Conditional PD-1/PD-L1 Probody Therapeutics Induce Comparable Antitumor Immunity but Reduced Systemic Toxicity Compared with Traditional Anti-PD-1/PD-L1 Agents.

Immune-checkpoint blockade has revolutionized cancer treatment. However, most patients do not respond to single-agent therapy. Combining checkpoint inhibitors with other immune-stimulating agents increases both efficacy and toxicity due to systemic T-cell activation. Protease-activatable antibody prodrugs, known as Probody therapeutics (Pb-Tx), localize antibody activity by attenuating capacity to bind antigen until protease activation in the tumor microenvironment. Herein, we show that systemic administration of anti-programmed cell death ligand 1 (anti-PD-L1) and anti-programmed cell death protein 1 (anti-PD-1) Pb-Tx to tumor-bearing mice elicited antitumor activity similar to that of traditional PD-1/PD-L1-targeted antibodies. Pb-Tx exhibited reduced systemic activity and an improved nonclinical safety profile, with markedly reduced target occupancy on peripheral T cells and reduced incidence of early-onset autoimmune diabetes in nonobese diabetic mice. Our results confirm that localized PD-1/PD-L1 inhibition by Pb-Tx can elicit robust antitumor immunity and minimize systemic immune-mediated toxicity. These data provide further preclinical rationale to support the ongoing development of the anti-PD-L1 Pb-Tx CX-072, which is currently in clinical trials.

The tumor targeting performance of anti-CD166 Probody drug conjugate CX-2009 and its parental derivatives as monitored by 89Zr-immuno-PET in xenograft bearing mice.

Probody® therapeutics are recombinant masked monoclonal antibody (mAb) prodrugs that become activated by proteases present in the tumor microenvironment. This makes them attractive for use as Probody drug conjugates (PDCs). CX-2009 is a novel PDC with the toxic drug DM4 coupled to an anti-CD166 Probody therapeutic. CD166 is overexpressed in multiple tumor types and to a lesser extent by healthy tissue. Methods: The tumor targeting potential of CX-2009 was assessed by performing 89Zr-immuno-PET/biodistribution/therapy studies in a CD166-positive H292 lung cancer mouse model. Head-to-head comparisons of CX-2009 with the Probody therapeutic without DM4 (CX-191), the unmasked antibody drug conjugate (ADC) CX-1031, and the parental mAb CX-090 were performed. All constructs were 89Zr labeled in a GMP compliant way, administered at 10, 110, or 510 µg, and ex vivo biodistribution was assessed at 24, 72, and 168 hours post-injection. Results: Comparable biodistribution was observed for all constructs, confirmed with PET/CT. Tumors showed the highest uptake: 21.8 ± 2.3 ([89Zr]Zr-CX-2009), 21.8 ± 5.0 ([89Zr]Zr­CX-191), 18.7 ± 2.5 ([89Zr]Zr-CX-1031) and 20.8 ± 0.9 %ID/g ([89Zr]Zr-CX-090) at 110 µg injected. Increasing the dose to 510 µg resulted in lower tumor uptake and higher blood levels for all constructs, suggesting receptor saturation. In addition, CX-2009 and CX-1031 showed similar therapeutic potential. Conclusions: CX-2009 is optimally capable of targeting CD166-expressing tumors when compared with its derivatives, implying that enzymatic activation inside the tumor, required to allow CD166 binding, does not limit tumor targeting. Because CX-2009 does not bind to mouse CD166, however, reduced targeting of healthy organs should be confirmed in ongoing clinical 89Zr-immuno-PET studies.

Thermodynamic, kinetic, and equilibrium parameters for the removal of lead and cadmium from aqueous solutions with calcium alginate beads.

The sorption of cadmium (Cd) and lead (Pb) by calcium alginate beads (CAB) from aqueous solutions in batch systems was investigated. The kinetic and thermodynamic parameters, as well as the sorption capacities of CAB in each system at different temperatures, were evaluated. The rate of sorption for both metals was rapid in the first 10 minutes and reached a maximum in 50 minutes. Sorption kinetic data were fitted to Lagergren, pseudo-second-order and Elovich models and it was found that the second-order kinetic model describes these data for the two metals; comparing kinetic parameters for Cd and Pb sorption a higher kinetic rate (K2) for Pb was observed, indicating that the interaction between lead cations and alginate beads was faster than for cadmium. Similarly, isotherm data were fitted to different models reported in literature and it was found that the Langmuir-Freundlich (L-F) and Dubinin-Radushkevich (D-R) models describe the isotherms in all cases. CAB sorption capacity for cadmium was 27.4 mg/g and 150.4 mg/g for lead, at 25 °C. Sorption capacities of Cd and Pb increase as temperature rises. According to the thermodynamic parameters, the cadmium and lead adsorption process was spontaneous and endothermic. It was also found that pH has an important effect on the adsorption of these metals by CAB, as more were removed at pH values between 6 and 7.

Peripheral sensory deafferentation affects olfactory bulb neurogenesis in zebrafish.

The potential effects of the removal of olfactory input on adult neurogenesis in the olfactory bulb were examined. Olfactory organs of adult zebrafish were permanently and completely ablated by cautery and animals were exposed to bromodeoxyuridine then examined following short (4-hour) or long (3-week) survival periods. Short survival times allowed analysis of cell proliferation in the olfactory bulb. Long survival times permitted investigation of survival of adult-formed cells. Deafferentation did not immediately affect the dividing cells in the bulb but did affect the number of adult-formed cells, some of which expressed a neuronal marker, present in the bulb 3 weeks later. Thus, afferent removal influenced the fate of newly formed cells by impacting subsequent divisions, maturation, or survival of those cells. One week of deafferentation altered the pattern of cell genesis, with a significant increase in the number of dividing cells located in the olfactory bulb and also in the ventral telencephalic proliferation zone. Sham surgery did not impact either proliferation or survival of adult-formed cells in the olfactory bulb, suggesting that the deafferentation effect is specific. Thus, afferent innervation is necessary for normal cell proliferation and maintenance of the olfactory bulb in adult zebrafish.

Changes in glutamate receptor subunit 4 expression in the deafferented olfactory bulb of zebrafish.

The distribution of ionotropic glutamate receptor subunit 4 (iGluR4) was examined in both normal and deafferented olfactory bulbs of adult zebrafish, Danio rerio. With the exception of the olfactory nerve layer, there was extensive labeling with antibodies to iGluR4 in the olfactory bulbs, specifically in juxtaglomerular cell bodies and their processes. These results are consistent with previous work, which has suggested differential distribution of glutamate receptors in the vertebrate olfactory system. Analysis of bulbs following olfactory organ removal revealed a significant loss of iGluR4 immunoreactivity by 24 h post-deafferentation. At 48 h after denervation, iGluR4 labeling had returned to normal levels and was retained through 3 weeks post-surgery. Thus, afferent input plays a role in reduced labeling of this protein immediately following injury, but return of immunoreactivity can occur even without sensory innervation.