RNA vaccines are efficient preventive measures to combat the SARS-CoV-2 pandemic. High levels of neutralizing SARS-CoV-2-antibodies are an important component of vaccine-induced immunity. Shortly after the initial two mRNA vaccine doses, the IgG response mainly consists of the pro-inflammatory subclasses IgG1 and IgG3. Here, we report that several months after the second vaccination, SARS-CoV-2-specific antibodies were increasingly composed of non-inflammatory IgG4, which were further boosted by a third mRNA vaccination and/or SARS-CoV-2 variant breakthrough infections. IgG4 antibodies among all spike-specific IgG antibodies rose on average from 0.04% shortly after the second vaccination to 19.27% late after the third vaccination. This induction of IgG4 antibodies was not observed after homologous or heterologous SARS-CoV-2 vaccination with adenoviral vectors. Single-cell sequencing and flow cytometry revealed substantial frequencies of IgG4-switched B cells within the spike-binding memory B-cell population (median 14.4%; interquartile range (IQR) 6.7–18.1%) compared to the overall memory B-cell repertoire (median 1.3%; IQR 0.9–2.2%) after three immunizations. Importantly, this class switch was associated with a reduced capacity of the spike-specific antibodies to mediate antibody-dependent cellular phagocytosis and complement deposition. Since Fc-mediated effector functions are critical for antiviral immunity, these findings may have consequences for the choice and timing of vaccination regimens using mRNA vaccines, including future booster immunizations against SARS-CoV-2.

Dual targeting of surface receptors with bispecific antibodies is attracting increasing interest in cancer therapy. Here, we present a novel bivalent and bispecific antagonistic molecule (Dab-Fc) targeting human epidermal growth factors 2 and 3 (HER2 and HER3) derived from the Db-Ig platform, which was developed for the generation of multivalent and multispecific antibody molecules. Dab-Fc comprises the variable domains of the anti-HER2 antibody trastuzumab and the anti-HER3 antibody 3–43 assembled into a diabody-like structure stabilized by C H 1 and C L domains and further fused to a human γ1 Fc region. The resulting Dab-Fc 2 × 3 molecule retained unhindered binding to both antigens and was able to bind both antigens sequentially. In cellular experiments, the Dab-Fc 2 × 3 molecule strongly bound to different tumor cell lines expressing HER2 and HER3 and was efficiently internalized. This was associated with potent inhibition of the proliferation and migration of these tumor cell lines. Furthermore, IgG-like pharmacokinetics and anti-tumoral activity were demonstrated in a xenograft tumor model of the gastric cancer cell-line NCI-N87. These results illustrate the suitability of our versatile Db-Ig platform technology for the generation of bivalent bispecific molecules, which has been successfully used here for the dual targeting of HER2 and HER3.

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Repeated mRNA vaccinations are an efficient tool to combat the SARS-CoV-2 pandemic. High levels of neutralizing SARS-CoV-2-antibodies are an important component of vaccine-induced immunity. Shortly after the first or second mRNA vaccine dose, the IgG response mainly consists of the pro-inflammatory isotypes IgG1 and IgG3 and is driven by T helper (Th) 1 cells. Here, we report that several months after the second vaccination, SARS-CoV-2-specific antibodies were increasingly composed of non-inflammatory IgG2 and particularly IgG4, which were further boosted by a third mRNA vaccination and/or SARS-CoV-2 variant breakthrough infections. While IgG antibodies were affinity matured and of high neutralization capacity, the switch in constant domains caused changes in fragment crystallizable (Fc)-receptor mediated effector functions, including a decreased capacity to facilitate phagocytosis. IgG4 induction was neither induced by Th2 cells nor observed after homologous or heterologous SARS-CoV-2 vaccination with adenoviral vectors. In addition, IgG2- and IgG4-producing memory B cells were phenotypically indistinguishable from IgG1- or IgG3-producing cells. Since Fc-mediated effector functions are critical for antiviral immunity, the described class switch towards non-inflammatory IgG isotypes, which otherwise rarely occurs after vaccination or viral infection, may have consequences for the choice and timing of vaccination regimens using mRNA vaccines.

Targeting costimulatory receptors of the tumor necrosis factor superfamily (TNFSF) to activate T-cells and promote anti-tumor T-cell function have emerged as a promising strategy in cancer immunotherapy. Previous studies have shown that combining two different members of the TNFSF resulted in dual-acting costimulatory molecules with the ability to activate two different receptors either on the same cell or on different cell types. To achieve prolonged plasma half-life and extended drug disposition, we have developed novel dual-acting molecules by fusing single-chain ligands of the TNFSF to heterodimerizing Fc chains (scDuokine-Fc, scDk-Fc). Incorporating costimulatory ligands of the TNF superfamily into a scDk-Fc molecule resulted in enhanced T-cell proliferation translating in an increased anti-tumor activity in combination with a primary T-cell-activating bispecific antibody. Our data show that the scDk-Fc molecules are potent immune-stimulatory molecules that are able to enhance T-cell mediated anti-tumor responses.

Vaccines are the most important means to overcome the SARS–CoV–2 pandemic. They induce specific antibody and T–cell responses but it remains open how well vaccine-induced immunity is preserved over time following homologous and heterologous immunization regimens. Here, we compared the dynamics of humoral and cellular immune responses up to 5 months after homologous or heterologous vaccination with either ChAdOx1–nCoV–19 (ChAd) or BNT162b2 (BNT) or both. Antibody responses significantly waned after vaccination, irrespective of the regimen. The capacity to neutralize SARS-CoV–2 – including variants of concern such as Delta or Omicron – was superior after heterologous compared to homologous BNT vaccination, both of which resulted in longer–lasting humoral immunity than homologous ChAd immunization. T–cell responses showed less waning irrespective of the vaccination regimen. These findings demonstrate that heterologous vaccination with ChAd and BNT is a potent approach to induce long–term humoral and cellular immune protection.