CA2090317A1 - Homoconjugated immunoglobulins - Google Patents

Abstract

Homoconjugated antibodies with high avidity for antigen have increased therapeutic activies and are utilized in pharmaceutical and diagnostic compositions. The homoconjugates, typically prepared from monoclonal antibodies of the IgG class which bind to the same antigenic determinant, are covalently linked by synthetic cross-linking. The homoconjugates are comprised of at least two immunoglobulin monomers so as to provide an IgG-like molecule which is tetravalent, hexavalent or more for the selected antigen. The homoconjugates are able to cross the placenta. Methods of treatment using these homoconjugates are also provided.

Description

W092/~053 PCT/US9t/06~9~

2~ 9 ~7 HOMOCONJUGATED IMMUNOçLOBULINS
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~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.

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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 :
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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.

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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

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W092/04053 PCT/~S91/06~95 ~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~

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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~.

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-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 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 . .

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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, ~'. ' : . : : ' .

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~ ~ 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 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 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 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.

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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 .. . . .

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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 ,' ' .
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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 . .

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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 ~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 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.

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'~" :' ~ ' ' ' 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 .. . . . ..

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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) ..... . . .

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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 ~ . ~,. .
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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

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Claims (22)

WHAT IS CLAIMED IS:

1. A pharmaceutical composition which comprises covalently cross-linked homoconjugated monoclonal antibodies having at least two IgG antibody molecules which bind to the same antigenic determinant, and a pharmaceutically acceptable carrier.

2. The pharmaceutical composition of claim 1, the homoconjugated monoclonal antibodies having two antibody molecules.

3. The pharmaceutical composition of claim 1, the homoconjugated monoclonal antibodies having three antibody molecules.

4. The pharmaceutical composition of claim 1, wherein the antibodies are cross-linked by disulfide bonds.

5. The pharmaceutical composition of claim 1, wherein the antibodies are human.

6. The pharmaceutical composition of claim 1, wherein the antibody molecules are murine.

7. The pharmaceutical composition of claim 1, wherein the antibody molecules are murine-human chimerics.

8. The pharmaceutical composition of claim 5, wherein the human antibody heavy chain is an IgG1.

9. The pharmaceutical composition of claim 1, which is protective against infection due to E. coli Kl.

10. The pharmaceutical composition of claim 1, which is protective against infection due to group B
streptococci.

11. The pharmaceutical composition of claim 1, wherein the homoconjugated monoclonal antibodies bind to a tumor associated antigen and inhibit growth of breast tumor cells

12. The pharmaceutical composition of claim 1, wherein the homoconjugated monoclonal antibodies are capable of crossing the placenta.

13. The pharmaceutical composition of claim 1, wherein the constant regions of the light and heavy chains of the antibody molecules are human.

14. The pharmaceutical composition of claim 1, wherein the cross-linked antibodies are derived from the same cell line.

15. A method of treating a patient having a disease related to an antigen, the method comprising administering to the patient a therapeutically effective amount of homoconjugated monoclonal antibodies including at least two covalently cross-linked IgG antibody molecules which bind to the same determinant of the antigen.

16. The method of claim 15, wherein the antigen related disease is group B streptococcal infection.

17. The method of claim 15, wherein the antigen related disease is E. coli Kl infection.

18. The method according to claim 15, wherein the homoconjugated monoclonal antibodies are administered to a pregnant patient and the homoconjugates are able to pass through the placenta into fetal circulation.

19. The method according to claim 18, wherein the homoconjugated monoclonal antibodies are able to treat the fetus for the antigen related disease.

20. The method of claim 19, wherein the antigen related disease is infection by group B
streptococci or E. coli Kl.

21. The method of claim 15, wherein the antigen related disease is breast tumor and the homoconjugated monoclonal antibodies bind to a breast tumor associated antigen.

22. In a method for therapeutic administration of monoclonal antibodies to a patient for treatment of a disease related to an antigen, the improvement which comprises administering to the patient homoconjugated monoclonal antibodies including at least two covalently cross-linked IgG antibody molecules which bind to the same antigenic determinant of the antigen.