In Situ Conversion of Melanoma Lesions into Autologous Vaccine by Intratumoral Injections of α-gal Glycolipids

Abstract

Autologous melanoma associated antigens (MAA) on murine melanoma cells can elicit a protective anti-tumor immune response following a variety of vaccine strategies. Most require effective uptake by antigen presenting cells (APC). APC transport and process internalized MAA for activation of anti-tumor T cells. One potential problem with clinical melanoma vaccines against autologous tumors may be that often tumor cells do not express surface markers that label them for uptake by APC. Effective uptake of melanoma cells by APC might be achieved by exploiting the natural anti-Gal antibody which constitutes ~1% of immunoglobulins in humans. This approach has been developed in a syngeneic mouse model using mice capable of producing anti-Gal. Anti-Gal binds specifically to α-gal epitopes (Galα1-3Galβ1-4GlcNAc-R). Injection of glycolipids carrying α-gal epitopes (α-gal glycolipids) into melanoma lesions results in glycolipid insertion into melanoma cell membranes, expression of α-gal epitopes on the tumor cells and binding of anti-Gal to these epitopes. Interaction between the Fc portions of bound anti-Gal and Fcγ receptors on APC induces effective uptake of tumor cells by APC. The resulting anti-MAA immune response can be potent enough to destroy distant micrometastases. A clinical trial is now open testing effects of intratumoral α-gal glycolipid injections in melanoma patients.

Figure 1

Ceramide pentahexoside (CPH) (left) and ceramide decahexoside (CDecaH) (right) as representative α-gal glycolipids. CPH is the most abundant glycolipid in rabbit RBC and is presented as a schematic α-gal glycolipid with five carbohydrates. CDecaH is a glycolipid with 10 carbohydrate branched chain. α-gal epitopes (Galα1-3Galβ1-4GlcNAc-R) are marked by the broken line rectangles. The terminal α-galactosyl (Gal) is linked α1,3 to the penultimate Gal of the carbohydrate chain by the glycosylation enzyme α1,3galactosyltransferase (α1,3GT). The carbohydrate chain is linked to the lipid portion (ceramide) embedded in the cell membrane via the two fatty acid tails. Anti-Gal binding to α-gal epitopes is presented as schematic IgG molecules. α-gal glycolipids in rabbit RBC (with the exception of ceramide heptahexoside) increase in size in increments of five carbohydrates, each forming an additional branch that is capped by α-gal epitopes.

Figure 2

Insertion of α-gal glycolipids into the lipid bilayer of tumor cell membranes. α-gal glycolipid molecules dissolve in water or saline as micelles (ball like structures with the cross-section described in this figure) in which the hydrophobic (lipophilic) ceramide chains are clustered in the core. When these micelles are adjacent to cells, individual α-gal glycolipid molecules “jump” into the outer leaflet of the cell lipid bilayer, because the energetic state of the ceramide tail surrounded by phospholipids is much more stable than in micelles surrounded by water. α-gal glycolipid insertion into the tumor cell membrane results in expression of α-gal epitopes on the cell. Binding of the natural anti-Gal antibody to these epitopes leads to destruction of tumor cells and their uptake by APC.

Figure 3

In vivo effect of injection of the α-gal glycolipids into B16 melanoma. Lesions reaching subcutaneous size of ~5 mm received one injection of α-gal glycolipids and were resected at different time points for histological analysis. (A) On day 2 post injection, mononuclear cells migrating from blood vessels (BV) are detected already; (B) On day 7 post injection, the infiltration is more extensive as indicated by the number of mononuclear cells migrating from blood vessels into the tumor; and (C) On day 7 post PBS injection, control tumors displayed no infiltration of mononuclear cells (×200).

Figure 4

Effect of injected 1.0 mg α-gal glycolipids on tumor growth. (A) Pre-treated tumor; (B) Tumor on day 5 post α-gal glycolipids injection; (C) Tumor on day 15 post injection. Note the gradual regression of the tumor. The black spot developing in the skin near the regressing tumor was caused by a subcutaneous administration of α-gal glycolipids into normal skin, inducing local inflammation that results in transient activation of normal melanocytes.

Figure 5

Anti-Gal mediated targeting of tumor cells to APC in lesions injected with α-gal glycolipids. Anti-Gal IgG binds in situ to α-gal epitopes on α-gal glycolipids inserted into tumor cell membranes. Subsequent interaction between the Fc portion of the bound anti-Gal and FcγR on the APC (schematic illustration of a dendritic cell) induces uptake of intact or lysed tumor cells by APC and thus, effective internalization of the tumor antigens (MAA in melanoma lesions). Internalized tumor antigens are processed and various immunogenic tumor antigen peptides (●) are presented by the APC in association with class I and class II MHC molecules. These immunogenic peptides can activate tumor specific T cells and elicit a protective anti-tumor immune response.

Figure 6

Melanocytes activation following subcutaneous injection of 1.0 mg α-gal glycolipids into the KO mouse. A. The epidermis displays 4–5 layers of cells 7 days post injection in the black spot area. The apical region under the keratinous layer (stained in red) is filled with many melanin granules. B. The injected skin four weeks post injection. The epidermis displays normal structure of ~2 layers of epidermal cells, the amount of melanin granules is residual and the overall color of the skin returns to normal pink (x400).