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Recombinant PASylated nanobody probes with improved blood circulation and tumor targeting

Guangdong Provincial Key Laboratory of Advanced Biomaterials, Department of Biomedical Engineering, Southern University of Science and Technology, Shenzhen, Guangdong 518055, P. R. China

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Lingyue Jin

https://orcid.org/0009-0001-0165-1556

Guangdong Provincial Key Laboratory of Advanced Biomaterials, Department of Biomedical Engineering, Southern University of Science and Technology, Shenzhen, Guangdong 518055, P. R. China

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You Zhang

https://orcid.org/0000-0001-7968-8079

Department of Nuclear Medicine, Institute of Clinical Nuclear Medicine Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200127, P. R. China

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Siyu Zhou

https://orcid.org/0009-0000-2296-4001

Guangdong Provincial Key Laboratory of Advanced Biomaterials, Department of Biomedical Engineering, Southern University of Science and Technology, Shenzhen, Guangdong 518055, P. R. China

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Weijun Wei

https://orcid.org/0000-0003-3190-2480

Department of Nuclear Medicine, Institute of Clinical Nuclear Medicine Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200127, P. R. China

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Gang Huang

https://orcid.org/0000-0003-1939-3978

Department of Nuclear Medicine, Institute of Clinical Nuclear Medicine Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200127, P. R. China

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, and

Changfeng Wu

https://orcid.org/0000-0001-6797-9784

Guangdong Provincial Key Laboratory of Advanced Biomaterials, Department of Biomedical Engineering, Southern University of Science and Technology, Shenzhen, Guangdong 518055, P. R. China

E-mail Address: wucf@sustech.edu.cn

Corresponding author.

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https://doi.org/10.1142/S1793545825410019Cited by:0 (Source: Crossref)

Abstract

Nanobodies have been extensively demonstrated in biomedical imaging and therapy. However, nanobody probes often suffer from rapid renal clearance due to its small size. Herein, we reported a recombinant nanobody with a 200 amino-acid polypeptide chain consisting of Pro, Ala, and Ser (PAS) at the C-terminal, which can be easily expressed in Escherichia coli with a high yield. The PASylated nanobody was functionalized with a fluorescent dye and the cell labeling properties were characterized by flow cytometry and confocal microscopy. In vivo fluorescence imaging indicated that the PASylated nanobody showed comparable blood circulation time, but $\sim 1.5$ times higher tumor targeting ability as compared to the PEGylated nanobody of comparable molecular weight. Our findings demonstrate that nanobody PASylation is a promising approach to produce long-circulating nanobody probes for imaging and therapeutic applications.

Keywords:

1. Introduction

Nanobodies, also known as single-domain antibodies, are derived from the variable region (VHH) of heavy-chain only antibodies (HCAb), which was first identified in camelids in 1993.¹ Nanobodies are considered as promising targeting molecules due to their full affinity and specificity when expressed alone.² Compared to full-length antibodies and artificial antibody derivatives, nanobodies have several advantages including high affinity, low immunogenicity, high solubility, and low aggregation in vivo.^3,4 Additionally, the small size of nanobodies not only enables them to bind to some antigens that are difficult to access with full-length antibodies,^5,6,7 but also makes them easily engineered to produce polyvalent nanobodies or various fusion proteins.^8,9 These properties make nanobodies a promising targeting moiety in antibody-drug conjugates^10,11,12,13 and molecular imaging.^14,15,16 Nevertheless, the extensive use of nanobodies in medical imaging has been constrained by their small size and rapid filtration by the kidney, resulting in a short blood circulation and reduced targeting in vivo.¹⁷

The most established approach to improve blood circulation of a probe is chemical conjugation of poly-(ethylene glycol) (PEG), a hydrophilic and uncharged synthetic polymer.^18,19 The PEG polymer exhibits an expanded random coil structure in solution that can increase the hydrodynamic volume of the biomolecules, which results in reducing renal filtration.^20,21,22 However, the nonbiodegradable properties of PEG have raised increasing concern in its biological applications. Frequent and high doses of PEGylated drugs have been shown to induce PEG deposition in phagocytes, causing cellular vacuolation in multiple organs.^23,24,25 Furthermore, the coupling and purification of PEGylated molecules are often associated with the high heterogeneous products and low yield.^26,27 These issues not only result in the loss of activity and instability of PEGylated drugs, but also significantly increased their production cost.^28,29,30

PASylation describes the genetic fusion or chemical coupling of proteins or drug molecules with biopolymers made of the small l-amino acids Pro, Ala, and/or Ser.³¹ Such proline/alanine-rich sequences are highly soluble in physiological solution and typically adopt a random coil conformation, which results in an expanded hydrodynamic volume. As a biological alternative of PEGylation, PASylated conjugates show retarded kidney filtration and drastically prolonged pharmacokinetics in vivo.^32,33,34 Besides, it can also improve the solubility and stability in plasma.³⁵ By constructing of fusion proteins, PASylated peptides can avoid chemical reactions, purification, and loss of activity during the PEGylation process. In addition, the Pro, Ala, and Ser (PAS) polypeptides can be degraded by intracellular proteases after cellular uptake, so that the PASylated peptides or drugs show lower immunogenicity and viscosity in blood plasma.^36,37,38 These superior properties highlight the considerable potential of PASylation of nanobody probes for clinical application.

Multiple myeloma (MM) is a hematological neoplasm of B cell lineage and is characterized by uniform overexpression of CD38³⁹ In this study, we modified the anti-CD38 nanobody Nb1053. To address the limitation of the short half-life of nanobodies and improve the signal accumulation in tumors, this study describes the development of a PASylated nanobody probe with a 200 amino acid PAS sequence at the C-terminal (Nb1053PAS). For comparative studies, we also synthesized the PEGylated nanobody (Nb1053PEG) with similar length by a mTGase-mediated site-specific modification. We observe that Nb1053PAS has a significantly increased hydrodynamic volume and extended circulating half-life which is similar to Nb1053PEG. In mice bearing myeloma, the Nb1053PAS probe shows increased accumulation in tumor, resulting in enhanced fluorescence intensity in tumor, as well as higher tumor-to-background and tumor-to-kidney signal ratios than those of Nb1053PEG and Nb1053. This study demonstrates the potential of PASylated nanobodies for tumor-targeted imaging.

2. Materials and Methods

2.1. Materials

The human MM Daudi and MM.1S cell lines and the human glioma U87 cell line was purchased from Procell (Wuhan, China). RPMI1640, dulbecco’s modified eagle medium (DMEM) medium, and phosphate-buffered saline (PBS) were purchased from Cytiva (Logan, USA). Fetal bovine serum (FBS), penicillin/streptomycin, and poly-l-lysine were purchased from Thermo Fisher Scientific (Waltham, USA). Bovine serum albumin (BSA) was purchased from J&K Chemical (Beijing, China). Anti-CD38 nanobody Nb1053 with Q-tag was expressed in GenScript (Nanjing, China). Sulfo-Cy5.5 NHS ester was purchased from Ruixi Biological Technology Company (Xi’an, China). FITC was purchased from Bide Pharmatech Company (Shanghai, China). mPEG-NH₂ (molecular weight (MW), 20kDa) was purchased from Tansh-Tech (Guangzhou, China). Microbial transglutaminase (mTGase) was purchased from Yiming Biological (Taixing, China). Ultrapure H₂O was used throughout the study.

2.2. Expression and purification of PASylated nanobody

The PAS sequence [ASPAAPAPASPAAPA PSAPA] $_{10}$ was genetically fused to the C-terminus of nanobody gene. The construct was then synthesized by Genscript (Nanjing, China) and subsequently cloned into pET-30a(+) expression vector using NdeI and HindIII restriction sites. Nb1053PAS expression plasmid was transformed to BL21 (DE3) Escherichia coli and cultured in terrific broth media with 50mg/mL kanamycin. The Nb1053PAS was purified by Ni–NTA column chromatography (Genscript, China) according to the manufacture’s instruction. The purified protein was buffer exchanged to PBS and concentrated to 3mg/mL by ultrafiltration using Amicon Ultra centrifugal filter units (3000MWCO; 50mL; Millipore, USA).

2.3. Synthesis of PEGylated nanobody

Nb1053PEG was generated as described previously.³⁸ Briefly, Nb1053-LLQS was diluted to 0.9mg/mL and mixed with 24mg/mL mPEG-NH₂ (MW, 20kDa) in PBS, then mTGase was added to 1U/mL for 2h at room temperature. Then the Nb1053PEG was purified by Ni–NTA column chromatography and buffer exchanged to PBS and concentrated to 3mg/mL using Amicon Ultra centrifugal filter.

2.4. Characterizations of Nb1053 analogs

The MW of the Nb1053 and Nb1053PAS was measured by an ultrafleXtreme MALDI-TOF mass spectrometer at the Jingxiang Pharmaceutical Technology Co. LTD in Science and Technology Park of Xiamen University (Xiamen, China). The proteins were mixed with $5 \times$ sample buffer (Epizyme, China) and loaded onto 12% SDS-PAGE gels (Epizyme, China) (10 $μ$ g/well). Size distributions of Nb1053, Nb1053PEG, and Nb1053PAS were measured using Zetasizer Nano ZS instrument (Malvern, UK) at room temperature, the purified proteins were diluted to 0.5mg/mL of each sample. Absorbance of Cy5.5 labeled Nb1053, Nb1053PEG, and Nb1053PAS was measured by UV-2600 spectrometer (Shimadzu, Japan), the purified proteins were diluted to 3 $μ$ M of each sample.

2.5. In vitro cell targeting ability assay

Daudi, MM.1S cells were cultured in RPMI1640 medium containing 10% (v/v) FBS and 1% (v/v) penicillin/streptomycin. U87 cells were cultured in DMEM medium containing 10% (v/v) FBS and 1% (v/v) penicillin/streptomycin. The cells were incubated at $3 7^{\circ}$ C in a humidified environment with 5% CO₂. For confocal microscopy analysis, Daudi and U87 cells were seeded in a 10mm glass bottom culture dish (NEST, China) coated with poly-l-lysine at a density of 3 $\times 1 0^{5}$ cells per dish and incubated with FITC-labeled Nb1053, Nb1053PEG, and Nb1053PAS (5 $μ$ M) for 1h on ice. After washing with 4^∘C PBS for three times, the cells were observed under 63× objective lens of the confocal laser scanning microscope (Zeiss, Germany). For flow cytometry, MM.1S cells were harvested and suspended in a centrifuge tube ( $2 \times 1 0^{6}$ cells per tube) and stained with Cy5.5 labeled Nb1053, Nb1053PEG, and Nb1053PAS at 0, 62.5, 125, 250, 500, 1000, 2000nM concentration for 1h on ice. After washing with 4^∘C PBS for three times, the cells were harvested for flow cytometry analysis using a FACSCanto SORP (BD, USA).

2.6. Animals

The female BALB/c nude and NCG mice (∼20g, 6∼8 weeks) were purchased from Gempharmatech Co., Ltd (Foshan, China) and housed in Laboratory Animal Platform of Southern University of Science and Technology. All the animal studies were approved by Institutional Animal Care and Use Committee (IACUC) of Southern University of Science and Technology.

2.7. Pharmacokinetic studies

BALB/c nude mice were randomly divided into three groups (n = 3). A single intravenous dose of Cy5.5 labeled Nb1053 (1.5mg/kg), Nb1053PAS (3mg/kg), or Nb1053PEG (3.5mg/kg) was administered. After intravenous injection, 20 $μ$ L blood was collected from the tail vein at predetermined time points post nanobody injection. The plasma fluorescence intensity was measured by a microplate reader (BioTek, USA). The pharmacokinetics of nanobodies were analyzed through one-compartment model by DAS2.0 software.

2.8. In vivo and ex vivo tumor-targeting and biodistribution imaging

$5 \times 1 0^{6}$ human MM.1S myeloma cells were cultured and subcutaneously implanted into NCG mice. When the tumor diameter reached ∼5mm, mice were randomly divided into three groups (n = 5). Before the experiment, their back hair was removed using a hair removal cream. A single intravenous dose of Cy5.5 labeled Nb1053 (1.5mg/kg), Nb1053PAS (3mg/kg), or Nb1053PEG (3.5mg/kg) was administered. Fluorescence dorsal images of mice (excitation at 660nm and emission at 710nm) were obtained at different time points post nanobody injection using an IVIS imaging system (PerkinElmer, USA). Mice were sacrificed after 72h and the major tissues and organs (tumor, heart, kidney, liver, spleen, lung, muscles) were harvest and imaged ex vivo with the IVIS system.

3. Results

3.1. Expression and characterizations of PASylated nanobody

To extend the blood circulation of nanobody for tumor imaging in vivo, we designed a PASylated 1053 nanobody that targets human CD38, which is widely used as a target for myeloma treatment and diagnosis. In this study, each PAS sequence is composed of a 20-amino acid repeating fragment (ASPAAPAPASPAAPAPSAPA), and 10 PAS sequence of total 200 amino acids was added to the C-terminus of the nanobody by genetic engineering (Fig. 1(a)). The expression yield of PASylated nanobody is around 8mg/L E. coli. As PASylation technology was developed as a biological alternative to PEGylation, we also synthesized PEGylated nanobody (Nb1053PEG) with a similar molecular weight to that of Nb1053PAS by the site-specific conjugation of mPEG amine (MW, 20kDa) to the C-terminus of Nb1053 nanobody. The Nb1053PEG conjugates were synthesized by the mTGase mediated reaction and purified by affinity chromatography to remove free mTGase and unreacted PEG.⁴⁰

Fig. 1. Characterizations of PASylated and PEGylated nanobody. (a) Expression and synthesis of Nb1053, Nb1053PEG, and Nb1053PAS. (b) Gel electrophoresis analysis of the nanobody conjugates. Lane 1: protein marker; lane 2: Nb1053; lane 3: Nb1053PEG; lane 4: Nb1053PAS. (c) Hydrodynamic diameter distribution of Nb1053, Nb1053PEG, and Nb1053PAS measured by DLS. (f) Mass spectra of Nb1053 and Nb1053PAS.

The Nb1053PAS and Nb1053PEG conjugates were analyzed by gel electrophoresis (SDS-PAGE). Because both PEG and PAS sequences can increase the hydrodynamic volume of proteins,⁴¹ the migration bands of Nb1053PEG and Nb1053PAS appeared in the range of 75–100kDa (Fig. 1(b)). It is clear that Nb1053PAS shows a narrow migration band as compared to the Nb1053PEG, indicating the uniform molecular weight of Nb1053PAS probes. Both Nb1053PAS and Nb1053PEG were characterized by dynamic light scattering (DLS). The two probes exhibit similar hydrodynamic diameters (∼7.5nm), which is ∼2 times higher than that of pure Nb1053 (Fig. 1(c)). These results demonstrate that PASylation has a comparable effect to PEGylation in increasing the hydrodynamic volume of proteins. The molecular weight of Nb1053 and Nb1053PAS were further characterized using mass spectrometry (Fig. 1(d)). The results of mass spectrometry showed that the molecular weight of Nb1053PAS is around 31kDa, which corresponds to the sum of the molecular weight of the nanobody and PAS sequences, indicating that the PAS sequence was successfully fused to the C-terminus of Nb1053 nanobody. Nb1053 and Nb1053PAS have mass spectral peaks corresponding to only one molecular weight, indicating that both samples have highly homogeneous fractions. These results demonstrated the successful construction and purification of PASylated nanobodies.

3.2. In vitro bioactivity of PASylated nanobody

To analyze the targeting properties of Nb1053 nanobody, we synthesized FITC-labeled Nb1053, Nb1053PEG, and Nb1053PAS probes. Our previous study demonstrated that both Daudi and MM1S cells are CD38 overexpressing cells.¹⁴ The targeting ability of nanobody conjugates was examined by fluorescent staining of Daudi cells with high CD38 expression and U87 cells with low CD38 expression. Confocal microscopy revealed that all three nanobody probes exhibited strong fluorescence on the cell membrane of Daudi, whereas very weak signals appeared on the U87 cell surface (Fig. 2(a)), indicating that the specific labeling properties of the PASylated and PEGylated nanobody probes are comparable to those of the original nanobody. We further analyzed the targeting ability of Cy5.5 labeled Nb1053, Nb1053PEG, and Nb1053PAS by flow cytometry (Fig. 2(b)). The Cy5.5 labeled Nb1053, Nb1053PEG, and Nb1053PAS probes at the same nanobody concentration have comparable absorbance at 680nm (Fig. S1), indicating that they were labeled by the same amounts of Cy5.5 molecules. The results of flow cytometry showed that the fluorescence intensity of Nb1053PAS was slightly lower than Nb1053, likely due to the physical shielding effect on the nanobody. In contrast, the fluorescence intensity of Nb1053PEG is significantly lower than Nb1053 and Nb1053PAS at the same nanobody concentrations (Fig. 2(c)), revealing that Nb1053PAS retains higher targeting activity as compared to Nb1053PEG.

Fig. 2. Specific cell labeling of Nb1053, Nb1053PEG, and Nb1053PAS probes. (a) Immunofluorescence imaging of Daudi and U87 cells by FITC-conjugated Nb1053 analogs (scale bar: 15 $μ$ m). (b) Flow cytometry results of the three Nb1053 analogs at different concentrations. MM.1S cells treated with gradient concentration of Cy5.5-conjugated Nb1053 analogs (62.5, 125, 250, 500, 1000, 2000nM). (c) Mean fluorescence intensity (MFI) of the three Nb1053 analogs (n = 3).

3.3. Pharmacokinetics of PASylated nanobody

We investigated the pharmacokinetics of the three nanobody probes in mouse study. The plasma level of nanobody probes was analyzed by measuring the fluorescence intensity of blood within 48h after intravenous injection of Cy5.5 labeled nanobodies (Fig. 3(a)). As seen from the results, the plasma level of the Nb1053 probes decreased rapidly, while the plasma level of Nb1053PEG and Nb1053PAS probes decreased slowly due to their increased hydrodynamic volume. The pharmacokinetic profiles of Nb1053, Nb1053PEG, and Nb1053PAS probes show significant differences (Table S1). Specifically, the Nb1053 probe has a very short half-life of 0.25h, while the half-life times of Nb1053PEG (1.34h) and Nb1053PAS (0.96h) are 5.4- and 3.8-times of Nb1053, respectively (Fig. 3(b)). Similar trend is also observed in the elimination rate constant (Fig. 3(c)), as the elimination rate constant of Nb1053PEG and Nb1053PAS are 5.8- and 4.1-fold lower than that of Nb1053. The area under the curve (AUC) values of Nb1053PEG and Nb1053PAS is 4.5- and 3.4-times that of Nb1053 (Fig. 3(d)). These results demonstrate that PASylation can significantly prolong the blood circulation times of nanobody just like PEGylation.

Fig. 3. Pharmacokinetics of mice post intravenous administration of Cy5.5 labeled Nb1053PAS. (a) Plasma fluorescence intensity of Nb1053, Nb1053PEG, and Nb1053PAS (300nmol/kg, n = 3). (b) Half-life values of Nb1053, Nb1053PEG, Nb1053PAS. (c) Elimination rate constant of Nb1053, Nb1053PEG, and Nb1053PAS. (d) AUC values of Nb1053, Nb1053PEG, and Nb1053PAS. ** $P < 0.01$ and *** $P < 0.001$ .

3.4. In vivo tumor targeting and biodistribution of PASylated nanobody

Finally, we investigate the tumor-targeting properties of the nanobody probes in a subcutaneous xenograft model of myeloma. Cy5.5-conjugated Nb1053, Nb1053PEG, and Nb1053PAS probes were intravenously injected into the tumor-bearing mice. Fluorescence imaging of the dorsal side of mice was obtained over a period of 72h post-injection. As shown in Fig. 4(a), fluorescence intensities of the mice injected with Nb1053PEG and Nb1053PAS at different time points were higher than those injected with Nb1053. The fluorescence intensities of the tumor and a selected dorsal area as the background in the three mice groups were analyzed (Fig. 4(b)). As indicated, both the tumor and background signal intensity decreased over time, the Nb1053PAS and Nb1053PEG groups exhibited a similar background signal intensity, for approximately 1.2-fold higher than that of Nb1053 group at 72h post-injection. Again, this observation indicated that Nb1053PAS and Nb1053PEG have improved pharmacokinetic behaviors. It is worth noting that the fluorescence intensity of tumor position of Nb1053PAS was 1.5-fold higher than that of Nb1053 and 1.3-fold higher Nb1053PEG at 72h post-injection, indicating that Nb1053PAS has the best tumor targeting and enrichment capabilities. The tumor to background ratio of Nb1053PAS increased over time and reached a maximum of 4.6 at 72h post-injection, which is much higher than the ratios of 3.2 for Nb1053PEG and 3.7 for Nb1053 groups (Fig. S2). As PASylation prolongs the pharmacokinetics in vivo by retarding the kidney filtration, we analyzed the tumor-kidney signal ratios of the mice injected with three nanobody probes, respectively (Fig. 4(c)). The tumor-kidney signal ratios in all three groups increased over time post-injection, and the ratio of Nb1053PAS (2.1 at 72h post-injection) was significantly higher than those of Nb1053 and Nb1053PEG (1.3 for both at 72h post-injection).

Fig. 4. Fluorescence imaging of the mice bearing subcutaneous MM.1S tumors. (a) *In vivo* fluorescence imaging of the tumor-bearing mice post administration of Cy5.5 labeled Nb1053, Nb1053PEG, and Nb1053PAS (100nmol/kg, n = 5), Near-infrared fluorescence *in vivo* imaging was obtained at 0.5, 1, 2, 4, 8, 12, 24, 36, 48, 60, 72h post-injection. (b) MFI of tumor site and background (left hind limb) of Nb1053, Nb1053PEG, and Nb1053PAS. (c) Tumor-to-kidney signal ratio of Nb1053, Nb1053PEG, and Nb1053PAS. (d) *Ex vivo* fluorescence imaging of tumor and organs of mice 72 h post-injection of Nb1053, Nb1053PEG, and Nb1053PAS. (e) Fluorescence intensities of tumor and organs of mice 72 h post-injection of Nb1053, Nb1053PEG, Nb1053PAS (n = 5). *** $P < 0.001$ .

Fluorescence imaging of the resected organs 72h post-injection confirmed the specific targeting of Nb1053PAS in the tumors (Figs. 4(d) and 4(e)). The resected tumor of Nb1053PAS showed a fluorescence intensity 1.6-fold higher than that of Nb1053PEG and 1.5-fold higher than Nb1053. All the three groups exhibited high renal and hepatic signals, indicating that clearance of nanobody probes occurs mainly through the renal and hepatic pathways. Furthermore, the hepatic signal of Nb1053PAS and Nb1053PEG was elevated as compared with Nb1053, indicating that the large molecular weights of the Nb1053PAS and Nb1053PEG probe likely contribute to the hepatic clearance pathway.

4. Discussion

Nanobodies have been extensively investigated as a potential targeting molecule. However, the administration of nanobody probes is impeded by their rapid kidney clearance due to their small size. Therefore, it is essential to develop nanobody probes with prolonged circulation times in vivo. In this study, we designed a recombinant PASylated nanobody, whereby a PAS sequence comprising 200 amino acids was fused to the C-terminus of the nanobody. The objective of this study was to investigate the targeting ability of PASylated nanobody probes, as well as their half-life and targeting tumor imaging capabilities in vivo.

The mass spectrometry analysis showed the expected molecular weights of the PASylated nanobody. As a biological alternative to PEGylation, PAS200 has been reported to exhibit comparable half-life extension effects to a PEG chain of molecular weight 20,000.³¹ Consequently, we synthesized the PEGylated nanobody for comparison. The SDS-PAGE analysis indicated much higher molecular weights of both PEGylated and PASylated nanobodies due to their extended hydrodynamic volume in gel.²⁰ These results indicated the successfully construction of the PASylated nanobody. Given that the PEGylation reaction has been demonstrated to reduce the biological activity of proteins,^42,43 we proceeded to investigate the ability of PASylated, PEGylated and unmodified nanobodies to target cells by antibody titration experiments. The results of flow cytometry showed that PASylation resulted in a slight loss of targeting ability, but retained significantly higher targeting ability than PEGylation, suggesting that PASylation is a better nanobody modification strategy for retaining bioactivity.

Subsequently, we performed pharmacokinetic experiments in mice. The half-life of PASylated nanobody was found to be prolonged 3.8-times while the PEGylated nanobody prolonged it by 5.4-fold. The PEG chain is not biodegradable, it makes the protein harder to be taken up by cells and slower to be degraded in vivo,⁴⁴ which may be the reason why PEGylated nanobodies have a longer half-life. However, the shielding effect of PEG chains also impairs the penetration of the nanobody into the tumor and targeting ability, thus restricting the accumulation of the nanobody probe in the tumor. This was demonstrated by our in vivo imaging experiments of tumors in mice. Despite comparable fluorescent dye loading ratios for PASylated, PEGylated and unmodified nanobody probes, there was no significant difference in either the fluorescence intensity at the tumor site or the tumor background signal ratios for the PEGylated nanobody probes in comparison to those of the unmodified nanobodies. However, the PASylated nanobodies increased the fluorescence signal at the tumor site by approximately 1.5-fold 72 h post-injection, as demonstrated by subsequent ex vivo organ imaging experiments. This emphasized PASylation as a superior modification strategy to PEGylation to produce long-circulating protein or peptide probes or drugs. Furthermore, the PAS sequence can be easily edited to access additional functional peptides. By incorporating reactive amino acids, such as cysteine or enzymatic recognition sequences at the C-terminus of the PAS sequence, various functional modifications of PASylated nanobodies can be achieved, which can be used in diverse imaging techniques including optical, PET, MRI, etc., as well as in the construction of antibody-drug conjugates.

5. Conclusion

In this study, we designed a recombinant PASylated nanobody with a C-terminal PAS fusion sequence and demonstrated it has a comparable effect to PEGylation in increasing the hydrodynamic volume of proteins. As the PASylated nanobody can be expressed directly by E. coli, the bioconjugation, purification, and loss of bioactivity caused by PEGylation process were avoided. The in vitro characterizations showed that the PASylated nanobody retained the targeting ability, with a higher cell labeling brightness as compared with the PEGylated nanobody. As demonstrated by in vivo results, the PASylated nanobody exhibited an enhanced targeting ability in tumor-bearing mice as compared to the original and PEGylated nanobody, indicating the great potential of PASylated nanobody probes in tumor imaging. We envision that PASylation may be a superior modification strategy to PEGylation to produce long-circulating protein or peptide probes or drugs.

Acknowledgments

The authors are grateful for the financial support by the National Key R&D Program of China (Grant No. 2020YFA0909000), National Natural Science Foundation of China (Grant Nos. 62235007 and 22204070), Shenzhen Science and Technology Program (Grant Nos. KQTD20170810111314625, JCYJ20210324115807021, and SGDX20211123 114002003), Shenzhen Bay Laboratory (SZBL 2021080601002), and Guangdong Provincial Key Laboratory of Advanced Biomaterials (2022 B1212010003).

Supplemental Materials

The Supplemental Materials are available at: https://www.worldscientic.com/doi/suppl/10.1142/S1793545825410019.

Conflicts of Interest

The authors declare that there is no conflict of interest relevant to this paper.

ORCID

Yicheng Yang https://orcid.org/0009-0009-7337-4373

Lingyue Jin https://orcid.org/0009-0001-0165-1556

You Zhang https://orcid.org/0000-0001-7968-8079

Siyu Zhou https://orcid.org/0009-0000-2296-4001

Weijun Wei https://orcid.org/0000-0003-3190-2480

Gang Huang https://orcid.org/0000-0003-1939-3978

Changfeng Wu https://orcid.org/0000-0001-6797-9784

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Received 4 October 2024

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