Journal: Oncoimmunology

Article Title: Mono- and dual-targeting triplebodies activate natural killer cells and have anti-tumor activity in vitro and in vivo against chronic lymphocytic leukemia

doi: 10.1080/2162402X.2016.1211220

Figure Lengend Snippet: Specificity of ULBP2-aCD19, ULBP2-aCD33, ULBP2-aCD19-aCD33 and ULBP2-aCD19-aCD19 immunoligands to their respective target moieties. (A) CD19+ (MEC1) and CD19− (HL60) cell lines were incubated with 10 µg/mL of ULBP2-aCD19 or ULBP2-aCD19-aCD19 and binding was detected by either anti c-Myc tag antibody (upper panel—“c-Myc”) or recombinant human NKG2D receptor (lower panel—“NKG2D”) where the latter confirms that immunoligands can bind to CD19 and NKG2D simultaneously. Gray-filled area depicts the background staining. Pre-blocking of CD19 antigen by anti-CD19scFv (20 µg/mL) inhibited the binding of immunoligands (dashed line—“MEC1”). (B) CD19+CD33+ cell line BV173 was incubated with 10 µg/mL of ULBP2-aCD19, ULBP2-aCD33 and ULBP2-aCD19-aCD33 and binding was detected by recombinant human NKG2D receptor (“No blocking”). Pre-blocking of CD19 or CD33 antigen by respective blocking construct (20 µg/mL) prevented the binding of ULBP2-aCD19 and ULBP2-aCD33, respectively (“Block + Bispecific ILs”) but not of ULBP2-aCD19-aCD33 (“Block + ULBP2-aCD19-aCD33”). Only simultaneous blocking of both CD19 and CD33 antigens on BV173 cells could completely inhibit the binding of ULBP2-aCD19-aCD33 (“Block + ULBP2-aCD19-aCD33”).

Article Snippet: For detection of simultaneous binding of a dual-targeting triplebody ULBP2-aCD19-aCD33, immunoligands were incubated with CD19 + MEC1 cells and bound immunoligands were detected using recombinant human NKG2D-Fc receptor and recombinant human CD33-FLAG (Origene, Cat. TP307023).

Techniques: Incubation, Binding Assay, Recombinant, Staining, Blocking Assay, Construct

Journal: Oncoimmunology

Article Title: Mono- and dual-targeting triplebodies activate natural killer cells and have anti-tumor activity in vitro and in vivo against chronic lymphocytic leukemia

doi: 10.1080/2162402X.2016.1211220

Figure Lengend Snippet: Simultaneous and specific antigen binding of a triplebody ULBP2-aCD19-aCD33. Simultaneous binding of ULBP2-aCD19-aCD33 to all three target moieties CD19, CD33 and NKG2D was detected. CD19+ MEC1 cell line was incubated with ULBP2-aCD19-aCD33 and its CD19 specific binding to MEC1 cells was detected using recombinant human NKG2D-Fc and CD33-FLAG receptors followed by AF647 labeled anti-Fc and PE labeled anti-FLAG antibodies, respectively (U-19-33). Binding of ULBP2-aCD19 to CD19 on MEC1 cells was detected only using NKG2D-Fc as it lacked anti-CD33scFv (U-19). ULBP2-aCD33 was used as a negative control that failed to bind CD19+ MEC1 cells (U-33) whereas NKG2D-Fc and CD33-FLAG background binding was minimal (No IL).

Article Snippet: For detection of simultaneous binding of a dual-targeting triplebody ULBP2-aCD19-aCD33, immunoligands were incubated with CD19 + MEC1 cells and bound immunoligands were detected using recombinant human NKG2D-Fc receptor and recombinant human CD33-FLAG (Origene, Cat. TP307023).

Techniques: Binding Assay, Incubation, Recombinant, Labeling, Negative Control

Journal: Oncoimmunology

Article Title: Mono- and dual-targeting triplebodies activate natural killer cells and have anti-tumor activity in vitro and in vivo against chronic lymphocytic leukemia

doi: 10.1080/2162402X.2016.1211220

Figure Lengend Snippet: Enhancement of primary NK cell effector functions by bispecific immunoligands and triplebodies. Cytotox assays. Primary NK cells were purified from peripheral blood mononuclear cells (PBMC) of healthy donors by negative selection and were cultured with IL-2 (200 U/mL) and IL-15 (10 ng/mL) overnight before the experiments on the following day. For all experiments, each N represents independent healthy donor. (A, Upper panel) Purified NK cells were co-incubated with DiR labeled CD19+ (MEC1) and CD19− (HL60) cell lines at indicated effector to target (E:T) ratios either alone (•) or in presence of 10 nM ULBP2-aCD19 (▪) or ULBP2-aCD19-aCD19 (▴) immunoligand for 3 h and dead target cells were measured by 7-AAD staining on FACS. (A, lower lane) Purified NK cells were co-incubated with DiR labeled CD19+CD33+ cell lines (BV173 and SEM) at indicated effector to target (E:T) ratios either alone (•) or in presence of 100 nM ULBP2-aCD19 (▪), ULBP2-aCD33 (▾) or ULBP2-aCD19-aCD33 (▴) immunoligand for 3 h and dead target cells were measured by 7-AAD staining on FACS. For simplicity, selected statistical significances are shown in comparison with “No construct” group (*p < 0.05; **p < 0.01). Error bars indicate SEM (MEC1 (N = 4), HL60 (N = 3), BV173 (N = 5) and SEM (N = 4)). (B) Degranulation assay. Purified NK cells were co-incubated with MEC1 and SEM cells at E:T ratio of 2.5:1 either alone or in presence of indicated immunoligand for 6 h and CD107a/LAMP-1 staining within NK cells (stained and gated with anti-CD56 and anti-NKp46 antibodies) were measured to determine degranulated NK cell population. Error bars indicate SEM and ** represents p < 0.01; *** represents p < 0.001 (MEC1 (N = 3) and SEM (N = 5)) (C) ELISA-based IFNγ assay. Left: Purified NK cells were co-incubated with MEC1 cells at E:T ratio of 1:1 either alone or in presence of 10 nM immunoligand for 24 h and supernatant was collected for IFNγ detection by ELISA. IFNγ secretion by MEC1 cells (with or without immunoligand) was carefully controlled and was found to be negative (data not shown). Experiments were conducted with two independent NK donors and one example is shown where error bars indicate SEM of duplicates. (C, right) Purified NK cells were cultured in plate pre-coated with indicated immunoligands for 48 h and supernatant was collected for IFNγ detection by ELISA. Experiments were conducted with three independent NK cell donors and one example is shown where error bars indicate SEM of duplicates.

Article Snippet: For detection of simultaneous binding of a dual-targeting triplebody ULBP2-aCD19-aCD33, immunoligands were incubated with CD19 + MEC1 cells and bound immunoligands were detected using recombinant human NKG2D-Fc receptor and recombinant human CD33-FLAG (Origene, Cat. TP307023).

Techniques: Purification, Selection, Cell Culture, Incubation, Labeling, Staining, Construct, Degranulation Assay, Enzyme-linked Immunosorbent Assay

Journal: Cells

Article Title: Shape-Shifted Red Blood Cells: A Novel Red Blood Cell Stage?

doi: 10.3390/cells7040031

Figure Lengend Snippet: Protein cell markers for shRBCs. ( a ) RBCs incubated at 25 °C for 3 days were stained with different antibodies and analyzed by flow cytometry. The dot plot shows RBCs (red) and shRBCs (yellow). Flow cytometry results are represented as MRFI (mean relative fluorescence intensity = fluorescence in shRBC/RBC). ( b ) Protein cell markers: erythrocyte marker (glycophorin C [GYPC]), myeloid cell surface antigen (CD33), hematopoietic progenitor cell surface antigen (CD34), and B cell marker (immunoglobulin M [IgM]). Data are displayed as black circles showing the mean ± SD (n = 5). The Mann–Whitney Test was performed for statistical analysis between shRBCs and RBCs. RBCs are represented by the dashed line.

Article Snippet: Myeloid cell surface antigen (CD33) and hematopoietic progenitor cell surface antigen (CD34) antibodies were obtained from CUSABIO Life Science (Houston, TX, USA).

Techniques: Incubation, Staining, Flow Cytometry, Fluorescence, Marker, MANN-WHITNEY

Journal: Molecules

Article Title: In Vitro and In Vivo Characterization of 89 Zirconium-Labeled Lintuzumab Molecule

doi: 10.3390/molecules27196589

Figure Lengend Snippet: Radiolabeling of lintuzumab with 89 Zr and its in vitro binding to CD33. ( A ) Binding of Lintuzumab−DFO conjugate to recombinant human CD33 protein is demonstrated via ELISA. ( B ) Binding of Lintuzumab−DFO conjugate to human cancer cell lines that express CD33 is demonstrated via immunofluorescence staining using a flow cytometer. ( C ) HPLC trace chromatograms ran on purified antibody conjugate (upper panel) and radiolabeled conjugate at a wavelength of 280 nm (middle panel UV trace, lower panel radioactivity trace).

Article Snippet: ELISA: Nunc MaxiSorp flat-bottomed 96-well plates were coated with 100 ng/well of human recombinant CD33 His-Tag protein (R&D Systems) in PBS and incubated overnight at 4 °C.

Techniques: Radioactivity, In Vitro, Binding Assay, Recombinant, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining, Flow Cytometry, Purification

Journal: Molecules

Article Title: In Vitro and In Vivo Characterization of 89 Zirconium-Labeled Lintuzumab Molecule

doi: 10.3390/molecules27196589

Figure Lengend Snippet: 89 Zr-lintuzumab Selectively Accumulates in CD33 Expressing Tumors. ( A ) PET/CT imaging of mice administered with 5.55 MBq 89 Zr-DFO-Lintuzumab only ( n = 4). Images were taken on Days 1, 2, 3, and 7 post administration. The radioconjugate was cleared from blood and other organs except for the tumor after Day 1 and accumulated in CD33-positive OCI-AML-3 tumors. ( B ) Cohort of mice ( n = 4) pre-blocked with cold Lintuzumab 24hr prior to administration of radioconjugate. PET/CT images were taken on Days 3 and 7 post radioconjugate administration. The clearance of radioconjugate from blood and other organs in mice pre-blocked with Lintuzumab was not as effective as in non-blocked mice: radioactivity was detected in various organs by PET/CT imaging even on Day 7 post administration. ( C ) Standardized uptake values (SUV) analysis of PET/CT images taken on Days 3 and 7. Tumor volumes of interest (VOI) were drawn, and SUV were calculated. 89 Zr-DFO-Lintuzumab showed a significantly higher uptake in the tumors of non-blocked mice than in the tumors of pre-blocked mice. ( D ) Time–activity curves (TAC) show that the radiolabeled antibody remains mostly constant over the 7-day period after initial uptake for both non-blocked and pre-blocked tumors, with the pre-blocked has significantly less uptake. All images are displayed as maximum intensity projections (MIP). ** and *** mean p = 0.001 and p < 0.0001, respectively.

Article Snippet: ELISA: Nunc MaxiSorp flat-bottomed 96-well plates were coated with 100 ng/well of human recombinant CD33 His-Tag protein (R&D Systems) in PBS and incubated overnight at 4 °C.

Techniques: Expressing, Positron Emission Tomography-Computed Tomography, Imaging, Radioactivity, Activity Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: Developing a membrane-proximal CD33-targeting CAR T cell

doi: 10.1136/jitc-2024-009013

Figure Lengend Snippet: Identification of a membrane-proximal CD33 antibody. (A) Heatmap showing relative binding of selected antibodies to human full-length CD33, CD33-IgC, and U937 CD33-expressing tumor lines. (B) Schematic representation of retroviral vector encoding 5′ and 3′ long terminal repeats sequence (LTR), truncated EGFR, linked with a T2A element to a fully human CD33-targeted single-chain variable fragment, human CD28 hinge, transmembrane, and intracellular domains (CD28), and human zeta chain signaling domain (z chain). A Myc-tag is included for detection of the chimeric antigen receptor (CAR). (C) Summary data of retroviral transduction efficiency of CAR constructs as determined by flow cytometry using cetuximab. P values determined by repeated measures one-way analysis of variance. Data are a mean±SEM of three biological replicates conducted in three independent experiments; ns, non-significant. (D) Representative flow cytometry plot demonstrating CAR expression following human T cell transduction, detected with fluorescently labeled Cetuximab and Myc-Tag-specific antibodies. MFI, mean fluorescence intensity.

Article Snippet: Recombinant human CD33 protein was sourced commercially from R&D Systems and Acro Biosystems.

Techniques: Membrane, Binding Assay, Expressing, Retroviral, Plasmid Preparation, Sequencing, Transduction, Construct, Flow Cytometry, Labeling, Fluorescence

Journal: Journal for Immunotherapy of Cancer

Article Title: Developing a membrane-proximal CD33-targeting CAR T cell

doi: 10.1136/jitc-2024-009013

Figure Lengend Snippet: Membrane-proximal targeting chimeric antigen receptor (CAR) T cells demonstrate enhanced functionality and proliferative capacity in vitro. (A) Flow cytometry histograms of CD33 expression on acute myeloid leukemia (AML) cells detected with isotype control or fluorescently labeled CD33-specific antibody. (B) Quantitative geometric mean fluorescence intensity (MFI) of CD33 expression on AML cells detected with either isotype control or fluorescently labeled CD33-specific antibody. (C) 24-hour D-luciferin assay demonstrating lysis of CD33-expressing tumor cells (n=4 biological replicates; ****p<0.0001; ***p<0.001; **p<0.01; *p<0.05 by two-way analysis of variance (ANOVA)). Data are a mean±SEM of four biological replicates. (D) 96-hour D-luciferin assay demonstrating lysis of CD33 expressing tumor cells (n=4 biological replicates; ****p<0.0001; ***p<0.001; **p<0.01; *p<0.05 by two-way ANOVA). Data are a mean±SEM of four biological replicates. (E) Quantification of flow cytometric analysis demonstrating enhanced proliferation by membrane-proximal CD33 targeting CAR T cell in the presence of U937-CD33 high tumor cells at E:T ratios of 1:2.5 (left) or 1:10 (right) (n=3 biological replicates; ****p<0.0001 by two-way ANOVA at day 21). Arrows indicate when additional target cells were added. Data are a mean±SEM of three biological replicates.

Article Snippet: Recombinant human CD33 protein was sourced commercially from R&D Systems and Acro Biosystems.

Techniques: Membrane, In Vitro, Flow Cytometry, Expressing, Control, Labeling, Fluorescence, Lysis

Journal: Journal for Immunotherapy of Cancer

Article Title: Developing a membrane-proximal CD33-targeting CAR T cell

doi: 10.1136/jitc-2024-009013

Figure Lengend Snippet: Membrane-proximal targeting chimeric antigen receptor (CAR) T cells are characterized by a unique activation profile in vitro. (A) 24-hour cytokine secretion profile of Tc1/Th1 cytokines when co-cultured with U937-CD33 high , OCiAML3-CD33 low , U937-CD33 IgC , and U937-CD33 KO tumor as detected by human 12-plex Luminex panel (n=3; ****p<0.0001; *p<0.05 by Dunnett’s multiple comparison tests against H195DEL control). Data are a mean±SEM of three biological replicates conducted in three independent experiments. (B) Quantification of flow cytometric analysis showing CAR T cell intracellular production of Tc1/Th1 activation cytokines when cultured with U937-CD33 high tumor (n=4; ****p<0.0001; **p<0.01; *p<0.05 by two-way analysis of variance (ANOVA); ns, non-significant). Data are a mean±SEM of four biological replicates conducted in three independent experiments. (C) Qualitative representation of CD4 + CAR + and CD8 + CAR + Tc1/Th1 activation cytokines secretion. Cytokines evaluated were IFN-γ, IL-2, granzyme B, and TNF-α. (D) Quantification of flow cytometric analysis showing CD4 + CAR T cell intracellular production of Tc1/Th1 activation cytokines when cultured with U937-CD33 high tumor (n=4; ****p<0.0001, *p<0.05 by two-way ANOVA). Data are a mean±SEM of four biological replicates conducted in three independent experiments. Cytokines evaluated were IFN-γ, IL-2, granzyme B, and TNF-α. (E) Quantification of flow cytometric analysis showing CD8 + CAR T cell intracellular production of Tc1/Th1 activation cytokines when cultured with U937-CD33 high tumor (n=4; ***p<0.001; **p<0.01; *p<0.05 by two-way ANOVA). Data are a mean±SEM of four biological replicates conducted in three independent experiments. Cytokines evaluated were IFN-γ, IL-2, granzyme B, and TNF-α. (F) Quantification of flow cytometric analysis demonstrating CAR T cell activation status 7 days post-antigen stimulation with U937-CD33 high tumor (n=4; ****p < 0.0001 by two-way ANOVA). Data are a mean±SEM of four biological replicates conducted in three independent experiments. (G) Qualitative representation of flow cytometric analysis comparing CAR T cell activation profiles. Data are pooled from four biological replicates conducted in three independent experiments.

Article Snippet: Recombinant human CD33 protein was sourced commercially from R&D Systems and Acro Biosystems.

Techniques: Membrane, Activation Assay, In Vitro, Cell Culture, Luminex, Comparison, Control

Journal: Journal for Immunotherapy of Cancer

Article Title: Developing a membrane-proximal CD33-targeting CAR T cell

doi: 10.1136/jitc-2024-009013

Figure Lengend Snippet: Membrane-proximal CD33-targeting chimeric antigen receptor (CAR) T cells provide enhanced survival in xenograft mouse model. (A) Schematic diagram of in vivo experimental setup. NCG mice were inoculated with U937-CD33 high tumor and subsequently treated with CAR T cells. (B) Survival of NCG mice bearing U937-CD33 high tumors and treated with titrated doses of CAR T cells (n=5; **p<0.01, *p<0.05). P values for survival determined by log-rank Mantel-Cox test, with 95% CI. (C) Tumor regression of NCG mice inoculated with U937-CD33 high tumor and subsequently treated with 5.0×10 5 of CAR T cells. (D) Bioluminescence over time of U937-CD33 high in tumor-bearing NCG mice treated with 5.0×10 5 CAR T cells. (E) Schematic diagram of in vivo experimental setup. NCG mice were inoculated with OCiAML3-CD33 low tumor and subsequently treated with CAR T cells. (F) Survival of NCG mice inoculated with OCiAML3-CD33 low tumors and treated with 5.0×10 5 CAR T cells (n=5; *p<0.05). P values for survival determined by log-rank Mantel-Cox test, with 95% CI. (G) Tumor regression of NCG mice bearing OCiAML3-CD33 low tumors and treated with 5.0×10 5 CAR T cells. (H) Bioluminescence over time of OCiAML3-CD33 low in tumor-bearing NCG mice treated with 5.0×10 5 CAR T cells.

Article Snippet: Recombinant human CD33 protein was sourced commercially from R&D Systems and Acro Biosystems.

Techniques: Membrane, In Vivo

Journal: Journal for Immunotherapy of Cancer

Article Title: Developing a membrane-proximal CD33-targeting CAR T cell

doi: 10.1136/jitc-2024-009013

Figure Lengend Snippet: Membrane-proximal CD33-targeting chimeric antigen receptor (CAR) T cells decrease tumor burden in patient-derived acute myeloid leukemia (AML) xenograft model. (A) Flow cytometry histograms of CD33 expression on AML60B patient sample detected with isotype control or fluorescently labeled CD33-spcific antibody. (B) Quantitative geometric mean fluorescence intensity (MFI) of CD33 expression on AML60B patient sample detected with either isotype control or fluorescently labeled CD33-specific antibody. (C) Schematic diagram of experimental setup of a patient-derived xenograft model. NCG mice were inoculated with patient-derived AML blasts and treated with allogeneic CAR T cells. Bone marrow aspirates were analyzed 28 days post tumor inoculation. (D) Quantification of flow cytometric analysis demonstrating decreased tumor burden in mice treated with membrane-proximal CD33-targeting CAR T cells (n=6; *p<0.05 by unpaired t-test). (E) Survival of NCG mice-bearing AML60B patient-derived tumor and treated with membrane-distal or membrane-proximal CAR T cells (n=6; **p<0.01; *p<0.05). P values for survival determined by log-rank Mantel-Cox test, with 95% CI.

Article Snippet: Recombinant human CD33 protein was sourced commercially from R&D Systems and Acro Biosystems.

Techniques: Membrane, Derivative Assay, Flow Cytometry, Expressing, Control, Labeling, Fluorescence