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  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
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  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
  • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
  • C07K2317/622—Single chain antibody (scFv)
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  • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
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  • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
  • C12N2799/026—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a baculovirus

Definitions

• NGF assumes a particular importance in the study of Alzheimer's Disease: in the hippocampus of these patients, there is an increase in the expression of NGF, whilst a decrease in NGF levels is observed in the basal forebrain (Crutcher et al., 1993; Scott et al., 1995; Fahnestock et al., 1996). This results depends on an alteration in the retrograde transport of NGF from the post-synaptic cortical territory to the cellular body of the basal forebrain.
• NGF Tumor Necrosis Factor
• NGF pre-pro-protein of 27 kDa
• the doses are between 0.5 and 15.0 ⁇ g/kg of body weight.
• exposure takes place for at least 15 days.
• anti-NGF mice are the sole animal model for Alzheimer's Disease which shows lesions at the level of the cholinergic neurons of the basal forebrain and of the Meynert basal nucleus (Ruberti et al., 2000; Capsoni et al., 2000, 2002a).
• the anti-NGF ( ⁇ Dll) antibody produced by hybridomas binds the NGF molecule at the epitope responsible for its interaction with the high affinity TrkA receptor and blocks interaction with the p75 receptor. Consequently, blocking this binding, it is an antibody characterised by neutralising activity on both the TrkA and p75 receptors. It was expected that the administration of said molecules would anticipate and/or enhance the degree of neurodegeneration in anti-NGF transgenic mice.
• LAP The expression of LAP is regulated by different factors, such as "tumour necrosis factor ⁇ " (Chu et al., 1997), the growth factor of vascular endothelium (Tran et al., 1999), and NGF (Wiese et al. 1999) .
• NGF determines an increase in the expression of ITA, the homologue of c-IAP-2 in chickens (Wiese et al., 1999). Since, in chickens, both NGF and ITA determine the survival of sympathetic and sensorial neurons, it is presumed that NGF is able to promote the survival of these neuronal populations by the induction of high levels of ITA.
• FIG. 1 Schematic representation of the interaction of anti-NGF oDll antibodies (Cattaneo et al., 1988), anti-NGF 27/21 antibodies (Nanduri et al., 1994), anti-TrkA MNAC13 antibodies (Cattaneo et al., 1999), anti-p75 LLG 17 antibodies; and immunoadhesin LLG 65 with the receptors of NGF TrkA and p75.
• FIG. 1 Coloration for cholin acetyltransferase (ChAT) of the basal forebrain of control mice (A), anti-NGF transgenic mice (B), anti-NGF transgenic mice after implant with hybridoma P3U (C), anti-NGF transgenic mice after implant with hybridoma secreting ⁇ Dll (D).
• ChAT cholin acetyltransferase
• FIG. 4 Quantification of the cholinergic deficit in anti-NGF mice treated with hybridomas secreting ⁇ Dl l, MNAC13, 27/21, LLG 17 and LLG 65.
• the chart shows the prevention of the reduction in the number of cholinergic neurons of the basal forebrain in anti-NGF mice treated with the oDll, 27/21, LLG 17 antibodies, and with the immunoadhesin LLG 65.
• the anti-TrkA antibody MNAC13 is not able to prevent cholinergic deficit in the basal forebrain.
• Figure 9 Quantification of the number of positive c-IAP-1 neurons in the entorhinal cortex after treatment with hybridomas secreting anti-NGF ⁇ Dl l and 27/21, MNAC13, LLG 17 and of LLG 65 immunoadhesin.
• Figure 14 A) Series of successive injections of h- prongf on the channel with AD 11 at low concentration. Increasing concentration of antigen, from 100 to 500 nm. The white was removed from the curves (fc2-fcl). B) Series of successive injections of h-proNGF on the channel with AD 11 at high concentration. Increasing concentration of antigen, from 100 to 500 nm. The white was removed from the curves (fc4-fcl). C) Series of successive injections of h- prongf on the channel with asv5. Increasing concentration of antigen, from 100 to 500 nm. The white was removed from the curves (fc3-fcl). Figure 15.
• Immunoadhesin LLG65 was prepared by fusion of the extracellular portion of the human p75 receptor with the Fc portion of camel IgG 2 comprising the 35 amino acids of the hinge region of the antibody followed by the C H 2 and C H 3 domains according to the protocol published by Hamers-Casterman (1993).
• the resulting coding sequence for immunoadhesin p75 was inserted in the genome of Baculovirus (Autographa californica virus) using the pAcGP67B vector for expression in insect cells. Sf9 insect cells were used to amplify the viruses. H5 cells were infected with amplified recombining
• the ⁇ Dll hybridomas secreting anti-NGF monoclonal antibodies which block the interaction between NGF and its receptors TrkA and p75, Fig. 1), 27/21 (secreting antibodies which inhibit the binding of NGF to p75, Fig. 1), MNAC13 (secreting anti- TrkA antibodies; Fig. 1), LLG17 (secreting p75 neutralising antibodies, Fig. 1) and LLG 65 (secreting immunoadhesin which prevents the binding of NGF to p75; Fig.
• the brain was removed and post-fixed in the same fixative. After cryoprotection in 30% saccharose, the brains were sectioned using a refrigerated slide microtome.
• the sections of the brain of the anti-NFG mice obtained according to Example 1 were incubated with the following primary antibodies according to the protocol for immunohistochemistry described in example 1: anti-phospho CREB (serl33) (Cell Signaling, Beverly, MA), anti phospho-c- jun (ser63) (Cell Signaling, Beverly, MA), anti-fas (epitope corresponding to amino acids 1-335), anti-p53 (epitope corresponding to amino acids 1-393), anti-NfKB (epitope corresponding to the carboxy terminal of NfkB), anti- TRAF2 (epitope to the terminal carboxy of TRAF2; Santa Cruz, Santa Cruz, CA), anti-ARMS (epitope to the terminal carboxy; provided by Dr. Moses Chao, New York Medical Center, New York).
• Table 1 The results are summarised in Table 1.
• the sections containing the basal forebrain and the hippocampus were processed to highlight the number of positive cells for c-IAP-1 and c-IAP-2.
• the sections were washed twice in TBST buffer. After blocking the endogenous peroxidase in a 3% solution of hydrogen peroxide, non specific bindings were blocked in 10% of bovine foetal serum dissolved in TBST. After half an hour, the sections were incubated with the primary antibody directed against c-IAP-2 and c-IAP-2 (antibodies in anti-ChAT rabbit; 1: 100, Santa Cruz, Santa Cruz, CA). Incubation took place at 4°C for 12 hours.
• proNGF All the proNGF from 348 to 698 (pro+ first 14 aa of NGF) from SEQ LD No. 1: gaa ccg ta
• Pro-NGF was cloned in the polylinker downstream of lexA as a product of PCR cut with the BamHI-Pstl enzymes.
• the Pro-41 was cloned in the polylinker downstream of lex A as a product of PCR cut with the BamHI-Pstl enzymes (primers used: pro Bam for 5 ' TAT AAT GGG ATC CGT
• the alfaDll antibody interacts differently with NGF and with proNGF.
• NGF has a greater affinity for the antibody, both because of a very rapid association, and because of a very slow dissociation.
• the proNGF whose association is slower and whose dissociation is much more rapid, has a different behaviour.
• the behaviour of both antigens is specific, since in the case of the channel with aSV5 the same type of effect does not take place.
• the interaction of the proNGF with alfaDll is due solely to the NFG part, since the negative GSTpro (containing the part of the precursor in fusion with the GST) does not respond to the antibodies in any way.
• the alfaDl 1 antibody when expressed in vivo, is able to alter the balance between pro-survival action of NGF and the pro-apoptotic function of pro-NGF because a determines a practically irreversible sequestration of mature NGF. Therefore this antibody can be used to set up systems for selecting molecules with a proNGF blocking action.
• the selection may be conducted in vitro and in vivo, through the expression of recombinant antibodies in murine models. The prototype of these models is represented by the anti-NGF AD11 mouse.
• Example 8 Prevention of the cholinergic deficit in anti-NGF mice by implanting hybridomas secreting antibodies neutralising pro-NGF
• Anti-proNGF hybridomas secreting monoclonal antibodies neutralising the pro-10 mutated form (see Example 5) of proNGF, selected as in Example 6 and myeloma P3X63Ag8 (P3U, cellular control line) were grown Dulbecco's modified Eagle's culture medium supplemented with 10% of bovine foetal serum. Before injection, cells were washed four times in the Hank saline solution (HBSS) and re-suspended in HBSS at the concentration of 2 x 10 5 cells/ ⁇ l.
• HBSS Hank saline solution
• the signal deriving from the reaction between primary and secondary antibody was amplified by means of incubation with a peroxidase (Vector Labtek). The reaction was then developed by incubation with the diaminobenzidine chromogen (Sigma, St. Louis, MO).
• the volume of the basal forebrain was calculated using Cavalieri's method (Michel and
• Example 9 Inhibition of the binding of proNGF to sortilin by incubation with anti- proNGF.
• the inhibition by the anti-proNGF antibody of the binding of proNGF to sortilin was studied according to the method described in Mahadeo et al. (1994) and Esposito et al.
• the human melanoma cells A875 (American Type Culture collection), transfected to determine the expression of the sortilin receptor (2x10 /ml) were incubated (4 °C, 40 min) with I 125 -proNGF (2-20 x 10 "10 M) in the presence or absence of an excess of anti-proNGF (500 times in excess relative to the concentration of pro-NGF).
• the ligand bound to the receptor was then separated from the free pro-NGF ligand in the ⁇ • 19 middle by centrifuging in the presence of bovine serum. The radioactivity of I -proNGF bound to the cell was determined in the pellet.
• the p75/sortilin receptor complex is involved in the pathogenesis of the Alzheimer phenotype observed in anti-NGF mice and in ADll-VK mice.
• the ADll-VK mice have a neurodegenerative phenotype which can be superposed to that of ADll mice.
• the ADll-VK mice have been crossed with p75NTR exonIII (-/-) mice, in which the genie exon corresponding to the extracellular domain of the p75NTR receptor was deleted by gene knockout with homologous recombination (Jackson Laboratories, Lee et al., 1992).
• the sections were washed twice in Tris/HCl 0.1 M buffer to which were added 0.15 M of NaCl and 0.3% of Triton-X 100 (TBST). After blocking the endogenous peroxidase in a 3% solution of hydrogen peroxide, non specific bindings were blocked in
• AD11 VK mice have a high number of beta-amyloid deposits
• AD12 mice have a number of deposits comparable to that of non transgenic mice.
• amyloid component of the neurodegenerative phenotype in ADll mice and in ADll-VK mice disappears if signalling through p75NTR is inhibited.

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