Chimeric CD19 antibody mediates cytotoxic activity against leukemic blasts with effector cells from pediatric patients who received T-cell–depleted allografts

Transplantation of T-cell–depleted peripheral stem cells from unrelated and mismatched related donors has become an option for the treatment of children with high-risk acute lymphoblastic leukemias (ALLs).^1-4  Graft-versus-host disease (GvHD) has been reduced significantly by positive selection of CD34⁺ progenitors^5,6  and other T-cell–depletion techniques, but relapses still remain a major problem.⁷  Recovery of T cells has been reported to be delayed because of graft manipulation.⁸  This was also observed in our patients who received transplants with CD34⁺-selected stem cells.^2,5  Therefore, we investigated the feasibility of recruiting the more rapidly regenerating donor-derived natural killer (NK) cells for antileukemic effects.

Natural killer cells are capable of lysing several types of malignant cells, including lymphomas and leukemias, but high expression of HLA class I antigens on some B-lineage ALLs may impair NK cell lysis.^9-11  Moreover, NK cell alloreactivity because of killer cell immunoglobulin-like receptor (KIR) ligand incompatibility has been reported in acute myeloid leukemia (AML) but not in ALL.^11,12  However, both high HLA class I expression and resistance of ALL cells to alloreactive NK lysis may be overcome by deploying antibody-dependent cellular cytotoxicity (ADCC).^9,13 

ADCC is generally accepted to be an important mechanism for tumor cell elimination by therapeutic antibodies.^14,15  However, to our knowledge, no systematic trials addressing the usefulness of antibody therapy for the elimination of minimal residual disease (MRD) after allogeneic stem cell transplantation have been reported. Consequently, we investigated whether NK cells of patients who received transplants were capable of exerting ADCC against B-lineage ALL blasts in combination with a chimeric CD19 antibody produced in insect cells.

ADCC was carried out with immunomagnetically selected NK cells (CD56⁺ microbeads; Miltenyi-Biotec, Gladbach, Germany) from 7 healthy donors and with mononuclear cells (MNCs) from 16 pediatric patients with ALL, AML/MDS (myelodysplastic syndrome) and others after transplantation. The patients received positive-selected CD34⁺ peripheral stem cells (CLINIMACS; MiltenyiBiotec) from unrelated or mismatched related donors with a median number of only 7000 residual T cells/kg body weight. No posttransplantation immunosuppression was administered to patients with completely depleted grafts. Six patients received T-cell add-backs and a short course of cyclosporin A. This study was approved by the ethical committee of the University of Tuebingen. Informed consent was provided according to the Declaration of Helsinki.

Variable heavy- and variable light-chain sequences were amplified by polymerase chain reaction (PCR) from the DNA of CD19-reactive scFvs previously generated from the hybridoma 4G7¹⁶  by the phage display technique using standard procedures.^17,18  Cleavage sites for restriction enzymes were introduced and subsequently used for the insertion of these fragments into the baculoviral expression vector pAc-K-CH3.¹⁹  Sf21 insect cells were cotransfected with the baculoviral expression construct and Sapphire Baculovirus DNA (Orbigen, San Diego, CA). Purification of the secreted recombinant protein from culture supernatants was performed by protein A agarose affinity chromatography.

Effector cells were stored overnight, either without or with interleukin-2 (IL-2) at 40 or 1000 international units (IU)/mL. The MHH4 cell line (DSMZ, Braunschweig, Germany) and cryopreserved primary B-lineage ALL blasts from 4 patients were used as target cells in a 2-hour BATDA europium release assay (Wallac, Turku, Finland). The blasts expressed CD10, CD19, and CD34 (> 90%), but all were CD20^–. MHH4 cells expressed both CD19 and CD20 (> 95%). Cytolytic assays were performed as published⁹  by using antibody concentrations of 0.15 to 0.4 μg/mL (CD19-4G7chim or chimeric CD20 IgG1 antibody; Mabthera; Roche, Basel, Switzerland). For blocking experiments the murine IgG2a antibody W6/32 (Dako, Hamburg, Germany), specific for human HLA class I, or W6/32 Fab fragments were used.^20,21  Only those assays were evaluated for which the spontaneous release was less than 25% (MHH4) or less than 30% (blasts).

Differences in specific lysis of targets were analyzed by the paired Wilcoxon rank sum test.

Childhood B-lineage ALLs consistently express CD19.²²  In our experience, only 12% of pro-B, pre-B, and common ALLs expressed CD20, but all leukemic cells were uniformly positive for CD19. Therefore, a high-affinity chimeric CD19 antibody (CD19-4G7chim; K_D, 5.5 ± 1.7 × 10^–9 M) was constructed and expressed in Sf21 insect cells.

CD19-4G7chim mediated specific lysis of cryopreserved primary pre-B/common ALL blasts with enriched NK cells from 7 healthy donors (Figure 1A). NK cells from each donor showed significantly enhanced lysis after addition of CD19-4G7chim. Preincubation of the effector cells with IL-2 at 40 IU/mL increased specific lysis to its maximum value. Thus, both these IL-2 and antibody concentrations fall into a range, which may be reached in clinical applications.²³  As expected, the murine 4G7 antibody did not induce ADCC. Specificity of CD19-4G7chim was shown by addition of the chimeric CD20 IgG1 antibody, which failed to raise specific lysis. Blocking of HLA class I antigens on target cells relevant for NK cell inhibitory receptors^20,21  with complete W6/32 antibody or Fab fragments increased specific lysis comparable to CD19-4G7chim (Figure 1B). This suggests that masking of HLA class I on ALL blasts may abolish possible HLA class I–mediated inhibition of NK cell lysis. However, these inhibitory effects did not prevent ADCC by CD19-4G7chim.

ADCC by CD19-4G7chim produced in insect cells was more effective compared with ADCC by the same antibody produced in mammalian cells (K.B., unpublished data, April 2003). This may be due to different glycosylation, which has been shown to be critical for antibody-mediated effector functions. In particular, fucosylation of the Asn297-linked IgG1 carbohydrate, which plays a critical role in enhancement of ADCC, may have contributed to these results.^24,25 

Next, we investigated whether patient-derived effector cells were also capable of exerting ADCC. MNCs from the majority of patients (66%) produced substantial lysis of MHH4 cells in combination with CD19-4G7chim. Lysis was enhanced by incubation of effector cells with IL-2 (Figure 2A, right panel). Mabthera was more effective than CD19-4G7chim against CD19⁺CD20⁺ targets (Figure 2B). However, for the treatment of pediatric B-lineage leukemias, often lacking CD20 expression, a CD19-directed antibody therapy may be more appropriate. Effector cells from the other one third of patients produced specific lysis below 10%, although the percentage of NK cells among MNCs was not significantly different for both groups (Figure 2A, left panel). This may be attributable to a polymorphism of FcγRIIIa, which has been reported to influence therapeutic response to rituximab.²⁶  Therefore, genotype determinations of this receptor on effector cells should be included in future experiments.

In contrast to MHH4 cells, cryopreserved primary B-lineage blasts were more susceptible to NK lysis, although leukemic blasts from a patient with refractory disease were predominantly used as targets. In repeated experiments with effector cells from 7 patients against these allogeneic blasts and from 1 patient for whom autologous blasts were available, a consistent pattern emerged: specific lysis by unstimulated MNCs was less extensive than specific lysis by unstimulated MNCs plus CD19-4G7chim. IL-2 stimulation of effector cells increased specific lysis. Combination of antibody and IL-2 stimulation significantly enhanced specific lysis to a maximum (Figure 2C). As expected, both the parental murine 4G7 antibody and Mabthera, used as controls, failed to raise specific lysis, consistent with the fact that these target cells were CD20^–. Blocking of HLA class I with W6/32 antibody raised specific lysis to the same extent as treatment with CD19-4G7chim (Figure 2D), suggesting that NK cells were the major effectors and that CD19-4G7chim mediated ADCC also in the posttransplantation setting, despite possible HLA class I–induced inhibition of effector cells. ALL patients in particular may benefit from such antibody-augmented GVL effects, because NK cell alloreactivity alone was shown to have no effect on adult ALL even in mismatched transplantations.^11,12 

Additional advantages of the use of the antibody in a posttransplantation situation include the following. First, the leukemia cell load is reduced to a minimum. Thus, an early-acting immunotherapy based on the recruitment of rapidly regenerating donor-derived NK cells can be focused on MRD, for which high effector–target cell ratios can be expected. Second, although CD19 antibodies may also lead to a deficiency of donor-derived nonmalignant B cells, additional infusions of donor stem cells after immunotherapy are expected to compensate for this lack.

In summary, our data encourage clinical trials with chimeric CD19 antibodies for patients with B-lineage ALL in an attempt to control MRD after transplantation.

We thank Drs M. Liang and S. Dübel for the baculoviral expression vector pAc-K-CH3, Dr R. Levy for the 4G7 hybridoma, Dr G. Jung for providing monoclonal antibodies, and D. Saul and A. Babarin-Dorner for excellent technical assistance. Constructive criticism of the manuscript by Drs T. Allen, G. Jung and B. Emmerich is gratefully acknowledged.