T cell-Dependent Bispecific Therapy Enhances Innate Immune Activation and Antibody-Mediated Killing.

T cell-retargeting therapies have transformed the therapeutic landscape for hematologic diseases. T cell-dependent bispecific antibodies (TDB) function as conditional agonists that induce a polyclonal T-cell response, resulting in target cell destruction and cytokine release. The relationship between this response and its effects on surrounding innate immune populations has not been fully explored. Here we show that treatment with mosunetuzumab in patients results in natural killer (NK) cell activation in the peripheral blood. We modeled this phenomenon in vitro and found that TDB-mediated killing activated NK cells, increasing NK function and antibody-dependent cellular cytotoxicity (ADCC), and enhanced the capability of macrophages to perform antibody-dependent cellular phagocytosis (ADCP). This enhancement was triggered by cytokines released through TDB treatment, with IL2 and IFNγ being major drivers for increased ADCC and ADCP, respectively. Surprisingly, cytolytic ability could be further augmented through neutralization of IL10 for NK cells and TNFα for macrophages. Finally, we showed that TDB treatment enhanced the efficacy of Fc-driven killing to an orthogonal solid tumor target in vivo. These results provide rationale for novel antibody therapy combinations that take advantage of both adaptive and innate immune responses.

Loss of CD20 expression as a mechanism of resistance to mosunetuzumab in relapsed/refractory B-cell lymphomas.

ABSTRACT: CD20 is an established therapeutic target in B-cell malignancies. The CD20 × CD3 bispecific antibody mosunetuzumab has significant efficacy in B-cell non-Hodgkin lymphomas (NHLs). Because target antigen loss is a recognized mechanism of resistance, we evaluated CD20 expression relative to clinical response in patients with relapsed and/or refractory NHL in the phase 1/2 GO29781 trial investigating mosunetuzumab monotherapy. CD20 was studied using immunohistochemistry (IHC), RNA sequencing, and whole-exome sequencing performed centrally in biopsy specimens collected before treatment at predose, during treatment, or upon progression. Before treatment, most patients exhibited a high proportion of tumor cells expressing CD20; however, in 16 of 293 patients (5.5%) the proportion was <10%. Analyses of paired biopsy specimens from patients on treatment revealed that CD20 levels were maintained in 29 of 30 patients (97%) vs at progression, where CD20 loss was observed in 11 of 32 patients (34%). Reduced transcription or acquisition of truncating mutations explained most but not all cases of CD20 loss. In vitro modeling confirmed the effects of CD20 variants identified in clinical samples on reduction of CD20 expression and missense mutations in the extracellular domain that could block mosunetuzumab binding. This study expands the knowledge about the occurrence of target antigen loss after anti-CD20 therapeutics to include CD20-targeting bispecific antibodies and elucidates mechanisms of reduced CD20 expression at disease progression that may be generalizable to other anti-CD20 targeting agents. These results also confirm the utility of readily available IHC staining for CD20 as a tool to inform clinical decisions. This trial was registered at www.ClinicalTrials.gov as #NCT02500407.

Targeting MCL-1 and BCL-2 with polatuzumab vedotin and venetoclax overcomes treatment resistance in R/R non-Hodgkin lymphoma: Results from preclinical models and a Phase Ib study.

The treatment of patients with relapsed or refractory lymphoid neoplasms represents a significant clinical challenge. Here, we identify the pro-survival BCL-2 protein family member MCL-1 as a resistance factor for the BCL-2 inhibitor venetoclax in non-Hodgkin lymphoma (NHL) cell lines and primary NHL samples. Mechanistically, we show that the antibody-drug conjugate polatuzumab vedotin promotes MCL-1 degradation via the ubiquitin/proteasome system. This targeted MCL-1 antagonism, when combined with venetoclax and the anti-CD20 antibodies obinutuzumab or rituximab, results in tumor regressions in preclinical NHL models, which are sustained even off-treatment. In a Phase Ib clinical trial (NCT02611323) of heavily pre-treated patients with relapsed or refractory NHL, 25/33 (76%) patients with follicular lymphoma and 5/17 (29%) patients with diffuse large B-cell lymphoma achieved complete or partial responses with an acceptable safety profile when treated with the recommended Phase II dose of polatuzumab vedotin in combination with venetoclax and an anti-CD20 antibody.

Development of T-cell engagers selective for cells co-expressing two antigens.

T cell-engaging bispecific antibodies (TCEs) are clinically effective treatments for hematological cancers. While the utility of TCEs in solid malignancies is being explored, toxicities arising from antigen expression on normal tissues have slowed or halted several clinical trials. Here, we describe the development of TCEs that preferentially drive T cell-mediated death against target cells co-expressing two tumor-associated antigens. We show that Ly6E and B7-H4 are simultaneously expressed on approximately 50% of breast cancers, whereas normal tissue expression is limited and mostly orthogonal. Traditional bispecific TCEs targeting a singular antigen, either Ly6E or B7-H4, are active when paired with high-affinity CD3-engagers, but normal tissue expression presents a toxicity risk. Treatment with a murine cross-reactive B7-H4-TCE results in rapid and severe weight loss in mice along with damage to B7-H4-expressing tissues. To overcome on-target toxicity, we designed trispecific antibodies co-targeting Ly6E, B7-H4, and CD3 and characterized the impact of dual-antigen binding and the relative placement of each binding domain on tumor killing in vitro and in vivo. In vitro killing of tumor cells co-expressing both antigens correlates to the placement of the higher affinity B7-H4 binding domain, with only modest enhancements seen upon addition of Ly6E binding. In xenograft models, avid binding of appropriately designed trispecific TCEs enables tumor growth inhibition while evading the poor tolerability seen with active bispecific TCEs. Collectively these data highlight the potential for dual-antigen targeting to improve safety and efficacy, and expand the scope of tumors that may effectively be treated by TCEs.Abbreviations: Chimeric antigen receptor T cells (CAR-Ts), dual-antigen targeted T cell engagers (DAT-TCE), Fragment antigen-binding (Fab), Hematoxylin and eosin (H&E), Institutional Animal Care and Use Committee (IACUC), Immunoglobulin G (IgG), immunohistochemistry (IHC), NOD SCID gamma (NSG), peripheral blood mononuclear cells (PBMCs), surface plasmon resonance (SPR), T cell-engagers (TCEs).

Novel Anti-LY6G6D/CD3 T-Cell-Dependent Bispecific Antibody for the Treatment of Colorectal Cancer.

New therapeutics and combination regimens have led to marked clinical improvements for the treatment of a subset of colorectal cancer. Immune checkpoint inhibitors have shown clinical efficacy in patients with mismatch-repair-deficient or microsatellite instability-high (MSI-H) metastatic colorectal cancer (mCRC). However, patients with microsatellite-stable (MSS) or low levels of microsatellite instable (MSI-L) colorectal cancer have not benefited from these immune modulators, and the survival outcome remains poor for the majority of patients diagnosed with mCRC. In this article, we describe the discovery of a novel T-cell-dependent bispecific antibody (TDB) targeting tumor-associated antigen LY6G6D, LY6G6D-TDB, for the treatment of colorectal cancer. RNAseq analysis showed that LY6G6D was differentially expressed in colorectal cancer with high prevalence in MSS and MSI-L subsets, whereas LY6G6D expression in normal tissues was limited. IHC confirmed the elevated expression of LY6G6D in primary and metastatic colorectal tumors, whereas minimal or no expression was observed in most normal tissue samples. The optimized LY6G6D-TDB, which targets a membrane-proximal epitope of LY6G6D and binds to CD3 with high affinity, exhibits potent antitumor activity both in vitro and in vivo. In vitro functional assays show that LY6G6D-TDB-mediated T-cell activation and cytotoxicity are conditional and target dependent. In mouse xenograft tumor models, LY6G6D-TDB demonstrates antitumor efficacy as a single agent against established colorectal tumors, and enhanced efficacy can be achieved when LY6G6D-TDB is combined with PD-1 blockade. Our studies provide evidence for the therapeutic potential of LY6G6D-TDB as an effective treatment option for patients with colorectal cancer.

Anti-CD79B Antibody-Drug Conjugate DCDS0780A in Patients with B-Cell Non-Hodgkin Lymphoma: Phase 1 Dose-Escalation Study.

PURPOSE: Targeting CD79B using antibody-drug conjugates (ADC) is an effective therapeutic strategy in B-cell non-Hodgkin lymphoma (B-NHL). We investigated DCDS0780A, an anti-CD79B ADC with THIOMAB technology (TDC) that consistently conjugates two anti-neoplastic molecules per antibody, in contrast with ADCs with heterogeneous loads. PATIENTS AND METHODS: This phase 1 study enrolled 60 patients with histologically confirmed B-NHL that had relapsed/failed to respond following ≥1 prior treatment regimens; 41 (68%) had diffuse large B-cell lymphoma (DLBCL). Fifty-one patients received DCDS0780A monotherapy once every 3 weeks (0.3-4.8 mg/kg); 9 received combination therapy (3.6-4.8 mg/kg) with rituximab. RESULTS: Fifty-four (90%) patients experienced adverse events related to study drug, the most common of which were blurred vision, fatigue, corneal deposits, neutropenia, nausea, and peripheral neuropathy. 4.8 mg/kg was the highest dose tested and the recommended phase II dose. The pharmacokinetic profile was linear at doses ≥1.2 mg/kg. Response rate in all-treated patients (N = 60) was 47% (n = 28), including 17 complete responses (28%) and 11 partial responses (18%). The median duration of response (15.2 months) was the same for all responders (n = 28) and patients with DLBCL (n = 20). CONCLUSIONS: DCDS0780A as the TDC format for CD79B was tested at higher doses than its ADC counterpart investigated earlier, leading to deep responses. However, dose intensity was limited by ocular toxicities seen at the higher doses indicating that the TDC format was unable, in the current study, to expand the therapeutic index for the CD79B target. The encouraging antitumor activity advocates continuation of investigations into novel ADC technologies.

A BCMA/CD16A bispecific innate cell engager for the treatment of multiple myeloma.

Despite the recent progress, multiple myeloma (MM) is still essentially incurable and there is a need for additional effective treatments with good tolerability. RO7297089 is a novel bispecific BCMA/CD16A-directed innate cell engager (ICE®) designed to induce BCMA+ MM cell lysis through high affinity binding of CD16A and retargeting of NK cell cytotoxicity and macrophage phagocytosis. Unlike conventional antibodies approved in MM, RO7297089 selectively targets CD16A with no binding of other Fcγ receptors, including CD16B on neutrophils, and irrespective of 158V/F polymorphism, and its activity is less affected by competing IgG suggesting activity in the presence of M-protein. Structural analysis revealed this is due to selective interaction with a single residue (Y140) uniquely present in CD16A opposite the Fc binding site. RO7297089 induced tumor cell killing more potently than conventional antibodies (wild-type and Fc-enhanced) and induced lysis of BCMA+ cells at very low effector-to-target ratios. Preclinical toxicology data suggested a favorable safety profile as in vitro cytokine release was minimal and no RO7297089-related mortalities or adverse events were observed in cynomolgus monkeys. These data suggest good tolerability and the potential of RO7297089 to be a novel effective treatment of MM patients.

Calicheamicin Antibody-Drug Conjugates with Improved Properties.

Calicheamicin antibody-drug conjugates (ADCs) are effective therapeutics for leukemias with two recently approved in the United States: Mylotarg (gemtuzumab ozogamicin) targeting CD33 for acute myeloid leukemia and Besponsa (inotuzumab ozogamicin) targeting CD22 for acute lymphocytic leukemia. Both of these calicheamicin ADCs are heterogeneous, aggregation-prone, and have a shortened half-life due to the instability of the acid-sensitive hydrazone linker in circulation. We hypothesized that we could improve upon the heterogeneity, aggregation, and circulation stability of calicheamicin ADCs by directly attaching the thiol of a reduced calicheamicin to an engineered cysteine on the antibody via a disulfide bond to generate a linkerless and traceless conjugate. We report herein that the resulting homogeneous conjugates possess minimal aggregation and display high in vivo stability with 50% of the drug remaining conjugated to the antibody after 21 days. Furthermore, these calicheamicin ADCs are highly efficacious in mouse models of both solid tumor (HER2+ breast cancer) and hematologic malignancies (CD22+ non-Hodgkin lymphoma). Safety studies in rats with this novel calicheamicin ADC revealed an increased tolerability compared with that reported for Mylotarg. Overall, we demonstrate that applying novel linker chemistry with site-specific conjugation affords an improved, next-generation calicheamicin ADC.

Preclinical optimization of Ly6E-targeted ADCs for increased durability and efficacy of anti-tumor response.

Early success with brentuximab vedotin in treating classical Hodgkin lymphoma spurred an influx of at least 20 monomethyl auristatin E (MMAE) antibody-drug conjugates (ADCs) into clinical trials. While three MMAE-ADCs have been approved, most of these conjugates are no longer being investigated in clinical trials. Some auristatin conjugates show limited or no efficacy at tolerated doses, but even for drugs driving initial remissions, tumor regrowth and metastasis often rapidly occur. Here we describe the development of second-generation therapeutic ADCs targeting Lymphocyte antigen 6E (Ly6E) where the tubulin polymerization inhibitor MMAE (Compound 1) is replaced with DNA-damaging agents intended to drive increased durability of response. Comparison of a seco-cyclopropyl benzoindol-4-one (CBI)-dimer (compound 2) to MMAE showed increased potency, activity across more cell lines, and resistance to efflux by P-glycoprotein, a drug transporter commonly upregulated in tumors. Both anti-Ly6E-CBI and -MMAE conjugates drove single-dose efficacy in xenograft and patient-derived xenograft models, but seco-CBI-dimer conjugates showed reduced tumor outgrowth following multiple weeks of treatment, suggesting that they are less susceptible to developing resistance. In parallel, we explored approaches to optimize the targeting antibody. In contrast to immunization with recombinant Ly6E or Ly6E DNA, immunization with virus-like particles generated a high-affinity anti-Ly6E antibody. Conjugates to this antibody improve efficacy versus a previous clinical candidate both in vitro and in vivo with multiple cytotoxics. Conjugation of compound 2 to the second-generation antibody results in a substantially improved ADC with promising preclinical efficacy.