Localization of Therapeutic Fab-CHP Conjugates to Sites of Denatured Collagen for the Treatment of Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation in synovial joints and protease-induced cartilage degradation. Current biologic treatments for RA can effectively reduce symptoms, primarily by neutralizing the proinflammatory cytokine TNFα; however, continued, indiscriminate overinhibition of inflammatory factors can significantly weaken the host immune system, leading to opportunistic infections and interrupting treatment. We hypothesize that localizing anti-TNFα therapeutics to denatured collagen (dCol) present at arthritic joints, via conjugation with collagen-hybridizing peptides (CHPs), will reduce off-site antigen binding and maintain local immunosuppression. We isolated the antigen-binding fragment of the clinically approved anti-TNFα therapeutic infliximab (iFab) and prepared iFab-CHP conjugates via lysine-based conjugation with an SMCC linker. After successful conjugation, confirmed by LC-MS, the binding affinity of iFab-CHP was characterized by ELISA-like assays, which showed comparable antigen binding relative to infliximab, comparable dCol binding relative to CHP, and the hybrid ability to bind both dCol and TNFα simultaneously. We further demonstrated localization of Fab-CHP to areas of high dCol in vivo and promising therapeutic efficacy, assessed by histological staining (Safranin-O and H&E), in a pilot mouse study.

Antibody and antibody derivatives as cancer therapeutics.

Antibodies are an important class of therapeutic for treating a wide range of diseases. These versatile macromolecules can be engineered to target many different antigens and to utilize several mechanisms of action to produce a pharmacological effect. The most common antibody platform used for therapeutics is immunoglobulin G (IgG). Advances in protein-display and genetic engineering have enabled the construction and manipulation of IgG to enhance desired activity such as increasing antigen affinity, modulating pharmacokinetics, and enhancing effector functions. IgGs can also be altered to suppress undesired effects, such as immunogenicity. The main approaches to control IgG behavior include engineering the protein sequence and glycosylation of intact IgG; constructing IgG-based derivatives, including bispecific and multivalent binders; and fusing small-drug molecules or proteins to IgG-derived scaffolds. Often, a single modification applied to a given IgG can alter more than one property. The desired effects of an antibody therapeutic should be carefully tailored to the physiology and characteristics of each disease condition. This article is categorized under: Therapeutic Approaches and Drug Discovery > Nanomedicine for Oncologic Disease Therapeutic Approaches and Drug Discovery > Emerging Technologies Biology-Inspired Nanomaterials > Peptide-Based Structures.

Biophysical Properties and Heating-Induced Aggregation of Lysine-Conjugated Antibody-Drug Conjugates.

The commercially available antibody-drug conjugate (ADC) product, Kadcyla® is synthesized using a 2-step reaction, wherein the linker is conjugated to native lysines on the mAb in step 1, followed by drug conjugation to the linker-modified antibody in step 2. In our study, we synthesized a lysine-conjugated ADC (Syn-ADC) on the same trastuzumab scaffold as Kadcyla® using a 1-step reaction. Mass spectrometry of both products revealed a subpopulation of Kadcyla® containing free linkers conjugated to the mAb, but not conjugated to the drug, which were absent in the 1-step reaction ADC product. Differential scanning calorimetry thermograms showed that the drug and linker conjugation significantly reduced the thermal stability and energies of activation for the denaturation of the CH2 domain of the ADCs. The heating induced aggregation events started as early as ∼57°C and ∼45°C for Kadcyla® and Syn-ADC, respectively, compared with 71°C for Herceptin®. The colloidal stability measurements clearly showed that the hydrophobic drug payload on ADCs significantly reduced the repulsive interprotein interactions when compared to the unconjugated antibody under formulation buffer conditions (pH 6.0). Attaching hydrophobic drug and linker moieties onto the antibody lowered the thermal and colloidal stabilities and increased the aggregation propensity of the ADCs.

In-Depth Comparison of Lysine-Based Antibody-Drug Conjugates Prepared on Solid Support Versus in Solution.

Antibody drug conjugates are a rapidly growing form of targeted chemotherapeutics. As companies and researchers move to develop new antibody-drug conjugate (ADC) candidates, high-throughput methods will become increasingly common. Here we use advanced characterization techniques to assess two trastuzumab-DM1 (T-DM1) ADCs; one produced using Protein A immobilization and the other produced in solution. Following determination of payload site and distribution with liquid chromatography-mass spectrometry (LC/MS), thermal stability, heat-induced aggregation, tertiary structure, and binding affinity were characterized using differential scanning calorimetry (DSC), dynamic light scattering (DLS), Raman spectroscopy, and isothermal titration calorimetry (ITC), respectively. Small differences in the thermal stability of the CH2 domain of the antibody as well as aggregation onset temperatures were observed from DSC and DLS, respectively. However, no significant differences in secondary and tertiary structure were observed with Raman spectroscopy, or binding affinity as measured by ITC. Lysine-based ADC conjugation produces an innately heterogeneous population that can generate significant variability in the results of sensitive characterization techniques. Characterization of these ADCs indicated nominal differences in thermal stability but not in tertiary structure or binding affinity. Our results lead us to conclude that lysine-based ADCs synthesized following Protein A immobilization, common in small-scale conjugations, are highly similar to equivalent ADCs produced in larger scale, solution-based methods.

In vitro and in vivo assessment of controlled release and degradation of acoustically responsive scaffolds.

Spatiotemporally controlled release of growth factors (GFs) is critical for regenerative processes such as angiogenesis. A common strategy is to encapsulate the GF within hydrogels, with release being controlled via diffusion and/or gel degradation (i.e., hydrolysis and/or proteolysis). However, simple encapsulation strategies do not provide spatial or temporal control of GF delivery, especially non-invasive, on-demand controlled release post implantation. We previously demonstrated that fibrin hydrogels, which are widely used in tissue engineering and GF delivery applications, can be doped with perfluorocarbon emulsion, thus yielding an acoustically responsive scaffold (ARS) that can be modulated with focused ultrasound, specifically via a mechanism termed acoustic droplet vaporization. This study investigates the impact of ARS and ultrasound properties on controlled release of a surrogate payload (i.e., fluorescently-labeled dextran) and fibrin degradation in vitro and in vivo. Ultrasound exposure (2.5MHz, peak rarefactional pressure: 8MPa, spatial peak time average intensity: 86.4mW/cm2), generated up to 7.7 and 21.7-fold increases in dextran release from the ARSs in vitro and in vivo, respectively. Ultrasound also induced morphological changes in the ARS. Surprisingly, up to 2.9-fold greater blood vessel density was observed in ARSs compared to fibrin when implanted subcutaneously, even without delivery of pro-angiogenic GFs. The results demonstrate the potential utility of ARSs in generating controlled release for tissue regeneration. STATEMENT OF SIGNIFICANCE: Simple encapsulation of a molecular payload within a conventional hydrogel scaffold does not provide spatial or temporal control of payload release. Yet, spatiotemporally controlled release of bioactive payloads is critical for tissue regeneration, which often utilizes hydrogel scaffolds to facilitate processes such as angiogenesis. This work investigates the design and performance (both in vitro and in vivo) of hydrogel scaffolds where release of a fluorescent payload is non-invasively and spatiotemporally-controlled using focused ultrasound. We also quantitatively characterize the degradation and vascularization of the scaffolds. Our results may be of interest to groups working on controlled release strategies for implants, especially within the field of tissue engineering.

Design and Characterization of Fibrin-Based Acoustically Responsive Scaffolds for Tissue Engineering Applications.

Hydrogel scaffolds are used in tissue engineering as a delivery vehicle for regenerative growth factors. Spatiotemporal patterns of growth factor signaling are critical for tissue regeneration, yet most scaffolds afford limited control of growth factor release, especially after implantation. We previously found that acoustic droplet vaporization can control growth factor release from a fibrin scaffold doped with a perfluorocarbon emulsion. This study investigates properties of the acoustically responsive scaffold (ARS) critical for further translation. At 2.5 MHz, acoustic droplet vaporization and inertial cavitation thresholds ranged from 1.5 to 3.0 MPa and from 2.0 to 7.0 MPa peak rarefactional pressure, respectively, for ARSs of varying composition. Viability of C3H/10T1/2 cells, encapsulated in the ARS, did not decrease significantly for pressures below 4 MPa. ARSs with perfluorohexane emulsions displayed higher stability versus those with perfluoropentane emulsions, while surrogate payload release was minimal without ultrasound. These results enable the selection of ARS compositions and acoustic parameters needed for optimized spatiotemporally controlled release.