Journal: STAR Protocols

Article Title: Optimized protocol for naive human pluripotent stem cell-derived trophoblast induction

doi: 10.1016/j.xpro.2021.100921

Figure Lengend Snippet:

Article Snippet: Recombinant anti-human CD327 (SIGLEC6), biotin conjugated (clone REA852) (1:100 dilution) , Miltenyi Biotec , Cat#130-112-708; RRID: AB_2725881.

Techniques: Recombinant, Membrane, Knock-Out, Red Blood Cell Lysis, Software, Cell Culture, Inverted Microscopy

Journal: Frontiers in Oncology

Article Title: A Unique Human Immunoglobulin Heavy Chain Variable Domain-Only CD33 CAR for the Treatment of Acute Myeloid Leukemia

doi: 10.3389/fonc.2018.00539

Figure Lengend Snippet: Identification of VH binders targeting human CD33 antigen. (A) Schema illustrating the workflow of VH binders screening using a phage display library. (B) Non-reducing and reducing SDS-PAGE of the selected VH binders. Molecular masses of standards are shown on the left. (C) ELISA binding to recombinant human CD33. (D) FACS binding of the selected binders to CD33-negative CHO and acute lymphoblastic leukemia cell line RS4;11, and CD33-positive acute myeloid leukemia cell line MV4-11.

Article Snippet: Briefly, the phage library was cycled through three rounds of panning against biotinylated recombinant human CD33-Fc fusion protein (R&D Biosystems, Minneapolis, MN) and specific binders were identified from the third round of panning using soluble expression-based monoclonal enzyme-linked immunosorbent assay (semELISA) as described previously ( ).

Techniques: SDS Page, Enzyme-linked Immunosorbent Assay, Binding Assay, Recombinant

Journal: Frontiers in Oncology

Article Title: A Unique Human Immunoglobulin Heavy Chain Variable Domain-Only CD33 CAR for the Treatment of Acute Myeloid Leukemia

doi: 10.3389/fonc.2018.00539

Figure Lengend Snippet: Structure and surface expression of anti-CD33 CAR constructs in primary human T cells. CAR33VH and the positive control My96CAR , are represented (A) Each binder sequence was connected in frame to CD8 linker (H), CD8 transmembrane domain (TM), CD137/4-1BB and CD3 zeta signaling domains. Lentiviral vectors encoding each of the constructs under the control of EF1a promoter were generated and used to transduce primary human T cells at 10%v/v. (B) Flow cytometric analysis of CAR T expression. Transduced live singlet cells were stained with primary CD33-Fc followed by anti Fc–APC F(ab)′2 (light blue). Non-transduced cells (pink) served as negative controls. Results are representative of five transduction experiments from five separate donors. (C) Summary of CAR surface expression as a function of MOI. The expression of CAR33VH or My96CAR for five separate donors transduced at MOI of 20 or 60 was measured by flow cytometry. Average CAR expression ±SEM for each determination is shown.

Article Snippet: Briefly, the phage library was cycled through three rounds of panning against biotinylated recombinant human CD33-Fc fusion protein (R&D Biosystems, Minneapolis, MN) and specific binders were identified from the third round of panning using soluble expression-based monoclonal enzyme-linked immunosorbent assay (semELISA) as described previously ( ).

Techniques: Expressing, Construct, Positive Control, Sequencing, Generated, Transduction, Staining, Flow Cytometry

Journal: Frontiers in Oncology

Article Title: A Unique Human Immunoglobulin Heavy Chain Variable Domain-Only CD33 CAR for the Treatment of Acute Myeloid Leukemia

doi: 10.3389/fonc.2018.00539

Figure Lengend Snippet: Heavy chain only anti-CD33 CAR demonstrates tumor-specific cytokine responses in vitro . Cytokine analysis was performed on supernatants from 5 × 10 4 CAR T cells cultured with CD33 high cell lines MOLM-14, or HL60, or CD33 low cell line KG-1a at E:T ratio of 1:1 in triplicate. CAR alone group was included to control for spontaneous CAR T cytokine release. Levels of cytokines in culture supernatants were measured by ELISA. Mean values + SEM for CAR33VH, My96CAR and untransduced control (UTD) from three independent donors are shown. Groups were compared by Two Way ANOVA followed by Tukey's multiple comparisons test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns-non significant. UTD, untransduced T cells.

Article Snippet: Briefly, the phage library was cycled through three rounds of panning against biotinylated recombinant human CD33-Fc fusion protein (R&D Biosystems, Minneapolis, MN) and specific binders were identified from the third round of panning using soluble expression-based monoclonal enzyme-linked immunosorbent assay (semELISA) as described previously ( ).

Techniques: In Vitro, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Oncology

Article Title: A Unique Human Immunoglobulin Heavy Chain Variable Domain-Only CD33 CAR for the Treatment of Acute Myeloid Leukemia

doi: 10.3389/fonc.2018.00539

Figure Lengend Snippet: Heavy chain only anti-CD33 CAR demonstrates antigen-specific cytotoxicity in vitro . (A–E) CD33-targeting CAR constructs CAR33VH, My96CAR or untransduced T cells (UTD) were incubated with tumor lines (listed above each plot) for 18 h at effector to target ratios of 5, 10, or 20, x-axis, in triplicate. CAR cytotoxic effect was measured by luminometry. Mean values ± SEM from four independent donors are shown. ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Briefly, the phage library was cycled through three rounds of panning against biotinylated recombinant human CD33-Fc fusion protein (R&D Biosystems, Minneapolis, MN) and specific binders were identified from the third round of panning using soluble expression-based monoclonal enzyme-linked immunosorbent assay (semELISA) as described previously ( ).

Techniques: In Vitro, Construct, Incubation

Journal: Frontiers in Oncology

Article Title: A Unique Human Immunoglobulin Heavy Chain Variable Domain-Only CD33 CAR for the Treatment of Acute Myeloid Leukemia

doi: 10.3389/fonc.2018.00539

Figure Lengend Snippet: Heavy chain only anti-CD33 CAR T persist and eliminate tumors in a long-term co-incubation assay. VH33CAR persistence and long-term cytotoxic activity were evaluated by co-incubating CAR cells with CD33 hi HL-60 leukemia cells at low E:T ratios (5:1-0.04:1) for 11 days. My96CAR served as a positive control, and untransduced cells (UTD) as negative controls. On days 5 and 11, co-cultures were stained with anti-CD3, and anti-CD33 antibody, and signal from 7-AAD (–) cells acquired by flow cytometry to determine the percentage of live effector and target cells in each culture. (A) Representative plots from one of three donors at E:T ratio of 5:1 on day 5 is shown. HL-60 leukemia cells are gated in the upper left box of each plot, HL60 targets bound to T cells (HL60+T cells) are gated at the upper right box of each plot, activated T cells are gated at the bottom left box, and resting CD3 + effector T cells are gated in the lower right box of each plot, and percentage of gated cells for each population is noted next to corresponding box. (B) Mean %Live HL-60 +SEM from three independent co-incubation experiments performed with CAR T cells from three separate donors. **** p < 0.0001, ** p < 0.01, NS-non-significant, two way ANOVA followed by Dunnett's multiple comparisons test.

Article Snippet: Briefly, the phage library was cycled through three rounds of panning against biotinylated recombinant human CD33-Fc fusion protein (R&D Biosystems, Minneapolis, MN) and specific binders were identified from the third round of panning using soluble expression-based monoclonal enzyme-linked immunosorbent assay (semELISA) as described previously ( ).

Techniques: Incubation, Activity Assay, Positive Control, Staining, Flow Cytometry

Journal: Frontiers in Oncology

Article Title: A Unique Human Immunoglobulin Heavy Chain Variable Domain-Only CD33 CAR for the Treatment of Acute Myeloid Leukemia

doi: 10.3389/fonc.2018.00539

Figure Lengend Snippet: Heavy chain only CAR CD33VH targets V domain-containing the full length isoform of CD33. The CD33-targeting CAR constructs CAR33VH, My96CAR or negative controls were incubated with tumor lines (A) A431 (CD33-), (B) A431v1 (containing the full length CD33 isoform) or (C) A431v2 (containing the V domain-truncated CD33 isoform) for 18 h at effector to target ratios of 5, 10 or 20 in triplicate. All target lines stably expressed firefly luciferase, and CAR cytotoxic effect was measured by luminometry. Data from one representative experiment out of three experiments utilizing CART cells derived from different donors is shown. (D) Supernatants from co-cultures of CAR cells with tumor lines in (A) were harvested after 18 h co-incubation and analyzed for IFN-gamma by ELISA in triplicate. Mean values +SEM from four independent experiments performed in different donors are shown. UTD-untransduced T cells, GFP-transduced T cells. NS-non-significant.

Article Snippet: Briefly, the phage library was cycled through three rounds of panning against biotinylated recombinant human CD33-Fc fusion protein (R&D Biosystems, Minneapolis, MN) and specific binders were identified from the third round of panning using soluble expression-based monoclonal enzyme-linked immunosorbent assay (semELISA) as described previously ( ).

Techniques: Construct, Incubation, Stable Transfection, Luciferase, Derivative Assay, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Oncology

Article Title: A Unique Human Immunoglobulin Heavy Chain Variable Domain-Only CD33 CAR for the Treatment of Acute Myeloid Leukemia

doi: 10.3389/fonc.2018.00539

Figure Lengend Snippet: Heavy chain only construct VH33CAR activity in vivo . (A) Study design schema: NOD- scid IL2Rg null (NSG) mice were injected with 0.5 × 10 6 luciferase-enabled MOLM-14 tumor cells i.v . on day 0. On day 6, mice were dosed i.v . with 5 × 10 6 CART + cells. Tumor burden was evaluated weekly by bioluminescence, between days 14-35. Blood was collected for analysis on Day 19. (B) Immunofluorescent Imaging of experimental groups was performed on study days 14, 21, 28, 35. TA- tumor alone, UTD-untransduced T cell control, CAR33VH–single chain only anti CD33 CAR, My96CAR–positive control CAR. Images were acquired on IVIS Lumina and analyzed by Living Image software (PerkinElmer). (C) Tumor burden was assessed by bioluminescent imaging on study days 14, 21, 28, 35. N = 6 mice per group, average radiance ± SEM was plotted for each time point. TA- tumor alone, UTD-untransduced T cell control. (D) Survival analysis of treated mice. While all animals survived to day 35 in CAR33VH and My96CAR groups, animals in the control groups survived only up to day 21. Retro-orbital bleeds were obtained from surviving mice on study day 19. (E) MOLM-14 tumor cells (CD45 + /Singlets/Live/GFP + ) and (F) . CAR T + cells (CD45 + /Singlets/Live/CD3 + /CAR + ) in blood samples were analyzed by flow cytometry. Total cell count was determined by volumetric flow cytometry, normalized using CountBright beads added during sample preparation. N = 6 for CAR groups, N = 4 for UTD; mean ± SEM. One way ANOVA with multiple comparisons analysis, **** p < 0.0001. Survival data were compared by log-rank Mantel-Cox test, *** p < 0.001.

Article Snippet: Briefly, the phage library was cycled through three rounds of panning against biotinylated recombinant human CD33-Fc fusion protein (R&D Biosystems, Minneapolis, MN) and specific binders were identified from the third round of panning using soluble expression-based monoclonal enzyme-linked immunosorbent assay (semELISA) as described previously ( ).

Techniques: Construct, Activity Assay, In Vivo, Injection, Luciferase, Imaging, Positive Control, Software, Flow Cytometry, Cell Counting, Sample Prep

Journal: International Journal of Molecular Sciences

Article Title: Identification of Nanobodies against the Acute Myeloid Leukemia Marker CD33

doi: 10.3390/ijms21010310

Figure Lengend Snippet: ( A ) Schematic representation of an Nb in the phagemid vector pMECS. Downstream of the PelB secretion sequence, the Nb-sequence is followed by a triple alanine linker, a hemagglutinin (HA), and hexa-histidine (His) tags. ( B ) Amino acid sequences of the anti-CD33 Nbs (numbering according to IMGT) . The CDR1, CDR2, and CDR3 regions are highlighted in cyan, green, and pink, respectively. The amino acid sequence of the CDR3 region is displayed in alphabetical order. Position 10 in the framework region-1 (FR1-IMGT) and position 73 in the FR3-IMGT are gaps introduced to align to other V-GENE groups or subgroups. For Nb_7, Nb_21, and Nb_22, an amino acid deletion relative to other sequences occurred at position 85 (represented by a dash).

Article Snippet: The biotinylated CD33 recombinant protein (Acro Biosystems, CD3-H82E7, Asp 18–His 259) was coupled to a streptavidin (SA)-coated sensor chip and used to evaluate the binding of a serial dilution series of the anti-CD33 Nbs.

Techniques: Plasmid Preparation, Sequencing

Journal: International Journal of Molecular Sciences

Article Title: Identification of Nanobodies against the Acute Myeloid Leukemia Marker CD33

doi: 10.3390/ijms21010310

Figure Lengend Snippet: Purity of the anti-CD33 Nb preparations. ( A ) SEC profile of the Nb_12, showing a single peak of protein. (All other Nbs gave comparable chromatograms). ( B , C ) SDS-PAGE under reducing conditions, where proteins are revealed after staining with Coomassie blue ( B ) or by western blot, using a mouse anti-HA tag monoclonal antibody and a goat anti-mouse IgG horse radish peroxidase (HRP)-conjugated antibody ( C ). For both staining conditions and for each Nb preparation, only one single band was revealed.

Article Snippet: The biotinylated CD33 recombinant protein (Acro Biosystems, CD3-H82E7, Asp 18–His 259) was coupled to a streptavidin (SA)-coated sensor chip and used to evaluate the binding of a serial dilution series of the anti-CD33 Nbs.

Techniques: SDS Page, Staining, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Identification of Nanobodies against the Acute Myeloid Leukemia Marker CD33

doi: 10.3390/ijms21010310

Figure Lengend Snippet: Six anti-CD33 Nbs bind native CD33 protein expressed on THP-1 cells, without affecting the cells’ in vitro proliferative capacity. ( A ) Individual histogram plots of flow cytometry analysis from the selected Nbs (clear peak) versus a non-targeting Nb (tinted peak). An anti-CD33 monoclonal antibody was used as positive control (clear peak), with an isotype-matched antibody as negative control (tinted peak). ( B ) Graphical representation of the ΔMFI values for the generated Nbs. The ΔMFI is defined as the MFI signal from THP-1 cells treated with Nb and HA-labeled monoclonal antibody subtracted with the MFI signal from cells and labeled monoclonal, but without Nb. An Nb was selected as a binder if its ΔMFI signal was at least three times higher than the one obtained with non-targeting binder (αBabA Nb_19), which defined the threshold (dashed line). ( C ) The impact of ant-CD33 Nbs on the proliferation of THP-1 cells was determined by the alamarBlue assay, in which the proliferative status measured by absorbance is translated into a bar plot. THP-1 cells were incubated for 48 h, with 5 µg of the selected anti-CD33 Nbs (gray bars) or a non-targeting Nb (αBabA Nb_19; light grey bar), or they were left untreated (patterned bar). Medium alone was also included as extra control condition (black bar).

Article Snippet: The biotinylated CD33 recombinant protein (Acro Biosystems, CD3-H82E7, Asp 18–His 259) was coupled to a streptavidin (SA)-coated sensor chip and used to evaluate the binding of a serial dilution series of the anti-CD33 Nbs.

Techniques: In Vitro, Flow Cytometry, Positive Control, Negative Control, Generated, Labeling, Alamar Blue Assay, Incubation, Control

Journal: International Journal of Molecular Sciences

Article Title: Identification of Nanobodies against the Acute Myeloid Leukemia Marker CD33

doi: 10.3390/ijms21010310

Figure Lengend Snippet: Kinetic association (k a ), dissociation (k d ), and equilibrium (K D ) constants of the six anti-CD33 Nbs for CD33 ectodomain measured by SPR. The Tm value representing the thermal stability of the six Nbs was measured with the ThermoFluor ® method.

Article Snippet: The biotinylated CD33 recombinant protein (Acro Biosystems, CD3-H82E7, Asp 18–His 259) was coupled to a streptavidin (SA)-coated sensor chip and used to evaluate the binding of a serial dilution series of the anti-CD33 Nbs.

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Identification of Nanobodies against the Acute Myeloid Leukemia Marker CD33

doi: 10.3390/ijms21010310

Figure Lengend Snippet: SPR sensorgram of Nb_12 on the CD33 ectodomain and epitope binning by SPR: ( A ) sensorgram of different concentrations (as indicated in the graph) of anti-CD33 Nb_12 binding to biotinylated recombinant human CD33 ectodomain. Kinetics were measured with a two-fold dilution series of Nbs (250–1.95 nM). The fitting of the binding curves using the 1:1 binding mathematical model calculated a K D of 3.9 nM. ( B , C ) Examples of a noncompetitive (independent) and a competitive (overlapping) epitope binding of two Nbs for the same antigen, respectively.

Article Snippet: The biotinylated CD33 recombinant protein (Acro Biosystems, CD3-H82E7, Asp 18–His 259) was coupled to a streptavidin (SA)-coated sensor chip and used to evaluate the binding of a serial dilution series of the anti-CD33 Nbs.

Techniques: Binding Assay, Recombinant

Journal: International Journal of Molecular Sciences

Article Title: Identification of Nanobodies against the Acute Myeloid Leukemia Marker CD33

doi: 10.3390/ijms21010310

Figure Lengend Snippet: Overview of the epitope binning experiments. Each pair of anti-CD33 Nbs is classified as noncompetitive if it recognizes a non-overlapping epitope, or as competitive if it binds to an overlapping epitope.

Article Snippet: The biotinylated CD33 recombinant protein (Acro Biosystems, CD3-H82E7, Asp 18–His 259) was coupled to a streptavidin (SA)-coated sensor chip and used to evaluate the binding of a serial dilution series of the anti-CD33 Nbs.

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Identification of Nanobodies against the Acute Myeloid Leukemia Marker CD33

doi: 10.3390/ijms21010310

Figure Lengend Snippet: An example of Tm measurement of anti-CD33 Nb_12, as determined by ThermoFluor ® assay. The samples were heated from 10 to 95 °C, with stepwise increments of 0.5 °C per 30 s. AU stands for arbitrary units. The Tm for this Nb (58.00 ± 0.23 °C) was determined by using Boltzmann’s Equation.

Article Snippet: The biotinylated CD33 recombinant protein (Acro Biosystems, CD3-H82E7, Asp 18–His 259) was coupled to a streptavidin (SA)-coated sensor chip and used to evaluate the binding of a serial dilution series of the anti-CD33 Nbs.

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Identification of Nanobodies against the Acute Myeloid Leukemia Marker CD33

doi: 10.3390/ijms21010310

Figure Lengend Snippet: Biodistribution of 99m Tc-labeled anti-CD33 Nbs, and the non-targeting 99m Tc-ctrl_Nb in mice bearing THP-1 tumors. Micro-SPECT/CT images were obtained 1 h after intravenous injection of 99m Tc-labeled Nbs. Arrows indicate THP-1 tumors. K: kidney; B: bladder; and L: liver.

Article Snippet: The biotinylated CD33 recombinant protein (Acro Biosystems, CD3-H82E7, Asp 18–His 259) was coupled to a streptavidin (SA)-coated sensor chip and used to evaluate the binding of a serial dilution series of the anti-CD33 Nbs.

Techniques: Labeling, Micro-SPECT, Injection

Journal: International Journal of Molecular Sciences

Article Title: Identification of Nanobodies against the Acute Myeloid Leukemia Marker CD33

doi: 10.3390/ijms21010310

Figure Lengend Snippet: Graphical presentation of the biodistribution of the 99m Tc-labeled anti-CD33 Nbs and non-targeting control Nb (ctrl_Nb) in mice bearing subcutaneous THP-1 tumors ( n = 3). ( A ) Ex vivo biodistribution results obtained 1.5 h after injection. Results are presented as mean %IA/g ± standard deviation. ( B ) Mice injected with 99m Tc-labeled Nb_12, Nb_87, Nb_21, and Nb_7 showed significant higher tumor uptake compared to the group of mice injected with non-targeting 99m Tc labeled ctrl_Nb.

Article Snippet: The biotinylated CD33 recombinant protein (Acro Biosystems, CD3-H82E7, Asp 18–His 259) was coupled to a streptavidin (SA)-coated sensor chip and used to evaluate the binding of a serial dilution series of the anti-CD33 Nbs.

Techniques: Labeling, Control, Ex Vivo, Injection, Standard Deviation