Journal: Nature Communications

Article Title: An antibody cocktail targeting two different CD73 epitopes enhances enzyme inhibition and tumor control

doi: 10.1038/s41467-024-55207-9

Figure Lengend Snippet: A Flow cytometry analysis of HB0038 and HB0039 binding to CHO-S cells stably overexpressing human CD73 protein. For clarity, “38” refers to HB0038 and “39” refers to HB0039 in subsequent references. Results represent two technical replicates from one of three independent experiments; data are presented as mean values ± SD. B Soluble CD73 activity inhibition by HB0038, HB0039, HB0045, Oleclumab, Mupadolimab, and AK119. Inhibition efficiency was calculated using Eq. as described in the Methods section. Results represent three technical replicates from one of three independent experiments; data are presented as mean values ± SD. C Membrane-bound CD73 activity inhibition by HB0038, HB0039, HB0045, Oleclumab, and lgG control. Inhibition efficiency was calculated using Eq. as described in the Methods section. mCD73: membrane-bound CD73. Results represent two technical replicates from one of three independent experiments; data are presented as mean values ± SD. D CD73 activity inhibition by HB0038, HB0039, and HB0045, CD73 MIX indicates a mixture of soluble and membrane-bound CD73. Inhibition efficiency was calculated using Eq. as described in the Methods section. Results represent two technical replicates from one of three independent experiments; data are presented as mean values ± SD. E , F Antibody-mediated proliferation in CD4 + and CD8 + T cells with dose-dependent HB0038, HB0039, or HB0045. Stim: anti-CD3 antibody + anti-CD28 antibody + IL-7. Workflow refers to Supplementary Fig. . Results represent two technical replicates from one of three independent experiments; data are presented as mean values ± SD.

Article Snippet: BALB/c mice were immunized with recombinant human CD73 protein (Sino Biological, HPLC-10904-H08H), and spleen cells were isolated and fused with SP2/0 myeloma cells for generation of hybridomas according to standard methods .

Techniques: Flow Cytometry, Binding Assay, Stable Transfection, Activity Assay, Inhibition, Membrane, Control

Journal: Nature Communications

Article Title: An antibody cocktail targeting two different CD73 epitopes enhances enzyme inhibition and tumor control

doi: 10.1038/s41467-024-55207-9

Figure Lengend Snippet: A Hydrogen-deuterium exchange (HDX) identified the epitopes of HB0038 and HB0039 on CD73. These were then mapped onto the CD73 structure (PDB code: 4H1Y) via PyMOL. B – K Time-resolved SPR analysis detailing affinity between HB0038 and CD73 wide type (WT) and its mutants. All CD73 residues identified by HDX analysis potentially involved in HB0038 binding were mutated to Ala individually and were assessed for their contribution to HB0038 binding. The SPR profile of HB0038 against specific CD73 WT and CD73 mutations in the HDX epitope study. L – Q As in ( B – K ), but between HB0039 and CD73.

Article Snippet: BALB/c mice were immunized with recombinant human CD73 protein (Sino Biological, HPLC-10904-H08H), and spleen cells were isolated and fused with SP2/0 myeloma cells for generation of hybridomas according to standard methods .

Techniques: Binding Assay

Journal: Nature Communications

Article Title: An antibody cocktail targeting two different CD73 epitopes enhances enzyme inhibition and tumor control

doi: 10.1038/s41467-024-55207-9

Figure Lengend Snippet: A Visualization of the CD73 antigen (6TVG), Fab (1M71), and complete IgG (1IGY) docked within the electron density map derived from negative staining of the CD73-HB0039 IgG-HB0038 Fab complex. B Enzymatic assay results display the concentration-dependent inhibitory effects on soluble CD73 enzymatic activity by HB0038 IgG, HB0039 IgG, HB0038 Fab, and HB0039 Fab. Results represent two technical replicates from one of three independent experiments; data are presented as mean values ± SD.

Article Snippet: BALB/c mice were immunized with recombinant human CD73 protein (Sino Biological, HPLC-10904-H08H), and spleen cells were isolated and fused with SP2/0 myeloma cells for generation of hybridomas according to standard methods .

Techniques: Derivative Assay, Negative Staining, Enzymatic Assay, Concentration Assay, Activity Assay

Journal: Nature Communications

Article Title: An antibody cocktail targeting two different CD73 epitopes enhances enzyme inhibition and tumor control

doi: 10.1038/s41467-024-55207-9

Figure Lengend Snippet: A Cryo-EM visualization of the CD73-HB0038 Fab-HB0039-Fab complex. Within the CD73 dimer, one monomer is depicted in magenta while its counterpart is in purple. The fragment variable (Fv) domains of HB0039 are illustrated in varying shades of green, with one HB0038 Fv domain in blue (with the second in cyan). The CH1 domains for both HB0038 and HB0039 Fabs are represented in gray. B Cartoon representation of the CD73-HB0038 Fab-HB0039-Fab complex, colored as in ( A ). C Close-up views of the interaction interfaces: Interface I between HB0039 (in green) and CD73 (in magenta) and Interface II between HB0038 (in blue) and CD73. Interacting residues are depicted as sticks. D Comparative alignment of different CD73 conformations when bound to various antibodies (referenced antibodies omitted for clarity). CD73 conformations bound to TB38 and TB19 are shown in shades of cyan, while its conformation with HB0045 is represented in magenta. The fully open and fully closed conformation of CD73 were shown in gray and blue, respectively.

Article Snippet: BALB/c mice were immunized with recombinant human CD73 protein (Sino Biological, HPLC-10904-H08H), and spleen cells were isolated and fused with SP2/0 myeloma cells for generation of hybridomas according to standard methods .

Techniques: Cryo-EM Sample Prep

Journal: Nature Communications

Article Title: An antibody cocktail targeting two different CD73 epitopes enhances enzyme inhibition and tumor control

doi: 10.1038/s41467-024-55207-9

Figure Lengend Snippet: A Workflow illustrating CD73 antibody treatment across different tumors in PBMC-humanized NPG-immunodeficient mice. “I.P.” denotes intraperitoneal injections; “BIW” indicates administration twice weekly. It was created in BioRender. Zhifeng, Y. (2024) https://BioRender.com/r91t302 . B Impact of varying anti-CD73 antibody doses on MDA-MB-231 tumor growth and weight in PBMC-humanized NPG mice. Once tumors reached an average size of 50 ~ 100 mm³, mice bearing tumors were grouped randomly into six segments ( n = 8) and administered i.v with 1 × 10 7 PBMCs. Treatments were inclusive of the IgG negative control (3 mg/kg), in-house Oleclumab (1 mg/kg), HB0038 (1 mg/kg), HB0039 (1 mg/kg), and HB0045 at two dosages (1 mg/kg and 3 mg/kg). Treatment commenced on the same day with bi-weekly intraperitoneal antibody dosing for 4 weeks. P_volume (38 1 mg/kg vs 45 1 mg/kg) = 0.0087; P_volume (Oleclumab 1 mg/kg vs 45 1 mg/kg) <0.0001; P_volume (Oleclumab 1 mg/kg vs 45 3 mg/kg) <0.0001; P_weight (Oleclumab 1 mg/kg vs control 3 mg/kg) <0.0001; P_weight (Oleclumab 1 mg/kg vs 45 1 mg/kg) = 0.0141; P_weight (45 1 mg/kg vs 45 3 mg/kg) = 0.004. C NPG mice received a subcutaneous inoculation of BxPC-3 (5 × 10 6 ) in Matrigel on their right flank. Upon tumors achieving an average size of 68 mm³, mice with tumors were grouped randomly into four segments ( n = 6) and administered i.v with 1 × 10 7 PBMCs. Treatments began on day one, with bi-weekly intraperitoneal antibody dosing spanning four weeks. P < 0.0001. D The HB0045 antibody cocktail (anti-CD73), coupled with the HB0025 antibody (anti-PD-L1/VEGF bispecific antibody) and HB0027 (anti-4-1BB antibody), demonstrated potent antitumor capabilities in the A375 melanoma models using PBMC-humanized NPG mice ( n = 6). Unless otherwise specified, the concentration of antibody used in samples was 1 mg/kg. P_volume (Vehicle vs 45) < 0.0001; P_volume (45 vs 25 + 45) = 0.0013; P_volume (25 + 45 vs 25 + 45 + 27) = 0.008; P_weight (Vehicle vs 45) < 0.0001; P_weight (27 vs 25 + 45 + 27) = 0.0054. Prism was employed to compute P values for tumor volumes using two-way ANOVA coupled with Tukey’s multiple-comparison test. For tumor weights, P values were determined using a one-way ANOVA. * P < 0.05, ** P < 0.01, **** P < 0.0001. Data are presented as mean values ± SEM.

Article Snippet: BALB/c mice were immunized with recombinant human CD73 protein (Sino Biological, HPLC-10904-H08H), and spleen cells were isolated and fused with SP2/0 myeloma cells for generation of hybridomas according to standard methods .

Techniques: Negative Control, Control, Concentration Assay, Comparison

Journal: Biomedicines

Article Title: A Novel Anti-CD73 Antibody That Selectively Inhibits Membrane CD73 Shows Antitumor Activity and Induces Tumor Immune Escape

doi: 10.3390/biomedicines10040825

Figure Lengend Snippet: 22E6 is CD73-specific. ( A ) 22E6 binds to HEK293 cells stably transfected with a CD73 expression plasmid (293/CD73) but not to parental HEK293 cells (293). ( B ) 22E6 specifically precipitates CD73 from lysates. ( C ) 22E6 binds to various permanent cancer cell lines and cells isolated from primary ascites. Plain histograms in ( A , C ) = isotype control antibody.

Article Snippet: The 293T cells were transfected with an expression plasmid, coding for human CD73 (human CD73 ORF cDNA clone expression plasmid, HIS-tagged, (Sino Biologicals, Beijing, China) using LipofectamineTM 2000 transfection reagent (ThermoFisher Scientific, Menzel, Germany) according to manufacturer’s instructions.

Techniques: Stable Transfection, Transfection, Expressing, Plasmid Preparation, Isolation, Control

Journal: Biomedicines

Article Title: A Novel Anti-CD73 Antibody That Selectively Inhibits Membrane CD73 Shows Antitumor Activity and Induces Tumor Immune Escape

doi: 10.3390/biomedicines10040825

Figure Lengend Snippet: 22E6 inhibits CD73 enzyme activity. ( A ) U138 MG glioblastoma cells were incubated with APCP, 22E6 or an isotype control antibody in the presence of AMP. The amount of inorganic phosphate was measured in a Malachite Green assay. ( B ) The IC50 of 22E6 was calculated to be 0.5 µg/mL, corresponding to approximately 3.5 nM. ( C ) The generation of inorganic phosphate from ADP is CD73-dependent and effectively blocked by 22E6 as shown on MDA-MB231 and GBM20 cells. CD73-negative T47-D do not produce detectable amounts of phosphate. Experiments were performed three times. ** p < 0.01; **** p < 0.0001.

Article Snippet: The 293T cells were transfected with an expression plasmid, coding for human CD73 (human CD73 ORF cDNA clone expression plasmid, HIS-tagged, (Sino Biologicals, Beijing, China) using LipofectamineTM 2000 transfection reagent (ThermoFisher Scientific, Menzel, Germany) according to manufacturer’s instructions.

Techniques: Activity Assay, Incubation, Control, Malachite Green Assay

Journal: Biomedicines

Article Title: A Novel Anti-CD73 Antibody That Selectively Inhibits Membrane CD73 Shows Antitumor Activity and Induces Tumor Immune Escape

doi: 10.3390/biomedicines10040825

Figure Lengend Snippet: 22E6 blocks CD73 enzyme activity on tumor-derived extracellular vesicles. ( A ) TEVs from GBM20 cells were floated into a Optiprep gradient and separated into eight fractions, which were subsequently tested for the presence of the EV markers CD63, CD81 and TSG-101, as well as of CD73. ( B ) Particle numbers in fractions 1 to 8 were counted by NTA, and the protein content was measured in a Bradford assay. ( C ) Fraction 3 contains vesicles which stain positive for CD63, CD81 and CD73, while it is negative for Calnexin indicative for a cell-free preparation. ( D ) APCP and 22E6 block CD73 activity on TEVs from GBM20 cells and ( E ) isolated from primary ascites from a patient with ovarian cancer. ( F ) enzyme activity can be transferred by CD73-positive TEVs from GBM20 cells onto CD73-negative T47-D cells. Experiments were performed at least three times. * p < 0.05; ** p < 0.01; **** p < 0.0001.

Article Snippet: The 293T cells were transfected with an expression plasmid, coding for human CD73 (human CD73 ORF cDNA clone expression plasmid, HIS-tagged, (Sino Biologicals, Beijing, China) using LipofectamineTM 2000 transfection reagent (ThermoFisher Scientific, Menzel, Germany) according to manufacturer’s instructions.

Techniques: Activity Assay, Derivative Assay, Bradford Assay, Staining, Blocking Assay, Isolation

Journal: Biomedicines

Article Title: A Novel Anti-CD73 Antibody That Selectively Inhibits Membrane CD73 Shows Antitumor Activity and Induces Tumor Immune Escape

doi: 10.3390/biomedicines10040825

Figure Lengend Snippet: 22E6 is a non-competitive inhibitor of CD73. ( A ) Incubation of U138 MG cells with 22E6 and increasing concentrations of AMP resulted in a decreases Vmax. ( B ) The 22E6 antibody but not the 22E6 Fab fragments inhibits CD73 activity. One representative experiment of three is shown. *** p < 0.001; **** p < 0.0001.

Article Snippet: The 293T cells were transfected with an expression plasmid, coding for human CD73 (human CD73 ORF cDNA clone expression plasmid, HIS-tagged, (Sino Biologicals, Beijing, China) using LipofectamineTM 2000 transfection reagent (ThermoFisher Scientific, Menzel, Germany) according to manufacturer’s instructions.

Techniques: Incubation, Activity Assay

Journal: Biomedicines

Article Title: A Novel Anti-CD73 Antibody That Selectively Inhibits Membrane CD73 Shows Antitumor Activity and Induces Tumor Immune Escape

doi: 10.3390/biomedicines10040825

Figure Lengend Snippet: 22E6 does not inhibit enzyme activity of soluble CD73. ( A ) specific precipitation of recombinant CD73 by 22E6. ( B ) 22E6 does not detectably inhibit enzyme activity of soluble CD73. Error bars = SD; p < 0.001 for APCP, n.s. for Isotype and 22E6. This experiment was performed at least four times.

Article Snippet: The 293T cells were transfected with an expression plasmid, coding for human CD73 (human CD73 ORF cDNA clone expression plasmid, HIS-tagged, (Sino Biologicals, Beijing, China) using LipofectamineTM 2000 transfection reagent (ThermoFisher Scientific, Menzel, Germany) according to manufacturer’s instructions.

Techniques: Activity Assay, Recombinant

Journal: Biomedicines

Article Title: A Novel Anti-CD73 Antibody That Selectively Inhibits Membrane CD73 Shows Antitumor Activity and Induces Tumor Immune Escape

doi: 10.3390/biomedicines10040825

Figure Lengend Snippet: 22E6 induces downregulation of CD73 on PDX ALL cells in vivo. ( A ) PDX ALL cells express surface CD73 before transplantation. Black line = 22E6; tinted histogram = isotype control. ( B ) CD73 on PDX ALL cells is enzymatically active, and this activity can be inhibited with 22E6 and APCP. ( C ) NSG mice were transplanted with ALL-272 or ALL-1124 cells. Tumor burden was monitored by bioluminescence imaging (BLI). Mice were treated with 100 µg 22E9 twice per week ( n = 5 for ALL-272, n = 6 for ALL-1124; black triangles) or left untreated ( n = 3 for ALL-272, n = 4 for ALL-1124; grey dots). Tumor burden (BLI signal) relative to treatment start was calculated. Data of two independent experiments were integrated. Shown are individual mice (dots) as well as mean ± standard deviation (line). Tumor burdens of ALL-1124 between control and 22E6 treated mice differed significantly at days 35 ( p < 0.05) and 42 ( p < 0.01). At high tumor burden, mice were sacrificed, and cells were isolated. ( D ) Flow Cytometric analysis of isolated xenograft ALL cells from 22E6 treated (solid line) and control mice (dashed line) checking for expression of CD73. Black line = 22E6 anti-CD73; Tinted = control. ( E ) AMPase assay with ALL cell lines testing for enzymatic activity of CD73 of isolated xenograft ALL cells from 22E6 treated and control mice. *** p < 0.001.

Article Snippet: The 293T cells were transfected with an expression plasmid, coding for human CD73 (human CD73 ORF cDNA clone expression plasmid, HIS-tagged, (Sino Biologicals, Beijing, China) using LipofectamineTM 2000 transfection reagent (ThermoFisher Scientific, Menzel, Germany) according to manufacturer’s instructions.

Techniques: In Vivo, Transplantation Assay, Control, Activity Assay, Imaging, Standard Deviation, Isolation, Expressing