In vitro methods for developing binding domains have been well-established for many years, owing to the cost-efficient synthesis of DNA and high-throughput selection and screening technologies. However, generating high-affinity binding domains often requires the development of focused maturation libraries for a second selection, which typically demands a detailed understanding of the binding surfaces from the initial selection, a process that can be time-consuming. In this study, we accelerated this process by using deep sequencing data from the first selection to guide the design of the maturation library. Additionally, we employed a high-throughput screening system using flow cytometry based on Escherichia coli display to identify conditional binding domains from the selection output. This approach enabled the development of a high-affinity binder targeting the cancer biomarker HER3, with a binding affinity of 3.3 nM at extracellular pH 7.4, 100 times higher than the first-generation binding domain. Notably, the binding domain features a pH-dependent release mechanism, enabling rapid release in slightly acidic environments (pH ≈6), which resemble endosomal conditions. When conjugated to the cytotoxin mertansine (DM1), the binding domain demonstrated specific cytotoxic activity against HER3-expressing cell lines, with an IC50 of 2–5 nM. The presented approach enables the efficient development of conditional binding domains which hold promise for therapeutic applications.

The emerging strategy of protein–drug conjugates (PDCs) for targeted cancer therapy holds great potential to improve treatment efficacy by specifically targeting cancer biomarkers and delivering toxic payloads directly to tumor cells, minimizing off-target toxicity. The success of this approach depends on the internalization and retention of the payload in target cells. This study introduces a method using a small protein domain engineered for conditional target affinity, enabling lysosomal trafficking independent of the biological fate of the receptor. Specifically, we describe the development of an EGF receptor binder, CaRAEGFR, with calcium-regulated affinity (CaRA), meaning the target binding strength is tailored by the available calcium concentration. This allows for endosomal dissociation, as calcium levels are lower in endosomes than in the bloodstream. Affinity measurements and structural modeling reveal the molecular basis of the calcium modulated affinity. Live cell imaging demonstrates efficient internalization and lysosomal trafficking of the calcium-dependent domain, while the EGF receptor is recycled to the membrane. When used as a drug carrier, CaRAEGFR effectively delivers the toxin to the lysosomes, resulting in potent cytotoxicity with an IC50 of 0.8 nM in EGFR-expressing cancer cells

Designing proteins with tunable activities from easily accessible external cues remains a biotechnological challenge. Here, we set out to create a small antibody-binding domain equipped with a molecular switch inspired by the allosteric response to calcium seen in naturally derived proteins like calmodulin. We have focused on one of the three domains of Protein G that show inherent affinity to antibodies. By combining a semi-rational protein design with directed evolution, we engineered novel variants containing a calcium-binding loop rendering the inherent antibody affinity calcium-dependent. The evolved variants resulted from a designed selection strategy subjecting them to negative and positive selection pressures focused on conditional antibody binding. Hence, these variants contains molecular “on/off” switches, controlling the target affinity towards antibody fragments simply by the presence or absence of calcium. From NMR spectroscopy we found that the molecular mechanism underlying the evolved switching behavior was a coupled calcium-binding and folding event where the target binding surface was intact and functional only in the presence of bound calcium. Notably, it was observed that the response to the employed selection pressures gave rise to the evolution of a cooperative folding mechanism. This observation illustrates why the cooperative folding reaction is an effective solution seen repeatedly in the natural evolution of fine-tuned macromolecular recognition. Engineering binding moieties to confer conditional target interaction has great potential due to the exquisite interaction control that is tunable to application requirements. Improved understanding of the molecular mechanisms behind regulated interactions is crucial to unlock how to engineer switchable proteins useful in a variety of biotechnological applications.

Engineering naturally occurring proteins enables us to customize affinity domains according to our specific needs, tailoring them to become an important tool in a wide array of applications limited merely by our creativity. One of nature’s ways to regulate protein activity is by creating a functional change through alteration of a protein’s tertiary structure upon interaction with metal ions. Inspired by this elegant solution, this thesis has focused on engineering calcium-regulated affinity proteins using two different strategies and protein scaffolds.

The first strategy revolved around designing and selecting a calcium- binding motif that can render the inherent target affinity of a naturally occurring protein domain to be turned on or off depending on whether calcium is present or not. The subject of this part of the thesis was one of the immunoglobulin-binding domains derived from Streptococcal Protein G. A library of various loops with prerequisites for attracting calcium was inserted between the IgG-binding surfaces of the domain prior to performing cell display selections aimed for rendering the inherent target interaction dependent on the presence of calcium. Successful selections resulted in a calcium-dependent version of the IgG-binding protein and its structure could be solved using NMR. A deeper investigation of the incorporated structural calcium-dependency could explain the underlying mechanisms giving rise to the functional on-and-off switch in target affinity and show how it derived from the evolutionary selection pressures applied.

The second strategy included the creation of a combinatorial library based on a calcium-dependent protein scaffold, derived from Staphylococcal Protein A, for development of small calcium-regulated affinity – CaRA –imolecules with novel target specificities. Mimicking the multifaceted usefulness of naturally occurring metalloproteins, this second part of the thesis aimed at performing phage display selection campaigns towards a diverse set of targets relevant for various applications from bioprocessing (e.g. scFv) to biological therapies (e.g. TNFa, IL-23, EGFR). When evaluating the binding properties in the presence and absence of calcium, all discovered CaRA variants display calcium-dependent binding and target affinities in the nanomolar range.

Engineering conditional binding can enhance the potential of next generation therapies in several ways. When used as calcium-dependent affinity ligands, it enables mild purification at neutral pH of therapeutic antibodies and antibody fragments that was previously limited by harsh acidic elution conditions. Reducing the risk of aggregated product by eluting at neutral pH would result in improved safety as well as the possibility to manufacture a greater repertoire of antibody formats. Furthermore, the conferred calcium- dependency of the CaRA scaffold can be used in a therapeutic approach envisioned to result in increased tissue penetration due to its small size and improved intracellular delivery by taking advantage of the existing calcium- gradient across the endosomal membrane of cells. This could lead to higher therapeutic efficacy by enabling lower doses or dosing frequency, further advancing a more patient-friendly future.

När biologi kombineras med ingenjörers strukturerade tankesätt kan naturligt förekommande proteiner förvandlas till användbara verktyg, skräddarsydda för en mängd olika applikationer som endast verkar begränsas av vår fantasi.

Naturligt förekommande proteiner har oftast en dedikerad funktion, en uppgift att utföra, och aktivitetsgraden kan styras genom förändringar i proteinets struktur via interaktion med andra småmolekyler, såsom exempelvis metalljoner. Inspirerad av naturens eleganta lösningar utforskar denna avhandling olika tillvägagångssätt för att konstruera metallreglerade proteiner vars interaktion med ett målprotein kan slås på eller stängas av genom tillgång till kalcium.

Den första strategin kretsar kring att förädla ett existerande protein som redan kan känna igen och interagera med antikroppar till att endast kunna upprätthålla den förmågan i närvaro av kalcium. Tack vare banbrytande molekylärbiologiska tekniker som möjliggjort att vi idag kan klippa och klistra i arvsmassa (DNA) så finns möjligheten att infoga och kombinera gener som uttrycker olika proteinfunktioner. I detta fall kombinerades ett protein som molekylärt kan känna igen och har affinitet för vissa antikroppar med diverse strukturella motiv som kan binda kalcium. För att lyckas utveckla ett kalciumbindande motiv som kan påverka den existerande affiniteten till antikroppar byggdes ett bibliotek med olika motiv med olika förutsättningar för att attrahera kalciumjoner och detta infogades mellan de antikroppsbindande ytorna på proteinet, i hopp om att strukturen och funktionen hos proteinet skulle kunna regleras genom inbindningen av kalcium. Biblioteket genomsöktes efter varianter som uppvisade den eftersökta förmågan, kalciumberoende interaktion med antikroppar, och viiiihittade flera varianter som uppfyllde detta. Genom att undersöka proteinstrukturen på en av dessa nya kalciumberoende antikroppsbindande varianter så kunde vi förklara de underliggande mekanismerna i strukturen som gör att proteinets funktion regleras med hjälp av kalcium.

Vår andra strategi för att konstruera kalciumberoende proteiner är mer generell och syftar till att använda ett bibliotek med proteinvarianter som redan har förmågan att binda kalcium men som kan utvecklas till att interagera med andra molekyler än antikroppar. Vår vision är att kunna utveckla kalciumreglerad affinitet för vilket målprotein som än önskas och hittills har vi utvecklat flera proteiner vars affinitet för sin specifika målmolekyl kan regleras med hjälp av kalcium.

Det finns många tillämpningar där det kan vara användbart att ha ett protein vars funktion kan regleras. En stor del av den moderna läkemedelsutvecklingen drivs av cellfabriker som producerar proteiner, ofta antikroppar, som kan känna igen och hämma cancer eller inflammation när de injiceras i patienter. Cellerna producerar dock andra molekyler som krävs för deras överlevnad samtidigt som de tillverkar antikropparna och därför krävs efterföljande reningssteg som separerar de intressanta proteinerna ifrån biprodukterna. Ett kalciumberoende protein som binder antikroppar kan därför användas för att isolera det potentiella läkemedlet ifrån biprodukterna och därefter lösgöras ifrån antikroppen igen genom borttagning av kalcium.

Proteinbaserad läkemedelsutveckling innebär även fantastiska möjligheter för kreativa terapeutiska strategier. Ett kalciumberoende biologiskt läkemedel kan styras av de naturliga skiftningar i kalciumkoncentration som återfinns i kroppen och exempelvis designas på ett finurligt sätt till att leverera en sjukdomsframkallande molekyl till kroppens egna celler för nedbrytning.ivSammanfattningsvis presenterar denna avhandling olika tillvägagångssätt för att utveckla proteiner vars funktion kan regleras genom tillgången på kalcium för diverse applikationer. Förhoppningsvis kommer dessa kunna bidra till nya tillverkningsprocesser av antikroppsbaserade läkemedel och inspirera till utveckling av framtidens proteinbaserade läkemedel.

Highly accurate serological tests are key to assessing the prevalence of SARS-CoV-2 antibodies and the level of immunity in the population. This is important to predict the current and future status of the pandemic. With the recent emergence of new and more infectious SARS-CoV-2 variants, assays allowing for high throughput analysis of antibodies able to neutralize SARS-CoV-2 become even more important. Here, we report the development and validation of a robust, high throughput method, which enables the assessment of antibodies inhibiting the binding between the SARS-CoV-2 spike protein and angiotensin converting enzyme 2 (ACE2). The assay uses recombinantly produced spike-f and ACE2 and is performed in a bead array format, which allows analysis of up to 384 samples in parallel per instrument over seven hours, demanding only one hour of manual handling. The method is compared to a microneutralization assay utilising live SARS-CoV-2 and is shown to deliver highly correlating data. Further, a comparison with a serological method that measures all antibodies recognizing the spike protein shows that this type of assessment provides important insights into the neutralizing efficiency of the antibodies, especially for individuals with low antibody levels. This method can be an important and valuable tool for large-scale assessment of antibody-based neutralization, including neutralization of new spike variants that might emerge.

Protein activity regulated by interactions with metal ions can be utilized for many different purposes, including biological therapies and bioprocessing, among others. Calcium ions are known to interact with the frequently occurring EF-hand motif, which can alter protein activity upon binding through an induced conformational change. The calcium-binding loop of the EF-hand motif has previously been introduced into a small protein domain derived from staphylococcal Protein A in a successful effort to render antibody binding dependent on calcium. Presented here, is a combinatorial library for calcium-regulated affinity, CaRA, based on this domain. CaRA is the first alternative scaffold library designed to achieve novel target specificities with metal-dependent binding. From this library, several calcium-dependent binders could be isolated through phage display campaigns towards a set of unrelated target proteins (IgE Cε3-Cε4, TNFα, IL23, scFv, tPA, PCSK9 and HER3) useful for distinct applications. Overall, these monomeric CaRA variants showed high stability and target affinities within the nanomolar range. They displayed considerably higher melting temperatures in the presence of 1 mM calcium compared to without calcium. Further, all discovered binders proved to be calcium-dependent, with the great majority showing complete lack of target binding in the absence of calcium. As demonstrated, the CaRA library is highly capable of providing protein-binding domains with calcium-dependent behavior, independent of the type of target protein. These binding domains could subsequently be of great use in gentle protein purification or as novel therapeutic modalities.