Concomitant Administration of Gene-Modified T Cells Expressing a Chimeric CD16-CD3ζ Receptor with Mogamulizmab Synergistically Suppresses Adult T Cell Leukemia Cells in Vivo
Authors: Tanimoto Kazushi , Fujiwara Hiroshi , Tanaka Hiroki , Ochi Fumihiro , Asai Hiroaki , Okamoto Sachiko , Mineno Junichi , Kuzushima Kiyotaka , Shiku Hiroshi , Barrett John , Yasukawa Masaki 
Published: November 2019

Abstract
[Background and purpose] Mogamulizumab, a newly developed monoclonal antibody (mAb) targeting the receptor for C-C chemokine 4 (CCR4) has initially demonstrated a promising clinical outcome for the treatment of therapy-resistant Adult T cell leukemia (ATL) of which tumor cells broadly express CCR4. However, in practice, we sometimes encounter unsuccessful cases of ATL treated with mogmulizumab, especially in the setting of combination therapy with multiple anticancer agents. In those cases, ATL tumor cells were still positive for CCR4, but the chemotherapy-induced lymphocytopenia was reproducibly noticeable. Considering the pharmacologic action of mogamulizumab, the defucosylation-enhanced antibody-dependent cellular cytotoxicity (ADCC) exerted by FcγRIIIa (CD16) expressing effector cells in vivo, as observed for Natural Killer (NK) cells, we hypothesized that adoptive transfer of ADCC effector cells might be able to regain the anti-ATL efficacy of mogamulizumab. Accordingly, we examined in vivo the feasibility of T cells gene-modified to express a newly generated chimeric CD16-CD3ζ receptor as a transferable alternative effector cell for mogamulizumab.
[Methods] A novel affinity-matured chimeric CD16 with a 158V/V-CD3ζ (cCD16ζ ) gene construct was lentivirally introduced into CD3+ T cells from both healthy individuals (n=4) and patients with adult T cell leukemia (ATL) (n=3) (cCD16ζ-T cells). Using ATL or HTLV-1 infected cell lines variably expressing CCR4 on the cell surface (n=7), and primary ATL tumor cells (n=3), in the context of ADCC activity, functional properties of cCD16ζ-T cells were extensively examined both in vitro and in vivo, compared with those of NK cells from healthy donors (n=3). Next, we examined the in vivo therapeutic efficacy of concomitantly infused cCD16ζ-T cells with mogamulizumab via tail vein using a xenografted mouse model. Finally, we examined the feasibility of double gene-modified T cells to express human telomerase reverse transcriptase (hTERT)-specific TCR and cCD16ζ receptor, because we recently demonstrated that HLA-A*24:02-restricted and hTERT461-469 (VYGFVRACL)-specific TCR gene-modified CD8+ T cells displayed the cytocidal activity against ATL tumor cells (Blood, 2014).
[Results] cCD16ζ-T cells were readily expandable in ex vivo culture using anti-CD2/CD3/CD28 beads and recombinant human (rh)IL-2, and they successfully displayed ADCC-mediated tumoricidal activity against CCR4+ MT-4 and ATN-1 (ATL cell lines) cells, but not CCR4- K562 cells with mogamulizumab, in a effector cell number and an antibody dose dependent manner. Pharmacological dose of 0.1μg/ml mogamulizumab could bind to ATL cell line cells expressing variable extent of CCR4 ranging from MFI 1.01 for HUT102 cell line to 12.3 for MT-2 (0.57 for K562 as a negative control), and could mediate the similar degree of ADCC activity exerted by cCD16ζ-T cells. The magnitude of ADCC activity mediated by cCD16ζ-T cells against opsonized ATN-1 with mogamulizumab was almost similar to that by activated NK cells using rhIL-2. This cytotoxicity was inhibited by anti-CD16 mAb. During ADCC, ligation of opsonized cancer cells with mogamulizumab to cCD16ζ receptor stimulated cCD16ζ-T cells to release toxic granules shown by CD107a expression. cCD16ζ-T cells generated from patients with ATL (n=3) successfully displayed ADCC activity against autologous tumor cells in vitro. Human cCD16ζ-T cells infused concomitantly with mogamulizumab synergistically inhibited the growth of disseminated luciferase gene-modified ATN-1 cells in immunodeficient mice, demonstrated using an in vivo bioluminescence assay. Additionally, this tumor suppressive effect contributed to the longer survival of treated mice. Finally, the double-gene modified CD3+ T cells could successfully recognize ATN-1 through both the mogamulizumab-opsonized CCR4 and hTERT epitope/HLA-A24*02 complex on the cell surface.

Disclosures
Okamoto: CDM Center, Takara Bio Inc.: Employment. Mineno:Takara Bio Inc.: Employment. Shiku:Takara Bio Inc.: Research Funding.