Human Membrane Proteins Research Articles

Parameters for sensing lipid loosening at the inner nuclear membrane by amphipathic helices.

Amphipathic helices (AHs) detect differences in bulk membrane properties, but how AHs detect the nuclear membrane surrounding the genome is not well understood. Here, we computationally screened for candidate AHs in a curated list of characterized and putative human inner nuclear membrane (INM) proteins. Cell biological and in vitro experimental assays combined with computational calculations demonstrate that AHs detect lipid packing defects over electrostatics to bind to the INM, indicating that the INM is loosely packed under basal conditions. Membrane tension resulting from hypotonic shock further promoted AH binding to the INM, whereas cell-substrate stretch did not recruit membrane tension-sensitive AHs. Thus, distinct mechanical inputs enhance lipid loosening at the INM to different degrees, which AHs in INM proteins may harness for downstream biochemical functions. Our resource provides a framework for future studies on the contributions of lipid-protein interactions at the INM and enables exploration of the membrane properties of the INM under different conditions.

Atomistic Insights into gp82 Binding: A Microsecond, Million-Atom Exploration of Trypanosoma cruzi Host-Cell Invasion.

Chagas disease, caused by the protozoan Trypanosoma cruzi , affects millions globally, leading to severe cardiac and gastrointestinal complications in its chronic phase. The invasion of host cells by T. cruzi is mediated by the interaction between the parasite's glycoprotein gp82 and the human receptor lysosome-associated membrane protein 2 (LAMP2). While experimental studies have identified a few residues involved in this interaction, a comprehensive molecular-level understanding has been lacking. In this study, we present a 1.44-million-atom computational model of the gp82 complex, including over 3,300 lipids, glycosylation sites, and full molecular representations of gp82 and LAMP2, making it the most complete model of a parasite-host interaction to date. Using microsecond-long molecular dynamics simulations and dynamic network analysis, we identified critical residue interactions, including novel regions of contact that were previously uncharacterized. Our findings also highlight the significance of the transmembrane domain of LAMP2 in stabilizing the complex. These insights extend beyond traditional hydrogen bond interactions, revealing a complex network of cooperative motions that facilitate T. cruzi invasion. This study not only confirms key experimental observations but also uncovers new molecular targets for therapeutic intervention, offering a potential pathway to disrupt T. cruzi infection and combat Chagas disease.

A novel co-receptor with mutated TIGIT to enhance PSCA CAR-T therapy for bladder cancer.

e14574 Background: TIGIT plays a crucial role in immune regulation, particularly in tumor environments, by acting as an inhibitory receptor. Leveraging its interaction with ligands like CD155 on tumor cells, we developed an innovative CAR-T cell therapy using a mutated TIGIT co-receptor to overcome inhibitory effect induced by CD155, together with targeting Prostate Stem Cell Antigen (PSCA) CAR, chosen for its high expression in bladder and other cancers versus its minimal presence in normal tissues. Methods: We initiated our approach by inducing strategic mutations in TIGIT, selecting a variant with heightened affinity for CD155. Bio-Layer Interferometry (BLI) analysis confirmed the variant's high binding affinity (KD = 1.635 nM). This mutated TIGIT, combined with CD28's signaling components, was integrated as a co-receptor in our CAR construct. For PSCA targeting, we isolated a high-affinity ScFv against PSCA from a fully human phage display library. The specificity of this ScFv for PSCA was rigorously validated using Cell Membrane Protein Targeting Catcher (CMPTC) technology, covering over 6000 human cell membrane proteins. Results: In our comparative analyses, CAR-T cells featuring a mutated TIGIT co-receptor (T-PSCA-CAR-T) matched the cytotoxicity of traditional second-generation BBz-structured CAR-T cells (PSCA-CAR-T) against PSCA-expressing HT-1376 bladder cancer cells. However, T-PSCA-CAR-T cells distinguished themselves by producing significantly higher IFN-γ levels (3805.683±65.506 vs 1271.712±39.768 pg/mL). Our in vivo tumor model, utilizing luciferase-expressing HT-1376 cells, demonstrated T-PSCA-CAR-T's enhanced anti-tumor efficacy, as evidenced by a more significant reduction in bioluminescence signal intensity (average decreasing ratio: 0.02 for T-PSCA-CAR-T vs 0.35 for PSCA-CAR-T; p < 0.05). These results underscore the efficacy of the mutated TIGIT co-receptor in improving CAR-T cell therapy. Conclusions: Our study signifies a transformative advancement in CAR-T cell therapy by incorporating a mutated TIGIT co-receptor, markedly improving therapeutic outcomes in bladder cancer treatment. Utilizing PSCA as a model target not only validates the efficacy of this novel approach but also paves the way for its application in a broader range of cancers, representing a significant leap in the field of immunotherapy.

Optimizing NMR fragment-based drug screening for membrane protein targets

NMR spectroscopy has played a pivotal role in fragment-based drug discovery by coupling detection of weak ligand-target binding with structural mapping of the binding site. Fragment-based screening by NMR has been successfully applied to many soluble protein targets, but only to a limited number of membrane proteins, despite the fact that many drug targets are membrane proteins. This is partly because of difficulties preparing membrane proteins for NMR—especially human membrane proteins—and because of the inherent complexity associated with solution NMR spectroscopy on membrane protein samples, which require the inclusion of membrane-mimetic agents such as micelles, nanodiscs, or bicelles. Here, we developed a generalizable protocol for fragment-based screening of membrane proteins using NMR. We employed two human membrane protein targets, both in fully protonated detergent micelles: the single-pass C-terminal domain of the amyloid precursor protein, C99, and the tetraspan peripheral myelin protein 22 (PMP22). For both we determined the optimal NMR acquisition parameters, protein concentration, protein-to-micelle ratio, and upper limit to the concentration of D6-DMSO in screening samples. Furthermore, we conducted preliminary screens of a plate-format molecular fragment mixture library using our optimized conditions and were able to identify hit compounds that selectively bound to the respective target proteins. It is hoped that the approaches presented here will be useful in complementing existing methods for discovering lead compounds that target membrane proteins.

Identification of a Fully Human Antibody VH Domain Targeting Anaplastic Lymphoma Kinase (ALK) with Applications in ALK-Positive Solid Tumor Immunotherapy.

The anaplastic lymphoma kinase (ALK, CD247) is a potential target for antibody-based therapy. However, no antibody-based therapeutics targeting ALK have entered clinical trials, necessitating the development of novel antibodies with unique therapeutic merits. Single-domain antibodies (sdAb) bear therapeutic advantages compared to the full-length antibody including deeper tumor penetration, cost-effective production and fast washout from normal tissues. In this study, we identified a human immunoglobulin heavy chain variable domain (VH domain) (VH20) from an in-house phage library. VH20 exhibits good developability and high specificity with no off-target binding to ~6000 human membrane proteins. VH20 efficiently bound to the glycine-rich region of ALK with an EC50 of 0.4 nM and a KD of 6.54 nM. Both VH20-based bispecific T cell engager (TCE) and chimeric antigen receptor T cells (CAR Ts) exhibited potent cytolytic activity to ALK-expressing tumor cells in an ALK-dependent manner. VH20 CAR Ts specifically secreted proinflammatory cytokines including IL-2, TNFα and IFNγ after incubation with ALK-positive cells. To our knowledge, this is the first reported human single-domain antibody against ALK. Our in vitro characterization data indicate that VH20 could be a promising ALK-targeting sdAb with potential applications in ALK-expressing tumors, including neuroblastoma (NBL) and non-small cell lung cancer.

Decrypting the multi-genome data for chimeric vaccine designing against the antibiotic resistant Yersinia pestis

Yersinia pestis, the causative agent of plague, is a gram-negative bacterium that can be fatal if not treated properly. Three types of plague are currently known: bubonic, septicemic, and pneumonic plague, among which the fatality rate of septicemic and pneumonic plague is very high. Bubonic plague can be treated, but only if antibiotics are used at the initial stage of the infection. But unfortunately, Y. pestis has also shown resistance to certain antibiotics such as kanamycin, minocycline, tetracycline, streptomycin, sulfonamides, spectinomycin, and chloramphenicol. Despite tremendous progress in vaccine development against Y. pestis, there is no proper FDA-approved vaccine available to protect people from its infections. Therefore, effective broad-spectrum vaccine development against Y. pestis is indispensable. In this study, vaccinomics-assisted immunoinformatics techniques were used to find possible vaccine candidates by utilizing the core proteome prepared from 58 complete genomes of Y. pestis. Human non-homologous, pathogen-essential, virulent, and extracellular and membrane proteins are potential vaccine targets. Two antigenic proteins were prioritized for the prediction of lead epitopes by utilizing reverse vaccinology approaches. Four vaccine designs were formulated using the selected B- and T-cell epitopes coupled with appropriate linkers and adjuvant sequences capable of inducing potent immune responses. The HLA allele population coverage of the T-cell epitopes selected for vaccine construction was also analyzed. The V2 constructs were top-ranked and selected for further analysis on the basis of immunological, physicochemical, and immune-receptor docking interactions and scores. Docking and molecular dynamic simulations confirmed the stability of construct V2 interactions with the host immune receptors. Immune simulation analysis anticipated the strong immune profile of the prioritized construct. In silico restriction cloning ensured the feasible cloning ability of the V2 construct in the expression system of E. coli strain K12. It is anticipated that the designed vaccine construct may be safe, effective, and able to elicit strong immune responses against Y. pestis infections and may, therefore, merit investigation using in vitro and in vivo assays.

Abstract 724: MPSA-AB5000: A high-throughput membrane proteins screening array for therapeutic biologics specificity profiling

Abstract The development of novel antibody drugs is a lengthy journey. Many factors can lead to failures during clinical development. According to reports, off-target binding of antibodies could also be a significant cause. It has been stated that about 25% of preclinical candidates might have off-target issues. With the emergence of new modalities like CD3 bispecific antibodies, ADCs, and CAR-T therapies, early-stage off-target screening of antibodies has become even more crucial. Although techniques such as immunohistochemistry, ELISA, and flow cytometry (FACS) have been used for off-target testing of antibodies, these methods still lack sensitivity and specificity, and their throughput is limited. To address the need for antibody target deconvolution and receptor identification, we developed a novel membrane protein screening array-AB5000 (MPSA-AB5000). MPSA-AB5000 is a high-throughput cell-based platform for identifying the targets of antibodies and other ligands that bind to membrane proteins. MPSA-AB5000 is an excellent solution for antibody specificity screening, which enables early off-target screening and can de-risk the antibody drug development program. The MPSA-AB5000 makes use of the full coverage of unique human membrane proteins, including receptors, transporters, enzymes, miscellaneous, and others. Each membrane protein is expressed in live cells to ensure native conformation. In addition, the same membrane protein clones with a C-terminal epitope tag were constructed, allowing confirmation of protein expression and cellular location. To provide the highest level of sensitivity, MPSA-AB5000 uses luciferase reporter cell line detection. The wash-free strategy reduces false negatives, and signal amplification makes remarkable discrimination. Compared to similar technologies, we possess significant advantages in the following aspects: a. High sensitivity and sigh throughput: our technology platform offers not only high sensitivity, capable of detecting subtle interactions but also supports high-throughput screening, allowing for the evaluation of a large number of samples in a short timeframe. b. Avoidance of false negative signals: by eliminating washing steps, our method reduces the risk of false negative signals, ensuring reliable detection of non-specific binding. c. Comprehensive validation approaches: We employ various validation methods such as FACS and ELISA to eliminate false positive signals and enhance the accuracy and credibility of our results. All the advantages ensure that MPSA-AB5000 is more rapid, simple, and highly sensitive than traditional assays. Citation Format: dan Li, Junyi Xiong, Jinying Ning, Feng Hao. MPSA-AB5000: A high-throughput membrane proteins screening array for therapeutic biologics specificity profiling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 724.

Abstract 6743: Identifying new cellular adjuvants for cancer vaccines and immunotherapies

Abstract Background: Cancer vaccines represent a promising strategy for antitumor immunotherapy. Despite considerable research efforts and progress, the effectiveness of cancer vaccines remains a challenge. This is primarily due to relatively low immune responses against tumors, stemming from issues of poor immunogenicity and the immunosuppressive microenvironment commonly found in many solid tumors. Use of an optimal adjuvant can augment immune responses against tumor antigens and guide them towards a specific adaptive immune enhancement. Dendritic cells (DCs) play a primary role in initiating immunes response to vaccines, often through activation of toll-like receptors (TLRs). Identifying novel immunomodulatory proteins that regulate DCs or TLR activation can identify novel adjuvant candidates with applications for cancer immunotherapy. Methods: We constructed a Membrane Proteome Array (MPA) encompassing ~6,000 structurally intact human membrane proteins in their native conformation (94% of the entire membrane proteome) and used it to screen for cellular proteins that can modulate response to vaccination through DCs and TLRs. To identify proteins that interact with TLRs, the library was expressed in live TLR reporter cells, and these cells were tested for response to known activators. In addition, cells expressing the MPA were presented to DCs to identify membrane proteins that modulate DC activity. TLR-expressing cell activation was measuring using an NF-kB-activated luciferase reporter, while DC assays used upregulation of cell surface markers (CD80, CD83, and MHCII) to measure the activation of immature monocyte-derived dendritic cells. Results: Screening of TLR2, TLR3, and TLR5 reporter cell lines identified both known and novel regulators of TLR activation. Activators common to several TLRs were identified as well as activators unique to individual TLRs. Screening of primary human DCs likewise identified known regulators of activation (for example CD40L) and several novel targets. Putative novel regulators of DC activation are being further investigated to determine their biological significance. Conclusion: Discovering novel immunomodulatory targets and proteins that regulate DCs and TLR activation can identify novel adjuvant candidates with targeted immune outcomes, such as immune stimulating and anti-cancer responses. This study identified novel regulators of immune activation which are being further investigated as adjuvant candidates for cancer vaccines. Citation Format: Joseph Rucker, Nnenna Nwogu, Nicholas M. Molino, Ami Snyder, Jonathan T. Sullivan, Benjamin J. Doranz. Identifying new cellular adjuvants for cancer vaccines and immunotherapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6743.

Abstract 5798: Preclinical safety assessment for specificity of biotherapeutics using the membrane proteome array

Abstract Rigorous specificity analysis is critical for the development of antibody-based therapies, as even minimal off-target binding can lead to toxicity and clinical failures. Long believed to be exquisitely specific, recent preclinical data and our own work indicate that monoclonal antibodies (MAbs) frequently (~25%) display cross-reactivity. In many cases, off-target interactions occur with unrelated proteins that cannot be predicted by protein sequence homology. Cross-reactivity can lead to serious or even life-threatening consequences especially when MAbs are configured as bispecifics, antibody-drug conjugates (ADC) or CAR-T cell therapies, and IND applications for biotherapeutics require cross-reactivity assessment to prevent adverse events. Tissue cross-reactivity (TCR) studies have traditionally been used to screen for off-target binding, however, with poor predictive value for in vivo safety and toxicity. We developed the Membrane Proteome Array (MPA) platform to de-risk MAb-based therapeutics by testing for specificity across 6,000 human membrane proteins expressed in unfixed cells. The MPA assesses binding interactions by high-throughput flow cytometry allowing for high sensitivity detection and rapid analysis. All targets identified on the MPA screen are validated by secondary titration analysis. In contrast to TCR studies, proteins in the MPA exist in their native conformations and are not altered by fixation. Recently, we used the MPA to select a lead antibody candidate targeting the oncotherapeutic target Claudin 6 (CLDN6). Through MPA screening, we successfully identified a lead candidate devoid of cross-reactivity against other CLDN family members or any other membrane protein across the human genome. The MPA has also been used to rapidly evaluate the specificity of novel CAR-T cell therapies for cancer and autoimmunity. The MPA has been used for lead selection and data from the MPA has been accepted by the FDA as a part of IND applications for antibody-based therapies. The MPA is currently under consideration for being developed as an FDA-qualified Drug Development Tool. Citation Format: Kristopher S. Raghavan, Jonathan T. Sullivan, Carmen Navia, Rachel H. Fong, Benjamin J. Doranz. Preclinical safety assessment for specificity of biotherapeutics using the membrane proteome array [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5798.

Purification and characterisation of the platelet-activating GPVI/FcRγ complex in SMALPs

The collagen/fibrin(ogen) receptor, glycoprotein VI (GPVI), is a platelet activating receptor and a promising anti-thrombotic drug target. However, while agonist-induced GPVI clustering on platelet membranes has been shown to be essential for its activation, it is unknown if GPVI dimerisation represents a unique conformation for ligand binding. Current GPVI structures all contain only the two immunoglobulin superfamily (IgSF) domains in the GPVI extracellular region, so lacking the mucin-like stalk, transmembrane, cytoplasmic tail of GPVI and its associated Fc receptor γ (FcRγ) homodimer signalling chain, and provide contradictory insights into the mechanisms of GPVI dimerisation. Here, we utilised styrene maleic-acid lipid particles (SMALPs) to extract GPVI in complex with its two associated FcRγ chains from transfected HEK-293T cells, together with the adjacent lipid bilayer, then purified and characterised the GPVI/FcRγ-containing SMALPs, to enable structural insights into the full-length GPVI/FcRγ complex. Using size exclusion chromatography followed by a native polyacrylamide gel electrophoresis (PAGE) method, SMA-PAGE, we revealed multiple sizes of the purified GPVI/FcRγ SMALPs, suggesting the potential existence of GPVI oligomers. Importantly, GPVI/FcRγ SMALPs were functional as they could bind collagen. Mono-dispersed GPVI/FcRγ SMALPs could be observed under negative stain electron microscopy. These results pave the way for the future investigation of GPVI stoichiometry and structure, while also validating SMALPs as a promising tool for the investigation of human membrane protein interactions, stoichiometry and structure.

Protein-Protein Docking Approach to GPCR Oligomerization.

G-protein-coupled receptors (GPCRs), the largest family of human membrane proteins, play a crucial role in cellular control and are the target of approximately one-third of all drugs on the market. Targeting these complexes with selectivity or formulating small molecules capable of modulating receptor-receptor interactions could potentially offer novel avenues for drug discovery, fostering the development of more refined and safer pharmacotherapies. Due to the lack of experimentally derived X-ray crystallography spectra of GPCR oligomers, there is growing evidence supporting the development of new in silico approaches for predicting GPCR self-assembling structures. The significance of GPCR oligomerization, the challenges in modeling these structures, and the potential of protein-protein docking algorithms to address these challenges are discussed. The study also underscores the use of various software solutions for modeling GPCR oligomeric structures and presents practical cases where these techniques have been successfully applied.

Abstract A173: ASP2074, a novel tetraspanin 8 x CD3 bispecific antibody, demonstrates selectivity and antitumor activity in preclinical cancer models

Abstract ASP2074 is a novel bispecific antibody that targets the tumor-associated antigen tetraspanin 8 (TSPAN8) and CD3, which is expressed on the plasma membrane of all T cells. By engaging with both TSPAN8 and CD3, ASP2074 enables the retargeting of cytotoxic T cells specifically against TSPAN8-expressing tumor cells in a human leukocyte antigen-independent manner. TSPAN8 is a member of the tetraspanin superfamily of proteins that plays a crucial role in intracellular signal transduction and regulates various cellular functions, including leukocyte trafficking, angiogenesis, and wound repair. TSPAN8 is highly expressed in various types of cancer, such as colorectal cancer, gastric cancer, pancreatic cancer, esophageal adenocarcinoma, and gastroesophageal junction (GEJ) adenocarcinoma. Its overexpression has been associated with tumor cell growth, angiogenesis, invasion, metastasis, and poor prognosis, making it an attractive target for antibody-based cancer immunotherapy. ASP2074 is currently undergoing phase 1 clinical trials for the treatment of patients with colorectal cancer, esophageal adenocarcinoma, pancreatic cancer, gastric cancer, and GEJ adenocarcinoma. In this study, we examined the selectivity and antitumor activity of ASP2074 in vitro and in vivo. Methods: Retrogenix Cell Microarray Technology was used to evaluate the selective binding of ASP2074 to TSPAN8 and CD3 in 6018 full-length human plasma membrane proteins, secreted and cell surface-tethered human secreted proteins, and 397 human heterodimers containing homologs and isoforms of tetraspanin. Cytotoxicity and T-cell activation of ASP2074 were examined using TSPAN8-expressing tumor cells in combination with human peripheral blood mononuclear cells (PBMCs) as effector cells in a redirected T-cell cytotoxicity assay. Furthermore, the antitumor effect of ASP2074 was examined in a human PBMC-engrafted NOG mouse model bearing human TSPAN8-expressing colorectal cancer cells. Results: Our results showed that ASP2074 selectively bound to TSPAN8 and CD3 without significant binding to other human plasma membrane proteins, secreted proteins, and heterodimers. ASP2074 exhibited cytotoxicity against TSPAN8-expressing cancer cells and increased interferon-gamma production and expression of T-cell activation markers. The cytotoxic activities were dependent on the TSPAN8 expression levels in tumor cells. In the NOG mouse model, ASP2074 exhibited an antitumor effect on human TSPAN8-expressing colorectal cancer cells in vivo. Conclusion: Our findings suggest that ASP2074 has potent antitumor activity through cytotoxicity against TSPAN8-expressing tumor cells by T-cell activation via selective binding to TSPAN8 and CD3. These preclinical results support the continued development of ASP2074 as a promising immunotherapy for the treatment of TSPAN8-expressing cancers. Citation Format: Yoshiyuki Tenda, Yuichi Hanada, Hiroaki Tanaka, Masashi Shimazaki, Kenna Shirasuna. ASP2074, a novel tetraspanin 8 x CD3 bispecific antibody, demonstrates selectivity and antitumor activity in preclinical cancer models [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A173.

Selectivity and Safety of VIP943: A Novel CD123-Targeting Antibody-Drug Conjugate (ADC) Using a Proprietary Linker and Payload Class

Introduction ADCs afford high efficacy though often have shortcomings in their safety profile. Most ADC payloads are cytotoxic agents that can inhibit general processes of cellular homeostasis, including microtubule polymerization or DNA damage repair. While these are good targets for cytotoxicity, they have the drawback of being nonselective for dividing cancer cells. Here, we report the potent, on-target activity and selectivity of our novel CD123-targeting ADC VIP943, which uses a kinesin spindle protein (KSP; also known as KIF11 or Eg5) inhibitor as the payload (VIP716). The kinesin superfamily (KIF) is comprised of motor-proteins involved in various cellular mechanisms, including formation of the bipolar mitotic spindle during cell division. Targeting KSP selectively inhibits dividing cells, while sparing nondividing, differentiated cells thereby increasing the therapeutic index of VIP943. Methods VIP716 selectivity and potency: The motor domain of the human KSP and the other KIF family members were incubated with stabilized microtubules in a biochemical assay. The freshly prepared mixture was then incubated VIP716 and ispinesib, a clinical stage KSPi. ATPase activity was directly analyzed by measuring the formed inorganic phosphate. VIP943 selectivity: The binding of VIP943 to 6,019 human plasma membrane proteins expressed on HEK cells was evaluated (Retrogenix cell microarray). Cell cycle/death analysis: The effect of VIP943 on the 3 phases of the cell cycle (G0/G1- S- G2/M) was measured with DNA staining and flow cytometry in MOLM-13 and MV-4-11 cell lines. In addition, cell death was measured by flow cytometry after a 4 h incubation with VIP943 in MOLM-13 cells. Isotype ADC was used as a control. In vitro cytotoxicity: 56 hematologic cell lines were treated in vitro with VIP331 (cell permeable analog of VIP716). IC 50 results were matched with the DNA/RNA profile of relevant oncogenes to determine correlations. Nonhuman primate safety and toxicokinetics: VIP943 was intravenously administered to Cynomolgus monkeys at 1, 3, or 9 mg/kg once weekly for 4 weeks in a Good Laboratory Practice (GLP) toxicology study. Serial plasma samples were collected on Day 1 and Day 22. Total antibody from VIP943 (as a surrogate for VIP943) and VIP716 concentrations were measured by LC/MS/MS. Toxicokinetics (TK) were assessed using noncompartmental methods. Results In a biochemical assay, VIP716 (payload), achieved comparable potency to ispinesib with IC 50 values of 1.4 and 1.6 nM, respectively, for KSP. VIP716 did not inhibit any of the 11 other KIF family members up to 10 µM. In the Retrogenix assay, VIP943 (ADC) had no interaction with any of the analyzed human cell surface proteins except for CD123. Cell cycle analysis in AML cells showed a dose- and time-dependent increase in G2/M arrest with VIP943 treatment, but not with isotype control. An increase in cell death with VIP943 treatment was also observed after a 4-hour incubation. In a broad hematologic cell line panel, the median cytotoxicity IC 50 for VIP331 (cell permeable KSPi) was 2.49 nM. Of note, no genomic alterations correlated with sensitivity, suggesting VIP943 may be effective even in hard-to-treat hematologic malignancies. VIP943 was well tolerated in the GLP monkey toxicology study. No mortality occurred in any monkeys. No neutropenia, thrombocytopenia or anemia were observed. VIP943 and VIP716 exposures increased with increasing dose in a dose-proportional or greater than dose-proportional manner. No consistent differences in TK were observed with sex. Based on area under the concentration curve (AUC), no accumulation was observed. Payload to parent ratios were consistent with a low level of non-specific release of VIP716 from VIP943. Conclusion High on-target activity of VIP716, the payload of our new CD123-targeting ADC, VIP943, was demonstrated in a biochemical assay and selectivity against other KIF family members was confirmed. This activity translated into cell cycle arrest in the G2/M phase and subsequent cell death in a dose- and time-dependent manner. Additionally, toxicology in non-human primates and in vivo TK studies confirm safety, favorable drug exposures, and little non-specific release of the payload. Based on these data, evaluation of VIP943 in human clinical trials is warranted.

Deep Learning Dynamic Allostery of G-Protein-Coupled Receptors.

G-protein-coupled receptors (GPCRs) make up the largest superfamily of human membrane proteins and represent primary targets of ∼1/3 of currently marketed drugs. Allosteric modulators have emerged as more selective drug candidates compared with orthosteric agonists and antagonists. However, many X-ray and cryo-EM structures of GPCRs resolved so far exhibit negligible differences upon the binding of positive and negative allosteric modulators (PAMs and NAMs). The mechanism of dynamic allosteric modulation in GPCRs remains unclear. In this work, we have systematically mapped dynamic changes in free energy landscapes of GPCRs upon binding of allosteric modulators using the Gaussian accelerated molecular dynamics (GaMD), deep learning (DL), and free energy prOfiling Workflow (GLOW). GaMD simulations were performed for a total of 66 μs on 44 GPCR systems in the presence and absence of the modulator. DL and free energy calculations revealed significantly reduced dynamic fluctuations and conformational space of GPCRs upon modulator binding. While the modulator-free GPCRs often sampled multiple low-energy conformational states, the NAMs and PAMs confined the inactive and active agonist-G-protein-bound GPCRs, respectively, to mostly only one specific conformation for signaling. Such cooperative effects were significantly reduced for binding of the selective modulators to "non-cognate" receptor subtypes. Therefore, GPCR allostery exhibits a dynamic "conformational selection" mechanism. In the absence of available modulator-bound structures as for most current GPCRs, it is critical to use a structural ensemble of representative GPCR conformations rather than a single structure for compound docking ("ensemble docking"), which will potentially improve structure-based design of novel allosteric drugs of GPCRs.

Photoradical-Mediated Catalyst-Independent Protein Cross-Link with Unusual Fluorescence Properties.

Photo-actively modified natural amino acids have served as lucrative probes for precise mapping of the dynamics, interaction networks, and turnover of cytosolic proteins both in vivo and ex vivo. In our attempts to extend the utility of photoreactive reporters to map the molecular characteristics of vital membrane proteins, we carried out site-selective incorporation of 7-fluoro-indole in the human mitochondrial outer membrane protein VDAC2 (voltage-dependent anion channel isoform 2), with the aim of generating Trp-Phe/Tyr cross-links. Prolonged irradiation at 282 nm provided us with a surprisingly unusual fluorophore that displayed sizably red-shifted excitation (λex-max =280 nm→360 nm) and emission (λem-max =330 nm→430 nm) spectra that was reversible with organic solvents. By measuring the kinetics of the photo-activated cross-linking with a library of hVDAC2 variants, we demonstrate that formation of this unusual fluorophore is kinetically retarded, independent of tryptophan, and is site-specific. Using other membrane (Tom40 and Sam50) and cytosolic (MscR and DNA Pol I) proteins, we additionally show that formation of this fluorophore is protein-independent. Our findings reveal the photoradical-mediated accumulation of reversible tyrosine cross-links, with unusual fluorescent properties. Our findings have immediate applications in protein biochemistry and UV-mediated protein aggregation and cellular damage, opening avenues for formulating therapeutics that prolong cell viability in humans.

Lysosomal LAMP proteins regulate lysosomal pH by direct inhibition of the TMEM175 channel

Maintaining a highly acidic lysosomal pH is central to cellular physiology. Here, we use functional proteomics, single-particle cryo-EM, electrophysiology, and invivo imaging to unravel a key biological function of human lysosome-associated membrane proteins (LAMP-1 and LAMP-2) in regulating lysosomal pH homeostasis. Despite being widely used as a lysosomal marker, the physiological functions of the LAMP proteins have long been overlooked. We show that LAMP-1 and LAMP-2 directly interact with and inhibit the activity of the lysosomal cation channel TMEM175, a key player in lysosomal pH homeostasis implicated in Parkinson's disease. This LAMP inhibition mitigates the proton conduction of TMEM175 and facilitates lysosomal acidification to a lower pH environment crucial for optimal hydrolase activity. Disrupting the LAMP-TMEM175 interaction alkalinizes the lysosomal pH and compromises the lysosomal hydrolytic function. In light of the ever-increasing importance of lysosomes to cellular physiology and diseases, our data have widespread implications for lysosomal biology.

Identification of glycolytic proteins as binding partners of Bri2 BRICHOS domain

Human integral membrane protein 2B (ITM2B or Bri2) is a member of the BRICHOS family, that can attenuate Aβ pathology in the brain. As a result, the identification of novel Bri2 BRICHOS client proteins has been sought to help elucidate signaling pathways and the potential identification of novel therapeutic targets. To identify Bri2 BRICHOS interacting partners, we carried out a 'protein fishing' experiment using recombinant human (rh) Bri2 BRICHOS-coated magnetic particles, in combination with proteomic analysis on cytosolic and membrane fractions of cortical homogenates from C57BL/6 J WT mouse. We identified 4 proteins from the cytosolic fractions and 44 proteins from the membrane fractions that had significant interactions (p < 0.05) with Bri2 BRICHOS domain, of which 11 proteins were previously identified as proteins that interacted with Bri2 BRICHOS domain. Enrichment analysis of the retained proteins identified glycolysis/gluconeogenesis as the most enriched pathway, with several proteins identified playing roles in carbon metabolism, amino acid synthesis. The data suggested that Bri2 BRICHOS may have a role in cellular energy demands in the brain via glycolysis and mitochondrial oxidative phosphorylation and may play a role in mitochondrial homeostasis.

Artificial Immunogenic Cell Death Lipid Nanoparticle Functions as a Therapeutic Vaccine for Cancer

AbstractAnticancer drug‐mediated induction of immunogenic cell death (ICD) blocks metastasis or recurrence in cancer cells by promoting specific immune activity against cancer antigens. However, this strategy has failed to afford adequate treatment efficiency. Overcoming the failure of ICD‐mediated cancer therapy, lipid nanoparticles (LNPs) containing cancer cell surface proteins are synthesized using sonication and extrusion without microfluidics. In addition, these LNPs are decorated with high‐mobility group box 1 protein and calreticulin, indicators of ICD, and named artificial ICD LNPs (AiLNPs). Administration of AiLNPs effectively targets dendritic cells (DCs) and induces DC activation in mice. Moreover, treating CT‐26 tumor‐bearing mice with AiLNPs inhibits tumor growth by inducing CT‐26 antigen‐specific T‐cell immunity. Furthermore, AiLNPs containing Lewis lung carcinoma (LLC1) membrane proteins can prevent metastatic LLC1 tumor growth in the lung via LLC1 antigen‐specific T‐cell activation. Finally, AiLNPs synthesized with human breast cancer membrane proteins activate DC‐mediated antigen‐specific T‐cell immunity, effectively killing tumor cells. Therefore, AiLNPs are expected to be developed as a patient‐specific cancer treatment to prevent cancer recurrence and metastasis.

Abstract 3967: CRISPR/Cas9 screening identified novel membrane targets sensitizing hepatocellular carcinoma cells to natural killer cell-mediated cytotoxicity

Abstract Natural killer cells (NK) play a central role in cancer immune surveillance by directly killing cancer cells and producing pro-inflammatory cytokines to stimulate and recruit other immune cells to elaborate long-lasting anti-tumor responses. However, little is known about the immune escape mechanisms and regulatory proteins, particularly those for NK, involved in hepatocellular carcinoma (HCC), one of the most common and lethal cancers. To identify the determinants of NK killing function, we performed an HCC cell line-based CRISPR/Cas9 loss-of-function genetic screen, using a human whole membrane protein library containing ~7000 genes curated from 3 public membrane protein datasets. The human SNU449 was chosen as a representative HCC cell line for CRISPR/Cas9 editing based on the genetic profile analysis, representing one HCC patient subpopulation (&amp;gt; 30%) in the TCGA cohort. The cell line was incubated with NK92 cells at ratios of 2:1, 1:1, and 0.5:1. Bioinformatics methods for prioritizing genes (e.g., MAGeCK) were applied on 3 tested groups with different ratios. Single guide RNAs targeting genes in the antigen presentation machinery were depleted, including HLA-C, B2M, and TAP1/2, whereas components of the tumor-immune synapse, such as NCR3LG1 and ICAM1, were enriched, confirming the validity of the screen. SLC33A1 (an endosomal acetylCoA transporter) and SLC31A1 (the major copper influx transporter) were also depleted, previously unknown as NK regulators. The effects of SLC33A1 and SLC31A1 were validated in individual gene knockout cells generated respectively by lentiviral sgRNA expression. Furthermore, knockout of either transporter in SNU449 cells increased IFNγ secretion, confirming NK92 activation. Our results revealed that CRISPR/Cas9-based functional genomics screens in HCC cell lines could identify novel targets modulating NK-mediated killing. Further analysis of mechanisms of action, and particularly functional validation in in vivo systems, will potentially provide novel targets for the treatment of HCC. Citation Format: Shuo Li, Lin Han, Siyao Zhang, Guangfeng Geng, Lei Huo, Dawei Huang, Zhaoren He, Shuoran Li, Zhaoshi Jiang, James X. Rong. CRISPR/Cas9 screening identified novel membrane targets sensitizing hepatocellular carcinoma cells to natural killer cell-mediated cytotoxicity. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3967.

Abstract 2646: Screening the membrane proteome to determine antibody specificity and de-risk CAR-T cell development

Abstract Rigorous specificity analysis is critical for the development of antibody-based therapies, as even minimal off-target binding can lead to toxicity and clinical failures. Long believed to be exquisitely specific, recent preclinical data and our own work indicate that monoclonal antibodies (MAbs) frequently (~25%) display cross-reactivity. In many cases, off-target interactions occur with unrelated proteins that cannot be predicted by protein sequence homology. Cross-reactivity can lead to serious or even life-threatening consequences especially when MAbs are configured as bispecifics, antibody-drug conjugates (ADC) or CAR-T cell therapies, and IND applications for biotherapeutics require cross-reactivity assessment to prevent adverse events. Tissue cross-reactivity (TCR) studies have traditionally been used to screen for off-target binding, however, with poor predictive value for in vivo safety and toxicity. We developed the Membrane Proteome Array (MPA) platform to de-risk MAb-based therapeutics by testing for specificity across 6,000 human membrane proteins expressed in live cells. In contrast to TCR studies, proteins in the MPA exist in their native conformations and are not altered by fixation. The MPA assesses binding interactions by high-throughput flow cytometry allowing for high sensitivity detection and rapid analysis. We will discuss the importance of early-stage specificity testing to expose possible toxicities and present case studies for antibody and cell therapy profiling. MPA data have been used in numerous successful regulatory applications and may be used to replace or complement other cross-reactivity studies. In particular, MPA profiling has been used in IND applications for CAR-T and cell-therapies where conventional immunohistochemical binding assays did not suffice. Citation Format: Joseph Rucker, Rachel H. Fong, Tabb Sullivan, Carmen Navia, Brad Screnci, Rona Wilf, Benjamin J. Doranz. Screening the membrane proteome to determine antibody specificity and de-risk CAR-T cell development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2646.