Journal: bioRxiv

Article Title: Engineering hyaluronic acid-binding cytokines for enhanced tumor retention and safety

doi: 10.64898/2026.04.06.716711

Figure Lengend Snippet: A , A panel of dimeric (Fc) and monomeric (MSA) cytokine fusions to untargeted, collagen-anchored, or HA-anchored proteins was engineered. B , Mice bearing B16F10 or MC38 tumors were treated days 6 and day 13 after tumor induction with 0.11 nmol IL-15 and 0.014 nmol IL-12. Mice were monitored for tumor burden and weight loss. C , Kaplan-Meier survival and ( D ) weight loss of mice bearing B16F10 tumors intratumorally injected with PBS or cytokine fusion proteins (mean ± SD, n = 11). E , Kaplan-Meier survival and ( F ) weight loss of mice bearing MC38 tumors treated with PBS or cytokine fusions (mean ± SD, n = 5). Statistics: analysis of survival was performed using log-rank Mantel-Cox test. Weights compared by two-way ANOVA with Tukey’s multiple comparison test. ns, not significant; ****P < 0.0001.

Article Snippet: B16F10 (ATCC), MC38 (ATCC), Expi293F (Gibco), HEK-Blue IL-12 (Invivogen), and CTLL-2 (ATCC) cells were cultured following vendor instructions.

Techniques: Injection, Comparison

Journal: bioRxiv

Article Title: Engineering hyaluronic acid-binding cytokines for enhanced tumor retention and safety

doi: 10.64898/2026.04.06.716711

Figure Lengend Snippet: A , B16F10- or MC38-tumor bearing mice were intratumorally injected with 0.22 nmol IL-15 and 0.028 nmol IL-12 fusion proteins 6 days after tumor induction. Weight loss was monitored for 5 days post-treatment (mean ± SD, n =5). Percent weight change measured from the start of treatment in B16F10 ( B ) and MC38 ( C ) tumor-bearing mice. D , Mice were treated as described above, and blood collected 1 and 3 days post-treatment for analysis of ALT activity and serum cytokine/chemokine levels (mean ± SD, n =5). E , Alanine aminotransferase activity in serum 1 and 3 days post-treatment. F , Z-scored expression of serum cytokines and chemokines for each cohort 1 and 3 days post-treatment. G-I, Serum concentrations of ( G ) IFNɣ, ( H ) IFNɑ, and ( I ) TNFɑ tested at both timepoints. J , B16F10 tumor-bearing mice were treated with 0.11 nmol IL-15 and 0.014 nmol IL-12 of indicated cytokine fusions 6 days post-tumor inoculation. Mice were injected both intratumorally and on their healthy contralateral flank. Seven days after the primary injection, mice were either euthanized for tissue collection or treated again. Six days after the second injection, the rest of the mice were euthanized for histopathological analysis of tissues (n=6). K , (Left) Macroscopic images of dosed contralateral flank after 2 cytokine injections. (Center, right) H&E stained skin sections of contralateral flank after 1 (center) or 2 (right) subcutaneous injections of cytokine fusions. Statistics: two-way ANOVA with Tukey’s multiple comparison test. ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Article Snippet: B16F10 (ATCC), MC38 (ATCC), Expi293F (Gibco), HEK-Blue IL-12 (Invivogen), and CTLL-2 (ATCC) cells were cultured following vendor instructions.

Techniques: Injection, Activity Assay, Expressing, Staining, Comparison

Journal: bioRxiv

Article Title: Engineering hyaluronic acid-binding cytokines for enhanced tumor retention and safety

doi: 10.64898/2026.04.06.716711

Figure Lengend Snippet: A , B16F10 tumor-bearing mice were intratumorally injected with 0.05 nmol of indicated IL-12 fusion proteins 8 days after tumor inoculation. Mice were euthanized and tumors collected 5 days later for downstream flow cytometric analysis (mean ± SD, n=5). B, Treatment effects on proportion and ( C ) count of tumor-infiltrating CD45+ leukocytes. D , Representative histograms and gating for pSTAT4+ cells, previously gated on CD45+ cells. E , Proportion and ( F ) count of pSTAT4+ cells in the whole CD45+ compartment. G , Treatment effects on proportion and ( H ) count of tumor-infiltrating T cells. I , Representative histograms and gating of pSTAT4+ cells in the CD3+ compartment. J , Proportion and ( K ) count of pSTAT4+ T cells in the tumor. Statistics: one-way ANOVA followed by Tukey’s multiple-comparison test. ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Article Snippet: B16F10 (ATCC), MC38 (ATCC), Expi293F (Gibco), HEK-Blue IL-12 (Invivogen), and CTLL-2 (ATCC) cells were cultured following vendor instructions.

Techniques: Injection, Comparison

Journal: Journal of Medicinal Chemistry

Article Title: A Rationally Designed Novel Bifunctional Human TNF-α- and Janus Kinase-Targeted soloMER Drug Conjugate (SDC) with a Neutrophil Elastase Cleavable Linker Delivering Inflammation Site-Specific Release of Payload

doi: 10.1021/acs.jmedchem.6c00532

Figure Lengend Snippet: SDC demonstrates dual functionality. (A) Neutralization of TNF-α-mediated cytotoxicity in mouse fibrosarcoma L929 cells. (B,C) Neutralization of human IL-23-induced Tyk2-JAK2 heterodimerization and STAT3 phosphorylation in HEK-Blue IL-23 Reporter Cell. HEK-Blue IL-23 cells were treated with recombinant human IL-23 (1 ng/mL) and dilution series of (B) tofacitinib and (C) ELN28-135-01 with and without HNE (50 nM). (D) Neutralization of cynomolgus IL-12-induced Tyk2-JAK2 heterodimerization and STAT4 phosphorylation in the HEK-Blue IL-12 Reporter Cell. Data are shown as mean ± SEM ( n = 2). (E) Inhibition of PHA-mediated proliferation of human PBMC samples. Human PBMCs were stimulated with PHA and treated with either tofacitinib, ELN28-135-01 in the presence or absence of HNE, ELN22-135, or ELN0-2V-135-01 in the presence or absence of HNE. (F) Inhibition of PHA-mediated proliferation of PBMCs from 4 additional donors. Human PBMCs were stimulated with PHA and treated with either tofacitinib or ELN28-135-01 in the presence of HNE. Data are shown as mean ± SEM ( n = 2), ND 50 values were calculated using four-parameter nonlinear regression.

Article Snippet: Actinomycin D (1229, R&D Systems), Healthy human PBMCs (Precision Medicine), HEK Blue Reporter Assay IL23 (hkb-il23, InvivoGen), HEK Blue Reporter Assay IL12 (hkb-il12, InvivoGen), L929 (Sigma), human IL23 (1290-IL/CF, R&D Systems), cynomolgus IL12 (10215-CL, R&D systems), human neutrophil elastase (324681, Merck), human TNF-α (TNFA-HA4211, Acro Bioscience), Phytohemagglutinin-L (PHA) (11249738001, Sigma), QUANTI-Blue solution (rep-qbs, InvivoGen), tofacitinib (FT32555, Biosynth), WST-1 reagent (11644807001, Merck), mIL-6 (Quantikine ELISA, M6000B, R&D Systems), MCP-1 (Quantikine ELISA, JE00B, R&D Systems), and CXCL1 (DuoSet ELISA, DY453, R&D Systems).

Techniques: Neutralization, Phospho-proteomics, Recombinant, Inhibition

Journal: bioRxiv

Article Title: BLAST: A blue light-assisted secretion toolkit tunable by reversible protein-protein interactions

doi: 10.64898/2026.03.30.715452

Figure Lengend Snippet: Secretion of therapeutic proteins with a-BLAST and d-BLAST. (a) The human preproinsulin (preproINS) construct contains a signal peptide, B-chain (yellow), C-peptide (dark gray), and A-chain (yellow), with engineered Furin cleavage sites flanking the C-peptide for maturation. To facilitate efficient processing within the Golgi apparatus, Furin protease was co-transfected with the BLAST modules. (b) Kinetic profiling of light-induced insulin secretion. Summary graphs of secreted C-peptide levels, quantified by ELISA, as a proxy for insulin secretion from a-BLAST (left) and d-BLAST (right). Both systems exhibited significant, time-dependent insulin release starting from 2 h of illumination (8.2-fold for a-BLAST, 8.4-fold for d-BLAST), reaching maximal induction at 24 h (13.8-fold for a-BLAST, 19.3-fold for d-BLAST). (c) Plasmid configurations for IL-12 secretion. Schematic of the heterodimeric cytokine IL-12-a-BLAST (left) and d-BLAST-IL-12 (right) constructs. (d) Kinetic profiling of light-induced IL-12 secretion. Summary graphs showing IL-12 secretion levels measured by ELISA. Significant secretion was observed starting at 3 h for a-BLAST (2.5-fold) and 2 h for d-BLAST (2.2-fold). At the 24 h time point, d-BLAST (4.7-fold) demonstrated a slightly higher dynamic range compared to a-BLAST (4.5-fold). Open circles represent individual measurements from three biologically independent samples. Data are presented as means ± S.D. Statistical significance was assessed using one-way ANOVA followed by Tukey’s multiple comparisons test (ns = not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001).

Article Snippet: HEK-BlueTM IL-12 reporter cells were purchased from InvivoGen.

Techniques: Construct, Transfection, Enzyme-linked Immunosorbent Assay, Plasmid Preparation

Journal: bioRxiv

Article Title: BLAST: A blue light-assisted secretion toolkit tunable by reversible protein-protein interactions

doi: 10.64898/2026.03.30.715452

Figure Lengend Snippet: Functional validation of optogenetically secreted IL-12 using a reporter cell assay. (a) Schematic of the experimental workflow for bioactivity validation. The protocol spans 5 days. HEK293T cells transfected with BLAST-IL-12 were subjected to dark or blue light conditions for 24 h (Day 3). On Day 4, the conditioned media containing secreted IL-12 was harvested and transferred to HEK-Blue™ IL-12 reporter cells (seeded on Day 3). After a 24 h incubation to allow for signal transduction, SEAP activity was quantified (Day 5). (b) Plasmid configurations. Schematics of the a-BLAST-IL-12 (upper) and d-BLAST-IL-12 (lower) constructs used for the assay. (c) Illustration of the JAK-STAT signaling pathway in the reporter cells. Binding of secreted IL-12 to the IL-12 receptor complex activates Tyk2/JAK2, leading to STAT4 phosphorylation. Phosphorylated STAT4 dimerizes and translocates to the nucleus to induce SEAP expression. (d) Summary graph of SEAP activity induced by the conditioned media. The results confirm that both systems secrete biologically active IL-12 upon blue light stimulation, exhibiting robust fold changes (12.4-fold for a-BLAST and 11.3-fold for d-BLAST) compared to the dark control. Data are presented as means ± S.D. Statistical significance was assessed using one-way ANOVA followed by Tukey’s multiple comparisons test (**** P < 0.0001).

Article Snippet: HEK-BlueTM IL-12 reporter cells were purchased from InvivoGen.

Techniques: Functional Assay, Biomarker Discovery, Transfection, Incubation, Transduction, Activity Assay, Plasmid Preparation, Construct, Binding Assay, Phospho-proteomics, Expressing, Control