Targeting the TNFR2: Biological Roles and Therapeutic Strategies

• Gene Information: The TNFRSF1B gene encodes human TNFR2, a key member of the tumor necrosis factor receptor superfamily that lacks a death domain and primarily drives cellular survival and proliferation.
• Protein Expression: TNFR2 is highly restricted and predominantly expressed on critical immunosuppressive cells within the tumor microenvironment—such as regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs)—as well as certain tumor cells.
• Signaling Pathway: Upon binding to membrane-bound TNF, TNFR2 recruits TRAF2 to activate the canonical and non-canonical NF-κB and MAPK pathways, promoting Treg stability, survival, and robust immune suppression.
• Therapeutic Inhibition: Targeted therapeutic inhibition using antagonistic anti-TNFR2 antibodies aims to selectively deplete or deactivate intra-tumoral Tregs, thereby restoring potent cytotoxic T-cell activity to eradicate tumors.

TNFR2

• The exons 2-6 of mouse Tnfr2 gene that  encode the extracellular domain was replaced by human TNFR2 exons 2-6 in B-hTNFR2 mice.

• Mouse Tnfr2 mRNA was detectable only in splenocytes of wild-type C57BL/6 mice (+/+). Human TNFR2 mRNA was detectable only in homozygous B-hTNFR2 mice (H/H), but not in wild-type C57BL/6 mice.

Strain specific analysis of TNFR2 gene expression in wild-type C57BL/6 mice and homozygous B-hTNFR2 mice by RT-PCR. The splenocytes RNA was isolated from wild-type C57BL/6 mice (+/+) and homozygous B-hTNFR2 mice (H/H), and then cDNA libraries were synthesized by reverse transcription, followed by PCR with mouse or human TNFR2 primers.

• The mRNA expression of TNFR2 in homozygous B-hTNFR2 mice (H/H) was similar to those in the wild-type C57BL/6 mice (+/+), demonstrating that introduction of human TNFR2 in place of its mouse counterpart does not change the expression level of TNFR2 mRNA.

Strain specific analysis of TNFR2 gene expression in wild-type C57BL/6 mice and homozygous B-hTNFR2 mice by RT-PCR. The splenocytes RNA was isolated from wild-type C57BL/6 mice (+/+) and homozygous B-hTNFR2 mice (H/H), and then cDNA libraries were synthesized by reverse transcription, followed by PCR with mouse or human TNFR2 primers.

• Mouse TNFR2 was detectable in wild-type C57BL/6 mice (+/+) but not in the in homozygous B-hTNFR2 mice (H/H). Human TNFR2 was exclusively detectable in homozygous B-hTNFR2 mice.

Strain specific TNFR2 expression analysis in wild-type C57BL/6 mice and homozygous B-hTNFR2 mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6 mice (+/+) and homozygous B-hTNFR2 mice (H/H), and analyzed by flow cytometry with species-specific anti-TNFR2 antibody (anti-mouse CD120b (TNF R Type II/p75) Antibody, Biolegend, 113405; anti-human CD120b, Biolegend, 358403).

• Mouse TNFR2 was detectable in wild-type C57BL/6 mice (+/+) but not in the in homozygous B-hTNFR2 mice (H/H). Human TNFR2 was exclusively detectable in homozygous B-hTNFR2 mice.

Strain specific TNFR2 expression analysis in wild-type C57BL/6 mice and homozygous B-hTNFR2 mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6 mice (+/+) and homozygous B-hTNFR2 mice (H/H) stimulated with anti-CD3ε in vivo, and analyzed by flow cytometry with species-specific anti-TNFR2 antibody (anti-mouse CD120b (TNF R Type II/p75) Antibody, Biolegend, 113405; anti-human CD120b, Biolegend, 358403).

• The frequencies of B cells, T cells, NK cells, granulocytes, DCs, macrophages, and monocytes in homozygous B-hTNFR2 mice were similar to those in C57BL/6 mice, demonstrating that humanization of TNFR2 does not change the frequency or distribution of these cell types in spleen or lymph nodes.

Analysis of leukocyte subpopulations by flow cytometry in immune organs. Splenocytes, and lymph nodes were isolated from female C57BL/6 and B-hTNFR2 mice (female, 6-week-old, n = 3). Single live cells were gated on the CD45⁺ population and analyzed by flow cytometry as indicated. Values are expressed as mean ± SEM.

Analysis of splenocytes of B-hTNFR2 mice by FACS. Splenocytes were isolated from B-hTNFR2 mice (female, 6-week-old, n=3) treated with anti-mCD3ε (0.2 or 1 μg/mL) and anti-mCD28 (1 μg/mL) in vitro. Single live cells were gated for CD45 population and used for further analysis as indicated here. Human TNFR2 expression was detectable on CD3 T cells in B-hTNFR2 mice as evidenced by hTNFR2 Ab2 binding vs isotype control (hIgG). mTNFα enhanced hTNFR2 Ab2 binding under mild anti-mCD3ε (0.2 μg/mL) stimulation, suggesting that mTNFα/hTNFR2 signaling pathway also works well in the B-hTNFR2 mice.

Establishment of a MC38 model and in vivo efficacy study of an anti-human TNFR2 antibodies. MC38 cells were implanted subcutaneously into homozygous B-hTNFR2 mice (male, 9-10-week-old, n=8).

Antitumor activity of anti-human TNFR2 antibodies in B-hTNFR2 mice. (A) Tumor growth curves. (B) Body weight changes during treatment. These results demonstrate that B-hTNFR2 mice provide a powerful preclinical model for in vivo evaluation of anti-human TNFR2 antibodies.

The overage of this tumor model is 40%.

Antitumor activity of anti-human TNFR2 antibodies in B-hTNFR2 mice. Tumor cells growth of individual mouse. These results demonstrate that B-hTNFR2 mice provide a powerful preclinical model for in vivo evaluation of anti-human TNFR2 antibodies.

Establishment of a MC38 model and in vivo efficacy study of an anti-human TNFR2 antibodies. MC38 cells were implanted subcutaneously into homozygous B-hTNFR2 mice (male, 8-9-week-old, n=8).

Antitumor activity of anti-human TNFR2 antibodies in B-hTNFR2 mice. (A) Tumor growth curves. (B) Body weight changes during treatment. (C) Tumor cells growth of individual mouse. These results demonstrate that B-hTNFR2 mice provide a powerful preclinical model for in vivo evaluation of anti-human TNFR2 antibodies. The overage of this tumor model is 40%.