Supplemental Materials
Supplemental Methods
Study Design
This was a single institution study approved by the JHMI Institutional Review Board
(IRB), the Food and Drug Administration (FDA) Center for Biologic Evaluation and Research
and the Recombinant DNA Advisory Committee (RAC).
A classical ‘3+3’ dose escalation design was adopted in this trial. Three patients were
initially enrolled at the lower dose level (DL1). All patients within this cohort were observed for a
minimum of three weeks after the second vaccination prior to administration of vaccine to a
patient at the higher dose level. If 0/3 patients experienced a DLT, then accrual continued at the
higher dose level (DL2). If 1/3 patients experienced a DLT, an additional 3 patients would have
been accrued at DL1. If 1/6 patients had experienced a DLT, then accrual continued at DL2. If 2
patients developed a DLT in this cohort of 3 or 6 patients, the study would have been closed to
further accrual.
Each vaccination consisted of three (DL1) to fifteen (DL2) intradermal injections,
distributed equally among the right and left thighs, and the non-dominant arm. In the event that
the
specified
limb
was
contraindicated,
the
dominant
arm
was
used.
Anesthetic
lidocaine/prilocaine cream was applied to the injection sites one to two hours prior to vaccination
to minimize patient discomfort. Patients received up to four monthly vaccinations.
Vaccine Production
SW837, SW620 and K562 cell lines were originally obtained from the American Type
Culture Collection (ATCC, Manassas, VA, USA). Master Cell Banks and subsequently clinical
lots of each cell line were manufactured by the Johns Hopkins cGMP facility. The SW837 cell
line expresses HLA-Class I alleles: A2, A24, B7, Bw6, Cw7; and the SW620 cell lines expresses
A32, B35, Bw6, Cw4. Neither cell line expresses HLA-Class II alleles.
According to the
literature, they share tumor-associated antigens (TAAs) including CEA, EpCAM, S100A4, and
MUC1, which many colorectal cancers are known to express1-2. K562 lacks expression of HLA
class I molecules and only expresses HLA class II when treated with interferon-α. The K562
cell line is transfected with a plasmid vector encoding human GM-CSF3. K562/GM-CSF grows
well in serum-free media, stably expresses > 1000 ng of GM-CSF / 106 cells/ 24 hours, and is
easily expanded to large numbers for clinical lot production3. The clinical lot of SW837 cells or
K562/GM-CSF cells was irradiated at 10,000 rads while the clinical lot of SW620 was irradiated
at 15,000 rads prior to cryopreservation in vapor phase liquid nitrogen.
Patient Selection and Eligibility Criteria
Patients were considered eligible if they met the following criteria including: histologically
confirmed adenocarcinoma of colon and/or rectum; radiographic evidence of metastatic
disease; patients may have received systemic chemotherapy for colorectal cancer; adequate
hematologic, hepatic and renal function; an age equal to or above 18, an Eastern Cooperative
Oncology Group (ECOG) performance score of 0-1; a life expectancy more than 12 weeks; no
childbearing potential or using a medically acceptable method of highly effective contraception
throughout the study period and for 28 days after study treatment administration; no surgery,
systemic chemotherapy, radiation therapy, biologic therapy, any other anti-cancer vaccine
therapy, investigational new drug treatment, or systemic corticosteroid treatment within 28 days
prior to the initiation of treatment on study; no unresolved chronic toxicity greater than National
Cancer Institute Common Terminology Criteria Version 3.0 (NCI CTC V3.0) grade 2 from
previous anticancer therapy (except alopecia); no prior or currently active autoimmune disease
requiring management with systemic immunosuppression; no active or uncontrolled medical or
psychosocial problems; no evidence of active acute or chronic infection; no history of other
malignancies within the prior five years; no known history or evidence of central nerve system
metastases
within
2
years;
no
known
or
suspected
hypersensitivity
to
GM-CSF,
cyclophosphamide, pentastarch, hetastarch, corn, or DMSO.
Statistical Consideration
This study was not designed to determine a maximum tolerated dose; however, a safety
evaluation was performed following the standard ‘3+3’ dose escalation approach with the two
dose levels defined (Table 1). The highest dose level at which no more than 1 out of 6 patients
developed a DLT was considered to have an acceptable safety profile and would be suggested
for subsequent trials. The sample size for this study was determined by clinical rather than
statistical considerations. However, the probabilities of detecting at least one DLT in 6 patients
are 0.26, 0.47, and 0.74 when the true rates are 5%, 10%, and 20%. All patients who received
at least one dose of treatment were included in the safety analysis. Safety was measured by
systemic toxicity. Local vaccination site reactions were anticipated, and therefore, were not
considered in the safety analysis. All statistical analyses were descriptive in nature. The
number, type and degree of toxicities for each round of vaccination were tabulated with
frequencies and percentages.
Assessment of toxicities
Toxicities were assessed by obtaining histories and performing physical examinations.
Monitoring for toxicity also included a complete blood count with differential white blood cell
count and a comprehensive metabolic panel on days the patients received cyclophosphamide
and days 7 and 28 of each vaccination cycle. Toxicities were characterized according to the
NCI CTC V3.0.
Immune monitoring studies
The patients underwent collection of peripheral blood for mononuclear cells and serum
archiving prior to the first vaccine administration, then one month after each vaccination to
perform the correlative studies.
Serum samples were frozen at -80 C until tested. Antibody responses to colon cancer
tumor antigens were first identified using western blot analysis of pre and post vaccination
patient serum. Investigated antigens were based on the antigenic characterization of the colon
cancer vaccine tumor cell lines SW620 and SW837. These included Ep-CAM (Abnova,
H0004072-G01), carcinoembryonic antigen (CEA) (Prospec, PRO-287), insulin like growth
factor-1 receptor (Prospec, PKA-237) ErbB-2 (Prospec, PKA-343), S100A4 (Protein
Biotechnologies, RP-179), and mucin 1 (MUC1) (Origene, TP321390). Influenza protein was
used as a positive control. In cases where Western blot analysis demonstrated post vaccination
induction of anti-tumor antigen humoral response, these subject samples were further
investigated using enzyme linked immune sorbent assay (ELISA) analysis.
Briefly, ELISA plates were coated with 100 l/well MUC1 (1 g/ml) protein and stored
overnight at 4C. Plates were subsequently washed three times with TBS plus 0.1% Tween
(TBST) and incubated with 150 l/well of ELISA blocking buffer (Pierce, Cat # N502) at room
temperature for 1 hour. After washing three times with TBST, 100 l/well of serum samples at
different dilutions were plated and incubated at room temperature for 2 hours. The plates were
washed as described above and incubated for 1 hour at room temperature in 100 l/well of a
1:30000 dilution of goat anti-human IgG-peroxidase conjugate secondary antibody (Sigma,
A8419). Plates were again washed as described above and 100 l/well of TMB ready to use
solution was added (Sigma, T0440) and incubated in the dark for 30 min. The reaction was
stopped with the addition of 100 l/well of 1 N H2SO4. Plates were read at an optical density of
450 nm. Each serum sample was tested in duplicate. The specific reactivity to the MUC1
antigen was calculated subtracting the averaged OD from the antigen and coating buffer alone.
Statistical analysis of comparing the titer of anti-MUC1 antibodies in pre-vaccination vs. postvaccination sera was done with t test.
References for Supplemental Materials
1.
2.
3.
Shawler DL, Bartholomew RM, Garrett MA, et al. Antigenic and immunologic
characterization of an allogeneic colon carcinoma vaccine. Clin Exp Immunol 2002;
129(1):99-106.
Takenaga K, Nakanishi H, Wada K, et al. Increased expression of S100A4, a
metastasis-associated gene, in human colorectal adenocarcinomas. Clin Cancer Res
1997; 3(12 Pt 1):2309-16.
Borrello I, Sotomayor EM, Cooke S, Levitsky HI. A universal granulocyte-macrophage
colony-stimulating factor-producing bystander cell line for use in the formulation of
autologous tumor cell-based vaccines. Hum Gene Ther 1999; 10(12):1983-91.