In vitro evolution of antibody affinity via insertional scanning mutagenesis of an entire antibody variable region.

We report a systematic combinatorial exploration of affinity enhancement of antibodies by insertions and deletions (InDels). Transposon-based introduction of InDels via the method TRIAD (transposition-based random insertion and deletion mutagenesis) was used to generate large libraries with random in-frame InDels across the entire single-chain variable fragment gene that were further recombined and screened by ribosome display. Knowledge of potential insertion points from TRIAD libraries formed the basis of exploration of length and sequence diversity of novel insertions by insertional-scanning mutagenesis (InScaM). An overall 256-fold affinity improvement of an anti-IL-13 antibody BAK1 as a result of InDel mutagenesis and combination with known point mutations validates this approach, and suggests that the results of this InDel mutagenesis and conventional exploration of point mutations can synergize to generate antibodies with higher affinity.

A CD80-Biased CTLA4-Ig Fusion Protein with Superior In Vivo Efficacy by Simultaneous Engineering of Affinity, Selectivity, Stability, and FcRn Binding.

Affinity- and stability-engineered variants of CTLA4-Ig fusion molecules with enhanced pharmacokinetic profiles could yield improved therapies with the potential of higher efficacy and greater convenience to patients. In this study, to our knowledge, we have, for the first time, used in vitro evolution to simultaneously optimize CTLA4 affinity and stability. We selected for improved binding to both ligands, CD80 and CD86, and screened as dimeric Fc fusions directly in functional assays to identify variants with stronger suppression of in vitro T cell activation. The majority of CTLA4 molecules showing the largest potency gains in primary in vitro and ex vivo human cell assays, using PBMCs from type 1 diabetes patients, had significant improvements in CD80, but only modest gains in CD86 binding. We furthermore observed different potency rankings between our lead molecule MEDI5265, abatacept, and belatacept, depending on which type of APC was used, with MEDI5265 consistently being the most potent. We then created fusions of both stability- and potency-optimized CTLA4 moieties with human Fc variants conferring extended plasma t1/2 In a cynomolgus model of T cell-dependent Ab response, the CTLA4-Ig variant MEDI5265 could be formulated at >100 mg/ml for s.c. administration and showed superior efficacy and significantly prolonged serum t1/2 The combination of higher stability and potency with prolonged pharmacokinetics could be compatible with very infrequent, s.c. dosing while maintaining a similar level of immune suppression to more frequently and i.v. administered licensed therapies.

Phenotypic screening-the fast track to novel antibody discovery.

The majority of antibody therapeutics have been isolated from target-led drug discovery, where many years of target research preceded drug program initiation. However, as the search for validated targets becomes more challenging and target space becomes increasingly competitive, alternative strategies, such as phenotypic drug discovery, are gaining favour. This review highlights successful examples of antibody phenotypic screens that have led to clinical drug candidates. We also review the requirements for performing an effective antibody phenotypic screen, including antibody enrichment and target identification strategies. Finally, the future impact of phenotypic drug discovery on antibody drug pipelines will be discussed.

Exosomal delivery of doxorubicin enables rapid cell entry and enhanced in vitro potency.

Doxorubicin is a chemotherapeutic agent that is commonly used to treat a broad range of cancers. However, significant cardiotoxicity, associated with prolonged exposure to doxorubicin, limits its continued therapeutic use. One strategy to prevent the uptake of doxorubicin into cardiac cells is the encapsulation of the drug to prevent non-specific uptake and also to improve the drugs' pharmacokinetic properties. Although encapsulated forms of doxorubicin limit the cardiotoxicity observed, they are not without their own liabilities as an increased amount of drug is deposited in the skin where liposomal doxorubicin can cause palmar-plantar erythrodysesthesia. Exosomes are small endogenous extracellular vesicles, that transfer bioactive material from one cell to another, and are considered attractive drug delivery vehicles due to their natural origin. In this study, we generated doxorubicin-loaded exosomes and demonstrate their rapid cellular uptake and re-distribution of doxorubicin from endosomes to the cytoplasm and nucleus resulting in enhanced potency in a number of cultured and primary cell lines when compared to free doxorubicin and liposomal formulations of doxorubicin. In contrast to other delivery methods for doxorubicin, exosomes do not accumulate in the heart, thereby providing potential for limiting the cardiac side effects and improved therapeutic index.

Harnessing phage and ribosome display for antibody optimisation.

Therapeutic antibodies have become a major driving force for the biopharmaceutical industry; therefore, the discovery and development of safe and efficacious antibody leads have become competitive processes. Phage and ribosome display are ideal tools for the generation of such molecules and have already delivered an approved drug as well as a multitude of clinical candidates. Because they are capable of searching billions of antibody variants in tailored combinatorial libraries, they are particularly applicable to potency optimisation. In conjunction with targeted, random or semi-rational mutagenesis strategies, they deliver large panels of potent antibody leads. This review introduces the two technologies, compares them with respect to their use in antibody optimisation and highlights how they can be exploited for the successful and efficient generation of putative drug candidates.

Phenotypic screening: the future of antibody discovery.

Most antibody therapeutics have been isolated from high throughput target-based screening. However, as the number of validated targets diminishes and the target space becomes increasingly competitive, alternative strategies, such as phenotypic screening, are gaining momentum. Here, we review successful phenotypic screens, including those used to isolate antibodies against cancer and infectious agents. We also consider exciting advances in the expression and phenotypic screening of antibody repertoires in single cell autocrine systems. As technologies continue to develop, we believe that antibody phenotypic screening will increase further in popularity and has the potential to provide the next generation of therapeutic antibodies.

A Shorter Route to Antibody Binders via Quantitative in vitro Bead-Display Screening and Consensus Analysis.

Affinity panning of large libraries is a powerful tool to identify protein binders. However, panning rounds are followed by the tedious re-screening of the clones obtained to evaluate binders precisely. In a first application of Bead Surface Display (BeSD) we show successful in vitro affinity selections based on flow cytometric analysis that allows fine quantitative discrimination between binders. Subsequent consensus analysis of the resulting sequences enables identification of clones that bind tighter than those arising directly from the experimental selection output. This is demonstrated by evolution of an anti-Fas receptor single-chain variable fragment (scFv) that was improved 98-fold vs the parental clone. Four rounds of quantitative screening by fluorescence-activated cell sorting of an error-prone library based on fine discrimination between binders in BeSD were followed by analysis of 200 full-length output sequences that suggested a new consensus design with a Kd ∼140 pM. This approach shortens the time and effort to obtain high affinity reagents and its cell-free nature transcends limitations inherent in previous in vivo display systems.

Cloning and characterization of Xenopus beta2-microglobulin.

cDNAs for Xenopus beta2-microglobulin (beta2m), the obligatory light chain of most vertebrate Major Histocompatibility Complex (MHC) class I molecules, were isolated and ESTs were identified. Alignment of the deduced amino acid sequence to other species' beta2m showed that the overall structure is evolutionarily conserved, and phylogenetic analysis showed that the Xenopus beta2m sequence is intermediate between fish and bird/mammal beta2m. The Xenopus beta2m mRNA is expressed ubiquitously with highest expression in intestine, spleen, and thymus, correlating well with classical class Ia expression. beta2m mRNA and protein were also detected in Xenopus thymic tumor and kidney cell lines. Segregation analysis on a tetraploid Xenopus laevis family revealed two independently segregating, non-MHC-linked loci. As expected, only one locus was found in the diploid Xenopus tropicalis, strongly suggesting that the two beta2m loci in the tetraploid species were generated by genome-wide duplication, and did not undergo diploidization unlike many other MHC genes.

Engineering therapeutic proteins for cell entry: the natural approach.

Owing to the challenges of cell entry, protein-based therapies have so far been restricted to extracellular targets, whereas intracellular targets have been almost exclusively addressed by small molecules. The specificity and potency of proteins would enable them to be effective intracellular drugs, provided that the proteins are delivered efficiently to appropriate intracellular compartments within specific cell types. By mimicking the natural mechanisms of toxins and other natural proteins, new intracellular delivery systems are being developed, the first of which are showing clinical efficacy. This review highlights a range of ingenious approaches designed to adapt natural entry mechanisms to facilitate delivery of proteins and open up a range of validated intracellular targets to modulation by potent and specific therapeutic drugs.

Combinatorial Screening Identifies Novel Promiscuous Matrix Metalloproteinase Activities that Lead to Inhibition of the Therapeutic Target IL-13.

The practical realization of disease modulation by catalytic degradation of a therapeutic target protein suffers from the difficulty to identify candidate proteases, or to engineer their specificity. We identified 23 measurable, specific, and new protease activities using combinatorial screening of 27 human proteases against 24 therapeutic protein targets. We investigate the cleavage of monocyte chemoattractant protein 1, interleukin-6 (IL-6), and IL-13 by matrix metalloproteinases (MMPs) and serine proteases, and demonstrate that cleavage of IL-13 leads to potent inhibition of its biological activity in vitro. MMP-8 degraded human IL-13 most efficiently in vitro and ex vivo in human IL-13 transgenic mouse bronchoalveolar lavage. Hence, MMP-8 is a therapeutic protease lead against IL-13 for inflammatory conditions whereby reported genetic and genomics data suggest an involvement of MMP-8. This work describes the first exploitation of human enzyme promiscuity for therapeutic applications, and reveals both starting points for protease-based therapies and potential new regulatory networks in inflammatory disease.

Human monomeric antibody fragments to TRAIL-R1 and TRAIL-R2 that display potent in vitro agonism.

Apoptosis through the TRAIL receptor pathway can be induced via agonistic IgG to either TRAIL-R1 or TRAIL-R2. Here we describe the use of phage display to isolate a substantive panel of fully human anti-TRAIL receptor single chain Fv fragments (scFvs); 234 and 269 different scFvs specific for TRAIL-R1 and TRAIL-R2 respectively. In addition, 134 different scFvs that were cross-reactive for both receptors were isolated. To facilitate screening of all 637 scFvs for potential agonistic activity in vitro, a novel high-throughput surrogate apoptosis assay was developed. Ten TRAIL-R1 specific scFv and 6 TRAIL-R2 specific scFv were shown to inhibit growth of tumor cells in vitro in the absence of any cross-linking agents. These scFv were all highly specific for either TRAIL-R1 or TRAIL-R2, potently inhibited tumor cell proliferation, and were antagonists of TRAIL binding. Moreover, further characterization of TRAIL-R1 agonistic scFv demonstrated significant anti-tumor activity when expressed and purified as a monomeric Fab fragment. Thus, scFv and Fab fragments, in addition to whole IgG, can be agonistic and induce tumor cell death through specific binding to either TRAIL-R1 or TRAIL-R2. These potent agonistic scFv were all isolated directly from the starting phage antibody library and demonstrated significant tumor cell killing properties without any requirement for affinity maturation. Some of these selected scFv have been converted to IgG format and are being studied extensively in clinical trials to investigate their potential utility as human monoclonal antibody therapeutics for the treatment of human cancer.

Probing a protein-protein interaction by in vitro evolution.

In this study, we used in vitro protein evolution with ribosome and phage display to optimize the affinity of a human IL-13-neutralizing antibody, a therapeutic candidate for the treatment of asthma, >150-fold to 81 pM by using affinity-driven stringency selections. Simultaneously, the antibody potency to inhibit IL-13-dependent proliferation in a cell-based functional assay increased 345-fold to an IC50 of 229 pM. The panoply of different optimized sequences resulting from complementarity-determining region-targeted mutagenesis and error-prone PCR using ribosome display was contrasted with that of complementarity-determining region-targeted mutagenesis alone using phage display. The data highlight the advantage of the ribosome-display approach in identifying beneficial mutations across the entire sequence space. A comparison of mutation hotspots from in vitro protein evolution to knockout mutations from alanine scanning demonstrated that in vitro evolution selects the most appropriate positions for improvements in potency without mutating any of the key residues within the functional paratope.

Generation and characterization of LymphoStat-B, a human monoclonal antibody that antagonizes the bioactivities of B lymphocyte stimulator.

OBJECTIVE: To identify and characterize a fully human antibody directed against B lymphocyte stimulator (BLyS), a tumor necrosis factor-related cytokine that plays a critical role in the regulation of B cell maturation and development. Elevated levels of BLyS have been implicated in the pathogenesis of autoimmune diseases. METHODS: A human phage display library was screened for antibodies against human BLyS. A human monoclonal antibody, LymphoStat-B, specific for human BLyS was obtained from the library screening and subsequent affinity optimization mutagenesis. The antibody was tested for inhibition of human BLyS in vitro and in an in vivo murine model. Additionally, the consequences of BLyS inhibition were tested in vivo by administration of LymphoStat-B to cynomolgus monkeys. RESULTS: LymphoStat-B bound with high affinity to human BLyS and inhibited the binding of BLyS to its 3 receptors, TACI, BCMA, and BLyS receptor 3/BAFF-R. LymphoStat-B potently inhibited BLyS-induced proliferation of B cells in vitro, and administration of LymphoStat-B to mice prevented human BLyS-induced increases in splenic B cell numbers and IgA titers. In cynomolgus monkeys, administration of LymphoStat-B resulted in decreased B cell representation in both spleen and mesenteric lymph nodes. CONCLUSION: A fully human monoclonal antibody has been isolated that binds to BLyS with high affinity and neutralizes human BLyS bioactivity in vitro and in vivo. Administration of this antibody to cynomolgus monkeys resulted in B cell depletion in spleen and lymph node. This antibody may prove therapeutically useful in the treatment of autoimmune diseases in humans.