Journal: bioRxiv

Article Title: Development of a LRRC15-Targeted Radio-Immunotheranostic Approach to Deplete Pro-tumorigenic Mechanisms and Immunotherapy Resistance

doi: 10.1101/2024.01.30.577289

Figure Lengend Snippet: A. Flow cytometry demonstrates that DUNP19 binds with picomolar affinity to LRRC15 molecules on various cell lines, exhibiting distinct expression levels and tissue origins. Detection of LRRC15 was based on the assessment of antigen-antibody equilibrium. B. Analysis across a diverse range of cell lines unveils correlations between LRRC15 mRNA expression and the abundance of LRRC15 molecules bound by DUNP19. C. Confocal microscopy of various cell lines incubated with AlexaFluor647-labeled DUNP19 at room temperature, followed by staining for plasma membrane-associated calcium ATPase (PCMA) and DNA (DAPI). Images reveal that DUNP19 binding corresponds with LRRC15 expression levels and co-localizes with LRRC15. D. Internalization rates of AlexaFluor647-labeled DUNP19 at 37°C were examined in LRRC15-expressing HuO9 and SAOS2 cells using live confocal microscopy. The endocytic process of DUNP19 is accelerated in cells with higher LRRC15 abundance. E. Confocal microscopy of SAOS2 cells incubated with AlexaFluor647-labeled DUNP19 at 4°C or 37°C, co-stained for lysosomes (LAMP1) and DNA (DAPI). DUNP19 is exclusively found in the plasma membrane at the lower temperature demonstrating that the rapid endocytosis after binding to LRRC15 is an active, energy-requiring process. F. LigandTracer sensorgram of [ 177 Lu]-DUNP19 binding to LRRC15-expressing HuO9 cells, measured at 1 nM and 3 nM concentrations. Cell-bound activity, presented as CPS, was used to determine association, dissociation rates, and equilibrium dissociation constants (K D ). The table shows K D values for various LRRC15-expressing cell lines.

Article Snippet: To overexpress LRRC15, HEK293T and K7M2 cells were transduced with a pLenti-LRRC15-GFP-Puro vector (Origene, NM_130830) with a multiplicity of infection of 5.

Techniques: Flow Cytometry, Expressing, Confocal Microscopy, Incubation, Labeling, Staining, Membrane, Binding Assay, Activity Assay

Journal: bioRxiv

Article Title: Development of a LRRC15-Targeted Radio-Immunotheranostic Approach to Deplete Pro-tumorigenic Mechanisms and Immunotherapy Resistance

doi: 10.1101/2024.01.30.577289

Figure Lengend Snippet: A. Representative PET images of s.c. SAOS2 osteosarcoma xenografts obtained at different time points post i.v. administration of [ 64 Cu]-DUNP19, highlighting significant tumor-specific uptake with minimal accumulation in normal tissues. In contrast, PET with the clinical bone scanning agent [ 18 F]-NaF showed low activity in tumor tissue, with the majority of the tracer dose observed in bone (Bn) and bladder (Bl). B. In vivo assessment of LRRC15 targeting specificity by [ 177 Lu]-DUNP19. At 48 h post i.v. injection, [ 177 Lu]-DUNP19 displayed significantly higher uptake (p < 0.001) in LRRC15+ U118MG (blue bar) and HuO9 (red bar) tumors compared to LRRC15-LNCaP tumors (light gray bar). The accumulation of non-specific [ 177 Lu]-IgG1 in LRRC15+ U118MG tumors (dark grey bar) was significantly lower than that of [ 177 Lu]-DUNP19. C. [ 177 Lu]-DUNP19 tumor uptake in multiple s.c. tumor models at 72 h p.i., correlating with the LRRC15 expression level in the respective model. D. + E . Kinetics of [ 177 Lu]-DUNP19 in healthy organs and LRRC15+ SAOS2 and HuO9 osteosarcoma lesions. Ex vivo tissue biodistributions of [ 177 Lu]-DUNP19 obtained at multiple time points after i.v. injection showed a continuous decline in activity levels in healthy organs, but sustained uptake by malignant lesions. F. + G. Microanatomy of tumor tissues obtained from animals treated with fluorescently labeled DUNP19. Confocal images of s.c. SAOS2 (LRRC15+ cancer cells / LRRC15+ CAF) and HCC1954 (LRRC15-cancer cells / LRRC15+ CAF) tumors harvested at 72 h post-i.v. injection of AF594-DUNP19 (yellow). Tumor sections were co-stained for Actin (red), DNA (DAPI, blue) and LAMP1 (lysosomal marker, green). Images show that DUNP19 accumulates in the cellular cytoplasm and co-localized with LAMP1 indicating intracellular trafficking of the mAb to the lysosomal compartments (arrow) after binding membranous LRRC15.

Article Snippet: To overexpress LRRC15, HEK293T and K7M2 cells were transduced with a pLenti-LRRC15-GFP-Puro vector (Origene, NM_130830) with a multiplicity of infection of 5.

Techniques: Activity Assay, In Vivo, Injection, Expressing, Ex Vivo, Labeling, Staining, Marker, Binding Assay

Journal: bioRxiv

Article Title: Development of a LRRC15-Targeted Radio-Immunotheranostic Approach to Deplete Pro-tumorigenic Mechanisms and Immunotherapy Resistance

doi: 10.1101/2024.01.30.577289

Figure Lengend Snippet: A I-II. Tumor volumes in mice bearing s.c. HuO9 xenografts (n=10 per arm) were randomized for a single i.v. administration of 30 MBq [ 177 Lu]-DUNP19 when tumors reached 203±64 mm3 (blue line; day 21) or 504±152 mm3 (red line; day 39) or received no treatment (black line). The results demonstrated a significant delay in disease progression, with treatment efficacy being influenced by tumor volume ( A I ). Kaplan-Meier survival analysis revealed that [ 177 Lu]-DUNP19 extended survival, with the impact varying based on the timing of intervention ( A II ). B I-II . Representative coronal SPECT/CT images showing orthotopic HuO9 osteosarcoma tumors (indicated by arrow) after initial (left) and follow-up (right) i.v. administrations of 20 MBq [ 177 Lu]-DUNP19. In all treated mice (right) (n=10), no tumor associated uptake was observed at 163 days after treatment (B I ) . Kaplan-Meier survival analysis revealed a significant increase in survival for the [ 177 Lu]-DUNP19 treated group during the observed 190-days period ( B II ). C I-V . Mice with s.c. HuO9 osteosarcoma xenografts (n=12 per arm) were randomized for three treatment cycles (red: 10+20+10 MBq; blue: 20+10+20 MBq) of i.v. [ 177 Lu]-DUNP19 (at day 0, 32, and 75), resulting in a total administered activity of 50 MBq, or no treatment (black; n=12). Assessment of tumor volumes demonstrated that repeated cycles of LRRC15-RIT effectively inhibit tumor growth ( C I ). Kaplan-Meier survival analysis confirmed significantly improved survival for mice randomized for [ 177 Lu]-DUNP19 over no treatment ( C II ). Four tumors from the treatment and control (non-treatment) arm, harvested 72 hours after administration of an imaging dose of [ 177 Lu]-DUNP19 (3 MBq), were imaged ex vivo by SPECT and CT ( C III ). Tissue activity levels (%IA/g), assessed by gamma counter and normalized to tissue weight, revealed significantly lower uptake of the antibody in treated vs. non-treated tumors (p < 0.001), reflecting reduction in total LRRC15-expressing cells post-treatment ( C IV ). Quantification of radiopacity in CT images showed significantly higher ossification levels in treated vs. non-treated tumor tissues (p < 0.001) ( C V ). Together, these findings illustrate that repeated cycles of [ 177 Lu]-DUNP19 effectively reduce tissue viability and calcification.

Article Snippet: To overexpress LRRC15, HEK293T and K7M2 cells were transduced with a pLenti-LRRC15-GFP-Puro vector (Origene, NM_130830) with a multiplicity of infection of 5.

Techniques: Single Photon Emission Computed Tomography, Activity Assay, Control, Imaging, Ex Vivo, Expressing

Journal: bioRxiv

Article Title: Development of a LRRC15-Targeted Radio-Immunotheranostic Approach to Deplete Pro-tumorigenic Mechanisms and Immunotherapy Resistance

doi: 10.1101/2024.01.30.577289

Figure Lengend Snippet: A, B. [ 177 Lu]-DUNP19 demonstrates antitumor activity in other cancer indications. BALB/c nude mice bearing s.c. U118MG glioblastoma xenografts (were treated with two fractions of [ 177 Lu]-DUNP19 at days 0 and 34 for a cumulative activity of 20 MBq (10+10 MBq, red, n=12) or 30 MBq (20+10 MBq, blue, n=11). Despite lower LRRC15 expression by U118MG tumors, treatment with [ 177 Lu]-DUNP19 significantly controlled tumor growth and prolonged survival in both [ 177 Lu]-DUNP19 doses (median survival; untreated = 74 days, 20 MBq = not reached, 30 MBq = not reached, p < 0.001). C, D. [ 177 Lu]-DUNP19 is effective in HCC1954 breast cancer models (LRRC15-cancer cells, LRRC15+ stroma). Results demonstrate delayed s.c. HCC1954 growth in female mice intravenously administered a single dose of [ 177 Lu]-DUNP19 (20 MBq; day 7, n=10). D. Median survival was not reached for the treated group by the end of the observation period (day 62), while median survival of treated mice was 30.5 days (p < 0.005).

Article Snippet: To overexpress LRRC15, HEK293T and K7M2 cells were transduced with a pLenti-LRRC15-GFP-Puro vector (Origene, NM_130830) with a multiplicity of infection of 5.

Techniques: Activity Assay, Expressing

Journal: bioRxiv

Article Title: Development of a LRRC15-Targeted Radio-Immunotheranostic Approach to Deplete Pro-tumorigenic Mechanisms and Immunotherapy Resistance

doi: 10.1101/2024.01.30.577289

Figure Lengend Snippet: DEGs overlapped in [ 177 Lu]-DUNP19-treated stroma from U118MG ( A , 26 genes), HuO9 ( B , 23 genes), and HCC1954 ( C , 26 genes) tumors. Relative expression (Z-score normalization) was plotted to indicate upregulated (red) or downregulated (blue) genes. D. Box- and-whisker plots representing relative transcript expression of LRRC15 (top) and TGFB1 (bottom), comparing untreated tumors to tumors after [ 177 Lu]-DUNP19 therapy. HuO9 transcripts are plotted in red (left), U118MG in blue (middle), and HCC1954 in black (right). Samples were separated by transcript signature based on PCA plots and hierarchical clustering (Supp. Fig. 7) into 2 (HCC1954) or 3 (U118MG, HuO9) clusters. Expression of LRRC15 and TGFB1 in treated samples from cluster 3 are significantly (p<0.005) decreased in U118MG and HuO9, while no changes are observed in the LRRC15-HCC1954 cancer cells. E, F. Transcript data from clustered (Supp. Fig. 7) cancer cells (E) or tumor stroma (F) show decreased expression of the LRRC15+ TGFβ signature. E. Untreated U118MG (top) and HuO9 (middle) cancer cells lose expression of the LRRC15+ TGFβ signature after [ 177 Lu]-DUNP19 treatment (red = high, blue = low expression). F. Loss of the LRRC15+ TGFβ signature is observed across all tumor stroma after [ 177 Lu]-DUNP19 RIT (green = high, orange = low expression) G. HCC1954 tumors that were resistant to [ 177 Lu]-DUNP19 treatment (defined as reaching 1000m 3 endpoint before conclusion of study) had no significant reduction of the 11-gene LRRC15+ TGFβ signature within tumor stroma.

Article Snippet: To overexpress LRRC15, HEK293T and K7M2 cells were transduced with a pLenti-LRRC15-GFP-Puro vector (Origene, NM_130830) with a multiplicity of infection of 5.

Techniques: Expressing, Whisker Assay