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-LYPD1/CD3 T-Cell-Dependent Bispecific Antibody for the Treatment of Ovarian Cancer.

Ovarian cancer is a diverse class of tumors with very few effective treatment options and suboptimal response rates in early clinical studies using immunotherapies. Here we describe LY6/PLAUR domain containing 1 (LYPD1) as a novel target for therapeutic antibodies for the treatment of ovarian cancer. LYPD1 is broadly expressed in both primary and metastatic ovarian cancer with ∼70% prevalence in the serous cancer subset. Bispecific antibodies targeting CD3 on T cells and a tumor antigen on cancer cells have demonstrated significant clinical activity in hematologic cancers. We have developed an anti-LYPD1/CD3 T-cell-dependent bispecific antibody (TDB) to redirect T-cell responses to LYPD1 expressing ovarian cancer. Here we characterize the nonclinical pharmacology of anti-LYPD1/CD3 TDB and show induction of a robust polyclonal T-cell activation and target dependent killing of LYPD1 expressing ovarian cancer cells resulting in efficient in vivo antitumor responses in PBMC reconstituted immune-deficient mice and human CD3 transgenic mouse models. Anti-LYPD1/CD3 TDB is generally well tolerated at high-dose levels in mice, a pharmacologically relevant species, and showed no evidence of toxicity or damage to LYPD1 expressing tissues.

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.

Avidity-based binding to HER2 results in selective killing of HER2-overexpressing cells by anti-HER2/CD3.

A primary barrier to the success of T cell-recruiting bispecific antibodies in the treatment of solid tumors is the lack of tumor-specific targets, resulting in on-target off-tumor adverse effects from T cell autoreactivity to target-expressing organs. To overcome this, we developed an anti-HER2/CD3 T cell-dependent bispecific (TDB) antibody that selectively targets HER2-overexpressing tumor cells with high potency, while sparing cells that express low amounts of HER2 found in normal human tissues. Selectivity is based on the avidity of two low-affinity anti-HER2 Fab arms to high target density on HER2-overexpressing cells. The increased selectivity to HER2-overexpressing cells is expected to mitigate the risk of adverse effects and increase the therapeutic index. Results included in this manuscript not only support the clinical development of anti-HER2/CD3 1Fab-immunoglobulin G TDB but also introduce a potentially widely applicable strategy for other T cell-directed therapies. The potential of this discovery has broad applications to further enable consideration of solid tumor targets that were previously limited by on-target, but off-tumor, autoimmunity.

Relative Target Affinities of T-Cell-Dependent Bispecific Antibodies Determine Biodistribution in a Solid Tumor Mouse Model.

Anti-HER2/CD3, a T-cell-dependent bispecific antibody (TDB) construct, induces T-cell-mediated cell death in cancer cells expressing HER2 by cross-linking tumor HER2 with CD3 on cytotoxic T cells, thereby creating a functional cytolytic synapse. TDB design is a very challenging process that requires consideration of multiple parameters. Although therapeutic antibody design strategy is commonly driven by striving for the highest attainable antigen-binding affinity, little is known about how the affinity of each TDB arm can affect the targeting ability of the other arm and the consequent distribution and efficacy. To our knowledge, no distribution studies have been published using preclinical models wherein the T-cell-targeting arm of the TDB is actively bound to T cells. We used a combined approach involving radiochemistry, invasive biodistribution, and noninvasive single-photon emission tomographic (SPECT) imaging to measure TDB distribution and catabolism in transgenic mice with human CD3ε expression on T cells. Using CD3 affinity variants, we assessed the impact of CD3 affinity on short-term pharmacokinetics, tissue distribution, and cellular uptake. Our experimental approach determined the relative effects of (i) CD3 targeting to normal tissues, (ii) HER2 targeting to HER2-expressing tumors, and (iii) relative HER2/CD3 affinity, all as critical drivers for TDB distribution. We observed a strong correlation between CD3 affinity and distribution to T-cell-rich tissues, with higher CD3 affinity reducing systemic exposure and shifting TDB distribution away from tumor to T-cell-containing tissues. These observations have important implications for clinical translation of bispecific antibodies for cancer immunotherapy. Mol Cancer Ther; 17(4); 776-85. ©2018 AACR.

Membrane-Proximal Epitope Facilitates Efficient T Cell Synapse Formation by Anti-FcRH5/CD3 and Is a Requirement for Myeloma Cell Killing.

The anti-FcRH5/CD3 T cell-dependent bispecific antibody (TDB) targets the B cell lineage marker FcRH5 expressed in multiple myeloma (MM) tumor cells. We demonstrate that TDBs trigger T cell receptor activation by inducing target clustering and exclusion of CD45 phosphatase from the synapse. The dimensions of the target molecule play a key role in the efficiency of the synapse formation. The anti-FcRH5/CD3 TDB kills human plasma cells and patient-derived myeloma cells at picomolar concentrations and results in complete depletion of B cells and bone marrow plasma cells in cynomolgus monkeys. These data demonstrate the potential for the anti-FcRH5/CD3 TDB, alone or in combination with inhibition of PD-1/PD-L1 signaling, in the treatment of MM and other B cell malignancies.

An anti-CD3/anti-CLL-1 bispecific antibody for the treatment of acute myeloid leukemia.

Acute myeloid leukemia (AML) is a major unmet medical need. Most patients have poor long-term survival, and treatment has not significantly changed in 40 years. Recently, bispecific antibodies that redirect the cytotoxic activity of effector T cells by binding to CD3, the signaling component of the T-cell receptor, and a tumor target have shown clinical activity. Notably, blinatumomab is approved to treat relapsed/refractory acute lymphoid leukemia. Here we describe the design, discovery, pharmacologic activity, pharmacokinetics, and safety of a CD3 T cell-dependent bispecific (TDB) full-length human IgG1 therapeutic antibody targeting CLL-1 that could potentially be used in humans to treat AML. CLL-1 is prevalent in AML and, unlike other targets such as CD33 and CD123, is not expressed on hematopoietic stem cells providing potential hematopoietic recovery. We selected a high-affinity monkey cross-reactive anti-CLL-1 arm and tested several anti-CD3 arms that varied in affinity, and determined that the high-affinity CD3 arms were up to 100-fold more potent in vitro. However, in mouse models, the efficacy differences were less pronounced, probably because of prolonged exposure to TDB found with lower-affinity CD3 TDBs. In monkeys, assessment of safety and target cell depletion by the high- and low-affinity TDBs revealed that only the low-affinity CD3/CLL1 TDB was well tolerated and able to deplete target cells. Our data suggest that an appropriately engineered CLL-1 TDB could be effective in the treatment of AML.

Preclinical Development of an Anti-NaPi2b (SLC34A2) Antibody-Drug Conjugate as a Therapeutic for Non-Small Cell Lung and Ovarian Cancers.

PURPOSE: Antibody-drug conjugates (ADC) selectively deliver a cytotoxic drug to cells expressing an accessible antigenic target. Here, we have appended monomethyl auristatin E (MMAE) to an antibody recognizing the SLC34A2 gene product NaPi2b, the type II sodium-phosphate cotransporter, which is highly expressed on tumor surfaces of the lung, ovary, and thyroid as well as on normal lung pneumocytes. This study evaluated its efficacy and safety in preclinical studies. EXPERIMENTAL DESIGN: The efficacy of anti-NaPi2b ADC was evaluated in mouse ovarian and non-small cell lung cancer (NSCLC) tumor xenograft models, and its toxicity was assessed in rats and cynomolgus monkeys. RESULTS: We show here that an anti-NaPi2b ADC is effective in mouse ovarian and NSCLC tumor xenograft models and well-tolerated in rats and cynomolgus monkeys at levels in excess of therapeutic doses. Despite high levels of expression in normal lung of non-human primate, the cross-reactive ADC exhibited an acceptable safety profile with a dose-limiting toxicity unrelated to normal tissue target expression. The nonproliferative nature of normal pneumocytes, together with the antiproliferative mechanism of MMAE, likely mitigates the potential liability of this normal tissue expression. CONCLUSIONS: Overall, our preclinical results suggest that the ADC targeting NaPi2b provides an effective new therapy for the treatment of NSCLC and ovarian cancer and is currently undergoing clinical developments.

Anti-CD20/CD3 T cell-dependent bispecific antibody for the treatment of B cell malignancies.

Bispecific antibodies and antibody fragments in various formats have been explored as a means to recruit cytolytic T cells to kill tumor cells. Encouraging clinical data have been reported with molecules such as the anti-CD19/CD3 bispecific T cell engager (BiTE) blinatumomab. However, the clinical use of many reported T cell-recruiting bispecific modalities is limited by liabilities including unfavorable pharmacokinetics, potential immunogenicity, and manufacturing challenges. We describe a B cell-targeting anti-CD20/CD3 T cell-dependent bispecific antibody (CD20-TDB), which is a full-length, humanized immunoglobulin G1 molecule with near-native antibody architecture constructed using "knobs-into-holes" technology. CD20-TDB is highly active in killing CD20-expressing B cells, including primary patient leukemia and lymphoma cells both in vitro and in vivo. In cynomolgus monkeys, CD20-TDB potently depletes B cells in peripheral blood and lymphoid tissues at a single dose of 1 mg/kg while demonstrating pharmacokinetic properties similar to those of conventional monoclonal antibodies. CD20-TDB also exhibits activity in vitro and in vivo in the presence of competing CD20-targeting antibodies. These data provide rationale for the clinical testing of CD20-TDB for the treatment of CD20-expressing B cell malignancies.

Antitumor efficacy of a bispecific antibody that targets HER2 and activates T cells.

Clinical results from the latest strategies for T-cell activation in cancer have fired interest in combination immunotherapies that can fully engage T-cell immunity. In this study, we describe a trastuzumab-based bispecific antibody, HER2-TDB, which targets HER2 and conditionally activates T cells. HER2-TDB specifically killed HER2-expressing cancer cells at low picomolar concentrations. Because of its unique mechanism of action, which is independent of HER2 signaling or chemotherapeutic sensitivity, HER2-TDB eliminated cells refractory to currently approved HER2 therapies. HER2-TDB exhibited potent antitumor activity in four preclinical model systems, including MMTV-huHER2 and huCD3 transgenic mice. PD-L1 expression in tumors limited HER2-TDB activity, but this resistance could be reversed by anti-PD-L1 treatment. Thus, combining HER2-TDB with anti-PD-L1 yielded a combination immunotherapy that enhanced tumor growth inhibition, increasing the rates and durability of therapeutic response.

Targeting FGFR4 inhibits hepatocellular carcinoma in preclinical mouse models.

The fibroblast growth factor (FGF)-FGF receptor (FGFR) signaling system plays critical roles in a variety of normal developmental and physiological processes. It is also well documented that dysregulation of FGF-FGFR signaling may have important roles in tumor development and progression. The FGFR4-FGF19 signaling axis has been implicated in the development of hepatocellular carcinomas (HCCs) in mice, and potentially in humans. In this study, we demonstrate that FGFR4 is required for hepatocarcinogenesis; the progeny of FGF19 transgenic mice, which have previously been shown to develop HCCs, bred with FGFR4 knockout mice fail to develop liver tumors. To further test the importance of FGFR4 in HCC, we developed a blocking anti-FGFR4 monoclonal antibody (LD1). LD1 inhibited: 1) FGF1 and FGF19 binding to FGFR4, 2) FGFR4-mediated signaling, colony formation, and proliferation in vitro, and 3) tumor growth in a preclinical model of liver cancer in vivo. Finally, we show that FGFR4 expression is elevated in several types of cancer, including liver cancer, as compared to normal tissues. These findings suggest a modulatory role for FGFR4 in the development and progression of hepatocellular carcinoma and that FGFR4 may be an important and novel therapeutic target in treating this disease.

Interactive effects of mechanical ventilation and kidney health on lung function in an in vivo mouse model.

We hypothesized that the influence of acute kidney injury (AKI) on the sensitivity of the lung to an injurious process varies with the severity of the injurious process. Thus, we thought that AKI would exacerbate lung injury from low degrees of lung trauma but attenuate lung injury from higher degrees of lung trauma. C57BL/6 mice underwent AKI (30-min kidney ischemia) or sham surgery, followed at 24 h by 4 h of spontaneous breathing (SB), mechanical ventilation with low tidal volume (7 ml/kg, LTV), or mechanical ventilation with high tidal volume (30 ml/kg, HTV). Compared with LTV, median bronchoalveolar lavage (BAL) protein leak was significantly lower with SB and greater with HTV in both sham and AKI mice. Compared with LTV, median Evans blue dye-labeled albumin extravasation in lungs (L-EBD) was also significantly lower with SB and greater with HTV. L-EBD showed a significant interaction between ventilatory mode and kidney health, such that AKI attenuated the L-EBD rise seen in HTV vs. LTV sham mice. An interaction between ventilatory mode and kidney health could also be seen in BAL neutrophil number (PMN). Thus, AKI attenuated the BAL PMN rise seen in HTV vs. LTV sham mice. These data support the presence of a complex interaction between mechanical ventilation and AKI in which the sensitivity of the lung to trauma varies with the magnitude of the trauma and may involve a modification of pulmonary neutrophil activity by AKI.

The role of natriuretic peptide receptor-A signaling in unilateral lung ischemia-reperfusion injury in the intact mouse.

Ischemia-reperfusion (IR) causes human lung injury in association with the release of atrial and brain natriuretic peptides (ANP and BNP), but the role of ANP/BNP in IR lung injury is unknown. ANP and BNP bind to natriuretic peptide receptor-A (NPR-A) generating cGMP and to NPR-C, a clearance receptor that can decrease intracellular cAMP. To determine the role of NPR-A signaling in IR lung injury, we administered the NPR-A blocker anantin in an in vivo SWR mouse preparation of unilateral lung IR. With uninterrupted ventilation, the left pulmonary artery was occluded for 30 min and then reperfused for 60 or 150 min. Anantin administration decreased IR-induced Evans blue dye extravasation and wet weight in the reperfused left lung, suggesting an injurious role for NPR-A signaling in lung IR. In isolated mouse lungs, exogenous ANP (2.5 nM) added to the perfusate significantly increased the filtration coefficient sevenfold only if lungs were subjected to IR. This effect of ANP was also blocked by anantin. Unilateral in vivo IR increased endogenous plasma ANP, lung cGMP concentration, and lung protein kinase G (PKG(I)) activation. Anantin enhanced plasma ANP concentrations and attenuated the increase in cGMP and PKG(I) activation but had no effect on lung cAMP. These data suggest that lung IR triggered ANP release and altered endothelial signaling so that NPR-A activation caused increased pulmonary endothelial permeability.

Strain-specific differences in sensitivity to ischemia-reperfusion lung injury in mice.

Ischemia-reperfusion (I/R) lung injury is characterized by increased pulmonary endothelial permeability and edema, but the genetic basis for this injury is unknown. We utilized an in vivo mouse preparation of unilateral lung I/R to evaluate the genetic determinants of I/R lung injury. An index of pulmonary vascular protein permeability was measured by the ratio of left-to-right lung Evans blue dye of eight inbred mouse strains after 30 min of left lung ischemia and 150 min of reperfusion. The order of strain-specific sensitivity to I/R lung injury was BALB/c < SJL/J < CBA/J < C57BL/6J < 129/J < A/J < C3H/H3J < SWR/J. The reciprocal F1 offspring of the BALB/c and SWR/J progenitor strains had intermediate phenotypes but a differing variance. A similar pattern of right lung Evans blue dye content suggested the presence of contralateral injury because baseline vascular permeability was not different. Lung I/R injury was attenuated by NADPH oxidase inhibition, indicating a role for NADPH oxidase-derived reactive oxygen species (ROS). There was no strain-dependent difference in lung NADPH oxidase expression. Strain-related differences in zymosan-stimulated neutrophil ROS production did not correlate with I/R lung injury in that neutrophil ROS production in SWR/J mice was greater than C57BL/6J but not different from BALB/c mice. These data indicate the presence of a genetic sensitivity to lung I/R injury that involves multiple genes including a maternal-related factor. Although neutrophil-derived ROS production is also modulated by genetic factors, the pattern did not explain the genetic sensitivity to lung I/R injury.

Effect of ischemia and reperfusion without airway occlusion on vascular barrier function in the in vivo mouse lung.

Ischemia-reperfusion (I/R) lung injury causes increased vascular permeability and edema. We developed an in vivo murine model of I/R allowing measurement of pulmonary vascular barrier function without airway occlusion. The left pulmonary artery (PA) was occluded with an exteriorized, slipknotted suture in anesthetized C57BL/6J mice. The effect of ischemic time was determined by subjecting mice to 5, 10, or 30 min of left lung ischemia followed by 150 min of reperfusion. The effect of reperfusion time was determined by subjecting mice to 30 min of left lung ischemia followed by 30 or 150 min of reperfusion. Changes in pulmonary vascular barrier function were measured with the Evans blue dye (EBD) technique, dual-isotope radiolabeled albumin (RA), bronchoalveolar lavage (BAL) protein concentration, and wet weight-to-dry weight ratio (WW/DW). Increasing left lung ischemia with constant reperfusion time or increasing left lung reperfusion time after constant ischemic time resulted in significant increases in left lung EBD content at all times compared with both right lung values and sham surgery mice. The effects of left lung ischemia on lung EBD were corroborated by RA but the effects of increasing reperfusion time differed, suggesting binding of EBD to lung tissue. An increase in WW/DW was only detected after 30 min of reperfusion, suggesting edema clearance. BAL protein concentrations were unaffected. We conclude that short periods of I/R, without airway occlusion, increase pulmonary vascular permeability in the in vivo mouse, providing a useful model to study molecular mechanisms of I/R lung injury.