Phase I trial of thymidylate synthase poly-epitope peptide (TSPP) vaccine in advanced cancer patients

Abstract

Thymidylate synthase (TS) poly-epitope peptide (TSPP) is a 27-mer peptide vaccine containing the amino acidic sequences of three epitopes with HLA-A2.1-binding motifs of TS, an enzyme overexpressed in cancer cells, which plays a crucial role in DNA repair and replication. Based on the results of preclinical studies, we designed a phase Ib trial (TSPP/VAC1) to investigate, in a dose escalation setting, the safety and the biological activity of TSPP vaccination alone (arm A) or in combination with GM-CSF and IL-2 (arm B) in cancer patients. Twenty-one pretreated metastatic cancer patients, with a good performance status (ECOG ≤ 1) and no severe organ failure or immunological disease, were enrolled in the study (12 in arm A, nine in arm B) between April 2011 and January 2012, with a median follow-up of 28 months. TSPP resulted safe, and its maximal tolerated dose was not achieved. No grade 4 toxicity was observed. The most common adverse events were grade 2 dermatological reactions to the vaccine injection, cough, rhinitis, fever, poly-arthralgia, gastro-enteric symptoms and, to a lesser extent, moderate hypertension and hypothyroidism. We detected a significant rise in auto-antibodies and TS-epitope-specific CTL precursors. Furthermore, TSPP showed antitumor activity in this group of pretreated patients; indeed, we recorded one partial response and seven disease stabilizations (SD) in arm A, and three SD in arm B. Taken together, our findings provide the framework for the evaluation of the TSPP anti-tumor activity in further disease-oriented clinical trials.

Electronic supplementary material

The online version of this article (doi:10.1007/s00262-015-1711-7) contains supplementary material, which is available to authorized users.

Introduction

Peptide vaccine-based immunotherapy is emerging as an investigational therapeutic strategy for human cancer [1–5]. It relies on the concept that synthetic tumor-associated antigen (TAA)-derived peptides may be recognized by cytotoxic T lymphocyte (CTL) precursors and used to generate a specific antitumor immune response [5–8]. Different well-known TAAs play critical functions in tumor cell proliferation and survival, and their loss, under selective pressure exerted by antigen-specific CTLs, might impair tumor progression. Thymidylate synthase (TS) is a 36-kDa enzyme commonly overexpressed by cancer cells that play an essential role for thymidylate synthesis, which is crucial for DNA synthesis and repair [9–11]. During the normal cell cycle, the TS expression is restricted at the beginning of S phase, and its synthesis is under control of genes involved in cell cycle progression, often deregulated in cancer cells [9, 10]. Additionally, TS has self-regulatory properties, and intracellular metabolites, cofactors and substrates can modulate its expression. Indeed, tumor cell exposure to anticancer drugs such as pemetrexed or 5′-fluorouracil (5′-FU), which act by inhibiting TS activity, generates a rapid positive feedback which leads to enzyme overexpression [9]. Tumor cells with high intracellular levels of TS become resistant to 5′-FU representing a major cause of 5′-FU treatment failure [9, 10]. TS overexpression in tumor tissues has been investigated and recognized as a marker of poor prognosis in colon and gastric cancer patients [10]. On this basis, we carried out a preclinical characterization of a novel immunogenic 27-mer poly-epitope peptide, designated as TSPP (thymidylate synthase poly-epitope peptide). This peptide was synthesized by assembling amino acidic sequences of three known CTL-specific TS-epitopes (TS-1; TS-2 and TS-3) with HLA-A2.1 anchorage motifs, and it was found to be immunogenic with an efficient antitumor T cell response in vitro [12, 13]. Indeed, preclinical findings revealed that TSPP-sensitized CTLs were able to kill HLA-A2.1⁺target cells pulsed with the three single TS-epitopes, and HLA-A2.1⁺human breast and colon carcinoma cells in vitro [12]. Furthermore, CTL toxicity was higher against tumor cells previously exposed to sublethal doses of 5′-FU which enhanced TS expression [12]. TSPP immunological and antitumor activity was also demonstrated in HLA-A(*)02.01/human-CD8α transgenic (HHD) mice [12] inoculated with syngeneic lymphoma EL4/HHD cells, showing antitumor effects in both preventive and therapeutic studies, with additive interaction with 5′FU-based chemotherapy. Pathology studies showed that TSPP vaccination ±5′-FU-based chemotherapy was associated with enhanced CTL (CD3⁺CD8⁺) tumor infiltration, decline in regulatory T cells (T_regs) (CD4⁺CD25⁺FoxP3⁺) and disappearance of TS-overexpressing tumor cells, as compared to controls. The latter finding, in particular, suggested that TSPP vaccination could potentially restore tumor cell sensitivity to 5-FU [12, 14]. This phase Ib trial evaluated the safety and biological activity of TSPP administered alone or in combination with an immune-adjuvant regimen (IG-1) containing granulocyte–macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-2 [15], in advanced cancer patients. The rationale to combine TSPP with IG-1 regimen derived from preclinical in vitro findings [12, 14, 16, 17] and previous clinical studies, where the IG-1 regimen alone [15] or in combination with 5′FU-based chemotherapy was found to be safe, able to generate activated dendritic cells (DCs) and to promote an efficient antigen-specific T cell response [18, 19].

Patients, materials and methods

TSPP vaccine

TSPP (YMIAHITGLFLDSLGFSTTLGDAHIYL) was synthesized and characterized according to the good manufacturing practice (GMP) procedures by the American Peptide (USA) and aseptically vialed by the Pharmacy of the “Azienda Ospedaliera Universitaria Senese” (Siena, Italy), which also performed the stability study, endotoxin evaluation and chemical-related toxicity analysis. TSPP (100, 200 or 300 μg), dissolved in dimethyl sulfoxide (DMSO) (1.5 mg peptide in 2 ml DMSO), was suspended in 250 μl of PBS and then diluted (1:2 ratio) with Montanide ISA 720 VG ST (Seppic, Milan, Italy) in a final injection volume of 500 μl. DMSO was approved for clinical use, and its dosage was within the safety limits [20].

Ethical considerations and study design

This trial was designed according to good clinical practice (GCP) recommendations and authorized by the Italian National Institute of Health (Istituto Superiore di Sanità) and by the Italian Ministry of Health, and approved by the University of Siena Ethical Committee (equivalent to Human Subject Committee of Investigational Review Board); the trial has been registered with the TSPP/VAC-1 code, Eudract: #2009-016897-33. TSPP/VAC-1 was a phase IB trial, planned on dose escalation setting and subdivided in two parallel and independent arms (A and B) with unmasked treatment allocation; patients were consequently enrolled in arm A (nine patients in three doses) and then in arm B (nine patients in three doses). Three additional patients, according to the trial protocol, were enrolled in the highest dose level of arm A after the end of arm B enrollment. Patients were grouped into three doses following a standard dose escalation using a Fibonacci series (n, 2n and 3n) [21].

The first cohort of patients received three-weekly subcutaneous (sc) injections of 100 μg of peptide [dose level (DL)1)], the second one, 200 μg (DL2), and the third one, 300 μg (DL3). New patients could be enrolled in higher-dose cohorts only if no grade 3/4 events had been recorded in lower-dose cohorts. Patients in arm A received peptide vaccination alone, while those in arm B received peptide vaccination (day 3/q21) combined with the IG-1 regimen [GM-CSF (Sagramostim/Leukine^®, Berlex, USA) (50 μg days 1–5) and IL-2 (Aldesleukine, Proleukin^®, Novartis, Switzerland) (0.5 MIU bi-daily, days 6–15)].

The study was designed to investigate the toxicity and biological activity of TSPP. Its primary endpoint was the definition of TSPP maximal tolerated dose (MTD) and most effective biological dose (MEBD). Evaluation of antitumor activity was a secondary endpoint.

Eligibility and outcome measures

The inclusion criteria were as follows: written informed consent, histological diagnosis of malignant disease, at least two previous treatments for advanced disease, clinical and imaging-confirmed progressive disease, ECOG performance status ≤1, no major organ or hematological impairment. The exclusion criteria were as follows: second malignancies, active infectious disease and acquired immunosuppression.

Standard clinical (clinical history, physical examination) and laboratory evaluation [blood cell counts and chemistry and carcinoembryonic antigen (CEA) monitoring] were performed prior to each TSPP injection. Chest X-rays, ultrasound abdominal scans and computerized tomography (CT) scans were carried out at baseline and after 3 months (or at the appearance of clinical signs of progression) to evaluate treatment response (complete response, CR; partial response, PR; disease stabilization, SD; progressive disease, PD) according to the RECIST criteria. Patients remained on treatment until disease progression, occurrence of unacceptable toxicity, clinical judgment or withdrawal of consent. Progression-free survival (PFS) was measured as the time elapsed between enrollment and PD or death, while overall survival (OS) was measured as the time elapsed between enrollment and death. A quality-of-life (QoL) evaluation test, a psychometric analysis designed to measure the patients’ compliance to the treatment and to detect subjective reactions to potential side effects, symptoms and changes in social interaction was performed at each immunization.

Serum inflammatory and autoimmune markers

Patient serum was withdrawn prior to each TSPP vaccination and immediately frozen. Serum levels of C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), lactate dehydrogenase (LDH) and rheumatoid factor were included in the routine blood chemistry analysis. Serum myeloperoxidase (MPX) was evaluated by enzyme-linked immune absorbance assay (ELISA) (Calbiochem, Milan, Italy), antinuclear antibodies (ANA) by indirect fluorescence antibody (SSA HEp2000, ImmunoConcepts) through EliA™ Symphony screening (Thermo Fisher Scientific); further EliA tests were performed for single ANA specificities; anti-neutrophil cytoplasmic antibodies (ANCA) to proteinase-3 (p-ANCA), ANCA to MPX (c-ANCA) and extractable nuclear antigen antibodies (ENA) were tested on Phadia250 instrument; ANCA and anti-smooth muscle antibodies (ASMA) were evaluated by indirect immunofluorescence using INOVA substrate. The presence of TS and TSPP peptide-specific antibodies in the patients’ serum was evaluated by ELISA on flat-bottom microtiter plates coated with 100 μl/well of rTS (5 μg/ml) or TSPP peptide (5 μg/ml) diluted in coating buffer (0.05 M NaHCO3/Na2CO3, pH 9.6). Anti-human IgG peroxidase- conjugated antibodies (Sigma, Milan, Italy) were added to each well. Colorimetric conversion of the substrate (tetramethylbenzidine, TMB) was measured at 450 nm in a microplate spectrophotometer. Sera were considered positive if showing an optical density at least twice the background represented by a pool of sera of healthy 1-year-old children. A positive control was represented by an anti-TS mAb (Chemicon, DE).

Blood samples

Peripheral blood was collected from patients at baseline and prior each TSPP vaccination. Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation using Ficoll-Hypaque (Celbio S.P.A., Italy), re-suspended in FBS with 10 % DMSO, and immediately stored in nitrogen liquid, as described elsewhere [22, 23]. Viability of the thawed cells, assessed by trypan blue stain before each experiment, was greater than 90 %. Plasma samples were also collected by centrifugation and stored at −80 °C.

Cytokine multiplex analysis

Serum levels of interferon (IFN)-γ, tumor necrosis factor (TNF)-α, IL10, IL4, IL12(p70) and IL17 cytokines were measured at baseline and 20 days after the third vaccination using Bio-Plex human cytokine multiplex kits (Bio-Rad Inc., Hercules, CA). The Beads’ fluorescence intensity was measured and analyzed by using the Bio-Plex array reader and the provided manager software with five-parametric-curve fittings.

Fluorescence-activated cell sorting (FACS) analysis

PBMCs were analyzed by FACS analysis (BD FACSCanto II flow cytometer, CA, USA) as described in previous studies [19, 22] after staining with different combinations of conjugated monoclonal antibodies (mAbs) (CD4-V450, CD45RA-PE, CD62L-FITC, CCR7-PE-Cy7, Pharmingen; CD8-PerCPCy 5.5, CD45Ra-APC, CD3-FITC, CD19-FITC, CD16-PE, Rat IgG2a-FITC, Mouse IgG1, Becton–Dickinson, Italy; CD56-APC, Mouse IgG2a-APC Immune-tools, DE; CD25-PE, FoxP3-FITC, eBioscence, UK). T_regs were analyzed after FoxP3 intracellular staining according to the manufacturer guidelines (eBioscience). Statistical analysis was carried out by using the FlowJo^® software.

ELISPOT analysis

IFN-γ ELISPOT (Enzyme-Linked ImmunoSPOT) assays were performed according to the provider guidelines (U-Cytech Bioscience CM-Utrech TCH the Netherlands #CT230-PB2, Human IFN-ɣ ELISPOT KIT) [24] and the results evaluated by an ELISPOT reader (A.EL.VIS Gmbh, Hannover, Germany; ELISPOT analysis software V 5.1, Roxio creator 2009 License #13413). The assay, as described in previous studies [12, 13, 22], evaluates a T cell response in TSPP-sensitized PBMCs withdrawn at baseline and before the fourth and sixth TSPP vaccination. Briefly, TSPP-sensitized PBMCs were co-cultured with autologous DCs (1:5, DC/lymphocyte ratio) loaded with 10 μg/ml of the following antigen peptides: recombinant TS (rTS), TS1, TS2, TS3 and TSPP, and Respiratory Syncytial Virus (RSV) antigen (Meridian, Bioscience, Milan, IT).

To enhance the frequency and the detectability of TSPP-specific T cell precursors in the assays, patients’ PBMCs were sensitized to this peptide by two in vitro stimulations (IVS) prior to carrying out the assay. Each IVS consisted of a 5-day PBMCs’ co-incubation with TSPP peptide-loaded autologous DCs, followed by 10-day culture in rIL-2 (10 IU/ml)-enriched medium [12, 22].

Dendritic cells used for the ELISPOT assay and IVS were generated as described in previous studies [12, 22]. In each assay, fresh AIM-V medium with 5 % AB human serum (medium) was used as negative background control; Respiratory Syncytial Virus Antigen peptide (RSV) was used as an irrelevant antigen peptide control, while PHA was used as an unspecific positive control.

Results were expressed as the mean number of spot-forming units (spots) per 10⁶ cells. According to Moodie et al. [25], we considered a cytokine response as positive if 40 spots/10⁶ cells were detected and the mean number of spots/10⁶ cells was at least twofold higher than that in the negative control wells [26]. Differences in response between time points were assessed by comparing spot number averages by using a paired Student’s T test.

Statistical analysis

Statistical analysis was carried out by using GraphPadInstat PRISM 3.2 statistical software. The results were analyzed by performing the Student’s t test (two-tailed), which was paired when used to evaluate variable modulation during the treatment in the same patients, and unpaired when used to compare intergroup differences.

Results

Patients’ characteristics

Twenty-one patients, whose baseline characteristics are summarized in Table 1, were enrolled in the TSPP/VAC1 trial between April 2011 and January 2012, with a median follow-up of 28 (±2.94) months. Twelve patients, six males and six females, with a median age of 57 years (range 46–70), were enrolled in arm A, and nine, five males and four females, with a median age of 65 years (range 56–76) in arm B. Eighteen patients enrolled in the study had previously received multiple treatment lines and presented clinical and imaging-confirmed PD. Three patients had received one previous chemotherapy line only, due to the lack of a standard second-line treatment (MA/A/DL3, gallbladder carcinoma; RV/B/DL1, gastric cancer) or to the refuse of a further chemotherapy regimen (AR/A/DL3, NSCLC). In agreement with ethical committee, we decided to offer to these patients the possibility to be enrolled in this trial.

Table 1.

Patients characteristics, adverse events and response

ID	Age/sex	HLA	Site of primary tumor	Metastatic sites	Previous treatments (number)	Vaccinations (number)	Adverse events (grading)	Clinical response	PFS/OS
Arm A
MF/A/DL1	48/F	A2	Colorectal cancer	Liver, lung	11	3	Erythema (G1), cough (G1), rhinitis (G2), hypertension (G2)	SD	5/6
RA/A/DL1	59/F	A2	Colorectal cancer	Abdomen, nodes	7	3	Erythema (G1), rhinitis (G2), diarrhea (G2), hypothyroidism (G1)	PD	3/3
AS/A/DL1	56/M	A2	Colorectal cancer	Liver, lung	3	3	Hypertension (G2)	PD	4/4
LV/A/DL2	70/M	A11	Colorectal cancer	Lung, nodes	7	6	Erythema (G2), headache (G2), vomiting (G2), fever (G1)	SD	7/30+
OA/A/DL2	53/M	A1	NSCLC	Brain, bone, liver, lung	3	5	Erythema (G2), cough (G2), rhinitis (G2)	SD	5/9
SG/A/DL2	46/M	A25	Colorectal cancer	Abdomen, nodes	4	6	Erythema (G1), hives (G2), fever (G2)	SD	7/16
ZS/A/DL3	70/F	A2	Colorectal cancer	Nodes, adrenal	3	32	Arthritis (G2), diarrhea (G2)	SD	30 +/30+
MA/A/DL3	54/F	A23	Gallbladder Carcinoma	Peritoneum, nodes	1	28	Erythema (G2), hives (G2), edema (G2), coughing (G2), rhinitis (G2)	PR	24/29+
CM/A/DL3	70/F	A2	Breast cancer	Lung, skin, pleura	3	12	Erythema (G1), hives (G2), arthritis (G2), diarrhea (G2), hypertension (G2)	SD	4/15
AR/A/DL3	66/M	A2	NSCLC	Lung, nodes	1	2	Erythema (G1), Infection (G2), cough (G2), rhinitis (G2) hypothyroidism (G2)	PD	3/7
PN/A/DL3	61/F	A1	Colorectal cancer	Lung, bone	3	3	Erythema (G1), cough (G2), rhinitis (G2) hypothyroidism (G2)	SD	4/8
SN/A/DL3	51/M	A2	Colorectal cancer	Liver, lung	3	3	Fever (G2), infection (G2), hypertension (G2)	PD	3/3
Arm B
PL/B/DL1	63/F	A1	NSCLC	Lung, nodes	4	5	Hypothyroidism (G2), asthenia (G2), vomiting (G2)	SD	5/6
RV/B/DL1	76/M	A2	Gastric cancer	Liver, peritoneum	1	3	Fever (G2)	PD	2/3
PG/B/DL1	62/M	A26	NSCLC	Brain, lung, nodes	3	3	Coughing (G2), fever (G2)	SD	3/5
GL/B/DL2	42/F	A24	Colorectal cancer	Liver, abdomen	3	3	No	PD	3/4
PP/B/DL2	70/F	A2	Colorectal cancer	Lung, liver, peritoneum	5	3	Fever (G1), vomiting (G2)	PD	3/4
FG/B/DL2	81/M	A24	Colorectal cancer	Peritoneum, nodes	3	3	Erythema (G1), hives (G2), hypothyroidism (G2), diarrhea (G2)	PD	3/5
SF/B/DL3	73/F	A24	NSCLC	Liver, abdomen	3	15	Hypertension (G2), cough (G2), rhinitis (G2), diarrhea (G2), vomiting (G2)	SD	13/16
MM/B/DL3	56/M	A1	Colorectal cancer	Lung, liver, peritoneum	4	3	Headache (G2), cough (G1)	PD	3/7
BA/B/DL3	50/F	A30	NSCLC	Peritoneum, nodes	2	3	Vomiting (G2), depression (G2)	PD	3/9

The enrollment code was composed by patient’s initials/arm of enrollment (A or B)/dose level (DL). NSCLC non-small cell lung cancer, SD stable disease, PR partial response, PD progression disease

Patients enrolled in arm A received TSPP vaccination every 3 weeks, while those enrolled in arm B received TSPP vaccination (on day 3) and immune-adjuvant cytokines according to IG-1 regimen [15]. Patients in both arms were subdivided into three cohorts, receiving escalating TSPP doses. A code, composed by name initials, enrollment arm (A or B) and DL (1, 2 or 3), was assigned to each patient. The study was not restricted to patients with a HLA-A2.1 haplotype. This decision was based on the knowledge that TSPP processing by antigen-presenting cells (APCs) produces multiple cryptic epitopes potentially able to bind different class I HLA molecules (as indicated by a HLA-binding prediction algorithm), with the potential of producing an immune response in 90 % of the human individuals ([12] and personal communication). HLA typing was, however, mandatory, and nine out 21 patients (seven in arm A and two in arm B) showed a HLA-A2.1⁺ haplotype.

Adverse events and clinical monitoring

No treatment-related death or life-threatening toxicity occurred during treatment. The most frequent adverse events included grade 1–2 hypersensitivity and skin reactions at the site of vaccine injection, rhinitis, cough, vomiting, diarrhea, fever and diffuse arthralgia (Table 1). Five patients, under treatment with angiotensin-converting enzyme inhibitors showed a grade 1–2 arterial hypertension episode, while other five presented slight hypothyroidism. A bacterial infection, with complete recovery upon antibiotic treatment, was diagnosed in four patients (two in arm a: one in DL2 and one in DL3; and two in arm B: one in DL1 and one in DL2). A patient in arm A (AR/A/DL3) presented an ab ingestis pneumonia due to previous lung surgery and refused to continue the vaccination program after he received the second vaccine dose. Finally, a NSCLC patient (OA/A/DL2) with irradiated brain metastases showed progressive cognitive impairment and a reversible episode of narcolepsy (fifth vaccine course). This patient received corticosteroid treatment and was withdrawn from the study.

TSPP vaccination resulted safe; there was no statistical correlation between frequency of adverse events and TSPP dose level; thus, we were unable to identify the MTD. However, it was observed a greater discomfort in arm B patients as evaluated by the quality-of-life (QoL) test [27]. Indeed, average QoL score in arm A, showed a late improvement [average QoL score 60.4 (±14.88) at baseline vs. 62.96 (±21.91), after three treatment courses, vs. 76.2 (±10.60) after six treatment courses (score at Baseline vs. sixth treatment course; p = 0.045)], while a score decrease was observed in arm B [average QoL score 60.4 (±27.55) at baseline vs. 59.37 (±26.67), after three treatment courses, vs. 50 (±31.43) after six treatment courses (score at Baseline vs. sixth treatment course; p = 0.06)]. This finding could be related to either effects of cytokine administration, or different tumor-type distributions in the two arms, or different responses to vaccine treatment.

Blood cell counts and serologic analysis

An initial increase in neutrophil counts was observed; no change in lymphocyte, basophil, and platelet counts, and a slight and progressive reduction in monocyte and eosinophil counts were recorded in arm A. Progressive increase in neutrophil and eosinophil counts, no change in lymphocyte, basophil and platelet counts and again a decline in monocyte counts were, instead, observed in arm B (Fig. 1). Monitoring of systemic inflammatory markers showed an opposite trend for CRP and ESR in the two arms (Fig. 1); indeed, both were found to decrease in arm A and to increase in arm B. Serum levels of MPX, a protein released during neutrophil and monocyte degranulation, which reflects tissue damage and inflammation [28], showed significant rise in both arms (arm A: values at baseline vs. third and vs. sixth treatment course = 1600 vs. 3000 vs. 2700 U/ml, p < 0.05; arm B: values at baseline vs. third and vs. sixth treatment course = 1800 vs. 1700 vs. 6000 U/ml, p < 0.05) (Fig. 1). Serum LDH levels finally showed a significant reduction in both arms with no inter-arm difference (data not shown).

Fig. 1.

a Peripheral blood cell counts, serum inflammation markers in cancer patients enrolled in the TSPP/VAC1 trial arm A (diamond) and arm B (dark gray square), at baseline and before each treatment course. Asterisk represents statistical significance when arm A and arm B values were compared (p < 0.05). CRP C-reactive protein, ESR erythrocyte sedimentation ratio, MPX myeloperoxidase. b Serological evaluation of c-ANCA, p-ANCA and ENA immunoglobulins in peripheral blood samples of patients enrolled in arms A and B at baseline (black square) and after the third (light gray square) and sixth (dark gray square) treatment course (±Standard error). Asterisks indicate statistical significance of post-treatment versus baseline values (p < 0.05)

The research of specific IgGs commonly associated with autoimmune disease revealed a progressive increase in the mean serum level of ENA, c-ANCA and p-ANCA in both arms (Fig. 1). We also detected the presence of ANA (Ab titer: 1/640; normal value titer: 1/160) in five patients of arm A and four patients of arm B, after three treatment courses, and the occurrence of ASMA in a good responder patient of arm A, after six TSPP immunizations (ZS/A/DL3). Only two patients, ZS/A/DL3 and MA/A/DL3 produced IgGs reacting with TSPP after 13 treatment courses.

Taken together, these data suggest that TSPP vaccination is associated with the occurrence of serological signs of autoimmunity with rise in multiple autoantibodies. However, the finding of a progressive and parallel rise in neutrophils, eosinophils, CRP, ESR and MPX in arm B [differences among arm A and arm B patients for these parameters were statistically significant (p < 0.05)] suggests that the use of TSPP in combination with immune-adjuvant cytokines compared with TSPP alone ignites a stronger systemic inflammatory response in cancer patients.

Analysis of lymphocyte subpopulations

A FACS analysis of patients’ PBMCs did not show any significant treatment-related changes of CD3⁺CD4⁺ and CD3⁺CD8⁺ lymphocytes, CD3⁺CD4⁺CD45Ra⁻ (memory) T cells, effector memory (CD8⁺CD45Ra⁻CCR7⁻) T cells (T_ems) or activated CTLs (CD3⁺CD8⁺CD62L⁺Tcells) in either arms (data not shown). Furthermore, we observed a significant increase in central memory (CD8⁺CD45Ra⁻CCR7⁺; baseline vs. third course values, p = 0.048) T cells (T_cms) and natural killer cells (NK, CD3⁻CD16⁺CD56⁺; baseline vs. third course values, p = 0.05) in arm A patients (Fig. 2). For what concerns possible changes in immunosuppressive blood cell lineages, we found a significant treatment-related increase in regulatory CD4⁺CD25⁺FoxP3⁺T cells (T_regs) (p = 0.035) in arm B patients (Fig. 2).

Fig. 2.

Baseline (pre) and post-TSPP vaccination (20 days after the third vaccination) (post) flow cytometric analysis of PBMCs from patients enrolled in arm A (black square) and B (gray square). NK natural killer cells (CD3⁻CD56^dimCD16⁺), T_cms central memory T cells (CD3⁺CD8⁺CD45RA⁻CCR7⁺), T _reg s regulatory T cells (CD3⁺CD4⁺CD25⁺FoxP3⁺). Asterisks indicate statistical significance of post-treatment versus baseline values (p < 0.05)

Cytokine analysis

The baseline serum levels of TNF-α, and IFN-γ, IL12(p70), IL17A, IL10 and IL4 in the enrolled patients resulted very heterogeneous. We then evaluated modulations in cytokine levels after three treatment courses. There was no treatment-related change in the serum levels of IL10 and IL4 (Th2 cytokines) in both treatment arms (Fig. 3). On the other hands, patients in arm A showed a slight decline in TNFα, and no change in IFN-γ, IL12 and IL17, while those in arm B, showed a significant increase in IFN-γ, IL12(p70) [Th1 cytokines], and IL17 (p < 0.05). Again these results suggest effective engagement of an inflammatory response associated with TSPP and IG cytokine treatment.

Fig. 3.

a Represents fold-change (FC) modulation (baseline vs. 20 days after the third vaccination) in serum level of TNFα, IFNγ, IL12, IL17, IL10 and IL4 of the patients enrolled in arms A and B (±Standard error). b Shows a gamma-interferon ELISPot assay performed on the PBMCs isolated at baseline (light gray square) and after three treatment courses (20 days after the last vaccination) (dark gray square), from patients enrolled in arm A and arm B of the trial. PBMCs derived from two different normal donors (solid lines) were used as a control group. Results are expressed as average of the number of spots × 10⁶ cells per group ± Standard error. PHA, phytohemagglutinin; rTS, recombinant thymidylate synthase; RSV, respiratory syncytial virus antigen peptide; TS1, TS2, TS3, thymidylate synthase epitopes 1, 2 and 3; TSPP, thymidylate synthase poly-epitope peptide

TS specific CTL precursors

IFN-γ ELISpot analysis performed on patients’ TSPP-sensitized PBMCs revealed a treatment-associated T cell response to either rTS or TSPP peptide (Fig. 3) in both arms. When performed in HLA-A2.1⁺ patients, this analysis showed a treatment-associated CTL response only to the TS-1 and TS-3 epitopes (Fig. 3). The small sample of evaluated patients which just includes four patients in arm A and two patients in arm B could have affected the sensitivity of this assay. Our test did not record any change when complete AIM-V medium with 5 % human AB serum (background), PHA (positive control) or RSV (negative antigen peptide control) were used to stimulate patients' TSPP-sensitized PBMCs in vitro (Fig. 3).

Antitumor activity

A PR and a SD were, respectively, recorded in one and seven cases in arm A (on a total of 12 patients), while a SD was recorded in three out nine patients in arm B (Table 1; Fig. 4). Nine patients, three in arm A (AS/A/DL1, SN/A/DL3 and AR/A/DL3) and six in arm B (RV/B/DL1, FG/B/DL1, GL/B/DL2, PP/B/DL2, BA/B/DL3 and MM/B/DL3) discontinued the treatment because of rapid PD or refuse. Mean PFS was 6.4 (95 % CI 3.66–9.2) months in arm A and 3.69 (95 % CI 1.55–5.82) months in arm B, while mean OS was 10.98 (95 % CI 7.56–14.4) months in arm A and 5.9 (95 % CI 4.11–7.69) months in arm B. Six patients in arm A (50 %) and one patient in arm B (11 %) survived more than 12 months (Table 1); in particular, two patients in arm A had a PFS of more than 20 months.

Fig. 4.

TSPP vaccination activity in terms of clinical responses. The two representative TC scans included in this picture showed the PR obtained in the patient affected by gallbladder carcinoma (MA/A/DL3) and an impressive 30-month disease stabilization in a patient affected by metastatic colorectal cancer (ZS/A/DL3)

Correlations between antitumor activity and inflammatory status

We further evaluated correlations between survival and inflammatory parameters in arms A and B. We found that patients in arm A surviving more than 10 months (median of the group) presented a low inflammatory status at baseline, with a significant lower serum CRP level, ESR and neutrophil-to-lymphocyte ratio (NLR) and a significant higher lymphocyte count. Furthermore, lymphocyte count at baseline linearly correlated with patients’ survival (Fig. 5). Conversely, in arm B, none of such parameters was found to correlate with patients’ survival (Supplementary Fig. 1). These preliminary findings are in line with the current idea that inflammatory status at baseline (or induced by the exogenous administration of pro-inflammatory cytokines) may impair the capability of patient’s immune system to mount an effective response against cancer [29].

Fig. 5.

Correlation analysis performed in arm A to evaluate the association between baseline inflammatory status and survival after vaccination. a Shows the linear correlation between baseline lymphocyte count and patients’ OS. b Evidences the differences between different baseline clinical laboratory parameters associated with inflammation in patients surviving more or less than 10 months (median OS of the group)

Discussion

In this phase I study, TSPP vaccine-based immunotherapy demonstrated to be relatively safe and did not affect patients’ quality of life as shown by our psychometric monitoring, thus fulfilling the primary endpoint of our trial. There were no G4 adverse events, while the most common (grades 1 and 2) side effects were local reactions to vaccine injections, acute inflammatory disease-related symptoms and/or self-limiting autoimmune reactions. Immune monitoring revealed direct and indirect signs of TSPP-dependent immune activation/autoimmunity (changes in peripheral blood cell lineages, serum inflammatory markers and specific cytokine production) in both arms, a finding in line with the results of other authors, who reported similar effects in different immunotherapy trials [30–34]. Indeed, the occurrence of autoimmunity and/or inflammatory disease following a specific immune-treatment appears to be consequent to active and complex immunological phenomena occurring in the tumor microenvironment [35]. These events can cause tissue damage, acute inflammation, antigen release/cross-presentation, which in turn trigger a process of antigen cascade with the final result of a more efficient multi-antigen-specific response [19, 22, 31, 33, 36]. Furthermore, the occurrence of autoimmunity following patient immunotherapy revealed to be a strong favorable outcome and prolonged survival predictor in different clinical trials [19, 31, 33].

IFN-γ ELISpot assays further confirmed the immunological activity of TSPP vaccination on patients CTL precursors. Indeed, the treatment was associated with a significant ex vivo T cell response to rTS, TSPP and TS1 and TS3 epitopes in patients’ PBMCs, with no significant differences between the two treatment arms. However, in spite of immune activation, we found different results, in terms of outcome, between patients enrolled in arm A and B. Specifically, the outcome of patients who received TSPP vaccine alone was found to be greatly influenced by the baseline inflammatory status; indeed, patients with low inflammatory status at baseline experienced the longest survival. Conversely, the administration of pro-inflammatory cytokines appeared to exacerbate patients’ systemic inflammation, in spite of their baseline status, with a potential detrimental effect on the therapeutic activity of TSPP vaccination.

Several studies in this field demonstrated that a systemic chronic inflammatory status with high neutrophil counts at baseline is predictive of poor prognosis in patients with different malignant disease [37–39] and in a recent phase III chemo-immunotherapeutic trial on metastatic colorectal cancer patients we observed that patients with low baseline neutrophil counts presented a survival advantage over those with high baseline neutrophil counts [40].

In our opinion, these results underline the dual and conflicting role of GM-CSF in both improving immune response, through eliciting DC maturation [41], and inducing immune suppression, by generating and mobilizing immune-suppressive populations such as myeloid-derived suppressor cells [42]. Such counterbalancing effects may indeed rely on patients’ systemic inflammatory status and tumor microenvironment. As a further observation, in arm B, the treatment-associated increase in systemic inflammation markers was coupled with an uncoordinated Th1/Th2/Th17 cytokine response. In this setting, the ability of Th1 cells to promote an efficient CTL response to cancer cells may be mostly counteracted by an enhanced presence of Th17 [43]. This important point, however, is presently under active investigation by our group, and further studies are needed to elucidate the potential predictive/prognostic role of the chronic inflammatory response on OS in vaccine patients. Furthermore, in this trial, we cannot exclude that part of the above-reported biological events are related to a higher frequency of NSCLC patients in arm B compared to arm A. Further differences in the two arms were also recorded in terms of specific T cell response. Indeed, we found a significant increase in T_cms and NK cells with cytotoxic phenotype in arm A and a significant increase in T_regs in arm B patients. T_cm lymphocytes represent a fresh source of CTLs expressing the CCR7 receptor, a surface molecule which allows them to follow the gradient of their ligands, the chemotactic chemokines (CCL) 19 and 21, produced by inflammatory cells in lymph nodes and tumor sites [44] where these cells may differentiate in highly cytotoxic effector cells or acquire long-lasting memory [45]. We have recently shown that their overexpression in the primary tumor is strongly predictive of favorable prognosis in metastatic colorectal cancer patients [46, 47]. The T_regs rise observed in patients on arm B could instead be dependent on the presence of IL-2 in the combined TSPP/IG-1 cytokine treatment arm [48]. Even though antitumor activity was a secondary endpoint of this trial, we recorded a disease control rate (PR + SD) of 66.7 % (t 12) in arm A and 33.3 % (three out nine) in arm B, with two patients (two in arm A and one in arm B) who remained free of progression for more than 12 months, and two additional patients (in arm A) who showed a survival longer than 12 months.

Taking account that the majority of these patients presented multiple metastatic sites and that they had received multiple treatment lines prior to receiving TSPP vaccination, we can consider our results encouraging in terms of antitumor activity. Additionally, the finding that patients experiencing the longest survival also presented a low baseline inflammatory status leads us to believe that the pharmacological modulation of systemic inflammatory response could be a promising candidate approach to improve anticancer immunotherapy.

In conclusion, the findings of this clinical study suggest that the TSPP vaccine is safe (its MTD could not be reached in both arms) and is associated with both bio-modulatory and antitumor activity. The immunological analysis showed that TSPP vaccination induces effects with potential anti-tumor activity at the dosage of 2–300 μg (MEBD), while the addition of GM-CSF and IL2 does not provide any clinical or immunological advantages. These results offer the rationale to investigate TSPP anti-tumor activity in disease-oriented phase II trials.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Abbreviations

5-FU

5-fluorouracil

ANA

Antinuclear antibodies

ANCA

Anti-neutrophil cytoplasmic antibodies

APC

Antigen-presenting cell

ASMA

Anti-smooth muscle antibodies

CEA

Carcinoembryonic antigen

CRP

C-reactive protein

CTL

Cytotoxic T lymphocyte

DC

Dendritic cell

DMSO

Dimethyl sulfoxide

ECOG

Eastern Cooperative Oncology Group

ELISA

Enzyme-linked immune absorbance assay

ELISPOT

Enzyme-linked immunospot assay

ENA

Extractable nuclear antigen antibodies

ESR

Erythrocyte sedimentation rate

FACS

Fluorescence-activated cell sorting

GCP

Good clinical practice

GM-CSF

Granulocyte–macrophage colony-stimulating factor

GMP

Good manufacturing practice

IVS

In vitro stimulation

LDH

Lactate dehydrogenase

mAb

Monoclonal antibody

MEBD

Most effective biological dose

MPX

Myeloperoxidase

MTD

Maximal tolerated dose

NK

Natural killer

NSCLC

Non-small cell lung cancer

OS

Overall survival

PBMCs

Peripheral blood mononuclear cells

PD

Progressive disease

PFS

Progression-free survival

PHA

Phytohemagglutinin

PR

Partial response

QoL

Quality of life

RECIST

Response evaluation criteria in solid tumors

RSV

Respiratory syncytial virus

SD

Stable disease

TAA

Tumor-associated antigens

T_cm

Central memory T cell

T_em

Effector memory T cell

T_reg

Regulatory T cell

TS

Thymidylate synthase

TSPP

Thymidylate synthase poly-epitope peptide