Abstract
Therapeutic and diagnostic efficacies of numerous small biomolecules and chemical compounds are hampered by the short half-lives. Here we report the development of a repertoire of diverse, high-affinity albumin-nanobodies (NbHSA) to facilitate drug delivery. By integrating biophysics, and hybrid structural biology, we have systematically characterized the NbHSA for albumin binding, mapped the epitopes, and resolved the architecture of a tetrameric Nb-albumin complex. We employed quantitative proteomics for accurate, multiplex Nb pharmacokinetic analysis. Using a humanized albumin mouse model, we found that the NbHSA has outstanding pharmacokinetics; the most stable NbHSA has a 771-fold T1/2 improvement compared with a control Nb. Interestingly, the pharmacokinetics of NbHSA is related to their biophysical and structural properties. To demonstrate the utility of NbHSA, we developed a highly stable NbHSA-hIL-2 cytokine conjugate “Duraleukin” and confirmed its improved anticancer properties than hIL-2 alone. We envision that this high-quality Nb resource will advance research into novel biotherapeutics.
Introduction
Numerous chemical compounds and small biologics, such as hormones, cytokines, and coagulation factors, have limited therapeutic efficacy due to their poor in vivo stabilities. Different strategies have been implemented to address this challenge, including chemical modifications, conjugation with the antibody Fc domain, and targeting to human serum albumin (HSA). Human serum albumin (HSA) is an abundant serum protein with an exceptionally long half-life of ~ three weeks 1. Unlike small biomolecules (< 50 kDa) rapidly eliminated (e.g., from mins to a few hours) via glomerular filtration, HSA can be retained due to the relatively large size (66.5 kDa). Importantly, HSA can bind with the neonatal Fc receptor (FcRn) at acidic pH and is continuously recycled via the FcRn-mediated transcytosis 2,3. Because of its stable interaction with the FcRn, it can escape the endo-lysosomal degradation pathway and obtain outstanding serum stability. Interestingly, although HSA is highly abundant in the serum, its intracellular concentration is likely very low 3,4.
The high in vivo solubility and stability of HSA make it an attractive endogenous carrier for drug delivery. Different HSA-based strategies, such as direct fusion to HSA, albumin-binding domains (ABDs) derived from gram-positive bacteria, and albumin nanoparticles, have been developed 1,3,5. Despite the encouraging progress, however, successful applications of these methods towards robust bioengineering and clinical uses remain limited 6. The development of robust agents, ideally easily accessible to the research community, will help advance drug development.
Nanobodies (Nbs) are small antigen-binding fragments (~15 kDa) derived from the camelid’s heavy chain only antibodies (HcAbs) 7. They are well known for the robust folds, excellent solubility, stability, and ease of bioengineering and manufacturing. Besides, they can efficiently penetrate tissues and are potentially low immunogenic for clinical applications due to the high sequence similarity with the human IgGs. Because of these characteristics, Nbs have emerged as a new class of antibodies for biomedical research and drug development 8,9.
We have recently discovered an extensive repertoire of anti-HSA Nbs (NbHSA) by camelid immunization of recombinant HSA in conjugation to proteomic identifications {Xiang et al., submitted}10. We selectively produced a set of diverse NbHSA sequences for systematic characterizations by multidisciplinary approaches, including biophysics, integrated proteomics, structural biology, and modeling. We then developed a strategy based on PRM (parallel reaction monitoring) mass spectrometry that enabled unbiased, parallel pharmacokinetic (PK) analysis of dozens of high-affinity NbHSA in a humanized albumin mouse model. Our studies revealed that the NbHSA PKs are related to their binding kinetics at the acidic pH environment and their epitopes. To demonstrate the utility of our NbHSA, we conjugated high-affinity, cross-species Nbs to a critical cytokine interleukin-2 (hIL-2), developed and characterized several stable Nb-cytokine conjugates (Duraleukins), and have confirmed the improved antitumor efficacy of a Duraleukin than hIL-2 in a melanoma mouse model. We envision that this high-quality Nb resource and the hybrid proteomic technologies will find broad utility in biomedical applications.
Results
Characterization of a highly diverse repertoire of high-quality NbHSA
A collection of 71 recombinant His6-tagged Nbs with diverse sequences was produced from E. coli cells (Fig 1a, Table S1). The selected NbHSA vary significantly on the CDR3 (complementarity-determining region 3) fingerprint regions, including amino acid compositions, sequence length, isoelectric points (pIs), and hydrophobicity potentially spanning a large spectrum of physicochemical structural properties (Fig 1b). We performed enzyme-linked immunosorbent assay (ELISA) to assess their relative affinities and cross-species selectivities. We ranked NbHSA based on the ELISA affinities to different albumins, including HSA, cynomolgus monkey albumin, and mouse albumin. HSA is highly conserved with cynomolgus monkey albumin (92% sequence identity) and is relatively conserved with mouse albumin (78% sequence identity). As expected, greater than 75% (53 NbHSA) of NbHSA can bind monkey albumin, and 11% (8 NbHSA) can bind mouse albumin (Fig 1c, Methods). Interestingly, 8% (6 NbHSA) strongly associated with all three albumin orthologs and were confirmed by immunoprecipitations (Fig 1c). For example, Nb132 binds both humans, monkeys, and mouse albumin but not bovine and llama albumin (Fig 1g). The high affinity, cross-species NbHSA are particularly useful for Nb-based drug development in the animal models.
We selected 20 NbHSA of high yields (from E. coli cell lysis) to assess the binding kinetics by surface plasmon resonance (SPR) (Fig 1d, Fig S1). We found a good correlation (Pearson r= 0.78, p<0.001) between the ELISA affinity and SPR KD (Fig 1e). 75% (15 out of 20) of NbHSA present sub-nM KD with different binding kinetics, while the remaining 25% have single-digit to hundreds of nM affinities (Fig 1f). Next, we evaluated the thermostability of NbHSA by differential scanning fluorimetry. The Nbs are generally thermostable, with melting temperatures between 39 □C to 70 □C (Fig 1h, Fig S2, Table S2). The excellent physicochemical properties, including solubility, target specificity, affinity, and stability, are essential for Nb therapeutic development 11,12.
Integrative structural proteomics reveals dominant epitopes for NbHSA binding
Epitope mapping provides insight into antigen-antibody interactions and is useful for rational drug designs 13–15. We employed an integrative structure approach by chemical cross-linking/mass spectrometry (CXMS) and computational modeling to map HSA epitopes for Nb binding. CXMS identifies residue-specific cross-link restraints that are highly informative to interrogate the binding interfaces of protein complexes. Cross-linking information can be readily integrated into other structural biology techniques and computational tools to derive accurate structural models 16–21.
We purified and reconstituted 38 different HSA-Nb complexes for disuccinimidyl subtracted (DSS) crosslinking, which can reach up to 30 Å between two cross-linked amines (e.g., lysine residues). A total of 125 intersubunit cross-links were confidently identified by high-resolution MS (Table S3, Methods). These cross-links were then used to compute structural models. Integrative modeling analysis revealed that 29 HSA-Nb complexes converged with an average RMSD (root-mean-square-deviation) of 7.4 Å (Fig 2c). Overall, 87.5% of cross-links were satisfied by the models (i.e., <30 Å) (Fig S3b).
Cross-linking based structural modeling identified 6 HSA epitope clusters (from A to F) of NbHSA (Fig 2a, Table S3). Despite the high sequence diversity of CDR3s, >75% of the NbHSA (21 Nbs) binds two dominant epitope clusters: clusters A (31.03%) and E (44.83%) (Fig 2b), indicating a convergent, CDR3-dependent contact between Nbs and HSA. We found excellent shape complementarities between the concave epitopes on HSA and the corresponding convex paratopes of NbHSA for high-affinity interactions (Fig 2d). Interestingly, a sub-nM affinity Nb (Nb126) was identified to localize at the major crevice on HSA that regulates FcRn binding (Fig S3a), raising a possibility of competitive binding between Nb126 and the FcRn.
Hybrid structural determination of a tetrameric HSA-Nb complex
To further verify our integrative structures, we selected Nb13, Nb29, and Nb80 that correspond to three epitope clusters A, C, and E, respectively, and employed size-exclusion chromatography (SEC) to confirm that they do not compete for HSA binding. The presence of three separate SEC peaks corresponding to HSA-Nb complexes of different molecular weights indicates that these Nbs bind distinct HSA epitopes (Fig 3a). After SEC, a homogenous fraction of the complex was selected for analysis by negative stain electron microscopy (EM). ~22,000 EM particles were selected to reconstruct a 3D density map (Fig 3b, Methods). Consistently, the cross-link models of the tetrameric Nb-HSA complex agreed well with EM density (Fig 3c). Based on our integrative structure, we found that sub-nM Nb80 (KD=166 pM) and Nb13 (KD=201 pM) have better shape complementarity with HSA than Nb29 (KD=1.2 nM) (Fig 3d-f). Our models revealed the formation of two crossed, salt bridge pairs between HSAK383 and HSAE400 with the respective CDR residues on Nb80. To validate this, we made two HSA mutants and evaluated their binding to NbHSA (Fig 3g). While a single-point mutation E400R significantly weakened the HSA-Nb80 association, a double-residue mutation (K383D-E400R) completely abolished the strong interaction (Fig 3h). Collectively, our integrative structure of the tetrameric HSA-Nb complex confirmed the epitope mapping results and provided the structural basis to understand the HSA-Nb interactions.
Development of a proteomic approach that enables specific, accurate and multiplex Nb pharmacokinetic analysis
ELISA has been extensively used for the analysis of antibody PK. However, this approach can be significantly limited by the availability of high-quality detection antibodies, rendering specific and accurate PK analysis from the complex serum background challenging. Such a method is particularly challenging for comparative PK analysis of a large set of antibodies as it requires a substantial cohort of animals and slow handling time that may complicate experiments and introduce bias. To alleviate these problems, here we developed a strategy by employing MS2-based quantitative proteomics (parallel reaction monitoring or PRM) to facilitate specific, accurate, and multiplex analysis of Nb PKs from the serum samples. This approach enables specific detections of at least three femtomoles of an Nb (10−15 mole) per μL of the serum and offers better quantification linearity than the MS-1 based approach (R2= 0.999) (Fig S5, Table S4). The high dynamic range of this approach is essential for accurate PK measurement.
Since most of our NbHSA do not strongly bind mouse albumin, we chose a humanized albumin mouse model to mimic the physiology of endogenous HSA in humans. This mouse model (B6.Cg-Tg(FCGRT)32Dcr Albem12Mvw Fcgrttm1Dcr/MvwJ) removed the native mouse albumin and replaced the mouse FcRn with a human ortholog. HSA can be later administered to study PK of HSA-based drugs 22.
Following HSA injection, we administered a mixture of 22 Nbs, including 20 well-characterized NbHSA (with SPR kinetic and thermostability measurements) and two non-binder control Nbs with equal molarity to this model (n = 3) via single bolus intravenous (i.v.) injections (Methods). We used a molar ratio of 1: 5 between NbHSA and HSA to ensure an excessive HSA, avoiding potential competitive binding among different NbHSA. The blood was sampled at different time points post-injection. Sera fractions containing NbHSA and other serum proteins were efficiently proteolyzed. The resulting signature NbHSA peptides and their fragment ions were accurately quantified by PRM 23,24 (Fig 4a, Table S4). The median CV (coefficient of variation) of our proteomic quantifications from three animals was 15.3% (Fig S6).
We observed that the control Nbs were rapidly cleared from the serum with the half-lives (T1/2s) of approximately 26-60 mins, consistent with the previous ELISA measurements 25. In sharp contrast, our NbHSA showed significantly increased T1/2s of up to 771-fold, from 1.6 days (Nb100) to 7.6 days (Nb75) (Fig 4b, 4c, Table S2). The T1/2s of NbHSA correlated with T1/2 of HSA (~ 8 days) that we measured in this model (Fig S7c). To explore the potential mechanism(s) that underlies the relatively fast T1/2 of HSA in this mouse model (the T1/2 of HSA in humans is ~21 days), we employed label-free quantitative proteomics 26,27 to monitor the longitudinal changes of the blood proteome of a mouse (up to 10 days) post-HSA injection. Four hundred forty-one blood proteins were accurately quantified and clustered based on their relative abundance changes (Fig S8). We found that a group of acute-phase inflammatory proteins, such as Serum Amyloid A and C-reactive protein, increased by hundreds of folds in abundance after 12-24 hours post-injection, before returning to the baseline level after 24-48 hours 28,29. Interestingly, we observed an increase of serum IgGs after 5-7 days indicating the potential anti-HSA immune response, which may explain the faster-than-expected clearance of the injected HSA in this model 30.
NbHSA pharmacokinetics are related to their physicochemical and structural properties
The findings that NbHSA has diverse PK prompted us to investigate the potential mechanism(s) contributing to such differences. We analyzed the correlations between the PK and the physicochemical properties of NbHSA, including affinity (at both neutral and acidic pHs), TM, pI, and hydrophobicity (Fig 4d). We found that NbHSA ELISA affinities at pH 5 (acidic pH) and SPR dissociation rates Kd, positively correlated with the elimination rates (p < 0.01). In general, the higher the affinities, the slower NbHSA was cleared from the blood. It is conceivable that both the slow dissociation-rates and high-affinity binding at the acidic pH are important for the extended T1/2 of the low abundance NbHSA-HSA-FcRn ternary complexes in the endosome. NbHSA with lower affinities at the acidic pH tend to dissociate faster from the complex and, therefore, have decreased T1/2. The only exception was Nb126, which binds strongly with HSA (KD of 251 pM) and has relatively fast T1/2s (41.4 hrs). The interaction with HSA might be interfered with by a structural change of the epitope induced by FcRn binding (Fig S3a) 2,30.
Development of Duraleukin - a novel application of the NbHSA platform for cancer immunotherapy
To demonstrate the utility of our Nbs for drug development, we applied them to modify a key cytokine interleukin-2 (IL-2). IL-2 functions primarily to regulate immune cells such as T cells and natural killer cells, thereby regulating immune responses that suppressor cancer 31. Human IL-2 (hIL-2) was the first FDA-approved cancer immunotherapy drug (in 1992) developed for effective treatment of renal cell carcinoma and metastatic melanoma 32. ~10% of these devastating late-stage cancer patients who received high dose hIL-2 treatment had a complete response. However, the clinical efficacy of hIL-2 has been significantly limited by its small size (~15 kDa) and poor PK (i.e., shorter than 30 minutes in humans) 31,32. High-dose, repetitive administration of hIL-2 (e.g., i.v. treatment every 8 hours), while critical for its antitumor efficacy, can lead to severe side effects such as liver toxicity and vascular leak syndrome 32,33.
We fused our NbHSA with hIL-2 to develop “Duraleukin” for mitigation of the poor PK of hIL-2 (Fig 5a). We chose three high-affinity NbHSA (Nb77, Nb80 and Nb158) that bind both human and mouse albumin to demonstrate the robustness of this approach. Large quantities of Duraleukins (DL77, DL80and DL158) were rapidly produced from E. coli (Fig 5b, Methods). All three DLs demonstrated excellent thermostability (Tm ≈ 60 °C, Fig 5c). They had comparable bioactivities to hIL-2 alone in the in vitro CD8+ T cell proliferation assay (Fig 5d), and were stable for days when incubated with mouse serum at 37°C (Fig 5e). Moreover, these DLs retained sub-nM affinities for HSA binding (Fig 5e). We selected DL80 for further in vivo evaluation because it has a high affinity for both mouse and human albumin binding, and remained associated with HSA at acidic pH (Fig 5g).
Duraleukin demonstrates superior therapeutic efficacy for melanoma treatment in a mouse model
We confirmed that DL80 had a 46-fold improvement of PK compared with hIL-2 in C57BL/6J mouse (Fig S9). This relatively moderate improvement corresponds to the half-life of endogenous mouse albumin (<1 day) 25. We then assessed its therapeutic efficacy in the B16F10 melanoma model 34. Tumor-bearing C57BL/6J mice were intraperitoneally administered with equimolar of either DL80 or hIL-2 at each injection. The animals were treated with different frequencies (every six days for DL80 and hIL-2 daily) for a 24-day treatment. TA99 monoclonal antibody that recognizes melanoma marker TRP1 and functions synergistically with hIL-2, was co-treated for both groups 35–38 (Methods). We continuously monitored tumor sizes and survival for 62 days starting from the first treatment (Fig 6a, 6b). We observed that both DL80 and hIL-2 treated groups exhibited significant reductions in tumor loads and better overall survival than the PBS controls. As little as 1/6 total therapeutic dose was applied to the DL80-treated animals, and due to the relatively short T1/2 of mouse albumin, only a moderate PK improvement was observed in the C57BL/6J mice. Despite this, we observed near-complete tumor recessions for the DL80 responsive animals. In contrast, clear rebounds of tumorigenesis in the hIL-2 treated mice were evident (Fig 6a), indicating the potential benefits of the sustainable antitumor activity of DL80 for improving melanoma treatment.
To explore the mechanisms that contribute to Duraleukin’s superior antitumor activity, we isolated tumors at different stages and analyzed their immune cells by flow cytometry (Fig 6c, Fig S10). We observed significant and persistent increases of tumor-infiltrating immune cells, including CD8+ T cells and natural killer (NK) cells in the DL80-treated tumor tissues at day three post-treatment. At the same time, hIL-2 appeared to lose its activity for promoting T cell expansion. Our results indicate that Duraleukin exerts its long-lasting antitumor activity by promoting tumor-infiltrating immune cells.
Discussion
The short serum half-lives limit the therapeutic and diagnostic efficacy of many small biotherapeutics, including cytokines, hormones, and Nbs. Here we develop a robust platform based on a large, diverse set of high-quality NbHSA to improve PK. We have employed and developed multidisciplinary approaches that span biochemistry, biophysics, structural proteomics, and integrative structural biology, longitudinal blood proteomics, and a quantitative proteomics-based PK assay that collectively enabled systematic characterizations of Nbs for advanced biomedical applications. We have positively evaluated the outstanding PK of NbHSA in a humanized HSA mouse model. To demonstrate the utility of this Nb platform, we applied several cross-species NbHSA to a key cancer-fighting cytokine (IL-2) and developed a specific Nb-based treatment (Duraleukin) for cancer. We demonstrated Duraleukin’s increased antitumor activity compared to IL-2 therapy alone in a melanoma mouse model.
The Nb platform that we have developed here likely represents a significant step towards tailored, optimal drug delivery. First, it permits minimal interference with the bioactivity of the small target drug compared with other approaches such as chemical modifications and direct fusion to HSA 1,5. The robustness of NbHSA is desirable for bioengineering. Second, it comprises a set of well-characterized NbHSA of excellent solubility, stability, and affinity. Their favorable physicochemical and structural properties are critical for drug development, administration, manufacturing, and storage 12. Rapid bioengineering and cost-effective production of Duraleukins, as demonstrated here, would be important for the future development of duraleukin technology towards clinical applications 32. Third, it offers a wide range of PK, biophysical and structural properties towards possible tailored drug development. Fourth, it does not elicit Fc-associated antibody-dependent cellular cytotoxicity (ADCC) that is likely toxic to the functional cells by which the Nb-fusion constructs target (such as IL-2R+ T cells). Combined with the high sequence similarity of Nbs to human IgGs, our approach may restrain the toxicity for clinical applications 9,39.
Our investigation leveraged a powerful combination of proteomic technologies for drug development. The integrative structural analysis revealed major HSA epitopes and provided a structural basis to understand NbHSA interactions with HSA and their PKs. Our analysis underlies the importance of high-affinity, acidic pH-dependent binding of NbHSA for half-life extensions. Our results indicate the presence of low intracellular concentration(s) of HSA-FcRn complex in vivo and the acidic microenvironment(s) of the FcRn receptor-mediated transcytosis 3,4,40. Moreover, we found that the PK of our most stable NbHSA is consistent with the half-life of HSA in the humanized mouse model. It is anticipated that NbHSA will have significantly improved PK in the non-human primate models and humans.
We developed Duraleukins by taking advantage of the cross-species NbHSA that help translate the preclinical data of animal models into clinical studies. Despite using a much smaller administration dose than hIL-2, Duraleukin80 outperformed it for melanoma treatment. The improved drug efficacy of Duraleukin is likely due to the robust cytokine activity and improved half-life. The retention of albumin in the tumor interstitium has been reported 3,41,42, raising a possibility that the superior efficacy of Duraleukin is related to its potentially favorable tumor localization as a cargo of albumin.
This proof-of-concept might be broadly applicable to other small antitumor cytokines such as hIL-15 43, ultimately leading to enhanced antitumor activity, optimal dosing regimen, better drug safety, and improved patient compliance. It is conceivable that a combination of Duraleukin and an immune checkpoint blockade therapy like PD-1/L1 or CTLA-4 may further improve cancer treatment in a synergistic manner 44,45. It is also feasible to produce a trifunctional construct by adding another Nb that binds a prominent tumor surface marker (e.g., EGFR and HER2) for a more target-specific, and potentially more efficient, and safer therapy 46. Future studies will be needed to investigate the NbHSA and Duraleukin for preclinical and clinical applications.
Supplemental Tables
Table S1: 71 NbHSA sequences, the pI, hydrophobicity, and the number of cysteine residues of NbHSA CDR3s, and the ELISA affinities of NbHSA towards different albumin species.
Table S2: SPR kinetic analysis, thermostability, and pharmacokinetic analysis of 20 different NbHSA.
Table S3: Cross-link information and the cross-link models of the HSA-NbHSA complexes.
Table S4: Ion information of MS1-based and PRM quantification of NbHSA.
Methods
Nb DNA synthesis and cloning
Nb genes were codon-optimized for expression in Escherichia coli and the nucleotides were in vitro synthesized (Synbio). The Nb genes were cloned into a pET-21b (+) vector at BamHI and XhoI restriction sites, or a pMAL-c5X vector at BamHI and EcoRI restriction sites.
Nb Purification
Nb DNA constructs were transformed into BL21(DE3) cells and plated on Agar with 50 μg/ml ampicillin at 37 °C overnight. A single bacterial colony was picked and cultured in LB broth to reach O.D. at ~ 450 nm. 0.1 or 0.5 mM IPTG was added to the E. coli cell culture for MBP fusion Nbs or his-tagged Nbs induction at 16°C overnight. Cells were then harvested, briefly sonicated, and lysed on ice with a lysis buffer (1xPBS, 150 mM NaCl, 0.2% TX-100 with protease inhibitor). After lysis, soluble protein extract was collected at 15,000xg for 10 mins, and recombinant his-tagged Nbs were purified by His6-Cobalt resin (Thermo) and eluted by imidazole; while MBP-fusion Nbs were purified by amylose resin (NEB) and eluted by maltose. The eluted Nbs were subsequently dialyzed in the dialysis buffer (e.g., 1x DPBS, pH 7.4). A HiLoad 16/600 Superdex 200 pg column (GE Healthcare) on an ÄKTA FPLC protein purification system (GE Healthcare) was used to further purify the Nbs and ensure their monomeric states. The purified Nbs were analyzed by SDS-PAGE. For the animal experiments, excessive endotoxin in the Nbs was removed (below 0.1 EU/dose) by the oxinEraser Endotoxin Removal column (Genscript) and measured by ToxinSensor Chromogenic LAL Endotoxin Assay Kit (Genscript). The purified Nbs were further sterilized by passing a 0.2 μm filter (Millex) and were stored at −80°C before use.
ELISA (Enzyme-linked immunosorbent assay)
Indirect ELISA was carried out to measure the relative affinities of Nbs. Albumin was coated onto a 96-well ELISA plate (R&D system) at 1-10 ng/well in coating buffer (15 mM sodium carbonate, 35 mM sodium bicarbonate, pH 9.6) overnight at 4°C and was blocked with a blocking buffer (DPBS, 0.05% Tween 20, 5% milk) at room temperature for 2 hrs. NbHSA were diluted in the blocking buffer and incubated with albumin for 2 hrs. HRP-conjugated secondary antibodies against His-tag (Genscript) were diluted 1:5,000-10,000 and incubated with the well for 1 hr at room temperature. After PBST (DPBS, 0.05% Tween 20) washes, the samples were further incubated under dark with freshly prepared w3,3′,5,5′-Tetramethylbenzidine (TMB) substrate for 10 mins to develop the signals. After the STOP solution (R&D system), the plates were read at multiple wavelengths (the optical density at 550 nm wavelength subtracted from the density at 450 nm) on a plate reader (Multiskan GO, Thermo Fisher). The relative affinity of each Nb was determined by the average of O.D. readouts. For human and monkey albumin, relative affinity of each Nb was tested at Nb concentration of 1 μM, 100 nM, 10 nM, 1 nM, and 100 pM. An average ELISA O.D. of >0.1 was used as the cutoff value to assign albumin binding Nbs. For mouse albumin, relative affinity was tested at one μM and a cutoff O.D. of >1 was used.
In vitro pull-down assay (Immunoprecipitation)
Nb or serum albumin was coupled to CNBr-activated sepharose resin (GE Healthcare). For the in vitro pull-down assay, different concentrations of Nbs were incubated with the human serum albumin coupled resin. Samples were incubated for 15-30 min at 4°C with gentle agitation. The resin was collected and washed three times with a washing buffer and was boiled in the LDS sample loading buffer (Thermo) before SDS PAGE analysis.
Nb Affinity measurement by surface plasmon resonance (SPR)
Surface plasmon resonance (SPR, Biacore 3000 system, GE Healthcare) was used to measure Nb affinities. Briefly, human serum albumin was immobilized to the flow channels of an activated CM5 sensor-chip. Protein analytes were diluted to 10 μg/ml in 10 mM sodium acetate, pH 4.5, and injected into the SPR system at 5 μl/min for 420 s. The surface was then blocked by 1 M ethanolamine-HCl (pH 8.5). For each Nb analyte, a series of dilution (spanning ~ 1,000-fold concentration range) was injected in duplicate, with HBS-EP+ running buffer (GE-Healthcare) at a flow rate of 20- 30 μl/min for 120- 180 s, followed by a dissociation time of 10 – 20 mins based on dissociation rate. Between each injection, the sensor chip surface was regenerated twice with a low pH buffer containing ten mM glycine-HCl (pH 1.5- 2.5) at a flow rate of 40-50 μl/min for 30 s - 1 min. Binding sensorgrams for each Nb were processed and analyzed using BIAevaluation by fitting with the 1:1 Langmuir model.
Clustering and phylogenetic tree analysis of NbHSA
A phylogenetic tree was generated by Clustal Omega 47 with the input of unique NbHSA CDR3 sequences and the adjacent framework sequences (i.e., YYCAA to the N-terminus and WGQG to the C-terminus of CDR3s) to help alignments. The data was plotted by ITol (Interactive Tree of Life) 48. Isoelectric points and hydrophobicities of the CDR3s were calculated using the BioPython library. The sequence logo was plotted using WebLogo 49.
Thermostability analysis of NbHSA
The thermal stability of NbHSA was measured by differential scanning fluorimetry (DSF). To prepare DSF samples, NbHSA were mixed with SYPRO orange dye (Invitrogen) in PBS to reach a final concentration of 2.5-15 μM. The samples were analyzed by a 7900HT Fast Real-Time PCR System (Applied Biosystems) in triplicate. The temperature was programmed to increase from 25°C to 95°C with a ramp rate of 1°C/min to generate the melting curves. The melting point was calculated by first derivatives method 50.
Chemical cross-linking and mass spectrometry (CXMS)
Nb was incubated with albumin in PBS and 1 mM dithiothreitol (DTT) to allow the formation of the complex. HSA-NbHSA complexes were crosslinked with 1 mM disuccinimidyl suberate (DSS, ThermoFisher Scientific) for 23 min at 25°C with gentle agitation. The crosslinking reaction was then quenched with 50 mM ammonium bicarbonate (ABC) for 10 min at room temperature. After protein reduction and alkylation, the cross-linked samples were separated by a 4–12% SDS-PAGE gel (NuPAGE, Thermo Fisher). The regions corresponding to the cross-linked species (~130 kDa) were cut and in-gel digested with trypsin and Lys-C as previously described 16,19,51. After proteolysis, the peptide mixtures were desalted and analyzed with a nano-LC 1200 (Thermo Fisher) coupled to a Q Exactive™ HF-X Hybrid Quadrupole-Orbitrap™ mass spectrometer (Thermo Fisher). The cross-linked peptides were loaded onto a picochip column (C18, 3□μm particle size, 300□Å pore size, 50□μm□×□10.5□cm; New Objective) and eluted using a 60□min LC gradient : 5% B–8% B, 0 – 5 min; 8% B – 32% B, 5 – 45 min; 32% B–100% B, 45 – 49 min; 100% B, 49 −54 min; 100% B - 5 % B, 54 min - 54 min 10 sec; 5% B, 54 min 10 sec - 60 min 10 sec; mobile phase A consisted of 0.1% formic acid (FA), and mobile phase B consisted of 0.1% FA in 80% acetonitrile. The QE HF-X instrument was operated in the data-dependent mode, where the top 8 most abundant ions (mass range 380–2,000, charge state□3 - 7) were fragmented by high-energy collisional dissociation (normalized collision energy 27). The target resolution was 120,000 for MS and 15,000 for MS/MS analyses. The quadrupole isolation window was 1.8□Th, and the maximum injection time for MS/MS was set at 120□ms. After the MS analysis, the data was searched by pLink for the identification of cross-linked peptides. The mass accuracy was specified as 10 and 20□p.p.m. for MS and MS/MS, respectively. Other search parameters included cysteine carbamidomethylation as a fixed modification and methionine oxidation as a variable modification. A maximum of three trypsin missed-cleavage sites was allowed. The initial search results were obtained using the default 5% false discovery rate, estimated using a target-decoy search strategy. The crosslink spectra were then manually checked to remove potential false-positive identifications essentially as previously described 16,19,51,52.
Integrative structural modeling of HSA-Nb complexes
Structural models for Nbs were obtained using a multi-template comparative modeling protocol of MODELLER53. Next, we refined the CDR3 loop and selected the top 5 scoring loop conformations for the downstream docking. Each Nb model was then docked to the HSA structure (PDB 4g03) by an antibody-antigen docking protocol of PatchDock software that focuses the search to the CDRs54 and optimizes CXMS-based distance restraints satisfaction55. A restraint was considered satisfied if the Ca-Ca distance between the cross-linked residues was within 30Å for DSS cross-linkers 16,56. The models were then re-scored by a statistical potential SOAP 57. The antigen interface residues (distance <6Å from Nb atoms) among the 10 best scoring models according to the SOAP score, were used to determine the epitopes. Convergence was measured as the average RMSD among the 10 top-scoring models. Once the epitopes were defined, we clustered the Nbs based on the epitope similarity using hierarchical clustering. The clusters reveal the most immunogenic surface patches on the antigens.
Size exclusion chromatography (SEC) and negative stain electron microscopy
HSA and MBP-Nbs were mixed in a HSA:Nb ratio of 1:1.2 and incubated at room temperature for 2 hours in PBS and 2 mM DTT. The complexes were analyzed by SEC (Superdex200, GE LifeSciences). HSA and the tetrameric HSA-NbHSA complex were visualized by transmission electron microscopy (TEM) under negative staining. For TEM analysis, the complexes were digested overnight at 4 °C with TEV protease to remove MBP tag. Protein was diluted to a final concentration of 30 μg/ml (HSA in PBS; HSA-Nb complexes in Tris 20 mM, 200mM NaCl, 3% Glycerol) and deposited on carbon-coated CF400-CU grids (EMS) which were freshly glow discharged. After 30 seconds of incubation, the excessive protein was removed, and the grid was stained with two drops of uranyl acetate 2 % w/v. Electron micrographs were recorded in an FEI TECNAI T12 operating at 120 kV with a 2k x 2k Gatan UltraScan 1000. Raw images were converted by IMOD 4.8 58and particles were selected using EMAN2 59. Particle processing and 3D reconstruction were done by Relion 3.0 60 with several rounds of auto-refine iterations. The final 3D structures were obtained with 4,000 particles for HSA and 21,400 particles for the tetrameric complex. Crystallographic structural models (PDB: 1AO6 and 5VNW) were used for the initial fitting by Chimera 61.
Site-directed mutagenesis
An HSA expression plasmid was obtained from Addgene (ALB-bio-His, Plasmid #52176). E400R and K383D point mutations were introduced to the HSA sequence by the Q5 site-directed mutagenesis kit. After sequence verification by Sanger Sequencing, plasmids bearing wild type HSA and the mutants were transfected to HeLa cells using Lipofectamine 3000 transfection kit (Invitrogen) and Opti-MEM (Gibco) according to the manufacturer’s protocol. The cells were cultured overnight before a change of medium to DMEM without FBS supplements to remove BSA. After a 48 h culture at 37°C, 5% CO2, the media expressing HSA were collected and stored at −20°C. The media were analyzed by SDS-PAGE and Western Blotting to confirm protein expression.
MS quantification of Nb pharmacokinetics
600 μg/g of HSA (w:w) was administered in B6.Cg-Tg(FCGRT)32Dcr Albem12Mvw Fcgrttm1Dcr/MvwJ mice (JAX, n = 3) by intravenous (i.v.) injection three days before the injection of Nbs. A mixture of Nbs including 20 NbHSA and 2 control Nbs in equal molarity were then injected at 30 μg/g (w:w, day 0). After injections, the whole blood samples were collected from the tail veins of the mice. The blood sampling time for HSA was pre-dose (0 min), 10 min, 24 h, 48 h. The sampling time for Nb mixture was pre-dose (0 min), 5, 15, 30 min, 1, 2, 4, 8, 16, 24 hrs, 2, 3, 4, 7, 10, 15, 21 days post-dose. Whole blood samples were allowed to clot at room temperature for 30 min and were centrifuged at 4°C, 20,000 × g for 3 min. Serum proteins from the supernatants were collected for the downstream proteomic analysis.
Serum samples were reduced in 8M urea digestion buffer (with 50 mM Ammonium bicarbonate, 5 mM TCEP, and DTT) at 57°C for 1 hr, and alkylated in the dark with 30 mM Iodoacetamide for 30 mins at room temperature. The samples were in-solution digested with 1:100 (w/w) trypsin and Lys-C at 37°C overnight, with incubation with another bolus of trypsin 4 hours. After proteolysis, the peptide mixtures were desalted and analyzed with a nano easyLC 1200 device coupled with a Q Exactive™ HF-X Hybrid Quadrupole Orbitrap™ mass spectrometer (Thermo Fisher). Briefly, Nb peptides were loaded onto a Picochip column (C18, 1.9 μm particle size, 120□Å pore size, 75□μm□×□25□cm; New Objective) and eluted using a 45-min liquid chromatography gradient (5% B–8% B, 0–3 min; 8% B–42% B, 3–35 min; 42% B–100% B, 35 – 40 min; 100% B, 40 - 45 min; mobile phase A consisted of 0.1% formic acid (FA), and mobile phase B consisted of 0.1% FA in 80% acetonitrile (ACN)). The flow rate was ~300 nl/min. The QE HF-X instrument was operated in the data-dependent mode, where the top 6 most abundant ions (mass range 300 – 2,000, charge state□2 - 8) were fragmented by high-energy collisional dissociation (HCD). The target resolution was 120,000 for MS and 60,000 for MS2 analyses. The quadrupole isolation window was 1.8□Th, and the maximum injection time for MS/MS was 120□ms.
Serum proteins including Nbs were first identified by MaxQuant version 1.6.1.0 62. Up to 4 unique and specific tryptic peptides with the preference of CDR3-containing peptides with high intensities were selected from each Nb.
For the MS1-based quantification, the area under the curve (AUC) of the selected peptides were calculated from the raw data by using Xcalibur Qual Browser. For each Nb, AUCs of the selected peptides were averaged to represent the abundance of the Nbs. Selected peptides were validated by MS/MS.
For the MS2-based quantification, an inclusion list of the Nb peptides to be monitored was generated. An LC RT window of −2~+3 min (for a 90-min LC gradient) was used to ensure that all the targeted peptides were included. The instrument was operated in a data-independent model where the targeted peptides were specifically selected for fragmentation (HCD normalized energy 27). Up to 5 abundant fragment ions from each peptide were chosen to increase the specificity of our quantification. AUCs of the fragment ions were calculated using Xcalibur Qual Browser. The relative abundance of Nbs was presented as the median AUC intensities of their respective MS2 fragment ions. It was further normalized based on the total MS1 ion current (TIC) of each LC run. An in-house script was developed to enable automatic quantification described above. The samples were analyzed in triplicates. The abundance of serum Nbs in pharmacokinetics analysis was determined by averaging technical and biological replicates; the resulting data was used to fit into a two-phase decay model by Prism GraphPad.
Serum proteomic quantification
1 mg HSA was administered to a B6.Cg-Tg(FCGRT)32Dcr Albem12Mvw Fcgrttm1Dcr/MvwJ mouse. Blood samples were collected from the tail vein at the following time: pre-dose, 10 min, 24, 48, 72, 96 h, 7, 10 days. Serum sample preparation and in-solution digestion of serum proteins were processed, as previously mentioned. Peptide samples were analyzed with a nano easy□C 1000 device coupled with a Q Exactive™ HF-X Hybrid Quadrupole Orbitrap™ mass spectrometer (Thermo Fisher). Briefly, Nb peptides were loaded onto an analytic column (1.6 μm particle size, 100□Å pore size, 75□μm□×□25□cm; IonOpticks) and eluted using a 90-min liquid chromatography gradient. A QE HF-X instrument was used for analysis. The quadrupole isolation window was 1.6□Th, and the maximum injection time for MS/MS was set at 100□ms. Serum protein levels were quantified using MaxQuant version 1.6.1.0. The resulting raw data were searched against a mouse Uniprot FASTA protein database (December 2018) with fixed modification of carbamidomethylation and variable modifications of methionine oxidation and N-terminal acetylation. Trypsin was set as a digestion enzyme, and a maximal missed cleavage of 2 was allowed. Protein’s false discovery rate was set to be 0.01. Minimal peptide length was 7, and maximal peptide mass was 4600 Da. Minimal ratio count of label-free quantification was set to be 2. Serum protein abundance at each time point is derived by averaging ion intensities of 3 technical replicates and normalized based on baseline abundance. Serum proteins were then clustered using the K-means clustering algorithm (K=10), and heatmap was generated by software Morpheus (https://software.broadinstitute.org/morpheus).
Purification of Duraleukins
hIL-2 (C125A) sequence was fused to C-termini of Nb sequences at the DNA level with a flexible linker (GGGGS)2 (Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser). The resulting DNA sequences were synthesized and cloned into pET-21b(+) (Synbio). Plasmids were transformed into BL21(DE3) E. coli cells to produce Duraleukins. After IPTG induction, Duraleukins were purified from the cell pallets. Cell pellets containing the inclusion bodies of Duraleukins were washed with a solubilization buffer A (0.1 M Tris, 2% sodium deoxycholate (SDC), 5 mM EDTA, pH 8) and sonicated on ice. Cell lysates were spun down at 12,000 × g for 20 min 4°C. Supernatants were discarded, and the pellets were saved for analysis. Cell pellets were washed with Milli-Q water and spun down at 12,000 × g for 20 min at 4°C. Solubilization buffer B (0.1 M Tris, 6 M guanidine hydrochloride (GuHCl), pH 8) was added to the pallets and incubated for 1 h incubation at room temperature with gentle agitation. After centrifugation 12,000 × g for 20 min at 4°C, the supernatants that contain soluble forms of Duraleukins were collected, and subsequently diluted with a Duraleukin refolding buffer (0.1 M Tris, pH 8, the final concentration of 10 mM reduced and 1 mM oxidized glutathione) to a GuHCl concentration of 2 M and a protein concentration of ~0.3 mg/mL. The solutions were then incubated at room temperature for 16 h with gently rotating to allow protein refolding. The insoluble fraction was removed by centrifugation at 16,000 × g for 20 min at 4°C, and refolded Duraleukins were obtained in the supernatants. IMAC and FPLC purification, detoxification, and sterilization were performed as previously described.
In vitro T cell proliferation assay
CTLL-2 cells (ATCC) were cultured, according to ATCC’s protocol. The complete culture medium was formulated as 10% FBS, an additional 2 mM L-glutamine, and 1 mM sodium pyruvate in ATCC-formulated RPMI-1640 containing 10% T-STIM with Con A (Corning). For CTLL-2 proliferation assay, cells were grown to a density of 1 × 105 cells/mL. Duraleukins or hIL-2 (PeproTech) were added into the wells of a flat-bottom 96-well tissue culture microplate. The final concentration of hIL-2 and Duraleukins was adjusted to 0.01~25 ng/mL and 0.025~50 ng/mL by 2-fold serial dilution. Every concentration group was tested in duplicate. 104 cells were seeded to each well of the plate, and the total volume in each well was 100 μL. The cells were then incubated at 37°C, 5% CO2 for 56 h. 10 μL of WST-1 reagent (Roche) was added to each well and the cells were incubated for another four h. The plate was shaken at 1,000 rpm for 1 min before analysis. A spectrometer read absorbance at 450 nm. 690 nm was used as a reference wavelength. A dose-response curve was fit via nonlinear regression using GraphPad Prism 7.
Generation and treatment of melanoma mouse model
B16F10 (ATCC) was cultured, according to ATCC’s protocol. The complete medium is formulated as 10% FBS in ATCC-formulated DMEM. C57BL/6J mice were purchased from The Jackson Laboratory. All mice were aged eight weeks before tumor inoculation. B16F10 melanoma cells were grown into the logarithmic growth phase (≤ 50% confluent), harvested, and adjusted to a concentration of 1 × 107 cells/ml in ice-cold HBSS. Mice were anesthetized with isoflurane and shaved in the middle of the back with a razor. 100 μl (106) of cells is subcutaneously (s.c.) injected to the middle of the shaved area with a 291/2-G insulin syringe (EXEL). For tumor growth experiments, intravenous (i.v.) injection of DL80 (17 μg), hIL-2 (7.5 μg, PeproTech), TA99 (125 μg, BioXCell) and PBS were done on indicated days. For flow cytometry analysis, a single i.v. administration of DL80 (17 μg) or hIL-2 (7.5 μg) was done on day 12.
Flow cytometry
The tumor was isolated from the mice (n = 5) 1 or 3 days after a single dose treatment. Cell suspensions were harvested by mechanical dissection and Collagenase IV Cocktail digestion (formulated as 7.4 kU Collagenase Type 4, 53 kU Deoxyribonuclease I, 20 mg Soybean Trypsin Inhibitor in 10 mL cocktail; Worthington Biochemical). The suspensions were passed through 40 μm cell strainers to ensure singularity. Cells were first treated with BioWhittaker ACK lysing buffer (Lonza), followed by TruStain FcX (BioLegend) and stained with antibodies against CD45 (clone 30-F11), CD3 (clone 17A2), CD8a (clone 53-6.7), NK1.1 (clone PK136). All antibodies are purchased from Biolegend. All samples were analyzed on a BD LSRFortessa cell analyzer. Data analysis was performed by Flowjo and Prism GraphPad.
Contributions
Y.S and Z.S conceived the research. Y.S. supervised the studies. Y.X and Y.S identified the Nbs. Z.S and Y.X performed major experiments and data analysis with help from Y.S, S.V, Z.X, A.V, C.J, G.C, B.H, G.C, Z. Sang, J.L and K.C. D.S and C.C performed structural modeling. Thank P. Duprex (Vaccine center, Pitt) for his assistance in the SPR experiments, D.Goebe, for critical reading of the manuscript. Y.S and Z.S drafted the manuscript with input from all the authors.
Completing Interests
The University of Pittsburgh has filed a provisional patent encompassing the technologies described in this manuscript.
Acknowledgment
This work was supported by the Pitt SOM start-up funds (Y.S.), a UPMC Aging Institute pilot fund (Y.S.), and a grant from the Alzheimer’s Association and Michael J. Fox Foundation (Y.S.).
Footnotes
Author affiliation information is corrected.