Targeting Lymphoma By the Bi-Functional Hexameric Fusion Protein CTLA4-Fasl; Mode-of-Action, Pre-Clinical Efficacy and Toxicology Studies
Authors: Aronin Alexandra , Amsili Shira , Prigozhina Tatyana , Baru Moshe , Makdasi Efi , Tzdaka Kobi , Shkedy Fanny , Shani Noam , Dranitzki Elhalel Michal 
Published: October 2019

Abstract
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 86) on immune cells, 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 Fas receptors. 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 shown before to improve survival in a human:mouse xenograft model of lymphoma.
In the present study we present for the first time CTLA4-FasL's efficacy in inducing apoptosis in different highly-resistant DLBCL cell-lines such as LY 7, 19 and SUHDL-4. We show that CTLA4-FasL inhibits viability of these cell-lines with EC50 of 0.02-0.06 nM by using MTS assay, and that it induces robust apoptosis in these cells as shown by the anexin-PI assay.
We show that in cells expressing both B7 and Fas receptors, CTLA4-FasL activates pro-apoptotic signaling, inhibits anti-apoptotic ones and interestingly prevents the activation of the non-canonical NFkB pathway at the level of p100 to P52 processing. The combined effects, which are in accordance with CTLA4-FasL ability to induce extremely effective apoptosis, cannot be elicited in B7 negative cells or in the presence of B7 blocking antibodies.
Importantly, in both a BCL1 mouse model of systemic lymphoma, and in a xenograft model of human to mouse systemic lymphoma, CTLA4-FasL significantly improves mouse survival when dosed 4 times by subcutaneous (SC) injections. Of note, the human CTLA4-FasL protein was shown to be as active on mouse cells as on human cells.
CTLA4-FasL's toxicity was evaluated in GLP toxicology studies in mice (following 3 repeated injections) and cynomolgus monkeys (following a single injection). No anatomical pathology findings 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 and elevation in AST, ALT and CPK was observed in monkeys only. Pharmacokinetic studies indicated maximal plasma concentrations (Cmax) after a single SC injection are reached at 4h in mice and 4-6h in monkeys, and T1/2 was calculated to be about 2h in mice and 10-20h in monkeys. Detectable amount of CTLA4-FasL were still found after 48h in moneys, but could not be found in mice at 24h.
In summary, the fusion protein CTLA4-FasL, with its unique hexameric structure and bi-functional mode of action, was shown to be an effective treatment for B cell lymphoma in multiple pre-clinical studies. Transient and dose-dependent leukopenia was the only significant adverse effect seen only in monkeys. A Phase I/IIa clinical study with CTLA4-FasL (KAHR-102) in lymphoma patients expressing both B7 and FasL on tumor cells, is planned to start in 2016.

Disclosures
Amsili: KAHR Medical LTD: Employment, Other: options. Baru:KAHR Medical ltd: Employment, Other: Options. Makdasi:KAHR Medical LTD: Other: Options. Tzdaka:KAHR Medical LTD: Other: Options. Shkedy:KAHR Medical LTD: Other: Options. Shani:KAHR Medical LTD: Employment, Other: Options, Patents & Royalties: Patents but no royalties. Dranitzki Elhalel:KAHR Mdical LTD: Consultancy, Other: Options, Patents & Royalties: patents but no royalties, Research Funding.