Marlies Sproll

Characterization of a high-affinity monoclonal antibody specific for CD44v6 as candidate for immunotherapy of squamous cell carcinomas.

Abstract Variant isoforms of CD44, a family of cell-surface glycoproteins generated by alternative splicing and post-translational modifications, are expressed in a variety of human tumors and play important roles in tumor progression and metastasis formation. The murine monoclonal IgG1 antibody VFF18, specific for an epitope encoded by human CD44 variant exon 6, binds with high affinity to the recombinant protein (Kd = 1.7×10–10 M) as well as to tumor cell lines in vitro, and is suitable for immunohistochemical analysis of human tumors. Screening of more than 500 tumor samples of different histogenesis showed that VFF18 most strongly and uniformly reacts with squamous cell carcinomas (SCC). Detailed analysis of 185 SCC (head and neck, lung, skin) confirmed reactivity of the antibody with 99% of the samples, with intense and homogeneous staining of the tumor cells in the majority of cases, whereas reactivity of VFF18 with normal tissues is limited to certain epithelia and activated lymphocytes. When radiolabelled VFF18 was administered to nude mice bearing human epidermoid carcinoma (A-431) xenograft, fast and selective tumor uptake of the radioimmunoconjugate with a maximum of 18% of the injected dose per gram of tissue was observed. Taken together, these data suggest that mAb VFF18 is a promising targeting vehicle for radioimmunotherapy of squamous cell carcinomas in humans.

Tumor targeting properties of monoclonal antibodies with different affinity for target antigen CD44V6 in nude mice bearing head-and-neck cancer xenografts.

The CD44 protein family consists of isoforms with tissue‐specific expression, which are encoded by standard exons and up to 9 alternatively spliced variant exons (v2–v10) of the same gene. The murine MAbs U36 and BIWA‐1, directed against overlapping epitopes within the v6 region of CD44, have previously been shown to efficiently target HNSCC. We herein report on the construction of 1 chimeric (BIWA‐2) and 2 humanized (BIWA‐4 and BIWA‐8) derivatives of BIWA‐1. Together with U36 and BIWA‐1, these new antibodies were evaluated for affinity to the antigen in vitro as well as for biodistribution and efficacy in RIT using nude mice bearing the HNSCC xenograft line HNX‐OE. As determined by surface plasmon resonance, the MAbs bound to CD44v6 with an up to 46‐fold difference in affinity (Kd ranging from 1.1 × 10−8 to 2.4 × 10−10 M) with the following ranking: mMAb U36 < hMAb BIWA‐4 < hMAb BIWA‐8 < mMAb BIWA‐1 ∼ cMAb BIWA‐2. To evaluate their in vivo tumor‐targeting properties, 2 MAbs with identical murine or human isotype were labeled with either 131I or 125I and administered simultaneously (50 μg/10 μCi each) as pairs showing a stepwise decrease in the difference in affinity: U36 vs. BIWA‐1 (35.0‐fold difference), BIWA‐4 vs. BIWA‐2 (14.0‐fold) and BIWA‐4 vs. BIWA‐8 (4.0‐fold). Biodistribution was assessed at 1, 2, 3 or 4 and 7 days after injection. Remarkably, for all 3 MAb pairs tested, the lower‐affinity MAb showed a higher degree and specificity of tumor localization. The difference in tumor localization was more pronounced when the difference in affinity was larger. For example, 3 days after injection, the lower‐affinity mMAb U36 showed a 50% higher tumor uptake than the higher‐affinity mMAb BIWA‐1, while blood levels and uptake in organs were similar. After labeling with 186Re (300 or 400 μCi), the same MAb pairs showed RIT efficacy consistent with the biodistribution data: 186Re‐U36 was more effective than 186Re‐BIWA‐1, 186Re‐BIWA‐4 was slightly more effective than 186Re‐BIWA‐2 and 186Re‐BIWA‐4 and 186Re‐BIWA‐8 demonstrated similar efficacy. Based on these data, we conclude that antibodies with markedly lower affinity to a given target antigen (e.g., U36, BIWA‐4) may show superior tumor targeting in comparison with higher‐affinity versions of these antibodies.