Coupling of IL-1β and Collagen II Targeting Antibodies using the SpyMask System
ABSTRACT
Osteoarthritis (OA) is a degenerative joint disease characterized by cartilage breakdown and inflammation, globally affecting millions of people and lacking any disease modifying therapies. Current treatments, such as pain relievers, can provide temporary relief but fail to address the molecular processes that advance the disease. Here, a bispecific molecule that binds degraded cartilage and reduces inflammation was created by coupling one previously engineered antibody targeting type II collagen (mAb-CII) to an IL-1β inhibiting antibody (Gevokizumab) using the SpyMask system. DNA from both SpyTagged and His-tagged antibody fragments (Fabs) were transfected into eukaryotic HEK293T cells while Double Catcher and Double Catcher H-Lock were produced in E. coli bacteria. All proteins were purified through affinity chromatography and confirmed via SDS-PAGE. Results suggested that using nickel resin significantly improved protein yield, and the SpyMask system successfully coupled the SpyCatcher proteins to SpyTagged antibodies, as confirmed by a distinct band at the expected molecular weight. These findings establish a foundation for developing potential localized therapies that address both cartilage degradation and inflammation in OA.
INTRODUCTION.
In osteoarthritis (OA), changes within the joint lead to cartilage breakdown and the development of inflammation, resulting in chronic pain, reduced mobility, and diminished quality of life [1]. In 2020, this condition affected almost 600 million individuals worldwide, representing more than a 100% increase since 1990, with prevalence predicted to continue rising [2]. Current clinical treatments, such as physical therapy and non-steroidal anti-inflammatory drugs (NSAIDs), can provide relief but have not been shown to effectively prevent cartilage deterioration or reverse existing joint damage, therefore many patients resort to complicated and expensive surgical procedures [1]. Due to the high prevalence and irrevocable nature, there is a critical need for new therapies that target cartilage degradation in OA joints instead of only suppressing the symptoms [2]. A healthy synovial joint can handle stress and absorb impact because the articular cartilage, covering the articular surfaces of bones, is primarily composed of type II collagen (CII) and an extracellular matrix (ECM) which provides the strength and flexibility required for support [1]. Additionally, pro-inflammatory cytokines such as Interleukin-1β (IL-1β) normally help maintain cartilage flexibility by promoting controlled ECM degradation; however, in individuals with OA, this balance becomes disrupted, leading to excessive ECM loss [3]. While IL-1α also binds to the IL-1 receptor, IL1β is more dominant in the facilitation of inflammation because it activates the NF- κB and MAPK signal transduction pathways, which drive positive feedback loops in the degradation of cartilage through enzymatic processes [3]. Antibody-based technologies offer a promising approach to target cartilage degradation and reduce inflammation, establishing a more localized treatment. The recombinant monoclonal antibody targeting type II collagen (mAb-CII) specifically recognizes and binds to collagen II, which is increasingly exposed in arthritic lesions [4]. This targeting strategy enables preferential localization of the therapeutic construct to cartilage-rich and damaged joint tissues, increasing local drug concentration at sites of pathology while minimizing systemic exposure. Additionally, Gevokizumab is an anti-inflammatory recombinant monoclonal antibody that selectively binds IL-1β, functioning as a competitive inhibitor [4]. Here, the goal was to create a bispecific molecule using antibody fragments (Fabs) that contain the single-chain variable fragments (scFvs) necessary for target-specific binding, as their smaller size improves molecular control. The SpyMask covalent coupling system was employed, in which a linker protein (Double Catcher) provides two sites for SpyTagged Fabs to form irreversible isopeptide bonds [5]. In this system, a SpyTag is a short peptide sequence genetically fused to Fab fragments that forms a spontaneous covalent isopeptide bond with SpyCatcher domains, a strategy that has been widely used in protein engineering and has been explored for assembling bispecific antibody constructs.[5] The amino-terminal site of the Double Catcher is reactive and binds the first SpyTagged Fab, whereas the carboxy-terminal site is masked with a removable tag that can be cleaved with TEV protease to expose the second reactive site for coupling. Using this strategy, a bispecific construct was produced to bind collagen II while simultaneously inhibiting IL-1β, enabling localized anti-inflammatory activity at sites of cartilage degeneration. Additionally, this work aims to optimize protein yield by improving the purification to maximize recombinant His-tagged protein production efficiency.
MATERIALS AND METHODS.
Protein Expression.
DNA expression plasmids for Double Catcher (DC) and Double Catcher H-Lock (HL) (Addgene, #216284 and #216285) were transfected into Escherichia coli BL21(DE3) by heat shock according to the manufacturer’s protocol (NEB:C2527) to produce two related protein constructs. 25 µL of the transfected bacteria were spread onto agar plates and incubated overnight at 37°C. Afterwards, single colonies were picked, and starter cultures were grown in 10 mL LB medium supplemented with 100 µg/mL of ampicillin at 37°C in a shaking (200 rpm) incubator overnight. Starter cultures were then diluted 100-fold into 1 L of LB containing 100 µg/mL of ampicillin and 0.8% w/v glucose, incubated at 37°C in a shaker (200 rpm). After this culture reached an optical density at 600 nanometers of 0.5-0.8, 0.42 mM of IPTG was added and cultures were incubated at 16°C overnight. Finally, cells were centrifuged at 4000 x g for 15 minutes and then stored frozen in a -80°C freezer. In addition to prokaryotic expression of Double Catcher constructs, Gevokizumab-SpyTag and CII-SpyTag Fab fragments were expressed in HEK293T cells, and conditioned media containing the secreted proteins was collected for downstream purification.
Protein Purification.
Frozen bacterial pellets were thawed and lysed by sonication (2 h), then centrifuged (1000 × g, 1 h) to remove debris. Conditioned media from HEK293T cells expressing Gevo-SpyTag Fab and CII-SpyTag Fab was collected. Proteins (Double Catcher, Double Catcher H-Lock, Gevo Fab, CII Fab) were bound to Cobalt-NTA or Ni-NTA resin at 4°C overnight, washed with Fridy Wash Buffers I and II, and eluted with Fridy Elution Buffer following the manufacture’s protocol.
Protein Coupling.
Protein coupling was performed to establish a permanent, bispecific, and covalently bound molecule with a molar ratio of DoubleCatcher:Gevokizumab Fab:CII Fab at 1:1.25:5. First, DoubleCatcher was mixed with a 1.25-fold excess of the Gevokizumab Fab for 2 hours at 25°C in 1X TEV reaction buffer. Then, a 5-fold excess of the CII Fab and 10 U of TEV protease (NEB #P8112) was added simultaneously to the reaction mixture and incubated at 34°C for 4 hours. The reaction products were analyzed by SDS-PAGE and stored at 4°C until further use.
SDS–PAGE Analysis.
Molecular weights were estimated with SDS-PAGE to assess protein purification and coupling. 4–20% Mini-PROTEANTM TGX precast polyacrylamide gels were used under reducing conditions (by adding β-mercaptoethanol) in Tris-Glycine SDS running buffer. Gels were stained with SimplyBlue™ SafeStain Coomassie Blue (Thermo Fisher), left shaking overnight, and imaged using the Bio-Rad imaging system.
RESULTS.
Double Catcher (DC) and Double Catcher H-Lock (HL) were previously purchased constructs originally developed by the Howarth laboratory [5]. These proteins contain an N-terminal His-Tag, which contains 6 histidine residues that specifically bind to immobilized metals on agarose resin, making them accessible for purification using affinity chromatography. Protein purification was performed from prokaryotic expression by binding the lysate of sonicated E. coli cells to Cobalt-NTA resin, followed by washing and eluting. Elution fractions 1-4 were pooled based on SDS-PAGE analysis, which was also used to confirm purification. A significant proportion of protein was seen remaining in the flow-through (FT), likely because the binding capacity of the resin was not high enough to capture all the protein in the cell extracts (Fig. 1A). To recover this protein, the flow-through was run through a Nickel-NTA resin column, which successfully captured the unbound protein and increased the overall yield (Fig. 1B).
For purification of the SpyTagged antibody fragments, the Gevokizumab (IL-1β Fab) and CII Fab were collected from HEK293T conditioned media and purified using Cobalt-NTA resin. Elutions 1-4 were pooled based on SDS-PAGE analysis (Fig. 2). This purification produced a much lower protein yield compared to the prokaryotic expressed proteins due to the nature of eukaryotic expression, which often results in a smaller culture volume. Purified proteins were concentrated using Amicon Ultra centrifugal filters with a 30 kDa molecular weight cutoff and stored at -80°C until further use.
Coupling reactions were conducted with a molar ratio of Double Catcher:Gevokizumab:CII Fab at 1:1.25:5, under controlled temperatures to ensure sufficient bonding. Isopeptide bond formation between the Double Catcher construct and SpyTagged antibody fragments was revealed through SDS-PAGE analysis (Fig. 3). Serial dilutions of the molecular weight standard were included to ensure band intensities remained within the linear detection range and to prevent signal saturation for accurate comparison. DC and HL were used as separate constructs and analyzed independently under identical experimental conditions to assess coupling behavior resulting from differences in coupling configuration within the SpyMask system. DC and HL were approximately 46.5 and 47 kDa, while the Gevokizumab Fab and CII Fab were 57.1 and 54.8 kDa, respectively (Fig. 3). The measured weight of the coupled protein (CP) was approximately 157 kDa, which closely matches the calculated weight of the sum of the individual proteins, with a difference corresponding to 0.25% of the total protein mass. DC and HL migrated at an experimental molecular weight of 45.5 kDa, which is higher than the expected molecular weight of approximately 31.2 kDa. Interestingly, similar discrepancies in molecular weight migration have also been reported by the Howarth laboratory [5].
DISCUSSION.
The goal of this study was to express, purify, and couple proteins using the SpyMask system to create a bispecific molecule targeting IL-1β (via Gevokizumab) and collagen II (via mAb-CII), which are both hallmarks of osteoarthritis. IL-1β is a pro-inflammatory cytokine that activates signal transduction pathways involved in cartilage degradation, and mAb-CII specifically targets collagen II, a molecule found in areas of damaged cartilage [1,4]. By creating a stable bispecific molecule with these functional traits, a foundation for possible therapeutic research can be formed.
SpyCatcher proteins (such as the Double Catcher and Double Catcher H-Lock) were successfully expressed, purified, and coupled. It can be concluded that the use of nickel resin for affinity chromatography protein purification significantly increased the protein yield of the Double Catcher constructs in comparison to cobalt resin. Additionally, the results indicated that the coupling reactions between Gevokizumab Fab and CII Fab produced distinct SDS-PAGE bands at the expected molecular weights, suggesting successful conjugation and formation of a new stable complex.
The cobalt and nickel purifications revealed that the protein that was remaining in the initial purification flow-through was successfully eluted in the subsequent experiment using nickel resin (Fig. 1). This analysis supports the theory that nickel resin possesses a higher affinity for histidine-tagged proteins because its chemical structure allows stronger bonding with imidazole [1]. This finding suggests that nickel resin is better suited for purifying DoubleCatcher constructs with histidine tags.
Eukaryotic proteins were successfully purified using cobalt resin, ensuring proper expression for coupling (Fig. 2). Here, the overall yield of protein was much lower in comparison to the prokaryotic expression, which may be due to the smaller culture volumes and the increasing risk of contamination for mammalian cell culture [1]. While this resulted in less overall protein, it was expected since it reflects culturing limitations rather than experimental biases. Coupling reactions between Double Catcher and the Fabs produced distinct bands on SDS-PAGE, confirming the successful formation of a new bispecific molecule (Fig. 3). The molecular weights of each individual protein were equal to the sum of the composite, validating this band as the coupled protein. The band directly below the coupled protein likely represents a part of the Double Catcher bound to only one Fab, due to its molecular weight. A smaller band below the unreacted protein is the size of the predicted TEV protease. The double catcher protein band migrated slightly further along the gel to be 44 kDa, but the predicted size from the manufacturer’s website was 31.2 kDa; however, these results were consistent with the Howarth paper, suggesting that this shift may be due to the construct rather than an experimental error [5].
It is not within the scope of this study to demonstrate the biological activity of the coupled protein, a limitation attributed to time constraints. Additionally, the quantification aspect using SDS-PAGE can introduce minor variations in protein quantification; however, this technique is commonly used and well-established in scientific practices [1]. These limitations are typical of early-stage molecular studies and will be addressed in future work.
Future steps will focus on further experimentation with the coupled protein. CII and IL-1 will be introduced in subsequent assays to test the new construct’s ability to bind to the biological markers of osteoarthritis. Additionally, Double Catcher and Double Catcher H-Lock proteins will be compared to determine how their structures affect function. Longterm studies will further this research from in vitro to in vivo models, such as rats, to evaluate the therapeutic potential this protein could have in living organisms.
This project shows the potential for engineered proteins in advancing targeted treatments for osteoarthritis, a condition that affects more than 500 million people worldwide [2]. Coupling OA-targeted Fabs allows for a localized treatment which is essential in treating conditions such as arthritis or other inflammatory diseases [4]. Millions of people live with these conditions, and current therapies only manage symptoms rather than addressing the root cause [1]. By successfully purifying and coupling SpyMask proteins, this study establishes a reliable and consistent method for creating stable protein complexes that could be used to further biological research or develop targeted treatments. Finding ways to repair or replace damaged cartilage at the molecular level could reduce pain, restore mobility, and improve the quality of life for countless individuals. This work lays a foundation for future research and the development of long-lasting solutions to conditions that impact so many lives.
ACKNOWLEDGMENTS.
This work is supported by NSF CAREER Award (CBET–2237639). Thank you to Dr. Rebecca Shattuck-Brandt, Zachary M. Eidman, Dr. Joanne C. Lee, Dr. Jonathan M. Brunger, and Dr. Angela M. Eeds. Thank you to the School for Science and Math at Vanderbilt, Hume-Fogg Academic Magnet High School, and the Department of Biomedical Engineering at Vanderbilt.
REFERENCES.
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- GBD 2021 Osteoarthritis Collaborators. Global, regional, and national burden of osteoarthritis, 1990–2020 and projections to 2050: a systematic analysis for the Global Burden of Disease Study 2021. Lancet Rheumatol. 7, e508–e522 (2023)
- F. R. Greten, et al. NF-κB Is a Negative Regulator of IL-1β Secretion as Revealed by Genetic and Pharmacological Inhibition of IKKβ. Cell. 130, 918–931 (2007).
- B. L. Walton et al., A programmable arthritis-specific receptor for guided articular cartilage regenerative medicine. bioRxiv : the preprint server for biology. 33, 231–240 (2024)
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Posted by buchanle on Thursday, May 14, 2026 in May 2026.
Tags: bispecific antibodies, collagen II targeting, IL-1β inhibition, inflammation, Osteoarthritis