Functional blockade of tyrosine kinase A in the rat basal forebrain by a novel antagonistic anti-receptor monoclonal antibody

Abstract

We have exploited a new monoclonal antibody against the tyrosine kinase A (TrkA) nerve growth factor (NGF) receptor to block the NGF-TrkA interaction in the rat basal forebrain. The monoclonal antibody MNAC13 is a potent antagonist that prevents the binding of NGF to TrkA in a variety of systems. This antibody was used to study the maintenance of the cholinergic phenotype in the rat basal forebrain in vivo, by the implant of antibody-secreting cells. Basal forebrain cholinergic neurons (BFCNs) are greatly affected by the antibody treatment, both in terms of cell number and of cell soma size. When antibody-secreting cells are implanted at postnatal day 2 (P2), the effects observed at P8 are as severe as those obtained with anti-NGF antibodies and, interestingly, are observed also if anti-TrkA cells are implanted at P8, when anti-NGF antibodies, delivered by the same route, are no longer effective (). The effects induced by anti-TrkA, as those induced by anti-NGF, are reversible, but the time required for recovery and the critical period in the sensitivity of BFCNs to the functional inactivation of TrkA is twice as long than that observed when NGF is intercepted. These results demonstrate that BFCNs are more sensitive to the block of TrkA activation than they are to the block of NGF. The cloning of MNAC13 variable regions and their assembly into a functional polypeptide of reduced size (single chain Fv fragment) will allow its use in gene transfer applications.

Fig. 1.

Inhibition of binding of ¹²⁵I-NGF to TrkA-BALB/C 3T3 cells. Hybridoma supernatants were preincubated with TrkA-BALB/C 3T3 cells before the addition of ¹²⁵I-NGF. The histogram reports the inhibition of specific NGF binding to TrkA-BALB/C 3T3 cells by different antibodies. Specific binding was evaluated by subtracting from the total binding that obtained in the presence of an excess of unlabeled NGF. The values reported are the mean ± SEM of triplicates.

Fig. 2.

mAb MNAC13 recognizes the extracellular domain of TrkA receptor and does not inhibit NGF binding to soluble TrkA receptors. A, Soluble TrkA and TrkB receptors, engineered as immunoadhesins (TrkA-IgG and TrkB-IgG, see Materials and Methods) were used as solid-phase antigens for an ELISA assay and incubated with 2 or 20 ng/ml of purified mAb MNAC13. B,TrkA-IgG was used as solid-phase antigen for an ELISA assay, preincubated with saturating NGF (left) or saturating MNAC13 (right) and then incubated with MNAC13 (left) or NGF (right). MNC13 does not block NGF binding to TrkA-IgG and vice versa.

Fig. 3.

mAb MNAC13 recognizes the TrkA receptor on living cells. BALB/C 3T3 or TrkA-BALB/C 3T3 cells were incubated with purified mAb MNAC13 and subjected to FACS analysis.

Fig. 4.

mAb MNAC13 labels the TrkA receptors on rat basal forebrain neurons and does not bind to TrkC. Coronal sections of P10 rat basal forebrain (A, B) were incubated in the presence (A) or in the absence (B) of mAb MNAC13. Coronal sections of P10 rat arcuate nucleus were incubated with mAb MNAC13 (C) or anti-TrkC (D). Scale bar, 98 μm.

Fig. 5.

mAb MNAC13 inhibits the NGF-induced differentiation of rat pheochromocytoma PC12 cells. PC12 cells were transferred to serum-free medium and incubated in the absence (A) or the presence (B–D) of 20 ng/ml NGF for 4 d. mAb MNAC13 (4 μg/ml) (C) completely inhibits NGF-induced survival and differentiation, whereas the control antibody 9E10 does not (D).

Fig. 6.

Implant of mAb MNAC13-secreting cells in the rat brain significantly reduces the number of cholinergic basal forebrain neurons. The cholinergic phenotype of P9 (A–C) and P16 (D–F) rat basal forebrain neurons was determined by immunoreactivity for ChAT after the intraventricular implant at P2 of cells secreting the anti-TrkA MNAC13 antibody (B,E), the anti-NGF αD11 antibody (C, F), or of control myeloma cells (A,D). Note the marked reduction of the number of ChAT-positive neurons in MNAC13-implanted rats (B). Scale bar, 75 μm.

Fig. 7.

Soma size distribution of ChAT-immunopositive cells in the medial septum (MS) and diagonal band (DB) of animals implanted with mAb MNAC13-secreting cells. The soma size distribution of ChAT-immunopositive cells in the MS and DB of animals implanted with mAb MNAC13-secreting cells (black bars) or with myeloma cells (white bars). Animals were injected at P2 and killed at P9, P16, and P22 (A) or injected at P8 or P15 and killed at P16 or P22 (B) (Figure 7continues).

Fig. 7.

Soma size distribution of ChAT-immunopositive cells in the medial septum (MS) and diagonal band (DB) of animals implanted with mAb MNAC13-secreting cells. The soma size distribution of ChAT-immunopositive cells in the MS and DB of animals implanted with mAb MNAC13-secreting cells (black bars) or with myeloma cells (white bars). Animals were injected at P2 and killed at P9, P16, and P22 (A) or injected at P8 or P15 and killed at P16 or P22 (B) (Figure 7continues).

Fig. 8.

Recombinant forms of mAb MNAC13 bind TrkA. Phage-displayed ScFvMNAC13 (A), soluble ScFvMNAC13 (B), and the parental mAb MNAC13 (C) were used in a TrkA ELISA assay, using TrkA immunoadhesin as a solid-phase antigen in the presence of increasing concentrations of competing soluble TrkA immunoadhesin.Squares are 10-fold dilutions of the input antibody, with respect to triangles.

Fig. 9.

Inhibition of NGF-induced neurite growth in PC12 cells by the recombinant ScFvMNAC13. Periplasmic fractions containing recombinant ScFvMNAC13 (B) or an irrelevant control ScFv fragment (anti-phox ScFv) were added, together with 10 ng/ml of NGF, to PC12 cells primed for 7 d with 50 ng/ml of mouse NGF, and replated at the beginning of the assay. The recombinant ScFvMNAC13 in B inhibits the regrowth of neurites in replated primed PC12 cells, mediated by the activation of TrkA by NGF.