Therapeutic efficacy of the humanized JAA-F11 anti-Thomsen-Friedenreich antibody constructs H2aL2a and H3L3 in human breast and lung cancer xenograft models.

The Thomsen-Friedenreich antigen (TF-Ag-α) is found on ~85% of human carcinomas but is cryptic on normal tissue. The humanized highly specific hJAA-F11-H2aL2a and -H3L3 antibodies target TF-Ag-α without binding to TF-Ag-beta (found on surface glycolipids of some normal cells). The relative affinity of H3L3 is 17 times that of H2aL2a, which would seem to favor superior efficacy, however, increased affinity can result in less tumor penetration. To assess the potential therapeutic efficacy of these antibodies, four human cancer- mouse xenograft models were treated with H2aL2a and H3L3. The tumor xenograft models used were human non-small cell lung cancer, H520, and small cell lung cancer, HTB171 in nude mice and human triple negative breast cancer, MDA-MB-231 and HCC1806 in SCID mice. H2aL2a significantly decreased tumor growth in both breast and both lung cancer models. H2aL2a showed statistically equal or better efficacy than H3L3 and has superior production capabilities. These results suggest that H2aL2a may be superior as a naked antibody, as an antibody drug conjugate or as a radiolabeled antibody, however the higher affinity of H3L3 may lead to better efficacy in bi-specific therapies in which the binding is decreased due to the presence of only one TF-Ag-α binding site.

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

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 studies with JAA-F11 anti-Thomsen-Friedenreich monoclonal antibody for human breast cancer.

AIM: The Thomsen-Friedenreich antigen (TF-Ag) is a disaccharide hidden on normal cells, but selectively exposed on the surface of breast, colon, prostate and bladder cancer cells. JAA-F11, a highly specific monoclonal antibody to TF-Ag, reduces metastasis and prolongs survival in a mouse model. In addition,(124)I-JAA-F11 localizes 4T1 tumors in mice. These studies continue translation of JAA-F11 to human breast cancer. MATERIALS & METHODS & RESULTS: Of the 41 human breast cancer cell lines tested, 78% were positive for reactivity with JAA-F11 by whole-cell enzyme immunoassay and positivity occurred unrelated to estrogen, progesterone or HER2 receptor status. JAA-F11 inhibited the growth rate of the human cancer cell lines tested. At 1 h, approximately 80% of JAA-F11 internalized in the three cell lines tested. (124)I-JAA-F11 specifically imaged human triple-negative tumors in mice by microPET. CONCLUSION: The results highlight the potential that humanized JAA-F11 may have for immunotherapy and drug conjugate therapy in breast cancer patients.

Design and synthesis of multifunctional gold nanoparticles bearing tumor-associated glycopeptide antigens as potential cancer vaccines.

The development of vaccines against specific types of cancers will offer new modalities for therapeutic intervention. Here, we describe the synthesis of a novel vaccine construction prepared from spherical gold nanoparticles of 3-5 nm core diameters. The particles were coated with both the tumor-associated glycopeptides antigens containing the cell-surface mucin MUC4 with Thomsen Friedenreich (TF) antigen attached at different sites and a 28-residue peptide from the complement derived protein C3d to act as a B-cell activating "molecular adjuvant". The synthesis entailed solid-phase glycopeptide synthesis, design of appropriate linkers, and attachment chemistry of the various molecules to the particles. Attachment to the gold surface was mediated by a novel thiol-containing 33 atom linker which was further modified to be included as a third "spacer" component in the synthesis of several three-component vaccine platforms. Groups of mice were vaccinated either with one of the nanoplatform constructs or with control particles without antigen coating. Evaluation of sera from the immunized animals in enzyme immunoassays (EIA) against each glycopeptide antigen showed a small but statistically significant immune response with production of both IgM and IgG isotypes. Vaccines with one carbohydrate antigen (B, C, and E) gave more robust responses than the one with two contiguous disaccharides (D), and vaccine E with a TF antigen attached to threonine at the 10th position of the peptide was selected for IgG over IgM suggesting isotype switching. The data suggested that this platform may be a viable delivery system for tumor-associated glycopeptide antigens.

Development, characterization, and immunotherapeutic use of peptide mimics of the Thomsen-Friedenreich carbohydrate antigen.

The tumor-associated carbohydrate Thomsen-Friedenreich antigen (TF-Ag; Galbeta1-3GalNAcalpha-O-Ser/Thr) is overexpressed on the cell surface of several types of tumor cells, contributing to cancer cell adhesion and metastasis to sites containing TF-Ag-binding lectins. A highly specific immunoglobulin G(3) monoclonal antibody (Ab) developed to TF-Ag (JAA-F11) impedes TF-Ag binding to vascular endothelium, blocking a primary metastatic step and providing a survival advantage. In addition, in patients, even low levels of antibodies to TF-Ag seem to improve prognosis; thus, it is expected that vaccines generating antibodies toward TF-Ag would be clinically valuable. Unfortunately, vaccinations with protein conjugates of carbohydrate tumor-associated Ags have induced clinically inadequate immune responses. However, immunization using peptides that mimic carbohydrate Ags such as Lewis has resulted in both Ab and T-cell responses. Here, we tested the hypothesis that vaccinations with unique TF-Ag peptide mimics may generate immune responses to TF-Ag epitopes on tumor cells, useful for active immunotherapy against relevant cancers. Peptide mimics of TF-Ag were selected by phage display biopanning using JAA-F11 and rabbit anti-TF-Ag Ab and were analyzed in vitro to confirm TF-Ag peptide mimicry. In vitro, TF-Ag peptide mimics bound to TF-Ag-specific peanut agglutinin and blocked TF-Ag-mediated rolling and stable adhesion of cancer cells to vascular endothelium. In vivo, the immunization with TF-Ag-mimicking multiple antigenic peptides induced TF-Ag-reactive Ab production. We propose that this novel active immunotherapy approach could decrease tumor burden in cancer patients by specifically targeting TF-Ag-positive cancer cells and blocking metastasis.

Inhibition of spontaneous breast cancer metastasis by anti-Thomsen-Friedenreich antigen monoclonal antibody JAA-F11.

Thomsen-Friedenreich antigen (TF-Ag) is expressed in many carcinomas, including those of the breast, colon, bladder, and prostate. TF-Ag is important in adhesion and metastasis and as a potential immunotherapy target. We hypothesized that passive transfer of JAA-F11, an anti-TF-Ag monoclonal antibody, may create a survival advantage for patients with TF-Ag-expressing tumors by cytotoxicity, blocking of tumor cell adhesion, and inhibition of metastasis. This was tested using in vitro models of tumor cell growth; cytotoxicity assays; in vitro, ex vivo, and in vivo models of cancer metastasis; and, finally, in vivo effects in mice with metastatic breast cancer. Unlike some anti-TF-Ag antibodies, JAA-F11 did not enhance breast carcinoma cell growth. JAA-F11 did not induce the killing of 4T1 tumor cells through complement-dependent cytotoxicity or apoptotic mechanisms. However, JAA-F11 blocked the stages of metastasis that involve the adhesion of human breast carcinoma cells to human endothelial cells (human umbilical vein endothelial cells and human bone marrow endothelial cells 60) in in vitro static adhesion models, in a perfused ex vivo model, and in murine lung vasculature in an in vivo metastatic deposit formation assay. JAA-F11 significantly extended the median survival time of animals bearing metastatic 4T1 breast tumors and caused a > 50% inhibition of lung metastasis.