Radiolabeled targeting agents have emerged as valuable tools for the treatment of disseminated cancer. Monoclonal antibodies (mAbs) are widely employed as carriers for diagnostic and therapeutic radionuclides due to their exceptional specificity and affinity. However, their prolonged circulatory half-life can diminish diagnostic efficacy and increase radiation exposure to non-target tissues in therapeutic applications, resulting in dose-limiting toxicities. To overcome this limitation, pretargeting technologies emerge as promising strategies to enhance tumor-to-background ratio and reduce radiation exposure of healthy tissues. Our previous work introduced a pretargeting concept leveraging the specific interaction between two peptide nucleic acid (PNA) probes, HP1 and HP2, as the recognition mechanism. This early iteration of the PNA-based concept showed limited efficacy when used with mAb-based vectors. To improve its performance, we re-engineered the primary and secondary targeting agents by incorporating newly designed PNA-probes. As the primary targeting agent, we functionalized trastuzumab (T), a well-characterized human epidermal growth factor receptor 2 (HER2)-targeting IgG1 mAb, with a 9-mer PNA probe (HP9). Both FcIII-based covalent UV-light crosslinking and enzyme-mediated glyco-engineering click-chemistry methods were applied to generate trastuzumab-PNA conjugates T-FcIII-HP9 and T-gly-HP9, respectively. As a radionuclide-carrying secondary agent, we utilized a 9-mer complementary PNA probe, HP16, which forms a stable duplex with HP9 as well as displaying favorable in vivo kinetics. Biacore and flow cytometry assessment of the HP9-conjugated trastuzumab agents demonstrated retained HER2-binding properties. The secondary HP16 probe, labeled with either a dye or a radionuclide, showed cell surface accumulation contingent on the presence of HP9 on the primary HER2-targeting agents. In vivo, T-gly-HP9 exhibited significantly longer blood circulation half-life and superior tumor uptake compared to T-FcIII-HP9. Further, therapeutic dosing with [177Lu]-HP16 of trastuzumab-HP9 pretargeted HER2+ tumor models resulted in significantly delayed disease progression and extended survival compared to untreated subjects. Furthermore, pretargeted [177Lu]-HP16 exhibited comparable efficacy to [177Lu]-trastuzumab in both delaying disease progression and prolonging survival. In conclusion, the optimization of our PNA-based pretargeting system has resulted in exceptional in vivo targeting characteristics and therapeutic efficacy, validating the potential of this novel approach.

Antibodies and affinity proteins are essential tools in research, diagnostics and therapy. Antibodies have the ability to bind to a large variety of protein targets with great specificity and selectivity, allowing them to be used for identification, or targeted therapy of devastating diseases such as cancer. Bioconjugation to other molecules enables the extension of antibody function, enhancing its capabilities beyond recognition and binding. By attachment of a cytotoxic molecule such as a cytotoxic drug or a radionuclide to the antibody, a cell-killing agent can be directly delivered to the cancer cells, eliciting localized effect and sparing healthy tissues. Selecting a suitable labelling technique to produce high-quality antibody conjugates is a crucial consideration for many applications. Site-specific labelling is an attractive approach, where the cargo is directed to a predefined location on the antibody, resulting in homogeneous and well-characterized conjugates, without impacting its tumour antigen binding properties. Many different methods have been explored; affinity-based strategies serve as the basis of this thesis. Ligands such as the Protein A- derived Z domain and the FcIII peptide bind to the Fc domain on the antibody framework, a suitable location for delivering a selected payload. 

In this thesis, I have explored affinity ligand-based and glycan engineering-based site-specific antibody labelling methods for the production of antibody conjugates for therapeutic purposes and as research tools. In Papers I-II, we adopted site-specific labelling methods based on the Fc- targeting ligands Z domain and FcIII peptide along with a glycan-modification approach for the preparation of antibody conjugates for radioimmunotherapy (RIT) purposes. Our methods resulted in well-characterized conjugates covalently labelled with a chelator suitable for radiolabelling. The radioimmunoconjugates were evaluated in vivo and showed great potential for future applications in RIT. 

In Paper III, we aimed to improve an earlier developed PNA-based antibody pretargeting system by adopting different labelling strategies for the production of antibody-PNA conjugates. Such systems hold great potential to increase the uptake of radioactivity in the tumour and decrease exposure of healthy tissues to radiation as well as improve imaging contrast for radioimaging. Here, we demonstrated that by carefully reengineering of both the primary and secondary targeting agents, considerable enhancement can be achieved with PNA-based pretargeting. 

Apart from their success as therapeutic and diagnostic agents, antibody conjugates are widely established reagents in basic research. As sequencing-based single-cell protein expression analysis assays are becoming more popular, the production of high-quality antibody-oligonucleotide conjugates gains high importance. In such assays, specific antibody-targeted antigens are identified following the amplification and next-generation sequencing of their corresponding barcodes. In Paper IV, we utilize the Z domain for decorating an antibody panel targeting key immune cell markers with unique barcodes. The barcoded antibodies were used in DBS-Pro, a high-throughput and multiplex single-cell protein analysis method. In this project, DBS-Pro was applied for the identification of major immune cell subpopulations in PBMC samples. i In Paper V, we explored as an alternative to RIT, the potential of using affinity ligands based on endogenous peptides for radionuclide therapy. The short peptide RM26, targeting the cancer antigen gastrin-releasing peptide receptor (GRPR), commonly overexpressed in prostate cancer, is a promising candidate for imaging and therapy. Being a short peptide, RM26 suffers from fast blood clearance, which is a challenge for the therapeutic application of such ligands. Our approach to overcome this issue and to extend the circulatory half-life of RM26 is to conjugate the peptide to an albumin-binding domain (ABD) that can bind to HSA, the most abundant protein in our blood, thus achieving prolonged blood residence time. By designing, producing and evaluating several ABD-RM26 conjugate variants, we were able to investigate the effect of molecular composition on the biodistribution and bioavailability of such molecules, and identifying candidates with the most favourable properties for future development. 

In conclusion, in this thesis I combined recombinant protein techniques with chemical synthesis to produce unique protein conjugates followed by their in vitro and in vivo characterization to evaluate functional and biophysical properties. The experimental work presented in this thesis provides a rationale for the development of novel bioconjugates for a variety of future applications. 

Antikroppar och affinitetsproteiner är viktiga verktyg inom forskning, diagnostik och terapi. Antikroppar har förmågan att binda till en mängd olika proteintarget med stor specificitet och selektivitet, vilket gör att de kan användas för identifiering eller riktad terapi av förödande sjukdomar såsom cancer. Biokonjugering med andra molekyler möjliggör utvidgning av antikroppsfunktionen, vilket förbättrar dess kapacitet bortom igenkänning och bindning. Genom att fästa en cytotoxisk molekyl som ett cytotoxiskt läkemedel eller en radionuklid till antikroppen kan ett cellförstörande reagens levereras direkt till cancercellerna, vilket ger en lokaliserad effekt och skonar friska vävnader. Att välja en lämplig inmärkningsmetod för att producera högkvalitativa antikroppskonjugat är en avgörande faktor för många tillämpningar. Platspecifik inmärkning är ett attraktivt tillvägagångssätt där lasten riktas till en fördefinierad plats på antikroppen, vilket resulterar i homogena och välkarakteriserade konjugat utan att påverka dess tumörantigenbindande egenskaper. Många olika metoder har undersökts; affinitetsbaserade strategier utgör grunden för denna avhandling. Ligander som Z-domänen, som härrör från Protein A, och FcIII-peptiden binder till Fc-domänen på antikroppsstrukturen, en lämplig plats för att leverera en utvald last. 

I denna avhandling utforskar jag affinitetsligand-baserade och glykankonstruerade platspecifika antikroppsinmärkningsmetoder för produktion av antikroppskonjugat för terapeutiska ändamål och som forskningsverktyg. I arbete I-II använder vi platspecifika inmärkningsmetoder baserade på de Fc-bindande liganderna Z och FcIII tillsammans med en glykanmodifieringsmetod för framställning av antikroppskonjugat för radioimmunoterapi (RIT). Våra metoder resulterade i välkarakteriserade konjugat kovalent märkta med en kelator lämplig för radioinmärkning. Radioimmunokonjugaten utvärderades in vivo och visade stor potential för framtida tillämpningar inom RIT. 

I arbete III syftade vi till att förbättra ett tidigare utvecklat PNA-baserat antikroppsadministrationssystem genom att använda olika inmärkningsstrategier för produktion av antikropps-PNA-konjugat. Sådana system har stor potential att öka upptaget av radioaktivitet i tumören och minska exponeringen av friska vävnader för strålning samt förbättra bildkontrasten vid molekylär avbildning. Här visade vi att genom noggrann modifiering av både de primära och sekundära reagensen kan betydande förbättringar uppnås med PNA-baserad leverans av radioaktivitet. 

Förutom deras framgång som terapeutiska och diagnostiska medel är antikroppskonjugat väletablerade reagenser inom grundforskning. När sekvenseringsbaserade singelcellproteinexpressionsanalyser blir allt mer populära blir produktionen av högkvalitativa antikropps-oligonukleotidkonjugat av stor betydelse. I sådana analyser identifieras specifika antikroppsbundna antigen efter amplifiering och sekvensering av deras motsvarande streckkoder. I arbete IV använde vi Z-domänen för att dekorera en antikroppspanel riktad mot viktiga immuncellmarkörer med unika streckkoder. De streckkodade antikropparna användes i DBS-Pro, en multiplex-singelcellproteinanalysmetod med hög genomströmning. I detta projekt tillämpades DBS-Pro för identifiering av större immuncellsubpopulationer i PBMC-prover. iii I arbete V utforskade vi som ett alternativ till RIT möjligheten att använda affinitetsligander baserade på endogena peptider för radionuklidterapi. Den korta peptiden RM26, riktad mot cancerantigenet gastrin-releasing peptide receptor, som vanligtvis överuttrycks i prostatacancer, är en lovande kandidat för avbildning och terapi. Som en kort peptid lider RM26 av snabb blodclearance, vilket är en utmaning för den terapeutiska tillämpningen av sådana ligander. Vårt tillvägagångssätt för att övervinna detta problem och förlänga cirkulationstiden för RM26 är att konjugera den med en albuminbindande domän (ABD) som kan binda till HSA, det mest förekommande proteinet i vårt blod, och därmed uppnå förlängd uppehållstid i blodet. Genom att designa, producera och utvärdera flera ABD-RM26-konjugatvarianter kunde vi undersöka effekten av molekylär sammansättning på biodistributionen och biotillgängligheten av sådana molekyler samt identifiera kandidater med de mest gynnsamma egenskaperna för framtida tillämpningar. 

Sammanfattningsvis använde jag i denna avhandling rekombinant proteinteknik kombinerat med kemisk syntes för att producera unika proteinkonjugat följt av karakterisering in vitro och in vivo för att utvärdera deras funktionella och biofysikaliska egenskaper. Det experimentella arbetet som presenteras i denna avhandling ger en grund för utvecklingen av nya biokonjugat för mångsidiga framtida tillämpningar. 

Antibody radionuclide conjugates are an emerging modality for targeted imaging and potent therapy of disseminated disease. Coupling of radionuclides to monoclonal antibodies (mAbs) is typically achieved by applying non-site-specific labelling techniques. With the ambition of reducing variability, increasing labelling efficacy and stability, several site-specific conjugation strategies have been developed in recent years for toxin- and fluorophore-mAb conjugates. In this study, we studied two site-specific labelling strategies for the conjugation of the macrocyclic chelating agent, DOTA, to the anti-Leucine Rich Repeat Containing 15 (LRRC15) mAb DUNP19. Specifically, one approach utilized a DOTA-bearing peptide (FcIII) with a strong affinity for the fragment crystallizable (Fc) domain of the human IgG1 of DUNP19 (DUNP19LF-FcIII-DOTASS), while the other leveraged a chemo-enzymatic technique to substitute the N-linked bi-antennary oligosaccharides in the human IgG1 Fc domain with DOTA (DUNP19LF-gly-DOTASS). To assess if these methods impact the antibody's binding properties and targeting efficacy, comparative in vitro and in vivo studies of the generated DUNP19-conjugates were performed. While the LRRC15 binding of both radioimmunoconjugates remained intact, the conjugation methods had different impacts on their abilities to interact with FcRn and FcγRs. In vitro assessments of DUNP19LF-FcIII-DOTASS and DUNP19LF-gly-DOTASS demonstrated markedly decreased affinity for FcRn and FcγRIIIa (CD16), respectively. DUNP19LF-FcIII-DOTASS demonstrated increased blood and tissue kinetics in vivo, confirming loss of FcRn binding. While the ablated FcγR interaction of DUNP19LF-gly-DOTASS had no immediate impact on in vivo biodistribution, reduced immunotherapeutic effect can be expected in future studies as a result of reduced NK-cells interaction. In conclusion, our findings underscore the necessity for meticulous consideration and evaluation of mAb labelling strategies, extending beyond mere conjugation efficiency and radiolabeling yields. Notably, site-specific labelling methods were found to significantly influence the immunological impact of Fc interactions. Therefore, it is of paramount importance to consider the intended diagnostic or therapeutic application of the construct and to adopt conjugation strategies that ensure the preservation of critical pharmacological properties and functionality of the antibody in use.

High heterogeneity in the membrane protein expression of small extracellular vesicles (sEVs) means that bulk methods relying on antibody-based capture for expression analysis have a drawback that each type of antibody may capture a different sub-population. An improved approach is to capture a representative sEV population, without any bias, and then perform a multiplexed protein expression analysis on this population. However, such a possibility has been largely limited to fluorescence-based methods. Here, we present a novel electrostatic labelling strategy and a microchip-based all-electric method for membrane protein analysis of sEVs. The method allows us to profile multiple surface proteins on the captured sEVs using alternating charge labels. It also permits the comparison of expression levels in different sEV-subtypes. The proof of concept was tested by capturing sEVs both non-specifically (unbiased) as well as via anti-CD9 capture probes (biased), and then profiling the expression levels of various surface proteins using the charge labelled antibodies. The method is the first of its kind, demonstrating an all-electrical and microchip based method that allows for unbiased analysis of sEV membrane protein expression, comparison of expression levels in different sEV subsets, and fractional estimation of different sEV sub-populations. These results were also validated in parallel using a single-sEV fluorescence technique.

Purpose: Site-specific approaches to bioconjugation produce well-defined and homogeneous immunoconjugates with potential for superior in vivo behavior compared to analogs synthesized using traditional, stochastic methods. The possibility of incorporating photoaffinity chemistry into a site-specific bioconjugation strategy is particularly enticing, as it could simplify and accelerate the preparation of homogeneous immunoconjugates for the clinic. In this investigation, we report the synthesis, in vitro characterization, and in vivo evaluation of a site-specifically modified, 89Zr-labeled radioimmunoconjugate created via the reaction between an mAb and an Fc-binding protein bearing a photoactivatable 4-benzoylphenylalanine residue. Procedures: A variant of the Fc-binding Z domain of protein A containing a photoactivatable, 4-benzoylphenylalanine residue — Z(35BPA) — was modified with desferrioxamine (DFO), combined with the A33 antigen-targeting mAb huA33, and irradiated with UV light. The resulting immunoconjugate — DFOZ(35BPA)-huA33 — was purified and characterized via SDS-PAGE, MALDI-ToF mass spectrometry, surface plasmon resonance, and flow cytometry. The radiolabeling of DFOZ(35BPA)-huA33 was optimized to produce [89Zr]Zr-DFOZ(35BPA)-huA33, and the immunoreactivity of the radioimmunoconjugate was determined with SW1222 human colorectal cancer cells. Finally, the in vivo performance of [89Zr]Zr-DFOZ(35BPA)-huA33 in mice bearing subcutaneous SW1222 xenografts was interrogated via PET imaging and biodistribution experiments and compared to that of a stochastically labeled control radioimmunoconjugate, [89Zr]Zr-DFO-huA33. Results: HuA33 was site-specifically modified with Z(35BPA)-DFO, producing an immunoconjugate with on average 1 DFO/mAb, high in vitro stability, and high affinity for its target. [89Zr]Zr-DFOZ(35BPA)-huA33 was synthesized in 95% radiochemical yield and exhibited a specific activity of 2 mCi/mg and an immunoreactive fraction of ~ 0.85. PET imaging and biodistribution experiments revealed that high concentrations of the radioimmunoconjugate accumulated in tumor tissue (i.e., ~ 40%ID/g at 120 h p.i.) but also that the Z(35BPA)-bearing immunoPET probe produced higher uptake in the liver, spleen, and kidneys than its stochastically modified cousin, [89Zr]Zr-DFO-huA33. Conclusions: Photoaffinity chemistry and an Fc-binding variant of the Z domain were successfully leveraged to create a novel site-specific strategy for the synthesis of radioimmunoconjugates. The probe synthesized using this method — DFOZ(35BPA)-huA33 — was well-defined and homogeneous, and the resulting radioimmunoconjugate ([89Zr]Zr-DFOZ(35BPA)-huA33) boasted high specific activity, stability, and immunoreactivity. While the site-specifically modified radioimmunoconjugate produced high activity concentrations in tumor tissue, it also yielded higher uptake in healthy organs than a stochastically modified analog, suggesting that optimization of this system is necessary prior to clinical translation.

Small extracellular vesicles (sEVs) have in recent years evolved as a source of biomarkers for disease diagnosis and therapeutic follow up. sEV samples derived from multicellular organisms exhibit a high heterogeneous repertoire of vesicles which current methods based on ensemble measurements cannot capture. In this work we present droplet barcode sequencing for protein analysis (DBS-Pro) to profile surface proteins on individual sEVs, facilitating identification of sEV-subtypes within and between samples. The method allows for analysis of multiple proteins through use of DNA barcoded affinity reagents and sequencing as readout. High throughput single vesicle profiling is enabled through compartmentalization of individual sEVs in emulsion droplets followed by droplet barcoding through PCR. In this proof-of-concept study we demonstrate that DBS-Pro allows for analysis of single sEVs, with a mixing rate below 2%. A total of over 120,000 individual sEVs obtained from a NSCLC cell line and from malignant pleural effusion (MPE) fluid of NSCLC patients have been analyzed based on their surface proteins. We also show that the method enables single vesicle surface protein profiling and by extension characterization of sEV-subtypes, which is essential to identify the cellular origin of vesicles in heterogenous samples.

An electrical immuno-sandwich assay utilizing an electrokinetic-based streaming current method for signal transduction is proposed. The method records the changes in streaming current, first when a target molecule binds to the capture probes immobilized on the inner surface of a silica micro-capillary, and then when the detection probes interact with the bound target molecules on the surface. The difference in signals in these two steps constitute the response of the assay, which offers better target selectivity and a linear concentration dependent response for a target concentration within the range 0.2-100 nM. The proof of concept is demonstrated by detecting different concentrations of Immunoglobulin G (IgG) in both phosphate buffered saline (PBS) and spiked in E. coli cell lysate. A superior target specificity for the sandwich assay compared to the corresponding direct assay is demonstrated along with a limit of detection of 90 pM in PBS. The prospect of improving the detection sensitivity was theoretically analysed, which indicated that the charge contrast between the target and the detection probe plays a crucial role in determining the signal. This aspect was then experimentally validated by modulating the zeta potential of the detection probe by conjugating negatively charged DNA oligonucleotides. The length of the conjugated DNA was varied from 5 to 30 nucleotides, altering the zeta potential of the detection probe from -9.3 +/- 0.8 mV to -20.1 +/- 0.9 mV. The measurements showed a clear and consistent enhancement of detection signal as a function of DNA lengths. The results presented here conclusively demonstrate the role of electric charge in detection sensitivity as well as the prospect for further improvement. The study therefore is a step forward in developing highly selective and sensitive electrokinetic assays for possible application in clinical investigations.

We present an approach to improve the detection sensitivity of a streaming current-based biosensor for membrane protein profiling of small extracellular vesicles (sEVs). The experimental approach, supported by theoretical investigation, exploits electrostatic charge contrast between the sensor surface and target analytes to enhance the detection sensitivity. We first demonstrate the feasibility of the approach using different chemical functionalization schemes to modulate the zeta potential of the sensor surface in a range -16.0 to -32.8 mV. Thereafter, we examine the sensitivity of the sensor surface across this range of zeta potential to determine the optimal functionalization scheme. The limit of detection (LOD) varied by 2 orders of magnitude across this range, reaching a value of 4.9 x 10(6) particles/mL for the best performing surface for CD9. We then used the optimized surface to profile CD9, EGFR, and PD-L1 surface proteins of sEVs derived from non-small cell lung cancer (NSCLC) cell-line H1975, before and after treatment with EGFR tyrosine kinase inhibitors, as well as sEVs derived from pleural effusion fluid of NSCLC adenocarcinoma patients. Our results show the feasibility to monitor CD9, EGFR, and PD-L1 expression on the sEV surface, illustrating a good prospect of the method for clinical application.

The targeting of gastrin-releasing peptide receptors (GRPR) was recently proposed for targeted therapy, e.g., radiotherapy. Multiple and frequent injections of peptide-based therapeutic agents would be required due to rapid blood clearance. By conjugation of the GRPR antagonist RM26 (D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2) to an ABD (albumin-binding domain), we aimed to extend the blood circulation of peptides. The synthesized conjugate DOTA-ABD-RM26 was labelled with indium-111 and evaluated in vitro and in vivo. The labelled conjugate was stable in PBS and retained specificity and its antagonistic function against GRPR. The half-maximal inhibitory concentration (IC50) of In-nat-DOTA-ABD-RM26 in the presence of human serum albumin was 49 +/- 5 nM. [In-111]In-DOTA-ABD-RM26 had a significantly longer residence time in blood and in tumors (without a significant decrease of up to 144 h pi) than the parental RM26 peptide. We conclude that the ABD-RM26 conjugate can be used for GRPR-targeted therapy and delivery of cytotoxic drugs. However, the undesirable elevated activity uptake in kidneys abolishes its use for radionuclide therapy. This proof-of-principle study justified further optimization of the molecular design of the ABD-RM26 conjugate.