Yield, redissolution and activity of desolvated monoclonal antibodies.

(a) Yield of trastuzumab desolvation with different sodium chloride concentrations. (n = 3) (b) DLS intensity distribution of redissolved and diluted trastuzumab nanoparticles (n = 3). (c) Percentage of trastuzumab nanoparticles that redissolved in PBS with 0.5 mg/mL polysorbate-20 (n = 3). (d) Relative activity of redissolved trastuzumab compared to dialyzed trastuzumab (prior to desolvation) in a HER2 specific ELISA (n = 3). (e) Representative histograms of redissolved and stock trastuzumab and atezolizumab binding to OVCAR-3 and ES-2 cells, respectively. (f) Binding study of redissolved and stock trastuzumab, atezolizumab and daratumumab to OVCAR-3, ES-2 and U2932 cells, respectively. The solid lines are guides to the eye (n = 3). Data are presented as mean with all shaded areas and error bars denoting standard deviation. Statistical analyses were performed using a paired, one-way Anova, followed by a Tukey’s test if significant (p48]. “*” and “**” indicates p<0.05 and p<0.005, respectively.</p

Effect of sodium chloride on monoclonal antibody desolvation particle size.

(a) Schematic illustration of the monoclonal antibody (mAb) desolvation process in which 1.1 mL of acetonitrile was drop-wise added (0.5 mL/min) to 200 μL of mAb (10 mg/mL) at 20°C under stirring (1300 rpm). (b) Dynamic light scattering (DLS) intensity distribution of desolvated commercial mAbs nanoparticles atezolizumab and trastuzumab (n = 4). The mAbs elotuzumab, rituximab, daratumumab and cetuximab flocculated out of solution. (c) DLS intensity distribution of desolvated trastuzumab at various sodium chloride (NaCl) concentrations (n = 3). (d) Influence of initial NaCl concentration on particle size after desolvation of trastuzumab, atezolizumab and daratumumab, with corresponding Table 2 (n = 3). The solid lines are guides to the eye. (e) Desolvated atezolizumab at indicated NaCl concentrations, including flocculated particles at 3 mM. Data are presented as mean with all shaded areas and error bars denoting standard deviation.</p

DLVO and desolvation theory.

(a) Schematic depiction of the interplay between the potential energy of electrostatic (EDL) and Van der Waals (VdW) forces over distance and the influence of ion concentration (n) and particle radius (R) on the aggregation barrier. (b) Schematic illustration of exposed protein surface area to the solvent when denatured or precipitated. In general, protein precipitation leads to reduced protein surface area, whereas denatured protein increases its surface area.</p

Numerical data used for plotting figures.

About 30% of the FDA approved drugs in 2021 were protein-based therapeutics. However, therapeutic proteins can be unstable and rapidly eliminated from the blood, compared to conventional drugs. Furthermore, on-target but off-tumor protein binding can lead to off-tumor toxicity, lowering the maximum tolerated dose. Thus, for effective treatment therapeutic proteins often require continuous or frequent administration. To improve protein stability, delivery and release, proteins can be encapsulated inside drug delivery systems. These drug delivery systems protect the protein from degradation during (targeted) transport, prevent premature release and allow for long-term, sustained release. However, thus far achieving high protein loading in drug delivery systems remains challenging. Here, the use of protein desolvation with acetonitrile as an intermediate step to concentrate monoclonal antibodies for use in drug delivery systems is reported. Specifically, trastuzumab, daratumumab and atezolizumab were desolvated with high yield (∼90%) into protein nanoparticles below 100 nm with a low polydispersity index (</div

Physicochemical properties and excipients of the commercial monoclonal antibodies atezolizumab, cetuximab, daratumumab, elotuzumab, rituximab and trastuzumab.

Physicochemical properties and excipients of the commercial monoclonal antibodies atezolizumab, cetuximab, daratumumab, elotuzumab, rituximab and trastuzumab.</p

Influence of initial sodium chloride concentration on particle size and polydispersity index after desolvation of trastuzumab, atezolizumab and daratumumab.

Influence of initial sodium chloride concentration on particle size and polydispersity index after desolvation of trastuzumab, atezolizumab and daratumumab.</p

Stability desolvated particles over time and cellular binding of redissolved trastuzumab.

(a) Dynamic light scattering (DLS) intensity distribution of desolvated trastuzumab particles 0 min (n = 4), 30 min (n = 2) and 60 min (n = 2) after production. (b) Binding study of redissolved and stock trastuzumab to SK-BR-3 cells, detected by recombinant FcR (n = 1). (c) Histogram of redissolved and stock trastuzumab binding MDA-MB-231 and MDA-MB-231.HER2 cells (n = 1). Data are presented as mean with all shaded areas denoting standard deviation. (PDF)</p

DataSheet_1_EGFR-selective activation of CD27 co-stimulatory signaling by a bispecific antibody enhances anti-tumor activity of T cells.pdf

A higher density of tumor infiltrating lymphocytes (TILs) in the tumor microenvironment, particularly cytotoxic CD8+ T cells, is associated with improved clinical outcome in various cancers. However, local inhibitory factors can suppress T cell activity and hinder anti-tumor immunity. Notably, TILs from various cancer types express the co-stimulatory Tumor Necrosis Factor receptor CD27, making it a potential target for co-stimulation and re-activation of tumor-infiltrated and tumor-reactive T cells. Anti-cancer therapeutics based on exploiting CD27-mediated T cell co-stimulation have proven safe, but clinical responses remain limited. This is likely because current monoclonal antibodies fail to effectively activate CD27 signaling, as this receptor requires higher-order receptor cross-linking. Here, we report on a bispecific antibody, CD27xEGFR, that targets both CD27 and the tumor antigen, epidermal growth factor receptor (EGFR). By targeting EGFR, which is commonly expressed on carcinomas, CD27xEGFR induced cancer cell-localized crosslinking and activation of CD27. The design of CD27xEGFR includes an Fc-silent domain, which is designed to minimize potential toxicity by reducing Fc gamma receptor-mediated binding and activation of immune cells. CD27xEGFR bound to both of its targets simultaneously and triggered EGFR-restricted co-stimulation of T cells as measured by T cell proliferation, T cell activation markers, cytotoxicity and IFN-γ release. Further, CD27xEGFR augmented T cell cytotoxicity in a panel of artificial antigen-presenting carcinoma cell line models, leading to Effector-to-Target ratio-dependent elimination of cancer cells. Taken together, we present the in vitro characterization of a novel bispecific antibody that re-activates T cell immunity in EGFR-expressing cancers through targeted co-stimulation of CD27.</p

DataSheet_2_EGFR-selective activation of CD27 co-stimulatory signaling by a bispecific antibody enhances anti-tumor activity of T cells.xlsx

A higher density of tumor infiltrating lymphocytes (TILs) in the tumor microenvironment, particularly cytotoxic CD8+ T cells, is associated with improved clinical outcome in various cancers. However, local inhibitory factors can suppress T cell activity and hinder anti-tumor immunity. Notably, TILs from various cancer types express the co-stimulatory Tumor Necrosis Factor receptor CD27, making it a potential target for co-stimulation and re-activation of tumor-infiltrated and tumor-reactive T cells. Anti-cancer therapeutics based on exploiting CD27-mediated T cell co-stimulation have proven safe, but clinical responses remain limited. This is likely because current monoclonal antibodies fail to effectively activate CD27 signaling, as this receptor requires higher-order receptor cross-linking. Here, we report on a bispecific antibody, CD27xEGFR, that targets both CD27 and the tumor antigen, epidermal growth factor receptor (EGFR). By targeting EGFR, which is commonly expressed on carcinomas, CD27xEGFR induced cancer cell-localized crosslinking and activation of CD27. The design of CD27xEGFR includes an Fc-silent domain, which is designed to minimize potential toxicity by reducing Fc gamma receptor-mediated binding and activation of immune cells. CD27xEGFR bound to both of its targets simultaneously and triggered EGFR-restricted co-stimulation of T cells as measured by T cell proliferation, T cell activation markers, cytotoxicity and IFN-γ release. Further, CD27xEGFR augmented T cell cytotoxicity in a panel of artificial antigen-presenting carcinoma cell line models, leading to Effector-to-Target ratio-dependent elimination of cancer cells. Taken together, we present the in vitro characterization of a novel bispecific antibody that re-activates T cell immunity in EGFR-expressing cancers through targeted co-stimulation of CD27.</p