Hendrik Knoetgen

Behavioural and functional characterization of Kv10.1 (Eag1) knockout mice

Kv10.1 (Eag1), member of the Kv10 family of voltage-gated potassium channels, is preferentially expressed in adult brain. The aim of the present study was to unravel the functional role of Kv10.1 in the brain by generating knockout mice, where the voltage sensor and pore region of Kv10.1 were removed to render non-functional proteins through deletion of exon 7 of the KCNH1 gene using the ‘3 Lox P strategy’. Kv10.1-deficient mice show no obvious alterations during embryogenesis and develop normally to adulthood; cortex, hippocampus and cerebellum appear anatomically normal. Other tests, including general health screen, sensorimotor functioning and gating, anxiety, social behaviour, learning and memory did not show any functional aberrations in Kv10.1 null mice. Kv10.1 null mice display mild hyperactivity and longer-lasting haloperidol-induced catalepsy, but there was no difference between genotypes in amphetamine sensitization and withdrawal, reactivity to apomorphine and haloperidol in the prepulse inhibition tests or to antidepressants in the haloperidol-induced catalepsy. Furthermore, electrical properties of Kv10.1 in cerebellar Purkinje cells did not show any difference between genotypes. Bearing in mind that Kv10.1 is overexpressed in over 70% of all human tumours and that its inhibition leads to a reduced tumour cell proliferation, the fact that deletion of Kv10.1 does not show a marked phenotype is a prerequisite for utilizing Kv10.1 blocking and/or reduction techniques, such as siRNA, to treat cancer.

Committing cytomegalovirus-specific CD8 T cells to eliminate tumor cells by bifunctional major histocompatibility class I antibody fusion molecules

Schmittnaegel and colleagues describe the generation of a novel tumor-peptide-MHCI–antibody fusion protein that redirects a highly functional subset of CMV-specific T cells to eliminate tumor cells by engaging a naturally occurring T-cell population in humans that controls cytomegalovirus infection. Tumor cells escape immune eradication through multiple mechanisms, including loss of antigenicity and local suppression of effector lymphocytes. To counteract these obstacles, we aimed to direct the unique cytomegalovirus (CMV)-specific immune surveillance against tumor cells. We developed a novel generation of fusion proteins composed of a tumor antigen–specific full immunoglobulin connected to a single major histocompatibility class I complex bearing a covalently linked virus-derived peptide (pMHCI–IgG). Here, we show that tumor antigen–expressing cancer cells, which are decorated with pMHCI–IgGs containing a HLA-A*0201 molecule associated with a CMV-derived peptide, are specifically eliminated through engagement of antigen-specific CD8+ T cells isolated from peripheral blood mononuclear cell preparations of CMV-infected humans. These CD8+ T cells act without additional expansion, preactivation, or provision of costimulatory signals. Elimination of tumor cells is induced at similar concentrations and with similar time kinetics as those seen with bispecific T-cell engagers (BiTE). However, while BiTE-like reagents indiscriminately activate T cells through binding to the T-cell receptor complex, pMHCI–IgGs selectively engage antigen-specific, constantly renewable, differentiated effector cytotoxic T lymphocytes to tumor cells, thereby representing a novel class of anticancer immunotherapeutics with potentially improved safety and efficacy profiles. Cancer Immunol Res; 3(7); 764–76. ©2015 AACR.

A New Class of Bifunctional Major Histocompatibility Class I Antibody Fusion Molecules to Redirect CD8 T Cells

Bifunctional antibody fusion proteins engaging effector T cells for targeted elimination of tumor cells via CD3 binding have shown efficacy in both preclinical and clinical studies. Different from such a polyclonal T-cell recruitment, an alternative concept is to engage only antigen-specific T-cell subsets. Recruitment of specific subsets of T cells may be as potent but potentially lead to fewer side effects. Tumor-targeted peptide–MHC class I complexes (pMHCI-IgGs) bearing known antigenic peptides complexed with MHC class I molecules mark tumor cells as antigenic and utilize the physiologic way to interact with and activate T-cell receptors. If, for example, virus-specific CD8+ T cells are addressed, the associated strong antigenicity and tight immune surveillance of the effector cells could lead to efficacious antitumor treatment in various tissues. However, peptide–MHC class I fusions are difficult to express recombinantly, especially when fused to entire antibody molecules. Consequently, current formats are largely limited to small antibody fragment fusions expressed in bacteria followed by refolding or chemical conjugation. Here, we describe a new molecular format bearing a single pMHCI complex per IgG fusion molecule characterized by enhanced stability and expression yields. This molecular format can be expressed in a full immunoglobulin format and can be designed as mono- or bivalent antibody binders. Mol Cancer Ther; 15(9); 2130–42. ©2016 AACR.

Activation of cytomegalovirus-specific CD8(+) T-cell response by antibody-mediated peptide-major histocompatibility class I complexes.

Imposing antigenicity on tumor cells is a key step toward successful cancer-immunotherapy. A cytomegalovirus-derived peptide recombinantly fused to a major histocompatibility class I complex and a monoclonal antibody can be targeted to tumor cells by antibody-mediated delivery and activate a strong and specific CD8+ T cell response.

Abstract B69: Novel MHC class I antibody fusions for cancer treatment.

MHC (major histocompatibility complex) class I-restricted CD8 cytotoxic T cells recognize tumor cells or virus infected cells as foreign and consequently initiate a cascade of events resulting in their destruction. An immunotherapeutic strategy based on antibody-mediated targeting of virus-derived peptide-MHC class I complexes to tumor cells aiming at the subsequent elimination of these tumor cells by peptide-specific cytotoxic T cells has been proposed a few years ago. A crucial limitation hampering advances of this therapeutic concept was the recombinant expression of MHC class I fused full IgG immunoglobulins. We have identified novel recombinant fusion formats that allow the expression of full-length peptide-MHC class I IgG fusion molecules. These formats contain a single recombinant human pMHC class I complex consisting of a viral peptide (CMV or EBV derived), beta-2-microglobulin and HLA heavy chain (A*0201, lacking the transmembrane domain) fused to one of the two immunoglobulin heavy chains of a complete antibody molecule. These fusions can be expressed at high levels in standard mammalian expression systems overcoming several former technical hurdles (e.g. prokaryotic expression, low refolding yield, chemical coupling of components). By flow cytometry we show that the new fusion proteins successfully deliver virus-peptide complexed MHC class I molecules to tumor cells. The fusion proteins are potent CD8 T cell recruiters for human donor derived specific T cells or human PBMCs from chronically infected donors. A low frequency of virus peptide specific CD8 T cells in PBMCs (0.25 to 3.8 % of all CD8 positive T cells) effectively triggers in a peptide specific manner the killing of tumor cells at sub-nanomolar concentrations (cytotoxic T cell assay using real time analysis with xCelligence confirmed by classical LDH release). No unspecific activation of T cells was observed even at high concentrations, indicating a favorable safety profile. Confocal time-lapse microscopy showed T cell synapse formation on the tumor cells and serial killing by the T cells. We experimentally compare the killing of tumor cells of the MHC class I fused antibodies with bispecific anti-CD3 mediated T cell recruiters and characterized the virus specific T cells from human donors in detail. Our results show for the first time that recombinant pMHC class I antibody fusions in our novel full human IgG format can be expressed with a significant yield in common mammalian production cell lines. The redirection of MHC class I mediated recruitment of pre-existing virus specific CD8 T cells from human PBMCs is potent mechanism to attack tumor cells with a low risk of unspecific T cell activation making the concept now suitable as a therapeutic option. Citation Format: Martina Schmittnaegel, Eike Hoffmann, Olaf Mundigl, Gerhard Niederfellner, Klaus Bosslet, Pablo Umana, Victor Levitsky, Christian Klein, Hendrik Knoetgen. Novel MHC class I antibody fusions for cancer treatment. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr B69.