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. 2024 Oct 16;146(43):29824–29835. doi: 10.1021/jacs.4c11648

Rapid Systematic Screening of Bispecific Antibody Surrogate Geometries for T-Cell Engagement Using DNA Nanotechnology

Sabrine Messaoudi †, Kevin Wai ‡, April Marple ‡, Seyed Farzad Baniahmad §, Ryan G Wylie ‡, Marianne Pelletier ∥,⊥, Morgan Craig ∥,⊥, Yves Durocher §, Andrea A Greschner †,*, Marc A Gauthier †,*
PMCID: PMC11529601  PMID: 39412838

Abstract

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Bispecific antibodies (bsAbs) are emerging immune-therapeutics, and many formats exist that differ considerably in structure. However, little systematic data exist about how the spatial organization of their components influences activity, requiring innovative approaches combining empirical and quantitative frameworks. This study presents a modular DNA nanotechnology platform to generate numerous bsAbs with surrogate geometries that span the structural features of the BiTE, IgG-like, and IgG-conjugate platforms to screen for T-cell engagement. Results highlight interesting structure–activity relationships regarding bsAb potency and selectivity and raise questions regarding the molecular phenomena underlying activity. To elucidate some effects, the platform was paired with a simple mathematical model. This work is thus one of the first to systematically investigate and reveal the importance of the spatial organization of bsAb components on activity and equally provides an accessible and convenient tool for rapidly mapping out such trends for other combinations of target epitopes.

Introduction

Since the approval of the first clinical bispecific “T-cell engager” (BiTE), blinatumomab, the field of bispecific antibody (bsAb) immune-therapeutics has rapidly developed. Blinatumomab simultaneously binds CD19+ B-cells and CD3+ T-cells, thereby bridging them and potentiating a T-cell-induced cytotoxic effect.1 While initially developed for B-cell acute lymphoblastic leukemia, promising results in phase I–III trials and real world data have prompted the testing of blinatumomab in several B-cell malignancies.2−4 Presently, nine bsAbs have been approved by the FDA,5 but over 200 others are in the pipeline (80% oncology and 9% autoimmune disorders). Sixty are undergoing clinical evaluation, and 11 are in phase II or above for Hodgkin’s lymphoma,6 pancreatic cancer,7 and solid tumors.8

Over 100 different formats of multispecific antibodies exist. These are generally based on the structure of immunoglobulin G (IgG) or its components, some having an Fc portion and others not.9 The vast majority of bsAb fall into broad categories such as IgG-like molecules, IgG conjugates, and conjugates of IgG fragments (Figure 1A).10,11 Intriguingly, while the structures of these platforms differ considerably, little systematic data exists about how their geometry influences activity. Moreover, considering the multifactorial nature of T-cell-mediated cytotoxicity (bsAb properties and concentration, antigen size(s) and expression level(s), epitope locations and accessibility, etc.), predicting performance is challenging. Hence, modular bsAb platforms would be of great importance to rapidly explore this parameter space empirically. To this end, seminal work has exploited DNA nanotechnology to tether antigen binding modules (ABM) onto peptide nucleic acid and left-handed DNA nanostructures to prepare “hybrid” bsAb by self-assembly.12−15 Wagenbauer and colleagues took innovation a step further by using origami DNA to assemble IgG antibodies, Fab, and scFv fragments onto large DNA nanocarriers, creating antibody variants forming distinct quadruplets to assess their potential to activate T cells in the presence of specific targets.16 These proof-of-concept studies demonstrated the stability of the hybrid bsAb in serum and in vitro/vivo T-cell engagement. However, while such platforms could be used to modulate bsAb geometry, the innovative character of the studies described above was placed on swapping different ABMs onto single nanostructures, thereby restricting antibody flexibility and orientation as design variables. Thus, an unexplored opportunity exists to use hybrid bsAb platforms to screen surrogate geometries spanning “conventional” bsAb formats and guiding the choice of the most suitable bsAb format from the onset of lead development.

Figure 1.

Figure 1

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Library of hybrid bsAb analogues of blinatumomab. (A) Illustrations drawn to scale of BiTE, IgG, and IgG-conjugate bsAb formats. (B) DNA–scFv conjugates used as building blocks to decorate DNA nanostructures. (C–F) Library of 1–3D hybrid bsAb with variable structures designed to test the effect of inter-scFv distance (C), flexibility (D), 2D topology (E), and 3D topology (F). Estimated distances between scFv and percentage of ssDNA in the structures are shown when relevant. Sequences and connectivity of all strands can be found in Table S1 and Figure S1 of the Supporting Information.

This study presents a new serum-stable platform based on inexpensive DNA oligomers to construct a library of hybrid bsAb leads with variable ABM distance, conformational flexibility, valency, and topology. The structures of the bsAb span values expected for the BiTE, IgG-like, and IgG-conjugate platforms to vastly explore this geometric space while fine-tuning within it. In vitro screening of 19 hybrid bsAb analogues of blinatumomab revealed multifactorial trends that influenced T-cell engagement. Some leads were substantially more active and CD19-selective than blinatumomab, while others performed poorly. This work is thus one of the first to systematically investigate and reveal the importance of the spatial organization of bsAb components on T-cell engagement and provides an accessible and convenient tool for rapidly mapping out such trends for other combinations of targets.

Results and Discussion

DNA nanotechnology17,18 enables the preparation of structurally distinct nanostructures onto which can be grafted scFvCD19 and scFvCD3, the ABMs of blinatumomab. Several variables were modulated by design, including inter-scFv distance, conformational flexibility, and the number/ratio of the ABMs (Figure 1). Inter-scFv distance is controlled by DNA oligomer length. Conformational flexibility is modulated via single-stranded DNA regions within the structure. The number/ratio of ABMs are controlled via the shape of the nanostructure (2, 3, and 4 for linear (L), triangular (Tg), and tetrahedral (Th) structures, respectively). Values for geometric parameters were based on the following criteria: IgG is a Y-shaped molecule with two ABMs separated by ∼13.7 nm.19 The flexibility of IgG comes from its hinge region, which allows it to adopt a range of conformations.19,20 The ABMs of the BiTE platform are separated by a ∼ 1.7 nm long pentapeptide linker.21,22 IgG conjugates can have an estimated maximal inter-ABM distance of ∼24.5 nm23,24 when hypothetically connected and extended end-to-end via their Fc. To mimic these features, linear double-stranded DNA (dsDNA) nanostructures (L1–L3) were designed with 20–71 DNA base pairs, which equates to ∼6.8–24.5 nm. Inter-ABM distances <6.8 nm were not tested due to the predicted instability of very short dsDNA at 37 °C.25−27 dsDNA sequences shorter than ∼147 base pairs are considered rigid/linear, whereas ssDNA sequences as short as 2–3 bases behave like hinges.28 ssDNA regions were introduced at different locations to mimic hinge regions (L4–L14; Figure 1). More complex Tg and Th nanostructures were designed to accommodate three or four ABMs on the same plane or that project in 3D, respectively. Tg and Th have fully duplexed rigid cores with ssDNA overhangs at each vertex designed to interface with unique complementary sequences on the ABMs (vide infra). For Tg, two bsAb were produced with scFvCD19/scFvCD3 ratios of 1/2 (Tg1) and 2/1 (Tg2). For Th, three bsAb were produced with scFvCD19/scFvCD3 ratios of 3/1 (Th1), 2/2 (Th2), and 1/3 (Th3). Thus, a library of 19 hybrid bsAb with precisely defined geometries and properties was prepared. All DNA sequences can be found in Table S1 and were either adapted from literature or designed de novo.29,30

To facilitate screening, the workflow was designed to minimize synthesis and purification. Two DNA–scFv conjugates (one with scFvCD19 and the other with scFvCD3) were prepared as ABMs and could then be hybridized to any of the preassembled DNA nanostructures consisting of inexpensive oligonucleotides (Figure 2). scFvCD19 and scFvCD3 with sequences identical to those in blinatumomab were produced with C-terminal cysteines (Table S2) and conjugated to unique DNA–maleimide oligonucleotides. The chromatograms of the reaction mixture following diafiltration show a shift of the scFv peaks to shorter retention times upon conjugation to DNA and no side reactions (Figure 2A). Gel electrophoresis corroborates this finding, and colocalization of protein/DNA stains supports successful conjugation. Thereafter, these DNA–scFv conjugates were mixed with equimolar amounts of the oligonucleotides required to produce a given bsAb (Figure S1). Because scFvs are temperature sensitive, several protocols were evaluated to maximize the yield. For the L structures, assembly was best achieved by incubation at 37 °C for 48 h. Because of their increased complexity, Tg and Th were prepared by first assembling the DNA nanostructure using a 95 to 4 °C gradient over 3.5 h (Figure S1), then grafting the DNA–scFv conjugates to the overhangs using a 37 to 4 °C ramp. The yields of the desired thermodynamic structures were generally very good (58–84%), with no monospecific antibodies remaining. This facilitated purification by diafiltration or size-exclusion chromatography before testing (Figure S2). Native gel electrophoresis of the library of L bsAb (Figure 2B) showed a single band for each compound, demonstrating purity, and the degree of migration of the bsAb band was consistent with that of the expected product. To characterize the number and type of scFv present on Tg/Th, the latter were mixed with different quantities and ratios of DNA–scFv to see how individual grafting events influence the migration of the band (Figures 2C and S3). Hence, for these bsAb, the correct number of predicted scFv can be validated. To test for serum stability, selected bsAb were incubated in 10% fetal bovine serum (FBS) for 24 h (identical conditions to cell assays) and analyzed by gel electrophoresis. Four linear structures (L4, L7, L10, and L11) alongside Tg2 and Th1 were tested. The linear bsAb possessed different length ssDNA regions, as presumably these would be most prone to digestion. Finally, the corresponding bare nanostructures (devoid of scFv) were tested to gauge the effect of scFv on the stability. As expected,31 the bare DNA nanostructures were digested over 24 h in partial serum (Figure 3). However, the bsAb were fully resistant to partial serum over this period, as seen by the persistence of the bsAb band and no appearance of additional bands. The scFv might have a shielding effect on the DNA nanostructure, rendering it less accessible to serum enzymes. In particular, the hexahistidine tag on the scFv (adjacent the cysteine residue used for conjugation) might complex and stabilize the DNA, as observed elsewhere with oligolysine.31 Overall, the approach used to produce hybrid bsAbs was simple and, by design, can be easily adapted to swap the scFv for other ABMs or to use the same ABMs to probe a multitude of structures.

Figure 2.

Figure 2

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Assembly of hybrid bsAb library. (A) Preparation of scFv–DNA building blocks. Reverse-phase chromatography and PAGE analysis of the conjugation of ssDNA to the scFv. Lane 1: DNA1, lane 2: DNA1–scFvCD19, lane 3: DNA2, and lane 4: DNA2–scFvCD3. GelRed and Coomassie were used to stain DNA and proteins, respectively. (B) PAGE analysis demonstrating the assembly and purity of the linear hybrid bsAb. Short horizontal arrows are used to identify the band of the bsAb. (C) Assembly of various intermediate 2D/3D hybrid bsAb to characterize assembly into the final desired Tg and Th bsAb. Native PAGE was used during optimization to validate the stepwise assembly of the different nanostructures.

Figure 3.

Figure 3

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Native gel electrophoresis of DNA nanostructures and corresponding hybrid bsAb incubated in 10% serum. Illustrative gels showing that the DNA nanostructures devoid of scFv were degraded over a 24 h period, while the hybrid bsAb were stable. Short horizontal arrows are used to identify the band of the bsAb.

In vitro T-cell engagement was assessed using a luciferase/luciferin proliferation assay at different effector to target cell ratios (E/T; Figure S4).32 T-cells (CD3+) expanded from peripheral blood mononuclear cells (PBMCs) were cocultured with either CD19+ Raji-luc B-cells or CD19– SKBR3-luc control cells alongside 1 nM bsAb for 24 h. In the absence of bsAb, the B-cell viability was not affected by T-cells at E/T 0–4:1, though a small loss of viability was observed at E/T 5–10:1. Experiments were thus limited to E/T 0–4:1. Five control groups were tested: absence of T-cells (E/T = 0:1), vehicle (no bsAb), DNA1–scFvCD19 alone, DNA2–scFvCD3 alone, and blinatumomab (positive control). All cell viability data is in Figures 4A and S5 and full pairwise means comparisons are in Tables S3 and S4. These data show that the presence of bsAb is required to observe cytotoxicity, as neither phosphate buffered saline (PBS) nor the monospecific DNA–scFv controls decreased viability in either cell line. Raji-luc cell viability varied strongly with both the bsAb structure and the E/T ratio, while SKBR3-luc cell viability was much less sensitive to these two parameters. Thus, the relative effect of each bsAb on SKBR3/Raji cell viability provides insight into the CD19-selectivity of their activity. Substantial differences in performance were observed within the library of bsAb, and a significant number of bsAb both more and less potent/selective than blinatumomab were observed, which testifies to the importance of bsAb geometry. To facilitate comparisons, data from Figures 4 and S5 for two E/T ratios, in addition to selectivity data, are transposed into Figure 5, which categorizes the bsAb based on inter-scFv distance, length of the ssDNA region(s), location of ssDNA region(s), and the number of appended scFv. The general trends observed were similar at both E/T ratios, with some minor exceptions. First, no obvious trend encompassing the entire hybrid bsAb library was observed as a function of any bsAb parameter considered alone. Hence, trends inevitably depended on more than one factor. Moreover, the increased potency correlated with increased CD19 selectivity. The most potent structures were the fully (L1–L3) and partially (L7 and L9) duplexed linear bsAb, as well as Tg1,2 and Th1–3, equally fully duplexed. Inter-scFv distance (6.8–37.5 nm) yielded no obvious trend among these bsAb. At E/T 2:1, the most potent structures were Tg, with superior cytotoxicity (∼20%) and CD19-selectivity (∼5) compared to blinatumomab (∼45%; ∼2), whose performance most resembled L2. At E/T 4:1, the most cytotoxic/selective bsAb became L2 (∼4%, ∼12), whereas the performance of blinatumomab (∼16%, ∼5) mostly resembled L1. The least potent bsAb were L4,6,8 that spanned similar inter-scFv distances as above (13–24.4 nm). These structures each possessed ssDNA regions whose lengths were inversely correlated with activity. Curiously, the location of the ssDNA regions was important for activity within the library at E/T 4:1. ssDNA regions near scFvCD3 were detrimental to activity while they had a lesser or no effect when located elsewhere (irrespective of length). No trend was observed with the number of appended scFv or with the relative proportion of scFvCD19/CD3 for a given geometry. Tg and Th had activity and selectivity within the range of the best performing linear structures, and Tg slightly outperformed Th in selectivity. To disentangle the effects of bsAb geometry and multivalency (number of scFv), a mathematical model for blinatumomab reported by Flowers et al.33 was adapted. For this, the binding/unbinding rates of the bsAb to their cellular targets were adjusted according to the number/type of scFv associated with their topology/configuration. This model does not consider the effect of bsAb geometry on these rates and thus purely simulates effects of multivalency. As seen in Figure 4B, Tg1,2 and Th1 form higher concentrations of effector cell–drug–T-cell trimer complexes (EDT) than L or Th2,3, without major alterations to their formation kinetics. The model also predicts that bsAb potency scales as Th1 > Th2 ∼ Tg1 > Th3 ∼ Tg2 ∼ L, which implies a trend with the number of scFVCD19. This trend is not observed empirically (Figure 5), which points to the contribution of the bsAb geometry on experimental results.

Figure 4.

Figure 4

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Influence of E/T ratio and hybrid bsAb structure on T-cell engagement for CD19+ Raji-luc cells. (A) Experimental data presented as the mean + SD, n = 3. Full ANOVA multiple pairwise comparison tables for E/T ratios 2:1 and 4:1 can be found in Tables S3 and S4 of the Supporting Information. (B) Predicted effector cell–drug–T-cell (EDT) ternary complex concentrations over time and predicted target cell viability with respect to the initial E/T ratio in the presence of bsAb using a mathematical model based on that of Flowers et al. Complete modeling details are provided in the Supporting Information.

Figure 5.

Figure 5

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Dependence of T-cell engagement on structural parameters of the hybrid bsAb library. Box charts of the mean viability and CD19-selectivity achieved using each bsAb tested for E/T ratios 2:1 (A) and 4:1 (B) using data from Figures 4 and S5, plotted as a function of structural variables. Certain bsAb useful for discussing trends are identified with labels. Full multiple pairwise means comparison tables of all viability data is found in Tables S3 and S4.

To explore the concentration-dependent differences in potency observed for selected structures (L2, Tg2, and Th1) in comparison to blinatumomab, IC50 values were measured at E/T 4:1 (Figure 6A). Tg2 (IC50 0.001 ± 0.001 nM) was substantially more potent than blinatumomab (IC50 0.05 ± 0.02 nM), L2 (the most potent linear; IC50 0.2 ± 0.1 nM), and Th1 (IC50 0.030 ± 0.004 nM), indicating an optimal geometry of this bsAb within the geometric space investigated. Different concentration-dependent profiles were equally observed: dose–response was sharp and reached a plateau at high concentrations of L2 and Tg1, while a gradual response not reaching a plateau was observed for blinatumomab and Th1. Finally, T-cell engagement assays were performed in the presence of inhibitors to confirm that all three hybrid bsAb induced cell death according to a mechanism similar to that of blinatumomab, which is mainly the perforin/granzyme B pathway (Figure 6B).34−36 Thus, the presence of the DNA component within the hybrid bsAb did not, a priori, alter the mechanism of action.

Figure 6.

Figure 6

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BsAb concentration-dependence and mechanism of T-cell-mediated cytotoxicity. (A) Determination of the 50% inhibitory concentration (IC50) of L2, Tg2, Th1, and blinatumomab toward Raji-luc cells at an E/T ratio 4:1. (B) T-cell engagement assays were performed in the presence of inhibitors for the perforin/granzyme B pathway (c: concanamycin A, b: brefeldin A, and m: monensin), antagonistic antibodies for the death ligand pathway (r: anti-TRAIL antibody RIK2 and i: anti-TNFα infliximab), and for the Fas/FasL interaction pathway (n: anti-FasL NOK1). Cell death is primarily achieved via the perforin/granzyme B pathway. Data presented as the mean + SD, n = 3.

While this study was not designed to characterize the molecular mechanisms of T-cell activation and B-cell lysis, the prevailing hypotheses from the literature warrant a brief presentation, as bsAb geometry may play a role in influencing them. For instance, the kinetic-segregation model37 suggests that T-cell activation is initiated by T-cell receptor (TCR) phosphorylation upon encounter with antigen-presenting cells. When the TCR and small adhesion/costimulatory proteins engage their ligands, this nucleates zones of “close contact” between the cells that locally deplete molecules with extracellular domains above ∼14 nm (via size-exclusion) such as tyrosine phosphatase CD45.38 Because TCR–peptide–major histocompatibility complexes (TCR–pMHC) trap TCR within these zones, segregation of TCR from CD45 occurs, leading to TCR phosphorylation. This process is multifaceted, and other mechanisms such as force-induced conformational changes to receptors and receptor clustering may equally be occurring.39,40 It is thus reasonable to speculate that the size, flexibility, and multivalency of a bsAb may influence these phenomena. For perspective, existing studies have demonstrated that increasing the dimensions of the TCR–pMHC complex resulted in an increased intermembrane separation distance (∼17.8 ± 4.6 nm). In these conditions, less depletion of CD45 was observed.41 This greatly reduced TCR triggering, though it did not affect TCR–pMHC ligation. From a different angle, Li et al. observed that targeting a membrane-proximal epitope using a bsAb, thereby shortening the intercellular distance, facilitated T-cell synapse formation, whereas efficacy was lost for a membrane distal epitope.42 Similar results were obtained for smaller bsAb based on the BiTE format, as compared to those with longer inter-scFv distances.43

In addition to size, the bsAb conformational flexibility could play a role in effectively targeting antigens. This has been explored via structural differences naturally present among IgG subclasses. The hinge regions of IgG follow the flexibility trend IgG3 > IgG1 > IgG4 > IgG2 and hybridoma technology has been used to prepare bsAb with hinge regions from each of these four subclasses, as well as bsAb with mixed hinge regions.44−49 For example, Kapelski et al. developed anti-CD19/CD3 bsAb based on IgG1, IgG2, and IgG4 and showed that the IgG2 variant did not bind both targets while the others did. Moreover, activity was restored for chimeric subclass IgG4:2 and IgG1:2 variants, indicating the importance of the hinge region.50 Overall, these results illustrate how bsAb size and flexibility can potentially influence potency and selectivity.

Within this context, the trends observed within the hybrid bsAb library studied herein are intriguing, especially the trends toward increased potency and CD19-selectivity for smaller, more rigid structures (at least for the pair of epitopes targeted in this study). For instance, a potent and selective bsAb was L2 with an inter-scFv distance of ∼13 nm that parallels the values of the intercellular gap discussed above. Nevertheless, the comparable performances of L3 (24.5 nm) and Tgs (37.5 nm) could suggest that larger bsAb might lie “flatly” on the cell, and that different bsAb formats may yield optimal performance. Because Th’s 3D structure prevents it from lying flat on the cell surface, it might be used in future work to explore the effect of bsAb size on intermembrane spacing and T-cell activation. In contrast, the least potent bsAb were the linear structures that possessed ssDNA regions near the scFvCD3. This was curious and could infer a relationship between bsAb flexibility and a possible binding-induced conformational change phenomenon, and this warrants further investigation. Multivalency studied using the L, Tg, and Th structures did not yield an obvious trend with cytotoxicity, which contrasted to simulated viability data obtained with the geometry-independent mathematical model. Model simulations predicted that the absolute and relative number of scFvCD19/CD3 should influence cytotoxicity, which was not observed experimentally. This underlines that structure (which varied a lot in this study) plays a key role in determining bsAb potency/selectivity, and future work should improve this model by considering multistep binding and parametrizing the model to binding rates specific to the bsAb in our library, which likely depend on geometry. The influence of bsAb structure on the concentration-dependent cytotoxicity profiles in Figure 6A is also an observation that merits additional examination.

Concluding Remarks

In summary, this work demonstrates the convenience of DNA platforms to generate libraries of hybrid bsAb with vastly different geometries spanning typical bsAb formats. It is the first systematic investigation of the effect of bsAb geometry on T-cell engagement and has revealed interesting trends that warrant additional scrutiny for their fundamental mechanistic importance. Based on findings, the optimal bsAb formats for engaging the epitopes of blinatumomab could be the IgG format (inter-ABM distance of ∼13 nm, like L2) or a rigid trivalent structure resembling Tg, such as an appended IgG format.9 These observations are specific to the epitope pair studied herein, and the modularity of the platform should be used to probe other epitope combinations, as suggested by others.12−14 It will be particularly interesting to explore different applications, such as targeting antigens on different cells, antigens on the same cell, epitopes on the same antigen, etc. The facility of implementing these studies makes this hybrid platform useful for accelerating the identification of surrogate bsAb leads and gaining mechanistic insight into their activity. Finally, one limitation of this work is that results may not necessarily reflect in vivo therapeutic activity, which depends on other factors. This, of course, is an inherent limitation of all in vitro screening approaches.

Methods

Preparation of DNA–scFv Conjugates

DNA–maleimide oligonucleotides were dissolved in PBS (0.137 M sodium chloride, 0.0027 M potassium chloride, 0.01 M sodium phosphate dibasic, and 0.0018 M potassium phosphate monobasic) pH 7 to yield 20 μM stock solutions. One molar equivalent of freshly concentrated monomeric (reduced) scFv (Figure S6) was added to the DNA solution, and sufficient conjugation buffer (0.237 M sodium chloride, 0.0027 M potassium chloride, 0.01 M sodium phosphate dibasic, 0.0018 M potassium phosphate monobasic, and 10 μM magnesium chloride) pH 7 was added to bring the final DNA concentration to 10 μM. The reaction was left at room temperature for 24 h. The samples were washed twice by centrifugal dialysis (10 kDa MWCO) with 50 μL of reaction buffer and then analyzed by reverse-phase high pressure liquid chromatography using an Agilent C18, 150 mm × 2.1 mm, 3.6 μm column. Analytes were eluted using gradients of water +0.1 vol % trifluoroacetic acid (TFA; solvent A) and acetonitrile +0.1% TFA (solvent B) at a flow rate of 0.5 mL/min. A typical elution profile was 41 to 49% (solvent A) over 15 min. To confirm conjugation, samples were analyzed using precast polyacrylamide electrophoresis gels (4–15%). 20 μL aliquots of each sample (total of 0.02 nmol of DNA) was mixed with 2 μL of glycerol and loaded into the gel. As controls, DNA/maleimide strands were mixed with 2 μL of glycerol and sufficient water to bring the final volume to 22 μL and were loaded into the same gels. Gels were run at 80 V for 60 min using TBE 1× buffer (90 mM Tris, 90 mM boric acid, and 1.15 mM EDTA) and then stained first with Gel RED to visualize DNA and then with Coomassie blue to visualize proteins.

Assembly of Linear Hybrid bsAb (L1–L14)

One hundred microliters of each 10 μM DNA–scFv conjugate stock solution (anti-CD19 and anti-CD3) were mixed with appropriate complementary DNA strands from their stock solutions in water (see Table S1 and Figure S1 for DNA strand sequences and connectivity) to yield the desired structures. Sixty microliters of assembly buffer (0.737 M sodium chloride, 0.0027 M potassium chloride, 0.01 M sodium phosphate dibasic, 0.0018 M potassium phosphate monobasic, 10 μM magnesium chloride hexahydrate) was added to bring the final volume to 260 μL. The mix was incubated at 37 °C for 48 h. Aliquots were taken at 24 and 48 h for verification of assembly (4–15% native PAGE, 95 V, 75 min, TBE 1× as running buffer). Extinction coefficients for all hybrid bsAb were calculated as described in the Supporting Information Methods. Hybrid bsAb were purified by size-exclusion chromatography with Hitrap desalting columns (GE Healthcare) using the assembly buffer as an eluent (Figure S2).

Assembly of the Triangular and Tetrahedral Hybrid bsAb

Bare triangular and tetrahedral DNA nanostructures were assembled by mixing appropriate DNA strands (Figure S1) in equimolar quantities in 50 μL TAMg buffer (final DNA concentrations ∼3.33–5 μM). The annealing protocol was as follows: 95 °C at 5 min, 80 °C at 3 min, 78–60 °C at 4 min/°C, and 57–4 °C at 9 min/°C. Following assembly, a 0.003 nmol aliquot of each assembly was mixed with 2 μL of glycerol and sufficient water to yield a 10 μL solution, which was then analyzed by native 4–15% PAGE (80 V, 1 h, TBE 1× buffer). The gel was then stained with GelRed and imaged using a BioDoc-It imaging system (Figure S1). Thereafter, DNA–scFv conjugates were added in 2-fold molar excess relative to the number of corresponding overhangs on the nanostructure to yield Tg1,2 and Th1–3. Stepwise addition of lesser amounts of DNA–scFv was performed to monitor the assembly process by gel electrophoresis (Figures 2C and S3). For annealing, a 20 μL solution containing 15 μL of assembly buffer (0.737 M sodium chloride, 0.0027 M potassium chloride, 0.01 M sodium phosphate dibasic, 0.0018 M potassium phosphate monobasic, and 10 μM magnesium chloride hexahydrate) and 5 μL of TAMg buffer 10× (0.4 M Tris base, 0.124 M magnesium chloride hexahydrate, 0.01 M EDTA, 0.2 M glacial acetic acid, with a pH of 7.8.) were added to 10 μL of the DNA nanostructure solution (3.33–5 μM) and exposed to a temperature program (37 to 30 °C at 10 min/°C; 29 to 25 °C at 7 min/°C; 24 to 20 °C at 5 min/°C; 19 to 4 °C at 3 min/°C). Afterward, the hybrid bsAb were purified by diafiltration (Amicon, MWCO 100 kDa) to remove excess DNA–scFv conjugates and were characterized by native PAGE (6% gels, 80 V, 16 h, 4 °C) using TAE 1× (0.04 M Tris base, 0.02 M glacial acid, and 0.001 M EDTA) as running buffer.

Stability of DNA Nanostructures and the Hybrid bsAb

The stability of the hybrid bsAb L4, L7, L10, L11, Tg2, and Th1, as well as their corresponding bare DNA nanostructures (i.e., without appended scFv), was investigated to determine their resistance to degradation in FBS. The experiments were conducted under conditions identical to the in vitro experiments (24 h at 37 °C). Solutions of the hybrid bsAb and bare DNA nanostructures were prepared (Figures S1A and 2) and then incubated with 10% FBS at 37 °C. Aliquots (0.02 nmol) were taken at specific intervals (0–24 h) and stored at −80 °C until analyzed using by 4–15% polyacrylamide gel and TBE migration buffer (80 V, 1 h). Tg2 and Th1 were analyzed using a 6% polyacrylamide gel and TAE migration buffer (80 V, 16 h, refrigerated). Subsequently, the gels were poststained with GelRed and imaged using a BioDoc-It imaging system from UVP, along with UVP imaging software.

Cytotoxicity Assays

Raji-luc and SKBR3-luc cells expressing firefly luciferase were purchased from FenicsBIO and the Japanese Collection of Research Bioresources Cell Bank, respectively, and cultured according to the protocol provided by the supplier. Human T-cells were isolated from PBMCs from healthy donors following a protocol provided by STEMCELLS Technologies. To initiate T-cell culture, 106 PBMCs were seeded in ImmunoCult-XF T Cell Exp Medium media supplemented with 15 μL of a 100 μg/mL stock of human recombinant IL-2 added for every 50 mL of XFT media (complete media). This mixture was combined with 25 μL of ImmunoCult HuCD3/CD28/CD2 T-Cell Act activator for every mL cell solution in a 96-well round-bottom microplate and incubated at 37 °C with 5% CO2 for 3 days. T-cell cultures were subjected to an 8-fold dilution after 2 days with the complete media above, followed by 4-fold dilution every two subsequent days. Cells were used between days 10 and 12.

Raji-luc and SKBR3-luc cells were diluted to 20,000 cells/mL in complete RPMI medium supplemented with 10% FBS. 90 μL of cell suspension was added to 90 μL of T-cell suspensions diluted in the same media used to reach different cell ratios between (0–10:1; E/T). Twenty microliters of 10 nM hybrid bsAb solutions was prepared in sterile PBS and added to the cocultures containing 180 μL of media (final hybrid bsAb concentration of 1 nM). Additional wells without cells or bsAb were filled with FBS-supplemented RPMI complete medium or PBS as negative controls. The plates were incubated at 37 °C (humidified incubator with 5% CO2) for 24 h. The next day, 50 μL of a 5× luciferin solution were added to each well to yield a 1× final concentration. The bioluminescence signal generated from the conversion of d-luciferin to oxyluciferin by luciferase was read immediately with a spectrophotometer (460–520 nm, in the blue range). The percentage of viable B-cells in a given well was calculated relative to wells devoid of bsAb and T cells at each E/T cell ratio (percentage of viable cells = 100 × (observed luminescence of sample) ÷ (luminescence of control (no bsAb)).

Mechanisms of T-Cell-Mediated Cytotoxicity

Protocols were adapted from the work of Gruen et al.51 Briefly, Raji-luc cell and T-cell cultures were diluted with RPMI medium supplemented with 10% FBS to reach final concentrations of 20,000 and 80,000 cells/mL, respectively. Perforin/granzyme B pathway: in each well of a 96-well plate, 85 μL of T-cell suspension was preincubated for 2 h with either 5 μL of 80 nM concanamycin A, 0.8 μg/mL brefeldin A, or 8 μg/mL monensin. Subsequently, 85 μL of the Raji-luc cell suspension was added, followed by 20 μL of either PBS or a 10 nM stock of bsAb. This was combined with 5 μL of either an 8 nM stock of concanamycin A, 0.8 μg/mL brefeldin, or 0.8 μg/mL monensin, as appropriate. To maintain a consistent total volume of 200 μL, 10 μL of an RPMI medium with 10% FBS was added to each well. Cytokine and Fas/FasL pathways: an identical amount and volume of T cells and Raji-luc cells were incubated with 5 μL of either a 0.8 μg/mL solution of infliximab (anti-TNFα), 0.8 μg/mL of RIK2 (anti-H CD253 TRAIL), or 0.8 μg/mL of NOK-1 (anti-H CD178 CD95L), as well as 20 μL of PBS or 10 nM of the different bsAb used in the previous tests. This was followed by the addition of 15 μL of RPMI medium mixed with 10% FBS to maintain a consistent total volume of 200 μL. Different combinations of inhibitors were tested using the protocols described above. Microplates were incubated at 37 °C in a humidified incubator with 5% CO2 for 24 h. The following day, 50 μL of a 5× luciferin solution was added to each well, resulting in a final 1× concentration. Subsequently, the bioluminescence signal was promptly measured using a spectrophotometer. To determine the percentage of viable cancer cells, the bioluminescence signal obtained in the presence of the different hybrid bsAb was normalized to the wells for the vehicle alone.

Mathematical Model of bsAb

Flowers et al.33 recently reported on the accuracy of models of T-cell engagers with trans-avidity. Their calibrated and validated 2D model, wherein reactant density and total cellular membrane area defined 2D rate constants, was shown to predict both in vitro trimer formation and cytotoxicity. We modified this base 2D model of blinatumomab to consider how the number and type of scFv units on the linear, triangular, and tetrahedral bsAb affect target cell viability. Briefly, bsAb binding and unbinding to cellular targets were considered to be affected by the reaction volume, effector and target cell surface area (incorporating antigen concentrations), and antibody avidity. For each bsAb, the binding and unbinding rates were modified, according to the number and type of scFv according to its topology and configuration. This model does not consider the effect of bsAb geometry on the intrinsic binding affinity of scFvCD19 or scFvCD3 to their corresponding cellular targets. Complete model equations are provided in the Supporting Information.

Acknowledgments

Critical reading of this manuscript by Prof. Maya Saleh and Dr. Scott McComb is gratefully acknowledged. S.M. acknowledges a doctoral scholarship from the INRS/Tunisian Ministry of Education and the NSERC-CREATE PrEEmiuM program. M.A.G. is a Research Scholar of the Fonds de Recherche du Québec Santé (FRQS), Canada.

Glossary

Abbreviations

bsAb

bispecific antibodies

BiTE

T-cell engager

Immunoglobulin G

IgG

ABM

antigen binding modules

ssDNA

single-stranded DNA

dsDNA

double-stranded DNA

L

linear structure

Tg

triangular structure

Th

tetrahedral structure

PBMCs

peripheral blood mononuclear cells

TCR

T-cell receptor

APCs

antigen-presenting cells

TCR–pMHC

TCR–peptide–major histocompatibility complexes.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/jacs.4c11648.

  • Materials, plasmid construction, protein expression and purification, preparation of DNA–maleimide oligonucleotides, assembly of bare DNA nanostructures, calculation of extinction coefficients, details of the mathematical model, results for the expressed proteins, assembled DNA nanostructures, purified hybrid bsAb, cell assays, oligonucleotide, protein sequences, and statistics (PDF)

Author Contributions

The manuscript was written through the contributions of all authors. All authors have given their approval to the final version of the manuscript.

This work was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC; RGPIN-2015-04254), the Prostate Cancer Canada-Cancer Research Society (400399), and the Canadian Institutes for Health Research (CIHR; 438050).

The authors declare no competing financial interest.

Supplementary Material

ja4c11648_si_001.pdf (2.7MB, pdf)

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Supplementary Materials

ja4c11648_si_001.pdf (2.7MB, pdf)

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