CD19-CD28: an affinity-optimized CD28 agonist for combination with glofitamab (CD20-TCB) as off-the-shelf immunotherapy.

ABSTRACT: Effective T-cell responses not only require the engagement of T-cell receptors (TCRs; "signal 1"), but also the availability of costimulatory signals ("signal 2"). T-cell bispecific antibodies (TCBs) deliver a robust signal 1 by engaging the TCR signaling component CD3ε, while simultaneously binding to tumor antigens. The CD20-TCB glofitamab redirects T cells to CD20-expressing malignant B cells. Although glofitamab exhibits strong single-agent efficacy, adding costimulatory signaling may enhance the depth and durability of T-cell-mediated tumor cell killing. We developed a bispecific CD19-targeted CD28 agonist (CD19-CD28), RG6333, to enhance the efficacy of glofitamab and similar TCBs by delivering signal 2 to tumor-infiltrating T cells. CD19-CD28 distinguishes itself from the superagonistic antibody TGN1412, because its activity requires the simultaneous presence of a TCR signal and CD19 target binding. This is achieved through its engineered format incorporating a mutated Fc region with abolished FcγR and C1q binding, CD28 monovalency, and a moderate CD28 binding affinity. In combination with glofitamab, CD19-CD28 strongly increased T-cell effector functions in ex vivo assays using peripheral blood mononuclear cells and spleen samples derived from patients with lymphoma and enhanced glofitamab-mediated regression of aggressive lymphomas in humanized mice. Notably, the triple combination of glofitamab with CD19-CD28 with the costimulatory 4-1BB agonist, CD19-4-1BBL, offered substantially improved long-term tumor control over glofitamab monotherapy and respective duplet combinations. Our findings highlight CD19-CD28 as a safe and highly efficacious off-the-shelf combination partner for glofitamab, similar TCBs, and other costimulatory agonists. CD19-CD28 is currently in a phase 1 clinical trial in combination with glofitamab. This trial was registered at www.clinicaltrials.gov as #NCT05219513.

Tissue-resident memory T cells in cytomegalovirus infection.

Herpesviruses establish life-long infection in their hosts and maintain latent reservoirs for sporadic reactivation at peripheral sites, such as skin and mucosae. For herpes simplex virus infection, experimental studies in mice revealed that immediate protection against local reactivation or superinfection events in the skin relies on tissue resident memory T cells (TRM) rather than on their circulating counterparts. Recent evidence extends this notion to cytomegalovirus infection, which potently induces TRM cells in both mice and humans particularly in mucosal tissues that constitute important viral sanctuaries and are relevant entry sites for challenge and superinfections. The discovery unravels promising opportunities to exploit cytomegalovirus based vaccine vectors for the specific induction of tissue resident T cell subsets.

The Salivary Gland Acts as a Sink for Tissue-Resident Memory CD8(+) T Cells, Facilitating Protection from Local Cytomegalovirus Infection.

Tissue-resident memory T cells (TRM) reside in barrier tissues and provide local immediate protective immunity. Here, we show that the salivary gland (SG) most-effectively induces CD8(+) and CD4(+) TRM cells against murine cytomegalovirus (MCMV), which persists in and spreads from this organ. TRM generation depended on local antigen for CD4(+), but not CD8(+), TRM cells, highlighting major differences in T cell subset-specific demands for TRM development. CMV-specific CD8(+) T cells fail to control virus replication upon primary infection in the SG due to CMV-induced MHC I downregulation in glandular epithelial cells. Using intraglandular infection, we challenge this notion and demonstrate that memory CD8(+) T cells confer immediate protection against locally introduced MCMV despite active viral immune evasion, owing to early viral tropism to cells that largely withstand MHC I downregulation. Thus, we unravel a yet-unappreciated role for memory CD8(+) T cells in protecting mucosal tissues against CMV infection.

NK cells gain higher IFN-γ competence during terminal differentiation.

NK cells are the main cells of the innate immune system that produce IFN-γ, and they express this cytokine at early stages of maturation in response to cytokine stimulation. Conversely, acquisition of IFN-γ-competence in CD4(+) T helper cells requires a differentiation process from naïve toward type 1 (Th1) cells, which is associated with epigenetic remodeling at the IFNG locus. In the present study, we show that the ability of NK cells to produce IFN-γ in response to activating receptor (actR) engagement is gradually acquired during terminal differentiation and is accompanied by progressively higher NF-κB activation in response to actR triggering. Moreover, during the differentiation process NK cells gradually display increasing expression of IFNG and TBX21 (encoding T-bet) transcripts and demethylation at the IFNG promoter. This study provides new insights in the molecular mechanisms underlying NK-cell ability to express IFN-γ upon actR engagement. Thus, we propose that in order to efficiently produce IFN-γ in response to infected or transformed cells, NK cells gain Th1-like features, such as higher IFN-γ competence and epigenetic remodeling of the IFNG promoter, during their terminal differentiation.

Salivary gland resident APCs are Flt3L- and CCR2-independent macrophage-like cells incapable of cross-presentation.

Cytomegaloviruses (CMVs) disseminate within the human population via mucosal excretions, for example, from the salivary glands (SGs), which represent a privileged site of viral immune evasion and persistence. The murine CMV (MCMV) model has served to identify factors that maintain a unique virus-host relationship in this organ. In contrast to all other organs, the SG is resistant to CD8(+) T-cell mediated control of MCMV replication due to virally induced MHC class I downregulation, which is exceptionally efficient in acinar glandular epithelial cells. Uniquely to the SG, IFN-γ producing CD4(+) T cells are required for virus control. While T-cell responses have been extensively characterized in the SG, the ontogeny and function of APCs in this organ remain to be assessed. Here, we show that macrophage-like cells constitute the population of SG-resident APCs in steady state and during MCMV-induced inflammation in mice. Inflammatory monocytes, monocyte-derived DCs as well as conventional, Flt3L-dependent DCs do not contribute to this population. Despite supporting contact formation to CD4(+) and CD8(+) T cells in principle, SG-resident APCs fail to activate the latter due to their inability to cross-present MCMV-derived antigen.