A cis-acting diversification activator both necessary and sufficient for AID-mediated hypermutation.

Hypermutation of the immunoglobulin (Ig) genes requires Activation Induced cytidine Deaminase (AID) and transcription, but it remains unclear why other transcribed genes of B cells do not mutate. We describe a reporter transgene crippled by hypermutation when inserted into or near the Ig light chain (IgL) locus of the DT40 B cell line yet stably expressed when inserted into other chromosomal positions. Step-wise deletions of the IgL locus revealed that a sequence extending for 9.8 kilobases downstream of the IgL transcription start site confers the hypermutation activity. This sequence, named DIVAC for diversification activator, efficiently activates hypermutation when inserted at non-Ig loci. The results significantly extend previously reported findings on AID-mediated gene diversification. They show by both deletion and insertion analyses that cis-acting sequences predispose neighboring transcription units to hypermutation.

Preclinical Characterization of a Novel Anti-Cancer PD-L1 Inhibitor RPH-120.

RPH-120 is a novel fully human anti-PD-L1 IgG1 monoclonal antibody with specifically designed Asn300Ala mutation in Fc fragment. Surface plasmon resonance assay showed that affinity of the RPH-120 to the dimeric form of human PD-L1-Fc fusion protein was much higher than affinity to the monomeric His-tagged PD-L1. Further binding studies demonstrated that RPH-120 is able to bind to human and monkey but not mouse PD-L1. Tissue cross-reactivity study showed good comparability of human and Cynomolgus monkeys tissue staining. Bioactivity was assessed using mixed lymphocyte reaction assay. This study revealed that RPH-120 was able to activate T cells preventing PD1/PD-L1 interaction. Antitumor efficacy was analyzed in HCC-827 lung cancer xenografts in humanized CD34+ mice at three dosage levels: 20, 80, and 200 mg/kg. RPH-120 demonstrated significant tumor growth inhibition, and this inhibition was comparable to that of atezolizumab. In a single dose toxicity, toxicokinetic and dose range finding study performed in Cynomolgus monkeys, RPH-120 was administered via intravenous (IV) bolus or 60-min IV infusion, followed by 8-weeks recovery period. An acceptable toxicokinetic profile was demonstrated and administration at doses of up to 200 mg/kg was well tolerated by all animals. In conclusion, RPH-120 revealed promising in vitro and in vivo activity and safety. RPH-120 is a potent anti-PD-L1 drug candidate for cancer immunotherapy.

Protein evolution by hypermutation and selection in the B cell line DT40.

Genome-wide mutations and selection within a population are the basis of natural evolution. A similar process occurs during antibody affinity maturation when immunoglobulin genes are hypermutated and only those B cells which express antibodies of improved antigen-binding specificity are expanded. Protein evolution might be simulated in cell culture, if transgene-specific hypermutation can be combined with the selection of cells carrying beneficial mutations. Here, we describe the optimization of a GFP transgene in the B cell line DT40 by hypermutation and iterative fluorescence activated cell sorting. Artificial evolution in DT40 offers unique advantages and may be easily adapted to other transgenes, if the selection for desirable mutations is feasible.

Understanding the immunoglobulin locus specificity of hypermutation.

The immunoglobulin (Ig) genes of B cells are diversified at high rate by point mutations whereas the non-Ig genes of B cells accumulate no or significantly fewer mutations. Ig hypermutations are critical for the affinity maturation of antibodies for most of jawed vertebrates and also contribute to the primary Ig diversity repertoire formation in some species. How the hypermutation activity is specifically targeted to the Ig loci is a long-standing debate. Here we describe a new experimental approach to investigate the locus specificity of Ig hypermutation using the chicken B-cell line DT40. One feature is the use of a green fluorescent protein (GFP) gene as a mutation reporter. Some nucleotide changes produced by somatic hypermutation can cripple the GFP gene which leads to a decrease or loss of the green fluorescence. Therefore such changes can be easily quantified by fluorescence-activated cell sorting (FACS). Another advantage of this approach is the targeted integration of the mutation reporter into a defined chromosomal position. This system allowed us to identify a 10 kb sequence within the Ig light chain (IgL) locus, which is both necessary and sufficient to activate hypermutation in the neighboring reporter gene. We have called this sequence Diversification Activator (DIVAC) and postulated that similar cis-acting sequences exist in the heavy and light chain Ig loci of all jawed vertebrate species. Our experimental system promises further insight into the molecular mechanism of Ig hypermutation. For example, it may be possible to identify smaller functional motifs within DIVAC and address the role of putative transacting binding factors by gene knock-outs.

Identification, genome mapping, and CTCF binding of potential insulators within the FXYD5-COX7A1 locus of human chromosome 19q13.12.

Identification of insulators is one of the most difficult problems in functional mapping of genomes. For this reason, up to now only a few insulators have been described. In this article we suggest an approach that allows direct isolation of insulators by a simple positive-negative selection based on blocking enhancer effects by insulators. The approach allows selection of fragments capable of blocking enhancers from mixtures of genomic fragments prepared from up to 1-Mb genomic regions. Using this approach, a 1-Mb human genome locus was analyzed and eight potential insulators were selected. Five of the eight sequences were positioned in intergenic regions and two were within introns. The genes of the alpha-polypeptide H+/K+ exchanging ATPase (ATP4A) and amyloid beta (A4) precursor-like protein 1 (APLP1) within the locus studied were found to be flanked by insulators on both sides. Both genes are characterized by distinct tissue-specific expression that differs from the tissue specificity of the surrounding genes. The data obtained are consistent with the conception that insulators subdivide genomic DNA into loop domains that comprise genes characterized by similar expression profiles. Using chromatin immunoprecipitation assay, we demonstrated also that at least six of the putative insulators revealed in this work could bind the CTCF transcription factor in vivo. We believe that the proposed approach could be a useful instrument for functional analysis of genomes.