CD169 (Siglec-1) as a Robust Human Cell Biomarker of Toll-Like Receptor 9 Agonist Immunotherapy

Immunotherapy is a promising therapeutic area in cancer and chronic viral infections. An important component of immunotherapy in these contexts is the activation of innate immunity. Here we investigate the potential for CD169 (Siglec 1) expression on monocytes to serve as a robust biomarker for activation of innate immunity and, particular, as a proxy for IFN-α production. Specifically, we investigated the effects of Toll-like receptor 9 agonism with MGN1703 (lefitolimod) across experimental conditions ex vivo, in humanized mice, and in clinical trial participants. Ex vivo we observed that the percentage of classical monocytes expressing CD169 increased dramatically from 10% pre-stimulation to 97% 24 hrs after MGN1703 stimulation (p\u3c0.0001). In humanized NOG mice, we observed prominent upregulation of the proportions of monocytes expressing CD169 after two doses of MGN1703 where 73% of classical monocytes were CD169 positive in bone marrow following MGN1703 treatment vs 19% in vehicle treated mice (p=0.0159). Finally, in a clinical trial in HIV-infected individuals receiving immunotherapy treatment with MGN1703, we observed a uniform upregulation of CD169 on monocytes after dosing with 97% of classical monocytes positive for CD169 (p=0.002). Hence, in this comprehensive evaluation ex vivo, in an animal model, and in a clinical trial, we find increases in the percentage of CD169 positive monocytes to be a reliable and robust biomarker of immune activation following TLR9 agonist treatment

Discovery of neutralizing SARS-CoV-2 antibodies enriched in a unique antigen specific B cell cluster.

Despite development of effective SARS-CoV-2 vaccines, a sub-group of vaccine non-responders depends on therapeutic antibodies or small-molecule drugs in cases of severe disease. However, perpetual viral evolution has required continuous efficacy monitoring as well as exploration of new therapeutic antibodies, to circumvent resistance mutations arising in the viral population. We performed SARS-CoV-2-specific B cell sorting and subsequent single-cell sequencing on material from 15 SARS-CoV-2 convalescent participants. Through screening of 455 monoclonal antibodies for SARS-CoV-2 variant binding and virus neutralization, we identified a cluster of activated B cells highly enriched for SARS-CoV-2 neutralizing antibodies. Epitope binning and Cryo-EM structure analysis identified the majority of neutralizing antibodies having epitopes overlapping with the ACE2 receptor binding motif (class 1 binders). Extensive functional antibody characterization identified two potent neutralizing antibodies, one retaining SARS-CoV-1 neutralizing capability, while both bind major common variants of concern and display prophylactic efficacy in vivo. The transcriptomic signature of activated B cells harboring broadly binding neutralizing antibodies with therapeutic potential identified here, may be a guide in future efforts of rapid therapeutic antibody discovery

Cluster distribution of drug development liabilities.

Cluster distribution of drug development liabilities.</p

Single-cell profiling of 7176 B cells from three donor groups by scRNA-seq and V(D)J-seq.

(A) UMAP projection and unsupervised clustering revealing 9 transcriptomic clusters annotated according to selected marker genes. (B) Somatic hyper-mutation percentage compared to inferred naïve germline stratified by isotype and transcriptomic cluster, highlighting one cluster of naïve B cells associated with particularly low mutation rate. The dotted line indicates the threshold used for the selection of mAbs for validation. The number of cells within each cluster or isotype is displayed below. (C) Fraction of cells with each isotype stratified by transcriptomic cluster highlighting the naïve cluster and three IgG-rich clusters. (D) mRNA expression levels given by the log of the normalized UMI counts of selected markers in each transcriptomic cluster. (E) Scaled average expression in each cluster indicating memory B cell, naïve B cell and activated B cell markers highlighting unique transcriptomic profiles of the clusters. The size of the dots indicates the percentage of cells expressing the given marker gene within the cluster.</p

Characteristics of the single-cell population.

Despite development of effective SARS-CoV-2 vaccines, a sub-group of vaccine non-responders depends on therapeutic antibodies or small-molecule drugs in cases of severe disease. However, perpetual viral evolution has required continuous efficacy monitoring as well as exploration of new therapeutic antibodies, to circumvent resistance mutations arising in the viral population. We performed SARS-CoV-2-specific B cell sorting and subsequent single-cell sequencing on material from 15 SARS-CoV-2 convalescent participants. Through screening of 455 monoclonal antibodies for SARS-CoV-2 variant binding and virus neutralization, we identified a cluster of activated B cells highly enriched for SARS-CoV-2 neutralizing antibodies. Epitope binning and Cryo-EM structure analysis identified the majority of neutralizing antibodies having epitopes overlapping with the ACE2 receptor binding motif (class 1 binders). Extensive functional antibody characterization identified two potent neutralizing antibodies, one retaining SARS-CoV-1 neutralizing capability, while both bind major common variants of concern and display prophylactic efficacy in vivo. The transcriptomic signature of activated B cells harboring broadly binding neutralizing antibodies with therapeutic potential identified here, may be a guide in future efforts of rapid therapeutic antibody discovery.</div

Monoclonal antibody screening.

(A) Neutralization percentage of SARS-CoV-2 pseudovirus shown for each individual mAb supernatant analyzed at 20 μg/ml, shown on y-axis. MAbs are ordered along the x-axis from best (left) to poorest (right) neutralizers. n = 455. Screening was performed once in duplicate determinations. (B) Visualization of the expressed mAbs B cell cluster origin and distribution within all isolated B cells. Neutralizers shown in green ( neutralization, n = 9). Top neutralizers shown in red (<95% neutralization, n = 24). Non-neutralizers shown in blue (<80% neutralization). Background shown in grey (cells not expressed for screening).(C) Distribution of successful neutralizing mAbs, between clusters 6, 8 and remaining clusters (cluster 7 excluded). Hit rate cut-off for defining successful neutralizations was set at 80% pseudovirus neutralization. Hit rates were calculated within each cluster group. n = 455 (D) Predictive performance of Ag scores used for SARS-CoV-2 specific B cell sorting towards neutralization capability in cluster 8. n = 108. Red = SARS-CoV-2 D614G mutant trimer, Blue = SARS-CoV-2 RBD, Orange = SARS-CoV-1 RBD, Green = SARS-CoV-2 trimer. The p-value is based on a Kruskal-Wallis test of the receiver-operator characteristics curve.</p

In vitro analysis of lead mAbs binding.

(A) Ranking of purified mAbs from lowest IC50 pseudovirus neutralization value at the top (best neutralization) to highest IC50 at the bottom (poorest neutralization). Each antibody IC50 value obtained from triplicate point determinations of the dilution curve. Mesoscale binding values are shown for each mAb (supernatant) towards SARS-CoV-2 Spike, N-terminal domain (NTD) and receptor binding domain (RBD), as a heat-map. The binding determinations were performed once in duplicate. Colors indicate normalization from 0–100 within each column. (B) Mesoscale binding values for binding to the RBD of viral variants Alpha (N501Y, A570D), Beta (K417N, E484K, N501Y), Gamma (K417T, E484K, N501Y) and Delta (L452R), shown as fold change from SARS-CoV-2 RBD binding within each mAb individually. The binding determinations were performed once in duplicate. (C) Heat-map showing percentage ACE2 blocking for each mAb binding viral variant spike proteins (CoV-2, Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2) and Omicron BA.1(B.1.1.529). The ACE2 blocking analysis was performed in duplicate determinations of a dilution curve.</p

FACS gating strategy.

Despite development of effective SARS-CoV-2 vaccines, a sub-group of vaccine non-responders depends on therapeutic antibodies or small-molecule drugs in cases of severe disease. However, perpetual viral evolution has required continuous efficacy monitoring as well as exploration of new therapeutic antibodies, to circumvent resistance mutations arising in the viral population. We performed SARS-CoV-2-specific B cell sorting and subsequent single-cell sequencing on material from 15 SARS-CoV-2 convalescent participants. Through screening of 455 monoclonal antibodies for SARS-CoV-2 variant binding and virus neutralization, we identified a cluster of activated B cells highly enriched for SARS-CoV-2 neutralizing antibodies. Epitope binning and Cryo-EM structure analysis identified the majority of neutralizing antibodies having epitopes overlapping with the ACE2 receptor binding motif (class 1 binders). Extensive functional antibody characterization identified two potent neutralizing antibodies, one retaining SARS-CoV-1 neutralizing capability, while both bind major common variants of concern and display prophylactic efficacy in vivo. The transcriptomic signature of activated B cells harboring broadly binding neutralizing antibodies with therapeutic potential identified here, may be a guide in future efforts of rapid therapeutic antibody discovery.</div

Cryo-EM refinement.

Despite development of effective SARS-CoV-2 vaccines, a sub-group of vaccine non-responders depends on therapeutic antibodies or small-molecule drugs in cases of severe disease. However, perpetual viral evolution has required continuous efficacy monitoring as well as exploration of new therapeutic antibodies, to circumvent resistance mutations arising in the viral population. We performed SARS-CoV-2-specific B cell sorting and subsequent single-cell sequencing on material from 15 SARS-CoV-2 convalescent participants. Through screening of 455 monoclonal antibodies for SARS-CoV-2 variant binding and virus neutralization, we identified a cluster of activated B cells highly enriched for SARS-CoV-2 neutralizing antibodies. Epitope binning and Cryo-EM structure analysis identified the majority of neutralizing antibodies having epitopes overlapping with the ACE2 receptor binding motif (class 1 binders). Extensive functional antibody characterization identified two potent neutralizing antibodies, one retaining SARS-CoV-1 neutralizing capability, while both bind major common variants of concern and display prophylactic efficacy in vivo. The transcriptomic signature of activated B cells harboring broadly binding neutralizing antibodies with therapeutic potential identified here, may be a guide in future efforts of rapid therapeutic antibody discovery.</div