Collagen-binding Domain Research Articles

Design and characterization of a tumor-retained interleukin-2 cytokine/antibody fusion protein for cancer immunotherapy

Abstract Interleukin-2 (IL-2) is a pleiotropic cytokine with clinical utility in stimulating anti-cancer immunity. However, its efficacy is limited by its short serum half-life, systemic toxicity, and concurrent activation of immunostimulatory and immunosuppressive immune cells. IL-2 can signal down an intermediate-affinity receptor consisting of IL-2 receptor β (IL-2Rβ) and the common gamma chain (γc) or a high-affinity receptor that also includes IL-2Rα. Regulatory T (Treg) cells express high levels of IL-2Rα making them more responsive to IL-2 than natural killer (NK), naïve/resting effector T (Teff), and memory T cells. To extend the half-life and selectively bias the immunostimulatory functions of IL-2 in a unimolecular format, we engineered a single-chain fusion protein, F10 immunocytokine (F10 IC), that intramolecularly fuses IL-2 to an anti-IL-2 antibody that sterically blocks IL-2Rα binding and allosterically enhances IL-2Rβ binding. However, preclinical data suggested a narrow therapeutic index (TI) for F10 IC. To improve the TI of F10 IC we designed a format of F10 IC poised for intratumoral delivery and retention. We fused collagen binding domains (CBD) to F10 IC in various topographies (F10 IC-CBD) to exploit the abundance and exposure of collagen in the tumor microenvironment. With intratumoral delivery, F10 IC-CBD is retained in the tumor and exhibits a therapeutically favorable pharmacokinetic profile. Furthermore, F10 IC-CBD increases and prolongs intratumoral immune cell infiltration, significantly slows tumor progression, minimizes F10 IC toxicity and synergizes with anti-PD-1 blockade in syngeneic mouse tumor models. We have illustrated the potential of F10 IC-CBD as a potent tumor-agnostic cancer therapy. Supported by grants from the NIH (R01 EB029341, T32 AI007417), Melanoma Research Alliance, Willowcroft Foundation and a Sanofi iAward.

Structural and biochemical characterization of Clostridium perfringens pili protein B collagen-binding domains.

Sortase-mediated pili are flexible rod proteins composed of major and minor/tip pilins, playing important roles in the initial adhesion of bacterial cells to host tissues. The pilus shaft is formed by covalent polymerization of major pilins, and the minor/tip pilin is covalently attached to the tip of the shaft involved in adhesion to the host cell. The Gram-positive bacterium Clostridium perfringens has a major pilin, and a minor/tip pilin (CppB) with the collagen-binding motif. Here, we report X-ray structures of CppB collagen-binding domains, collagen-binding assays and mutagenesis analysis, demonstrating that CppB collagen-binding domains adopt an L-shaped structure in open form, and that a small β-sheet unique to CppB provides a scaffold for a favourable binding site for collagen peptide.

Targeting advanced prostate cancer with STEAP1 chimeric antigen receptor T cell and tumor-localized IL-12 immunotherapy

Six transmembrane epithelial antigen of the prostate 1 (STEAP1) is a cell surface antigen for therapeutic targeting in prostate cancer. Here, we report broad expression of STEAP1 relative to prostate-specific membrane antigen (PSMA) in lethal metastatic prostate cancers and the development of a STEAP1-directed chimeric antigen receptor (CAR) T cell therapy. STEAP1 CAR T cells demonstrate reactivity in low antigen density, antitumor activity across metastatic prostate cancer models, and safety in a human STEAP1 knock-in mouse model. STEAP1 antigen escape is a recurrent mechanism of treatment resistance and is associated with diminished tumor antigen processing and presentation. The application of tumor-localized interleukin-12 (IL-12) therapy in the form of a collagen binding domain (CBD)-IL-12 fusion protein combined with STEAP1 CAR T cell therapy enhances antitumor efficacy by remodeling the immunologically cold tumor microenvironment of prostate cancer and combating STEAP1 antigen escape through the engagement of host immunity and epitope spreading.

Abstract 2331: Loss of LRRC15 in osteosarcoma cells disrupts extracellular matrix integration, intercellular communication, and differentiation

Abstract Background: Leucine rich repeat containing 15 (LRRC15) is a 581 amino acid type 1 transmembrane protein that contains 15 leucine rich repeats. Cysteine clusters flank the LRR domain that functions as the collagen binding domain. LRRC15 has been found to be upregulated during osteogenic differentiation of mesenchymal stem cells, induced by TGFβ, and highly expressed in multiple solid tumors, notably in osteosarcoma (1). Antibody drug conjugates targeting LRRC15 have shown promising activity in pre-clinical studies (2). Results: To investigate the function of LRRC15 in the osteosarcoma microenvironment, we generated inducible shRNA knockdown (KD) derivatives targeting 3’UTR and ORF regions of LRRC15 of four osteosarcoma cell lines. Using these models, we characterized the biological properties of LRRC15 knockdown under 2-D and 3-D growth conditions. We found that LRRC15 KD in 2-D culture resulted in reduced adhesion to extracellular matrix (ECM) components and impaired cell migration. In 3-D spheroid culture, LRRC15 KD severely impacted scaffold-free spheroid compaction and invasion of spheroids embedded in Matrigel. Dysregulation of multiple ECM, cell adhesion and communication pathways were revealed by transcriptomic analysis from samples collected at time points during the LRRC15 KD. Of interest, several key transcription factors including RUNX2, CTNNB1, and SP7 were downregulated in LRRC15 deficient cells. Conclusion: Our comprehensive results establish the importance of LRRC15 as a critical cell-adhesion molecule essential for the maintenance of the osteogenic intercellular communication, invasion and differentiation revealing novel aspects of osteosarcoma cell-biology. Reference:1.Purcell JW, Tanlimco SG, Hickson J, Fox M, Sho M, Durkin L, et al. LRRC15 Is a Novel Mesenchymal Protein and Stromal Target for Antibody-Drug Conjugates. Cancer Res 2018;78:4059-4072.2.Slemmons KK, Mukherjee S, Meltzer P, Purcell JW, Helman LJ. LRRC15 antibody-drug conjugates show promise as osteosarcoma therapeutics in preclinical studies. Pediatr Blood Cancer 2020:e28771. Citation Format: Sanjit Mukherjee, Ankit Jajoo, Jack Zhu, Marbin Pineda, Robert Walker, James Purcell, Lee J Helman, Paul S. Meltzer. Loss of LRRC15 in osteosarcoma cells disrupts extracellular matrix integration, intercellular communication, and differentiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2331.

The New General Biological Property of Stem-like Tumor Cells (Part II: Surface Molecules, Which Belongs to Distinctive Groups with Particular Functions, Form a Unique Pattern Characteristic of a Certain Type of Tumor Stem-like Cells).

An ability of poorly differentiated cells of different genesis, including tumor stem-like cells (TSCs), to internalize extracellular double-stranded DNA (dsDNA) fragments was revealed in our studies. Using the models of Krebs-2 murine ascites carcinoma and EBV-induced human B-cell lymphoma culture, we demonstrated that dsDNA internalization into the cell consists of several mechanistically distinct phases. The primary contact with cell membrane factors is determined by electrostatic interactions. Firm contacts with cell envelope proteins are then formed, followed by internalization into the cell of the complex formed between the factor and the dsDNA probe bound to it. The key binding sites were found to be the heparin-binding domains, which are constituents of various cell surface proteins of TSCs-either the C1q domain, the collagen-binding domain, or domains of positively charged amino acids. These results imply that the interaction between extracellular dsDNA fragments and the cell, as well as their internalization, took place with the involvement of glycocalyx components (proteoglycans/glycoproteins (PGs/GPs) and glycosylphosphatidylinositol-anchored proteins (GPI-APs)) and the system of scavenger receptors (SRs), which are characteristic of TSCs and form functional clusters of cell surface proteins in TSCs. The key provisions of the concept characterizing the principle of organization of the "group-specific" cell surface factors of TSCs of various geneses were formulated. These factors belong to three protein clusters: GPs/PGs, GIP-APs, and SRs. For TSCs of different tumors, these clusters were found to be represented by different members with homotypic functions corresponding to the general function of the cluster to which they belong.

37P Synthetic tumour-infiltrating interleukin (IL)-12 for targeted and tolerable immunotherapy reduces metastasis in pancreatic cancer

Metastatic Pancreatic Ductal Adenocarcinoma (PDAC) is a root cause of lethality in patients. The metastasis is promoted by abundant proteases in the PDAC microenvironment. Synthetic immuno-oncology opens new avenues towards overcoming limitations of standard immunotherapy agents, previously unsuccessful in PDAC. Anti-tumour cytokines such as IL-12 poorly infiltrate the stroma of solid tumours and trigger toxicity. Here, by exploiting the abundant proteases in PDACs, a targeted and tolerable masked Collagen-Binding Domain CBD-Il12 (mCBD-Il12) was produced by fusing the mask to the p35 subunit of Il-12 through a cleavable linker.

Enhancement of keratinocyte growth factor potential in inducing adipose-derived stem cells differentiation into keratinocytes by collagen-targeting.

Different growth factors can regulate stem cell differentiation. We used keratinocyte growth factor (KGF) to direct adipose-derived stem cells (ASCs) differentiation into keratinocytes. To enhance KGF bioavailability, we targeted KGF for collagen by fusing it to collagen-binding domain from Vibrio mimicus metalloprotease (vibrioCBD-KGF). KGF and vibrioCBD-KGF were expressed in Escherichia coli and purified to homogeneity. Both proteins displayed comparable activities in stimulating proliferation of HEK-293 and MCF-7 cells. vibrioCBD-KGF demonstrated enhanced collagen-binding affinity in immunofluorescence and ELISA. KGF and vibrioCBD-KGF at different concentrations (2, 10, and 20 ng/ml) were applied for 21 days on ASCs cultured on collagen-coated plates. Keratinocyte differentiation was assessed based on morphological changes, the expression of keratinocyte markers (Keratin-10 and Involucrin), and stem cell markers (Collagen-I and Vimentin) by real-time PCR or immunofluorescence. Our results indicated that the expression of keratinocyte markers was substantially increased at all concentrations of vibrioCBD-KGF, while it was observed for KGF only at 20 ng/ml. Immunofluorescence staining approved this finding. Moreover, down-regulation of Collagen-I, an indicator of differentiation commitment, was more significant in samples treated with vibrioCBD-KGF. The present study showed that vibrioCBD-KGF is more potent in inducing the ASCs differentiation into keratinocytes compared to KGF. Our results have important implications for effective skin regeneration using collagen-based biomaterials.

A functionalized collagen-I scaffold delivers microRNA 21-loaded exosomes for spinal cord injury repair

MicroRNA (miRNA)-based therapies have shown great potential in the repair of spinal cord injury (SCI). MicroRNA 21 (miR21) has been proven to have an essential protective effect on SCI. However, there are some challenges for miRNAs application due to their easy degradation and ineffective cell penetration. As natural vesicles, exosomes were considered ideal carriers for miRNAs delivery for their advantages of low immunogenicity, inherent stability and tissue/cell penetration. However, poor targeting and the low capacity of specific miRNAs impede their practical applications. This study aims to develop a type of genetically engineered miR21-loaded exosomes that can be entrapped in collagen-I (Col-I) scaffold to repair SCI. The collagen-binding domain (CBD)-fused lysosome-associated membrane glycoprotein 2b (Lamp2b) protein (CBD-LP) and miR21 were overexpressed in host HEK293T (293T) cells that were used to produce engineered miR21-loaded exosomes. The CBD peptide fused in Lamp2b on the exosome surface can stably tether exosomes to Col-I scaffold, facilitate the retention of miR21-loaded exosomes in lesion sites, promote the sustained release of miR21 to cells. Finally, a functionalized Col-I scaffold biomaterial enriched with miR21-loaded exosomes was developed and it could benefit the repair of SCI. Statement of significanceMiRNA-based therapeutics have promising potential in spinal cord injury (SCI) repair. However, easy degradation and ineffective cell penetration impede miRNAs application. Exosomes are natural vehicles for miRNAs delivery but face the challenge of diffusion in vivo. Here, the collagen-binding domain (CBD)-fused Lamp2b and miR21 were overexpressed in HEK293T cells to produce miR21-loaded and CBD-modified exosomes (CBD-LP-miR21-EXOs). The CBD modified on the exosome surface can stably tether exosomes to collagen-I scaffold to form functionalized CBD-LP-miR21-EXO-Col scaffold that can facilitate the retention of miR21-loaded exosomes, promote the sustained release of miR21 to cells and finally benefit SCI repair. Furthermore, this type of functionalized collagen-I materials can be widely applied for other tissue injury repairs by enriching the CBD-LP-EXOs loaded with appropriate miRNAs.

In silico design of fusion keratinocyte growth factor containing collagen-binding domain for tissue engineering application

Keratinocyte growth factor (KGF) is a potential therapeutic factor in wound healing. However, its applications have been restricted due to its low stability, short half-life, and limited target specificity. We aimed to immobilize KGF on collagen-based biomaterials for long-lasting and targeted therapy by designing fusion forms of KGF with collagen-binding domains (CBD) from natural origins. Twelve fusion proteins were designed consisting of KGF and CBDs with different lengths and amino acid compositions. Three-dimensional (3D) structures of the fusions were predicted by homology modeling. Physiochemical properties and secondary structure of the fusions were evaluated by bioinformatics tools. Moreover, the effect of the CBDs on the 3D structure and dynamic behavior of the fusions was investigated by molecular dynamics (MD) simulation. The binding affinity of the fusions to collagen, KGF receptor, and heparin was assessed using docking tools. Our results demonstrated that fusions with small CBDs like CBD of mammalian collagenase and decapeptide CBD of von Willebrand factor (VWF) were more stable and properly folded than those with larger CBDs. On the other hand, the insertion of bulky CBDs, including Fibronectin CBD and CBD of Clostridium histolyticum collagenase, into KGF resulted in stronger binding to collagen. Therefore, very small or large CBDs are inappropriate for constructing KGF fusions because they suffer from low collagen affinity or poor stability. By comparing the results of MD simulation and docking, this study proposed that CBDs belonging to Vibrio mimicus metalloprotease and A3 domain of VWF would be good candidates to produce stable fusions with proper affinities toward collagen and KGF receptors. Moreover, the secondary structure analysis showed that the overall structure of KGF and CBDs was better preserved when CBDs were inserted at the C-terminal of KGF. This computational information about novel KGF fusions may help find the best constructs for experimental studies.

Research on Polycaprolactone-Gelatin Composite Scaffolds Carrying Nerve Growth Factor for the Repair of Spinal Cord Injury.

Objective This study was to investigate the mechanism of action of polycaprolactone/gelatin (PCL/GE) composite fiber scaffold with nerve growth factor (NGF) in the recovery of spinal cord injury (SCI). Methods Sixty female Sprague-Dawley (SD) rats were randomly assigned to the negative control group, the positive control group, the PCL/GE scaffold group, and the collagen-binding structural domain nerve growth factor (CBD-NGF)/PCL/GE scaffold group, with 15 rats in each group. Spinal cord transection was used to establish SCI models in rats. The negative control group received sham surgery, while the other three groups were given spinal cord transection at the tenth thoracic vertebra (T10) segment. The rats in the PCL/GE scaffold group were implanted with a 4 mm PCL/GE composite fiber scaffold, and those in the CBD-NGF/PCL/GE scaffold group were implanted with a CBD-NGF/PCL/GE composite fiber scaffold. The Basso–Beattie–Bresnahan (BBB) locomotor rating scale was used to evaluate the locomotor ability of the hind limbs of the rats, and the amplitude and latency of motor evoked potentials (MEP) were recorded by neurophysiological testing at 12 w postoperatively. The levels of growth-associated protein 43 (GAP43) and neurofilament protein 200 (NF200) in the spinal cord tissue of the injury site were determined using Western Blot at 12 w after surgery. Spinal cord tissues of 2 cm within the injury site, the thoracic segment above the injury site, and the lumbar segment below the injury site were collected from the measurement of axonal transport using fluorescent retrograde tracer fluorogold, and the integrated absorbance (IA) values of FC-positive cells were calculated. Results After treatment, the negative control rats showed normal locomotion function of the hind limb with the highest BBB scores, while the positive control rats had the lowest BBB scores and showed paraplegia. The scaffold groups exhibited better locomotion function of the hind limb and higher BBB scores than the positive controls, with greater improvement observed in the CBD-NGF/PCL/GE scaffold group (P < 0.05). Compared with the positive controls, the PCL/GE scaffold group and CBD-NGF/PCL/GE scaffold group exhibited significantly shorter latency and increased amplitude of MEP, with more significant changes observed in the CBD-NGF/PCL/GE scaffold group (P < 0.05). Compared with the positive control group, the GAP43 and NF200 levels of spinal cord tissue were significantly elevated in both the PCL/GE scaffold group and the CBD-NGF/PCL/GE scaffold group, and the changes were more pronounced in the CBD-NGF/PCL/GE scaffold group (P < 0.05). The differences in the IA values of FC-positive cells in the spinal cord tissue of the lumbar segment below the injury site among the four groups did not come up to the statistical standard (P > 0.05). Compared with the positive control group, the FC-positive cell IA values of spinal cord tissue in the thoracic segment above the injury area were markedly increased in the PCL/GE scaffold group and the CBD-NGF/PCL/GE scaffold group, and the alterations were more significant in the CBD-NGF/PCL/GE scaffold group (P < 0.05). Conclusion PCL/GE composite fiber scaffold with NGF significantly improves motor and neurological functions in the hind limbs of SCI rats and promotes the recovery of axonal transport, and the mechanism may be associated with the upregulation of GAP43 and NF200 levels in spinal cord injury site tissues.

A Slotted Decellularized Osteochondral Scaffold With Layer-Specific Release of Stem Cell Differentiation Stimulators Enhances Cartilage and Bone Regeneration in Osteochondral Defects in a Rabbit Model

Background: Owing to the disappointing regenerative ability of osteochondral tissue, without treatment an osteochondral defect would progress to osteoarthritis. This situation motivates the need for new strategies to enhance the regeneration of osteochondral defects. Purpose: To develop a tissue-engineering scaffold by tethering bone morphogenetic protein 2 (BMP2) and transforming growth factor beta 3 (TGFβ3) in a layer-specific manner on a slotted decellularized osteochondral matrix (SDOM) and to evaluate the efficacy of this scaffold for osteochondral regeneration. Study Design: Controlled laboratory study. Methods: Normal osteochondral tissue from the rabbit patellofemoral groove was sectioned into a slot shape and decellularized for fabricating an SDOM. The collagen-binding domain (CBD) was fused into the N-terminus of BMP2 or TGFβ3 to synthesize 2 recombinant growth factors (GFs) (CBD-BMP2 or CBD-TGFβ3), which were tethered to the bone layer and cartilage layer, respectively, of the SDOM to prepare a tissue-engineering scaffold (namely, CBD-GFs/SDOM). After examining the influence of the CBD-GFs/SDOM on the viability and layer-specific differentiation of bone marrow mesenchymal stem cells in vitro, we determined the regeneration potential of the CBD-GFs/SDOM on osteochondral regeneration in a rabbit model. A total of 72 New Zealand White rabbits with a cylindrical osteochondral defect in the patellofemoral groove were randomly assigned to 3 groups: defect only (control [CTL] group), defect patched with an SDOM (SDOM group), and defect patched with the CBD-GFs/SDOM (CBD-GFs/SDOM group). At 6 or 12 weeks postoperatively, the rabbits were euthanized to harvest the knee joint, which was then evaluated via gross observation, micro–computed tomography, histological staining, and mechanical testing. Results: In vitro, the CBD-GFs/SDOM was noncytotoxic, showed high biomimetics with normal osteochondral tissue, was suitable for cell adhesion and growth, and had good layer-specific ability in inducing stem cell differentiation. Macroscopic images showed that the CBD-GFs/SDOM group had significantly better osteochondral regeneration than the CTL and SDOM groups had. Micro–computed tomography demonstrated that much more bony tissue was formed at the defect sites in the CBD-GFs/SDOM group compared with the defect sites in the CTL or SDOM group. Histological analysis showed that the CBD-GFs/SDOM group had a significant enhancement in osteochondral regeneration at 6 and 12 weeks postoperatively in comparison with the CTL or SDOM group. At 12 weeks postoperatively, the mechanical properties of reparative tissue were significantly better in the CBD-GFs/SDOM group than in the other groups. Conclusion: The CBD-GFs/SDOM is a promising scaffold for osteochondral regeneration. Clinical Relevance: The findings of this study indicated that the CBD-GFs/SDOM is an excellent candidate for reconstructing osteochondral defects, which may be translated for clinical use in the future.

LL37 and collagen-binding domain-mediated LL37 binding with type I collagen: Quantification via QCM-D

Antimicrobial peptide (AMP)-loaded biomaterials may represent a viable alternative for stimulating wound healing while protecting against infections. Previously, to develop an efficient delivery system for the cathelicidin antimicrobial peptide, LL37, our lab modified LL37 with a collagen-binding domain derived from collagenase (cCBD) as an anchoring unit to collagen-based wound dressings. However, a direct quantification of unmodified LL37 and cCBD-LL37 binding with collagen has not been performed. In this study, we used quartz crystal microbalance with dissipation monitoring (QCM-D), immunohistochemistry (IHC), and atomic force microscopy (AFM) to establish and characterize an adsorbed layer of type I collagen on the QCM-D sensor and quantify peptide-collagen binding. A collagen deposition protocol was successfully established by measuring concentration-dependent deposition of collagen in QCM-D, and collagen self-assembly was observed by IHC and AFM. Hydrophobicity is known to affect the behavior of collagen adsorption. Therefore, we compared the deposition of collagen on hydrophilic SiO2-coated sensors vs. hydrophobic polystyrene (PS)-coated sensors via QCM-D, and found that the hydrophobic surface yielded more collagen adsorption, which suggested that hydrophobic surfaces are preferable for collagen layer establishment. There was no significant difference between LL37 and cCBD-LL37 binding with collagen, but the cCBD-LL37 showed better retention on the collagen after washing with PBS, indicating that there is an advantage to using cCBD as an anchoring unit to collagen. Collectively, these results provide important information on cCBD-mediated AMP-binding mechanisms and establish an effective method for quantifying peptide-collagen binding.

Collagen‑binding vascular endothelial growth factor (CBD‑VEGF) promotes liver regeneration in murine partial hepatectomy.

Liver regeneration is a complex process that needs orchestration of multiple nonparenchymal cells including sinusoid endothelial cells. Vascular endothelial growth factor (VEGF) serves a crucial role in angiogenesis and liver regeneration. However, the lack of an high‑efficiency delivery system target to the injured site reduces the local therapeutic efficacy of VEGF. In our previous study, collagen binding VEGF (CBD‑VEGF) was established by fusing collagen binding domain (CBD) into the N‑terminal of native VEGF and improved cardiac function after myocardial infraction. The present study investigated the therapeutic effect of CBD‑VEGF on liver regeneration by a mouse model of partial hepatectomy. After injection through portal vein following 2/3 hepatectomy, CBD‑VEGF was largely retained in the hepatic extracellular matrix for 48h. Furthermore, CBD‑VEGF application significantly promoted sinusoidal regeneration and remodeling in remanent liver tissue 48h after hepatectomy. In addition, CBD‑VEGF treatment significantly enhanced the proliferation of hepatocytes at 2 and 3days post‑surgery compared with native VEGF, concomitant with attenuated liver injury. In conclusion, these results demonstrated that CBD‑VEGF could be a promising therapeutic strategy for liver regeneration.

Acellular dermal matrix decorated with collagen-affinity peptide accelerate diabetic wound healing through sustained releasing Histatin-1 mediated promotion of angiogenesis

Treating diabetic ulcers is a major challenge in clinical practice, persecuting millions of patients with diabetes and increasing the medical burden. Recombinant growth factor application can accelerate diabetic wound healing via angiogenesis. The local administration of recombinant growth factors has no robust clinical efficiency because of the degradation of append short duration of the molecules in the hostile inflammatoryenvironment.The present study focused on the pathophysiology of impaired neovascularization and growth factor short duration in the diabetic wound. We prepared a collagen-binding domain (CBD)-fused recombinant peptide (C-Histatin-1) that had both pro-angiogenesis capacity and collagen-affinity properties. Next, we created a biocompatible acellular dermal matrix (ADM) as a drug delivery carrier that featured collagen-richness, high porosity, and non-cytotoxicity. C-Histatin-1 was then tethered on ADM to obtain a sustained-release effect. Finally, a functional scaffold (C-Hst1/ADM) was developed. C-Hst1/ADM can sustain-release Histatin-1 to promote the adhesion, migration, and angiogenesisof vascular endothelial cells in vitro. Using a diabetic wound model, we showed that C-Hst1/ADM could significantly promote angiogenesis, reduce scar widths, and improve extracellular collagen accumulation. Therefore, the results of this study provide a foundation for the clinical application of C-Hst1/ADM covering scaffold in the treatment of diabetic wounds.

Abstract 3505: CLN-617 is an IL-2/IL-12 fusion protein with a collagen-anchoring domain that induces potent systemic anti-tumor immunity upon intra-tumoral administration

Abstract Despite strong anti-tumor immune responses in the clinic, cytokine therapy for cancer treatment, has remained limited in use due to its narrow therapeutic window. Moreover, anti-tumor immunity is typically driven through multiple cytokines acting in concert in a local tumor microenvironment (TME) via autocrine or paracrine signaling. Yet most cytokine therapies focus on systemic delivery of individual cytokines causing adverse events without achieving therapeutic doses at the tumor site. Recently, intratumoral (IT) delivery of cytokines such as IL-2 and IL-12 anchored to collagen in the TME has resulted in promising anti-tumor efficacy with minimal toxicity in multiple murine tumor models, and remarkably also in spontaneously occurring large canine tumors (unpublished data). Cullinan Amber is developing CLN-617, a fusion protein that uniquely combines IL-2 and IL-12 with albumin and a collagen-binding domain in a single polypeptide. CLN-617 aims to increase cytokine persistence and retention in the TME upon IT administration and thus improve tolerability. Data presented in this abstract is generated using a murine surrogate, mCLN-617. mCLN-617 retains bioactivity of both IL-2 and IL-12 in the presence of collagen binding. Upon IT injection into B16F10 tumor-bearing mice, systemic drug levels of mCLN-617 were &amp;lt 5% compared to mice dosed via intravenous (IV) administration. When IL-2/IL-12 fusion proteins lacking a collagen-binding domain were injected IT in B16F10-bearing mice, 60% of mice were euthanized due to severe body weight loss, whereas mCLN-617-treated mice maintained their body weight. In checkpoint-refractory B16F10 and MC38 models, mCLN-617 demonstrated 95% tumor growth inhibition and 100% CRs, respectively. All mice cured of their primary MC38 tumors were either protected from re-challenge or showed delayed tumor growth kinetics. Notably, 70% CRs were observed in the MC38 model after a single-dose treatment of mCLN-617. In mice bearing dual-flank MC38 tumors, only one of which was treated IT, 90% of treated tumors and 100% of distal untreated tumors were eliminated when mCLN-617 was combined with an anti-PD1 antibody, demonstrating a curative abscopal response. Of note, mCLN-617 monotherapy in the treated tumor also significantly delayed the growth of distal untreated tumors. Our human lead candidate CLN-617 is produced at high titers in CHO cells and is currently in IND enabling studies. We have demonstrated that the use of collagen-binding domains for tumor retention enables safe and effective delivery of a combination of IL-2 and IL-12 in a single multifunctional molecule. The strong anti-tumor efficacy of mCLN-617 as a monotherapy and significant deepening of responses in combination with checkpoint blockade, together with a favorable tolerability profile, supports future clinical development of CLN-617. Citation Format: Naveen K. Mehta, Bochong Li, Kavya Rakhra, K Dane Wittrup, Patrick A. Baeuerle, Jennifer S. Michaelson. CLN-617 is an IL-2/IL-12 fusion protein with a collagen-anchoring domain that induces potent systemic anti-tumor immunity upon intra-tumoral administration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3505.

Stiffness-responsive feedback autoregulation of DDR1 expression is mediated by a DDR1-YAP/TAZ axis

Objective: Increased matrix stiffness is sensed by the collagen-binding receptor tyrosine kinase discoidin domain receptor 1 (DDR1). We have previously shown that DDR1 stimulates a positive feedback loop to increase its own expression in vascular smooth muscle cells (VSMCs). The transcriptional co-factors YAP/TAZ are stiffness sensing molecules that have not previously been investigated in DDR1 signaling. Here, we test the hypothesis that DDR1 signals through YAP/TAZ to auto-regulate its own expression.Approach and Results: We used vascular smooth muscle cells (VSMCs) from wild-type and DDR1 knockout mice stimulated with collagen and/or substrates of different stiffness. We show that DDR1 controls YAP/TAZ nuclear localization and activity, whereas knockdown of YAP/TAZ attenuates DDR1 expression. In response to increased substrate stiffness, collagen stimulation, or RhoA activation, YAP/TAZ translocate to the nucleus and bind to chromatin. Finally, collagen stimulation promotes increased YAP/TAZ association with the Ddr1 promoter.Conclusions: These findings reveal the mechanism by which DDR1 regulates YAP/TAZ activity which can then mediate positive feedback regulation of DDR1 expression by promoting transcription of the DDR1 gene.

Development of chimeric forms of the matrix metalloproteinase 2 collagen binding domain as artificial membrane binding proteins for targeting stem cells to cartilage lesions in osteoarthritic joints

Targeting stem cells to cartilage lesions has the potential to enhance engraftment and chondrogenesis. Denatured type II collagen fibrils (gelatin) are exposed in lesions at the surface of osteoarthritic articular cartilage and are therefore ideal target sites. We have designed and investigated chimeric mutants of the three modules of the MMP-2 collagen binding domain (CBD) as potential ligands for stem cell targeting. We expressed full-length CBD for the first time and used it to identify the most important amino acid residues for binding to gelatin. Module 2 of CBD had the highest affinity binding to both Type I and Type II gelatin, whereas module 1 showed specificity for type II gelatin and module 3 for type I gelatin. We went on to generate chimeric forms of CBD consisting of three repeats of module 1 (111), module 2 (222) or module 3 (333). 111 lacked solubility and could not be further characterised. However 222 was found to bind to type II gelatin 14 times better than CBD, suggesting it would be optimal for attachment to cartilage lesions, whilst 333 was found to bind to type I gelatin 12 times better than CBD, suggesting it would be optimal for attachment to lesions in type I collagen-rich tissues. We coated 222 onto the external membrane of Mesenchymal Stem Cells and demonstrated higher attachment of the coated cells to type II gelatin than uncoated cells. We conclude that the three modules of CBD each have specific biological properties that can be exploited for targeting stem cells to cartilage lesions and other pathological sites.

A tumor-targeted cytokine/antibody fusion protein to stimulate anti-cancer immunity

Abstract Interleukin-2 (IL-2) is a pleiotropic cytokine that has clinical utility in stimulating anti-cancer immunity. However, the clinical efficacy of IL-2 is limited by its short serum half-life, systemic toxicity, and concurrent activation of immunostimulatory natural killer (NK) and effector T (Teff) cells and immunosuppressive regulatory T (Treg) cells. IL-2 can signal down an intermediate-affinity receptor consisting of IL-2 receptor beta (IL-2Rβ) and common gamma chain (γc) or a high-affinity receptor that also includes IL-2Rα. Tregs express high levels of IL-2Rα, making them much more responsive to IL-2 than naïve NK and Teff cells. To extend the serum half-life and selectively bias the immunostimulatory functions of IL-2 in a stable unimolecular format, we created a single-chain fusion protein, denoted F10 immunocytokine (F10 IC), that intramolecularly fuses human IL-2 to an anti-IL-2 antibody that sterically blocks the IL-2/IL-2Rα interaction and allosterically enhances the IL-2/IL-2Rβ interaction. To exploit the abundance and accessibility of collagen in the tumor microenvironment for targeting, we fused collagen binding domains to F10 IC in various topographies. These novel fusion proteins occlude IL-2Rα binding and enhance IL-2Rβ binding, thereby eliminating the Treg advantage conferred by IL-2Rα and preferentially activating immune effector cells. Furthermore, these proteins selectively bind purified collagen and are capable of homing to, and being retained in, the tumor in vivo. Building on these promising results, we are assessing the in vivo therapeutic efficacy of these fusion proteins in various murine tumor models to illustrate the potential of collagen binding F10 IC as a powerful anti-cancer therapeutic. Supported by a Sanofi Innovation Award and NIH grants (R01 EB029341, T32 AI007417)

A SIRPαFc Fusion Protein Conjugated With the Collagen-Binding Domain for Targeted Immunotherapy of Non-Small Cell Lung Cancer.

The SIRPαFc fusion protein can block the immunosuppressive CD47-SIRPα signal between macrophages and tumor cells as a decoy receptor and has demonstrated its immunotherapeutic efficacy in various tumors. However, its clinical application was limited because of the potential hematologic toxicity. The heptapeptide “TKKTLRT” is a collagen-binding domain (CBD) which can bind collagen specifically. Herein, we aim to improve the tumor targeting of SIRPαFc and therefore avoid its unnecessary exposure to normal cells through synthesizing a TKKTLRT–SIRPαFc conjugate. Experiments at molecular and cellular levels indicate that the TKKTLRT–SIRPαFc conjugate-derived collagen-binding affinity and the introduction of CBD did not impact the CD47-binding affinity as well as its phagocytosis-promoting effect on NSCLC cells. In vivo distribution experiments showed that CBD–SIRPαFc accumulated in tumor tissue more effectively compared to unmodified SIRPαFc, probably due to the exposed collagen in the tumor vascular endothelium and stroma resulting from the abnormal vessel structure. On an A549 NSCLC nude mouse xenograft model, CBD–SIRPαFc presented more stable and effective antitumor efficacy than SIRPαFc, along with significantly increased CD11b+F4/80+ macrophages especially MHC II+ M1 macrophages within tumors. All of these results revealed that CBD brought a tumor-targeting ability to the SIRPαFc fusion protein, which contributed to the enhanced antitumor immune response. Altogether, the CBD–SIRPαFc conjugate may have the potential to be an effective tumor immunotherapy with improved antitumor efficacy but less non-tumor-targeted side effect.

Novel Likely Pathogenic Variant in the A3 Domain of von Willebrand Factor Leading to a Collagen-Binding Defect.

Von Willebrand disease (VWD) is the most prevalent congenital bleeding disorder. Diagnosis and classification of VWD is complex due to its heterogeneity regarding clinical manifestations and molecular genetic analysis. Genetic investigations became an inherent part of diagnosis and help distinguish different types/subtypes of VWD. Although many variants have been listed being causative for VWD, the genetic etiology remains undefined in a lot of patients. We report about two siblings with severely reduced values for von Willebrand factor collagen-binding activity (VWF:CB). Genetic analysis using panel sequencing identified a heterozygous non-synonymous single nucleotide variant in exon 30. At the protein level, the alteration (p.Ser1731Leu) is located in the A3 collagen-binding domain. The amino acid position is already known to be important for collagen binding because p.Ser1731Thr has been reported to affect the VWF:CB.