Pre-Clinical Assessment of SAR442257, a CD38/CD3xCD28 Trispecific T Cell Engager in Treatment of Relapsed/Refractory Multiple Myeloma.

Current treatment strategies for multiple myeloma (MM) are highly effective, but most patients develop relapsed/refractory disease (RRMM). The anti-CD38/CD3xCD28 trispecific antibody SAR442257 targets CD38 and CD28 on MM cells and co-stimulates CD3 and CD28 on T cells (TCs). We evaluated different key aspects such as MM cells and T cells avidity interaction, tumor killing, and biomarkers for drug potency in three distinct cohorts of RRMM patients. We found that a significantly higher proportion of RRMM patients (86%) exhibited aberrant co-expression of CD28 compared to newly diagnosed MM (NDMM) patients (19%). Furthermore, SAR442257 mediated significantly higher TC activation, resulting in enhanced MM killing compared to bispecific functional knockout controls for all relapse cohorts (Pearson's r = 0.7). Finally, patients refractory to anti-CD38 therapy had higher levels of TGF-ß (up to 20-fold) compared to other cohorts. This can limit the activity of SAR442257. Vactoserib, a TGF-ß inhibitor, was able to mitigate this effect and restore sensitivity to SAR442257 in these experiments. In conclusion, SAR442257 has high potential for enhancing TC cytotoxicity by co-targeting CD38 and CD28 on MM and CD3/CD28 on T cells.

Design Strategy for Nanostructured Arrays of Metallodielectric Cuboids to Systematically Tune the Optical Response and Eliminate Spurious Bulk Effects in Plasmonic Biosensors.

Plasmonic biosensors are a powerful tool for studying molecule adsorption label-free and with high sensitivity. Here, we present a systematic study on the optical properties of strictly regular nanostructures composed of metallodielectric cuboids with the aim to deliberately tune their optical response and improve their biosensing performance. In addition, the patterns were tested for their potential to eliminate spurious effects from sensor response, caused by refractive index changes in the bulk solution. Shifts in the plasmonic spectrum are exclusively caused by the adsorbing molecules. For this purpose, nanopatterns of interconnected and separated cubes with dimensions ranging from 150 to 600 nm have been fabricated from poly(methyl methacrylate) using electron-beam lithography followed by metallization with gold. It is shown that a small lateral pattern size, a high aspect ratio, and short connection lengths are favorable to generate extinction spectra with well-separated and pronounced peaks. Furthermore, for selected nanostructures, we have been able to identify reflection angles for which the influence of the bulk refractive index on the position of the plasmonic peaks is negligible. It is shown that sensor operation under these angles enables monitoring of in situ biomolecule adsorption with high sensitivity providing a promising tool for high-throughput applications.

A microfluidic Braille valve platform for on-demand production, combinatorial screening and sorting of chemically distinct droplets.

Droplet microfluidics is a powerful tool for a variety of biological applications including single-cell genetics, antibody discovery and directed evolution. All these applications make use of genetic libraries, illustrating the difficulty of generating chemically distinct droplets for screening applications. This protocol describes our Braille Display valving platform for on-demand generation of droplets with different chemical contents (16 different reagents and combinations thereof), as well as sorting droplets with different chemical properties, on the basis of fluorescence signals. The Braille Display platform is compact, versatile and cost efficient (only ~US$1,000 on top of a standard droplet microfluidics setup). The procedure includes manufacturing of microfluidic chips, assembly of custom hardware, co-encapsulation of cells and drugs into droplets, fluorescence detection of readout signals and data analysis using shared, freely available LabVIEW and Python packages. As a first application, we demonstrate the complete workflow for screening cancer cell drug sensitivities toward 74 conditions. Furthermore, we describe here an assay enabling the normalization of the observed drug sensitivity to the number of cancer cells per droplet, which additionally increases the robustness of the system. As a second application, we also demonstrate the sorting of droplets according to enzymatic activity. The drug screening application can be completed within 2 d; droplet sorting takes ~1 d; and all preparatory steps for manufacturing molds, chips and setting up the Braille controller can be accomplished within 1 week.

Circulating Tumor and Immune Cells for Minimally Invasive Risk Stratification of Smoldering Multiple Myeloma.

PURPOSE: Early intervention in smoldering multiple myeloma (SMM) requires optimal risk stratification to avoid under- and overtreatment. We hypothesized that replacing bone marrow (BM) plasma cells (PC) for circulating tumor cells (CTC), and adding immune biomarkers in peripheral blood (PB) for the identification of patients at risk of progression due to lost immune surveillance, could improve the International Myeloma Working Group 20/2/20 model. EXPERIMENTAL DESIGN: We report the outcomes of 150 patients with SMM enrolled in the iMMunocell study, in which serial assessment of tumor and immune cells in PB was performed every 6 months for a period of 3 years since enrollment. RESULTS: Patients with >0.015% versus ≤0.015% CTCs at baseline had a median time-to-progression of 17 months versus not reached (HR, 4.9; P < 0.001). Presence of >20% BM PCs had no prognostic value in a multivariate analysis that included serum free light-chain ratio >20, >2 g/dL M-protein, and >0.015% CTCs. The 20/2/20 and 20/2/0.015 models yielded similar risk stratification (C-index of 0.76 and 0.78). The combination of the 20/2/0.015 model with an immune risk score based on the percentages of SLAN+ and SLAN- nonclassical monocytes, CD69+HLADR+ cytotoxic NK cells, and CD4+CXCR3+ stem central memory T cells, allowed patient' stratification into low, intermediate-low, intermediate-high, and high-risk disease with 0%, 20%, 39%, and 73% rates of progression at 2 years. CONCLUSIONS: This study showed that CTCs outperform BM PCs for assessing tumor burden. Additional analysis in larger series are needed to define a consensus cutoff of CTCs for minimally invasive stratification of SMM.

Hyaluronan hydrogels delivering BMP-6 for local targeting of malignant plasma cells and osteogenic differentiation of mesenchymal stromal cells.

Multiple myeloma is a malignant disease characterized by accumulation of clonal plasma cells in the bone marrow. Uncoupling of bone formation and resorption by myeloma cells leads to osteolytic lesions. These are prone to fracture and represent a possible survival space for myeloma cells under treatment causing disease relapse. Here we report on a novel approach suitable for local treatment of multiple myeloma based on hyaluronic acid (HA) hydrogels mimicking the physical properties of the bone marrow. The HA hydrogels are complexed with heparin to achieve sustained presentation and controlled release of bone morphogenetic protein 6 (BMP-6). Others and we have shown that BMP-6 induces myeloma cell apoptosis and bone formation. Using quartz crystal microbalance and enzyme-linked immunosorbent assay, we measured an initial surface density of 400 ng BMP6/cm2, corresponding to two BMP-6 per heparin molecule, with 50% release within two weeks. HA-hydrogels presenting BMP-6 enhanced the phosphorylation of Smad 1/5 while reducing the activity of BMP-6 antagonist sclerostin. These materials induced osteogenic differentiation of mesenchymal stromal cells and decreased the viability of myeloma cell lines and primary myeloma cells. BMP-6 functionalized HA-hydrogels represent a promising material for local treatment of myeloma-induced bone disease and residual myeloma cells within lesions to minimize disease relapse or fractures. STATEMENT OF SIGNIFICANCE: Multiple myeloma is a hematological cancer characterized by the accumulation of clonal plasma cells in the bone marrow and local suppression of bone formation, resulting in osteolytic lesions and fractures. Despite recent advances in systemic treatment of multiple myeloma, it is rare to achieve a targeted suppression of myeloma cells and healing of bone lesions. Here we present hydrogels which mimic the physico-chemical properties of the bone marrow, consisting of hyaluronic acid with crosslinked heparin for the controlled presentation of bioactive BMP-6. The hydrogels decrease the viability of myeloma cell lines and primary myeloma cells and induces osteogenic differentiation of mesenchymal stromal cells. The presentation of BMP-6 in the hyaluronan hydrogels enhances the phosphorylation of Smad1/5 while reducing the activity of the BMP-6 antagonist sclerostin. As such, BMP-6 functionalized hyaluronan hydrogels represent a promising material for the localized eradication of myeloma cells.

Copresentation of BMP-6 and RGD Ligands Enhances Cell Adhesion and BMP-Mediated Signaling.

We report on the covalent immobilization of bone morphogenetic protein 6 (BMP-6) and its co-presentation with integrin ligands on a nanopatterned platform to study cell adhesion and signaling responses which regulate the transdifferentiation of myoblasts into osteogenic cells. To immobilize BMP-6, the heterobifunctional linker MU-NHS is coupled to amine residues of the growth factor; this prevents its internalization while ensuring that its biological activity is maintained. Additionally, to allow cells to adhere to such platform and study signaling events arising from the contact to the surface, we used click-chemistry to immobilize cyclic-RGD carrying an azido group reacting with PEG-alkyne spacers via copper-catalyzed 1,3-dipolar cycloaddition. We show that the copresentation of BMP-6 and RGD favors focal adhesion formation and promotes Smad 1/5/8 phosphorylation. When presented in low amounts, BMP-6 added to culture media of cells adhering to the RGD ligands is less effective than BMP-6 immobilized on the surfaces in inducing Smad complex activation and in inhibiting myotube formation. Our results suggest that a local control of ligand density and cell signaling is crucial for modulating cell response.

Switchable Release of Bone Morphogenetic Protein from Thermoresponsive Poly(NIPAM-co-DMAEMA)/Cellulose Sulfate Particle Coatings.

Thermoresponsive coatings of poly(N-isopropylacrylamide-co-DMAEMA)/cellulose sulfate (PNIPAM-DMAEMA/CS) complexes are reported eluting bone-morphogenetic-protein-2 (BMP-2) on demand relevant for implant assisted local bone healing. PNIPAM-DMAEMA/CS dispersions contained colloid particles with hydrodynamic radii RH = 170⁻288 nm at T = 25 °C shrinking to RH = 74⁻103 nm at T = 60 °C. Obviously, PNIPAM-DMAEMA/CS undergoes volume phase transition (VPT) analogously to pure PNIPAM, when critical VPT temperature (VPTT) is exceeded. Temperature dependent turbidity measurements revealed broad VPT and VPTT 47 °C for PNIPAM-DMAEMA/CS colloid dispersions at pH = 7.0. FTIR spectroscopy on thermoresponsive PNIPAM-DMAEMA/CS particle coatings at germanium model substrates under HEPES buffer indicated both wet-adhesiveness and VPT behavior based on diagnostic band intensity increases with temperature. From respective temperature courses empirical VPTT ≈ 42 °C for PNIPAM-DMAEMA/CS coatings at pH = 7.0 were found, which were comparable to VPTT found for respective dispersions. Finally, the PNIPAM-DMAEMA/CS coatings were loaded with BMP-2 and model protein papain (PAP). Time dependent FTIR spectroscopic measurements showed, that for T = 37 °C there was a relative protein release of ≈30% for PAP and ≈10% for BMP-2 after 24 h, which did not increase further. Heating to T = 42 °C for PAP and to 47 °C for BMP-2 further secondary protein release of ≈20% after 24 h was found, respectively, interesting for clinical applications. BMP-2 eluted even at 47 °C was found to be still biologically active.

Target Expression, Generation, Preclinical Activity, and Pharmacokinetics of the BCMA-T Cell Bispecific Antibody EM801 for Multiple Myeloma Treatment.

We identified B cell maturation antigen (BCMA) as a potential therapeutic target in 778 newly diagnosed and relapsed myeloma patients. We constructed an IgG-based BCMA-T cell bispecific antibody (EM801) and showed that it increased CD3+ T cell/myeloma cell crosslinking, followed by CD4+/CD8+ T cell activation, and secretion of interferon-γ, granzyme B, and perforin. This effect is CD4 and CD8 T cell mediated. EM801 induced, at nanomolar concentrations, myeloma cell death by autologous T cells in 34 of 43 bone marrow aspirates, including those from high-risk patients and patients after multiple lines of treatment, tumor regression in six of nine mice in a myeloma xenograft model, and depletion of BCMA+ cells in cynomolgus monkeys. Pharmacokinetics and pharmacodynamics indicate weekly intravenous/subcutaneous administration.

Matrix-Immobilized BMP-2 on Microcontact Printed Fibronectin as an in vitro Tool to Study BMP-Mediated Signaling and Cell Migration.

During development, growth factors (GFs) such as bone morphogenetic proteins (BMPs) exert important functions in several tissues by regulating signaling for cell differentiation and migration. In vivo, the extracellular matrix (ECM) not only provides support for adherent cells, but also acts as reservoir of GFs. Several constituents of the ECM provide adhesive cues, which serve as binding sites for cell trans-membrane receptors, such as integrins. In conveying adhesion-mediated signaling to the intracellular compartment, integrins do not function alone but rather crosstalk and cooperate with other receptors, such as GF receptors. Here, we present a strategy for the immobilization of BMP-2 onto cellular fibronectin (cFN), a key protein of the ECM, to investigate GF-mediated signaling and migration. Following biotinylation, BMP-2 was linked to biotinylated cFN using NeutrAvidin as cross-linker. Characterization with quartz crystal microbalance with dissipation monitoring and enzyme-linked immunosorbent assay confirmed the efficient immobilization of BMP-2 on cFN over a period of 24 h. To validate the bioactivity of matrix-immobilized BMP-2 (iBMP-2), we investigated short- and long-term responses of C2C12 myoblasts, which are an established in vitro model for BMP-2 signaling, in comparison to soluble BMP-2 (sBMP-2) or in absence of GFs. Similarly to sBMP-2, iBMP-2 triggered Smad 1/5 phosphorylation and translocation of the complex to the nucleus, corresponding to the activation of BMP-mediated Smad-dependent pathway. Additionally, successful suppression of myotube formation was observed after 6 days in sBMP-2 and iBMP-2. We next implemented this approach in the fabrication of cFN micropatterned stripes by soft lithography. These stripes allowed cell-surface interaction only on the patterned cFN, since the surface in between was passivated, thus serving as platform for studies on directed cell migration. During a 10-h observation time, the migratory behavior, especially the cells' net displacement, was increased in presence of BMP-2. As such, this versatile tool retains the bioactivity of GFs and allows the presentation of ECM adhesive cues.