Allogeneic Granulocyte Macrophage Colony-Stimulating Factor–Secreting Tumor Immunotherapy Alone or in Sequence with Cyclophosphamide for Metastatic Pancreatic Cancer: A Pilot Study of Safety, Feasibility, and Immune Activation

Dan Laheru; Eric Lutz; James Burke; Barbara Biedrzycki; Sara Solt; Beth Onners; Irena Tartakovsky; John Nemunaitis; Dung Le; Elizabeth Sugar; Kristen Hege; Elizabeth Jaffee

doi:10.1158/1078-0432.CCR-07-0371

DOI: 10.1158/1078-0432.CCR-07-0371 Published March 2008

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Fig. 1.
Comparison of the magnitude of CD8⁺ T cells specific for mesothelin versus the CEF pool of peptides. An ELISPOT analysis was done to determine the number of IFN-γ–secreting T cells specific for mesothelin as described in Table 3. All patient lymphocytes were also assessed for recognition of a HIV-negative control peptide and a positive antigen control pool of peptides (CEF pool). Background spots ranged from 0 to 10 spots per well as described in Table 3. Mesothelin-specific T-cell response data presented in Table 3 and the corresponding CEF pool–specific CD8⁺ T-cell responses are graphed. A, immunotherapy-only cohort. B, cyclophosphamide + immunotherapy cohort. Striped columns, mesothelin data. Black columns, CEF pool data.

Tables

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Additional Files

Table 1.

Patient demographics

	Cohort A (n = 30)	Cohort B (n = 20)	Total (N = 50)
Median age (range)	56 (37-88)	61 (38-81)	56 (37-88)
Gender
Male	19 (63%)	11 (55%)	30 (60%)
Female	11 (37%)	9 (45%	20 (40%)
Ethnic origin
1	27 (90%)	20 (100%)	47 (94%)
2	1 (3.3%)	0	1 (2%)
3	1 (3.3%)	0	1 (2%)
4	1 (3.3%)	0	1 (2%)
KPS
70	3 (10%)	2 (10%)	5 (10%)
80	12 (40%)	5 (25%)	17 (34%)
≥90	15 (50%)	13 (65%)	28 (56%)
Stage
III	3 (10%)	0	3 (6%)
IV	27 (90%)	20 (100%)	47 (94%)
Prior surgery	24 (80%)	15 (75%)	39 (78%)
Prior XRT	7 (23%)	6 (30%)	13 (26%)
Prior chemotherapy regimens
0	6 (20%)	3 (15%)	9 (18%)
1	8 (27%)	12 (60%)	20 (40%)
2	11 (37%)	2 (10%)	13 (26%)
3	5 (17%)	3 (15%)	8 (16%)

NOTE: Ethnic origin: 1, White Caucasian; 2, African American; 3, Hispanic; 4, Asian.
Abbreviation: KPS, Karnofsky performance status.

Table 2.

Summary of treatment-related adverse events

(A) Injection site reactions (per vaccine)
Vaccine 1	Cohort A (n = 30)					Cohort B (n = 20)
Site reaction	Grade 1	Grade 2	Grade 3/4	Grade 1	Grade 2	Grade 3/4
Erythema/induration	18	8	0	18	2	0
Pruritus	20	0	0	16	0	0
Pain/soreness	11	0	0	7	0	0

Vaccine 2	Cohort A (n = 15)					Cohort B (n = 15)
Site reaction	Grade 1	Grade 2	Grade 3/4	Grade 1	Grade 2	Grade 3/4
Erythema/induration	11	4	0	15	0	0
Pruritus	8	0	0	9	0	0
Pain/soreness	8	0	0	3	0	0

Vaccine 3	Cohort A (n = 9)					Cohort B (n = 10)
Site reaction	Grade 1	Grade 2	Grade 3/4	Grade 1	Grade 2	Grade 3/4
Erythema/induration	8	1	0	9	1	0
Pruritus	5	0	0	5	0	0
Pain/soreness	2	0	0	2	0	0

Vaccine 4	Cohort A (n = 7)					Cohort B (n = 7)
Site reaction	Grade 1	Grade 2	Grade 3/4	Grade 1	Grade 2	Grade 3/4
Erythema/induration	5	0	0	6	0	0
Pruritus	5	0	0	6	0	0
Pain/soreness	0	0	0	2	0	0

Vaccine 5	Cohort A (n = 5)					Cohort B (n = 4)
Site reaction	Grade 1	Grade 2	Grade 3/4	Grade 1	Grade 2	Grade 3/4
Erythema/induration	4	1	0	4	2	0
Pruritus	5	0	0	2	0	0
Pain/soreness	1	0	0	1	0	0

Vaccine 6	Cohort A (n = 3)					Cohort B (n = 4)
Site reaction	Grade 1	Grade 2	Grade 3/4	Grade 1	Grade 2	Grade 3/4
Erythema/induration	2	1	0	4	0	0
Pruritus	2	0	0	3	0	0
Pain/soreness	1	0	0	0	0	0

Vaccine 6	Cohort A (n = 3)					Cohort B (n = 4)
Site reaction	Grade 1	Grade 2	Grade 3/4	Grade 1	Grade 2	Grade 3/4
Erythema/induration	2	1	0	4	0	0
Pruritus	2	0	0	3	0	0
Pain/soreness	1	0	0	0	0	0

(B) All other treatment-related adverse events
Organ class	All grades					Grade 3/4
		Cohort A	Cohort B	Total	Cohort A	Cohort B	Total
		n = 30 (%)	n = 20 (%)	N = 50 (%)	n = 30 (%)	n = 20 (%)	N = 50 (%)
Metabolism
Dehydration	1 (3.3)	0 (0)	1 (2)	1 (3.3)	0 (0)	1 (2)
Nervous system
Dizziness	0 (0)	1 (5)	1 (2)	0 (0)	0 (0)	0 (0)
Headache	0 (0)	1 (5)	1 (2)	0 (0)	0 (0)	0 (0)
Vascular
Hot flashes	0 (0)	1 (5)	1 (2)	0 (0)	0 (0)	0 (0)
Gastrointestinal
Nausea	1 (3.3)	1 (5)	2 (4)	0 (0)	0 (0)	0 (0)
Vomiting	0 (0)	1 (5)	1 (2)	0 (0)	0 (0)	0 (0)
Skin
Dry skin	1 (3.3)	0 (0)	1 (2)	0 (0)	0 (0)	0 (0)
Night sweats	1 (3.3)	1 (5)	2 (4)	0 (0)	0 (0)	0 (0)
Pruritus	1 (3.3)	1 (5)	2 (4)	0 (0)	0 (0)	0 (0)
Rash	1 (3.3)	2 (10)	3 (6)	0 (0)	0 (0)	0 (0)
Generalized itching	1 (3.3)	0 (0)	1 (2)	0 (0)	0 (0)	0 (0)
Musculoskeletal
Arthralgia	1 (3.3)	2 (10)	3 (6)	0 (0)	0 (0)	0 (0)
Myalgia	0 (0)	2 (10)	2 (4)	0 (0)	0 (0)	0 (0)
Extremity pain	0 (0)	1 (5)	1 (2)	0 (0)	0 (0)	0 (0)
General disorders
Asthenia	6 (20)	7 (35)	13 (26)	1 (10)	1 (5)	2 (4)
Chills	1 (3.3)	5 (25)	6 (12)	0 (0)	0 (0)	0 (0)
Fatigue	4 (13.3)	2 (10)	6 (12)	1 (3.3)	1 (5)	2 (4)
Inflammation	0 (0)	1 (5)	1 (2)	0 (0)	0 (0)	0 (0)
Injection site discoloration	0 (0)	1 (5)	1 (2)	0 (0)	0 (0)	0 (0)
Injection site vesicles	0 (0)	1 (5)	1 (2)	0 (0)	0 (0)	0 (0)
Pyrexia	5 (16.7)	3 (15)	8 (16)	0 (0)	0 (0)	0 (0)
Overall	24	34	58	3	2	5

Table 3.

Summary of mesothelin-specific T-cell responses in the HLA-A1⁺/HLA-A2⁺/HLA-A3⁺ patients treated with the vaccine alone or in sequence with cyclophosphamide

Patient/HLA	Mesothelin-specific T cells/10⁶ CD8 T cells							Survival (mo)
Patient/HLA		Pre	Vaccine 3	Vaccine 6	Follow-up	Follow-up 2	Follow-up 3	Survival (mo)
Cohort A (patients given immunotherapy alone)
1/(2)	40	110	NA	NA	NA	NA	3.36
2/(1)	10	0	0	NA	NA	NA	7.1
3/(3)	20	10	NA	NA	NA	NA	3.36
4/(3)	20	50	100	30	50	NA	7.9
5/(1)	90	160	110	NA	NA	NA	17.6
6/(2)	40	60	NA	NA	NA	NA	6.1
7/(2)	60	0	NA	NA	NA	NA	1.7
8/(2)	0	0	NA	NA	NA	NA	2.86
Cohort B (patients given immunotherapy + Cytoxan)
1/(2)	50	230	NA	NA	NA	NA	3.23
2/(1)	10	60	NA	NA	NA	NA	22.5
3/(2)	110	270	NA	NA	NA	NA	6
4/(2)	240	400	NA	NA	NA	NA	7.73
5/(3)	0	0	210	70	NA	NA	25+
6/(3)	70	130	190	NA	NA	NA	8.13
7/(2)	0	0	10	10	NA	NA	13.07
8/(2)	130	350	50	NA	NA	NA	3.7
9/(2)	30	60	50	30	40	110	12.3
10/(2)	50	100	NA	NA	NA	NA	2.6

NOTE: The ELISPOT assay was used to determine the number of mesothelin-specific CD8⁺ T cells specific for the HLA-A1⁺ epitope, MesoA1_(20-28), HLA-A2⁺ mesothelin epitope MesoA2_(530-539), or the HLA-A3 mesothelin_(225-234) epitope. All time points for each patient were assayed simultaneously in six replicates and reported as the mean number of mesothelin-specific CD8⁺ T cells per 10⁶ total CD8⁺ T cells. Background spots were determined using a negative peptide control known to bind to HLA-A1 HIV-Nef_(73-87) (QVPLRPMTY), HLA-A2 HIV-gag_(77-85) (SLYNTVATL), and HLA-A3 HIV-1NEF_(94-102) (QVPLRPMTYK6). Background spots ranged from 0 to 10 spots per well. A CEF pool was used as a positive control. The CEF pool contains epitopes from cytomegalovirus, EBV, and influenza A virus proteins that bind to most HLA class I molecules. The CEF pool was obtained from NIH AIDS Research and Reference Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases, NIH (CEF control peptide pool, 9808). Positive control spots ranged from 40 to 1,300 per well.
Abbreviations: Pre, pretreatment 1; treatment cycle 1, posttreatment 1; vaccine 3, posttreatment 3; vaccine 6, posttreatment 6; NA, not available due to patient progression.

Table 4.

Summary of HLA-A2 tetramer titrations in a subset of HLA-A2⁺ patients treated with the vaccine alone or in sequence with cyclophosphamide

Patient	MesoA2_(531-539)			Tyrosinase		Survival (mo)
Patient		Pre	Vaccine 3	Pre	Vaccine 3	Survival (mo)
Tetramer titration (patients without cytoxan)
4	1:60	1:60	1:10	1:10	7.9
6	1:40	>1:60	1:20	1:20	6.1
7	1:20	1:40	1:10	1:20	1.7
Tetramer titration (patients given cytoxan)
1	1:10	1:40	1:10	1:10	3.23
4	1:20	>1:40	1:20	1:20	7.73
7	1:40	1:60	1:20	1:20	13.07
8	>1:60	1:20	1:10	1:10	3.7
9	>1:60	>1:60	1:20	1:20	12.3
10	1:60	<1:60	1:10	1:10	2.6

NOTE: Patient PBL were labeled with HLA-A2 tetramers as described in Patients and Methods. The tetramer dilutions at which detectable tetramer staining was lost are shown.
Abbreviations: Pre, prevaccine 1; vaccine 3, prevaccine 3.