Research for elucidation of immune evasion mechanisms and development of a novel immunotherapy for cancer in dogs [an abstract of entire text]

概要

Title

Author(s)

Citation

Issue Date

Doc URL

Research for elucidation of immune evasion mechanisms and development of a novel immunotherapy for cancer in
dogs [an abstract of entire text]

竹内, 寛人

北海道大学. 博士(獣医学) 甲第15580号

2023-06-30

http://hdl.handle.net/2115/90418

Type

theses (doctoral - abstract of entire text)

Note

この博士論文全文の閲覧方法については、以下のサイトをご参照ください。

Note(URL)

File Information

https://www.lib.hokudai.ac.jp/dissertations/copy-guides/

Hiroto_Takeuchi_summary.pdf

Instructions for use

Hokkaido University Collection of Scholarly and Academic Papers : HUSCAP

学位論文の要約
博士の専攻分野の名称：博士（獣医学）

氏名：竹内 寛人

学位論文題名
Research for elucidation of immune evasion mechanisms and
development of a novel immunotherapy for cancer in dogs
(イヌ腫瘍における免疫回避機構の解明と
新規治療法の開発に向けた臨床研究)
While deaths from infectious diseases, which used to be a major cause of death,
have declined dramatically, cancer has become a major cause of death in dogs due to the
increasing life expectancy by improvements in the rearing environment and veterinary
medicine. Studies in spontaneous cancers in dogs have attracted attention as a good
model for cancers in humans because they share many features in their etiology and in
the response to therapy. Currently, surgery, chemotherapy, and radiotherapy serve as the
standard therapeutic modalities for canine cancers. However, in some cases, these
treatments cannot be applied for some reasons, such as limited access for surgical
resection and irradiation due to its anatomical sites as well as adverse effects and
complications of the treatment. Due to the success of antibody therapies targeting
immune checkpoint molecules, including programmed cell death 1 (PD-1) and PDligand 1 (PD-L1), immunotherapy has become one of the standard therapies for cancer
in humans. In the field of veterinary medicine, immunotherapy has also emerged as a
breakthrough therapy. Previous studies showed the expression of PD-L1 in various
types of canine cancers, suggesting the potential of using immunotherapy targeting the
PD-1/PD-L1 axis in dogs. However, the clinical trial in canine oral malignant melanoma
(OMM) using a chimeric anti-PD-L1 antibody (c4G12) revealed that the therapeutic
effect is limited despite PD-L1 expression in most of the cancer tissues. Thus, it is
necessary to investigate the resistance mechanisms to c4G12 treatment and to develop
combinational therapeutic strategies to enhance the efficacy of antibody therapy in
canine cancers. Additionally, to facilitate the development of antibody drugs in dogs, the
establishment of a cost-effective purification process is warranted.
In chapter I, for a better understanding of the regulation mechanisms of PD-L1

expression in canine cancers, the nucleotide sequences of canine CKLF-like MARVEL
transmembrane domain containing protein 6 (CMTM6) and CMTM4 genes were
identified. Interestingly, the deduced amino acid sequences of canine CMTM6 and
CMTM4 were highly similar to their orthologs in other mammalian species, and the
predicted functional domain, MAL and related proteins for vesicle trafficking and
membrane link domain, was conserved in dogs. The gene expression of CMTM6 and
CMTM4 was confirmed by quantitative reverse transcription polymerase chain reaction
in canine immune cells and cancer cells. Immunohistochemical analysis in canine
malignant melanoma and osteosarcoma (OSA) revealed that all tissue samples coexpressed CMTM6, CMTM4, and PD-L1. Gene knockdown of CMTM6 and CMTM4 in
a PD-L1-expressing cell line reduced the cell surface PD-L1 expression, suggesting that
both CMTM6 and CMTM4 maintain the cell surface expression of PD-L1.
In chapter II, as an additional therapeutic target for combination therapy with
anti-PD-L1 antibody, expression and functions of transforming growth factor (TGF)-β1
in dogs with cancer was examined. Canine melanoma cell lines produced TGF-β1 in the
culture supernatant, and the serum TGF-β1 levels in dogs with metastatic OMM were
elevated compared to those in healthy dogs. The immunosuppressive effects of TGF-β1
on canine peripheral blood mononuclear cells support the potential of TGF-β1 as a
therapeutic target for cancer treatment. To reverse immunosuppression exerted by TGFβ1, a decoy receptor for TGF-β, TGF-β receptor 2-Ig (TGF-βRII-Ig), was prepared as a
fusion protein of the extracellular region of canine TGF-βRII and the Fc region of
canine IgG-B. TGF-βRII-Ig activated canine immune cells in the presence of exogenous
TGF-β1, highlighting that its potential as a candidate therapeutic strategy for enhancing
anti-tumor immunity. The anti-tumor efficacy of inhibiting the TGF-β1 pathway should
be further investigated in clinical studies involving dogs with cancers.
In chapter III, to establish a robust and efficient purification method for canine
therapeutic antibodies, the molecular characterization of SpsQ from Staphylococcus
pseudintermedius was performed. From S. pseudintermedius isolates, the nucleotide
sequence of SpsQ, which contained five immunoglobulin binding domains (Ig-BDs),
was determined. To compare the binding characteristic of SpsQ and SpA, recombinant
proteins of Ig-BDs from SpsQ (r-SpsQ) or SpA (r-SpA) were prepared and subjected to
the binding assay. The binding capacity of r-SpsQ to canine IgG-A and IgG-D was
higher than that of r-SpA, supporting the potential of SpsQ as an affinity
chromatography ligand for a highly efficient purification process of canine therapeutic
antibodies whose subclass are IgG-A and IgG-D.
Taken together, this study contributes to (1) the elucidation of PD-L1

regulation mechanism and providing a clue to fully understand the resistance
mechanism to anti-PD-L1 therapy in dogs, (2) the development of a novel biologic in
the field of veterinary medicine with enhanced efficacy, and (3) the improvement of
purification process for canine therapeutic antibody with acceptable cost-effectiveness.
Further studies including the investigation of regulators of PD-L1 and TGF-β1, clinical
studies of a combination therapy involving anti-PD-L1 antibody and an TGF-β1
inhibitor in dogs with cancer, and the optimization of purification processes using SpsQ
affinity chromatography are required to accelerate the development of novel antibodybased immunotherapies for canine cancers. Given the similarity in antitumor immunity
between dogs and humans, the accumulated data from clinical studies in dogs with
cancer will also contribute to the advance of cancer research in humans. ...

この論文で使われている画像