Per
`
`WORLD INTELLECTUAL PROPERTY ORGANIZATION
`International Bureau
`
`INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCD
`wo 92/05274
`2 April1992 (02.04.92)
`
`(11} International Publication Number:
`
`(43) International Publication Date:
`
`(51} International Patent Oassification 5 :
`C12P 21/08, C12N 15/13
`A61K39/395
`
`At
`
`(21) International Application Number:
`
`PCT/GB91101578
`
`~
`
`(22) International Filing Date:
`
`16 September 1991 (16.09.91)
`
`(74) Agent: MARCHANT, James, Ian; Elkington and Fife,
`Prospect House, 8 Pembroke Road, Sevenoaks, Kent
`TNI3 1XR (GB).
`
`(30} Priority data:
`9020282.1
`
`17 September 1990 (17.09.90) GB
`
`(71X72) Applicants and Inventors: GORMAN, Scott, David
`[US/GB]; Flat 22, Abington House, Adrian Way, Long
`Road, Cambridge CB2 2SA (GB). CLARK, Michael,
`Ronald [GB/GB]; 108 York Street, Cambridge CBI 2PY
`(GB). COBBOLD, Stephen, Paul [GB/GB]; Lower Flat,
`22 Guest Road, Cambridge CBI 2AL (GB). WALD(cid:173)
`MANN, Herman [GB/GB]; 11 Gurney Way, Cambridge
`CB4 2ED (GB).
`
`(81) Designated States: AT (European patent), AU, BE (Euro(cid:173)
`pean patent}, CA, CH (European patent), DE (Euro(cid:173)
`pean patent), DK (European patent), ES (European pa(cid:173)
`tent), FR (European patent), GB (European patent), GR
`(European patent), IT (European patent), JP, KR, LU
`(European patent), NL (European patent), SE (Euro(cid:173)
`pean patent), US.
`
`Published
`With international search report.
`
`(54) Title: FRAMEWORK MUTATED ANTIBODIES AND THEIR PREPARATION
`
`(57) Abstract
`
`An altered antibody chain is produced in which the CDR's of the variable domain of the chain are derived from a first
`mammalian species. The framework-encoding regions of DNA encoding the variable domain of the first species are mutated so
`that the mutated framework-encoding regions encode a framework derived from a second different mammalian species. The or
`each constant domain of the antibody chain, if present, are also derived from the second mammalian species. An antibody which
`is capable of binding to human CD4 antigen is also provided together with a pharmaceutical composition comprising the antib(cid:173)
`ody.
`
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`FOR THE PURPOSES OF INFORMATION ONLY
`
`Codes used to identify Stales party to the Per on the front pages of pamphlets publishing international
`applications under the PCf.
`
`AT
`AU
`BB
`BE
`BF
`BC
`BJ
`BR
`CA
`CF
`cc
`CH
`Cl
`CM
`cs
`DE*
`DK
`
`Austria
`Australia
`Barbados
`Belgium
`Burkina Fa~
`Bulgaria
`Benin
`Brazil
`Canada
`Central African Republic
`Congo
`Switzcrland
`Cote d'lvoire
`Cameroon
`Czechoslovakia
`Germany
`Denmark
`
`F.S
`Fl
`FR
`CA
`CB
`CN
`CR
`HU
`IT
`JP
`KP
`
`KR
`Ll
`LK
`LU
`MC
`
`Spain
`Finland
`France
`Gabon
`United Kingdom
`Guinea
`Grcccc
`Hungary
`Italy
`Japan
`Democratic People's Republic
`of Korea
`Republic of Korea
`Liechtenstein
`Sri Lanka
`Luxembourg
`Monaco
`
`MC
`ML
`MN
`MR
`MW
`NL
`NO
`PL
`RO
`SD
`SE
`SN
`su+
`TD
`TC
`us
`
`Madagascar
`Mali
`Mongolia
`Mauritania
`Malawi
`Netherlands
`Norway
`Poland
`Romania
`Sudan
`Sweden
`Senegal
`Soviet Union
`Chad
`Togo
`United States of America
`
`+
`Any designation of "SU" has effect in the Russian Federation. It is not yet known whether
`any such designation has effect in other States of the former Soviet Union.
`
`~
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`·".
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`--....
`of
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`FRAMEWORK MUTATED ANTIBODIES AND THEIR PREPARATION
`
`5
`
`The present invention relates to altered antibodies and
`their preparation. The invention is typically applicable
`to the production of humanised antibodies.
`Antibodies typically comprise two heavy chains linked
`together by disulphide bonds and two light chains. Each
`light chain is linked to a respective heavy chain by
`disulphide bonds. Each heavy chain has at one end a
`variable domain followed by a number of constant domains.
`10 Each light chain has a variable domain at one end and a
`constant domain at its other end. The light chain variable
`domain is aligned with the variable domain of the heavy
`chain. The light chain constant domain is aligned with the
`first constant domain of the heavy chain. The constant
`15 domains in the light and heavy chains are not involved
`directly in binding the antibody to antigen.
`The variable domains of each pair of light and heavy
`chains form the antigen binding site. The domains on the
`light and heavy chains have the same general structure and
`20 each domain comprises a framework of four regions, whose
`sequences are relatively conserved, connected by three
`complementarity determining regions (CDRs). The four
`framework regions largely adopt a beta-sheet conformation
`and the CDRs form loops connecting, and in some cases
`forming part of, the beta-sheet structure. The CDRs are
`held in close proximity by the framework regions and, with
`the CDRs from the other domain, contribute to the formation
`of the antigen binding site.
`The preparation of an altered antibody in which the CDRs
`30 are derived from a different species than the framework of
`the antibody's variable domains is disclosed in
`EP-A-0239400. The CDRs may be derived from a rat or mouse
`monoclonal antibody. The framework of the variable
`domains, and the constant domains, of the altered antibody
`35 may be derived from a human antibody.
`such a humanised
`
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`15
`
`5
`
`10
`
`antibody elicits a negligible immune response when
`administered to a human compared to the immune response
`mounted by a human against a rat or mouse antibody.
`Humanised CAMPATH-1 antibody is disclosed in EP-A-0328404.
`We have now devised a new way of preparing an altered
`antibody.
`In contrast to previous proposals, this involves
`altering the framework of a variable domain rather than the
`CDRs. This approach has the advantages that it does not
`require a pre-existing eDNA encoding, for example, a human
`framework to which to reshape and that it is technically
`easier than prior methodologies.
`Accordingly, the present invention provides a process
`for the preparation of an antibody chain in which the CDRs
`of the variable domain of the antibody chain are derived
`from a first mammalian species and the framework of the
`variable domain and, if present, the or each constant
`domain of the antibody chain are derived from a second
`different mammalian species, which process comprises:
`(i) mutating the framework-encoding regions of DNA
`20 encoding a variable domain of an antibody chain of the said
`first species such that the mutated framework-encoding
`regions encode the said framework derived from the said
`second species: and
`(ii) expressing the said antibody chain utilising the
`25 mutated DNA from step (i).
`A variable domain of either or both chains of an
`antibody can therefore be altered by:
`(a) determining the nucleotide and predicted amino acid
`sequence of a variable domain of a selected antibody chain
`30 of the said first species:
`(b) determining the antibody framework to which the
`framework of the said variable domain is to be altered;
`(c) mutating the framework-encoding regions of DNA
`encoding the said variable domain such that the mutated
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`framework-encoding regions encode the framework determined
`upon in step (b);
`(d)
`linking the mutated DNA obtained in step (c) to DNA
`encoding a constant domain of the said second species and
`5 cloning the DNA into an expression vector; and
`(e)
`introducing the expression vector into a compatible
`host cell and culturing the host cell under such conditions
`that antibody chain is expressed.
`The antibody chain may be co-expressed with a
`10 complementary antibody chain. At least the framework of
`the variable domain and the or each constant domain of the
`complementary chain generally are derived from the said
`second species also. A light chain and a heavy chain may
`be co-expressed. Either or both chains may have been
`15 prepared by the process of the invention. Preferably the
`CDRs of both chains are derived from the same selected
`antibody. An antibody comprising both expressed chains can
`be recovered.
`The antibody preferably has the structure of a natural
`20 antibody or a fragment thereof. The antibody may therefore
`comprise a complete antibody, a (Fab 1 ) 2 fragment, a Fab
`fragment, a light chain dimer or a heavy chain. The
`antibody may be an IgG such as an IgGl, IgG2, IgG3 or IgG4
`IgM, IgA, IgE or IgD. Alternatively, the antibody may be a
`25 chimaeric antibody of the type described in wo 86/01533.
`A chimaeric antibody according to WO 86/01533 comprises
`an antigen binding region and a non-immunoglobulin region.
`The antigen binding region is an antibody light chain
`variable domain or heavy chain variable domain. Typically,
`the chimaeric antibody comprises both light and heavy chain
`variable domains. The non-immunoglobulin region is fused
`at its c-terminus to the antigen binding region. The non(cid:173)
`immunoglobulin region is typically a non-immunoglobulin
`protein and may be an enzyme region, a region derived from
`a protein having known binding specificity, from a protein
`
`30
`
`35
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`toxin or indeed from any protein expressed by a gene. The
`two regions of the chimaeric antibody may be connected via
`a cleavable linker sequence.
`The invention is preferably employed to humanise an
`5 antibody, typically a monoclonal antibody and, for example,
`a rat or mouse antibody. The framework and constant
`domains of the resulting antibody are therefore human
`framework and constant domains whilst the CDRs of the light
`andjor heavy chain of the antibody are rat or mouse CDRs.
`~o Preferably all CDRs are rat or mouse CDRs. The antibody
`may be a human IgG such as IgGl, IgG2, IgG3, IgG4; IgM;
`IgA; IgE or IgD carrying rat or mouse CDRs.
`The process of the invention is carried out in such a
`way that the resulting antibody retains the antigen binding
`15 capability of the antibody from which it is derived. An
`antibody is reshaped according to the invention by mutating
`the framework-encoding regions of DNA coding for the
`variable domains of the antibody. This antibody and the
`reshaped antibody should both be capable of binding to the
`same antigen.
`The starting antibody is typically an antibody of a
`selected specificity.
`In order to ensure that this
`specificity is retained, the variable domain framework of
`the antibody is preferably reshaped to about the closest
`25 variable domain framework of an antibody of another
`species. By "about the closest" is meant about the most
`homologous in terms of amino acid sequences. Preferably
`there is a homology of at least 50% between the two
`variable domains.
`There are four general steps to reshape a monoclonal
`antibody. These are:
`(1) determining the nucleotide and predicted amino acid
`sequence of the starting antibody light and heavy chain
`variable domains;
`
`20
`
`30
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`(2) designing the reshaped antibody, i.e. deciding
`which antibody framework region to use during the reshaping
`process;
`(3)
`the actual reshaping methodologies/techniques; and
`(4)
`the transfection and expression of the reshaped
`antibody.
`These four steps are explained below in the context of
`humanising an antibody. However, they may equally well be
`applied when reshaping to an antibody of a non-human
`10 species.
`Step 1: Determining the nucleotide and predicted amino acid
`sequence of the antibody light and heavy chain
`variable domains
`To reshape an antibody only the amino acid sequence of
`15 antibody's heavy and light chain variable domains needs to
`be known. The sequence of the constant domains is
`irrelevant because these do not contribute to the reshaping
`strategy. The simplest method of determining an antibody's
`variable domain amino acid sequence is from cloned eDNA
`20 encoding the heavy and light chain variable domain.
`There are two general methods for cloning a given
`antibody's heavy and light chain variable domain cDNAs:
`(1)
`via a conventional eDNA library, or (2) via the polymerase
`chain reaction (PCR). Both of these methods are widely
`25 known. Given the nucleotide sequence of the cDNAs, it is a
`simple matter to translate this information into the
`predicted amino acid sequence of the antibody variable
`domains.
`
`30 step 2: Designing the reshaped antibody
`There are several factors to consider in deciding which
`human antibody sequence to use during the reshaping. The
`reshaping of light and heavy chains are considered
`independently of one another, but the reasoning is
`35 basically similar for each.
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`This selection process is based on the following
`rationale: A given antibody's antigen specificity and
`affinity is primarily determined by the amino acid sequence
`of the variable region CDRs. Variable domain framework
`residues have little or no direct contribution. The
`primary function of the framework regions is to hold the
`CDRs in their proper spacial orientation to recognize
`antigen. Thus the substitution of rodent CDRs into a human
`variable domain framework is most likely to result in
`retention of their correct spacial orientation if the human
`variable domain is highly homologous to the rodent variable
`domain from which they originated. A human variable domain
`should preferably be chosen therefore that is highly
`homologous to the rodent variable domain(s).
`A suitable human antibody variable domain sequence can
`be selected as follows:
`1. Using a computer program, search all available protein
`(and DNA) databases for those human antibody variable
`domain sequences that are most homologous to the rodent
`antibody variable domains. This can be easily
`accomplished with a program called PASTA but other
`suitable programs are available. The output of a
`suitable program is a list of sequences most homologous
`to the rodent antibody, the percent homology to each
`sequence, and an alignment of each sequence to the
`rodent sequence. This is done independently for both
`the heavy and light chain variable domain sequences.
`The above analyses are more easily accomplished if
`customized sub-databases are first created that only
`include human immunoglobulin sequences. This has two
`benefits. First, the actual computational time is
`greatly reduced because analyses are restricted to only
`those sequences of interest rather than all the
`sequences in the databases. The second benefit is that,
`by restricting analyses to only human immunoglobulin
`
`5
`
`10
`
`15
`
`20
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`25
`
`30
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`35
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`sequences, the output will not be cluttered by the
`presence-of rodent immunoglobulin sequences. There are
`far more rodent immunoglobulin sequences in databases
`than there are human.
`5 2. List the human antibody variable domain sequences that
`have the most overall homology to the rodent antibody
`variable domain (from above). Do not make a distinction
`between homology within the framework regions and CDRs.
`Consider the overall homology.
`10 3. Eliminate from consideration those human sequences that
`have CDRs that are a different length than those of the
`rodent CDRs. This rule does not apply to CDR 3, because
`the length of this CDR is normally quite variable.
`Also, there are sometimes no or very few human sequences
`that have the same CDR lengths as that of the rodent
`antibody. If this is the case, this rule can be
`loosened, and human sequences with one or more
`differences in CDR length can be allowed.
`4. From the remaining human variable domains, the one is
`selected that is most homologous to that of the rodent.
`5. The actual reshaped antibody (the end result) should
`contain CDRs derived from the rodent antibody and a
`variable domain framework from the human antibody chosen
`above.
`
`15
`
`20
`
`25 Step 3: The actual reshaping methodologies/techniques
`A eDNA encoding the desired reshaped antibody is
`preferably made beginning with the rodent eDNA from which
`the rodent antibody variable domain sequence(s) was
`originally determined. The rodent variable domain amino
`30 acid sequence is compared to that of the chosen human
`antibody variable domain sequence. The residues in the
`rodent variable domain framework are marked that need to be
`changed to the corresponding residue in the human to make
`the rodent framework identical to that of the human
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`framework. There may also be residues that need adding to
`or deleting from the rodent framework sequence to make it
`identical to that of the human.
`Oligonucleotides are synthesised that can be used to
`5 mutagenize the rodent variable domain framework to contain
`the desired residues. Those oligonucleotides can be of any
`convenient size. One is normally only limited in length by
`the capabilities of the particular synthesizer one has
`available. The method of oligonucleotide-directed i~ vitro
`10 mutagenesis is well known.
`The advantages of this method of reshaping as opposed to
`splicing CDRs into a human framework are that (1) this
`method does not require a pre-existing eDNA encoding the
`human framework to which to reshape and (2) splicing CDRs
`is technically more difficult because there is usually a
`large region of poor homology between the mutagenic oligo(cid:173)
`nucleotide and the human antibody variable domain. This is
`not so much a problem with the method of splicing human
`framework residues onto a rodent variable domain because
`there is no need for a pre-existing eDNA encoding the human
`variable domain. The method starts instead with the rodent
`eDNA sequence. Also, splicing framework regions is
`technically easier because there is a high degree of
`homology between the mutagenic oligonucleotide and the
`rodent variable domain framework. This is true because a
`human antibody variable domain framework has been selected
`that is most homologous to that of the rodent.
`The advantage of the present method of reshaping as
`opposed to synthesizing the entire reshaped version from
`30 scratch is that it is technically easier. synthesizing a
`reshaped variable domain from scratch requires several more
`oligonucleotides, several days more work, and technical
`difficulties are more likely to arise.
`
`25
`
`15
`
`20
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`5
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`10
`
`Step 4: The transfection and expression of the reshaped
`antibody
`Following the mutagenesis reactions to reshape the
`antibody, the cDNAs are linked to the appropriate DNA
`encoding light or heavy chain constant region, cloned into
`an expression vector, and transfected into mammalian cells.
`These steps can be carried out in routine fashion. A
`reshaped antibody may therefore be prepared by a process
`comprising:
`a)
`preparing a first replicable expression vector
`including a suitable promoter operably linked to a DNA
`sequence which encodes at least a variable domain of an Ig
`heavy or light chain, the variable domain comprising
`framework regions from a first antibody and CDRs comprising
`15 at least parts of the CDRs from a second antibody of
`different specificity;
`b)
`if necessary, preparing a second replicable
`expression vector including a suitable promoter operably
`linked to a DNA sequence which encodes at least the
`20 variable domain of a complementary Ig light or heavy chain
`respectively;
`c)
`transforming a cell line with the first or both
`prepared vectors; and
`d)
`culturing said transformed cell line to produce
`25 said altered antibody.
`Preferably the DNA sequence in step a) encodes both the
`variable domain and the or each constant domain of the
`antibody chain, the or each constant domain being derived
`from the first antibody. The antibody can be recovered and
`30 purified. The cell line which is transformed to produce
`the altered antibody may be a Chinese Hamster ovary (CHO)
`cell line or an immortalised mammalian cell line, which is
`advantageously of lymphoid origin, such as a myeloma,
`hybridoma, trioma or quadroma cell line. The cell line may
`35 also comprise a normal lymphoid cell, such as a B-cell,
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`5
`
`which has been immortalised by transformation with a virus,
`such as the Epstein-Barr virus. Most preferably, the
`immortalised cell line is a myeloma cell line or a
`derivative thereof.
`Although the cell line used to produce the altered
`antibody is preferably a mammalian cell line, any other
`suitable cell line, such as a bacterial cell line or a
`yeast cell line, may alternatively be used.
`In particular,
`it is envisaged that ~ coli - derived bacterial strains
`10 could be used.
`It is known that some immortalised lymphoid cell lines,
`such as myeloma cell lines, in their normal state secrete
`isolated Ig light or heavy chains. If such a cell line is
`transformed with the vector prepared in step (a) it will
`15 not be necessary to carry out step (b) of the process,
`provided that the normally secreted chain is complementary
`to the variable domain of the Ig chain encoded by the
`vector prepared in step (a).
`However, where the immortalised cell line does not
`20 secrete or does not secrete a complementary chain, it will
`be necessary to carry out step (b). This step may be
`carried out by further manipulating the vector produced in
`step (a) so that this vector encodes not only the variable
`domain of an altered antibody light or heavy chain, but
`25 also the complementary variable domain.
`Alternatively, step (b) is carried out by preparing a
`second vector which is used to transform the immortalised
`cell line. This alternative leads to easier construct
`preparation, but may be less preferred than the first
`30 alternative in that it may not lead to as efficient
`production of antibody.
`In the case where the immortalised cell line secretes a
`complementary light or heavy chain, the transformed cell
`line may be produced for example by transforming a suitable
`35 bacterial cell with the vector and then fusing the
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`5
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`20
`
`bacterial cell with the immortalised cell line by
`spheroplast fusion. Alternatively, the DNA may be directly
`introduced into the immortalised cell line by
`electroporation or other suitable method.
`An antibody is consequently produced in which CDRs of a
`variable domain of an antibody chain are homologous with
`the corresponding CDRs of an antibody of a first mammalian
`species and in which the framework of the variable domain
`and the constant domains of the antibody are homologous
`10 with the corresponding framework and constant domains of an
`antibody of a second, different, mammalian species.
`Typically, all three CDRs of the variable domain of a light
`or heavy chain are derived from the first species.
`The present process has been applied to obtain an
`15 antibody against human CD4 antigen. Accordingly, the
`invention also provides an antibody which is capable of
`binding to human CD4 antigen, in which the CDRs of the
`light chain of the antibody have the amino acid sequences:
`CDRl:
`LASEDIYSDLA
`NTDTLQN
`CDR2:
`CDR3:
`QQYNNYPWT,
`in which the CDRs of the heavy chain of the antibody have
`the amino acid sequences:
`NYGMA
`CDRl:
`CDR2:
`TISHDGSDTYFRDSVKG
`QG7'IAGIRH, and
`CDR3:
`in which the framework of the variable domain and, if
`present, the or each constant domain of each chain are
`derived from a mammalian non-rat species.
`The antibody preferably has the structure of a natural
`antibody or a fragment thereof. The antibody may therefore
`comprise a complete antibody, a (Fab 1 ) 2 fragment, a Fab
`fragment,-a light chain dimer or a heavy chain.
`
`25
`
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`5
`
`15
`
`The antibody may be an IgG such as IgGl, IgG2, IgG3 or
`IgG4 IgM, IgA, IgE or IgD. Alternatively, the antibody may
`be a chimaeric antibody of the type described in WO
`86/01533.
`A chimaeric antibody according to WO 86/01533 comprises
`an antigen binding region and a non-immunoglobulin region.
`The antigen binding region is an antibody light chain
`variable domain or heavy chain variable domain. Typically
`the chimaeric antibody comprises both light and heavy chain
`10 variable domains. The non-immunoglobulin region is fused
`at its c-terminus to the antigen binding region. The non(cid:173)
`immunoglobulin region is typically a non-immunoglobulin
`protein and may be an enzyme region, a region derived from
`a protein having known binding specificity, from a protein
`toxin or indeed from any protein expressed by a gene. The
`two regions of the chimaeric antibody may be connected via
`a cleavable linker sequence.
`The invention is preferably employed to humanise a CD4
`antibody such as a rat or mouse CD4 antibody. The
`framework and the constant domains of the resulting
`antibody are therefore human framework and constant domains
`whilst the CDRs of the light and/or heavy chain of the
`antibody are rat or mouse CDRs. Preferably all CDRs are
`rat or mouse CDRs. The antibody may be a human IgG such as
`IgG1, IgG2, IgG3, IgG4; IgM; IgA; IgE or IgD carrying rat
`or mouse CDRs.
`Preferably the framework of the antibody heavy chain is
`homologous to the corresponding framework of the human
`antibody KOL (Schmidt et al, Hoppe-Seyler's z. Physiol.
`30 Chem., ~ 713-747, 1983). The sixth residue of
`framework 4 in this case is suitably Thr or Pro, preferably
`Thr. This residue is the 121st residue in the KOL antibody
`heavy chain variable region (Schmidt et al, 1983), and is
`identified as residue 108 by Kabat (Kabat et al, "Sequences
`35 of proteins of immunological interest", US Dept of Health
`
`20
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`and Human Services, US Government Printing Office, 1987).
`Alternatively, the framework of the antibody heavy chain is
`homologous to the corresponding framework of the human
`antibody NEW (Saul et al, J. Biol.Chem. 253: 585-597,
`5 1978). The final residue of framework 1 in this case is
`suitably Ser or Thr, preferably Ser. This residue is at
`position 30 (Kabat et al, 1987). Preferably the framework
`of the antibody light chain is homologous to the variable
`domain framework of the protein REI (Epp et al, Eur. J.
`10 Biochem., 45, 513-524, 1974).
`The framework regions of one or both chains of a CD4
`antibody can be reshaped by the present process.
`Alternatively, one or both chains of a CD4 antibody may be
`reshaped by the procedure described in EP-A-0239400. The
`15 procedure of EP-A-0239400 involves replacing CDRs rather
`than the replacement of frameworks. The CDRs are grafted
`onto a framework derived from a mammalian non-rat species,
`typically a human. This may be achieved by
`oligonucleotide-directed in vitro mutagenesis of the CDR-
`20 encoding regions of an antibody chain, light or heavy, from
`a mammalian non-rat species. The oligonucleotides in such
`an instance are selected so that the resulting CDR-grafted
`antibody has the light chain CDRs 1 to 3 and the heavy
`chain CDRs 1 to 3 shown above.
`The reshaped CD4 antibody can be used to induce
`tolerance to an antigen. It can be used to alleviate
`autoimmune diseases such as rheumatoid arthritis. It can
`be used to prevent graft rejection. Tolerance to a graft
`such as an organ graft or a bone marrow transplantation can
`30 be achieved. Also, the reshaped CD4 antibody might be used
`to alleviate allergies. Tolerance to allergens could be
`achieved.
`The CD4 antibody may be depleting or non-depleting. A
`depleting antibody is an antibody which depletes more than
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`SO%, for example from 90 to 99%, of target cells in vivo.
`A non-depleting antibody depletes fewer than 50%, for
`example, from 10 to 25% and preferably less than 10% of
`target cells in vivo. A CD4 antibody may be administered
`5 alone or may ~e co-administered with a non-depleting or
`depleting COS-antibody. The CD4 antibody, depleting or
`non-depleting, and CDS monoclonal antibody, depleting or
`non-depleting, may be administered sequentially in any
`order or may be administered simultaneously. An additional
`10 antibody, drug or protein may be administered before,
`during or after administration of the antibodies.
`A CD4 antibody and, indeed, a CDS antibody as
`appropriate are given parenterally, for example
`intravenously. The antibody may be administered by
`injection or by infusion. For this purpose the antibody is
`formulated in a pharmaceutical composition further
`comprising a pharmaceutically acceptable carrier or
`diluent. Any appropriate carrier or diluent may be
`employed, for example phosphate-buffered saline solution.
`The amount of non-depleting or depleting CD4 and, if
`desired, CDS antibody administered to a patient depends
`upon a variety of factors including the age and weight of a
`patient, the condition which is being treated and the
`In
`antigen(s) to which it is desired to induce tolerance.
`a model mouse system from 1~g to 2mg, preferably from 400~g
`to 1mg, of a mAb is administered at any one time.
`In
`humans from 3 to 500mg, for example from 5 to 200mg, of
`antibody may be administered at any one time. Many such
`doses may be given over a period of several weeks,
`typically 3 weeks.
`A foreign antigen(s) to which it is desired to induce
`tolerance can be administered to a host before, during, or
`after a course of CD4 antibody (depleting or non-depleting)
`and/or CDS antibody (depleting or non-depleting).
`35 Typically, however, the antigen(s) is administered one week
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`after commencement of antibody administration, and is
`terminated three weeks before the last antibody
`administration.
`Tolerance can therefore be induced to an antigen in a
`host by administering non-depleting or depleting CD4 and
`CDS mAbs and, under cover of the mAbs, the antigen. A
`patient may be operated on surgically under cover of the
`non-depleting or depleting CD4 and CDS mAbs to be given a
`tissue transplant such as an organ graft or a bone marrow
`transplant. Also, tolerance may be induced to an antigen
`already possessed by a subject. Long term specific
`tolerance can be induced to a self antigen or antigens in
`order to treat autoimmune disease such as multiple
`sclerosis or rheumatoid arthritis. The condition of a
`15 patient suffering from autoimmune disease can therefore be
`alleviated.
`The following Example illustrates the invention.
`accompanying drawings:
`Figure 1: shows the nucleotide and predicted amino acid
`sequence of rat CD4 antibody light chain variable region.
`The number of the first and last amino acid or nucleotide
`in each line is indicated in the left and right margins,
`respectively. Base pairs 1-269 (Hindiii-Pvuii) and 577-620
`([Bglii/Bcli]-BamHI) are part of the vector Ml3VKPCR3,
`25 while base pairs 270-576 are from the PCR product of the
`CD4 antibody light chain variable region (VL) . CDRs
`(boxes) were identified by comparison to known
`immunological sequences (Kabat et al, "Sequences of
`proteins of immunological interest, US Dept of Health and
`30 Human Services, US Government Printing Office, 19S7).
`Figure 2: shows the nucleotide and predicted amino acid
`sequence of the reshaped CAMPATH-1 antibody light chain
`eDNA. The number of the first and last amino acid or
`nucleotide in each line is indicated in the left and right
`35 margins, respectively. CDRs are identified by boxes.
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`Figure 3: shows the nucleotide and predicted amino acid
`sequence of the reshaped CD4 antibody light chain eDNA
`CD4VLREI. The number of the first and last amino acid or
`nucleotide in each line is indicated in the left and right
`5 margins, respectively. CDRs are identified by boxes.
`Figure 4: shows the nucleotide and predicted amino acid
`sequence of rat CD4 antibody heavy chain variable region.
`The number of the first and last amino acid or nucleotide
`in each line is indicated in the left and right margins,
`respectively. CDRs are identified by boxes. Base pairs 1-
`272 (Hindiii-Psti) and 603-817 (BstEII-BamHI) are part of
`the vector M13VHPCR1, while base pairs 273-602 are from the
`PCR product of the CD4 antibody he