Distant Epitopes Research Articles

CD3-engaging bispecific antibodies trigger a paracrine regulated wave of T-cell recruitment for effective tumor killing

The mechanism of action of bispecific antibodies (bsAbs) directing T-cell immunity to solid tumors is incompletely understood. Here, we screened a series of CD3xHER2 bsAbs using extracellular matrix (ECM) embedded breast cancer tumoroid arrays exposed to healthy donor-derived T-cells. An initial phase of random T-cell movement throughout the ECM (day 1–2), was followed by a bsAb-dependent phase of active T-cell recruitment to tumoroids (day 2–4), and tumoroid killing (day 4–6). Low affinity HER2 or CD3 arms were compensated for by increasing bsAb concentrations. Instead, a bsAb binding a membrane proximal HER2 epitope supported tumor killing whereas a bsAb binding a membrane distal epitope did not, despite similar affinities and intra-tumoroid localization of the bsAbs, and efficacy in 2D co-cultures. Initial T-cell-tumor contact through effective bsAbs triggered a wave of subsequent T-cell recruitment. This critical surge of T-cell recruitment was explained by paracrine signaling and preceded a full-scale T-cell tumor attack.

Abstract LB126: Generation of a novel ROR1 x CD3 bispecific T-cell engager for better tumor killing and minimal cytokine release

Abstract Receptor tyrosine kinase-like orphan receptor 1 (ROR1) expression is a highly specific tumor associated antigen expressed in multiple hematological malignancies and solid tumors. EMB-07 is a novel monovalent asymmetrical tandem Fab (MAT-Fab) antibody composed of two different Fab fragments targeting ROR1 and CD3ε respectively, and a “knob-in-hole” Fc fragment maintaining FcRn binding and Ig-like pharmacokinetics in vivo. To avoid light chain miss-pairing, the anti-ROR1 light chain was directly connected to the N-terminus of the anti-CD3 heavy chain. The ROR1 binding arm has a high affinity of 0.52 nM and recognizes a membrane distal epitope, which is different from a reference BiTE molecule currently in clinical trial. In comparison with this BiTE molecule, EMB-07 showed similar tumor killing efficacy to ROR1 expressing tumor cell lines while it induced much lower levels of cytokines both in vitro and in vivo. Moreover, comparing with a clinical stage ROR1-vc-MMAE ADC, EMB-07 demonstrated ~500-fold increased tumor lysis potency in vitro. In PD-1 resistant tumor xenograft mice models, EMB-07 showed potent tumor growth inhibition and synergistic activity in combination with PD-1 blockade. In a preclinical GLP toxicological study, EMB-07 was well tolerated in cynomolgus monkeys with no evidence of lethality, life-threatening toxicity, or obvious EMB-07-related macroscopic and histopathological adverse findings up to 50 mg/kg. By using a highly specific IHC clone, we validated membrane-associated ROR1 expression in multiple tumor types, including triple negative breast cancer, ovarian cancer, endometrial cancer, lung adenocarcinoma, and pancreatic cancer, while the observation of its expression in normal tissues is very limited. This study supports the clinical development of EMB-07 for treating multiple solid tumors with ROR1 expression. A FIH study of EMB-07 in locally advanced/metastatic solid tumors or relapse/refractory lymphoma is currently ongoing (NCT05607498). Citation Format: Danqing Wu, Shiyong Gong, Xuan Wu, Gaowa Naren, Liqin Dong, Stephan Lensky, Chengbin Wu. Generation of a novel ROR1 x CD3 bispecific T-cell engager for better tumor killing and minimal cytokine release [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB126.

Development of a Regulated, Optimized Mesothelin Chimeric Antigen Receptor (CAR) for the Treatment of Mesothelin Positive Cancers

Introduction: Mesothelin (MSLN) is an attractive target for chimeric antigen receptor T cell (CAR-T) immunotherapy as it is highly expressed on mesothelioma, ovarian, lung cancer, and some forms of leukemia, including approximately 30% of acute myeloid leukemia (AML); however, MSLN directed CAR-T cells are prone to exhaustion, and soluble proteins, such as shed MSLN and MUC16 can inhibit anti-tumor efficacy. To address these deficiencies, we developed a novel, fully-human MSLN-specific CAR using our OutSpacer™ technology to optimize the immune synapse geometry. Further, this MSLN CAR is expressed using a novel regulatory response element to dynamically express the CAR molecule to enhance CAR-T potency and durability. Methods: A set of fully-human single domain binders (VHH) were produced and screened against a spacer library to identify high-performing CARs. The spacer library is composed of human extracellular polypeptides selected for optimal biophysical properties (e.g., flexibility), enabling systematic sampling to identify CAR constructs with optimal immune synapse geometry. More than 300 MSLN-directed CAR candidates were evaluated in vitro using 2D or 3D (spheroid) culture assays to measure tumor killing and cytokine production against several MSLN + cell lines that express different levels of the target protein. These functional assays were also performed in the presence of recombinant MSLN (10ug/mL) or MUC16 (1ug/mL) to assess potential inhibition by shed target or ligand. Top performing MSLN CARs were subsequently tested in NSG mice (2e6, 4e6 and 8e6 cells/animal; 2 donors) subcutaneously engrafted with MSLN + H1650 tumor cells to identify the best CAR candidate. To further improve the durability of T cell response, the MSLN CAR was expressed under the control of an engineered promoter (OP1) to dynamically regulate expression, and compared to CARs expressed constitutively using the MND promoter. CAR-T cells were repetitively stimulated (4x) by plate-bound antigen in combination with CD58 and ICAM1, and subsequently tested in a tumor spheroid assay for killing, cytokine production, CAR expression and T cell activation/ exhaustion markers (i.e., PD-1, TIGIT, CD39 and TIM3). Results: Anti-MSLN VHH domains targeting region 1 or 3 (RI and RIII) of the MLSN extracellular domain were paired with a defined spacer library to define the optimal immune synapse. Contrary to the conclusions of prior studies, RI (membrane-distal) binders outperformed RIII (membrane-proximal) binders when paired with their optimal spacer. Based on tumor killing and cytokine production, RI targeted CARs preferred a spacer length of approximately 75 Å, consistent with our previously-reported optimal CAR synapse of ~200 Å. All spacer optimized MSLN-specific CARs resisted inhibition by soluble MSLN and MUC16. In vivo studies with two donors in NSG mice engrafted with MSLN + tumors showed that one CAR (B8S8) eliminated tumors at a dose of 2e6 and 4e6 CAR-T cells per animal, where other CAR constructs, including a clinically-used MSLN-specific CAR, failed to control tumors even at 8e6 cells per animal. Secondly, we assessed whether a novel “stim-on” promoter (OP1) could enhance CAR-T activity by preserving T cell function. Here, B8S8 CAR was expressed via OP1 or the constitutive MND promoter (n=3 donors). Following chronic antigen exposure by plate-bound antigen, OP1 regulated MSLN CAR T cells retained the capacity to eliminate MSLN + H226 tumor spheroids compared to MND-driven CAR T constructs. OP1-regulated CAR T cells also continued to produce high levels of cytokines (IFN-γ and IL-2) whereas MND CAR-T cells lost cytokine production ability. Further, phenotypic analyses showed that MND CAR-T cells expressed elevated levels of PD-1 and TIGIT compared to OP1 CAR-T cells. Conclusions: Contrary to the conclusions of prior studies with MSLN-targeted CARs, membrane distal epitopes were superior to membrane proximal epitopes when binders were paired with the correct spacer, and shed MSLN and MUC16 were not inhibitory for the best-performing CARs, regardless of target epitope. Furthermore, regulated expression enhanced the potency and durability of response compared to constitutive expression. This approach may improve CAR activity in patients with MSLN + solid tumors or leukemia.

Single-chain dimers from de novo immunoglobulins as robust scaffolds for multiple binding loops

Antibody derivatives have sought to recapitulate the antigen binding properties of antibodies, but with improved biophysical attributes convenient for therapeutic, diagnostic and research applications. However, their success has been limited by the naturally occurring structure of the immunoglobulin dimer displaying hypervariable binding loops, which is hard to modify by traditional engineering approaches. Here, we devise geometrical principles for de novo designing single-chain immunoglobulin dimers, as a tunable two-domain architecture that optimizes biophysical properties through more favorable dimer interfaces. Guided by these principles, we computationally designed protein scaffolds that were hyperstable, structurally accurate and robust for accommodating multiple functional loops, both individually and in combination, as confirmed through biochemical assays and X-ray crystallography. We showcase the modularity of this architecture by deep-learning-based diversification, opening up the possibility for tailoring the number, positioning, and relative orientation of ligand-binding loops targeting one or two distal epitopes. Our results provide a route to custom-design robust protein scaffolds for harboring multiple functional loops.

Human VH-based chimeric antigen receptor T cells targeting glypican 3 eliminate tumors in preclinical models of HCC.

Efficacy of chimeric antigen receptor (CAR) T cells for treating solid tumors, including HCC, remains a challenge. Nanobodies are emerging building blocks of CAR T cells due to their small size and high expression. Membrane proximal sites have been shown as attractive epitopes of CAR T cells. However, current CAR formats are not tailored toward nanobodies or targeting membrane distal epitopes. Using hYP7 Fv (membrane proximal) and HN3 VH nanobody (membrane distal) as GPC3 targeting elements, we sought to determine how hinges and transmembrane portions of varying structures and sizes affect CAR T-cell function. We generated multiple permutations of CAR T cells containing CD8, CD28, IgG4, and Fc domains. We show that engineered HN3 CAR T cells can be improved by 2 independent, synergistic changes in the hinge and transmembrane domains. The T cells expressing the HN3 CAR which contains the hinge region of IgG4 and the CD28 transmembrane domain (HN3-IgG4H-CD28TM) exhibited high cytotoxic activity and caused complete HCC tumor eradication in immunodeficient mice. HN3-IgG4H-CD28TM CAR T cells were enriched for cytotoxic-memory CD8+ T cells and NFAT signals, and reduced β catenin levels in HCC cells. Our findings indicate that altering the hinge and transmembrane domains of a nanobody-based CAR targeting a distal GPC3 epitope, in contrast to a membrane proximal epitope, lead to robust T-cell signaling and induce swift and durable eradication of HCC tumors.

Development of an 18F-labeled anti-human CD8 VHH for same-day immunoPET imaging.

Cancer immunotherapies (CITs) have revolutionized the treatment of certain cancers, but many patients fail to respond or relapse from current therapies, prompting the need for new CIT agents. CD8+ T cells play a central role in the activity of many CITs, and thus, the rapid imaging of CD8+ cells could provide a critical biomarker for new CIT agents. However, existing 89Zr-labeled CD8 PET imaging reagents exhibit a long circulatory half-life and high radiation burden that limit potential applications such as same-day and longitudinal imaging. To this end, we discovered and developed a 13-kDa single-domain antibody (VHH5v2) against human CD8 to enable high-quality, same-day imaging with a reduced radiation burden. To enable sensitive and rapid imaging, we employed a site-specific conjugation strategy to introduce an 18F radiolabel to the VHH. The anti-CD8 VHH, VHH5v2, demonstrated binding to a membrane distal epitope of human CD8 with a binding affinity (KD) of 500pM. Subsequent imaging experiments in several xenografts that express varying levels of CD8 demonstrated rapid tumor uptake and fast clearance from the blood. High-quality images were obtained within 1h post-injection and could quantitatively differentiate the tumor models based on CD8 expression level. Our work reveals the potential of this anti-human CD8 VHH [18F]F-VHH5v2 to enable rapid and specific imaging of CD8+ cells in the clinic.

Exploiting the Innate Plasticity of the Programmed Cell Death-1 (PD1) Receptor to Design Pembrolizumab H3 Loop Mimics.

Checkpoint blockade of the immunoreceptor programmed cell death-1 (PD1) with its ligand-1 (PDL1) by monoclonal antibodies such as pembrolizumab provided compelling clinical results in various cancer types, yet the molecular mechanism by which this drug blocks the PD1/PDL1 interface remains unclear. To address this question, we examined the conformational motion of PD1 associated with the binding of pembrolizumab. Our results revealed that the innate plasticity of both C'D and FG loops is crucial to form a deep binding groove (371 Å3 ) across several distant epitopes of PD1. This analysis ultimately provided a rational-design to create pembrolizumab H3 loop mimics [RDYRFDMGFD] into β-hairpin scaffolds. As a result, a 20-residue long β-hairpin peptide 1 e was identified as a first-in-class potent PD1-inhibitor (EC50 of 0.29 μM; Ki of 41 nM).

Abstract 5567: hYP218 CAR-T cells targeting a membrane-proximal epitope of mesothelin are highly effective against mesothelin expressing cancers

Abstract Background and Hypothesis: Mesothelin is an attractive target for CAR-T cell therapy since it is highly expressed in many solid tumors but has limited expression on normal human tissues. However, anti-mesothelin CAR-T cells have shown limited efficacy in patients. Since most antibodies to mesothelin bind the membrane distal epitopes we generated antibodies that bind mesothelin in a membrane-proximal region. We hypothesize that CAR-T cells recognizing a membrane proximal epitope on mesothelin will be more efficacious. Study Design: We developed CAR-T cells targeting a membrane-distal (SS1CAR-T) and a membrane-proximal epitope (hYP218 CAR-T) of mesothelin. Using mesothelin-expressing cancer cell lines, anti-tumor efficacy of CAR-T cells was assessed in vitro by measuring direct killing of tumor cells by the CAR-T cells and quantifying cytokine-release by the CAR-T cells. In vivo anti-tumor efficacy of CAR-T was determined in NSG mice using three different solid tumor models of high-mesothelin expression: ovarian cancer (OVCAR-8), pancreatic cancer (KLM-1), and mesothelioma patient-derived xenograft (NCI-Meso63). Blood and tumor infiltration of CAR-T cells in vivo was determined via flow cytometry analysis. Results: Upon co-culture with tumor cells with high mesothelin expression (A431-H9, OVCAR-8, KLM-1, H226, NCI-Meso63, NCI-Meso29), hYP218 CAR-T cells killed cancer cells more efficiently than SS1 CAR-T cells, with a 2-4-fold lower ET50 value (Effector to Target ratio for 50% killing of tumor cells). Additionally, compared to SS1 CAR-T cells, hYP218 CAR-T cells secreted significantly higher level of cytokines (IL-2, TNFα, IFNγ) in a co-culture with tumor cells. In three solid tumor models, single treatment of tumor-bearing mice with hYP218 CAR-T cells lead to improved tumor response and survival compared to SS1 CAR-T, with complete regression of OVCAR-8 and NCI-Meso63 tumors. Compared to SS1 CAR-T cells, higher hYP218 CAR-T cell infiltration was detected both in the blood and tumors isolated from hYP218 CAR-T treated mice seven days post-treatment. Conclusion: We demonstrate that hYP218 CAR-T cells targeting a membrane-proximal epitope of mesothelin are more potent than similar CAR-T cells targeting a membrane distal epitope. Increased accumulation of hYP218 CAR-T cells in the tumor may explain their increased efficacy. These results support its clinical development to treat patients with mesothelin expressing cancers. Citation Format: Sakshi Tomar, Jingli Zhang, Nan Li, Ira Pastan, Mitchell Ho, Raffit Hassan. hYP218 CAR-T cells targeting a membrane-proximal epitope of mesothelin are highly effective against mesothelin expressing cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5567.

Abstract 6343: A multispecific T cell engager binding to both membrane proximal and membrane distal epitopes of MSLN with low affinity CD3 for the treatment of AML and solid tumors

Abstract Purpose: The effective treatment of AML and solid tumors remains an unmet medical need. Mesothelin (MSLN) is a glycophosphatidylinositol-linked tumor antigen overexpressed in a variety of malignancies, including AML and solid tumors, such as mesothelioma, cholangiocarcinoma, ovarian, pancreatic, lung, esophageal, gastric, colorectal, endometrial and triple-negative breast cancer. Early signs of clinical efficacy with MSLN-targeting agents have validated MSLN as a promising target for therapeutic intervention, but therapies with improved efficacy are still needed. CD3-based T cell engagers are highly potent therapeutic molecules with T cell cytotoxicity activities in the picomolar range. But high CRS risk and T cell exhaustion has been observed for high affinity CD3-based T cell engagers in clinicals, so we used high affinity MSLN and low affinity CD3 to improve efficacy of T cell engager. Experimental Design: We designed SMTE-001, a 78-kDa, tetra-specific, T-cell-activating protein molecule, which binds to two epitopes (membrane proximal and membrane distal) of MSLN, CD3ε on T cells, and to serum albumin. Experiments were conducted to assess the potency, activity, and half-life of HPN536 in in vitro assays, rodent models, and in nonhuman primates (NHP). This molecule was tested in a cytotoxicity assay using human PBMCs co-cultured with OVCAR3, Hela and NCI-H292 cells, which express different levels of MSLN. Soluble MSLN and CA125 (ligand of MSLN) was added to determine effects on cytotoxicity. In vivo xenograft mouse studies were conducted using a PBMC model. Results: Here we report the design and the promising preclinical activity of SMTE-001 molecule in vitro and in vivo. We demonstrate that the bivalent MSLN T cell engager has increased in vitro potency in T cell activation and tumor cell killing, as compared to a monovalent counterpart on high MSLN expressing cells. We also demonstrate that T cell exhaustion is reduced for the low-affinity CD3, compared to the low-affinity CD3 molecule. Because soluble MSLN and CA125 is shed from cancer cells into cancer patient serum, we also demonstrate that soluble MSLN and CA125 does not interfere with the cytotoxic activity of SMTE-001. Importantly, we demonstrate in vivo that SMTE-001 significantly inhibits tumor growth in a dose-dependent manner, while high-affinity CD3 molecule’ efficacy reduces when higher dosing. In cynomolgus monkeys, SMTE-001 shows pharmacokinetics in support of weekly dosing in humans. Conclusions: Collectively, these data demonstrate strong anti-tumor efficacy by this novel multispecific bivalent T cell engager. These data indicate the therapeutic potential of this molecule to activate T cells and improve the clinical efficacy in AML and MSLN-expressing solid tumors. Citation Format: Cuiqing Yang, Xiaohui Shao, Feng Zhou, Lei Zhou, Qi Deng, Yun Zhang, Guangcun Cheng, Yingying Hu, Huaiyuan Ma, Yadan Wu, Shuai Wang, Jie Zang, Lei Liu, Wenqing Yang, Yang Liu, Chunlei Xia, Jianzhong Hu, Ande Luo, Yayuan Fu, Zhuoxiao Cao, Renhong Tang. A multispecific T cell engager binding to both membrane proximal and membrane distal epitopes of MSLN with low affinity CD3 for the treatment of AML and solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6343.

A nanobody toolbox targeting dimeric coiled-coil modules for functionalization of designed protein origami structures

Coiled-coil (CC) dimers are widely used in protein design because of their modularity and well-understood sequence-structure relationship. In CC protein origami design, a polypeptide chain is assembled from a defined sequence of CC building segments that determine the self-assembly of protein cages into polyhedral shapes, such as the tetrahedron, triangular prism, or four-sided pyramid. However, a targeted functionalization of the CC modules could significantly expand the versatility of protein origami scaffolds. Here, we describe a panel of single-chain camelid antibodies (nanobodies) directed against different CC modules of a de novo designed protein origami tetrahedron. We show that these nanobodies are able to recognize the same CC modules in different polyhedral contexts, such as isolated CC dimers, tetrahedra, triangular prisms, or trigonal bipyramids, thereby extending the ability to functionalize polyhedra with nanobodies in a desired stoichiometry. Crystal structures of five nanobody-CC complexes in combination with small-angle X-ray scattering show binding interactions between nanobodies and CC dimers forming the edges of a tetrahedron with the nanobody entering the tetrahedral cavity. Furthermore, we identified a pair of allosteric nanobodies in which the binding to the distant epitopes on the antiparallel homodimeric APH CC is coupled via a strong positive cooperativity. A toolbox of well-characterized nanobodies specific for CC modules provides a unique tool to target defined sites in the designed protein structures, thus opening numerous opportunities for the functionalization of CC protein origami polyhedra or CC-based bionanomaterials.

Mapping Binding Epitopes and Allosteric Effects in Staph Enterotoxins

Staphylococcal enterotoxins (SEs) cause food poisoning-like symptoms and can be lethal at microgram amounts. SEs elicit profound immune responses via the bridging of major histocompatibility complex class II (MHCII) molecules on antigen presenting cells with T-cell receptors (TCR). 20B1, 14G8, and 6D3 stand out as promising neutralizing monoclonal antibodies (mAbs) that have been co-crystalized with staphylococcus enterotoxin B (SEB). Although crystal structures have been solved with SEB and a variety of antibodies, it is still not understood why some antibodies effectively neutralize toxins while others do not. Paired with structures of MHC class II and TCR bound to SEB, it has been suggested that 20B1 inactivates SEB by sterically hindering the binding of the toxin to immune molecules. 14G8 and 6D3 also inactivate SEB, but through binding interactions at distal epitopes. Using Hydrogen Deuterium Exchange coupled to Mass Spectrometry (HDX-MS) and X-Ray Footprinting (XRF) in tandem with MS experiments, we observe that neutralizing antibodies binding to distal epitopes elicit allosteric effects in SEB that may drive neutralization.

Extended Recognition of the Histone H3 Tail by Histone Demethylase KDM5A.

Human lysine demethylase KDM5A is a chromatin-modifying enzyme associated with transcriptional regulation, because of its ability to catalyze removal of methyl groups from methylated lysine 4 of histone H3 (H3K4me3). Amplification of KDM5A is observed in many cancers, including breast cancer, prostate cancer, hepatocellular carcinoma, lung cancer, and gastric cancer. In this study, we employed alanine scanning mutagenesis to investigate substrate recognition of KDM5A and identify the H3 tail residues necessary for KDM5A-catalyzed demethylation. Our data show that the H3Q5 residue is critical for substrate recognition by KDM5A. Our data also reveal that the protein-protein interactions between KDM5A and the histone H3 tail extend beyond the amino acids proximal to the substrate mark. Specifically, demethylation activity assays show that deletion or mutation of residues at positions 14-18 on the H3 tail results in an 8-fold increase in the KMapp, compared to wild-type 18mer peptide, suggesting that this distal epitope is important in histone engagement. Finally, we demonstrate that post-translational modifications on this distal epitope can modulate KDM5A-dependent demethylation. Our findings provide insights into H3K4-specific recognition by KDM5A, as well as how chromatin context can regulate KDM5A activity and H3K4 methylation status.

A Chemical Approach for Profiling Intracellular AKT Signaling Dynamics from Single Cells.

We present here a novel chemical method to continuously analyze intracellular AKT signaling activities at single-cell resolution, without genetic manipulations. A pair of cyclic peptide-based fluorescent probes were developed to recognize the phosphorylated Ser474 site and a distal epitope on AKT. A Förster resonance energy transfer signal is generated upon concurrent binding of the two probes onto the same AKT protein, which is contingent upon the Ser474 phosphorylation. Intracellular delivery of the probes enabled dynamic measurements of the AKT signaling activities. We further implemented this detection strategy on a microwell single-cell platform, and interrogated the AKT signaling dynamics in a human glioblastoma cell line. We resolved unique features of the single-cell signaling dynamics following different perturbations. Our study provided the first example of monitoring the temporal evolution of cellular signaling heterogeneities and unveiled biological information that was inaccessible to other methods.

Tri‐Orthogonal Scaffolds for the Solid‐Phase Synthesis of Peptides

Multi‐orthogonal scaffolds can be useful for the attachment of several different compounds to the same central skeleton. Such compounds can find applications in the development of protein mimics because of their potential to mimic several distant epitopes in the protein structure. Three new tri‐orthogonal variants of a previously reported scaffold have been developed that are suitable for the solid‐phase synthesis of three different peptides on the same skeleton. Different chemical moieties were combined for the phased attachment of amino acids to the scaffolds: Fmoc‐ or Alloc‐protected amine, free or TIPS‐protected alkyne or azido group. Several model compounds were prepared, characterized, and compared for their suitability as new scaffolds for peptide synthesis. All three scaffolds provided peptides with satisfactory yields and purities, making them suitable for a synthesis of libraries of compounds.

Amino Acid Changes in the HIV-1 gp41 Membrane Proximal Region Control Virus Neutralization Sensitivity

Most HIV-1 vaccines elicit neutralizing antibodies that are active against highly sensitive (tier-1) viruses or rare cases of vaccine-matched neutralization-resistant (tier-2) viruses, but no vaccine has induced antibodies that can broadly neutralize heterologous tier-2 viruses. In this study, we isolated antibodies from an HIV-1-infected individual that targeted the gp41 membrane-proximal external region (MPER) that may have selected single-residue changes in viral variants in the MPER that resulted in neutralization sensitivity to antibodies targeting distal epitopes on the HIV-1 Env. Similarly, a single change in the MPER in a second virus from another infected-individual also conferred enhanced neutralization sensitivity. These gp41 single-residue changes thus transformed tier-2 viruses into tier-1 viruses that were sensitive to vaccine-elicited tier-1 neutralizing antibodies. These data demonstrate that Env amino acid changes within the MPER bnAb epitope of naturally-selected escape viruses can increase neutralization sensitivity to multiple types of neutralizing antibodies, and underscore the critical importance of the MPER for maintaining the integrity of the tier-2 HIV-1 trimer.

The N-terminal Region of the Ubiquitin Regulatory X (UBX) Domain-containing Protein 1 (UBXD1) Modulates Interdomain Communication within the Valosin-containing Protein p97

Valosin-containing protein/p97 is an ATP-driven protein segregase that cooperates with distinct protein cofactors to control various aspects of cellular homeostasis. Mutations at the interface between the regulatory N-domain and the first of two ATPase domains (D1 and D2) deregulate the ATPase activity and cause a multisystem degenerative disorder, inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia/amyotrophic lateral sclerosis. Intriguingly, the mutations affect only a subset of p97-mediated pathways correlating with unbalanced cofactor interactions and most prominently compromised binding of the ubiquitin regulatory X domain-containing protein 1 (UBXD1) cofactor during endolysosomal sorting of caveolin-1. However, how the mutations impinge on the p97-cofactor interplay is unclear so far. In cell-based endosomal localization studies, we identified a critical role of the N-terminal region of UBXD1 (UBXD1-N). Biophysical studies using NMR and CD spectroscopy revealed that UBXD1-N can be classified as intrinsically disordered. NMR titration experiments confirmed a valosin-containing protein/p97 interaction motif and identified a second binding site at helices 1 and 2 of UBXD1-N as binding interfaces for p97. In reverse titration experiments, we identified two distant epitopes on the p97 N-domain that include disease-associated residues and an additional interaction between UBXD1-N and the D1D2 barrel of p97 that was confirmed by fluorescence anisotropy. Functionally, binding of UBXD1-N to p97 led to a reduction of ATPase activity and partial protection from proteolysis. These findings indicate that UBXD1-N intercalates into the p97-ND1 interface, thereby modulating interdomain communication of p97 domains and its activity with relevance for disease pathogenesis. We propose that the polyvalent binding mode characterized for UBXD1-N is a more general principle that defines a subset of p97 cofactors.

Structure of the eukaryotic translation initiation factor eIF4E in complex with 4EGI-1 reveals an allosteric mechanism for dissociating eIF4G.

The interaction of the eukaryotic translation initiation factor eIF4E with the initiation factor eIF4G recruits the 40S ribosomal particle to the 5' end of mRNAs, facilitates scanning to the AUG start codon, and is crucial for eukaryotic translation of nearly all genes. Efficient recruitment of the 40S particle is particularly important for translation of mRNAs encoding oncoproteins and growth-promoting factors, which often harbor complex 5' UTRs and require efficient initiation. Thus, inhibiting the eIF4E/eIF4G interaction has emerged as a previously unpursued route for developing anticancer agents. Indeed, we discovered small-molecule inhibitors of this eIF4E/eIF4G interaction (4EGIs) that inhibit translation initiation both in vitro and in vivo and were used successfully in numerous cancer-biology and neurobiology studies. However, their detailed molecular mechanism of action has remained elusive. Here, we show that the eIF4E/eIF4G inhibitor 4EGI-1 acts allosterically by binding to a site on eIF4E distant from the eIF4G binding epitope. Data from NMR mapping and high-resolution crystal structures are congruent with this mechanism, where 4EGI-1 attaches to a hydrophobic pocket of eIF4E between β-sheet2 (L60-T68) and α-helix1 (E69-N77), causing localized conformational changes mainly in the H78-L85 region. It acts by unfolding a short 310-helix (S82-L85) while extending α-helix1 by one turn (H78-S82). This unusual helix rearrangement has not been seen in any previous eIF4E structure and reveals elements of an allosteric inhibition mechanism leading to the dislocation of eIF4G from eIF4E.

Hilary Koprowski and the viral immunity hub at the 1980s Wistar Institute: a T cell epitope perspective.

Hilary Koprowski and the viral immunity hub at the 1980s Wistar Institute: a T cell epitope perspective.

Independent Expansion of Epitope-Specific Plasma Cell Responses upon HIV-1 Envelope Glycoprotein Immunization

Abs that bind the functional envelope glycoprotein (Env) spike are considered critical for a broadly effective prophylactic HIV-1 vaccine. The difficulty in eliciting such Abs by vaccination is partially attributed to the immunodominance of hydrophilic, surface-exposed variable protein regions of Env. However, little is known about the potential for competition between B cells that recognize distinct and distal epitopes on Env during protein subunit vaccination. In this study, we address this basic question at the level of Ab-secreting cells and serum IgG using a pair of isogenic soluble Env trimers, designated wildtype and gV3, which differ only in their potential to activate B cell responses against the highly immunogenic V3 region of Env. Immunization of mice with gV3 resulted in a markedly lower Ag-specific response compared with that induced by wildtype Env and could be explained by a loss of V3-directed reactivities. There was no redistribution of the response to other regions of Env in gV3-inoculated mice, suggesting that the epitope-specific Ab-secreting cell responses measured after boost are independently regulated rather than dictated by direct or indirect competition between B cells recognizing different structural elements of Env. This information is relevant for ongoing efforts in Env immunogen design to focus responses on conserved neutralizing determinants and for our general understanding of B cell responses to large-protein Ags that display numerous B cell epitopes.

A New High Activity Anti-CD22 Chimeric Antigen Receptor (CAR) Targeting B Cell Leukemia.

AbstractAbstract 2611CD22 is expressed on the surface of B cell hematologic malignancies such as acute lymphoblastic leukemia (ALL). CD22 is a Siglec family lectin present on B cells, starting at the pre-B cell stage of development, but is not expressed on plasma cells. CD22 consists of 7 extracellular Ig domains and is found in 2 isoforms, one of which is missing the second and third N-terminal Ig domains. We generated CAR modified T cells containing anti-CD22 extracellular binding motifs fused to intracellular signaling domains for T cells activation (CD3 zeta) or costimulation (CD28 or 4-1BB). The binding motifs were derived from scFvs that targeted a membrane distal epitope of CD22, Ig domain 3, (BL22 and a higher affinity HA22 motif) or that bound a more membrane proximal, Ig domains 5–7, of CD22 (m971). The CAR constructs we generated were second-generation (CD28 and CD3 zeta; or, 4-1BB and CD3 zeta) or third generation (CD28, 4-1BB and CD3 zeta signaling domains). A CH2CH3 spacer domain from IgG1 was added in some constructs to examine the impact of extending the scFv-derived binding domain away from the transduced T cell membrane. In vitro cellular cytotoxicity and cytokine release experiments with 4 B cell-ALL cell lines (REH, SEM, NALM6, KOPN8) as well as the CD22 (+)ve Daudi and Raji cell lines were performed. Our results demonstrate that addition of the CH2CH3 domain did not improve tumor lysis and that standard affinity BL22 and higher affinity HA22-derived scFv epitopes were equivalent. With regard to signaling domains, second generation constructs were better than third generation constructs both in vitro and in vivo. In comparison between second generation constructs, CD28 containing domains outperformed 4-1BB with regard to lytic activity and cytokine release. Most surprising was the activity of the m971-derived scFv binding epitope. m971-CAR had significantly higher killing activity, a far more robust cytokine release profile, and superior in vivo activity. NSG mice were injected i.v. with 0.5× 106 NALM6-GL cells (pre-B cell ALL line engineered to express luciferase). Three days later, when disease was evident, mice were treated with 1×107 CAR+ T cells, and then followed by bioluminescent imaging to measure disease burden. The m971 CAR was significantly more potent at tumor clearance than our previously developed most active construct expressing the HA22-derived scFv domain (Figure 1). Disease progressed rapidly when non-transduced T cells were used (mock). We are currently examining the activity of different signaling domains on m971 CAR efficacy in vivo and directly comparing the anti-CD22 m971 CAR to the CD19 CAR currently being evaluated in clinical trials. These studies will guide future anti-CD22 CAR-based anti-leukemia immunotherapy trials. [Display omitted] Disclosures:No relevant conflicts of interest to declare.