We describe a generic approach to assemble correctly two heavy and two light chains, derived from two existing antibodies, to form human bivalent bispecific IgG antibodies without use of artificial linkers. Based on the knobs-into-holes technology that enables heterodimerization of the heavy chains, correct association of the light chains and their cognate heavy chains is achieved by exchange of heavy-chain and light-chain domains within the antigen binding fragment (Fab) of one half of the bispecific antibody. This “crossover” retains the antigen-binding affinity but makes the two arms so different that light-chain mispairing can no longer occur. Applying the three possible “CrossMab” formats, we generated bispecific antibodies against angiopoietin-2 (Ang-2) and vascular endothelial growth factor A (VEGF-A) and show that they can be produced by standard techniques, exhibit stabilities comparable to natural antibodies, and bind both targets simultaneously with unaltered affinity. Because of its superior side-product profile, the CrossMab CH1-CL was selected for in vivo profiling and showed potent antiangiogenic and antitumoral activity.

Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis, and new therapies are needed. RG7787 is a novel low-immunogenic anti-mesothelin recombinant immunotoxin (RIT), engineered to overcome the limitations of SS1P, a RIT now in clinical trials. In vitro activity was evaluated on five established PDAC cell lines (KLM-1, AsPC-1, BxPC-3, Panc 3.014 and PK-1) and on PDAC cells directly established from a patient tumor (GUMC108). RG7787 had subnanomolar IC50s in most cell lines, and was significantly more active than SS1P in GUMC108, KLM-1 and Panc 3.014 cells. GUMC108 was most sensitive, with RG7787 killing >99% of the cells. In a subcutaneous KLM-1 xenograft mouse model, two cycles of 3 × 2.5 mg/kg RG7787 QOD combined with two cycles of 1 × 50 mg/kg paclitaxel induced near-complete responses, with all tumors regressing below 5 mm3 within 30 days after therapy was initiated (>95% decrease) and no significant growth increase for at least another 3 weeks. RG7787 alone gave limited but significant regressions and paclitaxel by itself arrested tumor growth. Quantifying the uptake of Alexa647-labeled RG7787 in tumors showed that the RIT reached only 45% of KLM-1 cells, accounting in part for the limited responses. Paclitaxel did not improve RG7787 uptake, which thus cannot explain the beneficial effect of the combination therapy. In conclusion, RG7787 has high cytotoxic activity on PDAC cell lines as well as on primary patient cells. In vivo, this novel RIT gives durable near-complete tumor responses when combined with paclitaxel. RG7787 merits further evaluation for the treatment of PDAC.

PharmacokineticsLung cancer Patient-derived xenografts Targeted therapy A B S T R A C TMesothelin overexpression in lung adenocarcinomas correlates with the presence of activating KRAS mutations and poor prognosis. Hence SS1P, a mesothelin-targeted immunotoxin, could offer valuable treatment options for these patients, but its use in solid tumor therapy is hampered by high immunogenicity and non-specific toxicity. To overcome both obstacles we developed RG7787, a de-immunized cytotoxic fusion protein comprising a humanized SS1 Fab fragment and a truncated, B-cell epitope silenced, 24 kD fragment of Pseudomonas exotoxin A (PE24). Reactivity of RG7787 with sera from immunotoxin-treated patients was >1000 fold reduced. In vitro RG7787 inhibited cell viability of lung cancer cell lines with picomolar potency. The pharmacokinetic properties of RG7787 in rodents were comparable to SS1P, yet it was tolerated up to 10 fold better without causing severe vascular leak syndrome or hepatotoxicity. A pharmacokinetic/ pharmacodynamic model developed based on NCI-H596 xenograft studies showed that for RG7787 and SS1P, their in vitro and in vivo potencies closely correlate. At optimal doses of 2e3 mg/kg RG7787 is more efficacious than SS1P. Even large, well established tumors

(2016) Heavy and light chain pairing of bivalent quadroma and knobs-into-holes antibodies analyzed by UHR-ESI-QTOF mass spectrometry, mAbs, 8:1, 49-55, DOI: 10.1080/19420862.2015 To link to this article: https://doi.org/10. 1080/19420862.2015 Keywords: bispecific antibody, CrossMab, chain pairing, ESI-QTOF mass spectrometry, knobs-into-holes, quadromaAbbreviations: Ang2, angiopoietin-2; bsAbs, bispecific antibodies; ESI, electrospray ionization; GuA HCl, guanidine hydrochloride; HEK, human embryonic kidney; HC, heavy chain; IgG1, immunoglobulin G1; ISCID, ion source collision induced dissociation; KiH, knobs-into-holes; LC, light chain; MS, mass spectrometry; TCEP, tris(2-carboxyethyl)phosphine; UHR, ultrahigh-resolution; VEGF-A, vascular endothelial growth factor A; QTOF, quadrupole time-of-flightThe quadroma antibody represents the first attempt to produce a bispecific heterodimeric IgG antibody by somatic fusion of 2 hybridoma cells each expressing monoclonal antibodies with distinctive specificities. However, because of random heavy and light chain pairing, the desired functional bispecific antibody represents only a small fraction of the protein produced. Subsequently, the knobs-into-holes (KiH) approach was developed to enforce correct heavy chain heterodimerization. Assuming equimolar expression of 4 unmodified chains comprising 2 heavy and 2 light chains, the statistical distribution of all paired combinations can be calculated. With equimolar expression as the goal, we transfected HEK cells with 1:1:1:1 plasmid ratios and analyzed the protein A affinity-purified antibodies from the quadroma and KiH approaches qualitatively and quantitatively with regard to the estimated relative amounts of the products using electrospray quadrupole time-of-flight mass spectrometry. Our results show that all expected species are formed, and that, within the methodological limits, the species distribution in the mixtures corresponds approximately to the statistical distribution.

Bifunctional antibody fusion proteins engaging effector T cells for targeted elimination of tumor cells via CD3 binding have shown efficacy in both preclinical and clinical studies.