Collagenase

Patients underwent a total nephrectomy or, if the kidney was already removed before inclusion, a metastasectomy. The tissue that was not used for pathologic diagnosis and staging was transferred to our vaccine production laboratory within 48 h of the surgery. The tumor tissue was then dissociated as previously described [13 (link), 24 (link)]. Briefly, tumor tissue was cut into small pieces and subsequently incubated in a 0.1% DNase I, 0.14% collagenase (Boehringer) solution. After 45 min incubation at 37 °C, single cells were harvested and remaining tumor fragments again suspended in a DNase/collagenase solution; this cycle was repeated 3–4 times, after which single cells were harvested through a 100 µm gauze, a sample for bacteriology control was taken and viability was tested using trypan blue exclusion. Cells were aliquoted (at 15–20 × 10⁶ viable cells per vial) and cryopreserved using a linear freezer. Vials were stored in liquid nitrogen until vaccination. Prior to vaccination, the frozen tumor cells were irradiated (20,000 rad), thawed, counted and assessed for viability. For each patient, we aimed to produce as many vaccines as possible.

Spermidine-tetrahydrochloride (SPD), guinea pig liver TG2 (GPL-TG2), standard crystallins from bovine eye lens, trichloroacetic acid (TCA), dithiothreitol (DTT), ortho-phthalaldehyde (OPA), dansylchloride, trifluoroacetic acid (TFA), acetonitrile (C₂H₃CN), MDL 72527 (N,N′-Bis(2,3-butadienyl)-1,4-butanediamine dihydrochloride) and all reagents were from Sigma-Aldrich (St. Louis, MO, USA). [³H]-SPD (18 Ci/mmol) were obtained from NEN Radiochemicals (Italy). N¹-mono(γ-glutamyl)SPD, N⁸-mono(γ-glutamyl)SPD, N¹-N⁸bis-(γ-glutamyl)SPD were generously provided by J. E. Folk (National Institutes of Health, Bethesda, MD, USA). Pronase, collagenase, aminopeptidase M, carboxypeptidases A, B, and Y were obtained from Boehringer, (Mannheim, Germany). Usual laboratory chemicals were purchased from Merck (Darmstadt, Germany).