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Keywords:

  • antibody therapy;
  • minimal residual disease;
  • natural killer cells;
  • Fcγ receptor;
  • tumour immunotherapy

Summary

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

A recombinant bispecific single-chain fragment variable antibody (bsscFv), directed against the B-cell antigen CD19 and the low affinity Fc-receptor FcγRIII (CD16), was designed for use in the treatment of patients with leukaemias and lymphomas. The Fc-portions of whole antibodies were deliberately eliminated in this construct to avoid undesired effector functions. A stabilised bsscFv, ds[CD19 × CD16], was generated, in which disulphide bonds bridging the respective variable light (VL) and variable heavy (VH) chains were introduced into both component single-chain (sc)Fvs. After production in 293T cells and chromatographic purification, ds[CD19 × CD16] specifically and simultaneously bound both antigens. The serum stability of ds[CD19 × CD16] was increased more than threefold when compared with the unstabilised counterpart, while other biological properties were not affected by these mutations. In antibody-dependent cellular cytotoxicity experiments, ds[CD19 × CD16] mediated specific lysis of both CD19-positive malignant human B-lymphoid cell lines and primary tumour cells from patients with B-cell chronic lymphocytic leukaemia or B-cell acute lymphoblastic leukaemia. Natural killer cells, mononuclear cells (MNCs) from healthy donors and, in some instances, MNCs isolated from patients after allogeneic stem cell transplantation, were used as effectors. Thus, ds[CD19 × CD16] holds promise for the treatment of CD19+ B-lineage malignancies.

Relapse of leukaemias and lymphomas, caused by minimal residual disease (MRD) cells that were not eradicated by previous chemo- and radiotherapy, still remains a major problem after transplantation of haematopoietic stem cells (Handgretinger et al, 2003). The first months after allogeneic stem cell transplantation offer an advantageous window of time for the elimination of persisting MRD cells. Early reconstituted donor-derived effector cells, such as natural killer (NK) cells can be used to redirect cellular cytotoxicity against leukaemic blasts and to increase graft-versus-leukaemia effects, without the induction of graft-versus-host disease. Indeed, in two studies with completely T cell-depleted grafts, the relapse rates were not clearly increased, despite delayed T cell regeneration. This observation may be ascribed to the rapid reconstitution of NK cells in those patients (Eyrich et al, 2001; Lang et al, 2003; Lang et al, in press). Antibody-based therapies offer advantages, particularly antigen specificity and the recruitment of immune effector cells. A chimaeric antibody targeting CD19 was recently shown to mediate specific lysis of primary acute lymphoblastic leukaemia (ALL) blasts with donor-derived effector cells obtained from paediatric leukaemia patients after transplantation of purified allogeneic stem cells (Lang et al, 2004).

However, some limitations restricting the therapeutic efficacy of conventional monoclonal antibodies are known. Penetration of the tumour is limited by the size of the whole antibody (approximately 150 kDa). Interactions of the Fc domain with Fc receptors on non-cytotoxic cells, e.g. platelets or B cells, or non-activating Fc receptors, such as FcγRIIIb on granulocytes, may also reduce their therapeutic effects (Peipp & Valerius, 2002). Interaction with inhibitory Fc receptor isoforms, such as FcγRIIb on monocytes/macrophages, may further decrease their cytotoxic activity (Clynes et al, 2000). In addition, the glycosylation pattern of the IgG1 Fc-region at amino acid Asn297 influences binding to Fc receptors and the induction of antibody-dependent cellular cytotoxicity (ADCC) (Shields et al, 2002). Finally, Fc receptor polymorphisms may critically determine the clinical response to antibody therapy. This effect was demonstrated for the bi-allelic polymorphism of FcγRIIIA (Val 158 versus Phe 158) in clinical applications of the CD20 antibody Rituximab (Cartron et al, 2002; Weng & Levy, 2003).

By contrast, bispecific antibodies (bsAbs) have the potential to overcome at least some of the limitations associated with conventional antibodies (Peipp & Valerius, 2002). bsAbs combine two antigen-binding sites, one directed against a tumour-associated antigen, the other against a trigger molecule on effector cells. Thereby, bsAbs very efficiently recruit cytotoxic effector cells, such as NK cells, T-cells, monocytes/macrophages or granulocytes to the tumour cells and mediate elimination of the tumour cells via ADCC or phagocytosis. For the induction of cellular cytotoxicity, activation of effector cells is a critical requirement, which is achieved by antibody binding to cytotoxic trigger molecules, such as CD3 on T cells, CD16 on NK cells, CD64 on activated neutrophils and monocytes/macrophages, or CD89 on neutrophils (Peipp & Valerius, 2002). Initially, bsAbs were generated by the hybrid-hybridoma technique, but subsequently different types of genetically-engineered bispecific antibody-derivatives were designed, e.g. diabodies, mini-antibodies, single chain diabodies, and bispecific single chain fragment variable antibodies (bsscFvs) (Peipp & Valerius, 2002). Single-chain diabodies and bsscFvs have the particular advantage of being single-chain polypeptides and, therefore, are easier to produce in a homogeneous and defined final state.

One of the most interesting targets for antibody therapy on malignant human B cells is CD19 (Grossbard et al, 1992). This surface antigen is expressed on nearly all developmental stages of the B cell lineage. More importantly, CD19 is not shed from the cell surface, lost from tumour cells, or expressed on haematopoietic stem cells, T cells, or other non-lymphoid cells. Thus, CD19 is a particularly attractive target antigen for antibody therapy. So far, CD19 antibodies have been investigated in various formats for therapeutic studies in cell culture and in vivo (Hekman et al, 1991; Pietersz et al, 1995; Lang et al, 2004). The first CD19-directed conventional bsAbs used CD3 as the trigger molecule for the recruitment of T cells as effectors (Haagen et al, 1992; Weiner & De Gast, 1995; Kipriyanov et al, 1998; Loffler et al, 2000). Although cell culture data demonstrated significant lytic activity for these [CD19 × CD3] bsAbs, the bsAb-mediated cross-linking of CD3 led to non-specific T-cell activation, causing bsAb-associated toxicity in vivo (Segal et al, 1999). So far, a [CD19 × CD16]-directed recombinant diabody has been reported to trigger NK cell-mediated tumour cell lysis in vitro and in a mouse model (Kipriyanov et al, 2002).

CD16 is the low affinity receptor for IgG (FcγRIII), which is constitutively expressed as a transmembrane isoform on the surface of NK cells and macrophages (CD16a), and as a glycosyl phosphatidyl inositol (GPI)-anchored molecule on the surface of neutrophils (CD16b) (Ravetch & Kinet, 1991; van de Winkel & Anderson, 1991). For intracellular signalling, CD16a requires association with the FcRγ chain containing the immunoreceptor tyrosine-based activation motif (ITAM), which triggers downstream signalling. Studies with conventionally coupled bsAbs redirecting NK cells via CD16 demonstrated potent cytolysis of cultured malignant cells and in animal models (Garcia de Palazzo et al, 1992; Hombach et al, 1993; Kipriyanov et al, 2002). Therefore, clinical trials with CD16-directed bsAbs were initiated (Weiner et al, 1995; Hartmann et al, 1997). However, immunogenicity of hybrid-hybridoma antibodies, as well as undesired side effects caused by the presence of Fc-domains, and difficulties in producing sufficient amounts of clinical-grade material limited these clinical trials.

Lysis of CD19+ MRD cells by donor-derived NK cells, mediated by a chimaeric CD19 antibody, was previously reported (Lang et al, 2004). However, this chimaeric antibody is still afflicted with the limitations of conventional antibodies. To overcome some of these limitations, a recombinant bsscF was used instead of a whole antibody to eliminate the Fc-region, which was recognised as one of the causes of the problems in the past, and which is not strictly required for the present purpose. The instability of scFvs has been one of the main arguments precluding their use in therapeutic constructs to date. Therefore, a key innovation was utilised in the present study to enhance the serum stability of this recombinant bsscFv: disulphide bridges were introduced to stabilise both scFv components of our construct. The combination of these two advances promises to be useful, and in the preclinical studies reported here, ds[CD19 × CD16] triggered potent ADCC of lymphoma cell lines and primary human B-cell chronic lymphocytic leukaemia (B-CLL) and B-lineage ALL cells.

Materials and methods

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Antibodies and bispecific antibodies

The hybridoma cell line 3G8 (FcγRIII, CD16; mIgG1) (Fleit et al, 1982) was from the American Type Cell Culture Collection (ATCC, Manassas, VA, USA). The 4G7 hybridoma (CD19, mIgG1) (Meeker et al, 1984) was provided by Dr R. Levy (Stanford University, Palo Alto, CA, USA). The monoclonal antibodies used for detection of recombinant proteins were Penta-His (Qiagen, Hilden, Germany), horseradish peroxidase (HRP)-conjugated sheep anti-mouse IgG (Dianova, Hamburg, Germany), phycoerythrin (PE)-conjugated goat anti-mouse IgG (DAKO Diagnostica GmbH, Hamburg, Germany) and PE-conjugated donkey anti mouse IgG variable light (VL) + variable heavy (VH) (Dianova).

Culture of eukaryotic cells

Chinese hamster ovary (CHO) cells, stably transfected with a human CD16A cDNA expression construct, were provided by Dr Jan van de Winkel (University Medical Centre, Utrecht, The Netherlands). Leukaemia-derived SEM cells [t(4;11)-positive ALL], ARH-77 (mature B cell lymphoma; ATCC), and the hybridomas 3G8 and 4G7 were cultured in Roswell Park Memorial Institute (RPMI) 1640-Glutamax-I medium (Invitrogen, Karlsruhe, Germany), containing 10% fetal calf serum (FCS), 100 units/ml penicillin and 100 μg/ml streptomycin (RF10+-medium). Human 293T cells (ATCC) were cultured in Dulbecco's modified Eagles medium (DMEM)-Glutamax-I medium supplemented with 10% FCS, 100 units/ml penicillin and 100 μg/ml streptomycin.

Bacterial strains and plasmids

Escherichia coli XL-1-Blue (Stratagene, Amsterdam, The Netherlands) was used for the amplification of plasmids and cloning. The vector pSecTag2HygroC (Invitrogen) was used for expression in eukaryotic cells.

Construction of recombinant bsscFv [CD19 × CD16] and ds[CD19 × CD16]

To generate the expression vector for the bsscFv [CD19 × CD16], the CD19 4G7 scFv was excised from the vector pSecTag2HygroC-CD19 4G7-GFP (Peipp et al, 2004) as a SfiI cassette and inserted into the vector pSecTag2HygroC-Strep-CD16 (unpublished observations) linearised with SfiI, thus generating the vector pSecTag2HygroC-Strep-CD19 4G7 × CD16. Disulphide-stabilisation of the scFv components in the bsscFv was achieved by the introduction of cysteine residues into conserved framework regions (Reiter et al, 1994) in the vector pSecTag2HygroC-Strep-CD19 4G7 × CD16 using the Quikchange® Multi Site-Directed Mutagenesis Kit (Stratagene, Cedar Creek, TX, USA) following manufacturers instructions. Sequences were confirmed by dideoxynucleotide sequencing (Sambrook & Russel, 2001) on an Applied Biosystems automated DNA sequencer (ABI Prism 310 Genetic Analyzer; Perkin Elmer, Ueberlingen, Germany).

Expression and purification of bsscFv and GFP fusion proteins

BsscFvs and GFP fusion proteins were expressed in 293T cells. For this purpose, 10 μg of the expression vector were transfected using the calcium phosphate procedure including 5 mmol/l chloroquine (Sambrook & Russel, 2001). After 12 h, the transfection medium was replaced by fresh culture medium. Supernatants were collected every day for 5 d and dialysed at 4°C against a 2000-fold excess of a buffer containing 50 mmol/l NaH2PO4, 300 mmol/l NaCl, 10 mmol/l imidazole, pH 8·0. Purification of the 6 × His-tagged bsscFv was achieved by affinity chromatography with nickel-nitrilotriacetic acid (Ni-NTA) agarose beads (Qiagen) and a final dialysis against phosphate-buffered saline. Concentrations of the final purified proteins were determined by colorimetric assay using a Bradford Reagent (Sigma, Taufkirchen, Germany).

Expression of the chimaeric CD19 antibody

The CD19 4G7chim IgG1 antibody was expressed in Sf21 insect cells as previously published (Lang et al, 2004).

Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis

Reducing SDS-PAGE was carried out according to standard procedures (Sambrook & Russel, 2001). Gels were stained with Coomassie brilliant blue R250. BsscFvs were detected with a Penta–His antibody. Western blots were developed with secondary antibodies (sheep anti-mouse IgG coupled to horseradish peroxidase; Dianova), and developed using enhanced chemiluminescence reagents (Amersham Pharmacia Biotech, Freiburg, Germany).

Isolation of mononuclear cells, neutrophil effector cells, and CLL leukaemic cells

After receiving informed consent, 20 ml of peripheral blood was obtained from healthy volunteers, and both mononuclear cells (MNCs) and neutrophil effector cells (PMNs) were isolated as described (Elsasser et al, 1996). The purity of PMNs and MNCs was assessed by cytospin preparations and exceeded 95%. Viability of cells was >95%, as tested by trypan blue exclusion. Tumour cells from patients with CD5/CD19 positive CLL were isolated from citrate-buffered peripheral blood by centrifugation over Ficoll.

Paediatric leukaemia patients

Blood samples were obtained from three paediatric patients with acute leukaemias after transplantation of T cell-depleted grafts from unrelated human leucocyte antigen matched allogeneic or haploidentical donors. The myeloablative conditioning regimes were based on total body irradiation. Patients received a median of 12 × 106 CD34+ progenitor cells/kg of body weight. T cells were depleted on average by five logs, with less than 25·000 residual CD3+ cells in the grafts. No regular post-transplant pharmacological immunosuppression was administered. Ficollised MNCs were used as effector cells for ADCC reactions.

Positive selection of CD56+ cells

Peripheral MNC were enriched for NK cells by immuno-magnetic separation with CD56+ microbeads as previously described (Lang et al, 2002).

Flow cytometry

Immunofluorescence staining was performed as previously published on a FACSCalibur instrument using CellQuest software (Becton Dickinson) (Bruenke et al, 2004). For each sample, 1 × 104 events were collected and analyses of whole cells were performed using appropriate scatter gates to exclude cellular debris and aggregates. Reconstitution of CD3+, CD4+, CD8+, CD19+ and CD56+ lymphocytes after transplantation was assessed weekly by fluorescence-activated cell sorting (FACS) analysis until T cell recovery began and was subsequently assessed every 3 months.

Determination of affinity constants (KD) for antibodies and scFv components by flow cytometry

Determination of the affinity constants (KD) of the 4G7 antibody and both scFv components of the bsscFv, 4G7 and 3G8, was performed by flow cytometry using published procedures (Benedict et al, 1997). Experiments for the determination of antibody KD values were repeated three to four times and mean values are reported. Values and graphical analyses were generated using GraphPad Prism Software (GraphPad Software Inc., San Diego, CA, USA).

ADCC and complement-dependent cytotoxicity (CDC) assays

ADCC assays were performed as described (Elsasser et al, 1996; Lang et al, 2004). For analysis of effects induced by Fc-receptor blockade, antibody 3G8 F(ab)2 (FcγRIII,CD16) was added at a concentration of 10 μg/ml. Relative inhibition was calculated as follows: % inhibition = (% lysis without blocking antibody –% lysis with blocking antibody)/(% lysis without blocking antibody) × 100.

Statistical analyses

Group data are reported as mean ± SEM. Differences between groups were analysed by unpaired (or, when appropriate, paired) Student's t-test. P-values < 0·05 were considered significant.

The experiments reported here were approved by the Ethics Committee of the University of Erlangen-Nuremberg and the University of Tuebingen, in accordance with the Declaration of Helsinki.

Results

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Generation, expression and purification of the recombinant bsscFv ds[CD19 × CD16]

To redirect NK cells against malignant B cells, a recombinant tandem bsscFv was constructed targeting CD19 on malignant B-lymphoid cells and FcγRIII (CD16) on NK cells. To further enhance the stability of this recombinant protein, a stabilised form of this bsscFv ds[CD19 × CD16] was generated, in which each scFv component was stabilised by the introduction of a disulphide bond bridging the corresponding VL and VH chains (Fig 1A).

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Figure 1. Design and purification of the recombinant bsscFv ds[CD19 × CD16]. (A) Block-structure of the expression vector for the bsscFv. CMV, cytomegalovirus early promotor; Igκ, secretion leader sequence from the murine Ig kappa light chain; VL, VH, cDNA-segments coding for the immunoglobulin light chain or heavy chain variable regions, respectively; L, cDNA coding for a 20 amino acid flexible linker (Gly4Ser)4; Strep, c-myc, 6 × His, cDNA coding for a strep, c-Myc or a hexahistidine tag; stabilising S-S, disulphide bond. (B) Western blot analysis of the bsscFv after affinity chromatography with Ni-NTA agarose beads. Lanes 1, 2: consecutive elution fractions–revealed with an anti-His-tag antibody. (C) Evaluation of the purity and integrity of the purified bsscFv by SDS-PAGE and staining with Coomassie blue: St: size marker; lanes 1, 2: consecutive elution fractions from Ni-NTA agarose beads.

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Recombinant bsscFvs were expressed in 293T cells. The secreted bsscFvs were collected from culture supernatants and purified by affinity chromatography with Ni-NTA agarose beads. Protein analysis by SDS-PAGE showed a protein of Mr = 58–60 kDa, in close agreement with the calculated molecular mass Mr = 59·2 kDa (Fig 1B). The purified protein was intact, and neither higher molecular weight aggregates nor degradation products were observed. The purity of the enriched protein was estimated from Coomassie stained gels (Fig 1C). Yields ranged from 1 to 1·5 mg of purified bsscFv per litre of culture supernatant.

Binding and stability characteristics of the recombinant bsscFv ds[CD19 × CD16]

The recombinant bsscFv ds[CD19 × CD16] retained the ability to bind to each of the two antigens CD19 and CD16, as evidenced by its ability to specifically bind to the corresponding single-antigen-positive cells (Fig 2A). To demonstrate simultaneous binding of both scFv components within the same molecule, additional experiments were performed. CD19-positive SEM cells were incubated with the bsscFv and stained with a recombinant fusion protein, consisting of the extracellular domain of CD16 (CD16ex) linked to the green fluorescent protein (GFP) (CD16ex-GFP). In flow cytometry experiments, a fluorescent GFP signal was observed with CD16ex-GFP, but not with a control GFP-fusion protein (Fig 2B, i). Thus, both scFv-moieties of one bsscFv-molecule were capable of binding simultaneously to their respective antigens. This result was further supported by competition experiments. Incubation with a molar excess of one of the parental antibodies, directed against CD19 (Fig 2B, ii) or CD16 (Fig 2B, iii), resulted in a decrease of the fluorescence signal to baseline levels. Competition with a non-relevant antibody, added in the same molar excess, did not alter the fluorescence signal. Therefore, both binding sites of the bsscFv were antigen-specific.

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Figure 2. Specific and simultaneous antigen binding of the bsscFv ds[CD19 × CD16]. (A) Specificity of binding. Flow cytometric analyses of the bsscFv binding to (i) CD19-positive cells; (ii) CD19-negative cells; (iii) CD16-transfected cells; (iv) untransfected control cells. Black peaks: signals obtained with the bsscFv; white peaks: the signals obtained with a non-relevant scFv. (B) Simultaneous antigen binding of both scFv components contained in the bsscFv. Flow cytometry analyses were performed with the bsscFv on CD19-positive cells. Binding of the bsscFv was revealed by adding a fusion protein consisting of the extracellular domain of CD16 fused to GFP (CD16ex-GFP) (i); black peaks: fluorescent signal produced by CD16ex-GFP; white peaks: fluorescent signal after addition of a non-relevant GFP-fusion protein. The CD16ex-GFP-signal was blocked by addition of a 100-fold molar excess of the parental CD19 and CD16 antibodies 4G7 (ii); and CD16 3G8, respectively (iii); addition of a non-relevant antibody produced no reduction in fluorescence intensity (iv).

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To assess, whether stabilisation of the scFv components in the bsscFv resulted in an affinity alteration, binding of the scFv components was measured and compared with the unstabilised counterpart. For this purpose, KD was determined as the antibody concentration, at which half maximal binding (half-maximal fluorescence intensity associated with the cells) was reached (Table I). The ds[CD19 × CD16] displayed affinities in the same nanomolar range as the unstabilised counterpart. Thus, stabilisation had not affected the binding affinities to both antigens, CD19 and CD16 respectively. Furthermore, affinities of the scFvs incorporated into the bsscFv were in the range of other published recombinant bispecific antibodies (Kipriyanov et al, 1998; McCall et al, 1999; Bruenke et al, 2004).

Table I. Flow cytometry-based measurement of affinities for both CD19 and CD16 scFv components in the unstabilised [CD19 × CD16] and disulphide bond-stabilised bsscFv ds[CD19 × CD16] calculated by antibody concentrations, at which half maximal binding was observed (n = 3).
Antibody K D (M)
CD19 scFv componentCD16 scFv component
[CD19 × CD16]4·1 × 10−86·1 × 10−8
ds[CD19 × CD16]3·8 × 10−85·5 × 10−8

A critical and important factor contributing to the therapeutic usefulness of recombinant antibodies is their stability. Therefore, the plasma stability of the ds[CD19 × CD16] was investigated and compared with the unstabilised [CD19 × CD16] counterpart (Fig 3). For this purpose, both bsscFv-constructs were incubated in human serum at 37°C for prolonged periods of time. At different time points residual binding was quantified by flow cytometry. The half-lifes of the binding sites in the unstabilised bsscFv determined by this method were t1/2 = 18 h and t1/2 = 40 h for the CD19 and the CD16 scFv components respectively. In contrast, the ds[CD19 × CD16] retained ≈60% of its antigen binding capacity after 96 h for the CD19 scFv moiety and >90% for the CD16 scFv respectively. Thus, the introduction of a disulphide bond into each scFv of the bsscFv resulted in a more than threefold increase of serum stability.

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Figure 3. Serum stability of the disulphide-stabilised bsscFv ds[CD19 × CD16] in comparison with the unstabilised bsscFv. The unstabilised and stabilised [CD19 × CD16] bsscFvs were incubated at subsaturating concentrations of 1 μg/ml in human serum at 37°C for prolonged periods of time. The residual binding activity was estimated by flow cytometry on antigen-positive cells. (A) Stability of the CD19-scFv component in the unstabilised (○) and the stabilised (•) bsscFv. (B) Stability of the CD16-scFv component in the unstabilised (○) and the stabilised (•) bsscFv. All data are normalised to time-point t0 = 100%. Significant values of P < 0·05 are indicated by an asterisk (*). Data are presented as mean percentage of residual binding ± SEM of five independent experiments.

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The recombinant bsscFv ds[CD19 × CD16] mediates effector cell lysis (ADCC)

For functional studies, the CD19-positive mature B-cell lymphoma line ARH-77 was used as a target in 3 h 51Cr release assays with freshly isolated, unstimulated MNCs as effectors. The ds[CD19 × CD16] triggered specific lysis of target cells in a dose-dependent manner with optimal concentrations in the range of 0·4–2 μg/ml (Fig 4A), while the parental murine CD19 antibody was unable to induce ADCC. The bsscFv displayed effector cell-mediated cytotoxicity against target cells over a broad range of effector-to-target cell ratios down to ratios of total MNCs to target cells of 2·5:1. Target cell lysis in the presence of the parental murine CD19 antibody was not observed. ADCC of target cells by MNCs in the absence of any added antibody construct was only observed at high E/T ratios and specific lysis was always <10% (Fig 4B). Thus, antibody binding to CD19 alone is not sufficient to induce target cell lysis.

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Figure 4. ADCC against CD19-positive tumour cells: role of concentration and E/T ratio. ADCC against ARH-77 target cells mediated by the bsscFv and freshly isolated human peripheral MNCs as effector cells. (A) At a constant E/T ratio of 40:1, bsscFv-mediated lysis was concentration-dependent and saturable (black bars). The parental murine CD19 antibody used as a control was unable to induce ADCC (grey bars). (B) In the presence of constant amounts of 0·4 μg/ml of ds[CD19 × CD16] (black bars), the extent of specific lysis increased with the E/T ratio in an expected, saturable fashion. ‘Effector cells’ refers to total number of MNCs, of which only about 10% were NK cells. White bars: without antibody; grey bars: parental murine CD19 antibody. Significant values of P < 0·05 are indicated by an asterisk (*). Data are presented as mean percentage lysis ± SEM observed with isolated MNCs from at least three different donors.

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To identify the effector population responsible for this cytotoxicity, peripheral blood from healthy donors was fractionated into MNCs and PMNs and tested in ADCC reactions. Only the MNC fraction, containing the NK cells, demonstrated significant lysis of ARH-77 cells in the presence of the ds[CD19 × CD16], whereas PMNs were inactive. As expected by the design, no complement-dependent cytotoxicity (CDC) was observed when using the plasma fraction. Thus, the cell population mediating ADCC was enriched in the MNC fraction, suggesting that CD16a-positive NK cells were the effectors (data not shown).

To further confirm that lysis was dependent on CD16, blocking experiments were performed. In the presence of ds[CD19 × CD16], MNC-mediated lysis of ARH-77 cells was significantly inhibited by addition of F(ab)2 fragments of FcγRIII antibody 3G8 (83·9% ± 6·6%, Fig 5). Furthermore, neither the FcγRIII-directed F(ab)2 fragment nor a recombinant control bsscFv that also binds to CD16, but not to ARH-77 cells, triggered MNC-mediated lysis of target cells (Fig 5). Thus, non-specific activation of NK cells by ds[CD19 × CD16] was not observed under our experimental conditions.

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Figure 5. ADCC of ARH-77 cells is FcγRIII dependent. The bsscFv ds[CD19 × CD16] triggered the MNC-mediated lysis of ARH-77 target cells at concentrations of 2 μg/ml. Target cell lysis was significantly inhibited (83·9% ± 6·6%) by addition of F(ab)2 fragments of FcγRIII antibody 3G8 at concentrations of 10 μg/ml. Neither the 3G8 F(ab)2 fragments nor a CD16-directed control bsscFv at concentrations of 2 μg/ml were able to induce MNC-mediated lysis. MNCs as effectors also failed to trigger ADCC in the absence of antibody. Significant values of P < 0·05 are indicated by an asterisk (*). Data are presented as mean percentage lysis ± SEM observed with isolated MNCs from at least four different donors.

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Cytotoxic activity of the ds[CD19 × CD16] and a chimaeric CD19 antibody against primary CD19-positive B-CLL cells

Primary tumour cells are generally more difficult to lyse than lymphoma cell lines. Therefore, the ds[CD19 × CD16] was tested in ADCC reactions against freshly isolated and cryopreserved CD19-positive B-CLL cells, derived from bone marrow or peripheral blood, and compared with the corresponding chimaeric IgG1 CD19 antibody. Significant lysis of patient samples (P < 0·05) was observed in the presence of either the recombinant bispecific in all six cases or the chimaeric CD19 4G7chim antibody (4/6 cases), whereas effector cell-mediated lysis without antibody was consistently low (Fig 6).

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Figure 6. Lysis of primary B-CLL cells by bsscFv ds[CD19 × CD16]. CD5/CD19-positive human B-CLL cells were isolated from bone marrow (BM) or peripheral blood lymphocytes (PBL) of six different patients. Antibody-dependent lysis (P < 0·05 indicated by *) of B-CLL cells was evaluated in ADCC reactions using the recombinant bsscFv ds[CD19 × CD16] at concentrations of 0·4 μg/ml (black bars) or the corresponding chimaeric CD19 antibody (CD19 4G7chim) at concentrations of 1·5 μg/ml (open bars). Assays were performed with MNC effector cells from two different healthy donors at an E/T ratio of 40:1. The ds[CD19 × CD16] mediated the lysis of leukaemic cells from all six patients, while the chimaeric CD19 antibody induced ADCC in four of six patient samples. Data are presented as mean percentage lysis ± SEM. No attempt was made to search for, or avoid, MHC class I matching between effector and target cells.

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Cytotoxic activity of the ds[CD19 × CD16] and chimaeric CD19 antibody against primary CD19-positive ALL blasts

Furthermore, the ds[CD19 × CD16] produced specific lysis of primary cryopreserved CD19-positive ALL-blasts from paediatric patients in ADCC reactions mediated by enriched NK cells from unrelated healthy donors at different E/T ratios (Fig 7A). Although the donor/target pairs produced different levels of spontaneous lysis, NK cells from each donor mediated significantly enhanced lysis (P < 0·05) in the presence of both, bispecific ds[CD19 × CD16] or the chimaeric CD19 antibody. Under these experimental conditions at an E/T ratio of 20:1, specific lysis mediated by the ds[CD19 × CD16] was somewhat less efficient than by the chimaeric IgG1. Lytic activity <10% of NK cells alone was only observed at an E/T ratio of 20:1. As expected, NK cell-mediated cytotoxicity was not significantly enhanced in the presence of the murine 4G7 IgG1 hybridoma antibody, used as a control. Stimulation of NK cells with interleukin-2 at 40 units/ml further increased specific lysis mediated by ds[CD19 × CD16] or the chimaeric CD19 antibody (data not shown). Thus, both CD19-directed antibody constructs, ds[CD19 × CD16] and the chimaeric CD19 antibody, mediated enhanced ADCC against patient ALL blasts in the presence of NK cells from different unrelated donors.

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Figure 7. The recombinant ds[CD19 × CD16] mediates effector cell lysis of primary B-ALL blasts. (A) The recombinant ds[CD19 × CD16] induced ADCC of cryopreserved B-ALL blasts by enriched NK cells from different healthy donors at different E/T ratios. Lysis of target cells (<10%) by NK cells alone was only observed at an E/T ratio of 20:1. Significantly enhanced specific lysis (P < 0·05) was observed in the presence of the recombinant bsscFv ds[CD19 × CD16] at concentrations of 0·4 μg/ml or the chimaeric CD19 4G7chim at saturating concentrations of 0·15 μg/ml. Killing mediated by the ds[CD19 × CD16] was less efficient than by the chimaeric 4G7 under these experimental conditions, while the murine CD19 hybridoma antibody 4G7 triggered no ADCC. Data are presented as mean percentage lysis ± SEM observed with enriched NK cells from three donors. (B) MNCs isolated from three different patients after CD34+ stem cell transplantation mediated significant specific lysis (P < 0·05) of cryopreserved B-ALL blasts in the presence of ds[CD19 × CD16] or CD19 4G7chim. ADCC at different E/T ratios using ds[CD19 × CD16] at 0·4 μg/ml, or CD19 4G7chim at saturating 0·15 μg/ml. Lysis mediated by the ds[CD19 × CD16] was consistently stronger than lysis by CD19 4G7chim. No specific lysis was observed when a CD16-directed control bsscFv or MNCs alone were used. Significant differences (P < 0·05) between ds[CD19 × CD16] and CD19 4G7chim are indicated by (#). Data are presented as mean percentage lysis ± SEM observed with isolated MNCs from three different patients after transplantation.

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The recombinant bsscFv ds[CD19 × CD16] mediates specific lysis of primary leukaemic blasts in the presence of effector cells from transplanted patients

To address the question, whether donor-derived effector cells were capable of lysing primary leukaemic blasts in conjunction with our bispecific antibody, the ADCC-inducing potential of ds[CD19 × CD16] was investigated in ADCC reactions and compared with the chimaeric CD19 antibody. For this purpose, MNCs from three different patients were isolated after CD34+ stem cell transplantation and tested against cryopreserved ALL blasts at different E/T ratios (Fig 7B) in the presence of either the recombinant bsscFv ds[CD19 × CD16] or the chimaeric CD19 antibody. Although, the donor/target pairs produced different levels of effector cell-mediated lysis, both, ds[CD19 × CD16] and CD19 4G7chim, triggered significantly enhanced (P < 0·05) lysis of leukaemic blasts, while MNCs alone or a CD16-directed control bsscFv did not trigger cellular lysis. In this experimental setting at an E/T ratio of 20:1, killing obtained with the bsscFv was significantly more effective than with CD19 4G7chim (P < 0·05). The lytic activity was ascribed to NK cells, because the extent of ADCC obtained with MNCs was proportional to the fraction of CD56+/CD16+ cells in this mixture (data not shown). Thus, donor-derived effector cells from patients after transplantation were capable of mediating high lytic activity against leukaemic blasts in the presence of ds[CD19 × CD16].

Discussion

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

The main results of this report are: (a) a recombinant bispecific scFv molecule in the tandem format has an increased serum stability after disulphide-stabilisation of both of its scFv components; (b) the disulphide-stabilised bsscFv was more efficient in mediating specific lysis of primary ALL-blasts by donor-derived MNCs after transplantation than the chimaeric IgG1 antibody CD19 4G7chim.

One of the perceived shortcomings of scFv and bsscFv-proteins for clinical applications is their instability in human serum. BsscFvs usually have a serum stability of only a few hours to a few days, as demonstrated also by the non-stabilised controls in this study (Fig 3). To overcome this limitation, disulphide-stabilised Fvs have been generated. It had previously been shown that disulphide-stabilisation significantly increased the serum stability of individual scFv-molecules (Brinkmann et al, 1993). However, to our knowledge, there are no prior reports demonstrating that the simultaneous stabilisation of two different scFv components in a tandem format bsscFv increases the serum stability of the entire molecule.

In ADCC experiments with malignant cells obtained either from the bone marrow (BM) or peripheral blood (PBL) of six different adult B-CLL patients, MNCs from unrelated healthy human donors were used as effectors (Fig 6). Although no deliberate effort was made to ensure a mismatch of their MHC class I molecules, lysis was significantly enhanced by addition of the bsscFv molecule in all six cases. Furthermore, the lytic effect was consistently higher with the bsscFv molecule than with the chimaeric CD19 antibody, regardless of whether the malignant cells were taken from the bone marrow or peripheral blood (Fig 6). In four of six cases (patients 1, 2, 3 and 5) the cytotoxic effects obtained with the bsscFv were more than twice as large as those obtained with the chimaeric CD19 antibody. It is presently unknown whether this advantage will also hold true in human patients, but we anticipate certain advantages over the chimaeric CD19 antibody, including improved tissue penetration due to its smaller size.

Normally, lytic ability of NK cells is impaired by high expression levels or expression of matched MHC class I molecules on target cells. Apparently, this inhibitory effect, which is caused by killer inhibitory receptors (KIR) on the surface of NK cells, is overcome in ADCC situations. Here, the lysis-promoting effect produced by the therapeutic antibody apparently compensates the killer inhibitory effects (Lang et al, 2004). Rather large variations in the extent of lysis observed for individual pairs of tumour and effector cells (Fig 6) may be partly due to the variable extent of MHC class I mismatch between the target and effector cells. Other potential causes for this observation may include variations in the expression levels of activating NK cell receptors, such as NKG2D, DAP10 or DAP12.

The ADCC experiments with primary leukaemic ALL blasts from paediatric patients with the bsscFv in comparison with the chimaeric antibody produced an unexpected observation. When unstimulated enriched NK cells from healthy unrelated donors were used as effectors (Fig 7A), the chimaeric CD19 antibody produced somewhat higher lysis than the bsscFv. However, when unstimulated donor-derived MNCs from transplanted patients were used, then the bsscFv produced a greater extent of lysis (Fig 7B). In this situation, MNCs were used and no attempt was made to enrich NK cells, because the amount of blood cells available from transplanted paediatric patients was too limited to permit enrichment of NK cells. An explanation for this observation might be that transplanted patients regularly received standard human IgG infusions (200 mg/kg every 3 weeks) in order to prevent infectious complications. Such infusions are likely to block human Fc receptors as a side effect, and therefore may reduce free binding sites for the chimaeric antibody on the effector cells. By contrast, it is conceivable that the function of the bsscFv is not inhibited under these conditions, because the CD16-specific reading head binds FcγRIII at an epitope outside its Fc-binding site. This experimental setting using donor-derived MNCs comes closest to the in vivo situation for which our construct was primarily designed, namely the treatment of MRD cells in a post-transplantation setting. In this situation, the bsscFv format had superior properties over the chimaeric antibody, which remain to be confirmed by clinical investigations.

CD19 has long been recognised as a potentially very useful target antigen for the therapy of B-lymphoid malignancies, because of its restriction to the B cell lineage. It is expressed on most B-lineage ALLs, including infant pro-B ALLs, which usually lack CD20, and therefore appears to be particularly attractive for the treatment of CD20-negative paediatric leukaemias. Consequently, CD19-directed antibodies have been investigated for therapeutic use against human B-lymphoid malignancies, but until now, therapeutic IgG antibodies have not produced clinical benefits comparable with those of CD20 antibodies (Hekman et al, 1991). In addition, conventional bsAbs targeting CD19 were generated for the recruitment of T cells via CD3. These bsAbs were effective in vitro (Bohlen et al, 1993a,b; Haagen et al, 1994; Csoka et al, 1996) and in animal models (Demanet et al, 1992; Bohlen et al, 1997; Daniel et al, 1998), but not in first clinical trials (De Gast et al, 1995; Haagen, 1995).

More recently, recombinant bsscFvs comprising only antibody variable domains have been constructed. These molecules are expected to be less immunogenic than complete antibodies and can be produced at relatively high yields in a more defined final state. The currently most advanced recombinant protein in this format is a [CD19 × CD3] bsscFv (Loffler et al, 2000). For the particular purpose of our work, the elimination of MRD cells in paediatric ALL patients after transplantation of T cell-depleted grafts, T cells clearly are not the ideal class of effector cells, because of their delayed reconstitution (Eyrich et al, 2001). NK cells and granulocytes show much faster reconstitution, and, therefore, CD16 was the more promising choice of a trigger molecule on the available population of effector cells: the NK cells. It is also important to note that there are few MRD cells in the first few months after transplantation, and high effector-to-target cell ratios can be achieved.

CD16 has been appreciated by other groups as a potent trigger molecule on the surface of NK cells (Gessner et al, 1998). CD16 antibodies, such as 3G8 used in this study, bind FcγRIII outside of the Fc-binding pocket, activate NK cells, and induce cytotoxic responses. Some properties of CD16 may also limit its use as a trigger molecule. Among these is the inability of CD16 antibodies to discriminate between the CD16a isoform present on NK cells and macrophages, which is capable of triggering a cytolytic response, and the GPI-linked CD16b isoform present on neutrophilic granulocytes, which is unable to mediate ADCC. In addition, soluble CD16 is present in considerable concentrations in human plasma (Koene et al, 1996) and may compete with FcγRIII on the surface of NK cells. However, CD16-directed bsAbs have been successfully used in clinical trials, although none have advanced past stage II and none have yet been approved for clinical application. Interestingly, the cytotoxicity of these bsAbs was not inhibited by the presence of CD16-positive PMNs in ADCC assays, a still unexplained observation (Weiner et al, 1996). In addition, the in vivo cytotoxicity of CD16-directed bsAbs was not compromised by competition with CD16b on neutrophils. This effect was also observed in preclinical studies and phase I/II clinical trials of patients with refractory Hodgkin's disease treated with a [CD30 × CD16] bsAb (Hartmann et al, 2001). These reported properties of CD16-directed bsAbs provided the basis for our current study and it is anticipated that recombinant bsscFv constructs directed against CD16 as the trigger molecule may be therapeutically useful. Based on the results of this study, we concluded that the format of the tandem bsscFv may have distinct advantages over conventional bsAbs used to date. In fact, this particular format allows investigators to fully exploit the perceived benefits of CD19 as a target molecule, which had remained below expectations when other formats of CD19-directed antibody-derived therapeutics were used. The data presented here provide a clear impetus for further in vivo evaluation of [CD19 × CD16] bsscFvs.

Acknowledgements

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

We thank Dr R. Levy for the 4G7 hybridoma, Dr J.G. van de Winkel for the CD16-transfected CHO cells, D. Saul, B. Bock and A. Babarin-Doner for excellent technical assistance. Th. Lange is gratefully acknowledged for administrative assistance. Supported by research grant DFG RE 1276/2-1 to R. Repp, T. Valerius and G.H. Fey from the Deutsche Forschungsgemeinschaft (DFG) and the association of supporters of the University of Erlangen Children's Hospital (bequest of Dr Wilhelmine Fey).

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  5. Discussion
  6. Acknowledgements
  7. References
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