A bioactive soluble recombinant mouse LIGHT promotes effective tumor immune cell infiltration delaying tumor growth

The TNF family member LIGHT (TNFSF14) binds to two receptors, HVEM (TNFSFR14) and LTβR (TNFSFR3). HVEM functions as a costimulatory molecule, whereas LTβR is involved in the development of lymph nodes and ectopic tertiary lymphoid structures at chronic inflammation sites. The classical approach of fusing soluble recombinant proteins to the Fc fragment of IgG resulted in a functionally inactive Ig.mouse (m) LIGHT protein. However, in line with the fact that TNF family members cluster receptors as trimers, addition of a small homotrimeric domain (foldon) N-terminal of mLIGHT produced an Ig.Foldon-mLIGHT protein able to bind and engage HVEM and LTβR in a cell-based reporter bioassay. In the tumor model of B16.F10 melanoma cells implanted into syngeneic recipients, cells transduced with membrane-bound mLIGHT grew as aggressively as mock-transduced cells, but growth of tumors of B16.F10 cells expressing Ig.Foldon-mLIGHT was delayed and characterized by significant immune infiltration of dendritic cells and cytotoxic cells. This work unveils the potential of active soluble LIGHT, as a single agent, to recruit cytotoxic cells and dendritic cells at the tumor site to inhibit tumor growth. This effect may be further enhanced with immune checkpoint blockade therapies.

Key messages

• The classical approach of fusing soluble recombinant proteins to the Fc fragment of IgG resulted in a functionally inactive Ig.mouse (m) LIGHT (TNFSF14) protein.

• The addition of a small homotrimeric domain (foldon) N-terminal of mouse LIGHT produces a proper folded bioactive mouse LIGHT recombinant protein.

• Constitutive intratumor expression of secreted Ig-Foldon-LIGHT, but not membrane LIGHT, delays tumor growth.

• Tumors secreting LIGHT, as a single agent, promote beneficial anti-tumor responses through the recruitment and infiltration of cytotoxic cells and dendritic cells.

Graphical Abstract

The intrinsic source of native soluble LIGHT and LTα1β2 (produced by neutrophils and activated T cells and NK cells) along with the tumor secreting recombinant Ig.Foldon-LIGHT co-stimulate NK and T cells through HVEM and license DC for antigen presentation to promote anti-tumor T cell responses in the tumor draining lymph nodes. Furthermore, native LIGHT produced by neutrophils, NK, and T cells and recombinant LIGHT along with LTα1β2 secreted by recruited B cells (LTi, lymphoid tissue inducer cells) would together activate stromal cells of the tumor microenvironment (TME) through LTβR (LTo, lymphoid tissue organizing cells) to release chemokines driving endothelial activation and the upregulation of adhesion molecules that in turn would facilitate transmigration of immune cells from the blood vessels to the tumor site. It would also condition myeloid cells expressing LTβR to secrete cytokines such as TGF-beta that contribute to blood vessel normalization.