Abstract

Abstract 15

PR1 (VLQELNVTV) is an HLA-A2-restricted peptide derived from endogenous myeloid leukemia-associated antigens (LAA) proteinase 3 and neutrophil elastase. PR1/HLA-A2 is targeted by PR1-specific cytotoxic T lymphocytes (PR1-CTL) that contribute to cytogenetic remission in patients with CML after allogeneic stem cell transplantation or interferon therapy, and PR1 peptide vaccination induces specific CD8 immunity in AML, MDS, and CML patients. Because clinical effects after PR1 vaccination are associated with a low amount of leukemia and because the effects on leukemia stem cells (LSCs) are unknown, we sought to develop a PR1/HLA-A2-specific MAB that could be more effective in high disease burden cases and that might also target LSC. We previously reported a mouse IgG2A antibody (8F4) with PR1/HLA-A2 specificity. We now used 8F4 to study PR1 expression by FACS and confocal mircroscopy on leukemia subsets and normal peripheral blood mononuclear cells (PBMC). Circulating blasts from peripheral blood (PB) or leukapheresis products (LP) from AML patients (5 HLA-A2+, 2 control HLA-A2-,) were studied (see Table 1). PR1 expression (MFI±SEM) was higher in AML (23.7±5.18) compared to control (4.0±2.42) and normal PBMC (13.6±0.23; p = 0.046). Confocal microscopy confirmed bright heterogeneous surface expression of PR1 on AML, but was nearly absent on healthy donor PB. Interestingly, PR1 expression was higher on LSC (CD34+CD38-lin-) (76±44) compared to healthy donor HSC (CD34+CD38-lin-) (13.4), which suggests that only differentiated myeloid cells might be more susceptible to 8F4 toxicity. To study whether 8F4 mediated specific lysis of PR1+ cells, we determined cell-mediated cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) of 8F4 compared to isotype control. 8F4 induced 51% specific lysis by CDC and 15% specific lysis by ADCC of PR1-pulsed T2 cells, which was dose-dependent on both 8F4 and PR1, with no lysis of target cells by isotype control. Moreover, 8F4 induced CDC-mediated lysis of blasts from 5/5 HLA-A2+ AML patients, but not blasts from an HLA-A2- AML patient (see Table 1), or mononuclear cells from HLA-A2+ healthy donors. To study the in vivo effects of 8F4 on AML, we used an established xenogeneic model of human AML in NOD/SCID mice. 1 × 106 blasts from 4 HLA-A2+ AML patients (including the HLA-A2+ patients susceptible to 8F4-mediated lysis in vitro) and 1 HLA-A2- patient were mixed for one hour under serum-free conditions with 8F4 or isotype control antibody and adoptively transferred to NOD/SCID mice. Engraftment of AML was compared on day 14 and AML growth was confirmed on day 28 by comparing FACS analysis and histopathology of BM from sacrificed animals. 8F4 treatment of AML cells, but not isotype treatment, abrogated engraftment of 4/4 HLA-A2+ AML patients, but not of 1 HLA-A2- AML patient on d14. By day 28, AML expanded by 150% in one surviving isotype-treated animal. Thus, 8F4-mediated specific lysis of AML and LSC by CDC and ADCC mechanisms in vitro were confirmed by in vivo results of AML engraftment. Taken together, these results show that 8F4, a novel TCR-mimic antibody that targets only PR1/HLA-A2+ cells, mediates lysis of AML blasts and LSC, but not total normal BM cells or HSC. Therefore, 8F4 may be useful as an immunotherapeutic agent in the treatment of HLA-A2+ myeloid leukemia, and as the first antibody therapy that may target and eliminate LSC.

Table 1.

Patients' characteristics and 8F4 effects

UPN Diagnosis+ Disease status HLA-A2 Sex Age Sample* Blast 8F4 FCM In vitro CDC lysis In vivo Engraftment
 
iso 8F4 
AML1 AML-M1 secondary Myelofibrosis Untreated 79 LP 87% ++ − 
AML2 AML-M2 Relapsed 61 PB 66% +++ Low* − 
AML3 AML-M5 Refractory 73 PB 64% +++ − 
AML4 AML-M1 Untreated 55 LP 92% ++ N/D N/D 
AML5 AML-M2 Refractory relapsed 33 PB 98% N/D N/D − 
AML6 AML-M5 Untreated 53 LP 96% ++ N/D N/D N/D 
AML7 AML-M1 Untreated 24 LP 85% − − 
AML8 AML-M1 Untreated − 41 PB 91% − − Low Low 
+French-American-British AML subtype            
UPN Diagnosis+ Disease status HLA-A2 Sex Age Sample* Blast 8F4 FCM In vitro CDC lysis In vivo Engraftment
 
iso 8F4 
AML1 AML-M1 secondary Myelofibrosis Untreated 79 LP 87% ++ − 
AML2 AML-M2 Relapsed 61 PB 66% +++ Low* − 
AML3 AML-M5 Refractory 73 PB 64% +++ − 
AML4 AML-M1 Untreated 55 LP 92% ++ N/D N/D 
AML5 AML-M2 Refractory relapsed 33 PB 98% N/D N/D − 
AML6 AML-M5 Untreated 53 LP 96% ++ N/D N/D N/D 
AML7 AML-M1 Untreated 24 LP 85% − − 
AML8 AML-M1 Untreated − 41 PB 91% − − Low Low 
+French-American-British AML subtype            
*

Peripheral blood (PB) or leukopheresis (LP)

FCM flowcytometry staining intensity (+ indicates staining intensity close to those of normal PBMC, ++ and +++ indicate increased intensity)

N/D The experiment was not performed due to low viability of sample (less than 50%) or sample was not available

Low* - indicates engraftment <0.5% of viable mCD45- cells, effect of treatment is inaccessible.

Disclosures:

No relevant conflicts of interest to declare.

Author notes

*

Asterisk with author names denotes non-ASH members.