WO2010030203A1

WO2010030203A1 - Monoclonal antibody to human amyloidogenic and modified forms of transthyretin and its use in the detection and treatment of fap and pathologies presenting modified ttr

Info

Publication number: WO2010030203A1
Application number: PCT/PT2008/000034
Authority: WO
Inventors: Maria João SARAIVA; Anabela Clao Teixeira; António Bernardino GUIMARÃES PARADA
Original assignee: Biocodex - Incubação De Empresas De Ciências Da Vida, S.A.
Priority date: 2008-09-09
Filing date: 2008-09-09
Publication date: 2010-03-18

Abstract

The present invention relates to a monoclonal antibody (mab) for human mutant transthyretin (TTR), a kit for the detection of FAP comprising the referred mab, methods for its production and its uses such as in screening for familial amyloidotic polyneuropathy (FAP) and pathologies presenting modified forms of TTR, and in research and treatment of associated diseases thereof. This monoclonal antibody presents as main characteristic the recognition of amyloidogenic plasma mutant TTR and modified TTR. These properties are assigned to unique sequences of the mab that reacts with a cryptic epitope in TTR that is exposed by mutant and/or modified TTR implying a high specificity for modified forms of mutated and non-mutated plasma TTR. The mab of the invention finds use in epidemiological studies of TTR amyloidogenic mutations in the population and in biomarker studies associated with pathologies presenting modified TTR.

Description

DESCRIPTION

MONOCLONAL ANTIBODY TO HUMAN AMYLOIDOGENIC AND MODIFIED FORMS OF

TRANSTHYRETIN AND ITS USE IN THE DETECTION AND TREATMENT OF FAP

AND PATHOLOGIES PRESENTING MODIFIED TTR

Technical domain of the invention

The present invention relates to a monoclonal antibody (mab) for human mutant transthyretin (TTR) methods for its production and its uses such as in screening for familial amyloidotic polyneuropathy (FAP) , pathologies presenting modified forms of TTR and in research and treatment of associated diseases thereof. Therefore, the present invention applies to pharmaceutical and medical areas.

Background of the invention

In 1952, Corino de Andrade described, for the first time, a particular form of polyneuropathy characterized by the deposition of amyloid, in particular in peripheral nerve. This form of neuropathy is endemic in northern Portugal, and has a dominant autosomal mode of inheritance. Onset of symptoms occurs between 25 and 35 years, leading to death in 10 to 15 years . This form of neuropathy, which is designated by familial amyloidotic polyneuropathy - FAP (Andrade 1952) and was subsequently described in other populations in the world with focci in Sweden, Japan and Italy among other countries. Other FAP forms differing in clinical presentation, namely age of onset, progression, affected organs, had been described (for a revision, see Saraiva, 2001) .

The chemical nature of the protein deposited in tissues of Portuguese FAP patients was elucidated in 1984, with the demonstration of a mutant transthyretin (TTR) in plasma presenting a single amino acid substitution of methionine for valine in position 30 (Saraiva et al., 1984) . Since the description of TTR Val30Met, many other TTR mutations have been described, the great majority associated with FAP. Some forms are not neuropathic but rather cardiomyopathic, whereas other mutations are not pathogenic.

TTR is the main constituent of extracellular amyloid deposits in FAP, found in several organs and tissues, in particular, in nerve. TTR is produced mainly by the liver and choroid plexus, and can be found in the plasma and cerebrospinal fluid, where it circulates as a tetramer composed of four 127 amino acid sub-units. TTR has a physiological role in the transport of the hormone thyroxine (T₄) and of retinol (indirectly, through RBP - Retinol Binding Protein) . T₄ binds in a central channel formed by the interaction of the four monomers (Blake et al., 1976) and RBP attaches to the surface of the tetramer, as revealed by the structure of the complex (Monaco et al . , 2002) . Each TTR monomer is composed of eight β-strands designated A-H, organised in 2 β-sheets interacting in a face-to- face fashion, forming the hydrophobic core of the molecule. Sheets CBEF are oriented to the exterior of the molecule and sheets DAGH line the T₄ binding channel, with exclusively hydrophobic residues.

Several evidences regarding the amyloidogenic process in TTR amyloidoses have been pointed out and the prevalent theory postulates that tetramer dissociation into monomers is the triggering event. Quintas et al . (1999) studied the influence of TTR mutations on tetrameric stability, showing dissociation into non-native monomeric species at physiological pH, followed by self-association of this intermediate into amyloid fibrils. This was possibly due to subtle conformational modifications that occur in the TTR molecule before dissociation into monomers with altered tertiary structure. These authors had previously established a relationship between the amyloidogenic potential of TTR variants and a decreased stability of the tetramer, with subsequent dissociation to monomeric species (Quintas et al . , 1999) . This amyloidogenic intermediate was postulated to contain most of the native structure except for the rearrangement involving strands C and D. In this model, FAP mutations would not affect the structure of the folded state (tetramers) but would favour the denaturation pathway and/or degradation pathway (s) for TTR turnover.

There are more than 100 point amyloidogenic mutations described for TTR

(http://www.ibmc.up.pt/mjsaraiva/ttrmut.html); analyses of the distribution of TTR variants along the peptide chain identified a peak in the distribution of TTR variants along the peptide chain corresponding to a region of maximum mutational frequency ("hot-spot"), associated with the edge strands C and D (residues 45-58) of the two β-sheets that form the structural framework of the TTR molecule. Changes at the edges of the β-sheets of TTR could be so significant as to form, or expose, an amyloidogenic determinant, not present in the native TTR protein.

Leu55Pro TTR has been associated with early onset and highly aggressive amyloidosis and X-ray structural data showed a crystal with intermolecular contacts profoundly altered, resulting in the assembly of an oligomeric structure that might represent an intermediate in the amyloidogenesis cascade (Sebastiao et al., 1998) . The exchange of the amino acid Leu for the amino acid Pro at position 55 changes the TTR secondary structure by disruption of strand D; the molecule is rearranged and residues 54-56 are included in a long loop that connects β strands C and E, which is involved in the crystallographic packing observed in this crystal structure. This "amyloid-like" structure revealed additional positional differences such as the contacts of the α-helix and the AB loop. In particular, structural comparative studies of TTR native fold and the abnormal conformation of Leu55Pro variant, revealed that the OH group of Tyr 78 plays an important role in maintaining the tertiary structure of the AB loop, which to the design the TTR mutant Tyr78Phe (Redondo et al . , 2000) . It was found that this variant formed fibrils in the same pH range as Leu55Pro TTR and was reactive, in its tetrameric soluble form, to a monoclonal antibody described by Goldsteins and co-workers (1999) as specific for amyloid. A substitution of a Tyr for a Phe at position 78 might have caused a rearrangement in the tight tetrameric structure due to the breaking of key H-bonds, yielding a soluble amyloidogenic intermediate with exposed cryptic epitopes, present in an earlier step of the amyloidogenic process .

The reasons leading soluble plasma mutant TTR to aggregate and deposit in the extracellular space of tissues are not totally clear. No evidence exists for the circulation of TTR aggregates in plasma from FAP patients (Saraiva et al . , 1983), Sousa et al . , (2001) demonstrated, that mutant TTR can deposit early in an aggregated non-fibrilar form in asymptomatic carriers of TTR Val30Met, or in transgenic mice carrying this mutant human gene (Sousa et al . , 2002), before overt amyloid deposition. Deposition of the protein in this oligomeric, non- fibrillar form triggers the expression of inflammatory molecules (such as interleukine 1) and of oxidative stress molecules (such as nitric oxide synthase) (Sousa et al . , 2001a) .

Monoclonal antibodies (mabs) produced in mice against highly- aggressive amyloidogenic synthetic TTR mutants (Goldsteins et al . , 1999) were shown to react with high molecular weight TTR aggregates, and do not recognize soluble native TTR when tested under ELISA (enzyme-linked immunoassay) by direct procedures. It was hypothesized that these mabs recognize cryptic epitopes that are exposed in mutant TTRs resembling aggregated TTR. Interestingly, these mabs react to TTR from plasma of FAP patients and/or asymptomatic carriers of neuropathic TTR mutants, but not to plasma from normal individuals (Palha et al., 2001) .

The antibodies described in these publications discriminate mutant TTR of FAP patients and asymptomatic carriers from normal TTR of control subjects, but only by a direct ELISA procedure, making them useful to epidemiological studies for mutant neuropathic mutations .

In a previous report, Costa et al (1993) described a mab TTR with similar characteristics used in epidemiological studies for the Val30Met mutation in the Swedish population.

By contrast, the present invention discloses discriminating properties between mutant and normal TTR not only in direct, but also in indirect ELISA procedures. Furthermore, the mab TTR of the present invention detects by immunoblotting particular modified TTR bands in plasma of FAP patients. The presence > of modified forms of TTR in plasma has been speculated in the literature, but never documented to date, opening, for the first time the possibility to address pathogenesis and follow up of therapies in FAP.

By using powerful screening techniques, such as 2 -dimensional electrophoresis, TTR has been associated with a number of pathological conditions, including different types of cancers (Fung et al 2005, Moore et al . 2006, Escher et al . , 2007) , Alzheimer and other neurodegenerative diseases (Biroccio et al , 2006) , pre-eclampsia (Vascotto et al , 2007), moyamoya disease (Rϋggeberg et al . , 2008); in these conditions, modified forms of TTR are detected in plasma, cerebrospinal fluid or amniotic fluid (for pre-eclampsia) , most of them representing oxidized TTR. The used techniques require cumbersome procedures, expensive sophisticated mass spectrometry equipment and expertise not available in many institutions and laboratories, limiting their use on a routine basis .

By contrast, the present invention allows the detection of these modifications, such as oxidation, in a one dimensional electrophoretic separation followed by Western bloting with the mab, opening the possibility of the use of the mab as a biomarker in these pathologies.

General description of the invention

The present invention features a mab produced in mice against recombinant mutant Tyr78Phe TTR mutant that, selectively recognises under specific conditions: (i) plasma samples from carriers of certain known TTR mutants associated with FAP; (ii) slow migrating electrophoretic TTR band only present in the plasma of carriers of mutant TTR; (iii) faster migrating electrophoretic TTR bands detected in pathologies presenting modified TTR; the mab is able to remove TTR modified species from tissues of transgenic mice for human mutant Val30Met TTR presenting TTR extracellular deposition.

A second aspect of the present invention refers to a method to generate such preferred mab. The method involves (a)hybridoma production by fusion of spleens cells sample from immunized mice with Sp2/0 cells sample; (b) screening of hybridomas reactive to different mutant TTRs and non-reactive to normal non-mutated TTR, by ELISA immunoassay under native conditions; (c) re-cloning of the mab; and (d) isolation and sequence determination.

A third aspect of the present invention refers to a kit for the detection of FAP comprising the monoclonal antibody, according to the present invention.

A fourth aspect of the present invention refers to the use of the produced mab, according to the present invention, to detect the presence of known mutant amyloidogenic TTR in plasma samples and, for unknown mutant TTRs. The mab of the invention is useful as to give indication for the presence in plasma of an amyloidogenic mutation. The mab also finds use in screenings for epidemiological studies of TTR amyloidogenic mutations in the population once this mab has unique properties as recognizing cryptic epitopes/conformations associated with circulating plasma mutant TTR species, not present in normal TTR, which, to date, have never been documented.

A fifth aspect of the present invention is the use of the mab for detection of faster migrating electrophoretic TTR bands in pathologies presenting modified TTR, making the mab useful in biomarker studies associated with these pathologies. To test whether TTR deposition in FAP can be counteracted by- antibodies for cryptic epitopes, we have immunized with Tyr78Phe transgenic mice carrying the most common FAP associated TTR mutant - Val30Met - at selected ages that present normally with either non- fibrillar or TTR amyloid d deposition (Terazaki et al . , 2006) .

Compared to age matched control non- immunized mice, Tyr78Phe immunized mice had a significant reduction in TTR deposition usually found in this strain, in particular in stomach and intestine; by contrast, animals immunized with Val30Met did not show differences in deposition in comparison with non- immunized mice.

Immunohistochemical analyses of tissues revealed that immunization with Tyr78Phe lead to infiltration by lymphocytes and macrophages at common deposition sites, but not in tissues such as liver, choroid plexus, and Langerhans islets, in which TTR is produced. These results suggest that Tyr78Phe induced production of an antibody that reacts specifically with deposits and leads to an immune response effective in removing/preventing TTR deposition. Taken together, production of TTR mabs capable of removing modified TTR from FAP patients is most warranted; passive immunization procedures are the best approach to attain this goal. The present invention, when administered by passive immunization in transgenic mice carrying the Val30Met mutation removes TTR deposits from tissues.

1. Characterization of the mab

The monoclonal antibody to human amyloidogenic transthyretin, according to the present invention, comprises the following amino acid residues: SEQ. ID 1: Amino acid sequence of AD7F6 antibody VL regions)

SEQ. ID 2: Amino acid sequence of AD7F6 antibody VH regions

SEQ. ID 3: Nucleotide sequence of full-length heavy chain

AD7F6

SEQ. ID 4: Nucleotide sequence of full-length light chain

AD7F6

The monoclonal antibody of the invention produces an IgG monoclonal antibody of subclass IgGa_b* kappa light chain. The antibody has a molecular weight of 160 kDa, which upon reduction yields 50 kDa and 28 kDa fragments. It recognizes amyloidogenic plasma mutant TTR both in reducing and non- reducing conditions and modified plasma TTR. These properties are assigned to unique sequences of the mab that reacts with a cryptic epitope in TTR that is exposed by mutant and/or modified TTR.

2. Characterization of the process of mab production

Further, the present invention features a method to generate such preferred mab. This method involves the following steps:

(a) immunization of TTR null mice with TTR Tyr78Phe;

(b) hybridoma production by fusion of spleens cells from immunized mice with Sp2/0 cells;

(c) screening of hybridomas reactive to different mutant TTRs and non-reactive to normal non-mutated TTR, by ELISA immunoassay under native conditions.

3. Characterization of the uses of the mab

The mab reacts in ELISA immunoassay under native conditions with Val30Met TTR in its isolated form obtained either from the periplasmic space of recombinant bacteria or from the plasma of carriers of Val30Met but does not react with normal, non-mutated TTR isolated from bacteria or plasma. The monoclonal antibody of the present invention reacts with modified human plasma TTR from carriers of mutant TTR Val30Met. For unknown mutant TTRs, the mab of the invention is useful as to give indication for the presence in plasma samples of an amyloidogenic mutation.

Therefore, the mab of the present invention is applicable in the detection of known mutant amyloidogenic TTR in plasma samples and, for unknown mutant TTRs, to give indication for the presence in plasma of an amyloidogenic mutation. The mab also finds use in screenings for epidemiological studies of TTR.

The mab of the invention detects abnormal TTR bands representing modified non-mutated TTR, making the mab useful in biomarker studies associated with pathologies presenting modified TTR.

In the present invention we give in detail examples of properties of an antibody generated against Tyr78Phe.

3.1. One particular embodiment, related to the use of the mab according to the present invention, is the detection of amyloidogenic TTR mutants in plasma and involves the production of an ELISA kit comprising the mab of the present invention and the use of the following method:

(a) Coating of a plate with polyclonal rabbit anti-TTR; this step is omitted in direct procedures;

(b) application of the plasma sample ;

(c) contacting the sample with the mab;

(d) detection of positive signal by reaction with labelled secondary antibody against mouse immunoglobulins. 3.2. In one preferred embodiment, the mab reacts in ELISA immunoassay under native conditions with Val30Met TTR in its isolated form obtained either from the periplasmic space of recombinant bacteria or from the plasma of carriers of Val30Met but does not react with normal, non-mutated TTR isolated from bacteria or plasma.

3.3. In other preferred embodiments, the mutant TTR is isolated from the periplasmic space of recombinant bacteria producing:

(i) mutant Tyr78Phe (ii) mutant Leu55Pro

In one preferred embodiment of the present invention, the mab of the invention detects synthetically produced oligomeric or aggregated forms of TTR. One particular method of detection comprises :

(a) Treatment of Tyr78Phe by stirring, to produce oligomeric species or by acidification to produce protofibrils;

(b) Dot blotting after application of the samples to nitrocellulose filters;

3.4. Other preferred embodiments, aggregates of Val30Met TTR, are recognized by the mab.

3.5. In preferred embodiments, the mab of the invention detects a slower migrating electrophoretic band -SMT- present in plasma samples of carriers of Val30Met TTR, composed of intact modified TTR, absent in normal plasma samples. One more specific particular method of detection involves:

(a) Immunoprecipitation of the samples with the mab; (b) Application of immunoprecipitate to a denaturant Laemli SDS gel;

(c) Immunoblotting after denaturant SDS gel electrophoresis .

3.6. In other more specific preferred embodiments SMT is detected by:

• immunoblotting after native electrophoresis

• silver staining of the gel after immunoprecipitation of the samples with the mab

3.7. In preferred embodiments, the mab of the invention detects two faster migrating electrophoretic bands FMT - generated upon oxidation treatment of plasma samples of carriers, of Val30Met TTR, or normal controls. One particular method of detection involves:

(a) Treatment of plasma samples with oxidative agent;

(b) immunoprecipitation of the sample with the mab;

(c) application of immunoprecipitate to a denaturant Laemmli SDS gel;

(d) immunoblotting after denaturant SDS gel electrophoresis.

3.8. In other preferred embodiments FMT are detected in isolated TTR from plasma samples or recombinant bacteria after oxidation.

3.9. In other preferred embodiments FMT are detected in plasma of pathologies presenting modified TTR.

The invention also features therapeutic applications of the mab. In one preferred embodiment, the mab can remove TTR deposits from transgenic mice for human Val30Met TTR (hTTR Met30) . One particular method comprises:

• 6 transgenic mice for human Val30Met TTR in a heat -shock factor-1 null background (Santos et al . , 2008), aged 3 months, are given intraperitonealy weekly doses of 300 μg of mab in a final volume of 400 μl for 6 months.

• Five days later after the last dose, animals are sacrificed, and the sciatic nerve removed and fixed in PLP solution (paraformaldehyde : lysine : sodium periodate) and crioprotected in a 20% glycerol solution. 7 micron frozen sections are processed for immunohistochemistry .

• Endogenous peroxidase is destroyed before blocking with 1% BSA, 4% fetal bovine serum in PBS for 1 hour at 37 0C. Rabbit anti-human TTR antibody (1:1000, DAKO) is incubated in a humidified chamber at 4 ⁰C. Biotin- conjugated anti-rabbit (1:20 Sigma), followed by extravidin labelling (1:20, Sigma) are then, each incubated for 30 minutes at 37 ⁰C. The reaction is developed with 3-amino-9-ethyl carbaxole, AEC (Sigma) .

Advantages of the invention

The present invention provides a number of advantages such as the screening of amyloidogenic TTR mutations and the detection of SMT and FMT in:

(i) asymptomatic carriers of the Val30Met mutation;

(ii) FAP patients carriers of the Val30 Met mutation;

(iii) asymptomatic carriers of the Val30Met mutation subjected to pilot clinical trials; (iv) FAP patients carriers of the Val30Met mutation subjected to pilot clinical trials,- (v) FAP patients carriers of the Val30Met mutation subjected to liver transplantation; (vi) normal recipients of livers from Val30Met mutation individuals in domino transplantation procedures; (vii) patients with pathologies presenting modified TTR (viii) in studies aimed at using transgenic mice for human

Val30Met TTR as means to elucidate mechanisms of deposition and action of drugs on the mutant human

TTR metabolism.

And will allow:

(a) to monitor efficiently the outcome of hepatic transplant as a means of FAP treatment;

(b) to monitor efficiently the outcome of future FAP treatments ;

(c) to monitor the outcome of mutant TTR in recipients of FAP livers subjected to domino transplants;

(d) the application of FAP treatment by immunization protocols

(e) high through output screening of certain neuropathic TTR mutations;

(f) high through output biomarker screening of patients with pathologies presenting modified TTR.

Description of the Drawings

Figure 1 : Represents the comparison of the mab reactivity with the TTR isolated from plasma of Val30Met carriers and from control non-carriers individuals and, the reactivity to Val30Met -TTR obtained from recombinant bacteria.

Figure 2: Represents the specificity of the mab towards oligomeric and aggregated forms of TTR. Figures 3a and 3b: Represent the comparison of the mab reactivity towards plasma of Val30Met carriers and of control non-carriers individuals by direct (3a) and sandwich (3b) ELISA procedures.

Figures 4a and 4b: Represent the detection of soluble modified slow migrating TTR (arrow) in human sera of Val30Met carriers (4b) and in sera from transgenic mice (4a) for the same mutant after native Western blotting using the mab.

Figures 5 : Represent the demonstration of the slow migrating TTR (SMT) in sera of carriers of Val30Met TTR (5a) and of fast migrating TTR (5b) (FMT) after oxidation treatment following immunoprecipitation and Western blotting from a denaturant gel, using the mab.

Figure 6: Represents the demonstration of the effect of the mab by passive immunization on TTR deposition in the sciatic nerve of transgenic mice.

Detailed description of the invention

1. Process for the preparation of the antibody

1.1. Samples

Sera from carriers and control individuals were previously screened by DNA RFLP analyses with Nsi I enzyme to identify the carriers and non-carriers of the Val30Met mutation. Mice sera were obtained from transgenic mice for the human Val30Met mutation in a TTR null background (Kohno et al . , 1997) . 1.2. TTR expression and preparation of TTR oligomers and fibrils

TTR was expressed in E. coli strain Bl-21 after transformation with individual expression plasmids containing WT or mutated TTR (Val30Met and Tyr78Phe) and the periplasmic contents were obtained by osmotic shock. The supernatant was fractionated on DEAE-cellulose, as described by Almeida and colleagues and TTR containing peaks were dialysed against water and lyophilised. Further purification was achieved by preparative gel electrophoresis, in a native Prosieve agarose system (FMC, Mockland, ME) . TTR was purified from serum following an established procedure (Almeida et al . , 1996), involving chromatography on DEAE-cellulose, chromatography on Blue Sepharose and preparative gel electrophoresis as described above. A small volume of Val30Met TTR and Tyr78Phe TTR (1 mg ml^"1) were incubated in 0.05 M sodium acetate/0,1 M KCl buffer pH 3.7, for 48 hours, at room temperature, to form amyloid fibrils. Oligomers were produced by stirring at room temperature (Teixeira et al., 2006) The preparations were positive by Thioflavin-T spectrofluorometric assays.

1.3. Mice immunization

Mice used in the immunization were 16 months old TTR knockouts obtained from the Jackson's laboratory and were crossed to the 129Sl/Sv background for more than 10 generations, which were immunised with the recombinant mutant TTR Tyr78Phe by injecting 10 μg of antigen diluted in Freund's complete adjuvant (Sigma) intraperitonealy, in a final volume of 200 μl . Two weeks later, a new immunisation was performed, injecting 10 μg of antigen diluted in Freund's incomplete adjuvant (Sigma), in a final volume of 200 μl . This procedure was repeated twice every two weeks, until high levels of IgG reactivity was detected in ELISA assays. A final boost of 50 μg of antigen diluted in a final volume of 200 μl of PBS was given to the mice exhibiting positive results for TTR recognition (by direct ELISA) . Five days later, the animal was sacrificed, blood collected and spleen removed to perform fusion.

1.4. Fusion protocol

Spleen was homogenised in RPMI -1640 medium (Gibco BRL) , spun and then re-suspended in cold NH₄Cl (0.17 M) . After a short spin the pellet was mixed with approximately 10⁷ SP2/0 myeloma cells, spun, re-suspended in a small volume and incubated at 37 ⁰C. To induce cell fusion, a 50% PEG solution (Sigma) was added drop-wise for one minute and the mixture re-suspended in RPMI-1640 medium. After a final spin, the supernatant was discarded and cells were re-suspended in foetal bovine serum (FBS, Gibco BRL) . This suspension was then diluted in complete selection medium [RPMI -1640 supplemented with HEPES, L-Glutamine, FBS (10%) and hypoxanthine/aminopterin/thymidine (HAT) (GibcoBRL) ] to a final volume of 100 ml and seeded in microtiter plates (96 wells, Nunc, Denmark), 200 μl/well. The plates were left to incubate for five > days at 37 ⁰C in a 5% CO₂ humidified incubator. Feeding was then performed every third day, for one week, with the same medium. On day 7, the feeding protocol was repeated with hypoxanthine/thymidine (HT) selection medium and plates were returned to the incubator and checked every two or three days. Supernatants from wells with growing cells belonging to multiple clones were then screened by ELISA for antibodies reactive to human plasma from carriers of the Val30Met mutation but not to normal human plasma. The ELISA procedure was selective for IgG type molecules by using a sheep anti-mouse IgG-HRP conjugated (Pierce) , as secondary antibody. Positive clones, selected from the original plates, were re-cloned on new plates by a serial dilution starting from an undiluted well up to 10⁶, until isolated clones were obtained in isolated wells. A new ELISA screening was performed and positive, interesting clones were selected and grown to a larger scale in 75 cm² flasks .

1.5 Enzyme-Linked Immunosorbent Assay

In a direct approach, microtiter plates (96 wells, Nunc, Denmark) were coated with 1 μg of TTR or 100 μl of serum diluted 1:10 in coating buffer (0.1 M carbonate buffer, pH 9.6), using 100 μl/well. Washes were performed three times with PBST (phosphate buffered saline, 0.02% Tween) and twice with PBS. After blocking with 5% non-fat dry milk in PBS, 200μl/well and washed as mentioned above, plates were incubated with 100 μl/well of undiluted hybridoma culture supernatants, for one hour at room temperature. After washing, bound antibodies were detected with sheep anti -mouse immunoglobulins G-HRP conjugated (Pierce) (1:5000 dilution in PBST), left to incubate for one hour, at RT. Plates were developed using 5 mM 2 , 2 ' -azinobis (3-ethybenzthiazoline-6- sulfonic acid) (ABTS) (Sigma) , in 50 mM phosphate-citrate buffer, pH 4.3, and read at 405 nm on a Bio-Tek BL 800 microplate reader.

In a "sandwich approach" plates were first incubated at 4 ⁰C, with 100 μl/well of rabbit anti-TTR polyclonal antibody (DAKO) in a 1:500 dilution in coating buffer. After blocking with 5% non-fat dry milk in PBS, different , amounts of isolated TTR, ranging from 2 to 100 μg or 100 μl of serum diluted 1:10 were incubated 1 hr at RT, followed by incubation with 100 μl/well of undiluted hybridoma culture supernatants, for one hour at RT. After washing, bound antibodies were detected with sheep anti-mouse immunoglobulins G-HRP conjugated (Pierce) (1:5000 dilution in PBST) , left to incubate for one hour, at RT. Plates were developed using 5 mM 2 , 2 ' -azinobis (3-ethybenzthiazoline-6- sulfonic acid) (ABTS) (Sigma) , in 50 mM phosphate-citrate buffer, pH 4.3 and read at 405 nm on a Bio-Tek BL 800 microplate reader.

1.6. Mab purification / concentration

Protein G Sepharose High performance (MabTrap G II protein G Pharmacia) was used for purification and isolation of monoclonal antibody from cell culture supernatants . Mab was eluted from affinity column with Immuno Pure Gentle Elution Buffer (Pierce) .

2. Antibody characterization

2.1. Immunoprecipitation of TTR

Five μg of purified Mab pre-adsorved to 50 μl of Protein G Sepharose (Pharmacia) was mixed with 25 μl of human plasma (from healthy individuals or FAP patients) , 0/N, at 4 ⁰C. After short spins at 14,000 rpm, pellets were washed several times with 50 mM Tris, pH 7.5 with 0.05% (v/v) Tween-20. The last wash was performed in the same buffer, without detergent. After a short spin, the pellet was dissolved in 50 μl of SDS-PAGE loading buffer with β-mercaptoethanol . One tenth of this solution was loaded on 15% acrylamide denaturing gels. Protein bands were visualised after Silver staining of the gels or by immunoblotting using the mab.

2.2. Gel electrophoresis and Immunoblotting

Proteins were analysed either in native or denaturing conditions. Native electrophoresis was carried out on 10% (w/v) acrylamide gels system. Electrophoresis under denaturing conditions was performed in SDS-PAGE gels (15% acrylamide, 0.1% (w/v) SDS), after heat-treatment of samples and addition of β-mercaptoethanol (0.1 M) . Proteins were transferred from gels into nitro-cellulose membranes (Hybond™-C pure, Amersham) , using a Tris -Glycine system, for one hour, at 1 mA/ctn² of membrane. After blocking and washing with PBST, the immunodetection was performed with either a rabbit anti-TTR polyclonal antibody (DAKO, 1:1000 dilution), for one hour at room temperature and goat anti- rabbit immunoglobulins, HRP conjugated (Pierce, 1:5000 dilution), or the mab (pure hybridoma supernatants) , for one hour at room temperature and sheep anti-mouse immunoglobulins-HRP (Pierce, 1:5000 dilution) . TTR was visualised using either the ECL method (Pierce) or DAB substrate.

2.3. Nitrocellulose dot blot

Five μg of protein were immobilized in nitrocellulose membrane under vacuum.

Membranes were saturated in 5% skim milk in PBS 1 hour at RT followed by incubation 1 hour with monoclonal antibody (pure hybridoma supernatant) and sheep anti -mouse immunoglobulin G - HRP conjugated. Proteins were visualised with DAB.

2.4. Oxidation reaction

5 μg of TTR dissolved in 30 μl of PBS or 25 μl of serum in a final volume of 30 μl of PBS were incubated with 1 μl of 30% hydrogen peroxide for 20 minutes at room temperature. The reaction mixture was then quenched with SDS-PAGE loading buffer with β-mercaptoethanol. 2.5 Characterization of abnormal TTR bands

TTR from sera of Val30Met carriers was semi-purified by- affinity chromatography to isolate the slow TTR migrating (SMT) band detected by the mab on western blotting. 0,45 mg of rabbit anti-TTR polyclonal antibody (DAKO) was coupled to a HiTrap NHS - activated affinity column (Amersham) following instructions of the supplier.

800 μl of serum was used to perform purification. 20 mM phosphate buffer pH 7.0 buffer was used for binding/washing. Elution was performed using 0.1 M glycine buffer pH 2.7, fractions of 1 ml collected to 50 μl of 1 M Tris-HCl pH 9.0 per tube and dialyzed with phosphate buffer saline. Approximately 10 μg of protein, obtained by affinity purification, was applied to a SDS gels which was silver stained. The SMT band was excised from gel and digested with trypsin. MALDI mass spectroscopic analysis was then performed.

N- terminal sequencing of the SMT band was performed after SDS transfer to PVDF membrane and Coomassie staining. The fast migrating band (FMT) detected by the mab on western blotting after oxidation was characterized by mass spectrometry of tryptic digests and N- terminal sequence, as described above, after application of 5 μg of oxidized TTR to a SDS gel which was silver stained, followed by excision of the FMT band.

3. Antibody sequence determination

3.1 PCR cloning of mab immunoglobulin cDNA for Complementary Determining Regions (CDR' s) .

This protocol utilizes a signal sequence primers based on results from N-terminal sequence and specific gamma 2B or kappa reverse primers for PCR amplification of mouse immunoglobulin genes using l^sC strand cDNA, generated from hybridoma total RNA, as template.

3.2 Preparation of RNA

Approximately 5 million viable hybridoma cells were" harvested for the preparation of total RNA. RNA was prepared using the Tryzol reagent (Invitrogen) according to manufacturer's specifications. Briefly, 2 ml of Tryzol reagent was added to semi -confluent T-175 flask containing hybridoma cells and pipeted repeatedly to lyse the cells. The lysate was transferred to two centrifuge tubes and 0.2 ml of chlorophorm was added to each. After vigorous mixing the tubes were spun to separate the mixture into phases . The upper aqueous phase containing the RNA was transferred to fresh, tubes and precipitated with 1 ml of isopropanol. After centrifugation, the pellets were washed with 75% ethanol, and resuspended in 100 μl of DEPC-treated water.

3.3 Preparation of first strand cDNA

Complementary DNA was generated from the total RNA sample using MMLV reverse transcriptase according to manufacturer' s specifications (Clontech, Advantage RT for PCR) . Two microliters of RNA were used as template in a reaction containing oligo-dT primer. Prior to assembly of the reaction, the template and primer were briefly incubated at 70 ⁰C for denaturing of template and rapid annealing of the primer. To this mix 4 μl of 5x reaction buffer, 1 μl of 10 mM dNTPs. 0.5 μl of RNAse inhibitor, and 1 μl of RT was then added. The reaction was run for 1 hour at 42 ⁰C. The RT was then inactivated by heating at 94 °C for 5 minutes, then placed on ice. The cDNA preparation was then stored at -20 ⁰C for use in PCR. 3.4 PCR amplification of heavy and light chain gehes

In order to allow for PCR amplification of AD7F6 mouse immunoglobulin genes, we designed primers based on N-terminal sequence of the antibody for variable heavy and light chains. We utilized specific primers reverse primers complementary to the 3 'end of mouse IgG2b and kappa light chain constant region.

PCR reactions were assembled using the Expand PCR kit (Boerhinger Mannheim) according manufacture's specifications. Successful heavy chain amplification generated products of approximately 1.4 Kb in size. Successful light chain amplification generated products of approximately 0.7 Kb in size .

3.5 Cloning of PCR products

The pGEM-T easy vector System (Promega) was used for the cloning of PCR products obtained above according manufacture's specifications.

The PCR product to be ligated was gel purified using QIAEX kit (QIAGEN) . A 1:3 molar ratio of the pGem-T vector to a PCR product (heavy or light chain) was used.

Transformation of ligated pGEM-T vector and PCR products were performed using XL2-Blue Ultracompetent cells (Statagene)

EXAMPLE 1 - The mab does not recognize soluble normal TTR but rather amyloidogenic TTR in its soluble and/or aggregated form.

When soluble TTR isolated from sera from normal and Val30Met carrier individuals is assayed by direct ELISA using the mab (from hybridoma cultures or on a purified form) , a clear positive reaction is observed for TTR isolated from Val30Met carriers (heterozygous or homozygous) ; in contrast, normal serum TTR does not give a positive reaction, as exemplified in figure 1. Val30Met TTR isolated from recombinant bacteria was used as a positive control.

When soluble wild type and Tyr78Phe recombinant TTRs are applied to nitrocellulose filters and blotted with the mab, a positive reaction is only observed for the mutant TTR. A positive reaction is also observed for TTR aggregates (e.g., prepared from Tyr78Phe or Val30Met) , is exemplified in figure 2.

The mab is able to distinguish sera of val30Met carriers from control individuals both by direct and sandwich ELISA procedures, as depicted in figure 3. Val30Met carriers include asymptomatic and symptomatic individuals.

EXAMPLE 2 - The mab detects modified soluble TTR species with altered electrophoretic mobility in plasma from carriers of the Val30Met TTR mutation.

When sera from Val30Met carriers and normal controls were applied to a native electrophoresis gel, followed by Western blotting with the mab, a slower migrating TTR band (SMT) was detected in the Val30Met carriers, which was absent from controls; this SMT band is also visible in transgenic mice for human Val30Met TTR serum, as exemplified in figure 4. SMT is also differentially observed in sera of Val30Met carriers after immunoprecipitation by the mab and Western blotting after SDS-PAGE (Figure 5A) . SMT is intact TTR, as assessed by N-terminal sequence and MALDI-TOF mass spectrometry after triptic digestion and represents TTR with modifications that alter electrophoretic mobility. Figure 5B shows sera from Val30Met carriers and controls pre- treated under oxidative conditions before immunoprecipitation by the mab followed SDS-PAGE and Western blotting using the same mab; a faster migrating TTR (FMT) band is disclosed both in mutant carriers and normal controls. FMT is intact TTR, as assessed by N-terminal sequence and MALDI-TOF mass spectrometry after tryptic digestion and represents TTR with modifications that alter electrophoretic mobility.

EXAMPLE 3 - Passive immunization with the mab of transgenic mice for human Val30Met TTR, presenting tissue TTR deposition, results in removal of deposition.

The mab was administrated for 6 months to young 3 months old transgenic mice for human Val30Met TTR in a background deficient for heat-shock factor 1; this strain of mice normally presents deposition of human TTR in the peripheral nervous system, particularly in the nerve starting at 3 months of age,- at 9 months deposition in the nerve is highly penetrant. The animals receiving passive immunization through mab treatment presented virtually no TTR deposits as compared to mice receiving vehicle alone, as documented by semiquantitative immunohistochemistry in Figure 6.

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Claims

1. A monoclonal antibody to human amyloidogenic transthyretin which comprises the SEQ. ID 1 as the amino acid sequence of AD7F6 antibody VL regions, the SEQ. ID 2 as the amino acid sequence of AD7F6 antibody VH regions, the SEQ. ID 3 as the nucleotide sequence of full-length heavy chain AD7F6 and the SEQ. ID 4 as the nucleotide sequence of full-length light chain AD7F6.

2. A monoclonal antibody, according to claim 1, producing an IgG monoclonal antibody of subclass IgG₂b, kappa light chain.

3. A monoclonal antibody, according to the previous claims, having a molecular weight of 160 kDa, which upon reduction yields 50 kDa and 28 kDa fragments.

4. A monoclonal antibody, according to claims 1 a 3, which recognizes mutated and non-mutated plasma modified TTR.

5. A monoclonal antibody, according to the previous claims, which recognizes amyloidogenic plasma modified mutant TTR both in reducing and non-reducing conditions.

6. A monoclonal antibody, according to the previous claims, to be applicable in research and treatments for FAP and pathologies presenting modified TTR.

7. A hybridoma producing the monoclonal antibodies according to the previous claims.

8. A kit for the detection of FAP comprising the monoclonal antibody of claims 1 to 5.

9. A method for producing the monoclonal antibody of claims 1 to 5 comprising:

(a) hybridoma production, by fusion of spleens cells sample from immunized mice with Sp2/0 cells sample;

(b) screening of hybridomas reactive to different mutant TTRs and non-reactive to normal non-mutated TTR, by ELISA immunoassay under native conditions;

(c) re-cloning of the mab by dilution;

(d) isolation and sequence determination.

10. Use of the monoclonal antibody of claims 1 to 5 to produce a kit for the detection of FAP.

11. Use of the monoclonal antibody of claims 1 to 5 to detect mutated and non-mutated plasma modified TTR

12. Use of the monoclonal antibody of claims 1 to 5 in research and treatments for FAP and pathologies presenting modified_, TTR.

13. Use of a kit of claim 8 in the detection of FAP.

14. Use of the process of claim 9 to produce a hybridoma of claim 6.

15. Use of the process of claim 9 to produce a monoclonal antibody of claims 1 to 5.