CA2163345A1 - Antibodies - Google Patents

Abstract

The invention provides antibodies with altered ability to fix complement. The invention further relates to pharmaceutical, therapeutic and diagnostic compositions containing said antibodies and to methods of therapy and diagnosis using said antibodies. The invention additionally provides a method of modulating the function of cell surface associated antigens using said antibodies. Also provided are processes for preparing said antibodies.

Description

WO 94/29351 ~16 3 ~ 4 5 PCT/GB94/01290 ANTI~on I-IFI n OF THE INVF~mON
5 This invention relates to altered antibodies, to pharmaceutical, therapeutic and diagnostic compositions containing said antibodies; to processes for preparing said compositions; to methods of therapy and diagnosis using said antibodies, to a method of modulating the function of cell surface associa~ed antigens using said antibodies; to DNA sequences coding for 10 said antibodies; to cloning and expression vectors containing DNA
sequences coding for said antibodies; to host cells transformed with said vectors and to processes for preparing said anlil~od;es E~ACKGROUND OF THF INVF~TION
15 In order for an antibody to be eflective ther~pelJtic~lly it is desirable that it achieves the required physiological effect without producing any significant adverse toxic effects. Such toxic effects may be me~ led for example, via complement ~ixdlioll.

20 Antibody when bound to its coy"ate an~iye" can link to and activate the complement cascade. Complement consists of a complex series of proteins. The proteins of the complement system form two interrelated enzyme cascades, termed the classical and alternative pathways, providing two routes to the cleavage of C3, the central event in the 25 complement system. The sequence of events comprising the classical complement ,udli~ ay is recog"ition, enzymatic activation, and membrane attack leading to cell death. The recognition unit of the complement system is the C1 complex. The C1 complement protein complex is a unique feature of the classical complement c~-sc~de leading to C3 30 conversion. Complement ~ixaliol, occurs when the C1q subcomponent binds directly to immu, ~JIol.lJlin antigen immune complex. Whether or not complement fixation occurs depends on a number of constraints. For example, only certain sllhcl~-sses of immunoglobu~ can fix complement even under optimal conditions. These are IgG1, IgG3 and IgM in man and 3~ IgG2a, IgG2b and IgM in mice.

wos4/2s3sl 21633~ rcT/GBg4l0l290 The C1 q molecule is potentially multivalent for attachment to the complement ~ tio" sites of immunoglobulin. The CH2 domain of IgG and probably the CH4 domain of IgM contain binding sites for C1q.

5 Fc bearing cells also play a role in enhancing the effect of the immune response by binding to and opsonising, phagocytosing or killing target cells coated with antibody of the relevant class. Three IgG binding receptors (FcyR) have been described for murine and human leukocytes~ FcyRI has high binding affinity for monomeric IgG, while FcyRII and FcyRIII have low 10 affinity for mono IgG and interact mainly with antigen complexed IgG. The presence of Fc receptors confers on these immune cells the ability to mediate a number of effector mechanisms important in the effector phase of the humoral response.

1~ The gamma 1 isotype of human IgG, like IgG3, binds to FcRI and, when complexed with its cognate antigen, activates complement and binds to FcRli and FcRIII. Conversely, human IgG2 and IgG4 are relatively inactive isotypes; both fail to activate the c~-ssic~l complement pathway and IgG4 binds weakly to FcRI lBurton, D R and Woof, J M (1992) Adv. Immunol. 51, 20 1. Lucisano Valim, Y M and Lachmann, P J. (1991) Clin. exp. Immunol, 84,1].

Loc~lis~tion of amino acid residues of IgG that interact with FcRI in the CH2 domain of human IgG is well est~hlished [Woof, J M et al (1986) 25 Molec. Immunol. ~, 319. Lund, J et al(1991) J. Immunol, 147, 2657;
Canfield, S M and Morrison, S L (1991), J. exp. Med. 173, 1483; Chappel, S M ~1, (1991) Proc. Natl. Acad. Sci. ~, 9036; Chappel, S M et al . (1993), J. Biol. Chem 268, 25124; Alegre, M-L et al (1992) J. Immunol, 1~, 3461]. Amino acid sequence comparisons of the CH2 domains of 30 antibodies from different species and sl~hcl~sses that bind well to FcRI
suygested that a region at the N-terminal end of CH2 comprising residues Leu 234 - Ser 239 (using the Kabat Eu numbering system [Kabat, E A
al, (1987) Sequences of proteins of Immunological interest. US Dept. of Health and Human Services, Bethesda, MD, USA]) is critical for interaction 35 with FcRI. The motif Leu 234, Leu 235, Gly 236, Gly 237, Pro 23~, Ser 239, is present in all IgG isotypes with high affinity for FcRI [Woof, J M et al 2 ~ 6 ~ 3 4 ~ PCT/GB94/01290 (1986~, Molec. Immunoi. 2;~ 319]. Domain exchanges between Ig's with different Fc effector functions have demonstrated the importance of CH2 for FcRI binding [Canfield, S M and Morrison, S L (1991), J. exp. Med. ~, 7 483; Chappel, S M ~l (1991) Proc. Natl. Acad. Sci. ~, 9036; Chappel, 5 S M et al (1993), J. Biol. Chem ~, 25124] in particular the residue 235.
Replacement of the Leu residue at position 235 with a Glu residue reduces the affinity of 19G3 for FcRI by 100 fold [Lund, J et al (1991) J. Immunol, 147,2657; Canfield, S M and Morrison, S L (1991), J. exp. Med. 173, ~483]. The same Leu 235 to Glu change when performed on an IgG4 10 \rariant of OKT3 [Alegre, M-L ~1 (1992) J. Immunol, ~, 3461] abolished its FcRI binding and, conse~ luently, its mitogenic properties.

Although the sequence requirements for FcRIII binding has been less extensively studied, Sarmay et aL [(1992) Molec. Immunol. ~,, 633] have~
15 identified the CH2 domain residues 234 to 237 as important for IgG3 billdi.ly to all three Fc receptors. The relative importance of each residue differs with each Fc receptor with 235 and 237 being most important for FcRIII medr~ed cell killing.

20 111 contrast, another Fc mediated function, C1q binding and subsequent complement activation, appears to require the carboxyl terminal half of ths C H2 domain [Tao, M H., Canfield, S M., and MGn;SOII, S L(1991) J. EXP.
I\~led. 173, 1025]. Morrison's group, following sequence analysis of polymorphisms in the CH2 domain of human IgGs also identified the 25 importance of the C-terminal region of CH2. With a Pro to Ser change at 331 in IgG1 they abolished complement tixdLior, and re~luce~ C1 q binding [Tao, M H et a/ (1993), J. Exp. Med. 1~, 661]. Using inter- and intra-dlomain switch variants of CAMPATH-1, Greenwood et a/. (1993) [Eur. J.
Immunol. 2~, 1098] further endorsed the i"")G,lance of the C-terminal end 30 f CH2. Complement fixation could be restored to human IgG4 with just the carboxyl terminal of CH2 from residue 292 of IgG1 and not the N-terminal half or any other domain. Duncan & Winter (1988) [Nature, 332.
21] id~lllified a motif in CH2 of Glu 318, Lys 320 and Lys 322 of the mouse IgG2b isotype. Changing any of these residues abolished C1q binding, as 35 did the use of competitive peptides of sequences in this region. Howcvcr, the C1q motif residues are also found in antibodies that do not fix 3~ 3 3 ~ 5 PCT/GB94/01290 complement suggesting that these residues may well be necess~ry but not sut~icient for co,nplament activation.

We have found that amino acid residues necessary for C1q and FcR
binding of human IgG1 are located in the N-terminal region of the CH2 domain, residues 231 to 238, using a matched set of engineered antibodies based on the anti-HLA DR antibody L243. Changing the leucine 235 in the CH2 region of IgG3 and IgG4 to glutamic acid was already known to abolish FcRI binding, we have cor,~i""ed this for IgG1 and also 10 found a concomitant abolition of human complement ~i~dlion with ~ter,lio,.
of FcRIII mediated function. Changing the glycine at 237 to alanine of IgG1 also abolished FcRI binding and red~ce~l complement fixation and FcRIII mediated function. Exchanging the whole region 233 to 236, with the sequence found in human IgG2 abolished FcRI binding and-15 complement fixation and reduced FcRIII mediated function of IgG1. Incontrast, a change in the previously described Clq binding motif, from Iysine at 320 to alanine had no effect on IgG1-mediated complement alion.

20 The proposed site Leu 234 - Leu 235 - Gly 236 - Gly 237 - Pro 238 - Ser 239, is present in all IgG isotypes with high affinity for Fc~RI. Recent mutagenesis experiments on IgG3 anliLGJies have introduced point mutations in this region and the ability of the mutants to interact with FcyRI
has been examined [Lund ~t al (1991) J. Immunol 147. 2657-2662]. The 25 most marked effect is seen at position 235 where replacement of the naturally occurring Leu residue with a Glu residue produces an lg with a >100-fold decrease in affinity for FcyRI.

Our observation of the effect of this alteration at residue 235 on the ability 30 of the antibody to fix complement was highly surprising. Earlier protein engineering studies had introduced mutations at various positions in order to locate the C1q-binding site on IgG [Duncan & Winter (1988) Nature, ;~, 738-740]. The binding site for C1q was loc~lised to three side chains, Glu 318, Lys 320 and Lys 322 of the mouse IgG2b isotype. Res;rines Glu 318, 3~ Lys 320 and Lys 322 are conserved in all the human IgGs, rat IgG2b and IgG2c, mouse IgG2a, IgG2b and IgG3, guinea pig IgG1 and rabbit IgG.

wo 94/29351 ~ 16 3 3 ~ 5 PCT/GB9~/01290 Further experiments showed that the affinity of human Clq for mutant mouse IgG2b antibodies in which residue 235 was mutated was unaffected i.e. it was in the same range of values as that obtained with the wild type.
..
5 Although the fact that altering residue 235 of the CH2 region of IgG is known to abolish Fc~RI binding as we too observed, this concomitant substantial reduction in complement fixation has not been reported or suggested elsewhere and was completely unexpected.

10 SILIMMARY OF THF INVFI~ITION
The invention provides a method of treating diseases in which antibody therapy leads to unclesird61e toxicity due to anlibo.ly me~ l;'tecl complement fixation comprising administering an altered antibody wherein one or more arnino acid residues in the N-terminal region of the CH2 domain of said 15 antibody are altered characterised in that the ability of said antibody to fix complement is altered as compared to unaltered antibody.

In a preferred embodiment the altered antibody binds to one or more cellular Fc rec~ tors especi~lly FcRIII and exclùding FcRI i.e. the antibody 20 does not bind siy"i~icantly to FcRI, and more pr~erably binding to FcRI is abolished.

Accordingly in a further aspect the invention provides an altered antibody wherein one or more amino acid resi~uss in the N-terminal region of the 25 C1~2 domain of said alllibo.ly are altered characterised in that the ability of said anliL,ody to fix complement is altered, as compared to unaltered arlti60dy.

In a further preferred embodiment the invention therefore provides an 30 al~ered antibody wherein one or more amino acid residues in the N-terminal region of the CH2 domain of said antibody are altered characterised in that the ability of said antibody to fix complement is altered as compared to unaltered antibody and said altered antibody binds to one or more cellular Fc receptors especially FcRIII and does not bind 35 significantly to FcRI.

~ 33 ~ 6 The constant region of the antibodies to be altered according to the invention may be of animal origin and is preferably of human origin. It may also be of any isotype but is preferably human IgG and most preferably human IgG1.

In a p~e~er,ed embodiment of the invention the amino acid residue(s) which is altered lies within amino acid posilions 231 to 239 preferably within 234 to 239.

10 In a particularly preferred embodiment of the invention the amino acid residue(s) which is altered lies within the motif Leu 234 Leu 235 Gly 236 Gly 237 Pro 238 Ser 239.

In a most preferred embodiment the amino acid residue(s) which is altered 15 is either Leu 235 and/or Gly 237.

D~ Fn DF~CRIPTION OF THF INVF~lllON
As used herein the term 'altered' when used in conjunction with the ability of an antibody to fix complement most usually in-J;G~tes a decrease in the 20 ability of antibody to fix complement compared to the starting antibody. By choosing appropriate amino acids to alter it is possible to produce an antibody the ability of which to fix co""~le."ent is s~bst~ntially reduced such as for example by altering residue Leu 235. It is also possi'~le to produce an antibody with an intermediate ability to fix complsment by for example 25 altering amino acid residue Gl~ 237.

As used herein the phrase 'substantially reduce complement fixation' denotes that human complement fixation is preferably <30% more preferably <~0% and most preferably <10% of the level seen with the 30 starting wild type unaltered antil~-ly.

The term 'significantly' as used with res~.~cl to FcRI binding denotes that the binding of antibody to FcRI is typically <~0% and is most preferably <10% of that seen with unaltered antibody.

wo 94/29351 ~16 3 ~ ~ ~ . PCT/GB94/01290 The altered antibodies of the invention preferably bind to FcRIII as measured by their ability to mediate antibody dependent cellular cytotoxicity (ADCC) at a concentration no greater than ten times that of the wild type unaltered antibody.

The proteins encoded in the Major Histocompatibility Complex region of the genome are involved in many aspects of immunological recognition. It is known that all mammals and probably all vertebrates possess b~sic~lly equivalent MHC systems and that immune response genes are linked to 10 the MHC.

In man the major histocompatibility complex is the HLA gene cluster on chromosome 6. The main regions are D B C and A. The D region contains genes for class ll proteins which are involved in cooperation and 15 interaction between cells of the immune system. Many dise~ses have been found to be associated with the D region of the HLA gene cluster. Studies to date have shown associ~tio"s with an enormous variety of dise~ses including most autoimmune dise~ses (see for e,~a",ple, European Patent No. 68790). European Patent No. 68790 suggests controlling dise~ses 20 ~soci~ted with a particular allele of certain f~y:ollS of the MHC such as the HLA-D region in humans by selectively suppressing the immune response(s) controlled by a monoclonal antibody specific for an MHC-class Il a"liyen.

25 W3 have found that by altering an MHC-class ll specific antibody at position 2~5 in the N-terminal region of the CH2 domain it is possible to produce an antibody which fully retains its immunosuppressive properties but which has s~b~ tially reduced toxicity in vitro and is tolerated in vivo.

30 In a further preferred embodiment the invention provides an MHC specific antibody wherein one or more amino acid resid~es in the N-terminal region of the CH2 domain of said antibody are altered characterised in that the ability of said antibody to fix complement is altered as compared to unaltered antibody.

WO 94/~9351 2-1 6 ~3 ~ !~ PCT/GB94/01290 In a preferred embodiment the invention provides an MHC specific monoclonal antibody characterised in that said antibody has been altered at position 235 of the N-terminal region of the CH2 domain.

5 In some instances such as with MHC specific monoclonal antibodies it may be deslrable that the alteration in the N-terminal region of the CH2 domain of the antibody while altering the ability to fix complement additionally inhibits the binding to FcRI recb~tors.

10 The antibodies are preterdbly specific for MHC-class 11 antigens and due to the alteration of one or more amino acid residues in the N-~emminal region of the CH2 domain will not bind siy"i~icantly to FcRI.

In a further preferred embodiment the altered antibodies of the invention or-15 for use accordi"g to the invention are directed against an MHC class 11anligen characlerisecl in that said antibody has been altered at pOSi~iOI ~ 235 of the N-terminal region of the CH2 domain.

In a particularly preferred embodiment the altered antibodies of the 20 invention or for use according to the invention are ~;fecl6d against an MHC
class 11 antigen characterised in that said antibody has been altered at ~.ositior, 235 of the N-terminal region of the CH2 domain and the ability of said antibody to fix compiement is altered as compared to unaltered antibody and said altered anliL,o.ly binds to one or more cellular Fc 25 rece~lors especi~lly FcRIII and does not bind sig"i~icantly to FcRI.

In a further aspect the invention provides a method for producing an altered antibody with altered ability to fix complement comprising altering one or more amino acids in the N-terminal region of the CH2 domain of 30 said antibody altering the ability of said antibody to fix complement as compared with unaltered anlibody.

As used herein the term 'altered antibody' is used to denote an antibody which differs from the wild type unaitered antibody at one or more amino 35 acid residues in the N-terminal region of the CH2 domain of the Fc region of the antibody. The alteration may for example comprise the substitution WO 94/293~ 1 6 ~ 3 ~ J PCT/GB94/01290 .

or replacement of the starting wild type antibody amino acid by another amino acid, or the deletion of an amino acid residue The residue numbering used herein is according to the Eu index described 5 in Kabat etal[(1991) in: Sequences of Proteins of Immunological Interest, 5th Edition. United States Department of Health and Human Services.]

In human IgG1 and IgG3 antibodies the naturally occurring amino acid at position 235 of the N-terminal region of the CH2 domain is a leucine 10 residue. The alterations at position 235 of replacing leucine by glutamic acid or alanine have been found particularly effective at producing a potent irrlmuno-suppressive antibody with minimal toxicity in vitro and which is tolerated in vivo.

15 The alteration at position 237 of replacing glycine by alanine has been found to produce an antibody with an intermediate ability to fix human complement. i.e. the complement ~ix~lio" level is approximately 15-80%, preferably 20-60%, most pre~e,ably 20-40% of that seen with the starting wild type unaltered antibody.
The residue(s) could similarly be relJl~^ed using an analogous process to that describe.l herein, by any other amino acid residue or amino acid derivative, having for example an inappro~ridte functionality on its side chain. This may be achieved by for example changing the charge and/or 25 polarity of the side chain.

Tlhe altered a,~ ,ocJ;es of the invention may also be produced for example, b~r deleting residues such as 235, or by, for example, inserting a glycosylation site at a suitable position in the molecule Such techniques 30 are well known in the art, see for example the teaching of published European patent application EP-307434.

Tlhe altered antibodies of the invention may also be produced by exchanging lower hinge regions of antibodies of different isotypes. For 35 example a G1/G2 lower hinge exchange abolished complement fixation and is a further pre~er,e.J embodiment of the invention. This is described in WO 94/29351 ~16 3 3 ~ 5 ~ PCT/GB94/01290 I - ~ r~ ~

more detail in the accompanying examples. The G1/G2 lower hinge exchange results in an antibody with altered residues in the 231 to 238 region of the N-terminal region of the CH2 domain wherein one or more residues may be altered and/or deleted.

In a particularly preferred embodiment of the invention the antibody is a human IgG1 antibody directed against an MHC class ll antigen.

In a further aspect the invention provides a method of modulating the 10 function of cell surface associated a"~iye"s avoiding co",~l6",ent medi~ted toxicity comprising administering an altered antibody wherein one or more amino acid residues in the N-terminal region of the CH2 domain of said antibody are altered characterised in that the ability of said antibody to fix complement is altered as compared to unaltered antibody.
In a preferred embodiment of this aspect of the invention said altered antibody is able to bind to one or more cellular Fc rece~tors especially FcRlli while binding to FcRI is siy"i~icar,lly re~Ucerl 20 Examples of such cell surface antigens include for example adhesion molecules, T-cell receplor CD4, CD8 CD3 CD28 CD69 MHC Class 1, MHC Class ll and CD25.

The invention also includes therapeutic pharmaceutical and diagnostic 25 compositions comprising the altered antibodies according to the invention and the uses of these products and the compositions in therapy and diagnosis.

Thus in a further aspect the invention provides a therapeutic, 30 pharmaceutical or diagnostic composition comprising an altered antibody according to the invention, in combination with a pharmaceutically acceptable e~c;~ ie~ ll, diluent or carrier.

The invention also provides a process for the preparation of a therapeutic, 35 pharmaceutical or diagnostic composition comprising admixing an altered ~I WO94/29351 21633~5 PCTIGB9J/01290 antibody according to the invention together with a pharmaceutically acce~ ldble ex ;;piant, diluent or carrier.

The antibodies and compositions may be for administration in any 5 appropriate form and amount according to the therapy in which they are employed.
The altered antibodies for use in the therapeutic, diagnostic, or pharmaceutical compositions, pr for use in the method of treatment of 10 diseases in which antibody therapy leads to undesirable toxicity due to antibody mediated complement fixation are preferably MHC specific antibodies most preferably specific for MHC Class ll antigens, and most preferably have specificity for antigenic determinants dependent on the DRa chain.
The therapeutic, pharmaceutical or diagnostic composition may take any suitable form for administration, and, preferably is in a form suitable for parenteral adminisl,dlio" e.g. by i"jeclio" or infusion, for example by bolus injection or continuous infusion. Where the product is for injection or 20 infusion, it may take the form of a suspension, solution or emulsion in an oily or aqueous vehicle and it may contain formulatory agents such as suspending, preservative, st~nilis "y and/or dis~ersi,lg agents.
Alternatively, the antibody or composition may be in dry form, for 25 reconslilution before use with an a,u~Jropria~e sterile liquid.
If the antibody or composition is suitable for parental administration the formulation may contain, in addition to the active ingredient, additives such a~;: starch - e.g. potato, maize or wheat starch or cellulose - or starch 30 derivatives such as microcrystalline cellulose; silica; various sugars such as lactose; magnesium carbonate and/or calcium phosphate. It is desirable that, if the forrrlulation is for parental adminisl,dlio" it will be well tolerated by the, alie, ll's digestive system. To this end, it may be desirable to include in the formulation mucus formers and resins. It may also be 35 desirable to improve tolerance by formulating the antibody or compositions in a capsule which is insoluble in the gastric juices. It may also be WO 94/29351 ~I 6 3~ C~/GB94101290 preferable to include the antibody or composition in a controlled release formulation.
If the antibody or composition is suitable for rectal administration the 5 formulation may contain a binding and/or lubricating agent, for example polyme~ic glycols, gelatins, cocoa-butter or other vegetable waxes or fats.
The invention also provides methods of therapy and diagnosis comprising administering an effective amount of an altered antibody according to the invention to a human or animal S~ jeCt The antibodies and compositions may be for administration in any appropriate form and amount accordi"g to the therapy in which they are employed. The dose at which the anlibody is administered d6pel Ids on the nature of the condition to be treated and on whether the antibody is being.
15 used prophylactically or to treat an existing con.lilio". The dose will also be selected according to the age and conditions of the patient. A
therapeutic dose of the antibodies accGr~Ji"g to the invention may be, for example, preferably between 0.1-25mg/kg body weight per single therapeutic dose and most ~re~erably ~el~:sen 0.1-10mg/kg body weight 20 per single therapeutic dose.

Immunological dise~ses which may be l~ated with the antibodies of the invention include for example joint dise~.se such as ankylosing spondylitis, juvenile rheumatoid arthritis, rheumatoid arthritis; neurological disease 25 such as multiple sclerosis; pa"crealic ~ise~-se such as dial~etes, juvenile onset diabetes; gastrointestinal tract dise~c-e such as chronic active hep~titis, celiac ~ise~se, ulcerative colitis, Crohns dise~se, pernicious anaemia; skin ~ise~ses such as psoriasis; allergic dise~ses such as asthma and in trans~lal,~aliG" related conditions such as graft versus host 30 dice~se~ and allograft rejection. Other lise~ses include those described in European Patent No. 68790.

The altered antibodies of the invention may also be useful in the treatment of infectious dise~ses e.g. viral or bacterial infections and in cancer 35 immunotherapy.

WO 94/29351 2 16 3 3 4 ~ PCT/GB94/01290 As used herein the term 'antibody' is used to cover natural antibodies, chimeric antibodies and CDR~ afle.J or humanised antibodies. Chimeric antibodies are antibodies in which an antigen binding site comprising the complete variable domains of one antibody is linked to constant domains 5 derived from another antibody. Methods for carrying out such chimerisation procedures are described in EP 120694 (Celltech Limited), EP 125023 (Genentech Inc and City of Hope), EP 171496 (Res. Dev.
Corp. Japan), EP 173494 (Stanford University) and WO 86/01533 (Celltech Ltd). CDR grafted or humanised antibodies are antibody mol~cules having 10 an antigen binding site derived from an immunoglobulin from a non-human species and remaining immunogloblJlirl-derived parts of the molecule being derived from a human immunoglobulin. Procedures for generating CDR-gldfled or humanised antibodies are desc,il,ad in WO 91/09967 (Celltech Ltd), WO 90/07861 (r,olei" Design Labs. Inc) and WO 92/11383 (Celltech 15 Ltd).

In further aspects the invention also includes DNA sequences coding for the altered antibodies according to the inv~"liG"; clo"iny and expression vectors containing the DNA sequences, host cells transformed with the 20 DINA sequences and processss for producing the altered antibodies according to the invention CG~ .lisillg expressing the DNA sequences in the transformed host cells.

According to a further aspect of the invention there is provided a process 25 for producing an altered antibody of the invention which process comprises:

a. producing in an expression vector an operon having a DNA
sequence which encodes an antibody heavy or light chain.
30 b. producing in an expression vector an operon having a DNA
sequence which encodes a complementary antibody light or heavy chain.
c. transfecting a host cell with both operons, and d. culturing the transfec~ed cell line to produce the antibody molecule WO 94/293!;1 , PCT/GB94/01290 2 ~ ~ 3 3 ~ ~ 14 wherein at least one of the expression vectors contains a DNA sequence encoding an antibody heavy chain in which one or more amino acid residues in the N-terminal region of the CH2 domain of said antibody has been altered from that in the corresponding unaltered a~,libody.

As will be readily apparent to one skilled in the art, the alteration in the N-terminal region of the CH2 domain may be made using techniques such as site directed mutagenesis after the whole altered antibody has been expressed. To express unaltered antibody the DNA sequences should be 10 ex~ressed following the teaching described above for altered antibody.

The DNA sequences preferably encode a humanised antibody; a CDR-grafted heavy and/or light chain or a chimeric antibody.

15 The cell line may be tran~tec~ecl with two vectors, the first vector containing the operon encoding the light chain-derived poly,ue~c~ide and the second vector containing the operon encoding the heavy chain derived polylJe~tide. Preferably the vectors are identical except in so far as the coding sequences and select~hle markers are concerned so as to ensure 20 as far as possi'~'e that each poly~ Je chain is equally ex~ ssed.

Alternatively, a single vector may be used, the vector including a selectable marker and the operons encoding both light chain- and heavy chain-derived pol~3e~lides.
The general methods by which the vectors may be constructed, lld"s~ection methods and culture methods are well known ~er se. Such methods are shown, for instance, in Maniatis ~L Molecular Cloning, Cold Spring Harbor, New York 1989 and Primrose and Old, Principles of Gene 30 Manipulation, Blackwell, Oxford, 1980.

The altered antibody according to the invention is preferably derived from the anti-MHC antibody L243, which has been deposited at the American Type Culture Collection, Rockville, Maryland USA under Accession number 35 ATCC HB5~, and is most preferably a chimeric or a CDR-grafted derivative W0 94/29351 ~16 3 3 ~ ~ PCTIGBg4/0l290 thereof. L243 was previously described by Lampson and Levy [J.
Immunol. (1980) 125, 293].

The standard techniques of molecular biology may be used to prepare DNA sequences coding for the altered antibodies according to the invention. Desired DNA sequences may be synthesised completely or in part using oligonucleotide synthesis techniques. Site-directed mutagenesis and polymerase chain reaction (PCR) techniques may be used as appropriate. See for example "PCR Technology Principles and 10 Applications for DNA Ampli~ication~ (1989), Ed. H. A. Erlich, Stockton Press, N.Y. London. For example, oligonucleotide directed synthesis as described by Jones et a/ [Nature, 321, 522 (1986)~ may be used. Also oligonucleotide directed mutagenesis may be used as described by Kramer ~1 [Nucleic Acid Res. 12 9441 (1984)].
Any suitable host celUvector system may be used for the expression of the DNA sequences coding for the altered antibody. Bacterial e.g. E.coli and o~her microbial systems may be used. Eucaryotic e.g. mammalian host cell expression systems may also be used such as for example COS cells 20 and CHO cells [Bebbington, C R (1991) Methods 2. 136-145], and myeloma or hybridoma cell lines [Bebbington, C R ~ (1992) Bio/Technology 10. 169-175].

V~here the altered antibody is derived from L243 CHO based expression 25 systems are preferably used.

Assays for determining FcRIII binding indirectly via ADCC assays and for determining cor",~len,ent ~ixalioll and Clq LJ .,d;n9 are well known in the art,and are describe.l in detail in the following examples.
Immune function/immunosu~.~.ressio" by antibodies may be assayed using techniques well known in the art including for example: Mixed Lymphocyte Responses and T-cell antigen recall responses to Tetanus Toxoid. These assays are described in detail in the following examples.

WO 94/29351 21 6 3~ ~ ~ . PCT/GB94/01290 The invention is illustrated in the following non-limiting examples and with re~ere"ce to the following figures in which:
Figure 1 shows: a map of plasmid pMR15.1 Figure 2 shows: a map of plasmid pMR14 5 Figure 3 shows: the nucleotide sequence and predicted amino acid sequence of L243 heavy chain Figure 4 shows: the nucleotide and amino acid sequences of (a) clone 43, (b) clone 183 (c) clone 192 Figure 5 shows: the nucleotide sequence and ~,red;cted amino acid sequence of L243 light chain Figure 6 shows: a map of plasmid pGamma 1 Figure 7 shows: a map of plasmid pGamma 2 Figure 8 shows: the nucleotide sequence of hinge and CH2 region of human C-gamma 1 15 Figure 9 shows: Antigen binding potency of L243 human isotype series G1 ~ G4 ~L235q G1 [L235A] ~ G4 G1 [G237Al ~ 100%
+ G1 [K320A]
20 Figure 10 shows: FcRI binding of L243 isotype series + G1 [G237A] X G1 [L235A]
G2 ~ G1/G2-L-hinge ~ G1 [L235E] ~ G4 [L235q ~ G1 [K320A]
Figure 11 shows: human complement ~ dLiol I by L243 isotype series G1 ~ G4 G1 [G 237A] ~ G1 [L235A]
~ G2 ~ G1/G2 L-hinge ~ G1 [l~23~q ~ G4 [L235E
~ G1 [K320A]
Figure 12 shows: binding of human Clq to L243 human isotype series - I G1 ~ Cells alone ~ G4 [L235E] ~ Cells + C1q X G1 [L235E] + G1 [K320A]
~ G4 WO 94/29351 2 ~L 6 3 3 ~ 5 PCT/GB94/01290 F-igure 13 shows: human complement fi~alioi) by L243 isotype G1 7~ G1 ~L235A]
G2 ~ G4 [L235E]
~ G1 [L235E] ~ G1 [K320A]
~ G4 Figure 14 shows: guinea pig complement fixation by L243 isotype G1 ~ G1 [L235A]
G2 ~ G4 [L235E]
~ G1 [L235E] + G1 [K320A]
-~ G4 F:igure 15 shows: rabbit complement tiAtlLiCil I by L243 isotype G1 ~ G1 [L235A]
G2 ~ G4 [L235E]
~ G1 [L235E] ~ G1 [K320A]
~ G4 F:igure 16 sl ,o~r;s. FcRIII binding of L243 isotype series by ADCC
+ G1 ~ G4 + G1 [K320A] X G1 [G237A]
~ G2 v G1/G2 L-hinge ~_ G1 [L235A] ~ G4 [L235q ~ G1 [L235q Figure 17 shows: L243 Isotype Series Inl ,i~i~iorl of 1~ recall response ~ G2 ~ G4 C~ ;lhSil)C;I il l ~ medium control Figure 18 shows: L243 Isotype Series Inhil,ilior, of rr recall response ~ hG1 ~ G1/G2 L-hinge + hG1 [L235E] El G4 [L235E]
O medium control ~ cyclosporin Figure 19 shows: L243 Isotype Series ll Ih i~ iGn of Mixed Lymphocyte Re~ction.
~ hG1 ~ G1/G2 L-hinge ~ hG1 [L235E] ~ G4 [L235E]

~16334~ 18 cyclosporin ~ medium control Figure 20 shows: L243 Isotype Series Inhibition of TT response I G1 [L235A]
G1 [G237A]
O Cyclosporin ~ Medium control Figure 21 shows: L243 Isotype Series Inhibition of Mixed Lymphocyte Re~ction ~ G1 [L235y + G1 [L235A]

Cyclosporin ~ Medium control Figure 22 shows: the nucleotide and amino acid sequence of Vl region in L243-gL1 Figure 23 shows: shows the nucleotide and amino acid sequence of Vl region of L243-gL2 Figure 24 shows: the nucleotide and amino acid sequence of Vh region of L243-gH
Figure 25 shows: a graph of the results of a com~,elilior~ assay for L243 grafts vs FlTC-chimeric L243 ~ cH cL
cH gL1 gHcL
q gH gL1 Figure 26 shows: a graph of a Scatchard analysis for L243 gamma 4 _ cHcL Kd=4.1nM
gH gL1 Kd = 6.4nM
~ gH gL2 Kd = 9.6nM
Figure 27 shows: a graph of FcRIII binding of chimeric, gla~led and grafted [L235E] L243 as measured by ADCC
~ ChimericG1 wt ChimericG1 [L235E]

WO 94/29351 ~16 ~ 3 4 S PCT/GB94/01290 GraftG1wt El GraftG1 [L235E]
Figure 28 shows: a graph of immunosuppressive activity of CDR y~ ed L243 measured by MLR
~ GraftG1 wt + GraftG1 [L235E]
Cyclosporin Chimeric Gl wt ~ Chimeric G1 [L235E]
~ Medium Control Figure 29 shows: a graph of CDR yld~l~ L243 and 5J,atla~l [L235E]
L243 TT recall re~po"se GraftG1wt GraftG1 [L~235q ~ Cyclosporin Chimeric G1 wt 0 ChimericG1 [L235E]
~ Medium Control Figure 30 shows: a graph of complement "~ed;~ cytotoxic potency of CDR y~dtl6~ L243 and CDR y,alled [L235E] L243 Chimeric G1 wt ChimericG1 [L235q ~1~ GraftG1 wt ~ Graft G1 [L235E]

nFTAII Fn nF~:CRlpTloN OF SPFCIFIC EMBODIMENTS
OF Ti-lF INVFNTION

F~AMpl ~!;
hcample 1 Gene Cloning and Expr~s 9iG
RNA pr~r~r~tion from 1 ~43 hy~ri.lo",~ cells Total RNA was prepared from 3 x 10exp7 L243 hybridoma cells as 3~ described below. Cells were washed in physiological saline and dissolved in RNAzol (0.2ml per 1 Oexp6 cells). Chloroform (0.2ml per 2ml WO 94129351 ~63~ 4S PCT/GB9~101290 ~

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 centrifuge and RNA precipitated from the aqueous phase by the addition of an equal volume of isopropanol. After 15 minutes on ice, the RNA was peileted by centrifugation, washed with 70% ethanol, dried and dissolved in sterile, RNAase free water. The yield of RNA was 350 119.

Amino ~id se~uence of the 1~43 light ch~in.
The sequence of the first nine amino acids of the mature L243 light chain was determined to be NH2-DIQMTQSPAS.

PCR cloning of 1~43 Vh ~nd Vl The cDNA genes for the variable regions of L243 heavy and light chains were synthesised using reverse transcriptase to produce single stranded cDNA copies of the mRNA present in the total RNA, followed by Polymerase Chain Reaction (PCR) on the cDNAs with specific oligonucleotide prilller:j.

20 a) ~nNA synthesis cDNA was synthesisesl in a 20~LI reaction containing the following reagents: 50mM Tris-HCI PH8.3, 75mM KCI, 10mM dithiothreitol, 3mM MgCI2, 0.5mM each deoxyribonucleoside triphosphates, 20 units RNAsin, 75ng random hexanuclQolide primer, 2~Lg L243 RNA
and 200 units Moloney Murine Leukemia Virus reverse transo,i~lase. After incub~tion at 42C for 60 min the reaction was terminated by heating at 95C for 5 minutes.

b) ~B
Aliquots of the cDNA were suLjected to PCR using combinations of primers for the heavy and light chains. The nucleotide sequences of the 5' primers for the heavy and light chains are shown in Tables 1 and 2 respectively. These sequences, all of which contain a restriction site starting 6 nucleotides from their 5 ends, followed by the sequence GCCGCCACC to allow optimal translation of the resulting mRNAs, an initiator codon and a further 20 - 30 nucleotides, are a compilation based on the leader peptide sequences of known mouse antibodies [Kabat et al (1991) in Sequences of Proteins of Immunological Interest, 5th Edition -United States Department of Health and Human Services].

The 3' primers are shown in Table 3. The light chain primer spans the V - C junction of the antibody and contains a restriction 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 Immunol~Ji~l Interest, 5th Edition, US Department of Health and Human Services (1991)].

The combinations of primers described abovs enables the PCR
products for Vh and Vl to be cloned directly into the a~ rop,ia~e eA~.ression vector (see below) to produce chimeric (mouse - human) heavy and light chains and for these genes to be expressed in mammalian cells to produce chimeric antibodies of the desired isotype.
Incub~l;o,-s (20 ~I) for the PCR were set up as follows. Each reaction contained 10 mM Tris-HCI pH 8.3, 1.5 mM MgCI2, 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. Reactions were incub~ted 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 electrophoresis 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 216334~

expected 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 hybridoma cell.
c) Molecular cloning of the PCR fr~gments DNA fragments produced in reactions B2, B3 and B5 were digested with the enzymes BstB1 and Spl1, concentrated by ethanol precipitation, eleclropl,oresed on a 1.4 % agarose gel and DNA
bands in the range of 400 base pairs recovered. These were cloned by ligation into the vector pMR1~.1 (Figure 1) that had been restricted with BstB1 and Spll. After ligation, mixtures were transformed into E. coli LM1035 and plasmids from the resulting bacterial colonies screened for inserts by cl;geslio" with BstB1 and Spl1. Represel ,lali~es with inserts from each liyd~ n were analysed further by nucleotide sequencing.

In a similar manner, the DNA fragments produced in reaction B9 and digested with Hindlll and Apa1 were cloned into the vector pMR14 (Figure 2) that had been restricted with Hindlll and Apa1.
Again, re~r~s6ntali~e plasmids containing inserts were analysed by nucleotide sequencing.

d) Nucleotiule sequence ~n~lySjS
Plasmid DNA (pE1701 and pE1702) from two isol~es containing Vh inserts from reaction B9 was sequenced using the primers R1053 (which primes in the 3' region of the HCMV promoter in pMR14) and R720 (which primes in the 5' region of human C - gamma 4 and allows sequencing through the DNA insert on pMR14). The determined nucleotide sequence and predicted amino acid sequence of L243 Vh in pE1702 is given in Figure 3. The nucleotide sequence for the Vh insert in pE1701 was found to be identical to that in pE1702 except at nucleotide 20 (A in pE1701) and nucleotide 426 (A in pE1701). These two differences are in the signal peptide and J regions of Vh respectively and indicate that the two clones WO 94/29351 21~ 3 3 4 ~ ~ ; PCT/GB91/01290 1~

examined are independent isolates arising from the use of different primers from the mixture of oligonucleotides during the PCR stage.

To analyse the iight chain clones, sequence derived from priming with R1053 was examined. The nucleotide sequence and predicted amino acid sequence of the Vl genes arising from reactions B2 (clone 183), B3 (clone 43 and B5 (clone 192) are shown in Figure 4. Comparison of the predicted protein sequences shows the following:
i) clones 182, 183, 43 and 45 all code for a Vl gene which, when the signal peptide is removed, produces a light chain whose sequence is identical 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 ~e~ide of 20 amino acids, while the Vl gene in clones 43 and 45 results from priming with a different set of oligonucleotides and has a leader sequence of only 15 amino acids.
iii) Clone 192 does not code for L243 Vl. Instead, examination of the t~:3t~ ce of antibody sequences [Kabat, 1991] indicates that clone 192 contains the Vl gene for MOPC21, a light chain synthesised by the NS1 myeloma fusion partner used in the production of the L243 hyiJ, i.lGma.
iv) Clones 182 and 183 are id6"lical except at nucleotide 26 (T in clone 182, C in clone 183). This difference can be accounted for by the use of clitterei,l primers in the PCR and indicates that clones 182 and 183 are independent isolates of the same gene.
The nucleotide sequence and predicted amino acid sequence of the cG"".lete Vl gene from clone 183 is shown in Figure 5.
Construction Qf human gamma 1 and ~amma 2 isotyDes.
35 The L243 Vh gene was subcloned on a Hindlll - Apal fragment into pGamma 1 and pGamma 2, vectors containing the human C - gamma 1 and C - gamma 2 genes respectively (Figures 6 and 7).

WO94/~9351 21~33~5 PCT/GB9J/01290 Hum~n Isotype mut~nts PCR mutagenesis was used to change residue 235 in human C - gammal contained in the vector pGamma 1 from leucine to either glutamic acid or to 5 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 changes:

i) L235E change R4911 5' GCACCTGMCTCGAGGGGGGACCGTCAGTC3' R4910 5'CCCCCCTCGAGTTCAGGTGCTGAGGMG3' Il) L235A change R5081 5'GCACCTGMCTCGCAGGGGGACCGTCAGTC3' R5082 5'GACTGACGGTCCCCCTGCGAGTTCAGGTGC3' Ill) G237A change R5088 5'GCACCTGMCTCCTGGGTGCACCGTCAGTC3' R5087 5'GACTGACGGTGCACCCAGGAGTTCAGGTGC3' IV) Exchange of lower hinge regions R4909 5'GCACCTCCAGTGGCAGGACCGTCAG l ~;1 I CCTC3' R4908 5'CGGTCCTGCCACTGGAGGTGCTGAGGMGAG3' Other oligonucleotides used in the PCR mutagenesis are:

R4732 5'CAGCTCGGACACC I 1~; l c; I CCTCC3' R4912 5'CCACCACCACGCATGTGACC3' R4732 and R4912 prime between nucleotides 834 and 858 and between nucleotides 1156 and 1137 respectively in human C - gamma 1 (Figure 8).

The general strategy for the PCR mutagenesis was as follows. For each 35 amino acid change, two rounds of PCR were used to generate DNA
fragments containing the required substitutions. These fragments were ~I 633~5 then restricted with the enzymes Bgl ll and Sty1 and used to replace the corresponding fragments containing the wild type sequence in the pGamma 1 vector, (Figure 6).

For the first round PCR, reactions (20 1ll) 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 ng pQamma 1 DNA, 0.4 unit Taq polymerase and 6 pmoles of each of the primer. The following combinations 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 electrophoresad on a 1.4 % agarose gel. Gel slices containing the DNA
fragments were excicesl from the gel, the DNA recovered from the agarose using a "Mermaid~ kit (from ~;tldtecl, Scienliric Ltd., Luton, England) and eluted into 20~LI sterile water.

S~3cond round PCR was in a 100 1ll reaction containing 10 mM Tris - HCI
pH 8.3, 1.5 mM MgCI2, 50 mM KCI, 0.01 % gelatin, 0.25 mM each deoxyribonucleeside triphosphate, 2 units Taq polymerase, 1/20 of each pair of DNA fragments from the first round reaction and 30 pmoles of each of R4732 and R4912. After 30 cycles, see above, the reactions were extracted with phenol / chloroform (1/1) and precipitated with ethanol.
Fragments were tiigested with Bgl11 and Sty1, electrophoresed on a 1.4 %
agarose gel and DNA bands of 250 base-pairs recovered from gel slices as previously described.

WO 94/293~;1 21 ~ t ' PCT/GB94101290 These Bgl ll - Sty1 fragments were ligated in a 3 - way ligation to the 830 base-pair Sty1 - EcoR1 fragment, containing the C - terminal part of the CHZ domain and the entire CH3 domain of human C - gamma 1, and the 5 Bglll - EcoR1 vector fragment from pGammal (see Figure 6). After transformation into LM1035, plasmid minipreps from resulting colonies were screened for the presence of the Bgl ll - Sty1 fragment and representatives of each taken for nucleotide sequence analysis. From this, plasmids containing the desired sequence were i~le"li~ied and, for future 10 re~ere"ce, named as follows:

pGamma1 [L~23~E] containing glutamic acid at residue 235, pGamma1 ~L~35A] containing alanine at residue 235, pGamma1 [G237A] containing alanine at residue 237, 15 pGamma1 [91--~92] containing the C-gamma 2 lower hinge region.
The above plasmids were each resl~icted with Hind111 and Apa1 and the Hind111 - Apa1 fragment containing L243 Vh inserted to produce the follow;"g plasmids:

20 L243Gamma1 [L235E]
L243Gamma1 lL235A]
L243Gamma1 [G237A]
L243Gamma [91--,92]

25 a) Production of chimeric i ~43 ~nliL)Gdy Antibody for biological evaluation was produced by transient expression of the appropriate heavy and light chain pairs after co-transfection into Chinese Hamster Ovary (CHO) cells using calcium phosphate preci~Ji~li~".
On the day prior to transfection, semi - confluent flasks of CHO-L761 cells were tr~si"ised, the cells counted and T75 flasks set up each with 1 Oexp7 cells.

35 On the next day, the culture medium was changed 3 hours before transfection. For transfection, the calcium phosphate precipitate was WO 94/29351 216 33 ~ 5 ` ~ PCT/GB94/01290 prepared by mixing 1.25 ml of 0.25M CaCI2 containing 50 ~lg of each of heavy and light chain expression vectors with 1.25 ml of 2xHBS (16.36 gm ~laCI, 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 5 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 glycerol was removed, the cells washed once with PBS and inc~lh~ted for 48 - 96 hours in 25 ml medium containing 10 mM sodium butyrate. Antibody was 10 purified from the culture medium by binding to and elution from protein A -Sepharose and quar,lilatecl using an lg ELISA (see below).

b) ELISA
For the ELISA, Nunc ELISA plates were coated overnight at 4C with a 15 F(ab)2 fragment of a polyclonal goat anti-human Fc fragment specific antibody (Jackson Immuno-research, code 109-006-098) at 5 ~lg/ml in coating buffer (15mM sodium carbonate, 35mM sodium hydrogen carbonate, pH6.9). Unco~ted antiboJy was removed by washing 5 times with ~lislilled water. Samples and purified standards to be quantitated were 20 diluted to a~ ~.roxi-"ately 1 llg/ml in conjugate buffer (0.1M Tris-HCI pH7.0, 0.1M NaCI, 0.2% v/v Tween 20, 0,2% w/v Hammersten casein). The samples were lit.~ted in the microtil.e 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 shaking. After the first incuh~tion step the plates 25 were washed 10 times with distilled water and then incub~ted for 1 hr as b~fore with 0.1 ml of a mouse monoclonal anti-human kappa (clone GD12) p~roxid~s~ conjugated antibody (The Binding Site, code MP135) at a - d~lution of 1 in 700 in conjugate buffer. The plate was washed again and sul~sl-~dte solution (0.1 ml) added to each well. Sul)~l-ale solution contained 30 150 1ll N,N,N,N-tetramethylbenzidine (10 mg/ml in DMSO), 150 ~l hydrogen peroxide (30% solution) in 10 ml 0.1M sodium ~Get~t~/sodium citrate, pH6Ø The plate was developed for 5 -10 minutes until the albsorbance at 630nm was approximately 1.0 for the top standard.
Absorbance at 630nm was measured using a plate reader and the 35 conce"l,dtion of the sample determined by comparing the titration curves with those of the standard.

WO 94/29351~16 3 3 ~ 5 PCT/GB94/01290 T~RI F 1 Oli~onucleotide primers for the 5' region of mouse h~vv chains.

CH1: 5'ATGAMTGCAGCTGGGTCAT(G,C) ~ 3' 10 CH2: 5'ATGGGATGGAGCT(A,G)TATCAT(C,G)(C,T) ~ 13' CH3: 5'ATGMG(A,T)TGTGGTTAAACTGGGI I 1 1;3' CH4: 5'ATG(G,A)AC; I I I (iGG(T,C)TCAGCTTG(G,A)T3' CH5: 5'ATGGACTCCAGGCTCM I I I AG I I I 13' CH6: 5'ATGGCTGTC(C,T)T(G,A)G(G,C)GCT(G,A)C; I ~ G3' .

20 CH7: 5'ATGG(G,A)ATGGAGC(G,T)GG(G,A) 1~; l 1 1 (A,C) 1~; l 13' CH8: 5'ATGAGAGTGCTGA I ~ I I I GTG3' CH9: 5'ATGG(C,A)TTGGGTGTGGA(A,C)CTTGCTATT3' CH10: 5'ATGGGCAGACTTACA I 1~;1 CATTCCT3' CH11: 5'ATGGATmGGGCTGAI 1 ~ I ATTG3' 30 CH12: 5'ATGATGG l ~i I I MG l ~ i l ACCT3' Each of the above primers has the sequence 5'GCGCGCMGCTTGCCGCCACC3' added to its 5' end.

6 3 3 ~ S PCT/GB94/01290 T~RI F 2 Oli~onucleotide primers for the 5' region of mouse li~ht chains.

CLl: 5'ATGMGTTGC(; I G I i AGGC l (~ GGTGCT3' 1 0 CL2: 5'ATGGAG(T,A)CAGACACACTCCTG(T,C)TATGGGT3' CL3: 5'ATGAGTGTGCTCACTCAGGTCCT3' CL4: 5'ATGAGG(G,A)CCCCTGCTCAG(A,T)TT(C,T)TTGG3' CL5: 5'ATGGAI I I(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' CL7: 5'ATGGGC(T,A)TCMGATGGAGTCACA3' CL8: 5'ATGTGGGGA(T,C)CT(G,T) I I I (T,C)C(A,C)(A,C) I I I I I CA
AT3' CL9: 5'ATGGT(G,A)TCC(T,A)CA(G,C)CTCAGTTCCTT3' CL10: 5'ATGTATATA I (~i I I I ~i I I G I c; I AmC3' 30 CL11: 5'ATGGMGCCCCAGCTCAGC; I 1 Each of the above primers has the sequence 5'GGACTGTTCGMGCCGCCACC3' added to its 5' end.

WO 94/29351 . PCT/GB94/01290 21633~ 30 Oliqonucleotide Drimers for the 3' ends 5of mouse Vh and Vl genes.

Light chain ( CL12 ):
5'GGATACAGTTGGTGCAGCATCCGTACG I I l 3' Heavy chain ( R2156 ):

5'GCAGATGGGCCCTTCGTTGAGGCTG(A,C)(A,G)GAGAC(G,T,A)GTGA3' ~R~ ~ 4 5' Primer mixtures for PCR

B1: CL2.
B2: CL6.
B3: CL8.
25 B4 : CL4, CL9.
. B5 : CL1, CL3, CL5,CL7, CL10, CL11.
B6: CH6.
B7: CH7.
B8 : CH2, CH4.
B9 :CH1, CH3, CH5, CH8, CH9, CH10, CH11, CH12.

WO 94/29351 ~ 1 6 3 3 4 5 PCT/GB94/01290 .xample 2 r~iological ~ tiE~ of engineered~
l~he aim of the following experiments was to separate the immunosuppressive effects of anti-MHC-II antibodies from possible toxic 5 consequences of their use. In the process we hope to demonstrate which F:c effector functions are necess~ry for immunosuppression.

~Nllr~F~I BINDING POTF~CY BY INHIR'TION ASSAY
1~he principle of this experime,nt is that antibodies that have the same 10 binding will compete equally well with a l~helleçl antibody for their cognateantigen. Any changes in the antigen binding potency of the engineered L243 antibodies will be revealed in this system.

I\/lurine L243 (IgG2a) was labelled with fluorescein (FITC) using standard 15 techniques. All dilutions, manipulations and incubations were done in Phosphate Buffered Saline (Gibco UK) containing 0.1% Sodium Azide (3DH UK) and 5% Foetal Calf Serum (Sigma UK). Serial dilutions of engineered antihod;es in 100111 in RB polystyrene tubes (2052 12x75mm Falcon UK) were premixed with a constant amount in 100,u1 (at a previously 20 dletermined optimal concentration) of the labelled antibody on 5x104 i"d;cator cells (JY B Iymphoblastoid cell line bearing high levels of HLA-DR). Cells and antibody were incub~ted together at 4C for 30min, washed twice and binding revealed using a Fluorescence Activated Cell Scanner (FACS Becton Dickinson). After appropriate analysis, median 25 fluorescence intensity is plotted against antibody concenl,ation.

,Re~un~
~s eYrected, none of the changes in the Fc portion of the molecule allect6~ antigen binding ability (Figure 9).
/~55~ MENT OF FCy Rl BINDINt:
The ability of the engineered variants of L243 to bind to FcgRI was measured. The ~ri"c;ple of this experiment is that antibodies will bind to cells through Fc recertors and the affinity of this interaction is determined 35 by the subclass and hence the structure of the Fc of the antibody. The WO 94/29351 ~ 1 6 3 ~ 4 S : PCT/GB94/01290 -assay is based on the ability of the engineered antibodies to compete for binding with FITC labelled murine IgG2a to IFN~stimulated U937 cells.

U937 (myelomonocytic) cells when incubated with 50011/ml IFN~
5 (Genzyme UK) for 24 hours expresses high levels of FcgRI as ~sse.ssed by CD64 binding and monomeric IgG2a binding low levels of Fc~RII and no FcyRIII.

U937 cells are washed extensively in DMEM containing 25mM HEPES
10 (Gibco UK) incub~ted for 2 hours at 37C in RPMI 1640 (Gibco UK) and then washed again in DMEM containing 25mM HEPES (Gibco UK) to remove bovine IgG bound to Fc receplors. Serial dilutions of engineered antibodies were prepared in 50u1 in Phosphate Buffered Saline (Gibco UK) containing 0.1% sodium azide in V-bottom 96 well microtitre plates 15 (ICN/Flow UK) and were incuh~t.s~ with 5x104 U937 cells in 50111 for 30min at 4C. 50~1 of FITC labelled IgG2a a~ o.ly was then added to all wells at a previously determined optimal concenl, lio" for a further 90min at 4C. Cells were washed once in the microtll,e tray tra"sfer~ed to RB
polystyrene tubes (2052 12x75mm Falcon UK) washed once again and 20 binding was revealed using a FluGresce"ce Activated Cell Scanner (FACS
Becton Dickinson). After appro~criate analysis median fluorescence inle,)si~y is plotted against an~ilJody co"cen~l~lio,l.

~ A ~ ~ ItC
25 Cl,a"yes in the N-terminal region of the CH2 domain of IgG1 had profound effects on binding to FcRI (Figure 10). As exrecte~ wild type IgG1 bound well to FcRI IgG4 about 10 times less well and IgG2 did not bind at all.
We have confirmed that the Leu 235 to Glu change in human IgG4 reduced its low FcRI binding to nothing and that the same change in IgG1 30 completely abolishes FcRI binding. Ala at 235 reduced (by about 100 fold) but did not ablate FcRI binding. Changing Gly 237 to Ala of IgG1 also abolished FcRI as did exchanging the whole region 233 to 236, with the sequence found in human IgG2. The G1[K320A] change had no effect on FcRI binding.

~ WO 94/29351 216 3 3 ~ 5 PCT/GB94/01290 ~NTIBODY DEPF~IDENT COMPI FMENT
~FnlATFn C~Y I U I ~XICITY~
The ability of the engineered variants of L243 to fix human complement 5 was ~ssessed using the technique of antibody dependent complement mediated cytotoxicity.

The principle of the experiment is that antibodies will mediate complement Iysis of target cells bearing their cog"atQ antigen if the Fc of the antibody is10 able to interact with the co""uo"ents of the (usually cl~ssic~l) 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 endoloxi" free glass bottles which is then allowed to clot' 15 at 37C for 1 hour. The clot is detached from the glass and then incub~ted at 4C for 2 hours to allow it to retract. The clot is then removed and the serum separated from thQ remaining red cells by centrifugation at 10009.
Once prepared, the serum can be stored for up to one month at -20C
without nolice~l~le deLerioralio" of p~fle"cy but is best used fresh.
~ll manipulations, dilutions and incubations are done in RPMI 1640 medium (Gibco UK) containing 2mM Glutamine (Gibco UK) and 10% foetal calf serum (Sigma UK). Target cells (JY B Iymphoblastoid cell line bearing high levels of HLA-DR) are l~helled with 1mCi Na51Cr for 1 hour at room 25 temperature, ayilated every 15 min. The cells are then washed three times, to remove free radiolabel, and resuspended at 2xl06/ml. Serial antiL,o-ly dilutions are prepared in duplicate in V-bottom 96 well microlil,e plates (ICN/Flow UK) in 25~11. Control wells containing medium only are aLlso prepared to establish the spontaneous release of label giving the 30 aLssay background. Target 51Cr l~hellecl JY cells are added to all wells in 10~11. The same number of JY cells are also added to wells containing 2%
1'riton x100 in water to establish the 100% release value. Target cells and antibody are incubated together and, after 1 hour at room temperature, 25,ul serum as a source of complement is added to all wells (except the 35 100%) for a further 1 hour at room temperature. 100111 of EDTA saline at 4C is then added to stop any further cell killing, the microlil~e plates are WO 94/29351 Z ~. ;6 3 3 ~ ~ PCT/GB94101290 ' 34 centrifuged at 2009 to pellet the intact cells and 100~1 supernatant is removed and counted in a gamma counter.

Percent celi Iysis is calculated by subtracting the background from all 5 values and then expressing them as a percentage of the adjusted maximum release. Replicates vary by less than 5%. Percent cell Iysis is then plotted against antibody dilution.

Re~lts 10 The ability of L243 to fix human complement was not affected by all the changes made in the N-temminal region of the CH2 domain, resi~lJes 233 to 237 (Figure 11). Wild type IgG1 mediated potent killing with 600ng/ml giving half maximum cell killing (64% maximum). IgG2 and IgG4 caused no cell killing even at 20~Lg/ml. The Gly to Ala at 237 gave an intermediate~
15 level killing (20% maximum killing at 2~g/ml). Exchanging the whole lower hinge region with the sequence found in human IgG2 failed to cause Iysis even at 20~Lg/ml. Changes at 235 in IgG1 had unexpectedly profound effects on human complement ~ixdliGIl. Changing the Leu 235 to Glu abolished complement Iysis (no killing at 20~g/ml). Ala at 235 permitted 20 low levels of killing. In contrast, a change in the previously described Clq binding motif [Duncan A R and Winter G (1998), Nature, 332. 21.], from Lys to Ala at 320 effec~ed no cl,ange from the IgG1 wild type killing (70%
maximum cell killing and half the cells dead with 600ng/ml).

25 DIRECT BINDING OF C1~1 Measurement of the direct binding of human Clq to different engineered variants of L243 was established to confirm that complement mediated cytotoxicily was due to activation of the cl~sic~l pathway.

30 Purified human Clq (Sigma UK) was directly labelled with fluorescein isothiocyanate (FITC Sigma) using conventional methods. All dilutions, manipulations and incubations were done in Phosphate Buffered Saline (Gibco UK) containing 0.1% Sodium Azide (BDH UK) and 5% Foetal Calf Serum (Sigma UK). 5x104 indicator cells (JY B Iymphoblastoid cell line 35 bearing high levels of HLA-DR) were coated with the different engineered antibodies by incubating at saturating conce~ dtio~s for 1 hour at 4C in WO 94/29351 ~16 3 ~ ~ ~ PCT/GB91101290 RB polystyrene tubes (2052 12x75mm Falcon UK). After washing, serial dilutions of FITC labelled C1q in 100,u1 were added and were incubated together for a further 30 min at 4C. After washing, binding of C1q was revealed using a Fluorescence Activated Cell Scanner (FACS Becton 5 Dickinson). After appropriate analysis, median fluorescence intensity is plotted against C1q concenlr~tion.

Results Direct binding of human C1q to the L243 human isotype series confirmed 10 ~he results with complement me~i~ted cytotoxicity (Figure 12). Labelled human C1q bound well to wild type IgG1, when bound to JY cells, and bound poorly to IgG4. Equilibrium ~issoci~lio,l constants were determined essentially as described by Krause et al. ~Behring Inst. Mitt. 87 56 (1990)]
and were 1.2 x10-7M and 1.5 x10-8M for IgG4 and IgG1 respectively 15 These values compare favourably with those obtained for the mouse antibodies IgG1 and IgG2a which have similar functions [Leatherbarrow and Dwek (1984), Molec. Immunol. ~1, 321]. The Leu 235 to Glu change in IgG1 reduced the binding of C1q to the same level as IgG4. In contrast, a change in the previously desc,il,ed C1q binding motif [Duncan A R and 20 Winter G (1988) Nature 3~ 21], from Lys to Ala at 320 had no effect on ~1q binding. The Leu 235 to Glu change in IgG4 did not alter wild type binding.

~it and Guin~ Plg con~le...~..l 25 The G1[L235E] and G1[L235A] modifications behaved differently when rabbit or guinea pig serum was used as a source of complement instead of human. With rabbit C' they c~use-l the same level of Iysis as the wild type G1. With guinea pig they caused 40% and 4g% plateau level killing, respectively, compared with 80% killing by the IgG1 wild type. The 235 30 change only affects human complement binding inclicating that rabbit and s~uinea pig complement interact differently with human IgG1 (see Figures - 13-15).

~NTIBODY DE~PENDENT CFI ~ MEDIATEl~ CYTOTOXICITY.
35 The ability of the engineered variants of 1243 to bind to FcgRIII was ,3ssessed using antibody dependent cell mediated cytotoxicity (ADCC).

2~63~ ~~' ', . 3y;

The principle of the experiment is that antibodies will mediate Iysis of target cells bearing their cognate antigen if the Fc of the antibody is able to interact with Fc receptor bearing effector cells capable of cytotoxicity. The critical interaction is between antibody Fc and cellular Fc recep~ols.

Effector cells are prepared fresh for each experiment. Human venous blood is drawn into endotoxin free tubes containing heparin. Peripheral blood mononuclear cells (PBMC) are prepared by density gradient 10 centrifugation according to the manufacturers instructions (Pharmacia).
PBMC are adjusted to 1x107 cells/ml in RPMI 1640 medium (Gibco UK) containing 2mM Glutamine (Gibco UK) and 10% foetal calf serum (Sigma UK), in which all manipulations, dilutions and incubations are done.

15 Target cells (JY B Iymphobl~to.~ cell line bearing high levels of HLA-DR) are labelled with 1mCi Na51Cr for 1 hour at room temperature, ~git~te~
every 15 min. The cells are then washed three times, to remove free radiolabel, and resuspended at 2x106/ml. Serial antibody dilutions are prepared in ~u~lic~te in sterile U-bottom 96 well microlilre plates (Falcon 20 UK) in 25~LI. Control wells containing medium only are also prepared to establish the s,uo"t;;neous release of label giving the assay background.
Target 51Cr l~l~elled JY cells are added to all wells in 10~L1. 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 25 incubated together and, after 30min at room temperature, 25~11 effector cells are added to all wells (except the 100%) for a further 4 hours at 37C.
100~L1 of EDTA saline at 4C is then added to stop any further cell killing, the microlil,e plates are centrifuged at 2009 to pellet the intact cells and 100111 su~.e",atant 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 maximum release. Replicates vary by less than 5%. Percent cell Iysis is then plotted against antibody dilution.

WO 94/29351 21~ 3 3 ~ 5 ~ PCT/GB94/01290 ne~Ks Not all the changes made in the N-terminal region of the CH2 domain, residues 233 to 237, affected FcRIII mediated function (Figure 16 and Tables 5 and 7). L243 IgG2 was unable to mediate peripheral blood 5 mononuclear cell cytotoxicity (ADCC) of HLA-DR positive JY Iympho-blastoid cells at conce"l,alions up to 100pml. IgG4 caused a low level of ADCC (20% maximum killing at 1~/ml) which could be abrogated by the Leu 235 to Glu change. Wild type IgG1 was a potent mediator of cell killing giving 50% cell death at 5nglml alllibody. Gly to Ala at 237 reduced the 10 I~G1 wild type killing to the level seen with IgG4. Exchanging the whole lower hinge region with the sequence found in human IgG2 gave intermediate levels of killing with 500nglml needed for 50% cell death. In contrast, changes at 235 in IgG1 had minimal effect on ADCC.

15 Changing the Leu 235 to Ala gave levels of killing comparable with the G1 wild type (9ng/ml for 50% cell death)) and changing the Leu 235 to Glu reduced ADCC a little (40ng/ml for 50% cell death). A change in the previously described C1q binding motif, from Lys to Ala at 320 had no effect on the ability of IgG1 to mediate ADCC.
IMMU~IF FUNCTION
FX vivo T cell function experiments were pe,fol",ed where an interaction between MHC-II and the T cell receptor was an obli~J~tory requirement for T cell activation. The L243 isotype series was tested in mixed Iymphocyte 25 reactions, which measures both naive and memory T cell activation, and recall respo"ses to tetanus toxoid which only measures a memory T cell response.

MiY~ I yrn~ho~.~t~ Rea~tion.
30 Tlhe immunosuppressive potency of engineered variants of L243 was ~ssessed using a mixed Iymphocyte reaction.

The principle of the experiment is that when leucocytes from one individual are mixed with those of another which express different HLA alleles, they 35 will recognise each other as foreign and will become activated. This activation is dependent, primarily, on interactions between the CD31TcR

WO 94/29351 2~33 ~S PCT/GB94/01290 complex on T cells and the MHC-II molecule on antigen presenting cells.
Antibodies that bind to MHC-II are known to inhibit this reaction.

Leucocytes are prepared fresh for each experiment. Human venous blood 5 from two individuals is drawn into endotoxin free tubes containing heparin.
Peripheral blood mononuclear cells (PBMC) are prepared by density gradient centrifugation according to the manufacturers instructions (Pharmacia). PBMC are ~djusted to 2X106 cells/ml in RPMI 1640 medium (Gibco UK) containing 2mM Glutamine (Gibco UK) 100~1ml/10011g/ml 10 Penicillin/ Stlepto",~cin (Gibco) and 10% foetal calf serum (Sigma UK) in which all manipulations dilutions and incub~tions are done. PBMC from one individual are irradiated with 3000 rads. These cells will be stimulate a response from the other individual.

15 Serial antibody dilutions are prepared in ~riplic~te in sterile U-bottom 96 well microlil,e plates (Falcon UK) in 100~11. Control wells containing medium only and optimal Cyclosporin (Sandimmun~ Sandoz) levels (100nM) are also prepared to establish the maximum response and maximum inhibition respectively. Equal numbers of irr~ ted stimulators 20 and responders are mixed together and 100~L1 are added to each well.
Wells of stimulator alone and res~.o"ders alone are also set up as controls.
The experiment is incubated at 37C in 100% humidity and 5%CO2 for 5 days. Response is measured by ~ssessing proli~raLio" during the last 18 hours of culture by inc~lh~tion with 1~1C~well 3H-Thymidine (Amersham 25 UK) harvesting on to glass filter mattes and counting using a beta counter.

Results are plotted as CPM against antibody concentration. Replicates vary by less than 10%.

30 T cell Recall Re3uG.,sa to Tet~nus Toxoid The ability of the engineered variants of L243 to suppress a secondary response was ~-ssessed using a recall response to Tetanus Toxoid.

The principle of the experiment is that T Iymphocytes from an individual 35 previously immunised with Tetanus Toxoid (TT) will respond to TT when re-exposed ex vivo. This activation is dependent on the interaction WO 94/29351 ~16 3 3 ~ ~ PCT/GB94/01290 between the CD3/TcR complex on T cells and the MHC-II molecule on cells which process and present the antigen. Antibodies that bind to MHC-ll are known to inhibit this reaction.
-5 Lymphocytes are prepared fresh for each experiment. Human venousblood is drawn into endotoxin free tubes containing heparin. Peripheral blood mononuclear cells (PBMC) are prepared by density gradient centrifugation according to the manufacturers instructions (Pharmacia).
PBMC are adjusted to 2X106 cells/ml in RPMI 1640 medium (Gibco UK) 10 containing 2mM Glutamine (Gibco UK), 100,uJmU100~1g/ml Penicillin/
St,eploi"ycin (Gibco) and 10% foetal calf serum (Sigma UK), in which all rr~anipulations, dilutions and incub~tions are done.

Serial antibody dilutions are prepared in lliplicate in sterile U-bottom 96 15 vvell microtilre plates (Falcon UK) in 100~1. 50~11 containing an optimal conce"l,dlio" of rr previouslydete.".ined byexperi,ne"lalioll is added to all wells. Control walls containing medium only or Cyclosporin (Sandimmun, Sandoz) (1 OOnM) are also prepared to establish the maximum response and maximum inhibition, respectively. 50~1 PBMC are 20 then added to each well. The ex~eri",ent is incuh~ted at 37C in 100%
humidity and s%co2 for 7 days. Response is measured by ~-ssessing proliferation during the last 18 hours of culture by incub~tion with 1~Ci/well 3H-Thymidine, harvesting on to glass filter mattes and counting using a beta counter.
Results are plotted as CPM against antibody concentration. Replicates vary by less than 10%.

F~sult~ (Figures 17-21) 30 There were no significant or qu~lit~tive di~erences between the effects of the L243 human isotype series b~ oon the MLR and TT response.
Maximal inhibition was achieved with G1, G1[L235E] and G1[L235A].
Approximately two orders of magnitude more of G2, G4 and G1[G237A]
was required to give similar levels of inhibition. The G1/G2 L hinge 35 exchange mutant was intermediate in immuno-suppresser potency. There was no correlation between complement fixation or FcRI binding and WO 94/293~1 2~ 633 4~ PCT/GB94/01290 immuno-suppression, G1 binding well to FcRI and fixing complement and G1[L235E] doing neither, but both giving good immunosuppression. But, there was good correlation with FcRIII binding. Human G1 and G1[L235E]
interact with FcRIII and give good immunos~,p~.ression. The G1/G2 L
5 hinge is intermediate in FcRIII binding and immuno-suppression. In contrast, the G237A mutation in human G1, in agreement with published observations, reduces FcRIII binding. This antibody gave poor immunosuppression. (Table 5). Table 6 shows a number of L243 isotype mutants.
Conclusion We have found that amino acid residues necessary for Clq and FcR
binding of human IgG1 are located in the N-terminal region of the CH2 domain, residues 231 to 238, using a matched set of engineered~
15 ar~Lil~odias based on the anti-HLA DR antibody L243. Changing the leucine 235 in the CH2 region of 19G3 and 19G4 to glutamic acid was already known to abolish FcRI binding, we have co"ri-",ed this for 19G1 and also found a concomitant abolition of human complement ~ alio,l with ret6"lio,l of FcRIII mediated function. Changing the glycine at 237 to alanine of 20 19G1 also abolished FcRI binding and reduced complement fixation and FcRIII mediated function. Exchanging the whole region 233 to 236, with the sequence found in human IgG2 abolished FcRI binding and complement fixation and reduced FcRIII mediated function of 19G1. In contrast, a change in the previously described C1q binding motif, from 2~ Iysine at 320 to alanine had no effect on IgG1-mediated complem~nt ~iAdli~l 1.

The effect of these changes in IgG1 on FcRI binding are similar to published observations using IgG3 and IgG4 [Lund J ~l J. Immunol.
30 1991. 147, 265; and Alegre M-L ~L J. Immunol. 1992. 148, 3461] with changes at 235 and 237 in the lower hinge/N-terminal CH2 region markedly reducing FcRI binding. The similarities between these three isotypes strongly suggests that they interact with FcRI in a similar way.

35 We have found residlJes necessary for FcRIII binding of human IgG1 within the lower hinge/N-terminal end of the CH2 region. Modi~icaliG" at 237 and WO 94/29351 ~16 33 4S PCT/GB94/01290 exchanging the lower hinge for IgG2 residues caused low and intermediate levels, respectively, of FcRIII mediated killing. These effects are similar to those reported by Sarmay et a/ [Molec. Immunol. 1992. ;2~. 633] for human IgG3. In contrast to Sarmay ~ using IgG3, our changes at residue 235 5 of IgG1 had little effect on FcRIII binding.

Greenwood et al [Eur. J. Immunol. 1993. ~,1098], using inter and intra domain switch variants between IgG1 and IgG4, identify residues in IgG1 necessary for FcRIII binding in the C-terminal half of the CH2 domain 10 beyond 292. This incliG~tes that the resid~les we have identified within the lower hinge/N-terminal end of the CH2 region are necessary but not sufficient for FcRIII effector function mediated through binding of human IgG1 .

15 IgG1 variants with changes at 235 failed to mediate Iysis with human complement and did not bind purified human Clq. We also found that an IgG1 molecule containing a change at 320 gave complement mediated killing equivalent to the IgG1 wild type. Residues, GIU 318, LYS 320 and Lys 322 were idenlitied by protein engineering studies as necess~ry in 20 mouse IgG2b for C1q binding [Duncan, A R and Winter G, Nature, 1988.
. 21]. The same study also demonstrated that the 235 change in mouse IgG2b left unchanged its affinity for human C1q [Duncan, A R and V'l/inter G, Nature, 1988. 322,21]. The apparent contradiction between these observations is probably due to di~ter~"ces in C1 q contacts between 25 human IgG1 and mouse 19G26.

Vl~/e found that most changes in the lower hinge/N-terminal end of the CH2 domain affect C1q binding. The G1/G2 lower hinge exchange abolished complement ~ixdlion and the change at 237 also reduces it significantly. In 30 contrast, Greenwood et a/ [Eur. J. Immunol. 1993. ~, 1098], found residues necess~ry for human complement ~ alio" in the C-temminal half of the CH2 domain. Tao et al[J. exp. Med. 1993. 178. 661] also identify the C:-terminal half of the CH2 domain as necess~ry for complement ~ alion.
They are able to separate C1q binding from complement mediated Iysis.
35 IgG1 with a Pro to Ser change at 331, in the C-terminal half of the CH2 domain, is able to bind human C1 q as well as the wild type but is unable to WO 941293S1 2~G33 4S ~ - PCT/GB94/01290 activate complement. This predicts that the amino acids that we have identified within the lower hinge/N-terminal end of the CH2 region are necess~ry for C1q binding and that the C-terminal resid~les are necessary for the binding and activation of the antibody dependent complement 5 c~sc~de beyond Clq.

WO 9~129351~16 3 3 4 ~ PCT/GB94101290 T~RI F5 5Summary of 1 ~43 Isotype Series L.243 RI RIII Cl q MLP~ rr C~2 ++ ++

G4 + ++ ++
Gl ~ +++ ~ +

GlL235A + I-I I + I I I I +I m G1G237A + + + ++ ++

wos4/293sl ~Z1~33~5 rcT/GBg4lol290 TAE~I F 6 Human Iso~e Mutants Gene Residue From IQ NAME

Gl 235 L E Gl [L235E]
G1 235 L A G1[L235A]
G1 237 G A G1[G237A]
G1 320 K A G1[K320A]
G4 235 L E G4~L235E]
G1 231-238 APELLGGP AP-PVAGP G1/G2L-hinge WO 94/29351 ~16 3 3 4 5 PCT/GB94/01290 Summ~ry of 1 ~431sotyDe Series L2~ Rla BIDb Com~ lC

G2 >10 >100000 >20/0 G4 1.2 10000ex >20/0 G4[L~35E] >10 >100000 ~20/0 G1 0.13 5 0.6/65 G1/G2Lh >10 500 ~20/0 G1~L2~5E] >10 40 >20/0 G1[L235Al 5.0 9 >20/0 G1~G237A] >10 10000ex 2.0/20 G 1 [K320A] 0.1 10 0.6/70 10 aL) mg/ml antibody necessary for 50% inhibition of binding of FITC-labelled mouse IgG2a antibody to U937 cells.

b) ng/ml antibody necessary for half maximal cell killing in ADCC. (ex) extrapolated value.
c) mg/ml antibody necess~ry for half maximal cell killing by human complement and percent plateau cell killing.

WO 94/29351 ~ j PCT/GB94tO1290 FY~MP! F 3 L243 is a mouse monoclonal antibody raised against human MHC Class ll.
The nuc!~otide and amino acid sequences of L243 Vl and Vh are shown in Figures 5 and 3 respectively. The following examples describe the 5 humanisation of the L243 antibody (CDR grafting).

Clr)R grafting of 1 ~431~ght ch~ln Alignment of the framework regions of L243 light chain with those of the four human light chain subgroups [Kabat, E.A., Wu, T.T., Perry, H.M., 10 Gottesman, K.S. and Foeller, C. 1991, Sequences of Proteins of Immunological Interest, Fifth Editionl revealed that L243 was most homologous to antibodies in human light chain subgroup 1. Consequently, for constructing the CDR gr;~tl6d light chain, the framework regions chose"
corresponded to those of the human Group 1 consensus sequence. A~
15 comparison of the amino acid sequences of the framework regions of L243 and the consensus human group l light chains is given below and shows that there are 21 differences (u"derlL leJ) u~ on the two sequences.

Analysis of the co,~ ution that any of these framework differences might 20 have on antigen binding (see published l"ler"ali~nal patent application No.

7) ider,li~iecl 4 res~ ss for inv~ lion; these are at positions 46,49,70 and 71. Based on this analysis, two versions of the CDR gratl6.1 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, 25 L243-gL2, all re~ es are human consensus.

I ~ht ch~in Comparisons Hu group 1 consen~us : DIQMTQSPSSLSRSUGDRUTITC

L2~3 : DIQMTQSP_SLS_SUG _ UTITC

Hu Group 1 consensus : ~YQQKPGKRPKLLIY
L2~3 : ~Y_QKQGK=PQLLUF

WO 94/29351 ~16 3 3 ~ 5 PCT/GB94/01290 Hu Group l con~en~u~ : GUPSRFSGSGSGTDFTLTISSLQPEDFRTYYC
.
L.2~3 : GUPSRFSGSGSGTQYSL_INSLQ_EDFGDYYC

~u G~oup l ~on~en~u~ : FGQGTKUEIKR

L2~3 : FG_GTNLEIKR
Construction of CDR .~ tecl li~ht chain 1 ~43~
The construction of L243-gL1 is given below in detail. The following oligonucleotides were used in the Polymerase Chain Reactions (PCR) to introduce chal ,yes into the framework regions of the chimeric light chain:
R50~3 : 5'GTRGGRGRCCGGGTCRCCRTCRC~TGTCGRGCRR3' R50~ : 5'CTGRGGRGCTTTTCCT~GTTTCTGCTGRTRCCRTGCTRR~3' R50~5 : 5'RRRCCRGGRRRRGCTCCTCRGCTCCTGRTCTTTGCTGCRTC3' R50~6 : 5'CTTCTGGCTGCRGGCTGGRGRTRGTTRGGGTRTRCTGTGTGCC3' 20 R50~ : 5'CTTCRGCCTGCRGCCRGRRGRTTTTGCTRCTTRTTRCTGTCRR3' R50~8 : 5'GGGCCGCTRCCGTRCGTTTTRGTTCCRCTTTGGTGCCTTGRCCGRR3 Three reactions, each of 20 ~11, 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 25 deoxyribonucleoside triphos~Jl,ate, 0.1 ~ug chimeric L243 light chain DNA, 6 pmoles of R5043/R5044 or R5045/R5046 or R5047/R5048 and 0.25 units Taq polymerase. Re~ctions were cycled through 94C for 1 minute, 55C for 1 minute and 72C for 1 minute. After 30 cycles, each reaction u~as analysed by electrophoresis on an agarose gel and the PCR
30 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 ~LI, 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 35 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 - 2~ ~3~ 4S ! ; ' mixture was extracted with phenol / chloroform and then with chloroform and precipitated with ethanol. The ethanol precipitate was recovered by centrifugation, dissolved in the appropriate buffer and restricted with the enzymes BstEII and Spll. The resulting product was finally elec~,oplloresed 5 on an agarose gel and the 270 base pair DNA fragment recovered from a gel slice and ligated into the vector pMR15.1 (Figure 1) that had previously been digested with the same enzymes.

The ligation mixture was used to lrans~or", E. coli LM1035 and resulting 10 colonies analysed by PCR, resl,icliGn enzyme digests and nucleotide sequencing. The nucleotide and amino acid sequence of the Vl region of L243-gL1 is shown in Figure 22.

Construction of CDR 5"~1e.1 light ch~in 1 ~4~1 ~
15 L243-gL2 was constructed from L243-gL1 using PCR. The following oligonucleotides were used to introduce the amino acid cl ,anges:

R1053 : 5~GCTGRCRGRCTRRCRGRCTGTTCC3' R5350 :
20 5'TCTRGRTGGCRCRCCRTCTGCTRRGTTTGRTGCRGCRTRGRTCRGGRGCTTRGGR
GC3' R53~9 :
5'GCRGRTGGTGTGCCRTCTRGRTTCRGTGGCRGTGGRTCRGGCRCRGRCTTTRCCC
TRRC3' 25 R68t : 5'TTCRRCTGCTCRTCRGRT3' Two ~actiGl lsl each 20 ~11, 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 deoxyribonuc'~csi~le triphosphate, 0.1 ~19 L243-gL1, 6 pmoles of R1053/
30 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,opl,oresis on an agarose gel and the PCR fragments excised from the gel and recovered using a Mermaid Kit.

WO 94/29351 ~ I 633~ ~ PCT/GB94/01290 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/~ of each of the PCR fragments from the first set of reactions, 30 pmoles of R1053 and R684 and 2.5 units Taq polymerase.
5 Reaction temperatures were as above. After the PCR, the mixture was extract-ed with phenol / chloroform and then with chloroform and precipitated with ethanol. The ethanol precipitate was recovered by centrifugation, dissolved in the appropriate buffer and restricted with the enzymes BstEII and Spll. The resulting product was finally electrophoresed 10 on an agarose gel and the 270 base pair DNA fragment recovered from a gel slice and ligated into the vector pMR15.1 (Figure 1) that had previously been digesled with the same enzymes.

The ligation mixture was used to transform E. coli LM1035 and resulting 15 colonies analysed by PCR, restriction enzyme digests and nucleotide sequencing. The nllcleotide and amino acid sequence of the Vl region of L743-gL2 is shown in Figure 23.

C~graftin~ Of 1 ~43 haS~vy ch~
20 CDR grafting of L243 heavy chain was accomplished using the same strategy as descriLecl for the light chain. L243 heavy chain was found to be most homologous to human heavy chains belonging to subgroup 1 and therefore the consensus sequence of the human subgroup 1 frameworks was chosen to accept the ~43 heavy chain CDRs.
2~
A comparison of the framework regions of the two structures is shown below where it can be seen that L243 differs from the human consensus at 2~3 positions (underlined). After analysis of the cGnl,il)ution that any of these might make to antigen binding, only residues 27,67,69,71,72 and 75 30 were retained in the CDR y~arlecl heavy chain, L243-gH.

He~v~ ch~in comparisons 3~ HU Group 1 con3en~u~: QUQLUQSGREUKKPGRSUKUSCKRSGYTFT
L2~3 : Q_QLUQSG_E_KKPGETUK_SCKRSG_TFT

WO 94/29351 2~33 ~ PCTIGB94/01290 Hu Group 1 oon~en~u~ : WURQRPGQGLEWMG
L2~3 : WU_QRPG_GLKWMG
~ 6 77 7 Hu Group 1 consen~u~ : RUTITRDTSTSTRYMELSSLRSEDTRUYYCRR
L2~3 : RFRFSLETS_STRYLQINNLKNEDTR Y_CRR

Hu Group 1 con~en~u~ : WGQGTLUTUSS
L2~3 : ~GQGTTLTUSS

Construction of CDR grafted h~vy ch~in. 1 ~43~H
L243gH was assembled by su~jecting overlapping oligonucleotides to PCR
in the presence of the appropriate primers. The following oligonucleotides.
were used in the PCR:

R300~ : 5 7 GGGGGGRRGCTTGCCGCCRCCRTGG3' R3005 : 5'CCCCCCGGGCCCTTTGTRGRRGCRG3' R~90~ : 5'GRCRRCRGGRGTGCRCTCTCRGGTGCRGCTGGTGCRGTCTGGRGC
RGRGGTGRRGRRGCCTGGRGCRTCTG3' R~903 : 5'RCRTTCRCRRRTTRCGGRRTGRRTTGGGTGRGRCRGGCRCCTGGR
CRGGGRCTCGRGTGGR3' R~90~ : 5'CCTRCGTRCGCRGRCGRCTTCRRGGGRRGRTTCRCRTTCRCRCTG
GRGRCRTCTGCRTCTRCRGCRTRCRT3' R~905 : 5'CRGCRGTGTRCTRCTGTGCRRGRGRCRTTRCRGCRGTGGTRCCTR
CRGGRTTCGRCTRCTGGGGRCRGGGR3' R~897 : 5'TGRGRGTGCRCTCCTGTTGTCRCRGRCRGGRRGRRCRGGRRCRCC
CRRGRCCRCTCCRTGGTGGCGGCRRGCTTCCCCCC3' R~898 : 5'CRTTCCGTRRTTTGTGRRTGTGRRTCCRGRTGCCTTRCRRGRCRC
CTTCRCRGRTGCTCCRGGCTTCTTCR3' R~899 : 5'GRRGTCGTCTGCGTRCGTRGGCTCTCTTGTGTRTGTRTTRRTCCR
TCCCRTCCRCTCGRGTCCCTGTCCRG3' wo Y4n93sl 2 ~ 6 3 3 ~ ~ PCTIGB94/01~90 R't900 : 5'TTGCRCR~TRGTRCRCTGCTGTGTCCTCRGRTCTCRGRGRRGRCR
GCTCCRTGTRTGCT~TRGRTGCRGRT3' R4901 : ~'CCCCCCGGGCCCTTTGTRGRRGCRGRRGRCRCTGTCRCCRGTGTT
CCCTGTCCCCRGTRGTCGRR3~

The assembly reaction, 50 ~I, contained 10 mM Tris-HCI pH 8.3, 1.5 mM
MgCI2, 50 mM KCI, 0.01% w/v gelatin, 0.25 mM each deoxyribonucleoside 10 trhphosphate,1 pmole of each of R4897 - R4905, 10 pmoles of eacn of R3004 and R3005 and 2.~ units Taq polymerase. Reactions were cycled through 94 C for 1 minute, 55 C for 1 minute and 72 C for 1 minute. After 30 cycles, the reaction was extracted with phenol/chloroform (1/1), then with chloroform and precipitated with ethanol. After centrifugation, the DNA
15 was dissolvedl in the appru~.riate resl,iclio" buffer and ~I;gested with Hindlll and Apal. The resulting fragment was isolated from an agarose gel and ligated into pMR14 (Figure 2) that had previously been ~igested with the same enzymes. pMR14 contains the human gamma 4 heavy chain censtant region and so the heavv chain e3-~,ressed from this vector will be a 20 g;3mma 4 isotype. The ligation mixture was used to transform E. coli Ll\A1035 and resulting bacterial colonies screened by resl,iction digest and n~cleotide sequence analysis. In this way, a plasmid containing the correct sequence for L243gH was identified (Figure 24).

25 Construction of ~mm~ 1 versions of chimeric ~nd CDR ~ 43 h~vy ch~in Human Gamma 1 versions of L243 heavy chains were constructed by lr~n~ter,i,lg the variable regions of both the murine and the CDR grafted heavy chains as Hindlll to Apal fragments into the vector pGamma1 30 (F-igure 6).111is vector contains the human Gamma 1 heavy chain constant region.

Ev~l~ætion of activities of CDR gr~fted gene~
The activities of the CDR grafted genes were evaluated by expressing 35 them in mammalian cells and purifying and quantitating the newly synthesised antibodies. The methodology for this is described next, WO 94/29351 2~3~ ~ PCT/G1194/01290 followed by a description of the biochemical and cell based assays used for the biologiG~I characterisation of the antibodies.

a) Gene FXl ,ression in CHO cells 5 Chimeric and CDR grafted L243 was produced for biological ev~llJ~tion by transient expression of heavy and light chain pairs after co-transfection into Chinese Hamster Ovary (CHO) cells using calcium phosphate prec;~.ilalion as described above for production of chimeric L243.

10 Antibody concenl,~lio,) was determined using a human Ig ELISA (see below).

b) .ELISA
For the ELISA, Nunc ELISA plates were coated overnight at 4C with a 15 F(ab)2 fragment of a polyclonal goat anti-human Fc fragment specific antibody (Jackson Immuno-research, code 109-006-098) at 5 ~Lg/ml in coating buffer (15mM sodium carbonate, 35mM sodium hydrogen carbonate, pH6.9). Unco~tecl alllil.ody was removed by washing 5 times with distilled water. Samples and purKied standards to be quantitated were 20 diluted to a~proxi",ately 1 ~lg/ml in conjugate buffer (0.1M Tris-HCI pH7.0, 0.1M NaCI, 0.2% v/v Tween 20, 0,2% w/v Hammersten casein). The samples were lilldted in the micr~lit,~ 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 shaking. After the first incub~tion step the plates 25 were washed 10 times with distilled water and then incubated for 1 hr as bsfore with 0.1 ml of a mouse monoclonal anti-human kappa (clone GD12) peroxidase conjugated antibody (The Binding Site, code MP135) at a dilution of 1 in 700 in conjugate buffer. The plate was washed again and sub~l.dle solution (0.1 ml) added to each well. Subsl,ate solution contained 30 150111 N,N,N,N-tetramethylber,~idi"e (10 mg/ml in DMSO), 150~1 hydrogen 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 cGncenlralion of the sample 35 determined by comparing the lilr~lioll curves with those of the standard.

WO 94/29351 216 ~ i PCT/GB94/01290 c)l Competition ,~.~c:~y The principle of this assay is that if the antigen binding region has been correctly transferred from the murine to human frameworks, then the CDR
gratled antibody will compete equally well with a l~helled chimeric antibody 5 for binding to human MHC Class ll. Any changes in the antigen binding potency will be revealed in this system.

Chimeric L243 was labelled with fluorescein (FITC) using the method of Wood et al [Wood,T., Thompson, S and Goldstein, G 1965, J. Immunol ~i.
10 2~5-æg and used in the competition assay descril,ed above.

Figure 25 compares the ability of combinations of L243 heavy and light chains to compete with FlTC-labelled chimeric L243 for L il l.li, lg to JY cells.
All combinations were effective competitors although none of those 15 containing CDR grafted heavy or light chains were as effective as the clhimeric antibody itself. Thus, the combi"~lio"s cH/gL1, gH/cL and gH/gL1 were 89%, 78% and 64% respectively, as effective as chimeric L243 in this assay.

20 d) Deterrnin~tion of Affinity co~ nls by Scatchard Analysis L243 antibodies were titrated from 10~1g/ml in PBS, 5% fetal calf serum, 0.1% sodium azide in 1.5-fold dilutions (150~11 each) before incubation with 5x104 JY cells per titration point for 1 hour on ice. The cells were previously counted, washed and resuspended in the same medium as the 25 samples. After incub~tion, the cells were washed with 5ml of the above medium, spun down and the s~".er"atant discarded. Bound antibody was r3vealed by addition of 100~11 of a 1/100 dilution of FITC conjugated anti-human Fc monoclonal (The Binding Site; code MF001). The cells were tl1en incl~h~ted for 1 hour on ice and then the excess FITC conjugate 30 removed by washing as before. Cells were dispersed in 250~11 of the same buffer and the median fluoresce"ce intensity per cell was determined in a - F:ACScan (Becton Dickinson) and calibrated using standard beads (Flow C,ytometry standards CoI~oralio,,). The number of molecules of antibody bound per cell at each antibody concenl,alion was thus established and 35 used to generate Scatchard plots. For the purpose of calculation, it was 21~3345 ' ~

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 affinities of chimeric L243 (cH/cL) L243-gH/L243-gL1 and L243-gHlL243-gL2 is shown in Figure 26. Chimeric L243 was found to have an apparent Kd of 4.1 nM whiie the CDR grafted antibodies 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 re~lec~s the Col lll ibution made by resi~ues 45 49 70 and 71 that had been retained in L243-gL1 from the parent light chain.

e) Antibody de~e"~e,1l cell me~i~t~d cytotoxici~
The ability of chimeric and CDR grafted L243 to msdiate antibody dependent cell cylotoxicity (ADCC) was compared as descril-ecl previously.
The ~.,i"c;~le of the ex~.e,i",ent is that antibodies will mediate Iysis of target cells bearing their cognate antigen if the Fc of the ~nlibo.ly is able to interact with Fc rece~tor bearing ~ec~or cells capable of cytc,toxicity.

A comparison of the activities of chimeric (cH/cL) and CDR grafted (gH/gL1) L243 human gamma 1 isotypes in the above assay is shown in Figure 27. Both antibodies were effective mediators of cell Iysis with maximal activity being achieved at antibody concenlraliG"s of less than 100 ng/ml. There was no siy~itica~ itter~nce between the activities of the two antibodies fl Immune function tests FY vivo T cell function experiments were performed where an interaction be~ oon MHC-II and the T cell r~cel)tor was an obligatory requirement for T cell activation. Chimeric and CDR grafted L243 antibodies were compared in mixed Iymphocyte reactions which measures both naive and memory T cell activation and in recall res~onses to tetanus toxoid which only measures a memory T cell response.

WO 94/29351 2 ~ 6 3 3 ~ 5 PCT/GB94/01290 1 ~ Mixed Lymphocyte reaction - as described above Tlhe principle of the experiment is that when leucocytes from one individual are mixed with those of another individual which express different HLA
alleles, they will recognise each other as foreign and the Iymphocytes will 5 become activated. This activation is dependent primarily on interactions between the CD3/TcR complex on T cells and the MHC Class ll molecule on antigen ,~,resenling cells. L243 is known to inhibit this reaction.

When an MLR was carried out to compare the effectiveness of the Gamma 10 1 isotypes of chimeric and CDR grafted L243 as inhibitors of T cell activation, no significant differences were observed between the two antibodies (Figure 28). Greater than 90% inhibition of the MLR was observed using 100 ng/ml of either antibody.

15 2) T cell recall response to Tetanus toxoid The ability of chimeric and CDR yld~led L243 to suppress a secondary response was ~ssessed using a recall response to Tetanus toxin. The pri"c;~le of the eA~6ri,nenL is desc,ib~d above.

20 The results of an experiment comparing the ability of human gamma 1 isotypes of chimeric and CDR g,dtled L243 to inhibit the response to TT is shown in Figure 29. Both a"libodies were effective inhibitors of the T cell ~espo,)se to TT and produced litldLiol, curves that were indistinguishable.

25 FY~MPI F 4 The ability of CDR grafted L243 with the alteration at position 235 i.e.
Ll235E] to mediate antibody dependent cell cytoxicity (ADCC) was measured essei,lially as described in the previous examples. The results are shown in Figure 27.
Similarly the CDR grafted L243 [L235E] antibody was tested in a mixed Iymphocyte reaction and in recall response to tetanus toxoid essentially as desc,ibed in the previous Examples. The results are provided in Figures 2E~ and 29.

WO 94/29351 : PCT/GB94/01290 2163~4~ 56 The ability of the CDR-grafted L243 antibody [L235E] to fix human complement was assessed using the technique of antibody dependent complement mediated cytotoxicity as described in the previous Examples.
The results are shown in Figure 30.

Claims (19)

1. An altered antibody wherein one or more amino acid residues in the N-terminal region of the CH2 domain of said antibody are altered characterised in that the ability of said antibody to fix complement is altered as compared to unaltered antibody.

2. An antibody according to Claim 2 which binds to one or more cellular Fc receptors and does not bind significantly to FcR1.

3. An antibody according to Claim 1 or 2 wherein the amino acid residue which is altered lies within amino acid positions 231 to 239.

4. An antibody according to any of the preceding claims which is an MHC specific antibody.

5. A method for producing an altered antibody with altered ability to fix complement as compared to unaltered antibody comprising altering one or more amino acids in the N-terminal region of the CH2 domain of said antibody altering the ability of said antibody to fix complement as compared with unaltered antibody.

6. A method of modulating the function of cell surface associated antigens avoiding complement mediated toxicity comprising administration of an altered antibody wherein one or more amino acid residues in the N-terminal region of the CH2 domain of said antibody are altered characterised in that the ability of said antibody to fix complement is altered as compared to unaltered antibody and also said antibody.

7. A method according to Claim 6 wherein said altered antibody is able to bind one or more cellular Fc receptors especially FcRIII while binding to FcRI is significantly reduced

8. A therapeutic, diagnostic or pharmaceutical composition comprising an altered antibody according to any of the preceding claims.

9. A process for the preparation of a therapeutic, pharmaceutical or diagnostic composition comprising admixing an altered antibody according to any of the preceding claims together with a pharmaceutically acceptable excipient, diluent or carrier.

10. A method of therapy and diagnosis comprising administering an effective amount of an altered antibody according to any of the preceding claims to a human or animal subject.

11. A process for producing an altered antibody according to any of the preceding claims comprising:
a) producing in an expression vector an operon having a DNA
sequence which encodes an antibody heavy or light chain b) producing in an expression vector an operon having a DNA
sequence which encodes a complementary antibody light or heavy chain c) transfecting a host cell with both operons and d) culturing the transfected cell line to produce the antibody molecule.

12. A process according to Claim 11 wherein said DNA sequences encode a humanised antibody.

13. A process according to Claim 12 wherein said DNA sequences encode a CDR-grafted heavy and/or light chain, or a chimeric antibody

14. A process according to Claim 11, 12 or 13 wherein at least one of the expression vectors contains a DNA sequence encoding an antibody heavy chain in which one or more amino acid residues in the N-terminal region of the CH2 domain has been altered from that in the corresponding unaltered antibody.

15. A process according to Claim 11 or 12 wherein the alteration in the N-terminal region of the CH2 domain is made after the unaltered antibody has been expressed.

16. An altered antibody according to any of the preceding claims derived from the anti-MHC antibody L243 (ATCC HB55).

17. An altered antibody according to any of the preceding claims wherein the lower hinge of said antibody has been exchanged with an antibody of different isotype.

18. An altered antibody according to Claim 17 wherein an IgG1 lower hinge region has been exchanged with a IgG2 lower hinge region.

19. A method of treating diseases in which antibody therapy leads to undesirable toxicity due to antibody mediated complement fixation comprising administering an altered antibody wherein one or more amino acid residues in the N-terminal region of the CH2 domain of said antibody are altered characterised in that the ability of said antibody to fix component is altered as compared to unaltered antibody.