Journal: bioRxiv

Article Title: A strategically designed homogeneous antibody-drug conjugate improves safety and therapeutic index in renal cell carcinoma

doi: 10.64898/2026.01.12.699160

Figure Lengend Snippet: Characterization of transduction of KIM-1 on RENCA cells using ( a ) Western blotting and ( b ) flow cytometry. The mean fluorescence intensity (MFI) was plotted in the bar graph. FITC conjugated anti-mouse KIM-1 antibody was used to probe the KIM-1 expression ( c ) The KIM-1 AFC internalization in Normal Renca and Renca KIM-1++ by flow cytometry. The histogram shows a notable increase in the cellular internalization both at 1 and 4 h. ( d ) The bar graph represents the MFI of AFC internalization in Renca and Renca KIM-1++ by flow cytometry. The data show a significant increase in cellular internalization. The data were represented as mean ± SEM (n = 3, two-way ANOVA followed by Tukey’s multiple comparison test).

Article Snippet: Mouse-KIM-1 antigen (SinoBiological, 50321-M16H) was biotinylated using Thermo Scientific EZ-LinkTM Micro Sulfo-NHS-LC-Biotinylation Kit.

Techniques: Transduction, Western Blot, Flow Cytometry, Fluorescence, Expressing, Comparison

Journal: bioRxiv

Article Title: A strategically designed homogeneous antibody-drug conjugate improves safety and therapeutic index in renal cell carcinoma

doi: 10.64898/2026.01.12.699160

Figure Lengend Snippet: ( a ) Median expression of the KIM-1 gene in different cancer and normal tissues obtained from the TCGA dataset using Gene Expression Profiling Interactive Analysis (GEPIA). ( b ) Representative images for immunostaining of kidney tissue microarray by anti-KIM-1 (red) antibody and DAPI (blue). Tissue specimens were collected from randomized patients based on age, sex, and stage of cancer. The normal adjacent tissue (NAT) and tumor tissue were collected from the same patients.

Article Snippet: 50 μl solution 2 μg/mL solution (100 ng) of mouse KIM-1 (SinoBiological, 50321-M16H) in PBS was coated on an ELISA plate (ELISA Max, Biolegend) and incubated overnight at 4 °C.

Techniques: Expressing, Gene Expression, Immunostaining, Microarray

Journal: bioRxiv

Article Title: A strategically designed homogeneous antibody-drug conjugate improves safety and therapeutic index in renal cell carcinoma

doi: 10.64898/2026.01.12.699160

Figure Lengend Snippet: ( a ) Median expression of the KIM-1 gene in different cancer and normal tissues obtained from the TCGA dataset using Gene Expression Profiling Interactive Analysis (GEPIA). Inset: The pictorial representation of the KIM-1 expression in cancer (red) and normal (green) organs. The figure was generated using the GEPIA platform. ( b ) Immunostaining of kidney tissue microarray by anti-KIM-1 (red) antibody, and DAPI (blue). Tissue specimens were collected from randomized patients based on age, sex, and stage of cancer. The normal adjacent tissue (NAT) and tumor tissue were collected from the same patients. The image shows the majority of the tumor expressing high KIM-1. 16 such images were combined to form a single circular tissue image. ( c ) Bar plot showing relative KIM-1 expression in cancer and normal adjacent tissue (NAT). Each data point represents one patient sample. The data were represented as mean ± SEM (n = 16, paired t-test, P < 0.01 **).

Article Snippet: 50 μl solution 2 μg/mL solution (100 ng) of mouse KIM-1 (SinoBiological, 50321-M16H) in PBS was coated on an ELISA plate (ELISA Max, Biolegend) and incubated overnight at 4 °C.

Techniques: Expressing, Gene Expression, Generated, Immunostaining, Microarray

Journal: bioRxiv

Article Title: A strategically designed homogeneous antibody-drug conjugate improves safety and therapeutic index in renal cell carcinoma

doi: 10.64898/2026.01.12.699160

Figure Lengend Snippet: ( a ) Schematic representation of the synthesis of KIM-1 targeting ADC, LT-025, by using transglutaminase. First, the native monoclonal KIM-1 antibody was deglycosylated in the presence of the PNGase F enzyme, and then the payload-linker was attached to the antibody at Q295 in the presence of the microbial transglutaminase enzyme (MTGase). The payload used was DM1. ( b ) Data shows the difference in the retention time of KIM-1 antibody, deglycosylated KIM-1 antibody, and ADC (LT-025) using hydrophobic interaction chromatography (HIC). Data was recorded under physiological conditions in PBS (pH 7.4) at 280 nm. 10 μg of each sample was injected for analysis. The chromatograms were baseline corrected using Origin 8.0 and plotted using GraphPad Prism 10 software. ( c ) Mass spectral analysis of the antibody drug conjugate. The data were recorded using ESI-MS after DTT reduction of the LT-025. Deconvoluted mass spectra of light chain and heavy chain, of KIM-1 antibody, deglycosylated KIM-1 antibody, and LT-025. 5 μg of each sample was injected for ESI-MS analysis. ( d ) The schematic shows the importance of the deglycosylation of the antibody for ADC production. The upper histogram represents the HIC chromatogram of the native antibody. The lower histogram is the product after the conjugation reaction without performing the deglycosylation step. The chromatogram shows the presence of unreacted antibody with some additional product, which does not match the desired LT-025 chromatogram. ( e ) The data represent the stability of the LT-025 for 15 days. The area under the curve from the HIC chromatogram of the ADC was normalized to day 1 and plotted with increasing days. 10 μL of 1 mg/mL of each sample was injected for analysis. The data shows no significant change in the peak area, indicating the stability of the ADC.

Article Snippet: 50 μl solution 2 μg/mL solution (100 ng) of mouse KIM-1 (SinoBiological, 50321-M16H) in PBS was coated on an ELISA plate (ELISA Max, Biolegend) and incubated overnight at 4 °C.

Techniques: Hydrophobic Interaction Chromatography, Injection, Software, Conjugation Assay

Journal: bioRxiv

Article Title: A strategically designed homogeneous antibody-drug conjugate improves safety and therapeutic index in renal cell carcinoma

doi: 10.64898/2026.01.12.699160

Figure Lengend Snippet: Calculation of DAR using UV-visible spectroscopy. (a) The absorption of spectra of LT-025, DM1 linker, and KIM-1 antibody are given. (b) The equation for the calculation of DAR. (c) Table showing the calculation of DAR using the equation by measuring the absorbance of the antibody and linker from the ADC. The extinction coefficients of the antibody and the linker were calculated by measuring the absorbance at known concentrations of antibody and linker. The absorption maxima of the linker and antibody were obtained as 254 nm, and 280 nm, respectively. The DAR was finally calculated by the ratio of C drug to C mAb. Notation ‘l’ in the equation corresponds to the path length, which was considered as 1 cm.

Article Snippet: 50 μl solution 2 μg/mL solution (100 ng) of mouse KIM-1 (SinoBiological, 50321-M16H) in PBS was coated on an ELISA plate (ELISA Max, Biolegend) and incubated overnight at 4 °C.

Techniques: Spectroscopy

Journal: bioRxiv

Article Title: A strategically designed homogeneous antibody-drug conjugate improves safety and therapeutic index in renal cell carcinoma

doi: 10.64898/2026.01.12.699160

Figure Lengend Snippet: ( a ) The antigen-binding capacity of LT-025, and the KIM-1 antibody was studied using ELISA. The immobilization of the LT-025 was checked in a KIM-1 protein-coated ELISA plate. The presence of the LT-025 was measured using an HRP-conjugated secondary antibody. The data shows a dose-dependent increase in the absorption, representing the successful antigen binding by the LT-025. The antigen recognition of the LT-025 shows a similar dose response curve to that of the monoclonal KIM-1 antibody, signifying no loss of the antigen-binding capability of the ADC. ( b ) The comparison of the binding of LT-025 alone with ADC in the presence of KIM-1 free antibody was studied using ELISA. The data shows a 50% reduction in the immobilization of both ADC in the presence of free antibody. ( c ) Quantitative estimation of the antigen binding of the LT-025 using Bio-layer interferometry (BLI). The data shows dose-dependent increase in the association and dissociation events. ( d ) Comparison of the antigen binding of LT-025 and KIM-1 antibody using BLI. 50 nM of each sample was used for the study. ( e ) Equilibrium constants and rate constants of ADC and the antibody (K D : Dissociation equilibrium constant, k a : association rate constant, and k dis : dissociation rate constant.

Article Snippet: 50 μl solution 2 μg/mL solution (100 ng) of mouse KIM-1 (SinoBiological, 50321-M16H) in PBS was coated on an ELISA plate (ELISA Max, Biolegend) and incubated overnight at 4 °C.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Comparison

Journal: bioRxiv

Article Title: A strategically designed homogeneous antibody-drug conjugate improves safety and therapeutic index in renal cell carcinoma

doi: 10.64898/2026.01.12.699160

Figure Lengend Snippet: (a) Schematic structure of KIM-1 Cy5 AFC and the structure of Cy5 linker (b) Characterization data of AFC using HIC. Method used for the HPLC is a gradient method from 100 % of 25 mM phosphate buffer pH 7 with 1.5 M ammonium sulphate to 100 % of 25 mM phosphate buffer pH 7 with 20 % isopropanol. The absorption was recorded at 280 nm and 649 nm (absorption maxima of Cy5 linker). The distinguished peak in the 649 nm chromatogram represents the presence of the Cy5 fluorophore in the AFC.

Article Snippet: 50 μl solution 2 μg/mL solution (100 ng) of mouse KIM-1 (SinoBiological, 50321-M16H) in PBS was coated on an ELISA plate (ELISA Max, Biolegend) and incubated overnight at 4 °C.

Techniques:

Journal: bioRxiv

Article Title: A strategically designed homogeneous antibody-drug conjugate improves safety and therapeutic index in renal cell carcinoma

doi: 10.64898/2026.01.12.699160

Figure Lengend Snippet: ( a ) Schematic representation of the synthesis of an antibody fluorophore conjugate (AFC) by using the MTGase reaction as described for the ADC. First, the native monoclonal antibody was deglycosylated in the presence of the PNGase F enzyme, and then the Cy5-fluorophore-linker was attached to the antibody at Q295 in the presence of the MTGase. ( b ) SDS-PAGE (fluorescence imaging and Coomassie staining) of KIM-1 antibody, deglycosylated antibody, LT-025, and Cy5-AFC. The data shows attachment of the Cy5 fluorophore in the heavy chain of the antibody. No conjugation of Cy5 was observed in the light chain. ( c ) Schematic representation of the cellular internalization of the ADC and AFC. The AFC first binds with the KIM-1 receptor on the cell surface, and it gets endocytosed into the cell and is transported to the lysosomal compartment. ( d ) Internalization study of Cy5 AFC at different time points. 50 μg/ml of AFC. An increase in MFI (Cy5) over time means increased binding/internalization of AFC onto the Renca RCC cell line. ( e ) Confocal microscopy images showing the internalization of the AFC in Renca cells. The images show successful cellular internalization of AFC (50 μg/ml) within 0.5 h and 4 h. An increased cellular internalization and colocalization at the lysosomal compartment were observed in 4 h. Scale bar = 5 μm.

Article Snippet: 50 μl solution 2 μg/mL solution (100 ng) of mouse KIM-1 (SinoBiological, 50321-M16H) in PBS was coated on an ELISA plate (ELISA Max, Biolegend) and incubated overnight at 4 °C.

Techniques: SDS Page, Fluorescence, Imaging, Staining, Conjugation Assay, Binding Assay, Confocal Microscopy

Journal: bioRxiv

Article Title: A strategically designed homogeneous antibody-drug conjugate improves safety and therapeutic index in renal cell carcinoma

doi: 10.64898/2026.01.12.699160

Figure Lengend Snippet: Characterization of transduction of KIM-1 on RENCA cells using ( a ) Western blotting and ( b ) flow cytometry. The mean fluorescence intensity (MFI) was plotted in the bar graph. FITC conjugated anti-mouse KIM-1 antibody was used to probe the KIM-1 expression ( c ) The KIM-1 AFC internalization in Normal Renca and Renca KIM-1++ by flow cytometry. The histogram shows a notable increase in the cellular internalization both at 1 and 4 h. ( d ) The bar graph represents the MFI of AFC internalization in Renca and Renca KIM-1++ by flow cytometry. The data show a significant increase in cellular internalization. The data were represented as mean ± SEM (n = 3, two-way ANOVA followed by Tukey’s multiple comparison test).

Article Snippet: 50 μl solution 2 μg/mL solution (100 ng) of mouse KIM-1 (SinoBiological, 50321-M16H) in PBS was coated on an ELISA plate (ELISA Max, Biolegend) and incubated overnight at 4 °C.

Techniques: Transduction, Western Blot, Flow Cytometry, Fluorescence, Expressing, Comparison

Journal: bioRxiv

Article Title: A strategically designed homogeneous antibody-drug conjugate improves safety and therapeutic index in renal cell carcinoma

doi: 10.64898/2026.01.12.699160

Figure Lengend Snippet: Cell viability and IC 50 of ADC on ( a ) normal Renca and ( b ) Renca KIM-1++, obtained by MTT assay. The LT-025 exhibits significant cell death with the IC 50 values in the nanomolar range. A lower IC 50 value was observed in Renca KIM-1++ than in normal Renca because of increased receptor-mediated cellular internalization of the ADC. The data were represented as mean ± SEM (n = 3). ( c ) Comparison of the cell viability of LT-025, free antibody, and the KIM-1 MMAE ADC by MTT assay. The data showed significantly higher cell death by LT-025 in comparison to free KIM-1 antibody and KIM-1 MMAE ADC. The data were represented as mean ± SEM (n = 3, two-way ANOVA followed by Tukey’s multiple comparison test). ( d ) Confocal images of Renca with and without treatment of LT-025 (100 μg/ml) displaying tubulin disruption. The images showed disruption of the tubulin (green) crosslinking by DM1, a known microtubule inhibitor. Sacale bar = 10 μm. ( e ) Quantification of tubulin in untreated and LT-025 treated cells from the confocal images. The mean pixel intensity of the tubulin (green) was plotted after normalizing with the mean pixel intensity of actin (red). The data were represented as mean ± SEM (n = 23, Unpaired t -test with Welch’s correction). ( f ) Quantification of the area of the control and treated Renca cells. The data showed a significant reduction in the area of the cells. The data were represented as mean ± SEM (n = 23, Unpaired t -test with Welch’s correction). ( g ) The bar plot showing the % of apoptotic cells measured with flow cytometry using Annexin-V AF645 apoptosis kit. The data represent significantly higher apoptosis in normal Renca by LT-025 compared to the control and sunitinib-treated group. (n = 3, one-way ANOVA followed by Tukey’s multiple comparison test). ( h ) The bar plot showing the % of apoptotic sunitinib-resistant Renca measured with flow cytometry using Annexin V-AF645 apoptosis kit. The data represent significantly higher apoptosis in sunitinib-resistant Renca by treatment with dual therapy (LT-025 + sunitinib) compared to the control and monotherapy. 12.5 μM of sunitinib and 100 nM of ADC were used for the study (n = 3, one-way ANOVA followed by Tukey’s multiple comparison test).

Article Snippet: 50 μl solution 2 μg/mL solution (100 ng) of mouse KIM-1 (SinoBiological, 50321-M16H) in PBS was coated on an ELISA plate (ELISA Max, Biolegend) and incubated overnight at 4 °C.

Techniques: MTT Assay, Comparison, Disruption, Control, Flow Cytometry