Search

Botulinum neurotoxins (BoNT) are approved for a number of therapeutic indications, including blepharospasm, cervical dystonia and hyperhidrosis, and have also shown efficacy in a variety of pain disorders. The potency of any given BoNT preparation can be routinely assessed by using the Digit Abduction Score (DAS) assay, which measures the local muscle weakening efficacy of BoNT following injection into mouse hindlimb muscle. While most studies have employed mice to assess BoNT efficacy in the DAS, few have utilized rats. In this study, we applied the DAS assay to a rat model and compared the potency of IM-BOTOX(®) (onabotulinumtoxinA) injections between two separate hind limb muscles, gastrocnemius and tibialis anterior (TA). The results demonstrated that the DAS assay can be performed on rats with similar criteria and parameters as for mice. Moreover in the rat, BoNT can be injected into either the gastrocnemius or TA muscle to elicit similar DAS scoring responses. Interestingly, onabotulinumtoxinA potency in the rat DAS was ∼3-fold higher following TA injections than gastrocnemius injections. Additionally, our data showed that the durational kinetics of onabotulinumtoxinA in the rat DAS are approximately twice as long as in the mouse DAS. These results position the rat DAS as a more flexible model for examining the mechanisms of BoNT diffusion and muscle paralysis, while mouse DAS can be used for physiological screening of BoNT because of the potential for higher throughput. Overall, these data confirm the utility of the DAS assay for characterizing the physiological potency of BoNT and related compounds.

PSMA (Prostate-Specific Membrane Antigen) is a promising therapeutic target in prostate cancer. Exploiting the high and selective expression of PSMA in the prostate, capromab and J591 antibodies are used as imaging or therapeutic agents, and antibody-toxin conjugates such as MLN2704 are being developed. Such antibodies do not stimulate T cell-mediated killing of prostate cancer cells; however, promising clinical data with T cell-recruiting bispecific agents and chimeric antigen receptor (CAR) T cells have created new immunotherapy paradigms that may also have potential in prostate cancer. We designed long-acting humanized bispecific antibodies that coengage PSMA+ cells and CD3+ T cells to stimulate redirected T cell-mediated cytotoxicity (RTCC) of prostate cancer cells. Unlike other bispecific formats, our antibodies possess an Fc domain and form stable heterodimers that are easily manufactured. Binding to Fcγ receptors was also abolished (reducing the potential for nonselective T cell activation), yet binding to human FcRn was preserved to maintain long serum half-life. We screened several anti-PSMA x anti-CD3 bispecific antibodies in vitro, and selected XmAb14484 based on its potent (< 1ng/ml) stimulation of human T cell killing of the LNCaP prostate tumor cell line. RTCC activity required PSMA binding, because a non-specific control (XENP13245, anti-RSV x anti-CD3) was inactive. XmAb14484 crossreacts with monkey but not mouse targets; therefore, we evaluated its effects in cynomolgus monkeys. Peripheral blood cells do not express PSMA, so we were unable to monitor target cell depletion. However, T cell engagement of target cells induces distinct effects on peripheral T cells, which can be used as surrogate markers for activity in the prostate. After treatment with 0.03 mg/kg XmAb14484, CD4+ and CD8+ T cells were rapidly activated (quantified by CD69 upregulation) and redistributed from the periphery. Serum cytokines including IL-6 and TNF were also strongly induced. T cell and cytokine responses returned to baseline within 2-3 days. In marked contrast, a 100-fold higher dose (3 mg/kg) of the anti-RSV x anti-CD3 control antibody induced minimal effects, demonstrating that PSMA+ target cells are required for T cell activation by XmAb14484. In summary, the pharmacologic activities of XmAb14484 on human cells and in monkeys support its clinical assessment in prostate cancer. We also demonstrate that T cell effects readily measured in peripheral blood (T cell redistribution, activation and cytokine induction) are useful surrogate markers of target-specific engagement. Citation Format: Seung Y. Chu, Erik W. K. Pong, Rumana Rashid, Hsing Chen, Emily W. Chan, Sheryl Phung, Nancy A. Endo, Maria C. Ardila, Christine Bonzon, Irene W. L. Leung, Umesh S. Muchhal, Gregory L. Moore, Matthew J. Bernett, John R. Desjarlais, David E. Szymkowski. Immunotherapy with anti-PSMA x anti-CD3 bispecific antibody stimulates potent killing of a human prostate cancer cell line and target-mediated T cell activation in monkeys: A potential therapy for prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5000.

IncobotulinumtoxinA (Xeomin®) and onabotulinumtoxinA (BOTOX®) are unique botulinum neurotoxin type A (BoNT/A)-derived drugs. IncobotulinumtoxinA utilizes the naked 150 kDa holotoxin portion of BoNT/A, whereas onabotulinumtoxinA uses the complete native 900 kDa complex as drug substance. On the basis of purportedly similar pharmacological characteristics, these formulations were evaluated for potency by LD₅₀ and mouse Digit Abduction Score (DAS) bioassays. DAS was also used to assess antigenicity. Full-range DAS dose-response profiles were achieved with four lots of each product, with similar observations between lots for a given product. Between products, however, the mean DAS potency of incobotulinumtoxinA (ED₅₀ range 7.0-10.2 U/kg) was significantly lower than that of onabotulinumtoxinA (ED₅₀ range 4.4-6.4 U/kg), consistent with lower measured potencies in the LD₅₀ assay for incobotulinumtoxinA (potency range 62-82 U). In assessments of DAS duration of effect at similar unit doses, the observed lower potency of incobotulinumtoxinA translated into decreased peak efficacy and dose effect over time (i.e. shorter duration). In contrast, at equi-efficacious doses yielding near-maximal DAS responses, both toxin formulations were uniformly inhibited in a statistically significant manner when preincubated with rabbit-derived, onabotulinumtoxinA-neutralizing antibodies, supporting the position that inhibition of 150 kDa holotoxin serves as the common basis for neutralization and, therefore, incobotulinumtoxinA would not be expected to be effective in onabotulinumtoxinA-immunoresistant subjects (and vice versa). Further, with lower lot-to-lot relative potency, incobotulinumtoxinA is not dose-equivalent or interchangeable with onabotulinumtoxinA, suggesting that various aspects of drug product formulation may influence observed pharmacology.

CD38 is highly expressed on plasma cells and is an attractive target for multiple myeloma (MM) therapies. Several anti-CD38 antibodies including daratumumab and SAR650984 show promising results in clinical development, though such antibodies are not able to stimulate T cell-mediated killing of myeloma cells. To exploit a T cell immunotherapy mechanism while retaining the favorable drug properties of therapeutic antibodies, we designed bispecific antibodies that recruit T cells to CD38+ MM cells. Such bispecifics act via redirected T cell-cytotoxicity (RTCC) to stimulate T cell-mediated target cell killing regardless of T cell receptor antigen specificity. These anti-CD38 × anti-CD3 antibodies possess a full Fc domain and spontaneously form stable heterodimers that are readily manufactured. Their Fc domain lacks binding to Fcγ receptors to minimize nonselective T cell activation, yet retains binding to FcRn to maintain long serum half-life. We have previously reported that XmAb13551, a humanized and affinity-optimized anti-CD38 × anti-CD3 antibody, stimulates killing of the CD38+ MM cell line RPMI8226 by human T cells and suppresses human Ig levels in SCID mice engrafted with human PBMCs, showing much greater efficacy than daratumumab in these models (Blood 2014 124:4727). We also investigated efficacy of XmAb13551 in monkeys given a single dose of 2, 5, and 20 μg/kg. Within 1 hr after dosing, CD25 and CD69 activation markers were upregulated on T cells and within 8 hr, circulating CD38+ cells were depleted by > 95% at the 20 μg/kg dose. However, depletion of peripheral CD38+ cells was not sustained, suggesting that a large antigen sink was limiting drug exposure. Although higher dosing might overcome an antigen sink, higher doses of XmAb13551 (0.2 mg/kg or higher) resulted in a T cell-mediated cytokine release syndrome (CRS) in monkeys. We reasoned that an anti-CD38 × anti-CD3 antibody with reduced CD3 affinity would stimulate sufficient RTCC to deplete MM cells, yet would attenuate the acute T cell activation (and associated CRS) induced by high-affinity coengagement of T cells with CD38+ target cells. Using XmAb13551 as a starting point, we engineered a series of bispecifics retaining the same high-affinity (0.2 nM) binding to CD38, but with reduced affinity to CD3. We selected two antibodies - XmAb15426 and XmAb14702 - that have significantly reduced CD3 affinity. As expected, these molecules showed reduced potency in RTCC assays using T cells to kill RPMI8226 cells, with potency correlating with CD3 affinity (XmAb13551 > XmAb15426 >> XmAb14702). We next tested XmAb15426 and XmAb14702 at single doses of 0.5 mg/kg and 3 mg/kg, respectively, in cynomolgus monkeys. Both antibodies were well-tolerated at these higher doses, consistent with the moderate levels of IL6 observed in serum from the treated monkeys. Moreover, XmAb15426, with intermediate CD3 affinity, more effectively depletes CD38+ cells at 0.5 mg/kg compared to the original high-affinity XmAb13551 dosed at 2, 5 or 20 µg/kg. Depletion by XmAb15426 was more sustained compared to the highest dose of XmAb13551 in the previous study (7 vs. 2 days, respectively). Notably, although target cell depletion was greater for XmAb15426, T cell activation (CD69, CD25 and PD1 induction) was much lower in monkeys treated with XmAb15426 even dosed 25-fold higher than the 20 µg/kg XmAb13551 group. XmAb14702, with very low CD3 affinity, had little effect on CD38+ cells and T cell activation. Our results demonstrate that modulating T cell activation by attenuating CD3 affinity is a promising method to improve the therapeutic window of T cell-engaging bispecific antibodies. This strategy has potential to expand the set of antigens amenable to targeted T cell immunotherapy by improving tolerability and enabling higher dosing to overcome antigen sink clearance with targets such as CD38. We have shown that by reducing affinity for CD3, XmAb15426 effectively depletes CD38+ cells while minimizing the CRS effects seen with comparable doses of its high-affinity counterpart XmAb13551. Our preclinical data for XmAb15426 provide a rationale for clinical testing of this bispecific antibody in patients with multiple myeloma and other CD38+ malignancies. Disclosures Moore: Xencor, Inc.: Employment, Equity Ownership. Lee:Xencor, Inc.: Employment, Equity Ownership. Schubbert:Xencor, Inc.: Employment, Equity Ownership. Miranda:Xencor, Inc.: Employment, Equity Ownership. Rashid:Xencor, Inc.: Employment, Equity Ownership. Pong:Xencor, Inc.: Employment, Equity Ownership. Phung:Xencor, Inc.: Employment, Equity Ownership. Chan:Xencor, Inc.: Employment, Equity Ownership. Chen:Xencor, Inc.: Employment, Equity Ownership. Endo:Xencor, Inc.: Employment, Equity Ownership. Ardila:Xencor, Inc.: Employment, Equity Ownership. Bernett:Xencor, Inc.: Employment, Equity Ownership. Chu:Xencor, Inc.: Employment, Equity Ownership. Leung:Xencor, Inc.: Employment, Equity Ownership. Muchhal:Xencor, Inc.: Employment, Equity Ownership. Bonzon:Xencor, Inc.: Employment, Equity Ownership. Szymkowski:Xencor, Inc.: Employment, Equity Ownership. Desjarlais:Xencor, Inc.: Employment, Equity Ownership.

Both rHis6-BoNT/E and rBoNT/E-His6 were produced from a codon optimized gene within Escherichia coli. Similar to native BoNT/E isolated from Clostridium botulinum, the recombinant proteins were isolated as single-chain molecules. The His6 affinity tag facilitated a two-step IMAC-IEX purification. While reasonable yields were obtained, single-chain rHis6-BoNT/E was produced less efficiently than was rBoNT/E-His6. Treatment with recombinant trypsin efficiently converted both purified rBoNT/E constructs to the activated dichain forms, with concomitant removal of the His6 tag, and the nicking profile for each rBoNT/E molecule matched that of native BoNT/E. Following nicking with trypsin, it was observed that the resultant rHis6-BoNT/E dichain displayed reduced in vivo potency (mouse potency assay) compared to activated native BoNT/E dichain. In contrast, activated rBoNT/E-His6 displayed a specific potency equivalent to that of trypsin-nicked native BoNT/E in the mouse potency assay. These results demonstrate that rBoNT/E, containing a C-terminal His6 affinity tag, can be expressed and purified in high yield, with the activated molecule displaying equivalent in vivo potency to activated native BoNT/E.