8F4-Specific Lysis, % 1 UPN1 UPN3 8 UPN7 6 Pearson r =.69 UPN2 UPN5 P (one-tailed) =.192 4 UPN8 n = 9 2 UPN9 UPN4 UPN6 5 1 15 2 25 8 8F4, % Max MFI Supplementary Figure S1. AML samples UPN1-UPN9 show variable level of susceptibility to 8F4-mediated lysis in vitro. Cells from HLA-A2+ (filled circles) and HLA- A2- (open circles) patients were stained with Alexa 647-conjugated 8F4 as described in Methods. To test susceptibility to 8F4-mediated complement-dependent cytotoxicity (CDC, 8F4-specific Lysis), same samples were incubated with 8F4 or control antibodies in presence of rabbit complement. After one hour cells were stained with aqua viability marker. Viability was analyzed by flow cytometry for in presence of BD counting beads. 8F4 MFI (% of Max) and 8F4-Specific Lysis (%) were calculated as indicated in Methods. Filled circles show HLA-A2 + AML samples, open circles show show HLA-A2 - AML samples. One representative experiment out of three is shown.!
UPN1 AML cells! UPN1+IgG! UPN1+8F4! Control mouse! 4 weeks! 91.5 13.1.135 UPN5 AML cells! UPN5+IgG! UPN5+8F4! Control mouse! 1 3 1 2 1 1 1 3 92.5 1 1 1 2 1 3 99.8 1 3 1 2 1 1 1 3 1 2 1 1 1 3.38 1 1 1 2 1 3.861 1 1 1 2 1 3 2.77.348.775.61 UPN6 AML cells! UPN6+IgG! UPN6+8F4! Control mouse! <FL 9 Log>: CD45 M APC A75 1 3 1 2 1 1 1 3 1 2 1 1 1 3.278 1 1 1 2 1 3.118 1 1 1 2 1 3 <FL 8 Log>: CD45 APC.231.15 1 3 1 2 1 1 1 3 1 2 1 1 1 3.153.18.166 4.e-3 1 1 1 2 1 3 1 1 1 2 1 3.378 Bone marrow! Spleen! Liver! 3 weeks! Bone marrow! Spleen! 3 weeks! Supplementary Figure S2. 8F4 Ex vivo treatment prevents engraftment of primary HLA-A2+ AML in NOD scid/hla-a2 mice. HLA-A2 + AML (UPN1, UPN5) and HLA-A2 negative AML control (UPN6) were co-incubated with (2 µg/ml) 8F4 or isotype control mouse IgG2a anti-klh monoclonal antibody (IgG) and intravenously injected into sublethally-irradiated recipient mice. Three to four weeks later, mouse tissues were analyzed for the presence of leukemia cells. Flow cytometry plots depict leukemia cells in tissues of mice that received AML, treated ex vivo with 8F4 or control antibody. AML cells ( + /mocd45 - ) are shown within the gate in each plot. Freshly thawed AML is shown in left panels, and cells from control mice that did not receive AML are shown in right panels 1 2 1 1 1 2 1 1 1 2 1 1 1 2 1 1 Bone marrow! 1 1 1 2 1 3 1 1 1 2 1 3 1 1 1 2 1 3 1 1 1 2 1 3
A NOD scid/hla-a2 FMO! NOD scid/hla-a2! NOD/scid! 1 1 1 2 1 3 8F4 (PE)! B B6/HLA-A2 FMO! B6/HLA-A2! B6! 1 2 1 3 1 4 1 5 8F4 (Alexa 647)! Supplementary Figure S3. PR1/HLA-A2 is expressed in bone marrow of HLA-A2 transgenic mouse models. Bone marrow cells of NOD scid/hla-a2 (A) and B6/HLA-A2 (B) were stained with fluorophore-conjugated 8F4, and analyzed by flow cytometry. Bone marrow cells of non-hla-a2- transgenic NOD scid and B6 were used for control, respectively. Histograms show 8F4 staining of live singlet mononuclear cells, identified based on scatter characteristics. FMO (Fluorescence minus one) - bone marrow cells cells were stained with the same antibody panel except for 8F4.!
5 4 3 2 1 Serum 8F4, µg/ml! 2-phase exponential elimination model (R 2 =.95): t 1/2 (fast) = 2.9 hours t 1/2 (slow) = 93.4 hours Supplementary Figure S4.! Serum 8F4 binding concentration after single iv injection (1 mg/kg) in NSG mice. Serum 8F4 concentration was determined by PR1/HLA-A2 ligand ELISA by measuring OD from duplicate wells at the indicated time points. Concentration was determined from a standard curve. Each data point represents mean concentration ± SEM (n=3 mice per time point). The data was analyzed according to a two-phase exponential decay model with Prism 6 (Macintosh) software. Half-life was determined according to the model of double exponential elimination model.! 5 1 15 Hours Time, hours!
Blood chimerism, % 6 5 4 3 2 1 A. Peripheral Blood Chimerism! UPN9! 1mg/kg, 3x/ week, 2 weeks 5 11 12 14 16 18 2 22 Weeks Tissue engraftment, % B. Tissue Engraftment! 1 8 6 4 2 BM SP LV Supplementary Figure S5. 8F4 treatment does not affect the growth of established HLA-A2 negative leukemia (UPN9) in NSG. 8F4 treatment (1mg/kg 3 times weekly) was started at week 19 when AML engraftment was confirmed.(a) AML engraftment was monitored by flow cytometry analysis of human cells ( + /HLA-ABC + / - ) in the peripheral blood. (B)Three days after the sixth dose of 8F4, AML persistence was confirmed by FACS in bone marrow (BM), spleen (SP) and liver (LV).
Blood chimerism, % 6 5 4 3 2 1 UPN1!.5 mg/kg and 2. mg/kg; 1x/week *** *** 2 4 6 8 1 Time post transplant, weeks pre- Rx 2. mg/kg 2. mg/kg.5 mg/kg IgG 8F4! Supplementary Figure S6. Low dose 8F4 prevents growth of established leukemia. AML (UPN1, 7 x 1 6 cells) were injected iv (tail vein) into NSG mice and engraftment was confirmed by ~1-fold increase of AML in peripheral blood (.17% ±.8, n=4 on day 7; 1.7% ±.67, n=4 on day 14). Starting on day 15, mice were treated once weekly with low-dose 8F4 (.5 mg/kg or 2 mg/kg) or IgG isotype control (2 mg/kg). The percentages of gated human cells (mo CD45 - /hu CD45 + /HLA- A2 + ) are shown. Data shown is mean percent ± SEM of AML chimerism for each treatment group (n=2-5);! *** p<.5.!!
IgG-treated! 8F4-treated! Spinal cord, lumbar region, x4! 2µm l------l 2µm l------l Brain, x1! 1 mm l-------l 1 mm l-------l Supplementary Figure S7. Persistence of AML (UPN8) in central neural system (CNS) of 8F4treated mouse. Representative tissue histology (H&E staining) shows extensive AML infiltration in parenchyma and meninges (black arrow) of the brain and spinal cord, and into the other tissues surrounding the CNS, regardless of treatment.!
Live Dead Aqua- - + Lin - 1 5.18 6 1 5 8F4 - treated <APC-Cy7-A>: MCD45 1 4 1 3 1 2 # Cells 4 2 88.3 11.7 <PE-Cy7-A>: CD34 1 4 1 3 1 2 1 2 1 3 1 4 1 5 <Pacific Blue-A>: HCD45 1 2 1 3 1 4 1 5 <PE-Cy5-A>: LIN (4 14 16 19 8) 1 2 1 3 1 4 1 5 <PE-A>: CD38 IgG - treated <APC-Cy7-A>: MCD45 1 5 1 4 1 3 1 2 3.3 # Cells 6 4 2 77.8 22.2 <PE-Cy7-A>: CD34 1 5 1 4 1 3 1 2.633 1 2 1 3 1 4 1 5 <Pacific Blue-A>: HCD45 1 2 1 3 1 4 1 5 <PE-Cy5-A>: LIN (4 14 16 19 8) 1 2 1 3 1 4 1 5 <PE-A>: CD38 Supplementary Figure S8. Phenotype of residual human cells in bone marrow of mice, transplanted with AML UPN2, and treated with 8F4 or IgG, as shown on Figure 2. Cells were transplanted into secondary NSG, as shown on scheme Figure 4B. 16 weeks after secondary transplant tissues of recipients were analyzed (See Figure 4C)
A! Cells, x1 3 /mm 3 15 1 5 WBC 1 mg/kg, 3x /week, total 1 Cells, x1 3 /mm 3 5 4 3 2 1 GRA 1 mg/kg, 3x /week, total 1 g/dl 18 16 14 12 HGB 1 mg/kg, 3x /week, total 1 Cells, x1 3 /mm 3 15 1 5 PLT 1 mg/kg, 3x /week, total 1-1 1 2 3 4 6 8 1 12 14 16 Time post first treatment, weeks -1 1 2 3 4 6 8 1 12 14 16 Time post first treatment, weeks 1-1 1 2 3 4 6 8 1 12 14 16 Time post first treatment, weeks -1 1 2 3 4 6 8 1 12 14 16 Time post first treatment, weeks B! Mouse:! Treatment:! B6! B6/HLA-A2! B6/HLA-A2! NT! NT! 8F4! 15 1 Granulocytes, % 12.2 17.6 15.2 5 SSC-A FSC-A SSC-A FSC-A SSC-A FSC-A Mouse Treatment B6 B6/HLA-A2 B6/HLA-A2 NT NT 8F4 1 1 1 8 GR1 high, % 8 8 8 6 % of Max 6 4 2 % of Max 6 4 2 % of Max 6 4 2 4 2 1 2 1 3 1 4 1 5 GR-1 1 2 1 3 1 4 1 5 GR-1 1 2 1 3 1 4 1 5 GR-1 Mouse Treatment B6 B6/HLA-A2 B6/HLA-A2 NT NT 8F4 Supplementary Figure S9. Multiple doses of 8F4 transiently reduce normal hematopoiesis in HLA-A2 transgenic mice. C57BL/6-Tg(HLA-A2.1)1Enge/J (B6/HLA-A2) mice received 8F4 (1 mg/kg, 3 times per week for total 1 intravenous injections)! starting day. (A) Complete blood count (CBC) was assessed before 8F4 and then weekly beginning 1 week after the1 th dose. Data are expressed as mean ± SEM of n=3 mice. (B) At week 16 8F4-treated mice (n=3) were sacrificed and bone marrow cells were analyzed for presence of granulocytes by flow cytometry. For control, 5 untreated B6/HLA-A2 and 4 B6-WT mice were analyzed. Top panels show representative scatter plots for each group, second row shows overlays of histograms for each group. Right panels show mean±sem for each group.!