Note: Descriptions are shown in the official language in which they were submitted.
W093~18792 PCT/CA93/00110
SELECTIVE ALTERATION OF ANTIBODY IMMUNOGENICITY
BACRGROUND OF THE INVENTION
Field of th~ Invention
Thls invention relates to a method of al~erlng the
i~munogenicity of antibodies so tha~, upon administration to a
sui~able subject, an immune response is elicited which is
predominantly anti-idiotypic rather than anti-isotypic in
character.
Description of the Background Art
10All vertebrates possess a surveillance mechanism, called the
immune system, that protects them from pathogenic microorganisms
(including viruses), multicellular parasites, and cancer cells.
The immune system specifically recognizes and selectively
eliminates these undesirables by a process known as the immune
response. One of its two important subsystems is the humoral
immune system, which relies on antibodies, produced in quantity
by plasma cells, that circulate through the blood and the
lymphatic fluid.
The first step in the immune response is the recognition of
the presence of a foreign entity. Antigens are molecules which
are subject to immune recognition. The portion of an antigen to
which an antibody binds is called its antigenic determinant, or
epito?e. Not all antigens are capable of eliciting a response,
as opposed to simple molecular recognition, ~rom the immune
system. Antigens which can elicit an immune response are te~ned
immunogens, and are usually macromolecules, such as proteins,
nucleic acids, carbohydrates, and lipidC, of at lease 5000
Daltons molecular weight. However, many small nonimmunogenic
molecules, termed haptens, can stimulate an immune response if
associated with a large carrier molecule.
Antibodies, also known as immunoglobulins, are proteins.
They have two prin~ipal f ~tions. The first is to recognize
(bind) foreign antigens. The second is to mobilize other
elements of the immune system to destroy the foreign entity.
35The basic unit of immunoglobulin structure is a complex o~
four polypeptides -- two identical low molecular weight ~"lightn)
chains and two identical high molecular weight ("heavyn) chains,
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~ nked together by both nocovalenr associations and by àisulfide
bonds. Different antibodies will have anywhere from one tO five
of chese basic units. The immunoglobulin unit may be represented
schematically as a "Y". Each branch of the "Y" is formed by the
amino terminal portion of a heavy chain and an associated light
chain. The base of the "Y" is formed by the carboxy t~rminal
portions of the two heavy chains. The node of the "Y" is the so-
called hinge region, and is quite flexible. Five human antibody
classes (IgG, IgA, IgM, IgD and IgE), and within these classes,
various subclasses, are recognized on the basis of structural
differences, such as the number of immunoglobulin units in a
single antibody molecule, the disulfide bridge structure of the
individual units, and differences in chain length and sequence.
The ~lass and subclass of an antibody is its isotype.
The amino terminal regions of the heavy and light chains are
far more diverse in sequence than the carboxy terminal regions,
and hence are termed the variable domains. This is the part of
the antibody whose structure confers the antigen-binding
specificity of the antibody. A heavy variable domain and a light
variable domain together form a single antigen-binding site,
thus, the basic immunoglobulin unit has two antigen-binding
sites. The walls of the antigen-binding site are defined by
hypervariable segments of the heavy and light variable domains.
Binding site diversity is generated both by sequence variation
in the hypervariable region and by random combinatorial
association of a heavy chain with a light chain. Collectively,
the hypervariable segments are termed the paratope of the
antibody; this paratope is essentially complementary to the
epitope of the cognate antigen.
The carboxy terminal portion of the heavy and light chains
form the constant domains. While there is much less diversity
in these domains, there are, first of all, differences from one
animal species to another, and secondly, within the same
individual, there will be several different isotypes of antibody,
each having a different function.
The IgG molecule may be divided into homology units. The
light chain has two such units, the VL and CL, , and the heavy
chain has four, designated VH~ CH1, CH2 and CH3 All are about
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110 amino acids 'n length and have a c~ntrally locat~d intrachain
disulfide bridge that spans abou~ 60 amino acl~ residues. The
sequences of the two v-region homology units are similar, as are
the sequences of the four C-region homology units. These
- homology units in turn form domains The two variabl~ domains
have already been mentioned; cher~ are also four conscant
domains. Mild proteolytic digestion of IgG results in the
production of certain fragments of interest. V-C1 is Fab; CH2-
CH3 is Fc; (V-C1), is (Fab')" V-Cl-C2 is Fabc, and V alone is Fv.
While the variable domains are responsible for antigen
binding, the constant domains are charged with the various
effector functions: stimulation of B cells to undergo
proliferation and differentiation, activation of the complement
'5 cell lysis system, opsonization, attraction of macrophages to
ingest the invader, etc. Antibodies of different isotypes have
different constant domains and therefore have different effector
functions. The best studied isotypes are IgG and IgM.
If a specific antibody from one animal is injected as an
immunogen into a suitable second animal, the injected antibody
will elicit an immune response. Some of these anti-antibodies
will be specific for the unique epitopes (idiotopes) of the
variable domains of the injected anti~odies; these epitopes are
known collectively as the idiotype of the primary antibody and
~5 the secondary (anti-) antibodies which bind to these epitopes are
known as anti-idiotypic antibodies. Other secondary antibodies
will be specific for the epitopes of the constant domains of the
injected antibodies and hence are known as anti-isotypic
antibodies. ~The term "anti-isotypic" antibodies, as used
herein, includes antibodies that are merely species-specific as
well as antibodies which are also class or subclass-specific.)
The "network" theory states that antibodies produced
initially during an immune response will carry unique new
epitopes to which the organism is not tolerant, and therefore
3 ' will elicit production of secondary antibodie~ ~Ab2) directed
against the idiotypes of the primary antibodies (Abl). The-qe
secondary antibodies likewise will have an idiotype, which will
induce production of tertiary antibodies (Ab3), and so forth.
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~ ~iso sugges~s cha. som~ ~~ those secondary an~l~odi~s will
have a binding site which is the complement of ~he complement of
the original antigen, and thus will reproduce the "internal
image~ of the original antigen. In other words, an anti-
idiotypic antibody may be a surrogate antigen.
There are four major types of anti-idiocypic an~ibodies.
The alpha-type is one which binds an epitope remote from the
paratope of the primary antibody. The beta-type is one whose
paratope mimicks the epitope of the original antigen. The gamma-
type binds near enough to the paratope of the primary antibodyto interfere with antigen binding. The epsilon type recognizes
an idiotypic determinant that mimicks a constant domain antigenic
structure. Moreover, anti-isotypic antibodies may be heavy
chain-specific or light chain-specific.
"Active immunotherapy~ is the administration of an antigen,
in the form of a vaccine, to a patient, so as to elicit a
protective immune response. "Passive immunotherapy" involves the
administration of antibodies to a patient. Antibody therapy is
conventionally characterized as passive since the patient is not
20 the source of the antibodies. However, the term passive is -
misleading because the patient can produce anti-idiotypic
secondary antibodies which in turn provoke an immune response
which is cross-reactive with the original antigen.
As stated by Koprowski (3), a traditional approach to cancer
2S immunotherapy is to administer anti-tumor antibodies, i.e.,
antibodies which recognize an epitope on a tumor cell, to
patients. However, the development of the "network" theory led
her and others ~4) to suggest the direct administration of
exogenously produced anti-idiotype antibodies, that is antibodies
raised against the idiotype of an anti-tumor antibody. Koprowski
assumes that the patient's body will produce anti-aneibodies
which will not only recognize these anti-idiotype antibodies, but
also the original tumor epitope.
Koprowski ' 9 exogenous anti-idiotypic antibodies are the
product of a rather complex production process. Polyclonal anti-
idiotypic antibodies must be separated from other antibodies in
the serum of the animal. The use of monoclonal anti-idiotypic
antibodies simplifies purification to some degree, but at the
.
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cos~ of a laborious screening proc~dur~ ~c lden~iry hybridomas
secreting ~he desired anti-idiotypic antibody. Then these cells
must be expanded in culture. Finally, once a production culture
is developed, the antibodies still must be recovered, purified
and tested. Applicants believe it Co be preferable to stimulate
in vivo p~oduction of the anti-idio~ypic antibo~y.
It is of course true that Applicants' antibodies must also
be purified. However, Applicants need only distinguish between
antibodies which bind to the immunogen and those which do not.
The proponents of exogenous anti-idiotypic antibody therapy must
differentiate antibodies which bind to the same immunogen, but
in different places.
In a related vein, it has been suggested that one may
administer a synthetic polypeptide that substantially
immunologically corresponds to an idiotypic epitope of an
antibody directed against an antigen of interest (5). However,
this polypeptide must be synthesized and purified. Moreover,
this methodology requires knowledge of the sequence of the
antigen binding site of the anti-idiotypic antibody.
Sources of human antibodies are limited to subjects already
suffering from the disease of interest, as it is unethical to
introduce a disease into a subject merely so the subject will
begin producing antibodies which may be harvested. ~ecause of
the difficulties of collecting human antibodies, clinicians rely
on antibodies of nonhuman origin, such as mouse antibodies.
Unfortunately, besides eliciting an anti-idiotypic response,
these mouse antibodies, when administered to humans, also provoke
production of secondary human anti-mouse antibodies ~HAM~)
directed against mouse-specific and mouse isotype-specific
portions of the primary antibody molecule~ This immune reaction
occurs because of differences in the primary amino acid sequences
in the constant regions of the immunoglobulins of mice and
humans. Both IgG and IgM subclas9es of HAMA have been detected.
The IgG response appears later, is longex-lived than the typical
IgM response, and is more resistant to removal by pla9mapheresis.
Clinically, the development of HAMA increases the likelihood
of anaphylactic or serum sickness-like reactions to subsequent
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admlnistratlon of murin~ immunoglobulins Thes~ secondary
antibodies reduce the efficacy of repeat immunotherapy by
complexing subsequently administered mouse antibody (31). HAMA-
induced increases in the clearance of the injected antibody or
fragment can result in reduced tumor localization, enhanced
uptake into liver and spleen, and tumor escape from therapy.
HAMA can also cause interference with immunodiagnosis, and
thereby hinder monitoring of the progress of the disease and the
effectiveness of the course of treatment.
The anti-isotype response has been avoided in prior
immunoimaging work through the use of monovalent Fab fragments
or divalent (Fab'). fragments. These fragments lack most of the
constant region and therefore present only a very limited
oppdrtunity for anti-isotype binding (1). Moreover, they lack
the effector functions of a more intact antibody and therefore
will not activate complement, or bind to an Fc receptor on a
killer cell. Accordingly, such fragments, which lack most or
all of the constant region, are not normally used in
immunotherapy.
Another approach is to conjugate a tolerogen, such as
polyethylene glycol, to the antibody to reduce its immunogenicity
(2). Unfortunately, PEGging an antibody also diminishes its
ability to elicit an anti-idiotypic response.
Wagner, et al. (6) radioimmunoimaged 12 patients with
ovarian carcinomas using Iodine-131 labeled F(ab'). fragments of
the anti-CA125 mouse antibody OC125. All patients had been
treated in the same manner by surgery followed by chemotherapy.
Five of the patients developed anti-idiotypic antibodies against
the imaging antibody. In 1989, only these five patients were
still alive. Wagner, et al. suggested that their longterm
survival was attributable to their development of anti-idiotypic
antibodies against the OC125 fragments, and hence to induction
of the idiotypic network. While Wagner et al.'s fragments may
have exerted a serendipitou9 immunotherapeutic effect through
generation of Ab3, they nonethele~s lack the effector function8
of conventional immunotherapeutic agents. Moreover, because
these fragments are more rapidly cleared from the bloodstream,
they are less useful than intact antibody for immunotherapy.
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The us~ of intacc antibody (Abl) to act~vate the idio~ype-
anti-idio~ype network, while po~entially enhancing the
immunotherapeutic utility of the antibody, would raise the issue
of problems with anti-isotypic responses, as previously
_ mentionea. wagner et al. did not need to address the possibility
of an anti-isotypic response since he had administered fragments
lacking most of the constant region.
A methodology is urgently needed that allows use of animal
antibodies in human therapy, with in vi~o stimulation of an
endogenous anti-idiotypic response and without concomitant
stimulation of a substantial anti-isotypic re~ponse tthe term
here including a species-specific response), which does not
re~uire use of antibody fragments which lack constant regions.
i5 All references, including patents and patent applications,
which are cited anywhere in this specification are hereby
incorporated by reference. No admission is made that any cited
r~ference constitutes prior art, or pertinent prior art.
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SUMMARY OF T~E INVENTION
Applicants have discovered that the immunogenic character
of antibodies may be modified so as to substantially eliminate
the anti-isotype response while substantially preserving the
anti-idiotype response to the antibodies.
If the anti-isotype response is eliminated, it may be
possible to repeatedly administer an antibody to a patient
without fear of putting the patient into anaphylactic shock
brought on by an adverse immune reaction between the exogenous
antibody and previously elicited anti-isotype anti-antibodies.
Retention of the anti-idiotype response is advantageous, however,
as the anti-idiotype anti-antibody mimics the original antigen,
and thereby can elicit production in the patient of endogenous
antibodies which likewise recognize the original antigen.
Elimination of the anti-isotypic response will also facilitate
subsequent immunosurveillance of the patient by in vitro and ~a
vivo immunodiagnostic technigues, as interference from anti-
isotypic anti-antibodies will be avoided.
While simply removing the Fc portion of an antibody is
likely to substantially eliminate its ability to elicit an anti-
isotype response, the use of antibody fragments such as Fab alld
Fab' fragments has other disadvantages. These fragments have a
shorter residency time in the bloodstream, and therefore are less
desirable from a therapeutic standpoint than a whole antibody.
They also fail to pro~ide all of the effector functions
associated with intact antibody, which reduces their therapeutic
effectiveness. Indeed, they may actually interfere with the
action of endogenous antibodies, which have the effector
function, by blocking the antigenic determinants. Thus, while
they have some therapeutic value through eliciting production of
Ab3, in general they are not ~uitable as immunotherapeutic
agents~
Instead, applicants treat the antibody with a reagent that
is capable of reducing certain of the diQulfide ~-S-S-) bridge~
of the immunoglobulin, thereby generating free sulfhydryl groups,
but without fragmen~ting the antibody sufficiently to abolish
effector function.-
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Th~ reduc~ion also results ~n a dena~uratlon of the hea wchain conformationl and thereby ~ubstantially eliminates anti-
heavv chain or isotype antibody response. It is also believed
that under certain circumstances che a~ti-idiotypic response can
_ be increased in both an absolute as well as a relative sense.
While applicants do not wish to b~ bound to this theory, it is
believed that the cleavage of certain disul~ides resul_s in
greater conformational flexibility in the critical antigen
binding variable and hypervariable regions, exposing areas which
~-eviously were subject to steric hindrance, and therefore to a
greater propensity toward anti-idiotype responses. However, an
absolute increase in the anti-idiotypic response is not required
for the practice of this invention.
The present invention also relates to an improved method of
reducing, and, if desired, radiolabeling antibodies. These
antibodies may be used for radioimmunotherapy, or for
radioimmunoimaging (with a reduced isotypic HAMA response to
interfere with subsequent immunotherapy).
The appe~ded claims are here~y incorporated by reference as
a fur~cher recitation of 'che preferred errbodiments.
DETAILED DESCRIPrION OF T~E PREFERRED EMBODDMENTS
The present invention relates to the production of reduced
antibodies and their u~e, alone or in combination with other
agents, as immunotherapeutic agents.
All immunoglobulin G molecules consist of two heavy and two
light polypeptide chains covalently bound to each other through
several disulphide bridges between cysteine amino acids~ In
addition to these interchain bridges, there are a greater number
of intrachain disulphide bonds which also aid in the maintenance
of the tertiary structure of the molecule. Under reductive
conditions, these bridges can be cleaved to the corre3ponding
sulphydryl forms.
There are numerou8 techniques for preparing reduced
antibodies. In general, the compounds u~ed fall into three
categories - the classical reducing agents compri8ing organic
(for example, fonmamidine sulfonic acid) and inorganic (for
example, mercurous ion, stannous ion, cyanide ion, sodium
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~yanoborohydrid~, sodlum borohyàr~de, etc.) compounds, tn~ ~hiol
exchange reagents (for example, dithiothreltol, mercaptoethanol,
mercaptoethanolamine) and protein reductants (for example,
thioredoxin). Exposure of immunoglobulin-G molecules (or their
fragments) to these compounds results in somewhat selective
reduction of disulphides to form various sulphydryl ~roups.
Under continuing reductive conditions, these sulphydryl groups
remain, resulting in an at least partially disulphide reduced
protein molecule, and at least potentially chan~ing the tertiary
structure of the immunoglobulin. The effect of the reduction on
the conformation and immunoreactivity of the antibody molecule
is dependent on the degree of reduction.
The reduction results in a denaturation of the heavy chain
conformation, and there~y substantially reduces or even
eliminates anti-heavy chain or isotype antibody response.
While totally reduced antibody molecules are potentiall~-
usable, it is likely that their affinity for antigen will be
substantially diminished. Consequently, it is preferable to
control the degree of reduction of the antibody so that it
retains at least some of its intra- and/or inter-chain disulphide
bonds. The most susceptible disulphide bridges are those in the
hinge region and therefore under appropriate conditions these can
be preferentially c'eaved. This potentially allows greater
movement of the critical antigen bindin~- variable and
hypervariable regions and may expose previously hindered areas
of these regions. With some antibodies, this may lead to an
enhancement of the anti-idiotype human anti-mouse antibody
response.
Reducing agents potentially useful for the selective
elimination of the isotype immunogenicity of the antibody are
readily tested for suitability by the HAMA assay described in
this specification, or by other assays capable of differentiating
anti-idiotypic and anti-isotypic HAMA (31).
The HAMA assay described in the Examples is a two-step
indeirect radioimmunoassay. Beads which have been precoated with
goat anti-mouse antibody are incubated with a second murine
antibody or fragment to form the complex that captures HAMA. In
order to measure a generalized HAMA response, only a nonspecific
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an~ibody, ~ g. an i~levan~ murine IgG monoclonal antibody, i~
used as ~he second antibody. In order to measure an anti-
idiotypic HAMA response, the particular antibody administered
to the patients is used on some beads and the nonspecific control
antibody i~ used on others.
After the lncubation with the second murine antibody or
fragment, the beads are washed to remove any un~ound antibodies.
The beads are now considered ~primed~ to capture HAMA. After
washing, diluted test serum is added and incubated with the
primed beads. HAMA present in the serum is captured or linked
to the primed beads during this incubation. Following a second
wash, the beads are incubated with a radiolabeled tracer
antibody, e.g., Iodine-125 labeled polyclonal anti-human
antibodies, which binds to captured HAMA. Any unbound
radiolabeled antibody is removed by a final wash before measuring
the amount of bound radioactivity.
Results obtained using the positive (anti-mouse Ig serum)
and negati~e (human serum) controls supplied in the kit are used
to calculate the HAMA limit.
About 9~ of a normal population has been found to exhibit
positive HAMA responses before in vivo administration of murine
immunoglobulin. Certain patient groups have higher preinjection
HAMA responses, so it is desirable to obtain a pre-injection
baseline sample.
The present invention is not limited tO any particular
method of ~etermining anti-isotypic and anti-idiotypic HAMA, or
any particular reagents for use therein. It is believed that the
Behringerwerke ENZYGNOST HAMA micro assay has the components
needful for measuring both HAMA responses, though the kit does
not explain how to perform this calculation. Measurement of
anti-idiotypic response is reported in, e.g., Reinsberg, et al.,
Clin. Chem. ,36: 164-167 (1990); GOldman-Leikin, et al., Exp.
Hematol. 16: 861-864 (1988).
While we have spoken in terms of the HAMA re9pon9e, we could
as well have addre99ed any immune response of one animal to
antibodies deri~ed from a dif-ferent specie~ of animal.
The reduced antibody elicits at least some anti-idiotypic
anti-antibody response but no more than a substantially
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dec~ase~, ~- any, an~ so~ype r~s~onse, ~elaciv~ n~
unreduced antibody. Desirably, no more than 20%, and more
desirably, no more than 5~, of the antl-isotyplc response of the
subject to the antibody is r~tained after reduction. Most
~- desirably, the anti-isotypic response is essentially elimina~ed.
Preferably, at least 25~, more preferably at least 50~, still
more preferably at least 80~, and most preferably, at least 95~,
of the anti-idiotypic response of the subject to the antibody is
left under these circumstances. Preferably, the reduction in the
anti-isotypic response is substantially greater than the
reduction in the anti-idiotypic response.
While it is preferable that the reduced antibodies of the
present invention retain their Fc and hinge regions, it is also
poss ble tO reduce antibody frasments that possess only a portion
of the nonmal Fc region or hinge region, such as (Fab')..
If desired, the reduced antibody may be radiolabeled with
pertechnetate or perrhenate to produce a radiolabeled antibody
which may be used for radioimmunoimaging as well as
radioimmunotherapy. The radioisotope may be one with a
cytotoxic effect and therefore of therapeutic value if the
antibody is directed against an antigen of an undesirable cell,
such as a cancer cell.
A particularly preferred reduction method employs SnCl. as
the reducing agent. Preferably, the molar ratio of this reducing
agent to the antibody is in the range of 20:l to lOO:l; the most
preferred value is about 40:l. Use of a high level of stannous
ion increases the chance of damaging or fragmenting the antibody
and also increases the likelihood of Tc-~9m-Sn(II) fonmation
competing significantly with the MAb-Tc-9~m reaction.
The concentration of the antibody may be in the range of 1 to lO
mg/mL; preferably 5mg/mL.
The reaction buffer preferably is a tartrate (e.g., NaK
tartrate) buffer; the preferred tartrate concentration is greater
than 0.05 and less than about 0.2M; the most desirable value
being about O.lM. The use of phthalate, as suggested by Rhodes,
U.S. 4,424,200 and 5,078,985, is unnecessary. The high tartrate
concentration stabilizes the Sn(II) ions and retards the
oxidation to the SntIV) state. As a result, precipitation of
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Sn(IT) ~- colloidal fo ~ a~ion aurl~g buffer pr~Daratlon lS n~
usualiy observed. The pH or the buf~er may be 4-8; a pH which
results in excessive ~:recipltation or cloudiness of the buffer,
or which results in d~gradatlon and loss of immunoreactivity on
the par~ of the antibody, should be avoided. One of the
advan~ages of the present system is, however, the broad pH range
it accommodates, allowing selection of a pH to which the antibody
is insensitive. Degassing of the buffer is not essential. The
pretreatment buffer is compatible with MAb stored in either
normal saline or phosphate-buffered saline (P3S), and therefore
the researcher may select whichever storage buffer provides
better stability for the M~b.
The incubation is preferably from 8-24 hours and the
incubation temperature is preferably in the range of 18-40 deg.
C., and most desirably is 37 deg. C.
After this treatment, the reduced antibody may be frozen or
lyophilized for storage purposes. When desired, the reduced
antibody preparation may be reacted with a pertechnetate salt,
e.g., Na salt, for labeling purposes. Radiolabeling efficiencies
of over 90~ are routinely observed, and the immunoreactivity of
the antibody is essentially unaffected.
The antibody may also b_ incorporated into a conjugate
having desirable properties. An example of such a conjugate is
an immunotoxin, wherein one molety is an antibody and another is
a toxin. The antibody may target, e.g., a virus-infected cell,
and the toxin then kills the cell. Useful toxins include, e.g.,
ricin and abrin.
The antibody may be directed against any antigen of clinical
significance, but preferably is directed against a tumor-,
pathogen- or parasite-associated antigen. In the case of a
tumor-associated antigen ~TAA), the cancer may be of the lung,
colon, rectum, breast, ovary, prostate gland, head, neck, bone,
immune system, or any other anatomical location. The subject
may be a human or animal subject. The antibody may be a
polyclonal antibody or a monoclonal antibody. When tha subject
is a human subject, the antibody may be obtained by immNnizing
any animal capable of mounting a usable immune response to the
antigen. The animal may be a mouse, rat, goat, sheep, rabbit or
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o~her sul~able experlmental anima The an~lgen may ~ pres~nced
in the form of a naturally occurring lmmunogen, or a synthetic
immunogenic conjugate of a hap~en and an immunogenic carrier.
~~ the case of a monoclonal antibody, antibody producing cells
of the immunized animal may be fused with "immortal" or
"immortalized" human or animal cells to obtain a hybridoma which
produces the antibody. If desired, the genes encoding one or
more of the immunoglobulin chains may be cloned so that the
antibody may be produced in different host cells, and if desired,
0 the genes may be mutated so as to alter the sequence and hence
the immunological characteristics of the antibody produced.
The antibody may be administered to the patient by any
immunologically suitable route, such as intra~enous,
intraperitoneal, subcutaneous, intramuscular or intralymphatic
routes, however the intravenous route is preferred. The
clinician may compare the anti-idiotypic and anti-isotypic
responses associated with these different routes in detenmining
the most effective route of administration.
Example I
Reduction of Antibody
Stannous ion is a known sulphydryl reductant. We use a
stabilized stannous ion solution prepared from stannous chloride
and taxtrate salt. Controlled reduction with stannous ion of a
monoclonal antibody produced a modifi~d MAb preparation
containing an average of approximately one sulphydryl group per
molecule. Further evidence of sulphydryl creation is the ability
of the molecule to radiolabel with Tc-99m in the presence of
Tc-99m[(III),(IV)mtV)] complexes, known to fonm stable bonds with
thiol groups. This mild controlled process does not lead to any
significant loss of antigen binding properties of the MAb.
A solution containing 2.822 g of Sodium Potassium Tartrate
is prepared in ga ml of sterile water for injection and degassed
of dissolved oxygen by bubbling nitrogen gas ~5-10 pSi) ehrough
the solution for 30 minutes. A second solution is prepared
containing 1.13 g of stannous chloride in 10.0 ml of 1.0 ~ HCl.
A quantity of 400 ~1 of this solution is added to the tartrate
buffer solution and the mixture adjusted to pH=5.6~0.05 as
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m~asur~d b~ a calibraced p~ me~er by slow add~ rl~n 0l 1. O N NaOH.
A quantity of 40 ml of this ~ar~rate stabillzed stannous fon
solution lS added to 60 ml of a 5.0 mg/ml solut_on of M~b-170 or
MAb-B43 (contained in a pH 7.4 NaH.PO~ buffered matrix).
MAb-170 (more accurately, MAb170H.82) is a murine monoclonal
antibody of the IgGl kappa isotype that was produced by
immunizing BALB/c mice with a synthetic glycoconjugate consisting
of a Thomsen-Friedenreich (TF) beta (Galbetal->3GalNAc)
di~ccharide hapten coupled to an immunologically suitable
ca-:ier (serum albumin). It was selected based on its reactivity
wit human adenocarc;noma tissue in vitro. It clearly reaccs with
adenocarcinomata of the breast, ovary, endometrium, colon,
prostate and some bladder. It also reacts with adenosquamous,
small cell and squamous cell lung carcinoma tissue. It is
described in more detail in copending Ser. No. 07/153,162, filed
May 12, 1988, incorporated by reference herein, which is a :
continuation of Ser. No. 06/927,277, filed Oct. 27, 19~6. MAb-170
has been formulated into a Tc-99m radiolabeled antibody kit
(TRUSCINT AD, ~iomira, I~c., Edmonton, Alberta, Canada) for
2~ radioimmunodiagnos~ 3 of aienocarcinomas. See McEwan, et al.,
Nuclear Medicine Communications, 13: 11-19 (1992). A hybridoma
~170H82. R1808) secreting MAb 170 was deposited on July 16, 1991
with the American Type Culture Collection, 12301 Parklawn Drive,
Rock~ille, Maryland 20852 USA, an In~ernational Depository
Authority under the Budapest Treaty, and assigned the accession
nunb~er HB 10825. This deposit s~uld not be construed as a
license to make, use or sell the h- -idoma or MAb 170.
MAb-B43 (more accurate'y, B43.13) ,s a murine monoclonal
antibody of the IgG1 kapF~ isotype that was produced by
immunizing mice with the CA125 antigen. It was selected for its
reactivity to CA 125, an ovarian carcinoma-associaeed antigen.
It inhibits the binding of MAb OC125 to CA125. MAb ~43 is
reactive with CA125 antigen in biopsy tissue a;.d in serous and
endometroid carcinomas of the ovary. It has been formulated into
a Tc99m-radiolabeled antibody kit (TRUSCINT OV, ~iomira, Inc.
Edmonton, Alberta, Canada) for radioimmunodiagnosis of ovarian
carcinomas. See Capstick, et al., Int. J. Biol. Markers, 6: 129-
135 (1991).
SUeSTrrU~E SHEEI-
W093/18792 ~ PCT/CA93/00ll0
1 ~
Referenc~ ~o these cwo antibodies shouid no~ b~ ~ons~ue~
as a limitation on the generality of the present inven~ion.
The headspace of the reaction vessel con~aining this
comb~nation is purged with nltrogen gas and allowed ~o lncubac~
- for about 24 hours. Then, 0.67 ml aliquots of the solution are
filtered into 5 ml nitrogen purged sterile vials and froze~ at
-20C. Each vial contains nominally 2.0 mg of treated MAb-170
or MAb-B43. The final preparation is sterile, pyrogen-free and
suitable for human injection.
Example II
Human Anti-Mouse Antibody (HAMA) AssaYs
The Biomira TRUQUANT HAMA-RIA kit (~3iomiria, Inc.,
Edom~nton, ALberta, Canda) is an in vitro test for the detection
of anti-idiotypic and anti-isotypic human anti-mouse antibodies
(HAM~) of either the IgG or IgM subclasses, in human serum.
However, the principles of the kit are more broadly applicable
to the detection of anti-idiotypic and anti-isotypic antibodies.
. The Biomira kit utilizes goat anti-mouse capture reagent
on 1/4" polystyrene beads. Of course, other anti-mouse capture
reagents could be subtituted for the goat anti-mouse antibody.
This allows for capture of (a) idiotype and is~type matched or
(b) idiotype mismatched, isotype matched control MAbs. Patient
samples are then tested against beads that have been primed with
matched and mismatched mouse antibodies. By subtracting the
anti-isotype (control) response from the anti-idiotype (or
matched) response, the two types of HAMA responses can be
determined Formulae for the calculation of the Total, Control,
and Idiotype HAMA Indexes appear below:
Total HAMA Index (calculated using the specific or
matched antibody)=CPM Sample on idiotype-specific Ab / HAMA
Limit*
Control HAMA Index (calculated using the mismatched
antibody)=CPM Sample on idiotype mismatched, isotype-matched Ab
/ HAMA Limit*
SUBSrlT~E SHEET
WO93/18792 PCT/CA93/00110
17
Idiotype Index = Total HAMA Ind~x (specific) - Con~rol
HAMA Index (mismatched)
*The HAMA Limit [(0.2 x CPM of the Posltlve Control) t
CPM of the Negative Control] used in the HAMA kit was determined
5 tO be the upper limit of normal distribution of samples from
patients not injected with mouse antibodies. This run specific
cutoff value establishes a level above which a ~95~ confidence
can be used to determine that the result obtained is a crue
anti-mouse antibody response. The evaluation of the MAb-170
patients was based on a change of the HAMA Index from
pre-injection to post injection samples. A significant change
is a-difference greater than 1 HAMA Index valuc.
Example III
An~i-Idiotype Serum Assays
The present example shows a reduced antibody elicited
almost no anti-isotype response relative to an unreduced
antibody. While the reduced antibody also exhibited some
reduction of the anti-idiotype response, possibly as a result of
cleavage of disulfide bridges near the antigen-binding site, this
latter response was still substantial. MAb-170, as described
above, was labeled with either Tc-99m or In-lll. Labeling with
Tc-9~m was accomplished by first reducing the antibody as
described in Example I and then reacting it with sodium
pertechnetate as previously described. Labeling with In-lll,
25 tO act as a control for the reduced MAb 170, did not invol~e any
reductive process. Instead, MAb 170 was reaceed with DTPA
anhydride to produce a chelate attachment site for In-lll
labeling. The HAMA response to a single 4-8 mg dose was
determined.
The results are shown in Table 1 below.
While the HAMA kit used to measure the HAMA response used bead-
bound MAb 170 in unreduced form as the capture reagent for anti-
idiotype antibodies, substitution of bead-bound reduced MAb 170
did not lead to a significant change in the results obtained.
The HAMA response may also be quantified in terms of the
SOEISl~ITl~JlE SHEET
W093/18792 ,~ PCT/CA93/00ll0
~ i 18
number of patients s~roconvertln~ to produccion of an~ io~ype
or anti-isotype following injec~lon of the antibody. The r~sults
are shown in Table 2 below.
_xampl~ IV
Correlation of HAMA Idiotype with Cancer Survival
In Table III, ten ovarian cancer patients injected with
MAbs (fragment MAb OC 125 and reduced but unfragmented MAb B43)
had a mean survival time as of the date of compilation of about
three years. Of the ten patients, nine were still alive. Of
these nine, two have progressing disease and 7 are stable or free
of the disease. This is beyond normal expectations for these
patients and is attributed to the presence of anti-idotype MAbs
agai~st the injected MAbs.
OC-125 is a murine antibody generated by the immuni~ation
of BALB/c mice with a human serous papillary cystadenocarcinoma.
OC125 reacts with the CA125 antigen, which has been identified
as a high molecular weight glycoprotein found on the cell surfa~e
of many ovarian cancers.
For mo?ecular biology and immunology procedures not
described above, see Sambrook, et al., ~olecular Clon~na: A
Laboratory Manual (2nd ed., Cold Spring Harbor: 1989J; Harlow and
~ane, Antibodies: A Laboratory Manual tCold Spring Harbor: 1988);
Ausubel, et al., Current ~rotocols in Molecular Bioloqy (Wiley
Interscience: 1987, 1991).
Example V
Hama Analysis Post ~ 70 and MAb 174 Immunoscinti~raphy
In support of previous findings the nonspecific and anti-
isotype HAMA seroconversion rates after a single
immunoscintigraphy with the reduced antibodies of the present
inYention is significantly lower than historical results with
other antibodies~conjugates. Using the TRUQU~NT HAMA RIA to
measure the reQponse to a single 1 mg dose, and comparing pre-
infusion to p~5t infu5ion samples, 0/22 patients developed a
generalized or non-specific HAMA. Amongst patients infused with
partially reduced MAb 170 (n=16), no patients showed anti-isotype
or generalized HAMA responses and 2/16 seroconverted in an
SUeSmUTE SHEET
W093/18792 ~ PCT/C~93~00110
19
ià~otype speciflc manner. Amongs~ patients infused with
pa-tially reduced MAb 174 (n=6) no patients showed generalized
HAMA while l/6 did seroconvert in an idiotype specific manner.
While the idiotypic-specific HAMA was less pronounced than for
_ Example I, this may well be attribu~able to the lower dosage
employed. In any event, the isocypic HAMA response was
eliminated, while at least some idiotypic H~MA response was
retained.
SU~SlT~TE SHEET
WO 93/18792 PCT/CA93/001 10
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SU~ITUTE SHEET
WO 93tl8792 .~ 2 ~ PcTtcAg3tnol l0
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SU~T~JTE SHEET
W093/1879,~ ?~ 22 PCT/CA93/00110
Table 3: HAMA status and Sur~ival times of ovarian cancer
patients injected with OC-1~5 and B43.13
Stage Number HAMA Survlval
or of MAb Idiotype Time
Patent #Cancer InjectionsA Positiv~ (Months) B
l IV 2 YES 43+
2 III 2 YES 27+
3 I/II 2 YES 41+
4 , I/II 5 YES 27+
I/II 2 YES 441
6 III 2 YES 45+
7 IV 2 YES l4
8 I/II 2 YES 52+
9 III 2 YES 16+
III 2 YES 35+
A All patients were injected with 1 mg of MAb OC 125
F(ab')2 per dose. Patients marked with a also received 2 mg
of MAb B43, reduced, unfragmented antibody.
B Patients are listed with a + i~ they are ongoing in
the study. Patients listed without a + are deceased.
SUeSmUTE SHEE~T
w093/tx792 ~ PCT~CA93/001l0
23
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WO93/l8792 PCT/CA93J00110
.A ' 26
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~ -`
~ ~- : SUeSrll~ SHEET
