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: bioRxiv

Article Title: Chimeric Antigen Receptors Transmit Piconewton Forces that are Coupled with T Cell Function

doi: 10.1101/2025.10.23.684052

Figure Lengend Snippet: A. Proposed mechanism of action for dasatinib in CAR T cells which leads to Lck inhibition. B . Representative RICM and tension images of CAR T cells treated with escalating doses of dasatinib. C and D. Plots quantifying CAR T cell forces and cell spread area as a function of dasatinib treatment. N ≥ 162 from 5 independent transductions. ***= 0.0001, ****<0.0001. E . Plot showing cytotoxicity as a function of dose-dependent dasatinib CAR T cell treatment. Data is averaged from two technical replicates using the same batch of CAR T cells. F . Relationship between CAR T cell tension and cytotoxicity showing correlation. G . Schematic showing three additional engineered CARs with 4-1BB costimulatory domain, ITAM mutants lacking key tyrosine residues, and finally CAR lacking a cytoplasmic domain along with representative RICM and 8 pN locked tension (Cy3B) images. H . Plot of single cell tension levels for each CAR T cell construct tested. N ≥ 162 from 5 independent transductions. One-way ANOVA. ***= 0.0001 , ****<0.0001. I . Representative RICM and 8 pN tension images showing the tension and spread area of cells on CD19 and CD33 antigens. J. Quantification of cellular tension levels from n≥90 cells/condition from 3 separate transductions. Each color indicates a biological replicate from a unique transduction using the same human donor. The larger data points indicate the mean of each replicate while the small data points represent the single cell measurements. N ≥ 106 cells/condition from 3 independent transductions. Student’s Two-Tailed Paired T Test, ****<0.0001.

Article Snippet: Biotinylated CD19 protein (Cat#CD9-H82E9-25ug), biotinylated CD33 protein (Cat#CD3-H82E7-25ug), and AlexaFluor 647 Anti-FMC63 Ab (Cat#FM3-AM534-25tests) were purchased from AcroBioSystems (Newark, DE).

Techniques: Inhibition, Construct, Transduction, Two Tailed Test

Journal: bioRxiv

Article Title: Rational design of oxidation-resistant antibodies through local electrostatic modulation

doi: 10.1101/2025.06.29.662139

Figure Lengend Snippet: a) Cryo-EM structure of 15G15.3 Fab (gray) bound to CD33 (violet), showing Trp96 forming key interactions with Lys52 (CD33) and Asp101 (Fab). Trp96 oxidizes at 97% under AAPH stress; W96F mutation abolishes binding ( > 1000-fold loss). b) Electrostatic potential of the lead candidate, with 12 mutated residues shown as pink spheres. c) Scatter plot of Trp oxidation vs. Epot for the lead and 13 variants; point color reflects relative KD. d) Summary of 13 engineered variants. S11 and S13 show improved oxidation resistance with preserved binding.

Article Snippet: Briefly, each antibody variant was captured by Protein A sensor chip (Series S) on the different flow cell to achieve approximately 150 response units (RU), followed by the injection of fivefold serial dilutions of human CD33 protein (R&D Systems; 0.16 nM to 100 nM) in HBS-EP buffer.

Techniques: Cryo-EM Sample Prep, Mutagenesis, Binding Assay

Journal: Frontiers in Immunology

Article Title: Multi-targeted, NOT gated CAR-T cells as a strategy to protect normal lineages for blood cancer therapy

doi: 10.3389/fimmu.2025.1493329

Figure Lengend Snippet: Tmod can be adapted for blood cancer. (A) Diagram for Tmod system showing the two receptors that comprise the NOT gate targeting HLA loss of heterozygosity (LOH) in solid tumors. (B) Diagram for Tmod utilizing tandem receptors for blood cancer. (C) CD33 and CD16b mRNA expression in primary AML and healthy blood cells including T cells, neutrophils, monocytes, and hematopoietic stem cells (HSC) (data from sources shown; see Supplementary Table 1 ). (D) mRNA expression of targets in AML cell lines (n=43; DepMap).

Article Snippet: Engineered T cells were profiled via flow cytometry for construct expression using recombinant human CD33 (Acro Biosystems) and recombinant human CD16b (NA2) (Acro Biosystems).

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: Multi-targeted, NOT gated CAR-T cells as a strategy to protect normal lineages for blood cancer therapy

doi: 10.3389/fimmu.2025.1493329

Figure Lengend Snippet: CD33 | CD16b Tmod functions robustly in Jurkat and primary T cells. (A) Diagram of functional screen in Jurkat reporter cell line cocultured with K562 target cells transfected with different amounts of CD16b mRNA. Tmod transgene expression in Jurkat cells was detected by staining with recombinant human (rh) CD16b and CD33. (B) Diagram of functional parameters estimated from the Jurkat cell assay data. (C) Functional readout from 8-point mRNA titration curves. Three CARs combined with 4 blockers, that were selected for further analysis, are shown in color. Data are shown as mean ± standard deviation of technical replicates (n=2), normalized to each sample’s maximum activation. (D) Left, diagram of non-viral construct-screening in primary T cells using PiggyBac transposase and single vectors (BA vectors). Right, metrics used to quantify the potency and selectivity of the Tmod pair. (E) Flow cytometry analysis of stable integrants via staining with labeled recombinant human CD33 (see Methods). (E, F) T cell cytotoxicity curves generated from GFP signal at 48 hour time point with each well normalized to the zero time point. Tumor (CD33(+)CD16(-)) target-cell curves are shown with dashed lines and “normal” (CD33(+)CD16(+)) target-cell curves with solid lines. Black lines are CAR constructs and colored lines are Tmod constructs. Tumor cells are K562 cells engineered with CD33 and normal cells are K562 cells engineered to overexpress CD33 and CD16b. Data are shown as mean ± standard deviation of technical replicates (n=3). (G) Potency calculated as ET50 of Tmod cells cocultured with tumor cells. (H) Selectivity ratios are calculated as ET50 on normal cells divided by ET50 on tumor cells. (I) Kinetic cytotoxicity analysis of the most selective and potent construct compared to the CAR-T. GFP(+) area was used as proxy for target cell viability. Data are shown as mean ± standard deviation of technical replicates (n=3).

Article Snippet: Engineered T cells were profiled via flow cytometry for construct expression using recombinant human CD33 (Acro Biosystems) and recombinant human CD16b (NA2) (Acro Biosystems).

Techniques: Functional Assay, Transfection, Expressing, Staining, Recombinant, Titration, Standard Deviation, Activation Assay, Construct, Flow Cytometry, Labeling, Generated

Journal: Frontiers in Immunology

Article Title: Multi-targeted, NOT gated CAR-T cells as a strategy to protect normal lineages for blood cancer therapy

doi: 10.3389/fimmu.2025.1493329

Figure Lengend Snippet: CD33 | CD16b Tmod cells selectively kill tumor but not “normal” cells in vivo . (A) Schema for in vivo experiment. 2 million MV-4-11 AML cells or MV-4-11 cells that overexpress CD16b were injected into NSG-SGM3 mice and 6 days later 7.5 million T cells were injected. (B) Selectivity in vitro using MV-4-11 cells. Surrogate normal cells were generated by overexpression of CD16b in the AML cells. E:T cytotoxicity curves were generated from firefly luciferase bioluminescence at 48 hours. Data are shown as mean ± standard deviation of technical replicates (n=3). Inset: ET50 values of depicted curves. Data shown are interpolated values with 95% CI. (C) Flow cytometry analysis of construct expression by staining with labeled recombinant human CD16b and CD33. (D) Bioluminescence imaging (BLI) at 20 days post target-cell injection. (E) Flow cytometry analysis of MV-4-11 cells in the bone marrow 27 days post target-cell injection. (F) Quantification of data shown in panel. (E) Statistics were calculated using a non-parametric Kruskal-Wallis H test; *: 0.01 < adjusted p < 0.05; **: adjusted p value < 0.01; ns: not significant (adjusted p > 0.05).

Article Snippet: Engineered T cells were profiled via flow cytometry for construct expression using recombinant human CD33 (Acro Biosystems) and recombinant human CD16b (NA2) (Acro Biosystems).

Techniques: In Vivo, Injection, In Vitro, Generated, Over Expression, Luciferase, Standard Deviation, Flow Cytometry, Construct, Expressing, Staining, Labeling, Recombinant, Imaging

Journal: Frontiers in Immunology

Article Title: Multi-targeted, NOT gated CAR-T cells as a strategy to protect normal lineages for blood cancer therapy

doi: 10.3389/fimmu.2025.1493329

Figure Lengend Snippet: Tandem Tmod constructs for blood cancer. (A) Diagram of a Tmod cell with bispecific activator to target AML (CD33) and other blood cancers (SPN) and bispecific blocker to protect HSCs (CLEC9A) and neutrophils (CD16b). (B) Target expression in primary blood cancers and healthy blood cells (data from sources shown; see Supplementary Table 1 ). (C) Target expression in blood cancer cell lines (DepMap). (D) Jurkat cell (SPN KO) functional readout of SPN | CD16b Tmod with blocker titration curves. (E) Jurkat cell (SPN KO) functional readout of SPN-CD33 tandem CAR activation and blocking by CD16b blocker in the presence of SPN and CD33 antigens. (F) Jurkat functional readout of binders cloned as CARs with titration of primary HSCs. (G) Jurkat functional readout of CD33 CAR4 blocked by tandem CLEC9A-CD16b blocker. (H) Jurkat functional readout of CD33 and/or SPN monospecific or bispecific activators paired with CD16b and/or CLEC9A monospecific or bispecific blockers. Data are shown as mean ± standard deviation of technical replicates (n=2).

Article Snippet: Engineered T cells were profiled via flow cytometry for construct expression using recombinant human CD33 (Acro Biosystems) and recombinant human CD16b (NA2) (Acro Biosystems).

Techniques: Construct, Expressing, Functional Assay, Titration, Activation Assay, Blocking Assay, Clone Assay, Standard Deviation

Journal: ACS Omega

Article Title: In Vitro and In Vivo Comparison of Random versus Site-Specific Conjugation of Bifunctional Chelating Agents to the CD33-Binding Antibody for Use in Alpha- and Beta-Radioimmunotherapy

doi: 10.1021/acsomega.4c09450

Figure Lengend Snippet: Immunoreactivity and binding data of modified HuM195 immunoconjugates. (A) Indirect enzyme linked immunosorbent assay (ELISA) of HuM195 immunoconjugates against recombinant human Siglec-3 (CD33). (B) Titration flow cytometry binding of HuM195 immunoconjugates to OCI-AML3 cells. (C) Comparison of the total binding capacity (Bmax) of immunoconjugates to that of naïve HuM195. (D) Binding curve of HuM195 immunoconjugates to Daudi cells as a negative control. Flow cytometry controls shown in Supporting Information Figure S1 .

Article Snippet: Corning High Binding 96-well plate (Cat#9018, Corning, NY, USA) was coated with 0.1 μg/well of recombinant human Siglec-3 (CD33, SinoBiologicals Cat# 12238-HCCH) in carbonate/bicarbonate coating buffer (100 mM, pH 9.6) overnight at 4 °C.

Techniques: Binding Assay, Modification, Indirect ELISA, Enzyme-linked Immunosorbent Assay, Recombinant, Titration, Flow Cytometry, Comparison, Negative Control

Journal: ACS Omega

Article Title: In Vitro and In Vivo Comparison of Random versus Site-Specific Conjugation of Bifunctional Chelating Agents to the CD33-Binding Antibody for Use in Alpha- and Beta-Radioimmunotherapy

doi: 10.1021/acsomega.4c09450

Figure Lengend Snippet: Internalization into OCI-AML3 and in vitro cytotoxicity data of 225 Ac-labeled HuM195 immunoconjugates and free 225 Ac-DTPA control against OCI-AML3 cells. (A–C) Cytotoxicity curves for high (18.5 kBq), medium (4.63 kBq), and low dose (0.29 kBq) of 225 Ac-labeled HuM195 immunoconjugates against CD33 + cells over 24 h. (D) 24 h dose–response curve of 225 Ac-labeled HuM195 immunoconjugates against OCI-AML3 cells with calculated inhibitory constant ( K i ) values. (E) Comparison of 225 Ac-labeled HuM195 immunoconjugates effect on cell confluency compared to untreated control; and (F) internalization of HuM195 and of immunoconjugates into OCI-AML3 cells.

Article Snippet: Corning High Binding 96-well plate (Cat#9018, Corning, NY, USA) was coated with 0.1 μg/well of recombinant human Siglec-3 (CD33, SinoBiologicals Cat# 12238-HCCH) in carbonate/bicarbonate coating buffer (100 mM, pH 9.6) overnight at 4 °C.

Techniques: In Vitro, Labeling, Control, Comparison