U.S. patent application number 09/125460 was filed with the patent office on 2002-04-25 for antibody variants.
Invention is credited to FREWIN, MARK R, GILLILAND, LISA K, TONE, MASAHIDE, WALDMANN, HERMAN, WALSH, LOUISE.
Application Number | 20020048578 09/125460 |
Document ID | / |
Family ID | 10789047 |
Filed Date | 2002-04-25 |
United States Patent
Application |
20020048578 |
Kind Code |
A1 |
WALDMANN, HERMAN ; et
al. |
April 25, 2002 |
ANTIBODY VARIANTS
Abstract
This invention relates to an antibody which is a modified
version of a therapeutic antibody with affinity for a cell-surface
antigen, said antibody having reduced affinity for the antigen
compared with the therapeutic antibody as a result of a
modification or modifications to the antibody molecule, wherein the
antibody is capable of inducing immunological tolerance to the
therapeutic antibody. The invention further relates to a method of
inducing immunological tolerance to a therapeutic antibody
comprising administering to a patient an antibody which is a
modified version of the therapeutic antibody and which has reduced
affinity for the antigen as compared with the therapeutic
antibody.
Inventors: |
WALDMANN, HERMAN; (OXFORD,
GB) ; GILLILAND, LISA K; (OXFORD, GB) ; TONE,
MASAHIDE; (OXFORD, GB) ; FREWIN, MARK R;
(OXFORD, GB) ; WALSH, LOUISE; (CAMBRIDGE,
GB) |
Correspondence
Address: |
MR LEE CHENG
WENDEROTH LIND AND PONACK LLP
2033 K STREET NW
SUITE 800
WASHINGTON
DC
20006
US
|
Family ID: |
10789047 |
Appl. No.: |
09/125460 |
Filed: |
August 19, 1998 |
PCT Filed: |
February 20, 1997 |
PCT NO: |
PCT/GB97/00472 |
Current U.S.
Class: |
424/133.1 ;
424/135.1; 424/141.1; 424/144.1; 424/153.1; 424/154.1; 424/173.1;
530/387.3; 530/388.1; 530/388.2; 530/388.22; 530/388.73;
530/388.75 |
Current CPC
Class: |
A61K 38/00 20130101;
C07K 16/2893 20130101; C07K 16/465 20130101; C07K 2317/24 20130101;
A61P 37/00 20180101; C07K 2319/00 20130101; A61P 37/02
20180101 |
Class at
Publication: |
424/133.1 ;
424/135.1; 424/144.1; 424/154.1; 424/173.1; 424/153.1; 424/141.1;
530/388.1; 530/388.2; 530/388.73; 530/387.3; 530/388.22;
530/388.75 |
International
Class: |
A61K 039/395; A61K
039/40; A61K 039/42; C12P 021/08; C07K 016/00 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 20, 1996 |
GB |
9603507.6 |
Claims
1. An antibody which is a modified version of a therapeutic
antibody with affinity for a cell-surface antigen, said antibody
having reduced affinity for the antigen compared with the
therapeutic antibody as a result of a modification or modifications
to the antibody molecule, wherein the antibody is capable of
inducing immunological tolerance to the therapeutic antibody and
wherein the antibody is not a mixed molecule antibody having an H
or L chain of the therapeutic antibody paired with an L or H chain
of an unrelated antibody.
2. An antibody as claimed in claim 1, wherein the framework regions
of the variable domains of the antibody have the same or
substantially the same amino acid sequence as the therapeutic
antibody framework regions.
3. An antibody as claimed in claim 1 or claim 2, wherein the
modification comprises an alteration in at least one of the
complementarity determining regions (CDRs).
4. An antibody as claimed in claim 3, wherein the alteration is
achieved by genetic manipulation of a nucleic acid coding for the
CDR.
5. An antibody as claimed in any one of claims 1 to 4, wherein the
affinity of the antibody for the antigen is reduced to 50% or less
of the affinity of the therapeutic antibody.
6. An antibody as claimed in any one of claims 1 to 5, wherein the
CDRs are foreign with respect to the constant region of the
antibody.
7. An antibody as claimed in any one of claims 1 to 6, wherein the
CDRs are foreign with respect to the heavy and light chain variable
domain framework regions.
8. An antibody as claimed in claim 7, which is of substantially
human origin other than the CDRs.
9. An antibody as claimed in any one of claims 1 to 8, wherein the
therapeutic antibody has affinity for CD52.
10. An antibody as claimed in claim 9, wherein the therapeutic
antibody is a humanised Campath-1 antibody
11. An antibody as claimed in claim 10, wherein the modification
comprises an alteration in (VH) CDR2.
12. An antibody as claimed in claim 11, wherein the modification
comprises a single or a double amino acid substitution in (VH)
CDR2.
13. An antibody as claimed in any one of claims 1 to 12, wherein
the constant domains of the antibody have substantially the same
amino acid sequence as the therapeutic antibody constant
regions.
14. A fragment of an antibody according to any one of claims 1 to
13, which fragment retains tolerance-inducing capability of the
antibody.
15. An antibody fragment as claimed in claim 14, which is a
modified version of a therapeutic antibody fragment.
16. An antibody or fragment as claimed in any one of claims 1 to
15, which is monovalent.
17. An antibody or fragment as claimed in any one of claims 1 to
16, for inducing tolerance to the therapeutic antibody in a
patient.
18. A cell line which expresses an antibody or fragment as claimed
in any one of claims 1 to 17.
19. A method of producing an antibody which is a modified version
of a therapeutic antibody with affinity for a cell-surface antigen,
said antibody having reduced affinity for the antigen compared with
the therapeutic antibody as a result of a modification or
modifications to the antibody molecule, wherein the antibody is
capable of inducing immunological tolerance to the therapeutic
antibody, comprising maintaining a cell line as claimed in claim 18
under conditions suitable for expression of said antibody or
fragment thereof.
20. The method of claim 19, further comprising recovering the
antibody or fragment.
21. The method of claim 20, further comprising isolating the
antibody or fragment.
22. A composition for administration to a patient, comprising an
antibody or fragment as claimed in any one of claims 1 to 17 or as
produced by the method according to any one of claims 19 to 21,
together with a physiologically acceptable diluent or carrier.
23. The use of an antibody or fragment as claimed in any one of
claims 1 to 17 or as produced by the method according to any one of
claims 19 to 21, in the manufacture of a medicament for the
induction of tolerance.
Description
[0001] This invention relates to modified antibodies for inducing
immunological tolerance in human beings or animate.
[0002] Antibodies, or immunoglobulins, comprise two heavy chains
linked together by disulphide bonds and two light chains, each
light chain being 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. Each light chain has a variable
domain at one end and a constant domain at its other end, the light
chain variable domain being aligned with the variable domain of the
heavy chain and the light chain constant domain being aligned with
the first constant domain of the heavy chain. The constant domains
in the light and heavy chains are not involved directly in binding
the antibody to antigen.
[0003] The variable domains of each pair of light and heavy chains
form the antigen binding site. The variable domains of the light
and heavy chains have the same general structure; each domain
comprises four framework regions, whose sequences are relatively
conserved, connected by three complementarity determining regions
(CDRs). The CDRs are held in close proximity by the framework
regions. CDRs from adjacent light and heavy chain variable domains
together contribute to the formation of the antigen binding
site.
BACKGROUND OF THE INVENTION
[0004] Antibodies directed to specifically chosen antigens have
been used in the treatment of various conditions. For example,
Campath-1 monoclonal antibodies (mAb) have been used successfully
to induce remissions in lymphoma and leukemia patients and for the
treatment of rheumatoid arthritis and vasculitis. The target
antigen, CD52 (also referred to as CDw52; see e.g. Xia et al.,
1991), is a GPI-anchored glycoprotein of lymphocytes and monocytes
(and parts of the male reproductive system). It has an
exceptionally short peptide sequence of 12 amino acids and a
single, complex, N-linked oligosaccharide at Asn3 (Hale et al,
1990; Xia et al, 1991). CD52 is a good target for antibody-mediated
killing and is therefore an effective cell surface molecule for
various therapeutic regimens in which reduction in lymphocytes is
an objective (e.g. removal of cells from donor bone marrow to
prevent graft-versus-host disease, treatment of leukemia and
lymphoma, and immuno-suppression).
[0005] Several rat anti-human CD52 Campath-1 mAb were generated by
fusion of the Y3 rat myeloma line with spleen cells from a rat
immunized with human T lymphocytes (Hale et al, 1983). Although the
clinical effectiveness of rat Campath-1 mAb has been demonstrated
regularly, many patients mounted an anti-antibody (antiglobulin)
response against the xenogeneic protein that prevented retreatment
with the therapeutic antibody. Antibody therapy is often limited by
the antiglobulin response. The anti-idiotypic component (anti-Id;
directed against the Ab V regions and in particular the
Ab-combining site) inhibits the binding of the Ab to its target
while both the anti-Id and the anti-isotypic component (directed
against the constant regions) act to accelerate antibody clearance.
A major concern is the neutralizing effect of the antiglobulin
response. As with antiglobulin responses in general, anti-Id
responses interfere with the clinical potency of a therapeutic Ab
by forming Ab aggregates that are rapidly cleared from the
circulation, reducing the chance for interaction with target
antigen. Unfortunately, most antiglobulin sera contain anti-Id
antibodies. This has been demonstrated for a number of therapeutic
mAb and is especially noted after repeated treatments.
[0006] To reduce the immunogenicity of the rat IgG2b Campath-1
antibody, YTH34-5, the gene fragments encoding the VL and VH were
humanized by "CDR grafting" of the rodent hypervariable regions
onto human framework regions (Jones et al, 1986; Reichmann et al,
1988). This was carried out by splicing the CDR sequences encoding
the rat Campath-1 antibody onto sequence encoding human framework
backbone provided by the crystallographically solved myeloma
proteins NEW (for the VH) and REI (for the VL). The resulting
protein had low antigen-binding titre and modelling of the
humanized V-region showed that residue 27 in the VH framework
sequence was critical for preserving the loop structure of CDR1.
This residue was changed from the residue found in NEW (Ser) back
to the rat residue Phe which resulted in restoration of antigen
binding. During the modelling, an additional change (NEW residue
Ser to the rat residue Thr) was also suggested. However, in
functional assays this substitution had no effect on antigen
binding, but the double mutant (Ser27 to Phe27 and Ser30 to Thr30)
expressed the most protein and therefore was used to produce
therapeutic humanized Campath-1 Ab, designated Campath-1H
(Reichmann et al, 1988). As many human VH frameworks have threonine
at position 30, this change was not considered an additional risk
to the antibody's immunogenicity. The humanized VL and VH were then
genetically fused with human light chain and heavy chain constant
regions, respectively. In summary, the humanized Campath-1 antibody
consists of human residues at all positions except those encoding
the 3 CDRs of the light chain, the 3 CDRs of the heavy chain, and
residues Phe27 and Thr30 in VH of the heavy chain.
[0007] In clinical trials, the humanized version (Campath-1H) was
found to be much less immunogenic than the rat IgG2b Campath-1
antibody. Humanization reduces the immunogenicity of rodent mAb,
although both the idiotype and the allotype of a humanized mAb
might still be targets for humoral responses. Sensitization to
idiotype has indeed been documented in some allotype-matched
recipients of Campath-1H (Isaacs et al, 1992; Lockwood et al,
1993). These responses were revealed by the presence of anti-Id in
the patients' sera. One patient generated high-titre anti-Id that
crossreacted on the entire panel of CD52 mAb. Humanized Campath-1
antibodies are described in EP 0 328 404 the teachings of which are
incorporated herein by reference, in their entirety.
[0008] One strategy to further reduce the immunogenicity of
Campath-1H might be to re-graft the 6 CDR loops onto
well-characterized human germline framework regions. The majority
of the humanized V regions so far have used rearranged V-genes as
acceptor framework sequence. This was the case for Campath-1H as
framework sequences from myeloma proteins were used to provide
acceptor sequences for both VH and VL. Rearranged V-genes often
contain somatic mutations, acquired during the process of affinity
maturation. These will be unique to the individual from which the
rearranged genes were derived and therefore may be seen as foreign
in another individual. However, there is a possibility that
regrafting may introduce new idiotypic epitopes, formed by the
junctional regions encompassing CDR residues and new framework
residues. Furthermore, humanization alone may not solve the problem
of anti-Id responses because the human population is outbred and it
is unlikely that all patients will be tolerant to a given humanized
mAb. Even in antibody constant regions, there are a number of
different alleles which carry allotypic markers to which naturally
occurring antiglobulin responses can be demonstrated. The problem
is more complex for V-region segments, which show a higher degree
of variation both in allotype and haplotype in comparison to
constant regions.
[0009] Another approach is to induce tolerance to the potentially
foreign peptides contained within the Campath-1H V-region. We know
that the antiglobulin response is itself a B-cell response which is
CD4+ T-cell dependent. Isaacs and Waldmann (1994) demonstrated that
mice deprived of CD4+ T-cells were unable to respond to a foreign
cell-binding mAb (rat anti-mouse CD8 mAb). CD4+ T-cell depletion
was carried out by adult thymectomy combined with administration of
a depleting CD4 mAb. In these mice, the response to subsequently
administered mAb or SRBC was measured. CD4+ T-cell deficient mice
failed to make either an antiglobulin response or an anti-SRBC
response, demonstrating that the anti-Ig response, like the
anti-SRBC response, is classically CD4+ T-cell dependent. in order
to generate T-cell help and to get the appropriate T-cell response,
the adminstered Ab must be processed as a protein antigen and
presented, presumably in the context of an MHC class II molecule,
by a suitable antigen presenting cell. Therefore, two main
strategies can be adopted to decrease the immunogenicity of a
humanized V-region. (1) We can "silence" the antibody molecule
itself, adopting strategies to eliminate any potential T helper
epitopes, or (2) we can present all the potential T helper epitopes
in a manner that induces tolerance instead of reactivity to those
epitopes.
[0010] "Silencing the Antibody Molecule:
[0011] a) In theory, we might be able to silence the antibody
itself so that the immune system will not recognize foreign
determinants. This would be possible if we could scan the VL and VH
amino acid sequences for motifs that could bind to MHC class II
molecules. If we could thus identify key residue(s) in a potential
class II peptide that were not involved in antibody specificity or
affinity, then it/they could be changed by site-directed
mutagenesis to residue(s) that did not allow association with class
II molecules. T helper peptides are not random, and any protein has
only a limited number of peptides capable of binding to MHC class
II molecules, and also to T-cell antigen receptors. However, this
is not possible at present because class II-binding peptides are
not yet characterized to a sufficient degree to be identified by
scanning protein sequences. This is in part due to the
heterogeneous nature of class II peptides. Naturally processed
peptides isolated from MHC class II molecules are generally larger
in size, variable in length and have both ragged ends at C- and
N-termini in comparison to processed peptides isolated from MHC
class I molecules. Whereas class I-derived peptides are mostly of
uniform length of 8-9 amino acid residues, MHC class II-associated
peptides range from 12-24 amino acids (Rudensky et al., 1991; Hunt
et al, 1992; Rudensky and Janeway, 1993). Class I-derived peptides
have sequence motifs with specific anchor residues in certain
positions allowing their side chains to fit in the binding pockets
of the peptide-binding groove, and the peptide-binding groove is
closed at both ends. In contrast, class II peptides are bound in an
extended conformation that projects from both ends of an
"open-ended" antigen-binding groove; a prominent non-polar pocket
into which an anchoring peptide side chain fits near one end of the
binding groove (Brown et al., 1993).
[0012] b) Other strategies that might be adopted to "silence" the
antibody if we could predict class II peptide-binding motifs. For
example, one could include insertion of a protease cleavage site
within any potential class II epitope to increase the chance of
peptide degredation before they could be presented in the context
of class II. Alternatively, insertion of motifs into a V-region
such as Gly-Ala repeats may inhibit the degradation of the V-region
into peptides that could associate with class II molecules. In one
system, it was shown that EBNA1 Gly-Ala repeats generated a
cis-acting inhibitory signal that interfered with antigen
processing during MHC class I-restricted presentation such that CTL
recognition was inhibited (Levitskaya et al, 1995). Although either
of these approaches may hold some promise in the future, they again
rely on prediction of potential MHC class II peptides from protein
sequence of humanized VL and VH regions and are therefore limited
by insufficient knowledge regarding consensus motifs for class II
peptides.
[0013] Inducing Tolerance to T Helper Epitopes
[0014] In lieu of sufficient knowledge regarding class II peptide
motifs, we have turned our attention toward induction of tolerance
to therapeutic antibodies. In 1986, Benjamin et al and Cobbold et
al described an unexpected property of cell-binding mAb: whereas it
was possible to induce tolerance to the Fc region (anti-isotype
tolerance), the idiotype remained antigenic under equivalent
conditions. Moreover, it was relatively easy to induce tolerance to
non-cell binding mAb but cell-binding mAb were found to be very
immunogenic.
[0015] Isaacs and Waldmann (1994) in a preliminary study used a
non-cell-binding "mixed molecule" derivatives of a cell-binding Ab
to induce tolerance to the wild-type form. The cell-binding
antibody was an anti-CD8 mAb in a mouse model. The non-cell-binding
derivatives were made by pairing the relevant L- and H-chains with
an irrelevant H- or L-chain, respectively. The relevant H-chain
paired with an irrelevant L-chain was obtained by limiting dilution
cloning of the original hybridoma that was expressing a myeloma
light chain (from the Y3 fusion partner), as well as the specific
anti-CD8 H- and L-chains. A variant of the hybridoma that expressed
the myeloma L-chain and the specific anti-CD8 H-chain but no
anti-CD8 L-chain was obtained. A clone expressing the relevant
L-chain only was also obtained in this manner. That clone was then
fused to a hybridoma expressing an irrelevant specificity
(anti-human CD3) and a variant was selected that expressed the
relevant anti-CD8 L-chain with the irrelevant anti-CD3 H-chain.
Because proteins are processed into peptides prior to presentation
to T-cells, helper peptides from antigen-specific Hand L-chains
would be "seen" by T-cells, regardless of their partner chain.
However, in this case, there was no advantage in tolerance
induction using non-cell-binding mixed molecule derivatives of a
therapeutic mAb in vivo compared to an isotype-matched control,
suggesting that in the strain of mice used, most (or all) of the
helper epitopes were located within the constant region.
[0016] In practice, using these "mixed molecules" of
antigen-specific and irrelevant immunoglobulin chains for human
therapy would not be feasible because the irrelevant H- and
L-chains would carry some helper epitopes themselves, thus
complicating the ability to achieve tolerance to the relevant H-
and L-chains. Nor would one expect to toterize those B-cells which
"see" idiotypic determinants formed by the combination of the
relevant H- and L-chains of the antibody.
[0017] Campath-1 is a cell-binding mAb, and an effective tolerogen
for use with it, such as a non-cell-binding form of the therapeutic
mAb would therefore be advantageous. The same goes for other
therapeutic antibodies which have cell-binding properties, and
non-cell-binding variants thereof.
[0018] The Invention
[0019] The invention therefore provides an antibody which is a
modified version of a therapeutic antibody with affinity for a
cell-surface antigen, said antibody having reduced affinity for the
antigen compared with the therapeutic antibody as a result of a
modification or modifications to the antibody molecule, wherein the
antibody is capable of inducing immunological tolerance to the
therapeutic antibody.
[0020] Preferably, the affinity of the antibody according to the
invention for the antigen is reduced to 50% or less of the affinity
of the therapeutic antibody for the antigen. More preferably, the
affinity is reduced to 10% or less, or to 1% or less of the
affinity of the therapeutic antibody. The affinity needs to be
sufficiently reduced to allow the antibody according to the
invention to act as a tolerogen with respect to the therapeutic
antibody. The term "non-cell-binding variant" is used herein to
refer to antibodies according to the invention, although antibodies
according to the invention may still have some binding affinity for
the cell surface antigen.
[0021] The ability of the antibody according to the invention to
induce immunological tolerance to a therapeutic cell-binding
antibody relies on the presence in the non-cell-binding antibody of
at least one epitope also present in the therapeutic antibody,
which induces an immune response in the intended patient.
[0022] The non-cell-binding antibody is preferably capable of
tolerising to anti-idiotypic responses, at least to the V domain
hypervariable regions of the therapeutic antibody and preferably
also to the framework regions. Thus it is desirable that the
tolerising antibody has an amino acid sequence similar to the
therapeutic antibody in those regions. Preferably there is >90%,
or >95% or >99% amino acid sequence identity between the
variable domains of the non-cell-binding antibody and the
therapeutic antibody. Most preferably the differences are
restricted to any amino acid substitution(s) required to
sufficiently reduce antigen binding affinity in the
non-cell-binding antibody.
[0023] Preferably also the non-cell-binding antibody is capable of
inducing tolerance to the constant regions of the therapeutic
antibody. Thus, it is preferred that the constant domains of the
non-cell-binding antibody are similar to those of the therapeutic
antibody, having for example >90% or >95% or >99% amino
acid sequence identity. Most preferably, the constant domains of
the non-cell-binding antibody and the therapeutic antibody are
identical and are thus matched allotypically.
[0024] The invention further provides fragments of an antibody
described herein, the fragments having tolerance-inducing
capability. Such fragments include monovalent and divalent
fragments such as Fab, Fab', and F(ab').sub.2 fragments. Also
included are single chain antibodies. The preferred features of
such fragments are as described herein in relation to
non-cell-binding antibodies according to the invention. The
non-cell-binding fragments may be for use with corresponding
therapeutic antibody fragments, or with therapeutic antibody
molecules.
[0025] The reduced binding affinity of the non-cell-binding
antibodies may be achieved in a variety of ways. In the preferred
embodiment described herein, an alteration in the CDRs comprising
one or two or more amino acid substitutions reduces binding
affinity. Alternatively, amino acid substitutions in other parts of
the antibody molecule may be used to reduce binding affinity. For
example, amino acid substitutions in the framework regions are
known to significantly affect binding affinity (Reichmann et al
1988). Another alternative is a monovalent form of the therapeutic
antibody. Monovalent antibodies have reduced binding affinity
compared to their bivalent counterparts. Monovalent forms may be
for example Fab fragments, or single chain antibodies, or any other
genetically engineered antibody fragments retaining a single
binding site. Monovalent variants can also be produced by mutating
the cysteine residue which participates in interchain (H-H)
disulphide formation (e.g., cys.fwdarw.ser or cys.fwdarw.ala). The
reduction in binding affinity of a monovalent antibody compared to
its bivalent counterpart may be sufficient to enable tolerance
induction. Preferably, the monovalent antibody is either incapable
of binding Fc receptors, or incapable of binding complement
component C1q, or both. Either or both of these properties can be
introduced by suitable mutations (see e.g., Morgan et al., WO
94/29351, published Dec. 22, 1994 and Winter et al., EP 0 307 434
B1).
[0026] The non-cell-binding antibodies or fragments according to
the invention may thus be one of a variety of types of antibodies
or fragments, including genetically engineered antibodies or
antibody fragments. In addition, the antibodies or fragments will
generally be from a mixture of origins. For example, they may be
chimeric e.g. human constant regions with rat variable domains; or
humanised or CDR grafted or otherwise reshaped (see, e.g., Cabilly
et al., U.S. Pat. No. 4,816,567; Cabilly et al., European Patent
No. 0 125 023 B1; Boss et al., U.S. Pat. No. 4,816,397; Boss et
al., European Patent No. 0 120 694 B1; Neuberger, M. S. et al., WO
86/01533; Neuberger, M. S. et al., European Patent No. 0 194 276
B1; Winter, U.S. Pat. No. 5,225,539; Winter, European Patent No. 0
239 400 B1; Queen et al., U.S. Pat. No. 5,585,089; Queen et al.,
European Patent No. 0 451 216 B1; Adair et al., WO 91/09967,
published Jul. 11, 1991; Adair et al., European Patent No. 0 460
167 B1; and Padlan, E. A. et al., European Patent No. 0 519 596 A1.
See also, Newman, R. et al., Biotechnology, 10:1455-1460 (1992),
regarding primatized antibody, and Huston et al., U.S. Pat. No.
5,091,513; Huston et al., U.S. Pat. No. 5,132,405; Ladner et al.,
U.S. Pat. No. 4,946,778 and Bird, R. E. et al., Science, 242:
423-426 (1988) regarding single chain antibodies). Campath-1H is
considered humanised although it contains two amino acid
substitutions in the framework regions.
[0027] Ideally, the antibody according to the invention is as close
as possible to the therapeutic antibody on which it is based.
Administration of such a "minimal mutant" prior to injection of the
cell-binding therapeutic mAb can be used to tolerise to all T- and
most B-cell epitopes in the therapeutic mAb. Classic experiments
indicate that tolerance is maintained more effectively by T- cells
than by B-cells. But since most B-cell responses including the
anti-Id response require T-cell help, even if a B-cell is
responsive to a given antigen, antibody production will be
determined by the state of responsiveness of the T-cells (Chiller
et al, 1971). Thus, it will be preferable to use a non-cell-binding
variant which contains the minimum differences needed to reduce its
affinity for the cell-surface antigen sufficiently to enable it to
be used as a tolerogen. By using techniques such as X-ray
crystallography, computer modeling and site-directed mutagenesis,
and also genetic methods such as phage display, it will be possible
to design suitable non-cell-binding variants for any cell-binding
therapeutic antibody.
[0028] The antibody according to the invention is preferably in
biologically pure form, desirably being at least 95% (by weight)
free of other biological materials.
[0029] As used herein, the term "cell-surface antigen" means an
antigen which is found on cell surfaces, but not necessarily
exclusively on cell surfaces.
[0030] The term "therapeutic antibody" is used herein to refer to
an antibody which may be administered to humans or animals to have
a desired effect in particular in the treatment of disease. Such
therapeutic antibodies will generally be monoclonal antibodies and
will generally have been genetically engineered.
[0031] In another aspect the invention comprises a composition for
administration to a patient comprising an antibody as described
herein, together with a physiologically acceptable diluent or
carrier.
[0032] In a further aspect, the invention provides a host cell or
cell line which expresses an antibody as herein described and use
of such a host cell or cell line for the production of such an
antibody.
[0033] Additional aspects of the invention include the use of an
antibody as described herein in the manufacture of medicament for
the induction of tolerance, in particular tolerance to a
therapeutic antibody.
[0034] In attached figures:
[0035] FIG. 1 shows the Campath-1H heavy chain minimal mutant
constructs prepared as described in the Examples.
[0036] FIG. 2 shows the PCR mutagenesis strategy for preparing the
mutant constructs of FIG. 1.
[0037] FIG. 3 shows pGEM9zf containing wild type Campath-1H heavy
chain, and substitution of mutant fragments in the heavy chain.
[0038] FIG. 4 shows a schematic representation of one embodiment of
a monovalent non-cell-binding therapeutic antibody.
[0039] Using Rational Design to Create a "Minimal Mutant"
[0040] In one embodiment for producing a non-cell-binding variant
of a therapeutic mAb, amino acid residues which are involved in
binding to target antigen are identified. Relative to the number of
residues that comprise the VL and VH domains, those that are
directly involved in interactions with antigen are small in number
(Novotny et al, 1983). And although the Ab-combining site is made
up of 6 hypervariable loops, 1 or 2 of those loops may dominate in
that interaction. If a key residue or residues can be identified,
it/they can be changed by site-directed mutagenesis to a residue
that will reduce (reduce or abolish) antigen-binding. Because these
residues will most likely be found within the hypervariable loop
structures and not in the framework sequence supporting those
loops, small changes may not significantly disrupt the overall
structure of the Ab.
[0041] Model Building of Ag-Binding Sites to Define Key Residues
for Mutagenesis:
[0042] Because the constant regions and variable regions of Ab
molecules are very similar in sequences and structures, general
principles regarding Ab structure have been defined using
relatively few solved crystal structures. To date, approximately 50
structures of Ab fragments have been included in the Brookhaven
Protein Data Bank, and of these, 20% have been refined to a
resolution of 2.0 angstroms or better. As the structural knowledge
base increases, comparative Ab modelling (modelling by homology)
becomes more reliable since there is a greater choice of structural
templates. Variable regions (VL and VH) of different Ab structures
can be combined as a structural template after superimposing their
most conserved residues. Side chain conformations of buried
residues are then modelled. The CDR loops are modelled by
identification of structurally similar loop templates (often loops
with the same length and similar sequence have similar backbone
conformations). These CDR sequences often fall into canonical loop
motifs (excluding H3, between 50 and 95% of murine VL (kappa) and
VH have loop sequences consistent with classified canonical
motifs). Canonical loop backbones can then be spliced onto the
model of the framework and CDR side-chain conformations can be
modelled based on conformations of residues found at corresponding
positions in other loops of the same canonical structure. Finally
the model is often refined using computer programs that minimize
troublesome stereochemical constraints.
[0043] Comparative model building is becoming widely used as the
size of the structural database increases, providing a greater
range of structural templates. Also, the greater range of computer
programs available ensures that models are becoming increasingly
accurate. For example, the solved crystal structure of Campath-1H
was very close to the structure predicted by molecular modelling.
We could predict from the modelling data that mutations 1 and 2
described in the Examples were likely to have a detrimental effect
on binding to CD52. The crystal structure confirmed these
predictions and also predicted that mutation 3 could disrupt
binding to CD52.
[0044] Obviously, to create a non-cell-binding version of a
therapeutic Ab, it is desirable to start with a solved crystal
structure, preferably co-crystallized with antigen so that the key
contact residue(s) can be identified and substituted for residue(s)
that destroy antigen binding. However, in many cases, a good
molecular model could provide the necessary information. In cases
where the molecular model is of poor quality (for example, if the
appropriate structural templates do not exist in the databank), CDR
swapping experiments (as described in the Examples) will provide
information on which CDRs must be targeted for mutation. Alanine
scanning mutagenesis (mutating each residue sequentially to Ala)
through those regions could identify the key residue(s) involved in
antigen-binding (Cunningham and Wells, 1989). If changing a single
residue to Ala reduced but did not destroy binding, that position
could be targetted for more drastic mutations (for example, a
substitution that created in a charge difference) to further reduce
binding, if desired.
[0045] Alternative methods for obtaining non-cell-binding versions
of therapeutic antibodies include genetic techniques such as phage
display using error prone PCR (Gram et al, 1992) and cycling of
V-region genes (e.g. as sFv constructs) through a bacterial mutator
strain (e.g. mutD5) (Low et al., 1996). Such genetic methods can
provide powerful screening systems.
[0046] The invention will now be further described in the examples
which follow. Although the specific example of Campath-1H is given
in this document, the invention is not limited to antibodies based
on Campath-1H. It is anticipated that other cell-binding
therapeutic antibodies, especially those which would be given in
repeated doses, will become more widely accepted using this
strategy.
EXAMPLES
[0047] A. Creating a "Minimal Mutant"
[0048] We have devised a method to determine which of the CDR loops
of the humanized Campath-1 mAb are the most important ones for
binding to CD52. Mutant VL or VH were genetically constructed in
which each of the 6 hypervariable regions (as defined by Kabat et
al (1987) using amino acid sequence alignments of V-regions in the
protein databases) was individually swapped for the corresponding
CDR from the V-region that had provided the human VL or VH acceptor
sequence during humanization (REI and NEW, respectively). The
engineered V-regions were expressed as Fab fragments in E. coli
using the pHEN vector (Hoogenboom et al, 1991). In this system, the
peIB leader sequence was used to direct protein expression to the
periplasm, where association of L-chain and truncated H-chain
occurs (Hoogenboom et al, 1991). When these Fab fragments were
assayed for binding to immobilized CD52, it was found that swapping
the (VH) CDR2 of NEW into the humanized Campath-1 Fab completely
destroyed binding to CD52. Replacing (VH) CDR3 reduced binding to
CD52 8-fold while replacing (VH) CDR1 and (VL) CDR3 reduced binding
3-fold. No change in binding was detected when (VL) CDR1 or (VL)
CDR2 were replaced. From these results, it appeared that (VH) CDR2
contained a key residue(s) necessary for antigen-binding. DNA
encoding the "wild-type" humanized Campath-1H-chain (Reichmann et
al, 1988) was used as PCR template for site-directed mutagenesis.
This heavy chain sequence encodes human protein at all positions
except the three VH CDR regions and positions 27 and 30 of the
first framework region. We focussed on the H2 loop within VH CDR2
to make mutations which would abolish binding of the Ab to CD52. H2
is the actual loop structure (Chothia and Lesk, 1987) that is found
within the 19 amino acid VH CDR2 denoted "hypervariable" by Kabat
et al's definition (Kabat et al, 1987) (see FIG. 1). It is known
that a few key residues in the loop and/or framework regions
determine relatively few CDR loop conformations and canonical loop
motifs have been identified for most CDR including VH CDR2 (Chothia
and Lesk, 1987). Since it is the loop structures that stick out
from the V-region .beta.-barrel framework to make contact with
antigen, mutations in the loop would have the greatest chance of
destroying antigen binding whilst preserving Ab structure. In
general, we restricted the changes to the H2 loop except for
H-chain mut6 which contained an additional mutation in the residue
immediately preceeding H2 as discussed in more detail below.
[0049] Summary of Campath-1H Heavy Chain Minimal Mutant Constructs
(FIG. 1)
[0050] Mutation 1 is a single charge difference at residue 52b from
Lys to Asp. It was predicted from the molecular modelling of
Campath-1H Ab, and supported by the crystal structure, that the
side chain of this residue is pointing out of the Ag binding
pocket, towards the approach of antigen. Since the positive charge
of the Lys is thought to interact with the negatively charged
phosphate groups of the GPI anchor of CD52, it is possible that
this single mutation will destroy antigen-binding.
[0051] Mutation 2 is a single charge difference at residue 52a from
Asp to Lys. It was predicted from the molecular modelling of
Campath-1H Ab that this change could interfere with
antigen-binding.
[0052] Mutation 3 is a single charge difference at residue 53 from
Lys to Asp. From the crystal structure of Campath-1H Ab, it is
clear that the majority of this residue side chain is solvent
accessible and therefore may be involved in the interaction with
the negatively charged phosphate groups of the GPI anchor of CD52,
as for mutation 1.
[0053] Mutation 4 is a double mutation encompassing the individual
substitutions of mutant 1 and 3 (Lys52b and Lys53 to Asp).
[0054] Mutation 5 is a triple mutation encompassing the individual
substitutions of mutant 1, 2 and 3 (three charge differences:
Asp52a to Lys; Lys52b and Lys53 to Asp).
[0055] Mutation 6 is a triple mutation encompassing the individual
substitutions of mutant 1 and 2 (two charge differences Asp52a to
Lys; Lys52b to Asp), and an additional mutation of Arg52 to Ala.
Residue 52 has been shown to differ between 3 different Campath-1
Ab of high, low and moderate affinity and may be therefore directly
involved in affinity maturation. This in turn might suggest a role
in antigen binding.
[0056] For each of these heavy chain mutations, the change(s)
was/were encoded on oligonucleotide primers 1B and 2A (FIG. 2). PCR
was carried out on "wild-type" Campath-1 heavy chain DNA using a 5'
primer annealing to the leader sequence and containing an upstream
HindIII site (primer 1A) and primer 1B to generate a 200 bp
fragment. Similarly, primer 2B (annealing to CH1 and containing a
BstXI site followed by an EcoRI site) and primer 2A, a 440 bp
fragment was generated. These fragments were gel purified and then
combined in a single PCR reaction. Primer 1A and primer 2B were
added after the first cycle (thus allowing the 2 pieces of
overlapping DNA to anneal before amplification). Following PCR, the
fragments were gel purified and digested with HindIII and EcoRI and
were transferred into intermediate sequencing vectors (PUC19 or
pGEM3zf) for verification of sequence. A unique PstI site located
in the Campath-1H VH and a unique BstXI site in the CH1 region
allowed the mutant V-regions (and partial CH1 sequence) to be
isolated as PstI-BstXI fragments such that the mutation(s) were
encoded in six different DNA cassettes flanked by PstI and BstXI
sites. To create the mutant Campath-1H heavy chains, the original
heavy chain construct (in intermediate vector pGEM9zf) was cut with
PstI and BstXI and the fragment was removed. The remaining DNA
(encoding the Campath-1H heavy chain leader sequence and the
V-region upsteam of the PstI site, plus the CH1 region downstream
of the BstXI site followed by the hinge, CH2, CH3 in pGEM9zf) was
gel purified (FIG. 3). Ligations were then set up in which each of
the DNA cassettes containing the mutation(s) described above was
joined to the gel purified pGEM9zf/PstI-BstI cut Campath-1H heavy
chain DNA. These six mutagenized Campath-1H heavy chains were then
isolated by digestion with HindIII and gel purification, followed
by ligation into HindIII cut mammalian expression vector pBAN-2.
This vector is derived from the pNH316 vector that contains a
neomycin selectable marker under the control of the mouse
metallothionein promoter and the strong human .beta.-actin
promoter/polyadenylation signals for expression of the desired gene
product (Page and Sydenham, 1991). As these fragments were
introduced into a single HindIII restriction site, orientation of
each fragment was checked by DNA sequencing.
[0057] Campath-1H Light Chain Construct for Co-Transfection:
[0058] DNA encoding the humanized "wild type" Campath-1H light
chain (human sequence at all residues except the three CDR in the
V-region) was isolated from an intermediate vector as a HindIII to
EcoRI fragment. This fragment was gel purified and then ligated
into HindIII-EcoRI cut mammalian expression vector pRDN-1. This
vector is derived from the pLD9 vector that contains a "crippled"
dihydrofolate reductase (dhfr) selectable marker (the enhancer
element of the SV40 promoter has been removed to allow for
increased levels of gene expression in the presence of
methotrexate) and the strong human .beta.-actin
promoter/polyadenylation signals for expression of the desired gene
product (Page and Sydenham, 1991).
[0059] Co-Transfection of Campath-1H Light Chain DNA and Mutant
Heavy Chain DNA:
[0060] The expression system used to produce high levels of
humanized Campath-1H Ab in the past is the widely used mammalian
expression system featuring gene amplification by the use of
dihydrofolate reductase (dhfr) deficient Chinese hamster ovary
(CHO) cells and the use of strong .beta.-actin promoters for
selection and amplification of the desired gene products (Page and
Sydenham, 1991).
[0061] The following transfections (TF) were carried out:
[0062] TF1: mock ("empty" pRDN-1 plus "empty" pBAN-2)
[0063] TF2: Light chain only (Light-chain/pRDN-1 plus "empty"
pBAN-2)
[0064] TF3: Light chain/pRDN-1 plus H chain mutant 1/pBAN-2
[0065] TF4: Light chain/pRDN-1 plus H chain mutant 2/pBAN-2
[0066] TF5: Light chain/pRDN-1 plus H chain mutant 3/pBAN-2
[0067] TF6: Light chain/pRDN-1 plus H chain mutant 4/pBAN-2
[0068] TF7: Light chain/pRDN-1 plus H chain mutant 5/pBAN-2
[0069] TF8: Light chain/pRDN-1 plus H chain mutant 6/pBAN-2
[0070] TF9: Light chain/pRDN-1 plus H chain "wild-type"/pBAN-2 DNA
(20 .mu.g of light chain/pRDN-1 plus 20 .mu.g of heavy
chain/pBAN-2) was mixed in a sterile eppendorf, ethanol
precipitated, and rinsed twice with 70% ethanol. DNA pellets were
resuspended in sterile Tris-EDTA.
[0071] For each transfection, the DNA was diluted with 60 .mu.l of
20 mM HEPES (pH 7.4) in a 5 ml polystyrene tube. In another tube,
120 .mu.l of DOTAP liposomal transfection reagent (Boehringer
Mannheim) was diluted with 80 .mu.l of 20 mM HEPES (pH 7.4). Then
the DNA/HEPES was added to the diluted DOTAP, mixed gently, and
left at room temperature for 15 min. Culture medium (IMDM+5%
FCS+HT) was aspirated from a T75 flask containing dhfr deficient
CHO cells growing at approximately 50% confluency. The DNA/DOTAP
was then added to the flask along with 10 ml fresh culture medium.
The flask was cultured for 24 h at 37.degree. C. in 5% CO.sub.2.
The DNA/DOTAP was then aspirated from the flask and the cells were
given 15 ml fresh culture medium. After a further 24 h, selection
was initiated by removing the culture medium and adding selection
medium (IMDM+5% dialysed FCS+1 mg/ml G418). The cells were cultured
at 37.degree. C. in 5% CO.sub.2 and fresh selection medium was
added as necessary. Culture supernatants were then tested by ELISA
for the presence of antibody as described below.
[0072] Detection of Secreted Ab in Transfection Supernatants by
ELISA:
[0073] Microtitre plates were coated with 50 .mu.l/well anti-human
Ig Fc (Sigma, catalogue number I-2136) in PBS at 2.5 .mu.g/ml
overnight at 4.degree. C. The coating Ab was removed and the plates
were blocked by addition of 100 .mu.l/well blocking buffer (PBS+1%
BSA+5% FCS+1% heat-inactivated normal rabbit serum (NRS) overnight
at 4.degree. C. The transfection supernatants were added (50
.mu.l/well) for at least 1 h at room temperature. The wells were
washed with PBS/0.5% Tween-20 (PBS/Tween). Biotinylated sheep
anti-human Ig (Amersham, catalogue number RPN 1003) diluted 1/5000
in blocking buffer or biotinylated goat anti-human kappa light
chain (Sigma, catalogue number B-1393) diluted 1/1000 in blocking
buffer was added (50 .mu.l/well) for 1 h at room temperature. The
wells were washed with PBS/Tween and 50 .mu.l/well
ExtrAvidin-peroxidase (Sigma, catalogue number E-2886) was added
for 30 min at room temperature. The wells were washed once more and
100 .mu.l/well of substrate o-phenylenediamine dihydrochloride
(Sigma, catalogue number P-7288) was added. Colour change was
measured at 492 nM using a Multiskan Plus microtitre plate
reader.
[0074] Detection of Binding to Campath-1 Antigen by ELISA:
[0075] Microtitre plates were coated with 50 .mu.l/well anti-mouse
Ig Fc (Sigma, catalogue number M4280) in PBS at 2.5 .mu.g/ml
overnight at 4.degree. C. The coating Ab was removed and the plates
were blocked by addition of 100 .mu.l/well blocking buffer (PBS+1%
BSA+5% FCS+1% heat-inactivated NRS) overnight at 4.degree. C.
Purified recombinant Campath-1 Ag-fusion protein (sequence encoding
the CD52 peptide backbone fused to sequence encoding mouse CH2 and
CH3 domains, and purified on a protein A column) was then added at
4 .mu.g/ml to each well (50 .mu.l/well) in PBS overnight at
4.degree. C. The wells were then washed with PBS/Tween and the
transfection supernatants were added (50 .mu.l/well) for at least 1
h at room temperature. The wells were washed with PBS/Tween and
biotinylated sheep anti-human Ig (Amersham, catalogue number RPN
1003) diluted 1/5000 in blocking buffer was added (50 .mu.l/well)
for 1 h at room temperature. The wells were washed with PBS/Tween
and 50 .mu.l/well ExtrAvidin-peroxidase (Sigma, catalogue number
E-2886) was added for 30 min at room temperature. The wells were
washed once more and 100 .mu.l/well of substrate o-phenylenediamine
dihydrochloride (Sigma, catalogue number P-7288) was added. Colour
change was measured at 492 nM.
[0076] Assessment of Non-Binding Mutants by ELISA:
[0077] "Wild-type" purified Campath-1H Ab binds strongly to
recombinant Campath-1 Ag-fusion protein in ELISA assays. To provide
a comparison for assessing non-cell-binding antibodies, the
wild-type Ab can be titrated down in concentration until binding is
just detectable. This may be referred to as "1 Ab binding unit". A
suitable non-binding mutant of the wild-type will not show
detectable binding at many times this concentration e.g. 100 times,
or 1000 times, or preferably 10,000 times this concentration of
wild-type Campath-1H Ab.
[0078] Assessment of Non-Binding Mutants In Vivo:
[0079] An alternative method which can be applied to assess
non-binding potential is described. Because we know that the
wild-type Campath-1H Ab elicits a strong anti-immunoglobulin
response in transgenic mice expressing human CD52 (see next section
for details on these mice), purified deaggregated preparations of
the mutants can be used in vivo to assess whether they are
immunogenic. If an anti-globulin response cannot be detected at
doses between 1 .mu.g and 1 mg deaggregated mutant per mouse, this
is a good indication that the mutant is unable to bind CD52.
[0080] B. In Vivo Models of Tolerance Induction:
[0081] To test the ability of the minimal mutants of Campath-1H to
tolerize to the wild-type Campath-1H Ab in vivo, transgenic mice
are used. For example, transgenic mice expressing human CD52 behind
a murine CD2 promoter to mimic the expression of CD52 on T-cells
can be used.
[0082] To create such mice, a 2.8 kb genomic fragment containing
the 2 exons of the human CD52 gene as well as 4.5 kb upstream and
3' flanking sequence of the human CD2 gene can be introduced into
the genome of transgenic mice. It is thought that strong control
regions are present 3' to the human CD2 gene that determine the
high levels and tissue-specific expression of the gene (Greaves et
al, 1989). By this method, four CD52/CBA founders were established
that transmitted the transgene. Indeed, when peripheral blood
staining of their offspring was analysed by fluorescence activated
cell sorting and 2-colour staining, it was shown that the cells
expressing mouse CD3 (T-cells) also expressed human CD52 (D.
Kioussis, unpublished data). These mice were bred to homozygosity
and greater than 95% of their T-cells express high levels of human
CD52 on the cell surface.
[0083] These mice produce a vigorous anti-globulin response (titre
of 1/1000 or greater) to wild-type Campath-1H at doses of 1 to 10
mg/mouse. This anti-globulin response includes an anti-Id component
as the CDR loops are rat sequence. The effectiveness of the minimal
mutants to tolerize to subsequent challenge of wild-type Campath-1H
Ab may be assessed in the following ways:
[0084] 1. Intravenous administration of a single dose (0.5 to 1
mg/mouse day 0) of each non-cell-binding mutant or irrelevant
control Ab (deaggregated by ultracentrifugation) followed by
challenge with 1 to 10 mg wild-type Campath-1H Ab at 4 to 6 wks.
Tail bleeds 10 days post challenge are tested by ELISA for
Campath-1H anti-Id specificity.
[0085] 2. Intravenous administration of multiple doses (0.5 to 1
mg/mouse) of deaggregated non-cell-binding mutant or control Ab
over 2 months prior to challenge with 1 to 10 mg wild-type
Campath-1H Ab. Tail bleeds 10 days post challenge are tested by
ELISA for Campath-1H anti-Id specificity. In both cases, the
irrelevant control Ab is be an isotype-matched non-cell-binding Ab
in mice such as Campath-9 which is a humanized anti-CD4 Ab (Gorman
et al, 1991).
[0086] C. How the Strategy Could be Adopted for Human Therapy:
[0087] An amount (e.g. 500 mg) of the non-cell-binding form of the
therapeutic antibody would be administered to a patient awaiting
treatment with the therapeutic antibody. Preferably the
non-cell-binding antibody as administered is freshly deaggregated
(for example by passage through a fine filter). A period of time
later (for example 7 days), during which time the T-cells and
B-cells would become tolerised, the wild-type form of the
therapeutic antibody would be given.
[0088] 1:). Additional Considerations:
[0089] 1. It should be easier to tolerize to a minimal mutant than
to HGG or to mixed chain Ab molecules.
[0090] In the tolerance models of Benjamin et al (1986), tolerance
to polyclonal HGG was induced in mice following depletion of CD4+
T-cells, but also using deaggregated material. It was found that
tolerance to these soluble proteins could be achieved relatively
easily. Also, in the work of Isaacs and Waldmann (1994), CD4 Ab
were given during tolerance induction to the non-cell-binding mixed
chain Ab molecules (irrelevant and antigen specific H- and
L-chains), or non-cell-binding forms were used as tolerogens in
their own right following their deaggregation.
[0091] In our modified approach to inducing tolerance using a
minimal mutant, the foreigness of the protein will be less than
that of polyclonal HGG or of the mixed chain Ab molecules in mice.
In cases where the therapeutic mAb is humanized, only the CDR loops
(and in some cases, some framework positions) are comprised of
rodent sequence. It therefore may be possible to tolerize with a
deaggregated minimal mutant in the absence of CD4 mAb. However,
even if CD4 administration was required, a humanized therapeutic
CD4 is available (CAMPATH-9; Gorman et al, 1991). The studies in
transgenic animals should address these details.
[0092] 2. Creation of a Monovalent Form of the Minimal Mutant (FIG.
4).
[0093] Thus far we have considered tolerance induction using a
minimal mutant that is essentially like the wild-type therapeutic
except for minimal residue change(s) that will disrupt antigen
binding. We also propose a monovalent form that is also
significantly smaller than the minimal mutant.
[0094] In one embodiment, the monovalent form is a single-chain Fv
[formed by the VL, a short peptide linker (such as those reviewed
in Huston et al, 1991) and the mutated VH] genetically fused with
the sequence encoding the hinge-CH2-CH3 of human IgG1. This
construct is expressed in association with a truncated heavy chain
(hinge-CH2-CH3 only; Routledge et al, 1991) such that a protein is
expressed that is composed essentially of a single Ab-combining
site and a functional Ig Fc domain. The immunogenicity of the
different peptide linkers is expected to be negligible given their
small size (generally 14 to 18 residues in length) and abundance of
small residues (eg Gly and Ser) making up the linkers. A popular
choice is the 15-residue linker (Gly.sub.4Ser).sub.3 in which the
serine residues confer extra hydrophilicity on the peptide backbone
(to inhibit its intercalation between the variable domains during
folding) and which is otherwise free of side chains that might
complicate domain folding (Huston et al, 1988).
[0095] SFv have been expressed in mammalian cells from a number of
different antibodies and have been shown to fold into the correct
conformation for antigen-binding by functional activity (Gilliland
et al, 1996). The Fc portion is a preferred feature which should
ensure serum half-life comparable to the minimal mutant and to the
wild-type therapeutic Ab, whilst monovalency will ensure that
binding to CD52 is greatly reduced due to the decrease in avidity.
We have already shown from the CDR-swapping experiments (section
A1) that the Campath-1 Ab binds poorly to CD52 in a monovalent
form. In addition to reducing the avidity of the molecule, the
smaller size may be a bonus: in classical tolerance experiments, it
was found that the smaller the molecule, the better it was at
inducing tolerance (Parish and Ada, 1969; Anderson, 1969; Miranda
et al, 1973). By combining monovalency with a non-cell-binding
mutant, a highly effective tolerogen may be obtained.
REFERENCES
[0096] Anderson B. 1969. Induction of immunity and immunologic
paralysis in mice against polyvinyl pyrrolidone. J Immunol 102,
1309-1313.
[0097] Benjamin R J, Cobbold S P, Clark M R and Waldmann H. 1986.
Tolerance to rat monoclonal antibodies: implications for
serotherapy. J Exp Med 163, 1539-1552.
[0098] Bird R E, Hardman K D, Joacobson J W. 1988. Single-chain
antigen-binding proteins. Science 242, 423-426.
[0099] Brown J H, Jardetzky T S, Gorga J C, Stern", Urban R G,
Strominger J L, Wiley D C. 1993. Three-dimensional structure of the
human class II histocompatability antigen HLA-DR1. Nature 364,
33-39.
[0100] Chiller J M, Habicht G S, Weigle W O. 1971. Kinetic
differences in unresponsiveness of thymus and bone marrow cells.
Science 171, 813-815.
[0101] Chothia C and Lesk A M 1987. Canonical structures for the
hypervariable regions of immunoglobulins. J Mol. Biol. 196,
901-917.
[0102] Chothia C, Lesk A M, Tramontano A, Levitt M, Smith-Gill S J,
Air G, Sheriff S, Padlan E A, Davies D, Tulip W R, Colman P M,
Spinelli S, Alzari P M, Poljak R J. 1989. Conformations of
immunoglobulin hypervariable regions. Nature 342, 877-883.
[0103] Chothia C, Lesk A M, Gherardi E, Tomlinson I M, Walter G,
Marks J D, Llewelyn, M B, Winter G. 1992. Structural repertoire of
the human VH segments. J Mol Biol 227, 799-817.
[0104] Cobbold S P, Clark M R, Benjamin R J, Waidmann H. Monoclonal
antibodies and their use. EP 0 536 807 and EP 0 240 344. Wellcome
Foundation Ltd.
[0105] Cunningham, B. C. and Wells, J. A. 1989. High resolution
epitope mapping of hGH-receptor interactions by alanine-scanning
mutagenesis. Science, 244, 1081-1085.
[0106] Gilliland L K, Norris N A, Marquardt H, Tsu T T, Hayden M S,
Neubauer M G, Yelton D E, Mittler R S, Ledbetter J A. 1996. Rapid
and reliable cloning of antibody variable regions and generation of
recombinant single chain antibody fragments. Tissue Antigens, 47,
1-20.
[0107] Gorman S D, Clark M R, Routledge, E G, Cobbold S P, Waldmann
H. 1991. Reshaping a therapeutic CD4 antibody. Proc Natl Acad Sci
USA 88, 4181-4185.
[0108] Gram H, Marconi L A, Barbas C F 3rd, Collet T A, Lerner R A,
Kang A S. 1992. In vitro selection and affinity maturation of
antibodies from a naive combinatorial immunoglobulin library. Proc
Natl Acad Sci USA 89, 3576-3580.
[0109] Greaves D R, Wilson F D, Lang G, Kioussis D. 1989. Human CD2
3'-flanking sequences confer high-level, T-cell specific,
position-independent gene expression in transgenic mice. Cell 56,
979-986.
[0110] Hale G, Xia M-Q, Tighe H P, Dyer M J S, Waldmann H. 1990.
The CAMPATH-1 antigen (CDw52). Tissue Antigens 35, 118-127.
[0111] Hale G, Hoang T, Prospero T, Watt S M, Waldmann H. 1983.
Removal of T cells from bone marrow for transplantation: comparison
of rat monoclonal anti-lymphocyte antibodies of different isotypes.
Mol Biol Med 1, 305-319.
[0112] Huston J S, Mudgett-Hunter M, Tai M-S, McCartney J, Warren
F, Haber E, Oppermann H. 1991. Protein engineering of single-chain
Fv analogs and fusion proteins. Methods Enzymol 203, 46-88.
[0113] Hoogenboom H R, Griffiths A D, Johnson K S, Chiswell D J,
Hudson P, Winter G. 1991. Multi-subunit proteins on the surface of
filamentous phage: methodologies for displaying antibody (Fab)
heavy and light chains. Nucl Acids Res 19, 4133-4137.
[0114] Hunt D F, Michel H, Dickinson T A, Shabanowitz J, Cox A L,
Sakaguchi K, Apella E, Grey H M, Sette A. 1992. Peptides presented
to the immune system by the murine class II major
histocompatability molecule I-Ad. Science 256, 1817-1820.
[0115] Huston J S, Levinson D, Mudgett-Hunter M et al. 1988.
Protein engineering of antibody binding sites: recovery of specific
activity in an anti-digoxin single-chain Fv analogue produced in
Escherichia coli. Proc Natl Acad Sci USA, 85, 5879-5883.
[0116] Isaacs J D, Watts R A, Hazleman B L et al. 1992. Humanized
monoclonal antibody therapy for rheumatoid arthritis. Lancet 340,
748-752.
[0117] Isaacs J D, Waldmann H. 1994. Helplessness as a strategy for
avoiding antiglobulin responses to therapeutic monoclonal
antibodies. Therapeutic Immunol 1, 303-312.
[0118] Jones P T, Dear P H, Foote J, Neuberger M S, Winter G. 1986.
Replacing the complementarity-determining regions in a human
antibody with those from a mouse. Nature 321, 522-525.
[0119] Kabat E A, Wu T T, Reid-Miller M, Perry H M, Gottesman K S.
1987. Sequences of Proteins of Immunological Interest, 4th ed.
Washington D.C., Public Health Service, NIH.
[0120] Levitskaya J, Coram M, Levitsky V, Imreh S,
Steigerwald-Mullen P M, Klein G, Kurilla M G, Masucci M G. 1995.
Inhibition of antigen processing by the internal repeat region of
the Epstein-Barr virus nuclear antigen-1. Nature 375, 685-688.
[0121] Lockwood C M, Thiru S, Isaacs J D, Hale G, Waldmann H. 1993.
Long-term remission of intractable systemic vasculitis with
monoclonal antibody therapy. Lancet 341, 1620-1622.
[0122] Low N M, Hollinger P H, Winter G. 1996. Mimicking somatic
hypermutation: affinity maturation of antibodies displayed on
bacteriophage using a bacterial mutator strain. J Mol Biol 260,
359-368.
[0123] Miranda J J Zola H, Howard J G. 1973. Studies on
immunological paralysis. X. Cellular characteristics of the
induction and loss of tolerance to leva (polyfructose). Immunology
23, 843-855.
[0124] Novotny J, Bruccoleri R E, Newell J, Murphy D, Haber E,
Karplus M. 1983. Molecular anatomy of the antibody binding site. J
Biol Chem 258, 14433-14437.
[0125] Page M J, Sydenham M A. 1991. High level expression of the
humanized Biotechnol. 9, 64-68.
[0126] Parish C R, Ada G L. 1969. The tolerance inducing properties
in rats of bacterial flagellin cleaved at the methionine residues.
Immunology 17, 153-164.
[0127] Riechmann L, Clark M, Waldmann H, Winter G. 1988. Reshaping
human antibodies for therapy. Nature 332, 323-327.
[0128] Routledge E G, Gorman S D, Clark M R. 1993. Reshaping
antibodies for therapy. In Protein Engineering of Antibody
Molecules for Prophylactic and Therapeutic Applications in Man. ed.
M Clark, Academic Titles, Nottingham, UK. pp 13-44.
[0129] Routledge E G, Lloyd I, Gorman S D, Clark M, Waldmann H.
1991. A humanized monovalent CD3 antibody which can activate
homologous complement. Eur J Immunol 21, 2717-2725.
[0130] Rudensky A Y, Preston-Hurlburt P, Hong S, Barlow A, Janeway
C A Jr. 1991. Sequence analysis of peptides bound to MHC class II
molecules. Nature 353, 622-627.
[0131] Rudensky A, Janeway C A Jr. 1993. Studies on Naturally
processed peptides associated with MHC class II molecules. In Sette
A (ed.) Naturally Processed Peptides. Basel, Karger, pp
134-151.
[0132] Xia M-Q Tone M, Packman L, Hale G, Waldmann H. 1991.
Characterization of the CAMPATH-1 (CDw52) antigen: biochemical
analysis and cDNA cloning reveal an unusually small peptide
backbone. Eur J Immunol 21, 1677-1684.
* * * * *