Multispecific Antibody Development Platform Based on Human Heavy Chain Antibodies

Heavy chain-only antibodies (HCAbs) do not associate with light chains and their VH regions are functional as single domains, forming the smallest active antibody fragment. These VH regions are ideal building blocks for a variety of antibody-based biologics because they tolerate fusion to other molecules and may also be attached in series to construct multispecific antibodies without the need for protein engineering to ensure proper heavy and light chain pairing. Production of human HCAbs has been impeded by the fact that natural human VH regions require light chain association and display poor biophysical characteristics when expressed in the absence of light chains. Here, we present an innovative platform for the rapid development of diverse sets of human HCAbs that have been selected in vivo. Our unique approach combines antibody repertoire analysis with immunization of transgenic rats, called UniRats, that produce chimeric HCAbs with fully human VH domains in response to an antigen challenge. UniRats express HCAbs from large transgenic loci representing the entire productive human heavy chain V(D)J repertoire, mount robust immune responses to a wide array of antigens, exhibit diverse V gene usage and generate large panels of stable, high affinity, antigen-specific molecules

Evolutionary Selection on Barrier Activity: Bar1 Is an Aspartyl Protease with Novel Substrate Specificity.

UnlabelledPeptide-based pheromones are used throughout the fungal kingdom for coordinating sexual responses between mating partners. Here, we address the properties and function of Bar1, an aspartyl protease that acts as a "barrier" and antagonist to pheromone signaling in multiple species. Candida albicans Bar1 was purified and shown to exhibit preferential cleavage of native α pheromone over pheromones from related fungal species. This result establishes that protease substrate specificity coevolved along with changes in its pheromone target. Pheromone cleavage by Bar1 occurred between residues Thr-5 and Asn-6 in the middle of the tridecapeptide sequence. Surprisingly, proteolytic activity was independent of the amino acid residues present at the scissile bond and instead relied on residues at the C terminus of α pheromone. Unlike most aspartyl proteases, Bar1 also exhibited a near-neutral pH optimum and was resistant to the class-wide inhibitor pepstatin A. In addition, genetic analysis was performed on C. albicans BAR1 and demonstrated that the protease not only regulates endogenous pheromone signaling but also can limit interspecies pheromone signaling. We discuss these findings and propose that the unusual substrate specificity of Bar1 is a consequence of its coevolution with the α pheromone receptor Ste2 for their shared peptide target.ImportancePheromones are important for intraspecies communication across the tree of life. In the fungal kingdom, extracellular proteases play a key role in antagonizing pheromone signaling in multiple species. This study examines the properties and function of Candida albicans Bar1, an aspartyl protease that cleaves and thereby inactivates α pheromone. We demonstrate that Bar1 plays important roles in regulating both intra- and interspecies pheromone signaling. The fungal protease shows preferential activity on the endogenous pheromone, but, surprisingly, cleavage activity is dependent on amino acid residues distal to the scissile bond. We propose that the unusual substrate specificity of Bar1 is a direct result of coevolution with Ste2, the receptor for α pheromone, for recognition of the same peptide target. The novel specificity of Bar1 reveals the complex forces shaping the evolution of mating pathways in fungi and uncovers a protease with potentially important applications in the biotechnology industry

Evolutionary Selection on Barrier Activity: Bar1 Is an Aspartyl Protease with Novel Substrate Specificity.

UnlabelledPeptide-based pheromones are used throughout the fungal kingdom for coordinating sexual responses between mating partners. Here, we address the properties and function of Bar1, an aspartyl protease that acts as a "barrier" and antagonist to pheromone signaling in multiple species. Candida albicans Bar1 was purified and shown to exhibit preferential cleavage of native α pheromone over pheromones from related fungal species. This result establishes that protease substrate specificity coevolved along with changes in its pheromone target. Pheromone cleavage by Bar1 occurred between residues Thr-5 and Asn-6 in the middle of the tridecapeptide sequence. Surprisingly, proteolytic activity was independent of the amino acid residues present at the scissile bond and instead relied on residues at the C terminus of α pheromone. Unlike most aspartyl proteases, Bar1 also exhibited a near-neutral pH optimum and was resistant to the class-wide inhibitor pepstatin A. In addition, genetic analysis was performed on C. albicans BAR1 and demonstrated that the protease not only regulates endogenous pheromone signaling but also can limit interspecies pheromone signaling. We discuss these findings and propose that the unusual substrate specificity of Bar1 is a consequence of its coevolution with the α pheromone receptor Ste2 for their shared peptide target.ImportancePheromones are important for intraspecies communication across the tree of life. In the fungal kingdom, extracellular proteases play a key role in antagonizing pheromone signaling in multiple species. This study examines the properties and function of Candida albicans Bar1, an aspartyl protease that cleaves and thereby inactivates α pheromone. We demonstrate that Bar1 plays important roles in regulating both intra- and interspecies pheromone signaling. The fungal protease shows preferential activity on the endogenous pheromone, but, surprisingly, cleavage activity is dependent on amino acid residues distal to the scissile bond. We propose that the unusual substrate specificity of Bar1 is a direct result of coevolution with Ste2, the receptor for α pheromone, for recognition of the same peptide target. The novel specificity of Bar1 reveals the complex forces shaping the evolution of mating pathways in fungi and uncovers a protease with potentially important applications in the biotechnology industry

Evolutionary Selection on Barrier Activity: Bar1 Is an Aspartyl Protease with Novel Substrate Specificity

Peptide-based pheromones are used throughout the fungal kingdom for coordinating sexual responses between mating partners. Here, we address the properties and function of Bar1, an aspartyl protease that acts as a “barrier” and antagonist to pheromone signaling in multiple species. Candida albicans Bar1 was purified and shown to exhibit preferential cleavage of native α pheromone over pheromones from related fungal species. This result establishes that protease substrate specificity coevolved along with changes in its pheromone target. Pheromone cleavage by Bar1 occurred between residues Thr-5 and Asn-6 in the middle of the tridecapeptide sequence. Surprisingly, proteolytic activity was independent of the amino acid residues present at the scissile bond and instead relied on residues at the C terminus of α pheromone. Unlike most aspartyl proteases, Bar1 also exhibited a near-neutral pH optimum and was resistant to the class-wide inhibitor pepstatin A. In addition, genetic analysis was performed on C. albicans BAR1 and demonstrated that the protease not only regulates endogenous pheromone signaling but also can limit interspecies pheromone signaling. We discuss these findings and propose that the unusual substrate specificity of Bar1 is a consequence of its coevolution with the α pheromone receptor Ste2 for their shared peptide target

Integrated Activity and Genetic Profiling of Secreted Peptidases in Cryptococcus neoformans Reveals an Aspartyl Peptidase Required for Low pH Survival and Virulence.

The opportunistic fungal pathogen Cryptococcus neoformans is a major cause of mortality in immunocompromised individuals, resulting in more than 600,000 deaths per year. Many human fungal pathogens secrete peptidases that influence virulence, but in most cases the substrate specificity and regulation of these enzymes remains poorly understood. The paucity of such information is a roadblock to our understanding of the biological functions of peptidases and whether or not these enzymes are viable therapeutic targets. We report here an unbiased analysis of secreted peptidase activity and specificity in C. neoformans using a mass spectrometry-based substrate profiling strategy and subsequent functional investigations. Our initial studies revealed that global peptidase activity and specificity are dramatically altered by environmental conditions. To uncover the substrate preferences of individual enzymes and interrogate their biological functions, we constructed and profiled a ten-member gene deletion collection of candidate secreted peptidases. Through this deletion approach, we characterized the substrate specificity of three peptidases within the context of the C. neoformans secretome, including an enzyme known to be important for fungal entry into the brain. We selected a previously uncharacterized peptidase, which we term Major aspartyl peptidase 1 (May1), for detailed study due to its substantial contribution to extracellular proteolytic activity. Based on the preference of May1 for proteolysis between hydrophobic amino acids, we screened a focused library of aspartyl peptidase inhibitors and identified four high-affinity antagonists. Finally, we tested may1Δ strains in a mouse model of C. neoformans infection and found that strains lacking this enzyme are significantly attenuated for virulence. Our study reveals the secreted peptidase activity and specificity of an important human fungal pathogen, identifies responsible enzymes through genetic tests of their function, and demonstrates how this information can guide the development of high affinity small molecule inhibitors

Integrated Activity and Genetic Profiling of Secreted Peptidases in <i>Cryptococcus neoformans</i> Reveals an Aspartyl Peptidase Required for Low pH Survival and Virulence

<div><p>The opportunistic fungal pathogen <i>Cryptococcus neoformans</i> is a major cause of mortality in immunocompromised individuals, resulting in more than 600,000 deaths per year. Many human fungal pathogens secrete peptidases that influence virulence, but in most cases the substrate specificity and regulation of these enzymes remains poorly understood. The paucity of such information is a roadblock to our understanding of the biological functions of peptidases and whether or not these enzymes are viable therapeutic targets. We report here an unbiased analysis of secreted peptidase activity and specificity in <i>C</i>. <i>neoformans</i> using a mass spectrometry-based substrate profiling strategy and subsequent functional investigations. Our initial studies revealed that global peptidase activity and specificity are dramatically altered by environmental conditions. To uncover the substrate preferences of individual enzymes and interrogate their biological functions, we constructed and profiled a ten-member gene deletion collection of candidate secreted peptidases. Through this deletion approach, we characterized the substrate specificity of three peptidases within the context of the <i>C</i>. <i>neoformans</i> secretome, including an enzyme known to be important for fungal entry into the brain. We selected a previously uncharacterized peptidase, which we term <b>M</b>ajor <b>a</b>spart<b>y</b>l peptidase 1 (May1), for detailed study due to its substantial contribution to extracellular proteolytic activity. Based on the preference of May1 for proteolysis between hydrophobic amino acids, we screened a focused library of aspartyl peptidase inhibitors and identified four high-affinity antagonists. Finally, we tested <i>may1Δ</i> strains in a mouse model of <i>C</i>. <i>neoformans</i> infection and found that strains lacking this enzyme are significantly attenuated for virulence. Our study reveals the secreted peptidase activity and specificity of an important human fungal pathogen, identifies responsible enzymes through genetic tests of their function, and demonstrates how this information can guide the development of high affinity small molecule inhibitors.</p></div

DMEM conditioned media contains a metallopeptidase and trypsin-like endopeptidase activity.

<p>(A) The peptidase substrate specificity profile of DMEM media conditioned by wild type. Residues are significantly favored or disfavored as determined by the frequency of detection in substrates versus the frequency of the residue in the peptide library, p < 0.05. (B) Substrate specificity profile of peptidase activity from <i>mpr1Δ</i> conditioned media, p < 0.05. (C) Peptidase substrate specificity profile constructed from cleavage events detected in wild type but not <i>mpr1Δ</i>, p < 0.05. (D) A representative peptide cleaved by peptidases in both wild type and <i>mpr1Δ</i> conditioned media.</p

Peptidase deletion strains generated in this study.

<p>Gene names were determined where possible by following the recommended naming guidelines for <i>C</i>. <i>neoformans</i> [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006051#ppat.1006051.ref049" target="_blank">49</a>]. <i>Nat</i>^<i>R</i> is nourseothricin resistance. An asterisk indicates the observation of a phenotype in subsequent mutant characterization studies (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006051#ppat.1006051.s008" target="_blank">S8</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006051#ppat.1006051.s009" target="_blank">S9</a> Figs). Evidence for activity in YNB or DMEM conditioned media was determined in subsequent experiments analyzing proteolytic activity in media conditioned by the peptidase deletion strains (Figs <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006051#ppat.1006051.g002" target="_blank">2</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006051#ppat.1006051.g003" target="_blank">3</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006051#ppat.1006051.s004" target="_blank">S4</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006051#ppat.1006051.s005" target="_blank">S5</a> Figs). Proteins identified in the present study’s secretome proteomics are indicated.</p