Discovery of a novel human antibody VH domain with potent activity against mesothelin expressing cancer cells in both CAR T-cell and antibody drug conjugate formats

Abstract

Antibody based therapeutics targeting mesothelin (MSLN) have shown limited anti-tumor activities in clinical trials. Novel antibodies with high affinity and better therapeutic developability properties are needed as preclinical candidates. In the present study, we have isolated and characterized a novel VH domain 3C9 from a large size human immunoglobulin heavy chain variable (VH) domain library. 3C9 exhibited high affinity [KD (dissociation constant) < 3nM] and demonstrated good specificity in a membrane proteome array (MPA). Both CAR-T cells and antibody domain drug conjugations (DDCs) generated with 3C9, showed effective killing of MSLN positive cells in vitro without off-target effects. This is also the first report of the crystal structure of MSLN based on the analysis of the MSLN-3C9 complex which was solved at 2.9 A resolution. The newly identified antibody domain is a promising candidate therapeutic against cancer.

Assessment of Novel Mesothelin-Specific Human Antibody Domain VH-Fc Fusion Proteins-Based PET Agents

“…The resulting DDC demonstrated promising therapeutic responses in mice bearing MSLN-expressing tumors. 16 While the initial results were promising, signs of toxicity (weight loss) were observed at higher doses, highlighting the need to develop tools to assess and compare a variety of doses or alternative domain-based targeting agents. The development of anti-MSLN companion PET-agents in parallel to anti-MSLN therapies provides tools to identify tumors that will best respond to anti-MSLN therapy.…”

“…Low aggregation of both the 2A10 VH domain (3%) and VH-Fc protein fusion (<1%) was observed, which is consistent with the anti-MSLN 3C9 VH domain and VH-Fc fusion protein. 16 Conjugation and Radiolabeling of Anti-MSLN Antibody and VH-Fc Fusion Protein. The ratios of DFO chelators to VH-Fc fusion proteins are as follows: 1.37 ± 0.17 (2A10), 1.52 ± 0.18 (3C9), and 1.07 ± 0.40 (Ab6).…”

“…The addition of the anti-MSLN antibody, m912 IgG1, increased the EC 50 value to 70 nM, indicating that the 2A10 VH domain competes with the m912 IgG1 for binding (SI Figure 1B). However, the addition of the 3C9-VH-Fc 16 did not impact the binding of 2A10 VH, indicating that they target nonoverlapping epitopes (SI Figure 1C). SPR analysis demonstrated that the 2A10 VH domain and VH-Fc fusion protein had high affinities to human MSLN, with a K D of 2.4 ± 0.01 nmol and 2.0 ± 0.1 nmol (SI Figure 2), which were slightly lower than those of the 3C9 VH and VH-Fc fusion proteins as reported previously, 2.6 and 7.4 nmol, respectively.…”

“…Previously, we generated a large fully human VH domain phage-displayed library. − From this library we were able to isolate a variety of human immunoglobulin variable heavy chain (VH) domains that target SARS-CoV-2, CD22, and PD-L1 as well as MSLN. An anti-MSLN VH domain 3C9, modified as a VH-Fc fusion protein drug conjugate, showed promising therapeutic results in MSLN-expressing xenograft tumors . However, the current tools to assess and optimize novel targeting agents for anti-MSLN therapies are limited.…”

“…An anti-MSLN VH domain 3C9, modified as a VH-Fc fusion protein drug conjugate, showed promising therapeutic results in MSLN-expressing xenograft tumors. 18 However, the current tools to assess and optimize novel targeting agents for anti-MSLN therapies are limited. In the present study, we aim to utilize PET-imaging to assess and compare anti-MSLN targeting agents, including a VH-Fc fusion protein utilizing a newly identified MSLN specific human antibody domain, 2A10; our previously developed VH-Fc 3C9 fusion protein; and a fully intact IgG1 (m912; a clinical benchmark antibody with an overlapped epitope as 2A10).…”