January 16, 2022

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Intratumoral injection of cytokines bound to alum adjuvant elicits more potent and safer local and systemic antitumor responses

Intratumoral injection of cytokines bound to alum adjuvant elicits more potent and safer local and systemic antitumor responses



 

Intratumoral injection of cytokines bound to alum adjuvant elicits more potent and safer local and systemic antitumor responses.

 

Immune checkpoint blockade therapy, while improving progression-free survival in cancer patients , typically elicits durable effects in only a minority of patients due to high levels of immunosuppression in the tumor microenvironment ( TME ) .

 

Although combinations with inflammatory cytokines or immune agonists can alleviate immunosuppression, systemic administration often leads to severe immune-related adverse events, and therefore, therapeutic strategies that target cytokine effects to the tumor microenvironment have received much attention .

 

With advances in interventional radiology, endoscopy, and laparoscopic surgical procedures, intratumoral ( it ) drug delivery is now available for most lesions in the human body.

 

However, it injection of therapeutics does not ensure drug persistence in the TME, as free drug is rapidly cleared through lymphatics or tumor vasculature, leading to toxic accumulation in the circulation [1, 2] .

 

Previous studies have reported a strategy to fuse cytokines to collagen-binding proteins to enhance maintenance in the TME following it administration and reduce the toxicity of these potent drugs, but the drugs are spatiotemporally regulated by the distribution of collagen in the tumor. and updated controls.

 

The FDA-approved vaccine adjuvant aluminum hydroxide (alum adjuvant) has a nearly 100-year history of safe use in humans and is administered to millions of people each year in more than 20 vaccine formulations.

Aluminum hydroxide adjuvants consist of micron-sized aggregates of nanorod-like nanocrystals that form a “depot” that persists for weeks at the injection site in the tissue [3] .

In addition, phosphorylated proteins bind tightly to aluminum hydroxide through ligand exchange reactions with surface hydroxyl groups, thereby enabling the bound molecules to exist stably in vivo [4, 5] .

 

On January 10, 2021, K. Dane Wittrup ‘s  team and Darrell J. Irvine ‘s team from MIT collaborated to publish a paper titled Intratumourally injected alum-tethered cytokines elicit potent and safer local and systemic anticancer immunity in the journal Nature Biomedical Engineering .

Anticancer immunity article, researchers developed a method for intracellular site-specific protein phosphorylation to synthesize biologically active proteins fused to phosphorylated alum-binding peptide ( ABP ) tags .

This method is used to generate a series of ABP-labeled cytokines, which can be retained in the tumor for more than a week after being adsorbed on alum, which can increase the anti-tumor efficacy and eliminate the systemic toxicity caused by the injection of free drugs. and control of distant untreated lesions.

Thus, intratumoral therapy with alum-anchored cytokines represents a safer strategy to improve local and systemic anticancer immunity.

 

Intratumoral injection of cytokines bound to alum adjuvant elicits more potent and safer local and systemic antitumor responses

 

The kinase Fam20C is responsible for the phosphorylation of most mammalian secreted phosphorylated proteomes, and co-expression of Fam20C with a therapeutic protein containing a kinase consensus motif (ABP) will result in ABP-specific phosphorylation (Figure 1) .

Based on this principle, the authors purified monomeric phosphorylated IL-12-ABP protein, and the splenocyte activation assay after co-incubation in vitro showed that IL-12-ABP-p could still maintain biological activity and a high level of activity when adsorbed to alum particles. stability.

 

Intratumoral injection of cytokines bound to alum adjuvant elicits more potent and safer local and systemic antitumor responses

Figure 1. Schematic diagram of purified phosphorylated IL-12-ABP fusion protein

 

To test the biodistribution and pharmacokinetics of alum-bound IL-12-ABP-p in vivo, the authors labeled alum particles and IL-12-ABP-p with different AlexaFluor dyes and injected them into small cells by subcutaneous injection. in murine B16F10 melanoma.

Alum particles co-localized with IL-12-ABP-p and were distributed throughout the tumor bed, and IL-12 remained in tumors 144 hours after injection compared to the injected free IL-12-ABP-p group increased >400-fold (Fig. 2) . Mice injected with alum-conjugated IL-12-ABP-p did not experience the significant increases in body weight, serum IFN-γ, and alanine aminotransferase (ALT, indicating liver toxicity) exhibited by the free group .

Furthermore, intratumoral injection of alum-IL-12-ABP-p combined with systemic anti-PD1 treatment elicited complete responses in 12 of 23 mice, whereas treatment not anchored with alum caused only modest tumor growth delays and There was no long-term survival, and similar effects were achieved in the MC38 colon cancer model.

These data suggest that anchoring of IL-12 to alum via phosphorylated ABP can achieve potent it retention and therapeutic efficacy, and significantly enhance systemic tolerance.

 

Intratumoral injection of cytokines bound to alum adjuvant elicits more potent and safer local and systemic antitumor responses

Figure 2. AlexaFluor dye showing co-localization and persistent retention of alum particles with IL-12-ABP-p in B16F10 melanoma

 

Beyond the primary tumor, clinical efficacy also depends on its ability to promote systemic antitumor responses to control distant untreated lesions and metastases.

We established Ag104A tumors or B16F10 tumors on both sides of mice and treated only one of these tumors with a single dose of alum-IL-12-ABP-p, notably, in the absence of systemic immune checkpoint blockade therapy.

In cases where this treatment can eliminate established distal untreated tumors.

 

To further identify the cellular and molecular effectors associated with persistent it IL12, the authors quantified cytokines and chemokines produced in the TME of B16F10 tumors.

After three days of alum-IL-12-ABP-p treatment, IL-6, TNF-α, IL-1β, CXCL9 and CXCL10 were significantly up-regulated, and the production of IFN-γ increased 5 times compared with the control group.

The efficacy and control of tumor growth of Alum-IL-12-ABP-p were completely abolished if IFN-γ neutralizing antibodies were added.

In addition, adaptive immunity-related CD8+ T cells, CD4+ T cells, and innate immunity-related NK cells and NKT cells were significantly induced. Next, the authors analyzed B16F10 tumor-draining lymph nodes (dLNs) stably expressing the fluorescent protein Zsgreen43 by flow cytometry .

Several antigen-presenting myeloid cell types remained highly activated after treatment with alum-IL-12-ABP-p, while presenting antigens in tumor dLNs and enhancing tumor-specific T cell priming.

 

Collectively, this study developed an intracellular phosphorylation strategy for transient transfection of Fam20C-expressing stable cell lines with any ABP fusion protein (cytokine) , in situ seeding in combination with alum particles, and Durable and effective local and systemic antitumor responses and reduced toxicity in a variety of mouse tumor models.

The customizability of the ABP approach could also allow one to precisely control and tune protein release from alum in vivo, and ultimately to program the duration of treatment for the most potent antitumor effects.

 

 

Reference:

https://doi.org/10.1038/s41551-021-00831-9

Intratumoral injection of cytokines bound to alum adjuvant elicits more potent and safer local and systemic antitumor responses

(source:internet, reference only)


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