A tumor-associated macrophage-targeted immunocytokine leveraging T and NK cell synergy for potent cancer immunotherapy [CITE-seq]

Tumor-associated macrophages (TAMs) expressing the myeloid checkpoint TREM2 are key immunosuppressive cells in the tumor microenvironment (TME), driving tumor progression and contributing to poor prognosis in cancer patients. Due to their pivotal role, TAMs have emerged as a promising target for immunotherapies. However, current TAM-targeting monotherapies show limited efficacy, highlighting the need for strategies engaging multiple immune modalities. Here, we developed a new class of cancer immunotherapies: myeloid-targeted immunocytokines and NK-T cell enhancers (MiTEs). MiTEs are trans-acting immunocytokines with tumor-specific activation, allowing dual targeting of TAMs and lymphocytes by TREM2 antagonism and cytotoxic effector cell activation through IL-2. To avoid off-target toxicities, MiTEs contain an IL-2 masking moiety, which is cleaved by a TAM-specific protease. MiTEs demonstrate high efficacy in preclinical tumor models through extensive immune reprogramming spanning TAM, T and NK cell compartments. MiTEs show transformative potential for treating solid-cancers by inducing potent multi-arm anti-tumor immunity and minimizing toxicities. Overall design: Fresh tumor samples were obtained from discarded surgical resection specimens collected from consented patients under a Cooperative Human Tissue Network-CHTN IRB approved protocol. Tumor samples were cut into 1-2mm3 Patient-Derived Tumor Fragments (PDTFs) and cryopreserved in 1ml CryoStor CS10). Cryopreserved PDTFs from five distinct renal cell carcinoma patient tumors were rapidly thawed in a 37°C water bath and immediately placed in RPMI 1640 medium supplemented with 5% fetal bovine serum (FBS). Following thawing, individual tumor fragments were cultured for 48 hours as previously detailed73. For each donor, a total of 16 fragments per treatment condition were allocated to each of the four experimental arms: Isotype Control: anti-Respiratory Syncytial Virus (RSV) IgG1 (10 nM) and anti-Hen Egg Lysozyme (HEL) IgG4 (10 µg/mL), anti-PD-1 Monotherapy: anti-PD-1 (10 µg/mL; Cat: SIM0010) and anti-RSV-IgG1 (10 nM), MiTE144 Monotherapy: MITE144 (1 0nM) and anti-HEL-IgG4 (10 µg/mL), Combination Therapy: MITE144 (10 nM) and anti-PD-1 (10 µg/mL). Upon completion of the 48-hour culture period, 90ul of supernatants were collected and frozen for secreted protein analysis. Next, all fragments from the same donor and treatment arm were pooled and digested into single cell suspensions. Immune cells were enriched from the digested fragments using CD45 magnetic-activated cell sorting (MACS). The resulting CD45-enriched cell fractions were subsequently stained with a custom Immunai CITE-seq antibody panel (see STAR methods) and subjected to fluorescence-activated cell sorting (FACS) to isolate viable CD45 cells. Single-cell gene expression (GEX) and feature (CITE-seq) libraries were prepared from the sorted cells using the 10x Genomics Chromium Next GEM Single Cell 5' Kit v2. Libraries were sequenced on a Novaseq X Plus sequencer using a 300 cycle kit.