Journal: Science Advances

Article Title: A nonviral, nonintegrating DNA nanovector platform for the safe, rapid, and persistent manufacture of recombinant T cells

doi: 10.1126/sciadv.abf1333

Figure Lengend Snippet: The efficacy of targeting tumor cells expressing the human epitopes CEA, NY-BR1, and CD19 was evaluated in preclinical models. For the CEA model, 1 × 10 6 cells were implanted subcutaneously in NSG mice, and on day 7, the mice were treated with mock, and CAR-T cells were generated with lentivirus and nS/MARt. Tumor growth ( A ) and mice survival ( B ) were monitored. nS/MARt CAR-T cells showed a higher tumor infiltration when compared to Lenti CAR-T cells ( C ), while the human T cells engraftment in both groups appeared similar ( D ). The outgrown tumors showed an antigen loss in the treated groups, while it remains expressed in the control group ( E ). CAR-T cells from the spleens, tumors, and total blood of the treated mice were isolated, and an RCA demonstrated the presence of the episomal plasmids [( F ). M. DNA molecular marker; V, nS/MARt-CEA DNA restriction analysis; +, RCA on purified nS/MARt-CEA subsequentially digested with restriction enzymes]. Similarly, for the NY-BR1 model, cells expressing the epitope were xenografted into NSG mice. The efficacy of tumor killing was recorded as tumor growth ( G ) and survival ( H ). CAR-T cells in the tumors, spleens, and blood of treated mice were found 30 days after injection ( I ), and the episomal maintenance of the vector was demonstrated by RCA ( J ). Nalm-6 cells (1 × 10 5 ) modified to express the luciferase constitutively were injected into NSG mice by tail vein injection. Three days after tumor cell injection, 1 × 10 6 CD19-CAR-T cells generated with mock, nS/MARt, or lentivirus were administered. The tumor growth was monitored through bioluminescent imaging ( K ), as the radiance (p/s/cm 2 /sr), and the statistical analysis was performed with a one-way ANOVA followed by post hoc analysis (** P < 0.001 and *** P < 0.001).

Article Snippet: The expression of the CEA-CAR was detected with the anti-human FCg antibody (polyclonal and R-PE conjugated; Jackson ImmunoResearch), while biotinylated human CD19 (Acro Biosystems) followed by Streptavidin–Alexa Fluor 488 (Life Technologies) was used for the detection of the expression of the CD19-CAR.

Techniques: Expressing, Generated, Control, Isolation, Marker, Purification, Injection, Plasmid Preparation, Modification, Luciferase, Imaging

Journal: Nature Cancer

Article Title: ISB 2001 trispecific T cell engager shows strong tumor cytotoxicity and overcomes immune escape mechanisms of multiple myeloma cells

doi: 10.1038/s43018-024-00821-1

Figure Lengend Snippet: a , Cartoon and structural model of ISB 2001 BEAT. On the cartoon, immunoglobulin domains are shown as rectangles. VH domains of the anti-CD38, anti-BCMA and anti-CD3ε binders are depicted in blue, orange and magenta, respectively. All binders make use of a cLC depicted in gray. Fc-silencing mutations are depicted by the orange dots. The BEAT interface shown in the CH3 domains is depicted by the green and black dots. Chain A encompasses an engineered human IgG1 CH2 domain with an engineered human IgG3 CH3 domain. Chain B has engineered human IgG1 CH2 and CH3 domains. CHx, constant domain x; TCR Cα or TCR Cβ, BEAT interface proprietary mutations based on the T cell receptor constant domain α or β, respectively. ISB 2001 BEAT was generated by homology modeling. b , Human T cell activation of anti-CD3ε produced as human IgG1 LALA and control isotype by incubating with a dose–response of the cLC Fab bound to the plate. Graph shows mean ± s.d. ( n = 6 independent T cell donors from two independent experiments). c , SPR sensorgrams from a single replicate show blockade of BCMA/APRIL interaction (top sensorgram) and blockade of BCMA/BAFF interaction (bottom sensorgram) upon binding of anti-BCMA-E6 Fab to recombinant human BCMA protein. Curves are colored by anti-BCMA-E6 Fab concentration in BCMA/anti-BCMA premix solution. Data provided are from a single experiment (no repeats). d , Competition binding assay by Octet BLI. Curves represent injection of anti-CD38-B3 Fab/daratumumab Fab premix (dashed line) or daratumumab at twofold concentration of saturating solution (solid line) over CD38 immobilized surface saturated with daratumumab Fab from a single replicate in one experiment (no repeats). e , Binding sensorgrams of respective representative measurements from three independent replicates show the binding of ISB 2001 to human CD3εδ, human BCMA and human CD38 by SPR. Colored curves represent single concentration injections with serial dilutions of 1:3. Black curves represent 1:1 kinetic fits (BCMA and CD38). For binding to CD3εδ, the K d was inferred from a steady-state affinity model.

Article Snippet: For binding to CD3εδ, BCMA and CD38, biotinylated human CD3ε&CD3δ protein (Creative Biomart, CD3E & CD3D-377H), biotinylated human CD38 protein (Acrobiosystems, CD8-H82E7) or biotinylated human BCMA protein (Acrobiosystems, BCA-H82E4) were immobilized on a Biotin Capture (CAP) sensor chip (Cytiva) and increasing concentrations of ISB 2001 were flushed onto the immobilized ligand.

Techniques: Generated, Activation Assay, Produced, Control, Binding Assay, Recombinant, Concentration Assay, Injection

Journal: Nature Cancer

Article Title: ISB 2001 trispecific T cell engager shows strong tumor cytotoxicity and overcomes immune escape mechanisms of multiple myeloma cells

doi: 10.1038/s43018-024-00821-1

Figure Lengend Snippet: a – c , Cytotoxicity of KMS-12-BM cells at different concentrations of CD3 ×DU × CD38 and CD3 × CD38 × DU ( a ), CD3 × BCMA × DU and CD3 × DU × BCMA ( b ) and ISB 2001 and CD3 × CD38 × BCMA ( c ). RDL assays were performed at a 5:1 effector to target ratio for 48 h with purified T cells. Graphs show four-parameter logistic curve fitting with symbols representing mean ± s.d. ( n = 6 independent T cell donors from two independent experiments). d – f , T cell activation in a HD-PBMC at different concentrations of CD3 × DU × CD38 and CD3 × CD38 × DU ( d ), CD3 × BCMA × DU and CD3 × DU × BCMA ( e ) and ISB 2001 and CD3 × CD38 × BCMA ( f ). Graphs show four-parameter logistic curve fitting with symbols representing mean ± s.d. ( n = 6 independent T cell donors from two independent experiments). g , Cytotoxicity of the KMS-12-BM cell line at different concentrations of ISB 2001, CD3 × BCMA × DU, CD3 × DU × CD38 and the combination of CD3 × BCMA × DU and CD3 × DU × CD38. Graphs show four-parameter logistic curve fitting with symbols representing mean ± s.d. ( n = 6 independent T cell donors from three independent experiments). h – j , EC 50 values for cytotoxicity on KMS-12-BM ( h ), NCI-H929 ( i ) and MOLP-8 ( j ). log 10 (EC 50 ) ( n = 6 independent T cell donors from three independent experiments; EC 50 values for CD3 × DU × CD38 were not quantifiable except for n = 2 on MOLP-8) were compared using repeated measure ANOVA followed by Tukey’s multiple comparison in h – j . k , Representative confocal image (from three independent experiments) of ISB 2001 (white) and the synapse between a T cell (green) and a KMS-12-BM cell (blue), acquired with a Zeiss LSM 800 inverted confocal microscope, magnification ×40. l , Quantification of T cell and KMS-12-BM tumor cell interaction over time using Incucyte live imaging for ISB 2001, CD3 × BCMA × DU and CD3 × DU × CD38. Quantification of T cell and tumor interaction using Incucyte. Graph shows mean of n = 6 (ISB 2001) or 5 (CD3 × BCMA × DU and CD3 × DU × CD38) technical replicates from two independent experiments. Statistical differences from post-hoc comparison are shown in the graphs as exact P value when statistically significant ( P < 0.05). NQ, not quantifiable. WT, wild-type.

Article Snippet: For binding to CD3εδ, BCMA and CD38, biotinylated human CD3ε&CD3δ protein (Creative Biomart, CD3E & CD3D-377H), biotinylated human CD38 protein (Acrobiosystems, CD8-H82E7) or biotinylated human BCMA protein (Acrobiosystems, BCA-H82E4) were immobilized on a Biotin Capture (CAP) sensor chip (Cytiva) and increasing concentrations of ISB 2001 were flushed onto the immobilized ligand.

Techniques: Purification, Activation Assay, Comparison, Microscopy, Imaging

Journal: Nature Cancer

Article Title: ISB 2001 trispecific T cell engager shows strong tumor cytotoxicity and overcomes immune escape mechanisms of multiple myeloma cells

doi: 10.1038/s43018-024-00821-1

Figure Lengend Snippet: (a-c) Quantification of cytokines and cytolytic factors (granzyme B, IFN-γ, IL-2, IL-6, IL-10, perforin and TNF-α) in a high-density PBMC assay comparing the secretion induced by ( a ) CD3 × DU ×CD38 and CD3 × CD38 × DU, ( b ) CD3 × BCMA ×DU and CD3 × DU × BCMA and ( c ) ISB 2001 and CD3 × CD38 × BCMA. Graphs show the maximum secretion in pg/mL. LLOD for each cytokine are the following: granzyme B = 43.4 pg/ml, IFN-γ = 13.7 pg/ml, IL-2 = 39.5 pg/ml, IL-6 = 3.4 pg/ml, IL-10 = 3 pg/ml, perforin = 9.3 pg/ml and TNF-α = 2.9 pg/ml. Each dot corresponds to a different PBMC donor (n = 6 individual PBMC donors from 3 independent experiment). Samples were compared for each cytokines using multiple paired T-Test (two-sided, using false discovery with the two-stage step-up method) and statistical differences are shown in the graphs when statistically significant (P < 0.05). LLOD=Lower limit of detection ( d-e ) BCMA and CD38 human antibody binding capacity (BioCytex) on three MM cell lines (KMS-12-BM, NCI-H929 and MOLP-8) and on MM tumor cells from five patients. Each dot represents one patient sample, bars the mean and error bars the SD. The staining on the cell lines were done in parallel to each patient sample. Dotted line corresponds to the mean of all patients tested ( f ) Representative histograms (from 5 repeats) of sABC staining for BCMA (top, orange) and CD38 (bottom, blue) on KMS-12-BM, NCI-H929 and MOLP-8 MM cell lines compared to the isotype antibody (gray). Numbers show the mean of the experiments for specific antigen binding capacity with mouse anti-BCMA and anti-CD38 antibodies followed by anti-mouse FITC-labeled secondary antibody (QIFIKIT). See Supplementary Figure for gating strategy.

Article Snippet: For binding to CD3εδ, BCMA and CD38, biotinylated human CD3ε&CD3δ protein (Creative Biomart, CD3E & CD3D-377H), biotinylated human CD38 protein (Acrobiosystems, CD8-H82E7) or biotinylated human BCMA protein (Acrobiosystems, BCA-H82E4) were immobilized on a Biotin Capture (CAP) sensor chip (Cytiva) and increasing concentrations of ISB 2001 were flushed onto the immobilized ligand.

Techniques: PBMC Assay, Binding Assay, Staining, Labeling

Journal: Nature Cancer

Article Title: ISB 2001 trispecific T cell engager shows strong tumor cytotoxicity and overcomes immune escape mechanisms of multiple myeloma cells

doi: 10.1038/s43018-024-00821-1

Figure Lengend Snippet: ( a-c ) Binding to MM cell lines of ISB 2001 (pink), CD3 × BCMA ×DU (orange) and CD3 ×DUxCD38 (blue). Graphs show 4-parameters logistic curve fitting using variable slope with symbols representing the mean ± SD of 3 experiments (top) and maximum binding and K D for the three molecules are shown (bottom), each dot represents one independent experiment. Log 10 (K D ) and Max binding of 3 experiments were compared using repeated measure ANOVA model and Tukey HSD comparison. Statistical differences are shown on the graph when statistically significant (P < 0.05) ( d ) Curves showing the binding of ISB 2001 to NCI-H929 cell line expressing CD38 and BCMA (WT, full line), not expressing CD38 (CD38 KO, dashed line) or not expressing BCMA (BCMA KO, dotted line). Graph shows 4-parameters logistic curve fitting using variable slope with symbols representing mean ± SD (n = 3 independent experiments). ( e ) EC 50 values for cytotoxicity of ISB 2001, CD3xDUxCD38 and CD3 × BCMAxDU on MM cell lines expressing different sABC levels of BCMA and CD38. Log 10 (EC 50 ) (n = 6 individual T cell donors from 2 experiments; before exclusion based on acceptance criteria) were compared using repeated measure ANOVA or mixed-effect model (for RPMI-8226 only) followed by Tukey’s multiple comparisons test. (f ) Representative images captured at 20X of Incucyte data (from 2 independent experiments) from one donor after 18 h of incubation with ISB 2001 (left), CD3 × BCMAxDU (middle) and CD3xDUxCD38 (right), at 2000 pM. Green show T cells, red KMS-12-BM and yellow the interaction between effector and target cells. (g-i) Quantification of T cell and KMS-12-BM tumor cell interaction over time using Incucyte live imaging for ISB 2001, CD3 × BCMAxDU and CD3xDUxCD38 at (g) 200 pM, (h) 20 pM and (i) 2 pM. Graphs show mean (n = 6 technical replicates performed on 2 independent experiments using 2 different T cell donors).

Article Snippet: For binding to CD3εδ, BCMA and CD38, biotinylated human CD3ε&CD3δ protein (Creative Biomart, CD3E & CD3D-377H), biotinylated human CD38 protein (Acrobiosystems, CD8-H82E7) or biotinylated human BCMA protein (Acrobiosystems, BCA-H82E4) were immobilized on a Biotin Capture (CAP) sensor chip (Cytiva) and increasing concentrations of ISB 2001 were flushed onto the immobilized ligand.

Techniques: Binding Assay, Comparison, Expressing, Incubation, Imaging

Journal: Nature Cancer

Article Title: ISB 2001 trispecific T cell engager shows strong tumor cytotoxicity and overcomes immune escape mechanisms of multiple myeloma cells

doi: 10.1038/s43018-024-00821-1

Figure Lengend Snippet: a – d , Cytotoxicity of the KMS-12-BM at different concentrations of ISB 2001, teclistamab, alnuctamab and EM801 ( a ) and EC 50 values for cytotoxicity of KMS-12-BM ( b ), NCI-H929 ( c ) and MOLP-8 cell lines ( d ). Graph in a shows four-parameter logistic curve fitting with symbols representing mean ± s.d. of six independent donors. log 10 (EC 50 ) ( n = 6 independent T cell donors from three independent experiments) were compared using repeated measure ANOVA model and Dunnett’s comparison in b – d . e , f , CD8 + T cell activation ( e ) and proliferation ( f ) after treatment in an RDL assay against KMS-12-BM. log 10 (EC 50 ) ( n = 6 independent T cell donors from two independent experiments) were compared using repeated measure ANOVA model and Dunnett’s comparison. g , h , ISB 2001, teclistamab, alnuctamab and EM801 killing curves ( g ) and EC 50 values for cytotoxicity ( h ) of NCI-H929 WT, CD38 −/− and BCMA −/− cell lines. log 10 (EC 50 ) ( n = 4 independent T cell donors (before acceptance criteria exclusion) from two independent experiments) were compared using repeated measure ANOVA model and Dunnett’s comparison to ISB 2001. Graphs in g show four-parameter logistic curve fitting with symbols representing mean ± s.d. ( n = 4 independent T cell donors). i , EC 50 of cytotoxicity, IL-6, IFNγ, TNF and IL-10 release in an RDL assay against KMS-12-BM. Boxplots show 25th to 75th percentile and whiskers minimum and maximum values of n = 6 donors from two independent experiments. j , k , Cytotoxicity in presence (+) or absence (−) of soluble factors after treatment with ISB 2001, teclistamab, alnuctamab and EM801 ( j ) or ISB 2001 and CD3 × BCMA × BCMA molecule ( k ) in an RDL assay. log 10 (EC 50 ) ( n = 6 independent PBMC donors from two experiments) were compared using two-way ANOVA and Tukey’s multiple comparisons in k (only differences between ISB 2001 and TCEs with soluble factors are shown). RDL assays were performed at a 5:1 effector to target ratio with purified T cells or PBMCs with six donors for 48 or 72 h. Statistical differences from post-hoc comparison are shown in the graphs as exact P value when statistically significant ( P < 0.05). NQ, not quantifiable.

Article Snippet: For binding to CD3εδ, BCMA and CD38, biotinylated human CD3ε&CD3δ protein (Creative Biomart, CD3E & CD3D-377H), biotinylated human CD38 protein (Acrobiosystems, CD8-H82E7) or biotinylated human BCMA protein (Acrobiosystems, BCA-H82E4) were immobilized on a Biotin Capture (CAP) sensor chip (Cytiva) and increasing concentrations of ISB 2001 were flushed onto the immobilized ligand.

Techniques: Comparison, Activation Assay, Purification

Journal: Nature Cancer

Article Title: ISB 2001 trispecific T cell engager shows strong tumor cytotoxicity and overcomes immune escape mechanisms of multiple myeloma cells

doi: 10.1038/s43018-024-00821-1

Figure Lengend Snippet: a , b , Representative curves of cytotoxicity of KMS-12-BM cell line at different concentrations of ISB 2001 and teclistamab by T cells isolated from HD- or PS-PBMCs ( n = 5 and n = 2, respectively) or BMA ( n = 5 and n = 2 donors, respectively) ( a ) and cytotoxicity at 10 pM ( b ). Percentage of cytotoxicity of n = 3 (PS-BMMC donors) or n = 5 (HD- and PS-PBMC and HD-BMMC donors, before acceptance criteria application) from n = 5 independent experiments were compared using REML followed by Šidák’s multiple comparison for each population of T cells. c , Experimental setup schema to assess ISB 2001 and teclistamab cytotoxic capacity of CD138 + tumor cells in BMA and T cell activation. d , Representative dot plots of CD138 + cell killing (top) and CD69 + of CD8 + T cells (bottom). e , f , Cytotoxicity of CD138 + tumor cells ( e ) or T cell activation (CD69 + ) ( f ) on samples from patients with MM treated with ISB 2001 or teclistamab at 0.01 ( n = 10 PS for cytotoxicity and n = 9 PS for T cell activation), 0.1 ( n = 6 PS for cytotoxicity and T cell activation) and 1 nM ( n = 10 PS for cytotoxicity and n = 9 PS for T cell activation). CD138 + cell killing and T cell activation were compared using REML followed by Šidák’s multiple comparison for each concentration. g , Cytotoxicity of CD138 + tumor cells on samples from newly diagnosed (left, n = 4 PS) or patients with r/r MM (right, n = 6 PS), treated with ISB 2001 or teclistamab at 0.1 nM. Graph shows dots for individual samples. Previous treatments are stated in the graph as § for anti-CD38-treated or # for anti-BCMA-treated PS. Percentage of CD138 + cell killing was compared using a Holm–Šidák’s multiple two-sided paired t -test. h , Cytotoxicity curve of CD138 + MM cells by ISB 2001, teclistamab and isotype controls at 20 h in a sample from PCL ( n = 1 PS). Graph shows four-parameter logistic curve fitting and symbols represent the mean of replicates ( n = 2 replicates for ISB 2001 and teclistamab and n = 4 replicates for isotype controls). PCL, plasma cell leukemia. Statistical differences are shown in graphs as exact P value when statistically significant ( P < 0.05).

Article Snippet: For binding to CD3εδ, BCMA and CD38, biotinylated human CD3ε&CD3δ protein (Creative Biomart, CD3E & CD3D-377H), biotinylated human CD38 protein (Acrobiosystems, CD8-H82E7) or biotinylated human BCMA protein (Acrobiosystems, BCA-H82E4) were immobilized on a Biotin Capture (CAP) sensor chip (Cytiva) and increasing concentrations of ISB 2001 were flushed onto the immobilized ligand.

Techniques: Isolation, Comparison, Activation Assay, Concentration Assay, Clinical Proteomics

Journal: Nature Cancer

Article Title: ISB 2001 trispecific T cell engager shows strong tumor cytotoxicity and overcomes immune escape mechanisms of multiple myeloma cells

doi: 10.1038/s43018-024-00821-1

Figure Lengend Snippet: Tumor cell killing of bone marrow aspirates (BMA) of ISB 2001 (pink), CD3 × BCMA ×DU (orange) and CD3 ×DU ×CD38 (blue). Graphs show three Multiple Myeloma patients performed in 3 independent experiments. Each dot represents one dose for each molecule.

Article Snippet: For binding to CD3εδ, BCMA and CD38, biotinylated human CD3ε&CD3δ protein (Creative Biomart, CD3E & CD3D-377H), biotinylated human CD38 protein (Acrobiosystems, CD8-H82E7) or biotinylated human BCMA protein (Acrobiosystems, BCA-H82E4) were immobilized on a Biotin Capture (CAP) sensor chip (Cytiva) and increasing concentrations of ISB 2001 were flushed onto the immobilized ligand.

Techniques:

Journal: Nature Cancer

Article Title: ISB 2001 trispecific T cell engager shows strong tumor cytotoxicity and overcomes immune escape mechanisms of multiple myeloma cells

doi: 10.1038/s43018-024-00821-1

Figure Lengend Snippet: a , Workflow for developing QSP model. b , Simplified binding schematic with accompanying equations below, where [ D ] is ISB 2001 concentration; [ T 1], [ T 2] and [ T 3] are the concentrations of free CD3, BCMA and CD38; [ D : T 1] is a concentration of dimer complex of ISB 2001-CD3; [ D : T 1: T 2] is a concentration of trimer complex of ISB 2001–CD3–BCMA; [ D : T 1: T 2: T 3] is a concentration of tetramer complex of ISB 2001–CD3–BCMA–CD38; k on T 1, k on T 2 and k on T 3 are the association rate constants for CD3, BCMA and CD38, respectively; and k off T 1, k off T 2 and k off T 3 are the dissociation rate constants for CD3, BCMA and CD38, respectively. c , d , Goodness-of-fit plots for ISB 2001 and teclistamab dose–response data in vitro and in vivo post-calibration of killing models. RSE for in vitro (CD8, CD4 and tumor) ISB 2001 (30%, 29% and 12%) and teclistamab (20%, 33% and 10%). RSE in vivo ISB 2001 4.7% teclistamab 9.9%. Symbols represent individual experimental data points (tumor, CD4 + T and CD8 + T cell counts for n = 1 representative donor out of 6 ( c ) and tumor volume for n = 8 mice per group ( d )). Lines show model simulations. e , nACT plots showing maximum nACT for a range of doses. f , Repeated dose nACT levels reach the EC 50 and EC 90 thresholds (ISB 2001) and EC 50 threshold for teclistamab. g , MPAD predictions for ISB 2001 and teclistamab. All RSE values are calculated at the EC 50 of the effect modeled . Graphs in e – g show model predictions of nACT levels in simulated patients.

Article Snippet: For binding to CD3εδ, BCMA and CD38, biotinylated human CD3ε&CD3δ protein (Creative Biomart, CD3E & CD3D-377H), biotinylated human CD38 protein (Acrobiosystems, CD8-H82E7) or biotinylated human BCMA protein (Acrobiosystems, BCA-H82E4) were immobilized on a Biotin Capture (CAP) sensor chip (Cytiva) and increasing concentrations of ISB 2001 were flushed onto the immobilized ligand.

Techniques: Binding Assay, Concentration Assay, In Vitro, In Vivo

Journal: Journal of Translational Medicine

Article Title: CD19/CD20 dual-targeted chimeric antigen receptor-engineered natural killer cells exhibit improved cytotoxicity against acute lymphoblastic leukemia

doi: 10.1186/s12967-024-04990-6

Figure Lengend Snippet: Preparation and mechanism of CD19/CD20 dual-targeted CAR-NK cell. ①: Ex-vivo expanded natural killer cells are derived from umbilical cord blood. ②: The mRNA encoding anti-CD19 CARs (FMC63 scFv-CD8α-4-1BB-CD3ζ) and anti-CD20 CARs (LEU16 scFv-CD8α-4-1BB-CD3ζ) were constructed by IVT. ③-④: CD19/CD20 dual-targeted CAR-NK cells were generated by simultaneous electroporation of CAR-mRNA into UCB-NK cells derived from umbilical cord blood, which specifically recognizes CD19 + and/or CD20 + ALL cells. ⑤: Once activated, CAR-NK binds to the target antigen and then lyses tumor cells by releasing perforin and IFN-γ. CAR chimeric antigen receptor, IVT in vitro transcription, UCB umbilical cord blood, NK natural killer cells

Article Snippet: This was followed by two washes in FACS buffer (PBA solution with 2% bovine serum albumin) and subsequent staining with allophycocyanin (APC)-conjugated Streptavidin Protein (stock: 200 μg/mL, 1:200 dilution, ACRO Biosystems) and FITC-Labeled Monoclonal Anti-FMC63 Antibody (stock: 50 μL, 1:50 dilution, ACRO Biosystems, clone: Y45) (or PE-Labeled Monoclonal Anti-FMC63 Antibody (stock: 50 μL, 1:50 dilution, ACRO Biosystems, clone: Y45)) for 60 min at 4 °C.

Techniques: Ex Vivo, Derivative Assay, Construct, Generated, Electroporation, In Vitro

Journal: Journal of Translational Medicine

Article Title: CD19/CD20 dual-targeted chimeric antigen receptor-engineered natural killer cells exhibit improved cytotoxicity against acute lymphoblastic leukemia

doi: 10.1186/s12967-024-04990-6

Figure Lengend Snippet: Construction and identification of the second-generation CARs targeting CD19 and CD20 antigen. A Schematic illustration of the construction of mRNA encoding CD19 CARs and CD20 CARs utilized IVT. B Schematic diagrams of the CD19 CAR-mRNA and the CD20CAR-mRNA. FMC63(anti-CD19) scFv and LEU16 (anti-CD20) scFv were linked to the CD8α hinge and transmembrane domain, the 4-1BB (CD137) signaling domain, and the CD3 zeta signaling domain, respectively. C Denatured agarose gel electrophoresis of the CAR-mRNA. Lane (1) represents the CD19 CAR-mRNA group, lane (3) represents the CD20 CAR-mRNA group, and lane (2) represents mRNA Marker (6000nt). D Detection of CAR expression on NK cells by flow cytometry. CAR expression was detected by using one or two of four staining reagents: PE-Labeled monoclonal anti-FMC63 Antibody (column 1), Biotinylated Human CD20/MS4A1 full length protein followed by Streptavidin Protein-Alexa Fluor 647 (column 2), or FITC-Labeled Monoclonal Anti-FMC63 Antibody (column 3). The expression percentage of CAR on NK cells is noted on the right of each histogram. EGFP-transduced cells served as an additional negative control. E CD19 CARs and CD20 CARs expression kinetics on dual-target CAR-NK over 4 days. IVT in vitro transcription, scFv single-chain fragment variable regions, Cap1 Cap1 structure, UTR untranslated region

Article Snippet: This was followed by two washes in FACS buffer (PBA solution with 2% bovine serum albumin) and subsequent staining with allophycocyanin (APC)-conjugated Streptavidin Protein (stock: 200 μg/mL, 1:200 dilution, ACRO Biosystems) and FITC-Labeled Monoclonal Anti-FMC63 Antibody (stock: 50 μL, 1:50 dilution, ACRO Biosystems, clone: Y45) (or PE-Labeled Monoclonal Anti-FMC63 Antibody (stock: 50 μL, 1:50 dilution, ACRO Biosystems, clone: Y45)) for 60 min at 4 °C.

Techniques: Agarose Gel Electrophoresis, Marker, Expressing, Flow Cytometry, Staining, Labeling, Negative Control, In Vitro

Journal: Nature Cancer

Article Title: ISB 2001 trispecific T cell engager shows strong tumor cytotoxicity and overcomes immune escape mechanisms of multiple myeloma cells

doi: 10.1038/s43018-024-00821-1

Figure Lengend Snippet: a , Cartoon and structural model of ISB 2001 BEAT. On the cartoon, immunoglobulin domains are shown as rectangles. VH domains of the anti-CD38, anti-BCMA and anti-CD3ε binders are depicted in blue, orange and magenta, respectively. All binders make use of a cLC depicted in gray. Fc-silencing mutations are depicted by the orange dots. The BEAT interface shown in the CH3 domains is depicted by the green and black dots. Chain A encompasses an engineered human IgG1 CH2 domain with an engineered human IgG3 CH3 domain. Chain B has engineered human IgG1 CH2 and CH3 domains. CHx, constant domain x; TCR Cα or TCR Cβ, BEAT interface proprietary mutations based on the T cell receptor constant domain α or β, respectively. ISB 2001 BEAT was generated by homology modeling. b , Human T cell activation of anti-CD3ε produced as human IgG1 LALA and control isotype by incubating with a dose–response of the cLC Fab bound to the plate. Graph shows mean ± s.d. ( n = 6 independent T cell donors from two independent experiments). c , SPR sensorgrams from a single replicate show blockade of BCMA/APRIL interaction (top sensorgram) and blockade of BCMA/BAFF interaction (bottom sensorgram) upon binding of anti-BCMA-E6 Fab to recombinant human BCMA protein. Curves are colored by anti-BCMA-E6 Fab concentration in BCMA/anti-BCMA premix solution. Data provided are from a single experiment (no repeats). d , Competition binding assay by Octet BLI. Curves represent injection of anti-CD38-B3 Fab/daratumumab Fab premix (dashed line) or daratumumab at twofold concentration of saturating solution (solid line) over CD38 immobilized surface saturated with daratumumab Fab from a single replicate in one experiment (no repeats). e , Binding sensorgrams of respective representative measurements from three independent replicates show the binding of ISB 2001 to human CD3εδ, human BCMA and human CD38 by SPR. Colored curves represent single concentration injections with serial dilutions of 1:3. Black curves represent 1:1 kinetic fits (BCMA and CD38). For binding to CD3εδ, the K d was inferred from a steady-state affinity model.

Article Snippet: Pre-mixed solutions of 50 nM human BCMA FcTag protein (Acrobiosystems, BC7-H5254) and of anti-BCMA Fab at various concentrations (0, 25, 50 and 200 nM), were individually flushed over immobilized APRIL or BAFF.

Techniques: Generated, Activation Assay, Produced, Control, Binding Assay, Recombinant, Concentration Assay, Injection

Journal: Nature Cancer

Article Title: ISB 2001 trispecific T cell engager shows strong tumor cytotoxicity and overcomes immune escape mechanisms of multiple myeloma cells

doi: 10.1038/s43018-024-00821-1

Figure Lengend Snippet: a – c , Cytotoxicity of KMS-12-BM cells at different concentrations of CD3 ×DU × CD38 and CD3 × CD38 × DU ( a ), CD3 × BCMA × DU and CD3 × DU × BCMA ( b ) and ISB 2001 and CD3 × CD38 × BCMA ( c ). RDL assays were performed at a 5:1 effector to target ratio for 48 h with purified T cells. Graphs show four-parameter logistic curve fitting with symbols representing mean ± s.d. ( n = 6 independent T cell donors from two independent experiments). d – f , T cell activation in a HD-PBMC at different concentrations of CD3 × DU × CD38 and CD3 × CD38 × DU ( d ), CD3 × BCMA × DU and CD3 × DU × BCMA ( e ) and ISB 2001 and CD3 × CD38 × BCMA ( f ). Graphs show four-parameter logistic curve fitting with symbols representing mean ± s.d. ( n = 6 independent T cell donors from two independent experiments). g , Cytotoxicity of the KMS-12-BM cell line at different concentrations of ISB 2001, CD3 × BCMA × DU, CD3 × DU × CD38 and the combination of CD3 × BCMA × DU and CD3 × DU × CD38. Graphs show four-parameter logistic curve fitting with symbols representing mean ± s.d. ( n = 6 independent T cell donors from three independent experiments). h – j , EC 50 values for cytotoxicity on KMS-12-BM ( h ), NCI-H929 ( i ) and MOLP-8 ( j ). log 10 (EC 50 ) ( n = 6 independent T cell donors from three independent experiments; EC 50 values for CD3 × DU × CD38 were not quantifiable except for n = 2 on MOLP-8) were compared using repeated measure ANOVA followed by Tukey’s multiple comparison in h – j . k , Representative confocal image (from three independent experiments) of ISB 2001 (white) and the synapse between a T cell (green) and a KMS-12-BM cell (blue), acquired with a Zeiss LSM 800 inverted confocal microscope, magnification ×40. l , Quantification of T cell and KMS-12-BM tumor cell interaction over time using Incucyte live imaging for ISB 2001, CD3 × BCMA × DU and CD3 × DU × CD38. Quantification of T cell and tumor interaction using Incucyte. Graph shows mean of n = 6 (ISB 2001) or 5 (CD3 × BCMA × DU and CD3 × DU × CD38) technical replicates from two independent experiments. Statistical differences from post-hoc comparison are shown in the graphs as exact P value when statistically significant ( P < 0.05). NQ, not quantifiable. WT, wild-type.

Article Snippet: Pre-mixed solutions of 50 nM human BCMA FcTag protein (Acrobiosystems, BC7-H5254) and of anti-BCMA Fab at various concentrations (0, 25, 50 and 200 nM), were individually flushed over immobilized APRIL or BAFF.

Techniques: Purification, Activation Assay, Comparison, Microscopy, Imaging

Journal: Nature Cancer

Article Title: ISB 2001 trispecific T cell engager shows strong tumor cytotoxicity and overcomes immune escape mechanisms of multiple myeloma cells

doi: 10.1038/s43018-024-00821-1

Figure Lengend Snippet: (a-c) Quantification of cytokines and cytolytic factors (granzyme B, IFN-γ, IL-2, IL-6, IL-10, perforin and TNF-α) in a high-density PBMC assay comparing the secretion induced by ( a ) CD3 × DU ×CD38 and CD3 × CD38 × DU, ( b ) CD3 × BCMA ×DU and CD3 × DU × BCMA and ( c ) ISB 2001 and CD3 × CD38 × BCMA. Graphs show the maximum secretion in pg/mL. LLOD for each cytokine are the following: granzyme B = 43.4 pg/ml, IFN-γ = 13.7 pg/ml, IL-2 = 39.5 pg/ml, IL-6 = 3.4 pg/ml, IL-10 = 3 pg/ml, perforin = 9.3 pg/ml and TNF-α = 2.9 pg/ml. Each dot corresponds to a different PBMC donor (n = 6 individual PBMC donors from 3 independent experiment). Samples were compared for each cytokines using multiple paired T-Test (two-sided, using false discovery with the two-stage step-up method) and statistical differences are shown in the graphs when statistically significant (P < 0.05). LLOD=Lower limit of detection ( d-e ) BCMA and CD38 human antibody binding capacity (BioCytex) on three MM cell lines (KMS-12-BM, NCI-H929 and MOLP-8) and on MM tumor cells from five patients. Each dot represents one patient sample, bars the mean and error bars the SD. The staining on the cell lines were done in parallel to each patient sample. Dotted line corresponds to the mean of all patients tested ( f ) Representative histograms (from 5 repeats) of sABC staining for BCMA (top, orange) and CD38 (bottom, blue) on KMS-12-BM, NCI-H929 and MOLP-8 MM cell lines compared to the isotype antibody (gray). Numbers show the mean of the experiments for specific antigen binding capacity with mouse anti-BCMA and anti-CD38 antibodies followed by anti-mouse FITC-labeled secondary antibody (QIFIKIT). See Supplementary Figure for gating strategy.

Article Snippet: Pre-mixed solutions of 50 nM human BCMA FcTag protein (Acrobiosystems, BC7-H5254) and of anti-BCMA Fab at various concentrations (0, 25, 50 and 200 nM), were individually flushed over immobilized APRIL or BAFF.

Techniques: PBMC Assay, Binding Assay, Staining, Labeling

Journal: Nature Cancer

Article Title: ISB 2001 trispecific T cell engager shows strong tumor cytotoxicity and overcomes immune escape mechanisms of multiple myeloma cells

doi: 10.1038/s43018-024-00821-1

Figure Lengend Snippet: ( a-c ) Binding to MM cell lines of ISB 2001 (pink), CD3 × BCMA ×DU (orange) and CD3 ×DUxCD38 (blue). Graphs show 4-parameters logistic curve fitting using variable slope with symbols representing the mean ± SD of 3 experiments (top) and maximum binding and K D for the three molecules are shown (bottom), each dot represents one independent experiment. Log 10 (K D ) and Max binding of 3 experiments were compared using repeated measure ANOVA model and Tukey HSD comparison. Statistical differences are shown on the graph when statistically significant (P < 0.05) ( d ) Curves showing the binding of ISB 2001 to NCI-H929 cell line expressing CD38 and BCMA (WT, full line), not expressing CD38 (CD38 KO, dashed line) or not expressing BCMA (BCMA KO, dotted line). Graph shows 4-parameters logistic curve fitting using variable slope with symbols representing mean ± SD (n = 3 independent experiments). ( e ) EC 50 values for cytotoxicity of ISB 2001, CD3xDUxCD38 and CD3 × BCMAxDU on MM cell lines expressing different sABC levels of BCMA and CD38. Log 10 (EC 50 ) (n = 6 individual T cell donors from 2 experiments; before exclusion based on acceptance criteria) were compared using repeated measure ANOVA or mixed-effect model (for RPMI-8226 only) followed by Tukey’s multiple comparisons test. (f ) Representative images captured at 20X of Incucyte data (from 2 independent experiments) from one donor after 18 h of incubation with ISB 2001 (left), CD3 × BCMAxDU (middle) and CD3xDUxCD38 (right), at 2000 pM. Green show T cells, red KMS-12-BM and yellow the interaction between effector and target cells. (g-i) Quantification of T cell and KMS-12-BM tumor cell interaction over time using Incucyte live imaging for ISB 2001, CD3 × BCMAxDU and CD3xDUxCD38 at (g) 200 pM, (h) 20 pM and (i) 2 pM. Graphs show mean (n = 6 technical replicates performed on 2 independent experiments using 2 different T cell donors).

Article Snippet: Pre-mixed solutions of 50 nM human BCMA FcTag protein (Acrobiosystems, BC7-H5254) and of anti-BCMA Fab at various concentrations (0, 25, 50 and 200 nM), were individually flushed over immobilized APRIL or BAFF.

Techniques: Binding Assay, Comparison, Expressing, Incubation, Imaging

Journal: Nature Cancer

Article Title: ISB 2001 trispecific T cell engager shows strong tumor cytotoxicity and overcomes immune escape mechanisms of multiple myeloma cells

doi: 10.1038/s43018-024-00821-1

Figure Lengend Snippet: a – d , Cytotoxicity of the KMS-12-BM at different concentrations of ISB 2001, teclistamab, alnuctamab and EM801 ( a ) and EC 50 values for cytotoxicity of KMS-12-BM ( b ), NCI-H929 ( c ) and MOLP-8 cell lines ( d ). Graph in a shows four-parameter logistic curve fitting with symbols representing mean ± s.d. of six independent donors. log 10 (EC 50 ) ( n = 6 independent T cell donors from three independent experiments) were compared using repeated measure ANOVA model and Dunnett’s comparison in b – d . e , f , CD8 + T cell activation ( e ) and proliferation ( f ) after treatment in an RDL assay against KMS-12-BM. log 10 (EC 50 ) ( n = 6 independent T cell donors from two independent experiments) were compared using repeated measure ANOVA model and Dunnett’s comparison. g , h , ISB 2001, teclistamab, alnuctamab and EM801 killing curves ( g ) and EC 50 values for cytotoxicity ( h ) of NCI-H929 WT, CD38 −/− and BCMA −/− cell lines. log 10 (EC 50 ) ( n = 4 independent T cell donors (before acceptance criteria exclusion) from two independent experiments) were compared using repeated measure ANOVA model and Dunnett’s comparison to ISB 2001. Graphs in g show four-parameter logistic curve fitting with symbols representing mean ± s.d. ( n = 4 independent T cell donors). i , EC 50 of cytotoxicity, IL-6, IFNγ, TNF and IL-10 release in an RDL assay against KMS-12-BM. Boxplots show 25th to 75th percentile and whiskers minimum and maximum values of n = 6 donors from two independent experiments. j , k , Cytotoxicity in presence (+) or absence (−) of soluble factors after treatment with ISB 2001, teclistamab, alnuctamab and EM801 ( j ) or ISB 2001 and CD3 × BCMA × BCMA molecule ( k ) in an RDL assay. log 10 (EC 50 ) ( n = 6 independent PBMC donors from two experiments) were compared using two-way ANOVA and Tukey’s multiple comparisons in k (only differences between ISB 2001 and TCEs with soluble factors are shown). RDL assays were performed at a 5:1 effector to target ratio with purified T cells or PBMCs with six donors for 48 or 72 h. Statistical differences from post-hoc comparison are shown in the graphs as exact P value when statistically significant ( P < 0.05). NQ, not quantifiable.

Article Snippet: Pre-mixed solutions of 50 nM human BCMA FcTag protein (Acrobiosystems, BC7-H5254) and of anti-BCMA Fab at various concentrations (0, 25, 50 and 200 nM), were individually flushed over immobilized APRIL or BAFF.

Techniques: Comparison, Activation Assay, Purification

Journal: Nature Cancer

Article Title: ISB 2001 trispecific T cell engager shows strong tumor cytotoxicity and overcomes immune escape mechanisms of multiple myeloma cells

doi: 10.1038/s43018-024-00821-1

Figure Lengend Snippet: a , b , Representative curves of cytotoxicity of KMS-12-BM cell line at different concentrations of ISB 2001 and teclistamab by T cells isolated from HD- or PS-PBMCs ( n = 5 and n = 2, respectively) or BMA ( n = 5 and n = 2 donors, respectively) ( a ) and cytotoxicity at 10 pM ( b ). Percentage of cytotoxicity of n = 3 (PS-BMMC donors) or n = 5 (HD- and PS-PBMC and HD-BMMC donors, before acceptance criteria application) from n = 5 independent experiments were compared using REML followed by Šidák’s multiple comparison for each population of T cells. c , Experimental setup schema to assess ISB 2001 and teclistamab cytotoxic capacity of CD138 + tumor cells in BMA and T cell activation. d , Representative dot plots of CD138 + cell killing (top) and CD69 + of CD8 + T cells (bottom). e , f , Cytotoxicity of CD138 + tumor cells ( e ) or T cell activation (CD69 + ) ( f ) on samples from patients with MM treated with ISB 2001 or teclistamab at 0.01 ( n = 10 PS for cytotoxicity and n = 9 PS for T cell activation), 0.1 ( n = 6 PS for cytotoxicity and T cell activation) and 1 nM ( n = 10 PS for cytotoxicity and n = 9 PS for T cell activation). CD138 + cell killing and T cell activation were compared using REML followed by Šidák’s multiple comparison for each concentration. g , Cytotoxicity of CD138 + tumor cells on samples from newly diagnosed (left, n = 4 PS) or patients with r/r MM (right, n = 6 PS), treated with ISB 2001 or teclistamab at 0.1 nM. Graph shows dots for individual samples. Previous treatments are stated in the graph as § for anti-CD38-treated or # for anti-BCMA-treated PS. Percentage of CD138 + cell killing was compared using a Holm–Šidák’s multiple two-sided paired t -test. h , Cytotoxicity curve of CD138 + MM cells by ISB 2001, teclistamab and isotype controls at 20 h in a sample from PCL ( n = 1 PS). Graph shows four-parameter logistic curve fitting and symbols represent the mean of replicates ( n = 2 replicates for ISB 2001 and teclistamab and n = 4 replicates for isotype controls). PCL, plasma cell leukemia. Statistical differences are shown in graphs as exact P value when statistically significant ( P < 0.05).

Article Snippet: Pre-mixed solutions of 50 nM human BCMA FcTag protein (Acrobiosystems, BC7-H5254) and of anti-BCMA Fab at various concentrations (0, 25, 50 and 200 nM), were individually flushed over immobilized APRIL or BAFF.

Techniques: Isolation, Comparison, Activation Assay, Concentration Assay, Clinical Proteomics

Journal: Nature Cancer

Article Title: ISB 2001 trispecific T cell engager shows strong tumor cytotoxicity and overcomes immune escape mechanisms of multiple myeloma cells

doi: 10.1038/s43018-024-00821-1

Figure Lengend Snippet: Tumor cell killing of bone marrow aspirates (BMA) of ISB 2001 (pink), CD3 × BCMA ×DU (orange) and CD3 ×DU ×CD38 (blue). Graphs show three Multiple Myeloma patients performed in 3 independent experiments. Each dot represents one dose for each molecule.

Article Snippet: Pre-mixed solutions of 50 nM human BCMA FcTag protein (Acrobiosystems, BC7-H5254) and of anti-BCMA Fab at various concentrations (0, 25, 50 and 200 nM), were individually flushed over immobilized APRIL or BAFF.

Techniques:

Journal: Nature Cancer

Article Title: ISB 2001 trispecific T cell engager shows strong tumor cytotoxicity and overcomes immune escape mechanisms of multiple myeloma cells

doi: 10.1038/s43018-024-00821-1

Figure Lengend Snippet: a , Workflow for developing QSP model. b , Simplified binding schematic with accompanying equations below, where [ D ] is ISB 2001 concentration; [ T 1], [ T 2] and [ T 3] are the concentrations of free CD3, BCMA and CD38; [ D : T 1] is a concentration of dimer complex of ISB 2001-CD3; [ D : T 1: T 2] is a concentration of trimer complex of ISB 2001–CD3–BCMA; [ D : T 1: T 2: T 3] is a concentration of tetramer complex of ISB 2001–CD3–BCMA–CD38; k on T 1, k on T 2 and k on T 3 are the association rate constants for CD3, BCMA and CD38, respectively; and k off T 1, k off T 2 and k off T 3 are the dissociation rate constants for CD3, BCMA and CD38, respectively. c , d , Goodness-of-fit plots for ISB 2001 and teclistamab dose–response data in vitro and in vivo post-calibration of killing models. RSE for in vitro (CD8, CD4 and tumor) ISB 2001 (30%, 29% and 12%) and teclistamab (20%, 33% and 10%). RSE in vivo ISB 2001 4.7% teclistamab 9.9%. Symbols represent individual experimental data points (tumor, CD4 + T and CD8 + T cell counts for n = 1 representative donor out of 6 ( c ) and tumor volume for n = 8 mice per group ( d )). Lines show model simulations. e , nACT plots showing maximum nACT for a range of doses. f , Repeated dose nACT levels reach the EC 50 and EC 90 thresholds (ISB 2001) and EC 50 threshold for teclistamab. g , MPAD predictions for ISB 2001 and teclistamab. All RSE values are calculated at the EC 50 of the effect modeled . Graphs in e – g show model predictions of nACT levels in simulated patients.

Article Snippet: Pre-mixed solutions of 50 nM human BCMA FcTag protein (Acrobiosystems, BC7-H5254) and of anti-BCMA Fab at various concentrations (0, 25, 50 and 200 nM), were individually flushed over immobilized APRIL or BAFF.

Techniques: Binding Assay, Concentration Assay, In Vitro, In Vivo