Antibody Engineering & Therapeutics, held in December 2022, offered many opportunities to hear exciting and informative presentations by experts in the field, including K. Dane Wittrup, Professor of Chemical Engineering and Biological Engineering, Koch Institute for Integrative Cancer Research at the Massachusetts Institute of Technology, who discussed “Intratumorally Anchored Cytokine Therapy”.
As a result of advances in interventional radiologic, endoscopic, and laparoscopic procedures, most cancer tissues can now be accessed for a local injection directly into the tumor, with the aim of triggering an immune response that will act globally against cancer. Because of their anti-tumor activity and synergistic behavior, cytokines such as IL-2, IL-12, and interferons are currently considered for intratumoral therapies. Cytokine exposure is spatiotemporally programmed during immune responses, which means cytokines are present at certain places at certain times and in a particular order. As therapeutic agents, optimally they should be supplied in particular tissues at particular times for a specific duration. Direct cytokine injection into tumors has been attempted, but this approach was previously unsuccessful because of leakages out of the target tissue and systemic toxicity.
In his presentation, Prof. Wittrup described two strategies to localize cytokines to the target tissue that could allow for efficacious levels to be reached without overall toxicity. Both methods take advantage of retaining cytokines at the site of injection through interaction with collagen. [1,2,3] One approach relies on anchoring cytokines to the collagen-binding extracellular protein Lumican and the other relies on anchoring cytokines to the vaccine adjuvant Alum (aluminum hydroxide). Alum forms clusters of nanocrystals that are positively charged, which, when injected, tend to stay at the injection site. Because phosphorylated proteins bind very strongly to Alum, cytokines are fused to a proprietary peptide called alum peptide. The construct is co-expressed with kinase Fam20C, which attaches multiple phosphates to the peptide. Phosphorylated cytokine is then mixed with Alum and injected into the tumor site. Lumican binds to collagen type 1 and type 4, and Alum binds to collagen type 1. Lumican anchored molecules stay in place for 2 to 3 days. Alum anchoring increases tumor exposure to more than three weeks.
In Prof. Wittrup’s lab, one group of anti-PD-1-treated mice bearing B16-F10 tumors were injected intratumorally with IL-12 and IL-2, both linked to mouse serum albumin (MSA). The survival of this group was compared with the survival of a group of mice injected intratumorally with constructs containing Lumican-MSA-IL-12 and Lumican-MSA-IL-2. Mice were dosed every few days. Mice treated with anchored cytokines showed 80% survival, vs ~40% for those treated with the unanchored cytokine. Similar results were obtained with Alum, which was only given once. In both cases, no toxicity was observed with the anchored cytokines.
In collaboration with Prof. Timothy Fan, University of Illinois, Wittrup’s lab tested various anchored-cytokine therapies on companion animals. Dogs with oral melanoma were subject to experimental treatment with IL-12 fused to Lumican and albumin and IL-2 also fused to the same components. Prof. Wittrup highlighted results from three dogs, first treated with radiation and then with six doses of anchored cytokines, in which oral melanoma was cured. Gene expression analysis of the dogs showed signatures of enhanced inflammatory gene expression and signatures predictive of checkpoint blockade response.
The approach of targeted injection of anti-tumor agents was recently extended to type I interferon anchored on Alum  and to the alternatively spliced EIIIB (EDB) domain of fibronectin, which is not expressed in healthy adult tissue, but is widely expressed in a variety of cancers as a component of the tumor extracellular matrix. 
The technologies invented in Wittrup’s lab were licensed to Cullinan Oncology (Lumican-based anchoring) and Ankyra Therapeutics (Alum-based anchoring). Cullinan Oncology is also pursuing a combination of IL-2 and IL-12 because preclinical tumor models have shown they work synergistically. Both cytokines Induce orthogonal JAK and STAT signaling pathways, enhancing the production of other anti-tumoral cytokines, such as Interferon-gamma and TNF. Administered systemically, they both show profound toxicities. Cullinan Oncology used their platform technology in which IL-2 and IL-12 are fused to collagen-binding protein LAIR2 and albumin to produce the fusion protein CLN-617. The murine construct binds to collagen with nanomolar activity and shows the biological activity of both cytokines in free and collagen-bound states. Using the B16F10 tumor model in mice, it was demonstrated that intratumoral delivery was essential for efficacy. Intratumoral injection of the construct with the retention domain was not associated with toxicity (no weight loss). In other models, CT26 and MC38, which like B16F10, are checkpoint-refractory tumors, mice showed excellent response to CLN-617. Studies also demonstrated that treatment with CLN-617 in combination with anti-PD1 showed robust synergy.
Ankyra Therapeutics is pursuing intratumoral immunotherapy with aluminum hydroxide-tethered cytokines. Efforts are focused on IL-12, a dimer composed of p40 and p35 subunits. Alum-binding peptide is fused to p35. There are about five phosphates for protein. Alum retains phosphorylated cytokine for several days in the presence of serum. Cell-based assays revealed that IL-12 bound to Alum retains almost complete biological activity. Fluorescently labeled Alum complexed IL-12 stays at the injection site for up to 28 days. mIL-12-ABP causes regression of CT26 tumors in mice in a reproducible manner, while unmodified IL-12 injected intratumorally showed no efficacy in the same study. Efficacy was associated with a significant increase in the intratumoral CD8/Treg ratio. In mice, regression was observed in CT-26, MC38, A20, and B16F10 syngeneic tumor models. Alum-anchored IL-12 upregulated PD-1, suggesting that it may enhance the efficacy of checkpoint blockade in mice. Neither body weight loss nor immunogenicity was not seen in various mouse tumor models upon injection of anchored Il-12. Ankyra’s lead product candidate is ANK-101, an IL-12-based locally administered immunotherapy. Ankyra is currently advancing ANK-101 through Investigational New Drug (IND) enabling studies for the treatment of solid tumors and plans to submit an IND to the U.S. Food and Drug Administration in the third quarter of 2023. The company is also evaluating intratumoral alum-tethered canine interleukin-12 (cANK-101) in dogs with measurable oral malignant melanoma.
- Momin N, Mehta NK, Bennett NR, Ma L, Palmeri JR, Chinn MM, Lutz EA, Kang B, Irvine DJ, Spranger S, Wittrup KD. Anchoring of intratumorally administered cytokines to collagen safely potentiates systemic cancer immunotherapy. Sci Transl Med. 2019 Jun 26;11(498):eaaw2614. doi: 10.1126/PMID: 31243150.
- Lutz EA, Jailkhani N, Momin N, Huang Y, Sheen A, Kang BH, Wittrup KD, Hynes RO. Intratumoral nanobody-IL-2 fusions that bind the tumor extracellular matrix suppress solid tumor growth in mice. PNAS Nexus. 2022 Nov 3;1(5):pgac244. doi: 10.1093/pnasnexus/pgac244.
- Chang JYH, Agarwal Y, Rodrigues KA, Momin N, Ni K, Read BJ, Moyer TJ, Mehta NK, Silva M, Suh H, Melo MB, Wittrup KD, Irvine DJ. Co-Anchoring of Engineered Immunogen and Immunostimulatory Cytokines to Alum Promotes Enhanced-Humoral Immunity. Adv Ther (Weinh). 2022 Jul;5(7):2100235. doi: 10.1002/adtp.202100235.
- Lutz EA, Agarwal Y, Momin N, Cowles SC, Palmeri JR, Duong E, Hornet V, Sheen A, Lax BM, Rothschilds AM, Irvine DJ, Spranger S, Wittrup KD. Alum-anchored intratumoral retention improves the tolerability and antitumor efficacy of type I interferon therapies. Proc Natl Acad Sci U S A. 2022 Sep 6;119(36):e2205983119. doi: 10.1073/pnas.2205983119.