Note: Descriptions are shown in the official language in which they were submitted.
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- 1 -
METHODS FOR INDUCING IMMUNE RESPONSIVENESS IN A SUBJECT
- Technical Field
The present invention relates to improved methods for in-luçing an imm~ln~
response in a subject to cells or tissues, particularly tumor cells and/or T or B-cells that
5 cause an a~-toimm~ne disorder. The present invention specifically provides methods
and compositions for the extracorporeal tre~tmPnt of blood and for a~lministration of
an extracorporeally treated blood mixture to a subject, to induce an immune response
to cells or tissues that express a target antigen. In particular, the methods include
using extracorporeal lle~ ..P~.I agents, such as photochelllolll~lap~ tic agents, that
10 increase MHC Class I peptide expression, and using/adding dendritic cells and/or other
antigen plesf,..li~g cells to the extracorporeal blood stream during lle~ with such
agents.
Background Art
Tmm~ln~ system responses may be classified as humoral or cell-medi~ted A
humoral response is medi~ted by B Iymphocytes in the form of freely diffusible
antibody molecules. A cell-m~ ted response is medi~ted by specifically reactive
Iymphocytes, such as T Iymphocytes ("T cells"). T cells react with foreign ~ntig~nc via
surface receptors that are distinctive for each T cell clone. The T cell surface receptors
generally are composed of two tli.culfide-linked protein chains having unique amino
2 0 acid sequences (Edelson, R., Annals of N. Y. Acad. of Sciences 636:154-164 (1991)).
The physical pl opel ~ies of these receptors confer specific binding capabilities and
permit each of the several million clones of T cells in an individual to operateindependently.
T cells function in the regulation of an imrnune response via recognition by the2 5 immllne system of the T cell surface receptor. In the initiation of an imm~lne response,
the T cell receptor is capable of recognizing a particular antigen only when it is
associated with a surface marker on an antigen presenting cell, such as a dendritic cell.
These surface markers most commonly belong to a group of molecules known as the
major histocompatibility complex (MHC). Binding of the T cell receptor to the
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antigen on the antigen pres~nting cell induces ch~nges in the T cell, which changes
collectively initiate a cascade of events leading to the cell-mediated immlmnlogic
response.
Cutaneous T cell Iymphoma (CTCL) is an immune system disease that is
5 caused by a massive expansion of a single clone of aberrant T cells. Extracorporeal
photochemotherapy "photopheresis" for the tre~tm~nt of cutaneous T cell Iymphomahas been described (Edelson, R., Scientif~cAmerican 2~6(8):68-75 (1988); Edelson,
R., supra. (1991)). Photopheresis trç~tm~nt involves isolating the subject's white
blood cells (inr.lll~ing the T cells), irM~i~ting the cells in the presence of a1 0 photoactivatable agent (8-methoxypsoralen, "8-MOP") and r~infilcin~ the damaged
cells. The 8-MOP is activated by the ultraviolet light to form a transiently energized
molecule capable of photomodifying cellular DNA. This therapy reportedly results in
selective destruction of the m~lign~nt T cell clone. It is believed that exposure of as
little as five percent of the members of the m~lign~nt T cell clone to the
1 5 8-MOP/irradiation tre~tm~nt followed by return of the irr~ tetl, ~l~m~ged cells to the
subject, elicits a specific re~ollse to the abe,~ L T cells that is me~ ted by the T cell
surface receptors, i.e., the ~l~m~ged cells of the m~lign~nt clone in effect prime the
immune system to specifically destroy the untreated rçm~in~çr of the aberrant clone.
Photopheresis also has been used for the trç~tm~nt of several autoimm~ln~: disorders,
2 o incl~ltling pemphigus vulgaris, systemic sclerosis, rhe~lm~toid arthritis, HIV infection
and rejection oftransplanted organs.
U.S. Patent Nos. 4,321,919; 4,398,906; 4,428,744, and 4,464,166, all issued to
Edelson, describe photopheresis methods for treating the blood of a diseased subject
where the disease-producing blood cells have been naturally stimlll~ted as a
2 5 consequence of the disease state, either as an immunologically reactive state or a
m~lign~ncy. Specifically, the methods involve treating such naturally stim~ ted
specific human blood cells with a dissolved photoactivatable drug, such as a psoralen
which is capable of forming photoadducts with the DNA of the dice~ced cells, in the
presence of ultraviolet or visible light irradiation. Following extracorporeal irradiation,
3 0 the damaged lymphocytes are returned to the subject. The (~m~ged Iymphocytes are
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cleared from the subject's system by natural processes, but at an accelerated pace,
presumably because of disruption of cell n.t;lllbl ane integrity, alteration of the DN~
within the cells, or related modifications.
More recently, methods and pharm~eutical compositions for specifically
5 modifying an immllne response to a specific antigen have been reported. These
methods include treating an antigen-presenting cell to enh~nce eA~I e~;on by the cell of
empty major histocompatibility complex molec~lles, followed by reacting the treated
antigen pres~nting cell with an antigen extracorporeally in the presence of a
photoactivatable agent and irradiation to form an antigen-associated antigen presenting
cell. (See, e.g., PCT application number US93/11220, publication number
WO 94/11016). None ofthe references and/or patents disclosed herein describes
extracorporeal blood tre~tm~nf methods that increase immune reactivity to specific
target ~ntigen~ and methods that can be used to ~ugm~nt existing photopheresis
methods. Accordingly, there is still a need for improved methods and pharm~ce~ltic~l
15 coll'~)Gsilions for in~lu~.ing an imm~lne response to a target ~ntigen; for methods of
prtpa,hlg d;sease associated antigen plepalnlions which are specific for a subject and
methods for augm~nting existing photopheretic methods.
Summary of the Invention
The methods and compositions of the present invention are based on the
2 0 identific~tion that: I ) current agents used in photopheretic methods to induce an
immllne response to one or more target ~ntig~n~ are effective because they increase the
level of MHC ~A~,r~ss;on in the treated cell, 2) blood, col~t~ g disease effector cells,
that is extracorporeally treated in known photopheretic methods results in the
transport of disease associated antigens to the surface of the treated cells as weakly
2 5 bound ~ntigen~ to MHC molecules, 3) known photopheretic methods can be
substantially improved by adding dendritic cells, or other antigen presenting cells, to
the treated blood prior to re-infusion and 4) isolated and cultured dendritic cells can be
used to boost a subject's immune system response in most context where an immnn~.
system response in desired.
.. . .. .
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Based on these observations, the present invention provides: 1) methods and
pharm~ceutical compositions for plepa-ing a disease-associated antigen plepal~lion;
2) methods for inducing an immllne response in a subject using extracorporeal
llealmenl of blood; 3) methods for a~lgm~nting existing extracorporeal blood L~ea~lllt;
methods, such as photoçhemir~l therapy; 4) methods for identifying agents that can be
used in the extracorporeal tre~tm~nt of blood; 5) methods to optimize extracorporeal
blood treatment methods; and 6) methods for augmenting existing immlmotherapeutic
methods.
One embodiment of the present invention is based, at least in part, on the
discovery that disease-associated ~ntig~ni contained in unfractionated blood that has
been subjected to tre~tm~nt with an agent that increases MHC Class I t;~,res~ion (such
as in photopheresis using a photoactivated chemical), can be presented by exogenously
- added dendritic cells in vivo to elicit an antigen-specific imml-ne response. More
particularly, one embodiment of the invention provides methods and compositions for
an improved method for extracorporeal blood Ireallllenl in which dendritic cells are
introduced into the treated blood during the extracorporeal treatment, such as during
photopheresis. Also provided are methods for in~ r.ing immllnologic tolerance toautologous or exogenous antigens and compositions useful in su~uplessillg clinically
undesirable immunologic reactions.
2 0 In general, these methods rely on the use of dendritic cells. Dendritic cells,
preferably peripheral blood dendritic cells, or other antigen presenting cells, are first
removed from a subject and cultured in vi~ro. The cultured dendritic cells can be
added to an extracorporeal treated blood sample, such as that described above, to
increase the degree of jmml~ne response obtained. In addition, cultured dendritic cells
2 5 can be added in combinalion with a subunit vaccine to f~cilit~te and increase vaccine
presentation. Further, cultured dendritic cells can be used as a booster to prolong the
effectiveness of methods that rely on the induction of an immune response, such as in
photopheresis and vaccination protocols.
The present invention is further based on the observation that photochemical
3 0 agents used in photopheresis are effective in photopheretic methods because they
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- 5 -
increase the level of MHC Class I e,~ression on the treated cells. Based on thisobservation the present invention provides methods for identifying agents for use in
extracorporeal tre~tment methods in addition to the presellLly used photochemical
agents. Further, this observation provides a means to optimize a treatmlo.nt protocol by
assaying for an increase in MHC Class I eA,~1~ t;ssion during agent ll e~ c
The methods and compositions of the present invention are used to treat a
subject that has a disease that is medi~ted by or is conditioned upon the presence of
cirCul~ting "disease effector" cells. Examples of disease effector cells include, but are
not limited to, T cells, B cells, and/or infected white blood cells, such as virally or
bacterially infected cells. Exemplary tlicç~ces that can be treated using the methods of
the present invention inCl~ldç~ but are not limited to, leuk~mi~ Iymphoma, autoimm--ne
disease, graft versus host disease, and transplanted tissue rejection. In these
conrlitionc, an antigen that medi~tes the disease state (i.e., the "disease-associated
antigen") is a peptide that is associated with (binds to) an MHC Class I site, an MHC
Class II site or, to a heat shock protein that is involved in transporting peptides to/from
MHC sites (i.e., a chaperone). Other conditions that can be treated using the present
methods include condition in which a disease-associated antig~n such as a viral or
bacterial peptide, is expressed on the surface of an infected white blood cell, usually in
association with an MHC Class I or Class II molecule.
2 0 The methods and compositions of the present invention are useful for
improving the effectiveness and specificity of therapeutic strategies that involve antigen
presentation on any type of antigen presçll~ g cell and in providing methods forin~u~in~ and ~ugm~onting an immllne les~,onse to specific ~ntig~nc~ The invention is
particularly useful for improving the efficacy of extracorporeal blood tre~tm~.nt
2 5 methods, such as photopheresis, in subject populations for which photopheresis has
proven of little or no value as a tre~tmçnt modality, e.g., the 25% of subjects
diagnosed with cutaneous T cell Iymphoma for whom photopheresis has proven to becompletely ineffective and the 50% of subjects in which the effect is transient and/or
incomplete. The invention also provides a substantial cost savings by providing
., , . , . . ~ .. . ~ ....... . . .
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methods and compositions that permit the generation of multiple therapeutic
compositions for subsequent ~1ministration from a single procedure.
According to still another aspect of the invention, an antigen composition for
enh~ncing a cellular immllne system response is provided. The antigen composition
5 contains disease-associated anti~çns that have been released from disease effector cells
contained in blood and a detect~hle arnount of a tre.~tm.o.nt agent, such as psoralen or
other photoactivatable agent. The composition is forml-l~ted to contain an amount of
disease-associated ~ntig~n.c for mixing with a single dose of dendritic cells to form a
cellular vaccine.
10According to yet another aspect of the invention, a process for producing a
product for P.nh~nr.inf~ an immune response and the product produced thereby aredisclosed. The process involves: (a) acidifying a prepal~lion co,~ il-g a plurality of
disease effector cells for a period of time sufficient for the disease effector cells to
- release disease-associated antigens without Iysing the cells; and (b) neutralizing the
15 acidified prepa.~Lion to form the product. In one embodiment of this method, beta2-
microglobin is added to stabilize the prepared antigens.
The present invention further provides a titration point for determining when tostop agent tre~tm~tlt during the extracorporeal tre~tm~nt and when to ~lmini~ter the
treated blood to a subject. In general, these methods rely on art known methods to
2 0 determine the course of MHC eA~ression during the course of agent trP~tm~nt of the
extracorporeal blood. A treated blood mixture is ready for ati" ~ ion or
colllbini--g with dendritic cells when an increase in MHC class I eA~.ession, as a result
of the agent tre~tm~nt, is observedldetectecl
These and other aspects of the invention, as well as various advantages and
2 5 utilities will be more appa-enl with reference to the detailed description of the
plefel-ed embodiments.
Brief Description of the Fi~ures
Figure 1 is a graph that demonstrates the ~ntitllmor response of vaccinations
against tumor growth using a therapeutic mixture comprising irradiated 2B4. 11 tumor
3 0 cells and dendritic cells.
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Figure 2 shows the inhibition of tumor growth using dendritic cells (DAPC)
alone.
Figure 3 shows the impact of 8-MOPfUVA tre~tm~nt on MHC Class I
,res~ion.
Detailed Description of the Invention
A. General Description
The present invention provides improved methods for use in extracorporeal
blood tre~tmPnt for use in in~cing an imm~lne les~,onse in a subject. The improved
methods use a ~y"e,~lic combination of two therapeutic methods, extracorporeal
Ll ~ rnt of blood with agents that h,c, ~,ase MHC Class I c~ression (such as
photophelesis) and dendritic or other antigen plrse,.l;t-g cell-me~ ted immnn~
- therapy.
One embodiment of the present invention relates to the discovery that when
cor"bined, these two methods exert a synergistic I~ utic effect in l~eali"g a subject
that is diagnosed as having a disease state that is medi~ted by circ~ ting T cells,
B cells or infected circul~tin~ cells, such as white blood cells infected by an infectious
agent, such as, but not limited to, virus, bacteria, protozoa, etc.
B. Specific Descriptions
I) Extracorporeal Blood Tre~l-.,e .l
2 0 According to one aspect of the present invention, an improved method for
extracorporeal tre~tm~nt of the blood of a dice~qed subject to induce an imm~me
system response to one or more disease-associated antigens is provided.
In one embodiment, the methods of the invention comprise the steps of:
1) obtaining blood co~ -g disease effector cells that express one or more disease
2 5 associated ~ntig~nc from the subject; 2) oblainh~g dendritic cells, or other antigen
presenting cells, from the subject; 3) treating the blood co..l~in;~g the disease effector
cells with an agent that increases MHC Class I e.~ ssion (extracorporeal blood
tre~tm~nt); 4) introducing the dendritic cells, or other antigen pres~nting cells, into the
treated blood mixture to form a therapeutic mixture; and (5) r~infi-cing or otherwise
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introducing the therapeutic mixture into the ~ise~ced subject as a mixture of treated
cells and dendritic cells or as a vaccine Col-t~ g purified, antigen loaded dendritic
cells.
The present invention further provides methods for identifying agents that can
be used in the methods of the present invention as well as other extracorporeal blood
treatment methods that are used to induce an immllne response to a target antigen.
Specifically, it has been found that the effectiveness of photopheresis is pot~nti~ted~ in
part, by the ability of the photoçhP.mic~l agent/tre~tm~nt to induce MHC cA~ression on
the treated cells. The ability to increase MHC eA,uression can be used as an assay point
for identifying agents for use in the present method. Further, MHC ~ ,res~ion can be
used as a titration point for determining the optimum tre~tm~nt agent/protocol for the
present methods.
II) Disease Effector Cells and Conditions Treatable with the
Present Methods
The methods of the present invention are useful for treating a diseased subject,i.e., a subject who has been diagnosed as having a disease that is me~ ted by "disease
effector cells," ''circ~ ting aberrant cells," or "non-circl-l~ting disease effector cells,"
cells that express an antigen that is associated with a pathological condition, i.e. solid
tumor cells.
2 0 As used herein, "disease effector cells" or "circul~ting aberrant cells" refers to
cells that (1) are present in peripheral blood (prefel~bly a T cell, a B cell, or a virally or
bacterially infected white blood cell), (2) merli~te a pathological or disease state and 3)
express peptides or proteins that can be used to dietin~ h them from other similar
cells that are not associated with the pathological condition. For example, a disease
2 5 effector cell can be a T-cell that has undergone a transformation to be a tumor cell, a
T-cell that mediates an autoimmune disorder or can be a cell infected with a virus,
bacteria or other microorganism that results in the cell eAI~I esshlg one or more
proteins/peptides of viral, bacterial, protozoan or microorganism origin. As used
herein, "non-circ~ ting disease effector cells" refers to cells that (1) are not present in
3 0 peripheral blood (preferably a solid tumor or infected organ), (2) m~di~te a
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9_
pathological or disease state and 3) express peptides or proteins that can be used to
~lictin~]i~h them from other similar cells that are not associated with the pathological
condition. For example, a non-circul~ting disease effector cell can be a solid tumor.
Thus, disease effector cells (circ~ tinE and non-circ~ tine) include, but are not limited
to, m~lign~nt T cells, m~lign~nt B cells, T cells or B cells that metii~te an autoimmune
or transplanted tissue rejection response, virally or bacterially infected white blood
cells or tissues that express viral or bacterial proteins/peptides and solid tumor cells.
Preferably, the disease effector cells will express an antigen on the cell surface and will
be T cells or B cells, more preferably T cells. The pre~el I ed disease effector cells a're
T cells belonging to a single clone. More preferably, the disease effector cells are
T cells that are polyclonal or are tumor cells obtained from a solid tumor.
As described below, the agent tre~tmçnt step of the methods of the present
invention, for example photopheresis, ~m~ges the disease effector cells to the extent
- that the agent treated cells increase MHC Class I eAI)less.on, and/or transport/release
15 disease-associated ~ntig~n~, but are not ;~-,e~i~tely Iysed or killed. The
transported/released peptides then are passed "baton-fashion" to the dendritic cell
major histoco.l")aLibility complex molecules, either by entering empty MHC sites or by
~iepl~çin~ peptides that are present in the added dendritic cell MHC Class I or Class II
sites for presentation.
2 0 Thus, the methods of the invention are useful for treating dice~ces such as
le lk~mi~c, Iymphomas, solid tumors, met~t~tic tumors, autoimml-ne tliee~es~
transplanted tissue rejection, and graft versus host disease. In addition, the methods of
the present invention are useful for treating viral or bacterial conditions including HIV,
malaria, etc. and other blood borne infections that are metli~ted by intrac.ol~ r
parasites or other factors, including e.g., listeria, Epstein Barr virus, HTLV-1, herpes
simplex, varicella, hepatitis A, B and C virus, protozoan, such as leichm~ni~ donovans.
See, e.g., Goodman and Gilman's "The Pharmacological Basis of Therapeutics, W.A.Goodman Gilman e~ al., Pergamon Press, N.Y., N.Y. (1990) for a description of
- protozoan infections which can be treated in accordance with the methods disclosed
,
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herein. These infections include malaria, amebiasis, guardiasis, trichomoniasis,lP.i~hm~ni~ic, trypanosomiasis, and toxoplasmosis.
III) Subject
The methods of the present invention are inten~ed to be used in treating any
5 m~mm~ n subject, so long as the subject is in need of intl~lçing an immune response
to disease effector cells. The methods of the present invention are preferably used to
treat humans.
Because modulation of the cellular immune response is important to the
execution ofthe invention, it is plerelled that the recipient subject ofthe methods of
10 the present invention have a competent immnne system, as evidenced by, for example,
near normal absolute levels of CD8 positive T cells.
- IV) Blood removal or Tissue Removal
The first step used in practicing one aspect of the present invention is to
remove blood from a subject that contains disease effector cells as defined above or
15 tissue that contains the non-circ ~l~ting disease effector cells. A variety of methods are
known in the art for removing blood co"~ g disease effector cells, for isolatingdisease effector cells for removed blood, for example by using centrifugation, and for
removing non-circul~ting cells, such as solid tumors. Some of these methods are
described in detail below. A skilled artisan can readily adapt any of the blood
2 0 removaVtissue removal, separation and culturing methods known in the art for use in
the present methods to obtain blood co"l~ il-g disease effector cells, a population of
disease effector cells that express one or more target ~ntig~n~, or a population of non-
circ-ll~ting disease effector cells.
The amount of blood or tissue removed will be based primarily on the disorder
2 5 being treated and the therapeutic protocol that is employed. For example, for treating
CTCL using a therapeutic protocol calling for from a single injection, to weekly or
monthly injection injections of treated cells, approximately from about 200 cc to about
750 cc of blood is removed. A skilled artisan can readily determine the amount of
blood that needs to be removed for tre~tment based on the number of disease effector
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cells per cc of blood. As can be readily appreciated, the blood used in the
extracorporeal tre~tm~nt may be removed over a course of several sessions.
Alternatively the blood can be removed, treated and re-infused in a continuous process.
For treating a solid tumor, as much of the tumor is removed for both therapeuticpurposes as well as to provide a source of antigen for dendritic cell loading.
V) Agent Treatment of the Disease Effector Cells
After removal of blood from the subject, the blood co~ g disease effector
cells, or purified disease effector cells, is subjected to extracorporeal lle~ e ~l using a
1l~1l~l~1 agent. As used herein, "extracorporeal blood lle~ çl-l" refers to the
process in which the blood of a ~ e~ed subject is removed and is treated with anagent to forrn an agent-treated blood sample.
One observation of the present invention is that photochemical agents that have
been used in photopheretic methods cause a previously known increase in MHC
eA~"es~ion, particularly MHC Class I, on the treated cells. Accordingly, the agent used
in the present method can be any agent that will act to increase MHC expression,particularly MHC Class I tAIures~ion. For cells that do not express MHC proteins, any
agent that leads to an increase in cellular proteolysis can be used. The tre~tm~nt agent
may further be an agent that has an affinity for an important component of blood cells
or for a particular disease effector cell.
2 0 The agent used in the present method can be, but is not limited to, chemical
agents and physical agents. For example, the agent may be a chemical compound that
induces MHC ~;A~re~ion and/or cellular proteolysis, such as a photoactivatable drug
such as psoralen. Alternatively, the agent may be a physical tre~tmPnt that the blood is
subjected to. For example, W light, heat shock and other envirol""~"l~l stresses have
2 5 been shown to induce MHC Class I expression in other experimental contexts. As
outlined below, a skilled artisan can readily identify agents for use in the present
methods based on the ability of the agent to induce MHC expression on treated
(disease effector) cells.
Exemplary photoactivatable chemical agents that can be used with the present
3 o methods include, but are not limited to, psoralens, porphyrins, pyrenes, phthalocyanine,
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photoactivated cortisone, photoactivated antibodies specifically reactive with the
disease effector cells present in the blood, photoactivatable dyes, and monoclonal
antibodies which have been linked to porphyrin molecules. Exemplary non-
photoactivated chemical agents inchl~lç7 but are not limited to, chemotherapeutic
5 agents, such as cyclophospharnide or methotrexate, and cytokines, such as TNF-alpha,
and interferon-gamma. Exemplary non-chemical agents inclllde, but are not lirnited to,
WA iradiation, X-ray irradiation, gamma-ray iradiation, hydrostatic or other pressure,
heat or cold shock and ultrasound.
The psoralens are a pr~elled class of photoactivatable agent that are used in
10 current photopheretic methods. Following oral ~mini~tration, psoralens are absorbed
from the digestive tract, reaching peak levels in the blood and other tissues in one to
four hours and are excreted almost entirely within 24 hours following oral
~d,.,;~ lion. These agents can alternatively or additionally be added directly to the
extracorporeal bloodstream.
The psoralen molecules are inert prior to exposure to irradiation and are
transiently activated to an excited state following irradiation. The transiently activated
psoralen molecules are capable of forming photoaddition products with cellular DNA,
proteins, or lipids and generating other reactive species, such as singlet oxygen, which
are capable of modifying other cellular components, e.g., cell Ill~ ne and
Z O cytoplasmic components such as proteins and aromatic amino acids. Although other
agents such as mitomycin C and cis-platinum compounds also damage DNA by cross-
linking strands of the nucleic acid, such altemative agents remain in an active state
following reinfusion, cause systemic adverse effects and thus are not as desirable as
psoralens for achieving the purposes of the invention.
2 5 The prere- I ed psoralens include 8-methoxypsoralen (8-MOP), 4' aminomethyl-
4,5', 8 trimethylpsoralen (AMT), 5-methoxypsoralen (5-MOP) and trimethyl psoralen
(TMP). These and other analogs of 8-MOP are described in Berger, ef al., Annals
N.Y. Acad. Science 453:80-90 (1985). The conditions for oral admini~tration of
8-MOP are described in U.S. Patent No. S,147,289. 8-MOP is the preferred psoralen
3 0 for use in accordance with the methods of the invention.
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When using a photoactivatable agent, the agent-treated blood sample is further
irradiated with ultraviolet or visible light during the agent ~- ea~ l step, for example
- see U. S. Patent No. 5,462,733, issued to Edelson et al., for a discussion of the
irradiation conditions for activating photoactivatable agents such as psoralen
compounds. Photopheresis procedures also are described in U.S. Patent Nos.
4,321,919; 4,398,906; 4,428,744; 4,464,166; and 5,147,289, all issued to Edelson e~
al.
The ~ .,enl of the blood or purified disease effector cells with the tre~tmçnt
agent can be done, as is known in the art, on a continuous stream of blood or in a
batch wise manner. Continuous extraco~uoreal tre~tm~nt can be divided into five
stages: (1) blood collection; (2) centrifugation; (3) agent tre~tmPnt; (4) cell pooling
and (5) reinfusion. The choice of the agent tre~tmPnt method used will be based
primarily on the disorder being treated, the agent used and the f~c.ilities that are
- available.
During the agent trÇ~tment step, such as in the use of a photoactivatable agent,the agent can be present within or on the surface of the cells of the blood sample. This
is typically accomplieh~d by ~minist~ring the agent to the subject prior to obtaining
the blood for extracorporeal tre~tm~nt or by injecting the agent directly into the
extracorporeal blood stream when using a continuous stream Lle,-l...ç~.l method.2 0 In contrast to the literature that sllgge.ets that an underlying mec~ lll of
photopheresis involves subtly modifying antigen pres~ g cells to enhance their
ability to induce an immllne system response, as shown in the Examples, ~le~l."r.nt of
the extracorporeal blood leads to an increase in MHC Class I t~AlJI es~;on and allows for
an increase in the rate and extent that ~ntig~n.c are transported and bound (weakly) to
2 5 surface MHC molecules. Such antigens then become available for presentation by
dendritic or other antigen pres~nting cells, which are added to the blood sometime
prior to reinfusion.
VI. Dendritic Cells Addition
The extracorporeal treatment methods of the present invention rely on the use
3 0 of dendritic cells, or other antigen presenting cells, in combination with the
~, .... .
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extracorporeal agent tre~tm~nt. Dendritic cells, or other antigen plesenLi"g cells, are
added directly or indirectly to the blood co~ g the disease effector cells to form a
therapeutic mixture comprising agent treated disease effector cells and added dendritic
cells prior to re-infusion into the subject.
Dendritic cells are added to the blood or purified disease effector cells (before
or after agent tre~tment) in an amount sufficient to enhance the immune system
response of the subject to the one or more disease-associated ~ntig~n~ Such an
increase is measured relative to the level of jmm~1ne system response that would have
been intl~1ce~ had the extracorporeal blood lle~ been performed in the absence of
the added dendritic or antigen pl es~ g cells. In general, the amount of dendritic
cells contained in a single dose to achieve this purpose is appro~il"a~ely 1 million cells,
but can range from about one thousand cells to about one hundred million cells per
dose. However, as described below, larger numbers of dendritic cells can be prepared
and introduced into the treated blood to obtain multiple doses of antigen-loaded and
non-antigen loaded dendritic cells. In general, these cell numbers are consistent with
the cell numbers described in Zitvogel, L., et al., JExp Med 184:87-97 (1996) for an
animal model in which peptide-loaded dendritic cells were ~imini~tered to a tumor-
challenged mouse to enhance the animal's specific immnne system response to a solid
tumor. According to Zitvogel, et al., in a weakly immnnogenic tumor model, animals
2 0 were injected three to four times, starting at day 4 or day 8 after tumor establi~hm~nt
and subsequently, every 4 days, with 3-5 x 105 dendritic cells pulsed with peptides. In
the more immnnogenic tumor model, animals were injected on days 14, 21, and 28
after initial intradermal (i.d). tumor inoculation. In an analogous manner, booster
immnni7~tions for human subjects are designed to take into consideration the
2 5 immunological state of the subject in accordance with standard clinical practice.
Methods for obtaining dendritic cells are well known in the art and are
described in detail below. In general, the dendritic cells used will be obtained from the
subject sometime prior to reinfusion of the treated blood. The dendritic cells can be
obtained at the same time as the blood is removed for trç~tm~nt or can be obtained
3 0 prior to of after blood removal.
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In one application, the dendritic cells can be activated in vivo prior to their
removal from the subject. A variety of method can be used to activate dendritic cells
in vivo prior to their removal. For example, activation can be accomplished by
~rlln;~ e~ illg a sufficient dosage of GM-CSF to the subject prior to removing of the
5 dendritic cells. As used herein a '~sufficient dosage of GM-CSF" is the amount and
frequency of a~l~...n~ alion of GM-CSF that is sufficient to increase the number and/or
activation state of the dendritic cells in the subject. Exemplary dosages of GM-CSF
for increasing the number and/or activation state ofthe subject's dendritic cells are
provided in the Fx~mplç~ (see, "Isolation of Dendritic Cells from Human Blood").In such a use, the steps of the present method comprise~ minist~ring GM-
CSF to the subject prior to removal of dendritic cells, wherein the dosage of GM-CSF
is sufficient to increase the number and/or activation state of the dendritic cells in the
subject; 2) obtaining blood co..l~ g disease effector cells that express one or more
- disease associated ~ntigen~ from the subject; 3) obtaining and culturing, in vitro,
15 dendritic cells, or other antigen pres~nting cells, from the subject; 4) treating the blood
cc.,.l~;r~ the disease effector cells with an agent that increases MHC Class I
t;A~"~ssion (extracorporeal blood l,eal",elll); S) introducing the dendritic or other
antigen prese.~ cells into the treated blood mixture to form a therapeutic mixture;
and (6) reinfi-~ing or otherwise introducing the therapeutic rnixture into the diseased
2 0 subject.
In general, the isolated dendritic cells can be introduced at any stage during the
extracorporeal trç~tment process: (1) during the blood or tissue collection step;
(2) prior to or after the disease effector cell isolation steps (i.e. centrifugation);
(3) prior to or after the agent tl~ step; and (4) prior to or after disease effector
2 5 cell pooling. Thus, the dendritic cells can be introduced to the treated blood/tissue
before, during, or after agent tre~tm~nt. The advantage of introducing the dendritic
cells during the blood collection stage is that a high dendritic cell concentration can be
achieved by adding the dendritic cells directly to the blood collection bag. However, if
centrifugation procedures are used to separate blood into plasma, white blood cell and
30 red blood cell components it is not 100% efficient and some dendritic cells may enter
,, .. .~.. ... . ...... . .. .. . . ... .
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the plasma and/or red blood cell fractions and not be proximal to the antigens
immetii~tçly upon their release from the agent treated cells in the white blood cell
fraction. Accordingly, it is pl ~Ç~I I ed that the dendritic cells be introduced into the
extracorporeal blood stream during one or more of the agent tre~tment stages to
5 ensure sufficient numbers of peptide-loaded dendritic cells for subsequent reinfusion
(e.g., injection).
It is believed that centrifugation forces, alone, or in conlbindlion with agent
tre~tm~nt, such as during irradiation in the presence of a photoactivatable agent,
f~nilit7~tes the release and llansr~r of disease-associated ~ntigen~ from the disease
10 effector cells by increasing contact between the dendritic cells and the disease effector
cells. Accordingly, introducing the dendritic cells during centrifugation
advantageously places the released disease-associated antigens in close proxirnity to
the added dendritic cells, thereby f~cilit~ting transfer of the released peptides from the
disease effector cells to the MHC sites of the dendritic cells and, presumably,
15 ~ g enzyrnatic digestion of the released peptides.
Alternatively (or additionally), the dendritic cells can be introduced into the
treated blood/tissue during the agent tre~tment stage. For I A...I,le, the agenttre~tmrnt stage of photopheresis is pel rOI Illed by passing the disease effector cell
cot-~ g fraction through an irradiation exposure field that is positioned between
2 0 opposing irradiation sources. Introducing the dendritic cells during the irradiation
stage places the dendritic cells in close pr~xill~ily to the disease effector cells at the
time of their irradiation, thereby f~ lit~ting ll~nsrer of the released peptides to the
dendritic cells and ...;Il;,~ g enzymatic digestion.
Further, the dendritic cells can be added prior to agent tre~tm~.nt and are then2 5 treated along with the disease effector cells. Although not infçntlin~ to be bound to a
particular theory, it is believed that agent L- e~ L of the dendritic cells (i.e., the
photopheretic irradiation of the dendritic cells in the presence of a photoactivatable
agent) may also activate the dendritic cells to release cytokines (e.g., IL-l2, IFN-
gamma, FNF-alpha, GM-CSF, IL-3), an effect that further ~nh~nces the immlmç
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system response following rei&sion of the antigen-loaded dendritic cells to the
subject.
Alternatively (or additionally), the dendritic or other antigen presenting cellscan be added to the blood admini~tration bag prior to reinfusing the agent treated
5 disease effector cells. Methods for introducing the dendritic or other cells at any stage
in the treatment process are based on conventional procedures and can be readilyadapted for adding dendritic cells into a treated blood prepalaLion. For example, at
each stage of ll~a~ process, conventional intravenous tubing connections provideaccess ports through which the dendritic cells can be injected into, for example, the
10 blood collection bag, the centrifugation app~ alus, the tubing located in theagent/irr;~ tion Ll e~ f nl chamber and the blood re-i&sion bag. Additional
re~nts, such as cytokines, also can be introduced via these same infusion ports.In the above methods, the dendritic or other antigen presçnting cells can be
added directly or indileclly to the blood or tissue. As used herein, direct ad~hion
15 refers to adding the dendritic cells directly to the blood or tissues (before or after
ln,~.nl) under condition in which there can be direct cell-to-cell contact between the
added dendritic cells and the disease effector cells.
As used herein, indirect addition refers to adding dendritic cells to the blood or
tissues (before or after L~e~,n~.-l) such that the dendritic cells do not come into direct
2 0 contact with the disease effector cells. For example, a filter membrane, dialysis
membrane or other partitioning ,~ llbl~lle can be placed in between the dendritic cells
and the disease effector cells. Such a partition acts to allow the transfer of disease
associated antigçn~ to the dendritic cells but does not allow mixing of the cell types.
The pl~r~lled partitions will have a pour size of no greater than about 6 microns since
2 5 this is the appro~hnaLe size of the small cell that would need to be prevented from
passing through the partition. There is no lower limit, however, the pour size must be
large enough to allow passage of antigens and other cytokines released from the
treated disease effector cells. Alternatively, the treated cells can be remove from the
treatment solution, for example by centrifugation, leaving released di~ç~ed associated
30 antigens, and the dendritic cells can be added to the rçsl-ltin~ cell free solution.
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Indirect addition is p~ ere. l ed in most methods because it avoids potential problems that
may be associated with reintroduction of treated disease effector cells to a subject.
The dendritic cells preferably are obtained from peripheral blood but may be
obtained from bone marrow, Iymph nodes, infiltrated tumors and/or rejected organs.
5 The dendritic cells should be "genetically identical" to the cells of the subject.
Accordingly, the dendritic cells of the invention are preferably autologous cells: being
obtained from the subject or an identical twin of the subject, or are genetically
engineered to be recognized as an autologous cell by the subject's immune system.
Theoretically it is possible to isolate large numbers of dendritic cells from
1 0 peripheral blood (e g., by an affinity method in which the dendritic cells are specifically
absorbed from the blood and concentrated), for example see Radmayer et al., Int. J.
Cancer 63:627-632 (1995). However, it is preÇelled that the dendritic cells be
cultured in vitro to expand their number prior to introducing the cells to the treated
- extracorporeal blood stream. In general, the cultured dendritic cells have the same or
1 5 greater pl esen~a~ion characteristics (i.e, the number and type of MHC molecules) on
their surfaces a naturally-occurring dendritic cells that have been freshly isolated from
the subject.
The dendritic cells can be altered, for example, by increasing the number of
empty Class I or Class I sites on the surface of the cell, prior to introducing the cells to
2 0 the treated blood. This may be accopli~l-ed in accordance with the methods
disclosed in PCT application number US93/11220, publication number WO 94/11016,
entitled "Specific Tmmllne System Modulation" (Edelson, R. et al). In addition,
culturing dendritic cells provides a source of dendritic cells that can be used for
booster inoculations.
2 5 Numerous references have described culturing dendritic cells, cont~cting the
cultured dendritic cells with a purified antigen to form an antigen-loaded dendritic cell
and a~mini~t~ring the antigen-loaded dendritic cell to an animal to enhance a specific
immlme response to the antigen. For example, St~inm~n et al. (PCT/US93/03141,
publication no. WO 93/20185) disclose a method for producing proliferating cultures
3 0 of dendritic antigen pl ese~ -g cell (DAPC) precursors The method involves isolating
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the precursors and culturing the precursors in the presence of a cytokine. Stç-nman et
al. report that GM-CSF is an ess~nti~l cytokine for dendritic cell culturing in vifro.
Accordingly, Stçinm~n ef al. recommend ~1mini~tration of GM-CSF to a subject prior
to s~mpline the subject's blood to obtain dendritic cell precursors for proliferation in
5 vifro. stçinm~n e~ al. further report that the cultured, imm~tllre dendritic cells can be
pulsed with antigen in vifro and will phagocytose the antigen and process it into a form
which is presented on the dendritic cell surface, i.e., the Sttoinm~n dendritic cells must
phagocytose the antigen for proper antigen presel~lalion in Class II.
The Steinm~n method involves (a) providing a tissue source (e.g., blood, bone
10 marrow) CO~Ail~ g dendritic cell precursors; (b) treating the tissue source to increase
its proportion of dendritic cell precursors to obtain a population of cells which is
suitable for culture in vifro (e.g., by cont~cting the tissue source with GM-CSF);
(c) culturing the tissue source on a substrate and in a culture media co~ e GM-
- CSF, or a biologically active derivative of GM-CSF, to obtain prolir~l aling
15 non~lh~rent cells and cell clusters; (d) subculturing the non~h~rent cells and cell
cultures to produce cell aggregates cGn~,risiilg proliferating dendritic cell precursors;
and (e) serially subculturing the cell aggregates one or more times to enrich the
proportion of dendritic cell precursors.
Mature dendritic cells are produced from the prolirel~ling cell cultures by
2 0 continlling to culture the dendritic cell precursors for a period of time sufficient to
allow these cells to mature into mature dendritic cells. Mature dendritic cells are
identified by cell markers such as, for example, high MHC Class II, 2AI positivegranules, and interdigit~ting cell (NLDC) antigen. In contrast to the Steinman ef al.
teaching~, a plefelled embodiment ofthe instant invention involves introducing mature
2 5 dendritic cells into the treated blood to form disease-associated antigen-loaded
dendritic cells. Thus, there is no requirements that the dendritic cells of the instant
invention be in an ;~."~ e state and capable of phagocytosis to present the disease-
associated antigens of the invention.
Dendritic cells (or precursors) are cultured in the presence of GM-CSF, IL-4
3 0 and fibroblast growth factor at a concentration that is sufficient to promote the survival
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and proliferation of the dendritic cell precursors. This amount depends on the amount
of competition from other cells (e.g., macrophages ad granulocytes) for the GM-CSF,
etc., as well as on the presence of GM-CSF, etc., inactivators in the cell population. In
general, dendritic cells are cultured in the presence of between about 1 and 1,000 U/ml
of GM-CSF. More pl erel ~bly, dendritic cells that are obtained from blood are cultured
in the presence of GM-CSF at a concentration of between about 30 and 800 U/ml.
Most preferably, the GM-CSF concentration is between about 400-800 U/ml for
culturing proliÇel~ing human dendritic cells from blood. Higher conce,~ lions o
GM-CSF (e.g., between 500-1,000 U/ml) are preÇelled for culturing dendritic cells
1 0 obtained from bone Illallow. The GM-CSF may be isolated from natural sources,
produced using the I econ-bin~ DNA techniques or prepared by chemical synthesis."GM-CSF" is defined as any bioactive analog, fragment or derivative of the naturally
occurring (native) GM-CSF. This definition inr.ludes fr~gment~ and derivatives of
GM-CSF provided that the fr~gment~ or derivatives promote the proliferation and
1 5 culture of dendritic cell ~ ;UI :~Ol ~ and, in addition, can be i~entified by their ability to
bind to GM-CSF receptors on the appr~pliate cell types.
Additional cytokines may be optionally inclll~led in the culture medinm to
further increase the yield of dendritic cells. These include such cytokines as IL-4 (at
app~ ,lllately the same U/ml as GM-CSF (See, e.g., L. Zitvogel, et al., J E;xp Med
183:87-97 (1996)); IL-1 alpha and beta (1-100 LAF U/l); TNF-a (5-500 U/m); IL-3
(25-500 U/ml); monocyte-macrophage colony-stim~ ting factor (M-CSF, 100-1,000
U/ml); granulocyte colony-sfiml-l~ting factor (G-CSF, 25-300 U/ml); stem cell factor
(SINGLE-CHAIN FORMS, 10-100 ng/ml); IL-6 (10-100 ng/ml); and FGF (lng-500
U/ml). TNFa at concentrations from about 10-50 U/ml reportedly increases dendritic
2 5 cell yields several fold. A skilled artisan can readily adapt know dendritic cell culturing
methods for using with the present invention.
A panel of monoclonal antibodies may be used to identify and characterize the
cells in the GM-CSF-e~cp~ntled cultures to ensure that they are dendritic or other
antigen ~les~ g cells. Antibodies that are suitable for identifying mature dendritic
cells include, but are not limited to: (1) those which bind to the MHC Class I antigen
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-21-
(Ml/42 anti-M:HC Class I, ATCC number TIB 126); (2) those which bind to the MHC
Class II antigen, B21-2 anti-MHC Class II, ATCC number TIB 229), (M5/114 anti-
MHC Class II, ATCC number TIB 120); (3) those which bind to heat-stable antigen
(M1/69 anti-heat stable antigen, HSA~ ATCC number TIB 125); (4) 33DI anti-
5 dendritic cell antibodies, ATCC number TIB 227; (5) those which bind to the
interdigit~ting cell antigen (NLDC 145 anti-interdigit~ting cell, Kraal, G., et al., JExp
Med 163:981 (1986)); and (6) those which bind to ~ntig~nc in granules in the
perinuclear region of mature dendritic cells (monoclonal antibodies 2A1 and M342,
Agger, R., et al., Int Rev Immunol 6:89 (1990)). Additional ~ntig~nc that are
1 0 e,.~,lessed by dendritic cells that can be used to identify mature dendritic cells include
CD44 (iA~ntified with monoclonal antibody 2D2C), and CD1 lb (identified with
monoclonal antibody M1/70). (See, e.g., Monoclonal Antibodies~ New York, Plenum
1980,Ed. R. Kennett etal., pp. 185-217foradescriptionofsomeofthemonoclonal
antibodies which are used to identify ~ntig~nc which are eApressed on mature dendritic
1 5 cells). One skilled in the art will recognize that other antibodies may be used to
characterize and identif~ mature dendritic cells and also to characterize and identify
precursor dendritic cells and to di~tin~lich these stages of dendritic cell growth.
Although mature dendritic cells are pre~.led for introduction into the treated
blood, either during or following agent tre~tmPnt, imm~tl~re dendritic cells also can be
2 0 pulsed with the disease-associated antigen p~ lions of the invention. Thus,
cont~cting the mature or imm~t~lre dendritic cells in vitro with the antigen preparations
of the instant invention results in a composition cont~ining antigen-loaded dendritic
cells in which the antigen is presented on the surface of the dendritic cells. Although
not intending to be bound to a particular theory, it is believed that the mature dendritic
2 5 cells present antigen by loading the (released, disease-associated) antigen directly into
the empty MEIC sites or, alternatively, by exch~nging the disease-associated antigens
for peptides that already are present in the MHC sites of the mature dendritic cells. In
contrast, the imm~t~1re dendritic cells present antigen by the foregoing mech~nism, as
well as by phagocytosing released antigens, processing the released ~ntig~?n.c into
", ,, . _. ... , ~ .. ,
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- 22 -
smaller fragments, and eApless;ng the smaller fr~Em~nts in association with MHC
molecules on the surface ofthe antigen plese..~ E cells.
Although various references have disclosed culturing dendritic cells and the useof such cultured cells in modifying an imm--ne system response, the introduction of an
5 exogenous antigen pres~ntinE cell, such as a dendritic cell, particularly a mature
dendritic cell, into an extracorporeally treated blood stream to enhance an immllne
system response has not been previously described. Further, the art did not teach that
antigen loaded or non-antigen loaded dendritic cells can be used for booster
inocluations following reinfusion of extracorporeally treated blood. It is believed that
10 the absence of such a te~çhinE within the art is consistent with the lack of a complete
underst~nr~inE ofthe m~ch~nicm underlying extracorporeal blood lle~l...~..l methods
such as photopheresis, i.e., the failure by those skilled in the art to recognize that
~ photopheresis induces release of disease-associated peptides. In the absence of
recognition of this effect of photopheresis, one would not be motivated to add
exogenous antigen ples~ E cells to the treated blood for the purpose of enh~n~inE
an immllne response to the released disease-associated ~ntig~n~
In addition to dendritic cells, other types of antigen p~s~ E cells can be used
in accordance with the methods of the invention, i.e., by sub~ -E these alternative
antigen plt;sr~ -E cells for the dendritic cells in the methods and compositionsdisclosed herein. For example, C~n~di~n patent application 2,069,541, entitled
"Induction of an antigen-specific T-lymphocyte response" (inventors, Melief, et al).
describes antigen p,ese~ E cells which are incapable of loading peptide into MHCsites, i.e., the cells have an antigen processing defect which prevents proper antigen
prese.,lalion at the cell surface. As a result, the M:HC sites of these defective cells are
2 5 empty and available for binding to the disease-associated antigens of the instant
invention. In general, these cells have a defect in one of the cellular gene products
responsible for peptide transport into the subcellular co",pa, L..l~ , (endoplasmic
reticulum and golgi appa~ ~LIls) where peptide loading into MHC Class I or MHC
Class II molecules takes place. The exemplary processing defective cell lines include
30 RMA-S cells of murine origin and 174.CEM T2 cells of human origin (Salter, R.D. et
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- 23 -
al., EMBO J 5:943-949 (1986)). There is no requirement that a processing defect in
the antigen pres~ntine cells-ofthe invention be complete, provided that the cells
express an increased population of cell surface MHC Class I or Class II molecules
which are devoid of endogenously processed peptides. Such cells are capable of
5 inr~llçin~ a primary CTL response when appropliately loaded with MHC Class I
binding peptides.
The antigen plesP...~ g cells used in the present methods can also be antigen
presentine cells that have been treated with ~ntiC~n~e oligonucleotides to inactivate one
or more genes responsible for proper antigen processing and presentation at the cell
10 surface. This approach increases the number of empty Class I molecules on theantigen presç~ g cells, thereby making these cells more capable of binding antigenic
peptides released from treated disease effector cells. Thus, for t;~nl~,le, dendritic cells
or other antigen pres~ g cells are incub~ted with ~nti~en~e oligonucleotides under
con~lition.~ to permit hybridization of the ~nticet~e oligonucleotide to the processing
15 gene or mRNA (e.g., the human TAP-2 gene). The TAP genes encode proteins which
are necess~ry for the ll ~InSpGll of relevant cytoplasmic peptides to Class I molecules,
prior to their joint transport to the cell surface. Therefore, inhibition of the formation
of TAP proteins, climini~hes filling of Class I molecules with peptides. This
circn.,.~l~nce will, hence, increase the amount of surface "empty" Class I. Exemplary
2 0 conditions and oligonucleotides for inactivating the TAP-2 gene in cultured RMA and
EL4 cells or freshly isolated splenocytes are provided in Nair, S. et al., Jlmmunology
156:1772-1780 (1996). In particular, S. Nair report that MHC Class I e"plession was
decrcased in a~)pro~ hndlely 30% of the cells which had been treated with the AS-1 or
AS-2 ~ntisen~e oligonucleotides. These oligonucleotides are complementary to two25 dirrelelll regions ofthe TAP-2 mRNA and were synthesi7ed as 25 nucleotide long
phosphorothioate derivatives. (See, Nair, S. et al., ibid. for sequence and storage
information for these ~ntisen~e oligonucleotides).
The following procedure, based upon that described by Nair, S. et al., ibid., isused for pl~p~ing a pleÇ~led antigen presenting cell for use in the present method.
3 o Briefly, antigen presenting cells (preferably in log phase) are washed in medium (e.g.,
.
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- 24 -
Opti-MEM medium, Life Technologies), resuspended in medium at 5 to 10 x 1 o6
cells/ml and added to 24-well or 6-well plates. A cationic lipid, e.g., Lipofectin (Life
Technologies), is used to deliver the antieçn~e oligonucleotides into cells as described
by Chiang et al., JB~ol Chem 266:18162 (1991). Other methods known in the art for
5 delivering oligonucleotides into cells (e.g., receptor-m~ ted delivery, electroporation)
can be substituted for the Lipofectin method described herein. The oligonucleotides
and Lipofectin are added to medillm at the desired concenllalion (see below) andmixed in a 12 x 75 rnm polystyrene tube at room temperature for 20 min. The
complex is added to the cells to achieve a final concentration of 400 nM
oligonucleotide and 15 ~lg/ml Lipofectin and incubated at 37~C for 6 to 8 hours. The
~nti.ce.n~e-treated cells are washed, incub~ted at subphysiologic temperature (preferably
in the range 23 to 30~C) for 24 to 48 hours, and analyzed for MHC Class I e~ ession
by flow cytometry, used as stin~ tors for CTL induction using standard procedures
(e.g., chromium release assay) and/or used as antigen ples~ , cells for subsequent
presentation in vivo of disease-associated peptides (e.g., by r~infi~sing or otherwise
introducing the ~nti~çn~e-treated cells to the extracorporeal blood system or byincub~ting the ~nticçn~e-treated cells in vitro with disease-associated peptides and
subsequently introducing the peptide-loaded ~nti~P.n~e-treated cells to the subject.
Preferably, the ~nti~en~e-treated cells are incub~te~ with beta2-microglobulin prior to,
2 0 or concurrent with, incubating the ~nti~en~e-treated cells with disease-associated
peptides. lt is believed that prior trç~tment or co-incubation ofthe ~nti~n~e-treated
cells with beta2-microglobulin and disease-associated peptide f~cilit~tes loading of the
peptides into the empty MHC Class I sites ofthe anti~çnse-treated antigen plese~.l;g
cells.
2 5 In a particularly prerel ~ ed embodiment, one or more cDNAs (or gene
sequences) encoding the following proteins are introduced (e.g., transfected) into a
processing defective cell line (e.g., a T2 cell line) to obtain an improved antigen
pres~nting cell for use in accordance with the methods ofthe invention: (1) a
cytokine(s) (e.g., GM-CSF, IL-12); (2) an accessory molecule such as a costim~ tory
3 o molecule(s) (e.g., B7-ltCD80 and B7-2/CD86 (Mayordomo, J., et al., Nature Med
CA 02249412 1998-09-18
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- 25 -
1:1297-1302 (1995)) or an adhesion molecule (e.g., ICAM-l/CD54; ICA-3/CD50
(Young, J. etal. JExpMed 183:7-11 (1996) and references cited therein); and (3) one
or more ofthe MHC Class I molecules ofthe subject. Human B7-1 and B7-2 are
described in Free~lm~n, A.S., et al., JImmunol 137:3260-3267 (1987), Freeman, G.J.,
etal., Jlmmunol 143:2714-2722 (1989), Freeman, G.J., etal., Science 262:909-911
(1993) and Azuma, M., et al., Nature 366:76-79 (1993). Thus, an improved dendritic
stock cell line can be pl~pared by introducing one or more cDNAs encoding a cytokine
(plt;rel~bly, GM-CSF) and an accessory molecule (preferably, a B7 and/or ICAM-1
molecule) into a processing defective cell line (p~ bly, a T2 cell line). (See, for
c~llple, Paglia, P., etal., JExpMed 183:317-322 (1996), for an exemplary
procedure for tr~n.cduçing dendritic cells with the gene encoding GM-CSF in a mouse
model system). The improved dendritic stock cell line can be prepared and ~ it-tAil-ed
in accordance with slal~dard procedures known in the art for introducing and
e~lessing genetic material in m~mm~ n (preferably, human) cells. Moreover, in the
1 5 prt;r~,led embo~lim~nt~J the dendritic stock cell line is used to prepare subject specific
antigen pre~ g cells by, for example, introducing the cDNA encoding the subject's
MHC Class I molecules into the stock cell line using standard genetic engine~ring
procedures. Of course, such ll~nsrolllled antigen ples~ ;ng cells are treated (e.g.,
gamma-irradiated) to prevent further cell division prior to ~ a~ion to the subject
2 0 The amount and nature of irradiation that is sufficient to prevent further cell division is
determined empirically by irradi~ting the cells with a preselected radiation source (e.g.,
gamma-irradiation, 8-MOP and ultraviolet irra~i~tion, X-irradiation, 8-MOP and
visible irradiation) over a range of intensities (e.g., 1000 to 3000 rads) for preselected
time periods (e.g., 0.5 minutes to 24 hours) and observing whether any clones develop
2 5 over a period of time, usually a one month period. The amount and nature of the
irradiation is selected which is sufficient to prevent any clones from developing during
this time period. Typically, 2000 rads of gamma-irradiation reportedly is sufficient to
achievethispurpose. (See,e.g.,ZitvogeLL.,etal.,JExpMed183:87-97(1996)).
In contrast to the above-described transformed cell lines which must be
3 o irradiated to prevent further cell division in vivo, cultured dendritic cells that are
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-26-
derived from the subject do not require irradiation prior to reintroduction to the
subject. However, dendritic cells which have been loaded with disease-associatedantigens preferably are irradiated (e.g., with gamrna-irradiation or within the
extracorporeal stream during photopheresis) prior to reintroduction (e.g., i.v. infusion)
5 to the subject to prevent in vivo processing and undesired p~esentaLion by the dendritic
cells of autologous (non-disease-associated) peptides (e.g., peptides which could
medi~te an autoimmllne response) following ~dminietration to the subject.
GM-CSF and IL-4, cytokines that are hlll)ol ~anl for dendritic cell culturing,
optionally are co~-~minietPred with the antigen-loaded dendritic cells to the subject to
10 further çnh~nce the subject's immune response to the presented antigen. In addition to
these key cytokines, TNF-a and IL-12 also reportedly are important for dendritic cell
medi~ted imml-n~ system modulation, presumably by intlllc.ing a CD8 T cell response.
Accordingly, the methods of the invention optionally include the step of introducing
the dendritic cells to the treated blood in the presence of one or more of the following
cytokines, GM-CSF, IL-4, TNF-a and IL-12. Preferably, a TNF-a and/or IL-12 are
introduced with the dendritic cells to the treated blood or at some stage prior to
reinfusion.
At any stage in the extracorporeal tre~tment process, following introduction of
the dendritic cells, a sample of the therapeutic mixture may be taken and assayed to
2 0 determine, for example, the number of viable dendritic cells and/or the number of
disease-associated antigen-loaded dendritic cells, using the markers listed above. By
using these markers in conjunction with fluorescein di~cet~te, which cytopl~emic.~lly
labels non-viable cells, the number of viable dendritic antigen pres~ g cells can be
determined. The number of viable dendritic cells can be determined in accordance with
2 5 standard practice, e.g., by trypan blue exclusion assay. The number of antigen-loaded
dendritic cells can be deterrnined, for example, using a cytotoxic T cell assay as
described in PCT publications WO 94/02156 or WO 94/21287. Alternative
procedures for ~eseseing the viability and/or functional activity of the dendritic cells are
known to those of ordinary skills in the art and can be perforrned using routine3 o ~pe,;,.,~nt~tion. See, e.g., PCT publications WO 93/20185, WO 93/03766,
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W O 95/29698, W O 94/02156, W O 94/20127, W O 91/13632, W O 95/28479,
WO 94/21287 and CA patent application 2,069,541.
VII. Reinfusion
After the dendritic or other antigen pre~enting cells are added to the treated
5 blood (directly or indirect}y) to form a therapeutic mixture, the mixture can be re-
infused into the subject as a treated dendritic cell/treated cell mixture or can be further
processed to obtain isolated, antigen-loaded dendritic cells. The former provides an
efflcient method that can further provide an additional source of disease cell antigen
(i.e. t~e agent treated disease cell antigens), while the later removes potentially viable
10 disease effector cells.
Re-isolating the dendritic cells prior to reinfusion provides a means for
obtaining multiple re-infusion doses and a source of antigen-loaded dendritic cells that
can be used for immllnotherapy. Specifically, the peptide-loaded dendritic cellsgenerated by cont~ctin~ dendritic cells with agent treated blood, can be used as an
15 immlmogen by a~mini~tering the cells to a subject in accordance with methods known
in the art for eliciting an immune response. Preferably, the dendritic cells are injected
into the same individual from whom the source cells were obtained. The injection site
~ may be subcutaneous (s.c.), intraperitoneal (i.p.), intr~mllsc~ r (i.m.), intraderrnal
(i.d.), or intravenous (i.v.). Intravenous ~(lmini.efration ofthe antigen-loaded dendritic
2 0 cell is the p, efe-led route of a~lminictration.
The number of antigen-loaded dendritic cells that are atiministered to the
subject varies as a function of the antigen, the imm.lne status of the subject the size of
the subject and the disease that is treated. In a plerelled embodiment, blood is used as
the tissue source and preferably, the subject is first treated with cytokine to stimlllate
2 5 hematopoiesis. Following isolation and expansion of the dendritic precursor cells, the
precursors are contacted with the antigen prepal ~ions made from agent treated
disease effector cells and/or alternatively are stim-ll~ted by cytokines (e.g., GM-CSF)
to maturity before introducing the antigen. The antigen-loaded dendritic cells are
reintroduced to the subject in sufficient quantity to invoke an immlme response. In
general, between lx106 and 10x106 dendritic cells constitute a single dose for injection
. . ~
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into the subject. Preferably, between about 1 to 100 micrograms of antigen in its
presented form, is a-lmini~tered per dose.
For example, to prepare a sufficient number of dendritic cells (with loaded
antigen) for between ten and one-hundred doses, from about lx108 to about 1x109
dendritic cells are introduced into the extracorporeal blood sometime prior to, during
or following agent tre~tmP.nt. Following contact with the disease-associated antigens
contained in the extracorporeal treated blood, the dendritic cells either process
(phagocytose) the disease-associated antigens and present the processed antigen in
association with MH:C Class I or II molecules or directly load the disease-associated
antigens into MHC Class I or II molecules. Eventually, these antigen-loaded dendritic
cells are pooled during the final stage of the extracorporeal tre~tmçnt and prior to re-
infusion. The pooled dendritic cells, with or without the treated disease effector cells
can be stored for subsequent booster imml~ni7~tions Preferably, these pooled antigen-
- loaded dendritic cells and other treated disease effector cells are distributed in aliquots
prior to storage, each aliquot co~ g an amount of dendritic cells suffficient for a
single dose for injection into the subject. The cells can be stored in accordance with
slalldal-d methods to retain cell viability. Preferably, the cells are stored at -70~C.
VIII) Non-Anti~en Loaded Dendritic Cells
2 0 In another embodiment of the present invention, it was found that non-antigen
loaded dendritic cells can be used for one or more booster inoculations for subjects
that are being treated by the methods of the present invention, are being treated by
other methods in which it is desired to elicit an imml-ne response (for example during
routine vaccination protocols) or are in need of an increase in immlme activity (for
2 5 example to inhibit tumor cell growth). Specifically, isolated or cultured dendritic cells
that have not been contacted with treated disease effector cells, can be introduced into
a subject as a means for increasing cellular and humoral immune responses. In anapplication of this methods, the dendritic cells are treated with an agent in a manner
analogous to the tr~atm~nt described for the disease effector cells. As shown in the
3 0 Examples, dendritic cells that are treated with a photochemical agent, greatly reduced
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or çlimin~te~l tumor growth in an animal model without neetling to contact the
dendritic cell with a treated disease effector cell or disease associated antigen..
IX Antigen Con~ Compositions
Although the prior art reports antigen-loaded dendritic cells for enh~nring
5 cellular jmmllnity to a mixture of solid tumor ~ntigçnC or to purified peptide antigens,
methods for plepa hlg disease-associated antig~nc by subjecting blood to agent
Ll ealnlcllt (such as photopheresis) and using the treated blood as an antigen source
have not been described. Thus, according to another aspect of the invention, antigen
compositions for enhancing an immllne system response are disclosed. The antigen10 compositions have in common a plurality of disease-associated antigens that have been
released from disease effector cells contained in blood by extracorporeal agent
tre~tm~nt as herein described. In addition, the compositions optionally contain a
~letect~kle arnount of one or more protease or peptidase inhibitors. Exemplary
protease or peptidase inhibitors and compositions C~ g the same include: (l) a
15 mixture co~ g bestatin (30 uM), thiorphan (lO uM) and captopril (lO uM);
(2) phenylmethylsulfonyl fluoride (a serine protease inhibitor); (3) n-ethylm~leim;de
and various nonselective peptidase inhibitors (e.g., EDTA, o-ph~nA..Ihroline,
bacitracin); (4) ben7~ ine (2x 1 o2 mol/L); (5) a mixture of peptidase inhibitors
inr.ll~ding am~ct~tin, captopril, phosphoramidon); (6) a mixture of peptidase inhibitors
2 0 such as actinonin (6 uM), arphamenine B (6 uM), bestatin ( l O uM), captopril ( l O uM)
and thirophan (0.3 uM); and (7) one or more of the protease inhibitors that are useful
for treating HIV infection (e.g., ritonavir, saquinavir, indinovir).
The antigen compositions of the invention can be preserved at reduced
te--.pel~L-Ires (e.g., frozen to prevent bacterial growth) or alternatively, can be
2 5 Iyophilized for prolonged storage. Preferably, the antigen compositions are forrn~ ted
to contain an amount of disease-associated antigens for mixing with a single dose of
dendritic cells. In general, each dendritic cell contains apploxi...ately lOO,OOO MHC
sites for binding to antigenic peptides. Accordingly, a single dose of dendritic cells is
introduced into an excess of disease-associated ~ntig~n.c to drive the reaction to
3 0 completion, i.e., to ensure that as many disease-associated antigens as possible are
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loaded onto the MHC sites of the dendritic cells. Thus, a suff1cient number of disease-
associated ~ntigçn~ are allowed to react with each dendritic cell to fill between 300 and
300,000 Class I sites and thereby elicit a specific immllne system response to the
presented antigen. It is well known that as few as three hundred MHC sites occupied
5 by a particular anti~nic peptide are sufficient to elicit an immune response. In general,
this is accomplished by incubating the eluate from ten-fold to one hundred-fold disease
effector cells with one-fold number of dendritic antigen presçnting cells.
According to yet another aspect of the invention, an alternative process for
producing an antigen product for use in ~nh~n~ing an immllne response is provided.
10 The process involves two steps: (a) acidifying a prepa~lion co,~ g a plurality of
disease effector cells for a period of time sufflcient for the disease effector cells to
release disease-associated antigens without immediately Iysing the cells; and
(b) neutralizing the acidified prepal ~lion to form the product. The disease effector
~ cells are obtained from peripheral blood and inr.h~de, for example, m~lign~nt T cells,
15 m~lign~nt B cells, T cells or B cells which me~ te an autoimmllne response, T cells or
B cells which mPrli~te transplanted tissue rejection, and virally, bacterially or
protozoally infected disease effector cells which express on their surface viral, bacterial
or protozoan proteins and/or peptides. Pl ~rerably, the disease effector cells express on
their surface an antigen that is associated with (bound to) an M:HC Class I protein, an
2 0 MIIC Class II protein or a heat shock protein that is capable of transporting peptide to
or from an MHC site. More preferably, the disease effector cells are isolated from
peripheral blood prior to acidification. In the pl ~rel l ed embodimP.nt~, the disease
effector cells are T cells or B cells. More preferably, the disease effector cells are
T cells, preferably within a single family. In the most plerell~d embo-lim~nt~, the
2 5 T cells are of a single clone.
The acidification step of the foregoing process is based upon published
procedures. See, e.g., Storkus, W., etal., Jlmmunotherapy 14:94-103 (1993) and
L. Zitvogel, et al., JExpMed 183:87-97 (1996). An exemplary protocol for acid
eluting antigens from the MHC Class I sites of antigen presentin~ cells is provided in
3 0 the Examples. In the pi~rel I ed embodiments, the acid elution procedure is performed
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at room temperature. In general, the acidific~tion step involves subjecting the
p,epalalion of disease effector cells to a pH of between about pH 2 and pH 6
(preferably between pH2 and pH 4) for between about 0.5 to about 20 minutes In the
prefe:l~ed embodimçnts~ acidification involves subjecting the preparation to a pH of
5 about 3.3 for about one minute. Thereafter, the cell prepal~lion is neutralized in
accordance with standard practice (e.g., by washing pelleted or flask-adherent cells
with buffered tissue culture meclil-m), and the eluted peptides plefél~bly are further
concentrated (e.g., by chlol"d~ography and/or Iyophilization). Performing the
acidification step under these conditions, results in release by the disease effector cells
10 of their disease-associated ~ntie~n~ without immedi~tçly Iysing the disease effector
cells. Optionally, the acid-eluted antigen pleparalion is divided into aliquots, each
aliquot cont~ining an amount of antigen s~fficiçnt for mixing with a single dose of
dendritic cells to ~nh~nce the immune system. Preferably, the aliquots are Iyophilized
to f~çilit~te storage and shipping. Although not inten~iing to be bound by a particular
15 theory, it is believed that inc~lb~tion of cells a pH 3 . 3 in citrate-phosphate buffer
denatures Class I complçYes, res..lting in the release of beta2 microglobulin and
previously Class I-bound peptides into the extracçll-ll~r media (Storkus, W., et aL (J
Immuno~herapy 14:94-103 (1993)). Because the mild pH tre~tment does not
im metli~tely Iyse the disease effector cells, the cells regenerate their Class I peptide
2 0 co~"?!~ es in culture, thereby providing a meçll~ni~m whereby mllltirle batches of
disease-associated antigens can be harvested from the disease effector cells in culture.
In a particularly p,efell~d embodiment, dendritic cells or other antigen
plesç.~ g cells, are subjected to the above-described acid elution/neutralization
protocol prior to cont~ting the cells with the disease-associated ~ntigen~. In this
2 5 manner, the MHC molecules of the dendritic cells are emptied of their endogenous
peptides prior to exposure to the disease-associated antigens, thereby increasing the
number of empty M:HC molecules available for association with the disease-associated
antigens and rendering the acid-eluted dendritic or other antigen presçntin~ cells more
efficient antigen plesç~ll;tlg cells, presumably, by providing an increased number of
3 0 empty MHC sites into which the disease-associated antigens can be loaded. It is
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believed that the above-described acid elution/neutralization protocol also results in
release of B2-microglobulin from the MHC molecules. Accordh~,ly, it is ple~elledthat the acid-eluted, neutralized antigen presenting cells be incubated with B2-microglobulin prior to, or concurrent with, cont~cting the cells with disease-associated
antigens to increase the efficiency of antigen presentation by the acid-eluted,
neutralized antigen plese~ . cells. Further, in the ple~lled embo~im~nts, the acid-
eluted, neutralized dendritic cells are contacted with the disease-associated ~nti~n~ at
a temperature that is less than physiological temperature to further stabilize the empty
MHC sites of these antigen presenting cells.
1 0 In a related aspect of the invention, a product for ~nh~nr.ing an immnne
response is disclosed. The product is produced by the process of: (a) acidifying a
p~ ion co..l~ -g a plurality of disease effector cells for a period of time sufficient
for the disease effector cells to release disease associated ~ntig~n~ without imm~ tely
- lysing the cells; and (b) neutralizing the acidified pl epal alion to form the product.
Pl~r~lably~ the disease effector cells are obtained from pclil~helal blood. As disclosed
above in reference to the process, the product can be an aliquoted product in which
each aliquot cont~in~ an amount of neutralized product sufficient to mix with a single
dose of dendritic cells for ~lmini~tration to a subject to enhance the subject's specific
immllne system response to the presented ~ntig~n
2 0 The specific examples presented below are illustrative only and are not
int~.n-led to limit the scope of the invention.
Example 1
~t;p~lion of Dendritic Cells
Various methods have been reported for the isolation of dendritic cells from,
2 5 e.g., human peripheral biood, bone marrow and spleen cells. For example, PCTApplication No. PCT/US93/06653 having publication Number WO 94/02156, entitled
"Methods for Using Dendritic Cells to Activate T Cells" (Engleman et a/., hereinafter
"WO 94/02156") describes methods for isolating dendritic cells from human blood and
for using the isolated dendritic cells to present antigens for the induction of an antigen-
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specific T-cell-medi~ted imm~me response. More recently, methods have been
reported for the isolation of precursor dendritic cells and their expansion in vitro. For
example, PCT Application No. PCT/US93/03141 having publication Number
WO 93/20185, entitled "Method for in vitro Proliferation of Dendritic cell Precursors
and their use to produce Tmmllnogens" (St~inm~n et al., hereinafter WO 93/20185)describes methods for isolating dendritic cell precursors from human blood, expanding
the i~ol~ted cell precursors in vitro in the presence of GM-CSF, and pulsing theexp~nAed cell precursors with peptide antigen in vitro to obtain peptide-loaded
dendritic cells that are suitable for inAucting an immlme system response. The
1 0 following procedures for i~ol~tin~ and culturing dendritic cells/dendritic cell precursors
from human peripheral blood are based upon the protocols for culturing such cells that
are described in WO 94/02156 and WO 93/20185.
(A) Isolation and Culture of Dendritic Cell Precursors obtained from
Human Blood
1 5 The procedure described herein is adapted from the isolation and culturing
protocols provided in WO 93/20185 (stPinm~n et al). . Briefly, blood mononuclearcells are isolated by sediment~tion in standard dense medium, such as Lymphoprep(Nycomed, Oslo). The isolated mononuclear cells are depleted of cells that are not
dendritic cell progenitors. For example, these CO~ lAlll~ are coated with
2 0 monoclonal antibodies to CD3 and HLA-DR antigens and depleted on petri dishes
coated with affinity-purified, goat anti-mouse IgG ("p~ ing' ) Appro?~hl.ately lo6
cells in one ml of culture me~ m are plated in 16 mm ~i~metçr plastic culture wells
(Co-star, New York). The mçAillm (e.g., RPMI-1640) is supplçmented with typical
growth nutrients (e.g., 50 uM 2-mercaptoethanol, 10 mM gl~1t~mine, 50 llg/ml
2 5 gentamicin, 5% serum from cord blood without heat inactivation or 5% fetal calf
serum (with inactivation)) and human recombinant GM-CSF (preferably 400 U/ml).
Optionally, serum-free me~ m that is apl)ic.pliate for m~mm~ n cell culture can be
used. Every second day thereafter and for a total of 16 days, the cultures are fed by
removing 0.3 ml of the me~ lm and replacing this with 0.5 ml of fresh medium
3 0 supplçm~.nted with the cytokines. Preferably, the cells are cultured n the presence of
, . ~ . ,
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additional cytokines, such as IL-4,~ 2,IL-1 alpha, TNF alpha, D-3, FGF and/or
LAF. Typically, these additional cytokines are added during the last 24 hours ofdendritic cell culturing. These same cytokines optionally are added during
~(imini~tration ofthe antigen-loaded dendritic cells to the subject.
Characteristic prolir~l~ling dendritic cell aggregates (termed "balls" by
St~inm~n et al. in WO 93/20185) appear by the fifth day, as evident by ~Y;.,,~;.,AIion
with an inverted phase contrast microscope. The balls expand in size over the course
of a week. Some balls appear in the original wells, but typically these do not enlarge to
the same extent as the non-adherent wells. The wells are subcultured, e.g., one well is
split into two or three wells, as cell density increases.
Two alternative approaches can be used to isolate mature dendritic cells from
the growing cultures. The first method involves removing cells that are non-adherent
and sepa~aLing the balls from non-balls by 1 g se-l;.n~ ;on. Dendritic cells then are
~ released in large numbers from the balls over an additional one or two days of culture
and the mature dendritic cells are isolated from the non-balls by flotation on dense
metrizamide as previously described (Freudçnth~l and Steinm~n~ ProcNatlAcadSci
USA 87:7698-7702(1990)). Alternatively, mature dendritic cells are isolated by
harvesting the non-adherent cells when the balls are very large. The cells are then left
on ice for 20 minllteS7 resuspended vigorously with a pipette to t~ ntegrate the balls,
2 0 and the mature dendritic cells are floated on m~LIi~llide columns.
GM-CSF reportedly is an ess~.nti~l cytokine for the development of dendritic
cell balls. IL-4 and to a lesser extent, IL-12, also f~rilit~te dendritic cell culture. The
addition of TNF alpha at 10-50 U/ml increases dendritic cell yields approxillla~ely two-
fold. Starting with 60 ml of blood culturing in the presence of GM-CSF alone, the
2 5 yield of mature dendritic cells is between 6 to 12xl o6 cells, leprese~ g 40-~0 percent
of the cells. Alternatively, a sufficient number of dendritic cells for achieving the
purposes of the invention can be isolated from blood without prior culture (see below,
"Isolation of Dendritic Cells from Human Blood").
Other sources of dendritic cell progenitors, e.g., bone marrow, spleen cells, and
3 0 fetal or umbilical cord blood, also can be used. For example, PCT application number
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PCT/US91/01683, publication number WO 91/13632, entitled "Idiotypic Vaccination
Against B Cell Lymphoma" ("Hohlen et al.") describes a protocol for isolating
dendritic cells from spleen. The Hohlen et al. protocol is based upon the methodpreviously reported by Steinm~n and Cohen, JExpMed 139:380-397 (1974).
(B) Isolation of Dendritic Cells from Human Blood
The procedure described herein is adapted from the isolation and culturing
protocols provided in WO 94/02156 (Engleman et al.). Although dendritic cells are
found in both Iymphoid and nonlymphoid tissues, the most readily acceseible source of
dendr~tic cells in man is peripheral blood, which contains less than about 1 dendritic
1 0 cell per 100 disease effector cells. To obtain a sufficient number of dendritic cells
directly from blood without neces~ ;ng dendritic cell culture, a disease effector cell
concentrate is prepared in accordance standard leukaphelesis practice. In general,
appl o~ a~ely two billion disease effector cells are collected during leuka~here~is.
Thus, ~ee~lmin~ that the dendritic cells represent one percent of the total disease
1 5 effector cell population collected by le -k~rhçresis, applo~ alely 20 million dendritic
cells are present in the leukaphelesis disease effector cell concentrate. As diec~lssed
below, this number of cells is sufficient to perform multiple treatment~ in accordance
with the methods disclosed herein. In addition, further culture of these dendritic cells
can be performed to increase further the total number of dendritic cells for therapy.
2 0 For the in vivo priming of an immllne system response, a highly purified dendritic cell
population (of at least about 80%, preferably of at least about 90%) is recon...,ended.
The number of dendritic cells present in blood and, hence, in a le--k~rheresis
disease effector cell concentrate, can be increased by a~lminietering one or more agents
which stim~ te hematopoiesis prior to photopheresis or leukapheresis. Such agents
25 include G-CSF, GM-CSF and may include other factors which promote hematopoiesis.
The amount of hematopoietic agent to be a-lminietered is determined by monitoring the
cell differential of subjects to whom the factor(s) are a~1mini.etçred. Typically, dosages
of cytokine agents, such as G-CSF and GM-CSF, are similar to the dosages of these
agents that are administered to treat subjects recovering from treatmlont with cytotoxic
3 0 agents. Preferably, GM-CSF or G-CSF is ~minietçred for 4 to 7 days at standard
,, . ,.. ~ .. .,~ .. . . .. .
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doses prior to removal of the source tissue (e.g., blood, bone "lal l ow) to increase the
proportion of dendritic cells. (Editorial, Lancet 339:648-649 (March 14, 1992)).Exemplary dosages are provided in St~inm~n et al. (WO 93/20185). For example,
dosages of G-CSF of 300 '~lg daily for 5 to 13 days and dosages of GM-CSF of 400 llg
5 daily for 4 to 19 days reportedly result in a significant increase in dendritic cell
precursors in vivo. It is believed that GM-CSF activates the dendritic cells in vivo,
thereby causing the dendritic cells to release their own cytokines which fi~rther activate
CD8+ cells in vivo. Accordingly, cytokines in addition to GM-CSF (e.g., IL-12 and
IL-4) optionally are coa~mini~tered to the subject to fa~.ilit~te this process.
1 0 In general, human peripheral blood mononuclear leukocytes (PBML) are
isolated from blood s~mples, particularly buffy coat or leukocytes prepared by, for
example, aphereses (optional), Ficoll Hypaque gradient centrifugation followed by
Percoll density centrifugation. Twenty-five to five-hundred milliliters of blood that is
- processed by Ficoll Hypaque gradient centrifugation and Percoll density centrifugation
1 5 will provide a sufficient number of dendritic cells for further expansion in vitro. The
high buoyant density fraction (HD) contains the T cells, B cells and dendritic cells,
whereas the monocytes are co,llailled in the low buoyant density (LD) fraction.
Centrifugation of the HD fraction in Nycodenz/Nycoprep (Nycomed Pharma, Oslo,
Norway) separates the dendritic cells (present in the LD fraction) from the T and
2 0 B cells (present in the HD fraction). The dendritic cells optionally are further enriched
using additional protocols (described below).
Alternatively, dendritic cells are isolated using procedures which involve
repetitive density gradient centrifilgation, positive selection, negative selection, or a
combination thereof. For example, negative selection of dendritic cells can be
2 5 accomplished by panning using antibodies to remove nondendritic cells to result in a
prepal~lion cont~ining approximately 80-90% dendritic cells. Alternatively, positive
selection can be performed in which affinity chromatography is employed wherein
antibodies to dendritic cell surface markers are used as the affinity ~hrolllalography
ligand to remove dendritic cells from a complex mixture. Exemplary antibodies that
3 0 are useful for negative and/or positive selection are described in WO 94/02156.
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Briefly, human dendritic cells can obtained from buffy coats using the followingprocedure. Peripheral blood mononuclear leukocytes (PBML) are isolated by Ficoll-
Hypaque gradient centrifugation (Bouyam, Scand J Clin Lab Invest 21 :21 -29 (1968)).
Blood dendritic cells optionally are further separated by, for example, the methods
described in WO 94/02156. (See, in particular, WO 94/02156, Fig. 1, for an overview
of the separation process). Briefly, PBML are separated into LD and HD fractions in a
four-step discontinuous Percoll gradient (Pharmacia Uppsala, Sweden) (Markowicz
and Engleman, J Clin Inves~ 85 :955-961 (1990)). The HD fraction cont~ining the
dendritic cells is collected and cultured in culture media in Teflon vessels for 16-28
1 0 hours at 37~C. Thereafter, the cells are centrifuged over a Nycodenz/Nycopret
discontinuous gradient (Nycomed Pharma, Oslo, Norway). The dendritic cells are
coll~ained entirely in the LD fraction and occupy ap~ro~lllalely 30-40% of the total
cell population. This partially purified dendritic cell population can be used for T cell
plh~ g and activation ~GIilllents in vivo or in vitro.
1 5 In the p.erell ed embodiments, the dendritic cells are further purified for in vivo
applications. Further purification of the dendritic cell population is achieved by
pelrolll~ing a second round of Nycodenz/Nycoprep centrifugation and collecting the
LD fraction obtained therefrom. The LD fraction contains applo~illlately 80-90%
dendritic cells. Alternatively, the LD fraction following the first Nycodenz/Nycoprep
2 0 step is inc~1bated with antibody-coated petri dishes to remove CD3+, CD14+, CD16+,
and CD20+ cells to obtain a nonadherent cell population CO~ .;.,g between
app~uxilnately 80-90% dendritic cells. In general, these procedures produce a yield of
l-2.5x106 cells from about 400-500 ml of whole blood.
~sessm~nt of dendritic cell purity following enrichment is determined by
2 5 staining with an anti-HLA-DR antibody (e.g., an anti-MHC Class II antibody such as
CA141) which is conjugated to a detectable reagent (e.g., fluorescein), and an
anL.ll.onocyte antibody such as phycoerythrin-conjugated anti-CD14.
Cytofluorographic analysis of the cell population is ~c~es.ced by fluorescence-activated
cell sorters. HLA-DR+ CD14- cells represent the dendritic cell population. In general,
3 0 dendritic cells are readily ~ictin~ hed from other PBML on the basis of their high
.... . ~ , . ... ..
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levels of expression of MHC-Class II dete~ inallls and their lack of CD14 expression.
Further definitive analysis of the cell population is accomplished by determining
whether the putative dendritic cells are also negative for a variety of known T cell and
B cell markers and are positive for a variety of known dendritic cell markers (discussed
above).
Example 2
Preparation of Disease-Associated Anti~ens for In Vitro Loadin~
Disease-associated ~nti~en~ for in vitro loading into dendritic or other antigenpresenting cells ofthe invention are plepared in accordance with procedures known to
1 0 those of ordinary skill in the art. The procedure described herein is based upon the
acid elution protocol described in Zitvogel, L. et aL JExp Med I83:87-97 (1996).(See also, e.g., Storkus, W. e~ al. JImmunotherapy 14:94-103 (1993) for buffer and
reagent preparation for the acid elution procedure).
Disease effector cells (appro~sill-alely l-5x109 cells) obtained from the
1 5 extracol~oreal blood stream are washed three times in HBSS (GIBCO-BRL), and the
cell pellet is treated with rnild acid buffer. Briefly, 10 ml of citrate-phosphate buffer,
pH=3.3, is added at room te,llpel~ re, and the cell pellets are imm~di~t.o,ly
resuspended by, for example, pipetting, and centrifuged for 5 min. at 1,000 g. The
cell-free s~ lllalalll is harvested, and peptides in the acid-extracted supernatants are
concentrated, e.g., on activated SepPak C18 cartridges (Millipore Corp., Bedford,
MA). The bound material is eluted with 2-3 ml of 60% acetonitrile in water and
Iyophilized to near complete dryness (e.g., 20-50 ~ll). The peptides are then
recon~tit~-ted in 1 ml HBSS (GIBCO-BRL) and stored frozen (e.g., at -70~C) untilloading onto dendritic or other antigen pres~nting cells of the invention. In general,
2 5 one million dendritic cells are allowed to react with peptides derived from 1 o8 to 109
effector cell equivalents in a total volume of 1-2 ml of dendritic cell culture merlil-m
(e.g., overnight incubation at 37~C, 5% carbon dioxide). In the preferred
embo~imP.nt~ the loading reaction is performed at a temperature less than
physiological temperature (e.g., between about 22~C and 27~C).
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Example 3
Loading Disease-Associated Antigens onto Dendritic Cells
In the plerelled emborlimçntc, dendritic cells are introduced into the
extracorporeal blood stream at any stage during photophelesis. More preferably, the
5 dendritic cells are acid-eluted or agent treated as diccl-csed above prior to introduction
to the extracorporeal blood stream. Acid elution or-agent tre~tm~n1 (such as using ~-
MOP/UVA) of the dendritic cells induces release by the cells of peptides that may have
become associated with their MHC Class I molecules during cell isolation and/or
culture. Accordingly, in the most pl~r~lled embo(~iments, the acid-eluted dendritic
10 cells are introduced to the extracorporeal blood stream at a temperature less than
physiological temperature (between about 22~C and 27~C) to enh~nce dendritic cell
empty MHC stability and ~ .e enzymatic ~ntiE~nic peptide degradation.
Introduction of the dendritic cells into the extracol yoreal blood stream is
accomplished using standard injection ports (i.e., ports on the intravenous tubing sets)
15 known to those of ordinary skill in the art. Preferably, between lxlO6 and 1 OxlO6
dendritic cells constitute a single dose for injection into the subject. However, as
~isc~c.ced above, a number of dendritic cells to support multiple doses (e.g., 100-fold
the number of cells for a single dose) can be introduced into the extracorporeal blood
stream to prepare a stock prepal~Lion of antigen-loaded dendritic cells for storage and
20 subsequent booster immllni7~tions. In the prefel-ed embo~lim~ntc) this pr~l)alalion of
antigen-loaded dendritic cells is stored in aliquots cont~ining a single dose for re-
injection into the subject. In general, in a single dose of antigen-loaded dendritic cells,
300 to 300,000 MHC sites per cell are occupied by disease-associated peptides. More
preferably, between about 1000 and 200,000 MHC site per cell are occupied by
2 5 disease-associated peptides.
Following injection of the dendritic cells, the disease-associated antigens which
are present in the extracorporeal blood stream following their release from the disease
effector cells are loaded onto the MHC sites of the exogenously added dendritic cells.
The photopheresis process steps (e.g., centrifugation) provide sufficient agitation and
3 0 mixing of the dendritic cells and the disease effector cells from which the antigens are
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derived to facilitate rapid loading of the release antigens onto the dendritic cell MHC
sites, thereby further reduçing the likelihood of enzymatic degradation of the release
antigens.
Alternatively, the treated blood co..~ the disease-associated antigen is
loaded into the MHC sites of dendritic cells in vitro, as opposed to introducing the
cells to the extracorporeal blood stream. Preparation of the disease-associated
antigens by acid elution of disease effector cells is described in Example 2, above. The
antigen compositions of the invention which are useful for this purpose are disease-
associated ~ntigen~ which have been released from disease effector cells that are
contained in blood. The compositions contain a detect~ble amount of a
photoactivatable agent (e.g., a psoralen), such as the amount that would be present in
the blood of a subject who has been subjected to photopheresis therapy. The
composition is plepared by aliquoting the photopheresed blood into portions which
contain an amount of disease-associated antigens suitable for mixing with one or more
doses of dendritic cells. Preferably, the antigen composition for enh~nrin~ an imm~me
response is p.epared by acidifying a preparation co~ i"il-~ a plurality of disease
effector cells (e.g., disease effector cells contained in the extracoll,o,eal blood stream)
for a period of time sufficient for the disease effector cells to release the disease-
associated ~ntig~n~ without Iysing the disease effector cells. The pl~pa~a~ion is
2 0 neutralized prior to loading the released ~ntig~n~ onto the MHC sites of the dendritic
cells.
In general, the disease-associated antigens which are prepal ed in accordance
with the methods of the invention (by photopheresis alone or together with acid elution
of antigens) are loaded onto dendritic cells in acco~ dance with the procedures
2 5 described by Steinm~n et a/. ~0 93/20185). Briefly, dendritic cells prepared as
described above are plated at a concentration of approximately lx105 cells per well of
a 24-well plastic culture plate. For antigen preparations in which the peptides are
intentled to be directly loaded into the MHC sites (without phagocytosis), mature
dendritic cells are p.e~l-ed. The cells are incubated and culture medium (e.g., RPM:I
3o 1640) preferably cont~inin~ additional nutrients (e.g., 5% fetal calfserum), and
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GM-CSF (p~ bly at 30 U/ml). Preferably, the dendritic cells are subjected to acid
elution (as described in Example 2, above), washed and placed in an appropriate
loading medium at less than physiological temperature prior to cont~cting the dendritic
cells with the disease-associated antigens. Loading the dendritic cells at a temperature
5 less than physiological temper~ re çnh~nces empty MHC stability and thereby
m~imi~es the number of MHC sites available for association with the disease-
associated ~ntigen~ educed temperature loading also ~ i7es the likelihood of
enzymatic digestion of the released peptide ~ntigenc
The disease-associated antigen pl epa. ~lion of the invention is added to the
10 dendritic cell cultures and the cultures are incub~ted with the antigen for several hours
or for s~lfficie-nt time to allow the dendritic cells to present the antigen in a form which
is recognized by T cells. Preferably, the cultures are inrllb~ted at a temperature less
than physiological te,llpe,~lul e to maximize the number of empty MHC sites available
for antigen loading. Following loading of the disease-associated antigens into the
15 dendritic cell MHC sites, the cells are collected from the culture, washed extensively
and are used to immllni7e the subject. Of course, a known control antigen can beinclude.~ in the prepalalion as a control and a portion ofthe collected cells can be
devoted to a quality control assay to determine (1) the viability ofthe dendritic cells
and/or (2) the functional activity of the antigen-loaded dendritic cells with respect to
2 0 their ability to induce an antigen-specific imml-ne response in vitro (e.g., a cytotoxic
T cell assay) or in vivo. For example, the peptide-loaded dendritic cells can be injected
subcutaneously into a mouse in an arnount s~-fficient to induce an immune response to
the known control antigen to estim~te the ~ffiçiçncy of antigen loading for the tested
cells. In the prer~l l ed embo~im~nt~, the antigen-loaded dendritic cells are irradiated
2 5 (3000 rads gamma irradiation) before injection (preferably, i.v., or i.d. injection).
More preferably, the antigen-loaded dendritic cells are coa~lminietered with one or
more cytokines (e.g., GM-CSF, IL-12, IL-4) to further enh~nce a specific imml-neresponse to the disease-associated antigen. (See, e.g., Zitvogel e~al., JExpMed
184:87-97 (1996) which reports that co-administration of peptide-pulsed dendritic
30 cells with low doses of IL-12 may favor the priming oftumor-specific T cells).
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Optional booster imm1lni7~tions are con~1cted in accordance with standard
practice using the above-described disease-associated antigen-loaded dendritic cell
,~. epa. aLion. In general, the cells are tested for activity, viability, toxicity and sterility
prior to ~(lminictration to the subject. The cytotoxic activity of the peptide-loaded
dendritic cells can be determined in a cytotoxic T cell assay, e.g., a chromium release
assay, using target cells that express the appropliate MHC molecule, in the presence
and absence of the disease-associated peptide or other known peptide control that is
known to be capable of loading onto dendritic cells and invoking a cytotoxic T cell
response in vitro. (See, e.g., WO 94-20127 and Zitvogel, L., et al., JExp Med
0 183:87-97(1996)). Zitvogel, et aL also describe an animal model for testing the ability
of antigen-loaded dendritic cells (pulsed with acid-eluted peptides derived fromautologous solid tumors) to inhibit tumor progression in vivo. The Zitvogel animal
model is adapted for use in the current invention to further oplillliGe the procedures for
- obla;l~ng the disease-associated ~ntigenc of the invention, loading these ~nti~enc into
the MHC sites of the dendritic or other antigen presçnting cells of the invention and
selectin~ the optimum dose and booster frequency for the subject. However, in
contrast to the literature reports, increased serum levels of CD4 and/or CD8+ T cells
(rather than solid tumor size) are used as indicator(s) of disease inhibition.
Cell viability is determined by the exclusion of trypan blue dye by live cells.
2 0 Cells are tested for the presence of endotoxin and for bacterial or fungal co"l~;~tion
by conventional methods known to those of ordinary skill in the art. Cells which have
passed these safety and activity criteria are washed and placed in the approplia~e
solution (e.g., an infusion solution such as Ringer/glucose lactate for i.v. infusion) and
~lminict~red to the subject.
2 5 Additional methods for pulsing dendritic cells with antigen and/or procedures
for using antigen-loaded dendritic cells to induce a cytotoxic T cell response in vitro or
in vivo are described in WO 93/20185, WO 94/21287, WO 94/02156; Mayordomo, J.
e~ al., Nature Medicine 1(12):127-1302(1995); and Hsu, F. et al., Nature Medicine
2(1):52-58(1996). See, also, WO 94-21287, WO 94-20127 and Hsu, F. e~ al., NatureMedicine 2(1):52-58(1996) for exemplary protocols for irra~i~ting antigen-loaded
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dendritic cells to prevent cell proliferation when the cells are reintroduced into the
subject.
Example 4
Demonstrated Efficacy of Photopheresis/Dendritic Cell Treatment
To demostrate the effectiveness of combining extracorporeal tre~tment of
disease effector cells and the addition of dendritic cells, murine 2B4.11 tumorigenic T
cells were treated as described above using 8-MOP and WA. The 2B4.11 tumor cellswere derived by hybridizing or co~llbil~ing two original cell types: normal AKR mouse
T cel~s with a BW5147 mouse m~lign~nt T cells. The AKR parental cell provides
1 0 specific ~ntig~n.~, incl~-ling a T cell receptor, which can appare,-~ly serve as a tumor
specific antigen, to be targeted by an intl~ced anti-tumor immllnologic reaction. The
BW5147 parental cell contribution permits the 2B4.11 cells to act like a cancer cell,
dividing without check until they kill the animal.
Following 8-MOP/UVA Ll e~ , dendritic cells were added to the treated
1 5 cell mixture.
Two groups of 5 test mice were v~crin~ted with 5 million 2B4.11 cells that had
been inactivated with 8-MOP/UVA L~e~ fnt and mixed with dendritic cells (DPAC).
The 8-MOP/UVA irradiated tumorigenic cells were shaken overnight with 200,000
dendritic cells (a 25 to 1 ratio), to maximize cell-to-cell contact between the DAPCs
2 0 and the 2B4.11 cells. One group of cells (irradiated 2B4.11 plus DAPCs) was
incubated at 23~ C, to maximize stability of empty class I MHC molecules on the
DAPCs. The other group of cells was incllbated at 37~ C, to .,,~x;..,;~e normal cellular
metabolism. The combined irradiated 2B4.11/DAPC cell mix was injected into the test
mice one week prior to challenging the animals with viable, tumorigenic 2B4.11 cells.
2 5 In Figure 1, reading from left to right, tumor growth is first depicted in five
control mice, which received only skin injections of tumor cells on day 0 and no anti-
tumor vaccination. All of these mice had visible tumors by day 8, which progressively
grew until day 21, the last day of observation in these experiments.
ll I I
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Of the va~.~in~ted mice, all ten (both groups that received DAPCs) developed
tumors that grew more slowly than those in the control mice, or did not grow at all.
Three of the mice receiving the 23~C cell mix and two of the mice receiving the 37~C
cell mix did not grow tumors at all. The other two mice in the 23~C group had small
5 tumors which stopped enlarging after day 11. One of the other mice in the 37~C group
also had a very small tumor which stopped growing by day 11. Of great interest, two
of the other mice in the 37~C group developed small, slowly growing tumors, which
then completely resolved.
This data demonstrates that direct contact between 8-MOP/UVA irradiated
10 tumorigenic cells and DAPCs leads to formation of an effective cellular vaccine, which
not only prevents or slows tumor growth, but reverses growth of certain tumors. The
Figure 1 demonstrates that the combination of genetically identical dendritic antigen
ples~ g cells (DAPCs) plus 8-MOP~UVA p.~tlealed murine 2B4.11 tumorigenic T
cells con~titutes an effective vaccine against the specific tumor. (It has previously been
15 shown that inoculation of mice with the tumorigenic 2B4.11 cells kills .the mice within
40 days, unless the animals have been succes~fi-lly v~cr.in~ted against the tumor).
Alternatively, the antigen loaded dendritic cells can be isolated following mixing.
Further, a membrane partition, for example a .45 micron filter, can be place between
the dendritic cells and the disease e~,lor cells to allow passage of the antigens to the
2 0 dendritic cells but keeps the two cell population separated.
Example S
Demonstrated Efficacy of Dendritic Cell Therapy
Figure 2 shows results that d~mon~trate the impact of exogenously supplied
dendritic antigen pres~nting cells, that are supplied without prior contact with treated
2 5 disease effector cells, on tumor growth. Large tumors grew in control ~nim~l~, with
initial appearance of tumor on day 7 and continuous enlal~,e---elll through the
observation period to day 28. Vaccination with DAPCs cultured overnight at either
23~C or 37~C prevented tumor growth, respectively, in 4 and 3 mice out of both
groups of 5, and reversed initial limited growth in the ~ ining tumors. This effect is
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substantial, but does not lead to the specific immunologic memory which follows
vaccination with the 2B4. 1 l/DAPC mix shown in the Figure 1. For example, although
not shown in these graphs, if a second 2B4. 11 tumor is ~lmini~tered after the
resolution of the tumors, immllnoprotection is only seen in the group getting the
5 co...bination vaccination of 2B4. 11 mixed with DAPCs.
Example 6
Demonstrated Increase in M:HC Expression Following Photopheresis
Figure 3 shows the impact of 8-MOP/UVA on Class I eA~. ~ssion. Mean
fluorescence channel qu~ntifi~s the amount of Class I protein on the cell surface, as
10 identified with a fluorescent antibody against the Class I protein. Normally, at least
200,000 Class I molecules are displayed, as shown by a value of 2.4 in the control cell
population. After exposure to 8-MOP/UVA (1 joule per cm2 WA and 200 ng/ml of
8-MOP) and overnight inr.ubation, Class I ~AI,ression doubles. Since this massive
increase in Class I eA~,ression is prevented by çmetine~ it results from new protein
1 5 synthesis.
It should be understood that the preceding is merely a detailed description of
certain preferred embodiments. It therefore should be apparent to those of ordinary
skill in the art that various modifications and equivalents can be made without
departing from the spirit and scope of the invention. All references, patents and patent
2 0 publications that are identified in this application are incorporated in their entirety
herein by reference.
