Phase I/II study of treatment with dendritic cell vaccines in patients with disseminated melanoma

Abstract

Previous studies have suggested that immunotherapy with dendritic cell (DC) vaccines may be effective in treatment of patients with AJCC stage IV melanoma. We examined this treatment in phase I/II studies in 33 patients with good performance status and low volume disease. Nineteen patients received DCs plus autologous lysates and 14 patients DCs plus peptides from the melanoma antigens MAGE-3.A2, tyrosinase, gp100, and MART-1. Keyhole limpet hemocyanin (KLH) was used as a helper protein and influenza peptide was given as a positive control. DCs were produced from adherent cells in blood lymphocytes (monocytic DCs), grown in IL-4 and GM-CSF without a maturation step. The DCs were injected into inguinal lymph nodes at weekly intervals (×4), 2 weeks (×1), and 4-weekly intervals (×2). There were 3 responses (3 partial responses) and 1 mixed response in the 19 patients treated with DCs plus autologous lysates. No responses were seen in the group treated with DCs plus peptides. Stable disease (defined as no progression over a period of 3 months) was seen in 4 patients in group 1 and 5 patients in group 2. Treatment was not associated with significant side effects. We examined whether DTH skin tests or assays of IFN-γ cytokine production may be useful predictors of clinical responses. Twenty-two of 30 patients had DTH responses to KLH and 12 of 13 patients had DTH responses to the influenza peptide. Five of 15 DTH responses were seen against autologous lysates. This was strongly correlated with clinical responses. Approximately half the patients had responses to MART-1 peptide and a third to the other melanoma peptides. Similarly, cytokine production assays showed responses to influenza in 7 of 13 patients, and approximately one third of patients had responses to the other peptides. No IFN-γ responses were seen in 5 patients against their autologous lysates. There was no correlation between assays of IFN-γ production and clinical responses. The present studies suggest that autologous lysates may be more effective than the melanoma peptides used in the study as the source of antigen for DC vaccines. DTH responses to autologous lysates appear useful predictors of clinical responses, but further work is needed to identify other measures associated with clinical responses.

Introduction

Treatment of disseminated melanoma remains unsatisfactory with median survivals from date of diagnosis ranging from 4 to 9 months. Surgical removal is the treatment of choice for isolated metastases but chemotherapy with dacarbazine (DTIC) remains the standard treatment for widespread disease. Responses are seen in approximately 20% of patients but are usually short lived [1]. Immunological responses are believed to play a role in the natural history of the disease, and immunotherapy with melanoma vaccines in several large phase II studies and one randomized study was associated with similar response rates and survival to that seen with chemotherapy [2, 3]. This has prompted a number of different approaches to increase the effectiveness of immunotherapy and in particular, the use of dendritic cell (DC) melanoma vaccines.

DCs are specialized antigen-presenting cells that are derived from cells in the bone marrow [4, 5]. They are particularly localized to tissues that comprise the body’s external barrier, such as skin and mucosal surfaces. A number of studies in animal models have shown that DCs exposed to tumor antigens in vitro and administered to the animal can induce protection against tumor growth and T-cell cytotoxic responses [6, 7]. Mice bearing sarcomas or Lewis lung carcinomas were rendered tumour free in 80% of cases by treatment with DCs that had been ‘pulsed’ with tumour-associated peptides [8]. This was associated with the development of CTL with specificity to tumour antigen [9]. Similar therapeutic efficacy was shown by treatment with DC pulsed with peptides stripped by acid elution from the surface of tumour cells [10].

Similar studies in patients with melanoma and other malignancies have given encouraging results. Nestle et al. [11] reported 2 complete responses (CRs) and 3 partial responses (PRs) in 16 patients immunized with DCs plus autologous lysates (4 patients) or melanoma peptides (12 patients). There was 1 CR and 1 PR in those treated with DCs plus lysates, and 1 CR and 2 PRs in the 12 patients treated with DCs + peptides. Thurner et al. [12] immunized 11 patients with DCs plus a HLA-A*0101-restricted, MAGE-3-derived peptide and reported regression of individual metastases in 6 of 11 patients. Two of 14 patients developed antitumour responses when immunized with DCs plus melanoma peptides [13]. One PR was seen in 17 patients treated with DCs plus peptides from gp100 and MART-1 given i.v. by Panelli et al. [14], and 1 PR was seen in studies by Chakraborty et al. [15] on 15 patients treated with DCs plus autologous lysates. Banchereau et al. [16] treated 17 patients with DCs plus melanoma peptides and reported 3 CRs and 3 PRs.

In view of these reports, we initiated a clinical trial to assess the clinical and immunological response rates of therapy with DCs exposed to autoogous tumor cell lysates or melanoma peptides.

Methods and materials

Patients

Patients entered into the study had AJCC stage IV melanoma that was clinically or radiologically measurable. Inclusion criteria were Eastern Co-Operative Oncology Group (ECOG) Performance Status of 0 or 1, low volume disease, no other concomitant malignancy, no previous chemotherapy or immunotherapy in the past 4 weeks and no other serious illness.

Study design

DC vaccines were given by intranodal injection under ultrasound control at weekly intervals for the first four injections then once 2 weeks later and twice at 4-week intervals, as described elsewhere [11]. Delayed hypersensitivity tests were carried out pretreatment, at 4 weeks and at the end of the study. Tumour measurements were carried out prior to treatment and at 8 and 16 weeks. Autoantibody tests were carried out at similar intervals. Metastatic lesions and clinical responses were evaluated by response evaluation criteria in solid tumors (RECIST), as described elsewhere [17]. In patients with PR or stable disease (SD; no progression over 3 months), DC vaccines were continued at 4-week intervals and tumour measurements reevaluated every 14 weeks. Observation of progressive disease (PD) led to cessation of treatment.

Preparation of DC vaccines

The methods were as described by Nestle et al. [11]. Mononuclear cells were separated from 80–100 mls of heparinized blood taken from the patient by centrifugation on Ficoll-Hypaque in the GLP facility, Department of Dermatology, University of Sydney, and resuspended in RPMI + 10% fetal calf serum (FCS) (CSL, Parkville, Victoria; Australasian source gamma irradiated). The cells were allowed to adhere to plastic for 2 h and nonadherent cells removed. Nonadherent cells were recultured overnight and nonadherent cells were removed. The adherent cells were cultured with GM-CSF (Schering Plough, Baulkham Hills, NSW) 800 μg/ml and IL-4 (Schering Plough, Baulkham Hills) 500 μg/ml for 7 days (complete media).

For patients receiving DCs plus peptides at 7 days, the DCs were washed and resuspended in RPMI and approximately 10⁶ DCs pulsed with each peptide at 50 μg/ml and keyhole limpet hemocyanin (KLH) (Calbiochem, Merck, Melbourne; Victoria, Australia, Catalogue No. 374816) 50 μg/ml for 2 h at 37°C in serum-free media. The DCs were washed in PBS plus 1% autologous sera, and all DCs were pooled together and resuspended in 1 ml of PBS plus 1% autologous sera prior to injection into two uninvolved inguinal lymph nodes (LNs) of 0.5 ml under ultrasound control.

For patients receiving DCs + autologous melanoma lysates, the DCs were harvested on day 7, washed and pulsed with lysate at 600 μg/ml (melanoma cells frozen and thawed 3 times) and KLH (50 μg/ml) for 4 h, washed and resuspended for injection, as for the peptide-pulsed DCs.

Peptides used in the studies

Peptides from MAGE-A3, tyrosinase, gp100, and MART-1 were used in the studies, as described elsewhere [18]. The MART-1 peptide was the modified decamer described by Valmori et al. [19] and the gp100 peptides were the modified gp209 2M and gp280 9V peptides described by Parkhurst et al. [20]. The tyrosinase peptide was the posttranslationally modified variant with aspartic acid in place of asparagine [21]. These peptides were supplied by the University of Pittsburgh Peptide Facility according to the instructions of current ‘Good Manufacturing Practice’, part 21 of the Code of Federal Regulations, Food and Drug Administration, USA (cGMP 21 CFR, FDA). The influenza matrix peptide used as a positive control was kindly supplied by Dr Andrew Scott, Ludwig Cancer Institute, Heidelberg, Victoria, Australia. The sequences of the HLA-A2-restricted peptides are as follows: Melan A/Mart-1, 26-35-ELAGIGILTV; tyrosinase, YMDGTMSQV; MAGE-3.A2, FLWGPRALV; gp100 280-9V, YLEPGPVTV; gp100 209-2M, IMDQVPFSV; influenza matrix, GILGFVFTL.

Skin tests

Skin tests with the peptides used for treatment and the control influenza peptide were carried out prior to and at 4 and 14 weeks after commencement of therapy, as described by Jaeger et al. [22]. Peptides (100 μg) were given in 100 μl of PBS by intradermal injection on the volar aspect of the forearm. DTH reactions were evaluated at 48 h after injection. Reactions were considered positive when palpable skin induration was 2 mm or greater in diameter.

IFN-γ cytokine production assays

Blood samples from the patients were taken pretreatment and at 4 and 14 weeks during treatment, separated on Ficoll-Hypaque, resuspended in DMEM (Trace Bioscience, Castle Hill, NSW, Australia) at 5×10⁶/ml and then added to an equal volume of FCS + 20% DMSO. Vials of 1 ml were placed in a Handy freeze tray (Taylor Wharton) in the neck of a 35-l VHC (Taylor Wharton) liquid nitrogen container overnight and then stored in liquid nitrogen. After thawing they were cultured overnight in DMEM plus 10% heat-inactivated human AB serum at 37°C. Assay procedures are as described previously [23]. T2 cells [24] were pulsed with individual peptides (10 μg/ml) in AIM-V (serum free) media (Gibco BRL, Invitrogen, Melbourne, Victoria, Australia) overnight at 37°C. PBLs (5×10⁵/ml) were then incubated with T2 peptide-pulsed cells in DMEM plus 10% human AB serum at a ratio of 1:1 for 2 days at 37°C, as described by Salgaller et al. [25] and Parkhurst et al. [20]. Positive controls were either 5×10⁵ PBLs plated onto anti-CD3 (OKT3)-coated wells or 5×10⁵ PBL stimulated with PHA-P (Sigma, Catalogue No. L8754). Negative controls were non-peptide-pulsed T2 cells and HIV reverse transcriptase_476–484-pulsed T2 cells. Cultures were in duplicate. Supernatants were harvested after centrifugation at 600 g to remove cells, then stored at –80°C. Assay of IFN-γ in the supernatants was carried out by ELISA using a commercially available kit (PharMingen, Becton Dickinson, North Ryde, NSW, Australia) and read in a plate reader (model 450, Bio-Rad) with a minimal detectable concentration of 2 pg/ml. Similar methods were used to assay IL-10.

Results

Patient details and clinical responses

In total, 33 patients were entered into the study. Nineteen patients received DCs plus autologous lysates (group 1) and 14 patients DCs plus melanoma peptides (group 2). Details of the patients and clinical responses to treatment are summarized in Tables 1 and 2. In group 1 there were 10 females and 9 males aged from 30 to 73. All but one had an ECOG status of 0. One patient (No. 15) had only 3 vaccines because of PD. All others had a minimum of 4 vaccines. However, in total, 6 patients did not complete the full course of treatment because of PD. Ten patients had received prior chemotherapy with DTIC or a new phase II agent. Side effects were predominantly tiredness, tenderness of LNs and/or subcutaneous metastases. Best responses seen were 3 PRs in female patients (numbers 4, 9 and 18), and 1 mixed response (MR) in a male patient (number 3). Stable disease (no progression over 3 months) was seen in 4 patients (3 females, 1 male).

Table 1.

Clinical responses in patients treated with dendritic cells + autologous melanoma cell lysates. AWD alive with disease, A&W alive and well, VMCL vaccinia melanoma cell lysate vaccine, DTIC dacarbazine, SC subcutaneous, Abdo abdomen

Patient no.	Age & sex	Time from diagnosis to metastases (months)	Site of metastases	Previous therapy	Time from first metastasis to DC treatment (months)	Adverse effects during treatment	No. of vaccines	Best response	Subsequent therapy	Duration to death/follow-up
										From diagnosis (months)	From first metastasis (months)
1	73, F	83	LNs abdo/pelvis		2	Tiredness	5	SD	Palliation	102	19
		84	Pulmonary mets		5		7	SD
		90	Regional LNs		9		5	PD
		91	Brain
2	46, M	22	Lung mets	DTIC	15	Tenderness, inguinal LNs	7	PD	Palliation	47	25
			Inguinal LNs	Cis Platin		Emotional lability
3	44, M	27	Lung mets	Surgery	0	LN tenderness	6	SD	DTIC	46	19
			SC trunk						Radiotherapy
		32	Adrenal, axil. LNs		5	LN tenderness	7	MR
		35	Inguinal LNs		9	Fatigue, purpuric rash	5	PD
		41	Bone			Flu-like symptoms
		44	Brain
4	57, F	0	Lung, SC mets	Surgery	4	Tender SC mets	7	SD	DTIC	55	48
		5	SC back		11	Tiredness	7	MR
		10	L Psoas region		15		5	SD
		13	SC chest, back		19		4	PR
						23		4	PR
						30		5	PD
5	69, F	89	Lung, abdo. & pelvic LNs		0	Tiredness	6	PD	Surgery, radiotherapy	117	28
		92	Brain
6	71, M	49	Inguinal LNs	Surgery	4	Pulmonary embolus	7	PD	Palliation	57	18
		51Intransit SC mets	IL-1		Chest infection
7	42, F	110	LNs abdo & pelvis	Surgery	2	Fatigue, nausea	5	PD	Surgery	120	10
			Lung			Lack of appetite			DTIC
		112	Brain			Decreased Hb			Radiotherapy
		113	Pharynx			Abdo cramping
		115	Small bowel
8	38, M	0	SC mets	Surgery	26	Vaccine site red/swollen	7	PD	Palliation	38	38
		13	Brain	Radiotherapy		Emotionally labile
		16	Small intestine	DTIC		Tiredness
		21	Spleen
9	54, F	117	Linguinal LNs	DTIC	10	Abdo & throat discomfort	7	PR	Surgery	137	30
		118	Brain	Radiotherapy	14	Tenderness at vac sites	4	MR	Palliation
		119	Small bowel	Surgery	17	Leg cramps	4	PR
		122	SC trunk			Itchy nevi		PD
		125	R lung
10	51, M	1	Lung, SC mets, stomach	DTIC	12	Nil	6	SD	Surgery	30	30
					19			6	PD	Palliation
11	56, F	98	L lung	DTIC	6	Tiredness	6	PD	Palliation	116	18
			Liver	IFN
			SC chest
12	61, M	231	SC	Surgery	3	Muscle tenderness and aching	6	PD	DTIC	10	10
		236	Lung
			Bone, brain
13	57, M	17	Lung, SC	DTIC	7	Nil	7	PD	Palliation	28	11
14	41, F	15	SC mets, liver	Chemotherapy	5	Nil	2	SD	Palliation	52	39
			Breast		8		7	PD
15	52, F	136	R neck	VMCL	20	Tiredness	3	PD	Surgery	161	25
		155	L thigh, bone	DTIC					Radiotherapy
			Bowel, L neck	Radiotherapy
16	51, M	60	Lung	Surgery	16	Nil	7	PD	Surgery	96 AWD	36
		74	Spleen	Temozolomide
		76	Brain	Radiotherapy
17	70, M	14	R anterior chest	Surgery	1	Nil	6	PD	DTIC radiotherapy	26 AWD	12
18	68, F	36	R post neck	Surgery	19	Nil	7	PR	Surgery	64 A&W	28
		43	LN, liver	DTIC	22		4	PR
		51	SC mets		25		4
		58	SC					PRa
19	30, F	33	Brain	Surgery	8	Nil	7	SD		45 AWD	12
		35	R Lower lobe lung	Radiotherapy

^aRendered tumour-free by surgery

Table 2.

Clinical responses in patients treated with dendritic cells + melanoma peptides. AWD alive with disease, A&W alive and well, VMCL vaccinia melanoma cell lysate vaccine, DTIC dacarbazine, SC subcutaneous, Abdo abdomen

Patient no.	Age & sex	Time from diagnosis to metastases (months)	Site of metastases	Previous therapy	Time from first metastasis to DC treatment (months)	Adverse effects during treatment	No. of vaccines	Best response	Subsequent therapy	Duration to death/follow-up
										From diagnosis (months)	From first metastasis (months)
20	44, M	27	Lung, SC trunk	Surgery	0	Muscle aches			DTIC
		32	Adrenal, LNs		5	LN tenderness	6	SD	Radiotherapy	46	19
			L&R axilla		9	Fatigue, fevers	7	SD
		35	L inguinal LN			Sore throat	5	PD
		41	Bone			Purpuric rash, both legs
		44	Brain			Flu-like symptoms
21	63, M	117	LN neck	VMCL	15	Nil	7	PD	DTIC	143	26
		131	Lung						Radiotherapy
		134	Bone		18
		140	Abdo LN
			Adrenal
22	46, M	55	Lung, ribs		0	Pain at vaccine sites	7	PD	Radiotherapy	59	4
			R axilla LN						DTIC
			Abdominal LNs
23	77, F	26	SC leg	Surgery	6	Tiredness	7PD		41	15
		29	Lung			Breathlessness
		31	Spleen			R leg pain
		31	Brain mets	Radiotherapy		Rash on arms, hands, feet
24	59, M	0	LN abdo/pelvis	Radiotherapy	9	Tiredness	7	PD		13	13
		6	Lung mets	DTIC		Body aches
						Emotional lability
						Pain in knees & ankles
25	81, M	265	R&L lung mets	DTIC	0	Tiredness	6	SD		302 AWD	37
					5	Weight loss	5	PD
						Diarrhoea
26	68, M	185	L lung mets	VMCL	2	General tiredness	7	SD	Surgery	225 A&W	40
27	44, M	19	R humerus mets, liver & SC chest	Surgery, radiotherapy	0	Nil	7	PD	DTIC	32	13
									CC5013
28	52, M	23	SC mets, R chest	Surgery	17	Nil	4	SD	DTIC + G3139	59 AWD	36
		37	R&L lung
		R para trach LN			21	Nil	7	PD	CC5013
29	72, M	6	SC head		0	Nil	7	SD	Chemotherapy	27 AWD	21
			R lobe liver, lung		5		5	PD
			Porta LN
30	66, M	16	R lung	C Vax	0	Nil	7	PD	Surgery	23 AWD	9
			L lung
31	54, M	0	Abdo wall	Temozolomide	12	Nil	7	PD	Chemotherapy	17 AWD	17
			Chest wall	Radiotherapy
32	51, M	30	R axilla	C Vax	14	Nil	7	PD	Chemotherapy	60 AWD	30
		36	R neck	Radiotherapy
		37	R axilla	VMCL
33	66, M	135	Liver, lungs	DTIC	4	Fatigue	5	PD		141	6
			Mediastinal LNs
			Adrenal

There were 13 males and 1 female aged from 44 to 81 years in group 2 (DCs plus HLA-A2 restricted peptides). All were HLA-A*0201. ECOG status was 0 in all but 2 with ECOG status of 1. Four patients had received chemotherapy with DTIC or temozolomide. One patient (No. 14) did not complete the full course of injections. There were no clinical responses in the patients treated with DCs plus melanoma peptides. Five patients had stable disease for periods ranging from 3 to 6 months. The difference in clinical response rates between the two groups was not significant by Fisher exact test (p=0.119).

The median number of DCs given per injection for all patients was 8.3×10⁶ ±6.3 with a range of 2.1×10⁶ to 10.3×10⁶ in those receiving DCs plus melanoma lysates and a range of 4.9×10⁶ to 15.6×10⁶ in those receiving DCs plus peptides. There was no difference in the median numbers of DCs given to the patients in groups 1 and 2. The mean percentage MHC class II CD11c and CD1a expression for all patients was 55.9±19, 65.2±19 and 31.7±21. The low number of patients with clinical responses made correlations with number of DCs difficult to assess. Patients 4, 9 and 18 with PRs had DC injections of 7.1±0.6, 3.9±0.8 and 8.7±3.2 million. These values were not significantly different from the mean for all patients (8.3×10⁶ ±6.3×10⁶) by t-test. The percentages of HLA-DR expression on DCs in these patients were 43±16.7, 42±13.2 and 60±16×10⁶. These values were not significantly different from the mean for all patients (55.9×10⁶ ±19×10⁶) by t-test.

CD1a expression on the DCs given to the patients with PRs was 26.3% ±13, 29.5% ±22 and 12.8% ±7, respectively. As before, these values were not significantly different from the mean (31.7±21) by t-test. CD1a expression was shown by others not to increase on maturation of DCs [26], and immature DCs were efficient in presentation of lipid and glycolipid antigens. CD11c is expressed during maturation of monocytes [27]. Not all DC preparations were assessed for CD11c expression but those that were, indicated relatively high levels of expression. Attempted correlation between clinical responses and other parameters is shown in Table 3.

Table 3.

Correlates with clinical responses. Figures in bold relate to patients with PR (progressive disease), MR (mixed response) or SD (stable disease)

Patient no.	Clinical response	DC yield x106	DC% MHC class II	DTH				IFN-γ
				Any DTH	MART	gp100 280	gp100 209	IFN-γ against any mel pep	MART-1	gp100 280	gp100 209	Tyrosinase
Group 1
1	SD	5.6	62	0				+
2	PD	4.4	43					+
3	MR	5.2	28	0				+
4	PR	7.1	43	+				+
5	PD	6.7	45	0				0
6	PD	6.2	23	0				0
7	PD	2.1	39	0				0
8	PD	5.0	50	0				0
9	PR	3.9	42	+				0
10	SD	-	-	0				-
11	PD	-	-	0				0
12	PD	13.9	74	0				0
13	PD	5.8	71	0				+
14	SD	9.5	79	+				-
15	PD	9.7	94	-				-
16	PD	6.7	51	0				+
17	PD	4.0	44	-				-
18	PR	8.7	60	0				0
19	SD	10.3	63	+				0
p Values				p=.002				ns
Group 2
20	SD	5.2	28		+	0	0		0	0	0	0
21	PD	5.3	29		+	+	+		+	+	+	+
22	PD	7.2	40		+	0	0		0	0	0	0
23	PD	6.3	44		0	+	0		0	0	0	0
24	PD	5.2	32		+	0	0		0	0	0	+
25	SD	4.9	11		0	+	+		+	+	0	0
26	SD	3.5	65		+	0	0		0	0	0	0
27	PD	12	80		0	0	0		0	+	0	0
28	SD	12.5	67		+	0	0		+	+	+	0
29	SD	6.7	68		+	0	0		0	0	0	0
30	PD	15.3	50		0	0	0		0	0	0	+
31	PD	14.9	65		+	0	0		0	0	0	0
32	PD	15.6	70		0	0	0		0	0	+	0
33	PD	-	-		-	-	-		-	-	-	-
All patients, mean ± SD		(8.3±6.3)	(55.9±19)
p Values					ns	ns	ns		ns	ns	ns	ns

Delayed hypersensitivity skin test (DTH)

Eleven of 17 patients in group 1 immunized with DCs plus autologous lysates had responses to KLH. Two patients (Nos. 15 & 17) were not evaluable. Five of 17 had weak responses to the autologous lysates given alone. In 4 patients tested, responses to the melanoma lysates were stronger when given with DCs but similar DTH responses were seen against DCs alone, suggesting that the responses may have been mainly against fetal calf serum in the culture medium that was adherent to the DCs. Tests were therefore done only with lysates alone in the subsequent 15 patients. Two of 3 patients with PR, 1 with MR and 2 of 4 with SD, had positive skin responses. None of those with progressive disease had positive skin responses. The association with clinical responses, including stable disease, was significant by Fisher exact test (p=0.0018).

In group 2, immunized with DCs plus peptides, 11 of 13 patients responded to KLH and 12 of 13 patients responded to the influenza peptide. Seven of 13 patients had responses to MART-1 and 1 patient had a strong erythemal response without induration. Two of 13 and 4 of 13 had responses to gp100 209-2M and gp100 280-9V, respectively. One patient was not evaluable. Two patients had responses to tyrosinase but none had responses to MAGE-3.A2. There was no correlation of responses to individual or combinations of peptides and clinical responses as defined as stable disease (Table 3).

IFN-γ production from PBL of patients treated with DC vaccines

Patients treated with DCs plus autologous lysates were tested against known peptide epitopes and when available the autologous lysates. IFN-γ production in response to the peptides was low. Increased responses (greater than 50 pg above background) against MART-1 were seen in patients 1, 2, 3, 13 and 16. Using the same criteria, responses were seen against MAGE-3.A2 in patients 1, 4 and 13, against tyrosinase in patients 4, 10 and 13, against gp100 280-9V in patients 10 and 16, and gp100 209-2M in patients 2, 3, 4 and 16. No increases in responses were seen against the influenza peptides. In some of the patients, responses to PHA or anti-CD3 were low and lack of responsiveness may have indicated generalized lack of responsiveness (e.g. patients 2, 3, 4, 8, 12, 13, 16). IFN-γ in response to the individual peptides or combinations of peptides did not correlate with clinical responses (Table 3) in patients 4, 9 and 18.

In group 2, IFN-γ production was also low. In some patients this could be attributed to generalized lack of responsiveness, as shown by low responses to anti-CD3 or PHA by patients 1, 3 and 4. Increase in IFN-γ production (greater than 50 pg) was seen, however, against the influenza peptides in 7/13 patients, and MART-1peptides in 4/13 patients. There were 4, 5 and 5 responses in 13 patients immunized with peptides from tyrosinase, gp100 280-9V and gp100 209-2M respectively. Three responses were seen against MAGE-3.A2. There was no significant association with clinical responses (defined as stable disease) by Fisher exact test. Cox stratified regression analysis taking into account patients with low responses to anti-CD3 or PHA did not show a correlation between cytokine and clinical responses.

In 5 patients (9, 10, 17, 18 and 19) it was possible to test responses of their PBL to DCs sensitized with autologous lysates. There was no evidence of specific responses to the DC plus lysates (data not shown).

IL-10 production was very low and there were no clear increases in production of IL-10 in response to the peptides in patients immunized with DCs plus peptides or melanoma cell lysates (data not shown).

Discussion

The protocol selected for this study was based largely on that reported by Nestle et al. [11] that was associated with 2 CR and 3 PR in a group of 16 patients. Results from the present study were not so impressive but nevertheless, 3 significant clinical responses (PR) were seen in the 19 patients receiving DCs plus autologous lysates (response rate approximately 16%). In both groups combined there were 11 patients (4 in group 1 and 5 in group 2) who had no significant progression of their disease over periods ranging from 3 to 6 months. The significance of the latter is difficult to assess in phase I/II studies, particularly as melanoma may not progress over similar time periods without active treatment. A randomized trial assessing time to progression and overall survival would be needed to assess the significance of stable disease in those treated by DC vaccines. Six patients in total did not complete the full course of injections due to disease progression, consistent with the natural history of stage IV melanoma. As noted in previous trials, side effects from the treatment were minimal and were sometimes difficult to distinguish from symptoms of the disease itself. Tiredness, aching of muscles and tender lymph nodes were the commonest symptoms and in most patients, were related in time to the injections. Vitiligo or autoantibodies were not induced in the patients. The responses that were seen could not be related to the number of DCs given per injection or to the phenotype of the DCs.

The DC preparations were effective in inducing DTH responses to KLH in 22 of 30 patients tested. In those receiving DC plus peptides, responses to the influenza peptide were seen in 12 of 13 patients. The DCs used were therefore functionally competent. Relatively few DTH responses (5 of 17) were induced against autologous lysates but these were significantly associated with clinical responses (p=0.0018). DTH responses were induced in over half the patients immunized with DCs plus MART-1 peptides. DTH responses against the other peptides were less frequent and were seen in only approximately one third of the patients. This is consistent with previous studies that have shown MART-1 to be more immunogenic than other common melanoma antigens [23]. Similarly, assays of cytokine production showed that immunization with peptides from influenza were associated with an increase in IFN-γ production in 7/13 patients. Responses in these assays were also seen in approximately one third of the patients against peptides from tyrosinase and gp100 209-2M and gp100 280-9V. In both groups combined there were 10 patients in which IFN-γ production in response to PHA or anti-CD3 was low and hence generalized low immune responsiveness may have accounted in part for the low response to melanoma antigens in DTH and IFN-γ production assays.

Despite the evidence of immune responses to the peptides in patients immunized with DCs plus peptides, this was not associated with clinical responses even though 5 patients had stable disease for at least 3 months. The difference in clinical response rates between the two groups was not significant by Fisher exact test (p=0.119), but there was a predominance of males (13/14) in the DC plus peptide group compared with those immunized with DCs plus lysates (9/19). All 3 clinical responses in the latter group were in females, with a mixed response in a male patient. Stable disease was seen in 3 females and 1 male. Factors other than the antigen source may therefore have influenced the differences in clinical response rates between the groups. An association between clinical responses and sex was, however, not evident in other reported studies [11, 12, 28].

In group 1 patients, we examined by IFN-γ production assays whether immunization with DCs plus autologous lysates induced responses to the known melanoma peptide antigens used in this study. With 3 exceptions, there was no convincing evidence of such responses. In 5 patients tested against their own DCs + lysates we were also unable to detect an increase in IFN-γ production. These tests included PBL from patient 9 and 18, who had a PR during treatment. We conclude that the assays used to detect responses against peptides, including DTH tests, were useful in determining whether the immunization procedures were able to induce immune responses but the presence or absence of detectable immune responses against the melanoma peptides were not reliable indicators of clinical responses. Previous authors have drawn attention to the lack of correlation between assays on PBL and lymphocytes infiltrating into tumours [29], perhaps due to selective homing of specific T cells to the tumour sites. Others have also noted the lack of correlation with clinical responses [30, 31], which was considered due in part to the low frequency of clinical responses. Some studies suggest that measures of T-cell receptor affinity [32] or of particular T-cell subsets [33] or of regulatory T cells [34, 35] may be important correlates of clinical responses. Studies on tumour biopsies have been suggested as more accurate surrogates of clinical responses [36].

Since initiation of this study, evidence has been presented that ‘mature’ DCs are more effective in induction of immunity than ‘immature’ DCs, as used in this trial [37]. Jonuleit [38] reported that ‘mature’ DCs compared with ‘immature’ DCs sensitized with different peptide antigens and injected into different LNs in the same patients induced more peptide-specific CD8 T-cell IFN-γ and CTL responses. Similar results were reported from in vitro studies [39]. Adema et al. [40] reported low reactivity to KLH and peptides from gp100 and tyrosinase in patients treated with immature DCs compared with those treated with mature DCs. These authors reported that mature but not immature DCs migrated to adjacent LNs when injected into lymph nodes [41]. Immature DCs were found in some studies to induce tolerance [42] or inhibit induction of effector T cells [43] and induce induction of regulatory T cells [44, 45]. Studies by others using immature DCs were also not impressive. Smithers et al. [28] treated 19 patients with immature DCs sensitized with autologous melanoma peptides and reported 1 CR and 2 PRs. Panelli et al. [14] in a dose-finding phase I study with DCs + gp100 209-2M reported one transient PR in 10 patients and Ranieri [46] reported 2 CR and 1 PR in 23 patients treated with immature DCs. These and other studies are summarized in Table 4.

Table 4.

Summary of dendritic cell vaccine trials against melanoma. CR complete response, MR mixed response, PR partial response, N no, Y yes, Mono monocyte derived, CD34 myeloid progenitors

Investigator	Source of antigen	DCs	Matured	Route	No. of patients	Response
Nestle et al. [16]	Lysates, peptidesa and KLH	Mono	N	LNs	16	2 CR, 3 PR
Chakraborty et al. [14]	Melanoma lysates	Mono	N	i.d.	15	1 PR
Ranieri et al. [46]	gp100, MART-1, tyrosinase	Mono	N	i.v.	23	2 CR, 1 PR
Thurner et al. [11]	MAGE-3A.1	Mono	Y	s.c. and i.d. x3	13	6 PR or MR
Schuler-Thurner et al. [54]	MAGE-3A.1	Mono	Y	i.v. x3	24	1 CR
Gajewsky et al. [55]	MAGE-3, MART-1	PBL	N	s.c. & IL-12 s.c.	15	1 PR, 3 MR
Panelli et al. [13]	MART-1, gp100	Mono	N	i.v.	17	1 PR
Jonuleit et al. [38]	MART-1, MAGE-1	Mono	Y	LNs	11	3 PR
Smithers et al. [28]	Lysates	Mono	N	s.c.	22	1 CR, 2 PR
Mackenson et al. [12]	MART-1, gp100, tyrosinase	CD34	Y	i.v.	14	1 CR, 1 PR
Banchereau et al. [15]	MART-1, gp100, MAGE-3, tyrosinase	CD34	Y	s.c.	17	3 CR, 3 PR
Chang et al. [56]	Melanoma lysates	Mono	N	id.	17	1 PR
Lau et al. [57]	gp100, tyrosinase	Mono	N	iv.	16	1 CR, 2 PR

^aPeptides were tyrosinase, gp100 and MART-1 for HLA-A2 patients, and MAGE-1.1 for HLA-A1 patients

Whether use of mature DCs is the only factor needed to improve clinical response rates is doubtful. There is a consensus that addition of foreign helper proteins, such as KLH, improves immune responses to peptides and that this can be further increased by injections of IL-2 [47, 48, 49] or IL-12 [50]. There is also some evidence that lymphocyte depletion prior to immunization may increase specific T-cell responses to tumours [51, 52]. These hypotheses will need to be tested in well planned clinical studies. We have hypothesized that immunotherapy may be limited by the induction of resistance to apoptosis induced by effector cells [53] and if this is correct, important gains may be made by combining immunotherapy with agents that sensitize melanoma cells to apoptosis [54].

Abbreviations

DC

dendritic cells

DTH

delayed hypersensitivity skin tests

KLH

keyhole limpet hemocyanin

CTL

cytotoxic T lymphocytes