Low Stability of Integrin-Binding Deficient Mutant of FGF1 Restricts Its Biological Activity

Fibroblast growth factor 1 (FGF1) has been shown to interact with integrin αvβ3 through a specific binding site, involving Arg35 residue. The FGF1 mutant (R35E) with impaired integrin binding was found to be defective in its proliferative response, although it was still able to interact with FGF receptors (FGFR) and heparin and induce the activation of downstream signaling pathways. Here, we demonstrate that the lack of mitogenic potential of R35E mutant is directly caused by its decreased thermodynamic stability and susceptibility to proteolytic degradation. Introduction of three stabilizing mutations into R35E variant compensated the effect of destabilizing R35E mutation and restored the proliferation potential of FGF1. Moreover, the stabilized R35E variant regained both anti-apoptotic and wound healing activities, while remaining defective in binding to integrin αvβ3. Our results suggest that the thermodynamic stability and resistance to degradation, rather than the interaction with integrin are required for mitogenic response of FGF1.

The in vitro effect of kynurenic acid on the rainbow trout (Oncorhynchus mykiss) leukocyte and splenocyte activity

Kynurenic acid (KYNA), an endogenous neuroprotectant formed along the kynurenine pathway of tryptophan degradation, is a selective ligand of the GPR35 receptor, which can be found on the surface of various populations of human immune cells. In infections and inflammations, KYNA produces an anti-inflammatory effect through this receptor, by depressing the synthesis of reactive oxygen species and pro-inflammatory cytokines. However, it is still unrecognized whether receptors for kynurenic acid are also localized on immune cells of poikilothermic animals, or whether KYNA is able to affect these cells. The objective of this study has been to determine the effect of different concentrations of kynurenic acid (12.5 μM to 10 mM) on the viability and mitogenic response of lymphocytes and on the activity of phagocytic cells isolated from blood and the spleen of rainbow trout. The results imply low toxicity of kynurenic acid towards fish immune cells, and the proliferative effect observed at the two lowest concentrations of KYNA (12.5 μM and 25 μM) seems indicative of endogenous kynurenic acid being capable of activating fish lymphocytes. Non-toxic, micromole concentrations of KYNA, however, had no influence on the mitogenic response of lymphocytes nor on the activity of phagocytes in rainbow trout under in vitro conditions. There is some likelihood that such an effect could be observed at lower, nanomole concentrations of KYNA.

Assessment of the Effect of Nilotinib (Tasigna) Maintenance Therapy After Allogeneic Stem Cell Transplantation in Patients with Advanced CML and Ph+ ALL On Immune Reconstitution and Lymphocyte Function

Abstract 4478 Allogeneic stem cell transplantation (AlloSCT) is the treatment of choice in advanced chronic myelogenous leukemia (CML) and Ph+ acute lymphoblastic leukemia (ALL). However, post transplant relapse rate is high and outcome is often poor in this setting. Reduction of tumor mass pre-transplant and maintenance therapy post alloSCT, may improve response rate and reduce relapse rate. We speculated that the second-generation tyrosine-kinase inhibitor (TKI) Nilotinib (Tasigna, Novartis Pharmaceuticals) would be effective in achieving these goals. In the current study Nilotinib, was administered as maintenance therapy post alloSCT in patients (pts) with advanced CML and Ph+ ALL (study CAMN107AIL03T). However, TKIs have been demonstrated in previously published literature to affect T cells proliferation and signal transduction and to potentiate LGL and NK cell activity. Furthermore, in recent studies TKIs have also been demonstrated to ameliorate chronic GVHD. We therefore assessed immunological reconstitution and function including flow analysis of lymphocyte subsets, T-mitogenic response to αCD3 and PHA, thymic activity as determined by the quantification of the T cell receptor excision circles (TREC), TCR repertoire and NK cells cytotoxic activity against K562 cell line. In all, the study included 24 pts. Patients engrafted in a median day +15 (range, 10–38) with 100% donor chimerism. Acute GVHD grade 3/4 was reported in 3 pts (14%) and the rate of extensive chronic GVHD at last follow up was 50%. At 6 months after alloSCT, 11 of 15 pts with advanced CML had attained CCyR, 11 of 15 pts with advanced CML had attained a MMR or better, and 5 of 7 pts with Ph+ ALL attained a CR. The median OS was 16 months, with predicted 1- and 2- year rates of 55% (95% CI, 32% – 72%) and 50% (95% CI, 28% – 68%), respectively. The median PFS was 11 months, with predicted 1- and 2- year rates of 50% (95% CI, 28% – 68%) and 38% (95% CI, 17% – 59%), respectively. Immunological testing was performed pre- and post Nilotinib maintenance therapy in 12 pts (advanced CML-8, Ph+ ALL-4) who received Nilotinib for at least 90 days following alloSCT. The median age was 34.5 years (range, 21–57) and 75% were males. Six pts underwent alloSCT from an HLA-matched sibling donor, 4 from matched unrelated and 2 from an alternative donor (cord blood-1, haploidentical-1). All had myeloablative conditioning. GVHD prophylaxis included CSA and MMF. The relative percentage of T- lymphocyte subsets (assessed by FACS) and total lymphocytes number were stable during Nilotinib maintenance administration after alloSCT, while a 7.8±2.5 fold increase in B cells was noted. T cell mitogenic response with αCD3 and PHA (stimulation index ratio) was sustained (2.5±1.0, vs. 2.8±1.05 and 3.3±1.3 vs. 5.3±2.9 stimulation, pre- and post Nilotinib therapy, respectively). Mean thymic output determined by TREC quantification pre-, during and post Nilotinib administration was 81.8±108, 81.2±90.3 and 142.8±197.4 copies per 0.5ug DNA indicating continuous thymopoiesis. Similarly, no significant change of the TCR repertoire was observed during Nilotinib treatment. Specifically, normal expression of the TCR repertoire was detected in 15.1±5.5 and 15.3±5.6 of the examined TCRs, clonal expression was detected in 2.5±2.2 and 2.9±3 of the examined receptors, while reduced expression was detected only in 6.4±4.3 and 5.8±4.5 of the examined receptors pre-and post Nilotinib treatment, respectively. NK cytotoxic activity against K562 expressed as fold of change from baseline, also remained stable during Nilotinib treatment (2.8±1.1 and 2.3±0.8, respectively). In summary, Nilotinib maintenance therapy post alloSCT in pts with advanced CML and Ph+ALL did not interfere or jeopardized immune reconstitution and function including the number of immune cell subsets, T cell mitogenic response, TCR repertoire, thymic output and NK cytolytic activity post alloSCT. Based on this immunological data we would further recommend Nilotinib maintenance therapy post alloSCT in pts with advanced CML and Ph+ALL. Disclosures: Nagler: Novartis: Honoraria, Research Funding.