Journal: Materials Today Bio

Article Title: Conjugation strategy shapes antitumor efficacy and enables dose-sparing in non-antibody protein nanoconjugates

doi: 10.1016/j.mtbio.2025.102698

Figure Lengend Snippet: Design of modular CXCR4 targeted protein nanocarriers . A) In silico 3D structure prediction of parental T22-GFP-H6 and its variants adapted for site-directed conjugation: T22-GFP-H6-SORT and THIO-T22-GFP-H6. The main physicochemical properties of each protein are indicated in their respective tables. Conjugation sites are highlighted for each protein: solvent-exposed lysines in T22-GFP-H6 (black), the LPTEGLE Sortase A recognition tag in T22-GFP-H6-SORT (blue), and a reactive cysteine in THIO-T22-GFP-H6 (green). In all cases the targeting ligand T22 is also highlighted in red. B) MALDI-TOF spectra of T22-GFP-H6, T22-GFP-H6-SORT and THIO-T22-GFP-H6 proteins, with determined full-length molecular weight.

Article Snippet: CXCR4 + Toledo cells (CRL-2631, ATCC) were cultured in 24-well plates in RPMI medium supplemented with 10 % fetal bovine serum (FBS), 10 mM L-glutamine, 100 U/mL penicillin and 10 mg/mL streptomycin (Gibco) at 37 °C and 5 % CO2.

Techniques: In Silico, Conjugation Assay, Solvent, Molecular Weight

Journal: Materials Today Bio

Article Title: Conjugation strategy shapes antitumor efficacy and enables dose-sparing in non-antibody protein nanoconjugates

doi: 10.1016/j.mtbio.2025.102698

Figure Lengend Snippet: In vivo biodistribution of T22-GFP-H6-MMAE, T22-GFP-H6-SORT-MMAE and THIO-T22-GFP-H6-MMAE in a SC CXCR4 + DLBCL mouse model. A) Experimental design of the in vivo study. B) Representative ex vivo images of the fluorescence (FLI) emitted in SC Toledo tumors and normal organs 48 h post-treatment in mice treated with buffer, T22-GFP-H6-MMAE (n = 3), T22-GFP-H6-SORT-MMAE (n = 3), or THIO-T22-GFP-H6-MMAE (n = 3). Results are expressed as average radiant efficiency [p/s/cm 2 /sr]/[μW/cm 2 ] ± SEM. C) FLI quantification in SC Toledo tumors and normal organs in nanoconjugate-treated mice 48 h post-injection. FLI values, expressed as radiant efficiency, were calculated by subtracting autofluorescence from corresponding tissues in buffer-treated mice (n = 9). A correction factor, derived from the FLI measurements of each nanoconjugate, was applied to all data. Unpaired t -test was used to determine significant differences: ∗p < 0.05.

Article Snippet: CXCR4 + Toledo cells (CRL-2631, ATCC) were cultured in 24-well plates in RPMI medium supplemented with 10 % fetal bovine serum (FBS), 10 mM L-glutamine, 100 U/mL penicillin and 10 mg/mL streptomycin (Gibco) at 37 °C and 5 % CO2.

Techniques: In Vivo, Ex Vivo, Fluorescence, Injection, Derivative Assay

Journal: Materials Today Bio

Article Title: Conjugation strategy shapes antitumor efficacy and enables dose-sparing in non-antibody protein nanoconjugates

doi: 10.1016/j.mtbio.2025.102698

Figure Lengend Snippet: CXCR4-binding and receptor-dependent internalization . A) Intracellular mean fluorescence intensity (MFI) of T22-GFP-H6-MMAE (GFP: grey), T22-GFP-H6-SORT-MMAE (SORT: blue) THIO-T22-GFP-H6-MMAE (THIO: green) and GFP-H6-MMAE (C: red) nanoconjugates upon incubation in Toledo cell line at 100 nM for 24h. B) Relative internalization of each nanoconjugate in absence (-AMD) or presence (+AMD) of the CXCR4 antagonist AMD3100. Internalization values were normalized from each nanoconjugate incubation at 100 nM for 1h. ∗∗: p < 0.01 C) Summary table with the dissociation constants (KD) of each protein comparing the effect of conjugation in each strategy. The fold change reflects the impact of MMAE conjugation (+MMAE) versus the unconjugated nanoparticle (-MMAE). KD values were determined experimentally using a saturation binding assay for T22-GFP-H6, T22-GFP-H6-SORT, and THIO-T22-GFP-H6 variants with and without MMAE.

Article Snippet: CXCR4 + Toledo cells (CRL-2631, ATCC) were cultured in 24-well plates in RPMI medium supplemented with 10 % fetal bovine serum (FBS), 10 mM L-glutamine, 100 U/mL penicillin and 10 mg/mL streptomycin (Gibco) at 37 °C and 5 % CO2.

Techniques: Binding Assay, Fluorescence, Incubation, Conjugation Assay, Saturation Assay

Journal: Materials Today Bio

Article Title: Conjugation strategy shapes antitumor efficacy and enables dose-sparing in non-antibody protein nanoconjugates

doi: 10.1016/j.mtbio.2025.102698

Figure Lengend Snippet: In vivo therapeutic efficacy of T22-GFP-H6-MMAE, T22-GFP-H6-SORT-MMAE and THIO-T22-GFP-H6-MMAE in a disseminated CXCR4 + AML mouse model. A) Representative images of AML cells dissemination in vehicle or nanoconjugate treated animals 15 days after THP-1-Luci cells injection. B) Follow up of total bioluminescence signal throughout the experiment until all vehicle-treated animals were euthanized. Results are expressed as total flux (photons/second; radiance photons) ± SEM. C) Cumulative BLI signal of the different treated groups during the study, calculated as the area under the curve of AML progression for each mouse. Results are presented as mean area in total flux units [p/s] ± SEM. Unpaired t -test was used to determine significant differences: ∗p < 0.05 and ∗∗p < 0.01.

Article Snippet: CXCR4 + Toledo cells (CRL-2631, ATCC) were cultured in 24-well plates in RPMI medium supplemented with 10 % fetal bovine serum (FBS), 10 mM L-glutamine, 100 U/mL penicillin and 10 mg/mL streptomycin (Gibco) at 37 °C and 5 % CO2.

Techniques: In Vivo, Drug discovery, Injection