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Laminin, as a key component of the basement membrane extracellular matrix (ECM), regulates tissue morphogenesis. Here, we show that multiple laminin isoforms promiscuously bind to growth factors (GFs) with high affinity, through their heparin-binding domains (HBDs) located in the α chain laminin-type G (LG) domains. These domains also bind to syndecan cell-surface receptors, promoting attachment of fibroblasts and endothelial cells. We explore the application of these multifunctional laminin HBDs in wound healing in the type-2 diabetic mouse. We demonstrate that covalent incorporation of laminin HBDs into fibrin matrices improves retention of GFs and significantly enhances the efficacy of vascular endothelial cell growth factor (VEGF-A165) and platelet-derived growth factor (PDGF-BB) in promoting wound healing in vivo, under conditions where the GFs alone in fibrin are inefficacious. This laminin HBD peptide may be clinically useful by improving biomaterial matrices as both GF reservoirs and cell scaffolds, leading to effective tissue regeneration. Laminins are important regulators of epidermal wound healing. Here, the authors show that laminins bind to multiple growth factors via their heparin-binding domains, and that incorporation of these domains into fibrin matrices increases growth factor retention, promoting wound healing in type 2 diabetic mouse models.

Checkpoint-inhibitor (CPI) immunotherapy has achieved remarkable clinical success, yet its efficacy in ‘immunologically cold’ tumours has been modest. Interleukin-12 (IL-12) is a powerful cytokine that activates the innate and adaptive arms of the immune system; however, the administration of IL-12 has been associated with immune-related adverse events. Here we show that, after intravenous administration of a collagen-binding domain fused to IL-12 (CBD–IL-12) in mice bearing aggressive mouse tumours, CBD–IL-12 accumulates in the tumour stroma due to exposed collagen in the disordered tumour vasculature. In comparison with the administration of unmodified IL-12, CBD–IL-12 induced sustained intratumoural levels of interferon-γ, substantially reduced its systemic levels as well as organ damage and provided superior anticancer efficacy, eliciting complete regression of CPI-unresponsive breast tumours. Furthermore, CBD–IL-12 potently synergized with CPI to eradicate large established melanomas, induced antigen-specific immunological memory and controlled tumour growth in a genetically engineered mouse model of melanoma. CBD–IL-12 may potentiate CPI immunotherapy for immunologically cold tumours. A collagen-binding interleukin-12 formulation intravenously injected into mice bearing established immunologically cold mouse tumours led to marked tumour remission, particularly when combined with checkpoint-inhibitor immunotherapy.

Background Although disease in a majority of rheumatoid arthritis (RA) patients is often initially limited to one or a few joints, currently approved medications including anti-tumor necrosis factor-α antibody (α-TNF) are injected systemically. Given that α-TNF systemic injection typically does not cure RA and involves risk of treatment-related adverse events, one possible approach to enhance therapeutic efficacy and reduce α-TNF systemic exposure is to retain the antibodies in arthritic joints after local administration. The aim of this study was to evaluate the approach of conferring extracellular matrix (ECM) binding affinity to α-TNF antibodies in a RA model. Methods α-TNF was chemically conjugated with a promiscuous ECM-binding peptide derived from placenta growth factor 2 (PlGF-2 123-144 ). The binding activity of PlGF-2 123-144 -conjugated α-TNF (PlGF-2 123-144 -α-TNF) against ECM proteins was assessed by ELISA and by immunostaining on human cartilage specimens. The effect of conjugation on antibody function was assessed as a neutralizing activity against osteoclast differentiation. Retention at the injection site and therapeutic efficacy of PlGF-2 123-144 -α-TNF were tested in a collagen antibody-induced arthritis (CAIA) model in the mouse. Results PlGF-2 123-144 peptide conjugation conferred α-TNF with affinity to ECM proteins without impairment of antigen recognition. PlGF-2 123-144 -α-TNF locally injected at a paw in the CAIA model was retained for at least 96 h at the injection site, whereas unmodified α-TNF was dispersed rapidly after injection. Local treatment with unmodified α-TNF did not suppress the arthritis score relative to isotype controls. By contrast, local administration of PlGF-2 123-144 -α-TNF suppressed arthritis development almost completely in the treated paw even at a 1000× lower dose. Conclusion These data demonstrate that retention of α-TNF in arthritic joints can suppress arthritis development and enhance therapeutic efficacy. This simple bioengineering approach of ECM-binding peptide conjugation offers a powerful and clinically translational approach to treat RA.

Interleukin-4 (IL-4) suppresses the development of multiple sclerosis in a murine model of experimental autoimmune encephalomyelitis (EAE). Here, we show that, in mice with EAE, the accumulation and persistence in the lymph nodes and spleen of a systemically administered serum albumin (SA)–IL-4 fusion protein leads to higher efficacy in preventing disease development than the administration of wild-type IL-4 or of the clinically approved drug fingolimod. We also show that the SA–IL-4 fusion protein prevents immune-cell infiltration in the spinal cord, decreases integrin expression in antigen-specific CD4 + T cells, increases the number of granulocyte-like myeloid-derived suppressor cells (and their expression of programmed-death-ligand-1) in spinal cord-draining lymph nodes and decreases the number of T helper 17 cells, a pathogenic cell population in EAE. In mice with chronic EAE, SA–IL-4 inhibits immune-cell infiltration into the spinal cord and completely abrogates 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. The enhanced accumulation and residence time of systemically administered interleukin-4 fused to serum albumin in lymph nodes and in the spleen prevents the development of multiple sclerosis in mice.

Non-healing wounds have a negative impact on quality of life and account for many cases of amputation and even early death among patients. Diabetic patients are the predominate population affected by these non-healing wounds. Despite the significant clinical demand, treatment with biologics has not broadly impacted clinical care. Interleukin-4 (IL-4) is a potent modulator of the immune system, capable of skewing macrophages towards a pro-regeneration phenotype (M2) and promoting angiogenesis, but can be toxic after frequent administration and is limited by its short half-life and low bioavailability. Here, we demonstrate the design and characterization of an engineered recombinant interleukin-4 construct. We utilize this collagen-binding, serum albumin-fused IL-4 variant (CBD-SA-IL-4) delivered in a hyaluronic acid (HA)-based gel for localized application of IL-4 to dermal wounds in a type 2 diabetic mouse model known for poor healing as proof-of-concept for improved tissue repair. Our studies indicate that CBD-SA-IL-4 is retained within the wound and can modulate the wound microenvironment through induction of M2 macrophages and angiogenesis. CBD-SA-IL-4 treatment significantly accelerated wound healing compared to native IL-4 and HA vehicle treatment without inducing systemic side effects. This CBD-SA-IL-4 construct can address the underlying immune dysfunction present in the non-healing wound, leading to more effective tissue healing in the clinic.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Checkpoint inhibitor (CPI) immunotherapy has achieved remarkable clinical success, yet its efficacy in ‘immunologically cold’ tumours has been modest. Interleukin (IL)-12 is a powerful cytokine that activates the innate and adaptive arms of the immune system, yet its administration has been associated with immune-related adverse events. Here, we show that the intravenous administration of a collagen-binding domain fused to IL-12 (CBD–IL-12) in mice bearing aggressive murine tumours accumulates in the tumour stroma, owing to exposed collagen in the disordered tumour vasculature. In comparison with the administration of unmodified IL-12, CBD–IL-12 induced sustained intratumoral levels of interferon-γ, markedly reduced its systemic levels as well as organ damage, and led to superior anticancer efficacy, eliciting complete regression of CPI-unresponsive breast tumours. Furthermore, CBD–IL-12 potently synergized with CPI to eradicate large established melanoma, induced antigen-specific immunological memory, and controlled tumour growth in a genetically engineered mouse model of melanoma. CBD–IL-12 may potentiate CPI immunotherapy for immunologically cold tumours.

Laminin, as a key component of the basement membrane extracellular matrix (ECM), regulates tissue morphogenesis. We show that multiple laminin isoforms promiscuously bind to growth factors (GFs) with high affinity, through their heparin binding domains (HBDs) located in the a chain LG domains. Interestingly, these domains also bind to syndecan cell-surface receptors, promoting attachment of fibroblasts and endothelial cells. We next explore application of these multifunctional laminin HBDs in skin healing in the type 2 diabetic mouse. We demonstrate that covalent incorporation of laminin HBDs into fibrin matrix enables the slow-release of GFs. Incorporation of the 33043-3067 laminin HBD significantly enhances in vivo wound-healing efficacy of vascular endothelial cell growth factor (VEGF)-A165 and platelet-derived growth factor (PDGF)-BB, under conditions where the GFs alone in fibrin are inefficacious. This laminin HBD peptide may be clinically useful by improving biomaterials as both GF reservoirs and cell scaffolds, leading to effective tissue regeneration.