CTLA4-FasL, the Hexameric Targeted FasL Fusion Protein, As Potential Novel Treatment for DLBCL

The bi-functional drug CTLA4-FasL (KAHR-102) is a fusion protein composed of the extracellular domains of CTLA4 and FasL. It naturally forms a stable homo-hexamer capable of targeting two different cellular receptors; the CTLA4 domains target B7 receptors (CD80 and CD86) on cells bearing these receptors, while the FasL domains target functional Fas-receptors (CD95) to induce apoptosis. Although CTLA4-FasL can induce apoptosis in all cells expressing functional Fas receptors, it is at least 100 times more potent on cells expressing both B7 and functional Fas receptors. The higher efficacy in cells expressing both receptors is adjoined by robust activation of pro-apoptotic intracellular pathways in conjunction to abrogation of anti-apoptotic ones. This unique combined activity cannot be detected in cells bearing just Fas receptors and can be blocked by preventing the binding of the protein to CD80 and CD86. Both B7 and functional Fas receptors are expressed on lymphoma cells, mainly of the B cell lineage, making them exceptionally sensitive to the CTLA4-FasL apoptotic effect.

CTLA4-FasL was found to be highly efficient in inducing apoptosis in different resistant DLBCL cell-lines bearing translocations such as , FARAGE, OCI-LY7, OCI-LY19 and SUD-HL-4. Now we show that CTLA4-FasL specifically binds to CD80 and CD86 on these cells, hinders their viability with EC50 of 0.02-0.06 nM (MTS assay), and induces robust apoptosis in these cells (annexin V-PI assay). CTLA4-FasL's potency is correlated with CD80, CD86 and CD95 expression on the cell's surface.

Importantly, CTLA4-FasL significantly improves mouse survival in a systemic lymphoma model induced by IV injection of FARAGE DLBCL cells into the tail vein of irradiated NUDE mice (4 SC injections, every other day). When given at a dose of 10mg/mice/injection its effect was comparable to that of 350 mg/mice of Rituximab.

Previously, we evaluated CTLA4-FasL's toxicity in GLP studies in mice and cynomolgus monkeys. No gross pathology or adverse clinical symptoms were observed in mice (n=76) or monkeys (n=10) treated with the highest dose of CTLA4-FasL (0.5 mg/kg in mice, 0.125 mg/kg in monkeys). Transient and dose-dependent leukopenia was observed in monkeys only and this was resolves in 7 days. As leukopenia was observed in monkeys only and was not evident in mice treated with the same adjusted dose, we now compared Fas receptor level of expression and CTLA4-FasL 's binding to mice, monkeys and human PBMCs, and also CTLA4-FasL 's ability to induce apoptosis of the different PBMCs. Interestingly, the monkeys' cells express more Fas receptors and bind the protein better than both human and mice cells. In accordance with that, significantly more apoptosis of monkeys' PBMCs was evident when cells were treated with CTLA4-FasL.

Though leukopenia was not observed in mice, it is still possible that some population of cells are affected by CTLA4-FasL. In order to test CTLA4-FasL's effect on different immune system cells in immune competent mice, BALB/C mice were treated with CTLA4-FasL and 24h later peripheral blood and spleens were harvested and cells were immuno-stained and analyzed by flow-cytometry. A significant decrease in percentage and actual counts of B and T lymphocytes (CD4 and CD8) as well as macrophages and monocytes was importantly accompanied by an increase in percentage and actual cell number of neutrophils and NK cells.

In summary, the fusion protein CTLA4-FasL, with its unique hexameric structure and bi-functional mode of targeted action, was shown to be an effective treatment for human-mouse xenograft model of DLBCL. Transient and dose-dependent leukopenia was the only significant adverse effect, seen only in monkeys, and is maybe explained by the higher sensitivity of monkeys' cells to the protein's apoptotic effect. It might also be the result of the longer half-life of the protein in monkeys when compared to mice. The sparing and even increase in the number of neutrophils and NK cells might indicate that the innate immune system will be preserved after treatment with CTLA4-FasL.