Communication Cite This: Bioconjugate Chem. 2019, 30, 1314−1322
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Development of Bispecific NT-PSMA Heterodimer for Prostate Cancer Imaging: A Potential Approach to Address Tumor Heterogeneity Xiaofen Ma,#,†,‡ Mengzhe Wang,#,‡ Hui Wang,‡ Tao Zhang,‡ Zhanhong Wu,‡ Mariia V. Sutton,§ Vladimir V. Popik,§ Guihua Jiang,*,† and Zibo Li*,‡
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†
Department of Medical Imaging, Guangdong Second Provincial General Hospital, Guangzhou City, Guangdong Province 510317, P. R. China ‡ Biomedical Research Imaging Center, Department of Radiology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, United States § Department of Chemistry, University of Georgia, Athens, Georgia 30602, United States S Supporting Information *
ABSTRACT: Prostate cancer is a heterogeneous disease with a poor survival rate at late stage. In this report, a dual targeting PET agent was developed to partially address the tumor heterogeneity issue. The heterodimer F-BCN-PSMA-NT was designed to target PSMA and neurotensin receptor1 (NTR1), both of which have demonstrated great potential in prostate cancer management. The heterodimer was synthesized through the conjugation of Glu-urea-lys(Ahx) (PSMA targeting motif) and NT20.3 (NTR1 targeting motif) to a symmetric trifunctional linker, bearing an azide group for further modification. Radio-labeling was performed using strain promoted azide−alkyne click reaction with high yield. Cell based assays suggested that F-BCN-PSMA-NT has comparable or only slightly reduced binding affinity with the corresponding monomers. Small animal PET clearly demonstrated that the heterodimer probe has prominent uptake not only in NTR1 positive/PSMA negative PC-3 tumors (1.4 ± 0.3%ID/g), but also in the PSMA positive/NTR1 negative LnCap tumors (1.3 ± 0.2% ID/g). The tracer showed comparable tumor to background ratio with each monomer. In summary, prostate cancer is a heterogeneous disease in need of improved diagnostics and treatments. The PSMA-NT heterodimer represents a new class of molecules that can be used to target two distinct antigens related to prostate cancer. In addition to the imaging applications demonstrated in this study, the agent also holds great potential on the treatment of heterogeneous prostate cancer.
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regardless of androgen status, PSMA targeted PET agents are also clinically attractive due to its ability to image recurrent or metastatic prostate cancer lesions with high contrast and potentially higher sensitivity compared with Choline-PET/ CT.10−15 However, the limitation of PSMA as a biomarker is its low expression in tumor tissues with low Gleason scores, which has been demonstrated by 68Ga-PSMA-11 imaging.16,17 Tumor heterogeneity is another factor that has to be considered in prostate cancer imaging and therapy. In addition to PSMA, G-protein-coupled neurotensin receptor (NTR) and its ligand neurotensin peptide (NT) were recently suggested to play important roles in a variety of cancers, especially prostate cancer.18−21 Studies have shown that NTRs affect the signaling on IL-8 expression and subsequent CXCR1/STAT3 signaling pathway activation in malignant tumors and induce gelsolin mediated invasion of cancer cell through NTR1 activation.22,23 Overexpression of NTR has been discovered in androgen-independent human
espite recent improvements in prognosis and treatment, prostate cancer remains one of the most common malignancies and a leading cause of cancer-related death in the United States and Europe.1 Due to its high occurrence rate and low (29%) 5-year survival rate at late stage, there is clearly an urgent need to develop new prognostic and therapeutic options.2 Prostate cancer is a highly heterogeneous pathology in need of improved diagnostics and therapeutics.3 Randomized trials and epidemiological data indicate that the current standard for prostate cancer diagnosisdetection of prostate-specific antigen (PSA) levelshas had little survival benefit for patients and has contributed to widespread overdiagnosis and overtreatment of prostate cancer.4 Recently, prostatespecific membrane antigen (PSMA) has become one of the most widely studied prostate cancer biomarkers and its expression has been proven to be associated with progression to androgen independence of prostate cancer.5−8 It was also validated by several methods that increased PSMA expression was found in benign prostatic hyperplasia, high-grade prostatic intraepithelial neoplasia, and prostatic adenocarcinoma.9 In spite of the reasonable expression in primary prostate tumors, © 2019 American Chemical Society
Received: April 6, 2019 Revised: April 25, 2019 Published: April 29, 2019 1314
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Bioconjugate Chemistry Scheme 1. Synthesis and 18F Labeling of the N3-NT-PSMA Heterodimer
Scheme 2. Synthesis and 18F Labeling of the N3-NT Monomer
prostate tissues and, thus, provides a potential target for prostate cancer diagnosis and therapy.24−26 Additionally, NTR1 was also reported to be expressed in neuroendocrine prostate cancers, in which PSMA expression was low.23,27 Clearly, NTR1 could be another important biomarker that may complement PSMA. Due to the heterogeneity seen in receptor
expression in prostate tumors, targeting more than one receptor may have an advantage over single receptor targeting. In this study, we report the development of a NT-PSMA heterodimer, which was then radiolabeled with 18F via strainpromoted azide−alkyne reaction (Scheme 1). The targeting efficacy was evaluated in both androgen-dependent LnCap and 1315
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Bioconjugate Chemistry Scheme 3. Synthesis and 18F Labeling of the N3-PSMA Monomer
Figure 1. HPLC profile of (a) 18F-BCN-NT-PSMA at radio channel, (b) 18F-BCN-NT-PSMA at 210 nm UV channel, (c) 19F-BCN-NT-PSMA at 210 nm UV channel, (d) 18F-BCN-NT at radio channel, (e) 18F-BCN-NT at 210 nm UV channel, and (f) 19F-BCN-NT at 210 nm UV channel.
could be obtained at 85.7% yield and 99% purity. In brief, 18FBCN was synthesized in 8% isolation yield based on our previous report.28 Through strain-promoted azide alkyne click reaction, the radiolabeling of the N3-NT-PSMA heterodimer and N3-NT monomers is performed and the decay-corrected isolation yield was 39.5% and 34.1%, respectively, with radiochemical purity over 99% (Figure 1). The purity was double confirmed by isocratic HPLC using 32% acetonitrile/ water 0.1% TFA as mobile phase (Figures S4 and S5). The identity of the product was confirmed by coinjection with the 19 F compounds. The 18F-BCN-N3-PSMA was prepared as previously reported.28 The logP value of 18F-BCN-NT-PSMA and 18F-BCN-NT was −1.06 ± 0.02 and −0.30 ± 0.02, respectively. The logP of 18F-BCN-PSMA was −1.90 ± 0.01, which was consistent with the previously reported value.28 In Vitro Cell Binding Assay. The NTR1 binding affinity of 19F-BCN-NT-PSMA and 19F-BCN-NT was evaluated in PC-3 cells and compared with the parent peptide NT (8−13). As can be seen in Figure 2a, both compounds showed dose-
androgen-independent PC-3 prostate cancer xenografts and compared with its corresponding NT and PSMA monomers (Schemes 2 and 3).
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RESULTS Chemistry and Radiochemistry. The N3-NT-PSMA heterodimer was synthesized by sequentially coupling NTR1 ligand (NT20.3) and PSMA ligand (Glu-urea-lys(Ahx)) to trifunctional linker N-(Azido-PEG3)-N-bis(PEG3-NHS ester). One of the two NHS ester groups reacts with NT first, leading to NT-trifunctional linker-NHS intermediate, unreacted trifunctional linkers, and some decomposed NHS esters. Excess amount of PSMA ligand was then added and N3-NTPSMA heterodimer was obtained with a yield of 44.3% based on NT20.3 after HPLC purification. Other impurities in final reaction mixtures may include N3-(PSMA)2 and N3-NTCOOH, which could be successfully removed to get N3-NTPSMA heterodimer with 99% purity. The azide group in heterodimer was left for further radiolabeling. Similarly, N3-NT 1316
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Figure 2. (a) Inhibition of 125I-NT (8−13) binding to NTR1 on PC3 cells by 19F-BCN-NT and 19F-BCN-NT-PSMA heterodimer (n = 3, mean ± SD). (b) In vitro binding assay of 19F-BCN-PSMA and 19FBCN-NT-PSMA heterodimer (n = 3, mean ± SD).
dependent binding curve with IC50 value of 50.2 nM for 19FBCN-NT-PSMA and 43.7 nM for 19F-BCN-NT. These values demonstrated that the NT-PSMA heterodimer has slightly lower but still comparable NTR1 binding affinity compared with the corresponding monomer. For the PSMA binding moiety, Figure 2b showed that the 19F-BCN-NT-PSMA had IC50 value of 156.4 nM, whereas 19F-BCN-PSMA had IC50 value of 26.1 nM in LnCaP cells. The heterodimer had 6-fold higher IC50 value, which indicated slightly reduced binding affinity than the corresponding monomer. Small Animal PET Imaging Studies. To evaluate the dual receptor targeting efficiency of 18F-BCN-NT-PSMA in vivo, we performed static PET imaging studies in both NTR1_positive/PSMA_negative PC-3 tumor model and NTR1_negative/PSMA_positive LnCaP tumor model at 40 and 120 min post injection. As shown in Figure 3, 18F-BCNNT-PSMA showed reasonable tumor uptake at 40 min post injection in both tumor models with uptake of 1.4 ± 0.3%ID/g in PC-3 and 1.3 ± 0.2%ID/g in LnCaP, respectively. The tracer was then cleared through the renal pathway and the tumor uptake was reduced to 0.5 ± 0.2%ID/g in PC-3 and 0.3 ± 0.1%ID/g in LnCaP at 120 min post injection. The specificity of each targeting moiety was confirmed by blocking studies where either an excess amount of NT peptide was coinjected in PC-3 tumor models or excess amount of PSMA ligand was coinjected in LnCaP models. Both blocking groups showed significant decrease of tumor uptake at 40 min post injection (Figure S6). As for the corresponding monomers, 18 F-BCN-NT only showed apparent tumor uptake in NTR1_positive PC-3 tumor xenografts with 1.2 ± 0.3%ID/g at 40 min post injection. The NTR1_negative/PSMA_positive LnCaP tumor did not have obvious tracer uptake (Figure 4). 18 F-BCN-PSMA, on the other hand, showed visible uptake in LnCap tumors but undetectable uptake in NTR1_positive/ PSMA_negative PC-3 tumors (Figure 5) similar to a previous
Figure 3. Representative PET/CT images of 18F-BCN-NT-PSMA heterodimer in PC-3 tumor models at (a) 40 min and (b) 120 min post injection. Representative PET/CT images of 18F-BCN-NTPSMA heterodimer in LnCaP tumor models at (c) 40 min and (d) 120 min post injection. Orange circled area is the tumor.
report.28 18F-BCN-NT-PSMA was mainly cleared through the renal pathway, similar to the two monomers. However, the absolute kidney uptake number was significantly reduced for the heterodimer, compared with PSMA monomer in both tumor models, which could be partially caused by the decreased water solubility (the logP value of 18F-BCN-NTPSMA and 18F-BCN-PSMA was −1.06 ± 0.02 and −1.90 ± 0.01, respectively). The heterodimer has comparable liver or muscle uptake with monomers at both time points. The quantitative uptake of other major organs was shown in Figure 6.
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DISCUSSION Prostate cancer is the most common cancer affecting men in the United States.2 Although the incidence has leveled off in recent years, the American Cancer Society estimates that approximately 181,000 men were diagnosed with prostate cancer in 2016.29 Approximately 60% of incident prostate cancer cases are diagnosed at ≥65 years of age.30 The initial treatment and subsequent monitoring of these large numbers of prostate cancer cases places a burden on U.S. health care systems. Prevalence based treatment costs for 2010 alone have been estimated at $11.9 billion.31,32 The current standard for prostate cancer screening involves a digital rectal examination and the determination of prostatespecific antigen (PSA) levels in patient blood samples followed by biopsy confirmation.33 However, high PSA level has been 1317
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Figure 4. Representative PET/CT images of 18F-BCN-NT in PC-3 tumor models at (a) 40 min and (b) 120 min post injection. Representative PET/CT images of 18F-BCN-NT in LnCaP tumor models at (c) 40 min and (d) 120 min post injection. Orange circled area is the tumor.
Figure 5. Representative PET/CT images of 18F-BCN-PSMA in PC-3 tumor models at (a) 40 min and (b) 120 min post injection. Representative PET/CT images of 18F-BCN-PSMA in LnCaP tumor models at (c) 40 min and (d) 120 min post injection. Orange circled area is the tumor.
observed in benign prostatic hyperplasia (BPH), and patients with low PSA value could have prostate cancer metastases.34 Clearly, developing new biomarkers for accurate prostate cancer diagnosis is still in great need. Recently, PSMA has attracted extensive attention due to its great potential in detecting metastasis of recurrent prostate cancer with high quality imaging results.10 However, prostate cancer is a highly heterogeneous disease, and some prostate metastases could have low or negative PSMA expression based on recent studies.35 Clearly, the heterogeneity of prostate cancer needs to be addressed when developing novel prognosis and therapeutic options for prostate cancer. One strategy to improve the sensitivity of an imaging agent is to add another targeting moiety and combine the two specific targeting agents into one dual-targeting molecule. Here we utilized a trifunctional linker with two N-hydroxysuccinimide groups for targeting moiety conjugation and one azide branch for radiolabeling (Scheme 1). Compared with some previous studies on heterodimer synthesis, our method did not involve isomers which may form using asymmetric linkers. Furthermore, by introducing azide group for radiolabeling, strain-promoted azide−alkyne reaction could be easily applied. This would improve the radiolabeling yield and make the synthesis procedure straightforward as long as the ligands to be screened bear a N3 group. We first evaluated the in vitro cell binding affinity of the heterodimer. The NTR1 targeting moiety retained its binding
property compared with the corresponding monomer, whereas the PSMA moiety showed reduced binding affinity. The reduced binding affinity for the PSMA moiety might be caused by the introduction of a relatively large NT peptide motif. The NT peptide accounted for roughly half of the molecular weight of 19F-BCN-NT-PSMA, while PSMA ligand only contributed to 1/10. In fact, the binding affinity was not altered much between NT monomer and heterodimer but showed a large decrease for heterodimer compared with PSMA monomer. This was further confirmed by microPET imaging studies. Although the heterodimer accumulated in both NTR1 and PMSA positive tumor models, the heterodimer uptake in LnCaP tumor models was less than PSMA monomer which may be partially due to the reduced binding affinity for the PSMA moiety. The specificity was confirmed by performing imaging studies of monomers in both NTR1 positive/PSMA negative and NTR1 negative/PSMA positive tumor xenografts. Our results showed that monomers could only be observed in either one of the tumor models, which indicated the specificity of each targeting moiety. However, the heterodimer tracer could successfully visualize both tumor models, which implied it could serve as a diagnostic tool in heterogeneous prostate cancer. Another impact that needs to be considered when studying heterodimer is the pharmacokinetic properties compared with each specific monomer. The heterodimers should at least show comparable pharmacokinetic properties with the separate 1318
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Figure 6. PET derived quantitative uptake of BCN-NT-PSMA heterodimer and corresponding monomers in (a) PC-3 at 40 min, (b) PC-3 at 120 min, (c) LnCaP at 40 min, and (d) LnCaP at 120 min.
further supported by the observation that PSMA negative prostate cancer (for example PC-3) could have high NTR1 expression, suggesting the complementary role of these two targets. We would like to point out that these agents would need to be further optimized before clinical application. The absolute tumor uptake for the current construct is still low and may not meet the therapeutic requirements. Our future design would focus on increasing absolute tumor uptake and reducing the uptake in normal tissues.
application of both monomer compounds. In our scenario, the heterodimer showed similar tumor uptake with the corresponding monomer N3-NT in NTR1 positive/PSMA negative PC-3 tumor xenografts. Normal organ distribution such as liver, kidney, and muscles is also comparable. Regarding the comparison between heterodimer and N3-PSMA monomer, both compounds showed similar liver uptake but the heterodimer exhibit significantly reduced kidney uptake. Since Glu-urea-lys(Ahx) was much more hydrophilic than NT peptide, the combination of the two moieties in heterodimer may increase the lipophilicity and thus reduce the kidney uptake. However, we would like to point out that all three tracers showed high signals from the abdomenal area; thus, the uptake reduction in kidney did not change too much on the tumor to background contrast. Compared with other commonly used PSMA PET probes such as 68Ga-PSMA11, 18 F-DCFPyL, and 18F-PSMA-1007, the LnCaP tumor uptake of 18F-BCN-NT-PSMA heterodimer was lower along with reduced uptake in other major organs such as liver and kidney.36−38 However, integration of the NTR1 targeting moiety could not only increase prostate cancer sensitivity, but also combine two NTR1 and PSMA PET scans into one, which would potentially reduce the radiation exposure to patients. This dual targeting approach could also provide a treatment option that would target more cancer lesions once the heterodimer is loaded with therapeutic moieties. In addition to guiding radiation therapy after detecting the lesions, therapeutic radionuclide or chemo-drugs could be conjugated with the NT-PSMA heterodimer for targeted delivery to prostate cancer. The 18F labeled heterodimer could be used as a companion diagnosis agent. The dual targeting rationale was
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CONCLUSION
We have developed a heterodimeric peptide that binds to two distinct antigens related to prostate cancer: neurotensin receptor 1 and prostate-specific membrane antigen. In vitro and in vivo experiments demonstrated that PSMA-NT heterodimer has promising bispecific targeting capability, which would benefit prostate cancer imaging by enhancing the sensitivity. In addition to the imaging applications demonstrated in this study, the agent also holds great potential in the treatment of heterogeneous prostate cancer.
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MATERIALS AND METHODS All chemicals obtained from commercial source were used without further purification. NT peptide Lys-NT20.3 was purchased from C.S. Bio (CA,USA) and PSMA ligand Gluurea-lys(Ahx) was purchased from FutureChem (Seoul, Korea). A Kintex 5 μ C18 column (250 mm × 4.6 mm) was used on analytical reversed-phase HPLC and Gemini 5 μ C18 column (250 mm × 10 mm) was used for semiprep reversedphase HPLC. Solvent A was 0.1% TFA in water and solvent B was 0.1% TFA in acetonitrile. The flow was 3 mL/min. 1319
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HPLC program A: The mobile phase was 95% solvent A and 5% solvent B from 0 to 2 min and jumped to 70% solvent A and 30% solvent B at 4 min. Then the gradient went to 60% solvent A and 40% solvent B at 24 min. HPLC program B: The mobile phase was 95% solvent A and 5% solvent B from 0 to 2 min and ramped to 5% solvent A and 95% solvent B in 20 min. Preparation of N3-NT-PSMA Heterodimer. N-(AzidoPEG3)-N-bis(PEG3-NHS ester) was purchased from Broadpharm (CA, USA). To a solution of N-(Azido-PEG3)-Nbis(PEG3-NHS ester) (2.5 mg, 3.1 μmol) in 50 μL of anhydrous dimethyl sulfoxide (DMSO), 0.3 equiv of LysNT20.3 (1.0 mg, 0.9 μmol) was added. After adding 7.4 mg (57.4 μmol) of diisopropylethyl amine, the mixture was incubated at room temperature for 2 h. Without purification, 1.5 equiv of Glu-urea-lys(Ahx) (1.5 mg, 4.7 μmol) was added and the reaction was stirred for another 2 h. The desired product was isolated by semiprep HPLC using HPLC program A. Preparation of N3-NT and N3-PSMA. Azido-PEG-NHS (1.0 mg, 3.3 μmol) was dissolved in 50 μL of anhydrous DMSO. 0.3 equiv of Lys-NT20.3 (1.1 mg, 1.0 μmol) was added to the solution and incubated at room temperature for 2 h. The product was isolated with semiprep HPLC using HPLC program B. N3-PSMA was synthesized according to our previously reported protocol.28 Radiochemistry. 18F-bicyclo[6.1.0]nonyne (18F-BCN) was synthesized according to our previously reported protocol.28 To prepare corresponding PET agents, 50 μg of N3-NTPSMA, N3-NT, or N3-PSMA was added to the solution containing 185 MBq of 18F-BCN respectively and incubated at 80 °C for 15 min. The mixture was loaded on HPLC for further analysis and purification. The desired product collected from HPLC was reconstituted in 1× PBS and rotary evaporated to remove the organic solvent before being subjected to further in vivo experiments. The octanol/water partition coefficients were measured based on the literature report.28 Cell Lines and Animal Models. Prostate cancer cell lines PC-3 and LnCaP were obtained from the Tissue Culture Facility of UNC Lineberger Cancer Center. Cell culture was performed as described.39 Animal procedures were performed according to a protocol approved by the University of North Carolina Institutional Animal Care and Use Committee. In brief, PC-3 xenograft model was established by subcutaneous inoculation of 5 × 106 PC-3 cells resuspended in 100 μL PBS into 4- to 6-week-old male athymic mice (BALB/nu/nu), while LnCaP xenografts were established by subcutaneous inoculation cells 100 μL mixture of Matrigel and 5 × 106 LNCaP cells at 1:1 volume into NSG mice. In Vitro Cell Binding Assay. The in vitro cell binding assays toward NTR1 and PSMA were performed according to previously reported protocol.28,40 Small Animal PET Imaging Studies. The PET scan and analysis were performed as previously reported.28 PC-3 or LnCaP tumor model mouse was injected with either 18F-BCNN3-NT-PSMA, 18F-BCN-N3-NT, or 18F-BCN-N3-PSMA and subjected to static PET scan at 40 and 120 min post injection. Statistical Analysis. Quantitative data are expressed as mean ± SD. Means were compared using one-way ANOVA and the Student’s t test. P < 0.05 were considered statistically significant.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.9b00252.
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HPLC profile of starting materials and radiolabeling reaction crude; imaging results of blocking study (PDF)
AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected]. *E-mail:
[email protected]. ORCID
Vladimir V. Popik: 0000-0001-9112-3935 Zibo Li: 0000-0002-1063-0298 Author Contributions #
X.M. and M.W. contributed equally.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by NSF grant (CHE-1565646), UNC Department of Radiology and BRIC, the science foundation of Guangdong Second Provincial General hospital (grant number: YN2018-001), and the Science and Technology Program of Guangzhou, China (grant number: 201804010448). Small animal PET were obtained with instrumentation supported by NIH grants 1S10OD023611.
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