Cytotoxicity of the CD3×CD20 bispecific antibody epcoritamab in CLL is increased by concurrent BTK or BCL-2 targeting.

Chronic lymphocytic leukemia (CLL) is an immunosuppressive disease characterized by increased infectious morbidity and inferior antitumor activity of immunotherapies. Targeted therapy with Bruton's tyrosine kinase inhibitors (BTKis) or the Bcl-2 inhibitor venetoclax has profoundly improved treatment outcomes in CLL. To overcome or prevent drug resistance and extend the duration of response after a time-limited therapy, combination regimens are tested. Anti-CD20 antibodies that recruit cell- and complement-mediated effector functions are commonly used. Epcoritamab (GEN3013), an anti-CD3×CD20 bispecific antibody that recruits T-cell effector functions, has demonstrated potent clinical activity in patients with relapsed CD20+ B-cell non-Hodgkin lymphoma. Development of CLL therapy is ongoing. To characterize epcoritamab-mediated cytotoxicity against primary CLL cells, peripheral blood mononuclear cells from treatment-naive and BTKi-treated patients, including patients progressing on therapy, were cultured with epcoritamab alone or in combination with venetoclax. Ongoing treatment with BTKi and high effector-to-target ratios were associated with superior in vitro cytotoxicity. Cytotoxic activity was independent of CD20 expression on CLL cells and observed in samples from patients whose condition progressed while receiving BTKi. Epcoritamab induced significant T-cell expansion, activation, and differentiation into Th1 and effector memory cells in all patient samples. In patient-derived xenografts, epcoritamab reduced the blood and spleen disease burden compared with that in mice receiving a nontargeting control. In vitro, the combination of venetoclax with epcoritamab induced superior killing of CLL cells than either agent alone. These data support the investigation of epcoritamab in combination with BTKis or venetoclax to consolidate responses and target emergent drug-resistant subclones.

Patient-derived Siglec-6-targeting antibodies engineered for T-cell recruitment have potential therapeutic utility in chronic lymphocytic leukemia.

BACKGROUND: Despite numerous therapeutic options, safe and curative therapy is unavailable for most patients with chronic lymphocytic leukemia (CLL). A drawback of current therapies such as the anti-CD20 monoclonal antibody (mAb) rituximab is the elimination of all healthy B cells, resulting in impaired humoral immunity. We previously reported the identification of a patient-derived, CLL-binding mAb, JML-1, and identified sialic acid-binding immunoglobulin-like lectin-6 (Siglec-6) as the target of JML-1. Although little is known about Siglec-6, it appears to be an attractive target for cancer immunotherapy due to its absence on most healthy cells and tissues. METHODS: We used a target-specific approach to mine for additional patient-derived anti-Siglec-6 mAbs. To assess the therapeutic utility of targeting Siglec-6 in the context of CLL, T cell-recruiting bispecific antibodies (T-biAbs) that bind to Siglec-6 and CD3 were engineered into single-chain variable fragment-Fc and dual-affinity retargeting (DART)-Fc constructs. T-biAbs were evaluated for their activity in vitro, ex vivo, and in vivo. RESULTS: We discovered the anti-Siglec-6 mAbs RC-1 and RC-2, which bind with higher affinity than JML-1 yet maintain similar specificity. Both JML-1 and RC-1 T-biAbs were effective at activating T cells and killing Siglec-6+ target cells. The RC-1 clone in the DART-Fc format was the most potent T-biAb tested and was the only anti-Siglec-6 T-biAb that eliminated Siglec-6+ primary CLL cells via autologous T cells at pathological T-to-CLL cell ratios. Tested at healthy T-to-B cell ratios, it also eliminated a Siglec-6+ fraction of primary B cells from healthy donors. The subpicomolar potency of the DART-Fc format was attributed to the reduction in the length and flexibility of the cytolytic synapse. Furthermore, the RC-1 T-biAb was effective at clearing MEC1 CLL cells in vivo and demonstrated a circulatory half-life of over 7 days. CONCLUSION: Siglec-6-targeting T-biAbs are highly potent and specific for eliminating Siglec-6+ leukemic and healthy B cells while sparing Siglec-6- healthy B cells, suggesting a unique treatment strategy for CLL with diminished suppression of humoral immunity. Our data corroborate reports that T-biAb efficacy is dependent on synapse geometry and reveal that synapse architecture can be tuned via antibody engineering. Our fully human anti-Siglec-6 antibodies and T-biAbs have potential for cancer immunotherapy. TRIAL REGISTRATION NUMBER: NCT00923507.

BTK inhibitors, irrespective of ITK inhibition, increase efficacy of a CD19/CD3-bispecific antibody in CLL.

Bruton tyrosine kinase inhibitors (BTKis) are a preferred treatment of patients with chronic lymphocytic leukemia (CLL). Indefinite therapy with BTKis, although effective, presents clinical challenges. Combination therapy can deepen responses, shorten treatment duration, and possibly prevent or overcome drug resistance. We previously reported on a CD19/CD3-bispecific antibody (bsAb) that recruits autologous T-cell cytotoxicity against CLL cells in vitro. Compared with observations with samples from treatment-naïve patients, T cells from patients being treated with ibrutinib expanded more rapidly and exerted superior cytotoxic activity in response to the bsAb. In addition to BTK, ibrutinib also inhibits interleukin-2 inducible T-cell kinase (ITK). In contrast, acalabrutinib, does not inhibit ITK. Whether ITK inhibition contributes to the observed immune effects is unknown. To better understand how BTKis modulate T-cell function and cytotoxic activity, we cultured peripheral blood mononuclear cells (PBMCs) from BTKi-naive and ibrutinib- or acalabrutinib-treated CLL patients with CD19/CD3 bsAb in vitro. T-cell expansion, activation, differentiation, and cytotoxicity were increased in PBMCs from patients on treatment with either BTKi compared with that observed for BKTi-naïve patients. BTKi therapy transcriptionally downregulated immunosuppressive effectors expressed by CLL cells, including cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) and CD200. CTLA-4 blockade with ipilimumab in vitro increased the cytotoxic activity of the bsAb in BTKi-naïve but not BTKi-treated PBMCS. Taken together, BTKis enhance bsAb-induced cytotoxicity by relieving T cells of immunosuppressive restraints imposed by CLL cells. The benefit of combining bsAb immunotherapy with BTKis needs to be confirmed in clinical trials.