ImmunoTox LetterDecember 2021

Report on the 28th Annual Meeting of the Japanese Society of Immunotoxicology(JSIT 2021)

Reiko Teshima
President of the 28th Annual Meeting of the JSIT
Faculty of Veterinary Medicine, Okayama University of Sciences (OUS), JapanThe 28th Annual Meeting of the Japanese Society of Immunotoxicology was held in theform of a web conference for two days, September 6th (Sat) and 7th (Sun), 2021. Initially, it wasplanned to be held at the lecture building of the Imabari Campus, Faculty of Veterinary Medicine,
Okayama University of Science, but since the spread of coronavirus infections continued to beuncontrollable, the regular holding was abandoned in early June after discussions. We havedecided to hold it as a web society. This is the second year since last year, and as with last year,
posters are delivered on demand and oral presentations are delivered live. The final number ofabstract submissions was not so different from last year due to the fact that it was held on the web,and we are truly grateful that we were able to hold the annual meeting successfully with thecooperation and support of many people.
At this meeting, symposium, special lectures and educational lectures were planned with themain theme of "relationship between innate immunity and acquired immunity andimmunotoxicity".
Symposium was titled “The development status of various vaccine and their safety
evaluation“. Dr. Hiroyuki Oshiumi (Kumamoto University) gave a lecture on "Micro RNAs
associated with adverse reactions of vaccine”, Dr. Yoshikazu Yuki (HanaVax Co., Ltd.) gave alecture on "Safety assessment of nasal vaccine", and Dr. Eita Sasaki (National Institute ofInfectious Diseases) gave a lecture on "Screening for a safe and effective novel vaccine adjuvant
using genomics technology”.
Special lectures of the annual meeting were “Dendritic cell- based new immunotherapy" byDr. Shinichiro Fujii (RIKEN Center for Integrative Medical Sciences) and “Immunogeniityrelated toxicity” by Dr.Jeanine Bussiere (Amgen Inc.), who was sent by SOT-ITSS, with a video
distribution.
Educational lectures of the annual meeting were “The significance of allergy and innate
lymphocytes (ICL2) from the evolutionary perspectives” by Kenji Matsumoto (National ResearchInstitute for Child Health and Development) and “New Corona Vaccine” by Dr. Yasuhiro
Yoshikawa (Okayama University of Sciences).
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Workshop on the test methods was titled “Toward the development and guidance of in vitro
immunotoxicity test method”, Drs. Setsuya Aiba (Tohoku University), Masaharu Fujita (Fujifilm
Safety Evaluation Center), and Takao Ashikaga (National Institute of Health Sciences) presented
on the development of in vitro immunotoxicology test methods for an immunosuppression or skinsensitization and the status of inclusion in the OECD test guidelines or guidance.
General session included 7 oral presentations and 15 posters, and Young Scientist Session
included 8 presentations which were to be presented as posters as well as oral presentations.
Fifteen minutes was given for each talk to ensure enough discussion time as previous annualmeetings.
Dr.Michihiko Aoyama (National Institute of Health Sciences) received the Best Presenter
Award and Mr.Takashi Kato (Wakayama Medical University) received the best Student andYoung Scientist Presentation Award.
We would like to express sincere appreciation for attendants who actively discuss both oraland poster presentations. Totally 96 people attended the meeting.
We really feel the success of the meeting was thanks to your understanding and cooperation,as well as support from organizations and companies. We wish the further development of theSociety.

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The Best Presentation AwardFcγ receptor-dependent internalization andoff-target cytotoxicity of antibody-drug conjugate aggregatesMichihiko Aoyama
Division of Biological Chemistry and Biologicals
National Institute of Health Sciences
Antibody-drug conjugates (ADCs), which are monoclonal antibodies (mAbs) conjugatedto highly toxic small molecules (payloads) via linkers, are one of the fastest growing classes ofnext-generation therapeutic mAbs. ADCs combine the advantages of the target-specificity ofmAbs and the high tumor-killing efficacy of payloads, and achieve wider therapeutic windows
than traditional chemotherapy. On the other hand, conjugation of payloads often increases thehydrophobicity of mAbs, resulting in reduced stability and increased aggregation. It is known thatmAb aggregates have a potential risk for immunogenicity, because they can activate Fcγ receptors(FcγRs) on immune cells, and are internalized via FcγRs. However, the impacts of aggregationon the off-target toxicity of ADCs have not been well studied. In this study, we investigated theimpacts of aggregation of ADCs on off-target cytotoxicity.
We generated aggregates of two commercial anti-HER2 mAb (trastuzumab)-based ADCs (TDM1 and T-DXd) by stirring stress (600 rpm, 20 h) or thermal stress (90ºC, 3 min). A qLD size
distribution analysis showed that aggregates between 0.1 and 50 µm were generated by the stresstreatment, and these aggregates were removed after filtration of ADC aggregates with a 0.45 µmfilter. We evaluated the cytotoxicity of ADC aggregates in HER2-positive cells and -negative cells.
In several HER2-negative cell lines, the aggregation of ADCs enhanced the off-target cytotoxicity
compared with non-stressed ADCs, whereas the target-dependent cytotoxicity was reduced byaggregation of ADCs. The enhancement of off-target toxicity was suppressed by filtrating thesamples with a 0.45 µm filter. These results strongly suggested that the aggregates, which wereremoved by the 0.45 µm filter, were responsible for the enhancement of off-target cytotoxicity.

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To examine the mechanism of off-target cytotoxicity, we focused on FcγR-activation
properties of ADC aggregates, because ADC aggregates dramatically enhanced the off-target
cytotoxicity in FcγR-expressing cell lines (such as THP-1 cells and MEG01-S cells). We
evaluated the FcγR-activation properties of ADC aggregates using FcγR-expressing reporter cells.
The results showed that the FcγR-activation properties of ADC aggregates varied depending on
the kind of mAb/ADCs (trastuzumab, T-DM1, or T-DXd) and the stress treatment (stirring stressor thermal stress), and the FcγR-activation properties of ADC aggregates were related to theinternalization and enhanced cytotoxicity of ADC aggregates in FcγR-expressing reporter cells.
Additionally, FcγRIIa-blocking reduced the off-target cytotoxicity of ADC aggregates in anFcγRIIa-expressing cell line (MEG01-S), whereas the cytotoxicity of non-stress ADCs or thefiltered sample of ADC aggregates was not affected by FcγRIIa-blocking. Fc-engineering forsilencing Fc-mediated effector functions also reduced FcγR-mediated off-target cytotoxicity ofADC aggregates. These results indicate that FcγRs play an important role in the off-target toxicityof ADC aggregates.
Our findings indicated that the aggregation of ADCs increases the potential risk for not only
immunogenicity but also off-target toxicity of ADCs, and FcγR-dependent internalization of ADC
aggregates may be one of the mechanisms underlying the off-target toxicity of ADCs.

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The Student and Young Scientists Award

Molecular mechanisms of type I interferonopathy
in a novel COPA syndrome model mouse

Takashi Kato
Department of Immunology,
Institute of advanced Medicine, Wakayama Medical University
COPA syndrome is an autoinflammatory disease with autoimmune disorders, featured byinterstitial lung disease, arthritis and glomerulonephritis caused by heterozygous variants in thecoatomer subunit alpha (COPA) gene. COPA is a subunit of the coatomer protein complex I (COPI), a carrier complex required for retrograde protein trafficking from the Golgi apparatus to theendoplasmic reticulum (ER). In COPA syndrome, expression of interferon (IFN)-stimulated genes(ISGs) was elevated, what we call type I interferonopathy. At present, however, it is not clear how
the genetic variant of COPA leads to the type I interferonopathy and manifestations of COPAsyndrome.
Dr. Matsubayashi of the Seirei Hamamatsu hospital and Dr. Izawa of Kyoto University
found a novel COPA variant (c.725T>G, p.Val242Gly) in four patients in one Japanese family.
V242 is variant localized in the hotspot of the variants causing COPA syndrome, the WD40
repeat domain, and well conserved beyond species. In order to clarify whether or how the COPA
V242G mutant contributes to the patient manifestations, we generated and analyzed the mutantmice.
Copa V242G heterozygous mutant mice manifested interstitial lung disease, which wassimilar to that of COPA syndrome patients. Furthermore, ISGs expression was elevated insplenocytes from the mutant mice. Dendritic cells from the mutant mice showed elevation of type
I IFN production in response to a cytoplasmic DNA sensor, STING. Importantly, phosphorylationof STING and its signaling molecule, TBK1, was enhanced and STING localization was deviatedfrom the ER to the Golgi apparatus in the mutant DCs when stimulated with a STING agonist.
These findings suggest that Copa V242G heterozygous mutant led to defects in protein
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ImmunoTox Lettertransportation by COP I, thereby causing localization shift of STING from ER to Golgi andhyperactivation of STING-induced type I IFN production. Copa V242G mutant mice are novel
model mice for COPA syndrome and further study on the mice should contribute to clarificationof the pathogenesis, generation of the new therapeutic strategy for COPA syndrome andelucidation of regulatory mechanisms for STING pathway.

The Student and Young Scientists Award

Journey with “Immunotoxicity”

Atsuhito Kubota
School of Pharmaceutical Sciences,
Health Sciences University of Hokkaido
Inflammatory bowel disease (IBD) is an inflammatory disease of unknown aetiology, andthe number of patients in Japan has been increasing. One of the problems of IBD is that themechanism of pathogenesis is unknown and the curative therapy has not been established.
Furthermore, the pressure on medical costs caused by the increase in the number of patients ismaking it difficult to continue social security, and in 2017, some patients with mild disease wereexcluded from public subsidies. Against this such background, I started research using clinicalbig data when I was working in the Pharmaceutical Department, and I continue to do so today. In
these analyses, I focused on 5-aminosalicylic acid (5-ASA), a key drug for the treatment of IBD.
5-ASA has been reported to have several mechanisms, including inhibition of reactive oxygen
species production, activation of PPARγ, and inhibition of NF-κB. In addition, it has beensuggested that 5-ASA induces regulatory T cells (Tregs) via the aryl hydrocarbon receptor (AhR),and we have investigated whether 5-ASA is a direct ligand for AhR using DR-EcoScreen cells, ahighly sensitive reporter cell line. In silico docking simulation was used to calculate the affinityof 5-ASA to the ligand binding site. As a result, we recently reported that 5-ASA acts as a ligand
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ImmunoTox Letterfor AhR at high concentrations, and has Treg-inducing ability in a primary spleen culture system(Kubota et al. Pharmacology. in press).
Similarly, Elemental diet (ED) therapies, which are formulations of complete
gastrointestinal nutrition, have been used empirically to protect the mucosa damaged byinflammation and reduce the burden on intestinal function. Therefore, we focused on thecomponents of ED therapy and its intestinal microflora metabolites and investigated whether they
have anti-inflammatory effects using a DSS-induced colitis model. As a result, we found thatseveral metabolites of the components in ED therapy functioned as AhR ligands and induced
splenic Tregs in mice treated with ED therapy. Furthermore, we found that ED therapy withenhanced active ingredients significantly suppressed DSS-induced colitis, and we are now
focusing on the transport mechanism of the metabolites.
In the future, we would like to establish a method to control immunotoxicity throughmodification of existing therapies targeting AhR and drug repositioning, and strive to establish
basic evidence, which is useful for clinical use.

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