Lub-de Hooge, Marjolijn Nicolette

English

A new generation of innovative receptor targeted drugs rapidly enters the market. However, since these drugs target a specific receptor, they are not of value for all cancer patients, but only for specific tumor types or a subset within a tumor type. This makes it increasingly important to select the patients that might benefit from the targeted therapy and offers a potential role for receptor imaging wih radiolabeled variants of these drugs. ... Zie: Summar

Dissertations of the University of Groningen

   University of GroningenNew receptor targeted drugsLub-de Hooge, Marjolijn NicoletteIMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:2006Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Lub-de Hooge, M. N. (2006). New receptor targeted drugs: pharmaceutical aspects and molecular imaging.s.n.CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 04-06-2022CHAPTER 9SUMMARYA new generation of innovative receptor targeted drugs rapidly enters the market.However, since these drugs target a specific receptor, they are not of value for allcancer patients, but only for specific tumor types or a subset within a tumor type. Thismakes it increasingly important to select the patients that might benefit from thetargeted therapy and offers a potential role for receptor imaging with radiolabeledvariants of these drugs.This thesis aimed to study receptor targeted drugs and focused on recombinanthuman TNF-re lated apoptosis  inducing l igand ( rhTRAlL)  and t rastuzumab and thei rradiolabeled counterparts, intended to target and visualize the death recePtor and thehuman epidermal growth factor receptor 2 (HER2/erb82) respectively.TRAIL is a member of a subfamily within the TNF-superfamily of cytokines, also calledthe death receptor l igands or death factors, that are able to induce apoptosis uponbinding to thei r  death receptors.  Trastuzumab is  a humanized lgGl  monoclonalantibody directed against HER2. HER2 is a receptorfor regulation of cell survival. TheHER2 receptor is overexpressed in a wide variety of human cancers, including 25-30%of primary breast cancers. Trastuzumab, by blocking the growth signal, and rhTRAlL,by directly init iating apoptosis, are both in different ways able to change the apoptosisbalance in tumor cells after binding to their cognate cell surface receptor.In chapter 2 an overview is presented concerning regulation of cell death and cellsurvival by death receptor l igand and HER signaling pathways. Attention is paid torhTRAlL and trastuzumab and to novel opportunities for receptor imaging. Dataregarding death receptor imaging and imaging of HER-receptor family have beenreviewed and c lear ly  i l lust rate that  molecular  imaging techniques can be used instaging and restaging, evaluation and prediction of treatment response andcharacterization of lesions, as they permit total body imaging in a non-invasive way.With the development of these new generation of targeted agents, molecular imagingmay become an important part of future clinical studies and clinical practice,support ing indiv idual ized cancer therapy.The first part of the thesis (chapters 3-6) is dedicated to TRAIL. To learn more aboutthe physiological role of TRAIL we have studied soluble (s)TRAIL concentrations intwo non cancer settings namely, sepsis and systemic lupus erythematosis (SLE).SUMMARYIntensive research has brought us an enormous body of knowledge about sepsis andthe mechanisms behind it. The involvement of the immune and the coagulationsystem has received much attention. Currently, dysregulation of the immune systemis considered to be the most important factor that determines outcome. The role ofapoptosis in the regulation of the immune response is well recognized, but its role inthe dysfunction of other organs and in the mortality due to sepsis is less clear. TNFcr isone of the bridges between the inflammatory reaction and apoptosis. The role thatthe other death l igands Fas l igand (FasL) and TRAIL play, is far less known. There arehowever increasing indications that TRAIL is also involved in inflammatory processes.These data raised the question whether sTRAIL is involved in sepsis.A human endotoxemia model is available in which some aspects of sepsis can bestudied. In chapter 3, the effect of endotoxin administration (4 ng/kg body weight(10,000 endotoxin U/pg)  as a 1-min in fus ion)  on the response of  sTRAlL,  and the ro leof  p38 MAP k inase (K)  inh ib i t ion was studied in  21 human volunteers.  p38 MAPKinhibitors are considered potential drugs in inflammatory diseases. Thirty minutesbefore the endotoxin infusion, the volunteers received a single oral dose of placebo orthe selective p38 MAPK inhibitor drug, RWJ-67657. We tested the RWJ-67657 doselevels 1400 mg (n = 4) ,700 mg (n = 6) ,350 mg (n = 5) ,  and 0 mg (n = 6) .  B lood forsTRAIL analys is  was drawn predose,  and 1,  1 .5,2,2.5,3.5,4.5,  6.5,8,  and 24 hours af terendotoxin infusion. Plasma levels of sTRAIL were determined with a validated solid-phase sandwich enzyme-linked immunosorbent assay (ELISA). At t = 0 before theendotoxin infusion, the median sTRAIL level in the placebo group was 681 t 86.8pg/ml  (mean t  SEM, n = 6) .  This  level  can be considered as the normal  va lue in  youngrestingmales in the morning. Plasma sTRAIL increased 10-fold to6564 t 5'11 pg/mLafter 2.5 hours. This increase was blocked completely by the highest dose of RW-67657 (1400 mg). The sTRAIL levels were already lowering ' l hour later (p=0.003,Student's f test) and were normalized 6.5 hours after endotoxin infusion (p=0.98).After a dose of 350 mg or 750 mg RWJ-67657, sTRAIL levels 24 hours after endotoxininfusion were elevated. The peak levels (t = 3.5 h) were 2729.2 t 328.0 pg/ml and3155.4 t 645.3 pg/mL respectively. However no dose response relation was found.In conclusion, sTRAIL is responsive to endotoxemia. However, the precise role insepsis remains to be elucidated. lf sTRAIL turns out to participate in the pathogenesisof sepsis, there is already a tool available, namely p38 MAPK inhibit ion, to inhibit thesTRAIL resDonse.1 5 9CHAPTER 9.Increased apoptosis  may induce auto immune condi t ions.  SLE is  an auto immunedisease with a wide spectrum of clinical and immunological abnormalit ies. Thepresence of autoantibodies, especially those directed to double stranded DNA' ischaracteristic for the disease. SLE may affect different organ systems, including theskin, joints, central and peripheral nervous system, kidneys, and liver' The etiology ofSLE remains unknown. There is however increasing evidence that the Presence andaccumulation of apoptotic cells play a role in autoimmunity. Apoptosis is induced bybinding of death recePtor l igands to their cognate receptors' The Fas-FasL pathwayhas been studied extensively in relation to SLE. However, other death pathways arealso considered important and we hypothesized that secreted sTRAIL might beimportant in the induction of apoptosis in SLE'The aim of chapter 4 was to assess TRAIL concentrations in sera of SLE patients.Forty SLE patients were studied (20 with active disease and 20 with inactive disease).Serum sTRAIL levels were measured by a validated ELISA. Serum sTRAIL levels in SLEpatients were comPared with those in patients with rheumatoid arthrit is (n=20),Wegener's granulomatosis (n=20) and healthy controls (n=20). Mean serum sTRAILconcentration (t SEM) in SLE patients (936.0 t 108.2 pgimL) was higher than inheal thy contro ls  (509.4 t  33.8 pg/mL,  p<0.01)and in d isease contro l  pat ients wi thrheumatoid arthrit is (443.5 t 36.' l  pg/mL, p<0.001) or wegener's granulomatosis(357.1 x 32.2 pglmL, p<0.001). The mean serum sTRAIL concentration was 1010.2 t168.0 pg/mL for patients with inactive disease and 861.8 t 138.7 Pg/mL for patientswith active disease. Soluble TRAIL values were not correlated with specificmani festat ions of the d isease,  such as leucopenia or  lymphopenia,  orwi th SLE diseaseactivity index.In conclusion, this study reports that serum sTRAIL concentrations are increased inSLE patients. This seems to be disease specific and could indicate a role for TRAIL inSLE pathophysiology.Of the death receptor l igands, especially TRAIL might be an interesting candidate tofacil i tate the apoptotic process in combination with chemotherapeutic drugs, as it caninduce apoptosis in transformed cell l ines, but not in normal cells, by bindingto its cellmembrane receptors. A non-commercial production of rhTRAlL was set up to allow acl in ica l  phase I s tudy.SUMMARYIn chapter 5 we described an approach for the production of clinical grade rhTRAlLmeeting requirements of Good Manufacturing Practice and regulatory guidelines ofthe European Agency for the Evaluation of Medicinal Products assuring quality andsafety of the product. RhTRAIL was produced by recombinant DNA techniques inEscherichia coli BL21-S| cells. Competent BL21-sl cells were transformed withpET15b-TRAlL114-281plasmid, that harbors the gene encoding soluble human TRAIL,corresponding to the extracel lu lar  par t  of  the TRAIL molecule ( i .e .  amino acids 114_281)' Soluble rhTRAlL was recovered from host BL21-Sl cell lysates by sonication andwas subsequently purif ied by sp-sepharose Fast Frow cation exchangechromatography, nickel-nitri lotriacetic acid affinity chromatography and dialysis.Three consecutive production batches were run. An extensive range of in-process andcharacterization tests was performed to show batch-to-batch consistency with regardto identity, purity and biorogicar activity. The recombinant protein was thenformulated in a TRIS buffer containing zinc, glycerol, Nacl, and human serum arbuminand stored in polypropylene vials at -g0 "C.Because of the shown anti-tumor activity in tumor xenograft moders and the rack ofsystemic toxicity in mice, cynomolgus monkeys and chimpanzees, rhrRArL is nowunder investigation as a therapeutic agent. The physiological role of TRA|L and thepharmacokinetic behavior of rhrRAlL are complex and sti l l  not fully understood.Availabil ity of radiolabeled rhTRAlL would offer the possibil i ty of molecular imaging inhumans to study biodistribution and whole body pharmacokinetics. Furthermoreimaging enables to i l lustrate whether the drug reaches the target.In chapter 6 radioiodinated rhTRAlL was developed to study in_vitro and in_vivodeath receptor targeting and pharmacological behavior in a human tumor bearingmice model. In order to deverop a tracer suitabre for imaging in patients, muchattention was paid to optimization and validation of the labeling process, to stabil itytesting and to show maintenance of receptor binding properties.RhTRAIL radioiodination was optimized using chloramine T as oxidizing agent.Radiochemical purity and stabil ity of 12sl-rhTRAlL in TRls-buffer and human serumwere determined by size exclusion chromatography HpLC. lmmunoreactivity andreceptor binding properties were examined using different TRAIL receptor positivetumor cel l  l ines.CHAPTER 912sl-rhTRAlL biodistribution was assessed in a biodistribution study using athymicmice bearing human SKBR3 or SW948 xenografts with different TRAIL receptorexpression levels.12sl-rhTRAlL (labeling yield 70.3 x 1.1 %, radiochemical purity > 98%, specific activity0.75 t 0.05 MBq/Ug) was stable in TR|S-buffer and human serum for 24 h althoughfree iodine and h igh molecular  weight  compounds gradual ly  developed (16 and 19 %respectively after 72 h). The immunoreactive fraction, determined using the Colo320tumor cell l ine, was 0.80 t 0.08 (n=4). A concentration of 9.3x10-8 M rhTRAlL wasneeded to reduce t2sl-TRAIL binding with 50%. Due to competit ion with soluble DR5(5 ptg) binding of 12sl-rhTRAlL (5 ng) to various cell l ines was considerably reducedwith 56% in SW948 and up to 74% in Colo320. t2sl-rhTRAll biodistribution ischaracterized by fast renal elimination. Uptake rapidly decreased in all normal t issues.SW948 tumor uptake however, although only 1.1% of the injected doseig, increasedover time.In conclusion, rhTRAlL can be efficiently radioiodinated. Radioiodinated rhTRAlL bindsto the TRAIL receptors in-vitro and in-vivo and can be used in humans for deathreceptor imaging.The second part of this thesis (chapters 7 and 8) is dedicated to the anti-HER2monoclonal antibody trastuzumab. Trastuzumab is an exciting new targeted drug withan unique mechanism of action, that is currently approved for the treatment ofpatients with HER2 overexpressing metastatic breast cancer. In addition to its use inthe metastatic breast cancer setting, impressive results of large randomizedmulticenter trials, designed to evaluate the use of adjuvant rastuzumab, were recentlyreported. The studies showed highly significant reductions in the risk of recurrence,prolongation of disease free and overall survival in the adjuvant reatment of patientswith HER2 oositive breast cancer.Unfortunately, even when strong HER2 overexpression (3+) is present, part of thepatients do not respond to trastuzumab therapy. Moreover, during trastuzumabtreatment patients can develop severe cardiac dysfunction. The mechanism of thetrastuzumab-induced cardiac toxicity is sti l l  unclear, but might be related to specificbinding to HER2 receptors expressed in the myocardium. A reliable test for theprediction of tumor response and the risk of developing cardiac failure is thereforeurgently needed. This was the main motivation for the development of radiolabeledtrastuzumab that is described in chapter 7.162SUMMARYThe aim of  th is  s tudy was to develop c l in ica l  gradel l l lnd ium 1111lny radio labeledtrastuzumab, to evaluate the stabil ity and immunoreactivity of the tracer and toperform a biodistribution study in human tumor-bearing mice. Trastuzumab wasradio labeled wi th 1111n using cyc l ic  anhydr ide d iethy lenetr iamine pentaacet ic  ac id(cDTPA) as a chelator . t t l ln-DTPA-trastuzumab ( label ing y ie ld 92.3 x 2.3%,radiochemical purity 97.0 + 1.5%) is stable in phosphate buffered saline when stored at4"C for more than 14 days. The immunoreactive fraction determined by cell-bindingassays,  us ing the HER2-overexpressing human ovar ian SKOV3 tumor cel l  l ine,  was0.87 t 0.06. Eiodistribution and tumor targeting were studied in HER2 receptor-positive and HER2-negative tumor-bearing athymic mice. The HER2-positive tumorshowed (9.77 x 1.14% iniected dose per gram) substantial uptake of the labeledantibody already after 5 hours. The difference in uptake between HER2-positive versusHER2-negative tumors was even more pronounced 3 days after injection (16.30 t0.64% iniected dose per gram), and was visualized by radioimmunoscintigraphy. Liver,spleen, and kidney showed marked tracer uptake.In summary,  we showed that  t rastuzumab can be ef f ic ient ly  radio labeled wi th111lnresul t ing in  h igh label ing y ie lds and h igh stabi l i ty .  t " ln-DTPA-trastuzumab select ive lybinds to the human HER2 receptor both in-vitro and in-vivo in animals. Therefore,1 1 1 ln-DTPA-trastuzumab a ppea red suita ble for clinical use.Chapter  8 descr ibed the c l in ica l  s tudy that  a imed to evaluate whether l l l ln- labeledtrastuzumab scintigraphy can predict cardiotoxicity and identifo tumor lesions. Inaddition, we evaluated whether plasma markers for cardiac dysfunction can be used topredict ca rdiotoxicity.Patients with HER2-positive metastatic breast cancer underwent gammacameraimaging f rom 15 minutes to 7 days af ter  in ject ion of  150 MBqt" ln-DTPA-trastuzumab, prior to and after 12 weekly trastuzumab doses and concomitant 3-weekly paclitaxel. Cardiac assessments were performed before treatment, after 4 and6 cycles. Plasma N-terminal proB-type natriuretic peptide (NT-proBNP) and serumtroponin |  (Tnl )  were measured wi th immunoassay.Fifteen of the 17 patients were available for cardiac and tumor uptake analysis. Pre-treatment myocardial l l t ln-DTPA-trastuzumab uptake was observed in one patientwith pre-existent cardiac arrhythmias, who did not develop heart failure duringtreatment. Severe cardiotoxicity occurred in three patients, without init ial myocardial1111n-DTPA-trastuzumab uptake, while one showed weak myocardial uptake after 4't63CHAPTER 9.cycles. The detection rate of single tumor lesions was 45%. New tumor lesions werediscovered in 13 115 patients. Pre-treatment plasma NT-proBNP levels were higher inpatients with, than without heart failure (mean 534 t SD 236 vs. 105 + 79 nglL'p=0.009).In  conclus ion,  radio labeled t rastuzumab sc int igraphy was not  va luable in  predict ingtrastuzumab-related cardiotoxicity in metastatic breast cancer patients, but canidentify HER2-positive tumors. Measurement of plasma NT-proBNP is promisingregarding prediction of trastuzumab-related cardiotoxicity.

New receptor targeted drugs:pharmaceutical aspects and molecular imaging

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New receptor targeted drugs:pharmaceutical aspects and molecular imaging

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