A BCMA/CD16A bispecific innate cell engager for the treatment of multiple myeloma.

Despite the recent progress, multiple myeloma (MM) is still essentially incurable and there is a need for additional effective treatments with good tolerability. RO7297089 is a novel bispecific BCMA/CD16A-directed innate cell engager (ICE®) designed to induce BCMA+ MM cell lysis through high affinity binding of CD16A and retargeting of NK cell cytotoxicity and macrophage phagocytosis. Unlike conventional antibodies approved in MM, RO7297089 selectively targets CD16A with no binding of other Fcγ receptors, including CD16B on neutrophils, and irrespective of 158V/F polymorphism, and its activity is less affected by competing IgG suggesting activity in the presence of M-protein. Structural analysis revealed this is due to selective interaction with a single residue (Y140) uniquely present in CD16A opposite the Fc binding site. RO7297089 induced tumor cell killing more potently than conventional antibodies (wild-type and Fc-enhanced) and induced lysis of BCMA+ cells at very low effector-to-target ratios. Preclinical toxicology data suggested a favorable safety profile as in vitro cytokine release was minimal and no RO7297089-related mortalities or adverse events were observed in cynomolgus monkeys. These data suggest good tolerability and the potential of RO7297089 to be a novel effective treatment of MM patients.

Method for Measurement of Antibody-Dependent Cellular Phagocytosis.

Antibody-based therapeutics are powerful tools to treat disease. While their mechanism of action (MOA) always involves binding to a specific target via the antibody-binding fragment (Fab) region of the antibody, the induction of immune-mediated effector functions through the fragment crystallizable (Fc) region is a vital aspect of antibody therapeutics targeting tumor cells. Cross-linking of the Fc gamma receptors (FcγRs) via cell-bound antibodies activate immune effector cells, leading to antibody-dependent cellular cytotoxicity via natural killer (NK) cells. Linking of FcγRs on macrophages triggers the process of antibody-dependent cellular phagocytosis (ADCP), where antibody-opsonized target cells are internalized in phagosomes and degraded through the process of phagosome maturation and acidification. ADCP activity can be challenging to measure accurately due to the difficulty in differentiating target cells that are bound to a macrophage versus those that are internalized within phagosomes. In this chapter, we describe a protocol that measures ADCP activity by labeling target cells with a pH-sensitive dye that fluoresces brightly in mature phagosomes. The ADCP activity of therapeutics is then measured via flow cytometry. This assay is capable of detecting glycosylation differences arising from manufacturing processes and is suitable for evaluation of ADCP activity of monoclonal antibodies (mAb) to support in vitro biological characterization of drug candidates and lead candidate selection for desirable effector functions.

An integrated approach for characterizing immunogenic responses toward a bispecific antibody.

Bispecific antibodies (bsAbs) recognize and bind two different targets or two epitopes of the same antigen, making them an attractive diagnostic and treatment modality. Compared to the production of conventional bivalent monospecific antibodies, bsAbs require greater engineering and manufacturing. Therefore, bsAbs are more likely to differ from endogenous immunoglobulins and contain new epitopes that can increase immunogenic risk. Anti-A/B is a bsAb designed using a 'knobs-into-holes' (KIH) format. Anti-A/B exhibited an unexpectedly high immunogenicity in both preclinical and clinical studies, resulting in early termination of clinical development. Here, we used an integrated approach that combined in silico analysis, in vitro assays, and an in vivo study in non-human primates to characterize anti-A/B immunogenicity. Our findings indicated that the immunogenicity is associated with epitopes in the anti-B arm and not with mutations engineered through the KIH process. Our results showed the value of this integrated approach for performing immunogenicity risk assessment during clinical candidate selection to effectively mitigate risks during bsAb development.

Immunogenicity risk assessment for biotherapeutics through in vitro detection of CD134 and CD137 on T helper cells.

Biotherapeutics, which are biologic medications that are natural or bioengineered products of living cells, have revolutionized the treatment of many diseases. However, unwanted immune responses still present a major challenge to their widespread adoption. Many patients treated with biotherapeutics develop antigen-specific anti-drug antibodies (ADAs) that may reduce the efficacy of the therapy or cross-react with the endogenous counterpart of a protein therapeutic, or both. Here, we describe an in vitro method for assessing the immunogenic risk of a biotherapeutic. We found a correlation between clinical immunogenicity and the frequency with which a biotherapeutic stimulated an increase in CD134, CD137, or both cell surface markers on CD4+ T cells. Using high-throughput flow cytometry, we examined the effects of 14 biotherapeutics with diverse rates of clinical immunogenicity on peripheral blood mononuclear cells from 120 donors with diverse human leukocyte antigen class II-encoding alleles. Biotherapeutics with high rates of ADA development in the clinic had higher proportions of CD4+ T cells positive for CD134 or CD137 than biotherapeutics with low clinical immunogenicity. This method provides a rapid and simple preclinical test of the immunogenic potential of a new candidate biotherapeutic or biosimilar. Implementation of this approach during biotherapeutic research and development enables rapid elimination of candidates that are likely to cause ADA-related adverse events and detrimental consequences.

A novel method for determining antibody-dependent cellular phagocytosis.

Antibody-based therapeutics are powerful tools to treat disease. While their mechanism of action (MOA) always involves binding to a specific target via the Fab region of the antibody, the induction of effector functions through the Fc region of the antibody is equally important for antibody therapeutics designed to deplete tumor cells. By binding of the Fc region to Fc gamma receptors (FcγRs) on the surface of immune cells or complement factors, antibody therapeutics exert effector functions such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), both of which induce target cell death and aid in the efficacy of treatment. Another major Fc effector function is antibody-dependent cellular phagocytosis (ADCP). ADCP is the mechanism by which antibody-opsonized target cells activate the FcγRs on the surface of macrophages to induce phagocytosis, resulting in internalization and degradation of the target cell through phagosome acidification. ADCP has been implicated as a major MOA of several biologics, but this activity is difficult to measure in in vitro. Most assays measure the association of target cells and macrophages; however, co-localization can represent cell attachment rather than internalization. Here, we describe the development of a novel method to accurately measure ADCP activity. By labeling target cells with a pH sensitive dye that only fluoresces in mature phagosomes, the ADCP activity of antibody therapeutics can be accurately quantitated via flow cytometry.

Development of a kinetic antibody-dependent cellular cytotoxicity assay.

Antibody-dependent cellular cytotoxicity (ADCC) is an important mechanism of action (MOA) of monoclonal antibody (mAb) therapeutics. Target cells opsonized with therapeutic antibody bind and activate FcγR-bearing immune effector cells, resulting in target cell lysis. A key step in mAb drug development is the characterization of ADCC activity for its potential to inform mAb efficacy and safety. A number of in vitro assays are commonly used to assess ADCC. Most are endpoint assays that measure a surrogate marker of cell lysis. Newer imaging technologies allow direct measurement of ADCC-mediated cell lysis over time. In this study, we detail the development and characterization of a kinetic ADCC assay applicable to multiple target and effector cell types. This kinetic assay shows comparable sensitivity to an endpoint fluorescence release ADCC assay, while offering the advantages of a simpler set up and shorter assay time. Our results demonstrate that kinetic ADCC activity is a valid alternative assay format for measuring in vitro ADCC of mAbs.

Tenofovir disoproxil fumarate loaded PLGA nanoparticles for enhanced oral absorption: Effect of experimental variables and in vitro, ex vivo and in vivo evaluation.

In this study, PLGA based nanoparticles of tenofovir disoproxil fumarate (TDF) were designed for enhancing its oral absorption. To develop PLGA based TDF nanoparticles with the goal of minimum particle size and maximum entrapment efficiency statistical optimization techniques (factorial design and response surface methodology) were employed. The optimized nanoparticles were characterized for size, shape, charge and physical state. Further, the stability, cytotoxicity and metabolic protective effect of the nanoparticles were evaluated. Single dose pharmacokinetic study in rats was conducted to evaluate the oral absorption of the designed nanoparticles. Ex vivo everted gut sac studies were performed to evaluate the role of active uptake mechanisms in the absorption of the designed nanoparticles. The results showed that the statistical models employed could determine the interaction effects of the critical factors which were used in the optimization of the nanoparticles. The optimized nanoparticles with a particle size of 218±3.85nm and an entrapment efficiency of 57.3±1.6%. The nanoparticles were able to increase the AUC of tenofovir by 5.8 fold. It was observed that active uptake mechanisms predominantly via clathrin-mediated uptake played a key role in increasing the oral absorption of TDF.

Oral pharmacokinetic interaction of ester rich fruit juices and pharmaceutical excipients with tenofovir disoproxil fumarate in male Wistar rats.

1. The aim of this study was to evaluate the role of intestinal esterases on the absorption process of tenofovir disoproxil fumarate (TDF). 2. The esterase inhibition capacity of fruit juices (FJs) rich in ester linkages and pharmaceutical excipients (having ester bonds) was performed in vitro by incubating TDF with each FJ and excipient in the intestinal washings. The ex vivo everted gut sac model was also used to evaluate the absorption enhancement capacity of these FJs and excipients. Single-dose oral pharmacokinetic studies were performed by concomitant administration of TDF with each of the selected FJs and excipients. 3. The in vitro and ex vivo studies showed that cremophor-EL and all FJs prevented the metabolism of TDF with grapefruit juice (GFJ) having the highest level of inhibition. Further, the permeability flux of the monoester form of tenofovir was increased by 113% and 212% by cranberry juice (CBJ) and GFJ, respectively. The in vivo studies also showed that both CBJ and GFJ enhanced the oral bioavailability of TDF as the AUC was increased by 24% and 97%, respectively. 4. These results indicate that the prevention of the metabolic conversion of TDF to its monoester form is crucial in increasing the oral absorption of TDF.

Curcumin Delivery by Poly(Lactide)-Based Co-Polymeric Micelles: An In Vitro Anticancer Study.

PURPOSE: This work describes the synthesis of block co-polymeric micelles, methoxy-poly(ethylene glycol)-poly(D,L-lactide) (mPEG-PLA) to encapsulate Curcumin (CUR), thereby improving the dispersibility and chemical stability of curcumin, prolonging its cellular uptake and enhancing its bioavailability. METHODS: CUR-mPEG-PLA micelles, was prepared using the thin-film hydration method and evaluated in vitro. The preparation process was optimized with a central composite design (CCD). Micelles were characterized by size, transmission electron microscopy, loading capacity, and critical micelle concentration (CMC). The cytotoxicity of CUR-mPEG-PLA micelles was investigated against murine melanoma cells, B16F10 and human breast cancer cells, MDA-MB-231. RESULTS: The average size of the CUR-mPEG-PLA micelles was 110 ± 5 nm with polydispersity index in the range of 0.15-0.31, and the encapsulating efficiency for CUR was 91.89 ± 1.2, and 11.06 ± 0.8% for drug-loading. Sustained release of CUR from micelles was observed with 9.73% CUR release from micelles compared to 64.24% release of free curcumin in first 6 h under sink condition. The CUR-mPEG-PLA was efficiently taken up by the cancer cells, B16F10 and MDA-MB-231. Following 24 h incubation, CUR-mPEG-PLA induced higher cytotoxicity compared to free CUR in MDA-MB-231 cell lines indicating exposure of higher dose of free CUR to cells lead to up-regulation of drug efflux mechanisms leading to decreased cell death in case of free CUR administration. CONCLUSION: Our results indicate that the proposed micellar system has the potential to serve as an efficient carrier for CUR by effectively solubilizing, stabilizing and delivering the drug in a controlled manner to the cancer cells.