Introduction

Renal cell carcinoma (RCC) is a rare and orphan disease responsible for 90% of kidney cancers, which stand as the 9th most common cancer in men and 14th most common cancer in women worldwide.1 The clear cell RCC (ccRCC) comprises approximately 75% of cases, which in the USA has risen to 55,000 annually. Problematically, one-third of patients are already metastatic on diagnosis due to a mild symptomatic nature that slows diagnosis, and unlike earlier grades that are effectively treated with surgical resection, advanced lesions are inoperable.1 2 Intermediate-risk and poor-risk patients go on to acquire resistance to traditional chemotherapies, radiation, and targeted antiangiogenics. With only 10%–15% of these patients surviving 5 years, advanced ccRCC represents a cancer population in significant need of new treatment options.3

Immunotherapy has transformed the landscape of cancer care by empowering the immune system to detect and kill cancers, with particular potential for advanced ccRCC. Checkpoint inhibitors that unleash T cells on tumors serve as a first-line therapy where they offer a relatively high response rate of 50%–60%. However, beyond still leaving nearly half of patients nonresponsive, progression-free survival is only extended to 1–2 years for responders due to discontinuations from acquired resistance and side effects.3 Resistant tumors subsequently acquire a more aggressive and metastatic phenotype and with no treatment options left, approximately 10,000 ccRCC patients die annually in the USA.1 New therapies could leverage and expand this cancer’s immune responsivity by driving alternative mechanisms of action, like focusing immune activity directly on tumors through the select targeting of tumor-associated antigens (TAAs), more effectively eradicate ccRCC.

Bispecific T cell engagers (BTEs) are a powerful, antibody-based immunotherapy designed to simultaneously bind immune and cancer cells to trigger cancer’s selective destruction. To accomplish this, they are equipped with multiple binding domains that recognize distinct epitopes or antigens, with most research directed at binding to the CD3 complex of T cells alongside a relevant TAA.4 The dual specificity forces a proximal relationship between the target and T cells, with the goal of engaging the T cell-mediated cellular cytotoxicity (CMC) of cancer cells through the formation of artificial, MHC-independent synapses. While clinically successful, first-generation BTEs given their simplified, dual single-chain variable fragment (scFv) design suffer a short half-life of ~2 hours (hrs) owing to the absence of an Fc region that would otherwise facilitate an extended circulation time from a higher molecular weight (MW) and neonatal Fc receptor (FcRn)-mediated recycling.5 Dramatic improvements to circulation time have been made by numerous second-generation formats that reintroduce an Fc domain, such as the Persistent BTE (PBTE) that is equipped with a single-chain Fc domain (scFc) linked to the conventional BTE molecule.6 There have since been at least eight PBTEs in clinical development, among them an anti-CD19 PBTE which demonstrated a well-extended half-life of 210 hours.5 7 Our group has also leveraged this format with a recently reported PBTE against CD45, made possible through epitope base editing, to engineer a universal blood cancer therapy.8 However, detailed comparative characterizations between first-generation and second-generation formats that shed light on potential trade-offs for clinically relevant formats like this one have yet to be reported. Nevertheless, the known benefits of PBTEs promoted the application of this format not only for additional targets, but to the creation of new structures that advance the scFc concept. For instance, PBTEs are still limited by their 1:1 valency that detracts from the higher avidity naturally afforded to antibodies. Increasing valency with additional binding domains has endowed other therapies with more functional affinity to wield greater potency and efficacy.9 10 The longer residence times enabled by higher valency can also drive better tumor distribution and help distinguish between high-target-expressing and low-target-expressing cells to better mitigate toxicity.11–13 We, therefore, hypothesize that bispecific designs integrating both scFc and valency adaptations could give rise to new and effective, antibody therapies for advanced ccRCC if provided a highly targetable TAA by the disease.

90% of RCC cases involve notable overexpression of the TAA, carbonic anhydrase 9 (CA9).14 Its high and homogeneous expression is observed at both primary and metastatic sites and its expression patterns correlate with poor prognoses.14–16 This contrasts with its otherwise scarce expression in normal tissue, as its aberrant upregulation arises from the inactivation of a specific regulatory von Hippel-Lindau tumor-suppressor gene.17 Functioning as a modulator of extracellular pH by catalyzing the interconversion of carbon dioxide to protons and bicarbonate, CA9 serves cancer cells by acidifying the tumor microenvironment (TME). In doing so, it drives the cancer growth, metabolic adaptation, invasion, and metastasis associated with poor patient prognoses.17 Eliminating CA9-expressing cells in cancer or inhibiting its enzymatic activity could thus dismantle these effects. Given its consistent expression in advanced ccRCC and the immune responsivity of this disease, CA9 represents an ideal antigen for targeted immunotherapies aimed at the isolated eradication of cancer and pH equilibration of the TME, wherein relief from acidic conditions could further facilitate immune function and tumor clearance.18

Despite the promising alignment of these parameters, there are no approved therapies targeting CA9 and clinical testing is largely confined to radiotherapies and chemotherapies.19 Vaccination strategies have demonstrated limited clinical efficacy for RCC and the phase III failure of girentuximab, an IgG1 monoclonal antibody relying on an antibody-dependent cellular cytotoxicity mechanism of action, emphasizes the need for newer preclinical approaches to better harnessing the power of the immune system in targeting CA9.20 A recent investigation of a CA9-directed BTE supported this technology’s potential for advanced ccRCC therapy after it exhibited tumor control in patient-derived xenograft models; the first-generation format described, however, would face limited translatability in the modern clinical landscape and CA9-targeted BTEs that instead adopt from advancing bioengineering strategies would maximize the clinical impact for ccRCC patients.21 In addition, the simultaneous advancement of the antibody delivery platform could further extend a meaningful reach to ccRCC patients.

Nucleic acid medicines such as synthetic DNA (synDNA) and messenger RNA (mRNA) represent innovative strategies for delivering self-synthesized, durably expressed biologics for cancer. In contrast to the manufacturing and infusion of recombinant medicines, synDNA leverages one’s own cellular machinery in expressing and continually disseminating anticancer biologics.22 Pronounced improvements to pharmacokinetic (PK) parameters and efficacy have been reported with in vivo DNA-launched, BTE (dBTE) targeting HER2, IL13Ra2, follicle-stimulating hormone receptor (FSHR), and EGFRViii in ovarian and brain cancers.23–26 The physiological benefits stand alongside manufacturing ones that include reduced costs and long-term temperature stability, which could eliminate the requirement for cold-chain storage and expand patient access.22 Coupling bispecific antibodies to synDNA through the development of in vivo-produced dBTEs is thus a promising approach for the potential improvement of patient clinical outcomes, as well as the upstream manufacturing and expanded distribution of antibody therapies.

Here, we present two novel, T cell engager molecules targeting CA9 for ccRCC, one of which is designed in a novel format for multivalent or multispecific T cell engagers. The first of these molecules is a PBTE while the second, referred to as a persistent multivalent T cell engager (PMTE), builds off the PBTE with an additional binding domain to generate a multivalent format, providing CA9 bivalency in this iteration. We characterize and compare the functional differences in recombinant form between the prototypical 1:1 BTE, 1:1 PBTE, and 2:1 PMTE formats as it pertains to avidity and potency in vitro using ccRCC cell lines, before studying their PK/pharmacodynamic (PD) relationships in mouse models. We then transform the biologics into nucleic acid medicine for a multidisciplinary examination of their therapeutic potential. Lastly, we test the reproducibility of these formatting trends beyond the CA9-targeted molecules with novel, FSHR-targeted antibodies for ovarian cancer. This study ultimately determines that the PMTE could be a superior agent to existing single-chain formats, warranting its further investigation for medicinal use against ccRCC and other cancers or antigens. Further, it supports the application of synDNA for the sustained delivery of these bispecific antibody therapeutics.

ResultsPBTE displays reduced affinity and potency compared with BTE

Early testing began with only the BTE and PBTE formats bispecific for CA9 and CD3. To generate the PBTE format, an scFc was linked to the conventional BTE N-terminus (online supplemental figure 1A). The antibody DNA was codon-optimized and inserted into a pVax1 expression vector for Expi293 cell expression and purification (online supplemental figure 1B). Flow cytometry binding analyses against target cell lines revealed an interesting consequence of linking an scFc to the BTE to generate a PBTE. Doing so attenuated binding affinity to target cells, evidenced by right-shifted median fluorescent intensity (MFI) curves for ACHN-CA9, 293T-CA9, and T cells, with corresponding dissociation constant (KD) values declining approximately twofold for all cell types (online supplemental figure 1C). To next assess the effects of the PBTE’s binding affinity reductions on cytotoxic potency in vitro, we leveraged xCELLigence technology that proxies cell death as a function of electrical impedance by adherent, target cells. Increasing cell index over time is a measure of unprohibited proliferation, unlike low indices resulting from the cytotoxic removal of impeding target cells, indicative of an effective therapy (online supplemental figure 1D). At 30 nanomolar (nM), both the BTE and PBTE engaged T cell killing of the primary RCC cell line 786-O with over 50% cell death by the 24-hour time point. While the BTE maintained this activity at 200 picomolar (pM), PBTE activity declined nearlytwofold (online supplemental figure 1E). Trending reductions in potency for the PBTE were similarly observed against the primary cell line A-498 and accompanying microscopic images were taken at the terminal time point (online supplemental figure 1F).

PMTE overcompensates for PBTE’s reduced CA9 avidity

To attempt recovering this lost activity, a second CA9 binding domain was then linked to the PBTE to generate the PMTE, whose anti-CD3 domain was placed medially to maintain equidistantly tight synapses for either engagement arm, to render a maximally potent molecule.27 28 The full panel of single-chain formats to undergo further testing in unison for assessment of the functional effects of compounding domain appendages is visually represented (figure 1A, online supplemental figure 2A). Western blotting presented bands at appropriate MWs for each antibody (online supplemental figure 2B). The synDNA therapies are also visually represented by their plasmid map, which describes the insertion of antibody sequences into individual, codon-optimized pVax1 expression vectors, together with Kozak and IgE leader sequences for enhanced expression in humans and mice, as previously described26 (figure 1B).

Figure 1

PMTE restores avidity for target cells from PBTE levels. (A) Graphic of the single-chain BTE, PBTE, and PMTE formats. Gray lines represent GS linkers. (B) Plasmid map for the antibody-encoding pVax1 vectors used for synDNA therapies. (C) Flow cytometry binding curves to ACHN-CA9 (n=3), 293T-CA9 (n=4), and T cells (n=3). An irrelevant IgG1 was used as a negative control. (D) KD calculations from respective cell binding curves were plotted as donor mean±SEM. E) z-Movi workflow depicting insertion of adherent cancer cells, suspended T cells, and compounds to a flow chamber. Ultrasound waves pull T cells from cancer cells toward acoustic nodes. Force required to separate cells is recorded at single cell resolution, and detachment force represents synaptic strength generated by bispecific antibodies. (F) Curves represent the percent of cells remaining bound with increasing detachment force over time. Antibodies were analyzed at 30 nM (n=5), 3 nM (n=3), and 300 pM (n=3). An irrelevant BTE was used as a negative control. (G) Area under curve (AUC) analyses of adjacent force curves at 30 nM (n=5), 3 nM (n=3), and 300 pM (n=3) were plotted as mean±SEM. (H) Single cell resolution of force required to detach T cells from cancer cells for individual antibodies for respective antibody concentrations. Points represent individual T cells and data represent all replications collectively. (I) Representative images of the flow chamber at 1000 pN display localization of detached T cells at acoustic nodes for lower avidity antibodies at 30 nM. BTE, bispecific T cell engager; PBTE, persistent bispecific T cell engager; PMTE, persistent multivalent T cell engager. Significance is assigned as * (p<.05), ** (p<0.005), *** (p<0.001), or **** (p<0.0001).

Binding was first reexamined where the PBTE reproducibly displayed right-shifted MFI curves against CA9-expressing cells compared with the BTE, amounting to significant twofold and fourfold higher KD values for ACHN-CA9 and 293T-CA9, respectively (figure 1C). However, equipped with CA9 bivalency, the PMTE rescued this lost binding strength with additional enhancement over the conventional BTE. This overcompensation is illustrated in the PMTE’s left-most shifted MFI curves for binding both target cells. Significantly lower KD values reflect this binding difference, which compared with the PBTE were 12.9-fold and 17.6-fold for ACHN-CA9 and 293T-CA9, respectively. Compared with the BTE, the PMTE displayed a 4.7-fold lower KD for ACHN-CA9 and a significant 4.4-fold lower KD for 293T-CA9. On testing of T cell binding kinetics, both the PBTE and PMTE displayed right-shifted curves with trending 2.5-fold higher KD values than the BTE (figure 1D). Pronounced binding at 500 nM is represented by 3-log population shifts for all formats, unlike the irrelevant-IgG1, negative control (online supplemental figure 2C). This analysis divulged a dynamic relationship between structural appendage and receptor interactions, notably with the PMTE experiencing conflicting effects on CA9 and CD3 binding. Characterization was, therefore, limited by testing avidities in antigenic isolation, and clarifying the net effect on intercellular, and synaptic strength required integrating both antigen-binding kinetic analyses in a single assay.

zMovi technology is an emerging tool that leverages force-generating, ultrasonic waves to measure the collective, intercellular-synapse strength generated by immunotherapies. It has provided valuable insight into the field of CAR T-cell therapy, yet its application to bispecific antibody research herein is novel.29 30 The assay is initiated by target cell monolayer adhesion within a microfluidic chip prior to the introduction of T cells and the bispecific antibody of interest to then precipitate forceful, immunological synapses. Ultrasonic waves then pull and disconnect T cells, allowing measurement of the force required to overcome bispecific avidity at single-cell resolution. T cells in strong relationships due to higher avidity bispecific antibodies can resist ultrasonic forces without release from target cells. Contrarily, those bound by lower avidity bispecific antibodies succumb to detachment at lower forces to be visualized in clusters at acoustic nodes (figure 1E).

The CA9xCD3 engagers were tested at 30 nM, 3 nM, and 300pM while a FAPxCD3 bispecific-negative control was tested at 30 nM with the consideration that lower concentrations of the control would only have equal or less background signal. Approximately 60% of cells remained bound to target 293T-CA9 cells at peak forces for both the BTE and PMTE at 30 nM while only 20% remained for the PBTE. At 3 nM, approximately 30% of cells remained bound with the BTE and PMTE, with almost no bound cells remaining for the PBTE. Neither the BTE, PBTE, or PMTE resisted detachment forces at 300 pM (figure 1F). Transformed to the area under curve (AUC) for statistical analysis, both the BTE and PMTE signaled significantly higher force resistance compared with the negative control, but not the PBTE. In fact, the conversion of the BTE to a PBTE induced a threefold decline in force resistance at 30 nM. Converting the PBTE to the PMTE, however, restored this synaptic strength by threefold. At 3 nM, the BTE generated significant resistance to detachment compared with 30 nM of negative control. This effect was lost with the PBTE but recovered by the PMTE, which demonstrated a twofold improvement in comparative force resistance. The AUC analysis indicates concentration-dependent reductions in force resistance with no antibodies showing a meaningful effect at 300 pM (figure 1G). These data can be observed at single-cell resolution where points represent each T cell at conceding forces, with violin density expressing trends for each treatment (figure 1H). Images taken at max force during the 30 nM tests illustrate clusters of detached T cells from PBTE and negative control treatments, but a lack thereof with BTE and PMTE treatments (figure 1I). Ultimately, this comprehensive assay examined the dual nature of the engagers to form a holistic readout on bispecific avidity. In doing so, the repercussions of the PBTE’s parallel reductions in flow binding appeared to take shape with weaker synapses while the PMTE’s higher avidity for CA9 lent itself to stronger synapses. The ensuing activation of T cells would offer functional appraisals for the engagement capacities of these formats.

PMTE activates T cells with the highest potency in co-culture with RCC cells

To model T cell activation with translational relevance, we acquired SKRC-52 cells which are primary cells derived from a metastatic lesion in the mediastinum of a ccRCC patient. SKRC-52 cells were co-incubated with donor T cells and antibodies for 24 hours before the T cell staining of CD69 and CD25 activation markers. At 10 nM, all three CA9xCD3 engagers induced pronounced upregulation of CD69 in approximately 75% of CD4+ and CD8+ T cell populations, compared with a FAPxCD3 bispecific-negative control. Potency differences began revealing themselves at lower concentrations, such as at 1 pM, where the PBTE lost all activity unlike its BTE predecessor. However, the PMTE significantly restored CD69 expression on CD4+cells with a 14-fold improvement from the PBTE, and twice the activity of the BTE. It too recovered CD8+T cell activation by measure of its 10-fold higher CD69 upregulation than the PBTE, which itself had no activity. Only the PMTE maintained activity at 100 femtomolar (fM), a 100,000-fold dilution from the initial 10 nM, with meaningful differences to the PBTE and negative control in both populations (figure 2A). Potency differences are similarly represented by CD25 expression, whose significant upregulation in CD4+ and CD8+ populations from control levels is distinctly observed with the PMTE at 1 pM (figure 2B).

Figure 2

PMTE activates T cells with the most potency in vitro. (A) CD69+ and (B) CD25+expression on CD4+ (top row) and CD8+ (bottom row) T cells after 24 hours of co-incubation with antibodies and SKRC-52 cells (n=5). (C) CD69+and D) CD25+expression on CD4+ (top row) and CD8+ (bottom row) T cells after 24 hours of co-incubation with antibodies and 293T-CA9 (n=5). An irrelevant BTE was used as a negative control. Data are expressed as mean±SEM. BTE, bispecific T cell engager; PMTE, persistent multivalent T cell engager. Significance is assigned as * (p<.05), ** (p<0.005), *** (p<0.001), or **** (p<0.0001).

T cell activation driven by their bispecific union to CA9-expressing target cells is recapitulated with 293T-CA9, further magnifying the observable effects of multivalent extension. At 10 pM, only the PMTE induced a significant, 17-fold upregulation of CD69 on CD4+ and CD8+ T cells compared with control; its upregulation on CD8+T cells was also fourfold higher than the PBTE. With further examination, the PMTE drove significant CD69 expression compared with all other treatments at 1 pM. Specifically, fivefold and eightfold higher expression was measured on CD4+T cells than those treated with the BTE and PBTE, respectively. For CD8+T cells, these significant, respective differences were 3.5-fold and 10-fold. Only the PMTE drove CD69 upregulation on CD4+ and CD8+ T cells at the very low 300 fM concentration, with particularly meaningful differences to the BTE and PBTE treatments for the latter population (figure 2C). CD25 expression again co-represented activation states and its measurement on CD4+T cells at 1 pM significantly differentiated the PMTE from all other treatments. A meaningful difference from the PBTE was further maintained at 300 fM. Similar behavior trended for CD8+T cells, with differences at 300 fM instead most apparent between BTE and PMTE formats (figure 2D). 293T absent of transduced CA9 failed to induce CD69 or CD25 upregulation under these conditions, as did the absence of co-cultured target cells, which supports the specificity for CA9 antigen in observable T cell activation (online supplemental figure 3A,B). The gating strategy used to capture activated T cell populations is demonstrated with or without 10 nM BTE treatment in co-culture with 293T-CA9 (online supplemental figure 3C,D).

PMTE induces highly potent killing of RCC cells at femtomolar-range concentrations

CMC is a powerful tool in the immune repertoire and its comparative examination among the panel once again leveraged xCELLigence technology. For ccRCC, an effective therapy would exercise high potency at both primary and metastatic sites, the latter of which is here first represented by SKRC-52. Mirroring the observed T cell activation and avidity, the right-shifted curve from the BTE to PBTE represented a 10-fold decline in potency due to scFc linkage. Engrafting the second CA9 binding domain to form the PMTE, however, led to a 44-fold increase in potency from the PBTE to demonstrate an impressive 340 fM half-maximal effective concentration (EC50) against these metastatic cells at this time point. The primary cell lines A-498 and 786-O experienced 33-fold and 14.5-fold reductions in potency with the PBTE compared with the BTE, to be recovered by the PMTE that went on to maintain 25% cytotoxicity at a low 100 fM in both cases. Milder reductions in PBTE potency from the BTE format were observed with ACHN and 293T transduced to overexpress CA9, ranging from 2.5-fold to 4-fold. However, greater potencies were still attained by the PMTE, which in comparison to the PBTE, improved EC50 values by 41-fold and 23-fold for ACHN-CA9 and 293T-CA9, respectively (figure 3A, online supplemental table 1).

Figure 3

PMTE induces the T cell-mediated cytotoxicity of target cells with the highest potency. (A) Percent of terminal cell population death at increasing concentrations of antibody treatment for the following target cell lines: SKRC-52 (n=4), A-498 (n=3), 786-O (n=2), ACHN-CA9 (n=3), and 293T-CA9 (n=2). Percent cytotoxicity was calculated as (1–(treatment/target cell control))×100) at the terminal experimental time point. (B) Real-time analyses of cell growth over time for 10 nM and 1 pM antibody treatments for the adjacent cell lines illustrate potent cytotoxicity. (C) Representative images taken at the terminal time point of single experiments for each treatment group at 10 nM, illustrating the cytotoxicity of the adjacent target cell lines. PMTE, persistent multivalent T cell engager.

Killing data were acquired in real-time and the suppression of cell growth in units of cell index over time is best represented by the saturating, 10 nM condition. Negative controls do not show activity and include an irrelevant bispecific, treatments without T cells, and T cells without treatments. The 1 pM time analysis showed higher PMTE activity across target cells compared with the BTE and PBTE (figure 3B). Unlike with their CA9-transduced counterparts, naïve ACHN and 293T did not succumb to CMC, nor did the CA9-negative OVCAR3 or DAOY (online supplemental figure 4A). This reinforces the on-target selectivity for CA9-presenting cells only. Visual recording accompanies impedance in this system, allowing images at terminal time points to illustrate the effects of 10 nM treatments on cell viability and density compared with control (figure 3C). In contrast, images of CA9-negative cell lines show no effects on growth or viability from treatments (online supplemental figure 4B). The domain-annexing effects on cytotoxic potency with the PMTE closely followed the trends observed with activation markers and prior CA9 binding kinetics. While the scFc came at a functional consequence to the PBTE’s mechanism of action, the multivalent PMTE restored cytotoxic potency in vitro.

PMTE displays improved tumor distribution and tumor control compared with PBTE

PKs were modeled using Balb/c mice at a relatively high, intravenous antibody dose of 2.5 mg/kg, based on the literature describing a common dose range of 0.1–5 mg/kg for studies of this kind.31 32 The low MW-BTE fell to undetectable levels in just after 30 hours which aligns closely with the known kinetics of this first-generation format. However, the PBTE and PMTE comparably extended circulation times out to nearly 1000 hours before escaping detection, providing 13-fold higher exposure by AUC. These data are presented in both units of μg/mL and nM to correct for MW differences (figure 4A). A one-compartment model helped reduce molecular natures into fundamental, pharmacological parameters including volume of distribution (VD), which was highest for the PBTE. This suggests that the PBTE maintains better tissue distribution than the PMTE, although both had approximately twofold higher VD than the original BTE. The PBTE and PMTE shared similarly low clearance (CL) calculations in relation to the BTE, whose faster clearance and consequent 3.4-hour half-life explain the original format’s clinical requirement of continuous intravenous infusion. Comparatively, the PBTE exhibited an improved half-life of 4.5 days for a 32-fold extension over the BTE while the PMTE experienced a mild reduction of half-life from PMTE levels to 3.9 days. While the decline is in the error range, it is possible that changes in the PMTE’s physicochemical properties and MW alter its permeability and excretion, and additional studies are required to draw firmer conclusions. Ultimately, the PMTE’s half-life amounted to a 27-fold extension beyond the first-generation BTE within an overall PK profile with similarity to the PBTE (online supplemental table 2).

Figure 4

PMTE displays improved tumor distribution and tumor control in vivo. (A) Balb/c mice were administered a single 2.5 mg/kg (intravenous) dose of antibodies and circulating antibody was quantified from sera via His-capture ELISA for pharmacokinetic analysis (n=3). Serum concentrations are displayed in both μm/mL and nM units. Corresponding pharmacokinetic calculations are in online supplemental table S2. (B) The tumor distribution PK analysis workflow began with an ACHN-CA9 xenograft in NSG mice (n=4). Single 0.3 mg/kg (intravenous) doses in PBS were administered when tumors reached 200 mm3. Tumors and serum were harvested after 24 hours, tumors homogenized, and antibody concentrations analyzed by His-capture ELISA. An irrelevant BTE was used as negative control. (C) A tumor distribution summary plot of intratumoral concentrations against serum. The tumor/serum concentration ratio (×10−3) is represented by point size. (D) Serum antibody concentrations at 24 hours are given in both ng/mL and nM units. (E) Intratumoral antibody concentrations at 24 are given in both ng/mL and nM units. (F) Relative distribution of antibodies into the tumor compartment is considered as the tumor/serum concentration ratio (×10−3). (G) Nu/J mice harboring subcutaneous ACHN-CA9 tumors were administered 106 donor T cells (IP) and 3 doses of recombinant antibodies at 1 mg/kg, once every 5 days (n=5). Arrows represent doses and data are presented individually and as grouped mean. Bar graphs display average tumor volumes (mm3) at days 25 and 30. Data were plotted as mean±SEM. BTE, bispecific T cell engager; PMTE, persistent multivalent T cell engager. Significance is assigned as * (p<.05), ** (p<0.005), *** (p<0.001), or **** (p<0.0001).

Changes to tumor distribution were next evaluated with a PK analysis in a mouse tumor model. Using ACHN-CA9, NSG mice were implanted with subcutaneous tumors that would be recollected alongside plasma samples 24 hours after a lower antibody dose of 0.3 mg/kg. This dose was selected for its therapeutic relevance given the common dosing range of 0.01–0.3 mg/kg for T cell engagers in tumor efficacy models.12 33 Tumor dissociation enabled the ELISA-based quantification of antibody concentrations in the original tumors, and their ratio to plasma concentrations would grant additional insight into relative shifts in compartmental distribution with domain attachments (figure 4B). All antibodies were detected in plasma samples after 24 hours with concentrations ranging from 750 to 1000 ng/mL for the PBTE, PMTE, and an Fc-containing, FAPxCD3 negative control. However, in line with the plasma PK, the BTE was measured at a significantly lower 100 ng/mL than the Fc-containing formats by this time point. Concentrations are also provided in molar units (figure 4C,D).

Antibodies were detected in tumors at much lower concentrations, characteristic of limited antibody tumor penetration from poor blood flow due to high cell density, abnormal vasculature, and high interstitial pressure that can dramatically limit exposure.34 35 The negative control established background levels of 0.5 ng/mL compared with which all formats demonstrated significant distribution. The BTE had a tumor concentration of 2 ng/mL that was higher than the plasma, demonstrating a distribution that endured systemic clearance. This could be due to its low MW expediating deeper diffusion, coupled with CA9 adherence that slows clearance. PBTE levels of 1.5 ng/mL were below the BTE to indicate a counterbalance between its higher circulating levels and distribution capacity, and this difference was significantly magnified to 2.5-fold with molar unit normalization. The PMTE measured higher than both the BTE and PBTE in units of ng/mL, with a respective 2-fold and 2.7-fold higher tumor concentration. Molar unit normalization flipped the script to highlight the BTE, which retained significantly higher concentrations than both the PBTE and PMTE to create an interesting dichotomy between plasma and tumor PK characteristic desirability while the PMTE demonstrated a 2-fold higher tumor concentration than the PBTE (figure 4C,E). For the Fc-containing formats of comparable plasma PK behavior, relative differences in tumor distribution were examined using the concentration ratios between tumor and plasma compartments. This proportional analysis calculated the PMTE’s significantly higher tumor distribution than both the PBTE and irrelevant bispecific, with respective 2.8-fold and 6.5-fold differences (figure 4C,F).

Translating these PK insights and functional projections into proof of efficacy required mouse tumor models that introduce effector T cells, a critical test first performed using immunodeficient, Nu/J mice harboring subcutaneous, ACHN-CA9 tumors. These mice were treated with three doses of compound at 1 mg/kg intraperitoneally in a 5-day interval, beginning at the time of T cell administration. Despite its previously observed tumor retention of at least 1 day, BTE treatment had little effect on tumor growth compared with vehicle, given this challenging 3×5 dosing regimen in place of the typical once-daily dosing required to derive meaningful, long-term effects with this format. However, both scFc-containing formats had substantially improved tumor control, represented with day 25 comparisons to the BTE. Importantly, significant differences between the PBTE and PMTE also became evident by day 25, with average tumor sizes, respectively, measuring 700 mm3 and 140 mm3. Day 30 marked 20 days since final treatment, by which point mice in both remaining groups harbored regrowing tumors. However, tumor burden was 4.5-fold lower in the PMTE treatment group, signaling significantly more potent tumor control with this multivalent format.

synDNA-delivered PMTE sustains durable activity in vitro with superior tumor control in mice

synDNA delivery of BTEs (dBTEs) is visually represented, whereby DNA vectors and hyaluronidase (HYA) are intramuscularly administrated and accompanied by electroporation for the employment of host cellular machinery to autosynthesize therapeutics.22 The sustained expression and systemic dissemination of dBTEs that follow continually equips T cells to track and kill cancers like ccRCC. Blood from dosed mice is also sampled to confirm dBTE expression and function in vitro (figure 5A).

Figure 5

synDNA-delivered PMTE demonstrates better durability and tumor control in vivo. (A) DNA-based BTEs administered as plasmid DNA with hyaluronidase (50 U/mouse) and electroporation for enhanced cellular uptake. Myocytes synthesize antibodies in situ for secretion into circulation. In tumor models, donor T cells are administered (IP) and treatment efficacy assessed. Serum can be collected from antibody-treated mice, whether naïve or from tumor models, for in vitro functional assessments of in vivo-synthesized antibodies. (B) Naïve, balb/c mice were administered 200 µg of dBTE, dPBTE, or dPMTE DNA (n=5). Sera collected over time were used to stain ACHN and ACHN-CA9 cells by flow cytometry. Untranduced ACHN cells are represented in gray while colored shifts represent ACHN-CA9 cells up to day 21. (C) Sera were also used to stain donor T cells by flow cytometry from days 0 to 21. (D) Sera from synDNA-treated balb/c mice (n=5) were tested for their ability to induce cytotoxicity of SKRC-52 cells in vitro, using an xCELLigence system. Diminished cell indices represent target cell death. Negative controls included sera from empty pVax1-treated mice while recombinant BTE (10 nM) was used as a positive control. (E) NSG mice harboring subcutaneous A-498 tumors were administered 106 donor T cells (IP) and a single 10 ug dose of synDNA on day 0, represented by arrow (n=5). Mice are line-plotted individually while bar graph displays tumor volumes (mm3) at day 33 as mean±SEM. (F) NSG mice harboring subcutaneous SKRC-52 tumors were administered 106 donor T cells (intraperitoneally) and a single 10 ug dose of synDNA, represented by arrow (n=4–5). Mice are line-plotted individually while bar graph displays tumor volumes (mm3) at day 30 as mean±SEM. BTE, bispecific T cell engager; dBTE, delivery of BTE; dPBTE, delivery of persistent BTE; dPMTE, delivery of persistent multivalent T cell engager. Significance is assigned as * (p<.05), ** (p<0.005), *** (p<0.001), or **** (p<0.0001).

200 µg of expression vectors encoding the CA9-targeted dBTE, dPBTE, and dPMTE were administered to naïve, Balb/c mice whose plasma was sampled over time and target cell staining with pooled-group plasma samples illustrate antibody concentrations by MFI shift. Using samples from days 0 to 21, ACHN-CA9 cells were stained alongside their non-transduced counterparts as negative controls. MFI increases were observed for the ACHN-CA9 population compared with ACHN for all formats and given the nature of the dBTE’s half-life in particular, its consistent shift through day 21 represents the persistent in vivo expression achievable with synDNA. dPBTE expression levels peaked more acutely on day 4 before returning to lower maintenance expression levels resembling the dBTE while the dPMTE induced a later and more prominent shift that peaked on day 14 (figure 5B). T cells were next stained to confirm functional CD3 binding, and the patterns of MFI among formats were comparable to those of ACHN-CA9 (figure 5C).

The in vivo-produced T cell engagers were next tested for their induction of SKRC-52 CMC through xCELLigence analyses. Plasma from conventional dBTE-treated mice produced maximal cytotoxicity through day 14 post-treatment, with half-maximal effects from days 28 to 48. Its effects were observed on day 90 before falling by day 180. The dPBTE instead saw earlier, gradual reductions from maximum killing beginning at day 14, with effects also persisting out to day 90 to demonstrate similar activity to the dBTE, most likely due to differences in enduring-synDNA expression profiles. The dPMTE appeared to maintain maximum activity through day 90 without losing its effects until day 180 to outperform both the dBTE and dPBTE in synDNA form (figure 5D).

The reintroduction of mouse tumor modeling next enabled translationally relevant comparisons of efficacy. Subcutaneous xenografts of A-498 and IP human T cells in NSG mice first modeled a primary RCC condition, and a single 100 µg dose of dBTE expression vector elicited complete tumor control (online supplemental figure 5). At a low dose of 10 µg for distinguishment, both the dBTEs and dPBTEs displayed limited tumor control with respective measurements of approximately 1100 mm3 and 1800 mm3 tumors by day 33, with a significant difference between the two formats. dPMTE treatment appeared to induce the early elimination of tumors and enforce their ongoing suppression with a terminal 80 mm3 mean measurement. Conversely, control mice did not survive beyond day 30. Meaningful differences in terminal mean volumes were ultimately calculated for the dPMTE compared with both the dBTE and dPBTE (figure 5E).

To model metastatic ccRCC, SKRC-52 xenografts were implanted in NSG mice, after which the mice were infused with human T cells and administered a single, low dose of 10 µg dBTE or empty vector control. Control mice experienced rapid tumor growth with mice reaching the terminal volumes between days 15 and 27. The dPBTE produced noticeable yet marginal tumor control with a 2000 mm3 mean tumor volume by this day, with only one mouse surviving to day 33. The conventional dBTE trended toward higher activity with mouse tumors measuring 1250 mm3. In contrast, dPMTE treatment eliminated SKRC-52 tumors soon after administration, with three of the five mice remaining completely tumor-free by day 33 and a terminal mean volume of 190 mm3. Day 30 statistics, powered by at least an n=2 per group, demonstrate the dPMTE’s significant tumor suppression compared with b