U.S. patent application number 12/219100 was filed with the patent office on 2008-11-06 for method for enhancing immune responses in mammals.
Invention is credited to Mark Douglas Howell, Leland Charles Leber, Cheryl Lynn Selinsky.
Application Number | 20080275376 12/219100 |
Document ID | / |
Family ID | 23763686 |
Filed Date | 2008-11-06 |
United States Patent
Application |
20080275376 |
Kind Code |
A1 |
Howell; Mark Douglas ; et
al. |
November 6, 2008 |
Method for enhancing immune responses in mammals
Abstract
The present invention provides a method for enhancing an immune
response in a mammal to facilitate the elimination of a chronic
pathology. The method involves the removal of immune system
inhibitors from the circulation of the mammal, thus, enabling a
more vigorous immune response to the pathogenic agent. The removal
of immune system inhibitors is accomplished by contacting
biological fluids of a mammal with one or more binding partner(s)
capable of binding to and, thus, depleting the targeted immune
system inhibitor(s) from the biological fluids. Particularly useful
in the invention is an absorbent matrix composed of an inert,
biocompatible substrate joined covalently to a binding partner,
such as an antibody, capable of specifically binding to the
targeted immune system inhibitor.
Inventors: |
Howell; Mark Douglas; (Ft.
Collins, CO) ; Selinsky; Cheryl Lynn; (Ft. Collins,
CO) ; Leber; Leland Charles; (Ft. Collins,
CO) |
Correspondence
Address: |
Brad Pedersen;Patterson, Thuente, Skaar & Christensen, PA
4800 IDS Center, 80 South 8th Street
Minneapolis
MN
55402-2100
US
|
Family ID: |
23763686 |
Appl. No.: |
12/219100 |
Filed: |
July 16, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10071829 |
Feb 7, 2002 |
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12219100 |
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09444144 |
Nov 20, 1999 |
6379708 |
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10071829 |
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Current U.S.
Class: |
604/5.04 |
Current CPC
Class: |
A61P 37/04 20180101;
A61M 1/3486 20140204; Y10T 442/2525 20150401; A61K 35/14 20130101;
A61K 2039/505 20130101; C07K 16/2878 20130101 |
Class at
Publication: |
604/5.04 |
International
Class: |
A61M 37/00 20060101
A61M037/00 |
Claims
1-49. (canceled)
50. An extracorporeal system for reducing the amount of a targeted
immune system inhibitor in blood, comprising: a) an absorbent
matrix comprising an inert medium attached to at least one binding
partner capable of specifically binding to a targeted immune system
inhibitor; and b) a conduit for conducting the blood to the
absorbent matrix to produce altered blood having a reduced amount
of the targeted immune system inhibitor, wherein all of said
binding partners in said extracorporeal system are selected from
the group consisting of binding partners to: soluble receptors for
tumor necrosis factor .alpha. and .beta., interleukin-1 receptor
antagonist, soluble receptors for interleukin-1, and soluble
receptors for interleukin-6.
51. The extracorporeal system of claim 50, wherein the targeted
immune system inhibitor is present in a plasma component of the
blood or fraction thereof.
52. The extracorporeal system of claim 50, wherein the inert medium
is selected from the group consisting of: a hollow fiber, a
macroporous bead, a cellulose-based fiber, a synthetic fiber, a
flat membrane, a pleated membrane, and a silica-based particle.
53. The extracorporeal system of claim 50, wherein the binding
partner is covalently joined to the inert medium.
54. The extracorporeal system of claim 50, wherein the binding
partner is a binding partner to which the targeted immune system
inhibitor binds to in nature, or a fragment of the binding partner
to which the targeted immune system inhibitor binds to in nature,
wherein the fragment specifically binds to the targeted immune
system inhibitor.
55. The extracorporeal system of claim 50, wherein the binding
partner or fragment is produced recombinantly.
56. The extracorporeal system of claim 50, wherein the binding
partner is a monoclonal antibody or a fragment of a monoclonal
antibody that specifically binds to the targeted immune system
inhibitor.
57. The extracorporeal system of claim 50, wherein the binding
partner comprises a plurality of different monoclonal antibodies or
fragments thereof, wherein the plurality of monoclonal antibodies
or fragments thereof are capable of specifically binding to the
targeted immune system inhibitor.
58. The extracorporeal system of claim 50, wherein a plurality of
binding partners are collectively capable of specifically binding
to a plurality of targeted immune system inhibitors.
59. The extracorporeal system of claim 50, wherein the binding
partner comprises a polyclonal antibody preparation or fragments of
a polyclonal antibody preparation that specifically bind to the
targeted immune system inhibitor.
60. The extracorporeal system of claim 50, wherein the binding
partner comprises a plurality of different polyclonal antibody
preparations or fragments thereof, wherein the polyclonal
antibodies or fragments thereof are capable of specifically binding
to the targeted immune system inhibitor.
61. The extracorporeal system of claim 50, wherein the binding
partner comprises at least one synthetic peptide.
62. The extracorporeal system of claim 61, wherein the at least one
synthetic peptide is conjugated to a carrier.
63. The extracorporeal system of claim 50, wherein the binding
partner comprises a plurality of synthetic peptides capable of
specifically binding to a plurality of targeted immune system
inhibitors.
64. An extracorporeal system for reducing the amount of a targeted
immune system inhibitor in whole blood, comprising: a) a means for
separating whole blood into a cellular component and an acellular
component or fraction of the acellular component, wherein the
acellular component or the fraction of the acellular component
contains a targeted immune system inhibitor; b) a means for
providing a binding partner capable of specifically binding to the
targeted immune system inhibitor in (a); c) a means for conducting
the acellular component or fraction of the acellular component to
the means for providing the targeted immune system inhibitor to
produce an altered acellular component or fraction of the acellular
component; d) a means for conducting the altered acellular
component or fraction of the acellular component from the absorbent
matrix to the cellular component to produce an altered whole bloods
wherein all of said binding partners in said extracorporeal system
are selected from the group consisting of binding partners to:
soluble receptors for tumor necrosis factor .alpha. and .beta.,
interleukin-1 receptor antagonist, soluble receptors for
interleukin-1, and soluble receptors for interleukin-6.
65. An extracorporeal system for reducing the amount of a targeted
immune system inhibitor in whole blood, comprising: a) a means for
separating whole blood into a cellular component and an acellular
component or fraction of the acellular component, wherein the
acellular component or the fraction of the acellular component
contains a targeted immune system; b) a means for providing a
binding partner capable of specifically binding to the targeted
immune system inhibitor in (a); c) a means for conducting the
acellular component or fraction of the acellular component to the
means for providing the targeted immune system inhibitor to produce
an altered acellular component or fraction of the acellular
component; d) a means for conducting the altered acellular
component or fraction of the acellular component from the absorbent
matrix to the cellular component to produce an altered whole blood,
wherein the means for providing a binding partner comprises an
absorbent matrix comprising an inert medium attached to a binding
partner capable of specifically binding to the targeted immune
system inhibitor, wherein all of said binding partners in said
extracorporeal system are selected from the group consisting of
binding partners to: soluble receptors for tumor necrosis factor
.alpha. and .beta., interleukin-1 receptor antagonist, soluble
receptors for interleukin-1, and soluble receptors for
interleukin-6.
66. The extracorporeal system of claim 50, further comprising an
apparatus for separating whole blood into a cellular component and
an acellular component or a fraction of the acellular
component.
67. The extracorporeal system of claim 66, wherein the acellular
component or the fraction of the acellular component contains the
targeted immune system inhibitor.
68. The extracorporeal system of claim 67, wherein the conduit
conducts the acellular component or fraction of the acellular
component to the absorbent matrix to produce an altered acellular
component or altered fraction of the acellular component having a
reduced amount of the targeted immune system inhibitor.
69. The extracorporeal system of claim 68, further comprising a
conduit for conducting the altered acellular component or fraction
of the acellular component from the absorbent matrix to the
cellular component to produce an altered whole blood.
Description
[0001] This invention relates generally to the field of
immunotherapy and, more specifically, to methods for enhancing host
immune responses.
BACKGROUND OF THE INVENTION
[0002] The immune system of mammals has evolved to protect the host
against the growth and proliferation of potentially deleterious
agents. These agents include infectious microorganisms such as
bacteria, viruses, fungi, and parasites which exist in the
environment and which, upon introduction to the body of the host,
can induce varied pathological conditions. Other pathological
conditions may derive from agents not acquired from the
environment, but rather which arise spontaneously within the body
of the host. The best examples are the numerous malignancies known
to occur in mammals. Ideally, the presence of these deleterious
agents in a host triggers the mobilization of the immune system to
effect the destruction of the agent and, thus, restore the sanctity
of the host environment.
[0003] The destruction of pathogenic agents by the immune system
involves a variety of effector mechanisms which can be grouped
generally into two categories: innate and specific immunity. The
first line of defense is mediated by the mechanisms of innate
immunity. Innate immunity does not discriminate among the myriad
agents that might gain entry into the host's body. Rather, it
responds in a generalized manner that employs the inflammatory
response, phagocytes, and plasma-borne components such as
complement and interferons. In contrast, specific immunity does
discriminate among pathogenic agents. Specific immunity is mediated
by B and T lymphocytes and it serves, in large part, to amplify and
focus the effector mechanisms of innate immunity.
[0004] The elaboration of an effective immune response requires
contributions from both innate and specific immune mechanisms. The
function of each of these arms of the immune system individually,
as well as their interaction with each other, is carefully
coordinated, both in a temporal/spatial manner and in terms of the
particular cell types that participate. This coordination results
from the actions of a number of soluble immunostimulatory mediators
or "immune system stimulators" (Reviewed in, Trinchieri, et al., J.
Cell. Biochem. 53:301-308 (1993)). Certain of these immune system
stimulators initiate and perpetuate the inflammatory response and
the attendant systemic sequelae. Examples of these include, but are
not limited to, the proinflammatory mediators tumor necrosis
factors .alpha. and .beta., interleukin-1, interleukin-6,
interleukin-8, interferon-.gamma., and the chemokines RANTES,
macrophage inflammatory proteins 1-.alpha. and 1-.beta., and
macrophage chemotactic and activating factor. Other immune system
stimulators facilitate interactions between B and T lymphocytes of
specific immunity. Examples of these include, but are not limited
to, interleukin-2, interleukin-4, interleukin-5, interleukin-6, and
interferon-.gamma.. Still other immune system stimulators mediate
bidirectional communication between specific immunity and innate
immunity. Examples of these include, but are not limited to,
interferon-.gamma., interleukin-1, tumor necrosis factors .alpha.
and .beta., and interleukin-12. All of these immune system
stimulators exert their effects by binding to specific receptors on
the surface of host cells, resulting in the delivery of
intracellular signals that alter the function of the target cell.
Cooperatively, these mediators stimulate the activation and
proliferation of immune cells, recruit them to particular
anatomical sites, and permit their collaboration in the elimination
of the offending agent. The immune response induced in any
individual is determined by the particular complement of immune
system stimulators produced, and by the relative abundance of
each.
[0005] In contrast to the immune system stimulators described
above, the immune system has evolved other soluble mediators that
serve to inhibit immune responses (Reviewed in, Arend, W. P., Adv.
Int. Med. 40:365-394 (1995)). These "immune system inhibitors"
provide the immune system with the ability to dampen responses in
order to prevent the establishment of a chronic inflammatory state
with the potential to damage the host's tissues. Regulation of host
immune function by immune system inhibitors is accomplished through
a variety of mechanisms as described below.
[0006] First, certain immune system inhibitors bind directly to
immune system stimulators and, thus, prevent them from binding to
plasma membrane receptors on host cells. Examples of these types of
immune system inhibitors include, but are not limited to, the
soluble receptors for tumor necrosis factors .alpha. and .beta.,
interferon-.gamma., interleukin-1, interleukin-2, interleukin-4,
interleukin-6, and interleukin-7.
[0007] Second, certain immune system inhibitors antagonize the
binding of immune system stimulators to their receptors. By way of
example, interleukin-1 receptor antagonist is known to bind to the
interleukin-1 membrane receptor. It does not deliver activation
signals to the target cell but, by virtue of occupying the
interleukin-1 membrane receptor, blocks the effects of
interleukin-1.
[0008] Third, particular immune system inhibitors exert their
effects by binding to receptors on host cells and signalling a
decrease in their production of immune system stimulators. Examples
include, but are not limited to, interferon-.beta., which decreases
the production of two key proinflammatory mediators, tumor necrosis
factor-.alpha. and interleukin-1 (Coclet-Ninin et al., Eur.
Cytokine Network 8:345-349 (1997)), and interleukin-10, which
suppresses the development of cell-mediated immune responses by
inhibiting the production of the immune system stimulator,
interleukin-12 (D'Andrea, et al., J. Exp. Med. 178:1041-1048
(1993)). In addition to decreasing the production of immune system
stimulators, certain immune system inhibitors also enhance the
production of other immune system inhibitors. By way of example,
interferon-.alpha..sub.2b inhibits interleukin-1 and tumor necrosis
factor-a production and increases the production of the
corresponding immune system inhibitors, interleukin-1 receptor
antagonist and soluble receptors for tumor necrosis factors .alpha.
and .beta. (Dinarello, C. A., Sem. in Oncol. 24(3 Suppl. 9):81-93
(1997).
[0009] Fourth, certain immune system inhibitors act directly on
immune cells, inhibiting their proliferation and function, thereby,
decreasing the vigor of the immune response. By way of example,
transforming growth factor-.beta. inhibits a variety of immune
cells, and significantly limits inflammation and cell-mediated
immune responses (Reviewed in, Letterio and Roberts, Ann. Rev.
Immunol. 16:137-161 (1998)). Collectively, these various
immunosuppressive mechanisms are intended to regulate the immune
response, both quantitatively and qualitatively, to minimize the
potential for collateral damage to the host's own tissues.
[0010] In addition to the inhibitors produced by the host's immune
system for self-regulation, other immune system inhibitors are
produced by infectious microorganisms. For example, many viruses
produce molecules which are viral homologues of host immune system
inhibitors (Reviewed in, Spriggs, M. K., Ann. Rev. Immunol.
14:101-130 (1996)). These include homologues of host complement
inhibitors, interleukin-10, and soluble receptors for
interleukin-1, tumor necrosis factors .alpha. and .beta., and
interferons .alpha., .beta. and .gamma.. Similarly, helminthic
parasites produce homologues of host immune system inhibitors
(Reviewed in, Riffkin, et al., Immunol. Cell Biol. 74:564-574
(1996)), and several bacterial genera are known to produce
immunosuppressive products (Reviewed in, Reimann, et al., Scand. J.
Immunol. 31:543-546 (1990)). All of these immune system inhibitors
serve to suppress the immune response during the initial stages of
infection, to provide advantage to the microbe, and to enhance the
virulence and chronicity of the infection.
[0011] A role for host-derived immune system inhibitors in chronic
disease also has been established. In the majority of cases, this
reflects a polarized T cell response during the initial infection,
wherein the production of immunosuppressive mediators (i.e.,
interleukin-4, interleukin-10, and/or transforming growth
factor-.beta. dominates over the production of immunostimulatory
mediators (i.e., interleukin-2, interferon-.gamma., and/or tumor
necrosis factor-.beta.) (Reviewed in, Lucey, et al., Clin. Micro.
Rev. 9:532-562 (1996)). Over-production of immunosuppressive
mediators of this type has been shown to produce chronic,
non-healing pathologies in a number of medically important
diseases. These include, but are not limited to, diseases resulting
from infection with: 1) the parasites, Plasmodium falciparum
(Sarthou, et al. Infect. Immun. 65:3271-3276 (1997)), Trypanosoma
cruzi (Reviewed in, Laucella, et al. Revista Argentina de
Microbiologia 28:99-109 (1996)), Leishmania major (Reviewed in,
Etges and Muller, J. Mol. Med. 76:372-390 (1998)), and certain
helminths (Riffkin, et al., supra); 2) the intracellular bacteria,
Mycobacterium tuberculosis (Baliko, et al., FEMS Immunol. Med.
Micro. 22:199-204 (1998)), Mycobacterium avium (Bermudez and
Champsi, Infect. Immun. 61:3093-3097 (1993)), Mycobacterium leprae
(Sieling, et al. J. Immunol. 150:5501-5510 (1993)), Mycobacterium
bovis (Kaufmann, et al., Ciba Fdn. Svmp. 195:123-132 (1995)),
Brucella abortus (Fernandes and Baldwin, Infect. Immun.
63:1130-1133 (1995)), and Listeria monocytogenes (Blauer, et al.,
J. Interferon Cytokine Res. 15:105-114 (1995)); and, 3) the
intracellular fungus, Candida albicans (Reviewed in, Romani, et
al., Immunol. Res. 14:148-162 (1995)). The inability to
spontaneously resolve infection is influenced by other host-derived
immune system inhibitors as well. By way of example, interleukin-1
receptor antagonist and the soluble receptors for tumor necrosis
factors .alpha. and .beta. are produced in response to
interleukin-1 and tumor necrosis factor .alpha. and/or .beta.
production driven by the presence of numerous infectious agents.
Examples include, but are not limited to, infections by Plasmodium
falciparum (Jakobsen, et al. Infect. Immun. 66:1654-1659 (1998),
Sarthou, et al., supra), Mycobacterium tuberculosis
(Balcewicz-Sablinska, et al., J. Immunol. 161:2636-2641 (1998)),
and Mycobacterium avium (Eriks and Emerson, Infect. Immun.
65:2100-2106 (1997)). In cases where the production of any of the
aforementioned immune system inhibitors, either individually or in
combination, dampens or otherwise alters immune responsiveness
before the elimination of the pathogenic agent, a chronic infection
may result.
[0012] In addition this role in infectious disease, host-derived
immune system inhibitors contribute also to chronic malignant
disease. Compelling evidence is provided by studies of soluble
tumor necrosis factor receptor type I (sTNFRI) in cancer patients.
Nanomolar concentrations of sTNFRI are synthesized by a variety of
activated immune cells in cancer patients and, in many cases, by
the tumors themselves (Aderka et al., Cancer Res. 51: 5602-5607
(1991); Adolf and Apfler, J. Immunol. Meth. 143: 127-36
(1991)).
[0013] In addition, circulating sTNFRI levels often are elevated
significantly in cancer patients (Aderka, et al., supra; Kalmanti,
et al., Int. J. Hematol. 57: 147-152 (1993); Elsasser-Beile, et
al., Tumor Biol. 15: 17-24 (1994); Gadducci, et al., Anticancer
Res. 16: 3125-3128 (1996); Digel, et al., J. Clin. Invest. 89:
1690-1693 (1992)), decline during remission and increase during
advanced stages of tumor development (Aderka, et al., supra;
Kalmanti, et al., supra; Elsasser-Beile, et al., supra; Gadducci,
et al., supra) and, when present at high levels, correlate with
poorer treatment outcomes (Aderka, et al., supra). These
observations suggest that sTNFRI aids tumor survival by inhibiting
anti-tumor immune mechanisms which employ tumor necrosis factors
.alpha. and/or .beta. (TNF), and they argue favorably for the
clinical manipulation of sTNFRI levels as a therapeutic strategy
for cancer.
[0014] Direct evidence that the removal of immune system inhibitors
provides clinical benefit derives from the evaluation of
Ultrapheresis, a promising experimental cancer therapy (Lentz, M.
R., J. Biol. Response Modif. 8: 511-27 (1989); Lentz, M. R., Ther.
Apheresis 3: 40-49 (1999); Lentz, M. R., Jpn. J. Apheresis 16:
107-14 (1997)). Ultrapheresis involves extracorporeal fractionation
of plasma components by ultrafiltration. Ultrapheresis selectively
removes plasma components within a defined molecular size range,
and it has been shown to provide significant clinical advantage to
patients presenting with a variety of tumor types. Ultrapheresis
induces pronounced inflammation at tumor sites, often in less than
one hour post-initiation. This rapidity suggests a role for
preformed chemical and/or cellular mediators in the elaboration of
this inflammatory response, and it reflects the removal of
naturally occurring plasma inhibitors of that response. Indeed,
immune system inhibitors of TNF .alpha. and .beta., interleukin-1,
and interleukin-6 are removed by Ultrapheresis (Lentz, M. R., Ther.
Apheresis 3: 40-49 (1999)). Notably, the removal of sTNFRI has been
correlated with the observed clinical responses (Lentz, M. R.,
Ther. Apheresis 3: 40-49 (1999); Lentz, M. R., Jpn. J. Apheresis
16: 107-14 (1997)).
[0015] Ultrapheresis is in direct contrast to more traditional
approaches which have endeavored to boost immunity through the
addition of immune system stimulators. Pre-eminent among these has
been the infusion of supraphysiological levels of TNF (Sidhu and
Bollon, Pharmacol. Ther. 57: 79-128 (1993)), and of interleukin-2
(Maas, et al., Cancer Immunol. Immunother. 36: 141-148 (1993)),
which indirectly stimulates the production of TNF. These therapies
have enjoyed limited success (Sidhu and Bollon, supra; Maas, et
al., supra) due to the fact: 1) that at the levels employed they
proved extremely toxic; and, 2) that each increases the plasma
levels of the immune system inhibitor, sTNFRI (Lantz, et al.,
Cytokine 2: 402-406 (1990); Miles, et al., Brit. J. Cancer 66:
1195-1199 (1992)). Together, these observations support the utility
of Ultrapheresis as a biotherapeutic approach to cancer--one which
involves the removal of immune system inhibitors, rather than the
addition of immune system stimulators.
[0016] Although Ultrapheresis provides advantages over traditional
therapeutic approaches, there are certain drawbacks that limit its
clinical usefulness. Not only are immune system inhibitors removed
by Ultrapheresis, but other plasma components, including beneficial
ones, are removed since the discrimination between removed and
retained plasma components is based solely on molecular size. An
additional drawback to Ultrapheresis is the significant loss of
circulatory volume during treatment, which must be offset by the
infusion of replacement fluid. The most effective replacement fluid
is an ultrafiltrate produced, in an identical manner, from the
plasma of non-tumor bearing donors. A typical treatment regimen (15
treatments, each with the removal of approximately 7 liters of
ultrafiltrate) requires over 200 liters of donor plasma for the
production of replacement fluid. The chronic shortage of donor
plasma, combined with the risks of infection by human
immunodeficiency virus, hepatitis A, B, and C or other etiologic
agents, represents a severe impediment to the widespread
implementation of Ultrapheresis.
[0017] Because of the beneficial effects associated with the
removal of immune system inhibitors, there exists a need for
methods which can be used to specifically deplete those inhibitors
from circulation. Such methods ideally should be specific and not
remove other circulatory components, and they should not result in
any significant loss of circulatory volume. The present invention
satisfies these needs and provides related advantages as well.
SUMMARY OF THE INVENTION
[0018] The present invention provides a method for stimulating
immune responses in a mammal through the depletion of immune system
inhibitors present in the circulation of said mammal. The depletion
of immune system inhibitors can be effected by removing biological
fluids from said mammal and contacting these biological fluids with
a binding partner capable of selectively binding to the targeted
immune system inhibitor.
[0019] Binding partners useful in these methods can be antibodies,
both polyclonal or monoclonal antibodies, or fractions thereof,
having specificity for a targeted immune system inhibitor.
Additionally, binding partners to which the immune system inhibitor
naturally binds may be used. Synthetic peptides created to attach
specifically to targeted immune system inhibitors also are useful
as binding partners in the present methods. Moreover, mixtures of
binding partners having specificity for multiple immune system
inhibitors may be used.
[0020] In a particularly useful embodiment, the binding partner is
immobilized previously on a solid support to create an "absorbent
matrix" (FIG. 1). The exposure of biological fluids to such an
absorbent matrix will permit binding by the immune system
inhibitor, thus, effecting a decrease in its abundance in the
biological fluids. The treated biological fluid can be returned to
the patient. The total volume of biological fluid to be treated and
the treatment rate are parameters individualized for each patient,
guided by the induction of vigorous immune responses while
minimizing toxicity. The solid support (i.e., inert medium) can be
composed of any material useful for such purpose, including, for
example, hollow fibers, cellulose-based fibers, synthetic fibers,
flat or pleated membranes, silica-based particles, or macroporous
beads.
[0021] In another embodiment, the binding partner can be mixed with
the biological fluid in a "stirred reactor" (FIG. 2). The binding
partner-immune system inhibitor complex then can be removed by
mechanical or by chemical or biological means, and the altered
biological fluid can be returned to the patient.
[0022] The present invention also provides apparatus incorporating
either the absorbent matrix or the stirred reactor.
BRIEF DESCRIPTION OF THE FIGURES
[0023] FIG. 1 schematically illustrates the "absorbent matrix"
configuration described herein. In this example, blood is removed
from the patient and separated into a cellular and an acellular
component, or fractions thereof. The acellular component, or
fractions thereof, is exposed to the absorbent matrix to effect the
binding and, thus, depletion of the targeted immune system
inhibitor. The altered acellular component, or fractions thereof,
then is returned contemporaneously to the patient.
[0024] FIG. 2 schematically illustrates the "stirred reactor"
configuration described herein. In this example, blood is removed
from the patient and separated into a cellular and an acellular
component, or fractions thereof. A binding partner is added to the
acellular component, or fractions thereof. Subsequently, the
binding partner/immune system inhibitor complex is removed by
mechanical or by chemical or biological means from the acellular
component, or fractions thereof, and the altered biological fluid
is returned contemporaneously to the patient.
[0025] FIG. 3 shows the depletion of sTNFRI from human plasma by
absorbent matrices constructed with monoclonal and polyclonal
anti-sTNFRI antibody preparations, and with a monoclonal antibody
of irrelevant specificity.
DETAILED DESCRIPTION OF THE INVENTION
[0026] The present invention provides novel methods to reduce the
levels of immune system inhibitors in the circulation of a host
mammal, thereby, potentiating an immune response capable of
resolving a pathological condition. By enhancing the magnitude of
the host's immune response, the invention avoids the problems
associated with the repeated administration of chemotherapeutic
agents which often have undesirable side effects (e.g.,
chemotherapeutic agents used in treating cancer).
[0027] The methods of the present invention generally are
accomplished by: (a) obtaining a biological fluid from a mammal
having a pathological condition; (b) contacting the biological
fluid with a binding partner capable of selectively binding to a
targeted immune system inhibitor to produce an altered biological
fluid having a reduced amount of the targeted immune system
inhibitor; and, thereafter (c) administering the altered biological
fluid to the mammal.
[0028] As used herein, the term "immune system stimulator" refers
to soluble mediators that increase the magnitude of an immune
response, or which encourage the development of particular immune
mechanisms that are more effective in resolving a specific
pathological condition.
[0029] As used herein, the term "immune system inhibitor" refers to
a soluble mediator that decreases the magnitude of an immune
response, or which discourages the development of particular immune
mechanisms that are more effective in resolving a specific
pathological condition, or which encourages the development of
particular immune mechanisms that are less effective in resolving a
specific pathological condition. Examples of host-derived immune
system inhibitors include interleukin-1 receptor antagonist,
transforming growth factor-.beta., interleukin-4, interleukin-10,
or the soluble receptors for interleukin-1, interleukin-2,
interleukin-4, interleukin-6, interleukin-7, interferon-.gamma. and
tumor necrosis factors .alpha. and .beta.. Immune system inhibitors
produced by microorganisms are also potential targets including,
for example, complement inhibitors, and homologues of
interleukin-10, soluble receptors for interleukin-1, interferons
.alpha., .beta., and .gamma., and tumor necrosis factors .alpha.
and .beta.. As used herein, the term "targeted" immune system
inhibitor refers to that inhibitor, or collection of inhibitors,
which is to be removed from the biological fluid by the present
method.
[0030] As used herein, the term "mammal" can be a human or a
non-human animal, such as dog, cat, horse, cattle, pig, or sheep
for example. The term "patient" is used synonymously with the term
"mammal" in describing the invention.
[0031] As used herein, the term "pathological condition" refers to
any condition where the persistence, within a host, of an agent,
immunologically distinct from the host, is a component of or
contributes to a disease state. Examples of such pathological
conditions include, but are not limited to those resulting from
persistent viral, bacterial, parasitic, and fungal infections, and
cancer. Among individuals exhibiting such chronic diseases, those
in whom the levels of immune system inhibitors are elevated are
particularly suitable for the treatment of the invention. Plasma
levels of immune system inhibitors can be determined using methods
well-known in the art (See, for example, Adolf and Apfler, supra).
Those skilled in the art readily can determine pathological
conditions that would benefit from the depletion of immune system
inhibitors according to the present methods.
[0032] As it relates to the present invention, the term "biological
fluid" refers to the acellular component of the circulatory system
including plasma, serum, lymphatic fluid, or fractions thereof. The
biological fluids can be removed from the mammal by any means known
to those skilled in the art, including, for example, conventional
apheresis methods (See, Apheresis: Principles and Practice, McLeod,
B. C., Price, T. H., and Drew, M. J., eds., AABB Press, Bethesda,
Md. (1997)). The amount of biological fluid to be extracted from a
mammal at a given time will depend on a number of factors,
including the age and weight of the host mammal and the volume
required to achieve therapeutic benefit. As an initial guideline,
one plasma volume (approximately 5-7 liters in an adult human) can
be removed and, thereafter, depleted of the targeted immune system
inhibitor according to the present methods.
[0033] As used herein, the term "selectively binds" means that a
molecule binds to one type of target molecule, but not
substantially to other types of molecules. The term "specifically
binds" is used interchangeably herein with "selectively binds".
[0034] As used herein, the term "binding partner" is intended to
include any molecule chosen for its ability to selectively bind to
the targeted immune system inhibitor. The binding partner can be
one which naturally binds the targeted immune system inhibitor. For
example, tumor necrosis factor .alpha. or .beta. can be used as a
binding partner for sTNFRI. Alternatively, other binding partners,
chosen for their ability to selectively bind to the targeted immune
system inhibitor, can be used. These include fragments of the
natural binding partner, polyclonal or monoclonal antibody
preparations or fragments thereof, or synthetic peptides.
[0035] The present invention further relates to the use of various
mixtures of binding partners. One mixture can be composed of
multiple binding partners that selectively bind to different
binding sites on a single targeted immune system inhibitor. Another
mixture can be composed of multiple binding partners, each of which
selectively binds to a single site on different targeted immune
system inhibitors. Alternatively, the mixture can be composed of
multiple binding partners that selectively bind to different
binding sites on different targeted immune system inhibitors. The
mixtures referred to above may include mixtures of antibodies or
fractions thereof, mixtures of natural binding partners, mixtures
of synthetic peptides, or mixtures of any combinations thereof.
[0036] For certain embodiments in which it would be desirable to
increase the molecular weight of the binding partner/immune system
inhibitor complex, the binding partner can be conjugated to a
carrier. Examples of such carriers include, but are not limited to,
proteins, complex carbohydrates, and synthetic polymers such as
polyethylene glycol.
[0037] Additionally, binding partners can be constructed as
multifunctional antibodies according to methods known in the art.
For example, bifunctional antibodies having two functionally active
binding sites per molecule or trifunctional antibodies having three
functionally active binding sites per molecule can be made by known
methods. As used herein, "functionally active binding sites" refer
to sites that are capable of binding to one or more targeted immune
system inhibitors. By way of illustration, a bifunctional antibody
can be produced that has functionally active binding sites, each of
which selectively binds to different targeted immune system
inhibitors.
[0038] Methods for producing the various binding partners useful in
the present invention are well known to those skilled in the art.
Such methods include, for example, serologic, hybridoma,
recombinant DNA, and synthetic techniques, or a combination
thereof.
[0039] In one embodiment of the present methods, the binding
partner is attached to an inert medium to form an absorbent matrix
(FIG. 1). As used herein, the term "inert medium" is intended to
include solid supports to which the binding partner(s) can be
attached. Particularly useful supports are materials that are used
for such purposes including, for example, cellulose-based hollow
fibers, synthetic hollow fibers, silica-based particles, flat or
pleated membranes, and macroporous beads. Such inert media can be
obtained commercially or can be readily made by those skilled in
the art. The binding partner can be attached to the inert medium by
any means known to those skilled in the art including, for example,
covalent conjugation. Alternatively, the binding partner may be
associated with the inert matrix through high-affinity,
non-covalent interaction with an additional molecule which has been
covalently attached to the inert medium. For example, a
biotinylated binding partner may interact with avidin or
streptavidin previously conjugated to the inert medium.
[0040] The absorbent matrix thus produced can be contacted with a
biological fluid, or a fraction thereof, through the use of an
extracorporeal circuit. The development and use of extracorporeal,
absorbent matrices has been extensively reviewed. (See, Kessler,
L., Blood Purification 11:150-157 (1993)).
[0041] In another embodiment, herein referred to as the "stirred
reactor" (FIG. 2), the biological fluid is exposed to the binding
partner in a mixing chamber and, thereafter, the binding
partner/immune system inhibitor complex is removed by means known
to those skilled in the art, including, for example, by mechanical
or by chemical or biological separation methods. For example, a
mechanical separation method can be used in cases where the binding
partner, and therefore the binding partner/immune system inhibitor
complex, represent the largest components of the treated biological
fluid. In these cases, filtration can be used to retain the binding
partner and immune system inhibitors associated therewith, while
allowing all other components of the biological fluid to permeate
through the filter and, thus, to be returned to the patient. In an
example of a chemical or biological separation method, the binding
partner and immune system inhibitors associated therewith, can be
removed from the treated biological fluid through exposure to an
absorbent matrix capable of specifically attaching to the binding
partner. For example, a matrix constructed with antibodies reactive
with mouse immunoglobulins (e.g., goat anti-mouse IgG) would serve
this purpose in cases where the binding partner were a mouse
monoclonal IgG. Similarly, were biotin conjugated to the binding
partner prior to its addition to the biological fluid, a matrix
constructed with avidin or streptavidin could be used to deplete
the binding partner and immune system inhibitors associated
therewith from the treated fluid.
[0042] In the final step of the present methods, the treated or
altered biological fluid, having a reduced amount of targeted
immune system inhibitor, is returned to the patient receiving
treatment along with untreated fractions of the biological fluid,
if any such fractions were produced during the treatment. The
altered biological fluid can be administered to the mammal by any
means known to those skilled in the art, including, for example, by
infusion directly into the circulatory system. The altered
biological fluid can be administered immediately after contact with
the binding partner in a contemporaneous, extracorporeal circuit.
In this circuit, the biological fluid is (a) collected, (b)
separated into cellular and acellular components, if desired, (c)
exposed to the binding partner, and if needed, separated from the
binding partner bound to the targeted immune system inhibitor, (d)
combined with the cellular component, if needed, and (e)
readministered to the patient as altered biological fluid.
Alternatively, the administration of the altered biological fluid
can be delayed under appropriate storage conditions readily
determined by those skilled in the art.
[0043] It may be desirable to repeat the entire process. Those
skilled in the art can readily determine the benefits of repeated
treatment by monitoring the clinical status of the patient, and
correlating that status with the concentration(s) of the targeted
immune system inhibitor(s) in circulation prior to, during, and
after treatment.
[0044] The present invention further provides novel apparatus for
reducing the amount of a targeted immune system inhibitor in a
biological fluid. These apparatus are composed of: (a) a means for
separating the biological fluid into a cellular component and an
acellular component or fraction thereof; (b) an absorbent matrix or
a stirred reactor as described above to produce an altered
acellular component or fraction thereof; and (c) a means for
combining the cellular fraction with the altered acellular
component or fraction thereof. These apparatus are particularly
useful for whole blood as the biological fluid in which the
cellular component is separated either from whole plasma or a
fraction thereof.
[0045] The means for initially fractionating the biological fluid
into the cellular component and the acellular component, or a
fraction thereof, and for recombining the cellular component with
the acellular component, or fraction thereof, after treatment are
known to those skilled in the art. (See, Apheresis: Principles and
Practice, supra.)
[0046] In one specific embodiment, the immune system inhibitor to
be targeted is sTNFRI (Seckinger, et al., J. Biol. Chem. 264:
11966-73 (1989); Gatanaga, et al., Proc. Natl. Acad. Sci. 87:
8781-84 (1990)), a naturally occurring inhibitor of the pluripotent
immune system stimulator, TNF. sTNFRI is produced by proteolytic
cleavage which liberates the extracellular domain of the membrane
tumor necrosis factor receptor type I from its transmembrane and
intracellular domains (Schall, et al., Cell 61: 361-70 (1990);
Himmler, et al., DNA and Cell Biol. 9: 705-715 (1990)). sTNFRI
retains the ability to bind to TNF with high affinity and, thus, to
inhibit the binding of TNF to the membrane receptor on cell
surfaces.
[0047] The levels of sTNFRI in biological fluids are increased in a
variety of conditions which are characterized by an antecedent
increase in TNF. These include bacterial, viral, and parasitic
infections, and cancer as described above. In each of these disease
states, the presence of the offending agent stimulates TNF
production which stimulates a corresponding increase in sTNFRI
production. sTNFRI production is intended to reduce localized, as
well as systemic, toxicity associated with elevated TNF levels and
to restore immunologic homeostasis.
[0048] In tumor bearing hosts, over-production of sTNFRI may
profoundly affect the course of disease, considering the critical
role of TNF in a variety of anti-tumor immune responses (Reviewed
in, Beutler and Cerami, Ann. Rev. Immunol. 7:625-655 (1989)). TNF
directly induces tumor cell death by binding to the type I
membrane-associated TNF receptor. Moreover, the death of vascular
endothelial cells is induced by TNF binding, destroying the
circulatory network serving the tumor and further contributing to
tumor cell death. Critical roles for TNF in natural killer cell-
and cytotoxic T lymphocyte-mediated cytolysis also have been
documented. Inhibition of any or all of these effector mechanisms
by sTNFRI has the potential to dramatically enhance tumor
survival.
[0049] That sTNFRI promotes tumor survival, and that its removal
enhances anti-tumor immunity, has been demonstrated. In an
experimental mouse tumor model, sTNFRI production was found to
protect transformed cells in vitro from the cytotoxic effects of
TNF, and from cytolysis mediated by natural killer cells and
cytotoxic T lymphocytes (Selinsky, et al., Immunol. 94: 88-93
(1998)). In addition, the secretion of sTNFRI by transformed cells
has been shown to markedly enhance their tumorigenicity and
persistence in vivo (Selinsky and Howell, unpublished). Moreover,
removal of circulating sTNFRI has been found to provide clinical
benefit to cancer patients, as demonstrated by human trials of
Ultrapheresis as discussed above (Lentz, M. R., supra). These
observations affirm the importance of this molecule in tumor
survival, and suggest the development of methods for more specific
removal of sTNFRI as promising new avenues for cancer
immunotherapy.
[0050] The following examples are intended to illustrate but not
limit the invention.
EXAMPLE 1
Production, Purification, and Characterization of the Immune System
Inhibitor, Human sTNFRI
[0051] The sTNFRI used in the present studies was produced
recombinantly in cell culture. The construction of the eukaryotic
expression plasmid, the methods for transforming cultured cells,
and for assaying the production of sTNFRI by the transformed cells
have been described (Selinsky, et al., supra). The sTNFRI
expression plasmid was introduced into HeLa cells (American Type
Culture Collection #CCL 2), and an sTNFRI-producing transfectant
cell line was isolated by limiting dilution. This cloned cell line
was cultured in a fluidized-bed reactor at 37.degree. C. in
RPMI-1640, supplemented with 2.5% (v/v) fetal bovine serum and
penicillin/streptomycin, each at 100 micrograms per milliliter.
sTNFRI secreted into the culture medium was purified by affinity
chromatography on a TNF-Sepharose-4B affinity matrix essentially as
described (Engelmann, et al., J. Biol. Chem. 265:1531-1536).
[0052] sTNFRI was detected and quantified in the present studies by
capture ELISA (Selinsky, et al., supra). In addition, the
biological activity of recombinant sTNFRI, i.e., its ability to
bind TNF, was confirmed by ELISA. Assay plates were coated with
human TNF-A (Chemicon), blocked with bovine serum albumin, and
sTNFRI, purified from culture supernatants as described above, was
added. Bound sTNFRI was detected through the sequential addition of
biotinylated-goat anti-human sTNFRI, alkaline
phosphatase-conjugated streptavidin, and
p-nitrophenylphosphate.
EXAMPLE 2
Production of Absorbent Matrices
[0053] Binding partners used in the present studies include an IgG
fraction of goat anti-human sTNFRI antisera (R&D Systems, Cat.
#AF-425-PB) and a monoclonal antibody reactive with sTNFRI
(Biosource International, Cat. #AHR3912). An additional monoclonal
antibody, OT145 (Cat. #TCR1657), reactive with a human T cell
receptor protein, was purchased from T Cell Diagnostics (now,
Endogen) and was used as a control binding partner. Each of these
respective binding partners was covalently conjugated to cyanogen
bromide-activated Sepharose-4B (Pharmacia Biotech), a macroporous
bead which facilitates the covalent attachment of proteins.
Antibodies were conjugated at 1.0 milligram of protein per
milliliter of swollen gel, and the matrices were washed extensively
according to the manufacturer's specifications. Matrices were
equilibrated in phosphate buffered saline prior to use.
EXAMPLE 3
Depletion of the Immune System Inhibitor, sTNFRI, from Human Plasma
Using Absorbent Matrices
[0054] Normal human plasma was spiked with purified sTNFRI to a
final concentration of 10 nanograms per milliliter, a concentration
comparable to those found in the circulation of cancer patients
(Gadducci, et al., supra). One milliliter of the spiked plasma was
mixed with 0.25 milliliter of the respective absorbent matrices at
0.degree. C. and a plasma sample was removed at time=0. The samples
were warmed rapidly to 37.degree. C., and incubated with agitation
for an additional 45 minutes. Plasma samples were removed for
analysis at 15 minute intervals and, immediately after collection,
were separated from the beads by centrifugation. Samples were
analyzed by ELISA to quantify the levels of sTNFRI, and to permit
the determination of the extent of depletion.
[0055] FIG. 3 shows the results of the sTNFRI depletion. The
absorbent matrix produced with the goat anti-human sTNFRI
polyclonal antibody rapidly removed the sTNFRI from the plasma
sample; 90% of the sTNFRI was depleted within 15 minutes. The
residual 1 nanogram per milliliter of sTNFRI in these samples is
within the range of sTNFRI concentrations found in healthy
individuals (Aderka, et al., supra; Chouaib, et al., Immunol. Today
12:141-145 (1991)). The matrix produced with the monoclonal
anti-human sTNFRI antibody, in contrast, only removed approximately
one-fifth of the plasma sTNFRI. The differences in the ability of
these two matrices to deplete sTNFRI likely reflect the influence
of avidity which is enabled by the heterogeneity of epitope
specificities present in the polyclonal antibody preparation. The
control matrix produced no reduction in sTNFRI levels, confirming
the specificity of the depletion observed with the anti-sTNFRI
antibody matrices.
[0056] Although the invention has been described with reference to
the presently preferred embodiments, it should be understood that
various modifications can be made without departing from the spirit
of the invention. Accordingly, the invention is limited only by the
following claims.
* * * * *