GLYPICAN 3 – SPECIFIC CAR T IN COMBINATION WITH SORAFENIB AS A NOVEL THERAPEUTIC TREATMENT FOR HEPATOCELLULAR CARCINOMA TL. Trinha,b, Q. Wua, L. Changa, Mitchell Hoc, C. Liua,d* aDepartment of Pathology, Immunology and Laboratory Medicine, University of Florida, Gainesville, FL, USA bDepartment of Immunology, Moffitt Cancer Center, Tampa, FL, USA CNational Cancer Institute, Bethesda, MD, USA dDepartment of Pathology and Laboratory Medicine, Rutgers Robert Wood Johnson Medical School, Biscataway, NJ, USA
RESULTS
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(A) GPC3-specific CAR T construct using anti-GPC3 HN3 scFv sequence. (B) GPC3 expression in HCC cell lines (Huh7, HepG2) and primary hepatocyte (Hu1545V) by FLOW (left) and W.B. (right).
(A) Lentiviral transduction efficiency. (B) Affinity of CAR lentiviral transduced T cells. (C) Killing capacity of CAR Ts showing specific lysis towards GPC3positive HCC cells (Huh7 and HepG2) but not GPC3-negative cell (Hu1545V). CAR Ts were incubated with cells for 24h with different E:T ratio.
Fig. 4 Synergistic Effect of Sorafenib and CAR T Treatment 1 .5
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GPC3-specific CAR T Effectively Lyses HCC but Not Normal Liver Cell Lines After 16h of incubation at the E (αGPC3 CAR T cells) : T (Huh7 or HepG2 cells) ratio of 1:1, α-GPC3 CAR T cells effectively lysed Huh7 (39%) and HepG2 (77%) but not primary hepatocytes ( 9%), while the control CAR T cells (α-CD30 CAR) could not initiate a specific lysis on those cells. At the E:T ratio of 3:1, the specific killing increased to 78% (Huh7) and 99% (HepG2) while remaining low with primary hepatocytes (11%)’ Combination of Sorafenib and CAR T Treatment Show Synergistic Effect At the very low E:T ratio of 1:10, combination with Sorafenib priming (24h, IC10 concentration) prior to CAR T incubation boosted the specific lysis up to 25% (p=0.0416).
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Conclusions
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References To whom correspondence should be addressed: Chen Liu,
[email protected]
Fig.3: PD-L1 is Upregulated in Cancer Cells Treated with CAR T
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The fourth generation of GPC3specific chimeric antigen receptor (CAR) was constructed using a lentiviral vector containing CD28, CD27 and 41BB costimulatory domains, a CD3ζ signaling domain fused to a FKBP-iCasp9 apoptosis inducing gene, and a GPC3-specific sequence derived from HN3, a human single-domain VH monoclonal antibody.
Fig.2: Specificity of GPC3 CAR T
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Methods
Fig.1: GPC3 CAR T Construct
R e la t iv e G P C 3 e x p r e s s io n
FDA-approved targeted therapy for advanced HCC is limited with the only option of Sorafenib. Glypican 3 (GPC3) is a cell surface heparan sulfate proteoglycan, shown to be up-regulated in HCC but not normal tissues. In our study, we developed T cells expressing the fourth generation of GPC3-specific chimeric antigen receptor (α-GPC3 CAR) that efficiently eliminated GPC3positive HCC cells without killing GPC3-negative primary liver cell.
Principal Findings
(A) GPC3 expression is not altered by Sorafenib treatment. (B) PD-L1 expression is down-regulated in long-term Sorafenib treatment (7days). (C) Synergistic effect of the combination between Sorafenib and CAR T treatment.
(A) CAR Ts proliferates when incubating with cancer cell. (B) Cytokines are upregulated showing CAR T is activated and ready to kill.
(A) PD-L1 is upregulated in HCC cells treated with GPC3specific CAR T (α-GPC3) but not in primary hepatocyte or cells treated with non-specific CAR Ts (mock or αCD30). Anti-PD-L1 treatment helps reduce PD-L1 expression and facilitates better cell lysis. In both Huh7 (B) and HepG2 (C).
These results indicate that combination of GPC3-specific CAR T transfer and Sorafenib regimen could be a promising therapeutic option for the treatment of HCC, with the doses of both CAR T and Sorafenib reduced up to 10 times, hencelower side effects while preserving the efficacy of tumorspecific cytotoxicity.
