Functional Epitopes Research Articles

Imprinting and Editing of the Human CD4 T Cell Response to Influenza Virus.

Immunity to influenza is unique among pathogens, in that immune memory is established both via intermittent lung localized infections with highly variable influenza virus strains and by intramuscular vaccinations with inactivated protein-based vaccines. Studies in the past decades have suggested that the B cell responses to influenza infection and vaccination are highly biased by an individual's early history of influenza infection. This reactivity likely reflects both the competitive advantage that memory B cells have in an immune response and the relatively limited diversity of epitopes in influenza hemagglutinin that are recognized by B cells. In contrast, CD4 T cells recognize a wide array of epitopes, with specificities that are heavily influenced by the diversity of influenza antigens available, and a multiplicity of functions that are determined by both priming events and subsequent confrontations with antigens. Here, we consider the events that prime and remodel the influenza-specific CD4 T cell response in humans that have highly diverse immune histories and how the CD4 repertoire may be edited in terms of functional potential and viral epitope specificity. We discuss the consequences that imprinting and remodeling may have on the potential of different human hosts to rapidly respond with protective cellular immunity to infection. Finally, these issues are discussed in the context of future avenues of investigation and vaccine strategies.

Cytomegalovirus memory inflation and immune protection.

Cytomegalovirus (CMV) infection induces powerful and sustained T-cell responses against a few selected immunodominant antigenic epitopes. This immune response was named memory inflation, because it does not contract in the long term, and may even expand over months and years of virus latency. It is by now understood that memory inflation does not occur at the expense of the naïve T-cell pool, but rather as a competitive selection process within the effector pool, where viral antigens with higher avidity of TCR binding and with earlier expression patterns outcompete those that are expressed later and bind TCRs less efficiently. It is also understood that inflationary epitopes require processing by the constitutive proteasome in non-hematopoietic cells, and this likely implies that memory inflation is fuelled by direct low-level antigenic expression in latently infected cells. This review proposes that these conditions make inflationary epitopes the optimal candidates for adoptive immunotherapy of CMV disease in the immunocompromised host. At present, functional target CMV epitopes have been defined only for the most common HLA haplotypes. Mapping the uncharacterized inflationary epitopes in less frequent HLAs may, thus, be a strategy for the identification of optimal immunotherapeutic targets in patients with uncommon haplotypes.

Oxidation of human plasma fibronectin by inflammatory oxidants perturbs endothelial cell function

Dysfunction of endothelial cells of the artery wall is an early event in cardiovascular disease and atherosclerosis. The cause(s) of this dysfunction are unresolved, but accumulating evidence suggests that oxidants arising from chronic low-grade inflammation are contributory agents, with increasing data implicating myeloperoxidase (MPO, released by activated leukocytes), and the oxidants it generates (e.g. HOCl and HOSCN). As these are formed extracellularly and react rapidly with proteins, we hypothesized that MPO-mediated damage to the matrix glycoprotein fibronectin (FN) would modulate FN structure and function, and its interactions with human coronary artery endothelial cells (HCAEC). Exposure of human plasma FN to HOCl resulted in modifications to FN and its functional epitopes. A dose-dependent loss of methionine and tryptophan residues, together with increasing concentrations of methionine sulfoxide, and modification of the cell-binding fragment (CBF) and heparin-binding fragment (HBF) domains was detected with HOCl, but not HOSCN. FN modification resulted in a loss of HCAEC adhesion, impaired cell spreading and reduced cell proliferation. Exposure to HCAEC to HOCl-treated FN altered the expression of HCAEC genes associated with extracellular matrix (ECM) synthesis and adhesion. Modifications were detected on HCAEC-derived ECM pre-treated with HOCl, but not HOSCN, with a loss of antibody recognition of the CBF, HBF and extra-domain A. Co-localization of epitopes arising from MPO-generated HOCl and cell-derived FN was detected in human atherosclerotic lesions. Damage was also detected on FN extracted from lesions. These data support the hypothesis that HOCl, but not HOSCN, targets and modifies FN resulting in arterial wall endothelial cell dysfunction.

Enhancing Natural Killer and CD8+ T Cell-Mediated Anticancer Cytotoxicity and Proliferation of CD8+ T Cells with HLA-E Monospecific Monoclonal Antibodies.

Cytotoxic NK/CD8+ T cells interact with MHC-I ligands on tumor cells through either activating or inhibiting receptors. One of the inhibitory receptors is CD94/NKG2A. The NK/CD8+ T cell cytotoxic capability is lost when tumor-associated human leukocyte antigen, HLA-E, binds the CD94/NKG2A receptor, resulting in tumor progression and reduced survival. Failure of cancer patients to respond to natural killer (NK) cell therapies could be due to HLA-E overexpression in tumor tissues. Preventing the inhibitory receptor–ligand interaction by either receptor- or ligand-specific monoclonal antibodies (mAbs) is an innovative passive immunotherapeutic strategy for cancer. Since receptors and ligands can be monomeric or homo- or heterodimeric proteins, the efficacy of mAbs may rely on their ability to distinguish monospecific (private) functional epitopes from nonfunctional common (public) epitopes. We developed monospecific anti-HLA-E mAbs (e.g., TFL-033) that recognize only HLA-E-specific epitopes, but not epitopes shared with other HLA class-I loci as occurs with currently available polyreactive anti-HLA-E mAbs. Interestingly the amino acid sequences in the α1 and α2 helices of HLA-E, critical for the recognition of the mAb TFL-033, are strikingly the same sequences recognized by the CD94/NKG2A inhibitory receptors on NK/CD8+ cells. Such monospecific mAbs can block the CD94/NKG2A interaction with HLA-E to restore NK cell and CD8+ anticancer cell cytotoxicity. Furthermore, the HLA-E monospecific mAbs significantly promoted the proliferation of the CD4−/CD8+ T cells. These monospecific mAbs are also invaluable for the specific demonstration of HLA-E on tumor biopsies, potentially indicating those tumors most likely to respond to such therapy. Thus, they can be used to enhance passive immunotherapy once phased preclinical studies and clinical trials are completed. On principle, we postulate that NK cell passive immunotherapy should capitalize on both of these features of monospecific HLA-E mAbs, that is, the specific determination HLA-E expression on a particular tumor and the enhancement of NK cell/CD8+ cytotoxicity if HLA-E positive.

Functional Evolution of Proteins.

The functional evolution of proteins advances through gene duplication followed by functional drift, whereas molecular evolution occurs through random mutational events. Over time, protein active-site structures or functional epitopes remain highly conserved, which enables relationships to be inferred between distant orthologs or paralogs. In this study, we present the first functional clustering and evolutionary analysis of the RCSB Protein Data Bank (RCSB PDB) based on similarities between active-site structures. All of the ligand-bound proteins within the RCSB PDB were scored using our Comparison of Protein Active-site Structures (CPASS) software and database (http://cpass.unl.edu/). Principal component analysis was then used to identify 4431 representative structures to construct a phylogenetic tree based on the CPASS comparative scores (http://itol.embl.de/shared/jcatazaro). The resulting phylogenetic tree identified a sequential, step-wise evolution of protein active-sites and provides novel insights into the emergence of protein function or changes in substrate specificity based on subtle changes in geometry and amino acid composition.

Playing Chess with HIV

HIV envelope glycoprotein (Env) exhibits extreme antigenic variation that can be countered by an amazing class of immunoglobulins known as broadly neutralizing antibodies. Dingens etal. (2019) use saturating mutagenesis of Env to play out all of the potential bnAb escape strategies and in doing so define the functional epitopes of these important vaccine and immunotherapeutic targets.

An Antigenic Atlas of HIV-1 Escape from Broadly Neutralizing Antibodies Distinguishes Functional and Structural Epitopes

Anti-HIV broadly neutralizing antibodies (bnAbs) have revealed vaccine targets on the virus's envelope (Env) protein and are themselves promising immunotherapies. The efficacy of bnAb-based therapies and vaccines depends in part on how readily the virus can escape neutralization. Although structural studies can define contacts between bnAbs and Env, only functional studies can define mutations that confer escape. Here, we mapped how all possible single amino acid mutations in Env affect neutralization of HIV by nine bnAbs targeting five epitopes. For most bnAbs, mutations at only a small fraction of structurally defined contact sites mediated escape, and most escape occurred at sites near, but not in direct contact with, the antibody. The Env mutations selected by two pooled bnAbs were similar to those expected from the combination of the bnAbs's independent action. Overall, our mutation-level antigenic atlas provides a comprehensive dataset for understanding viral immune escape and refining therapies and vaccines.

Discovery and characterization of potent IL-21 neutralizing antibodies via a novel alternating antigen immunization and humanization strategy.

Interleukin-21 (IL-21), a member of the common cytokine receptor γ chain (γc) family, is secreted by CD4+ T cells and natural killer T cells and induces effector function through interactions with the IL-21 receptor (IL-21R)/γc complex expressed on both immune and non-immune cells. Numerous studies suggest that IL-21 plays a significant role in autoimmune disorders. Therapeutic intervention to disrupt the IL-21/IL-21R/γc interaction and inhibit subsequent downstream signal transduction could offer a treatment paradigm for these diseases. Potent neutralizing antibodies reported in the literature were generated after extensive immunizations with human IL-21 alone and in combination with various adjuvants. To circumvent the laborious method of antibody generation while targeting a conserved functional epitope, we designed a novel alternating-antigen immunization strategy utilizing both human and cynomolgus monkey (cyno) IL-21. Despite the high degree of homology between human and cyno IL-21, our alternating-immunization strategy elicited higher antibody titers and more potent neutralizing hybridomas in mice than did the immunization with human IL-21 antigen alone. The lead hybridoma clone was humanized by grafting the murine complementarity-determining regions onto human germline framework templates, using a unique rational design. The final humanized and engineered antibody, MEDI7169, encodes only one murine residue at the variable heavy/light-chain interface, retains the sub-picomolar affinity for IL-21, specifically inhibits IL-21/IL-21R–mediated signaling events and is currently under clinical development as a potential therapeutic agent for autoimmune diseases. This study provides experimental evidence of the immune system’s potential to recognize and respond to shared epitopes of antigens from distinct species, and presents a generally applicable, novel method for the rapid generation of exceptional therapeutic antibodies using the hybridoma platform.

A combination of bioactive and nonbioactive alkyl-peptides form a more stable nanofiber structure for differentiating neural stem cells: a molecular dynamics simulation survey

Self-assembling alkyl-peptides are important molecules due to their ability to construct nano-level structures such as nanofibers to be utilized as tissue engineering scaffolds. The bioactive epitope of FAQRVPP which acts as neural stem cells (NSCs) outgrowth inducing factor is used in nanofiber structures. Based on previous experimental studies the density and distribution pattern of the epitopes on the surface of the nanofibers plays an important role in the differentiation function efficiency. We decided to survey and compare the stability of two pre-constructed fiber structures in the forms of all-functionalized nanofiber (containing only bioactive alkyl-peptides) and distributed functionalized nanofiber (a combination of nonbioactive and bioactive alkyl-peptides with ratio 2:1). Our findings reveal that the all-functionalized fiber shows an unstable structure and is split into intermediate micelle-like structures to reduce compactness and steric hindrance of functional epitopes whereas the distributed functionalized fiber shows an integrated stable nanofiber with a more amount of beta sheets that are well-organized and oriented around the hydrophobic core. The hydrogen bonds and energy profiles of the structures indicate the role of hydrophobic interactions during the alkyl-chain core formation and the important role of electrostatic interactions and hydrogen bond network in the stability of the final structures. Finally, it seems that the possibility of the presence of intermediate structure is increased in the all-functionalized nanofiber environment, and it can reduce functional efficiency of the scaffolds. These findings can help to design more efficient nanofiber structures with different goals in scaffolds for tissue engineering.AbbreviationsMDMolecular DynamicsNSCsNeural Stem CellsPMEParticle mesh EwaldRDFRadial Distribution FunctionRGRadius of gyrationRASARelative Accessible Surface AreaRMSDRoot Mean Square DeviationsSASASolvent Accessible Surface Area.Communicated by Ramaswamy H. Sarma

Class II HLA Epitope Level Mismatch Can Predict Chronic Graft-Versus-Host Disease and Survival Following Haploidentical Transplant Using Post-Transplant Cyclophosphamide

Post-transplant cyclophosphamide (PTCy) has improved the outcomes and expanded the use of haploidentical hematopoietic cell transplantation (haplo-HCT). Unlike many other allogeneic HCT settings, the impact of HLA disparity on graft-versus-host disease (GVHD) and transplant outcome in this setting remains unclear. HLAMatchmaker is a computer algorithm that assesses HLA compatibility at the structural level by determining what and how many functional epitopes (eplets), defined as patches of polymorphic residues within a radius of 3.0-3.5 Ångstroms, are shared between donor and recipient. It has been useful in the identification of acceptable mismatches (mm) for alloimmunized kidney transplant candidates. In order to determine the effects of HLA class I (HLA-A, B, C) and II (HLA-DR, DQ, DP) epitope mm on transplant outcome, we retrospectively analyzed 208 consecutive donor-recipient pairs receiving haplo-HCT with PTCy for hematologic malignancy. The impact of epitope mm (GVH direction) on GVHD and survival endpoints was evaluated by Cox multivariate analysis (MVA), controlling for other significant patient, donor and transplant-related factors. Median (range) recipient and donor age was 52 (19-75) and 38 (15-73) years respectively. Patients were transplanted for AML (34%), MDS/MPS/CML (20%), ALL (17%), NHL/HD/CLL (25%). PBSC was used as the stem cell source in 66% of patients, and conditioning intensity was myeloablative in 41%. The donor was a child, sibling, or parent in 47%, 38%, and 14% respectively. Median (range) follow-up for surviving patients was 33 (7-130) months. HLA class I epitope mm had no effect on GVHD or survival. In contrast, increased HLA class II epitope mm (>16) was significantly correlated to an increased frequency of chronic GVHD (figure 1). In MVA, higher degree of class II epitope mm was associated with chronic GVHD, total (HR 1.91, p=0.012) and moderate-to-severe (HR 2.37, p=0.006). The positive effect of increased class II epitope mm on chronic GVHD was driven mostly by HLA-DQ epitope mm (HR 1.7 for >7 vs. ≤7, p=0.047) with a non-significant contribution from HLA-DP (HR 1.36 for >2 vs. ≤2, p=0.24). In contrast, increased HLA-DR epitope mm had a protective effect on chronic GVHD (HR 0.52 for >7 vs. ≤7, p=0.021). Epitope mm was not significantly associated with acute GVHD, grade 2-4 or 3-4. There was also no effect of allele-level mm at any HLA loci on acute or chronic GVHD. We next tested the impact of class I and II epitope mm on survival, including the individual impact of HLA-DR, -DQ and -DP epitope mm. Although class I epitope mm had no impact in univariate analysis, a higher number of class II epitope mm (>16) was correlated with better overall survival and the effect was primarily driven by HLA-DQ epitope mm (figure 2). To better assess the impact of class II epitope mm on survival, we analyzed this variable in the context of a previously published MVA (Solomon et al. Biol Blood Marrow Transplant. 2018;24:789-798). Controlling for other significant variables (age, race, CMV status, donor relationship, HLA-DR mm, nonpermissive HLA-DP mm, KIR receptor-ligand mm and KIR haplotype), only increased HLA-DQ epitope mm (>7) was independently associated with decreased non-relapse (HR 0.34, p=0.021) and overall mortality (HR 0.60, p=0.039). These results indicate a significant effect of class II epitope mm on chronic GVHD and survival following haplo-HCT with PTCy. Higher level of class II epitope mm and HLA-DQ epitope mm is associated with increased chronic GVHD incidence, whereas HLA-DR epitope mm is protective. Higher HLA-DQ epitope mm is independently associated with better survival, when controlling for the presence of HLA-DR allele-level mm or a nonpermissive HLA-DP mm, which have been shown previously to improve survival. Class II HLA epitope level matching provides important prognostic information in the setting of haplo-HCT and PTCy, which is not reflected by conventional allele-level matching. [Display omitted] DisclosuresSolh:Amgen: Speakers Bureau; Celgene: Speakers Bureau; ADC Therapeutics: Research Funding.

Wnt5a Induces ROR1 Dependent Tyrosine Phosphorylation of DOCK2, and Enhanced Activation of ERK to Promote Proliferation of CLL Cells

ROR1 (receptor tyrosine kinase-like orphan receptor 1) is a conserved, oncoembryonic surface antigen expressed in chronic lymphocytic leukemia (CLL) B cells, but not on most normal post-partem tissues. Patients with high-level leukemia-cell expression of ROR1 have been found to have a shorter median time from diagnosis to therapy and shorter median survival than patients with CLL cells that express low-to-negligible amounts of ROR1 (Cui B. et al., Blood, 128:2931, 2016). In prior studies we found that Wnt5a could induce ROR1 to recruit DOCK2 at proline 808 and activate Rac1/2, leading to enhanced leukemia-cell proliferation (Hasan M.K. et al., Blood, 132:170, 2018). Moreover, Wnt5a-enhanced proliferation could be inhibited by cirmtuzumab, a first-in-class humanized mAb specific for a functional epitope of the ROR1; this mAb is undergoing phase II clinical testing in patients with CLL (Choi MY, et al, Cell Stem Cell22:951, 2018). However, the molecular mechanism(s) of ROR1/DOCK2 signaling in the activation of Rac1/2, or downstream signaling molecules such as ERK, or AKT remains elusive. Here, we report that Wnt5a can induce tyrosine phosphorylation of DOCK2 in serum-starved primary CLL cells. The capacity of Wnt5a to induce DOCK2 phosphorylation could be inhibited by cirmtuzumab, but not by ibrutinib, even when used at concentrations that exceeded those required to inhibit B-cell receptor signaling. Phosphorylation of DOCK2 was associated with activation of ERK1/2. Furthermore, siRNA mediated knockdown of DOCK2 could inhibit Wnt5a-induced activation of ERK1/2, as did treatment of the CLL cells with cirmtuzumab. We corroborated these findings in CLL-cell line MEC1, which expresses endogenous Wnt5a but lacks expression of ROR1. We stably transfected ROR1 into MEC1 cells to generate MEC1-ROR1 cells. We found higher-levels phosphorylated DOCK2 and activation of ERK1/2 in MEC1-ROR1 cells than in MEC1 cells lacking ROR1. Treatment of MEC1-ROR1 cells with neutralizing antibodies to Wnt5a or cirmtuzumab suppresed DOCK2 phosphorylation and activation of ERK1/2. Moreover, siRNA mediated knockdown of DOCK2 reduced the level of activation of ERK1/2 in MEC1-ROR1 cells. We examined whether the proline-rich-domain (PRD) of ROR1, and SH3-binding motifs with the PRD that were necessary for these effects. We found that PRD and more specifically proline at 808 position of ROR1 was required to enhance phosphorylation of DOCK2 and activation of ERK1/2 over that seen in MEC1 cells lacking ROR1. We also found that inhibition of Rac using a small molecule inhibitor of Rac1/2 could suppress the capacity of Wnt5a to induce activation of ERK, suggesting that Wnt5a induced activation of Rac was required for activation of ERK. Finally, Wnt5a could not enhance the proliferation of CLL cells when treated with an ERK inhibitor or citmuzumab. Collectively, our results support that Wnt5a/ROR1/DOCK2 signaling axis contributes enhanced activation of ERK to promote proliferation of CLL cells independently of BTK. DisclosuresNo relevant conflicts of interest to declare.

Ligand design for somatostatin receptor isoforms 4 and 5

The somatostatin receptor (SSTR) isoforms, SSTR-4 and SSTR-5 are targets in numerous disorders and diseases. Although there has been some success in achieving selective isoform inhibition, structure-based drug design and development in this area has faced a challenge, mainly attributed to the lack of availability of SSTR-4 and SSTR-5 crystal structures. Previous structure activity relationship (SAR) studies have included work on non-peptide peptidomimetics or β-turn peptidal peptidomimetics where side chains of lysine, tryptophan, and phenylalanine (i.e. functional epitopes) are presented on a scaffold or molecular framework. However, there could be more structural information that would help design ligands selective for one or more of these isoforms. Here, we include synthesis of new mimetics and include their evaluation as ligands for SSTR-4 and SSTR-5. Inhibitors based on small to larger sized scaffolds (ManNAc, iminosugars, Eannaphane macrocycles, acyclic and cyclised peptide structures) are compared. These scaffolds have been grafted with side chains of lysine, tryptophan, and phenylalanine or similar bioisosteres/pharmacophoric groups. A new macrocycle as well as an iminosugar derivative show 5-fold or greater selectivity for SSTR-4 over SSTR-5. A new glycopeptide presenting GlcNAc showed ∼6 fold selectivity for SSTR-5, which contrasted with the non-glycosylated peptide. A number of non-peptide dual inhibitors (Ki values of 0.58 μM to 5 μM) were also identified. Conceivable molecular interactions of these inhibitors were studied with newly constructed homology models of SSTR-4 and SSTR-5 isoforms.

Multifaceted characterization of recombinant protein-based vaccines: An immunochemical toolbox for epitope-specific analyses of the hepatitis E vaccine

The integrity of functional epitopes is a critical quality attribute for recombinant protein based vaccines since the presence of these native-like epitopes is the structural basis for vaccines to elicit functional antibodies. To demonstrate the quality and quantity of functional epitopes on vaccine antigens, a toolbox of assessing antigen characteristics is essential. Among the physicochemical, biophysical, immunochemical and in vivo potency analyses, the epitope-specific assays are most critical assessment of the antigen functionality. In this study, we used hepatitis E virus vaccine as an example to illustrated how the monoclonal antibody (mAb) based immunochemical assays were established for in-depth and multifaceted antigen characterization. A large panel of mAbs were developed and characterized using epitope clustering analysis. A subset of these mAbs recognizing non-overlapping epitopes were chosen to be used for assay development. Orthogonal methods, including surface plasma resonance-based BIAcore, solution competitive ELISA and sandwich ELISA, were developed for the antigenicity assessment. The sandwich ELISA with a pair of mAbs, recognizing two different epitopes, was used to assess the accelerated antigen stability, showing enhanced stability with adjuvant adsorption. Such a sandwich ELISA with robust performance has the potentials to be used for in vitro potency analysis to replace animal-based potency assay as product release test. In summary, using hepatitis E vaccine as an example, we demonstrated the importance and establishment of a mAb-based immunochemical toolbox for multifaceted antigen characterization. This is particularly important to demonstrate the successful reconstruction of the native-like and functional epitopes on a recombinant antigen post expression and purification. These epitope-specific and multifaceted assays serve as critical tools for process monitoring or lot consistency tests in support of vaccine development and manufacturing.

Computational Design of Epitope-Specific Functional Antibodies

The ultimate goal of protein design is to introduce new biological activity. We propose a computational approach for designing functional antibodies by focusing on functional epitopes, integrating large-scale statistical analysis with multiple structural models. Machine learning is used to analyze these models and predict specific residue-residue contacts. We use this approach to design a functional antibody to counter the proinflammatory effect of the cytokine interleukin-17A (IL-17A). X-ray crystallography confirms that the designed antibody binds the targeted epitope and the interaction is mediated by the designed contacts. Cell-based assays confirm that the antibody is functional. Importantly, this approach does not rely on a high-quality 3D model of the designed complex or even a solved structure of the target. As demonstrated here, this approach can be used to design biologically active antibodies, removing some of the main hurdles in antibody design and in drug discovery.

Identification of amino acid residues critical for the B cell growth-promoting activity of HIV-1 matrix protein p17 variants

BackgroundHIV-1 matrix protein p17 variants (vp17s) detected in HIV-1-infected patients with non-Hodgkin's lymphoma (HIV-NHL) display, differently from the wild-type protein (refp17), B cell growth-promoting activity. Biophysical analysis revealed that vp17s are destabilized as compared to refp17, motivating us to explore structure-function relationships. MethodsWe used: biophysical techniques (circular dichroism (CD), nuclear magnetic resonance (NMR) and thermal/GuHCL denaturation) to study protein conformation and stability; Surface plasmon resonance (SPR) to study interactions; Western blot to investigate signaling pathways; and Colony Formation and Soft Agar assays to study B cell proliferation and clonogenicity. ResultsBy forcing the formation of a disulfide bridge between Cys residues at positions 57 and 87 we obtained a destabilized p17 capable of promoting B cell proliferation. This finding prompted us to dissect refp17 to identify the functional epitope. A synthetic peptide (F1) spanning from amino acid (aa) 2 to 21 was found to activate Akt and promote B cell proliferation and clonogenicity. Three positively charged aa (Arg15, Lys18 and Arg20) proved critical for sustaining the proliferative activity of both F1 and HIV-NHL-derived vp17s. Lack of any interaction of F1 with the known refp17 receptors suggests an alternate one involved in cell proliferation. ConclusionsThe molecular reasons for the proliferative activity of vp17s, compared to refp17, relies on the exposure of a functional epitope capable of activating Akt. General significanceOur findings pave the way for identifying the receptor(s) responsible for B cell proliferation and offer new opportunities to identify novel treatment strategies in combating HIV-related NHL.

Autoantibodies against aquaporin-5, a novel biomarker of primary Sjögren's syndrome

The pathophysiology of exocrine dysfunction observed in Sjögren's syndrome (SS) is not fully understood. We identified autoantibodies against human AQP5 in the sera of primary SS patients. The screening of 53 control and 112 SS samples by an indirect immunofluorescence (IIF) assay using the AQP5-expressing MDCK cells revealed the presence of significantly higher levels of anti-AQP5 IgG in the SS samples than in the control samples with sensitivity of 0.73 and a specificity of 0.68. Furthermore, the presence of anti-AQP5 autoantibodies was associated with low resting salivary flow in SS patients. Subsequently, epitope analysis of the anti-AQP5 autoantibodies was performed by IIF assay in the presence or absence of peptides corresponding to the second transmembrane helix and extracellular loops A, C and E of AQP5. Overall, SS samples (n = 27) were inhibited by peptides corresponding to A, C, and E by approximately 40 to 50%, whereas control samples (n = 14) were inhibited only by peptides corresponding to E by less than 20%. A cyclized peptide mimicking E(E1) was most frequently recognized and best differentiated between SS and control samples. To determine functional epitopes, the effects of purified IgG and neutralizing peptides on the transepithelial osmotic permeability (PfT) of MDCK cells expressing AQP5 were measured. Incubation of MDCK-AQP5 cells with SS-, but not with control-IgG, significantly decreased PfT, which was reversed by the neutralization of IgG binding to any of the extracellular loops. In conclusion, antiAQP5 autoantibodies may be a potential biomarker and therapeutic target in SS.

Vaccine based on antibody-dependent cell-mediated cytotoxicity epitope on the H1N1 influenza virus increases mortality in vaccinated mice

Antibody-dependent cell-mediated cytotoxicity bridges humoral immunity and cellular immunity. Thus vaccine candidates which can elicit both broadly neutralizing antibodies and potent antibody-dependent cell-mediated cytotoxicity (ADCC) are recommended. Previously, a panel of functional epitopes that can elicit ADCC effects is isolated and characterized on the H1N1 Influenza Virus. Based on these identified epitopes, an epitope vaccine against H1N1 infection has been designed. The serum of vaccine immunized mice show potent ADCC activities in comparison with vector control group and HA ecto domain vaccinated group. However, the release of IL-6 and TNFα is higher in lung of epitope vaccine immunized mice. The viral load is also higher in epitope vaccine immunized mice. In addition, the epitope vaccine immunized mice showed lower survive rate than both empty vector immunized mice and HA ectodomain immunized mice. Passive transfer of serum from epitope vaccine immunized mice to healthy adult mice can decrease the survival rate of recipients after viral challenge. Our data suggested that ADCC epitope based vaccine has a mortality promoting effect rather than protective effect after H1N1 viral challenge. This result provides indications in future vaccine design with a consideration of balancing humoral immune response and cellular immune response.

Complete functional mapping of infection- and vaccine-elicited antibodies against the fusion peptide of HIV.

Eliciting broadly neutralizing antibodies (bnAbs) targeting envelope (Env) is a major goal of HIV vaccine development, but cross-clade breadth from immunization has only sporadically been observed. Recently, Xu et al (2018) elicited cross-reactive neutralizing antibody responses in a variety of animal models using immunogens based on the epitope of bnAb VRC34.01. The VRC34.01 antibody, which was elicited by natural human infection, targets the N terminus of the Env fusion peptide, a critical component of the virus entry machinery. Here we precisely characterize the functional epitopes of VRC34.01 and two vaccine-elicited murine antibodies by mapping all single amino-acid mutations to the BG505 Env that affect viral neutralization. While escape from VRC34.01 occurred via mutations in both fusion peptide and distal interacting sites of the Env trimer, escape from the vaccine-elicited antibodies was mediated predominantly by mutations in the fusion peptide. Cryo-electron microscopy of four vaccine-elicited antibodies in complex with Env trimer revealed focused recognition of the fusion peptide and provided a structural basis for development of neutralization breadth. Together, these functional and structural data suggest that the breadth of vaccine-elicited antibodies targeting the fusion peptide can be enhanced by specific interactions with additional portions of Env. Thus, our complete maps of viral escape both delineate pathways of resistance to these fusion peptide-directed antibodies and provide a strategy to improve the breadth or potency of future vaccine-induced antibodies against Env’s fusion peptide.

Abstract 5620: Multispecific antibodies targeting CD38 and PD-L1 show potent tumor cytotoxicity

Abstract Multivalent antibodies targeting either CD38 alone or CD38 in conjunction with PD-L1 may yield therapeutics with superior biologic activities and provide benefit for treating malignancies expressing low levels of CD38 (DLCBL, daratumumab refractory MM, and NSCLC). Multivalent, multispecific antibodies kill CD38low cells through a variety of mechanisms including stronger and more specific engagement of CD38. Potent and directed immune checkpoint inhibition is realized by adding an anti-PD-L1 binding domain. Teneobio's discovery platform utilizes VH domains (UniDabs) of fully human heavy chain antibodies (UniAbs) to develop bi-, tri-, and tetravalent antibodies. Individual UniDabs targeting CD38 and PDL1 were identified using our unique sequence-based discovery platform and high-throughput lead evaluation pipeline (TeneoSeek). This robust screening workflow enables evaluation of a large diversity of natural fully human antibodies, targeting multiple epitopes on a single antigen and uncovering important sequence activity relationships. We have identified UniDabs that bind five different functional epitopes on human, cynomolgus and mouse CD38 including potent inhibitors of hydrolase and cyclase enzymatic functions. In addition, we generated a large panel of PD-L1 inhibitors that bind with high affinities human and cynomolgus PD-L1. Using different combinations and arrangements of UniDabs, a variety of multivalent antibodies were constructed and evaluated in in vitro models. Data from a range of assay types show that multivalent UniAbs targeting CD38 can be engineered to display superior tumor cell cytotoxicity through multiple mechanisms of action. Summary: Teneobio's discovery platform utilizes VH domains (UniDabs) of fully human heavy chain antibodies (UniAbs) to develop bi-, tri-, and tetravalent antibodies. Multivalent UniAbs targeting CD38 and PD-L1 can be engineered to display superior tumor cell cytotoxicity through multiple mechanisms of action. Citation Format: Wim van Schooten. Multispecific antibodies targeting CD38 and PD-L1 show potent tumor cytotoxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5620.

Abstract 1729: Integrative immune profiling of syngeneic tumor models provides predictive immune signatures for treatment response with HMBD002, a novel anti-VISTA neutralizing antibody

Abstract Myeloid-derived suppressor cells (MDSCs) are a group of immature myeloid cells with potent immune suppressive activity. Accumulation of pathologically activated MDSCs within the tumor microenvironment has emerged as an important mechanism of primary and acquired resistance to immunotherapy. MDSCs have high surface expression levels of V-domain immunoglobulin (Ig)-containing suppressor of T-cell activation (VISTA), a co-inhibitory negative checkpoint regulator (NCR). Immune suppression by VISTA+ MDSCs has been implicated in the failure of, and resistance to, anti-PD1/PDL1 therapy and represents a unique axis for NCR targeting in the non-responder population. Elucidating the dynamics of VISTA+ MDSCs within tumors is imperative for predicting response and patient stratification. HMBD-002 is a novel anti-VISTA neutralizing monoclonal antibody, developed using Hummingbird Bioscience's proprietary Rational Antibody Discovery platform, against a specific functional epitope on VISTA predicted to be involved in ligand binding. HMBD002 binds human, cyno and murine VISTA protein with high affinity (kD <5nM) and demonstrated a strong, dose dependent inhibition for the interaction of VISTA with its putative ligand VSIG3. In ex vivo PBMC models of immune activation, HMBD002 treatment showed potent induction of cytokine release. Further, HMBD002 monotherapy in a CT26 syngeneic BALB/C model showed significant delay on tumor progression with no observable toxicity. To investigate predictive immune signatures for HMBD002 antibody efficacy, as a monotherapy and in combination with anti- PD-L1 therapy, we established multiple mouse models of tumor immune subtypes using syngeneic cell lines. Tumor biopsies were analyzed before and during treatment, to determine the evolution of immune response and its effect on tumor burden within responders and non-responders. The combination of HMBD-002 and anti-PD-L1 was more effective than either antibody alone, especially in tumors that showed abundant MDSC infiltration. Immune phenotyping revealed that this was associated with a significant increase in activated CD8+ T effector cells and concomitant increase in IFN-gamma. Profiling syngeneic models for deeper insights into the interplay between the tumor, immune system and therapy, will facilitate the clinical development of immune checkpoint and combination therapies. Hummingbird Bioscience anticipates commencing First-in-Human trials of HMBD002 in 2019. Citation Format: Jerome D. Boyd-Kirkup, Dipti Thakkar, Konrad Paszkiewicz, Piers J. Ingram. Integrative immune profiling of syngeneic tumor models provides predictive immune signatures for treatment response with HMBD002, a novel anti-VISTA neutralizing antibody [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1729.