Michael Schwenkert

Targeting of DEC-205 on human dendritic cells results in efficient MHC class II-restricted antigen presentation

The use of dendritic cells (DCs) in therapeutic cancer vaccination requires their loading with tumor-specific antigen(s). DEC-205, a phagocytosis receptor mediating antigen uptake, is associated with CD8(+) T-cell responses in mice. Here we fused an anti-DEC-205scFv to an HLA-DP4-restricted epitope from the tumor antigen MAGE-A3, and examined the suitability and efficacy of DEC-205 to deliver a helper epitope to human monocyte-derived DCs (moDCs). The construct specifically bound DEC-205 on human moDCs without negative impact on DC phenotype and function. We measured antigen presentation with specific autologous CD4(+) T cells, generated by TCR-RNA transfection. DEC-205 targeting resulted in significant major histocompatibility complex class II-restricted antigen presentation, and was superior to loading DCs by electroporation of mRNA encoding endosome-targeted MAGE-A3-DCLAMP or by direct peptide pulsing. Anti-DEC-205scFv-MAGE-A3 was presented 100 times more efficiently than the control constructs. DC maturation before or during incubation with anti-DEC-205scFv-MAGE-A3 reduced the interleukin-10/interleukin-2 ratio. Moreover, we successfully applied the DEC-205 targeting strategy to moDCs from malignant melanoma patients. Again, DEC-205-targeted mature DCs (mDCs) presented the antigen more efficiently than peptide-pulsed DCs and maintained their stimulatory capacity after cryoconservation. Thus, DEC-205 targeting represents a feasible and effective method to deliver helper epitopes to DCs in anticancer vaccine strategies, which may also be suitable for DC targeting in vivo.

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.

A novel CD19-directed recombinant bispecific antibody derivative with enhanced immune effector functions for human leukemic cells.

A novel bispecific antibody-derived recombinant protein targeting leukemias and lymphomas was designed, a single-chain Fv triple body (sctb) consisting of 1 polypeptide chain with 3 scFvs connected in tandem. The distal scFvs were specific for the tumor antigen CD19, and the central scFv for the trigger molecule CD16 (FcγRIII) on natural killer (NK) cells and macrophages. We had previously built a disulphide stabilized (ds) bsscFv [19×16] with monovalent binding for CD19 from ds components. The sctb ds[19×16×19] also used ds components and displayed 3-fold greater avidity for CD19 than the bsscFv (KD=13 vs. 42 nM), whereas both had equal affinity for CD16 (KD=58 nM). Plasma half-lives in mice were 4 and 2 hours for the sctb and the bsscFv, respectively. In antibody-dependent cellular cytotoxicity reactions with human mononuclear cells as effectors, the sctb promoted equal lysis of leukemic cell lines and primary cells from leukemia and lymphoma patients at 10-fold to 40-fold lower concentrations than the bsscFv. This new format may also be applicable to a variety of other tumor antigens and effector molecules. With half-maximum effective concentrations (EC50) in the low picomolar range, the sctb ds[19×16×19] is an attractive candidate for further preclinical evaluation.

Novel conjugates of single-chain Fv antibody fragments specific for stem cell antigen CD123 mediate potent death of acute myeloid leukaemia cells

Four new single‐chain Fv antibody fragments (scFvs) specific for the human leucocyte surface antigen CD123 (interleukin‐3 receptor α) were generated to achieve preferential targeting of leukaemia stem cells (LSCs) in acute myeloid leukaemia (AML). The scFvs were isolated from a phage display library generated with spleen RNA from mice, immunized with a fusion protein consisting of the extracellular domain of CD123 and the Fc domain of a human immunoglobulin G1. The scFvs displayed CD123‐specific binding on tumour cells (binding constants (KD) 4·5–101 nmol/l). The scFv with the highest affinity was used to design two cell death‐inducing molecules. First, an immunotoxin, a fusion protein with truncated Pseudomonas Exotoxin A, induced potent apoptosis of AML‐derived MOLM‐13 and SKNO‐1 cells at nanomolar concentrations. Second, the fusion to another scFv, specific for the low affinity Fcγ‐receptor III (CD16), created a bispecific single chain Fv (bsscFv). This bsscFv [123 × ds16] mediated potent lysis of AML‐derived MOLM‐13, THP‐1 and SKNO‐1 cells in antibody‐dependent cellular cytotoxicity (ADCC) reactions at picomolar concentrations. The recruitment of CD16‐positive effector cells for the lysis of AML cells via CD123 represents a novel combination with attractive prospects for future clinical testing.

Melanoma-associated Chondroitin Sulfate Proteoglycan (MCSP)-targeted delivery of soluble TRAIL potently inhibits melanoma outgrowth in vitro and in vivo

BackgroundAdvanced melanoma is characterized by a pronounced resistance to therapy leading to a limited patient survival of ~6 - 9 months. Here, we report on a novel bifunctional therapeutic fusion protein, designated anti-MCSP:TRAIL, that is comprised of a melanoma-associated chondroitin sulfate proteoglycan (MCSP)-specific antibody fragment (scFv) fused to soluble human TRAIL. MCSP is a well-established target for melanoma immunotherapy and has recently been shown to provide important tumorigenic signals to melanoma cells. TRAIL is a highly promising tumoricidal cytokine with no or minimal toxicity towards normal cells. Anti-MCSP:TRAIL was designed to 1. selectively accrete at the cell surface of MCSP-positive melanoma cells and inhibit MCSP tumorigenic signaling and 2. activate apoptotic TRAIL-signaling.ResultsTreatment of a panel of MCSP-positive melanoma cell lines with anti-MCSP:TRAIL induced TRAIL-mediated apoptotic cell death within 16 h. Of note, treatment with anti-MCSP:sTRAIL was also characterized by a rapid dephosphorylation of key proteins, such as FAK, implicated in MCSP-mediated malignant behavior. Importantly, anti-MCSP:TRAIL treatment already inhibited anchorage-independent growth by 50% at low picomolar concentrations, whereas > 100 fold higher concentrations of non-targeted TRAIL failed to reduce colony formation. Daily i.v. treatment with a low dose of anti-MCSP:TRAIL (0.14 mg/kg) resulted in a significant growth retardation of established A375 M xenografts. Anti-MCSP:TRAIL activity was further synergized by co-treatment with rimcazole, a σ-ligand currently in clinical trials for the treatment of various cancers.ConclusionsAnti-MCSP:TRAIL has promising pre-clinical anti-melanoma activity that appears to result from combined inhibition of tumorigenic MCSP-signaling and concordant activation of TRAIL-apoptotic signaling. Anti-MCSP:TRAIL alone, or in combination with rimcazole, may be of potential value for the treatment of malignant melanoma.

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.

Heterodimeric bispecific antibody-derivatives against CD19 and CD16 induce effective antibody-dependent cellular cytotoxicity against B-lymphoid tumor cells

Bispecific scFv antibody-derivatives (bsscFvs) recruiting natural killer (NK) cells for the lysis of malignant cells have therapeutic potential. However, a bsscFv specific for the B-lymphoid tumor antigen CD19 and the trigger molecule CD16 on NK cells had similar affinities for both antigens (42 and 58nM, respectively) and was not optimal for cytotoxicity. Therefore, a bispecific tribody (bsTb) was constructed with two binding sites for CD19 and one for CD16. This bsTb contained a CD19-specific Fab fragment carrying a CD16-specific scFv fused to its light chain and a CD19-specific scFv fused to its heavy chain. The bsTb was compared with a bispecific bibody (bsBb) lacking the CD19-specific scFv. The bsTb had 3-fold greater avidity for CD19 than the bsBb (8 and 24nM, respectively), while both had equal affinity for CD16 (56nM). Both molecules mediated antibody-dependent cellular cytotoxicity (ADCC) of leukemia-derived SEM cells and primary cells from leukemia patients. The bsTb showed half-maximum effective concentrations (EC(50)) of 55pM and promoted equal lysis as the bsBb and the bsscFv at 6- and 12-fold lower concentrations, respectively. Among these three molecules the bsTb showed the most promising in vitro properties which are anticipated to be displayed also in vivo.

A single chain immunotoxin, targeting the melanoma-associated chondroitin sulfate proteoglycan, is a potent inducer of apoptosis in cultured human melanoma cells.

A recombinant immunotoxin was constructed by fusing a single chain fragment variable antibody fragment, specific for the melanoma-associated chondroitin sulfate proteoglycan (MCSP), to a truncated variant of Pseudomonas exotoxin A (ETA′), carrying a C-terminal KDEL-peptide for improved retrograde intracellular transport. The resulting immunotoxin MCSP-ETA′ was periplasmatically expressed in Escherichia coli and purified under native conditions by affinity chromatography resulting in a yield of approximately 30 μg/l bacterial culture. This immunotoxin induced antigen-specific apoptosis in the cultured human melanoma-derived cell lines A2058 and A375M, and treatment with a single dose of the agent eliminated up to 80% of these cells within 72 h. The dose needed for half-maximum killing (EC50) was approximately 1 nmol/l for both cell lines. MCSP-ETA′ also displayed cytotoxic activity against cultured primary melanoma cells from patients with advanced disease (pathologic stages IIIC and IV), with net cell death reaching up to 70% within 96 h after treatment with a single dose of 14 nmol/l. MCSP-ETA′ induced cell death synergistically with cyclosporin A, both in established human melanoma cell lines and cultured primary melanoma cells. The distinctive antigen-restricted induction of apoptosis and the synergy with cyclosporin A justify further evaluation of this novel agent with regard to its potential application for the treatment of malignant melanoma.

CD19 and CD20 targeted vectors induce minimal activation of resting B lymphocytes.

B lymphocytes are an important cell population of the immune system. However, until recently it was not possible to transduce resting B lymphocytes with retro- or lentiviral vectors, making them unsusceptible for genetic manipulations by these vectors. Lately, we demonstrated that lentiviral vectors pseudotyped with modified measles virus (MV) glycoproteins hemagglutinin, responsible for receptor recognition, and fusion protein were able to overcome this transduction block. They use either the natural MV receptors, CD46 and signaling lymphocyte activation molecule (SLAM), for cell entry (MV-LV) or the vector particles were further modified to selectively enter via the CD20 molecule, which is exclusively expressed on B lymphocytes (CD20-LV). It has been shown previously that transduction by MV-LV does not induce B lymphocyte activation. However, if this is also true for CD20-LV is still unknown. Here, we generated a vector specific for another B lymphocyte marker, CD19, and compared its ability to transduce resting B lymphocytes with CD20-LV. The vector (CD19ds-LV) was able to stably transduce unstimulated B lymphocytes, albeit with a reduced efficiency of about 10% compared to CD20-LV, which transduced about 30% of the cells. Since CD20 as well as CD19 are closely linked to the B lymphocyte activation pathway, we investigated if engagement of CD20 or CD19 molecules by the vector particles induces activating stimuli in resting B lymphocytes. Although, activation of B lymphocytes often involves calcium influx, we did not detect elevated calcium levels. However, the activation marker CD71 was substantially up-regulated upon CD20-LV transduction and most importantly, B lymphocytes transduced with CD20-LV or CD19ds-LV entered the G1b phase of cell cycle, whereas untransduced or MV-LV transduced B lymphocytes remained in G0. Hence, CD20 and CD19 targeting vectors induce activating stimuli in resting B lymphocytes, which most likely renders them susceptible for lentiviral vector transduction.