Anti-CD99 Antibody Therapy Triggers Macrophage-Dependent Ewing Cell Death In Vitro and Myeloid Cell Recruitment In Vivo.

BACKGROUND: Ewing sarcoma is a rare tumor of the bone or soft tissues characterized by diffuse membranous staining for CD99. As this tumor remains incurable in the metastatic, relapsed, and refractory settings, we explored the downstream immune implications of targeting CD99. METHODS: We discovered a human anti-CD99 antibody (NOA2) by phagemid panning and investigated NOA2 immune cell-mediated cytotoxicity in vitro and in vivo focusing on the myeloid cell compartment, given that M2 macrophages are present in human tumors and associated with a poor prognosis. RESULTS: NOA2 is capable of inducing immune effector cell-mediated Ewing death in vitro via engagement of macrophages. Mice with metastatic Ewing tumors, treated with NOA2, experience tumor growth arrest and an associated increase in intratumoral macrophages. Further, incubation of macrophages and Ewing cells with NOA2, in conjunction with anti-PILRα antibody blockade in vitro, results in the reactivation of previously dormant macrophages possibly due to interrupted binding of Ewing CD99 to macrophage PILRα. CONCLUSIONS: These studies are the first to demonstrate the role of human immune effector cells in anti-CD99-mediated Ewing tumor death. We propose that the engagement of CD99 by NOA2 results in the recruitment of intratumoral macrophages. In addition, interruption of the CD99:PILRα checkpoint axis may be a relevant therapeutic approach to activate tumor-associated macrophages.

The variable conversion of neutralizing anti-SARS-CoV-2 single-chain antibodies to IgG provides insight into RBD epitope accessibility.

Monoclonal antibody (mAb) therapies have rapidly become a powerful class of therapeutics with applications covering a diverse range of clinical indications. Though most widely used for the treatment of cancer, mAbs are also playing an increasing role in the defense of viral infections, most recently with palivizumab for prevention and treatment of severe RSV infections in neonatal and pediatric populations. In addition, during the COVID-19 pandemic, mAbs provided a bridge to the rollout of vaccines; however, their continued role as a therapeutic option for those at greatest risk of severe disease has become limited due to the emergence of neutralization resistant Omicron variants. Although there are many techniques for the identification of mAbs, including single B cell cloning and immunization of genetically engineered mice, the low cost, rapid throughput and technological simplicity of antibody phage display has led to its widespread adoption in mAb discovery efforts. Here we used our 27-billion-member naïve single-chain antibody (scFv) phage library to identify a panel of neutralizing anti-SARS-CoV-2 scFvs targeting diverse epitopes on the receptor binding domain (RBD). Although typically a routine process, we found that upon conversion to IgG, a number of our most potent clones failed to maintain their neutralization potency. Kinetic measurements confirmed similar affinity to the RBD; however, mechanistic studies provide evidence that the loss of neutralization is a result of structural limitations likely arising from initial choice of panning antigen. Thus this work highlights a risk of scFv-phage panning to mAb conversion and the importance of initial antigen selection.

IgG-like bispecific antibodies with potent and synergistic neutralization against circulating SARS-CoV-2 variants of concern.

Monoclonal antibodies are a promising approach to treat COVID-19, however the emergence of SARS-CoV-2 variants has challenged the efficacy and future of these therapies. Antibody cocktails are being employed to mitigate these challenges, but neutralization escape remains a major challenge and alternative strategies are needed. Here we present two anti-SARS-CoV-2 spike binding antibodies, one Class 1 and one Class 4, selected from our non-immune human single-chain variable fragment (scFv) phage library, that are engineered into four, fully-human IgG-like bispecific antibodies (BsAb). Prophylaxis of hACE2 mice and post-infection treatment of golden hamsters demonstrates the efficacy of the monospecific antibodies against the original Wuhan strain, while promising in vitro results with the BsAbs demonstrate enhanced binding and distinct synergistic effects on neutralizing activity against circulating variants of concern. In particular, one BsAb engineered in a tandem scFv-Fc configuration shows synergistic neutralization activity against several variants of concern including B.1.617.2. This work provides evidence that synergistic neutralization can be achieved using a BsAb scaffold, and serves as a foundation for the future development of broadly reactive BsAbs against emerging variants of concern.

Immune Evasion of SARS-CoV-2 Omicron Subvariants.

Since the SARS-CoV-2 Omicron variant (B.1.1.529) was declared a variant of concern (VOC) by the WHO on 24 November 2021, it has caused another global surge of cases. With extensive mutations in its spike glycoprotein, Omicron gained substantial capabilities to evade the antiviral immunity provided by vaccination, hybrid immunity, or monoclonal antibodies. The Omicron subvariants BA.1, BA.2, BA.2.12.1, BA.4 and BA.5 extended this immune evasion capability by having additional unique mutations in their respective spike proteins. The ongoing Omicron wave and emergence of new Omicron subvariants leads to additional concerns regarding the efficacy of the current antiviral measurements. To have a better understanding of the Omicron subvariants, this review summarizes reports of the immune evasion of subvariants BA.1, BA.2, BA.2.12.1, BA.4, and BA.5 as well as the molecular basis of immune evasion.

Analysis of a SARS-CoV-2 convalescent cohort identified a common strategy for escape of vaccine-induced anti-RBD antibodies by Beta and Omicron variants.

BACKGROUND: Evolutionary pressure has led to the emergence of SARS-CoV-2 variants, with the most recent Omicron variant containing an unparalleled 30 mutations in the spike protein. Many of these mutations are expected to increase immune evasion, thus making breakthrough cases and re-infection more common. METHODS: From June 2020 to December 2021 serial blood samples (initial post recovery, 6 months, 12 months) were collected from a COVID-19 convalescent cohort in Boston, MA. Plasma was isolated for use in Mesoscale Discovery based antibody binding assays. Unvaccinated donors or those vaccinated prior to the primary blood draw were excluded from this analysis, as were those who did not have at least two blood draws. Wilcoxon signed rank tests were used to compare pre- and post-vaccination titers and antibody response against different variants, while McNemar tests were used to compare the proportions of achieving ≥ 4 fold increases against different variants. FINDINGS: Forty-eight COVID convalescent donors with post-infection vaccination (hybrid immunity) were studied to evaluate the levels of cross-reactive antibodies pre- and post- vaccination against various SARS-CoV-2 Spike and receptor binding domain (RBD) proteins. Vaccination with BNT162b2, mRNA-1273 or Ad26.COV2.S led to a 6·3 to 7·8 fold increase in anti-Spike antibody titers and a 7·0 to 7·4 fold increase in anti-WT, Alpha and Delta RBD antibody. However, a lower response was observed for Beta and Omicron RBDs with only 7/48 (15%) and 15/48 (31%) donors having a ≥4 fold increase in post-vaccination titers against Beta and Omicron RBDs. Structural analysis of the Beta and Omicron RBDs reveal a shared immune escape strategy involving residues K417-E484-N501 that is exploited by these variants of concern. INTERPRETATION: Through mutations of the K417-E484-N501 triad, SARS-CoV-2 has evolved to evade neutralization by the class I/II anti-RBD antibody fraction of hybrid immunity plasma as the polyclonal antibody response post-vaccination shows limitations in the ability to solve the structural requirements to bind the mutant RBDs. FUNDING: Massachusetts Consortium on Pathogen Readiness (280870.5116709.0016) and the National Institute of Allergy and Infectious Diseases (1R01AI161152-01A1).