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

Protein therapeutics hold great potential in cancer treatment as they combine specificity with effective delivery to tumor sites, but traditional antibodies face limitations related to size, production cost, and stability. As an alternative, smaller protein scaffolds present a promising approach, offering reduced costs due to production in bacterial hosts, greater stability, and versatile engineering potential for diverse functionalities.

This thesis aims to contribute to the field of engineered scaffold proteins, optimizing the discovery workflow, introducing a new conditional binding scaffold, and evaluating its applicability as protein-drug conjugates for targeted cancer therapies.

The first part of this thesis aims to streamline the discovery pipeline for protein scaffolds. Study I presents an optimized high-throughput phage display system incorporating automated selection and sequencing techniques to discover protein binders efficiently, as demonstrated with the albumin binding domain-derived affinity protein (ADAPT) and Calcium-regulated affinity (CaRA) libraries. This semi-automated system reduces hands-on time and increases robustness, making the discovery process more accessible for labs without high-cost equipment. Results show the possibility of generating high-affinity binders with broad applications in diagnostics and therapeutics. Following the foundation laid by the workflow optimization, Study II introduces the CaRA library more deeply. The library is engineered to provide calcium-dependent and pH-dependent binding capabilities. The library’s design enables conditional interactions, where calcium levels modulate binding. This feature is particularly beneficial for therapeutic applications requiring precise targeting and controlled binding release. The CaRA scaffold demonstrated stability, nanomolar affinities, and calcium-dependent binding across diverse targets, with potential in both therapeutic and biotechnological settings. Study III introduces an accelerated maturation process for conditional binders to further enhance the therapeutic potential of small scaffold proteins. Utilizing deep sequencing data from initial selections with the CaRA library and E. coli display screening, a high-affinity binder for HER3 with pH-dependent binding, CaRA_HER3, was developed. This characteristic allows for rapid release in acidic environments, mimicking endosomal conditions, which could be advantageous for intracellular drug delivery. The final paper, Study IV, focuses on applying CaRA binders in developing protein-drug conjugates for targeted cancer treatment. Specifically, a CaRA-based EGFR binder (CaRA_EGFR) was engineered to bind EGFR conditionally, depending on calcium levels. This calciumregulated binding allows the protein to dissociate in the low-calcium environment of endosomes, potentially enhancing cytotoxic drug delivery directly to tumor cells. Confocal microscopy confirmed that the CaRA_EGFR binder effectively internalizes and trafficks to lysosomes, achieving targeted cytotoxicity in EGFR-expressing cells. This approach highlights the value of conditional affinity in challenges related to the biological fate of receptors, paving the way for more effective, receptor-specific drug delivery systems. This thesis advances protein engineering for small scaffold therapeutics through new discovery workflows and calcium- and pH-dependent binding mechanisms. By advancing new ways to engineer these scaffolds, the findings contribute to developing safer, more effective proteinbased therapies for cancer treatment.

Proteinbaserade läkemedel har stor potential inom cancerbehandling eftersom de kombinerar specificitet med effektiv leverans till tumörområden. Traditionella antikroppar har dock begränsningar kopplade till storlek, produktionskostnad och stabilitet. Som ett alternativ erbjuder mindre proteinscaffolds en lovande metod, med lägre kostnader tack vare produktion i bakterieceller, högre stabilitet och mångsidig möjlighet till anpassning för olika funktioner. 

Denna avhandling syftar till att bidra till området för konstruerade proteinscaffolds genom att optimera upptäcktsprocessen, introducera ett nytt scaffold med villkorlig bindning och utvärdera dess användbarhet som protein-läkemedelskonjugat för riktad cancerbehandling. 

Den första delen av denna avhandling fokuserar på att effektivisera selektionsprocessen för proteinscaffolds. Studie I presenterar ett optimerat high-throughput-fagdisplaysystem som inkluderar automatiserade selektions- och sekvenseringstekniker för att effektivt identifiera proteinerabindare, som exemplifieras med bibliotek av albuminbindande domäner (ADAPT) och kalciumreglerade affinitetsproteiner (CaRA). Detta halvautomatiserade system minskar den manuella arbetsinsatsen och ökar robustheten, vilket gör selektiopnsprocessen mer tillgänglig för laboratorier utan högkostnadsutrustning. Resultaten visar möjligheten att generera högaffinitetsbindare med breda användningsområden inom diagnostik och terapi. Efter optimering av arbetsflödet introducerar Studie II CaRA-biblioteket mer ingående. Biblioteket är designat för att ge kalcium- och pH-beroende bindningsegenskaper, där kalciumnivåerna modulerar bindningen. Denna egenskap är särskilt fördelaktig för terapeutiska applikationer som kräver exakt målstyrning och kontrollerad dissociation. CaRA-scaffoldet uppvisade stabilitet, nanomolära affiniteter och kalciumberoende bindning till olika måll, med potential både inom terapi och bioteknologiska applikationer. Studie III introducerar en accelererad affinitetsmatureringsprocess för villkorliga bindare för att ytterligare förbättra de terapeutiska möjligheterna för små proteinscaffolds. Med hjälp av djupsekvenseringsdata från initiala selektioner med CaRA-biblioteket och E. coli display-screening utvecklades en högaffinitetsbindare för HER3 med pH-beroende bindning, CaRA_HER3. Denna egenskap möjliggör snabb frisättning i sura miljöer, som efterliknar endosomala förhållanden, vilket kan vara fördelaktigt för intracellulär läkemedelsleverans. 

Den sista studien, Studie IV, fokuserar på tillämpningen av CaRA-bindare i utvecklingen av protein-läkemedelskonjugat för riktad cancerbehandling. Specifikt utvecklades en CaRA-baserad EGFR-bindare (CaRA_EGFR) för att binda EGFR villkorligt beroende på kalciumnivåer. Denna kalciumreglerade bindning möjliggör att proteinet dissocierar i den låga kalciumhalten i endosomer, vilket potentiellt ökar cytotoxisk läkemedelsleverans direkt till tumörceller. Konfokalmikroskopi bekräftade att CaRA_EGFR-bindaren effektivt internaliseras  och transporteras till lysosomer, vilket uppnår riktad cytotoxicitet i EGFR-uttryckande celler. Detta tillvägagångssätt understryker värdet av villkorlig affinitet i utmaningar kopplade till receptorernas biologiska öde, och banar väg för effektivare, receptor-specifika läkemedelsleveranssystem. Denna avhandling främjar proteiningenjörskonst för små scaffold-baserade terapeutiska proteiner genom nya upptäcktsarbetsflöden och kalcium- och pH-beroende bindningsmekanismer. Genom att utveckla nya s.tt att konstruera dessa scaffolds bidrar resultaten till säkrare och effektivare proteinbaserade terapier för cancerbehandling.

Targeted cancer therapy is a promising alternative to the currently established cancer treatments, aiming to selectively kill cancer cells while sparing healthy tissues. Hereby, molecular targeting agents, such as monoclonal antibodies, are used to bind to cancer cell surface markers specifically. Although these agents have shown great clinical success, limitations still remain such as low tumor penetration and off-target effects. To overcome this limitation, novel fusion proteins comprised of the two proteins ADAPT6 and Horseradish Peroxidase (HRP) were engineered. Cancer cell targeting is hereby enabled by the small scaffold protein ADAPT6, engineered to specifically bind to human epidermal growth factor receptor 2 (HER2), a cell surface marker overexpressed in various cancer types, while the enzyme HRP oxidizes the nontoxic prodrug indole-3-acetic acid (IAA) which leads to the formation of free radicals and thereby to cytotoxic effects on cancer cells. The high affinity to HER2, as well as the enzymatic activity of HRP, were still present for the ADAPT6-HRP fusion proteins. Further, in vitro cytotoxicity assay using HER2-positive SKOV-3 cells revealed a clear advantage of the fusion proteins over free HRP by association of the fusion proteins directly to the cancer cells and therefore sustained cell killing. This novel strategy of combining ADAPT6 and HRP represents a promising approach and a viable alternative to antibody conjugation for targeted cancer therapy.

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.

Albumin-binding fusion partners are frequently used as a means for the in vivo half-life extension of small therapeutic molecules that would normally be cleared very rapidly from circulation. However, in applications where small size is key, fusion to an additional molecule can be disadvantageous. Albumin-derived affinity proteins (ADAPTs) are a new type of scaffold proteins based on one of the albumin-binding domains of streptococcal protein G, with engineered binding specificities against numerous targets. Here, we engineered this scaffold further and showed that this domain, as small as 6 kDa, can harbor two distinct binding surfaces and utilize them to interact with two targets simultaneously. These novel ADAPTs were developed to possess affinity toward both serum albumin as well as another clinically relevant target, thus circumventing the need for an albumin-binding fusion partner. To accomplish this, we designed a phage display library and used it to successfully select for single-domain bispecific binders toward a panel of targets: TNFα, prostate-specific antigen (PSA), C-reactive protein (CRP), renin, angiogenin, myeloid-derived growth factor (MYDGF), and insulin. Apart from successfully identifying bispecific binders for all targets, we also demonstrated the formation of the ternary complex consisting of the ADAPT together with albumin and each of the five targets, TNFα, PSA, angiogenin, MYDGF, and insulin. This simultaneous binding of albumin and other targets presents an opportunity to combine the advantages of small molecules with those of larger ones allowing for lower cost of goods and noninvasive administration routes while still maintaining a sufficient in vivo half-life. 

In vitro methods for developing binders have been well established for many years, owing to the costefficient synthesis of DNA and high-throughput selection and screening technologies. However, achieving high-affinity binders often requires focused maturation libraries developed 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 binders from the selection output. This approach allowed us to develop a conditional high affinity binder, targeting the cancer biomarker HER3, exhibiting an affinity of 3.3 nM at extracellular pH 7.4. Interestingly, it features a pH-dependent release mechanism that enables rapid release in a slightly acidic environment (pH ≈ 6) resembling the endosomes. Further development of this novel binder holds the prerequisites for enhanced intracellular delivery of cytotoxic drugs.