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Photodynamic therapy of squamous cell carcinoma : an evaluation of a new photosensitizing agent and photoimmunoconjugate Hemming, Alan William 1993

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"PHOTODYNAMIC THERAPY OFSQUAMOUS CELL CARCINOMAAN EVALUATION OF A NEW PHOTOSENSITIZING AGENTAND PHOTOIMMUNOCONJUGATE"byALAN WILLIAM HEMMINGM.D., The University of British Columbia, 1987A THESIS SUBMITTED IN PARTIAL FULFILLMENT OFTHE REQUIREMENTS FOR THE DEGREE OFMASTER OF SCIENCEinTHE FACULTY OF GRADUATE STUDIES(Department of Surgery)We accept this thesis as conformingto the required standardTHE UNIVERSITY OF BRITISH COLUMBIAAPRIL 1993© Alan William HemmingIn presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.(Signature)Department of ^ry_r F0. c-.)^oZNi'edl•c_\A_The University of British ColumbiaVancouver, CanadaDate ^koscti■ a L rk ck.r-SDE-6 (2/88)iiABSTRACT:Photodynamic therapy for cancer depends on the relatively selectivedistribution of photosensitizing agents to malignant as compared to normaltissues, rendering the malignant cells more susceptable to light mediateddamage. Photodynamic therapy has been used with only moderate successto date. The purpose of this study was to compare a new photosensitizingagent, Benzoporphyrin derivative (BPD), to the standard agent presentlyin use, Photofrin II, in a hamster cheek pouch model of squamous cellcarcinoma. As well we have investigated the potential of using a tumorspecific monoclonal antibody - BPD conjugate to improve the tumorlocalizing properties of BPD.Treatment consisted of photodynamic therapy with either PhotofrinII, BPD, or a tumor specific antiepidermal growth factor receptor -BPDconjugate. Control groups of light alone, antiEGFr, tumor nonspecificantiCEA, and tumor nonspecific antiCEA-BPD conjugate were includedwith the contralateral cheek pouch of each animal acting as a darkcontrol. An assessment of differential delivery of BPD to tumor and tonormal mucosa was undertaken using a spectrophotometric assay.Parametric statistical analysis included student t tests and linear regressionwhile non-parametric analysis was undertaken using Fisher's exact test.Animals receiving BPD alone demonstrated tumor to tissue levelsof approximately 2:1 while animals receiving the tumor specificiiiantiEGFr-BPD conjugate had significantly better tumor:tissue ratios of26:1. (p < 0.005)Animals treated with Photofrin II had a one month cancer freesurvival of 27% while animals treated with BPD had an improvedsurvival of 67% (p =0.03) The group treated with the tumor specificantiEGFr-BPD conjugate at a twentieth the total dose of BPD had an 80%one month cancer free survival which was not statistically different fromthe group treated with BPD alone.Benzoporphyrin appears to be a more effective photosensitizingagent than Photofrin II and its tumor selectivity can be improved using atumor specific monoclonal antibody conjugate.ivTABLE OF CONTENTS:^ pageABSTRACT ^  iiLIST OF TABLES  viLIST OF FIGURES  viiACKNOWLEDGEMENT ^  viiiI. INTRODUCTION1. Photodynamic Therapy : History1.1 Early Development of PDT ^ 11.2 Porphyrins as Photosensitizers  12. Mechanism of Action2.1 Photochemistry ^  22.2 Cellular Effects  43. Properties of an Ideal Photosensitizer3.1 Absorbtion ^  53.2 Tumor Selectivity  54. Light Delivery & Dosage4.1 Light Delivery ^  74.2 Light Dosage  85. Clinical Trials5.1 Overview of Clinical Applications ^ 95.2 Cutaneous and Subcutaneous Malignancies . . . ^ 105.3 Head and Neck Cancer ^  105.4 Genitourinary Cancer  115.5 Endobronchial Tumors  125.6 Esophageal and Gastrointestinal Tumors ^ 135.7 Miscellaneous Malignancies ^ 156. Benzoporphyrin Derivative6.1 Synthesis ^  166.2 Photosensitizing Properties of BPD ^ 16v7. Immunoconjugates^ page7.1 History and Principles ^  177.2 Photoimmunoconjugates  208. Epidermal Growth Factor Receptor ^ 21II. EXPERIMENTAL RATIONALE & PURPOSE1. Experimental Rationale ^  232. Purpose ^  25III. MATERIALS & METHOD1. Animal Model ^  262. Monoclonal Antibody Selection ^ 273. Photosensitizers & Preparation of Immunoconjugates3.1 Photosensitizers ^  283.2 Antibody Purification and Conjugation ^ 294. Assay of BPD Delivery to Tissues ^ 305. Photodynamic Therapy ^  326. Statistics ^  34IV. RESULTS1. Antibody Specificity ^  352. BPD Assay ^  353. Photodynamic Therapy  39V. DISCUSSION ^  42VI. SUMMARY  45VII. CONCLUSION ^  46VIII. BIBLIOGRAPHY  47viLIST OF TABLES^ pageTable I: Photochemical Reactions Involved in PDT . . . ^ 3Table II: Standard Curve : Raw Data ^ 37Table III: BPD Assay : Raw Data  38Table IV: Results of Photodynamic Therapy ^ 41Figure 2:Figure 3:Figure 4:Figure 5:Figure 6:Figure 7:Figure 8:Figure 9a:Figure 9b:Figure 9c:Figure 10:page55Excitation Curve of BPD ^ 56Hamster Cheek Pouch SCC  57Immunohistochemistry : Method ^ 58+ve Expression of EGFr  59Porphyrin Assay : Fluorescence Curves ^ 60Standard Curve ^  61Assay: BPD Levels  62SCC Before Photodynamic Therapy ^ 63SCC 1 Week Post PDT ^  64SCC 1 Month Post PDT  651 Month Cancer Free Survivals ^ 66viiLIST OF FIGURESFigure 1: Structure of Photofrin II and BPD ^ACKNOWLEDGEMENTI would like to thank my research supervisor , Dr. Noelle Davis forher support and guidance throughout the year spent in the laboratory andduring preparation of this thesis. In addition I wish to acknowledge thedirection given to this thesis by Dr. Charles Scudamore and Dr.StevenLam. Dr. Andrew Seal, director of the MSc. program has earned myappreciation for the ongoing encouragement and assistance he providedthroughout the year. Dr. Richard Finley deserves special mention forwithout his assistance this project would never have been completed.Finally I would like to thank Rendi Yan for her assistance and expertisein the laboratory and QuadraLogic Technologies for providing both theBPD and the assistance in creating a workable conjugate.viiipage^1I. INTRODUCTION.1 Photodynamic Therapy: History1.1 Early Development of Photodynamic TherapyThe first known use of phototherapy occurred over 6000 years agowhen the ancient Egyptians used the technique to treat depigmented areasof skin [1]. They applied crushed leaves from plants, containing what wenow know to be psoralens, to areas of depigmented skin and on exposureto sunlight this resulted in sunburn and eventual pigmentation of theunderlying skin [2]. In more recent times chemical sensitization of livingtissues was first reported in 1900 by Raab using the aniline dye, acridineto render unicellular organisms sensitive to light [3].Tappenier and Jesionek (1903) were the first to utilizephotodynamic therapy for the treatment of malignant disease when theytreated skin cancers using topical eosin as a photosensitizer along withwhite light [4]. Over the next 75 years several compounds includingmethylene blue, tetracycline, chlorophylls and other porphyrins werefound to be cytotoxic in combination with light [5,6].1.2 Porphyrins as PhotosensitizersAuler and Banzer described uptake of hematoporphyrin (HP) byneoplastic tissues in 1942 and this was later confirmed using fluorescenceby Figge et al [7,8]. Hematoporphyrin derivative (HPD), the porphyrinpage^2initially popularized for photodynamic therapy is a synthetic derivative ofhemoglobin demonstrated by Lipson et al in 1960 to have tumorlocalizing properties [9]. HPD, marketed as Photofrin, in fact consists ofa mixture of different porphyrins and has been further purified to thecomponents that are thought to localize to tumor, ethers and esters ofdihematoporphyrin (DHE) [10,11]. DHE also consists of a complexmixture of hematoporphyrin dimers, trimers, tetramers, pentamers, andtheir dehydration byproducts with both ether and ester linkages[12,13,14]. This tumor localizing fraction marketed as Photofrin II(Quadralogic Technologies, Vancouver, Canada) has enhancedphotodynamic cytotoxic effects and is presently the standardphotosensitizing agent in use now [2].2. Mechanism of Action2.1 PhotochemistryPorphyrins and photosensitizers in general have a commonmechanism of action. After absorbing light of an appropriate wavelengththe sensitizer is converted from its stable electronic ground state (S o) toa short lived excited state known as the singlet (S 1 ) state that may undergoconversion to a longer-lived excited state known as the triplet state (T 1 ).The triplet state is responsible for forming cytotoxic species or mayundergo several competing processes including fluorescence decay [15].page^3It is this fluorescence decay of photosensitizers that has led to theiradditional use in the detection of malignancy [16,17,18].Table IPhotochemistry involved in PDT.So + light -> S I^AbsorbenceS 1 -> So + light^FluorescenceS 1 -> So + heat^Internal conversionS 1 + M -> So + M^QuenchingS 1 -' T i^Intersystem crossoverWhere^So = Sensitizer ground stateS I = Sensitizer singlet stateT 1 = Sensitizer triplet stateM = SubstrateInteraction of the triplet state sensitizer with tissues can proceed viaeither a type I or type II reaction. Type I reactions involve hydrogenabstraction from the sensitizer to produce free radicals and are oxygenindependent. The type II reaction exclusively involves the interactionbetween molecular oxygen and the triplet state to form singlet oxygenpage^4( 102) which is highly reactive in living tissue [19].Type II: T 1 + 02 —> So + ' 02'02 + M .- photo-oxidationIt is this type II reaction that is thought to be responsible for the cytotoxiceffects of PDT and in fact the absolute requirement for oxygen inphotosensitization has been documented in solution, in culture and in vivo[20,21,22].An important concept is that the excited sensitizer after creatingsinglet oxygen, returns to its ground state and can again be excited. Thisallows a single sensitizer molecule to produce many times its ownconcentration of singlet oxygen.2.2 Cellular EffectsAt the cellular level, singlet oxygen causes lipid peroxidation anddamage to both mitochondrial and outer cell membranes resulting in celldeath [23]. At the nuclear level single strand breaks in DNA can beproduced and as well it has been shown that both RNA-dependant DNApolymerase and DNA dependant RNA polymerase can be inactivated byporphyrin photosensitization [24,25,26].page^53 Properties of an Ideal Photosensitizer3.1 AbsorptionFor photodynamic therapy a sensitizer with absorption in the red orinfrared region of the spectrum is desirable [14]. Absorption andscattering of red light by human tissue are much less extensive than ofblue light [27]. Living tissues are best penetrated by light of awavelength above 600 nm and in fact it has been shown by Bown et althat significantly improved tissue penetration is achieved as thewavelength of activating light is increased from 630 nm to 675nm andbeyond [28]. As well as having an absorption peak at this preferred bandthe sensitizer must also have an efficient quantum yield at this excitationwavelength , i.e., the predominant result of activating the sensitizershould be the production of the excited triplet state and therefore singletoxygen rather than the production of fluorescence decay [29]. PhotofrinII has intense absorption in the violet region (400nm) and severaladditional absorption bands between 500nm and 600nm. Unfortunately itabsorbs relatively poorly at 630 nm the only absorption band above600nm and yet it is this wavelength that is most often used clinically [23].Photofrin II although the standard photosensitizer in present use is not anideal photosensitizer in terms of its absorption characteristics.3.2 Tumor selectivityThe ideal photosensitizer should distribute to malignant as opposedpage 6to normal tissues, thus in theory, causing maximum tumor destructionwhile minimizing the destruction of normal surrounding tissues andsystemic side effects. Pharmacokinetics of Photofrin II have beenanalyzed using radiolabelled tracers and it has been shown that theporphyrin is retained in tumor more than in normal tissues such as skin,muscle, brain and lungs. However much larger concentrations are foundin organs such as liver, spleen and kidney [30]. While the mechanism fordelivery and retention of porphyrins in tumor is not known, evidencesuggests that there is a high affinity between porphyrins and lipoproteins[31,32]. Porphyrins bind strongly to both HDL and LDL. It is interestingto note that LDL receptors are elevated in neoplastic cells as well as inthose organs where there are relatively high concentrations of porphyrin[33].Barel et al have shown that the amount of porphyrin retained intumor is higher when porphyrin is bound to isolated LDLs pre-injectionas opposed to free drug or to drug bound to isolated HDLs [34]. Kesselhas shown that tumor nonlocalizing porphyrins are bound to albumin andalso found a correlation between the distribution of porphyrin and thenumber of LDL receptors in various tissues [35].An alternative hypothesis has been suggested by Korbelik et al thatsuggests that macrophages have a high affinity for porphyrins and thatrelatively high levels of photosensitizers found in tumor as well as inpage^7liver, spleen and kidney are due to the high number of functioningmacrophages at each of these sites [36].While the mechanism of tumor selectivity is not completely defined,it is certain that the lack of complete selectivity accounts for the main sideeffect reported with photodynamic therapy which is cutaneousphotosensitivitity [37,38,39].4 Light Delivery & Dosage4.1 Light DeliveryEnergy delivered to the target site is the product of the power ofthe light source and the time of exposure to this light source. Sufficientenergy for photodynamic activity can be delivered over a prolonged timeusing an ordinary low-watt light bulb equipped with a red filter and infact in early studies this method was used [40,41]. This method is quitetime consuming and therefore the majority of preclinical studies have usedhigh intensity arc lamps equipped with filters in order to adjust thewavelength to the appropriate band [14,42,43 ,]. While any source oflight with the appropriate light band may be used, clinical applicationsgenerally involve the transmission of the light into an enclosed cavity andtherefore a very coherent light source, such as laser light that can betransmitted via optical fibres, is required. Argon pumped dye lasers arepresently the most common light source for clinical use and have thepage^8advantage of being tunable to various desired wavelengths [44].4.2 Light DosageTo a large extent light dosage in photodynamic therapy remainsquite empiric and tumor type dependant with doses of between 50 - 300J/cm2 used with success [45,46]. It has been demonstrated that for agiven wavelength of light increasing the light dose increases the depth oftissue penetration and tumor necrosis. Mang et al demonstrated that skinlesions 3-4mm in depth were adequately treated with Photofrin II 2.0mg/kg and 72 J/cm2 while lesions 5-10mm in depth required light dosagein excess of 108 J/cm 2 [47].Patterson et al have validated the concept of a threshold dose fortumor necrosis in photodynamic therapy that states that tissue necrosis dueto photodynamic therapy will occur if the number of photons absorbed bythe photosensitizer per unit volume of tumor exceeds a critical value [48].Grossweiner has gone on to develop a light dosimetry model for PhotofrinII where the concentration of DHE in the tumor is 2-4 pt,g/g and thenecrosis threshold is on the order of 0.4 - 0.7 J/cm 3 or ..--', 2 X 10 18photons/cm' at 630 nm. [49]. This model can be used as a generalguideline for light dosage in most animal models using Photofrin II,however it should be noted that changes in photosensitizer concentrationresult in a proportionately inverse change in the required light doseneeded to achieve the necrosis threshold.page^95 Clinical Trials5.1 Overview of Clinical ApplicationsA large number of different tumors have been treated withPhotofrin or Photofrin II and photodynamic therapy since the first clinicalstudies were initiated in 1978 by Dougherty et al [38]. In early studiespatients treated by photodynamic therapy had long histories of cancer,either metastatic or local recurrences, which had failed more conventionaltreatments. Debulking of large tumors has in general been unsatisfactoryand while interstitial treatment of solid tumors has been accomplished ithas had limited success due to lack of sufficient light penetration [50].Overall, complete response rates using Photofrin and Photofrin II havebeen approximately 20-40%.The major side effect of photodynamic therapy seen in all of theclinical trials has been skin photosensitivity resembling that seen inpatients with porphyria [51]. Patients are advised to avoid both direct andindirect sun exposure for a period of 4-8 weeks after intravenous injection[52]. Other side effects reported include nausea, vomiting, metallic taste,eye photosensitivity and liver toxicity [53]. Toxicity does not appear tobe additive in patients previously treated with either chemotherapy orradiation and can apparently be used in conjunction with other modalities[54]. Photodynamic therapy has been disadvantageous in situations wheremajor complications can be predicted with treatment of the tumor, as inpage 10treatment of a full thickness esophageal tumor resulting in an esophagealfistula [50].5.2 Cutaneous and Subcutaneous MalignanciesCutaneous and subcutaneous malignancies represent the mostaccessible of all malignancies. Various cutaneous malignancies have beentreated including malignant melanoma, squamous cell carcinoma, basalcell carcinoma, Kaposi's sarcoma and dermal metastatic breast carcinoma[55]. Response rates have varied according to tumor morphology withsmall superficial lesions usually showing complete response in 4-6 weeksand larger more invasive lesions often requiring multiple treatments forcomplete response. Phototoxicity was the primary side effect in all trials.Toxicity was reduced apparently without compromising effect by reducingthe dose of photosensitizer used [56].5.3 Head and Neck CancerMost patients with head and neck malignancies treated withphotodynamic therapy have had squamous cell carcinomas refractory totraditional treatment however in several recent trials patients with earlycarcinoma in situ and patients with "condemned mucosa" consisting ofmultiple areas of malignant and premalignant change have been included[2,57]. In cases where the purpose of therapy was only to providepalliation success was achieved in a high percentage of cases, howevercomplete response was only obtained in 20-30% of cases [58,59].page 11Gluckman reported on 13 patients with early cancers of the aerodigestivetract treated with photodynamic therapy, 11 of which demonstrated acomplete response. However, on follow-up 4 of these recurred between8 and 12 months. More encouraging results were obtained in the sameseries in patients with condemned mucosa, with 8 of 8 patients having anexcellent response to treatment with only 1 patient going on to have apositive biopsy with 12 month follow-up [2].Complications in these series were again mainly due to skinphotosensitization and, in fact, in some palliative cases symptoms of skinphotosensitization were of such severity to worsen the quality of life. Inone patient, the carotid artery, which was encased with tumor, rupturedthree days post photodynamic therapy with resultant death [2].5.4 Genitourinary CancerPhotodynamic therapy has probably had its best result in treatmentof transitional cell carcinoma of the bladder. The lesions that havebenefitted most are diffuse carcinoma in situ or very superficial bladdercancers, especially if multicentric [60]. Tsuchiya et al and Hisazumi etal report 75 % and 83 % complete response rates in treating superficialbladder cancers with 6-18 month disease free follow-up [61,62]. Multipleother studies report essentially the same result with complete responserates ranging from 60 -85 % however several of these studies have beencomplicated by troublesome bladder volume contraction that in at leastpage 12two cases required subsequent cystectomy [63,64,65]. This has led toattempt photodynamic therapy at 514 nm which is a less penetratingwavelength and at least in theory should result in less bladder fibrosis andcontracture [66].5.5 Endobronchial TumorsPhotodynamic therapy has been used for both curative and palliativetreatment of endobronchial lesions. Balchum et al treated 100 patientsthat presented with 40 -90% obstruction due to squamous cell, large cell,and carcinoid tumors, with palliation achieved in the majority, howeversurvival was not affected. All patients required bronchoscopy to removenecrotic debris 2 to 4 days after photodynamic therapy at which timetreatment was repeated if any gross tumor was left behind [67]. Thismode of therapy for obstructing endobronchial lesions should beconsidered in light of ablative laser therapy, the primary alternative forthese lesions, which has immediate response, carries no complications ofphotosensitivity and generally does not require a second bronchoscopy.Hayata et al have reported on photodynamic therapy in eight casesof early stage, central lung cancers that would otherwise have requiredextensive resection or been unresectable [68]. Six of the eight cases hadcomplete response after photodynamic therapy and remained disease freeby endoscopic, cytologic and histologic evaluation with follow-up between11 and 36 months. The resected specimens of two patients with partialpage 13response were found to have tumor in areas that were thought to havebeen inadequately irradiated.Preoperative photodynamic therapy has been used by Kato et al toconvert inoperable to operable lung cancer in four of five patients withone patient remaining tumor free four years after resection. As wellseven of ten patients with planned pneumonectomy underwent lessextensive resection after photodynamic therapy [69].Complications of treatment encountered included excessivebronchial mucous secretions, mucosal sloughing, fever, pneumonia, andpneumothorax. In addition there are several reports of death fromhemoptysis four to five weeks after treatment [67]. Many of thesecomplications have apparently been eliminated by adding the routine useof bronchoscopy two to four days after photodynamic therapy.5.6 Esophageal and Gastrointestinal TumorsPhotodynamic therapy has been used for both cure and palliation ofesophageal carcinoma. McCaughan et al reported seven cases ofcomplete or near complete esophageal obstruction who were treated withphotodynamic therapy with all patients being adequately palliated [70].More recently photodynamic therapy has been applied to earlier,superficial lesions with curative result. Okunaka et al demonstratedcomplete response in four of six patients with early superficial squamouscell carcinoma with 5 of the six patients alive at 2 years [71]. Calzavarapage 14et al reported complete response in eight of twenty-one patients withsuperficial esophageal carcinoma with three of the eight patients alive anddisease free at two years post treatment [53].Early stage gastric cancer has been treated with photodynamictherapy with Hayata et al treating four of sixteen cases with photodynamictherapy alone. Complete response was obtained in all four patients asdemonstrated by endoscopy, cytology and histology however three of thefour had recurrences between five and 27 months after treatment. Theother 12 patients underwent surgical resection after photodynamic therapywith the resection specimen showing complete response in five, howeverseven of the twelve showed residual tumor [72].Photodynamic therapy has been used for the treatment of colorectalcancer predominantly in unresectable cases for palliation or on thetreatment of pelvic recurrences. Relief of pain from unresectable pelvicrecurrences seems to be obtained in 50 % of patients [73]. In a pilot studyby Barr et al 2 of ten patients with colorectal cancer unsuitable foroperation were disease free two years after photodynamic therapy [74].Kashtan et al have as well documented significant palliation with markeddecrease in tumor bulk in five of six patients with inoperable rectalcarcinoma [75].Complications associated with treatment of gastrointestinalmalignancies include fistulas, hemorrhage especially from the bulkierpage 15tumors and of course skin photosensitivity.5.7 Miscellaneous MalignanciesPhotodynamic therapy has been attempted in several other clinicalsettings however reports for the most part are few. One area that seemsof particular interest to surgeons is intraoperative use of photodynamictherapy to improve clearance of resection margins. Nambisan et al havereported on intraoperative photodynamic therapy for advancedretroperitoneal sarcomas in ten patients and have as well used thefluorescence of the photosensitizer to guide resection. Two of the tenpatients treated were tumor free at two years with no reportedcomplications [76]. This intraoperative approach has also been attemptedfor intracranial malignancies. McCulloch et al reported on primaryresection of glioblastoma with treatment of the tumor bed withphotodynamic therapy, however noted significant cerebral edema posttreatment [77]. This intraoperative technique appears promising and mayoffer advantages in situations where wide resection margins are difficultto achieve.page 166 Benzoporphyrin Derivative6.1 SynthesisWhile clinical results of photodynamic therapy have shown somesuccesses the results overall have been somewhat disappointing. Allclinical trials have used either Photofrin or Photofrin II, which aspreviously pointed out have less than ideal properties forphotosensitization in the clinical setting. The search for an idealphotosensitizer has led to the synthesis of a customized molecule,benzoporphyrin derivative or BPD. (Quadralogic Technologies,Vancouver, Canada.) The synthesis of BPD has been described by Richteret al in 1987 and its structure is shown in Figure 1. BPD as originallysynthesized consists of four isomeric forms of which BPD monoacid Ahas the most desirable tumor localizing effects [78].6.2 Photosensitizing Properties of BPDBPD absorbs extremely well at 692 nm and therefore is activatedby light that penetrates living tissue better than the wavelength thatactivates Photofrin II (Figure 2). BPD has been utilized in vitro and hasbeen shown to be a more potent photosensitizer than is Photofrin II [79].The lethal effects of photodamage with BPD are associated withmembrane damage and there is no evidence for "dark toxicity" [80].Biodistribution of BPD appears to be similar to that of Photofrin IIwith marked binding to plasma lipoproteins, predominantly HDL,page 17however as well to LDL. Precomplexing BPD to LDL increases thedeposition of BPD to tumor. Tumor to skin ratios of BPD given aloneare between 2-3:1, which is less than ideal, however this is increased toan acceptable 5:1 when precomplexed to LDL [81].BPD undergoes inactivation of its photoactivity in the tissues withup to 60% of its activity lost at 24 hours [82]. This may actually be ofbenefit in reducing the duration of skin photosensitivity. Richter et alhave reported on a mouse model in which skin photosensitivity with BPDis higher than Photofrin II in the first 24 hours, however animals exposedto light after 24 hours showed only minimal effects of photosensitivity[83].BPD also offers the advantage of being a single compound withoutthe problems associated with Photofrin II, which consists of a mixture ofporphyrins. BPD in theory appears to approach the ideal photosensitizermore closely than does Photofrin II.7 Immunoconjugates7.1 History and PrinciplesKohler and Milstein first described a general procedure for theproduction of monoclonal antibodies (MoAb) in 1975 known as thehybridoma technique [84]. Briefly mice are immunized with a source ofantigen and then splenocytes from the immunized animals that includepage 18immune B lymphocytes are fused with an established mouse myeloma cellline. Hybrid cells grow out under selective conditions that killnonhybridized mouse myeloma cells. Hybrid cells combine the ability toproduce a specific antibody from the B lymphocytes with the property ofimmortality from the myeloma cell line. This allows the large scaleproduction of specific monoclonal antibodies to desired antigens [85].The development of monoclonal antibodies has led to efforts toidentify tumor specific antigens to which MoAbs can be directed.Initially it was hoped that simply binding the MoAb to the malignant cellswould be sufficient to achieve tumor destruction, or at least inhibition, viaa complement mediated system or antibody dependant cell mediatedcytotoxicity and in fact passive administration of unmodified antibodieshas been shown to have some anti-tumor activity [86]. In generalhowever, unmodified antibodies have not proven to have a major effecton tumor destruction and the search has largely turned elsewhere.The identification of oncogenes and their products has led toanother mechanism to interrupt tumor biology. A fundamental differencebetween cancer cells and normal cells is the ability of the transformedcancer cell to go on dividing forever and to have reduced requirementsfor exogenous growth factors [87]. It has been shown that the oncogenev-erb B codes for a protein product homologous to the receptor forepidermal growth factor [88]. It is thought that cancer cells can producepage 19growth factors that promote the growth of that same cell. For examplea cancer cell that over expresses EGF receptor would initially bestimulated by normal amounts of EGF resulting in the same cells releaseof EGF as well as possible up regulation of the EGF receptor [89]. Thisso-called autocrine mechanism of stimulation is thought to provide agrowth advantage and suggests a possible step at which to intervene witha monoclonal antibody directed to EGFr. In animal models growth oftumors is inhibited by the treatment of the animals with a MoAb to EGFr,however cure is not achieved [90].The next step was to modify the tumor specific antibody in orderto make it more toxic. In theory this produces the "magic bullet " so longsought after, concentrating the toxic agent in the malignant tissue whilesparing normal tissues. Initial attempts with immunoconjugates weremade by adding potent toxins or chemotherapeutic agents to tumorspecific MoAbs. Ricin a potent cytotoxin has been linked to MoAbs toboth carcinoembryonic antigen and EGFr with excellent resulting tumorcell destruction [91,92]. Radiolabelled antibodies to CEA have beenused clinically in both localization and treatment of human colon cancerwith some success, demonstrating the possibilities of using otherimmunoconjugates in the treatment of cancer [93].page 207.2 PhotoimmunoconjugatesIn 1985 Mew et al demonstrated the feasibility of using specificmonoclonal antibody - hematoporphyrin conjugates to photosensitize anddestroy selected cancer cell lines in culture [94]. Later in 1986 Oseroffet al created a monoclonal antibody-chlorin conjugate that was effectivein selective destruction of human T-cell leukemia in vitro [95].Interestingly the chlorin used had no tumor selectivity of itself howeverwas chosen as a photosensitizer due to its absorption in the 680-690 nmrange demonstrating the potential for future applications of thistechnology.A problem that continued to arise was the difficulty in loading anappropriate number of photoactive molecules onto the tumor specificantibody. One solution suggested by Jiang et al was to use a polyvinylalcohol carrier on which to preload the photosensitizer(BPD) and then linkthe polyvinyl alcohol to the MoAb. This technique was found to retainantibody specificity with up to 50 molecules of BPD bound to eachantibody [96]. Using this method a monoclonal antibody - BPD conjugatedirected towards a human squamous cell carcinoma antigen 5E8 wasproduced which successfully localized and destroyed tumor cells inculture [97].page 218 Epidermal Growth Factor ReceptorIn order to use a photoimmunoconjugate an appropriate cell surfacemust be expressed by the malignant tissue. Epidermal growth factorreceptor would appear to be a good marker to target with some possibilityof future clinical use as it has been shown to be over-expressed in avariety of human neoplasms including squamous cell carcinomas of thehead and neck, esophagus and lung [98,99,100]. A number of othernon-squamous cell neoplasms such as colon, thyroid, breast and bladderhave demonstrated an increase in expression of EGFr as well[101,102,103].Epidermal growth factor receptor is a 170,000 MW protein thatspans the cell membrane and mediates the cell's initial response to EGFand perhaps TGFa [104]. It has an extracellular region that binds EGFand an intracellular region that possesses tyrosine specific protein kinaseactivity [105]. The erb-b oncogene codes a product homologous for aportion of the EGF receptor [88]. When activated by EGF the EGFreceptor promotes DNA synthesis and cell growth. The overexpressionof EGFr appears to be a step in malignant transformation of cells and isthought to confer a growth advantage to those cells via an autocrinemechanism of growth factor stimulation [89].EGF receptor, if not bound, is generally shed into the surroundingcell matrix during normal receptor turnover. When binding occurspage 22however, the receptor and substance bound to it are internalized viaendocytosis [106].Both the overexpression of EGFr by certain neoplasms, its role inmalignant cell growth and its mechanism of action suggest that EGFrwould be an ideal marker for a photoimmunoconjugate.page 23II. Experimental Rationale & Purpose.1. Experimental RationaleAs outlined above photodynamic therapy has been used in both thepreclinical and clinical stages with varying amounts of success. Photofrinand Photofrin II have been the primary photosensitizing agents used inphotodynamic therapy of tumors and yet neither possessesphotosensitizing properties that can be considered to approach in any waywhat might be considered ideal. Recent advances in biochemistry haveallowed photosensitizing agents to be custom designed to providephotochemical properties that more closely approximate that of the idealphotosensitizer, specifically a modified chlorin structure, benzoporphyrinderivative or BPD.The development of monoclonal antibodies to tumor specific cellsurface markers suggests an elegant method of delivering aphotosensitizing agent selectively to malignant tissue, in theory sparingthe surrounding normal tissues illuminated during treatment as well asreducing if not eliminating the systemic toxicities associated with otherphotosensitizers during photodynamic therapy.The theoretical benefits of using benzoporphyrin derivative may beseen to have basis in reality by comparing the response and cure rates ofa specific tumor after photodynamic therapy with either Photofrin II, thestandard photosensitizing agent in use, or benzoporphyrin derivative.page 24The ability in vivo of a monoclonal antibody - photosensitizerconjugate to distribute to malignant and not normal tissues can be assessedin two ways. One method is to treat a specific malignancy and infer fromlack of side effects, particularly skin photosensitivity, that the agent isselective. The other and more objective method is to construct an assayfor the specific photosensitizing agent in both normal and malignanttissues to assess relative levels of agent in the tissues.Lastly the photodynamic ability of the monoclonal antibody -photosensitizer conjugate, specifically an anti-EGFr - BPD conjugate, toeffect tumor destruction may be assessed in the same model as incomparing Photofrin II to the new agent, benzoporphyrin derivative. Ifresponse and cure rates of the monoclonal antibody - photosensitizerconjugate are equivalent to, or better than, the best of the other twoagents and a strong tumor selective effect can be demonstrated this wouldbe strong supportive evidence that photoimmunoconjugates offeradvantages in photodynamic therapy and deserve consideration for theclinical setting.page 252. PurposeThe purpose of this thesis is to:1) evaluate a new photosensitizing agent, benzoporphyrin derivativeagainst the standard photosensitizing agent Photofrin II.2) assess the tumor localizing properties of a photoimmunoconjugate.3) evaluate the effectiveness of a photoimmunoconjugate in photodynamictherapy.page 26III. MATERIALS & METHOD.1. Animal Model:The Syrian golden hamster cheek pouch model has been describedpreviously [18]. In brief, Dimethylbenzanthracene (Sigma Chemical St.Louis) impregnated, silicone coated sutures were prepared and insertedsubmucosally into both cheek pouches of 95 male outbred Syrian goldenhamsters (Charles River, Montreal, Canada) age 4-6 wks. A separategroup of 15 male Syrian golden hamsters had a similarly placed suture inonly one cheek pouch. This has previously been shown to inducesquamous cell carcinomas in 85% of animals by 12 weeks(Figure 3) [18].In our experience all animals developed tumor by 18 wks although 8animals required reimplantation with suture at 4 weeks when it becameevident that the original sutures had fallen out with initial inflammationcaused by the DMBA. Animals were housed at room temperature witha 12-hr light /dark cycle. Water and laboratory chow were given adlibitum. Procedures were done under intraperitoneal barbiturateanesthetic (Sodium pentobarbital, 5 mg/kg). Euthanasia was performedusing an overdose of the same barbiturate one month post photodynamictherapy. At the time of photodynamic therapy animals weighed between175 and 200 grams. All procedures received approval of the Universityof British Columbia animal care committee prior to the institution of thestudy.page 272. Monoclonal Antibody SelectionBoth a tumor specific antibody as well as a nonspecific controlantibody were required for our purposes. In order to identify the relativespecificities of our chosen antibodies the following was undertaken(Figures 4 +5).Tumors along with samples of normal mucosa from 3 separate animalswere harvested after euthanasia by sharp dissection away from underlyingtissues. Specimens were embedded in OCT (polyvinyl alcohol/ethyleneglycol/nonreactive ingredients) and snap frozen in liquid nitrogen.Standard hematoxylin and eosin frozen sections were done to confirm thepresence of squamous cell carcinoma. 5 micron frozen sections were cutfrom both tumor and normal mucosa, air dried and fixed in acetone. Thesections were then rehydrated with Tris buffer (pH 7.6) and incubated atroom temperature for 5 minutes with non immune normal goat serum(Cedarlane Laboratories, Hornby, ONT, Canada) to block nonspecificbinding. The primary antibodies used were a mouse monoclonal IgG1 tothe extracellular domain of the EGFr (Sigma St. Louis Mo.) [107] anda mouse monoclonal IgG1 to Carcinoembryonic Antigen ( PierceLaboratories Rockford Illinois). Dilutions of 1:100 and 1:50 respectivelywere applied for one hour and then washed with Tris buffer. Endogenousperoxidase was blocked by applying 1.5 % peroxide for 20 minutes. Aftera second wash with Tris buffer the secondary antibody-peroxidasepage 28conjugate was applied for 90 minutes (goat anti-mouse/horseradishperoxidase, Biocan Scientific, Mississauga,ONT, Canada). The slideswere then washed with Tris buffer, placed in an acetate buffer (0.1 M,pH 5.2) for 3 minutes and developed over 15 minutes with a substratesolution of 3-amino-9-ethyl carbazole(6 mg AEC in 1.5 ml N,Ndimethylformamide, 28.5 ml 0.1 M acetate buffer and 0.3 ml 3% H 202).Subsequently the sections were washed with distilled water, counterstained with Carrazzi's hematoxylin, mounted with an aqueous mediumand dried. For each set of slides and for each primary antibody used, anegative control omitting the primary antibody and a positive control ofa known EGFr expressing human oral squamous cell carcinoma or aknown CEA expressing human colonic carcinoma were simultaneouslystained. The staining was rated as positive if any portion of the slideshowed staining.3. Photosensitizers & Preparation of Monoclonal AntibodyConjugates3.1 PhotosensitizersBoth Photofrin II and Benzoporphyrin derivative were kindlydonated by Quadralogic Technologies (Vancouver, Canada). BPD and itssynthesis have been well described elsewhere [79]. Crystallized BPDmonoacid A was made into solution by dissolution in DMSO to make apage 29stock solution of 5mg/ml BPD/DMSO. This was kept frozen at -40degrees Celsius until use at which time it was diluted with sterilephosphate buffered saline (PBS) for injection. The solution was kept inthe dark until injected. The Photofrin II supplied as a crystalline powderwas dissolved in 5% dextrose water immediately prior to use.3.2 Antibody Purification and ConjugationBoth the tumor specific EGFr-BPD conjugate and the nonspecificCEA-BPD conjugate were prepared using a method that has beendescribed in detail previously and is outlined below [14].The antiEGFr IgG1 was supplied in ascites (Sigma Immunochemicals St.Louis. Mo.) purified over a recombinant protein G column(PierceLaboratories, Rockford Illinois), lyophilized and stored in PBS. Theconcentration of antibody in solution was assayed using the Biuret methodand found to be 3.8 mg/ml. A portion of this sample was used as theprimary antibody in immunohistochemical staining of a known EGFrpositive oral squamous cell carcinoma to confirm the maintenance ofantibody specificity. The antiCEA IgG1 was supplied as a purifiedlyophilized powder.Benzoporphyrin derivative is loaded onto modified polyvinylalcohol carrier, which is then further substituted with 3-mercaptopropionicacid such that three free thiol(SH) groups are introduced per carriermolecule. The PVA-BPD-SH was supplied by Quadralogicpage 30Technologies. (Vancouver, Canada).Both specific antiEGFr conjugate and nonspecific antiCEAconjugate were prepared in the same manner. Antibody, initially inphosphate buffered saline, underwent volume reduction and bufferexchange to carbonate buffer(pH 8.5). Antibody was then transferred toan amber vial flushed with nitrogen. SMBS(sulfo-m-maleimido-benzoyl-N-hydroxysulfosuccinimide ester, 5mg/m1)was added to give a molar ratio of SMBS to antibody of 30:1, the vialagain flushed with nonreactive nitrogen gas and the mixture stirred fortwo hours in the dark. The carbonate buffer was then exchanged to a0.05M acetate buffer pH(5.4) and the BPD-PVA-SH added to the MoAb-SMBS with 5% PVA added to make a final concentration of 0.8% PVA.The mixture was stirred overnight at 4°C in the dark.This results in a molar ratio of BPD: monoclonal antibody of 25:1and has been shown in vitro to maintain both antibody specificity as wellas the photosensitizing properties of BPD [97]. The BPD-monoclonalantibody conjugates were mixed with sterile PBS immediately prior touse.4 Assay of BPD Delivery to Tissues.The method of tissue porphyrin assay used has been adapted fromthat described by Straight [108]. The 15 animals with tumor in onepage 31cheek pouch were divided into four groups for intravenous injection ofeither 2.5 mg/kg BPD, 1 mg/kg BPD-anti EGFr conjugate(containing120/Ag BPD/mg antibody), 1 mg/kg BPD-antiCEA conjugate(120p,gBPD/mg antibody) or 1.5 cc PBS(control). Three animals were in eachgroup except the control group that consisted of six animals. Intravenousaccess was achieved via a left internal jugular cutdown. The animalswere then kept in the dark for 6 hrs and sacrificed. Tumor and normalmucosa were harvested and divided by group into lgm wet weightsamples. Pooling of control group samples was undertaken prior todivision in order to achieve control samples representing the averagebaseline characteristics of all tumors collected. The samples were thenminced and 1.2 ml of distilled water was added to each 1 gm sample andthe resulting mix homogenized mechanically (Brinkman HomogenizerModel PT 10/35). For the control (PBS) group a known amount of BPDwas added to each sample prior to homogenization in order to constructa standard curve. Samples were then lyophilized overnight (Lab-ConCo., Freeze dry-3, Fisher Scientific Vancouver, Canada). The dryweight of the samples was determined after which each sample wasreconstituted with 1.0 ml of reagent grade water to each 50mg dry weightof tumor and sonicated. (Bransonic 52, Branson Equipment Shelton CT).Each 1 gm wet weight sample would provide 100 - 150 mg dry weight oftumor. 100 microL aliquots of homogenized tissue were then added topage 321.0 ml methylbenzonium hydroxide (1.0 M in methanol, Sigma Chemical,St. Louis Mo.) and hydrolysed at 60 ° C for two hours. This step extractsthe porphyrin from the tissue and monomerizes any porphyrin polymersthat may be present. The samples were then cooled to room temperatureand solubilized with 2.0 ml of DMSO added to each sample. Thefluorescence of each sample was then assessed on a fluorometer(PerkinElmer LS-5, Oakbrook Illinois) using an excitation wavelength of 418nmand an emission wavelength of 691nm (Figure 6). A standard curve(Figure 7, Table II) was constructed using the control samples withknown amounts of BPD added while the concentrations of porphyrin inthe BPD, BPD-antiEGFr and BPD-antiCEA treated animals werecalculated by measuring the fluorescence at 691 nm and comparing thisto the standard curve.5. Photodynamic TherapyThe 90 animals with tumor in both cheek pouches were divided intogroups receiving the following treatments; PBS(control n=15), Photofrin10 mg/kg(n =15), BPD 2.5 mg/kg(n =15), BPD-antiEGFr conjugate,1 mg/kg,(120pg BPD/mg antibody n=15) BPD-antiCEA conjugate1mg/kg(120pg/mg antibody n =10), antiCEA alone 1 mg/kg(n =10), andantiEGFr alone 1 mg/kg(n =10). Each animal received photodynamictreatment to only one cheek pouch with the other side acting as a darkcontrol. All tumors were 6-8 mm in diameter at time of treatment.page 33Intravenous access was achieved via a left internal jugular cutdown underpentobarbital anesthetic. The animals were then kept in the dark for 6 hrsat which time they underwent photodynamic therapy. The exception tothis was the group receiving Photofrin II which was left in the dark for24 hrs as it has been shown that the maximum tumor to normal tissuelevels for Photofrin II are achieved at around 24-48 hrs, while for BPDthis is achieved between 3-6 hrs. [82,109] Animals were then treated byeverting the tumor bearing cheek pouch and delivering 200 J/cm2 of lightto the tumor using a high intensity xenon arc lamp equipped with highand low band pass filters ( Oriel Corp., Stratford CT, USA). The widthof the wavelength band used for the Photofrin II group was between600-630 nm while for all other groups it was 680-710 nm. Energydelivered at tumor level was measured using a surface radiometer( GentecModel TPM-310, Gentec, Sainte-Foy, Quebec, Canada). The incidentlight density was 320 milliwatt/cm 2 . The tissue being treated was keptmoist using room temperature normal saline and the tissue temperaturemonitored with a temperature probe throughout the treatment.Temperature did not rise more than 0.5 degrees Celsius during treatment.The animals were then returned to their cages and had both cheekpouches examined at 24 and 48 hours after treatment with biopsies takenat one week and then repeat biopsies taken at sacrifice one month aftertreatment. Biopsies were snap frozen in OCT and underwent standardpage 34hematoxylin and eosin staining . Slides were evaluated for the presenceor absence of residual cancer.6. STATISTICS:Data was stored on a 386 PC using Dbase IV and statistical analysisperformed using SPSS 4.0 statistical software. Non-parametric analysisof treatment differences was undertaken using Fisher's exact test, whilestudent t tests were used for parametric analysis of difference betweenmeans. The equation of the standard curve for the porphyrin assay wasgenerated using a "best fit" regression analysis. Significance wasspecified as p < 0.05. Values are reported as mean ± S.E.M. whenappropriate.page 35IV. RESULTS:1. Antibody SpecificityMultiple sections of the three squamous cell carcinomas revealedpositive staining for Epidermal growth factor receptor in all sections. Thepattern of staining( i.e., diffuse or focal) varied from tumor to tumor aswell as between different sections of the same tumor, however wasstrongly positive in all cases. Sections cut from the same areas as forEGFr staining showed a complete absence of staining using theanti-CEA monoclonal as a primary antibody. Normal mucosa revealedno areas of positive staining for either EGFr or CEA.2. BPD AssayThe standard curve generated for the tissue porphyrin assay isshown in figure 7. A linear regression model provided the "best fit"with a correlation coefficient of 0.97 with 1.0 being a perfectcorrespondence to the equation generated. The animals given 2.5 mg/kgof BPD showed a tumor tissue concentration of BPD of 7.8 ± 0.7 ,ug/gwith normal mucosa of the same animals containing 5.0 ± 0.8 µg/g, adifference that was significant at p= 0.046. The animals receiving theBPD-antiEGFr conjugate demonstrated a tumor tissue concentration ofBPD of 6.8 ± 0.6 Ag/g while normal mucosa taken from the sameanimals had a much lower level of BPD of 0.26 ± 0.09 Agig (p =page 360.0016)(Figure 8). No statistical difference was demonstrated betweenBPD and the BPD-antiEGFr conjugate in terms of the concentration ofBPD delivered to the tumor although the amount of BPD given to theBPD-antiEGFr conjugate receiving animals was approximately 20 timesless than the animals receiving BPD alone. BPD concentration in normalmucosa of animals receiving the BPD-antiEGFr conjugate wassignificantly lower than levels found in the normal mucosa of the animalsreceiving BPD alone (0.26/Ag/g vs. 5.0 µg/g, p = 0.002). The levels ofBPD in tumor and normal mucosa of animals receiving the BPD-antiCEAconjugate were below the limits of our assay and could not bedistinguished from the zero Ag/g BPD controls of the standard curve.Page^37Standard Curve Dataµg BPD Added Fluorescence0 5.20 4.51 22.51 212 292 37.62 283 49.13 47.73 36.54 67.54 69.94 68.76 77.86 71.78 94.28 9610 101.710 107.4TABLE IIpage^38Assay DataGroup Fluorescence pg BPD/gtumorBPD/Tumor 116.8 9.88BPD/Tumor 82.1 6.63BPD/Tumor 89.8 7.36BPD/Tumor 89.7 7.35BPD/Mucosa 56 4.19BPD/Mucosa 73 5.78BPD/Mucosa 65.1 5.03AntiEGFr/Tumor 81.8 6.61AntiEGFr/Tumor 84.3 6.84AntiEGFr/Tumor 70.6 5.56AntiEGFr/Tumor 99.7 8.28AntiEGFr/Mucosa 13 0.16AntiEGFr/Mucosa 15 0.35AntiCEA/Tumor 6 0AntiCEA/Tumor 12 0AntiCEA/Mucosa 8 0AntiCEA/Mucosa 9 0TABLE IIIpage^393. PHOTODYNAMIC THERAPYControl animals receiving light alone had no response to treatmentat 48 hours and all animals demonstrated squamous cell carcinoma at 1week biopsy as well as at 1 month biopsy at time of sacrifice. 2 of the15 animals required sacrifice at three weeks post treatment due toadvanced disease. These 3 week results are included as 1 month resultsfor those animals. Control animals receiving antiEGFr alone, antiCEAalone, and the nonspecific BPD-CEA conjugate uniformly had noresponse to treatment at 48 hrs, demonstrated squamous cell carcinomaat 1 week biopsy as well as at sacrifice 1 month post treatment. Again2 antiEGFr animals and 1 BPD-CEA animal required sacrifice atapproximately 3 weeks post treatment due to advanced disease and theseresults have been included as 1 month results. None of the dark controlson any animal showed any response at 48 hrs and all animals had tumorpresent at 1 week and 1 month.The 15 animals undergoing PDT with Photofrin II uniformlyexhibited a strong response at 48 hrs demonstrating tumor necrosis withmucosal sloughing and marked submucosal edema in the area exposed tolight. At one week only necrotic tumor bed was visible, however,biopsy of this area revealed residual squamous cell carcinoma in 11 ofthe 15 animals. At one month 27% (4 of 15) of animals had nohistologic evidence of persistent tumor.page 40The 15 animals undergoing PDT with BPD alone uniformlyexhibited a strong response at 48 hours, demonstrating an effect similarto that of Photofrin II with tumor necrosis and marked submucosal edemain the area exposed to light. At one week only necrotic tumor bed wasvisible(Fig 9a,9b,9c) however biopsy of the tumor bed revealed residualsquamous cell carcinoma in 5 of the 15 animals. One month biopsydemonstrated a 67% disease free rate(10 of 15).The 15 animals undergoing PDT with the BPD-EGFr conjugateuniformly exhibited a strong response at 48 hrs with marked tumornecrosis. It appeared that there was less submucosal edema and mucosalsloughing in the area around the tumor than had been evident in eitherthe BPD or Photofrin II treated animals. Biopsies taken at 1 weekrevealed no presence of squamous cell carcinoma in 12 of the 15 treatedanimals and these findings were confirmed at sacrifice one month aftertreatment.Photodynamic therapy with BPD gave a 1 month disease free rateof 67% which was significantly higher than the 27% disease free rateachieved with Photofrin II (p= 0.03 )Figure 10. Both BPD andPhotofrin II gave a better 1 month disease free rate than the lightreceiving control. (p < 0.0001 and p = 0.049 respectively)Animals treated with the BPD-antiEGFr conjugate showed a 1month disease free rate of 80% (12 of 15). This was significantly higherpage 41than either the group treated with light alone, (p = 0.02) or the othercontrol groups of nonspecific antibody-BPD conjugate, nonspecificantibody alone and tumor specific antiEGFr alone (p = 0.04). TheBPD-EGFr conjugate treated group showed a significantly higher curerate than did the Photofrin II group p =0.004. While on the surface thephotoimmunoconjugate appeared to give a better cure rate than BPDalone (80% vs. 67%) this was not statistically significant (p=0.23).GROUP CANCER FREEAT 1 MONTHCANCER AT1 MONTHLIGHT ALONE 0 15PHOTOFRIN II 4 11BPD 10 5antiEGFR 0 10antiCEA 0 10antiCEA-BPD 0 10antiEGFR-BPD 12 3Table IV: Results of photodynamic therapy intreatment and control groups.page^42V. DISCUSSION:The expression of EGFr in the hamster cheek pouch squamous cellcarcinoma model has been investigated recently by Shin et al whodemonstrated that normal cheek pouch mucosa did not expressidentifiable levels of EGFr while squamous cell carcinomas induced byDMBA expressed very high levels of EGFr [110]. This specificity fortumor and not normal mucosa makes an antiEGFr monoclonal antibodyan ideal delivery vehicle for photosensitizing agents in the hamster cheekpouch model.Immunohistochemical staining of the squamous cell carcinomas inour experiment confirmed overexpression of EGFr by SCC cells with noidentifiable staining of normal mucosa. In addition the stainingconfirmed the specificity of the particular antiEGFr monoclonal antibody,and nonspecificity of the antiCEA monoclonal antibody in use for theduration of the experiment.BPD biodistribution has been investigated by Richter et al, whofound that BPD, while accumulating in tumor shows no real specificityfor tumor tissue, with significant concentrations found in other normaltissue [82]. The results of our tissue assay confirm this finding, showingBPD concentrations of 7.8 ± 0.7 ttg/g of tumor vs. 5.0 ± 0.8 /Leg ofBPD in normal mucosa; a tumor /tissue ratio of 1.6:1. An idealphotosensitizing agent should localize to the tumor and not to thepage 43surrounding tissue, a property that in theory should be conferred uponBPD by its conjugation to a tumor specific monoclonal antibody such asantiEGFr. In practice this has in fact turned out to be the case withtumor concentrations of BPD remaining relatively high (6.8 ± 0.6 µg/g)and concentrations in normal tissue being reduced, (0.26 ± 0.09 µg/g)producing a tumor/tissue ratio of BPD of approximately 26:1. This tumorspecificity appears to be a function of the specificity of the antibodyrather than of the BPD binding to IgG as the BPD-antiCEA non specificantibody conjugate showed no increased affinity for the tumor. Thisagrees with findings of Mew et al for a hematoporphyrin - monoclonalantibody conjugate used in M1 tumor bearing mice [111].Photofrin II is the present standard for photosensitizing agents andhas been used in photodynamic therapy for a variety of cancers includingcolorectal, bladder, esophagus and head & neck tumors, however it hashad variable success [112,60,71,2]. Benzoporphyrin derivative offerstheoretical advantages over Photofrin II in that its peak absorption andmaximum activation is at a longer wavelength (691) nm which penetratesliving tissue better than does the corresponding wavelength for PhotofrinII (418nm) [79]. The wavelength (630nm) at which Photofrin II isactivated in clinical use produces much less than maximal activation ofthe porphyrin [19]. In our experiment we have evaluated the relativeeffectiveness of both BPD and Photofrin II in photodynamic therapy ofpage 44squamous cell carcinoma. BPD appears a more effective agent forphotodynamic therapy producing a 67% one month disease free rateversus a disease free rate of only 27% for Photofrin II. The rate ofcomplete response obtained for Photofrin II is similar to that reported inthe literature of between 10 and 40% [38,45]. There is to date little invivo work with the recently developed BPD although in one series,treatment of M-1 tumors induced in DBA/2J mice produced an 83% curerate although no histologic confirmation was obtained [78].Several reports of monoclonal antibody-photosensitizing agentconjugates have been made in the past [113-115]. A BPD-monoclonalantibody conjugate has recently been demonstrated to be effective in vitroin killing human squamous cell carcinomas [97]. Our preliminary resultsin treating squamous cell carcinoma in a hamster cheek pouch modelsuggest that the BPD-monoclonal antibody conjugates are effective incancer cell destruction in vivo as well.Patrice et al have suggested a clonal selection mechanism forfailure in photodynamic therapy [44]. Clonal selection would appear tobe even more likely when using a monoclonal antibody-BPD conjugateand may well explain the treatment failures seen in thephotoimmunoconjugate treated group.page^45VI. SUMMARY.Photodynamic therapy using Photofrin for malignant disease hasbeen used with disappointing success to date. Benzoporphyrin derivativeis modification of the chlorin structure designed specifically to enhanceits photoactive properties and improve the results of photodynamictherapy. We have shown that BPD is more effective at eradicatingsquamous cell cancer than is Photofrin in a hamster cheek pouch model.Unfortunately BPD itself has little tumor localizing properties, asshown by our assay, where tumor levels of BPD were only 1.6 times ashigh as in normal tissues. We have dramatically improved the tumorlocalizing ability of BPD by creating a monoclonal antibody-BPDconjugate directed at a tumor specific antigen, Epidermal Growth Factorreceptor. When twenty times less BPD was given as a tumor specificconjugate, tumor levels of BPD remained high while drastically reducinglevels of BPD found in normal tissues. The tumor localizing propertiesof the antiEGFr-BPD conjugate allowed us to achieve a tumor: tissueratio of 26:1.This tumor specific antiEGFr-BPD conjugate when used inphotodynamic therapy in the hamster cheek pouch squamous cell cancermodel gave us a cure rate of 80%. This was better than the 27% curerate achieved with Photofrin. As well, although given at one twentieththe dose of BPD, the antiEGFr-BPD conjugate gave a cure ratepage^46statistically similar to that of BPD when given alone (67%).VII. CONCLUSIONS.Benzoporphyrin derivative is a more effective photosensitizingagent for photodynamic therapy than is the current standard agentPhotofrin II. Tumor to normal tissue ratios of BPD can be increased byusing a tumor specific monoclonal antibody-BPD conjugate which, intheory, will decrease the side effects clinically encountered withphotodynamic therapy. 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Photochem Photobiol1987;46: 83-96.CCCHFigure la: Photofrin II or DHEFigure lb: Benzoporphyrin derivative monoacid AAbsorbence100806040200300 350 400 450 500 550 600 650 700 750 800Wavelength (nm)Figure 2: Absorbence spectrum of BPDpage 57Figure 3:Squamous cell carcinoma arising in the cheekpouch of a hamster after being exposed toDMBA. [Hematoxylin & Eosin X 100]page 58FITC= Mouse monoclonalIgG1 to marker(EGFr or CEA)Aantimouse IgGlabelled withFITC orperoxidaseFigure 4:Immunohistochemical method fordetecting recptor expression. A primarymouse anti-receptor antibody is appliedafter which a secondary labelled antibodydirected against mouse IgG is added.page 59Figure 5:EGFr expression by Hamster SCC.Lighted areas represent binding offlurescent antimouse IgG to primarymouse anti-EGFr as shown in Figure 4.FLUORESCENCE0q"0Eloo806040200560 580 600 620 640 660 680 690 695 700 705 710 720 730WAVELENGTHFigure 6:Fluorescence peaks obtained using tumor samples containing knownamounts of BPD were compared to tumor samples of animals given BPDat therapeutic dosage.page 611201008060402000 1 2 3 4 5 6 7 8 9 10 11 12pg BPD/ g of tissueFigure 7:Standard curve generated for the BPD assay. A linearregression model gave the "best fit" with a correlationcoefficient of 0.97 for this regression line.Fluorescence140page 62BPD^MoAb-BPDFigure 8:BPD levels are higher in tumor than in normalmucosa when given alone. (p = 0.046) When thetumor specific BPD-MoAb conjugate is giventumor levels of BPD remain high however levelsof BPD in normal tissues are markedlyreduced. (p =0.0016)page 63Figure 9a:Squamous cell carcinoma prior to treatmentwith photodynamic therapy.Figure 9b:The same tumor as in Fig.9a one week postphotodynamic therapy. Notice the markedtumor necrosis.page 65Figure 9c :The same tumor as in Fig. 9a and 9b onemonth after photodynamic therapy.Histologic examination of the area showsno evidence of residual tumor.page 661 Month Cancer Free %Photofrin II BPD MoAb-BPDFigure 10Animals treated with BPD or the tumorspecific BPD-EGFr conjugate showedsignificantly better cancer free survival thandid those treated with Photofrin II.


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