An engineered NKp46 antibody for construction of multi-specific NK cell engagers.

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.

An engineered ultrahigh affinity bi-paratopic uPAR targeting agent confers enhanced tumor targeting.

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.

Engineered CD47 protects T cells for enhanced antitumour immunity.

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. 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.

Structural insights reveal interplay between LAG-3 homodimerization, ligand binding, and function.

Lymphocyte activation gene-3 (LAG-3) is an inhibitory receptor expressed on activated T cells and an emerging immunotherapy target. Domain 1 (D1) of LAG-3, which has been purported to directly interact with major histocompatibility complex class II (MHCII) and fibrinogen-like protein 1 (FGL1), has been the major focus for the development of therapeutic antibodies that inhibit LAG-3 receptor-ligand interactions and restore T cell function. Here, we present a high-resolution structure of glycosylated mouse LAG-3 ectodomain, identifying that cis-homodimerization, mediated through a network of hydrophobic residues within domain 2 (D2), is critically required for LAG-3 function.

Grand Challenges at the Interface of Engineering and Medicine.

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.

An engineered interleukin-11 decoy cytokine inhibits receptor signaling and proliferation in lung adenocarcinoma.

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.

De novo design of highly selective miniprotein inhibitors of integrins αvβ6 and αvβ8.

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.

design of highly selective miniprotein inhibitors of integrins αvβ6 and αvβ8.

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 the two closely related integrin proteins and other RGD integrins, stabilize specific conformational states, and have sufficient stability enabling tissue restricted administration could have considerable therapeutic utility. Existing small molecules and antibody inhibitors do not have all of these properties, and hence there is a need for new approaches.

Targeted TLR9 Agonist Elicits Effective Antitumor Immunity against Spontaneously Arising Breast Tumors.

Spontaneous tumors that arise in genetically engineered mice recapitulate the natural tumor microenvironment and tumor-immune coevolution observed in human cancers, providing a more physiologically relevant preclinical model relative to implanted tumors. Similar to many cancer patients, oncogene-driven spontaneous tumors are often resistant to immunotherapy, and thus novel agents that can effectively promote antitumor immunity against these aggressive cancers show considerable promise for clinical translation, and their mechanistic assessment can broaden our understanding of tumor immunology. In this study, we performed extensive immune profiling experiments to investigate how tumor-targeted TLR9 stimulation remodels the microenvironment of spontaneously arising tumors during an effective antitumor immune response.

Mutational screens highlight glycosylation as a modulator of colony-stimulating factor 3 receptor (CSF3R) activity.

The colony-stimulating factor 3 receptor (CSF3R) controls the growth of neutrophils, the most abundant type of white blood cell. In healthy neutrophils, signaling is dependent on CSF3R binding to its ligand, CSF3. A single amino acid mutation in CSF3R, T618I, instead allows for constitutive, ligand-independent cell growth and leads to a rare type of cancer called chronic neutrophilic leukemia.

Broad-spectrum CRISPR-mediated inhibition of SARS-CoV-2 variants and endemic coronaviruses in vitro.

A major challenge in coronavirus vaccination and treatment is to counteract rapid viral evolution and mutations. Here we demonstrate that CRISPR-Cas13d offers a broad-spectrum antiviral (BSA) to inhibit many SARS-CoV-2 variants and diverse human coronavirus strains with >99% reduction of the viral titer. We show that Cas13d-mediated coronavirus inhibition is dependent on the crRNA cellular spatial colocalization with Cas13d and target viral RNA.

Enhanced safety and efficacy of protease-regulated CAR-T cell receptors.

Regulatable CAR platforms could circumvent toxicities associated with CAR-T therapy, but existing systems have shortcomings including leakiness and attenuated activity. Here, we present SNIP CARs, a protease-based platform for regulating CAR activity using an FDA-approved small molecule. Design iterations yielded CAR-T cells that manifest full functional capacity with drug and no leaky activity in the absence of drug.

Anti-GD2 synergizes with CD47 blockade to mediate tumor eradication.

The disialoganglioside GD2 is overexpressed on several solid tumors, and monoclonal antibodies targeting GD2 have substantially improved outcomes for children with high-risk neuroblastoma. However, approximately 40% of patients with neuroblastoma still relapse, and anti-GD2 has not mediated significant clinical activity in any other GD2 malignancy. Macrophages are important mediators of anti-tumor immunity, but tumors resist macrophage phagocytosis through expression of the checkpoint molecule CD47, a so-called 'Don't eat me' signal.

Systemic delivery of a targeted synthetic immunostimulant transforms the immune landscape for effective tumor regression.

Promoting immune activation within the tumor microenvironment (TME) is a promising therapeutic strategy to reverse tumor immunosuppression and elicit anti-tumor immunity. To enable tumor-localized immunotherapy following intravenous administration, we chemically conjugated a polyspecific integrin-binding peptide (PIP) to an immunostimulant (Toll-like receptor 9 [TLR9] agonist: CpG) to generate a tumor-targeted immunomodulatory agent, referred to as PIP-CpG. We demonstrate that systemic delivery of PIP-CpG induces tumor regression and enhances therapeutic efficacy compared with untargeted CpG in aggressive murine breast and pancreatic cancer models.

Use of Outpatient-Derived COVID-19 Convalescent Plasma in COVID-19 Patients Before Seroconversion.

Background: Transfusion of COVID-19 convalescent plasma (CCP) containing high titers of anti-SARS-CoV-2 antibodies serves as therapy for COVID-19 patients. Transfusions early during disease course was found to be beneficial. Lessons from the SARS-CoV-2 pandemic could inform early responses to future pandemics and may continue to be relevant in lower resource settings.

Heterogeneous delivery across the blood-brain barrier limits the efficacy of an EGFR-targeting antibody drug conjugate in glioblastoma.

Background: Antibody drug conjugates (ADCs) targeting the epidermal growth factor receptor (EGFR), such as depatuxizumab mafodotin (Depatux-M), is a promising therapeutic strategy for glioblastoma (GBM) but recent clinical trials did not demonstrate a survival benefit. Understanding the mechanisms of failure for this promising strategy is critically important.

Methods: PDX models were employed to study efficacy of systemic vs intracranial delivery of Depatux-M.

An engineered ligand trap inhibits leukemia inhibitory factor as pancreatic cancer treatment strategy.

Leukemia inhibitory factor (LIF), a cytokine secreted by stromal myofibroblasts and tumor cells, has recently been highlighted to promote tumor progression in pancreatic and other cancers through KRAS-driven cell signaling. We engineered a high affinity soluble human LIF receptor (LIFR) decoy that sequesters human LIF and inhibits its signaling as a therapeutic strategy. This engineered 'ligand trap', fused to an antibody Fc-domain, has ~50-fold increased affinity (~20 pM) and improved LIF inhibition compared to wild-type LIFR-Fc, potently blocks LIF-mediated effects in pancreatic cancer cells, and slows the growth of pancreatic cancer xenograft tumors.

LYTACs that engage the asialoglycoprotein receptor for targeted protein degradation.

Selective protein degradation platforms have afforded new development opportunities for therapeutics and tools for biological inquiry. The first lysosome-targeting chimeras (LYTACs) targeted extracellular and membrane proteins for degradation by bridging a target protein to the cation-independent mannose-6-phosphate receptor (CI-M6PR). Here, we developed LYTACs that engage the asialoglycoprotein receptor (ASGPR), a liver-specific lysosome-targeting receptor, to degrade extracellular proteins in a cell-type-specific manner.

CD52 Is Elevated on B cells of SLE Patients and Regulates B Cell Function.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by B cell dysregulation and breaks in tolerance that lead to the production of pathogenic autoantibodies. We performed single-cell RNA sequencing of B cells from healthy donors and individuals with SLE which revealed upregulated CD52 expression in SLE patients. We further demonstrate that SLE patients exhibit significantly increased levels of B cell surface CD52 expression and plasma soluble CD52, and levels of soluble CD52 positively correlate with measures of lupus disease activity.

Identification of N-Terminally Diversified GLP-1R Agonists Using Saturation Mutagenesis and Chemical Design.

The glucagon-like peptide 1 receptor (GLP-1R) is a class B G-protein coupled receptor (GPCR) and diabetes drug target expressed mainly in pancreatic β-cells that, when activated by its agonist glucagon-like peptide 1 (GLP-1) after a meal, stimulates insulin secretion and β-cell survival and proliferation. The N-terminal region of GLP-1 interacts with membrane-proximal residues of GLP-1R, stabilizing its active conformation to trigger intracellular signaling. The best-studied agonist peptides, GLP-1 and exendin-4, share sequence homology at their N-terminal region; however, modifications that can be tolerated here are not fully understood.

PET Reporter Gene Imaging and Ganciclovir-Mediated Ablation of Chimeric Antigen Receptor T Cells in Solid Tumors.

Imaging strategies to monitor chimeric antigen receptor (CAR) T-cell biodistribution and proliferation harbor the potential to facilitate clinical translation for the treatment of both liquid and solid tumors. In addition, the potential adverse effects of CAR T cells highlight the need for mechanisms to modulate CAR T-cell activity. The herpes simplex virus type 1 thymidine kinase (HSV1-tk) gene has previously been translated as a PET reporter gene for imaging of T-cell trafficking in patients with brain tumor.

An engineered antibody binds a distinct epitope and is a potent inhibitor of murine and human VISTA.

V-domain immunoglobulin (Ig) suppressor of T cell activation (VISTA) is an immune checkpoint that maintains peripheral T cell quiescence and inhibits anti-tumor immune responses. VISTA functions by dampening the interaction between myeloid cells and T cells, orthogonal to PD-1 and other checkpoints of the tumor-T cell signaling axis. Here, we report the use of yeast surface display to engineer an anti-VISTA antibody that binds with high affinity to mouse, human, and cynomolgus monkey VISTA.