LTßR Agonism Promotes Anti-Tumor Immune Responses via Modulation of the Tumor Microenvironment.

The presence of high endothelial venules (HEV) and tertiary lymphoid structures (TLS) in solid tumors is correlated with favorable prognosis and better responses to immune-checkpoint blockade (ICB) in many cancer types. Elucidation of the molecular mechanisms underlying intratumoral HEV and TLS formation and their contribution to anti-tumor responses may facilitate development of improved treatment strategies. Lymphotoxin beta receptor (LTßR) signaling is a critical regulator of lymph node organogenesis and can cooperate with antiangiogenic and ICB treatment to augment tumor-associated HEV formation. Here, we demonstrated that LTßR signaling modulates the tumor microenvironment via multiple mechanisms to promote anti-tumor T cell responses. Systemic activation of the LTßR pathway via agonistic antibody treatment induced tumor-specific HEV formation, upregulated the expression of TLS-related chemokines, and enhanced dendritic cell (DC) and T cell infiltration and activation in syngeneic tumor models. In vitro studies confirmed direct effects of LTßR agonism on DC activation and maturation and associated DC-mediated T cell activation. Single agent LTßR agonist treatment inhibited syngeneic tumor growth in a CD8+ T cell- and HEV-dependent manner, and the LTßR agonist enhanced anti-tumor effects of anti-PD-1 and CAR T cell therapies. An in vivo tumor screen for TLS-inducing cytokines revealed that the combination of LTßR agonism and lymphotoxin alpha (LT⍺) expression promoted robust intratumoral TLS induction and enhanced tumor responses to anti-CTLA-4 treatment. Collectively, this study highlights crucial functions of LTßR signaling in modulating the tumor microenvironment and could inform future HEV/TLS-based strategies for cancer treatments.

Generation of T-cell-redirecting bispecific antibodies with differentiated profiles of cytokine release and biodistribution by CD3 affinity tuning.

T-cell-redirecting bispecific antibodies have emerged as a new class of therapeutic agents designed to simultaneously bind to T cells via CD3 and to tumor cells via tumor-cell-specific antigens (TSA), inducing T-cell-mediated killing of tumor cells. The promising preclinical and clinical efficacy of TSAxCD3 antibodies is often accompanied by toxicities such as cytokine release syndrome due to T-cell activation. How the efficacy and toxicity profile of the TSAxCD3 bispecific antibodies depends on the binding affinity to CD3 remains unclear. Here, we evaluate bispecific antibodies that were engineered to have a range of CD3 affinities, while retaining the same binding affinity for the selected tumor antigen. These agents were tested for their ability to kill tumor cells in vitro, and their biodistribution, serum half-life, and anti-tumor activity in vivo. Remarkably, by altering the binding affinity for CD3 alone, we can generate bispecific antibodies that maintain potent killing of TSA + tumor cells but display differential patterns of cytokine release, pharmacokinetics, and biodistribution. Therefore, tuning CD3 affinity is a promising method to improve the therapeutic index of T-cell-engaging bispecific antibodies.

A BCMAxCD3 bispecific T cell-engaging antibody demonstrates robust antitumor efficacy similar to that of anti-BCMA CAR T cells.

CD3-engaging bispecific antibodies (bsAbs) and chimeric antigen receptor (CAR) T cells are potent therapeutic approaches for redirecting patient T cells to recognize and kill tumors. Here we describe a fully human bsAb (REGN5458) that binds to B-cell maturation antigen (BCMA) and CD3, and compare its antitumor activities vs those of anti-BCMA CAR T cells to identify differences in efficacy and mechanism of action. In vitro, BCMAxCD3 bsAb efficiently induced polyclonal T-cell killing of primary human plasma cells and multiple myeloma (MM) cell lines expressing a range of BCMA cell surface densities. In vivo, BCMAxCD3 bsAb suppressed the growth of human MM tumors in murine xenogeneic models and showed potent combinatorial efficacy with programmed cell death protein 1 blockade. BCMAxCD3 bsAb administration to cynomolgus monkeys was well tolerated, resulting in the depletion of BCMA+ cells and mild inflammatory responses characterized by transient increases in C-reactive protein and serum cytokines. The antitumor efficacy of BCMAxCD3 bsAb was compared with BCMA-specific CAR T cells containing a BCMA-binding single-chain variable fragment derived from REGN5458. Both BCMAxCD3 bsAb and anti-BCMA CAR T cells showed similar targeted cytotoxicity of MM cell lines and primary MM cells in vitro. In head-to-head in vivo studies, BCMAxCD3 bsAb rapidly cleared established systemic MM tumors, whereas CAR T cells cleared tumors with slower kinetics. Thus, using the same BCMA-binding domain, these results suggest that BCMAxCD3 bsAb rapidly exerts its therapeutic effects by engaging T cells already in place at the tumor site, whereas anti-BCMA CAR T cells require time to traffic to the tumor site, activate, and numerically expand before exerting antitumor effects.

Cytokine-producing effector B cells regulate type 2 immunity to H. polygyrus.

Immunity to the intestinal parasite Heligomosomoides polygyrus is dependent on the successful generation of T helper 2 (Th2) memory cells. We showed that B cells contribute to immunity against H. polygyrus by producing antibody (Ab) and by promoting expansion and differentiation of primary and memory Th2 cells. We also demonstrated that cytokine-producing effector B cells were essential for effective immunity to H. polygyrus. Tumor necrosis factor alpha production by B cells was necessary for sustained Ab production, whereas interleukin 2 production by B cells was necessary for Th2 cell expansion and differentiation. These results show that B cells mediate protection from pathogens not only by presenting antigen and secreting antibody but also by producing cytokines that regulate the quality and magnitude of humoral and cellular immune responses.

Mast cells, basophils, and eosinophils acquire constitutive IL-4 and IL-13 transcripts during lineage differentiation that are sufficient for rapid cytokine production.

Mast cells, basophils, and eosinophils are myeloid cells that are distinguished by their capability to produce IL-4 and IL-13. However, it is not clear how this potential is related to the lineage differentiation of these subsets. In the present study we used bicistronic IL-4 reporter (4get) mice to directly visualize IL-4 expression by nonlymphoid cells in vitro and in vivo at the single-cell level. Our data show that frequent expression of both Il4 alleles is initiated and maintained during ontogeny by an IL-4Ralpha- or Stat6-independent mechanism. Despite the constitutive presence of cytokine transcripts in differentiated cells under steady state conditions, cytokine production is not detectable in the absence of stimulation. Moreover, mature mast cells, basophils, and eosinophils also constitutively express IL-13. Both preformed IL-4 and IL-13 mRNAs are sufficient for rapid cytokine production upon stimulation. Our data show that mast cells, basophils, and eosinophils are programmed for IL-4 and IL-13 expression early in ontogeny. These novel findings have important implications for the prevention and therapeutic intervention of allergic and asthmatic diseases.