Jennifer R. Cochran

Antibody–drug conjugates (ADCs) are standard-of-care therapies for many types of cancer, with an expanding representation in clinical development pipelines. ¹ A key determinant of efficacy for most ADCs is their ability to internalize into the target cell and traffic to the lysosome, where the ADC payload is released. However, this internalization process is target- and antibody-dependent, limiting therapeutic results and ADC target scope. Here we extend the lysosome-targeting chimera (LYTAC) ^2,3 concept to ADCs. We conjugated a lysosome-targeting polyspecific integrin-binding peptide (PIP) ^4,5 to ADCs, generating lysosomally targeted ADCs (LytADCs). Across three targets (HER2, EGFR, CA9), two cancer cell lines (SKOV‑3, U-87 MG), and three payloads (MMAE, MMAF, Dxd), LytADCs were significantly more potent than the corresponding ADC in cell culture, 3D spheroid culture and in a murine xenograft model. Notably, the LytADC strategy converted an anti-CA9 ADC, a poor internalizer with no detectable cytotoxicity in 2D cell culture despite target expression, into a potent therapeutic in vitro and in vivo . LytADCs therefore expand the ADC-druggable target space to include cell surface antigens with poor intrinsic internalization properties.

Antibody-drug conjugates (ADCs) are standard-of-care therapies for several types of cancer, with an expanding representation in clinical development pipelines. A key determinant of efficacy for most ADCs is their ability to internalize into the target cell, traffic to the lysosome, and release the toxic payload. This internalization and trafficking, however, is target- and antibody-dependent. Here we describe an approach to increase both the target selectivity and internalization ability of ADCs, leveraging recent advances in the extracellular targeted protein degradation space. Lysosome-targeting chimeras (LYTACs) are a class of molecules where a target binder is conjugated to a ligand for a lysosome trafficking receptor. LYTACs thereby deliver the target protein to the lysosome, leading to its degradation. We generated lysosomally-targeted ADCs (LytADCs) by attaching a polyspecific integrin-targeting peptide (PIP) to ADCs targeting several cancer-associated antigens. LytADCs were ~10x more efficient at cell-killing than the corresponding ADC, across three targets (HER2, EGFR, CA9), two different cancer cell lines (SKOV-3, U-87 MG), and in both 2D and 3D spheroid cell culture. These data suggest that LytADCs are a promising modality as next-generation ADCs.

Urokinase‐type plasminogen activator receptor (uPAR) is overexpressed on tumor cells in multiple types of cancer and contributes to disease progression and metastasis. In this work, we engineered a novel bi‐paratopic uPAR targeting agent by fusing the binding domains of two native uPAR ligands: uPA and vitronectin, with a flexible peptide linker. The linker length was optimized to facilitate simultaneous engagement of both domains to their adjacent epitopes on uPAR, resulting in a high affinity and avid binding interaction. Furthermore, the individual domains were affinity‐matured using yeast surface display and directed evolution, resulting in a bi‐paratopic protein with affinity in the picomolar to femtomolar range. This engineered uPAR targeting agent demonstrated significantly enhanced tumor localization in mouse tumor models compared to the native uPAR ligand and warrants further investigation as a diagnostic and therapeutic agent for cancer.

Adoptively transferred T cells and agents designed to block the CD47–SIRPα axis are promising cancer therapeutics that activate distinct arms of the immune system^1,2. Here we administered anti-CD47 antibodies in combination with adoptively transferred T cells with the goal of enhancing antitumour efficacy but observed abrogated therapeutic benefit due to rapid macrophage-mediated clearance of T cells expressing chimeric antigen receptors (CARs) or engineered T cell receptors. Anti-CD47-antibody-mediated CAR T cell clearance was potent and rapid enough to serve as an effective safety switch. To overcome this challenge, we engineered the CD47 variant CD47(Q31P) (47_E), which engages SIRPα and provides a ‘don’t eat me’ signal that is not blocked by anti-CD47 antibodies. TCR or CAR T cells expressing 47_E are resistant to clearance by macrophages after treatment with anti-CD47 antibodies, and mediate substantial, sustained macrophage recruitment to the tumour microenvironment. Although many of the recruited macrophages manifested an M2-like profile³, the combined therapy synergistically enhanced antitumour efficacy. Our study identifies macrophages as major regulators of T cell persistence and illustrates the fundamental challenge of combining T-cell-directed therapeutics with those designed to activate macrophages. It delivers a therapeutic approach that is capable of simultaneously harnessing the antitumour effects of T cells and macrophages, offering enhanced potency against solid tumours.

Tumors comprise a complex ecosystem consisting of many cell types that communicate through secreted factors. Targeting these intercellular signaling networks remains an important challenge in cancer research. Cardiotrophin-like cytokine factor 1 (CLCF1) is an interleukin-6 (IL-6) family member secreted by cancer-associated fibroblasts (CAFs) that binds to ciliary neurotrophic factor receptor (CNTFR), promoting tumor growth in lung and liver cancer^1,2. A high-affinity soluble receptor (eCNTFR-Fc) that sequesters CLCF1 has anti-oncogenic effects³. However, the role of CLCF1 in mediating cell-cell interactions in cancer has remained unclear. We demonstrate that eCNTFR–Fc has widespread effects on both tumor cells and the tumor microenvironment and can sensitize cancer cells to KRAS inhibitors or immune checkpoint blockade. After three weeks of treatment with eCNTFR-Fc, there is a shift from an immunosuppressive to an immunostimulatory macrophage phenotype as well as an increase in activated T, NKT, and NK cells. Combination of eCNTFR-Fc and αPD1 was significantly more effective than single-agent therapy in a syngeneic allograft model, and eCNTFR-Fc sensitizes tumor cells to αPD1 in a non-responsive GEM model of lung adenocarcinoma. These data suggest that combining eCNTFR-Fc with KRAS inhibition or with αPD1 is a novel therapeutic strategy for lung cancer and potentially other cancers in which these therapies have been used but to date with only modest effect. Overall, we demonstrate the potential of cancer therapies that target cytokines to alter the immune microenvironment.

Over the past two decades Biomedical Engineering has emerged as a major discipline that bridges societal needs of human health care with the development of novel technologies. Every medical institution is now equipped at varying degrees of sophistication with the ability to monitor human health in both non-invasive and invasive modes. The multiple scales at which human physiology can be interrogated provide a profound perspective on health and disease. We are at the nexus of creating “avatars” (herein defined as an extension of “digital twins”) of human patho/physiology to serve as paradigms for interrogation and potential intervention. Motivated by the emergence of these new capabilities, the IEEE Engineering in Medicine and Biology Society, the Departments of Biomedical Engineering at Johns Hopkins University and Bioengineering at University of California at San Diego sponsored an interdisciplinary workshop to define the grand challenges that face biomedical engineering and the mechanisms to address these challenges. The Workshop identified five grand challenges with cross-cutting themes and provided a roadmap for new technologies, identified new training needs, and defined the types of interdisciplinary teams needed for addressing these challenges. The themes presented in this paper include: 1) accumedicine through creation of avatars of cells, tissues, organs and whole human; 2) development of smart and responsive devices for human function augmentation; 3) exocortical technologies to understand brain function and treat neuropathologies; 4) the development of approaches to harness the human immune system for health and wellness; and 5) new strategies to engineer genomes and cells.

Recent developments in cancer immunotherapy have highlighted the potential of harnessing natural killer (NK) cells in the treatment of neoplastic malignancies. Of these, bispecific antibodies, and NK cell engager (NKCE) protein therapeutics in particular, have been of interest. Here, we used phage display and yeast surface display to engineer RLN131, a unique cross-reactive antibody that binds to human, mouse, and cynomolgus NKp46, an activating receptor found on NK cells. RLN131 induced proliferation and activation of primary NK cells, and was used to create bispecific NKCE constructs of varying configurations and valency. All NKCEs were able to promote greater NK cell cytotoxicity against tumor cells than an unmodified anti-CD20 monoclonal antibody, and activity was observed irrespective of whether the constructs contained a functional Fc domain. Competition binding and fine epitope mapping studies were used to demonstrate that RLN131 binds to a conserved epitope on NKp46, underlying its species cross-reactivity.

The RGD (Arg-Gly-Asp)-binding integrins αvβ6 and αvβ8 are clinically validated cancer and fibrosis targets of considerable therapeutic importance. Compounds that can discriminate between homologous αvβ6 and αvβ8 and other RGD integrins, stabilize specific conformational states, and have high thermal stability could have considerable therapeutic utility. Existing small molecule and antibody inhibitors do not have all these properties, and hence new approaches are needed. Here we describe a generalized method for computationally designing RGD-containing miniproteins selective for a single RGD integrin heterodimer and conformational state. We design hyperstable, selective αvβ6 and αvβ8 inhibitors that bind with picomolar affinity. CryoEM structures of the designed inhibitor-integrin complexes are very close to the computational design models, and show that the inhibitors stabilize specific conformational states of the αvβ6 and the αvβ8 integrins. In a lung fibrosis mouse model, the αvβ6 inhibitor potently reduced fibrotic burden and improved overall lung mechanics, demonstrating the therapeutic potential of de novo designed integrin binding proteins with high selectivity.

The cytokine interleukin (IL)‐11 has been shown to play a role in promoting fibrosis and cancer, including lung adenocarcinoma, garnering interest as an attractive target for therapeutic intervention. We used combinatorial methods to engineer an IL‐11 variant that binds with higher affinity to the IL‐11 receptor and stimulates enhanced receptor‐mediated cell signaling. Introduction of two additional point mutations ablates IL‐11 ligand/receptor association with the gp130 coreceptor signaling complex, resulting in a high‐affinity receptor antagonist. Unlike wild‐type IL‐11, this engineered variant potently blocks IL‐11‐mediated cell signaling and slows tumor growth in a mouse model of lung cancer. Our approach highlights a strategy where native ligands can be engineered and exploited to create potent receptor antagonists.