cd33 recombinant ectodomain  (Sino Biological)

Journal: Molecular Therapy Oncology

Article Title: Discovery and preclinical development of a SdAb-based CAR-T technology for targeting CD33 in AML

doi: 10.1016/j.omton.2025.200949

Figure Lengend Snippet: Identification of CD33-specific SdAbs (A) CD33 indirect ELISA curves of llama plasma at different stages during immunization (percentage of signal vs. plasma dilution factor [DF]). (B) Output/input (O/I) phage ratio for each panning round. (C) Phage ELISA of the original library and the final output AO3. (D) Preliminary ELISA screening of isolated E. coli clones infected with bacteriophages. As positive control, one positive clone from a previous round output (C+) was offered. (E) Final ELISA screening of isolated E. coli clones expressing the SdAbs from the expression vector pETMod. The positive control (C+) was C4 from the preliminary screening, and the negative control was an irrelevant SdAb-expressing clone culture supernatant from a panning round against another target. (F) Sequence alignment of the five candidate SdAbs selected for further characterization. (G) Phylogenetic tree of the identified SdAb sequences against CD33, clustered into five families based on half distance. (H) Coomassie blue-stained SDS-PAGE and anti-HA Western blot of the IMAC and IEC purified SdAbs. (I) Cross reactivity test (ELISA) of the five candidate SdAbs against antigens from the same llama library.

Article Snippet: A male llama was immunized with the CD33 recombinant ectodomain (SinoBiological #12238-H08H) four times in-between 20 and 30 days at doses of 150 and 200 μg, alongside other antigens (SLAMF7, CD70 and EDA) using Freund’s adjuvants, while monitoring anti-CD33 humoral immune response by indirect enzyme-linked immunosorbent assay (ELISA), using CD33-coated plates, plasma samples, and the Goat anti-Llama IgG (H + L) Secondary Antibody, horseradish peroxidase (HRP) (Thermo Fisher Scientific Cat# A16060, RRID: AB_2534733 ).

Techniques: Indirect ELISA, Enzyme-linked Immunosorbent Assay, Isolation, Clone Assay, Infection, Positive Control, Expressing, Plasmid Preparation, Negative Control, Sequencing, Staining, SDS Page, Western Blot, Purification

Journal: Molecular Therapy Oncology

Article Title: Discovery and preclinical development of a SdAb-based CAR-T technology for targeting CD33 in AML

doi: 10.1016/j.omton.2025.200949

Figure Lengend Snippet: Characterization of SdAbs (A) ELISA sigmoidal curves for each candidate SdAb and the “My96” ScFv as relative A450 vs. log of SdAb concentration (nM). (B) SPR sensorgrams for each SdAb and the reference ScFv from SCK runs, along with their respective fitting curves (χ 2 < 10% Rmax, tc > 100∗Kon, and U < 15) (C) BLI epitope binding curves of the secondary binding of each SdAb, the primary SdAb or ScFv to bind is specified in each graph title. (D) Histograms of anti-HA PE stained MOLM13 CD33+ cells pre-incubated with 1 μg of each SdAb for 1 h, alongside control staining with antiCD33 BV510 (WM53).

Article Snippet: A male llama was immunized with the CD33 recombinant ectodomain (SinoBiological #12238-H08H) four times in-between 20 and 30 days at doses of 150 and 200 μg, alongside other antigens (SLAMF7, CD70 and EDA) using Freund’s adjuvants, while monitoring anti-CD33 humoral immune response by indirect enzyme-linked immunosorbent assay (ELISA), using CD33-coated plates, plasma samples, and the Goat anti-Llama IgG (H + L) Secondary Antibody, horseradish peroxidase (HRP) (Thermo Fisher Scientific Cat# A16060, RRID: AB_2534733 ).

Techniques: Enzyme-linked Immunosorbent Assay, Concentration Assay, Binding Assay, Staining, Incubation, Control

Journal: Molecular Therapy Oncology

Article Title: Discovery and preclinical development of a SdAb-based CAR-T technology for targeting CD33 in AML

doi: 10.1016/j.omton.2025.200949

Figure Lengend Snippet: CD33 binding affinity and kinetic constants for the five SdAb candidates and the reference ScFv

Article Snippet: A male llama was immunized with the CD33 recombinant ectodomain (SinoBiological #12238-H08H) four times in-between 20 and 30 days at doses of 150 and 200 μg, alongside other antigens (SLAMF7, CD70 and EDA) using Freund’s adjuvants, while monitoring anti-CD33 humoral immune response by indirect enzyme-linked immunosorbent assay (ELISA), using CD33-coated plates, plasma samples, and the Goat anti-Llama IgG (H + L) Secondary Antibody, horseradish peroxidase (HRP) (Thermo Fisher Scientific Cat# A16060, RRID: AB_2534733 ).

Techniques: Binding Assay

Journal: Molecular Therapy Oncology

Article Title: Discovery and preclinical development of a SdAb-based CAR-T technology for targeting CD33 in AML

doi: 10.1016/j.omton.2025.200949

Figure Lengend Snippet: Functional characterization of SdAb-CAR-T cells (A) Population doublings during 14 days of CAR-T cell expansion. (B–D) FACS characterization of untransduced T cells (UTD) and CAR-T cells at baseline and after 12–14 days. Repeated measures ANOVA with Tukey’s multiple comparisons (FDR correction) was performed for all compositional analyses. (B) T cell populations (CD8/CD4) and (C) subpopulations (Te: effector, Tem: effector memory, Tcm: central memory, Tscm: stem cell memory, Tn: naive) of CD4 (left) and CD8 (right) T cells. (D) CAR+ % over total T cells. (E) Activation and exhaustion markers of CD4+ CAR-T cells. (F) CAR-T cytotoxicity on three different AML cell lines expressing different levels of CD33 (MFI of stained cells on left panel) evaluated via luciferase activity. (G) Cytokine expression in the supernatant of 1:1 E:T co-cultures: IL2 (left) and IFNγ (right). n = 6 independent healthy donors aged between 18 and 26 years old n = 4 for Nb1 (not included in the first experiments). Statistical analysis was performed using two-way ANOVA for repeated measurements with Tukey's multiple comparisons with FDR correction. Results are shown as mean and error bars represent the standard deviation (SD) derived from biological (A–F) or technical (G) replicates. ∗ p < 0.05,∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: A male llama was immunized with the CD33 recombinant ectodomain (SinoBiological #12238-H08H) four times in-between 20 and 30 days at doses of 150 and 200 μg, alongside other antigens (SLAMF7, CD70 and EDA) using Freund’s adjuvants, while monitoring anti-CD33 humoral immune response by indirect enzyme-linked immunosorbent assay (ELISA), using CD33-coated plates, plasma samples, and the Goat anti-Llama IgG (H + L) Secondary Antibody, horseradish peroxidase (HRP) (Thermo Fisher Scientific Cat# A16060, RRID: AB_2534733 ).

Techniques: Functional Assay, Activation Assay, Expressing, Staining, Luciferase, Activity Assay, Standard Deviation, Derivative Assay

Journal: Molecular Therapy Oncology

Article Title: Discovery and preclinical development of a SdAb-based CAR-T technology for targeting CD33 in AML

doi: 10.1016/j.omton.2025.200949

Figure Lengend Snippet: In vivo evaluation of CD33 targeted SdAb-based CAR-T cells on mouse xenograft AML model (A) Schematic representation of the optimized procedure on NGS mice created with BioRender.com . (B) Kaplan-Meier survival curves of low-dose (0.5 × 10 6 CAR-T cells/mice), intermediate-dose (1.5 × 10 6 CAR-T cells/mice), and high-dose (3 × 10 6 CAR-T cells/mice) treatments of AML (MOLM13) xenografted mice ( n = 8). For statistical analysis, survival curves were compared using the Log rank (Mantel-Cox) test, ∗∗ p < 0.01, ∗∗∗ p < 0.001. (C) In vivo tumoral progression in AML (MOLM13 luciferase+) xenografted mice treated with SdAb/ScFv-based CAR-T cells, and UTD T cells. Top panel: luminescence images of a sample of the treated mice (two males on the left and two females on the right), injected with luciferin at different time points. Bottom panel: progression of MOLM13 cells on each mouse, measured as luciferase activity (Total Flux), and corresponding survival curves, analyzed using the Log rank (Mantel-Cox) test, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: A male llama was immunized with the CD33 recombinant ectodomain (SinoBiological #12238-H08H) four times in-between 20 and 30 days at doses of 150 and 200 μg, alongside other antigens (SLAMF7, CD70 and EDA) using Freund’s adjuvants, while monitoring anti-CD33 humoral immune response by indirect enzyme-linked immunosorbent assay (ELISA), using CD33-coated plates, plasma samples, and the Goat anti-Llama IgG (H + L) Secondary Antibody, horseradish peroxidase (HRP) (Thermo Fisher Scientific Cat# A16060, RRID: AB_2534733 ).

Techniques: In Vivo, Luciferase, Injection, Activity Assay

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: Briefly, a llama ( Lama glama, housed in Lamasté, Leopoldsburg, Belgium) was immunized six times, at weekly intervals, with 150 μg of recombinant human CD33 ectodomain (Acro Biosystems, CD3-H5226, Asp 18–His 259) supplemented with Gerbu LQ 3000 adjuvant (Gerbu Biotechnik GmbH).

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: Briefly, a llama ( Lama glama, housed in Lamasté, Leopoldsburg, Belgium) was immunized six times, at weekly intervals, with 150 μg of recombinant human CD33 ectodomain (Acro Biosystems, CD3-H5226, Asp 18–His 259) supplemented with Gerbu LQ 3000 adjuvant (Gerbu Biotechnik GmbH).

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: Briefly, a llama ( Lama glama, housed in Lamasté, Leopoldsburg, Belgium) was immunized six times, at weekly intervals, with 150 μg of recombinant human CD33 ectodomain (Acro Biosystems, CD3-H5226, Asp 18–His 259) supplemented with Gerbu LQ 3000 adjuvant (Gerbu Biotechnik GmbH).

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: Briefly, a llama ( Lama glama, housed in Lamasté, Leopoldsburg, Belgium) was immunized six times, at weekly intervals, with 150 μg of recombinant human CD33 ectodomain (Acro Biosystems, CD3-H5226, Asp 18–His 259) supplemented with Gerbu LQ 3000 adjuvant (Gerbu Biotechnik GmbH).

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: Briefly, a llama ( Lama glama, housed in Lamasté, Leopoldsburg, Belgium) was immunized six times, at weekly intervals, with 150 μg of recombinant human CD33 ectodomain (Acro Biosystems, CD3-H5226, Asp 18–His 259) supplemented with Gerbu LQ 3000 adjuvant (Gerbu Biotechnik GmbH).

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: Briefly, a llama ( Lama glama, housed in Lamasté, Leopoldsburg, Belgium) was immunized six times, at weekly intervals, with 150 μg of recombinant human CD33 ectodomain (Acro Biosystems, CD3-H5226, Asp 18–His 259) supplemented with Gerbu LQ 3000 adjuvant (Gerbu Biotechnik GmbH).

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: Briefly, a llama ( Lama glama, housed in Lamasté, Leopoldsburg, Belgium) was immunized six times, at weekly intervals, with 150 μg of recombinant human CD33 ectodomain (Acro Biosystems, CD3-H5226, Asp 18–His 259) supplemented with Gerbu LQ 3000 adjuvant (Gerbu Biotechnik GmbH).

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: Briefly, a llama ( Lama glama, housed in Lamasté, Leopoldsburg, Belgium) was immunized six times, at weekly intervals, with 150 μg of recombinant human CD33 ectodomain (Acro Biosystems, CD3-H5226, Asp 18–His 259) supplemented with Gerbu LQ 3000 adjuvant (Gerbu Biotechnik GmbH).

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: Briefly, a llama ( Lama glama, housed in Lamasté, Leopoldsburg, Belgium) was immunized six times, at weekly intervals, with 150 μg of recombinant human CD33 ectodomain (Acro Biosystems, CD3-H5226, Asp 18–His 259) supplemented with Gerbu LQ 3000 adjuvant (Gerbu Biotechnik GmbH).

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