Target arm affinities determine preclinical efficacy and safety of anti-HER2/CD3 bispecific antibody.

Systemic cytokine release and on-target/off-tumor toxicity to normal tissues are the main adverse effects limiting the clinical utility of T cell-redirecting therapies. This study was designed to determine how binding affinity for CD3 and tumor target HER2 impact the efficacy and nonclinical safety of anti-HER2/CD3 T cell-dependent antibodies (TDBs). Affinity was found to be a major determinant for the overall tolerability. Higher affinity for CD3 associated with rapidly elevated peripheral cytokine concentrations, weight loss in mice, and poor tolerability in cynomolgus monkeys. A TDB with lower CD3 affinity was better tolerated in cynomolgus monkeys compared with a higher CD3-affinity TDB. In contrast to tolerability, T cell binding affinity had only limited impact on in vitro and in vivo antitumor activity. High affinity for HER2 was critical for the tumor-killing activity of anti-HER2/CD3 TDBs, but higher HER2 affinity also associated with a more severe toxicity profile, including cytokine release and damage to HER2-expressing tissues. The tolerability of the anti-HER2/CD3 was improved by implementing a dose-fractionation strategy. Fine-tuning the affinities for both the tumor target and CD3 is likely a valuable strategy for achieving maximal therapeutic index of CD3 bispecific antibodies.

CD3 bispecific antibody-induced cytokine release is dispensable for cytotoxic T cell activity.

T cell-retargeting therapies have transformed the therapeutic landscape of oncology. Regardless of the modality, T cell activating therapies are commonly accompanied by systemic cytokine release, which can progress to deadly cytokine release syndrome (CRS). Because of incomplete mechanistic understanding of the relationship between T cell activation and systemic cytokine release, optimal toxicity management that retains full therapeutic potential remains unclear. Here, we report the cell type-specific cellular mechanisms that link CD3 bispecific antibody-mediated killing to toxic cytokine release. The immunologic cascade is initiated by T cell triggering, whereas monocytes and macrophages are the primary source of systemic toxic cytokine release. We demonstrate that T cell-generated tumor necrosis factor-α (TNF-α) is the primary mechanism mediating monocyte activation and systemic cytokine release after CD3 bispecific treatment. Prevention of TNF-α release is sufficient to impair systemic release of monocyte cytokines without affecting antitumor efficacy. Systemic cytokine release is only observed upon initial exposure to CD3 bispecific antibody not subsequent doses, indicating a biological distinction between doses. Despite impaired cytokine release after second exposure, T cell cytotoxicity remained unaffected, demonstrating that cytolytic activity of T cells can be achieved in the absence of cytokine release. The mechanistic uncoupling of toxic cytokines and T cell cytolytic activity in the context of CD3 bispecifics provides a biological rationale to clinically explore preventative treatment approaches to mitigate toxicity.

IFNγ-induced Chemokines Are Required for CXCR3-mediated T-Cell Recruitment and Antitumor Efficacy of Anti-HER2/CD3 Bispecific Antibody.

PURPOSE: The response to cancer immune therapy is dependent on endogenous tumor-reactive T cells. To bypass this requirement, CD3-bispecific antibodies have been developed to induce a polyclonal T-cell response against the tumor. Anti-HER2/CD3 T-cell-dependent bispecific (TDB) antibody is highly efficacious in the treatment of HER2-overexpressing tumors in mice. Efficacy and immunologic effects of anti-HER2/CD3 TDB were investigated in mammary tumor model with very few T cells prior treatment. We further describe the mechanism for TDB-induced T-cell recruitment to tumors. EXPERIMENTAL DESIGN: The immunologic effects and the mechanism of CD3-bispecific antibody-induced T-cell recruitment into spontaneous HER2-overexpressing mammary tumors was studied using human HER2 transgenic, immunocompetent mouse models. RESULTS: Anti-HER2/CD3 TDB treatment induced an inflammatory response in tumors converting them from poorly infiltrated to an inflamed, T-cell abundant, phenotype. Multiple mechanisms accounted for the TDB-induced increase in T cells within tumors. TDB treatment induced CD8+ T-cell proliferation. T cells were also actively recruited post-TDB treatment by IFNγ-dependent T-cell chemokines mediated via CXCR3. This active T-cell recruitment by TDB-induced chemokine signaling was the dominant mechanism and necessary for the therapeutic activity of anti-HER2/CD3 TDB. CONCLUSIONS: In summary, we demonstrate that the activity of anti-HER2/CD3 TDB was not dependent on high-level baseline T-cell infiltration. Our results suggest that anti-HER2/CD3 TDB may be efficacious in patients and indications that respond poorly to checkpoint inhibitors. An active T-cell recruitment mediated by TDB-induced chemokine signaling was the major mechanism for T-cell recruitment.

Lgr5+ stem cells are indispensable for radiation-induced intestinal regeneration.

The intestinal epithelium continually self-renews and can rapidly regenerate after damage. Lgr5 marks mitotically active intestinal stem cells (ISCs). Importantly, intestinal homeostasis can be maintained after depletion of Lgr5(+) cells due to the activation of Lgr5(-) reserve ISCs. The Lgr5(-) ISC populations are thought to play a similar role during intestinal regeneration following radiation-induced damage. We tested this regeneration hypothesis by combining depletion of Lgr5(+) ISCs with radiation exposure. In contrast to the negligible effect of Lgr5(+) ISC loss during homeostasis, depletion of Lgr5(+) cells during radiation-induced damage and subsequent repair caused catastrophic crypt loss and deterioration of crypt-villus architecture. Interestingly though, we found that crypts deficient for Lgr5(+) cells are competent to undergo hyperplasia upon loss of Apc. These data argue that Lgr5(-) reserve stem cells are radiosensitive and that Lgr5(+) cells are crucial for robust intestinal regeneration following radiation exposure but are dispensable for premalignant hyperproliferation.