Current address: The Laboratory of Dendritic Cell Biology, Department of Immunology, Institut Pasteur, Paris, France
The authors have declared that no competing interests exist.
Studies of patients with paraneoplastic neurologic disorders (PND) have revealed that apoptotic tumor serves as a potential potent trigger for the initiation of naturally occurring tumor immunity. The purpose of this study was to assess the feasibility, safety, and immunogenicity of an apoptotic tumor-autologous dendritic cell (DC) vaccine.
We have modeled PND tumor immunity in a clinical trial in which apoptotic allogeneic prostate tumor cells were used to generate an apoptotic tumor-autologous dendritic cell vaccine. Twenty-four prostate cancer patients were immunized in a Phase I, randomized, single-blind, placebo-controlled study to assess the safety and immunogenicity of this vaccine. Vaccinations were safe and well tolerated. Importantly, we also found that the vaccine was immunogenic, inducing delayed type hypersensitivity (DTH) responses and CD4+ and CD8+ T cell proliferation, with no effect on FoxP3+ regulatory T cells. A statistically significant increase in T cell proliferation responses to prostate tumor cells
An apoptotic cancer cell vaccine modeled on naturally occurring tumor immune responses in PND patients provides a safe and immunogenic tumor vaccine. (ClinicalTrials.gov number NCT00289341).
ClinicalTrials.gov
Tumor immunity in patients with paraneoplastic neurologic disorders (PND) have been studied with the hope of uncovering principles that can be applied to the general population of cancer patients
Several observations support the suggestion that apoptotic tumor cells may serve as a potent source of antigen for stimulating host immune responses
DCs presenting apoptotic tumor cells stimulate T cell responses in animals and
Twenty-four consecutive eligible patients were vaccinated with DC/LNCaP, together with control vaccinations. A total of 28.4–78.9 million (average 50.3 million) DCs cross-presenting apoptotic LNCaP tumor cells were given per patient, divided over 4 doses, each 2 weeks apart (
Patients were screened and randomized into 1 of 2 arms, each with 12 patients. Patients in both arms were blind during the vaccine/placebo phases until Week 8. Patients in Arm 1 continued into the post-vaccine phase while patients in Arm 2 crossed over into the vaccine phase before entering post-vaccine phase.
Pt | Age | Previous Treatments | Clinical Status | Gleason Score | PSA at Study Entry |
1 | 55 | Leuprolide Acetate, Bicalutamide, Nilutamide | CM/HR | 8 | 0.69 |
2 | 80 | RRP, Leuprolide Acetate, Bicalutamide | BCR/HR | 7 | 2.71 |
3 | 65 | RRP, Salvage RT | BCR | 7 | 0.79 |
4 | 58 | RRP | BCR | 6 | 0.49 |
5 | 64 | RRP | BCR | 7 | 0.59 |
6 | 75 | RRP | CM | 9 | 0.57 |
7 | 81 | RT | BCR | 6 | 8.66 |
8 | 54 | RRP, Salvage RT | BCR | 6 | 0.46 |
9 | 60 | RRP, IL-2 and J-591 Ab Study, Testosterone Gel/ Leuprolide or Goserelin Acetate/Docetaxel Study | BCR | 7 | 7.56 |
10 | 57 | Brachytherapy, Salvage RRP | BCR | 7 | 6.38 |
11 | 56 | RRP, Salvage RT, Leuprolide Acetate, Bicalutamide | BCR/HR | 8 | 5.24 |
12 | 69 | RRP, Salvage RT, Goserelin Acetate | BCR/HR | 7 | 0.86 |
13 | 63 | RRP, Salvage, RT, Testosterone Gel/Leuprolide or Goserelin Acetate/Docetaxel Study | BCR/HR | 8 | 0.48 |
14 | 53 | RRP, Salvage RT, Leuprolide Acetate | LR/HR | 8 | 0.94 |
15 | 57 | RRP, Salvage RT, Leuprolide Acetate, Bicalutamide | BCR | 9 | 0.29 |
16 | 64 | RRP | BCR | 7 | 15.27 |
17 | 68 | RT, Testosterone Gel/Leuprolide or Goserelin Acetate/Docetaxel Study | BCR | 8 | 4.61 |
18 | 74 | RRP | BCR | 6 | 40.25 |
19 | 67 | RRP, Salvage RT | BCR | 7 | 5.02 |
20 | 63 | RRP, Leuprolide Acetate, Bicalutamide | BCR | 7 | 2.15 |
21 | 59 | RRP, Salvage RT | BCR | 7 | 0.62 |
22 | 70 | RRP, Salvage RT | BCR | 7 | 1.75 |
23 | 55 | RRP, Salvage RT | BCR | 7 | 0.44 |
24 | 68 | RRP, Salvage RT | BCR | 7 | 1.27 |
Previous Treatments: RRP = Radical Retropubic Prostatectomy, RT = Radiation Therapy. Clinical Status: CM = Clinical Metastasis, HR = Hormone Refractory, BCR = Biochemical Relapse, LR = Local Recurrence.
DCs were cocultured with LNCaP or LNCaP-M1 cells that were >90% apoptotic (
UV irradiation (+UV) specifically induced apoptosis in LNCaP cells as indicated by 96% Caspatag+ TOPRO+ staining 38 hours post UV. DC cocultured with apoptotic LNCaP cells (Vaccine) are mature, with >96% CD83 positive cells. Data shown is representative of all 24 vaccines prepared.
Vaccine Groups | % CD14 + Median (Interquartile Range) | % CD83 + Median (Interquartile Range) | % PI + Median (Interquartile Range) |
DC/LNCaP | 0.13 (0.83) | 86.94 (8.86) | 4.14 (5.87) |
DC/LNCaP-M1 | 0.09 (0.48) | 87.82 (6.86) | 4.37 (4.69) |
DC/KLH | 0.09 (0.41) | 96.19 (3.59) | 1.25 (1.72) |
DCs alone | 0.17 (0.45) | 96.41 (4.94) | 1.52 (1.94) |
Vaccine groups were prepared from patient monocytes as described. DCs were assessed for maturation by staining for expression of surface markers, CD14 and CD83, and for viability using PI stain. Cells were assessed by flow cytometry on Day 8 prior to vaccine release. All vaccine groups administered met release criteria.
The incidence of injection-site and systemic reactions to vaccine are presented in
Single Blind | Unblinded | |||
Arm 2 Placebo Phase | Arm 1 Vaccine Phase | Arm 2 Vaccine Phase | Arm 1 and 2 Post-Vaccine Phase | |
80 | 120 | 90 | 76 | |
12 (12) | 12 (12) | 12 (12) | 22 (24) | |
injection site reaction | 2 (2) | 22 (11) | 29 (12) | 0 |
injection site reaction (grade 2) | 0 | 4 (2) | 0 | 0 |
0 | 0 | 0 | 0 | |
albumin, serum, low | 0 | 1 (1) | 4 (3) | 0 |
albumin, urine, high | 2 (1) | 3 (3) | 0 | 3 (3) |
ALT, serum, high | 3 (3) | 4 (2) | 0 | 2 (2) |
ALT, serum, high (grade 2) | 0 | 0 | 1 (1) | 0 |
ANA, high | 1 (1) | 1 (1) | 3 (3) | 4 (4) |
BUN, serum, high | 1 (1) | 4 (3) | 2 (2) | 1 (1) |
chloride, serum, high | 4 (4) | 1 (1) | 0 | 2 (2) |
CO2, serum, low | 4 (4) | 3 (2) | 2 (2) | 1 (1) |
creatinine, serum, high | 1 (1) | 3 (2) | 2 (2) | 2 (2) |
diarrhea/loose stools | 1 (1) | 5 (3) | 0 | 0 |
edema, lower extremities | 2 (2) | 0 | 3 (1) | 1 (1) |
eosinophils, high | 3 (3) | 1 (1) | 1 (1) | 1 (1) |
fatigue | 5 (5) | 6 (5) | 5 (4) | 0 |
glucose, serum, high, non-fasting | 5 (5) | 6 (6) | 2 (1) | 7 (6) |
ketones, urine, high | 2 (2) | 1 (1) | 1 (1) | 3 (3) |
potassium, serum, high | 3 (3) | 0 | 6 (5) | 1 (1) |
potassium, serum, high (grade 2) | 1 (1) | 0 | 0 | 0 |
rash | 2 (2) | 2 (1) | 0 | 3 (2) |
URI | 3 (3) | 3 (3) | 1 (1) | 0 |
0 | 1 (1) | 0 | 3 (1) | |
Hospitalization: Cardioversion for atrial fibrillation | 0 | 1 (1) | 0 | 0 |
Hospitalization: Urinary retention | 0 | 0 | 0 | 1 (1) |
Elective Hospitalization: Cholecystectomy | 0 | 0 | 0 | 1 (1) |
Elective Hospitalization: Inguinal hernia repair | 0 | 0 | 0 | 1 (1) |
Study visits during the placebo and vaccine phases are identical. Placebo injections consisted of vaccine vehicle only. All adverse events are Grade 1 unless otherwise specified. There were no vaccine related serious adverse events. The only statistically significant adverse event was the number of patients having injection site reactions between Arm 1 vaccine phase and Arm 2 pre-vaccine placebo phase (p = 0.001, Fisher's exact test). All other adverse events listed are not statistically significant (p≥0.2 in all cases, Fisher's exact test) between Arm 1 vaccine phase and Arm 2 pre-vaccine placebo phase.
All patients who received the DC/keyhole limpet hemocyanin (KLH) vaccine had a positive DTH response to KLH. None of the 24 patients had a response to LNCaP lysate at baseline (week 0). All 24 patients were given LNCaP lysate as part of DTH panels at weeks 3, 5, 7, and 9. Sixteen of 24 patients (67%) had a DTH response to LNCaP lysate in at least 1 of these 4 time points, with the highest proportion of patients (54.2%) responding to LNCaP lysate at 2 weeks after the last booster (week 9). Responses were maintained in 9 of 13 patients (69%) at 22 weeks after the last booster (week 29,
Vaccine induced DTH response to LNCaP cell lysates injected intradermally were measured at the indicated times. Week 1 was baseline, at which time no patients had DTH responses (data not shown). DTH responses were considered positive at ≥5 mm erythema read at 48 hours after placement. Bars indicate the number of patients with positive responses at each time point. Error bars represent 95% confidence intervals. Dotted line represents trend of percentage of patients with positive responses at each time point. Statistically significant positive DTH responses to LNCaP cell lysate appeared at Week 3 (first time point after baseline) and responses were still present in 9 of 13 patients (69%) at 22 weeks after the last booster dose (Week 29).
A 3H thymidine proliferation assay was used to assess the reactivity of T cells to KLH protein and to prostate tumor cells (either those used in vaccination (LNCaP) or to another prostate tumor cell line (PC3)). Negative control antigens included autologous monocytes and an irrelevant cell line (3T3). 3T3 infected with influenza was used as positive control. T cells were collected at week 0 (pre-vaccine) leukapheresis and week 13 (post-vaccine) leukapheresis (
Comparison of pre- and post-vaccine bulk T cell responses to prostate antigen. a. Apoptotic tumor cells (LNCaP and PC3, or an irrelevant cell line (3T3)) or KLH protein were co-cultured with patient peripheral blood monocytes and syngeneic bulk T cells obtained from patients pre- or post-vaccination. Monocytes without exogenous antigen (No Ag) or apoptotic 3T3 cells (Ctrl Ag), served as negative controls. Proliferation was assessed on day 5 after an 18-hour 3H thymidine pulse. Data is presented for 22 of 24 patients. The difference in proliferation (post- minus pre-vaccine) for each antigen group is shown in box plots. Values reported are average counts per minute (CPM) of triplicate wells. The median difference for each antigen group is shown by the line in the box. Each patient who is an outlier is indicated by a unique symbol. Statistically significant differences in pre-vaccine vs. post-vaccine T cell proliferative responses were found for KLH (p = 0.008), LNCaP (p = 0.017) and PC3 (p = 0.011). b. Bulk T cells obtained post-vaccination were stained with CFSE and cultured with DCs cross-presenting prostate antigens, LNCaP and PC3, or an irrelevant cell line (293 cells, Ctrl Ag). Cell proliferation on day 5, assessed by CFSE dye dilution, is shown on the x-axis and CD8 expression is shown on the y-axis. Percentages shown represent CD8+ cells that have divided within the bulk T cell population. Four of five additional patients tested showed similar CD8+ responses; data shown are for patient #15.
Group: | p-value | 95% CI |
No Ag | 0.160 | (−1703.275, 9649.413) |
KLH | 0.008* | (4681.672, 27308.539) |
LNCaP | 0.017* | (3759.725, 34618.214) |
PC3 | 0.011* | (5835.155, 38744.036) |
3T3 | 0.070 | (−862.319, 20294.713) |
Foxp3+ cells | 0.160 | (−0.978, 0.893) |
95% confidence intervals and p-values for T cell proliferation response (3H thymidine incorporation assay) and %Foxp3+ cells (FACS analysis) comparing cells collected pre-vaccine and post-vaccine using the paired t-test.
Based on the DTH data (
The proliferation response was also assessed by CFSE dye dilution assay
We considered the possibility that the increased T cell proliferation response post vaccination could relate to a decline in regulatory T cells in circulation. To determine the percent of CD4+ T cells that are T regulatory cells in pre- and post-vaccinated blood samples, PBMCs were stained and assessed for Foxp3 expression (
a. FACS profile of Foxp3 expression in pre- and post-vaccinated peripheral blood gated on CD4+ T cells. A representative patient (#13) is shown. b. Box plots of the percent Foxp3+ cells (gated on CD4+ T cells) pre and post-vaccination in 15 representative patients, including those across the whole range of proliferative responses and changes in PSA slope. The median is shown by the +. Outliers are indicated by •. No difference in pre-vaccine vs. post-vaccine T cell expression of Foxp3 (p = 0.924) was observed.
The prostate specific antigen doubling time (PSADT) was calculated for each patient during each phase of study. The median doubling time during the pre-vaccine phase was 4.5 months, and this increased to 5.4 and 8.9 months during the vaccine and post-vaccine phases respectively. There were statistically significant differences in PSADT between the pre- and post-vaccine phases (p = 0.003) and between the vaccine and post-vaccine phases (p<0.001) but not between the pre-vaccine and vaccine phases (p = 0.915).
We also compared the slope of the PSA rise between the 3 study phases. Eighteen of 23 (78%) evaluable patients had a decrease in PSA slope between the pre- and post-vaccine phases. When considering all 23 patients, there was a statistically significant decrease in the PSA slope between the pre-vaccine and post-vaccine phases of −0.093/month (p = 0.016,
Graph of the average log2 (PSA) slope per study phase (solid line); for comparison, an extrapolation of the pre-vaccine average log2 (PSA) slope is shown (dotted line). Based on the linear spline model, the average change in PSA slope of 23 patients from pre- to post-vaccine phases is −0.093/month (p = 0.016). One patient's PSA values were not included in the analysis as his pre-vaccine values were affected by other treatment near the start of study participation. Three other patients started other treatment either during or after vaccination; PSA values obtained after this point were not included in the analysis.
PSA Slopes by Study Phases: | p-value | 95% CI |
Pre- vs Post | 0.016 | (−0.1694, −0.0166) |
Pre- vs Vaccine | 0.681 | (−0.1044, 0.0682) |
Vaccine vs Post | 0.098 | (−0.1613, 0.0134) |
95% confidence intervals and p-values for PSA slopes comparing pre- vs vaccine, vaccine vs post-vaccine, and pre- vs post-vaccine phases from the linear mixed model.
We then stratified the study population with two more fixed effects on a mixed model and conducted a likelihood ratio test; the patients that had a DTH response to LNCaP lysate had a significantly different PSA slope change when compared to those that had no DTH response to LNCAP lysate (p = 0.004). Sixteen of 24 patients had DTH responses to LNCaP in at least one time point. This group of patients had a statistically significant change in PSA slope between the pre-vaccine and post-vaccine phases (−0.105/month, p = 0.020,
Stratified by: | p-value | 95% CI | |
Whole group | 0.016 | (−0.1694, −0.0166) | |
Response by DTH to LNCaP lysate | Non-Responders | 0.631 | (−0.1682, 0.1022) |
Responders | 0.020 | (−0.1932, −0.0168) | |
PSA at start of study | <1 ng/ml | 0.279 | (0.2191, 0.0631) |
≥1 ng/ml | 0.031 | (−0.1872, −0.0108) |
95% confidence intervals and p-values for PSA slopes comparing pre- vs vaccine, upon stratification by DTH response or no response to LNCaP lysate or upon stratification by PSA <1 ng/ml or 1 ng/ml at start of study.
Patients with PND develop effective tumor suppression of common cancer types
Despite the conceptual link between the development of apoptotic cells as a vaccine and tumor immunity in PND, we found no evidence that this approach triggered autoimmune disease in our patients. We did note small ANA elevations post vaccine in 5 patients (titer of 1∶160 in one patient, ≤1∶80 in four patients) which resolved over time in 4/5 patients. However, we also noted that of 7 patients with detectable ANA levels pre-vaccination, 5 became lower after vaccination. Statistical analysis of ANA changes pre versus post vaccine/placebo in Arm 1 versus Arm 2 revealed no significant differences (Fisher's exact test), and we conclude that ANA changes were not clinically meaningful; moreover, such transient responses have commonly been seen in DC based vaccines
Our dendritic cell/apoptotic tumor vaccine was immunogenic. Sixty-seven percent of patients developed DTH responses to LNCaP antigens. Furthermore, these DTH responses were positively correlated with post-vaccine bulk T cell proliferation responses. This high level of immunogenicity was similar to that reported in other studies of peptide-pulsed or tumor cell associated DC vaccines. Importantly, these responses included CD8+ T cell responses to prostate tumor cells (
Significantly, we found that PSA slopes decreased and PSADT increased after vaccination in our patient population as a whole (p = 0.016). We hypothesized that if this correlation was related to the immunogenicity of the vaccine, PSA changes should be present in the subset of patients showing immunogenic response to vaccine but not in those who do not. In fact, patients who had DTH responses to LNCaP after vaccination had significant decreases in PSA slope (p = 0.020), compared to patients who did not have DTH responses (p = 0.631). Taken together, our data suggests that the changes seen in PSA slope represent an immune response to patient tumor cells
Although variable immunologic and clinical responses have been reported to vaccines using dead tumor cells as a source of antigen, these studies have not focused on using pure, well-defined populations of apoptotic tumor cells. We used UV-B irradiation to induce apoptotic (not necrotic) death in >90% of the prostate cell line used for the vaccine; nonetheless, our side-effect profile was very low, similar to other tumor vaccines. Most other studies have used gamma irradiation or freeze-thawing, generating variable mixtures of apoptotic and necrotic cells, which may underlie differences in immunogenic potential
Taken together, the results presented in this study provide initial safety and immunogenicity data for a vaccine mimicking what we believe is a critical trigger for naturally occurring effective tumor immune responses seen in PND patients. These responses correlate with a clinically relevant response to patient tumor, as assessed by highly statistically significant effects on PSA slope and doubling time. These observations suggest that this vaccination approach warrants further exploration as a safe and potent means of triggering tumor immune response in the general population of cancer patients. Future vaccine modifications to be considered are the addition of immune adjuvants during vaccine preparation ex vivo or in conjunction with vaccine administration in vivo
The protocol for this trial and supporting CONSORT checklist are available as supporting information; see
The study was approved by The Rockefeller University Institutional Review Board (RDA-0466) and the FDA (IND 10710). Written consent was obtained from all patients. No
The study was conducted at the Rockefeller University in New York; twenty-four patients aged 53 to 81 were enrolled between November 2003 and February 2006. All authors vouch for the completeness and accuracy of the data and its analysis and participated in writing the article.
Twenty-four patients were randomly assigned to one of two arms for the purpose of assessing vaccine safety, our primary endpoint (
Patients were eligible to participate if they provided informed consent, had biopsy proven prostate cancer and progressive disease: PSA documented to be rising on 3 occasions, either despite castrate testosterone levels (below 50 ng/dl) or despite definitive local therapy (prostatectomy, radiation, etc.). Exclusion criteria included prior biologic therapy with dendritic cells, autoimmune disease, or significant major organ disease.
DC vaccines were given together with DTH panels and patients were closely observed for one hour. Patients returned to clinic at 48 hours, at which time they were examined clinically and their DTH responses were read. Vaccines were administered at a cell dose ranging from 2−10×106 DCs/vaccination, given subcutaneously in the inner aspect of the upper arm, approximately 6–8 cm from the axillary lymph nodes. Patients received one priming and three biweekly booster vaccinations. During the first two injections, patients also received 2−10×106 DC/KLH.
Vaccine was manufactured in a BSL-2 facility maintained and independently audited to meet Good Tissue Practice specifications. To prepare autologous DCs, peripheral blood mononuclear cells (PBMCs) were obtained by leukapheresis, adhered to endotoxin free tissue-culture dishes (Falcon), and differentiated
Patients were monitored at each study visit by history, physical examination, and by laboratory evaluations including CBC, chemistries, and urinalysis, for any adverse effects. The National Cancer Institute Common Toxicity Criteria version 3 was used to grade toxicities. Patients were also asked to maintain a diary of local injection site reactions and systemic adverse events for one week following each vaccination.
To assess for clinical response, PSA levels were measured in the serum using MSA Bayer Immuno I or IEA Tosoh Nexia assays at each study visit. The PSA slope was calculated for each phase of the study (pre-vaccine, vaccine, and post-vaccine phases) using a linear mixed model with two knots representing the change in slope in each phase of the study. In addition, for each phase of study for each patient, a PSA doubling time (PSADT) was calculated using the formula: 1 divided by the slope log2 PSA derived from this linear spline model.
A DTH panel was placed intradermally at weeks 1 (vaccine), 3 (Boost #1), 5 (Boost #2), 7 (Boost #3), 9, 17 and 29 to assess for T cell responses. The panel included lysate of 105 LNCaP cells (in 0.1 ml normal saline), 0.05 mg KLH, candida or tetanus toxoid (whichever the patient had responses to at baseline, as positive control), and saline (as negative control). Responses were considered positive if erythema was equal to or greater than 5 mm at 48 hours post implantation.
We evaluated immunogenicity outcomes against apoptotic LNCaP and PC3 (another prostate cancer tumor cell line) by 3H thymidine or CFSE proliferation assays as described
To assess for correlations between patients who had a DTH response to LNCaP lysate and those who had T cell proliferation responses to apoptotic LNCaP
FACS staining of regulatory T cells was done by surface staining with anti-CD4-FITC (Becton Dickinson) followed by intracellular staining with anti-Foxp3-APC (eBioscience, clone PCH101) per manufacturers instructions. For analysis, PBMCs were gated on CD4+ T cells and the percentage of Foxp3+ cells was determined (FlowJo).
The protocol called for 12 patients to be recruited to each of two arms, which would have 90% power to detect an increase in cumulative serious adverse events (SAEs) from 5% in the placebo group to 50% in the vaccine group at the 10% level. All adverse events were analyzed using Fisher's exact test to compare placebo and vaccine phase. Significance of DTH responses to LNCaP cell lysate was determined by the exact binomial test with 95% confidence intervals. To analyze pre- to post- T cell proliferation data (per antigen) for study subjects as a group, a two-sampled paired t-test was used with 95% confidence intervals. The Spearman rank correlation test was used to determine if those that had DTH responses to LNCaP lysate also had T cell proliferation responses to apoptotic LNCaP
To model the evolution of PSA (in log-scale) during the three study phases (pre-vaccine, vaccine, and post-vaccine phases), a mixed linear spline model was used. Two knots (one at the start of the vaccine phase and the other at the start of the post-vaccine phase) were used to directly quantify the differences in slopes between each phase. To account for the heterogeneous treatment effect and the repeated measures structure, random effects are incorporated into the model. For the general model, random effects for the intercept, slope and the first knot were considered. To determine if there was any difference in PSA slopes between those that did have an immunologic response (DTH responders to LNCaP lysate) and those that did not (DTH non-responders to LNCaP lysate), a second model, stratified by these response indicators (with separate knots for the two groups), was fitted and the likelihood ratio test was conducted. Similarly, a third model was fitted, this time, stratified by PSA at study start of <1 ng/ml or ≥1 ng/ml. The models with stratification were then compared to the general model. All models were fitted and hypotheses were tested using the
Consort checklist.
(0.13 MB DOC)
FDA-approved IND 10710 protocol.
(0.58 MB DOC)
We thank Michel Sadelain for helpful discussions, members of the Darnell laboratory for critical discussion and review of the manuscript, members of the Rockefeller University Hospital Nursing Staff for their compassionate care of the study participants, members of the MSKCC clinical and research nurses, Gabrielle Aruz, Anthony Delacruz, and Tracy Smart-Curley, for their assistance with study patients, Sarah Schlesinger and Mary Marovich for help drafting SOPs, Stephen Paget for advice regarding relevance of ANAs, and Denise Larone for help with release criteria. The authors are grateful for extraordinary support from David Koch. RBD is an Investigator of the Howard Hughes Medical Institute.