Pretargeted radioimmunotherapy with the novel anti-oxMIF/HSG bispecific antibody ON105 results in significant tumor regression in murine models of cancer.

Radioimmunotherapy (RIT) uses mAbs to deliver radionuclides to cancer cells or the tumor microenvironment and has shown promise in treating localized and diffuse tumors. While RIT agents have gained FDA/EMA approval for certain hematological malignancies, effectiveness of RIT in treating solid tumors remains limited. Here we present PreTarg-it®, a novel approach for pretargeted radioimmunotherapy, providing optimized delivery of payloads in a two-step regimen. The effectiveness of PreTarg-it® is demonstrated by a powerful combination of ON105, a novel bispecific antibody against both oxMIF and the histamine-succinyl-glycyl (HSG) hapten, as the first component and the radioactively labeled DOTA-di-HSG peptide as the second component in murine models of cancer. Mice bearing either subcutaneous mouse colorectal CT26 or human pancreatic CFPAC-1 tumors received an intravenous injection of ON105. After ON105 had accumulated in the tumor and cleared from circulation to approximately 1-3% of its peak concentration, 177Lu-DOTA-di-HSG peptide was administered. A single PreTarg-it® treatment cycle resulted in tumor regression when mice bearing CT26 tumors were given the highest treatment dose with a pretargeting delay of three days. Administered with a 5-day interval, the highest dose arrested tumor growth in both CT26 syngrafts and CFPAC-1 xenografts. In all cases, the highest treatment dose resulted in 100% survival at the study endpoint whereas the control cohorts showed 0% and 60% survival in the CT26 and CFPAC-1 models, respectively. Therefore, PreTarg-it® holds potential as a novel and potent therapy for patients with hard-to-treat solid tumors such as pancreatic cancer, as well as those with late-stage malignancies.

Identification of two subtilisin-like serine proteases engaged in the degradation of recombinant proteins in Nicotiana benthamiana.

The tobacco variant Nicotiana benthamiana has recently emerged as a versatile host for the manufacturing of protein therapeutics, but the fidelity of many recombinant proteins generated in this system is compromised by inadvertent proteolysis. Previous studies have revealed that the anti-HIV-1 antibodies 2F5 and PG9 as well as the protease inhibitor α1 -antitrypsin (A1AT) are particularly susceptible to N. benthamiana proteases. Here, we identify two subtilisin-like serine proteases (NbSBT1 and NbSBT2) whose combined action is sufficient to account for all major cleavage events observed upon expression of 2F5, PG9 and A1AT in N. benthamiana. We propose that downregulation of NbSBT1 and NbSBT2 activities could constitute a powerful means to optimize the performance of this promising platform for the production of biopharmaceuticals. DATABASES: NbSBT sequence data are available in the DDBJ/EMBL/GenBank databases under the accession numbers MN534996 to MN535005.

Steric Accessibility of the Cleavage Sites Dictates the Proteolytic Vulnerability of the Anti-HIV-1 Antibodies 2F5, 2G12, and PG9 in Plants.

Broadly neutralizing antibodies (bNAbs) to human immunodeficiency virus type 1 (HIV-1) hold great promise for immunoprophylaxis and the suppression of viremia in HIV-positive individuals. Several studies have demonstrated that plants as Nicotiana benthamiana are suitable hosts for the generation of protective anti-HIV-1 antibodies. However, the production of the anti-HIV-1 bNAbs 2F5 and PG9 in N. benthamiana is associated with their processing by apoplastic proteases in the complementarity-determining-region (CDR) H3 loops of the heavy chains. Here, it is shown that apoplastic proteases can also cleave the CDR H3 loop of the bNAb 2G12 when the unusual domain exchange between its heavy chains is prevented by the replacement of Ile19 with Arg. It is demonstrated that CDR H3 proteolysis leads to a strong reduction of the antigen-binding potencies of 2F5, PG9, and 2G12-I19R. Inhibitor profiling experiments indicate that different subtilisin-like serine proteases account for bNAb fragmentation in the apoplast. Differential scanning calorimetry experiments corroborate that the antigen-binding domains of wild-type 2G12 and 4E10 are more compact than those of proteolysis-sensitive antibodies, thus shielding their CDR H3 regions from proteolytic attack. This suggests that the extent of proteolytic inactivation of bNAbs in plants is primarily dictated by the steric accessibility of their CDR H3 loops.