The smallest functional antibody fragment: Ultralong CDR H3 antibody knob regions potently neutralize SARS-CoV-2.

Cows produce antibodies with a disulfide-bonded antigen-binding domain embedded within ultralong heavy chain third complementarity determining regions. This "knob" domain is analogous to natural cysteine-rich peptides such as knottins in that it is small and stable but can accommodate diverse loops and disulfide bonding patterns. We immunized cattle with SARS-CoV-2 spike and found ultralong CDR H3 antibodies that could neutralize several viral variants at picomolar IC50 potencies in vitro and could protect from disease in vivo. The independent CDR H3 peptide knobs were expressed and maintained the properties of the parent antibodies. The knob interaction with SARS-CoV-2 spike was revealed by electron microscopy, X-ray crystallography, NMR spectroscopy, and mass spectrometry and established ultralong CDR H3-derived knobs as the smallest known recombinant independent antigen-binding fragment. Unlike other vertebrate antibody fragments, these knobs are not reliant on the immunoglobulin domain and have potential as a new class of therapeutics.

Generation of orthogonal Fab-based trispecific antibody formats.

The clinical success of monoclonal antibodies to treat diseases across nearly every therapeutic area has spurred advances in bispecific antibody technology with the goal of cost-effectively combining various therapies or providing novel mechanisms for disease intervention. Many novel bispecific antibodies are now in clinical development or the late pre-clinical setting. A new horizon exists for novel molecular entities with the ability to bind three or more antigens. Here we describe the production and characterization of novel trispecific antibody-like proteins denoted 'OrthoTsAbs' that self-assemble through the application of engineered antibody domain interfaces.

Transient and stable CHO expression, purification and characterization of novel hetero-dimeric bispecific IgG antibodies.

IgG bispecific antibodies (BsAbs) represent one of the preferred formats for bispecific antibody therapeutics due to their native-like IgG properties and their monovalent binding to each target. Most reported studies utilized transient expression in HEK293 cells to produce BsAbs. However, the expression of biotherapeutic molecules using stable CHO cell lines is commonly used for biopharmaceutical manufacturing. Unfortunately, limited information is available in the scientific literature on the expression of BsAbs in CHO cell lines. In this study we describe an alternative approach to express the multiple components of IgG BsAbs using a single plasmid vector (quad vector). This single plasmid vector contains both heavy chain genes and both light chain genes required for the expression and assembly of the IgG BsAb, along with a selectable marker. We expressed, purified, and characterized four different IgG BsAbs or "hetero-mAbs" using transient CHO expression and stable CHO minipools. Transient CHO titers ranged from 90 to 160 mg/L. Stable CHO titers ranged from 0.4 to 2.3 g/L. Following a simple Protein A purification step, the percentage of correctly paired BsAbs ranged from 74% to 98% as determined by mass spectrometry. We also found that information generated from transient CHO expression was similar to information generated using stable CHO minipools. In conclusion, the quad vector approach represents a simple, but effective, alternative approach for the generation of IgG BsAbs in both transient CHO and stable CHO expression systems. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:469-477, 2017.

Parallel synthesis and screening of peptide conjugates.

Peptide conjugates represent an emerging class of therapeutics. However, in contrast to that of small molecules and peptides, the discovery and optimization of peptide conjugates is low in throughput, resource intensive, time-consuming, and based on educated decisions rather than screening. A strategy for the parallel synthesis and screening of peptide conjugates is presented that (1) reduces variability in the conjugation steps; (2) provides a new method to rapidly and quantitatively measure conversion in crude conjugation mixtures; (3) introduces a purification step using an immobilized chemical scavenger that does not rely on protein-specific binding; and (4) is supported by robust analytical methods to characterize the large number of end products. Copper-free click chemistry is used as the chemoselective ligation method for conjugation and purification. The productivity in the generation and screening of peptide conjugates is significantly improved by applying this strategy as is demonstrated by the optimization of the anti-Angiopoietin-2 (Ang2) CovX-body, CVX-060, a peptide-antibody scaffold conjugate that has advanced in clinical trials for oncology indications.

The Effect of Molecular Weight, PK, and Valency on Tumor Biodistribution and Efficacy of Antibody-Based Drugs.

Poor drug delivery and penetration of antibody-mediated therapies pose significant obstacles to effective treatment of solid tumors. This study explored the role of pharmacokinetics, valency, and molecular weight in maximizing drug delivery. Biodistribution of a fibroblast growth factor receptor 4 (FGFR4) targeting CovX-body (an FGFR4-binding peptide covalently linked to a nontargeting IgG scaffold; 150 kDa) and enzymatically generated FGFR4 targeting F(ab)2 (100 kDa) and Fab (50 kDa) fragments was measured. Peak tumor levels were achieved in 1 to 2 hours for Fab and F(ab)2 versus 8 hours for IgG, and the percentage injected dose in tumors was 0.45%, 0.5%, and 2.5%, respectively, compared to 0.3%, 2%, and 6% of their nontargeting controls. To explore the contribution of multivalent binding, homodimeric peptides were conjugated to the different sized scaffolds, creating FGFR4 targeting IgG and F(ab)2 with four peptides and Fab with two peptides. Increased valency resulted in an increase in cell surface binding of the bivalent constructs. There was an inverse relationship between valency and intratumoral drug concentration, consistent with targeted consumption. Immunohistochemical analysis demonstrated increased size and increased cell binding decreased tumor penetration. The binding site barrier hypothesis suggests that limited tumor penetration, as a result of high-affinity binding, could result in decreased efficacy. In our studies, increased target binding translated into superior efficacy of the IgG instead, because of superior inhibition of FGFR4 proliferation pathways and dosing through the binding site barrier. Increasing valency is therefore an effective way to increase the efficacy of antibody-based drugs.

Development of a potential high-throughput workflow to characterize sites of bioconjugation by immuno-affinity capture coupled to MALDI-TOF mass spectrometry.

The characterization of conjugation sites in bioconjugates is critical in the early discovery phase because site-specific conjugation improves in vivo stability and drug efficacy. We previously developed an engineered monoclonal antibody (mAb) scaffold which enables site-specific conjugation toward a reactive lysine (Lys) residue on each heavy chain (HC) by using an azetidinone (AZD) linker. In order to explore conjugations in other location which avoids potential interference with target binding, other chemical linkers have been studied and the investigation of N-hydroxysuccinimade (NHS) linker is reported here. The complexity of identifying the sites lies in part to the large number of Lys residues available for conjugation on the mAb scaffold. This has posed technical challenges to standard peptide mapping approaches. Therefore, an alternative strategy intended for a rapid analysis has been investigated by coupling immuno-affinity capture to matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). In this study, we have employed a novel application of two different capture formats: Surface enhanced laser dissociation/ionization (SELDI) and mass spectrometry immunoassay (MSIA) tips to reduce the analysis time. An antibody against the pharmacophore portion was immobilized to capture the conjugated peptides, and subsequently provide characterization of the conjugation sites on the scaffold. Multiple sites for the AZD and NHS linkers have been easily identified and confirmed by MS2 sequencing. Lysine99 is the predominant site for the AZD linker, and Lysine55 is the primary site for the NHS linker with Lysine193 and Tyrosine37 being minor sites as shown in the abstract figure. We have also demonstrated the use of conjugation mapping to compare the distribution pattern between the AZD and NHS linkers as well as to study the stability of conjugation sites in a rapid way.

Maleimide conjugation markedly enhances the immunogenicity of both human and murine idiotype-KLH vaccines.

The collection of epitopes present within the variable regions of the tumor-specific clonal immunoglobulin expressed by B cell lymphomas (idiotype, Id) can serve as a target for active immunotherapy. Traditionally, tumor-derived Id protein is chemically conjugated to the immunogenic foreign carrier protein keyhole limpet hemocyanin (KLH) using glutaraldehyde to serve as a therapeutic vaccine. While this approach offered promising results for some patients treated in early clinical trials, glutaraldehyde Id-KLH vaccines have failed to induce immune and clinical responses in many vaccinated subjects. We recently described an alternative conjugation method employing maleimide-sulfhydryl chemistry that significantly increased the therapeutic efficacy of Id-KLH vaccines in three different murine B cell lymphoma models, with protection mediated by either CD8(+) T cells or antibodies. We now define in detail the methods and parameters critical for enhancing the in vivo immunogenicity of human as well as murine Id-KLH conjugate vaccines. Optimal conditions for Id sulfhydryl pre-reduction were determined, and maleimide Id-KLH conjugates maintained stability and potency even after prolonged storage. Field flow fractionation analysis of Id-KLH particle size revealed that maleimide conjugates were far more uniform in size than glutaraldehyde conjugates. Under increasingly stringent conditions, maleimide Id-KLH vaccines maintained superior efficacy over glutaraldehyde Id-KLH in treating established, disseminated murine lymphoma. More importantly, human maleimide Id-KLH conjugates were consistently superior to glutaraldehyde Id-KLH conjugates in inducing Id-specific antibody and T cell responses. The described methods should be easily adaptable to the production of clinical grade vaccines for human trials in B cell malignancies.