Biology subjects

Lerner, R. A.

Publications and source records attributed to Lerner, R. A..

5 recordsLinked to original sources

bioRxiv2022

Enhancing the anti-tumor efficacy of Bispecific T cell engagers via cell surface glycocalyx editing

Bispecific T-cell engager (BiTE)-based cancer therapies that activate the cytotoxic T cells of a patients own immune system have gained momentum with the recent FDA approval of Blinatumomab for treating B cell malignancies. However, this approach has had limited success in targeting solid tumors. Here, we report the development of BiTE-sialidase fusion proteins that enhance tumor cell susceptibility to BiTE-mediated cytolysis by T cells via targeted desialylation at the BiTE-induced T cell-tumor cell interface. Targeted desialylation results in better immunological synapse formation, T-cell activation and effector function. As a result, BiTE-sialidase fusion proteins show remarkably increased efficacy in inducing T-cell-dependent tumor cell cytolysis in response to target antigens compared to the parent BiTE molecules alone. This enhanced function is seen both in vitro and in in vivo xenograft and syngeneic solid tumor mouse models. Our findings highlight BiTE-sialidase fusion proteins as promising candidates for the development of next-generation bispecific T-cell engaging molecules for cancer immunotherapy.

cancer biology↗

bioRxiv2021

Antigen-specific stimulation and expansion of CAR-T cells using membrane vesicles as target cell surrogates

Development of CAR-T therapy led to immediate success in the treatment of B cell leukemia and lymphoma. It also raised an opportunity to design new protocols to target solid tumors. Manufacturing of therapy-competent functional CAR-T cells needs robust protocols for ex vivo/in vitro expansion of modified T-cells. This step is challenging, especially if non-viral low efficiency delivery protocols are used to generate CAR-T cells. Modern protocols for CAR-T cell expansion are based on incubation with high doses of recombinant cytokines to support proliferation, non-specific stimulation with surface-bound antibodies to induce TCR cross-linking, or co-cultivation with antigen-expressing feeder cell lines. These approaches are imperfect since non-specific stimulation results in rapid outgrowth of CAR-negative T cells, and removal of feeder cells from mixed cultures necessitates additional purification steps. In an effort to develop a specific and improved protocol for CAR-T cell expansion, we took advantage of cell-derived membrane vesicles, and the simple structural demands of the CAR-antigen interaction. Our approach was to make antigenic microcytospheres from common cell lines stably expressing surface-bound CAR antigens (antigenic vesicles, AVs), and then use them for stimulation and expansion of CAR-T cells. We developed a rapid, simple, efficient, and inexpensive protocol to generate, stabilize and purify AVs. As proof-of-concept we tested the efficacy of our AV constructs on several CAR-antigen pairs. The data presented in this article clearly demonstrate that our protocol produced AVs with the capacity to induce stronger stimulation, proliferation and functional activity of CAR-T cells than is possible with existing protocols. We predict that this new methodology will significantly improve the ability to obtain improved populations of functional CAR-T cells for therapy. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=199 HEIGHT=200 SRC="FIGDIR/small/435976v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@abbe23org.highwire.dtl.DTLVardef@15007dforg.highwire.dtl.DTLVardef@134e506org.highwire.dtl.DTLVardef@1a7f1a2_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2020

Potent SARS-CoV-2 neutralizing antibodies selected from a human antibody library constructed decades ago

Combinatorial antibody libraries not only effectively reduce antibody discovery to a numbers game, but enable documentation of the history of antibody responses in an individual. The SARS-CoV-2 pandemic has prompted a wider application of this technology to meet the public health challenge of pandemic threats in the modern era. Herein, we used a combinatorial human antibody library constructed 20 years before the COVID-19 pandemic to discover three highly potent antibodies that selectively bind SARS-CoV-2 spike protein and neutralize authentic SARS-CoV-2 virus. Compared to neutralizing antibodies from COVID-19 patients with generally low somatic hypermutation (SHM), these antibodies contain over 13-22 SHMs, many of which are involved in specific interactions in crystal structures with SARS-CoV-2 spike RBD. The identification of these somatically mutated antibodies in a pre-pandemic library raises intriguing questions about the origin and evolution of human immune responses to SARS-CoV-2.

microbiology↗

bioRxiv2019

Influenza H3 and H1 hemagglutinins have different genetic barriers for resistance to broadly neutralizing stem antibodies

In the past decade, the discovery and characterization of broadly neutralizing antibodies (bnAbs) to the highly conserved stem region of influenza hemagglutinin (HA) have provided valuable insights for development of a universal influenza vaccine. However, the genetic barrier for resistance to stem bnAbs has not been thoroughly evaluated. Here, we performed a series of deep mutational scanning experiments to probe for resistance mutations. We found that the genetic barrier to resistance to stem bnAbs is generally very low for the H3 subtype but substantially higher for the H1 subtype. Several resistance mutations in H3 cannot be neutralized by stem bnAbs at the highest concentration tested, do not reduce in vitro viral fitness and in vivo pathogenicity, and are often present in circulating strains as minor variants. Thus, H3 HAs have a higher propensity than H1 HAs to escape major stem bnAbs and creates a potential challenge in the development of a bona fide universal influenza vaccine. ONE SENTENCE SUMMARYAcquisition of resistance by influenza virus to broadly neutralizing hemagglutinin stem antibodies varies tremendously depending on subtype.

microbiology↗

bioRxiv2018

Studies on the mechanism of general anesthesia

Inhaled anesthetics are a chemically diverse collection of hydrophobic molecules that robustly activate TWIK related K+ channels (TREK-1) and reversibly induce loss of consciousness. For a hundred years anesthetics were speculated to target cellular membranes, yet no plausible mechanism emerged to explain a membrane effect on ion channels. Here we show that inhaled anesthetics (chloroform and isoflurane) activate TREK-1 through disruption of palmitate-mediated localization of phospholipase D2 (PLD2) to lipid rafts and subsequent production of signaling lipid phosphatidic acid (PA). Catalytically dead PLD2 robustly blocks anesthetic TREK-1 currents in cell patch-clamp. Localization of PLD2 renders the anesthetic-insensitive TRAAK channel sensitive. General anesthetics chloroform, isoflurane, diethyl ether, xenon, and propofol disrupt lipid rafts and activate PLD2. In the whole brain of flies, anesthesia disrupts rafts and PLDnull flies resist anesthesia. Our results establish a membrane mediated target of inhaled anesthesia and suggest PA helps set anesthetic sensitivity in vivo.

neuroscience↗