Journal: bioRxiv

Article Title: Antibody-Based Targeting of the SPP1-CD44 Axis in Pediatric High-Grade Glioma through Single-Cell and Structural Bioinformatics

doi: 10.1101/2025.05.01.651763

Figure Lengend Snippet: Cellchat cell-cell communication analysis results. a) Outgoing signal heatmap. The intensity of color represents higher communication probability. b) incoming signal heatmap. The intensity of color represents higher communication probability. c) All outgoing signaling network from MGD TAMs. e) Network topology heatmap of SPP1. f) Violin plots of SPP1 transcript abundance across all clusters.

Article Snippet: Adding anti-SPP1 antibody i.e. bioXcell Clone 100D3 and clone MPIIIB10 showed reversal of immunosuppression TME and enhanced CAR T cell antitumor responses [ ].

Techniques:

Journal: bioRxiv

Article Title: Antibody-Based Targeting of the SPP1-CD44 Axis in Pediatric High-Grade Glioma through Single-Cell and Structural Bioinformatics

doi: 10.1101/2025.05.01.651763

Figure Lengend Snippet: pySCENIC results highlight that no one TF dominates SPP1 gene expression regulation in MGD TAM. a) TF Regulon activity profile of SPP1 transcription factors, here size of the dot shows regulon activity and color shows gene expression correlation of the corresponding TF with SPP1 gene expression. b) TF gene expression dot plot. The size of the dot represents number of cells expressing corresponding TF in each cell type population, and the color represents average expression of the gene within the cell type population, c) MAFB gene expression feature plot showing expression of this gene across all cell types. d) MAFB violin plot showing expression of this gene across all cell types.

Article Snippet: Adding anti-SPP1 antibody i.e. bioXcell Clone 100D3 and clone MPIIIB10 showed reversal of immunosuppression TME and enhanced CAR T cell antitumor responses [ ].

Techniques: Gene Expression, Activity Assay, Expressing

Journal: bioRxiv

Article Title: Antibody-Based Targeting of the SPP1-CD44 Axis in Pediatric High-Grade Glioma through Single-Cell and Structural Bioinformatics

doi: 10.1101/2025.05.01.651763

Figure Lengend Snippet: SPP1 protein structure and sequence analysis results. a) Root mean squared fluctuation of SPP1 protein residues under replica exchange molecular dynamics simulation starting at temperatures 283.15K (black), 303.15K (red), 333.15K (green), and 353.15K (blue). 8, b) Energy landscape-based conformation sampling from four replicate exchange runs. Here y-axis represents radius of gyration values across all 4 replicates and x axis represents RMSD values across all 4 replicates. c) Most stable SPP1 conformation across all 4 replica exchange trajectories. CD44 binding motif (residue 121-140) is shown in orange, 23C3 binding motif is shown in blue, and 2K1 and C2K1 binding motif is shown in cyan. and known SPP1 antibody binding interface shown in blue, and cyan, d) SPP1 protein residue phosphorylation score heatmap. Arrow highlights the top computationally predicted likely phosphorylation site 169 with score > 0.8, e) Sequence alignment of SPP1 across mammals. Orange bar highlights SPP1-CD44 binding motif, blue bar highlights 23C3 binding motif, cyan bar highlights 2K1 and C2K1 binding motif, and red arrow represent phosphorylation site 169 on SPP1 protein sequence. f) Root mean squared fluctuation quadratic mean of normal SPP1 (black) and residue 169 phosphorylated SPP1 (red) across all three independent MD simulation replicates, showing increase in stability in SPP1-CD44 binding interface upon phosphorylation.

Article Snippet: Adding anti-SPP1 antibody i.e. bioXcell Clone 100D3 and clone MPIIIB10 showed reversal of immunosuppression TME and enhanced CAR T cell antitumor responses [ ].

Techniques: Sequencing, Sampling, Binding Assay, Residue, Phospho-proteomics

Journal: bioRxiv

Article Title: Antibody-Based Targeting of the SPP1-CD44 Axis in Pediatric High-Grade Glioma through Single-Cell and Structural Bioinformatics

doi: 10.1101/2025.05.01.651763

Figure Lengend Snippet: Antibody directed evolution results obtained from RosettaAntibodyDesign tool. a) UMAP showing 2500 new antibodies shown as colored dots and the parental 23C3 antibody shown as blast star. Color of the dots highlight energy required to break interaction between SPP1 and the corresponding antibody variant. UMAP is calculated using cosine distance between amino acid sequence esm2 embeddings of CDR regions of two antibody variants. b) Hex plot showing 2500 antibody variants. Here energy required to break SPP1 binding with the corresponding antibody variant is shown as x axis, and cosine distance of amino acid sequence esm2 embeddings of CDR regions of an antibody variant with parental 23C3 amino acid sequence esm2 embeddings of CDR regions. c) Most stable variant (23C3-v1) protein structure (grey) bonded to SPP1 protein structure (wheat). Blue spheres are the residue changes in 23C3-v1 as compared to 23C3 amino acid sequence. d) 23C3-v1 heavy chain amino acid sequence aligned to 10 humanized antibody heavy chain amino acid sequence. Class I and class II epitope sequence region is highlighted using black line and corresponding residue location, e) 23C3-v1 light chain amino acid sequence aligned to 10 humanized antibody light chain amino acid sequence. Class II epitope sequence region is highlighted using black line and corresponding residue location, f) Heavy chain (light green) mutations (red spheres), and light chain (cyan) mutations (blue spheres) induced in 23C3-v1 to neutralize class I and class II epitope. Heavy chain residues 62-68 are shown as green spheres, g) Three independent MD simulation replicates RMSF quadratic mean of 23C3-v1 (black) and Hu23C3-v1 (red) heavy chain, showing no significant change across SPP1 binding region of heavy chain. A sudden increase in fluctuation was observed in residue 62-68, which is distant and upward from SPP1 binding region (shown as green spheres in ), h) Three independent MD simulation replicates RMSF quadratic mean of 23C3-v1 (black) and humanized 23C3-v1 (Hu23C3-v1) (red) light chain, showing no significant change across SPP1 binding region of light chain, i) Box-whiskers plot of 23C3-v1 and Hu23C3-v1 heavy and light chain binding affinity with SPP1 across all three independent MD simulation replicates calculated using gmx_MMPBSA, shows no significant change in Hu23C3-v1 as compared to 23C3-v1 upon epitope neutralization.

Article Snippet: Adding anti-SPP1 antibody i.e. bioXcell Clone 100D3 and clone MPIIIB10 showed reversal of immunosuppression TME and enhanced CAR T cell antitumor responses [ ].

Techniques: Variant Assay, Sequencing, Binding Assay, Residue, Neutralization

Journal: bioRxiv

Article Title: Antibody-Based Targeting of the SPP1-CD44 Axis in Pediatric High-Grade Glioma through Single-Cell and Structural Bioinformatics

doi: 10.1101/2025.05.01.651763

Figure Lengend Snippet: Cellchat cell-cell communication analysis results. a) Outgoing signal heatmap. The intensity of color represents higher communication probability. b) incoming signal heatmap. The intensity of color represents higher communication probability. c) All outgoing signaling network from MGD TAMs. e) Network topology heatmap of SPP1. f) Violin plots of SPP1 transcript abundance across all clusters.

Article Snippet: Recent work showed that using an anti-SPP1 antibody (bioXcell Clone: 100D3) and (clone MPIIIB10) in combination with mIL13Rα2 CAR T cell therapy shows enhanced CAR T cell antitumor response [ ].

Techniques:

Journal: bioRxiv

Article Title: Antibody-Based Targeting of the SPP1-CD44 Axis in Pediatric High-Grade Glioma through Single-Cell and Structural Bioinformatics

doi: 10.1101/2025.05.01.651763

Figure Lengend Snippet: pySCENIC results highlight that no one TF dominates SPP1 gene expression regulation in MGD TAM. a) TF Regulon activity profile of SPP1 transcription factors, here size of the dot shows regulon activity and color shows gene expression correlation of the corresponding TF with SPP1 gene expression. b) TF gene expression dot plot. The size of the dot represents number of cells expressing corresponding TF in each cell type population, and the color represents average expression of the gene within the cell type population, c) MAFB gene expression feature plot showing expression of this gene across all cell types. d) MAFB violin plot showing expression of this gene across all cell types.

Article Snippet: Recent work showed that using an anti-SPP1 antibody (bioXcell Clone: 100D3) and (clone MPIIIB10) in combination with mIL13Rα2 CAR T cell therapy shows enhanced CAR T cell antitumor response [ ].

Techniques: Gene Expression, Activity Assay, Expressing

Journal: bioRxiv

Article Title: Antibody-Based Targeting of the SPP1-CD44 Axis in Pediatric High-Grade Glioma through Single-Cell and Structural Bioinformatics

doi: 10.1101/2025.05.01.651763

Figure Lengend Snippet: SPP1 protein structure and sequence analysis results. a) Root mean squared fluctuation of SPP1 protein residues under replica exchange molecular dynamics simulation starting at temperatures 283.15K (black), 303.15K (red), 333.15K (green), and 353.15K (blue). 8, b) Energy landscape-based conformation sampling from four replicate exchange runs. Here y-axis represents radius of gyration values across all 4 replicates and x axis represents RMSD values across all 4 replicates. c) Most stable SPP1 conformation across all 4 replica exchange trajectories. CD44 binding motif (residue 121-140) is shown in orange, 23C3 binding motif is shown in blue, and 2K1 and C2K1 binding motif is shown in cyan. and known SPP1 antibody binding interface shown in blue, and cyan, d) SPP1 protein residue phosphorylation score heatmap. Arrow highlights the top computationally predicted likely phosphorylation site 169 with score > 0.8, e) Sequence alignment of SPP1 across mammals. Orange bar highlights SPP1-CD44 binding motif, blue bar highlights 23C3 binding motif, cyan bar highlights 2K1 and C2K1 binding motif, and red arrow represent phosphorylation site 169 on SPP1 protein sequence. f) Root mean squared fluctuation quadratic mean of normal SPP1 (black) and residue 169 phosphorylated SPP1 (red) across all three independent MD simulation replicates, showing increase in stability in SPP1-CD44 binding interface upon phosphorylation.

Article Snippet: Recent work showed that using an anti-SPP1 antibody (bioXcell Clone: 100D3) and (clone MPIIIB10) in combination with mIL13Rα2 CAR T cell therapy shows enhanced CAR T cell antitumor response [ ].

Techniques: Sequencing, Sampling, Binding Assay, Residue, Phospho-proteomics

Journal: bioRxiv

Article Title: Antibody-Based Targeting of the SPP1-CD44 Axis in Pediatric High-Grade Glioma through Single-Cell and Structural Bioinformatics

doi: 10.1101/2025.05.01.651763

Figure Lengend Snippet: Antibody directed evolution results obtained from RosettaAntibodyDesign tool. a) UMAP showing 2500 new antibodies shown as colored dots and the parental 23C3 antibody shown as blast star. Color of the dots highlight energy required to break interaction between SPP1 and the corresponding antibody variant. UMAP is calculated using cosine distance between amino acid sequence esm2 embeddings of CDR regions of two antibody variants. b) Hex plot showing 2500 antibody variants. Here energy required to break SPP1 binding with the corresponding antibody variant is shown as x axis, and cosine distance of amino acid sequence esm2 embeddings of CDR regions of an antibody variant with parental 23C3 amino acid sequence esm2 embeddings of CDR regions. c) Most stable variant (23C3-v1) protein structure (grey) bonded to SPP1 protein structure (wheat). Blue spheres are the residue changes in 23C3-v1 as compared to 23C3 amino acid sequence. d) 23C3-v1 heavy chain amino acid sequence aligned to 10 humanized antibody heavy chain amino acid sequence. Class I and class II epitope sequence region is highlighted using black line and corresponding residue location, e) 23C3-v1 light chain amino acid sequence aligned to 10 humanized antibody light chain amino acid sequence. Class II epitope sequence region is highlighted using black line and corresponding residue location, f) Heavy chain (light green) mutations (red spheres), and light chain (cyan) mutations (blue spheres) induced in 23C3-v1 to neutralize class I and class II epitope. Heavy chain residues 62-68 are shown as green spheres, g) Three independent MD simulation replicates RMSF quadratic mean of 23C3-v1 (black) and Hu23C3-v1 (red) heavy chain, showing no significant change across SPP1 binding region of heavy chain. A sudden increase in fluctuation was observed in residue 62-68, which is distant and upward from SPP1 binding region (shown as green spheres in ), h) Three independent MD simulation replicates RMSF quadratic mean of 23C3-v1 (black) and humanized 23C3-v1 (Hu23C3-v1) (red) light chain, showing no significant change across SPP1 binding region of light chain, i) Box-whiskers plot of 23C3-v1 and Hu23C3-v1 heavy and light chain binding affinity with SPP1 across all three independent MD simulation replicates calculated using gmx_MMPBSA, shows no significant change in Hu23C3-v1 as compared to 23C3-v1 upon epitope neutralization.

Article Snippet: Recent work showed that using an anti-SPP1 antibody (bioXcell Clone: 100D3) and (clone MPIIIB10) in combination with mIL13Rα2 CAR T cell therapy shows enhanced CAR T cell antitumor response [ ].

Techniques: Variant Assay, Sequencing, Binding Assay, Residue, Neutralization