Note: Descriptions are shown in the official language in which they were submitted.
1 1 341 277
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.
1 341 277
~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
1 341 X77
3
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
4 1 341 277
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
1 341 277
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.
1341277
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
1341277
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.
1 341 277
9
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.
1341277
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
1 341 277
11
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
1 341 X77
12
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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1 341 2 7 7
14
.ith 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
garatopes specific for HIV is 312. Put differently there are
on average thirty nine HIV paratopic bearing human
immunoglobulins per immunoglobin sub-class. Thus, for
=xample, it might eventuate that human IgGl has specificities
for all thirty nine epitopes ("haptens"), i.e., there would be
thirty nine IgGl molecules all absolutely identical except for
~~ne feature namely their Fab paratope would be different.
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.
1 341 277
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.
16 1341277
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.
E
1 341 277
17
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
1341277
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:
TABLE 3
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:
19 1 341 277
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.
1 341 277
21
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
22
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
1341277
23
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.
1 341 277
24
~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
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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).
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1 341 277
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.
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28
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.
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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.
