Current antibody-based immunotherapy depends on tumor antigen shedding for proper T cell priming. Here we select a novel human CD40 agonistic drug candidate and generate a bispecific antibody, herein named BiA9*2_HF, that allows for rapid antibody-peptide conjugate formation. The format is designed to facilitate peptide antigen delivery to CD40 expressing cells combined with simultaneous CD40 agonistic activity. In vivo, the selected bispecific antibody BiA9*2_HF loaded with peptide cargos induces improved antigen-specific proliferation of CD8+ (10-15 fold) and CD4+ T cells (2-7 fold) over control in draining lymph nodes. In both virus-induced and neoantigen-based mouse tumor models, BiA9*2_HF demonstrates therapeutic efficacy and elevated safety profile, with complete tumor clearance, as well as measured abscopal impact on tumor growth. The BiA9*2_HF drug candidate can thus be utilized to tailor immunotherapeutics for cancer patients.

Cancer immunotherapy, where a patient's immune system is harnessed to eradicate cancer cells selectively, is a leading strategy for cancer treatment. However, successes with immune checkpoint inhibitors (ICI) are hampered by reported systemic and organ-specific toxicities and by two-thirds of the patients being non-responders or subsequently acquiring resistance to approved ICIs. Hence substantial efforts are invested in discovering novel targeted immunotherapies aimed at reduced side-effects and improved potency. One way is utilizing the dual targeting feature of bispecific antibodies, which have made them increasingly popular for cancer immunotherapy. Easy and predictive screening methods for activation ranking of candidate drugs in tumor contra non-tumor environments are however lacking. Herein, we present a cell-based assay mimicking the tumor microenvironment by co-culturing B cells with engineered human embryonic kidney 293 T cells (HEK293T), presenting a controllable density of platelet-derived growth factor receptor β (PDGFRβ). A target density panel with three different surface protein levels on HEK293T cells was established by genetic constructs carrying regulatory elements limiting RNA translation of PDGFRβ. We employed a bispecific antibody-affibody construct called an AffiMab capable of binding PDGFRβ on cancer cells and CD40 expressed by B cells as a model. Specific activation of CD40-mediated signaling of immune cells was demonstrated with the two highest receptor-expressing cell lines, Level 2/3 and Level 4, while low-to-none in the low-expressing cell lines. The concept of receptor tuning and the presented co-culture protocol may be of general utility for assessing and developing novel bi-specific antibodies for immuno-oncology applications.

Abstract [ENG]

Proteins govern a multitude of biological functions vital to sustain life. The ability to withstand diseases development and harboring a defense against foreign pathogens is attributed to the wonders of the immune system and the proteins and cells its comprised of. Antibodies arguably stands as one of the most important protein classes involved in conferring immunity, able to recognize and engage, i.e., bind to pathogenic agents and exert their function. With the rise of engineered antibodies, the last three decades have ushered in an age of targeted therapeutics to address complex diseases with a more favorably efficacy and safety profile. However, an array of these diseases are governed by multifactorial variables often in interplay with each other, demanding a more broadened therapeutic strategy. Hence, significant efforts have been invested in the engineering of antibodies, expanding beyond a single speci- ficity to bi or multispecific molecules able to recognize more than one antigen within one and the same molecule. Naturally, this opens up for a multitude of functions i.e. mode of actions and harnessing of numerous diseases relevant pathways a multispe- cific potential drug compounds could carry out for a therapeutically beneficial out- come. The increased drug design complexity is accompanied with developability chal- lenges related to optimal drug design between the two or more binding specificities to achieve intended effect in a particular diseases’ biology setting. Moreover, the de- velopability profile of multispecific affinity proteins with regards to product yields and quality have long hampered the true translation-ability of these novel drug com- pounds into a clinical and industrial setting. The herein presented thesis aimed at highlighting the importance of a harmonized drug development pipeline taking into account aforementioned challenges and introducing toolboxes, platforms and work- flows to screen and optimize a variety of bispecific drug formats. Furthermore, we exploratively developed four unique bispecific constructs carrying out different mode- of-actions inhabiting partly rationally tailored design with respect to targeted dis- eases setting in the field of autoimmune diseases and oncology. 

The first bispecific molecule developed herein presented in paper I, aimed at target- ing the diseases condition in SLE from multiple fronts, with a dual blocking mode of action targeting two related ligands driving diseases progression. A lead bispecific AffiMab format chosen post screening of several candidates in molecular and in vitro systems was evaluated in ex vivo whole blood model assay demonstrating a signifi- cant effect by the dual blocking strategy to actively decrease the levels of the target ligands. The molecule warrants further evaluation in approriate in vivo models and ii ex vivo whole blood assay with patient derived material given the potential beneficial effect of the proposed therapeutic strategy based on the fundmenetal biology of the diseases and clinical observations. In paper II, a novel bispecific format able to de- liver cargo antigens to antigen presenting cells in a modular fashion was developed. Moreover, the bispecific exerts agonistic downstream signaling of targeted cells via CD40 engagement, synergistically priming immune cell activation whilst delivering the cargo antigen simultaneously. The delivery is based on an affinity interaction between a static peptide stretch synthesized with the antigen peptide sequence and a single chain attached to the structure of an anti-CD40 agonistic antibody. Employ- ment of the established adaptable drug affinity conjugate platform (ADAC) enabled the delivery of antigen cargo strictly dependent on the affinity interaction, inducing a significant anti-tumor response by expansion of antigen specific CD8+ T cells demonstrated in vivo. 

In paper III, we explore a HER2 and EGFR dual blocking strategy employing bis- pecific AffiMabs. The bispecifics demonstrated a significantly greater effect in an in vitro cell based assay compared to the combination treatment with the two monospe- cific molecules targeting respective antigen, indicating a potential synergistic effect conferred by the format. However, the effect of the molecule and potential benefit versus the monospecific or combination treatment need to be further evaluated in vivo. Paper IV aimed at harnessing the CD40 dendritic cell activation axis by a bispecific immune cell engager AffiMab, governing CD40 mediated activation depen- dent on the engagement with a stroma antigen upregulated in the tumor microenvi- ronment, platelet growth factor receptor B (PDGFRβ). The AffiMab demonstrated the intended mode of action in in vitro cell based model assays, and with isolated antigen presenting cells and B cells from healthy donor blood, albeit room for format optimization should be taken into consideration. The study warrants further investi- gation in appropriate in vivo models for treatment of solid tumors. 

In paper V we developed a modular platform to fine tune protein expression in mammalian cells on a translational level utilizing 5’UTR hairpin structure, herein coined as Regulatory elements (RgEs). Hypothesizing that “less is more” wherein a balanced expression of a proteins subunits was demonstrated to be of greater impor- tance than a maximum expression output of each component to apprehend correctly assembled protein product. The developed tool box holds possibility for multifaceted applications, and was extended in paper VI to the use in the establishment of an in vitro culture system to fine tune receptor densities on the cell surface of a defined iii cell line. The applications end-use would be functioning as an integrative part in the high throughput screening pipeline of bispecific immune cell engagers for early eval- uation and ranking of formats and access to target antigens impact on the function- ality of screened constructs. 

In summary, the herein presented work exploratively evaluated mode of actions, de- sign, format, and engineering of bispecific molecules to address both challenges in terms of achieving intended effect but equally important considerations and solutions to improve and evaluate product manufacturability early on in the drug development pipeline. 

Sammanfattning [SV]

Proteiner styr en mängd biologiska funktioner som är avgörande för att upprätthålla liv. Förmågan att motstå utveckling av sjukdomar och att aktivera ett försvar mot främmande patogener tillskrivs immunsystemets underverk och de proteiner och celler som det består av. Antikroppar står utan tvekan som en av de viktigaste proteinklasserna involverade i att skapa immunitet, genom att känna igen och binda med en viss affinitet till patogen samt utöva deras specialiserade biologiska funktioner. Den exponentiella tillväxten av rationellt konstruerade antikroppar har de senaste tre decennierna inlett en tid av målinriktad terapi för att hantera komplexa sjukdomar med en mer fördelaktig effektivitet och säkerhetsprofil. Men en rad av dessa sjukdomar styrs av multifaktoriella variabler, ofta i samspel med varandra, vilket kräver en mer breddad behandlings strategi. Därför har betydande ansträngningar investerats i konstruktionen av antikroppar, som expanderar bortom en enda specificitet till bi- eller multispecifika molekyler som kan känna igen mer än ett antigen inom en och samma molekyl. Detta öppnar upp för en mängd funktioner en multispecifik antikropp skulle kunna utföra. Den ökade komplexiteten av dessa syntetiska molekyler åtföljs av utvecklingsutmaningar relaterade till optimal design mellan de två eller flera bindnings-specificiteterna för att uppnå avsedd effekt i en viss sjukdoms biologiska kontext. Dessutom har utvecklingsprofilen för multispecifika affinitetsproteiner med avseende på produktutbyte och kvalitet länge hämmat den verkliga översättningsförmågan hos dessa nya läkemedelskandidater i en klinisk och industriell miljö. Den här presenterade avhandlingen syftar till att belysa vikten av en harmoniserad läkemedelsutvecklings-pipeline som tar hänsyn till ovannämnda utmaningar och introducerar verktygslådor, plattformar och arbetsflöden för att utvärdera och optimera en mängd olika bispecifika läkemedelsformat. Dessutom utforskade vi fyra unika bispecifika antikroppar som utför olika funktioner rationellt skräddarsydda med avseende på att rikta sig på komplexa sjukdomar inom området autoimmuna sjukdomar och onkologi. 

Den första bispecifika molekylen som utvecklats häri presenterad i artikel I, syftar till att adressera det komplexa sjukdomstillståndet i SLE från flera fronter, genom ett dubbelt blockerande verkningssätt riktat mot två sjukdomsrelaterade ligander som driver sjukdomsprogression. Ett optimerat bispecifikt AffiMab-format valt efter utvärdering av flera kandidater i molekylära och in vitro-system testades i ex vivo helblods-analyser som uppvisade en signifikant effekt av den dubbla blockerings- strategin genom att påverka nivåerna av båda mål ligander i ett representativt SLE- v modellsystem. Sammanfattningsvis, besitter molekylen förmågan att adressera två kritiska signal vägar i SLE för att avlasta den inflammatoriska processen. I artikel II utvecklades ett nytt bispecifikt format som kan leverera lastantigen till antigen- presenterande celler på ett modulärt sätt. Dessutom utövar den bispecifika antikroppen agonistisk nedströms-signalering av målceller via CD40-engagemang, vilket synergistiskt initierar immuncells-aktivering samtidigt som last-antigenet levereras till den aktiverade målcellen. Leveransen är baserad på en affinitets- interaktion mellan en statisk peptid-sträcka syntetiserad med en antigen-peptid sekvens och ett sekundärt affinitets protein fäst till strukturen av en CD40 inriktad agonistisk antikropp. Användning av den etablerade plattformen (ADAC) möjliggjorde leverans av last-antigen strikt beroende på affinitets-interaktionen, vilket inducerade ett anti-tumörsvar genom expansion av antigen-specifika CD8+ T- celler demonstrerat i en in vivo tumör djur-modell. 

I artikel III utforskar vi en dubbel blockerings-strategi av två cancer relaterade receptorer (HER2 och EGFR) med hjälp av bispecifika AffiMabs. Bispecifikernas visade en signifikant bättre effekt i termer av cytotoxisk effekt i en in vitro cell- baserad analys jämfört med kombinationsbehandlingen med de två monospecifika molekylerna riktade mot respektive antigen. Vilket indikerar en potentiell synergistisk effekt med det bispecifika formatet. Emellertid måste effekten av molekylen och dess potentiell nytta jämfört med den monospecifika eller kombinationsbehandlingen utvärderas ytterligare in vivo. Artikel IV syftade till att utnyttja CD40-dendritiska cell aktiverings-axeln med hjälp av en bispecifik immuncell-engagerande AffiMab, som styr CD40-medierad aktivering beroende på engagemanget med ett stroma-antigen upp-reglerat i tumörmikromiljön, trombocyt- tillväxtfaktor-receptor β (PDGFRβ). AffiMaben uppvisade dess avsedda verkningssättet i in vitro cell-baserade analyser, och med antigen-presenterande celler och B-celler isolerade från blod donerat från en frisk donator, även om utrymme för formatoptimering bör beaktas. Studien kräver ytterligare undersökning i lämpliga in vivo-modeller för behandling av solida tumörer. 

I artikel V utvecklade vi en modulär plattform för att finjustera proteinuttryck i däggdjursceller genom användning av ett modulärt genetiskt verktyg, här myntad som regulatoriska element (RgEs), med hypotesen att "less is more" där ett balanserat uttryck av ett proteins sub-enheter visade sig vara av större betydelse än en maximal uttrycksgrad av varje komponent för att erhålla en korrekt sammansatt proteinprodukt i höga mängder. Den utvecklade verktygslådan rymmer möjligheten vi för mångfacetterade tillämpningar, och utökades i artikel VI till att användas vid etablering av ett in vitro-odlingssystem för att finjustera receptor-densiteter på cellytan av en definierad cellinje. Applikationens slutanvändning skulle fungera som en integrerande del i en läkemedels utvärderings-pipeline av bispecifika immuncell- engagerare för tidig utvärdering och rangordning av format och påverkan av tillgången till mål-antigenet och dess påverkar på funktionaliteten hos utvärderade format. 

Sammanfattningsvis, det häri presenterade arbetet, explorativt utvärderade behandlingsformer, design, format och produktions utvärdering av bispecifika molekyler för att möta både utmaningar när det gäller att uppnå avsedd effekt men lika viktigt lösningar för att förbättra och utvärdera produkttillverkning av dessa komplexa molekyler tidigt i läkemedelsutvecklings-pipelinen. 

Biologics represent the fastest growing group of therapeutics, but many advanced recombinant protein moieties remain difficult to produce. Here, we identify metabolic engineering targets limiting expression of recombinant human proteins through a systems biology analysis of the transcriptomes of CHO and HEK293 during recombinant expression. In an expression comparison of 24 difficult to express proteins, one third of the challenging human proteins displayed improved secretion upon host cell swapping from CHO to HEK293. Guided by a comprehensive transcriptomics comparison between cell lines, especially highlighting differences in secretory pathway utilization, a co-expression screening of 21 secretory pathway components validated ATF4, SRP9, JUN, PDIA3 and HSPA8 as productivity boosters in CHO. Moreover, more heavily glycosylated products benefitted more from the elevated activities of the N- and O-glycosyltransferases found in HEK293. Collectively, our results demonstrate the utilization of HEK293 for expression rescue of human proteins and suggest a methodology for identification of secretory pathway components for metabolic engineering of HEK293 and CHO.

Background: Monoclonal antibodies (mAbs) have proved to be a valuable tool for the treatment of different cancer types. However, clinical use of an increasing number of mAbs, have also highlighted limitations with monotherapy for cancers, in particular for such with more complex mechanisms, requiring action on additional molecules or pathways, or for cancers quickly acquiring resistance following monotherapy. An example for the latter is the mAb trastuzumab, FDA approved for treatment of metastatic gastric carcinoma. To circumvent this, researchers have reported synergistic, anti-proliferative effects by combination targeting of HER2 and EGFR by trastuzumab and the EGFR-targeting mAb Cetuximab overcoming trastuzumab resistance. Methods: Maintaining the proven functionality of trastuzumab, we have designed bi-specific antibody molecules, called AffiMabs, by fusing an EGFR-targeting Affibody molecule to trastuzumab’s heavy or light chains. Having confirmed binding to EGFR and Her2 and cytotoxicity of our AffiMabs, we analyzed apoptosis rate, receptor surface levels, phosphorylation levels of receptors and associated signaling pathways as well as differentially expressed genes on transcriptome level with the aim to elucidate the mode of action of our AffiMabs. Results: The AffiMabs are able to simultaneously bind HER2 and EGFR and show increased cytotoxic effect compared to the original trastuzumab therapeutic molecule and, more importantly, even to the combination of trastuzumab and EGFR-targeting Affibody molecule. Analyzing the mode of action, we could show that bi-specific AffiMabs lead to reduced surface receptor levels and a downregulation of cell cycle associated genes on transcriptome level. Conclusion: Our study shows that transcriptome analysis can be used to validate the choice of receptor targets and guide the design of novel multi-specific molecules. The inherent modularity of the AffiMab format renders it readily applicable to other receptor targets. 

Regulatory T cells (Tregs) are the key cells regulating peripheral autoreactive T lymphocytes. Tregs exert their function by suppressing effector T cells. Tregs have been shown to play essential roles in the control of a variety of physiological and pathological immune responses. However, Tregs are unstable and can lose the expression of FOXP3 and suppressive functions as a consequence of outer stimuli. Available literature suggests that secreted proteins regulate Treg functional states, such as differentiation, proliferation and suppressive function. Identification of secreted proteins that affect Treg cell function are highly interesting for both therapeutic and diagnostic purposes in either hyperactive or immunosuppressed populations. Here, we report a phenotypic screening of a human secretome library in human Treg cells utilising a high throughput flow cytometry technology. Screening a library of 575 secreted proteins allowed us to identify proteins stabilising or destabilising the Treg phenotype as suggested by changes in expression of Treg marker proteins FOXP3 and/or CTLA4. Four proteins including GDF-7, IL-10, PAP and IFNα-7 were identified as positive regulators that increased FOXP3 and/or CTLA4 expression. PAP is a phosphatase. A catalytic-dead version of the protein did not induce an increase in FOXP3 expression. Ten interferon proteins were identified as negative regulators that reduced the expression of both CTLA4 and FOXP3, without affecting cell viability. A transcriptomics analysis supported the differential effect on Tregs of IFNα-7 versus other IFNα proteins, indicating differences in JAK/STAT signaling. A conformational model experiment confirmed a tenfold reduction in IFNAR-mediated ISG transcription for IFNα-7 compared to IFNα-10. This further strengthened the theory of a shift in downstream messaging upon external stimulation. As a summary, we have identified four positive regulators of FOXP3 and/or CTLA4 expression. Further exploration of these Treg modulators and their method of action has the potential to aid the discovery of novel therapies for both autoimmune and infectious diseases as well as for cancer.

Human embryonic kidney cells HEK293 can be used for the production of therapeutic glycoproteins requiring human post-translational modifications. High cell density perfusion processes are advantageous for such production but are challenging due to the shear sensitivity of HEK293 cells. To understand the impact of hollow filter cell separation devices, cells were cultured in bioreactors operated with tangential flow filtration (TFF) or alternating tangential flow filtration (ATF) at various flow rates. The average theoretical velocity profile in these devices showed a lower shear stress for ATF by a factor 0.637 compared to TFF. This was experimentally validated and, furthermore, transcriptomic evaluation provided insights into the underlying cellular processes. High shear caused cellular stress leading to apoptosis by three pathways, i.e. endoplasmic reticulum stress, cytoskeleton reorganization, and extrinsic signaling pathways. Positive effects of mild shear stress were observed, with increased recombinant erythropoietin production and increased gene expression associated with transcription and protein phosphorylation.

Predictably regulating protein expression levels to improve recombinant protein production has become an important tool, but is still rarely applied to engineer mammalian cells. We therefore sought to set-up an easy-to-implement toolbox to facilitate fast and reliable regulation of protein expression in mammalian cells by introducing defined RNA hairpins, termed 'regulation elements (RgE)', in the 5'-untranslated region (UTR) to impact translation efficiency. RgEs varying in thermodynamic stability, GC-content and position were added to the 5'-UTR of a fluorescent reporter gene. Predictable translation dosage over two orders of magnitude in mammalian cell lines of hamster and human origin was confirmed by flow cytometry. Tuning heavy chain expression of an IgG with the RgEs to various levels eventually resulted in up to 3.5-fold increased titers and fewer IgG aggregates and fragments in CHO cells. Co-expression of a therapeutic Arylsulfatase-A with RgE-tuned levels of the required helper factor SUMF1 demonstrated that the maximum specific sulfatase activity was already attained at lower SUMF1 expression levels, while specific production rates steadily decreased with increasing helper expression. In summary, we show that defined 5'-UTR RNA-structures represent a valid tool to systematically tune protein expression levels in mammalian cells and eventually help to optimize recombinant protein expression.