Importance of antibody isotypes in antitumor immunity by monocytes and complement using human-immune tumor models.

Monoclonal antibodies (mAbs) have revolutionized clinical medicine, especially in the field of cancer immunotherapy. The challenge now is to improve the response rates, as immunotherapy still fails for many patients. Strategies to enhance tumor cell death is a fundamental aim, but relevant model systems for human tumor immunology are lacking. Herein, we have developed a preclinical human immune - three-dimensional (3D) tumor model (spheroids) to map the efficiency of tumor-specific isotypes for improved tumor cell killing. Different anti-CD20 Rituximab (RTX) isotypes alone or in combination, were evaluated for mediating complement-dependent cytotoxicity and antibody-dependent phagocytosis by human monocytic cells in 3D spheroids, in parallel with monolayer cultures, of human CD20+ B-cell lymphomas. We demonstrate that the IgG3 variant of RTX has the greatest tumoricidal effect over other isotypes, and when combined with apoptosis-inducing RTX-IgG2 isotype the therapeutic effect can be substantially enhanced. The results show further that the treatment outcome by RTX isotypes is influenced by tumor morphology and expression of the complement inhibitor CD59. Hence, the human immune-3D tumor model is a clinical relevant and attractive ex vivo system to predict mAbs for best efficacy in cancer immunotherapy.

SIRPα-specific monoclonal antibody enables antibody-dependent phagocytosis of neuroblastoma cells.

Immunotherapy with anti-GD2 monoclonal antibodies (mAbs) provides some benefits for patients with neuroblastoma (NB). However, the therapeutic efficacy remains limited, and treatment is associated with significant neuropathic pain. Targeting O-acetylated GD2 (OAcGD2) by 8B6 mAb has been proposed to avoid pain by more selective tumor cell targeting. Thorough understanding of its mode of action is necessary to optimize this treatment strategy. Here, we found that 8B6-mediated antibody-dependent cellular phagocytosis (ADCP) performed by macrophages is a key effector mechanism. But efficacy is limited by upregulation of CD47 expression on neuroblastoma cells in response to OAcGD2 mAb targeting, inhibiting 8B6-mediated ADCP. Antibody specific for the CD47 receptor SIRPα on macrophages restored 8B6-induced ADCP of CD47-expressing NB cells and improved the antitumor activity of 8B6 mAb therapy. These results identify ADCP as a critical mechanism for tumor cytolysis by anti-disialoganglioside mAb and support a combination with SIRPα blocking agents for effective neuroblastoma therapy.

Enhancement of Antibody-Dependent Cellular Cytotoxicity and Phagocytosis in Anti-HIV-1 Human-Bovine Chimeric Broadly Neutralizing Antibodies.

No prophylactic vaccine has provided robust protection against human immunodeficiency virus type 1 (HIV-1). Vaccine-induced broadly neutralizing antibodies (bNAbs) have not been achieved in humans and most animals; however, cows vaccinated with HIV-1 envelope trimers produce bNAbs with unusually long third heavy complementarity-determining regions (CDRH3s). Alongside neutralization, Fc-mediated effector functions, including antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADP), may be critical for in vivo bNAb antiviral activity. Here, we aimed to augment the Fc-dependent effector functions of a chimeric human-bovine bNAb, NC-Cow1, which binds the CD4 binding site (CD4bs) and exhibits broader and more potent neutralization than most human CD4bs bNAbs by using an exceptionally long 60-amino acid (aa) CDRH3. The bovine variable region of NC-Cow1 was paired with a human IgG1 Fc region mutated to create the following three variants: G236R/L328R (GRLR) that abrogates Fc-gamma receptor (FcγR) binding, and two variants that enhance binding, namely, G236A/S239D/I332E (GASDIE) and G236A/S239D/A330L/I332E (GASDALIE). Both GASDIE and GASDALIE improved binding to human FcγRIIA and FcγRIIIA, enhanced human natural killer (NK) cell activation, and mediated higher levels of ADCC and ADP activity than the wild-type human IgG1 Fc. GASDALIE mediated higher phagocytic activity than GASDIE. As expected, GRLR eliminated binding to FcγRs and did not mediate ADCC or ADP. We demonstrated that mutations in the human Fc region of bovine chimeric antibodies with ultralong CDRH3s could enhance antibody effector functions while maintaining envelope binding and neutralization. This study will have significant implications in the development of multifunctional anti-HIV antibodies, which may be important to prevent HIV-1 transmission in an antibody-based topical microbicide. IMPORTANCE Despite successful antiviral chemotherapy, human immunodeficiency virus (HIV) is still a lifelong persistent virus, and no vaccine yet prevents HIV transmission. Topical microbicides offer an important alternative method to prevent sexual transmission of HIV-1. With the production of highly potent anti-HIV-1 broadly neutralizing antibodies (bNAbs) and multifunctional antibodies, monoclonal antibodies are now important prophylactic agents. Recently discovered anti-HIV-1 bovine bNAbs (with higher potency and breadth than most human bNAbs) could be novel candidates as potent topical microbicides. Our study is significant as it demonstrates the compatibility of combining bovine-derived neutralization with human-derived antibody-effector functions. This study is a new approach to antibody engineering that strengthens the feasibility of using high-potency bovine variable region bNAbs with augmented Fc function and promotes them as a strong candidate for antibody-mediated therapies.

The protective potential of Fc-mediated antibody functions against influenza virus and other viral pathogens.

In recent years, there has been a renewed interest in utilizing antibody fragment crystallizable (Fc) functions to prevent and control viral infections. The protective and therapeutic potential of Fc-mediated antibody functions have been assessed for some clinically important human viruses, including HIV, hemorrhagic fever viruses and influenza virus. There is mounting evidence that influenza-specific antibodies with Fc-mediated functions, such as antibody-dependent cellular cytotoxicity and antibody-dependent phagocytosis, can aid in the clearance of influenza virus infection. Recent influenza challenge studies and intravenous immunoglobulin G therapy studies in humans suggest a protective role for Fc effector functions in vivo. Broadly reactive influenza antibodies with Fc-mediated functions are prevalent in the human population and could inform the development of a universally protective influenza vaccine or therapy. In this review, we explore the utility of antibodies with Fc-mediated effector functions against viral infections with a focus on influenza virus.

Anti-CD20 monoclonal antibody-dependent phagocytosis of chronic lymphocytic leukaemia cells by autologous macrophages.

Unconjugated monoclonal antibodies (mAbs) are an important component of effective combination therapies for chronic lymphocytic leukaemia (CLL). Antibody-dependent phagocytosis (ADP) is a major mediator of mAb cytotoxicity, but there is limited knowledge of the determinants of ADP efficacy. We used macrophages derived in vitro from autologous circulating monocytes to test the effects of mAb structure and concentration, target : effector cell ratio, duration of co-incubation and CLL cell CD20 expression on ADP. Next-generation anti-CD20 mAbs (ofatumumab, ublituximab, obinutuzumab, ocaratuzumab) were significantly more effective at inducing ADP compared to rituximab, but none were as effective as the anti-CD52 mAb alemtuzumab. Ofatumumab (10 µg/ml) used as a representative next-generation anti-CD20 mAb achieved an ADP plateau at 3 h co-incubation with a target : effector ratio of 10 : 1 (mean = 2.1 CLL cells/macrophage, range = 1.5-3.5). At 0.156 µg/ml (the lowest concentration tested) ofatumumab ADP was significantly higher than alemtuzumab. However, ofatumumab-induced ADP did not increase significantly at higher mAb concentrations. We show that anti-CD20 mAb ADP efficacy is determined by the mAb characteristics, target : effector ratio and incubation time. We suggest that preclinical evaluation of anti-CD20 mAbs to understand the determinants of ADP could be useful in designing future combination therapies for CLL.

Myeloid cells as effector cells for monoclonal antibody therapy of cancer.

Monoclonal antibodies (mAbs) have become an important addition to chemo- and/or radiotherapy for the treatment of cancer. They have multiple effector functions that can lead to eradication of tumor, including induction of apoptosis, growth inhibition, and initiation of complement-dependent lysis. Furthermore, mAbs can recruit immune effector cells. Traditionally, natural killer cells have been considered as the main effector cell population in mAb-mediated tumor killing. Myeloid cells have potent cytotoxic ability, as well. Monocytes and macrophages have been shown to induce antibody-dependent cytotoxicity and phagocytosis of tumor cells in the presence of IgG anti-tumor mAb. Furthermore, neutrophils are the most abundant population of circulating white blood cells, and as such may constitute a formidable source of effector cells. However, when targeting neutrophils for tumor therapy, antibodies of the IgA subclass may be more effective. This article focuses on enlisting myeloid effector cells for mAb-based immunotherapy of cancer. Additionally, methods to study mAb-dependent phagocytosis of tumor cells by macrophages are compared.

Passive antibody transfer from pregnant women to their fetus are maximized after SARS-CoV-2 vaccination irrespective of prior infection.

Background: Pregnancy is associated with a higher risk of adverse symptoms and outcomes for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection for both mother and neonate. Antibodies can provide protection against SARS-CoV-2 infection and are induced in pregnant women after vaccination or infection. Passive transfer of these antibodies from mother to fetus in utero may provide protection to the neonate against infection. However, it is unclear whether the magnitude or quality and kinetics of maternally derived fetal antibodies differs in the context of maternal infection or vaccination. Objective: We aimed to determine whether antibodies transferred from maternal to fetus differed in quality or quantity between infection- or vaccination-induced humoral immune responses. Methods: We evaluated 93 paired maternal and neonatal umbilical cord blood plasma samples collected between October 2020 and February 2022 from a birth cohort of pregnant women from New Orleans, Louisiana, with histories of SARS-CoV-2 infection and/or vaccination. Plasma was profiled for the levels of spike-specific antibodies and induction of antiviral humoral immune functions, including neutralization and Fc-mediated innate immune effector functions. Responses were compared between 4 groups according to maternal infection and vaccination. Results: We found that SARS-CoV-2 vaccination or infection during pregnancy increased the levels of antiviral antibodies compared to naive subjects. Vaccinated mothers and cord samples had the highest anti-spike antibody levels and antiviral function independent of the time of vaccination during pregnancy. Conclusions: These results show that the most effective passive transfer of functional antibodies against SARS-CoV-2 in utero is achieved through vaccination, highlighting the importance of vaccination in pregnant women.

Impact of Fc receptors and host characteristics on myeloid phagocytic response to rituximab-treated 3D-cultured B-cell lymphoma.

Antibody-based immunotherapy is successful in treating cancer, but its effectiveness varies among patients. Therefore, understanding myeloid phagocytic responses to therapeutic antibodies is critical. Immunoglobulin Fc receptors and host characteristics were evaluated in phagocytosis of 3D-cultured CD20+ B-cell lymphoma (spheroids) treated with different anti-CD20 rituximab (RTX) monoclonal antibody isotypes. Monocytes from healthy donors of different ages and sexes were isolated, and their Fc receptors for IgG (FcγRI, FcγRIIa, FcγRIIIa) and IgA (FcαRI) were determined, as well as Fc receptor gene polymorphisms. Antibody-dependent phagocytosis was assessed using flow cytometry, confocal imaging, and Fc receptor blocking. RTX isotypes showed varying efficacy in stimulating the phagocytosis of spheroids. RTX-IgG3 proved to be the most efficient, followed by RTX-IgG1. Monocytes infiltrated RTX-treated spheroids at the periphery but migrated also into the core when stimulated with RTX-IgG3. Blocking FcγRI or FcγRIIa, but not FcγRIIIa, with antibodies inhibited RTX-IgG1 and RTX-IgG3-mediated phagocytosis. Monocytes from younger women demonstrated higher FcγRI and FcγRIIa levels compared to older women, while older men displayed increasing FcγRI and FcγRIIIa levels compared to younger men. Monocytes from younger women displayed greater phagocytic activity compared to older women, while older men had better IgG-mediated phagocytosis than younger men. Single Fc receptor levels, or FcγRIIa and FcγRIIIa genetic variants, had a low correlation with phagocytic intensity, likely as a result of multiple engagements of Fcreceptors for IgG-mediated phagocytosis. In conclusion, antibody isotype, Fc receptors, age, and sex influence tumor phagocytosis. This study exposes the relationship between host traits and the efficacy of therapeutic antibodies, providing insights into cancer immunotherapy treatment.

Expression Analysis of Outer Membrane Protein HPS_06257 in Different Strains of Glaesserella parasuis and Its Potential Role in Protective Immune Response against HPS_06257-Expressing Strains via Antibody-Dependent Phagocytosis.

HPS_06257 has been identified as an important protective antigen against Glaesserella parasuis infection. However, little is known about the role of HPS_06257 in the protective immune response. A whole-genome data analysis showed that among 18 isolates of Glaesserella parasuis, 11 were positive for the HPS_06257 gene, suggesting that not every strain contains this gene. We used PCR to investigate the presence of the HPS_06257 gene among 13 reference strains and demonstrated that 5 strains contained the gene. A polyclonal antibody against HPS_06257 was generated with a recombinant protein to study the expression of HPS_06257 in those 13 strains. Consistent with the PCR data, five strains expressed HPS_06257, whereas eight strains were HPS_06257 null. We also compared the protective effects of HPS_06257 against an HPS_06257-expressing strain (HPS5) and an HPS_06257-null strain (HPS11). Immunization with HPS_06257 only protected against HPS5 and not HPS11. Moreover, phagocytosis of antibody-opsonized bacteria demonstrates that the antibody against HPS_06257 increased the phagocytosis of the HPS5 strain by macrophages but not the phagocytosis of the HPS11 strain, suggesting that antibody-dependent phagocytosis is responsible for the protective role exerted by HPS_06257 in the immune response to HPS5. Our data also show that the antibody against HPS_06257 increased the phagocytosis of the other HPS_06257-expressing strains by macrophages but not that of HPS_06257-null strains. In summary, our findings demonstrate that antibody-dependent phagocytosis contributes to the protective immune response induced by immunization with HPS_06257 against HPS_06257-expressing strains.

SIRPα Suppresses Response to Therapeutic Antibodies by Nurse Like Cells From Chronic Lymphocytic Leukemia Patients.

Targeted antibody therapies improve outcomes for chronic lymphocytic leukemia (CLL) patients. However, resistance often develops. We have previously shown that resistance to therapeutic antibodies, by monocyte derived macrophages (referred to as nurse like cells, NLCs), from CLL patients is characterized by suppression of antibody dependent phagocytosis (ADP). The mechanism(s) contributing to the muted ADP responses remain unresolved. In this regard, an innate immune checkpoint was recently described that uses the CD47:SIRPα axis to suppress phagocytic responses by macrophages. In this study we examine whether the SIRPα axis regulates ADP responses to the anti-CD20 antibody, obinutuzumab, by NLCs. Using siRNA depletion strategies we show that SIRPα is a suppressor of ADP responses. Moreover, we show that this innate immune checkpoint contributes to the resistance phenotype in NLCs derived from CLL patients. Finally, we show that SIRPα suppression is mediated via the phosphatase, Shp1, which in turn suppresses SYK-dependent activation of ADP. Thus, we identify a druggable pathway that could be exploited to enhance sensitivity to existing therapeutic antibodies used in CLL. This is the first study to show that activation of the CD47:SIRPα innate immune checkpoint contributes to ADP resistance in NLCs from CLL patients.

Alveolar Macrophage Dysfunction and Increased PD-1 Expression During Chronic SIV Infection of Rhesus Macaques.

HIV infected individuals have been shown to be pre-disposed to pulmonary infections even while receiving anti-retroviral therapy. Alveolar macrophages (AMs) play a critical role in lung innate immunity, but contradictory results have been reported regarding their functionality following HIV infection. Here, using the SIV rhesus macaque model, we document the effect of SIV infection on the phenotypic and functional properties of AMs. Following infection with SIVmac251, AMs in bronchoalveolar lavage (BAL) sampled over 2- to 20-weeks post-infection (wpi) were compared to those in BAL samples from naïve macaques. AM expression of proinflammatory cytokines TNF-α, IL-6, IL-1ß, and chemokine RANTES drastically increased 2-wpi compared to AMs of naïve macaques (p < 0.0001 for all), but dropped significantly with progression to chronic infection. Phagocytic activity of AMs 2-and 4-wpi was elevated compared to AMs of naive animals (p = 0.0005, p = 0.0004, respectively) but significantly decreased by 12-wpi (p = 0.0022, p = 0.0019, respectively). By 20-wpi the ability of AMs from chronically infected animals to perform SIV-specific antibody-dependent phagocytosis (ADP) was also diminished (p = 0.028). Acute SIV infection was associated with increased FcγRIII expression which subsequently declined with disease progression. Frequency of FcγRIII+ AMs showed a strong trend toward correlation with SIV-specific ADP, and at 2-wpi FcγRIII expression negatively correlated with viral load (r = -0.6819; p = 0.0013), suggesting a contribution to viremia control. Importantly, PD-1 was found to be expressed on AMs and showed a strong trend toward correlation with plasma viral load (r = 0.8266; p = 0.058), indicating that similar to over-expression on T-cells, PD-1 expression on AMs may also be associated with disease progression. Further, AMs predominantly expressed PD-L2, which remained consistent over the course of infection. PD-1 blockade enhanced SIV-specific ADP by AMs from chronic infection indicating that the PD-1/PD-L2 pathway may modulate functional activity of AMs at that stage. These findings provide new insight into the dynamics of SIV infection leading to AM dysfunction and alteration of pulmonary innate immunity. Our results suggest new pathways to exploit in developing therapies targeting pulmonary disease susceptibility in HIV-infected individuals.

Antibody-dependent phagocytosis (ADP) responses following trivalent inactivated influenza vaccination of younger and older adults.

Globally the most commonly utilised immunisation against influenza is the trivalent inactivated influenza vaccine (TIV) derived from an A/H1N1, an A/H3N2 and aB type influenza virus. Vaccine effectiveness of TIV varies year to year, depending on how well antigenically matched the strains in the vaccine are compared to circulating strains [1,2]. Moreover, vaccine effectiveness can vary within certain subpopulations such as HIV-positive, young children and the elderly. Decreased vaccine effectiveness in the elderly is associated with impaired Ab production, as measured by standard hemagglutination inhibition (HAI) assays. We investigated the level of Antibody Dependent Phagocytosis (ADP)-mediating Abs induced by the 2008-TIV in healthy Australian adults aged over and under 60years to determine if this immune function was also reduced in the elderly. We utilised an ADP assay that measures the uptake of IgG-opsonised HA-coated fluorescent microspheres by a monocytic cell line. We also measured HA-specific Abs that are close enough to bind to dimeric FcγRIIa ectodomains in an ELISA-based assay. Furthermore, we compared the extent of cross-reactive recognition of diverse influenza strains by ADP-mediating Abs found in pre- and post-vaccination sera in both of these groups. We found that young adults and older adults mounted similar ADP activity against HAs contained in the 2008-TIV, despite older adults have diminished HI responses. The level of cross-reactive antibodies against other HAs was limited in both groups. We conclude that seasonal influenza vaccination elicits limited cross-reactive ADP to HA in both young and older adults. New influenza vaccination strategies that elicit cross-reactive and polyfunctional antibodies are needed.