WO1992016562A1

WO1992016562A1 - Humanised antibodies having modified allotypic determinants

Info

Publication number: WO1992016562A1
Application number: PCT/GB1992/000445
Authority: WO
Inventors: Michael Ronald Clark
Original assignee: Lynxvale Limited
Priority date: 1991-03-12
Filing date: 1992-03-12
Publication date: 1992-10-01
Also published as: CA2105980A1; GB9105245D0; JPH06510659A; EP0575407A1

Abstract

The invention relates to molecules which have an amino acid sequence derivable from part or all of the constant region of an immunoglobulin heavy chain. The constant regions are of a particular isotype and have one or more allotypic determinants. The amino acid sequence is substantially homologous to the amino acid sequence of the constant region. However, it has been altered so that it is without at least one of said allotypic determinants by making its sequence the site for an allotypic determinant identical to the amino acid sequence from the corresponding position in another equivalent immunoglobulin constant region of a different isotype. The invention provides synthetic immunoglobulins with reduced allotypic differences as compared to a given wild-type immunoglobulin.

Description

Humani sed antibodies havi ng modified a llotypic determinants

The present invention relates to binding molecules . In particular, it relates to recombinantly produced antibodies .

Owing to their high specificity for a given antigen, antibodies and particularly monoclonal antibodies ( Kohler , G . and Milstein C. , 1975 Nature 256 : 495 ) represented a significant technical break-through with important consequences scientifically, commercially and therapeutically.

Monoclonal antibodies are made by establishing an immortal cell line which is derived from a single immunoglobulin producing cell secreting one form of a biologically functional antibody molecule with a particular specificity.

Owing to their specificity , the therapeutic applications of monoclonal antibodies hold great promise for the treatment of a wide range of diseases ( Clinical Applications of Monoclonal Antibodies, edited by E. S . Lennox . British . Medical Bulletin 1984 , publishers Churchill Livingstone ) . Antibodies are generally raised in animals , particularly rodents , and therefore the immunoglobulins produced bear characteristic features specific to that species . The repeated administration of these foreign rodent proteins for therapeutic purposes to human patients can lead to harmful hypersensitivity reactions. In the main therefore, these rodent-derived monoclonal antibodies have limited therapeutic use. A further problem with these rodent derived antibodies, is that they are relatively ineffective at the depletion of cells in vivo, although the rat IgG2b antibody CAMPATH-1G is an exception to this rule.

Thus, there is a need for therapeutic antibodies which have characteristic features specific to human proteins. Unfortunately, immortal human antibody- producing cell lines are very difficult to establish and they give low yields of antibody (approximately 1 μg/ml). In contrast, equivalent rodent cell lines yield high amounts of antibody (approximately 100 μg/ml). Furthermore, where one wants to produce a human antibody with a particular specificity it is not practically or ethically feasible to immunise humans with an immunogen bearing the epitope of interest.

In part, this problem has been overcome in recent years by using the techniques of recombinant DNA technology to 'humanise' non-human antibodies. Structurally, the simplest antibody (IgG) comprises four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulphide bonds (see figure 1). The light chains are of two types, either kappa or lambda. Each of the H and L chains has a region of low sequence variability, the constant region (C) giving rise to allotypes and a region of high sequence variability, the variable region (V) giving rise to idiotypes. The antibody has a tail region (the Fc region) which comprises the C regions of the two H chains. The antibody also has two arms (the Fab region) each of which has a V_L and a V_H region associated with each other. It is this pair of V regions (V_L and V_H) that differ from one antibody to another, and which together are responsible for recognising the antigen. In even more detail, each V region is made up from three complementarity determining regions (CDR) separated by four framework regions (FR). The CDRs are the most variable part of the variable regions, and they perform the critical antigen binding function. The CDR regions are derived from many potential germ line sequences via a complex process involving recombination, mutation and selection. It has been shown that the function of binding antigens can be performed by fragments of a whole antibody. Binding fragments are the Fv fragment which comprises the V_L and V_H of a single heavy chain variable domain (V_H).

In creating "humanised" immunoglobulins, the Fc tail of a non-human antibody is exchanged for that of a human antibody. For a more complete humanisation, the FRs of the non-human antibody are exchanged for human FRs. This process is carried out at the DNA level using recombinant techniques. However, these humanised immunoglobulins do not solve all the problems, because an immune response may still be mounted against the treatment antibody even when a patient is treated with a human antibody, as it may show certain sequence differences in the V (ie idiotypic differences) and C (ie allotypic differences) regions when compared with the patients own equivalent antibodies. This is a particular problem where the patient's immune system has already seen, and therefore been primed against, antibodies having these sequence differences (eg a patient may have received a prior blood transfusion which contained allotypically different immunoglobulins). A model system of injecting "mouseised human antibodies" into mice indicated that the allotype matching could critically affect the anti-idiotype response (Bruggemann M., Winter G., Waldmann H., Neuberger M.S., (1989) J. Exp. Med. 170, 2153-2157).

The present applicants have realised that one way around this problem is to eliminate the allotypic variation from the constant region.

There are a range of different immunoglobulins IgG, IgM, IgA, IgD, IgE, known as isotypes, of which IgG is most commonly used therapeutically. It exists as isotypic sub-classes IgG1, IgG2, IgG3 and IgG4.

There are 24 recognised allotypes of human immunoglobulin distributed between the different isotypes as follows:

IgG1 × 4

IgG2 × 1

IgG3 × 13

IgA2 × 2

IgE × 1

Kappa × 3

The allotypes represent alternative amino acid substitutions found at discrete sites in the protein sequence. These different allotypic determinants are found in different combinations within given allelic forms of genes, but not all possible combinations which theoretically might exist are in practice observed.

For example, the four different allotypes of IgG1 can be seen (ie distinguished) by the immune system. These are G1m 1, 2, 3 and 17. Alternatively, combinations thereof, such as G1m (1, 17 ) , can also be distinguished. The four different single allotypes are depicted in figure 2.

Antisera can be raised in other non-human species which can see the alternative isoallotypes provided that the antibody is purified away from the other human isotypes. Such isoallotypes for which such an antisera exists have been called non-allotypes and given the designation for example, nG1m(1) which is the isoallotype of G1m(1). Thus, although a human isoallotype should not be immunogenic in humans, it can still potentially be recognized in a different species.

Of the above mentioned different allotypes of IgG1, three common allelic forms of human IgG1 occur with different frequencies within different racial groups, namely G1m (3), G1m (1, 17), and G1m (1, 2, 17) based upon their reactivities with human antisera directed against the determinants G1m 1, 2, 3 and 17. At some point in the future, it is likely that a patient with an existing anti-allotype response to one or more of these determinants will need treatment with a humanised antibody. The obvious solution and one which has been proposed in a letter to the Journal Nature (Mage, R.G., Nature (1988) 333, 807-808), is to make all the different allelic forms of an antibody and to allotype match each patient for therapy. The present applicants have realised that commercially this is not a good proposal because of increased production costs and the need to process several reagents in parallel through the regulatory requirements. Additionally, each patient would have to be tested for the response to different allotypes.

Thus, the present applicants propose eliminating the allotypes altogether from each therapeutic antibody. The sequence of the human allotype of IgG1 G1m (1, 2, 17) is shown aligned with sequences for the other human IgG, isotype sub-classes in figure 4 (a, b, c and d). It can be seen that the four isotypes are extremely homologous for the domains CH1, CH2 and CH3, and that the major isotypic differences are in the hinge region which varies in both, length and sequence between isotypes. The allotypic residues of IgG1 G1m (1, 2, 17) have been marked in figure 4. However, for the purposes of clarity the sequences around the allotypic sites G1m (1) (2) and (17) are shown below for each isotype.

Site (1)

355 356 357 358

Arg Asp or G1u G1u Leu or Met IgG1

Arg G1u G1u Met IgG2 Arg G1u G1u Met IgG3

Gin G1u G1u Met IgG4

Thus, at site (1), IgG1 may exist as several allotypes depending on whether aspartic acid or glutamic acid at position 356, or leucine or methionine at position 358 are present.

Site 2

430 431 432

G1u G1y or Ala Leu IgG1

G1u Ala Leu IgG2

G1u Ala Leu IgG3

G1u Ala Leu IgG4 Thus, at site (2), IgG1 may exist as either of two allotypes depending on whether glycine or alanine is present at position 431.

Site (17)/(3)

Sites (3) and (17) are alternative substitutions at the same site.

213 214 215

Lys Lys or Arg Val IgG1

Lys Thr Val IgG2

Lys Arg Val IgG3

Lys Arg Val IgG4

Thus, at site (17)/(3), IgG1 may exist as either of two allotypes depending on whether lysine or arginine is present. The allotypes (17) and (3) cannot co-exist as they represent alternative substitutions at the same position.

The alternative alleles of G1m (1) and (2) do not provoke a human allotype response because of the homology of these alleles with the other IgG sub-classes in this region. These alleles are therefore called isoallotypes because they are only recognisable by xenoantisera (antisera from a different species) and only when the isotype is purified away from the other sub-classes.

Therefore, the present applicants propose the creation of a new IgG1 allele by site-directed mutagenesis of the gene, for example, an existing CAMPATH-1H monoclonal antibody gene described below, so that the new allele consists entirely of isoallotypic determinants. The preparation of IgG1 mutants according to the teaching provided by the present applicants is shown schematically in figure 3.

For G1m (1) and G1m (2), the changes comprise simple substitution by the alternative isoallotypic residues. However, in the case of G1m (17) the conversion of lysine to arginine would in some cases merely change the allotype to an allotype that is recognised by certain individuals as a G1m (3) allotype despite the fact that this residue is homologous with IgG3 and IgG4. This apparent contradiction is thought to be because this arginine is seen in a tertiary epitope in the context of the other IgG1 specific residues in close proximity in the CH1 domain or hinge region. This indicates that in addition to changing lysine, other residues in CH1 or the hinge will need to be changed in order to create a new isoallotype.

Although the above and ensuing description is specifically directed to IgG1 and in particular, the CAMPATH-1H monoclonal antibody, the same approach can be used to create isoallotypes of the other human isotypes such as IgG2, IgG3 and kappa.

Thus, the present invention provides a first binding molecule derivable from a second binding molecule; which second binding molecule is an immunoglobulin, or a derivative, structural or functional analogue thereof, a member of a family of homologous molecules, and has one or more sites which are structurally distinctive from equivalent sites in the other family members;

wherein said first binding molecule is more closely homologous to the other family members than to said second binding molecule, at at least one of said one or more sites.

The first binding molecule may also be an immunoglobulin or a derivative, structural or functional analogue thereof. The one or more sites which are structurally distinctive from the equivalent sites in the other family members may be in the constant region giving rise to an allotypic difference. The first binding molecule may comprise entirely isoallotypic determinants.

The second binding molecule may be selected from the group consisting of IgG1, IgG2, IgG3, lgA2, IgE, kappa light chains or derivatives, structural or functional analogues thereof. Where the second binding molecule is IgG1, the allotypic differences may be present at one or more of sites (1) (2) (3) or (17) as described herein. Where the second binding molecule is IgG2, the allotypic difference may be present at site (23). Where the second binding molecule is IgG3, the allotypic differences may be present at one or more of the sites (11) (5) (13) (14)

(10) (6) (24) (21) (15) (16) (26) or (27). Where the second binding molecule is IgA2, the allotypic differences may be present at one or more of the sites (1) and (2). Where the second binding molecule is kappa light chain, the allotypic differences may be present at one or more of the sites (1) (2) or (3). The sites referred to above are well documented in the literature

(see e.g. Eur. J. Immunol. 1976.6:599-601. Review of the notation for the allotypic and related marks of human immunoglobulins).

The present invention also provides pharmaceutical preparations comprising a first binding molecule as defined above or described herein together with one or more excipients. The pharmaceutical preparation may comprise a cocktail of said first binding molecules.

Also provided by the present invention are methods for making a first binding molecule as defined above or described herein.

These methods comprise the steps of: a) identifying in said second binding molecule, one or more sites which are structurally distinctive from the equivalent sites in the other family members; b) making said first binding molecule whereby it is more closely homologous to the other family members than to said second binding molecule at at least one of said one or more sites. The first binding molecule may be made by providing a gene sequence encoding the second binding molecule and altering those parts of the gene sequence encoding said one or more sites. The gene sequence may be altered by site directed mutagenesis using oligonucleotide primers. The altered gene sequence may be incorporated into a cloning vector or expression vector. The expression vector may be used to transform a cell. The cell may be induced to express the altered gene sequence.

The present invention therefore provides cloning vectors and expression vectors incorporating the altered gene sequence. Also provided are cells transformed by expression vectors defined above. Also provided are cell cultures and products of cell cultures containing the first binding molecules. Also provided are recombinantly produced said first binding molecules.

Thus the present invention provides a molecule which comprises an amino acid sequence derivable from part or all of the constant region of an immunoglobulin heavy chain which constant regions are of a particular isotype and have one or more allotypic determinants

wherein said amino acid sequence is substantially homologous to the amino acid sequence of said constant region, but has been altered so that it is without at least one of said allotypic determinants, by making the amino acid residues at the site of an allotypic determinant identical to the amino acid residues from the corresponding position in another equivalent immunoglobulin constant region of a different isotype.

The molecule may comprise an amino acid sequence derivable from part or all of a human immunoglobulin constant region.

The molecule may also comprise one or more polypeptides together with said amino acid sequence.

The polypeptide may comprise a functional biological domain. The domain may be such that it mediates any biological function. The functional biological domain may comprise a binding domain. The binding domain will have an ability to interact with another polypeptide. The interaction may be non-specific or specific.

The polypeptide, biological domain, binding domain and immunoglobulin-like binding domain may derive from the same source or a different source to the constant region.

The constant region may be from an immunoglobulin of the isotype IgG. The isotype subclass may be IgG1 and the molecule may no longer have one or more of the allotypic determinants 1,2,3 and 17. The isotype subclass may be IgG2 and the molecule may no longer have the allotypic determinant 23. The isotype subclass may be IgG3 and the molecule may no longer have one or more of the allotypic determinants 11,5,13,14,10,6,24,21,15, 16, 26 and 27.

The constant region may be from an immunoglobulin of the isotype IgA2 and the molecule may no longer have either or both of the allotypic determinants 1 and 2.

The present invention also provides a pharmaceutical preparation which comprises a molecule as defined.

The present invention also provides a reagent which comprises a molecule as defined.

The present invention also provides a nucleotide sequence encoding a molecule as defined.

The present invention also provides cloning and expression vectors comprising a nucleotide sequence as delivered above.

The present invention also provides host cells comprising a cloning or expression vector as defined above.

The present invention also provides a method of preparing a molecule as defined above which comprises the steps of:

(a) identifying a constant region of an immunoglobulin heavy chain;

(b) comparing the identified constant region with constant regions from immunoglobulin heavy chains of the same isotype to locate allotypic determinants in the identified constant region;

(c) obtaining the coding sequence for the identified constant region having allotypic determinants;

(d) altering the coding sequence so that it codess for a molecule without at least one of said allotypic determinants and by making the amino acid residues at the site for an allotypic determinant identical to the amino acid residues from the corresponding position in an equivalent immunoglobulin constant region of an isotype different to that of said identified constant region;

(e) using said altered coding sequence in an expression system to produce a said molecule.

The present invention also provides a method of treating a patient which comprises administering a pharmaceutical preparation as defined above.

Of course, there are a number of different strategies which could be used in order to make the molecules with fewer allotypic determinants.

Genes encoding therapeutically useful antibodies are generally available in one of several different forms. They may be available as a cloned variable region DNA sequence with restriction sites at each end, suitable for recloning along with a chosen cloned constant region DNA sequence into a suitable expression vector. This is the strategy described herein for the constructs TF57-19, MTF121 and MTF123. Alternatively, they may be available as complete immunoglobulin DNA sequences including the V and C regions together, e.g. a cDNA clone of a complete humanised or human antibody.

Whatever the form in which the cloned imrπunoglobulion gene is obtained, the next step is to predict the amino acid sequence of the constant region from the DNA sequence. The DNA sequence can be obtained using a variety of strategies familiar to molecular biologists. The predicted amino acid sequence would then be checked for the amino acids known to vary as allotypes. Any isoallotypes present within the sequence can be left unaltered. Any allotypes present can be mutated.

The next step, is to decide what amino acid sequence to mutate the allotype to, in order to imitate an isoallotype. This is done by lining up the sequence with the corresponding region of the other immunoglobulin isotypes. For all known allotypes, it has been found that one or more of the other isotypes have invariant sequences for the homologous region. One of these sequences can then be chosen to form the basis for the changes to be made in the allotype in question. Having predicted the new amino acid sequence for the constant region, it is necessary to alter the existing DNA clone or to create a new DNA clone which will encode this sequence. Again there are several strategies available to molecular biologists in order to achieve this. In the case of the example CAMPATH-1H constructs described herein, the gamma-1 constant region was cloned in an M13TG131 single stranded phage vector. Mutagenic oligonucleotides were synthesised which were largely homologous to the single strand, but which contained base changes necessary to alter the codons for the critical amino acids. The mutagenesis was carried out using a commercial kit from Amersham International, High Wycombe, Bucks. Alternatively it would be possible to synthesise a complete artificial gene which encodes the predicted sequence.

Once mutated or newly synthesised, the gene is ready for expression. There are many different expression vectors available. Some of these are more suitable for expression in restricted cell types. Again it is within the standard technical expertise of one skilled in this field to choose and adapt expression vectors for this purpose. In the case of the CAMPATH-1H constructs described herein, modifications of the pSVgpt and pSVneo vectors have been used. These vectors have convenient cloning sites for the immunoglobulin variable and constant region, encoding DNA fragments adjacent to suitable promoter and enhancer sequences to allow expression in lymphoid cells. The vector allows the easy independent replacement of variable or constant region encoding DNA fragments. Thus, any suitable variable region can be subcloned into the vector, to give rise to a new specificity, or the variable region can be kept and the constant region changed to give rise to a new isotype or allotype. Alternative vector systems are readily available.

Having removed allotypes from heavy chain constant regions by mutating them all to isoallotypes, it may still be desirable to consider the light chain effect in stimulating an immune response.

The most common kappa light chain allotype is Km(3) in the general population. Therefore it may be sufficient to utilise this common kappa light chain allotype, as relatively few members of the population would see it as foreign.

Alternatively there are no lambda light chain allotypes. Therefore they could be used in combination with the de-allotyped molecules derivable from heavy chain constant regions.

Where one utilises the kappa light chain, the allotype Km(1,2) could first be mutated to the allotype Km(1). The light chain allotype Km(1) is often only weakly recognized in combination with certain heavy chain classes and subclasses, and so may not cause a major problem in therapeutic use.

In order that the present invention is more fully understood embodiments will now be described in more detail, by way of example only, and not by way of limitation. Reference will be made (and has already been made in the text above) to the following figures in which:

figure 1 illustrates the structure of an IgG antibody;

figure 2 shows the allotypes for the IgG1 antibody CAMPATH-1H;

figure 3 shows schematically the preparation of IgG1 mutants;

figure 4 shows the IgG1 G1m (1,2,17) allotype sequence aligned to single allotypic examples of IgG2, 3 and 4 (none of these other subclasses have allotypic residues which cover the same residues as for the IgG1 allotypes);

figure 5 shows the M13TG131 cloning vector containing the human gamma-1 constant region, showing cloning sites and modified polylinker;

figure 6 shows the original Hu4vH HuG1 pSVgpt expression vector and its modified version;

figure 7 shows the result of an ELISA assay testing different dilutions of the antibodies of mutants 1, 2 and wild type CAMPATH-1H for IgG1 specificity;

figure 8 shows the result of an autologous complement mediated lysis test using human peripheral blood lymphocytes; and

figure 9 illustrates an antibody-dependent cell- mediated cytotoxicity assay (ADCC) using CD3 activated interleukin-2 expanded human blastocytes cell effectors (E) and targets (T).

The starting antibody used for site-directed mutagenesis was CAMPATH-1H, a monoclonal antibody with a kappa light chain containing the human constant region sequence for IgG1 which carries the G1m (1, 17) allelic determinants. The whole IgG1 encoding region exists as approximately 2.3 kb HindIII-SphI restriction fragment cloned in an M13 vector. The M13TG131 cloning vector containing the human gamma-1 constant region showing cloning sites and modified polylinker is shown in figure 5.

The IgG1 encoding region is entered in the EMBL Sequence Database under the code number HS1GCC4. The accession number is AC J00228 (the printout from the database is provided herein as Appendix 1). This sequence is for the G1m (1, 17) allotype. It covers 2009 bases from the 5' Hindlll site (A)AGCTT including all of the coding region. It does not however, include some of the 3' non-coding region up to the SphI site. The sequence provided by the EMBL Database is that of the upper strand of DNA. The CH1 domain starts at nucleotide 210 and ends at nucleotide 503. The mutagenic oligonucleotides MO1 and MO4 hybridise to nucleotides 486 to 510. The hinge region starts at nucleotide 892 and ends at nucleotide 936. The CH2 domain starts at nucleotide 1481 and ends at nucleotide 1803. The mutagenic oligonucleotide MO2 hybridises to nucleotides 1515 to 1543. The essential signal for the poly A tail is provided by nucleotides 1902 to 1908.

In M13TG131, the IgG1 coding region exists as a 2260 nucleotide fragment, of which the final 251 nucleotides are non-coding and therefore, inessential. Therefore, an embodiment of the invention could be carried out using the sequence information provided by the EMBL Sequence Database. It should be noted however, that the Sph1 restriction site referred to above, is present in the 3' end inessential non-coding region. Therefore, if the sequence data as provided by the EMBL database were being used, alternative restriction sites would have to be utilised.

Using site-directed mutagenesis, (carried out using protocols and reagents as supplied in kit form, Amersham code RPN. 1523, Amersham International Pic, Amersham, UK) the sequence corresponding to the G1m ( 1 ) allele was converted to the corresponding sequence found in the other sub-classes for IgG (Asp G1u Leu to G1u G1u Met at positions 356-358 in the CH3 domain).

The mutagenic oligonucleotides used were:

a) MOl (to convert G1m (17) to G1m (3))

5' CTC TCA CCA ACT CTC TTG TCC ACC T 3 '; b) MO2 (to convert G1m (1) to its isoallotype nG1m (1)) 5' GGT TCT TGG TCA TCT CCT CCC GGG ATG GG 3'; and c) M04 (to eliminate G1m(3) by changing Lys to Thr in the CH1 region)

5' CTC TCA CCA ACA GTC TTG TCC ACC T 3'.

The oligonucleotides were synthesised and then purified using an automated synthesizer and oligo purification columns supplied by Applied Biosystems (Applied Biosystems, 850 Lincoln Drive, Foster City, California, 94404 USA) following the manufacturers recommended protocols. Mutations were checked by Sanger Dideoxy sequencing (Sanger, F.S., Nicklen, S., and Coulson, A.R., (1977) Proc. Natl. Acad. Sci., USA, 74, 5463) using standard protocols and kits. As this newly formed allotype sequence is found in all humans, there should be no immunological response to this alternative form of G1m (1). Additionally and similarly, the lysine residue responsible for the G1m (17) allotypic determinant was converted to an arginine residue corresponding to the G1m allele (Lys 214-Arg; mutant 1).

The gene for this new constant region of mutant 1 carrying these three changes has been sequenced, incorporated into an expression vector containing the CAMPATH-1H V-region and expressed together with the CAMPATH-1H light chain which had been introduced by co-transfection. A further mutant has been made by replacing the critical arginine residue associated with G1m (3) of mutant 1 with a threonine residue, to produce a heavy chain which is the equivalent of IgG2 and which should fail to react with both anti-G1m (17) and anti-G1m (3) antisera (mutant 2).

Mutant 2 has also been sequenced, re-cloned in an expression vector containing the CAMPATH-1H light chain.

The supernatants of growing cultures containing either of the two mutants were subsequently assayed for the expression of a human IgG1 kappa product.

The mutations were introduced with the oligonucleotides listed above. The modified Hu4vHG1pSVgpt vector shown in figure 6 was used to simplify the subcloning of all the new mutants into the expression vector, owing to the possibility of use of two different "sticky ends" Bam HI and NotI. The expression vectors and V_H region sequences and expression, along with the light chains, in YO rat plasmacytoma cells are all as described in Riechmann L., Clark, M.R. Waldman H., Winter G. (1988) Nature 332, 323-327.

From the positive cultures, the producers of the largest amount of the IgG1 product were selected to obtain purified antibody for biological assays to determine their allotypes and biological effector functions. Example 1

An Enzyme-linked Immuno Sorbent Assay (ELISA) was performed to verify that an IgG1 type antibody was produced by the mutants. This was tested with microtiter plates coated with anti-CAMPATH-idiotype antibody (YID 13.9). Wild type CAMPATH-1H antibody served as control. The bound antibody was detected with biotin-labelled anti-human kappa reagents or anti-human IgG reagent (monoclonals NH3/41 and NH3/130 respectively although other suitable reagents are commonly available) and subsequent development with streptavidin horseradish peroxidase. Figure 7 illustrates the results obtained for:

TF 57-19 ("wild type" CAMPATH-1H antibody, 0) MTF 121 (mutant 1,Δ )

MTF 123 (mutant 2,□)

and the wild type CAMPATH-1H (▼) in a known amount as standard. The concentrations had been estimated, and the starting dilutions adjusted to 50 μg/ml in PBS/10 mg/ml BSA. The starting dilution was used to prepare 8 twofold dilutions.

The slope of the graph shows clearly that the CAMPATH-idiotype antibodies recognises mutants 1 and 2 to an extent equivalent to that of the wild type CAMPATH-1H, and that all three antibodies tested are present in similar concentrations as the standard. Example 2

The mutants' capability of autologous complement mediated lysis of human peripheral blood lymphocytes was tested.

Human peripheral blood mononuclear cells from a healthy donor were isolated from 60 ml defibrinated blood on a Lymphoprep gradient (Nyeggard & Co., AS, Oslo, Norway). The cell pellet was washed in IMDM (Iscove's Modification of Dulbecco's Medium, Flow Laboratories, Scotland), and the cells were labelled with ⁵¹Cr. The starting dilution of antibodies used in the test was 50 μg/ml in PBS, 10 μg/ml BSA (dilution 1). Dilution 1 was further diluted 8 times two-fold to a final dilution of 1/128. Wild type antibody diluted in the same manner was used as a control.

The result is illustrated in figure 8. As can be seen, both antibody mutants show a very similar result in lysing the blood mononuclear cells as the wild type. The efficiency of the mutants is almost identical.

Example 3

Experiments were conducted to investigate whether or not, the mutant antibodies were capable of antibody-dependent cell-mediated cytotoxicity (ADCC) using CD3 activated interleukin-2 expanded human blastocytes as effectors (E) and targets (T). Cells were generated and used as both effectors and targets essentially as described in Riechmann L., Clark M.R., Waldmann H., Winter G., 1988, Nature 322, 323-327.

Preparation of Target Cells (T)

5 ml of blastocytes (3 x 10⁴ cells) were labelled with ⁵¹Cr for 1 h. After 1 h the cells were washed and transferred in 6 equal aliquots in 100 μl IMDM 1% BSA, to 6 x 10 ml tubes containing 100 μl of the antibodies of mutants 1 and 2, and the control. The tubes were incubated for 1.5 h at room temperature. The cells were then washed with 10 ml IMDM 1% BSA and resuspended in 1.5 ml IMDM 1% BSA.

Preparation of Effector Cells (E)

Unlabelled blastocytes (2 × 10⁶) were diluted 100:1 and 30:1 in IMDM 1% BSA medium. The ratios 100:1 and 30:1 refer to the final absolute ratios of effectors to 5¹Cr labelled targets in the assay. Assays were performed in microtitre plates with a total volume of 200 μl per assay well. Thus 100 μl of targets at a concentration of 2 × 10⁴ were put in each well ie 2 x 10³ total cells. For E:T of 100:1, 100 μl of effectors at 2 x 10⁴ were plated per well ie 2 × 10⁵. For E:T of 30:1 100 μl of effectors at 6 × 10⁵ were put into each well ie 6 x 10⁴ total cells.

The efficiency percentage of specific ⁵¹Cr release was calculated as follows:

% specific ⁵¹Cr release = (test release cpm - spontaneous (cpm) × 100

(total cpm - spontaneous cpm)

cpm = radioactive counts per minute as measured on a counter.

The result is shown in figure 9. The figure shows that all of the antibodies tested released chromium. Wild type TF 57-19 and mutant 2 (MTF 123) released at about equal levels, whereas mutant 1 (MTF 121) shows a slightly higher release.

These results clearly show that the mutants have biological activity comparable to the wild type CAMPATH- 1H antibody.

Example 4

The antibodies were tested in an assay specific for their G1m (3) allotypes reactivity using a monoclonal reagent from Oxoid (WHO/IVISS recognised agent, Study

Code No HP 6027). These tests were performed in replicates of two.

Microtiter plates were coated with the anti-CAMPATH idiotype YID 13.9.4 antibody captive, and divided into three arrays of 4 × 4 wells. Into each of the three arrays, 4 × 5 fold dilutions of the antibody TF 57-19, MTF 121 and MTF 123 (50 μg/ml) in PBS 1% BSA and a control solution of PBS/BSA each were added.

After an incubation of 45 minutes at room temperature, the antibody solution was removed, and (i) to the first array was added a 1:500 dilution of biotin-labelled anti-G1m (3);

(ii) to the second array was added a 1:100 dilution of biotin-labelled antibody (NH3/41) specific for the kappa light chain; and

(iii) to the third array was added a 1:1000 dilution of biotin-labelled antibody (NH3/130) specific for human IgG1.

The microtiter plate was developed with streptavidin horseradish peroxidase.

The result is illustrated in Table 1. The numbers in the results represent the optical density (O.D) as measured in an ELISA plate reader multiplied by 100 ie 12 represents an O.D of 0.12 and 70 an O.D of 0.70.

The result clearly shows, that samples 1-3 all react with the antibodies specific for IgG1 (see also Example 1 above) and the kappa light chains. The control is negative. However, in the assay for G1m (3) specificity, only MTF 121 (mutant 1) shows reactivity, whereas the wild type TF 57-19, MTF 123 (mutant 2) and the PBS/BSA control did not show any response.

This result illustrates clearly that the elimination of sites recognised in the allotype response by sitedirected mutagenis of these sites can overcome the problems of allotypic immuno-reactions. Although the examples refer to the mutagenesis of IgG1 only, it will be clear to the person skilled in the art that other immunoglobulin isotypes can be similarly modified.

Example 5

The antibodies were tested in a conventional allotyping experiment using inhibition of red cell agglutination. The experiment was carried out using reagents supplied by the Central Laboratory of the Netherlands Red Cross, Blood Transfusion Service (PO Box 9190, 1006 AD Amsterdam, Netherlands).

Human blood group O Rhesus D red cells were washed and then aliquots separately labelled as described below with one of the following three relevant anti-RhD human sera having antibodies of known allotype.

(1) anti-D G1m(az) = G1m (1,17)

(2) anti-D G1m(x) = G1m (2)

(3) anti-D G1m(f) = G1m (3)

Coating of Red Cells with Anti-Rh Antibodies

One volume of packed washed red blood cells were incubated with 4 volumes anti-Rh serum and 4 volumes (phosphate) buffered saline (PBS) at 37ºC during 60 minutes. Every 15 minutes the cells were mixed by shaking.

After incubation the coated cells were washed four times with PBS and stored at 4°C in preservation fluid (although coated red blood cells can be stored at 4°C in PBS for one week). These coated red blood cells were then agglutinated with four antisera to the IgG1 allotypes as follows using the recommended dilution for each antiserum.

(1) anti-G1m(z) = anti-G1m(17) 1 in 30 dilution (2) anti-G1m(a) = anti-G1m(l) 1 in 30 dilution

(3) anti-G1m(x) = anti-G1m(2) 1 in 20 dilution

(4) anti-G1m(f) = anti-G1m(3) 1 in 30 dilution The wild-type CAMPATH-1H TF57-19 or the different

CAMPATH-1H constructs (MTF 121, MTF 123) with the altered gamma-1 constant regions were then tested for their abilities to inhibit the agglutination of the red cells by the above antisera. The inhibiting antibodies were tried at concentrations of 0.5mg/ml, 0.25mg/ml and 0.125mg/ml in phosphate buffered saline containing 5% foetal bovine serum. Control sera containing IgG1 of allotype G1m(zax) or G1m(f) [G1m(1,2,17) or G1m(3)] were also included in the experiment and were used at dilutions of 1 in 10,20 and 40. Where it occurred the inhibition was most easily seen for the CAMPATH-1H antibodies at the 0.5mg/ml concentration and it was much weaker for 0.25mg/ml and no inhibition was seen at 0.125mg/ml. The control sera inhibited at all three dilutions tested. The results for the highest concentration are shown below.

Allotype CAMPATH-1H constructs Control sera TF57-19 MTF121 MTF123 G1m(1,2,17) G1m(3) G1m(1) + - - + - G1m(2) - - - +

- G1m(3) + - +

- - G1m(17) + - - +

-

The results are therefore consistent with the original wild type CAMPATH-1H antibody TF57-19 having allotype G1m(1,17). The new mutant MTF121 type as allotype G1m(3) whilst the mutant MTF123 fails to allotype for any of the IgG1 allotype markers G1m( 1, 2, 3, 17 ) i.e. it appears not to have an IgG1 allotype.

The skilled man will be able to use the binding molecules hereby provided to make pharmaceuticals according to standard techniques. Similarly the pharmaceuticals can be used in accordance with standard practices.

APPENDIX 1 - Sheet (a)

HSIGCC4 2009 bases

Human ig germline g-e-a region a: gamma-1 constant region

ID HSIGCC4 standard; DNA; PRI; 2009 BP.

AC J00228;

DT 23-APR-1990 (reference update)

DT 18-JUL-1985 (incorporated)

DE Human ig germline g-e-a region a: gamma-1 constant

DE region

KW constant region; gamma-immunoglobulin; germ line;

KW hinge exon; immunoglobulin; immunoglobulin heavy

KW chain.

OS Homo sapiens (human)

OC Eukaryota; Metazoa; Chordata; Vertebrata; Tetrapoda;

OC Mammalia; Eutheria; Primates.

RN [1] (bases 1-2009)

RA Ellison J.W., Berson B.J., Hood L.E.;

RT "The nucleotide sequence of a human immunoglobulin

RT c-gamma-1 gene";

RL Nucleic Acids Res. 10:4071-4079(1982).

RN [2] (bases 469-1070, 1465-1821)

RA Takahashi N., Ueda S., Obata M., Nikaido T.,

RA Nakai S., Honjo T.;

RT "Structure of human immunoglobulin gamma genes:

RT Implications for evolution of a gene family";

RL Cell 29:671-679(1982).

CC [1] and [2] report that nucleotide divergence among

CC the four gamma genes is much greater in the hinge

CC regions than anywhere else. [2] also reports the

CC hinge regions of gamma-2, gamma-3, gamma-4, a gamma

CC pseudogene, and the 5' flanking, ch2, and ch3

CC domains of the gamma genes.

CC

CC this entry is part of a multigene region (region a)

CC containing the gamma-3, gamma-1, pseudo-epsilon, and

CC alpha-1 genes. see segment 1 for more comments.

Key Location/Qualifiers

FT CDS 210..503

FT /note= " Ig gaπuna-1 heavy chain APPENDIX 1 - cont. Sheet (b)

FT c-region chl domain (aa at 212)"

FT conflict 563..563

FT /citation=([1],[2])

FT /note="T in [1]; c in [2]"

FT conflict 593..593

FT /citation=([1],[2])

FT /note="C in [1]; t in [2]"

FT conflict 614..614

FT /citation=([1],[2])

FT /note="G in [1]; a in [2]"

FT conflict 633.-633

FT /citation= ([1],[2])

FT /note="G in [1]; gg in [2]"

FT conflict 643..643

FT /citation=([1],[2])

FT /note="G in [1]; a in [2]"

FT conflict 654..654

FT /citation=( [1] , [2] )

FT /note="G in [1]; a in [2]"

FT conflict 684..684

FT /citation=([1],[2])

FT /note="C in [1]; cc in [2]"

FT conflict 692..692

FT /citation=([1],[2])

FT /note="G in [1]; a in [2]"

FT conflict 765..766

FT /citation=([1],[2])

FT /note="Aa in [1]; a in [2]"

FT CDS 892..936

FT /note="Ig gamma-1 heavy chain

FT c-region hinge"

FT CDS 1055..1384

FT /note="Ig gamma-1 heavy chain

FT c-region ch2 domain"

FT conflict 1475..1475

FT /citation=([1],[2])

FT /note="C in [1]; cc in [2]"

FT CDS 1481..1803

FT /note="Ig gamma-1 heavy chain

FT c-region ch3 domain"

FT conflict 1578..1578

FT /citation---([1],[2])

FT /note="T in [1]; c in [2]"

SQ Sequence 2009 BP; 418 A; 698 C; 566 G; 327 T; O

SQ Other; PPENDIX 1 - cont. Sheet (c)

10 20 30 40 50 60

1 AGCTTTCTGG GGCAGGCCAG GCCTGACCTT GGCTTTGGGG CAGGGAGGGG GCTAAGGTGA

61 GGCAGGTGGC GCCAGCAGGT GCACACCCAA TGCCCATGAG CCCAGACACT GGACGCTGAA

121 CCTCGCGGAC AGTTAAGAAC CCAGGGGCCT CTGCGCCTGG GCCCAGCTCT GTCCCACACC

181 GCGGTCACAT GGCACCACCT CTCTTGCAGC CTCCACCAAG GGCCCATCGG TCTTCCCCCT

241 GGCACCCTCC TCCAAGAGCA CCTCTGGGGG CACAGCGGCC CTGGGCTGCC TGGTCAAGGA

310 320 330 340 350 360

301 CTACTTCCCC GAACCGGTGA CGGTGTCGTG GAACTCAGGC GCCCTGACCA GCGGCGTGCA

361 CACCTTCCCG GCTGTCCTAC AGTCCTCAGG ACTCTACTCC CTCAGCAGCG TGGTGACCGT

421 GCCCTCCAGC AGCTTGGGCA CCCAGACCTA CATCTGCAAC GTGAATCACA AGCCCAGCAA 481 CACCAAGGTG GACAAGAAAG TTGGTGAGAG GCCAGCACAG GGAGGGAGGG TGTCTGCTGG

541 AAGCAGGCTC AGCGCTCCTG CCTGGACGCA TCCCGGCTAT GCAGCCCCAG TCCAGGGCAG

610 620 630 640 650 660 601 CAAGGCAGGC CCCGTCTGCC TCTTCACCCG GAGCCTCTGC CCGCCCCACT CATGCTCAGG 661 GAGAGGGTCT TCTGGCTTTT TCCCAGGCTC TGGGCAGGCA CAGGCTAGGT GCCCCTAACC 721 CAGGCCCTGC ACACAAAGGG GCAGGTGCTG GGCTCAGACC TGCCAAGAGC CATATCCGGG 781 AGGACCCTGC CCCTGACCTA AGCCCACCCC AAAGGCCAAA CTCTCCACTC CCTCAGCTCG 841 GACACCTTCT CTCCTCCCAG ATTCCAGTAA CTCCCAATCT TCTCTCTGCA GAGCCCAAAT

910 920 930 940 950 960

901 CTTGTGACAA AACTCACACA TGCCCACCGT GCCCAGGTAA GCCAGCCCAG GCCTCGCCCT

961 CCAGCTCAAG GCGGGACAGG TGCCCTAGAG TAGCCTGCAT CCAGGGACAG GCCCCAGCCG

1021 GGTGCTGACA CGTCCACCTC CATCTCTTCC TCAGCACCTG AACTCCTGGG GGGACCGTCA

1081 GTCTTCCTCT TCCCCCCAAA ACCCAAGGAC ACCCTCATGA TCTCCCGGAC CCCTGAGGTC

1141 ACATGCGTGG TGGTGGACGT GAGCCACGAA GACCCTGAGG TCAAGTTCAA CTGGTACGTG

APPENDIX 1 - cont. Sheet (d)

1210 1220 1230 1240 1250 1260 1201 GACGGCGTGG AGGTGCATAA TGCCAAGACA AAGCCGCGGG AGGAGCAGTA CAACAGCACG

1261 TACCGGGTGG TCAGCGTCCT CACCGTCCTG CACCAGGACT GGCTGAATGG CAAGGAGTAC

1321 AAGTGCAAGG TCTCCAACAA AGCCCTCCCA GCCCCCATCG AGAAAACCAT CTCCAAAGCC 1381 AAAGGTGGGA CCCGTGGGGT GCGAGGGCCA CATGGACAGA GGCCGGCTCG GCCCACCCTC 1441 TGCCCTGAGA GTGACCGCTG TACCAACCTC TGTCCTACAG GGCAGCCCCG AGAACCACAG

1510 1520 1530 1540 1550 1560 1501 GTGTACACCC TGCCCCCATC CCGGGATGAG CTGACCAAGA ACCAGGTCAG CCTGACCTGC 1561 CTGGTCAAAG GCTTCTATCC CAGCGACATC GCCGTGGAGT GGGAGAGCAA TGGGCAGCCG 1621 GAGAACAACT ACAAGACCAC GCCTCCCGTG CTGGACTCCG ACGGCTCCTT CTTCCTCTAC 1681 AGCAAGCTCA CCGTGGACAA GAGCAGGTGG CAGCAGGGGA ACGTCTTCTC ATGCTCCGTG 1741 ATGCATGAGG CTCTGCACAA CCACTACACG CAGAAGAGCC TCTCCCTGTC TCCGGGTAAA 1810 1820 1830 1840 1850 1860 1801 TGAGTGCGAC GGCCGGCAAG CCCCGCTCCC CGGGCTCTCG CGGTCGCACG AGGATGCTTG 1861 GCACGTACCC CCTGTACATA CTTCCCGGGC GCCCAGCATG GAAATAAAGC ACCCAGCGCT 1921 GCCCTGGGCC CCTGCGAGAC TGTGATGGTT CTTTCCACGG GTCAGGCCGA GTCTGAGGCC 1981 TGAGTGGCAT GAGGGAGGCA GAGCGGGTC

Claims

1. A molecule which comprises an amino acid sequence derivable from part or all of the constant region of an immunoglobulin heavy chain which constant regions are of a particular isotype and have one or more allotypic determinants

2. A molecule according to claim 1 which comprises an amino acid sequence derivable from part or all of a human immunoglobulin constant region.

3. A molecule according to claim 1 or claim 2 which comprises one or more polypeptides together with said amino acid sequence.

4. A molecule according to claim 3 wherein the polypeptide comprises a functional biological domain.

5. A molecule according to claim 4 wherein the functional biological domain comprises a binding domain.

6. A molecule according to claim 5 wherein the binding domain is an immunoglobulin-like binding domain.

7. A molecule according to claim 6 in which said immunoglobulin-like binding domain and said amino acid sequence are derivable from the same or different sources.

8. A molecule according to any one of claims 1 to 7 wherein the constant region is from an immunoglobulin of the isotype IgG.

9. A molecule according to claim 8 wherein the isotype subclass is IgG1 and the molecule no longer has one or more of the allotypic determinants 1,2,3 and 17.

10. A molecule according to claim 8 wherein the isotype subclass is IgG2 and the molecule no longer has the allotypic determinant 23.

11. A molecule according to claim 8 wherein the isotype subclass is IgG3 and the molecule no longer has one or more of the allotypic determinants 11,5,13,14,10,6,24,21, 15,16,26 and 27.

12. A molecule according to any one of claims 1 to 7 wherein the constant region is from an immunoglobulin of the isotype IgA2 and the molecule no longer has either or both of the allotypic determinants 1 and 2.

13. A pharmaceutical preparation which comprises a molecule according to any one of claims 1 to 12.

14. A reagent which comprises a molecule according to any one of claims 1 to 12.

15. A nucleotide sequence encoding a molecule according to any one of claims 1 to 12.

16. A cloning or expression vector comprising a nucleotide sequence according to claim 15.

17. A host cell comprising a cloning or expression vector according to claim 16.

18. A method of preparing a molecule according to any one of claims 1 to 12 which comprises the steps of:

(a) identifying a constant region of an immunoglobulin heavy chain;

(d) altering the coding sequence so that it codes for a molecule without at least one of said allotypic determinants and by making the amino acid residues at the site for an allotypic determinant identical to the amino acid residues from the corresponding position in an equivalent immunoglobulin constant region of an isotype different to that of said identified constant region;

19. A method of treating a patient which comprises administering a pharmaceutical preparation according to claim 13.