..by. Winter).~.Such CDR-grated antibodies have been
successfully
constructed against various antigens, for erhmplr::,:antibodies against IL-2
receptor as
. ... described in.Queen .et al., 1989, Proc...Natl:: Acaa'. Sci US.4
8_6:10029; antibodies against cell
surface.receptors-CAMPAT'H as described is Rieohntann et al.. l 988. :"Jature
X32:323:
antibodies against hepatitis B iri Cti'et al., 1.991, Pros. Natl. Acad fci
llfA >~:2869; as well
i was against viral antigens of the respirator~msyneitial virus in Tempest et
al., 1991. Bio-
.Technoloy x:267.
CDR-grafted'variable region genes have b~er~ construrtcd by various rz~ethads
such
as site-directed mutagenesis as describua iau Jane of al.. :986, Nature
~2~:.522: ltiechmann
et al.,1988. Nature X2_:323; in vitro,xssembly of erttirc CDR-gr~d ~ v
ariable. regi<ms
:y0 .(t?u,~n et al.;~ 1989, ~Proc:. Natl. Acad Se#. US.~ .~6:1 U()29); an~1
the use of PCIt. to synthrsix
-::~:.~,~:ed~genes~(vaugherty~et~al-.19~11;..Nxr;,lri~ ~~:cids RE~c. IQ:2471
). CDR=gra:~ed
~antihodios are generated in which~thc CDI~.~ of the .marine monoclonal
antibody are grafted
=ontathe..framz~ncork~regions.ofahuman antibirdy:,,Follnwir~ grafting, most
antibodies
-: be~fit'from additional.amino acid cha:~ge:,~ift
~hrvcrm~ew~rk:2egicn:~tomc~itarai.~ affinity,
25 ~ presttcnably. because framework residues ar~necessa~y..to
maintaiir~C:Dit~ cotrformation, and
. . some framework.residues.have been dcmoctstrated to be part of the antigun
combining site.
- Thus. in specific.embodiments of the itivtutie:~~:. tla~. modit~c~i antibody
comprises a variable
- :~~ciomain ~irt ~whieh~at-least one of the framework regions hag one or more
arninu a;:id residues
that differ.from the residue at that position in the ndttraliy occurring
&~nework region.
w . ~ ~ 30 . . ~...,~. In a preferred embodiment ofthe invention, the modifies
antibody is derived from a
. htm~an monoclonal. antibody. T'he creation of completely human mona.;lonal
antibodies is
possible through the use of transgenic mica. Transgenic mice in which the
mouse
immunoglobulin gene loci have been replaced with human immunoglobulin loci
provide in
v'rvo~ affinity-maturation machinery .for~tlte-pnxiuction ~of human
immunoglobulins.
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In certain embodiments, the modified immunoglobulin (or fragment thereof] is
fused
via a covalent bond (for example, but not by way of limitation, 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 50 amino acid portion thereofy that
is not the
modified immunoglobulin. Preferably, the modified immunoglobulin is covalently
linked
.- . to the other protein at the N=terminus of the constant domain of the
modified
immunoglobulin. In preferred embodiments, the invention provides fusion
proteins in which
w the modified iirununoglobulin is covalently linked to a portion of a
growt't~ enhancing factor
or; a portion of an immun~~stimulator,.~ factor, including interleukin-2.
interleukir~-4,
~.~: interleukin-S,. interleukin-6,.interleukin-7; interleukin-10, interleukin-
12, interleukin-15, G
colony stimulating factor, tumor necrosis factar;~.po~_ in,: interferon-gamma,
and NK cell .
antigen or Ml'IC derived peptide. .
The modif ed imm~.moglobulin may. he further modified, e.g, by the covalent
attachment of any type of molecule, as long as such covalent attfchment does
not prevent or
~ 5~-inhibit immunosgecific binding of the imznunoglobulin to its target
antigen. For example.
:. but not by v~~ay of.limic,~tic~n, the modified imlnwloghihulin may be
further modified, e.g.,
by glycosylation, acetytation, pegylation; ph~~snharylation, amidation,
derivatization.by
knov~m protecting.~ocking groups, proteolptic cieavage, linkage to a cellular
ligand or other
. protein, ere. , Any of numerous chemical modifications may be carried out by
known
. 20. ~o~iques,~ imiading, but.not limited to specifiv chernical cleavage,
acetylation,
~~formylation;nnetabolic synthesis of tuni~,amycin: ecc.~ v~dditionally, the
modified antibody
=may contain one or more nori-classia~l amino acid::. e.g:, as listed above in
this~Section.
.,:In.specific~embodiments oftl:e invention. the.modified immunoglobulin (or a
...fragment thereofl~is~covalently linked to a
th~rapeutic~an~sle~;ul~;~or~e~uimple, to target the
25 ~~peutic molecule to a particular cell type~oraissue,:
e:g:;;a.cancer~~r~ucnor cell. The
.- .Ttherapeutic inulecuJe can be anytype of therape~itic molecule known in
tree ari, for examyte,. .
~. bus not limited ta, a chamotherapeutic agent. :a toxin, such as ricin, an
antisense
:~oligonucleotide; w radionuclide; an antibiotic; anti-viral, or anti-
parasitic, etc.
35
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~rp 99~~3~g PCT/US98/24302
5.2. METHODS OF PRODUCING THE MODIFIED IMMUNOGLOBULIN
The modified immunoglobulins of this 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 thereof, requires construction of a nucleic acid encoding the
modified
immunogiobulin. Such an isolated nucleic acid which contains a nucleotide
sequence
encoding the modified immunoglobulin can be produced using any method known in
the art,
for example, recombinant techniques or chemical synthesis {e.g., see
Creighton, 1983,
"Proteins: Structures and Molecular Principles", V~'.H. Freeman & Co., N.Y..
pp.34-49; and
Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual. Cold Springs
Harbor
Press, N.Y.). or using PCR on known immunoglobulin genes to engineer the
nucleotide
sequence encoding the CDR sequence containing the binding site.
Accordingly, the invention provides nucleic acids that contain a nucleotide
sequence
encoding a modified immunoglobulin of the invention, or a functionally active
fragment
thereof.
Preferably, a nucleic acid that encodes a modified immunoglobulin may be
assembled from chemically synthesized oligonlicleoti.des (e.g., as described
in Kutmeier et
al., 1994, Biotechniques I7:242), which, briefly, involves the synthesis of a
set of
overlapping oligonucleotides containing portions of the sequence encoding the
modified
immunoglobulin. annealing and ligation of those oligonucleotides, and then
amplification of
the ligated oligonucleotides by PCR,~e.g., as exemplified in Se~tion~6, inj~a.
Accordingly, the invention provides~a method of producing a nucleicacid
encoding a
modified immunoglobulin, said method comprising: (a) synthesizing a set of
oligonucleotides, said set comprising oligonucleotides containing a portion of
the nucleotide
sequence that encodes the synthetic modified immunoglobulin and
oligonucleotides
containing a portion of the nucleotide sequence that is complementary to the
nucleotide
~quence that encodes the synthetic modified immunoglobulin, and each of said
oligonucleotides having overlapping terminal sequences with another
oligonucleotide of said
set, except for those oligonucleotides containing the nucleotide sequences
encoding the N-
terminal and C-terminal portions of the synthetic modified immunoglobulin; (b)
allowing
the oligonucleotides to hybridize or anneal to each other; and (c) ligating
the hybridized
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WO 99/25378 PCT/US98/Z4302
oligonucleotides, such that a nucleic acid containing the nucleotide sequence
encoding the
synthetic modified immunoglobulin is produced.
Alternatively, a nucleic acid containing a nucleotide sequence encoding a
modified
immunoglobulin can be constructed from a nucleic acid containing a nucleotide
sequence
encoding, e.g., an antibody molecule, or at least a variable region of an
antibody molecule.
Nucleic acids containing nucleotide sequences encoding antibody molecules can
be obtained
either from existing clones of antibody molecules or variable domains or by
isolating a
nucleic acid encoding an antibody molecule or variable domain from a suitable
source,
preferably a cDNA library e.g., an antibody DNA library or a cDNA library
prepared from
cells or tissue expressing a repertoire of antibody molecules or a synthetic
antibody library
(see, e.g., Clackson et al., 1991, Nature 352:624; Hane et al., 1997, Proc.
Natl. Acad Sci
LaSA 94:4937), for example, by hybridization using a probe specific for the
particular
antibody molecule or by PCR amplification using synthetic primers hybridizable
to the 3'
and 5' ends of the sequence.
Once a nucleic acid containing a nucleotide sequence encoding at least a
variable
region of an antibody molecule has been cloned, then the binding site sequence
can be
inserted into the nucleotide sequence coding for one or more of the CDRs. Such
engineering
of the particular CDR ceding sequence can be accomplished by routine
recombinant DNA
techniques known in the art. For example, the nucleotide sequence encoding the
CDR can
2G be replaced by a nucleotide sequence encoding the L DR containing the
particular binding
site sequence, for example, using PCR based meth«ds, in vitro site. directed
mutagenesis,
etc. If a convenient restriction enzyme site is available in the nucleotide
sequence of the
CDR, then the sequence can be cleaved with the restriction enzyme and a
nucleic acid
fragment containing the nucleotide sequence encoding~the binding site can be
ligated into
the restriction site. The nucleic acid fragment containing the binding site
can be obtained
either from a nucleic acid encoding all or a portion of the protein containing
the binding site
or can be generated from synthetic oligonucleotides containing the sequence
encoding the
binding site and its reverse complement.
The nucleic acid encoding the modified antibody optionally contains a
nucleotide
sequence encoding a leader sequence that directs the secretion of the
synthetic modified
antibody molecule.
Once a nucleic acid encoding at least the variable domain of the modified
antibody is
obtained, it may be introduced into a vector containing the nucleotide
sequence encoding the
constant region of the antibody (see, e.g., PCT Publication WO 86/05807; PCT
Publication
WO 89/01036; and U.S. Patent No. 5,122,464). Vectors containing the complete
light or
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CA 02310269 2000-OS-12
wo ~ns3~s pcTius9sna3oz
heavy chain for co-expression with the nucleic acid to allow the expression of
a complete
antibody molecule are also available and are known in the art, for example,
pMRR010.1
and pGammal -(see also Bebbington, 1991, Methods in EnzymoloRy 2:136-145) .
The expression vector can then be transferred to a host cell by conventional
techniques and the transfected cells can be cultured by conventional
techniques to produce
the antibody of the invention. Specifically, once a nucleic variable region of
the modified
antibody has been generated, the modified antibody can be expressed, for
example, by the
method exemplified in Section 6. (See also Bebbington, 1991, Methuds in
Enrymology
2_:136-145.) For example, by transient transfection of the expression vector
encoding the
modified immunoglobulin into COS cells, culturing the cells for an appropriate
period of
time to permit immunoglobulin expression, and then taking the supernatant from
the COS
cells, which supemztant contains the secreted, expressed modified
immunoglobulin.
The host cells used to express the recombinant antibody of the invention may
be
either bacterial cells such as Escherichia coli, particularly for the
expression of recombinant
~tibody fragments or, preferably, eukaryotic cells, particularly for the
expression of
recombinant antibody molecules. In particular, mammalian cells such as Chinese
hamster
ovary cells (CHO) or COS cells, used in conjunction with a vector in which
expression of
the antibody is under control of the major intermediate early gene promoter
element from
human cytomegalovirus is an effective expression system for immunoglobulins
(Foecking et
al., 1986, Gene 45:101; Cockett et al., 1990, BiolTechnology 8_:662).
A variety of hose-expression vector systems may be utilirxd to express the
antibody
coding sequences of the invention. Such host-expression systems represent
vehicles by
which the coding sequences of interest may be produced and subsequently
purified, but also
produce cells which may, when transformed or transfected with.the appropriate
nucleotide
coding sequences, exhibit the antibody product:of the invention in situ. These
systems
include, but are nut limited to, microorganisms such as bacteria (e.g., E.
coli, B. subtilis)
transformed with recombinant bacteriophage DNA, plasmid DI~'A or cosmid DNA
expres-
sion vectors containing antibody coding sequences; yeast (e.g., Saccharomyces,
Pichia)
transformed with recombinant yeast expression vectors containing antibody
coding
sequences; insect cell systems infected with recombinant virus expression
vectors (e.g.,
baculovirus) containing the antibody 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 pla~mid expression vectors
(e.g., Ti
plasmid) containing antibody coding sequences; or mammalian cell systems
(e.g., COS,
CHO, BHK, 293, and 3T3 cells) harboring recombinant expression constructs
containing
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WO 99/25378 PGTNS98/24302
promoters derived from the genome of mammalian cells (e.g., the
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 antibody being expressed. For example,
when a
large quantity of such a protein is to be produced, for the generation of
pharmaceutical
compositions of an antibody, 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 pUR278 (Ruther et al., 1983, E:1~IB0
J. 2:1791 ), in
which the antibody 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 Ileeke & Schuster, 1989,
J. Biol.
Chem. 264:5503-5509); and the like. pGEX vectors may also be used to express
foreign
polypeptides as fusion proteins with glutathione S-transferase (GST). In
general, such
lion proteins are soluble and can easily be purified from lysed cells by
adsorption and
binding to a matrix glutathione-agarose beads followed by elution in the
presence of free
glutathione. T'he 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, Autographa californica nuclear polyhedrosis virus (AcNPV)
is
used as a vector to express foreign genes. The virus grows in Spodoptera
frug_iperda cells.
T'he antibody 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).
Ir~ 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
antibody coding
sequence of interest may be iigated 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 E 1 or E3) will result
in a recombinant
virus that is viable and capable of expressing the antibody in infected hosts
(e.g., see Logan
& Shenk, 1984, Proc. Natl. Acad. Sci. USA 8_x:3655-3659). Specific initiation
signals may
also be required for efficient translation of inserted antibody 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 translational control
signals and
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WO 99/Z5378 PCT/US98/24302
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 Enzymol.
153:516-544).
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-traclslational 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.
CUS,
MDCK, 293, 3T3, WI38.
1 S For long-term, high-yield production of recombinant proteins, stable
expression is
preferred. For example, cell lines which stably express the antibody 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 ;vhich in turn can be cloned and expanded into cell lines. .This method
may
advantageously be used to engineer cell lines which express the antibody. Such
engineered
celi lines may be particularly useful in screening and evaluation of compounds
that interact
directly or indirectly with the antibody.
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, 1962, Proc. Natl. Acad Sci.
USA
48:2026), and adenine phosphoribosyltransferase (Lowy et al., 1980, Cell
22:817) genes can
be employed in tk', hgprC or aprt- cells, respectively. Also, antimetabolite
resistance can be
used as the basis of selection for the following genes: dhfi, which confers
resistance to
methotrexate -(Wigler et al., 1980, Natl. Acad. Sci. USA 77'3567; O'Hare et
al., 1981, Proc.
Nat1 Acad Sci. USA 78:1527); gpt, which confers resistance to mycophenolic
acid
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WO 99/25378 PCT/US98/24302
(Mulligan & Berg, 1981, Proc. Natl. Acad Sci. USA 7$:2072); neo, which confers
resistance
to the aminoglycoside G-418 (Colberre-Garapin et al., 1981, J. Mol. Biol.
150:1); and hygro,
which confers resistance to hygromycin (Santerre et al., 1984, Gene 30:147).
The expression levels of the synthetic modified antibody can be increased by
vector
S amplification (for a review, see Bebbington and Hentschel, The Use of
Vectors Based on
Gene Amplification 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 immunoglobulin is ampliftable, increase in the level of inhibitor
present in
culture of host cell will increase the number of copies of the marker gene.
Since the
~plified 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
~,~~h 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 322:562; Kohler, 1980, Proc.
lfutl. Acad
Sci. LISA x:2197). The coding sequences for the heavy and light chains may
comprise
cDNA or genomic DNA.
The invention provides a recombinant cell that contain a vector which encodes
a
synthetic antibody that has a CDR that contain the amino acid sequence of an
active binding
site from a member of a binding pair.
5,3. TH1ERAPEUTIC USE OF SYNTHETIC MODIFIED Ai\1T~IBODIES
The invention also provides methods for treating or preventing diseases and
disorders associated with the expression of a particular molecule by
administration of a
therapeutic of the invention (termed herein "Therapeutic"). Such Therapeutics
include the
modified immunoglobulins of the invention, and functionally active fragments
thereof, (e.g.,
~ described in Section 5.1, supra), and nucleic acids encoding the modified
immunoglobulins of the invention, and functionally active fragments thereof
(e.g., as
described in Section 5.2, 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 preferred
embodiments, the
~empeutic methods of the invention use a modified antibody that is derived
from a human
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WO 99/25378 PCT/US98/24302
antibody; in other embodiments, the methods of the invention use a modified
antibody that
is derived from a chimeric or humanized antibody.
Specifically, pharmaceutical compositions containing the modified antibodies
(or
functionally active fragment thereof) of the invention that immunospecifically
bind a
particular molecule can be used in the treatment or prevention of diseases or
disorders
associated with the expression of the particular molecule, e.g., an antigen.
In particular, in
embodiments discussed in more detail in the subsections that follow, modified
antibodies
that immunospecifically bind a tumor or cancer antigen or an antigen of an
infectious
disease agent or a cellular receptor for an infectious disease agent can be
used to treat or
prevent tumors, cancers or infectious diseases associated with the expression
of the
particular antigen. Modified immunoglobulins that immunospecificaily bind a
ligand or
receptor can be used to treat or prevent a disease associated with a defect in
decrease in or
increase the amount of the particular ligand receptor. In certain embodiments.
the modified
immunoglobulins are used to treat or prevent autoimmune disease, including but
not limited
to rheumatoid arthritis, lupus, ulcerative colito, or psoriasis. The modified
icnmunoglobulins may also be used to treat allergies.
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
h~~, In a preferred embodiment, the subject is a human.
5.3.1. TREATI~iENT AND PREVENTION OF CANCERS
The invention provides methods of treating or preventing cancers characterized
by
the presence of a particular cancer antigens~which are a merilber ~f a binding
pair. The
method includes administering to a subject in need~of~such~treatment ur
prevention a
Therapeutic of the invention, e.g., a synthetic modified antibody (or
functionally active
ti~agment thereof) that immunospecifically binds to the particular cancer
antigen, which
antibody comprises a variable domain with a CDR containing the amino acid
sequence of a
binding site for the cancer antigen.
Cancers, including, but not limited ~to, neoplasms, tumors, metastases, or any
disease
or disorder characterized by uncontrolled cell growth, can be treated or
prevented by
administration of the synthetic modified antibody of the invention, which
modified antibody
immunospecifically binds one or more antigens associated with the cancer cells
of the cancer
to be treated or prevented. Whether a particular Therapeutic is effective to
treat or prevent a
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WO 99/25378 PCTNS98/24302
certain type of cancer can be determined by any method known in the art, for
example but
not limited to, these methods described in Section 5.6, infra.
For example, but not by way of limitation, cancers and tumors associated with
the
following cancer and tumor antigens may be treated or prevented by
administration of a
synthetic antibody of the invention containing in its CDR the sequence that
recognizes these
cancer antigens: KS 1 /4 pan-carcinoma antigen (Perez and Walker. 1990, J.
Immunol.
142:3662-3667; Bumal, 1988, Hybridoma 7(4):407-415), ovarian carcinoma antigen
(CA125) (Yu et al., 1991, Cancer Res. 51(2):468-475), prostatic acid phosphate
(Tailor et
al., 1990, Nucl. Acids Res. 1$(16):4928), prostate specific antigen (Henttu
and Vihko, 1989,
Biochem. Biophys. Res. Comm. 160(2):903-910; Israeli et al., 1993, Cancer Res.
53:227-230), melanoma-associated antigen p97 (Estin et al., 1989, J. Natl.
Cancer Instil.
81 (6):445-446), melanoma antigen gp75 (Vijayasardahl et al., 1990, J. Exp.
Med
171(4):1375-1380), high molecular weight melanoma antigen (HMW-MAA) (Natali et
al.,
1987, Cancer 59:55-63; Mittelman et al., 1990, J. Clin. Invest. 86:2136-2144),
prostate
specific membrane antigen, carcinoembryonic antigen (CEA) (Foon et al., 1994,
Proc. Am.
Soc. Clin. Oncol. 13:294), polymorphic epithelial mucin antigen, human milk
fat globule
antigen, colorectal tumor-associated antigens such as: CEA, TAG-72 (Yokata et
al., 1992,
Cancer Res. 52:3402-3408), C017-lA (Ragnhammar et al.. 1993, Int. J. Cancer
53:751-
758); GICA 19-9 (Herlyn et al., 1982, J. Clin. Immunol. x:135), CTA-1 and LEA,
Burkitt's
lymphoma antigen-38.13, CD19 (Ghetie et al., 1994, Blood 83:1329-1336), human
B-
lymphoma antigen-CD20 (Reff et al., 1994, Blood 83:435-445), CD33 (Sgouros et
al., 1993,
J. Nucl. Med 34:422-430), melanoma specific antigens such as ganglioside GD2
(Saleh et
al., 1993, J.Immunol., 1 S 1, 3390-3398), ganglioside GD3 (Shitara et al.,
1993, Cancer
Immunol. Immunother X6_:373-380), ganglioside GM2 (Livingston et al., 1994,
.i. Clin.
Oncol. 12:1036-1044), ganglioside GM3 (Hoon et al., 1993, Cancer Res. 53:5244-
5250),
tumor-specific transplantation type of cell-surface antigen (TSTA) such as
virally-induced
tumor antigens including T-antigen DNA tumor viruses and Envelope antigens of
RNA
tumor viruses, oncofetal antigen-alpha-fetoprotein such as CEA of colon,
bladder tumor
oncofetal antigen (Hellstrom et al., 1985, Cancer. Res. 45:2210-2188),
differentiation
~tigen such as human lung carcinoma antigen L6, L20 (Hellstrom et al., 1986,
Cancer Res.
46:3917-3923), antigens of fibrosarcoma, human leukemia T cell antigen-Gp37
(Bhattacharya-Chatterjee et al., 1988, J. of Immunospecifically. 141:1398-
1403),
neoglycoprotein, sphingolipids, breast cancer antigen such as EGFR (Epidermal
growth
factor receptor), HER2 antigen (p185'~~), polymorphic epithelial mucin (PEM)
(Hilkens et
al., 1992, Trends in Bio. Chem. Sci. 17:359), malignant human lymphocyte
antigen-APO-1
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(Bernhard et al., 1989, Science 2:301-304), differentiation antigen (Feizi,
1985, Nature
314:53-57) such as I antigen found in fetal erythrocytes, primary endoderm, I
antigen found
in adult erythrocytes, preimplantation embryos, I(Ma) found in gastric
adenocarcinomas,
M18, M39 found in breast epithelium, SSEA-1 found in myeloid cells, VEPB,
VEP9, Myl,
VIM-D5, D, 56-22 found in colorectal cancer, TRA-1-85 (blood group H), C 14
found in
colonic adenocarcinoma, F3 found in lung adenocarcinoma, AH6 found in gastric
cancer, Y
hapten, Ley found in embryonal carcinoma cells, TLS (blood group A), EGF
receptor found
in A431 cells , E, series (blood group B) found in pancreatic cancer, FC10.2
found in
embryonal carcinoma cells, gastric adenocarcinoma antigen, CO-S 14 (blood
group Le8)
found in Adenocarcinoma, NS-10 found in adenocarcinomas, CO-43 (blood group
Leb), G49
found in EGF receptor of A431 cells, MH2 (blood group ALeb/Ley) found in
colonic
adenocarcinoma, 19.9 found in colon cancer, gastric cancer mucins, TSA, found
in myeloid
cells, R~4 found in melanoma, 4.2, Gp3, D1.1, OFA-1. GM2, OFA-2, Gp,, and
M1:22:25:8
found in embryonal carcinoma cells, and SSEA-3 and SSEA-4 found in 4 to 8-cell
stage
embryos. In one embodiment, the antigen is a Tcell receptor derived peptide
from a
Cutaneous Tcell Lymphoma (see, Edelson, 1998, The Cancer Journal 4:62).
In other embodiments of the invention, the subject being treated with the
modified
antibody may, optionally, be treated with other cancer treatments such as
surgery, radiation
therapy or chemotherapy. In particular, the Therapeutic of the invention used
to treat or
prevent cancer may be administered in conjunction with one or a combination of
chemotherapeutic agents including, but not limited to, methotrexate, taxol,
mercaptopurine,
thioguanine, hydroxyurea, cytarabine, cyclophosphamide, ifosfamide,
nitrosoureas,
cisplatin, carboplatin, mitomycin, dacarbazine, procarbizine, etoposides,
campathecins,
bleomycin, doxorubicin, idarubicin, daunorubicin. dactinornycin,
plicamycin,.mitoxantrone,
~p~gi~e, vinblastine, vincristine, vinorelbine, paclitaxel, docetaxel, etc. In
a preferred
embodiment, the synthetic modified antibody is conjugated to a
chemotherapeutic agent or
other type of toxin, e.g., a ricin toxin, or a radionuclide, or any other
agent effective to kill
cancer or tumor cells or to arrest cancer cell growth. In another preferred
embodiment, the
modified immunoglobulin has one CDR containing a binding site for a cancer
antigen and
~o~er CDR containing a binding site for molecule on the surface of an immune
cell, such
as but not limited to a T cell, a B cell, NK cell, K cell, TIL cell or
neutrophil.
In certain embodiments of the invention where the CDR of the synthetic
modified
antibody includes an amino acid sequence that immunospecifically binds a human
colon
carcinoma-associated protein antigen, it is prefen:ed that the antibody has
the following
ceristics: (i) the antibody recognizes epitopes of a protein component of the
antigen,
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but does not recognize the epitopes of the carbohydrate components) of the
antigen; (ii) the
antigen is not detectable on normal human tissue; and (iii) the antigen is not
detectable on
human carcinoma cells other than colon carcinoma cells. In other embodiments,
the CDR of
the synthetic modified antibody includes an amino acid sequence that
immunospecifically
binds an antigen which is not detectable on human carcinoma cells other than
breast
carcinoma cells. In yet other embodiments, the CDR of the synthetic modified
antibody
includes an amino acid sequence that immunospecifically binds an antigen is
not detectable
on human carcinoma cells other than ovarian carcinoma cells.
5.3.1.1. MALIGNANCIES
Malignancies and related disorders that can be treated or prevented by
administration
of a Therapeutic of the invention include but are not limited to those listed
in Table 2 (for a
review of such disorders, see Fishman et al., 1985, Medicine, 2d Ed., J.B.
Lippincott Co.,
Philadelphia):
TABLE 3
MALIGNANCIES AND RELATED DISORDERS
Leukemia
acute leukemia
acute lymphocytic leukemia
acute myelocytic leukemia
myeloblastic
promyelocytic
myelomonocytic
monocytic
erythroleukemia
chronic leukemia
chronic myelocytic (granulocytic) leukemia
chronic lymphocytic leukemia
Polycythemia vera
Lymphoma
Hodgkin's disease
non-Hodgkin's disease
Multiple myeloma
Waldenstrom's macroglobulinemia
Heavy chain disease
Solid tumors
sarcomas and carcinomas
fibrosarcoma
myxosarcoma
liposarcoma
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chondrosarcoma
osteogenic sarcoma
chordoma
angiosarcoma
endotheliosarcoma
lymphangiosarcoma
lymphangioendotheliosarcoma
synovioma
mesothelioma
Ewing's tumor
Ieiomyosarcoma
rhabdomyosarcoma
colon carcinoma
pancreatic cancer
breast cancer
ovarian cancer
prostate cancer
squamous cell carcinoma
basal cell carcinoma
adenocarcinoma
sweat gland carcinoma
sebaceous gland carcinoma
papillary carcinoma
papillary adenocarcinomas
cystadenocarcinoma
medullary carcinoma
bronchogenic carcinoma
renal cell carcinoma
hepatoma
bile duct carcinoma
choriocarcinoma
seminoma
embryonal carcinoma
Wilms' tumor
cervical cancer
uterine cancer
testicular tumor
lung carcinoma
small cell lung carcinoma
bladder carcinoma
epithelial carcinoma
glioma
astrocytoma
medulloblastoma
craniopharyngioma
ependymoma
pinealoma
hemangioblastoma
acoustic neuroma
oligodendroglioma
meningioma
melanoma
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neuroblastoma
retinoblastoma
In specific embodiments, malignancy or dysproliferative changes (such as
metaplasias and dysplasias), or hyperproliferative disorders, are treated or
prevented in the
ovary, bladder, breast, colon, lung, skin, pancreas, prostate, uterus,
gastrointestinal tract, B
lymphocytes or T lymphocytes. In other specific embodiments. sarcoma.
melanoma, or
leukemia is treated or prevented.
5.3.1.2. PREMALIGNANT CONDITIONS
The Therapeutics of the invention can also be administered to treat
premalignant
conditions and to prevent progression to a neoplastic or malignant state,
including, but not
limited to. those disorders listed in Table 3. Such prophylactic or
therapeutic use is
indicated in conditions known or suspected of preceding progression to
neoplasia or cancer,
in particular, where non-neopiastic cell growth consisting of hyperplasia.
metaplasia, or
most particularly, dysplasia has occurred (for review of such abnormal growth
conditions,
see Robbins and Angell, 1976, Basic Pathology, 2d Ed., W.B. Saunders Co.,
Philadelphia,
pp. 68-79.) Hyperplasia is a form of controlled cell proliferation involving
an increase in
cell number in a tissue or organ, without significant alteration in structure
or function. As
but one example, endometrial hyperplasia often precedes endometrial cancer.
Metapiasia is
a form of controlled cell growth in which one type of adult or fully
differentiated cell
substitutes for another type of adult cell. Metaplasia can occur in epithelial
or connective
tissue cells. Atypical metaplasia involves a somewhat disorderly metaplastic
epithelium.
Dysplasia .is frequently a forerunner of cancer, and is found mainly in the
epithelia; it is the
most disorderly form of non-neoplastic cell growth, involving a loss in
individual cell
uniformity and in the architectural orientation of cells. Dysplastic cells
often have
abnormally large, deeply stained nuclei, and exhibit pleomorphism. Dysplasia
characteristically occurs where there exists chronic irritation or
inflammation, and is often
found in the cervix, respiratory passages, oral cavity, and gall bladder.
Alternatively or in addition to the presence of abnormal cell growth
characterized as
hyperplasia, metaplasia, or dysplasia, the presence of one or more
characteristics of a
transformed phenotype, or of a malignant phenotype, displayed in vivo or
displayed in vitro
by a cell sample from a patient, can indicate the desirability of
prophylactic/therapeutic
administration of a Therapeutic. As mentioned supra, such characteristics of a
transformed
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phenotype include morphology changes, looser substratum attachment, loss of
contact
inhibition, loss of anchorage dependence, protease release, increased sugar
transport,
decreased serum requirement, expression of fetal antigens; disappearance of
the 250,000
dalton cell surface protein, etc. (see also id., at pp. 84-90 for
characteristics associated with a
S transformed or malignant phenotype).
In a specific embodiment, leukoplakia, a benign-appearing hyperplastic or
dysplastic
lesion of the epithelium, or Bowen's disease, a carcinoma in situ, are pre-
neoplastic lesions
indicative of the desirability of prophylactic intervention.
In another embodiment, fibrocystic disease (cystic hyperplasia, mammary
dysplasia,
p~icularly adenosis (benign epithelial hyperplasia)) is indicative of the
desirability of
prophylactic intervention.
In other embodiments, a patient which exhibits one or more of the following
predisposing factors for malignancy is treated by administration of an
effective amount of
the Therapeutic of the invention: a chromosomal translocation associated with
a malignancy
I 5 (e,g. ~ ~e Philadelphia chromosome for chronic myelogenous leukemia. t(
14;18) for
follicular lymphoma, etc.), familial polyposis or Gardner's syndrome (possible
forerunners
of colon cancer), benign monoclonal gammopathy (a possible forerunner of
multiple
myeloma), and a first degree kinship with persons having a cancer or
precancerous disease
showing a Mendelian (genetic) inheritance pattern (e.g., familial polyposis of
the colon,
Gardner's syndrome, hereditary exostosis, polyendocrine adenomatosis,
medullary thyroid
carcinoma with amyloid production and pheochromocytoma, Peutz-Jeghers
syndrome,
neurofibromatosis of Von Recklinghausen, retinoblastoma, carotid body tumor,
cutaneous
melanocarcinoma, intraocular melanocarcinoma, xeroderma pigmertosum, ataxia
telangiectasia, Chediak-Higashi syndrome, albinism, Fanconi's aplastic anemia,
and Bloom's
syndrome; see Robbins and Angell, 1976, Basic Pathology, 2d Ed., Vi'.B.
Sounders Co.,
Philadelphia, pp. I 12-113) etc.)
In another specific embodiment, Therapeutics of the invention. is administered
to a
human patient to prevent progression to ovary, breast, colon, lung,
pancreatic. bladder, skin,
prostate, colon, gastrointestinal, B lymphocyte, T lymphocyte or uterine
cancer, or
3U melanoma or sarcoma.
5.3.2. TREATMENT OF INFECTIOUS DISEASE
The invention also provides methods of treating or preventing an infectious
disease
by administration of a Therapeutic of the invention, in particular, a
synthetic modified
i~~oglobulin (or the functionally active fragment thereof] that
immunospecifically binds
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an antigen of the agent causing the infectious disease or a cellular receptor
for the infectious
disease agent, or an enzyme expressed by the infectious diseases agent. As
discussed in
detail below, the infectious agents include, but are not limited to, viruses,
bacteria, fungi,
protozoa, and parasites.
In specific embodiments, infectious diseases are treated or prevented by
administration of a modified antibody of the immunoglobin (or functionally
active fragment
thereof) that immunospecifically recognizes one of the following antigens of
an infectious
disease agent: influenza virus hemagglutinin {Genbank accession no. J02132;
Air, 1981,
Prvc. Natl. Acad Sci. USA 78:7639-7643; Newton et al., 1983, V irology 128:495-
501 ),
human respiratory syncytial virus G glycoprotein (Genbank accession no.
233429; Garcia et
al., 1994, J. Virol. ; Collins et al., 1984, Proc. Natl. Acad. S'ci. L:SA
81:7683 j, core protein,
matrix protein or other protein of Dengue virus (Genbank accession no. M
19197; Hahn et
al., 1988, Virology 162:167-180), measles virus hemagglutinin (Genbank
accession no.
M81899; Rota et al., 1992, Virology 188:135-142), herpes simplex virus type 2
glycoprotein
I S gB (Genbank accession no. M14923; Bzik et al., 1986, Virology 1 5,5_:322-
333), poliovirus I
VP1 ,(Emini et al., 1983, Nature X04:699), envelope glycoproteins of HIV (
{Putney et al.,
1986, Science 234:1392-1395), hepatitis B surface antigen (Itoh et al., 1986,
Nature 308:19;
Neurath et al., 1986, Vaccine 4:34), diptheria toxin (Audibert et al., 1981,
Nature 289:543),
streptococcus 24M epitope (Beachey, 1985, Adv. Exp. Med. Biol. 185:193),
gonococcal pilin
(Rotht~ard and Schoolnik, 1985, Adv. Exp. Med. Biol. 1:247), pseudorabies
virus g50
(gpD), pseudorabies virus II (gpB), pseadorabies virus gIII (,gpC),
p:~eudorabies virus
glycoprotein H, pseudorabies virus glycoprotein E, transmissible
gastroenteritis glycoprotein
. 195, transmissible gastroenteritis matrix protein, swine rotavirus
glycopro:ein 38, swine
parvovirus capsid protein, Serpulina hydodysenteriae:~rotectiveiantigen,
bo~~ine viral
diarrhea glycoprotein 55, Newcastle disease virus~hemagglutinii~-
neuraminiJase, swine flu
hemagglutinin, swine flu neuraminidase, foot and mouth disease virus, hog
colera virus,
swine influenza virus, African swine fever virus, Mycoplasnla hyopneumoniae,
infectious
bovine rhinotracheitis virus (e.g., infectious bovine rhinotracheitis virus
glycopro~.ein E or
glycoprotein G), or infectious laryngotracheitis virus (e.g., infectious
laryngotracheitis virus
glycoprotein G or glycoprotein I), a glycoprotein of La Crosse virus (Gonzales-
Scarano et
al., 1982, Virology 120:42), neonatal calf diarrhea virus (Matsuno and Inouye,
1983,
Injection and Immunity X9:155), Venezuelan equine encephalomyelitis virus
(Mathews and
Roehrig, 1982, J. Immunol. x:2763), punts toro virus (Dalrymple et al., 1981,
in
Replication of. Negative Strand Viruses, Bishop and Compans (eds.), Elsevier,
NY, p. 167),
mine leukemia virus (Steeves et al., 1974, J. ViroL 14:187), mouse mammary
tumor virus
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(Massey and Schochetman, 1981, virology 115:20), hepatitis B virus core
protein and/or
hepatitis B virus surface antigen or a fragment or derivative thereof (see,
e.g., U.K. Patent
Publication No. GB 2034323A published June 4, 1980; Ganem and Varmus, 1987,
Ann.
Rev. Biochem. 56:651-693; Tiollais et al., 1985, Nature 317:489-495), antigen
of equine
influenza virus or equine herpesvirus (e.g., equine influenza virus type
A/Alaska 91
neuraminidase, equine influenza virus type A/Miami 63 neuraminidase, equine
influenza
virus type A/Kentucky 81 neuraminidase equine herpesvirus type 1 glycoprotein
B, and
equine herpesvirus type 1 glycoprotein D. antigen of bovine respiratory
syncytial virus or
bovine parainfluenza virus {e.g., bovine respiratory syncytial virus
attachment protein
(gR.SV G), bovine respiratory syncytial virus fusion protein f,BRSV F), bovine
respiratory
syncytial virus nucleocapsid protein (BRSV N), bovine parain:'luenza virus
type 3 fusion
protein, and the bovine parainfluenza virus~type 3 hemagglutinin
r_euraminidase), bovine
viral diarrhea virus glycoprotein 48 or glycoprotein 53.
Cellular receptors that can be targeted for treatment of an infectious disease
are listed
in Table 4. along with the pathogen which binds to the cellular receptor.
TABLh; 4
Pathogen Cellular Receptor
B-lymphotropic papovavirusLPV receptor un B-cells
,0(LPV)
_. _
_Bordatella pertussis .Adenylate cyclase
Borna Disease virus (BDV)BDV surface glycogroteins
_ Bovine coronavirus 1'T-acetyl-9-O-acetylneuraminic acid
. receptor
.
Churiomeningitis virus CD4~~
.Dengue virus Highly sniphatad.typealeparin sulphate
p6~
E. coli ~ .Gal alpha 1-4Ctal-containing isoreceptors
Ebola CD l 6b
30Echovirus 1 Integrin VLA-2 receptor
Echovirus-11 (EV) EV receptor
Endotoxin (LPS) CD 14
Enteric bacteria Glycoconjugate receptors
35Enteric Orphan virus alpha/beta T-cell receptor
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Pathogen Cellular Receptor
Enteroviruses Decay-accelerating factor receptor
Feline leukemia virus Extracellular envelope glycoprotein
(Env-SU)
receptor
Foot and mouth disease Immunoglobulin Fc receptorPoxvirusM-T7
virus
Gibbon ape leukemia virusGALV receptor
(GALV)
Gram-negative bacteria CD 14 receptor
Heliobacter pylori Lewis(b) blood group antigen receptor
hepatitis B virus (HBV) T-cell receptor
Herpes Simplex Virus Heparin sulphate glycoaminoglycan
receptor
Fibroblast growth factor receptor
1 HIV-1 ~ GC-Chemokine receptor CCRS
S
CDlla
CD2
G-protein coupled receptor
CD4
Htunan cytomegalovina Heparin. sulphate protecglycan
Annexin 1I
CD13 (aminopeptidase N)
Human coronovirus Humsn alninopeptidase N receptor
25Influenza A, B & C . Hemagglutinin receptor
Legionella GR:3 receptor
Protein kinase receptor
Galactose -?~1-acetylgalactosamine
((ial,JGaINAc)-
inhibitab'.e iectin receptor
30. Chemokine receptor
Leishmania mexicana Annexin I
Listeria monocytogenes ActA protein
Measles virus CD46 receptor
35Meningococcus Meningococcal virulence associated
Opa receptors
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Pathogen Cellular Receptor
Morbilliviruses CD46 receptor
Mouse hepatitis virus Carcinoembryonic antigen family receptors
Carcinoembryonic antigen family Bgla
receptor
Marine leukemia virus Envelope glycoproteins
Marine gamma herpes gamma interferon receptor
virus
Marine retrovirus Glycoprotein gp70
Rmc-1 receptor
Marine coronavirus Carcinoembryonic antigen family receptors
mouse
hepatitis virus
Mycobacterium avium-M Human Integrin receptor alpha v beta
3
Neisseria gonorrhoeae Heparin sulphate proteoglycan receptor
t CD66 receptor
5
lntegrin receptor
Membrane cofactor protein
CD46
GM 1
liM2
GM3
CD3
Ceramide
Newcastle disease virus Hemagglutinin-nEUraminidase protein
Fusion protein
Parvuvirus B 19 Erytlwocyte P antigen receptor
. Plasmodium falciparum CD36 receptor
~
Glycophor in A receptor
Pox Virus Interferon gamma receptor
Pseudomonas KDEL receptor
Rotavirus ~Mucosal homing alpha4beta7 receptor
Samonella typhiurium Epidermal growth factor receptor
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Pathogen Cellular Receptor
Shigella alpha5betal integrin protein
S Streptococci Nonglycosylated J774 receptor
T-helper cells type 1 Chemokine receptors including:
6.CXCR1-4
7.CCR1-5
8.CXCR3
1 ' 9.CCR5
U
I-cell Iymphotropic virusgp46 surface glycoprotein
1
Vaccinia virus TNFRp55 receptor
TNF Rp75 receptor
Soluble Interleukin-1 beta receptor
I5-
Viral diseases that can be treated or prevented by the methods of the present
invention include, but are not limited to, those caused by hepatitis type A,
hepatitis type B,
hepatitis type C, influenza, varicella, adenovirus, herpes simplex type I (l
ISV-I); herpes
simplex type I1 (HSV-I1), rinderpest, rhinovirus, echovirus, rotavirus,
respiratory syncytial
'~~ vi:us, papilloma virus, papova virus, cytomegalovirus, echinouirus,
arbovirus, hantavirus,
coxsachie vints. mumps virus, measles virus, rubella virus, polio virus, human
immunodeficicncy virus type 1 (HIV-I), and human immunodeficiency virus type
II (HIV-
. -1I),~ any. picornaviridae, enteroviruses, caliciviridae, any of the Norwalk
group ~f vintses,
tcgaviruses, such as Dengue vinis,.alphaviruses; flaviviruses,
coronaviruses,~rabies virus,
15 Marburg viruses, ebola viruses, paraintluenza virus, orthomyxoiiinWes:
bunYaviruses.
arenaviruses, reovirises, rotaviruses, orbiviruses, human T cell leukemia
virus ty tie I, human
T cell leukemia virus type II, .simian immunodeficiency virus, lertiviruses.
pohomaviruses,
w parvoviruses, Epstein-Barr virus, human herpesvirus-6, cercopithecine herpes
virus 1 (~~
virus), poxviruses, and encephalitis.
30 Bacterial diseases that can be treated or prevented by the methods of the
present
invention are caused by bacteria including, but not limited to, gram negative
and gram
positive bacteria, mycobacteria rickettsia, mycoplasma, Shigella spp.,
Neisseria spp. (e.g.,
Neisseria mennigitidis and Neisseria gonorrhoeae), legionella, Vibrio
cholerae,
Streptococci, such as Streptococcus pneumoniae, corynebacteria diphtheriae,
clostridium
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tetani, bordetella pertussis, Haemophilus spp. (e.g., influenzae), Chlamydia
spp.,
Enterotoxigenic Escherichia coli, etc. and bacterial diseases Syphillis.
Lyme's desease, ete.
Protozoal diseases that can be treated or prevented by the methods of the
present
invention are caused by protozoa including, but not limited to, plasmodia,
eimeria,
leishmania, kokzidioa, and trypanosoma, fungi, such as Candida, etc.
In specific embodiments of the invention, the Therapeutic is administered in
conjunction with an appropriate antibiotic, antifilngal, anti-viral or any
other drug useful in
treating or preventing the infectious disease. In a preferred embodiment, the
synthetic
modified antibody is conjugated to a compound effective against the infectious
disease agent
lO :to which the synthetic modified antibody is directed. for example, an
antibiotic, antifungal
or anti-viral agent. In another preferred embodiment;vtl~e:riodiFed
immunoglobulin has one
CDR containing a binding site for an antigen of an infectious~;iisease agent
and another
CDIZ containing a binding site for a molecule on the surface of an immune
;,ell, such as but
not limited to a T cell, a B cell, NK cell, K cell, TIL yell or neatrophii.
5.3.3. GENE THERAPY
In a specific embodiment, nucleic acids comprising a sequence: encoding a
synthetic
modified antibody of the invention are administers d. to treat or pre'~ent a
disease or disorder
asso~:iated with the expression of a molecule to which the synthetic modified
antibody
immunospecifically binds.
;~ ~Iwthis~embodimentof the invention;-the~therapeQti.cwucleic avid encodes a
sequence
tl.at produces intxaceilularly l without. a leader sequenc,: j or
interce:lulariy ( with a leader .
sequence! a modified immunoglobulin of the invention.
Any of the methods for gene therapy. avsilable~in -roe an can ~b~ used
acnording to the
present invention. Exemplary methods are described below.
For general reviews of the methods of gene therapy, see Uokispiel et al., l
993,
t'.'linicai .Pharmucy 12:488-SOS; Wu and W~i, 191, ;Rzorhera.Fy 3_:87-9:5;
Tolstoshev, 1993,
w;4nn.~iRev: Pharmacol:~Toxicol.w32:573~596;'Mulligan, 1993, .Science 260:926-
932: and
~Morgan and Anderson, 1993, Ann. Rev. Biochem. 62:191-217: May, ! 993, TIBTECH
11 (5):155-215). 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, 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.
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In one aspect, the therapeutic nucleic acid comprises an expression vector
that
expresses the modified immunoglobulin (or fragment thereof) in a suitable
host. In
particular, such a nucleic acid has a promoter operably linked to the coding
sequence for the
modified synthetic antibody, said promoter being inducible or constituitive,
and, optionally,
tissue-specific. In another embodiment, a nucleic acid molecule is used in
which the
antibody coding sequences and any other desired sequences are flanked by
regions that
promote homologous recombination at a desired site in the genome, thus
providing for
intrachromosomal expression of the modified antibody (holler and Smithies,
1989, Proc.
Nat'!. Acad. Sc~i. USA 86:8932-8935; Zijlstra et al., 1989, .~Vuture 342:435-
438).
~ - w Delivery of the nucleic acid intc a patient may be either direct, in
which case the
patient is directly exposed to the nucleic acid yr nucleic acid-carrying
vector or a delivery
complex, or indirect, in which case,.cells are firs transformed with the
nucleic acid in vitro.
then transplanted into the.patient. These two approaches are known,
respectively, a$ ir_ vivo
or ex vivo gene therapy.
~ In a specific embodiment, the nucleic acid is directly admrnistered in vivo,
where it is
expressed to produce the antibodies. This can be accomplished by any of
numerous
methods known in the art, e.~ , by constructing it as part of an appropriate
nucleic acid
expression vector and administering ~t 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 o:~naked DI''A., or by use of
rnicropartic;le bombardment
. .(Q.g.,~a gene gun; Biolistic,wDupont;; or coating with lipids or cell-
sarface receptors or
transfecting agents, encapsulation in biopolymers (e.g.. poly-13-1->4-N-
acetylglucosamine
. polysaccharide: see U.S..Patent No. S,ti35,493>, encapsulatio:~ in
iiposomes, rricroparticles,
or microcapsules, or by administering it in linkage~ta a prptid~° which
is kao~vrr ~to enter the
nucleus, by administering it in linkage to a ligand awhich isvnown to enter-
the nucleus, by
administering it in linkage to a ligand subject t~ receptor-m~di,3ted
endoc;vtosis (See e.g., Vl'u
and Wu, 1987, J. Biol. Chem. 262:442,9-4432 i, ctc. In another embodiment, a
nucleic acid-
.. v ligand complex can be formed in which tl-~ev-ligand c;oropr~ises r:
Fusvgenic viral peptide to
disrupt endosomes, allowing tt~e 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
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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 can also be administered, for example,
by
expressing nucleotide sequences encoding single-chain antibodies within the
target cell
population by utilizing, for example, techniques such as those described in
Marasco et al.
(Marasco et al., 1993, Proc. Natl. Acad. Sci. USA X0:7889-7893). 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-lU demo:tstrated 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 R.osenfeld 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 (AA'') has also been proposed far use in gene
therapy (Walsh
et al., 1993, Proc. .Soc..Exp. Biol. Med. 204:289-300).
The form and amount of therapeutic nucleic acid envisioned for use depends on
the
type of disease and the severity of the desired effect; patient state, etc.,
and can be
determined by one skilled in the ari.
5.3.4. VACCINE, Mf MUNIZ-ATION
The modified antibody of the present.invention maybe used as a vaccine in a
subject
in which immunity for the binding site for the particular molecule ur antigen
is desired. The
vaccines and methods of the present invention may be ~~.sed either to prevent
a disease or
disorder, or to treat a particular disease ur disorder; where an anti-idiotype
response against a
. particular synthetic antibody is therapeutically or prophylactically useful.
The methods and compositions of the present invention may be used to elicit a
h~oral and/or a cell-mediated response against the synthetic antibody of the
vaccine in a
subject. In one specific embodiment, the methods and compositions elicit a
humoral
response against the administered synthetic antibody in a subject. In another
specific
embodiment, the methods and compositions elicit a cell-mediated response
against the
administered synthetic antibody in a subject. In a preferred embodiment, the
methods and
compositions elicit both a humorai and a cell-mediated response.
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5.4. PHARMACEUTICAL PREPARATIONS AND METHODS OF
ADMINISTRATION
5.4.1. FORMULATIONS AND ADMINISTRATION
Therapeutic compositions containing a modified immunoglobulin for use in
accordance with the present invention can be formulated in any conventional
manner using
one or more physiologically acceptable carriers or excipients.
Thus, the modified immunoglobulins (or functionally active fragments thereof
or
nucleic acids encoding the antibodies or fragments) and their physiologically
acceptable
salts and solvents can be formulated for administration by inhalation or
insufflation (either
~.ough the mouth or the nose) or oral, buccal, parenteral or rectal
administration.
For oral administration, the Therapeutics can take the form of, for example,
tablets or
capsules prepared by conventional means with pharmaceutically acceptable
excipients such
as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone or
hydroxypropyl
methylcellulose); fillers {e.g., lactose, microcrystalline cellulose or
calcium hydrogen
phosph te); lubricants (e.g., magnesium stearate, talc or silica);
disintegrants (e.g., potato
starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl
sulphate). The
tablets can be coated by methods well known in the art. Liquid preparations
for oral
administration can take the form of, for example, solutions, syrups or
su.Spensions, or they
can be presented as a dry product for constitution with water or other
suitable vehicle before
use. Such liquid preparations can be prepared by conventional means with
pharmaceutically
acceptable additives such as suspending agents (e.g., sorbitol sy:up,
cellulose derivatives or
hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacial; non-
aqueous vehicles
(e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetable oils);
and preservatives
(e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations
can also
contain buffer salts, flavoring, coloring and sweetening agents as
appropriate.
Preparations for oral administration can be suitably formulated to give
controlled
release of the active compound.
For buccal administration the Therapeutics can take the form of tablets or
lozenges
formulated in conventional manner.
For administration by inhalation, the Therapeutics according to the present
invention
are conveniently delivered in the form of an aerosol spray presentation from
pressurized
packs or a nebulizer, with the use of a suitable propellant, e.g.,
dichlorodifluoromethane,
trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other
suitable gas. In
the case of a pressurized aerosol the dosage unit can be determined by
providing a valve to
deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in
an inhaler or
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insufflator can be formulated containing a powder mix of the compound and a
suitable
powder base such as lactose or starch.
The Therapeutics can be formulated for parenteral administration (i.e.,
intravenous or
intramuscular) by injection, via, for example, bolus injection or continuous
infusion.
Formulations for injection can be presented in unit dosage form, e.g., in
ampoules or in
mufti-dose containers, with an added preservative. The compositions can take
such forms as
suspensions, solutions or emulsions in oily or aqueous vehicles, and can
contain formulatory
agents such as suspending, stabilizing and/or dispersing agents.
Alternatively, the active
ingredient can be in powder form for constitution with a suitable vehicle,
e.g., sterile
pyrogen-free water, before use.
The Therapeutics can also be formulated in rectal compositions such as
suppositories
or retention enemas, e.g., containing conventional suppository bases such as
cocoa butter or
other glycerides.
In addition to the formulations described previously, the Therapeutics can
also be
formulated as a depot preparation. Such long acting formulations can be
administered by
implantation (for example subcutaneously or intramuscularly) or by
intramuscular injection.
Thus, for example, the compounds can be formulated with suitable polymeric or
hydrophobic materials (for example as an emulsion in an acceptable oil) or ion
exchange
resins, or as sparingly soluble derivatives, for example, as a sparingly
soluble salt.
The modified immunoglobulins of the invention may be administered as separate
compositions or as a single composition with more than one antibodies linked
by
conventional chemical or by molecular biological methods. Additionally, the
diagnostic and
therapeutic value of the antibodies of the invention may be augmented by their
use in
combination with radionuclides or with toxins such as ricin oe with
chemotherapeutic agents
such as methotrexate.
The composition, if desired, can also contain minor amounts of wetting or
emulsifying agents, or pH buffering agents. 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,
h~ magnesium stearate, sodilun 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.
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The invention also provides a pharmaceutical pack or kit comprising one or
more
containers filled with one or more of the ingredients off' 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 be
prepared, placed in an appropriate container, and labelled for treatment of an
indicated
condition.
Many methods may be used to introduce the vaccine formulations of the
invention;
1 S these include but are not limited to oral, intracerebrai, intradermal,
intramuscular,
intraperitoneal, intravenous, subcutaneous, intranasal routes, and via
scarification
(scratching through the top layers of skin, e.g., using a bifurcated needle)
or any other
standard routes of immunization.
The precise dose of the modified irnmunoglobulin 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 synthetic antibody
in the host
to which the vaccine preparation is administered. : ~t~ective doses may also
be extrapolated .
from dose-response curves derived from animal model test systems.
5.4.2. EFFECTIVE DOSE
The compounds and nucleic acid sequences described herein can be administered
to
a patient at therapeutically effective doses to treat pertain diseases or
disorders such as
cancers or infectious diseases. A therapeutically effective dose refers to
that amount of a
compound sufficient to result in a healthful benefit in the treated subject.
Toxicity and therapeutic efficacy of compounds can be determined by standard
pharmaceutical procedures in cell cultures or experimental animals, e.g., for
determining the
LDso (the dose lethal to 50% of the population) and the EDso (the dose
therapeutically
e~~tive in 50% of the population). The dose ratio between toxic and
therapeutic effects is
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the therapeutic index and it can be expressed as the ratio LDso/EDso.
Compounds which
exhibit large therapeutic indices are preferred. While compounds that exhibit
toxic side
effects can be used, care should be taken to design a delivery system that
targets such
compounds to the site of affected tissue in order to minimize potential damage
to uninfected
cells and, thereby, reduce side effects.
The data obtained from the cell culture assays and animal studies can be used
in
formulating a range of dosage for use in humans. The dosage of such compounds
lies
preferably within a range of circulating concentrations that include the EDso
with little or no
toxicity. The dosage can vary within this range depending upon the dosage form
employed
~d the route of administration utilized. For any compound used in the method
of the
invention, the therapeutically effective dose can be estimated initially from
cell culture
assays. A dose can be formulated in animal models to achieve a circulating
plasma
concentration range that includes the ICso (i.e., the concentration of the
test compound which
achieves a half maximal inhibition of symptoms) as determined in cell culture.
Such
i~ormation can be used to more accurately determine useful doses in humans.
Levels in
plasma can be measwed, for example, by high performance liquid chromatography.
5.4.3. ~A~'CINE 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 (humoral and/or cell mediated) response against certain antigens ,
e.g., for the
treatment and prevention of diseases.
Suitable preparations of such vaccines include injectables, either as liquid
solutions
or suspensions; solid forms suitable for solution in, 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, for example, water, saline, buttered 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-
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nor-muramyl-L-alanyl-D-isoglutamine, N-acetylmuramyl-L-alanyl-D-isoglutaminyl-
L-
alanine-2-( 1'-2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine.
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.
I S 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 tilled with one cr 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 fail, such as a blister pack. The pack
or dispenser
device may be accompanied by instructions for administration. (:omposition
comprising a
compound of the invention. formulated in a compatible pharmaceutical carrier
may also be
prepped, 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.
3S
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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
(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 modified 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.
5.5. DIAGNOST1C METHODS
Modified immunoglobulins, particularly antibodies, (and functionally active
fragments thereof) that bind a specific molecule that is a member of a binding
pair may be
used as diagnostics and prognostics, as described herein. In various
embodiments, the
present invention provides the measurement of a member of the binding pair,
and the uses of
. such measurements in clinical applications. The modified immunoglobulins in
the present
invention may be used, for example, in the detection of an antigen in a
biological sample
whereby patients may be tested for aberrant levels of the molecule to which
the modified
immunoglobulin binds, and/or for the presence of abnormal forms of such
molecules. By
"aberrant levels" is meant increased or decreased relative to that present, or
a standard level
representing that present; in an analogous sample from a portion of the body
or from a
subject not having the disorder. The modified antibodies of this invention may
also be
included as a reagent in a kit for use in a diagnostic or prognostic
technique.
In the specific embodiments of the invention, a modified antibody of the
invention
that immunospecifically binds to a cancer or tumor antigen or an antigen of an
infectious
disease agent may be used to diagnose, prognose or screen for a cancer or
tumor or an
infectious disease associated with the expression of the cancer or tumor
antigen or the
antigen of the infectious disease agent. In a preferred aspect, the invention
provides a
method of diagnosing or screening for the presence of or a predisposition for
developing a
c~cer characterized by the increased presence of a cancer antigen, which is a
first member
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of a binding pair consisting of said first member and a second member. said
method
comprising measuring in a subject the level of immunospecific binding of a
modified
antibody to a sample derived from the subject, in which said modified antibody
immunospecifically binds said cancer antigen and in which said modified
antibody
comprises a variable domain having at least one CDR containing portion of said
second
member, said portion containing a binding site for said cancer antigen and not
being found
naturally within said CDR, in which an increase in the level of said
immunospecific binding,
relative to the level of said immunospecif~c binding in an analogous sample
from a subject
not having the cancer or a predisposition for developing the cancer, indicates
tl-.e presence of
the cancer or a predisposition for developing the cancer.
In another preferred aspect, the invention provides a method of diagnosing or
screening for the presence of an infectious disease agent. characterized by
the presence of an
antigen of said infectious disease agent, which antigen is a first member of a
binding pair
consisting of said first member and a second member, said method comprising
measuring in
a subject the level of immunospecific binding of a modified antibody to a
sample derived
from the subject, in which said modified antibody immunospecifically binds
said antigen
and in which said modified antibody comprises a variable domain having at
least one CDR
containing an at least four amino acid portion of said second member, said
portion
containing a binding site for said antigen and not being found naturally
within said CDR, in
v,~oh an increase in the level of said immunospeci6.c binding, relative to the
level of'said
~.im~ospecific binding in an analogous sample-from a subject not having th~.;
infectious
disease agent. indicates the presence of said infectious disease agent.
In another preferred embodiment, the invention provides a method for detecting
abnormal levels of a particular ligand er receptor in a sample derived from a
subject by
comparing the immunospecific binding of a modified antibodythat binds .the
particular
ligand or receptor to the sample with the immunospecific binding of the
modified antibody
to a sample having normal levels of the ligand or receptur.
'The measurement of a molecule that is bound by a modified antibody can be
valuable in detecting and/or staging diseases related to the molecule in a
subject, in
screening of such diseases in a population, in differential diagnosis of the
physiological
condition of a subject, and in monitoring the effect of a therapeutic
treatment on a subject.
The following assays are designed to detect molecules to which the modified
antibodies immunospecifically bind.
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In specific embodiments, these diagnostic methods may be used to detect
abnormalities in the level of gene expression, or abnormalities in the
structure and/or
temporal, tissue, cellular, or subcellular location of the particular molecule
to be assayed.
The tissue or cell type to be analyzed will generally include those which are
known,
or suspected, to express the particular molecule. The protein isolation
methods employed
herein may, for example, be such as those described in Harlow and Lane
(Harlow, E. and
Lane, D., 1988, "Antibodies: A Laboratory Manual", Cold Spring Harbor
Laboratory Press,
Cold Spring Harbor, New York). The isolated cells can be derived from cell
culture or from
a patient. The modified antibodies (or func:ionally active fragments thereof]
useful in the
present invention may, additionally, be employed histologically, as in
immunofluorescence
or immunoelectron microscopy, for in situ detection of the molecule. In situ
detection may
be accomplished by removing a histological specimen from a patient, such as
paraffin
embedded sections of affected tissues and applying thereto a labeled modified
antibody of
the present invention. The modified antibody (or functionally active fragment
thereof) is
preferably applied by overlaying the labeled modified antibody onto a
biological sample. If
the molecule to which the antibody binds is present in the cytoplasm, it may
be desirable to
introduce the modified antibody inside the cell, .for example. by making the
cell membrane
permeable. Through the use of such a procedure, it is possible to determine
not only the
presence of the particular molecule. but also its distribution in the examined
tissue. Using
'0 the present invention, those of ordinary .skill will readily perceive that
any of a wide variety
of histological methods (such as staining procedures) can be modified in order
to achieve
such in situ detection.
Immunoassays for the particular molecule will typically comprise incubating a
sample, such as a biological fluid, a tissuevxtract;~~ieshly harvested cells.
or lysates of
cultured cells, in the presence of a detectably labeled modified antibody~and
detecting the
bound antibody by any of a number of techniques well-known in the art.
The biological sample may be brought in contact with and in~,cnobiliz~ed unto
a solid
phase support or carrier such as nitrocellulose, or ether solid support which
is capable of
immobilizing cells, cell particles or soluble proteins. The support may then
be washed with
suitable buffers followed by treatment with the detestably labeled modified
antibody. The
solid phase support may then be washed with the buffer a second time to remove
unbound
antibody. The amount of bound label on solid support may then be de:ected by
conventional
means.
By "solid phase support or carrier" is intended any support capable of binding
an
~flgen or an antibody. Well-known supports or carriers include glass,
polystyrene,
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WO 99/2538 PCT/US98/24302
polypropylene, polyethylene, dextran, nylon, amylases, natural and modified
celluloses,
polyacrylamides, gabbros, and magnetite. The nature of the carrier can be
either soluble to
some extent or insoluble for the purposes of the present invention. The
support material
may have virtually any possible structural configuration so long as the
coupled molecule is
capable of binding to an antigen or antibody. Thus, the support configuration
may be
spherical, as in a bead, or cylindrical, as in the inside surface of a test
tube, or the external
surface of a rod. Alternatively, the surface may be flat such as a sheet, test
strip, etc.
Preferred supports include polystyrene beads. Those skilled in the art will
know many other
suitable can-iers for binding antibody or antigen, or will be able to
ascertain the same by use
of routine experimentation.
The binding activity of a given modified antibody may be determined according
to
well known methods. Those skilled in the art will he able to determine
operative and
optimal assay conditions for each determination by employing routine
experimentation.
One of the ways in which a modified antibody can be detectably labeled is by
linking
the same to an enzyme and use in an enzyme immunoassay (EIA) (Voller, A., "The
Enzyme
Linked Immunosorbent Assay (ELISA)", 1978, Diagnostic Horizons 2_:1-7,
Microbiological
Associates Quarterly Publication, Walkersville. hiDl; Voller et al., 1978, J.
Clin. Path~l.
X1_:507-520; Butler, 1981, Meth. Lnzymol. 73:482-523; Maggio, E. (ed.), 1980,
Enzyme
Immunoassay, CRC Press, Boca Raton, FL,; Ishikawa et al., (eds.), 1981, Enzyme
Immunoassay, Kgaku Shoin, Tokyo)). The enzyme which is bound to the modified
antibody: will .react with an appropriate substrate, preferably a chromogenic
substrate, in
such a manner as to produce a chemical moiety which can be detected, for
example, by
.spectrophotometric, fluorimetric or by visual means. Fnzymrs which can be
used to
detectably label the modified antibody include, but are nit limited to; malate
dehydrogcnase,
staphylococcal nuclease, delta-5-steroid isomerase. y east alcohol
dehydrogenase, alpha-
glycerophosphate, dehydrogenase, triose phosphate isomerase, horseradish
peroxidase,
alkaline phosphatase, asparaginase, glucose oxida~;, beta-galactosidase,
ribonuclease,
urease;rcatalase;.glucose-6-phosphate-dehydrogenase; gluc;oamylase znd
acetylcholinesterase. The detection can be accomplished by colorimetric
methods which
employ a chromogenic substrate for the enzyme. Detection may also be
accomplished by
visual comparison of the extent of enzymatic reaction of a substrate in
comparison with
similarly prepared standards.
Detection may also be accomplished using any of a variety of other
immunoassays.
For example, by radioactively labeling the synthetic antibodies or fragments,
it is possible to
detect the protein that the antibody was designed for through the use of a
radioimmunoassay
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(RIA) (see, for example, Weintraub, 1986, Principles of Radioimmunoassays,
Seventh
Training Course on Radioligand Assay Techniques, The Endocrine Society). The
radioactive isotope can be detected by such means as the use of a gamma
counter or a
scintillation counter or by autoradiography.
It is also possible to label the modified antibody with a fluorescent
compound.
When the fluorescently labeled antibody is exposed to light of the proper wave
length, its
presence can then be detected due to fluorescence. Among the most commonly
used
fluorescent labeling compounds are fluorescein isothiocyanate, rhodamine,
phycoerythrin,
phycocyanin, allophycocyanin, o_-phthaldehyde and fluorescamine.
The modified antibody can also be delectably labeled using fluorescence
emitting
metals such as ~5-'Eu, or others of the lanthanide series. These metals can be
attached to the
antibody using such metal chelating groups as diethylenetriaminepentacetic
acid (DTPA) or
ethylenediaminetetraacetic acid (EDTA?.
The modified antibody also can be delectably labeled by coupling it to a
chemiluminescent compound. 'the presence of the chemilumineseent-tagged
antibody us
then determined by detecting the presence of luminescence that arises during
the course of a
chemical reaction. Examples of particularly useful chemiluminescent labeling
compounds
are luminol, isoluminol, theromaiic acridinium ester, imidawle, acridinium
salt and oxalate
ester.
2U Likewise, a.bioluminescent compound may be used to label the synthetic
modified
antibody of the present invention. Bioluminescence is a type of
chemiluminescence found
in biological sy stems, in which a catalytic protein increases the et~iciency
of the
chemiiuminescent reaction. The presence of a bioluminescent protein is
determined by
detecting the presence of luminescence. Important tiioluminescant~;,ompaunds
for purposes
Z5 of labeling are luciferin, luciferase and aequorin.
5.6. DEMONSTRATION OF THERAPEiITIC UT~ITY
The Therapeutics of the invention are preferably tested in vary, and then in
vivo, for
the desired therapeutic or prophylactic activity, prior to use in humans. For
example, in
30 vitro assays that can be used to determine whether administration of a
specific Therapeutic is
indicated include in vitro cell culture assays in which appropriate cells from
a cell line or
cells cultured from a patient having a particular disease or disorder are
exposed to or
otherwise administered a Therapeutic, and the effect of the Therapeutic on the
cells is
observed.
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Where the Therapeutic is a modified immunoglobulin that recognizes a cancer or
tumor antigen, the potential efficacy of the modified immunoglobulin may be
assayed by
contacting the Therapeutic to cultured cells (either from a patient or
cultured cell line) and
then assaying for cell survival or growth using any method known in the art,
for example,
cell proliferation can be assayed by measuring'H-thymidine incorporation, by
direct cell
count, by detecting changes in transcriptional activity of known genes such as
proto-
oncogens e.g., fos. myc) or cell cycle markers; cell viability can be assessed
by trypan blue
staining, differentiation can be assessed visually based on changes in
morphology; etc.
Where the Therapeutic is a modified antibody that recognizes an antigen of an
l0 i~'ectious disease agent or a cellular receptor for an infectious disease
agent, the potential
efficacy of the antibody may be assayed by contacting the Therapeutic to
cultured cells
(,either from a patient or cultured cell line) that are infected with the
infectious disease agent
and then assaying the cells for reduction in the infectious disease agent or
for reduction in
physiological indicators of infection with the infectious disease agent.
Alternatively, the
. 15 Therapeutic may be assayed by contacting the Therapeutic to cells (either
cultured from a
patient or from a cultured cell line) that are susceptible to infection by the
infectious disease
agent but that are not infected with the infectious disease agent, exposing
the cells to the
infections disease agent, and then determining whether the infection rate of
cells contacted
with the 'Therapeutic was lower than the infection rate of cells not so
cr~ntacted with the
~0 Therapeutic. Infection of cells.with an infectious disease agent may be
assayed by any
method known in the art.
Wherr the Therapeutic is a modified immunogl~bolin specific for a particular
ligand
or receptor, the potential efficacy of the mwdified immur~oglabulin may be
tested by
contacting the Therapeutic to cultured cells (eiti:er from a patient~.or
culnir~d cell line) that
..
~'S express the receptor member of the binding p?.iz..and.det~rmining whether
the Therapeutic
prevents ligand binding to the receptor and/or receptor ~ignaiing or if the
'fher-apeutic
stimulates receptor signaling. These indicators can be measured by any method
known in
. :. the art for measuring ligand-receptor binding and~receptor signaling-
~~.g., a.S exemplified in
Section 6).
30 'fhe Therapeutics may also be tested for efficacy in appropriate animal
models, and
in clinical trials, in humans. The efficacy of the Therapeutic may be
determined by any
method in the art, for example, after administration of the Therapeutic to the
animal model
or to the humann subject, the animal or human subject is evaluated for any
indicator of the
disease or disorder that the Therapeutic is intended to treat. For example,
the efficacy of the
35 ~empeutic can~be assessed by measuring the level of the molecule against
which the
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modified antibody is directed in the animal model or human subject at suitable
time intervals
before, during, or after therapy. Any change or absence of change in the
amount of the
molecule can be identified and correlated with the effect of the treatment on
the subject. The
level of the molecule can be determined by any method known in the art, e.g.,
by any of the
S immunoassay methods described in Section 5.5, supra, or 5.7, infra.
In other aspects, the modified antibodies may be tested for efficacy by
monitoring
the subject for improvement or recovery from the particular disease or
condition associated
with the molecule against which the synthetic modified antibody is directed.
When the
modified antibody is directed against a tumor or a cancer antigen, the
progress of the
1 U particular tumor or cancer may be followed by any diagnostic or screening
method known
for monitoring cancer or a tumor. For example, but not by way of limitation,
the process of
the cancer or tumor may be monitored by assaying the levels of the particular
cancer or
tumor antigen (or another antigen associated with the particular cancer or
tumor) either in
the serum of the subject or by injecting a labeled antibody specific for the
antigen.
'~ 5 Additionally, other imaging techniques, such as computer tomographic (CT)
scan or
sonograms, or any other imaging method, may he used to monitor the progression
of the
cancer or tumor. Biopsies may also be performed. Before carrying out such
trials in
humans, the tests for efficacy of the modified imlnunogiobulins can be
performed in animal
rxrodels of the particular cancer or tumor.
2U Where the Therapeutic is specific for an antigen or an infectious disuse
agent or a
. _ ~ : cellular receptor of an infectious disease agent, the efficacy of the
modified antibody can be
assayed by administering the modified antibody to a subject;eithe: a humar_
subject or an
anima! model for the disease) and then monitoring either the levels of the
particular
infectious disease agent or symptoms of th~.~par!ical~r infection d.iseasP.
The levels of the
25 infectious disease agent max be deterrnineit by_anyxn~thi~d know~nin-the
ari, for assaying the
levels of an infectious disease agent, e.g., the viral titer, in the case of a
vims, or bacterial
levels (for example, by culturing of a sair~ple from the patient), etc. The
levels of the
infectious disease agentvmay:also be determined bymeasuring the levels of the
antigen
against which the modified immunoglobulin was directed. A decrease in the
levels of the
30 infectious disease agent or an amelioration of the symptoms of the
infectious disease
indicates that the modified antibody is effectivE.
Where the therapeutic is administered as a vaccine, 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
35 vaccine. Generation of a humoral response may be taken as an indication of
a generalized
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immune response, other components of which, particularly cell-mediated
immunity, may be
important for protection against a disease. 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
immunoglobulin molecules or best combinations of immunoglobuiin molecules to
use in
chimpanzee efficacy studies.
The immune response of the test subjects can be analyzed by various approaches
.such as the reactivity.of the resultant immune serurn to antibodies, as
assayed by known
techniques, e.g., enzyme linked immunosor~~nt assa~~ (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
° may-be tested in rabbits far the ability-to induce an anti-iciiotypic
response to tl-~e modified
immlutoglubulin 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 thz vaccine. A co ytr«1 group of rabbits receives an
injection is i l:zM
Tris-HCl pH 9.0 of the vaccine containing a naturally occurring antibody.
Blood samples
2p may be drawn from the rabbits every one or two ~~eeks. and serum analyzed
for anti-
. idiorypic.antibadies to.the.modified immmoglobulm molecule and anti-amt-
idiotypic
antibodies specific for the antigen against whicf. the mo3ified antibody was
directed using,
e.g., by a radioimmunoassay (Abbott Laboratori:a). T'he presence of anti-
idiotyhic
antibodies may be assayed, using an ELISA.v i3EC;~use rabbits may give a
variabiz res~nse
due to their outbred nature. it may aiso.beuseful to test the vaccines in
mice.
5.7. ASSAYS OF THE MODiFiED I~VIMUNOGLOBULINS
. After constructing an immun~globulin having one or more CDRs containing a
binding site for a particular molecule. any binding assay !mown in the art can
be used to
~~ss the binding between the resulting modified antibody and the particular
molecule.
These assays may also be performed to select antibodies that exhibit a higher
affinity or
specificity for the particular antigen.
For example, but not by way of limitation, binding of the modified antibody to
the
particular molecule can be assayed using various immunoassays known in the art
including
but not limited to, competitive and non-competitive assay systems using
techniques such as
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radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich"
immunoassays, immunoradiometric assays, gel diffusion precipitin reactions,
immonodiffusion assays, in situ immunoassays (using colloidal gold, enzyme or
radioisotope Tables, for example), western blots, precipitation reactions,
agglutination assays
(e.g., gel agglutination assays, hemaggiutination assays), complement fixation
assays,
immunofluorescence assays, protein A assays, and immunoelectrophoresis assays,
etc. In
one embodiment, antibody binding is detected by detecting a label on the
primary antibody.
In another embodiment, the primary antibody is detected by detecting binding
of a
secondary antibody or reagent to the primary- antibody. In a further
embodiment, the
1 fl secondary antibody is Labelled. Many means are known in the art for
detecting binding in an
immunoassay and are within the scope of~the present invention.
An in vitro assay system useful in:assessing the binding of the modified
antibody to
its target molecule is described below. Briefly, a reaction mixture of the
modified antibody
and the test sample is incubated under conditions and for a time sufficient to
allow the two
1 ~ components to interact with, e.g., bind to each other, thus forming a
complex, which can
represent a transient complex, which can be remo~~ed and/or detected in the
reaction mixture.
These assays can be conducted in a variety of ways. For example, one method to
conduct
such an assay would involve anchoring the modified antibody or the test
substance onto a
solid phase and detecting the antibodyimolecule complexes anchored on the
solid phase at
2~' .the end of the reaction. ~In one embodiment of such a method, the modif
ed antibody may be
-~,iabeled, either directly c~rindirectly; and the test sample be anchored
onto a solid surface. In
practice, microtiter plates may conveniently be utilized as thz solid phase.
The anchored
. . component may be immobilized by non-covalent or covalent attachments. Nun-
covalent
attachment may be accomplished by simply:coating the solid surf~cv with a
solution of the
25 test sample and drying.
In order to conduct the assay, the nonimmobilized component is added to the
coated
surface containing the anchored component. Afl:er the reaction a cc~mplcte,
uiueacted
=.:°components are removed (v.g.; bywashing) under conditions such~that
any complexes
. formed will remain imrriobilized on the solid surface. The detection of
complexes anchored
34 on ~e solid surface can be accomplished in a number of ways. Where the
previcusly-
~onimmobilized component is pre-labeled, the detection of label immobilized on
the surface
indicates that complexes were formed. Where the previously nonimmobilized
component is
not pre-labeled, an indirect label can be used to detect complexes anchored on
the surface.
Alternatively, a reaction can be conducted in a liquid phase, the reaction
products
35 separated from unreacted components, and complexes detected.
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5.8. TRANSGENIC ANIMALS
The invention also provides animals that are transgenic for (i.e., contain a
nucleic
acid encoding) a modified immunoglobulin of the im~ention (or a functional
fragment
thereof). Animals of any species, including, but not limited to, mice, rats,
rabbits, guinea
pigs, sheep, pigs, micro-pigs, goats, and non-human primates, e.g., baboons,
monkeys, and
chimpanzees, may be used to generate transgenic animals of the invention.
Accordingly, in specific embodiments, the invention provides recombinant non-
human animals containing a recombinant nucleic acid that contains a nucleotide
sequence
encoding a modified immunoglobulin of the invention:, in particular, a
recombinant nun-
I(~ human animal that is transgenic for ~ nucleic acid ewoding a modified
antibody that
imlnunospeeifically binds a cancer or tumor antigen cr that is transgenic for
a nucleic acid
encoding a modified antibody that immunospecifically binds as antigen of an
infectious
disease agent or a cellular receptor of an infectious disease agent.
Any technique known in the art may be used :o introduce the antibody transgene
into
1 ~ animals to produce the founder lines of transgenic animals. Such
techniques include. but are
not limited to pronuciear microinjection (Hupp~ and ~~agner, 1y89, L;.S. Pat.
No.
4,8?3,191 ): retrovirus mediated gene transfer into germ lines ( Van den
Fatten et al., I 985,
Froc. Natl. Acua: Sci. C'S.~ 82:6148-6152); g~n.° targeting in
embryonic stem cells
(?'hompson et al., 1989, Cell 56:313-321 ); electropo;ation of embryos (Lo.
1983, rdol Cell.
20 t~rvl. 6:1803-1814); and sperm-mediated gene transfer (L a~; itrano et ai.,
19fc9, Cell.57:71'7-
. :.123);:ete.. For a review of such Techniques; see Gc~rdoa, 1989. :
rcr~sgenic ~:nimuls, Intl.
ttev. C.ytul. I 1_5:171-229, which is incorporated ov reference lerein in its
entirety.
The present invention provides for.tran.Sgenic animals that carry the
nucleotide
sequewe encoding the modified antibody:a.~ transgene in alf their cells, ;a
~r~ell as ar~imsl~
25 which cant' the transgene in some, but not ali:their cells, i.e., mosaic
animals. The transgene
may be integrated as a single transgene or in cuncatamers, e.R., head-to-head
tandems or
head-to-tail tandems. The transgene may also be selectively introduced into
and activated in
.~~ a particular cell type by follow ng, for example, the teaching of Lasko et
acl. (Lasko et al.,
1992, Froc. Natl. Acad Sci. USA 89:6232-6236). The regulatory sequences
required for
30 such a cell-type specific activation will depend upon the particular cell
type of interest, and
will be apparent to those of skill in the art. When it is desired that the
nucleotide encoding
the synthetic antibody transgene be integrated into the chromosomal site of
the endogenous
immunoglobulin, gene targeting is preferred. Briefly, when such a technique is
to be
utilized, vectors containing some nucleotide sequences homologous to the
endogenous
35 i~~oglobulin are designed for the purpose of integrating, via homologous
recombination
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with chromosomal sequences, into and disrupting the function of the nucleotide
sequence of
the endogenous immunoglobulin gene. The transgene may also be selectively
introduced
into a particular cell type, thus inactivating the endogenous immunoglobulin
in only that cell
type, by following, for example, the teaching of Gu et al. (Gu et al., 1994,
Science 265:103-
106). The regulatory sequences required for such a cell-type specific
inactivation will
depend upon the particular cell type of interest, and will be apparent to
those of skill in the
art.
Methods for the production of single-copy transgenic animals with chosen sites
of
integration are also well known to those of skill in the art (see, for
example, Bronson et al.,
1996, Proc. Natl. Acad. Sci. (1SA 93:9067-9072).
Once transgenic animals have been generated, the expression of the recombinant
antibody gene may be assayed utilizing standard techniques. Initial screening
may be
accomplished by Southern blot analysis or PCR techniques to analyze animal
tissues to
assay whether integration of the transgene has taken place. The level of mRNA
expression
of the transgene in the tissues of the transgenic animals may also be assessed
using
techniques which include but are not limited to Northern blot analysis of
tissue samples
obtained from the animal, in situ hybridization analysis, and RT-PCR. Samples
of gene-
expressing tissue, may also be evaluated immunocytochemically using antibodies
specific
for the antibody transgene product.
6. EXAMPLE: BRADYKININ-CONTAINING SYNTHETIC MODIFIED
ANTIBODIES
This example describes the construction of synthetic modified antibodies that
immunospecifically bind to the bradykinin receptor (BR). The bradykinin
receptor binds to
a ~tive ligand called bradylCinin. The BR-bradykinin interaction is one
example of a
binding pair that may be used in the methods of the invention. The BR-
bradykinin
interaction occurs when amino acids in bradykinin, known as the binding site,
contact the
bradykinin receptor. The synthetic modified antibodies of this example,
contain amino acids
derived from the bradykinin binding site. These synthetic modified antibodies,
therefore,
mimic the bradykinin ligand and predictably bind to the bradykinin receptor
(BR). Six
synthetic modified antibodies containing bradykinin sequences were constructed
and
demonstrated to bind BR as constructed as described below.
The strategy for producing synthetic modified antibodies containing bradykinin
binding sequences is outlined as follows:
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1 ) using oligonucleotides, a variable region gene was engineered to contain a
CDR
with a bradykinin binding sequence;
2) the engineered variable region gene was then inserted into a mammalian
expression vectors containing the appropriate constant regions;
3) a vector containing both light and heavy chains was transfected into a
mammalian
cell and the synthetic modified antibody was expressed; and
4) the synthetic modified antibodies were assayed for BR binding.
6.1. CONSTRUCTION OF THE VARIABLE REGION GENE CONTAINING
BRpDYKININ BINDING SITE
In order to construct the variable region gene encoding a CDR containing the
binding
site of bradykinin, the following strategy was performed.
First, single strand oligonucleotides were annealed to create cohesive double
stranded DNA fragments (as diagramed in Figure 5, Step 1; see also, Kutemeier
et al., 1994
BioTechnigues 17:242). 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
these oligonucleotides are presented in Figures 6A and B (for construction of
the light and
heavy chain variable regions, respectively). Figure 6A lists the sequences of
the
oligonucleotides used in engineering the light chain variable region genes
containing a
bradykinin binding sequence. Figure 6B lists the sequences of the
oligonucleotides used in
engineering the heavy chain variable region genes containing a bradykinin
binding
sequence. The combination of oligos used to engineer the six bradykinin CDRs
(BKCDR1,
BKCDR2, BKCDR3, BKCDR4, BKCDRS, BKCDR6) as well as the two consensus region
(ConVLI and ConVHI) are listed in Table 5.
35
-60-
CA 02310269 2000-OS-12
WO PCT/US9$/24302
99IZ5378
N
0
O O O O
.-...r..... '
O~
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pam m m ca
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oe
~
000
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~o
00
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...
w
o h
p o U U U U U U ~ U
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CA 02310269 2000-OS-12
WO 99/25378 PCT/US98/24302
In order to combine the oligos into the desired gene, groups of 10 or 12
oligos were
used to engineer a variable region gene as described below. Each
oligonucleotide was 5'
phosphorylated as follows: 25 ul of each oligo was incubated for 1 hour in the
presence of
T4 polynucleotide kinase and 50 mM ATP at 37°C. The reactions were
stopped by heating
for 5 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 5 + oligo 6) as shown in Figure S, were then mixed in
sterile
microcentrifuge tubes 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
resulted in
short double strand DNA fragments with cohesive ends.
Next, the cohesive double stand DNA fragments were ligated into longer strands
(Figure 5. 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 Iigase and 10 mM 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 oIigo
3/8/4/7.
Next, oligo 3/8/4/7 was ligated to oligo 5/6. The resulting oligo 3/8/4/7/5/6
was then ligated
to oligo 1/10/2/9 which resulted in a full length variable region gene.
Alternatively, when a group of 12 oligos were used, the order of addition was
oligo
numbers 1+12 = 1/I2, 2+11=2/11, 3+10=3!lU, 4+9---4/9, 5+8=5/8, 6+?=6/7,
1/12+2/11=1/12/2/11, 3;10+4/9=3/10/x/9, 5/8+6/7=5/8/6/7, 1//2/2/11+3/10/4/9 =
1/12/2/11/3/10/4/9, 1/12/2/11/3/10/4/9+5/8/6/7= full length variable region
gene. Eight
variable region genes were constructed by this method. Four genes were light
chain variable
region and four genes were heavy chain variable region. The engineered light
chain genes
included ConVLI, a consensus light chain variable region without a bradykinin
sequence;
BKCDR1, a light chain variable region containing bradykinin sequence in CDRl;
BKCDR2,
a light chain variable region containing bradykinin sequence in CDR2; and
BKCDR3, a
light chain variable region containing bradykinin sequence in CDR3. The
engineered heavy
can variable region genes included ConVHI, a consensus heavy chain variable
region
without a bradykinin sequence; BKCDR4, a heavy chain variable region
containing
bradykinin sequence in CDR4; BKCDRS, a heavy chain variable region containing
bradykinin sequence in CDRS; and BKCDR6, a heavy chain variable region
containing
bradykinin sequence in CDR6. The sequences of the eight engineered variable
region genes
is shown in Figures 4A to 4F.
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Each one of the engineered gene made by combining oligonucleotides was treated
as
follows:
The resulting engineered variable region gene was purified by gel
electrophoresis.
To remove unligated excess of oligonucleotides and other incomplete DNA
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
sterile 1.S 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 i hour followed by centrifugation at
12,000 rpm for 1 S
minutes. The purified DNA pellet was resuspended in SO pl 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 2SmM dNTPs, 200 ng of primers
and S U
of high fidelity thermostable Pfu DNA polymerase in buffer. DNA was amplified
for 28
cycles. Resulting PCR product was analyzed on 1 % agarose gel.
1 S Each purified DNA corresponding to the engineered variable region genes
was
subsequently inserted into the pUC 19 bacterial vector. pUC 19, is a 2686 base
pair, a high
copy number E. coli plasmid vector containing a S4 base pair poly linker
cloning site in lacZ
and an Amp selection marker. In order to prepare the vector for insertion of
the engineered
variable region gene, 10 pg of pUC 19 was linearized with Hinc ll (50 U) for 3
hours at
37°C resulting in a vector with blunt end sequence S' GTC. To prevent
self re-ligation,
linear vector DNA was dephosphorylated with 2S U of calf intestine alkaline
phosphatase
(CIP) for 1 hour at 37°C. In order to insert the engineered variable
region gene into the
pt)C19 vector. approximately O.S pg of dephosphorylated linear vector DNA was
mixed
with 3 ~g of phosphorylated variable region gene in the presence of T4 DNA
ligase ( 1000
2S U)~ ~d incubated at 16°C for 12 hours.
The bacterial vector containing the engineered variable region gene was then
used to
transforrn bacterial cells. Specifically, freshly prepared competent DHS-a
cells, SO ~l. were
mixed with 1 p.g of pUCl9 containing the engineered variable region gene and
transferred to
an electroporation cuvette (0.2 cm gap; Bio-Rad). Each cuvette was pulsed at
2.S kV/200
o~2S 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 pg/ml).
The plates
were incubated at 37°C overnight due to the presence of the Amp marker.
Only
3 S t~sformants containing pUC 19 vector grew on LB/Amp plates.
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A single transformant colony was picked and grown overnight in a 3 mi 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 pl of TE buffer, pH
7.5. To
confirm the presence of gene insert in pUCl9, 25 pl of plasmid DNA from each
colony was
digested with Hinc !I 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
(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 M 13/pLJC reverse primer
(5'AACAGCTATGACCATG 3') for the clones as well as PCR gene products using 5'
end
base primer ( 5' GAATTCATGGCTTG GGTGTG 3') on automated ABI 377 DNA
15 Sequences. All clones were confirmed to contain correct sequences.
Six engineered variable region genes that contained bradykinin sequence were
constructed by the methods of this example. Shown ir. Table 6 is the name of
the synthetic
modified antibody and the location corresponding bradykinin binding sequence
within the
variable region gene. For example, the synthetic antibody named hAbBKCDRI
contained
20 bradykinin binding sequence (BK) in the CDR1 of the variable region light
chain gene (V~).
This synthetic antibody had a consensus sequence (coa) in the variable region
heavy chain
gene (VH).
Table 6. Bradvrkinin-containing synthetic rnudified antibodies
Nine of Synthetic
Modified Antibody V~ VH
lIAbBKCDR1 BKCDR1 ConVHI
hAbBKCDR2 BKCDR2 Cor_VH 1
hAbBKCDR3 BKCDR3 ConVH 1
hAbBKCDR4 ConVLI BKCDR4
hAbBKCDRS ConVLI BKCDRS
hAbBKCDR6 ConVLI BKCDR6
The amino acid sequences corresponding to variable regions of each of the six
synthetic modified antibodies of this example are listed in Table 7. CDRs are
shown in
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bold. The Bradykinin binding site amino acids are: ArgProProGlyPheSerProPheArg
and
are indicated in the CDRs by underlines. Table 5 also illustrates the
consensus sequence of
a human kappa light chain V~ subgroup I and human heavy chain VH subgroup I
gene. In
cases where the consensus CDR was too short to include the complete bradykinin
binding
site sequence, the amino terminal residues from the bradykinin binding site
were deleted
since the carboxyterminal residues were known to be more important in receptor
binding
(Stewart and Vavrek, Chemistry of peptide B2 bradykinin antagonists, pp. 5196,
Burch,
R.M., editor, Bradykinin Antagonists, Basic and Clinical Research, New York:
Marcel
Dekker, 1991; hereby incorporated by reference).
Table 7. Amino acid sequences of engineered variable reEion genes.
Human kappa Light Chain V~ Subgroup (Kabat et a1,1991)
Amino Region Consensus BKCDR1 BKCDR2 BKCDR3
Acid
1 FR1 Asp Asp Asp Asp
2 Ile Ile Ile
153 Gln Gln Gln Gln
4 Met Met Met Met
5 Thr Thr Thr Thr
6 Gln Gln Gln Gln
7 Ser Ser Ser Ser
8 Pro Pro Pro Pro
209 Ser Ser Ser Ser
10 Ser Ser Ser Ser
11 Leu Leu Leu Leu
12 Ser Ser Ser Ser
13 Ala ~ Ala Ala Ala
14 Ser Ser Ser Ser
Val Val Val Val
2516 Gly Gly Gly Gly
17 Asp Asp Asp Asp
18 Arg Arg Arg Arg
19 Val Val Val Val
Thr Thr Thr Thr
21 Ile Ile Ile Ile
22 Thr Thr Thr Thr
3023 Cys Cys Cys Cys
24 CDR1 Arg A_gr Arg Arg
Ala Pro Ala Ala
26 Ser Pro Ser Ser
27 {A-F) Gin fly Gln Gln
28 Ser Phe Ser Ser
29 Ile Ser Ile Ile
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Amino Region Consensus BKCDR1 BKCDR2 BKCDR3
Acid
30 Ser Pro ' Ser Ser
31 Asn Phe Asn Asn
32 Tyr A-gr Tyr Tyr
33 Leu Leu Leu Leu
34 Ala Ala Ala Ala
35 FR2 Trp Trp Trp Trp
36 Tyr Tyr Tyr Tyr
37 Gln Gln Gln Gln
38 Gln Gln Gln Gln
39 Lys Lys Lys Lys
40 Pro Pro Pro Pro
41 Gly Gly Gly Gly
42 Lys Lys Lys Lys
43 Ala Ala Ala Ala
44 Pro Pro Pro Pro
45 Lys Lys Lys Lys
46 Leu Leu Leu Leu
47 Leu Leu Leu Leu
48 Ile Ile Iie Ile
49 Tyr Tyr Tyr Tyr
50 CDRZ Ala Ala Pro Ala
51 Ala Ala Gly Ala
52 Ser Ser Phe Ser
53 Ser Ser Seer Ser
54 Leu Leu ro Leu
55 Glu Glu Phe Glu
56 Ser Ser erg Ser
57 FR3 Gly Gly Gly Gly
58 Val Val Val Val
59 Pro Pro Pro Pro
60 Ser Ser Ser Ser
b 1 Arg Arg Arg Arg
62 Phe Phe Phe Phe
63 Ser Ser Ser Ser
64 Gly Gly Gly Gly
65 Ser Ser Ser Ser
66 Gly Gly Gly Gly
67 Ser Ser Ser Ser
6g Gly Gly Gly Gly
69 Thr Thr Thr Thr
70 Arg Arg Arg Arg
71 Phe Phe Phe ~ Phe
72 Thr Thr Thr Thr
73 Leu Leu Leu Leu
?4 Thr Thr Thr Thr
3
5
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Amino Region Consensus BKCDR1 BKCDR2 BKCDR3
Acid
75 Ile Ile - Ile Ile
76 Ser Ser Ser Ser
77 Ser Ser Ser Ser
78 Leu Leu Leu Leu
79 Gln Gln Gln Gln
80 Pro Pro Pro Pro
81 Glu Glu Glu Glu
82 Asp Asp Asp Asp
83 Phe Phe Phe Phe
84 Ala Ala Ala Ala
85 Thr Thr Thr Thr
86 Tyr Tyr Tyr Tyr
87 Tyr Tyr Tyr Tyr
88 Cys Cys Cys Cys
89 CDR3 Gln Gln Gln A-gr
90 Gln Gln Gln Pro
91 Tyr Tyr Tyr Pro
92 Asn Asn Asn
93 Ser Ser Ser _Phe
94 Leu Leu Leu Ser
95 {A-F) Pro Pro Pro Pro
96 Trp Trp Trp Phe
97 Thr Thr Thr ArE
98 FR4 Phe Phe Phe Phe
99 Gly Gly Gly Gly
100 Gin Gin Gin Gin
101 Gly G1y Gly Gly
102 Thr Thr Thr Thr
103 Lys Lys Lys Lys
104 Val Val Val Val
105 Glu Glu Glu Glu
106 Ile Ile Ile Ile
107 Lys I,ys Lys Lys
108 Arg Arg Arg Arg
109 Thr Thr Thr Thr
Human Heavy Chain VH Subgroup I (Kabat et a1,1991)
Amino Acid RegionConsensus BKCDR4 BKCDRS BKCDR6
1 FRl Gln Gln Gln Gln
2 Val Val Val Val
3 Gln Gln Gln Gln
4 Leu Leu Leu Leu
5 Val Val Val Val
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Amino AcidRegion Consensus BKCDR4 BKCDRS BKCDR6
6 Gln Gln - Gln Gln
7 Ser Ser Ser Ser
8 Gly Gly Gly Gly
9 Ala Ala Ala Ala
Glu Glu Glu Glu
11 Val Val Val Val
12 Lys Lys Lys Lys
13 Lys Lys Lys Lys
14 Pro Pro Pro Pro
Gly Gly Gly Gly
16 Ala Ala Ala Ala
1017 Ser Ser Ser Ser
18 Val VaI Val Val
19 Lys Lys Lys Lys
Val Val Val Val
21 Ser Ser Ser Ser
22 Cys Cys Cys Cys
23 Lys Lys Lys Lys
1524 Ala Ala Ala Ala
Ser Ser Ser Ser
26 Gly Gly Gly Gly
27 Tyr Tyr Tyr Tyr
28 Thr Thr Thr Thr
29 Phe Phe Phe Phe
2030 Thr Thr Thr Thr
31 CDR4 Ser Pro Ser Ser
32 Tyr fly Tyr Tyr
33 Ala Phe Ala Ala
34 Ile Ser Ile Ile
(A-B) Ser Pro Ser Ser
35A Trp P ie Trp Trp
25 35B Asn A-gr Asn Asn
36 FR2 Trp Trp Trp Trp
37 Val Val Val Val
3 8 Arg Arg Arg Arg
39 Gln Gln Gln Gln
Ala Ala Ala Ala
3041 Pro Pro Pro Pro
42 Gly Gly Gly Gly
43 Gln Gln Gln Gln
44 Gly Gly Gly Gly
Leu Leu Leu Leu
46 Glu Glu Glu Glu
47 Trp Tip Trp Trp
3548 Met Met Met Met
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Amino AcidRegion Consensus BKCDR4 BKCDRS BKCDR6
49 Gly Gly - Gly Gly
50 CDRS Trp Trp Trp Trp
51 Ile Ile Ile Ile
52 (A-C) Asn Asn Asn Asn
53 Gly Gly Gly Gly
54 Asn Asn Asn Asn
39 Lys Lys Lys Lys
40 Pro Pro Pro Pro
41 Gly Gly Gly Gly
42 Lys Lys Lys Lys
43 Ala Ala Ala Ala
1044 Pro Pro Pro Pro
45 Lys Lys Lys Lys
46 Leu Leu Leu Leu
47 Leu Leu Leu Leu
48 Ile Ile Ile Ile
49 Tyr Tyr Tyr Tyr
50 CDR2 Ala Ala Pro Ala
15S 1 Ala Ala ~ Ala
52 Ser Ser Phe Ser
53 Ser Ser Ser Ser
54 Leu Leu _Pro Leu
55 Glu Glu Phe Glu
56 Ser Ser A-"fir Ser
2057 FR3 Gly Gly Gly Gly
58 Val Val Val Val
59 Pro Pro Pro Pro
60 Ser Ser Ser Ser
61 Arg Arg Arg Arg
62 Phe Phe Phe Phe
63 Ser Ser Ser Ser
2564 Gly Gly Gly Gly
65 Ser Ser Ser Ser
66 Gly Gly Gly Gly
67 Ser Ser Ser Ser
68 Gly Gly Gly Gly
69 Thr Thr Thr Thr
70 Arg Arg Arg Arg
3071 Phe Phe Phe Phe
72 Thr Thr Thr Thr
73 Leu Leu Leu Leu
74 Thr Thr Thr Thr
75 Ile Ile Ile Ile
76 Ser Ser Ser Ser
77 Ser Ser Ser Ser
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Amino Region Consensus BKCDR4 BKCDRS BKCDR6
Acid
78 Leu Leu ~ Leu Leu
79 Gln Gln Gln Gln
80 Pro Pro Pro Pro
81 Glu Glu Glu Glu
82 Asp Asp Asp Asp
83 Phe Phe Phe Phe
84 Ala Ala Ala Ala
85 Thr Thr Thr Thr
86 Tyr Tyr Tyr Tyr
87 Tyr Tyr Tyr Tyr
88 Cys Cys Cys Cys
89 CDR3 Gln Gln Gln Arg
90 Gin Gln Gln _Pro
55 Gly Gly Pro Gly
56 Asp Asp Pro Asp
57 Thr Thr Gly Thr
58 Asn Asn Phe Asn
59 Tyr Tyr Ser Tyr
60 Ala Ala Pro Ala
61 Gln Gln Phe Gln
62 Lys Lys A_gi- Lys
63 Phe Phe Phe Phe
64 Gln Gln Gln Gln
65 Gly Gly Gly Gly
66 FR3 Arg Arg Arg Arg
67 Val Val Val Val
68 Thr Thr Thr Thr
69 Ile Ile Ile Ile
70 Thr Thr Thr Thr
71 Ala Ala Ala Ala
72 Asp Asp Asp Asp
73 Thr Thr Thr Thr
74 Ser Ser Ser Ser
75 Thr Thr Thr Thr
76 Ser Ser Ser Ser
77 Thr Thr Thr Thr
78 Ala Ala Ala Ala
79 Tyr Tyr Tyr Tyr
80 Met Met Met Met
81 Glu Glu Glu Glu
82 (A-C) Leu Leu Leu Leu
82A Ser Ser Ser Ser
82B Ser Ser Ser Ser
82C Leu Leu Leu Leu
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Amino AcidRegion Consensus BKCDR4 BKCDRS BKCDR6
83 Arg Arg - Arg Arg
84 Ser Ser Ser Ser
85 Giu Glu Glu Glu
86 Asp Asp Asp Asp
87 Thr Thr Thr Thr
88 Ala Ala Ala Ala
89 Val Val Val Val
90 Tyr Tyr Tyr Tyr
91 Tyr Tyr Tyr Tyr
92 Cys Cys Cys Cys
93 Ala Ala Ala Ala
1094 Arg Arg Arg Arg
95 CDR6 AIa Ala Ala Ala
96 Pro Pro Pro Pro
97 Gly Gly Gly Gty
98 Tyr Tyr Tyr Phe
99 Gly Gly Gly Ser
100 (A-K) Ser Ser Ser Pro
15101 Asp Asp Asp Phe
102 Tyr Tyr Tyr Arg
i 03 FR4 Trp Trp Trp Trp
91 Tyr Tyr Pro Pro
92 Asn Asn Asn
93 Ser Ser Ser ~,e
2094 Leu Leu Leu Ser
95 (A-F) Pro Pro Pro
96 Trp Trp Trp he
97 Thr Thr Thr Ark
98 FR4 Phe Phe Phe Phe
99 Gly Gly Gly Gly
100 Gln Gln Gln Gln
25101 Gly Gly Gly Gly
102 Thr Thr Thr Thr
103 Lys Lys Lys Lys
104 Val Val Val Val
105 Glu Glu Glu Glu
106 Ile Ile Ile Ile
107 Lys Lys Lys Lys
30108 Arg Arg Arg Arg
109 Thr Thr Thr Thr
104 Gly Gly Gly Gly
105 Gln Gln Gln Gln
106 Gly Gly Gly Gly
107 Thr Thr Thr Thr
108 Leu Leu Leu Leu
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Amino Acid Region Consensus BKCDR4 BKCDRS BKCDR6
109 Val Val ~ Val Val
110 Thr Thr Thr Thr
111 Val Val Val Val
II2 Ser Ser Ser Ser
113 Ser Ser Ser Ser
6.2. INSERTION OF THE ENGINEERED VARIABLE REGION GENE INTO
A MAMMALIAN EXPRESSION VECTOR
A complete antibody light chain has both a variable region and a constant
region. A
c°mplete antibody heavy chain contains a variable region, a constant
region, and a hinge
region. In order to construct complete light chains and heavy chains, the
modified variable
region genes engineered above were then inserted into vectors containing the
appropriate
constant region. Engineered variable region genes with bradykinin sequence
inserted into a
light chain CDR, were inserted into the pMRR010.1 vector (Figure 3A), which
contains 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,
engineered
variable region genes with bradykinin sequence inserted into a heavy chain
CDR, were
inserted into the pGAMMAI vector (Figure 3B),which contains the human IgGI
constant
region and hinge region sequences. Insertion of the engineered heavy chain
variable region
gene into this vector resulted in a complete heavy chain sequence.
In order to engineer a mammalian vector encoding a complete antibody, both a
complete heavy chain sequence and a light chain sequence were inserted into a
single
mammalian expression vector (Bebbington, C.R., 1991, In METHODS: A Companion
to
Methods in Enzymology, vol. 2, pp. 136-145). The resulting vector encoded both
a light
chain and heavy chain of antibody and was named pNEPuDGV (Figure 3C).
6.3. E~SSION OF SYNTHETIC MODIFIED ANTIBODIES IN
MAMMALIAN CELLS
To examine the production of assembled antibodies the pNEPuDGV vector was
~fected 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 transfected into COS7 cells (obtained from the American
Type
C~ture Collection) using calcium precipitation (Sullivan et al., FEBS Lett.
285:120-123,
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1991 ). The transfected cells were grown in Dulbecco's modified Eagle's Medium
and
cultured for 72 hours after which supernatants containing the bradykinin-
containing
antibodies were collected. Supernatants from transfected COS cells were
assayed using
ELISA method for assembled IgG. The ELISA method involved 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 horseradish peroxidase
(HRP) and
the substrate tetramethylbenzidine (TMB). Color development was proportional
to the
amount of assembled antibody present in the sample.
6.4. BRADYKININ-CONTAINING SYNTHETIC MODIFIED
ANTIBODIES MIMIC BRADYKININ LIGANDS AND BIND TO
BRADYKININ RECEPTOR
The synthetic modified antibodies engineered to contain bradykinin binding
sequences were predicted to mimic the bradykinin ligand and bind the
bradykinin receptor
(gR), In order to confirm that these synthetic modified antibodies bound BR,
the synthetic
antibodies were assayed in a bradykinin receptor binding assay. The assay to
examine
synthetic antibody binding to BR was performed in the following manner. SV-T2
cells were
transformed fibroblasts that express approximately 3;000 bradykinin receptors
(BR) per cell.
Stimulation of bradykinin receptors on SV-T2 cells leads to a rapid increase
in PGE2
synthesis that is proportional to bradykinin binding. Therefore, PGE2 released
into the
medium is indicative of receptor binding.
As shown in Figure 7A, PGE2 synthesis was stimulated approximately four folds
by
the addition of 1 nM bradykinin (ligand). PGE2 synthesis was quantitated by
ELISA. Also
examined in Figure 7A was the receptor antagonist HOE-140. Addition of both
HOE-140
~d bradykinin or HOE-140 alone did not lead to PGE2 synthesis.
Further, as shown in Figure 7B, the expressed modified antibodies were assayed
for
their ability to bind and stimulate the bradykinin receptor. Medium from COS
cells
transfected with an antibody expression vector pNEPuDGV 1 encoding either
hABBKCDR3,
hABBKCDR4, hABBKCDRS, or consensus was used to stimulate bradykinin receptors
on
SV-T2 cells. The synthetic antibodies having the variable chain regions BKCDR3
and
BKCDRS stimulated PGE2 synthesis in a dose dependent manner. BKCDR4, ConVH
media alone, HOE-140 did not stimulate PGE2 synthesis (Figures 7B). The lack
of PGE2
synthesis by cells exposed to BKCDR4 was likely attributed to the fact that
the CDR4
consensus sequence was too short to accommodate the entire bradykinin binding
sequence.
Table 6 shows the comparison of consensus CDR amino acid sequences and BKCDR
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sequences. The synthetic modified antibodies BKCDR3 and BKCDRS were
demonstrated
to complete for receptor binding against the native ligand bradykinin. As
shown in Figure
7C addition of bradykinin stimulated PGE2 synthesis four fold (second bar from
left).
Addition of either BKCDR3 or BKCDR 5 to cells prestimulated with native
bradykinin
inhibited the bradykinin-stimulated PGE2 synthesis.
Table 8
Consensus CDR3: Gln Gln Tyr Asn Ser Leu Pro Trp Thr
BKCDR3: Arg Pro Pro Gly Phe Ser Pro Phe Arg
Consensus CDR4: Ser Tyr Ala Ile Ser Trp Asn
BKCDR4: Pro Gly Phe Ser Pro Phe Arg
Consensus CDRS: Trp Ile Asn Gly Asn Gly Asp Thr Asn Tyr Ala Gln Lys Phe Gln
Gly
BKCDRS: Trp Ile Asn Gly Arg Pro Pro Gly Phe Ser Pro Phe Arg Phe Gln Gly
Taken together, these results indicate that the modified antibodies containing
the
bradykinin binding site were able to bind the bradykinin receptor.
The present invention is not to be limited in scope by the specific
embodiments
described herein. Indeed, various modifications 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.
30
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