Recombinant antibodies specific for tnf-.alpha.

W O 92lll38~ 2129~ _ RECOMBINANT ANTIBODIES SPECIFIC FOR TNF-~<

This application is a division of patent application Serial No, 2,076,540-2 filed December 20, 1991 as International PCT Application Serial No. PCT/GB91/02300 ~ield of the In~ention This invention relates to recom~inant, in particular hu~anised, antibody molecules ha~ing specificity lor antigenic deter~in~ts o~ tumour necrosis factor alpha (TNF-~), to processes for t~eir production using recom~inant DNA technology, and to their the~aDeutic uses.

.

~r the purposes of the Present descri~tion the term ~recombinant antibody molecule~ is u~ed to describe an anti~ody molecule produced by any process involving the use of recombinant DNA technology, including any analosues of ~atural ~mmmlnoslobulins or thelr frag~ent~.

Also for the purposes of the prese~t description the term ~hl~m~;sed a~tibody mole~ule~ i5 u~ed to describe a mole~ule having an antigen binding site de-i~ed from an noglobulin from a non-human species, and remA;ni~s ~ oglobulin derived parts of the molecule being derived from a h-~m~n i~munoglobulin. Thus hu~ni~ed anti~ody molecules include h-lm~ ed chime~ic anti~ody mclecules comDrising co~plete non-human heavy and~or light chain varia~le region dom~; ns linked to human consta~t region domains. Humani~ed antibody 30t e~ules zlso comprise CDR-gralted h~~nised antiDody molecules comDrising one o~ more CDRs from a non-human antibody grafted intD a he~vy and/or light chain hl1m~n variable region framewor~.

The antigen ~inding s~ecificity of anti~odies is determined by thei~ ~omplementari~y dete~m;~;~g regions (CDRs) which are relatively short peotiàe seouences carried on th~ framewor~ regions of the v2-iab~e dom~ins. There are 3CD~s, ~CDR1, CD~2 and CDR3) ~n eac:G- the heavy and li~n~ cnain varia~le aomzins.

- 2 - ~2129~ The a~reviation "MAb" is used to indicate a monoclonal antibody. In the present description reference is made to a number of publications by nllmher~ and the~e publications are listed in numerical order at the end of the description.

Bac~round of the Invention Natural immunoglobulins have been known for many years, as have the various fragments thereof, such as the Fab, Fv, (Fab')2 and Fc fragments, which can be derived by enzymatic cleavage. Natural immunoglobulins comprise a generally Y-shaped mole~ule having an antigen-binding site towards the end of each upper arm. The rem~ r of the structure, and particularly the stem of the Y, mediates the effector functions associated with ;mmn~oglobulins.

Natural immunoglobulins have been used in assay, diagnosi~ and, to a more limited extent, therapy. ~owever, such uses, e~pecially in therapy, were h i~ered until recently by the polyclonal nature of natural ;mm-lnoglobulins. A significant step towards the realisation of the potential of immunoglobulins as therapeutic agents was the discovery of procedures for the reproducible production of monoclonal antibodies (MAbs) of defined spe~ificity (1).

~owever, most MAbs are produced by hybridoma~ which are fusions of rodent spleen cells with rodent myeloma cells. They are therefore essentially rodent proteins.

There are very few reports of the production of httm~n MAbs.

Since most availàble MAbs are of rodent origin, they are naturally antigenic in humans and thus can give rise to an undesirable immune response termed the ~AMA (~m~n Anti-Mouse Antibody) response if the MAb is ~m; n; ~tered to a h~m~n. Therefore, the use of rodent MAbs as therapeutic agents in humans is inherently l;~ited by the WO92/11383 r~~ 7,~.vv _ ~ 3 - 2129~t~ ~ ~ fact that the human su~ject will mount an imml~nological response to the MAb and will either remove it entirely or at least reduce its effectiveness. In practice, MAbs of rodent origin may not be u~ed in patients for more than one or a f ew treatments as a ~AMA response soon develops rendering the MAb ineffecti~e as well as giving rise to undesirable reactions. For instancP, ORT3 a mouse IgG2a/k MAb which recognises an antigen in the T-cell receptor-CD3 complex has been approved for use in many countries throughout the world as an immunosuppressant in the treatment of acute allograft rejection [Chatenoud et al (2) and Jeffers et al (3)~. However, in view of the rodent nature of this and other such MAbs, a significant ~AMA response which may include a major anti-idiotype component, may build up on use. Clearly, it would be highly desirable to ~im; ni ~h or abolish this undesirable ~AMA response and thus enlarge the areas of use of such antibodies.

Proposals have therefore been made to render non-h-~m~n MAbs lesc antigenic in humans. Such techniques can be generically termed ~humanisation" techni~ues~ These techniques typically involve the use of recombinant DNA technology to manipulate DNA sequence~ encoding the polypeptide chains of the antibody molec~le.

Early methods for h~nising MAbs involved production of chimeric antibodies in which an antigen binding site comprising the complete variable ~om~ins of one antibody is linked to constant dnm~ i nS derived from another antibody. Methods for carrying out such chimerisation procedures are descri~ed in EP0120694 (Celltech L;m~ted), EP0125023 (Genentech Inc. and City of ~ope), EP-A-0 171496 : (Res.

De~.

Corp.

Japan), EP-A-0 173 494 (Stanford Uni~ersity), and WO 86/01533 (Celltech LLmited). These prior patent applications generally disclose processes for ~ v- 2129~ preparing antibody molecules having the variable dom~ i ns from a mouse MAb and the constant domains from a human ;mml~noglobulin. Such humanised rh;meric antibodies, however, still contain a sign; f; C~nt proportion of non-human ~m; no acid sequence, i.e. the complete non-human variable dom~; nc ~ and thus may sti~l elicit some ~AMA response, particularly if adm;nistered over a prolonged period [Begent et al (ref. 4)].

In an alternative approach, described in EP-A-0239400 (Winter), the complementarity determining regions (CDRs) of a mouse MAb have been grafted onto the framewor~ regions of the variable domains of a human immunoglobulin by site directed mutagenesis using long oligonucleotides.

Such CDR-grafted h~m~ni~ed antibodies are much less likely to give rise to a ~AMA response than hn~nlcet c~imeric antibodies in view of the much lower proportion of non-human amino acid sequence which they contain.

The earliest work on hllm~nising MAbs by C~R-grafting was carried out on MAbs recognising synthetic antigens, such as the NP or NIP antigens. ~owever, examples in which a mouse MAb recognising lysozyme and a rat MAb recognising an antigen on ht~n T-cells were hllm~niced by CDR-grafting have ~een described by Verhoeyen et al (5) and Riechm~nn et al (6) respectively. The preparation of CDR-grafted antibody to the antigen on hl~m~n T cells is also described in WO 89/07452 (Medical Research Council).

In Riechmann et al~Medical Research Council it was found that transfer of the CDR regions alone [as defined by Xabat refs. (7) and (8)] was not sufficient to provide satisfactory antigen binding activity in the C~R-grafted product. Riechmann et al found that it was necessary to convert a serine residue at position 27 of the human heavy chain sequence to the corresponding rat phenylalanine WO92/1l383 A ~ - 5 - ~129~4 ~.., residue to obtain a CDR-grafted product ha~ing improved antigen binding activity. This residue at position 27 of the heavy chain is within the structural loop adjacent to CDRl. A further construct which additionally contained a human serine to rat tyrosine change at position 3Q of the heavy chain did not have a significantly altered bi~;ng activity over the humanised antibody with the serine to phenylalanine change at position 27 alone. These results indicated that changes to residues of the human sequence outside the CDR regions, in particular in the structural loop adjacent to C~Rl of the heavy chain, may be necessary to obtain effective antigen bin~ing acti~ity for CDR-grafted antibodies which recognise more complex antigens. E~en so the bi n~; ng affinity of the best CDR-grafted antibodies obtained was still significantly less than the original MAb.

Recently Queen et al (9) have described the preparation of a humanised antibody that binds to an interleukin 2 receptor, by combining the CDRs of a murine MAb (anti-Tac) with human immunoglobu}in framework and constant regions. The human framework regions were chosen to m~Yim;se homology with the anti-Tac MAb se~uence. In addition computer modelling was used to identify framewor~ amino acid residues which were likely to interact with the CDRs or antigen, and mouse ~m;no acids were used at these positions in the humanised antibody.

In WO90/07861 Queen et al propose four criteria for designing humanised ;m~noglobulins. The first criterion is to use as the human acceptor the framework from a particular human immunoglobulin that is unusually homologous to the non-human donor ;mml~noglobulin to be humanised, o_ to use a consensus framework from many h~n antibodies. The second criterion is to use the donor amino acid rather than the acceptor if the h~lm~n acceptor -- 6 - 2 1 2 ~ residue is unusual and the donor residue is typical for hn~n sequences at a specific residue of the framework.

The third criterion is to use the donor framewor~ amino acid re~idue rather than the acceptor at po~itions ;mme~;~tely adjacent to the CDRs. The fourth criterion is to use the donor amino acid resi~ue at framework positions at which the Am;no acid is predicted to have a side chain atom within about 3 ~ of the CD~s in a three-~;m~nsional imm-lnoglobulin model and to be capable of interacting with the antigen or with the CDRs of the hnm~nised immllnoglobulin. It is proposed that the second, third or fourth criteria may be applied in addition or alternatively to the first criterion, and m~y be applied singly or in any com~ination.

WO90/07861 describes in detail the preparation of a single CDR-grafted humanised antibody, a hllm~ni~ed antib~dy having specificity for the pS5 Tac protein of the I~-2 receptor. The combination of all four criteria, as above, were employed in designing this h~ n;sed antibody, the variable region framewor~s of the human antibody EU (7) being used as acceptor. In the resultant hl~m~n;sed antibody the donor CDRs were as defined by Rabat et al (7 and 8) and in addition the mouse donor residues were used in place of the human acceptor residues, at positions 27, 30, 48, 66, 67, 8g, 91, 94, 103, 104, 10~ and 107 in the heavy chain and at positions 48, 60 and 63 in the light chain, of the variable region frameworks. The humanised anti-Tac antibody obtained is reported to have an affinity for p55 of 3 x 109 M-l, about one-third of that of the murine MAb.

We have further investigated the preparation of CDRgrafted humanised antibody molecules and have identified a hierarchy of positions within the framewor~ of the variable regions (i.e. outside both the Rabat CDRs and _ 7 ~ ~ ~g 5 5 4 structural loops of the variable regions) at which the amino acid identities of the residues are important for obtaining CDR-grafted products with satisfactory binding affinity.

This has enabled us to establish a protocol for obtaining satisfactory CDR-grafted products which may be applied very widely irrespective of the level of homology between the donor immunoglobulin and acceptor framework.

The set of residues which we have identified as being of critical importance overlaps but does not coincide with the residues identified by Queen et al (9).

Our copending International patent application WO91/09967 describes this protocol for the preparation of CDR-grafted, in particular humanised, antibody heavy and light chains and complete molecules of any desired specificity.

Tempest et al (10) have very recently described the preparation of a reshaped human monoclonal antibody for use in inhibiting human respiratory syncytial virus (RSV) infection in vivo.

This reshaped antibody was prepared by grafting synthetic oligo nucleotides coding for the CDRs of a murine MAb, which neutralises RSV infection, by site - directed mutagenesis into DNA coding for the frameworks of a human IgGl, monoclonal antibody.

However the simple reshaped antibody in which the CDRs alone had been transferred between mouse and human antibodies had only very poor binding for RSV which was not significantly above background.

In order to partially restore binding ability it proved necessary to additionally convert human residues to mouse residues in a framework region adjacent to CDR3 of the heavy chain.

Tempest et al did not convert human residues to mouse residues at important positions identified in the protocol of WO91/09967.

- 8 ~ ¦ 2 9 5 TNF~ is a cytokine which is released by and interacts with cells of the immune system. Thus TNF~ is released by macrophages which have been activated by lipopolysaccharide (LPS) of gram negative bacteria. As _uch TNF~ appears to be an endogenous mediator of central importance involved in the development and pathogenesis of endotoxic shoc~ associated with bacterial sepsis. Antibodies to TNF~ has been proposed for the prophylaxis and treatment of endotoxic shock (Beutler et al (11)). ~owever the antibodies to TNF~ currently available for use in such treatment are typically murine MAbs. As such these murine MAbs are of only li mi ted ùse for treatment of hnmAns in view of the undesirable ~AMA (~nman Anti-Mouse Antibody) response which they can elicit if used for more than one or a few treatments. It is thus a highly deqirable objective to prepare ht1mAnised anti-TNF~ products for use in hum2n therapy.

Our co-~n~;ng International patent application WO91/09967 describes, among other things, the preparation of hl~m~n;ced CDR-grafted antibody products which have specificity for TNF~. In particular WO91/09967 describes, in ~Y~mrle 5, preparation of specific hllmAn;sed CDR grafted anti~odies to human TNF~ derived from the murine anti-h-~m~n TNF~ MAbs identified as 61E71 (alternatively known as CB0006), hTNFl (alternatively known as CB0010), hTNF3 and 101.4. The present application relates specifically to recombinant, in particular h~m~nised antibodies to human TNF~, including those described in WO91/09967 and further improved hnmAn;eed CDR-grafted antibodies to h~m~n TNF~ based upon the hTNFl (CB0010) and 101.4 murine MAbs.

Further studies of various anti-human TNF~ murine MAbs have revealed that hTNF1 and 101.4 have particularly desirable properties for use in anti-TNF therapy.

WO 92/11383 - r~ v~ v--vv 9 2129~ ~4 Summary of the Invention Accordingly the present invention provides recombinant antibody molecules which have specificity for h~ n TNF~.

The recombinant antibody molecules of the invention are preferably TNF neutralising, i.e. are capable of reducing or inhibiting a biological activity of human TNF~ as measured by an in vitro or in vivo test.

Preferably the invention provides recombinant antibodymolecules having antigen b; n~; ng sites derived from the murine MAbs C~0006. CB0010, hTNF3 or 101.4, especially from the murine MAbs CBQ010 or 101.4.

Preferably the recombinant antibody molecules of the invention are h~m~n;sed antibody molecules including both chimeric h~m~ ed antibody molecules a~d CDR-grafted humanised antibody molecules.

For the purposes of the present description a n chimeric hllmAn;sed antibody molecule~ comprises complete non-human (e.g. murine MAb) variable dom~;n~ linked to hl-m~n constant ~om~; ns ~ and a ~CDR-grafted hl~m~nised antibody molecule~ comprises an antibody heavy and/or light chain cont~;ning one or more CDRs from a non-hu~an antibody (e.g. a murine MAb) grafted into a hl~man heavy and/or light chain variable region framework.

The CDR-grafted hl~m~n;sed anti-TNF~ antibody products of this invention include anti-~l~m~n TNF~ antibody heavy and light chain and molecule products as defined in the first, second, third and fourth aspects of the invention described in WO91/09967.

WO92~11383 r~l/~D~rl~u~Jvv - lo - 2129~4 Detailed Description of the Invention Thus in first preferred embodiments, the invention provides a CDR-grafted h~ nised anti-hTNF~ antibody heavy chain having a variable region domain comprising human acceptor framework and donor antiqen binding regions wherein the framewor~ comprises donor residues at at least one of positions 6, 23 and/or 24, 48 and/or 49, 71 and/or 73, 75 and/or 76 and/or 78 and 88 and/or 91.

Preferably in these first preferred embodiments, the heavy chain framework comprises donor residues at positions 23, 24, 49r 71, 73 and 78 or at positions 23, 24 and 49. The residues at positions 71, 73 and 78 of the heavy chain framework are preferably either all acceptor or all donor residues.

Especially in these first preferred emboA;m~ts the heavy chain framewor~ additionally comprises donor residues at one, some or all of positions 6, 37, 48 and 94. Also it is particularly preferred that residues at positions of the heavy chain framework which are commonly conserved across species, i.e. positions 2, 4, 25, 36, 39, 47, 93, 103, 104, 106 and 107, if not con~er~ed between donor and acceptor, additionally comprise donor residues. Most preferably the heavy chain framework additionally comprises donor residues at positions 2, 4, 6, 25, 36, 37, 39, 47, 48, 93, 94, 103, 104, 106 and 107.

In addition the heavy chain framewor~ optionally comprises donor residues at one, some or all of positions:

1 and 3, 72 and 76, 69 (if 48 is different between donor and acceptor), 38 and 46 (if 48 is the donor residue), 80 and 20 (if 69 is the donor residue), 67, WO92111383 PCT/GB9t/023~ -- 11 - 2129~ 82 and 18 (if 67 is the donor residue), 9 1 , 88, and any one or more of 9, ll, 41, 87, 108, llO and 112.

In the present description, typically the donor antibody is a non-human anti-hTNF~ antibody, such as a rodent MAb, and the acceptor antibody is a h~mAn antibody.

In the CDR-grafted humanised anti-hTNF2 antibodies of the present invention, the donor antigen b;~ g region typically comprises at least one CDR from the donor antibody. Usually the donor antigen bi n~; ng region comprises at least two and preferably all three CDRs of each of the heavy chain and/or light chain variable regions. The CDRs may compri~e the Rabat CDRs, the structural loop CDR~ or a compo~ite of the Rabat and structural loop CDRs and any comb~nation of any of these.

Prefera~ly, the antigen binding regions of the CDR-grafted heavy chain variable domain comprise C~Rs corresponding to the Rabat CDRs at CDR2 (residues 50-65) and CDR3 ~residues 9j-102) and a composite of the Rabat and structural loop CDRs at CDRl (residues 26-35). These preferred CDR designations are preferably used for the CDR-grafted heavy chains of the first preferred em~odiments, i.e. residues 26-30 are included within CDRl.

The residue designations given above and elsewhere in the present application are num~ered according to the Rabat nllmhering [refs. (7) and (8)3. Thus the residue designations do not always correspond directly with the linear n~bering of the ~mi no acid residues. The actual linear amino acid se~uence may contain fewer or additional Amino acids than in the strict Kabat num~ering corresponding to a shortening of, or insertion into, a WU ~ v----vv - 12 - 2129~54 .~ structural component, whether framewor~ or CDR, of the basic variable domain structure. For example, the heavy chain variable region of the anti-Tac antib~dy described by Queen et al (9) contains a single amino acid insert (residue 52a) after residue 52 of CDR2 and a three amino acid insert (residues 82a~ 82b and 82c) after framework residue 82, in the ~a~at numbering. The correct ~abat numbering of residues may be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a ~stAn~Ard" Rabat numbered sequence.

. .

It will be appreciated that when the CDR-grafted humanised antibody molecule embodiments of the invention, as described above and elsewhere in the present description, are applied to a particular donor/acceptor antibody pair, in some ca~es the donor and acceptor Amino acid residues may be identical at a particular position identified for change to the donor residue, and thus no change or acceptor framework residue is re~uired.

The invention also provides in ~econd preferred embodiments a CDR-grafted humanised anti-hTNF~ antibody lisht chain having a variable region ~s~i n comprising h-lmAn acceptor framework and donor antigen binding regions wherein the framewor~ comprises donor residues at at least one of positions 1 and/or 3 and 46 and/or 47.

Preferably the CDR grafted light chain of the second preferred em~odiment comprises donor residues at positions 46 and/or 47.

The invention also provides in third preferred Pmbo~;ments a CDR-grafted h-lm~n;ced anti-hTNF~ antibody light chain having a variable region domain comprising human acceptor framewor~ and donor antigen b; n~; ~g regions wherein the ~ W~92tl1383 PCT/GB91/02~ ~1295~ . - 13 framewor~ comprises donor residues at at least one of positions 46, 48, 58 and 71.

In the third preferred embo~iments, the framework preferably comprises donor residues at all of positions 46,-48, 58 and 71.

In particularly preferred embodiments of the second and third preferred embo~im~nts, the framework additionally comprises donor residues at positions 36, 44, 47, 85 and 87. Similarly positions of the light ch~; n framework which are commonly conserved across species, i.e.

positions 2, 4, 6, 35, 49, 62, 64-69, 98, 99,.101 and 102, if not conserved between donor and human acceptor, additionally comprise donor residues. Most preferably the light chain framewor~ additionally comprises donor residues at positions 2, 4, 6, 35, 36, 38, 44, 47, 49, 62, 64-69, 85, 87, 98, 99, lO1 and 102.

In addition the framewor~ of the second or third pr~ferred embo~;ments optionally comprises donor residues at one, some or all of positions:

1 and 3, 63, 60 (if 60 and 54 are able to form at potential saltbridge), 70 (if 70 and 24 are able to form a potential saltbridge), 73 and 21 (if 47 is different between donor and acceptor), 37 and 45 (if 47 is different between donor and acceptor), and any one or more of 10, 12, 40, 80, 103 and 105.

Preferably, the antigen binding regions of the CDR-grafted light chain variable domain, including those of the second and third prererred embodiments described above, comprise CDRs corresponding to the Rabat CDRs at CDRl (residue 24-34~, CDR2 (residues 50-56) and CDR3 (residues 89-97).

WO92/11383 r~~ 7.~v~vv - 14 21295~ i The invention further provides in a fourth preferred embodiment a CDR-grafted antibody molecule comprising at least one CDR-grafted heavy chain and at least one CDR-grafted light rh~;~ according to the first and second or first and third preferred embo~;mP~ts of the invention.

In a first particularly preferred embodiment, however, the invention provides a CDR-grafted hu_anised antibody heavy chain having a variable region d~m~;n comprising human acceptor framework (especially EU human acceptor framework) and hTNFl donor antigen binding regions wherein the framework comprise~ hTNFl donor residues at positions 12, 27, 30, 38, 46, 48, 66, 67, 69, 71, 73, 7~, 83, 89, 9l and 94.

The EU heavy chain framewor~ has residues in framework 4 (FR4) of the heavy chain which are anomalous for human heavy chain frzmewor~s. Thus preferably hllm~n consensus residues are used in place of EU residues in FR4 of the heavy chain. In particular, the h~lm~n con~ensus residue threonine (T) may be used at position 108. Fortuitously the murine hTNFl residue at position 108 is also threonine.

Ir. a second particularly preferred emboA;ment the invention provides a CDR-grafted h-lm~nised antibody light chain ha~ing a variable domain comprising human acceptor framewor~ (especially EU h~ n acceptor framewor~) and hTNFl donor antigen binding regions wherein the framework comprises hTNFl donor re~idues at positions 3, 42 and 49.

When the EU human framewor~ is used for the li~ht chain it is also desirable to change residues from EU residues at positions 48, 83, 106 and 108, as the EU residues at these positions are anomalous for human antibodies. Thus the hllm~n consensus residues may be used at some or preferably all of these residues, i.e. isoleucine (I) at position 48, WO92~ll383 PCT/G~I/uz~uo - 15 - 2i29~ valine (V) at position 83, isoleucine (I~ at position 106 and arginine (R) at position 108. Fortuitously the murine hTN~1 residues are the same as the human consensus residues at positions 48 (I), 106 (I) and 108 (R).

~owever, the human consensus residue valine (V) at position 83 differs from both the EU residue (F) and the hT~F1 residue (L) at this position.

Especially the invention includes CDR-grafted htlm~n;sed anti~ody molecules comprising at least one CDR-grafted humanised heavy chain according to the first particularly preferred embodiment and at least one CDR-grafted humanised light chain according to the second particularly preferred embodimen~.

Also in a third particularly preferred Pmho~ i ~ent the invention provides a CDR-grafted hnm~niced antibody heavy chain having a variable region dom~ i n compriBing human acceptor framework (especially ROL human acceptor framework) and 101.4 donor antigen binding regions wherein the framewor~ comprises 101.4 donor residues at positions 4, 11, 23, 24, 28, 73, 77, 78, 79, 91, 93 and 94.

The ~OL residue proline (P) at position 108 of the heavy chain is anomalous for human antibodies. Thus preferably the human consensus residue leucine (~) is at this position if ROL is used as the htlmAn acceptor framework.

Fortl~itously the murine 101.4 antibody has the human consensus residue (L) at this position.

Moreover in a fourth particularly preferred emboAi~ent the invention provides a CDR-grafted humanised antibody light chain having a variable region domain comprising human acceptor framewor~ (especially REI human acceptor framewor~) and 101.4 donor residues at positions 1, 3, 4 and 73.

~V 7~ ~ IJ~J - 16 ~129~ ~ The REI light chain human framework has residues which are anomalous for hum2n antibodies at positions 39 (threonine, T), 104 (leucine, L), 105 (glutamine, Q), and 107 (threonine, T). Thus when REI is used as the light chain framework, hum~n consensus residues are used at positions 39 (lysine, ~), 104 (valine, V), 105 (gluta_ic acid, E) and 107 (lysine, R). Fortuitously the murine 101.4 residues are the same as the human consensus residues at po~itions 39 (R), 105 (E) and 107 (R). ~owever, the human consensus residue at position 104 (V) differs from the leucine (L) REI and murine 101.4 residues at this position.

, Especially also the invention includes CDR grafted h~mAn;sed antibody molecules comprising at least one CDR-grafted h~m~ni~ed heavy chain according to the third particularly preferred emboA; m~nt and at least one CDR-grafted hllm~nised light chain according to the fourth particularly preferred embodiment.

Preferably the ~abat CDRR are used for all of t~e CDRs (CDRl, CDR2 and CDR3) of both the heavy and light chains of the first, second, third and fourth particularly preferred embodiments described above.

The recombinant and h~m~n;~ed antibody molecules and ~h~; n~ of the present invention ~ay comprise: a complete antibody molecule, having full length heavy and light ch~; nc; a fragment thereof, such as a Fab, Fab', F(ab')2 or Fv fragment; a light chain or heavy chain monomer or dimer; or a single chain antibody, e.g. a single chain Fv in which heavy and light chain variable regions are joined by a peptide linker; or any other recombinant, chimeric or CDR-grafted molec~le with the same specificity as the original donor antibodies. Sim~larly the heavy and light chain variable region may be combined with other antibody dom~ i n S as appropriate.

- 17 Also the heavy or light chains or recombinant or humanised complete antibody molecules of the present invention may have attached to then an effector or reporter molecule.

For instance, it may have a macrocycle, for chelating a heavy metal atom, or a toxin, such as ricin, attached to it by a covalent bridging structure.

Alternatively, the procedures of recombinant DNA technology may be used to produce an immunoglobulin molecule in which the Fc fragment or CH3 domain of a complete immunoglobulin molecule has been replaced by, or has attached thereto by peptide linkage, a functional non-immunoglobulin protein, such as an enzyme or toxin molecule.

The amino acid sequences of the heavy and light chain variable domains of the CB0010, 101.4, CB0006 and hTNF3 murine MAbs, CDR-grafted variants thereof and human acceptor antibodies are given in the accompanying diagrams Figures 1, 2, 3 and 4 respectively.

The recombinant and humanised antibody products of the invention may be prepared using recombinant DNA techniques, for instance substantially as described in WO91/09967.

Any appropriate human acceptor variable region framework sequenced may be used having regard to class/type of the donor antibody from which the antigen binding regions are derived.

Preferably, the type of human acceptor framework used is of the same/similar class/type as the donor antibody.

Conveniently, the framework may be chosen to maximise/ optimise homology with the donor antibody sequence particularly at positions close or adjacent to the CDRs.

However, a high level of homology between donor and acceptor sequences is not critical for application of the present invention.

The present invention identifies a hierarchy of framework residue positions at which donor residues may be important or desirable for obtaining a CDR-grafted antibody product W092/tl383 .~ - 18 - 2129351 ', ..

having satisfactory binding properties. The CDR-grafted products usually have binding affinities of at least 105 M-l, preferably at least about 108 M-l, or espe~ially in the range 108-1012 M-1. In principle, the pre~ent invention is applicable to any combination of anti-hTNF~ donor and hl~mAn acceptor antibodies irrespective of the level of homology between their sequences. Examples of h~ n framewor~s which may be used are ROL, NEWM, REI, EU, TUR, ~EI, LAY and POM (refs. 7 and 8) and the like; for instance ROL and NEWM for the heavy chain and REI for the li~ht chain and EU, LAY and POM for both the heavy chain and the light chain.

Also the constant region ~omA i n~ of the products of the invention may be selected haYing regard to the proposed function of the antibody in particular the effector functions which _ay be required. For example, the constant region dom~in~ may be human IgA, IgE, IgG or ~gM ~m~;~c.

In particular, Ig~ human constant region ~om~in~ may be used, especially of the IgG1 and IgG3 isotypes, when the hu_anised antibody molecule is intended for therapeutic uses, and antibody effector functions are required.

Alternatively, IgG2 and IgG4 isotypes may be used when the humanised antibody molecule is intended for therapeutic purposes and antibody effector functions are not required, e.g. for simple bloc~ing of TNF acti~ity.

~owe~er, the remainder of the antibody molecules need not comprise only protein sequences from immunoglobulins.

~or instance, a gene may be constructed in which a DNA sequence encoding part of a human ;m~unoglobulin chain is fused to a DNA sequence encoding the ~mi no acid sequence of a functional polypeptide such as an effector or reporter molecule.

PCT/GB91t02~n W092/11383 2129S~ ;~ .

In further aspects the invention also includes DNA sequences coding for the recombinant and h1~m~n;~ed antibody, e.g. CDR-grafted, heavy and light ch~;n~, cloning and expression vectors cont~i n; ng the D~A sequences, host cells transformed with the DNA sequences and processes for producing the recombinant and h~1m~n;sed, e.g. CDR-grafted, chains and antibody molecules comprising expressing the DNA sequences in the transformed host cells.

The general methods by which the vectors may beconstructed, transfection methods and culture methods are well known per se. Such ~ethods are shown, for instance, in references 12 and 13.

The DNA sequences which encode the anti-hTNF~ antibody molecule amino acid sequences may be obtained by methods well known in the art. For eY~m~le the anti-TNF coding sequences may be obtained by genomic cloning, or cDNA cloning from suitable anti-hTNF~ producing hybridom~ cell lines.

Positive clones may be screened using appropriate probes for the heavy and light chain genes in question.

Also PCR cloning may be used. DNA sequence coding for part or all of the antibody heavy and light ~h~; n~ may be synthetised as desired from the determined DNA sequence or on the basis of the corresponding amino acid se~uence.

DNA coding for acceptor, e.g. h~ n acceptor, sequences may be obtained in any appropriate way. For example DNA sequences coding for preferred human acceptor frameworks such as ROL, REIj EU and NE~M, are widely available to wor~ers in the art, or may be readily synthetised on the basis of their known amino acid sequences (see refs.

7 & 8).

W092/1l3~3 - 20 - 2129 _ .

The stAn~ rd techniques of molecular biology may be used to prepare DNA sequences coding for the chimeric and CDR-grafted hnm;~nised antibody products. 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. For example oligonucleotide dire~ted synthesis as described by Jones et al (ref. 14) may be used. Also oligonucleotide directed mutagenesis of a pre-existing variable region as, for example, described by Verhoeyen et al (ref. 5) or Riechm;nn et al (ref. 6) may be used. Also enzymatic filling in of gapped oligonucleotides using T4 DNA polymerase as, for example, described by Queen et al ~ref. 9) may be used.

Any suitable host cell/vector system may be used for expression of the DNA sequence~ ~Ai ng for the recombinant, ~h;meric and CDR-grafted ht7m~ni-ed ~ntibody heavy and light chains. Bacterial e.g. E. coli, and other microbial systems may be used, in particular for expression of antibody fragments such as Fab and F(ab')2 fr~qm~ts, and e~pecially Fv fragments and single chain antibody fragments e.g. single rhA i n Fvs. ~ucaryotic e.g. ~mm~l ian host cell expression systems may be used for production of larger CDR-grafted anti~ody products, including complete antibody molecules, and/or if glycosylated products are required. Suitable ~7~mm-l i,7n host cells include CH0 cells and myeloma or hybridoma cell lines.

Thus, in a furthèr aspect the present invention provides a process for producing a recombinant or h..m;7ni ~ed anti-hTNF~ antibody product comprising:

r~ D~ ~ v~7vv WO 9Z/1 1383 - 21 - 2i295~ a) producing in an expression vector an operon having a DNA se~uence which encodes an anti-hTNF2 antibody heavy chain;

and/or (b) producing in an expresfiion vector an operon having a DNA sequence which encodes a complementary anti-h~NF~ antibody light chain;

(c) transfecting a host cell with the or each vector; and (d) culturing the transfected cell line to produce the recombinant anti-hTNF~ antibody product.

The recombinant or hllm~n;~ed anti-hTNF~ product may comprise only heavy or light chain derived polypeptide, in which case only a heavy chain or light chain polypeptide coding sequence is used to transfect the host cells.

For production of products comprising both heavy and light rh~;rs, the cell line may be transfected with two vectors, a first vector cont~;nirtg an operon encoding a light ~hrt;n-derived polypeptide and a second vector cont~;n;~g an operon encoding a heavy chain-derived polypeptide.

Preferably, the vectors are identical, except in so far as the coding sequences and selectable mar~ers are concerned, so as to ensure as far as possible that each polypeptide chain is equally expressed. Alternatively, a single vector may be used, the vector including the se~uences encoding both light chain- and heavy chain-derived polypeptides.

The DNA in the coding sequences for the light and heavy chains may comprise cDNA or genomic D~A or both.

The invention also includes therapeutic and diagnostic compositions comprising the recom~inant and h~m~n;~ed antibody products of the invention and the uses of these products and the compositions in therapy and diagnosis.

.

W092/11383 .~. ~ .

- 22 ~ 2129~4 Thus in a further aspect the in~ention provides a therapeutic or diagnostic composition comprising a recombinant or hllm~n;sed antibody according to the invention in combination with a ph~rm~eutically acceptable excipient, diluent or carrier.

The invention also provides a process for the preparation of a therapeutic or diagnostic composition comprising A~m;Y;ng a recombinant or hllm~n;sed antibody according to the invention together with a ph~rm~ceutically acceptable excipient, diluent or carrier.

The recombinant or humanised antibody may be the sole active ingredient in the therapeutic or diagnostic composition or may be accompanied by one or more other active ingredients includi~g other antibody ingredients, e.g. anti-T cell, anti-IFN~ or anti-TPS ~ntibodies, or non-antibody ingredients ~uch as xanthi~e~. The therapeutic and diagnostic compositions m2y be in unit dosage form, in which caRe each unit dose comprises an effective amount of the recombinant or hnm~ised antibody of the invention.

Furthermore, the in~ention also provides methods of therapy and diagnosis comprising ~m; n; ctering an effective amount of a recombinant or humanised antibody according to the invention to a human or ~nim~l subject.

The antibodies and compositions may be utilised in any therapy where it is desired to reduce the level of TNF present in the human or ~nim~l body. The TNF may be in circulation in the body or present in an undesirably high level localised at a particular site in the body.

PCI /GBYlJU~w WO 92J1 1383 -- 23 - ;~129~5 i For e~mrle, elevated levels of TNF are implicated in imml~noregulatory and infl~mm~tory dis~rders and in septic, or endotoxic, and cardiovascular shoc~. The antibody or composition m~y be utilised in therapy of conditions which include sepsis, septic or endotoxic shoc~, cachexia, adult respiratory distress syndrome, AIDS, allergies, psoriasis, T.B., inflammatory bone disorders, blood coagulation disorders, burns, rejection episodes following organ or tissue transplant and auto;mml~ne disease e.g. organ specific disease such as thyroiditis or non-specific organ diseases such as rheumatoid and osteo-arthritis.

Additionally, the antibody or composition may be used to ameliorate side effects associated with TNF generation during neoplastic therapy and also to eli~;nate or ameliorate shoc~ related symptoms associated with the treatment or prevention of graft rejection by u5e of an antilymphocyte antibody, or may be u~ed for treating multi-organ failure (MOF~.

The recombinant and humani~ed antibodies and compositions of the invention are preferably for treatment of sepsis or septic/endotoxic shock.

The antibodies and compositions may be for A~m; ni stration in any appropriate form and amount according to the therapy in which they are employed. This may be for prophylactic use, for example where circ1tmstances are such that an ele~ation in the level of TNF might be expected or alternatively, they may be for use in reducing the level of TNF after it has reached an undesirably high level or as the level is rising.

The therapeutic or diagnostic composition may take any suitable form for ~mi n;stration, and, preferably is in a form suitable for parentera~ a~m; ni stration e.g. by W092/113~3 - 24 ~ 2129~ .. . .

injection or infusion, for example by bolus injection or continuous infusion. Where the product is for injection or 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, stabilising and/or dispersing agents.

Alternatively, the antibody or composition may be in dryform, for reconstitution before use with an appropriate sterile liquid.

If the antibody or composition is suitable for oral a~;nictration, e.g. in the case of antibody frasments, the formulation may contain, in addition to the active ingredient, additives such as: starch - e.g. potato, maize or wheat starch or cellulose - or starch derivatives such as microcryst~11;ne cellulose; silica; various sugars such as lactose; m~gnesium carbonate and/or calcium phosphate. It is desirable that, if the for~ Ation is for oral ~m; ni stration it will be well tolerated by the patient~s digestive system. To this end, it may be desirable to include in the formulation mucus formers and resins. It may also be desirable to improve tolerance by forml11~ting the antibody or compositions in a capsule which is insoluble in the gastric juices. It may also be preferable to include the antibody or composition in a controlled release formulation.

In a still further aspect of the invention, there is provided a method of treatment of a human or An;m~l subject suffering from or at risk of a disorder associated with an undesirably high level of TNF, the method comprising A~m; n; stering to the subject an effective amount of the antibody or composition of the invention. In particular, the human or ~n;m~l su~ject may be suffering from, or at risk from, sepsis, or septic or endotoxic shock.

PCI-/G B9 1 /UZ3UU Wp 92/1 1383 - 2~i - 21295~4 .

The dose at which the antibody is a~mi n; stered depends on the nature of the condition to be treated, the degree to which the TNF to be neutralised is, or is expected to be, raised above a desirable level, and on whether the antibody is being used prophylacti~A lly or to treat an existing condition. The dose will also be selected according to the age and conditions of the patient.

Thus, for example, where the product is for treatment or prophylaxis of septic shock suitable doses of antibody to TNF lie in the range O.001-30mg/kg/day, preferably O.Ol-lOmg/~g/day and particularly preferably 0.1-2mg/kg/day.

The antibody products may be used in diagnosis e.g. in in vivo diagnosis and imaging of disease states involving elevated TNF levels.

The invention is further described by way of illustration only in the following Examples which refers to the accompanying diagrams, Figures 1 - 6.

Brief De~cription of the Fiqure~ Figure 1 shows amino acid sequences for the variable do~;ns of the heavy and light chAin~ for the h~m~n acceptor antibody EU (lEU), the murine MAb CBOO10 (h t n f 1) and humanised CDR grafted light (gEU) and heavy (2hEUg) chains;

Figure 2 shows ~m; no acid sequences for the variable region ~om~in~ of the h~man acceptor ant;ho~;es REI ( 1 r e i ) for the light chain and ROL (ROL) for the heavy chain, of the heavy and light chains of the murine MA~ 101.4 (10!/4J and humanised grafted light and heavy chains (both designated glO14);

w~J y--~ l l~o~ ~ - 26 - 2129~4 Figure 3 shows amino acid sequences for the variable region dom~ins of the human acceptor antibodies REI (REI) for the light chain and ~OL (ROL) for the heavy chain, of the heavy and light chA;~C of the murine MAb CB0006 (CB6) and humanised grafted light and heavy chains (both designated gCB6);

Figure 4 shows ~m; no acid sequences for the variable region ~om~;n~ of the human acceptor antibodies REI (REI) for the light chain and ROL (~OL) ~or the heavy ~h~; n ~ of the heavy (~TNF3) and light (hTNF3) chains of the murine MAb HTNF3 and humanised grafted light (g~TNF3) and heavy (ghTNF3) chains; . Figure ~ shows a graph comparing the ability of murineCB0010 (hTNF1) and CDR-grafted CB0010 (GrhTNF1; CDP571) to compete with ~RP-conjugated murine ~TNFl for b;n~;ng to recombinant human TNF~, and Figure 6 shows a graph comparing the ability of murine ~TNFl (CB0010) and CDR-grafted ~TNFl tCP571) to neutralise recombinant T~F~ in the ~929 bioassay.

WO92/11383 P~ /u~w - 27 - ~129~ .. ..

Detailed Description of Embodiments of the In~ention e 1 CDR-Graftinq of murine anti-TNF~ antibodies A n~mher of murine anti-human TNF2 MAbs were CDR-grafted substantially as described in detail in WO91/09967 for the CDR-grafting of the murine anti-CD3 antibody ORT3. In this and subsequent ~xAm~les, the chimeric and CDR-grafted hl~m~n;sed antibodies were prepared using hl~m~n IgG4 constant regi~n dgm~ i ns, substantially as described for preparation of ~4 chimeric and CDR-grafted ORT3 antibodies in WO91/09967. It will be appreciated, however, that human constant region dom~ i n~ of other types and isotypes, e.g.

IgG1, IgG2 and IgG3, could also have been used without significantly altering the proc~dures described.

These anti-hTNF~ antibodies included the murine MAbs designated C90006 (also ~nown as 61E71), CB0010 (also known as hTNF1), hTNF3 and 101.4 A brief snmmAry of the CDR-grafting of each of theqe antibodies is gi~en below.

CB0006 A s; m; lar analysis as described in Example 1, Section 12.1. of WO91/09967 was carried out for CB0006 and for the heavy chain 10 framewor~ residues were identified at positions 23, 24, 48, 49, 68, 69, 71, 73, 75 and 88 as residues to potentially retain as murine. The hllm~n frameworks chosen for CDR-grafting of this antibody, and the hTNF3 and 101.4 antibodies were RE1 for the light chain and ROL for the heavy chain. The amino acid se~uences of the murine CB0006 (CB6) (heavy and Light) REI (REI) light and ROL (ROT) heavy chain variable ~om~ins are gi~en in Figure 3.

Three genes were built, the first of which coded for amino acid residues 23, 24, 48, 49, 71 and 73 [g~341(6)~ as murine residues. The ~mi no acid sequence of the ~ariable - 28 - ~ 1 2 9 ~ ~ ~ AomA; n coded by this first gene is shown as gCB6 in the heavy chain sl~mmAry in Figure 3. The ~e~ond gene also had amino acid residues 75 and 88 as murine residues [g~341(8)~ while the third gene addit;onAlly had ~m;no acid residue~ 68, 69, 75 and 88 as murine residues [g~341(10)]. Each was co-expressed with gL221, the m;~;mllm grafted light chain (CDRs only) shown as gCB6 in the hea~y chain summary in Figure 3. The gL221~g~341(6) and g~221/g~341(8) antibodies both bound as well to TNF as murine 61E71. The gL2~1/g~341(10) antibody did not express and this combination was not taken further.

Subsequently the gL221/gH341(6) antibody was assessed in an L929 cell competition assay in which the a~tibody competes against the TNF receptor on L929 cells for binding to TNF in solution. In this assay the gL221/g~341(6) antibody was apprnximAtely 10% as active as murine CB0006.

C80010 (also known as hTNF1) CB0010 is a monoclonal antibody which recognises an epitope of human TNF-~. The EU human framewor~ was used for CDR-grafting of both the heavy and light variable domains. The amino acid sequences of the heavy and light variable domAins of EU (EU), CB0010 (h t n f 1) and grafted versions of CB0010 (gEU, light; 2h~Ug, heavy) are shown in Figure 1.

~eavy Chain ~n the CDR-grafted heavy chain mouse CDRs were used at positions 26-35 ~CDR1), 50-65 (CDR2) and 95-102 (CDR3).

Mouse residues were also used in the fr~meworks at positions 48, 67, 69, 71, 73, 76, 89, 91, 94 and 108.

romr~rison of the TNFl mouse and ~U hnm~n heavy chAi n residues reveals that these are identical at positions 23, 24, 29 and 78.

PCTJGB9l/O~oo W0 92/1 1383 - 29 - ~129 '.

~i~ht Chain In the CDR-grafted light chain mouse CDRs were used at positions 24-34 (CDRl), 50-56 (CDR2) and 89-97 (CDR3).

In addition mouse residues were used in the framewor~s at positions 3, 42, 48, 49, 83, 106 and 108. Comparison of the hTNFl mouse and EU human light chain residues reveals that these are identical at positions 46, 58 and 71.

The grafted CB0010 heavy chain was co-expressed with the rh;meric light chain and the bin~;ng a~ility of the product compared with that of the chimeric light chain/chimeric heavy chain product in a TNF binding assay.

The grafted heavy chain product appeared to.have b;n~ing ability for TNF slightly ~etter than the fully chim~ric product.

S;~;larly, a grafted heavy chain/grafted light chain product was co-expressed and compared with the fully chimeric product and found to have closely ~; m; lar b; n~; ng properties to the latter product. ~owever when the grafted heavy chain/grafted light chain product was assayed in the Lg29 assay (see ~Y~mFle 4), it was found to have an activity only half that of the ~; m~riC product.

Thus further CDR-grafting experiments were carried out as described in Example 2.

hTNF3 hTNF3 recognises an epitope on human TNF-~. The seguence of hTNF3 shows only 21 differences compared to CB0006 in the light and heavy chain variable regions, lO in the light chain (2 in the CDRs at positions.50, 96 and 8 in the framework at 1, 19, 40, 45, 46, 76, 103 and 106) and 11 in the heavy chain (3 in the CDR regions at positions 52, 60 and 95 and 8 in the framework at 1, lO, 38, 40, 67, 73, 87 and 105). The light and heavy chain variable domain amino acid se~uences of hTNF3 (~tnf3, light;

- 30 '~129~ ~ hTNF3, heavy), CDR-grafted hTNF3 (gHTNF3, light; ghTNF3, heavy) and R~I (REI, light) and KOL (ROL, heavy) are shown in Figure 4. The light and heavy chains of the CB0006 and hTNF3 chimeric antibodies can be exchanged without loss of activity in the direct b; n~ assay. However CB0006 iB an order of ~agnitude less a~le to compete with the TNF receptor on L929 cells for TNF-a compared to hTNF3. Based on the CB0006 CDR grafting data gL221 and gH341(+23, 24, 48, 49 71 and 73 as mouse) genes have been built for hTNF3 and tested and the resultant grafted antibody binds well to TNF-a, ~ut competes very poorly in the L929 assay. The gL221 gene codes for the gHTNF3 and the gH341 etc. gene codes for the ghTNF3 variable dom~i n sequences as shown in Fig~re 4. It is likely that in this case other framework residues may need to be changed to improve the competitive b; n~i ng ability of this antibody.

101.4 101.4 is a further murine MAb able to recognige hllm~n TNF-~. The heavy chain of this antibody shows good homology to ROL and so the CDR-grafting ha~ been based on REl for the light ch~in and ROL for the heavy chain. The heavy and light variable dom~ mi no Acid sequences of 101.4 (101~4) and a CDR-grafted version of 101.4 (glO14) and the REI light chain ( 1 r e f) and ROL heavy chain (ROL) variable ~om~; n~ are given in Figure 2. Several grafted he~vy chain genes have been constructed with con~ervative choices for the CDR's (g~341) and which have one or a small nn~her o~ non-CDR residues at positions 73, 78 or 77-7g inclusive, as the mouse amino acids. These have been co-expressed with the chimeric light chain or the Rabat CDR-grafted light chain. In all ca~es binding to TNF equivalent to the chimeric antibody is seen and when co-expressed with cL the resultant antibodies are able to compete well in the L929 assay. However, with W092/11383 PCT/GB91/UZ3~ - 31 - ~129~ _ gL221 the resultant antibodies are at least an order of magnit~de less able to compete for TNF against the TNF receptor on L929 cells.

Mouse residues at other positions in the heavy chain, for example, at 23 and 24 together or at 76 have been demonstrated to provide no improvement to the competitive ability of the grafted antibody in the L929 assay.

- 32 ~129~ ~Y~le 2 Further CDR-Graftin~ of Murine anti-human TNP3 Antibodies CB0010 and 101.4 ~urine anti-human TNF~ monoclonal antibodies CB0010 and 101.4 were further CDR-grafted substantially as described in WO91/09667.

CB0010 CB0010 is a monoclonal antibody which recognises an epitope on human TNF-~. The EU human framework was used for CDR-grafting of both the heavy and light variable domains.

The ~;no acid sequences of the heavy and light chain variable domains of the EU acceptor, CB0010 (h t n f 1) murine donor and CDR-grafted (gEU, light chain and 2hEUg, heavy chain) antibodies are given in Figure 1.

~eavy chain In the CDR-grafted heavy chain (2hEUg), mouse CDRs were used at positions 31-35 (CDR1), 50-65 (CDR2) and 95-102 (CDR3).

Mouse residues were also used in the frameworks at positions 12, 27, 30, 38, 46, 48, 66, 67, 69, 71, 73, 76, 83, 89, 91, g4 and 108. Comparison of the CB0010 mouse and EU human heavy chain residues reveals that these are identical as positions 23, 24, 29 and 78.

Li~ht chain In the CDR-grafted light chain (gEU) mouse CDRs were used at positions 24-34 (CDR1), 50-65 (CDR2) and 89-97 (CDR3).

In addition mouse residues were used in the frameworks at positions 3, 42, 48, 49, 106 and 108. The human consensus residue (valine) was used at position 83. Comparison of the CB0010 mouse and EU human light chain residues reveals that-these are identical at positions 46, 58 and 71.

WO92/11383 r~/~7~ vv ~12g~ 1 The grafted CB0010 heavy chain was co-expressed with the chimeric light chain and the bin~;ng ability of the product compared with that of the chLmeric light chain/chimeric heavy chain product in a TNF b;n~;ng assay. The grafted heavy chain product appeared to have b; n~; ng ability for TNF slightly better than the fully chimeric product.

S;m; 1 ~rly~ a grafted heavy chain/grafted light chain product was co-expressed and compared with the fully chimeric product and found to have closely S;mi 1 ~r binding propert-ies to the latter product. The specific combination of grafted light chain (gEU) and grafted heavy chain (2hEUg), as shown in Figure 1, provides the antibody known as CDP571. The murine CB0010 (CB0010), chimeric CB0010 (chimeric CB0010) and the grafted heavy chain/grafted light chain product(CDP571) were compared for b;~;ng to human TNF~ in a st~n~Ard assay. The results obtained are given in the table below in terms of the ~D (pM) measured for each antibody.

Antibody gD (pM) CB0010 80 rh; m~riC CB0010 81 CDP571 87 The fully grafted antibody product (CDP571) is currently in pre-clinical development for treatment of sepsis syndrome and acute transplant rejection.

101.4 10~.4 is a further murine MAb able to recognise human TNF-~. The heavy chain of this antibody shows good homology to ROL and so the CDR-grafting has been based on REI for the light chain and ~OL for the heavy chain. An improved CDR-grafted product has been prepared. Variable - 34 212gj'5~ domain amino acid sequences for REI (rei, light chain), ROL (~OL, heavy chain) murine 101.4 (101/4, heavy and light chain) and fully grafted antibody (glO14, heavy and -light chainJ are shown in Figure 2.

~eavy chain In the CDR-grafted heavy chain ~glO14) mouse CDRC were used at position 31-35 (CDR1), 50-65 (CDR2) and 95-102 (CDR3). Mouse residues were also used in the framework at positions 4, 11, 23, 24, 28, 73, 77, 78, 79, 91, 93, 94 and 108.

Liqht chain In the CDR-grafted light chain (glO14) mouse CDRs were used at positions 24-34 (CDR1), 50-~6 (CDR2) and 89-97 (CDR3).

In addition mouse residues were used in the framework at positions 1, 3, 4, 39, 73, 105 and 107. The human consen~u~ residue (valine) was used at position 104.

The fully grafted heavy and light chain (glO14) were co-expressed and their binding to TNF compared with-murine and chimeric 101.4 and also the fully grafted (gEU/2hEUg, CDP571) CBOO10 antibody. The fully grafted 101.4 antibody was found to having bi~;ng properties for human T~F~ similar to the murine, chimeric, and grafted CBOO10 antibodies.

W~92Jl13~3 PCT~GBgl/0230~ - 35 - ~129~ .~, tr~le In vitro comparison of ~uri~e a~d CD~-qrafted Antibodies A.

Affinity Measurements for Murine CBOO10 and CDP571 Materials and Methods Materials:

PBS/BSA: Dulbeccos PBS I 1% (w/~) ~ovine serum alb-~;n.

_ : 50nM rec. human TNF-alpha (Bissendorf Biochemicals), 0.8~g/ml in PBS/BSA.

Stoc~ l2sI-TN~ Ci, 185kBq (Amersham International) dissolved in 500~1 water and stored at -70~C.

Workin~ Solution ~2~I-TNF: -62pM for titration curYe and 124pM for Scatchard analysis, in PBS/BSA.

Antibodies: PurifLed murine CBOQ10 (m~TNFl) and CDP571 were quantified by A280nm (Elmg/ml, 280nm'l '), and diluted to a conc2ntration of l~g/ml for titration, or 20Ong/ml for Scatchard analysis.

Tmm-~no~eads: Goat anti-murine IgG whole mole~ule-agarose or goat anti-human IgG whole molec~le-agarose (Sigma) were used undiluted.

Method:

Antibody tltration: m~TNF1 and CDP571 were titrated in doubling dilutions (100~1 eachJ to giYe a total of 16 samples and l25I-TNF (100~1, 62pM) wa~ added. The final top concentration of anti~ody was 500ngJml and l25I-TNF was ~lp~. Control tubes (8) contained I 2 6 I-TNF and PBS/BSA only. The samples were left to equilibrate overnight at room temperature, with shaking. After e~uilibration, 25~1 goat anti-mouse-agarose was added to the m~TNFl samples, and 50~1 goat anti-ht~ n beads were added to the CDP~71 samples except for the total ~25I-TN~ controls. Non-speci~ic absorption of I-TNF to the agarose beads was corrected for ~y adding beads to w~ yz/ ~ - 36 2129~ 4 of the controls and comparing supernatant counts for these samples with those cont~in;ng PBS/BSA instead o~ beads. After 1 hour equilibration at room temperature PBS/BSA (O.5ml) was added and the samples were centrifuged at 1500rpm for 10 mins at 20~C. The supernatant (O.5ml) was removed and radioactivity was counted in a gamma counter.

Confirmation that 12 sI-TNF behaved s; m; 1 ~ rly to the unlabelled material in this assay was made by performing the antibody titration in the presence of mixtures of ~26I-TNF and unlabelled TNF ~at 25% and 75% ~26I-TNF) at the same total concentration.

Scatchard analysis: For both antibodies, unlabelled TNF (100~1, 50nM) was titrated in,~uplicate, in 13 doubling dilutions. One sample cont~;~ing PBS/3SA in place of TNF was included for each antibody. l25I_TNF (50~1, 124pM) was added to each sample. A constant amount of ant~body, determined from the titration cur~e (50~1, 200ng/ml) was then added.

This gave the following final concentrations: antibody, 50ng/ml; TNF, 25nM top concentration; ~2sI-TNF, 31p~.

The samples were left to equilibrate overnight and then treated exactly as for the antibody titration samples.

Calculations Titration Cur~es Bound 12 5 I-TNF cpm = NSB cpm - supernatant cpm Bound 12 5 I-TNF cpm = B/T Total 12 S I -TNF NSB = non-speci~ic absorption blank, supernatant cpm Total = total counts for l2sI-TNF only WO ~2/11383 PCT/GB91/023 - 37 - 2~ , B/T was plotted against antibody concentration and the appropriate antibody concentration for use in Scatchard analy~es was chosen at B/T = 0.6 Scatchard analysis The mean of duplicate determination was used throughout NSB z Total cpm-NSB supernatant cpm Free cpm = sample cpm + NSB Proportion of free-TNF = Free/Total (F/T) = sample cpm + NSB cpm l-F/T = B~F = Total cpm F/T B/F was plotted against Bound TNF to give a slope of -l/Rd from which Rd was calculated RESULTS Di~sociation constants for murine C~0010 and CDP571 Antibody Rd:~ Murine ETNF1 1.3 x 10-1~ CDP571 1.4 x 10-~~ 38 ~ 5 5 ~ B. Competition of murine CB0010 (Muhtnfl) and CDP571 (GrhTNFl) with HRP-coniuqated in Murine CB0010 for binding to rhuTNF Method A 96 well microtitre plate (Nunc, Maxisorb*) was coated with 100~1/well TNF at 0.5~g/ml.

Serial dilutions of murine or grafted antibody were prepared using PBS/1~ BSA diluent, from 200~g/ml to 0.01~g/ml. 50~1 of antibody was added to each well followed by 50~1 HRP-murine CB0010 at 3 concentrations (0.625, 0.315 and 0.16~g/ml).

Plates were incubated for 2 hours at room temperature with agitation, washed 4 times with PBS and 100~1 of TMB substrate added.

Optical Density was measured and OD plotted against antibody concentration.

Conclusions Curves for both murine antibody (MuhTNFl) and grafted antibody (GrhTNFl) are superimposable, indicating both antibodies compete with similar affinity for binding to TNF (see Figure 5).

* TRADE-MARK WO 92/1 1383 t~ /u~uu . ~ .

- 39 - ~1295~ '_ le 4 ~omr~rison of Murine CB0010 and CDR-qrafted C~P571 Antibodies in Bioassay and ~nim~l Mo~el Experiments A.

Neutralisation of TNF ~y CB0010 and CDP571 in the L929 Assay The ability of the parent murine antibody CB0010 (hTNF1) and the CDR-grafted antibody CDP571 to neutralise recombinant hu~an TNF was determine~ using the L929 bioassay. The assay uses the L929 mouse fibroblastoid cell line which is killed by ~NF. The assay is performed in the presence of 1 ug/ml actinomycin D which renders the cells more sensitive to TNF. Serial dilution of the two antibodies were mixed with a constant amount of recom~inant human TNF (100 pg/ml) and ~e~ to a ~929 monolayer in 96 well flat bottomed plates. After a 16 hour incubation the cells which had not been killed by TNF were revealed using the stain crystal violet. The apparent amount of TNF not neutralised (residual T~F) was det~rmined by comparison with a recombinant TNF st~n~rd c~rve.

Results from a representative experiment where residual TNF is plotted against antibody concentration are shown in Figure 6. It can ~e seen that CB0010 and CDP571 have S i m; lar neutralisation activities.

.

B.

Effect of CDP571 in Baboon Sepsis Model In this study the effect of the prior treatment with CDP571 on the physiological congequences of severe sepsis (including death) was asse~sed. Baboons were chosen as a relevant species to study since CDP~71 is known to neutralise baboon TNF.

~ale adult ba~oons, Pa~io ursinus, weighting 20-25 kg were anaesthetised with ketamine hydrochloride and sodium pentabar~itone and instrumented for the measurement of bl~od pressure, cardiac index (by thermodilution), ECG and right atrial filling pressures. An infusion of either w~) y~ o~ ~ 40 - 2129~4 saline only or antibody was then given for 120 min at a rate of 2.5 ml/kg/h following which they were given a further 120 min infusion of live E.coli at the same infusion rate. The bacterial straLn used was ~inshaw's strain B7 ([086a:61~, ATCC 33985) ~m;ni~tered whilst in the log growth phase at a do5e of 2x109 CFU/kg giving a plasma concentration of 2-2.5xlO~ CPU/ml at the end of the infusion. Following a further 120 min, ~n;m~l S were returned to their home cages, given free access to food and water and mnnitored for cardiovascular changes twice a day for 3 days. All ~ ls were given constant fluid replacement infusion of 5 ml/kg/h which was adjusted, where necessary, to maintain adequate right heart filling pressures. Baboons that had died during treatment or that had survived the 72h experimental period, and then killed were post-mortemed. All major organs were assessed for gross macro-pathalogical damage according to ~emi-quantitative scale (+f~ being the most severel.

~nim~l S were randomly assigned to one of 4 treatment groups;

-saline only -CDP571 0.1 mg/kg -CDP571 1.0 mg/~g -CB0010 0.1 mg/kg (parent murine antibody) The survival and cumulative organ damage scores are shown in table 1. CDP571 at l.0 mg/kg prevented death and significantly (P<0.005) reduced the incidence of organ damage in this model; furthermore, these effects were dose-related (P<0.005). In addition, the survival rate and organ damage score seen with CB0010 were s;mi 1 ~r to those seen with CDPS71 at the same dose, indicating a maintained in vivo potency of C~P571 compared to its parent murine antibody.

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