1. Introduction
Immune checkpoints, which mitigate immune activities under physiological conditions, can be hijacked by cancer cells, thereby overcoming the patient’s anti-tumor immune response. Amongst the immune checkpoints, programmed death-ligand 1 (PD-L1) and its receptor programmed death-1 (PD-1), have drawn the most attention during the last 10 years and monoclonal antibody (mAb)-based immunotherapies antagonizing PD-1/PD-L1 have revolutionized the cancer treatment paradigm [
1,
2]. Recent clinical trials showed long-lasting responses and provided evidence for their importance in cancer therapy [
3]. However, only a subset of patients benefits from existing treatments, typically 20% [
4]. This low response rate can be explained, at least partially, by the lack of a precise technique to select patients with PD-L1 expression [
5,
6]. Immunohistochemistry (IHC) is currently employed in order to assay PD-L1 expression by staining a biopsy sample, which is not representative for the heterogeneous expression within the primary tumor lesion, nor within the metastases [
7]. In addition, PD-L1 expression within the tumor microenvironment is a dynamic process that can be influenced by routine cancer treatments, such as radiotherapy [
8]. Moreover, PD-L1 expression is not limited to tumor cells, as immune cells within the tumor can also express PD-L1 and contribute to immune escape [
9]. To date, several antibodies are available for IHC with variable ability to stain PD-L1 on tumor cells and immune cells, implying that the pathologist’s interpretation during analysis is important to categorize samples as positive or not [
10].
Pre-clinical and clinical studies with radiolabeled mAbs showed that the assessment of the PD-L1 status by positron emission tomography (PET) imaging is a promising strategy for patient stratification [
11,
12,
13]. However, mAbs have very slow clearance kinetics from the blood through the hepatobiliary system and, therefore, imaging is performed several days post-injection (p.i.), in order to achieve a sufficiently low background signal to visualize the molecular target.
Nanobodies (Nbs), also called single domain antibody fragments, can overcome limitations linked to the use of mAbs for PET imaging. Their high affinity and specificity, as well as their small size (~15 kDa), make Nbs ideal probes for molecular imaging. Their fast blood clearance allows for imaging as early as 1 h p.i. and radiolabeling with short-lived radioisotopes, lowering the radiation burden for the patients [
14]. We previously selected a lead high affinity PD-L1-specific Nb that allowed non-invasive SPECT/CT imaging of PD-L1 expression in murine tumor models with varying PD-L1 expression. [
15] The
99mTc-labeled Nb revealed high signal-to-noise ratios, strong ability to specifically detect PD-L1 in melanoma and breast tumors, and surprisingly low kidney retention, unlike many other radiolabeled Nbs.
For further clinical translation, we aimed to radiolabel and validate the Nb with a PET radioisotope, such as Gallium-68 (
68Ga) (radioactive half-life 67.7 min.), which is commonly used in clinical settings. To allow for chelation of
68Ga, Nbs are typically conjugated with the bifunctional chelating agent 2-
S-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (
p-NCS-Bn-NOTA) on the primary amino groups of the lysines of the Nb’s structure [
16]. Although this strategy is straightforward and already applied to functionalize Nbs undergoing clinical trials [
16,
17], it can present a problem when there are lysines in one of the three complementarity determining regions (CDRs) of the Nb sequence, as is the case for the PD-L1-specific lead compound [
18], as these are hypervariable regions that are likely involved in antigen-binding.
In the current study, we aimed to determine whether the lysine in the CDR could hamper binding capacity of the Nb, which is critical information before selecting a bioconjugation technique suitable for clinical translation. To this end, we compared the impact on the Nb of two bioconjugation strategies: a classical approach using conjugation on lysines (further referred to as random approach) versus a site-specific approach while using the Sortase-A-mediated transpeptidation [
19,
20].
2. Materials and Methods
2.1. Reagents
All of the reagents and solvents were purchased from Sigma–Aldrich (Overijse, Belgium) or VWR (Oud-Heverlee, Belgium). Buffers used for coupling reactions and for radiolabeling were prepared with metal free water (Honeywell, Fluka, Brussels, Belgium) and purified from metal contamination using Chelex 100 resin. p-SCN-Bn-NOTA was purchased from Macrocyclics (Plano, USA). 68Ga was obtained from a 68Ge/68Ga generator (Galli Eo™, IRE ELiT, Fleurus, Belgium) eluted with 0.1 N HCl. [67Ga]Ga-citrate solution was purchased from Mallinckrodt (Amsterdam, The Netherlands).
2.2. Chromatographic Analysis
Size-exclusion chromatography (SEC) columns were purchased from GE Healthcare (Diegem, Belgium). SEC purification of the site-specifically functionalized Nb was performed on a Superdex 75 Increase 10/300 GL column using 0.1 M NH4OAc Ph 7, at a flow rate of 0.8 mL/min. SEC purification of randomly functionalized Nb was performed on a Superdex Peptide 10/300 GL column while using 0.1 M NH4OAc pH 7, at a flow rate of 0.5 mL/min. For quality control (QC), radiochemical purity (RCP) was assayed with binderless glass microfiber paper that was impregnated with silica gel (instant thin layer chromatography, iTLC-SG) (Agilent Technologies, Diegem, Belgium) using 0.1 M sodium citrate buffer pH 4.5–5 as eluent. RCP was also assayed by SEC on a Superdex Peptide 3.2/300 GL using a 2× Phosphate-buffered saline (2× PBS: 5.36 mM KCl, 273.8 mM NaCl, 2.94 mM KH2PO4, 16.2 mM Na2HPO4) at a flow rate of 0.150 mL/min. Serum and urine samples were analyzed on a Superdex 5/150 GL using 2× PBS at a flow rate of 0.3 mL/min.
2.3. Production and Purification of the hPD-L1 Nb and Sortase-A Enzyme
The lead hPD-L1 Nb K2 with a his
6-tag and/or a sortag (LPETG) and the Sortase-A enzyme were produced as described before [
15,
19,
20].
2.4. Synthesis of GGGYK-NHCS-Bn-NOTA
2.5. Site-Specific Nb Functionalization
These procedures are essentially described elsewhere [
20]. To the (hPD-L1)-sortag-his
6-tag Nb (1 eq., 50 μM in final volume) in Tris-buffered Saline (TBS) pH 7, was added GGGYK-NHCS-Bn-NOTA (20 eq., 1 mM in final volume) and his
6-tagged Sortase-A enzyme (2 eq., 100 μM in final volume). 150 µL (10% of final reaction volume) of 10× Sortase buffer (500 mM Tris-HCl, 150 mM NaCl, 100 mM CaCl
2, pH 7.7) was added and the reaction volume was topped up to 1.5 mL with metal free water. The reaction mixture (RM) was incubated 16 h at 37 °C. The his
6-tagged compounds (unreacted Nb, Sortase-A enzyme, cleaved
C-terminus of the Nb) were removed by immobilized metal affinity chromatography (IMAC) by adding Ni-NTA resin (500 µL in TBS, Thermo Fisher Scientific, Belgium) and then moderately shaken for 2 h at room temperature (RT). After filtration, the flow-through was incubated with excess EDTA (50 mM final concentration) for 1 h at RT. RM was concentrated on a vivaspin 2 (MWCO 5KDa, Sartorius, Belgium) and then purified by SEC.
2.6. Random Conjugation of the NOTA Chelator to the Lysines of the Nb
Conjugation was performed, as described elsewhere [
16]. Briefly, the his
6-tag hPD-L1 Nb in 0.05 M sodium carbonate buffer (1.2 mg/mL, 2 mL) was incubated for 2.5 h at RT with
p-NCS-Bn-NOTA (20 eq.) at pH 8.5–8.7. RM was neutralized by the addition of 1 N HCl, concentrated, and purified by SEC.
2.7. Quality Controls of NOTA-Nbs
The purity of the functionalized hPD-L1 Nbs was assessed by SEC, SDS-PAGE, and Western Blot (WB). The equilibrium dissociation constant (K
D) of the unmodified and NOTA-functionalized Nbs was measured by surface plasmon resonance SPR. The procedures are described in the
Supplementary Information.
2.8. Nanobody Radiolabeling
The site-specifically or randomly conjugated NOTA-(hPD-L1) Nb (7.0–9.0 nmol) in 1 mL of 1 M NaOAc buffer pH 5 was incubated 10 min. at RT with 1 mL of 68Ga eluate (340-950 MBq). The radiolabeled-Nb solution was purified by SEC on a PD-10 column (GE Healthcare, Belgium) pre-equilibrated with freshly prepared 0.9% NaCl containing 5 mg/mL vitamin C pH 5.8–6.1 (injection buffer). The final solution was filtered through a 0.22 µm filter (Millipore, Belgium). RCP was determined before and after purification by radio-iTLC ([68Ga]Ga-NOTA-Nb Rf = 0, [68Ga]Ga-citrate Rf = 1).
For studies that require later time point analysis, labeling with Gallium-67 (
67Ga, t
1/2 = 78.3 h) was performed. The NOTA-(hPD-L1) Nb solution (7.0 nmol) was brought to pH 5 with 5 M NH
4OAc pH 5–5.2 and incubated for 10 min. at RT with 200 μL (100–400 MBq) of [
67Ga]GaCl
3, obtained from [
67Ga]Ga-citrate, as previously described [
21]. The radiolabeled Nb was purified by SEC NAP-5 column (GE Healthcare, Belgium), eluted with 1 mL of injection buffer, and filtered. RCP was determined by radio-iTLC.
Decay-corrected radiochemical yield (DC-RCY) was calculated for the time point after SEC purification.
2.9. Stability Studies
The stability of the 68Ga or 67Ga-labeled Nbs (15–50 MBq, after filtration) was tested over 4 h at RT, in human serum (HS) at 37 °C, and in the presence of a 1000-fold excess of competitor (DTPA chelator, which is able to chelate 68Ga at RT) to assay transchelation that could occur with transferrin in vivo before going to mouse models. At different time points, the aliquots were analyzed by radio-iTLC and radio-SEC.
2.10. Animal Models and Cell Lines
Dr. S.L. Topalian (National Cancer Institute, USA) provided HLA-A*0201
+ 624-MEL cells. The 624-MEL cells were stably transduced to express hPD-L1 and they have been characterized, as previously described [
15]. The 624-MEL cells were cultured in RPMI1640 medium supplemented with 10% Fetal clone I serum (Thermo Scientific, Belgium), 2 mM L-Glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin, 1 mM sodium pyruvate, and nonessential amino acids. Female, five to six weeks old C57BL/6 mice (for biodistribution, biological half-life in blood and stability studies), and athymic nude Crl:NU(NCr)-Foxn1nu mice (for tumor targeting) were purchased from Charles River. All of the experiments were performed in accordance with the European guidelines for animal experimentation under the license LA1230272. Experiments were approved by the Ethical Committee for the use of laboratory animals of the Vrije Universiteit Brussel (17-272-6 and 20-272-4). Intravenous injections were performed in the tail vein. The animals were anesthetized with 2.5% isoflurane in oxygen (Abbott) for injections, samplings, imaging, and euthanasia.
2.11. Cell Binding Study
The radiolabeled Nb’s specificity was tested on transduced hPD-L1 positive (hPD-L1
POS) 624-MEL cells [
15]. Excesses of unmodified, unlabeled Nb, or untransduced hPD-L1 negative (hPD-L1
NEG) 624-MEL cells were used as control conditions. The procedures are detailed in the
Supplementary Information.
2.12. Affinity Measure (KD) by Cell Saturation Assay
The affinity of the radiolabeled Nb was tested on hPD-L1POS 624-MEL cells. 5x104 cells in 1 mL of medium per well were allowed to attach in a 24 well plate at 37 °C two days prior to the experiment. The plate was cooled to 4 °C 1 h prior to the experiment. The supernatant was removed and the cells were incubated for 1 h at 4 °C with 500 μL of a 68Ga-labeled Nb solution at different concentrations (300 nM, 100 nM, 33.3 nM, 11.1 nM, 3.7 nM, 1.2 nM, 0.4 nM, and 0.1 nM) in unsupplemented medium (N = 3 wells per condition). The wells were processed the same way as the cell binding study. To correct for nonspecific binding, the same procedure was simultaneously applied to a second plate containing 100-molar excess of unlabeled competitor in each well. The KD was calculated while using a “One site—total and nonspecific binding” analysis in Prism software.
2.13. Biological Half-Life in Blood of the 68Ga-Labeled Nbs
C57BL/6 mice were injected intravenously (N = 6 per group) with 6.6 ± 1.2 MBq of randomly or site-specifically 67Ga-labeled Nb (10 μg NOTA-Nb) and blood samples from different time points (5, 10, 20, 30, 40, 60, 120, and 180 min.) were counted against a standard of known activity using a γ-counter. Blood sample volume was calculated and activity in blood was expressed as a percentage of injected activity per total blood volume (%IA/TBV). The biological half-life was calculated using a one phase decay model in the Prism software. The experiment was repeated with 28.6 ± 2.1 MBq of 68Ga-labeled Nbs (10 μg NOTA-Nb).
2.14. In Vivo Stability Studies
C57BL/6 female mice (N = 8 per group) were intravenously injected with 9.4 ± 1.2 MBq of randomly or site-specifically 67Ga-labeled Nb (10 μg NOTA-Nb). At each time points (5, 15, 45, and 120 min.), two animals were euthanized to collect blood and urine samples to determine the percentage of intact 67Ga-labeled Nb in the samples. Samples were diluted with 0.1 M sodium citrate buffer pH 4.5–5 containing 0.1% Tween 80, filtered with 0.22 μm filter and analyzed by radio-SEC. The experiment was repeated with 68Ga-labeled Nbs with the same experimental settings (10 μg NOTA-Nb, 30.5 ± 7.1 MBq).
2.15. Biodistribution and Comparative Tumor Targeting Studies
Athymic nude mice (seven weeks old, 42 animals) were subcutaneously injected in the right leg with 5x106 hPD-L1POS 624-MEL cells. Tumor volume was measured twice weekly with an electronic caliper and calculated using the following formula: (length × width2)/2. The animals were randomized (N = 21/group, two groups). After three to six weeks post inoculation, the tumor volume reached 99 ± 54 mm3 for the first group and 87 ± 44 mm3 (NS) for the second group, injected respectively with site-specifically 68Ga-labeled Nb (4.5 µg NOTA-Nb; 20.5 ± 2.1 MBq, 64.8 GBq/μmol) and randomly 68Ga-labeled Nb (6.0 μg NOTA-Nb; 19.4 ± 2.1 MBq, 45.8 GBq/μmol). Injected activities and apparent molar specific activities are reported for the time of injection.
As a control group, athymic nude mice (six weeks old, N = 6/group) were injected with 4.2 × 106 hPD-L1NEG 624-MEL cells, allowing for reaching 303 ± 303 mm3 in three weeks.
Biodistribution was evaluated at 1 h 20 p.i. After euthanasia, main organs and tissues were isolated, weighed, and counted against a standard of known activity using a γ-counter. The amount of radioactivity in organs and tissues was expressed as percentage of injected activity per gram (%IA/g), corrected for decay. A single cell suspension from the tumors was prepared and flow cytometry analysis was performed in order to characterize hPD-L1 expression (procedure in the
Supplementary Information).
2.16. PET/CT Iimaging and Analysis
hPD-L1POS 624-MEL xenografted athymic nude mice (nine weeks old), with a tumor size of (615 ± 502) mm3 (N = 2) were injected with site-specifically 68Ga-labeled Nb (11 μg NOTA-Nb, 17.8 ± 2.2 MBq, 23.0 GBq/µmol). The acquisition was performed with a β-CUBE PET/CT system (MOLECUBES, Ghent, Belgium) 1 h 20 min. p.i. Total PET/CT scanning time was 17 min. The PET images were acquired over 15 min. and reconstructed into a matrix of 193 × 192 × 384 voxels with 400 μm voxel size. The CT images were iteratively reconstructed using the ISRA reconstruction algorithms into 200 μm voxels (matrix 200 × 200 × 393). The animals injected with randomly 68Ga-labeled Nb or animals bearing negative tumors were not scanned for logistical reasons.
2.17. Statistical Analyses
The calculation of the amount of necessary animals for the comparative tumor targeting study between randomly and site-specifically labeled Nbs was performed using a Wilcoxon–Mann–Whitney test (two groups) analysis in G POWER (considering that 1% difference uptake would be a relevant difference between the two groups for this model, with 95% confidence, with a pooled standard deviation (stdev) of 1.09087 based on preliminary targeting studies.
The results are expressed as mean ± stdev. A non-parametric Mann–Whitney U test was carried out to compare the data sets. Sample sizes and number of repetitions of experiments are indicated in the figure legends or in the materials and methods section. The number of asterisks in the figures indicates the statistical significance, as follows: * p < 0.05; ** p < 0.01; *** p < 0.001; Non-significant (NS).
4. Discussion
In the past years, antibody-based treatments blocking the interactions between PD-1 and its ligand PD-L1 have been developed to restore the patient’s anti-cancer immune activity, however providing clinical benefits in only a fraction of all patients. Many novel PET imaging agents are being developed in order to improve upfront patient selection as well as follow up changes in PD-L1 expression during treatments, based on full antibodies or smaller targeting moieties, such as peptides [
24], affibodies [
25], adnectins [
26], and Nanobodies [
27,
28]. Here, we present our results on a Nb targeting human PD-L1 that was previously selected based on its theranostic capacity [
15], and that we now prepare for patient use by the development of
68Ga labeling strategies suitable for clinical applications.
The hPD-L1 Nb was functionalized with NOTA in a site-specific manner using an enzymatic coupling reaction at the C-terminal end of the Nb and in a random manner on exposed lysine residues throughout the Nb protein. The Nb protein contains in total four lysines, including a lysine residue located in one of its binding region. It is unknown which lysine or how many lysines will be coupled to NOTA after a random coupling, and thereby this strategy could affect the Nb’s binding potential. Both strategies resulted in good recovery yields, high radiochemical yields and excellent radiochemical purity. However, the site-specific strategy allowed 68Ga-labeling with less Nb, resulting in an injectable product with higher apparent molar specific activity than for the random strategy.
Both radiolabeled Nbs were stable in vitro in injection buffer over 4 h. In human serum at 37 °C, both
68Ga-labeled Nbs were stable over 1 h (>95% RCP). A smaller fragment could be observed at later time points (4 h) and it is most likely due to radiolysis, which is a result of combined positron emission and high temperatures [
22]. As the biological half-life of the compounds is very short and since the product is diluted in the blood, radiolysis effects will be lower, and these effects will not be clinically relevant.
The stability of both probes was also evaluated in vivo up to 2 h post injection. The radioactive metabolites, as observed in urine, were relatively higher for the random than for the site-specific compound, but no signs of recirculation in the blood could be detected, which makes those metabolites not relevant for imaging purposes.
In vitro and in vivo, the site-specific strategy could theoretically provide better targeting properties, since the attached chelator does not interfere with the CDRs interacting with the antigen. Experimental data however demonstrated that tumor uptake of the randomly radiolabeled Nb is as good as the site-specifically radiolabeled analogue.
When considering that the lead compound hPD-L1 Nb was found to be not cross-reactive to the murine PD-L1 [
15], no specific uptake in other organs or tissues (besides the tumor) in mice was expected. In patients, the biodistribution of the hPD-L1 Nb in healthy tissues will have to be assessed. Uptake can mainly be expected in the spleen as for the
18F-labeled Adnectin [
12] and brown fat as reported for the murine PD-L1 binder in wild mice [
29]. Kidney and bladder retention are due to the excretion route of Nbs. Kidney retention of the randomly labeled Nb was higher than for the site-specifically radiolabeled Nb, which can be attributed to the presence of a his
6-tag at the
C-terminus of the randomly radiolabeled Nb, affecting the
C-terminal charge as already observed [
30,
31], while the his
6-tag is removed by the Sortase-A enzyme during the site-specific coupling [
32]. Nevertheless, kidney uptake of the site-specifically radiolabeled hPD-L1 Nb is the lowest ever reported for a Nb. The rationale for this effect is still unknown and could be further investigated in the future.
Preliminary studies involving six animals per group bearing hPD-L1POS tumors indicated higher tumor uptake for the site-specifically labeled Nb. However, high variation in uptake was observed, which was likely due to variations in the tumor model. Based on these data, an additional experiment was designed involving 42 randomized animals (21/group). Tumor uptake at 80 min. p.i. of the site-specifically and randomly labeled Nbs in the hPD-L1POS 624-MEL tumors was not significantly different, thereby statistically proving the absence of a relevant difference. These results indicate that the position of the NOTA chelator does not negatively impact the affinity, and that the lysine in the CDR does not present a problem for this particular Nb.
This was, to our knowledge, the first time that the role of a random coupling on lysines was investigated using a Nb with a lysine in its CDR. Our observations are only valid for this 68Ga-NOTA labeling, but the effects on affinity and biodistribution might be different if this Nb would be coupled while using lysines to other molecules (e.g., chelator, prosthetic groups, fluorescent dye) or other radioisotopes, as the accessibility of the lysine in the CDR and the effect on binding properties might be very different. Moreover, lysines in the framework structure of Nbs can also participate in the binding, therefore when the crystal structure of the lead Nb is not available, a side-by-side comparison is necessary for such coupling and labeling methods. Nbs with lysines in their CDRs should not be immediately excluded for lead compound selection and for random conjugation, but should rather be evaluated, especially when no information on their crystal structure is available.
The here reported tumor uptake values were in the same range as for previously pre-clinically tested anti-PD-L1 small-sized PET probes. For example, 2.4 ± 0.3%ID/g uptake was reported for the
18F-labeled Adnectin in L297 xenografts at 90 min. p.i. [
26] and 1.7 to 5%ID/g for the
68Ga-labeled Nb109 at 1 h p.i. in A375 and MCF-7 xenografts, respectively [
27]. Tumor-to-blood ratios were in the same range as other probes (6 vs. 5 vs. 10 vs. 3 vs. 5 for site-specifically
68Ga-labeled Nb, randomly labeled Nb 80 min. p.i.,
89Zr-labeled Atezolizumab mAb 72 h p.i. [
11],
18F-labeled Adnectin 90 min. p.i. [
26], and
68Ga-labeled Nb109 [
27], respectively). Tumor-to-muscle ratios were even higher for the here tested compounds than those that were reported for other anti-PD-L1 PET probes (34 and 28 for our labeled Nbs, compared to only 2–10 for the mAb, 12 for the Adnectin, and 9 for the Nb109) adding to the potential of our compounds. As compared with the
68Ga-labeled Nb109, the biological half-life of our Nb variants were shorter (12.4 vs. 10.8 vs. 49.8 min. for the site-specifically
68Ga-labeled Nb, randomly labeled Nb and
68Ga-labeled Nb109, respectively), as well as lower kidney retention resulting in images with low background. Low-level background activity is especially important to correctly assess low-levels of expression, as it is the case for PD-L1, with for example cut-offs of only 1 to 5% positive cells in immunohistochemistry for optimal treatment selection in lung carcinoma patients [
33].
Taking together all of the results obtained for each of the functionalization strategies, their high production yields, high purity, specific tumor targeting, and excellent tumor-to-background ratios make them both excellent candidates for future clinical translation. The site-specific coupling method offers the advantage that it yields a homogenous pharmaceutical product, which is not the case for the random lysine conjugation. Its implementation in a GMP radiopharmacy is however less straightforward as compared with the random strategy due to the use of an enzyme. Nevertheless, the use of enzymes in pharmaceutical productions is increasing, for example in antibody drug conjugate production for clinical trials, and such a development might also facilitate the use of the Sortase strategy for radiopharmaceutical production [
34]. Since the in vivo study showed no impact on the uptake of the randomly radiolabeled Nb in positive tumors as compared with the site-specifically radiolabeled Nb and, given their similar K
D in vitro, the random strategy is currently the easiest strategy for clinical translation in the case of this hPD-L1 Nb. This strategy was also applied to the anti-HER2 and -MMR Nbs that are currently undergoing clinical trials, so that the translation of the hPD-L1 Nb should be similar and straightforward. The radiation burden for the hPD-L1 Nb will be considerably lower when compared with other Nbs in clinical trials due to it low kidney retention. Moreover, as the use of Nbs targeting PD-L1 enables PET imaging already at early time points after injection with patient procedures very similar to current clinical practice with [
18F]FDG, implementation in routine patient care should also be very straightforward.