Weekly Digests
‹ Back to October

TIM-3 blockade offers new hope for incurable childhood cancer, DIPG

October 11, 2023

Mainly due to its location in the pons, a part of the brainstem that controls many of the body's vital functions, and its infiltrative nature, diffuse intrinsic pontine glioma (DIPG) is one of the most devastating and challenging childhood cancers. Currently, oncologists can only offer radiotherapy to stabilize the tumor and reduce symptoms. Sadly, over 90% of the young patients die within two years of diagnosis. In a recent paper published in Cancer Cell, Ausejo-Mauleon et al. suggest TIM-3 blockade as a possible effective treatment option for DIPG, and push for future clinical evaluation.

First, to confirm the potential for therapeutic targeting of TIM-3, Ausejo-Mauleon and team performed in silico analysis of DIPG patient samples, validating TIM-3 expression in 94% of cases and no expression in healthy human brains. Relative expression levels of TIM-3 were higher than those of other immune checkpoint molecules, such as CTLA-4, PD-1, LAG-3, and TIGIT, and expression of the main TIM-3 ligand, galectin-9, was also increased. Single-cell analysis revealed TIM-3 expression in all three different DIPG tumor cell populations: astrocyte-like, oligodendrocyte-like, and oligodendrocyte precursor cell (OPC)-like. OPC-like cells showed the highest expression of TIM-3, and gene enrichment analysis suggested a direct role of TIM-3 in tumor cell survival and tumorigenesis.

Three murine DIPG cells lines with TIM-3 knockout (KO) were viable, but showed decreased metabolic activity, proliferation, migratory activity, and capability to form colonies. Immunocompetent mice orthotopically implanted with TIM-3 KO cell lines in the pons of the brainstem had a longer median survival than mice implanted with parental cells. This effect was due to a slower growth rate of TIM-3 KO DIPG cells, and not due to differences in immune infiltrates. Transcriptomic analysis revealed downregulation and/or decreased phosphorylation of components of the MAPK and PI3K-AKT signaling pathways in TIM-3 KO DIPG cells compared to parental cells.

In human DIPG cell lines, genetic ablation of TIM-3 was lethal, and knockdown of TIM-3 expression with doxycycline-inducible shRNA or TIM-3 blockade with anti-TIM-3 (BMS-986258) reduced viability of the evaluated human DIPG cell lines. In vivo, immunodeficient Rag2 KO mice orthotopically implanted with a human DIPG cell line and treated with anti-TIM-3 showedda longer median survival of 78 days, compared to 63 days for mice treated with a control antibody.

In addition to the DIPG tumor cells themselves, TIM-3 is also expressed, at even higher levels, in the immune compartment of DIPG, as revealed by in silico analysis of scRNAseq data for DIPG patients. The tumor microenvironment (TME) of DIPG is mainly composed of microglia and macrophages, with nearly negligible numbers of T cells. Multiplex immunofluorescence analysis of 9 patient samples demonstrated TIM-3 expression in 54% of CD68+ myeloid cells, 39% of CD3+ T cells, and 44% of H3K27M+ DIPG tumor cells. Of note, TIM-3 displayed a differential expression pattern depending on H3 status (higher in mutated H3). In microglia, TIM-3 expression correlated with genes involved in the activation of an immune response. When the TME of DIPG in an immunocompetent model was assessed 10 days after implantation, TIM-3 expression was found on 85% of CD8+ T cells, 79% of DCs, 69% of microglia, 66% of macrophages, 63% of Tregs, 61% of CD4+ T cells, 55% of monocytes, and less than 50% of NK cells and granulocytes. The highest expression intensity was observed for DCs, macrophages, microglia, and Treg populations.

Ausejo-Mauleon and colleagues treated immunocompetent DIPG mice with anti-TIM-3 on a hybrid schedule, in which an initial intratumoral administration three days after tumor implantation was followed by two systemic doses. Biodistribution analysis by SPECT scan revealed antibody in the brainstem, which declined over the course of one week, and antibody in the tumor-draining cervical lymph nodes, which are located in the neck region. Treatment led to an increase in median overall survival, with 50% long-term survivors, which were also able to reject a new tumor implantation. When this tumor rechallenge experiment was performed in the presence of FTY720, abrogating T cell traffic from the lymph nodes, survivors were still able to reject the repeated tumor challenge, suggesting that resident memory cells were responsible for tumor rejection. Of note, systemic-only administration also resulted in a significant, but less pronounced, therapeutic benefit. Treatment was well tolerated, and weight loss or hepatic toxicity were not observed. Seven days after the first treatment dose, the tumor site was characterized by a 3-fold increase in microglia with a proliferative phenotype and by increases in the percentage of proliferative, non-exhausted NK cells and proliferative DCs. Intratumoral levels of IL-1β and IFNγ, and chemoattractants CCL2, CCL5, and CXCL10 were increased, as were the number of infiltrating tumor-specific CD8+ T cells. CD8+ and CD4+ T cells displayed an activated, cytotoxic IFNγ+TNFα+ or GrzB+TNFα+ phenotype upon restimulation. By day 14, augmentation of an adaptive immune response was observed, with increases in the ratios of proinflammatory CD8+ T cell:Treg, CD4+ T cell:Treg and CD8+ T cell:macrophage.

Concurrent depletion of NK, CD4+, and CD8+ T cells resulted in a partial loss of the therapeutic effect of anti-TIM-3 treatment, and only administration of the CSF1R inhibitor, PLX-3397, led to a total loss of survival benefit. PLX-3397 preferentially depleted macrophages and decreased the number of active, proliferating Ki67+MHC-II+ microglia, and CD4+ and CD8+ T cells. At the same time, the upregulated levels of IL-1β, IFNγ, CCL2, CCL5, and CXCL10 were lost. Thus, TIM-3 blockade resulted in the activation of proinflammatory macrophages and microglia, which induced pro-inflammatory cytokines and chemokines that in turn enhanced antitumor T cell responses.

Next, the researchers set out to study the kinetics of different immune populations in the deep cervical and superficial cervical tumor draining lymph nodes (LN). At day 7 after the first treatment dose, deep cervical LNs of anti-TIM-3-treated mice had significantly higher numbers of CD45+ immune cells due to increases in the CD4+ and B cell populations, and to a lesser extent, in CD8+ T cells and DCs. DCs in the deep cervical LN displayed a significantly lower expression of PD-L1, and DCs in the superficial cervical LN had a mature, antigen-presenting CD80+MHCII+ phenotype. In particular, the deep, but also the superficial cervical LN demonstrated significantly higher percentages of activated, cytotoxic CD4+ and CD8+ T cells with a Ki67+TNFα+ and GrzB+TNFα+ phenotype upon anti-TIM-3 treatment. The researchers assumed that effective antigen presentation by DCs mediated T cell activation and proliferation.

Interestingly, the value of targeting TIM-3 appeared to be unique to DIPG. Despite expression of TIM-3 in GBM patients, valuation of anti-TIM-3 in one orthotopic TIM-3+ glioblastoma model (CT-2A) led to no therapeutic benefit. And although PD-1 expression was also confirmed in the orthotopic DIPG model used here, anti-PD-1 treatment was not effective.

Here, Ausejo-Mauleon and colleagues showed that TIM-3 blockade can have a direct effect on DIPG tumor cells. Furthermore, in immunocompetent, orthotopic DIPG models, anti-TIM-3 transformed the TME toward a more proinflammatory milieu and promoted a coordinated action of mainly macrophages, microglia, and CD8+ T cells to eliminate tumor cells and produce a durable memory response. This study establishes TIM-3 blockade as a new therapeutic approach for DIPG, and urges for the clinical evaluation of this new glimmer of hope.

Write-up by Ute Burkhardt, image by Lauren Hitchings and Ute Burkhardt

Meet the researcher

This week, first author Iker Ausejo-Mauleon answered our questions. 

Laboratory of Advanced Therapies for Pediatric Solid Tumors and collaborators of the Cancer Center Clínica Universidad de Navarra that helps in this work. First author Iker Ausejo-Mauleon (navy blue t-shirt) and lead author Marta M Alonso (yellow t-shirt) in the center of the photo

What was the most surprising finding of this study for you?
What surprised us most was the high expression of a typical T cell immune checkpoint molecule such as TIM-3 in these pediatric brain tumors in comparison with other molecules, such as PD-1, CTLA-4, LAG-3, or TIGIT. Although we already knew about TIM-3 expression in myeloid cells, we were surprised by its high expression in tumor-infiltrating macrophages and microglia. Moreover, it was fascinating to find out that this high expression in many immune cell types meant that blocking it increased survival and led to long-term survivors and immune memory in orthotopic models of DIPG, where we had not previously seen these good results with other therapies.

What is the outlook?
TIM-3 blockade emerges as an exciting alternative to classical immune checkpoints, such as PD-1, that did not obtain the desired results in clinical trials for DIPG and other pediatric brain tumors. Moreover, the lack of other effective therapies for these devastating pediatric brain cancers makes these preclinical results especially promising in the field, and offers a strong rationale for initiating a clinical trial with an anti-TIM-3 antibody for the treatment of DIPG.

What was the coolest thing you’ve learned (about) recently outside of work?
In the last months, I have learned how important it is to take care of myself mentally and to disconnect from my science life as well. For this, I like to go running, biking, climbing, and skiing in the mountains near my village (Arroniz, Spain) and in the Pyrenees. Discovering new paths, adventures, and new challenges every day helps me to disconnect from the pressure that science research puts on us.


Ausejo-Mauleon I, Labiano S, de la Nava D, Laspidea V, Zalacain M, Marrodán L, García-Moure M, González-Huarriz M, Hervás-Corpión I, Dhandapani L, Vicent S, Collantes M, Peñuelas I, Becher OJ, Filbin MG, Jiang L, Labelle J, de Biagi-Junior CAO, Nazarian J, Laternser S, Phoenix TN, van der Lugt J, Kranendonk M, Hoodendijk R, Mueller S, De Andrea C, Anderson AC, Guruceaga E, Koschmann C, Yadak VN, Gállego Pérez-Larraya J, Patiño-García A, Pastor F, Alonso MM. TIM-3 blockade in diffuse intrinsic pontine glioma models promotes tumor regression and antitumor immune memory. Cancer Cell. 2023 Oct 3.

In the Spotlight...

Cooperative CAR targeting to selectively eliminate AML and minimize escape

AML cells are heterogeneous and share protein expression with healthy HSPCs. To target AML and spare HSPC toxicity, Haubner et al. combined a sensitivity-tuned CAR for ADGRE2 (an antigen expressed poorly on HSPCs and more highly on AML) with a 4-1BB chimeric costimulatory receptor (CCR) binding CLEC12A (not expressed on HSPCs) to specifically augment CAR signaling toward ADGRE2loCLEC12A+ AML, but not CLEC12A- HPSCs. Compared to ADGRE2-alone CAR T cells, the combinatorial CAR T cells better controlled ADGRE2loCLEC12A+ AML cell lines and patient-derived xenografts in humanized mice, while minimally reducing HSPC numbers.

Contributed by Alex Najibi

Antigen-loaded Monocyte Administration and Flt3 Ligand Augment the Antitumor Efficacy of Immune Checkpoint Blockade in a Murine Melanoma Model

In a mouse OVA⁺ melanoma model, D’Anniballe et al. showed that the effects of OVA-monocyte vaccination, anti-PD-1, anti-PD-L1 or anti-CTLA-4 on s.c. tumor volume were greatest when ICB was combined with monocyte vaccination. Flt3 ligand (FltL3) treatment prior to OVA-monocyte vaccination expanded splenic DCs (>40X) and circulating OVA-specific T cells (2X). Flt3 + OVA-monocyte vaccination inhibited OVA⁺ tumor growth more than ICB alone. OVA-monocyte vaccination + Flt3L + anti-PD-1 induced the greatest increases in the frequency of circulating Ag-specific CD8⁺ T cells (including those secreting IFNγ) and reductions of OVA⁺ tumor growth.

Contributed by Paula Hochman

Camel nanobody-based B7-H3 CAR-T cells show high efficacy against large solid tumours

Li et al. showed aberrant high expression of the B7-H3 Ig4 isoform by multiple solid human tumor types, and isolated nanobodies specific for major B7-H3 domains from camel VHH phage libraries. Human CAR T cells based on nanobodies specific for novel B7-H3 IgC epitopes showed the strongest antigen binding and lysis of B7-H3+ human neuroblastoma and PDAC cells in vitro. In mice, CAR T cells infiltrated and mediated strong antitumor activity against large and metastatic neuroblastoma and PDAC xenografts. CAR T cells collected from spleens showed antigen-specific upregulation of transcripts related to translation, protein synthesis, and T cell metabolism ex vivo.

Contributed by Paula Hochman

Identification of neoepitope reactive T-cell receptors guided by HLA-A*03:01 and HLA-A*11:01 immunopeptidomics

By performing mass spectrometry (MS) to survey peptides eluted from cell lines transduced to express a single HLA-I allele frequently expressed in the US population, and a minigene encoding one of 10 commonly occurring tumor-specific mutations, four HLA-A*11:01- and five HLA-A*03:01-restricted naturally processed neoepitopes were identified. Corresponding HLA-I Tg mice were immunized with MS-defined minimal peptides, and murine TCRs were isolated that were active at nM peptide concentrations and were highly mutation-specific for KRAS G13D, PIK3CA E545K, EGFR L858R, BRAF V600E (HLA- A*11:01), and KRAS G12V, EGFR L858R, BRAF V600E (HLA-A*03:01).

Contributed by Paula Hochman

Epitope-engineered human hematopoietic stem cells are shielded from CD123-targeted immunotherapy

Most antibody-based treatments for myeloid malignancies, including AML, result in myelotoxicity due to sharing of target antigens by human hematopoietic stem and progenitor cells (HSPCs). To shield HSPCs, Marone, Landmann, Devaux, and Lepore et al. engineered CD123 (IL-3Rα) variants with a single aa substitution in a mAb epitope. The variants were structurally and functionally similar to wtCD123, but were resistant to mAb-targeted ADCC, Ab–drug conjugates and CAR T cell therapy. Transplanted into mice, epitope-engineered HSPCs engrafted with long-term reconstitution potential, suggesting this approach could enable targeted immunotherapy while rebuilding a functional hematopoietic system.

Contributed by Katherine Turner

Engineering of dendritic cell bispecific extracellular vesicles for tumor-targeting immunotherapy

Xu et al. engineered PD-L1-deleted DCs to produce bispecific extracellular vesicles (EVs) that expressed anti-human CD19 scFv and mouse PD-1 (EV-PD1-aCD19). Upon i.v. injection of mice implanted s.c. with human CD19-overexpressing murine CT6 colorectal carcinoma, which generally responds poorly to anti-PD-1, the nano-sized EV-PD1-aCD19, but not anti-CD19 scFV-engineered DCs, infiltrated and were retained in huCD19+ solid tumors, reshaped the TME and tumor draining LN milieu from immunologically cold to reactive, and induced tumor regression. EV-PD1-aCD19 also targeted circulating tumor-derived (PD-L1+) EVs to reverse pre-metastatic lung niches.

Contributed by Paula Hochman

Everything New this Week In...

Close Modal

Small change for you. Big change for us!

This Thanksgiving season, show your support for cancer research by donating your change.

In less than a minute, link your credit card with our partner RoundUp App.

Every purchase you make with that card will be rounded up and the change will be donated to ACIR.

All transactions are securely made through Stripe.