Weekly Digests
‹ Back to January

New year, new IL-12

January 19, 2022

Strategies to activate tumor-infiltrating lymphocytes (TILs) can support existing cancer therapies and overcome resistance mechanisms such as exhaustion. One such approach, the inflammatory cytokine IL-12, can activate T cells and may be linked to patient survival, but its application is limited by toxicity and a short half-life. Recently published in Science Immunology, Xue and Moon et al. overcame these drawbacks by engineering an improved IL-12 therapy.

Xue, Moon, and the team began by increasing the stability of IL-12 in circulation. To do so, they linked IL-12 to a human Fc (IL-12–Fc) in two forms: a heterodimer and homodimer. These modifications did not alter IL-12 bioactivity (via induction of STAT4 signaling in a reporter cell line), and in fact, improved MC38 antitumor efficacy over recombinant mouse IL-12. Hetero-IL-12–Fc more effectively treated MC38 and B16F10 tumors and maintained its serum concentration compared to homo-IL-12–Fc, and was selected for further studies. However, both versions induced inflammatory serum cytokines after i.p. delivery.

To tackle the toxicity issue, the researchers aimed to formulate hetero-IL-12–Fc as a prodrug that would ideally circulate in an inactive state, but become activated in the tumor microenvironment (TME). Matrix metalloproteinases (MMPs), a class of enzymes overexpressed in the TME, presented an opportunity to enable tumor-specific IL-12 activity through their local enzymatic activity. The team linked hetero-IL-12–Fc through an MMP14-cleavable peptide to a masking domain, based upon the IL-12 receptor, which would already bind IL-12 with high affinity. Theoretically, this would block IL-12 functionality, unless the linker was cleaved. This construct, named “pro-IL-12,” indeed had reduced bioactivity, which was restored after incubation with MMP14 and was dependent on the presence of the cleavable linker.

Xue and Moon et al. next characterized how pro-IL-12 behaved in vivo. The engineered IL-12 extended survival in mice bearing MC38, B16F10, or 4T1 tumors, comparable to the non-masked hetero-IL-12–Fc. However, relative to hetero-IL-12–Fc, pro-IL-12 reduced serum cytokine levels, weight loss, and liver damage, providing the same therapeutic benefit with a clear improvement of toxicity.

Next, the researchers questioned which cell types could be responding to pro-IL-12. In a series of depletion experiments, they found that NK cells, neutrophils, and macrophages were not necessary for pro-IL-12’s efficacy, suggesting that innate immune cells are not the key responders. On the other hand, pro-IL-12 was ineffective in Rag1-/- mice, or mice depleted of CD8+ (but not CD4+) T cells, pointing to CD8+ T cells as the target. The likely responders were more specifically CD8+ T cells within the TME, as pro-IL-12 was still effective in mice treated with the lymphocyte egress inhibitor FTY720 (which would prevent migration from lymphoid organs to the tumor). The team found that pro-IL-12 increased tumor antigen-specific and IFNγ-expressing CD8+ T cells, and decreased exhaustion markers such as PD-1 and Tim3. Interestingly, co-administration of an anti-IFNγ antibody abrogated the therapeutic effect, demonstrating that the therapeutic response to pro-IL-12 involves IFNγ and CD8+ T cells.

The team next investigated the specific cellular source of IFNγ. Bulk splenocytes were treated with hetero-IL-12-Fc ex vivo, stimulating IFNγ production as expected. Consistent with the in vivo depletion results, depletion of NK cells from the splenocytes did not reduce IFNγ production, while loss of CD4+ T cells led to a slight decrease, and loss of CD8+ T cells led to a substantial decrease, again highlighting CD8+ T cells as the main responders. To test whether CD8+ T cells bound IL-12 themselves, the researchers co-cultured mixtures of CD4+ and CD8+ T cells taken either from WT or IL-12R-/- (IL-12 non-responsive) mice. Interestingly, WT CD8+ T cells mixed with IL-12R-/- CD4+ T cells and stimulated with hetero-IL-12-Fc had comparable IFNγ production to the fully WT control, while WT CD4+ T cells mixed with IL-12R-/- CD8+ T cells did not secrete IFNγ. These results were substantiated in vivo. When the cell mixtures were transferred into Rag1-/- mice bearing MC38 tumors, only the mice with IL-12R-competent CD8+ T cells responded to pro-IL-12 therapy. Altogether, these results suggested that CD8+ T cells directly sense IL-12, triggering IFNγ production.

Given the ability of pro-IL-12 to activate IFNγ production in the TME, the researchers tested whether pro-IL-12 could supplement other cancer therapies. In the TUBO HER2+ breast cancer model, pro-IL-12 along with an EGFR tyrosine kinase inhibitor (TKI) substantially improved tumor control and survival over pro-IL-12 or TKI alone. Furthermore, the combination of pro-IL-12 and anti-PD-L1 reduced tumor growth and improved survival in MC38-bearing mice – again better than either therapy alone – indicating pro-IL-12’s potential in combination therapies to activate the TME.

Finally, the team considered the application of this approach to human IL-12. They constructed a human hetero-IL-12–Fc, which induced IFNγ production from human PBMCs, and developed a prodrug version that became activated after MMP14 exposure. In a humanized mouse model, the human IL-12 prodrug slowed growth of a human colon cancer cell line, with reduced toxicity compared to the hetero-IL-12–Fc version. These results suggested potential for this same therapeutic strategy in human cancer patients.

Altogether, Xue, Moon, and the team engineered a more stable and less toxic IL-12 that could regress tumors and synergize with other cancer therapies. The approach to make this cytokine fit for use could be extended to a variety of other cytokines and chemokines to support antitumor efficacy. More broadly, the utility of a strategy that can heat up the TME may support a variety of combination therapies in treating diverse cancers.

Write-up by Alex Najibi, image by Lauren Hitchings

References:

Xue D., Moon B., Liao J., Guo J., Zou Z., Han Y., Cao S., Wang Y., Fu Y.X., Peng H. A tumor-specific pro-IL-12 activates preexisting cytotoxic T cells to control established tumors. Sci Immunol. 2022 Jan 7.

In the Spotlight...

CLINICAL TRIAL: Relatlimab and Nivolumab versus Nivolumab in Untreated Advanced Melanoma

Based on a randomized, double-blind phase 2-3 trial, Tawbi et al. reported that relatlimab (anti-LAG3) and nivolumab (anti-PD-1) as combination therapy induced longer PFS (10.1 months) compared to nivolumab alone (4.6 months) in patients with previously untreated metastatic or unresectable melanoma. At 12 months, the PFS was 47.7% for the combination and 36% for nivolumab, with grade 3 or 4 treatment-related adverse events slightly higher in the combination group (18.9% vs 9.7%). Treatment with the combination therapy showed longer PFS regardless of BRAF mutation status, LAG3 or PD-L1 expression, metastasis stage, or tumor burden.

Contributed by Shishir Pant

Temporal single-cell tracing reveals clonal revival and expansion of precursor exhausted T cells during anti-PD-1 therapy in lung cancer

Using scRNAseq with TCR-based lineage tracking, Liu, Hu, and Feng et al. characterized the temporal dynamics of T cells from dissociated tumor biopsies of 36 patients with NSCLC following combination therapy with anti-PD-1 and chemotherapy. Two precursor exhausted T cell states (Texp) with high GZMK expression and low expression of exhaustion markers were identified. Responsive tumors showed an increase in Texp cells with high expression of CXCL13. Increased Texp cells in responsive tumors following treatment was likely due to the local expansion and infiltration of pre-existing and new clonotypes from peripheral Texp cells, but not reinvigoration of terminally exhausted T cells.

Contributed by Shishir Pant

An In Vivo Screen to Identify Short Peptide Mimotopes with Enhanced Antitumor Immunogenicity

He et al. developed a two-step screening method using antitumor immunity as a readout to identify preferred substitution position and substituted amino acids to generate enhanced TAA mimotopes (e-mimotopes) following immunization with a CPQ adjuvant system. E-mimotopes (Trp2-8C and 8Y) identified using the Trp2180–188 peptide showed improved Trp2-specific cytotoxic T cell phenotypes, TCR affinity (but not MHC-I affinity), antitumor immunity in prophylactic and therapeutic tumor models, and enhanced tumor control with ICB using anti- PD-1 and anti-CTLA-4 antibodies. The screening method also generated e-mimotopes for AH1 and WT1 epitopes.

Contributed by Shishir Pant

A novel oral metronomic chemotherapy provokes tumor specific immunity resulting in colon cancer eradication in combination with anti-PD-1 therapy

Using oral oxaliplatin and pemetrexed formulations with improved bioavailability, Maharjan and Choi et al. showed that immunogenic death of a colon carcinoma line was induced better by metronomic (MCT) than maximum tolerated dosing, with less immuno- and overall toxicity. MCT dosing more effectively boosted tumor cell MHC expression, tumor sensitivity to cytotoxic T cells, activation of and antigen presentation by DCs in tumor-dLNs, numbers of PD-1- and proliferating TILs, IFNγ expression by tumor-specific TILs and splenocytes, macrophage migration to LNs, and polarization to M1 TAMs in tumor-bearing mice. MCT combined with anti-PD-1 eradicated tumors and induced specific memory.

Contributed by Paula Hochman

Local radiotherapy and E7 RNA-LPX vaccination show enhanced therapeutic efficacy in preclinical models of HPV16+ cancer

Salomon et al. showed in two mouse models that a single subtherapeutic i.v. dose of HPV-E7 RNA lipoplex vaccine synergized with high-dose local radiotherapy (LRT) to promote rejection of HPV16 E6/E7+ tumors. This depended on total LRT dose, not dose fractionation. Vaccine with or without LRT increased intratumoral CD8+ cells, CD4+ cells, NK cells, and PD-L1+CD11b+ myeloid cells, and MHC-I and PD-L1 on tumor cells. E7-specific CD8+ T cells were enriched in tumors, spleens, and LNs, and upon ex vivo antigen restimulation, made IFNγ/TNFα. LRT increased tumor cell apoptosis and reduced tumor mass and hypoxia to sensitize tumor cells to antigen-specific T cell killing.

Contributed by Paula Hochman

Anticancer efficacy of monotherapy with antibodies to SIRPα/SIRPβ1 mediated by induction of antitumorigenic macrophages

Sakamoto et al. studied the antitumor mechanism of mAb M1, which binds murine SIRPα and SIRPβ1 on myeloid cells and blocks the interaction of SIRPα with CD47 on cancer cells. Monotherapy with M1 inhibited growth of bladder and mammary cancer cells in immunocompetent mice by promotion of macrophage-mediated phagocytosis and killing and by polarization of tumor-infiltrating macrophages towards a TNFα-expressing anti-tumorigenic M1-like phenotype. SIRPα ablation had no effect on efficacy, whereas knockdown of SIRβ1 in macrophages attenuated M1 activity, revealing SIRPβ1 as a potential cancer target.

Contributed by Katherine Turner

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.