Microfluidic Squeezing Enables MHC Class I Antigen Presentation by Diverse Immune Cells to Elicit CD8+ T Cell Responses with Antitumor Activity.

CD8+ T cell responses are the foundation of the recent clinical success of immunotherapy in oncologic indications. Although checkpoint inhibitors have enhanced the activity of existing CD8+ T cell responses, therapeutic approaches to generate Ag-specific CD8+ T cell responses have had limited success. Here, we demonstrate that cytosolic delivery of Ag through microfluidic squeezing enables MHC class I presentation to CD8+ T cells by diverse cell types. In murine dendritic cells (DCs), squeezed DCs were ∼1000-fold more potent at eliciting CD8+ T cell responses than DCs cross-presenting the same amount of protein Ag. The approach also enabled engineering of less conventional APCs, such as T cells, for effective priming of CD8+ T cells in vitro and in vivo. Mixtures of immune cells, such as murine splenocytes, also elicited CD8+ T cell responses in vivo when squeezed with Ag. We demonstrate that squeezing enables effective MHC class I presentation by human DCs, T cells, B cells, and PBMCs and that, in clinical scale formats, the system can squeeze up to 2 billion cells per minute. Using the human papillomavirus 16 (HPV16) murine model, TC-1, we demonstrate that squeezed B cells, T cells, and unfractionated splenocytes elicit antitumor immunity and correlate with an influx of HPV-specific CD8+ T cells such that >80% of CD8s in the tumor were HPV specific. Together, these findings demonstrate the potential of cytosolic Ag delivery to drive robust CD8+ T cell responses and illustrate the potential for an autologous cell-based vaccine with minimal turnaround time for patients.

Investigating receptor-mediated antibody transcytosis using blood-brain barrier organoid arrays.

BACKGROUND: The pathways that control protein transport across the blood-brain barrier (BBB) remain poorly characterized. Despite great advances in recapitulating the human BBB in vitro, current models are not suitable for systematic analysis of the molecular mechanisms of antibody transport. The gaps in our mechanistic understanding of antibody transcytosis hinder new therapeutic delivery strategy development. METHODS: We applied a novel bioengineering approach to generate human BBB organoids by the self-assembly of astrocytes, pericytes and brain endothelial cells with unprecedented throughput and reproducibility using micro patterned hydrogels. We designed a semi-automated and scalable imaging assay to measure receptor-mediated transcytosis of antibodies. Finally, we developed a workflow to use CRISPR/Cas9 gene editing in BBB organoid arrays to knock out regulators of endocytosis specifically in brain endothelial cells in order to dissect the molecular mechanisms of receptor-mediated transcytosis. RESULTS: BBB organoid arrays allowed the simultaneous growth of more than 3000 homogenous organoids per individual experiment in a highly reproducible manner. BBB organoid arrays showed low permeability to macromolecules and prevented transport of human non-targeting antibodies. In contrast, a monovalent antibody targeting the human transferrin receptor underwent dose- and time-dependent transcytosis in organoids. Using CRISPR/Cas9 gene editing in BBB organoid arrays, we showed that clathrin, but not caveolin, is required for transferrin receptor-dependent transcytosis. CONCLUSIONS: Human BBB organoid arrays are a robust high-throughput platform that can be used to discover new mechanisms of receptor-mediated antibody transcytosis. The implementation of this platform during early stages of drug discovery can accelerate the development of new brain delivery technologies.

Proteolysis-Targeting Chimeras Enhance T Cell Bispecific Antibody-Driven T Cell Activation and Effector Function through Increased MHC Class I Antigen Presentation in Cancer Cells.

The availability of Ags on the surface of tumor cells is crucial for the efficacy of cancer immunotherapeutic approaches using large molecules, such as T cell bispecific Abs (TCBs). Tumor Ags are processed through intracellular proteasomal protein degradation and are displayed as peptides on MHC class I (MHC I). Ag recognition through TCRs on the surface of CD8+ T cells can elicit a tumor-selective immune response. In this article, we show that proteolysis-targeting chimeras (PROTACs) that target bromo- and extraterminal domain proteins increase the abundance of the corresponding target-derived peptide Ags on MHC I in both liquid and solid tumor-derived human cell lines. This increase depends on the engagement of the E3 ligase to bromo- and extraterminal domain protein. Similarly, targeting of a doxycycline-inducible Wilms tumor 1 (WT1)-FKBP12F36V fusion protein, by a mutant-selective FKBP12F36V degrader, increases the presentation of WT1 Ags in human breast cancer cells. T cell-mediated response directed against cancer cells was tested on treatment with a TCR-like TCB, which was able to bridge human T cells to a WT1 peptide displayed on MHC I. FKBP12F36V degrader treatment increased the expression of early and late activation markers (CD69, CD25) in T cells; the secretion of granzyme ß, IFN-γ, and TNF-α; and cancer cell killing in a tumor-T cell coculture model. This study supports harnessing targeted protein degradation in tumor cells, for modulation of T cell effector function, by investigating for the first time, to our knowledge, the potential of combining a degrader and a TCB in a cancer immunotherapy setting.

Vaccine-induced CD8 T cells are redirected with peptide-MHC class I-IgG antibody fusion proteins to eliminate tumor cells in vivo.

Simulating a viral infection in tumor cells is an attractive concept to eliminate tumor cells. We previously reported the molecular design and the in vitro potency of recombinant monoclonal antibodies fused to a virus-derived peptide MHC class I complex that bypass the peptide processing and MHC loading pathway and directly displays a viral peptide in an MHC class I complex on the tumor cell surface. Here, we show that a vaccination-induced single peptide-specific CD8 T cell response was sufficient to eliminate B16 melanoma tumor cells in vivo in a fully immunocompetent, syngeneic mouse tumor model when mice were treated with mouse pMHCI-IgGs fusion proteins targeting the mouse fibroblast activation protein. Tumor growth of small, established B16 lung metastases could be controlled. The pMHCI-IgG had similar potency as an analogous pan-CD3 T-cell bispecific antibody. In contrast to growth control of small tumors, none of the compounds controlled larger solid tumors of MC38 cancer cells, despite penetration of pMHCI-IgGs into the tumor tissue and clear attraction and activation of antigen-specific CD8 T cells inside the tumor. pMHCI-IgG can have a similar potency as classical pan-T-cell recruiting molecules. The results also highlight the need to better understand immune suppression in advanced solid tumors.

Quantitative fluorescence imaging determines the absolute number of locked nucleic acid oligonucleotides needed for suppression of target gene expression.

Locked nucleic acid based antisense oligonucleotides (LNA-ASOs) can reach their intracellular RNA targets without delivery modules. Functional cellular uptake involves vesicular accumulation followed by translocation to the cytosol and nucleus. However, it is yet unknown how many LNA-ASO molecules need to be delivered to achieve target knock down. Here we show by quantitative fluorescence imaging combined with LNA-ASO microinjection into the cytosol or unassisted uptake that ∼105 molecules produce >50% knock down of their targets, indicating that a substantial amount of LNA-ASO escapes from endosomes. Microinjected LNA-ASOs redistributed within minutes from the cytosol to the nucleus and remained bound to nuclear components. Together with the fact that RNA levels for a given target are several orders of magnitude lower than the amounts of LNA-ASO, our data indicate that only a minor fraction is available for RNase H1 mediated reduction of target RNA. When non-specific binding sites were blocked by co-administration of non-related LNA-ASOs, the amount of target LNA-ASO required was reduced by an order of magnitude. Therefore, dynamic processes within the nucleus appear to influence the distribution and activity of LNA-ASOs and may represent important parameters for improving their efficacy and potency.

A New Class of Bifunctional Major Histocompatibility Class I Antibody Fusion Molecules to Redirect CD8 T Cells.

Bifunctional antibody fusion proteins engaging effector T cells for targeted elimination of tumor cells via CD3 binding have shown efficacy in both preclinical and clinical studies. Different from such a polyclonal T-cell recruitment, an alternative concept is to engage only antigen-specific T-cell subsets. Recruitment of specific subsets of T cells may be as potent but potentially lead to fewer side effects. Tumor-targeted peptide-MHC class I complexes (pMHCI-IgGs) bearing known antigenic peptides complexed with MHC class I molecules mark tumor cells as antigenic and utilize the physiologic way to interact with and activate T-cell receptors. If, for example, virus-specific CD8(+) T cells are addressed, the associated strong antigenicity and tight immune surveillance of the effector cells could lead to efficacious antitumor treatment in various tissues. However, peptide-MHC class I fusions are difficult to express recombinantly, especially when fused to entire antibody molecules. Consequently, current formats are largely limited to small antibody fragment fusions expressed in bacteria followed by refolding or chemical conjugation. Here, we describe a new molecular format bearing a single pMHCI complex per IgG fusion molecule characterized by enhanced stability and expression yields. This molecular format can be expressed in a full immunoglobulin format and can be designed as mono- or bivalent antibody binders. Mol Cancer Ther; 15(9); 2130-42. ©2016 AACR.

Activation of cytomegalovirus-specific CD8+ T-cell response by antibody-mediated peptide-major histocompatibility class I complexes.

Imposing antigenicity on tumor cells is a key step toward successful cancer-immunotherapy. A cytomegalovirus-derived peptide recombinantly fused to a major histocompatibility class I complex and a monoclonal antibody can be targeted to tumor cells by antibody-mediated delivery and activate a strong and specific CD8+ T cell response.

Committing Cytomegalovirus-Specific CD8 T Cells to Eliminate Tumor Cells by Bifunctional Major Histocompatibility Class I Antibody Fusion Molecules.

Tumor cells escape immune eradication through multiple mechanisms, including loss of antigenicity and local suppression of effector lymphocytes. To counteract these obstacles, we aimed to direct the unique cytomegalovirus (CMV)-specific immune surveillance against tumor cells. We developed a novel generation of fusion proteins composed of a tumor antigen-specific full immunoglobulin connected to a single major histocompatibility class I complex bearing a covalently linked virus-derived peptide (pMHCI-IgG). Here, we show that tumor antigen-expressing cancer cells, which are decorated with pMHCI-IgGs containing a HLA-A*0201 molecule associated with a CMV-derived peptide, are specifically eliminated through engagement of antigen-specific CD8(+) T cells isolated from peripheral blood mononuclear cell preparations of CMV-infected humans. These CD8(+) T cells act without additional expansion, preactivation, or provision of costimulatory signals. Elimination of tumor cells is induced at similar concentrations and with similar time kinetics as those seen with bispecific T-cell engagers (BiTE). However, while BiTE-like reagents indiscriminately activate T cells through binding to the T-cell receptor complex, pMHCI-IgGs selectively engage antigen-specific, constantly renewable, differentiated effector cytotoxic T lymphocytes to tumor cells, thereby representing a novel class of anticancer immunotherapeutics with potentially improved safety and efficacy profiles.

Behavioural and functional characterization of Kv10.1 (Eag1) knockout mice.

Kv10.1 (Eag1), member of the Kv10 family of voltage-gated potassium channels, is preferentially expressed in adult brain. The aim of the present study was to unravel the functional role of Kv10.1 in the brain by generating knockout mice, where the voltage sensor and pore region of Kv10.1 were removed to render non-functional proteins through deletion of exon 7 of the KCNH1 gene using the '3 Lox P strategy'. Kv10.1-deficient mice show no obvious alterations during embryogenesis and develop normally to adulthood; cortex, hippocampus and cerebellum appear anatomically normal. Other tests, including general health screen, sensorimotor functioning and gating, anxiety, social behaviour, learning and memory did not show any functional aberrations in Kv10.1 null mice. Kv10.1 null mice display mild hyperactivity and longer-lasting haloperidol-induced catalepsy, but there was no difference between genotypes in amphetamine sensitization and withdrawal, reactivity to apomorphine and haloperidol in the prepulse inhibition tests or to antidepressants in the haloperidol-induced catalepsy. Furthermore, electrical properties of Kv10.1 in cerebellar Purkinje cells did not show any difference between genotypes. Bearing in mind that Kv10.1 is overexpressed in over 70% of all human tumours and that its inhibition leads to a reduced tumour cell proliferation, the fact that deletion of Kv10.1 does not show a marked phenotype is a prerequisite for utilizing Kv10.1 blocking and/or reduction techniques, such as siRNA, to treat cancer.

Regulation of geminin functions by cell cycle-dependent nuclear-cytoplasmic shuttling.

The geminin protein functions both as a DNA rereplication inhibitor through association with Cdt1 and as a repressor of Hox gene transcription through the polycomb pathway. Here, we report that the functions of avian geminin are coordinated with and regulated by cell cycle-dependent nuclear-cytoplasmic shuttling. In S phase, geminin enters nuclei and inhibits both loading of the minichromosome maintenance (MCM) complex onto chromatin and Hox gene transcription. At the end of mitosis, geminin is exported from nuclei by the exportin protein Crm1 and is unavailable in the nucleus during the next G(1) phase, thus ensuring proper chromatin loading of the MCM complex and Hox gene transcription. This mechanism for regulating the functions of geminin adds to distinct mechanisms, such as protein degradation and ubiquitination, applied in other vertebrates.

The cell-cycle regulator geminin inhibits Hox function through direct and polycomb-mediated interactions.

Embryonic development is tightly controlled. The clustered genes of the Hox family of homeobox proteins play an important part in regulating this development and also proliferation. They specify embryonic structures along the body axis, and are associated with normal and malignant cell growth. The cell-cycle regulator geminin controls replication by binding to the licensing factor Cdt1, and is involved in neural differentiation. Here, we show that murine geminin associates transiently with members of the Hox-repressing polycomb complex, with the chromatin of Hox regulatory DNA elements and with Hox proteins. Gain- and loss-of-function experiments in the chick neural tube demonstrate that geminin modulates the anterior boundary of Hoxb9 transcription, which suggests a polycomb-like activity for geminin. The interaction between geminin and Hox proteins prevents Hox proteins from binding to DNA, inhibits Hox-dependent transcriptional activation of reporter and endogenous downstream target genes, and displaces Cdt1 from its complex with geminin. By establishing competitive regulation, geminin functions as a coordinator of developmental and proliferative control.