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 x 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.

The CD44 protein family consists of isoforms with tissuespecific 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 (K d 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 ϳ cMAb BIWA-2. To evaluate their in vivo tumortargeting properties, 2 MAbs with identical murine or human isotype were labeled with either 131 I or 125 I and administered simultaneously (50 g/10 Ci 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 186 Re (300 or 400 Ci), the same MAb pairs showed RIT efficacy consistent with the biodistribution data: 186 The use of radiolabeled MAbs has been recognized as a realistic option for improvement of diagnosis and treatment of cancer. While in the last decade MAbs have been administered to thousands of patients with various types of tumor for both diagnostic and therapeutic purposes, the application of MAbs in head-andneck oncology has not kept pace. One of the main reasons for this slow progress has been the lack of MAbs with a high specificity for head-and-neck cancer, in particular for HNSCC, which accounts for approximately 90% of head-and-neck tumors.In recent years, a panel of MAbs has been developed that is capable of selective HNSCC targeting, as demonstrated in clinical radioimmunoscintigraphy/biodistribution studies with HNSCC patients. Among the best-qualified antibodies are the mMAbs U36 and BIWA-1 (formerly VFF18). 1,2 U36 and BIWA-1 bind to overlapping epitopes in the variable domain v6 of the cell-surface antigen CD44 and have been characterized extensively. 3,4 CD44 isoforms, which arise from differential splicing of up to 9 variant exons, show a tissue-specific expression pattern. Homogenous expression of v6-containing CD44 isofo...

Summary:anical, chemical or immunohistochemical procedures. 4 This results in a decrease of malignant cells with values from 3 up to more than 5 log, 4-6 but does not eradicate malignant CD34-positive cells were isolated from cord blood (n ‫؍‬ 8), bone marrow (n ‫؍‬ 4) and leukapheresed material cells in any case. A more recently applied method is enrichment and reinfusion of hematopoietic stem cells. In humans (n ‫؍‬ 7), using an immunomagnetic isolation technique, MACS (Miltenyi Biotec, Bergisch Gladbach, Germany).hematopoietic stem cells express CD34. Several approaches have been developed successfully to isolate CD34-positive In flow cytometric analysis, cell populations after enrichment revealed a fraction of 96.1% (cord blood), cells using immunoabsorbent, immunomagnetic or centrifugational methods. 7 Until now, on the clinical scale it is 96.2% (bone marrow) and 98.6% (leukapheresis material) CD34-positive cells. Cells were further stained hardly possible to enrich the desired cell population to homogeneity. This requirement should be met to omit the risk with antibodies specific for CD44 isoforms: CD44s (SFF-2), CD44v5 (VFF-8) and CD44v6 (VFF-18). CD44-of contaminating cells. Therefore, albeit using purging methods as well as stem cell enrichment strategies, the risk positive cells were detected by directly (FITC, fluorescein isothiocyanate) or indirectly (streptavidin-PE, of minimal residual disease in autologous transplantation still remains. for the generation of several splice variants. 8,10 Until now, 15 different spliced types of CD44 are known. To disKeywords: autologous bone marrow transplantation; CD44 standard receptor (CD44s); CD44 isoforms (CD44v); tinguish between the standard receptor phenotype and spliced phenotypes, the commonly expressed hyaluronic purging methods; hematopoietic stem cell; stem cell enrichment; magnetic cell separation acid receptor was termed CD44s, all splice variants were termed CD44v. 8,11 All splice variants are longer in size and bear additional extracellular protein domains. The generation of CD44 splice variants seems to be correlated with Autologous bone marrow transplantation is of increasing processes of activation, inflammation and tumor proimportance not only in the therapy of leukemia, but also in gression. [12][13][14][15] Possibly by analogy to its function in lymphothe therapy of solid tumors. The benefit of autologous bone cyte migration, CD44 variant molecules have been shown marrow transplantation is hampered by an elevated risk of to confer metastatic capability on rat tumor cell lines. 16relapse in the course of bone marrow reconstitution. This Homologues of these variants, especially CD44v5 und relapse may result from malignant cells, which contaminate CD44v6, have been detected in several human maligthe bone marrow to varying extents.

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