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1. CA2163344 - HUMANISED ANTIBODIES

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[ EN ]
HUMANIsFn ANTlRonlf~:
FUFI n OF lbF INVENTION 5 TlliS invention relates to humanised antibodies having specificity for the epilope recoy"ised by the murine mo, loclo"al antibody L243, to processes for preparing said antibodies, to pharmaceutical compositions containing said ar,liLocJies and to medical uses of said antibodies.
10 The term humanised antibody molecule is used to describe a molecule having an a,niy6" binding site derived from an immu"Gglobulin from a non-human species, the remaining immunoglobulin-derived parts of the molecule being derived from a human immunoglobulin. The anligen binding site may co"".rise either complete variable regions fused onto 15 human cGIl:Jnl domains or only the comple",e,ltarity d~6r",ining regions (CDRs) yld~leJ onto appropriate human framework rig;ons in the variable domains.
R~cKGRouhln OF ll-lf INVENTlON 20 Tle proteins ellCG~ J in the Major I lislocGr",,dlibility Complex region of this genome are involved in many ~-see-,1s of immunological lecog"itiG". It is known that all ."a,n",als and probably all v6,Perales ~,assess hæC~cs~lly equivalent MHC systems and that immune res,uonse genes ars linked to thls MHC.
In man the major hi:,toc~"",~ ilit~ complex is the HLA gene cluster on cl,r~",osome 6. The main regions ara D, B C, and A. The D region cGIltai--s genes for Class ll ,vrot6i-,s which are involved in cOO~&IdliOll and interaction tJ~l~ree.l cells of the immune system. Many ~ise~ses have 30 been found to be ~ssoci te.J with the D region of the HlA gene cluster.
Studies to date have shown ~-association,-s with an enormous variety of rlise~Ses including most autoimmune ~Ji3~ ''03 (see for example Europe)
Patent No. 68790). European Patent No. 68790 suggests controlling diseases ~-associated with a particular allele of certain regions of the MHC 35 such as the HLA-D region in humans by selectively suppressing the immune respo,)se(s) controlled by a monoclonal antibody specific for an
MlC-Class ll antigen.
L243 is a murine IgG2A anti-HLA DR antibody which we believe to be of particular use in treatment of diseases such as autoimmune diseases since it shows particularly potent suppressio" of fn vitro immune function 5 and is monomorphic for all HLA-DR.
Since most available monoclonal antibodies are of rodent origin, they are naturally antigenic in humans and thus can give risa to an undesirable immune response termed the HAMA (Human Anti-Mouse Antibody) 10 response. Therefore, the use of rodent monoclonal antibodies as therapeutic agents in humans is inherently limited by the fact that the human subject will mount an immunological rea~o"se to the anli~ody and will either remove it e, lti, ely or at least reduce its effectiveness.
15 Plor-osAI~ have been made for "laki"y non-human MAbs less a"lige"ic in humans. Such techniques can be generically termed humanisation' tecl",i~ues. These tecl",i~ues ~"ert 11y involve the use of recombinant
DNA technology to manirlJI~te DNA sequences e"co,ring the pol~l,eptide chains of the a"libGIly molecule. A simpla form of hu".al,isation involves 20 the rerl^^ement of the consl~n~ regions of the murine a,ltiLo.ly with those from a human antibody [Morrison et al (1984) Proc. Natl. Acad. Sci. USA 6851-55; Whittle at a/ (1987) Prot. Eng. 1 499-505]. The lowering of the level of the HAMA rest,G"se to the cl,i",eric antibodies leads to the eel-ect~lion that further hu",al,isalio" of the variable region outside of the antigen binding site may abolish the rest.ol,se to these regions and further reduce any adverse r~spGnse.
A more complex form of humanisation of an antibody involves the l~e5IeS-J~ ~ of the variable region domain so that the amino acids CGI ,sliluting 30 the murine antibody binding site are integrdted into the framework of a human antibody variable region. Humanisation has led to the reconstitution of full antigen binding activity in a number of cases 2Co et al (1990) J. Immunol. 148 1149-1154; Co eta/(1992) Proc. Natl. Acad. Sci.
USA 88 2869-2873; Carter et al (1992) Proc. Natl. Acad. Sci. ~L 428535 4289; Routledge et al (1991) Eur. J. Immunol. 27 2717-2725 and
Intel"alicsnal Patent Speci~icalio"s Nos. WO 91/09967; WO 91/09968 and
V\lrO 92/1 1383].
It can therefore be a~ ted that the humanisation of L243 may lead to 5 reduced immunogenicity in man and overcome the potential problem of the HAMA response previously associated with the use of murine antibodies in humans.
We have now pre.ared recombinant antibody molecules having specificity 10 forthe e~ilol,e ieccjgn;sell bythe murine monoclonal antibody 1~43.
SIJMMARY OF Tl IF INYF~mON
Thus according to a first aspect the invention provides a recci"~binant antibody molecule having s,uecificity for a"liye"ic determinants dE3p6 15 on the DRoc chain.
The term recombinant a,ltiLo.ly molecule is used to de,~e an antibody pro~llJced using recombinant DNA tech,-i~ues. The a,l~il,Gdy is ~rtl~ldbly a humaniseci antibody e.g. a chimeric or CDR-y,d~led an~i~c;Jy.
In a preferred embodiment of the first aspect the invention provides a recombinant a, Itil~Jy molecule have s~ specificity for the epitope recognised by the murine l"G"ocl~nal antibody L243.
25 In a ,~,r~fe"eJ e",lcs.li,ne"t of the first aspect of the ,urease,l invention there is provided a humanised anlil~ody molecule having s~ ec tidily for the e~,itel,e recGy"ised by the murine monoclonal antibody L243 and having an a,-tiye,n binding site wherein at least one of the complementarity determining regions (CDRs) of the variable domain is derived from the 30 mouse monoclonal antibody L243 (MAb L243) and the remaining imrnunoglobulin-derived parts of the humanised antibody molecule are derived from a human immunoglobulin or an analogue thereof said humanised antibody molecule being optionally conjugated to an effector or r e,ucj, ler molecule.
The humanised antibody molecule may cor",l.rise a chimeric humanised antibody or a CDR-grafted humanised antibody. When the humanised antibody molecule comprises a CDR-grafted humanised antibody, the heavy and/or light chain variable domains may comprise only one or two 5 MAb L243 derived CDRs; though pr~teraLly all three heavy and light chain
CDRs are derived from MAb L243.
As describecl above L243 is a monoclonal a~ ody previously described by Lampson & Levy [J. Immunol. (1980) 125 293]. The amino acid 10 sequences of the light and heavy chain variable regions of the antibody are shown in Figures 1 and 2 hereinafter. L243 has been deoiled at the
American Type Culture Collection, Rockville, Maryland USA under accession, number ATCC HB55.
15 n~ Fn rTF-~cRlfrnoN OFTH~ ~VF~ITInN
The humanised antibody of the present invention may have attached to it an vector or red.o, ler molecule. For inalance a macrocycle for chelating a heavy metal atom, or a toxin such as ricin, may be attached to the humanised antibody by a covalent bridging structure. Alternatively, the 20 procedure of recombinant DNA technology may be used to produce a humanised a,ltibody molecule in which the Fc fragment, CH3 or CH2 domain of a complete antibody molecule has been red,l3.ce.1 by or has attached thereto by peptide linkage a fu"clional non-immunoglobulin protein such as an enzyme or toxin molecular
The humanised antibody of the prese"t invention may comprise a complete allLibo.ly molecule, having full length heavy and light chains; a fragment l~,ereof, such as a Fab, Fab', (Fab')2, or Fv fragment; a single chain alllibGdy fragment, e.g. a single chain Fv, a light chain or heavy 30 chain monomer or dimer; multivalent monospecific antigen binding proteins comprising two, three, four or more antibodies or fragments thereof bound to each other by a connecting structure; or a fragment or analogue of any of these or any other molecule with the same specificity as MAb L243.
In a preferred embodiment the antibody comprises a eomplete antibody molecule having full length heavy and light ehains.
The remaining non-L243 immunoglobulin derived parts of the humanised 5 antibody moleeule may be derived from any suitable human immu"oylol.ulin. For instanee where the humanised antibody molecule is a ICDR-~ led humanised anlilJe-by moleeule, appropriate variable region framework sequenees may bs used having regard to elass/type of the donor antibody from whieh the antigen binding regions are derived. 10 Preferably the type of human framework used is of the same/similar elass/type as the donor a~ o.ly. Advantageously the framework is ehosen to maximise/optimise homology with the donor anlibolJy sequenee partieularly at positions spaeially elose or adjaeent to the CDRs.
Examples of human frameworks whieh may be used to eonstruet CDR15 grSIrled and odies are LAY, POM, TUR, TEI, KOL, NEWM, REI and EU;for instanee KOL and NEWM for the heavy ehain and REI for the light eh~in or EU for both the heavy ehain and light ehain.
An alle",alive ,vroee~lure for the selection,) of a suitable human framework 20 involves aligning the framework rey;ons of the light ehain of the nonhuman framework with those of the four human light ehain subgroups identified by Kabat et al (1991) [in: Sequenees of Proteins of
Imrnunologieal Inleresl, Fifth Editionl. The eol,se"sus sequenee for the light ehain subgroup most homologous to the non-human antibody light 25 chain is c hos6l I.
Any differenees between the framework residues of the non-human a-llil/Gdy and the eonsensus human group light chain se~uenee are analysed for the ,uote"lial eo"l, ibutton they may have to antigen binding as 30 ulesc,ibed in PubWIe.l I"te",aliG"al Patent Apl~lie~lio" No. WO91/09967.
Based on this analysis some or all of the residues identified may be altered. The same ,urocedure is carried out for the selection of a suitable frameworlc to aeeept the non-human heavy ehain CDRs.
35 For eonstrueting the L243 CDR g,a~led light ehain, the human subgroup 1 consensus sequence was found to be partieularly suitable, and for constructing the L243 grafted heavy chain the human subgroup 1 co"sensus sequence was also found to be particularly suitable.
The light or heavy chain variable domains of the humanised antibody 5 molecule may be fused to human light or heavy chain constant domains as appropriate, (the term heavy chain constant domains' as used herein are to be understood to include hinge r6y;0ns unless specified otherwise).
The human constant domains of the humanised antibody molecule, where present, may be celscted having regard to the ,I.roposed function of the 10 antibody, in particular the lack of et~eclor functions which may be required.For example, the heavy chain consta"L domains fused to the heavy chain variable region may be human IgA, IgG or IgM domains. Preferably human IgG domains are used. Light chain human constant domains which may be fused to the light chain variable region include human 15 Lambda or human Kappa chains.
Analogues of human constant domains may alternatively be advantageously used. These include those consla"t domains containing one or more Addit;o!,al amino acids than the co"espG"di,)g human domain 20 or those constant domains wherein one or more ex;-~i"g amino acids of the correspGI~li"y human domain has been deleted or altered. Such domains may be obtained, for example, by oligonucleotide directed mul~ye"esis.
25 The remainder of the humanised antibody molecule need not comprise only pn~t~i., sequences from human imm globulins. For instance, a gene may be constructed in which a DNA sequence e"coding part of a
- human imml,l,Gylol,ulin chain is fused to a DNA sequence encoding the amino acid sequence of a poly,uelfl;de Ceclor or red.o, ler molecule.
We have found that by modifying one or more residues in the N-terminal region of the CH2 domain of the L243 antibody we produce an antibody with an altered ability to fix complement as compared to unaltered allti~- y.
The amino acid residue which is altered preferably lies within amino acid oil;ons 231 to 239 preferably within amino acid positio,ls 234 and 239.
In a particularly preferred embodiment the amino acid residue which is 5 altered is either Leu 235 and/or Gly 237.
As used herein the term altered' when used in conjunction with the ability of an antibody to fix complement most usually indicates a decrease in the ability of antibody to fix complement compared to the starting unaltered 10 antibGJy. By choosing an aypropriale amino acid to alter it is possih'Q to produce an antibody the ability of which to fix complement is sub lidlly reduced such as for example by altering residue Leu 235. It is also possible to produce an antibody with an intermediate ability to fix complement as compared to unaltered antibody by for example altering 15 amino acid residue Gly 237.
As used herein the phrase substantially' reduce complement fixation denotes that human complement lixation is preferably ~30% more preferably s20% and is most pre~e,ally <10% of the level seen with wild 20 type a"liL,o ly.
Due to the alle,~lio" of one or more amino acid residoes in the N-te,lllil ,al region of the CH2 domain the alltiLGCIy will preferably not bind siy"i~ica,-lly to FcRI and will bind to FcRIII ,decelerator.
The residue nlu~lbe~iny used herein is according to the Eu index desc,il,ed in IKabat ~1[(1991) in: Ser ences of Proteins of Immunological Interest 5th Edition, United States Department of Health and Human Services].
30 The alterations at posPion 235 of replacing leucine by glutamic acid or alanine have been found particularly effective at producing a potent immunosuppressive L243 ~ iLolJywith minimal toxicity in vftro.
The alteration at position 237 of replacing glycine by alanine has been 35 found to produce an antibody with an intermediate ability to fix complement i.e. the complement fixation level is approximately 15-80%
re~7r~bly 20-60% most preferably 20-40% of that seen with the wild type anliL~dy.
The residue(s) could similarly be replaced using an analogous process to 5 that described herein, by any other amino acid residue or amino acid derivative, having for example an inapt,o,criate functionality on its side chain. This may be achieved by for example changing the charge and/or polarity of the side chain.
10 The term 'siyr,i~ica"ll~ as used with reset to FcRn binding denotes that the bi.,d;.,g of ~Illibo.ly to FcRI is typically s20%, and is most preferably
S10% of that seen with unaltered antibody.
The standard ten;t"~ ues of molecular biology may be used to prepare 15 DNA sequences coding for the anliL.odies according to the invention.
Desired DNA sequences may be s~,ltl,devised co""~letely or in part using oligonucleotide synthesis techniques. Site-directed mutagenesis and polymerase chain reacts,) (PCR) techniques may be used as approx, iate.
20 Suitable processes include the PCR strand overlap procecure and PCR mutagenesis as desc-ibed in for example PCR Technology Principles and
ApplicaliGI,s for DNA Amp ic~liG"~ (1989), Ed. H.A. Erlich, Stockholm
Press, N.Y., London, and oligonucleotide ~iirecte~i mutagenesis [Kramer et al, Nucleic. Acid. Res. 12 9441 (1984)]. Suitable techniques are also 25 .disclosed in Pul,list,ed Eu,opean Patent No. EP307434B.
The altered L243 with altered complement fixing ability may also be pro~we~ by for example, deleting residues such as 235, or by for example, inserting a glycosylation site at a suitable position in the 30 molecule. Such techniques are well known in the art, see for example the teaching of published Eun,,uean patent ~P~JI;CII;OII EP-307434.
The altered L243 may also be produced by exchanging lower hinge reg;o"s of antibodies of different isotypes. For example a G1/G2 tower 35 hinge exchange abolished complement fixation.
The G1/G2 lower hinge exchange results in an antibody with altered residues in the 231-238 region of the N-terminal region of the CH2 domain v:: ,erei" one or more residl-ss may be altered or delete 5 According to a seco, Icl aspect of the invention there is provided a process for producing the humanised antibody of the first aspect of the invention which process comprises:
a) producing in an emission vector an ol-eron having a DNA sequence which 6ncoJ13s an antibody heavy or light chain comprising a variable domain wherein at least one of the CDRs of the variable domain is derived from the L243 MAb and the remaining immunoglobulinderived parts of the anlibGJy chain are derived from a human immunoglob! Clin; b) producing in an expression vector an G~ eron having a DNA sequence which encGcles a complementary a"~ Gdy light or heavy chain co.,-~)fising a variable domain wherein at least one of the CDRs of the variable domain is derived from the MAb L243 and the remaining immunoglobulin-derived parts of the hlllibody chain are derived from a human immunoglobulin; c) lra,s~e~.1i,,y a host cell with both opero"s, and 25 d) culturing the l.~nsteclecl cell line to produce the humanised antibody molecule.
In a yre~er,ed embodiment of this aspect of the invention at least one of the expression vectors contains a DNA sequence encoding an antibody heavy chain in which one or more amino acid residues the N-terminal region of the CH2 domain of said antibody has been altered from that in the correslJo"di"s~ unaltered antibody.
The alteration in the N-terminal region of the CH2 domain may be made after the whole unmodified antibody has been expressed using techniques such as site JirecteJ mutagenesis.
The cell line may be transfected with two vectors, the first vector containing the operon encoding the light chain-derived polyA.e,side and the second vector containing the operon encoding the heavy chain derived 5 polypeptide. Preferably the vectors are identical except in so far as the coding sequences and selective markers are concemed so as to ensure as far as possi'Ile that each polypeptide chain is equally exp.resse.l.
Altematively, a single vector may be used, the vector including the 10 operons encoding both light chain- and heavy chain-derived polypeptides, and a s~ hle marker.
The alleralio" in the N- terminal region of the CH2 domain, e.g. at position 235 of the CH2 domain of the molecule may be introduced at any 15 convenient stage in the humanisation e.g. CDR-$~r ~lil,g ,,.rucess. It is conveniently introducer after the variable domains have been ylatlecl onto the heavy chains.
In further aspects the invention also includes DNA sequences coding for 20 the heavy and light chains of the a,-liboc;:as of the present invention, clot,ing and expression vectors containing these DNA sequences, host cells l,d"s~or,ned with these DNA sequences and processes for producing the heavy or light chains and anli~Gdy molecules comprising expressing these DNA sequences in a l,hn~fo",fed host cell.
The ganeral methods by which the vectors may be constructed,
transfection methods and culture methods are well known per se lsee for example Maniatis ~1(1982) (Molecular Cloning, Cold Spring Harbor,
New York) and Primrose and Old (1980) (Principles of Gene Manipulation, 30 Blackwell, Oxford) and the examples hereinafter].
The DNA sequences which encode the L243 light and heavy chain variable domain amino acid sequences (and the cor,espo"ding deduced amino acid sequences) are given hereafter in Figures 1 and 2 35 respectively.
C)NA coding for human immunoglobulin sequences may be obtained in any appropriate way. For example, amino acid sequences of preferred hluman Accel.tor frameworks such as, LAY, POM, KOL, REI, EU, TUR,
TEI and NEWM are widely available to workers in the art. Similarly the 5 consensus sequences for human light and heavy chain subgroups are available to workers in the art.
The standard techniques of molecular biology may be used to prepare
DNA sequences coding for CDR-grafted products. Desired DNA 10 sequences may be synthesise-~ completely or in part using oligonucleotide synthesis techniques. Site-di,æted mutaye"esis and polymerase chain re~c~iGn (PCR) techniques may be used as approx ridte. For example, oligonucleotide directed synll,ests lJones etal(1986) Nature 321 522-525] and also oligonucleotide .li.ected mu~e"esis of a pre-exisliny variable 15 domain ragion [Verhoeyen et al (1988) Science ~ 1534-1536; naicl ""an" et al (1988) Nature 332 32~327].
Enzymatic filling-in of 9~5.l)ed oligonuc'~otides using T4 DNA polymerase [Queen ~ (1989) Proc. Natl. Acad. Sci. USA 86 10029-10033; 20 I"te",slio,)al Patent A~)PIjÇ~1;GIF No. WO 90/078611 may be used.
Any suitable host celUvector system may be used for egress;G,) of the
DNA sequences coding for the chimeric or CDR-filed heavy and light chains. Bacterial e.g. E.coli and other microbial systems may be used 25 adva~ eously in particular for ~ressior, of ~ lil,ody fragments, e.g. Fv,
Fab and Fab' fragment and single chain antibody fragments e.g. single chain Fvs. Eucaryotic e.g. l"am",alian host cell e,to.r~ssio" systems may also be used to obtain antibodies accorcl;.,g to the invention, particularly for production of larger chimeric or CDR-grafted antibody products. 30 Suitable mammalian host cells include COS cells and CHO cells [Bebbin.Jtell C R (1991) Methods 2 136-145] and myaloma or hybridG",a cell lin2s, for example NSO cells lBebbington C R et al (1992)
Bio/Technology 10 169-175]. The use of CHO cells is especially preferred.
In the humanised antibody according to the invention, the heavy and light chain variable domains may cor"prise either the entire variable domains of
MAb L243, or may comprise frarnework regions of a human variable domain having yldtleJ thereon one, some or all of the CDRs of MAb L243. 5 Thus the humanised antibody may comprise a chimeric humanised antibody or a CDRy-d~led humanised antibody.
When the humanised antibody is a CDR-graded humanised antibody, in addition to the CDRs, s~.eci~ic variable region framework r6si~1ues may be 10 altered to cor,est.ond to non-human i.e. L243 mouse r6sidues. r,preferably the CDR-gra(led humanised A"lil,o~ies of the ~.resent invention include
CDR-y,clad humanised a"li~,o.J;ss as defined in our Inl~",~liGnal Patent
Speciricalion No. W0-A-91/09967. The disclosure of W0-A-91/09967 is inco".Vrecl herein by reference.
PleferdL,ly the CDRs of the heavy chain cor,spo-,d to the L243 residues at all of CDR1 (31 to 35), CDR2 (50 to 6~) and CDR3 (95 to 102).
Preferably the CDRs of the light chain cor,es~o"d to L243 r~siduss at all of CDR1 (24 to 34) CDR2 (50 to 56) and CDR3 (89 to 9n. In ~d~ ion the 20 heavy chain may have mouse L243 resides at one or more of resitlues 27, 67, 69, 71, 72 and 75. Similarly the light chain may have mouse L243 residues at one or more Nos;Bio, ls 45, 49, 70 and 71.
The invention further provides a CDR-grafted humanised antibody heavy 25 chain having a variable regibn domain comprising acceptor frameworks derived frorn human subgroup CGIlSellsus sequence 1 and L243 donor antigen bind;.by regions wherein the framework co."~.rises L243 donor residues at one or more of positio"s 27, 67, 69, 71 72 and 75.
30 The invention further provides a CDRs d~lecl humanised antibody light chain having a variable region domain comprising acceptor frameworks darived from human subgroup COnSel)SUS sequence 1 and L243 donor antigen binding regions wherein the framework comprises L243 donor residues at one or more of ~,ositiG,-s 45, 49, 70 and 71.
The heavy chain may further have mouse L243 residues at one or more of residues2, 9, 11, 16, 17, 20, 38, 43, 46, 80, 81, 82, 82a, 82b, 83, 84, 89, 91,108 and 109.
The light chain may further have mouse L243 residues at one or mora of residlJes 9, 13, 17, 18, 37, 40, 43, 45, 48, 49, 72, 74, 76, 80, 84, 85, 100, 103 and 104.
The antibody according to the invention may be a complete antibody or as 10 explained above, a fragment ll,er~of, a mG"o",er or dimer or a multivalent monospecific antigen binding ~.ratei.,. Certain compounds of this latter group are particularly advanlayeous in that they l,assess high avidity. See for example Published International Patent Specification No. WO 92101472 the teaching of which is i"co"Joraled herein.
Thus accGr~ling to a further particular aspect of the invention we provide a multivalent monose.ecitic allyls binding protei" comprising two, three, four or more ar,l;bodies or fragments thereof bound to each other by a connecting structure which ,I.rotat.~ is not a natural immunoglobulin, each 20 of said a"liboJies or fragments having a :",eciticity for the epitope recGg,.ised by murine MAb L243 said antigen binding protein being opliGnally conjugate.sd with an e~tec~or or red, ler molecule.
In this aspect of the invention each antibody or fragment is preferably a 25 humanised a"liLoJy or a fragment thereof as defined above and the multivalent monose,ecitic antigen binding protein is thus a humanised muJtivalent monospecrfic antigen binding protein. Non-humanised e.g. murine, multivalent monospecitiG anlagen binding proteins can, howevor, be co,-~e--,~ ~d and the invention is to be uncJerslood to also extend to 30 these.
The multivalent antigen binding protein, ,ureterably col-,~u,ises two, three or four anlibGJies or fragments Ihareof bound to each other by a connecting structure.
Immunological ~ise~ses which may be treated with the antibodies of the invention include for example joint diseases such as ankylosing spondylitis, juvenile rheumatoid arthritis, rheumatoid arthritis; neu,~ .J ~l disease such as multiple sclerosis; pancreatic disease such as ~ betes, 5 juvenile onset diabetes; gast-ointesli"al tract disease such as chronic active hepatitis, celiac disease, ulcerative colitis, Crohns disease, pernicious anaemia; skin licenses such as psoriasis; allergic diseases such as asthma and in transplantation related conditions such as graft versus host dise~-se and allograft reject;on. Other diseases include those 10 .less ed in Eur.e~ Patent No. 68790.
The present invention also includes therapeutic and diagnostic compositions containing the antibodies of the invention. Such compositions typically comprise an antil,Gdy according to the invention 15 toy~ther with a pharmaceutically accel.lable excipient, diluent or carrier, e.g. for in vlvo use.
Thus in a further aspect the invention provides a therapeutic, pharmaceutical or diagnostic composition comprising an antibody 20 according to the invention, in combination with a pharmaceutically ~cepPble excipient, diluent or carrier.
The invention also provides a process for the preparation of a therapeutic, pharmaceutical or diagnostic composition comprising admixing an 25 a,~ ody according to the invention together with a pharmaceutically ~c~pt~ble excipient, diluent or carrier.
The antil,odies and compositions may be for administration in any approx.riate form and amount acconJi.,y to the therapy in which they are 30 employed.
The therapeutic pharmaceutical or diagnostic composition may take any suiPhle form for adminislldlion, and, preferably is in a form suitable for pa,~,lte~dl admini~lralio" e.g. by inj&'~ioll or infusion, for example by bolus 35 injection or continuous infusion. Where the product is for injection of infusion, it may take the form of a suspsllsion, solution or emulsion in an oily or aqueous vehicle and it may contain formulatory agents such as suspending, preservative, stabilising and/or cJis~er~i"g agents.
Alternatively, the antibody or composition may be in dry form, for 5 reconstitution before use with an ~ J~,criate sterile liquid.
If the antibody or composition is suitable for oral administration the forrnulation may contain, in addition to the active iny,edie"t, additives such as: starch e.g. potato, maize or wheat starch or cellulose or starch 10 d3rivatives such as microcrystalline cql' close silica; various sugars such as lactose; magnesium carbonate and/or calcium phosphate. It is desirable that, if the oral forrnulation is for administration it will be well tolerated by the patient's digestive system. To this end, it may be desirable to include in the formulation mucus formers and resins. It may 15 also be desirable to improve tolerance by formulating the ar,Tibo.ly or compositions in a c~pslJIs which is insoluble in the gastric juices. It may also be ~Jreter~ble to include the antibody or cGy position in a cG"l,olled r01ease formulation.
20 If the antibody or CGIllyOSiliol~ is suitable for rectal administration the formulation may contain a binding and/or lul~ri~dli"g agent; for example polymeric glycols, geldli"s, cocoa-butter or other veyetdble waxes or fats.
Therapeutic and diagnostic uses typically comprise administering an 25 effective amount of an antibody according to the invention to a human subject. The exact dose to be administered will vary according to the use of the anlil~ody and on the age, sex and co"di~ion of the palient but may tylDically be varied from about 0.1mg to 1000mg for e~r"ple from about 1M9 to 500mg. The antibody may be administered as a single dose or in 30 a continuous manner over a period of time. Doses may be repeated as apprG~I iate.
The antibodies and compositions may be for administration in any appropridle form and amount accordi"g to the therapy in which they are 35 employed. The dose at which the antibody is administered depends on the nature of the condition to be treated and on whether the antibody is being used prophylactically or to treat an existing co"dition. The dose will also be selected according to the age and conditions of the patient. A therapeutic dose of the anli~oclies according to the invention may be, for example, between preferably 0.1-25mg/kg body weight per single 5 therapeutic dose and most preferably l)el~oon 0.1-10 mg/kg body weight for single therapeutic dose.
The antibody may be formulated in accordance with convel,lio"al practice for administration by any suitable route and may generally be in a liquid 10 form (e.g. a solution of the antibody in a sterile physio'Dgic~lly ~ccert~'q buffer) for adminislf~LiG" by for example an intravenous, i,intraperitoneal or intramuscu'~r route.
The pres6-,t invention is now ~lesc,iLled by way of example only, by 15 reference to the accG---~,anying dry IY-~ in which:
Figure 1: shows the nucleotide and amino acid sequence of L243
Vl region
Figure 2: shows the nucleotide and amino acid sequence of L243
Vh region
Figure 3: shows a diag.~."",lic map of plasmid pMR15.1
Figure 4: shows the nucleotide and amino acid sequence of Vl region in L243 gL1
Figure 5: shows the nucleotide and amino acid sequence of Vl region of L243-gL2
Figure 6: shows a diag,d.. -n,atic map of plasmid pMR14.
Figure 7: shows the nucleotide and amino acid sequence of Vh region of L243-gH.
Figure 8: shows a diagrammatic map of plasmid pGamma 1.
Figure 9: shows a graph of the results of a competition assay for
L243 grafts vs FlTC-chimeric L243 gHgL1
Figure 10: shows a graph of a Scatchard analysis for L243 gamma 4 cH cL Kd = 4.1nM gH gL1 Kd = 6.4nM gH gL2 Kd = 9.6nM 5 Figure 11: shows a graph of FcRIII binding of chimeric and grafted
L243 as measured by ADCC
Figure 12: shows a graph of L243 Isotype series MLR
G1 L243 gH gL1
Cys,h~ipo,i,. A
Medium control n&s,.~o~ Ider alone
Figure 13: shows a graph of L243 Isotype Series TT Rest.onse
+ G1 L243 gH gL1
Cyclone o,i,. A
Medium control n~s~"d~r alone
Fil~ure 14: shows the nucleotide and amino acid sequence of the hinge and part of the CH2 region of human C-ga",r"a 1
Figure 15: shows a graph of FcRIII binding of chimeric, grafted and grafted ll ~35F] L243 as measured by ADCC
ChimericG1 wt
Cilill-e.icG1 [L235El
Gran G1 wt e Graft G1 [L235q
Figure 16: shows a graph of immunos~ passive activity of CDR ~lafle~l L243 measured by MLR
Graft G1 wt
Graft G1 [l~35q
Cyclospo,in A
Chimeric G1 wt " - ChimericG1 [L235E
Medium Control
Figure 17: shows a graph of CDR 9ra~1ed L243 and gla~l6d [L235E]
L243 rr recall resin, .se
GraftG1wt
Graft G1 L235E]
Cyclosporin A
Chimeric G1 wt
O Chimeric G1 [L235El
Medium Control
Figure 18: shows a graph of co,--,~ ."ent mediated C~r~OlO~iC pot-)-;y of CDR gl~a~led L243 and CDR 91a~led [L235E]
Chimeric G1 wt
Chimeric G1 [L235q
Graft G1 wt
Graft G1 L235E]
Figure 19: shows the nucleotide and amino acid sequences of a) Clone 43 b) Clone 183 and c) Clone 192 20 Figure 20: shows a d;aglallll"dtic map of plas",i~ pGamma 2.
DETAII ~:n DESCRIPTlON OF Tl IF SPFCIFIC EMBODIMENTS OF TIFF
INVENTION
EXAMPLE 1 gene Cloning ~n~ Expression
RNA rrR~r~tinrl from 1~43 hyL"i.h,..~ cells
Total RNA was prepared from 3 x 10exp7 L243 hyl.rido",a cells as 30 describe below. Cells were washed in physiological saline and dissolved in RNAzol (0.2ml per 10exp6 cells). Chloroform (0.2ml per 2ml homogenate) was added the mixture shaken vigorously for 15 seconds and then left on ice for 15 minutes. The resulting aqueous and organic phases were separated by centrifugation for 15 minutes in an Eppendorf 35 centrifuge and RNA prec;~ ten from the aqueous phase by the addition of an equal volume of isolJropa,Vol. After 15 minutes on ice the RNA was pelleted by centrifugation, washed with 70% ethanol, dried and dissolved in sterile, RNAase free water. The yield of RNA was 350 SLg.
Amino ~ se~uenee of the 1~ t eh~in.
The sequence of the first nine amino acids of the mature L243 light chain was determined to be NH2-DIQMTQSPAS.
PCR eloning of ! ~43 Vh and Vl the eDNA genes for the variable ,eyio"s of L243 heavy and light ehains 10 severe synthesised using reverse lldnsc,mutase to produee single strande eDNA eopies of the mRNA present in the total RNA, followed by
Polymerase Chain Reaetion (PCR) on the eDNAs with speeifie oligonucleotide primers.
15 a) eDNA synthesis eDNA was s~",ll,esise~l in a 20111 reaelion eontaining the following reagents: 50mM Tris-HCI PH8.3, 75mM KCI, 10mM dithiothreitol, 3mM MgC12, 0.5mM eaeh deoxyribonueleoside triphosphates, 20 units RNAsin, 75ng rhlldGIll hexan!~el~30ti~le primer, 2~19 L243 RNA and 200 units Moloney Murine Leukemia Virus reverse lle,se,i~Jtase. After inu~b~tion at 42C for 60 mins the reaetion was temlinated by l,eali"g at 95C for 5 minutes.
by PCR
Aliqvots of the eDNA ~rere subjeeted to PCR using eombinations of primers for the heavy and light ehains. The nueleotide sequenees of the 5' primers for the heavy and light ehains are shown in Tables 1 and 2 respeetively. These sequenees, all of whieh eontain a restriction site starting 6 nueleot;des from their 5' ends, followed by the sequenee GCCGCCACC to allow optimal translation of the resulting mRNAs, an initiator codon and a further 20 - 30 nueleotides, are a eompilation based on the leader peptide sequenees of known mouse antibodies [Kabat ~l (1991) in
Sequenees of Proteins of Immunological Interest, 5th Edition United States Department of Health and Human Serviees].
The 3' primers are shown in Table 3. The light chain primer spans the V - C junction of the antibody and contains a real,icliol) site for the enzyme Spl1 to facilitate cloning of the Vl PCR fragment. The heavy chain 3' primers are a mixture designed to span the J - C
Junction of the antibody. The first 23 nucleotides are identical to those found at the start of human C - gamma 1, 2, 3 and 4 genes and include the Apa1 restriction site common to these human isotypes. The 3' region of the primers contain a mixed sequence based on those found in known mouse antibodies [Kabat E A, Wu,
T.T.; Perry H M, Gottesman K S, and Foeller L; In: Sequences of
Proteins of Immuno.o5lical Interest, 5th Edition, US Department of
Health and Human Services (1991)].
The combinations of primers described above enables the PCR products for Vh and Vl to be cloned directly into the appropriate expression vector (see below) to produce chimeric (mouse human) heavy and light chains and for these genes to be e~.esse.J in mammalian cells to produce chimeric antibodies of the dest,~d isotype.
IncubAtiG"s (20 ~11) for the PCR were set up as follows. Each reaction contained 10 mM Tris-HCI pH 8.3l 1.5 mM MgC12, 50 mM
KCI, 0.01% w/v gelatin, 0.25 mM each deoxyribonucleoside triphosphate, 1 - 6 pmoles 5' primer mix (Table 4), 6 pmoles 3' primer, 1 ~11 cDNA and 0.25 units Taq polymerase. Rear,tions were incubated at 95C for 5 minutes and then cycled through 94C for 1 minute, 55C for 1 minute and 72C for 1 minute. After 30 cycles, aliquots of each reaction were analysed by electfo~Jl,oresis on an agarose gel. Reactions containing 5' primer mixes B1, B2, B3 and
B5 produced bands with sizes consistent with full length Vl fragments while reaction B9 produced a fragment with a size collect.ed of a Vh gene. The band produced by the B1 primers was not followed up as previous results had shown that this band corresponds to a light chain pseudogene produced by the hybrido",a cell.
e~ Molecular cloning of the PCR fragments.
DNA fragments produeed in reaetions B2, B3 and B5 were digested with the enzymes BstB1 and Spl1, coneentrated by ethanol reeipilalion, eleetlopl,oresed on a 1.4 % agarose gel and DNA bands in the range of 400 base pairs reeovered. Tnese were eloned by ligation into the veetor pMR15.1 (Figure 3) that had been restrieted with BstB1 and Spl1. After ligation, mixtures were transformed into E. eoli TM1035 and plasmids from the resulting
L)aclerial eolonies sereerled for inserts by ~ eslicill with BstB1 and
Spl1. Res s~"ld~ es with inserts from eaeh liyalioll were analysed further by nueleotide sequene;"~.
In a similar manner, the DNA fragments produeed in reaetion B9 and dides~etl with Hindlll and Apa1 were eloned into the veetor pMR14 (Figure 6) that had been real,ieted with Hindlll and Apa1.
Again, red,rese"lali~e plasmids eontaining inserts were analysed by nueleotide sequeneing.
d) Nueleotide sequenee analysis
Plasmid DNA (pE1701 and pE1702) from two isolates eontaining
Vh inse,ls from reaetion B9 was sequeneed using the primers
R1053 Shieh ,u,i,nes in the 3' region of the HCMV promoter in pMR14) and R720 (whieh primes in the 5' region of human C gamma 4 and allows sequeneing through the DNA insert on pMR14). The dete""ined nueleotide sequenee and pre ted amino aeid sequenee of L243 Vh in pE1702 is given in Figure 2. The nueleotide sequenee for the Vh insert in pE1701 was found to be ide"lical to that in pE1702 exeept at nueleoti.le 20 (A in pE1701) and nueleotide 426 (A in pE1701). These two dittere"ces are in the signal ~,ç~t;~ b and J regions of Vh leg,selectively and indicate that the two elones examined are indepe"d6t,~ isol-Res arising from the use of di~tere"l primers from the mixture of oligonucleotides during the
PCR stage.
To analyse the light chain clones, sequence derived from priming with R1053 was examined. The nucleotide sequenee and prediGted amino acid sequence of the Vl genes arising from reactions B2 (clone 183), B3 (clone 43 and B5 (clone 192) are shown in Figure 19. Comparison of the predicted protein sequences shows the following:
i) clones 182, 183, 43 and 4~ all code for a Vl gene which, when thesignal peptide is removed, produces a light chain whose sequence is ider,tical to that determined by amino acid sequence analysis for
L243 light chain (see above). ii) clones 182 and 183 contain a Vl gene that codes for a signal per e of 20 amino acids, while the Vl gene in clones 43 and 45 results from priming with a dir~ar~n~ set of oligonucleoli.les and has a leader sequence of only 15 amino acids. iii) Clone 192 does not code for L243 Vl. Instead, examination of the d~t~h~se of a"li~G.ly sequences (Kabat, 1991) indicates that clone 192 conldins the Vl gene for MOPC21, a light chain synthesised by the NS1 myeloma fusion ,ua,l"er used in the prodlJction of the L243 hy~rido".a.
iv) Clones 182 and 183 are identical except at nucleotide 26 (T in clone 182, C in clone 183). This .lirte~nce can be accounted for by the use of different primers in the PCR and in~ic~tes that clones 182 and 183 are independent isolates of the same gene. The nucleotide sequence and ~re~l;ctep amino acid sequence of the complete Vl gene from clone 183 is shown in Figure 1.
Construction of human ~mm~ 1 and ~rnm~ ~ isotypes. 30 The L243 Vh gene was subcloned on a Hindlll - Apa1 fragment into pGamma 1 and pGamma 2, vectors containing the human C - gamma 1 and C - gamma 2 genes res,Qectively (Figures 8 and 20).
Human IsotyFe mutants 3~ PCR mutagenesis was used to change residue 23~ in human C - gamma1 contained in the vector pGamma 1 from leucine to either glutamic acid or to alanine and to change residue 237 from glycine to alanine. The lower hinge region of human C-gamma 1 was also replaced by the corresponding region of human C-gamma 2. The following oligonucleotides were used to effect these chal,ges;
I) L235E change
R4911 5' GCACCTGMCTCGAGGGGGGACCGTCAGTC3'
R4910 5'CCCCCCTCGAGrrCAGGTGCTGAGGMG3'
Il)~ L235A cl ,a"ge
R5081 5'GCACCTGMCTCGCAGGGGGACCGTCAGTC3'
R5082 5'GACTGACGGTCCCCCTGCGAGTTCAGGTGC3'
Ill) G237A change
R5088 5'GCACCTGMCTCCTGGGTGCACCGTCAGTC3'
R5087 5'GACTGACGGTGCACCCAGGAGTTCAGGTGC3'
IV) E~;l ,al ,ye of lower hinge regions
R4909 5'GCACCTCCAGTGGCAGGACCGTCA~i I t; l I CCTC3'
R4908 5'CGGTCCTGCCACTGGAGGTGCTGAGGMGAG3'
Other oligonucleotides used in the PCR mutage, Issis are:
R4732 5'CAGCTCGGACACC I I C 1~ l CCTCC3'
R4912 5'CCACCACCACGCATGTGACC3'
R4732 and R4912 prime between nucleotides 834 and 858 and between nucleotides 1156 and 1137 respectiYely in human C - gamma 1 (Figure 30 Tne general ~ teyy for the PCR mutagenesis was as follows. For each amino acid change, two rounds of PCR were ussd to generate DNA fragments containing the required substitutions. These fragments were then rest,icteJ with the enzymes Bgl ll and Siy1 and used to replace the corresponding fragments containing the wild type sequence in the 35 pGamma 1 vector, (Figure 8).
For the first round PCR, reactions (20~LI) were prepared containing the following reagents: 10 mM Tris - HCI pH 8.3,1.5 mM MgCI2, 50 mM KCI, 0.01% gelatin, 0.25 mM each deoxyribonucleoside triphosphate. 50~Lg pGamma 1 DNA, 0.4 unit Taq polymerase and 6 pmoles of each of the 5 primer The following c~, nL.. IdliOllS of primers were used:
R4911/R4912,
R4910/R4732,
R5081/R4912,
R5082/R4732,
R5088/R4912,
R5087/R4732,
R4909/R4912,
R4908/R4732.
After 30 cycles through 94C for 1 minute, 55C for 1 minute and 72C for 1 minute, the reactions were extracted with chloroform, the newly synthesised DNA precipitated with ethanol, dissolved in water and electro,vl,oresed on a 1.4 % agarose gel. Gel slices containing the DNA 20 fragments were e~.c;~e-J from the gel, the DNA recovered from the agarose using a ~I\la.,naid~ kit ffrom St-dtecll Scientific Ltd., Luton, England) and eluted into 20~11 sterile water.
Second round PCR was in a 100 1l1 rsaction containing 10 mM Tris - HCI 25 pH 8.3, 1.5 mM MgCI2, 50 mM KCI, 0.01 % gelatin, 0.25 mM each dsoxyribonucleoside triphosphate, 2 units Taq polymerase, 1/20 of each pair of DNA fragments from the first round read,1iG" and 30 pmoles of each of R4732 and R4912. After 30 cycles, see above, the reactions were extracted with phenol / chloroform (1/1) and prec;~.ilated with ethanol. 30 Fragments were dj~JeSIed with Bgl11 and Styl, elem,url,oresed on a 1.4 % agarose gel and DNA bands of 250 base-pairs recovered from gel slices as previously .lescribed.
These Bgl ll - Siy1 fragments were ligated in a 3 - way li!Jaliûl) to the 830 35 base-pair Siy1 - EcoR1 fragment, containing the C - terminal part of the
CH2 domain and the entire CH3 domain of human C - gamma 1, and the
B~ EcoR1 vector fragment from pGamma1 (see Figure 8). After transformation into TM1035, plasmid minipreps from resulting colonies were screened for the presence of the Bgl ll - Siy1 fragment and representatives of each taken for nucleotide sequence analysis. From
S this, p!asmids containing the desired sequence were identified and, for fulure reference, named as follows:
pGamma11L235E] containing glutamic acid at residue 235, pGamma1[L235A] containing alanine at residue 235, 10 pGamma1[G237A] containing alanine at residue 237, pGamma 1 [91--~92] containing the C-gamma 2 lower hinge region.
The above plasmids were each rest.icted with blindly and Apa1 and the
Hindlll - Apa1 fragment containing L243 Vh inserted to produce the 1~ following plasmids:
L243Gamma1~1 ~3~9
L243Gamma1 [1~35A]
L243Gamma1 [G237A] 20 L243Gamma [91--~92]
Production of chimeric 1 ~43 ~n1i~
Antibody for biological ev~lu~tion was produced by lfa"sie- lt e~.ressio, - of the appropriate heavy and light chain pairs after co-transfection into 25 Chinese Hamster Ovary (CHO) cells using calcium phosphate precipitation.
On the day prior to l.~l ,stectior" semi - confluent flasks of CHO-L761 cells were trypsinised, the cells counted and 175 flasks set up each with 30 1 Oe~xp7 cells.
On the next day, the culture medium was changed 3 hours before transfection. For transection, the calcium phos l,ate precipitate was prepared by mixing 1.25 ml of 0.25M CaC12 containing 50 ,ug of each of 35 heavy and light chain expression vectors with 1.25 ml of 2xHBS (16.36 gm
NaCI, 11.9 gm HEPES and 0.4 gm Na2HPO4 in 1 litre water with the pH adjusted to 7.1 with NaOH) and adding immediately into the medium on the cells. After 3 hours at 37 C in a C02 incubator, the medium and precipitate were removed and the cells shocked by the addition of 15 ml 15 % glycerol in phosphate buffered saline (PBS) for 1 minute. The 5 glycerol was removed, the cells washed once with PBS and incubated for 48- 16 hours in 25 ml medium containing 10 mM sodium butyrate.
Antibody was purified from the culture medium by binding to and elution from protein A - Sepharose. Antibody conce"lralion was determined using a human lg ELISA (see below).
For the ELISA, Nunc ELISA plates were coated over at 4C with a
F(ab)2 fragment of a polyclonal goat anti-human Fc fragment specific anlibo.ly (Jackson Immuno-research, code 109-006-098) at 5 ~Lg/ml in 15 coating buffer (15mM sodium carbonate, 35mM sodium hydrogen carbonate, pH6.9). U. ,com a~ ody was removed by washing 5 times with cli-axilla.l water. Sa",Kulas and purified standards to be quantitated were diluted to a~,r~,xi,-.ately 1 ~g/ml in conjugate buffer (0.lM Tris-HCI pH7.0, 0.1M NaCI, 0.2% v/v Tween 20, 0,2% w/v Hammersten casein). 20 The samples were litl~dled in the "3cr~it.a wells in 2-fold dilutions to give a final volume of 0.1 ml in each well and the plates incubated at room temperature for 1 hr with ~ dl~illy. After the first incubation step the plates were washed 10 times with 1iC~ 6',1 water and then incubated for 1 hr as before with 0.1 ml of a mouse ",onoclo"al anti-human kappa (clone GD12) 25 peroxide-se conjugated antibody (The Binding Site, code MP135) at a dilution of 1 in 700 in conjugate buffer. The plate was washed again and sul,slfate solution (0.1 ml) added to each well. SuLslrdte solution
- contained 150 ~I N,N,N,N-tetramethylba,l~i.li"e (10 mg/ml in DMSO), 150 111 hyd~oge" peroxide (30% solution) in 10 ml 0.1M sodium acetate/ 30 sodium citrate, pH6Ø The plate was developed for 5-10 minutes until the absorbance at 630nm was approximately 1.0 for the top standard.
Absorbance at 630nm was measured using a plate reader and the co"ce"lrdlion of the sample determined by comparing the li~rdliorl curves with those of the standard.
Oligonucleotide primers for the 5' region of mouse heavy chains.
CHl : 5'ATGAAATGCAGCTGGGTCAT(G,C) I I C I I ~'
10 CH2 : 5'ATGGGATGGAGCT(A,G)TATCAT(C,G)(C, I ) I ~; I 1~'
CIH3 : 5'ATGMG(A,T)TGTGGTTAAACTGGG I ~
CIH4: 5'ATG(G,A)ACI I IGGG(T,C)TCAGCTTG(G,A)T3'
CH5: 5'ATGGACTCCAGGCTCM I I IAG I I I ~
CH6: 5'ATGG~ i(C,T)T(G,A)G(G,C)GCT(G,A)~ ~ G3'
20 CH7 : 5'ATGG(G,A)ATGGAGC(G,T)GG(G,A) 1~; l 1 1 (A,C) 1 CH8: 5'ATGAGAGTGCTGA I 1~ G3'
CH9 : 5'ATGG(C,A)TTGGG I ci I GGA(A,C)CTTGCTATT3'
CHl 0: 5'ATGGGCAGACTTACA I I ~; I CATTTCCTGG
Ctlll: 5'ATGGAI 1 l ACTGGCTGAA 1~ ATTG3'
30 CHT2: 5'ATGATGGTGTTM~ GTACCT3'
Each of the above primers has the sequence 5'GCGCGCMGCTTGCCGCCACC3' added to its 5' end.
Oligonucleotide primers for the 5' region of mouse light chains.
CL1: 5'ATGMGTTGCCTGTTAGGCTGTTGGTGCT3'
1 0 CL2: 5'ATGGAG(T,A)CAGACACACTCCTG(T,C)TATGGGT3'
CL3: 5'ATGA~ GCTCACTCAGGTCCT3'
CL4: 5'ATGAGG(G,A)CCCCTGCTCAG(A,T)TT(C,T)TTGG3'
CL5: 5'ATGGAm(T,A)CAGGTGCAGATT(T,A)TCAGCTT3'
CL6: 5'ATGAGGT(T,G)C(T,C)(T,C)TG(T,C)T(G,C)AG(T,C)
T(T,C)CTG(A,G)G3'
CU: 5'ATGGGC(T,A)TCMGATGGAGTCACA3'
CL8: 5'ATGTGGGGA(T,C)CT(G,T) I I I (T,C)C(A,C)(A,C) l l I
TTGMT3'
CL9: 5'ATGGT(G,A)TCC(T,A)CA(G,C)CTCAGTTCCTT3'
CL10: 5'ATGTATATA I ~ I CàTCTA I I I C3'
30 CL11: 5'ATGGMGCCCCAGCTCAGC ~ ; I l 3'
Each of the above primers has the sequence 5'GGACTGTTCGMGCCGCCACC3' added to its 5' end.
Oligonucleotide primers for the 3' ends of mouse Vh and Vl genes.
Light chain ( CL12 ): 5'QGATACAGTTGGTGCAGCATCCGTACG I I i :~'
10 Heavy chain ( R2155 ): 5'S3CAGATGGGCCCl~CGTTGAGGCTG(A,C)(A,G)GAGAC(G,T,A)GTGA3'
5' Primer miYtures for PCR
B5 : CL1, CL3, CL5,CU, CL10, CLl 1.
B9 : CH1, CH3, CH5, CH8, CH9, CH10, CH11, CH12.
FY~Mpl F ~?
L243 is a mouse monoclonal antibody raised against human MHC Class
Il. The nucleotide and amino acid sequences of L243 Vl and Vh are 5 shown in Figures 1 and 2 respectively. The following examples describe the humanisation of the L243 antibody (CDR g~ ink).
CDR ~.~tlT~--J of ~ ~43 ligbLçh~
Alignment of the framework rey;o"s of L243 light chain with those of the 10 four human light chain subgroups [Kabat, E. A., Wu, T. T., Perry, H. M.,
Gottesman, K. S. and Foeller, C. 1991, Sequences of Proteins of
Immunological Interest, Fifth Editionl revealed that L243 was most ho",oloyous to a"liL,odies in human light chain subgroup 1. Consequently, for constructing the CDR yratl6.1 light chain, the framework regions 15 chosen corresponded to those of the human Group 1 consensus sequence. A comparison of the arnino acid sequences of the framework regions of L243 and the co"se"sus human group l light chains is given below and shows that there are 21 differences (u"derlined) between the two sequences.
Analysis of the co"l-iLution that any of these framework differences might have on antigen binding (see published Intemational patent Rrlrlic~libll
No. WO91/09967) iclentiti6~1 4 resin es for invesligatio,); these are at positions 45,49,70 and 71. Based on this analysis, two versions of the 25 CDR y~ ed light chain were constructed. In the first of these, L243-gL1, residues 45,49,70 and 71 are derived from the L243 light chain while in the second, L243-gL2, all residues are human consensus.
Ligb~b~ln Residue 30 llesides 37 (Gin to Arg) and 48 (lle to Val) would be included in any future y~a~led molecules.
I tht chain Comparisons
Hu group 1 consensus : D I QMTQSPSSLSRSUGDRUT I TC
L243 : D I Qt1TQSP_SLS_SUGETUT I TC HCl Group l consensus : WYQQKPGKRPKLLIY
L243 ' : WY_QKQGK_PQLLUF
Hu Group l consensus . GUPSRFSGSGSGTDFTLTISSLQPEDFRTYYC LXt3 : GUPSRFSGSGSGTQYSLKI_SLQSEDFGDYYC
Hu Group l ~on~en~u~ : FGQGTKUEIKR
L243 : FGGGTNLEIKR
ConstructionofCnR gra~ledlig~tch~inl~43-~ 1
The construction of L243-gLt is given below in detail. The following oligonucleotides were used in the Polymerase Chain n~-clie"s (PCR) to introduce cl ,~"ges into the framework regions of ths chimeric light chain:
R50~3 : 5'GTRGGRGRCCGGGTCRCCRTCRCRTGTCGRGCRR3'
R5044 : 5'CTGRGGRGCTTTTCCTGGTTTCTGCTGRTRCCRTGCTRRR3'
R5045 : 5-RRRCCRGGRRRRGCTCCTCR5CTCCTG~TCTTTGCTGCRTC3'
R50t6 : 5 ' CTTCTGGCTGCRGGCTGGRGRTRGTTRGGGTRTRCTGTGTGCC3'
R50~7 : 5 ' CTTCRGCCTGCRGCCRGRRGRTTTTGCTRCTTRTTRCTGTCRR3'
R50~8 : 5'GGGCCGCTRCCGTRCGTTTTRGTTCCRCTTTGGTGCCTTGRCCGRR3
Three reactio-,s, each of 20 lli, were set up each containing 10 mM TrisHCI pH 8.3, 1.5 mM MgCI2, 50 mM KCI, 0.01% w/v gelatin, 0.25 mM each deoxyribonucleoside triphos ale, 0.1 1l9 chimeric L243 light chain DNA, 6 pmoles of R5043/R5044 or R5045/R5046 or R5047/R5048 and 0.25
- units Taq polymerase. Reactions were cycled through 94C for 1 minute, 55~C for 1 minute and 72C for 1 minute. After 30 cycles, each reaction was analysed by electrophoresis on an agarose gel and the PCR fra3ments excised from the gel and recovered using a Mermaid Kit (suprlied by St~al6cl~ Sciontific Ltd., Luton, England).
Aliquots of these were then suLj~cted to a second round of PCR. The reaction, 100 1lll, contained 10 mM Tris-HCI pH 8.3,1.5 mM MgCI2, 50 mM
KCI, 0.01% w/v gelatin, 1/10 of each of the three PCR fragments from the 5 first set of reactions, 30 pmoles of R5043 and R5048 and 2.5 units Taq polymerase. Reaction temperatures were as above. After the PCR, the mixture was extracted with phenol / chlorofor", and then with chlGro~or", and precipitated with ell,anol. The ethanol precise .rate was recovered by centrifugation, dissolved in the appropriate buffer and rest,iced with the 10 enzymes BstEII and Spl. The resulting product was finally electrophoresed on an agarose gel and the 270 base pair DNA fragment recovered from a gel slice and ligated into the vector pMR15.1 (Figure 3) that had previously been digested with the same enzymes.
15 The li~aliGn mixture was used to l~-lso,." E. coli TM1035 and resulting colonies analysed by PCR, r~slri~iGIl enzyrne cligesls and nucleotide sequencing. The nucleotide and amino acid sequence of the Vl region of
L243-gL1 is shown in Figure 4.
20 Construction of cnR 9f~lb-1 liSht chs~in 1 ~43-ol ~
L243-gL2 was cG,-~ ucted from L243-gL1 using PCR. The following oligonucleotides were used to introduce the amino acid cl ,anges:
R1053 : 5'GCTGRCRGRCTRRCRGRCTGTTCC3 5~TCTRGRTGGCRCRCCRTCTGCTRRGTTTGRTGCRGCRTRGRTCRGGRGCTTRGG 5~GCRGRTGGTGTGCCRTCTRGRTTCRGTGGCRGTGGRTCRGGCRCRGRCTTTRCC 30 CTRRC3J
R68L : 5'TTCRRCTGCTCRTCRGRT3~
Two reactions, each 20 ~I, were set up each containing 10 mM Tris-HCI pH 8.3, 1.5 mM MgCI2, 50 mM KCI, 0.01% w/v gelatin, 0.25 mM each 36 deoxyribonucleoside tri-phosphate, 0.1 ~19 L243-gL1, 6 pmoles of
R1053/R5350 or R5349/R684 and 0.25 units Taq polymerase. Reactions were cycled through 94C for 1 minute, 55C for 1 minute and 72C for 1 minute. After 30 cycles, each reaction was analysed by elect,opt)oresis on an agarose gel and the PCR fragments excised from the gel and recovered using a Mermaid Kit.
Aliquots of these were then subjected to a second round of PCR. The reaction, 100 ~11, contained 10 mM Tris-HCI pH 8.3, 1.5 mM MgCI2, 50 mM
KCI, 0.01% w/v gelatin, 1/5 of each of the PCR fragments from the first set ol reactions, 30 pmoles of R1053 and R684 and 2.5 units Taq polymerase. 10 Re~c~io" temperatures were as above. After the PCR, the mixture was extracted with phenol/ chloroform and then with chloroform and proc;~ At6d with ethanol. The ethanol precipitate was recovered by centrifugation, d;ssolv0d in the a~upro~.riate buffer and rest,depicted with the enzymes BstEII and Spl. The resulting product was finally 15 elect,~"Jl,ores on an agarose gel and the 270 base pair DNA fragment recovered from a gel slice and ligated into the vector pMR15.1 (Figure 3) thial had previously been diy6~le~ with the same enzymes.
The ligation mixture was used to l,~ to"n E. coli TM1035 and resulting 20 colonies analysed by PCR, reslliclio" enzyme digests and nucleotide sequ6"c;.)g. The nl~cl~ol;Ile and amino acid sequence of the Vl region of
L~243-gL2 is shown in Figure 5.
12~ffiriç~ of 1 ~43 h~vy chain 5 CDR grafting of L243 heavy chain was accomplished using the same as cJ~s~ribed for the light chain. L243 heavy chain was found to by most homologous to human heavy chains belonging to subgroup 1 and therefore the consa"sus sequence of the human subgroup 1 frameworhs was chose" to accept the L243 heavy chain CDRs.
A comparison of the framework regions of the two structures is shown below whera it can be seen that L243 differs from the human consensus at 28 positions (underlined). After analysis of the col,l,ibutton that any of these might make to a"lig6n binding, only re~id~les 27, 67, 69, 71, 72, and 35 75 were retained in the CDR yl d~led heavy chain, L243-gH.
Heavy Chain Resin residues 2 (Val to lle) and 46 (Glu to Lys) would be incorporated into any future grafted molecules. In addition to these two residues the murine residua 67 which is present in L243 gH would be changed back to the 5 human consensus residue i.e. Phe to Val change.
H~vy chain comparisons
Hu Group 1 con~en~uQ QUQLUQSGREUKKPGRSUKUSCKRSGYTFT
L243 : QQLUQSG=ELKKPGETUK_SCKRSG_TFT
Hu Group 1 con3en~u~ WVRQAPGQGLEWMG
L243 : WVKQRPGQGLEWIG
Hu Group 1 consensus RVTMTTDTSTSTAYMELRSLRSDDTAVYYCAR
L2L3 : RFRFSLETS_STRYLQINNLKNEDTR=YFCRR
Hu Group 1 consensus : WGQGTLUTUSS
L2L3 : WGQGTTLTUSS
Construction of CDR y~Hr~ heavy chain. 1 ~43 ~H
L243gH was assembled by substantilly overlapping oligonucleotides to 25 PCR in the presence of the appropriate primers. The following oligonucleotides were used in the PCR:
R3004 : 5'GGGGGGRRGCTTGCCGCCRCCRTGG3'
30 R3005 : 5'CCCCCCGGGCCCTTTGTRGRRGCRG3'
R4902 : 5'GRCRRCRGGRGTGCRCTCTCRGGTGCRGCTGGTGCRGTCTGGRGC
RGRGGTGRRGRRGCCTGGRGCRTCTG3'
R4903 : 5'RCRTTCRCRRRTTRCGGRRTGRRTTGGGTGRGRCRGGCRCCTGGR
CRGGGRCTCGRGTGGR3'
R~gO~ : 5'CCTRCGTRCGCRGRCGRCTTCRRGGGRRGRTTCRCRTTCRCRCTG
GRGRCRTCTGCRTCTRCRGCRTRCRT3'
R~905 : 5 ' CRGCRGTGTRCTRCTGTGCRRGR~RCRTTRCRGCRGTGGTRCCTR
CRGGRTTCGRCTRCTGGGGRCRGGGR3' 5 R~897 : 5'TGRGflGTGCRCTCCTGTTGTCRCRGRCRGGRRGRRCRGGRRCRCC
CRRGRCCRCTCCRTGGTGGCGGCRRGCTTCCCCCC3~
R~898 : 5 ' CRTTCCGTRRTTTGTGRRTGTGRRTCC~GRTGCCTTRCRRGRCRC
CTTCRCRGRTGCTCCRGGCTTCTTCR3'
Rt~99 : 5 ' GRRGTCGTCTGCGTRCGT~GGCTCTCTTGTGTRTGTRTTRRTCCR
TC~RTC~R~TC~RGTCC~TGTCCRG3'
R9 900 : 5 ' TTGCRCRGTRGTRC~CTGCTGTGTCCTCRGRTCTCRGRGRRGRCR
GCTCCRTGTRTGCTGTRGRTGCRG~T3'
RL90 1 : 5 ' CCCCCCGGGCCCTTTGTRGR~GCRGRRGRCRCTGTC~CCRGTGTT
CCCTGTCCCCRGTRGTCGRR3'
20 The assembly reaetiGn 50 111 eontained 10 mM Tris-HCI pH 8.3 1.5 mM
MgCI2 50 mM KCI, 0.01% w/v gelatin 0.25 mM eaeh deoxyribonucleoside l.ith.oil.ate 1 pmole of eaeh of R4897 - R4905, 10 pmoles of eaoh of R3004 and R3005 and 2.5 units Taq pol~",ease. Reaetions were eyeled through 94C for 1 minute, 55C for 1 minute and 72C for 1 25 minute. After 30 eyeles the n3aoticil~ was e~ eted with phenol/~l,lorofo" (111), then with ehloroform and preeipitated with ethanol. After eentrifugation, the DNA was dissolved in the ap,urc,~uliate .est-ictio-l buffer and diye~led with Hindlll and Apal. The resulting fragment was isolated from an agarose gel and ligated into pMR14 (Figure 6) that had previously 30 been di~sst~rl with the same enzymes. pMR14 eontains the human gar"lna 4 heavy ehain eonslant region and so the heavy ehain eA,..ressed frorn this veetor will be a gamma 4 isotype. The ligP~io" mixture was used to lf~nstc"" E. eoli TM1035 and resulting bacterial eolonies sereened by re:jl,ricin,. digest and nueleotide sequenee analysis. In this way a plasmid 35 eo.Lai,.ing the CGIId~:t sequenee for L243gH was idenlitied (Figure 7).
Construction of t~mm~ 1 versions of ehimerie and CDR ~Jr~tled 1 24 heavy eh~in
Human Gamma 1 versions of L243 heavy chains were constructed by l,a"ste"i,-~ the variable re~io"s of both the murine and the CDR grafted heavy chains as Hindlll to Apal fragments into the vector pGamma1 (Figure 8). This vector contains the human Gamma 1 heavy chain 5 conslar,t region.
Evaluation of activities of CnR gr~ft~l genes
The activities of the CDR y~tl6d genes were evaluated by expressing them in mammalian cells and purifying and qua"lilali"g the newly 10 synthesised a.,li~o~Jies. The methodology for this is described next, followe;l by a desc,iplio" of the bioch~",ical and cell based assays used for the biological characte,isdlio" of the antibodies.
ne F~ ~SSiOI I in CH2 cells 15 Cl,i",~,ic and CDR 9~ 6d L243 was produced for biological ev~lu~tion by transient e,~Jre~si~n of heavy and light chain pairs after co-transfection into Chinese Hamster Ovary (CHO) cells using calcium phosphate precipitation.
20 On the day prior to lr~"s~eclion, semi-confluent flasks of CHO-U61 cells [Cockett, M. I. BeLILi.,~to", C. R. and Ya,-anto" G. T. 1991, Nucleic
Acids Research 19, 31~325] were trypsinised the cells counted and 175 flasks set up each with 107 cells. On the next day, the culture medium was changed 3 hours before transfection. For trans~eclio" the calcium 25 phosphate precir ate was ,prepared by mixing 1.25 ml of 0.25M CaCI2 containing 50 ~19 of each of heavy and light chain expression vectors with 1.25 ml of 2xHBS ( 16.36 gm NaCI 11.9 gm HEPES and 0.4 gm
Na2HP04 in 1 litre water with the pH adjusted to 7.1 with NaOH ) and adding i",l"e~ ely into the medium on the cells. After 3 hours at 37C in 30 a C02 incubator the medium and p,ecto te were removed and the cells shocked by the addition of 15 ml 15 % glycerol in phosphate buffered saline (PBS ) for 1 minute. The glycerol was removed the cells washed once with PBS and incubated for 48 - 96 hours in 25 ml medium containing 10 mM sodium butyrate. Antibody was purified from the culture 35 medium by binding to and elution from proteinA - Sepharose. Antibody cor,c~"l~lion was deler",ined using a human lg EUSA (see below).
b) ELISA
For the ELISA Nunc ELISA plates wore coated overnight at 4C with a
F(ab)2 fragment of a polyclonal goat anti-human Fc fragment specific antibody (Jackson Immuno-research code 109-006-098) at 5 llg/ml in coating buffer (1 5mM sodium carbonate 35mM sodium hydrogen carbonate, pH6.9). Uncoated-l allti~G~Jy was removed by washing 5 times with distilled water. Samples and purified standards to be quar,lilac were diluted to ap~Jr~xi",ately 1 ,~Lg/ml in conjugate buffer (O.lM TrisHCl pH7.0 10 0.1M NaCI 0.2% v/v Tween 20, 0.2% w/v Hammersten cassin). The samples were til,ated in the microti,e wells in 2-fold dilutions to give a final volume of 0.1 ml in each well and the plates inundated at room temperature for 1 hr with shaking. After the first incubation step the plates were washed 10 times with ~lisl;lle water and then incubated for 1 hr as 15 before with 0.1 ml of a mouse ",o"o~lo,)al anti-human kappa (clans GD12) pel`OXid~Se conjugated a~,lit,o.ly (The Binding Site, code MP135) at a dilution of 1 in 700 in conjugate buffer. The pîate was washed again and substrate solution (0.1 ml) added to sach well. Sul)strate solution contained 150 ~11 N,N,N,N ten.a"-etl,yl~e"~i~ine (10 mg/ml in DMS0) 150 20 ul hydrOy6" peroxide (30% solution) in 10 ml 0.1M sodium acetate/ sodium citrate pH6Ø the plate was developed for 5-10 minutes until the absorbance at 630nm was approximately 1.0 for the top standard.
Absorbance at 630nm was measured using a plate reader and the
Col~C~lf~liGIl of the sample .late.",ined by comparing the ti~ld~iGn curves 25 with those of the standard.
The principle of this assay is that if the a"li~" binding region has been cGy ly t,a"sf~r,~d from the murine to human framework, then the CDR 30 grafted antibody will compete equally well with a labelled chimeric antibody for binding to human MHC Class ll. Any ~hanyes in the antigen bin-li"g potency will be revealed in this system.
Chimeric L243 was l~hslled with fluoresc6i., (FITC) using the method of 35 Wood et al lWood T. Thompson S and Gcl~sl~i.l G (1965) J. Immunol ~g 225-229]. All dilutions manipulations and incubations were done in phosphate buffered saline (PBS, Gibco, UK) containing 0.1% sodium azide and 5% Fetal Calf serum (Sigma, UK). Serial dilutions of anlibo-lies in 100~1 in RB polystyrene tubes (2052 12x75mm Falcon, UK) were premixed with a constant amount of the FlTC-labelled antibody (at a 5 previously determined optimal concentration) and added to 5x104 indicator cells (JY B lymphoblastoid cell line bearing high levels of HLADR). Cells and antibody were inc~b~tecl together at 4C for 30 minutes, washed twice and binding revealed using a Fluorescence Activated Cell
Scanner (FACS Becton Dickinson). After approx.state analysis, median 10 fluGr~sce"ce intensity is plotted against antibody concenlfalio".
Figure 9 compares the ability of combinations of L243 heavy and light chains to compete with FlTC-labelled chimeric L243 for ~ dil to JY cells.
All combinations were effective competitors although none of those 15 containing CDR titled heavy or light chains were as effective as the chimeric a-,Tibo.ly itself. Thus, the combinations cH/gL1, gH/cL and gH/gL1 were 89%, 78% and 64% rest,selectively, as effective as chimeric
L243 in this assay.
20 d) ~)ete""i,.~ .n of Affinity con.counts by .Allard Analysis
L243 antibodies were titrated from 10~1g/ml in PBS, 5% fetal calf serum, 0.1% sodium azide in 1.5-fold dilutions (150~ each) before inclination with 5x104 JY cells per titration point for 1 hour on ice. The cells were previously counted, washed and resulted,der in the same medium as the 25 samples. After incubation, the cells were washed with 5ml of the above medium, spun down and the sl".er"atal,l discarded. Bound antibody was revealed by addition of 100~11 of a 1/100 dilution of FITC coniu9~teri antihuman Fc monoclonal (The Binding Site; code MF001). The cells were then incubated for 1 hour on ice and then tha excess FITC conjugate 30 removed by washing as before. Cells were dispersed in 250~11 of the same buffer and the median fluorescence intensity per cell was determined in a
FACScan (Becton Dickinson) and calibrated using standard beads (Flow
Cytometry standards COI~JGral;On). The number of mo'Qcllles of antibody bound per cell at each antibody concentration was thus established and 35 used to generate Scatchard plots. For the purpose of calcd tion, it was assumed that the valency of binding of the FITC conjugate to L243 was 1 :1 and that the F/P ratio was 3.36 (as given by the manufacturer).
A Scatchard plot comparing the a~i"ilies of chimeric L243 (cH/cL). L243gH/L243-gL1 and L243-gH/L243-gL2 is shown in Figure 10. Chimeric
L243 was found to have an apparent Kd of 4.1 nM while the CDR grafted anliboclies containing gL1 and gL2 light chains had apparent Kd of 6.4nM and 9.6nM respectively. The difference in Kd values of the antibodies with the two CDR grafted light chains reflects the co"l,dilution made by residues 45 49 70 and 71 that had been retained, in L243-gL1 from the parent light chain.
e) Antibody dependent cell med;ated cytotoxic
The ability of chimeric and CDR glalle~ 1 243 to mediate antibody dependent cell cy~otoxi~ily (ADCC) was compared. The principle of the e3~eri",ent is that antibodies will mediate Iysis of target cells bearing their coynate anliye" if the Fc of the a~ by is able to i,n6racl with Fc receptor bearing t,vector cells c~r~hl~ of cytotoxicity.
Effector cells are prepared fresh for each experiment. Human venous blood is drawn into endoto3ci" free tubes containing heparin. Peripheral blood mononuclear cells (PBMC) are prepared by density gradient centrifugation accGrdi"g to the manufacturers instructions (Pharmacia).
PBMCs are adjusted to 1x107 cells/ml in RPMI 1640 medium (Gibco) conlai,li"g 2 mM glutamine (Gibco, UK) and 10% fetal calf serum in which all manir~ tions dilutions are incut-millions are done.
Target cells (JY see above) are labelled with lmCi Na51Cr for 1 hour at room temperature ~ telJ every 15 minutes. The cells are then washed three times to remove free Mygliol bel and resusper,d6d at 2X106 cells/ml.
Serial antibody dilutions are prepared in due,licate in sterile U-bottomed 96 well microtitre plates (Falcon UK) in 25~11. Control wells containing medium only are also prepared to est sh the spontaneous release of label giving the assay background. Target 51Cr labelled JY cells are added to all wells in 1 OLI. The same number of JY cells are also added to wells containing 2% Triton X100 in water to establish the 100% release value. Target cells and antibody are incubated together and after 30 minutes at room temperature 25~11 effector cells are added to all wells (except the 100%) for a further 4 hours at 37C. 100111 of EDTA/saline at 4C is then added to stop any further cell killing the microtilrt3 plates are 5 centrifuged at 200xg to pellet the intact cells and 100111 of the supernatant is removed and counted in a gamma counter.
Percent cell Iysis is calculated by subtracting the background from all values and then expressing them as a percentage of the adjusted 10 ma%imum release. Re,ulic~es vary by less than 5%. Percent cell Iysis is then plotted against antibody conce"lldlioll.
A comparison of the activities of chimeric (cH/cL) and CDR g~atled (glH/gL1) L243 human gamma 1 isotypes in the above assay is shown in 15 Figure 11. Both antibodies were effective mediators of cell Iysis with maximal activity being achieved at antibody cGIlcelllldtiGlls of less than 100 ng/ml. There was no Styl ,i~icant ~litfere"ce ~el~/oon the activities of thetwo antibodies.
20 f) Immune function tests
FY vivo T cell function e34.eri",e"ts were ~e,run,,ed where an interaction by ocn MHC-II and the T cell rec6p~0r was an obligatory requirement for
T cell activation. Chimeric and CDR g~led L243 antibodies were compared in mixed lymphocyte reactions which measures both naive and 25 memory T cell activation and in recall responses to tetanus toxoid which only measures a memory T cell r~s~.o"se.
1) Mixed L~""~ ;yte reaction
The principle of the e,~,.eri",ent is that when leucocytes from one individual 30 are mixed with those of another individual which express different HLA alleles they will recoy"ise each other as foreign and the lymphocytes will become activated. This activation is depende"t primarily on interactions between the CD3/TcR complex on T cells and the MHC Class 11 molecule on a, lige,) presenliny cells. L 243 is known to inhibit this reaction.
1 sucocytes are prepared fresh for each exp.elt,),ent. Human venous blood from two individuals is drawn into elldoloxill free tubes containing heparin.
Peripheral blood mononuclear cells (PBMC) are prepared by density centrifugation according to the manufacturers instructions (Pharmacia). 5 PBMC are ~djnsted to 2X106 cells/ml in RPMI 1640 medium (Gibco UK) containing 2 mM glutamine 100 ~lg/ml penicillin/s~,ep~G",ycin and 10% fetal calf serum in which all manipulations, dilutions and incubations are done. PBMC from one individual are i"~d;~ l with 3000 rads. These cells will be used to stimulate a response from those of the other individual.
Serial dilutions of anli~odie3 are prepared in ll;l~lic~te in sterile U-bottom 96 well microtti,e plates (Falcon, UK) in 100u1. Control wells containing either medium alone or 100nM C~ OSI (S81ldi-llmun, Sandoz are also prepared to establish the maximum respGnse and maximum inhibition 1~ respectively. Equal nu"l~er~ of i"~-J;ate-l stimulators are less.Anders are mixed togetl,er and 100~1 is added to each well. Wells with stimulator alone and re:,po"ders alone are also set up as cGIltl~ols. The experi,nent is incubated at 37C in 100% humidity and 5% C02 for 5 days. Response is measured by ~sses~i"y cell proline liell during the last 18 hours of culture 20 by incubation with 1 Lowell 3H-Thymidine (Ame,sl,a"" UK) harvesting
OlltO glass filter mats and counting using a beta counter.
When an MLR was carried out to co-"pare the effectiveness of the
Gamma 1 isotypes of chimeric and CDR grafted L243 as inhibitors of T 25 cell activation no siy"i~ica"l di~f~re"ces were observed ~t~e~n the two antibodies (Figure 12). Greater than 90% inhibition of the MLR was observed using 100 ng/ml of either al,liL~dy.
2) T cell recall respo"se to Tetanus toxoid 30 The ability of chimeric and CDR grafted L243 to s~ ress a secondarv res,uonse was ~-ssessed using a recall response to Tetanus toxin. The principle of the experiment is that T lymphocytes from an individual previously immunised with Tetanus toxoid (rr) will respond to TT when reexposed ex vivo. This activation is dependent on the interaction between 3~ the CD3/TcR complex on T cells and the MHC Class 11 molecules on cells which process and present the antigen. L243 is known to inhibit this re~tio".
PBMC are prepared as descril)ed above. Serial dilutions of a"li60dies are 5 prepared in triplicate in sterile U-bottom 96 well microtil,e plates in 100~1.50~11 containing an optimal concentration of TT previously determined by experimentation is added to all wells. Control wells containing medium only or 100nM cyclos~.ori,l are also prepared to establish the maximum response and maximum inhibition respectively. ~0~11 PBMC are then 10 added to each well. The experiment is incubated at 37C in 100% humidity and 5% C02 for 7 days. Response is measured by assessing cell proliferation during the last 18 hours of culture by incubation with 1~1Ci/well 3H-thymidine harvesting onto glass filter mats and counting using a beta counter.
The results of an experiment comparing the ability of human gamma 1 isotypes of cl,i",eric and CDR g~h~le~l L243 to inhibit the ~spo"se to TT is shown in Figure 13. Both antibodies were effective inhibitors of the T cell response to 1~ and produced ~ dliOII curves that were i"di.,ti"y,Jishable.
FY'~MPl F 3
The ability of CDR 9~l6d L243 with the alleralio,l at position 235 i.e. [L235El to mediate antibody dependent cell cytoxicity (ADCC) was measured essentially as desc,ibed in Example 2. The results are shown 25 in Figure 15.
Similarly the CDR yldtlecl L243 [L235q antibody was tested in a mixed
I~,l.lst ,ocyte reaction and in recall rest.o"se to tetanus toxoid essentially as desk, ibe~ in Example 2. The results are provided in Figures 16 and 17.
ANTIBODY DFPENDFI~IT COMPI ~ME~NT
MFn~Tlfn CY I U I ~ cmr
The ability of the engineered variants of L243 to fix human complement was assessed using the technique of antibody dependent complement 35 meclizine cytotoxicity.
The principle of the experiment is that antibodies will mediate complement
Iysis of target cells bearing their cog"ate antigen if the Fc of the antibody i~; able to interact with the components of the (usuaily classical) complement c~sc~de The critical interaction is with the Clq molecule.
The source of complement in these experiments is human venous blood freshly drawn into endotoY;~ I free glass bottles which is then allowed to clot at 37C for 1 hour. The clot is detached from the glass and then incubated at 4C for 2 hours to allow it to retract. The clot is then removed 10 and the serum separated from the remaining red cells by centrifugation at 10009. Once prepared, the serum can be stored for up to one month at -20C without notice deteriordliGn of pole,-cy.
All manipulations, dilutions and incubations are done in RPM1 1640 15 medium (Gibco UK) containing 2mM Glutamine (Gibco UK) and 10% foetal calf serum (Sigma UK). Target cells (JY B Iy,n,uho!)l~stloid cell line bearing high levels of HLA-DR) are l~-le1 with 1mCi Na51Cr for 1 hour at room temparature, agitated every 16 min. The cells are then washed three times, to remove free r~ ol~el, and resuspended at 2x106/ml. 20 Serial antibody dilutions are prepared in duplicate in V-~tlon, 96 well microtip.d plates (ICNlflow UK) in 25ml. Control wells containing medium only are also prepared to establish the spo"l~,.eous release of label giving the assay back-ground. Target 51Cr l~h~lled JY cells are added to all wells in 10ml. The same number of JY cells are also added to wells 25 containing 2% Triton x100 in water to est~hlisll the 100% release value.
Talrget cells and antibody are incubated together and, after 1 hour at room tempsrature, 25ml serum as a source of co,-"~le,nent is added to all wells (e;~e~t the 100%) for a further 1 hour at room temperature. 100ml of
EC~TA saline at 4C is then added to stop any further cell killing, the 30 micr~til,e plates are centrifuged at 200g to pellet the intact cells and 100ml super"at~"t is removed and counted in a gamma counter.
Percent cell Iysis is collated by subtracting the background from all values and then expressing them as a percentage of the adjusted 35 maximum release. Replicates vary by less than 5%. Percent cell Iysis is then plotted against antibody dilution,
The results (without subtraction of background) are shown in Figure 18.