Biophysical and Sequence-Based Methods for Identifying Monovalent and Bivalent Antibodies with High Colloidal Stability.

In vitro antibody discovery and/or affinity maturation are often performed using antibody fragments (Fabs), but most monovalent Fabs are reformatted as bivalent IgGs (monoclonal antibodies, mAbs) for therapeutic applications. One problem related to reformatting antibodies is that the bivalency of mAbs can lead to increased antibody self-association and poor biophysical properties (e.g., reduced antibody solubility and increased viscosity). Therefore, it is important to identify monovalent Fabs early in the discovery and/or optimization process that will display favorable biophysical properties when reformatted as bivalent mAbs. Here we demonstrate a facile approach for evaluating Fab self-association in a multivalent assay format that is capable of identifying antibodies with low self-association and favorable colloidal properties when reformatted as bivalent mAbs. Our approach (self-interaction nanoparticle spectroscopy, SINS) involves immobilizing Fabs on gold nanoparticles in a multivalent format (multiple Fabs per nanoparticle) and evaluating their self-association behavior via shifts in the plasmon wavelength or changes in the absorbance values. Importantly, we find that SINS measurements of Fab self-association are correlated with self-interaction measurements of bivalent mAbs and are useful for identifying antibodies with favorable biophysical properties. Moreover, the significant differences in the levels of self-association detected for Fabs and mAbs with similar frameworks can be largely explained by the physicochemical properties of the complementarity-determining regions (CDRs). Comparison of the properties of the CDRs in this study relative to those of approved therapeutic antibodies reveals several key factors (net charge, fraction of charged residues, and presence of self-interaction motifs) that strongly influence antibody self-association behavior. Increased positive charge in the CDRs was observed to correlate with increased risk of high self-association for the mAbs in this study and clinical-stage antibodies. We expect that these findings will be useful for improving the development of therapeutic antibodies that are well suited for high concentration applications.

Neutralization of macrophage migration inhibitory factor (MIF) by fully human antibodies correlates with their specificity for the ß-sheet structure of MIF.

The macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine that recently emerged as an attractive therapeutic target for a variety of diseases. A diverse panel of fully human anti-MIF antibodies was generated by selection from a phage display library and extensively analyzed in vitro. Epitope mapping studies identified antibodies specific for linear as well as structural epitopes. Experimental animal studies revealed that only those antibodies binding epitopes within amino acids 50-68 or 86-102 of the MIF molecule exerted protective effects in models of sepsis or contact hypersensitivity. Within the MIF protein, these two binding regions form a ß-sheet structure that includes the MIF oxidoreductase motif. We therefore conclude that this ß-sheet structure is a crucial region for MIF activity and a promising target for anti-MIF antibody therapy.

Transcriptome and proteome profiling of activated cardiac fibroblasts supports target prioritization in cardiac fibrosis.

Background: Activated cardiac fibroblasts (CF) play a central role in cardiac fibrosis, a condition associated with most cardiovascular diseases. Conversion of quiescent into activated CF sustains heart integrity upon injury. However, permanence of CF in active state inflicts deleterious heart function effects. Mechanisms underlying this cell state conversion are still not fully disclosed, contributing to a limited target space and lack of effective anti-fibrotic therapies. Materials and methods: To prioritize targets for drug development, we studied CF remodeling upon activation at transcriptomic and proteomic levels, using three different cell sources: primary adult CF (aHCF), primary fetal CF (fHCF), and induced pluripotent stem cells derived CF (hiPSC-CF). Results: All cell sources showed a convergent response upon activation, with clear morphological and molecular remodeling associated with cell-cell and cell-matrix interactions. Quantitative proteomic analysis identified known cardiac fibrosis markers, such as FN1, CCN2, and Serpine1, but also revealed targets not previously associated with this condition, including MRC2, IGFBP7, and NT5DC2. Conclusion: Exploring such targets to modulate CF phenotype represents a valuable opportunity for development of anti-fibrotic therapies. Also, we demonstrate that hiPSC-CF is a suitable cell source for preclinical research, displaying significantly lower basal activation level relative to primary cells, while being able to elicit a convergent response upon stimuli.

BTN2A1, an immune checkpoint targeting Vγ9Vδ2 T cell cytotoxicity against malignant cells.

The anti-tumor response of Vγ9Vδ2 T cells requires the sensing of accumulated phosphoantigens (pAgs) bound intracellularly to butyrophilin 3A1 (BTN3A1). In this study, we show that butyrophilin 2A1 (BTN2A1) is required for BTN3A-mediated Vγ9Vδ2 T cell cytotoxicity against cancer cells, and that expression of the BTN2A1/BTN3A1 complex is sufficient to trigger Vγ9Vδ2 TCR activation. Also, BTN2A1 interacts with all isoforms of BTN3A (BTN3A1, BTN3A2, BTN3A3), which appears to be a rate-limiting factor to BTN2A1 export to the plasma membrane. BTN2A1/BTN3A1 interaction is enhanced by pAgs and, strikingly, B30.2 domains of both proteins are required for pAg responsiveness. BTN2A1 expression in cancer cells correlates with bisphosphonate-induced Vγ9Vδ2 T cell cytotoxicity. Vγ9Vδ2 T cell killing of cancer cells is modulated by anti-BTN2A1 monoclonal antibodies (mAbs), whose action relies on the inhibition of BTN2A1 binding to the Vγ9Vδ2TCR. This demonstrates the potential of BTN2A1 as a therapeutic target and adds to the emerging butyrophilin-family cooperation pathway in γδ T cell activation.

Development of ICT01, a first-in-class, anti-BTN3A antibody for activating Vγ9Vδ2 T cell-mediated antitumor immune response.

Gamma delta T (γδ T) cells are among the most potent cytotoxic lymphocytes. Activating anti­butyrophilin 3A (BTN3A) antibodies prime diverse tumor cell types to be killed by Vγ9Vδ2 T cells, the predominant γδ T cell subset in peripheral circulation, by mechanisms independent of tumor antigen­major histocompatibility complex (MHC) complexes. In this report, we describe the development of a humanized monoclonal antibody, ICT01, with subnanomolar affinity for the three isoforms of BTN3A. We demonstrate that ICT01-activated Vγ9Vδ2 T cells kill multiple tumor cell lines and primary tumor cells, but not normal healthy cells, in an efficient process requiring approximately 20% target occupancy. We show that ICT01 activity is dependent on BTN3A and BTN2A but independent of the phosphoantigen (pAg)­binding B30.2 domain. ICT01 delays the growth of hematologic and solid tumor xenografts and prolongs survival of NOD/SCID/IL2rγnull (NSG) mice adoptively transferred with human Vγ9Vδ2 T cells. In single- and multiple-dose safety studies in cynomolgus macaques that received up to 100 mg/kg once weekly, ICT01 was well tolerated. With respect to pharmacodynamic endpoints, ICT01 selectively activated Vγ9Vδ2 T cells without affecting other BTN3A-expressing lymphocytes such as αß T or B cells. A first-in-human, phase 1/2a, open-label, clinical study of ICT01 was thus initiated in patients with advanced-stage solid tumors (EVICTION: NCT04243499; EudraCT: 2019-003847-31). Preliminary results show that ICT01 was well tolerated and pharmacodynamically active in the first patients. Digital pathology analysis of tumor biopsies of a patient with melanoma suggests that ICT01 may promote immune cell infiltration within the tumor microenvironment.

Engineering antibody heavy chain CDR3 to create a phage display Fab library rich in antibodies that bind charged carbohydrates.

A number of small charged carbohydrate moieties have been associated with inflammation and cancer. However, the development of therapeutic Abs targeting these moieties has been hampered by their low immunogenicity and their structural relationship to self-Ag. We report the design of an Ab repertoire enriched in Abs binding to small charged carbohydrates and the construction of a human Fab phagemid library, "FAB-CCHO." This library combines L chain Ig sequences from human donors and H chain synthetic diversity constructed in key Ag contact sites in CDRs 1, 2, and 3 of the human framework V(H)3-23. The H chain CDR3 has been engineered to enrich the library in Abs that bind charged carbohydrates by the introduction of basic residues at specific amino acid locations. These residues were selected on the basis of anti-carbohydrate Ab sequence alignment. The success of this design is demonstrated by the isolation of phage Abs against charged carbohydrate therapeutic target Ags such as sulfated sialyl-Lewis X glycan and heparan sulfate.

Screening isolates from antibody phage-display libraries.

Antibody phage display, coupled with automated screening, facilitates and potentiates the mining of complex combinatorial libraries and the identification of potent drug leads. In managing phage screening data, the behavior of individual phage isolates in binding assays must be linked to their antibody identities as deduced from DNA sequencing. Reviewed here are recently reported approaches for high-throughput screening of clones isolated from phage antibody libraries after selection on a defined antigen. Specific information management challenges, and possible solutions, are described for organizing screening data to enable rapid lead discovery using these antibody libraries.

Generation of high-affinity human antibodies by combining donor-derived and synthetic complementarity-determining-region diversity.

Combinatorial libraries of rearranged hypervariable V(H) and V(L) sequences from nonimmunized human donors contain antigen specificities, including anti-self reactivities, created by random pairing of V(H)s and V(L)s. Somatic hypermutation of immunoglobulin genes, however, is critical in the generation of high-affinity antibodies in vivo and occurs only after immunization. Thus, in combinatorial phage display libraries from nonimmunized donors, high-affinity antibodies are rarely found. Lengthy in vitro affinity maturation is often needed to improve antibodies from such libraries. We report the construction of human Fab libraries having a unique combination of immunoglobulin sequences captured from human donors and synthetic diversity in key antigen contact sites in heavy-chain complementarity-determining regions 1 and 2. The success of this strategy is demonstrated by identifying many monovalent Fabs against multiple therapeutic targets that show higher affinities than approved therapeutic antibodies. This very often circumvents the need for affinity maturation, accelerating discovery of antibody drug candidates.

Oligonucleotide-assisted cleavage and ligation: a novel directional DNA cloning technology to capture cDNAs. Application in the construction of a human immune antibody phage-display library.

The use of oligonucleotide-assisted cleavage and ligation (ONCL), a novel approach to the capture of gene repertoires, in the construction of a phage-display immune antibody library is described. ONCL begins with rapid amplification of cDNA ends to amplify all members equally. A single, specific cut near 5' and/or 3' end of each gene fragment (in single stranded form) is facilitated by hybridization with an appropriate oligonucleotide adapter. Directional cloning of targeted DNA is accomplished by ligation of a partially duplex DNA molecule (containing suitable restriction sites) and amplification with primers in constant regions. To demonstrate utility and reliability of ONCL, a human antibody repertoire was cloned from IgG mRNA extracted from human B-lymphocytes engrafted in Trimera mice. These mice were transplanted with peripheral blood lymphocytes from Candida albicans infected individuals and subsequently immunized with C.albicans glyceraldehyde-3-phosphate dehydrogenase (GAPDH). DNA sequencing showed that ONCL resulted in efficient capture of gene repertoires. Indeed, full representation of all V(H) families/segments was observed showing that ONCL did not introduce cloning biases for or against any V(H) family. We validated the efficiency of ONCL by creating a functional Fab phage-display library with a size of 3.3 x 10(10) and by selecting five unique Fabs against GAPDH antigen.

Selective inhibition of matrix metalloproteinase-14 blocks tumor growth, invasion, and angiogenesis.

Inhibition of specific matrix metalloproteinases (MMP) is an attractive noncytotoxic approach to cancer therapy. MMP-14, a membrane-bound zinc endopeptidase, has been proposed to play a central role in tumor growth, invasion, and neovascularization. Besides cleaving matrix proteins, MMP-14 activates proMMP-2 leading to an amplification of pericellular proteolytic activity. To examine the contribution of MMP-14 to tumor growth and angiogenesis, we used DX-2400, a highly selective fully human MMP-14 inhibitory antibody discovered using phage display technology. DX-2400 blocked proMMP-2 processing on tumor and endothelial cells, inhibited angiogenesis, and slowed tumor progression and formation of metastatic lesions. The combination of potency, selectivity, and robust in vivo activity shows the potential of a selective MMP-14 inhibitor for the treatment of solid tumors.