Theranostic Targeting of GRPR and PSMA in Prostate Cancer

This thesis is based on five original articles that investigated the theranostics of prostate cancer by gastrin-releasing peptide receptor (GRPR) and prostate-specific membrane antigen (PSMA) targeting. GRPR and PSMA are two extensively evaluated prostate cancer cell markers due to their overexpression in the majority of prostate cancer samples. Theranostic targeting of GRPR and PSMA is an attractive strategy to improve the management of prostate cancer patients. Papers I and II focused on the dual targeting of GRPR and PSMA. The effect of linker modification on the affinity for GRPR and PSMA and the pharmacokinetic profile was evaluated. In Paper III, the effect of the GRPR antagonist RM26 conjugation to an albumin-binding domain on the pharmacokinetic profile and its potential use in therapy was investigated. Paper IV focused on developing a GRPR antagonist that was suitable for single-photon emission computed tomography (SPECT) using technetium-99m. In Paper V, the GRPR antagonist developed in Paper IV was translated into a phase I clinical trial to assess safety and dosimetry. Modifying the linkers in GRPR and PSMA heterodimers can largely impact the affinity for both targets. This modification influenced the in vivo targeting specificity and biodistribution, with [125I]I-BO530 in Paper I and [111In]In-BQ7812 in Paper II outperforming other analogues. Our findings in Paper III indicated that the conjugation of an albumin-binding domain to RM26 increased the blood concentration of the radiotracer. This increase led to elevated and stable tumour uptake of [111In]In-DOTA-ABD-RM26 after several days of injection. However, [111In]In-DOTA-ABD-RM26 was also increasingly taken up by various healthy organs. The GRPR antagonist [99mTc]Tc-maSSS-PEG2-RM26, studied in Paper IV, showed high specificity and affinity for GRPR. This resulted in elevated GRPR-mediated uptake. Additionally, maSSS-PEG2-RM26 could be radiolabelled via a straightforward radiolabelling protocol. Clinical evaluation of [99mTc]Tc-maSSS-PEG2-RM26 in prostate and breast cancer patients (Paper V) demonstrated the safety and tolerability of the radiotracer, with favourable dosimetry and no side effects. In conclusion, this thesis evaluated different tools for the theranostic targeting of GRPR and PSMA. The findings warrant further investigation to optimise the reported radiotracers

Theranostic Targeting of GRPR and PSMA in Prostate Cancer

This thesis is based on five original articles that investigated the theranostics of prostate cancer by gastrin-releasing peptide receptor (GRPR) and prostate-specific membrane antigen (PSMA) targeting. GRPR and PSMA are two extensively evaluated prostate cancer cell markers due to their overexpression in the majority of prostate cancer samples. Theranostic targeting of GRPR and PSMA is an attractive strategy to improve the management of prostate cancer patients. Papers I and II focused on the dual targeting of GRPR and PSMA. The effect of linker modification on the affinity for GRPR and PSMA and the pharmacokinetic profile was evaluated. In Paper III, the effect of the GRPR antagonist RM26 conjugation to an albumin-binding domain on the pharmacokinetic profile and its potential use in therapy was investigated. Paper IV focused on developing a GRPR antagonist that was suitable for single-photon emission computed tomography (SPECT) using technetium-99m. In Paper V, the GRPR antagonist developed in Paper IV was translated into a phase I clinical trial to assess safety and dosimetry. Modifying the linkers in GRPR and PSMA heterodimers can largely impact the affinity for both targets. This modification influenced the in vivo targeting specificity and biodistribution, with [125I]I-BO530 in Paper I and [111In]In-BQ7812 in Paper II outperforming other analogues. Our findings in Paper III indicated that the conjugation of an albumin-binding domain to RM26 increased the blood concentration of the radiotracer. This increase led to elevated and stable tumour uptake of [111In]In-DOTA-ABD-RM26 after several days of injection. However, [111In]In-DOTA-ABD-RM26 was also increasingly taken up by various healthy organs. The GRPR antagonist [99mTc]Tc-maSSS-PEG2-RM26, studied in Paper IV, showed high specificity and affinity for GRPR. This resulted in elevated GRPR-mediated uptake. Additionally, maSSS-PEG2-RM26 could be radiolabelled via a straightforward radiolabelling protocol. Clinical evaluation of [99mTc]Tc-maSSS-PEG2-RM26 in prostate and breast cancer patients (Paper V) demonstrated the safety and tolerability of the radiotracer, with favourable dosimetry and no side effects. In conclusion, this thesis evaluated different tools for the theranostic targeting of GRPR and PSMA. The findings warrant further investigation to optimise the reported radiotracers

Co-injection of anti-HER2 antibody Trastuzumab does not increase efficacy of [177Lu]Lu-PSMA-617 therapy in an animal model of prostate cancer

One novel option for treating metastatic castration resistant prostate cancer is radionuclide therapy targeting prostate-specific membrane antigen (PSMA), e.g. [177Lu]Lu-PSMA-617. Overexpression of HER2 has been found in 80% of metastatic cases of prostate cancer. Previous research showed that HER2 is elevated post irradiation in PC-3 prostate cancer cells. Co-treating with anti-HER2 antibody Trastuzumab gave less proliferation of irradiated tumor cells in vitro, and when using radionuclide therapy, also in vivo. The aim of this study is to determine whether the same holds true in PSMA-expressing PC-3 PIP cells using [177Lu]Lu-PSMA-617 radionuclide therapy. PC-3 PIP and 22Rv1 prostate cancer cells were tested in vitro, treated with 6 Gy of x-rays with or without Trastuzumab incubation. We measured uptake of HER2-targeting affibody [68Ga]Ga-ABY-025 and cell survival, e.g. using the WST-1 assay. Three groups (n=10 each) of male nude Balb/c mice were inoculated with PC-3 PIP xenograft tumors and treated with just [177Lu]Lu-PSMA-617 (20 MBq), [177Lu]Lu-PSMA-617 (20 MBq) and Trastuzumab (4 × 5 mg/kg), or left untreated. Tumor sizes and animal survival was observed. In vitro, x-ray irradiation did reduce survival in 22Rv1 but not PC-3 PIP cells, and there was no significant effect of Trastuzumab treatment. Cells expressed HER2 but not significantly elevated post irradiation. In vivo, mice co-treated with Trastuzumab had significantly longer survival than untreated mice, but not than only [177Lu]Lu-PSMA-617. Staining of tumor sections showed similar HER2 and PSMA expression across groups. In conclusion, these results give no support for any benefit from co-treatment with anti-HER2 antibody for PSMA-targeted radioligand therapy

Increasing thermal stability and improving biodistribution of VEGFR2-binding affibody molecules by a combination of in silico and directed evolution approaches

The family of vascular endothelial growth factor (VEGF) ligands and their interactions with VEGF receptors (VEGFRs) play important roles in both pathological and physiological angiogenesis. Hence, agonistic and antagonistic ligands targeting this signaling pathway have potential for both studies on fundamental biology and for development of therapies and diagnostics. Here, we engineer VEGFR2-binding affibody molecules for increased thermostability, refolding and improved biodistribution. We designed libraries based on the original monomeric binders with the intention of reducing hydrophobicity, while retaining high affinity for VEGFR2. Libraries were displayed on bacteria and binders were isolated by fluorescence-activated cell sorting (FACS). In parallel, we used an automated sequence- and structure-based in silico algorithm to identify potentially stabilizing mutations. Monomeric variants isolated from the screening and the in silico approach, respectively, were characterized by circular dichroism spectroscopy and biosensor assays. The most promising mutations were combined into new monomeric constructs which were finally fused into a dimeric construct, resulting in a 15 degrees C increase in melting temperature, complete refolding capability after heat-induced denaturation, retained low picomolar affinity and improved biodistribution profile in an in vivo mouse model. These VEGFR2-binding affibody molecules show promise as candidates for further in vivo studies to assess their suitability as molecular imaging and therapeutic agents

Two Novel [68Ga]Ga-Labeled Radiotracers Based on Metabolically Stable [Sar11]RM26 Antagonistic Peptide for Diagnostic Positron Emission Tomography Imaging of GRPR-Positive Prostate Cancer

Gastrin releasing peptide receptor (GRPR) is overexpressed in prostate cancer (PC-3) and can be used for diagnostic purposes. We herein present the design and preclinical evaluation of two novel NOTA/NODAGA-containing peptides suitable for labeling with the positron emission tomography (PET) radionuclide Ga-68. These analogs are based on the previously reported GRPR-antagonist DOTAGA-PEG2-[Sar11]RM26, developed for targeted radiotheraostic applications. Both NOTA-PEG2-[Sar11]RM26 and NODAGA-PEG2-[Sar11]RM26 were successfully labeled with Ga-68 and evaluated in vitro and in vivo using PC-3 cell models. Both, [68Ga]Ga-NOTA-PEG2-[Sar11]RM26 and [68Ga]Ga-NODAGA-PEG2-[Sar11]RM26 displayed high metal-chelate stability in phosphate buffered saline and against the EDTA-challenge. The two [68Ga]Ga-labeled conjugates demonstrated highly GRPR-mediated uptake in vitro and in vivo and exhibited a slow internalization over time, typical for radioantagonistis. The [natGa]Ga-loaded peptides displayed affinity in the low nanomole range for GRPR in competition binding experiments. The new radiotracers demonstrated biodistribution profiles suitable for diagnostic imaging shortly after administration with fast background clearance. Their high tumor uptake (13 ± 1 and 15 ± 3% IA/g for NOTA and NODAGA conjugates, respectively) and high tumor-to-blood ratios (60 ± 10 and 220 ± 70, respectively) 3 h pi renders them promising PET tracers for use in patients. Tumor-to-normal organ ratios were higher for [68Ga]Ga-NODAGA-PEG2-[Sar11]RM26 than for the NOTA-containing counterpart. The performance of the two radiopeptides was further supported with the PET/CT images. In conclusion, [68Ga]Ga-NODAGA-PEG2-[Sar11]RM26 is a promising PET imaging tracer for visualization of GRPR-expressing lesions with high imaging contrast shortly after administration

Ga-66-PET-imaging of GRPR-expression in prostate cancer : production and characterization of [Ga-66]Ga-NOTA-PEG(2)-RM26

Molecular imaging of the gastrin-releasing peptide receptor (GRPR) could improve patient management in prostate cancer. This study aimed to produce gallium-66 (T-1/2=9.5 h) suitable for radiolabeling, and investigate the imaging properties of gallium-66 labeled GRPR-antagonist NOTA-PEG(2)-RM26 for later-time point PET-imaging of GRPR expression. Gallium-66 was cyclotron-produced using a liquid target, and enriched [Zn-66]Zn(NO3)(2). In vitro, [Ga-66]Ga-NOTA-PEG(2)-RM26 was characterized in GRPR-expressing PC-3 prostate cancer cells. In vivo, specificity test and biodistribution studies were performed 3 h and 22 h pi in PC-3 xenografted mice. microPET/MR was performed 3 h and 22 h pi. Biodistribution of [Ga-66]Ga-NOTA-PEG(2)-RM26 was compared with [Ga-68]Ga-NOTA-PEG(2)-RM26 3 h pi. [Ga-66]Ga-NOTA-PEG(2)-RM26 was successfully prepared with preserved binding specificity and high affinity towards GRPR. [Ga-66]Ga-NOTA-PEG(2)-RM26 cleared rapidly from blood via kidneys. Tumor uptake was GRPR-specific and exceeded normal organ uptake. Normal tissue clearance was limited, resulting in no improvement of tumor-to-organ ratios with time. Tumors could be clearly visualized using microPET/MR. Gallium-66 was successfully produced and [Ga-66]Ga-NOTA-PEG(2)-RM26 was able to clearly visualize GRPR-expression both shortly after injection and on the next day using PET. However, delayed imaging did not improve contrast for Ga-labeled NOTA-PEG(2)-RM26

Design, Synthesis, and Evaluation of Linker-Optimised PSMA-Targeting Radioligands

Prostate-specific membrane antigen (PSMA) is overexpressed in the majority of prostate cancer cells and is considered to be an important target for the molecular imaging and therapy of prostate cancer. Herein, we present the design, synthesis, and evaluation of 11 PSMA-binding radioligands with modified linker structures, focusing on the relationship between molecular structure and targeting properties. The linker design was based on 2-naphthyl-L-alanine-tranexamic acid, the linker structure of PSMA-617. X-ray crystal-structure analysis of PSMA and structure-based design were used to generate the linker modifications, suggesting that substitution of tranexamic acid could lead to interactions with Phe546, Trp541, and Arg43 within the binding cavity. After synthesis through SPPS, analogues were labelled with indium-111 and evaluated in vitro for their specific binding, affinity, and cellular retention. Selected compounds were further evaluated in vivo in PSMA-expressing tumour-bearing mice. Based on the results, 2-naphthyl-L-alanine appears to be crucial for good targeting properties, whereas tranexamic acid could be replaced by other substituents. [111In]In-BQ7859, consisting of a 2-naphthyl-L-alanine-L-tyrosine linker, demonstrated favourable targeting properties. The substitution of tranexamic acid for L-tyrosine in the linker led to an improved tumour-to-blood ratio, highlighting [111In]In-BQ7859 as a promising PSMA-targeting radioligand.De två sista författarna delar sistaförfattarskapet</p

Evaluation of Tumor-Targeting Properties of an Antagonistic Bombesin Analogue RM26 Conjugated with a Non-Residualizing Radioiodine Label Comparison with a Radiometal-Labelled Counterpart

Radiolabelled antagonistic bombesin analogues are successfully used for targeting of gastrin-releasing peptide receptors (GRPR) that are overexpressed in prostate cancer. Internalization of antagonistic bombesin analogues is slow. We hypothesized that the use of a non-residualizing radioiodine label might not affect the tumour uptake but would reduce the retention in normal organs, where radiopharmaceutical would be internalized. To test this hypothesis, tyrosine was conjugated via diethylene glycol linker to N-terminus of an antagonistic bombesin analogue RM26 to form Tyr-PEG(2)-RM26. [In-111]In-DOTA-PEG(2)-RM26 was used as a control with a residualizing label. Tyr-PEG(2)-RM26 was labelled with I-125 with 95% radiochemical purity and retained binding specificity to GRPR. The IC50 values for Tyr-PEG(2)-RM26 and DOTA-PEG(2)-RM26 were 1.7 +/- 0.3 nM and 3.3 +/- 0.5 nM, respectively. The cellular processing of [I-125]I-Tyr-PEG(2)-RM26 by PC-3 cells showed unusually fast internalization. Biodistribution showed that uptake in pancreas and tumour was GRPR-specific for both radioconjugates. Blood clearance of [I-125]I-Tyr-PEG(2)-RM26 was appreciably slower and activity accumulation in all organs was significantly higher than for [In-111]In-DOTA-PEG(2)-RM26. Tumor uptake of [In-111]In-DOTA-PEG(2)-RM26 was significantly higher than for [I-125]I-Tyr-PEG(2)-RM26, resulting in higher tumour-to-organ ratio for [In-111]In-DOTA-PEG(2)-RM26 at studied time points. Incorporation of amino acids with hydrophilic side-chains next to tyrosine might overcome the problems associated with the use of tyrosine as a prosthetic group for radioiodination

177Lu-labeled PSMA targeting therapeutic with optimized linker for treatment of disseminated prostate cancer; evaluation of biodistribution and dosimetry

&lt;bold&gt;Introduction:&lt;/bold&gt; Prostate specific membrane antigen (PSMA), highly expressed in metastatic castration-resistant prostate cancer (mCRPC), is an established therapeutic target. Theranostic PSMA-targeting agents are widely used in patient management and has shown improved outcomes for mCRPC patients. Earlier, we optimized a urea-based probe for radionuclide visualization of PSMA-expression in vivo using computer modeling. With the purpose to develop a targeting agent equally suitable for radionuclide imaging and therapy, the agent containing DOTA chelator was designed (BQ7876). The aim of the study was to test the hypothesis that Lu-177-labeled BQ7876 possesses target binding and biodistribution properties potentially enabling its use for radiotherapy.&lt;bold&gt;Methods:&lt;/bold&gt; BQ7876 was synthesized and labeled with Lu-177. Specificity and affinity of [Lu-177]Lu-BQ7876 to PSMA-expressing PC3-pip cells was evaluated and its processing after binding to cells was studied. Animal studies in mice were performed to assess its biodistribution in vivo, target specificity and dosimetry. [Lu-177]Lu-PSMA-617 was simultaneously evaluated for comparison.&lt;bold&gt;Results:&lt;/bold&gt; BQ7876 was labeled with Lu-177 with radiochemical yield &gt;99%. Its binding to PSMA was specific in vitro and in vivo when tested in antigen saturation conditions as well as in PSMA-negative PC-3 tumors. The binding of [Lu-177]Lu-BQ7876 to living cells was characterized by rapid association, while the dissociation included a rapid and a slow phase with affinities K-D1 = 3.8 nM and K-D2 = 25 nM. The half-maximal inhibitory concentration for Lu-nat-BQ7876 was 59 nM that is equal to 61 nM for Lu-nat-PSMA-617. Cellular processing of [Lu-177]Lu-BQ7876 was accompanied by slow internalization. [Lu-177]Lu-BQ7876 was cleared from blood and normal tissues rapidly. Initial elevated uptake in kidneys decreased rapidly, and by 3 h post injection, the renal uptake (13 +/- 3%ID/g) did not differ significantly from tumor uptake (9 +/- 3%ID/g). Tumor uptake was stable between 1 and 3 h followed by a slow decline. The highest absorbed dose was in kidneys, followed by organs and tissues in abdomen.&lt;bold&gt;Discussion:&lt;/bold&gt; Biodistribution studies in mice demonstrated that targeting properties of [Lu-177]Lu-BQ7876 are not inferior to properties of [Lu-177]Lu-PSMA-617, but do not offer any decisive advantages

Preclinical Evaluation of 99mTc-Labeled GRPR Antagonists maSSS/SES-PEG2-RM26 for Imaging of Prostate Cancer

Background: Gastrin-releasing peptide receptor (GRPR) is an important target for imaging of prostate cancer. The wide availability of single-photon emission computed tomography/computed tomography (SPECT/CT) and the generator-produced 99mTc can be utilized to facilitate the use of GRPR-targeting radiotracers for diagnostics of prostate cancers. Methods: Synthetically produced mercaptoacetyl-Ser-Ser-Ser (maSSS)-PEG2-RM26 and mercaptoacetyl-Ser-Glu-Ser (maSES)-PEG2-RM26 (RM26 = d-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2) were radiolabeled with 99mTc and characterized in vitro using PC-3 cells and in vivo, using NMRI or PC-3 tumor bearing mice. SPECT/CT imaging and dosimetry calculations were performed for [99mTc]Tc-maSSS-PEG2-RM26. Results: Peptides were radiolabeled with high yields (&gt;98%), demonstrating GRPR specific binding and slow internalization in PC-3 cells. [99mTc]Tc-maSSS-PEG2-RM26 outperformed [99mTc]Tc-maSES-PEG2-RM26 in terms of GRPR affinity, with a lower dissociation constant (61 pM vs 849 pM) and demonstrating higher tumor uptake. [99mTc]Tc-maSSS-PEG2-RM26 had tumor-to-blood, tumor-to-muscle, and tumor-to-bone ratios of 97 ± 56, 188 ± 32, and 177 ± 79, respectively. SPECT/CT images of [99mTc]Tc-maSSS-PEG2-RM26 clearly visualized the GRPR-overexpressing tumors. The dosimetry estimated for [99mTc]Tc-maSSS-PEG2-RM26 showed the highest absorbed dose in the small intestine (1.65 × 10−3 mGy/MBq), and the effective dose is 3.49 × 10−3 mSv/MBq. Conclusion: The GRPR antagonist maSSS-PEG2-RM26 is a promising GRPR-targeting agent that can be radiolabeled through a single-step with the generator-produced 99mTc and used for imaging of GRPR-expressing prostate cancer