Endocytosis Inhibition in Humans to Improve Responses to ADCC-Mediating Antibodies.

A safe and controlled manipulation of endocytosis in vivo may have disruptive therapeutic potential. Here, we demonstrate that the anti-emetic/anti-psychotic prochlorperazine can be repurposed to reversibly inhibit the in vivo endocytosis of membrane proteins targeted by therapeutic monoclonal antibodies, as directly demonstrated by our human tumor ex vivo assay. Temporary endocytosis inhibition results in enhanced target availability and improved efficiency of natural killer cell-mediated antibody-dependent cellular cytotoxicity (ADCC), a mediator of clinical responses induced by IgG1 antibodies, demonstrated here for cetuximab, trastuzumab, and avelumab. Extensive analysis of downstream signaling pathways ruled out on-target toxicities. By overcoming the heterogeneity of drug target availability that frequently characterizes poorly responsive or resistant tumors, clinical application of reversible endocytosis inhibition may considerably improve the clinical benefit of ADCC-mediating therapeutic antibodies.

Immune Checkpoint Inhibition Overcomes ADCP-Induced Immunosuppression by Macrophages.

Antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) critically contribute to the efficacy of anti-tumor therapeutic antibodies. We report here an unexpected finding that macrophages after ADCP inhibit NK cell-mediated ADCC and T cell-mediated cytotoxicity in breast cancers and lymphomas. Mechanistically, AIM2 is recruited to the phagosomes by FcγR signaling following ADCP and activated by sensing the phagocytosed tumor DNAs through the disrupted phagosomal membrane, which subsequently upregulates PD-L1 and IDO and causes immunosuppression. Combined treatment with anti-HER2 antibody and inhibitors of PD-L1 and IDO enhances anti-tumor immunity and anti-HER2 therapeutic efficacy in mouse models. Furthermore, neoadjuvant trastuzumab therapy significantly upregulates PD-L1 and IDO in the tumor-associated macrophages (TAMs) of HER2+ breast cancer patients, correlating with poor trastuzumab response. Collectively, our findings unveil a deleterious role of ADCP macrophages in cancer immunosuppression and suggest that therapeutic antibody plus immune checkpoint blockade may provide synergistic effects in cancer treatment.

A Fc engineering approach to define functional humoral correlates of immunity against Ebola virus.

Protective Ebola virus (EBOV) antibodies have neutralizing activity and induction of antibody constant domain (Fc)-mediated innate immune effector functions. Efforts to enhance Fc effector functionality often focus on maximizing antibody-dependent cellular cytotoxicity, yet distinct combinations of functions could be critical for antibody-mediated protection. As neutralizing antibodies have been cloned from EBOV disease survivors, we sought to identify survivor Fc effector profiles to help guide Fc optimization strategies. Survivors developed a range of functional antibody responses, and we therefore applied a rapid, high-throughput Fc engineering platform to define the most protective profiles. We generated a library of Fc variants with identical antigen-binding fragments (Fabs) from an EBOV neutralizing antibody. Fc variants with antibody-mediated complement deposition and moderate natural killer (NK) cell activity demonstrated complete protective activity in a stringent in vivo mouse model. Our findings highlight the importance of specific effector functions in antibody-mediated protection, and the experimental platform presents a generalizable resource for identifying correlates of immunity to guide therapeutic antibody design.

Inter-cellular CRISPR screens reveal regulators of cancer cell phagocytosis.

Monoclonal antibody therapies targeting tumour antigens drive cancer cell elimination in large part by triggering macrophage phagocytosis of cancer cells1-7. However, cancer cells evade phagocytosis using mechanisms that are incompletely understood. Here we develop a platform for unbiased identification of factors that impede antibody-dependent cellular phagocytosis (ADCP) using complementary genome-wide CRISPR knockout and overexpression screens in both cancer cells and macrophages. In cancer cells, beyond known factors such as CD47, we identify many regulators of susceptibility to ADCP, including the poorly characterized enzyme adipocyte plasma membrane-associated protein (APMAP). We find that loss of APMAP synergizes with tumour antigen-targeting monoclonal antibodies and/or CD47-blocking monoclonal antibodies to drive markedly increased phagocytosis across a wide range of cancer cell types, including those that are otherwise resistant to ADCP. Additionally, we show that APMAP loss synergizes with several different tumour-targeting monoclonal antibodies to inhibit tumour growth in mice. Using genome-wide counterscreens in macrophages, we find that the G-protein-coupled receptor GPR84 mediates enhanced phagocytosis of APMAP-deficient cancer cells. This work reveals a cancer-intrinsic regulator of susceptibility to antibody-driven phagocytosis and, more broadly, expands our knowledge of the mechanisms governing cancer resistance to macrophage phagocytosis.

The S2 subunit of spike encodes diverse targets for functional antibody responses to SARS-CoV-2.

The SARS-CoV-2 virus responsible for the COVID-19 global pandemic has exhibited a striking capacity for viral evolution that drives continued evasion from vaccine and infection-induced immune responses. Mutations in the receptor binding domain of the S1 subunit of the spike glycoprotein have led to considerable escape from antibody responses, reducing the efficacy of vaccines and monoclonal antibody (mAb) therapies. Therefore, there is a need to interrogate more constrained regions of spike, such as the S2 subdomain. Here, we present a collection of S2 mAbs from two SARS-CoV-2 convalescent individuals that target multiple regions in S2, including regions outside of those commonly reported. One of the S2 mAbs, C20.119, which bound to a highly conserved epitope in the fusion peptide, was able to broadly neutralize across SARS-CoV-2 variants, SARS-CoV-1, and closely related zoonotic sarbecoviruses. The majority of the mAbs were non-neutralizing; however, many of them could mediate antibody-dependent cellular cytotoxicity (ADCC) at levels similar to the S1-targeting mAb S309 that was previously authorized for treatment of SARS-CoV-2 infections. Several of the mAbs with ADCC function also bound to spike trimers from other human coronaviruses (HCoVs), such as MERS-CoV and HCoV-HKU1. Our findings suggest S2 mAbs can target diverse epitopes in S2, including functional mAbs with HCoV and sarbecovirus breadth that likely target functionally constrained regions of spike. These mAbs could be developed for potential future pandemics, while also providing insight into ideal epitopes for eliciting a broad HCoV response.

Engineering Dimeric EGFR-directed IgA Antibodies Reveals a Central Role of CD147 during Neutrophil-mediated Tumor Cell Killing of Head and Neck Squamous Cancer Cells.

Human IgA Abs engage neutrophils for cancer immunotherapy more effectively than IgG Abs. Previous studies demonstrated that engineering approaches improved biochemical and functional properties. In this study, we report a novel, to our knowledge, IgA2 Ab against the epidermal growth factor receptor generated by protein engineering and polymerization. The resulting molecule demonstrated a covalent linkage of L and H chains and an effective polymerization by the joining chain. The engineered dimer outperformed its monomeric variant in functional experiments on Fab-mediated modes of action and binding to the Fc receptor. The capacity to engage neutrophils for Ab-dependent cell-mediated cytotoxicity (ADCC) of adherent growing target cancer cells was cell line dependent. Although the engineered dimer displayed a long-term efficacy against the vulva carcinoma cell line A431, there was a notable in-efficacy against human papillomavirus (HPV)- head and neck squamous cell carcinoma (HNSCC) cell lines. However, the highly engineered IgA Abs triggered a neutrophil-mediated cytotoxicity against HPV+ HNSCC cell lines. Short-term ADCC efficacy correlated with the target cells' epidermal growth factor receptor expression and the ability of cancer cell-conditioned media to enhance the CD147 surface level on neutrophils. Notably, the HPV+ HNSCC cell lines demonstrated a significant increment in releasing soluble CD147 and a reduced induction of membranous CD147 on neutrophils compared with HPV- cells. Although membranous CD147 on neutrophils may impair proper IgA-Fc receptor binding, soluble CD147 enhanced the IgA-neutrophil-mediated ADCC in a dose-dependent manner. Thus, engineering IgA Abs and impedance-based ADCC assays provided valuable information regarding the target-effector cell interaction and identified CD147 as a putative critical parameter for neutrophil-mediated cytotoxicity.

Sequence Matters: Primary COVID-19 Vaccination after Infection Elicits Similar Anti-spike Antibody Levels, but Stronger Antibody Dependent Cell-mediated Cytotoxicity than Breakthrough Infection.

Infection before primary vaccination (herein termed "hybrid immunity") engenders robust humoral immunity and broad Ab-dependent cell-mediated cytotoxicity (ADCC) across SARS-CoV-2 variants. We measured and compared plasma IgG and IgA against Wuhan-Hu-1 and Omicron (B.1.1.529) full-length spike (FLS) and receptor binding domain after three mRNA vaccines encoding Wuhan-Hu-1 spike (S) and after Omicron breakthrough infection. We also measured IgG binding to Wuhan-Hu-1 and Omicron S1, Wuhan-Hu-1 S2 and Wuhan-Hu-1 and Omicron cell-based S. We compared ADCC using human embryonic lung fibroblast (MRC-5) cells expressing Wuhan-Hu-1 or Omicron S. The effect of Omicron breakthrough infection on IgG anti-Wuhan-Hu-1 and Omicron FLS avidity was also considered. Despite Omicron breakthrough infection increasing IgG and IgA against FLS and receptor binding domain to levels similar to those seen with hybrid immunity, there was no boost to ADCC. Preferential recognition of Wuhan-Hu-1 persisted following Omicron breakthrough infection, which increased IgG avidity against Wuhan-Hu-1 FLS. Despite similar total anti-FLS IgG levels following breakthrough infection, 4-fold higher plasma concentrations were required to elicit ADCC comparable to that elicited by hybrid immunity. The greater capacity for hybrid immunity to elicit ADCC was associated with a differential IgG reactivity pattern against S1, S2, and linear determinants throughout FLS. Immunity against SARS-CoV-2 following Omicron breakthrough infection manifests significantly less ADCC capacity than hybrid immunity. Thus, the sequence of antigenic exposure by infection versus vaccination and other factors such as severity of infection affect antiviral functions of humoral immunity in the absence of overt quantitative differences in the humoral response.

Structural basis for RIFIN-mediated activation of LILRB1 in malaria.

The Plasmodium species that cause malaria are obligate intracellular parasites, and disease symptoms occur when these parasites replicate in human blood. Despite the risk of immune detection, the parasite delivers proteins that bind to host receptors on the cell surfaces of infected erythrocytes. In the causative parasite of the most deadly form of malaria in humans, Plasmodium falciparum, RIFINs form the largest family of surface proteins displayed by erythrocytes1. Some RIFINs can bind to inhibitory immune receptors, and these RIFINs act as targets for unusual antibodies that contain a LAIR1 ectodomain2-4 or as ligands for LILRB15. RIFINs stimulate the activation of and signalling by LILRB15, which could potentially lead to the dampening of human immune responses. Here, to understand how RIFINs activate LILRB1-mediated signalling, we determine the structure of a RIFIN bound to LILRB1. We show that this RIFIN mimics the natural activating ligand of LILRB1, MHC class I, in its LILRB1-binding mode. A single mutation in the RIFIN disrupts the complex, blocks LILRB1 binding of all tested RIFINs and abolishes signalling in a reporter assay. In a supported lipid bilayer system, which mimics the activation of natural killer (NK) cells by antibody-dependent cell-mediated cytotoxicity, both RIFIN and MHC are recruited to the immunological synapse of NK cells and reduce the activation of NK cells, as measured by the mobilization of perforin. Therefore, LILRB1-binding RIFINs mimic the binding mode of the natural ligand of LILRB1 and suppress the function of NK cells.

Cross-neutralization of SARS-CoV-2 by a human monoclonal SARS-CoV antibody.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a newly emerged coronavirus that is responsible for the current pandemic of coronavirus disease 2019 (COVID-19), which has resulted in more than 3.7 million infections and 260,000 deaths as of 6 May 20201,2. Vaccine and therapeutic discovery efforts are paramount to curb the pandemic spread of this zoonotic virus. The SARS-CoV-2 spike (S) glycoprotein promotes entry into host cells and is the main target of neutralizing antibodies. Here we describe several monoclonal antibodies that target the S glycoprotein of SARS-CoV-2, which we identified from memory B cells of an individual who was infected with severe acute respiratory syndrome coronavirus (SARS-CoV) in 2003. One antibody (named S309) potently neutralizes SARS-CoV-2 and SARS-CoV pseudoviruses as well as authentic SARS-CoV-2, by engaging the receptor-binding domain of the S glycoprotein. Using cryo-electron microscopy and binding assays, we show that S309 recognizes an epitope containing a glycan that is conserved within the Sarbecovirus subgenus, without competing with receptor attachment. Antibody cocktails that include S309 in combination with other antibodies that we identified further enhanced SARS-CoV-2 neutralization, and may limit the emergence of neutralization-escape mutants. These results pave the way for using S309 and antibody cocktails containing S309 for prophylaxis in individuals at a high risk of exposure or as a post-exposure therapy to limit or treat severe disease.

Three families of CD4-induced antibodies are associated with the capacity of plasma from people living with HIV to mediate ADCC in the presence of CD4-mimetics.

CD4-mimetics (CD4mcs) are small molecule compounds that mimic the interaction of the CD4 receptor with HIV-1 envelope glycoproteins (Env). Env from primary viruses normally samples a "closed" conformation that occludes epitopes recognized by CD4-induced (CD4i) non-neutralizing antibodies (nnAbs). CD4mcs induce conformational changes on Env resulting in the exposure of these otherwise inaccessible epitopes. Here, we evaluated the capacity of plasma from a cohort of 50 people living with HIV to recognize HIV-1-infected cells and eliminate them by antibody-dependent cellular cytotoxicity (ADCC) in the presence of a potent indoline CD4mc. We observed a marked heterogeneity among plasma samples. By measuring the levels of different families of CD4i Abs, we found that the levels of anti-cluster A, anti-coreceptor binding site, and anti-gp41 cluster I antibodies are responsible for plasma-mediated ADCC in the presence of CD4mc. IMPORTANCE: There are several reasons that make it difficult to target the HIV reservoir. One of them is the capacity of infected cells to prevent the recognition of HIV-1 envelope glycoproteins (Env) by commonly elicited antibodies in people living with HIV. Small CD4-mimetic compounds expose otherwise occluded Env epitopes, thus enabling their recognition by non-neutralizing antibodies (nnAbs). A better understanding of the contribution of these antibodies to eliminate infected cells in the presence of CD4mc could lead to the development of therapeutic cure strategies.

SARS-CoV-2 BA.4/5 infection triggers more cross-reactive FcγRIIIa signaling and neutralization than BA.1, in the context of hybrid immunity.

SARS-CoV-2 variants of concern (VOCs) differentially trigger neutralizing and antibody-dependent cellular cytotoxic (ADCC) antibodies with variable cross-reactivity. Omicron BA.4/5 was approved for inclusion in bivalent vaccination boosters, and therefore the antigenic profile of antibodies elicited by this variant is critical to understand. Here, we investigate the ability of BA.4/5-elicited antibodies following the first documented (primary) infection (n = 13) or breakthrough infection after vaccination (n = 9) to mediate neutralization and FcγRIIIa signaling across multiple SARS-CoV-2 variants including XBB.1.5 and BQ.1. Using a pseudovirus neutralization assay and a FcγRIIIa crosslinking assay to measure ADCC potential, we show that unlike SARS-CoV-2 Omicron BA.1, BA.4/5 infection triggers highly cross-reactive functional antibodies. Cross-reactivity was observed both in the absence of prior vaccination and in breakthrough infections following vaccination. However, BQ.1 and XBB.1.5 neutralization and FcγRIIIa signaling were significantly compromised compared to other VOCs, regardless of prior vaccination status. BA.4/5 triggered FcγRIIIa signaling was significantly more resilient against VOCs (<10-fold decrease in magnitude) compared to neutralization (10- to 100-fold decrease). Overall, this study shows that BA.4/5 triggered antibodies are highly cross-reactive compared to those triggered by other variants. Although this is consistent with enhanced neutralization and FcγRIIIa signaling breadth of BA.4/5 vaccine boosters, the reduced activity against XBB.1.5 supports the need to update vaccines with XBB sublineage immunogens to provide adequate coverage of these highly antibody evasive variants. IMPORTANCE: The continued evolution of SARS-CoV-2 has resulted in a number of variants of concern. Of these, the Omicron sublineage is the most immune evasive. Within Omicron, the BA.4/5 sublineage drove the fifth wave of infection in South Africa prior to becoming the dominant variant globally. As a result this spike sequence was approved as part of a bivalent vaccine booster, and rolled out worldwide. We aimed to understand the cross-reactivity of neutralizing and Fc mediated cytotoxic functions elicited by BA.4/5 infection following infection or breakthrough infection. We find that, in contrast to BA.1 which triggered fairly strain-specific antibodies, BA.4/5 triggered antibodies that are highly cross-reactive for neutralization and antibody-dependent cellular cytotoxicity potential. Despite this cross-reactivity, these antibodies are compromised against highly resistant variants such as XBB.1.5 and BQ.1. This suggests that next-generation vaccines will require XBB sublineage immunogens in order to protect against these evasive variants.

Single-shot dendritic cell targeting SARS-CoV-2 vaccine candidate induces broad, durable and protective systemic and mucosal immunity in mice.

Current coronavirus disease 2019 vaccines face limitations including waning immunity, immune escape by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants, limited cellular response, and poor mucosal immunity. We engineered a Clec9A-receptor binding domain (RBD) antibody construct that delivers the SARS-CoV-2 RBD to conventional type 1 dendritic cells. Compared with non-targeting approaches, single dose immunization in mice with Clec9A-RBD induced far higher RBD-specific antibody titers that were sustained for up to 21 months after vaccination. Uniquely, increasing neutralizing and antibody-dependent cytotoxicity activities across the sarbecovirus family was observed, suggesting antibody affinity maturation over time. Consistently and remarkably, RBD-specific follicular T helper cells and germinal center B cells persisted up to 12 months after immunization. Furthermore, Clec9A-RBD immunization induced a durable mono- and poly-functional T-helper 1-biased cellular response that was strongly cross-reactive against SARS-CoV-2 variants of concern, including Omicron subvariants, and with a robust CD8+ T cell signature. Uniquely, Clec9A-RBD single-shot systemic immunization effectively primed RBD-specific cellular and humoral immunity in lung and resulted in significant protection against homologous SARS-CoV-2 challenge as evidenced by limited body weight loss and approximately 2 log10 decrease in lung viral loads compared with non-immunized controls. Therefore, Clec9A-RBD immunization has the potential to trigger robust and sustained, systemic and mucosal protective immunity against rapidly evolving SARS-CoV2 variants.

Engineered human antibodies for the opsonization and killing of Staphylococcus aureus.

Gram-positive organisms with their thick envelope cannot be lysed by complement alone. Nonetheless, antibody-binding on the surface can recruit complement and mark these invaders for uptake and killing by phagocytes, a process known as opsonophagocytosis. The crystallizable fragment of immunoglobulins (Fcγ) is key for complement recruitment. The cell surface of S. aureus is coated with Staphylococcal protein A (SpA). SpA captures the Fcγ domain of IgG and interferes with opsonization by anti-S. aureus antibodies. In principle, the Fcγ domain of therapeutic antibodies could be engineered to avoid the inhibitory activity of SpA. However, the SpA-binding site on Fcγ overlaps with that of the neonatal Fc receptor (FcRn), an interaction that is critical for prolonging the half-life of serum IgG. This evolutionary adaptation poses a challenge for the exploration of Fcγ mutants that can both weaken SpA-IgG interactions and retain stability. Here, we use both wild-type and transgenic human FcRn mice to identify antibodies with enhanced half-life and increased opsonophagocytic killing in models of S. aureus infection and demonstrate that antibody-based immunotherapy can be improved by modifying Fcγ. Our experiments also show that by competing for FcRn-binding, staphylococci effectively reduce the half-life of antibodies during infection. These observations may have profound impact in treating cancer, autoimmune, and asthma patients colonized or infected with S. aureus and undergoing monoclonal antibody treatment.

Levels and functionality of Pacific Islanders' hybrid humoral immune response to BNT162b2 vaccination and delta/omicron infection: A cohort study in New Caledonia.

BACKGROUND: Pacific Islanders are underrepresented in vaccine efficacy trials. Few studies describe their immune response to COVID-19 vaccination. Yet, this characterization is crucial to re-enforce vaccination strategies adapted to Pacific Islanders singularities. METHODS AND FINDINGS: We evaluated the humoral immune response of 585 adults, self-declaring as Melanesians, Europeans, Polynesians, or belonging to other communities, to the Pfizer BNT162b2 vaccine. Anti-spike and anti-nucleoprotein IgG levels, and their capacity to neutralize SARS-CoV-2 variants and to mediate antibody-dependent cellular cytotoxicity (ADCC) were assessed across communities at 1 and 3 months post-second dose or 1 and 6 months post-third dose. All sera tested contained anti-spike antibodies and 61.3% contained anti-nucleoprotein antibodies, evidencing mostly a hybrid immunity resulting from vaccination and SARS-CoV-2 infection. At 1-month post-immunization, the 4 ethnic communities exhibited no significant differences in their anti-spike IgG levels (p value = 0.17, in an univariate linear regression model), in their capacity to mediate omicron neutralization (p value = 0.59 and 0.60, in an univariate logistic regression model at 1-month after the second and third dose, respectively) and in their capacity to mediate ADCC (p value = 0.069 in a multivariate linear regression model), regardless of the infection status. Anti-spike IgG levels and functionalities of the hybrid humoral immune response remained equivalent across the 4 ethnic communities during follow-up and at 6 months post-third dose. CONCLUSIONS: Our study evidenced Pacific Islander's robust humoral immune response to Pfizer BNT162b2 vaccine, which is pivotal to re-enforce vaccination deployment in a population at risk for severe COVID-19. TRIAL REGISTRATION: This trial has been register in ClinicalTrials.gov (ID: NCT05135585).

The relevance of tumor target expression levels on IgA-mediated cytotoxicity in cancer immunotherapy.

Recent advances in cancer immunotherapy, particularly the success of immune checkpoint inhibitors, have reignited interest in targeted monoclonal antibodies for immunotherapy. Antibody therapies aim to minimize on-target, off-tumor toxicity by targeting antigens overexpressed on tumor cells but not on healthy cells. Despite considerable efforts, some therapeutic antibodies have been linked to dose-limiting side effects. Our hypothesis suggests that the efficacy of IgG leads to a lower target expression threshold for tumor cell killing, contributing to these side effects. Earlier, therapeutic IgG antibodies were reformatted into the IgA isotype. Unlike IgG, which primarily engages Fc gamma receptors (FcγR) to induce antibody-dependent cellular cytotoxicity (ADCC) by NK cells and antibody-dependent cellular phagocytosis (ADCP) by monocytes/macrophages, IgA antibodies activate neutrophils through the Fc alpha receptor I (CD89, FcαRI). In previous studies, it appeared that IgA may require a higher target expression threshold for effective killing, and we aimed to investigate this in our current study. Moreover, we investigated how blocking the myeloid checkpoint CD47/SIRPα axis affect the target expression threshold. Using a tetracycline-inducible expression system, we regulated target expression in different cell lines. Our findings from ADCC assays indicate that IgA-mediated PMN ADCC requires a higher antigen expression level than IgG-mediated PBMC ADCC. Furthermore, blocking CD47 enhanced IgA-mediated ADCC, lowering the antigen threshold. Validated in two in vivo models, our results show that IgA significantly reduces tumor growth in high-antigen-expressing tumors without affecting low-antigen-expressing healthy tissues. This suggests IgA-based immunotherapy could potentially minimize on-target, off-tumor side effects, improving treatment efficacy and patient safety.

Therapeutic antibodies in oncology: an immunopharmacological overview.

The biotechnological development of monoclonal antibodies and their immunotherapeutic use in oncology have grown exponentially in the last decade, becoming the first-line therapy for some types of cancer. Their mechanism of action is based on the ability to regulate the immune system or by interacting with targets that are either overexpressed in tumor cells, released into the extracellular milieu or involved in processes that favor tumor growth. In addition, the intrinsic characteristics of each subclass of antibodies provide specific effector functions against the tumor by activating antibody-dependent cellular cytotoxicity, complement-dependent cytotoxicity, and antibody-dependent cellular phagocytosis, among other mechanisms. The rational design and engineering of monoclonal antibodies have improved their pharmacokinetic and pharmacodynamic features, thus optimizing the therapeutic regimens administered to cancer patients and improving their clinical outcomes. The selection of the immunoglobulin G subclass, modifications to its crystallizable region (Fc), and conjugation of radioactive substances or antineoplastic drugs may all improve the antitumor effects of therapeutic antibodies. This review aims to provide insights into the immunological and pharmacological aspects of therapeutic antibodies used in oncology, with a rational approach at molecular modifications that can be introduced into these biological tools, improving their efficacy in the treatment of cancer.

A recombinant herpes virus expressing influenza hemagglutinin confers protection and induces antibody-dependent cellular cytotoxicity.

Despite widespread yearly vaccination, influenza leads to significant morbidity and mortality across the globe. To make a more broadly protective influenza vaccine, it may be necessary to elicit antibodies that can activate effector functions in immune cells, such as antibody-dependent cellular cytotoxicity (ADCC). There is growing evidence supporting the necessity for ADCC in protection against influenza and herpes simplex virus (HSV), among other infectious diseases. An HSV-2 strain lacking the essential glycoprotein D (gD), was used to create ΔgD-2, which is a highly protective vaccine against lethal HSV-1 and HSV-2 infection in mice. It also elicits high levels of IgG2c antibodies that bind FcγRIV, a receptor that activates ADCC. To make an ADCC-eliciting influenza vaccine, we cloned the hemagglutinin (HA) gene from an H1N1 influenza A strain into the ΔgD-2 HSV vector. Vaccination with ΔgD-2::HAPR8 was protective against homologous influenza challenge and elicited an antibody response against HA that inhibits hemagglutination (HAI+), is predominantly IgG2c, strongly activates FcγRIV, and protects against influenza challenge following passive immunization of naïve mice. Prior exposure of mice to HSV-1, HSV-2, or a replication-defective HSV-2 vaccine (dl5-29) does not reduce protection against influenza by ΔgD-2::HAPR8 This vaccine also continues to elicit protection against both HSV-1 and HSV-2, including high levels of IgG2c antibodies against HSV-2. Mice lacking the interferon-α/ß receptor and mice lacking the interferon-γ receptor were also protected against influenza challenge by ΔgD-2::HAPR8 Our results suggest that ΔgD-2 can be used as a vaccine vector against other pathogens, while also eliciting protective anti-HSV immunity.

Innate immune signatures to a partially-efficacious HIV vaccine predict correlates of HIV-1 infection risk.

The pox-protein regimen tested in the RV144 trial is the only vaccine strategy demonstrated to prevent HIV-1 infection. Subsequent analyses identified antibody and cellular immune responses as correlates of risk (CoRs) for HIV infection. Early predictors of these CoRs could provide insight into vaccine-induced protection and guide efforts to enhance vaccine efficacy. Using specimens from a phase 1b trial of the RV144 regimen in HIV-1-uninfected South Africans (HVTN 097), we profiled innate responses to the first ALVAC-HIV immunization. PBMC transcriptional responses peaked 1 day post-vaccination. Type I and II interferon signaling pathways were activated, as were innate pathways critical for adaptive immune priming. We then identified two innate immune transcriptional signatures strongly associated with adaptive immune CoR after completion of the 4-dose regimen. Day 1 signatures were positively associated with antibody-dependent cellular cytotoxicity and phagocytosis activity at Month 6.5. Conversely, a signature present on Days 3 and 7 was inversely associated with Env-specific CD4+ T cell responses at Months 6.5 and 12; rapid resolution of this signature was associated with higher Env-specific CD4+ T-cell responses. These are the first-reported early immune biomarkers of vaccine-induced responses associated with HIV-1 acquisition risk in humans and suggest hypotheses to improve HIV-1 vaccine regimens.

A new synthesis for antibody-mediated immunity.

The view that immunoglobulins function largely by potentiating neutralization, cytotoxicity or phagocytosis is being replaced by a new synthesis whereby antibodies participate in all aspects of the immune response, from protecting the host at the earliest time of encounter with a microbe to later challenges. Perhaps the most transformative concept is that immunoglobulins manifest emergent properties, from their structure and function as individual molecules to their interactions with microbial targets and the host immune system. Given that emergent properties are neither reducible to first principles nor predictable, there is a need for new conceptual approaches for understanding antibody function and mechanisms of antibody immunity.

Human Cytomegalovirus Infection Promotes Expansion of a Functionally Superior Cytoplasmic CD3+ NK Cell Subset with a Bcl11b-Regulated T Cell Signature.

Human CMV (HCMV) is a ubiquitous pathogen that indelibly shapes the NK cell repertoire. Using transcriptomic, epigenomic, and proteomic approaches to evaluate peripheral blood NK cells from healthy human volunteers, we find that prior HCMV infection promotes NK cells with a T cell-like gene profile, including the canonical markers CD3ε, CD5, and CD8ß, as well as the T cell lineage-commitment transcription factor Bcl11b. Although Bcl11b expression is upregulated during NK maturation from CD56bright to CD56dim, we find a Bcl11b-mediated signature at the protein level for FcεRIγ, PLZF, IL-2Rß, CD3γ, CD3δ, and CD3ε in later-stage, HCMV-induced NK cells. BCL11B is targeted by Notch signaling in T cell development, and culture of NK cells with Notch ligand increases cytoplasmic CD3ε expression. The Bcl11b-mediated gain of CD3ε, physically associated with CD16 signaling molecules Lck and CD247 in NK cells is correlated with increased Ab-dependent effector function, including against HCMV-infected cells, identifying a potential mechanism for their prevalence in HCMV-infected individuals and their prospective clinical use in Ab-based therapies.