Peptide Binder to Glypican-3 as a Theranostic Agent for Hepatocellular Carcinoma.

Glypican-3 (GPC3) is a membrane-associated glycoprotein that is significantly upregulated in hepatocellular carcinomas (HCC) with minimal to no expression in normal tissues. The differential expression of GPC3 between tumor and normal tissues provides an opportunity for targeted radiopharmaceutical therapy to treat HCC, a leading cause of cancer-related deaths worldwide. Methods: DOTA-RYZ-GPC3 (RAYZ-8009) comprises a novel macrocyclic peptide binder to GPC3, a linker, and a chelator that can be complexed with different radioisotopes. The binding affinity was determined by surface plasma resonance and radioligand binding assays. Target-mediated cellular internalization was radiometrically measured at multiple time points. In vivo biodistribution, monotherapy, and combination treatments with 177Lu or 225Ac were performed on HCC xenografts. Results: RAYZ-8009 showed high binding affinity to GPC3 protein of human, mouse, canine, and cynomolgus monkey origins and no binding to other glypican family members. Potent cellular binding was confirmed in GPC3-positive HepG2 cells and was not affected by isotope switching. RAYZ-8009 achieved efficient internalization on binding to HepG2 cells. Biodistribution study of 177Lu-RAYZ-8009 showed sustained tumor uptake and fast renal clearance, with minimal or no uptake in other normal tissues. Tumor-specific uptake was also demonstrated in orthotopic HCC tumors, with no uptake in surrounding liver tissue. Therapeutically, significant and durable tumor regression and survival benefit were achieved with 177Lu- and 225Ac-labeled RAYZ-8009, as single agents and in combination with lenvatinib, in GPC3-positive HCC xenografts. Conclusion: Preclinical in vitro and in vivo data demonstrate the potential of RAYZ-8009 as a theranostic agent for the treatment of patients with GPC3-positive HCC.

Identifying a predictive relationship between maximal flow rate and viscosity for subcutaneous administration of macromolecules with recombinant human hyaluronidase PH20 in a miniature pig model.

Subcutaneous (SC) infusion of large volumes at rapid flow rates has historically been limited by the glycosaminoglycan hyaluronan (HA), which forms a barrier to bulk fluid flow in the SC space. Recombinant human hyaluronidase PH20 (rHuPH20) depolymerizes HA, temporarily eliminating this barrier to rapid SC delivery of large volume co-administered therapeutics. Using a miniature pig model, in-line pressure and applied force to the delivery hardware were measured when subcutaneously infusing a representative macromolecule (human polyclonal immunoglobulin [Ig]), at varying concentrations and viscosities (20-200 mg/mL), co-formulated with and without rHuPH20 (2000 U/mL and 5000 U/mL). Maximal flow rate (Qmax) was calculated as the flow rate producing a statistically significant difference in mean applied force between injections administered with or without rHuPH20. There was a significant reduction in mean applied force required for SC delivery of 100 mg/mL Ig solution with 5000 U/mL rHuPH20 versus Ig solution alone. Similar significant reductions in mean applied force were observed for most Ig solution concentrations, ranging from 25-200 mg/mL when administered with or without 2000 U/mL rHuPH20. Qmax was inversely proportional to Ig solution viscosity and Qmax for solutions co-formulated with 5000 U/mL rHuPH20 was approximately double that of 2000 U/mL rHuPH20 solutions. Mathematical simulation of a hypothetical 800 mg Ig dose co-formulated with rHuPH20 showed that delivery times <30 s could be achieved across a broad range of concentrations. Addition of rHuPH20 can help overcome volume and time constraints associated with SC administration across a range of concentrations in a dose-dependent manner.

RYZ101 (Ac-225 DOTATATE) Opportunity beyond Gastroenteropancreatic Neuroendocrine Tumors: Preclinical Efficacy in Small-Cell Lung Cancer.

Overexpression of somatostatin receptors (SSTR), particularly SSTR2, is found in gastroenteropancreatic neuroendocrine tumors (GEP-NET), and subsets of other solid tumors such as small-cell lung cancer (SCLC). SCLC accounts for approximately 13% to 15% of lung cancer and lacks effective therapeutic options. IHC analysis indicates that up to 50% of SCLC tumors are SSTR2-positive, with a substantial subset showing high and homogenous expression. Peptide receptor radionuclide therapy with radiolabeled somatostatin analogue, Lu-177 DOTATATE, has been approved for GEP-NETs. Different strategies aimed at improving outcomes, such as the use of alpha-emitting radioisotopes, are currently being investigated. RYZ101 (Ac-225 DOTATATE) is comprised of the alpha-emitting radioisotope actinium-225, chemical chelator DOTA, and octreotate (TATE), a somatostatin analogue. In the cell-based competitive radioligand binding assay, RAYZ-10001-La (lanthanum surrogate for RYZ101) showed high binding affinity (Ki = 0.057 nmol/L) to human SSTR2 and >600-fold selectivity against other SSTR subtypes. RAYZ-10001-La exhibited efficient internalization to SSTR2-positive cells. In multiple SSTR2-expressing SCLC xenograft models, single-dose intravenous RYZ101 3 µCi (0.111 MBq) or 4 µCi (0.148 MBq) significantly inhibited tumor growth, with deeper responses, including sustained regression, observed in the models with higher SSTR2 levels. The antitumor effect was further enhanced when RYZ101 was combined with carboplatin and etoposide at clinically relevant doses. In summary, RYZ101 is a highly potent, alpha-emitting radiopharmaceutical agent, and preclinical data demonstrate the potential of RYZ101 for the treatment of patients with SSTR-positive cancers.

Targeting Adenosine with Adenosine Deaminase 2 to Inhibit Growth of Solid Tumors.

Extracellular adenosine in tumors can suppress immune responses and promote tumor growth. Adenosine deaminase 2 (ADA2) converts adenosine into inosine. The role of ADA2 in cancer and whether it can target adenosine for cancer therapy has not been investigated. Here we show that increased ADA2 expression is associated with increased patient survival and enrichment of adaptive immune response pathways in several solid tumor types. Several ADA2 variants were created to improve catalytic efficiency, and PEGylation was used to prolong systemic exposure. In mice, PEGylated ADA2 (PEGADA2) inhibited tumor growth by targeting adenosine in an enzyme activity-dependent manner and thereby modulating immune responses. These findings introduce endogenous ADA2 expression as a prognostic factor and PEGADA2 as a novel immunotherapy for cancer. SIGNIFICANCE: This study identifies ADA2 as a prognostic factor associated with prolonged cancer patient survival and introduces the potential of enzymatic removal of adenosine with engineered ADA2 for cancer immunotherapy.

Removal of interstitial hyaluronan with recombinant human hyaluronidase improves the systemic and lymphatic uptake of cetuximab in rats.

Interstitial, e.g. subcutaneous (SC) or intradermal (ID), administration of monoclonal antibodies (mAb) is less invasive than intravenous administration and leads to mAb uptake into both lymphatic and blood capillaries draining the injection site. Interstitial administration, however, is hindered by the presence of hyaluronan (HA), a glycosaminoglycan that is a major fluid barrier in the interstitial space. The transient removal of HA with recombinant human hyaluronidase (rHuPH20) helps facilitate the interstitial administration of often high therapeutic doses of mAb in the clinic. rHuPH20's impact on the systemic pharmacokinetics of several mAbs has been previously described, however effects on route of absorption (lymph vs blood) are unknown. The current study has therefore explored the lymphatic transport and bioavailability of cetuximab and trastuzumab after SC and ID coadministration in the presence and absence of rHuPH20 in rats. After SC administration cetuximab absolute bioavailability increased from 67 % to 80 % in the presence of rHuPH20. Cetuximab recovery in the lymphatics also increased after SC (35.8 % to 49.4 %) and ID (26.7 % to 58.8 %) administration in the presence of rHuPH20. When the injection volume (and therefore dose) was increased 10-fold in the presence of rHuPH20 cetuximab plasma exposure increased approximately linearly (12- and 8.9-fold respectively after SC and ID administration), although the proportional contribution of cetuximab lymphatic transport reduced slightly (6.2-fold increase for both administration routes). In contrast, co-administration with rHuPH20 did not lead to increases in plasma exposure for trastuzumab after SC or ID administration, most likely reflecting the fact that the reported absolute bioavailability of trastuzumab (in the absence of rHuPH20) is high (∼77-99 %). However, lymphatic transport of trastuzumab did increase when coadministered ID with rHuPH20 in spite of the lack of change to overall bioavailability. The data suggest that co-administration with rHuPH20 is able to increase the volume of mAb that can be administered interstitially, and in some instances can increase the amount absorbed into both the blood and the lymph. In the current studies the ability of rHuPH20 to enhance interstitial bioavailability was higher for cetuximab where intrinsic interstitial bioavailability was low, when compared to trastuzumab where interstitial bioavailability was high.

Remodeling the Tumor Microenvironment Sensitizes Breast Tumors to Anti-Programmed Death-Ligand 1 Immunotherapy.

Immunotherapies targeting immune checkpoint inhibitors have changed the landscape of cancer treatment, however, many patients are resistant or refractory to immunotherapy. The sensitivity of tumor cells to immunotherapy may be influenced by hyaluronan (HA) accumulation in the tumor microenvironment (TME). Enzymatic degradation of HA by pegvorhyaluronidase alfa (PEGPH20; PVHA) remodels the TME. This leads to reduced tumor interstitial pressure and decompressed tumor blood vessels, which are both associated with increased exposure of tumor cells to chemotherapy drugs. Here, we demonstrate PVHA increased the uptake of anti-programmed death-ligand 1 (PD-L1) antibody in HA-accumulating animal models of breast cancer. The increased levels of anti-PD-L1 antibody were associated with increased accumulation of T cells and natural killer cells and decreased myeloid-derived suppressor cells. PD-L1 blockade significantly inhibited tumor growth when combined with PVHA, but not alone. Our results suggest that PVHA can sensitize HA-accumulating tumors to anti-PD-L1 immunotherapy. SIGNIFICANCE: These findings show removal of hyaluronan in the tumor microenvironment improves immune cells and checkpoint inhibitors access to tumors. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/79/16/4149/F1.large.jpg.