Bench to Bedside: Albumin Binders for Improved Cancer Radioligand

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Bench to Bedside: Albumin Binders for Improved Cancer Radioligand Therapies Joseph Lau, Orit Jacobson, Gang Niu, Kuo-Shyan Lin, François Bénard, and Xiaoyuan Chen Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00919 • Publication Date (Web): 08 Jan 2019 Downloaded from http://pubs.acs.org on January 9, 2019

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Bioconjugate Chemistry

Bench to Bedside: Albumin Binders for Improved Cancer Radioligand Therapies Joseph Lau†, Orit Jacobson†, Gang Niu†, Kuo-Shyan Lin‡, François Bénard‡, Xiaoyuan Chen†* †Laboratory

of Molecular Imaging and Nanomedicine (LOMIN), National Institute of Biomedical Imaging and Bioengineering (NIBIB), National Institutes of Health (NIH), Bethesda, Maryland 20892, United States ‡Department of Molecular Oncology, BC Cancer, Vancouver, British Columbia, V5Z 1L3, Canada *Corresponding

Author Prof. Xiaoyuan Chen LOMIN, NIH BG 35A Rm GD937 35A Convent Dr. Bethesda MD, 20892 Email: [email protected] Phone: 1-301-451-4246 First Author Dr. Joseph Lau, Postdoctoral Fellow LOMIN, NIH BG 35A Rm GD959 35A Convent Dr. Bethesda MD, 20892 E-mail: [email protected] Phone: 1-301-451-9830 Word Count: 7217 (excluding Title Page and References) Financial Support: Support for this work was provided by the Intramural Research Program of the National Institute of Biomedical Imaging and Bioengineering (NIBIB), National Institutes of Health (NIH). Competing Interest: The authors declare that no competing financial interest exists.

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Bioconjugate Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Table of Contents Graphic

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ABSTRACT Radioligand therapy (RLT) relies on the use of pharmacophores to selectively deliver ionization energy to cancers to exert its tumoricidal effects. Cancer cells that are not directly targeted by a radioconjugate remain susceptible to RLT because of the crossfire effect. This is significant given the inter- and intra-heterogeneity of tumors. In recent years, reversible albumin binders have been used as simple ‘add-ons’ for radiopharmaceuticals to modify pharmacokinetics and to enhance therapeutic efficacy. In this review, we discuss recent advances in albumin binder platforms used in RLT, with an emphasis on 4-(p-iodophenyl)butyric acid and Evans blue derivatives. We focus on four biological systems pertinent to oncology that utilize this class of compounds: folate receptor, integrin αvβ3, somatostatin receptor, and prostate specific membrane antigen. Finally, we offer our perspectives on albumin binders for RLT, highlighting future areas of research that will help propel the technology further for clinical use. Keywords: Radioligand therapy, albumin binders, pharmacology, cancer, personalized medicine



INTRODUCTION Radioligand therapy (RLT) has become one of the most important treatment modalities for cancer. In RLT, an antigen recognition molecule (e.g. antibody, peptide, or drug inhibitor) is radiolabeled with a therapeutic radionuclide (e.g. alpha-emitter, beta-emitter, or auger electron emitter) to deliver lethal ionization energy to cancer cells.1–3 RLT is particularly useful in cases of oligometastatic disease, where conventional treatments such as surgery or external beam radiation therapy are no longer effective. Systemic chemotherapy can be efficacious; however, most chemotherapeutic agents lack selectivity and thus render an anticipated level of toxicity to normal tissues. By design, RLT targets specific biomarkers overexpressed in malignancies. This cancer targeting property endows a wider therapeutic window for RLT.1–3 The radiopharmaceuticals used for RLT are often the consequence of repurposed imaging agents. As such, RLT is usually accompanied by a companion diagnostic counterpart that can be used to predict clinical benefit.4,5 An established biological system where RLT has changed management and improved outcomes is neuroendocrine tumors (NETs) with somatostatin (SST) analogues.6,7 In the NETTER-1 phase III trial,

177Lu-DOTA-TATE

treatment led to higher progression free survival

rate (65.2% vs 10.8% at 20 mo) and response rate (18% vs 3%) compared to octreotide long-acting repeatable for patients with advanced progressive SST-receptor positive midgut neuroendocrine tumors.8 In early 2018,

177Lu-DOTA-TATE

received US Food and Drug Administration (FDA)

approval. While these clinical results are encouraging, there is room for improvement given the low response rate. An approach to ameliorate treatment efficacy is to modify the pharmacokinetics of the agents used in RLT. Specifically, the incorporation of an albumin binding moiety can be used to prolong the bioavailability of a radiopharmaceutical to enhance tumor uptake. In this


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review, we summarize the preclinical and clinical developments of albumin binder platforms for RLT.

ALBUMIN AND ALBUMIN BINDERS Albumin is the predominant plasma protein in the body, making up approximately 55-60% of the serum proteins.9 The 66.5 kDa tertiary polypeptide is comprised of 585 amino acids and 17 disulphide bonds.9 Albumin has various physiological functions including the regulation of oncotic pressure within the vascular system, as well as the transport of endogenous and exogenous compounds (Figure 1A).9 Notably, albumin is a carrier for lipids, hormones, metal ions, and lipophilic drugs. The biological half-life of albumin is reportedly 19 days, which puts it on similar standing with immunoglobulins.9 Previously, we and others have reviewed different strategies that leverage the circulation half-life of albumin to produce long-acting therapeutics.10–13 Aldoxorubicin (covalent binding to Cys 34 of albumin),14,15 insulin detemir (hydrophobic interaction of long fatty acids),16,17 and abliglutide (fusion protein)18,19 are examples of therapeutics that utilize albumin as a carrier. In recent years, two classes of reversible albumin binding molecules, 4-(p-iodophenyl)butyric acid and Evans blue derivatives, have seen extensive use for RLT, vide infra. The class of 4-(p-iodophenyl)butyric acid derivatives was first identified by Neri’s group at ETH Zurich (Figure 1B).20 These binders exhibited stable non-covalent binding with albumin, and dissociation constants (Kd) in the low micromolar range. The research group applied a Systematic Evolution of Ligands by Exponential Enrichment (SELEX)-like approach to a DNAencoded chemical library of 619 oligonucleotide-compound conjugates. The candidate pool was subjected to two selections against albumin and inactivated resin. Following polymerase chain reaction amplification, the identities of the enriched binders were decoded using an oligonucleotide 5 ACS Paragon Plus Environment


microarray. The enriched binders were structurally similar: a 4-phenylbutanoic acid motif in conjunction with hydrophobic substituent(s) on the aromatic ring. In the paper, the authors demonstrated that conjugation of a fluorescein derivative to one of the lead candidates significantly prolonged circulation by 100-fold compared to the free dye alone (495 min vs 4.6 min). Evans blue (EB) is an azo dye that has good affinity for albumin (Kd: 2.5 µM) (Figure 1C).11,21 Based on kinetic and equilibrium studies, it is predicted that there are 14 binding sites for EB on albumin.22 The binding of EB to albumin and its subsequent retention in blood, lends itself to different physiological applications. EB was administered in patients to determine blood plasma volumes, though it has been replaced with radiologic techniques.23,24 A secondary application of EB is the assessment of blood-brain-barrier (BBB) integrity.25,26 Under normal conditions, macromolecules cannot permeate the BBB. However, when BBB is compromised in situations of trauma or morbidity, the EB-albumin complex can accumulate freely in the brain. EB can also be used to measure vasculature permeability in tumors.27 Yamamoto et al. used a truncated EB derivative conjugated to the polyaminocarboxylate chelator diethylenetriamine pentaacetate (DTPA) for magnetic resonance imaging after Gd-labeling.28 Following this approach, our research group replaced DTPA with 1,4,7-triazacyclononane-1,4,7-trisacetic acid (NOTA) for positron emission tomography (PET) applications. 68Ga-NEB was translated into the clinic where it enabled high contrast blood pool and lymphatic imaging in healthy volunteers,29 and detection of lymphatic malformations in patients.30 Prompted by its success, our research group subsequently developed second generation EB constructs containing both a chelator and a maleimide functional group, paving the way for EB-based theranostics.31


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Figure 1. (A) The crystal structure of human albumin. Albumin is composed of three α-helical domains (DI, DII, and DII) which in turn are divided into subdomains (A and B). The domains and subdomains are binding sites for metal ions, fatty acids, and endogenous and exogenous ligands (listed in green and red). Image was adapted with permission from Front. Immunol. 2015, 5:682,9 in accordance with the Creative Commons Attribution License (CC BY) . (B) The chemical structure of 4-(p-iodophenyl)butyric acid. The carboxylic group can be used as a functional group to attach a ligand of interest. (C) The chemical structure of Evans blue (EB). Truncation of EB enables functionalization. BIOLOGICAL SYSTEMS In this section, we review 4-(p-iodophenyl)butyric acid or EB-based radiopharmaceuticals that have been developed for folate receptor (FR), integrin αvβ3, somatostatin receptor (SSTR), and prostate specific membrane antigen (PSMA). These biological systems are overexpressed in different cancer subtypes. Whereas RLT for FR and integrin αvβ3 has been limited to preclinical settings, SSTR2 and PSMA are well-established RLT targets in nuclear medicine. The inclusion of an albumin binder in the design of the radioconjugates is aimed at improving tissue-specific pharmacokinetics (e.g. reducing renal uptake and increasing tumor uptake).



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FOLATE RECEPTOR FRs are glycoproteins found on the extracellular surface anchored by a glycosylphosphatidylinositol domain.32,33 FRs exist as different isoforms (38-44 kDa), with the α-isoform being associated with malignant progression.32,33 FR-α is overexpressed in ovarian, cervical, breast, lung, kidney, colorectal, and brain cancers, while minimal expression is observed in normal tissues except for lungs, placenta, and kidneys.32,33 Notably, the use of methotrexate, an anti-folate, to treat gestational choriocarcinoma in the late 1950s has been credited as the impetus for advancing chemotherapies for solid tumors.34,35 Derivatives of folic acid have been used for drug delivery, pre- and intra-operative imaging.36–38 However, the application of radiolabeled folates for RLT has not been realized due to concerns of radionephropathy as FR is highly expressed in renal proximal tubules.39 Müller et al. hypothesized that prolonging the circulation of radiolabeled folates would reduce clearance and subsequent renal retention.39 The albumin binder 4-(p-iodophenyl)butyric acid was conjugated to folic acid and a 1,4,7,10-tetraazacyclododecane-N,N′,N″,N‴-tetraacetic acid (DOTA) chelator for

177Lu

radiotherapy (Figure 2). The resulting radiopharmaceutical,

177Lu-

cm09, was evaluated in athymic mice bearing KB human cervical carcinoma cells. At 1 nmol dose and 4 h post-injection (p.i.), tumor uptake for 177Lu-cm09 was 20.0 ± 3.22 percentage injected dose per gram (%ID/g) compared to 7.52 ± 1.15 %ID/g for

177Lu-EC0800,

a radiofolate without an

albumin binder motif. There was good retention of radioactivity in tumor for up to 48 h p.i. The radioactivity in kidneys was significantly lower for 177Lu-cm09 (28.1 ± 1.35 %ID/g), compared to 177Lu-EC0800

(73.7 ± 3.50 %ID/g) at 4 h p.i. Administered as a single dose (1 × 20 MBq), 177Lu-

cm09 led to complete remission in 4/5 mice.

177Lu-cm09

significantly prolonged survival time

(>84 d) compared to untreated cohort (27 d). A fractionated-dose approach (2 × 10 or 3 × 7 MBq)


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was explored but had minimal efficacy. This study represents the first application of an albumin binder for RLT.

Figure 2. (A) The chemical structure of 177Lu-cm09. (B) SPECT/CT images of KB-tumor bearing mice injected with 177Lu-cm09 (top) and 177Lu-EC0800 (bottom) at selected time points. Arrows indicate position of tumors and kidneys respectively. Higher tumor uptake and retention is observed for 177Lu-cm09 compared to 177Lu-EC0800. (C) Radioligand therapy with 177Lu-cm09. Of the five cohorts, treatment with 1x 20 MBq 177Lu-cm09 yielded the greatest therapeutic efficacy. Figure was adapted with permission from J. Nucl. Med. 2013, 54(1): 124-131.39 Copyright 2013 Society of Nuclear Medicine and Molecular Imaging. In a follow up study, the group investigated the tumor growth inhibition effect at different dosages of 177Lu-cm09 (1 × 10, 1 × 20, and 1 × 30 MBq) and 177Lu-EC0800 (1 × 10, and 1 × 20



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MBq), as well as long-term (8 mo) renal toxicity.40 RLT was performed in the previously established KB tumor model, while toxicity was assessed in nontumor-bearing mice. In agreement with earlier results,

177Lu-cm09

administration delayed tumor growth and resulted in complete

tumor regression in some animals (3/5 mice for 1 × 20 MBq, and 4/5 mice for 1 × 30 MBq). At 1 × 20 MBq dose, the calculated absorbed doses for tumor and kidneys were ~28 and ~46 Gy for 177Lu-cm09,

and ~8.8 and ~96 Gy for

177Lu-EC0800.

following perimeters were assessed: body weight,

For assessment of renal toxicity, the

99mTc-DMSA

imaging,41 blood urea nitrogen,

creatinine, WBC count, kidney size and morphology. Mice treated with 1 × 20 MBq 177Lu-cm09 had decreased uptake of 99mTc-DMSA and higher plasma levels of urea nitrogen. Histopathology showed morphological changes (e.g. interstitial fibrosis, and tubular atrophy) that were indicative of impaired renal function. More recently, the Müller group demonstrated that the pharmacokinetics of the albumin binder folate derivatives can also be modified by introducing different linkers (Figure 3).42 The four derivatives (cm10: no linker; cm12: PEG-11 linker; cm13: alkane linker; and cm14: no linker and no albumin binder) maintained good binding affinity for FR (Kd: 4.0-7.5 nM). In vivo studies were limited to single photon emission computed tomography (SPECT) imaging and biodistribution studies. The derivatives with albumin binders had higher blood retention and tumor uptake than internal control at all time points. The peak tumor uptake for the three albumin derivatives were similar, but they were achieved at different time points: 17.6 ± 2.58 %ID/g at 24 h p.i. for 177Lu-cm10, 19.5 ± 3.21 %ID/g at 4 h p.i. for 177Lu-cm12, and 16.0 ± 1.58 %ID/g at 4 h p.i. for 177Lu-cm13. 177Lu-cm12 with a PEG-11 linker exhibited the fastest clearance, while 177Lucm13 with an alkane linker cleared the slowest. Nonetheless, the tumor-to-kidney ratios observed


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in this study (0.30 ± 0.03 to 0.63 ± 0.11 at 120 h p.i.) were comparable with value obtained with 177Lu-cm09

(0.61 ± 0.15 at 120 h p.i.).39

Since linker modification alone was insufficient at improving tumor-to-kidney ratio, the group tried a combinational approach,43 administering pemetrexed (PMX), an antifolate metabolite, an hour before injecting

177Lu-cm13.

While pre-injection of PMX reduced kidney

uptake and increased tumor-to-kidney ratio by 2-fold, it required administration of a high therapeutic dose (400 µg/mouse). The study reaffirmed that the addition of an albumin binder was beneficial for radiofolates for RLT; however, further interventions are required to reduce renal uptake in order to find a satisfactory therapeutic index (TI).



Figure 3. (A) The chemical structure of 177Lu-cm13. 177Lu-cm13 contains an alkane linker indicated in blue. (B) SPECT/CT images of KB-tumor bearing mice injected with 177Lu-labeled folates at 24 h p.i. Images were scaled to show comparable renal uptake. (C). Time activity curve of the four different radiolabeled folates. Reduced renal uptake is observed with the albumin binder derivatives. Figure was adapted with permission from Mol. Pharm. 2017, 14(2): 523-532.42 Copyright 2017 American Chemistry Society. INTEGRIN αvβ3 Integrins are transmembrane proteins that promote cell-cell and cell-extracellular matrix (ECM) adhesion.44,45 They exist as heterodimers (α and β subunits) with 24 possible configurations or assemblies.46 In addition to their role as cell-adhesion molecules, integrins are involved in signaling pathways mediating survival, differentiation, gene transcription, and apoptosis. Integrin αvβ3 is important in cancer as it promotes angiogenesis – a fundamental hallmark of cancer biology.47 Integrin αvβ3 is expressed in neovasculature and activated endothelial cells, but not in latent endothelium or normal tissues. Therefore, the pharmacological inhibition of integrin αvβ3 has been proposed for anti-angiogenic therapy. Moreover, the expression of integrin αvβ3 has also been observed in selected cancers like glioblastoma multiforme (GBM).48 GBM is the most common (16% of all brain cancers) and aggressive (median survival of 15 mo) brain malignancy. 49,50

Treatment for GBM entails surgical resection, and postoperative radiation therapy concurrent

with temozolomide (TMZ); however, response is generally transient with most patients having disease progression.49,50 Cilengitide, an integrin inhibitor,48 advanced to phase III trials for GBM patients with methylated MGMT promoter.51 According to the study, the combination of cilengitide and TMZ chemoradiation did not improve outcome compared to control (TMZ chemoradiation alone).51 The lack of drug activity due to rapid clearance was postulated as one of the reasons why cilengitide failed to elicit favourable response.52


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Chen et al. used the integrin αvβ3 system as proof-of-concept for EB-based theranostics (Figure 4). NOTA-maleimide-EB (NMEB) and DOTA-maleimide-EB (DMEB) were synthesized to afford thiol-maleimide chemistry.31 To target integrin αvβ3, a functional thiol group was incorporated to the Lys ε-amino group of cyclic Arg-Gly-Asp-D-Phe-Lys c(RGDfK) peptide, and conjugated to NMEB and DMEB. The precursors NMEB-RGD and DMEB-RGD were subsequently used to chelate 64Cu and 90Y, respectively. PET imaging studies were performed with 64Cu-NMEB-RGD

in human glioma (U87-MG), melanoma (MDA-MB-435), and colorectal

cancer (HT-29) xenograft models, while RLT was performed with 90Y-DMEB-RGD in the U87MG and HT-29 models. The uptake of 64Cu-NMEB-RGD was approximately 10-fold higher than 64Cu-RGD

(16.6 ± 1.99 vs 1.06 ± 0.03 %ID/g at 24 h p.i.) for the U87-MG model. Notably, this

generated higher contrast PET images compared to 64Cu-RGD at late time points (4 and 24 h p.i.). MDA-MB-435 and HT-29 tumors also had higher uptake of 64Cu-NMEB-RGD than 64Cu-RGD, albeit at reduced levels compared to U87-MG due to lower expression of integrin αvβ3. For RLT, a dose escalation study (2 × 1.85, 2 × 3.7, and 2 × 7.4 MBq) was performed for 90Y-DMEB-RGD in the U87-MG model and a dose-dependent response was observed. The administration of 2 × 7.4 MBq of 90Y-DMEB-RGD led to complete tumor regression and extended survival, while the lower dosages were only able to retard tumor growth.



Figure 4. (A) The chemical structure of 90Y-DMEB-RGD. c(RGDfK) was conjugated to tEB by thiol maleimide chemistry. (B) Maximum intensity projection PET images of 64Cu-NMEB-RGD, 64Cu-NOTA-c(RGDfK), and 64Cu-NEB in mice bearing U87-MG xenografts (white arrow). 64CuNEB is an EB derivative without the targeting peptide. (C) Tumor volume change. Mice were injected with saline (Group A), 7.4 MBq 90Y-DMEB-RGD (Group B), 3.7 MBq 90Y-DMEB-RGD (Group C), 1.85 MBq 90Y-DMEB-RGD (Group D), 7.4 MBq 90Y-DOTA-RGD (Group E), and 1.85 MBq 90Y-DOTA-RGD (Group F). Figure was adapted with permission from J. Nucl. Med. 2017, 58(4): 590-597.31 Copyright 2017 Society of Nuclear Medicine and Molecular Imaging. SOMATOSTATIN RECEPTOR SSTRs are G-protein coupled receptors that regulate secretion of growth hormones. Somatostatin receptor subtype 2 (SSTR2) is overexpressed in NETs, a heterogenous group of lowincidence tumors that commonly present with metastatic disease at diagnosis.53 Short-lived and long-acting SST analogues are given palliatively to control acromegaly associated symptoms and 14 ACS Paragon Plus Environment

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to slow disease progression.53,54 As mentioned, radiolabeled SST analogues have become a standard of care for NETs. While these compounds are effective in disease control, the overall response rate remains relatively low at ~30%.55 A primary reason is the rapid renal clearance of the

177Lu-labeled

SST analogues from blood; relatedly; the kidneys are the dose limiting organs

for this class of compounds due to reabsorption by proximal tubules. Tian et al. conjugated thiolated octreotate to DMEB to synthesize DOTA-EB-TATE.55 DOTA-EB-TATE was subsequently radiolabeled with 86Y/90Y for theranostic targeting of SSTR2 (Figure 5). 86Y-DOTA-EB-TATE generated high-contrast PET images in the transfected human colorectal cancer HCT116/SSTR2 model and in the rat pancreatic cancer AR42J model. Image acutance was unequivocal for

86Y-DOTA-EB-TATE

and

86Y-DOTA-TATE.

For the AR42J

model, 86Y-DOTA-EB-TATE was able to attain 60.5 ± 8.03 and 65.8 ± 7.59 %ID/g 24 and 48 h p.i., respectively. Based on AUC calculations, HCT116/SSTR2 tumors received six-fold higher radiation exposure from 86Y-DOTA-EB-TATE than 86Y-DOTA-TATE. For RLT, 90Y-DOTA-EBTATE (1 × 3.7 or 1 × 7.4 MBq) was able to significantly inhibit tumor growth and more than double survival compared to control groups. At 1 × 7.4 MBq,

90Y-DOTA-EB-TATE

led to

complete tumor ablation in both models. Haematological analysis of complete blood count at 90120 d p.i., revealed no differences between mice that received 1 × 7.4 MBq 90Y-DOTA-EB-TATE or saline.



Figure 5. (A) The chemical structure of 90Y-DOTA-EB-TATE. (B) Maximum intensity projection PET images of 86Y-DOTA-EB-TATE in AR42J xenograft mice (white arrow). For blocking group, >50-fold excess of DOTA-EB-TATE was co-injected with the radiopharmaceutical. (C) Tumor volume response following 90Y-DOTA-EB-TATE administration. (D) Survival analysis in mice after 90Y-DOTA-EB-TATE administration. Figure was adapted with permission from Theranostics. 2018, 8(3): 735-745,55 in accordance to the Creative Commons Attribution License (CC BY-NC). Bandara et al. reported the preclinical data for 177Lu-DOTA-EB-TATE in comparison with 177Lu-DOTA-TATE.56

Studies were performed in mice bearing A427-7 human non-small cell lung

carcinoma xenografts. Tumor uptake of 177Lu-DOTA-EB-TATE was 78.8 ± 4.07 and 64.5 ± 7.39 %ID/g at 24 and 48 h p.i., respectively. The tumor uptake of 177Lu-DOTA-TATE peaked at 9.25 ± 0.81 %ID/g at 4 h p.i. before decreasing to 3.02 ± 0.20 %ID/g at 24 h p.i. A higher dose of 177Lu16 ACS Paragon Plus Environment

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DOTA-EB-TATE (1 × 18.5 MBq) was required to achieve complete tumor regression compared to

90Y-DOTA-EB-TATE

(1 × 7.4 MBq for AR42J model).

177Lu-DOTA-EB-TATE

was

significantly better than 177Lu-DOTA-TATE in terms of therapeutic efficacy. Due to differences in particle energy and range, 177Lu (Emax: 0.49 MeV; 2 mm) is better suited than 90Y (Emax: 2.27 MeV; 11 mm) for treating small lesions and minimizing energy deposition beyond malignant boundaries.57–59 However, neither dosimetry nor toxicity data were reported in the study. Although not investigated,

177Lu-DOTA-EB-TATE

may be useful in tandem with

90Y-DOTA-EB-TATE.

Previously, Kunikowska et al. reported that the combination of 90Y/177Lu-DOTA-TATE improved overall survival of NET patients compared to 90Y-DOTA-TATE alone.60 The safety, pharmacokinetics, and dosimetry of 177Lu-DOTA-EB-TATE was evaluated by Zhang et al. in a small cohort of patients with advanced metastatic NETs.61 Five patients received a single-dose administration of received a single dose of

177Lu-DOTA-EB-TATE

177Lu-DOTA-TATE

(0.35-0.70 GBq), while three patients

(0.28-0.41 GBq). Patients underwent whole-body

scintigraphy and SPECT/CT imaging. As expected, 177Lu-DOTA-EB-TATE showed significantly higher retention in circulation compared to 177Lu-DOTA-TATE (1.75 ± 0.58 vs 0.09 ± 0.03 SUV in blood at 24 h p.i.). Consequently, tumor than

177Lu-DOTA-TATE;

177Lu-DOTA-EB-TATE

delivered 7.9-fold higher dose to

however, the kidney and bone marrow doses also increased by

3.2 and 18.2-fold, respectively. In this study, no adverse events were observed during treatment or within the 3 mo follow up period. Wang et al. reported the pilot study of

177Lu-DOTA-EB-TATE

treatment in NET

patients.62 Four patients received a low dose of 177Lu-DOTA-EB-TATE (0.66 ± 0.06 GBq), while 3 patients received 177Lu-DOTA-TATE (3.98 ± 0.17 GBq). Treatment response was evaluated on a per-lesion basis with 68Ga-DOTA-TATE PET/CT at baseline, 1 and 3 mo post-treatment (Figure



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6). The ∆SUV for lesions that had comparable baseline SUVmax (10.0-35.0), was similar between 177Lu-DOTA-EB-TATE

(-7.9 ± 5.4%) and 177Lu-DOTA-TATE (-5.8 ± 3.9%). Based on ∆SUVmax

of the lesions with highest 68Ga-DOTA-TATE uptake (up to five lesions per patient), 3 patients were classified with partial response (PR) and 1 had stable disease (SD) after

177Lu-DOTA-EB-

TATE treatment. For the 177Lu-DOTA-TATE group, 2 patients had PR and 1 patient had SD. In line with the previous study, no abnormal findings were observed for hematology parameters, renal and hepatic functions.

Figure 6. 68Ga-DOTA-TATE PET/CT images of a NET patient (A-C) before and (D-F) 3 mo after a single low-dose (0.72 GBq) injection of 177Lu-DOTA-EB-TATE. Patient had primary pancreatic tumor and metastatic liver disease. Treatment with 177Lu-DOTA-EB-TATE reduced SUVmax of the pancreatic primary tumor (arrow; from 26.7 to 13.0), and of the highest-uptake liver lesion (triangle; from 50.6 to 28.6). Figure was adapted with permission from Theranostics. 2018, 8(12): 3308-3316,62 in accordance to the Creative Commons Attribution License (CC BY-NC). Rousseau et al. reported the preclinical evaluation of two 177Lu-DOTA-TATE derivatives conjugated to 4-(p-iodophenyl)butyric acid (177Lu-GluAB-DOTATATE and

177Lu-AspAB-

DOTATATE).63 Unlike previous albumin binder conjugates, the compounds were synthesized on


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solid-phase peptide synthesis. Both compounds showed extended circulation in blood (8.08 ± 2.05 and 11.8 ± 3.88 %ID/g at 1 h p.i.) compared to 177Lu-DOTA-TATE (0.32 ± 0.03 %ID/g at 1 h p.i.). However, only

177Lu-AspAB-DOTATATE

showed marginal improvement over

177Lu-DOTA-

TATE with respect to uptake in AR42J xenografts at late time points (16.9 ± 8.97 vs 10.1 ± 5.78 %ID/g at 120 h p.i.). 177Lu-AspAB-DOTATATE increased radiation dose delivered to tumor by 1.5-fold, but this was accompanied by a 3.7-fold increase to kidneys. As the calculated TI of 177LuAspAB-DOTATATE was inferior to

177Lu-DOTA-TATE,

no RLT studies were performed.

Comparing the results of this study with DOTA-EB-TATE, it can be concluded that the selection of albumin binder greatly affects tumor uptake and absorbed dose.55 PROSTATE SPECIFIC MEMBRANE ANTIGEN PSMA is a membrane-bound glutamate carboxypeptidase that is overexpressed in prostate cancer. The expression of PSMA is correlated with tumor grade and metastatic propensity. PSMA is also expressed in neovasculature of solid tumors.64 Subsequently, PSMA has emerged as an attractive theranostic target for prostate cancer, especially for patients with advanced metastatic castration resistant prostate cancer (mCRPC) who progress following androgen deprivation therapies.64 Pharmacophores that have been used for RLT include monoclonal antibodies as well as small molecular weight inhibitors.64

177Lu-PSMA-617,

a lysine-urea-glutamate derivative, is

currently in phase III clinical trials. Treatment with 177Lu-PSMA-617 induced ≥50% reduction in PSA in 30-60% of patients.65,66 However, there is a substantial portion of patients (~30%) that do not respond to PSMA RLT.66 By and large, PSMA is the biological system with the most extensive use of albumin binder derivatives for RLT. Kelly et al. reported the first class of albumin binder conjugated glutamate-urea derivatives targeting PSMA.66 The Babich group synthesized six derivatives, each containing the 419 ACS Paragon Plus Environment


Page 20 of 42

iodophenyl albumin binding motif but with different linkers. The compounds were directly radiolabeled with

131I,

obviating the need for a chelator group (Figure 7). The compounds

exhibited nanomolar affinity (IC50: 4-40 nM) for PSMA, and micromolar affinity for human serum albumin (HSA; Kd: 1-57 µM). Biodistribution studies were performed in mice bearing LNCaP human prostate adenocarincoma xenografts. For the reference compound (131I-MIP-1095),67 rapid clearance from blood was observed (12.7 %ID/g at 48 h p.i.). However, the slow clearance from blood led to elevated uptake in most organs except for tumor. The lead candidate

131I-RPS-027

(Kd: 11 µM) showed initial

retention in blood (3.91 ± 0.48 %ID/g at 3 h p.i.) that gradually decreased to 20 and >50%), while the remaining 2 patients had increase of 15-25%. For 177Lu-PSMA-617, only 1 patient had a decrease in PSA level (25%). Treatment with 177Lu-EB-PSMA-617 led to ∆SUV in 68Ga-PSMA-617 of -32.4 ± 0.14%, while 177Lu-PSMA617 yielded a ∆SUV of 0.21 ± 0.37%. For the 177Lu-EB-PSMA-617 group, 3 patients showed PR (fourth patient failed to receive a PET scan at 1 mo). For the

177Lu-PSMA-617

group, 1 patient

had PR while the remaining 4 patients had SD. Based on dosimetric analysis, tumor dose for bone metastases was 5.7-fold higher for 177Lu-EB-PSMA-617. Concurrently, 177Lu-EB-PSMA-617 also delivered approximately 6-fold higher dose to bone marrow and kidneys. Two patients developed grade 1 and grade 2 leukopenia after 177Lu-EB-PSMA-617 administration, but the condition was reversible.



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Figure 11. 68Ga-PSMA-617 PET/CT images of a mCRPC patient (A) before and (B) 1 mo after a single low-dose (1.11 GBq) injection of 177Lu-EB-PSMA-617. Treatment with 177Lu-EB-PSMA617 reduced SUVmax of the right scapular lesion (red arrow; from 10 to 3), and of the T6 bone metastasis (blue arrow; from 21 to 7). 68Ga-PSMA-617 PET/CT images of a second mCRPC patient (C) before and (D) 1 mo after a single low-dose (1.27 GBq) injection of 177Lu-PSMA-617. The SUVmax of the S1 bone metastasis (green arrow; from 5 to 12) and right ilium (orange arrow; from 4 to 8) increased despite treatment. Figure was adapted with permission from Eur. J. Nucl. Med. Mol. Imaging 2019, 46(1): 148-158.75 Finally, the Babich group developed a

225Ac-labeled

PSMA-targeting agent,

225Ac-RPS-

074, with a 4-(p-iodophenyl)butyric acid derivative as the albumin-binding moiety (Figure 12).76 225Ac

decays to yield four alpha-particles with high linear energy transfer (LET) that causes

irreparable double-stranded DNA breaks. Because of higher LET, alpha-emitters are more damaging than beta-emitters. Instead of a DOTA chelator, 225Ac-RPS-074 incorporates a macropa chelator77 which facilitates quantitative 225Ac labeling at room temperature. The uptake of 225Ac30 ACS Paragon Plus Environment

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RPS-074 in blood, LNCaP tumor and kidney at 24 h p.i. were 2.5 ± 0.2, 15.9 ± 4.3, and 5.2 ± 0.7 %ID/g, respectively. The activity in tumor persisted to 14 d p.i. (11.9 ± 1.5 %ID/g). For RLT, mice were injected with 225Ac-RPS-074 (1 × 37, 1 × 74, or 1 × 148 kBq) or control. A dose-dependent response was observed. Treatment with 1 × 74 kBq led to PR for 7/7 mice, but regrowth was observed after 42 d p.i., while treatment with 1 × 148 kBq led to complete tumor regression in 6/7 mice without any observed toxicity. According to the authors, 225Ac-RPS-074 would deliver 10fold less radiation dose to kidneys than 225Ac-PSMA-617;78,79 however, tumor dose estimate was not provided. Notably, this is the first example of an albumin binder derivative used in conjunction with targeted alpha therapy.



Figure 12. (A) The chemical structure of 225Ac-RPS-074. The complex uses a macropa chelator in place of the commonly used DOTA chelator. (B) Survival of LNCaP xenograft mice after treatment with 225Ac-RPS-074. ## indicates 225Ac-DOTA-Lys-IPBA, a control group without PSMA targeting moiety. (C) 68Ga-PSMA-11 PET/CT images 75 d p.i. of 225Ac-RPS-074. The mice on the left received 148 kBq of 225Ac-RPS-074, while the mice on the right received 74 kBq of 225Ac-RPS-074. Figure was adapted with permission from J. Nucl. Med. 2018, [epub ahead of print].76 Copyright 2018 Society of Nuclear Medicine and Molecular Imaging.


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PERSPECTIVES AND FUTURE DIRECTIONS In the last five years, we have seen significant advancements in albumin binder derivatives for RLT, with two compounds being translated from bench to bedside. For this class of radiopharmaceuticals, reversible binding to albumin imparts significant gains in circulation halflife, which enhances tumor uptake and dose delivery. This is evidenced by the favourable therapeutic responses obtained by these radiopharmaceuticals in preclinical studies relative to controls. With the maturation of the synthetic chemistry, peptides and small molecule inhibitors can be readily appended to either 4-iodophenyl or EB motifs provided that they have good metabolic stability. It is easy to envision the use of albumin binders in concert with other antigen recognition molecules like aptamers, affibodies, or antibody mimetics that show relatively quick clearance. An undervalued aspect of albumin binders with RLT is their potential cost-effectiveness as health interventions. With albumin binder derivatives, lower amounts of radioactivity are needed to achieve equivalent therapeutic efficacy compared to an unmodified ligand. Since less activity is needed to prepare individual doses, production costs can be reduced while access to care can be concomitantly increased for patients. This holds true for any therapeutic isotope, but its impact is particularly far-reaching with rare radionuclides such as

225Ac-actinium.

As mentioned, alpha

particles are more potent than beta emitters at inducing DNA damage and cell death. For example, full biochemical response can be achieved by refractory to

177Lu-PSMA-617

225Ac-PSMA-617

in mCRPC patients that are

treatment.78,79 However, the current global production of

225Ac

greatly lags behind the clinical demand. Albumin binders may be a viable solution for next generation 225Ac-based radiopharmaceuticals.



As with any technological platform, it is important to identify limitations. The prolonged circulation of these radiopharmaceuticals creates a double-edged situation where radiation dose becomes elevated for normal tissues as well. The toxicity to sensitive tissues like bone marrow or kidneys may negate the benefits of increased tumor uptake. Albumin binding does not uniformly increase uptake or retention across all tissues; thus, the AUC ratio (e.g. tumor/kidney or tumor/blood) can still be improved in certain cases. Initial clinical experience with 177Lu-DOTAEB-TATE and

177Lu-EB-PSMA-617

have been promising, and treatments were generally well-

tolerated. It should be noted that both reports were single-low dose studies conducted in small patient cohorts.62,75 Needless to say, caution must be taken when escalating doses and frequencies of administration for these radiopharmaceuticals. Personalized dosing based on imaging dosimetric analyses, following recommendation of EANM/MIRD guidelines,80 is a promising approach for improving treatment safety and efficacy. Albumin binder-conjugated radiopharmaceuticals are often compared to antibodies because of their prolonged residence time. There is also the fact that the catabolism of albumin and immunoglobulins are jointly regulated by the neonatal Fc receptor.9 As albumin binder derivatives are continuously binding to and disassociating from circulating albumin, they exhibit faster clearance than antibodies. From our experience with EB derivatives, we are able to increase the half-life of peptides and small molecules from minutes to hours, while achieving high tumorbackground uptake ratio at 24 h p.i. or earlier time points, in contrast to antibodies that stay longer in the blood circulation. Other intrinsic advantages of albumin binder constructs over antibodies include lack of immunogenicity and lower cost of production. An interesting study would be to perform a side-by-side comparison with 177Lu-EB-PSMA-617 and 177Lu-labeled PSMA-targeting


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monoclonal antibody J59181 to discern differences in pharmacokinetics, treatment efficacy, dosimetry, and hematological toxicity. As articulated though the works of Kelly et al.66 and Umbricht et al.,70 binding affinity to albumin is a crucial parameter in determining blood retention and tumor uptake. When a bioconjugate binds too tightly with albumin, it appears that tumor uptake may become depressed. One way to improve the TIs of albumin binder derivatives is to seek out lower affinity albumin binders. Different substituent groups on 4-phenylbutanoic acid and linker composition are known to affect pharmacokinetics; however, they are often studied independently of one another. Developing a high-throughput means for screening different albumin binder/ligand combinations in vivo would be valuable endeavour. An alternative approach to improve TI is to administer radioprotective agents before or clearing agents after treatment. While a two-step approach is a barrier to clinical translation as it is more complex than existing RLT strategies and requires the optimization of both dosing components, it is feasible. For instance, amino acids infusions given to patients receiving peptide receptor radionuclide therapy can be administered over the course of 2 days.82 RLT is a burgeoning field for personalized medicine and oncology. As more radiopharmaceuticals matriculate into clinical settings, we anticipate that more research groups will embrace the albumin binder technology, expanding the RLT repertoire for other biological systems in cancer beyond those reviewed in this paper. It remains to seen if albumin binding strategy will compete with or complement the current state-of-the-art RLT agents. In either case, comparative clinical studies in larger patient cohorts with appropriate follow up for outcome and toxicity assessment will be required. In summary, albumin binders are simple yet powerful pharmacokinetic modulators that have the potential to improve RLT efficacy and patient outcomes.



Financial Support Support for this work was provided by the Intramural Research Program of the National Institute of Biomedical Imaging and Bioengineering (NIBIB), National Institutes of Health (NIH). Competing Interest The authors declare that no competing financial interest exists.


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Bench to Bedside: Albumin Binders for Improved Cancer Radioligand

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