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. Author manuscript; available in PMC: 2017 Jan 7.
Published in final edited form as: Cell Stem Cell. 2015 Dec 5;18(1):144–155. doi: 10.1016/j.stem.2015.10.004

Phase I/II Trial of StemRegenin-1 Expanded Umbilical Cord Blood Hematopoietic Stem Cells Supports Testing as a Stand Alone Graft

John E Wagner Jr 1,2,*, Claudio G Brunstein 1, Anthony E Boitano 3, Todd E DeFor 1, David McKenna 2, Darin Sumstad 2, Bruce R Blazar 1, Jakub Tolar 1, Chap Le 1, Julie Jones 4, Michael P Cooke 3, Conrad C Bleul 4
PMCID: PMC4881386  NIHMSID: NIHMS782552  PMID: 26669897

SUMMARY

Clinical application of umbilical cord blood (UCB) as a source of hematopoietic stem cells for transplantation is limited by low CD34+ cell dose, increased risk of graft failure and slow hematopoietic recovery. While the cell dose limitation is partially mitigated by using two UCB units, larger dosed single units would be preferable. We have evaluated the feasibility and safety of StemRegenin-1 (SR-1), an aryl hydrocarbon receptor antagonist that expands CD34+ cells, by placing one of the two units in expansion culture. SR-1 produced a 330-fold increase in CD34+ cells and led to engraftment in 17/17 patients at a median of 15 days for neutrophils and 49 days for platelets, significantly faster than in patients treated with unmanipulated UCB. Taken together, the marked expansion, absence of graft failure and enhanced hematopoietic recovery support testing of SR-1 expansion as a stand-alone graft and suggest it may ameliorate a limitation of UCB transplantation.

Graphical Abstract

graphic file with name nihms782552u1.jpg

INTRODUCTION

Unrelated donor umbilical cord blood (UCB) is a major source of hematopoietic stem cells (HSC) for transplantation with more than an estimated 30,000 recipients to date (Ballen et al., (2013)). While UCB has the advantages of rapid availability, absent donor attrition and relatively less HLA restriction as compared to HSC from adult volunteer donors (Smith and Wagner, (2009), Cheuk, (2013)) the use of UCB is substantially limited by the low finite number of HSC and progenitor cells that can be collected from a placenta, resulting in prolonged periods of neutropenia, thrombocytopenia and suboptimal engraftment and impeding its widespread application (Ballen et al., (2013), Smith and Wagner, (2009), Cheuk, (2013), Rubinstein et al., (1998), Scaradavou and National Cord Blood Program, (2010)). Furthermore, studies have demonstrated an association between infused CD34+ and colony forming unit-granulocyte macrophage (CFU-GM) cell doses and pace of hematopoietic recovery, non-relapse mortality and survival (Wagner et al., (2002), Migliaccio et al., (2000), Page et al., (2011)). As a result, there is considerable interest in finding ways to increase the absolute number of hematopoietic cells in an UCB graft, such as with of ex vivo expansion culture prior to transplantation.

StemRegenin-1 (SR-1) was first identified in an unbiased screen for compounds that promoted expansion of CD34+ hematopoietic progenitors (Boitano et al., (2010)). SR-1 expanded HSC retained multi-lineage potential and significantly augmented early and late engraftment of human cells in immune deficient murine recipients. SR-1’s effect on CD34+ cell expansion is mediated through direct binding and inhibition of the aryl hydrocarbon receptor which normally promotes HSC differentiation during cytokine driven expansion culture. Preclinical data demonstrate that SR-1 in the presence of stem cell factor (SCF), Fms-related tyrosine kinase 3 ligand (FLT-3L), thrombopoietin (TPO) and interleukin-6 (IL-6) leads to greater numbers of CD34+ cells when compared to previously reported expansion methods being evaluated in clinical trials (de Lima et al., (2008), Delaney et al., (2010), de Lima et al., (2012), Horwitz et al., (2014)). To explore the clinical utility of SR-1 mediated expansion, a GMP-compliant expansion protocol was developed using SR-1 to expand UCB CD34+ cells and manufacture the product, referred to as HSC835, in order to speed hematopoietic recovery after transplantation.

The ‘double’ UCB transplant platform, i.e., the infusion of two partially HLA matched UCB units, was pioneered at the University of Minnesota (Barker et al., (2001), Barker et al., (2005), Brunstein et al., (2007)) as a clinical strategy for evaluating the safety and effectiveness of graft manipulations, including the testing of expanded CD34+ cells. With this approach, one unit could be left unmanipulated while the other is placed in expansion culture, offering two significant advantages: 1) enhanced safety by incorporating an unmanipulated unit as a ‘back-up’ should the expansion culture fail or interfere with engraftment, and 2) a means of tracking the relative contributions of the expanded and unmanipulated UCB units to hematopoietic recovery over time based on the inherent genetic differences between donors. Therefore, as in other recent trials evaluating hematopoietic cell expansion (de Lima et al., (2008), Delaney et al., (2010), de Lima et al., (2012), Horwitz et al., (2014)), we used the double UCB platform to explore the safety and efficacy of HSC835.

RESULTS

Patient and donor graft characteristics

Twenty patients were enrolled and 17 completed the prescribed treatment plan receiving HSC835 after a myeloablative conditioning in the double UCBT setting (Figure 1A). Demographics and graft characteristics for the recipients of HSC835 are shown (Table 1) along with the characteristics of a comparison historical control cohort selected on the basis of disease type and similarities in treatment plan (i.e., conditioning and GVHD prophylaxis) in order to assess the safety profile of HSC835 in terms of hematopoietic recovery and engraftment. All patients had leukemia or advanced myelodysplastic syndrome and similar performance status. Donor-recipient HLA match and ABO compatibility were similar as well. By study design, the most desirable of the two units based on cell dose and HLA match was left unmanipulated (unit 1) while the second best unit with a lower cell dose (unit 2) was selected for ex vivo expansion culture. As expected, HSC835 had significantly greater numbers of CD34 cells compared to unmanipulated units (Table 2) with a median of 17.5 × 106 CD34 (range, 1.4–48.3) per kilogram actual body weight for HSC835 units in contrast to the median number of 0.2 × 106 CD34+ cells/kg present in unmanipulated UCB units. In contrast, the CD3 cell dose was significantly less in recipients of HSC835 both because it was derived from the smaller unit 2 and resultant nonspecific losses with re-cryopreservation of the CD34 negative fraction collected 15 days prior to transplantation.

Figure 1. Trial design and engraftment outcome.

Figure 1

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(A) Schematic of the treatment plan. On day −15 prior to transplant, CD34+ cells were enriched from the lower cell dosed UCB unit (UCB 2) and placed in expansion culture for 15 days, including 50 ug/mL each of SCF, FLT-3L, TPO and IL-6 as well as SR-1. CD34 negative cells were collected and re-cryopreserved. Between days −8 and day −1, the patient was conditioned with cyclophosphamide (CY) 60 mg/kg/day (days −8 and −7), fludarabine (FLU) 25 mg/m2/day (days −8, −7 and −6) and total body irradiation (TBI) 165 cGy twice daily (days −4, −3, −2, and −1. On the day of transplant (day 0), the unmanipulated higher cell dosed unit (unit 1) was infused first with HSC835 infused four hours later. CD34 negative cells were infused 4 to 24 hours after HSC835. Dose levels are shown; however, only dose levels 1 and 2 were evaluated in this trial.

(B) Incidence of neutrophil recovery for patients transplanted with HSC835 (n=17) compared to that in the historical cohort (n=111). Neutrophil recovery was defined as the absolute neutrophil count ≥0.5 × 109/L for three consecutive days.

(C) Incidence of platelet recovery for patients transplanted with HSC835 (n=17) compared to that in the historical cohort (n=111). Platelet was recovery defined as the platelet count ≥0.5 × 1010/L for seven consecutive days without transfusion.

(D) Correlation between HSC835 CD34+ cell dose and time to neutrophil recovery. For those with HSC835 predominance in the CD15/CD33 myeloid cell population, neutrophil recovery is rapid with a strong correlation between the HSC835 CD34+ cell dose and time to recovery (left panel). For those with predominance of the unmanipulated unit, recovery is slower with no correlation between the HSC835 CD34+ cell dose and time to recovery (right panel).

(E) Patterns of chimerism in the CD3+ T cell and CD15/CD33+ myeloid cell populations are shown for each patient identified by their unique patient number (UPN). In the uppermost panel, chimerism in myeloid (CD15/CD33) and T cell (CD3) lineages is principally derived from the HSC835 unit. In the middle panel, chimerism in the myeloid lineage is mixed with contributions from both HSC835 and unmanipulated unit and lymphoid lineage almost exclusively derived from the unmanipulated unit. In the lower panel, chimerism in both lineages is nearly completely derived from the unmanipulated units with brief HSC835 predominance at day 7.

Table 1.

Characteristics of Patients

Demographics Expansion Cohort
N=17		 Historical Conventional Cohort
N=111		 P value
Median Recipient Age (Range) 29.9 years (12–54) 25.6 years (11–51) 0.07
Median Recipient Weight (Range) 87.1 kg (42–130) 69.6 kg (32–149) 0.09
Diagnosis 0.01
 Acute Myelocytic Leukemia
  1st CR 4 (23.7.6%) 33 (29.7%)
  2nd CR - 24 (21.6%)
  3rd CR - 4 (3.6%)
 Acute Lymphocytic Leukemia
  1st CR 10 (58.8%) 30 (27.0%)
  2nd CR - 14 (12.6%)
  3rd CR 1 (5.9%) 2 (1.8%)
 Myelodysplastic Syndrome 2 (11.8%) 4 (3.6%)
Cytomegalovirus Serostatus 0.88
 Recipient Positive 9 (52.9%) 61 (55.0%)
 Recipient Negative 8 (47.1%) 50 (45.0%)
HLA Match with Recipient (maximum disparity) Unit 1* Unit 2* Unit 1* Unit 2* 0.27
 6/6 3 (17.7%) 1 (5.9%) 9 (8.1%) 12 (10.8%)
 5/6 9 (52.9%) 9 (52.9%) 34 (30.6%) 52 (46.9%)
 4/6 5 (29.4%) 7 (41.2%) 68 (61.3%) 47 (42.3%)
ABO Match with Recipient Unit 1* Unit 2* Unit 1* Unit 2* 0.07
 Match 5 (29.4%) 7 (41.2%) 36 (32.4%) 23 (20.7%)
 Major Mismatch 12 (70.6%) 10 (58.8%) 53 (47.8%) 53 (47.8%)
 Minor Mismatch 0 0 22 (19.8%) 35 (31.5%)
Performance Score 0.59
 100 8 (47.1%) 54 (48.6%)
 90 8 (47.1%) 55 (49.5%)
 80 1 (5.9%) 2 (1.8%)
Conditioning and GVHD Prophylaxis NS
 Cy 120 mg/kg, Flu 75 mg/m2, TBI 1320 cGy 100% 100%
 Cyclosporin A and Mycophenolate Mofetil 100% 100%
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Abbreviations: CR = complete remission.

*

Unit 1 is the larger of the two units.

Cy = cyclophosphamide; Flu = fludarabine; TBI = total body irradiation (total doses shown)

Table 2.

Graft characteristics

HSC835 Cohort
N=17		 Historical Conventional Cohort
N=111		 P value
Cells Infused Median (Range) Unit 1 Unit 2 Combined+ Unit 1# Unit 2 Combined+
Pre Expansion Post Expansion
Nucleated cells x108/kg 0.2 (0.2–0.5) 0.2 (0.1–0.6) 0.5 (0.1–1.2) 0.7 (0.3–1.7) 0.2 (0.1–0.5) 0.2 (0.1–0.5) 0.4 (0.2–1.0) <0.001
CD34 cells × 106/kg 0.4 (0.2–0.9) 0.2 (0.0–1.0) 17.5 (1.4–48.3) 18.2 (2.3–48.5) 0.2 (0.0–1.1) 0.3 (0.1–1.0) 0.5 (0.1–2.1) <0.001
CD3 cells × 106/kg* 8.4 (4.6–28.8) ND 2.9Ω (0.4–5) 12.3 (5–33) 7.0 (2–26) 9.0 (3–21) 16 (5–47) 0.28
CFU-GM colonies × 104/kg 3.3 (1.2–7.4) ND 389.1 (40.7–1335.8) 394.5 (47.8–1338.4) 1.9 (0.1–8.9) 2.0 (0.0–14.0) 3.9 (0.1–22.9) <0.001
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#

Unit 1 is the larger of the two units.

*

CD3+ cells were derived solely from the cryopreserved CD34 depleted fraction.

Ω

p value <0.001 comparing HSC835 CD3 cell dose to that in the unmanipulated unit.

+

Combined cell dose were compared by general Wilcoxon test.

Abbreviations: CFU-GM = colony forming unit-granulocyte macrophage; ND = not determined

SR-1 expansion culture reliably provides greater than 200-fold expansion of CD34+ cells

Cell numbers at each step of the HSC835 manufacturing process (CD34 selection and expansion culture) are shown for each patient (Table 3). After CD34+ cell selection, a median of 4.4 × 106 total CD34+ cells (range, 2.1–14.3) were placed in expansion culture. After 15 days, a median of 1440 × 106 total CD34+ cells (range, 140–6361) were recovered. Based on these results, SR-1 expansion cultures yielded a median of 330-fold (range, 67–848) increase in the number of CD34+ cells, and 854-fold (range, 168–2121) increase in numbers of total nucleated cells (TNC). In three patients (UPN04, UPN17, UPN20), the final HSC835 product exceeded the maximum allowable cell dose permitted resulting in the infusion of only a proportion of the cells available (see Methods – Dose Capping). Importantly, the fold expansion of CD34+ cells was independent of starting CD34 number. In all but one case (UPN 03), CD34+ cell expansion exceeded >200-fold. For UPN03, expansion kinetics differed from all other cultures in that there was no evidence of expansion in nucleated cells after the first 3 days of culture.

Table 3.

Selection and Expansion Profiles

UPN Patient Weight kg Cryopreserved After Thawing After CD34 Selection HSC835 (After Expansion) CD34 Depleted (Infused)
Total NC (x 108) Total CD34+ (x 106) Total NC (x 108) Total CD34+ (x 106) % CD34 Recov* Total NC (x 106) Total CD34+ (x 106) Total NC (x 108) Total CD34+ (x 106) Fold CD34 Expansion Fold NC Expansion Total NC (x 108) Total CD34+ (x 106) Total CD3+ (x 106)
01 86.2 14.2 4.5 11.6 6.6 146.9 2.8 2.6 23.9 965.8 371 854 4.8 1.5 227.7
02 119.9 31.2 9.6 24.5 14.7 153.3 11.5 10.5 49.6 2496.9 238 431 14.1 6.2 592.7
03 64.5 13.4 5.2 12.0 7.4 142.4 2.2 2.1 3.7 140.2 67 168 4.0 0.2 185.7
04Ψ 110.9 20.6 14.3 17.6 31.7 221.9 15.6 14.3 118.4 6361.9 445 759 7.9 3.1 308.9
05 102.5 27.5 12.4 21.6 15.1 122.0 9.2 8.2 90.4 3066.5 374 982 13.1 5.8 393.0
06 89 14.2 8.7 9.6 8.5 97.9 5.2 4.9 41.7 1191.5 243 802 2.5 0.2 39.4
07 131.2 22.9 11.5 22.1 7.1 61.7 5.2 4.3 30.4 1123.0 261 585 9.5 0.8 85.1
08 90.7 16.6 3.3 13.5 9.5 284.0 2.8 2.3 22.3 461.8 201 796 5.3 2.4 237.6
09 63.1 11.6 7.5 10.4 13.0 171.9 7.0 6.0 58.4 1483.0 247 834 5.0 0.8 246.1
10 68.9 21.1 12.7 17.7 17.2 135.8 5.8 2.9 97.9 2460.3 848 1688 8.8 1.3 210.8
11 84.3 14.4 5.8 11.9 6.4 112.0 2.9 2.6 38.7 746.1 287 1334 7.2 2.7 229.2
12 63.1 18.1 9.0 15.7 13.2 146.9 4.6 4.2 97.6 2557.8 609 2121 5.3 0.5 257.3
13 52.4 10.1 7.7 8.4 10.6 137.1 4.7 4.5 95.3 2175.9 484 2028 4.7 0.7 102.4
14 113.5 19.0 5.4 18.1 12.5 231.8 4.5 3.7 50.0 1440.3 389 1111 5.9 0.5 199.0
15 48.0 22.5 9.2 19.1 17.4 188.9 6.4 6.0 NA NA NA NA NA NA NA
16 105.9 20.8 6.4 17.5 13.6 211.4 4.1 3.4 32.4 1183.7 348 790 8.7 2.5 339.8
17Ψ 40.5 29.7 21.4 22.4 41.2 192.7 12.2 9.7 115.0 3196.6 330 943 6.8 1.9 190.0
18 44.9 17.7 7.8 14.3 13.8 177.3 4.0 3.6 43.2 1212.7 337 1080 NA NA NA
19 113.2 23.3 5.8 20.0 17.6 304.3 6.1 5.9 57.5 1241.1 210 943 NA NA NA
20Ψ 49 19.1 19.9 16.6 34.1 171.6 8.0 7.8 43.3 1677.5 215 541 7.8 3.4 195.3
Median 86.2 19.1 8.3 17.1 13.4 162.5 5.2 4.4 49.6 1440.3 330 854 6.8 1.5 227.7
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Abbreviations: HSC835 = expanded unit; CD34 depleted = residual cells after CD34 selection; these were recryopreserved and infused 4–24 hours after HSC835 infusion; UPN = unique patient number; NC = nucleated cell count; NA = not available

*

%CD34 Recovery: Methodology in University of Minnesota Medical Center Clinical Laboratory differs from Cord Blood Banks resulting in uniformly higher %CD34 as previously reported (Flores et al., 2009).

Ψ

Cell expansion exceeded dose allowed; patients received maximum allowed at dose level 1

Note: Numbers of cells infused do not match the total numbers manufactured as samples were sent for lot release/quality control and research assays

Eighteen HSC835 products met lot release (see Methods – Unit Processing, Table S1). Two products (for UPN15 and UPN19) failed lot release due to a positive bacterial culture. In addition, one product (UPN13) that met lot release had a positive surveillance culture reported 18 hours after infusion. While this patient’s blood cultures remained negative after transplant, transient hypotension occurred 4 hours after infusion which was the only observed infusional toxicity event. For the three positive cultures the isolates differed (Proprionibacterium acnes [UPN13], coagulase negative Staphylococcus [UPN15] and Leifsonia aquatica [UPN19]). Process improvements in product manufacture, specifically batch production of expansion media and a reduction in in-process testing, resulted in 13 of 13 validation study and clinical products met lot release.

HSC835 promotes rapid hematopoietic recovery

For the 17 recipients of HSC835, neutrophil recovery was achieved in all of patients at a median of 15 days (6–30) while 86% patients in the control group recovered at a median of 24 days (Figure 1B). Like neutrophil recovery, platelets also recovered more rapidly at a median of 49 days (28–136) versus 89 days (p=0.001), respectively. Six months after transplantation, 76% (CI, 50–100) of HSC835 recipients achieved platelet transfusion independence as compared to 61% (CI, 50–72) of historical controls (Figure 1C).

After double UCBT, we and others have previously shown that one unit typically predominates and the second unit is lost (Barker et al., (2001), Barker et al., (2005), Brunstein et al., (2007)). In this study, the origin of hematopoietic recovery (unit 1 versus unit 2 versus host) was determined at fixed time points by discriminating short tandem repeat loci in sorted CD15+/CD33+ myeloid cells, the most prevalent population in the peripheral blood after transplant (Figure 1D). Hematopoiesis was principally derived from the expanded unit (HSC835) in 11 patients and the unmanipulated unit in six. In those engrafting with HSC835, time to neutrophil recovery was more rapid at a median 11 days (range 6–23) and correlated with CD34+ cell dose (Figure 1D, left panel). In contrast, no correlation was observed in the 6 patients engrafting with the unmanipulated unit and speed of recovery was significantly slower (median 23 days, range 14–30). For those engrafting with HSC835, myeloid engraftment was durable (median follow-up 272 days, range 35–688) (Figure 1E).

A detailed analysis of chimerism, including an evaluation of sorted CD3 cells in the peripheral blood, revealed three patterns: 1) in six patients, the CD15/33 (myeloid) and CD3 (T cell) populations are entirely derived from HSC835 (Figure E upper panel); 2) in six patients, the CD15/33 and CD3 populations are entirely derived from the unmanipulated unit (lower panel); and 3) in 5 patients (middle panel), a unique chimerism pattern was observed with the CD15/33 population predominantly derived from HSC835 and the CD3 population almost exclusively derived from the unmanipulated unit. These 5 patients (UPN04, UPN12, UPN14, UPN17, UPN20) with myeloid chimerism from both HSC835 and the unmanipulated unit had particularly rapid neutrophil recovery (median 7 days, range 6–10).

Unit predominance is associated with graft-versus-graft immune reactivity

In all patients, HSC835 myeloid chimerism was near complete on day 7. However, it was not sustained in six. As shown in Figure 2A, a more detailed analysis of the HSC835 product demonstrated expansion of the CD34+CD133+CD90+ in all products, the population enriched for the HSC (Radtke et al., (2015), Wong et al., (2013)) in addition to even greater expansion of committed progenitors (CD34+CD133+CD90− and CD34+CD133−CD90−), suggesting that the expansion culture did not have a deleterious effect on the engraftment potential of HSC835 even in the six ultimately engrafting with the unmanipulated unit. Based on the report by Gutman et al (Gutman et al., (2010)) documenting a specific CD8+ T cell response of the predominating unit against the non-engrafting unit, we examined the peripheral blood of each patient early after transplant for evidence of alloreactive T cells directed against the non-engrafting unit. Of 7 patients in whom sufficient numbers of CD8+ T cells could be recovered, 5 patients had interferon-gamma (IFNγ) producing T cells in the peripheral blood derived from the predominating unit in response to the non-engrafting unit (Figure 2B). Specifically, UPN02 and UPN05 showed reactivity against the unmanipulated unit and engrafted with HSC835; and UPN03, UPN09 and UPN10 showed reactivity against HSC835 and engrafted with the unmanipulated unit. In the two remaining patients, one had no reactivity to either the HSC835 or unmanipulated unit and engrafted with both HSC835 and the unmanipulated unit (UPN04) and one had borderline reactivity (UPN08) to HSC835 and engrafted with the unmanipulated unit.

Figure 2. Analysis of risk factors potentially influencing HSC835 reconstitution.

Figure 2

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(A) Fold expansion of CD34+ subpopulations based on co-expression of CD133+and CD90+ in recipients of HSC835.

(B) Graft-versus-graft immune reactivity. Proportion of interferon-γ secreting T cells obtained from the peripheral blood of 7 patients between day 14 and 56 after UCB transplantation in response to EBV-transformed HSC835, unmanipulated unit and third party donor cells. The box shows the UPN for each patient with a sufficient number of peripheral blood mononuclear cells for evaluation which wereobtained on day 14 (triangles), 28 (circles) or 56 (squares) after transplant with colors indicating individual patients.

(C) Probability of engraftment in recipients of double UCB transplant with and without SR-1 expanded hematopoietic cells. Engraftment of the lower cell dosed unit 2 is shown with and without SR-1 expansion (black bar). Overall engraftment by day 42 after double UCB transplant (regardless of which unit predominates is also shown (gray bar).

(D) Panels show the telomere length of mobilized peripheral blood (mPB) from normal donors and UCB CD34+ cells which were evaluated prior to and following culture in SCF,FLT-3L, TPO, IL6 (SFT6) with and without SR-1. Each plot is an individual experiment from two independent donors. Tables beneath each plot indicate the telomere length as measured in kilo-base pairs.

Having previously shown an association between the unit’s CD3+ cell dose and not nucleated or CD34+ cell dose and unit predominance (Brunstein et al., (2007), Radtke et al., (2015), Wong et al., (2013), Gutman et al., (2010), Ramirez et al., (2012)) corroborated by preclinical studies demonstrating the importance of T cells on unit predominance in NOD-scid recipients engraftment (Lund et al., (2015), Yahata et al., (2004)), we hypothesized that the frequency of unit 2 predominance would be similar regardless of expansion culture. Therefore we compared the frequency of unit 2 predominance after expansion culture (i.e., HSC835 and its associated CD34 negative fraction) with unit 2 without expansion culture in the historical cohort. As shown (Figure 2C), unit 2 was more likely to expand after expansion culture (p=0.05), particularly remarkable considering the additional losses of CD3+ T cells with re-cryopreservation of the CD34 negative fraction. While no patient in the study cohort experienced graft failure, there were insufficient numbers of patients to document a reduction in graft failure after the transplantation of two unmanipulated units (p=0.12). Together, these data suggest that an immune response between units accounted for unit predominance and expansion culture did not interfere with the unit’s repopulation potential.

Effect of HSC835 on other transplant outcomes

The primary endpoint of the clinical trial was to determine the safety of HSC835. Other than transient hypotension with an occult bacterial contamination of the HSC835 product in one patient (UPN13), no infusional toxicities were noted within the first 24 hours after transplant and no other adverse effects were observed that could be attributed to the HSC835 infusion. As shown in Figure S1, the adverse event profiles between day 0 and day 30 were comparable to those commonly observed after UCB transplantation. In addition, no patient had late graft failure within the follow-up period. Of patients engrafting with HSC835, the longest follow-up is 687+ days (UPN05). On this point, we also examined telomere length before and after UCB expansion culture as a potential risk factor for early HSC senescence. As shown in Figure 2D, expansion culture with SR-1 resulted in an approximately 500 base-pair reduction in mean telomere length. However, it is notable that the telomere length of expanded UCB CD34+ cells compared favorably to Neupogen-mobilized adult peripheral blood CD34+ cells without expansion culture which is the most commonly used HSC source for transplantation in adults. In terms of other transplant outcomes, risk of grade II–IV and grade III–IV acute GVHD, transplant-related mortality and overall survival was similar compared to the historical cohort (Figure 3A, B and C, respectively). Of note, the length of hospitalization was significantly reduced at a median of 30 days compared to 46 days in the control population (p<0.001).

Figure 3. Effects of HSC835 on clinical transplant outcomes and immune recovery.

Figure 3

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(A) Incidence of grade III–IV acute GVHD in recipients of HSC835 (n=17) relative to the historical control cohort (n=111).

(B) Incidence of transplant related mortality (TRM) defined as mortality due to any cause other than relapse.

(C) Probability of survival defined as death from any cause, including relapse.

(D) Patterns of immune recovery. Mean cell numbers (+/− standard error) per microliter of peripheral blood are shown for CD3, CD4, CD4/CD45RA/CCR7 and CD19 lymphocyte subsets on days 28, 60, 98, 180 and 360 after UCB transplantation. Patients are grouped on the basis of which units contributed to hematopoietic recovery; specifically, HSC835 only (n=5), unmanipulated (UNM) unit only (n=4) or both HSC835 and unmanipulated units (mixed, n=4).

Lastly, exploratory assessments of T and B cell recovery were performed. As shown (Figure 3D), the absolute number of CD3+, CD4+ and CD4+CD45RA+CCR7+ cells is lower at day 28 in those patients where the T cells emanate from the CD34 negative fraction associated with HSC835, as compared to other patients where the T cells are derived from the unmanipulated product. As early T cell recovery reflects homeostatic expansion of the mature T cells infused (Politikos and Boussiotis, (2014), Krenger et al., (2011)), the initial lag may reflect the lower number of T cells in the recryopreserved CD34 negative fraction associated with HSC835. As opportunistic infection and relapse have been associated with poor immune recovery, CMV interstitial pneumonitis was the primary cause of death in one patient and relapse was not observed within the follow-up period. Other primary causes of death were acute GVHD (n=4), chronic GVHD (n=1), idiopathic (n=1) and diffuse alveolar hemorrhage (n=2).

DISCUSSION

Low stem cell dose resulting in prolongation or failure of lympho-hematopoietic recovery after UCB transplant has markedly limited the success HSC source. Today, however, there are multiple promising strategies for enhancing the pace of hematopoietic recovery after UCB transplantation. Notch-ligand, adherent mesenchymal stromal cells and nicotinamide-based expansion cultures are each associated with rapid neutrophil recovery at a median of 16, 15 and 13 days, respectively (Delaney et al., (2010), de Lima et al., (2012), Horwitz et al., (2014)). However, neutrophil recovery from the expanded product is transient after Notch-ligand and mesenchymal stromal cell culture with long-term engraftment emanating from the unmanipulated unit in nearly all instances (Delaney et al., (2010), de Lima et al., (2012)). In contrast, nicotinamide-cultured cells, like SR-1 in this study, not only promote rapid recovery but also lead to sustained engraftment in a substantial proportion of patients (Horwitz et al., (2014)). While mechanism of action for nicotinamide remains to be elucidated, emerging data suggest that it may also enhance homing to the marrow microenvironment. Other methods to improve homing, as with the inhibition of dipeptidylpeptidase 4/CD26, treatment of cells with 16,16 dimethyl prostaglandin E2 (dmPGE2), and fucosylation, are also promising with neutrophil recovery reported at a median of 17.5, 21 and 14 days, respectively, in initial pilot trials (Farag et al., (2013), Cutler et al., (2013), Popat et al., (2015)).

In this study, several important findings emerge: SR-1 in the presence of specific cytokines 1) promotes remarkable CD34+ cell expansion, greater than that observed by all other methods previously tested in clinical trials (de Lima et al., (2008), Delaney et al., (2010), de Lima et al., (2012), Horwitz et al., (2014)), albeit in the progenitor compartment predominantly, 2) infusion of HSC835 is well tolerated, and 3) the pace of neutrophil and platelet recovery is significantly accelerated, correlating with HSC835 CD34 cell dose. Despite potential adverse effects with such marked expansion, clonal cytogenetic abnormalities or excess telomere shortening was not observed in any expanded product and engraftment has been durable, further supporting its safety profile.

In patients with HSC835 predominance, the pace of hematopoietic recovery was remarkably rapid, especially in those with a CD34 cell dose >10 million/kg. In addition, HSC835 contributed to sustained hematopoiesis in 11 of 17 patients which contrasts with UCB hematopoietic progenitors expanded in the presence of Notch-ligand (Delaney et al., (2010)) or adherent mesenchymal progenitor cells (de Lima et al., (2012)) which contributes to hematopoiesis only transiently. While HSC exhaustion cannot be ruled out, it is possible that the transient recovery observed with these methods was due to the absence of CD3+ cells, leaving the expanded unit at an immunological disadvantage. Like us, Horwitz et al (Horwitz et al., (2014)) infused the re-cryopreserved fraction containing T cells and also observed sustained engraftment. It is known that graft-versus-graft immune responses play a role in unit predominance after double UCB transplantation (Gutman et al., (2010), Ramirez et al., (2012)). We have previously shown that the unit with the larger CD3+ cell dose is more likely to predominate (odds ratio 4.4 [95% CI 1.8–10.6], p<0.01) (20). This pattern, however, did not hold true for HSC835. After expansion, the HSC835 product out-competed the unmanipulated unit in 11 of 17 transplants despite lower CD3 cell doses in all cases. This suggests that ‘mega doses’ of CD34+ cells may overcome the engraftment barrier, as suggested in preclinical models (Reisner et al., (2003)).

While transient engraftment has had a demonstrable clinical effect with some expansion methodologies (Delaney et al., (2010), de Lima et al., (2012)), the transient burst of myeloid cells derived from HSC835 detected on day 7 had no obvious impact on shortening the period of neutropenia in those that ultimately engrafted with the unmanipulated unit. In about 1/3 of patients, however, mixed myeloid chimerism derived from both units was associated with very rapid neutrophil recovery. Chimerism from two units (beyond 60 days) rarely occurs after double UCBT. Perhaps larger numbers of CD34+ cells and/or the reduction in the T cell dose influenced this outcome. For the entire cohort, pace of platelet recovery was accelerated, although perhaps less dramatically than that observed for neutrophil recovery. However, chimerism from both units had no demonstrable impact on speed of platelet recovery, although small patient numbers prevent definitive conclusion. Perhaps the full benefit of a higher CD34+ cell dose on platelet recovery was diminished by the higher rates of GVHD and graft-versus-graft reactions generally observed after double UCB transplant (Wagner et al., (2014)).

The data reported here suggest that graft-versus-graft interactions are likely responsible for the loss of HSC835 in the 6 patients rather than a loss in engraftment potential. Like Gutman et al. (Gutman et al., (2010)), we found interferon-γ secreting T cells in patients directed against the losing unit in 5 of 6 patients in whom one unit predominated. Interestingly, in one patient with myeloid chimerism from both units and T cells derived from the unmanipulated unit, interferon-γ secreting T cells could not be detected against either HSC835 or the unmanipulated unit, suggesting tolerance between unit 1 and unit 2.

The marked expansion of CD34+ cells with SR-1 suggests that the limitation of cell dose in recipients of UCB may have been ameliorated. In contrast to methods with a lower fold expansion of CD34+ cells, SR-1 obviates the need to target UCB units with higher CD34 doses and permits the utilization of UCB units previously considered to be unsuitable for general use due to low cell dose. Lowering the cell dose threshold for UCB transplant from 2.5 to 1 × 107 nucleated cells/kg, for example, not only markedly increases the size of the ‘useable’ inventory and therefore the chance of identifying a better HLA matched unit, but also significantly reduces the cell count threshold for banking which is currently at >1 billion nucleated cells. This would be especially important for patients of ethnic and racial minorities where there is a tendency for lower cell counts in the units collected (Cairo et al., (2005)). The availability of SR-1 based expansion could also have a profound effect on the economics of cord blood banking by improving collection efficiencies and availability of suitable units for transplantation.

The primary aim of the trial was to document the safety profile of HSC835. While higher cell dose levels have yet to be evaluated in children, the toxicity profile is encouraging. Thus far, there has been a single infusional toxicity that was related to an occult bacterial contamination. After procedural changes in the manufacturing process, including process improvements in batch production of expansion media and reductions in in-process testing, all subsequent products have met lot release. Safety profile, rapidity of neutrophil and platelet recovery, frequency of HSC835 predominance and reduction in the number of hospital days are sufficiently encouraging to justify further evaluation of HSC835 (in combination with its CD34 negative fraction) as a ‘stand-alone’ graft. The infusion of a single expanded UCB unit will eliminate the confounding graft-versus-graft effects observed in the current study. While the co-infusion of an unmanipulated unit served as a safety ‘back-up’ should the expansion procedure have a deleterious effect on the manipulated unit’s engraftment potential, double UCB transplant is associated with higher risks of GVHD and delayed platelet recovery. Therefore, future studies with a single expanded unit will more precisely define the impact of SR-1 expansion culture on the pace of hematopoietic recovery, engraftment, GVHD, transplant-mortality and survival as well as pace of immune recovery, resource utilization, health quality of life and hospital days. The demonstration that SR-1 promotes marked expansion of hematopoietic stem and progenitor cells also suggests it may be a useful strategy for amplifying the number of genetically-modified HSC, a consideration for future studies (Naldini, (2011)).

EXPERIMENTAL PROCEDURES

Study Design

This trial was an open-label, phase 1/2 dose-escalation study with four pre-defined maximum cell dose levels (Figure 1A). The primary end point was safety as measured by the absence of infusional toxicity or deleterious effects on hematopoietic recovery. Pre-specified secondary/exploratory endpoints included time to and incidence of engraftment, rate of neutrophil and platelet recovery, relative contribution of the two units to early and late hematopoietic recovery and immune reconstitution. Other endpoints included GVHD, transplant-related mortality and survival. Adverse event data (Figure S1) were reviewed by an independent Data and Safety Monitoring Board appointed by Novartis.

Consent from patients or guardians of minors was obtained in accordance with the Declaration of Helsinki. The work was approved by the Institutional Review Board of the University of Minnesota and the clinical protocol was registered with ClinicalTrials.Gov (NCT01474681) prior to study initiation.

Eligibility

To participate in this trial, patients had to be aged 10 to 55 years, inclusive, with high risk hematological cancer as described previously (Wagner et al., (2002), Barker et al., (2005)). Performance and organ function criteria included: good general health defined as Karnofsky/Lansky score ≥ 80%, creatinine < 2.0 mg/dL, bilirubin, AST, ALT and ALP <3x upper limit of normal, DLCO corrected >50% upper limit of normal, and left ventricular ejection fraction >45%. In addition, the patients could not have an active infection at time of transplantation (including active infection with Aspergillus or other mold within 30 days of conditioning for transplant), history of HIV infection, be pregnant or breast feeding, be a woman of child-bearing potential without acceptable contraception. In addition, patients were excluded if they had a prior myeloablative allogeneic transplant or autologous transplant, extensive prior therapy including >12 months alkylator therapy or > 6 months alkylator therapy with extensive radiation, or received Y-90 ibritumomab (Zevalin) or I-131 tostumomab (Bexxar).

Treatment

Patients were treated with cyclophosphamide 60 mg/kg/day IV (days −8 and −7), fludarabine 25 mg/m2/day IV (days −8, −7, and −6), and total body irradiation 165 cGy twice a day (days −4, −3, −2, and −1) with the infusion of two UCB units on day 0 (Figure 1A). GVHD immunoprophylaxis consisted of cyclosporine (targeting trough levels 200–400 μg/L) on days −3 to +100 with a 10% taper per week thereafter, and mycophenolate mofetil 15 mg/kg IV three times a day on days −3 to +45 if no GVHD was present on day +45.

Three UCB units were identified prior to study enrollment with one as ‘back-up’ should the expanded product’HSC835 not meet lot release. All units had a minimum dose of 1.5 × 107 nucleated cells/kg recipient body weight and were HLA matched at a minimum of 4 of 6 loci with the patient and between units, considering HLA A and B at antigen level and HLA DRB1 at allele level typing. Unit 1 was the unit with highest cell dose except in one instance when the unit was HLA matched at a comparable cell dose. Units were disqualified if donor-specific cross-reactive anti-HLA antibodies were identified.

Unit Processing

All UCB units were thawed using standard methods (Rubinstein et al., (1995)). On day −15, the lower dosed unit was CD34 enriched using the CliniMACS Cell Selection Device (Miltenyi Biotec) following manufacturer’s instructions and placed in expansion media and the CD34 negative fraction was cryopreserved. The expansion culture media consisted of SCF, FLT-3L, TPO and IL-6 (all at 50 ng/mL) and SR-1 without the addition of antibiotics. Prior to lot release, the product was required to have a cell viability ≥70%, negative gram stain, endotoxin <5EU/kg, and negative in-process aerobic and anaerobic bacterial/fungal cultures (Table S1). On day 0, the unmanipulated unit was infused followed 4 hours later by the infusion of HSC835. On day +1, the cryopreserved CD34 depleted cells were thawed and infused.

Dose Capping

The goal was to infuse the maximum dose achieved by the expansion culture. While the lowest acceptable dose of total nucleated cells and CD34+ cells has been defined for UCB transplantation, it is not known if there is an upper limit above which expanded cell infusions might be associated with unacceptable toxicities. Therefore, a staggered dose capping approach was adopted to limit the maximum number of cells in the HSC835 product and potentially identify the maximal tolerable dose (Figure 1A). Dose limiting toxicity (DLT) was defined as ≥ grade 3 CTCAE hypersensitivity reactions to HSC835 infusion or ≥ grade 4 CTCAE organ toxicities observed within the first 30 days after transplant. Excessive toxicity was defined on the basis of the observed rate of Bearman Grade III and IV AEs in the Cord Blood Transplantation (COBLT) trial (Kurtzberg et al., (2008)). To this end, a minimum of 3 adult and 3 pediatric (aged 10–17 years) patients had to be enrolled at a given dose level before moving to the next dose level if there were no DLTs. As not all expansion cultures exceeded the maximum dose at a given level, a total of 15 patients (13 adults and 2 children) were treated at dose level 1 (3–9 × 107 cells/kg body weight) or below and two adults were treated at dose level 2 (9.1–27 × 107 cells/kg). Three patients (1 adult and 2 children) achieved levels of cell expansion that exceeded the dose permitted (i.e., not all available cells were infused).

Correlative Studies

Chimerism analysis. Peripheral blood from the recipient was collected before transplant and approximately 7, 14, 21, 28, 60, 100, 180, and 365 days after transplant. Peripheral blood mononuclear cells were prepared by mixing 10 mL of peripheral blood with an equal volume of a dextran/saline solution (3% dextran in 0.9% NaCl) for 30 min at room temperature. The leukocyte-rich plasma (upper) layer was pelleted and the red blood cells were lysed. CD3 and CD15/CD33 cell populations in the peripheral blood were isolated by FACS. Chimerism testing was performed by a DNA-based assay for short tandem repeat loci using standard techniques (Brunstein et al., (2007)). Cord blood product analysis. Hematopoietic subset analyses based on surface antigen expression and colony forming unit enumeration were performed as previously described (Nawrot et al., (2011)). Analysis of telomeric DNA. DNA was analyzed for telomere length as described previously (Celli and de Lange, (2005)). Mobilized peripheral blood from normal donors or UCB was CD34 enriched and then cultured with stem cell factor, thrombopoietin, Flt-3-ligand, and interleukin-6 (STF6) with and without SR-1 (10 days for mobilized peripheral blood and 15 days for UCB). T cell allo-reactivity assay. Residual cells in the wash supernatant were collected when the cord blood units were thawed and washed. CD19+cells in the supernatant were transformed with Epstein-Barr virus (EBV) and the resulting lymphoblastoid cell lines (LCLs) were cultured as described (Rickinson et al., (1984)). On day 14, 28 and 56 after transplantation, 10 mL of peripheral blood was obtained from patients. Peripheral blood mononuclear cells were isolated by density gradient separation and cryopreserved. Interferon-γ secreting CD8+ cells following a 5-hour stimulation period with LCL from each UCB unit as well as third party cells were analyzed as described previously (Gutman et al., (2010)).

Data Collection and Statistical Analysis

Clinical outcome data were prospectively collected and endpoints defined using standard criteria (Wagner et al., (2002), Przepiorka et al., (1995), Weisdorf et al., (2003)). In addition, outcomes in patients treated with HSC835 were compared to 111 patients with acute leukemia and myelodysplastic syndrome aged 10–55 years treated with the same conditioning and post-transplant immune suppression (historical control arm). The two groups were similar in terms of recipient age, height, weight, gender, cytomegalovirus (CMV) serostatus, HLA match, ABO match and Karnofsky status. While patients in the historical cohort were more likely to have had acute myeloid leukemia (p=0.04) and been transplanted over a broader time period (2000–2014, p<0.001) with 64% after 2006, these factors were not expected to impact the primary endpoint. However, as expected, recipients of the expanded product HSC835 received a graft containing greater numbers of nucleated and CD34+ cells and CFU-GM colonies relative to those in the historical comparison cohort (p <0.001 each, Table 2). In terms of transplant outcomes, neutrophil and platelet recoveries were determined as the day after transplant to achieve an absolute neutrophil count (ANC) ≥ 0.5 × 109/L for 3 consecutive days and ≥20.0 × 109/L for 7 consecutive days without transfusion, respectively. Categorical variables were compared using the Pearson chi-square statistic while comparisons with smaller numbers were analyzed with the Fisher Exact test. The general Wilcoxon test was used to analyze continuous parameters between groups. Continuous factors across UCB units are compared by the Wilcoxon sign-rank test for paired data (Snedecor, (1989)). The Kaplan-Meier method was used to analyze the probability of overall survival (Kaplan, (1958)). The log-rank statistic was used to complete comparisons. Cumulative incidence treating non-event death as a competing risk was used to estimate the probabilities of neutrophil and platelet recovery, acute and chronic GVHD. The probability of regimen-related mortality was estimated by treating relapse a competing risk (Lin, (1997)). All p-values were reported as two-sided and a p-value <0.05 was considered statistically significant. All analyses were conducted using SAS 9.3 (SAS Institute, Cary, NC) and R 3.0.2.

Supplementary Material

supplement

Table 4.

Graft Characteristics and Transplant Outcomes

UPN (dose level)Ϯ Infused TNC (107/kg)* Infused CD34 (106/kg)* Infused CFU-GM (104/kg)* Infused CD3 (106/kg)* Days to ANC ≥ 5 108/L Predominant Unit+ Days to PLT ≥ 20 109/L Max Grade Acute GVHD Survival+ (Days)
UNM HSC835 UNM HSC835 UNM HSC835 UNM HSC835 CD3 CD15
01 (1) 1.8 2.8 0.3 8.9 1.9 121.7 7.2 2.6 23 HSC835 HSC835 80 3 140
02 (1) 2.0 5.0 0.4 19.1 3.3 639.4 7.3 5.0 14 HSC835 HSC835 136 3 442
03 (1) 2.4 1.0 0.9 1.4 7.0 40.7 6.6 2.9 22 UNM UNM NA 0 40
04 (1) 1.8 9.7Ψ 0.2 48.3 2.4 1325.1 8.4 2.8 6 UNM HSC835 54 3 688
05 (1) 2.8 6.4 0.7 17.5 3.6 523.2 9.1 3.8 16 HSC835 HSC835 72 2 687+
06 (1) 2.1 4.0 0.5 10.7 2.1 275.0 6.6 1.1 23 HSC835 HSC835 57 0 623+
07 (1) 1.5 2.6 0.2 7.0 2.0 231.0 4.6 2.3 30 UNM UNM NA 0 45
08 (1) 3.5 3.1 0.3 5.3 5.4 389.1 10.5 2.6 24 UNM UNM 35 0 545+
09 (1) 2.0 9.1 0.3 21.2 2.6 1335.8 8.9 4.0 24 UNM UNM 49 0 539+
10 (1) 2.9 9.6 0.6 21.0 7.4 1005.2 12.5 3.2 14 UNM UNM 33 3 391+
11 (1) 3.4 3.8 0.7 5.7 5.0 255.1 7.3 2.8 15 HSC835 HSC835 NA 0 46
12 (2) 5.2 12.1 0.6 29.6 4.5 144.8 28.8 4.3 6 UNM HSC835 31 2 281
13 (2) 3.7 12.3 0.5 26.1 2.6 662.2 13.7 2.2 11 HSC835 HSC835 29 2 272+
14 (1) 2.0 4.2 0.3 10.6 1.2 292.1 7.1 1.9 10 UNM HSC835 28 3 102
16 (1) 1.8 3.3 0.3 8.9 3.0 148.4 6.0 3.2 16 UNM UNM 71 2 238+
17 (1) 2.5 10.7Ψ 0.2 25.2 4.8 780.8 14.6 4.7 7 UNM HSC835 NA 2 35
20 (1) 4.2 10.6Ψ 0.4 34.9 6.0 637.5 13.9 4.4 7 UNM HSC835 31 0 43+
Median 2.4 5.0 0.4 17.5 3.3 389.1 8.4 2.9 15 49
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Abbreviations: HSC835 = expanded unit; UNM = unmanipulated unit; UPN = unique patient number; TNC = total nucleated cell count; CFU-GM = colony forming unit-granulocyte macrophage; ANC = absolute neutrophil count; PLT = platelet count; NA = not achieved

*

HSC835 Graft = Sum of cell populations from HSC835 and CD34 depleted fraction

Ϯ

Patients were eligible for dose level 2 if no dose limiting toxicities were observed at the prior dose level in at least 3 patients and the expansion culture yielded sufficient cells. Pediatric recipients were evaluated at each dose level separate from adults.

Ψ

Cell expansion exceeded dose allowed at the prescribed dose level

+

Predominant unit on day 60 (or last follow up) after UCB transplant for CD3 (T) and CD15/CD33+ (myeloid) cell subsets

Acknowledgments

The project was supported by Novartis Corporation and a grant from the National Cancer Institute P01 CA065493-20 (J.E.W.J., C.G.B., T.E. D., D.M., B.R.B., J.T., C.L.). Specific authors are employees and shareholders of Novartis (J.J, C.C.B.) and Genomics Institute of the Novartis Foundation (A.E.B, M.P.C.). We also wish to thank Diane Kadidlo, MT(ASCP), Fran Rabe, MS and staff of the Cell Therapy Laboratory at the Molecular and Cellular Therapeutics Facility for their efforts in the product manufacturing, development and monitoring, Eros Lazzerini Denchi, Ph.D. for his investigations on telomere length, Stefanie Hage, BSN of the University of Minnesota and Suzanne Maahs, Pharm D and Shazia Ali, Pharm D of the Novartis Institutes for Biomedical Research for their critical assistance in patient monitoring and moving the clinical trial forward, Albert E Parker, for performing the T cell allo-reactivity assays, Stephanie Fiola, for qualification of the media and cytokines, and the faculty, advanced practice providers, unit and clinic nurses, pharmacists and social workers of the Blood and Marrow Transplant Program who cared for the 20 patients on this phase I clinical trial at the University of Minnesota Medical Center and Children’s Hospital.

Footnotes

AUTHOR CONTRIBUTIONS

Contributions: J.E.W.J. and C.C.B. designed and coordinated the study. T.E.D. and C.L. performed the statistical analyses with independent review of the analyses by J.J. D.M. and D. S. oversaw the manufacture of the HSC835. A.E.B., D.S. and M.P.C. designed and coordinated the correlative assays. J.E.W.J. wrote the manuscript, and all coauthors had access to the primary data and contributed to the final report.

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