Journal: Journal of Clinical Investigation

Article Title: Tumor endothelial marker 1–specific DNA vaccination targets tumor vasculature

doi: 10.1172/jci67382

Figure Lengend Snippet: Figure 4 Tem1-TT vaccination inhibits CT26 tumor vascularization. (A) Tem1-TT vaccination reduced tumor hemoglobin content. Tumors at approximately 200 mm3 were excised from TT- or Tem1-TT–vaccinated mice and inspected grossly. Tumors from Tem1-TT–vaccinated mice appeared pale relative to control tumors. Reduced hemoglobin levels in tumors from Tem1-TT–vaccinated mice were observed by ELISA. (B) Tem1-TT vaccine reduces tumor vascularity. Tumors from TT- or Tem1-TT–vaccinated mice were analyzed by Doppler ultrasound. Perfused tumor area and real blood flux are shown, measured and calculated by Doppler image analysis. (C) CT26 tumors from Tem1-TT–immunized mice had significantly decreased CD31 expression compared with TT vaccination. Also note the abnormal blood vessel shape. Original magnification, ×20. (D) CAIX expression was increased in tumors from Tem1-TT–immunized animals. CAIX expression was visualized by immunohistochemistry, and Caix was independently quantified by qRT-PCR in tumors from mice vaccinated with either TT or Tem1-TT. Original magnification, ×20. Data in A–D are mean ± SD of a representative experiment (n = 5 per group). Statistical analyses were performed with Student’s t test.

Article Snippet: Sections (6 μm thick) were stained for mouse CAIX (R&D Systems; catalog no. AF2344, DAB chromogen), CD31 (BD Biosciences — Pharmingen; clone 390, catalog no. 558737, DAB chromogen), and CD3 (BD Biosciences — Pharmingen; clone 15.5-2C11, catalog no. 550275, DAB chromogen) with hematoxylin as a counterstain.

Techniques: Control, Enzyme-linked Immunosorbent Assay, Expressing, Immunohistochemistry, Quantitative RT-PCR

Journal: mAbs

Article Title: Isolation and characterization of monoclonal antibodies against human carbonic anhydrase-IX

doi: 10.1080/19420862.2021.1999194

Figure Lengend Snippet: Binding of selected recombinant anti-CAIX antibodies by Surface Plasmon Resonance. Six (6) hybridoma derived mAbs were selected through screening (see , Suppl. Fig. S2A-C), sequenced, recloned in the appropriate IgG framework and recombinantly expressed in CHO cells (for details see text). (a) Purified recombinant antibodies (c11H9, c12H8, c2C7, m4A2, m9B6, c2D7) were captured with the appropriate anti-Fc surface (anti-human Fc: c11H9, c12H8, c2C7, c2D7; anti-mouse Fc: m4A2, m9B6). cG250 was used as a benchmark. Serial dilutions (0.74–60 nM) of rhCAIX monomer (CAIX-M) and dimer (CAIX-D) were then injected, followed by a buffer injection. Sensorgrams were aligned, double-referenced, and fitted to the 1:1 binding model to calculate ka, kd, KD and RUmax when flowing CAIX-M and apparent ka, kd, KD and RUmax when flowing CAIX-D (see ). (b) Graphs depicting changes in the calculated ka, kd, KD and RUmax when flowing rhCAIX-M versus rhCAIX-D over the immobilized antibodies

Article Snippet: Mouse monoclonal anti-human CAIX MAB2188 was obtained from R&D Systems (Cat# MAB2188), goat anti-human and goat anti-mouse AlexaFluor(AF)488-labeleled IgG-Fab2 were purchased from Jackson ImmunoResearch (Cat# 109–547-003 and 115–547-003, respectively), recombinant human (rh)CAIV (Cat# 510472-H08S), CAXII (Cat# 10617-H08H) and CAXIV (Cat# 10458H08H) and recombinant murine (rm; Cat# 50660-M08H) and dog (rd; Cat# 70028D08H) CAIX were purchased from Sino Biologics Inc., and cynomolgus CAIX was purchased from ACRO Biosystems (Cat# CA9-C83E6).

Techniques: Binding Assay, Recombinant, SPR Assay, Derivative Assay, Purification, Injection

Journal: mAbs

Article Title: Isolation and characterization of monoclonal antibodies against human carbonic anhydrase-IX

doi: 10.1080/19420862.2021.1999194

Figure Lengend Snippet: Representative kinetic and apparent kinetic values for binding of recombinant anti-CAIX antibodies obtained by SPR analysis and by flowing rhCAIX monomer or dimer, respectively. Antibodies suitable for ADC, CAIX enzyme inhibitor and imaging/detection are listed in green, blue and red, respectively. The cG250 mAb was used as control (black)

Article Snippet: Mouse monoclonal anti-human CAIX MAB2188 was obtained from R&D Systems (Cat# MAB2188), goat anti-human and goat anti-mouse AlexaFluor(AF)488-labeleled IgG-Fab2 were purchased from Jackson ImmunoResearch (Cat# 109–547-003 and 115–547-003, respectively), recombinant human (rh)CAIV (Cat# 510472-H08S), CAXII (Cat# 10617-H08H) and CAXIV (Cat# 10458H08H) and recombinant murine (rm; Cat# 50660-M08H) and dog (rd; Cat# 70028D08H) CAIX were purchased from Sino Biologics Inc., and cynomolgus CAIX was purchased from ACRO Biosystems (Cat# CA9-C83E6).

Techniques: Binding Assay, Recombinant, Imaging, Control

Journal: mAbs

Article Title: Isolation and characterization of monoclonal antibodies against human carbonic anhydrase-IX

doi: 10.1080/19420862.2021.1999194

Figure Lengend Snippet: Binding of recombinant antibodies to hCAIX-expressing SK-RC-52 cells. Dose-dependent binding (0–100 nM) to SK-RC-52 cells of recombinantly expressed (a) chimeric antibodies (c11H9, c12H8, c2C7, c2D7) and (b) murine antibodies (m4A2, m9B6), together with the appropriate negative (hIgG1, mIgG2) and positive (cG250, M75) controls. Experiments were carried out in duplicate and repeated twice. Representative KD, Hill slope and Bmax (see ) values were calculated using Graphpad Prism v8

Article Snippet: Mouse monoclonal anti-human CAIX MAB2188 was obtained from R&D Systems (Cat# MAB2188), goat anti-human and goat anti-mouse AlexaFluor(AF)488-labeleled IgG-Fab2 were purchased from Jackson ImmunoResearch (Cat# 109–547-003 and 115–547-003, respectively), recombinant human (rh)CAIV (Cat# 510472-H08S), CAXII (Cat# 10617-H08H) and CAXIV (Cat# 10458H08H) and recombinant murine (rm; Cat# 50660-M08H) and dog (rd; Cat# 70028D08H) CAIX were purchased from Sino Biologics Inc., and cynomolgus CAIX was purchased from ACRO Biosystems (Cat# CA9-C83E6).

Techniques: Binding Assay, Recombinant, Expressing

Journal: mAbs

Article Title: Isolation and characterization of monoclonal antibodies against human carbonic anhydrase-IX

doi: 10.1080/19420862.2021.1999194

Figure Lengend Snippet: Apparent equilibrium binding constants (EC50) and maximum specific binding (Bmax) values for the recombinant antibodies using SK-RC-52 cells expressing hCA-IX. cG250 and M75(both in black) were used as positive controls; negative controls (not listed) showed no binding (ADC candidates, green; enzyme inhibitors, blue and imaging/detection antibody, red)

Article Snippet: Mouse monoclonal anti-human CAIX MAB2188 was obtained from R&D Systems (Cat# MAB2188), goat anti-human and goat anti-mouse AlexaFluor(AF)488-labeleled IgG-Fab2 were purchased from Jackson ImmunoResearch (Cat# 109–547-003 and 115–547-003, respectively), recombinant human (rh)CAIV (Cat# 510472-H08S), CAXII (Cat# 10617-H08H) and CAXIV (Cat# 10458H08H) and recombinant murine (rm; Cat# 50660-M08H) and dog (rd; Cat# 70028D08H) CAIX were purchased from Sino Biologics Inc., and cynomolgus CAIX was purchased from ACRO Biosystems (Cat# CA9-C83E6).

Techniques: Binding Assay, Recombinant, Expressing, Imaging

Journal: mAbs

Article Title: Isolation and characterization of monoclonal antibodies against human carbonic anhydrase-IX

doi: 10.1080/19420862.2021.1999194

Figure Lengend Snippet: Assessment of recombinant anti-hCAIX antibodies as tools for immunohistochemistry and in vivo imaging applications. (a) Immunohistochemical staining for expression of CAIX in FFPE tissue sections from PK-8 human PDAC xenografts using recombinant antibodies c11H9, m9B6 and c2D7. Commercial anti-CAIX mAb, M75, was used as benchmark-based positive control. Scale bar, 100 μm; inset, 20 μm. Anti-CAIX antibody 11H9 (b) and a (c) control antibody (IgG) were conjugated to the chelator pSCN-Bn-DTPA and radiolabelled with 111 In. Conjugates were administered to NODSCID IL2RKO mice bearing subcutaneous hCAIX-positive HT-29 colorectal cancer xenografts (100 mm 3 ). Uptake, accumulation and retention of the radiolabelled 11H9 and IgG control were monitored 24–168 h post-injection by SPECT/CT imaging (B, C; see also ) (t, tumor; l, lung; s, stomach; h, heart)

Article Snippet: Mouse monoclonal anti-human CAIX MAB2188 was obtained from R&D Systems (Cat# MAB2188), goat anti-human and goat anti-mouse AlexaFluor(AF)488-labeleled IgG-Fab2 were purchased from Jackson ImmunoResearch (Cat# 109–547-003 and 115–547-003, respectively), recombinant human (rh)CAIV (Cat# 510472-H08S), CAXII (Cat# 10617-H08H) and CAXIV (Cat# 10458H08H) and recombinant murine (rm; Cat# 50660-M08H) and dog (rd; Cat# 70028D08H) CAIX were purchased from Sino Biologics Inc., and cynomolgus CAIX was purchased from ACRO Biosystems (Cat# CA9-C83E6).

Techniques: Recombinant, Immunohistochemistry, In Vivo Imaging, Immunohistochemical staining, Staining, Expressing, Positive Control, Control, Injection, Single Photon Emission Computed Tomography, Imaging