Targeting the Antibody Checkpoints to Enhance Cancer Immunotherapy–Focus on FcγRIIB

Immunotherapy with therapeutic antibodies has increased survival for patients with hematologic and solid cancers. Still, a significant fraction of patients fails to respond to therapy or acquire resistance. Understanding and overcoming mechanisms of resistance to antibody drugs, and in particular those common to antibody drugs as a class, is therefore highly warranted and holds promise to improve response rates, duration of response and potentially overall survival. Activating and inhibitory Fc gamma receptors (FcγR) are known to coordinately regulate therapeutic activity of tumor direct-targeting antibodies. Similar, but also divergent, roles for FcγRs in controlling efficacy of immune modulatory antibodies e.g., checkpoint inhibitors have been indicated from mouse studies, and were recently implicated in contributing to efficacy in the human clinical setting. Here we discuss evidence and mechanisms by which Fc gamma receptors–the “antibody checkpoints”–regulate antibody-induced antitumor immunity. We further discuss how targeted blockade of the sole known inhibitory antibody checkpoint FcγRIIB may help overcome resistance and boost activity of clinically validated and emerging antibodies in cancer immunotherapy

Interleukin 8 Receptor Deficiency Confers Susceptibility to Acute Experimental Pyelonephritis and May Have a Human Counterpart

Neutrophils migrate to infected mucosal sites that they protect against invading pathogens. Their interaction with the epithelial barrier is controlled by CXC chemokines and by their receptors. This study examined the change in susceptibility to urinary tract infection (UTI) after deletion of the murine interleukin 8 receptor homologue (mIL-8Rh). Experimental UTIs in control mice stimulated an epithelial chemokine response and increased chemokine receptor expression. Neutrophils migrated through the tissues to the epithelial barrier that they crossed into the lumen, and the mice developed pyuria. In mIL-8Rh knockout (KO) mice, the chemokine response was intact, but the epithelial cells failed to express IL-8R, and neutrophils accumulated in the tissues. The KO mice were unable to clear bacteria from kidneys and bladders and developed bacteremia and symptoms of systemic disease, but control mice were fully resistant to infection. The experimental UTI model demonstrated that IL-8R–dependent mechanisms control the urinary tract defense, and that neutrophils are essential host effector cells. Patients prone to acute pyelonephritis also showed low CXC chemokine receptor 1 expression compared with age-matched controls, suggesting that chemokine receptor expression may also influence the susceptibility to UTIs in humans. The results provide a first molecular clue to disease susceptibility of patients prone to acute pyelonephritis

Fc Effector Function Contributes to the Activity of Human Anti-CTLA-4 Antibodies.

With the use of a mouse model expressing human Fc-gamma receptors (FcγRs), we demonstrated that antibodies with isotypes equivalent to ipilimumab and tremelimumab mediate intra-tumoral regulatory T (Treg) cell depletion in vivo, increasing the CD8+ to Treg cell ratio and promoting tumor rejection. Antibodies with improved FcγR binding profiles drove superior anti-tumor responses and survival. In patients with advanced melanoma, response to ipilimumab was associated with the CD16a-V158F high affinity polymorphism. Such activity only appeared relevant in the context of inflamed tumors, explaining the modest response rates observed in the clinical setting. Our data suggest that the activity of anti-CTLA-4 in inflamed tumors may be improved through enhancement of FcγR binding, whereas poorly infiltrated tumors will likely require combination approaches

Role of Neutrophils and Innate Immune Mechanisms in Urinary Tract Infections

Urinary tract infections (UTI) are among the most common infections in man. Despite their prevalence, information on the molecular mechanisms defects that explain the increased susceptibility to disease are lacking. This study focused on mucosal inflammation and the molecular mechanisms by which uropathogenic Escherichia coli activate this response. P fimbriae are expressed by most E. coli strains that cause acute pyelonephritis. By binding to Gala1-4Galb containing motifs in the globoseries of glycosphingolipids P fimbriae trigger inflammatory responses that underlie disease pathology. P fimbriae were shown here to utilize the Toll-like receptor 4 (TLR4) pathway to activate inflammation. P fimbriated E. coli induced a strong inflammatory response in TLR4+ mice, but not in TLR4- mice. In contrast to previously described activation of TLR4, the P fimbriae induced signal was independent of the LPS/LBP/CD14 pathway. The TLR4 deficiency was shown to impair resistance to UTI through an effect on neutrophil recruitment. Uropathogenic E. coli induced a rapid and strong neutrophil recruitment in TLR4+ mice, but only a weak response in TLR4- mice. TLR4+ mice cleared infection, while in TLR4- mice bacterial numbers increased with time. Neutrophil depletion converted the TLR4+ mice to the TLR4- susceptible phenotype. Neutrophil recruitment was shown to depend on IL-8 and IL-8 receptors. In vitro neutrophil transepithelial migration was blocked by antibodies to the interleukin-8 receptor CXCR1, and neutrophils of IL-8R knockout mice failed to cross the urinary tract epithelium and accumulated in subepithelial tissue compartments. mIL-8Rh knock-out mice failed to clear infection, developed symptoms of disease, and showed evidence of renal scarring. Subsequent studies showed that children prone to acute pyelonephritis have decreased neutrophil CXCR1 expression. The results suggest that TLR and CXCR expression determine neutrophil recruitment to the infected urinary tract, and suggest that a CXCR1 deficiency might underlie increased susceptibility to acute pyelonephritis

Resistance is futile: Targeting the inhibitory FcγRIIB (CD32B) to maximize immunotherapy

Monoclonal antibodies (mAb) are central to the treatment of several types of malignancy. However, these reagents are subject to particular types of resistance. Several resistance mechanisms are regulated by the inhibitory FcγRIIB. We recently developed mAbs to block FcγRIIB and provided in vivo proof-of-concept for their ability to overcome FcγRIIB-mediated resistance.</p

Sequence enrichment profiles enable target-agnostic antibody generation for a broad range of antigens

Phenotypic drug discovery (PDD) enables the target-agnostic generation of therapeutic drugs with novel mechanisms of action. However, realizing its full potential for biologics discovery requires new technologies to produce antibodies to all, a priori unknown, disease-associated biomolecules. We present a methodology that helps achieve this by integrating computational modeling, differential antibody display selection, and massive parallel sequencing. The method uses the law of mass action-based computational modeling to optimize antibody display selection and, by matching computationally modeled and experimentally selected sequence enrichment profiles, predict which antibody sequences encode specificity for disease-associated biomolecules. Applied to a phage display antibody library and cell-based antibody selection, ∼105 antibody sequences encoding specificity for tumor cell surface receptors expressed at 103–106 receptors/cell were discovered. We anticipate that this approach will be broadly applicable to molecular libraries coupling genotype to phenotype and to the screening of complex antigen populations for identification of antibodies to unknown disease-associated targets

Accelerating target deconvolution for therapeutic antibody candidates using highly parallelized genome editing

Therapeutic antibodies are transforming the treatment of cancer and autoimmune diseases. Today, a key challenge is finding antibodies against new targets. Phenotypic discovery promises to achieve this by enabling discovery of antibodies with therapeutic potential without specifying the molecular target a priori. Yet, deconvoluting the targets of phenotypically discovered antibodies remains a bottleneck; efficient deconvolution methods are needed for phenotypic discovery to reach its full potential. Here, we report a comprehensive investigation of a target deconvolution approach based on pooled CRISPR/Cas9. Applying this approach within three real-world phenotypic discovery programs, we rapidly deconvolute the targets of 38 of 39 test antibodies (97%), a success rate far higher than with existing approaches. Moreover, the approach scales well, requires much less work, and robustly identifies antibodies against the major histocompatibility complex. Our data establish CRISPR/Cas9 as a highly efficient target deconvolution approach, with immediate implications for the development of antibody-based drugs