No abstract

RNAi represents a powerful technology to specifically downregulate the expression of target genes. For cancer research and therapy, an efficient in vivo delivery system is supposed to distribute RNAi to all tumour cells upon systemic administration. We present replication-competent murine leukaemia virus (MLV) vectors, which deliver RNAi to tumour tissue upon tail vein injection. In HT1080 cells stably expressing GFP or luciferase, GFP expression was suppressed by more than 80% and luciferase (luc) activity by more than 90%, even when only 0.1% of the cells were initially infected with reporter gene specific vectors. To demonstrate its potential, PLK1-and MMP14-specific small hairpin RNA expression cassettes were applied in the system. Upon infection, PLK1 and MMP14 levels were reduced on mRNA and protein level. MLV-shPLK1-infected cells were arrested in the G2-phase and underwent apoptosis. MLV-shMMP14-infected cells showed reduced MMP2 activity, as well as substantially reduced invasion and tumour growth. In vivo, MLV-shLuc silenced luc expression in HT1080-luc tumour tissue by more than 80% and MLV-shPLK1 reduced tumour growth substantially, demonstrating the therapeutic relevance of this system. This RNAi vector system allows long-term downregulation of target gene expression as well as efficient delivery to and distribution throughout tumour tissue in vivo.

Retroviruses distinguish themselves from all other mammalian viruses by their abilities to infect and propagate in mammalian cells without causing a cytopathic effect and to stably integrate their genetic information into the genome of the host cell. These unique properties make them an ideal platform for the display and directed evolution of proteins in a mammalian cell environment. This review will describe the essentials about retrovirus biology and then discuss in detail display and screening strategies that have been developed during the past 15 years of retroviral display technology.

Background & Aim: T-cell bispecific antibodies (TCBs) binding to a target on tumor cells and CD3 on T cells induce potent T-cell mediated killing of cells carrying the target. In contrast to targets like e.g. CD38 or CD138, B-cell maturation antigen (BCMA) is suggested to be only expressed on plasma cells (PCs) and multiple myeloma (MM) PCs. Therefore, a BCMA-TCB should specifically act on these cell types. We report on a new class of BCMA-TCBs designed for effective and convenient therapy of MM. Molecular structure & its rationale (fig. 1A): The new class of BCMA-TCBs are asymmetric two-arm IgG-based human antibodies, bivalent to BCMA, monovalent to CD3 and comprising an engineered Fc. To achieve tumor specificity of the BCMA-TCBs and avoid unspecific T-cell activation, monovalent binding to CD3 was fixed at an affinity of KD= 70 nM, whereas various BCMA-TCBs with binding affinities to BCMA ranging from KD= 50 pM to 10 nM have been investigated (measurement of binding affinities by surface plasmon resonance and flow cytometry (FC)). For long elimination half-life, an Fc was introduced to enable once a week intravenous or subcutaneous administration. Fc is engineered to abolish binding to FcgR and C1q to minimize risk of infusion reactions without interfering with FcRn binding. The BCMA-TCBs can be well-manufactured and have no tendency to aggregation. In vitro profile: Potency of BCMA-TCBs to activate T cells and to induce killing of human MM cell lines was measured in a 24h co-culture assay with human PBMCs and MM cells in a ratio of 10:1. Results: (i) NCI-H929 cells expressing on cell surface up to 100 times more BCMA than primary patient MM cells were killed in presence of BCMA-TCBs with EC50 ranging from 5 to 50 pM, but not in presence of a control-TCB binding to CD3 only. (ii) As next, RPMI-8226 cells were used as target cells because surface BCMA expression was found to be only slightly higher than on primary patient MM cells (specific antigen binding capacity SABC as measured by FC between 1165 and 5461 per RPMI-8226 cell compared to 116 to 4479 per primary patient MM cell). Effective killing of RPMI-8226 MM cells was observed; the killing potency was higher respectively EC50 lower with the BCMA-TCBs having binding affinities below 1 nM (fig. 1B; EC50 from 50 pM to 1000 pM). (iii) Killing of U266 and L363 human MM cell lines was also observed with BCMA-TCBs. (iv) T-cell activation and increased T-cell function go in parallel with lysis of MM target cells as observed by an upregulation of CD69 and CD25 expression, release of granzyme B (>20 ng/mL at 3 nM vs. 20 pg/mL for control) and proinflammatory cytokines e.g. IFN-g, TNF-a, IL-2, IL-6, and IL-10. (v) At 120h incubation, BCMA-TCBs induced concentration dependant CD4 and CD8 T-cell proliferation as observed by CFSE dilution. A small exploratory study in whole bone marrow (BM) aspirates from MM patients (n=3) suggested a concentration dependent killing of MM PCs induced by BCMA-TCBs in presence of autologous T cells, thus justifying for a much larger trial to investigate one of the BCMA-TCB of this new class to induce killing and T-cell activation/function in MM patient BM aspirates performed by two clinical groups (Abstract also submitted to ASH). In vivo profile: New BCMA-TCBs bind to BCMA as well as CD3 of cynomolgus monkeys (cyno). Single dose pharmacokinetics (PK) and pharmacodynamics of a BCMA-TCB was studied in cyno at 0.003, 0.03, 0.3 mg/kg intravenously. Dose linear PK was observed. Mean serum concentrations in the first 7 days after 0.03 mg/kg were ≈1 to 2 nM, BM aspirates collected at 96h showed also concentrations of ≈1 to 2 nM. Elimination between 24 h and 504 h was found to be first order with a half-life of approximately 6 to 8 days. These data suggest a convenient clinical dosing schedule, e.g. once a week. Peripheral blood T-cell redistribution was observed 24h after dosing. Reduction of PCs could be observed by FC while total B cells and other cell types were unaffected. Summary: Effective and specific killing of MM cells was demonstrated with the BCMA-TCBs. Killing goes in parallel with T-cell activation and increased function. TCB with binding affinity to BCMA below 1 nM have higher potency/lower EC50 than those with affinity above 1 nM. TCBs could be produced with high purity and were stable with no tendency to aggregation. In cynomolgus monkeys, a PK profile has been found, suitable for once a week administration. This new class of BCMA-TCB has promises for clinical development. Disclosures Vu: EngMab AG: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Moser:Roche: Employment. Delon:Roche: Employment. Latzko:Roche: Employment. Gianotti:Roche: Employment. Lüoend:Roche: Employment. Friang:Roche: Employment. Murr:Roche: Employment. Duerner:Roche: Employment. Weinzierl:Roche: Employment. Fauti:Roche: Employment. Bacac:Roche: Employment. Ast:Roche: Employment. Freimoser-Grundschober:Roche: Employment. Rodriguez Diaz:Roche: Employment. Zielonka:Roche: Employment. van Puijenbroek:Roche: Employment. Hosse:Roche: Employment. Bruenker:Roche: Employment. Mössner:Roche: Employment. Klein:Roche: Employment. Umaña:Roche: Employment. Strein:EngMab AG: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; BB Biotech AG: Membership on an entity's Board of Directors or advisory committees; Novimmune SA: Membership on an entity's Board of Directors or advisory committees.

Selective binding of TCR-based therapies that target a single tumour-specific peptide epitope presented by human leukocyte antigens (HLA) is the absolute prerequisite for their therapeutic suitability and patient safety. To date, selectivity assessment has been limited to peptide library screening and predictive computational modeling. We developed the first experimental platform to de novo identify interactomes of TCR-like molecules directly in human tissues using mass spectrometry. As proof of concept, we confirm the target epitope of a novel MAGE-A4-specific TCR-like antibody. We further determine 16 cross-reactive sequences for ESK1, a TCR-like bispecific antibody recognizing WT-1 with known off-target activity, in healthy liver tissue. We observe strong, off-target-induced T cell activation for 8/16 sequences, demonstrating the high specificity of the approach. Off-target sequences define a previously missed amino acid compensation motif that structurally mimics the target peptide groove coordination and allows for peptide interaction with the engager molecule. We establish the importance of the identified off-target activity by demonstrating 3D liver spheroid killing in the presence of ESK1 and healthy donor PBMC. Finally, we utilize our approach to de-risk our novel MAGE-A4 targeting TCR-like bispecific antibody prior to entering now ongoing clinical trials. We conclude that our strategy offers an accurate, scalable route for de-risking TCR-based therapeutics prior to first-in-human clinical application.