Journal: Antibodies

Article Title: Hydrophilic Auristatin Glycoside Payload Enables Improved Antibody-Drug Conjugate Efficacy and Biocompatibility

doi: 10.3390/antib7020015

Figure Lengend Snippet: Chromatographic evaluation of ADC aggregation and hydrophilicity. ( a – f ) Aggregation was analyzed by size-exclusion chromatography (SEC) after two days’ storage at +4 °C (normal storage temperature) or +40 °C (temperature stress) in formulation buffer. ( a , b ) DAR = 8 trastuzumab MMAE ADC was moderately aggregated into HMWCs already at +4 °C in two days ( a ) and mostly (>95%) aggregated at +40 °C eluting as a broad peak between 7–12 mL ( b ). ( c , d ) Trastuzumab, the naked antibody control, was not aggregated at either +4 °C ( c ) or +40 °C ( d ) during storage and eluted at 12.1 mL. ( e , f ) DAR = 8 MMAU ADC was not aggregated at +4 °C eluting at 11.9 mL ( e ) and only 2% aggregated into high-molecular weight components (HMWCs) eluting at 7–8 mL at +40 °C ( f ). ( g , h ) Hydrophobic interaction chromatography (HIC) was performed for evaluating the relative hydrophilicity of DAR = 8 trastuzumab-MMAU ADC ( g ); and a standard mixture of the naked antibody trastuzumab as well as trastuzumab-MMAE ADCs with DAR = 1–8 constructed from reduced trastuzumab using standard MC-Val-Cit-PABC linker ( h ). The method separated the molecules in the order of relative hydrophobicity, with the most hydrophilic component (naked antibody) eluting first and the most hydrophobic component (DAR = 8 ADC) eluting last. DAR = 8 MMAU ADC eluted at position corresponding to DAR = 3–4 MMAE ADC. Detection was by absorbance at 280 nm. drug, MMAE; G, β- D -glucuronic acid glycoside.

Article Snippet: For analysis of the drug-to-antibody ratio (DAR), 30 μg sample of each ADC was digested with 34 U FabRICATOR enzyme (Genovis, Lund, Sweden) at +37 °C for 2 h, purified with Poros R1 tips (ThermoFisher Scientific, Vantaa, Finland) and analyzed by MALDI-TOF mass spectrometry using sinapinic acid matrix.

Techniques: Size-exclusion Chromatography, Formulation, Control, High Molecular Weight, Hydrophobic Interaction Chromatography, Construct

Journal: Antibodies

Article Title: Hydrophilic Auristatin Glycoside Payload Enables Improved Antibody-Drug Conjugate Efficacy and Biocompatibility

doi: 10.3390/antib7020015

Figure Lengend Snippet: In vitro cytotoxicity of anti-HER2 ADCs against HER2-expressing cancer cell lines. ( a ) For high copy number HER2-expressing cells, both anti-HER2 DAR = 8 MMAU ADC and DAR = 2 duostatin-3 ADC showed high cytotoxic activity, while both anti-HER2 naked antibody and an isotype IgG control antibody-MMAU conjugate showed only no or low cytotoxicity, respectively. All experiments were performed in triplicate except for anti-HER2 naked antibody; ( b ) For low copy number HER2-expressing cells, the anti-HER2 DAR = 8 MMAU ADC had superior activity compared with DAR = 2 duostatin-3 ADC. Both experiments were performed in triplicate; ( c ) To demonstrate bystander kill activity of MMAU ADCs, non-HER2-expressing Jurkat cells were co-cultured with HCC1954 cells that express HER2, and viability of Jurkat cells was measured after incubation with fluorescence-assisted cell sorting (FACS)-based assay. The anti-HER2 DAR = 8 MMAU ADC showed bystander kill activity, while neither the anti-HER2 DAR = 2 duostatin-3 ADC nor the free MMAU payload had cytotoxic activity towards Jurkat cells. The results are from a single representative experiment. IC 50 values are marked in the figure panels and they were determined using curve fitting by nonlinear regression as the concentration of the drug that causes 50% inhibition of cell viability compared to maximum inhibition. Error bars show the standard error of the mean, where applicable.

Article Snippet: For analysis of the drug-to-antibody ratio (DAR), 30 μg sample of each ADC was digested with 34 U FabRICATOR enzyme (Genovis, Lund, Sweden) at +37 °C for 2 h, purified with Poros R1 tips (ThermoFisher Scientific, Vantaa, Finland) and analyzed by MALDI-TOF mass spectrometry using sinapinic acid matrix.

Techniques: In Vitro, Expressing, Activity Assay, Control, Low Copy Number, Cell Culture, Incubation, Fluorescence, FACS, Concentration Assay, Inhibition

Journal: Antibodies

Article Title: Hydrophilic Auristatin Glycoside Payload Enables Improved Antibody-Drug Conjugate Efficacy and Biocompatibility

doi: 10.3390/antib7020015

Figure Lengend Snippet: In vivo experiments. ( a , b ) EGFR + HSC-2 tumor xenografts in mice were effectively treated with anti-EGFR DAR = 8 MMAU ADCs based on cetuximab. Mice were inoculated with tumor cells and treatment was given intravenously either with ( a ) 10 mg/kg or ( b ) 3 mg/kg ADC once weekly for four weeks (arrows). With the larger dose ( a ) the tumors shrunk permanently (follow-up of 100 days) and with the lower dose ( b ) the growth of the tumors was arrested in all but one mice (recurrence after 80 days). There were three mice in each treatment group. Error bars show the standard error of the mean. ( c ) Trastuzumab-MMAU ADCs (T-MMAU) were given to nude mice as single 10 mg/kg i.v. injection. DAR = 8 MMAU ADC showed 31% smaller area under curve (AUC) than DAR = 4 MMAU ADC. There were three mice in each treatment group. Error bars show the standard deviation.

Article Snippet: For analysis of the drug-to-antibody ratio (DAR), 30 μg sample of each ADC was digested with 34 U FabRICATOR enzyme (Genovis, Lund, Sweden) at +37 °C for 2 h, purified with Poros R1 tips (ThermoFisher Scientific, Vantaa, Finland) and analyzed by MALDI-TOF mass spectrometry using sinapinic acid matrix.

Techniques: In Vivo, Injection, Standard Deviation

Journal: Antibodies

Article Title: Hydrophilic Auristatin Glycoside Payload Enables Improved Antibody-Drug Conjugate Efficacy and Biocompatibility

doi: 10.3390/antib7020015

Figure Lengend Snippet: In vitro 3D patient-derived xenograft (PDX) model. ( a ) GXA3067 is a HER2 overexpressing gastric tumor PDX model; ( b ) Size-filtered tumoroids were seeded in 384-well plates and treated with increasing concentrations of ADCs, the trastuzumab-based DAR = 8 MMAU ADC (T-MMAU ADC, upper row) and T-DM1 (Kadcyla ® , middle row); as well as the naked antibody trastuzumab (bottom row). ( c ) The DAR = 8 MMAU ADC was 10× more effective in reducing tumoroid size than T-DM1, while trastuzumab had only a moderate effect. ( d ) The DAR = 8 MMAU ADC was 18× more effective in inducing apoptosis than T-DM1, while trastuzumab did not have an effect. IC 50 values are marked in the figure panels and they were determined using curve fitting by nonlinear regression as the concentration of the drug that causes 50% inhibition of cell viability compared to maximum inhibition. The experiments were made with eight replicates. Error bars show the standard error of the mean.

Article Snippet: For analysis of the drug-to-antibody ratio (DAR), 30 μg sample of each ADC was digested with 34 U FabRICATOR enzyme (Genovis, Lund, Sweden) at +37 °C for 2 h, purified with Poros R1 tips (ThermoFisher Scientific, Vantaa, Finland) and analyzed by MALDI-TOF mass spectrometry using sinapinic acid matrix.

Techniques: In Vitro, Derivative Assay, Concentration Assay, Inhibition