Christoph Uherek

Expression of a CD20-specific chimeric antigen receptor enhances cytotoxic activity of NK cells and overcomes NK-resistance of lymphoma and leukemia cells

Despite the clinical success of CD20-specific antibody rituximab, malignancies of B-cell origin continue to present a major clinical challenge, in part due to an inability of the antibody to activate antibody-dependent cell-mediated cytotoxicity (ADCC) in some patients, and development of resistance in others. Expression of chimeric antigen receptors in effector cells operative in ADCC might allow to bypass insufficient activation via FcγRIII and other resistance mechanisms that limit natural killer (NK)-cell activity. Here we have generated genetically modified NK cells carrying a chimeric antigen receptor that consists of a CD20-specific scFv antibody fragment, via a flexible hinge region connected to the CD3ζ chain as a signaling moiety. As effector cells we employed continuously growing, clinically applicable human NK-92 cells. While activity of the retargeted NK-92 against CD20-negative targets remained unchanged, the gene modified NK cells displayed markedly enhanced cytotoxicity toward NK-sensitive CD20 expressing cells. Importantly, in contrast to parental NK-92, CD20-specific NK cells efficiently lysed CD20 expressing but otherwise NK-resistant established and primary lymphoma and leukemia cells, demonstrating that this strategy can overcome NK-cell resistance and might be suitable for the development of effective cell-based therapeutics for the treatment of B-cell malignancies.

NK cells engineered to express a GD2 -specific antigen receptor display built-in ADCC-like activity against tumour cells of neuroectodermal origin.

Treatment of high‐risk neuroblastoma (NB) represents a major challenge in paediatric oncology. Alternative therapeutic strategies include antibodies targeting the disialoganglioside GD2, which is expressed at high levels on NB cells, and infusion of donor‐derived natural killer (NK) cells. To combine specific antibody‐mediated recognition of NB cells with the potent cytotoxic activity of NK cells, here we generated clonal derivatives of the clinically applicable human NK cell line NK‐92 that stably express a GD2‐specific chimeric antigen receptor (CAR) comprising an anti‐GD2 ch14.18 single chain Fv antibody fusion protein with CD3‐ζ chain as a signalling moiety. CAR expression by gene‐modified NK cells facilitated effective recognition and elimination of established GD2 expressing NB cells, which were resistant to parental NK‐92. In the case of intrinsically NK‐sensitive NB cell lines, we observed markedly increased cell killing activity of retargeted NK‐92 cells. Enhanced cell killing was strictly dependent on specific recognition of the target antigen and could be blocked by GD2‐specific antibody or anti‐idiotypic antibody occupying the CAR’s cell recognition domain. Importantly, strongly enhanced cytotoxicity of the GD2‐specific NK cells was also found against primary NB cells and GD2 expressing tumour cells of other origins, demonstrating the potential clinical utility of the retargeted effector cells.

DNA-carrier proteins for targeted gene delivery

The development of vectors for cell-specific gene delivery is a major goal of gene therapeutic strategies. Significant progress has been made in the construction of non-viral vectors that combine different functions required for gene transfer in an artificial complex. To some extent this can be achieved by complexing plasmid DNA with synthetic compounds such as lipids and polycations. Alternative approaches rely on the activities of natural or recombinant DNA-carrier proteins to achieve uptake and intracellular delivery of plasmid DNA. Nuclear proteins such as histones and members of the high mobility group protein family have been shown to condense DNA and transfect cultured cells. Some structural proteins of DNA viruses spontaneously assemble with plasmid DNA and form transfection-competent pseudocapsids. In addition, chimeric fusion proteins have been engineered that incorporate in a single polypeptide chain heterologous protein domains which facilitate binding to plasmid DNA, specific recognition of target cells, induction of receptor-mediated endocytosis, and DNA transport through intracellular compartments.

CD19‐CAR engineered NK‐92 cells are sufficient to overcome NK cell resistance in B‐cell malignancies

Many B‐cell acute and chronic leukaemias tend to be resistant to killing by natural killer (NK) cells. The introduction of chimeric antigen receptors (CAR) into T cells or NK cells could potentially overcome this resistance. Here, we extend our previous observations on the resistance of malignant lymphoblasts to NK‐92 cells, a continuously growing NK cell line, showing that anti‐CD19‐CAR (αCD19‐CAR) engineered NK‐92 cells can regain significant cytotoxicity against CD19 positive leukaemic cell lines and primary leukaemia cells that are resistant to cytolytic activity of parental NK‐92 cells. The ‘first generation’ CAR was generated from a scFv (CD19) antibody fragment, coupled to a flexible hinge region, the CD3ζ chain and a Myc‐tag and cloned into a retrovirus backbone. No difference in cytotoxic activity of NK‐92 and transduced αCD19‐CAR NK‐92 cells towards CD19 negative targets was found. However, αCD19‐CAR NK‐92 cells specifically and efficiently lysed CD19 expressing B‐precursor leukaemia cell lines as well as lymphoblasts from leukaemia patients. Since NK‐92 cells can be easily expanded to clinical grade numbers under current Good Manufactoring Practice (cGMP) conditions and its safety has been documented in several phase I clinical studies, treatment with CAR modified NK‐92 should be considered a treatment option for patients with lymphoid malignancies.

Tracking of [18F]FDG-labeled natural killer cells to HER2/neu-positive tumors

INTRODUCTION The objective of this study was to label the human natural killer (NK) cell line NK-92 with [(18)F]fluoro-deoxy-glucose (FDG) for subsequent in vivo tracking to HER2/neu-positive tumors. METHODS NK-92 cells were genetically modified to NK-92-scFv(FRP5)-zeta cells, which express a chimeric antigen receptor that is specific to the tumor-associated ErbB2 (HER2/neu) antigen. NK-92 and NK-92-scFv(FRP5)-zeta cells were labeled with [(18)F]FDG by simple incubation at different settings. Labeling efficiency was evaluated by a gamma counter. Subsequently, [(18)F]FDG-labeled parental NK-92 or NK-92-scFv(FRP5)-zeta cells were intravenously injected into mice with implanted HER2/neu-positive NIH/3T3 tumors. Radioactivity in tumors was quantified by digital autoradiography and correlated with histopathology. RESULTS The NK-92 and NK-92-scFv(FRP5)-zeta cells could be efficiently labeled with [(18)F]FDG by simple incubation. Optimal labeling efficiencies (80%) were achieved using an incubation period of 60 min and additional insulin (10 IU/ml). After injection of 5x10(6) [(18)F]FDG-labeled NK-92-scFv(FRP5)-zeta cells into tumor-bearing mice, digital autoradiography showed an increased uptake of radioactivity in HER2/neu-positive tumors at 60 min postinjection. Conversely, injection of 5x10(6) NK-92 cells not directed against HER2/neu receptors did not result in increased uptake of radioactivity in the tumors. Histopathology confirmed an accumulation of the NK-92-scFv(FRP5)-zeta cells, but not the parental NK cells, in tumor tissues. CONCLUSION The human NK cell line NK-92 can be directed against HER2/neu antigens by genetic modification. The genetically modified NK cells can be efficiently labeled with [(18)F]FDG, and the accumulation of these labeled NK cells in HER2/neu-positive tumors can be monitored with autoradiography.

Recombinant immunotoxins and retargeted killer cells: employing engineered antibody fragments for tumor-specific targeting of cytotoxic effectors

Over the past years, monoclonal antibodies have attracted enormous interest as targeted therapeutics, and a number of such reagents are in clinical use. However, responses could not be achieved in all patients with tumors expressing high levels of the respective target antigens, suggesting that other factors such as limited recruitment of endogenous immune effector mechanisms can also influence treatment outcome. This justifies the search for alternative, potentially more effective reagents. Antibody-toxins and cytolytic effector cells genetically modified to carry antibody-based receptors on the surface, represent such tailor-made targeting vehicles with the potential of improved tumor localization and enhanced efficacy. In this way, advances in recombinant antibody technology have made it possible to circumvent problems inherent in chemical coupling of antibodies and toxins, and have allowed construction via gene fusion of recombinant molecules which combine antibody-mediated recognition of tumor cells with specific delivery of potent protein toxins of bacterial or plant origin. Likewise, recombinant antibody fragments provide the basis for the construction of chimeric antigen receptors that, upon expression in cytotoxic T lymphocytes (CTLs) or natural killer (NK) cells, link antibody-mediated recognition of tumor antigens with these effector cells’ potent cytolytic activities, thereby making them promising cellular therapeutics for adoptive cancer therapy. Here, general principles for the derivation of cytotoxic proteins and effector cells with antibody-dependent tumor specificity are summarized, and current strategies to employ these molecules and cells for directed cancer therapy are discussed, focusing mainly on the tumor-associated antigens epidermal growth factor receptor (EGFR) and the closely related ErbB2 (HER2) as targets.

Chimeric antigen receptors for the retargeting of cytotoxic effector cells.

T lymphocytes recognize specific antigens through interaction of the T cell receptor (TCR) with short peptides presented by major histocompatibility complex (MHC) class I or II molecules. For initial activation and clonal expansion, naïve T cells are dependent on professional antigen-presenting cells (APCs) that provide additional co-stimulatory signals. TCR activation in the absence of co-stimulation can result in unresponsiveness and clonal anergy. To bypass immunization, different approaches for the derivation of cytotoxic effector cells with grafted recognition specificity have been developed. Chimeric antigen receptors have been constructed that consist of binding domains derived from natural ligands or antibodies specific for cell-surface antigens, genetically fused to effector molecules such as the TCR alpha and beta chains, or components of the TCR-associated CD3 complex. Upon antigen binding, such chimeric receptors link to endogenous signaling pathways in the effector cell and generate activating signals similar to those initiated by the TCR complex. Since the first reports on chimeric antigen receptors, this concept has steadily been refined and the molecular design of chimeric receptors has been optimized. Aided by advances in recombinant antibody technology, chimeric antigen receptors targeted to a wide variety of antigens on the surface of cancer cells and of cells infected by human immunodeficiency virus (HIV) have been generated. In initial clinical studies, infusion of such cells into patients proved to be safe and transient therapeutic effects have been observed.

Indatuximab ravtansine (BT062) combination treatment in multiple myeloma: pre-clinical studies

Indatuximab ravtansine is a monoclonal antibody-linked cytotoxic agent that specifically targets CD138-expressing cells. Monotherapy has been shown to significantly inhibit multiple myeloma tumour growth in vivo and improve host survival. Here, we show that in most cell lines tested, indatuximab ravtansine acts additively or even synergistically with clinically approved therapies for treatment of multiple myeloma. In addition, in vivo mouse xenograft models confirmed the activity of indatuximab ravtansine in combination with lenalidamide and lenalidomide/dexamethasone. Indatuximab ravtansine may therefore be a suitable combination partner for multiple myeloma, and a clinical study is ongoing.

Modular Fusion Proteins for Receptor-mediated Gene Delivery

The development of vectors for target-cell specific gene delivery is a major goal of gene therapeutic strategies. Thereby non-viral gene delivery vectors are gaining increasing interest [1]. Progress has been made in the understanding how individual activities of viruses can be mimicked and methodologies have been developed which allow to combine different functions required for gene transfer into an artificial complex. An attractive approach for the design of such modular self-assembling systems for gene delivery is based on fusion proteins engineered to incorporate in a single polypeptide chain several cooperating functions [2–4]. Each of these domains can account for a distinct activity required for DNA-binding, cell recognition and intracellular delivery. Such fusion proteins in contrast to similar chemical conjugates can be produced in suitable expression systems in their final form and the resulting products are generally homogeneous in their composition.

Antibody Fusion Proteins for Targeted Gene Delivery

Molecular conjugates which employ antibodies or other ligands chemically coupled to polycations to deliver DNA into cells constitute an important group of non-viral vectors which are investigated as tools for therapeutic gene delivery (Wagner, 1998; Kircheis et al., 1999). The protein and DNA components of such artificial compounds assemble into stable complexes with defined target cell tropism. Thereby interaction with DNA is mediated by the binding of a polycationic reagent such as poly-L-lysine to negatively charged plasmid DNA resulting in a so-called polyplex (Felgner et al., 1997). The addition of a target cell-specific ligand facilitates cell recognition and uptake of the complex via receptor-mediated endocytosis. An important improvement of this concept was the incorporation of endosomolytic activities which upon internalization into cells greatly enhances the release of DNA from endocytic vesicles and the expression of the transferred gene (Wagner, 1998).