Ephrin receptor A10 monoclonal antibodies and the derived chimeric antigen receptor T cells exert an antitumor response in mouse models of triple-negative breast cancer.

Expression of the receptor tyrosine kinase ephrin receptor A10 (EphA10), which is undetectable in most normal tissues except for the male testis, has been shown to correlate with tumor progression and poor prognosis in several malignancies, including triple-negative breast cancer (TNBC). Therefore, EphA10 could be a potential therapeutic target, likely with minimal adverse effects. However, no effective clinical drugs against EphA10 are currently available. Here, we report high expression levels of EphA10 in tumor regions of breast, lung, and ovarian cancers as well as in immunosuppressive myeloid cells in the tumor microenvironment. Furthermore, we developed anti-EphA10 monoclonal antibodies (mAbs) that specifically recognize cell surface EphA10, but not other EphA family isoforms, and target tumor regions precisely in vivo with no apparent accumulation in other organs. In syngeneic TNBC mouse models, we found that anti-EphA10 mAb clone #4 enhanced tumor regression, therapeutic response rate, and T cell-mediated antitumor immunity. Notably, the chimeric antigen receptor T cells derived from clone #4 significantly inhibited TNBC cell viability in vitro and tumor growth in vivo. Together, our findings suggest that targeting EphA10 via EphA10 mAbs and EphA10-specific chimeric antigen receptor-T cell therapy may represent a promising strategy for patients with EphA10-positive tumors.

Emerging Roles for Eph Receptors and Ephrin Ligands in Immunity.

Eph receptors are the largest family of receptor tyrosine kinases and mediate a myriad of essential processes in humans from embryonic development to adult tissue homeostasis through interactions with membrane-bound ephrin ligands. The ubiquitous expression of Eph receptors and ephrin ligands among the cellular players of the immune system underscores the importance of these molecules in orchestrating an optimal immune response. This review provides an overview of the various roles of Eph receptors and ephrin ligands in immune cell development, activation, and migration. We also discuss the role of Eph receptors in disease pathogenesis as well as the implications of Eph receptors as future immunotherapy targets. Given the diverse and critical roles of Eph receptors and ephrin ligands throughout the immune system during both resting and activated states, this review aims to highlight the critical yet underappreciated roles of this family of signaling molecules in the immune system.

[Development of a Fundamental Technology to Seek Drug Targets, and Its Application to Cancer Targeting Therapy].

　Human epidermal growth factor receptor 2 (Her2)-targeting antibodies and anti-hormone therapy are effective for most breast cancer patients. However, such approaches are not viable with resistant cases or in triple-negative breast cancer (TNBC) patients, given the lack of Her2 and estrogen and progesterone receptors in these patients. Thus, new drug targets are urgently required. From this perspective, we searched for novel drug targets using proteomic analysis, and identified Eph receptor A10 (EphA10), which is elevated in breast cancer cells as compared to normal breast tissue. Here, we evaluated the potential of EphA10 as a drug target by analyzing its protein expression profile/function in cancer cells, and then by using an anti-EphA10 antibody to treat EphA10-expressing tumor-bearing mice. Protein expression profile analysis showed that EphA10 was expressed in various breast cancer subtypes, including TNBCs, with no expression observed in normal tissues, apart from the testes. Moreover, functional analysis of the cancer cells revealed that ligand-dependent proliferation was observed in EphA10-expressed cancer cells. Thus, we developed our novel anti-EphA10 antibody, which binds to EphA10 with high specificity and affinity at the nanomolar level. Finally, therapeutic analysis indicated that tumor growth was significantly suppressed in the mAb-treated mice in a dose-dependent manner. These results suggest that the EphA10-targeting therapy may be a novel therapeutic option for the management of breast cancer, including in TNBCs which aren't currently treated with molecular-targeted agents. Consequently, we hope that these findings will contribute to the development of a new targeting therapy for refractory breast cancer patients.

Juxtacrine Signaling Inhibits Antitumor Immunity by Upregulating PD-L1 Expression.

Programmed death-ligand 1 (PD-L1) is a well-known immune checkpoint protein that helps cancer cells evade immune response. Anti-PD-L1 immune therapy has been approved for the treatment of several advanced human cancers. Therefore, further understanding of the regulatory mechanisms of PD-L1 is critical to improve PD-L1-targeting immunotherapy. Recent studies indicated that contact-dependent pathways may regulate anticancer immunity, highlighting the importance of cell contact-induced signaling in cancer immunity. Here, we show that tumor cell contact upregulates PD-L1 expression and reduces T-cell-mediated cell killing through the membrane receptor tyrosine kinase ephrin receptor A10 (EphA10), which is not expressed in normal tissues except testis and is known to mediate cell contact-dependent juxtacrine signaling. Knockout of EphA10 in tumor cells increased T-cell-mediated antitumor immunity in syngeneic mouse models. EphA10 expression also correlated positively with PD-L1 in human breast tumor tissues. Together, our data reveal that in addition to paracrine/autocrine signaling, cell contact-mediated juxtacrine signaling also promotes PD-L1 expression, implying that tumor cells may escape immune surveillance via this mechanism and that targeting EphA10 to boost antitumor immunity may be a new immune checkpoint blockade strategy for female patients with breast cancer.Significance: Regulation of PD-L1 expression by cell contact-mediated signaling promotes immune escape in breast cancer and may lead to the development of an immunotherapy with less adverse effects in female patients. Cancer Res; 78(14); 3761-8. ©2018 AACR.

Targeted therapies in hematological malignancies using therapeutic monoclonal antibodies against Eph family receptors.

The use of monoclonal antibodies (mAbs) and molecules derived from them has achieved considerable attention and success in recent years, establishing this mode of therapy as an important therapeutic strategy in many cancers, in particular hematological tumors. mAbs recognize cell surface antigens expressed on target cells and mediate their function through various mechanisms such as antibody-dependent cellular cytotoxicity, complement-dependent cytotoxicity, or immune system modulation. The efficacy of mAb therapy can be improved when they are conjugated to a highly potent payloads, including cytotoxic drugs and radiolabeled isotopes. The Eph family of proteins has received considerable attention in recent years as therapeutic targets for treatment of both solid and hematological cancers. High expression of Eph receptors on cancer cells compared with low expression levels in normal adult tissues makes them an attractive candidate for cancer immunotherapy. In this review, we detail the modes of action of antibody-based therapies with a focus on the Eph family of proteins as potential targets for therapy in hematological malignancies.

Eph Receptor Tyrosine Kinases in Tumor Immunity.

The family of Eph receptor tyrosine kinases and their ephrin ligands regulate a diverse array of physiologic processes, such as axonal guidance, bone remodeling, and immune cell development and trafficking. Eph/ephrin interactions have also been implicated in various pathologic processes, including inflammation, cancer, and tumor angiogenesis. Because Eph receptors play prominent roles in both the immune system and cancer, they likely impact the tumor immune microenvironment, an area in which Eph receptors remain understudied. Here, we provide the first comprehensive review of Eph receptors in the context of tumor immunity. With the recent rise of cancer immunotherapies as promising therapeutic interventions, further elucidation of the roles of Eph receptors in the tumor immune microenvironment will be critical for understanding and developing novel targets against tumor immune evasion. Cancer Res; 76(22); 6452-7. ©2016 AACR.

Anti-EphA10 antibody-conjugated pH-sensitive liposomes for specific intracellular delivery of siRNA.

Therapeutic delivery of small interfering RNA (siRNA) is a major challenge that limits its potential clinical application. Here, a pH-sensitive cholesterol-Schiff base-polyethylene glycol (Chol-SIB-PEG)-modified cationic liposome-siRNA complex, conjugated with the recombinant humanized anti-EphA10 antibody (Eph), was developed as an efficient nonviral siRNA delivery system. Chol-SIB-PEG was successfully synthesized and confirmed with FTIR and (1)H-NMR. An Eph-PEG-SIB-Chol-modified liposome-siRNA complex (EPSLR) was prepared and characterized by size, zeta potential, gel retardation, and encapsulation efficiency. Electrophoresis results showed that EPSLR was resistant to heparin replacement and protected siRNA from fetal bovine serum digestion. EPSLR exhibited only minor cytotoxicity in MCF-7/ADR cells. The results of flow cytometry and confocal laser scanning microscopy suggested that EPSLR enhanced siRNA transfection in MCF-7/ADR cells. Intracellular distribution experiment revealed that EPSLR could escape from the endo-lysosomal organelle and release siRNA into cytoplasm at 4 hours posttransfection. Western blot experiment demonstrated that EPSLR was able to significantly reduce the levels of MDR1 protein in MCF-7/ADR cells. The in vivo study of DIR-labeled complexes in mice bearing MCF-7/ADR tumor indicated that EPSLR could reach the tumor site rather than other organs more effectively. All these results demonstrate that EPSLR has much potential for effective siRNA delivery and may facilitate its therapeutic application.

[Challenge to the Development of Molecular Targeted Therapy against a Novel Target Candidate Identified by Antibody Proteomics Technology].

Disease proteomics that systemically analyzes and identifies differentially expressed proteins between healthy and diseased samples is a potentially useful approach for obtaining target proteins for drug development. To date, however, very few target proteins have been identified from this field. A key issue that remains to be resolved is how to correctly identify target proteins from a number of potential candidates. To circumvent this problem, we have developed "antibody proteomics technology" in which a single chain Fv phage antibody library is utilized for proteome analysis. Here, we describe the application of this technology by primarily focusing on Eph receptor A10 (EphA10), a novel breast cancer-related protein that is a promising target for antibody drugs. To establish an effective and safe targeted cancer therapy, it is important that the target is specifically expressed in cancer tissues. Therefore, we attempted to analyze the EphA10 expression profiles. Tissue microarray analysis showed that EphA10 was expressed in all subtypes of breast cancer containing triple negative breast cancer cases. On the other hand, EphA10 was only expressed in testis tissue among 36 kinds of normal tissues. Thus, EphA10 could be a highly cancer-specific protein, making it a promising target for female breast cancer patients. Finally, we examined the anti-tumor effect by anti-EphA10 antibody, aiming for the development of a novel EphA10 targeting therapy. Administration of the antibody showed that tumor volumes were significantly inhibited. Our results suggest that targeting EphA10 in breast cancer cases might be a promising new form of therapy.

A Novel Bispecific Antibody against Human CD3 and Ephrin Receptor A10 for Breast Cancer Therapy.

Ephrin receptor A10 (EphA10), a transmembrane receptor that binds to ephrin, is a newly identified breast cancer marker protein that has also been detected in HER2-negative tissue. In this study, we report creation of a novel bispecific antibody (BsAb) binding both EphA10 and CD3, thereby forming a bridge between antigens expressed on both tumor and immune cells and promoting recognition of tumor cells by immune cells and redirection of cytotoxic T cells (CTL). This BsAb (EphA10/CD3) was expressed in supernatants of BsAb gene-transfected cells as monomeric and dimeric molecules. Redirected T-cell lysis was observed when monomeric and dimeric BsAb were added to EphA10-overexpressing tumor cells in vitro. Furthermore, dimeric BsAb (EphA10/CD3) was more cytotoxic than monomeric BsAb, with efficient tumor cell lysis elicited by lower concentrations (≤10(-1) µg/mL) and a lower effector to target (E/T) cell ratio (E/T = 2.5). Dimeric BsAb (EphA10/CD3) also showed significant anti-tumor effects in human xenograft mouse models. Together, these results revealed opportunities to redirect the activity of CTL towards tumor cells that express EphA10 using the BsAb (EphA10/CD3), which could be tested in future clinical trials as a novel and potent therapeutic for breast cancer tumors.

Generation and characterization of a bispecific diabody targeting both EPH receptor A10 and CD3.

The EPH receptor A10 (EphA10) is up-regulated in breast cancer but is not normally expressed in healthy tissue, thus it has been suggested that EphA10 may be a useful target for cancer therapy. This study reports a diabody, an antibody derivative binding two different target molecules, EphA10 expressed in tumor cells and CD3 expressed in T cells, which showed T cell dependent-cytotoxicity. The diabody, which has His-tagged and FLAG-tagged chains, was expressed in Escherichia coli and purified in both heterodimer (Db-1) and homodimer (Db-2) formulations by liquid chromatography. Flow cytometry analysis using EphA10-expressing cells showed that binding activity of heterodimers was stronger than that of homodimers. Addition of diabodies to PBMC cultures resulted in T-cell mediated redirected lysis, and the bioactivity was consistent with the stronger binding activity of heterodimeric diabody formulations. Our results indicate that diabodies recognizing both EphA10 and CD3 could have a range of potential applications in cancer therapy, such as breast cancers that express the EPH receptor A10, especially triple negative breast cancer.

Ephrin receptor A10 is a promising drug target potentially useful for breast cancers including triple negative breast cancers.

Ephrin receptor A10 (EphA10) is a relatively uncharacterized protein which is expressed in many breast cancers but not expressed in normal breast tissues. Here, we examined the potential of EphA10 as a drug target in breast cancer. Immunohistochemical staining of clinical tissue sections revealed that EphA10 was expressed in various breast cancer subtypes, including triple negative breast cancers (TNBCs), with no expression observed in normal tissues apart from testis. Ligand-dependent proliferation was observed in EphA10-transfected MDA-MB-435 cells (MDA-MB-435(EphA10)) and native TNBC cells (MDA-MB-436). However, this phenomenon was not observed in parental MDA-MB-435 cells which express a low level of EphA10. Finally, tumor growth was significantly suppressed by administration of an anti-EphA10 monoclonal antibody in a xenograft mouse model. These results suggest that inhibition of EphA10 signaling may be a novel therapeutic option for management of breast cancer, including TNBCs which are currently not treated with molecularly targeted agents.

Eph receptor A10 has a potential as a target for a prostate cancer therapy.

We recently identified Eph receptor A10 (EphA10) as a novel breast cancer-specific protein. Moreover, we also showed that an in-house developed anti-EphA10 monoclonal antibody (mAb) significantly inhibited proliferation of breast cancer cells, suggesting EphA10 as a promising target for breast cancer therapy. However, the only other known report for EphA10 was its expression in the testis at the mRNA level. Therefore, the potency of EphA10 as a drug target against cancers other than the breast is not known. The expression of EphA10 in a wide variety of cancer cells was studied and the potential of EphA10 as a drug target was evaluated. Screening of EphA10 mRNA expression showed that EphA10 was overexpressed in breast cancer cell lines as well as in prostate and colon cancer cell lines. Thus, we focused on prostate cancers in which EphA10 expression was equivalent to that in breast cancers. As a result, EphA10 expression was clearly shown in clinical prostate tumor tissues as well as in cell lines at the mRNA and protein levels. In order to evaluate the potential of EphA10 as a drug target, we analyzed complement-dependent cytotoxicity effects of anti-EphA10 mAb and found that significant cytotoxicity was mediated by the expression of EphA10. Therefore, the idea was conceived that the overexpression of EphA10 in prostate cancers might have a potential as a target for prostate cancer therapy, and formed the basis for the studies reported here.

Effect of reduced EPHB4 expression in thymic epithelial cells on thymocyte development and peripheral T cell function.

The Eph kinase (EPH) and ephrin (EFN) families are involved in a broad range of developmental processes. Increasing evidence is demonstrating the important roles of EPHBs and EphrinBs in the immune system. In this study on epithelial cell-specific Ephb4 knockout (KO) mice, we investigated T-cell development and function after EPHB4 deletion. KO mice presented normal thymic weight and cellularity. Their thymocyte subpopulation percentages were in the normal range. KO mice had normal T-cell numbers and percentages in the spleen, and T cells were activated and proliferated normally upon TCR ligation. Furthermore, naïve spleen CD4 cells from KO and wild type mice were capable of differentiating, in a comparable manner, into Th1, Th17 and Treg cells. In vivo, KO mice mounted effective delayed type hypersensitivity responses, indicating that thymocytes develop normally in the absence of TEC EPHB4, and T cells derived from EPHB4-deleted thymic epithelian cells (TEC) have normal function. Our data suggest that heavy redundancy and promiscuous interaction between EPHs and EFNs compensate for the missing EPHB4 in TECs, and TEC EPHB4's role in T cell development might only be revealed if multiple EPHs are ablated simultaneously. We cannot exclude the possibility that (1) some immunological parameters not examined in this study are affected by the deletion; (2) the deletion is not complete due to the leaky Cre-LoxP system, and the remaining EPHB4 in TEC is sufficient for thymocyte development; or (3) EPHB4 expression in TEC is not required for T cell development and function.

A directed miniscreen for genes involved in the Drosophila anti-parasitoid immune response.

Drosophila larvae react against eggs from the endoparasitoid wasp Leptopilina boulardi by surrounding them in a multilayered cellular capsule. Once a wasp egg is recognized as foreign, circulating macrophage-like cells, known as plasmatocytes, adhere to the invader. After spreading around the wasp egg, plasmatocytes form cellular junctions between the cells, effectively separating the egg from the hemocoel. Next, a second sub-type of circulating immunosurveillance cell (hemocyte), known as lamellocytes, adhere to either the wasp egg or more likely the plasmatocytes surrounding the egg. From these events, it is obvious that adhesion and cell shape change are an essential part of Drosophila's cellular immune response against parasitoid wasp eggs. To date, very few genes have been described as being necessary for a proper anti-parasitization response in Drosophila. With this in mind, we performed a directed genetic miniscreen to discover new genes required for this response. Many of the genes with an encapsulation defect have mammalian homologues involved in cellular adhesion, wound healing, and thrombosis, including extracellular matrix proteins, cellular adhesion molecules, and small GTPases.

Activation of ephrin A proteins influences hematopoietic stem cell adhesion and trafficking patterns.

OBJECTIVE: To determine if Eph receptors and ephrins can modulate the homing of hematopoietic cells in a murine bone marrow transplantation model. MATERIALS AND METHODS: EphA and ephrin A gene expression by mouse hematopoietic stem cells and the progenitor cell line FDCP-1 was determined by real-time reverse transcription polymerase chain reaction and flow cytometry. The effect of ephrin A activation on adhesion of hematopoietic progenitors was determined by in vitro adhesion assays in which cells were exposed to fibronectin or vascular cell adhesion molecule-1 (VCAM-1) and an increasing gradient of immobilized EphA3-Fc. Adhesion to fibronectin and VCAM-1 was further investigated using soluble preclustered EphA3-Fc. We used soluble unclustered EphA3-Fc as an antagonist to block endogenous EphA-ephrin A interactions in vivo. The effect of injecting soluble EphA3-Fc on the mobilization of hematopoietic progenitor cells was examined. We determined the effect on short-term homing by pretreating bone marrow cells with EphA3-Fc or the control IgG before infusion into lethally irradiated mice. RESULTS: Preclustered and immobilized EphA3-Fc increased adhesion of progenitor cells and FDCP-1 to fibronectin and VCAM-1 (1.6- to 2-fold higher adhesion; p < 0.05) relative to control (0 µ/cm(2) EphA3-Fc extracellular molecule alone). Injection of the antagonist soluble EphA3-Fc increased progenitor cell and colony-forming unit-spleen cells in the peripheral blood (42% greater colony-forming unit in culture; p < 0.05, 3.8-fold higher colony-forming unit-spleen) relative to control. CONCLUSION: Treating bone marrow cells with EphA3-Fc resulted in a reduction by 31% in donor stem cells homing to the bone marrow and accumulation of donor cells in recipient spleens (50% greater than control) and greater recovery of donor stem cells from the peripheral blood.

Alterations in the thymocyte phenotype of EphB-deficient mice largely affect the double negative cell compartment.

In the present study, we have analysed the phenotype of EphB2 and/or EphB3 deficient thymocytes confirming and extending previous studies on the role of this family of molecules in T-cell differentiation. In all mutant thymuses statistically significant reduced cell contents were observed. This reduction of thymic cellularity correlated with increased proportions of apoptotic cells, largely both double negative (DN; CD4- CD8-) and double positive (CD4+ CD8+) cells, and decreased proportions of DN cycling cells. Adult deficient thymuses also showed increased proportions of DN cells but not significant variations in the percentages of other thymocyte subsets. In absolute terms, the thymocyte number decreased significantly in all thymocyte compartments from the DN3 (CD44- CD25+) cell stage onward, without variations in the numbers of both DN1 (CD44+ CD25-) and DN2 (CD44+ CD25+) cells. Remarkably, all these changes also occurred from the 15-day fetal EphB2 and/or EphB3 deficient mice, suggesting that adult phenotype results from the gradual accumulations of defects appearing early in the thymus ontogeny. As a reflection of thymus condition, a reduction in the number of T lymphocytes occurred in the peripheral blood and mesenteric lymph nodes, but not in spleen, maintaining the proportions of T-cell subsets defined by CD4/CD8 marker expression, in all cases.

EphrinB1-EphB signaling regulates thymocyte-epithelium interactions involved in functional T cell development.

The Eph and ephrin families are involved in numerous developmental processes. Recently, an increasing body of evidence has related these families with some aspects of T cell development. In the present study, we show that the addition of either EphB2-Fc or ephrinB1-Fc fusion proteins to fetal thymus organ cultures established from 17-day-old fetal mice decreases the numbers of both double-positive (CD4(+)CD8(+)) and single-positive (both CD4(+)CD8(-) and CD4(-)CD8(+)) thymocytes, in correlation with increased apoptosis. By using reaggregate thymus organ cultures formed by fetal thymic epithelial cells (TEC) and CD4(+)CD8(+) thymocytes, we have also demonstrated that ephrinB1-Fc proteins are able to disorganize the three-dimensional epithelial network that in vivo supports the T cell maturation, and to alter the thymocyte interactions. In addition, in an in vitro model, Eph/ephrinB-Fc treatment also decreases the formation of cell conjugates by CD4(+)CD8(+) thymocytes and TEC as well as the TCR-dependent signaling between both cell types. Finally, immobilized EphB2-Fc and ephrinB1-Fc modulate the anti-CD3 antibody-induced apoptosis of CD4(+)CD8(+) thymocytes in a process dependent on concentration. These results therefore support a role for Eph/ephrinB in the processes of development and selection of thymocytes as well as in the establishment of the three-dimensional organization of TEC.

Ephrin-A1 stimulates migration of CD8+CCR7+ T lymphocytes.

We have previously demonstrated that binding of ephrin-A1 to Eph receptors on human CD4+ T cells stimulates migration. Here, we show that a distinct population of CD8+ T lymphocytes, expressing the chemokine receptor CCR7, also binds ephrin-A1 and is stimulated to migrate after binding. The Eph receptor signaling pathway taking part in the migration event was here investigated. Induced tyrosine phosphorylation of several proteins was seen after ephrin-A1 binding. In particular, induced phosphorylation and kinase activity of the Src kinase family member Lck was observed. An Lck inhibitor inhibited ephrin-A1-induced migration, indicating the involvement of Lck in the migration event. In addition, we observed an induced association of the focal adhesion-like kinase proline-rich tyrosine kinase 2 (Pyk2) and the guanidine exchange factor Vav1 with Lck. PI3K inhibitors also inhibited migration, and studies in transfectants indicate an association of PI3K with EphA1. Further, ephrin-A1-induced migration could be related to the activation of Rho GTPases. This was also observed by using an inhibitor of the Rho-associated kinase ROCK, a downstream effector of Rho. Our results suggest that stimulation of Eph receptors on CD8+CCR7+ T cells leads to migration involving activation of Lck, Pyk2, PI3K, Vav1 and Rho GTPase.

EphA receptors inhibit anti-CD3-induced apoptosis in thymocytes.

The EphA receptor tyrosine kinases interact with membrane-bound ligands of the ephrin-A subfamily. Interaction induces EphA receptor oligomerization, tyrosine phosphorylation, and, as a result, EphA receptor signaling. EphA receptors have been shown to regulate cell survival, migration, and cell-cell and cell-matrix interactions. However, their functions in lymphoid cells are only beginning to be described. We show in this study that functional EphA receptors are expressed by murine thymocytes, including CD4(+)CD8(+), CD4(+)CD8(-), and CD4(-)CD8(+) subpopulations. We demonstrate that activation of EphA receptors by the ephrin-A1 ligand inhibits the anti-CD3-induced apoptosis of CD4(+)CD8(+) double-positive thymocytes. Furthermore, ephrin-A1 costimulation suppresses up-regulation of both the IL-2R alpha-chain (CD25) and early activation Ag CD69 and can block IL-2 production by CD4(+) single-positive cells. In agreement, EphA receptor activation in thymocytes also inhibits TCR-induced activation of the Ras-MAPK pathway. Our findings suggest that EphA receptor activation is antithetical to TCR signaling in thymocytes, and that the level of engagement by ephrin-A proteins on thymic APCs regulates thymocyte selection.

Mediolateral compartmentalization of the cerebellum is determined on the "birth date" of Purkinje cells.

The adult cerebellum is functionally compartmentalized into clusters along the mediolateral axis (M-L clusters), and a variety of molecular makers are expressed in specific subsets of M-L clusters. These M-L clusters appear to be the basic structure in which cerebellar functions are performed, but the mechanisms by which cerebellar mediolateral compartmentalization is established are still unclear. To address these questions, we examined the development of M-L clusters using replication-defective adenoviral vectors. The adenoviral vectors effectively introduced foreign genes into the neuronal progenitor cells of the cerebellum in a birth date-specific manner, allowing us to observe the native behavior of each cohort of birth date-related progenitor cells. When the adenoviral vectors were injected into the midbrain ventricle of mouse embryos on embryonic days 10.5 (E10.5), E11.5, and E12.5, the virally infected cerebellar progenitor cells developed into Purkinje cells. Notably, the Purkinje cells that shared the same birth date formed specific subsets of M-L clusters in the cerebellum. Each subset of M-L clusters displayed nested and, in part, mutually complementary patterns, and these patterns were unchanged from the late embryonic stage to adulthood, suggesting that Purkinje cell progenitors are fated to form specific subsets of M-L clusters after their birth between E10.5 and E12.5. This study represents the first such direct observation of Purkinje cell development. Moreover, we also show that there is a correlation between the M-L clusters established by the birth date-related Purkinje cells and the domains of engrailed-2, Wnt-7B, L7/pcp2, and EphA4 receptor tyrosine kinase expression.