Humanization of JAA-F11, a Highly Specific Anti-Thomsen-Friedenreich Pancarcinoma Antibody and In Vitro Efficacy Analysis

Abstract

JAA-F11 is a highly specific mouse monoclonal to the Thomsen-Friedenreich Antigen (TF-Ag) which is an alpha-O-linked disaccharide antigen on the surface of ~80% of human carcinomas, including breast, lung, colon, bladder, ovarian, and prostate cancers, and is cryptic on normal cells. JAA-F11 has potential, when humanized, for cancer immunotherapy for multiple cancer types. Humanization of JAA-F11, was performed utilizing complementarity determining regions grafting on a homology framework. The objective herein is to test the specificity, affinity and biology efficacy of the humanized JAA-F11 (hJAA-F11). Using a 609 target glycan array, 2 hJAA-F11 constructs were shown to have excellent chemical specificity, binding only to TF-Ag alpha-linked structures and not to TF-Ag beta-linked structures. The relative affinity of these hJAA-F11 constructs for TF-Ag was improved over the mouse antibody, while T20 scoring predicted low clinical immunogenicity. The hJAA-F11 constructs produced antibody-dependent cellular cytotoxicity in breast and lung tumor lines shown to express TF-Ag by flow cytometry. Internalization of hJAA-F11 into cancer cells was also shown using a surface binding ELISA and confirmed by immunofluorescence microscopy. Both the naked hJAA-F11 and a maytansine-conjugated antibody (hJAA-F11-DM1) suppressed in vivo tumor progression in a human breast cancer xenograft model in SCID mice. Together, our results support the conclusion that the humanized antibody to the TF-Ag has potential as an adjunct therapy, either directly or as part of an antibody drug conjugate, to treat breast cancer, including triple negative breast cancer which currently has no targeted therapy, as well as lung cancer.

Preclinical Analysis of JAA-F11, a Specific Anti–Thomsen-Friedenreich Antibody via Immunohistochemistry and In Vivo Imaging

“…The characterization of the hJAA-F11 H2aL2a has been detailed elsewhere [37] . Briefly, two vectors, encoding the heavy and light chains of H2aL2a, were transfected into the Chinese hamster ovary cell line CHO-K1 (ATCC, Manassas, VA).…”

“…Recently, the chemical specificity of humanized forms of JAA-F11 has been characterized. JAA-F11 was humanized to an IgG 1 in a manner which 1) retained the tumor-associated Galb1-3GalNAc alpha specificity, 2) added antibody-dependent cellular cytotoxicity activity, and 3) retained the ability to internalize for use as an antibody drug conjugate and added in vivo efficacy against a human triple-negative breast cancer xenograft [37] . These promising preclinical data are helping move JAA-F11 towards clinical trials, thus increasing the importance of these immunohistochemical (IHC) and imaging studies.…”

Glycan Microarrays as Chemical Tools for Identifying Glycan Recognition by Immune Proteins

“…As noted above, anti-glycan antibodies can be characterized on the array platforms, and many have been studied over the years on the CFG glycan array (Agrawal-Gamse et al, 2011;Zipser et al, 2012;Noble et al, 2013;Falkowska et al, 2014;Chua et al, 2015;Mickum et al, 2016;Tati et al, 2017;Nkurunungi et al, 2019), such as characterization of a new Lewis x antibody (Mandalasi et al, 2013) and multiple iterations of an anti-Tn antigen antibody (Chaturvedi et al, 2008;Tati et al, 2017). While antibody specificities are not the main focus of this review, it is clear from past work that the glycan microarrays have been and will continue to be very useful tools for their characterization, especially as the glycans and the types of linkers diversify.…”

Profiling of Naturally Occurring Antibodies to the Thomsen-Friedenreich Antigen in Health and Cancer: The Diversity and Clinical Potential

“…A general conclusion that can be made from the above findings is that TF Abs reveal cancer-specific changes in their level and glycosylation. Encouraging metastasis inhibition experiments by TF-specific MAbs clearly indicate that TF could be an important target for passive and active immunotherapies in TF-expressing tumors [35,40]. The glycodiversity of Abs is now a topic of interest because of a possibility to construct Ab glycoforms with the predicted potential [71,121,146,147], thus improving cancer immunotherapy potential.…”

“…Interestingly, up to date, there is no clear understanding of the pathophysiological role of natural TF Abs in health and disease though it is difficult to imagine that the rather high levels of these Abs of various isotypes would not have any important role for the host. Clinically observed prognostic improvements in cancer patients with a high level of TFspecific IgG Abs [17,[32][33][34] and encouraging experiments with TF-specific monoclonal antibodies (MAbs) [35,36] indicate that the TF-specific innate and/or adaptive immune response is an important part of cancer immunosurveillance, and the TF antigen is a promising molecular target for cancer immunotherapy [14,[37][38][39][40]. This short review focuses mostly on the clinical potential of natural TF-specific Abs in cancer diagnostics and prognosis.…”