B cell-reactive triad of B cells, follicular helper and regulatory T cells at homeostasis.

Autoreactive B cells are silenced through receptor editing, clonal deletion and anergy induction. Additional autoreactive B cells are ignorant because of physical segregation from their cognate autoantigen. Unexpectedly, we find that follicular B cell-derived autoantigen, including cell surface molecules such as FcγRIIB, is a class of homeostatic autoantigen that can induce spontaneous germinal centers (GCs) and B cell-reactive autoantibodies in non-autoimmune animals with intact T and B cell repertoires. These B cell-reactive B cells form GCs in a manner dependent on spontaneous follicular helper T (TFH) cells, which preferentially recognize B cell-derived autoantigen, and in a manner constrained by spontaneous follicular regulatory T (TFR) cells, which also carry specificities for B cell-derived autoantigen. B cell-reactive GC cells are continuously generated and, following immunization or infection, become intermixed with foreign antigen-induced GCs. Production of plasma cells and antibodies derived from B cell-reactive GC cells are markedly enhanced by viral infection, potentially increasing the chance for autoimmunity. Consequently, immune homeostasis in healthy animals not only involves classical tolerance of silencing and ignoring autoreactive B cells but also entails a reactive equilibrium attained by a spontaneous B cell-reactive triad of B cells, TFH cells and TFR cells.

Nasal spray of an IgM-like ACE2 fusion protein HH-120 prevents SARS-CoV-2 infection: Two investigator-initiated postexposure prophylaxis trials.

HH-120, an IgM-like angiotensin converting enzyme 2 (ACE2) fusion protein, has been developed as a nasal spray against Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and is currently undergoing human trials. HH-120 nasal spray was assessed for postexposure prophylaxis (PEP) in two investigator-initiated (NS01 and NS02) trials with different risk levels of SARS-CoV-2 exposure. NS01 enrolled family caregiver participants who had continuous contacts with laboratory-confirmed index cases; NS02 enrolled participants who had general contacts (Part 1) or close contacts (Part 2) with index cases. The primary endpoints were safety and laboratory-confirmed and/or symptomatic SARS-CoV-2 infection. In NS01 trial (14 participants), the SARS-CoV-2 infection rates were 25% in the HH-120 group and 83.3% in the external control group (relative risk reduction [RRR]: 70.0%). In NS02-Part 1 (193 participants), the infection rates were 4% (HH-120) versus 11.3% (placebo), symptomatic infection rates were 0.8% versus 3.5%, hence with a RRR of 64.6% and 77.1%, respectively. In Part 2 (76 participants), the infection rates were 17.1% (HH-120) versus 30.4% (placebo), symptomatic infection rates were 7.5% versus 27.3%, with a RRR of 43.8% and 72.5%, respectively. No HH-120-related serious adverse effects were observed. The HH-120 nasal spray used as PEP was safe and effective in preventing laboratory-confirmed and symptomatic SARS-CoV-2 infection.

An IgM-like inhalable ACE2 fusion protein broadly neutralizes SARS-CoV-2 variants.

Many of the currently available COVID-19 vaccines and therapeutics are not effective against newly emerged SARS-CoV-2 variants. Here, we developed the metallo-enzyme domain of angiotensin converting enzyme 2 (ACE2)-the cellular receptor of SARS-CoV-2-into an IgM-like inhalable molecule (HH-120). HH-120 binds to the SARS-CoV-2 Spike (S) protein with high avidity and confers potent and broad-spectrum neutralization activity against all known SARS-CoV-2 variants of concern. HH-120 was developed as an inhaled formulation that achieves appropriate aerodynamic properties for rodent and monkey respiratory system delivery, and we found that early administration of HH-120 by aerosol inhalation significantly reduced viral loads and lung pathology scores in male golden Syrian hamsters infected by the SARS-CoV-2 ancestral strain (GDPCC-nCoV27) and the Delta variant. Our study presents a meaningful advancement in the inhalation delivery of large biologics like HH-120 (molecular weight (MW) ~ 1000 kDa) and demonstrates that HH-120 can serve as an efficacious, safe, and convenient agent against SARS-CoV-2 variants. Finally, given the known role of ACE2 in viral reception, it is conceivable that HH-120 has the potential to be efficacious against additional emergent coronaviruses.

PreS1- targeting chimeric antigen receptor T cells diminish HBV infection in liver humanized FRG mice.

Current therapies control but rarely achieve a cure for hepatitis B virus (HBV) infection. Restoration of the HBV-specific immunity by cell-based therapy represents a potential approach for a cure. In this study, we generated HBV specific CAR T cells based on an antibody 2H5-A14 targeting a preS1 region of the HBV large envelope protein. We show that the A14 CAR T cell is capable of killing hepatocytes infected by HBV with high specificity; adoptive transfer of A14 CAR T cells to HBV infected humanized FRG mice resulted in reductions of all serum and intrahepatic virological markers to levels below the detection limit. A14 CAR T cells treatment increased the levels of human IFN-γ, GM-CSF, and IL-8/CXCL-8 in the mice. These results show that A14 CAR T cells may be further developed for curative therapy against HBV infection by eliminating HBV-infected hepatocytes and inducing production of pro-inflammatory and antiviral cytokines.

Nasal spray of an IgM-like ACE2 fusion protein HH-120 accelerates SARS-CoV-2 clearance: A single-center propensity score-matched cohort study.

HH-120, a recently developed IgM-like ACE2 fusion protein with broad-spectrum neutralizing activity against all ACE2-utilizing coronaviruses, has been developed as a nasal spray for use as an early treatment agent to reduce disease progression and airborne transmission. The objective of this study was to evaluate the safety and efficacy of the HH-120 nasal spray in SARS-CoV-2-infected subjects. Eligible symptomatic or asymptomatic SARS-CoV-2-infected participants were enrolled in a single-arm trial to receive the HH-120 nasal spray for no longer than 6 days or until viral clearance at a single hospital between August 3 and October 7, 2022. An external control was built from real-world data of SARS-CoV-2-infected subjects contemporaneously hospitalized in the same hospital using a propensity score matching (PSM) method. After PSM, 65 participants in the HH-120 group and 103 subjects with comparable baseline characteristics in the external control group were identified. The viral clearance time was significantly shorter in participants receiving the HH-120 nasal spray than that in subjects of the control group (median 8 days vs. 10 days, p < 0.001); the difference was more prominent in those subgroup subjects with higher baseline viral load (median 7.5 days vs. 10.5 days, p < 0.001). The incidence of treatment-emergent adverse events and treatment-related adverse events of HH-120 group were 35.1% (27/77) and 3.9% (3/77), respectively. All the adverse events observed were mild, being of CTCAE grade 1 or 2, and transient. The HH-120 nasal spray showed a favorable safety profile and promising antiviral efficacy in SARS-CoV-2-infected subjects. The results from this study warrant further assessment of the efficacy and safety of the HH-120 nasal spray in large-scale randomized controlled clinical trials.

A pH-dependent anti-CD47 antibody that selectively targets solid tumors and improves therapeutic efficacy and safety.

BACKGROUND: The antiphagocytic molecule CD47 is overexpressed in a wide variety of cancer cells, and antibodies targeting CD47 for cancer therapies are currently under intensive investigation. However, owing to the ubiquitous expression of CD47 on healthy cells, anti-CD47 therapies often achieve only weak therapeutic benefits and can induce severe side effects. Here, we report the generation of a pH-dependent anti-CD47 antibody (BC31M4) which selectively binds to tumors under the acidic solid tumor microenvironment. METHODS: BC31M4 was generated using antibody phage display and a pH-dependent selection strategy. The pH-dependent binding and blocking activities of BC31M4 were verified using in vitro assays, and the structural basis of the pH-dependent binding property was characterized. BC31M4's antitumor effect was confirmed by both phagocytosis assays and studies in xenograft models. The tumor selectivity, mechanism of action, PK properties, side effects, and therapeutic efficacy were further evaluated in humanized (hCD47 and its receptor hSIRPα) immunocompetent syngeneic mouse models. RESULTS: The crystal structure reveals that two histidines locate within the CDRs of the light chain directly contribute to the pH-dependent binding of BC31M4. BC31M4 promotes macrophage phagocytosis of tumor cells more potently at acidic-pH than at physiological-pH. Our hCD47/hSIRPα humanized syngeneic mouse model results demonstrated that BC31M4 selectively accumulates in tumors but not in normal tissues. BC31M4 causes minimal side effects and exhibits superior PK properties as compared to the other examined anti-CD47 antibodies. When combined with adoptive T cell transfer, BC31M4 efficiently promotes adaptive immune responses against tumors and also induces immune memory. Moreover, we show that BC31M4's antitumor effects rely on an Fc that mediates strong effector functions. CONCLUSIONS: Our study illustrates that the development of a tumor-selective, pH-dependent anti-CD47 antibody safely confers strong therapeutic effects against solid tumors, thus providing a promising therapeutic strategy to overcome the challenges of anti-CD47 therapy.

An anti-CD98 antibody displaying pH-dependent Fc-mediated tumour-specific activity against multiple cancers in CD98-humanized mice.

The cell-surface glycoprotein CD98-a subunit of the LAT1/CD98 amino acid transporter-is an attractive target for cancer immunotherapies, but its widespread expression has hampered the development of CD98-targeting antibody therapeutics. Here we report that an anti-CD98 antibody, identified via the screening of phage-display libraries of CD98 single-chain variable fragments with mutated complementarity-determining regions, preserves the physiological function of CD98 and elicits broad-spectrum crystallizable-fragment (Fc)-mediated anti-tumour activity (requiring Fcγ receptors for immunoglobulins, macrophages, dendritic cells and CD8+ T cells, as well as other components of the innate and adaptive immune systems) in multiple xenograft and syngeneic tumour models established in CD98-humanized mice. We also show that a variant of the anti-CD98 antibody with pH-dependent binding, generated by solving the structure of the antibody-CD98 complex, displayed enhanced tumour-specific activity and pharmacokinetics. pH-dependent antibody variants targeting widely expressed antigens may lead to superior therapeutic outcomes.

A cross-reactive pH-dependent EGFR antibody with improved tumor selectivity and penetration obtained by structure-guided engineering.

The clinical use of anti-EGFR antibody-based cancer therapy has been limited by antibody-EGFR binding in normal tissues, so developing pH-dependent anti-EGFR antibodies that selectively bind with EGFR in tumors-by taking advantage of the acidity of tumor microenvironment relative to normal tissues-may overcome these limitations. Here, we generated pH-dependent anti-EGFR antibodies with cross-species reactivity for human and mouse EGFR, and we demonstrate that pH-dependent antibodies exhibit tumor-selective binding by binding strongly to EGFR under acidic conditions (pH 6.5) but binding weakly under neutral (pH 7.4) conditions. Based on screening a non-immune human antibody library and antibody affinity maturation, we initially generated antibodies with cross-species reactivity for human and mouse EGFR. A structure model was subsequently constructed and interrogated for hotspots affecting pH-dependent binding, which supported development of a cross-reactive pH-dependent anti-EGFR antibody, G532. Compared with its non-pH-dependent antibody variant, G532 exhibits improved tumor selectivity, tumor penetration, and antitumor activity. Thus, beyond showing that pH-dependent anti-EGFR antibodies can overcome multiple limitations with antibody-based cancer therapies targeting EGFR, our study illustrates a structure-guided antibody-antigen binding pH-dependency engineering strategy to enhance antibody tumor selectivity and tumor penetration, which can inform the future development of antibody-based cancer therapies targeting other ubiquitously expressed molecules.

Structural mechanism of protein recognition by the FW domain of autophagy receptor Nbr1.

Neighbor of BRCA1 (Nbr1) is a conserved autophagy receptor that provides cargo selectivity to autophagy. The four-tryptophan (FW) domain is a signature domain of Nbr1, but its exact function remains unclear. Here, we show that Nbr1 from the filamentous fungus Chaetomium thermophilum uses its FW domain to bind the α-mannosidase Ams1, a cargo of selective autophagy in both budding yeast and fission yeast, and delivers Ams1 to the vacuole by conventional autophagy in heterologous fission yeast. The structure of the Ams1-FW complex was determined at 2.2 Å resolution by cryo-electron microscopy. The FW domain adopts an immunoglobulin-like ß-sandwich structure and recognizes the quaternary structure of the Ams1 tetramer. Notably, the N-terminal di-glycine of Ams1 is specifically recognized by a conserved pocket of the FW domain. The FW domain becomes degenerated in fission yeast Nbr1, which binds Ams1 with a ZZ domain instead. Our findings illustrate the protein binding mode of the FW domain and reveal the versatility of Nbr1-mediated cargo recognition.

Anatomically distinct fibroblast subsets determine skin autoimmune patterns.

The skin serves as a physical barrier and an immunological interface that protects the body from the external environment1-3. Aberrant activation of immune cells can induce common skin autoimmune diseases such as vitiligo, which are often characterized by bilateral symmetric lesions in certain anatomic regions of the body4-6. Understanding what orchestrates the activities of cutaneous immune cells at an organ level is necessary for the treatment of autoimmune diseases. Here we identify subsets of dermal fibroblasts that are responsible for driving patterned autoimmune activity, by using a robust mouse model of vitiligo that is based on the activation of endogenous auto-reactive CD8+ T cells that target epidermal melanocytes. Using a combination of single-cell analysis of skin samples from patients with vitiligo, cell-type-specific genetic knockouts and engraftment experiments, we find that among multiple interferon-γ (IFNγ)-responsive cell types in vitiligo-affected skin, dermal fibroblasts are uniquely required to recruit and activate CD8+ cytotoxic T cells through secreted chemokines. Anatomically distinct human dermal fibroblasts exhibit intrinsic differences in the expression of chemokines in response to IFNγ. In mouse models of vitiligo, regional IFNγ-resistant fibroblasts determine the autoimmune pattern of depigmentation in the skin. Our study identifies anatomically distinct fibroblasts with permissive or repressive IFNγ responses as the key determinant of body-level patterns of lesions in vitiligo, and highlights mesenchymal subpopulations as therapeutic targets for treating autoimmune diseases.

Transcriptionally inactive hepatitis B virus episome DNA preferentially resides in the vicinity of chromosome 19 in 3D host genome upon infection.

The hepatitis B virus (HBV) infects 257 million people worldwide. HBV infection requires establishment and persistence of covalently closed circular (ccc) DNA, a viral episome, in nucleus. Here, we study cccDNA spatial localization in the 3D host genome by using chromosome conformation capture-based sequencing analysis and fluorescence in situ hybridization (FISH). We show that transcriptionally inactive cccDNA is not randomly distributed in host nucleus. Rather, it is preferentially accumulated at specialized areas, including regions close to chromosome 19 (chr.19). Activation of the cccDNA is apparently associated with its re-localization, from a pre-established heterochromatin hub formed by 5 regions of chr.19 to transcriptionally active regions formed by chr.19 and nearby chromosomes including chr.16, 17, 20, and 22. This active versus inactive positioning at discrete regions of the host genome is primarily controlled by the viral HBx protein and by host factors including the structural maintenance of chromosomes protein 5/6 (SMC5/6) complex.

Molecular and structural mechanisms of ZZ domain-mediated cargo selection by Nbr1.

In selective autophagy, cargo selectivity is determined by autophagy receptors. However, it remains scarcely understood how autophagy receptors recognize specific protein cargos. In the fission yeast Schizosaccharomyces pombe, a selective autophagy pathway termed Nbr1-mediated vacuolar targeting (NVT) employs Nbr1, an autophagy receptor conserved across eukaryotes including humans, to target cytosolic hydrolases into the vacuole. Here, we identify two new NVT cargos, the mannosidase Ams1 and the aminopeptidase Ape4, that bind competitively to the first ZZ domain of Nbr1 (Nbr1-ZZ1). High-resolution cryo-EM analyses reveal how a single ZZ domain recognizes two distinct protein cargos. Nbr1-ZZ1 not only recognizes the N-termini of cargos via a conserved acidic pocket, similar to other characterized ZZ domains, but also engages additional parts of cargos in a cargo-specific manner. Our findings unveil a single-domain bispecific mechanism of autophagy cargo recognition, elucidate its underlying structural basis, and expand the understanding of ZZ domain-mediated protein-protein interactions.

Rab22a-NeoF1 fusion protein promotes osteosarcoma lung metastasis through its secretion into exosomes.

It remains unknown for decades how some of the therapeutic fusion proteins positive in a small percentage of cancer cells account for patient outcome. Here, we report that osteosarcoma Rab22a-NeoF1 fusion protein, together with its binding partner PYK2, is sorted into exosomes by HSP90 via its KFERQ-like motif (RVLFLN142). The exosomal Rab22a-NeoF1 fusion protein facilitates the pulmonary pre-metastatic niche formation by recruiting bone marrow-derived macrophages. The exosomal PYK2 activates RhoA in its negative recipient osteosarcoma cells and induces signal transducer and activator of transcription 3 activation in its recipient macrophages to increase M2 phenotype. Consequently, lung metastases of its recipient osteosarcoma cells are promoted by this exosomal Rab22a-NeoF1 fusion protein, and this event can be targeted by disrupting its interaction with PYK2 using a designed internalizing RGD peptide.

A histone H3K27me3 reader cooperates with a family of PHD finger-containing proteins to regulate flowering time in Arabidopsis.

Trimethylated histone H3 lysine 27 (H3K27me3) is a repressive histone marker that regulates a variety of developmental processes, including those that determine flowering time. However, relatively little is known about the mechanism of how H3K27me3 is recognized to regulate transcription. Here, we identified BAH domain-containing transcriptional regulator 1 (BDT1) as an H3K27me3 reader. BDT1 is responsible for preventing flowering by suppressing the expression of flowering genes. Mutation of the H3K27me3 recognition sites in the BAH domain disrupted the binding of BDT1 to H3K27me3, leading to de-repression of H3K27me3-enriched flowering genes and an early-flowering phenotype. We also found that BDT1 interacts with a family of PHD finger-containing proteins, which we named PHD1-6, and with CPL2, a Pol II carboxyl terminal domain (CTD) phosphatase responsible for transcriptional repression. Pull-down assays showed that the PHD finger-containing proteins can enhance the binding of BDT1 to the H3K27me3 peptide. Mutations in all of the PHD genes caused increased expression of flowering genes and an early-flowering phenotype. This study suggests that the binding of BDT1 to the H3K27me3 peptide, which is enhanced by PHD proteins, is critical for preventing early flowering.

A structure of human Scap bound to Insig-2 suggests how their interaction is regulated by sterols.

The sterol regulatory element-binding protein (SREBP) pathway controls cellular homeostasis of sterols. The key players in this pathway, Scap and Insig-1 and -2, are membrane-embedded sterol sensors. The 25-hydroxycholesterol (25HC)-dependent association of Scap and Insig acts as the master switch for the SREBP pathway. Here, we present cryo-electron microscopy analysis of the human Scap and Insig-2 complex in the presence of 25HC, with the transmembrane (TM) domains determined at an average resolution of 3.7 angstrom. The sterol-sensing domain in Scap and all six TMs in Insig-2 were resolved. A 25HC molecule is sandwiched between the S4 to S6 segments in Scap and TMs 3 and 4 in Insig-2 in the luminal leaflet of the membrane. Unwinding of the middle of the Scap-S4 segment is crucial for 25HC binding and Insig association.

A bispecific antibody targeting GPC3 and CD47 induced enhanced antitumor efficacy against dual antigen-expressing HCC.

Glypican-3 (GPC3) is a well-characterized hepatocellular carcinoma (HCC)-associated antigen, yet anti-GPC3 therapies have achieved only minimal clinical progress. CD47 is a ubiquitously expressed innate immune checkpoint that promotes evasion of tumors from immune surveillance. Given both the specific expression of GPC3 in HCC and the known phagocytosis inhibitory effect of CD47 in liver cancer, we hypothesized that a bispecific antibody (BsAb) that co-engages with GPC3 and CD47 may offer excellent antitumor efficacy with minimal toxicity. Here, we generated a novel BsAb: GPC3/CD47 biAb. With the use of both in vitro and in vivo assays, we found that GPC3/CD47 biAb exerts strong antitumor activity preferentially against dual antigen-expressing tumor cells. In hCD47/human signal regulatory protein alpha (hCD47/hSIRPα) humanized mice, GPC3/CD47 biAb had an extended serum half-life without causing systemic toxicity. Importantly, GPC3/CD47 biAb induced enhanced Fc-mediated effector functions to dual antigen-expressing HCC cells in vitro, and both macrophages and neutrophils are required for its strong efficacy against xenograft HCC tumors. Notably, GPC3/CD47 biAb outperformed monotherapies and a combination therapy with anti-CD47 and anti-GPC3 monoclonal antibodies (mAbs) in a xenograft HCC model. Our study illustrates a strategy for improving HCC treatment by boosting innate immune responses and presents new insights to inform antibody design for the future development of innovative immune therapies.

A Cross-Species Reactive TIGIT-Blocking Antibody Fc Dependently Confers Potent Antitumor Effects.

The T cell immunoreceptor with Ig and ITIM domains (TIGIT) has been shown to exert inhibitory roles in antitumor immune responses. In this study, we report the development of a human mAb, T4, which recognizes both human and mouse TIGIT and blocks the interaction of TIGIT with its ligand CD155 in both species. The T4 Ab targets the segment connecting F and G strands of TIGIT's extracellular IgV domain, and we show in studies with mouse tumor models that the T4 Ab exerts strong antitumor activity and induces durable immune memory against various tumor types. Mechanistically, we demonstrate that the T4 Ab's antitumor effects are mediated via multiple immunological impacts, including a CD8+ T immune response and Fc-mediated effector functions, through NK cells that cause significant reduction in the frequency of intratumoral T regulatory cells (Tregs). Notably, this Treg reduction apparently activates additional antitumor CD8+ T cell responses, targeting tumor-shared Ags that are normally cryptic or suppressed by Tregs, thus conferring cross-tumor immune memory. Subsequent engineering for Fc variants of the T4 Ab with enhanced Fc-mediated effector functions yielded yet further improvements in antitumor efficacy. Thus, beyond demonstrating the T4 Ab as a promising candidate for the development of cancer immunotherapies, our study illustrates how the therapeutic efficacy of an anti-TIGIT Ab can be improved by enhancing Fc-mediated immune effector functions. Our insights about the multiple mechanisms of action of the T4 Ab and its Fc variants should help in developing new strategies that can realize the full clinical potential of anti-TIGIT Ab therapies.

Autocrine INSL5 promotes tumor progression and glycolysis via activation of STAT5 signaling.

Metabolic reprogramming plays important roles in development and progression of nasopharyngeal carcinoma (NPC), but the underlying mechanism has not been completely defined. In this work, we found INSL5 was elevated in NPC tumor tissue and the plasma of NPC patients. Plasma INSL5 could serve as a novel diagnostic marker for NPC, especially for serum VCA-IgA-negative patients. Moreover, higher plasma INSL5 level was associated with poor disease outcome. Functionally, INSL5 overexpression increased, whereas knockdown of its receptor GPCR142 or inhibition of INSL5 reduced cell proliferation, colony formation, and cell invasion in vitro and tumorigenicity in vivo. Mechanistically, INSL5 enhanced phosphorylation and nuclear translocation of STAT5 and promoted glycolytic gene expression, leading to induced glycolysis in cancer cells. Pharmaceutical inhibition of glycolysis by 2-DG or blockade of INSL5 by a neutralizing antibody reversed INSL5-induced proliferation and invasion, indicating that INSL5 can be a potential therapeutic target in NPC. In conclusion, INSL5 enhances NPC progression by regulating cancer cell metabolic reprogramming and is a potential diagnostic and prognostic marker as well as a therapeutic target for NPC.