AU628286B2 - Anti-paratopic antibody as an immunogen - Google Patents

Description

AU-At-15449/88 WORLD INTELLECTUAL PROPERTY ORGANIZATION International Bureau (Ail0

*PCT

INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (51) International Patent Classification 4 (11) International Publication Number: WO 88/ 07058 C07K 15/12, C12P 21/00 Al C17K 15/12, 2P 21/00 (43) International Publication Date: CI2N 15/00, 5/00 22 September 1988 (22.09.88) (21) International Application Number: PCT/AU88/00074 (81) Designated States: AT (European patent), AU, BE (European patent), CH (European patent), DE (Euro- (22) International Filing Date: 16 March 1988 (16.03,88) pean patent), FR (European patent), GB (European patent), IT (European patent), JP, LU (European patent), NL (European patent), SE (European patent), (31) Priority Application Number' PI 0864 US.

(32) Priority Date: 16 March 1987 (16,03.87) Published (33) Priority Country: AU With international search report.

_____Before the expiration of the time limitfor amending the ient of the receipt SECTION 34(4)(a) DIRECTION SEE FOLIO NAME DIRECTED Rc L(~s l. t1 4 'Cr, C. -i)Y OV 1988 (72) Sir,' v14¢:5 cC,"m uf^tv.oSn!( o Asf yc land, Ketth7 AU/AT'r; 97 Belmiont Street, Mosman; FA A NSW 2088 OCT, i.

(74) Agent: SHELSTON WATERS; 55 Clarence Street, Sydney, NSW 2000 NPATEIT OF; 'EIC 62828 (54) Title: ANTI-PARATOPIC ANTIBODY AS AN IMMUNOGEN (57) Abstract The present invention provides a method of manufacture of an antiparatopic antibody comprising the steps of: (1) selecting from a pool of antibodies occurring in one species a prrotypic set the members of which are effective in binding a specific antigen (or antigen epitope), and utilizing one or more members of said prototypic set, or paratopic fragments thereof, as an immunogen in a host of a different species, c- in an in vitro incubation system comprising cells de, rived from the same or a different species, to produce antibodies having a char' "istic which is anti-paratopic with respect to said immunogen to produce a synthetic repiicate of the specific antigen J. tope, Antigen (or antigen epitope), and monoclonal antibodies, vaccines and processes of immunisation employing the product of the method of manufacture are also described.

WO 88/07058 PCT/A U88/00074 I- 1 "ANTI-PARATOPIC ANTIBODY AS AN IMMUNOGEN" Field of the Invention This invention relates to immunology anid more particularly to a method of manufacture of immunogenic, compositions, to immunogens manufactured by the method, and to antibodies manufactured therefrom.

Background Art An immunogen is a molecule capable of eliciting an immune response in a vertebrate. The response elicited is believed to be determined by topographical shape characteristics of the immunogen. Immunogens are also called antigens i.e. ANTIhody GENerators because one aspect of the induced response involves the production of antibody molecules whose function is to lock onto the immunogen, Those areas of the immunogen to which the antibody molecule binds are variously referred to as the antigenic determinants, epitores or haptens. The last term, namely hapten, is generally associated with the term carrier and this term refers to that part of the I I I I wo 88/07058 7CT/A 88/00074 -2immunogen/antigen which interacts with cellular components of the vertebrate immune system.

These regions on the immunogen and the names used to define them should not be regarded as absolute. Thus the genus of vertebrates has immune systems which will recognize immunogens; but not all species necessarily recognize the same molecular areas as being haptenic areas or carrier areas. Within a species this can only be determined experimentally. Thus mice will not necessarily process immunogens in the same way as would, for example, the immune system of Man. Furthermore, within a species, individual specimens will not respond to the same degree. This is because the immune response to an immunogen has a genetic (hereditary) component.

Thus some individuals will respond better to an immunogen while others may not respond at all.

The iiamune response to an immunogen is an integrated phenomenon in that a class of white blood cells called T lymphocytes, for example, reacts with the carrier determinants which in turn allows a class of white blood cells called B lymphocytes to transform and start producing antibodies to the antigenic determinants.

Each cell recognizes only one determinant and each antibody producing B cell (plasma cell) generates only antibody molecules of one given specificity. Hence the immune system is said to be highly specific. Upon stimulation these plasma cells multiply and thereby give WO 88/07058 PCT/AU88/00074 3 rise to a clone of identical antibody secreting cells.

If it were possible to isolate these identical antibody secreting cells, they would be referred to as monoclonal and the antibodies referred to as monoclonal antibodies.

Figure 1 is a diagrammatic illustration of the response of a mouse to an immunogen/antigen. Under normal conditions each monoclonal antibody generated by the mouse in vivo mixes with other monoclonal antibodies so that a polyclonal antibody response eventuates.

Each antibody comprises a glycoprotein molecule.

The portion of an antibody molecule embodying the characteristic of shape or molecular topography, or code sequence which enables it to bind and so for example neutralise the antigenic determinant-or epitope of an antigen is known as a "paratop". The paratope is conceptually a molecular region of a shape complimentary to the epitope or to a part of the epitope of the antigen and is thought to reside in the so called hypervariable region of the antibody glycoprotein molecule.

Antibody producing lymphocytes are present in high concentration in the spleen but antigen reactive spleen lymphocytes cannot readily be cultured in isolation.

However mono-clonal antibodies may be manufactured and isolated therefrom by use, for example, of techniques of hybridoma technology. In one such technique mice are first exposed to an antigen whereby the mouse develops antibodies. With reference to Fig. 2, spleen cells of WO.88. .758-PCI/ U WO 88/07058 PCT/A U88/000'74 4 the immunised mouse are fused with mouse myeloma cells.

The growth of hybrid cells is promoted and the hybrids are screened for specific antibody secretion. Those useful are cultured or undergo further genetic stabilisation procedures. By this means specific mono-clonal antibodies may be produced and isolated.

Selected antibodies, or mixtures thereof such as are produced in the method of Fig. 2, may be used to neutralise an antigen in an organism, a paratope of each antibody in effect forming a complex with an epitope of the antigen.

In anti-idiotypic immunology a second stage process shown in Fig. 3 is involved, Mouse 1 is first immunised with an antigen. Thereby giving rise to several clones of antibody producing cells. One cell line is chosen on the basis of the characteristics of the generated antibody and the antibody is referred to as Abl. Abl is then used to immunise a second mouse mouse 2. The latter must have a genetic constitution very similar to, or identical with that of mouse 1. Mouse 2 generates monoclonal antibodies to Abl, a subset of which may be directed against the paratope of Abl. All the antibody subsets generated by mouse 2 against Abl may be referred to as Ab2 though the Ab2 subset specific for the paratope of subset 1 is sometimes referred to as Ab2 beta. The second mouse mono-clonal antibody, Ab2, has an anti-paratope, that is to say having a molecular portion

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5 *o o *o*oo *oo oo e with a shape characteristic complementary to the paratope of the first antibody. If the epitope of the original antigen is considered to be "mould positive", then the paratope of the mono-clonal antibody Abl can be considered to be a counterpart or "mould negative" and the paratope of the anti-Abl antibody that is the paratope of the Ab2 mono-clonal antibody can be considered to replicate the "mould positive". It will be understood, however, that in each case, the replication is not exact. When used in a vaccine, the T cond mono-clonal antibody, Ab2, functions as a harmless immunogen which stimulates production of Ab3 antibodies in the vaccinated animal effectively producing immunity to the first antigen.

Disclosure of the Invention According to one aspect the present invention consist of a method of treating a vertebrate comprising the steps of: selecting from a pool of antibodies occurring in a first species of vertebrate a prototypic set the members of which are antibodies effective in binding a specific antigen or antigen epitope; utilizing one or more members of said prototypic set, or one or more paratopic fragments thereof, as an immunogen in a host of a different species from the first, or in an in vitro incubation system comprising cells derived from the same or a different species to produce one or more antibodies having a characteristic which is ps~ ~i 1 1, 6 anti-paratopic with respect to the specific antigen or antigen epitope; and introducing anti-paratopic antibodies produced in step into a subject of the same species selected in step wherein said subject has not been previously exposed to the immunogen which possess said specific antigen or antigen epitooe referred to in step 1.

In a preferred embodiment of the invention the anti-paratopic mono-clonal antibodies are then used to immunise a member of the same species as that from which the prototypic set was selected.

For preference the pool of antibodies consists of naturally occurring human antibodies.

The prototypic set is a set of antibodies selected on the basis of effectiveness against a particular antigen, or epitope thereof, for example is a set of human antibodies obtained from humans carrying antibodies

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resulting from exposure to HIV.

The antibodies, or prototypic paratope bearing segments of them, are utilized as an immunogen in a mouse host to produce mouse antibodies having anti-paratope characteristics.

The mouse antibodies are then screened for effectiveness for inducing, in humans, antibodies which bind the HIV.

A A*l- C L r~ 6a In a second embodiment of the invention the anti-paratopic mono-clonal antibodies are then used to immunise a member of a third species differing from that from which the prototypic set was selected or from which e e.

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iO 88/07058 PCT/AU88/00074 -7 the anti-paratopic monoclonal antibodies were derived.

Brief Description of the Drawings Figure 1 is a diagrammatic illustration of the response of a mouse to an immunogen/antigen.

Figure 2 is a diagrammatic representation of monoclonal antibody production.

Figure 3 illustrates anti-idiotypic antibody Ab2 production.

Figure 4 is a schematic representation of the method of manufacture of anti-paratopic antibodies according to the invention.

Figure 5 illustrates a general procedure for the purification of HIV positive human antibodies..

Figure 5 (II) illustrates a general procedure for the purification of HIV antigen specific human antibodies.

Figure 6 illustrates purifcation of human IgG prior to delineation into HIV/HIV antigen specific antibodies.

Figure 6 illustrates purification of human IgA prior to delineation into HIV/HIV antigen specific antibodies.

Figure 6 illustrates purification of human IgM prior to delineation into HIV/HIV antigen specific antigens.

Preferred embodiments of the invention have a number of advantages over the prior art.

Firstly, the invention produces a mouse WO 88075 p CT/AU88/O'i,~i~,~' WO 38/07058s P CT/A U88/0637,7 8 anti-paratope which is a counterpart of a naturally occurring human antibody paratope for a specified antigen. Upon inoculation the mouse anti-paratope mono-clonal antibody produces in a human an antibody bearing a replica of the naturally occurring human paratope.

In the prior art there was produced a mouse antiparatope which was a counterpart of an artificially generated mouse antibody. Such a mouse anti-paratope mono-clonal antibody would produce in a human an antibody bearing a replica of an artificially created mouse paratope (in contrast to a human paratope) and which may not be as effective in binding the specific antigen in a human. In relation to the prior art the invention does not rely upon the assumption inherent within the prior art that the mouse processes antigen in exactly the same way as humans.

Secondly, in comparison with the prior art scheme illustrated in Fig. 3, the invention provides a more direct general route shown schematically in Fig. 4 to the production of an anti-idiotypic antibody.

Thirdly, since preferred embodiments of the invention use widely available and naturally occurring, i.e. endogenous, antibodies as the starting material rather than antigens, the process is expected to be less costly to conduct.

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WO 88/07058 PCT/A U88/00074 -9- Fourthly, the process is safer to conduct than processq- requiring handling for example of potentially harmful virus antigens, Best Modes of Performing the Invention An embodiment of the invention will now be described by way of example only. The embodiment concerns the manufacture of a vaccine to confer immunity against Acquired Immune Deficiency Syndrome (AIDS), The invention is not however limited to use for production of any particular vaccine, and has uses other than for the production of vaccines.

The manufacture may be considered as involving the steps of: selecting a prototypic set of antibodies; preparing one or more imunogens therefrom; inoculating hosts with the one or more immunogens; generating a monoclonal antibody pool from each host; screening the monoclonal antibody pools; testing the screened antibodies for effectiveness as a vaccine, In the example under consideration the fivst stage is to select from the nool of human antibodies a prototypic set, in this case a set of immunoglobulins which effectively bind the aetiologic agent for Acquired Immune Difficiency Syndrome (AIDS), The generally wo 88/07058 PCT/AU88/00074 10 accepted aetiological agent for AIDS is currently known as Human Immunodeficiency Virus hereinafter referred to as HIV.

That is accomplished by obtaining human immunoglobulins from individuals exposed to HIV. AL,.

of such individuals have antibodies to HIV.

The antibodies from these individuals are screened for effectiveness in binding HIV antigens and/ov antigenic fragments, Those antibodies effective at this function are retained as members of the prototypic set i.e. they are a subset of the pool of human immunoglobulins.

If desired the retained immunoglobin members so selected may be purified and used directly in step Preferably, however, in a second step the human immunoglobulins are subdivided into classes G, A, M# D, E (to use the WHO designation) and more particularly identified in Taele I.

WO 88/07058 PCT/A U88/00074 WO 88/07058 PCT/AU88/00074 11 Table 1 PHYSICAL PROPERTIES (F MAJOR HUMAN IMMUNOCGLQBL.LIN CLASS IN SERUM WHO Designation Sedimentation Coefficient IgG IgA IgM 19S# IgD IgE 75 75,95,11S* Molecular Weight 150,000 160,000+ dimer 900,000 185,000 200,000 Number of Ig Units Number of Antigen Binding Sites 2-4 Identity of Heavy Chain Carbohydrates Content (gamma) (alpha) (mu) (delta) (epsilom) Total, Inmmunoglobulin 80 in normal human serum Concentration range 8-16 in normal human serum mg/ml 8 13 1.4-4 mg/mil 12 6 0,5-2 mg/ml 13 0-1 0-0.4 mg/ml 12 .002 17-450 mg/ml 19A dimer found in rmucosal (secretory) immune system.

It is complexed with a secretory component (MW=60,0v0) and J chain (MW=15,000).

1gM contains J chain.

Source: 1,M. Roitt, Essential hmmunolgoy, 4th Ed. Blackwell 1980 I WO 88/0)7058 PCT/AU88/00074 12 More desirably still the immunoglobulins are further divided into sub-classes, for example, IgG being divided four sub-classes, IgA being divided into two classes and IgM into two sub-classes. In the preferred embodiment each of sub-classes IgG 1-4, IgA 1-2 and IgM 1-2 are purified and isolated from each other.

IgD and IgE sub-classes are present in immunoglobin in small concentration and their inclusion is optional.

The human IgG/A/M is drawn from the three main groups affected by the AIDS viral infection, viz male homosexuals bisexual/female/heterosexual AIDS carriers haemophaelics The blood plasma is heated to 56 0 C to kill the virus, Cellular components and serum debris are removed either by aspiration of the serum component or by centrifugation (in the case of plasma).

Human IgG can be purified free of all non-IgG contaminants by affinity chromatography. Othjr procedures such as ion-exchange chromatography may be used but affinity chromatography is preferred for speed and selectivity. More particularly purification is generally effected by means of chromatography using PROTEIN-A SEPHAROSE beads (obtainable from e.g. Pharmacia Biotechnology Pty. Ltd.) Subclasses of IgG may also be isolated by chromatography.

WO 88/07058 PCT/AU88/00074 13 In a similar manner human IgA purification may be carried out by anti-IgA affinity chromatography.

Human IgM may be purified by a combination of Protamine sulphate chromatography, and Column chromatography, or IgM affinity chromatography.

The purified prototypic immunoglobulins set may be used directly as an immunogen for inoculati,. of mice in stage 3.

Alternatively the Ig subclasses may be screened to select antigen specific antibodies for use as the immunogen, In this case, the Ig sub-classes are next screened for effectiveness against HIV antigen to select the most effective sub-classes in binding the antigen.

More preferably the antigen is first divided into sub-c1'""~t known as pl8, p24, gp41, p55, gpl20 and These antigen sub-classes differ from each other in molecular structure and can be separated by SDS-polyacrylamide gel electrophoresus. Each Ig subclass is then ncreened against ea-ch antigen subclass to select the most effective Ig's, i i TABLE 2 !1VLV III HUMAN SEI4JM n PARAMIPE GR kwman Serum SgG lqA Ig (61) Antigen ILJ' 1 Spcific t 2 No:. of aw 111 1 2 3 4 1 2 1 2 Human Antigens Epitopes Paratps p24 2 16 gl4 32

P

5 3 5_ gpl2o 12 96 16 120 Carrier Specif i 39 39 39 39 39 39 39 39 Plratope Tbtal Nwitbr r -I Class 166 78 I Spraad of Pwatpe 50% 25% TsD ad MgE not inclrudi c I WO 88/07058 PCT/AU88/00074 15 With reference to Table 2 there is shown a "paratope grid". If it is assumed that there is one antigenic group anchored to a ten thousand dalton carrier group then the total number of antigenic groups (epitopes) available among the five antigen subclasses would be thirty nine. With eight potential antibody classes in the grid that can respond to the thirty nine antigens the total number of possible antibodies carrying paratopes specific for HIV is 312. Put differently there are on average thirty nine HIV paratopic bearing human immunoglobulins per immunoglobin sub-class. Thus, for example, it might eventuate that human IgGI hias specificities for all thirty nine epitopes ("haptens"), there would be thirty nine IgG1 molecules all absolutely identical except for one feature namely their Fab paratope would be different, In the third step of the embodiment, one or more members of the prototypic set are used as an immunogen in a non-human host for example by being injected into a mouse, The one or more members are preferably the most effective of the immunoglobin sub-classes. The criteria of effectiveness may be effectiveness against a specific antigen or effectiveness against a spectrum of antigen sub-classes or other criteria.

Human antibodies are excellent immunogens when injected into mice. The antigenic sites on the human antibody molecules are spread right across the length of WO 88/07058 PCT'/AU88/00074 16 the molecule from the NH2 terminii-ie the Fab end to the carboxylic acid terminus -ie the Fc end. The Fab NH2 end carries the paratope, Other antigenic components of the Fab are present for structural or "carrier" purposes.

For the purposes of the vaccine the Fc exclusively exhibits "carrier" as opposed to paratope antigens.

Immunization studies have demonstrated that not all the antigenic sites on the intact human immunoglobin molecule are of equal value in that a greater proportion of induced antibodies tend to be directed against the Fc region. This phenomena is described as entigenic competition or more accurately as intramolecular antigenic competition. When developing an antiparatopic antibody however the part of the molecule of most interest is the Fab area that io to say the paratope bearing :'egion. A simple way to overcome the problem of Fc dominance is to enzymatically cleave the immunoglobin molecule and isolate the Fab fragment, When used to immunize a mouse this will cause all the induced immunoglobulins to be directed against the Fab fragment.

A subset of the anti Fab antibodies generated by the mouse, irrespective of whether an intact immunoglobin molecule or a Fab/F(ab)'2 fragment has been used, will be directed against the internal idiotope i.e. paratopic image of the human immunolgen. Thus the member of the anti HIV prototypic setc used as an immunsgen in the mouse may be either the mixed intact human immunoglobin WO 88/07058 PCT/AU88/00074 I t- 17 specific for the AIDS virus, selected classes or subclasses of the intact immunoglobin, a Fab/F(ab)'2 fragment of one or a combination of the AIDS specific immunoglobulins or a Fab/F(ab)'2 fragment of one or a combination of the AIDS specific immunoglobulins complexed to carries eg. Keyhole Limpet Haemocyanin or human albumin.

The stage of preparation of immunogen may thus include enzymatic digestion or chemical cleavage of the human ari HIV immunoglobin and conjugation of the Fab/F(ab)'2 to microspheres or the like.

As will be apparent from the foregoing, it is conceivable that when injected into the mouse, the HIV IgG1 subgroup could provide all the relevant paratopes on one type of carrier. This regime would favour the generation of anti-idiotypes in the mouse (as opposed to the generation of anti-"carrier" molecules).

There is a possibility though of inter-molecular antigenic competition so that only a small variety of the human paratopes directed against HIV will end up being antigenic in the mouse. If this occurs then there are various ways of proceeding: After the screening step those paratopes that are dominant could be isolated from the immunogen population and a second immunization carried out to develop mouse anti-paratope antibodies to the remaining paratopes.

(ii) The anti-idiotypes/idiotypic reagents arising from 18 the first immunization could be screened and tested to see if the anti-idiotypes/idiotypic reagents cover the known HIV antigens/antigenic fragments. If all the known antigens are covered by the generation of anti-idiotypes/idiotypic reagents then a second immunization protocol may not be necessary.

(iii) A different mouse strain could be employed. The eventual manufacturing route thus depends on whether antiidiotypes/idiotypic reagents to all reagents are required.

It may suffice to have, say, one or two of the haptens from each antigen group covered. To a great extent though, this is an issue that will be resolved by the mouse itself in that it may only be able to raise anti-idiotypes/idiotypic reagents against a restricted idiotype range.

How all these factors are weighted will determine the nature of the immunogen that is preferred for injection into the mouse.

Thus it may be preferable to choose a particular class/subclass of HIV+ve human immunoglobulin which expresses several specificities and use this to immunize the mouse.

Alternatively (ii) given the diversity of the immnunoglobulin response the antibody range may not be restricted and a more general immunization routine adopted. In the latter case 88008PT/ 8/07 WO 88/07058 PCT/AUL88/00074 19 (iii) subclass purification may be called for coupled to several primary immunizations.

An excellent starting position though would be to opt for ang. then remove the Fc prior to further development of the immunogen by linking it to adjuvants such as precipitated immunoglobulins or microspheres.

While stating a preference for an outline of the various alternative pathways for the purification and preparation of the immunogen is shown in Figs. 5 and 6.

After injection of the immunogen into mice, preferably after a second immunization, mouse spleen cells are harvested by normal methods and fused to NSI in accordance with conventional hybridoma technology.

Hybrids are then grown and screened and positive hybrids cloned and re-tested. The clones are then adapted and grown in serum-free media and specific antibodies purified and ready for testing in humans.

The monoclonal antibody pool may be generated using for example the standard method or the "LOTTO" method as outlined in Table 3 below: WO 88/07058 P CT/A U 88/00074 WO 88/07058 PCT/AU88/00074 20 TABLE 3 STANDARD METHOD one-hit 11LOTTO 1 (multi-chance) (ii) (iii) spleen immunize 4 wks later boost (ii) 4 days later (ii) spleen cell prepn Hybiridoma productn (iii) Preliminary screen (iv) CLONE (v) 2-3 days later boost 2-3 days later cell prepn Hybridize Preliminary screen Clone (iv) (v) (vi) The shown in F .b pool may be screened by conventional means as Table 4: TABLE 4 SCREENING FOR FAB POOL Antigen Mc Ab Configuration (+Ve/-Ve) 1 2 3 4 5 6 Bence-Jones Human Ig Imiunogen

ACTION

Discard/Retain D D D 0 0 R WO 88/07058 PCT/AU88/00074 I t 21 The anti-idiotype pool may be screened by conventional means. For example HIV on a tray is mixed with human anti-HIV antibodies before and after incubation with mouse HIV idiotype complexed to microsphere/eupergit spheres, then chased with anti-mouse Ig-PO, +Ve is discarded, and -Ve is retained.

Alternatively HIV on beads is mixed with human anti-HIV PO-enzyme mouse HIV idiotype +Ve response is discarded.

As will be appreciated by those skilled in the art the antibodies may be selected from a pool occurring in a different species 'of veterbrate and the prototypic set may be selected for effectiveness against a different antigen. The antibodies may not be free in plasma but may be bound to cells B cells) or may exist as immune complex. The prototypic set may be divided into members using different criteria from that exemplified.

Other methods may be used for separation such as use of dyes bound to inert supports, or the use of monoclonal antibodies, etc, and purification of the immunogen without departure herefrom.

The immunogen, or fragments thereof may be utilized in host species other than mice, The antibodies so obtained may be used in various ways for example for immunization of the verterbrate from which the antibodies were obtained, in test methods and for other purposes.

WO 88/07058 PCT/A U88/00074 22 The invention will now be described more specifically by way of the following Example.

PREPARATION OF HUMAN IMMUNODEFICIENCY VIRUS SPECIFIC HUMAN

ANTIBODIES

A. PREPARATION AND PURIFICATION OF HIV ANTIGENS.

Native and recombinant antigens can be purified by affinity chromatography using human antibodies or antibodies from another species such as mous- monoclonal antibodies specific for the HIV antigens, By way of illustration the procedure described will be that using human antibodies. There is very little difference between the two approaches though the benefit is that with the appropriate mouse monoclonal antibodies specific antigens can be purified if the antigen source is the native one.

If the antigen source is a recombinant one then human antibodies will allow for the specific purification of the recombinant antigen. When human antibodies are used the steps involved are the preparation of human IgG from HIV infected individuals the preparation of the human antibody (IgG) column and the purification of the viral antigens using the aforementioned column, 1. Preparation of a human antibodies.

According to this procedure human antibodies were first purified by either hydroxyapatite chromatography, WO 88/07058 PCT/A U88/00074 23 ion-exchange chromatography (DEAE-cellulose) or protein-A affinity chromatography, By way of example the method for the purification described is that of protein-A agarose column chromatography.

Pooled human sera was obtained from patients positive for the AIDS virus as determined by both an AIDS antibody ELISA assay and subsequently confirmed by the Western Blot assay. Prior to use the serum had been heat treated (569C mins). A 2,0ml protein-A agarose column was washed with 20m of the Monopure bindng buffer (Pierce). 4mls of the pooled serum was diluted with 8mls binding buffer and centrifuged (2000xg:10 min:RT), The supernatant was applied to the column, allowed to percolate through and exhaustively washed in the binding buffE4r. The human IgG was specifically eluted using the commercially obtained elution buffer (Pierce). Following dialysis and concentration, the A 2 80 data was used to determine the concentration of protein which was calculated to be milligrams as determines by the E =1,43(280nm), Western Blot and ELISA data confirmed the presence of HIV specific antibodies in the IgG fraction purified in this mannert 2. The preparation of the IgG affinity column.

mgs of the human IgG was equilibrated in the coupling buffer M NaHCO 3 pH8.3 0,5M NaCd) and mixed with 4 gms CnBr-Sepharose4B (Pharmacia) which had been pre-washed in 1mM HC1, swollen and equilibrated in WO 88/07058 PcT/A o88/00074 24 the coupling buffer. The mixture was mixed end-over-end in a sealed coupling vessel (2hrs, RT) Unreactive groups on the matrix wEre blocked using 0.24 glycine In the coupking buffer (16 hrs, 4 0 C) and the ensuing IgG-Sepharose matrix exhaustively washed in high salt and variable pH buffers prior to the purification of the HIV antigens.

3. The purification of the native/recombinant antiqons.

By way of illustration the method described is that for the recombinant Iv antigens in particular recombinant '9gp120'.

Sub genomic clones of ITV cDNA encoding gp2O, 9p41, p24, and p18 were cloned and amplified In E.Coli using X gtJll. The E, Coli )ysates were screaened with in-house and by commercial HIV antigen ZLISA's, Radioimmunoprecipitation studies confirmed the presence of recombinant HIV antigens and the molecular weights of the recombinant antigens were as predicted e.g. 60OkD for the recombinant Following precipitation of 1. Coli antigens with

(NH

4 2 SO4 the supernate was concentrated (Amicon)dialysed against distilled water and then against 0.05M Phosphate buffer pH7i2(l6hrs;4*C), 40 mls of the dialysed concentrate was combined with apptoximata3ly 2 ml of the TgG-Sepharose and the mixture incubated end-over-end for 2 hrs(RT), The matrix was exhaustively washed and the recombinant protein eluted using 4M WVO 88/07058 PCT/A UJ88/00074 rKgC :11 pH 8.3. The presence of recombinant antigen was confirmed as outlined above.

B, THE PURIFICATION OF THE EOMAN HIV SPECIFIC ANTIBODIES.

The purification of HIV specific human antibodies involved two steps. These are outlined below.

i The preparation of the H-IV antigen column.

ii The purification of the F.11 spec-,ific human antibodies.

1. Preparation of the HIV antigen-Sepharose column.

mls of the eluted protein was mixed with 2 gins swollen, pre-washed and appropriately equilibrated CnBr-Sepharose (pH8.3), The mixture was mixed end-over-end (2hrstPT). Unrzeactive sites were blocked using 0.2M glycine (l6hrs, 4 0 C) atid the matrix exhaustively washed as outlined for the igG-Sepharose column.

2. Purif~ication of HTV specific human antibody.

4 mis of pooled human HIV serum heat treated as outlined above was passed through a PD-10 column equilibrated with freshly prepared 0,05M Phosphate buffer pH7.2 0.5K NaC1, The first 3 ml fraction (void volume) was discarded and the next 7.5 mis was collected, 10 mls otE the gpl2O-Seph arose matrix and 7.5 mis of the equilibrated serum were mixed end-over-end for 2hrs at RT4 Following extensive washing HIV specific Ig's were resorbed using buffer containing 4M MgCl 2 pH8.3, Approximately mg of HIV specific Ig was obtained uzing this method, WO 88/07058 P CT/A U 88/00074 WO 88/07058 PCT/A U88/00074 26 C. THE PRODUCTION OF MOUSE MONOCLONAL ANTIBODY TO THE HUMAN AB1.

The production of Mouse monoclonal antibodies firstly involves the induction of antibodies either by in vivo methods or by in vitro methods.

By way of illustration the in vitro method is described.

Two groups of Balb/c mice were used in this experiment. The first group consisted of mice which had been tolerized to human IgGl. This had been achieved by injecting mice intraperitoneally, 7 days previously, with milligrams of human IgGl. The second group cohsisted of untolerized mice.

Mouse Ab2 antibodies were induced in the following way. 1.3 x 10 mouse spleen cells were recovered and washed in the incubation medium (Iscoves DMEM medium containing 20% foetal calf serum (FCS), 40% thymus conditioned medium (TCM), 5 x 10 4 2-mercaptoethanol, 4mML-glutamine 50 IU penicillin and 50 IU streptomycin).

HIV specific human immunoglobulins at a concentration of micrograms/ml incubation medium was added to the mouse spleen cells. The total volume used in the incubation of the spleen cells with human antibody varied between 10 and mls. In this example the incubation was allowed to proceed for 7 days in a heated (37°C) CO 2 incubator.

Following incubation the cells were recovered for fusion to either SP2, NSl or X63-Ag*.653 mouse myeloma WO 88/07058 PCT/AU88/00074 1 f' 27 cells. The viability of the spleen cells was found to vary between 70 and 99% and the viability of the myeloma was generally 99%. For the sake of illustration SP2 mouse spleen cells were used though other cells such as rat or human myeloma cells could be used in this procedure.

Spleen cells were fused to the myeloma cells using polyethylene glycol 1500/4000 (Boehringer/Mannheim) using standard prpcedures and following 24 hrs incubation in a

CO

2 incubator at 370C the hybrids were plated out in the incubation medium now containing HAT.

D. RECOVERY OF ANTI-HIV ANTIBODY CLONES PRODUCED IN SITU AS A RESULT OF NATURAL INFECTION.

In addition to the serum HIV antibodies purified by the abovementioned methods it is possible to obtain the human Abl by Epstein-Barr virus (EBV) transformation of human B cells obtained from individuals exposed to the AIDS virus.

By way of illustration the following method was used.

Human periphezal blood lymphocytes (PBL's) were diluted 1:1 in phosphate buffered saline and the red cells removed by centrifugation through a Ficoll-hypaque cushion (Pharmacia).

The PBL's either depleted or not depleted of monocytes and lymphocytes using methods familiar to those skilled in the art, were then transformed using for example the EBV isolate B95-8 in sterile .issue culture media (RPMI-1640 5% FCS). In a simple example the B95-8 WO ~S/tJ7U5~ PCT/A U88/00074 wU 8/0U7U58 PCT/A U88/00074 28 isolate is made available as a supernate which is mixed with the monocyte/T cell depleted fraction enriched for the B lymphocytes. The cells are grown in this mixture, fed as required, and expanded in 96-well flat bottomed plates prior to fusion with the mouse myeloma cell line such as X63-Ag*.653. Screening is by a commercially available HIV antibody ELISA. Cloning and feeding (Medium containing HAT/HT) is by the usual methods except that non transformed will be selected out by feeding with 1 micromolar Oubain.

All these methods must be carried out in hybridoma facilities suitable for work involving HIV as virus may be shed under these conditions.

E. PRODUCTION OF HUMAN AB1 USING IN VITRO IMMUNIZATION OF HUMAN PERIPHERAL BLOOD LYMPHOCYTES AND/OR SPLENIC

LYP~HOCYTES.

HIV specific human Abl may also be obtained by in vitro immunization using whole virus or native, recombinant HIV antigens and antigens bound to nitrocellulose. According to one method 3-4 x 10 4 human PBL's or human splenic lymphocytes depleted of monocytes/T lymphocytres using L-Leucine methyl ester can be immunized with small amounts (1 nanogram 10 micrograms) of HIV antigen. The human Abl are monoclonal when the techniques of hybridoma technology as outlined in D. are used.

Human Abl obtained in this way may be used as the immunogen to produce the Ab2 by either in vivo or in vitro WO 88/07058 PCT/AU88/00074 1 r 29 culture techniques using human cells or cells of other species as the human Abl would house the prototypic paratopes as defined by the foregoing.

Such variations as will be apparent to those skilled in the art from the teaching hereof are deemed to be within the scope of the invention h3rein disclosed.

Claims (7)

1. A method of treating a vertebrate comprising the steps of: selecting from a pool of antibodies occurring in a first species of vertebrate a prototypic set the members of which are antibodies effective in binding a specific antigen or antigen epitope; utilizing one or more members of said prototypic set, or one or more paratopic fragments thereof, as an immunogen in a host of a different species from the first, or in a in vitro incubation system comprising cells derived from the same or a different species, to produce one or more P" antibodies having a characteristic which is :anti-paratopic with respect to the specific antigen or antigen epitope; and introducing anti-paratopic antibodies produced in step into the same or a different

2..member of the first species selected in step 1. 2. A method according to claim 1 wherein the oprototypic antibodies are human antibodies.

3. A method according to claim 2 wherein the prototypic human antibodies are naturally occurring antibodies.

4. A method according to any one of the preceding claims wherein the antibodies are immunoglobulins.

A method according to any one of the preceding claims wherein the antibodies are human antibodies to HIV. I I 11 1 11 11 1 -1 1.1 1 'll, 1- 31

6. A method of immunising a subject comprising the steps of: selecting from a pool of antibodies occurring in a first species of vertebrate a prototypic set the members of which are antibodies effective in binding a specific antigen or antigen epitope; utilizing one or more members of said prototypic set, or one or more paratopic fragments thereof, as an immunogen in a host of a different species from the first, or in an in vitro incubation system comprising cells derived from the same or a different species to produce one or more antibodies having a characteristic which is anti-paratopic with respect to the specific antigen or antigen epitope; and introducing anti-paratopic antibodies C produced in step into a subject of the same species selected in step wherein said subject has not been previously exposed to the immunogen which possess said specific antigen or antigen epitope referred to in step 1.

7. A method according to claim 1 substantially as goes herein described. DATED this 14th day of JULY, 1992 ROLAND KEITH McGREADY Attorney: IAN T. ERNST Fellow Institute of Patent Attorneys of Australia of SHELSTON WATERS