Note: Descriptions are shown in the official language in which they were submitted.
~VO 92/16562 ~ 8 l~ PCT/GB92/OQ445
Human1sPd antlbod1es hav~ng mod1f~ed allotypic determlnants
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 particular~y monoclonal antibodies
(Kohler, G. and Milstein CO, 1975 Nature 256:495)
represented a significant ~echnical break-throush with
important consequences scientifically, commercially and
therapeutically.
Monoclonal antibodies are made by establishing an
immortal cell line whi~h is derived from a single
immunoglobulin producing cell secreting one form of a
biologically functional antibody molecule with a
particular specificity.
Owing to their sp~cificity, the therapeutic
applications of monoclonal an~ibodies 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
~1059~
W092/]6562 " i PCT/GB92/0~5
human patients can lead to harmful hypersensitivity
raactions. 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-lG
is an exception to this rule.
Thus, there is a need for therapeutic antibodies
which have characteristic features speciic to human
proteins. Unfortunately, immortal human antibody-
producing cell lines are very difficult to establish and
they give low yields of antibody (approximately 1 ,ug/ml).
In contrast, equivalent rodent cell lines yield high
amounts o~ antibody (approximately 100 ~g/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 l). The light chains are of two types, either
kappa or la~bda. Each of the H and L chains has a region
,: ~
:
~092/16~62 ~1 Q ~ PCT/GB92/0~5
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 th~! C regions of the two H
chains. The antibody also has two arms (the Fab region)
each of which has a VL and a VH region associated with
each other. It is this pair of V regions (VL and VH)
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 VL and VH of a single heavy chain variable
domain (VH). -
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
:.
~ 1 ~3 ~ U
WO92/1fi~62 PCT/Gs92/0~45 -~
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 dif~erences 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 r antibodies having these sequence
differences (eg a patient may have received a prior blood
transfusion which containèd allotypically di~ferent
immunoglobulins). A model system of in~ecting "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 IgGl, IgG2, IgG3 and IgG4.
There are 2 A recognised allotypes of human
~092/ ~ 8 ~
16562 PCT/GB92/0~i5
immunoglobulin distributed between the different isotypes
as follows: -
IgG1 x 4
IgG2 x
IgG3 x 13
IgA2 x 2
IgE x
.
Kappa x 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 Glm 1, 2, 3 and 17. Alternatively,
combinations thereof, such as Glm (1, 17), can also be
distinguished. The four different single allotypes are
depicted in fisure 2.
Antisera can be raised in other non-human species
whlch 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, nGlm(1) which is the isoallotype
2 1 0 ~ 0
WO92/16~62 PCT/GB92/0~i4s _
of Glm(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 IgGl,
three common allelic forms of human IgGl occur with
different irequencies within different racial ~roups,
namely Glm (3~, Glm (1, 17), and Glm (1, 2, 17) based
upon their reactivities with human antisera directed
against the determinants Glm 1, 2, 3 and 17. At some
10 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 patlent
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 IgGl Glm (1, 2, 17) is
." ~ ..";. ..
~092/16562 ~1- Q ~ 9 8 0 PCT/GB92/0~45
shown aligned with sequences for the other human IgG,
iso~ype sub-classes in figure 4 (a, b, c and d). It can
be seen that the four isotypes are extrsmely homologous
for the domains CHl, 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 IgGl Glm (1, 2, 17) have been
marked in figure 4. However, for the purposes of clarity
the sequences around the allotypic sites Glm (1) (2) and
(17) are shown below for each isotype.
Site (l)
355 356 357 358
Arg Asp or Glu Glu Leu or Met IgGl
Arg Glu Glu Met IgG2
lS Arg Glu Glu Met IgG3
Gln Glu Glu Met IgG4
Thus, at site (l), IgGl may exist as several
allotypes depending on whether aspartic acid or glutamic r
acid at position 356, or leucine or methionine at
20 position 358 are present.
Site 2
430 431 432
Glu Gly or Ala Leu IgGl
Glu Ala Leu IgG2
25 Glu Ala Leu IgG3
Glu Ala Leu IgG4
9 ~ 0
WO92/1656~ PCT/GB92/0~5 -~
Thus, at site (2), IgGl may exist as either of two
allotypes depending on whether glycine or alanine is
present at position 431.
Site (17)/~3)
~ , .
5 Sites (3) and (17) are alternative substitutions at the ~
same site. `
213 Z14 2l5
Lys Lys or Arg Val IgGl
Lys Thr Val IgG2
10 Lys Arg Val IgG3
Lys Arg Val IgG4
Thus, at site (17)/(3), IgGl 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 Glm (l) 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 IyGl allele by site-directed
mutagenesis of the gene, for example, an existing
~092/16562 ~ PCT/GB9t/0~45
CAMPATH-lH monoclonal antibody gena described below, so
that the new allele consists entirely of isoallotypic
determinants. The preparation of IgGl mutants according
to the teaching provided by the present applicants is
shown schematically in figure 3.
For Glm (1) and Glm (2), the changes comprise simple
substitution by the alternative isoallotypic residues.
However, in the case of Glm (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 Glm (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 IgGl specific residues in close proximity in
the CHl domain or hinge region. This indicates that in
addition to changing lysine, other residues in CHl 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 IgGl and in particular, the
CAMPATH-lH monoclonaI 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 ~inding
molecule der:ivable from a second binding molecule;
2105~
WO92/16562 PCT/GB92/0~45 -
which second binding molecule is an immunoglobulin,
or a derivative, stru~tural 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 blnding
molecule may comprise entirely isoallotypic determinants.
The second binding molecule may be selected from the
group consisting of IgGl, IgG2, IgG3, IgA2, IgE, kappa
light chains or-derivatives, structural or functional
analogues thereof. Where the second binding molecule is
IgGl, 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
~0 92/lfi~62 ~ .1 n 5 ~ ~ Q PCT~GB92/0~45
11
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.
.. . . . . . . .. ...
~10~80
W092/16~62 PCT/GB92/O~S
12
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 incorporatin~ 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
`', .' . '
.
;' '
v092/]6562 ~1 Q~ PCT/GB92/0~45
13
determinant identlcal to the amino acid r~sidues fro~ 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 Aave
the allotypic determinant 23. The isotype subclass may
be IgG3 and the molecule may no longer have one or ~ore
of the allotypic determinants 11,5,13,14,10,6,24,21,15,
.. . . . .
~lO~Q
WO92/16562 pcT/Gs92fo~4
14
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
,! , . -. ' . ' . ' .:, ' ' ~ ' ' ` ', ! ' . ' ' ' ' '; ' ' ' ` ~ ' ' `
~092/16562 ~1 Q 5 ~ ~ O PiCT/GB~2/0~5
-
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 miake 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
- : ~ .. : :.. :: : . .
~10~) 98 0
WO 92/16562 PCr/GB92/00~5
16
humanised or human antibody.
Whatever the f orm in which the cloned
immunoglobulion gene is obtained, the next step is to
predict the amino acid sequence of the constant region
5 from the DNA sequence. The DNA sequence can be obtained
using a variety of strategies familiar to molecular
biologists. The predicted amino acid segiuence would then
be checked for the amino acids known to vary as
allotypes. Any isoallotypes present within the seguence
lO 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 wlth
15 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
...
20 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 Dl~1A clone which will encode this
sequence. Again there are several strategies available
25 to molecular biologists in order to achieve this. In the
~ ` ,
case of the example CAMPATH-lH constructs described
`;
"., ' ` ' .'. . ', ` , ' :~ i '' ' i ' ':~ . ' ' . . ` i ' i , ,,-. `
W092~16562 ? t O ~ PCT/GB92/0~45
17
herein, the gamma-l constant region was cloned in an
Ml3TGl31 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-lH 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
- , . . : - - . . ~ , "
210a98~
W092/16562 PCT/GB92/0
18
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 ara 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 combinatlon
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(l). The light chain allotype Km(l) 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
W092~16~62 ~ ~ 5 ~ ~ o PCT/GB92/0~45
- 1~
limitation. Reference will be made (and has already been
made in the text above) to the following figures in
which:
figure 1 illustratas the structure of an IgG
antibody;
figure 2 shows the allotypes for the IgG1 antibody
CAMPATH-lH;
figure 3 shows schematically the preparation of IgG1
mutants;
figure 4 shows the IgG1 Glm (1,2,17) allotype
seguence 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 IgGl
allotypes);
figure 5 shows the M13TGl31 cloning vector
contai~ing the human gamma-l constant region, showing
cloning sites and modified polylinker;
figure 6 shows the original Hu4vH HuGl pSVgpt
expression vector and its modified version;
figure 7 shows the result of an ELISA assay testing
different dilu-tions of the antibodies of mutants 1, 2 and
wild type CAMPATH-lH 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-
~10 2 ~ 8 0
W092/l6562 PCT/GB92/0~5
20mediated 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-lH, a monoclonal antibody with a
kappa light chain containing the human constant region
sequence for IgG1 which carries the Glm (1, 17) allelic
determinants. The whole IgGl 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-l constant region showing
cloning sites and modified polylinker is shown in figure
5.
The IgGl encoding region is entered in the EMBL
Sequence Database under the code number HSlGCC4. The
accession number is AC J00228 (the printout from the
database is provided herein as Appendix 1). This
sequence is for the Glm (1, 17) allotype. It covers 2009
bases from the 5' HindIII 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 pro~ided 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 M01 and M04 hybridise to nucleotides 4B6
to 510. The hinge region starts at nucleotide 892 and
~092J16562 ~ 3 0 ' ~ ,~ n PCT/GB92/0~45
21
ends at nucleotide 936. The CH2 domain starts at
nucleotide 1481 and ends at nucleotide 1803. The
mutagenic oligonucleotide M02 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 fragmant, 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 E~L Sequence
Database. It should be noted however, that the Sphl
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 sltes 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 Plc, Amersham, UK)
the sPquence corresponding to the Glm (1~ allele was
converted to the corresponding sequence found in the
other sub-classes for IgG (Asp Glu Leu to Glu Glu Met at
- positions 356-358 in the CH3 domain).
The mutagenic oligonucleotides used were-
a) M01 (to convert Glm (17) to Glm (3))
5' CTC TCA CCA ACT CTC TTG TCC ACC T 3';
' ~ `,
2~0~t381)
W092/~6~62 PCT/GB92/0~45 -
22
b) M02 (to convert Glm (1) to its isoallotype nGlm (1))
5 ' GGT TCT TGG TCA TCT CCT CCC GGG ATG GG 3'; and
c) M04 (to eliminate Glm(3) by chan~ing Lys to Thr in
the C~1 ragion)
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
sequenclng (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 ~ormed
allotype sequence is found in all humans, there should be
no immunological response to this alternative form of Glm
(1). Additionally and similarly, the lysine residue
responsible for the Glm (17) allotypic determinant was
converted to an arginine residue corresponding to the Glm
allele (Lys 214-Arg; mutant 1).
The gene for this new constant region of mutant 1
carrying these three changes has been se~uenced,
incorporated into an expression vector containing the
CAMPATH-lH V-region and expressed together with the
CAMPATH-lH light chain which had been introduced by co-
transfection.
~/O 92/16562 2 ~ O ~ ~ 8 ~ PCl`/GB92/00445
A further mutant has been made by replacing the
critical arginine residue associated with Glm (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-Glm (17) and anti-Glm (3)
antisera (mutant 2).
Mutant 2 has also bsen sequenced, re-cloned in an
expression vector containing the CAMPATH-lH 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
Hu4vHGlpSVgpt 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 Notl. The expression
vectors and VH region sequences and expression, along
with the light chains, in Y0 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
laryest amount of the IgGl product were selected to
obtain purified antibody for biological assays to
determine their allotypes and biological effector
functions.
: '
~, .
2~0.~9~0
W092/]6562 PCT/GB92/0~4~ -
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 coatPd with anti-CAMPATH-idiotype antibody (YID
13.9). Wild type CAMPATH-lH 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:
~F 57-19 ("wild type" CAMPATH-lH antibody, 0)
MTF 121 (mutant 1,~)
MTF 123 (mutant 2, a )
and the wild type CAMPATH-lH (~) in a known amount as
standard. The concentrations had been estimated, and the
starting dilutions adjusted to 50 ,ug/ml in PBS/lO mg/ml
BSA. The starting dilution was used to prepare 8 two-
fold 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-lH,
and that all three antibodies tested are present in
similar concentrations as the standard.
~092/l6~62 PCT~GB92/O~S
0~9~)
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). ~he cell pellet was washed in IMDM (Iscove's
Modification of Dulbecco's Medium, Flow Laboratories,
Scotland), and the cells were labelled with 5lCr. The
starting dilution of antibodies used in the test was 50
~g/ml in PBS, 10 ~g/ml BSA (dilution 1). Dilution 1 was
further diluted 8 times two-fold to a final dllution o~
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.
Exam~le 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 ~lastocytes as
effectors (E) and targets (T). Cells were generated and
- used as both effectors and targets essentially as
'.,':.;' ,
:' ' '
9 8 0
W092/16~62 PCT/GB92/0~5
26
described in Riechmann L., Clark M.R., Waldmann H.,
Winter G., lsa8, Nature 322, 323-327.
PreDaration of Target Cells ( T )
5 ml of blastocytes (3 x 106 cells) were labelled
with 51cr for 1 h. After 1 h the cells were washed and
transferred in 6 equal aliquots in 100 ~1 IMDM 1~ BSA, to
6 x 10 ml tubes containing 100 ~1 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.
Pre~aration of Effector Cells (E)
Unlabelled blastocytes (2 x 106) were diluted 100:1
and 30:1 in IMDM 1~ ~SA 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
,ul per assay well. Thus 100 ~1 of tar~ets at a
concentration of 2 x 104 were put in each well ie 2 x 103
total cells. For E:T of 100:1, 100 ~1 of effectors at 2
x 106 were plated per well ie 2 x 105. For E:T of 30:1
100 ~1 of effectors at 6 x 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 =
~092tl6~62 2 ~ 8 n PCT/GB92/0~5
27
(test release cpm - spontaneous (cpm) x 100
(total cpm - spontaneous cpm)
cpm = radioactive counts per minute as measured on a
counter.
The result is shown in fisure 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 ~he wild type CAMPATH-
lH antibody.
Example 4
The antibodies were tes~ed in an assay specific for
their Glm ~3) allotypes reactivlty 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 x 4 wells. Into each of the three
arrays, 4 x 5 fold dilutions of the antibody TF 57-l9,
MTF 121 and MTF 123 (50 ~g/ml~ in PBS l~ BSA and a
control solution of PBS/BSA each were added.
After an incubation of 45 minutes at room
temperature, th~ antibody solution was removed, and
, ~ .
210:?~80
W092/]6~62 ~ PCT/~B92/0~5
28
(i) to the first~ array was added a 1:~00
dilution o biotin-labelled anti-Glm (3~;
(ii) to the second array was added a 1:100
dilution of biotin-labelled antibody (NH3/41) specific
5 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 IgGl (see also Example 1
above) and the kappa light chains. The control i5
negative. However, in the assay for Glm (3) specificity,
only MTF 121 (mutant 1) shows reactivity, whereas the
20 wild type TF 57-19, MTF 123 (mutant 2) and the P~S/BSA
control did not show any response.
This result illustrates clearly that the elimination
of sites recognised in the allotype response by site-
directed mutagenis of these sites can overcome the
problems of allotypic immuno-reactions. Although the
examples refer to the mutagenesis of IgGl only, it will
- . . ; ,. , , - : :
~092/16562 ~1 n ~ 9 8 0 PCT/GB92/0~45
29
be clear to ~he person skilled in the art that other
immunoglobulin isotypes can be similarly modified.
Example 5
The antibodies were t:ested 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 Glm(az) = Glm (1,17)
(2) anti-D Glm(x) = Glm (2)
(3) anti-D Glm(f) = Glm (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 volwnes
(phosphate) buffered saline (PBS) at 37C during 60
minutes. Every 15 minutes the cells were mixed by
shakin~.
After incubation the coated cells were washed four
times with PBS and stored at 4C in preservation fluid
(although coated red blood cells can be stored at ~C in
PBS for one week).
'~10~80
~092/16562 PCT/GB92/O~
These coated red blood cells were then ag~lutina~ed
with four antisera to the IgGl allotypes as follows using
the recommended dilution for each antiserum.
( 1 ) anti-Glm( z ) = antl--Glm( 17 ) 1 in 30 dilution
(2) anti-Glm(a) = anti-Glm(l) 1 in 30 dilution
(3) anti-Glm(x) = anti-Glm(2) 1 in 20 diluti~n
(4) anti-Glm(f) = anti-Glm(3) 1 in 30 dilution
- The wild-type CAMPATH-lH TF57-19 or the different
CAMPATH-lH 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 IgGl of
allotype Glm(zax) or Glm(f) ~Glm(1,2,17) or Glm(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-lH
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-lH constructs Control sera ~r -~
~092/16562 ~ ~ PCT/GB92/0~5
TF57-19 MTFlZl MTF123 Glm(1,2,17) Glmt3)
Glm(l) + - _ +
Glm(2) - - - + _
Glm(3) - + _ _ +
5 Glm(17) + - _ +
~',
The results are therefore consistent with the ~ ;
oriyinal wild type CAMPATH-lH antibody TF57-19 having
allotype Glm(l, 17). The new mutant MTF121 type as
allotype Glm(3) whilst the mutant MTF123 fails to
allotype for any of the IgGl allotype markers
Glm(1,2,3,17) i.e. it appears not to have an IgGl
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.
21~8~
wo 92/16~62 PCI`/~B92/00445
32
_ ~ ~ ' ' -:'
Z- O ~ . `~ `
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C O ~
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t~ ~3 _ ~ ' ... N
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SUB~i~3 ~ E ~EET `
`~
~092/16562 21 0 :~ 9 8 ~ PCT/GB92/0~45
33
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-l 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., ~erson B.J., Hood L.E.;
RT "The nucleotide sequence of a human immunoglobulin
RT c-gamma-l 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-l, pseudo-epsilon, and
CC alpha-l genes. see segment 1 for more comments.
Key Location/Qualifiers
FT CDS 210............... 503 ;
FT ~note="Ig gamma-1 heavy chain
.:
210 ~98~
WO92/16562 PCT/GB92/0~5
34
APPENDIX 1 - COIlt . Sheet (b)
FT c-region chl domain (aa at 212)"
FT conflict 563... 563
FT /citation=([1],[2]) : :
FT /note="T in [l]; 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 [l]; 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 [l]; 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=([l]~t2]) ~,
FT /note="Aa in [1]; a in [2]"
FT CDS 892... 936
FT /note="Ig gamma-1 heavy chain
FT c-region hinge" r . .
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 /not-e="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, 0
SQ Other;
, ~,..
- . , .
WO 92/16562 2~ :~ Q ~PCr/GB92/oo~
o ~ O ~ U ~O O O ~O ~ D C U U ~ ~
~ o u u; ~ U ~ 5, e ~ ~;
o ~ ~ ~ y . yO y ~ y
,I y ~: u b U ~ '~~ ~ y ~ y ~ j a a
o c~ ~., o .c U, ~ o U ~ y o 5 U a
¢ ~ , ¢ ~ J i
~ 1 o ~ rO ~D ~ es:) ~ ~ ~ ~ ~
~lQ~8~
WO 92/16562 PCI~/GB92/00445
36
-I U a ~ U
5 ~ ~ U ~
O ~ E~ ~ o ~ C~ o
~ ~ ~ V ~ E~
o ~ E~ ~ ~ ~ o ~ E~ o
U ~ U
O j ~ ~ ~ u a
U ~ ¢ ~ ¢ ~ U ¢ U: ~ ¢ ~ ¢ U
U t~ h ~ ¢ ~ U c~ u ~ c~
U ~ ~ ~ E - ~ ¢ ~ ~ U ¢ _,, ~ U ~g U
~ U E~ ~ V E~ ~ ¢ ~ ~ U U C~
¢ ~U ~ ~ U E~ E~ ¢ V h ~ ~ u ~
u h E~ t3 u ~ ¢ ~: h ~ ~ h
O ~D ~ CO ~ O ~ r O ~ ~ CO
SUE3ST~T~ EFT
.
. ~ . .
