Immunological evaluation of personalized peptide vaccination in refractory small cell lung cancer

Abstract

Since the prognosis of small cell lung cancer (SCLC) remains poor, development of new therapeutic approaches, including immunotherapies, would be desirable. In the current study, to evaluate immunological responses in refractory SCLC patients, we conducted a small scale phase II clinical trial of personalized peptide vaccination (PPV), in which vaccine antigens are selected based on pre‐existing host immunity. Ten refractory SCLC patients, who had failed to respond to chemo‐ and/or chemoradiotherapies (median number of regimens, 2.5; median duration, 20.5 months), were enrolled. A maximum of four human leukocyte antigen (HLA)‐matched peptides showing higher antigen‐specific humoral responses were subcutaneously administered (weekly for six consecutive weeks and then bi‐weekly thereafter). PPV was terminated before the 3rd administration in four patients because of rapid disease progression, whereas the remaining six patients completed at least one cycle (six times) of vaccinations. Peptide‐specific immunological boosting was observed in all of the six patients at the end of the first cycle of vaccinations, with their survival time of 25, 24.5 (alive), 10 (alive), 9.5, 6.5, and 6 months. Number of previous chemotherapy regimens and frequency of CD3⁺ CD26⁺ cells in peripheral blood were potentially prognostic in the vaccinated patients (hazard ratio [HR] = 2.540, 95% confidence interval [CI] = 1.188–5.431, P = 0.016; HR = 0.941, 95% CI = 0.878–1.008, P = 0.084; respectively). Based on the feasible immune responses in refractory SCLC patients who received at least one cycle (six times) of vaccinations, PPV could be recommended for a next stage of larger‐scale, prospective clinical trials. (Cancer Sci 2012; 103: 638–644)

Although recent advances in chemotherapies contributed to improved clinical outcomes in refractory small cell lung cancer (SCLC) patients, their prognosis still remains very poor with a median survival time of 6–10 months.1, 2, 3 Several clinical trials of immunotherapies have been attempted in refractory SCLC patients,4, 5 but none of them demonstrated a meaningful therapeutic benefit to patients. We have developed a novel regime of personalized peptide vaccination (PPV), in which vaccine antigens are selected and administered based on the pre‐existing host immunity before vaccination.6, 7, 8, 9, 10, 11, 12, 13 For example, a recently conducted randomized clinical trial in advanced prostate cancer patients showed a promising clinical benefit of PPV.7 In the current study, to address if refractory SCLC patients have the capability to respond to cancer vaccines, we conducted a small scale phase II study of PPV and evaluated immunological responses in the vaccinated patients.

Materials and Methods

Patients

Patients with histological diagnosis of SCLC were eligible for inclusion in the current study, if they had failed to respond to previous chemotherapies and/or chemoradiotherapies. They also had to possess positive humoral responses to at least two of the 31 different vaccine candidate peptides (Table S1), determined by both human leukocyte antigen (HLA) class I types and the titers of IgG against each peptide. The other inclusion criteria as well as exclusion criteria were not largely different from those of the previously reported clinical studies;6, 7, 8, 9 an age between 20 and 80 years; an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1; positive status for HLA‐A2, ‐A3, ‐A11, ‐A24, ‐A26, ‐A31, or ‐A33; life expectancy of at least 12 weeks; adequate hematologic, renal, and hepatic function. Patients with lymphocyte counts of <1000 cells/μL were excluded from the study, since we previously reported that pre‐vaccination lymphopenia is an un‐favorable factor for overall survival (OS) in cancer patients receiving PPV.11 Other exclusion criteria included pulmonary, cardiac, or other systemic diseases; an acute infection; a history of severe allergic reactions; pregnancy or nursing; or other inappropriate conditions for enrollment judged by clinicians. The protocol was approved by the Kurume University Ethical Committee and conforms to the provisions of the Declaration of Helsinki in 1995 (as revised in Tokyo 2004). It was registered in the UMIN Clinical Trials Registry (UMIN# 2984). After full explanation of the protocol, written informed consent was obtained from all patients before enrollment.

Clinical protocol

This was an open‐label phase‐II study, in which the primary and secondary endpoints were to identify biomarkers for OS and to evaluate safety in refractory SCLC patients who received PPV, respectively. Thirty‐one peptides (PolyPeptide Laboratories, San Diego, CA, USA; American Peptide Company, Vista, CA, USA), whose safety and immunological effects had been confirmed in previously conducted clinical studies,6, 7, 8, 9, 10, 11, 12, 13 were used for vaccination (Table S1). The frequencies of expression of the parent proteins, from which the vaccine peptides were derived, in SCLC tissues were examined by immunohistochemistry (Fig. S1) and shown in Table S1. The right peptides for vaccination to individual patients were selected in consideration of the pre‐existing host immunity before vaccination, assessed by the titers of IgG specific to each of the 31 different vaccine candidates, as previously described.14 Although the prostate‐related antigens, including prostate‐specific antigen (PSA), prostatic acid phosphatase (PAP), and prostate‐specific membrane antigen (PSMA), have been reported to be expressed not only by prostate cancer but also by other types of cancers,15, 16, 17, 18 the expression frequencies of these molecules in SCLC tissues were low (Table S2). Therefore, the peptides derived from them were selected only when pre‐existing IgG responses to other remaining peptides were absent. A maximum of four peptides (3 mg/each peptide), which were selected based on the results of HLA typing and peptide‐specific IgG titers, were subcutaneously administered with incomplete Freund's adjuvant (Montanide ISA51; Seppic, Paris, France) once a week for consecutive 6 weeks. After the first cycle of six vaccinations, up to four antigen peptides, which were re‐selected according to the titers of peptide‐specific IgG at every cycle of six vaccinations, were administered every 2 weeks up to four cycles (24 vaccinations). Combined chemotherapy and/or radiotherapy were allowed during the vaccination. Adverse events were monitored according to the National Cancer Institute Common Terminology Criteria for Adverse Events version 3.0 (NCI‐CTC Ver 3.0). The clinical responses were evaluated using the Response Evaluation Criteria in Solid Tumors (RECIST 1.1) after the first cycle of vaccinations or at premature termination from the study. Pre‐vaccination blood samples (PBMCs and plasma) were available from all of the enrolled patients (n = 10). Post‐vaccination blood samples were available from six and four patients, who completed the first and second cycles of vaccinations, respectively.

Measurement of humoral and T cell responses

The humoral responses specific to each of the 31 peptide candidates (Table S1) were determined by peptide‐specific IgG levels using the Luminex system (Luminex, Austin, TX, USA), as previously reported.14 If the titers of peptide‐specific IgG to at least one of the vaccine peptides in the post‐vaccination plasma were more than twofold higher than those in the pre‐vaccination plasma, the changes were considered to be significant.

T cell responses specific to the vaccine peptides were evaluated by interferon (IFN)‐γ ELISPOT assay (MBL, Nagoya, Japan). Briefly, PBMCs (2.5 × 10⁴ cells/well) were incubated in 384‐well microculture plates (IWAKI, Tokyo, Japan) with 25 μL of medium (OpTmizer T Cell Expansion SFM; Invitrogen, Carlsbad, CA, USA) containing 10% FBS (MP Biologicals, Solon, OH), interleukin (IL)‐2 (20 IU/mL; AbD serotec, Kidlington, UK), and each peptide (10 μM). Half of the medium was replaced with new medium containing the corresponding peptide (20 μM) at day 3. After incubation for the following 6 days, the cells were harvested and tested for their ability to produce IFN‐γ in response to either the corresponding peptides or negative control peptides from human immunodeficiency virus (HIV). Antigen‐specific IFN‐γ secretion after 18‐h incubation was determined by ELISPOT assay with an ELISPOT reader (ImmunoSpot S5 Versa Analyzer; Cellular Technology Ltd, Shaker Heights, OH, USA). Means of the triplicate samples were used for analyses. Antigen‐specific T cell responses were evaluated by the differences between the spot numbers in response to the corresponding peptides and those to the control peptide; differences of at least 10 spot numbers per 10⁵ PBMCs were considered as positive. If the spot numbers in response to at least one of the vaccine peptides in the post‐vaccination PBMCs were more than twofold higher than those in the pre‐vaccination PBMCs, the changes were considered as significant.

Measurement of C‐reactive protein, serum amyloid A, and cytokines

C‐reactive protein (CRP), serum amyloid A (SAA), and IL‐6 in plasma were examined by ELISA using the kits from R&D systems (Minneapolis, MN, USA), Invitrogen, and eBioscience (San Diego, CA, USA), respectively. Multiplexed bead‐based Luminex assays were used to measure Th1/Th2 cytokines, including IL‐2, IL‐4, IL‐5, and IFN‐γ (Invitrogen). Frozen plasma samples were thawed, diluted, and assayed in duplicate in accordance with the manufacturer's instructions. Means of the duplicate samples were used for analyses.

Flow cytometric analysis of immune cell subsets in PBMCs

A suppressive immune cell subset, myeloid‐derived suppressor cells (MDSCs), in PBMCs was examined by flow cytometry. For analysis of MDSCs, PBMCs (0.5 × 10⁶) were incubated for 30 min at 4°C with mAbs against lineage markers (CD3, CD14, CD19, CD56), CD33, and HLA‐DR. In the cell subset negative for the lineage markers and HLA‐DR, MDSCs were identified as positive for CD33. The frequency of MDSCs in the mononuclear cell gate defined by the forward scatter and side scatter was calculated. In addition, the expression of CD26 in PBMCs was analyzed, since the gene expression level of this molecule assessed by DNA microarray analysis was prognostic for OS in the prostate cancer patients receiving PPV (Sasada T , Komatsu N, Itoh K, unpublished observation). PBMCs were stained with anti‐CD26 and anti‐CD3 mAbs followed by calculation of the frequencies of CD26⁺ subset in CD3⁺ cells. The samples were run on a FACSCanto II (BD biosciences, San Diego, CA, USA), and data were analyzed using the Diva software (BD biosciences). All mAbs were purchased from Biolegend (San Diego, CA, USA).

Immunohistochemistry

Anti‐tumor immune responses were examined by immunohistochemistry (IHC) in tumor tissues resected from SCLC patients treated with PPV (n = 1, Patient No. 5) or without PPV (n = 3). Paraffin‐embedded tissue samples were cut into 4‐μm sections, and labeled on the BenchMark XT (Ventata Automated Systems Inc., Tucson, AZ, USA) with anti‐CD3 (clone LN10; Novocastra, Newcastle, UK), anti‐CD4 (clone 4B12, Novocastra), and anti‐CD8 (clone 4B11, Novocastra) mAb. The streptavidin‐biotin complex method with 3,3′‐diaminobenzidine tetrachloride (DAB) was used as a chromogen (Ventana iVIEW DAB Detection Kit). The expressions of vaccine antigens SART3 and p56lck in the tumor tissue from the patient treated with PPV (Patient No. 5) were also examined by IHC with anti‐SART3 (rabbit polyclonal; Abcam, Cambridge, UK) and anti‐p56lck (rabbit polyclonal, Abcam) Abs.

Statistical analysis

The Wilcoxon test was used to compare differences between pre‐ and post‐vaccination measurements. All tests were two‐sided, and differences at P < 0.05 were considered to be statistically significant. OS time was calculated from the first day of peptide vaccination until the date of death or the last date when the patient was known to be alive. Curves for OS were estimated by the Kaplan–Meier method. Potentially prognostic factors were evaluated by the Cox proportional hazards model. A value of P < 0.1 was used to identify potentially significant variables. All statistical analyses were conducted using the JMP version 9 or SAS version 9.1 software package (SAS Institute Inc., Cary, NC, USA).

Results

Patients’ characteristics

Between March 2009 and October 2010, 10 patients with histology of SCLC were enrolled in this study. Table 1 shows the clinicopathological characteristics of the enrolled patients. All patients were male subjects with a median age of 63.5 years, ranging from 48 to 69. They had advanced stages of cancer (limited‐stage disease [LD] at diagnosis, n = 5; extended‐stage disease [ED] at diagnosis, n = 5), which had been refractory to previous treatments. Before enrollment, they failed to respond to one (n = 3), two (n = 2), three (n = 2), or more than 4 (n = 3) regimen(s) of chemotherapies and/or chemoradiotherapies. Median duration of these preceding regimens prior to the PPV was 20.5 months, ranging from 1 to 51. Performance status at the time of enrollment was grade 0 (n = 7) or grade 1 (n = 3). The numbers of peptides vaccinated to the patients at the first cycle of vaccinations were four peptides in eight patients and two in two patients. Of the 10 patients, six completed the first cycle of six vaccinations, whereas the remaining four patients failed before the 3rd vaccinations due to rapid disease progression. The median number of vaccinations was 10.5 with a range of 1–24. During the PPV, seven patients were treated in combination with chemotherapies and/or radiotherapy, and the remaining three patients did not tolerate them. None had a complete response (CR) or partial response (PR). The best response, seen in two patients, was stable disease (SD), whereas seven patients had progressive disease (PD). A patient without measurable lesions (Patient No. 6) had Non‐CR/non‐PD.

Table 1.

Characteristics of the enrolled patients with refractory SCLC (n = 10)

Patient No.	HLA Type	Gender	Age	Stage at diagnosis	PS	No. previous regimens	Previous treatment period (months)	Disease location (tumor size) before vaccination	No. vaccinations	Combined therapy	Treatment response†	OS (days)
1	A2/A26	M	58	ED	0	2	32	Mediastinal LN (28 mm), cervical LN‡, brain‡	24	CBDCA, PTX	PD	771
2	A24	M	68	LD	0	3	26	Pleural dissemination‡	2	(–)	PD	17
3	A24	M	62	LD	0	4	19	Cervical LN‡, liver (13 mm)	11	VNR	PD	178
4	A24/A26	M	52	ED	1	6	22	Liver (30 mm), bone (spine)‡, atelectasis‡	2	CBDCA, PTX	PD	16
5	A31/A33	M	67	LD	0	1	51	Lung (36 mm), brain‡	24	CDDP, VP16, WBRT	SD	746§
6	A2/A26	M	51	ED	0	2	5	Mediastinal LN‡, bone (spine)‡	10	AMR	Non‐CR/non‐PD	285
7	A26/A31	M	65	LD	0	5	31	Lung (39 mm), adrenal (40 mm, 18 mm), brain (10 mm), mediastinal LN‡	2	CPT11, PTX	PD	33
8	A2/A24	M	69	ED	1	3	10	Pancreas (19 mm), mediastinal LN (15 mm)	14	(–)	PD	195
9	A2/A26	M	69	ED	1	1	3	Lung (50 mm), brain‡	1	(–)	PD	89
10	A2/A24	M	48	LD	0	1	1	Mediastinal LN (16 mm)	21¶	AMR, TPT, SRT	SD	306§

Evaluated by the Response Evaluation Criteria in Solid Tumors (RECIST 1.1). ‡Non‐measurable lesion. §Patients alive (censored data). ¶Under treatment. AMR, amrubicin; CBDCA, carboplatin; CDDP, cisplatin; CPT11, irinotecan; CR, complete response; ED, extensive‐stage disease; LD, limited‐stage disease; LN, lymph node; M, male; OS, overall survival; PD, progressive disease; PS, performance status; PTX, paclitaxel; SCLC, small cell lung cancer; SD, stable disease; SRT, stereotactic radiotherapy; TPT, topotecan; VNR, vinorelbine; VP16, etoposide; WBRT, whole brain radiotherapy.

Toxicities

Toxicities are shown in Table 2. The most frequent adverse events were dermatological reactions at injection sites (n = 7), hematological toxicity (n = 10), and hypoalbuminemia (n = 8). Grade 3 serious adverse events (SAE) were as follows: dyspnea (n = 1), anemia (n = 1), leukocytopenia (n = 1), and lymphopenia (n = 1). The Grade 3 hematological SAE, including anemia, leukocytopenia, and lymphopenia, were transiently observed in the Patient No. 1 during PPV, just after he started receiving a concomitant chemotherapy with carboplatin and paclitaxel. But these SAE disappeared soon after stopping the concomitant chemotherapy, and did not recur even if he restarted the vaccinations after his recovery from the SAE. In addition, he showed no hematological SAE before this episode, while he received no concomitant chemotherapies. Based on these observations, the independent safety evaluation committee for this trial concluded that these SAE might not be directly associated with the vaccinations, but with the concomitant chemotherapy. The Grade 3 dyspnea was observed in Patient No. 2, who rapidly developed pleural effusion due to pleural dissemination and required hospitalization for oxygen supplementation. Since this symptom was highly likely to be caused by the rapidly progressing disease, the independent safety evaluation committee concluded that it might not be directly associated with the vaccinations.

Table 2.

Toxicities

	Grade 1	Grade 2	Grade 3	Grade 4	Total
Injection site reaction	3	4	0	0	7
Constitutional symptom
Fever	0	1	0	0	1
Fatigue	2	0	0	0	2
Gastrointestinal
Anorexia	2	0	0	0	2
Nausea	1	0	0	0	1
Pulmonary/Upper respiratory
Dyspnea	0	0	1	0	1
Blood/Bone marrow
Anemia	8	1	1	0	10
Leukocytopenia	3	0	1	0	4
Neutropenia	0	1	0	0	1
Lymphopenia	3	0	1	0	4
Thrombocytopenia	1	0	0	0	1
Laboratory
AST elevation	0	1	0	0	1
ALT elevation	1	1	0	0	2
γ‐GTP elevation	1	0	0	0	1
Creatinine elevation	1	1	0	0	2
Hypoalbuminemia	8	0	0	0	8
Hyperkalemia	1	0	0	0	1
Hyponatremia	1	0	0	0	1
Hyperglycemia	1	0	0	0	1
Hyperuricemia	1	0	0	0	1

ALT, alanine aminotransferase; AST, aspartate aminotransferase; GTP, glutamyl transpeptidase.

Immune responses to the vaccine peptides

Both IgG and T cell responses specific to the vaccine peptides were analyzed in blood samples before and after vaccinations (Table 3). Plasma samples were obtained from 10, six and four patients before and at the end of the first (six vaccinations) and second (12 vaccinations) cycles of vaccinations, respectively. For monitoring of humoral responses, the titers of peptide‐specific IgG reactive to each of 31 different peptides were measured by bead‐based multiplex assay. The IgG responses specific to at least one of the vaccine peptides were augmented in five of six patients (83%) and in all of four patients (100%) examined at the end of the first and second cycles of vaccinations, respectively.

Table 3.

Immunological responses to the vaccine peptides

Patient No.	Peptide	IgG response†			T cell response‡
		Before	1st	2nd	Before	1st	2nd
1	Lck‐422	185	252	0	0	1000	2050
	HNRPL‐140	428	723	1155	0	119	447
	SART3‐109	224	657	2028	1309	294	186
	WHSC2‐103	554	1332	16987	0	264	543
	MAP‐432§	176	290	0	0	53	949
2	SART2‐93	6609	NA	NA	0	NA	NA
	PSA‐248	8975	NA	NA	0	NA	NA
	SART2‐161	7979	NA	NA	0	NA	NA
	PSMA‐624	7555	NA	NA	0	NA	NA
3	SART2‐93	80	0	NA	146	0	NA
	MRP3‐503	410	3040	NA	0	2389	NA
	SART2‐161	166	0	NA	125	0	NA
	Lck‐486	76	413	NA	0	364	NA
	PAP‐213§	0	146	NA	NA	NA	NA
	PSMA‐624§	38	42	NA	NA	NA	NA
4	PAP‐213	552	NA	NA	0	NA	NA
	PSMA‐624	266	NA	NA	333	NA	NA
	MAP‐432	200	NA	NA	1333	NA	NA
	WHSC2‐103	591	NA	NA	0	NA	NA
5	SART3‐734	2142	11371	54795	1833	188	5390
	Lck‐449	45	31	21708	600	944	9500
	SART3‐109§	0	50	1854	NA	NA	0
	SART3‐511§	0	28	1328	NA	NA	107
6	MAP‐432	43	0	NA	0	227	NA
	HNRPL‐501	104	446	NA	0	444	NA
	UBE2V‐43	241	0	NA	157	71	NA
	SART3‐109	2075	2621	NA	0	694	NA
7	SART3‐109	174	NA	NA	117	NA	NA
	SART3‐511	25	NA	NA	42	NA	NA
	Lck‐90	85	NA	NA	0	NA	NA
	HNRPL‐501	294	NA	NA	41	NA	NA
8	SART2‐93	20	22	9222	0	56	NA¶
	PAP‐213	208	187	12293	86	0	NA¶
	PSA‐248	25	3856	18849	6	33	NA¶
	Lck‐486	35	67	17704	15	16	NA¶
9	CypB‐129	136	NA	NA	121	NA	NA
	Lck‐422	34	NA	NA	13	NA	NA
10	Lck‐246	74	63	3725	0	729	515
	WHSC2‐141	77	58	455	0	75	0
	PAP‐213	25	0	16345	0	89	166
	Lck‐486	41	0	1378	0	102	0
	CypB‐129§	70	86	81	0	0	19
	HNRPL‐140§	43	48	24	0	34	64

Values indicate the fluorescence intensity unit (FIU) of plasma IgG reactive with the corresponding peptides before and after the 1st and 2nd cycles of vaccinations. The augmented IgG responses are underlined. ‡Values indicate the number of spots per 10⁵ peripheral blood mononuclear cells (PBMCs) reactive with the corresponding peptides in IFN‐γ ELISPOT assay before and after the 1st and 2nd cycles of vaccinations. When the number of spots was <10 per 10⁵ PBMCs, the data are shown as “0”. The augmented T cell responses are underlined. §Peptides used for the 2nd cycle of vaccinations. ¶PBMCs unavailable. NA, not assessed.

T cell responses to the vaccine peptides were also measured by IFN‐γ ELISPOT assay (Table 3). PBMCs were available from 10, six and three patients before and at the end of the first and second cycles of vaccinations, respectively. Antigen‐specific T cell responses to at least one of the vaccine peptides were detectable in eight of 10 patients (80%) before vaccination, and augmented in five of six patients (83%) and in all of three patients (100%) tested at the end of the first and second cycles of vaccinations, respectively.

Collectively, at the end of the first cycle of six vaccinations, peptide‐specific immunological boosting assessed by IgG and/or T cell responses was observed in all of the six patients who received at least six vaccinations, with their survival time of 25, 24.5 (alive), 10 (alive), 9.5, 6.5, and 6 months.

Cytokines and inflammation markers

We then measured cytokines (IL‐2, IL‐4, IL‐5, IL‐6, and IFN‐γ) and inflammation markers (CRP and SSA) in the plasma before and at the end of the first cycle of vaccinations (Table 4). IL‐6 was detectable in five of 10 patients (50%) before vaccination with median of 0.5 pg/mL, ranging from 0 to 7 pg/mL. IL‐6 levels were increased, decreased, or unchanged in 2, 1, or 3 patients tested, respectively. There was no significant difference in the level of IL‐6 between before and after vaccinations (P = 0.500; Wilcoxon test). Other cytokines, including IL‐2, IL‐4, IL‐5, and IFN‐γ, were rarely detectable in either pre‐ or post‐vaccination plasma (data not shown).

Table 4.

Laboratory data before and after vaccination^a

Patient No.	IL‐6 (pg/mL)		CRP (mg/dL)		SAA (mg/dL)		MDSCs (%)		CD3+CD26+ (%)
	Before	After	Before	After	Before	After	Before	After	Before	After
1	0	0	0.39	0.56	8.58	7.78	0.3	0.6	48.2	58.4
2	7	NA	0.92	NA	12.65	NA	0.1	NA	29.8	NA
3	3	1	0.54	0.52	3.10	0.00	0.0	0.0	15.3	24.6
4	0	NA	0.47	NA	1.17	NA	0.1	NA	21.0	NA
5	1	2	0	0.56	0.28	3.99	0.2	0.1	32.9	34.8
6	3	9	0.39	0.61	5.47	11.95	0.2	0.5	49.7	57.3
7	1	NA	0.40	NA	5.48	NA	0.8	NA	19.0	NA
8	0	0	1.04	0.17	12.36	6.73	0.6	0.9	51.1	39.0
9	0	NA	0.94	NA	15.37	NA	0.4	NA	15.6	NA
10	0	0	0.45	0.53	0.13	0.55	0.1	0.1	39.4	28.3

^aValues before and after the 1st cycle of vaccinations are shown. CRP, C‐reactive protein; MDSCs, myeloid‐derived suppressor cells; NA, not assessed; SAA, serum amyloid A.

An inflammation marker, CRP, was detectable in pre‐vaccination plasma from the majority of patients (nine of 10 patients [90%]), with median value of 0.46 mg/dL (ranging from 0 to 1.04 mg/dL). Plasma CRP levels were increased or decreased in four or two patients, respectively. Another inflammation marker, SAA, was also detected in pre‐vaccination plasma from all of the patients (100%) with median value of 5.475 mg/dL (ranging from 0.13 to 15.37 mg/dL). Plasma SAA levels were increased or decreased in three or three patients, respectively. There were no significant differences in the levels of CRP as well as SAA between before and after vaccinations (P = 0.910 and P = 0.924, respectively; Wilcoxon test).

Flow cytometric analysis of immune subsets in PBMCs

Immune cell subsets in both pre‐vaccination and post‐vaccination PBMCs were examined by flow cytometry (Table 4). The median frequency of MDSCs in pre‐ and post‐vaccination PBMCs was 0.2% (range from 0 to 0.8%, n = 10) and 0.3% (range from 0 to 0.9%, n = 6), respectively. The median frequency of CD3⁺CD26⁺ cells in pre‐ and post‐vaccination PBMCs was 31.35% (range from 15.3 to 51.1%) and 36.9% (range from 24.6 to 58.4%), respectively. No significant differences were found in the frequencies of MDSCs and CD3⁺CD26⁺ between before and after the vaccinations (P = 0.140 and P = 0.825, respectively; Wilcoxon test).

Potentially prognostic factors in SCLC patients undergoing PPV

Median OS of the 10 patients was 186.5 days, with 1 year survival rate of 30% (Fig. 1). To identify potentially prognostic factors in refractory SCLC patients undergoing PPV, statistical analyses were carried out by the Cox proportional hazards model with clinical findings or laboratory data. As shown in Table 5, the number of previous chemotherapy regimens and frequency of CD3⁺CD26⁺ cells in PBMCs before vaccination were potentially prognostic in the patients receiving PPV (hazard ratio [HR] = 2.540, 95% confidence interval [CI] = 1.188–5.431, P = 0.016; HR = 0.941, 95% CI = 0.878–1.008, P = 0.084; respectively).

Figure 1.

Kaplan–Meier survival analysis in the enrolled patients. The median overall survival of patients who received personalized peptide vaccination (PPV) (n = 10; solid line) was 186.5 days and the 1 year survival rate was 30%. Dotted lines show 95% confidence intervals.

Table 5.

Statistical analysis with clinical findings and laboratory data

Factor	Hazard ratio (95% CI)a	P‐valuea
Age	1.047 (0.943–1.163)	0.393
Limited‐stage disease at diagnosis	1.250 (0.278–5.625)	0.771
Performance status (PS)	3.270 (0.651–16.427)	0.150
Number of previous treatment regimens	2.540 (1.188–5.431)	0.016
Previous treatment period (months)	0.989 (0.945–1.035)	0.637
Combined treatment (+)	0.336 (0.066–1.698)	0.187
IL‐6 (pg/mL)	1.299 (0.900‐1.877)	0.163
CRP (mg/dL)	7.459 (0.608–91.517)	0.116
SAA (mg/dL)	1.095 (0.940–1.275)	0.246
MDSCs (%)	2.872 (0.094–87.379)	0.545
CD3+CD26+ (%)	0.941 (0.878–1.008)	0.084

^aEvaluated by the Cox proportional hazards model. CI, confidence interval; CRP, C‐reactive protein; IL, interleukin; MDSCs, myeloid‐derived suppressor cells; SAA, serum amyloid A.

Accumulation of tumor‐infiltrating lymphocytes in a patient undergoing tumor resection after PPV

A patient (Patient No. 5), who had good immune responses to vaccine antigens and showed stable disease (24.5 months alive), underwent resection of the primary tumor after 24 vaccinations. The parent proteins for the used peptides, SART3 and p56lck, were expressed in the tumor tissue resected after the vaccinations (Fig. 2). To know the immune responses to the tumor following the vaccinations, tumor‐infiltrating lymphocytes were assessed by IHC using antibodies specific to immunological markers, including CD3, CD4, and CD8. In the tumor from this patient treated with PPV, CD3⁺ cells infiltrated densely not only within the cancer stroma but also within the cancer cell nest (Fig. 3a). These tumor‐infiltrating lymphocytes consisted of both CD4⁺ and CD8⁺ cells (Fig. 3b,c). In contrast, when the tumors from SCLC patients without PPV treatment (n = 3) were examined by IHC as a control, only a few cells positive for CD3, CD4, or CD8 accumulated within the tumors from all patients examined (representative data were shown in Fig. 3d–f). These results suggest the possibility that PPV induced anti‐tumor immunity mediated by CD4⁺ and CD8⁺ T cells, leading to better clinical outcomes.

Figure 2.

Expression of the vaccine antigens in the tumor from a small cell lung cancer (SCLC) patient undergoing surgery after personalized peptide vaccination (PPV) treatment. The vaccine antigens SART3 and p56lck were detected by immunohistochemistry (IHC) with the antibodies specific to these molecules in the tumor tissue from a patient undergoing surgery after PPV treatment (Patient No. 5). Both sections, ×200.

Figure 3.

Detection of tumor‐infiltrating lymphocytes in tumors from small cell lung cancer (SCLC) patients treated with or without personalized peptide vaccination (PPV). Immune cells infiltrating within tumors were detected by immunohistochemistry (IHC) with the antibodies against CD3 (a and d), CD4 (b and e), and CD8 (c and f). All sections, ×100. (a–c) Tumor from a SCLC patient after PPV treatment (Patient No. 5). (d–f) Tumor from a SCLC patient without PPV treatment. Since the tumors from three SCLC patients without PPV treatment showed similar findings, representative data are shown.

Discussion

Despite recent advances in chemotherapies for refractory SCLC patients, novel treatment modalities, including immunotherapies, still remain to be developed.1, 2, 3 However, there have been a few reports available regarding immunotherapies against SCLC.4, 5 For example, a DC‐based vaccine targeting p53 was reported to show a feasible result in a subset of SCLC patients, who had positive immune responses against p53. However, the induction rate of anti‐p53 immunity was relatively low.19, 20 Vaccinations with cell surface glycolipid antigens to induce antigen‐specific Ab responses were also attempted in several clinical studies.21, 22 However, only a limited number of patients developed a detectable Ab response, and there was no impact on clinical outcomes. In the current study, we addressed if refractory SCLC patients could have pre‐existing IgG responses to 31 different vaccine candidates and well respond to these peptide vaccines. Notably, our results demonstrated that pre‐vaccination plasma from all of the refractory SCLC patients had detectable levels of IgG specific to the cancer vaccine candidates, suggesting that they had the capability to show secondary immune responses to vaccine antigens. Furthermore, immunological boosting of T cell or IgG responses was observed in all of the patients, who completed at least one cycle of six vaccinations. Toxicity of PPV was mainly skin reactions at injection sites, and no SAE directly associated with the vaccinations were observed. These findings suggest the feasibility of PPV for refractory SCLC.

Interestingly, in a patient undergoing tumor resection after PPV, both CD4⁺ and CD8⁺ T cells infiltrated densely not only within the cancer stroma but also within the cancer cell nest. Since the vaccine antigens SART3 and p56lck were expressed in the tumor cells, it may be possible that T cells specific to these molecules infiltrated and accumulated within tumors. SART3 was strongly and homogeneously expressed in the tumor cells, whereas expression of p56lck was weak and heterogeneous. This heterogeneous expression of p56lck may be attributed to the immune escape mechanism of tumor cells following PPV, although the pre‐vaccination tumor tissue of this patient was unavailable to demonstrate this possibility.

The prognosis of refractory SCLC patients remains very poor with a median survival time of around 6–10 months.1, 2, 3 Therefore, it could be worthwhile to discuss the clinical efficacy of PPV, although it was not the main objective of this study. In 10 refractory SCLC patients receiving PPV, the median OS was 186.5 days, with 1 year survival rate of 30%. In particular, six patients who received at least one cycle of six vaccinations survived for 25, 24.5 (alive), 10 (alive), 9.5, 6.5, and 6 months (median OS, 528 days), although survival time of the remaining four patients without completing six vaccinations was only 0.5, 0.5, 1, and 3 months (median OS, 25 days). Statistically analyses with clinical findings and laboratory data were performed to identify potentially prognostic factors, although the result was preliminary due to the small number of patients and its clinical utility needs to be confirmed in future studies. In the analysis of clinical findings, greater numbers of previous chemotherapy regimens might be associated with worse prognosis, suggesting that PPV should be considered before repeated failures of multiple chemotherapeutic regimens. Similar to our finding, the ability to mount an immune response to therapeutic vaccines was reported to be directly correlated with fewer prior chemotherapy regimens.23 In addition, the statistical analysis with pre‐vaccination laboratory data demonstrated that the frequency of CD3⁺CD26⁺ cells in PBMCs was potentially prognostic in patients receiving PPV. The frequency of CD3⁺CD26⁺ cells has not been previously reported as a biomarker in SCLC patients. CD26 is a cell surface glycoprotein that functions as a proteolytic enzyme, dipeptidyl peptidase IV (DPP IV), and has been reported to play a critical role in signal transduction.24 Since this molecule is highly expressed on activated T cells,24 the increased frequency of CD3⁺CD26⁺ might contribute to better immune responses against the vaccine antigens. The role of CD26⁺ activated T cells in cancer vaccines remains to be determined.

In summary, the current study demonstrated that immune responses to the vaccine antigens were substantially induced without SAE in refractory SCLC patients who received at least one cycle (six times) of vaccinations. Nevertheless, due to the small number of patients and the short term of observation in this early phase trial, clinical efficacy of PPV for refractory SCLC remains to be confirmed in a next step of larger‐scale, prospective trials.

Disclosure Statement

The authors have no conflict of interest.

Supporting information

Fig. S1. Immunohistochemical analysis of vaccine antigens in small cell lung cancer (SCLC) tissues.

Table S1. Peptide candidates for cancer vaccination.

Table S2. Frequency of expression of vaccine antigens in small cell lung cancer (SCLC) tissues.