Immunological efficacy of glypican-3 peptide vaccine in patients with advanced hepatocellular carcinoma

Figures & data

Table 1. Patient characteristics and clinical response.

Patient No.	HLA-A	Age/Sex		Stage^a(UICC)	PS^b	Child-Pugh	Hepatic Virus infection^c	Prior therapy^d	Number of vaccinations	Tumor response^e	PFS^f (months)
1	0207	71	M	IIIA	0	A	C	RFA,TACE	9	PD	2
2	0206	55	M	IVB	1	A	B	SOR,RT	3	PD	2
3	0207	62	M	IIIB	0	B	C	TACE,SOR	2	PD	1
4	2402	54	F	IVB	0	A	B	Ope,RFA,SOR	5	PD	2
5	0201	69	M	IVB	0	A	—	TACE,S-1	4	PD	2
6	2402	65	M	IIIB	0	A	—	TAI	13	SD	4
7	2402	63	M	IVB	0	A	C	Ope,RT,SOR,UFT	3	PD	2
8	0201/0206	73	M	II	0	A	C	Ope,TACE,SOR,RAM	5	PD	2
9	2402	71	M	IIIB	0	A	C	Ope,RFA,TACE	4	PD	2
10	2402	70	F	IVB	0	A	—	Ope,RT,TACE	3	PD	2
11	2402	71	M	II	0	A	C	TACE,Ope,SOR	3	PD	2

^a Stage: Staging was performed according to the TNM classification for HCC (Union for International Cancer Control: UICC)

^b PS, performance status.

^c Hepatic virus infection B. HBsAg was examined by radioimmunoassay. C: HCV was detected by RT-PCR.

^d Prior therapy. RFA, radiofrequency ablation; TACE, transcatheter arterial chemoembolization; SOR, sorafenib; RT, radiotherapy; Ope, surgery; S-1, tegafur, gimeracil, oteracil potassium; proton, proton beam therapy; TAI, transcatheter arterial injection; UFT, tegafur plus uracil; RAM, ramucirumab.

^e Tumor response. Tumor responses were evaluated according to Response Evaluation Criteria in Solid Tumors (RECIST) guidelines and modified RECIST (mRECIST) assessment. The assessment of tumor response according to mRECIST was the same as that according to RECIST in all 11 patients.

^f PFS, progression free survival.

Table 2. GPC3 specific CTL response.

	Expression in the primary tumor^a
	GPC3				The spot number ofGPC3 specific CTL^c
Patient No.	Pre vaccine	Post vaccine	HLA class I	Plasma GPC3^b (pg/ml)	Pre vaccine	Post vaccine	increased CTL	OS^d(month)
1	+++	++	+	228.8	0	207	+	17
2	++		–	201.9	2	127	+	2
3	+	Weak	+	19.3	0	84	+	2
4	++		+	849.2	0	2	+	3
5	+		+	6.4	0	9	+	4
6	+	+/− hetero	–	46.5	0	290	+	21
7	++		+	107.9	1	33	+	5
8	++	+	–	47.1	0	2	+	12
9	++		+	691.7	1	1	–	3
10	++		+	19.9	0	81	+	20
11	++		+	1976.1	3	1	–	7

^a Expression in the primary tumor. Expression of GPC3 and HLA class I was determined by immunohistochemistry. Staining of tumor cells for GPC3, HLA class I: -, non-reactive; +, reactive.

^b Plasma GPC3, TheGPC3 plasma level before vaccination

^c Number of GPC3-specific CTL spots. The number of GPC3 peptide-specific CTL spots (post-vaccination) was the maximum number of spots in an ex vivo IFN-γ ELISPOT assay for GPC3 peptide, performed after vaccination and using 5 × 10⁵ PBMCs.

^d OS, overall survival.

Figure 1. The plasma and serum levels of 3 tumor markers—GPC3 (pg/mL), AFP (mg/mL), and PIVKA-II (AU/mL)—in 11 patients during follow-up. The cut-off thresholds for AFP and PIVKA-II were 10 ng/mL and 40 mAU/mL, respectively.

Figure 2. Skin reactions at the injection site in patients after third vaccination. The numbers within the parentheses represent the maximum spot number of GPC3-specific CTLs after vaccination. The 2 patients on the left exhibited stronger injection site reactions relative to those shown on the right.

Figure 3. GPC3 peptide vaccine improved overall survivals correlated with peptide-specific CTLs. (A) Kaplan-Meier curves for overall survival. Patients with GPC3-specific CTL frequencies ≥ 50 exhibited longer survival than those with GPC3-specfic CTL frequencies < 50 (p = 0.178). (B) Correlation between GPC3-specfic CTL frequencies and overall survival. GPC3-specfic CTL frequencies after vaccination were significantly correlated with overall survival (p = 0.032, r = 0.645).

Figure 4. Immunological monitoring of GPC3 peptide-specific T cell responses (A) Ex vivo IFN-γ ELISPOT assay for GPC3 in 5 × 10⁵ PBMCs was performed before and after vaccination in Patient 6. The spot number indicates the number of GPC3 peptide-specific CTLs. The number of IFN-γ-positive spots increased from 0 to 290 in wells preincubated with GPC3 peptide. (B) Ex vivo GPC3 Dextramer staining before and after vaccination in Patient 6. GPC3 peptide-specific CTL frequency is indicated as the percentage of Dextramer-positive CTLs to CD8-positive cells in PBMCs and tumor specimens. (C) Establishment of GPC3 peptide-specific CTL clones in the tumor specimen. Dextramer analysis (left) and IFN-γ ELISPOT assay (right) of the established clones are shown. (D) CTL clone reactivity (TIL 1) against cancer cell lines. Cytotoxic effects of CTL clones against peptide-pulsed T2A24 target cells. HIV_583–591 peptide-pulsed targets were used as negative controls. (E) IFN-γ ELISPOT assay against SK-Hep-1/vec, SK-Hep-1/hGPC3, and peptide-pulsed T2A24. Effector/target (E/T) ratio = 0.2. (F-G) Cytokine production by CTL clones (1.0 × 10⁵ cells/well) after 24-h co-culture with the indicated target cells (5 × 10⁴ cells/well). Data represent mean ± SD of triplicate cultures.

Table 3. Sequence analysis of GPC3 specific CTLs sorted from PBMCs.

No.	BV	BD	BJ	CDR3 amino acids	frequency^†	same sequence with TIL clone
1	TRBV5–1	TRBD2	TRBJ2–7	CASQQSSGVAIHEQYF	3/77 (3.9%)	TIL clone 1
2	TRBV5–1	TRBD2	TRBJ2–3	CASSVTSGRTHTDTQYF	10/77 (13.0%)
3	TRBV5–4	TRBD1	TRBJ1–3	CASSPGTFSGNTIYF	12/77 (15.6%)
4	TRBV6–1	TRBD1	TRBJ2–7	CASSRPLLGGGLYEQYF	15/77 (19.5%)	TIL clone 2
5	TRBV9	TRBD1	TRBJ1–2	CASRGTGSMYGYTF	3/77 (3.9%)
6	TRBV9	TRBD2	TRBJ2–1	CASSVGSGGGNEQFF	8/77 (10.4%)

^† Frequency indicate number of cells that have same TCR sequence in 77 sorted GPC3-dextramer⁺ cells from PBMCs.