{
  "abstract": "Background CD40, a TNFR family member, is a critical mediator of humoral and cellular immunity and agonist CD40 antibodies have shown therapeutic benefit in preclinical tumor models. However, clinical development of traditional CD40 antibodies has been hampered by systemic toxicity. We hypothesized that alum anchoring of CD40 agonist mAbs would promote receptor clustering needed for T cell activation and result in an improved therapeutic window. ANK-201 is a next-generation CD40 monoclonal agonistic antibody (CD40 mAb) structured to reduce toxicity and improve efficacy.Methods CD40 mAb was genetically fused at its c-terminus to a short alum-binding peptide (ABP) and co-expressed with the kinase Fam20C. Phosphorylation of selected ABP amino acids was measured by malachite green assay. The phosphorylated CD40-ABP was complexed with alum, and the complexes were characterized for in vitro potency in reporter assay and in vivo for safety (body weight and liver enzymes), immune responses, and efficacy in hCD40 C57BL/6 mice using a bilateral flank MC38 tumor model. Various doses were tested (range, 20-40 mg) by intratumoral delivery either one or two doses separated by 7 days. Independent experiments harvested tumor and blood at various time points for PK, cytokine and IHC assays. Tumor size was compared using Student’s t-test between groups and multiple comparisons utilized two-way ANOVA testing. For survival studies, log-rank (Mantel-Cox) tests were used.Results CD40-ABP is phosphorylated when co-expressed with Fam20C and the level of phosphorylation is proportional to the number of ABP repeats. Addition of ABP attenuated signaling compared to parental CD40mAb in a phosphate-level dependent manner. However, anchoring of CD40mAb-ABP fusions to alum potentiated CD40 signaling, leading to enhanced signaling vs. parental mAb suggesting the role of alum anchoring in promoting receptor clustering. Two doses of IT administered CD40-ABP alone or in complexation with alum is sufficient to induce robust monotherapy efficacy in syngeneic MC38 mouse model in hCD40 C57BL/6 mice (p<0.01 by day 9 post first dose). Locally administered CD40-ABP is further able to prime a systemic immune response leading to abscopal effects. At these doses, non-anchored antibody induced transient elevations in AST/ALT, whereas ANK-201 did not.Conclusions This is the first report of anchoring a humanized mAb to alum. ANK-201 was associated with enhanced CD40 signaling in vitro, generation of anti-tumor immunity, and an improved therapeutic window. These findings validate alum anchoring as a novel delivery strategy and position ANK-201 as a promising candidate for clinical translation.",
  "authors": [
    {
      "affiliations": [
        "Ankyra Therapeutics, Cambridge, MA, USA"
      ],
      "name": "Sailaja Battula"
    },
    {
      "affiliations": [
        "Ankyra Therapeutics, Quincy, MA, USA"
      ],
      "name": "Kanika Jain"
    },
    {
      "affiliations": [
        "Memorial Sloan Kettering Cancer Center, New York, NY, USA"
      ],
      "name": "Danny Khalil"
    },
    {
      "affiliations": [
        "Weill Cornell Medicine, New York, NY, USA"
      ],
      "name": "Taha Merghoub"
    },
    {
      "affiliations": [
        "Weill Cornell Medicine, New York, NY, USA"
      ],
      "name": "Jedd D Wolchok"
    },
    {
      "affiliations": [
        "Ankyra Therapeutics, Cambridge, MA, USA"
      ],
      "name": "Howard Kaufman"
    }
  ],
  "title": "999 Alum-anchored CD40 antibody drug conjugate (ANK-201) promotes local anti-tumor immunity and therapeutic activity without systemic toxicity in preclinical tumor models",
  "uid": "1f0c25cf-cf27-5fff-bed0-82561588daf5"
}