Isolation of Two Novel Human Anti-CTLA-4 mAbs with Intriguing Biological Properties on Tumor and NK Cells

Abstract

The cytotoxic T lymphocyte-antigen 4 (CTLA-4) has been considered an IC exclusively expressed on T cells, where it counteracts the co-stimulatory CD28 receptor, by competing for its binding to CD-80 and CD-86. We recently found that it is expressed also on tumor and NK cells, suggesting other possible unknown roles of CTLA-4. To shed light on these novel aspects of CTLA-4, we used Ipilimumab, the first FDA approved human antibody targeting CTLA-4, in parallel studies with two novel human mAbs we isolated by using an efficient phage display selection strategy on live activated lymphocytes and purified mouse and human CTLA-4. The selection for cross-reactive mAbs was guaranteed by a high throughput sequencing to identify the sequences commonly enriched by two parallel pannings on human and mouse CTLA-4. Two isolated antibodies were found to bind with high affinity to both human and mouse CTLA-4 and lymphocytes, showing nanomolar or sub-nanomolar Kd values. They were able to kill Treg cells by ADCC, and to activate both human and mouse PBMCs, by strongly increasing cytokines secretion. Interestingly, they activated NK cells, exhibited cytotoxicity against cancer cells by inducing ADCC and inhibited tumor cell growth by affecting CTLA-4 downstream pathways in a similar fashion to CD-80 and CD-86 ligands and differently from Ipilimumab. Moreover, the novel mAbs showed a reduced ability to interfere in the binding of CD-80 ligands to CTLA-4 on T cells with respect to Ipilimumab, suggesting that they could allow for anti-tumor effects without the irAEs associated with the potent antagonistic activity of Ipilimumab.

Interactions of Spike-RBD of SARS-CoV-2 and Platelet Factor 4: New Insights in the Etiopathogenesis of Thrombosis

“…To verify the binding of the anti-PF4 polyclonal antibody to Spike-RBD, ELISA assays were performed by testing the rabbit anti-PF4 polyclonal antibody (0.1-50 nM) on the immobilized chimeric Spike RBD-Fc protein, PF4 or Fc used in parallel as controls. The assays were performed as previously reported [19,[21][22][23][24]. Briefly, NuncTM flat bottom 96-well plates (Thermo Fisher Scientific 439454 Ferentino, Italy) were coated with 5 µg/mL Spike RBD-Fc, PF4 or Fc recombinant proteins in a solution of 0.05 M NaHCO3 for 72 h at 4 • C. After blocking with 5% nonfat dry milk in PBS for 1 h at 37 • C, the purified Abs were added at increasing concentrations to the plates in 3% Bovine Serum Albumin (BSA Sigma A7030, St Louise, MO, USA) in PBS and incubated for 90 min by gently shaking.…”

“…To test the binding of the novel human monoclonal or polyclonal anti-Spike mAbs to the immobilized PF4 or to RBD/Fc proteins, ELISA assays were performed by testing increasing concentrations of D3, S96 mAbs or the anti-Spike polyclonal Ab. Similarly, aliquots (15 or 30 µL) of human plasma from vaccinated donors were tested in the same conditions [19,[21][22][23][24].…”

Novel human neutralizing mAbs specific for Spike-RBD of SARS-CoV-2

“…Phagemid particles were isolated from the library by using the M13-K07 helper phage (Invitrogen, Thermo Fisher Scientific Carlsbad, CA 92008, USA), as previously reported 42 . To isolate anti-Spike scFv-phages, a phagemid library of up 10 12 different clones was used for selection on human chimeric Spike RBD-Fc protein 43 . For each round of selection, phages were firstly blocked with 5% (wt/vol) Skim Milk Powder in PBS (Fluka Analytical, Sigma-Aldrich, A08300500 USA) and then two negative pannings were performed by incubating them on immobilized rhIgG1-Fc protein domain in 2.5% (wt/vol) Skim Milk Powder for 2 h at 4 °C by gently rotation.…”

“…The unbound phages were collected in the supernatant by centrifugation at 1200 rpm for 10 min and then incubated with immobilized Spike RBD-Fc protein (20 μg/ml) in 2.5% (wt/vol) Skim Milk Powder over night at 4 °C by gently rotation for the positive selection. After extensive washes with PBS, the bound phages were eluted from the Spike RBD-Fc protein by using two parallel elution methods: the classical elution with 76 mM citric acid (pH 2.5) in PBS for 5 min followed by the addition of 1 M Tris-HCl (pH 8.0) neutralization buffer 23 , 43 , or selective competitive elution, by using the chimeric receptor ACE2-Fc (100 μg/ml in PBS) which binds to Spike-RBD with high affinity to obtain those phages that specifically recognize the same epitope. The eluted phages were used to infect E. coli TG1 cells for amplification and further rounds of selection on the purified chimeric protein.…”

“…The clones of interest were isolated from competitive elution cycle_3 and acidic elution cycle_2 by overlapping PCR, as previously described 43 and reported in the Supplementary Information .…”