SUMMARY Highly effective HIV-1-neutralizing antibodies could have utility in the prevention or treatment of HIV-1 infection. To improve the potency of 10E8, an antibody capable of near pan-HIV-1 neutralization, we engineered 10E8-surface mutants and screened for improved neutralization. Variants with the largest functional enhancements involved the addition of hydrophobic or positively charged residues, which were positioned to interact with viral membrane lipids or viral glycan-sialic acids, respectively. In both cases, the site of improvement was spatially separated from the region of antibody mediating molecular contact with the protein component of the antigen, thereby improving peripheral semi-specific interactions while maintaining unmodified dominant contacts responsible for broad recognition. The optimized 10E8 antibody, with mutations to phenylalanine and arginine, retained the extraordinary breadth of 10E8 but with ~10-fold increased potency. We propose surface-matrix screening as a general method to improve antibodies, with improved semi-specific interactions between antibody and antigen enabling increased potency without compromising breadth.

Stabilization and formulation of therapeutic proteins against physical instability, both structural alterations and aggregation, is particularly challenging not only due to each protein's unique physicochemical characteristics but also their susceptibility to the surrounding milieu (pH, ionic strength, excipients, etc.) as well as various environmental stresses (temperature, agitation, lyophilization, etc.). The use of high-throughput techniques can significantly aid in the evaluation of stabilizing solution conditions by permitting a more rapid evaluation of a large matrix of possible combinations. In this mini-review, we discuss both key physical degradation pathways observed for protein-based drugs and the utility of various high-throughput biophysical techniques to aid in protein formulation development to minimize their occurrence. We then focus on four illustrative case studies with therapeutic protein candidates of varying sizes, shapes and physicochemical properties to explore different analytical challenges in monitoring protein physical instability. These include an IgG2 monoclonal antibody, an albumin-fusion protein, a recombinant pentameric plasma glycoprotein, and an antibody fragment (Fab). Future challenges and opportunities to improve and apply high-throughput approaches to protein formulation development are also discussed.

The vaccine elicitation of broadly neutralizing antibodies against HIV-1 is a long-sought goal. We previously reported the amino-terminal eight residues of the HIV-1-fusion peptide (FP8) -when conjugated to the carrier protein, keyhole limpet hemocyanin (KLH) -to be capable of inducing broadly neutralizing responses against HIV-1 in animal models. However, KLH is a multi-subunit particle derived from a natural source, and its manufacture as a clinical product remains a challenge. Here we report the preclinical development of recombinant tetanus toxoid heavy chain fragment (rTTHC) linked to FP8 (FP8-rTTHC) as a suitable FP-conjugate vaccine immunogen. We assessed 16 conjugates, made by coupling the 4 most prevalent FP8 sequences with 4 carrier proteins: the aforementioned KLH and rttHc; the H. influenzae protein D (HiD); and the cross-reactive material from diphtheria toxin (CRM197). While each of the 16 FP8-carrier conjugates could elicit HIV-1-neutralizing responses, rTTHC conjugates induced higher FP-directed responses overall. A Sulfo-SIAB linker yielded superior results over an SM(PEG)2 linker but combinations of carriers, conjugation ratio of peptide to carrier, or choice of adjuvant (Adjuplex or Alum) did not significantly impact elicited FP-directed neutralizing responses in mice. Overall, SIAB-linked FP8-rTTHC appears to be a promising vaccine candidate for advancing to clinical assessment.The fusion peptide (FP) site of vulnerability on the HIV-1 envelope (Env) glycoprotein has recently been shown to be a promising vaccine target 1-3 . FP, a hydrophobic region of ~15 residues at the N terminus of the gp41 transmembrane glycoprotein, is an essential component of the HIV entry machinery 4 . FP embeds in the target cell membrane during the pre-hairpin intermediate stage of entry, where it serves to anchor the rearranging viral spike and to facilitate the merging of viral and cell membranes. The N-terminal portion of FP is solvent accessible and recognized by broadly neutralizing antibodies PGT151 5,6 , N123-VRC34.01 3 , and ACS202 7 . Because FP is a short linear peptide, it has low inherent immunogenicity due to its lack of helper T cell epitopes. Coupling peptides to highly immunogenic carrier proteins is a well-established approach for providing T cell help to peptide immunogens [8][9][10][11] . When the N-terminal 6-10 residues of FP are coupled to keyhole limpet hemocyanin (KLH), a standard protein carrier widely used in biotechnology, the resultant FP-KLH conjugate immunogens are able to induce broadly neutralizing FP-directed immune responses in mice, guinea pigs, and rhesus macaques 1,2,12 . Vaccine-induced FP-directed antibodies from mice or NHP neutralize up to 31% or 59%, respectively, of a cross-clade panel of 208 HIV-1 strains 2 .These results (illustrated in Fig. 1a) indicate FP coupled to a carrier protein to be a promising candidate immunogen. However, KLH is a multi-subunit metalloprotein derived from natural sources 13-15 with both sequence and glycan heterogeneity, which pose manufacturin...

Structure-based vaccine design has been used to develop immunogens that display conserved neutralization sites on pathogens such as HIV-1, respiratory syncytial virus (RSV), and influenza. Improving the immunogenicity of these designed immunogens with adjuvants will require formulations that do not alter protein antigenicity. Here, we show that nanoparticle-forming thermoresponsive polymers (TRP) allow for co-delivery of RSV fusion (F) protein trimers with Toll-like receptor 7 and 8 agonists (TLR-7/8a) to enhance protective immunity. Although primary amine conjugation of TLR-7/8a to F trimers severely disrupted the recognition of critical neutralizing epitopes, F trimers site-selectively coupled to TRP nanoparticles retained appropriate antigenicity and elicited high titers of prefusion-specific, T1 isotype anti-RSV F antibodies following vaccination. Moreover, coupling F trimers to TRP delivering TLR-7/8a resulted in ∼3-fold higher binding and neutralizing antibody titers than soluble F trimers admixed with TLR-7/8a and conferred protection from intranasal RSV challenge. Overall, these data show that TRP nanoparticles may provide a broadly applicable platform for eliciting neutralizing antibodies to structure-dependent epitopes on RSV, influenza, HIV-1, or other pathogens.