In vitro evolution of antibody affinity via insertional scanning mutagenesis of an entire antibody variable region

We report a systematic combinatorial exploration of affinity enhancement of antibodies by insertions and deletions (InDels). Transposon-based introduction of InDels via the method TRIAD (transposition-based random insertion and deletion mutagenesis) was used to generate large libraries with random in-frame InDels across the entire single-chain variable fragment gene that were further recombined and screened by ribosome display. Knowledge of potential insertion points from TRIAD libraries formed the basis of exploration of length and sequence diversity of novel insertions by insertional-scanning mutagenesis (InScaM). An overall 256-fold affinity improvement of an anti–IL-13 antibody BAK1 as a result of InDel mutagenesis and combination with known point mutations validates this approach, and suggests that the results of this InDel mutagenesis and conventional exploration of point mutations can synergize to generate antibodies with higher affinity.

Powerful selection technologies have made in vitro evolution of protein binders more efficient and paved the way for the use of tailor-made antibodies in therapy. After initial selections of antibody candidates with desired specificity, lead antibodies are typically improved by affinity maturation in multiple rounds of randomization and selection (1) to reach the subnanomolar affinities ideally required for targeting soluble ligands (2–4). This is usually attempted by introduction of point substitutions, either at random positions across the entire V-gene (5, 6) or in the complementary-determining regions (CDRs; e.g., by CDR walking mutagenesis) (7).In Nature, diversification of the primary antibody repertoire occurs by several mechanisms that generate variation in the regions forming the antigen-binding site, the CDRs, including considerable length variation (8–11) that is initially introduced by recombination of V(D)J gene segments. Length variations are concentrated in the CDR3 region (12), at the junctions of the joined segments, where additional diversity is produced by N- or P-nucleotide additions that can further extend the CDR3. The length of the CDRs considerably affects the topography of the combining site, as different shapes brought about by extension or shortening can form pockets, grooves, or fill space (13, 14).Following B cell stimulation by the antigen, further diversification of the antigen-binding interface is generated through somatic hypermutation (SHM) (15), involving mainly point mutagenesis that preferentially targets hotspots in the CDRs (16, 17). This process is initiated through deamination of cytosine to uracil by activation-induced cytidine deaminase (AID), leading to uracil:guanine mismatches (16). Upon removal of these uracil bases by base excision-repair enzymes, error-prone DNA polymerases are then recruited to fill in the gaps and introduce mutations around the position of the deaminated cytosines. Interestingly, up to 6% of the mutations generated by SHM are insertions and deletions (InDels) (18), which occur due to misalignment of repeated DNA sequences (19, 20). Thus, insertions occur by duplication, while deletions are brought about by removal of repeated sequences (21, 22).A small percentage of antibodies selected by in vivo SHM contain InDels in the CDRs 1 and 2 (1.6 to 6.5%) (21–24), while junctional diversity by N- or P-nucleotide additions in the CDR3 confounds the analysis of SHM-derived InDels, leading to an underestimation of the total percentage of affinity-improving InDels. In vitro-directed evolution has been unsuitable for introduction of InDels at random positions into an antibody gene, because of restrictions in the diversity of InDels that could be introduced (i.e., insertions by duplication in in vitro SHM) (22, 25). Rational (26) or computational (27) strategies have been successful at introducing InDels in a few, carefully chosen positions instead of random sampling. In contrast, an unusually high percentage of InDels with a functional role among in vivo affinity matured broadly neutralizing antibodies (bnAbs) to HIV-1 (28–30): ∼40% of the reported anti–HIV-1 bnAbs contain InDels that accumulate during in vivo SHM (28). Based on the frequent occurrence of InDels among multispecific, cross-reactive antibodies, one could infer that they provide a molecular solution for recognizing multiple targets by providing an altered interface (enlarged or tightened), possibly even involving conformational diversity (31). The accumulation of InDels in bnAbs has been attributed to extensive in vivo SHM, so that even positions that are rarely modified by SHM are also altered (17, 28).Insertions in the V-genes occur only by duplication of adjacent sequences (21, 22), so that the actual sequence diversity of the resulting insertions is limited because they repeat existing modules. To introduce more diversity in the inserted sequences, point mutations are required in subsequent rounds of SHM. However, since the CDRs can tolerate considerable length variation, it is likely that the antibody fold can accommodate a larger number of affinity-enhancing InDels compared to those observed in antibodies affinity-matured by SHM.Affinity gains by introduction of InDels have indeed been recognized (22, 25, 26, 32, 33) in in vitro-directed evolution, but often were by-products of campaigns focused on point mutations and not elicited systematically (32, 33). Only in mammalian cell surface display does the action of AID lead to InDels, just as AID brings about InDels in SHM in vivo (22, 25). In a seminal study by Bowers et al. (22), overexpression of AID enabled in vitro SHM of 53 antibodies against 21 antigens to identify InDels in multiple regions likely to improve binding, in particular to variable heavy domain (V_H) and variable light domain (V_L) CDR1, where 9 of 53 antibodies contained InDels. Despite the comprehensive nature of this study, AID-enabled insertions mirrored in vivo SHM and were therefore limited to direct duplication of adjacent sequences, not allowing the full exploration of length and sequence diversity in the insertions, and the low frequency of incorporation of in-frame InDels by AID (<0.1%) limited the combinatorial diversity explored. Finally, InDels have been introduced rationally based on structural analysis and natural length variation (26, 27). Taken together, only limited diversity of InDels in terms of length, position, and insert sequence across the variable domains has been explored thus far.Here we address this omission and explore libraries with in-frame InDels of different lengths and high diversity of inserted sequences at random positions across the entire antibody variable regions (Fig. 1). We applied a new transposon-based mutagenesis approach, dubbed TRIAD (transposition-based random insertion and deletion mutagenesis) (34) that introduces short in-frame insertions and deletions randomly across a gene (in sequences of steps following transposition that excise the transposon, religate the plasmid, and insert designed cassettes) (SI Appendix, Figs. S1 and S2). TRIAD was used here to build libraries with InDels at random positions across an entire single-chain variable fragment (scFv) gene. The antibody chosen for this campaign was the anti–IL-13 antibody BAK1 (35), a derivative of which, tralokinumab, is under clinical investigation for asthma (36). In addition, we built libraries that explore diversity in the different lengths of insertions in a semirandom approach, insertional-scanning mutagenesis (InScaM). These InDel libraries were starting points for antibody affinity evolution in vitro, leading to insertions in two loops that, together with two previously known point mutations, brought about a 256-fold affinity improvement. The observation of alternative routes to affinity maturation validate our strategy and suggest that InDel mutagenesis can complement existing approaches.Open in a separate windowFig. 1.Overview of the affinity maturation of the antibody BAK1 by transposon-based TRIAD and subsequent insertional scanning mutagenesis. TRIAD (Left) was applied to make libraries with deletions of one to three amino acids (step 1a) or single amino acid insertions (step 1b) at random positions across the scFv gene. These libraries were recombined (step 2) and four rounds of ribosome display selections for improved affinity to IL-13 were carried out by panning (step 3). The best binder was carrying an insertion in the V_L FWR3 (BAK1-INS1). Scanning (Right) was used to guide the design of libraries with different lengths of insertions at targeted positions. A fraction of the insertion library generated in step 1b (5,632 variants) was screened by HTRF to identify variants with insertions that retained binding to IL-13 (step 4). Based on sequencing analysis, regions able to tolerate single amino acid insertions were identified (Fig. 4) and the V_L CDR3 was chosen for targeted insertional mutagenesis. Libraries with zero to five amino acid insertions in targeted positions in the V_L CDR3 were constructed (step 5), followed by four rounds of phage display selections for improved affinity to IL-13 (step 6).     

Intermittent therapy with high-dose 5-aminosalicylic acid enemas maintains remission in ulcerative proctitis and proctosigmoiditis

PURPOSE: The aim of this study was to compare the efficacy of intermittent therapy with mesalazine enemas and continuous oral mesalazine to maintain remission of distal ulcerative colitis or proctitis. METHODS: Thirt-yeight patients with distal ulcerative colitis (n=17) or ulcerative proctitis (n=21) in clinical, endoscopic, and histologic remission were randomly assigned to receive either oral mesalazine (0.5 g three times/day, Eudragit L coating, n=19) or intermittent therapy with mesalazine enemas (4 g of 5-aminosalicylic acid enema every third night, n=19). Both groups were comparable in regard to sex, age, age at disease onset, extent and duration of disease, number and mode of treatment of previous attacks, and time in remission. Patients were reviewed at the beginning of the study and, subsequently, at two-month intervals for 24 months or until a relapse occurred. At each visit, diaries were reviewed and clinical and laboratory assessments were performed. Sigmoidoscopy was carried out and biopsies were obtained by a blinded observer. Histology was assessed without knowledge of the patient's clinical state or treatment category. RESULTS: At the end of the study, 6 of 19 patients on oral mesalazine (32 percent) and 14 of 19 patients on mesalazine enemas (74 percent) were still in full remission (log rank test: 15.280,P <0.001). Differences in relapse rates between groups were significant even when data were stratified by extent of disease (P <0.01). In the oral group, six and seven patients relapsed at 12 and 24 months, respectively. In the enema group, three and two relapses occurred in the first and second year of the study, respectively. All patients complied with the treatment satisfactorily and there were no dropouts. CONCLUSION: These results suggest that intermittent therapy with mesalazine enemas is more effective than continuous oral mesalazine in maintaining remission in patients with distal ulcerative colitis and proctitis.Read at the meeting of the First United European Gastroenterology Week, Athens, Greece, September 25 to 30, 1992.     

Effects of IVIg treatment on autoantibody testing in neurological patients: marked reduction in sensitivity but reliable specificity

Journal of Neurology - Therapy of autoimmune diseases of the central and peripheral nervous system with intravenous IgG immunoglobulin (IVIg) is well established. Since IVIg is produced from pooled...     

Physical activity and sedentary behavior thresholds for identifying childhood hypertension and its phenotypes: The Healthy Growth Study

Hypertension phenotypes may represent differential pathophysiologic mechanisms and clinical impact, yet they have been poorly investigated. The study aimed to examine the associations of physical activity and sedentary behavior with hypertension phenotypes in a large group of Greek children and to identify thresholds regarding risk of hypertension. This was a cross-sectional study with a regionally representative sample of 2473 schoolchildren aged 9–13 years, with full data on physical activity and sedentary behavior indices, as well as arterial blood pressure measurements, physical examination, and anthropometry. Hypertensive children of both sexes had lower levels of physical activity (steps/d). Hypertensive girls had lower moderate-to-vigorous physical activity (MVPA), whereas hypertensive boys with isolated systolic hypertension (ISH) had more screen time than their normotensive counterparts. Increased levels of physical activity was associated with 33%–54% lower risk of all hypertension phenotypes in both sexes, whereas increased MVPA was associated with 41%–65% lower risk of all phenotypes in girls and with ISH and systolic and diastolic hypertension (SDH) in boys. In boys, higher sedentary time was associated with 11%–13% higher risk for SDH and ISH. Cutoff points of 12,378 steps/d, 47.3 min/d of MVPA, and 2.9 h/d of sedentary behavior were determined for identifying children at increased risk of hypertension. Physical activity is inversely associated with all hypertension phenotypes, whereas sedentary behavior is positively associated with ISH and SDH in boys. More studies should confirm the hypertension-specific cutoff values identified to be used in future prevention programs for childhood hypertension.     

The ARVD/C Genetic Variants Database: 2014 Update

Arrhythmogenic cardiomyopathy (ACM) is an inherited cardiac disease characterized by myocardial atrophy, fibro‐fatty replacement, and a high risk of ventricular arrhythmias that lead to sudden death. In 2009, genetic data from 57 publications were collected in the arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C) Genetic Variants Database (freeware available at http://www.arvcdatabase.info ), which comprised 481 variants in eight ACM‐associated genes. In recent years, deep genetic sequencing has increased our knowledge of the genetics of ACM, revealing a large spectrum of nucleotide variations for which pathogenicity needs to be assessed. As of April 20, 2014, we have updated the ARVD/C database into the ARVD/C database to contain more than 1,400 variants in 12 ACM‐related genes (PKP2, DSP, DSC2, DSG2, JUP, TGFB3, TMEM43, LMNA, DES, TTN, PLN, CTNNA3) as reported in more than 160 references. Of these, only 411 nucleotide variants have been reported as pathogenic, whereas the significance of the other approximately 1,000 variants is still unknown. This comprehensive collection of ACM genetic data represents a valuable source of information on the spectrum of ACM‐associated genes and aims to facilitate the interpretation of genetic data and genetic counseling.     

Solid extraintestinal malignancies in patients with inflammatory bowel disease

Malignancies constitute the second cause of death in patients with inflammatory bowel diseases (IBD), after cardiovascular diseases. Although it has been postulated that IBD patients are at greater risk of colorectal cancer compared to the general population, lately there has been evidence supporting that this risk is diminishing over time as a result of better surveillance, while the incidence of extraintestinal cancers (EICs) is increasing. This could be attributed either to systemic inflammation caused by IBD or to long-lasting immunosuppression due to IBD treatments. It seems that the overall risk of EICs is higher for Crohn’s disease patients and it is mainly driven by skin cancers, and liver-biliary cancers in patients with IBD and primary sclerosing cholangitis. The aims of this review were first to evaluate the prevalence, characteristics, and risk factors of EICs in patients with IBD and second to raise awareness regarding a proper surveillance program resulting in early diagnosis, better prognosis and survival, especially in the era of new IBD treatments that are on the way.