U.S. patent application number 10/066021 was filed with the patent office on 2002-08-22 for methods for inducing the differentiation of monocytes into functional dendritic cells and immunotherapeutic compositions including such dendritic cells.
Invention is credited to Berger, Carole, Edelson, Richard Leslie, Hanlon, Douglas.
Application Number | 20020114793 10/066021 |
Document ID | / |
Family ID | 42357263 |
Filed Date | 2002-08-22 |
United States Patent
Application |
20020114793 |
Kind Code |
A1 |
Edelson, Richard Leslie ; et
al. |
August 22, 2002 |
Methods for inducing the differentiation of monocytes into
functional dendritic cells and immunotherapeutic compositions
including such dendritic cells
Abstract
A method for inducing differentiation of monocytes contained in
an extracorporeal quantity of a subject's blood into functional
dendritic antigen presenting cells is provided. The monocytes are
induced to differentiate into dendritic cells by activation forces
resulting from flow of the monocytes through a plastic channel,
such as the plastic channel in a conventional photopheresis
apparatus. Functional dendritic cells generated from induced
monocytes are incubated together with apoptotic or inactivated
disease effector agents to enhance the presentation of at least one
disease-causing antigen expressed by the disease effector agents.
Compositions including dendritic cells derived from induced
monocytes and compositions including such dendritic cells incubated
with disease effector agents are also provided for use in
immunotherapeutic treatment.
Inventors: |
Edelson, Richard Leslie;
(Westport, CT) ; Berger, Carole; (Bronx, NY)
; Hanlon, Douglas; (Branford, CT) |
Correspondence
Address: |
Cummings & Lockwood
Granite Square
700 State Street
P.O. Box 1960
New Haven
CT
06509-1960
US
|
Family ID: |
42357263 |
Appl. No.: |
10/066021 |
Filed: |
January 31, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10066021 |
Jan 31, 2002 |
|
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09294494 |
Apr 20, 1999 |
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Current U.S.
Class: |
424/93.21 ;
435/448; 514/185; 514/410; 514/455 |
Current CPC
Class: |
A61K 2039/5158 20130101;
A61K 41/0066 20130101; C12N 2501/22 20130101; A61P 35/00 20180101;
A61K 39/0011 20130101; A61P 37/04 20180101; Y10S 977/904 20130101;
A61K 2039/5154 20130101; A61P 43/00 20180101; C12N 2501/23
20130101; C12N 2501/25 20130101; C12N 5/0639 20130101; A61P 35/02
20180101; Y10S 977/924 20130101; C12N 2501/999 20130101 |
Class at
Publication: |
424/93.21 ;
514/185; 514/410; 514/455; 435/448 |
International
Class: |
A61K 048/00; A61K
031/555; A61K 031/37; A61K 031/409; C12N 015/01 |
Claims
We claim:
1. A method for producing functional antigen presenting dendritic
cells from an extracorporeal quantity of a subject's blood, said
method comprising the steps of: (a) treating the extracorporeal
quantity of blood with a photoactivatable agent capable of inducing
apoptosis in disease effector agents contained in the blood; (b)
flowing the the extracorporeal quantity of blood through a
photopheresis apparatus having plastic channels with a diameter of
about 1 mm or less; (c) irradiating the the extracorporeal quantity
of blood as it flows though the photopheresis apparatus; and (d)
incubating the the extracorporeal quantity of blood after treatment
in the photopheresis apparatus.
2. The method of claim 1, wherein prior to step (b) the method
further comprises the step of: separating the leukocytes and
monocytes from the the extracorporeal quantity of blood by
subjecting the the extracorporeal quantity of blood to a
leukapheresis process.
3. The method of claim 2, wherein the photoactivatable agent is a
psoralen.
4. The method of claim 3, wherein the photoactivatable agent is
8-MOP.
5. The method of claim 4, wherein the disease effector agents are
malignant T-cells.
6. The method of claim 1, wherein the disease effector agents are
cancer cells from solid tumors which are contained in the the
extracorporeal quantity of blood.
7. The method of claim 1, wherein incubation is conducted for a
period of from about 6 to about 48 hours.
8. The method of claim 7, wherein incubation is conducted for a
period of from about 12 to about 24 hours.
9. A method for producing functional antigen presenting dendritic
cells from an extracorporeal quantity of a subject's blood, said
method comprising the steps of: (a) inducing apoptosis of disease
effector agents contained in the the extracorporeal quantity of
blood; (b) flowing the the extracorporeal quantity of blood through
plastic channels having a diameter of between about 0.5 mm and
about 5 mm; and (c) incubating the the extracorporeal quantity of
blood following passage though the plastic channel.
10. The method of claim 9, wherein the step of flowing the the
extracorporeal quantity of blood through plastic channels is
performed in a photopheresis apparatus having channels with a
diameter of about 1 mm or less.
11. The method of claim 9, wherein the step inducing apoptosis of
disease effector agents contained in the extracorporeal quantity of
blood is comprised of the steps of: (d) adding a photoactivatable
agent to the the extracorporeal quantity of blood; and (e)
irradiating the the extracorporeal quantity of blood with
ultraviolet light.
12. The method of claim 11, wherein the photoactivatable agent is
8-MOP.
13. The method of claim 9, further comprising the step of treating
the the extracorporeal quantity of blood in a leukapheresis device
to prepare a white blood cell concentrate.
14. The method of claim 9, wherein incubation is conducted for a
period of from about 6 to about 48 hours.
15. The method of claim 14, wherein incubation is conducted for a
period of from about 12 to about 24 hours.
16. A method for producing functional antigen presenting dendritic
cells from an extracorporeal quantity of a subject's blood, said
method comprising the steps of: (a) coating disease effector agents
in the the extracorporeal quantity of blood with monoclonal
antibodies having a free Fc segment; (b) flowing the the
extracorporeal quantity of blood through plastic channels having a
diameter of from about 0.5 mm to about 5 mm; and (c) incubating the
the extracorporeal quantity of blood following passage though the
plastic channel.
17. The method of claim 16, wherein the disease effector agents are
solid tumor cancer cells which are contained in the extracorporeal
quantity of the subject's blood.
18. The method of claim 16, further comprising the step of inducing
apoptosis of the disease effector agents contained in the the
extracorporeal quantity of blood.
19. The method of claim 18, wherein the disease effector agents are
malignant T-cells.
20. The method of claim 16, wherein incubation is conducted for a
period of from about 6 to about 48 hours.
21. The method of claim 17, wherein incubation is conducted for a
period of from about 12 to about 24 hours.
22. A method for producing functional antigen presenting dendritic
cells from an extracorporeal quantity of a subject's blood, said
method comprising the steps of: (a) inducing apoptosis of disease
effector agents isolated from the subject; (b) flowing the the
extracorporeal quantity of blood through plastic channels having a
diameter of about 1 mm or less; (c) combining the apoptotic disease
effector agents with the extracorporeal quantity of blood; and (d)
incubating the combined apoptotic disease effector agents and
treated blood.
23. The method of claim 19, further comprising the step of coating
the apoptotic disease effector agents with monoclonal antibodies
having a free Fc segment.
24. The method of claim 19, wherein incubation is conducted for a
period of from about 6 to about 48 hours.
25. The method of claim 21, wherein incubation is conducted for a
period of from about 12 to about 24 hours.
Description
[0001] The present application is a continuation-in-part of patent
application Ser. No. 09/294,494 filed on Apr. 20, 1999.
FIELD OF THE INVENTION
[0002] The present invention relates to in vivo methods for
inducing the differentiation of monocytes into functional dendritic
antigen presenting cells and, more particularly, to extracorporeal
methods for treating and incubating monocytes to induce such
differentiation. The present invention further provides methods for
producing immunotherapeutic compositions including these dendritic
cells. In particular, the present invention provides
immunotherapeutic compositions comprising functional dendritic
cells derived from induced monocytes presenting at their surface
antigens from apoptotic or inactivated disease effector agents.
BACKGROUND OF THE INVENTION
[0003] The use of dendritic cells in cancer immunotherapy is
presently an area of significant clinical inquiry. Dendritic cells
are highly effective in presenting antigens to responding T-cells;
however, dendritic cells normally constitute less than one percent
of blood mononuclear leukocytes. Accordingly, a number of in vitro
methods have been developed to expand populations of dendritic
cells to augment anti-cancer immunity. By exposing increased
numbers of dendritic cells to antigens on tumor or other
disease-causing cells, followed by reintroduction of the
antigen-loaded dendritic cells to the patient, presentation of
these antigens to responding T-cells can be enhanced
significantly.
[0004] For example, culturing blood mononuclear leukocytes for
eight days in the presence of granulocyte-monocyte colony
stimulating factor (GM-CSF) and interleukin-4 (IL-4) produces large
numbers of dendritic cells. These cells can then be externally
loaded with tumor-derived peptide antigens for presentation to
T-cells. Alternatively, the dendritic cells can be transduced to
produce and present these antigens themselves. Expanding
populations of dendritic cells transduced to produce and secrete
cytokines which recruit and activate other mononuclear leukocytes,
including T-cells, may be an even more effective method of
generating anti-tumor immune responses.
[0005] Transducing cultivated dendritic cells to produce a
particular generic tumor antigen and/or additional cytokines is
labor intensive and expensive. More importantly, this procedure
likely fails to produce and present those multiple tumor antigens
that may be most relevant to the individual's own cancer. Several
approaches have been proposed to overcome this problem.
Hybridization of cultivated autologous dendritic cells with tumor
cells would produce tetraploid cells capable of processing and
presenting multiple unknown tumor antigens. In a second proposed
approach, acid elution of Class I and Class II major
histocompatability complexes (MHC) from the surface of malignant
cells would liberate a broad spectrum of tumor-derived peptides.
These liberated peptides could then be externally loaded onto MHC
complexes of autologous cultivated dendritic cells.
[0006] Conventional photopheresis is a method of vaccinating
patients against leukemic lymphocytes, even when the distinctive
tumor antigen(s) is not known. In this method, malignant cells are
exposed to photo-activated 8-methoxypsoralen (8-MOP) which enhances
cell surface display of Class I MHC-associated tumor antigens.
After intravenous return of these altered malignant lymphocytes to
the original patient, a potent anti-tumor response may be generated
in about 25% of the patients, leading to diminution of the
malignant cell population and occasionally long-standing
remissions. Experimental studies in mice, in which autologous
dendritic cells are first grown in tissue culture and then admixed
with the 8-MOP-treated tumor cells, appears to increase the
efficacy of conventional photopheresis. In this experimental
protocol, tumorigenic mouse T-cells are rendered apoptotic by
photopheresis using 8-MOP and exposure to ultraviolet (UV) energy.
Following this chemical alteration of the malignant leukeocytes,
autologous cultured dendritic cells are added to the apoptotic
T-cells, and the cell mix is incubated overnight with shaking to
maximize contact between the T-cells and the dendritic cells. The
apoptotic T-cell/dendritic cell mix has proven to be an effective
cellular vaccine in test mice challenged with viable tumorigenic
2B4.11 cells.
[0007] While the above-described experimental protocol is
apparently more efficient and comprehensive than alternative
approaches, it requires extensive ex vivo cellular manipulations
over a period of several days. Accordingly, an in vivo procedure
which could in a single day provide large numbers of functional
dendritic cells and expose those cells to apoptotic tumor cells
would greatly simplify the means by which the anti-tumor cellular
vaccine could be prepared.
SUMMARY OF THE INVENTION
[0008] The present invention is based on the convergence of two
disparate phenomena: treating monocytes in a manner which induces
their differentiation into functional dendritic antigen presenting
cells, and treating disease effector agents, such as tumor cells,
to render them apoptotic or to inactivate them. By incubating these
treated populations together for a period of time sufficient to
optimize processing and presentation by the dendritic cells of
disease associated antigens distinctive to the disease effector
agents, prior to returning the dendritic antigen presenting cells
to the patient, clinically enhanced immunity to the disease
associated antigens is achieved.
[0009] As used herein, the term "disease effector agents" refers to
agents that are central to the causation of a disease state in a
subject and which express disease-associated antigens. In certain
circumstances, these disease effector agents are disease-causing
cells which may be circulating in the bloodstream, thereby making
them readily accessible to extracorporeal manipulations and
treatments. Examples of such disease-causing cells include
malignant T-cells, malignant B cells, T-cells and B cells which
mediate an autoimmune response, and virally or bacterially infected
white blood cells which express on their surface viral or bacterial
peptides or proteins. Exemplary disease categories giving rise to
disease-causing cells include leukemia, lymphoma, autoimmune
disease, graft versus host disease, and tissue rejection. Disease
associated antigens which mediate these disease states and which
are derived from disease-causing cells include peptides that bind
to a MHC Class I site, a MHC Class II site, or to a heat shock
protein which is involved in transporting peptides to and from MHC
sites (i.e., a chaperone). Disease associated antigens also include
viral or bacterial peptides which are expressed on the surface of
infected white blood cells, usually in association with an MHC
Class I or Class II molecule.
[0010] Other disease-causing cells include those isolated from
surgically excised specimens from solid tumors, such as lung,
colon, brain, kidney or skin cancers. These cells can be
manipulated extracorporeally in analogous fashion to blood
leukocytes, after they are brought into suspension or propagated in
tissue culture. Alternatively, in some instances, it has been shown
that the circulating blood of patients with solid tumors can
contain malignant cells that have broken off from the tumors and
entered the circulation. [Kraeft, et al., Detection and analysis of
cancer cells in blood and bone marrow using a rare event imaging
system, Clinical Cancer Research, 6:434-42, 2000.] These
circulating tumor cells can provide an easily accessible source of
cancer cells which may be rendered apoptotic by the methods of the
present invention and presented to the dendritic cells formed by
the method described and claimed herein.
[0011] In addition to disease-causing cells, disease effector
agents falling within the scope of the invention further include
microbes such as bacteria, fungi and viruses which express
disease-associated antigens. It should be understood that viruses
can be engineered to be "incomplete", i.e., produce distinguishing
disease-causing antigens without being able to function as an
actual infectious agent, and that such "incomplete" viruses fall
within the meaning of the term "disease effector agents" as used
herein.
[0012] The present invention provides a method for treating an
extracorporeal quantity of a patient's blood to induce the
differentiation of monocytes contained in the blood into functional
antigen presenting dendritic cells. In a preferred embodiment of
the present invention, the extracorporeal quantity of the patient's
blood is treated using a conventional photopheresis apparatus to
induce differentiation of the monocytes into dendritic cells. While
not wishing to be limited to any particular mechanism, the
inventors believe that monocytes in the blood are attracted to and
stick to the plastic surfaces of the channels in the photopheresis
apparatus, and they are subsequently released from the plastic
surfaces by shearing forces from the flow of fluid through the
channel. Thus, as the monocytes pass through the photopheresis
apparatus, they undergo sequential adherence to and release from
the plastic surface. The physical forces of these events send
activation signals through the cell membrane and induce the
monocytes to differentiate into functional dendritic cells.
[0013] After treatment in the photopheresis device, the functional
dendritic cells are incubated in the presence of apoptotic disease
effector agents to allow the dendritic cells to phagocytize the
disease effector agents and present antigens from the disease
effector agents to T-cells in a subject's immune system. In a
particularly preferred embodiment of the present invention, as the
blood is passed through the photopheresis apparatus to induce
differentiation of monocytes into functional dendritic cells,
disease effector agents in the blood are rendered apoptotic by
treating the disease effector agents with a photoactivatable
substance and irradiating the blood as it passes through the
photopheresis apparatus. By rendering the disease effector agents
apoptotic as the monocytes are induced to form new dendritic cells,
the method of the present invention results in an enhanced number
of antigen presenting dendritic cells which can be reinfused into
the patient to trigger an immunotherapeutic response.
[0014] After the extracorporeal quantity of the patient's blood has
been treated in the photopheresis device, the composition is
incubated for a period of from about 6 to about 48 hours, most
preferably from about 12 to about 24 hours. During this period, the
dendritic cells phagocytize the apoptotic disease effector agents
and present antigens from the phagocytized cells at their surface,
where they will be recognized by T-cells in the patient's immune
system, thereby inducing an immunological response to the disease
effector agents in the patient.
[0015] In another embodiment of the present invention, which is
particularly effective in treating malignant T-cells contained in
the blood, the disease effector agents in the blood are rendered
apoptotic using monoclonal antibodies. The monoclonal antibodies
may include a free F.sub.c segment at the end of the antibody,
which can bond with a complementary receptor on the surface of the
dendritic cells. The antibodies thus form bridges between the
apoptotic disease cells and the dendritic cells, increasing the
likelihood that the apoptotic cells will be phagocytized and
processed by the dendritic cells. Alternatively, the disease
effector cells can be rendered apoptotic by other methods, and
coated with monoclonal antibodies with available F.sub.c receptors
to enhance uptake and processing of apoptotic disease effector
agents by the functional dendritic cells. Also, non-apoptotic
disease effector agents, such as cancer cells from solid tumors
which have broken off and are circulating in the blood, may be
coated with antibodies to enhance uptake of the cancer cells by
functional dendritic cells.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1 is a graph which illustrates the generation of both
dendritic antigen presenting cells and apoptotic T-cells following
overnight incubation of blood exposed to 8-MOP and ultraviolet A
energy.
[0017] FIG. 2 is a cross-sectional view of a plastic channel
containing the subject's blood illustrating a CTCL cell with a
class 1 associated peptide antigen and a blood monocyte.
[0018] FIG. 3 is a cross-sectional view of a plastic channel
containing the subject's blood illustrating a blood monocyte
adhered to the wall of the plastic channel.
[0019] FIG. 4 is a cross-sectional view of a plastic channel
containing the subject's blood illustrating a blood monocyte
partially adhered to the wall of the channel.
[0020] FIG. 5 is an illustration of dendritic cell produced by
differentiation of a blood monocyte by the method of the present
invention.
[0021] FIG. 6 is a cross-sectional view of a plastic channel
illustrating a CTCL cell with a class 1 associated peptide antigen
being irradiated to render the CTCL apoptotic.
[0022] FIG. 7 is an illustration of an apoptotic CTCL cell in the
process of being phagocytized by a dendritic cell.
[0023] FIG. 8 is an illustration of a dendritic cell which has been
reinfused into the subject's bloodstream presenting the class 1
associated peptide antigen to a T-cell.
[0024] FIG. 9 is an illustration of the class 1 associated peptide
antigen presented on the surface of the dendritic cell as it is
received by a complementary receptor site on the T-cell.
[0025] FIG. 10 is an illustration of a clone of the activated
T-cell attacking a CTCL cell displaying the class 1 associated
peptide antigen.
DETAILED DESCRIPTION OF THE INVENTION
[0026] As noted above, monocyte differentiation is initiated by
exposing the monocytes contained in an extracorporeal quantity of a
subject's blood to the physical forces resulting from the
sequential adhesion and release of the monocytes on plastic
surfaces, such as the surfaces of the channels of a conventional
photopheresis device. In a preferred embodiment of the invention, a
white blood cell concentrate is prepared in accordance with
standard leukapheresis practice using a leukapheresis/photopheresis
apparatus of the type well known to those skilled in the art. The
white blood cell concentrate includes monocytes, lymphocytes and
some red blood cells and platelets. Typically, up to two billion
white blood cells are collected during leukapheresis. Assuming that
monocytes comprise from about 2% to about 50% of the total white
blood cell population collected, approximately 40 million to 1
billion monocytes are present in the white blood cell
concentrate.
[0027] Following separation by leukapheresis, monocyte
differentiation is induced by pumping the blood cell concentrate
through a device which has a plurality of plastic channels.
Preferably, the plastic channels have a diameter of between about
0.5 mm and 5.0 mm. Most preferably, a conventional photopheresis
apparatus having a channel diameter of 1 mm or less is used. The
narrow channel configuration of the photopheresis apparatus
maximizes the surface area of plastic to which the blood cell
concentrate is exposed as it flows through the photopheresis
apparatus. The invention is not limited in this regard, however,
and any appropriate device having plastic channels may be used to
induce monocyte differentiation.
[0028] In a preferred embodiment of the present invention wherein
the blood cell concentrate is treated using a photopheresis
apparatus, monocyte differentiation is induced by the physical
forces experienced by the monocytes as they flow through the
plastic channels in the photopheresis apparatus. While the
invention is not limited to any particular mechanism, the inventors
believe that monocytes in the blood cell concentrate are attracted
to the plastic channel walls of the photopheresis apparatus, and
the monocytes adhere to the channel walls. The fluid flow through
the channel imposes shearing forces on the adhered monocytes that
cause the monocytes to be released from the plastic channel walls.
Accordingly, as the monocytes pass through the photopheresis
apparatus, they may undergo several episodes of adherence to and
release from the plastic channel walls. These physical forces send
activation signals though the monocyte cell membrane, which results
in induction of differentiation of monocytes into functional
dendritic cells.
[0029] Inducing monocytes to form dendritic cells by this method
offers several advantages for immunotherapeutic treatment. Because
all of the dendritic cells are formed from the monocytes within a
very short period of time, the dendritic cells are all of
approximately the same age. Dendritic cells will phagocytize
apoptotic cells during a distinct period early in their life cycle.
In addition, the antigens present in the phagocytized apoptotic
cells are processed and presented at the surface of the dendritic
cells during a later distinct period. By creating dendritic cells
with a relatively narrow age profile, the method of the present
invention provides an enhanced number of dendritic cells capable of
phagocitizing apoptotic disease effector agents and subsequently
presenting antigens from those disease effector agents for use in
immunotherapeutic treatment.
[0030] Following treatment to initiate differentiation of
monocytes, the treated blood cell concentrate is sequestered for
incubation in the presence of apoptotic or inactivated disease
effector agents. The incubation period allows the dendritic cells
forming and maturing in the blood concentrate to be in relatively
close proximity to the apoptotic disease effector agents, thereby
increasing the likelihood that the apoptotic disease agents will be
consumed and processed by the dendritic cells. As described below,
the disease cells may be induced to apoptosis as the blood
concentrate is being passed through the photopheresis apparatus, or
the disease cells may be treated separately to induce apoptosis and
added to the blood concentrate before or after passage of the blood
concentrate through the photopheresis device. A standard blood bag
may be utilized for incubation of the cells, as is typical in
photopheresis. However, it has been found to be particularly
advantageous to use a blood bag of the type which does not leach
substantial amounts of plasticizer and which is sufficiently porous
to permit exchange of gases, particularly CO.sub.2 and O.sub.2.
Such bags are available from, for example, the Fenwall division of
Baxter Healthcare Corp. under the name Amicus.TM. Apheresis Kit.
Various plasticizer-free blood bags are also disclosed in U.S. Pat.
Nos. 5,686,768 and 5,167,657, the disclosures of which are herein
incorporated by reference.
[0031] The blood cell concentrate and disease effector cells are
incubated for a period of time sufficient to maximize the number of
functional antigen presenting dendritic cells in the incubated cell
population. Typically, the treated blood cell concentrate and
disease effector cells are incubated for a period of from about 6
to about 48 hours, with the preferred incubation time extending
over a period of from about 12 to about 24 hours. By treating
monocytes in the manner described above and then incubating the
treated cell population, a large number of functional antigen
presenting dendritic cells can be obtained. It has been found to be
particularly advantageous to add a buffered culture medium to the
blood bag and one or more cytokines, such as GM-CSF and IL-4,
during the incubation period.
[0032] In a preferred embodiment of the present invention, which is
particularly useful where the disease effector agent is circulating
in the subject's blood, such as for example when the disease
effector cells are malignant T-cells, the disease effector agents
are rendered apoptotic in the photopheresis apparatus as the
monocytes are induced to form dendritic cells by the physical
forces they experience in the photopheresis apparatus. A
photoactivatable agent capable of inducing apoptosis in the disease
effector cells is added to the blood cell concentrate prior to
passage through the photopheresis apparatus, and the blood cell
concentrate is irradiated as it passes through the photopheresis
apparatus to render the disease cells apoptotic. By rendering the
disease cells apoptotic in the photopheresis apparatus, these cells
are immediately available to be phagocytized as the monocytes are
differentiating to form dendritic cells.
[0033] In this embodiment of the present invention, saline is added
to the white blood concentrate prior to photopheresis to dilute the
red blood cell concentration to about 2% by volume, thereby
permitting more effective penetration of the activating radiation
to the target disease cells. The photoactivatable agent can be
administered to the subject prior to obtaining a quantity of blood
from the subject for leukapheresis and photopheresis.
Alternatively, or additionally, the photoactivatable agent can be
added directly to the extracorporeal bloodstream, typically by
injecting the agent into the tubing leading to the
leukapheresis/photopheresis apparatus. Regardless of when and how a
particular agent is administered, the disease cells must be exposed
to the photoactivatable agent for a period of time sufficient for
the agent to react with cellular components in the disease
cells.
[0034] Exemplary photoactivatable agents which may be used in the
present invention are psoralens, porphyrins, pyrenes,
phthalocyanine, retinoid derivatives, photoactivated cortisone,
photactivatable dyes, and monoclonal antibodies which have been
linked to porphyrin molecules. The invention is not limited in this
regard, and any appropriate photoactivatable agent known to those
skilled in the art may be used.
[0035] The psoralens are a preferred class of photoactivatable
agents for use in the photopheresis procedure. Psoralens are
readily absorbed from the digestive track, reaching peak levels in
the blood and other tissues in one to four hours following oral
administration, and these agents are excreted almost entirely
within 24 hours. Accordingly, the psoralens are particularly
suitable for oral administration prior to obtaining an
extracorporeal quantity of the subject's blood. The psoralen
molecules are inert prior to exposure to irradiation and are
transiently activated to an excited state following irradiation.
The preferred psoralens include 8-methoxypsoralen (8-MOP), 4'
aminomethyl-4, 5', 8 trimethyl-psoralen (AMT), 5-methoxypsoralen
(5-MOP), and trimethyl-psoralen (TMP). 8-MOP is the most preferred
photoactivatable agent for use with the methods of the invention,
and the conditions for oral administration of this psoralen are
described in U.S. Pat. No. 5,147,289, the disclosure of which is
incorporated herein by reference.
[0036] The irradiation stage of photopheresis is performed as the
blood cell concentrate is passed through the photopheresis
apparatus. The preferred exposure device includes a transparent
plastic channel having a diameter of about 1 mm disposed between
opposed irradiation sources. Referring again to the preferred
embodiment, as the blood cell concentrate passes through the
plastic channel, the disease cells are never separated from the
irradiation sources by more than about 0.5 mm of blood. Maintaining
the disease cells in such close proximity to the irradiation
sources has proven particularly effective in ensuring adequate
exposure of the disease cells to the activating radiation. In the
case where a psoralen such as 8-MOP is used as the photoactivatable
agent, the irradiation sources emit ultraviolet A radiation (UVA)
as the activating radiation. To activate the psoralen, the treated
disease cells are typically exposed to about 1-2 joules/cm.sup.2 of
UVA for a period of from about 15 to about 150 minutes.
[0037] The application of one embodiment of the method described
above is illustrated in FIGS. 2-10 for the treatment of a
particular type of cancer called Cutaneous T-Cell Lymphoma (CTCL).
FIGS. 2-10 illustrate treatment of individual cells, but it should
be understood that in practice the subject's blood will contain a
plurality of the various cells described below, and that the
plurality of cells are treated simultaneously. Referring to FIG. 2,
a plastic channel 10 contains a quantity of the subject's blood, or
the blood cell concentrate if the subject's blood is first treated
by leukopheresis. The blood contains blood monocytes 12 and
malignant CTCL cells 14. The malignant CTCL cells display class 1
associated peptide antigens 16 comprised of a plurality of amino
acids 18. The subject's blood is pumped through the plastic channel
to induce differentiation of the monocytes into dendritic
cells.
[0038] As shown in FIG. 3, as the subject's blood is pumped though
the plastic channel, monocytes 12 adhere to the walls 15 of the
plastic channel 10. Shear forces are imposed on the adhered
monocytes by the fluid flowing past the monocytes and, as shown in
FIG. 4, the monocytes 12 become dislodged from the wall 15. As the
monocytes flow through the plastic channel, they may undergo
several episodes of adherence and removal from the channel walls.
As a result of the forces experienced by the monocyte, activation
signals are transmitted which cause the monocyte to differentiate
and form an immature dendritic cell 20, illustrated in FIG. 5.
[0039] As discussed above, in a particularly preferred embodiment,
the plastic channel is part of a conventional photopheresis
apparatus. This allows the malignant CTCL cells to be rendered
apoptotic as the blood is passed through the plastic channels. The
subject's blood is treated prior to passage though the channels
with a photoactivatable agents, such as 8-MOP. As illustrated in
FIG. 6, as the treated CTCL cell 14 passes though the plastic
channel 10 of the photopheresis device (not shown), ultraviolet
light 22 is transmitted though the transparent plastic channel
walls 15 of the photopheresis apparatus. The ultraviolet light 22
activates the photoactivatable agent, thereby inducing apoptosis of
the malignant CTCL cell 14.
[0040] After the blood has been passed though the photopheresis
apparatus, the subject's blood is incubated to allow maturation of
the dendritic cells and phagocytization of the apoptotic CTCL
cells. As illustrated in FIG. 7, the dendritic cell 20 ingests the
apoptotic CTCL cell 14 during the incubation period. As the
dendritic cell continues to mature during the incubation period, it
processes the apoptotic malignant CTCL cell. As shown in FIG. 8, at
the end of the incubation period, after the the dendritic cell
digests the malignant CTCL cell, the associated class 1 peptide
antigen 16 is presented at the surface of the dendritic cell 20.
After the incubation period, the composition containing the antigen
presenting dendritic cells is reinfused into the subject for
immunotherapy.
[0041] Referring now to FIGS. 8 and 9, which illustrate the antigen
presenting dendritic cell after reinfusion into the subject's blood
stream, the dendritic cell 22 presents at its surface the class 1
peptide antigen 16 from the malignant CTCL cell to a healthy T-cell
24 which has a receptor site 26 for the class 1 peptide antigen.
When the healthy T-cell 24 receives the class 1 peptide antigen
from the dendritic cell, as shown in FIG. 9, the healthy T-cell is
activated and induces the formation of T-cell clones which will
recognize and attack malignant T-cells displaying the same class 1
peptide antigen. As a result, as shown in FIG. 10, the healthy
T-cell clones 24 of the subject's immune system are triggered to
recognize the class 1 peptide antigen displayed by the maliganant
CTCL cell clones, and to attack and kill malignant CTCL cell clones
28 in the subject which display the class 1 peptide antigen.
[0042] While the foregoing description refers to the method for
treating CTCL, it should be understood that the invention is not
limited in this regard, and the method may be used to treat other
types of cancer or disease. In addition, as described further
herein, the method can be performed using any type of device having
plastic channels to induce monocyte differentiation. Moreover, the
cancer cells or other disease effector agents can be rendered
apoptotic by any method known to those skilled in the art and
incubated with the dendritic cells formed by the method of the
present invention.
[0043] As described above, by inducing apoptosis in disease cells
in the photopheresis apparatus at the same time that monocytes are
induced to differentiate into dendritic cells, the dendritic cells
are more likely to phagocytize the disease cells and present
antigens from the disease cells for use in immunotherapeutic
treatment. This embodiment of the invention is particularly useful
when the disease effector agents are present in the subject's
blood, such as, for example, where the disease cells are malignant
T-cells. This embodiment of the invention may also be used where
the disease cells are cells from a solid tumor. It has been shown
that, in at least some cases, cells from solid tumors can break off
and circulate in the blood. Under these circumstances, it may be
preferable to induce apoptosis of the tumor cells in the
photopheresis apparatus at the same time that monocytes undergo
differentiation into dendritic cells.
[0044] In another embodiment of the present invention, the disease
effector agents in the subject's blood are coated with monoclonal
antibodies which selectively bind to the surface of the disease
cell. The bound monoclonal antibodies are long chained proteins
which include a free Fc segment at the end of the protein chain. As
described in Dhodapker et al., [Antitumor monoclonal antibodies
enhance cross-penetration of cellular antigens and the generation
of myeloma-specific killer T cells by dendritic cells, Journal of
Experimental Medicine, 195:125-33, 2002], the monoclonal antibodies
attach to the disease cells, and the free F.sub.c segment is
attracted to, and bonds with, a complementary receptor on the
surface of the dendritic cell. This bond between the F.sub.c
segment on the monoclonal antibody and complementary receptor on
the dendritic cell essentially forms a bridge to the apoptotic
disease effector cell, thereby increasing the likelihood and speed
of the uptake of the apoptotic disease effector cells by the
dendritic cells. The antibodies also appear to direct the ingested
cancer antigens to a pathway which culminates in the antigens
stimulating a CD8 anti-tumor immune response. Preferably, the
disease effector agent is rendered apoptotic prior to being coated
with the monoclonal antibodies. The invention is not limited in
this regard, however, monoclonal antibodies may be used to enhance
uptake and processing of non-apoptoic disease effector agents.
[0045] The method of the present invention may be used, for
example, for immunotherapeutic treatment of subjects having solid
tumors without the need for invasive procedures to obtain cancer
cells. In some instances, the circulating blood of patients with
solid tumors may contain cancer cells that have broken off from the
tumors and entered the circulation. [Kraeft, et al., Detection and
analysis of cancer cells in the blood and bone marrow using a rare
event imaging system, Clinical Cancer Research, 6:434-42, 2000].
These circulating cancer cells may be present in the circulating
blood at relatively low levels, as little as 10-100 cancer cells
per million cells. Antibodies which react with specific types of
cancer cells, and do not react with white blood cells, can be added
to the blood to bind with the cancer cells. Such antibodies which
distinguish and bind to particular types of cancer cells are well
known to those skilled in the art. The free segment at the end of
the antibody protein chain preferentially bonds to a complementary
receptor site on the dendritic cell. Thus, the antibodies bound to
the cancer cells can preferentially direct the cancer cells to
dendritic cells, thereby enhancing uptake of the cancer cells by
the dendritic cells. This procedure can eliminate the need to
remove the cancer cells from the patient prior to treatment, as the
antibodies act to direct those cancer cells present in the blood to
the dendritic cells. The cancer cells are preferably rendered
apoptotic prior to coating with the antibody, but the invention is
not limited in this regard, and non-apoptotic cancer cells may be
used.
[0046] In the case of cutaneous T-cell lymphoma, coating the
malignant T-cells with monoclonal antibodies induces apoptosis of
the T-cells, increases the uptake of the dying T-cells by the
dendritic cells, and increases the rate of processing of the T-cell
antigens by the dendritic cells. Monoclonal antibodies may also be
used with other types of disease causing cells, such as cancer
cells or disease causing T and B lymphocytes (such as in autoimmune
disorders, organ transplant rejection and graft versus host disease
following stem cell transplants), to increase the uptake and
processing of the cancer cells by the dendritic cells. For example,
antibodies against breast cancer, colon cancer and prostate cancer
are available and could be used to coat the relevant cancer cells.
The cancer cells may be rendered apoptotic by any method known to
those skilled in the art, and the apoptotic cancer cells can be
coated with antibodies having free F.sub.c fragments. The free
F.sub.c fragment bonds to the complementary receptor on the
dendritic cells, thereby forming a bridge between the dendritic
cell and the apoptotic disease effector agent, and forming a bridge
between the disease cell and the dendritic cell to enhance the
uptake and processing of the apoptotic disease cell by the
dendritic cell.
[0047] Preferably, the disease effector agents are induced to
apoptosis and coated with antibodies prior to the passage of the
blood through the photopheresis device to induce monocyte
differentiation into dendritic cells. Alternatively, the disease
effector agents contained in the subject's blood may be induced to
apoptosis and coated with antibodies after passage of the blood
though the photopheresis apparatus and prior to incubation. If
desired, the disease effector agents can be treated separately from
the blood cell concentrate which is passed though the photopheresis
apparatus and added to the processed blood prior to incubation.
[0048] It should also be understood that it is not absolutely
necessary to separate the monocytes from the extracorporeal
quantity of the patient's blood by leukapheresis prior to
treatment. As long as the monocytes contained in the blood are
sufficiently exposed to physical forces imposed by flow through
plastic channels to initiate differentiation into dendritic cells
followed by subsequent incubation, separation of the monocyte
population is not required.
[0049] Inducing monocyte differentiation according to the invention
provides dendritic cells in numbers which equal or exceed the
numbers of dendritic cells that are obtained by expensive and
laborious culture of leukocytes in the presence of cytokines such
as GM-CSF and IL-4 for seven or more days. The large numbers of
functional dendritic cells generated by the method of the present
invention provide a ready means of presenting selected disease
associated antigens and are thereby conducive to efficient
immunotherapy. Antigen preparations selected to elicit a particular
immune response and derived from, for example, tumors,
disease-causing non-malignant cells, or microbes such as bacteria,
viruses and fungi, can be added directly to the blood bag during
incubation. The microbes may preferably be inactivated by prior
exposure to 8-MOP or other agents. It is known that 8-MOP can cause
apoptosis in bacteria and fungi and can inactivate viruses.
Bringing mature dendritic cells into close contact with such
antigen preparations within the confines of the blood bag provides
large numbers of antigen-loaded dendritic cells. The antigen-loaded
dendritic cells can be used as immunogens by reinfusing the cells
into the subject or by otherwise administering the cells in
accordance with methods known to elicit an immune response, such as
subcutaneous, intradermal or intramuscular injection. As described
below, it is also possible to generate antigen-loaded dendritic
cells by treating and co-incubating monocytes and disease effector
agents which are capable of expressing disease associated
antigens.
[0050] In another aspect of the present invention, monocytes may be
induced to -differentiate into functional dendritic cells, and the
disease effector agents can be rendered apoptotic or inactive, or
may be otherwise treated, separately from the blood or blood cell
concentrate used to form the dendritic cells. As discussed above,
such disease effector agents comprise microbes, such as bacteria,
fungi, and complete and incomplete viruses, and disease-causing
clonal populations of cells, including clones of malignant cells or
clones of non-malignant T- or B-cells attacking the individual's
own tissues or transplanted tissues. Since these agents have
distinctive antigens on their surface that permit them to be
distinguished from most other cells, immune reactions can be
ideally developed against their distinctive antigens. These immune
reactions can then suppress or eliminate the disease effector agent
populations. Through the generation of dendritic antigen-presenting
cells capable of effectively introducing the relevant antigens to a
responding immune system, this invention substantially enhances the
likelihood of such a disease-controlling immunologic response.
[0051] Central to this aspect of the invention is the
co-cultivation of increased numbers of antigen presenting dendritic
cells, generated as described above, with clones of apoptotic
disease-causing cells or inactivated or incomplete microbes which
bear distinctive antigens. In the case of disease-causing cells,
bacteria and fungi, other means of inducing apoptosis, in addition
to exposure to photo-activated drugs, may be applicable.
[0052] For example, synthetic peptides with the
arginine-glycine-aspartate (RGD) motif could be added to cell
suspensions of the disease-causing cells isolated from the
patient's blood, from excised solid tumors or tissue cultures of
the same. RGD has been shown (Nature, Volume 397, pages 534-539,
1999) to induce apoptosis in tumor cells, possibly by triggering
pro-capase-3 autoprocessing and activation. Similarly, apoptosis
could be induced in cells having Fas receptors, by stimulating with
antibodies directed against this receptor, in this way sending
signals to the inside of the cell to initiate programmed cell
death, in the same way that normally Fas ligand does. In addition,
apoptosis can be induced by subjecting disease-causing cells to
heat or cold shock, certain viral infections (i.e., influenza
virus), bacterial toxins, and x-ray or gamma-irradiation.
Alternatively, certain infectious agents such as influenza virus
can cause apoptosis and could be used to accomplish this purpose in
cell suspensions of disease-causing cells.
[0053] Hence, these approaches, although not as usually preferred
as the induction of apoptosis by photo-activated 8-MOP, could
accomplish the purpose of initiating apoptosis or inactivation in
disease-causing cellular populations, prior to their co-cultivation
with the induced dendritic antigen-presenting cells and return to
the patient for purposes of immunization. Of course, it should be
understood that since viruses are not cells, they cannot undergo
apoptosis as that term is generally understood and used by those
skilled in the art. It is known, however, that viruses can be
inactivated by exposure to 8-MOP and other photo-activated drugs
and therefore can be treated in this manner prior to their
co-cultivation with induced dendritic antigen presenting cells.
Protocol and Clinical Results of Application of the Invention
[0054] An example of the application of the present invention will
be described with particular reference to an enhanced therapy for
treating cutaneous T-cell lymphoma. However, it should be
understood that the invention is not limited to this particular
application and that the invention may be employed to treat any
disease state which includes as a component disease effector agents
distinguishable by their own surface antigens. A number of such
disease states, component effector agents and disease associated
antigens have been discussed above.
[0055] Cutaneous T-cell lymphoma (CTCL) is an immune disease that
is caused by a massive expansion of a single clone of aberrant
T-cells. These malignant cells are distinguished by clone-specific
or tumor-specific cell surface antigens, at least one set of which
are derived from clone specific protein components of the
clone-specific T-cell receptor. Cytotoxic T-cell responses can be
generated selectively against these clone-specific antigens. During
the past decade, photopheresis has become a standard immunotherapy
for advanced CTCL and works, at least in part, by generating such
anti-CTCL immune responses. In standard CTCL treatment using
photopheresis, leukocytes and monocytes are separated by
leukapheresis from an extracorporeal quantity of a subject's blood.
The monocytes and leukocytes are circulated through an ultraviolet
A exposure system of the type described above, in which
biologically inert 8-MOP is activated to covalently bond to DNA and
cytoplasmic proteins in the malignant lymphocytes. This is a highly
directed therapy, since the drug remains active for only millionths
of a second, thereby chemically altering only those cells in the
exposure field and explaining the paucity of systemic side effects.
Photopheresis provides increased immunogenicity of the exposed
leukocytes, without causing general immunosuppression. Thus,
returning the treated cells to the subject can lead to a
"vaccination" effect which, in the most responsive subjects,
results in a sustained immunologic response to the chemically
altered and reintroduced leukocytes. Alteration and return of less
than 5% of the body burden of malignant T-cells can induce a
meaningful anti-tumor response which in some subjects has resulted
in complete remissions lasting more than fifteen years. Methods for
applying photopheresis to the treatment of CTCL are disclosed in
U.S. Pat. Nos. 5,114,721 and 4,838,852 and published PCT
applications WO 97/34472 and WO 94/11016, the disclosures of which
are incorporated herein by reference.
[0056] The clinical results achieved through the application of
photopheresis to CTCL have encouraged a search for the treatment's
underlying mechanism for two major reasons. First, if the mechanism
by which photopheresis vaccinates patients against their malignant
cells could be better understood, it should then be possible to
refine the methodology and enhance its efficacy. For example, only
25% of the patients with advanced CTCL have a major persistent
response to photopheresis. While these positive responses are
profound and their frequency exceeds those produced by prior
conventional chemotherapy, it would be desirable to increase the
efficiency of the procedure. Second, if the mechanism could be
better understood, it should then also be possible to extend the
revised therapy to other types of malignancies and disease
processes. This application is based on the new recognition of the
role of dendritic antigen presenting cells in the response to
photopheresis, and more particularly on methodology of enhancing
this role. Studies in experimental systems and with transformed
human cells lines have yielded four lines of evidence. First, the
treatment stimulates CD8T-cells to suppress the activity of
pathogenic clones of T-cells. Second, these CD8 cells, at least in
CTCL where there is only a single clone of pathogenic T-cells,
recognize tumor-specific peptide antigens in the context of Class I
MHC complexes at the tumor cell surface. Third, exposure of human
lymphoblasts to photo-activated 8-MOP triples the display of Class
I complexes, peaking after overnight incubation. Finally, the
treatment also causes apoptosis in lymphocytes and their ingestion
by phagocytic mononuclear cells.
[0057] Multiple lines of clinical and experimental evidence have
confirmed the "vaccination" phenomenon which is associated with the
induction of potent CD8 responses capable of selectively
suppressing aberrant T-cell populations. In the case of CTCL, at
least some of the anti-cancer CD8 T-cells selectively targeted
tumor-specific peptides derived from the T-cell receptor proteins
of the malignant cells. Since the T-cell receptors of CD8 T-cells
recognize antigenic peptides in the context of Class I MHC,
attention has focused on the impact of 8-MOP on the display of
these complexes. It has recently been reported that 8-MOP triples
the display of Class I MHC at the cell surface of transformed human
lymphocytes, maximizing about 22 hours after exposure, and that
this effect is dependent on the degradation of cytoplasmic proteins
and the transport of the generated peptide fragments across the
endoplasmic reticulum through TAP pores. This effect appears to be
initiated by binding of 8-MOP to aromatic amino acids of cytosolic
proteins rather than the drug's other main molecular target,
pyrimidine bases of DNA.
[0058] The present invention is based on the assumption that if an
immune response is to be generated against weakly immunogenic
complexes containing the relevant antigens, then such a response
might be maximized if the complexes are maximized on the antigen
presenting cells. In conventional photopheresis, T-cells are
immediately returned to the subject at a point when apoptosis is
only modestly elevated over baseline and when Class I complexes are
also only modestly enhanced. In the present method, the treated
leukocytes are incubated overnight, typically for a period of from
about 6 to 48 hours. An unexpected finding was that overnight
incubation of the treated cells not only enhances the expression of
Class I complexes by the apoptotic T-cells, but also maximized the
maturation of monocytes into functional dendritic cells. Thus, the
convergence of these two phenomena made the incubation phase a
simple means of bringing large numbers of apoptotic malignant cells
into apposition with increased numbers of functional dendritic
cells capable of ingesting apoptotic cells or fragments of
apoptotic cells. It has previously been shown the mononuclear cells
in the photopheresis bag have already begun to phagocytose
apoptotic T-cells, although these mononuclear cells do not have the
properties of dendritic cells. Typically, antigen presenting cells
process endocytosed antigens through the Class II MHC pathway,
which ordinarily stimulates expression of CD4T-cells rather than
the desired CD8 cytoxic cells which "see" antigens only in the
context of Class I MHC. However, it is important to note that it
has recently been reported that dendritic cells have a special
capacity to process and present antigens derived from apoptotic
cells through the Class I MHC system.
[0059] An enhanced photopheresis protocol based on the present
invention has provided encouraging clinical results in a pilot
study which included four subjects suffering from advanced CTCL.
However, before discussing the clinical results of the study, a
treatment protocol describing an embodiment of the present
invention will be set forth in the following examples.
[0060] Photopheresis Protocol
[0061] The first step, which is the photopheresis protocol, is
essentially the same as the protocols currently approved by the
FDA. Subjects receive either oral 8-MOP (0.6 mg/kg) or intravenous
8-MOP directly into the photopheresis apparatus, to yield a
concentration of 50-200 ng/ml of drug. Next, the blood is
leukapheresed to obtain a buffy coat and is then passed through a
contiguous closed circuit ultraviolet A exposure device, which
delivers about 1-2 joules /cm.sup.2 of ultraviolet A energy (320
nm-400 nm). In this manner, about 1 to 100 molecules of 8-MOP are
induced to covalently bind to each million base pairs of DNA. A
nearly equal amount of 8-MOP is induced to covalently bind to
aromatic amino acids of cytoplasmic proteins. The treated leukocyte
fraction, comprising a total volume of approximately 250 cc, is
combined with 500 cc saline and then sequestered in a standard
blood bank bag, as is typical for the photopheresis procedure.
Following photopheresis, the treated fraction is subjected to the
following novel incubation phase protocol.
[0062] Incubation Phase Protocol
[0063] Following collection of the post photopheresis sample after
ultraviolet A activation with 8-MOP, the treated cell populations
are incubated as follows:
[0064] 1. Remove two Amicus platelet storage bags (Baxter Fenwall
PL 2410) from an apheresis kit (Baxter Fenwall 4R 23-12) by heat
sealing the tubing and cutting the tubing at the end connecting to
the kit.
[0065] 2. Insert a sharp catheter into the pheresis bag (spike),
thereby breaking the seal, with a Charter Medical 3-leg transfer
set (#O3-220-02) and clamp the tubing. Spike the two Amicus bags
with the other piercing pins of the same transfer set thereby
establishing a passageway for the transfer of the cell
suspension.
[0066] 3. Hang the pheresis bag on an IV pole and open the clamp
allowing 1/2 of the pheresis to drain into each Amicus bag by
gravity, and then clamp the tubing.
[0067] 4. Remove the spikes and replace with sampling site
couplers.
[0068] 5. Place each Amicus bag in a separate Fenwall centrifuge
bag and into a centrifuge carrier.
[0069] 6. Centrifuge for 10 minutes, at 1000 rpm, 23.degree. C., to
concentrate the cells as a pellet at the bottom of each bag to
permit removal of a large fraction of the plasma, which contains
traces of plasticizer.
[0070] 7. After centrifugation, insert a needle attached to the
tubing on a transfer pak into the sampling coupler on one of the
Amicus bags.
[0071] 8. Carefully place the Amicus bag in a plasma extractor to
avoid resuspending the cell pellet. Close the extractor and express
the plasma into the transfer bag by slowly tipping the extractor
forward. When approximately 50 cc has drained into the transfer bag
and/or the pellet begins to resuspend, return the extractor to an
upright position and remove the needle.
[0072] 9. Remix the contents of the bags by gentle agitation being
careful to resuspend any adherent cells attached to the bag
wall.
[0073] 10. Spike one 500 cc bottle containing 100 cc of colorless
RPMI 1640 media with Hepes Buffer with a Baxter vented medication
set and clamp the tubing. Insert the attached needle into the
sampling coupler port on the first Amicus bag. Hang the bottle on
the IV pole and open the tubing allowing the media to drain into
the bag.
[0074] 11. Clamp the tubing and remove the needle and discard the
medication set. Mix the bag by gentle inversion and place the bag
in a 370.degree. C. incubator on a shelf with the Abel side down,
overnight.
[0075] 12. Repeat steps 8-12 for the second bag.
[0076] 13. Following incubation for a period of 6-24 hours, remove
one bag from the incubator, gently mix by agitation and inversion,
making sure that all adherent cells are resuspended. Take out 60 cc
of blood in a syringe. Inject one aerobic, and one anaerobic blood
culture bottle for microbiology. Inject one lavender top tube for
WBC and differential to be sent to hematology.
[0077] 14. Resuspend the second Amicus bag and place both bags in
individual centrifuge bags and centrifuge.
[0078] 15. Remove and transfer the supernatant fluid as described
in steps 8-10.
[0079] 16. Return well mixed blood to the patient.
[0080] FIG. 1 is a composite graph which illustrates the generation
of both dendritic antigen presenting cells and apoptotic T-cells
following treatment by the photopheresis and incubation protocols
set forth above. As shown in FIG. 1, pre-treatment blood contained
nearly undetectable numbers of dendritic cells, using either the
.alpha.V.beta.5 or CD11c markers for identification. After
incubation for about 22 hours, both of these markers revealed large
numbers of mature dendritic cells. Similarly, the pre-treated blood
contained very few apoptotic T-cells. Only after overnight
incubation did apoptotic T-cells become significantly evident, as
illustrated by the simultaneous identification of the T-cells with
the CD3 marker and the apoptotic cells with the APO2 markers.
[0081] The fourth set of bars at the far right of the graph
illustrates the differentiation of monocyte into mature dendritic
cell by means of physical perturbation and incubation only, without
exposure to ultraviolet light. Differentiation was initiated by
isolating monocytes and T-cells from an extracorporeal quantity of
blood by leukapheresis. The isolated monocytes and T-cells were not
subjected to photopheresis but were exposed only to the centrifugal
forces associated with leukapheresis. The isolated cell populations
were then incubated for a period of about 22 hours according to the
incubation protocol set forth above. As shown in FIG. 1, the
physical forces applied during leukapheresis, together with
overnight incubation, caused the monocytes to efficiently evolve
into functional dendritic cells, as identified by the
.alpha.V.beta.5 and CD11c markers. No significant apoptosis of the
T-cells was observed, indicating that treatment with 8-MOP followed
by exposure to UV, or some other form of treatment as described
above, is required to induce T-cell apoptosis.
[0082] The Y axis of the graph gives the number of functional
dendritic cells per cubic centimeter. Since the total volume
incubated over the 22 hour period was 250 cc, 32.5 million
dendritic cells (130,000.times.250) were generated, as indicated in
the third set of bars by the CD11c marker. It has been shown that
dendritic cells having this level of maturity phagocytose apoptotic
cells and are efficient presentors of antigens derived from such
cells. Monocytes may also ingest apoptotic cells or fragments of
such cells, but monocytes cannot efficiently present antigen
material processed from the apoptotic cells to CD8 cytoxic T cells.
CD8 T cells only recognize antigens which are associated with Class
I MHC at the surface of the antigen presenting cell. Monocytes
primarily present antigens derived from ingested cells in
association with Class II MHC molecules, which CD8 T cells cannot
recognize. Dendritic cells, on the other hand, in part because they
include the .alpha.V.beta.5 integrin, have the special ability to
"cross-prime" CD8 T cells by presenting the antigens derived from
the digestion of apoptotic cells and displaying the processed
antigens in association with the Class I MHC molecules that CD8
cytoxic T cells can recognize. This is a major reason why
functional dendritic cells are so useful in stimulating tumor
immunity, or suppressing undesirable immunologic processes by
attacking the aberrant T cells that cause them.
[0083] The graph illustrated in FIG. 1 further demonstrates an
effective means of determining the optimum incubation time for the
mixed cell populations. Since the particular markers employed
permit the numbers of dendritic cells and apoptotic T-cells to be
quantified simultaneously, the incubation time that results in the
optimal combination of apoptotic cells and newly formed dendritic
cells can be readily determined. This is the controlling
determinant establishing when to terminate incubation and reinfuse
the incubated cells into the subject.
[0084] As noted previously, an incubation time of from about 12 to
about 48 hours results in a maximum number of dendritic cells. The
apoptotic T-cells maximize in a period of about 6 to about 40
hours. Accordingly, an incubation period of from about 6 to about
24 hours provides the most advantageous combination of apoptotic
T-cells and dendritic cells. After an incubation period of this
duration, the number of apoptotic cells is at a maximum and large
numbers of functional dendritic cells are also present in the
incubation bag. Thus, a maximum number of apoptotic cells capable
of expressing disease-associated antigens are present and a large
number of functional dendritic cells capable of processing and
presenting those antigens are also present. In the case where the
disease effector agent is derived from an exogenous source and is
added to the incubation bag, the incubation period required for
maximizing the number of apoptotic cells is obviously not a factor.
In such instances, the time period required for maximizing the
number of induced dendritic cells is the factor which determines
the duration of the co-incubation.
[0085] Clinical Efficacy of Combined Treatment and
Co-Incubation
[0086] The treatment method taught by the present invention has
been tested in a pilot study involving four CTCL subjects whose
disease had been advancing while on standard photopheresis. The
four patients in the pilot study were carefully selected from a
large CTCL population based on three criteria: (1) increasing tumor
burden despite continued conventional photopheresis; (2) malignant
clones that could be quantified in blood; and (3) low absolute
blood CD8 levels. The leukemic cells in three of the subjects could
readily be distinguished from normal T-cells, since their clonal
T-cell receptor phenotype was recognizable using fluorescein-tagged
anti-family V T-cell receptor monoclonal antibodies (V mAb). Values
above 5% indicate expansion of the malignant clone. Although the
clonal T-cell receptor of the fourth patient's CTCL cells does not
bind any currently available V mAb, the CD4/CD8 ratio permits
quantification of that patient's leukemic population as well. The
unresponsiveness of the four patients to conventional photopheresis
likely reflects their CD8 T cell deficiency, since clinical
responders usually require an intact CD8 compartment. Therefore,
these patients present a significant challenge for the new
treatment approach. Although the study population was small, it was
easy to quantify reversal of disease progression in this poor
prognosis patient group.
[0087] Following treatment with the above-described protocol, each
of the four patients had a diminution in the absolute circulating
malignant pool over the twelve months of the protocol. Whereas none
experienced complete hematologic remission, the previous rapid
increases in blood CTCL cells were reversed. Those symptomatic
infections common in individuals whose immune systems have been
compromised by their CTCL, and the therapy for this disease, were
not encountered. Measurements of tumor burden and clinical response
centered on blood determinations and quantitation of the number of
infiltrating T cells in biopsies of the clinically most severe skin
lesions. It is important to note that the severity and distribution
of skin lesions in three of the four patients lessened. In one
patient, long-standing, maximal, generalized exfoliative
erythroderma associated with intractable pruritus was transformed
to low grade, nearly asymptomatic erythroderma, and two of the
other patients had nearly complete cutaneous remissions.
[0088] The photopheresis/incubation protocol tested in this study,
like conventional photopheresis, appears to be safe, since no side
effects were encountered in these subjects. Further, the capacity
of the protocol to bring together malignant apoptotic cells bearing
the relevant immunizing antigens with functional dendritic cells
capable of presenting these antigens to a responding immune system
offers additional opportunities for immunotherapy beyond the
treatment of CTCL. For example, in a recently reported randomized,
controlled trial, the combination of photopheresis with
conventional immunosuppressive drugs proved effective in reducing
the number of rejection episodes experienced by heart transplant
recipients. Preliminary, studies have also suggested the efficacy
of conventional photopheresis in certain autoimmune diseases, such
as rheumatoid and psoriatic arthritis, lupus erythematosus,
scleroderma and graft-versus-host disease (following allogeneic
bone marrow transplantation). The present invention's capacity to
provide an in vivo source of large numbers of dendritic cells
should enhance these therapies. Modifications to the protocol may
also permit co-cultivation of dendritic cells derived from induced
monocytes with suspended apoptotic solid tumor cells, apoptotic
infectious microbes or inactivated or incomplete viruses.
[0089] Accordingly, it should be understood, as noted above, that
while certain aspects of the invention has been described in
connection with an enhanced therapy for CTCL, the invention is
applicable to a broad range of immune diseases without departing
from the spirit and scope of the invention.
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