The targeting of gastrin-releasing peptide receptors (GRPR) was recently proposed for targeted therapy, e.g., radiotherapy. Multiple and frequent injections of peptide-based therapeutic agents would be required due to rapid blood clearance. By conjugation of the GRPR antagonist RM26 (D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2) to an ABD (albumin-binding domain), we aimed to extend the blood circulation of peptides. The synthesized conjugate DOTA-ABD-RM26 was labelled with indium-111 and evaluated in vitro and in vivo. The labelled conjugate was stable in PBS and retained specificity and its antagonistic function against GRPR. The half-maximal inhibitory concentration (IC50) of In-nat-DOTA-ABD-RM26 in the presence of human serum albumin was 49 +/- 5 nM. [In-111]In-DOTA-ABD-RM26 had a significantly longer residence time in blood and in tumors (without a significant decrease of up to 144 h pi) than the parental RM26 peptide. We conclude that the ABD-RM26 conjugate can be used for GRPR-targeted therapy and delivery of cytotoxic drugs. However, the undesirable elevated activity uptake in kidneys abolishes its use for radionuclide therapy. This proof-of-principle study justified further optimization of the molecular design of the ABD-RM26 conjugate.

Pretargeting is a strategy to improve the tumor-to-healthy tissue contrast in targeted nuclear imaging and therapy. The strategy relies on separating the tumor-targeting agent from the radioactive payload and combine the two in vivo. In the pretargeting approach previously studied by our group, the tumor targeting was mediated by an Affibody functionalized with a peptide nucleic acid (PNA) probe and the radionuclide was carried by a complementary PNA probe. Affibody-mediated PNA-based pretargeting was shown to increase the tumor-to-kidney ratio when evaluated in HER2-overexpressing tumor-bearing mice. The aim of the current study is to further optimize the design of the PNA probes to achieve better biodistribution properties and preconditions for a more cost-efficient production. The first important feature of the PNA pretargeting system is the tumor-to-kidney ratio, where the kidney retention is the dose-limiting factor for a clinical therapeutic application. The second aspect is the production of PNA, where the synthesis of PNA strands can be a challenge due to the steric repulsion between two PNA residues’ side chain and poor solubility in the synthesis solvent. In order to simplify the synthesis, we optimized the automation of the process using a microwave-assisted peptide synthesizer. Once the automated synthesis protocols were set up, we designed and synthesized a panel of new PNA probes, aimed at reducing the length of the PNA strands. The reduction in length was expected to simplify the synthesis workflow, but also to possibly decrease the kidney retention of the radioactive payload, as was shown in a previous study when reducing the length of the secondary PNA strand could improve the tumor-to-kidney ratio. The PNA duplexes were studied by CD and UV spectroscopy, and the binding kinetics of the interaction were studied by SPR to identify the limit in terms of number of base pairs needed to reach the high affinity expected to be required for an efficient pretargeting system. Our results showed that high affinity duplexes are formed between PNA probes having only 8 to 9 complementary bases, but that PNA probes with 6 or 7 complementary bases give rise to less stable duplexes having lower melting temperatures and faster dissociation rates.

Treatment of patients with human epidermal growth factor receptor 2 (HER2)-expressing tumors using the monoclonal antibody trastuzumab increases survival. The Affibody-based peptide nucleic acid (PNA)-mediated pretargeted radionuclide therapy has demonstrated efficacy against HER2-expressing xenografts in mice. Structural studies suggest that Affibody molecules and trastuzumab bind to different epitopes on HER2. The aim of this study was to test the hypothesis that a combination of PNA-mediated pretargeted radionuclide therapy and trastuzumab treatment of HER2-expressing xenografts can extend survival compared with monotherapies. Methods: Mutual interference of the primary pretargeting probe Z(HER2:342)-SR-HP1 and trastuzumab in binding to HER2-expressing cell lines was investigated in vitro. Experimental therapy evaluated the survival of mice bearing HER2-expressing SKOV-3 xenografts after treatment with vehicle, trastuzumab only, pretargeting using Affibody-PNA chimera Z(HER2:342)-SR-HP1 and complementary probe Lu-177-HP2, and combination of trastuzumab and pretargeting. The ethical permit limited the study to 90 d. The animals'weightsweremonitored during the study. After study termination, samples of liver and kidneys were evaluated by a veterinary pathologist for toxicity signs. Results: The presence of a large molar excess of trastuzumab had no influence on the affinity of Z(HER2:342)-SR-HP1 binding to HER2-expressing cells in vitro. The affinity of trastuzumab was not affected by a large excess of Z(HER2:342)-SR-HP1. Themedian survival of mice treated with trastuzumab (75.5 d) was significantly longer than the survival of mice treated with a vehicle (59.5 d). Median survival of mice treated with pretargeting was not reached by day 90. Six mice of 10 in this group survived, and 2 had complete remission. All mice in the combination treatment group survived, and tumors in 7 mice had disappeared at study termination. There was no significant difference between animal weights in the different treatment groups. No significant pathologic alterations were detected in livers and kidneys of treated animals. Conclusion: Treatment of mice bearing HER2-expressing xenografts with the combination of trastuzumab and Affibody-mediated PNA-based radionuclide pretargeting significantly increased survival compared with monotherapies. Cotreatment was not toxic for normal tissues.

Riktad strålbehandling av cancer är ämnad att selektivt leverera cytotoxiska radionuklider till tumörceller. Affinitetsproteiner av olika slag utforskas för detta ändamål, och olika utmaningar uppstår beroende på vilket affinitetsprotein som används. Fullängdsantikroppar har lång halveringstid i blod, vilket leder till hög systemisk toxicitet, medan mindre affinitetsligander, såsom antikroppsfragment eller peptider, vanligtvis uppvisar högt radioaktivt upptag i njurarna. Vid användande av små affinitetsligander för riktad strålbehandling finns dessutom problem med låg terapeutisk effekt då de försvinner snabbt ur cirkulationen. För att uppnå en klinisk terapeutisk effekt med dessa små radioinmärkta affinitetsligander krävs ofta upprepade och frekventa administreringar.

Problemen med högt njurupptag och låg terapeutisk effektivitet för radioinmärkta små affinitetsproteiner är något som adresseras i denna avhandling. Små affinitetsproteiner (en Affibody-molekyl, ett en-domäns-antikroppsfragment och en peptid) har studerats med avsikten att använda dem för riktad strålbehandling mot de cancerassocierade proteinerna HER2, CD38 och GRPR. Affibody-molekylen och en-domän-antikroppsfragmentet användes i pre-targeting, där selektiv hybridisering mellan två peptidnukleinsyre (PNA)-prober användes som igenkänningsmärken på den tumörsökande primära molekylen och den radioinmärkta sekundära molekylen. I artikel I och II utvärderades uppsättningar av PNA-hybridiseringsprober, in vitro och in vivo. I artikel I visar vi att den kortaste testade sekundära PNA-proben (9-meren HP16) gav den bästa biodistributionsprofilen med en kombination av högt tumörupptag och det lägsta njurupptaget. I artikel II producerade vi dels en uppsättning kortare primära PNA-prober, med avsikten att förenkla dess produktion, samt nya uppsättningar av ännu kortare sekundära PNA-prober. En sekundär 8-mer identifierades som lämplig att testa i cellförsök och in vivo tillsammans med HER2-bindande Affibody-PNA-konjugat med varierande längd av den primära PNA-proben, för att avgöra om detta skulle förbättra biodistributionen ytterligare. I artikel III utvärderades den Affibody-medierade PNA-pretargeting-strategin som enskild terapi och som en kombinerad behandling tillsammans med trastuzumab, för att behandla möss med HER2-positiva tumörer. Möss som behandlats med kombinationsbehandlingen hade signifikant längre överlevnad jämfört med andra grupper. I artikel IV utvärderades möjligheten att använda PNA-pretargeting-strategin i kombination med ett annat affinitetsprotein (en-domän-antikroppsfragment) på en CD38-uttryckande cellinje. I artikel V konjugerades den GRPR-bindande peptiden RM26 till ett albuminbindande protein, med målet att uppnå ett högt tumörupptag över tid. RM26-ABD-konjugatet visade bra tumörupptag över tid, men också högt njurupptag, vilket i nuläget begränsar dess användning i en terapeutisk tillämpning.

Sammanfattningsvis visar arbetet som presenteras i denna avhandling strategier för selektiv strålningsterapi av tumörer med användning av affinitetsproteiner och PNA-medierad pretargeting.

Simple Summary Affibody molecules are small, engineered affinity proteins based on a nonimmunoglobulin scaffold. Affibody-based radionuclide imaging probes have demonstrated excellent tumor targeting. However, the renal clearance of affibody molecules is accompanied by high reabsorption and retention of activity in the kidney, which prevents their use for radionuclide therapy. We have previously shown the feasibility of overcoming the high renal uptake using a pretargeting approach for affibody-mediated therapy based on peptide nucleic acid (PNA) hybridization. In this study, we test the hypothesis that shortening the PNA pretargeting probes would further increase the difference between the accumulation of radiometals in tumor xenografts and in kidneys. A series of novel PNA probes has been designed and evaluated in vitro and in vivo. We have found that a variant containing 9 nucleobases enables a two-fold increase of the tumor-to-kidney dose ratio compared with a variant containing 15 nucleobases. This creates preconditions for more efficient therapy of cancer. Affibody-mediated PNA-based pretargeting is a promising approach to radionuclide therapy of HER2-expressing tumors. In this study, we test the hypothesis that shortening the PNA pretargeting probes would increase the tumor-to-kidney dose ratio. The primary probe Z(HER2:342)-SR-HP15 and the complementary secondary probes HP16, HP17, and HP18, containing 9, 12, and 15 nucleobases, respectively, and carrying a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelator were designed, synthesized, characterized in vitro, and labeled with Lu-177. In vitro pretargeting was studied in HER2-expressing SKOV3 and BT474 cell lines. The biodistribution of these novel probes was evaluated in immunodeficient mice bearing SKOV3 xenografts and compared to the previously studied [Lu-177]Lu-HP2. Characterization confirmed the formation of high-affinity duplexes between HP15 and the secondary probes, with the affinity correlating with the length of the complementary PNA sequences. All the PNA-based probes were bound specifically to HER2-expressing cells in vitro. In vivo studies demonstrated HER2-specific uptake of all Lu-177-labeled probes in xenografts in a pretargeting setting. The ratio of cumulated radioactivity in the tumor to the radioactivity in kidneys was dependent on the secondary probe's size and decreased with an increased number of nucleobases. The shortest PNA probe, [Lu-177]Lu-HP16, showed the highest tumor-to-kidney ratio. [Lu-177]Lu-HP16 is the most promising secondary probe for affibody-mediated tumor pretargeting.

Natural backbone-cyclized proteins have an increased thermostability and resistance towards proteases, characteristics that have sparked interest in head-to-tail cyclization as a method to stability-enhance proteins used in diagnostics and therapeutic applications, for example. In this proof-of principle study, we have produced and investigated a head-to-tail cyclized and HER2-specific Z(HER2:342) Affibody dimer. The sortase A-mediated cyclization reaction is highly efficient (>95%) under optimized conditions, and renders a cyclic Z(HER3:342)-dimer with an apparent melting temperature, T-m, of 68 degrees C, which is 3 degrees C higher than that of its linear counterpart. Circular dichroism spectra of the linear and cyclic dimers looked very similar in the far-UV range, both before and after thermal unfolding to 90 degrees C, which suggests that cyclization does not negatively impact the helicity or folding of the cyclic protein. The cyclic dimer had an apparent sub-nanomolar affinity (K-d similar to 750 pM) to the HER2-receptor, which is a similar to 150-fold reduction in affinity relative to the linear dimer (K-d similar to 5 pM), but the anti-HER2 Affibody dimer remained a high-affinity binder even after cyclization. No apparent difference in proteolytic stability was detected in an endopeptidase degradation assay for the cyclic and linear dimers. In contrast, in an exopeptidase degradation assay, the linear dimer was shown to be completely degraded after 5 min, while the cyclic dimer showed no detectable degradation even after 60 min. We further demonstrate that a site-specifically DyLight 594-labeled cyclic dimer shows specific binding to HER2-overexpressing cells. Taken together, the results presented here demonstrate that head-to-tail cyclization can be an effective strategy to increase the stability of an Affibody dimer.