Note: Descriptions are shown in the official language in which they were submitted.
W092/~053 PCT/US9t/06~9~
2~ 9 ~7
HOMOCONJUGATED IMMUNOçLOBULINS
S
~elA~e~ A~Lis~ion
The present application i~ a continuation-in-
p~rt.of Ser. No. o7/575~725! f~led August 31, 1990.
Backqrou~d Of The Invention
Monoclonal antibo~ies offer great promise as
exquisitely specific im~unotherapeutic agents with
potentially minimal side effects. Thus, monoclonal
antibodies are being developed for 8 wide variety of
applications, ~uch as t~e treatment of tumors, infectious
diseases ~nd autoimmune disord~rs, regulation of the
immune system, and other~. ~nfortunately, few ~onoclonal
antibodies have the gualities that enable them to
successfully make the transition from research ~nd
development t~ c~inical regimen.
The therapeutic or diagnostic usefulness of a
monoclonal antibody results from several factors, in
addition t~ s~mply b~nding the desired antigen. The
antibody must possess su~ficient bindi~g affinity, a
~easure of the inherent ~trength o~ the antibody binding
to its corresponding epitope. It ~ust also have a
relatively high level of avidity, which reflects the
overall stability of t~e antibody-antigen compl~x and is
based on the valency of the antibody (and antigen) ~nd
the geometric arrangement of the interact~ng components.
The af~inity and avidity o~ different antibodie~ can vary
widely.
,
.. ;
.
. .
W092/~0~3 PCT/US91/06195
- ~,Q~,~3~ ;` 2
Often the monoclonal antibody w~ic~ is ~elected
must be of an appropriate isotype or ~ubclass thereof to
efficiently initiate desired effector function~. These
functions include fixation of comple~ent, bin~ing to
effector ~acrop~age~ or poly~orphonucle~r leukocytes, or
other propertie~ that ~ay be required in a~ particular
therapeutic application. Isotype slso af~ect~ ~ntibody
bio-dlstribution, half-life, tran~placental pas6age, and
other characteristics.
In general, IgG antibodles would be pre~err~d
over IgM antibodies for most therapeutic uses. When
compared to IgMs, IgGs typically possess longer in ~ivo
half-lives, are able to cro~6 the placenta to the fetus,
and when formulated as ~ pharmaceutical composit~on may
have a longer shelf life. IgG molecules are monomeric,
however, and have only two antigen binding sites s~ the
avidity is much lower than with a comparable IgM
antibody, which is pentavalent and has ten antigen
binding sites.
With conventi~nal technology lt is frequently
very difficult t~ identify monoclonal antibodies ha~ing
the desired 2ntigen bindinq 6pecificity,-affinity,
avidity and effector ~unctions. Recombinant DNA
techniques have been developed to avoid the unpredictable
and labor intensive method of 6imply ~creening large
numbers of antibody-producing fused or transformed cells.
Genes encoding the ant~gen binding ~ariabl~ lor
hypervariable) regions of an antibody havlng a desired
binding specificity have been cloned next to genes
encodinq antib~dy constant regions which mediate desired
effector functions. See, for example, U.S. Pa
4,816,397, European Patent Ofice publications ~P 173,494
and 239,400 and PCT publication WO S9/07142. Such
procedures can also be quite laborious and have had only
limited experimental validation. Even with t~ese
procedures one may still be faced wlth a reco~binant Ig~
antibody not having sufficient avidity to initiate
.
:' ' -
W092/04053 , PCT/US91/06~
~;`. 209~17 -
. 3
bioloqically important effector ~unction6, or with IgM
molecules which have ~ de~red therapeutic activity but
suffer ~rom the general di6advantages as~ociated with
IqM~ ~a ~e~tlone~ above.
The avidity of IgG ~ntibodies could be i~proved
by increasing the v~lency of the ~olecule to greater than
two. More interaction~ betw~en ~ntibody ~Ind antigen
would result in tighter binding and would ~tabilize ~he
antibody-antigen interaction, generally an important
attribute for therapeutic use. IgG antibodie~ of high
avi~ity ~via multivalent attachment) and which have the
desired effector functions would be greatly preferred
over comparable antibodies of low avidity, but to date
antibodies having these characteristics have not been
described.
Accordingly, what is needed in the art is a
means for producing high avidity IgG antibodies hav~ng
desired effector functions whlle avoiding many of the
difficulties inherent in working with IgHs. Qulte
remarkably, the present invention fulfills this ~nd other
related needs.
~5
summarv of the Invention
Ho~ocon~ugated antibodies possess increased
therapeutic effectiveness when compared to the
corresponding pa~ental antibody monomer. This activity
may be due to, inte~ alia, interactions of ~igher a~idity
and increased effector functions. Accordingly,
antibodies which bind to the ~a~e antiqen, and more
particularly to the same antigenic determinant, are
covalently bonded via cross-linking to one another by
synthetic chemical coupling to produce such
homoconjugates. Generally, t~e homoconjugates comprise
:
., ~ .
': ~
W092/04053 ~ PCT/VS91/06195 : ,
~ 4 ~
at least two to three ~ntibody ~olecules, typlcally of
the IgG class. ~he ant~bodies are preferably ~onoclonal
antibodies, and ~ay be any of a var~ety of ~pecies. For
administration to hu~an~ the ~ntlbodie~ will usually ~e
human or murin~ in orlgin or hav~ human const~nt regions.
~ccordingly, pharmaceuticnl co~po~ltions ~re
provided which comprise a pharmaceutically ~ccep~able
carrier and at lea~t two IgG antibody ~oleculesO whlch
bind to substankially the ~ame ~ntigenic determinant,
c~emically bonded to one another by 6ynthetic covalent
linkage. The homocon~ugated antibodies and
pharmaceutical compositions thereof can be used
therapeutically in methods of treatment of antigen
related diseases to, e,g., protect aga~n~t lnfection,
such as by ~ li or group ~ streptococci, in~ibit the
growth of tumors, including breast an~ other tumors,
regulate the immune response, and the like. A~
homoconjugates of IgG antibod~es 2re able to p2SS the
placenta the preparations can be used to treat n fetus in
utero.
In another related ~spect the invent~on
provides a ~ubstantial i~provement in met~od~ for
therapeutic administration of ~onoclonal antibo~ies to 2
patient for treatment of an ant~gen related disease. The
improvement compri es administering to the patient
co~alently cross-linked homoconjugated monoclonal
antibodies having at least two IgG ant~body ~olecules
which bind to the 6ame antigenic determinant of the
antigen related to the disease. In preferred e~bodiments
the antibodies are cross-linked via disulf~de bonds.
... .. .
W092/~0~3 PCT/US91tO6D9
~ 5 2090317
~çg De~ç~iP~lon Q~-~hç-~is~L~es
Fig. 1 show~ chromatogram~ of FPLC profiles of
the IgG ho~ocon~ugate mixtures, with retention ti~e alonq
x-~xis ~nd A280 along y-axi~; Peaks labell~d ~, B ~d C,
represent tri~er, dlmer snd ~ono~er fractlon~,
r~spectively;
Fig. 2 illu~trate~ ~e increa~ed binding
activity ~n EIA~ o~ ho~ocon~ugates (dimer3 or tri~er~)
compared to ~nltial ~onomer~ of monoclonal antibody D31 a
~uman IgG monoclonal ~nt$body which binds to the group
carbohydrate of group ~ streptococci;
Fig. 3 illustrates the increased binding
activity of homoconjugates tdi~ers or trimer~ co~pared
to initial monomers of ~onoclonal antibody 5El-G, a human
IgG monoclonal antibody which binds to the capsular
carbohydrate of ~. Ç~li Kl;
Fig. 4 ~llustrates the increased binding
activity in EIAs of homoconjugates (d~mers) compared to
initial monomers of B~64, a ~urine IgG ~onoclonal
antibody which binds to a human breast tumor ~s~ciated
antigen;
Fig. 5 illustrate6 the comparative binding
actiYity of homocon~ugated chimeric BR96 ant~body.against
tu~or cell lines, where Fig. 5A shows binding activity
against human breast tumor cell llne H3760B, Fig. 5B
shows binding activlty against human lung tumor cell line
H2707, Fig. 5C ~hows binding activ~ty ~gainst human lung
tumor cell line H2987, ~nd Fig. 5D is binding activity
against human breast tumor cell lin~ H3396;
Fig. 6 hows the lncreased opsonic activity
against group B ~treptococci by di~er and trimer
homoconjugates of human monoclonal antibody D3 compared
to the initial IgG monomer;
Fig. 7 ~hows t~e enhanced opsonophagocy~osis by
monoclonal antibody D3 homoconjugates against group B
.
.
' :: , : , : .'
: . ~ . :
W092/04053 PCT/~S91/06~95
J 6
~streptococcal ~traln~ M94 ~nd I334 compared to the
activity by the D3 ~onomer antlbody:
Fi~. 8 çhow~ the lncreased opsonic ~ctlvity
against ~. ~Qll K1 of di~er ~nd trimer ho~ocon~ugates of
human ~onoclonal anti~o~ 5El-G compared to the in1tial
IgG monomer;
Fig. 9 depicts enhanced opsonophla~ocyto6i~
conferred by homocon~ugates of monoclonal iantibody 5El-G
againet ~train6 H16 and Al~ of ~ Ç~l Rl compared to the
ant~body monomer;
F~g. 10 ~hows increased co~plement dependent
cytotoxicity against breast tumor cell line H3630 by
dimer homoconjugates of monoclonal antibody BR64 compared
to the initial IgG ~onomer;
Fig. 11 illustrates the cytotoxicity shown by
BR96 homoconjugates and monomeric ~onoclonal antibody
against breast tumor cell l~ne H3396; and
Fig. 12 shows the ~a vivo protection conferred
by homoconjugates of monoclonal antibody D3 and control
monomer at different concentrations of antibody.
DescriPtion of the-Specific Embodimen~
The present in~ention provides homoconjugates
of monoclonal antibodies again6t selected antigens, and
methods for preparing 6uch homocon~ugates. By chemically
linking antibody molecules, ho~ocon~ugates are prepared
which possess increased ~alency and two or more Fc
regi~ns. By this means a Yar~ety of e~fects ~ay be
acco~plished, including, inter ~ , increase~ in binding
avidity, complement fixation, cellul~r 2ctivation,
opsonophagocytosis, etc. Thus the invention provides the
ability to convert antibodies o~ perhaps llmited in vivo
utility to antibodies having characteristics
significantly more conducive to a desired therapeutic
, , ' ,
W092/~0s3 PCT/US9~1~6~9~
2~90~:~ 7
activity. For example, homocon~ugation may BerYe to
convert an IgG monomer of low bind~n~ ~vid:Lty to one of
higher avidity and better able to promote effector
func~ions that were perhaps not prev~ously attainable,
By homocon~ugate is ~eant the co~valent
association or linking of two, three or ~ore antibody
molecules which bind to the ~ame antigenic determlnant,
thereby form~ng nntibody ho~odimers, homotrlmers, etc.
The homocon~ugate~ ~ay be prepared from two, three or .
~ore dif~erent ~onoclonal ~ntibodies (1.e., t~o~e
produced by different i~mortalized cell lines) ~hich bind
to the same antigenic determinants (epitopes) on t~e
antigen. The monoclonal antibodie6 w~ich comprise the
homoconjugate may be different (produced by di~tinct cell
lS lines) but preferably they are the same, l.e., obtained
from the same cell line, and thus constitute a relatively
homogeneous preparation of monoclonal nnt~bodles with
virtually identical antigen binding 6pecificity. By
binding to the ~ame or sub~tantially the 6~e epitope is
meant to re~er to monoclonal ~ntibo~ie~ wh~ch are capable
of reciprocal or non-reciprocal competition with the
other for binding to the antigen. One ~lled in the ~rt
will know how to conduct competition l~munoassays, ~uch
as by radioi~munoassay or enzyme immunoassay, as
generally described in, e.g., U.S. Pat. 3,817,837; Harlow
and Lane, ~ntibodiesl A aboratorY ~anual, Cold Spring
Harbor Press, Cold Spr~ng Harbor, NY (1988); ~d Day,
Advanced ImmunochemistrY~ 2d ed., Wiley-Llss
Publications, NY (1990), each incorporated herein by
reference.
The Fc regions of the monoclonals used or
homoconjugation, or other a~pect6 o~ the i~unoglobulin
~olecule which do not ~ub~tantially affect antigen
binding ~pecificity, ~ay also be ~ltered to produce
3~ desired e~fector function~. For example, it may be
~esirable to ~ubstltute a Fc domain for protein A binding
into a molecule not having tha~ capacity, for ease o~
:. .: .: . . . .
., .. .: .
. . ~ . ,
.
.~ .
.
' . . . ~ .
W092/~053 PCT/US91/06195 '
'' ~' , ' '' ~. `
pur ~ cati~ or tbe like. Other subst~tut~ons may
provide for decreased immunogenicity, increa6ed or
decreased co~plement activation, cell receptor blnding,
control o~ catabolic rate, placental and ~It trans~2r,
abil$ty to participate in antibody-dependent cQllular
cytotoxicity, and oth~r ~pect~ of lmmune regul~tion.
num~er of ant$body functions have been loclllized to a
constant ragion domain or domains. $çe, Paul,
Fundamental I~munoloqY, Raven Pre~s, New York, NY, 1984,
io incorporated herein ~y reference. A wide varl~ty of
techniques are available to produce recom~nant
immunoglobulins, e.g., U.S. Pat. 4,816,397, Eur~pean
Patent Office publications EP 173,494 and 239,4~0 and PCT
publicatio~ WO 89/07142, each incorporated herein by
reference. Accordingly, the ho~oconjugated
im~unoglobulins may be any of the heavy chains and
subclasses thereof. The light ~hains ~ay be either kappa
or lambda.
Part~cularly preferred in the pre~ent invention
are homocon~ugate~ of antibo~ies hav$ng g~mn hea~y
chains, 50 as to form homoconjugated ~ultiv~lent IgG
~olecules. Withln the IgG subclasses o~ 1, 2, 3 ~nd 4
(human) and 1, 2a, 2b and 3 (~ur$ne), human 6ubclasses 1
and 3 and muri~e ~ubclasses 1, 2a and 2b are generally
preferred for applications requiring ~ax~um comple~ent
fixation, binding to ~onocyte~, macrophages and
polymorphonuclear cel}~, and the ability to cro~6 the
placenta. The effector functions of human IgG2 and IgG~
antibodies may also be substa~ti~lly increased by the
homoconjugation procedures described herein.
It is al~o contemplated that under certain
circumstances, depending on the intended use, antibodies
having alpha, ~u, epsilon or delta type heavy chain~ may
al~o be employed for ho~ocon~ugation as de~cribed ~erein.
The binding a~inlty of the antibodie~ ~or use
in homoconjugates will vary, but will gen~rally ~e at
least 10 4 M, typically at least about 10 6 M to 10 7 M,
... .
,
W092/~053 , PCT/US9~/06~
f ' ~
~i- 9 20903~7
and preferably at least about 10 8 to 10 9 M or greater.
The avidity of the homocon~ugates prepared ~rom ~uch
antibodies 6hould generally be at least about 10 6 M to
10 7 ~, and preferably at lea~t about 10 8 to ~o 1~ M or
greater. Means for determining affinity and ~vidity are
known, as described in Day, ~dv ~e~ I~munQhç~ls5ry~
supra. While t~e homocon~ugate6 ~ay have quantitatlve
~ncreases in avidity, generally the hom w on~ug~tes 6hould
al~o have qualitative lncrea~e~ in avid~ty and effector
functions, e.g., those evidenced by ~ntigen blnding test~
and other functional as~ays as described herein and 2S
will generally be known to one of ordinary skill in the
art.
The homocon~ugated immunoglobulins may be of
lS any species or combination thereof from which monoclonal
antibodies may be prepared. Although ~t has generally
been relatively easy to produce murine monoclonal
antibodies of a desired antigen binding specificity, it
has been much more difficult to produce human ~onoclonal
antibodies of the desired specificity and having t~e
desired constant region properties. ~u~an ~onoclonal
antibodies are preferable for many applic~tions,
especially L~ Xi~Q diagnosis and therapy of humans to
minimize their recognition as foreign by a patient's
im~une system.
While murine and human immunoglobulins are most
commonly produced, ~onoclonal antibodies or portions
thereof originating w~th other species, such as
lagomorpha, bovine, ovine, r~uine, porcine, avian or the
like may be employed. It should be understood that the
monoclonal antibody art and genetic engineering
techniques ha~e advanced 6ufficiently such that antlbody
sequences of one ~pecies ~ay be interchanged with those
of another ~pecies. Thus, as used herein, a ~human~'
antibody, for example, refer~ to one that is
substantiaily human in origin but may also contain 60me
non-human and/or non-immunoglobulin sequence~.
.
.
.
.
~ , : - . ,
,
' : ' ' ' : .
-
Wo92/o4ns3 ~ ~ ~ PCT/US91/06195
Similarly, when referring to immunoglobulln, used
~ynonymously herein with antibody, lt w1ll be understood
that some non-immunoglobulin sequence~ may be present ~n
the molecule while reta~ning the ability to bind ~ntigenO
Immunoglobulin refer~ to b~th whole i~mun~lobulin~ and
binding fragments thereo~.
The antibodies whlch are u6ed fo:r
homoconjugatlon may be ~ubstantially ~onos]peclfic, i.e.,
relatively pure preparations of ~ubstantially homogeneous
antibodie~ obtained from polyclonal antisera, or ~ay b~
monoclonal antlbodies. Monoclonal antibodies ~hich b~nd
to a desired antigen or epitope thereof ~re obtained from
an established cell line which secrete~ them. The
antibody-producing cell line may be isolated from B cells
of several 6pecies using conventional ~usion, viral
transformation or other immortal~zation techniques well
known to those s~illed in the art. For instance, human
monoclonal antibodles may be generated using Epstein-
Barr viru6 (EBV) transformation, hybridoma fusion
techniques, or co~binatlons thereo~. See, for exa~ple,
Rozbor et ~l., Proc ~atl. Acad. Sci~ ~A 79:6651 (1982~,
and U.S. Pat~ Nos. 4,464,465 and ~,624,921, which are
incorporated herein by referenoe. By ~onoclonal antibody
is ~eant an antibody produced by ~ clonal, immortalized
cell line separate from cells producing antibodies with a
different antigen binding specificity. ~hus ~uch
monoclonal antibodies are produced and ~solated from
other monoclonal antibodies and, accordingly, in
substantially pure form (relative to other antibodies)
and at a concentration generally greater t~an normally
occurring in sera from the animal species which serves as
the B cell source.
Thus, it should be understood that the
invention i~ not limited by the antigen binding
specificity of t~e particular homocon~ugates exe~plified
herein, but rather, it can be used in t~e treatment of a
variety of antigen related diseases, particularly tho~e
.. ' ' ~ .
W092/04053 PCT/~S91/~6~
~; 20903~7
11
for which monoclonal antibodies have been therapeutically
administered. By antigen related disea~e is meant ~
disease whose manifestation coincide with the clinical
presence of a foreign antigen ~e.g., bacteria, virus,
tumor or tumor associated antigen) or ~el~E antigen tas
with autoimmune diseases)~ A wide variety of monoclonal
anti~odies have been de3cribed in the technical and
patent literature, ~any of wh~ch ~r~ publicly ~vailable
from cell depositories, such as the ~merican ~ype ~ultur~
Collection, 12301 Parklawn Dr., Parkville, MD 20852,
whose catalogue, ~TCC Catalo~ue of Cell Lines and
Hvbridomas, 6th ed. (1988), is incorpora~ed herein by
reference. Representative examples of monoclonal
antibodies are described in, e.g., U.S~ Pat. Nos.
4,596,769, 4,689,299, 4,753,894, 4,834,975, 4,834,976,
4,925,800, and 4,958,009, each of which i5 incorporated
herein by reference. The methods described herein
provide the ability to produce novel cross-linked
homoconjugates from immunoglobulins obtained from such
cell lines.
The chemically lin~ed homoconjugated
immunoglobulins will be produced by che~i~al con~ugation
of antibodies using well known laboratory procedures,
such as by employing cross-linking reagents. By
chemically linked is meant that the imm~noglobulin
~olecules are synthetically lin~ed, i.e., not produced as
such by a cell, to one another by covalent bonds. A
preferred method of conjugation is the formation of at
least onP covalent bond between the immunoglobulin
molecules.
The immunoglobulin molecules are co~plexed or
chemically bonded toget~er by any of a variety of well
known chemical linking pr~cedures. The Fc regions or Fab
regions may serve as the site of the lin~age. The
linkage may be direct, which includes linkages con aining
a synthetic linking group, or indirect, by which is meant
a link having an intervening moiety, ~uch as a protein or
. .
. . .. . . . .
. ' ' '' ' ~
W092/04053 ~ PCT/US91~0~9S
~ "` 12
peptide, e.g., plasma nlbumin, or other 6pacer molecule.
For example, the linkage ~ay be by way of
heterobifunctional or homobifunctional cross-linkers,
e.g., carbodiimide, glutaraldehyde, N-~uccinimidyl 3 ~2-
` 5 pyridydithio) propion~t~e~(SPDP) and derivatlve~, bi~-
maleimide, 4-(N maleimidomethyl)cyclohexane-l-carboxylate
(SMCC), cross-linking wit~out exogenous cross-linkers by
means of groups reactive with the indlvidual ~olecules,
such a~ carbohydrate, disulfide, carboxyl or amino groups
via oxidation or reduction of ~he native protein, or
treatment with an enzyme or the like. Methods for
chemically cross-linking antibody molecules are generally
known in t~e art, and a number of hetero- and
homobifunctional agent~ are described in, e.g., U.S~ Pat.
Nos. 4,355,023, 4,657,853, 4,676,980, 4,925,921, and
4,970,156, and in Harlow and L2ne, ~ntibodies A
aborator~ Manual, Cold Spring Harbor Press, 1988, Cold
Spring Harbor, NY and ImmunoTechn~loqy Çataloque and
Handbook, Pierce Chemical Co. (l98g), each of which
patents and publications i5 inc~rporated herein by
referenceO In general, such synthetic cross-linking
should not substantially affect the antiqen binding
region of the molecules nor the desired effector
functions.
Detection and purification of the
homoconjugated i~munoglobulins ~ay be accomplished by a
variety of techniques, including liquid and a~finity
chromatography, gradient centrifugation, ~nd gel
electrophoresis, among others. Increased activity of the
homoconjugates may be measured by quantitati~e antigen
binding assays, antibody competition experiments,
opsonophagocytic assays, complement dependent
cytotoxicity assays, and the like. These techniques are
familiar to those skilled in the art, and are described
in, for example, Harlow and LRne, ~uPra.
Xomoconjugated antibody preparations with
increased binding ability will likely be useful in the
W092/~053 PCT/US91/06195
~,
`~ 13 2090317
treatment and diagnosis of a wide variety of conditions
referred to herein as antigen related d~sea~es. ~he
h~moconjugates wlll offer significantly i~proved
therapeutic and diagnostic characteristics compared to
the uncon~ugated mono~eric antibody. Due to the
increased av~dity of the homocon~ugates, it i~ now
possible in certain ~nstances to convert ~ previously
non-protective or weakly protective IgG ~ntibody to be
protecti~e against ~n~ection or tumors, for example, or
to act as an i~munomodulator by potentiat~ng or otherwise
regulating ~ host's immune response to a particular
antigen. Where an IgM antibody to an ant~gen or
particular epitope of the antigen is protective and
monomeric IqG antibody is non-protective or weakly
protective, a homocon~ugate produced using the methods
described herein may provide ~ufficient avidity to confer
significant protection against infection, cell ~illing,
etc. For instance, an IgG di~er or trimer homocon~ugate
may possess therapeutic anti-infective quallties ~hat may
be fou~d with certain multivalent ~ntibodies ~uch as
IgMs, but also have qualities inherent to IgG monomers,
such as their ability to cross the placenta, to bind to
macrophages and PMNs, and the lack o~ ~ requirement for
complement to mediate opsonization. The lgG
homoconjugates may possess other attributes typically
associated with IgGs, such as ease of purif~cation,
lncreased stability, increased shelf life, and 1ncreased
half-life in vivo.
Although the homoconjugate preparations will be
useful against a range of targets, such as bacterial and
Yiral antigens, d~pending of course o~ ~he partlcular
specificity ~f ~ homoconjugate' antigen binding region,
they will be especially usef~l where the kill~ng of
mammalian cells is reguired. For example, the
homoconjugates can be used for t~e treatment of cancer
cells which display particular tumor-associated antigens
(e.g., breast or lung tumor associated antigens), the
.- ,
W092/~053 PCT/US91/06195
f~:~
~9~ 14
in~ bition or killing of mammalian cells infected with
viruses or bacteria or ce~ls wh~ch express antigens
associated with a part~cular autoimmune disease. The
homocon~ugates can ~150 be u~ed to eliminate 6elect~d
c~lls fr~m bone marrow or in the i~mun~upF~res~n o~
qraft reclpients, etc.
Qf course, it i~ under~tood that the present
invention is not l~mited to nntlbody ~omocon~ugates which
are protective or 6how other such functional attributes
0 ~ ViVQ, a6 increased avidity ~lso makes fea~ible an
array of diagnostic procedures perhaps not otherwise
available to a bivalent monomer of low affinity and~or
low avidity.
The ab~lity of the resultant antibodies to
lS inhibit a tumor, such as a breast or lung tumor, to act
as an immunomodulator, or to protect against challenge by
a pathogen, for example, can be measured in a wide
variety of in vitro and ln YiVQ 6ystems, as will be known
to the skilled artisan. An exemplary protocol for
protection against ~ ÇQli Xl, uslng a homocon~ugated
antibody which was non-protective or weakly protective as
an IgG, appears in Example III below.
The novel ho~oconjugates of monoclon~l
. antibodies and pharmaceutical compositions prepared
therefrom are particularly ~seful or administration for
prophylactic and/or therapeutic treatment of an antigen
related disease. Preferably, the pharmaceutical
compositions can be admini~tered parenterally, i.e.,
subcutaneously, intramuscularly or intravenously, or
3~ orally. Thus, this ~vention provldes compositions for
parenteral administration which comprise a solution of
the ~omoconjugated ~onoclonal ~ntibody preparat~ons or a
cocktail of homocon~ugated and m~nomeric antibodies
dissolved in an acceptable carrier, preferably an aqueous
carrier. A variety of aqueous carriers can be used,
e.g., water, buffered water, 0.4~ ~aline, 0.3% glycine
and the like. These compositions may be sterilized by
W092/~053 ., PCT/US91/06195
2~317 ~
convent~onal, well known ~ter~lization technique~. The
compositions may contain phar~aceutically acceptable
aux~ ry ~ubst~nce~ a~ required to approxi~ate
phy6iological condition6 6uch as pH ad~usting and
~uffer~ng agent6, toxicity ad~ustin~ agents ~nd the like,
for example, sodium acstate, ~odlu~llactate, ~odiu~
chloride, potassiu~ chloride, c~lcium chloride, etc. The
concentration ~f antibody ln the5e formulations can vary
widely, ~.e., from less than about 0.5%, usually at or at
}east about 1% to as ~uch ~6 15 or 20S by weight ~nd
will be selected primarily by fluid volumes, viscos~ties,
etc., in accordance with the particular mode o~
administration selected, the condition being treated,
e.g., an infectious disease such as a group B
streptococcal or ~ çoli infection, a tumor, such as
breast carcinoma, etc., and the subject being treated,
i.e., an adult, child or neonate.
Thus, a typical pharmaceutical composition for
intravenous infusion to treat an infection in ~n adult
could be made up to contain 250 ml of 6terile ~nger's
solution, and about 100 ~g to lO gra~s of antibody.
Actual methods for prepari~g parenterally or orally
administrable compounds will be known or apparent to
those ~killed in the art and are described in more detail
in for example, emin~ton's_Phar~aceutiçal S~enç~, 16th
ed., Mac~ Publishing Company, Easton, PA (1982), wh~oh is
incorporated herein by reference.
The co~positions containing the present
homoconjugated antibodies or a cocktail thereof can be
administered for prophylacti~ and/or therapeutic
treatments. In therapeutls applications, compositions
are administered to a patient already suffering from a
disease, in an amount ~ufficient to cure or at leas
partially arrest the disease and its complications. An
amount adequate to accomplish th~s is defined as a
"therapeutically effecti~e dose.~ Amounts effective for
this use will depend on the severity o~ the disease,
~'. ' : . : : '
.
. . .
.: . . . ~. . .
. . .
~ ~ 16 PC1/US91/06195
i.e., infection, tumor, etc., the ~ge of the patient and
the general sta~e of the patlent'~ immun~ s~ystem.
Generally, the amountB will range fr~n about O.l to about
50 mg of antibody per kil~gram of body weis~ht per dose,
S with dosaqes of from S to 25 mg of ~ntibody per kilogram
per patient belng more commonly used. It ~ust ba Xept in
mind that the ~aterlals of the present invent~on may
genera~ly be employed in serious di~ease ~tates, that l~
lifç-threatening or potentially life threatening
situations. In ~uch cases, ~n view of the minimization
of extraneous substances and the possibility of lower
"foreign substance" rejections which may be achieved by,
e.g., administering allogeneic homoconjugated antibodies
or chimeric homoconjugated antibodies ~ade feasible by
lS this invention, it is possible and may be felt desirable
by the treating physician to administer substanti~l
excesses of these ant~bodies.
In prophylactic applications, compositions
containing the present antibodies or cocktails thereo~
are administered to a patient not already in a dis~ase
state to enhance the patient' 6 resistance. Such an
amount is defined to be a "prophylactically effective
d~se." In this use, the precise amounts again depend on
the patient's state of health and general leYel of
immunity, but generally range from O.l to 25 mg per
kilogram, especially 0.5 to 2.5 ~g per kilogra~. A
preferred prophylactic use is for treatment of fetuses
and neonates at risk from infection through their
mothers. When treatment is dependent on passage through
the placenta, the dosage ~ay require adjustment to
reflect the percentage of antibody which is able to pass
from the blood of tbe pregnant female to t~at of the
fetus.
Single or multipl0 administrations of the
compositions can be carried out with dose levels and
pattern being selected by the treatiny physician~ In any
event, the pharmaceutical formulations should provide a
WOg2/040~3 ~ PCT~US91/~6195
2~903~7
17
quantity of homocon~ugated antibody sufficient to treat
the patient.
The homocon~ugated antlbodies of the invent~on
~ay also find several uses ~n Vit~o. By way of example,
the homocon;ugated IgG antibodies o~ ~xa~ple I below can
be used for detscting the presence of group B
~treptococci or ~. çQll X1, for vaccine preparation, or
the like.
For in vitro diagnostic purposes, the
antibodies may be e~ther labeled or unlabeled. Unlabeled
homoconjugated ant$bodies may find particular use in
agglutination assays, or they ~ay be used in combination
with other labeled antibodies (~econd antibodies) that
are reactive with the homoconjugated ~ntibodies, such as
antibodies specific for the Fc regions. Alternatively,
the antibody may be directly labeled. A wide variety of
labels may be employed, such as rad~onuclides, particles
~e.g. gold, ferritin, magnetic particles, red blood
cells), fluors, enzymes, enzyme ~ubstrates, enzyme
cofactors, enzyme inhibitors, ligands (particularly
haptens), etc. Numerous types of i~munoassays are
available and are known to those ~killed ~ the art, such
as competitive and sandwich assays as Bescribed in, e.gO,
U.5. Pat. 4,376,110, incorporated by reference herein,
and Harlow and Lane, supra.
Kits can also be supplied for use with the
subject antibodies in the protection against or detection
of the presence of a selected ant~gen. Thus, the subject
antibody compositions of the present invention may be
provided, usually in lyophilized form in a container,
either alone or in conjunction with additional
antibodies. The antibodies, which ~ay be conjugated to a
label or toxin, or uncon~ugated, are included in the kits
with buffers, such as Tris, phosphate, carbonate, etc.,
stabilizers, biocides, inert proteins, e.g., ~erum
albumin, or the like, and a ~et of instructions ~or use.
Generally, these ~aterials will be present in less than
. .
.
,
W092/04053 ~ PCT/US91/0619
18
about 5S wt. based on the amount of active antibody, and
usually present in total amount of at least about .OOOl~
wt., based on the antibody concentration. Frequently it
will be desirable to include an inert extender or
excipient to dilut~ the active $ngredient;s, where the
excipient ~ay be present ln ~rom about l to 99S of the
total composition. Where a second antib~ly capable of
binding to the homoconjugated antlbodies ~ employed in
an assay, this will be pre~ent in a ~eparate ~ial. ~he
second antibody is typically conjugated to a label and
formulated in an analogou~ manner with the antibody
formulations described above.
The following examples are offered by way of
illustration, not by li~itation.
~XAMP~E I
PreDaration of ~onoclonal AntibodY Hom~coniuaates
This example demonstrates means for preparing
homoconjugates of several representative ~onoclonal
antibodies to selected tumor and bacterial antigens. The
homoconjugates were then tested in functional assays
described in the examples which follow.
Homoconjugates of the Pollowing monoclonal
antibodies were prepared: Monoclonal antibody D3, a
human IgGl antibody w~ich bind~ to the group B
carbohydrate o~ qroup ~ streptococci. 5El-G, a human IgG
monoclsnal antibody which binds to the capsular
carbohydrate of ~. coli Xl. BR64, a murine IgGl
~onoclonal antibody which binds to human carcinoma
associated antigen, including colon, breast, ovary and
lung carcinomas. BR64 is on deposit with the ~merican
Type Culture Collection, 1230l Parklawn Drive, Rockville,
M.D, as ATCC No. HB 9895. And BR96, also on deposit with
the American Type Culture Collecti~n as ATCC No. HB
W092/~053 . PCT/US9l/06195
19 209031 7
10036, is an IgG ~uman-murlne chlmeric IgG ~onoclonal
antibody which bind~ to ~uman lung and brea~t tumor
associated antigens.
Homocon~ugate~ of each of the Antibodies were
prepared uslng malei~idobutyrloxysucc~n~lde and
iminothiolane ~ccording to the following protocol.
Antibodies (1 ~g/ml) were dialyzed overnight against a
coupling buffer ~0.1M Na2HPO4-dibasic, ~even-hydrate,
0.1M.NaCl, pH 7.5). One m~llillter of antibody was
thiolated wit~ 2-iminoth~ol~ne-HCl (Pierce Chemical Co.,
50 ~1 (0.5 m~) of 2-iminoth~olane solution (10 ~gJml in
coupling buffer) added while mixing. A ~econd aliguot of
the antibody (1 ml) was treated with N-~-
maleimidobutyryloxy-succinimide (GM~S)(Calbiochem, La
Jolla, CA), 5 ~1 (14 ~g) of ~MBS solution (1 mg in 360 ~1
dimethylformamide (DMF). Each treated aliquot of
antibody was incubated 1 hr. at room temp. and then the
antibodies were run over PD-10 columns ~Phar~acia) pre-
equilibrated in coupling buffer. After a void volume of
2.6 ml total, antibodies were collected in double the
original Yolu~e. The thiolated and GMBS-treated al~uo~s .
of antibodies were then mixed and incubated at roo~ temp.
for 5 hrs. The reactions were quenched by adding 1 ~1 of
25mM ~-mercaptoethanol (1 ~1 in 560 ~1 coupling buffer)
and incubating for 15 min. at room temp. Tb~ ~-ME was
stopped ~y adding 11 ~ g) ~; ethyl~aleimide tsigma
Chemical ~o., St. L4ui6) made up to 1 ~g/ml in DMF. The
homoconjugate preparations were dialyzed overn~ght in
phosphate buffered 6aline (PBS) and ~eparated ~y size- -
exclusion chromatography using Superose-6 and S~perose-
12 FPLC columns (Pharmacia, Uppsala, Sweden). The
chromatograms of the FPLC columns for monoclonal
antibodies D3, 5El-G, and BR64 are shown in Fig. 1.
.
.
.. ..
' ,' . -. ' ': ' ' : .
W092/~4~53 PCT/VS91/~619~ ~
~9~ ` 20
EXAMPhE II
Bindinq Açtivl~v of ~omo~oni w a~ç~
i . .
The ab~lity o~ the tetravalent and hexavalent
monoclonal antibody homocon~ugates to bind antiqen was
compared to the binding ~ctivity of the bivalent IqG
~onomer antibodies. The binding of the anti-GBS
homoconjugates was measured against ~ GBS ~train (I334)
bound to microtiter wells u~ing poly-L-lysine (PLL).
Equivalent protein concentration~ of untreated antlbody
~3 monomer were co~pared to FPLC fractionated IgG dimer
and trimer homoconjugates. Binding was ~ssayed with
biotin labeled anti-hu~an gamma-chain ~pecific
antibodies. The results are ~hown in Fig. 2, where the
relative binding activities of the di~er or trimer
homoconjugate preparations were ~ignlficantly greater
than the initial IgG monomer.
To measure the binding of the ant~-E, çol ;L Xl
antibody homocon~ugates, ~ coli strain Hl6 was bound to
microtiter wells using poly-L-lysine. Untreated antibody
5~1-G was compared to homoconjugates of IgG dimer and
tri~er, prepared as described abo~e. Equivalent pr~tein
concentrations of antibodies were reacted with the E.
coli. Binding was assayed with b~otin labeled anti~huma~
gamma-ch~in ~pecific antibodies. The results are shown
in Fig. 3, where the relative binding activities of the
dimer and trimer homoconjugate preparations were
significantly greater than the in~tial IgG monomer.
To ~easure the binding of t~e ant~breast tumor
antibody, BR64, and homoconjugates thereof, a breast
tumor cell line, 3396, was grown adherently to micro~iter
wells. Untreated antibody ~R64 was compared to
homoconjugates of IgG dimer and trimer. Equivalen~
protein concentrations of antlbodies were reacted with
the breast tumor cells. Binding was assayed wit~ biotin
labeled anti-murine gamma-chain specific antibodies. The
'
.
. ~ . -
W092/~53 . PCT/US9~tO619~ ~
~.Y.~ 21 209~3 ~ 7
results are ~hown in Flg. 4, where the relative binding
act~vit~es of the dimer and trlmer ~omocon~ugate
preparations were ~lgnif~cantly greater t~lan the in~tial
IgG monomer.
~o ~eaEure the binding of the anti-breast and
lun~ tumor antibody, human-mouse ch~meric BR96, and
~omoconjugates thereo~, two breast cell lines ~3396 and
~3760B) and two lung cell lines (H2987 anld H2707~ were
~sed as target~. Freshly trypsinized cells were attached
to microt~ter plates using PLL and t~e ELISAs performed
as follows. 2LL, made up at 1 ~g/ml in PBS, was adsorbed
to Immulon 96-well microtiter plates by incubating 75
~l/well of the PLL ~olution for l hour at room temp.
Carcinoma cell lines (cultured in IMDM with 15% FCS) were
trypsinized, washed twice, and resuspended in PBS at 2 x
105 cells/ml. The PLL treated ELISA plates were washed 3
times with saline/Tween (all wasb ~teps done with ~
gravlty flow wash system). The cell suspension wac added
at lOO ~l/well ~about 20,000 cells/well) and incubated
for l hr at 37-C. The plates were then was~ed 3 ti~es
with sa}ine/Tween. Antibodies were diluted in ~pecimen
diluent (5S nonfat dry milk, lOO ~l/L Foam A, O.Ol~ w/v
thimerosal in PBS) then added to the ELISA plates (lOO
~l/well) and incubated for 1 hr at room temp. Following
incubation, the plates were washed 3 ~imes with
saline/tween, and peroxidase-conjugated goat anti-hu~an
or ~ouse IgG (Tago) dil~ted in specimen diluent was used
as a second step reagent, ~lOO ~l/well) and incubated for
l hr at room temp. The plates were then washed 5 times
with saline/Tween, and tetra~ethylbenzidine (T~B)
chromogen (IMB), diluted l:lOO in buffered substrate, was
added ~lOO ~l/well), and plates incubated ~or 20 ~inutes.
The reactions wera ~t~pped with lOO ~l/well o~ 3N ~2S04
and the plates read at dual wavelength, 450/63Dnm.
~ntreated ~onoclonal antibody BR96 was compared
to homocon~ugated BR96 IgG dimers using approximately
equivalent proteln concentrations of antibody. The
.. . . .
,: , ,
. .
W092/040~3 9~3~ PCT/US91/06195
22 ~-~
results, ~own in Flg. 5A-D for each of the tested tumor
cell l~nes, indicate that the relatlve binding actlvity
against the four tumor cell lines by the predominantly
dimer homoconjugate preparation was greater than by the
initial IgG BR96 monomer.
O EXAMPLE I I I
Increased In Vi~ro-~ctivity of ~Dmoco~i~q~tes
As an indication of 1~ vivo effectiveness, the
monoclonal antibody h~moconjugate~ to GBS were tested in
an in vitro opsonophagocytic assay. Ho~oconjugates to E.
coli Xl were tested for functional activity in two types
of opsonization assays described below. ~omoco~jugates
of BR64 were tested for in vi~Q function in a complement
dependent cytotoxicity assay, and homocon~ugates of BR96
were tested in a co~plement independent cytotoxicity
assay.
Opsonization of GBS b Homocon~uqates o~ P3
The opsonophagocytic assays for GBS were
performed as follows. Bacteria were prepared by
inoculating 10 ml of tryptic ~oy broth (TSB) with 50 ~1
of an overnight broth culture. The tubes were incubated
at 37-C on a shaker for 3 hours at which ti~e 1.5 ml of
the culture was centrifuged for 1 nin. at 10,000 x g, the
spent culture ~edia discarded, and the pellet was
suspended in 3.5 ml of ~ank's balanced salt olution
containing 0.1~ gelatin and 5 mM HEPES (HBSS/Gel). ~he
bacterial concentrations were adjusted to about 3 x 104
bacteria/~l by neasurl~g the O.D.60~ and making the
appropriate dilutions ~approximately 1:50,000). Human
neutrophils were isola~ed according to van Furth and Van
Zwet ("In Vitro Determination of Phagocytosis and
W092/~053 ~ PCT/US91/06195
~ ~, . . .
23 2~9~317
Intracellular ~ ng by Polymorphonuclear and
Mononuclear Phagocytes, n in Hand~ook of E~perimçn~al
ImmunolQ~v, Vol. 2, D.M. Weir, ed., 2nd edition, .
~lackwell Scientific Publications, Oxford, 36.1-36.24
(19~3)) with ~everal ~odifications. Bufy co~t from 5 ml
of heparinized blood d~luted 1:2 wlth PBS wa~ underlayed
with Lymphocyte Separation Medium and centrifuged. The
red blood cell (R~C) pellet was was~ed once w~th ~PMI
1640 medium and resuspended in an egual volume of 37~C
PBS. Twenty-five ml of thl6 suspension was added to 25
ml of 2S dextran (in 37~C PBS) a~d the contents gently
but thoroughly mixed end over end. After a 20 min.
incubation at 37'C to allow the RBC's to 6ediment, the
supernatant (containing neutrophils) was removed, washed
twice in 4~C PBS, once in HBSS/Gel, ~nd ~u pended in same
to 5x107 neutrophils/ml. For the complement 60ur~e used
with GBS, human serum was thrice adsorbed with live
~acteria (Bjornson, A.B. and Michael, J.G., J. Inf, pis.,
130 Suppl:S119-S126 (1974)) corresponding to the
organisms used in the assay.
For the assay, into 1.5 ml sterile
polypropylene microfuge tubes were added_250 ~1 antibody
(test ~omoconjugate~ or ~onomer) preparation in 10~ ~etal
calf serum in HBSS/gel with IIEPES and 100 ~l bacterial
suspension (about 3 x 104 bacteria/~l). After 30 minutes
~t 37~C, 150 ~1 containing 75 ~1 complement, 50 ~1
neutrophils (5 x 107 ml), ~nd 25 yl HBSS~gel were added.
The mixtures were incubated on a rotator for 60 minutes
at 37-C, after which they were placed ~nto an ice water
slurry. After 10 ~inutes, 20 ~1 from each tube was added
to a 100 ~m petri dish containing 3 ml of ~olidified 0.5%
tryptic ~oy broth agarose, followed by ~ncubation ~t
37-C. After 18 hours the colonies were ~numerated and
the data was reported as colony forming units (CFU) for
each condition.
The results for homoconjugates oî D3 are shown
in Fig. 6, where the dimer and trimer required much less
,' ' .
. . . .
W092/~0s3 ~ PCT/US9l/06195
antibody, on a nan~gra~ protein ba~i~, to opsonize the
GBS strain tested w~en compared to the initial IgG
monomer.
AB a furt~er lndication Of ~n Y~YQ .
effectivene~, homoconjugate~ prepared with ~n addition~l
monoclonal ~nt~bo~y to the group ~ c~rbohyclr~te of GBS
(D3, produced aB generally described in Rai'~ ~t ~1., 2.
nfect. Dl~. ~63:346 (1991) ~nd PCT patent publlcation W0
91/06305, each of whioh iB incorporated herei~ by
refer~nce) were tested in in ~1~EQ opsonophagocytic
assays against two GBS strain~, M94 and I334. The
results of the assays are 6hown in Fig. 7, where it ~s
evident that the antl-GBS D3 ~omocon~ugates resulted in
increased opsonization of the GBS human clinical
isolates. Again, these result~ sugge~t that the
homoconjugates w~ll significantly increase the ~n vivo
protective act~vities of the antlbod~es when compared to
the parental IgG monomeric monoclonal antibodies.
oPsonization o~ Ql i XL bY 5El~Ç ~om~ç~n~uqates
To isolate ~u~an neutroph~ls, heparinized human
blood (5 ml) was layered onto 3.0 ml of ~ono-Poly
Resolving Medium (MPRM, Flow Labs) in polystyrene tubes
and centrifuged for 30 minutes at 300 x y at room temp.
After centrifugation, three cell layers ~ere evident,
wit~ t~e middle layer containing neutrophils. The serum
and top cell layer were removed a~ discarded, the
neutrophils collected and added to a 50 ~1 tube
containing pre-warmed PBS. The n~utrophils were
centrifuged for 10 minutes at 300 x g ~t room tempO, ~he
supernatant discarded and the cell pellet resuspended
with 10 ~1 tissue culture media ~RPMI-1640) containing
0.5S gelatin, and the cell concentration adjusted t~ 5 x
106 cells/ml.
The assays were performed as follows. To
luminometer tubes (LKB Nuclear) were added 100 ~1
W092/~0~3 P~T/US91/06195
` 25 209~3~7
containing appropriate test (SEl-G) or control IgG
monoclonal antibody ~onomer to ~ ae~uq~nos~ ~lagella,
100 ~l log phase growth bacterial suspens~on COD660
-0.02), and lO0 ~l diluted bacteria-adsorbed Auman serum
co~plement, final concentration 3.3~. Th~ complement was
thawed just prior to use and rece~ved 5 ~1 of 2 M
CaCl2~l. The tubes were plnced ~nto a prewarmed LKB
Luminometer whic~ allows 24 tubes to be ~ on a
continuous reading cycle. After 30 ~inut~s in which the
tubes were warmed and periodically ~ixed, lO0 ~l of
neutrophils (5 x l06/ml) and 600 ~l of lO 4 ~ Luminol in
Hank's Balanced Salt Solution were added. Counting
sessions for 25 continuous cycles, which corresponded to
-80 minutes for 24 sample tubes, were ~nitiated. The
15 chemiluminescence intensity was displayed as millivolts
(mV) with
mV va~ues f~r nlbes con~aining the test anobody
signal:noise ~
~v~rage of n~bes containing negadve annbody,
The results of the assays are shown in ~ig. 8,
where ~t is evident that t~e ~o~ocon~ugates re~ulted in
increased opsonization of t~e ~ ~Qli organis~s than the
initial IgG monomers. The homodimer and homotrimer of
5~1-G were significantly more opsonic than the 5El-G IgG
monomeric form. As the opsonophagocytic assays are
typically predictive of in Yi~ ability to protect
ani~a~ls (see, e.g., U.S. Pat~ No. 4,970,070, incorporated
herein by reference), these result~ suggest that the
dimer and trimer homoconjugates will significantly
increase the in ViVQ pro~ective activities of the
antibodies when compared to the parental IgG monomeric
antibody.
As a ~urther confirmation of in vitro efficacy,
3~ and thus ~n ~ivo act~vity, the ~onoclonal antibody
homocon~ugates to ~. çoli Kl were tested ~n in vltro
opsonophagocytic assays, as described above, against two
. .
:,
'. ~ ' " ` .
.
`
W092/~0~3 PCTtUS91/061g5
9~3~ 26
additional ~. çQll Kl strains, H16 and A14. As shown in
Fig. 9, the anti-~. ÇQll Xl homoconjugate~ refiulted in
increased opsonization of the human clinical l~olates,
suggesting that the predominantly dimer ho~oconjugate
preparations wlll signiflcantly increase t'he ln YiYQ
protective ~ctivitles of anti-El Ql1 Xl ~onoclonal
antibodies.
~om~lement pependent ÇYtotoxic~tv BR64 Ho~oc~ntuqa~es
In vitro functional assays were also used to
demonstrate the increased functional activity o the
anti-tu~or antigen homocon~ugated monoclonal antibodies.
For testing ~R64 homocon~ugates, target tumor cells
(H3630) were labeled with 51Cr by incubat~on 1 x 106
cells/0.3 ml tissue culture media in 100 ~Ci of 51Cr for
1 hour at 37C, 6S CO2. After.washing to remove excess
5lcr, 2 x 104 ~abeled cell~ ln 67 ~1 medi~ (RPMI-1640
plus lS% fetal bovine serum) were added per microtiter
plate well. Next, 67 ~1 of the appropriately diluted
test monomer (BR64), a negative control monomer (Mab
96.5), or homoconjugated (dimer) ~onoclo~al antibody was
added to duplicate we~ls. Finally, 67 ~1 of freshly
thawed human serum complement was added to each well, the
plates covPred with parafilm and incubated at 37'C for 4
hours. After incubation, plates were centrifuged at 400
g for 10 minutes, ~nd 100 ~1 of supernatant was removed
from each well and placed in 12 x 75 mm polystyrene
tubes. The tubes were counted in a gamma counter. The
following controls were included in each assay:
. ~ :
-
WO9~/04053 PCT/US91/06195
27 2~9031 7
~able I
D$1uted Target
Well Media Seru~ Ant~body Cell
Spontaneous Release 134~ - - 67
Complemen~ Toxicity 67 67 - 67
Total Incorpora~ion 134 - - 67
Maximu~ Release67 - - 67 ~ . -
Antibody Alone 67 - 67 67
- . _
. ~Amounts are expressed as ~l/well.
Prior to incubation of the assay, these are the only
wells which contain less than 201 ~l, because the wells
~0 later receive.67 ~l ~riton X-lO0 to lyse labelled target
cells.
The percentage ~ill (S ~ill) was calculated from the
following formula:
[Test (mean CPM)- Hc' ccn~ol tmearl CPM)]
--------------------------------------- X 100 ~ S kill
Total incorpora~on [mcan CPM Hc' con~ol (mean CPM)~
where CPM is counts per minute ~s average o~ duplicate
samples obtained from measurement in gamma counter and
Hc' is Comple~ent Toxicity control.
The results of the assays ar~ shown in Fig. lO,
where it is evident that the homoconjugated BR64 resulted
in eight times greater killing of t~e targeted tumor
cells than the initial IgG BR64 ~onomer. The CDC assay
is generally predictive of ln vivo abil$ty to protect
animals against tumors. These results suggest that the
homoconjugates will ~ignificantly increase the utility of
such antibodies i~ vivo ~gain~t tumors, particularly when
compared to parental IgG monomer antibodies.
: ;
.. , -
W09t/04053 ~ ~ PCT/US91/06195
28
~ncreased Çomplement IndePenden~ CYtotoxlci~y of Chimeri~
~R96 ~omoçon~uqa~es
~arget tumor cells (H3396) at 5 x 105 cells/tube
s were mixed wlth 100 ~l o~ te~t antlbody and were
incubated ~t 37-C fo.~ 30 ~inUtes. Cells were pelleted
and mixed with the appropriate concentratiLon of propidium
iodide (5igma, 10 ~gttube)- Prop~dium iodlde is ~ DNA
- reactlve ~taln that only penetrate~ the membra~e of dead
or dying cells. Therefore, by quantitating the number of
fluorescent cells w~thin the populatlon, the number of
dead cells can be determined (Hellstrom et ~ anceF
Res., 50:2183-2190 (1990)). After incubation for 1~
minutes, the cells were washed ~n tissue culture media
containing 15% fetal calf ~erum, resuspended in same, and
placed on ice. The cells were analyzed for fluorescence
on an EPICS Fluorescence Activated Cell Sorter which
quantitates live and dead cells on the bas~s of
fluorescence and size (~mall and large represent dead and
live cells, respectively). The resul~s ~Fig. ~1) showed
that the BR96 homoconjugate dimer~ were dramatically ~ore
effecti~e in killing the tu~or cells ~ha~ the initial
monomer. ~hese resu}ts ~uggest that the homocon~ugates
will significantly increase ~he utility of such
antibodies ~n YiVo against tu~or6.
W092/04~53 2 0 9 0 3 PCT/US91/0619
29
EXAMP~E IV
In Vivo Pr~eç~ion ~qainst E. çoli X1 Infection_ln
Nçonat~l R~5 ~sinq IqG Ho~oç~niu~ate~
. Outbred Sprague-Dawley rat pups less than 48
hours old ~housed with their mot~er6) were injected
~ntraperitoneally with approximately 72 ~, Ç~ll Kl
organisms, and 2 hour~ later rece~ved 1 o~e 5 ~g of dimer
homoconjugate~ of 5E1-G, or 100 ~g o~ mono~eric 5E1-G
antibody, or control IgG and IgM antibodies. In all
experiments, the rat pUp5 were examlned da$1y for
symptoms and were ~cored for ~urvival. The results of
the experiments, shown in Table II below, demonstrate
that 5 ~g of the dimer homoconjugates of 5El-G antibody
protected significantly more animals from death when
co~pared to animals receiving twenty times the a~ount
(100 ~g) of monomeric antibody.
Table II
Protection by Homoconjugates Against
E. ~li Xl In~çc~ion~
2 5 Antibody Dose n ~Sur~
(per rat) tper rat) (rats/group) (Sun~ivors/a allenged) p value
5El-lgM 20 ng 26 100% <0.~
5E1-lgG Monomer 100 ~g 15 40% cO.01
5El lg~ Conjugate5 ~,g 14 - 78% <0.01
5El-lgG Conjugate1 ~tg 14 2~ c0.05
21B8 (Negativc control) 100 llg24 0
No antibody control 25 0
Based on survival in experimental group versus
~ urvival in negative oontrol and control~ recei~ing no
antibody.
.... .. . . . . . .. .
, ~ . .
,~ ~ - . . ' . i.
'~" :' ~ ' ' '
W092/04053. PCT/VS91/06195 1 ~
~ 3~ ` 30
EXAMPLE V
TransDlaceT~l Pas6aqe 0~ Homoconiuqated
~ bodv ~o.~ç~use~ Qf Pr~qnant ~Ra~ !
.
The ability of the ~omocon~ugated IgG ~ntibody
to pass through the-placenta and into the fetus, and thus
into the ~ubs~quently del$Yered o~spring, was compared
with the monomeric antibody. An infant rat ~odel was
used as an animal model. Sl~ilar rat model6 have been
used to predict the tra~splacental passage of ant~body
and other molecules to human fetuses. Se~ 9@ne~9llY
Brambell, ~rontiers Biol. 18:234-276 (1970).
Two to three days prior to their anticipated
delivery date, pregnant rats were lnjected intravenously
with 40 ~g of either ~onomeric 5E1-G IgG (~onomar) (Dams
1 and 2) or homoconjugated dimeric IgG (Dams 3 and 4).
Blood samples were collected from the dams two hours
after antibody administration and on the day o~ delivery,
and fro~ the neonatal xats just after birth. Total hu~a~
IgG and ~uman IgG an~ . coli X1 ant~body were
determined in each blood sample ~sing individually
designed quantitative binding assays ~ELISA's). By using
anti-human IgG-specific enzyme-labeled secondary
antibodies, rat IgG was neither detected nor interfered
with the quantitation of the injected human IgG.
The amount of transplacentally passaged
antibody was determined as follow6. Anti-human gamma
chain antibody was attached to microtiter plates using
carbonate buffer. After adding diluted ~erum 6amples
from the dams or pups, binding wa~ assayed with biotin
labeled anti-human gamMa chain-speci~ac a~tibodies.
Sinoe one group of dams received only conjugated
antibody, any hu~an IgG detected in pup sera should be
transplacentally passed homoconjugate.
The experl~ents showed that the monomeric and
homoconjugated IgG antibodies were transplacentally
.. . . . ..
' ' ' ' " ' ~, ' ' '' '' .::
:
.
W092/040s3 PCTtUS91/061~5
31 ~ 3~
pa~sed with approximately equal efficiency. Therefore,
the homocon~ugated IgG monoclonal ~ntibody should be
useful when admlni6tered prophylactically t:o pregnant
female~ at risk of having a neonate with an increased
likel~hood of de~eloping a life-threatenin~ infec~lon,
such as by ~ çQl1 K1 in the case of the present
embodiment. The data al60 ~upport the u~e of these
homoconjugate6 in transplacental treatment of a variety
of other infections and tumors.
EXAMPLE VI
Transplacental Passaqe o~ Homoconi~qated Monoclonal
AntibodY ~o Grou~ B Streptococci
This Exa~ple demonstrates the transplacental
passaye o~ homoconjugated monoclonal ant~bo~y D3.
The experiments were performed ~g generally
described in Example V ~or the homocon~ugated ~onoclonal
antibody to ~ oli Xl.
The result~, ~hown in Table III, below, I
indicated that both the monomeric and homocon~ugated IgG
antibodies were transplacentally passed.
TABLE III: Transplacental Passage o~ Homoconjugated
_ . ~ Anti~odv From Preqnant Rats to TheiF ~eonates
Time Post Antibody Injected
Source Iniection Homocon~uqate Monomer
Dams 2 Hours 1.45+0.4a 2.4+0.3
Dams 3 Day~ 0.14~0.04 0.11+0.03
(Day of Delivery)
Pups Day of Delivery 0.4110.1 0.70+0.2
~ O _ __ _ _
a Concentration of human IgG in rat ierum (~g/ml)
..... . . .
,, . ~ . ~
W092/04053 PCT/US91/0619~ ~
Q~;33~ : 32
Accordingly, the homocon~ugated IgG monoclonal
antibody is useful admini5tered prophyl~ctically or
therapeutically to pregnant females l~kely to dellver a
neonate 6uscepti~1e to developing or alr~ady ~aving an
infection, such as by group ~ ~treptococal or E~ ÇQli Xlo
The present invention also ~akes possible t~e use of the
ho~oconjugates in transplacental treatment of a variety
of other infections and tumor6.
EXAMPLE VII
In Vivo ~rotection ~qainst Group B Stre~tococcal
Infection Wi~h IqG Ho~ocon~uaa~es
Thi6 Example describes the use o~
homoconjugates of the D3 monoclonal antlbody to protect
against group B ~treptococcal infection in vivo,
consistent with and confirming the r~sults of the
vitro opsonophagocytic assay~.
As generally described for the E~ Ç~ l protection
studies described in Example IV above, out~red Sprague-
Dawley rat pups less than 48 hour6 old (hou6ed with their
mothers) were injected intraperitoneally wit~
approximately lO0 GBS organisms two hours ~fter receiving
an intraperitoneal ~njection of either 20, 4, 0.8 or 0.2
~g of predominantiy dimer homoconjugate preparations, 80,
20, or 4 ~g of monomeric D3, or control IgG. In the
experiments, rat pups were examined daily for seven days
and were sc~red for symptoms and survival. The results
from two experiments ~data pooled, 25 animal~/group),
shown in Fig. 12, demonstrate the increased ln vivo
protective activity against G~S of the dimer
homoconjugates of human monoclonal antibody D3 c~mpared
~ . ~,. .
, ~ . ~ . . . . . .
.: . ... ~ , : : .
.. . : . ... , -
, , . . : , . . ~
W09~/~0s3 ~ PCT/US91J06195
~ 33 2~9~3~ 7
to the initial IgG monomer. As llttle as 4 ~g of
~omoconjugated dimer protected animal~ nearly as well as
.that conferred by 80 ~g of monomer.
Although the present invention has been
described in some detail by way of illustration and
example for purposes of clarity And under~tanding, it
will be apparent that certain changes and modifications
may be practiced within the ~cope of the appended clai~sO
. ~ .. .- .; ~ .- ., . :- . .
: . .. . : .:
. ' . ~': ' : ' . .
,.. . . .
.. ~
..
. . ,:
.
