Novel knock‐in mouse model for the evaluation of the therapeutic efficacy and toxicity of human podoplanin–targeting agents

Ukaji, Takao; Takemoto, Ai; Shibata, H; Kakino, Mamoru; Takagi, Satoshi; Katayama, Ryohei; Fujita, Naoya

doi:10.1111/cas.14891

Cited by 3 publications

(6 citation statements)

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“…However, we previously found that a high-dose single administration (100 mg/kg) of 2F7 surrogate anti-monkey PDPN antibody harboring PG4D2-like neutralizing activity did not demonstrate acute toxicity in cynomolgus monkeys ( 33 ). Moreover, we confirmed that there was no toxicity or change in hematologic and biochemical parameters after repeated PG4D2 administration in Pdpn KI/KI mice expressing human/mouse chimeric PDPN in which the mouse PLAG4 domain was replaced with the human homologous PLAG4 domain ( 28 ).…”

Section: Resultssupporting

confidence: 69%

“…S1; ref. 28 ). We also assessed PDPN expression in osteosarcoma cell lines obtained from samples surgically resected from patients with osteosarcoma after obtaining informed consent (Sa-xeno-147-P0 and GCB53-G3; Fig.…”

Section: Resultsmentioning

confidence: 99%

“…NSG (NOD.Cg- Prkdc scid Il2rg tm1Wjl /SzJ), NOD-SCID (NOD.CB17- Prkdc scid /J), and SCID beige (CB17.Cg- Prkdc scid Lyst bg-J /CrlCrlj), ICR (Crl:CD1(ICR)) mice were purchased from Charles River Laboratories, Inc. Pdpn KI/KI C57BL/6N mice were developed and maintained in our laboratory ( 28 ). All animal procedures were performed following the protocol approved by the Committee for the Use and Care of Experimental Animals of JFCR in accordance with relevant guidelines and regulations.…”

Section: Methodsmentioning

confidence: 99%

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Targeting Podoplanin for the Treatment of Osteosarcoma

Takemoto

Takagi

Ukaji

et al. 2022

Clinical Cancer Research

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Purpose: Osteosarcoma (OS), the most common bone malignancy in children, has a poor prognosis, especially when the tumor metastasizes to the lungs. Therefore, novel therapeutic strategies targeting both proliferation and metastasis of OS are required. Podoplanin (PDPN) is expressed by various tumors and is associated with tumor-induced platelet activation via its interaction with C-type lectin-like receptor 2 (CLEC-2) on platelets. We previously found that PDPN contributed to OS growth and metastasis through platelet activation; thus, in this study, we developed an anti-PDPN humanized antibody and evaluated its effect on OS growth and metastasis. Experimental Design: 9 OS cell lines and 2 OS patient-derived cells were collected, and evaluated the efficacy of anti-PDPN-neutralizing antibody, PG4D2 and the humanized anti-PDPN antibody AP201, which had IgG4 framework region. The antitumor and antimetastasis effect of PG4D2 and AP201 were examined in vitro and vivo. In addition, growth signaling by the interaction between PDPN and CLEC-2 was analyzed using phospho-RTK array, growth assay, or immunoblot analysis under the supression of RTKs by siRNA and inhibitor treatment. Results: We observed that PG4D2 treatment significantly suppressed tumor growth and pulmonary metastasis in OS xenograft models highly expressing PDPN. The contribution of PDGFR activation by activated platelet releasates to OS cell proliferation was confirmed, and the humanized antibody, AP201, suppressed in vivo OS growth and metastasis without significant adverse events. Conclusions: Targeting PDPN with a neutralizing antibody against PDPN-CLEC-2 without ADCC and CDC is a novel therapeutic strategy for PDPN-positive OS.

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Section: Resultssupporting

confidence: 69%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Targeting Podoplanin for the Treatment of Osteosarcoma

Takemoto

Takagi

Ukaji

et al. 2022

Clinical Cancer Research

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“…In addition to OSCC, PDPN promotes a variety of other cancers including mammary carcinoma [ 62 , 63 ], glioma [ 64 – 67 ], melanoma [ 59 , 68 , 69 ], ovarian cancer [ 70 ], and pulmonary adenocarcinoma [ 71 – 73 ]. Indeed, PDPN has been identified as an enticing target for chemotherapy [ 74 , 75 ]. The roles of cadherins and PDPN in contact normalization described in this study should yield insight into fundamental mechanisms that affect the general process of cancer development and progression at the cellular level of the tumor microenvironment.…”

Section: Discussionmentioning

confidence: 99%

Heterocellular N-cadherin junctions enable nontransformed cells to inhibit the growth of adjacent transformed cells

et al. 2022

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Background The Src tyrosine kinase phosphorylates effector proteins to induce expression of the podoplanin (PDPN) receptor in order to promote tumor progression. However, nontransformed cells can normalize the growth and morphology of neighboring transformed cells. Transformed cells must escape this process, called “contact normalization”, to become invasive and malignant. Contact normalization requires junctional communication between transformed and nontransformed cells. However, specific junctions that mediate this process have not been defined. This study aimed to identify junctional proteins required for contact normalization. Methods Src transformed cells and oral squamous cell carcinoma cells were cultured with nontransformed cells. Formation of heterocellular adherens junctions between transformed and nontransformed cells was visualized by fluorescent microscopy. CRISPR technology was used to produce cadherin deficient and cadherin competent nontransformed cells to determine the requirement for adherens junctions during contact normalization. Contact normalization of transformed cells cultured with cadherin deficient or cadherin competent nontransformed cells was analyzed by growth assays, immunofluorescence, western blotting, and RNA-seq. In addition, Src transformed cells expressing PDPN under a constitutively active exogenous promoter were used to examine the ability of PDPN to override contact normalization. Results We found that N-cadherin (N-Cdh) appeared to mediate contact normalization. Cadherin competent cells that expressed N-Cdh inhibited the growth of neighboring transformed cells in culture, while cadherin deficient cells failed to inhibit the growth of these cells. Results from RNA-seq analysis indicate that about 10% of the transcripts affected by contact normalization relied on cadherin mediated communication, and this set of genes includes PDPN. In contrast, cadherin deficient cells failed to inhibit PDPN expression or normalize the growth of adjacent transformed cells. These data indicate that nontransformed cells formed heterocellular cadherin junctions to inhibit PDPN expression in adjacent transformed cells. Moreover, we found that PDPN enabled transformed cells to override the effects of contact normalization in the face of continued N-Cdh expression. Cadherin competent cells failed to normalize the growth of transformed cells expressing PDPN under a constitutively active exogenous promoter. Conclusions Nontransformed cells form cadherin junctions with adjacent transformed cells to decrease PDPN expression in order to inhibit tumor cell proliferation. Plain English Summary Cancer begins when a single cell acquires changes that enables them to form tumors. During these beginning stages of cancer development, normal cells surround and directly contact the cancer cell to prevent tumor formation and inhibit cancer progression. This process is called contact normalization. Cancer cells must break free from contact normalization to progress into a malignant cancer. Contact normalization is a widespread and powerful process; however, not much is known about the mechanisms involved in this process. This work identifies proteins required to form contacts between normal cells and cancer cells, and explores pathways by which cancer cells override contact normalization to progress into malignant cancers. Graphical abstract

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“…Not only does this double the effort required for antibody discovery, but depending on the antibody fine epitope specificity, the impact of the antibody on disease in the different model systems may not be identical. This has been a challenge in developing antibodies to bind mouse CD3 to evaluate T cell retargeting antibodies and to evaluate antibody therapeutics to treat pediatric medulloblastoma in mice (43)(44)(45). In support of this future effort for myocilin, 2H2 has similar (within ~50%) K D values for each homolog and can easily be expressed with mouse or human constant domains to faithfully engage host Fc receptors (Table 3).…”

Section: Discussionmentioning

confidence: 99%

Recombinant antibodies recognize conformation-dependent epitopes of the leucine zipper of misfolding-prone myocilin

Patterson-Orazem

Qerqez

Azouz

et al. 2021

Journal of Biological Chemistry

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Novel knock‐in mouse model for the evaluation of the therapeutic efficacy and toxicity of human podoplanin–targeting agents

Abstract: This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.

Cited by 3 publications

References 36 publications

Targeting Podoplanin for the Treatment of Osteosarcoma

Targeting Podoplanin for the Treatment of Osteosarcoma

Heterocellular N-cadherin junctions enable nontransformed cells to inhibit the growth of adjacent transformed cells

Recombinant antibodies recognize conformation-dependent epitopes of the leucine zipper of misfolding-prone myocilin

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