Generation of a highly stable, internalizing anti‐CD22 single‐chain Fv fragment for targeting non‐Hodgkin's lymphoma

Abstract

The generation of a single chain Fv (scFv) fragment derived from the anti-CD22 monoclonal antibody LL2 resulted in a molecule with good antigen binding but very poor stability properties, thus hampering its clinical applicability. Here we report on the construction of an engineered LL2 scFv fragment by rational mutagenesis. The contribution of uncommon wild-type sequence residues for providing stability to the conserved common core structure of immunoglobulins was examined. Aided by computer homology modeling, 3 destabilizing residues within the core of the wild-type V H domain were identified. Owing to the conserved nature of the buried core structure, mutagenesis of these sites to respective consensus residues markedly stabilized the molecule but did not influence its antigen binding properties: the engineered scFv MJ-7 exhibited exceptional biophysical stability with a half-life not reached after 6 days of incubation in human serum at 37°C, while fully retaining the epitope specificity of the monoclonal antibody, and antigen binding affinity of the wild-type scFv. Furthermore, both the monoclonal antibody LL2 and the engineered scFv fragment became fully internalized after only 30 min of incubation at 37°C with CD22 ؉ tumor cells. These properties predict scFv MJ-7 could become a novel powerful tool to selectively deliver cytotoxic agents to malignant CD22 ؉ cells. NonHodgkin's lymphoma (NHL) accounts for about 5% of all newly diagnosed malignancies in the United States. Unlike other frequently occurring cancers only NHL and female lung cancer show continuously increasing mortality rates. 1,2 Less than 50% of patients with high-grade NHL eventually survive their disease and no curative therapeutic options for patients with low-grade NHL are currently available. 3 Moreover, patients in long-term remission face a high risk of developing long-term complications due to administered aggressive chemo-and radio-therapy regimens, which include secondary malignancies, cardiac dysfunction, endocrine dysfunctions, post-transfusion hepatitis and infertility. 4,5 Novel treatment concepts are therefore urgently needed. More than 80% of all NHLs are of B cell origin. The B cell lineage specific surface antigen CD22 is only present on a subset of normal mature B cells 6 but strongly expressed by the majority of malignant B cells in B-NHL. Thus CD22 represents an attractive target for immunotherapeutic approaches. The monoclonal anti-CD22 antibody LL2 7 recognizes the CD22 antigen with high specificity and is not cross-reactive with other tissues including hematopoetic progenitor cells. 7 LL2 is rapidly internalized into the cell upon binding to CD22. 8 To exploit this unique property, LL2 was employed as a targeting moiety to selectively deliver radionuclides, toxins or ribonucleases into CD22 ϩ tumor cells. 9 -11 Objective responses after administration of even diagnostic radiation doses of 131 I-labeled LL2 to patients with refractory B-NHL were achieved. 9,[12][13][14] . Recently, a humanized version of LL2 (Epratuzumab)...

Antigen binding and stability properties of non‐covalently linked anti‐CD22 single‐chain Fv dimers

“…The cDNA encoding the previously constructed anti-CD22 scFv MJ-7 [20] [20]. For cloning of scFv MLT-7 with variable domains directly joined in the opposite orientation (V L -0-V H ), the variable domain encoding genes were PCR amplified in separate reactions and assembled by overlap extension PCR as described in [22].…”

“…ScFv fragments were expressed using the E. coli strain TG1 (Stratagene, La Jolla, CA), isolated from the periplasm and purified by immobilized metal affinity chromatography (IMAC) as previously described [20]. Affinity purified scFv fragments were fractionated by size-exclusion chromatography using either a calibrated Superdex 75 HR 10/30 column (Amersham Pharmacia, Piscataway, NJ) or a Superdex 200 HR 10/30 column (Amersham Pharmacia) in phosphate buffered saline and 50 mM imidazole, pH 7.4, with a flow rate of 0.3 ml/min.…”

“…Staining was performed as previously described [20]. Stained cells were analyzed on a FACScan Flow Cytometer (BD Bioscience, San Jose, CA) and the median fluorescence intensity (MFI) was calculated using the CellQuest ä software (BD Bioscience).…”

“…Stained cells were analyzed on a FACScan Flow Cytometer (BD Bioscience, San Jose, CA) and the median fluorescence intensity (MFI) was calculated using the CellQuest ä software (BD Bioscience). Binding affinity constants (K D ) were measured as previously described [20,23].…”

“…We recently described the generation of the highly stable scFv fragment MJ-7 (V H -15-V L ) [20] that derived from the clinically established, internalizing anti-CD22 monoclonal antibody (mAb) LL2 [21]. This construct, obtained after rational mutagenesis of three destabilizing residues within the variable region from antibody heavy chain (V H ) domain core structure, retained similar antigen binding properties as the wild-type scFv but exhibited several orders of magnitude greater stability when incubated in human serum at 37°C.…”