Development of an antibody-like T-cell engager based on VH-VL heterodimer formation and its application in cancer therapy.

Following the clinical success of immunotherapeutic antibodies, bispecific antibodies for cytotoxic effector cell redirection, tumor-targeted immunomodulation and dual immunomodulation, have received particular attentions. Here, we developed a novel bispecific antibody platform, termed Antibody-Like Cell Engager (ALiCE), wherein the Fc domain of each heavy chain of immunoglobulin G (IgG) is replaced by the VH and VL domains of an IgG specific to a second antigen while retaining the N-terminal Fab of the parent antibody. Because of specific interactions between the substituted VH and VL domains, the C-terminal stem Fv enables ALiCE to assemble autonomously into hetero-tetramers, thus simultaneously binding to two distinct antigens but with different avidities. This design strategy was used to generate ACE-05 (two anti-PD-L1 Fab × anti-CD3 Fv) and ACE-31 (two anti-CD3 Fab × anti-PD-L1 Fv), both of which bound PD-L1 and CD3. However, ACE-05 was more effective than ACE-31 in reducing off-target toxicity caused by the indiscriminate activation of T cells. Moreover, in cell-based assays and PBMC-reconstituted humanized mice harboring human non-small-cell lung cancer tumors, ACE-05 showed marked antitumor efficacy, causing complete tumor regression at a dose of 0.05 mg/kg body weight. The dual roles of ACE-05 in immune checkpoint inhibition and T-cell redirection, coupled with reduced off-target toxicity, suggest that ACE-05 may be a promising anti-cancer therapeutic agent. Moreover, the bispecific ALiCE platform can be further used for tumor-targeted or multiple immunomodulation applications.

Leeia aquatica sp. nov., isolated from freshwater.

A Gram-stain-negative, rod-shaped, obligately aerobic, motile by a single polar flagellum, chemoheterotrophic bacterium, designated strain IMCC25680T, was isolated from surface water in Chungju Lake, Republic of Korea. 16S rRNA gene sequence analysis revealed that strain IMCC25680T was most closely related to Leeia oryzae HW7T with 95.5% sequence similarity and formed a robust clade with L. oryzae HW7T. Whole genome sequencing showed that strain IMCC25680T had a genome 3.6 Mbp long with 60.7 mol% DNA G+C content. Average nucleotide identity and digital DNA-DNA hybridization values between strain IMCC25680T and L. oryzae HW7T were 72.4% and 18.5%, respectively, indicating that the novel strain represents a novel species of the genus Leeia. The major cellular fatty acids of strain IMCC25680T were iso-C16:0 and summed feature 3 (comprising C16:1 ω6c and/or C16:1 ω7c). The respiratory quinone detected in the strain was ubiquinone-8. The major polar lipids were found to be phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol and two unidentified polar lipids. On the basis of the phylogenetic and phenotypic characterization, strain IMCC25680T was considered to represent a novel species within the genus Leeia, for which the name Leeia aquatica sp. nov. is proposed. The type strain is IMCC25680T (=KACC 19487T =NBRC 113132T).

Ferrimonas sediminicola sp. nov. and Ferrimonas aestuarii sp. nov., Fe(III)-reducing bacteria isolated from marine environments.

Two Gram-stain-negative, Fe(III)-reducing, facultatively anaerobic, motile via a single polar flagellum, rod-shaped bacterial strains, designated IMCC35001T and IMCC35002T, were isolated from tidal flat sediment and seawater, respectively. Results of 16S rRNA gene sequence analysis showed that IMCC35001T and IMCC35002T shared 96.6 % sequence similarity and were most closely related to Ferrimonas futtsuensis FUT3661T (98.6 %) and Ferrimonas kyonanensis Asr22-7T (96.8 %), respectively. Draft genome sequences of IMCC35001T and IMCC35002T revealed 4.0 and 4.8 Mbp of genome size with 61.0 and 51.8 mol% of DNA G+C content, respectively. Average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between the two strains were 73.1 and 19.8 %, respectively, indicating that they are separate species. The two genomes showed ≤84.4 % ANI and ≤27.8 % dDDH to other species of the genus Ferrimonas, suggesting that the two strains each represent novel species. The two strains contained both menaquinone (MK-7) and ubiquinones (Q-7 and Q-8). Major fatty acids of strain IMCC35001T were iso-C15 : 0, C18 : 1 ω9c, C17 : 1 ω8c and C16 : 0 and those of strain IMCC35002 T were C18 : 1 ω9c, C16 : 0 and summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c). Major polar lipids in both strains were phosphatidylethanolamine, phosphatidylglycerol, unidentified phospholipid, unidentified aminophospholipid and unidentified lipids. The two strains reduced Fe(III) citrate, Fe(III) oxyhydroxide, Mn(IV) oxide and sodium selenate but did not reduce sodium sulfate. They were also differentiated by several phenotypic characteristics. Based on the polyphasic taxonomic data, IMCC35001T and IMCC35002T were considered to represent each novel species in the genus Ferrimonas, for which the names Ferrimonas sediminicola sp. nov. (IMCC35001T=KACC 21161T=NBRC 113699T) and Ferrimonas aestuarii (IMCC35002T=KACC 21162T=NBRC 113700T) sp. nov. are proposed.

Halioglobus maricola sp. nov., isolated from coastal seawater.

A Gram-stain-negative, rod-shaped, aerobic, non-flagellated, chemoheterotrophic bacterium, designated IMCC14385T, was isolated from surface seawater of the East Sea, Republic of Korea. The 16S rRNA gene sequence analysis indicated that IMCC14385T represented a member of the genus Halioglobus sharing 94.6-97.8 % similarities with species of the genus. Whole-genome sequencing of IMCC14385T revealed a genome size of 4.3 Mbp and DNA G+C content of 56.7 mol%. The genome of IMCC14385T shared an average nucleotide identity of 76.6 % and digital DNA-DNA hybridization value of 21.6 % with the genome of Halioglobus japonicus KCTC 23429T. The genome encoded the complete poly-ß-hydroxybutyrate biosynthesis pathway. The strain contained summed feature 8 (C18 : 1 ω7c and/or C18 : 1 ω6c), summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c) and C17 : 1 ω8c as the predominant cellular fatty acids as well as ubiquinone-8 (Q-8) as the respiratory quinone. The polar lipids detected in the strain were phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, five unidentified phospholipids, an unidentified aminolipid, an unidentified aminophospholipid and four unidentified lipids. On the basis of taxonomic data obtained in this study, it is suggested that IMCC14385T represents a novel species of the genus Halioglobus, for which the name Halioglobus maricola sp. nov. is proposed. The type strain is IMCC14385T (=KCTC 72520T=NBRC 114072T).

Isolation, cultivation, and genome analysis of proteorhodopsin-containing SAR116-clade strain Candidatus Puniceispirillum marinum IMCC1322.

Strain IMCC1322 was isolated from a surface water sample from the East Sea of Korea. Based on 16S rRNA analysis, IMCC1322 was found to belong to the OCS28 sub-clade of SAR116. The cells appeared as short vibrioids in logarithmic-phase culture, and elongated spirals during incubation with mitomycin or in aged culture. Growth characteristics of strain IMCC1322 were further evaluated based on genomic information; proteorhodopsin (PR), carbon monoxide dehydrogenase, and dimethylsulfoniopropionate (DMSP)-utilizing enzymes. IMCC1322 PR was characterized as a functional retinylidene protein that acts as a light-driven proton pump in the cytoplasmic membrane. However, the PR-dependent phototrophic potential of strain IMCC1322 was only observed under CO-inhibited and nutrient-limited culture conditions. A DMSP-enhanced growth response was observed in addition to cultures grown on C1 compounds like methanol, formate, and methane sulfonate. Strain IMCC1322 cultivation analysis revealed biogeochemical processes characteristic of the SAR116 group, a dominant member of the microbial community in euphotic regions of the ocean. The polyphasic taxonomy of strain IMCC1322 is given as Candidatus Puniceispirillum marinum, and was confirmed by chemotaxonomic tests, in addition to 16S rRNA phylogeny and cultivation analyses.

Nibricoccus aquaticus gen. nov., sp. nov., a new genus of the family Opitutaceae isolated from hyporheic freshwater.

A yellow-coloured, Gram-strain-negative, non-motile, cocci-shaped, strictly aerobic bacterium, designated HZ-65T, was isolated from hyporheic freshwater in the Republic of Korea. Strain HZ-65T grew at 15-37 °C (optimum, 25-30 °C), pH 5.5-9.0 (optimum, pH 7.0) and 0-0.5 % NaCl (w/v; optimum at 0 % NaCl). Phylogenetic analysis based on the 16S rRNA gene showed that strain HZ-65T is a member of family Opitutaceae and is closely related to Opitutus terrae PB90-1T (94.0 % similarity), Cephaloticoccus primus CAG34T (93.0 %), and Cephaloticoccus capnophilus CV41T (92.7 %), while the similarities to other Opitutaceae-type strains were lower than 90.0 %. The DNA G+C content was 62.2 mol% and the quinone present was menaquinone-7. The predominant fatty acids were iso-C14 : 0, anteiso-C15 : 0, C16 : 0, and iso-C16 : 0, representing 70 % of the total fatty acids. The major polar lipid profile consisted of phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. Analysis of the HZ-65T genome revealed the presence of 300 genes that are involved in carbohydrate-active enzymes, which indicates the metabolic potential to degrade polysaccharides. The phenotypic, chemotaxonomic, genetic, and phylogenetic properties suggest that strain HZ-65T represents a novel species in a new genus within the family Opitutaceae, for which the name Nibricoccus aquaticus gen. nov., sp. nov., is proposed. The type strain of Nibricoccus aquaticus is HZ-65T (KACC 19333T=NBRC 112907T).

Pedobacter aquicola sp. nov., isolated from freshwater.

A non-motile, pink-pigmented bacterial strain designated IMCC25679T, was isolated from freshwater Lake Chungju of Korea. Phylogenetic trees based on 16S rRNA gene sequences showed that the strain IMCC25679T formed a lineage within the genus Pedobacter. The strain IMCC25679T was closely related to Pedobacter daechungensis Dae 13T (96.4% sequence similarity), Pedobacter rivuli HME8457T (95.3%) and Pedobacter lentus DS-40T (94.3%). The major fatty acids of IMCC- 25679T were iso-C15:0, iso-C16:0 and summed feature 3 (comprising C16:1ω6c and/or C16:1ω7c). The major respiratory quinone was MK-7. The major polar lipids were phosphatidylethanolamine (PE), an unidentified sphingolipid (SL), an unidentified aminolipid (AL) and three unidentified polar lipids (PL). The DNA G + C content of IMCC25679T was 32.2 mol%. Based on the evidence presented in this study, the strain IMCC25679T represents a novel species within the genus Pedobacter, with the proposed name Pedobacter aquicola, sp. nov. The type strain is IMCC25679T (= KACC 19486T = NBRC113131T).

Deinococcus lacus sp. nov., a gamma radiation-resistant bacterium isolated from an artificial freshwater pond.

A Gram-stain-negative, pink-coloured, non-motile and gamma radiation-resistant bacterium, designated strain IMCC1711T, was isolated from a freshwater sample collected from an artificial pond (Inkyong Pond). The 16S rRNA gene sequence analysis showed that strain IMCC1711T was most closely related to Deinococcus piscis 3axT (94.2 %) and formed a robust phylogenetic clade with other species of the genus Deinococcus. Optimal growth of strain MCC1711T was observed at 25 °C and pH 7.0 without NaCl. Strain IMCC1711T exhibited high resistance to gamma radiation. The DNA G+C content of strain IMCC1711T was 59.1 mol% and MK-8 was the predominant isoprenoid quinone. Major fatty acid constituents of the strain were C17 : 1ω8c, C16 : 0, summed feature 3 (C16 : 1ω6c and/or C16 : 1ω7c) and C15 : 1ω6c. The major polar lipids constituted phosphatidylethanolamine, one unidentified phosphoglycolipid and two unidentified glycolipids. On the basis of taxonomic data obtained in this study, it was concluded that strain IMCC1711T represented a novel species of the genus Deinococcus, for which the name Deinococcus lacus sp. nov. is proposed. The type strain of Deinococcus lacus is IMCC1711T (KCTC 52494T=KACC 18979T=NBRC 112440T).

Emticiciafontis sp. nov., isolated from a freshwater pond.

A bacterial strain, designated IMCC1731T, was isolated from an eutrophic freshwater pond and characterized using a polyphasic taxonomic approach. Cellular growth occurred at pH 6-9 (optimum, pH 8.0), at 10-30 °C (optimum, 25 °C) and with 0-1 % (w/v) NaCl (optimum, 0 %). Phylogenetic analyses based on 16S rRNA gene sequences indicated that strain IMCC1731T belonged to the genus Emticicia in the family Cytophagaceae and was most closely related to Emticicia ginsengisoli Gsoil 085T (98.1 %) followed by Emticicia paludis HMF3850T (95.0 %), Emticicia oligotrophica DSM 17448T (94.7 %), Emticicia aquatica HMF2925T (94.4 %) and Emticicia sediminis JBR12T (94.0 %). The DNA-DNA relatedness between strain IMCC1731T and E. ginsengisoli Gsoil 085T was 47±4 %. The DNA G+C content of strain IMCC1731T was 37.7 mol%. The predominant cellular fatty acids were summed feature 3 (C16 : 1ω6c and/or C16 : 1ω7c) and iso-C15 : 0. Based on the physiological and chemotaxonomic characteristics, DNA-DNA relatedness and 16S rRNA gene phylogeny, stain IMCC1731T is considered to represent a novel species of the genus Emticicia, for which the name Emticicia fontis sp. nov. is proposed. The type strain is IMCC1731T (=KCTC 52248T=JCM 31373T).

Rubrivirga marina gen. nov., sp. nov., a member of the family Rhodothermaceae isolated from deep seawater.

Two aerobic, Gram-stain-negative, pale-red-pigmented and rod-shaped bacterial strains, designated SAORIC-26 and SAORIC-28(T), were isolated from seawater (3000 m depth) from the Pacific Ocean. Phylogenetic analysis based on their 16S rRNA gene sequences revealed that the novel isolates could be affiliated with the family Rhodothermaceae of the class Cytophagia. Strains SAORIC-26 and SAORIC-28(T) shared 99.7% pairwise sequence similarity with each other and showed less than 92.6% similarity with other cultivated members of the class Cytophagia. The strains were found to be non-motile, oxidase-positive, catalase-negative and able to hydrolyse gelatin and aesculin. The DNA G+C contents were determined to be 64.8-65.8 mol% and MK-7 was the predominant menaquinone. Summed feature 9 (iso-C17:1ω9c and/or C16:0 10-methyl), summed feature 3 (C16:1ω6c and/or C16:1ω7c) and iso-C15:0 were found to be the major cellular fatty acids. On the basis of this taxonomic study using a polyphasic approach, it was concluded that strains SAORIC-26 and SAORIC-28(T) represent a novel species of a new genus in the family Rhodothermaceae, for which the name Rubrivirga marina gen. nov., sp. nov. is proposed. The type strain of the type species of is SAORIC-28(T) (=KCTC 23867(T)=NBRC 108816(T)). An additional strain of the species is SAORIC-26.

Soonwooa buanensis gen. nov., sp. nov., a member of the family Flavobacteriaceae isolated from seawater.

A Gram-negative, non-motile, non-gliding, strictly aerobic, pale yellow colony-forming, rod-shaped bacterial strain, designated HM0024(T), was isolated from coastal seawater of the Yellow Sea and subjected to a polyphasic taxonomy study. Based on 16S rRNA gene sequence similarities, strain HM0024(T) was most closely related to Chryseobacterium balustinum LMG 8329(T) (94.4 %), Chryseobacterium scophthalmum LMG 13028(T) (94.4 %), Chryseobacterium piscium LMG 23089(T) (94.3 %) and Elizabethkingia meningoseptica ATCC 13253(T) (94.0 %) and shared less than 92 % sequence similarity with other members of the family Flavobacteriaceae. Phylogenetic analyses showed that strain HM0024(T) formed an independent phyletic line of descent within the family Flavobacteriaceae. The DNA G+C content of the strain was 29.6 mol% and its major cellular fatty acids (>10 %) were iso-C(15 : 0), iso-C(17 : 0) 3-OH, iso-C(17 : 1)ω 7c and summed feature 3 (C(16 : 1)ω 6c and/or C(16 : 1)ω 7c). The major isoprenoid quinone was MK-6 and the major polar lipids were phosphatidylethanolamine and several aminolipids. Strain HM0024(T) was differentiated from phylogenetically related members of the family by having lower DNA G+C content, larger proportions of summed feature 3, anteiso-C(15 : 0) and iso-C(16 : 0) 3-OH and particular phenotypic characteristics. On the basis of phenotypic and phylogenetic data, strain HM0024(T) is classified as a representative of a novel genus and species, for which the name Soonwooa buanensis gen. nov., sp. nov. is proposed. The type strain of Soonwooa buanensis is HM0024(T) (=KCTC 22689(T) =CECT 7503(T)).

Azonexus hydrophilus sp. nov., a nifH gene-harbouring bacterium isolated from freshwater.

Three Gram-negative, non-pigmented, rod-shaped, facultatively aerobic bacterial strains, designated d8-1T, d8-2 and IMCC1716, were isolated from a freshwater spring sample and a eutrophic freshwater pond. Based on characterization using a polyphasic approach, the three strains showed highly similar phenotypic, physiological and genetic characteristics. All of the strains harboured the nitrogenase gene nifH, but nitrogen-fixing activities could not be detected in nitrogen-free culture media. The three strains shared 99.6-99.7 % 16S rRNA gene sequence similarity and showed 89-100 % DNA-DNA relatedness, suggesting that they represent a single genomic species. Phylogenetic analysis based on 16S rRNA gene sequences showed that strains d8-1T, d8-2 and IMCC1716 formed a monophyletic branch in the periphery of the evolutionary radiation occupied by the genus Azonexus. Their closest neighbours were Azonexus caeni Slu-05T (96.7-96.8 % similarity) and Azonexus fungiphilus BS5-8T (96.3-96.6 %). The DNA-DNA relatedness of the novel strains to these two species of the genus Azonexus was less than 70 %. The isolates could also be differentiated from recognized members of the genus Azonexus on the basis of phenotypic and biochemical characteristics. It is evident, therefore, that the three strains represent a novel species of the genus Azonexus, for which the name Azonexus hydrophilus sp. nov. is proposed. The type strain is d8-1T (=LMG 24005T=BCRC 17657T).