Science

Science has implicated certain chemokine and receptor pairs in pathogenesis from cancer to TH-17 mediated diseases such as chronic GVHD, ocular GVHD, Sjögren’s Disease and many others.

STRUCTURAL ANALYSIS LEADS
TO A KEY DISCOVERY

The foundational observation by the founders of XLock is that dimers of certain chemokines inhibit cell movement. To understand the potential for modifying the biology of chemokines, our scientists explored the effects of chemokine dimers first in in vitro models and then in in vivo disease models.

IN VITRO STUDIES

There are two initial effects of chemokines at their GPCR that are commonly used in in vitro studies: calcium flux and chemotaxis. After much structural biology research, stable dimers of CXCL12 were produced that inhibit chemotaxis, while retaining calcium flux activity. (Veldkamp CT et al (2008) Science Signaling)

Figure 1 below shows the biological activity of chemokine monomers and dimers. The upper panels: Wild-type CXCL12 shows a sigmoidal dose-response profile for intracellular Ca2+-flux, and a biphasic “bell-shaped” curve for transwell chemotaxis. Lower panels: An engineered covalently linked homodimer of CXCL12 (CXCL12 locked dimer; CXCL12LD) induces no cell migration while leaving the Ca2+-flux response intact.

Fig. 1 Veldkamp CT et al (2008) Science Signaling

Figure 2 shows the blocking of native CXCL12-induced transwell chemotaxis of THP-1 cells by CXCL12LD with a low nanomolar potency.

Fig. 2 Getschman et al (2017), PNAS

It was these observations, combined with a previous understanding that higher concentrations of CXCL12 lead to CXCL12 dimerization, that was critical to the understanding that obligate dimers might have a very different effect on chemotaxis.

Similar to the CXCL12/CXCL12LD results, a stable dimer of the chemokine CCL20, termed CCL20 S64C or CCL20LD, is unable to drive CCR6+ Jurkat cell migration as shown in figure 3A below. Figure 3B shows that CCL20LD is also able to inhibit cell migration in the presence of 30 nM WT CCL20.

Fig. 3 Getschman et al (2017), PNAS

The creation of obligate, covalently bound dimers of these two chemokines, CXCL12 and CCL20, reverses the CHEMOTACTIC function of these molecules and blocks the deleterious effects of the chemokine-receptor interaction; malignant tumor metastasis and Th-17 mediated inflammation. These two molecules, CXCL12LD and CCL20LD, form the basis of XLock’s current therapeutic leads.

APPLICATION IN ANIMAL MODELS

While it was interesting and important to demonstrate this dimer effect in vitro, to treat chemokine driven diseases it is necessary to show that the locked dimers can inhibit pathological processes. These pathological functions are tumor metastasis in the case of CXCR4/CXCL12 and Th-17 autoimmune disease for CCL6/CCL20. Both locked dimers were tested for in vivo efficacy in animal models.

CXCR4 DRIVEN METASTASIS

Published results by our founders have shown that the CXCR12LD has the ability to block CXCR4 mediated metastasis in murine models of colon cancer and melanoma tumors.

Fig. 3 (Drury L. et al (2011) PNAS) shows that systemic administration of CXCL12LD is able to inhibit migration/metastasis of orthotopically administered fluorescently labelled HCT-116 hepatocarcinoma cells in vivo. (A) Experimental protocol (B) Liver metastases were decreased in mice administered 63 ug/kg and 638 ug/kg CXCL12LD.

Representative images at 4 weeks post engraftment show localized tumor growth and decreased hepatic metastases.

Fig. 3 (Drury L. et al (2011) PNAS)

Fig. 4 (Drury L. et al (2011) PNAS) CXCR4-B16 melanoma cells were premixed with 5 uM CXCL12LD or vehicle immediately prior to tail-vein injection. The next day, mice received a second CXCL12LD or control treatment. After 14 days, the lungs were recovered and photographed, revealing markedly less melanoma in CXCL12LD treated animals.

Fig. 4 (Drury L. et al (2011) PNAS)

CCR6 DRIVEN INFLAMMATION

XLock Biosciences has shown that our lead molecule, CCL20 Locked Dimer (CCL20LD), has the ability to INHIBIT the development of psoriasis in a murine model.

Fig 5. (Getschman et al (2017), PNAS)

In a much more difficult animal model, our investigators also demonstrated the ability of CCL20LD to TREAT a murine model of Psoriatic Arthritis as shown in the pictures below.

Fig 6. (Shi Z et al (2021), Arthritis and Rheumatology)

XLock Biosciences' preclinical animal model data show inhibition of CCL20/CCR6 signaling can effectively block a number of Th17 mediated diseases.

XLock is also exploring the use of CCL20LD in treatment of chronic GVHD with particular interest in the treatment of ocular cGVHD.

Unlike the currently available drugs that target TNF-α, IL-17, and IL-23 and systemically lower human immune responses, we have a unique molecule that blocks the migration of cells to areas where they cause disease. CCL20LD engages a different part of the immune response cycle and offers the potential peace of mind with the knowledge that the immune system is still able to respond to new and additional challenges.

XLOCK BIOSCIENCES' PRECLINICAL ANIMAL MODEL DATA SHOW INHIBITION OF CCL20/CCR6 SIGNALING CAN EFFECTIVELY BLOCK A NUMBER OF TH17 MEDIATED DISEASES.

These in vivo data show the power and utility of targeting TH17 diseases with CCL20LD. Thus, while CCL20LD should work in the treatment of psoriasis and psoriatic arthritis, there appears to be an overabundance of therapies for these diseases. We are therefore targeting complex, poorly treated, TH17-driven diseases such as Sjörgren’s Syndrome and ocular graft versus host disease. These conditions have significant unmet medical needs and are a more tractable path forward to clinical trials and human data.

We have recently collaborated with investigators at City of Hope to determine the effects of CCL20LD in chronic graft versus host disease, now understood to be significantly TH17 driven. In a murine minor mismatch model, CCL20LD was able to slightly prolong survival and to dramatically reduce the number of TH17 cells in target organs such as lung, liver, and skin. As shown in Figure 4, the marked reduction of TH17 cells in the treated population is evident in flow cytometry analysis of the liver in a murine model of cGVHD.

FIGURE 4. Reduced TH17 cell recruitment in GVHD. Flow cytometry analysis of liver tissue in no GVHD control mice or mice with chronic GVHD, either untreated (control) or treated with CCL20LD.

This 4-fold reduction of TH17 cells in a target organ of GVHD, the liver, raises our confidence that CCL20LD will be effective in treating ocular GVHD administered either topically in the eye or systemically, quite possibly both. We plan to begin studies in a murine model of ocular GVHD in October of 2023.

Unlike the currently available drugs that target TNF-α, IL-17, and IL-23 and systemically lower human immune responses, we have a unique molecule that blocks the migration of TH17 inflammatory cells to areas where they cause disease. CCL20LD engages a different part of the immune response cycle and offers the potential peace of mind with the knowledge that the immune system is still able to respond to new and additional challenges.

If you would like more information about the role of CCL20 in GVHD and Dry Eye Disease, please review the following scientific references.

Varona R, Cadenas V, Lozano M, Moreno-Ortiz MC, Kremer L, Martínez AC, et al. CCR6 regulates the function of alloreactive and regulatory CD4+ T cells during acute graft-versus-host disease. Leuk Lymphoma. 2006;47: 1469–1476. DOI: 10.1080/10428190500513819

van der Waart AB, van der Velden WJ, van Halteren AG, Leenders MJ, Feuth T, Blijlevens NM, et al. Decreased levels of circulating IL17-producing CD161+CCR6+ T cells are associated with graft-versus-host disease after allogeneic stem cell transplantation. PloS One (2012) 7(12):e50896. PMCID: PMC3514180

Dohlman TH, Chauhan SK, Kodati S, et al. The CCR6/CCL20 axis mediates Th17 cell migration to the ocular surface in dry eye disease. Invest Ophthalmol Vis Sci. 2013; 54: 4081–4091. PMCID: PMC3681477

Oh JY, Kim MK, Choi HJ, et al. Investigating the relationship between serum interleukin-17 levels and systemic immune-mediated disease in patients with dry eye syndrome. Korean J Ophthalmol. 2011; 25: 73–76. PMCID: PMC3060396

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