"Negative" Impact: The Role of Payload Charge in the Physicochemical Stability of Auristatin Antibody-Drug Conjugates.

Antibody-drug conjugates (ADCs) tend to be less stable than their parent antibodies, which is often attributed to the hydrophobic nature of their drug payloads. This study investigated how the payload charge affects ADC stability by comparing two interchain cysteine ADCs that had matched drug-to-antibody ratios and identical linkers but differently charged auristatin payloads, vcMMAE (neutral) and vcMMAF (negative). Both ADCs exhibited higher aggregation than their parent antibody under shaking stress and thermal stress conditions. However, conjugation with vcMMAF increased the aggregation rates to a greater extent than conjugation with uncharged but more hydrophobic vcMMAE. Consistent with the payload logD values, ADC-vcMMAE showed the greatest increase in hydrophobicity but minor changes in charge compared with the parent antibody, as indicated by hydrophobic interaction chromatography and capillary electrophoresis data. In contrast, ADC-vcMMAF showed a decrease in net charge and isoelectric point along with an increase in charge heterogeneity. This charge alteration likely contributed to a reduced electrostatic repulsion and increased surface activity in ADC-vcMMAF, thus affecting its aggregation propensity. These findings suggest that not only the hydrophobicity of the payload, but also its charge should be considered as a critical factor affecting the stability of ADCs.

Evaluating the Potential of Cyclodextrins in Reducing Aggregation of Antibody-Drug Conjugates with Different Payloads.

Cyclodextrins (CDs) are versatile agents used to solubilize small drugs and stabilize proteins. This dual functionality may be particularly beneficial for antibody-drug conjugates (ADCs), as CDs may "mask" the hydrophobicity of the drug payloads. In this study, we explored the effect of CDs on the physical stability of ADCs composed of the same antibody but with different payloads (maytansinoid, auristatin, and fluorophore payloads). The aggregation of ADCs was evaluated under shaking stress conditions and elevated temperatures using size-exclusion chromatography, turbidity, and backgrounded membrane imaging. Our results showed that hydroxypropyl-(HP)-CDs effectively stabilized all ADCs during shaking stress, with increasing stabilization in the order of HPαCD < HPγCD < HPßCD at concentrations of 7.5 mM and (near) complete stabilization at 75 mM. Native CDs without surface activity also stabilized certain ADCs, although less effectively than HP-CDs under agitation stress. During quiescent incubation, the HP-CD effects were small for most ADCs. However, for an ADC with a fluorophore payload that rapidly aggregated after conjugation, HPγCD substantially reduced aggregate levels, in line with fluorescence data supporting CD-ADC interactions. In contrast, sulfobutylether-ß-CD (SBEßCD) increased the aggregation rates in all ADCs under all stress conditions. In conclusion, this study highlights the potential of appropriate CD formulations to improve the physical stability of ADCs.

Agitation-Induced Aggregation of Lysine- And Interchain Cysteine-Linked Antibody-Drug Conjugates.

Drug conjugation to an antibody can affect its stability, which depends on factors such as the conjugation technique used, drug-linker properties, and stress encountered. This study focused on the effects of agitation stress on the physical stability of two lysine (ADC-K) and two interchain cysteine (ADC-C) conjugates of an IgG1 monoclonal antibody (mAb) linked to either ∼4 MMAE or DM1 payloads. During agitation, all antibody-drug conjugates (ADCs) exhibited higher aggregation than the mAb, which was dependent on the conjugation technique (aggregation of ADC-Ks > ADC-Cs) and drug-linker (aggregation of ADCs with MMAE > ADCs with DM1). The aggregation propensities correlated well with higher self-interaction, hydrophobicity, and surface activity of ADCs relative to the mAb. The intermediate reduced mAb (mAb-SH) showed even higher aggregation than the final product ADC-Cs. However, blocking mAb-SH's free thiols with N-ethylmaleimide (NEM) strongly reduced its aggregation, suggesting that free thiols should be minimized in cysteine ADCs. Further, this study demonstrates that a low-volume surface tension method can be used for estimating agitation-induced aggregation of ADCs in early development phases. Identifying liabilities to agitation stress and their relationship to biophysical properties may help optimize ADC stability.

Miniaturized Forced Degradation of Therapeutic Proteins and ADCs by Agitation-Induced Aggregation Using Orbital Shaking of Microplates.

Microplate-based formulation screening is a powerful approach to identify stabilizing excipients for therapeutic proteins while reducing material requirements. However, this approach is sometimes not representative of studies conducted in relevant container closures. The present study aimed to identify critical parameters for a microplate-based orbital shaking method to screen biotherapeutic formulations by agitation-induced aggregation. For this purpose, an in-depth methodological study was conducted using different shakers, microplates, and plate seals. Aggregation was monitored by size exclusion chromatography, turbidity, and backgrounded membrane imaging. Both shaker quality and liquid-seal contact had substantial impacts on aggregation during shaking and resulted in non-uniform sample treatment when parameters were not suitably selected. The well volume to fill volume ratio (Vwell/Vfill) was identified as an useful parameter for achieving comparable aggregation levels between different microplate formats. An optimized method (2400 rpm [ac 95 m/s2], Vfill 60-100 µL [Vwell/Vfill 6-3.6], 24 h, RT, heat-sealed) allowed for uniform sample treatment independent of surface tension and good agreement with vial shaking results. This study provides valuable guidance for miniaturization of shaking stress studies in biopharmaceutical drug development, facilitating method transfer and comparability between laboratories.

Sortase-A mediated chemoenzymatic lipidation of single-domain antibodies for cell membrane engineering.

PURPOSE: Membrane engineering has versatile applications in adoptive cell therapies, immune therapy or drug delivery. Incorporation of lipidated antibody-derived ligands into cells may enforce supraphysiological cell interactions that offer new therapeutic approaches. A challenge is the defined synthesis of lipidated ligands that effectively interact with such membranes. METHODS: Sortase-A was used to attach a PEGylated, dimyristyl lipid-anchor on single-domain antibodies (VHH). The membrane insertion was investigated on liposomal bilayers, myeloid-derived suppressor cells (MDSC) and T cells. RESULTS: The lipidated VHHs remodeled liposomal as well as cellular membranes. The VHH carrying liposomes were successfully targeted towards antigen-positive cells. MDSC and T cells were both modified with lipidated VHHs as detected with an FITC-anti-llama antibody. T cells that carried an anti-CD11b VHH showed cellular association in vitro with CD11b+Gr-1+ MDSC in a two-dimensional magnetic activated cell sorting / flow-cytometry assay. CONCLUSION: The applied combination of chemoenzymatic ligation, PEGylated lipid anchors and single-domain antibodies delivers water-soluble and chemically defined lipidated ligands, which readily associate with liposomal and cellular membranes. This enables liposomal drug targeting and artificial cell-cell interactions. Hence, the presented concept for lipidation of single-domain antibodies is promising for further application in the field of drug delivery or cell-based therapies.

Sortagged anti-EGFR immunoliposomes exhibit increased cytotoxicity on target cells.

PURPOSE: Conventional chemotherapy is associated with therapy-limiting side effects, which might be alleviated by targeted chemotherapeutics such as immunoliposomes. The targeting ligands of immunoliposomes are commonly attached by unspecific chemical conjugation, bearing risk of structural heterogeneity and therewith related biological consequences. Chemoenzymatic methods may mitigate such risks through site-specific conjugation. METHODS: The formulation parameters for pentaglycine-modified, doxorubicin-loaded liposomes and the reaction conditions for a site-specific, Sortase-A mediated conjugation with monoclonal antibodies were thoroughly evaluated. The cytotoxicity of such sortagged, epidermal growth factor receptor (EGFR)-specific immunoliposomes was tested on human breast cancer cells. RESULTS: Sortaggable liposomes with a defined size (140 nm, PDI < 0.25) and high encapsulation efficiency (>90%) were obtained after manufacturing optimization. A ratio of 1.0-2.5 µM mAb/100 µM pentaglycine yielded stable dispersions and circumvented carrier precipitation during ligand grafting. The cytotoxicity on EGFR+ MDA-MB-468 was up to threefold higher for EGFR-specific immunoliposomes than for the nontargeted controls. CONCLUSIONS: Sortase-A is suitable to generate immunoliposomes with a site-specific ligand-carrier linkage and hence improves chemical homogeneity of targeted therapeutics. However, the sweet spot for manufacturability utilizing mAbs with two Sortase-A recognition sites is narrow, making mono-reactive binders such as scFvs or Fab's preferable for a further development. Despite this, the immunoliposomes demonstrated a targeted delivery of doxorubicin, indicating the potential to increase the therapeutic window during the treatment of EGFR+ tumors.

Sortagging of liposomes with a murine CD11b-specific VHH increases in vitro and in vivo targeting specificity of myeloid cells.

The therapeutic index of drugs can be increased via drug encapsulation in actively targeted, meaning ligand modified drug delivery systems. The manufacturing of such targeted drug delivery systems, in particular the conjugation between drug carrier and ligand, can be done by enzymatic conjugation methods, exploiting the site-specific, bioorthogonal nature of these reactions. The use of such enzymes like Sortase-A transpeptidase requires efficient purification methods, as residuals of the enzyme may be responsible for immunogenic potential and drug product instabilities. These instabilities may be based on the enzymatic reverse reaction, meaning here a cleavage between ligand and drug carrier. In the presented work, two differently PEGylated formulations were modified with variable fragments of camelid heavy chain-only antibodies (VHH) via Sortase-A, purified by different methodologies and tested for ligand cleavage upon storage. Strongly PEGylated liposomes (PEGhigh-LS) were found to retain higher amounts of Sortase-A than lowly PEGylated ones (PEGlow-LS) after dialysis purification. Surprisingly, this did not correlate with ligand stability during storage. PEGhigh-LS were less prone for degradation, compared to PEGlow-LS, which showed a ligand cleavage of 20% after an 8 weeks storage at 2-8 °C. Nonetheless, overall degradation could be minimized by an additional affinity bead purification procedure. Liposomes modified with a CD11b-specific VHH were tested for their in vitro and in vivo targeting ability towards CD11b+ cells. Specific targeting of CD11b was achieved in vitro and in vivo on various cell types. PEGylation decreased the targeting effect in vitro, however no differences between PEGhigh or PEGlow formulations were observed in vivo. The obtained results underline the need for a thorough characterization of novel conjugation strategies as well as an early in vivo characterization of such targeted drug delivery systems.

Sortaggable liposomes: Evaluation of reaction conditions for single-domain antibody conjugation by Sortase-A and targeting of CD11b+ myeloid cells.

Active targeting with ligand coated liposomal drug delivery systems is a means to increase the therapeutic index of drugs. Stable ligand coating requires bilayer anchorage of the commonly proteinaceous ligands and hence a conjugation of lipid structures towards amino acids. This often leads to heterogeneous reaction products especially when chemical coupling methods are employed. Chemoenzymatic Sortase-A mediated transpeptidation (sortagging) is a useful tool to avoid this protein heterogeneity through its site-specific, bioorthogonal ligation mechanism. Manufacturing of such sortaggable, pentaglycine modified liposomes was developed by adaption of a scalable solvent injection technique. The pentaglycine liposomes were prepared with different degrees of PEGylation and steric accessibility of the pentaglycine motif. Comparable hydrodynamic diameters (146-188 nm) of the different formulations were obtained after a flow rate screening. The sortagging reactivity of a single-domain antibody (VHH) towards the pentaglycine liposomes was strongly dependent on the steric accessibility of the pentaglycine nucleophile. Adjusting the pentaglycine to ligand ratio improved conversion rates up to 80%. The liposome bound VHH was accessible for its soluble antigen as shown by a chromatography-based binding assay. Mono- and granulocytes could be selectively targeted in vitro by conjugation of BMX1, a VHH directed towards human myeloid cell surface marker CD11b. Confocal microscopy revealed intracellular localization of the targeted liposomes. The developability of those pentaglycine liposomes as well as their proof of principle for targeted drug delivery shows their potential for further investigation, for example as delivery platform for diagnostics or drugs into the tumor microenvironment.

Pentaglycine lipid derivates - rp-HPLC analytics for bioorthogonal anchor molecules in targeted, multiple-composite liposomal drug delivery systems.

The quantification of lipids and assessment of lipid composition is an indispensable step during the pharmaceutical development of novel lipid based drug delivery systems such as liposomes. Broad excipient screenings of such formulations raise the need for versatile analytical methods. Even more demanding complexity is generated by introduction of targeted systems requiring functionalized lipids. We addressed this demand by developing an rp-HPLC based analytical method with evaporative light scattering detection (ELSD) for the simultaneous analysis of commonly used phosphatidylcholines, cholesterol and bilayer surface-modifying cationic, anionic or PEGylated lipids, which can be analyzed in combination with novel pentaglycine lipids suitable as targeting ligand anchor. The method was validated for specificity, precision, accuracy and sample stability. We monitor the continuous and scalable manufacturing of two pentaglycine-modified liposomal formulations and track the modification of these drug delivery systems with a single-domain antibody utilizing bioorthogonal Sortase-A technology. Both the presented analytical and preparative techniques can help to improve the quality control and to accelerate the pharmaceutical development of such targeted drug delivery systems.