Methods and compositions for improving the effectiveness of X-irradiation therapy for the treatment of an internal solid tumor

Methods and pharmaceutical compositions for improving the effectiveness of radiation therapy for treating a subject having an internal solid tumor malignancy are disclosed. The methods include irradiating the tumor to release tumor-derived antigens in vivo, preparing a cellular vaccine including the isolated antigens admixed with a preparation of altered antigen presenting cells and administering the cellular vaccine to the subject. In the preferred embodiments, the antigen presenting cells are leukocytes that have been photochemically altered by subjecting the cells to photopheresis.

FIELD OF THE INVENTION 
This invention relates to an improved method of cancer therapy. More 
particularly, the invention relates to methods and pharmaceutical 
compositions for improving the effectiveness of radiation therapy for 
treating a subject having an internal solid tumor malignancy by combining 
irradiation therapy with photopheresis. 
BACKGROUND OF THE INVENTION 
Cutaneous T cell lymphoma (CTCL) is a malignancy of the immune system that 
is caused by a massive expansion of a single clone of aberrant T cells. 
The choice of treatment for CTCL depends upon the extent of the disease 
state, as well as the general health and age of the patient. In general, 
early stage disease limited to the skin is treated with sequential topical 
therapies such as topical nitrogen mustard, psoralen phototherapy ("PUVA", 
i.e., administration of oral psoralen followed by UVA irradiation of the 
skin) and total-skin electron beam therapy (TSEB). Later stage disease, 
e.g., Sezary syndrome, is treated with extracorporeal photochemotherapy 
("photopheresis"). 
Photopheresis for the treatment of cutaneous T cell lymphoma was introduced 
by Edelson in 1987 (Edelson, R., et al., N. Engl. J. Med. 316:297-303 
(1987)). Although relatively new, photopheresis is now considered standard 
therapy for the erythrodermic variants of CTCL (Edelson, R., 
"Light-activated Drugs", Scientific American 256(8): 68-75 (1988); 
Edelson, R., "Photopheresis: A Clinically Relevant Immunobiologic Response 
Modifier", Annals of N.Y. Academy of Sciences 636:154-164 (1991). The 
therapy includes two steps: (1) irradiating a preparation of the patient's 
leukocytes in the presence of a photoactivatable agent (e.g., 
8-methoxypsoralen, "8-MOP") to photochemically alter the cells and (2) 
reinfusing the photochemically altered cells. 
Not all CTCL patients with erythroderma respond to photopheresis therapy. 
Those CTCL patients who do respond have a median survival time of more 
than 62 months, i.e., approximately twice as long as patients treated with 
other modalities (Edelson, R., et al., Prog. Dermatol. 25(3):1-6 (1991)). 
However, because up to 25% of patients with CTCL have a limited response 
to photopheresis, adjunctive TSEB therapy, chemotherapy (e.g., oral 
methotrexate (Rook, A., et al., Arch. Dermatol. 127:1535-1540 (1991)) or 
subcutaneous interferon alfa-2b (Heald, P. W., et al., Yale J. Biol. Med. 
62:629-638 (1989) has recently been introduced. 
The mechanism of action of photopheresis has not completely been 
elucidated. Exposure of a malignant T cell clone to 8-MOP and ultraviolet 
light, followed by return of the irradiated, damaged cells to the patient, 
appears to elicit a specific response to the aberrant T cells, which 
response is mediated by T cell surface receptors. Consistent with this 
hypothesis is the production of a heightened immunity against the 
pathogenic clone(s) of T cells following reinfusion of irradiated blood 
leukocytes (Edelson R., et al., N. Engl. J. Med. 316:297-303 (1987); 
Edelson, R., Yale J. Biol. Med. 62:565-577 (1989); Rook, A. H., et al., 
Ciba Foundation Symposium 146, New York, John Wiley & Sons, (1989) 
171-177). 
T cell receptors mediate a cellular immune response by recognizing a 
particular antigen only when the antigen is associated with a surface 
marker on an antigen presenting cell. The surface markers belong to a 
group of molecules known as the major histocompatibility complex (MHC). 
Binding of the T cell receptor to the antigen/MHC molecule on the antigen 
presenting cell induces changes in the T cell. These changes collectively 
comprise a cell-mediated immune response. 
Two signals are primarily responsible for inducing the T cell mediated 
response to an antigen that is associated with an antigen presenting cell. 
A first signal is generated following binding of the T cell to the antigen 
on the antigen presenting cell. A second, co-stimulatory signal is sent by 
"accessory" membrane molecules or soluble messengers from the antigen 
presenting cell to the responding T cell. These soluble intercellular 
messengers regulate the amplitude and duration of the immune response and 
are given the generic term, cytokines. Cytokines include the group 
previously referred to in the literature as lymphokines, monokines, 
interleukins, interferons and tumor necrosis factor (Essential Immunology, 
seventh edition, Blackwell Scientific Publications, Oxford, Great Britain, 
1991, pp. 140-150). If the antigen presenting cell does not send the 
second signal, the T cell is effectively paralyzed, i.e., unable to mount 
an immune response to the antigen. Certain types of antigen presenting 
cells, e.g., resting T cells, are unable to send the second signal. 
Accordingly, in the absence of exogenous cytokine or other second signal, 
such resting T cells which also function as antigen presenting cells 
down-regulate an immune response to the presented antigen and lead to 
antigen specific immunologic paralysis of the T cell whose membrane 
receptor has been engaged. Other types of antigen presenting cells, e.g., 
monocytes, are able to release cytokines. Accordingly, monocytes which 
have been stimulated to release cytokines up-regulate an immune response 
to the presented antigen. 
In addition to CTCL and scleroderma, photopheresis has been used for the 
treatment of several other autoimmune disorders, including pemphigus 
vulgaris, systemic sclerosis (Rook, A., "Photopheresis in the Treatment of 
Autoimmune Disease: Experience with Pemphigus Vulgaris and Systemic 
Sclerosis", Annals of N.Y. Academy of Science 636:209-216 (1991) and 
rheumatoid arthritis (Malawista, S., et al., "Photopheresis for Rheumatoid 
Arthritis", Annals of N.Y. Academy of Science 636:217-226 (1991). Other 
preliminary trials currently in progress which show promising results for 
photopheresis therapy include autoimmune or type I insulin-dependent 
diabetes, cardiac transplant rejection, AIDS-related complex and acute 
graft-vs.-host disease. 
In summary, photopheresis has been demonstrated to produce a generalized 
clinical benefit for a variety of autoimmune diseases that are 
characterized by a disorder in T cell regulation. Absent from the list of 
disease states reported in the prior art to be amenable to photopheresis 
therapy are diseases for which clonal expansion of circulating, aberrant 
T-cells has not been implicated including, for example, solid tumor 
malignancies associated with cancers that are difficult to treat, e.g., 
cancers of the lung, breast, ovaries, uterus, prostrate, testicles, liver, 
pancreas, stomach, squamous cell carcinoma of the oral phalynx, 
fibrosarcomas, kidney, bladder, brain, spinal cord, malignant melonoma and 
metastatic squamous cell carcinoma of the skin. To date, treatment of such 
solid tumor malignancies is limited to surgery, irradiation, and/or 
chemotherapy (see e.g., Harrison's Principles of Internal Medicine, 12th 
ed., eds. J. D. Wilson et al., McGraw-Hill, Inc., N.Y., N.Y. (1991)). 
Although the use of X-irradiation in combination with chemotherapy for the 
treatment of internal solid tumors may have a generalized therapeutic 
benefit, e.g., by destroying or inhibiting the proliferation of at least 
some tumor cells, such conventional treatment regimens are far from ideal. 
In particular, these adjunct therapies are limited by the potentially 
toxic side effects which stem from the lack of specificity of the 
chemotherapeutic agents. 
SUMMARY OF THE INVENTION 
The present invention overcomes the problems of conventional approaches for 
the treatment of solid tumor malignancies by combining with radiation 
therapy a method which specifically targets the tumor for destruction by 
the patient's cellular immune system. In essence, the method of the 
invention is a synergistic combination of two therapeutic methods, 
X-irradiation and photopheresis, each of which is known to be efficacious 
for treating a different disease state. Applicants' invention is the 
discovery that when combined, these two methods exert a synergistic 
therapeutic benefit in treating a subject having a solid tumor malignancy. 
Because modulation of the cellular immune response is important to the 
execution of the invention, it is preferred that the recipient subject of 
the cellular vaccine which mediates the cellular immune response 
(described below) have a competent immune system, as evidenced by, for 
example, near normal absolute levels of CD8 positive T cells. 
According to one aspect of the invention, a method for improving the 
effectiveness of radiation therapy for treating a subject having an 
internal solid tumor is provided. The method involves irradiating the 
internal solid tumor to release tumor-derived antigens, isolating a 
plurality of these antigens and contacting the tumor-derived antigens with 
a leukocyte preparation that previously has been subjected to 
photopheresis, i.e., irradiated in the presence of a photoactivatable 
agent to form a photoinactivated leukocyte preparation. The tumor-derived 
antigens are contacted with the photoinactivated leukocyte preparation 
under conditions for forming the cellular vaccine, which vaccine is then 
administered to the subject to improve the effectiveness of the radiation 
therapy. Alternative methods can be employed to release, in vivo, antigens 
from the solid tumor. These include physical manipulation of the tumor 
(e.g., at the time of surgery), such as squeezing the tumor, perfusing or 
injecting the tumor with a high concentration of a saline solution or a 
chemotherapeutic agent(s). Additional methods for effecting the in vivo 
release of antigens from the tumor include arterial chemoperfusion (i.e., 
perfusing a tumor by injecting a high concentration(s) of a 
chemotherapeutic agent(s) into an artery leading into the tumor-containing 
organ); photofrin (i.e., administering to the tumor via systemic or 
perfusion routes, a photoactivatable agent such as a porphyrin, followed 
by photoactivating the agent in situ), and damaging the tumor with laser 
light of a sufficient wavelength and intensity to effect the release of 
antigens from the tumor in vivo. 
In the preferred embodiments, the tumor-derived antigens are released from 
the tumor in viva following X-ray irradiation ("X-irradiation") of the 
tumor. Alternative methods for site-directed tumor necrosis, which methods 
are sufficient to release circulating tumor-derived antigens in viva, are 
contemplated as being within the scope of the instant invention. 
According to another aspect of the invention, a cellular vaccine for 
improving the effectiveness of radiation therapy is provided. The cellular 
vaccine includes an effective amount of an admixture containing a 
plurality of solid tumor-derived antigens admixed with a plurality of 
antigen presenting cells that have been treated to induce expression of 
empty class I and/or empty class II major histocompatibility complex 
molecules. Typically, such induction is accomplished by photopheresis and 
the induced cells are referred to as "photoinactivated" antigen presenting 
cells. The admixture is placed in a pharmaceutically-acceptable carrier. 
As used herein, an "effective amount" of the admixture is that amount 
sufficient for improving the effectiveness of radiation therapy for 
treating the subject. Whether the amount is an "effective amount" is 
determined according to criteria known to one of ordinary skill in the 
art. In the preferred embodiments, the antigen presenting cells used for 
preparing the cellular vaccine are autologous cells, i.e., isolated from 
the recipient subject, that have been photoinactivated by subjecting a 
leukocyte preparation to photopheresis. However, alternative sources of 
antigen presenting cells and methods for producing the functional 
equivalent of photoinactivated leukocytes also are provided. Because the 
solid tumor-derived antigens are isolated from body fluids such as whole 
blood, lymphatic fluid and possibly urine, the cellular vaccine optionally 
includes a detectable amount of the body fluid from which the antigen was 
isolated. 
According to another aspect of the invention, a method for making the 
above-described cellular vaccine is provided. The method includes 
isolating a plurality of tumor-derived antigens, exposing a leukocyte 
preparation to irradiation in the presence of a photoactivatable agent to 
form a photoinactivated leukocyte preparation and contacting the plurality 
of tumor-derived antigens with the photoinactivated leukocyte preparation 
under conditions for forming the cellular vaccine. If the admixture is not 
already contained in a pharmaceutically acceptable carrier, it is placed 
in such a carrier prior to administration to the subject. 
An alternative method for improving the effectiveness of radiation therapy 
for treating a subject having an internal solid tumor also is provided. 
The method includes irradiating the solid tumor to release tumor-derived 
antigens in vivo, isolating a plurality of the tumor-derived antigens, 
treating a cellular preparation (containing antigen presenting cells that 
are suitable for administration to the subject) to enhance surface 
expression by the cells of a major histocompatibility complex molecule, 
contacting the tumor-derived antigens with the treated cellular 
preparation under conditions for forming a plurality of antigen-associated 
antigen presenting cells, and administering the antigen-associated antigen 
presenting cells to the subject. Methods for treating the cellular 
preparation to enhance expression of the major histocompatibility 
molecules include, for example, subjecting the cellular preparation to one 
or more of the following extracorporeal procedures or conditions: (1) 
photopheresis, (2) temperatures less than physiological temperatures and 
(3) contacting the cellular preparation with one or more cytokines known 
to induce expression of the major histocompatibility molecules. In a 
preferred embodiment, the antigen presenting cells are autologous cells, 
i.e., the cells are isolated from the recipient subject. More preferably, 
the cells are monocytes or B-cells. 
According to another aspect of the invention, a method for enhancing an 
immune system response to a tumor specific antigen is disclosed. The 
method includes releasing a plurality of tumor-derived antigens in vivo, 
isolating the tumor-derived antigens and contacting the isolated antigens 
with a cellular preparation containing antigen presenting cells, which 
preparation has been treated to enhance expression by cells of empty major 
histocompatibility complex molecules. Preferably, the antigen presenting 
cells are leukocytes, more preferably monocytes or B-cells. In the 
preferred embodiments, the cellular preparation is treated to enhance 
expression of empty major histocompatibility complex molecules by any of 
the above-mentioned extracorporeal procedures, e.g., subjecting the 
cellular preparation to photopheresis and/or to a temperature less than a 
physiological temperature. The cellular vaccine for enhancing the immune 
system to a tumor specific antigen is formed by contacting the 
above-described cellular preparation with the plurality of tumor-derived 
antigens under conditions known to enhance association of the antigens 
with the empty major histocompatible molecules present on the surface of 
the antigen presenting cells. Thereafter, the vaccine is administered to 
the subject in accordance with methods known to one of ordinary skill in 
the art.

DETAILED DESCRIPTION OF THE INVENTION 
A method for improving the effectiveness of radiation therapy for treating 
a subject having an internal solid tumor malignancy is provided. The 
method synergistically combines two therapeutic methods: X-irradiation for 
the treatment of an internal solid tumor malignancy and a method for 
stimulating the immune system to specifically recognize tumor-specific 
antigens. The tumor-specific antigens are released from the solid tumor 
following irradiation. The specific immune response is mediated by 
administration of a cellular vaccine (described below) containing the 
tumor-derived antigens. In its simplest form, the cellular vaccine 
includes an effective amount of an admixture containing a plurality of the 
tumor-derived antigens admixed with a plurality of photoinactivated 
leukocytes. The admixture is contained in a pharmaceutically acceptable 
carrier. 
The preferred method for improving the effectiveness of radiation for 
treating a subject having an internal solid tumor involves irradiating the 
internal solid tumor to release the tumor-derived antigens in vivo, 
isolating a plurality of the tumor-derived antigens and contacting the 
isolated antigens with a photoinactivated leukocyte preparation to form 
the cellular vaccine. In the most preferred embodiments, the 
photoinactivated leukocyte preparation is formed by irradiating the 
leukocyte preparation with ultraviolet A radiation ("UVA") or visible 
light in the presence of a photoactivatable agent, i.e., by subjecting the 
leukocyte preparation to photopheresis. Conditions for activating 
psoralens in the presence of visible light are disclosed in U.S. 
application Ser. No. 08/013,831, filed Feb. 4, 1993, now issued as U.S. 
Pat. No. 5,462,733, the entire contents of which patent application are 
incorporated herein by reference. The cellular vaccine is formed by 
admixing the solid tumor-derived antigens with the photoinactivated 
leukocyte preparations under conditions for loading the tumor-derived 
antigens into the empty class I or class II major histocompatibility 
complex molecules of the antigen presenting cells. In general, such 
conditions include admixing the cells at temperatures between about 
22.degree. C. and about 30.degree. C. to enhance the stability of the 
empty class I or class II molecules. Exemplary conditions for loading the 
antigens into empty class I major histocompatibility complex molecules are 
disclosed in Example 3. 
As recited in the above-identified U.S. application Ser. No. 08/013,831, 
now issued as U.S. Pat. No. 5,462,733, methods other than 
photoinactivation with UVA light in the presence of a psoralen are useful 
for preparing a preparation of cells having enhanced expression of empty 
class I and/or empty class II major histocompatibility complex molecules. 
Exemplary alternative methods include ultraviolet B ("UVB") irradiation, 
contacting the cells with a chemotherapeutic agent(s) and/or a cytokine(s) 
such as tumor necrosis factor (TNF), exposing the cells to temperatures in 
the range of about 22.degree. C. to about 30.degree. C., exposing the 
cells to hypotonic or hypertonic saline solution or tissue culture medium 
and exposing the cells to hydrostatic pressure. 
Empty class I molecules are thermodynamically unstable at physiological 
temperatures. Accordingly, it is believed that exposure of the cellular 
preparation to a temperature that is less than physiological temperatures 
results in "enhanced expression" of the empty major histocompatibility 
complex class I molecules by stabilizing the "empty" molecules. As used 
herein, the term "enhanced expression" refers to a cell having on its 
surface substantially more empty major histocompatibility complex 
molecules than would be present on the surface of a corresponding, 
naturally occurring antigen presenting cell. 
The cellular vaccine is administered to the subject according to any 
appropriate mode of administration known in the art, e.g., introduction 
into the blood stream or the immune system of the patient. Intradermal 
injection is a preferred method of administration. However, subcutaneous 
injection, intramuscular injection and/or any other mode of depositing the 
cellular vaccine in a reservoir such that the tumor-derived antigens are 
exposed to the subject's cellular immune system, i.e., the cells 
comprising the cellular immune system can recognize and react to the 
presence of the tumor-derived antigen, can be used. Accordingly, the 
cellular vaccine further includes a pharmaceutically acceptable carrier 
that is appropriate for the selected mode of administration. 
Pharmaceutically acceptable carriers are known in the art and include, for 
example, normal saline for vaccines intended for administration by 
injection into the blood system. 
In the preferred embodiments, the subject is a human having an internal 
solid tumor that is amenable to X-irradiation therapy. As used herein, the 
phrase "amenable to X-irradiation therapy" in reference to a solid tumor 
means a tumor which contains at least some tumor cells that are 
susceptible to cellular damage in response to irradiation, which damage is 
sufficient to release tumor-derived antigens in vivo. 
The term "irradiation" as used herein, has its conventional meaning and is 
only limited to the extent that the X-irradiation have sufficient energy 
to penetrate the body and be capable of inducing the release of 
tumor-specific antigens in vivo. The optimal radiation intensity for 
damaging a particular type of tumor is known to one of ordinary skill in 
the art. In general, any form or intensity of radiation that is acceptable 
in the art for treating internal solid tumors is useful for releasing 
tumor-derived antigens in vivo for the purposes of the instant invention. 
Radiation is measured in RADS, with a typical dosage of X-irradiation 
falling between about 1000 to about 4000 RADS for tumor irradiation 
therapy. The radiation dosage range for the instant invention falls within 
the range of about 100 to about 6000 RADS. Since less radiation is needed 
to damage the tumor to induce the release of tumor-derived antigens in 
vivo than is needed to destroy the tumor (the objective of conventional 
radiation therapy), the instant invention advantageously permits the use 
of lower dosages of radiation, thereby avoiding the toxic side effects 
associated with high doses of X-irradiation. Typically, the radiation is 
administered in fractioned doses over several weeks with no more than 300 
RADS being administered per treatment. 
Because the combination therapy of the instant invention enlists the 
subject's immune system to specifically target and destroy the internal 
tumor, it is preferred that the recipient subject of the combined therapy 
have a competent immune system. In general, the immunocompetence of a 
particular subject is determined by measuring the amount of CD8 positive T 
cells in a blood sample taken from the subject. Subjects having near 
normal absolute levels of CD8 positive T cells are deemed to be 
immunocompetent for the purposes of the instant invention. In general, a 
subject having a CD8 level that is at least about 100 CD8 cells/100 ml 
blood is deemed to be immunocompetent. 
The phrase "internal solid tumor" refers to an extracutaneous, i.e., 
non-cutaneous T-cell lymphoma, neoplasm. Whether a particular treatment or 
combination therapy is efficacious in halting progression of the neoplasm 
is determined by criteria known to one of ordinary skill in the art. For 
example, efficacy can be demonstrated by observing a change in the rate of 
tumor growth (e.g., a static or reduced rate of growth as evidenced by the 
rate of change in the size of the tumor with time) and/or by observing a 
slowing in the progression of the disease state (e.g., a reduction in 
disease-associated symptoms with time). Such criteria are useful for 
determining whether the solid tumor is amenable to radiation therapy, as 
well as for assessing whether the methods and/or compositions of the 
instant invention improve the effectiveness of radiation therapy for 
treating the subject. Exemplary solid tumor malignancies amenable to 
treatment by the methods and compositions of the instant invention are 
listed above. 
X-irradiation induced cell damage or death results in the in vivo release 
of a myriad of oligopeptides, proteins and/or other cellular components 
from the irradiated cells. These previously immobile (i.e., associated 
with the solid tumor) cellular components enter the lymphatic and/or blood 
systems from which they can be isolated according to procedures well known 
in the art. As used herein, the term "isolated" refers to a preparation 
(e.g., a collection of tumor-derived antigens or a leukocyte preparation) 
that has been removed from its naturally occurring environment. Thus, for 
example, a blood sample withdrawn from a subject is "isolated" as defined 
herein, even if the blood sample is part of a continuous stream that is 
later reinfused into the subject. 
The term "tumor-derived antigens" refers collectively to components which 
are released from the solid tumor following irradiation. There is no 
requirement that the tumor-derived antigens be purified or concentrated 
prior to their use in accordance with the methods or compositions of the 
instant invention. However, the concentration of tumor-derived antigens in 
the isolated preparation optionally is enriched prior to storage to 
enhance the stability of the antigen upon storage. By "enriched" it is 
meant that the tumor-derived antigen is present at a higher concentration 
than that which it was present when isolated from the subject. In general, 
an enriched preparation of tumor-derived antigens is preferred because the 
higher concentration of antigen, when admixed with the treated antigen 
presenting cells, kinetically favors the loading of tumor-derived antigen 
into the empty class I or empty class II major histocompatibility complex 
molecules of the treated antigen presenting cells. 
Antigen storage conditions are known to those of ordinary skill in the art 
and include, for example, storage at a reduced temperature (e.g., at 
-70.degree. C.) in the presence of protein stabilizing agents such as 
protease inhibitors and/or agents known to stabilize the native 
conformation of peptide and/or protein antigens (e.g., dimethyl sulfoxide 
(DMSO)). Such agents are known to one of ordinary skill in the art and 
include, for example, glycerol, sucrose and urea. 
The cellular vaccine is prepared by contacting the tumor-derived antigens 
with a cellular preparation that has been "altered" (e.g., 
photoinactivated) to enhance interaction (e.g., association) of the 
tumor-derived antigens with the cells contained in the cellular 
preparation. As used herein, the phrase "cellular preparation" refers to a 
preparation of antigen presenting cells. Antigen presenting cells are a 
class of cells capable of presenting antigen to other cells of the immune 
system that are capable of recognizing antigen when it is associated with 
a major histocompatibility complex molecule. Antigen presenting cells 
include such diverse cell types as leukocytes (e.g., monocytes, 
macrophages and lymphocytes such as T cells and B cells), as well as 
synthetic ("artificial") cells such as those described in U.S. patent 
application Ser. No. 07/977,672, now issued as U.S. Pat. No. 5,651,993, 
the contents of which patent application are incorporated herein by 
reference. These diverse cell types have in common the ability to present 
antigen in a form that is recognized by specific T cell receptors. The 
preferred antigen presenting cells are leukocytes, more preferably, 
monocytes or B-cells. The leukocyte preparation is isolated from, for 
example, blood, lymph fluid, bone marrow, lymphatic organ tissue or tissue 
culture fluid. Optionally, the monocytes or B-cells are cultured in vitro 
to expand the number of cells available for forming the cellular vaccine 
in vitro. 
As previously discussed, each T lymphocyte clone, e.g., T-helper cell clone 
or cytotoxic T cell clone, expresses a different surface receptor which 
recognizes an antigen only when it is associated with a major 
histocompatibility complex molecule on the surface of the antigen 
presenting cell. In general, the term "major histocompatibility complex 
molecule" refers to a molecule on an antigen presenting cell that has the 
ability to associate with the antigen to form an antigen-associated 
antigen presenting cell. Recognition of the antigen-associated presenting 
cell by the T cell is mediated by the T cell surface receptor. In the 
preferred embodiments, the major histocompatibility complex molecule is a 
class I or class II molecule. A cytotoxic T cell binds antigen when the 
antigen is associated with a major histocompatibility complex class I 
molecule. A T helper cell recognizes and binds antigen when the antigen is 
associated with a major histocompatibility complex class II molecule. Each 
of these T cell types functions to mediate the subject's immune response 
to the tumor. 
The class I molecule, composed of a heavy chain and a noncovalently linked 
beta-2-microglobulin molecule, includes a cleft or crevice for receiving 
the tumor-derived antigen. Accordingly, the preferred tumor-derived 
antigen has a size and dimension that permit entry of the antigen into the 
crevice of the class I molecule. (See e.g., F. Latron, "A Critical Role 
for Conserved Residues in the Cleft of HLA-A2 in the Presentation of a 
Nonapeptide to T-cells", Science 257:964-967 (1992) for a discussion of 
class I molecule cleft dimensions). Although the tumor-derived antigen 
fits substantially within the crevice, it is still accessible to a T cell 
capable of recognizing the antigen when it is associated with the class I 
molecule. Thus, in a preferred embodiment, the tumor-derived antigen is a 
peptide having between about eight and about sixteen amino acids. In a 
most preferred embodiment, the tumor-derived antigen is a peptide having 
between eight and ten amino acids, two of which amino acids are 
hydrophobic residues for retaining the peptide in the crevice. Association 
of the tumor-derived antigen with the class I molecule is determined using 
screening assays that are capable of distinguishing between empty and full 
class I molecules. An exemplary screening assay is disclosed in Example 3. 
Association of a tumor-derived antigen with a major histocompatibility 
molecule of an antigen presenting cell is a prerequisite for inducing the 
cellular immune response to the tumor. Accordingly, various methods are 
disclosed herein for enhancing expression of empty major 
histocompatibility molecules on the surface of the antigen presenting 
cells. 
The preferred method for enhancing expression of empty major 
histocompatibility molecules is photopheresis. Photopheresis procedures 
are described in U.S. Pat. No. 5,147,289 ("Edelson '289") and U.S. Pat. 
No. 4,838,852 (Edelson '852), the entire contents of which patents are 
incorporated herein by reference. Photopheresis may be performed on a 
continuous stream, as described in the Edelson '289 patent, or may be 
performed batchwise. It is believed that subjecting the preparation to 
photopheresis disrupts the cells' metabolic pathways responsible for 
processing intracellular antigen into a form that fits within the crevice 
defined by the major histocompatibility complex molecule, thereby yielding 
a plurality of antigen presenting cells having unfilled (i.e., "empty") 
major histocompatibility complex molecules on their surface. These 
photoinactivated cells bind tumor-derived antigens having the 
above-described requisite size and charge characteristics. Once bound, the 
tumor-derived antigen-associated antigen presenting cells are administered 
to the subject in the form of a cellular vaccine. 
If the photoactivatable agent is a psoralen (described below), the 
radiation for photoactivation is ultraviolet A irradiation or visible 
light having a wavelength greater than about 420 nm (Gasparro, F., et al., 
"The Excitation of 8-methoxypsoralens with Visible Light. Reversed Phase 
HPLC Quantitation of Monoadducts and Crosslinks", Photochemistry and 
Photobiology 57:1007-1010 (1993)). 
Yet another method for enhancing expression of empty major 
histocompatibility complex molecules, is to contact the cellular 
preparation with a cytokine. The term cytokine denotes the molecules 
previously referred to in the literature as lymphokines, monokines, 
interleukins, interferons and tumor necrosis factor (TNF) (Essential 
Immunology, 7th edition, Blackwell Scientific Publications, Oxford, Great 
Britain, pp. 140-150 (1991)) and includes, for example, gamma-interferon, 
tumor necrosis factor alpha and granulocyte monocyte colony stimulating 
factor, as well as molecules in the family of interleukins. Cytokines are 
known to increase expression of the major histocompatibility complex 
molecules on some antigen presenting cells, e.g., monocytes or B-cells. 
The above-described altered antigen presenting cells, e.g., altered by 
photopheresis or exposure to a cytokine, are contacted with the 
tumor-derived antigens under conditions for forming the cellular vaccine. 
As used herein, the phrase "cellular vaccine" refers to a preparation of 
cells which, when introduced into a subject, elicits a cellular immune 
response that is specific for a component (e.g., the tumor-derived 
antigen) present in the cellular vaccine. The term "vaccine" is used 
herein because although only a small portion of the subject's total 
leukocytes are treated, a far-reaching therapeutic effect is obtained with 
respect to the irradiated tumor following infusion of the vaccine. 
In the preferred embodiments, the cellular vaccine is stored prior to 
administration to the subject. Preferably, the vaccine is stored in 
aliquots containing an amount of tumor-derived antigen-associated antigen 
presenting cells sufficient to boost the cellular immune response of the 
subject to the solid tumor. Determination of the amount of vaccine 
necessary to boost the patient's immune response is within the ordinary 
skill of the art. Preferably, an amount of cells ranging from a minimum of 
about 10,000 to a maximum of about 200.times.10.sup.6 antigen presenting 
cells is sufficient to boost the immune response of the subject. The 
amount of cells used will, in part, be dependent upon whether the antigen 
presenting cells are efficient, e.g., B cells or monocytes, or 
inefficient, e.g., T cells. 
The antigen and cellular components of the cellular vaccine differ from 
their corresponding natural counterparts in several respects. The 
antigen-associated antigen presenting cells of the instant invention 
represent a relatively homogeneous population of cells. This is primarily 
because the tumor-derived antigen is not further processed by the antigen 
presenting cells prior to association with the major histocompatibility 
complex molecule on the surface of the antigen presenting cell. Second, 
the antigen presenting cells of the present invention optionally have an 
elevated concentration of major histocompatibility complex molecules on a 
per cell basis. Moreover, the pharmaceutical composition optionally 
includes beta-2 microglobulin to facilitate association of the antigen 
with the major histocompatibility complex molecules. The concentration of 
beta-2 microglobulin in the preparation is greater than that which would 
be found in vivo. Selection of the concentration of beta-2 microglobulin 
necessary to augment association of the tumor-derived antigen with the 
major histocompatibility complex molecule is within the ordinary skill in 
the art. In general, the in vivo concentration of beta-2 microglobulin is 
in the range of about 0.2 to about 100 ug/ml, more preferably between 
about 2.0 to about 10 ug/ml (Rock et al., Proc. Natl. Acad. Sci. (USA) 
87:7517-7521 (1990). 
The cellular vaccine stimulates the immune system to specifically recognize 
the tumor of the recipient subject, thereby improving the effectiveness of 
radiation therapy for treating the subject. Although the cellular 
preparation typically is a leukocyte-containing preparation, other types 
of antigen presenting cells are deemed to be within the scope of the 
instant invention. Accordingly, the term "functional equivalent" of a 
photoinactivated leukocyte preparation refers to an antigen presenting 
cell-containing preparation that has been treated by photochemical (e.g., 
photopheresis) or non-photochemical (e.g., exposure of the cell to reduced 
temperatures) methods to enhance expression of the empty major 
histocompatibility complex molecules on the surface of the cells present 
in the preparation. 
The irradiation step takes place in the presence of a photoactivatable 
agent. Other methods (described above) for inducing expression of the 
empty major histocompatibility-complex molecules can be used. The 
photoactivatable agent may be any agent which has an affinity for an 
important component of the leukocyte or other antigen presenting cell and 
which, upon binding to the component, enhances and/or stabilizes 
expression of the major histocompatibility complex molecules. Exemplary 
photoactivatable agents are psoralens, porphyrins, pyrenes, 
phthalocyanine, photoactivated cortisone, photoactivated antibodies 
specifically reactive with the antigen presenting cell, and monoclonal 
antibodies which have been linked to porphyrin molecules. 
The psoralens are a preferred class of photoactivatable agents. The 
interactions of psoralens with the DNA, protein and lipid components of T 
cells have been described ("T Cell Molecular Targets for Psoralens", 
Annals of N.Y. Academy of Science 636:196-208 (1991), Malane, M. and 
Gasparro, F. Following oral administration, 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 administration. These agents can also be added directly to 
the extracorporeal cell preparation. The psoralen molecules are inert 
prior to exposure to ultraviolet or visible light irradiation and are 
transiently activated into an excited state following irradiation. These 
transiently activated molecules are capable of photomodifying biological 
molecules (e.g., DNA, protein) and generating other reactive species, 
e.g., singlet oxygen, which are capable of modifying other cellular 
components. Other agents, e.g., mitomycin C and cis-platinum compounds, 
damage DNA by crosslinking strands of the nucleic acid. However, such 
agents remain in an active state when returned to the patient and thus are 
not as desirable as the psoralens for altering cells to enhance expression 
of empty major histocompatibility complex molecules. 
Preferred psoralens include 8-methoxypsoralen (8-MOP), 
4'-aminomethyl-4,5',8-trimethyl-psoralen (AMT), 5-methoxypsoralen (5-MOP) 
and trimethylpsoralen (TMP). 8-MOP is both an anti-cancer drug, an immune 
system modulator and a prototype for the development of a class of drugs 
that are photoactivatable. AMT is a synthetic, water soluble analogue of 
8-MOP. This and other synthetic water soluble analogues of 8-MOP are 
described in Berger et al., "The Medical and Biological Effects of Light", 
Annals of N.Y. Academy of Science 453:80-90 (1985). Some investigators 
have reported that 5-MOP is not as efficacious as 8-MOP in the treatment 
of psoriasis (Calzavara-Pinton, et al., Exptl. Dermatol. 1:46-51 (1992)). 
TMP is widely used to treat vitiligo patients resulting in the 
repigmentation of depigmented areas of skin. 
Monoclonal antibodies which recognize 8-MOP-DNA photoadducts in irradiated 
cells may be used to determine the optimum amount of ultraviolet or 
visible light irradiation to achieve optimal cell photoinactivation (see 
Yang et al., "8-MOP DNA Photoadducts in Patients Treated with 8-MOP and 
UVA", J. Invest. Dermatol. 92:59-63 (1989). 
Each of the references, patents and patent applications recited in this 
specification are incorporated herein by reference. It should be 
understood that the preceding is merely a detailed description of certain 
preferred embodiments. It therefore should be apparent to those skilled in 
the art that various modifications and equivalents can be made without 
departing from the spirit or scope of the invention. 
EXAMPLES 
Example 1 
Case Studies of Three Patients Diagnosed with Cutaneous T Cell Lymphoma 
(CTCL) 
Tumor stage Cutaneous T Cell Lymphoma (CTCL) is well known to be 
unresponsive, in terms of survival of patients and prolonged disease-free 
periods, to conventional chemotherapy and Total Body Electron Beam (TBEB) 
radiotherapy. In general, the median survival time of a CTCL patient with 
two or more tumors greater than 3 cm in diameter is eighteen months. 
Three case studies are presented below. In each study, a CTCL patient was 
treated with Total Body Electron Beam (TBEB) radiation and photopheresis 
therapy. Following treatment, each patient was examined monthly for skin 
tumors and received periodic skin biopsies and regular CAT Scans to 
determine if internal lymphomas had developed. In contrast to the expected 
progression of the disease, each of the CTCL patients described below has 
remained skin tumor-free. 
(a) Patient M. K. 
Patient Profile and Diagnosis: Patient M. K., a 68 year old white male 
dairy farmer, presented to us in February 1990 with wide spread skin 
tumors that had developed over a period of approximately one year. Skin 
biopies were performed and were diagnostic of CTCL; A CAT scan ruled out 
the presence of obvious internal lymphomas. 
Therapy: M. K. received a total of 3600 RADS of Total Body Electron Beam 
(TBEB) radiation in two groups of 18 treatments (i.e., 100 RADS/treatment) 
over a nine week period. Approximately mid-way through TBEB radiation 
therapy, M. K. received photopheresis therapy (on two successive days) for 
the first time. M. K. continues to receive monthly photopheresis therapy 
on two successive days. 
Patient Evaluation: Since treatment, M. K. has remained skin tumor-free. 
(b) Patient C. Q. 
Patient Profile and Diagnosis: Patient C. Q., a 64 year old white male 
executive, presented to us in 1986 with wide-spread, large skin tumors 
(i.e., tumors having a diameter greater than 3 cm). Skin biopies were 
performed and were diagnostic of CTCL; A CAT scan ruled out the presence 
of obvious internal lymphomas. 
Therapy: C. Q. received a total of 3600 RADS of Total Body Electron Beam 
(TBEB) radiation in 36 treatments (i.e., 100 RADS/treatment; four 
treatments per week) over a nine week period. Approximately midway through 
TBEB radiation therapy, C. Q. received his first photopheresis therapy on 
two successive days. C. Q. continued to receive monthly photopheresis 
therapy on two successive days until 1992 when photopheresis therapy was 
discontinued. 
Patient Evaluation: Since treatment (seven years), C. Q. has remained skin 
tumor-free. 
(c) Patient C. D. 
Patient Profile and Diagnosis: Patient C. D., a 48 year old white male 
executive, presented to us in 1988 with more than fifty skin tumors, 
including a very large (i.e., a tumor having a diameter greater than 7 cm) 
ulcerated tumor on his right thigh. Skin biopies were performed and were 
diagnostic of CTCL; A CAT scan ruled out the presence of obvious internal 
lymphomas. 
Therapy: C. D. received a total of 3600 RADS of Total Body Electron Beam 
(TBEB) radiation in 36 treatments (i.e., 100 RADS/treatment; four 
treatments/week) over a nine week period. Approximately midway through the 
TBEB radiation therapy, C. D. received his first photopheresis therapy on 
two successive days. C. D. continues to receive monthly photopheresis 
therapy on two successive days. 
Patient Evaluation: Since treatment, C. D. has remained skin tumor-free. 
Example 2 
Case Study of a Patient Diagnosed With Metastatic Colon Carcinoma 
Patient Profile and Diagnosis: Patient S. C., a 78 year old white male 
presented to us with a severe productive cough (coughing up bright red 
blood) and was diagnosed as having metastatic colon carcinoma and lesions 
in the lung and liver. Biopsy of the lung lesion by bronchoscopy 
demonstrated that the large mass in the patient's right lung was colon 
carcinoma. 
Therapy: S. C. initially was treated with 5-fluorouracil chemotherapy but 
failed to respond. Subsequently, S. C. received a total of 3000 RADS of 
X-irradiation to the lung lesion in ten divided doses of 300 RADS each 
over a 12 day period. The liver lesion did not receive X-irradiation. 
Approximately midway through the X-irradiation therapy, S. C. received his 
first photopheresis therapy on two successive days. Monthly photopheresis 
therapy on two consecutive days was continued for four months. 
Patient Evaluation: At the end of the fourth month of photopheresis 
therapy, the liver metastases was still present (as determined by CAT 
scan), but the lung lesion was no longer evident. As assessed by periodic 
CAT scans, the lung remained tumor-free until the patient's death 
approximately three years following the initial 
X-irradiation/photopheresis treatment. 
Example 3 
8-MOP/UVA Induction of Empty MHC Class I Molecules at the Cell Surface: 
Association of Empty MHC Class I Molecules with Exogenous Peptide. 
EXPERIMENTAL DESIGN: 
Overview: RMA cells (Ljunggren et al., Nature 346:476-480 (1990)) were 
assayed by cytofluometry for empty class I expression following treatment 
with 8-MOP/UVA to determine whether 8-MOP/UVA uncouples the transport of 
peptide associated-MHC class I complexes to the cell surface. The 
phototreated cells were exposed to temperatures that specifically enhance 
the appearance of empty class I MHC (about 28.degree. C.). Following 
photoinactivation, the empty class I molecules were quantitated by adding 
either of two peptides (Sequence I.D. Nos. 1 and 2), each of which was 
known to bind and stabilize MHC molecules (two specific influenza 
nucleoprotein fragments). To determine whether the treated cells released 
oligopeptides that bind to class 1, the empty class I molecules also were 
quantitated initially following treatment. 
Cells. Murine RMA cells that contain only a few empty class I molecules and 
RMA-S cells, a mutant cell line, in which empty class I molecules have 
been shown to be stable at room temperature but labile at body temperature 
were provided by P. Cresswell (Yale Immunobiology). 
8-MOP/UVA Treatment. RMA cells suspended in PBS were exposed to therapeutic 
doses of 8-MOP (20-200 ng/ml) and UVA (1-10 J/cm.sup.2) in order to 
determine the specific sensitivity of these cells to phototreatment. 
Viability was assayed by trypan blue exclusion immediately after 
treatment. 
Immunochemicals. Monoclonal antibodies with specific reactivities toward 
class I MHC (K.sup.b, Y3 murine hybridoma ATCC No. HB176 and D.sup.b, 
28148S murine hybridoma ATCC No. HB27) were obtained from ATCC (Rockville 
Md.). Influenza virus nucleoprotein oligopeptides (NP365-380 and 
NP345-360) were prepared at the Keck Protein Center. The K.sup.b binding 
16 mer (Sequence I.D. No. 2 having the sequence SFIRGTKVSPRGKLST) and the 
D.sup.b optimally binding 9 mer (Sequence I.D. No. 1 having the sequence 
AENENMETM) were dissolved in IMDM (Iscove's modified Dulbecco's medium, 
GIBCO, Grand Island N.Y.) media in PBS at 50 uM and stored at 4.degree. C. 
FACS Analysis of MHC class I molecules. RMA-S cells served as positive 
controls for the expression of class I MHC molecules. The cells were 
cultured in 5% IMDM, 10% fetal calf serum (FCS) supplemented with 1% 
pen-strep antibiotics (GIBCO). Cells were removed from incubation 
(37.degree. C., 5% C0.sub.2), cultured in tissue flasks (10.sup.6 /ml) 
with tightened caps. To determine the thermal lability/stability of class 
I molecules, the cells were incubated in an adjustable water bath for 48 
hrs at a given temperature. For cytofluorometric assays, 2.times.10.sup.6 
cells were incubated on ice with 10% human serum type AB (GIBCO), then 
with 0.15 ml anti-class I monoclonal antibody tissue culture supernatant 
for 30 min on ice, washed twice with PBS, and then incubated with 0.15 ml 
fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse 
immunoglobulin (Sigma, St Louis Mo.) on ice for 30 min, washed twice with 
PBS, fixed in 1% formaldehyde and analyzed on a FACS cell sorter. To 
stabilize class I MHC molecules, influenza nucleoproteins fragments 
(Sequence I.D. Nos. 1 or 2) were added (50 uM). 
RESULTS: 
FIG. 1 shows the effects of 8-MOP (10-300 ng/ml) and UVA (1 J/cm.sup.2) on 
the survival of RMA cells (as determined by trypan blue exclusion). The 
viability of cells which were not exposed to 8-MOP or UVA are designated 
"no Tx" (i.e., no treatment) in the figure. The viability of cells which 
were exposed to UVA but which were not exposed to 8-MOP are designated "1 
J/cm" in the figure. A dose-dependent increase in cell damage was observed 
with increasing doses of 8-MOP used in combination with 1 J/cm.sup.2. The 
increasing doses are indicated in the figure (i.e., the designations of 
10, 30, 100 and 300 refer to the concentration of 8-MOP in ng/ml). 
The effects of 8-MOP (100 ng/ml) and UVA (1 J/cm.sup.2) on class I 
expression and the binding of specific oligopeptides (Sequence I.D. No. 1) 
to RMA class I molecules was determined. FIG. 2 represents a composite set 
of experiments showing the time course for enhancement of D.sup.b class I 
MHC molecules on RMA cells treated with 100 ng/ml 8-MOP and 1 J/cm.sup.2 
UVA. In this series of experiments, the 8-MOP/UVA treated cells were 
compared to cells exposed to UVA alone (1 J/cm.sup.2 ). 
FACS analysis was used to gauge the extent of class I expression using ATCC 
antibodies specific for D.sup.b class I molecules. The change in mean 
channel fluorescence (.DELTA.% MCF) was calculated by taking the 
difference in signal for cells incubated with and without the class I 
oligopeptide (Sequence I.D. No. 2, i.e., AENENMETM). FIG. 2 illustrates 
that the optimal signal (i.e., greatest increase in class I molecule 
expression) was detected within 10 hrs of 8-MOP/UVA treatment. An 
additional set of untreated control cells (designated "No Tx" in the 
figure) also was assayed. The optimal time for inducement of class I 
expression is determined by performing more detailed kinetic studies, 
e.g., by assaying the cells at more frequent intervals and at several 
different temperature conditions following 8-MOP/UVA treatment. 
Antigens are selected for their ability to associate with empty class I 
molecules using the above-described FACS method. Thus, the FACS assay 
serves as a screening protocol for selecting the optimum conditions for 
inducing empty class I molecule expression, as well as a screening 
protocol for selecting antigens that are capable of binding to and 
stabilizing the empty class I molecules. In this manner, various peptides, 
and the optimum concentrations for each, are screened for their ability to 
stabilize empty class molecules. 
Each of the above-recited patents and references is incorporated herein by 
reference. It should be understood that the preceding is merely a detailed 
description of certain preferred embodiments. It therefore should be 
apparent to those skilled in the art that various modifications and 
equivalents can be made without departing from the spirit or scope of the 
invention. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 2 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 9 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
AlaGluAsnGluAsnMetGluThrMet 
15 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 16 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
SerPheIleArgGlyThrLysValSerProArgGlyLysLeuSerThr 
151015 
__________________________________________________________________________