Note: Descriptions are shown in the official language in which they were submitted.
WO 95/10302 CA o2622o18 2oo8-o2-22 PCTIUS94/10547
CELLULAR AND SERUM PROTEIN ANCHORS AND CONJUGATES
INTRODUCTION
This application is a division of application No. 2,172,630
filed in Canada on September 16, 1994
Technical Field
The field of this-i-nvention is the extended lifetime of
physiologically active agents in a host.
Background
It is frequently desirable to limit the biologic
effects of agents present in the mammalian blood stream.
For example in the case of exposure to a toxin/poison,
acute mitigation of the effects of the toxin/poison may be
indicated. Alternatively, the agent may be cellular as an
infected, auto-, allo-, or xenoreactive or tumorous host
cell or an infectious organism.
Present blood therapies involve removing the agent
from the blood by for example, plasmapheresis or
adsorption with activated charcoal, disruption of the
agent with for instance, antibiotics or enzymes, or the
selective binding of the target, for example with
antibodies. These therapies are normally employed after
the patient has been exposed to the deleterious effects of
the toxin/poison'or the cells, so that substantial damage
to the host has already occurred. In many situations,
there is a need for long time administration of a drug, so
as to maintain a therapeutic level in the blood stream.
The oral administration or intravenous injection of drugs
can result in subtherapeutic dosages for extended periods
of time, followed by dosages exceeding the therapeutic
level and involving serious side effects. Also, there are
situations where one wishes to maintain an agent in the
blood stream for an extended period of time, for example,
where one wishes to obtain a strong immune response, where
the immunogen is continuously present_ in a form which
provides for continual stimulation of the immune system.
There is, therefore, substantial interest in being
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able to provide for improved methods of providing for the
continued maintenance of agents in the blood stream, where
the function of the agent may be for limiting the toxicity
or pathogenicity of blood-borne agents, long term
maintenance of a physiologically active agent, or long term delivery of an
active agen.t.
SUMMARY OF THE INVENTION
Bifunctional reagents or conjugates are provided
comprising an anchor and a physiologically active entity,
where the reagent is bound through the anchor to a long-
lived moiety associated with the blood, either cellular or
a mobile blood protein, so as to provide for long-lived
maintenance of the physiologically active entity in the
host. These reagents find broad applications in
prophylactic and therapeutic applications, production of
antibodies, reduction of the biologically effective
concentration or activity of an endogenous or exogenous
blood component, and in other situations where long term
administration of a physiologically active compound is of
interest.
Targets may be host derived or foreign. Host targets
include cells and blood compounds present in undesirable
concentrations, such as auto-, allo- or xenoreactive white
cells, i.e. leukocytes, including macrophages, infected
cells, platelets, tumorous cells, and overexpressed
cytokines and hormones. Foreign targets include toxins,
poisons, drugs of abuse, pathogenic infectious microbes,
or the like.
The long-lived blood associated entities include
long-lived serum proteins, erythrocytes, platelets or
endothelial cells, where the anchor binds specifically or
non-specifically, to one or more surface membrane
proteins, predominantly associated with the particular
cell, or to a soluble protein. In the case of
erythrocytes, platelets and proteins, the conjugate may be
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administered in vivo or ex vivo. In the case of
endothelial cells, the conjugate may be administered in
vivo.
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
Methods and compositions are provided for extending
the life of a target binding member, which is a
physiologically active agent, in a mammalian host by
providing for binding said target binding member to a
long-lived blood associated entity, normally a protein.
A reagent or conjugate is used which will have at least
two active moieties: (1) an anchor for binding the reagent
to the long-lived blood associated entity; and (2) a
target binding member, which is bound, directly or
indirectly, to the long-lived blood associated entity by
means of the anchor. The long-lived blood associated
entity may be cellular, e.g. a mobile cell, such as an
erythrocyte or platelet, namely, anuclear cells, or a
fixed cell, such as an endothelial cell; or soluble or
mobile proteins which are present in the blood for
extended periods of time and are present in a minimum
concentration of at least about 0.1 g/ml. Proteins which
fulfill this concentration requirement include, serum
albumin, transferrin, ferritin and immunoglobulins,
particularly IgM and IgG. The half-life of the protein
should be at least about 12 hours.
The target binding member or physiologically active
agent may provide for a wide variety of functions. It may
serve as an immunogen for the production of antisera or
monoclonal antibodies, where one is interested in a
diagnostic or therapeutic reagent, for the production of
vaccines, or for the protection, prophylactic or
therapeutic against a deleterious blood-borne target, such
as a pathogen, noxious agent or endogenous factor. The
physiologically active agent may react directly with the
deleterious blood-borne agent, have antiproliferative
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activity, particularly cytotoxic activity, by itself or in
conjunction with an endogenous system, such as with
antibodies to a pathogen or deleterious endogenous cell,
e.g. T cell, or to a toxin, may have specific binding
activity with a surface membrane receptor, so as to reduce or inhibit the
signal transduced by such receptor, may
bind to a homing receptor, integrin or addressin, so as to
inhibit extravasation or diapedesis, or may change the
distribution of the target, including enhancing the
excretion of the target by the host or reducing the
concentration of the target in one compartment of the body
in contrast to a different compartment of the body.
The physiologically active agent is provided at a
biologically effective concentration, where the
physiologically active agent may act in the bound form as
part of the reagent or may act independently of the long-
lived blood associated entity, as a soluble agent after
release from the long lived blood component. The long-
lived blood component may be part of the physiological
activity, for example, where erythrocyte binding to the
target may enhance the excretory processing of the target.
"Biologically effective concentration" of the
physiologically active agent means the agent concentration
-that is bioavailable to the target during the course of
the procedure. Biologically effective concentration of
the target will mean the immediately bioavailable
concentration of the target at the in vivo site of its
action. For the most part, the target will be at least
partially dispersed in the blood stream, may be in
solution or associated with components of the blood
stream. Thus the effective concentration of the target
may be reduced by limiting the volume of distribution of
the target as to sites at which the target is segregated,
by limiting diffusion, mobility, or migration of the
target, etc. Thus the physiologically active agent will
interact with a component in the blood stream, so as to
provide the desired result, where the interaction may be
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direct or indirect to achieve the desired result, or may
ka a specific or non-specific interaction, where for the
most part a non-specific interaction will provide for a
specific result with a subset of the entities 'with which
5 the physiological agents interacts.
The disclosed therapeutic methods are applicable to
a broad range of targets, both host derived and foreign
(meaning exogenous or non-host), which may be present in
the blood and have a deleterious physiological effect, due
to an undesirably high effective concentration, or as in
the case of neoplastic cells, being present in any amount.
Host derived cellular targets include, (with parenthetical
clinical indication): T cell or subsets, such as aIFN+,
CD4+, CD8+, LFA1+, etc., cells (autoimmune disease,
alloreactivity, xenoreactivity and inflammation), B cells
or subsets such as pre-B cells, CD5+, IgE+, IgM+ etc. (B
cell lymphoma, xenograft, autoimmunity, anaphylaxy),
leukocytes, such as macrophages and monocytes
(inflammation, myelomonocytic leukemia), other leukocytes
such as neutrophils, basophils, NK cells, eosinophils, or
allo- or xeno-reactive leukocytes, etc. (inflammation,
anaphylaxis, transplant rejection), stem cells such as
CD34+ cells (polycythemia), fetal red cells, such as Rh+
red cells (prophylaxis of anti-Rh immunization after
pregnancy in a Rh- mother), malignant cells (malignancies;
CALLA) or infected cells, particularly retroviral, e.g.
HIV, infected host cells, or the like.
Host derived non-cellular tarcTets include soluble
HLA, class I and class II, and non-classical class I HLA
(E, F and G) for modulating immunoregulation, soluble T or
B cell surface proteins, cytokines, interleukins and
growth factors such as IL1, 2, 3, 4, 6, 10, 13, 14 and 15,
soluble IL2 receptor, M-CSF, G-CSF, GM-CSF, platelet
growth factors, alpha, beta, and gamma- interferons, TNF,
NGFS, arachadonic acid metabolites such as prostaglandins,
leukotrienes, thromboxane and prostacyclin for
cardiovascular diseases, immunoglobulins such as total IgE
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for anaphylaxy, specific anti-allergen IgE, auto or allo-
antibodies for autoimmunity or allo- or xenoimmunity, Ig
Fc receptors or Fc receptor binding factors, carbohydrates
(gal), natural antibodies involved in allo- or
xenorejection, erythropoietin, angiogenesis factors, adhesion molecules, MIF,
MAF, complement factors
(classical and alternate pathways, including regulatory factors), PAF, ions
such as calcium, potassium, magnesium,
aluminum, iron, etc, enzymes such as proteases, kinases,
phosphatases, DNAses, RNAses, lipases and other enzymes
affecting cholesterol and other lipid metabolism,
esterases, dehydrogenases, oxidases, hydrolases,
sulphatases, cyclases, transferases, transaminases,
atriopeptidases, carboxylases and decarboxylases and their
natural substrates or. analogs, superoxide dismutase,
hormones such as TSH, FSH, LH, thyroxine (T4 and T3),
renin, insulin, apolipoproteins, LDL, VLDL, cortisol,
aldosterone, estriol, estradiol, progesterone,
testosterone, dehydroepiandrosterone (DHEA) and its
sulfate (DHEA-S), calcitonin, parathyroid hormone (PTH),
human growth hormone (hGH), vasopressin and antidiuretic
hormone (ADH), prolactin, ACTH, LHRH, THRH, VIP,
cathecolamines (adrenaline, vanillylmandelic acid, etc.),
bradykinins and corresponding prohormones, metabolites,
ligands or natural cell or soluble receptors thereof,
cofactors including atrionatriuretic factor (ANF),
vitamins A, B, C, D, E and K, serotonin, coagulation
factors, e.g. prothrombin, thrombin, fibrin, fibrinogen,
Factor VIII, Factor XI, Willebrand factor, plasminogen
factors, e.g. plasmin, complement activation factors, LDL
and ligands thereof, uric acid, etc. In some instances
one may provide specific effects associated with
complement, by having inhibitors such as DAF, CD59, etc., compounds regulating
coagulation, such as hirudin,
hirulog, hementin, TPA, etc. or other compounds, such as
tissue factor, nucleic acids for gene therapy, etc.,
compounds which are enzyme antagonists, compounds binding
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ligands, such as cytokines, hormones, inflammation factors
(PAF, complementation factors), etc.
Foreign targets include drugs; especially drugs
subject to abuse such as heroin and other opiates, PCP,
barbiturates, cocaine and derivatives thereof,
benzodiazepins, ecstasy, etc.,poisons, toxins such as
heavy metals like mercury and lead, chemotherapeutic
agents, paracetamol, digoxin, free radicals, arsenic,
bacterial toxins such as LPS and other grain negative
toxins, Staphylococcus toxins, Toxin A, Tetanus toxins,
Diphtheria toxin and Pertussis toxins, plant and marine
toxins, snake and other venoms, virulence factors, such as
aerobactins, radioactive compounds or pathogenic microbes
or fragments thereof, including infectious viruses, such
as hepatitis , A, B, C, E and delta, CMV, HSV (types 1, 2
& 6), EBV, varisella zoster virus (VZV), HIV-1, -2 and
other retroviruses, adenovirus, rotavirus, influenzae,
rhinovirus, parvovirus, rubella, measles, polio, reovirus,
orthomixovirus, paramyxovirus, papovavirus, poxvirus and
picornavirus, prions, plasmodia tissue factor, protists
such as toxoplasma, filaria, kala-azar, bilharziose,
entamoeba histolitica and giardia, and bacteria,
particularly gram-negative bacteria responsible for sepsis
and nosocomial infections such as E. coli, Acynetobacter,
Pseudomonas, Proteus and Klebsiella, but also gram
positive bacteria such as staphylococcus, streptococcus,
etc. Meningococcus and Mycobacteria, Chlamydiae,
Legionnella and Anaerobes, fungi such as Candida,
Pneumocystis carini, and Aspergillus, and Mycoplasma such
as Hominis and Ureaplasma urealyticum.
As already indicated, the subject invention may be
used for the production of antibodies, where the
antibodies are harvested or splenocytes or other B cells
are used for immortalization and the production of
monoclonal antibodies. Thus, the antibodies may be
directed to many of the targets indicated above or may be
directed to various drugs, for therapeutic dosage
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monitoring, treatments for overdosage of drugs or drugs of
abuse, or the like. Also, the subject invention may be
used to vaccinate against a pathogen or other deleterious
entity, where various unicellular microorganisms and
viruses have been described above. In addition, the subject invention can be
employed to activate T cells
toward particular targets, by providing for appropriate
targets for antigen presenting cells, which will then
present to the T cells or providing for direct activation
of T cells
The choice of the long-lived blood component will be
affected by the purpose for binding to the target. Thus
depending on the choice of the long-lived blood component,
the target may be rapidly segregated and excreted from the
body, segregated in a biologically inactive form to be
slowly eliminated, or degraded over time. The long-lived
blood component may be fixed or mobile, that is,
substantially fixed in position, as in the case of the
endothelial cells, or mobile in the vascular system,
having a substantially uniform or variant distribution in
the vascular system, where the long-lived blood component
may be preferentially present in particular compartments,
including solid tissue.. The choice of long-lived blood
component will also depend in part on the nature of the
target. For instance, red blood cells are preferred for
cellular pathogenic agents for efficient clearance, while
serum protein components, such as albumin or
immunoglobulins, are particularly useful for segregating
soluble toxins and endothelial cells are particularly
useful for restenosis and thrombosis.
A long-lived blood component has a half-life of at
least about 12 hrs, usually at least about 48 hrs,
preferably at least about 5 days, desirably at least about
10 days. Generally, half-lives are determined by serial
measurements of whole blood, plasma or serum levels of the
compound following labelling of the compound with an
isotope (e.g. 131I, 'ZSI, Tc, 5' Cr 3H etc) or fluorochrome
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and injection of a known quantity of labelled compound
I.V. Included are red blood cells (half life ca. 60
days), platelets (half life ca. 4-7 days), endothelial
cells lining the blood vasculature, and long lived blood
serum proteins, such as albumin, steroid binding proteins,
ferritin, a-2-macroglobulin, transferrin, thyroxin binding
protein, immunoglobulins, especially IgG, etc. In
addition to preferred half-lives, the subject components
are preferably in cell count or concentration sufficient
to allow binding of therapeutically useful amounts of the
conjugate. For cellular long lived blood components, cell
counts of at least 2,000/ l and serum protein
concentrations of at least l g/ml, usually at least about
0.01 mg/ml, more usually at least about 1 mg/ml, are
preferred.
The selected cellular long-lived blood components are
present in high number in the vascular system. Platelets
.are present in from about 1-4x10s/ l, while red blood cells
are present in about 4-6xl06/ l. The cells have a long
half-life and by appropriate choice of binding site of a
surface membrane protein, e.g. epitope for binding to
antibodies, endocytosis is avoided. The erythrocyte and
platelets lack a nucleus and cell division capability.
Preferred cells have a wide distribution in capillaries
and tissue and express specific binding sites on the cell
surface associated with their specific differentiation.
In addition to in vivo administration of the subject
conjugate, in the case of red blood cells and platelets,
these cells may be readily collected, combined with the
conjugate, and then administered to the host. The cells
may be autologous or allogeneic.
The choice of the long-lived blood component also
depends on the desired route of limiting the target's
toxicity/pathogenicity. For instance, it is often
desirable to modulate a target's access to the central
nervous system, interstitial spaces, etc. to limit
toxicity, infection, etc. By selecting the long-lived
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blood component based on location, distribution, specific
surface markers, or other differentiating characteristic
associated with the long-lived blood component, one can
modulate the ultimate sites and volume of distribution of
5 the target. For example, in some situations cellular activation results in
up-regulation of a surface membrane
protein. For endothelial cells, addressins, e.g. ELAM-1,
or integrins, e.g. VLA-4, may be up-regulated in the case
of inflammation. In this way, the agent may be segregated
10 to a site of inflammation or lymphoid organ such as the
spleen to enhance the agents exposure to a concentrated
immune response including phagocytic white cells. In
another example, the immune complexes of a complement
binding antibody can provide for complement mediated of
lysis cells or microorganism. Another example is the
modification of the distribution of an endogenous or
exogenous agent between compartments, such as the CNS and
the blood. By providing for high affinity receptors
for the agent in the blood, the concentration of the agent
in the CNS may be reducecd in proportion to the binding
capacity of the blood for the agent. In this way, one may
be able to reduce the concentration of a drug in the
brain, by enhancing the binding capacity of the blood for
the drug, e.g. using antibodies for the drug, so as to
withdraw the drug across the blood-brain barrier into the
blood. The conjugate may be bound to the long-lived
blood component specifically by means of complementary
specific binding sites or non-specifically, usually
involving covalent bonding. In referring to a specific
binding site is intended a particular spatial conformation
and charge distribution which has a high affinity to a
complementary molecule, which has the reciprocal
conformation and charge distribution. Thus in the case of
the long-lived blood component and anchor or the target
and the target binding moiety, the conjugate will be
selected to bind to its complementary binding member, as
compared to the numerous other molecules which may be
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encountered associated with the vascular system.
Examples of binding sites include immunological
epitopes (an epitope to which an antibody or TCR binds),
sugar moieties (binding sites to which a lectin binds),
peptide-specific binding sites (binding sites to which
peptides bind, as in ligand and cell surface membrane
receptor), synthetic or naturally occurring organic
compound specific binding sites (haptens binding to
antibodies or organic agonists or antagonists to naturally
occurring ligands), etc.
Suitable erythrocyte binding site containing
molecules include glycophorin A, B and C, Band 3 and
Rhesus blood group antigens. Preferred erythrocyte
binding sites are abundantly expressed on the erythrocyte
with copy numbers of at least 1,000, preferably at least
10,000, more preferably at least 100,000 per cell,
desirably are tethered at least about 0.5, preferably at
least about 1 nm above the bilayer surface and do not
facilitate per se cell deformation when the conjugate is
bound to the cell (e.g. the binding will be selected so as
not to be a key component of the cytoskeleton). Binding
sites of the erythrocyte surface glycoprotein glycophorin
A and erythrocyte binding sites comprising sialic acid are
examples of preferred binding sites. Preferred platelet
binding sites include GPIIa, GPIIb, GPIIIa and GPIV.
Desirably, upon binding to the the target, deformation of
the long-lived blood component, e.g. erythrocyte or
platelet, does not occur.
Binding to the long-lived blood component may be specific
or non-specific, covalent or non-covalent. By "specific
to" is meant that on a total availability basis the
binding site is present on a given target or long-lived
blood component and substantially absent from blood
associated components present during the presence of the
conjugate at the affinity of the conjugate moiety. For
non-covalent specific binding to the long-lived blood
component, the presence of a given binding. site is
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generally determined by the binding of a binding site
specific reciprocal binding member. Long-lived blood
component-specific binding sites or target specific
binding sites are readily identified empirically by
exploiting the ability of a binding site specific
reciprocal binding member to distinguish blood associated
components or targets containing the long-lived blood
component specific binding site or target specific binding
site from blood associated components present in the host
at the time of treatment. For example immunofluorescent
microscopy, flow cytometry, cell panning, enzyme
immunoassay (EIA), affinity chromatography, bead
separation, NMR, crystallography, etc. may be used to
assay binding site specificity for cellular targets and
for molecular (proteins, glycoproteins, carbohydrates,
lipids, organics, molecular toxins, etc) targets,
respectively. By employing blood free of the long-
lived blood component or target in a competitive assay
with labeled long-lived blood component or target, a
reduction in the level of binding of the long-lived blood
component or target in the presence of blood as compared
to the level of binding in the absence of blood would
indicate cross-reactivity of a blood component with the
long-lived blood component or target, respectively. Where
a reciprocal specific binding member (reciprocal to the
long-lived blood component or target) is introduced into
the host, generally, more than about fifty percent (50%),
preferably more than about seventy-five percent (75%) of
the reciprocal binding members, which are bound to the
binding site of the long-lived blood component or target
after administration, will be bound to the long-lived
blood component or target. Where binding assays are
optimized, specific receptor-epitope binding will have a
binding affinity of at least about 10-6M, preferably at
least about 10'?M, but usually will vary in the range of
about 10'8M to 10'12M under physiological conditions.
The reciprocal binding site members of the.conjugate
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may be derived from any molecule capable of providing the
requisite long-lived blood component or target binding
specificity that is compatible with in vivo use. The two
binding sites of the conjugate will be directed to
different complementary binding members, normally on
different targets. The conjugate binding members may be
derived from a natural receptor or ligand of the long-
lived blood component or target: Examples include enzymes
and substrates or cofactors, lectins and sugars,
antibodies or T-cell antigen receptors and immunological
epitopes, cytokines (e.g interleukins) or hormones (e.g.
LH) or drugs (e.g.opiates) or viruses (e.g. HIV) or
immunoglobulin (e.g. IgE) and their respective receptors,
(e.g CD4, Fc receptor, etc.) , etc., or truncated versions
thereof or fragments of these molecules. For targeting
some toxins such as heavy metals, chelators such as EDTA
or EGTA, provide useful receptors.
Alternatively, conjugate binding members may be
specifically selected or synthesized to specifically bind
the complementary binding member. For example, useful
peptides, carbohydrates, nucleic acids, poly- and
monoheterocyclic natural organics and synthetic molecules
are isolated by screening natural or synthetic libraries
for the requisite binding specificity. Typically,
specific binding molecules are assayed by EIA, RIA,
fluorescence, luminescence, chemiluminescence, direct
binding assays or competition assays by displacement of
labelled agonist, as described previously.
The conjugate may take many forms, being comprised of
one or two members. In the single member conjugate, both
the anchor and the target binding moiety will be bound
together when administered. Usually the two moieties will
be connected by covalent bonds, although in some
situations it will be satisfactory or desired to have a
non-covalent linkage between the two moieties. In other
situations, the conjugate may have two members. The first
member is added first and binds to the long-lived blood
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component, so as to provide a population of first member
long-lived blood components in the host. Subsequently,
one may add the second member, which will bind
specif ical ly to the first member to bind the second member
to the long-lived blood component. The anchor may be
specific or non-specific. As indicated, one may use
specific binding members to bind to an epitope present on
the long-lived blood component. Alternatively, one may
use reactive compounds, which will non-specifically react
with active functionalities on the long-lived blood
component as well as other molecules present in the host
blood stream. In this situation, one selects a long-lived
blood component which is in very high concentration in the
blood stream, as compared to most other components, is
present under conditions where it is reactive with the
anchor, and because of its long life, within a short time,
usually fewer than 7, more usually fewer than 5 days, the
first binding member will be primarily associated with the
desired long-lived blood component in the host.
For non-covalent binding to the long-lived blood
component and target, in one approach, the conjugate
binding member includes from one to two monoclonal
antibodies or fragments thereof. The antibody or fragment
thereof may be any one of the subtypes or isotypes, only
being one that does not participate in complement effected
lysis, where one does not wish to involve complement
mediated cytotoxicity. To avoid complement mediated
cytotoxicity, the whole antibody may be IgA, IgD, IgGl or
IgG4 or corresponding isotypes of other species, while
fragments may be from any isotype. Normally, the antibody
will be one which is allogeneic, although xenogeneic
antibodies may be employed, e.g. in those situations where
the host is immunocompromised, or the antibody or fragment
is non-antigenic, despite its being xenogeneic. To
minimize the immune response while maintaining the
convenience of using non-human antibodies, it is often
desirable to generate chimeric antibodies. The whole
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antibody need not be used, fragments being frequently
useful, such as Fv, Fab, F(ab')2, the heavy chain, a
single-chain antigen binding protein, a peptide conformer
mimicking an antibody binding site, or the like. Thus,
5 only the binding site of the antibody may be used, which
will usually comprise the variable regions of the heavy
and light chains, although in some instances, only the
heavy or light subunit variable region may suffice. The
fragment which is used, should retain at least a
10 substantial portion, preferably at least about ten percent
of the original affinity of the antibody; or following
selected mutations, may exhibit a higher affinity.
Monoclonal antibodies may be obtained in accordance
with conventional ways. For monoclonal antibodies
15 specific for red blood cells, lymphocytes may be collected
from an individual with a positive direct Coombs test,
particularly of the IgG type, (without complement) without
a showing of hemolysis. The lymphocytes may be
immortalized by any convenient means, e.g. Epstein-Barr
virus transformation, cell fusion, cell transfection or
the like, followed by screening for antibodies having the
desired specificity and affinity, as well as the desired
isotype. The antibodies may then be modified in a variety
of ways, by enzymatic cleavage to provide for fragments,
using papain, chymotrypsin, pepsin, trypsin, reduction
with cleavage of intramolecular disulfide linkages or the
like. The antibodies may be from any source, such as
primate, particularly human, murine, lagomorpha, canine,
ovine, porcine, equine, or the like or genes coding for at
least one region of the antibody may be cloned and
expressed in procaryotic or eukaryotic expression systems.
Conjugate binding members which bind to red blood
cells may be further characterized by binding to 0 Rh
negative erythrocytes or to panels of erythrocytes of
known phenotypes. Those conjugate binding members which
react with substantially all the cells (at least 80%) of
the panel and, furthermore, show negative result.s with the
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direct Coombs test using anti-complement globulin are
selected. Ref: Stratton, F.; Rawlinson, Vi; Merry, A.H.;
Thomson, E.E.; Clin. Lab. Haematol. 1983, 5:17-21. In
addition, the antibodies are screened as to cross-
reactivity with blood associated components, particularly cells, such a
lymphocytes, myelomonocytes, platelets, and
the like, and serum proteins. Similarly, for antibodies
specific for the long-lived blood components comprising
long-lived serum proteins, platelets or endothelial cells,
the antibodies are selected to be specific for such
proteins/cells and not,for other proteins/cells which may
be encountered in the host. For the most part, with red
blood cells the number of conjugates bound per cell (when
not bound to the blood borne target) will be below the
level that causes hemolysis.
If desired, the antibodies can be prepared or
modified in a variety of ways. Chimeric antibodies may be
prepared, where the constant region may be modified as to
isotype or species. For example, murine monoclonal
antibodies may be prepared, the genes encoding the heavy
and light chains isolated, and the constant regions of the
heavy and light chains substituted with the appropriate
constant regions of human constant regions to provide for
a chimeric antibody which lacks the antigenicity of the
murine constant region. Alternatively, variable regions
obtained from a host may be cloned and mutated and then
screened to identify specific binding affinities. A
further alternative is to exchange not only the constant
region of the heavy and light chain, but also exchange the
framework regions of the variable domains, so as to
further reduce the antigenicity of the antibody. Numerous
techniques are described in the literature, for example,
"The synthesis and in vivo assembly of functional
antibodies in yeast," Wood, C.R., Boss, M.A., Kenten,
J.H., Calvert, J.E., Roberts, N.A., Emtage, J.S., Nature,
Apr 4-10, 1985, 3 4(6010):446-9; "Construction of
chimaeric processed immunoglobulin genes containing mouse
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17
variable and human constant region sequences," Takeda, S.,
Naito, T., Hama, K., Noma, T., Honjo, T., Nature, Apr 4-
10, 1985, 314 (6010) :452-4; "A recombinant immunotoxin
consisting of two antibody variable domains fused to
Pseudomonas exotoxin," Chaudhary, V.K., Queen, C.,
Junghns, R.P., Waldmann, T.A., FitzGerald, D.J., Pastan,
I, Laboratory of Molecular Biology, DCBD, National Cancer
Institute, Bethesda, Maryland 20892, Nature, Jun 1, 1989,
339(6223):394-7; "Binding activities of a repertoire of
single immunoglobulin variable domains secreted from
Escherichia coli [see comments]," Ward, E.S., Gussow, D.,
Griffiths, A.D., Jones, P.T., Winter, G., MRC Laboratory
of Molecular Biology, Cambridge, UK, Na ur (ENGLAND),
Oct 12, 1989, 341(6242):544-6, ISSN 0028-0836, Comment in
Nature, 1989, Oct 12, 341(6242):484-5.
Instead of antibodies, other binding molecules may be
employed which are specific for the long-lived blood
component. The anchor may be a naturally occurring or
synthetic molecule or modified, e.g. truncated or mutated,
derivative thereof. Compounds, such as drugs, e.g.
penicillin, TZ, T4, cortisol, cholesterol, etc., hormones,
glycoproteins from microorganisms, e.g. viruses, such as
reovirus, rubella virus, influenzae virus, HIV, CMV,
lectins, etc., have binding sites which are specific for
such long-lived blood components as red cells, serum
albumin, thyroxine binding protein, steroid binding
protein, etc. In some instances, it will be desirable to
modify the binding molecule to maintain its binding
affinity, while diminishing other biological activity.
This will be frequently achieved by the choice of site for
linking the anchor to the target binding member.
In some instances, it will be desirable to allow for the
consecutive addition of first and second compounds, where
the first compound has the anchor and the second compound
has the target binding member. The first and second
compounds also have the complementary binding members of
a specific binding pair, so that upon addition of the
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second compound to the first compound, the two compounds
will be non-covalently bound to provide the conjugate.
The first compound will comprise an active functionality,
a linking group, and the first binding entity. The
functionalities which are available on proteins are primarily amino groups,
carboxyl groups and thiol groups.
While any of these may be used as the target of the
reactive functionality, for the most part, bonds to amino
groups will be employed, particularly formation of amide
bonds. To form amide bonds, one may use a wide variety of
active carboxyl groups, particularly esters, where the
hydroxyl moiety is physiologically acceptable at the
levels required. While a number of different hydroxyl
groups may be employed, the most convenient will be N-
hydroxysuccinimide, and N-hydroxy sulfosuccinimide,
although other alcohols, which are functional in an
aqueous medium such as blood, may also be employed. In
some cases, special reagents find use, such as azido,
diazo, carbodiimide anhydride, hydrazine, dialdehydes,
thiol groups, or amines to form amides, esters, imines,
thioethers, disulfides, substituted amines, or the like.
Usually, the covalent bond which is formed should be able
to be maintained during the lifetime of the target binding
member, unless it is intended to be the agent release
site.
A large number of bifunctional compounds are
available for linking to entities. Illustrative entities
i n c 1 u d e: a z i d o b e n z o y 1 h y d r a z i d e,
N-[ 4-( p- a z i d o s a l i c y l a m i n o) b u t y 1]-
3'-[2'-pyridyldithio]propionamid), bis-sulfosuccinimidyl
suberate,dimethyladipimidate, disuccinimidyltartrate,N-y-
maleimidobutyryloxysuccinimide ester, N-
hydroxy sulfosuccinimidyl-4-azidobenzoate,
N-succinimidyl [4-azidophenyl]-1,3'-dithiopropionate, N-
succinimidyl (4-iodoacetyl)aminobenzoate, glutaraldehyde,
and succinimidyl 4-[N-maleimidomethyl]cyclohexane-l-
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carboxylate.
When one or both of the linking groups is permanent,
the linking group(s) will not be- critical to this
invention and any linking group which is convenient,
physiologically acceptable at utilized doses, and fills
the requirements of the molecule, such as being stable in
the blood stream, effectively presenting the target
binding member or first binding entity, allowing for ease
of chemical manipulation, and the like, may be employed.
The linking group may be aliphatic, alicyclic, aromatic,
or heterocyclic, or combinations thereof, and the
selection will be primarily one of convenience. For the
most part, any heteroatoms will include nitrogen, oxygen,
sulfur or phosphorus. Groups which may be employed include
alkylenes, arylenes, aralkylenes, cycloalkylenes, and the
like. Generally the linking group will be of from 0-30,
usually 0-10, more usually of from about 0-6 atoms in the
chain, where the chain will include carbon and any of the
heteroatoms indicated above. For the most part, the
linking group will be straight chain or cyclic, since
there will normally be no benefit from side groups. The
length of the linking group will vary, particularly with
the nature of the target binding member and the first
binding entity, since in some instances, the target
binding member or the first binding entity may naturally
have a chain or functionality associated with it. In some
instances, amino acids, generally from 1-3 amino acids
may serve as the linking chain, particularly where the
carboxyl group of the amino acid may be the reactive
functionality. Thus, the amino group may serve to bond to
the target binding member or first binding entity.
The length of the arms may be used to provide for
flexibility, rigidity, polyfunctionality, orientation, or
other characteristic for improved function of the
molecule. The covalent linking group may be a
functionality which has an unequal affinity for different
blood proteins, or which has a high affinity for a given
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protein epitope or sequence, such as an IgG or albumin
epitope.
The first binding entity will generally be a small
molecule, where the molecule is likely to minimize any
5 immune response. Any physiologically acceptable molecule
may be employed, where there is a convenient reciprocal
binding member. Thus, of particular interest is biotin,
where avidin may be the reciprocal binding member, but
other molecules such as metal chelates, molecules
10 mimicking a natural epitope or receptor or antibody
binding site, also may find use, where the reciprocal
binding member may be an antibody or a fragment thereof,
particularly a Fab fragment, an enzyme, a naturally
occurring receptor, or the like. Thus, the first binding
15 entity may be a ligand for a naturally occurring receptor,
a substrate for an enzyme, or a hapten with a reciprocal
receptor.
The manner of producing the first compound will vary
widely, depending upon the nature of the various elements
20 comprising the first compound. The synthetic procedures
will be selected so as to be simple, provide for high
yields, and allow for a highly purified product.
Normally, the reactive functionality will be created as
the last stage, for example, with a carboxyl group,
esterification to form an active ester will be the last
step of the synthesis, unless one wishes to deprotect some
functionality of the target binding member or the first
binding entity as the last step.
Usually, the first compound when it comprises the
first binding entity will have a molecular weight of at
least about 200 D and not more than about 2.5 kD, usually
not more than about 1.5 kD and frequently less than about
1 kD.
Illustrative compounds include N-hydroxysuccinimidyl
biotin ester, N-hydroxysulfosuccinimidyl biotin ester, N-
hydroxysulfosuccinimidyl ester of N-biotinyl 6-
aminohexanoic acid, N-hydroxysulfosuccinimidyl ester of N-
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biotinyl 4-butyryl, 3-aminopropyl disulfide, and the like.
A large number of water soluble biotin derivatives for
functionalizing proteins are available and to the extent
that such compounds have linkers which are physiologically
acceptable, these compounds may find application in this
invention.
The first compound will usually be administered as a
bolus, but may be introduced slowly over time by infusion
using metered flow, or the like. Alternatively, although
less preferable, blood may be removed from the host,
treated ex vivo, and supplied to the host. The first
compound will be administered in a physiologically
acceptable medium, e.g. deionized water, phosphate
buffered saline, saline, mannitol, aqueous glucose,
alcohol, vegetable oil, or the like. Usually a single
injection will be employed although more than one
injection may be used, if desired. The first compound may
be administered by any convenient means, including
syringe, trocar, catheter, or the like. The particular
manner of administration, will vary depending upon the
amount to be administered, whether a single bolus or
continuous administration, or the like. For the most part
the administration will be intravascularly, where the site
of introduction is not critical to this invention,
preferably at a site where there is rapid blood flow,
e.g. intravenously, peripheral or central vein. Other
routes may find use where the administration is coupled
with slow release techniques or a protective matrix. The
intent is that the first compound be effectively
distributed in the blood, so as to be able to react with
the blood components.
For the most part, reaction will be with mobile
components in the blood, particularly blood proteins and
cells, more particularly blood proteins and red cells. By
"mobile" is intended that the component does not have a
fixed situs for any extended period of time, generally not
exceeding 5, more usually one minute. For the most part,
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reaction will be with plasma proteins, such as the
immunoglobulins, particularly IgM and IgG, albumin,
ferritin, and to a lesser degree other proteins which are
present in substantially reduced amount. There niay also be
reaction with platelets, endothelial cells and white blood
cells. There will, therefore,.initially be a relatively
heterogeneous population of functionalized proteins and
cells. However, for the most part, the population within
a few days will vary substantially from the initial
population, depending upon the half-life of the
functionalized proteins in the blood stream. Therefore,
usually within about three days or more, IgG will become
the predominant functionalized protein in the blood
stream. This means that after a few days, the target
binding member will be conjugated to, or the second
compound will, for the most part, become conjugated with
and bound to IgG.
In many situations, one may use a single first
compound comprising the first binding entity, once such
compound has been thoroughly tested in hosts, particularly
human hosts, since its physiology will be well
established, its pharmacokinetics will be established, and
its safety over an extended period of time may be also
established. In some instances the first compound will be
physiologically and/or therapeutically active, where it
may find use independent of addition of the second
compound. However, to the extent that there may be
idiosyncratic individuals, or that chronic administration
of the first compound may result in some immune reaction,
it may be desirable to have more than one first compound
to be used for administration. However, since the role of
the first compound may be somewhat restricted when used in
combination with a second compound, it is not necessary
that one develop numerous alternatives, although there
will be numerous alternatives which will be useful for the
same purpose.
For the most part, the half-life of the first
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compound will be at least about five days, more usually at
least about 10 days and preferably 20 days or more. The
period for providing an effective concentration may be
much longer, since one may introduce a substantial excess
of the first compound so that even after two or three
half-lives, there may still be, a useful amount of the
first compound in the blood stream.
Generally, it will be satisfactory to have the target
binding member as part of the first compound. However,
there will be situations where it will be desirable to use
the combination of the first and second compounds. For
example, if one wishes to have a relatively homogeneous
population of components carrying the target binding
member, one can wait till the short lived blood components
have dissipated, before adding the second compound.
Particularly, where the initial concentration of the first
compound may be high, it may be desireable to add the
second compound subsequently which will primarily bind to
the first compound, rather than react with a variety of
blood components. In some instances the synthesis of the
first compound with the target binding member may be
difficult, due to the highly functionalized nature of the
agent of interest. This could be particularly true with
oligopeptides which are synthesized and require removal of
protecting groups at the end of the synthesis. Also,
where the target binding member is to be released from the
blood component, it may be easier to design a combination
of first and second compounds which allow for ef f icient
release, rather than depending on the linking group of the
first compound.
The dosage of the first compound will depend upon
whether it comprises the target binding member and will
therefore be dependent on the adverse effects of the
target binding member, its activity when bound to blood
components, the time necessary to reduce the free
concentration of the first compound containing the target
binding member, the dosage necessary for therapeutic
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activity, the indication being treated, the sensitivity of
the agent to .blood enzymes, the route and mode of
administration, and the like. As necessary, the dosage
may be determined empirically, initially using a small
multiple of the therapeutic dosage normally administered,
and as greater experience is. obtained, enhancing the
dosage. Where the target binding member is used to elicit
an immune response, the target binding member will be an
antigen, and will not rely on the conjugation to the blood
component for enhancing the immune response. In this
case, relatively large dosages may be employed, where one
is interested in producing antibodies as a product, where
the treated host will normally be a domestic or laboratory
animal. Where the target binding member is acting as a
vaccine, the dosage may be smaller and may be determined
empirically. For the most part, as a vaccine the dosage
will generally be in the range of about 10 ng to 100 g.
Normally, any additional dose will be administered after
the original dose has been diminished by at least 50%,
usually at least 90%, of the original dose.
As compared to the first compound, in some ways, the
second compound may be much more sophisticated. This
compound will have a second binding entity, which will be
determined by the nature of the first binding entity. As
already indicated, this entity may take numerous forms,
particularly as binding proteins, such as immunoglobulins
and fragments thereof, particularly Fab, Fc, or the like,
particularly monovalent fragments, naturally occurring
receptors, such as surface membrane proteins, enzymes,
lectins, other binding proteins, such as avidin or
streptavidin, or the like.
Particularly where the first binding entity is used
with a second compound, the first binding entity will be
naturally found at low concentration, if at all, in the
host blood stream, so there will be little if any
competition between the first binding entity and naturally
occurring compounds in the blood for the second binding
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entity. The second binding entity should not bind to
compounds which it may encounter in the blood or
associated cells. Therefore, enzymes which are used will
usually be active on substrates which do not appear in the
5 blood. Lectins which are used will usually bind to sugars
which do not appear in the blood and are not present on
endothelial or other cells which line the vascular system.
For the most part, the second binding entity will be
a protein, although other molecules which can provide for
10 relatively high specificity and affinity may also be
employed. Combinatorial libraries afford compounds other
than proteins which will have the necessary binding
characteristics. Generally, the affinity will be at least
about 104, more usually about 104 M, e.g. binding
15 affinities normally observed with specific monoclonal
antibodies. Of particular interest is avidin or
streptavidin, although other receptors of particular
interest include receptors for steroids, TSH, LH, FSH, or
their agonists, as well as sialic acid and viral
20 hemagglutinins, and superantigens. The second binding
entity will usually be a macromolecule, generally of at
least about 5 kD, more usually of at least about 10 kD and
usually less than about 160 kD, preferably less than about
80 kD, which may be mono- or divalent in binding sites,
25 usually monovalent.
One or more agents of interest may be present in the
first or second compound, usually multiple agents of
interest being bound through first or second linking
groups to a central core, e.g. a protein, nucleic acid,
polysaccharide, or other multifunctional entity. Not only
does a linking group serve to covalently bond the target
binding member, but it also serves to determine whether
the target binding member remains bound to the blood
component or if released, the manner and rate of release.
Therefore, the nature of the linking group will vary
widely depending upon its role.
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Where the target binding member is to be retained
bound to the blood component, any of a wide variety of
convenient linking groups, including a bond, may be
employed. Thus, the same types of linking groups employed
in the first compound will find acceptance for the second
compound. However, where the linking group is to be
cleaved and release the target binding member, the linking
group will vary depending upon the nature of the target
binding member, the desired rate of release, the valency
or the functionality on the target binding member which is
to be released, and the like. Thus, various groups may be
employed, where the environment of the blood, components
of the blood, particularly enzymes, activity in the liver,
or other agent may result in the cleavage of the linking
group with release of the target binding member at a
reasonable rate.
Functionalities which may find use include esters,
either organic or inorganic acids, particularly carboxyl
groups or phosphate groups, disulfides, peptide or
nucleotide linkages, particularly peptide or nucleotide
linkages which are susceptible to trypsin, thrombin,
nucleases, esterases, etc., acetals, ethers, particularly
saccharidic ethers, or the like. Generally, the linking.
group for cleavage will require at least two atoms in the
chain, e.g. disulfide, and may require 50 atoms, usually
not more than about 30 atoms, preferably not more than
about 20 atoms in the chain. Thus, the chain may comprise
an oligopeptide, oligosaccharide, oligonucleotide,
disulfide, organic divalent groups which are aliphatic,
aromatic, alicyclic, heterocyclic or combinations thereof,
involving esters, amides, ethers, amines, or the like.
The particular linking group will be selected in
accordance with physiological acceptance, desired rate of cleavage, synthetic
convenience, and the like.
The conjugate may be prepared in any convenient way,
depending on the nature of the binding member components
of the conjugate, the need to maintain the binding
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capability of the binding members and such other
considerations as are relevant to the use of the conjugate
in vivo. Depending on the nature of the conjugate, the
coupling ratio between the two binding members of the
conjugate may vary from one to having a plurality of one
or both of the binding members, there usually being on the
average not more than 6, more usually not more than three
of either of the binding members in the conjugate.
Generally the anchor will be one, but may be more than one
where enhanced affinity for the long-lived blood component
is desired.
As previously indicated, there can be three types of
association between the anchor and the target binding
member: (1) the two may be covalently linked by an
appropriate chain, which may take many different forms;
(2) the two may be linked by a combination of chains,
each chain terminating in a reciprocal complementary
member of a specific binding pair, so as to be joined non-
covalently; or (3) the two may be added successively, so
that the anchor is added to the long-lived blood component
initially, followed by the addition of the target binding
member, where each member has a chain terminating in a
reciprocal complementary member of a specific binding
pair, so as to be joined non-covalently.
The nature of the linkage between the binding members
of the conjugate will vary widely depending upon the
chemical composition of the binding members, the fate of
the target binding member, the nature of the target, and
the like. Generally, stable linkages will be employed and
covalent linkages find general applicability. By "stable"
is intended that the linkage will not be preferentially
cleaved as compared to other bonds in the molecule;
"unstable" intends preferential cleavage. However,
occasionally one may wish to permit limited time-course
35- degradation of the linkage. For example, in some cases
one may wish to have controlled release of the complex of
the conjugate and the target, where the affinities of the
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binding members of the conjugate are very high. By
providing for cleavage or degradation of the conjugate the
target will be released in accordance with the rate of
cleavage or degradation of the conjugate, rather than the
off rate of the target from the complex. A wide variety
of linkages will be unstable under physiological
conditions, where the instability may be as a result of
enzymatic or non-enzymatic reactions. For example, ester
linkages can be provided which are relatively labile and
will be cleaved over time in the blood stream. By
providing for varying degrees of steric hindrance,
different degrees of lability can be achieved.
Alternatively, sequences can be provided, which are
recognized by a wide variety of enzymes such as proteases.
For example, a series of arginine-lysine dimeric units
will be sensitive to trypsin. Other protease labile
linkages may be employed, where the protease may be found
in the bloodstream. Alternatively, one may employ
disulfide linkages, which will be subject to reductive
cleavage. Other functionalities which may find use
include oligosaccharides, thiol esters, nucleic acids, or
the like. Alternatively, one may rely upon the nature of
one or both of binding members to provide for release of
the target from the long-lived blood component, e.g.
peptide nature of antibodies subject to proteases.
There are extensive reports in the literature of
procedures for covalently joining a wide variety of
compounds to prepare conjugates with different functional
groups. Various synthetic schemes may be envisioned,
again depending upon the nature of the conjugate binding
members. Thus, where sulfhydryl groups are present, these
may be readily activated to provide for linkage to a
sulfhydryl group to provide a disulfide or with a
maleimide group, where a thiol may bind to the maleimide
to provide for a thioether. Carboxyl groups may be
readily activated with a wide variety of hydroxyl
compounds or carbodiimide, where the ester or anhydride is
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allowed to react with hydroxyl or amino groups to provide
esters or amides. Analogously, saccharide moites can be
activated with periodate. Other lirikages may also find
application, such as imines, hydrazines, etc.
Alternatively, non-covalent linkages may be used, such as
biotin-avidin linkages, double antibody linkages, lectin-
saccharide linkages, and the like. Where the conjugate
binding members are both proteins, such as antibodies or
fragments, the conjugate may be genetically engineered as
a single construct, cloned and expressed using a variety
of vectors and host cells. Alternatively, the two
conjugate binding members can be recombinantly or
chemically synthesized and subsequently coupled,
particularly by affording unique functionalities available
for coupling. Chemical synthesis may employ commercially
available synthesizers using liquid or solid phase
synthesis, where the synthesis may be all or a part of the
conjugate. The subject compositions may be used for
the treatment, prophylactic or therapeutic, of a wide
variety of cellular diseases, toxicities and environmental
exposures and may be administered in a wide variety of
ways, depending on the indication. If desired, the
conjugate may be first bound to the long-lived blood
component in appropriate proportion followed by
administration. Alternatively, the conjugate may be
administered to the host for binding to the long-lived
blood component cells or proteins. The amount of the
conjugate which is administered will vary widely,
depending upon the nature of the conjugate, the purpose
for the conjugate, the therapeutic dosage, the
physiological activity of the compound when present as a
conjugate, and when bound to a cell, and the like.
Therefore, the amount of conjugate administered to a host
may vary from 1 g to 50 mg/kg of host.
The subject compositions will, for the most part, be
administered parenterally, such as intravascularly (IV),
intraarterially, intramuscularly (IM), subcutaneously
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(SC), or the like. Administration will normally be by
transfusion if the conjugate is bound to cells. If the
conjugate is unbound, administration will normally be IV,
IM or SC. Where the compositions are of low molecular
5 weight (less than about 10 kD) or resistant to digestive
enzymes, conjugate administration may be oral, nasal,
rectal, transdermal or aerosol, where the nature of the
conjugate allows for transfer to the vascular system.
Physiologically acceptable carriers will usually be
10 employed, such as water, saline, phosphate buffered
saline, aqueous ethanol, plasma, proteinaceous solutions,
glucose or mannitol solutions, or the like. The
concentration of the conjugate will vary widely, generally
ranging from about 1 pg/ml to 50 mg/ml. Other additives
15 which may be included include buffers, where the media are
generally buffered at a pH in the range of about 5 to 10,
where the buffer will generally range in concentration
from about 50 to 250 mM, salt, where the concentration of
salt will generally range from about 5 to 500 mM,
20 physiologically acceptable stabilizers, and the like. The
compositions may be lyophilized for convenient storage and
transport.
The choice of the long-lived blood component will
affect the manner in which the biological activity of the
25 target is modified. Depending on the nature of the target
different long-lived blood components will be employed.
Where the target is a molecule, such as a small organic
molecule or peptide, serum proteins will be satisfactory
long-lived blood components, and elimination of the target
30 will accompany elimination of the serum protein. The
target must be substantially inactivated while bound to
the conjugate, particularly where the target has cytotoxic
effects. Where the target is a virus particle or cell,
usually the long-lived blood component will be a cell, the
choice of cell depending upon whether one wishes to have
the target cell rapidly eliminated or maintained in the
host segregated to particular cellular compartments. For
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example, if one wishes to eliminate virus infected cells
or neoplastic cells, the long-lived blood component would
be erythrocytes or platelets, so that the target cell
would become bound to the erythrocytes or platelets to
prevent the target cell to enter small vessels and the
complex recognized by the spleen for elimination. With
erythrocytes, the deformation of the erythrocytes on
binding to the target cell aids in the recognition of the
complex for elimination. A similar mechanism is operative
for platelets.
The subject invention may be used in chronic or acute
situations, either prophylactic or therapeutic. For acute
situations, the conjugate will normally be administered in
anticipation of the acute situation. For example with
drug addicts, one may wish to prevent the use of a
particular drug of abuse, particularly where the patient
is on a methadone program. By administering the subject
conjugate, where the conjugate may exert an effective
binding capability of weeks or months, taking of the drug
of abuse would result in its inactivation, inhibiting the
euphoric effect. Similarly, where one is concerned about
nosocomial infection, one would administer the conjugate
in anticipation of the infection, so that upon infection,
the toxin or tumor necrosis factor would become bound to
diminish its effective concentration below a pathological
level.
For therapeutic use, one could administer the
conjugate to a cancer patient, so that the blood would be
monitored for metastatic cells or growth factor excess.
in this case, one would use an long-lived blood component
which provides for rapid elimination, such as
erythrocytes. Where cells are not normally found in the
vascular system, such as mammary cells, the conjugate
would have as the target protein, a mammary cell surface
protein, so that any mammary cells in the vascular system
could be detected and eliminated. In the case of T cells
involved with autoimmune disease, where such cells have a
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common epitope, the conjugate would supervise the blood
for such cells and be able to bind to such cells and
eliminate the cells by the mechanisms indicated above.
The subject invention can be used in anticipation of
one or more events which may have deleterious
physiological effects, e.g. drug ingestion or adverse
physiological response of a host to trauma or surgery, or
for a chronic situation where the continued production of
toxins, virus particles or the like, as a result of an
intractable infection, is continuously monitored by the
conjugate bound to a long lived blood component. The
subject invention provides a long-lived source of binding
capability, where upon binding of a target, the
deleterious biological effects of the target are rapidly
diminished and the target may be slowly or rapidly
eliminated from the host by a variety of natural
mechanisms associated with degradation, chemical
modification, elimination of modified or defective
erythrocytes and platelets.
Because of the extended delivery time or availability
of the subject agents, the subject invention may be used
in a wide variety of situations. The target binding
member may be an antibody to a toxin, where there is
concern about nosocomial infection in a hospital or other
situation where disease may be spread. The subject
invention may be employed before surgery so as to ensure
that a level of drug is maintained during and subsequent
to the surgery without requiring repetitive
administration, avoiding the disturbance of the patient.
For example, one may use anticlotting agents, where the
nature of the surgery and indication is susceptible to the
formation of clots. One may use inhibitors of leukocyte
homing to prevent perfusion injury. One may use the
subject invention with cardiovascular drugs, where a
patient is particularly susceptible during an extended
period to myocardial infarction. Other treatments which
will benefit from long term availability of drugs include
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hormonotherapy, infertility therapy, immunosuppressive
therapy, neuroleptic therapy, drug of abuse prophylaxy,
treatment of diseases caused. by infectious agents,
treatment of hemophilia, and the like.
By conjugating a biologically active target binding
member to IgG in the blood of a mammalian host, many
advantages ensue, in providing for a new activity for the
immunoglobulins, while retaining many of the desirable
features of the immunoglobulins, in addition to the
extended life-time. For example, the immunoglobulins may
still have F. effector function, such as its role in
complement fixation, or the action of antibody dependent
cytotoxic cells, effect on inactivation and secretion, and
the like. Similarly, serum albumin and other long lived
serum proteins can act to inactivate target entities and
aid in their rapid elimination. Thus, the blood
components to which the target binding member is bound
impart their physiological activities to the activity of
the target binding member. In this way cellular targets
may be inactivated or eliminated by having immunoglobulins
directed to a cellular or soluble target or by coating the
cellular or soluble target with blood proteins.
The following examples are offered by way of
illustration and not by way of limitation.
EXPERIMENTAL
xample 1: Modification of Cocaine Volume of Distribution
A monoclonal antibody raised against a cocaine-BSA
conjugate (molar ratio, 10:1) using BALB/c mice and SP20
myeloma is screened by solid phase microtiter plate ELIS:-
against Cocaine-BSA and irrelevant peptide-BSA as control
antigen. Selected antibodies specificity is verified by
= competition assay against cocaine-BSA in which binding of
antibodies is inhibited in a dose dependent manner by free
cocaine. Selected antibody is IgGl, with a supernatant
titer by ELISA of 1:64. Mab is produced in ascites fluid
and purified by protein A affinity chromatography.
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Anti-RBC Mab is an IgGl Mab specific for human
glycophorin A and reacting with blood group antigen M
(obtained from Hospital Saint-Louis, Paris). The Mab
cross-reacts with chimpanzee and Macaccus muliata
erythrocytes. Fab fragments (produced by papain digestion monitored
by SDS-PAGE) from both anti-RBC and anti-cocaine Mabs are
conjugated using the Nakane method, in which the first
member of the conjugate is activated by sodium periodate,
before addition of the second member of the conjugate,
resulting in a carbohydrate-NH2 residue covalent coupling.
pH is monitored during coupling reaction and ratios of
both members are optimized to provide a mean molar ratio
close to 1:1. Conjugate is purified by gel filtration.
The pics corresponding to molecular weight between 80 KD
and 120 KD (determined by SDS PAGE) are selected.
Reactivity of conjugate with cocaine is verified by ELISA
as above, using an anti-mouse kappa chain specific
peroxidase conjugate, and as control an anti-mouse Fc
specific peroxidase conjugate (which does not react with
cocaine bound Fab). Reactivity of conjugate with human
RBC is tested by flow cytometry using anti-mouse Fab FITC
conjugate and as control an anti-mouse Fc specific FITC
conjugate (which does not react with cocaine bound Fab).
The conjugate is shown not to agglutinate RBCs and not to
induce hemolysis (between 100 ng/mL and 10 g/mL) when
incubated in human and monkey plasma with addition of
fresh rabbit complement.
The biodistribution of '25I labelled cocaine is
evaluated with and without pretreatment of animals with
anti-RBC, cocaine specific conjugate. Animals (n=3)
receive IV by slow injection (5 minutes) 50 g/Kg of
conjugate diluted in glucose 5% with 1% of human serum albumin. One day after
the injection, animals receive 10
curie of 125I-labelled cocaine IV (100 g per animal). 1
hour after the cocaine injection, animals are sacrificed,
and various blood and tissue specimens are collected,
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including brain specimens (frontal and occipital cortex).
Control animals (n=3) receive only 125I labelled
cocaine and no conjugate. Specific radioactivity is
measured using a gamma counter, and expressed in cpm per
5 ml of blood or cpm per gram of tissue. Animals which
receive the conjugate pre-treatment have an average brain
1251 specific activity of 2.2% (+/- 0.7%) the average brain
1211 specific activity of control animals, and a mean whole
blood specific activity of 25 times (+/- 4) that of
10 control animals (with a ratio of erythrocyte to plasma 1251
specific activity of 20:1 in the conjugate treated group,
and 0.2 in the control group).
The results demonstrate that pre-treatment with an
erythrocyte bound to a conjugate specific for cocaine
15 modifies cocaine -volume of distribution, decreases
diffusion of cocaine into the central nervous system, and
decreases the ratio of plasma to erythrocyte cocaine
concentration (with a net increase in whole blood
concentration).
20 Examole 2: Anti-T cell Subset Conjuaate
A rat anti-mouse RBC specific Fab fragment is
conjugated to purified recombinant HIV1 gp120 (Molar ratio
1:2). Following IV administration to SCID Hu mice (10
per mouse) of the RBC/gp 120 long-lived blood component,
25 whole blood specimens are collected serially in the
following 48 hours, and analyzed by fluorescent microscopy
and flow cytometry. In the first 6 hours, rosettes of
erythrocytes and CD3+, CD4+, CD8- T cells are observed in
the animals pretreated with the Anchor, and are absent in
30 control animals.
Example 3: Anti-Viral Protein Anchor
A rat anti-mouse RBC specific Fab fragment is
conjugated to purified recombinant CD4 (Molar ratio 1:3).
48 hours following IV administration to BALB/c mice (10 g
35 per mouse) of the RBC/CD4 conjugate, 125I labelled
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recombinant HIV1 gp120 is injected IV to conjugate
pretreated or control animals. Half-life (whole blood,
erythrocyte and plasma) concentration of gp120 is measured
in the following 24 hours and 8 days. The asma half-
life of gp120 is dramatically decreased in treated
animals, whereas the erythrocyte and whole blood half
lifes are increased.
Example 4: Two-stacre Addition of Lifetime Extended
Immunoaen
I. Two rabbits were injected with a solution of N-
succinimidyl biotin ester (NHS-biotin)(5 or 50 mg in 200
l or 1 ml of DMSO) i.v. on day 0. Blood samples were
taken at initially hourly, followed by daily intervals and
the samples analyzed for the presence of biotin by gel
electrophoresis (SDS-PAGE) employing 60 g of sample,
detecting the proteins to which biotin is bound by avidin-
peroxidase conjugate, using a luminescent substrate (ECL
kit, Amersham). For biotin bound to red blood cells, the
cells were isolated by centrifugation, washed, lysed by
hypotonic lysis, and the proteins separated by SDS-PAGE in
the same manner as the plasma proteins. For the plasma
proteins, the electrophoresis was run under reducing and
non-reducing conditions, where the IgM and IgG were
reduced to sub-units under the reducing conditions.
The gels for the plasma proteins of one of the
rabbits showed that biotin was bound to IgM, IgG, p90, p75
(transferrin), serum albumin, and p38. The duration for
serum albumin for detection in the gel electrophoresis
under reducing conditions was 9 to 12 days for the serum
albumin, and up to 33 days for IgM and IgG, while for the
non-reducing conditions, where the time for exposure was
substantially less, bands could be observed for the serum
albumin for 2 days and for the IgG for 9 days. With the
red blood cells, a number of proteins could be observed
for 12 days, the next point being 33 days, at which time
no bands could be observed. The major labled component
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had an electrophoretic pattern suggesting Band 3.
II. To demonstrate that the subject invention can be used
to enhance the immune response to a heterologous protein,
after adminstration of the biotin conjugate as described
above, 250 g or 1 mg of egg white avidin was injected
intravenously into each of two rabbits. Within about 2
days the titer began to rise rapidly and by 10 days a high
anti-avidin titer was observed by ELISA, using avidin
coated to microplates, as evidenced by an OD value of
about 600. By comparison, when avidin was administered as
described above to rabbits to which the biotin conjugate
had not been previously administered, there was
substantially no production of antibodies until an
intravenous booster injection of 50 g of avidin was made
on day 6, at which time there was a rapid increase in
anti-avidin titer, approximating the titer observed with
the biotin conjugate modified rabbits by day 12.
Example 5. Binding of Biotin-NHS to RBCs and Plasma
Proteins.
5 mg (-1.5 mg/kg) and 50 mg (- 15 mg/kg) of NHS-
biotin solubilized in DMSO were injected into rabbits 3,
or A and 8, respectively. Blood samples were then taken
0.5, 1, 2 and 4 h on the same day after injection and then
on 1, 2, 3, 6, 9, 13, 20, 27, 34, 41, 48 and 55 days after
injection.
minutes after injection, all RBCs were
biotinylated as shown by flow cytometry performed with
phycoerythrin-conjugated avidin. The mean fluorescence
was much lower for rabbit 3 than for rabbit 8 (26 and 320,
30 respectively), showing a direct relationship between the
labeling of RBCs and the dose of NHS-biotin. Half-lives
of 17 or 15 days were calculated from the curves obtained
with rabbit 3 or 8, respectively. A life span of 55 days
was obtained with rabbit 3. After 49 days, 6 % of
biotinylated RBCs survived for rabbit 8. As measured by
conventional methods, the life span of rabbit RBCs is 45
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to 70 days.
The plasma proteins of rabbits 3 and 8 were analysed
by immunoblotting. The plasma proteins were separated
under reducing conditions (10 mM DTT) in a 10%
polyacrylamide gel (Coomassie blue staining). Major
components were identiifed as p180, p90, p75, albumin, and
the heavy and light chains of immunoglobulins. All plasma
proteins revealed with Ponceau red were able to capture
avidin-phycoerythrin, showing that they are biotinylated.
For rabbit 3, only serum albumin could be detected on day
20. The staining was much more intense with samples from
rabbit8, showing biotinylation of high molecular weight
components (- 200 kDa) and the light chains of
immunoglobulins. No staining was observed day 19.
The pattern of plasma proteins separated under non-
reducing conditions in a 8% polyacrylamide gel (Coomassie
blue staining) showed IgM, IgG, p90, p75, and serum
albumin as the major components. On immunoblots, albumin
(60 kDa), transferrin, p90, IgG (160 kDa) and high
molecular weight components corresponding in part to IgM
were detected. The half-life of high molecular weight
components and IgG appeared longer than that of albumin,
since the latter could not be visualized from samples
taken day 12 from rabbit S.
Example 6. Production of Anti-avidin with Avidin Bound to
Biotinvlated Plasma Components
A rabbit was injected on day 0 with 50mg NHS-biotin
in 1 ml DMSO followed 30 min later with an injection of
peroxidase labeled avidin (1 ml/250 g). Seven days later
the rabbit was injected with 'uI-avidin (1 x lo'cpm: 50 g).
Blood samples were taken periodically thereafter for the
presence of 125I and the presence of anti-avidin antisera.
On day 53, 50 mg of NHS-biotin in 600 l DMSO was injected
followed 30 min later with 1 mg/500 1 of avidin and blood
samples taken periodically thereafter to measure the anti-
avidin level. Anti-avidin appeared as soon as day 2, with
the level maximizing around day 12 and decreasing slowly
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thereafter. The level of antibodies was not significant
after 33 days. After boosting with NHS-biotin and avidin,
high titered antibodies were obtained. Based on OD value
as determined using avidin coated ELISA plates, on day 70,
the titer was about 8-fold greater than on day 12 (606 vs.
4790).
In a control rabbit that did not receive NHS-biotin,
no significant level of antibodies was observed within 7
days after injection of 500 g of peroxidase labeled
avidin. However, a further injection of 125I-avidin (15 g)
did result in an immuogenic response 5 days later.
The disclosed invention provides for extending the
lifetime of an agent in the blood strream of a mammalian
host for a wide variety of purposes. The disclosed
invention provides an improved method for limiting the
pathogenic or toxic effects of agents present in the
mammalian blood stream. In addition the subject invention
allows for continual presentation of antigens, providing
molecules which can act as surveillants, or maintaining a
therapeutic modality for an extended period of time to
avoid repetitive administsration of a physiologically
active agent.
The method finds application where surveillance is
desired for the presence of physiologically detrimental
agents in the blood stream. By providing for a conjugate
which binds specifically to such agents and is bound to a
long-lived blood associated component which enhances the
life of the conjugate in the blood, the conjugate can act
to bind and inactivate the agent, and by appropriate
choice of the long-lived blood associated component, the
agent can be slowly or rapidly eliminated from the host,
using the normal physiological mechanisms for such
elimination.
All publications and patent applications mentioned in
' 35 this specification are indicative of the level of skill of
those skilled in the art to which this invention pertains.
All publications and patent applications are herein
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incorporated by reference to the same extent as if each
individual publication or patent application was
specifically and individually indicated to be incorporated
by reference.
5 The invention now being fully described, it will be
apparent to one of ordinary skill in the art that many
changes and modifications can be made thereto without
departing from the spirit or scope of the appended claims.
