IL-15/IL-15Rα-Fc fusion protein XmAb24306 potentiates activity of CD3 bispecific antibodies through enhancing T cell expansion.

Insufficient quantity of functional T cells is a likely factor limiting clinical activity of T cell bispecific antibodies, especially in solid tumor indications. We hypothesized that XmAb24306 (efbalropendekin alfa), a lymphoproliferative interleukin (IL)-15/IL-15 receptor α (IL-15Rα) Fc-fusion protein, may potentiate the activity of T cell dependent (TDB) antibodies. Activation of human peripheral T cells by cevostamab, an anti-FcRH5/CD3 TDB, or anti-HER2/CD3 TDB resulted in upregulation of IL-2/15Rß (CD122) receptor subunit in nearly all CD8+ and majority of CD4+ T cells, suggesting that TDB treatment may sensitize T cells to the IL-15. XmAb24306 enhanced T cell bispecific antibody induced CD8+ and CD4+ T cell proliferation and expansion. In vitro combination of XmAb24306 with cevostamab or anti-HER2/CD3 TDB resulted in significant enhancement of tumor cell killing, which was reversed when T cell numbers were normalized, suggesting that T cell expansion is the main mechanism for the observed benefit. Pre-treatment of immune competent mice with a mouse-reactive surrogate of XmAb24306 (mIL-15-Fc) resulted in significant increase of T cells in blood, spleen and in tumors and converted transient anti-HER2/CD3 TDB responses to complete durable responses. In summary, our results support the hypothesis where the number of tumor infiltrating T cells is rate limiting for the activity of solid tumor targeting TDBs. Upregulation of CD122 by TDB treatment and the observed synergy with XmAb24306 and T cell bispecific antibodies supports clinical evaluation of this novel immunotherapy combination.

T cell-dependent bispecific antibodies alter organ-specific endothelial cell-T cell interaction.

Preclinical and clinical studies demonstrate that T cell-dependent bispecific antibodies (TDBs) induce systemic changes in addition to tumor killing, leading to adverse events. Here, we report an in-depth characterization of acute responses to TDBs in tumor-bearing mice. Contrary to modest changes in tumors, rapid and substantial lymphocyte accumulation and endothelial cell (EC) activation occur around large blood vessels in normal organs including the liver. We hypothesize that organ-specific ECs may account for the differential responses in normal tissues and tumors, and we identify a list of genes selectively upregulated by TDB in large liver vessels. Using one of the genes as an example, we demonstrate that CD9 facilitates ICAM-1 to support T cell-EC interaction in response to soluble factors released from a TDB-mediated cytotoxic reaction. Our results suggest that multiple factors may cooperatively promote T cell infiltration into normal organs as a secondary response to TDB-mediated tumor killing. These data shed light on how different vascular beds respond to cancer immunotherapy and may help improve their safety and efficacy.

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.

Immunization associated with primary tumor growth leads to rejection of commonly used syngeneic tumors upon tumor rechallenge.

The recent success of multiple immunomodulating drugs in oncology highlights the potential of relieving immunosuppression by directly engaging the immune system in the tumor bed to target cancer cells. Durable responses to immune checkpoint inhibitors experienced by some patients may be indicative of the formation of a T cell memory response. This has prompted the search for preclinical evidence of therapy-induced long-term immunity as part of the evaluation of novel therapeutics. A common preclinical method used to document long-term immunity is the use of tumor rechallenge experiments in which tumor growth is assessed in mice that have previously rejected tumors in response to therapy. Failure of rechallenge engraftment, typically alongside successful engraftment of the same tumor in naive animals as a control, is often presented as evidence of therapy-induced tumor immunity. Here, we present evidence that formation of tumor immunity often develops independent of therapy. We observed elevated rates of rechallenge rejection following surgical resection of primary tumors for four of five commonly used models and that such postexcision immunity could be adoptively transferred to treatment-naïve mice. We also show that tumor-specific cytolytic T cells are induced on primary tumor challenge independent of therapeutic intervention. Taken together these data call into question the utility of tumor rechallenge studies and the use of naïve animals as controls to demonstrate therapy-induced formation of long-term tumor immunity.

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.

IRE1α Disruption in Triple-Negative Breast Cancer Cooperates with Antiangiogenic Therapy by Reversing ER Stress Adaptation and Remodeling the Tumor Microenvironment.

Cancer cells exploit the unfolded protein response (UPR) to mitigate endoplasmic reticulum (ER) stress caused by cellular oncogene activation and a hostile tumor microenvironment (TME). The key UPR sensor IRE1α resides in the ER and deploys a cytoplasmic kinase-endoribonuclease module to activate the transcription factor XBP1s, which facilitates ER-mediated protein folding. Studies of triple-negative breast cancer (TNBC)-a highly aggressive malignancy with a dismal posttreatment prognosis-implicate XBP1s in promoting tumor vascularization and progression. However, it remains unknown whether IRE1α adapts the ER in TNBC cells and modulates their TME, and whether IRE1α inhibition can enhance antiangiogenic therapy-previously found to be ineffective in patients with TNBC. To gauge IRE1α function, we defined an XBP1s-dependent gene signature, which revealed significant IRE1α pathway activation in multiple solid cancers, including TNBC. IRE1α knockout in TNBC cells markedly reversed substantial ultrastructural expansion of their ER upon growth in vivo. IRE1α disruption also led to significant remodeling of the cellular TME, increasing pericyte numbers while decreasing cancer-associated fibroblasts and myeloid-derived suppressor cells. Pharmacologic IRE1α kinase inhibition strongly attenuated growth of cell line-based and patient-derived TNBC xenografts in mice and synergized with anti-VEGFA treatment to cause tumor stasis or regression. Thus, TNBC cells critically rely on IRE1α to adapt their ER to in vivo stress and to adjust the TME to facilitate malignant growth. TNBC reliance on IRE1α is an important vulnerability that can be uniquely exploited in combination with antiangiogenic therapy as a promising new biologic approach to combat this lethal disease. SIGNIFICANCE: Pharmacologic IRE1α kinase inhibition reverses ultrastructural distension of the ER, normalizes the tumor vasculature, and remodels the cellular TME, attenuating TNBC growth in mice.

The Tumor Suppressor BAP1 Regulates the Hippo Pathway in Pancreatic Ductal Adenocarcinoma.

The deubiquitinating enzyme BAP1 is mutated in a hereditary cancer syndrome with a high risk for mesothelioma and melanocytic tumors. Here, we show that pancreatic intraepithelial neoplasia driven by oncogenic mutant KrasG12D progressed to pancreatic adenocarcinoma in the absence of BAP1. The Hippo pathway was deregulated in BAP1-deficient pancreatic tumors, with the tumor suppressor LATS exhibiting enhanced ubiquitin-dependent proteasomal degradation. Therefore, BAP1 may limit tumor progression by stabilizing LATS and thereby promoting activity of the Hippo tumor suppressor pathway. SIGNIFICANCE: BAP1 is mutated in a broad spectrum of tumors. Pancreatic Bap1 deficiency causes acinar atrophy but combines with oncogenic Ras to produce pancreatic tumors. BAP1-deficient tumors exhibit deregulation of the Hippo pathway.See related commentary by Brekken, p. 1624.

PD-L1 expression by dendritic cells is a key regulator of T-cell immunity in cancer.

Inhibiting the programmed death-1 (PD-1) pathway is one of the most effective approaches to cancer immunotherapy, but its mechanistic basis remains incompletely understood. Binding of PD-1 to its ligand PD-L1 suppresses T-cell function in part by inhibiting CD28 signaling. Tumor cells and infiltrating myeloid cells can express PD-L1, with myeloid cells being of particular interest as they also express B7-1, a ligand for CD28 and PD-L1. Here we demonstrate that dendritic cells (DCs) represent a critical source of PD-L1, despite being vastly outnumbered by PD-L1+ macrophages. Deletion of PD-L1 in DCs, but not macrophages, greatly restricted tumor growth and led to enhanced antitumor CD8+ T-cell responses. Our data identify a unique role for DCs in the PD-L1-PD-1 regulatory axis and have implications for understanding the therapeutic mechanism of checkpoint blockade, which has long been assumed to reflect the reversal of T-cell exhaustion induced by PD-L1+ tumor cells.

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.

Disruption of IRE1α through its kinase domain attenuates multiple myeloma.

Multiple myeloma (MM) arises from malignant immunoglobulin (Ig)-secreting plasma cells and remains an incurable, often lethal disease despite therapeutic advances. The unfolded-protein response sensor IRE1α supports protein secretion by deploying a kinase-endoribonuclease module to activate the transcription factor XBP1s. MM cells may co-opt the IRE1α-XBP1s pathway; however, the validity of IRE1α as a potential MM therapeutic target is controversial. Genetic disruption of IRE1α or XBP1s, or pharmacologic IRE1α kinase inhibition, attenuated subcutaneous or orthometastatic growth of MM tumors in mice and augmented efficacy of two established frontline antimyeloma agents, bortezomib and lenalidomide. Mechanistically, IRE1α perturbation inhibited expression of key components of the endoplasmic reticulum-associated degradation machinery, as well as secretion of Ig light chains and of cytokines and chemokines known to promote MM growth. Selective IRE1α kinase inhibition reduced viability of CD138+ plasma cells while sparing CD138- cells derived from bone marrows of newly diagnosed or posttreatment-relapsed MM patients, in both US- and European Union-based cohorts. Effective IRE1α inhibition preserved glucose-induced insulin secretion by pancreatic microislets and viability of primary hepatocytes in vitro, as well as normal tissue homeostasis in mice. These results establish a strong rationale for developing kinase-directed inhibitors of IRE1α for MM therapy.

The tumor suppressor BAP1 cooperates with BRAFV600E to promote tumor formation in cutaneous melanoma.

The deubiquitinating enzyme BAP1 is mutated in a hereditary cancer syndrome with a high risk of mesothelioma and melanocytic tumors. Here, we show that Bap1 deletion in melanocytes cooperates with the constitutively active, oncogenic form of BRAF (BRAFV600E ) and UV to cause melanoma in mice, albeit at very low frequency. In addition, Bap1-null melanoma cells derived from mouse tumors are more aggressive and colonize and grow at distant sites more than their wild-type counterparts. Molecularly, Bap1-null melanoma cell lines have increased DNA damage measured by γH2aX and hyperubiquitination of histone H2a. Therapeutically, these Bap1-null tumors are completely responsive to BRAF- and MEK-targeted therapies. Therefore, BAP1 functions as a tumor suppressor and limits tumor progression in melanoma.

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.

The Hippo pathway effector TAZ induces TEAD-dependent liver inflammation and tumors.

The Hippo signaling pathway regulates organ size and plays critical roles in maintaining tissue growth, homeostasis, and regeneration. Dysregulated in a wide spectrum of cancers, in mammals, this pathway is regulated by two key effectors, YAP and TAZ, that may functionally overlap. We found that TAZ promoted liver inflammation and tumor development. The expression of TAZ, but not YAP, in human liver tumors positively correlated with the expression of proinflammatory cytokines. Hyperactivated TAZ induced substantial myeloid cell infiltration into the liver and the secretion of proinflammatory cytokines through a TEAD-dependent mechanism. Furthermore, tumors with hyperactivated YAP and TAZ had distinct transcriptional signatures, which included the increased expression of inflammatory cytokines in TAZ-driven tumors. Our study elucidated a previously uncharacterized link between TAZ activity and inflammatory responses that influence tumor development in the liver.

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.

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.

Tumour and host cell PD-L1 is required to mediate suppression of anti-tumour immunity in mice.

Expression of PD-L1, the ligand for T-cell inhibitory receptor PD-1, is one key immunosuppressive mechanism by which cancer avoids eradication by the immune system. Therapeutic use of blocking antibodies to PD-L1 or its receptor PD-1 has produced unparalleled, durable clinical responses, with highest likelihood of response seen in patients whose tumour or immune cells express PD-L1 before therapy. The significance of PD-L1 expression in each cell type has emerged as a central and controversial unknown in the clinical development of immunotherapeutics. Using genetic deletion in preclinical mouse models, here we show that PD-L1 from disparate cellular sources, including tumour cells, myeloid or other immune cells can similarly modulate the degree of cytotoxic T-cell function and activity in the tumour microenvironment. PD-L1 expression in both the host and tumour compartment contribute to immune suppression in a non-redundant fashion, suggesting that both sources could be predictive of sensitivity to therapeutic agents targeting the PD-L1/PD-1 axis.

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.

Antitherapeutic antibody-mediated hepatotoxicity of recombinant human Apo2L/TRAIL in the cynomolgus monkey.

Apo2L/TRAIL is a member of the tumor necrosis factor superfamily and an important inducer of apoptosis. Recombinant human (rhu) Apo2L/TRAIL has been attractive as a potential cancer therapeutic because many types of tumor cells are sensitive to its apoptosis-inducing effects. Nonclinical toxicology studies were conducted to evaluate the safety of rhuApo2L/TRAIL for possible use in humans. The cynomolgus monkey was chosen for this safety assessment based on high protein sequence homology between human and cynomolgus Apo2L/TRAIL and comparable expression of their receptors. Although hepatotoxicity was observed in repeat-dose monkey studies with rhuApo2L/TRAIL, all animals that displayed hepatotoxicity had developed antitherapeutic antibodies (ATAs). The cynomolgus ATAs augmented the cytotoxicity of rhuApo2L/TRAIL but not of its cynomolgus counterpart. Of note, human and cynomolgus Apo2L/TRAIL differ by four amino acids, three of which are surface-exposed. In vivo studies comparing human and cynomolgus Apo2L/TRAIL supported the conclusion that these distinct amino acids served as epitopes for cross-species ATAs, capable of crosslinking rhuApo2L/TRAIL and thus triggering hepatocyte apoptosis. We describe a hapten-independent mechanism of immune-mediated, drug-related hepatotoxicity - in this case - associated with the administration of a human recombinant protein in monkeys. The elucidation of this mechanism enabled successful transition of rhuApo2L/TRAIL into human clinical trials.