The VH framework region 1 as a target of efficient mutagenesis for generating a variety of affinity-matured scFv mutants

In vitro affinity-maturation potentially generates antibody fragments with enhanced antigen-binding affinities that allow for developing more sensitive diagnostic systems and more effective therapeutic agents. Site-directed mutagenesis targeting “hot regions,” i.e., amino acid substitutions therein frequently increase the affinities, is desirable for straightforward discovery of valuable mutants. We here report two “designed” site-directed mutagenesis (A and B) targeted the N-terminal 1–10 positions of the VH framework region 1 that successfully improved an anti-cortisol single-chain Fv fragment (Ka, 3.6 × 108 M−1). Mutagenesis A substituted the amino acids at the position 1–3, 5–7, 9 and 10 with a limited set of substitutions to generate only 1,536 different members, while mutagenesis B inserted 1–6 random residues between the positions 6 and 7. Screening the resulting bacterial libraries as scFv-phage clones with a clonal array profiling system provided 21 genetically unique scFv mutants showing 17–31-fold increased affinity with > 109 M−1Ka values. Among the mutants selected from the library A and B, scFv mA#18 (with five-residue substitutions) and mB1-3#130 (with a single residue insertion) showed the greatest Ka value, 1.1 × 1010 M−1.

Exploration and Modulation of Antibody Fragment Biophysical Properties by Replacing the Framework Region Sequences

In order to increase the successful development of recombinant antibodies and fragments, it seems fundamental to enhance their expression and/or biophysical properties, such as the thermal, chemical, and pH stabilities. In this study, we employed a method bases on replacing the antibody framework region sequences, in order to promote more particularly single-chain Fragment variable (scFv) product quality. We provide evidence that mutations of the VH- C-C′ loop might significantly improve the prokaryote production of well-folded and functional fragments with a production yield multiplied by 27 times. Additional mutations are accountable for an increase in the thermal (+19.6 °C) and chemical (+1.9 M) stabilities have also been identified. Furthermore, the hereby-produced fragments have shown to remain stable at a pH of 2.0, which avoids molecule functional and structural impairments during the purification process. Lastly, this study provides relevant information to the understanding of the relationship between the antibodies amino acid sequences and their respective biophysical properties.

Molecular Biclonality: Analysis of Clonotypic IgH VDJ Reveals the Frequent Occurrence of a Second Clone in Multiple Myeloma.

Abstract 1813 Poster Board I-839 Malignant clones in multiple myeloma (MM) express unique clonotypic VDJ sequences associated with the clinical isotype. It has been widely assumed that MM patients have only one clone. However, our analysis reveals that a high frequency of MM patients have two clones. Analysis of complementary determining region 3 (CDR3) in bone marrow (BM) by DNA fragment analysis using florescent labeled consensus primers that bind to the framework region 3 and the joining region (FR3/JHc) or the framework region 3 and the constant region (FR3/CH1) related to the clinical isotype typically yields a single CDR3 peak, corresponding to the clonotypic VDJ gene segments. Signature sequences identified from CDR2 and CDR3 of clonotypic VDJ provides a molecular tool to monitor the malignant clone within a given patient. Study of CDR3 in BM and peripheral blood (PB) provides valuable information about the IgH VDJ repertoire and Ig expression profile, especially in cases where two clonal sequences are molecularly identified or when validation by single cell analysis did not show high frequency for a presumptive clonotypic IgH VDJ. Here, we analysed CDR3 expression profile in 23 MM patients. The results indicated a high incidence of MM having a second clone (6/23 patients, 26%). Reverse transcription-polymerase chain reaction (RT-PCR) of FR3/JHc and FR3/CH1 reveals a CDR3 peak representing the major MM clone in all 23 BM analysed. Among 23 patients, 6 (4 IgG, 2 IgA) exhibit a second monoclonal peak which can be detected both in PB and BM. Fluorescent in situ hybridization was unable to detect an increased incidence of IgH translocations within these 6 patients. Characterization of VDJ sequences indicated that the second clone mainly utilizes VH3 gene family (5/6), undergoes somatic hypermutation (5/6 at 2% cutoff) and does not derive from MM clone (6/6) as shown by different combinatorial and junctional diversity. Repertoire analysis determined by FR3/JHc amplification of genomic DNA excluded biallelic expression of the MM clone. In patient MM1, the second clone (μ) predominates in PB while the MM clone (γ) predominates in BM. In MM2, the second clone has a very short CDR3 (18 nt), predominates in blood compartment and has undergone isotype switching (μ, γ, á). In MM3, the M protein is of IgG type, consistent with transcripts in an FR3/Cγ RT-PCR peak in BM, but repertoire analysis of genomic DNA showed that the majority of B lineage cells in bone marrow and blood have a rearranged μ isotype. In MM4, having a homogeneous MM (á) clone, the second clone (á) exhibited intraclonal diversity, as defined by sequencing of VDJ subclones. In MM5, clonal transcripts of both dominant γ clone and the second clone (á) persisted in blood following initial chemotherapy. In MM6, the MM clone (γ) predominates over the second clone (á) both in BM and PB. Overall, our studies indicate a high incidence of biclonal expansion in MM. The unrelated VDJ origin of sister clones detected in MM patients exhibiting biclonality suggests that they may have arisen from different transformation event, which may be promoted when cumulative genetic abnormalities reach a critical threshold. Although clinical manifestations in a patient may reflect a take-over by the most aggressive clone, our studies suggest that transformation may be an ongoing event that occurs at a heretofore underestimated rate in MM B lineage cells. Disclosures No relevant conflicts of interest to declare.