PAT-SM6 binds to C1q and mediates complement deposition and activation on MM cell lines and patients material.

<p>A, Sandwich ELISA: For C1q binding analysis, plates were coated with PAT-SM6 or controls (isotype or Rituximab). After blocking, plates were incubated with human C1q followed by sheep anti human C1q-HRP. PAT-SM6 showed clear but moderate binding to C1q compared to the isotype control. Rituximab displayed a stronger C1q binding capacity. B, PAT-SM6 mediated deposition of C1q on the surface of OPM-2 cells assessed by using human C1q and detecting antibodies in FACS. C1q deposition was clearly improved with PAT-SM6 compared to cells without antibody treatment or isotype control. C, CDC activity was determined using the alamar blue assay. OPM-2 cells were incubated with human complement (huC), PAT-SM6 or controls for 2 hours followed by alamar blue solution and the amount of lysed cells was analysed with a fluorescence reader. A dose dependent killing kinetic was observed. D, CD138-purified primary MM cells at primary diagnosis (n = 8, dots) or relapse (n = 2, rectangle) were incubated with human complement, PAT-SM6 or controls (isotype, Rituximab) for 2 h and the amount of lysed was assessed by FACS (Propidium iodide/LDS75). In the PAT-SM6 treated samples a moderate killing was observed, whereas Rituximab showed no significant cytotoxicity.</p

PAT-SM6 mediates cytotoxicity to patient MM cells and MM cell lines by induction of apoptosis.

<p>A, MM cell lines were incubated with various concentrations of PAT-SM6 or control (unrelated IgM at highest concentration) for 72 h. Cell death was determined by FACS (Propidium iodide/LDS75 double positive cells). B, CD138-purified primary MM cells at primary diagnosis (n = 8, dots) or relapse (n = 3, rectangle) were incubated for 48 h with PAT-SM6 or isotype in media containing IL-6 and 10% FCS. Dead cells were determined by FACS analysis (Propidium iodide/LDS75 double positive cells). As control, PBMCs from healthy volunteers were treated under the same conditions. In contrast to controls PAT-SM6 significantly induced programmed cell death. C, MM1.S cells were incubated with PAT-SM6 or controls (staurophorin as positive control) for 72 h. AnnexinV and 7AAD positive cells were assessed by flow cytometry. Percentage of 7-AAD/Annexin V double positive cells, Isotype: 14%, PAT-SM6∶79% and positive control: 76%. D, MM1.S cells were incubated with PAT-SM6 or controls (staurophorin or isotype) for 6 h and caspase 3 activation was measured using the AC-DEVD-AMC protease assay. E, Bone marrow cells from a MM patient were CD33-purified to obtain myeloid progenitors and stimulated with PAT-SM6 or controls. No specific binding and subsequently no specific cytotoxicity was observed. F, Cells from the same patient as in E were CD138-isolated and incubated with PAT-SM6 (200 µg/mL) for 24 h. Specific PAT-SM6 binding and killing was observed.</p

PAT-SM6 specifically binds to MM cell lines and primary human MM samples.

<p>A, INA-6, NCI H929, MM1.S, OPM-2 and U266 MM cell lines or CD138-purified patient MM cells were stained with PAT-SM6 (5 µg/mL, open histogram) or isotype control (gray histogram) followed by fluorescein isothiocyanate (FITC)-conjugated secondary antibodies and assessed by flow cytometry. Both, cell lines as well as primary MM cells displayed specific PAT-SM6 binding. B, Immunohistochemistry with PAT-SM6, CD138-positive control antibody or isotype on bone marrow biopsies of MM patients. PAT-SM6 specifically stained MM cells with a homogenous binding pattern. In samples without MM infiltration no specific binding was detected. Images were captured using a Leica DM BL microscope, the Leica ICC HD digital camera and the Leica LAS EZ V2.1.0 software.</p

Immunohistochemical analysis of the PAT-SM6 target expression pattern in bone marrow biopsies of MM patients.

<p>Immunohistochemical analysis of the PAT-SM6 target expression pattern in bone marrow biopsies of MM patients.</p

Target validation, PAT-SM6 reacts with cell surface expressed GRP78.

<p>A, Sandwich ELISA with recombinant GRP78 and PAT-SM6. Plates were coated with GRP78, blocked and incubated with PAT-SM6 or other natural IgM antibodies as controls followed by incubation with anti-human IgM-HRP. PAT-SM6 showed binding to recombinant GRP78. B, Competitive ELISA: Plates were coated with GRP78 and increasing amounts of GRP78 or control protein were added to a solution of PAT-SM6 followed by anti-human IgM-HRP. Unbound GRP78 reduced binding of PAT-SM6 on a GRP78 coated plate. C, Surface expression GRP78 (black histogram) in MM cell lines is shown using an anti GRP78 IgG (rabbit) antibody. D, GRP78 expression in bone marrow biopsies was assessed by immunohistochemistry with anti GRP78 IgG, PAT-SM6 or positive control CD138. Primary MM cells were positive for GRP78 and the staining pattern correlates with the pattern displayed by PAT-SM6. In contrast to PAT-SM6, anti-GRP78 IgG also reacted with non-malignant cells. Images were captured using a Leica DM BL microscope, the Leica ICC HD digital camera and the Leica LAS EZ V2.1.0 software.</p

Supplement from A GRP78-Directed Monoclonal Antibody Recaptures Response in Refractory Multiple Myeloma with Extramedullary Involvement

Supplementary Table 1 Figure S1A, S1B</p

DataSheet1_Reliable and Interpretable Mortality Prediction With Strong Foresight in COVID-19 Patients: An International Study From China and Germany.PDF

Cohort-independent robust mortality prediction model in patients with COVID-19 infection is not yet established. To build up a reliable, interpretable mortality prediction model with strong foresight, we have performed an international, bi-institutional study from China (Wuhan cohort, collected from January to March) and Germany (Würzburg cohort, collected from March to September). A Random Forest-based machine learning approach was applied to 1,352 patients from the Wuhan cohort, generating a mortality prediction model based on their clinical features. The results showed that five clinical features at admission, including lymphocyte (%), neutrophil count, C-reactive protein, lactate dehydrogenase, and α-hydroxybutyrate dehydrogenase, could be used for mortality prediction of COVID-19 patients with more than 91% accuracy and 99% AUC. Additionally, the time-series analysis revealed that the predictive model based on these clinical features is very robust over time when patients are in the hospital, indicating the strong association of these five clinical features with the progression of treatment as well. Moreover, for different preexisting diseases, this model also demonstrated high predictive power. Finally, the mortality prediction model has been applied to the independent Würzburg cohort, resulting in high prediction accuracy (with above 90% accuracy and 85% AUC) as well, indicating the robustness of the model in different cohorts. In summary, this study has established the mortality prediction model that allowed early classification of COVID-19 patients, not only at admission but also along the treatment timeline, not only cohort-independent but also highly interpretable. This model represents a valuable tool for triaging and optimizing the resources in COVID-19 patients.</p

Additional file 1 of Bortezomib induced peripheral neuropathy and single nucleotide polymorphisms in PKNOX1

Supplementary Material