Science

SCIENCE HAS IMPLICATED CERTAIN CHEMOKINE AND RECEPTOR PAIRS IN DISEASE PATHOGENESIS FROM CANCER TO THE TH-17 DRIVEN INFLAMMATION IN PSORIASIS AND PSORIATIC ARTHRITIS.

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STRUCTURAL ANALYSIS LEADS
TO A KEY DISCOVERY

The foundational observation by the founders of XLock is that dimers of chemokines inhibit cell movement. This can be seen in figure 1 below which shows increasing concentrations of CXCR12 lead to INHIBITION of CXCL4 driven chemotaxis. After much structural biology research, stable dimers of CXCL12 were produced that inhibit chemotaxis, while retaining calcium flux measurements. (Veldkamp CT et al (2008) Science Signaling)

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 effects of chemokines at their GPCR that are used in in vitro studies: calcium flux and chemotaxis.

The first two graphs below (figure 1) show calcium flux assays. The left graph shows calcium flux with native CXCL12, a classic sigmoidal curve. The right shows both native and, in green, CXCL12LD. While there is a slight right shift with CXCL12LD, the maximum effect is unchanged.

The lower set of graphs show chemotaxis. The left graph is a dose response curve of chemotaxis with increasing doses of CXCL12. Note the reduction in chemotaxis at higher doses. It was this observation, 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. After a great deal of work, the obligate, covalently bound dimer of CXCL12 (CXCL12LD) was produced.

The right graph shows that, as hoped, CXCL12LD fully inhibits chemotaxis.

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

Subsequently a stable dimer of CCL20 was produced which similarly inhibits CCR6 driven chemotaxis as shown in figure 2 below. The panels below show that the CCL20LD is also able to inhibit the migration of CCR6-expressing Jurkat cells.

Fig. 2 Getschman et al (2017), PNAS

The creation of obligate, covalently bound dimers of these two chemokines, CXCL12 and CCL20, reverses the function of these molecules and inhibits 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.

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