Loss of host TNFR1 perturbs the immunologic control of Panc02 tumors.

<p>Panc02-tumors were explanted one month after tumor cell inoculation, sectioned, and stained for different immune cells and blood vessels (B6.WT n = 4, B6.TNF KO n = 5, B6.TNFR1 KO n = 5, B6.TNFR2 KO n = 6, B6.TNFR1R2 KO n = 4).</p>*<p>p≤0.05, ** p≤0.01.</p

DataSheet_1_A TNFR2-Specific TNF Fusion Protein With Improved In Vivo Activity.pdf

Tumor necrosis factor (TNF) receptor-2 (TNFR2) has attracted considerable interest as a target for immunotherapy. Indeed, using oligomeric fusion proteins of single chain-encoded TNFR2-specific TNF mutants (scTNF80), expansion of regulatory T cells and therapeutic activity could be demonstrated in various autoinflammatory diseases, including graft-versus-host disease (GvHD), experimental autoimmune encephalomyelitis (EAE) and collagen-induced arthritis (CIA). With the aim to improve the in vivo availability of TNFR2-specific TNF fusion proteins, we used here the neonatal Fc receptor (FcRn)-interacting IgG1 molecule as an oligomerizing building block and generated a new TNFR2 agonist with improved serum retention and superior in vivo activity.MethodsSingle-chain encoded murine TNF80 trimers (sc(mu)TNF80) were fused to the C-terminus of an in mice irrelevant IgG1 molecule carrying the N297A mutation which avoids/minimizes interaction with Fcγ-receptors (FcγRs). The fusion protein obtained (irrIgG1(N297A)-sc(mu)TNF80), termed NewSTAR2 (New selective TNF-based agonist of TNF receptor 2), was analyzed with respect to activity, productivity, serum retention and in vitro and in vivo activity. STAR2 (TNC-sc(mu)TNF80 or selective TNF-based agonist of TNF receptor 2), a well-established highly active nonameric TNFR2-specific variant, served as benchmark. NewSTAR2 was assessed in various in vitro and in vivo systems.ResultsSTAR2 (TNC-sc(mu)TNF80) and NewSTAR2 (irrIgG1(N297A)-sc(mu)TNF80) revealed comparable in vitro activity. The novel domain architecture of NewSTAR2 significantly improved serum retention compared to STAR2, which correlated with efficient binding to FcRn. A single injection of NewSTAR2 enhanced regulatory T cell (Treg) suppressive activity and increased Treg numbers by > 300% in vivo 5 days after treatment. Treg numbers remained as high as 200% for about 10 days. Furthermore, a single in vivo treatment with NewSTAR2 upregulated the adenosine-regulating ectoenzyme CD39 and other activation markers on Tregs. TNFR2-stimulated Tregs proved to be more suppressive than unstimulated Tregs, reducing conventional T cell (Tcon) proliferation and expression of activation markers in vitro. Finally, singular preemptive NewSTAR2 administration five days before allogeneic hematopoietic cell transplantation (allo-HCT) protected mice from acute GvHD.ConclusionsNewSTAR2 represents a next generation ligand-based TNFR2 agonist, which is efficiently produced, exhibits improved pharmacokinetic properties and high serum retention with superior in vivo activity exerting powerful protective effects against acute GvHD.</p

Exogenous TNF treatment increases orthotopic Panc02 tumor growth.

<p>10⁴ tumor cells were injected into the spleen of albino B6.WT mice. The mice were either left untreated or were treated every other day with 5 µg of recombinant human TNF. Tumor growth was determined by <i>in vivo</i> BLI. A: Tumor growth displayed as total radiance (untreated n = 11, TNF n = 10). B: <i>Ex vivo</i> imaging 23 days after tumor cell inoculation. Internal organs were imaged for the presence of tumor cells. Pancreatic tumor size is displayed as total radiance (untreated n = 11, TNF n = 10). ** p≤0.01. Combined data from two independent experiments.</p

Loss of host TNFR1 perturbs the immunologic control of pancreatic ductal carcinoma.

<p>Panc02-tumors were explanted one month after tumor cell inoculation, consecutive histological sections were stained for indicated immune cell populations and blood vessels (CD31). Pancreatic tumors resulted in an influx of immune cell populations of the innate and adaptive immune system that were not observed in healthy pancreatic tissue under steady-state conditions. Of note, deficiency of TNFR1 resulted in a reduced cytotoxic CD8⁺ T cell infiltration but increased T_reg cell infiltration. Exemplary photomicrographs are shown. Scale bar indicates 100 µm.</p

Loss of host TNFR1 abrogates spontaneous rejection of orthotopic Panc02 tumors.

<p>Murine pancreatic ductal adenocarcinoma (Panc02) cells were transduced to stably express eGFP and firefly luciferase and 10⁴ tumor cells were injected orthotopically into albino C57Bl/6 mice. Tumor growth in wild type mice (B6.WT) and mice that were deficient for TNF or its receptors was determined by <i>in vivo</i> BLI. A: Tumor growth displayed as total radiance (B6.WT n = 8, B6.TNF KO n = 8, B6.TNFR1 KO n = 6, B6.TNFR2 KO n = 9, B6.TNFR1R2 KO n = 7). B: Exemplary pictures of the imaging time course of a mouse that spontaneously rejected the tumor (left) and a mouse that could not control tumor progression (right). C: <i>Ex vivo</i> imaging one month after tumor cell inoculation. Internal organs were imaged for the presence of tumor cells. Exemplary pictures of a mouse that spontaneously rejected the tumor (I), a mouse with low tumor burden (II), and a mouse with high tumor burden (III). D: Pancreatic tumor size one month after Panc02 inoculation is displayed as total radiance (B6.WT n = 8, B6.TNF KO n = 8, B6.TNFR1 KO n = 6, B6.TNFR2 KO n = 9, B6.TNFR1R2 KO n = 5). * p≤0.05, ** p≤0.01. Combined data from four independent experiments.</p

Loss of host TNFR1 does not affect the activation status of tumor-infiltrating T cells.

<p>Pancreata and spleens from naïve and tumor-bearing mice one and two weeks after tumor cell inoculation were explanted and prepared as single cell suspensions. T cells were analyzed for the expression of activation-associated surface receptors by flow cytometry. Furthermore, the percentage of T_regs, myeloid cells and tumor cells was determined by flow cytometry (w/o tumor: B6.WT n = 8, B6.TNFR1 KO n = 6; d+7: B6.WT n = 7, B6.TNFR1 KO n = 6; d+15: B6.WT n = 7, B6.TNFR1 KO n = 7). *p≤0.05, **p≤0.01. Combined data from four independent experiments.</p

Panc02 cells show little <i>in vitro</i> capabilities for metastasis.

<p>Panc02 cells were treated with 1.67: Adhesion to different extracellular matrix proteins (n = 4). B: Flow cytometric determination of the expression of proteins involved in adhesion and migration (n = 3). C: Invasive capabilities of Panc02 cells. Left panel: <i>In vitro</i> invasion of the basement membrane (n = 3). Right panel: Gelatin zymography of tumor cell samples. Infarcted mouse heart lysate was used as a positive control (n = 4).</p

Exogenous TNF treatment perturbs the immunologic control of Panc02 tumors.

<p>Panc02-tumors from untreated and human TNF treated mice were explanted 23 days after tumor cell inoculation, sectioned, and stained for different immune cells and blood vessels (untreated n = 7, TNF n = 9). * p≤0.05.</p

Loss of host TNFR1 affects the expression of immunosuppressive genes and IL-4.

<p>Panc02-tumors were explanted one month after tumor cell inoculation and total RNA was isolated from the tumor tissue. RNA was reverse transcribed and amplified by qRT-PCR. Data is presented as relative expression within tumors derived from B6.TNFR1 KO mice compared to tumors derived from wild type (WT) mice (B6.WT n = 3, B6.TNFR1 KO n = 4). * p≤0.05.</p

Primer sequences used for qRT-PCR.

<p>Primer sequences used for qRT-PCR.</p