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Improved candidate screening through tailored co-culture assays and precise tuning of protein expression
KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Technology.ORCID iD: 0000-0001-7987-6600
2024 (English)Licentiate thesis, comprehensive summary (Other academic)
Abstract [en]

The field of biopharmaceuticals is a rapidly growing one. In the last ten years the number of approved biopharmaceuticals has more than doubled. A major hurdle to overcome for increased availability of all the new, effective biopharmaceuticals is the cost of treatment. Much of this can be attributed to the sheer time required for their development. Owing to this, interest in improvements to the biopharmaceuticals and their development process has also rapidly increased. As costs increase the further into development a drug candidate progresses, increasing the fidelity of screening at early stages could alleviate some of the exorbitant costs of development.

In paper I, we showcase a novel way of targeting the tumor microenvironment (TME) to allow for TMElocalized CD40 activation. This is of interest as CD40 agonists have shown great potential for immune activation, but with systemic activation leading to severe adverse effects. The localized activation is achieved through the construction of an affinity fusion protein termed an AffiMab through fusion of a platelet derived growth factor receptor beta (PDGFRβ) targeting affibody to the heavy chain of a CD40 agonistic monoclonal antibody (mAb). We demonstrate PDGFRβ-dependent activation in a variety of assays, showing that the approach merits further investigation.

Building on the activation assays set up in paper I, we aim to generate an in vitro screening platform for immune cell engagers in paper II. Screening candidates for on-target off-tumor activation is essential, as such activation would lead to adverse effects and be a doselimiting factor. To screen for this, we construct a series of plasmids which upon transfecting cells allow for different levels of a cell-surface target protein to be expressed, a so-called target density panel. This is achieved through the use of hairpin forming elements in the 5’ untranslated region of the mRNA dubbed regulatory elements (RgEs). Through use of different RgEs, we show that a target density panel can be generated and validate it in activation assays with the AffiMab developed in paper I. The platforms’ uniform cell surface background due to all different levels of target being expressed in the same host cell line and tunability through use of different RgEs are features that make it interesting for further research.

Finally in paper III, we construct and test an improved translation initiation site (TIS) sequence. Using previous studies on the impact of the nucleotides in the sequence on the efficacy of the TIS, we constructed a novel sequence, TISNOV. This sequence enhanced titer and quality for recombinant production of IgG1 and IgG4 in both stable and transient settings. Further research into other TIS sequences and their uses in regulating protein expression, as well as usage of the TISNOV to improve expression of difficult to express proteins such as bispecifics remain interesting.

In conclusion this thesis focuses on different manners to improve and hasten development of new biopharmaceuticals through usage of new workflows, platforms, and genetic engineering strategies.

Abstract [sv]

Det biologiska läkemedelsfältet är i snabb tillväxt. De senaste tio åren har antalet godkända biologiska läkemedel mer än fördubblats. Den höga kostnaden för behandlingar med biologiska läkemedel är dock ett stort hinder som måste överkommas för att öka tillgängligheten till nya, effektiva behandlingar. Mycket av kostnaden kan attribueras till den långa utvecklingstiden för dem. Som en följd av detta har intresset för förbättringar av biologiska läkemedel och deras framställningsprocess även det ökat kraftigt. Eftersom kostnaderna ökar desto längre in i processen en läkemedelskandidat tar sig är förbättringar av tidiga tester av läkemedelskandidater en god kandidat till att minska de stora kostnaderna för läkemedelsutveckling.

I artikel I visar vi ett nytt sätt för att rikta läkemedel mot tumörmikromiljön (TME) och möjliggöra TMEriktad CD40-aktivering. Detta är av intresse då CD40- agonister har visat stor potential för immunaktivering, men lidit av bieffekter som uppkommit av systemisk immunaktivering. Den lokaliserade immunaktiveringen uppnås genom ett fuserat affinitetsprotein benämnt AffiMab, där en affibody riktat mot trombocytrelaterad tillväxtfaktor beta (PDGFRβ) fuserats till den tunga kedjan av en CD40-agonistisk monoklonal antikropp (mAb). Vi visar PDGFRβ-beroende aktivering i ett flertal av aktiveringsanalyser, vilket visar att tillvägagångssättet meriterar fortsatt forskning.

Som en påbyggnad till arbetssättet för utvärdering i artikel I avser vi att generera en in vitro platform för utvärdering av immune cell engagers i artikel II. Att utvärdera kandidater för aktivering som är on-target off tumor är essentiellt, då sådan aktivering leder till bieffekter som begränsar doseringen av läkemedlet. För att utvärdera detta konstruerar vi en serie plasmider som efter transfektion leder till olika uttrycksnivåer att ett målprotein på cellytan, en så kallad target density panel. Vi uppnår detta genom att använda oss av hårnålsbildande element i den otranslaterade 5’ regionen av mRNAt benämnda regulatoriska element (RgEs). Genom att använda oss av olika RgEs kan vi visa att olika målproteinsdensiteter kan genereras samt validera dem i aktiveringsanalyser med AffiMaben som utvecklades i artikel I. Den uniforma bakgrunden på cellytorna som följd av att alla nivåer av målprotein uttrycks i samma cellinje samt plattformens reglerbarhet genom användande av olika RgEs är egenskaper som gör att plattformen är intressant för vidare forskning.

Slutligen konstruerar vi en förbättrad sekvens för translationinitieringssstället (TIS) och testar den i artikel III. Med grund i tidigare studier kring vilken inverkan olika nukleotider i sekvensen har på effektiviteten hos en TIS konstruerar vi en ny sekvens, TISNOV. Denna sekvens uppvisar ökad titer och kvalitet för rekombinant produktion av IgG1 och IgG4 i transienta och stabila miljöer. Det är av fortsatt intresse att forska djupare kring andra sekvenser av TIS samt deras användning för att förbättra uttrycket av svåruttryckta proteiner såsom bispecifiker.

Sammanfattningsvis har denna avhandling fokuserat på olika tillvägagångssätt för att förbättra och påskynda utveckling av nya biologiska läkemedel, såsom nya arbetssätt, nya analysplattformar, och strategier för genmanipulation.

Place, publisher, year, edition, pages
KTH Royal Institute of Technology, 2024. , p. 51
Series
TRITA-CBH-FOU ; 2024:17
National Category
Biochemistry and Molecular Biology
Research subject
Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-346648ISBN: 978-91-8040-963-6 (print)OAI: oai:DiVA.org:kth-346648DiVA, id: diva2:1859315
Presentation
2024-06-14, Sal Q2, Malvinas väg 10, via Zoom: https://kth-se.zoom.us/j/64260982077, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

QC 2024-05-21

Available from: 2024-05-21 Created: 2024-05-21 Last updated: 2024-06-10Bibliographically approved
List of papers
1. A PDGFRB- and CD40-targeting bispecific AffiMab induces stroma-targeted immune cell activation
Open this publication in new window or tab >>A PDGFRB- and CD40-targeting bispecific AffiMab induces stroma-targeted immune cell activation
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2023 (English)In: mAbs, ISSN 1942-0862, E-ISSN 1942-0870, Vol. 15, no 1, article id 2223750Article in journal (Refereed) Published
Abstract [en]

CD40 agonism by systemic administration of CD40 monoclonal antibodies has been explored in clinical trials for immunotherapy of cancer, uncovering enormous potential, but also dosing challenges in terms of systemic toxicity. CD40-dependent activation of antigen presenting cells is dependent on crosslinking of the CD40 receptor. Here we exploited this requisite by coupling crosslinking to cancer-receptor density by dual-targeting of CD40 and platelet-derived growth factor receptor beta (PDGFRB), which is highly expressed in the stroma of various types of tumors. A novel PDGFRBxCD40 Fc-silenced bispecific AffiMab was developed to this end to test whether it is possible to activate CD40 in a PDGFRB-targeted manner. A PDGFRB-binding Affibody molecule was fused to each heavy chain of an Fc-silenced CD40 agonistic monoclonal antibody to obtain a bispecific "AffiMab". Binding of the AffiMab to both PDGFRB and CD40 was confirmed by surface plasmon resonance, bio-layer interferometry and flow cytometry, through analysis of cells expressing respective target. In a reporter assay, the AffiMab displayed increased CD40 potency in the presence of PDGFRB-conjugated beads, in a manner dependent on PDGFRB amount/bead. To test the concept in immunologically relevant systems with physiological levels of CD40 expression, the AffiMab was tested in human monocyte-derived dendritic cells (moDCs) and B cells. Expression of activation markers was increased in moDCs specifically in the presence of PDGFRB-conjugated beads upon AffiMab treatment, while the Fc-silenced CD40 mAb did not stimulate CD40 activation. As expected, the AffiMab did not activate moDCs in the presence of unconjugated beads. Finally, in a co-culture experiment, the AffiMab activated moDCs and B cells in the presence of PDGFRB-expressing cells, but not in co-cultures with PDGFRB-negative cells. Collectively, these results suggest the possibility to activate CD40 in a PDGFRB-targeted manner in vitro. This encourages further investigation and the development of such an approach for the treatment of solid cancers.

Place, publisher, year, edition, pages
Informa UK Limited, 2023
Keywords
Cancer, CD40, immuno-oncology, microenvironment, PDGFRB
National Category
Immunology in the medical area Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:kth:diva-331823 (URN)10.1080/19420862.2023.2223750 (DOI)001015010200001 ()37332119 (PubMedID)2-s2.0-85162719453 (Scopus ID)
Note

QC 20230714

Available from: 2023-07-14 Created: 2023-07-14 Last updated: 2024-05-21Bibliographically approved
2. Co-culture platform for tuning of cancer receptor density allows for evaluation of bispecific immune cell engagers
Open this publication in new window or tab >>Co-culture platform for tuning of cancer receptor density allows for evaluation of bispecific immune cell engagers
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2024 (English)In: New Biotechnology, ISSN 1871-6784, E-ISSN 1876-4347, Vol. 79, p. 120-126Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
Elsevier BV, 2024
Keywords
Expression tuning, Receptor density, Regulatory elements, Screening platform
National Category
Cancer and Oncology Immunology in the medical area
Identifiers
urn:nbn:se:kth:diva-342383 (URN)10.1016/j.nbt.2023.12.012 (DOI)38159596 (PubMedID)2-s2.0-85181763897 (Scopus ID)
Note

QC 20240122

Available from: 2024-01-17 Created: 2024-01-17 Last updated: 2024-05-21Bibliographically approved
3. Enhanced titer and quality of IgGs through alterations at the translation initiation sequence
Open this publication in new window or tab >>Enhanced titer and quality of IgGs through alterations at the translation initiation sequence
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Enhancing recombinant expression in mammalian cells remains an important and valuable goal. Much work has been done to develop cell lines, growth conditions, and expression vectors. However, some parts of the expression vector have been comparatively neglected in this quest for improvement. One such part is the translation initiation site (TIS) sequence, which drives translation initiation by increasing ribosomal recognition of the start codon. Using earlier work on what nucleotides could be exchanged for increased expression, we here present a novel TIS sequence. This TIS sequence increased titer in transient settings 4-fold while yielding 13-33 % increase in titer in final selected stable clones. This increase also comes with an increase in quality through reduction in non-paired chains, shift in charged species distribution, whilst maintaining the glycosylation profile. This increase in both titer and quality in transient and stable settings showing that TIS sequence engineering is of great interest for optimizing recombinant production in mammalian cells.

National Category
Biochemistry and Molecular Biology
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-346439 (URN)
Funder
Vinnova, 2017-02105
Note

QC 20240515

Available from: 2024-05-14 Created: 2024-05-14 Last updated: 2024-05-21Bibliographically approved

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