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.

Perspective on a Modified Developmental and Reproductive Toxicity Testing Strategy for Cancer Immunotherapy.

The intent of cancer immunotherapy (CIT) is to generate and enhance T-cell responses against tumors. The tumor microenvironment establishes several inhibitory pathways that lead to suppression of the local immune response, which is permissive for tumor growth. The efficacy of different CITs, alone and in combination, stems from reinvigorating the tumor immune response via several mechanisms, including costimulatory agonists, checkpoint inhibitors, and vaccines. However, immune responses to other antigens (self and foreign) may also be enhanced, resulting in potentially undesired effects. In outbred mammalian pregnancies, the fetus expresses paternally derived alloantigens that are recognized as foreign by the maternal immune system. If unchecked or enhanced, maternal immunity to these alloantigens represents a developmental and reproductive risk and thus is a general liability for cancer immunotherapeutic molecules. We propose a tiered approach to confirm this mechanistic reproductive liability for CIT molecules. A rodent allopregnancy model is based on breeding 2 different strains of mice so that paternally derived alloantigens are expressed by the fetus. When tested with a cross-reactive biotherapeutic, small molecule drug, or surrogate molecule, this model should reveal on-target reproductive liabilities if the pathway is involved in maintaining pregnancy. Alternatively, allopregnancy models with genetically modified mice can be interrogated for exquisitely specific biotherapeutics with restricted species reactivity. The allopregnancy model represents a relatively straightforward approach to confirm an expected on-target reproductive risk for CIT molecules. For biotherapeutics, it could potentially replace more complex developmental and reproductive toxicity testing in nonhuman primates when a pregnancy hazard is confirmed or expected.

Current nonclinical approaches for immune assessments of immuno-oncology biotherapeutics.

Harnessing the immune system to kill tumors has been revolutionary and, as a result, has had an enormous benefit for patients in extending life and resulting in effective cures in some. However, activation of the immune system can come at the cost of undesirable adverse events such as cytokine release syndrome, immune-related adverse events, on-target/off-tumor toxicity, neurotoxicity and tumor lysis syndrome, which are safety risks that can be challenging to assess non-clinically. This article provides a review of the biology and mechanisms that can result in immune-mediated adverse effects and describes industry approaches using in vitro and in vivo models to aid in the nonclinical safety risk assessments for immune-oncology modalities. Challenges and limitations of knowledge and models are also discussed.

Changing the drug development and therapeutic paradigm with biologic drug combinations and bispecifics: How to choose between these two approaches?

Biologics are increasingly being co-developed in combination or as novel constructs like bispecific antibodies (BsAbs) with the goal of targeting multiple, non-redundant mechanisms of action. Rational design of combinations and dual-targeting approaches that consider disease complexities have the potential to improve efficacy and safety, to increase duration of clinical benefit, and to minimize clinical resistance mechanisms. Here we summarize examples of BsAbs and biologic combinations that have been approved by health authorities and present drug development considerations when deciding between these two strategies. These include an understanding of target biology, nonclinical safety risks, dose optimization strategies, the regulatory framework, pharmacokinetic, immunogenicity, and bioanalytical assay considerations. The disease biology, target dynamics, and pharmacology objectives were identified as important factors in early drug development to decide between a BsAb versus a combination. Nonclinical safety assessment and dose optimization strategies can also pose challenges for BsAb versus combinations. High unmet medical needs and lack of treatment options are often the common denominators for deciding to develop a BsAb or a combination. Future development of biologic triple combinations and BsAbs combinations with other biologics will further increase drug development complexities and hold promise for more effective treatment options for patients.

Development of a nonhuman primate challenge model to evaluate CD8+ T cell responses to an adenovirus-based vaccine expressing SIV proteins upon repeat-dose treatment with checkpoint inhibitors.

Targeting immune checkpoint receptors expressed in the T cell synapse induces active and long-lasting antitumor immunity in preclinical tumor models and oncology patients. However, traditional nonhuman primate (NHP) studies in healthy animals have thus far demonstrated little to no pharmacological activity or toxicity for checkpoint inhibitors (CPIs), likely due to a quiescent immune system. We developed a NHP vaccine challenge model in Mauritius cynomolgus monkey (MCMs) that elicits a strong CD8+ T cell response to assess both pharmacology and safety within the same animal. MHC I-genotyped MCMs were immunized with three replication incompetent adenovirus serotype 5 (Adv5) encoding Gag, Nef and Pol simian immunodeficiency virus (SIV) proteins administered 4 weeks apart. Immunized animals received the anti-PD-L1 atezolizumab or an immune checkpoint-targeting bispecific antibody (mAbX) in early development. After a single immunization, Adv5-SIVs induced T-cell activation as assessed by the expression of several co-stimulatory and co-inhibitory molecules, proliferation, and antigen-specific T-cell response as measured by a Nef-dependent interferon-γ ELIspot and tetramer analysis. Administration of atezolizumab increased the number of Ki67+ CD8+ T cells, CD8+ T cells co-expressing TIM3 and LAG3 and the number of CD4+ T cells co-expressing 4-1BB, BTLA, and TIM3 two weeks after vaccination. Both atezolizumab and mAbX extended the cytolytic activity of the SIV antigen-specific CD8+ T cell up to 8 weeks. Taken together, this vaccine challenge model allowed the combined study of pharmacology and safety parameters for a new immunomodulatory protein-based therapeutic targeting CD8+ T cells in an NHP model.

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.

In Vivo Assessment of Antibody-Dependent Enhancement of Influenza B Infection.

A theoretical safety concern proposed in the influenza literature is that therapeutic antiviral antibodies could have the potential for antibody-dependent enhancement (ADE) of infection and disease. ADE may occur when virus-specific antibodies at subtherapeutic, nonneutralizing concentrations facilitate virus uptake and, in some cases, enhance replication, which can lead to an exacerbation of virus-mediated disease. Alternatively, ADE may occur due to antibody-dependent complement activation exacerbating virus-mediated disease in the absence of increased replication. As a result of this theoretical safety concern, safety assessment of anti-influenza antibodies may include an in vivo evaluation of ADE of infection and/or disease. These studies were conducted to investigate the potential of MHAB5553A, a broadly specific, neutralizing therapeutic anti-influenza B antibody, to elicit ADE of infection and disease in mouse models of influenza B infection. In parallel studies, female DBA/2J mice were infected with either influenza B/Victoria/504/2000 or influenza B/Brisbane/60/2008 representing distinct lineages. Assessment of ADE was based on an integration of results from multiple endpoints, including infectious lung viral titers and genomes, body weight, mortality, lung weight, and histopathology. In these studies, the high dose of 15 mg/kg MHAB5553A resulted in substantial attenuation of influenza pneumonia, with more modest effects at 1.5 mg/kg; whereas MHAB5553A treatment at 0.15 or 0.015 mg/kg was generally comparable to vehicle-treated controls. Our results demonstrate that MHAB5553A across a broad range of doses did not enhance primary influenza B infection or disease in this model, and represent a nonclinical de-risking of the ADE potential with this antibody.

Resolution of unexpected pregnancy-related findings in a rat embryofetal development and toxicokinetic study of monoclonal antibodies specific for hCMV.

BACKGROUND: Developmental and reproductive toxicity testing is not uniformly warranted for biopharmaceuticals that lack relevant targets in test species. However, RG7667, consisting of two monoclonal antibodies specific for human cytomegalovirus (hCMV), was intended for administration to pregnant women to prevent transmission of CMV to the developing fetus. METHODS: Considering the target indication, a Pilot Embryo Fetal Development/Toxicokinetic study was conducted to assess toxicokinetics in the dam and fetuses and general tolerability. RESULTS: RG7667 administered intravenously to presumed pregnant Sprague-Dawley rats was well tolerated with no clinical signs in any dam and comparable litter sizes and viability across groups. However, at cesarean section, hepatic necrosis and pancreatic edema were identified in two dams administered RG7667, with no clear dose relationship. Investigation of total protein, albumin, and transaminase activity in residual serum from TK samples demonstrated striking hypoproteinemia and elevated transaminases limited to these two dams. Overall, these pathology findings in dams were considered of uncertain relationship to RG7667; therefore, a subsequent Pivotal EFD study was conducted, which did not repeat the liver or pancreatic findings. CONCLUSIONS: The results of the Pivotal study confirmed the lack of overt toxicity, teratogenicity, or effects on litter size and viability when human or humanized monoclonal antibodies that lack an endogenous target are administered IV to rats during pregnancy. With these additional data, we concluded that the unexpected pathology findings in the Pilot study were not specific to RG7667, but rather highlight some clinical pathology and macroscopic/microscopic findings that can occur during pregnancy in rats.

Placental and Fetal Effects of Onartuzumab, a Met/HGF Signaling Antagonist, When Administered to Pregnant Cynomolgus Monkeys.

Onartuzumab is an engineered single arm, monovalent monoclonal antibody that targets the MET receptor and prevents hepatocyte growth factor (HGF) signaling. Knockout mice have clearly demonstrated that HGF/MET signaling is developmentally critical. A pre- and postnatal development study (enhanced design) was conducted in cynomolgus monkeys to evaluate the potential developmental consequences following onartuzumab administration. Control or onartuzumab, at loading/maintenance doses of 75/50 mg/kg (low) or 100/100 mg/kg (high), was administered intravenously once weekly to 12 confirmed pregnant female cynomolgus monkeys per group from gestation day (GD) 20 through GD 174. Onartuzumab administration resulted in decreased gestation length, decreased birth weight, and increased fetal and perinatal mortality. A GD147 C-section was conducted for a subset of Control and High Dose monkeys, and identified placental infarcts with hemorrhage in the chorionic plate, chorionic villus and/or decidual plate. These findings were limited to placentas from onartuzumab-treated animals. In addition, decreased cellularity of the hepatocytes with dilated hepatic sinusoids was inconsistently observed in the liver of a few fetal or infant monkeys that died in the perinatal period. Surviving offspring had some evidence of developmental delay compared with controls, but no overt teratogenicity. Overall, effects on the perinatal fetuses were consistent with those reported in knockout mice, but not as severe. Onartuzumab concentrations were low or below the level of detection in most offspring, with cord blood concentrations only 1%-2% of maternal levels on GD 147. Malperfusion secondary to onartuzumab-induced placental injury could explain the adverse pregnancy outcomes, fetal growth restriction and relatively low fetal exposures.

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.

Vascular endothelial growth factor blockade reduces intratumoral regulatory T cells and enhances the efficacy of a GM-CSF-secreting cancer immunotherapy.

PURPOSE: The purpose of the present study was to evaluate granulocyte macrophage colony-stimulating factor (GM-CSF)-secreting tumor cell immunotherapy in combination with vascular endothelial growth factor (VEGF) blockage in preclinical models. EXPERIMENTAL DESIGN: Survival and immune response were monitored in the B16 melanoma and the CT26 colon carcinoma models. VEGF blockade was achieved by using a recombinant adeno-associated virus vector expressing a soluble VEGF receptor consisting of selected domains of the VEGF receptors 1 and 2 (termed sVEGFR1/R2). Dendritic cell and tumor infiltrating lymphocyte activation status and numbers were evaluated by fluorescence-activated cell sorting analysis. Regulatory T cells were quantified by their CD4+CD25hi and CD4+FoxP3+ phenotype. RESULTS: The present study established that GM-CSF-secreting tumor cell immunotherapy with VEGF blockade significantly prolonged the survival of tumor-bearing mice. Enhanced anti-tumor protection correlated with an increased number of activated CD4+ and CD8+ tumor-infiltrating T cells and a pronounced decrease in the number of suppressive regulatory T cells residing in the tumor. Conversely, overexpression of VEGF from tumors resulted in elevated numbers of regulatory T cells in the tumor, suggesting a novel mechanism of VEGF-mediated immune suppression at the tumor site. CONCLUSION: GM-CSF-secreting cancer immunotherapy and VEGF blockade increases the i.t. ratio of effector to regulatory T cells to provide enhanced antitumor responses. This therapeutic combination may prove to be an effective strategy for the treatment of patients with cancer.

Administration of IFN-alpha enhances the efficacy of a granulocyte macrophage colony stimulating factor-secreting tumor cell vaccine.

IFN-alpha is approved for the treatment of multiple cancers. Its pleiotropic properties include inhibition of proliferation and angiogenesis and induction of apoptosis. Type I IFNs also exert immunomodulatory effects, which make it an appropriate candidate to combine with cancer vaccines. The studies reported herein show that 50% of mice reject established B16 tumors following treatment with the combination of a granulocyte macrophage colony-stimulating factor-secreting tumor cell vaccine (B16.GM) and subclinical doses of recombinant murine IFN-alpha delivered at the vaccine site. Similarly, 80% of mice treated with the combination reject established B16 tumors when recombinant murine IFN-alpha is given at the challenge site, suggesting that in the latter case its antiproliferative, proapoptotic, and antiangiogenic properties may be involved in controlling tumor growth. In contrast, fewer than 10% of mice reject the tumors when either one is used as a monotherapy. Furthermore, a 30-fold increase in the frequency of melanoma-associated antigen (Trp-2 and gp100) specific T cells was observed in mice treated with the combination when compared with unvaccinated controls. These data show that IFN-alpha combined with a granulocyte macrophage colony-stimulating factor-secreting tumor cell vaccine significantly enhances vaccine potency and may represent a potential new approach for tumor immunotherapy.

The generation of T cell memory: a review describing the molecular and cellular events following OX40 (CD134) engagement.

OX40 (CD134), a membrane-bound member of the tumor necrosis factor-receptor superfamily, is expressed primarily on activated CD4(+) T cells. Following engagement on the cell surface, OX40 delivers a costimulatory signal that leads to potent, proinflammatory effects. Engagement of OX40 during antigen (Ag)-specific stimulation of T cells leads to increased production of memory T cells, increased migration of Ag-specific T cells, enhanced cytokine production by effector T cells, and the ability to break peripheral T cell tolerance in vivo. Therefore, OX40 engagement in vivo could have important ramifications for the enhancement of vaccine strategies and inhibition of unwanted inflammation. This review summarizes the molecular and cellular events that occur following OX40 engagement during Ag-specific T cell activation.

A two-in-one antibody against HER3 and EGFR has superior inhibitory activity compared with monospecific antibodies.

Extensive crosstalk among ErbB/HER receptors suggests that blocking signaling from more than one family member may be essential to effectively treat cancer and limit drug resistance. We generated a conventional IgG molecule MEHD7945A with dual HER3/EGFR specificity by phage display engineering and used structural and mutational studies to understand how a single antigen recognition surface binds two epitopes with high affinity. As a human IgG1, MEHD7945A exhibited dual action by inhibiting EGFR- and HER3-mediated signaling in vitro and in vivo and the ability to engage immune effector functions. Compared with monospecific anti-HER antibodies, MEHD7945A was more broadly efficacious in multiple tumor models, showing that combined inhibition of EGFR and HER3 with a single antibody is beneficial.

Activation of immature hepatic NK cells as immunotherapy for liver metastatic disease.

NK cells can identify and eliminate emerging tumors due to altered expression of activating and inhibitory ligands on aberrant cells, a process that is greatly enhanced following NK cell activation. As a principal site of both tumor metastases and immature NK cells, the liver represents a unique anatomic location in which activation of the innate immune system could provide substantial therapeutic benefit. We describe here the NK cell-dependent destruction of a primary hepatic tumor following infection with an attenuated intracellular bacterium derived from Listeria monocytogenes. NK cell-mediated immunity correlated with the ordered migration and maturation of NK cells within the liver. Cytolytic activity was partially dependent on NKG2D-mediated tumor cell recognition, but surprisingly was still effective in the absence of type I IFN. Significantly, NK cell-mediated destruction of a primary hepatic tumor in infected mice led to long-lived CD4- and CD8 T cell-dependent tumor-specific adaptive immunity. These findings establish that activation and differentiation of immature NK cells using complex microbial stimuli can elicit potent anti-tumor activity within the liver, promote cross-presentation of tumor-derived Ags leading to long-lived systemic anti-tumor immunity, and suggests a paradigm for clinical intervention of liver metastatic carcinoma.

The anti-tumor efficacy of a GM-CSF-secreting tumor cell vaccine is not inhibited by docetaxel administration.

Docetaxel has demonstrated therapeutic efficacy against breast, prostate, and ovarian cancer and other solid tumors. The tumoricidal activity of docetaxel is mainly attributed to its ability to block microtubule depolymerization, thus inducing G(2)-M arrest and apoptosis. Mounting evidence indicates that docetaxel also possesses immunomodulatory activity such as augmenting macrophage and lymphokine activated killer activity and inducing pro-inflammatory cytokines, suggesting that docetaxel may be a good chemotherapeutic agent to combine with cancer immunotherapies, assuming that it does not inhibit the vaccine-induced immune response. The anti-tumor activity of the combination of docetaxel and a GM-CSF-secreting B16F10 tumor cell vaccine (B16.GM) was evaluated in the murine B16 melanoma model. Dose levels of docetaxel and the B16.GM vaccine known to be ineffective when used as single agents were selected. Three iv treatments of 6 mg/kg docetaxel per injection given on days 5, 9, and 13 after tumor challenge or a single vaccination with 2-3 x 10(6) B16.GM cells on day 3 were ineffective at inhibiting tumor growth when used as single agents [median survival time (MST)=24 days in both treatment groups and in control animals]. However, combination of docetaxel and B16.GM vaccine significantly delayed tumor growth, increasing MST to 45 days. A similar improvement in anti-tumor efficacy was observed using multiple treatment cycles of the B16.GM vaccine/docetaxel combination. Administration of docetaxel every 4 days between bi-weekly B16.GM vaccinations increased the median survival of tumor-bearing mice from 31 to 52 days compared to multiple B16.GM vaccinations alone. In summary, these data demonstrate that rather than inhibiting the anti-tumor effects of a GM-CSF-secreting tumor cell vaccine, docetaxel combined with a whole cell vaccine significantly inhibits tumor growth, increases survival time and does not impede T-cell activation in the murine B16F10 melanoma tumor model. GM-secreting tumor cell vaccines in combination with docetaxel may represent a new strategy for combining chemo and immunotherapy for cancer.

Efficacy of GM-CSF-producing tumor vaccine after docetaxel chemotherapy in mice bearing established Lewis lung carcinoma.

In this report, we evaluated the efficacy of a GM-CSF-producing tumor vaccine given before and after docetaxel in mice bearing established lung tumors. Mice bearing established 3LL tumors were treated with docetaxel and tumor vaccines transduced with either control or GM-CSF adenoviral vectors. Docetaxel (5-20 mg/kg) treatment alone had only a minimal effect on growth of established 3LL tumors in vivo, although docetaxel was cytotoxic to 3LL cells in vitro. When mice bearing established 3LL tumors were pretreated with docetaxel followed by vaccination with irradiated GM-CSF- transduced 3LL tumor cells, significant tumor regression and prolonged survival were observed compared with chemotherapy alone. Delaying docetaxel treatment until after tumor vaccination abrogated the vaccine's anti-tumor effects. Mice that survived treatment were able to resist a lethal rechallenge of 3LL tumor cells. Memory CTL specific for an epitope (MUT-1) derived from 3LL were detected in surviving mice. Docetaxel induced a mild lymphodepletion in mice, both CD4 and CD8 subsets were reduced in LN and spleens. Interestingly, docetaxel also diminished the number of memory CD8+ T cells (CD122+) and possible CD4+ CD25+ Foxp3+ natural Treg cells. Docetaxel treatment did not affect antigen-driven proliferation of naive T cells but significantly promoted survival of activated T cells. Thus, augmentation of vaccine induced antitumor immunity in docetaxel-treated mice primarily due to the enhanced survival of antigen-experienced T cells.

OX40-mediated memory T cell generation is TNF receptor-associated factor 2 dependent.

Tumor necrosis factor receptor-associated factor 2 (TRAF2), an adapter protein that associates with the cytoplasmic tail of OX40, may play a critical role in OX40-mediated signal transduction. To investigate the in vivo role of TRAF2 in OX40-mediated generation of Ag-specific memory T cells, we bred OVA-specific TCR transgenic mice to TRAF2 dominant-negative (TRAF2 DN) mice. Following Ag stimulation and OX40 engagement of TRAF2 DN T cells in vivo, the number of long-lived OVA-specific T cells and effector T cell function was dramatically reduced when compared with wild-type T cells. We also demonstrate that CTLA-4 is down-regulated following OX40 engagement in vivo and the OX40-specific TRAF2 DN defect was partially overcome by CTLA-4 blockade in vivo. The data provide evidence that TRAF2 is linked to OX40-mediated memory T cell expansion and survival, and point to the down-regulation of CTLA-4 as a possible control element to enhance early T cell expansion through OX40 signaling.