An IgG-Based Bispecific Antibody for Improved Dual Targeting in PSMA-Positive Cancer

Authors

Latifa Zekri^1,2,3, Fabian Vogt¹, Lukas Osburg¹, Stefanie Müller^2,3, Joseph Kauer^1,2,3, Timo Manz¹, Martin Pflügler^1,2,3, Andreas Maurer⁴, Jonas S Heitmann^2,3, Ilona Hagelstein^2,3, Melanie Märklin^2,3, Sebastian Hörner¹, Tilmann Todenhöfer⁵, Carsten Calaminus⁴, Arnulf Stenzl⁵, Bernd Pichler^3,4, Christian Fougère^3,6, Marc A Schneider⁷, Hans-Georg Rammensee^1,3, Lars Zender^3,8, Bence Sipos^3,8,9, Helmut R Salih^2,3 and Gundram Jung^1,3

Organizations

1. Department of Immunology, Institute for Cell Biology, Eberhard Karls University Tuebingen, German Cancer Consortium (DKTK), Partner Site Tuebingen, Tuebingen, Germany
2. Clinical Collaboration Unit Translational Immunology, German Cancer Consortium (DKTK), Department of Internal Medicine, University Hospital Tuebingen, Tuebingen, Germany
3. DFG Cluster of Excellence 2180 “Image-guided and Functional Instructed Tumor Therapy” (IFIT), Eberhard Karls University Tuebingen, Tuebingen, Germany
4. Department for Preclinical Imaging and Radiopharmacy, Werner Siemens Imaging Center, Eberhard Karls University Tuebingen, Tuebingen, Germany
5. Department of Urology, University Hospital Tuebingen, Tuebingen, Germany
6. Department of Nuclear Medicine and Clinical Molecular Imaging, Eberhard Karls University Tuebingen, German Cancer Research Center (DKFZ), Partner Site Tuebingen, Tuebingen, Germany
7. Translational Research Unit, Thorax Clinic at University Hospital Heidelberg, Translational Lung Research Center (TLRC) Heidelberg, Member of the German Center for Lung Research (DZL), Heidelberg, Germany
8. Department of Internal Medicine VIII, University Hospital Tuebingen, Tuebingen, Germany
9. Department of Pathology and Neuropathology, University Hospital Tuebingen, Tuebingen, Germany

Abstract

The prostate-specific membrane antigen (PSMA) has been demonstrated in numerous studies to be expressed specifically on prostate carcinoma cells and on the neovasculature of several other cancer entities. However, the simultaneous expression of PSMA on both, tumor cells as well as tumor vessels remains unclear, even if such “dual” expression would constitute an important asset to facilitate sufficient influx of effector cells to a given tumor site. We report here on the generation of a PSMA antibody, termed 10B3, which exerts superior dual reactivity on sections of prostate carcinoma and squamous cell carcinoma of the lung. 10B3 was used for the construction of T-cell recruiting bispecific PSMAxCD3 antibodies in Fab- and IgG-based formats, designated Fabsc and IgGsc, respectively. In vitro, both molecules exhibited comparable activity. In contrast, only the larger IgGsc molecule induced complete and durable elimination of established tumors in humanized mice due to favorable pharmacokinetic properties. Upon treatment of three patients with metastasized prostate carcinoma with the IgGsc reagent, marked activation of T cells and rapid reduction of elevated PSA levels were observed.

Insufficient penetration of immune cells and therapeutic antibodies into the tumor core is a major limitation in the immunotherapy field. This study reports the development of a novel bispecific antibody, named CC-1, for improved dual targeting of tumor- and vascular cells in PSMA positive tumors.

• A novel PSMA antibody (10B3) exhibiting enhanced reactivity with tumor- and vascular cells in samples from prostate carcinoma and squamous cell carcinoma of the lung was generated.
• Two different bispecific antibodies comprising 10B3 and anti-CD3 single chain in a Fabsc- and IgGsc-format were constructed and characterized.
• In vivo application of both bispecific antibodies revealed that only the IgGsc-molecule localized at a given tumor site, resulting in effective tumor cell destruction.
• A first-in-man application of the IgGsc-molecule, designated CC-1, in three patients with metastasized prostate carcinoma, demonstrated profound T cell activation and a rapid decline of elevated PSA levels.
• A first-in-man clinical study in patients with prostate carcinoma is currently ongoing (NCT04104607).

CovalX Technology Used

XL Kits
MALDI-ToF
Crosslinking Mass Spectrometry (XL-MS)

Outcomes

In this research, the prostate-specific membrane antigen (PSMA) antibody, named 10B3, was generated due to its ability for dual targeting of tumor cells and associated tumor vessels in the sections of prostate carcinoma and lung squamous cell carcinoma (SSC). 10B3 has superior performance compared to J591 antibody may be because 10B3 contains an unusual conformational epitope. To understand the structure of 10B3 antibody, CovalX’s crosslinking mass spectrometry (XL-MS) technology was utilized. A specialized crosslinking mixture (CovalX Crosslinking Kits) was created to stabilize non-covalent conjugates between recombinant PSMA and purified 10B3 antibody. The mixture was analyzed using High Mass MALDI-TOF Mass Spectrometry including CovalX’s HM4 high-mass detection system. Results revealed that 10B3 antibody is involved in conjugate formation with PSMA molecule via four stretches of amino acids from PSMA.

Source