Sabine Imhof-Jung

Immunoglobulin domain crossover as a generic approach for the production of bispecific IgG antibodies

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 CrossMabCH1-CL was selected for in vivo profiling and showed potent antiangiogenic and antitumoral activity.

In Vitro and In Vivo Activity of the Low-Immunogenic Antimesothelin Immunotoxin RG7787 in Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis, and new therapies are needed. RG7787 is a novel low-immunogenic antimesothelin 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 Alexa Fluor 647–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. Mol Cancer Ther; 13(8); 2040–9. ©2014 AACR.

Crystal structure of an anti-Ang2 CrossFab demonstrates complete structural and functional integrity of the variable domain.

Bispecific antibodies are considered as a promising class of future biotherapeutic molecules. They comprise binding specificities for two different antigens, which may provide additive or synergistic modes of action. There is a wide variety of design alternatives for such bispecific antibodies, including the “CrossMab” format. CrossMabs contain a domain crossover in one of the antigen-binding (Fab) parts, together with the “knobs-and-holes” approach, to enforce the correct assembly of four different polypeptide chains into an IgG-like bispecific antibody. We determined the crystal structure of a hAng-2-binding Fab in its crossed and uncrossed form and show that CH1-CL-domain crossover does not induce significant perturbations of the structure and has no detectable influence on target binding.

Heavy and light chain pairing of bivalent quadroma and knobs-into-holes antibodies analyzed by UHR-ESI-QTOF mass spectrometry

The 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.

Characterization of a re-engineered, mesothelin-targeted Pseudomonas exotoxin fusion protein for lung cancer therapy.

Mesothelin 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 2–3 mg/kg RG7787 is more efficacious than SS1P. Even large, well established tumors (600 mm3) underwent remission during three treatment cycles with RG7787. Also in two patient‐derived lung cancer xenograft models, Lu7336 and Lu7187, RG7787 showed anti‐tumor efficacy. In monotherapy two treatment cycles were moderately efficacious in the Lu7336 model but showed good anti‐tumor activity in the KRAS mutant Lu7187 model (26% and 80% tumor growth inhibition, respectively). Combination of RG7787 with standard chemotherapies further enhanced efficacy in both models achieving near complete eradication of Lu7187 tumors.

A New Class of Bifunctional Major Histocompatibility Class I Antibody Fusion Molecules to Redirect CD8 T Cells

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. Different from such a polyclonal T-cell recruitment, an alternative concept is to engage only antigen-specific T-cell subsets. Recruitment of specific subsets of T cells may be as potent but potentially lead to fewer side effects. Tumor-targeted peptide–MHC class I complexes (pMHCI-IgGs) bearing known antigenic peptides complexed with MHC class I molecules mark tumor cells as antigenic and utilize the physiologic way to interact with and activate T-cell receptors. If, for example, virus-specific CD8+ T cells are addressed, the associated strong antigenicity and tight immune surveillance of the effector cells could lead to efficacious antitumor treatment in various tissues. However, peptide–MHC class I fusions are difficult to express recombinantly, especially when fused to entire antibody molecules. Consequently, current formats are largely limited to small antibody fragment fusions expressed in bacteria followed by refolding or chemical conjugation. Here, we describe a new molecular format bearing a single pMHCI complex per IgG fusion molecule characterized by enhanced stability and expression yields. This molecular format can be expressed in a full immunoglobulin format and can be designed as mono- or bivalent antibody binders. Mol Cancer Ther; 15(9); 2130–42. ©2016 AACR.

Abstract 3285: Ang-2-VEGF CrossMab, a novel bispecific human IgG1 antibody blocking VEGF-A and Ang-2 function mediates potent anti-tumoral and anti-angiogenic efficacy

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL VEGF-A blockade has been validated clinically as a treatment for human cancers. Angiopoietin-2 (Ang-2) expression has been shown to function as a key regulator of blood vessel remodeling and tumor angiogenesis. In tumors Ang-2 is up-regulated and a bad prognostic factor. Recent data demonstrated that Ang-2 inhibitors, both as a single agent or in combination with chemo- or anti-VEGF therapy mediate anti-tumoral effects. We have recently described a novel generic method for the production of bivalent bispecific human IgG1 antibodies (CrossMabs) based on the crossover of the CH1 and CL domains within the Fab region of one half of a bispecific antibody combined with the knobs-into-holes technology to enforce heterodimerization of the Fc portion. Subsequently, we have applied the CrossMab technology for the generation of a bispecific antibody recognizing VEGF-A with one arm based on bevacizumab and recognizing Ang-2 with the other arm based on LC06, a highly Ang-2 selective human IgG1 antibody. The Ang-2-VEGF CrossMAb could be produced with good yields and purity by eukaryotic. Surface Plasmon resonance studies showed that the two different arms of the Ang-2-VEGF CrossMab retained their antigen binding affinity for VEGF-A and Ang-2 and interfered with VEGF-induced HUVEC proliferation and Ang-2 induced Tie2-phosphorylation in a similar manner than the parental antibodies. Crossmab showed very potent tumor growth inhibition in orthotopic (KPL-4) and in subcutaneous xenograft tumors (Colo205). In the orthotopic KPL-4 and the s.c. Colo205 xenograft we observed a strong inhibition of angiogenesis by ex vivo analysis. In the VEGF-induced cornea pocket assay Crossmab resulted in a complete shutdown of angiogenesis. We have generated a bispecific human IgG1 antibody blocking VEGF-A and Ang-2 function simultaneously. Our data indicate that the Ang-2-VEGF CrossMab mediates potent anti-tumoral, anti-metastatic and anti-angiogenic efficacy and represents a promising therapeutic agent for the therapy of cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3285. doi:10.1158/1538-7445.AM2011-3285

Variable heavy–variable light domain and Fab-arm CrossMabs with charged residue exchanges to enforce correct light chain assembly

Abstract Technologies for the production of bispecific antibodies need to overcome two major challenges. The first one is correct heavy chain assembly, which was solved by knobs-into-holes technology or charge interactions in the CH3 domains. The second challenge is correct light chain assembly. This can be solved by engineering the Fab-arm interfaces or applying the immunoglobulin domain crossover approach. There are three different crossovers possible, namely Fab-arm, constant domain and variable domain crossovers. The CrossMabCH1–CL exchange does not lead to the formation of unexpected side products, whereas the CrossMabFab and the CrossMabVH–VL formats result in the formation of typical side products. Thus, CrossMabCH1–CL was initially favored for therapeutic antibody development. Here, we report a novel improved CrossMab design principle making use of site-specific positional exchanges of charged amino acid pairs in the constant domain of these CrossMabs to enable the correct light chain assembly in the CrossMabVH–VL and improvements for the CrossMabFab design.

Abstract 4510: RG7787 - a novel de-immunized PE based fusion protein for therapy of mesothelin-positive solid tumors

Attaching toxic payloads to antibodies has recently been established as a breakthrough in cancer therapy. Most of the currently developed antibody drug conjugate programs represent targeted chemotherapy with microtubule polymerization inhibiting drugs and hence share resistance mechanisms and side effects with classical chemotherapeutics like Taxol. Inhibition of protein synthesis by Pseudomonas Exotoxin (PE) is a more powerful mode of action that interferes with all hallmarks of cancer cells, not just with proliferation. However, its clinical use has been limited by immunogenicity as shown for SS1P, a mesothelin targeted immunotoxin. We have developed a novel de-immunized PE fusion protein (RG7787) for treatment of mesothelin-positive tumors. In order to de-immunize PE, point mutations have been introduced into domain III and the entire domain II has been deleted reducing the size of the effector moiety to approximately 24 kD. PE24 fusions with a disulfide-stabilized Fv fragment have previously been shown to be much better tolerated in rodents, but this could, at least in part, also be attributed to a much reduced serum half-life and exposure. In order to also de-immunize the targeting moiety of RG7787 and restore PK parameters similar to those of SS1P, we substituted the mouse dsFv moiety by a humanized Fab fragment. We show that RG7787 has indeed similar PK properties to SS1P in mouse and cyno. Cell viability assays show that RG7787 has similar cytotoxic potency as SS1P on different cell lines. In-vivo equipotency to SS1P in different xenograft models has been achieved at ∼3 fold higher doses indicating that tumor penetration of SS1P might be slightly better. However, RG7787 is up to tenfold better tolerated in rodents and cynomolgus monkeys, indicating that the therapeutic window is improved. RG7787 achieves potent tumor growth inhibition and even tumor regressions in several xenograft models. We also observed clearly synergistic efficacy with Taxol treatment in different tumor models making this a promising combination for clinical trials. Citation Format: Gerhard Niederfellner, Frieder Bauss, Sabine Imhof-Jung, Friederike Hesse, Sven Kronenberg, Roland Staak, Martin Lechmann, Ben Krippendorff, Wolfgang Richter, Rita Mateus, Gwendlyn Kollmorgen, Ulli Brinkmann, Masanori Onda, Ira Pastan, Klaus Bosslet. RG7787 - a novel de-immunized PE based fusion protein for therapy of mesothelin-positive solid tumors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4510. doi:10.1158/1538-7445.AM2014-4510