CA2376001A1 - Photodynamic therapy compounds - Google Patents

Abstract

Compounds for photodynamic therapy comprising tetra (hydroxyaryl) porphyrins , chlorins and bacteriochlorins conjugated with antibodies reactive to antigen s of cancerous or other diseased cells.

Description

TITLE
Photodynamic Therapy Compounds FIELD OF INVENTION
The invention relates to compounds for photodynamic therapy (PDT) of cancerous and other diseased tissue.
Among known PDT compounds are those of USP 4,837,221 and USP 4,992,257 the disclosure of which is incorporated herein by reference.
The disclosure of '221' includes porphyrins of the formula:
A
Rn Rn nn where n=1 to 3 and each substituent R in the phenyl rings, or other aryl groups replacing phenyl, is a hydroxy group. Particularly noted are those compounds where each substituent is o-hydroxyphenyl, m-hydroxyphenyl or p-hydroxyphenyl.
The disclosure of '257' includes di-hydro porphyrins (chlorins), and corresponding tetra-hydro porphyrins (bacteriochlorins) of the formulae:

Ar III
IV
wherein each Ar is an aromatic group with one or more hydroxy substituent groups but as with '221' is desirably phenyl. Preferred compounds are m-THPC, meta tetra(hydroxyphenyl) chlorin, currently in FDA trials, and the corresponding bacteriochlorin.
As will be recognized, the above formulae show in each case one only of the possible imino-nitrogen tautomers; possible tautomers include for chlorins:-VA VB
VC VD
for bacteriochlorins:-v1A v18 and for isobacteriochlorins:-VIIA VIIB

THE INVENTION
We have sought improvements to PDT compounds and propose linking (conjugating) them to antibodies including active antibody fragments, specific to antigens of a diseased tissue. In particular, a number of monoclonal antibodies (Mabs) are available targeted to cancerous cells and we have found that they can be chemically linked to PDT compounds to give valuable PDT
therapies.
More particularly the invention provides compounds where a ring structure Ar representing a porphyrin, chlorin or bacteriochlorin/isobacteriochlorin ring in any of its imino-nitrogen tautomeric forms, carnes four aromatic preferably phenyl substituents Ar each themselves carrying one or more hydroxy groups, one or more of which hydroxy groups is in turn linked to an antibody (in which are included antibody fragments) reactive to a cancer or other diseased -cell antigen, giving a conjugate valuable in PDT therapy of the disease. The preferred aromatic substituent is m-hydroxy phenyl, as in m-THPC.
In principle the antibodies can be linked direct to the hydroxy groups but generally the presence of a linking group is preferred. Examples are ether-linked groups for example O
(CH2)n ~ or (CH2)n-A
OH
where n is 1 to 4 and A is -OH or NHZ

or, less preferred, ester-linked groups for example:--c o 0 \(CHz)n \
OH
-C O
(CHz)nA
C O
\CH(R~)NH~nH
where n and A are as before and R' is hydrogen or a hydrocarbon or carboxylated amino-acid side chain, representing particularly glycine, alanine, lysine or glutamic acid.
Ester, amide or other links are then readily formed with the antibody according to the free reactive groups present on it.
The invention extends to compounds as in VIII but carrying such linking groups, as intermediates, as well as to the antibody conjugates themselves.
The invention further relates to the treatment of disease by PDT using the antibody conjugates and to the use of antibody conjugates in the preparation of medicaments for PDT.
It is believed, though the invention is not restricted to any mechanism, that in use of the conjugates they or at least the PDT active are internalized by the cells of diseased tissue after administration of the conjugate.
The mechanism of action of PDT agents is believed to be that they destroy mitochondria) function by the generation of free radicals and/or reactive oxygen species.
Such free radicals or reactive oxygen species need to reach the mitochondria, which are within the cytoplasm, and it is supposed that for photodynamic therapy to be active, the PDT molecule has to penetrate the cell surface membrane and be energized in close proximity to the mitochondria. The free radicals and reactive oxygen species then do the damage.
However, it may equally be that if adequate PDT conjugate binds to cell membranes and is energized there, then the free radicals and reactive oxygen species can penetrate the cell surface membrane thereafter destroy the mitochondria.
The antibodies will normally be monoclonal antibodies of which mMAB 425 discussed in detail herein is an example. There are many such antibodies known, directed against cell surface antigens of cancer or other disease cells. Examples include Antigen Target CD 19 Lymphoma CD20 LymphomaJchronic lymphocytic leukemia/non-Hodgkin lymphoma (IVHL) CD33 Acute pro myelocytic leukemia CD45 Leukemia CD40 Leukemia Target (solid tumorsl HLADR (lyml) Lymphoma/CLL

Lewis Y Breast Integrin receptorBreast PEM (MCTCI) Breast HER2/erB2/neu Breast CEA Epithelial (colon) VEGF Epithelial a cell surface glycoprotein Epithelial (colon) EGF-r Breast, renal, pancreatic, H&N, lung TNT Lung, pancreas, gastric, colon, breast PSMA Prostate TAG-72 Ovarian CD56 Small-cell lung carcinoma PLAP Ovarian, cervical, testis The chemistry of the conjugation of the actives and antibodies depends on their nature and on whether a linking group is used and is not central to the invention in its broad sense.
However, chemistry as described herein for m-THPC is suitable for other chlorins and also for porphyrins and bacteriochlorins/isobacteriochlorins, particularly the m-hydroxy phenyl derivatives corresponding to m-THPC.
Particular aspects of the invention are described in detail below, giving first a summary and general discussion, largely with reference to m-THPC conjugates, and then going on to materials and methods, to results, and to further discussion, all again largely in relation to m-THPC.
SUNINIARY
A limitation of photodynamic therapy (PDT) is the lack of tumor selectivity of the photosensitizer. To overcome this problem, a protocol has been developed for coupling of meta-tetrahydroxyphenylchlorin (mTHPC), one of the most promising photosensitizers, to tumor selective monoclonal antibodies (MAbs). mTHPC was radiolabeled with 13'I
to facilitate the assessment of the in vitro and in vivo behavior. After modification to '31I-mTHPC-(CHzCO-OH)a, thus increasing the water solubility and creating a functional moiety suitable for coupling, conjugation was performed using a labile ester. Insoluble aggregates were not formed when mTHPC-MAb conjugates with a molar ratio of up to 4 were prepared.
These conjugates showed a minimal impairment of the integrity on SDS-PAGE, full stability in serum in vitro and an optimal immunoreactivity.
For testing of the in vivo behavior of the mTHPC-MAb conjugates the head and neck squamous cell carcinoma (HNSCC) selective chimeric MAb (cMAb) U36 was used in HNSCC
bearing nude mice. Biodistribution data showed that the tumor selectivity of cMAb U36-conjugated mTHPC was increased in comparison with free mTHPC, despite the fact that conjugates with a higher mTHPC:MAb ratio were more rapidly cleared from the blood.

Preliminary results on the in vitro efficacy of PDT with MAb-conjugated mTHPC
showed that mTHPC coupled to the internalizing murine MAb (mMAb) 425 exhibited more phototoxicity than when coupled to the non-internalizing cMAb U36 GENERAL DISCUSSION
Photodynamic therapy (PDT) is a therapeutic modality for the treatment of superficially localized tumors. In this approach a photosensitive dye (photosensitizer) is injected intravenously, whereafter it accumulates more or less selectively in the tumor. After exposure to laser light in the red or near-infrared region, the sensitizer is excited and is able to produce singlet oxygen, a cytotoxic form of oxygen. Direct cell killing, occlusion of tumor blood vessels as well as a strong acute inflammatory reaction can occur. These combined effects result in tumor necrosis. PDT has been applied for non-invasive treatment of many types of cancer, including colon, bladder, lung, and head and neck cancer.
Various photosensitizers have recently become available. One of the most promising second generation photosensitizers is meta-tetrahydroxyphenylchlorin (mTHPC).
mTHPC is a pure and well-defined compound. It has a strong absorption band at 652 nm (absorption coefficient 22400 l.mol-'.crri', compared with 3000 for Photofrin at 630 nm).
The longer wavelength light used to excite mTHPC can penetrate deeper into the tissue than 630 nm light, thus allowing treatment of larger tumors. The high photochemical efficiency of mTHPC means that lower light doses (and shorter illumination times) are required for a tumoricidal PDT
effect.
Preliminary results of PDT with mTHPC in head and neck cancer patients are encouraging (1). The largest study has been performed by Savary et al. using optimized protocols for PDT with mTHPC for the treatment of early second primary squamous cell carcinoma of the esophagus, bronchi and mouth (2). All lesions were carcinoma in situ or microinvasive carcinoma which had been detected by rigid endoscopy and toluidine blue as a vital stain. Of the 33 lesions treated in this trial, 28 showed no recurrence during the mean follow-up of 14 months. In comparison to surgery and radiotherapy, PDT shows a low morbidity with little fibrosis and scarring.
s Despite these promising results, mTHPC-based PDT leaves room for improvement.
A
limitation is the lack of tumor selectivity, which can result in severe normal tissue damage after PDT of large surface areas. An option to overcome this problem is to couple mTHPC to monoclonal antibodies (MAbs) directed against tumor-associated antigens. In this way the photosensitizer will be targeted selectively to the tumor. These mTHPC-MAb conjugates are especially suitable for the treatment of multiple tumor foci in large areas, as is the case in minimal residual disease after surgical resection of thoracic and peritoneal tumors. The problem of phototoxicity is also reduced since the accessibility of the skin is limited for MAbs.
In the development of photoimmunoconjugates for therapy the synthesis of mTHPC-MAb conjugates has not yet been described. A serious problem in this respect is the poor water solubility of mTHPC. Other factors expected to hamper the development of mTHPC-MAb conjugates suitable for tumor targeting are the potential chemical crosslinking during conjugation, as well as the impairment of the immunoreactivity and pharmacokinetic behavior of the MAb and the photochemical activity of the conjugates.
We have focused on the use of MAbs for selective targeting of squamous cell carcinoma of the head and neck (HNSCC). To this end MAbs E48 and U36 have been developed (3,4).
Radioimmunoscintigraphy/biodistribution studies in HNSCC patients showed that these MAbs are highly capable of selective tumor targeting (5,6,7). This observation justifies a study for the use of these MAbs as transport vehicle for selective delivery of mTHPC to HNSCC.
In this application we describe a protocol for the reproducible synthesis of mTHPC-MAb conjugates and their biodistribution after administration to HNSCC-bearing nude mice.
Conjugation and biodistribution studies were performed with dual labeling using '3'I-labeled mTHPC and 1'~I-labeled MAb. Preliminary data on the irc vitro efficacy of mTHPC-MAb mediated PDT are provided.
MATERIALS AND METHODS

mTHPC. Meta-tetrahydroxyphenylchlorin (mTHPC; Mr=680.76) was obtained from Scotia Pharmaceuticals (Stirling, UK) as a pure solid. ['4C]mTHPC (also provided by Scotia Pharmaceuticals) was synthesized by American Radiolabeled Chemicals Inc. (St.
Louis, USA).
Monoclonal Antibodies. Selection and production of MAb U36 and its chimeric (mouse/human) IgGl derivative (cMAb U36) have been described previously (4, 8). MAb U36 recognizes the v6 domain of the 200 kDa CD44 splice variant epican (9), which is highly expressed in squamous cell carcinoma of the head and neck, lung, skin, oesophagus and cervix, adenomacarcinomas of breast and lung, as well as in normal stratified epithelium. A
clinical RIS study with ~"'Tc-labeled U36 revealed that U36 IgG accumulates selectively and to a high level in HNSCC (7), and therefore the MAb is currently evaluated in RIT
studies.
Murine monoclonal antibody 425 (mMAb 425) is a IgG2a MAb developed and characterized by Murthy et al. (10). The epitope recognized by mMAb 425 is localized on the external domain of the EGF receptor (EGFR), which has been shown to be highly expressed by various tumor types including HNSCC, renal cell cancer, gliomas and carcinoma of the oesophagus, bladder, cervix, stomach, lung and breast. After binding to this antigen, anti-EGFR MAbs are internalized and catabolized by A431 cells (11). Anti-EGFR MAbs, MAb 425 included, have been extensively studied in clinical trials (12,13).
Cell Lines. Characteristics of the squamous cell carcinoma cell lines UM-SCC-11B, UM-SCC-22A and A431 and their culturing conditions are known in the art.
Analyses. HPLC analysis was performed by using a LKB 2150 HPLC-pump (Pharmacia Biotech, Roosendaal, The Netherlands), a LKB 2152 LC controller (Pharmacia Biotech) and a 25-cm Lichrosorb 10 RP 18 column (Chrompack, Middelburg, The Netherlands) at a flow-rate of 2 ml/min. The eluant consisted of a 9:1 (v/v) mixture of MeCN and 0.1 %
trifluoroacetic acid. Absorption was measured at 230 nm and 415 nm by a Pharmacia LKB VWM 2141 UV
detector. Radioactivity was measured by an Ortec 406A single-channel analyzer connected to a Drew 3040 Data collector (Betron Scientific, Rotterdam, The Netherlands).
Proton nuclear magnetic resonance (IH-NMR) spectra were recorded in [ZH6]MezSO
on a Bruker ARX 400 (400.14 MHz) spectrometer and a Bruker AC 200 (200.13 MHz) to spectrometer. Chemical shifts (8) are given in ppm relative to tetramethylsilane as the internal standard. For description of the NMR spectra of mTHPC and its derivatives see Table 1.
TABLE 1. Chemical Shifts (ppm) of mTHPC, mTHPC-(CHzCOOIT)a and mTHPC-(CHzCO-TFP)a OH pyrrole-CH TFP-H bent.--CHzCO- pyrrole-NH
(s) (s) (m) -H (d) CHz (m) (s) number of protons4 6 4 16 8 4 2 mTHPC 9.81 8.681/8.42/ 7.65- 4.20 -1.60 8.28z(d/s/d) 7.12 mTHPC-(CHzCOOH)a 11.308.623/8.37/ 7.66- 4.838 4.17 -1.64 8.244 (d/s/d) 7.20 mTHPC-(CHzCO-TFP)a 8.605/8.356/8.217.96 7.79- 5.579 4.13 -1.60 ' (dd/d/dd) 7.39 Note: s = ringlet; d = doublet; dd = double doublet; m = multiplet.
Observed coupling constants (Hz) : 1: J = 4. 9; 2: J =4. 9; 3 : J = 5 . 4; 4:
J = 5 .4; S : J = 5 .2; 6:
J=4.3; 7: J=5.1; 8: J=10.1; 9: J=12.7.
The absorption spectra of mTHPC and mTHPC-MAb conjugates were measured using a Ultrospec III spectrophotometer (Pharmacia Biochrom). The mTHPC concentration in the conjugate preparations was assessed with the same apparatus at a wavelength of 415 nm. The absorption of a range of dilutions (1-9 pg/ml) of mTHPC in MeCN was measured and graphically depicted using the least square method. The mTHPC concentration in the conjugate preparations was determined using this calibration curve.
The integrity of the mTHPC-MAb conjugates was analyzed by electrophoresis on a Phastgel System (Pharmacia Biotech) using preformed 7.5 % SDS-PAGE gels under non-reducing conditions. After running, gels were stained with 0.2 % Coomassie Brilliant Blue (CBB, Sigma) and exposed to a Phosphor plate for 1-3 h and analyzed with a Phosphor Imager (B&L-Isogen Service Laboratory, Amsterdam) for localization of the protein bands.
Quantitative information was obtained by cutting the lanes into pieces and dual label counting in a gamma counter (LKB-Wallac 1282 CompuGamma, Pharmacia, Woerden, The Netherlands) .
Dual Label Counting of u5I and 1311. The amounts of 'z5I (E,, 35 keV) and '3'I
(F.~ 364 keV) were measured simultaneously in a gamma counter in the corresponding window settings (channels 35-102 and 155-185, respectively) with automatic correction for the '3'1-comptongammas in the 'z5I-window setting; in our case this correction corresponded to 15.2 %
of the '31I-photopeak counts present in the sample.
~zsl_Labeling of MAbs. Radioiodination of cMAb U36 and mMAb 425 with 'z5I was performed using Iodogen (Brunschwig Chemie, Amsterdam) as described by Haisma et al. . 1-2 mg MAb dissolved in 500 ~l PBS (pH 7.4) and 1 mCi lzsl (100 mCi/ml, Amersham, Aylesbury, England) were mixed in a vial coated with 50 pg Iodogen. After 5 min incubation at room temperature the reaction mixture was filtered through a 0.22 pM
Acrodisc filter (Gelman Sciences Inc., Ann Arbor, MI) and unbound lzSI was removed using a PD-10 column (Pharmacia Biotech, Woerden) with 0.9 % NaCI as eluant. After removal of unbound izsl, the radiochemical purity always exceeded 98 % .
1311-Labeling of mTHPC. To facilitate the analysis of the stability of the mTHPC-MAb conjugates in vitro and in vivo, and their pharmacokinetic behavior, in most of the experiments mTHPC was trace-labeled with '3'I. This labeling and subsequent reaction steps with mTHPC
were carried out in the dark and under a Nz atmosphere to prevent unwanted photochemical reactions during the synthesis of the mTHPC-MAb conjugates.

13'I_Labeling of mTHPC was performed using Iodo-beads (Brunschwig Chemie) as follows: the appropriate amount of 13'I was added to 50 p,1 of 1 mM NaOH
containing 10 ~g NazS03. This '31I-solution was added to 4 Iodo-beads covered with 450 ~l of a MeCN/H20 mixture (10:1; v/v) followed by 100 p1 (734 nmol) of a mTHPC-solution (5 mg/ml in MeCN).
After labeling during 30 min the reaction mixture was diluted with 400 ~,l H20, loaded on a conditioned Sep-pak Cps cartridge (Waters, Millipore, MA) and washed with 50 ml H20. The 13~I_labeled mTHPC (actually consisting of a small proportion of '3~I_mTHPC
and an excess of unlabeled mTHPC) was eluted with 3 ml MeCN. The solvent was evaporated under a stream of Nz.
The radiochemical purity of '3'I_mTHPC was determined by HPLC analysis. The HPLC retention times were: 9.8 min for '3'I-mTHPC, between 5-9 min for '3'I-labeled minor impurities and 9.6 min for mTHPC (For the 'H-NMR data of mTHPC see Table 1).
Preparation of the Tetrafluorophenol (TFP) Ester. Preparation of the ester (either in labeled or unlabeled form) was performed in two steps. The first step was tetra-carboxymethylation of 131I_mTHPC/mTHPC. To 13'I_mTHPC/mTHPC, dissolved in 600 ~l of a DMF/H20 mixture (5:1; v/v), 150 mg (3.7 mmol) powdered NaOH were added and the mixture was stirred until the solution was green (2-3 min). Hereafter, 70 mg (380 ~,mol) of iodoacetic acid (Janssen Chimica, Beerse, Belgium) were added and stirring was continued for another 90 min. The pH was adjusted to 5.0 with 3 ml of 1 N HCI, and the tetracarboxymethylated product was isolated by extraction with four portions of 0.5 ml CHzCIz. The HPLC retention times were: 7.1 min for '3'I-mTHPC-(CHzCOOH)a and 7.3 min for mTHPC-(CHzCOOH)4 ('H-NMR data of mTHPC-(CHzCOOH)4 are given in Table 1).
In the next reaction step the four carboxylic acid groups were esterified using an excess of TFP (Janssen Chimica). To the tetracarboxymethylated product in CHzCIz 150 w1 of a TFP-solution (100 mg/ml in DMF) and 50 mg solid EDC (Janssen Chimica) were added.
The pH
was adjusted to 5.7-5.9 with 1 N NazC03. After reaction for 30 min, column chromatography was performed to remove all impurities. This purification was performed with a 24 cm LiChroprep Si 60 (40-63 pm) column (Merck, Darmstadt, Germany) using CHZCIz/MeCN

(97:3; v/v) as the eluant (flow rate of 1 ml/min). Fractions of 0.5 ml were collected and analyzed by HPLC with absorption measurement at 230 and 415 em. The pure tetra-ester fractions (under our conditions fractions 20-23) were pooled and the solvent was evaporated.
The HPLC retention times were: 17.2 min for 131I_mTHPC-(CH2C0-TFP)4 (Fig. 2B) and 15.9 min for mTHPC-(CHzCO-TFP)4 (Fig. 2A; IH-NMR data of mTHPC-(CHZCO-TFP)4 are given in Table 1).
Preparation of 1311-mTHPC_(CHzCOOI-~3CHzCONH_lzsl_MAb Conjugates. For the coupling reaction with the luI-labeled MAb the 131I_mTHPC-(CHzCO-TFP)a (Fig.
2A and B) was partly hydrolyzed in order to leave one reactive ester function thus preventing crosslinking of MAbs during conjugation. The partial hydrolysis was performed by dissolving the tetra-ester in 300 p1 MeCN and by stepwise addition of 10-25 p1 10 mM NazCOs. The degree of hydrolysis was monitored by radio-HPLC analysis with simultaneous absorption measurement at 415 em (Fig. 2C and D). When the percentage of mono-ester was optimal for conjugation (no tetra- and tri-ester, less than 5 % di-ester, 45 % mono-ester together with 50 % completely hydrolyzed '31I-mTHPC-(CHZCOOH)4, Fig. 2E and F), this mixture was added to 2 mg "~I-labeled MAb in 1 ml 0.9 % NaCI at pH 9.5. After 30 min incubation, the '3'I-mTHPC-(CHZCOOH)sCHzCONH-"~I-MAb conjugate was purified by gel filtration using a PD-column with 0.9 % NaCI as eluant.
The conjugation efficiency was determined from the "~I:13'I ratio before and after PD-10 purification using dual label counting in a gamma counter. The 13'I-mTHPC:"~I-MAb molar ratio was determined by measuring the absorbance at 415 em to calculate mTHPC
concentration and "~I measurement for MAb quantitation. The integrity of the conjugate was checked by gel electrophoresis.
The chemistry is summarised in Figure 1. Repeating the chemistry but starting with the porphyries of USP 4,837,221, particularly o-, p- or m-THPP corresponding to o-, p- or m-THPC, or with the bacteriochlorins or isobacteriochlorins of USP 4,992,257, particularly o-, p- or m-THPB or THPiB corresponding to o-, p- or m-THPC, gives the mMAB 425 conjugates of those compounds.
In Vitro Stability and Immunoreactivity of 1311-mTHPC lzsl_MAb Conjugates'-.
For measurement of the serum stability of the 1311-mTHPC-'~I-MAb conjugates, 0.5 ~,g conjugate in 10 ~1 0.9 % NaCI was added to 40 ~1 serum. Stability was measured in mouse and human serum, while 0.9 % NaCI served as a control. After 20 h incubation in the dark at 37°C, samples were analyzed with SDS-PAGE. Quantitative information was obtained by cutting the lanes into pieces and dual label counting.
In vitro binding characteristics of '3'I-mTHPC-'z5I-MAb conjugates were determined in an immunoreactivity assay as described by Lindmo et al. and compared with those of the unconjugated 'z5I-MAb. UM-SCC-11B cells were used for 'z5I-cMAb U36 and A431 cells for 'z5I-mMAb 425.
Biodistribution Studies. The biodistribution of 'z5I-cMAb U36 and '3'I-mTHPC-~zsl-cMAb U36 conjugate was studied in nude mice bearing s.c. xenografts of the HNSCC cell line HNX-OE, tumor size ranging from 50 to 200 mm3. For a comparison the distribution of '3'I-mTHPC-(CHzCOOH)4, 'z3I-mTHPC and ['4C]mTHPC (as a reference compound used by others) was studied in the same animal model. Because the latter two compounds were coinjected in the same group of animals, the short-living 'z3I-isotope was used instead of '3'I to facilitate the assessment of the '4C activity.
The cMAb U36 samples were injected i.v. in 0.9% NaCI; the 'z3I- and ['4C]mTHPC
derivatives in a mixture consisting of 20 % EtOH, 30 % polyethylene glycol 400 and 50 % water (v/v). At indicated time points post-injection, mice were anesthetized, bled, killed and dissected. The urine was collected and the organs were removed. After weighing, the amount of gamma-emitting radioactivity in organs, blood and urine was measured in a gamma counter.
For the weak (3-emitter '4C, the blood, urine and organs were treated as follows: after complete decay of 'z3I, tissue samples were placed in counting vials and 1 ml of Soluene-350 (Packard Instrument Company, Groningen, The Netherlands) was added to dissolve the organs. The vials were subsequently heated at 50°C for 24 h, after which 250 p1 of a 1:1 (v/v) mixture of 30% HzOz and acetic acid was added for decolorization of the solutions. After 1 h Ultima Gold liquid scintillation cocktail (15 ml, Packard Instrument Company) was added to the samples prior to counting in a LKB-Wallac 1410 Liquid Scintillation Counter (Pharmacia, Woerden). Radioactivity uptake in the tissues was expressed as the percentage of the injected dose per gram of tissue ( % ID/g).
Photoimmunotherapy in vitro. Phototoxicity of the mTHPC-cMAb U36 conjugates and the unconjugated mTHPC was assessed in UM-SCC-22A cells using the sulforhodamine B
(SRB, Sigma) assay, which measures the cellular protein content. Cells were plated in 96-well plates (2500 per well) and grown for 3 days before incubating with mTHPC or mTHPC-cMAb U36 conjugates (0.1 nM to 1.0 ~M mTHPC equivalent) in DMEM supplemented with 2 mM
L-glutamine, 5 % FCS and 25 mM HEPES at 37 °C. After 20 h remaining free mTHPC-cMAb U36 and mTHPC were removed by washing twice with medium. Fresh medium was added and cells were illuminated at 652 nm with a 6 W Diode Laser (AOC Medical Systems) at a dose of 25 J/cm2. Three days after illumination, growth was assessed by staining the cellular proteins with SRB and spectrophotometric measurement of the absorption at 540 nm with a microplate reader. ICso values were estimated based on the absorption values and defined as the concentration that corresponded to a reduction in growth of 50 % compared with values for control cells (no mTHPC-MAb conjugates or mTHPC added but illuminated in the same way).
Phototoxicity of the mTHPC-mMAb 425 conjugates was assessed in A431 cells (2000 cells/well) in a similar way.
RESULTS
Iodination of mTIiPC. The first step in the synthesis of '3'I-mTHPC-(CH2COOH)3CHzCONH-luI-MAb conjugates (5, Fig. 1) was trace-labeling of mTHPC
with '3'I using Iodo-beads. After 30 min incubation at room temperature, HPLC
analysis revealed 70-75 % 13'I-mTHPC (1, Fig. 1), less than 10 % 13'I-labeled impurities and about 20 % unreacted '3II. After purification on a Sep-pak cartridge the final preparation contained > 94 % 13'I-mTHPC, less than 4 % impurities and about 2 % unbound '3'I.
Synthesis of 1311-mTHPC-(CHzCO-TFP)a. In the next step, '31I-mTHPC was tetra-carboxymethylated. The reaction with an excess of iodoacetic acid at pH 13, followed by extraction with CHzCIz, gave 95 % ~ 5 % (HPLC analysis) tetracarboxymethylated product (2, Fig. 1). Esterification was performed with TFP and purification of the crude product with a LiChroprep column gave the desired product (3, Fig. 1) with > 95 % purity, also containing < 5 % 13'I-mTHPC-(CHzCO-TFP)s.
The purity of the fractions (0.5 ml) that were recovered from the LiChroprep column was analyzed using HPLC analysis at 415 nm for detection of mTHPC-(CHzCO-TFP)a and at 230 nm for detection of ICHaCO-TFP, formed as a side-product. On the LiChroprep column this latter ester had a retention time slightly longer than mTHPC-(CHzCO-TFP)a. The fractions that only contained mTHPC-(CHzCO-TFP)a (under our conditions fractions 20-23) were, after collection, evaporated under a stream of N2 and stored in the dark at 4 °C until use. The LiChroprep purification also removed unbound '3'I, and any unreacted TFP, EDC
or ICHZCOOH. As a result, the mTHPC-ester was obtained in an overall yield of 60%
with a purity > 95 % .
Conjugation. The 1311-mTHPC-(CHzCO-TFP)4 ester was dissolved in 300 p1 MeCN
before starting of the HPLC-monitored hydrolysis with 10 mM NazC03-buffer.
This base was added in portions of 10-25 p1 with intervals of 10 min. Approximately 125-150 ~.1 of this buffer was used to reach the optimum mixture for conjugation (4, Fig. 1).
For conjugation the mixture was added to a solution of '~I-MAb in 0.9 % NaCI
at pH
9.5. After 30 min at room temperature the 131I_mTHPC-(CHZCOOH)3CHZCONH-"~I-MAb conjugate (5, Fig. 1) was purified on a PD 10 column. When 2 mg 'ZSI-MAb in a conjugation volume of 1.8 ml was used, the 13'I-mTHPC-l2sl_MAb molar ratio was about 2.0-2.5. The conjugation efficiency was 60 % ~ 10 % (corrected for completely hydrolyzed ester, which is unable to couple), while the recovery of the MAb always exceeded 95 %
(measured by 1'~I
activity).
By adapting the ester concentration during conjugation, conjugates with a ratio > 4 could be obtained. However, under these conditions the recovery of the MAb from the column dropped significantly, indicating an impairment of the solubility of the MAb.

SDS-PAGE Analysis of 1311-mTHPC-lzsl_MAb Conjugates. SDS-PAGE and subsequent CBB staining and Phosphor Imager analysis (Fig. 3) of the conjugate revealed one major protein band and a minor band probably consisting of high molecular ~~~eight complexes.
Cutting of the gel and dual label counting of the gel pieces showed > 90 % of the 1'~I-MAb and > 80 % of the 1311-mTHPC to be localized in the main band, when conjugates with a ratio of up to 4 were analyzed. The remaining radioactivity was predominantly localized in the high molecular weight band. A typical example is shown by Figure 3.
In Vitro Stability and Immunoreactivity of '31I-mTHPC-lzsl_MAb Conjugates.
After 20 h incubation in serum, the 1311-mTHPC-1'~I-MAb conjugates were analyzed by SDS-PAGE.
Cutting of the gel and subsequent dual label counting showed that the "~I;13'I
ratio of the IgG
peak after incubation in mouse and human serum did not differ from that of the control incubation in 0.9 % NaCI. So 1311-mTHPC-1~I-MAb conjugates were fully stable in both serum sources .
Lindmo assays were performed to determine whether coupling of mTHPC to cMAb or mMAb 425 influenced the immunoreactivity of the MAb. For conjugates with a mTHPC:MAb ratio of up to 4 no effect on the immunoreactivity was seen in comparison to the unconjugated MAb. Immunoreactivity was > 93 % in all cases, irrespective whether assessed by 1'~I or 1311 counting.
Biodistribution of 131I_mTHPC lzsl_cMAb U36 conjugates. Dual label experiments were performed to determine whether coupling of '3'I-mTHPC-(CHaCOOH)4 to 1'~I-cMAb U36 resulted in improved targeting of the sensitizer to the tumor. To this end the biodistribution of unconjugated 1'~I-cMAb U36 and 1311-mTHPC-(CHZCOOH)a were first determined.
For evaluation of "~I-cMAb U36, 5 ~Ci 1'~I-cMAb U36 (100 fig) were injected in 5 HNX-OE
xenograft bearing nude mice. The mice were sacrificed 48 h after injection and the biodistribution was determined. The mean uptake in tumor tissue was 19.5 %ID/g, while the mean blood level was 13.9 %ID/g. Uptake in all other organs was less than 4 %
ID/g (Fig.
4A) .
For evaluation of mTHPC-(CHzCOOH)a, 2.5 ~Ci (5 fig) of 1311-mTHPC-(CHzC00H)4 was injected in 5 HNX-OE xenograft bearing nude mice. Figure 4B shows the biodistribution ~8 after 24 h. The compound was cleared very rapidly from the circulation. The mean blood level was 1.5 % ID/g, while the uptake in the tumor was 0.5 % ID/g. Uptake in all other organs was less than 1.5 % ID/g, except for the liver. Because the levels of '3'I were already very low after 24 h, the biodistribution after 48 h was not determined.
The biodistribution of '31I-mTHPC-1~I-cMAb U36 conjugates with a ratio of 0.9 and 1.8 is shown in Figure 4C (1'~I-data) and D ('3'I-data). Conjugates (100 pg;l0 ~,Ci "~I/ 2.5 pCi 13'I) were injected in 2 groups of 5 mice and mice were killed 48 h after injection.
The results depicted in Figure 4 revealed that coupling of mTHPC-(CH2COOH)4 to cMAb U36 resulted in selective targeting of the sensitizes to the tumor (compare Fig. 4D and B). However, tumor uptake levels of the sensitizes appeared to be lower than could be expected on the basis of the biodistribution of the unconjugated MAb (compare Fig. 4D and A). The fact that tumor uptake of the transporter of the sensitizes, i.e. the conjugated MAb, was also lower than expected, indicated that a proportion of the conjugate became rapidly eliminated from the blood (compare blood levels Fig. 4C and A). This elimination was more pronounced for conjugates with the higher mTHPC:MAb ratio. For both the free mTHPC-(CH2COOH)a and the conjugate, high mTHPC levels were found in the liver (Fig.
4B and D).
To establish the overall efficiency of sensitizes targeting by the MAb, the biodistribution of unmodified mTHPC was assessed in the same model as '3'I-mTHPC-(CHzCOOH)4.
Externally labeled 'Z3I-mTHPC (5.0 ~g per mouse; specific activity 11.3 Ci/mmol) and internally labeled ['4C]mTHPC (5.0 p.g per mouse; specific activity 74 mCi/mmol) were coinjected in 6 HNX-OE bearing nude mice. As previously found by others in BALB/c mice, the free sensitizes showed a random distribution in the organs, and no selective tumor uptake (Fig. 5A and B). For both mTHPC compounds the highest accumulation was observed in liver, spleen and lung, and the lowest uptake in muscle. Besides this, small differences were observed in the distribution pattern of the compounds, partly originating from the difficulty to assess the ['4C] radioactivity in solid/colored tissue.
Photoimmunotherapy in vitro. The efficacy of photoimmunotherapy with mTHPC-cMAb U36 conjugates was tested in 22A cells using the SRB-assay. After exposure to the relatively high concentration of 1 pM conjugated mTHPC, about 25 % growth inhibition was observed (Fig. 6A). In the same assay unconjugated mTHPC showed an ICso value of 0.75 nM. Conjugated and free mTHPC appeared to be non-toxic without illumination.
To investigate the possibility that the sensitizer must be internalized for phototoxicity to occur, we coupled mTHPC to mMAb 425, an internalizing MAb directed against EGFR.
Internalization of the mTHPC-MAb 425 conjugate was confirmed according by known methods. The efficacy of these conjugates was tested in A431 cells. The ICso value using mTHPC-mMAb 425 conjugates was 7.3 nM, while in this cell line the ICso value for free mTHPC was 2.0 nM (Fig. 6B). Once again, conjugated and free mTHPC were non-toxic without illumination.
Unconjugated cMAb U36 or mMAb 425 did not result in growth inhibition with or without illumination (data not shown).
Favourable results corresponding to the above may be expected from conjugates of m-THPP, m-THPB and m-THPiB and the other porphyries, chlorins, bacteriochlorins and isobacteriochlorins discussed herein.
COMPOSITIONS AND METHODS OF USE
PDT-immunoconjugates can be parenterally administered in pharmaceutical compositions. Such compositions, comprising of PDT-immunoconjugate e.g.(m-THPC-MAb or m-THPBC-MAb) and a parenterally administrable medium are formulated by methods commonly used in pharmaceutical chemistry. The effective concentration of immunoconjugates of the present invention is dictated by the PDT agent used in the conjugate.
One skilled in the art of preparing such compositions will be able to consider optimal ratio of composition of pharmaceutical components and PDT immunoconjugate.
However as one particular example of an injectable solution 20% EtOH, 30%
ethylene glycol 400, and 50 % water (v/v) with NaC 1 to give an 0.9 % solution (w/v) is made up with m-THPC-MAb to give a dose of 15 mg for an adult (70kg bodyweight) related to the m-THPC.
Broadly, suitable dose ranges of the m-THPC and the other PDT actives discussed herein are for example 0.1 to 5 mg/kg related to the active.
FURTHER DISCUSSION
Several attempts to use MAbs for selective delivery of photosensitizers to tumors have been made. However, none of these has led to conjugates suitable for therapeutic use. In 1983 the group of Levy described the synthesis of hematoporphyrin-MAb conjugates, but the in vivo efficacy of these conjugates appeared to be minimal (14). The same research group developed benzoporphyrin derivative monoacid ring A (BPD)-MAb conjugates, using polyvinyl alcohol as a linker (15), but no data on the therapeutic applicability of these conjugates have been reported. The photosensitizer chlorin es was conjugated to MAbs by the group of Hasan using polyglutamic acid as a linker. Preliminary results of PDT after intraperitoneal injection of these conjugates in a murine intraperitoneal ovarian cancer model showed an improved survival (16). No data on the intravenous use of these conjugates, or for chlorin e6-MAb conjugates using a dextran polymer linker (17), have been published so far.
Although mTHPC is one of the most promising photosensitizers available for clinical use, no reports on mTHPC-MAb conjugates have been published. In this paper a reproducible procedure for conjugation of mTHPC to MAbs is provided in detail. It is preferred that all reactions, including the modification of mTHPC, conjugation and subsequent purification, are performed in the dark and that solvents used are saturated with nitrogen.
Figure 7 illustrates the phototoxic effect of free mTHPC, when these precautions are not taken. In this case the integrity of the MAb was impaired in such a way, that it could not penetrate a 7.5 % SDS-PAGE gel.
The final route to the reproducible production of mTHPC-(CHzCOOH)3CHZC0-TFP
was the synthesis of the tetra-esterified compound, followed by partial hydrolysis to leave the mono-ester. The esterification of mTHPC-(CHzCOOH)4 using an excess of TFP and EDC gave after column chromatography pure mTHPC-(CHZCO-TFP)a in a reasonable yield (60 % ).
During the following partial hydrolysis procedure, the formation of fully hydrolyzed mTHPC-(CHZCOOH)4 is unavoidable. When hydrolysis was performed until no di-ester was left, only 20 % of the mixture consisted of mono-ester. In our experiments we hydrolyzed till 45 % of the mixture consisted of mono-ester. Under these conditions less than 5 % di-ester was left, which was judged acceptable for conjugation. As a result, the overall amount of mTHPC available for conjugation was about 30 % , owing to the loss during the modification, esterification and subsequent hydrolysis.
mTHPC-MAb conjugates prepared according to the method described herein showed a minimal impairment of the integrity on SDS-PAGE ( < 10% aggregate formation), full stability in serum in vitro and an optimal immunoreactivity, provided that not more than 4 mTHPC
molecules were coupled to the MAb. Nevertheless, the pharmacokinetics of mTHPC-MAb conjugates in xenograft bearing nude mice differed from that of unconjugated MAb. For conjugates with a mean ratio of 0.9 and 1.8, the "~I-levels of the '3'I-mTHPC-'uI-MAb in the blood at 48 h p.i. were 69% and 52% , respectively, of that of an unconjugated "~I-MAb. In addition, the '3'I-levels decreased more extensively than the "~I-levels.
These data indicate that conjugates with a higher ratio are more susceptible for removal from the blood. Our biodistribution data therefore indicate hepatic extraction with retention of the sensitizes in this organ after catabolism. Rapid blood clearance and extensive liver accumulation has also been observed for MAbs coupled with other chemical groups to their lysine residues.
The '3'I-mTHPC-(CHzCOOH)a was cleared more rapidly from the circulation than the unmodified mTHPC. The tumor selectivity of MAb-conjugated mTHPC was increased in comparison with both of these, despite the more rapid elimination of the conjugates with a higher ratio. For the conjugates with a ratio of 0.9 and 1.8 the tumor levels of '3'I-mTHPC
were 5.7 and 4.4 % LD./g, respectively. In absolute amounts this corresponds with 23 and 36 ng/g tumor, respectively. Given the fact that increasing the MAb dose to 400 ~g per mouse does not result in antigen saturation, this implies that about 150 ng mTHPC
per g tumor can be delivered.

Another aspect of evaluation is the uptake in the skin, in view of the problem of skin photosensitization. At 48 h after injection the levels of the MAb-conjugated mTHPC in the skin were much lower than in the tumor (tumor:skin ratio's were 3.5; Fig. 4D). For the unconjugated '23I-mTHPC and the reference compound ['4C]mTHPC the levels in the skin and tumor were almost the same, 24 h after injection (tumor:skin ratio's were 0.8 and 0.9, respectively; Fig. 5). This is in agreement with data of Whelpton et al., who showed that tumor:skin ratio's remained about 1 between l and 4 days after administration of ['4C]mTHPC
to Colo 26 bearing mice. Westermann et al. showed that in colon carcinoma bearing nude mice these ratio's were improved by using 'uI-mTHPC-PEG conjugates instead of I'~I-mTHPC. The improved selectivity of mTHPC directed by the MAb does not guarantee improved efficacy.
Therefore, in vitro studies were performed to compare the phototoxicity of internalizing and non-internalizing MAb-conjugated mTHPC with that of free mTHPC at equimolar doses. Our data on the photodynamic efficacy of the mTHPC-MAb conjugates revealed a remarkable difference between internalizing and non-internalizing MAbs. When coupled to mMAb 425, which was internalized by the cell after conjugation, mTHPC exhibited more phototoxicity than when coupled to the non-internalizing cMAb U36. Sobolev et al. also reported that the photosensitizer chlorin e6 was more effective when localized intracellularly (18). For BPD-MAb conjugates, produced by the group of Levy, internalization enhanced the cell killing by 10-fold (19). These data strongly suggest that the critical target for photodynamic damage is localized intracellularly. When this is true, it is clear that the kinetics of cellular uptake of free mTHPC versus mMAb 425-conjugated mTHPC are crucial parameters, and might have influenced the relative efficacy as observed in our SRB experiments (Fig. 6).
ABBREVIATIONS
The abbreviations used are: PDT, photodynamic therapy; mTHPC, meta-tetrahydroxyphenylchlorin; mMAb/cMAb, murine/chimeric monoclonal antibody;
CEA, carcinoembyryonic antigen; HNSCC, head and neck squamous cell carcinoma; RIS, radioimmunotherapy; EGFR, epidermal growth factor receptor; 'H-NMR: proton nuclear magnetic resonance; PBS phosphate-buffered saline; HPLC, high-performance liquid chromatography; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis;
TFP, 2,3,5,6-tetrafluorophenol; EDC, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide; BSA, bovine serum albumin; % ID/g, percentage of injected dose/g of tissue; FCS, fetal calf serum;
SRB, sulforhodamine B; HNX-OE, head and neck xenograft line OE.
It may be noted that mTHPC-(CHzCOOH)3CHZCONH-MAb conjugates are designated as mTHPC-MAb conjugates if the modification of mTHPC is not relevant for understanding LEGENDS OF FIGURES
Figure 1. Schematic representation of the synthesis of '3'I-mTHPC-(CHzCOOH)4, its esterification, partial hydrolysis and conjugation to a 1'~I-labeled MAb. It is of note that '3'I can occupy 12 positions (both ortho-positions and the para-position relative to the OH, in each of the 4 phenyl rings). In the mono-TFP ester 4, 1 of the 4 possible mono-TFP
esters is depicted.
Figure 2. HPLC profiles (absorbance (415 nm) and radioactivity) during the partial hydrolysis of '3'I-mTHPC-(CH2C0-TFP)4. At the start, HPLC analysis showed A (peak 1:
mTHPC-(CHZCO-TFP)4) and B (peak 1; 13'I-mTHPC-(CHiCO-TFP)a, peak 1': '3'I-mTHPC-(CHZCO-TFP)s). During hydrolysis, C (peak 1: mTHPC-(CHzCO-TFP)4, peak 2: mTHPC-(CHzCOOH)(CHzCO-TFP)3, peak 3/4: 2 isomers of mTHPC-(CHaCOOH)z(CHzCO-TFP)z, peak 5: mTHPC-(CH2COOH)sCH2C0-TFP and peak 6: mTHPC-(CHzCOOH)4) and D
(identical to C, corresponding 13'I-labeled compounds). Conjugations were performed with mixtures E (peak 3/4: 2 isomers of mTHPC-(CHzCOOH)z(CHZCO-TFP)z, peak 5: mTHPC-(CHzCOOH)3CHzC0-TFP and peak 6: mTHPC-(CHzCOOH)4) and F (identical to E, corresponding'3'I-labeled compounds).
Figure 3. Example of an SDS-PAGE and Phosphor Imager analysis of a '3'I-mTHPC-luI-cMAb U36 conjugate with ratio 1.8. Quantitative assessment of the radioactivity was obtained by cutting the lane and dual label counting.
Figure 4. Biodistribution of '~I-cMAb U36 and 13'I-mTHPC-(CHzCOOH)a before and after conjugation. Each preparation was intravenously injected in 6 HNX-OE bearing nude mice. A:
48 h p.i. of 1'~I-cMAb U36 (100 pg; 5 ~,Ci). B: 24 h p.i. of 13'I-mTHPC-(CH2COOH)4 (5 ~.g;
~,Ci). C (1~I) and D (131I): 48 h p.i. of '31I-mTHPC-(CHz.COOH)sCHzCONH-"~I-cMAb U36 (100 p.g; 5 p,Ci '~I, 1 p.Ci '31I) at a molar ratio of 0.9 (open bars) or 1.8 (hatched bars). At indicated time points mice were bled, sacrificed, dissected and the radioactivity levels ( % ID/g + SE) of blood, tumor and several organs were assessed. Tu: tumor, Bl: blood, He: heart, Ki:
kidney, Sto: stomach, Il: ileum, Co: colon, Ste: sternum, Lu: lung, Mu:
muscle, Sk: skin, To:
tongue, Li: liver, Sp: spleen.
Figure 5. Comparison of the biodistribution of ['4C]mTHPC (A) and 'z3I_mTHPC
(B), 24 h after intravenous injection. Six mice received ['4C]-mTHPC (5.0 p,g; 0.54 p.Ci) and lz3l-mTHPC (5.0 p.g; 83 ~Ci). At 24 h p.i. mice were bled, sacrificed, dissected and the radioacti-vity levels ( % ID/g + SE) of blood, tumor and several organs were assessed.
For abbreviations see legend Figure 4.
Figure 6. The SRB assay was used to assess the antiproliferative effect of mTHPC and mTHPC-MAb conjugates upon illumination. A) 22A cells, mTHPC + 25 J/cm2 (0), mTHPC
not illuminated (~), mTHPC-cMAb U36 + 25 J/cmz (O), mTHPC-cMAb U36 not illuminated (A). B) A431 cells, mTHPC + 25 J/cm2 (~), mTHPC not illuminated (~), mTHPC-mMAb 425 + 25 J/cmz (O), mTHPC-mMAb 425 not illuminated (A). Results of 3 experiments are indicated (means ~ SD).
Figure 7. Illustration of the phototoxicity of mTHPC to the integrity of "~I-cMAb U36. 50 ~,g 'uI-cMAb U36 was incubated in 500 p1 MeCN/0.9% NaCI (1/4 v/v) at pH 9.5: with 25 ~g mTHPC in dark (lane A), with 25 pg mTHPC in light under a NZ atmosphere (lane B), with 25 p.g mTHPC in light (lane C), without mTHPC in light as a control (lane D).
After 1 h incubation SDS-PAGE and Phosphor Imager analysis was performed.
REFERENCES
1. Dilkes, M. G., DeJode, M. L., Rowntree-Taylor, A., McGilligan, J. A., Kenyon, G.
S., and McKelvie, P. mTHPC photodynamic therapy for head and neck cancer.
Lasers Med. Sci. , Il: 23-29, 1996.
2s 2. Savary, J-F., Moonier, P., Fontolliet, C., Wagnieres, G., Braichotte, D., and Van den Bergh, H. Photodynamic therapy for early squamous cell carcinomas of the esophagus, bronchi, and mouth with meta-tetrahydroxyphenylchlorin. Arch. Otolaryngol.
Head Neck Surg., 123: 162-168, 1997.

3. Quak, J. J., Van Dongen, G. A. M. S., Balm, A. J. M., Brakkee, J. P. G., Scheper, R.
J., Snow, G. B., and Meijer, C. J. L. M. A 22-kDa surface antigen detected by monoclonal antibody E48 is exclusively expressed in stratified squamous and transitional epithelia. Am. J. Pathol., 136: 191-197, 1990.

4. Schrijvers, A. H. G. J., Quak, J. J., Uyterlinde, A. M., Van Walsum, M., Meijer, C. J.
L. M., Snow, G. B., and Van Dongen, G. A. M. S. MAb U36, a novel monoclonal antibody successful in immuno-targeting of squamous cell carcinoma of the head and neck. Cancer Res., 53: 4383-4.390, 1993.

5.. De Bree, R., Roos, J. C., Quak, J. J., Den Hollander, W., Van den Brekel, M. W. M., Van de Wal, J. E., Snow, G. B., and Van Dongen, G. A. M. S. Clinical imaging of head and neck cancer with ~'"'Tc-labeled monoclonal antibody E48 IgG or F(ab')z. J.
Nucl. Med., 35: 775-783, 1994.

6. De Bree, R., Roos, J. C., Quak, J. J., Den Hollander, W., Wilhehn, A. J., Van Lingen, A., Snow, G. B., and Van Dongen, G. A. M. S. Biodistribution of radiolabeled monoclonal antibody E48 IgG and F(ab')2 in patients with head and neck cancer.
Clin.
Cancer Res., l: 277-286, 1995.

7. De Bree, R., Roos, J. C., Quak, J. J., Den Hollander, W., Snow, G. B., and Van Dongen, G. A. M. S. Radioimmunoscintigraphy with ~'''"Tc-labeled monoclonal antibody U36 and its biodistribution in patients with head and neck cancer. Clin.
Cancer Res., 1:
591-598, 1995.

8. Brakenhoff R. H., Van Gog, F. B., Looney J. E., Van Walsum, M., Snow G. B., and Van Dongen G. A. M. S. Construction and characterization of the chimeric monoclonal antibody E48 for therapy of head and neck cancer. Cancer Immunol. Immunother., 40:
191-200, 1995.

9. Van Hal N. L. W., Van Dongen, G. A. M. S., Rood-Knippels, E. M. C., Van der Valk, P., Snow, G. B., and Brakenhoff, R. H. Monoclonal antibody U36, a suitable candidate for clinical immunotherapy of squamous-cell carcinoma, recognizes a isoform. Int. J. Cancer, 68: 520-527, 1996.

10. Murthy, U., Basu, A., Rodeck, U., Herlyn, M., Ross, A. H., and Das, M.
Binding of an antagonistic monoclonal antibody to an intact and fragmented EGR-receptor polypeptide Arch Biochem Biophys., 252: 549-560, 1987.

11. Sunada, H., Magun, B. E., Mendelsohn, J., and MacLeod, C. L. Monoclonal antibody against epidermal growth factor receptor is internalized without stimulating receptor phosphorylation. Proc. Natl. Acad. Sci. USA, 83: 3825-3829, 1986.

12. Modjtahedi, H., Hickish, T., Nicolson, M., Moore, J., Styles, J., Eccles, S., Jackson, E., Salter, J., Sloane, J., Spencer, L., Priest, K., Smith, I., Dean, C., and Gore, M.
Phase I trial and tumor localization of the anti-EGFR monoclonal antibody ICR62 in head and neck or lung cancer. Br. J. Cancer, 73: 228-235, 1996.

13. Bier, H., Reiffen, K. A., Haas, L, and Stasiecki, P. Dose-dependent access of marine anti-epidermal growth factor receptor monoclonal antibody to tumor cells in patients with advanced laryngeal and hypopharyngeal carcinoma. Eur. Arch. Otorhin., 252:

14. Mew, D., Wat, C.-K., Towers, G. H. N., and Levy, J. G. Photoimmunotherapy:
treatment of animal tumors with tumor-specific monoclonal antibody-hematoporphyrin conjugates. J.
Immunol., 130: 1473-1477, 1983.

15. Davis, N., Liu, D., Jain, A. K., Jiang, S.-Y., Jiang, F., Richter, A., and Levy, J. G. Modified polyvinyl alcohol-benzoporphyrin derivative conjugates as phototoxic agents.
Photochem.
Photobiol., 57.~ 64I-647, 1993.

16. Goff, B. A., Blake, J., Bamberg, M. P., and Hasan, T. Treatment of ovarian cancer with photodynamic therapy and immunoconjugates in a marine ovarian cancer model.
Br. J.
Cancer, 74: 1194-1198, 1996.

17. Oseroff, A. R., Ohuoha, D., Hasan, T., Bommer, J. C., and Yarmush, M. L.
Antibody-targeted photolysis: Selective photodestruction of human T-cell leukemia cells using monoclonal antibody-chlorin e6 conjugates. Proc. Natl. Acad. Sci. USA, 83:
8744-8748, 1986.

18. Sobolev A. S., Akhlynina, T. V., Yachmenev, S. V., Rosenkranz, A. A., and Severin, E. S.
Internalizable insulin-BSA-chlorin e6 conjugate is a more effective photosensitizer than chlorin e6 alone. Biochem. Int., 26: 445-450, 1992.

19. Jiang, F. N., Allison, B., Liu, D., and Levy J. G. Enhanced photodynamic killing of target cells by either monoclonal antibody or low density lipoprotein mediated delivery systems.
J. Controlled Release, 19: 41-58, 1992.

Claims (12)

1. Compounds where a ring structure (VIII) representing a porphyrin, chlorin or bacteriochlorin/isobacteriochlorin ring in any of its imino-nitrogen tautomeric forms carries four aromatic preferably phenyl substituents Ar each themselves carrying one or more hydroxy groups, one or more of which hydroxy groups is in turn linked to an antibody directed against a cell surface antigen of cancer or other diseased cells.

2. Compounds according to claim 1 where the aromatic groups are m-hydroxy phenyl groups.

3. Compounds according to claim 1 or claim 2 where the antibody is linked to the aromatic hydroxy group through a linking group, suitably an ether linked group particularly:- where n is 1 to 4 and A is -OH or -NH2, or alternatively an ester-linked group particularly:- where n and A are as before and R1 is hydrogen or a hydrocarbon or carboxylated amino-acid side chain representing particularly glycine, alanine, lysine or glutamic acid.

4. As intermediates, compounds as set out in claim 3, but lacking the antibody.

5. Compounds according to claim 1, 2 or 3 wherein the antibody is an MAb, particularly mMAb 425 or an antibody to an antigen as listed herein.

6. Compounds according to claim 1,2 or 3, where VIII represents a porphyrin particularly m-THPP.

7. Compounds according to claim 1, 2 or 3 where VIII represents a chlorin

8. Compounds according to claim 7 where VIII represents m-THPC.

9. Compounds according to claim 7, where VIII represents a chlorin other than m-THPC, particularly p-THPC or o-THPC.

10. Compounds according to claim 1, 2 or 3, where VIII represents a bacteriochlorin or isobacteriochlorin, particularly m-THPB or m-THPiB.

11. The treatment of disease using an antibody conjugate according to any one of claims 1 to 3 or 5 to 10 or prepared from an intermediate according to claim 4.

12. Use of a compound according to any one of claims 1 to 3 or 5 to 10 or prepared from an intermediate according to claim 4, in the preparation of a medicament for PDT, and a medicament for PDT in which such a compound is incorporated in a pharmaceutical diluent or carrier in an amount to enable administration of an 0.1 to 5 mg/kg (7 to 350 mg related to a 70 kg adult and calculated as the m-THPC or other PDT active) unit dose of said compound to a person in need of same.