Journal: Molecular Cancer Therapeutics

Article Title: Targeted Nanofitin-drug Conjugates Achieve Efficient Tumor Delivery and Therapeutic Effect in an EGFR pos Mouse Xenograft Model

doi: 10.1158/1535-7163.MCT-22-0805

Figure Lengend Snippet: Intratumoral infiltration 90 minutres after systemic administration. A, Intratumoral infiltration of antiEGFR Nanofitins or Cetuximab, revealed by anti-HA and anti-IgG IHC, respectively. Host vasculature is revealed by anti-CD31 staining of consecutive slice sections. Zoom of selected regions illustrates EGFR labeling at the vessel proximity. B, Labeling index, on the basis of cells positively labeled, in the whole tumor. C, Labeling index relative to the distance from the closest blood vessel. ****, P < 0.0001; ***, P < 0.0005.

Article Snippet: Affinities were also determined for cysteine-free and HA-tagged ( ) Nanofitins (500, 250, 125, 62.5, 31.25, 15.63, 7.81, and 0 nmol/L), either on human EGFR as described above, or on murine EGFR by using Recombinant Mouse EGFR Fc chimera protein (1280-ER, R&D Systems) for the loading step.

Techniques: Staining, Labeling

Journal: Molecular Cancer Therapeutics

Article Title: Targeted Nanofitin-drug Conjugates Achieve Efficient Tumor Delivery and Therapeutic Effect in an EGFR pos Mouse Xenograft Model

doi: 10.1158/1535-7163.MCT-22-0805

Figure Lengend Snippet: Biochemical profiles of Nanofitin-drug conjugates. A, Schematic representation of a Nanofitin-drug conjugate. The single chain of the Nanofitin scaffold (rainbow cartoon) is engineered to target EGFR by randomizing up to 14 amino acids (spheres in lieu of carbon alpha). Each Nanofitin is genetically fused to a C-terminal cysteine (gray/yellow stick) to allow the regioselective chemistry on the only thiol group. The vc-MMAE payload (structural formula) is coupled via its maleimide-based moiety (black) and releases the MMAE toxin (red) after proteolytic cleavage of the valine-citrulline linker (orange). B, UPLC-RP/MS profiles. Peaks were identified by ESI-MS spectral deconvolution to determine their mass. Percentages of corresponding species were determined from the area under the absorbance curves. C, Determination of the binding characteristics of the antiEGFR Nanofitin-drug conjugates D8-vc-MMAE (left) and B10-vc-MMAE (right) by biolayer interferometry on human EGFR, using the antiEGFR Nanofitin at concentrations of 500, 125, 31.25, and 7.81 nmol/L. Fittings are represented as solid red lines.

Article Snippet: Affinities were also determined for cysteine-free and HA-tagged ( ) Nanofitins (500, 250, 125, 62.5, 31.25, 15.63, 7.81, and 0 nmol/L), either on human EGFR as described above, or on murine EGFR by using Recombinant Mouse EGFR Fc chimera protein (1280-ER, R&D Systems) for the loading step.

Techniques: Binding Assay

Journal: Molecular Cancer Therapeutics

Article Title: Targeted Nanofitin-drug Conjugates Achieve Efficient Tumor Delivery and Therapeutic Effect in an EGFR pos Mouse Xenograft Model

doi: 10.1158/1535-7163.MCT-22-0805

Figure Lengend Snippet: Affinity determination against human and mouse EGFR.

Article Snippet: Affinities were also determined for cysteine-free and HA-tagged ( ) Nanofitins (500, 250, 125, 62.5, 31.25, 15.63, 7.81, and 0 nmol/L), either on human EGFR as described above, or on murine EGFR by using Recombinant Mouse EGFR Fc chimera protein (1280-ER, R&D Systems) for the loading step.

Techniques:

Journal: Chemistry of Materials

Article Title: Nanobodies and Antibodies for Duplexed EGFR/HER2 Immunoassays Using Terbium-to-Quantum Dot FRET

doi: 10.1021/acs.chemmater.6b03198

Figure Lengend Snippet: Figure 2. Representative normalized PL intensity decay curves (measured at 659 ± 10 nm) of AB−QD and Tb−AB conjugates mixed with increasing concentrations of EGFR (0 nM, black; 0.9 nM, red; 2.25 nM, blue; 4.5 nM, orange; and 9 nM, green) for the Tb-Cet/Mat-QD650 (A), Tb-Cet/MatFab-QD650 (B), and Tb-EgA1/EgB4-QD650 (C) FRET pairs. Yellow arrows indicate QD FRET sensitization with increasing EGFR concentration. The magenta curves present the mathematical sum of only AB-QD650 and only the Tb−AB conjugate. Although the optical bandpass filters (cf. Figure 1B) were selected to minimize Tb crosstalk into the QD detection channels, there was still significant Tb PL detectable. For intensity normalization, all curves within one graph where multiplied by the value that led to unity intensity for the magenta curve at 0.5 ms. PL decay curves (Tb donors and QD acceptors) of all immunoassays can be found in the Supporting Information (Supporting Figures S1−S8).

Article Snippet: Recombinant human EGFR Fc chimera (#344-ER-050) and HER2 Fc chimera (#1129-ER-050) dimers were purchased from R&D system.

Techniques: Concentration Assay

Journal: Chemistry of Materials

Article Title: Nanobodies and Antibodies for Duplexed EGFR/HER2 Immunoassays Using Terbium-to-Quantum Dot FRET

doi: 10.1021/acs.chemmater.6b03198

Figure Lengend Snippet: Figure 3. Homogeneous FRET immunoassay calibration curves against EGFR using different Tb−AB and AB−QD conjugates (blue, Tb-Cet/MatFab-QD605; green, Tb-Cet/MatFab-QD650; cyan, Tb- Cet/Mat-QD605; black, Tb-Mat/Cet-QD650; red, Tb-Cet/Mat- QD650; orange, Tb-EgA1/EgB4-QD650; brown, Tb-EgA1/EgB4- QD605; magenta, Tb-Cet/MatFab-QD650 measured in serum samples). [EGFR] corresponds to the variable EGFR dimer concentrations (recombinant human EGFR Fc chimera) in the 50 μL EGFR samples (in buffer or serum), whereas the overall measuring volume of 150 μL also contained 100 μL of a constant assay solution (50 μL of Tb−AB conjugate with 9 nM AB and 50 μL of AB−QD conjugate with 1.5 nM of QD650 or 3 nM of QD605 for all samples). Individual curves for the Tb and QD signals (that lead to the calculation of FR) and for the determination of LODs can be found in the Supporting Information (Supporting Figures S11−S18).

Article Snippet: Recombinant human EGFR Fc chimera (#344-ER-050) and HER2 Fc chimera (#1129-ER-050) dimers were purchased from R&D system.

Techniques: Recombinant

Journal: Chemistry of Materials

Article Title: Nanobodies and Antibodies for Duplexed EGFR/HER2 Immunoassays Using Terbium-to-Quantum Dot FRET

doi: 10.1021/acs.chemmater.6b03198

Figure Lengend Snippet: Figure 5. Specificity of the different AB pairs for EGFR (red) or HER2 (blue). Tb-Cet/Mat-QD650 (A) and Tb-Tras/Pert-QD650 (B) showed a concentration-dependent FRET-ratio increase only for their respective receptors. Tb-Pert/Mat-QD650 (C) and Tb-Tras/Mat-QD650 (D) showed a concentration-dependent FRET-ratio increase for HER2, which indicates a cross-reactivity of the EGFR-specific Mat to HER2. Tb-Cet/Pert-QD650 did not lead to any concentration dependent FRET-ratio increase (Supporting Figure S27).

Article Snippet: Recombinant human EGFR Fc chimera (#344-ER-050) and HER2 Fc chimera (#1129-ER-050) dimers were purchased from R&D system.

Techniques: Concentration Assay

Journal: Chemistry of Materials

Article Title: Nanobodies and Antibodies for Duplexed EGFR/HER2 Immunoassays Using Terbium-to-Quantum Dot FRET

doi: 10.1021/acs.chemmater.6b03198

Figure Lengend Snippet: Figure 6. Duplexed immunoassay (all samples containing Tb-Cet, Tb-Tras, Mat-QD605, and Pert-QD650) calibration curves of EGFR (FRET-ratio of QD605 and Tb PL intensities, A) and HER2 (FRET-ratio of QD650 and Tb PL intensities, B) for increasing (0 nM, black; 0.3 nM, green; 0.6 nM, magenta; 1.2 nM, red; 1.8 nM, cyan; 2.4 nM, brown; 3 nM, blue) HER2 (A) and EGFR (B) concentrations.

Article Snippet: Recombinant human EGFR Fc chimera (#344-ER-050) and HER2 Fc chimera (#1129-ER-050) dimers were purchased from R&D system.

Techniques:

Journal: Chemistry of Materials

Article Title: Nanobodies and Antibodies for Duplexed EGFR/HER2 Immunoassays Using Terbium-to-Quantum Dot FRET

doi: 10.1021/acs.chemmater.6b03198

Figure Lengend Snippet: Figure 7. Duplexed FRET immunoassay for the quantification of EGFR (blue squares) and HER2 (red dots) within 19 different samples. Dotted lines represent the known concentrations, and data points represent the measured concentrations (±10% error bars).

Article Snippet: Recombinant human EGFR Fc chimera (#344-ER-050) and HER2 Fc chimera (#1129-ER-050) dimers were purchased from R&D system.

Techniques:

Journal: Biomedicines

Article Title: A Rational Designed Novel Bispecific Antibody for the Treatment of GBM

doi: 10.3390/biomedicines9060640

Figure Lengend Snippet: ( a ) EGFRvIII antigen expression level on mutant GBM cell line U87MG.ΔEGFR and wild-type GBM cell line U87MG. ( b ) A binding activity comparison of EGFRvIII-BsAb and CD3 mAb with Jurkat cells (CD3-positive) (upper), as well as a binding activity comparison of the EGFRvIII-BsAb and the EGFRvIII mAb with U87MG.ΔEGFR cells (EGFRvIII-positive) (lower). ( c ) Photographs of the redirection of T cells to cancer cells by 0.01 ng/mL EGFRvIII-BsAb or EGFRvIII mAb. ( d ) FACS analysis of the redirection of CD3+ Jurkat cells to cancer cells by EGFRvIII-BsAb. Jurkat (CD3+) cells labeled by PKH26 (PE-A), as well as U87MG.ΔEGFR cells labeled by CFSE (FITC-A).

Article Snippet: The EGFRvIII antigen (AVI10494; R&D System, Minneapolis, MN, USA) and extracellular domain of human CD3D/CD3E heterodimer (CT038-H2508H; Sino Biological, Beijing, China) were immobilized to a CM5 chip (29149603; GE Healthcare, Chicago, IL, USA) surface using standard protocols with 1-ethyl-3 (3-dimethylaminopropyl) carbodiimide (EDC)/N-hydroxysuccinimide (NHS) amine.

Techniques: Expressing, Mutagenesis, Binding Assay, Activity Assay, Comparison, Labeling

Journal: Biomolecules

Article Title: A Multispecific Checkpoint Inhibitor Nanofitin with a Fast Tumor Accumulation Property and Anti-Tumor Activity in Immune Competent Mice.

doi: 10.3390/biom15040471

Figure Lengend Snippet: Figure 1. Cell labeling efficiency of the bispecific B10–B11 Nanofitin and its cytotoxic effect on the A431 on tumor cell line. (A) Cell labeling efficiency evaluation by flow cytometry of B10, B11, and bispecific B10–B11 Nanofitins on A431 cell line. Dotted lines represent alignment with the isotype control. (B) Expression level of programmed death-ligand 1 (PD-L1) (left) and Epithelial Growth Factor Receptor (EGFR) (right) on the A431 cell line. In grey: isotype control; in black: anti-PD-L1 or anti-EGFR antibody. (C) The real-time proliferation of A431 cells co-cultured with Jurkat cells exhibiting background activity. In black: A431 cells co-cultured with a mix of Jurkat cells and anti-EpCAM x CD3 BiTE without Nanofitin treatment; in red: A431 cells co-cultured with a mix of Jurkat cells and anti-EpCAM x CD3 BiTE and treated by the B11 Nanofitin (10 µM); in purple: A431 cells co-cultured with a mix of Jurkat cells and anti-EpCAM x CD3 BiTE and treated by the B10–B11 Nanofitin (10 µM). Significance was assessed by two-way ANOVA using 6 Prism software (GraphPad). **** p < 0.0001 (n = 3). (D) The real-time proliferation of A431 cells. In black: A431 cells alone; in red: A431 cells treated by the B11 Nanofitin (10 µM); in purple: A431 cells treated by the B10–B11 Nanofitin (10 µM). Significance was assessed by two-way ANOVA using 6 Prism software (GraphPad). NS: not significant (n = 3).

Article Snippet: The binding kinetic parameters of Nanofitins were defined by loading of 10 μg/mL mouse EGFR Fc chimera protein (#1280-ER-050, R&D Systems, Minneapolis, MN, USA) and 5 μg/mL recombinant mouse PDL1/B7-H1 Fc chimera protein (#1019-B7-100, R&D Systems) at 2 nm on protein A biosensors (#18-5012, Sartorius).

Techniques: Labeling, Flow Cytometry, Control, Expressing, Cell Culture, Activity Assay, Software

Journal: Biomolecules

Article Title: A Multispecific Checkpoint Inhibitor Nanofitin with a Fast Tumor Accumulation Property and Anti-Tumor Activity in Immune Competent Mice.

doi: 10.3390/biom15040471

Figure Lengend Snippet: Figure 3. Mouse cross-reactivity of the bispecific B10–B11 Nanofitin. (A) Expression level of PD-L1 (left) and EGFR (right) on CT26 cell line. In grey: isotype control; in black: anti-PD-L1 or EGFR antibody. (B) Biolayer interferometry sensorgrams and binding kinetic parameters of monomeric B10 and B11 Nanofitins and bispecific B10–B11 Nanofitin on mouse EGFR and PD-L1. Fittings (1:1 model) are represented as solid gray lines. (C) CT26 cell labeling efficiency evaluation by flow cytometry of monomeric B10 and B11 and bispecific B10–B11 Nanofitins. Dotted lines represent alignment with the isotype control.

Article Snippet: The binding kinetic parameters of Nanofitins were defined by loading of 10 μg/mL mouse EGFR Fc chimera protein (#1280-ER-050, R&D Systems, Minneapolis, MN, USA) and 5 μg/mL recombinant mouse PDL1/B7-H1 Fc chimera protein (#1019-B7-100, R&D Systems) at 2 nm on protein A biosensors (#18-5012, Sartorius).

Techniques: Expressing, Control, Binding Assay, Labeling, Flow Cytometry

Journal: Journal for Immunotherapy of Cancer

Article Title: Metabolic reprogramming via an engineered PGC-1α improves human chimeric antigen receptor T-cell therapy against solid tumors

doi: 10.1136/jitc-2022-006522

Figure Lengend Snippet: hEGFR-targeted CARs with 4-1BB co-stimulatory domains exhibit suppressed mitochondrial mass and exhaustion in the tumor microenvironment. (A) Structure of the anti-EGFR CAR that was used for the experiments, (B) in vitro killing assay of EGFR-specific CAR-T cells incubated overnight with A549 cells at the reported concentrations, (C) 10 7 hEGFR-targeted CARs were injected in NSG mice carrying 100 mm 3 tumors, (D) MitoTracker FM staining of CAR-T cells in tumor infiltrating lymphocyte (TIL) preparations from A549 tumors and respective spleens, (E) PD-1, Tim-3, and Lag-3 staining in CAR-T cells infiltrating A549 tumors and the respective spleens. (F) Cytokine production of TIL after 6 hours of ex vivo restimulation with phorbol 12-myristate 13-acetate and ionomycin. All experiments were repeated with three different donors. Plot B is representative of three different experiments and plots C–E contain all data from all donors. Each dot represents one mouse. Statistics are Wilcoxon matched-pairs signed-rank test. *p<0.05, **p<0.01. CAR, chimeric antigen receptors; IFN, interferon; PD-1, programmed cell death protein-1; TNF, tumor necrosis factor; ACT, adoptive cell therapy

Article Snippet: EGFR protein conjugated to Allophycocyanin was obtained by CREATIVE BIOMART (EGFR-692HA).

Techniques: In Vitro, Incubation, Injection, Staining, Ex Vivo

Journal: Journal for Immunotherapy of Cancer

Article Title: Metabolic reprogramming via an engineered PGC-1α improves human chimeric antigen receptor T-cell therapy against solid tumors

doi: 10.1136/jitc-2022-006522

Figure Lengend Snippet: Anti-EGFR PGC-1α S571A CAR-T cells, but not NT-PGC1α CAR-T cells, exhibit significant antitumor efficacy in vivo driven by increased cytokine production and memory formation. (A, B) Tumor growth curve and survival of A549-bearing NSG mice treated with 10 7 anti-EGFR PGC-1α S571A CAR-T cells when tumors reached 100 mm 3 (arrow). (C) Tumor growth curve as in A but using 10 7 anti-EGFR NT-PGC-1α CAR-T cells. (D) MitoTracker FM staining of TIL from tumors treated with unmodified or PGC-1α S571A CAR-T cells. (E) PD-1 and Tim-3 staining from TIL, (F) Cytokine from TIL after 6 hours ex vivo restimulation with phorbol 12-myristate 13-acetate and ionomycin, (G) Memory markers of TIL from day 10. The PGC-1α S571A growth curve was repeated with two donors while the TIL analysis was performed once across multiple mice. The NT-PGC-1α growth curve was repeated with three donors. Statistics are repeated-measures analysis of variance (A, C), log-rank test (B), and Wilcoxon rank-sum test (D–G). *p<0.05, **p<0.01 ***p<0.001. CAR, chimeric antigen receptors; IFN, interferon; PD-1, programmed cell death protein-1; PGC-1α, PPAR gamma coactivator 1α; TIL, tumor-infiltrating lymphocyte; TNF, tumor necrosis factor.

Article Snippet: EGFR protein conjugated to Allophycocyanin was obtained by CREATIVE BIOMART (EGFR-692HA).

Techniques: In Vivo, Staining, Ex Vivo