Tumor heterogeneity as a rationale for a multi-epitope approach in an autologous renal cell cancer tumor vaccine

Figures & data

Table 1 Patient characteristics (N=133)

Parameter
Age (mean ± standard deviation; median)	58±16.5; 60
Sex (n)
Male	80
Female	42
Unknown	11
Male/female ratio	60/32
pT stage (n)
pT 1	43
pT 2	21
pT 3	39
pT 4	1
Unknown/9	29
pN stage (n)
pN 0/pN x	85
pN 1	5
Unknown	43
Grading (n)
G 1	12
G 2	65
G 3, 4	29
Unknown	27
Histological cell type (n)
Clear cell carcinoma (ccRCC)	116
Papillary carcinoma (pRCC)	4
Chromophilic carcinoma	6
Chromophobic carcinoma	4
Oncocytoma RCC	2
Metastatic	1

Abbreviation: RCC, renal clear cell carcinoma.

Table 2 List of assays used and the respective sample dilutions

Antigens (tumor-associated antigen and cellular proteins) detected by ELISA
Antigen	Test name/manufacturer	Optimal sample dilution
CA-IX	Quantikine Human Carbonic Anhydrase IX/CA9 Immunoassay/R&D Systems, Minneapolis, MN, USA	1:4–1:16
CA-125	CA-125 II, Cobas/Roche Diagnostics, Mannheim, Germany	1:2
CA-50	RIA-gnost^® CA50/Schering, Berlin, Germany	1:2–1:4
CEA	CEA, Cobas/Roche Diagnostics, Mannheim, Germany	1:2
CYFRA21-1	CYFRA21-1, Cobas/Roche Diagnostics, Mannheim, Germany	1:12–1:16
FAS ligand	Human sFas Ligand ELISA/Bender Medsystems, Vienna, Austria	1:2
HSP-60	HSP60, R&D Systems, Wiesbaden, Germany; Catalog # DYC1800-2	1:2
HSP-70	HSP70, R&D Systems, Wiesbaden, Germany; Catalog # DYC1663-5	1:2
HMBG1	SHINO-TEST CORPORATION, Kanagawa, Japan	1:10
HER2/neu	HER-2/neu (H2n)/Siemens Medical Solutions Diagnostics, Deerfield, IL, USA	1:2–1:4
sICAM	Human sICAM-1 ELISA/Bender Medsystems, Vienna, Austria	1:5–1:20
MMP-2	Quantikine Human/Mouse MMP2/R&D Systems, Minneapolis, MN, USA	1:5–1:10
MMP-9	Human MMP-9-ELISA/Bender Medsystems, Vienna, Austria	1:2–1:5
NSE	Liaison^® NSE/DiaSorin S.p.a., Saluggia, Italy	1:2–1:8
NMP22	Matritech NMP22 Testkit/Freiburg, Germany	1:2–1:20
Survivin	Biomol (Enzo Life Sciences), Survivin (human total) EIA Kit; catalog # 900-111, Plymouth Meeting, PA, USA	1:2–1:8
SOD	Seramun, Wolzig, Germany	1:50
TPA	Liaison^® TPA/DiaSorin S.p.a., Saluggia, Italy	1:2–1:16
TPS	TPS (Cyk18) ELISA/DRG Instruments GmbH, Marburg, Germany	1:100–1:500
Antigens, detected by PCR-ELISA
hTERT	Quantitative Telomerase Detection Kit (QTD Kit)/US Biomax, Rockville, MD, USA	1:1–1:2
Cellular proteins detected by enzyme activity testing or spectroscopic tests
Hemoglobin (free)	Dr Lange Test freies Hämoglobin/Berlin, Germany	1:1
ALAT	RANDOX Laboratories Ltd., Ardmore, Antrim, UK	1:1
ASAT	RANDOX Laboratories Ltd., Ardmore, Antrim, UK	1:1
LDH	RANDOX Laboratories Ltd., Ardmore, Antrim, UK	1:1

Abbreviation: PCR, polymerase-chain-reaction.

Table 3 List of used primary and secondary antibodies, their manufacturers, and incubation conditions

Antigens detected by Western blotting
	Manufacturer primary	Dilution primary antibody	Incubation time primary antibody	Secondary antibody	Manufacturer secondary antibody	Dilution secondary antibody	Incubation time secondary antibody
CA-IX	Novus	1:500	Overnight (4°C)	Rabbit IgG-HRP	Dianova	1:6,000	1 hour (20°C–25°C)
CD10	Dianova	1:1,000
c-Met	Dianova	1:1,000
CK18	Dianova	1:1,000
CK19	Biozol	1:1,000
CXCR4	Dianova	1:1,000
N-Cadherin	Hypromatrix	1:1,000
Survivin	Novus	1:1,000
hTERT	Biomol	1:1,000
CD15	Biozol	1:1,000	Overnight (4°C)	Mouse IgG/IgM-HRP
EGFR	Biozol	1:1,000
EPAH2	Tebu-bio	1:1,000
GP200	Acris	1:1,000
MUC-1	Dianova	1:1,000
TPT1	Novus	1:1,000

Table 4 List of antigens and cellular proteins in tumor cell lysates

Cellular proteins/tumor-associated antigen/danger	Number tested				Rate positive (%)
	Total	ccRCC	Positive	Negative	Overall	Clear cell
CA-IX	16	15	16	0	100	100
CA50	14	13	10	4	71	69
CD15	17	17	0	17	0	0
EPHA 2	17	17	4	13	24	24
MUC-1	9	9	0	9	0	0
sFAS	11	11	0	11	0	0
CXCR4	21	20	18	3	86	85
Gp200	9	9	0	9	0	0
CD10	22	21	19	3	86	86
hTert	21	21	10	11	48	48
EGFR	17	17	4	13	24	24
C-Met	9	9	0	9	0	0
NMP22	14	13	11	3	79	85
CK8	6	5	6	0	100	100
CK18	5	4	5	0	100	100
HMGB1	3	2	3	0	100	100
TPT1	18	18	2	16	11	11
MMP2	14	13	10	4	71	77
NSE	11	10	11	0	100	100
CK19	5	4	5	0	100	100
Survivin	19	19	4	15	21	21
Her2neu	11	11	3	8	27	27
nCAD	17	17	7	10	41	41
SICAM	14	13	8	6	57	54
CEA	12	12	1	11	8	8
CA125	11	11	0	11	0	0
TPS	13	13	12	1	92	92
TPA	13	13	13	0	100	100
NMP9	14	13	12	2	86	85
CYFRA	17	15	15	2	88	12
Cellular proteins
HSP-60	13	13	13	0	100	100
HSP-70	13	13	12	1	92	100
ALAT	12	9	9	3	75	67
LDH	12	9	9	3	75	67
ASAT	13	10	10	3	77	70
SOD	12	9	9	3	75	78

Abbreviation: RCC, renal clear cell carcinoma.

Table 5 List of antigen/danger patterns in tumor cell lysates selected as typical indicators for renal clear cell carcinoma (frequency of occurrence >70%, n>10)

Tumor-associated antigen/danger	Number tested				Rate positive (%)
	Total	ccRCC	Positive	Negative	Overall	Clear cell
HSP-60	75	63	73	2	97	98
HSP-70	71	60	65	6	92	92
CA-IX	85	75	71	14	84	91
NSE	104	91	83	21	80	87
TPA	102	90	85	17	83	84
TPS	103	90	83	20	81	81
CYFRA	101	89	82	19	81	82

Abbreviation: RCC, renal clear cell carcinoma.

Figure 1 Pattern of antigen distribution in different RCCs: frequencies of occurrence were calculated for all RCC samples and stratified by sex.

Note: Red outline refers to danger signals, green outline refers to most frequent antigens available for potency testing.
Abbreviations: RCC, renal clear cell carcinoma; pos, positive; neg, negative.

Table 6 Absolute values of different tumor-associated antigens and cellular marker proteins (CYFRA, NSE, TPA, and TPS) in nontumor tissue (normal) and the respective tumor tissue (ccRCC) (ND, not determined)

Sample code	Tissue	CYFRA (mg/L)	NSE (μg/L)	TPA (U/L)	TPS (U/L)
840a	ccRCC	85.0	86.3	34,080	35,650
840b	Normal	311.6	16.9	39,296	13,500
799a	ccRCC	10.7	6.5	ND	<7,500
799b	Normal	66	1.7	ND	<7,500
806a	ccRCC	16.7	5.2	247,680	124,000
806b	Normal	177.5	3.6	ND	7,550
842a	pRCC	51.6	2.2	20,832	<7,500
842b	Normal	14.7	0.6	1,053	<7,500
811a	pRCC	1,942.4	4.6	669,120	118,000
811b	Normal	284.6	8.3	ND	42,550

Abbreviation: RCC, renal clear cell carcinoma.

Table 7 Absolute values of carbonic anhydrase IX (CA-IX) in nontumor tissue and the respective tumor tissue

Sample code	Tissue	CA-IX (μg/L)
596a	ccRCC	1.7
596b	Normal	<0.001
600a	ccRCC	49.5
600b	Normal	<0.001
905a	ccRCC	3.8
905b	Normal	<0.001

Abbreviation: RCC, renal clear cell carcinoma.

Figure 2 (A) Overlay immune-stains for an antibody panel in tumor tissue (examples): CD10 (purple), CD11b (green), CD21 (blue), CD4 (light green), CD40 (cyan), CD45RA (yellow), CD45RO (violet), CD8 (pink), CA IX (red), and propidium iodine (mint); (B) Overlay immune-stains for an antibody panel in single cell suspension (examples): NSE (red), p53 (green), and CD105 (blue), combined signals: NSE + p53 + CD105 (white), p53 + CD105 (yellow), and NSE + CD105 (violet); (C) Overlay immune stains for an antibody panel in tumor tissue for CA-IX (green), NSE (pink), and CXCR4 (blue); and (D) Immune stain for a NSE-specific antibody (white dots) in nontumor kidney tissue (negative control).

Abbreviations: CA-IX, carbonic anhydrase IX; NSE, neuron-specific enolase.

Table 8 Detection of a humoral immune response in mice (n=28) after two intradermal shots (200 μL) with a TCL; total protein and antigen levels in the respective TCL are given (missing values justified by sample availability)

Test item	Group	Protein content TCL (μg/mL)**	CA-IX (ng/mL)	NSE (ng/mL)	aCA-IX mean (AU/mL)	anti-NSE mean (AU/mL)	anti-CK mean (AU/mL)
TCL_1 (909) n=5	1	109	1.0	9.8	62.5 %naïve 352%	84.7 %naïve 188%	174.0 %naïve 239%
TCL_2 (868) n=5*	2	226	1.0	23.2	69.4 %naïve 390%	101.8 %naïve 226%	190.9 %naïve 262%
TCL_3 (867) n=5	3	78	ND	3.4	101.5 %naïve 571%	154.8 %naïve 344%	248.6 %naïve 341%
TCL_4 (865) n=5	4	ND	1.0	20.5	90.6 %naïve 510%	113.9 %naïve 253%	273.6 %naïve 375%
TCL_5 (859) n=3	5a	ND	0.03	ND	96.7 %naïve 544%	148.4 %naïve 330%	253.9 %naïve 348%
per subgroup	5b				104.7 %naïve 589%	248.0 %naïve 551%	286.6 %naïve 393%
	5c				117.2 %naïve 659%	144.7 %naïve 321%	255.5 %naïve 351%
Naïve, pooled (n=5)					17.8	45.0	72.9

Notes: TLC_5 was carried out in three subgroups to check repeatability.

* 1 died before bleeding.

** Measured by BCA assay.

Abbreviations: ND, not determined; TCL, tumor cell lysate; CA-IX, carbonic anhydrase IX; NSE, neuron-specific enolase.