Development of a Versatile Cancer Vaccine Format Targeting Antigen-Presenting Cells Using Proximity-Based Sortase A-Mediated Ligation of T-Cell Epitopes.

Cancer vaccines are a promising strategy to increase tumor-specific immune responses in patients who do not adequately respond to checkpoint inhibitors. Cancer vaccines that contain patient-specific tumor antigens are most effective but also necessitate the production of patient-specific vaccines. This study aims to develop a versatile cancer vaccine format in which patient-specific tumor antigens can be site-specifically conjugated by a proximity-based Sortase A (SrtA)-mediated ligation (PBSL) approach to antibodies that specifically bind to antigen-presenting cells to stimulate immune responses.

CRISPR-Cas9 screening reveals a distinct class of MHC-I binders with precise HLA-peptide recognition.

Human leukocyte antigen (HLA) class-I molecules present fragments of the cellular proteome to the T cell receptor (TCR) of cytotoxic T cells to control infectious diseases and cancer. The large number of combinations of HLA class-I allotypes and peptides allows for highly specific and dedicated low-affinity interactions to a diverse array of TCRs and natural killer (NK) cell receptors. Whether the divergent HLA class-I peptide complex is exclusive for interactions with these proteins is unknown.

CRISPR/Cas9-based Engineering of Immunoglobulin Loci in Hybridoma Cells.

Development of the hybridoma technology by Köhler and Milstein (1975) has revolutionized the immunological field by enabling routine use of monoclonal antibodies (mAbs) in research and development efforts, resulting in their successful application in the clinic today. While recombinant good manufacturing practices production technologies are required to produce clinical grade mAbs, academic laboratories and biotechnology companies still rely on the original hybridoma lines to stably and effortlessly produce high antibody yields at a modest price. In our own work, we were confronted with a major issue when using hybridoma-derived mAbs: there was no control over the antibody format that was produced, a flexibility that recombinant production does allow.

Thermal-exchange HLA-E multimers reveal specificity in HLA-E and NKG2A/CD94 complex interactions.

There is growing interest in HLA-E-restricted T-cell responses as a possible novel, highly conserved, vaccination targets in the context of infectious and malignant diseases. The developing field of HLA multimers for the detection and study of peptide-specific T cells has allowed the in-depth study of TCR repertoires and molecular requirements for efficient antigen presentation and T-cell activation. In this study, we developed a method for efficient peptide thermal exchange on HLA-E monomers and multimers allowing the high-throughput production of HLA-E multimers.

Identification of HLA-E Binding -Derived Epitopes through Improved Prediction Models.

Tuberculosis (TB) remains one of the deadliest infectious diseases worldwide, posing great social and economic burden to affected countries. Novel vaccine approaches are needed to increase protective immunity against the causative agent (Mtb) and to reduce the development of active TB disease in latently infected individuals. Donor-unrestricted T cell responses represent such novel potential vaccine targets.

Malaria parasite evades mosquito immunity by glutaminyl cyclase-mediated posttranslational protein modification.

Glutaminyl cyclase (QC) modifies N-terminal glutamine or glutamic acid residues of target proteins into cyclic pyroglutamic acid (pGlu). Here, we report the biochemical and functional analysis of QC. We show that sporozoites of QC-null mutants of rodent and human malaria parasites are recognized by the mosquito immune system and melanized when they reach the hemocoel.

Regulatory T Cell Depletion Using a CRISPR Fc-Optimized CD25 Antibody.

Regulatory T cells (T) are major drivers behind immunosuppressive mechanisms and present a major hurdle for cancer therapy. T are characterized by a high expression of CD25, which is a potentially valuable target for T depletion to alleviate immune suppression. The preclinical anti-CD25 (αCD25) antibody, clone PC-61, has met with modest anti-tumor activity due to its capacity to clear T from the circulation and lymph nodes, but not those that reside in the tumor.

Mesenchymal tumor cells drive adaptive resistance of Trp53 breast tumor cells to inactivated mutant Kras.

As precision medicine increases the response rate of treatment, tumors frequently bypass inhibition, and reoccur. In order for treatment to be effective long term, the mechanisms enabling treatment adaptation need to be understood. Here, we report a mouse model that, in the absence of p53 and the presence of oncogenic Kras , develops breast tumors.

Replicative history marks transcriptional and functional disparity in the CD8 T cell memory pool.

Clonal expansion is a core aspect of T cell immunity. However, little is known with respect to the relationship between replicative history and the formation of distinct CD8 memory T cell subgroups. To address this issue, we developed a genetic-tracing approach, termed the DivisionRecorder, that reports the extent of past proliferation of cell pools in vivo.

Multiscale imaging of therapeutic anti-PD-L1 antibody localization using molecularly defined imaging agents.

Background: While immune checkpoint inhibitors such as anti-PD-L1 antibodies have revolutionized cancer treatment, only subgroups of patients show durable responses. Insight in the relation between clinical response, PD-L1 expression and intratumoral localization of PD-L1 therapeutics could improve patient stratification. Therefore, we present the modular synthesis of multimodal antibody-based imaging tools for multiscale imaging of PD-L1 to study intratumoral distribution of PD-L1 therapeutics.

Inhibition of the Epigenetic Reader BRD4 Reduces SIRPα-Mediated Phagocytosis and Melanoma Invasion.

BRD4 acts as an epigenetic reader to regulate gene transcription. It represents a valid therapeutic target in cancer, and several selective and potent small molecule inhibitors have been discovered. A study by Le et al.

Dual Site-Specific Chemoenzymatic Antibody Fragment Conjugation Using CRISPR-Based Hybridoma Engineering.

Functionalized antibodies and antibody fragments have found applications in the fields of biomedical imaging, theranostics, and antibody-drug conjugates (ADC). In addition, therapeutic and theranostic approaches benefit from the possibility to deliver more than one type of cargo to target cells, further challenging stochastic labeling strategies. Thus, bioconjugation methods to reproducibly obtain defined homogeneous conjugates bearing multiple different cargo molecules, without compromising target affinity, are in demand.

Genetic Screening for Novel Regulators of Immune Checkpoint Molecules.

Inhibitory and stimulatory immune checkpoint molecules play important roles in regulating immune responses. An increasing number of these immune regulators are currently being evaluated as targets in putative anti-cancer therapies. Recently, sophisticated genetic screens have been performed to increase our understanding of immune checkpoint pathways and their immunomodulatory regulators.

The CD47-SIRPα Immune Checkpoint.

The cytotoxic activity of myeloid cells is regulated by a balance of signals that are transmitted through inhibitory and activating receptors. The Cluster of Differentiation 47 (CD47) protein, expressed on both healthy and cancer cells, plays a pivotal role in this balance by delivering a "don't eat me signal" upon binding to the Signal-regulatory protein alpha (SIRPα) receptor on myeloid cells. Here, we review the current understanding of the role of the CD47-SIRPα axis in physiological tissue homeostasis and as a promising therapeutic target in, among others, oncology, fibrotic diseases, atherosclerosis, and stem cell therapies.

Single-cell transcriptional diversity is a hallmark of developmental potential.

Single-cell RNA sequencing (scRNA-seq) is a powerful approach for reconstructing cellular differentiation trajectories. However, inferring both the state and direction of differentiation is challenging. Here, we demonstrate a simple, yet robust, determinant of developmental potential-the number of expressed genes per cell-and leverage this measure of transcriptional diversity to develop a computational framework (CytoTRACE) for predicting differentiation states from scRNA-seq data.

Functional diversification of hybridoma-produced antibodies by CRISPR/HDR genomic engineering.

Hybridoma technology is instrumental for the development of novel antibody therapeutics and diagnostics. Recent preclinical and clinical studies highlight the importance of antibody isotype for therapeutic efficacy. However, since the sequence encoding the constant domains is fixed, tuning antibody function in hybridomas has been restricted.

Metabolic stress in cancer cells induces immune escape through a PI3K-dependent blockade of IFNγ receptor signaling.

Background: T-cell mediated immunotherapy brought clinical success for many cancer patients. Nonetheless, downregulation of MHC class I antigen presentation, frequently occurring in solid cancers, limits the efficacy of these therapies. Unraveling the mechanisms underlying this type of immune escape is therefore of great importance.