Fc-mediated Effector Functions Research Articles

Fc-Effector-Independent in vivo Activity of a Potent Influenza B Neuraminidase Broadly Neutralizing Antibody.

Influenza B virus (IBV) contributes to substantial influenza-mediated morbidity and mortality, particularly among children. Similar to influenza A viruses (IAV), the hemagglutinin (HA) and neuraminidase (NA) of IBV undergo antigenic drift, necessitating regular reformulation of seasonal influenza vaccines. NA inhibitors, such as oseltamivir, have reduced activity and clinical efficacy against IBV, while M2 channel inhibitors are only effective against IAV, highlighting the need for improved vaccine and therapeutics for the treatment of seasonal IBV infections. We have previously described a potent human monoclonal antibody (hMAb), 1092D4, that is specific for IBV NA and neutralizes a broad range of IBVs. The anti-viral activity of MAbs can include direct mechanisms such as through neutralization and/or Fc-mediated effector functions that are dependent on accessory cells expressing Fc receptors and that could be impacted by potential host-dependent variability. To discern if the in vivo efficacy of 1092D4 was dependent on Fc-effector function, 1092D4 hMAb with reduced ability to bind to Fc receptors (1092D4-LALAPG) was generated and tested. 1092D4-LALAPG had comparable in vitro binding, neutralization, and inhibition of NA activity to 1092D4. 1092D4-LALAPG was effective at protecting against a lethal challenge of IBV in mice. These results suggest that hMAb 1092D4 in vivo activity is minimally dependent on Fc-effector functions, a characteristic that may extend to other hMAbs that have potent NA inhibition activity.

Coronavac inactivated vaccine triggers durable, cross-reactive Fc-mediated phagocytosis activities

ABSTRACT Although humoral responses elicited by infection or vaccine lost the ability to prevent transmission against Omicron, vaccine-induced antibodies may still contribute to disease attenuation through Fc-mediated effector functions. However, Fc effector function elicited by CoronaVac, as the most widely supplied inactivated vaccine globally, has not been characterized. For the first time, our study depicted Fc-mediated phagocytosis activity induced by CoronaVac, including antibody-dependent cellular phagocytosis (ADCP) and antibody-dependent neutrophil phagocytosis (ADNP) activities, and further compared with that from convalescent individuals and CoronaVac recipients with subsequent breakthrough infections. We showed that 2-dose of CoronaVac effectively induced both ADCP and ADNP, but was substantially lower compared to infection, whereas the booster dose further augmented ADCP and ADNP responses, and remained detectable for 52 weeks. Among CoronaVac recipients, ADCP and ADNP responses also demonstrated cross-reactivity against Omicron subvariants, and breakthrough infection could enhance the phagocytic response. Meanwhile, serum samples from vaccinees, convalescent individuals with wildtype infection, BA.2 and BA.5 breakthrough infection demonstrated differential cross-reactive ADCP and ADNP responses against Omicron subvariants, suggesting the different subvariants of spike antigen exposure might alter the cross-reactivity of Fc effector function. Further, ADCP and ADNP responses were strongly correlated with Spike-specific IgG responses and neutralizing activities, indicating coordinated neutralization activity, ADCP and ADNP responses triggered by CoronaVac. Of note, the ADCP and ADNP responses were more durable and cross-reactive than corresponding Spike-specific IgG titers and neutralizing activities. Our study has important implications for optimal boosting vaccine strategies that may induce potent and broad Fc-mediated phagocytic activities.

Antibody-dependent cellular cytotoxicity, infected cell binding and neutralization by antibodies to the SIV envelope glycoprotein.

Antibodies specific for diverse epitopes of the simian immunodeficiency virus envelope glycoprotein (SIV Env) have been isolated from rhesus macaques to provide physiologically relevant reagents for investigating antibody-mediated protection in this species as a nonhuman primate model for HIV/AIDS. With increasing interest in the contribution of Fc-mediated effector functions to protective immunity, we selected thirty antibodies representing different classes of SIV Env epitopes for a comparison of antibody-dependent cellular cytotoxicity (ADCC), binding to Env on the surface of infected cells and neutralization of viral infectivity. These activities were measured against cells infected with neutralization-sensitive (SIVmac316 and SIVsmE660-FL14) and neutralization-resistant (SIVmac239 and SIVsmE543-3) viruses representing genetically distinct isolates. Antibodies to the CD4-binding site and CD4-inducible epitopes were identified with especially potent ADCC against all four viruses. ADCC correlated well with antibody binding to virus-infected cells. ADCC also correlated with neutralization. However, several instances of ADCC without detectable neutralization or neutralization without detectable ADCC were observed. The incomplete correspondence between ADCC and neutralization shows that some antibody-Env interactions can uncouple these antiviral activities. Nevertheless, the overall correlation between neutralization and ADCC implies that most antibodies that are capable of binding to Env on the surface of virions to block infectivity are also capable of binding to Env on the surface of virus-infected cells to direct their elimination by ADCC.

Impact of pregnancy on polyfunctional IgG and memory B cell responses to Tdap immunization

BackgroundMaternal pertussis immunization using Tdap vaccine is recommended in many countries to protect newborns from severe post-natal infection. Immunological changes during pregnancy may influence the response to vaccines. The quality of IgG and memory B cell responses to Tdap immunization in pregnant women has not yet been described. MethodsThe impact of pregnancy on the response to Tdap vaccination was assessed by comparing humoral immune responses in 42 pregnant and 39 non-pregnant women. The levels of serum pertussis antigens and tetanus toxoid-specific IgG, IgG subclasses, IgG Fc-mediated effector functions, as well as memory B cell frequencies were assessed before and at several time points after vaccination. ResultsTdap immunization induced similar levels of pertussis and tetanus-specific IgG and IgG subclasses in pregnant and non-pregnant women. Pregnant women produced IgG promoting complement deposition, and neutrophils and macrophages phagocytosis at levels comparable to non-pregnant women. They were also able to expand pertussis and tetanus-specific memory B cells at similar frequencies as non-pregnant women, suggesting equivalent “boostability”. Higher levels of vaccine-specific IgG, IgG subclasses, and IgG Fc-mediated effector functions were detected in cord blood as compared to maternal blood, indicating efficient transport across the placenta. ConclusionsThis study demonstrates that pregnancy does not affect the quality of effector IgG and memory B cell responses to Tdap immunization and that polyfunctional IgG are efficiently transferred across the placenta. Registry's URL and the trial's registration numberClinicalTrials.Gov (NCT03519373).

ALVAC-HIV and AIDSVAX B/E vaccination induce improved immune responses compared with AIDSVAX B/E vaccination alone.

The RV144 phase III vaccine trial demonstrated that ALVAC-HIV and AIDSVAX B/E administration over 6 months resulted in 31% efficacy in preventing HIV acquisition, while administration of AIDSVAX B/E alone in both VAX003 and VAX004 studies failed to show efficacy. In this study, we aimed to understand the impact of ALVAC-HIV on the development of cellular, humoral, and functional immune responses compared to the administration of AIDSVAX B/E alone. ALVAC-HIV in combination with 3 doses of AIDSVAX B/E significantly increased CD4+ HIV-specific T cell responses, polyfunctionality, and proliferation compared with 3 doses of AIDSVAX B/E alone. Additionally, Env-specific plasmablasts and A244-specific memory B cells were identified with a significantly higher magnitude in the group that received ALVAC-HIV. Subsequently, data revealed increased magnitude of plasma IgG binding to and avidity for HIV Env in participants who received ALVAC-HIV compared with 3 doses of AIDSVAX B/E alone. Lastly, levels of the Fc-mediated effector functions antibody-dependent cellular cytotoxicity, NK cell activation, and trogocytosis were significantly increased in participants who received ALVAC-HIV compared with those receiving AIDSVAX B/E alone. Taken together, these results suggest that ALVAC-HIV plays an essential role in developing cellular and humoral immune responses to protein-boosted regimens relative to protein alone.

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

Abstract Pregnancy is associated with a higher risk of adverse symptoms and outcomes for most infectious diseases, including SARS-CoV-2. These adverse risks are also observed in neonates. Antibodies provide protection against SARS-CoV-2 infection, and have been reported after maternal vaccination, maternal infection, and through passive transfer from mother to fetus in utero. However, it is unclear whether the magnitude or quality of maternally-derived fetal antibodies differs in the context of infection or vaccination. Here, 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 the greater New Orleans area. Histories of SARS-CoV-2 infection or vaccination were obtained. Humoral immunity 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. We found that SARS-CoV-2 vaccination or infection during pregnancy increased the levels of antiviral antibodies from naïve subjects. Importantly, vaccinated mothers and cord samples had the highest anti-spike antibody levels and antiviral function independent of the time of vaccination during pregnancy. 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. Supported by grants from NIH (NCI U54CA260581-02, NHLBI Grant HL-061007) and USDA (APHIS NBAF NSTP)

Enhancing the Anti-tumor Potency of a Novel Siglec-15 Antibody by Engineering its Fc-mediated Effector Functions.

Siglec-15, an inhibitory immune checkpoint, is an emerging target in cancer immunotherapy. Blocking the function of Siglec-15 is an excellent strategy for cancer treatment and antibody blockade has been used to target Siglec-15. However, whether Fc-mediated effector functions contribute to the therapeutic effect of antibodies remains unclear. Herein, we generated a monoclonal antibody, 1-15D1, which had a high binding affinity with Siglec-15 and strongly activated T-cell immune response in vitro. Subsequently, the Fc-mediated effector functions of 1-15D1 were explored in a Siglec-15 humanized mouse model, and further improvement in antitumor efficacy was observed in the mouse IgG2a isotype group. Thus, we demonstrate that the antitumor effects of 1-15D1 were mediated via multiple factors. In addition to the T-cell immune response, 2 novel mechanisms were explored, including the internalization of the cell surface Siglec-15 and Fc-mediated effector functions. In conclusion, our studies not only provide a potential agent for the improvement of cancer immunotherapy but also suggest that a specific role of Fc-mediated immune regulation may improve the therapeutic potency of Siglec-15 monoclonal antibody.

Subclass-switched anti-spike IgG3 oligoclonal cocktails strongly enhance Fc-mediated opsonization

Antibodies play a central role in the immune defense against SARS-CoV-2. Emerging evidence has shown that nonneutralizing antibodies are important for immune defense through Fc-mediated effector functions. Antibody subclass is known to affect downstream Fc function. However, whether the antibody subclass plays a role in anti-SARS-CoV-2 immunity remains unclear. Here, we subclass-switched eight human IgG1 anti-spike monoclonal antibodies (mAbs) to the IgG3 subclass by exchanging their constant domains. The IgG3 mAbs exhibited altered avidities to the spike protein and more potent Fc-mediated phagocytosis and complement activation than their IgG1 counterparts. Moreover, combining mAbs into oligoclonal cocktails led to enhanced Fc- and complement receptor-mediated phagocytosis, superior to even the most potent single IgG3 mAb when compared at equivalent concentrations. Finally, in an invivo model, we show that opsonic mAbs of both subclasses can be protective against a SARS-CoV-2 infection, despite the antibodies being nonneutralizing. Our results suggest that opsonic IgG3 oligoclonal cocktails are a promising idea to explore for therapy against SARS-CoV-2, its emerging variants, and potentially other viruses.

LALAPG variant of the Human Contraception Antibody (HCA) reduces Fc-mediated effector functions while maintaining sperm agglutination activity.

High rates of unintended pregnancies worldwide indicate a need for more accessible and acceptable methods of contraception. We have developed a monoclonal antibody, the Human Contraception Antibody (HCA), for use by women in vaginal films and rings for contraception. The divalent F(ab')2 region of HCA binds to an abundant male reproductive tract-specific antigen, CD52g, and potently agglutinates sperm. Certain other antibody activities mediated by the Fc region such as mucus trapping, complement-dependent cytotoxicity (CDC) and antibody-dependent cellular phagocytosis (ADCP) could have beneficial or negative effects. The purpose of this study was to document HCA Fc effector functions and determine whether an engineered variant of HCA with a modified Fc region, HCA-LALAPG, retains desirable contraceptive activity while minimizing Fc-mediated effects. Fab and Fc functions were compared between HCA and HCA-LALAPG. Fab activity was assessed using sperm agglutination and modified swim-up ("sperm escape") assays. Fc functions were assessed by CDC (sperm immobilization), ADCP, and cervical mucus penetration assays. HCA and HCA-LALAPG showed equivalent activity in assays of Fab function. In the assays of Fc function, HCA supported strong CDC, ADCP, and sperm trapping in cervical mucus whereas HCA-LALAPG demonstrated little to no activity. HCA and the HCA-LALAPG variant were both highly effective in the sperm agglutination assays but differed in Fc mediated functions. Use of the HCA-LALAPG variant for contraception in women could reduce antibody-mediated inflammation and antigen presentation but may have reduced contraceptive efficacy due to much weaker sperm trapping in mucus and complement-dependent sperm immobilization activity.

Antibody dependent disease enhancement (ADE) after COVID-19 vaccination and beta glucans as a safer strategy in management

A potential risk associated with vaccines for COVID-19 is antibody-dependent disease enhancement (ADE) in which vaccine induced antibody mediated immune responses may lead to enhanced SARS CoV- 2 acquisition or increased disease severity. Though ADE has not been clinically demonstrated with any of the COVID-19 vaccines so far, when neutralizing antibodies are suboptimal, the severity of COVID-19 has been reported to be greater. ADE is presumed to occur via abnormal macrophages induced by the vaccine based immune response by antibody-mediated virus uptake into Fc gamma receptor IIa (FcγRIIa) or by the formation of Fc-mediated excessive antibody effector functions. Beta-glucans which are naturally occurring polysaccharides known for unique immunomodulation by capability to interact with macrophages, eliciting a specific beneficial immune-response and enhancing all arms of the immune system, importantly without over-activation are suggested as safer nutritional supplement-based vaccine adjuvants for COVID-19.

Antibody Fc-chimerism and effector functions: When IgG takes advantage of IgA.

Recent advances in the development of therapeutic antibodies (Abs) have greatly improved the treatment of otherwise drug-resistant cancers and autoimmune diseases. Antibody activities are mediated by both their Fab and the Fc. However, therapeutic Abs base their protective mechanisms on Fc-mediated effector functions resulting in the activation of innate immune cells by FcRs. Therefore, Fc-bioengineering has been widely used to maximise the efficacy and convenience of therapeutic antibodies. Today, IgG remains the only commercially available therapeutic Abs, at the expense of other isotypes. Indeed, production, sampling, analysis and related in vivo studies are easier to perform with IgG than with IgA due to well-developed tools. However, interest in IgA is growing, despite a shorter serum half-life and a more difficult sampling and purification methods than IgG. Indeed, the paradigm that the effector functions of IgG surpass those of IgA has been experimentally challenged. Firstly, IgA has been shown to bind to its Fc receptor (FcR) on effector cells of innate immunity with greater efficiency than IgG, resulting in more robust IgA-mediated effector functions in vitro and better survival of treated animals. In addition, the two isotypes have been shown to act synergistically. From these results, new therapeutic formats of Abs are currently emerging, in particular chimeric Abs containing two tandemly expressed Fc, one from IgG (Fcγ) and one from IgA (Fcα). By binding both FcγR and FcαR on effector cells, these new chimeras showed improved effector functions in vitro that were translated in vivo. Furthermore, these chimeras retain an IgG-like half-life in the blood, which could improve Ab-based therapies, including in AIDS. This review provides the rationale, based on the biology of IgA and IgG, for the development of Fcγ and Fcα chimeras as therapeutic Abs, offering promising opportunities for HIV-1 infected patients. We will first describe the main features of the IgA- and IgG-specific Fc-mediated signalling pathways and their respective functional differences. We will then summarise the very promising results on Fcγ and Fcα containing chimeras in cancer treatment. Finally, we will discuss the impact of Fcα-Fcγ chimerism in prevention/treatment strategies against infectious diseases such as HIV-1.

Penpulimab plus chemotherapy with or without anlotinib as first-line treatment for patients with advanced esophageal squamous cell carcinoma (ANSWER): A randomized, two-arm, clinical trial in progress.

TPS482 Background: For patients with advanced esophageal squamous cell carcinoma (ESCC), the combination of immune checkpoint inhibitors with chemotherapy has demonstrated synergistic antitumor activity and manageable safety profile. Anti-angiogenic targeted drugs for ESCC patients are still being explored. Penpulimab is a humanized high-affinity IgG1 anti-programmed cell death 1 (PD-1) monoclonal antibody engineered to completely eliminate Fcγ receptor binding and Fc-mediated effector functions that can compromise antitumor activity. Anlotinib, an oral small molecule tyrosine kinases inhibitor, targeting VEGFR 1/2/3, FGFR 1-4, PDGFR α/β and c-kit, have been recommended by the guidelines for advanced ESCC in China because of its good efficacy and tolerance. The purpose of the study was to investigate the efficacy and safety of penpulimab plus chemotherapy with or without anlotinib as first-line treatment for patients with advanced ESCC. Methods: This is a multi-center, two-arm, phase II study which enrolled patients with advanced ESCC. Eligible patients are aged 18-75 years, ECOG performance status 0~1. Approximately 100 patients will be randomly assigned 1:1 to induction with penpulimab (200mg, iv, d1, q3w) plus chemotherapy (TP: cisplatin [60-75mg/m2, iv, d1-d3, q3w] plus paclitaxel [135-175mg/m2, iv, d1, q3w] or albumin-bound paclitaxel [200-260mg/m2, iv, d1, q3w]) for 4-6 cycles followed by consolidation with penpulimab (200mg, iv, d1, q3w) (arm 1) or induction with penpulimab (200mg, iv, d1, q3w) plus anlotinib (10mg/d [<60kg] / 12mg/d [≥60kg], p.o., d1~14, q3w) plus chemotherapy (TP: cisplatin [60-75mg/m2, iv, d1-d3, q3w] plus paclitaxel [135-175mg/m2, iv, d1, q3w] or albumin-bound paclitaxel [200-260mg/m2, iv, d1, q3w]) for 4-6 cycles followed by consolidation with penpulimab (200mg, iv, d1, q3w) and anlotinib (10mg/d [<60kg)/12mg/d [≥60kg], p.o., d1~14, q3w) (arm 2). Randomization will be stratified by gender (male or female), age (<65, >65), PD-L1 combined positive score (CPS; <1, ≥1, ≥5, ≥10), metastatic site, history of surgery and disease. Patients in arm 1 were treated with second-line anlotinib combined with penpulimab after progression until further disease progression. Patients in arm 2 will continue treated with first-line anlotinib combined with penpulimab until disease progression. Tumor imaging assessment will be performed q6w for 1-6 cycle and q9w thereafter. The primary endpoint was objective response rate (per RECIST v1.1 assessed by investigator). Secondary endpoints included disease control rate, progression-free survival (per RECIST v1.1 assessed by investigator), overall survival, safety and quality of life score. Enrollment in this trial is ongoing. Clinical trial information: NCT05214222 .

Application of nucleic acid-encoded antibodies in prevention and treatment of emerging viral infectious diseases

<p indent="0mm">Emerging viral infectious diseases are diverse, fast-spreading and dangerous and have caused many outbreaks and epidemics worldwide, with serious and far-reaching effects on human health, social stability and economic development. At present, vaccines, small-molecule drugs and monoclonal antibodies (mAbs) are important tools for addressing new outbreaks of viral infectious diseases and have played key roles in the prevention and treatment of multiple viral infectious diseases. In particular, mAbs can effectively prevent a virus from entering host cells and block reinfection by progeny virus. mAbs can also facilitate the clearance of viruses and virus-infected cells via Fc-mediated effector functions. In addition, the effects of mAbs are related to several characteristics, such as their high specificity, ability to enhance immune responses and low toxicity. Thus, mAbs are appealing as potential therapeutics and prophylactics for viral infections. However, traditional mAb technology still has some limitations, such as complex production processes, high quality control requirements, high manufacturing costs, high treatment costs, and the need for strict cold chain storage and transportation, making it unsuitable for large-scale population use. Unfortunately, to date, only a small number of mAb drugs have been used to treat or prevent infectious diseases. Therefore, there is a need to rapidly develop a novel antiviral tool. As a type of novel biological agent, nucleic acid-encoded antibodies are DNA and mRNA molecules that encode virus-specific neutralizing antibodies that are appropriately sequenced and optimally regulated to obtain stably expressed DNA and mRNA molecules <italic>in vitro</italic>, which are then introduced into a host through a special delivery vehicle. Then, the host cells are used as individual bioreactors, and the protein synthesis mechanism is used to express structurally and functionally intact antibodies, which in turn act as neutralizing agents <italic>in vivo</italic>. Compared to traditional mAbs, nucleic acid-encoded antibodies have many potential advantages, such as ease and speed of design and testing, ease of rapid scale-up and manufacturing, ease of storage and transportation, and cost-effectiveness; consequently, they are considered an ideal alternative to traditional mAb technology. At present, nucleic acid-encoded antibodies have been used in preclinical studies on diseases caused by many viruses, such as human immunodeficiency virus, influenza virus, Chikungunya virus, Zika virus and SARS-CoV-2. Specifically, three nucleic acid-encoded antibodies have entered phase I clinical trials and have promising applications in the prevention and treatment of emerging viral infectious diseases. In this paper, we summarize the principles and current research status of nucleic acid-encoded antibodies in antiviral control and highlight the advantages of nucleic acid-encoded antibodies over traditional protein antibodies, the advantages and disadvantages of different nucleic acid-encoded antibodies, and the potential challenges and possible strategies for the future development of these agents to provide new research ideas for the application of nucleic acid-encoded antibodies in the control of future emerging viral infectious diseases.

Class switch toward noninflammatory, spike-specific IgG4 antibodies after repeated SARS-CoV-2 mRNA vaccination.

RNA vaccines are efficient preventive measures to combat the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. High levels of neutralizing SARS-CoV-2 antibodies are an important component of vaccine-induced immunity. Shortly after the initial two mRNA vaccine doses, the immunoglobulin G (IgG) response mainly consists of the proinflammatory subclasses IgG1 and IgG3. Here, we report that several months after the second vaccination, SARS-CoV-2-specific antibodies were increasingly composed of noninflammatory IgG4, which were further boosted by a third mRNA vaccination and/or SARS-CoV-2 variant breakthrough infections. IgG4 antibodies among all spike-specific IgG antibodies rose, on average, from 0.04% shortly after the second vaccination to 19.27% late after the third vaccination. This induction of IgG4 antibodies was not observed after homologous or heterologous SARS-CoV-2 vaccination with adenoviral vectors. Single-cell sequencing and flow cytometry revealed substantial frequencies of IgG4-switched B cells within the spike-binding memory B cell population [median of 14.4%; interquartile range (IQR) of 6.7 to 18.1%] compared with the overall memory B cell repertoire (median of 1.3%; IQR of 0.9 to 2.2%) after three immunizations. This class switch was associated with a reduced capacity of the spike-specific antibodies to mediate antibody-dependent cellular phagocytosis and complement deposition. Because Fc-mediated effector functions are critical for antiviral immunity, these findings may have consequences for the choice and timing of vaccination regimens using mRNA vaccines, including future booster immunizations against SARS-CoV-2.

PP 8.5 – 00080 Fc-engineering of anti-HIV-1 antibodies and nanobodies to improve Fc mediated effector functions

PP 8.5 – 00080 Fc-engineering of anti-HIV-1 antibodies and nanobodies to improve Fc mediated effector functions

Fc-Mediated Antibody Effector Function, Inflammation Resolution and Oligoclonality on TCR Rearrangements Predict Sustained MRD Negativity in Newly Diagnosed Multiple Myeloma Treated with Immunotherapy Regimens

Background: Targeted immunotherapy combinations including anti-CD38 monoclonal antibody daratumumab have significantly increased the depth of response and clinical outcome in newly diagnosed multiple myeloma (NDMM). Despite this improvement, 30-40% of patients still progress early and fail to achieve sustained minimal residual disease (MRD) negativity through largely unknown resistance mechanisms. Here we integrate whole genome sequencing (WGS) and single cell RNA (scRNA), surface protein (CITE), and TCR sequencing from patients with NDMM to characterize genomic and immune microenvironment features related to the failure to achieve sustained remission. Methods: 5'single-cell RNA-sequencing with an additional capture of the TCR and surface protein markers (CITEseq) was performed at both induction (T1) and at the end of induction (T2) with DKRd (Daratumumab-Carfilzomib, Lenalidomide and dexamethasone). A total of 148,280 cells were analyzed for RNA and CITE from 17 patients at T1 and 18 at T2. Twenty-seven cell types were identified and changes in their gene expression were examined. Data were integrated with 40,000 healthy bone marrow cells from the Human Cell Atlas. For TCR-seq, a total of 22,882 ⍺β clonotypes were analyzed. Fc-mediated antibody effector function is associated with sustained MRD negativity The microenvironment changed drastically between diagnosis and end of induction. The achievement of sustained MRD negativity were associated with an expansion of activated CD56-bright NK cells at T1 (chi-squared=4, p=0.044). Furthermore, significant changes in the monocyte profile were seen between timepoints and response groups. We identified a subset of resident-like macrophages expressing markers such as IL1B and CXCL2 suggesting that there are some immune hot tumors that are associated with suboptimal responses. Although there weren't absolute differences in T-cell subsets, PCA analysis showed that the combination of CD8 effector 1, CD16+monocytes and CD56+NK profiles could be used to identify a subset of good responders achieving sustained MRD negativity. Inflammation resolution is associated with sustained MRD negativity when complete response is achieved, significant changes in the microenvironment were observed and expansion of CD16+ monocytes associated with sustained MRD negativity. These data were validated prospectively using flow cytometry (n=31). These data suggest that the presence of cells mediating Fc-dependent immune effector mechanisms at diagnosis could predict good response and the resolution of tumor induced inflammation after treatment is associated with anti-inflammatory CD16+ monocytes. Oligoclonality in T-cell is associated with treatment and response TCR analysis was available for 22,882 cells and showed that there were significantly fewer clonotypes between T1 and T2 (Kruskal-Wallis chi-squared = 4.0, df = 1, p-value = 0.044) reflecting both the impact of treatment, possible antigen selection, and/or the disappearance of the chronic antigen in the form of the malignant plasma cells. Patients that failed to achieve MRD negativity at T2 had significantly more clonotypes suggesting an ineffective T-cell response to residual tumor. Diversity analysis is currently ongoing. Conclusion: In this multilayered analysis we show changes that the composition of the microenvironment is related to the level of response in the context of DKRd treatment. Fc-mediated antibody effector function and inflammation resolution are associated with sustained MRD negativity phenotypes response suggesting a significant contribution of the microenvironment to response, opening the way for therapeutic manipulation to enhance response. TCR analysis reveals for the first time that oligoclonal profiles seen on treatment may influence the fitness of the immune response.

The influenza hemagglutinin stem antibody CR9114: Evidence for a narrow evolutionary path towards universal protection

Human monoclonal antibodies (hmAbs) that protect against all influenza A and B strains are considered the road to universal influenza vaccines. Based on publicly-available data, we analyze the mechanistic and structural basis of pan-influenza protection by CR9114, a hemagglutinin (HA) stem-reactive antibody that protects against influenza subtypes from groups A1, A2, and B. The mechanistic basis of CR9114’s universal protection is not limited to in vitro neutralization, as CR9114 also protects in vivo from strains that escape its neutralizing activity: some H2 strains and influenza B. Fusion inhibition, viral egress inhibition, and activation of Fc-mediated effector functions are key contributors to CR9114’s universal protection. A comparative analysis of paratopes – between CR9114 (pan-influenza protection) and structurally similar VH1-69 hmAb CR6261 (influenza A1 protection) – pinpoints the structural basis of pan-influenza protection. CR9114’s heterosubtypic binding is conferred by its ability to bind HA with multiple domains: three HCDR loops and FR3. In contrast to other VH1-69 hmAbs, CR9114 uses a long and polar side chain of tyrosine (Y) residues on its HCDR3 for crucial H-bonds with H3, H5, and B HA. The recognition of a highly conserved epitope by CR9114 results in a high genetic barrier for escape by influenza strains. The nested, hierarchical structure of the mutations between the germline ancestor and CR9114 demonstrates that it is the result of a narrow evolutionary pathway within the B cell population. This rare evolutionary pathway indicates an immuno-recessive epitope and limited opportunity for vaccines to induce a polyclonal CR9114-like response.

HIV-1 Vpu restricts Fc-mediated effector functions invivo.

Non-neutralizing antibodies (nnAbs) can eliminate HIV-1-infected cells via antibody-dependent cellular cytotoxicity (ADCC) and were identified as a correlate of protection in the RV144 vaccine trial. Fc-mediated effector functions of nnAbs were recently shown to alter the course of HIV-1 infection invivo using a vpu-defective virus. Since Vpu is known to downregulate cell-surface CD4, which triggers conformational changes in the viral envelope glycoprotein (Env), we ask whether the lack of Vpu expression was linked to the observed nnAbs activity. We find that restoring Vpu expression greatly reduces nnAb recognition of infected cells, rendering them resistant to ADCC. Moreover, administration of nnAbs in humanized mice reduces viral loads only in animals infected with a vpu-defective but not with a wild-type virus. CD4-mimetics administration, known to "open" Env and expose nnAb epitopes, renders wild-type viruses sensitive to nnAbs Fc-effector functions. This work highlights the importance of Vpu-mediated evasion of humoral responses.

Enhanced delivery of antibodies across the blood-brain barrier via TEMs with inherent receptor-mediated phagocytosis

The near impermeability of the blood-brain barrier (BBB) and the unique neuroimmune environment of the CNS prevents the effective use of antibodies in neurological diseases. Delivery of biotherapeutics to the brain can be enabled through receptor-mediated transcytosis via proteins such as the transferrin receptor, although limitations such as the ability to use Fc-mediated effector function to clear pathogenic targets can introduce safety liabilities. Hence, novel delivery approaches with alternative clearance mechanisms are warranted. Binders that optimized transport across the BBB, known as transcytosis-enabling modules (TEMs), were identified using a combination of antibody discovery techniques and pharmacokinetic analyses. Functional activity of TEMs were subsequently evaluated by imaging for the ability of myeloid cells to phagocytose target proteins and cells. We demonstrated significantly enhanced brain exposure of therapeutic antibodies using optimal transferrin receptor or CD98 TEMs. We found that these modules also mediated efficient clearance of tau aggregates and HER2+ tumor cells via a non-classical phagocytosis mechanism through direct engagement of myeloid cells. This mode of clearance potentially avoids the known drawbacks of FcγR-mediated antibody mechanisms in the brain such as the neurotoxic release of proinflammatory cytokines and immune cell exhaustion. Our study reports a new brain delivery platform that harnesses receptor-mediated transcytosis to maximize brain uptake and uses a non-classical phagocytosis mechanism to efficiently clear pathologic proteins and cells. We believe these findings will transform therapeutic approaches to treat CNS diseases. This research was funded by Janssen, Pharmaceutical Companies of Johnson & Johnson.

Decoding human-macaque interspecies differences in Fc-effector functions: The structural basis for CD16-dependent effector function in Rhesus macaques.

Fc mediated effector functions of antibodies play important roles in immunotherapies and vaccine efficacy but assessing those functions in animal models can be challenging due to species differences. Rhesus macaques, Macaca mulatta (Mm) share approximately 93% sequence identity with humans but display important differences in their adaptive immune system that complicates their use in validating therapeutics and vaccines that rely on Fc effector functions. In contrast to humans, macaques only have one low affinity FcγRIII receptor, CD16, which shares a polymorphism at position 158 with human FcγRIIIa with Ile158 and Val158 variants. Here we describe structure-function relationships of the Ile/Val158 polymorphism in Mm FcγRIII. Our data indicate that the affinity of the allelic variants of Mm FcγRIII for the macaque IgG subclasses vary greatly with changes in glycan composition both on the Fc and the receptor. However, unlike the human Phe/Val158 polymorphism in FcγRIIIa, the higher affinity variant corresponds to the larger, more hydrophobic side chain, Ile, even though it is not directly involved in the binding interface. Instead, this side chain appears to modulate glycan-glycan interactions at the Fc/FcγRIII interface. Furthermore, changes in glycan composition on the receptor have a greater effect for the Val158 variant such that with oligomannose type glycans and with glycans only on Asn45 and Asn162, Val158 becomes the variant with higher affinity to Fc. These results have implications not only for the better interpretation of nonhuman primate studies but also for studies performed with human effector cells carrying different FcγRIIIa alleles.