An in vivo selection-derived d-peptide for engineering erythrocyte-binding antigens that promote immune tolerance

When displayed on erythrocytes, peptides and proteins can drive antigen-specific immune tolerance. Here, we investigated a straightforward approach based on erythrocyte binding to promote antigen-specific tolerance to both peptides and proteins. We first identified a robust erythrocyte-binding ligand. A pool of one million fully d-chiral peptides was injected into mice, blood cells were isolated, and ligands enriched on these cells were identified using nano-liquid chromatography–tandem mass spectrometry. One round of selection yielded a murine erythrocyte-binding ligand with an 80 nM apparent dissociation constant, Kd. We modified an 83-kDa bacterial protein and a peptide antigen derived from ovalbumin (OVA) with the identified erythrocyte-binding ligand. An administration of the engineered bacterial protein led to decreased protein-specific antibodies in mice. Similarly, mice given the engineered OVA-derived peptide had decreased inflammatory anti-OVA CD8+ T cell responses. These findings suggest that our tolerance-induction strategy is applicable to both peptide and protein antigens and that our in vivo selection strategy can be used for de novo discovery of robust erythrocyte-binding ligands.

An in vivo selection system with tightly regulated gene expression enables directed evolution of highly efficient enzymes

In vivo selection systems are powerful tools for directed evolution of enzymes. The selection pressure of the systems can be tuned by regulating the expression levels of the catalysts. In this work, we engineered a selection system for laboratory evolution of highly active enzymes by incorporating a translationally suppressing cis repressor as well as an inducible promoter to impart stringent and tunable selection pressure. We demonstrated the utility of our selection system by performing directed evolution experiments using TEM β-lactamase as the model enzyme. Five evolutionary rounds afforded a highly active variant exhibiting 440-fold improvement in catalytic efficiency. We also showed that, without the cis repressor, the selection system cannot provide sufficient selection pressure required for evolving highly efficient TEM β-lactamase. The selection system should be applicable for the exploration of catalytic perfection of a wide range of enzymes.

In vivo selection of nanobodies to target the tumor immune microenvironment for immunotherapy development

Cancer immunotherapy has changed the landscape of treatment for some cancers, but variable responses reflect limitations imposed by the tumor microenvironment (TME) . To facilitate the development of novel immunotherapies, we used an unbiased in vivo selection approach using a VHH nanobody library to identify immunologic targets in the TME. Using next generation sequencing (NGS), we demonstrate the parallel enrichment of immune cell-binding nanobodies that target immune cell subsets within the TME. We selected from enriched nanobodies - Cl.1 and Cl.2 - that bind to the CD11b+ and CD11c+ populations and we identified the desmosome complex as the target for Cl.1, suggesting the nanobodies can target key immune populations and may involve unvalidated targets. To demonstrate nanobodies could functionally alter target immune cells in vivo, we showed by single cell RNA sequencing (scRNAseq) that enriched nanobodies can increase activation transcripts in CD8+ T cells and dendritic cells. This approach is being used to develop novel cancer immunotherapies, but the methods can be broadly applicable to biologic discovery experiments in various physiologic or pathologic contexts.

Development of a highly pulmonary metastatic orthotopic renal cell carcinoma murine model

ABSTRACT The incidence of renal cell carcinoma (RCC) is high, and its outcomes remain poor. Mortality is attributable largely to metastatic disease and a dearth of effective therapeutic interventions. The lungs are the most common metastatic site. To elucidate the biological mechanisms underlying pulmonary metastasis and identify superior therapeutic strategies, we developed a novel and clinically relevant murine RCC model exhibiting enhanced pulmonary metastasis. Mice underwent intrarenal implantation using luciferase-expressing Renca, a murine renal adenocarcinoma cell line. Primary renal tumor progression and development of metastatic lung lesions were monitored in live mice using bioluminescent imaging, followed by post-mortem organ assessment. Cells were isolated from pulmonary metastases for reimplantation, followed by repeat monitoring and assessment. This process was repeated once more for a total of two in vivo passages to select for pulmonary metastatic Renca cell subpopulations. However, a single round of in vivo selection was sufficient to produce a near-maximally metastatic subpopulation. Relative to Renca cell-implanted mice, subpopulation-implanted mice exhibited shorter implantation-metastasis intervals (5 days), shorter implantation-moribundity intervals (sacrificed at 18.6±2.9 versus 22.3±1.1 days), a higher number of metastatic lung lesions at 23 days (183.9±39.0 versus 172.6±38.2) and poorer survival. Implantation of cells derived from the second round of in vivo selection produced no further significant differences in the above metrics. This model consistently and efficiently recapitulates RCC pulmonary metastasis while allowing in vivo monitoring of tumor progression, thereby facilitating elucidation of the molecular mechanisms underlying pulmonary metastasis and evaluation of therapeutic modalities.

The In Vivo Selection Method in Breast Cancer Metastasis

Metastasis is a complex event in cancer progression and causes most deaths from cancer. Repeated transplantation of metastatic cancer cells derived from transplanted murine organs can be used to select the population of highly metastatic cancer cells; this method is called as in vivo selection. The in vivo selection method and highly metastatic cancer cell lines have contributed to reveal the molecular mechanisms of cancer metastasis. Here, we present an overview of the methodology for the in vivo selection method. Recent comparative analysis of the transplantation methods for metastasis have revealed the divergence of metastasis gene signatures. Even cancer cells that metastasize to the same organ show various metastatic cascades and gene expression patterns by changing the transplantation method for the in vivo selection. These findings suggest that the selection of metastasis models for the study of metastasis gene signatures has the potential to influence research results. The study of novel gene signatures that are identified from novel highly metastatic cell lines and patient-derived xenografts (PDXs) will be helpful for understanding the novel mechanisms of metastasis.

In Vivo HSC Gene Therapy for Hemoglobinopathies: A Proof of Concept Evaluation in Rhesus Macaques

Current gene therapy or genome editing studies for hemoglobinopathies require highly sophisticated medical facilities to perform hematopoietic stem cell collections / selections and genetic modifications. In addition, patients receive high-dose chemotherapy to facilitate engraftment of gene-modified cells. Thus, current gene therapy protocols will not be accessible to most patients suffering from hemoglobinopathies. Here we describe a highly portable and scalable approach using in vivo hematopoietic stem cell (HSC) gene therapy to potentially overcome these limitations. The central idea of our in vivo HSC gene therapy approach is to mobilize HSCs from the bone marrow, and while they circulate at high numbers in the periphery, transduce them with an intravenously injected HSC-tropic, helper-dependent adenovirus HDAd5/35++ gene transfer vector system. Transduced cells return to the bone marrow where they persist long-term. Transgene integration is either achieved by a Sleeping Beauty transposase (SB100x) in a random pattern or by homology-directed-repair into a safe genomic harbor site. Currently, an in vivo selection system (involving the mgmtP140K gene/low-dose O6BG/BCNU) is employed to achieve 80-100% marking levels in peripheral blood cells. We demonstrated safety and efficacy of our approach in mouse models for thalassemia intermedia, Sickle Cell Disease, and hemophilia A, where we achieved a phenotypic correction. We now present data in 3 rhesus macaques. We show that treatment with G-CSF/AMD3100 resulted in efficient HSC mobilization into the blood circulation and subsequent intravenous injection of the HDAd5/35++ vector system (total 1-3 x1012 vp/kg, in two doses) was well tolerated. The longest follow up thus far is 24 weeks after in vivo HSC transduction with a human-gamma-globin expressing HDAd5/35++ vector. After in vivo selection with O6BG plus low dose (10 to 20 mg/m2) of BCNU, a dose that is up to 100-fold lower than what is used for autologous transplantation protocols, gamma-globin marking in peripheral red blood cells rose to ~90% and was stable for the duration of the study (see Figure). gamma-globin levels in red blood cells were ~18% of adult alpha1-globin (by HPLC). No abnormalities in genome and transcriptome analyses of animal #1 were found at the time of scheduled necropsy. We show that a new prophylaxis regimen (dexamethasone, IL-6R, IL-1bR antagonists, saline bolus IV) was able to mitigate all side effects associated with intravenous HDAd5/35++ vector administration. Analysis of day 3 bone marrow showed 30% transduced HSCs. Vector DNA biodistribution studies demonstrated very low or absent transduction of most tissues (including testes and CNS). Analysis of bone marrow showed efficient, preferential HSC transduction and re-homing of transduced CD34+/CD90+ cells to the bone marrow. At week 4, about 5% of progenitor colony-forming cells demonstrated stable transduction with integrated vector, and this frequency increased after starting the in vivo selection. The level of human mgmtP140K mRNA expression in PBMCs also increased after in vivo selection. In summary: Using a new and optimized prophylaxis regimen intravenous delivery of HDAd5/35++ was very well tolerated without any cytokine activation. We saw efficient transduction of HSCs and efficient in vivo selection of transduced progenitors with low dose O6BG/BCNU. This is the first proof-of-concept study that in vivo HSC gene therapy could be feasible in humans without the need of high-dose chemotherapy conditioning and without the need for highly specialized medical facilities. This approach would provide a major advance for the gene therapy and genome editing field and allow the necessary portability and accessibility to reach patients in places with limited medical resources. Figure 1 Disclosures Radtke: Forty Seven INC: Consultancy. Kiem:Umoja: Membership on an entity's Board of Directors or advisory committees; Rocket Pharma: Membership on an entity's Board of Directors or advisory committees; Vor Biopharma: Membership on an entity's Board of Directors or advisory committees; Enochian: Membership on an entity's Board of Directors or advisory committees; CSL: Consultancy; Magenta Therapeutics: Consultancy; Homology Medicines: Membership on an entity's Board of Directors or advisory committees. Lieber:Ensoma, Inc: Consultancy, Research Funding.