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.

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.

An Anti-CLL-1 Antibody-Drug Conjugate for the Treatment of Acute Myeloid Leukemia.

PURPOSE: The treatment of acute myeloid leukemia (AML) has not significantly changed in 40 years. Cytarabine- and anthracycline-based chemotherapy induction regimens (7 + 3) remain the standard of care, and most patients have poor long-term survival. The reapproval of Mylotarg, an anti-CD33-calicheamicin antibody-drug conjugate (ADC), has demonstrated ADCs as a clinically validated option to enhance the effectiveness of induction therapy. We are interested in developing a next-generation ADC for AML to improve upon the initial success of Mylotarg. EXPERIMENTAL DESIGN: The expression pattern of CLL-1 and its hematopoietic potential were investigated. A novel anti-CLL-1-ADC, with a highly potent pyrrolobenzodiazepine (PBD) dimer conjugated through a self-immolative disulfide linker, was developed. The efficacy and safety profiles of this ADC were evaluated in mouse xenograft models and in cynomolgus monkeys. RESULTS: We demonstrate that CLL-1 shares similar prevalence and trafficking properties that make CD33 an excellent ADC target for AML, but lacks expression on hematopoietic stem cells that hampers current CD33-targeted ADCs. Our anti-CLL-1-ADC is highly effective at depleting tumor cells in AML xenograft models and lacks target independent toxicities at doses that depleted target monocytes and neutrophils in cynomolgus monkeys. CONCLUSIONS: Collectively, our data suggest that an anti-CLL-1-ADC has the potential to become an effective and safer treatment for AML in humans, by reducing and allowing for faster recovery from initial cytopenias than the current generation of ADCs for AML.

Conjugation of Indoles to Antibodies through a Novel Self-Immolating Linker.

A novel strategy to attach indole-containing payloads to antibodies through a carbamate moiety and a self-immolating, disulfide-based linker is described. This new strategy was employed to connect a selective estrogen receptor down-regulator (SERD) to various antibodies in a site-selective manner. The resulting conjugates displayed potent, antigen-dependent down-regulation of estrogen receptor levels in MCF7-neo/HER2 and MCF7-hB7H4 cells. They also exhibited similar antigen-dependent modulation of the estrogen receptor in tumors when administered intravenously to mice bearing MCF7-neo/HER2 tumor xenografts. The indole-carbamate moiety present in the new linker was stable in whole blood from various species and also exhibited good in vivo stability properties in mice.

Is Digital Tomosynthesis on Par With Computed Tomography for the Detection and Measurement of Pulmonary Nodules?

Chest digital tomosynthesis (DT) has potential advantages compared to computed tomography (CT) such as radiation dose reduction. However, the role of DT in pulmonary nodule management remains investigative. We compared DT against CT for pulmonary nodule detection and size measurement. A clinical population comprising 54 nodules from 30 patients and a screening population comprising 42 nodules from 52 patients were included. Scans were independently read by two radiologists. Agreement in nodule measurements between readers and between modalities was assessed by Bland-Altman analysis using a 95% level of significance. The DT true positive fraction for the two readers was 0.44 and 0.39 in the clinical population, and 0.10 and 0.05 in the screening population. No significant inter-modality bias was observed between DT and CT measurements of nodule size, but the range of variation between modalities was approximately 30%. Inter-reader DT measurements also showed no significant bias, with a range of variation of approximately 15%. We conclude that DT has poor nodule detection sensitivity compared to CT. However, DT showed good measurement reproducibility and may be useful for monitoring growth of existing pulmonary nodules.

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.

Screen-detected subsolid pulmonary nodules: long-term follow-up and application of the PanCan lung cancer risk prediction model.

OBJECTIVE: To report the long-term follow-up of subsolid nodules (SSNs) detected in participants of a prospective low-dose CT lung cancer screening cohort, and to investigate the utility of the PanCan model in stratifying risk in baseline SSNs. METHODS: Participants underwent a baseline scan, two annual incidence scans and further follow-up scans for the detected nodules. All SSNs underwent a minimum of 2 years of follow-up (unless resolved or resected). Risk of malignancy was estimated using the PanCan model; discrimination [area under the receiver-operating characteristic curve (AUC)] and calibration (Hosmer-Lemeshow goodness-of-fit test) were assessed. The Mann-Whitney U-Wilcoxon test was used to compare estimated risk between groups. RESULTS: 70 SSNs were detected in 41 (16.0%) out of 256 total participants. Median follow-up period was 25.5 months (range 2.0-74.0 months). 29 (41.4%) SSNs were transient. Five (7.1%) SSNs were resected, all found to be Stage I lung adenocarcinoma, including one SSN stable in size for 3.0 years before growth was detected. The PanCan model had good discrimination for the 52 baseline SSNs (AUC = 0.89; 95% confidence interval 0.76-1); the Hosmer-Lemeshow goodness-of-fit test was non-significant (p = 0.27). Estimated risk was significantly higher in the baseline SSNs found to be cancer vs those not found to be cancer after 2-6 years of follow-up (p < 0.01). CONCLUSION: Our findings support a long-term follow-up approach for screen-detected SSNs for 3 years or longer. The PanCan model appeared discriminatory and well calibrated in this cohort. ADVANCES IN KNOWLEDGE: The PanCan model may have utility in identifying low-risk SSNs which could be followed with less frequent CT scans.

An anti-B7-H4 antibody-drug conjugate for the treatment of breast cancer.

B7-H4 has been implicated in cancers of the female reproductive system and investigated for its possible use as a biomarker for cancer, but there are no preclinical studies to demonstrate that B7-H4 is a molecular target for therapeutic intervention of cancer. We provide evidence that the prevalence and expression levels of B7-H4 are high in different subtypes of breast cancer and that only a few normal tissues express B7-H4 on the cell membrane. These profiles of low normal expression and upregulation in cancer provide an opportunity for the use of antibody-drug conjugates (ADCs), cytotoxic drugs chemically linked to antibodies, for the treatment of B7-H4 positive cancers. We have developed an ADC specific to B7-H4 that uses a linker drug consisting of a potent antimitotic, monomethyl auristatin E (MMAE), linked to engineered cysteines (THIOMAB) via a protease labile linker. We will refer to ADCs that use the THIOMAB format as TDCs to help distinguish the format from standard MC-vc-MMAE ADCs that are conjugated to the interchain disulfide bonds. Anti-B7-H4 (h1D11)-MC-vc-PAB-MMAE (h1D11 TDC) produced durable tumor regression in cell line and patient-derived xenograft models of triple-negative breast cancer. It also binds rat B7-H4 with similar affinity to human and allowed us to test for target dependent toxicity in rats. We found that our anti-B7-H4 TDC has toxicity findings similar to untargeted TDC. Our results validate B7-H4 as an ADC target for breast cancer and support the possible use of this TDC in the treatment of B7-H4(+) breast cancer.

Electromagnetic navigation bronchoscopy for the diagnosis of Aspergillus infection.

Electromagnetic navigation bronchoscopy (ENB) is a new diagnostic tool for the evaluation of pulmonary lesions inaccessible by conventional bronchoscopy. Most often, ENB is used for the diagnosis of lung cancer, but can be used to evaluate fungal conditions and other diseases. We present the case of a 44-year-old woman who was diagnosed with Aspergillus via ENB.

Mediastinal abscess after endobronchial ultrasound-guided transbronchial needle aspiration: a case report and literature review.

Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) is a minimally invasive technique that allows lung cancer nodal staging and biopsy of parabronchial and paratracheal tissue. Its simplicity, high diagnostic yield, ability to diagnose both benign and malignant conditions, and exceedingly low complication rate has resulted in rapid widespread adoption by surgeons and physicians. EBUS-TBNA-related complications, however, do occur and need to be considered when assessing the risk-benefit profile of performing the procedure, and if the patient represents with unexpected symptoms after the procedure. We describe a 64-year-old woman who presented with a mediastinal abscess 5 days after EBUS-TBNA. This case demonstrates the importance of considering EBUS-TBNA-related complications to guide relevant imaging decisions and antibiotic choices. We review the published literature regarding infective complications of EBUS-TBNA and propose possible pathophysiologies. These complications are likely to increase in frequency as the technique is more widely adopted.

Diagnostic bronchoscopy--current and future perspectives.

Lung cancer is the leading cause of cancer-related mortality worldwide. Standard bronchoscopy has limited ability to accurately localise and biopsy pulmonary lesions that cannot be directly visualised. The field of advanced diagnostic bronchoscopy is rapidly evolving due to advances in electronics and miniaturisation. Bronchoscopes with smaller outer working diameters, coupled with miniature radial and convex ultrasound probes, allow accurate central and peripheral pulmonary lesion localisation and biopsy while at the same time avoiding vascular structures. Increases in computational processing power allow three-dimensional reconstruction of computed tomographic raw data to enable virtual bronchoscopy (VB), providing the bronchoscopist with a preview of the bronchoscopy prior to the procedure. Navigational bronchoscopy enables targeting of peripheral pulmonary lesions (PPLs) via a "roadmap", similar to in-car global positioning systems. Analysis of lesions on a cellular level is now possible with techniques such as optical coherence tomography (OCT) and confocal microscopy (CM). All these tools will hopefully allow earlier and safer lung cancer diagnosis and in turn better patient outcomes. This article describes these new bronchoscopic techniques and reviews the relevant literature.

Electromagnetic navigation bronchoscopy: A descriptive analysis.

Electromagnetic navigation bronchoscopy (ENB) is an exciting new bronchoscopic technique that promises accurate navigation to peripheral pulmonary target lesions, using technology similar to a car global positioning system (GPS) unit. Potential uses for ENB include biopsy of peripheral lung lesions, pleural dye marking of nodules for surgical wedge resection, placement of fiducial markers for stereotactic radiotherapy, and therapeutic insertion of brachytherapy catheters into malignant tissue. This article will describe the ENB procedure, review the published literature, compare ENB to existing biopsy techniques, and outline the challenges for widespread implementation of this new technology.

The science behind the 7th edition Tumour, Node, Metastasis staging system for lung cancer.

The Tumour, Node, Metastasis (TNM) system for classifying lung cancer is the cornerstone of modern lung cancer treatment and underpins comparative research; yet is continuously evolving through updated revisions. The recently published Union for International Cancer Control 7th Edition TNM Classification for lung cancer addresses many of its predecessor's shortcomings and has been subject to rigorous evidence-based methodology. It is based on a retrospective analysis of over 80 000 lung cancer patients treated between 1990 and 2000 carried out by the International Association for the Study of Lung Cancer. The dataset was truly international and included patients treated by all modalities. Extensive internal and external validation of the findings has ensured that the recommendations are robust and generalizable. For the first time, a single classification system has been shown to be applicable not only to non-small cell lung cancer, but also to be of prognostic significance in small cell lung cancer and bronchopulmonary carcinoid tumours. We review the history of the Union for International Cancer Control TNM staging system, the changes in the most recent 7th edition and the strength of the scientific basis motivating these changes. Limitations of the current staging edition are explored, post-publication independent validation studies are reviewed, and the future of TNM staging for lung cancer is discussed.

Development of a two-part strategy to identify a therapeutic human bispecific antibody that inhibits IgE receptor signaling.

The development of bispecific antibodies as therapeutic agents for human diseases has great clinical potential, but broad application has been hindered by the difficulty of identifying bispecific antibody formats that exhibit favorable pharmacokinetic properties and ease of large-scale manufacturing. Previously, the development of an antibody technology utilizing heavy chain knobs-into-holes mutations and a single common light chain enabled the small-scale generation of human full-length bispecific antibodies. Here we have extended the technology by developing a two-part bispecific antibody discovery strategy that facilitates proof-of-concept studies and clinical candidate antibody generation. Our scheme consists of the efficient small-scale generation of bispecific antibodies lacking a common light chain and the hinge disulfides for proof-of-concept studies coupled with the identification of a common light chain bispecific antibody for large-scale production with high purity and yield. We have applied this technology to generate a bispecific antibody suitable for development as a human therapeutic. This antibody directly inhibits the activation of the high affinity IgE receptor FcepsilonRI on mast cells and basophils by cross-linking FcepsilonRI with the inhibitory receptor FcgammaRIIb, an approach that has strong therapeutic potential for asthma and other allergic diseases. Our approach for producing human bispecific full-length antibodies enables the clinical application of bispecific antibodies to a validated therapeutic pathway in asthma.