Use of photodynamic compositions for cytotoxic effects

Hematopoietic cells are selectively eliminated by cytotoxic agents which rely upon light activation. The cytotoxic agents are joined to ligands, particularly sugars, specific for myeloid monocyte and lymphocytic lineages. Further specificity can be achieved by limiting the light target. The method and compositions find particular use in organ transplants and arthritis.

INTRODUCTION 
1. Technical Field 
The subject invention is related to the use of photoactive compounds for 
cytotoxic therapeutic effects. 
2. Background 
There is a general interest in being able to target specific cells or 
tissue for cytotoxicity. The cells or tissue may be diseased tissue or 
cells or in many cases healthy cells. The healthy cells may be involved in 
inhibiting a therapeutic treatment, such as in transplants or causing a 
disease state, such as in autoimmune diseases. 
For example, organ transplantation can be lifesaving in diseases affecting 
the heart, kidneys, liver, and lung. It has also been shown that 
transplantation of dispersed pancreatic islet cells may completely cure 
diabetes, a common and debilitating disease. The major impediment to the 
more widespread use of transplantation is rejection of the donor tissue by 
the transplant recipient. In part, this problem has been overcome by the 
use of the immunosuppressive agent, cyclosporin-A. However, the renal 
toxicity, infection, and expense associated with cyclosporin-A treatment 
has impeded the widespread use of the drug. 
Experiments on animal model systems suggest that the transplantation 
rejection phenomenon is triggered by cells in the donor organ that express 
class II histocompatibility antigens. Therefore, if these cells could be 
removed from the donor organ, or inactivated, transplantation could be 
successfully accomplished even across major histocompatibility barriers. 
Experiments have suggested that besides T cells, monocytes and macrophages 
are the cells involved in triggering the rejection response. As a result, 
a number of laboratories are attempting to remove these cells from donor 
organs before transplantation. One method directs the A chain of the toxin 
ricin to the mannose receptors of the monocytes and macrophages. Using 
this technique, macrophages could be selectively removed from rat liver by 
treatment of the animals with the ricin-A chain. However, following cell 
death, the ricin-A chain is released into the plasma where it is 
conceivably toxic to other cell types. Another method uses monoclonal 
antibodies against macrophage cell surface proteins to remove the 
monocytes and macrophages from the donor organs. None of these procedures 
have found general usage, nor are they without undesirable side effects. 
There are also autoimmune diseases, which are believed to be associated 
with hematopoietic cell attack of native tissue. Exemplary of these 
diseases are various forms of joint inflammations, particularly rheumatoid 
arthritis and psoriatic arthritis. Many of the procedures used for the 
treatment of arthritis have been directed to the reduction of 
inflammation. The use of steroids has many side effects and frequently 
cannot be employed because of the adverse effects on the patient. Since 
the cells which may be involved with the inflammatory response in the case 
of the autoimmune disease may be of a general function, substantially 
depleting the host of such types of cells may have adverse effects on the 
ability of the host to fight off infectious agents. Therefore, generalized 
reagents which kill off all macrophages and monocytes, cells which may be 
involved with the arthritic condition, may subject the host to infection. 
There is therefore an interest in being able to selectively prevent the 
adverse effects resulting from activities of native tissue, where the 
activities may have both beneficial and debilitating effects on the host. 
Techniques which allow for elective responses will provide for protection 
for a transplanted organ, or from pathogens, while at the same time 
maintaining the immunological capability of the host. 
Relevant Literature 
Cell specific ligands which were found to be potent competitive inhibitors 
of the uptake of labeled D-mannose-bovine serum albumin conjugate by rat 
macrophages were disclosed by Ponpipom et al. J. Med. Chem. (1984) 24, 
1388-1395. Synthetic glycopeptide substrates for receptor-mediated 
endocytosis by macrophages were described by Robins et al. Proc. Natl. 
Acad. Sci. USA 78:7294-7298. The IME-thioglycosides for attaching sugars 
to proteins were described by Lee et al. Biochemistry (1976) 18:3956-3963. 
The use of ricin-A chain to delete mannose receptor bearing cells from rat 
liver was described by Simmons et al. (1987) Biochem Biophys. Research 
Comm. (1987) 146:849-854. The presence of a cell surface receptor on 
macrophages that binds glycoproteins having terminal sugars with the 
mannose or glucose configuration was disclosed by Stahl et al. (1978) 
Proc. Natl. Acad. Sci. USA (1978) 75:1399-1403. Compositions and methods 
for the treatment of organs to be transplanted are provided. The method 
involves removing phagocytic cells from the tissue to be transplanted by 
contacting the tissue for transplantation with a cytotoxic agent 
comprising a central nucleus, at least one saccharide moiety, and a toxic 
component specific for the inhibition of DNA replication. The method can 
be used to treat any organ for transplantation including heart, kidneys, 
liver, lung, and pancreas. Tupikin et al., Rev Matologian (Moskva) (1983), 
p. 32-32 describes the use of tetracycline as a photoactivator in exposure 
to argon laser rays in zymosan arthritis in rats. See also Tupikin et al., 
Voprosy Revmatizma (1982), p. 39-41. Oseroff et al., Proc. Natl. Acad. 
Sci. USA (1986) 83:8744-8748 describes the selective cytolysis of human 
T-cell leukemia cells in vitro using mAb conjugated to the photosensitizer 
chlorin. Ohnishi, et al., Jap. J. Ophthal. (1987), 31:160-170 described 
the use of argon-laser photoradiation on monkey retina treated with 
hematoporphyrin. 
Manyak, J. Clin. Oncol. (1988) 6:380-391 provides a review of photodynamic 
therapy. See also the references cited therein. Tshish and Bailey, Anal. 
Biochem. (1985) 144:132-141 describe a particular peptide photoaffinity 
agent which may be used for linking a polypeptide to a photoreceptor. 
SUMMARY OF THE INVENTION 
Photodynamic therapy is provided directed to hematopoietic cells involved 
with cellular attack on endogenous cells, particularly those cells 
involved with organ transplant rejection and autoimmune diseases such as 
arthritis. The method employs reagents having a ligand for binding to the 
cell target and a photoactivatable agent, which results in selective 
cytotoxicity upon exposure to light of the appropriate wavelength. As 
appropriate, light is specifically directed to the site of interest 
avoiding cytotoxicity of host cells removed from the site of interest. 
DETAILED DESCRIPTION OF THE INVENTION 
In accordance with the subject invention, novel methods and compositions 
are provided for destruction of hematopoietic cells associated with 
disease such as graft-versus-host disease, and autoimmune diseases. The 
method involves contacting the cells either directly or indirectly with a 
cytotoxic agent specific for hematopoietic cell targets, particularly 
macrophages and monocytes, and irradiating with light where the target 
cells are selectively killed. 
In the case of transplants, the organ to be transplanted, removed from the 
donor, is contacted under appropriate conditions with the cytotoxic agent, 
irradiated with the appropriate wavelength, and may then be washed free of 
cells with the assurance that macrophages or monocytes will have been 
substantially depleted and, desirably, totally eliminated as viable cells. 
In the case of autoimmune diseases where a specific site may be illuminated 
as in arthritis, the cytotoxic agent is generally administered to the 
host, and after sufficient time for dissemination of the agent throughout 
the host, the sites of the inflammation may be illuminated through the 
skin by employing an appropriate light source. 
For the most part, the compounds or cytotoxic agents will employ a toxic 
component, normally a photoactivatable agent, the ligand which directs the 
toxic component to the hematopoietic target cell and a linking moiety. The 
ligand will normally be a saccharide moiety which shows specificity for 
binding to surface receptors of cytotoxic cells, particularly phagocytic 
cells, such as macrophages and monocytes. 
Hematopoietic cells include phagocytic cells, helper T-cells and the like. 
Phagocytic cells are any cells which ingest microorganisms or other cells 
and foreign particles. In most cases, the ingested material is digested 
within the phagocyte. For purposes of the present invention, phagocytic 
cells include monocytes, macrophages, and other cells that are bound by 
the saccharide moiety and express class II histocompatibility antigens and 
trigger the transplantation rejection phenomenon. Monocytes include 
mononuclear phagocytic leucocytes which are formed in the bone marrow and 
are transported to the tissues, as of the lung and liver, where they 
develop into macrophages. 
Cytotoxic T-cells include those cells which are, for the most part, 
CD8-positive and are involved with Class I MHC antigen and may include 
lymphokine-activated killer cells, natural killer cells, 
antibody-dependent cytotoxic cells or the like. 
The instant method is effective in removing undesired hematocytes from cell 
tissue by directing an agent which is toxic to cells of the myeloid or 
T-cell lymphoid lineage but has no effect on other cell types. This is 
accomplished by directing the toxic agent to receptors which are specific 
to such cells. For example, mannose receptors are specific to phagocytic 
cells and are not detectable on any other cell type. Therefore, it is 
feasible to deplete mannose receptor bearing cells by directing a toxin to 
the mannose receptor containing cells. 
The cell surface receptors of the phagocytes follow a recycling pathway. 
That is, the receptor binds a ligand dissociates and takes it from the 
cell surface where it is bound to an intracellular, prelysosomal acidic 
compartment, where the ligand dissociates from the receptor. The 
unoccupied receptor is returned to the cell surface for binding of more 
ligand. Thus, any cell which employs this receptor mediated pinocytosis 
for the selective efficient uptake of macromolecules can be selectively 
killed by the instant method. The cell surface receptors include those 
which bind and internalize injected glycoproteins. For binding to 
monocyte-macrophage cells, the preferred glycoproteins are those having 
terminal sugars with mannose or glucose configuration, preferably with 
terminal mannose molecules. 
For cytotoxic T-cells, ligands other than sugars may be employed, which are 
specific for the cytotoxic T-cells. For example, various peptides may be 
employed which would bind to the CD8 protein or other receptors specific 
for the cytotoxic T-cells. These receptors include T-cell receptors and 
alpha-beta heterodimers. Alternatively monoclonal antibodies specific for 
cell types can be employed. 
The cytotoxic agents which are employed will comprise the specific 
cell-directing ligand, the linking moiety, and the toxic component 
normally a photoactivatable agent. For the most part, the linking moiety 
will be either a bond or a small group or molecule of about 1 to 60 atoms 
other than hydrogen, more usually of about 1 to 20 atoms other than 
hydrogen. Conveniently, the linking group may be comprised of one or more 
amino acids, particularly those amino acids which have an additional 
functionality other than the carboxy or amino group of glycine. These 
amino acids include the basic amino acids, lysine and arginine, aspartic 
and glutamic acid, serine and threonine, cystine and histidine. In some 
instances it may be desirable to combine one or more amino acids to 
provide oligopeptides of about 2 to 10 amino acids. Of particular interest 
are oligolysines of from 2 to 4 units. When amino acids are used, they may 
be the natural L-enantiomer or the unnatural D-enantiomer or a racemic 
mixture. The linking groups may include functionalities, such as amides, 
esters, ethers, amines, thioethers, or the like. The various methods for 
linking the ligand and cytotoxic agent to the linking moiety may be varied 
widely and will follow conventional techniques. Thus, sugars, esters, 
ethers, and the like may be employed, particularly with substitution at 
the 1, 4, or 6 positions, or the like. For preparation of sugar 
derivatives, see, for example, Lee, et al. Biochemistry (1976) 
15:3956-3963. 
As the ligand, of particular interest will be saccharide molecules. 
Attached to the linking moiety may be a saccharide monomer or a 
polysaccharide. It may be desirable to utilize more than one saccharide 
monomer, a polysaccharide, as more than one sugar may be involved in 
binding to the receptor. For the present invention, preferably from about 
1 to about 6 saccharide monomers will be utilized, more preferably about 3 
to about 4 saccharide monomers. 
Suitable saccharide molecules are any which bind specifically to the cell 
surface receptor site of interest. Since the present invention is involved 
in the elimination of particular cells, it will be desirable to utilize 
saccharide molecules which selectively bind to the cells of interest. 
Thus, as cells of the monocyte-macrophage lineage express specific 
receptors for mannose, and mannose receptors are not detectable on any 
other cell type, it will be particularly desirable to use mannose as the 
saccharide molecule. 
Where more than one saccharide monomer is used, the polysaccharide may 
contain backbone linkages of any possible variation. For example, the 
sugars may be linked by either alpha or beta linkages in 1-2, 1-3, 1-4, or 
1-6 fashion. However, as binding may be affected by the polysaccharide 
backbone, it may be desirable to construct the cytotoxic agent with a 
particular polysaccharide linkage. With mannose binding, although other 
constructions may be utilized, alpha-1,6 linkage is preferred. 
For the most part, homopolysaccharides, that is, polysaccharides containing 
only one type of monomeric unit, will be utilized. As indicated earlier, 
homopolysaccharides of mannose units are preferred for the removal of 
phagocytic cells. However, if desirable, heteropolysaccharides, that is, 
those saccharides which contain two or more different monomeric units, may 
find use. 
As disclosed earlier, recognition of carbohydrates by cells of binding 
proteins may require multivalency or clustering of saccharide residues, 
the various numbers of saccharide residues may be incorporated into the 
polysaccharide for binding to the linking moiety. These saccharide 
residues are introduced onto polypeptide backbones and these ligands used 
to deliver cytotoxic and therapeutic agents to saccharide receptor bearing 
cells. 
Derivative forms of the monosaccharides may find use, such as glycosides, 
N-glycosylamines, O-acyl derivatives, and O-methyl derivatives. Examples 
of derivative forms of saccharide molecules, useful for binding to 
macrophage receptors, can be found in Ponpipom et al. (1981), J. Med. 
Chem. 24, 1388-1395, which is incorporated herein by reference. Where 
desired, the thiol forms of the saccharide derivatives may be employed. 
The saccharide or polysaccharide molecule can be covalently attached to the 
linking moiety. For example, if trilysine is used as the backbone or 
linking moiety, saccharide molecules are covalently linked to the epsilon 
amino group. Alternatively, the polysaccharide molecule can be attached to 
the linking moiety by any number of techniques which have been developed 
for attaching sugars to proteins. Any of the known methods can be used 
including the mixed anhydride method, described by Ashwell (1972), Meth. 
Enzymology 28: 219, and the acyl azide method described by Lemieux et al. 
(1975), J. Am. Chem. Soc. 97, 4076. Also the use of diazonium salts, 
cyanoborohydride coupling agents, and 2-imino-2-methoxyethyl 
1-thioglycosides, IME-thioglycosides, as described by McBroom et al. 
(1972) Methods Enzymol. 28, 212; Gray (1974), Arch. Biochem. Biophys. 163, 
426; Moczar et al. (1975), FEBS Lett. 50, 300; and Lee et al. (1976), 
Biochemistry 15, 3956, respectively, may be employed. The conversion of 
the saccharide moiety to an IME-thioglycoside may be preferred as these 
IME-thioglycosides react readily with simple amines, amino acids, and 
proteins frequently without loss of any of the activity of the protein 
molecule. For use with macrophage receptor sites, mannosylating reagents 
that bind selectively to free amines have been described by Lee et al. 
supra, whose disclosure is incorporated herein by reference. 
Since, for the purpose of the subject invention, the primary concern will 
be selection between the target cells and the other cells in the vicinity 
of the target cells, the ligand need not be specific for the target cells 
as against all other cells. It will be sufficient for the subject 
invention that the ligand be specific for the target cells, so as to 
distinguish between the target cells and the cells subjected to the light 
irradiation. Therefore other ligands which may be used which are specific 
for T-cells may include interleukins such as IL-2, IL-4, IL-5, and IL-6. 
The toxic component will for the most part comprise photoactivatable agents 
which have found exemplification in the literature. These photoactivatable 
agents include coumarin, coumarin derivatives such as the psoralens, 
phthalocyanines, methylene blue, eosin, tetracycline, chlorophylls, 
various porphyrins, and the like. In many instances these compounds will 
have functionalities for linking to the sugars or to the linking moiety to 
provide for a stable linkage. The same types of linkages may be employed 
between the cytotoxic agent and the linking moiety as was described for 
linking the ligand to the linking moiety. 
In some instances, particularly for the removal of the myeloid cells from 
transplantation organs, cytotoxic agents other than the photoactivatable 
agents may be employed. Such agents are well-known and may include a 
variety of DNA inhibitors such as anthacycline, daunomycin and adriamycin, 
amino-acridine derivatives such as mAMSA, cis-platinum derivatives, or the 
like. 
In the case of transplantation, by transplantation is intended the transfer 
of tissue or organs from the body for grafting into another area of the 
same body or into another individual. Tissues to transplant include 
tissues, organs, and in some cases cells. Those transplants subject to the 
present method are any organs or tissues for transplantation, including 
but not limited to heart, kidneys, liver, lungs, pancreatic islet cells, 
bone marrow, stem cells, etc. The instant method is particularly useful 
for those organs that are very rich in monocytes and macrophages such as 
the pancreas, liver, and lung. 
After surgical removal of the tissue or organ for transplantation, the 
organ is flushed with a preservative liquid, the perfusion medium. At the 
same time the temperature of the tissue or organ is lowered and the organ 
is maintained in a cold state, usually less than about 25.degree. C., 
preferably about 4.degree. C., so that its metabolic needs are kept at a 
minimum. After the chilling procedure is completed, the cooled organ is 
maintained at the low temperature by ice or refrigeration. Just prior to 
transplantation, the organ is again flushed using the same procedure as in 
chilling but in reverse. The perfusion medium used to flush the organ or 
tissue is progressively raised to a temperature level close to normal body 
temperature. Upon completion, the organ is in condition for 
transplantation. 
By perfusion medium is intended the fluid which is pumped through the organ 
as well as the solution in which the organ or tissue is immersed. The 
cytotoxic agent is added to the perfusion medium used to maintain organs 
prior to transplantation to eliminate monocyte-macrophage cells. Cells of 
the monocyte-macrophage lineage ingest the conjugate, via their mannose 
receptors, and release the toxic agent, after digestion of the peptide in 
the endocytic vesicle. Uningested material and dead cells are washed out 
by perfusion, prior to transplantation of the organ or tissue. 
Thus, the cytotoxic agent is included in the perfusion medium which is used 
to flush and maintain the organ prior to transplantation for a time 
sufficient to eliminate phagocytic cells. The cytotoxic agent can be 
included in the perfusion medium at any stage of the flushing or washing 
of the organ to be transplanted. The agent can be added to the perfusion 
medium at a particular temperature during chilling or subsequent rewarming 
or, alternatively, it can be present during all phases of the washing and 
maintenance of the organ or tissue. In all cases, it will be desirable to 
wash the organ to be transplanted with liquid minus the cytotoxic agent 
prior to transplantation. As binding of the cytotoxic agent occurs more 
readily between about 25.degree. to about 37.degree. C., it will be 
preferable to include the cytotoxic agent in the preservative medium at 
this temperature. 
The tissue to be transplanted is washed with the perfusion medium 
containing the agent for about 1/2 hour to about 4 hours, preferably about 
1 hour. With photoactivatable agents, the organ may then be thoroughly 
irradiated with light of the appropriate wavelength. Various means for 
irradiation may be employed, depending upon the nature of the particular 
organ. The light wavelength will desirably be greater than about 300 nm 
and may have a wavelength as high as 900 nm. After thorough irradiation, 
which may require anywhere from about 1 min to about 1 hr, preferably not 
more than about 30 min, the organ may then be flushed with perfusion 
medium minus the cytoxic agent to wash out any uningested agent prior to 
transplantation. In some instances it may be desirable to use the 
photoactivatable agent in conjunction with a cytotoxic agent which does 
not require photoactivation. Alternatively, one or more perfusions may be 
employed using one or another of the reagents by themselves or in 
combination. 
The concentration of the cytotoxic agent contained in the perfusion medium 
during washing may vary according to the time of treatment, the 
temperature or pH of the medium, or the organ being treated. Normally the 
agent is included in the perfusion medium at a concentration of about 
100.mu.M to 10mM preferably about 1mM to 10mM. 
The cytotoxic agent can be utilized with practically any perfusion medium. 
Where the perfusion medium contains no calcium ions, it may be useful to 
add calcium to the medium to facilitate saccharide binding. Further, as 
non-specific binding is increased greatly in the absence of serum, it may 
be necessary to add HSA, BSA or other acceptable protein to the liquid. 
For the most part, the pH of the liquid will be suitable for binding of 
the cytotoxic agent. A pH from about 6.6 to about 7.4 will usually be 
employed for greater binding of the cytotoxic agent to the cell surface 
receptor. 
This method for treating organs to be transplanted can be used with any 
flushing procedure currently used. That includes those procedures which 
involve continuous gravity flow through the tissue, where the chilled 
liquid is dispersed from a solution bottle held at a height sufficient to 
produce a continuous gravity flow, or those procedures which involve 
mechanical washers, where the liquid is provided in pulsatory flow in a 
manner simulating the action of the circulatory system. After the donor 
organ or tissue has been treated with the cytotoxic agent and subsequently 
washed, the organ may be transplanted without releasing cytotoxic 
materials into the donor. In this manner, the phagocytic cells are 
eliminated from the tissue or organ to be transplanted and rejection of 
the tissue by the transplant recipient is prevented. 
For treatment of arthritis, the agent may be administered generally to the 
host through the vascular system, or be injected directly into the site to 
be treated. The determination of whether the drug should be generally or 
specifically administered will be a matter of the number of sites to be 
treated, the level of drug required and the like. Generally the 
concentration employed will provide for concentration at the site to be 
treated in the range of about 10.mu.M to about 1mM , more usually about 
10.mu.M to about 100.mu.M. Thus the concentration of the reagent will vary 
widely depending upon the manner of administration. Conveniently, the 
cytotoxic agent may be formulated in a suitable physiologically acceptable 
medium such as sterile water, phosphate-buffered saline, aqueous alcohol, 
or the like. 
After administration of the photoactivatable cytotoxic agent, the sites to 
be treated will be subjected to light for a time to sufficiently activate 
the toxic component. Optical fibres may be directed to the skin site or 
allowed to penetrate a small distance into the skin. With larger areas to 
be illuminated, a plurality of optical fibres may be employed or, 
alternatively, various irradiation lasers may be employed. The intensity 
of the light will vary depending upon the level required, the distance the 
light must penetrate, and the like. The energy delivered will vary, 
generally being in the range of about 0.1mJ/cm.sup.2 to about 
5000J/cm.sup.2. After illumination, depending upon the manner in which the 
cytotoxic agent is administered, the patient may be required to be 
protected from light after the treatment. 
The subject compositions may find alternative uses, particularly in 
research where it may be desirable to deplete various cellular 
preparations of a particular group of cells. Thus, rather than mechanical 
selection such as fluorescence-activated cell sorting, the subject 
methodology allows for relatively rapid specific removal of the undesired 
cells. The subject compositions may also find use in other applications 
besides research, where it is desired to remove a particular group of 
cells and the cells can be subject to irradiation. 
The use of the cytotoxic agent as described and the treatment of organs or 
tissues to be transplanted offer several advantages over current methods 
for the treatment of donor tissue. First, the agent is selectively toxic 
to cells of the monocyte-macrophage lineage, but will have little effect 
on other cell types. This is accomplished first by designing the cytotoxic 
agent to bind only to cell surface receptors of the monocyte-macrophage 
cells. Secondly, the cytotoxic component utilized is designed such that it 
interacts tightly with the DNA and/or needs the appropriate light wave 
length for activation. Further, after ingestion by the phagocytic cells, 
there is no release of toxic materials to other cells. 
Besides effectively eliminating the phagocytic cells, the cytotoxic agents 
are stable and easy to prepare. Furthermore, for use with transplants the 
drug would be used in vitro rather than in vivo and, therefore, would be 
washed out prior to transplantation. 
The following examples are offered by way of illustration and not by way of 
limitation.

EXPERIMENTAL 
Treatment of Arthritis 
First, chlorin monoethylenediamine monoamide (chlorin e.sub.6 -A) is 
prepared by sequential addition of triethylamine and ethylchloroformate to 
chlorin e.sub.6 dissolved in anhydrous dimethylformamide, as described by 
Oseroff et al. Proc. Nat'l. Acad. Sci. (1986) 83:8744. A 10-fold molar 
excess or ethylenediamine is then added to the monoactivated chlorin, and 
the product is purified by reverse phase HPLC on a C.sub.18 column eluted 
with 0-55% methanol gradient in 0.1 M sodium phosphate pH 6.8. CNM 
thioglycomannoside is purchased from E.Y. Laboratories, and is converted 
to the 2-amino-2-methoxyethyl-1-thiomannoside by incubation in sodium 
methoxide. After evaporation, the residue is mixed in 0.1 M borate buffer 
pH 9 with a synthetic di-peptide consisting of lysine-lysine. After 
overnight incubation, reactants are separated by passage through a column 
of Sephadex G25 to remove the free thioglycoside. In this way, a 
trimannosyldilysine peptide is generated in which the epsilon amino groups 
are coupled to mannose. The mannosylated peptide is then mixed with the 
chlorin e.sub.6 -monoethylenediamine monoamide in the presence of a water 
soluble carbodiimide to couple the chromophore to the free carboxyl group 
of the peptide. Conjugate is separated by reverse phase HPLC as described 
above. This chlorin-trimannosyl peptide the active moiety for elimination 
of macrophages and other cells having mannosyl receptors. 
In a typical in vitro experiment, human peripheral blood is obtained, and 
the mononuclear cell fraction is isolated by ficoll-hypaque sedimentation. 
Cells are suspended in RPMI 1640 medium supplemented with 5% autologous 
serum. Subsequently, the medium is supplemented with the 
chlorin-trimannosyl lysyl peptide. Then the cells are washed three times 
and suspended in RPMI 1640 medium supplemented with 10% autologous serum. 
The cells that have been incubated with the conjugate for 30 min at 
4.degree. C. are irradiated with a light source filtered to deliver light 
between 630-670 nm. The dose rate is typically 0.6 mJ/cm.sup.2. Controls 
include unirradiated cells and cells which have been exposed to light, but 
not the photosensitizer. After radiation, cells are washed, and both 
immediate and delayed photocytotoxicity is assayed using ethidium bromide 
staining and fluorescence microscopy. In some experiments, lymphocytes are 
distinguished from monocytes and macrophages using monoclonal antibodies 
to the CD5 Pan-T cell antigen, and to immunoglobulin. Monocytes and 
macrophages are visualized by the binding of anti-Macl (anti-CDllb) 
antibodies. Selectivity is demonstrated by the elimination of viable 
monocytes and macrophages and the preservation of viability in the 
lymphocyte population. 
In an in vivo experiment, adjuvant arthritis is induced in Spague Dawley 
rats by injection of complete Freund's adjuvant intradermally in the back. 
After the onset of joint swelling, the animals are injected 
intraperitoneally with the chlorin-peptide conjugates at dosages ranging 
from 0.1-10 mg/kg. At 4, 24, and 48 hrs after the injection the animals 
are anesthetized, and the immobilized limbs are selectively exposed to 
630-670 nm light to yield a total dosage of approximately 50 J/cm.sup.2. 
Control animals receive either light exposure alone, or treatment with the 
chlorin conjugate alone. After returning the animals to their cages, joint 
swelling is monitored daily. Efficacy of the treatment is indicated by 
accelerated reduction of joint swelling and inflammation in animals that 
have received the phototoxic therapy. 
Depletion of Immunogenic Monocytes and Macrophages From a Donor Organ 
Before Transplantation 
First, tri-mannosyl dilysine is coupled to daunomycin using a water-soluble 
carbodiimide as described earlier. Conjugate is purified by reverse phase 
HPLC This daunomycin-trimannosyl peptide is the active moiety for the 
elimination of macrophages and monocytes from donor organs. In a typical 
experiment, an explanated kidney or liver from a rat is perfused at 
4.degree. C. with cold Collins storage solution, or with the same solution 
supplemented with 10-100 .mu.M of the daunomycin peptide conjugate for 1 
hr. Then the organs are flushed vigorously with Collins solution and 
maintained in the same for up to 48 hrs prior to transplantation into 
nephrectomized rats. Following transplantation, the animals are followed 
for signs of graft rejection, as indicated by rising blood urea nitrogen 
and serum creatinine. Rats that have received kidneys treated with the 
daunomycin peptide conjugates have prolonged maintenance of renal 
function, compared to control rats. 
The subject invention provides for a convenient and efficient way to 
selectively remove cells which can be adverse to the host. By employing 
the subject technique, graft-versus-host disease can be avoided in the 
case of transplants, while substantial amelioration of inflammatory 
activity in joints may be substantially diminished without harm to the 
other tissue present and without substantially depleting the host of its 
immune capability. 
All publications and patent applications mentioned in this specification 
are indicative of the level of skill of those skilled in the art to which 
this invention pertains. All publications and patent applications are 
herein incorporated by reference to the same extent as if each individual 
publication or patent application was specifically and individually 
indicated to be incorporated by reference. 
Although the foregoing invention has been described in some detail by way 
of illustration and example for purposes of clarity of understanding, it 
will be obvious that certain changes and modifications may be practiced 
within the scope of the appended claim.