Weekly Digests
‹ Back to May

Hang on there! T cells bringing IL-12 are more tumor-reactive

May 11, 2022

IL-12 is of great interest for immunotherapy for its broad immune-activating effects. However, systemic treatment induces severe toxicities, limiting its application. Approaches to safely deliver cytokines to the tumor microenvironment are therefore warranted. Jones and Nardozzi et al. investigated a method to tether cytokines via antibodies to tumor-specific T cells used in adoptive therapy. Their results were recently published in Science Advances.

The researchers started by assessing surface receptors on T cells, including cytokine receptors, integrins and related surface adhesion molecules, and inhibitory receptors (e.g., PD-1) that could be used for tethering cytokines. Profiling T cells revealed that cytokine receptors and inhibitory receptors had low antibody binding capacity, even after activation, while integrin molecules showed a very high capacity, which further increased after stimulation. Several of these receptors may serve as ‘handles’ for cell surface cytokine tethering.

To assess this, cytokine “tethered fusion proteins” were constructed by linking a cytokine to a receptor-targeted antibody. IL-12 and IL-15 and antibodies directed against CD11a, CD18, or CD45 were created by fusing the cytokine to the C terminus of the antibody light chain in Fab fragments using a flexible polypeptide linker. Each cell surface receptor facilitated uniform dose-dependent loading of the cytokines onto the T cell surface. The highest tethering was achieved with CD45.

To assess whether the tethering resulted in sustained cytokine stimulus to the T cells, IL-15 tethered fusions were assessed for T cell expansion in vitro. Each fusion product resulted in dose-dependent T cell expansion, while blocking the target of the tethered fusion abolished proliferation. Higher affinity antibodies had stronger proliferation potency. Pulse incubation (1 r exposure followed by washing) with CD45-tethered IL-15, but not free IL-15, activated STAT5, and similar effects were found with IL-12 and STAT4. Conditioned supernatant from cells tethered with IL-12 showed that these cytokines could dissociate from the loaded T cell and activate other T cells.

IL-12 tethered to CD45 was assessed further to determine whether T cell activation could be enhanced without inducing off-target effects. Human CD8+ T cells specific for the HLA-A*02:01-restricted MART-1 peptide were generated by ex vivo stimulation of T cells from a healthy donor with autologous monocytic dendritic cells (mDCs) presenting the MART-1 peptide. After these cells were CD45-tethered with IL-12, they were co-cultured with a MART-1-expressing melanoma cell line (SK-MEL-5). This revealed a dose-dependent increase in cytolysis and IFNγ production, which did not occur when cells were cocultured with tumor cells lacking MART-1 expression.

To determine whether the cell-tethered IL-12 could improve antitumor activity in vivo, CD8+ T cells obtained from PMEL-1 TCR-transgenic mice were activated and expanded ex vivo and subsequently tethered with IL-12 before adoptive cell transfer (ACT) into mice bearing B16F10 tumors. These transferred cells were more efficacious than PMEL-1 T cells alone, and the delivery route did not affect this (intravenous or intratumoral). Jones and Nardozzi et al. then assessed the effects of tethered fusions with different CD45 binding affinities. Similar effects were found on tumor growth inhibition, suggesting the Fab antibody fragment used had sufficient affinity for antitumor effects.

Antitumor efficacy did not improve when untethered PMEL-1 T cells were coadministered with a four-fold higher dose of intravenous IL-12. Therefore, tethered IL-12 was more effective than coadministered systemic IL-12, and higher IL-12 loading resulted in more durable tumor regression. The tethered IL-12 also increased peak expansion of the transferred T cells and had increased long-term engraftment. The effects were dose-dependent on surface-tethering levels, as saturating and sub-saturating IL-12 loading had similar effects on engraftment.

These effects were observed in the absence of body weight loss, and systemic IFNγ production was moderate and transient. A higher cell dose resulted in transient peak body weight loss that reversed to baseline by day 7. Additionally, none of the doses resulted in liver or renal function blood marker increases, except for a temporary, moderate increase in alanine aminotransferase. Therefore, it was safe to repeat the treatment, which increased the durability of the antitumor response.

Next, the authors evaluated the antitumor effects of tethering T cells with endogenously stimulated T cells of variable reactivity obtained from tumor-draining lymph nodes of BALB/c mice bearing CT26 tumors. Isolated T cells were stimulated ex vivo to obtain a polyclonal population of tumor antigen-reactive T cells. CD45-tethering with IL-12 improved antitumor efficacy over ACT with untethered T cells. Smaller tumors were eradicated in 6/8 mice, while only 3/8 mice in the untethered group (systemic co-administration of IL-12) achieved complete remissions. Surviving mice also rejected a rechallenge, suggesting durable immunity was induced.

IL-12-tethered PMEL T cells demonstrated increased infiltration and tumoral IFNγ compared to T cells with untethered systemic IL-12. To assess how this affected the tumor immune microenvironment, myeloid lineage cells were profiled. Monocytic myeloid-derived suppressor cells (Mo-MDSCs) and tumor-associated macrophages (TAMs) were the most abundant myeloid populations in B16F10 tumors. Infiltration of T cells CD45-tethered with IL-12 reduced the infiltration of TAMs and recovered their expression of MHC-II, an activation marker associated with the M1-like phenotype. The tethered T cells did not affect Mo-MDSCs infiltration, but increased the expression of activation markers involved in antigen presentation and T cell costimulation (MHC-II and CD86) on these cells. Depleting Mo-MDSCs with anti-Ly6 antibody reduced the antitumor efficacy of the tethered T cells, suggesting the repolarization of Mo-MDSCs might be required for the efficacy of this therapy.

ACT with IL-12-tethered T cells resulted in the upregulation of PD-L1 on various immune cell populations, including Mo-MDSCs and TAMs. This effect was most pronounced in the repolarized MHC-II+CD86+ population of the Mo-MDSCs. When a neutralizing IFNγ antibody was used, there was less repolarization of Mo-MDSC and TAMs, and less PD-L1 expression induction.

The B16F10 model does not respond to PD-1/PD-L1 checkpoint blockade, even when combined with PMEL T cell ACT. However, combining ACT of IL-12-tethered T cells with anti-PD-L1 checkpoint blockade improved antitumor efficacy, resulting in more mice with complete responses.

This study shows the applicability of tethering IL-12 and IL-15 to adoptive T cell transfer (CARs, TILs, engineered TCRs) while overcoming toxicities associated with these cytokines, in particular, generating a pathway to capitalize on the immune-stimulating effect of IL-12. This provides a rationale for testing this therapy in combination with other immunotherapeutic strategies for various cancer types.

Write-up by Maartje Wouters, image by Lauren Hitchings.

Meet the researcher

This week, first author Douglas Jones 2nd answered our questions.

On the left, Douglas Jones 2nd; on the right, ruts from the Oregon Trail historic site.

What prompted you to tackle this research question?
IL-12 is a powerful cytokine that is ideally suited for combining with cell therapies. The challenge has been how to deliver it safely. We asked ourselves the question: could you control IL-12 dosing and focus its activity by directly tethering the cytokine to the surface of the T cell therapy?

What was the most surprising finding of this study for you?

We were impressed by how directly tethering IL-12 to the surface of the T cells prior to adoptive transfer drove such pronounced activity, specifically in the tumor. The activity was both more potent and more focused than what you get with conventional systemic IL-12 dosing. For example, it drove high levels of IFNγ (itself a potent immune-activating cytokine) specifically in the tumor, and repolarized mMDSCs and TAMs – two key suppressive tumor immune cell types – from suppressive to inflammatory phenotypes. Repertoire Immune Medicines, a clinical-stage cell therapy company, is currently investigating the use of cell-tethered IL-12 in an ongoing Phase 1 study of human papillomavirus-specific T cells.

What was the coolest thing you’ve learned (about) recently outside of work?

We recently took a road trip from Utah to Seattle and along the way we happened across a historic site of the Oregon Trail, which was a heavily used migration route in the mid-1800s. What was fascinating was that so many wagons had traveled this route that you could still see the ruts in the ground from this mass migration ~150 years ago. As someone who knew about the Oregon Trail mostly from the popular 1980s-era computer game, it was an intriguing piece of history brought to life.


Jones DS 2nd, Nardozzi JD, Sackton KL, Ahmad G, Christensen E, Ringgaard L, Chang DK, Jaehger DE, Konakondla JV, Wiinberg M, Stokes KL, Pratama A, Sauer K, Andresen TL. Cell surface-tethered IL-12 repolarizes the tumor immune microenvironment to enhance the efficacy of adoptive T cell therapy. Sci Adv. 2022 Apr 29.

In the Spotlight...

Addition of interleukin-2 overcomes resistance to neoadjuvant CTLA4 and PD1 blockade in ex vivo patient tumors

Kaptein, Jacoberger-Foissac, and Dimitriadis et al. showed that pretreatment biopsies of patients with stage III melanoma resistant to dual ICB (anti-CTLA-4 + anti-PD-1) had a low CD4+ T cell/IL-2 gene profile. Ex vivo, in fragments of dual ICB-resistant patients’ tumor biopsies, adding IL-2 to dual ICB induced T cell activation, particularly of FOXP3+ CD4+ T cells, and cytotoxic mediator production. In a metastatic triple-negative breast cancer mouse model, adding IL-2 to dual ICB boosted efficacy, activated and expanded the TCR repertoire of tumor-specific CD8+ Teff cells that were primed by CD4+ T cells, and increased proinflammatory TH1 cytokine production.

Contributed by Paula Hochman

Mesothelial cell-derived antigen-presenting cancer-associated fibroblasts induce expansion of regulatory T cells in pancreatic cancer

Huang et al. used scRNA sequencing and lineage tracing assays to determine the origin and functions of MHC-II+ antigen-presenting cancer-associated fibroblasts (apCAFs) in pancreatic ductal adenocarcinoma (PDA). As PDA progressed, the IL1-α and TGFβ pathways were responsible for inducing mesothelial cell differentiation to apCAFs that could directly induce naive CD4+ T cells to form suppressive Tregs in an antigen-specific manner. In a tumor model, treatment with a blocking mesothelin mAb inhibited apCAF formation, resulting in decreased Treg numbers, increased percentage of CD8+ T cells, and significantly lower tumor weight.

Contributed by Katherine Turner

Lymph node colonization induces tumor-immune tolerance to promote distant metastasis

Investigating the relationship between lymph node (LN) and distant metastases, Reticker-Flynn et al. developed a mouse model of LN metastasis by repeatedly inoculating B16 melanoma tumors, collecting LN-metastatic cells, and re-inoculating these cells. Mice with 6th-gen (versus parental) primary tumors had increased lung metastasis after i.v. injection of the parental line. Across generations, transcriptomic and epigenetic changes in IFN-related genes and increases in MHC-I/B2M and PD-L1 expression conferred resistance to NK and T cells. Tumor antigen-specific Tregs were expanded in metastatic LNs and supported lung colonization.

Contributed by Alex Najibi

In vivo labeling reveals continuous trafficking of TCF-1+ T cells between tumor and lymphoid tissue

Using the Kaede genetic model and photoactivation to directly label tumor immune cells in situ, Li, Tuong, and Dean et al. provided the first in vivo analysis of T cell recruitment, retention, and egress over time in a tumor model. CD8+ T cells retained within the tumors rapidly (<72 hrs) developed an exhausted phenotype. The TCF-1+ CD8+ T cell niche in tumors was highly dynamic, with constant recruitment of new cells, differentiation to effector cells, and egress of the “stem-like” cells back to lymphoid tissue. PD-L1 blockade reinvigorated exhausted CD8+ T cells retained within the tumor and enhanced effector functions of newly recruited CD8+ T cells to enhance the antitumor responses.

Contributed by Shishir Pant

Immunogenicity and therapeutic targeting of a public neoantigen derived from mutated PIK3CA

Chandran et al. isolated 4 distinct TCRs from 4 healthy donors following in vitro stimulation of naive T cells with autologous DCs expressing 4 mutations in PIK3CA (a driver oncogene). Each TCR recognized H1047L PIK3CA, a mutation harbored in 77.9% of 131 tumors surveyed. Recognition was restricted by HLA - A*03:01, an allele expressed in 20-28% of individuals. Signal transduction through these TCRs mediated inflammatory and lytic responses. H1047L PIK3CA-specific T cells were detected in 4 (29%) of the 14 patients analyzed. Cells expressing TCR that recognized H1047L PIK3CA inhibited tumor growth in NSG mice.

Contributed by Margot O’Toole

CAR T cell killing requires the IFNγR pathway in solid but not liquid tumours

Larson et al. conducted a genome-wide CRISPR knockout screen in a glioblastoma cell line, and identified that loss of interferon-γ receptor (IFNγR) signaling pathway genes (IFNGR1, JAK1 or JAK2) conferred resistance to CAR T cell killing. IFNγR1 blockade/IFNγR1-KO and JAK2-KO showed resistance to CAR T cells in several solid tumors, but not in hematologic malignancies, in a CAR T target-independent manner. Loss of IFNγR1 in glioblastoma cells downregulated ICAM1 expression and reduced CAR T cell–tumor cell binding duration and avidity. Antibody-mediated blockade of ICAM-1 increased resistance to CAR T cell killing in solid tumors.

Contributed by Shishir Pant

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.