WO2006082451A1

WO2006082451A1 - Bioactive compounds

Info

Publication number: WO2006082451A1
Application number: PCT/GB2006/050028
Authority: WO
Inventors: Deirdre Mcintosh; Stanley White; Angus George Dalgleish
Original assignee: Aimsco Limited
Priority date: 2005-02-02
Filing date: 2006-02-02
Publication date: 2006-08-10
Also published as: GB0502083D0

Abstract

Methods and compositions comprising bioactive molecules associated with antibodies are disclosed. The compositions are useful as medicaments. Preferred bioactive molecules include peptides of less than 30 kDa, and in particular may include corticotrophin releasing hormone (CRH) or adrenocorticotrophic hormone (ACTH).

Description

Bioactive compounds

FIELD OF THE INVENTION

The present invention relates inter alia to bioactive compounds for use as a medicament. Aspects of the invention also relate to methods for obtaining and / or amplifying such compounds; and to methods and preparations for delivery of such compounds.

BACKGROUND OF THE INVENTION

International patent applications WO03/ 004049 and WO03/ 064472 describe therapeutic agents and compositions which are based on a serum composition with many surprising beneficial effects. The respective content of each of these two texts is incorporated in full by specific reference. In particular, the reader is referred to them for an understanding of how the therapeutic agent can be prepared, and for the indications which can be treated.

Typically a goat is immunised with HIV-3B viral lysate raised in H9 cells. The resulting serum is believed to be active against HIV, and multiple sclerosis. The reader is further referred in particular to the section on pages 3 and 4 of WO03/ 004049 headed 'Example of Production of Goat Serum' for further details of the production of serum. This section is incorporated herein by reference.

It has now been surprisingly identified that the active ingredient of the composition may be a small active molecule, rather than an antibody or the like. For example, the active ingredient may include a peptide, carbohydrate, or the like. It has further been identified that the therapeutic agent may include an antibody which is associated with the active ingredient, the antibody serving simply to transport the active ingredient rather than acting as an antibody.

SUMMARY OF THE INVENTION

According to a first aspect of the present invention, there is provided a medicament comprising an antibody, and a bioactive molecule associated with the antibody. It has been surprisingly identified that antibodies obtained from the therapeutic composition of goat serum referred to above are capable of associating with bioactive molecules, so enhancing their use as a therapeutic agent.

By 'associated' we mean that the bioactive molecule forms a complex with the antibody, such that the bioactive molecule and antibody will both survive ammonium sulphate and protein G purification, as described herein. The bioactive molecule and antibody may be conjugated, but preferably are associated in some other way, for example hydrogen bonding, such that more stringent purification methods may separate the antibody and the bioactive molecule.

Preferably the bioactive molecule is not a pharmacologically active drug; preferably the bioactive molecule is not an anti-cancer agent.

The medicament may further comprise a pharmaceutically acceptable carrier. The medicament is preferably administered in a dosage of between 1 and 20 mg of composition to the subject; more preferably between 4 and 15 mg, and most preferably between 6 and 10 mg. The precise dosage to be administered may be varied depending on such factors as the species, age, sex, and weight of the recipient animal, the method and formulation of administration, as well as the nature and the severity of the condition to be treated, Other factors such as diet, time of administration, condition of the animal, drug combinations, and reaction sensitivity may be taken into account. The medicament may be administered to a human or non-human animal, including mammals.

The medicament may be administered by any effective route, preferably by subcutaneous or intramuscular injection, although alternative routes which may be used include oral, aerosol, parenteral, or topical. The formulation of the medicament will depend on the intended route of administration; embodiments of the invention may be provided as pills, capsules, gels, creams, ointments, oils, aerosols, liquids, dried formulations, and the like.

An effective treatment regimen may be determined by the clinician or veterinarian responsible for the treatment, and may depend on factors such as the age, sex, weight of the animal, the method of administration, and the nature and severity of the disorder to be treated. Other factors such as diet, time of administration, condition of the animal, drug combinations, and reaction sensitivity may be taken into account. One preferred regimen is the intramuscular injection of between 6 and 15 mg of composition in a liquid formulation once daily for one week.

The antibody is preferably an IgG molecule, and is conveniently a goat IgG molecule. Alternative sources of IgG may be used, for example, horses, sheep, and such like. The antibody may be derived from a serum composition obtained from a goat after challenge with an immunogen. The immunogen may comprise HIV. This may be presented in intact host cells, in cell-free extracts, as a viral lysate, or in a mixture thereof. Alternatively, in a variation of the invention, a supernatant of a viral culture or a medium which is capable of the same, but has not been used to grow a culture, may also be used following heat inactivation as an imrnunogen which will produce a suitable response. Any supernatant or other medium which is suitable for the in vitro growth of HIV or another virus may be used to produce an acceptable immunogen, which will produce an effective response. The supernatant of a cell culture growth medium such as PMBC or the cancer immortal cell line as used to grow HIV 3b are given as an example. The HIV or other selected virus does not need to be present to produce an effective immunogen to create the composition. Other suitable immunogens are recited on pages 12 and 13 of WO03/064472, the contents of which are incorporated herein by reference. It is possible that other immunogens may be suitable for production of the IgG antibody; for example, human cell line antigens, small peptides or fragments thereof, or the like. Alternatively, recombinant antibody technology may be used to produce a suitable antibody molecule.

The bioactive molecule may be less than 30 kDa, less than 20 kDa, less than 10 kDa, or less than 5 kDa. The bioactive molecule is conveniently a peptide, although alternative molecules may be used, such as carbohydrates, lipids, and the like. Modified peptides may be used, for example, glycosylated peptides. Examples of preferred peptides having a molecular weight of less than 5 kDa include those with peaks in any of the mass spectrograms of the accompanying drawings. In preferred embodiments, the bioactive molecule may be either or both of corticotrophin releasing hormone (CRHj or adrenocorticotrophic hormone (ACTH). Alternatively, or in addition, the bioactive molecule may be peptide fragments derived from either or both of CRH or ACTH. Further examples have a molecular weight below 2.5 kDa, or below 1 kDa. The peptides can be oligopeptides, notably 2 to 20-mers, especially 3, 4, or 5 to 10-rners. In certain embodiments of the invention, the bioactive molecule may be derived from a serum composition identical or similar to that from which the antibody may be obtained. Preferably, the bioactive molecule is obtained from the composition along with the antibody with which it is associated. Alternatively, the bioactive molecule may have a distinct origin from the antibody, and be subsequently associated therewith; for example, the bioactive molecule may be produced recombinantly, may^¬ be derived from micro organisms, plants, or other sources, or may be artificially synthesised.

The bioactive molecule is preferably releasably associated with the antibody. This permits the antibody to release the bioactive molecule over a period of time, so serving as a reservoir of the bioactive molecule. This has particular advantages for the controlled delivery of the bioactive molecule. Without wishing to be bound by theory, it is believed that in certain embodiments of the invention the bioactive molecule is received within a groove formed in the antibody molecule. Whether the bioactive molecule is retained or released within the groove may depend partially on the environment in which the antibody is placed; for example, pH, temperature, ion concentration, and the like. The medicament may be formulated to promote certain desired environmental conditions in vivo.

According to a further aspect of the present invention, there is provided the use of an antibody, and a bioactive molecule associated with the antibody, in the preparation of a medicament. The medicament may be useful in the treatment of various diseases, including HIV; multiple sclerosis; inflammatory diseases such as rheumatoid arthritis; optic neuritis; motor neurone disease; autoimmune diseases including lupus, psoriasis, eczema, thyroiditis, and polymyositis; axonal or nerve damage; cancers in particular myelomas, melanomas, and lymphomas; cardiovascular diseases; and neural disorders, both demyelinating and non-demyelinating. Examples of inflammatory disorders which may be treated in accordance with the present invention include cerebrovascular ischaernic disease; Alzheimer's disease; Huntingdon's chorea; mixed connective tissue diseases; scleroderma; anaphylaxis; septic shock; carditis and endocarditis; wound healing; contact dermatitis; occupational lung diseases; glornerulnephritis; transplant rejection; temporal arteritis; vasculitic diseases; hepatitis; and burns. All of these disorders may have an inflammatory component, but are believed to be additionally treatable based on the non-demyelinating neural aspect of the disorder. Further non-demyelinating disorders which may be treated, and which are considered to have a degenerative component include multiple system atrophy; epilepsy; muscular dystrophy; schizophrenia; bipolar disorder; and depression. Other non- demyelinating disorders which may be treated include channelopathies; myaesthenia gravis; pain due to malignant neoplasia; chronic fatigue syndrome; fibromyositis; irritable bowel syndrome; work related upper limb disorder; cluster headache; migraine; and chronic daily headache.

Demyelinating disorders which may be treatable include infections of the nervous system; nerve entrapment and focal injury; traumatic spinal cord injury; brachial plexopathy (idiopathic and traumatic, brachial neuritis, parsonage turner syndrome, neuralgic amyotrophy); radiculopathy; channelopathies; and tic douloureux.

The medicament may be useful in the treatment of all kinds of peripheral neuropathy of axonal and demyelinating type, including hereditary motor and sensor neuropathy of all types; Charcot-Marie- Tooth disease (CMT) types CMTlA, CMTlB, CMT2, CMT3 {Dejerine Sottas disease), CMT4 {Types A, B C and D), X-linked Charcot-Marie- Tooth disease (CMTX); Hereditary Neuropathy with liability to pressure palsies (HNPP) - also called Tomaculous neuropathy; Hereditary Motor and Sensory Neuropathy with Deafness - Lom (HMSNL); Proximal Hereditary Motor and Sensory Neuropathy / Neuronopathy (HMSNP); Hereditary Neuralgic Amyotrophy; Hereditary Sensory and Autonomic Neuropathies (HSANl, HSAN2, HSAN3 (also called Riley-Day syndrome or familial dysautonomia), HSAN4, HSAN5); Familial Amyloid polyneuropathies (Type I, Type II, Type III, Type IV); Metachromatic Leukodystrophy; Krabbe's Disease; Fabry's Disease;

Adrenoleukodystrophy; Refsum's disease (HMSN IV); Tangier Disease; Friedreich's ataxia; Spinal cerebellar ataxia (SCA) all types - SCAl , SCA2, SCA3, SCA4, SCA5, SCA6, SCA7, SCA8, SCAlO, SCAI l, SCA12, SCA13, SCA14, SCA16; Spinocerebellar Ataxia; Cockayne's syndrome; and Giant axonal neuropathy.

The medicament may also be useful in the treatment of chronic inflammatory demyelinating polyneuropathy (CIDP), and Guillain-Barre syndrome. It is believed that the medicament may also be effective for treatment of animals, in particular, but not exclusively, the treatment of canine atopic dermatitis, canine oral melanoma, and equine pulmonary disorders.

There is also provided a method of treatment of a human or non-human mammal, the method comprising administering a composition comprising an antibody, and a bioactive molecule associated with the antibody, to a recipient in need of treatment.

The present invention also provides a pharmaceutical delivery system comprising an antibody, and a bioactive molecule associated with the antibody.

A further aspect of the present invention provides a method of delivery of a bioactive molecule to a patient, the method comprising providing the patient with an antibody, and a bioactive molecule associated with the antibody. The method may also comprise the step of associating the bioactive molecule with the antibody prior to providing the molecule and antibody to the patient. The association step may comprise combining the antibody and the bioactive molecule under conditions suitable to promote association, and allowing the molecule and antibody to associate.

It has also surprisingly been determined that the production of serum for use in the present invention may promote amplification of certain bioactive molecules. Thus, the present invention yet further provides a method of amplifying a bioactive molecule, the method comprising : challenging a mammal with an imrnunogen; allowing an immune response to proceed; obtaining serum from the challenged mammal; and purifying a bioactive molecule from the serum.

Preferably the bioactive molecule is associated with an antibody, and the purification step comprises the step of purifying the antibody from the serum. The antibodies so obtained will be associated with the amplified bioactive molecule. By 'amplified' is meant an increase in concentration of the molecule compared with a pre»immune serum. Amplification of more than twice, conveniently five times, and preferably ten times is believed to be possible.

The bioactive molecules of the above-described first aspect of the invention may be used. For example, the bioactive molecule may be less than 30 kDa, less than 20 kDa, less than 10 kDa, or less than 5 kDa.

The bioactive molecule is conveniently a peptide, although alternative molecules may be used, such as carbohydrates, lipids, and the like.

Modified peptides may be used, for example, glycosylated peptides. Examples of preferred peptides having a molecular weight of less than 5 kDa include those with peaks in any of the mass spectrograms of the accompanying drawings. In preferred embodiments, the bioactive molecule may be either or both of corticotrophin releasing hormone (CRH) or adrenocortico trophic hormone (ACTH). Alternatively, or in addition, the bioactive molecule may be peptide fragments derived from either or both of CRH or ACTH. Further examples have a molecular weight below 2.5 kDa, or below 1 kDa. The peptides can be oligopeptides, notably 2 to 20-rners, especially 3, 4, or 5 to 10-mers.

Preferably the mammal is a goat. The amplified bioactive molecule is preferably contained in the immunogen, although certain embodiments of the invention may amplify bioactive molecules present in the pre- immune mammal. The immune response is preferably allowed to proceed for at least 3 days, preferably at least 5, 7, 9, 11, or 14 days.

The antibody purification may comprise ammonium sulphate purification. Additional or alternative purification may be performed, such as dialysis, filtration, concentration, and the like. Conveniently, any suitable method of purification may be used which will isolate IgG molecules.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other aspects of the present invention will now be described by way of example only with reference to the accompanying drawings, which show:

Figure 1 is a mass spectrogram of pre-immune H9 goat serum, dialysed with a 1OkD molecular weight cut-off membrane but nevertheless retaining molecules smaller than 10 kDa

Figures 2 to 4 are mass spectrograms of goat serum from the goat of Figure 1 , 14, 35, and 56 days post immunisation; Figures 5 and 6 are mass spectrograms of goat serum from a goat immunised with HIV 3B, without and with Fab digestion;

Figures 7 and 8 are mass spectrograms of goat serum from a goat immunised with H9 supernatant, after ammonium sulphate purification and protein G purification, respectively.

Figures 9 and 10 are mass spectrograms of goat serum from goats immunised with HIV 3B and H9 supernatant respectively.

Figure 11 is a protein gel showing proteins from various sera from goats immunised with H9, HIV 3B, or pre-immune;

Figure 12 is a protein gel showing various protein G purified samples of various sera from goats immunised with H9 or HIV 3B; and

Figures 13 and 14 show antibody capture data for antibodies to CRH and ACTH respectively.

DETAILED DESCRIPTION OF THE INVENTION

Example of Production of Goat Serum

A goat was inoculated by intramuscular injection with lysed HIV viral cocktail and formulated with Freund's adjuvant. The virus was previously heat killed at 60⁰C for 30 minutes. Blood samples were drawn after an appropriate interval, such as two weeks, for initial assessment. In the optimised procedure, the goat is injected every week for four weeks, then at six weeks the animal is bled to obtain the reagent. Approximately 400 ml of blood is drawn from the goat under sterile technique. The area for needle extraction is shaved and prepared with Betadine. An 18-gauge needle is used to draw approximately 400 ml of blood from the animal. Of note is that the animal can tolerate approximately 400 ml of blood drawn without the animal suffering any- untoward effects. The animal does not have to be sacrificed. The animal can then be re-bled in approximately 10 to 14 days after it replenishes its blood volume.

The presence of potentially useful antibodies was confirmed, having regard to the desired antibody activity. Once the presence of such reagents was confirmed, blood was then taken from the goat at between 4-6 weeks.

The base blood product in order to create the reagent is then centrifuged to create the serum. 300 ml of serum was then filtered to remove large clots and particulate matter. The serum was diluted 1 :2 with cold phosphate buffered saline pH7.4 (one part serurn:2 parts buffer). The diluted serum was then treated at 4⁰C with supersaturated ammonium sulphate to precipitate antibodies and other material. The resulting solution was centrifuged at 4⁰C and 5000 rpm for thirty five minutes, after which the supernatant fluid was removed. The precipitated immunoglobulin was resuspended in phosphate-buffered saline (PBS buffer, see Sambrook et al, 'Molecular Cloning: A Laboratory Manual', 1989) sufficient to redissolve the precipitate,

The solution was then dialysed at 4°C through a membrane with a molecular weight cut off of 10,000 Daltons. Dialysis was carried out in PBS buffer, changed every four hours over a period of 24 hours.

After 24 hours of dialysis the contents of the dialysis bag were emptied into a sterile beaker. The solution was adjusted such that the mass per unit volume = 10 mg per ml. The dilution was carried out using PBS. The resulting solution was then filtered through a 0.2 micron filter into a sterile container. After filtration, the solution was aliquoted into single dosages of ImI and stored at -22°C prior to use.

Serum obtained from goats immunised with HIV 3B is referred to as '3B' or ΗIV serum; serum obtained from goats immunised with H9 cell supernatant alone (that is, with no HlV component} is referred to as Η9' serum.

Research performed by the current inventors suggests the following findings based on the various sera.

1. We believe the bioactive component of the product may be peptide and/ or carbohydrate based as opposed to an antibody. We currently believe that several peptide components are responsible for the total bioactivity.

2. In the goat, we have found a way to amplify significantly certain peptides and possibly carbohydrates in our product through our immunisation process used on the goat. This is not an immune response in the traditional sense, but seems to be an alternative mechanism that amplifies peptides as opposed to creating antibodies specific to the irnmunogen.

3. These components, both amplified and un-amplified, are found on the goat IgG, and survive an ammonium sulphate and a protein G purification.

4. These components are destroyed through the digestion process when creating a Fab fragment.

5. It is believed that these components provide the therapeutic effects we observe. 6. The goat IgG is being used as a transport vehicle and not in its traditional sense as a binding molecule. 7. Some of these components will release from the IgG and can be fractionated according to size.

8. The goat IgG is secondly acting like a time-release or slow release mechanism for the components. 9. Different purification methods can alter the release rate of these components. We still need to determine if this is a result of the purification itself, or to the body's reaction to the different purifications. 10. Regarding the presence of the bioactive peptides, there is no significant difference between the H9 based product and the HIV-3B based product within the molecular weight range studied.

First Indications of a Bioactive Peptide

Early observations with the serum product suggested that something in the product entered the blood stream after oral administration. The product would immediately cross the Blood Brain Barrier. This suggested that the product may not be an antibody, but instead a small bioactive molecule(s) such as peptide(s) or carbohydrate(s).

Subsequently, we investigated small proteins and peptides within the product using mass spectrometry.

Peptide Amplification:

During our observations and testing of the pre-immune serum, we observed subtle activity just below the confidence threshold for detection.

When looking at the peptide profiles and amplitudes of the H9 serum pool via Mass Spectrometry, the amplification of peptide peaks become quite noticeable. The graphs of Figures 1 to 4 are based on purified samples taken at 2 week intervals. The "Y" axes of the graphs are related. A "50" is 10 x the amplitude of a "5". Scanning these graphs, one will notice that the levels of these peptides rapidly increase and remain quite stable for a long period of time. This response is not like a typical antibody response.

As mentioned above, the mechanism that allows for this peptide amplification is unknown. We are currently performing studies on production bleed procedures and immunization procedures of RIBI adjuvant alone to prove that it is not the procedures alone that amplify these peptides. Conversely, we have had success in informed consent patients with various activated PBMC cells and the H9 lymphocytes we used to produce this product.

Purification processes:

Fab: When antibody molecules are administered to humans Fab or (Fab)^J2 fragments are preferentially administered. In the production of Fab fragments the Fc fragment is cleaved off leaving the protein recognizing region of the molecule without the immunoreactive Fc portion.

When manufacturing our product, by contrast, we do not perform a Fab digestion procedure. As a result, the bioactive components of interest are not broken down with the enzymes needed to cleave off the Fc fragment. The resulting mass spectrometry graphs (see Figures 5 and 6) will detail the different profiles between our antibody as Fab antibody vs. our antibody forgoing this step.

Note that the peptide profiles shown in the two graphs are quite different, and peptides above a molecular weight of 5000 Da are virtually non-existent in the digested sample (Figure 6). The increased peptide noise at less than 4000 Da is most likely due to fragments that were digested during the Fab production process.

Ammonium Sulphate vs. Protein G Purification:

Figures 7 and 8 show two mass spectrometry graphs of the H9 product after ammonium sulphate and protein G purification respectively.

Though these graphs look quite different, one must remember that the protein G purified product on Figure 8 was 5x less concentrated. There was loss of -85% of the peptide at the 9210 Da and 4606 Da level (These are possibly dimers of the same peptide which is likely to be corticotropin-releasing hormone CRH). In another investigation using a procedure known as on chip antibody capture (see below) CRH and adrenocorticotropic hormone (ACTH, which has a molecular weight of 4540) were found to be present in the product. It is known that these proteins are amplified in the blood of patients being treated with the product. It is very likely that the 4606 / 9210 Da peptides are some of the bioactive molecules in the composition as described above.

The presence of these peptides in the product following dialysis can be explained by their adherence to IgG; this might permit their survival of both the ammonium sulphate and protein G purification processes and also maintain their presence in solution during the dialysis step.

Similarities Between The HIV-3B vs. H9 Derived Products

Figures 9 and 10 show mass spectrometry graphs detailing both the 1000 series (HIV-3B)-based immunogen and the H9-based immunogen. The peptide profiles between the two are almost identical. This suggests that the viral proteins found with the HIV-3B based immunogen have no significant effects on the peptide profiles observed. Origin of the peptides

These peptides could exist as soluble moieties in the product or they could be associated with a larger protein. The assumption we took was that they were associated with a larger protein. The gel shown in Figure 11 demonstrates the complexity of determining the identity of the carrier protein, as each band represents at least one protein.

Identified proteins to date include : 1. Band 1: Positive match for Fibronectin

2. Band 2: Positive match for Alpha-2-Macroglobulin,

3. Band 3: Ceruloplasmin:

4. Band 4: Bands in this region consistently identify with Plasminogen 5. Band 5: Gelsolin.

6. Band 6: Albumin

7. Band 7: Immunoglobulin Heavy chain.

8. Band 8: Fibrinogen Beta-Chain.

In addition, antibody capture and SELDI mass spectrometry has identified CRH and ACTH in the product.

The amplification of these peptides indicated that IgG, the levels of which change after immunisation might be a candidate carrier molecule.

The IgG as a Carrier Molecule:

Ammonium sulphate product (H9, day 56) was further purified via protein G purification. The First Fractionation and Peptide Identification:

1. Protein G purified H9 IgG that was eluted from the column was dialyzed using 10 kD dialysis tubing.

2. After filtering through a 0.2 um filter, the material was concentrated in a 30,000 MWCO Amicon ultra concentrator. The retentate was removed for later use. The material contains all the proteins greater than 30 KD and should contain the IgG.

3. The filtrate (<30 KD, without the IgG) was used to determine if there were any bioactive peptides present.

Two volunteers placed 0.5 ml of the <30 KD material under the tongue and noted activity within seconds. Four days later, two more volunteers performed the same experiment. Both noticed bioactivity in the <30 KD filtrate.

Implications of Peptides in the <30 KD Fraction:

The most plausible explanation for bioactivity in the <30 KD fraction was that our bioactive peptides survived the protein G purification process. Since the purification method is designed to only harvest the IgG, the logical explanation for the presence of these peptides is they were attached or associated with the IgG during the protein G purification process. If these peptides were found in the <30 KD fraction, one would assume bioactive peptides might still be found with the IgG in the >30 KD fraction.

Second Purification:

We then purified another batch of H9 via protein G purification. Steps were as follows: 1. Protein G purified IgG was eluted from the column as before. 2. After filtering through a 0.2 um filter, the material was concentrated in a 30,000 MWCO concentrator (filter). The retentate was removed and labelled as >30 kD. The filtrate was used for further processing. 3. The filtrate was then was concentrated in a 10,000 molecular weight cutoff (MWCO) filter. The retentate was removed and labelled as 3040 kD. This sample should contain material with molecular weights between 30,000 - 10,000 Da. The filtrate was used for further processing. 4. The filtrate from the 10,000 MWCO filter was then concentrated using a 5,000 MWCO Filter. The retentate was removed and labelled 10 - 5 kD.

The product at <5 kD was sampled and immediately the presence of a bioactive compound was noticed. This was repeated again with a second subject, and a similar effect was noted. Effects include;

1. Very rapid onset. Measured in seconds.

2. Feeling of "distance" .

3. Face was slightly flushed. 4. Effect wore off quite rapidly. Within 10- 15 minutes.

5. Subjects felt a little hungry and /or tired for a while after treatment.

A Gel was run on all of the above samples. This includes: 1. Dialyzed protein G product, unconcentrated.

2. Concentrated protein G (>30 kD)

3. Concentrate product <30 kD

4. Concentrate product < 10 kD

5. Product < 5kD

The gel {Figure 12) shows various protein G purified samples The gel also shows the various <30 KD fractions are pure and do not have any IgG. The two bands on the right are ammonium sulphate purified samples.

Lane 1 & 14: (extreme left}: Molecular weight markers Lane 2 & 3: Protein G purified product 1st and 2nd elution (unconcentrated)

Lane 4 & 5: Protein G purified product 1st and 2nd elution (concentrated & >30KD)

Lanes 6- 11: various <30 KD fractions Lane 12: Ammonium Sulphate purified H9 Lane 13; Ammonium Sulphate purified 3B

In lanes 6-11 of the gel had samples from the sequential filtration. All lanes are clear showing that the peptides are either too dilute to be detected, or possibly not a peptide (carbohydrate origin). Product was there as all of these gave the pharmacological effect.

Gel Analysis

The gel demonstrates that: 1. We were able to fractionate by molecular weight at least some of our bioactive entities as there is no IgG, and the samples are still bioactive.

2. These peptides are associated with the IgG because the product was protein G purified, so the peptides had to be attached to the IgG because they made it through the purification process.

3. These components are at a very low concentration due to their absence on the gel.

The IgG Fraction as a Transport Vehicle: We gave a test animal (dog) 0.5 rnl of the purified IgG fraction of the H9 > 30 kD subcutaneously. He received the same on the two subsequent days.

We noticed the effects seemed to wear off significantly faster than the Ammonium sulphate prep, thus the injection schedule was increased to daily. An improvement in his condition was noted.

The experiment with the dog was repeated over 2 days using protein G purified IgG from the 1000 Series (1016- 1019) goats (HIV-3B). Similar results were noted again.

The human experimenter tried 0.3 ml of the protein G purified S. C. and noticed the bioactivity within 10 seconds, and they noticed an increase in digital dexterity.

Purified Protein G Interpretations:

1. Small components are most likely still associated with the IgG as peptide like activities were noted even though the under 30 kD fractions were removed.

2. The antibody is indeed acting like a transport vehicle.

3. The antibody may also be acting like a "time release" mechanism.

Silver Staining:

The Coomassie Blue is not sensitive enough to detect the peptides in the <30 KD fractions. As a result, we decided to silver stain these samples. Initial results were similar with the silver stain as we had with the Coomassie. We have plans to use a more sensitive staining system. On chip antibody capture

We investigated the serum product by mass spectrometry for the presence of functional proteins of the HPA axis. Peptide capture assays and mass spectroscopy identified specific peaks present in the 2~6kDa. range. RSlOO Protein chip arrays coated with antibodies to CRH or

ACTH were incubated with the product and analysed by mass spectrometry (Fig. 13 and 14). The presence of CRH was confirmed

(Figure 13) by capture with a polyclonal antibody to CRH (but not 8 control antibodies); the peak at MH+/Z 4665.8 Da. corresponds to the complete caprine CRH molecule, molecular mass 4672 Da.

A monoclonal antibody to ACTH captured a peak (MH+/Z 4544) almost identical in mass to the calculated molecular weight of goat ACTH 1-39 (4540 Da.) demonstrating the presence of intact ACTH in the product (Figure 14).

Conclusion:

As a result of our work, we have a new appreciation of the versatility of the antibody molecule. Instead of a binding molecule, we are finding it being used as a transport molecule. While the use of immunoglobulins as carrier molecules has been used as a therapeutic approach to extending the plasma half-life of cancer therapeutic agents, there have been few if any published accounts of this occurring naturally in the body. It seems that we also have discovered a method of amplifying peptide(s) and/ or carbohydrate(s) through the immunisation process. Combining the two together, we have been able to produce both a new therapeutic product (goat based peptide(s) and /or carbohydrate(s)), and a delivery vehicle that leaves these components intact, and is able to deliver them in a productive and therapeutic manner,

Claims

1. A medicament comprising an antibody, and a bioactive molecule associated with the antibody.

2. The medicament of claim 1 wherein the bioactive molecule is not an anti-cancer agent.

3. The medicament of claim 1 or 2 wherein the antibody is an IgG molecule.

4. The medicament of claim 3 wherein the antibody is a goat IgG molecule.

5. The medicament of any preceding claim wherein the bioactive molecule is less than 30 kDa.

6. The medicament of claim 5 wherein the bioactive molecule is less than 10 kDa.

7. The medicament of any preceding claim wherein the bioactive molecule is a peptide.

8. The medicament of any preceding claim wherein the bioactive molecule is either or both of corticotrophin releasing hormone (CRH) or adrenocorticotrophic hormone (ACTH).

9. The medicament of any preceding claim wherein the bioactive molecule is a peptide fragment derived from either or both of CRH or ACTH.

10. The medicament of any preceding claim wherein the bioactive molecule is releasably associated with the antibody.

1 1. Use of an antibody, and a bioactive molecule associated with the antibody, in the preparation of a medicament.

12. A method of treatment of a human or non-human mammal, the method comprising administering a composition comprising an antibody, and a bioactive molecule associated with the antibody, to a recipient in need of treatment.

13. A pharmaceutical delivery system comprising an antibody, and a bioactive molecule associated with the antibody.

14. A method of delivery of a bioactive molecule to a patient, the method comprising providing the patient with an antibody, and a bioactive molecule associated with the antibody.

15. A method of amplifying a bioactive molecule, other than an antibody, the method comprising : challenging a mammal with an irnmunogen; allowing an immune response to proceed; obtaining serum from the challenged mammal; and purifying a bioactive molecule from the serum.

16. The method of claim 15 wherein the bioactive molecule is either or both of corticotrophin releasing hormone (CRH) or adrenocorticotrophic hormone (ACTH).