Folate-Hapten–Mediated Immunotherapy Synergizes with Vascular Endothelial Growth Factor Receptor Inhibitors in Treating Murine Models of Cancer

Abstract

The over-expression of folate receptors (FR) on many human cancers has led to the development of folate-linked drugs for the imaging and therapy of FR-expressing cancers. In a recent phase 1 clinical trial of late stage renal cell carcinoma patients, folate was exploited to deliver an immunogenic hapten, fluorescein, to FR+ tumor cells in an effort to render the cancer cells more immunogenic. Although >50% of the patients showed prolonged stable disease, all patients eventually progressed, suggesting that the folate-hapten immunotherapy was insufficient by itself to treat the cancer. In an effort to identify a companion therapy that might augment the folate-hapten immunotherapy, we explored co-administration of two approved cancer drugs that had been previously shown to also stimulate the immune system. We report that sunitinib and axitinib (VEGF receptor inhibitors that simultaneously mitigate immune suppression) synergize with the folate-hapten targeted immunotherapy to reduce tumor growth in three different syngeneic murine tumor models. We further demonstrate that the combination therapy not only enhances tumor infiltration of CD4+ and CD8+ effector cells, but surprisingly reduces tumor neovasculogenesis more than predicted. Subsequent investigation of the mechanism for this unexpected suppression of neovasculogenesis revealed that it is independent of elimination of any tumor cells, but instead likely derives from a reduction in the numbers of FR+ tumor-associated macrophages and myeloid derived suppressor cells, i.e. immunosuppressive cells that release significant quantities of VEGF. These data suggest that a reduction in stromal cells of myeloid origin can inhibit tumor growth by suppressing neovasculogenesis.

pH-Dependent Grafting of Cancer Cells with Antigenic Epitopes Promotes Selective Antibody-Mediated Cytotoxicity

“…We project that the exogenous approach may provide improved orthogonality and afford finer spatiotemporal control of patient antibody levels. We chose FITC as the exogenous epitope on the basis of the established precedence of folic acid–FITC conjugates being capable of inducing humoral and cellular immunity against FITC-decorated cancer cells in vitro and in vivo . ,,, Similar to DNP-modified agents, a panel of pHLIP conjugates were assembled by installing the FITC epitope at the N-terminus. A design deviation from the DNP series was the inclusion of a polyethylene glycol (PEG) spacer of variable lengths between pHLIP and FITC (Scheme S2).…”

“…Decoration of cancer cells with antigens leads to the recruitment of antibodies (opsonization) and subsequent killing of cancer cells through complement-dependent cytotoxicity (CDC) or antibody-dependent cellular cytotoxicity (ADCC) pathways. These agents have shown exciting anticancer activities in vitro and tumor reduction in vivo (1,(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15).…”

Covalent Stabilization of Antibody Recruitment Enhances Immune Recognition of Cancer Targets

“…ARMs against anti-DNP are often validated in cell assays using commercially available model antibodies with artificially high hapten binding affinity ( K d(Ab) ≤ 10 –8 M). ARM validation in mouse tumor models uses vaccination protocols to produce the hapten-specific antibody of interest. ,,, Vaccination, however, produces artificially high concentrations (≥10 –6 M) of high-affinity anti-hapten antibody, compared to what is found naturally in unboosted human serum. There exists an unmet need to both: (a) determine whether ARMs possess sufficient antibody binding affinity to maximize antibody recruitment to cancer cells and (b) define their maximal therapeutic potential, in the context of relevant human serum antibodies at native concentrations.…”

Methods to Validate Binding and Kinetics of “Proximity‐Inducing” Covalent Immune‐Recruiting Molecules

“…An emerging strategy in immunotherapy involves the use of synthetic small molecules to engage, redirect, or leverage the immune system for a therapeutic effect such as anticancer activity. One approach uses antibody engagers (AEs), also known as ARMs (Bandara, Bates, Lu, Hoylman, & Low, 2017; Murelli, Zhang, Michel, Jorgensen, & Spiegel, 2009; Sheridan, Hudon, Hank, Sondel, & Kiessling, 2014). These are bifunctional small molecules containing an antibody‐binding domain (ABD) and a target‐binding domain (TBD; Figure 1).…”