Immune checkpoint blockade exhibits considerable antitumor activity, but previous studies have reported instances of severe treatment-related adverse events. We sought to explore local immune checkpoint blockade, with an antibody (Ab) form that would be retained intra- or peritumorally, limiting systemic exposure. To accomplish this, we conjugated the checkpoint blockade Abs to an extracellular matrix (ECM)-super-affinity peptide derived from placenta growth factor-2 (PlGF-2). We show enhanced tissue retention and lower Ab concentrations in blood plasma after PlGF-2 conjugation, reducing systemic side effects such as the risk of autoimmune diabetes. Peritumoral injections of PlGF-2-anti-CTLA4 (cytotoxic T lymphocyte antigen 4) and PlGF-2-anti-PD-L1 (programmed death ligand 1) Abs delayed tumor growth and prolonged survival compared to the unmodified Abs in genetically engineered murine tumor models of melanoma and breast cancer. The PlGF-2-Abs increased tumor-infiltrating activated CD8 and CD4 T cells, resulting in a delay of distant tumor growth as well. This simple and translatable approach of engineered ECM-binding Abs may present a viable and safer approach in checkpoint blockade.

Cancer immunotherapy with immune checkpoint inhibitors (CPI) and interleukin (IL)-2 has demonstrated clinical efficacy but is frequently accompanied with severe adverse events caused by excessive and systemic immune system activation. Here, we addressed this need by targeting both the CPI antibodies anti-cytotoxic T-lymphocyte antigen 4 antibody (αCTLA4) + anti-programmed death-ligand 1 antibody (αPD-L1) and the cytokine IL-2 to tumors via conjugation (for the antibodies) or recombinant fusion (for the cytokine) to a collagen-binding domain (CBD) derived from the blood protein von Willebrand factor (VWF) A3 domain, harnessing the exposure of tumor stroma collagen to blood components due to the leakiness of the tumor vasculature. We show that intravenously (i.v.) administered CBD protein accumulated mainly in tumors. CBD conjugation or fusion decreases the systemic toxicity of both αCTLA4+αPD-L1 combination therapy and IL-2, for example eliminating hepatotoxicity with the CPI molecules and ameliorating pulmonary edema with IL-2. Both CBD-CPI and CBD-IL-2 suppressed tumor growth compared to their unmodified forms in multiple murine cancer models, and both CBD-CPI and CBD-IL-2 increased tumor-infiltrating CD8+ T cells. In an orthotopic breast tumor model, combination treatment with CPI and IL-2 eradicated tumors in 9 of 13 animals with the CBD-modified drugs, whereas it did so in only 1 of 13 animals with the unmodified drugs. Thus, the A3 domain of VWF can be used to improve safety and efficacy of systemically-administered tumor drugs with high translational promise.

Serum albumin (SA) is used as a carrier to deliver cytotoxic agents to tumors via passive targeting. To further improve SA’s tumor targeting capacity, we sought to develop an approach to retain SA-drug conjugates within tumors through a combination of passive and active targeting. SA was recombinantly fused with a collagen-binding domain (CBD) of von Willebrand factor to bind within the tumor stroma after extravasation due to tumor vascular permeability. Doxorubicin (Dox) was conjugated to the CBD-SA via a pH-sensitive linker. Dox-CBD-SA treatment significantly suppressed tumor growth compared to both Dox-SA and aldoxorubicin treatment in a mouse model of breast cancer. Dox-CBD-SA efficiently stimulated host antitumor immunity, resulting in the complete eradication of MC38 colon carcinoma when used in combination with anti–PD-1 checkpoint inhibitor. Dox-CBD-SA decreased adverse events compared to aldoxorubicin. Thus, engineered CBD-SA could be a versatile and clinically relevant drug conjugate carrier protein for treatment of solid tumors.

CD40 is an immune costimulatory receptor expressed by antigen-presenting cells. Agonistic anti-CD40 antibodies have demonstrated considerable antitumor effects yet can also elicit serious treatment-related adverse events, such as liver toxicity, including in man. We engineered a variant that binds extracellular matrix through a super-affinity peptide derived from placenta growth factor-2 (PlGF-2) to enhance anti-CD40's effects when administered locally. Peritumoral injection of PlGF-2-anti-CD40 antibody showed prolonged tissue retention at the injection site and substantially decreased systemic exposure, resulting in decreased liver toxicity. In four mouse tumor models, PlGF-2-anti-CD40 antibody demonstrated enhanced antitumor efficacy compared with its unmodified form and correlated with activated dendritic cells, B cells, and T cells in the tumor and in the tumor-draining lymph node. Moreover, in a genetically engineered melanoma model that does not respond to checkpoint inhibitors, PlGF-2-anti-CD40 antibody treatment enhanced T-cell infiltration into the tumors and slowed tumor growth. Together, these results demonstrate the marked therapeutic advantages of engineering matrix-binding domains onto agonistic anti-CD40 antibody as a therapeutic given by tumori-regional injection for cancer immunotherapy. Extracellular matrix-binding peptide conjugation to agonistic anti-CD40 antibody enhances antitumor efficacy and reduces treatment-related adverse events. .

Interleukin-4 (IL-4) suppresses the development of multiple sclerosis in a murine model of experimental autoimmune encephalomyelitis (EAE). Here, we show in mice with EAE that, compared with the administration of wild-type IL-4 or of the clinically approved drug fingolimod, the systemic injection of serum albumin (SA) fused to IL-4 better accumulates and persists in lymph nodes and in the spleen, leading to higher therapeutic efficacy and to the prevention of disease development in the majority of the mice. We also show that the SA-IL-4 fusion protein prevented immune-cell infiltration in the spinal cord, decreased integrin expression in antigen-specific CD4 + T cells, increased the number of granulocyte-like myeloid-derived suppressor cells (and their expression of programmed-death-ligand-1) in spinal-cord-draining lymph nodes, and decreased the number of T helper 17 cells, a pathogenic cell population in EAE. In mice with chronic EAE, SA-IL-4 inhibited immune-cell infiltration into the spinal cord and completely abrogated immune responses to myelin antigen in the spleen. The SA-IL-4 fusion protein may be prophylactically and therapeutically advantageous in the treatment of multiple sclerosis. Multiple sclerosis (MS) is a potentially disabling autoimmune disease that affects millions globally. Autoreactive immune cells home to the central nervous system (CNS) and cause demyelination and consequently focal damage to white matter 1 . Lymphocytes and macrophages that have infiltrated into the CNS cause axonal damage. Recent studies have shown that Th17 cells, activated in the secondary lymphoid organs (SLOs), migrate to the spinal cord and brain and play a crucial role in the disease development and severity of MS 2, 3 . Thus, inhibition of lymphocyte migration to the CNS and inducing an immune-suppressive microenvironment in the SLOs would provide an effective therapy for MS. FTY720 (fingolimod) and anti-integrin α4 antibody (natalizumab) are used in the clinic for treating MS 4, 5 , sequestering lymphocytes in the LNs and preventing them from reacting with autoantigens in target tissues. Experimental autoimmune encephalomyelitis (EAE) is a widely accepted murine model of MS, reflecting many features of disease progression and developmental mechanism, including lymphocyte migration to the CNS and demyelination.Interleukin (IL)-4 is a pleiotropic anti-inflammatory cytokine that differentiates naïve CD4 + T cells into a Th2 phenotype and results in decreased differentiation into Th1 and Th17 6 . IL-4 suppresses re-activation of committed Th17 cells 6 . Moreover, IL-4 polarizes macrophages toward the M2 phenotype, an antiinflammatory phenotype 7 . IL-4 has been reported to suppress EAE disease incidence and severity 8,9 ; this occurs not only through direct immuno-modulation, as a recent study has shown that intranasally administered IL-4 improved disease outcomes of EAE through IL-4 directly binding to neurons to promote regeneration 10 .Although IL-4 stimulates multiple pathways to suppress EAE, it has yet to be tr...