CA1341277C - Anti-paratopic antibody and a method of its manufacture - Google Patents

Abstract

The present invention provides a method of manufacture of an anti-paratopic antibody comprising the steps of:
(1) selecting from a pool of antibodies occurring in one species a prototypic set the members of which are effective in binding a specific antigen (or antigen epitope), and (2) utilizing one or more members of said prototypic set, or paratopic fragments thereof, as an immunogen in a host of a different species, or in an in vitro incubation system comprising cells derived from the same or a different species, to produce antibodies having a characteristic which is anti-paratopic with respect to said immunogen to produce a synthetic replicate of the specific antigen or epitope.
Antigen (or antigen epitope), and monoclonal antibodies, vaccines and processes of immunisation employing the product of the method of manufacture are also described.

Description

rrvtnnrmTnr~r, This invention relates to immunology and more particularly to a method of manufacture cf immunogenic, compositions, to immunogens manufactured by the method, and to antibodies manufactured therefrom.
An immunogen is a molecule capable of eliciting an immune response in a vertebrate. The response elicited is believed to oe determined by topographical shape characteristics of the immunogen. Immunogens are also called antigens i.e. ANTIbody GENerators because one aspect of the induced response involves the production of antibody molecules whose function is to lock unto the immunogen. Those areas of the immunogen to which the ,antibody molecule binds are variously referred to as the antigenic determinants, epitopes or haptens. The last term, ~zamely hapten, is generally associated with the term carrier and this term refers to that part of the immunogen/antigen which interacts with cellular components of the vertebrate immune system.
These regions on the immunogen and the names used to ~~efine them should not be regarded as absolute. Thus the genus ~~f 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.

~Chus some individuals will respond better to an immunogen i~hile others may not respond at all.
The immune response to an immunogen is an integrated phenomenon in that a class of white blood cel_Ls called T
lymphocytes, for example, reacts with the carrier determinants which in turn allows a class of white blood cells called B
_ymphocytes to transform and start produ~sing <~ntibodies 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 :>ystem is said to be highly specific. Upon stimulation these plasma cells multiply and thereby give rise to a clone of identical antibody secreting cells. If :it were possible to isolate these identical antibody secreting ce:Lls, they would be referred to as monoclonal and the antibodies referred to as monoclonal antibodies.
Under normal conditions of a mouse response to an s.mmunogen/antigen each monoclonal antibody generated by the mouse in vivo mixes with other monoclonal ant_bodies 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 "paratope".
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. Spleen cells of 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 as hereinafter described 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 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 ::haracteristics 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 oe 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 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 second 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.
According to one aspect the present invention consists of method of manufacture of an anti-paratopic antibody comprising the steps of:
(1) selecting from a pool of antibodies occurring in one species a prototypic set the members of which are effective in binding a specific antigen (or antigen epitope), and (2) utilizing one or more members of said prototypic set, or paratopic fragments thereof, as an immunogen in a host of a different species, or in an in vitro incubation system comprising cells derived from the same or a different species, to produce antibodies having a characteristic which is anti-paratopic with respect to said immunogen to produce a synthetic replicate of the specific antigen or epitope.
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 oasis of effectiveness against a particular antigen, or ~pitope thereof, for example is a set of human antibodies obtained from humans carrying antibodies 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.
In a second embodiment of the invention the anti-paratopic monoclonal 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 the anti-paratopic ;monoclonal antibodies were derived.

Brief Description of the Drawi:nas 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 (I) 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 (a) illustrates purification of human IgG prior ~o delineation into HIV/HIV antigen specific .antibodies.
Figure 6 (b) illustrates purification of human IgA prior ~o delineation into HIV/HIV antigen specific .antibodies.
Figure 6 (c) illustrates purification of human IgM prior vo delineation into HIV/HIV antigen specific .antigens.
Preferred embodiments of the v~nvention have a number of advantages over the prior art.
Firstly, the invention produces a mouse anti-paratope which is a counterpart of a naturally occurring human antibody ~~aratope for a specified antigen. Upon inoculation the mouse ~ 1 341 277 anti-paratope mono-clonal antibody produces i.n a human an antibody bearing a replica of the naturally occurring human paratope.
In the prior art there was produced a mouse anti-paratope which was a counterpart of an artificially generated mouse antibody. Such a mouse anti-paratope mono-cl.onal 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.
Fourthly, the process is safer to conduct than processes 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 s Deficiency Syndrome (AIDS). The invention is not however limited to use for production of any par_ticul.ar vaccine, and has uses other than for the production of vaccines.
The manufacture may be considered as involving the steps of:
(1) selecting a prototypic set of antibodies;
(2) preparing one or more immunogens therefrom;

(3) inoculating hosts with the one or more immunogens;

(4) generating a monoclonal antibody pool from each host;

(5) screening the monoclonal antibody pools;

(6) testing the screened antibodies for effectiveness as a vaccine.
In the example under consideration the first stage is to select from the pool of human antibodies a prototypic set, in this case a set of immunoglobulins which effectively bind the aetiologic agent for Acquired Immune Dif'ficiency Syndrome (AIDS). The generally 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. About 75~ of such individuals have antibodies to HIV.

The antibodies from these individuals are screened for effectiveness in binding HIV antigens and/or 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 nay be purified and used directly in step (3).
Preferably, however, in a second step the human immunoglobulins ("Ig") ire subdivided into classes G, A, M, D, (to use the WHO designation) and more particularly identified in Table I.

Table 1 PHYSICAL PROPERTIES OF MAJOR HUMAN IMNIUNOGLOBULIN
CLASS IN SERUM
WHO Designation IgG IgA IgM IgD IgE
Sedimentation 75 75,95,11 19S# 75 85 Coefficient S*
Molecular Weight 150,000 160,000+ 900,000 185,000 200,000 dimer Number of Ig 1 1-2 5 1 1 Units Number of 2 2-4 10 2 2 Antigen Binding Sites Identity of (gamma) (alpha) (mu) (delta) (epsilom) Heavy Chain Carbohydrates 3 8 12 13 12 Content o Total 80 13 6 0-1 .002 Immunoglobulin in normal human serum Concentration 8-16 1.4-4 0.5-2 0-0.4 17-450 range in normal mg/ml mg/ml mg/ml mg/ml mg/ml -human serum * IgA dimer found in mucosal (secretory) immune system. It is complexed with a secretory component (MW=60,000) and J chain (MW=15,000).
# IgM contains J chain.
Source: I.M. Roitt, Essential Immunolgoy, 4tt' Ed. Blackwell 1980 More desirably still the immunoglobulin~, are further divided into sub-classes, for example, I_gG being divided into four sub-classes, IgA being divided into two sub-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, vi.z - male homosexuals - bisexual/female/heterosexual AIDS carriers - haemophaelics The blood plasma is heated to 56°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. Other 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.
In a similar manner human IgA purification may be carried ~~ut by anti-IgA affinity chromatography.
* Trade-mark Human IgM may be purified by a combination of (a) Protamine sulphate chromatography, and (b) Column chromatography, or (c) IgM affinity chromatography.
The purified prototypic immunoglobulins set may be used directly as an immunogen for inoculation 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-classes known as p18, p24, gp4l, p55, gp120 and gp160. 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 screened against each antigen subclass to select the most effective Ig's.

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In the third step of the embodiment, one or more members of the prototypic set a:re used as an immunogen in a non-human )zost 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 he effectiveness against a specific antigen or effectiveness <~gainst a spectrum of antigen sub-classes or other criteria.
Human antibodies a:re excellent immunogens when injected into mice. The antigenic sites on the human antibody molecules a re spread right across the length of the molecule from the I~IH2 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 t:he Fc region. This phenomena is described as antigenic 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 is to say the paratope bearing region. 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 immunogen. Thus the member of the anti HIV prototypic set used as an immunogen in the mouse may be either (a) the mixed intact human immunoglobin specific for the AIDS virus, (b) selected classes or subclasses of the intact immunoglobin, (c) a Fab/F(ab)'2 fragment of one or a combination of the AIDS specific immunoglobulins or (d) 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 =nzymatic digestion or chemical cleavage of the human anti HIV
immunoglobin and conjugation of the Fab/F(ab)'2 to microspheres or the like.

As will be apparent. from the foregoing, _-_t is conceivable that when injected into the mouse, the H:IV IgC~l subgroup could provide all the relevant. paratopes on one type of carrier.
This regime would favour the generation of ant:i-idiotypes in t:he mouse (as opposed to the generation of anti-"carrier"
molecules).
There is a possibility though of inner-molecular antigenic competition so that only a sma:Ll variety of the human paratopes directed against HIV wil:L end up being antigenic in the mouse. If this occurs Then there are various ways of proceeding:
(i) After the screening step those paratopes that are dominant could be isolated from the immunogen population and a :>econd immunization carx:ied out to develop mouse anti-paratope antibodies to the remaining paratopes.
(ii) The anti-idiotypes/idiotypic reagen~s arusing from 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 emplo~led. The eventual manufacturing x:oute thus depends on whether anti-idiotypes/idiotypic reagents to all reagents are required.
It may suffice to have, say, one or two of the haptens f=nom each antigen group covered. To a great e:~tent though, this is an issue that will be resolved by the mouse itself in t:hat it may only be able to raise anti-idiotypes/idiotypic x:eagents against a restricted idiotype range.
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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 (i) 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 imrrunoglobulin response the antibody range may not be restricted and a more general immunization routine adopted. In the latter case (iii) subclass purification may be called for coupled to several primary immunizations.
An excellent starting position though would be to opt for (i) and 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 (i) an outline of the various alternative pathways for the purification and reparation 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:

STANDARD METHOD "LOTTO"
one-hit (multi-chance) ( i ) immuni ze (ii) 4 wks later boost (ii) 2-3 days later boost (iii) 4 days later: (ii) 2-3 days later :spleen spleen cell preen cell preen (iv) Hybridoma productn (iii) Hybridize (v) Preliminary screen (iv) Preliminary screen (vi) CLONE (v) Clone The Fab pool may be screened by conventional means as shown in Table 4:

SCREENING FOR FAB POOL
Antigen Mc Ab Configuration (+Ve/-Ve) 1 2. 3 4 S 6 Bence- + -- + - + -Jones Human Ig + + - + - -Immunoge - -- + + - +

n ?ACT I ON

Discard/

Retain D D D D D R

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 ~~ifferent 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 (e. g. B cells) or may exist as immune complex. The prototypic .set may be divided into members using different criteria from that exemplified.
=a Zo 1341277 Other methods may be used for separation such as use of eyes bound to inert supports, or the use of monoclonal ,antibodies, etc. and purification of the immunogen without ~~eparture herefrom.
The immunogens, or fragments thereof may be utilized in zost 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.
The invention will now be described more specifically by ~aay of the following Example.

PREPARATION OF HUMAN IMMUNODEFICIENCY VIRUS t;PECIFIC 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 mouse 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 (1) the preparation of human IgG from HIV infected individuals (2) the preparation of the human antibody (IgG) column and (3) 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, ion-exchange chromatography (DEAE-cellulose) or protein-A affinity chromatography. By way of example the method for the ~3412~7 purification described is that of protein-A agarose * column chromatography.
Pooled human sera was obtained from patients positive for t:he AIDS virus as determined by both an .SIDS antibody ELISA
assay and subsequently confirmed by the Weste=n Blot assay.
F?rior to use the serum had been heat treated (56°C 30 mins). A
2.Om1 protein-A agarosex column was washed wit=h 20m1 of the Monopure binding buffer (Pierce). 4mls of the pooled serum u~as 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 buffer. The human IgG was specifically eluted using the commercially obtained elution buffer (Pierce).
E,ollowing dialysis and concentration, the A28o data was used to determine the concentration of protein which was calculated to be 30 milligrams as determines by the E1%=1.43(280nm). Western Blot and ELISA data confirmed the presence of HIV specific antibodies in the IgG fraction purified :in th_s manner.
2. The preparation of the IgG affinity column.
30 mgs of the human IgG was equilibrated in the coupling buffer (O.1M NaHC03 pH8.3 + 0.5M NaCl) and mixed with 4 gms C;nBr-Sepharose*4B (Pharmacia) which had been pre-washed in 1mM
HCI, swollen and equilibrated in the coupling buffer. The mixture was mixed end-over-end in a sealed coupling vessel (2hrs, RT). Unreactive groups on the ma~rix were blocked using 0.2M glycine in the coupling buffer (16 hrs, 4°C) and * Trade-mark vhe ensuing IgG-Sepharose matrix exhaustively washed in high ;salt and variable pH buffers prior to the purification of the 1-IIV antigens.
3. The purification o:F the native/recombina:nt antigens.
By way of illustra~ion the method described is that for the recombinant HIV antigens in particular re~~ombinant 'gp120' Sub genomic clones of HiV cDNA encoding gp120, gp4l, p24, and p18 were cloned and amplified in E. Coli using ~ gtll.
~Che E. Coli lysates were screened with in-house and by commercial HIV antigen ELISA's. Radioimmunop:recipitation studies confirmed the presence of recombinant HIV antigens and l.he molecular weights of the recombinant antigens were as predicted e.g. 60kD for the recombinant 'gp120'.
Following precipitation of E. Coli antigens with (NH~)2504 1=he supernate was concentrated (Amicon)dialysed against distilled water and then against 0.05M Phosphate buffer pH7.2(l6hrs, 4°C). 40 mls of the dialysed co:zcentrate was combined with approximately 2 ml of the IgG-S~~pharose and the mixture incubated end-over-end for 2 hrs (RT). The matrix was exhaustively washed and the recombinant protein eluted using <~M MgCl2,pH 8.3. The presence of recombinant antigen was confirmed as outlined above.
B. THE PURIFICATION OF THE HUMAN HIV SPECIFIC ANTIBODIES.
The purification o.f HIV specific human antibodies :Lnvolved two steps. These are outlined below.
The preparation of the HIV antigen column.

~Che purification of the HIV specific human antibodies.
1. Preparation o' the HTV antigen-Sepharose column.

7.5 mls of the eluted protein was mixed ~Nith 2 gms swollen, pre-washed and appropriately equilib=rated CnBr-Sepharose (pH8.3). The mixture was mixed end-over-end (2hrs, RT). Unreactive sites were blocked using 0.2M glycine (l6hrs, ~6°C) and the matrix exhaustively washed as out=lined for the IgG-Sepharose column.
2. Purification of HIV specific human antibody.
4 mls of pooled human HIV serum heat treated as outlined above was passed through a PD-10 column equilibrated with f=reshly prepared 0.05M F?hosphate buffer pH7.2 + 0.5M NaCl. The f=first 3 ml fraction (void volume) was discarded and the next ~~.5 mls was collected. 10 mls of the gp120-Sepharose matrix rind 7.5 mls of the equilibrated serum were mixed end-over-end f:or 2hrs at RT. Following extensive washing HIV specific Ig's were desorbed using buffer containing 4M MgCl~; pH8.3.
~~pproximately 2 mg of HIV specific Ig was obtained using this method.
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.

Zs 1 34' 277 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 10 milligrams of human IgGl. The second group consisted of untolerized mice.
Mouse Ab2 antibodies were induced in the following way.
1.3 x 10a mouse spleen cells were recovered and washed in the incubation medium (Iscoves DMEM medium containing 20o foetal calf serum (FCS), 40o thymus conditioned medium {TCM), 5 x 10-9 2-mercaptoethanol, 4mML-glutamine 50 IU penicillin and 50 IU
streptomycin). HIV specific human immunoglobulins at a concentration of 10 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 15 ml_s. In this example the incubation was allowed to proceed for 7 days in a heated (37°C) C02 incubator.
Following incubation the cells were recovered for fusion to either SP2, NS1 or X63-Ag*.653 mouse myeloma cells. The viability of the spleen cells was found to vary between 70 and 99o and the viability of the myeloma was generally 990. 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 * Trade-mark ~ ,.,_ 1341277' myeloma cells using polyethylene glycol 1500/4000 (B.oehringer/Mannheim) using standard procedures and following 24 hrs incubation in a ~~02 incubator at 37°C t=he hybrids were plated out in the incubation medium now containing HA'T.
'J. 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 illustra~ion the following method was used.
Human peripheral b:Lood lymphocytes (PBL'.s) were diluted 1:1 in phosphate buffered saline and the red cells removed by centrifugation through <~ Ficoll-hypaque* cushion (Pharmacia).
*Trade-mark 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 tissue culture media (RPMI-1640 + 5o FCS).
In a simple example the B95-8 isolate is made available as a :~upernate 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 t~~ fusion with the mouse myeloma cell Line such as X63-Ag*.65.3. Screening is by a commercially available HIV antibody ELISA. Cloning and feeding (Medium ~~ontaining HAT/HT) is b:y the usual methods except that non transformed will be selected out by feeding with 1 micromolar Oubain.
*Trade-mark 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 1395-8 in sterile tissue culture media (RPMI-1640 + 5o FCS).
In a simple example the B95-8 isolate is made available as a aupernate 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 mous~= myeloma cell -Line such as X63-Ag*.65:3. Screening is by a commercially available HIV antibody ELISA. Cloning and feeding (Medium containing HAT/HT) is by the usual methods except that non 1=ransformed will be selected out by feeding with 1 micromolar Oubain.
*Trade-mark ~34~z»
All these methods T.ust be carried cut in hybridoma facilities suitable for work involving HIV as virus may be shed under these conditions.
E. PRODUCTION OF HUMAN ABl USING IN VITRO IMMUNIZATION OF
HUMAN PERIPHERAL BLOOD LYMPHOCYTES AND/OR SPLENIC
LYMPHOCYTES.
HIV specific human Abl may also be obtained by in vitro immunization using wholw virus or native, recombinant HIV
antigens and antigens bound to nitrocellulose. According to cane method 3-4 x 104 human PBL's or human splenic lymphocytes depleted of monocytes/T lymphocytres using L-Leucine methyl f=ster can be immunized with small amounts (1 nanogram - 10 micrograms) of HIV antigen. The human Abl are monoclonal when vhe 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 vivc or in vitro culture techniques using human cells or cells of other species as the human Abl would house the prototypic p~aratopes 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 :cope of the invention herein disclosed.

Claims (10)

1. A method of manufacture of an anti-paratopic antibody comprising the steps of:
(i) 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;
(ii) selecting from the prototypic set selected in step (i) an antibody class(es) or sub-class(es) or combination of classes or sub-classes which exhibit the highest level of immunological activity with the antigen in step (i);
(iii) subjecting the antibody class(es) or sub-class(es) selected in step (ii) to enzymatic cleavage to separate F (c) and F (ab) antibody fragments;
(iv) preparing one or more of the F (ab) antibody fragments of step (iii) as an immunogen for a host of a different species from the first species of in an in vitro incubation system comprising cells derived from the same or a different species, to produce one or more antibodies which are anti-paratopic with respect to the F (ab) fragments selected in step (iii); and (v) selecting, isolating and purifying the anti-paratopic antibodies produced in step (iv).

2. A method according to claim 1 wherein the antibodies in the prototypic set selected in step (ii) express the highest neutralising activity of all the antibodies in the pool of antibodies selected in step (i).

3. A method according to claim 1 wherein the antibodies selected in step (i) are human antibodies.

4. A method according to claim 1 wherein the antibodies selected, isolated and purified according to step (ii) are utilized to detect the presence of antibodies in other members of the first species selected in step (i).

5. A method according to claim 1, wherein the antibodies are human antibodies to HIV.

6. A method according to claim 1 wherein the step (iv) spleen cells from the host selected in step (iv) are harvested, and fused to myeloma cells to produce a hybridoma cell line which is capable of producing an anti-paratopic antibody.

7. A method according to claim 6 wherein the anti-paratopic antibodies-selected are monoclonal antibodies.

8. A method according to claim 6 wherein the anti-paratopic antibodies are polyclonal antibodies.

9. Purified non-human anti-paratopic antibodies expressing a binding affinity for the paratope of human antibodies generated against an antigen or antigen epitope.

10. Purified non-human anti-paratopic antibodies expressing a binding affinity for the paratope of human antibodies generated against epitope on a human immuno deficiency virus.