Evaluating the Combination of Radioimmunotherapy and Immunotherapy in a Melanoma Mouse Model.

Immunotherapy has changed the oncology landscape during the last decade and become standard of care for several cancers. The combinations of immunotherapy with other treatment modalities are also being investigated. One of the challenges to investigate such combinations is to identify suitable mouse models for the pre-clinical experiments. In the past, we and other researchers showed that murine B16-F10 melanoma in C57Bl6 mice is refractory to treatment with immune checkpoint inhibitors. In this work we studied the suitability of an alternative syngeneic model, Cloudman S91 murine melanoma in DBA/2 mouse (DBA/2NCrl), to study the combination of immunotherapy targeting PD-1 and radioimmunotherapy targeting melanin. DBA/2 male and female mice were injected subcutaneously with 3-6 million Cloudman S91 cells. When the tumors reached ~150 mm3 volume, the animals were treated intraperitoneally with PBS (sham), h8C3 unlabeled (cold) antibody to melanin, immunotherapy with anti-PD-1 antibody, radioimmunotherapy with 213Bismuth (213Bi)-labeled h8C3 antibody, or several combinations of immunotherapy and radioimmunotherapy. Treatments with immunotherapy alone produced very modest effect on the tumor size, while combination therapy resulted in significant slowing down of the tumor growth, increased animal survival, and no decrease in animal body weight. We conclude that Cloudman S91 murine melanoma in DBA/2 mouse is a suitable model to evaluate combination of immunotherapy of melanoma with tangentially targeted treatments.

Structure-function analysis and therapeutic efficacy of antibodies to fungal melanin for melanoma radioimmunotherapy.

Metastatic melanoma remains difficult to treat despite recent approvals of several new drugs. Recently we reported encouraging results of Phase I clinical trial of radiolabeled with 188Re murine monoclonal IgM 6D2 to melanin in patients with Stage III/IV melanoma. Subsequently we generated a novel murine IgG 8C3 to melanin. IgGs are more amenable to humanization and cGMP (current Good Manufacturing Practice) manufacturing than IgMs. We performed comparative structural analysis of melanin-binding IgM 6D2 and IgG 8C3. The therapeutic efficacy of 213Bi- and 188Re-labeled 8C3 and its comparison with anti-CTLA4 immunotherapy was performed in B16-F10 murine melanoma model. The primary structures of these antibodies revealed significant homology, with the CDRs containing a high percentage of positively charged amino acids. The 8C3 model has a negatively charged binding surface and significant number of aromatic residues in its H3 domain, suggesting that hydrophobic interactions contribute to the antibody-melanin interaction. Radiolabeled IgG 8C3 showed significant therapeutic efficacy in murine melanoma, safety towards healthy melanin-containing tissues and favorable comparison with the anti-CTLA4 antibody. We have demonstrated that antibody binding to melanin relies on both charge and hydrophobic interactions while the in vivo data supports further development of 8C3 IgG as radioimmunotherapy reagent for metastatic melanoma.

Blockade of both L-selectin and alpha4 integrins abrogates naive CD4 cell trafficking and responses in gut-associated lymphoid organs.

The recirculation of naive lymphocytes from blood to lymph that is initiated in high endothelial venules (HEV) of secondary lymphoid organs such as lymph nodes and Peyer's patches (PP) is regulated by multiple interactions of adhesion receptor/counter-receptor pairs involving both selectins and integrins. We showed previously that blocking of only L-selectin is sufficient to ablate trafficking of naive CD4 cells and the development of their responses in peripheral lymph nodes but not in PP where alpha4beta7 integrins are thought to primarily regulate entry. However, although antibody to alpha4 integrins partially inhibited homing of naive CD4 cells to PP and not to lymph nodes, there was no effect on the development primary responses in these tissues or spleens. Since previous studies indicate that both alpha4beta7 integrins and L-selectin regulate adhesion of naive cells to PP HEV, we examined the effect a blockade of both adhesion pathways on the recirculation of naive CD4 cells. There was no detectable homing of naive CD4 cells to PP or lymph nodes when interactions with both receptors were inhibited, resulting in a profound depletion of naive CD4 cells and loss of antigen responses in these sites. In contrast, increased numbers of naive CD4 cells and responses of higher magnitude were found in the spleen. The results demonstrate recirculation of naive CD4 cells through tissues where entry is controlled through HEV is essential for the local generation of primary responses.

L-selectin is not essential for naive CD4 cell trafficking or development of primary responses in Peyer's patches.

We showed previously that L-selectin-dependent recirculation of naive CD4 cells is essential for development of primary responses in peripheral lymph nodes. Recent studies suggest that L-selectin is also required for lymphocyte entry into gut mucosal lymphoid tissues that include Peyer's patches and mesenteric lymph nodes. Here we show that anti-L-selectin antibody, MEL-14, inhibited homing of a rigorously purified, homogenous population of naive CD4 cells into both of these tissues as well as peripheral lymph nodes, directly demonstrating a role for this receptor in regulating entry into gut-associated sites. However, in intact animals, treatment with MEL-14 resulted in the loss of naive CD4 cells (CD45RBhi, CD44lo from peripheral lymph nodes but not Peyer's patches, whereas mesenteric lymph nodes were intermediate in this regard. In mice primed by parenteral immunization with keyhole limpet hemocyanin (KLH), primary CD4 responses were readily detected in both. Peyer's patches and mesenteric lymph nodes, and were not affected by exposure to MEL-14. Indeed, similar frequencies of KLH-specific CD4 cells were recovered from both of these tissues irrespective of MEL-14 treatment. The results indicate that interactions with L-selectin can be circumvented to allow entry of naive CD4 cells into Peyer's patches but not peripheral lymph nodes.