Brianna Dalesandro

Adv Mater. 2021 Sep 12:e2103258. doi: 10.1002/adma.202103258. Online ahead of print.

ABSTRACT

Chimeric antigen receptor-T (CAR-T) cell immunotherapy has shown impressive clinical outcomes for hematologic malignancies. However, its broader applications are challenged due to its complex ex vivo cell-manufacturing procedures and low therapeutic efficacy against solid tumors. The limited therapeutic effects are partially due to limited CAR-T cell infiltration to solid tumors and inactivation of CAR-T cells by the immunosuppressive tumor microenvironment. Here, a facile approach is presented to in vivo program macrophages, which can intrinsically penetrate solid tumors, into CAR-M1 macrophages displaying enhanced cancer-directed phagocytosis and anti-tumor activity. In vivo injected nanocomplexes of macrophage-targeting nanocarriers and CAR-interferon-γ-encoding plasmid DNA induce CAR-M1 macrophages that are capable of CAR-mediated cancer phagocytosis, anti-tumor immunomodulation, and inhibition of solid tumor growth. Together, this study describes an off-the-shelf CAR-macrophage therapy that is effective for solid tumors and avoids the complex and costly processes of ex vivo CAR-cell manufacturing.

PMID:34510559 | DOI:10.1002/adma.202103258

Biol Chem. 2021 Sep 10. doi: 10.1515/hsz-2021-0243. Online ahead of print.

ABSTRACT

The gold standard for the diagnosis of bacterial infections in clinical samples is based on culture tests that are time-consuming and labor-intense. For these reasons, an extraordinary effort has been made to identify biomarkers as the tools for sensitive, rapid and accurate identification of pathogenic microorganisms. Moreover, biomarkers have been tested to distinguish colonization from infection, monitor disease progression, determine the clinical status of patients or predict clinical outcomes. This mini-review describes Pseudomonas aeruginosa and Staphylococcus aureus biomarkers, which contribute to pathogenesis and have been used in culture-independent bacterial identification directly from patient samples.

PMID:34505460 | DOI:10.1515/hsz-2021-0243

Proc Natl Acad Sci U S A. 2021 Aug 31;118(35):e2108894118. doi: 10.1073/pnas.2108894118.

ABSTRACT

A cell wall made of the heteropolymer peptidoglycan (PG) surrounds most bacterial cells. This essential surface layer is required to prevent lysis from internal osmotic pressure. The class A penicillin-binding proteins (aPBPs) play key roles in building the PG network. These bifunctional enzymes possess both PG glycosyltransferase (PGT) and transpeptidase (TP) activity to polymerize the wall glycans and cross-link them, respectively. In Escherichia coli and other gram-negative bacteria, aPBP function is dependent on outer membrane lipoproteins. The lipoprotein LpoA activates PBP1a and LpoB promotes PBP1b activity. In a purified system, the major effect of LpoA on PBP1a is TP stimulation. However, the relevance of this activation to the cellular function of LpoA has remained unclear. To better understand why PBP1a requires LpoA for its activity in cells, we identified variants of PBP1a from E. coli and Pseudomonas aeruginosa that function in the absence of the lipoprotein. The changes resulting in LpoA bypass map to the PGT domain and the linker region between the two catalytic domains. Purification of the E. coli variants showed that they are hyperactivated for PGT but not TP activity. Furthermore, in vivo analysis found that LpoA is necessary for the glycan synthesis activity of PBP1a in cells. Thus, our results reveal that LpoA exerts a much greater control over the cellular activity of PBP1a than previously appreciated. It not only modulates PG cross-linking but is also required for its cognate synthase to make PG glycans in the first place.

PMID:34429361 | DOI:10.1073/pnas.2108894118

Front Immunol. 2021 Jul 23;12:669103. doi: 10.3389/fimmu.2021.669103. eCollection 2021.

ABSTRACT

Targeted therapeutics for the treatment of coronavirus disease 2019 (COVID-19), especially severe cases, are currently lacking. As macrophages have unique effector functions as a first-line defense against invading pathogens, we genetically armed human macrophages with chimeric antigen receptors (CARs) to reprogram their phagocytic activity against SARS-CoV-2. After investigation of CAR constructs with different intracellular receptor domains, we found that although cytosolic domains from MERTK (CAR_MERTK) did not trigger antigen-specific cellular phagocytosis or killing effects, unlike those from MEGF10, FcRγ and CD3ζ did, these CARs all mediated similar SARS-CoV-2 clearance in vitro. Notably, we showed that CAR_MERTK macrophages reduced the virion load without upregulation of proinflammatory cytokine expression. These results suggest that CAR_MERTK drives an 'immunologically silent' scavenger effect in macrophages and pave the way for further investigation of CARs for the treatment of individuals with COVID-19, particularly those with severe cases at a high risk of hyperinflammation.

PMID:34367135 | PMC:PMC8343226 | DOI:10.3389/fimmu.2021.669103

Front Immunol. 2021 Jul 23;12:652223. doi: 10.3389/fimmu.2021.652223. eCollection 2021.

ABSTRACT

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is highly contagious and presents a significant public health issue. Current therapies used to treat coronavirus disease 2019 (COVID-19) include monoclonal antibody cocktail, convalescent plasma, antivirals, immunomodulators, and anticoagulants. The vaccines from Pfizer and Moderna have recently been authorized for emergency use, which are invaluable for the prevention of SARS-CoV-2 infection. However, their long-term side effects are not yet documented, and populations with immunocompromised conditions (e.g., organ-transplantation and immunodeficient patients) may not be able to mount an effective immune response. In addition, there are concerns that wide-scale immunity to SARS-CoV-2 may introduce immune pressure that could select for escape mutants to the existing vaccines and monoclonal antibody therapies. Emerging evidence has shown that chimeric antigen receptor (CAR)- natural killer (NK) immunotherapy has potent antitumor response in hematologic cancers with minimal adverse effects in recent studies, however, the potentials of CAR-NK cells in treating COVID-19 has not yet been fully exploited. Here, we improve upon a novel approach for the generation of CAR-NK cells for targeting SARS-CoV-2 and its various mutants. CAR-NK cells were generated using the scFv domain of S309 (henceforward, S309-CAR-NK), a SARS-CoV and SARS-CoV-2 neutralizing antibody (NAbs) that targets the highly conserved region of SARS-CoV-2 spike (S) glycoprotein and is therefore more likely to recognize different variants of SARS-CoV-2 isolates. S309-CAR-NK cells can specifically bind to pseudotyped SARS-CoV-2 virus and its D614G, N501Y, and E484K mutants. Furthermore, S309-CAR-NK cells can specifically kill target cells expressing SARS-CoV-2 S protein in vitro and show superior killing activity and cytokine production, compared to that of the recently reported CR3022-CAR-NK cells. Thus, these results pave the way for generating 'off-the-shelf' S309-CAR-NK cells for treatment in high-risk individuals as well as provide an alternative strategy for patients unresponsive to current vaccines.

PMID:34367128 | PMC:PMC8343231 | DOI:10.3389/fimmu.2021.652223

Int J Mol Sci. 2021 Jul 28;22(15):8108. doi: 10.3390/ijms22158108.

ABSTRACT

Staphylococcus aureus is a commensal bacterium that causes severe infections in soft tissue and the bloodstream. During infection, S. aureus manipulates host cell response to facilitate its own replication and dissemination. Here, we show that S. aureus significantly decreases the level of SUMOylation, an essential post-translational modification, in infected macrophages 24 h post-phagocytosis. The reduced level of SUMOylation correlates with a decrease in the SUMO-conjugating enzyme Ubc9. The over-expression of SUMO proteins in macrophages impaired bacterial intracellular proliferation and the inhibition of SUMOylation with ML-792 increased it. Together, these findings demonstrated for the first time the role of host SUMOylation response toward S. aureus infection.

PMID:34360873 | PMC:PMC8347835 | DOI:10.3390/ijms22158108

J Immunol. 2021 Aug 1;207(3):777-783. doi: 10.4049/jimmunol.2100378. Epub 2021 Jul 16.

ABSTRACT

Bactericidal/permeability-increasing protein (BPI) plays a major role in innate immunity through the ability of the N-terminal domain (NTD) to bind LPS, mediate cytotoxicity, and block LPS-induced inflammation. The C-terminal domain mediates phagocytosis of bacteria bound to the NTD. These two domains are linked by a surface-exposed loop at amino acids 231-249 for human BPI, known as the "hinge region." Autoantibodies to human BPI are prevalent in many chronic lung diseases; their presence is strongly correlated with Pseudomonas aeruginosa and with worse lung function in patients with cystic fibrosis and bronchiectasis. Although prior literature has reported BPI neutralization effect with autoantibodies targeting either NTD or C-terminal domain, the functionality of BPI Ab to the hinge region has never been investigated. Here, we report that Ab responses to the BPI hinge region mediate a remarkably selective potentiation of BPI-dependent phagocytosis of P. aeruginosa with both human and murine neutrophils in vitro and in vivo. These findings indicate that autoantibodies to the BPI hinge region might enhance bacterial clearance.

PMID:34272233 | PMC:PMC8354091 | DOI:10.4049/jimmunol.2100378

MAbs. 2021 Jan-Dec;13(1):1950264. doi: 10.1080/19420862.2021.1950264.

ABSTRACT

Epidermal growth factor receptor (EGFR)-targeted cancer therapy such as anti-EGFR monoclonal antibodies and tyrosine kinase inhibitors have demonstrated clinical efficacy. However, there remains a medical need addressing limitations of these therapies, which include a narrow therapeutic window mainly due to skin and organ toxicity, and primary and secondary resistance mechanisms of the EGFR-signaling cascade (e.g., RAS-mutated colorectal cancer). Using the redirected optimized cell killing (ROCK®) antibody platform, we have developed AFM24, a novel bispecific, IgG₁-scFv fusion antibody targeting CD16A on innate immune cells, and EGFR on tumor cells. We herein demonstrate binding of AFM24 to CD16A on natural killer (NK) cells and macrophages with K_D values in the low nanomolar range and to various EGFR-expressing tumor cells. AFM24 was highly potent and effective for antibody-dependent cell-mediated cytotoxicity via NK cells, and also mediated antibody-dependent cellular phagocytosis via macrophages in vitro. Importantly, AFM24 was effective toward a variety of EGFR-expressing tumor cells, regardless of EGFR expression level and KRAS/BRAF mutational status. In vivo, AFM24 was well tolerated up to the highest dose (75 mg/kg) when administered to cynomolgus monkeys once weekly for 28 days. Notably, skin and other toxicities were not observed. A transient elevation of interleukin-6 levels was detected at all dose levels, 2-4 hours post-dose, which returned to baseline levels after 24 hours. These results emphasize the promise of bispecific innate cell engagers as an alternative cancer therapy and demonstrate the potential for AFM24 to effectively target tumors expressing varying levels of EGFR, regardless of their mutational status.Abbreviations: ADA: antidrug antibody; ADCC: antibody-dependent cell-mediated cytotoxicity; ADCP: antibody-dependent cellular phagocytosis; AUC: area under the curve; CAR: chimeric-antigen receptor; CD: Cluster of differentiation; CRC :colorectal cancer; ECD: extracellular domain; EGF: epidermal growth factorEGFR epidermal growth factor receptor; ELISA: enzyme-linked immunosorbent assay; FACS: fluorescence-activated cell sorting; Fc: fragment, crystallizableFv variable fragment; HNSCC: head and neck squamous carcinomaIL interleukinm; Ab monoclonal antibody; MOA: mechanism of action; NK :natural killer; NSCLC: non-small cell lung cancer; PBMC: peripheral blood mononuclear cell; PBS: phosphate-buffered saline; PD: pharmacodynamic; ROCK: redirected optimized cell killing; RSV: respiratory syncytial virus; SABC: specific antibody binding capacity; SD: standard deviation; TAM: tumor-associated macrophage; TKI: tyrosine kinase inhibitor; WT: wildtype.

PMID:34325617 | DOI:10.1080/19420862.2021.1950264

Cell Mol Immunol. 2021 Jul;18(7):1835-1837. doi: 10.1038/s41423-021-00691-y. Epub 2021 May 18.

NO ABSTRACT

PMID:34007030 | PMC:PMC8245584 | DOI:10.1038/s41423-021-00691-y

Int J Mol Sci. 2021 Jun 17;22(12):6483. doi: 10.3390/ijms22126483.

ABSTRACT

Human epidermal growth factor receptor 2 (HER-2) is overexpressed in many malignant tumors. The anti-HER2 antibody trastuzumab has been approved for treating HER2-positive early and metastatic breast cancers. Pseudomonas exotoxin A (PE), a bacterial toxin of Pseudomonas aeruginosa, consists of an A-domain with enzymatic activity and a B-domain with cell binding activity. Recombinant immunotoxins comprising the HER2(scFv) single-chain Fv from trastuzumab and the PE24B catalytic fragment of PE display promising cytotoxic effects, but immunotoxins are typically insoluble when expressed in the cytoplasm of Escherichia coli, and thus they require solubilization and refolding. Herein, a recombinant immunotoxin gene was fused with maltose binding protein (MBP) and overexpressed in a soluble form in E. coli. Removal of the MBP yielded stable HER2(scFv)-PE24B at 91% purity; 0.25 mg of pure HER2(scFv)-PE24B was obtained from a 500 mL flask culture. Purified HER2(scFv)-PE24B was tested against four breast cancer cell lines differing in their surface HER2 level. The immunotoxin showed stronger cytotoxicity than HER2(scFv) or PE24B alone. The IC₅₀ values for HER2(scFv)-PE24B were 28.1 ± 2.5 pM (n = 9) and 19 ± 1.4 pM (n = 9) for high HER2-positive cell lines SKBR3 and BT-474, respectively, but its cytotoxicity was lower against MDA-MB-231 and MCF7. Thus, fusion with MBP can facilitate the soluble expression and purification of scFv immunotoxins.

PMID:34204265 | PMC:PMC8234717 | DOI:10.3390/ijms22126483

Int J Mol Sci. 2021 Jul 1;22(13):7099. doi: 10.3390/ijms22137099.

ABSTRACT

Staphylococcal aureus (S. aureus), a Gram-positive bacteria, is known to cause various infections. Extracellular vesicles (EVs) are a heterogeneous array of membranous structures secreted by cells from all three domains of life, i.e., eukaryotes, bacteria, and archaea. Bacterial EVs are implied to be involved in both bacteria-bacteria and bacteria-host interactions during infections. It is still unclear how S. aureus EVs interact with host cells and induce inflammatory responses. In this study, EVs were isolated from S. aureus and mutant strains deficient in either prelipoprotein lipidation (Δlgt) or major surface proteins (ΔsrtAB). Their immunostimulatory capacities were assessed both in vitro and in vivo. We found that S. aureus EVs induced pro-inflammatory responses both in vitro and in vivo. However, this activity was dependent on lipidated lipoproteins (Lpp), since EVs isolated from the Δlgt showed no stimulation. On the other hand, EVs isolated from the ΔsrtAB mutant showed full immune stimulation, indicating the cell wall anchoring of surface proteins did not play a role in immune stimulation. The immune stimulation of S. aureus EVs was mediated mainly by monocytes/macrophages and was TLR2 dependent. In this study, we demonstrated that not only free Lpp but also EV-imbedded Lpp had high pro-inflammatory activity.

PMID:34281154 | PMC:PMC8268867 | DOI:10.3390/ijms22137099

Eur J Pharm Sci. 2021 Jul 10;165:105941. doi: 10.1016/j.ejps.2021.105941. Online ahead of print.

ABSTRACT

Single-domain antibodies, VHHs or nanobodies, represent a promising set of alternatives to conventional therapeutic antibodies, gaining substantial attention in the field of cancer immunotherapy. However, inherent drawbacks of nanobodies such as fast clearance from blood circulation and lack of immune effector functions often led to unsatisfactory therapeutic efficacy. We previously reported that dinitrophenyl modification of an anti-EGFR VHH conferred Fc-dependent immune effector functions and elongated serum half-life on it through recruiting of hapten antibodies, resulting in improved immunotherapy efficacy in vivo. In the present work, we further tested the versatility of this approach in the case of an anti-PD-L1 blockade VHH (KN035). Site-specific dinitrophenyl conjugation did not impair the binding capacity of KN035 portion to PD-L1, but indirectly restored its immune effector functions, manifested by the observed antibody dependent cell-mediated cytotoxicity, antibody-dependent cellular phagocytosis and complement-dependent cytotoxicity against PD-L1 positive tumor cells. Significant delay of blood clearance of dinitrophenylated KN035 was evidenced by the prolonged half-life of ca. 22 h. This approach, using small hapten molecule conjugation, loaded additional antibody-mediated tumor killing mechanisms to PD-L1 blockade VHH and therefore improved efficacy is anticipated in the future in vivo therapeutic studies. Thus, our results underscore the power of this versatile approach for achieving desirable properties of VHH-based or similar therapeutics.

PMID:34256102 | DOI:10.1016/j.ejps.2021.105941

Microbiol Res. 2021 Jun 30;250:126810. doi: 10.1016/j.micres.2021.126810. Online ahead of print.

ABSTRACT

Plant pathogenic Gram-negative bacteria evade the host plant immune system by secreting Type III (T3E) and Type IV effector (T4E) proteins into the plant cytoplasm. Mostly T3Es are secreted into the plant cells to establish pathogenicity by affecting the vital plant process viz. metabolic pathways, signal transduction and hormonal regulation. Ubiquitin-26S proteasome system (UPS) exists as one of the important pathways in plants to control plant immunity and various cellular processes by employing several enzymes and enzyme components. Pathogenic and non-pathogenic bacteria are found to secrete effectors into plants with structural and/or functional similarity to UPS pathway components like ubiquitin E3 ligases, F-box domains, cysteine proteases, inhibitor of host UPS or its components, etc. The bacterial effectors mimic UPS components and target plant resistance proteins for degradation by proteasomes, thereby taking control over the host cellular activities as a strategy to exert virulence. Thus, the bacterial effectors circumvent plant cellular pathways leading to infection and disease development. This review highlights known bacterial T3E and T4E proteins that function and interfere with the ubiquitination pathway to regulate the immune system of plants.

PMID:34246833 | DOI:10.1016/j.micres.2021.126810

J Clin Invest. 2021 Jul 1;131(13):e141614. doi: 10.1172/JCI141614.

ABSTRACT

Clinical immunotherapy approaches are lacking efficacy in the treatment of glioblastoma (GBM). In this study, we sought to reverse local and systemic GBM-induced immunosuppression using the Helicobacter pylori neutrophil-activating protein (NAP), a potent TLR2 agonist, as an immunostimulatory transgene expressed in an oncolytic measles virus (MV) platform, retargeted to allow viral entry through the urokinase-type plasminogen activator receptor (uPAR). While single-agent murine anti-PD1 treatment or repeat in situ immunization with MV-s-NAP-uPA provided modest survival benefit in MV-resistant syngeneic GBM models, the combination treatment led to synergy with a cure rate of 80% in mice bearing intracranial GL261 tumors and 72% in mice with CT-2A tumors. Combination NAP-immunovirotherapy induced massive influx of lymphoid cells in mouse brain, with CD8+ T cell predominance; therapeutic efficacy was CD8+ T cell dependent. Inhibition of the IFN response pathway using the JAK1/JAK2 inhibitor ruxolitinib decreased PD-L1 expression on myeloid-derived suppressor cells in the brain and further potentiated the therapeutic effect of MV-s-NAP-uPA and anti-PD1. Our findings support the notion that MV strains armed with bacterial immunostimulatory antigens represent an effective strategy to overcome the limited efficacy of immune checkpoint inhibitor-based therapies in GBM, creating a promising translational strategy for this lethal brain tumor.

PMID:34196308 | PMC:PMC8245183 | DOI:10.1172/JCI141614

ChemMedChem. 2021 Jul 8. doi: 10.1002/cmdc.202100313. Online ahead of print.

ABSTRACT

Multivalent antibody-recruiting glycopolymers (MARGs) composed of hyaluronic acid (HA) grafted with multiple copies of dinitrophenol (DNP) were developed for targeted cancer immunotherapy. Structure-activity studies demonstrated that the MARGs were able to specifically recognize CD44-positive cancer cells and displayed remarkable antibody-recruiting capacities and tumor cell killing activities dependent on the introduced multivalent effect and the length of PEG linker. One of the MARGs, HA-[PEG₃ -DNP]₈ , showed the best capacity for clustering anti-DNP antibodies onto CD44-positive cancer cells and displayed potent in vitro anti-cancer activity by triggering complement dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC). Moreover, we found that HA-[PEG₃ -DNP]₈ significantly inhibited the xenograft tumor growth of Babl/c nude mice bearing triple negative breast cancer cells, while it did not cause detectable histological cytotoxicity. Given the easy access of this type of natural glycopolymer and the practical synthesis approach, these MARGs provide promising immunotherapeutics for cancer immunotherapy.

PMID:34235861 | DOI:10.1002/cmdc.202100313