Construction and Evaluation of murine Cytomegalovirus-targeting Bispecific T cell Engagers

Cytomegalovirus (CMV) is a ubiquitous herpesvirus from the family of -herpesviruses. Depending on socio-economic status, a seroprevalence rate of up to 90% can be found. After primary infection, which typically causes little to mild symptoms in healthy individuals, the infection enters a latent state. Symptomatic reactivation is usually limited to immunosuppressed patient groups, including (insufficiently treated) patients infected with Human Immunodeficiency Virus (HIV). The largest group affected newborns: An important feature of CMV is its transplacental transmission and conatal infection. This characteristic makes CMV the leading infectious cause of lifelong congenital disabilities. Neuronal development is particularly affected, leading to mental retardation and congenital hearing loss. Currently, active CMV infection (CMV disease) is treated with antiviral herpes drugs such as (Val-) Ganciclovir. This is only necessary for immunocompromised patients, such as those who have undergone solid organ or stem cell transplantation. These substances have diverse adverse effects and can induce viral resistance mutations. In particular, in high-risk settings involving CMV-negative donors (D-) and CMV-positive recipients (R+), reactivation of CMV infection can lead to severe adverse effects. In this context, the newly forming immune system is initially unable to recognise and combat CMV, as it has not yet fully matured or developed memory cells. As a result, this immunological immaturity can lead to CMV reactivation or uncontrolled primary infection. Most transplant centres, therefore, administer antiviral drugs preventively or monitor CMV reactivation by regularly measuring viral genome copies in blood, to immediately initiate antiviral treatment when a threshold is exceeded. A major challenge in the use of small antiviral substances is the induction of drug resistance. Hitherto, resistance mutations against all antiviral substances have been described in various CMV strains, even against Letermovir and the 2023 approved Maribavir. In this context, the investigation of alternative treatment options is urgently required. As primary and reactivated CMV infections are mainly controlled by T cell responses, this mechanism is a suitable starting point for research on new antiviral therapy. Since no memory T cells exist in a donor-negative (D-) relationship and cannot be transfused directly from the donor, T cell-engaging therapies can be adopted. Bispecific T cell engagers (BiTE) are a major histocompatibility complex (MHC)-independent, off-the-shelf option. They consist of two single-chain variable fragments (scFv) derived from antibodies directed against the cluster of differentiation (CD) 3 domain of T cells and against a specific antigen of the target cell. The BiTE molecule connects both effector and target cells in a specific manner, stimulating the native T cell activation mechanism, resulting in the secretion of pro-inflammatory, antiviral cytokines, and the induction of T cell-mediated target cell lysis. This concept has been clinically applied since 2015. The human CD19-directed BiTE Blinatumomab is used for B cell malignancies, such as acute lymphoblastic leukaemia (ALL). In addition, Elranatamab, a BCMA-directed BiTE, has been introduced in the treatment of multiple myeloma (MM) since 2023. My translational study examines whether BiTEs can also be used successfully for CMV therapy. Methods. Stable cell lines expressing the selected CMV target antigens were established for evaluation. Three murine CMV (MCMV) glycoprotein antigens, gB, gH, and gN, were selected as target antigens, and eight BiTEs with murine and eight with human -CD3 were constructed. Of these, 12 constructs were tested for their binding capacity using flow cytometry. Subsequently, target cell lines were established that expressed one MCMV glycoprotein complex in combination with nano-luciferase (nLuc) as a cytotoxicity indicator. In co-culture with BiTEs and T cells, nLuc activity was measured in the cell culture supernatant, reflecting the lysis capacity of human BiTEs. Results. All expressed BiTE constructs showed antigen-specific binding capacity in flow cytometry analysis. The nLuc experiments were performed using the human constructs. All constructs induced significant and antigen-specific lysis of the target cells when co-cultured with T cells. This is the first proof-of-concept study for BiTEs against various MCMV glycoproteins, especially novel targets gH and gN. Conclusion and discussion. The investigated constructs offer a previously unseen possibility for antiviral therapy against CMV in a recognised model. Although no fixed concentration was used in the experiments, the individual experiments achieved comparable results. In addition, the different target antigen offers the possibility of combination therapy, lowering the risk of resistance induction or immune evasion. To further validate the constructs for their application in therapy for MCMV infection, the constructs need to be tested in subsequent mouse experiments.