Domenica Saul

A recombinant trispecific single-chain Fv derivative directed against CD123 and CD33 mediates effective elimination of acute myeloid leukaemia cells by dual targeting

Two trivalent constructs consisting of single‐chain Fv antibody fragments (scFvs) specific for the interleukin‐3 receptor α chain (CD123), CD33 and the Fcγ‐receptor III (CD16) were designed and characterized for the elimination of acute myeloid leukaemia (AML) cells. The dual targeting single‐chain Fv triplebody (sctb) [123 × ds16 × 33] and the mono targeting sctb [123 × ds16 × 123] both specifically bound their respective target antigens and were stable in human serum at 37°C for at least 5 d. Both constructs induced potent antibody‐dependent cellular cytotoxicity (ADCC) of two different AML‐derived CD33‐ and CD123 double‐positive cell lines in the low picomolar range using isolated mononuclear cells (MNCs) as effector cells. In these experiments the dual targeting molecule produced significantly stronger lysis than the mono targeting agent. In addition, the sctbs showed a high potency in mediating ADCC of primary leukaemia cells isolated from peripheral blood or bone marrow of seven AML patients. Hence, these novel molecules displayed potent anti‐leukaemic effects against AML cells in vitro and represent attractive candidates for further preclinical development.

Mitochondrial metabolism contributes to oxidative stress and reveals therapeutic targets in chronic lymphocytic leukemia

Alterations of cellular metabolism represent a hallmark of cancer. Numerous metabolic changes are required for malignant transformation, and they render malignant cells more prone to disturbances in the metabolic framework. Despite the high incidence of chronic lymphocytic leukemia (CLL), metabolism of CLL cells remains a relatively unexplored area. The examined untreated CLL patients displayed a metabolic condition known as oxidative stress, which was linked to alterations in their lymphoid compartment. Our studies identified mitochondrial metabolism as the key source for abundant reactive oxygen species (ROS). Unlike in other malignant cells, we found increased oxidative phosphorylation in CLL cells but not increased aerobic glycolysis. Furthermore, CLL cells adapted to intrinsic oxidative stress by upregulating the stress-responsive heme-oxygenase-1 (HO-1). Our data implicate that HO-1 was, beyond its function as an antioxidant, involved in promoting mitochondrial biogenesis. Thus ROS, adaptation to ROS, and mitochondrial biogenesis appear to form a self-amplifying feedback loop in CLL cells. Taking advantage of the altered metabolic profile, we were able to selectively target CLL cells by PK11195. This benzodiazepine derivate blocks the mitochondrial F1F0-ATPase, leads to a surplus production of mitochondrial superoxide, and thereby induces cell death in CLL cells. Taken together, our findings depict how bioenergetics and redox characteristics could be therapeutically exploited in CLL.

Stabilization and humanization of a single-chain Fv antibody fragment specific for human lymphocyte antigen CD19 by designed point mutations and CDR-grafting onto a human framework

A single-chain Fv (scFv) fragment derived from the murine antibody 4G7, specific for human lymphocyte CD19, was engineered for stability and expression in Escherichia coli in view of future use as a therapeutic protein. We compared two orthogonal knowledge-based procedures. In one approach, we designed a mutant with 14 single amino-acid substitutions predicted to correct destabilizing residues in the 4G7-wt sequence to create 4G7-mut. In the second variant, the murine CDRs were grafted to the human acceptor framework huVkappa3-huV(H)3, with 11 additional point mutations introduced to obtain a better match between CDR graft and acceptor framework, to arrive at 4G7-graft. Compared to 4G7-wt, 4G7-mut showed greater thermodynamic stability in guanidinium chloride-induced equilibrium denaturation experiments and somewhat greater stability in human serum. The loop graft maintained the comparatively high stability of the murine loop donor, but did not improve it further. Our analysis indicates that this is due to subtle strain introduced between CDRs and framework, mitigating the otherwise highly favorable properties of the human acceptor framework. This slight strain in the loop graft is also reflected in the binding affinities for CD19 on leukemic cells of 8.4 nM for 4G7-wt, 16.4 nM for 4G7-mut and 30.0 nM for 4G7-graft. This comparison of knowledge-based mutation and loop-grafting-based approaches will be important, when moving molecules forward to therapeutic applications.

A single-chain triplebody with specificity for CD19 and CD33 mediates effective lysis of mixed lineage leukemia cells by dual targeting

A single-chain triplebody (sctb) 33-ds16-ds19 comprising two distal single-chain Fv fragments (scFvs) specific for the lymphoid antigen CD19 and the myeloid antigen CD33 flanking a central scFv specific for CD16, which is the low affinity Fc-receptor (FcγRIII) present on natural killer cells and macrophages, was produced and its properties were investigated. CD33 and CD19 in combina-tion are present on acute leukemiablasts with mixed lineage phenotype, but not on normal human hematopoietic cells. For comparison, two bispecific scFvs (bsscFvs), ds19-ds16 and 33-ds16, with monovalent binding to CD19 and CD33, respectively, were also studied. The sctb 33-ds16-ds19 specifically interacted with all 3 antigens. On the antigen double-positive cell line BV-173, the sctb bound with 2-fold greater avidity than bsscFv ds19-ds16 (KD = 21 vs. 42 nM) and with 1.4-fold greater avidity than bsscFv 33-ds16 (KD = 29 nM). All 3 fusion proteins had similar affinity for CD16 and sufficient thermic stability in human serum. In antibody-dependent cellular cytotoxicity (ADCC) reactions with human mononuclear cells as effectors, the sctb promoted lysis of BV-173 cells at 23-fold lower concentrations than bsscFv ds19-ds16 and at 1.4-fold lower concentrations than bsscFv 33-ds16. The sctb also mediated potent ADCC of the antigen double-positive mixed lineage leukemia cell line SEM, and the half-maximal concentration EC50 for BV-173 cells was 7 pM. Therefore, CD19 and CD33 are present on the surface of these leukemic cell lines such that they can be connected by a single sctb molecule, permitting the recruitment of NK cells via CD16 and tumor cell lysis.

A CD33‐specific single‐chain immunotoxin mediates potent apoptosis of cultured human myeloid leukaemia cells

A novel single‐chain immunotoxin was constructed by combining a CD33‐specific single chain Fv (scFv) antibody fragment with an engineered variant of Pseudomonas exotoxin A (ETA). The variant toxin carries the KDEL peptide at its C‐terminus, a cellular peptide mediating improved retrograde transport to the endoplasmic reticulum. The purified recombinant fusion protein induced potent apoptosis of the human myeloid cell lines U937, HL‐60 and THP‐1. Up to 98% of U937 cells were eliminated after treatment for 72 h with a single dose of 500 ng/ml (c. 7 nmol/l). Killing was antigen‐specific and occurred by apoptosis. A control protein, consisting of a CD19‐specific scFv antibody fragment fused to the ETA‐KDEL toxin, failed to induce death of the CD19‐negative cell lines U937, HL‐60 and THP‐1. The CD33‐ETA toxin also mediated apoptosis of fresh patient‐derived acute myeloid leukaemia cells from bone marrow and peripheral blood. The pronounced antigen‐restricted cytotoxicity of the novel fusion protein makes it a candidate for further evaluation of its therapeutic potential.

Influence of variable N-glycosylation on the cytolytic potential of chimeric CD19 antibodies.

To investigate the influence of N-linked oligosaccharides at asparagines-297 on the cytolytic potential of chimeric CD19 antibodies, three distinct variants were generated by production in different expression systems. The same chimeric CD19 antibody was produced in Sf21 insect cells, human 293 T cells, and 293 T cells expressing a co-transfected β1,4-N-acetylglucosaminyltransferase III (GnTIII). The N-glycan structures and the cytolytic potential of the antibodies produced in these three systems were directly compared. After expression in insect cells, the antibody carried paucimannosidic N-linked oligosaccharides, distinct from the complex biantennary carbohydrate moieties attached to the product from human cells. After co-expression with GnTIII in human cells, the antibody carried an eightfold greater percentage of oligosaccharides with a bisecting N-acetylglucosamine (78.7% versus 9.6%) and a 30-fold increased proportion of bisecting, defucosylated oligosaccharides (15.9% versus 0.5%). The insect cell product triggered stronger antibody-dependent cellular cytotoxicity (ADCC) of a human leukemia–derived cell line than the product from non-re-engineered 293 T cells and was equally effective at 50- to 100-fold lower concentrations. The antibody from glyco-engineered 293 T cells had comparable lytic activity as the insect cell product. Both mediated significant ADCC at lower effector-to-target cell ratios than the antibody from non-re-engineered 293 T cells, and both were highly effective against primary blasts from pediatric leukemia patients. The data demonstrate the influence of the N-glycosylation pattern on the ADCC activity of chimeric CD19 antibodies and point to the importance of suitable expression systems for the production of highly active therapeutic antibodies.

A recombinant triplebody with specificity for CD19 and HLA-DR mediates preferential binding to antigen double-positive cells by dual-targeting

To test the hypothesis that dual-targeting confers the novel ability of selective binding to antigen double-positive over antigen single-positive cells, a single-chain triplebody (sctb), HLA-ds16-hu19, was produced and characterized. The molecule carries three single-chain Fv (scFv) antibody fragments in a single polypeptide chain, the two distal ones specific for the human histocompatibility protein HLA-DR and the B-lymphoid cell surface protein CD19, the central one for CD16, the human low affinity Fc-receptor FcγRIII. For comparison, the bispecific scFvs (bsscFv) hu19-ds16 and HLA-ds16 were also produced. All CD16 binding modules are disulfide-stabilized (ds). The sctb bound simultaneously to both CD19 and HLA-DR on the same cancer cell and, thus, showed functional dual-targeting. In a mixing-experiment with HLA-DR single-positive HUT-78 cells and (HLA-DR plus CD19) double-positive SEM cells, the triplebody showed preferential binding to the double-positive cells, even when the single-positive cells were present in a numerical excess of up to 20-fold. In antibody-dependent cellular cytotoxicity experiments with mononuclear cells as effector cells, the sctb promoted equal lysis of Raji cells, an antigen double-positive cell line, at 130-fold lower concentrations than the bsscFv hu19-ds16, indicating that both distal scFvs of the sctb contributed to tumor cell lysis. A panel of stably-transfected HEK293 cell lines was generated that included CD19- and HLA-DR single-positive and (HLA-DR plus CD19) double-positive lines with antigen-surface densities varying over a broad range. Using a pair of cell lines with matching densities, the sctb eliminated double-positive target cells preferentially single-positive cells. This ability of preferential or selective targeting of antigen double-positive over single-positive cells opens attractive new perspectives for the use of dual-targeting sctbs in cancer therapy.

A dual-targeting triplebody mediates preferential redirected lysis of antigen double-positive over single-positive leukemic cells.

The single-chain triplebody HLA-ds16-hu19 consists of three single-chain Fv (scFv) antibody fragments connected in a single polypeptide chain. This protein with dual-targeting capacity mediated preferential lysis of antigen double-positive (dp) over single-positive (sp) leukemic cells by recruitment of natural killer (NK) cells as effectors. The two distal scFv modules were specific for the histocompatibility protein HLA-DR and the lymphoid antigen CD19, the central one for the Fc gamma receptor CD16. In antibody-dependent cellular cytotoxicity (ADCC) experiments with a mixture of leukemic target cells comprising both HLA-DR sp HuT-78 or Kasumi-1 cells and (HLA-DR plus CD19) dp SEM cells, the triplebody mediated preferential lysis of the dp cells even when the sp cells were present in ≤20-fold numerical excess. The triplebody promoted equal lysis of SEM cells at 2.5-fold and 19.5-fold lower concentrations than the parental antibodies specific for HLA-DR and CD19, respectively. Finally, the triplebody also eliminated primary leukemic cells at lower concentrations than an equimolar mixture of bispecific single-chain Fv fragments (bsscFvs) separately addressing each target antigen (hu19-ds16 and HLA-ds16). The increased selectivity of targeting and the preferential lysis of dp over sp cells achieved by dual-targeting open attractive new perspectives for the use of dual-targeting agents in cancer therapy.